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University of Groningen
Vascular reactivity in cardiopulmonary bypassSamarska, Iryna
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Vascular reactivity in
cardiopulmonary bypass
Iryna Samarska
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Financial support by the Groningen University Institute for Drug Exploration (GUIDE) and the University of Groningen for the publication of this thesis is gratefully acknowledged.
Samarska, I.V. Vascular reactivity in Cardiopulmonary bypass Proefschrift Groningen ISBN: 978-90-367-4789-9 ISBN: 978-90-367-4788-2
© Copyright 2011 I.V.Samarska All rights are reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, without permission of the author. Cover: Iryna Samarska. Source: Servier Medical Arts. Lay-out: Iryna Samarska Printed by Ipskamp Drukkers, Enschede
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RIJKSUNIVERSITEIT GRONINGEN !!!!
Vascular reactivity in cardiopulmonary bypass
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Proefschrift
ter verkrijging van het doctoraat in de Medische Wetenschappen
aan de Rijksuniversiteit Groningen op gezag van de
Rector Magnificus, dr. E.Sterken, in het openbaar te verdedigen op
woensdag 6 april 2011 om 11.00 uur
door
Iryna Samarska geboren op 8 januari 1979
te Kiev, Oekraïne
Promotores: Prof. dr. R.H. Henning Prof. dr. M.M.R.F. Struys Copromotores: Dr. J.H. Buikema Dr. A.H. Epema Beoordelingscommissie: Prof. dr. P. Wouters Prof. dr. G. Molema Prof. dr. J.G.R. de Mey
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Paranimfen: Magdalena Mazagova
Roelien Meijering
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Table of contents
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Chapter 1 Introduction and the aims of the thesis
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Chapter 2 Changes in vasomotor function of small coronary and mesenteric artery during 5-day follow-up after cardiopulmonary bypass in the rat. Characterization of endothelial mediators and contractility Submitted
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Chapter 3 Changes of vascular reactivity of rat thoracic aorta during prolonged recovery following cardiopulmonary bypass Manuscript in preparation
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Chapter 4 Microarray analysis of gene expression profiles in the kidney demonstrates a local inflammatory response induced by cardiopulmonary bypass in rat Submitted
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Chapter 5 Protracted pulmonary inflammation during long-term recovery following cardiopulmonary bypass in the rat Manuscript in preparation
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Chapter 6 Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia Anesthesiology 2009 Sep;111(3):600-8
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Chapter 7 S1P1 receptor modulation improves systemic vascular dysfunction after CPB in the rat independent of depletion of lymphocyte: a comparison between FTY720 and SEW2871 Manuscript in preparation
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Chapter 8 Summary and future perspectives
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Supplements Supplemental digital content to the Chapter 4 Editorial Views. Sanders RD, Maze M. Does correcting the numbers improve long-term outcome? Anesthesiology 2009 Sep;111(3):475-7
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Nederlandse Samenvatting
Ukrainian Summary
Acknowledgments
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Chapter 1
Introduction and the aims of the thesis
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Clinical aspects of cardiopulmonary bypass Cardiopulmonary bypass (CPB) is a widely used technique in cardiothoracic
surgery, including procedures such as coronary artery bypass grafting, valvular heart surgery, repair of aortic aneurysms, surgical treatment of congenital heart defects, heart-, lung or heart-lung transplant, and pulmonary thrombectomiy or tromboendarterectomy. The first successful open-heart surgery with extracorporeal circulation on a patient was performed in 1953, by John Gibbon in Philadelphia (USA) and consisted of an atrial septal defect repair. Surgery with CPB has now become a standard procedure.1 The principal components of the cardiopulmonary circuit include tubing, a pump, an oxygenator and cannulae. They enable clinicians’ to reach the main goal of CPB, namely temporarily taking over the function of heart and lung (fig. 1).2
However, CPB is also associated with serious postoperative clinical complications, including dysfunction of vital organs such as heart, kidney, lung, and liver.3;4 Several clinical syndromes are related to CPB, including increased pulmonary vascular resistance with postoperative pulmonary insult,5;6 pulmonary dysfunction,5;6 myocardial ischemia and atrial fibrillation,7 postoperative renal function deterioration,8-
11 renal ischemic injury up to dialysis-dependent renal failure,3;4;12;13 intestinal ischemia,12 stroke, neurocognitive dysfunction and neurologic complications.14. The post-CPB incidence of these syndromes and associated conditions is substantial, and associated with a high morbidity and mortality rate or prolonged hospital stay.15;16. Moreover, a transient organ dysfunction may be observed in patients with preoperatively normal function.10;11
Renal complications Acute kidney injury is thought to represent the most frequently occurring
adverse effect of CPB, present in up to 30% of the patients after cardiac surgery.17 Because of reduced clearance, decreased renal function results in metabolic disturbances with metabolic acidosis and hyperkalemia, which in turn affect function in other organs. Kidney injury post-CPB varies from mild renal dysfunction up to full blown renal failure requiring dialysis.18 The etiology of the CPB associated renal injury is multifactorial and includes perioperative renal hypoperfusion with possible ischemia-reperfusion injury, presence of nephrotoxins and microembolism.15;16;19. Moreover, activation of neutrophils, monocytes, endothelial cells and the complement system resulting from this systemic inflammatatory response, further adds to glomerular and tubular disturbances.18 In addition, postoperative renal dysfunction negatively affects long-term survival after cardiac surgery.8-11;18
Pulmonary complications Another severe complication of CPB is a postoperative lung dysfunction with
interstitial pulmonary edema and subsequent abnormal gas exchange. Clinical data demonstrated that approximately 25% of the patients following open heart surgery exhibited signs of pulmonary impairment for at least one week thereafter.20-22 Several CPB-related pathophysiological processes were described in the lung, namely, increased vascular permeability, water content, vascular resistance, neutrophils
Introduction and the aim of the thesis 11
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sequestration, arterial-alveolar gradient, decrease in pulmonary compliance, alveolar-capillary injury, rise in shunt fraction, and likelihood of atelectasis and pneumonia.15;16;23 The induction of such critical complications can be explained by the specifics of the lung perfusion during CPB, since lungs are deprived of the majority of their normal blood supply in this period. Thus extracorporeal circulation causes serious pulmonary injury with vascular injury and edema, atelectasis, collapse of non-ventilated lungs, and massive or submassive pulmonary embolism.17;24
CPB-related hypotension Although the phenomenon of CPB-induced hypotension has been addressed
during the last decade, the exact underlying mechanisms are still unknown. In this context, platelet-mediated serotonin release is thought to be one of the most prominent explanations as extracorporeal circulation, contact of blood with artificial surfaces and the roller pump induces platelet activation and triggers release of serotonin.25 Serotonin may act as a vasodilator through activation of the most sensitive and widespread 5-HT2B receptors, mediating enhanced NO-release.25 Prolonged anesthesia, which is common in surgery employing CPB, may have an additional impact on hemodynamic state during the operation. Volatile anesthetics have been shown to modulate vascular smooth muscle tone and depress the cardiovascular system. The systemic hypotensive effect of isoflurane reported previously26-29 has been ascribed to a direct inhibitory effect on vascular endothelial and/or smooth muscle cells, involving a decrease in myofilament calcium-sensitivity, intracellular calcium concentration and voltage-gated calcium influx.30 Lower arterial pressure under isoflurane anesthesia may also be explained by isoflurane-induced activation of ATP-sensitive potassium channels of vascular smooth muscle cells causing cellular membrane hyperpolarization and inhibition of calcium influx.29-32 Moreover, Pypendop et al. (2003) showed that addition of 70% nitrous oxide to isoflurane anesthesia improved arterial pressure and central venous pressure, but the mechanism of this effect was not investigated.31;33 Pathogenesis of the CPB-associated complications
Despite recent progress, the pathological mechanisms involved in CPB-associated complications, being complex and multifactorial, still are largely unknown. CPB circuit and CPB surgical procedure consist of different elements, each of them can be accounted as a contributory factor for the CPB-related complications.34 The main cause of the systemic inflammatory response is thought to be contact of blood with the artificial plastic surfaces of the circuit.34-37 The priming solutions of the CPB circuit induce a drop of hematocrit and colloid oncotic pressure that leads to decreased oxygen delivery and formation of tissue edema.34;38 The usage of the roller pump is responsible for blood cell damage, hemolysis and complement activation.34;39 Thus, CPB is associated with a wide range of the etiological factors that evoke different compensatory and/or pathological processes. Systemic inflammatory response syndrome, ischemia-reperfusion injury, hemodynamic abnormalities, and vascular dysfunction are thought to play a major role.
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CPB-related pathophysiological processes Surgical trauma, contact of blood with artificial surfaces of the extracorporeal circuit and ischemia-reperfusion injury due to insufficient perfusion cause stimulation of local and systemic cellular and humoral defense mechanisms.6;15;16;40-42 Particularly blood interaction with the extracorporeal circuit and air is thought to activate factor XII and the alternative complement pathway that in turn activate cells of the host defense system and stimulate coagulation, fibrinolysis and the kallikrein pathway.15;16;34 Activated white blood cells release cytokines, free oxygen radicals, and other mediators cause activation of the nuclear factor kB and cell adhesion molecules, and as a result initiate SIRS.6;15;16;20
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!Particularly, CPB is associated with release of inflammatory cytokines tumor
necrosis factor-! (TNF-!), interleukin-6 and 8, increased plasma neutrophils elastase level, but also increased anti-inflammatory cytokine interleukin-10 levels.15;16 The systemic inflammatory events may manifest itself as a decline of systemic vascular resistance, systemic hypotension, increased heart rate and cardiac output followed by myocardial failure and increased vascular permeability.34;43 Changes in systemic vascular resistance and arterial blood pressure may cause substantial hemodynamic alterations that lead to redistribution of the blood to the vital organs (heart and brain), decreased supply of other organs, and to microcirculatory disturbances herein. Such processes in the intestine may initiate release of endotoxins in the blood circulation, which activate both classical and alternative complement pathways.34 Activated leukocytes, through release of free radicals, inflammatory cytokines and proteolytic
Figure 1. The principal scheme of the cardiopulmonary circuit. The CPB circuit includes a venous reservoir, a pump, an oxygenator, and a heat exchanger. The venous blood is removed from the body to the venous reservoir. The roller pump propels it through the system. The membrane oxygenator enriches the blood with oxygen and removes carbon dioxide. Then oxygenated blood is returned to the body. The figure was produced using Servier Medical Art.
Introduction and the aim of the thesis 13
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enzymes, evoke endothelial abnormalities and tissue destruction.20;38;43 Upregulation of tumor necrosis factor-!, IL-6, IL-8, and vascular endothelial growth factor (VEGF) results in increased vascular permeability and attenuation of endothelial cell integrity.44 Capillary leak (due to increased vascular permeability) with decreased plasma colloid osmotic pressure (due to hemodilution) lead to accumulation of the fluid in the interstitial spaces and tissue edema, that diminish tissue perfusion, nutrients and oxygen delivery, and state the stage for organ dysfunction.38 Moreover, inflammatory events may cause rigidness of the erythrocytes, decrease their deformability, and affect their ability to flow in microcirculatory beds, which also participate in disturbances of tissue perfusion and oxygen delivery.34;38
Therefore, CPB is associated with hemodynamic abnormalities (centralisation of the circulation, decreased systemic vascular resistance and systemic hypotension), that result in ischemia-reperfusion injury, oxidative stress, and evoke local pathophysiological processes.32;44 Augmented local release of nitrous oxide, thromboxane A2 and products generated by inducible cyclooxygenase are thought also to affect vascular reactivity. Oxidative stress after ischemia-reperfusion injury may itself cause endothelial cells dysfunction. Moreover, formation of the peroxynitrite radicals, because of interaction of superoxide anion with NO, has an additional role in the free-radical induced injury.32;44
To summarise, systemic inflammatory events and ischemia-reperfusion injury trigger the development of systemic and/or local vascular dysfunction. Hemodynamic disorders and microcirculatory abnormalities, which initiate secondary tissue edema and tissue hypoperfusion, become the basis of the multiple organ dysfunction syndrome, and the serious co-morbidity and – mortality associated herewith.3;34;38;45;46
Changes in vascular motor function Vascular endothelium and vasculature represent an important aspect/feature
of CPB-related complications since major organ pathological conditions result from impaired vascular function.47 Alteration of cerebral blood flow with impairment of the mechanisms of cerebral vascular auto-regulation is thought to be the main cause of the CPB-mediated brain injury and early neuropsychological dysfunction.48-51 Impairment of the lung vascular function is believed to cause the pulmonary dysfunction of the CPB.24;52 CPB with deep hypothermic circulatory arrest is related to major renal and pulmonary artery dysfunction.53;54 Endothelial dysfunction was shown to be one of the major contributors to post-CPB intestinal complications.55 CPB is also associated with increased numbers of circulating endothelial cells, and studies suggested this to be a marker of the systemic endothelial dysfunction and injury after cardiac surgery with CPB.56;57
CPB is associated with both impairment of the endothelial function and the alteration of the myogenic tone. Short-term CPB (30 minutes) was found to be associated with loss in acetylcholine-induced vasodilatation in preparations of the middle cerebral artery.48 Short-term CPB with 90 minutes recovery period resulted in aggravated pulmonary vascular resistance with hyperreactivity to serotonin.58 In mesenteric arteries, 90 minutes of CPB followed by 6 hours recovery period induced
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endothelial dysfunction and increased vascular reactivity to !1-adrenoceptor agonists.59 Heart failure was shown to aggravate mesenteric artery endothelial and smooth muscle dysfunction after CPB due to increased oxidative stress.60 CPB was also shown to decrease acetylcholine-mediated relaxation in pulmonary resistances artery and seemed to have no effect on contractile reactivity of these arteries to norepinephrine, vasopressin, and the thromboxane A2.52 In another study pulmonary endothelial dysfunction with decreased relaxant response to acetylcholine was shown after fourth day of the post-operative period after CPB.61
The origin of the CPB-related endothelial dysfunction involves inflammatory mechanisms with neutrophils-mediated endothelial injury, while other processes also may contribute herein. Endothelial activation after CPB with increased circulatory levels of the soluble endothelial adhesion molecules (E-selectin and ICAM-1) was even confirmed after 48h of the postoperative period.62 The degradation of the endothelial and cardiomyocytes adherens junctions were shown to mediate CPB-related increased vascular permeability and cardiomyocyte dysfunction.63 Moreover, apoptosis of endothelial cells might represent another possible mechanism of the CPB-mediated vascular dysfunction and aggravated capillary permeability.56;57
CPB has also been shown to affect peripheral vasomotor tone. Decreased myogenic tone has been reported in CPB-related injury of skeletal microvascular beds.64-67 Inhibition of the mitogen-activated protein kinases was suggested to cause decreased coronary myogenic tone after CPB with cardioplegia.68 The activation of the large conductance calcium-activated potassium channels (BKCa) was shown to be the main cause of the reduced myogenic tone of the skeletal muscles arterioles after CPB in humans.64 Altered alpha-adrenergic receptors and protein kinase C-mediated contractions were observed in skeletal muscle microvessels.67 Together, these alteration in vascular tone and reduced peripheral vascular resistance are thought to be responsible for a systemic hypotensive response, which can be observed up to one week post-operatively.44 The suggested mechanisms involved in decreased intrinsic tone include increased circulating levels of vasoactive substances, increased expression of iNOS, adrenergic receptor desensitization and uncoupling from the second messenger system.43
In the majority of these studies, vascular responsiveness was evaluated in one vessel type and at one time point post-CPB only, mostly after a relatively short-term recovery period.43;48;58;64-67;69 It should be noted, however, that organ dysfunction post-CPB has been shown to influence not only in-hospital mortality and morbidity but also mid-term and long-term survival.8-11;19;64;65 However, data on alterations in vasoresponsiveness during the postoperative period that might account for the late effects of CPB are lacking.
Treatment and/or prevention of CPB-associated complications
Several anti-inflammatory pharmacological agents, including nonsteroid anti-inflammatory agents, corticosteroids, aprotinin, antioxidants, complement inhibitors and phosphodiesterase inhibitors have been proposed to inhibit the CPB-related inflammatory processes and vascular dysfunction.41;42;45 Though, none of them entirely
Introduction and the aim of the thesis 15
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prevented the adverse pathological and clinical outcomes that are associated with extracorporeal circulation. While a single dose of dexamethasone after the induction of anesthesia has been shown to reduce IL-6 related to CPB, it had no effect on clinical outcome.70 However, dexamethasone decreased the concentration of the circulating cytokines in plasma and had anti-inflammatory properties.71;72 Several clinical studies also showed that dexamethasone had no effect on perioperative abdominal organ damage.73 Conversely, recent data suggest that this compound offers a pulmonary protective effect.19 Another way to reduce the inflammatory response after CPB was to remove activated leukocytes from the circulating blood by leukocyte-depleting filters, but several clinical studies showed controversial data regarding its efficacy.19;74
Alternative therapeutic agents for the prevention of CPB-related complications are still under investigations. One of the possible interventions to limit CPB associated complication is the use of sphingosine-1 phosphate receptors antagonists. Fingolimod (FTY720) is a non-selective sphingosine 1-phosphate (S1P) receptor modulator that acts as an immunosuppressive agent. It was demonstrated to be effective in treatment of the autoimmune disorders (multiple sclerosis, autoimmune neuritis, autoimmune glomerulonephritis).75-79 and organ transplant.80 The main mechanism of its immunomodulatory action concerns activation of the S1P-receptors and is associated with inhibition of the egress of lymphocytes from the secondary lymphoid organs to the peripheral blood.81 Moreover, FTY720 has been shown to prevent or ameliorate ischemia-reperfusion injury,82-87 enhance endothelial barrier and decrease vascular permeability,75;88;89 and improve vascular function through modulation of the S1P1,3,4,5 receptors.90;91 Thus, taking into account the above described pharmacological properties, FTY720 might be a promising therapeutic agent to prevent CPB-related vascular dysfunction.
Aims of this thesis
Despite recent progress, there are major questions to be answered concerning the effects of CPB. First, with respect to changes in vascular function following CPB, it should be noted that previously mainly short-term changes (approximately 24 hours period) have been evaluated, while a substantial amount of critical post-operative events take place between the second and fifth day of the recovery period. Also, the association between vascular functionality and inflammatory events has not been addressed sufficiently. Finally, the possibilities of pre-operative and/or intraoperative intervention in vascular reactivity, as an approach to prevent the CPB-related complications, were not formerly evaluated. Thus, the principle aim of this thesis is to investigate the pathophysiology and novel therapeutic options with respect to CPB-related complications in a rat model of CPB.
The first part of this thesis investigates the changes in vascular function evoked by CPB. To this end, vasomotor responses were obtained in different types of vessels during a clinically relevant postoperative recovery period (up to 5 days) in rat. In addition, the relationship between endothelial activation and vascular responsiveness was studied in rat aorta throughout the whole recovery period.
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In the second part of this thesis, the pathophysiology of CPB related inflammation was further characterized by measurement of the expression of inflammatory markers in lung and kidney during the entire recovery period by PCR, Western blot and micro-array analysis in our rat model of CPB.
In the last part of the thesis, the effects of drugs are explored. Since anesthetics influence vascular tone, they may add to the CPB-related hypotension. Thus, the impact of different types of anesthesia was studied in a model of extreme hypotension in mice (hemorrhagic shock). Finally, the effect of the novel immunosuppressive drugs on CPB induced changes in vasomotor response was measured to define its therapeutic potential to prevent the CPB-related complications. To this end, rats were pretreated with selective and non-selective agonists of the S1P-receptors and contractile and relaxant vascular reactivity was assessed.
Reference List 1. Lewis FJ, Taufic M: Closure of atrial septal defects with the aid of hypothermia; experimental
accomplishments and the report of one successful case. Surgery 1953; 33: 52-9 2. Edmunds LH, Jr.: The evolution of cardiopulmonary bypass: lessons to be learned. Perfusion 2002;
17: 243-51 3. Murphy GJ, Angelini GD: Side effects of cardiopulmonary bypass: what is the reality? J.Card Surg.
2004; 19: 481-8 4. Murphy GJ, Lin H, Coward RJ, Toth T, Holmes R, Hall D, Angelini GD: An initial evaluation of post-
cardiopulmonary bypass acute kidney injury in swine. Eur.J.Cardiothorac.Surg. 2009; 36: 849-55 5. Ng CS, Wan S, Yim AP, Arifi AA: Pulmonary dysfunction after cardiac surgery. Chest 2002; 121:
1269-77 6. Ng CS, Wan S, Arifi AA, Yim AP: Inflammatory response to pulmonary ischemia-reperfusion injury.
Surg.Today 2006; 36: 205-14 7. Palin CA, Kailasam R, Hogue CW, Jr.: Atrial fibrillation after cardiac surgery: pathophysiology and
treatment. Semin.Cardiothorac.Vasc.Anesth. 2004; 8: 175-83 8. Loef BG, Epema AH, Navis G, Ebels T, van OW, Henning RH: Off-pump coronary revascularization
attenuates transient renal damage compared with on-pump coronary revascularization. Chest 2002; 121: 1190-4
9. Loef BG, Henning RH, Epema AH, Rietman GW, van OW, Navis GJ, Ebels T: Effect of dexamethasone on perioperative renal function impairment during cardiac surgery with cardiopulmonary bypass. Br.J.Anaesth. 2004; 93: 793-8
10. Loef BG, Epema AH, Smilde TD, Henning RH, Ebels T, Navis G, Stegeman CA: Immediate postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term survival. J.Am.Soc.Nephrol. 2005; 16: 195-200
11. Loef BG, Henning RH, Navis G, Rankin AJ, van OW, Ebels T, Epema AH: Changes in glomerular filtration rate after cardiac surgery with cardiopulmonary bypass in patients with mild preoperative renal dysfunction. Br.J.Anaesth. 2008; 100: 759-64
12. Mallick IH, Yang W, Winslet MC, Seifalian AM: Ischemia-reperfusion injury of the intestine and protective strategies against injury. Dig.Dis.Sci. 2004; 49: 1359-77
13. Elahi MM, Battula NR, Hakim NS, Matata BM: Acute renal failure in patients with ischemic heart disease: causes and novel approaches in breaking the cycle of self-perpetuating insults abrogated by surgery. Int.Surg. 2005; 90: 202-8
14. Hogue CW, Jr., Palin CA, Arrowsmith JE: Cardiopulmonary bypass management and neurologic outcomes: an evidence-based appraisal of current practices. Anesth.Analg. 2006; 103: 21-37
15. Asimakopoulos G: Mechanisms of the systemic inflammatory response. Perfusion 1999; 14: 269-77
Introduction and the aim of the thesis 17
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16. Asimakopoulos G: Systemic inflammation and cardiac surgery: an update. Perfusion 2001; 16: 353-60
17. Sadaba JR, Greco E, Alvarez LA, Pulitani I, Juaristi A, Goiti JJ: The surgical option in the management of acute pulmonary embolism. J.Card Surg. 2008; 23: 729-32
18. bu-Omar Y, Ratnatunga C: Cardiopulmonary bypass and renal injury. Perfusion 2006; 21: 209-13 19. Yasser MA, Elmistekawy E, El-Serogy H: Effects of dexamethasone on pulmonary and renal
functions in patients undergoing CABG with cardiopulmonary bypass. Semin.Cardiothorac.Vasc.Anesth. 2009; 13: 231-7
20. Apostolakis E, Filos KS, Koletsis E, Dougenis D: Lung dysfunction following cardiopulmonary bypass. J.Card Surg. 2010; 25: 47-55
21. Asimakopoulos G, Taylor KM, Smith PL, Ratnatunga CP: Prevalence of acute respiratory distress syndrome after cardiac surgery. J.Thorac.Cardiovasc.Surg. 1999; 117: 620-1
22. Asimakopoulos G, Smith PL, Ratnatunga CP, Taylor KM: Lung injury and acute respiratory distress syndrome after cardiopulmonary bypass. Ann.Thorac.Surg. 1999; 68: 1107-15
23. Li S, Price R, Phiroz D, Swan K, Crane TA: Systemic inflammatory response during cardiopulmonary bypass and strategies. J.Extra.Corpor.Technol. 2005; 37: 180-8
24. Groeneveld AB, Jansen EK, Verheij J: Mechanisms of pulmonary dysfunction after on-pump and off-pump cardiac surgery: a prospective cohort study. J.Cardiothorac.Surg. 2007; 2: 11
25. Borgdorff P, Fekkes D, Tangelder GJ: Hypotension caused by extracorporeal circulation: serotonin from pump-activated platelets triggers nitric oxide release. Circulation 2002; 106: 2588-93
26. Baumert JH, Hecker KE, Hein M, Reyle-Hahn SM, Horn NA, Rossaint R: Haemodynamic effects of haemorrhage during xenon anaesthesia in pigs. Br.J.Anaesth. 2005; 94: 727-32
27. Conzen PF, Habazettl H, Vollmar B, Christ M, Baier H, Peter K: Coronary microcirculation during halothane, enflurane, isoflurane, and adenosine in dogs. Anesthesiology 1992; 76: 261-70
28. Conzen PF, Vollmar B, Habazettl H, Frink EJ, Peter K, Messmer K: Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth.Analg. 1992; 74: 79-88
29. Preckel B, Bolten J: Pharmacology of modern volatile anaesthetics. Best.Pract.Res.Clin.Anaesthesiol. 2005; 19: 331-48
30. Qi F, Ogawa K, Tokinaga Y, Uematsu N, Minonishi T, Hatano Y: Volatile anesthetics inhibit angiotensin II-induced vascular contraction by modulating myosin light chain phosphatase inhibiting protein, CPI-17 and regulatory subunit, MYPT1 phosphorylation. Anesth.Analg. 2009; 109: 412-7
31. Ciofolo MJ, Reiz S: Circulatory effects of volatile anesthetic agents. Minerva Anestesiol. 1999; 65: 232-8
32. Sellke FW, Park KW, Lowenstein E: Vascular effects of isoflurane: no inconsistency between data. Anesthesiology 1999; 90: 919-20
33. Pypendop BH, Ilkiw JE, Imai A, Bolich JA: Hemodynamic effects of nitrous oxide in isoflurane-anesthetized cats. Am.J.Vet.Res. 2003; 64: 273-8
34. de VR, te MF, Eijsman L, Wildevuur WR, Wildevuur C, van OW: Induction and detection of disturbed homeostasis in cardiopulmonary bypass. Perfusion 2004; 19: 267-76
35. De SF: Optimal versus suboptimal perfusion during cardiopulmonary bypass and the inflammatory response. Semin.Cardiothorac.Vasc.Anesth. 2009; 13: 113-7
36. De SF, Van LA, Van NG, Delanghe J: Interaction of plasma proteins with commercial protein repellent polyvinyl chloride (PVC): a word of caution. Perfusion 2008; 23: 215-21
37. De SF: Optimization of the perfusion circuit and its possible impact on the inflammatory response. J.Extra.Corpor.Technol. 2007; 39: 285-8
38. Hirleman E, Larson DF: Cardiopulmonary bypass and edema: physiology and pathophysiology. Perfusion 2008; 23: 311-22
39. Graaff R, Gu YJ, Boonstra PW, van OW, Rakhorst G: Analysis of red blood cell aggregation in cardio-pulmonary bypass (CPB) surgery. Int.J.Artif.Organs 2004; 27: 488-94
40. Asimakopoulos G, Taylor KM: Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules. Ann.Thorac.Surg. 1998; 66: 2135-44
41. Asimakopoulos G: The inflammatory response to CPB: the role of leukocyte filtration. Perfusion 2002; 17 Suppl: 7-10
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42. Asimakopoulos G, Gourlay T: A review of anti-inflammatory strategies in cardiac surgery. Perfusion 2003; 18 Suppl 1: 7-12
43. Sellke FW: Vascular changes after cardiopulmonary bypass and ischemic cardiac arrest: roles of nitric oxide synthase and cyclooxygenase. Braz.J.Med.Biol.Res. 1999; 32: 1345-52
44. Ruel M, Khan TA, Voisine P, Bianchi C, Sellke FW: Vasomotor dysfunction after cardiac surgery. Eur.J.Cardiothorac.Surg. 2004; 26: 1002-14
45. Apostolakis EE, Koletsis EN, Baikoussis NG, Siminelakis SN, Papadopoulos GS: Strategies to prevent intraoperative lung injury during cardiopulmonary bypass. J.Cardiothorac.Surg. 2010; 5: 1
46. Murphy GS, Hessel EA, Groom RC: Optimal perfusion during cardiopulmonary bypass: an evidence-based approach. Anesth.Analg. 2009; 108: 1394-417
47. Verrier ED, Morgan EN: Endothelial response to cardiopulmonary bypass surgery. Ann.Thorac.Surg. 1998; 66: S17-S19
48. Modine T, Azzaoui R, Ouk T, Fayad G, Lacroix D, Warembourg H, Bordet R, Gourlay T: Changes in cerebral vascular reactivity occur early during cardiopulmonary bypass in the rat. Ann.Thorac.Surg. 2006; 82: 672-8
49. Gazzolo D, Abella R, Marinoni E, Di IR, Li VG, Galvano F, Pongiglione G, Frigiola A, Bertino E, Florio P: Circulating biochemical markers of brain damage in infants complicated by ischemia reperfusion injury. Cardiovasc.Hematol.Agents Med.Chem. 2009; 7: 108-26
50. Baba T, Goto T, Maekawa K, Ito A, Yoshitake A, Koshiji T: Early neuropsychological dysfunction in elderly high-risk patients after on-pump and off-pump coronary bypass surgery. J.Anesth. 2007; 21: 452-8
51. Ganushchak YM, Fransen EJ, Visser C, de Jong DS, Maessen JG: Neurological complications after coronary artery bypass grafting related to the performance of cardiopulmonary bypass. Chest 2004; 125: 2196-205
52. Glavind-Kristensen M, Brix-Christensen V, Toennesen E, Ravn HB, Forman A, Sorensen K, Hjortdal VE: Pulmonary endothelial dysfunction after cardiopulmonary bypass in neonatal pigs. Acta Anaesthesiol.Scand. 2002; 46: 853-9
53. Cooper WA, Duarte IG, Thourani VH, Nakamura M, Wang NP, Brown WM, III, Gott JP, Vinten-Johansen J, Guyton RA: Hypothermic circulatory arrest causes multisystem vascular endothelial dysfunction and apoptosis. Ann.Thorac.Surg. 2000; 69: 696-702
54. Nagashima M, Stock U, Nollert G, Sperling J, Shum-Tim D, Hatsuoka S, Mayer JE, Jr.: Effects of cyanosis and hypothermic circulatory arrest on lung function in neonatal lambs. Ann.Thorac.Surg. 1999; 68: 499-504
55. Andrasi TB, Buhmann V, Soos P, Juhasz-Nagy A, Szabo G: Mesenteric complications after hypothermic cardiopulmonary bypass with cardiac arrest: underlying mechanisms. Artif.Organs 2002; 26: 943-6
56. Schmid FX, Vudattu N, Floerchinger B, Hilker M, Eissner G, Hoenicka M, Holler E, Birnbaum DE: Endothelial apoptosis and circulating endothelial cells after bypass grafting with and without cardiopulmonary bypass. Eur.J.Cardiothorac.Surg. 2006; 29: 496-500
57. Schmid FX, Floerchinger B, Vudattu NK, Eissner G, Haubitz M, Holler E, Andreesen R, Birnbaum DE: Direct evidence of endothelial injury during cardiopulmonary bypass by demonstration of circulating endothelial cells. Perfusion 2006; 21: 133-7
58. Sato K, Li J, Metais C, Bianchi C, Sellke F: Increased pulmonary vascular contraction to serotonin after cardiopulmonary bypass: role of cyclooxygenase. J.Surg.Res. 2000; 90: 138-43
59. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7
60. Andrasi TB, Bielik H, Blazovics A, Zima E, Vago H, Szabo G, Juhasz-Nagy A: Mesenteric vascular dysfunction after cardiopulmonary bypass with cardiac arrest is aggravated by coexistent heart failure. Shock 2005; 23: 324-9
61. Nyhan D, Gaine S, Hales M, Zanaboni P, Simon BA, Berkowitz D, Flavahan N: Pulmonary vascular endothelial responses are differentially modulated after cardiopulmonary bypass. J.Cardiovasc.Pharmacol. 1999; 34: 518-25
62. Galea J, Rebuck N, Finn A, Manche A, Moat N: Expression of soluble endothelial adhesion molecules in clinical cardiopulmonary bypass. Perfusion 1998; 13: 314-21
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63. Bianchi C, Araujo EG, Sato K, Sellke FW: Biochemical and structural evidence for pig myocardium adherens junction disruption by cardiopulmonary bypass. Circulation 2001; 104: I319-I324
64. Feng J, Liu Y, Khabbaz KR, Sodha NR, Osipov RM, Hagberg R, Alper SL, Sellke FW: Large conductance calcium-activated potassium channels contribute to the reduced myogenic tone of peripheral microvasculature after cardiopulmonary bypass. J.Surg.Res. 2009; 157: 123-8
65. Feng J, Chu LM, Robich MP, Clements RT, Khabbaz KR, Hagberg R, Liu Y, Osipov RM, Sellke FW: Effects of cardiopulmonary bypass on endothelin-1-induced contraction and signaling in human skeletal muscle microcirculation. Circulation 2010; 122: S150-S155
66. Stamler A, Wang SY, Aguirre DE, Johnson RG, Sellke FW: Cardiopulmonary bypass alters vasomotor regulation of the skeletal muscle microcirculation. Ann.Thorac.Surg. 1997; 64: 460-5
67. Wang SY, Stamler A, Li J, Johnson RG, Sellke FW: Decreased myogenic reactivity in skeletal muscle arterioles after hypothermic cardiopulmonary bypass. J.Surg.Res. 1997; 69: 40-4
68. Khan TA, Bianchi C, Ruel M, Voisine P, Li J, Liddicoat JR, Sellke FW: Mitogen-activated protein kinase inhibition and cardioplegia-cardiopulmonary bypass reduce coronary myogenic tone. Circulation 2003; 108 Suppl 1: II348-II353
69. Putnam EA, Manners JM: Vascular resistance during cardiopulmonary bypass. Its effect on cardiac performance in the immediate post-bypass period. Anaesthesia 1983; 38: 635-43
70. Sobieski MA, Graham JD, Pappas PS, Tatooles AJ, Slaughter MS: Reducing the effects of the systemic inflammatory response to cardiopulmonary bypass: can single dose steroids blunt systemic inflammatory response syndrome? ASAIO J. 2008; 54: 203-6
71. Bronicki RA, Backer CL, Baden HP, Mavroudis C, Crawford SE, Green TP: Dexamethasone reduces the inflammatory response to cardiopulmonary bypass in children. Ann.Thorac.Surg. 2000; 69: 1490-5
72. El A, Sr., Rosseel PM, De Lange JJ, Groeneveld AB, Van SR, Van Wijk EM, Scheffer GJ: Dexamethasone decreases the pro- to anti-inflammatory cytokine ratio during cardiac surgery. Br.J.Anaesth. 2002; 88: 496-501
73. Morariu AM, Loef BG, Aarts LP, Rietman GW, Rakhorst G, van OW, Epema AH: Dexamethasone: benefit and prejudice for patients undergoing on-pump coronary artery bypass grafting: a study on myocardial, pulmonary, renal, intestinal, and hepatic injury. Chest 2005; 128: 2677-87
74. Boodram S, Evans E: Use of leukocyte-depleting filters during cardiac surgery with cardiopulmonary bypass: a review. J.Extra.Corpor.Technol. 2008; 40: 27-42
75. Brinkmann V, Cyster JG, Hla T: FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function. Am.J.Transplant. 2004; 4: 1019-25
76. Brinkmann V: FTY720 (fingolimod) in Multiple Sclerosis: therapeutic effects in the immune and the central nervous system. Br.J.Pharmacol. 2009; 158: 1173-82
77. Chun J, Hartung HP: Mechanism of Action of Oral Fingolimod (FTY720) in Multiple Sclerosis. Clin.Neuropharmacol. 2010;
78. Zhang Z, Schluesener HJ: FTY720: a most promising immunosuppressant modulating immune cell functions. Mini.Rev.Med.Chem. 2007; 7: 845-50
79. Zhang Z, Zhang ZY, Fauser U, Schluesener HJ: FTY720 ameliorates experimental autoimmune neuritis by inhibition of lymphocyte and monocyte infiltration into peripheral nerves. Exp.Neurol. 2008; 210: 681-90
80. Martini S, Peters H, Bohler T, Budde K: Current perspectives on FTY720. Expert.Opin.Investig.Drugs 2007; 16: 505-18
81. Mullershausen F, Zecri F, Cetin C, Billich A, Guerini D, Seuwen K: Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors. Nat.Chem.Biol. 2009; 5: 428-34
82. Delbridge MS, Shrestha BM, Raftery AT, El Nahas AM, Haylor JL: Reduction of ischemia-reperfusion injury in the rat kidney by FTY720, a synthetic derivative of sphingosine. Transplantation 2007; 84: 187-95
83. Egom EE, Ke Y, Musa H, Mohamed TM, Wang T, Cartwright E, Solaro RJ, Lei M: FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling. J.Mol.Cell Cardiol. 2010; 48: 406-14
84. Hasegawa Y, Suzuki H, Sozen T, Rolland W, Zhang JH: Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats. Stroke 2010; 41: 368-74
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85. Kaudel CP, Schmiddem U, Frink M, Bergmann S, Pape HC, Krettek C, Klempnauer J, Winkler M: FTY720 for treatment of ischemia-reperfusion injury following complete renal ischemia in C57/BL6 mice. Transplant.Proc. 2006; 38: 679-81
86. Kaudel CP, Frink M, van GM, Schmiddem U, Probst C, Bergmann S, Krettek C, Klempnauer J, Winkler M: FTY720 application following isolated warm liver ischemia improves long-term survival and organ protection in a mouse model. Transplant.Proc. 2007; 39: 493-8
87. Lee KD, Chow WN, Sato-Bigbee C, Graf MR, Graham RS, Colello RJ, Young HF, Mathern BE: FTY720 reduces inflammation and promotes functional recovery after spinal cord injury. J.Neurotrauma 2009; 26: 2335-44
88. Camp SM, Bittman R, Chiang ET, Moreno-Vinasco L, Mirzapoiazova T, Sammani S, Lu X, Sun C, Harbeck M, Roe M, Natarajan V, Garcia JG, Dudek SM: Synthetic analogs of FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] differentially regulate pulmonary vascular permeability in vivo and in vitro. J.Pharmacol.Exp.Ther. 2009; 331: 54-64
89. Dudek SM, Camp SM, Chiang ET, Singleton PA, Usatyuk PV, Zhao Y, Natarajan V, Garcia JG: Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor. Cell Signal. 2007; 19: 1754-64
90. Sarai K, Shikata K, Shikata Y, Omori K, Watanabe N, Sasaki M, Nishishita S, Wada J, Goda N, Kataoka N, Makino H: Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins. Am.J.Physiol Cell Physiol 2009; 297: C945-C954
91. Tolle M, Levkau B, Keul P, Brinkmann V, Giebing G, Schonfelder G, Schafers M, von Wnuck LK, Jankowski J, Jankowski V, Chun J, Zidek W, van der GM: Immunomodulator FTY720 Induces eNOS-dependent arterial vasodilatation via the lysophospholipid receptor S1P3. Circ.Res. 2005; 96: 913-20
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Chapter 2
Changes in vasomotor function of small coronary and mesenteric artery during 5-day follow-up after
cardiopulmonary bypass in the rat. Characterization of endothelial mediators and contractility
Iryna V. Samarska, Hendrik Buikema, Hubert Mungroop,
Martin C. Houwertjes, Fellery de Lange, Yumei Wang, Anne H. Epema, Robert H. Henning
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Abstract Background: Changes in vascular function may represent key elements of hemodynamic instability and organ injury after cardiopulmonary bypass (CPB). We investigated vascular contraction and endothelium dependent relaxation of small coronary and mesenteric arteries in a rat model of CPB with a follow-up period of 5 days. Materials and Methods: Male Wistar rats (n=78) were anesthetized with isoflurane (2.5 %) and fentanyl/midazolam during CPB. Animals were assigned to sham or CPB with normothermic extracorporeal circulation for 60 min at a flow of 120 mL kg-1 min-1. After recovery for 60 min to 5 days, constriction to phenylephrine (mesenteric artery) and serotonin (coronary artery), and relaxation to acetylcholine (both arteries) were assessed. The expression of nitrotyrosine was assessed by western blot. Results: Sham and CPB decreased the sensitivity to constricting agents at 1 day of recovery compared to control both in mesenteric (-log EC50; sham: 5.9±0.3, CPB: 5.9±0.1, control: 6.3±0.2) and in coronary arteries (-log EC50; sham: 5.8±0.3, CPB: 5.8±0.6, and control: 6.5±0.3). Moreover, CPB progressively decreased total ACh-mediated relaxation, the reduction becoming maximal at 2 days of recovery (AUC; sham: 248.8±48.8, CPB: 100.9±73.0), mainly due to decreased EDHF contribution. In addition, coronary artery of CPB animals displayed a biphasic change characterized by initial increased relaxation due to EDHF, followed by an inhibition of NO mediated relaxation. Nitrotyrosine content of mesenteric artery was about 8-fold higher at 2 days of recovery in CPB group compared to Sham. Conclusions: In Sham animals, vasoconstrictor responses were primarily inhibited, demonstrating profound changes in vascular function following a relative mild procedure of anesthesia and cannulation. In addition, CPB inhibited endothelial-mediated vasorelaxation related to changes in EDHF and NO. Notably, vascular responses after CPB were not normalized at 5 days after the procedure. The observed changes may contribute to hemodynamic instability and organ injury during a protracted period after CPB. Introduction
Cardiopulmonary bypass (CPB) enables complex cardiac surgical procedures. CPB however, is still associated with increased morbidity and mortality1 due to damage of various organ and systems including heart, kidney, lung, brain and gut.2-9 While the mechanisms initiating organ injury are complex and not fully understood, CPB induced activation of systemic inflammation and ischemia reperfusion injury are considered major factors. Previous research showed that this results in the impairment of vascular function immediately following CPB, consisting of both endothelial and smooth muscle dysfunction of small arteries.10-19 These changes affect organ blood supply contributing to the development of secondary tissue edema and tissue hypo-perfusion, setting the stage for multiple organ dysfunction. Thus, changes in vasoreactivity represent a pivotal component of the development of organ dysfunction following CPB. 20-22
Vascular reactivity of the small vessels 23
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Characteristic differences exist in the regulation of vascular tone in small artery beds of different organs. In general, vascular tone is controlled by neurohumoral factors acting through specific receptor pathways in the endothelium and smooth muscles, as well as by local hemodynamic factors including shear stress and intraluminal pressure.23;24 Further, vasodilation is governed mainly by the endothelium through the production of various components, including nitric oxide (NO), endothelium dependent hyperpolarizing factor (EDHF) and prostaglandins.25;26 The contribution of each component is dependent on the vascular bed studied, i.e. mesenteric artery is mainly dependent on EDHF, whereas coronary artery represents a mixed type dependent on NO and EDHF.27;28
Clinical and experimental data suggest that the CPB induced initial systemic inflammatory response (SIRS) weans off within 24 h following surgery.29 In contrast, organ injury following CPB is observed not only in the immediate post-operative period (in the ICU),30 but during a protracted time frame following surgery.31, 32 Thus, we hypothesized that acute CPB injury results in protracted dysfunction of vasomotor control. Therefore, our objective was to evaluate vascular reactivity of different small arteries in an experimental rat model of CPB. Thus, we measured changes in vascular contractility as well as endothelial relaxation function in mesenteric and coronary arteries, including assessment of the contribution of different endothelial dilative components. Further, vascular reactivity was evaluated during a clinically relevant post-operative period. Therefore, time-dependent changes in vascular reactivity were examined from 60 min up to 5 days of recovery.
Material and Methods
The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen. This study was performed in n=78 adult male Wistar rats with body weights of 507.4 ± 31.3 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study period.
Experimental groups
Experimental animals were randomly allocated to one of four experimental groups with different duration of the recovery period after the procedure, being 60 min and 1, 2 and 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated control group was examined. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5 %) only.
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Experimental protocol The experimental protocol consisted of the following parts: anesthesia, preparation, CPB, a weaning and a recovery period (see fig. 1). Anesthesia was induced with 2.5 % isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography 35-42 mmHg, and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.0 ± 0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for continuous blood pressure monitoring. The mean arterial pressure (MAP) was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.
Cardiopulmonary bypass Subsequently, CPB was initiated for 60 min. The set-up consisted of a glass
venous reservoir, a peristaltic pump (Pericor® SF70, Verder, Haan, Germany), a rat membrane oxygenator (M.Humbs, Valley, Germany) and a glass counter-flow heat exchanger with built-in bubble trap. The oxygenator carried a sterile, disposable three-layer artificial diffusion membrane, made from hollow polypropylene fibres (Jostra AG, Hirrlingen, Germany). All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of haes 60 mg ml-1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). No donor blood was used. Animals were additionally heparinized (250 IU kg-1) after the start of ECC. During CPB, rats were anesthetized with intravenous fentanyl (10 "g kg-1), atracurium (0.5 mg kg-1), and
Figure 1. Scheme of the experimental protocol in Sham and CPB groups regarding the duration of recovery period. The experimental protocol consisted of the following parts: anesthesia, preparation, extracorporeal circulation, a weaning and a recovery period (see methods). Abbreviations: CPB=cardiopulmonary bypass.
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Vascular reactivity of the small vessels 25
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midazolam (2 mg kg-1) and blood oxygen saturation was monitored continuously by a pulse oxymeter. Targeted CBP flow was 120 mL kg-1 min-1. During CPB ventilation was stopped and oxygenator gas flow was maintained at 800 ml min–1 of O2:air mixture (1:4). Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the end of preparation period (15 min before start of CPB), twice during the CPB (at 15 and 45 min), and at 45 min after the end of CPB.
Weaning from CPB and recovery At the end, CPB flow was gradually decreased and mechanical ventilation was
initiated. Protamine (150 IU kg-1 i.v.) was administered to neutralize heparin and cannules were removed and wounds sutured. Animals were kept ventilated under isoflurane anesthesia (0.8-1.0%) for 1 h to stabilize, followed by extubation. The total duration of the recovery period lasted 1 h, and 1, 2 and 5 days after the end of the CPB. Sacrification was performed under brief isoflurane anesthesia (2.5 %).
Vascular reactivity At sacrification, all mesenteric loops and the heart were removed and placed in
cold physiological saline solution. Several segments of the third branch of the mesenteric superior artery and the interseptal coronary artery were dissected, prepared as ring vessel preparations (1.8-2.0 mm in length) and mounted on two 40 µm stainless wires connected to force transducers in individual organ bath chambers for isometric tension recordings in a wire-myograph (Danish Myo Technology A/S, Aarhus, Denmark). The baths contained 6 ml of Kreb’s solution warmed to 37°C and bubbled continuously with 95%O2/5%CO2 at pH 7.4. Vessels were subjected to a standardized normalization procedure [33] and left to equilibrate for 40 min until they were at a steady baseline. In brief, the vessels were distended stepwise until effective pressure exceeded 100 mmHg (13.3 kPa). The internal circumference, IC100, was found from the Laplace’s equation and the experiments were performed at the IC1=0.9*IC100. Vascular segments were primed and checked for viability by two consecutive exposures to potassium chloride (60 mM).
The experimental protocol consisted of evaluation of contractile responses to phenylephrine (PE; 10 nM to 100 "M; mesenteric arteries) or serotonin (10 nM to 100 "M; coronary arteries). Endothelium-dependent relaxation to acetylcholine (ACh; 10 nM to 300 "M) was assessed in rings precontracted with PE (mesenteric artery) or serotonin (coronary artery). To assess the contribution of different endothelial mediators, ACh-induced relaxations were studied in the absence and presence of L-NMMA (0.1 mM; an inhibitor of NO-synthase) and/or indomethacine (1 "M; an inhibitor of cyclooxygenase) administered 20 min before application of phenylephrine.34 It has been shown previously that the remaining ACh induced relaxation in presence of both cyclooxygenase and NO-synthase inhibitors was fully dependent on EDHF.35 Following the final concentration of ACh, a maximal concentration of the NO-donor sodium nitroprusside (SNP; 0.1 mM) was applied to assess maximal endothelium-independent relaxation.
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Western blot The methods used were described previously.36;37 Briefly, after grinding, the
frozen mesenteric beds were placed in 300 "l of boiling 2% SDS followed by pounding by a polytron (Kinematica AG Littau, Switzerland). Then, samples were centrifuged (4000 rpm, 1 min) and boiled (95oC) for 5 min. After a second centrifugation (13000 rpm, 3 min), supernatant was collected and used for measurements. Protein concentration was determined by Bio-Rad Dc Protein Assay (Bio-Rad, Hercules, CA). Twenty µg of total protein from each sample was separated on 10% Tris-Glycine-SDS-polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membranes. Anti-nitrotyrosine antibody (ALEXIS Biochemicals, Lausen, Switzerland) and anti-GAPDH (Sigma, St. Louis, MO) were used as primary antibodies. Horseradish peroxidase-linked rabbit anti-mouse antibody (Sigma, St. Louis, MO) was applied as a secondary antibody. GAPDH served as a housekeeping protein.
Drugs The composition of Krebs solution was as follows (mM): 120.4 NaCl, 5.9 KCl,
2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: acetylcholine chloride (ACh), phenylephrine (PE), serotonin, indomethacine, SNP, NG-methyl-L-arginine (L-NMMA). Stock solution (10 mM) for indomethacine was prepared in 96% ethanol. All other drugs were dissolved in deionized water. L-NMMA was purchased from MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma (St. Louis, MO). The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the bath.
Data analysis Data are given as mean ± SD. Contractile responses to PE and serotonin are
presented in mN and relaxations to ACh and SNP were calculated as a percentage of pre-constriction. For each concentration-response curve the following parameters were determined: (1) the concentration at which half-maximal response was reached (EC50), (2) the maximal response (Emax) and (3) the Area Under the Curve (AUC) in arbitrary units (AU) (SigmaPlot 11, Systat Software, San Jose, CA, USA). The difference in AUC of concentration-response curves to ACh in the absence and presence of inhibitor(s) was used to quantify the contribution of different endothelial relaxing components.38 Differences were evaluated using Student’s t-test, one-way ANOVA, repeated measurements ANOVA combined with post-hoc Bonferroni test or with t-test (SPSS 16.0, Chicago, IL, USA). Differences were considered significant at P<0.05 (2-tailed). The relationship between different relaxant pathways and total acetylcholine-mediated relaxation was evaluated with regression analysis and Spearman correlation.
Vascular reactivity of the small vessels 27
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Results Hemodynamics and blood gas analysis At baseline, all parameters measured were similar and in the normal range.
During CPB, MAP was significantly lower compared with Sham (fig. 2). Blood gas analysis showed a decreased pH, bicarbonate (HCO-
3), and Base excess and increased levels of pCO2 and pO2 during CPB (fig. 2), indicating mild acidosis. Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with the priming solution. Intraoperative hyperglycemia was observed in both Sham and CPB groups during the first 2 hours. After weaning from CPB, all parameters, including MAP, returned to normal values and were similar in both Sham and CPB, except for the hematocrit (fig. 2).
Figure 2. Data on blood gas analysis and mean arterial pressure (MAP, femoral artery) and glucose concentration in Sham and CPB groups during the experimental protocol. Abbreviations: CPB= cardiopulmonary bypass. * indicates P<0.05, independent t-test. !
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Contraction studies in isolated arteries Full concentration-response curves to phenylephrine and serotonin were
constructed in mesenteric (fig. 3, left panel) and coronary arteries (fig. 3, right panel), respectively. EC50-values and Emax derived from these curves are presented in table 1. As compared to mesenteric arteries of control rats, concentration–response curves to PE in Sham rats were shifted to the right (fig. 3A) and statistical significance was reached at 1h, 2 days and 5 days of recovery (table 1). In contrast, Emax of PE was significantly decreased in Sham rats at 1 day of recovery, while normalizing again at longer periods of recovery (table 1). The time pattern of the changes in overall contractility to PE is shown in figure 3C as AUC for concentration-response curves to PE. In Sham rats, overall contractility was reduced most prominently at 1 day of recovery, followed by normalization at the 5th post-procedure day. Contractile reactivity in CPB treated animals followed the same pattern (fig. 3B, table 1). Collectively, our data show that changes in contractility of mesenteric arteries were due to the anesthetic and cannulation procedure.
Concentration–response curves to serotonin in coronary artery of Sham rats (fig. 3D) were significantly shifted to the right after short-term recovery compared to control (i.e. at 1 h and 1 day; table 1). In contrast, Emax for Serotonin in Sham was increased when studied after longer periods of recovery (i.e. at 2 days and 5 days), but not at short-term (table 1). Collectively, AUC of concentration-response curves show a tendency for a reduction in overall contractility in coronary arteries of Sham rats after acute recovery, followed by normalization towards the 5th post-procedural day, but variation was too large to reach statistical significance (fig. 3F). Contractile reactivity in the group of CPB rats largely followed the same pattern (fig. 3E). Comparison of
Table 1. Contractile response to phenylephrine in mesenteric arteries and to serotonin in coronary artery in Sham and CPB groups
Mesenteric artery Recovery Time
-Log EC50 Emax, µµM Control Sham CPB Control Sham CPB
-6.3 ±0.2
11.3±3.1
1hour -6.1±0.5 -6.0±0.4 10.5±3.6 10.8±2.8 1 day -5.9±0.3* -5.9±0.1* 5.8±2.7 * 6.9±3.6* 2 days -5.9±0.2* -6.2±0.3 10.4±3.1 9.7±5.9 5 days -6.2±0.3 -6.2±0.5 10.0±2.3 10.0±5.2
Coronary artery Recovery Time
-Log EC50 Emax, µµM Control Sham CPB Control Sham CPB
-6.5 ±0.3
1.4±1.0
1 hour -5.6±0.1* -5.7±0.3* 1.5±0.6 1.7±0.9 1 day -5.8±0.3* -5.8±0.6* 1.3±0.6 1.7±1.3 2 days -6.2±0.4 -6.4±0.5 3.4±1.9 1.4±0.8 5 days -5.8±0.3# -5.7±0.2* 2.7±0.9 2.4±1.8 Data are given as mean±SD. Abbreviations: CPB, cardiopulmonary bypass; Emax, the maximal response; -Log EC50, negative logarithm of the concentration at which half-maximal response was reached (EC50).*-P<0.05 vs Control, one-way ANOVA with post-hoc Bonferroni test #- P<0.05 vs Control, t-test
Vascular reactivity of the small vessels 29
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individual time points of Sham and CPB to controls showed a significant difference in EC50 values at almost all time points in both groups (table 1). These data indicate that the protracted changes in contractility in coronary arteries were also due to the anesthetic and cannulation procedure rather than ECC. Collectively, the above findings demonstrate that CPB-related surgical procedures and/or extended anesthesia induced extensive and protracted changes in the contractility of mesenteric and coronary arteries, without significant additional influence of CPB.
Relaxation studies in isolated arties Endothelium-dependent relaxation was assessed by construction of full
concentration-response curves to acetylcholine (ACh), shown in the figure in the Supplement Digital Content 1 for mesenteric and coronary arteries of normal control rats. The major endothelial dilative pathways were investigated by blocking the action of two components: vasoactive prostaglandins (PGs) and nitric oxide (NO) with indomethacin and additional pre-incubation with L-NMMA. The difference between AUCs of concentration-response curves in the absence and presence of inhibitors was used to calculate contribution of PGs, NO and endothelial derived hyperpolarizing factor (EDHF; Supplement Digital Content 1). Endothelium-independent relaxation was unaffected in both mesenteric and coronary artery since the relaxant response to sodium nitroprusside was similar in all groups (see table, Supplement Digital Content 2).
Preconstriction levels to phenylephrine were not significantly different among groups (see table, Supplement digital data 3, which is a table containing all preconstriction levels to phenylephrine). The changes in the AUC of total ACh-mediated relaxation and contributions of the endothelial dilative mediators in mesenteric artery were plotted against time of recovery (fig. 4). In Sham rats, total ACh-induced relaxation progressively increased during the recovery period to become even significantly larger as compared normal control rats (fig. 4A). In contrast, total ACh-mediated relaxation progressively decreased in CPB rats, the reduction becoming maximal at 2 days recovery (fig. 4A). Interestingly, the observed changes in total ACh-induced relaxation were similar to changes in EDHF contribution (fig. 4C), while contribution of NO and PGs did not change (fig. 4B, D). By the 5th day of recovery, AUC of total ACh-induced relaxation and the EDHF contribution had normalized again. Finally, EC50 and Emax of individual time points of Sham and CPB were compared to control by Student t-test (See table in Supplemental Digital Content 4), that showed significant changes in some point up to 5 days of recovery. The above findings indicate that the surgical procedure under general anesthesia induced a temporal increase in endothelium-dependent relaxation of mesenteric arteries via mobilization of EDHF, which is attenuated after CPB.
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Preconstriction levels of coronary artery to serotonin were not significantly different among groups (See table in Supplement Digital Content 3). Total relaxation of ACh mediated dilatation was unaltered in Sham compared to control rats (fig. 5 A,C). Regarding its components, the NO-contribution to ACh-induced relaxation was profoundly decreased at 1 h in Sham rats and to a lesser extent in later time points (fig.
Figure 3. Vascular contractility of rat mesenteric and coronary arteries at different periods of recovery (1h, 1day, 2 or 5 days) in Sham groups (A,B and D,F, respectively) and following subjection of the animals to CPB-related procedures (A,C and E,F, respectively). Contractile responses are given as full CR-curves (A,B,D,E) and the AUC (C). Data are mean ± SD (n=6-9). * indicates P<0.05 CPB vs Control, one way ANOVA with post-hoc Bonferroni test. # indicates P<0.05 Sham vs Control, repeated measurements ANOVA combined with post-hoc Bonferroni test. Abbreviations: PE= phenylephrine; CPB= cardiopulmonary bypass; SE= serotonine.
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Vascular reactivity of the small vessels 31
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5D). However, decreased NO contribution was compensated by the increase in the contribution of dilatory PGs (fig. 5B).
Following CPB a more complex pattern was observed, resulting in an enhanced total relaxation to ACh at 1 h of recovery, followed by a larger inhibition of relaxation at 2 days (fig. 5A). Enhanced relaxation was mainly due to a significant increase in EDHF (fig. 5C), whereas inhibition at 2 days seems caused by a persistent decrease of NO contribution (fig. 5D). Collectively, the above findings suggest that the unchanged relaxation observed following cannulation procedures and general anesthesia results from a balanced decrease in NO and increase in PGs. In addition, CPB mainly resulted in the acute mobilization of EDHF, thus enhancing relaxation at 90 min of recovery.
Western blotting Reactive oxygen species (ROS) have been implicated to impair EDHF
mediated relaxation of mesenteric artery.39;40 As CPB is associated with enhanced inflammatory response and oxidative stress [41], production of ROS was studied at 2 days of recovery, when relaxation in mesenteric artery was greatly enhanced in CPB compared to Sham (fig. 4). ROS production in mesenteric artery was quantified by measurement of nitrotyrosine content.42. Nitrotyrosine content was about 8-fold higher at 2 days of recovery in CPB group in comparison to Sham (fig. 6). Thus, these data suggest that CPB induced excess production of ROS at 2 days of recovery inhibiting the EDHF component of relaxation. !!
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Figure 4. Total acetylcholine (ACh-)-induced relaxation (A) - and the contribution of (B) prostaglandins (PG’s), (C) NO, and (D) EDHF hereto, all provided as AUC - in rat mesenteric arteries at different periods of recovery (1h, 1day, 2 or 5 days) following subjection of the animals to CPB-related procedures. Data are mean ± SD (n=5-8). * indicates P<0.05 CPB vs Control, t-test; # indicates P<0.05 Sham vs Control, one-way ANOVA with following Bonferroni test; # indicates P<0.05 CPB vs Sham, t-test. Abbreviations: CPB= cardiopulmonary bypass.
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Figure 5. Total acetylcholine (ACh-)-induced relaxation (A) - and the contribution of (B) prostaglandins (PG’s), (C) NO, and (D) EDHF hereto, all provided as AUC - in rat coronary arteries at different periods of recovery (1h, 1day, 2 or 5 days) following subjection of the animals to CPB-related procedures. Data are mean ± SD (n=5-8). *indicates P<0.05 Sham vs CPB, one-way ANOVA with following Bonferroni test; # indicates P<0.05 Sham vs Control, one-way ANOVA with following Bonferroni test; # indicates P<0.05 CPB vs Sham, one-way ANOVA with following Bonferroni test. Abbreviations: CPB=cardiopulmonary bypass.
Figure 6. Nitrotyrosine expression in mesenteric beds after 48h recovery period. representative blot (A) in Sham (n=3) and CPB (n=4) and Relative expressions patterns (B) in Sham and CPB samples. All data is normalized to expression level of GAPDH and present in arbitrary units. *P<0.05 CPB 2 days vs Sham 2 days, independent t-test.
Vascular reactivity of the small vessels 33
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Discussion Changes in the vascular responsiveness of small arteries after cardiopulmonary bypass were studied during 5 days of recovery. First, our data show that CPB affects the vasomotor response of isolated arteries during a protracted time period, as vascular function following CPB did not normalize during 5 days of follow-up. Second, Sham and CPB induce complex changes in vasomotor response of small arteries following CPB in rat, which differ between vascular beds. Sham operation primarily induced changes in vasoconstrictor responses, with additional changes in vasorelaxation caused by CPB.
Sham operation caused a temporal depression in contractility of mesenteric and coronary artery around day 1 of recovery. In addition, sham operation caused an increased relaxant responsiveness of mesenteric artery after 2 days of recovery. In contrast, CPB strongly inhibited the vascular relaxation of mesenteric artery at 2 days recovery caused by decreased EDHF contribution.
Coronary artery of CPB animals displayed a biphasic change characterized by initial increased relaxation due to EDHF, followed by an inhibition of NO mediated relaxation. Collectively, these data demonstrate the induction of profound changes in vascular function following a relative mild procedure of anesthesia and cannulation. Specific changes induced by CPB consist of a change in endothelial-mediated relaxation, mainly related to its EDHF component. Furthermore, since the relaxant response to sodium nitroprusside was similar in all groups, alterations in relaxation seem due to changes at the level of the endothelial cell rather than caused by altered vascular smooth muscle responsiveness. Finally, the inhibited EDHF response in mesenteric artery of CPB animals was accompanied by the upregulation of nitrotyrosine, suggesting a role for an inflammatory response and ROS. Vasoresponsiveness after CPB
Vascular reactivity after CPB has been investigated previously in various vascular beds43-49 although follow-up is limited to a few hours after CPB. In rat mesenteric artery, 90 min of CPB with 2.5 h recovery increased the contractile response to phenylephrine and evoked endothelial dysfunction.50 CPB at 280C body temperature with aortic cross-clamping and cardioplegic arrest in dog caused depressed endothelial mediated mesenteric vasodilation immediately following CPB.51 The differences from our results, in which vasorelaxation was unaltered shortly following CPB, may be explained by differences from our model, i.e. a longer duration of CPB, cardioplegic arrest, hypothermia and the use of donor blood. Data on vascular reactivity of coronary artery following CPB are limited. In particular, a decreased myogenic vascular tone has been reported in pig coronary and human atrial arteries.52-
54 However, data on changes in receptor mediated vasomotor responses following CPB are lacking, precluding comparison of our results to others.
Sham induced changes in vascular reactivity One of the main findings of our study constitutes of changes in vascular
reactivity following Sham procedure with a delay of 1 to 2 days following the procedure. Moreover, Sham induced changes appear more prominent in mesenteric artery. The impaired vascular function observed in isolated mesenteric artery favors vasodilation,
34 Chapter 2
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which in vivo may relate to a low flow state and splanchnicus hypoperfusion with increased incidence of gastrointestinal complications after major surgery.55
The underlying mechanism of changes in vascular reactivity in Sham remains unknown. In our study, a main candidate for the induction of these changes is isoflurane, but limited data are available. Volatile anesthetics, including isoflurane, are well known for their vasorelaxing effect.56 Additionally, volatile anesthetics have been shown to inhibit AngII-mediated contraction.57 Studies employing administration of isoflurane to isolated arterial segments indicate that inhibition of contraction by isoflurane, if any, is limited to a short time interval of the recovery (up to 25 min).58;59 Given the time course of our experiments, including the 40 min equilibration time in the absence of isoflurane, it seems unlikely that recovery from prolonged isoflurane administration accounts for inhibited contractility of isolated arteries following the Sham procedure.
A second cause of the Sham-mediated changes in vascular reactivity might be an inflammatory response evoked by the procedure. Indeed, a Sham procedure of CPB in the rat results in a substantial increased plasma concentration of TNF-alpha up to 6 hours post-procedure,60 a cytokine inducing endothelial dysfunction through various mechanisms (reviewed in Zhang et al., 2009). 61
Collectively our data suggest that the relatively mild procedure of anesthesia and cannulation have a long-lasting impact on vasoresponsiveness of small arteries and warrant further studies to its mechanism and the contribution of specific anesthetics.
CPB induced changes in vascular reactivity The observation that CPB selectively affects endothelium dependent relaxation
constitutes a second main finding of our study. Most likely, endothelial dysfunction is caused by the substantially aggravated systemic inflammation initiated by CPB,62-64 as increased plasma TNF-alpha concentrations relate to impaired endothelial mediated relaxation in rat.65 Further, impaired hemodynamics during CPB may add to vascular dysfunction, as demonstrated in a hypovolemic shock model in mice.66 Additionally, the observed changes in vascular reactivity might originate from metabolic disturbances during CPB. Slight changes in pH, HCO-
3, and BE during CPB were observed and likely represent mild metabolic acidosis, which normalized after weaning from the CPB.
Our data imply that CPB affects the relaxation of mesenteric artery predominantly. This may be caused by the mesenteric artery being dependent on a single component of relaxation (EDHF), compared to coronary artery, which is balanced between NO, EDHF and prostaglandins. Thus, the compensatory capacity of the coronary vascular bed seems intrinsically superior over mesenteric artery. Possibly, the limited capacity of adjustment of mesenteric function relates to gastrointestinal complications after CPB, which is one of the serious complications with high mortality rate.67
In summary, our study demonstrates that vascular responsiveness of small vessels was significantly altered during the entire follow-up period of 5 days following CPB in the rat. Not only CPB, but also Sham procedure causes protracted and
Vascular reactivity of the small vessels 35
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substantial alteration of vascular function. Changes in contractile and relaxation function of small arteries following CPB may lead to impaired control of vasomotor response, possibly related to hemodynamic instability and organ injury during the recovery period. Reference List 1. Loef BG, Epema AH, Smilde TD, Henning RH, Ebels T, Navis G, Stegeman CA: Immediate
postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term survival. J.Am.Soc.Nephrol. 2005; 16: 195-200
2. Apostolakis E, Filos KS, Koletsis E, Dougenis D: Lung dysfunction following cardiopulmonary bypass. J.Card Surg. 2010; 25: 47-55
3. bu-Omar Y, Ratnatunga C: Cardiopulmonary bypass and renal injury. Perfusion 2006; 21: 209-13 4. Groeneveld AB, Jansen EK, Verheij J: Mechanisms of pulmonary dysfunction after on-pump and off-
pump cardiac surgery: a prospective cohort study. J.Cardiothorac.Surg. 2007; 2: 11 5. Krishnadasan B, Morgan EN, Boyle ED, Verrier ED: Mechanisms of myocardial injury after cardiac
surgery. J.Cardiothorac.Vasc.Anesth. 2000; 14: 6-10 6. Modine T, Azzaoui R, Ouk T, Fayad G, Lacroix D, Warembourg H, Bordet R, Gourlay T: Changes in
cerebral vascular reactivity occur early during cardiopulmonary bypass in the rat. Ann.Thorac.Surg. 2006; 82: 672-8
7. Ranieri VM, Vitale N, Grasso S, Puntillo F, Mascia L, Paparella D, Tunzi P, Giuliani R, de Luca TL, Fiore T: Time-course of impairment of respiratory mechanics after cardiac surgery and cardiopulmonary bypass. Crit Care Med. 1999; 27: 1454-60
8. Ruel M, Khan TA, Voisine P, Bianchi C, Sellke FW: Vasomotor dysfunction after cardiac surgery. Eur.J.Cardiothorac.Surg. 2004; 26: 1002-14
9. Verrier ED, Morgan EN: Endothelial response to cardiopulmonary bypass surgery. Ann.Thorac.Surg. 1998; 66: S17-S19
10. Souza Neto EP, Loufouat J, Saroul C, Paultre C, Chiari P, Lehot JJ, Cerutti C: Blood pressure and heart rate variability changes during cardiac surgery with cardiopulmonary bypass. Fundam.Clin.Pharmacol. 2004; 18: 387-96
11. Boyle EM, Jr., Morgan EN, Kovacich JC, Canty TG, Jr., Verrier ED: Microvascular responses to cardiopulmonary bypass. J.Cardiothorac.Vasc.Anesth. 1999; 13: 30-5
12. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7
13. Mazer CD, Briet F, Blight KR, Stewart DJ, Robb M, Wang Z, Harrington AM, Mak W, Li X, Hare GM: Increased cerebral and renal endothelial nitric oxide synthase gene expression after cardiopulmonary bypass in the rat. J.Thorac.Cardiovasc.Surg. 2007; 133: 13-20
14. Modine T, Azzaoui R, Ouk T, Fayad G, Lacroix D, Warembourg H, Bordet R, Gourlay T: Changes in cerebral vascular reactivity occur early during cardiopulmonary bypass in the rat. Ann.Thorac.Surg. 2006; 82: 672-8
15. Ruel M, Khan TA, Voisine P, Bianchi C, Sellke FW: Vasomotor dysfunction after cardiac surgery. Eur.J.Cardiothorac.Surg. 2004; 26: 1002-14
16. Stamler A, Wang SY, Aguirre DE, Johnson RG, Sellke FW: Cardiopulmonary bypass alters vasomotor regulation of the skeletal muscle microcirculation. Ann.Thorac.Surg. 1997; 64: 460-5
17. Verrier ED, Morgan EN: Endothelial response to cardiopulmonary bypass surgery. Ann.Thorac.Surg. 1998; 66: S17-S19
18. Wang SY, Stamler A, Li J, Johnson RG, Sellke FW: Decreased myogenic reactivity in skeletal muscle arterioles after hypothermic cardiopulmonary bypass. J.Surg.Res. 1997; 69: 40-4
19. Wang SY, Friedman M, Franklin A, Sellke FW: Myogenic reactivity of coronary resistance arteries after cardiopulmonary bypass and hyperkalemic cardioplegia. Circulation 1995; 92: 1590-6
20. Boyle EM, Jr., Morgan EN, Kovacich JC, Canty TG, Jr., Verrier ED: Microvascular responses to cardiopulmonary bypass. J.Cardiothorac.Vasc.Anesth. 1999; 13: 30-5
36 Chapter 2
!
21. Krishnadasan B, Morgan EN, Boyle ED, Verrier ED: Mechanisms of myocardial injury after cardiac surgery. J.Cardiothorac.Vasc.Anesth. 2000; 14: 6-10
22. Verrier ED, Morgan EN: Endothelial response to cardiopulmonary bypass surgery. Ann.Thorac.Surg. 1998; 66: S17-S19
23. Luksha L, Agewall S, Kublickiene K: Endothelium-derived hyperpolarizing factor in vascular physiology and cardiovascular disease. Atherosclerosis 2009; 202: 330-44
24. Villar IC, Francis S, Webb A, Hobbs AJ, Ahluwalia A: Novel aspects of endothelium-dependent regulation of vascular tone. Kidney Int. 2006; 70: 840-53
25. Villar IC, Francis S, Webb A, Hobbs AJ, Ahluwalia A: Novel aspects of endothelium-dependent regulation of vascular tone. Kidney Int. 2006; 70: 840-53
26. Flammer AJ, Luscher TF: Human endothelial dysfunction: EDRFs. Pflugers Arch. 2010; 459: 1005-13
27. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
28. Gschwend S, Henning RH, de ZD, Buikema H: Coronary myogenic constriction antagonizes EDHF-mediated dilation: role of KCa channels. Hypertension 2003; 41: 912-8
29. Morariu AM, Loef BG, Aarts LP, Rietman GW, Rakhorst G, van OW, Epema AH: Dexamethasone: benefit and prejudice for patients undergoing on-pump coronary artery bypass grafting: a study on myocardial, pulmonary, renal, intestinal, and hepatic injury. Chest 2005; 128: 2677-87
30. Loef BG, Epema AH, Navis G, Ebels T, van OW, Henning RH: Off-pump coronary revascularization attenuates transient renal damage compared with on-pump coronary revascularization. Chest 2002; 121: 1190-4
31. Loef BG, Epema AH, Smilde TD, Henning RH, Ebels T, Navis G, Stegeman CA: Immediate postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term survival. J.Am.Soc.Nephrol. 2005; 16: 195-200
32. Loef BG, Epema AH, Navis G, Ebels T, Stegeman CA: Postoperative renal dysfunction and preoperative left ventricular dysfunction predispose patients to increased long-term mortality after coronary artery bypass graft surgery. Br.J.Anaesth. 2009; 102: 749-55
33. Xu Y, Henning RH, Lipsic E, van BA, van Gilst WH, Buikema H: Acetylcholine stimulated dilatation and stretch induced myogenic constriction in mesenteric artery of rats with chronic heart failure. Eur.J.Heart Fail. 2007; 9: 144-51
34. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
35. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
36. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF: Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ.Res. 2002; 90: 413-9
37. Samarska IV, van MM, Buikema H, Houwertjes MC, Wulfert FM, Molema G, Epema AH, Henning RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia. Anesthesiology 2009; 111: 600-8
38. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
39. Dal-Ros S, Oswald-Mammosser M, Pestrikova T, Schott C, Boehm N, Bronner C, Chataigneau T, Geny B, Schini-Kerth VB: Losartan prevents portal hypertension-induced, redox-mediated endothelial dysfunction in the mesenteric artery in rats. Gastroenterology 2010; 138: 1574-84
40. Inkster ME, Cotter MA, Cameron NE: Effects of trientine, a metal chelator, on defective endothelium-dependent relaxation in the mesenteric vasculature of diabetic rats. Free Radic.Res. 2002; 36: 1091-9
41. Deblier I, Sadowska AM, Janssens A, Rodrigus I, DeBacker WA: Markers of inflammation and oxidative stress in patients undergoing CABG with CPB with and without ventilation of the lungs: a pilot study. Interact.Cardiovasc.Thorac.Surg. 2006; 5: 387-91
Vascular reactivity of the small vessels 37
!
42. Yang YM, Huang A, Kaley G, Sun D: eNOS uncoupling and endothelial dysfunction in aged vessels. Am.J.Physiol Heart Circ.Physiol 2009; 297: H1829-H1836
43. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7
44. Khan TA, Bianchi C, Ruel M, Voisine P, Li J, Liddicoat JR, Sellke FW: Mitogen-activated protein kinase inhibition and cardioplegia-cardiopulmonary bypass reduce coronary myogenic tone. Circulation 2003; 108 Suppl 1: II348-II353
45. Mazer CD, Briet F, Blight KR, Stewart DJ, Robb M, Wang Z, Harrington AM, Mak W, Li X, Hare GM: Increased cerebral and renal endothelial nitric oxide synthase gene expression after cardiopulmonary bypass in the rat. J.Thorac.Cardiovasc.Surg. 2007; 133: 13-20
46. Modine T, Azzaoui R, Ouk T, Fayad G, Lacroix D, Warembourg H, Bordet R, Gourlay T: Changes in cerebral vascular reactivity occur early during cardiopulmonary bypass in the rat. Ann.Thorac.Surg. 2006; 82: 672-8
47. Modine T, Azzaoui R, Fayad G, Lacroix D, Bordet R, Warembourg H, Gourlay T: A recovery model of minimally invasive cardiopulmonary bypass in the rat. Perfusion 2006; 21: 87-92
48. Wang SY, Friedman M, Franklin A, Sellke FW: Myogenic reactivity of coronary resistance arteries after cardiopulmonary bypass and hyperkalemic cardioplegia. Circulation 1995; 92: 1590-6
49. Wang SY, Stamler A, Li J, Johnson RG, Sellke FW: Decreased myogenic reactivity in skeletal muscle arterioles after hypothermic cardiopulmonary bypass. J.Surg.Res. 1997; 69: 40-4
50. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7
51. Andrasi TB, Bielik H, Blazovics A, Zima E, Vago H, Szabo G, Juhasz-Nagy A: Mesenteric vascular dysfunction after cardiopulmonary bypass with cardiac arrest is aggravated by coexistent heart failure. Shock 2005; 23: 324-9
52. Khan TA, Bianchi C, Ruel M, Voisine P, Li J, Liddicoat JR, Sellke FW: Mitogen-activated protein kinase inhibition and cardioplegia-cardiopulmonary bypass reduce coronary myogenic tone. Circulation 2003; 108 Suppl 1: II348-II353
53. Wang SY, Friedman M, Franklin A, Sellke FW: Myogenic reactivity of coronary resistance arteries after cardiopulmonary bypass and hyperkalemic cardioplegia. Circulation 1995; 92: 1590-6
54. Wang SY, Stamler A, Li J, Johnson RG, Sellke FW: Decreased myogenic reactivity in skeletal muscle arterioles after hypothermic cardiopulmonary bypass. J.Surg.Res. 1997; 69: 40-4
55. Giglio MT, Marucci M, Testini M, Brienza N: Goal-directed haemodynamic therapy and gastrointestinal complications in major surgery: a meta-analysis of randomized controlled trials. Br.J.Anaesth. 2009; 103: 637-46
56. Kokita N, Stekiel TA, Yamazaki M, Bosnjak ZJ, Kampine JP, Stekiel WJ: Potassium channel-mediated hyperpolarization of mesenteric vascular smooth muscle by isoflurane. Anesthesiology 1999; 90: 779-88
57. Ishikawa A, Ogawa K, Tokinaga Y, Uematsu N, Mizumoto K, Hatano Y: The mechanism behind the inhibitory effect of isoflurane on angiotensin II-induced vascular contraction is different from that of sevoflurane. Anesth.Analg. 2007; 105: 97-102
58. Izumi K, Akata T, Takahashi S: Role of endothelium in the action of isoflurane on vascular smooth muscle of isolated mesenteric resistance arteries. Anesthesiology 2001; 95: 990-8
59. Akata T, Kanna T, Yoshino J, Takahashi S: Mechanisms of direct inhibitory action of isoflurane on vascular smooth muscle of mesenteric resistance arteries. Anesthesiology 2003; 99: 666-77
60. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7
61. Zhang H, Park Y, Wu J, Chen X, Lee S, Yang J, Dellsperger KC, Zhang C: Role of TNF-alpha in vascular dysfunction. Clin.Sci.(Lond) 2009; 116: 219-30
62. Larmann J, Theilmeier G: Inflammatory response to cardiac surgery: cardiopulmonary bypass versus non-cardiopulmonary bypass surgery. Best.Pract.Res.Clin.Anaesthesiol. 2004; 18: 425-38
63. Sistino JJ, Acsell JR: Systemic inflammatory response syndrome (SIRS) following emergency cardiopulmonary bypass: a case report and literature review. J.Extra.Corpor.Technol. 1999; 31: 37-43
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A
-Log [ACh]-8 -7 -6 -5 -4
rela
xatio
n, %
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-80
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-Log [ACh]-8 -7 -6 -5 -4
rela
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-120
-100
-80
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Mesenteric artery Coronary artery
64. Markewitz A, Lante W, Franke A, Marohl K, Kuhlmann WD, Weinhold C: Alterations of cell-mediated immunity following cardiac operations: clinical implications and open questions. Shock 2001; 16 Suppl 1: 10-5
65. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7
66. Samarska IV, van MM, Buikema H, Houwertjes MC, Wulfert FM, Molema G, Epema AH, Henning RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia. Anesthesiology 2009; 111: 600-8
67. McSweeney ME, Garwood S, Levin J, Marino MR, Wang SX, Kardatzke D, Mangano DT, Wolman RL: Adverse gastrointestinal complications after cardiopulmonary bypass: can outcome be predicted from preoperative risk factors? Anesth.Analg. 2004; 98: 1610-7, table
Supplement Digital Content 1. Acetylcholine (ACh-)induced relaxation in the absence and presence of 10 "M indomethacin (INDO; i.e. to inhibit cyclooxygenase-derived prostaglandins (PG’s)) only or with 100 "M L-NMMA (i.e. to inhibit NO production) additionally present, in arteries of normal control rats that underwent short anesthesia (for sacrifice) only. Full CR-curves are given for mesenteric (A) and coronary (B) arteries, as well as the individual contribution of different endothelial mediators hereto in both artery types (C), presented as the AUC (for further explanation and description, see text). Data are mean ± SD (n=5). Abbreviations: CPB, cardiopulmonary bypass.
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Vascular reactivity of the small vessels 39
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Supplement Digital Content 3 Table. Preconstriction level to phenylephrine of mesenteric and coronary arteries in different experimental groups Groups Preconstriction level, mN
Phenylephrine in Mesenteric artery
Serotonin in Coronary artery
Control 10.9±3.5 2.3±0.93
Sham 1h 10.8±5.9 1.7±0.7
CPB 1h 9.7±3.7 1.4±0.9
Sham 1day 7.3±4.0 1.3±0.6
CPB 1day 6.6±3.1 1.3±0.9
Sham 2 days 9.2±3.7 2.2±2.0
CPB 2days 10.5±4.9 1.5±0.9
Sham 5 days 10.0±3.8 2.7±1.0
CPB 5 days 8.8±5.7 2.7±1.9
Data are given as mean±SD. Abbreviations: CPB, cardiopulmonary bypass.
Supplement Digital Content 2 Table. Relaxant response to Sodium Nitroprusside of rat mesenteric and coronary arteries at different periods of recovery (1h, 1day, 2 and 5 days) in Sham and CPB groups Groups Response to Sodium Nitroprusside, %
Mesenteric artery Coronary artery Control 93.9±7.5 116.8±17.5
Sham 1h 95.2±12.6 99.1±16.5
CPB 1h 97.6±1.4 100.7±12.7
Sham 1day 95.7±2.8 110.5±13.5
CPB 1day 98.5±4.2 150.2±59.6
Sham 2days 94.6±9.0 119.7±29.9
CPB 2days 92.3±29.4 164.5±55.5
Sham 5days 92.4±9.8 107.3±10.6
CPB 5days 84.2±21.5 112.0±15.6
Data are given as mean±SD. Abbreviations: CPB=cardiopulmonary bypass.
Supplement Digital Content 4 Table. Characteristics of the ACh-mediated relaxation in coronary and mesenteric vessels during prolong recovery period. Comparisons were done with t-test vs Control group
Groups Coronary artery Mesenteric artery
Emax, % of the relaxation -log EC50 Emax, % of the relaxation -log EC50
Control 54.8±18.6 -6.6±0.2 58.6±16.3 -7.2±0.4
Sham 1h 58.0±8.3 -6.3±0.4 70.8±12.6* -6.3±0.6*
Sham 1d 51.5±22.6 -6.6±0.7 64.7±21.3 -7.1±0.4
Sham 2d 53.8±21.6 -6.4±0.3 70.7±25.5 -7.1±0.3
Sham 5d 39.9±9.3** -6.0±0.2** 47.9±33.5 -7.1±0.2
CPB 1h 72.55±11.0# -6.6±0.3 81.0±13.0* -6.9±0.7
CPB 1d 54.6±26.5 -6.3±0.7 66.8±38.2 -6.8±0.8
CPB 2d 41.9±21.3 -5.9±0.4** 51.1±32.8 -6.5±0.6*
CPB 5d 53.3±21.5 -6.4±0.3 49.54±25.3 -6.9±0.5
*P<0.05, t-test versus Control; **P<0.001, t-test versus Control; # P=0.051, t-test versus Control
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Chapter 3
Changes of vascular reactivity of rat thoracic
aorta during prolonged recovery following cardiopulmonary bypass
Iryna V. Samarska, Anouk Oldenburger, Hendrik Buikema, Hubert Mungroop Martin C. Houwertjes, Michel M.R.F. Struys,
Anne H. Epema, Robert H. Henning
42 Chapter 3
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Abstract Background: Changes in vascular reactivity may represent key elements of pathogenesis of cardiopulmonary bypass (CPB). The relationship between endothelial activation and the endothelial dependent vasomotor control following cardiopulmonary bypass has not been addressed. The aims of the present study were to examine vascular reactivity of rat thoracic aorta after CPB within a follow up from 60 min to 5 days and correlate the observed changes in vasoresponsiveness with the level of induction of markers of the endothelial activation. Materials and Methods: Male Wistar rats (n=78) were anesthetized with isoflurane (2,5-3%) and fentanyl/midazolam during CPB. Animals were assigned to sham or CPB with normothermic extracorporeal circulation (ECC) for 60 min at a flow of 100-110 mL kg-1 min-1. After recovery for 60 min to 5 days, constriction to phenylephrine (PE) and potassium chloride (KCl) and relaxation to acetylcholine (Ach) in thoracic aorta were assessed. The expression of E-selectin, VCAM-1, ICAM-1, TNF-!, HO-1 and TGF-! was assessed by PCR and of COX-2, p22phox and TxA2 receptors by western blot. Results: In both Sham and CPB the sensitivity to phenylephrine and potassium chloride were similarly and significantly decreased at day 5 of recovery compared to control (for PE AUC value in au: 322.0±152.7, 435.9±132.8 and 1200±245; Emax KCl in uM: 153.9±15.2, 183.6±10.9 and 331.4±71.3, respectively) At day 5 of recovery, EDHF-mediated relaxation to acetylcholine was significantly decreased in both Sham and CPB as compared to control, while total Ach-mediated relaxation was unchanged. Expressions of all molecular markers were upregulated in CPB group after 1hour recovery period. Conclusions: Extracorporeal circulation induced endothelial cell activation after short-term recovery period (1 hour). Sham-related procedures (surgical intervention and/or prolonged anesthesia) affected vasoresponsiveness by attenuating vascular contractility and elevation of EDHF-mediated reactivity after 5 days of recovery period. However, extracorporeal circulation did not additionally influence vascular reactivity. Hence, endothelial cell activation did not translate/result into impaired endothelium-dependent relaxation in rat aorta. Introduction
Cardiopulmonary bypass (CPB) represents a widely used technique in thoracic surgery enabling various surgical procedures such as coronary artery bypass grafting, valve surgery, heart-lung transplantation and pulmonary surgical interventions. CPB is associated with a systemic inflammatory response syndrome (SIRS), which is triggered by multiple factors such as surgical trauma, contact of blood with artificial surfaces of the extracorporeal circuit, and ischemia-reperfusion injury.1;2 The abovementioned processes may lead to the activation of the endothelium with accompanying up-regulation of the cell adhesions molecules.3-7 Induction of the soluble E-selectin, which mediates adhesion of the neutrophils to the endothelium, has been shown after cardiac surgery previously, and is thought to represent a marker of the neutrophil-evoked endothelial injury.3;8;9 CPB has been documented to change vascular reactivity in
Vascular reactivity and endothelial activation 43
!
mesenteric, cerebral, coronary, and skeletal muscle vessels.10-17 CPB caused endothelial dysfunction of the mesenteric artery after 6 hours of recovery,12 in cerebral artery directly after extracorporeal circulation,13 and reduced myogenic reactivity of the coronary and skeletal muscles arterioles.13;16;17 However, the relationship between endothelial activation and worsening of endothelial dependent vasomotor control following cardiopulmonary bypass has not been addressed.
The aims of the present study were 1) to examine vascular reactivity of rat thoracic aorta after CPB with a follow up from 60 min to 5 days and 2) correlate the observed changes in vasoresponsiveness with the level of induction of markers of the endothelial activation. Material and Methods
The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen. This study was performed in n=78 adult male Wistar rats (507.4±31.3 g; Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study.
Experimental groups
Animals were randomly divided in four experimental groups with different duration of the recovery period after the procedure: (1) an 1hour group where animals were allowed to recover for 60 min after the end of the extracorporeal circulation, (2) a 1day group in which recovery lasted one day, (3) a 2days group that was allowed to recover for 2 days; and (4) a 5days group in which animals were allowed to recover for 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated Control group was introduced. Animals in Control group were sacrificed under isoflurane anesthesia (2.5-3%) only.
Experimental protocol The experimental protocol consisted of the following parts: anesthesia,
preparation, extracorporeal circulation, weaning with a recovery period (see fig. 1). Anesthesia was induced (2-3% isoflurane in O2/air (1:1)) before the trachea was intubated and the lungs mechanically ventilated (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography and arterial blood gas analysis), with O2/air (0.5:1) at a respiration frequency of 50 min-1 (0.5 s inspiration time). During the preparation period, the left femoral artery was cannulated (26-gauge catheter) for blood pressure monitoring. Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The carotid artery was canulated for arterial inflow using a
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22-gauge catheter; a multi-orfice 4.5 French cannula (adapted from a Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the common jugular vein for access. The tail artery was canulated for the administration of intravenous anesthetics, heparin, and protamine sulfat.
Extracorporeal circulation In CPB groups extracorporeal circulation (ECC) was initiated with a duration of
60 min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70), a neonatal membrane oxygenator (0140GM, Polystan, Vaerlose, Denmark) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6mm inner diameter). The circuit was primed with 15 ml of Haes 60mg ml -1 solution (Voluven®, Fresenius Kabi, Bad Homburg, BRD)). Animals were also heparinised (250 IU) after start of ECC. During period of extracorporeal circulation, rats were anesthetized with intravenous fentanyl (125 "g kg-
1), atracurium (0.5mg kg-1), and midazolam (2 mg kg-1). During experimental procedure blood and venous saturation was continuously monitored. Targeted CBP flow was 100-110 mL kg-1 min-1, corresponding to 60-70% of normal cardiacoutput. Samples for the blood gas analysis (0.1"l) were withdrawn at four time-points: at the end of the preparation period (15 min before start of ECC), twice during the ECC period (15 min after the start of it and 15 min before the its ending), and after ECC (45 min after the end of ECC).
Weaning and recovery During the weaning period, ECC was terminated and mechanical ventilation
was initiated. Protamine (150 IU kg-1 i.v.) was administered in order to neutralize heparin after which the animals were decannulated and the wounds sutured. Following extubation during the recovery period thereafter, animals were first kept under isoflurane anesthesia (0.8-1.0%) for another 60 min in order to stabilize. The duration of the recovery period varied from 1 till 120 hours after the end of the extracorporeal circulation according the experimental design. Sacrification was performed in the end of assigned protocol under isoflurane anesthesia (2.5-3%) by exsanguinations due to taking blood and organs for the analysis.
Figure 1. Scheme of the experimental protocol in Sham and CPB groups regarding the duration of recovery period. The experimental protocol consisted of the following parts: anesthesia, preparation, extracorporeal circulation, a weaning and a recovery period (see methods). Abbreviations: ECC, extracorporeal circulation.
!
Vascular reactivity and endothelial activation 45
!
Vascular reactivity Immediately following sacrifice, thoracic aorta was removed and placed in cold
physiological saline solution, cleaned from connective tissue and cut into 8 rings of 2-2.5 mm, which were placed in the individual organ bathes for isotonic tension recording. The baths contained 15ml of Kreb’s solution at 37°C and bubbled continuously with 95%O2/5%CO2 at pH 7.4. Rings were allowed to equilibrate for 40 min to reach a steady baseline. Vascular segments were primed and checked for viability by two consecutive exposures to potassium chloride (60 mM).
The experimental protocol consisted of evaluation of contractile responses to phenylephrine (PE; 10 nM to 100 "M) and endothelium-dependent relaxation to acetylcholine (ACh; 10 nM to 300 "M) in rings precontracted with PE. To assess the contribution of different endothelial mediators, ACh-induced relaxation was additionally studied in the absence and presence of L-NMMA (0.1 mM; an inhibitor of NO-synthase) and/or indomethacine (1 "M; an inhibitor of cyclooxygenase) administered 20 min before application of PE [18]. Prostaglandin-mediated relaxation was determined as a difference between total Ach-mediated relaxation and response to Ach in the presence of indomethacin. NO-mediated contribution was evaluated as the portion of acetylcholine induced relaxation in presence of indomethacin additionally sensitive to eNOS-inhibition by L-NMMA. By exclusion, the rest relaxation was accounted to be EDHF-mediated [19]. To quantify the contribution of the above endothelial mediators, the area between the concentration-response curve in the absence and presence of one or more inhibitors was taken. Following the final concentration of ACh, a maximal concentration of the NO-donor sodium nitroprusside (SNP; 0.1 mM) was applied to assess total endothelium-independent relaxation [20]. Mainly two types of the impacts were evaluated: Sham-related affects (namely, anesthesia and cannulation) and influence of the extracorporeal circulation (ECC) on vascular reactivity.
Western blotting
The methods used were described previously.21 Briefly, after grinding, the frozen abdominal aortas were placed in 300 "l of boiling 2% SDS followed by pounding by a polytron (Kinematica AG Littau, Switzerland). Then, samples were centrifuged (4000 rpm, 1 min) and heated (95 oC) for 5 min. After a second centrifugation (13000 rpm, 3 min), supernatant was collected and used for measurements. Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Forty µg of total protein from each sample was separated on 4-20% Precise Protein Gels (Pierce, Rockford, IL) and transferred to nitrocellulose membranes. Anti-cyclooxygenase-2 antibody (BD Bioscience Pharmingen, San Diego, CA), p22 phox (BD Bioscience Pharmingen, San Diego, CA), thromboxane A2 (BD Bioscience Pharmingen, San Diego, CA), and anti-$-actin (Sigma, St. Louis, MO) were used as a primary antibody. Horseradish peroxidase-linked rabbit anti-mouse antibody was applied as a secondary antibody. The blots were analyzed using Super Signal assay (Pierce, Rockford, IL). $-actin served as a housekeeping protein. Experimental levels are expressed as ratios to $-actin protein levels.
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Real time PCR Total RNA was extracted from abdominal aorta using Trizol Reagent
(Invitrogen, Carlsbad, CA, USA) with subsequent quality control by ethidium bromide staining in 1% agarose gel. DNase treatment of the RNA samples prior to RT-PCR was performed with RQ1 RNase-Free DNase (Promega, Madison, WI, USA). First-Strand cDNA Synthesis from RNA (1"g) was performed by using Reverse Transcription reagents (Promega, Madison, WI, USA). The expressions of E-selectin, VCAM-1, ICAM-1, TNF!, HO-1 and TGF$ 1 (table 1) were analyzed by real-time PCR with Absolute QPCR SYBR Green reagents (Molecular Probes, Leiden, Netherlands) and CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR protocol consisted of 15 min at 95ºC, followed by 40 cycles with heating to 95ºC and cooling to 60ºC for 1 minute. The amplification product was evaluated by melting curves analysis and agarose gel (1.5%) electrophoresis. Sequence-specific primers (table 1) were obtained from Biolegio (Nijmegen, the Netherlands). Cycle threshold (CT) values for genes analyzed were normalized to their CT value of GAPDH.
Drugs The ionic millimolar composition of Krebs solution was as follows: 120.4 NaCl,
5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: acetylcholine chloride (ACh), phenylephrine (PE), indomethacine, SNP, NG-methyl-L-arginine (L-NMMA). Stock solution (10 mmol/L) for indomethacine was prepared in 96% ethanol. All other drugs were dissolved in deionized water. L-NMMA was purchased from MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma (St. Louis, Missouri, USA). The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the bath. Data analysis
Data are given as mean±SD and n refers to the number of animals in a corresponding group. Contractile responses to phenylephrine (PE) and potassium chloride (KCl) are presented in "m displacement and relaxations to ACh and SNP were calculated as a percentage of pre-constriction. For each concentration-response curve
Table 1. Primer pairs for RT-PCR!
Gene Forward Reverse
VCAM-1 TAAGTTACACAGCAGTCAAATGGA CACATACATAAATGCCGGAATCTT
ICAM-1 CTGCCACCATCACTGTGT CTGACCTCGGAGACATTCTT
TGF!1 AGAGCTGCGCCTGCAGAG GAAGCCGGTTACCAAGGT
HO-1 GTGCACATCCGTGCAGAGAA GAAGGCCATGTCCTGCTCTA
TNF" CACGCTCTTCTGTCTACTGA GTACCACCAGTTGGTTGTCT
E-selectin CCATTCGGCCTCTTCAAGCTA TGCAGCTCACAGAGCCATTC
GAPDH AAGGTCGGTGTCAACGGATTT CAATGTCCACTTTGTCACAAGAGAA
Vascular reactivity and endothelial activation 47
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the following parameters were determined: 1) the Area Under the Curve (AUC) in arbitrary units (AU) (Sigma, Systat Software, Inc, Germany), 2) the maximal response (Emax) and 3) the concentration at which half-maximal response was reached (EC50). The difference in AUC for concentration-response curves to ACh in the absence or presence of an inhibitor was used as an estimate for the contribution of different endothelial mediators, as described previously [18;19]. Differences were evaluated using one-way ANOVA or repeated measurements ANOVA combined with post-hoc Bonferroni test or with t-test where applicable. Differences were considered significant at P<0.05 (2-tailed). Results
Hemodynamics and blood gas analysis during the protocol During the procedure, hemodynamic measurements (MAP) and blood gas
analysis were performed in order to monitor the animals. At baseline all parameters were in the normal range. During CPB, MAP was significantly lower compared with Sham (65.4 ± 13.9 and 105.3 ± 4.0 mmHg, respectively). During weaning, MAP of CPB animals returned to baseline levels (75.5 ± 25.1 mmHg).
Blood gas analysis was performed before start of ECC, twice during ECC and after weaning (table 2). CPB decreased pH, bicarbonate ion (HCO-
3), and Base excesses and increased levels of pCO2 and pO2, which may represent moderate respiratory acidosis. Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with priming solution. Intraoperative hyperglycemia was observed in both SHAM and CPB groups during the first 2 hours.
Contractile responses in aorta In order to evaluate contractile properties of rat thoracic aorta, maximal
response to potassium chloride (KCl; fig.2) and full concentration-response curves to phenylephrine (PE; fig.3) were constructed. In both Sham and CPB, maximal contractile responses to KCl were significantly decreased at 5 days of recovery, both compared to controls and to shorter periods of recovery. After 1hour of the recovery period Sham-related procedures (anesthesia and cannulation) decrease contractile response to PE, while ECC counteract those changes in vascular reactivity. Similarly at 5 days of recovery, concentration-response curves for PE showed a right shift and decreased Emax, and hence a decrease in AUC (fig. 3, panel A: IV, table 3).
Thus, the above findings suggest that the Sham-related surgical procedures (extended anesthesia and cannulation) induced the depression in thoracic aorta contractility after prolonged recovery, without significant additional influence of extracorporeal circulation.
48 Chapter 3
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Tabl
e 2.
Dat
a on
blo
od g
ases
ana
lysi
s, g
luco
se c
once
ntra
tion
and
mea
n ar
teri
al p
ress
ure
(M
AP
, fem
oral
art
ery)
in S
ham
and
CP
B g
roup
s du
ring
the
expe
rim
enta
l pro
toco
l P
aram
eter
s S
HA
M
CP
B
Bef
ore
EC
C
15 m
in E
CC
45
min
EC
C
Aft
er E
CC
B
efor
e E
CC
15
min
EC
C
45 m
in E
CC
A
fter
EC
C
MA
P, m
mH
g 10
2.4±
32.8
10
9.8±
12.9
10
3.5±
22.4
92
.0±1
8.2
84.1
±11.
6 57
.9*±
12.9
81
.4*±
25.6
79
.5±2
5.1
pH
7.43
±0.0
5 7.
45±0
.05
7.46
±0.0
7 7.
41±0
.06
7.42
±0.0
5 7.
38 *
±0.0
7 7.
32 *
±0.0
8 7.
37±0
.05
pCO
2, k
Pa
4.74
±1.0
4.
51±0
.8
4.68
±1.0
5.
15±0
.9
4.77
±0.8
5.
07*±
1.1
5.72
*±1.
0 5.
22±0
.8
pO2,
kP
a 17
.4±4
.7
16.6
±3.9
17
.7±4
.2
16.9
±4.6
19
.6±7
.0
34.1
*±17
.1
35.8
*±18
.3
19.4
±10.
2
HC
O3,
mm
ol/l
22.9
±1.5
23
.3±1
.5
24.7
±1.5
24
.4±2
.2
22.6
±2.3
21
.5±3
.6
21.6
*±2.
4 22
.4*±
1.7
BE
, mm
ol/l
-0
.4±1
.0
0.5±
1.2
1.8±
1.5
0.5±
1.4
-0.6
±1.7
-1
.9*±
2.7
-3.6
*±3.
1 -2
.04±
1.9
Hct
c 0.
4±0.
02
0.
4±0.
03
0.
4±0.
03
0.
4±0.
03
0.4±
0.02
0.3*
±0.0
5
0.3*
±0.0
2
0.3*
±0.0
3
Glu
cose
, mm
ol/l
20.6
±5.4
15.2
±5.4
8.4±
4.3
6.
6±1.
0
22.9
±4.1
20.5
±5.1
11.6
±4.4
6.9±
3.0
Abb
revi
atio
ns: H
ctc,
hem
atoc
rit; C
PB
, car
diop
ulm
onar
y by
pass
. *
deno
tes
P<0
.05,
Sha
m v
s C
PB
, ind
epen
dent
t-te
st
!Ta
ble
3. P
aram
eter
s of
the
Phe
nyle
phri
ne-m
edia
ted
cont
ract
ion
Gro
ups
EC
50, u
Mol
/L
Hill
slo
pe
Max
imal
res
pons
e, u
M
Con
trol
2.
7±0.
5
0.9±
0.1
488.
9±13
.8
Sha
m1h
our
33.8
±4.2
**
0.7±
0.09
42
2.8±
23.5
* C
PB
1 h
our
6.8±
2.0
1.3±
0.6
402.
8±22
.9*
Sha
m 1
day
8.1±
2.7
1.2±
0.6
415.
6±31
.0
CP
B 1
day
6.5±
3.9
1.07
±0.7
39
7.5±
45.5
S
ham
2da
ys
6.4±
1.7
1.4±
0.5
427.
0±19
.7*
CP
B 2
days
5.
1±1.
1 1.
2±0.
3 42
7.3±
14.9
* S
ham
5da
ys
12.6
±3.1
* 1.
03±0
.3
210.
2±13
.0*
CP
B 5
days
18
.1±7
.6
0.76
±0.3
26
4.9±
26.6
* *-
P<0
.05,
vs
Con
trol,
t-tes
t; **
- P<0
.001
, vs
Con
trol,
t-tes
t
Vascular reactivity and endothelial activation 49
!
Relaxant response in aorta Aortic rings were additionally studied for endothelium-dependent responses to
ACh. Data are present as parameters of the concentration-response curves (table 4) and contribution of different relaxant pathways (fig. 4). Relaxant reactivity of thoracic aorta of the control rats were characterized by major contribution of NO in total acetylcholine mediated relaxation counteracted by contractile prostaglandins (fig. 4). Neither extracorporeal circulation, nor the duration of recovery period affected total acetylcholine-mediated relaxation. In contrast, various changes were observed in the contribution of PG-, NO- and EDHF to overall relaxation. Both in Sham and in CPB groups, NO-contribution was non-significantly reduced after 5 days of recovery period. Significantly increased contribution of EDHF-mediated pathways was found at 1day and 5 days time-points of the recovery period. Further, the “amount” of contractile prostaglandins seemed to be augmented during 1-2 days of the recovery period with normalization by the 5th day. Collectively, the findings showed that Sham-related procedures (prolonged anesthetic intervention and/or cannulation) increased the
Table 4. Parameters of the Acetylcholine-mediated relaxation Groups EC50, Mol/L Hill slope Maximal response, % Control 5.3±0.8*10-8 1.5±0.2 36.4±0.5 Sham1hour 4.7±6.2*10-6 * 0.3±0.2 * 66.07±11.0 * CPB 1 hour 17.7±1.2*10-8 * 0.8±0.03 * 42.7±0.4 * Sham 1day 9.8±2.02*10-8 1.5±0.4 38.05±1.4 CPB 1day 10.5±3.3*10-8 1.1±0.3 32.5±1.5 Sham 2days 7.6±1.02*10-8 1.5±0.3 45.9±1.1 * CPB 2days 7.3±2.5*10-8 1.3±0.5 50.6±2.3 * Sham 5days 30.3±9.4*10-8 * 0.6±0.1 * 60.6±2.4 * CPB 5days 47.7±9.3*10-8 * 0.5±0.1 * 52.7±1.7 * *-P<0.05, vs Control, t-test
Figure 2. Vascular contractility to potassium chloride of rat thoracic aorta at different periods of recovery (1 hour, 1day, 2 days, 5 days) in Sham groups and following subjection of the animals to CPB-related procedures. Contractile responses are given as Emax. Data are mean ± SD (n=6-9). * indicates P<0.05 vs Control, one way ANOVA with post-hoc Bonferroni test. Abbreviations: KCl=, potassium chloride; CPB=, cardiopulmonary bypass.
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contribution of EDHF-pathway in total acetylcholine-mediated relaxation to maintain the overall relaxation response to ACh. However, extracorporeal circulation did not add any influence herein. !
Figure 3. Vascular contractility to phenylephrine of rat thoracic aorta at different periods of recovery (1 hour, 1day, 2 days, 5 days) in Sham groups and following subjection of the animals to CPB-related procedures. Contractile responses are given as full CR-curves (A) and the AUC (B). Data are mean ± SD (n=6-9). * indicates P<0.05 vs Control, one way ANOVA with post-hoc Bonferroni test. # indicates P<0.05 vs Control, repeated measurements ANOVA combined with post-hoc Bonferroni test. Abbreviations: PE=phenylephrine; CPB=cardiopulmonary bypass.
Vascular reactivity and endothelial activation 51
!
Protein expression Because of the elevated contribution of contractile prostaglandins in ACh-induced responses in almost all groups we additionally assessed protein expression of COX-2 - hence, the isoform of inducible COX, might be responsible for the synthesis of contractile prostanoids- and of TxA2 receptors - hence, implied to be involved in cyclooxygenase-dependent vascular contraction [22] – in aortic segments. p22phox protein, a NAD(P)H oxidase subunit associated with increased ROS production and oxidative stress in vascular endothelium [23] , was also assessed. Both COX-2 and TxA2 receptor protein as well as p22phox were similarly expressed in all groups regardless of type of experimental protocol (fig.5, for TxA2 receptors and p22phox – data not shown). However, regression analysis revealed that the degree of relaxation-inhibition by (indomethacine-sensitive) COX-derived prostaglandins was proportionally correlated to COX-2 protein expression (Pearson correlation coefficient=-0.50, n=22, P=0.01), but not the TxA2 receptor or p22phox. These findings suggest the (overall)
Figure 4. Total acetylcholine (ACh-)-induced relaxation (A) - and the contribution of (B) prostaglandins (PG’s), (C) NO, and (D) EDHF hereto, all provided as AUC - in rat thoracic aorta at different periods of recovery (1h, 1day, 2days or 5days) following subjection of the animals to CPB -related procedures. Data are mean ± SD (n=5-8). * indicates P<0.05 vs control, one-way ANOVA with following Bonferroni test. # indicates P<0.05 vs control Mann-Whitney Rank Sum Test. $ indicates P<0.05 vs control; # independent t-test. Abbreviations: CPB= cardiopulmonary bypass.
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outcome for endothelial function in vascular reactivity in the present study to an important extent was determined by the level of vascular COX-2 protein expression.
Expression of markers of endothelial activation, anti-inflammatory marker, and hypoxia
To investigate changes in endothelial cells related to an inflammatory response, we the evaluated expression of the adhesion factors E-selectin, VCAM-1, ICAM-1 and of TNF-" in abdominal aorta of a subset of animals. HO-1, an inducible enzyme in response oxidative stress and hypoxia and TGF-!, which might modulate the cell adhesion molecules expression, were also evaluated. No expression of these 6 factors was detected in control animals and in Sham and CPB animals beyond 60 min of recovery (fig. 6). In contrast, a consistent upregulation of all factors tested was found in all tested CPB animals at 1 hour recovery period. In contrast, sham animals at 1 h of recovery showed consistently no increase in expression (VCAM-1 and ICAM-1) or only in 1 out of 4 animals (fig. 6). Discussion
The present work studied the changes in vascular responsiveness of thoracic aorta after cardiopulmonary bypass during 5 days of recovery in relation to endothelial cell activation. The main finding of this study is that endothelial cell activation did not result into impaired endothelium-dependent relaxation to ACh in rat aorta. We found that CPB induced endothelial cell activation only after a short-term recovery period (1 hour), as evidenced by a consistently increased expression of 6 markers studied. Secondly, changed vasoresponsiveness, characterized by attenuated vascular contractility and elevation of EDHF-mediated relaxation, was found only after 5 days of recovery. Moreover, these vasomotor changes were to a similar extent present in both CPB and Sham, indicating that they were mainly the result of the accompanying
Figure 5. COX-2 expression in thoracic aorta in all groups (at least 4 per group). Representative blot (A), relative expressions patterns (B) and relation between individual Pr’s-mediated contribution and COX-2 expression; Data were evaluated by regression analysis and Pearson correlation. Abbreviations: CPB= cardiopulmonary bypass.
Vascular reactivity and endothelial activation 53
!
procedures (cannulation procedures and/or prolonged anesthesia), but not of the extracorporeal circulation. Collectively, these data indicate that endothelial cell activation after extracorporeal circulation was not directly related to altered vascular properties of rat thoracic aorta.
Hemodynamics and blood gas analysis during the protocol Hemodynamic data showed that extracorporeal circulation significantly
decreased MAP to around 60-80 mmHg in CBP group, which corresponds to data in previously published studies of CBP in rat.12; 13 Although the phenomenon of CPB-induced hypotension has been addressed previously during last decade, the exact
Figure 6. Relative mRNA expression of E-selectin (A), VCAM-1 (B), ICAM-1 (C), TNF-! (D), HO-1 (E) and TGF-$ (F) in thoracic aorta. Abbreviations: CPB= cardiopulmonary bypass.
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underlying mechanisms are still unknown. In this context, platelet-mediated serotonin release is thought to be one of the most prominent explanations as extracorporeal circulation, contact of blood with artificial surfaces and the roller pump induces platelet activation and triggers release of serotonin. Serotonin acts as vasodilator through activation of the most sensitive and widespread 5-HT2B receptors mediating NO-release.24 Several other changes in parameters (pH, HCO-
3, and BE) indicate moderate metabolic acidosis in CPB animals. Since all levels returned to the normal range by the end of CPB, it is unlikely that this affected vascular reactivity. Previously severe acidosis has been shown to increase circulating TNF, IL-6, and IL-10 in sever septic shock.25 In our experimental setup only mild acidosis occurred, that turned to basal level after the end of extracorporeal circulation, hence it might doubtful modulate to some extend expression of the inflammatory cytokines. Intraoperative hyperglycemia was observed in both Sham and CPB groups during first 2 hours, indicating that this represents a reaction to the surgical intervention.
Vascular reactivity The present work showed an alteration of rat aorta vasoreactivity only after a
prolonged recovery period of 5 days in both Sham and CPB animals, demonstrating these changes to be caused by cannulation and/or prolonged anesthesia. Contractile response to PE and KCl at day 5 were decreased to a similar extent. Its decrease may be related to increased basal release of endothelium-mediated relaxing factors, particularly EDHF, which was found in the same time point of the 5th day of the recovery period. Previously, the role of the endothelium, in phenylephrine- and potassium chloride-mediated contraction has been shown, while mainly the role of COX, 20-HETE and NO have been investigated.25-28 Alternatively, inhibition of the contractile vascular reactivity may be mediated by decreased Ca2+ sensitivity, as both receptor dependent (PE) and independent (KCl) pathways seem affected. Possibly, cannulation and/or prolonged anesthesia inhibit contractile vascular reactivity through modulation of the MLC kinase and/or MLC phosphatase activity.
The main change in relaxant properties constitutes of an increase in the contribution of EDHF to maintain total endothelial dependent relaxation at 1day and 5 days of recovery in both Sham and CPB. Increased EDHF may serve a compensatory role in response to the depletion of the principal relaxant mediator in aorta, NO, as shown in several studies.29-31
Also our data showed that relaxant reactivity of thoracic aorta are characterized by involvement of contractile prostaglandins. Moreover the amount of contractile prostaglandins seemed to be augmented during first 2 days of the recovery period with normalization by the 5th day. Both types of cyclooxygenases (COX-1 and COX-2) can be involved in production of arachidonic acid contracting metabolites. COX-1 is accounted to be constitutively expressed in endothelium, while COX-2 is inducible form. In our study occurrence of contractile prostaglandins correlated with expression of COX-2. That corresponds to previously published data.22;32 Achieved results did not allow us to draw exact conclusion about the changes in COX-2 expression due to large inter-individual variability. However, correlation analysis
Vascular reactivity and endothelial activation 55
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showed relation between COX-2 expression and the value of the contractile prostaglandin’s participation in the total relaxation.
Endothelial activation and endothelial dysfunction To substantiate endothelial cell activation, we quantified the expression of
adhesion molecules, pro-inflammatory factors and oxidative stress by qPCR and western blot. Substantial upregulation of all these factors was found consistently in CPB and occasionally in Sham only after 60 min of recovery from CPB. Thus, we found ECC activation of E-selectin, which is a main marker of the endothelium activation in response to inflammatory cytokine (TNF-! and IL-1$)33 and the cell adhesions molecules of the immunoglobulin superfamily, ICAM-1 and VCAM-1, that serve as ligands for leukocyte integrins.34 In addition, CPB induced the upregulation of TNF- ! and TGF-$, which might modulate expression of adhesion molecules, provide a chemotactic gradient for leukocytes and other cells participating in an inflammatory response34-36 and HO-1, an enzyme induced in response to oxidative stress and hypoxia, with the same time-course. As a consequence, it is unlikely that endothelial activation, which is mainly present in the CPB group, accounts for vascular changes as these are present in both CPB and Sham to a similar extent. Nevertheless a direct relationship between the both has been suggested before in mouse thoracic aorta in diet-induced obesity model.34;37 Differences in pathophysiology of these two models (acute surgical intervention in our study and chronic disease state in later) do not allow us to make a comparison between two studies. Circulating cell adhesion molecules (E-selectin, VCAM-1, ICAM-1) have been shown to correlate with the development of the nephropathy, retinopathy, and cardiovascular disease in both type 1 and type 2 diabetes and with the degree of atherosclerosis and albuminuria in type-2 diabetic patients.18-21 Impaired acetylcholine-dependent vasodilation was recently associated with increased levels of the plasma soluble E-selectin and P-selectin in essential hypertension.38 However, in abovementioned studies E-selectin was measured in plasma (and therefore has unknown origin), while we evaluated its expression directly in rat abdominal aorta.
In conclusion, our study demonstrates CPB to induce an immediate and short-term endothelial activation in rat aorta. In addition, vasoresponsiveness is altered at the long run due to prolonged cannulation and/or anesthesia, without any additional effect of extracorporeal circulation. Achieved data suppose better understanding of the pathophysiological processes underlying CPB and possible therapeutical approaches of its complications. Reference List 1. Levy JH, Tanaka KA: Inflammatory response to cardiopulmonary bypass. Ann.Thorac.Surg. 2003;
75: S715-S720 2. Rinder C: Cellular inflammatory response and clinical outcome in cardiac surgery.
Curr.Opin.Anaesthesiol. 2006; 19: 65-8
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3. Galea J, Rebuck N, Finn A, Manche A, Moat N: Expression of soluble endothelial adhesion molecules in clinical cardiopulmonary bypass. Perfusion 1998; 13: 314-21
4. John AE, Galea J, Francis SE, Holt CM, Finn A: Interleukin-8 mRNA expression in circulating leucocytes during cardiopulmonary bypass. Perfusion 1998; 13: 409-17
5. Onorati F, Rubino AS, Nucera S, Foti D, Sica V, Santini F, Gulletta E, Renzulli A: Off-pump coronary artery bypass surgery versus standard linear or pulsatile cardiopulmonary bypass: endothelial activation and inflammatory response. Eur.J.Cardiothorac.Surg. 2009;
6. Onorati F, Santarpino G, Tangredi G, Palmieri G, Rubino AS, Foti D, Gulletta E, Renzulli A: Intra-aortic balloon pump induced pulsatile perfusion reduces endothelial activation and inflammatory response following cardiopulmonary bypass. Eur.J.Cardiothorac.Surg. 2009; 35: 1012-9
7. Paparella D, Yau TM, Young E: Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update. Eur.J.Cardiothorac.Surg. 2002; 21: 232-44
8. Kilbridge PM, Mayer JE, Newburger JW, Hickey PR, Walsh AZ, Neufeld EJ: Induction of intercellular adhesion molecule-1 and E-selectin mRNA in heart and skeletal muscle of pediatric patients undergoing cardiopulmonary bypass. J.Thorac.Cardiovasc.Surg. 1994; 107: 1183-92
9. Williams HJ, Rebuck N, Elliott MJ, Finn A: Changes in leucocyte counts and soluble intercellular adhesion molecule-1 and E-selectin during cardiopulmonary bypass in children. Perfusion 1998; 13: 322-7
10. Andrasi TB, Bielik H, Blazovics A, Zima E, Vago H, Szabo G, Juhasz-Nagy A: Mesenteric vascular dysfunction after cardiopulmonary bypass with cardiac arrest is aggravated by coexistent heart failure. Shock 2005; 23: 324-9
11. Boyle EM, Jr., Morgan EN, Kovacich JC, Canty TG, Jr., Verrier ED: Microvascular responses to cardiopulmonary bypass. J.Cardiothorac.Vasc.Anesth. 1999; 13: 30-5
12. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann.Thorac.Surg. 2004; 77: 2130-7
13. Modine T, Azzaoui R, Ouk T, Fayad G, Lacroix D, Warembourg H, Bordet R, Gourlay T: Changes in cerebral vascular reactivity occur early during cardiopulmonary bypass in the rat. Ann.Thorac.Surg. 2006; 82: 672-8
14. Ruel M, Khan TA, Voisine P, Bianchi C, Sellke FW: Vasomotor dysfunction after cardiac surgery. Eur.J.Cardiothorac.Surg. 2004; 26: 1002-14
15. Verrier ED, Morgan EN: Endothelial response to cardiopulmonary bypass surgery. Ann.Thorac.Surg. 1998; 66: S17-S19
16. Wang SY, Friedman M, Franklin A, Sellke FW: Myogenic reactivity of coronary resistance arteries after cardiopulmonary bypass and hyperkalemic cardioplegia. Circulation 1995; 92: 1590-6
17. Wang SY, Stamler A, Li J, Johnson RG, Sellke FW: Decreased myogenic reactivity in skeletal muscle arterioles after hypothermic cardiopulmonary bypass. J.Surg.Res. 1997; 69: 40-4
18. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
19. Gschwend S, Buikema H, Henning RH, Pinto YM, de ZD, van Gilst WH: Endothelial dysfunction and infarct-size relate to impaired EDHF response in rat experimental chronic heart failure. Eur.J.Heart Fail. 2003; 5: 147-54
20. Westendorp B, Schoemaker RG, van Gilst WH, Buikema H: Improvement of EDHF by chronic ACE inhibition declines rapidly after withdrawal in rats with myocardial infarction. J.Cardiovasc.Pharmacol. 2005; 46: 766-72
21. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF: Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ.Res. 2002; 90: 413-9
22. Feletou M, Verbeuren TJ, Vanhoutte PM: Endothelium-dependent contractions in SHR: a tale of prostanoid TP and IP receptors. Br.J.Pharmacol. 2009; 156: 563-74
23. Siekmeier R, Grammer T, Marz W: Roles of oxidants, nitric oxide, and asymmetric dimethylarginine in endothelial function. J.Cardiovasc.Pharmacol.Ther. 2008; 13: 279-97
24. Borgdorff P, Fekkes D, Tangelder GJ: Hypotension caused by extracorporeal circulation: serotonin from pump-activated platelets triggers nitric oxide release. Circulation 2002; 106: 2588-93
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25. Kellum JA, Song M, Venkataraman R: Effects of hyperchloremic acidosis on arterial pressure and circulating inflammatory molecules in experimental sepsis. Chest 2004; 125: 243-8
26. Aloamaka CP, Ezimokhai M, Morrison J: The role of endothelium in phenylephrine- and potassium-induced contractions of the rat aorta during pregnancy. Res.Exp.Med.(Berl) 1993; 193: 407-17
27. Chabot F, Mestiri H, Sabry S, l'Ava-Santucci J, Lockhart A, nh-Xuan AT: Role of NO in the pulmonary artery hyporeactivity to phenylephrine in experimental biliary cirrhosis. Eur.Respir.J. 1996; 9: 560-4
28. Guo Z, Su W, Allen S, Pang H, Daugherty A, Smart E, Gong MC: COX-2 up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice. Cardiovasc.Res. 2005; 67: 723-35
29. Ekse S, Clapp LH, Revhaug A, Ytrebo LM: Endothelium-derived hyperpolarization factor (EDHF) is up-regulated in a pig model of acute liver failure. Scand.J.Gastroenterol. 2007; 42: 356-65
30. Katz SD, Krum H: Acetylcholine-mediated vasodilation in the forearm circulation of patients with heart failure: indirect evidence for the role of endothelium-derived hyperpolarizing factor. Am.J.Cardiol. 2001; 87: 1089-92
31. Luksha L, Agewall S, Kublickiene K: Endothelium-derived hyperpolarizing factor in vascular physiology and cardiovascular disease. Atherosclerosis 2009; 202: 330-44
32. Wong SL, Leung FP, Lau CW, Au CL, Yung LM, Yao X, Chen ZY, Vanhoutte PM, Gollasch M, Huang Y: Cyclooxygenase-2-derived prostaglandin F2alpha mediates endothelium-dependent contractions in the aortae of hamsters with increased impact during aging. Circ.Res. 2009; 104: 228-35
33. Ulbrich H, Eriksson EE, Lindbom L: Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol.Sci. 2003; 24: 640-7
34. Asimakopoulos G, Taylor KM: Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules. Ann.Thorac.Surg. 1998; 66: 2135-44
35. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr.Opin.Pharmacol. 2009; 9: 447-53
36. Li MO, Wan YY, Sanjabi S, Robertson AK, Flavell RA: Transforming growth factor-beta regulation of immune responses. Annu.Rev.Immunol. 2006; 24: 99-146
37. Kim F, Pham M, Maloney E, Rizzo NO, Morton GJ, Wisse BE, Kirk EA, Chait A, Schwartz MW: Vascular inflammation, insulin resistance, and reduced nitric oxide production precede the onset of peripheral insulin resistance. Arterioscler.Thromb.Vasc.Biol. 2008; 28: 1982-8
38. de la SA, Larrousse M: Endothelial dysfunction is associated with increased levels of biomarkers in essential hypertension. J.Hum.Hypertens. 2009;
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Chapter 4
Microarray analysis of gene expression profiles in the kidney demonstrates a local inflammatory response
induced by cardiopulmonary bypass in rat
Hjalmar R. Bouma *; Iryna V. Samarska *; H. Maria Schenk; Marry Duin; Martin C. Houwertjes; Berthus G. Loef; Hubert E. Mungroop;
Michel M.R.F. Struys; Anne H. Epema; Robert H. Henning • these authors contributed equally
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Abstract Background: Cardiopulmonary bypass (CPB) is a commonly used technique in cardiac surgery. However, CPB is associated with acute renal dysfunction. Also, a temporary peri-operative decrease of renal function negatively influences long-term survival. To obtain an insight into the pathogenesis of renal dysfunction following CPB, we performed a microarray analysis of kidney gene expression in rat. Methods: Rats underwent CPB or Sham procedure and were sacrificed at 60 min, 1 and 5 days following the procedure. Microarray analysis was used to determine expression changes in the kidney following CPB and Sham. Results: Expression of 421 genes was significantly altered in CPB as compared to Sham, of which 407 genes in the acute phase (60 min) following CPB. Gene-ontology analysis revealed 28 of these genes being involved in inflammatory responses, with major activation of mitogen activated protein-kinase (MAP-kinase) signaling pathways. Potent inducers identified constitute of the interleukin-6 cytokine family that consists of interleukin-6 (IL6) and oncostatin M (OSM), which signal through the gp130-cytokine receptor. Downstream signaling leads to production of chemokines, adhesion molecules and molecules involved in coagulative pathways. Production of chemokines and adhesion molecules stimulate early influx of monocytes/macrophages, neutrophils and lymphocytes, thereby augmenting the renal inflammatory response. Conclusions: CPB induces an acute and local inflammatory response in the kidney, which might contribute to the induction of renal injury. The signaling pathways identified by microarray analysis of renal gene expression may represent pharmacological targets to limit renal injury following CPB. Introduction
Despite advances in anesthetic and surgical management, cardiac surgery with cardiopulmonary bypass (CPB) is still associated with increased morbidity and mortality. A serious and frequent complication of CPB is renal dysfunction, affecting up to 30% of patients.1-3 Importantly, this incidence has not changed in the last few decades. Serious renal dysfunction requiring renal replacement therapy has a high mortality rate, up to 80%.3 In addition, recent studies demonstrate that small changes in renal function across a broad spectrum of medical and surgical conditions resulted in increased in-hospital morbidity and mortality, and impaired long-term survival.4-6 For example, patients with a temporarily decline of renal function postoperatively, defined as a >25% increase of serum creatinine from baseline, have a substantially increased long-term mortality rate at 13 years of follow-up.3
The etiology of CPB-induced renal dysfunction is considered multifactorial involving hemodynamic changes, an inflammatory response and nephrotoxins.7 Although several contributing factors have been put forward, the most prominent being systemic inflammatory response syndrome (SIRS) and ischemia/reperfusion injury,8 the exact molecular mechanism of renal injury following CPB is still unexplored. To identify signaling pathways involved in the etiology of kidney injury following CPB, we
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investigated the genome-wide expression profile in the kidney during 5 days of follow-up of rats that had undergone CPB. Material and Methods
Animals The experimental protocol was approved by the Animal Ethics Committee of
the University of Groningen. This study was performed in n = 46 adult male Wistar rats with body weights of 510.4±31.0 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study period.
Experimental groups Experimental animals were randomly allocated to one of four experimental
groups with different duration of the recovery period after the procedure, being 60 min and 1 and 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated control group was examined. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5 %) only.
Experimental protocol The experimental protocol consisted of the following parts: anesthesia,
preparation, extracorporeal circulation, a weaning and a recovery period (fig. 1). Anesthesia was induced with 2.5 % isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography 35-42 mmHg, and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.0 ± 0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for continuous blood pressure monitoring. The mean arterial pressure (MAP) was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.
Extracorporeal circulation Subsequently, extracorporeal circulation was initiated in the CPB group for 60
min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70, Verder, Haan, Germany), a rat membrane oxygenator (M.Humbs, Valley, Germany). The oxygenator carried a sterile, disposable three-layer artificial diffusion
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membrane, made from hollow polypropylene fibres (Jostra AG, Hirrlingen, Germany) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of haes 60 mg ml-1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). No donor blood was used. Animals were additionally heparinized (250 IU kg-1) after the start of ECC. During CPB, rats were anesthetized with intravenous fentanyl (10 "g kg-1), atracurium (0.5 mg kg-1), and midazolam (2 mg kg-1) and blood oxygen saturation was monitored continuously by a pulse oxymeter. Targeted CBP flow was 120 mL kg-1 min-1. During CPB ventilation was stopped and oxygenator gas flow was maintained at 800 ml min–1 of O2:air mixture (1:4). Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the end of preparation period (15 min before start of CPB), twice during the CPB (at 15 and 45 min), and at 45 min after the end of CPB.
Weaning and recovery At the end of extracorporal circulation, CPB flow was gradually decreased and
mechanical ventilation was initiated. Protamine (150 IU kg-1 i.v.) was administered to neutralize heparin and cannules were removed and wounds sutured. Animals were kept ventilated under isoflurane anesthesia (0.8-1.0%) for 1 h to stabilize, followed by extubation. The total duration of the recovery period lasted 1 h, and 1, 2 and 5 days after the end of the CPB. Sacrification was performed under brief isoflurane anesthesia (2.5 %). Sacrification was performed in the end of the assigned protocol under isoflurane anesthesia (2.5-3%). Samples were snap-frozen in liquid nitrogen and stored at -80°C until further analysis.
Microarray RNA isolation was performed according to the manufacturer’s instructions
(RNA Isolation Kit, Bioké, The Netherlands). Preceding hybridization, RNA-quality was checked on a BioAnalyzer 2088 (Agilent, USA). In each group (n = 5-8), RNA-samples of two animals were pooled and randomized over the different arrays as shown in a table (Supplemental Digital Content 1, which is a table listing the randomization of samples on arrays). Microarrays were performed on RatRef-12 Expression Beadchips (Illumina, USA) according to the manufacturer’s protocol. After hybridization, iScan (Illumina, USA) was used to scan the arrays, and Genomestudio 2009.1 (Illumina, USA) was used to subtract background noise and normalize intensity values (quantile method). Next, Genespring GX 11.0 (Agilent, USA) was used to analyze these normalized values.
Statistics Differences in blood gas parameters between groups over time were
compared using a GLM Repeated Measures analysis. Prior to analysis of the microarray expression profiles, probes were filtered on normalized expression values from 20th to 100th percentile present in at least 50% of the arrays in any of the groups. Next, significant differences were calculated using a One-Way ANOVA with post-hoc
Gene expression profile in the kidney 63
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Tukey HSD corrected for multiple comparisons using Benjamini Hochberg FDR in Genespring GX 11.0 (Agilent, USA) to compare CPB to Sham (per time-point; P < 0.05).
Gene ontology analysis Gene ontology analysis is a statistical method that demonstrates which sets of
genes that are grouped to cellular component, biological process or molecular function (called “ontologies”) are significantly different expressed by the experiment. Of all genes being significantly expressed, the fold change versus pre-operative control was calculated and gene ontology analysis for biological processes was performed (cut-off P < 0.10).
Results Hemodynamic and blood gas analysis At baseline, all hemodynamic and blood gas parameters were similar and in
the normal range. During CPB, the mean arterial pressure (MAP) was significantly decreased compared to Sham treated animals. In addition, blood gas analysis demonstrated lower values for pH, bicarbonate (HCO3
-) and hematocrit (HCT), while arterial oxygen and carbon dioxide pressure (PaO2 and PaCO2) were increased as compared to Sham (P < 0.01; fig. 2).
Transcriptional alterations induced by CPB Microarray analysis demonstrated a substantial effect of CPB on genome-wide expression profiles in the kidney (fig. 3). Statistical analysis revealed 421 significant differences in expression of gene transcripts between CPB and Sham treated groups (P<0.05; Supplemental Digital Content 1, table, which is a table listing all genes different in expression). While 407 of these genes were different in the acute phase (60 min) between CPB and Sham, only ten and four genes were different at day 1 and 5 following the procedure, respectively. Of the genes that were differentially affected by CPB and Sham, 183 genes were downregulated in both, 158 genes were upregulated in both, while 80 genes were oppositely affected by Sham and CPB.
Figure 1. Time-line of the experimental procedure. Shown in this scheme are the induction of anesthesia using volatile anesthetics (2-3% isofluorane in O2/air), followed by preparation of the animals for extracorporeal circulation (±40 minutes). Animals are ventilated during the entire procedure except during extracorporeal circulation (60 minutes) when animals are anesthetized using intravenous anesthesia (fentanyl, atracurium, midazolam). After the weaning period (±15 minutes), animals are kept under very light anesthesia (0.8-1% isofluorane in O2/air) during the first hour of recovery in order to stabilize.
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Gene ontology analysis Gene ontology (GO) analysis was used to investigate classes of genes that
are involved in the same biological process among the differences between Sham and CPB across ~340,000 described assocations in the GO-database (cut-off P<0.10; tables 2–4). Herewith, gene ontology analysis provides initial information about signaling pathways that might be affected by CPB. Genes involved in cytokine biosynthetic processes and cytokine mediated signaling pathways (table 1; 11 genes), genes involved in defense and immune response (table 2; 17 genes), and genes involved in regulation of cellular metabolic processes (table 3; 23 genes) represented ontologies that were significantly affected by CPB.
Figure 3. Genomewide expression profiles following CPB (A) and Sham-surgery (B). Profile plots demonstrate the fold-change of gene expression for all genes with significant expression in the kidney as compared to healthy control for CPB (A) and Sham (B) during the acute phase (60 min), 1 and 5 days following the procedure. Changes are more profound following CPB and occur mainly in the acute phase.
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Gene expression profile in the kidney 65
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Figure 2. Perioperative hemodynamic values. Figure shows perioperative hemodynamic values of the different groups. MAP: mean arterial blood pressure; PaCO2: arterial carbon dioxide pressure; PaO2: arterial oxygen pressure; cHCO3-: bicarbonate; HCT: hematocrit. Blood gas analysis was performed on arterial blood samples drawn at different time-points during the experiment. Shown in the figures are mean ± standard deviation (SD). P=0.05, t-test
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Gene ontologies (biological processes) that were significantly altered in
expression by CPB were limited to the acute phase following surgery: no differences existed after 24 hours (tables 2 – 4). In the acute phase, profound (> 10-fold) upregulation occured of interleukin-6 (IL6; 283-fold), IL-1$ (19-fold), oncostatin M receptor (OSMR; 17-fold), IL-10 (14-fold) (table 1), chemokine (C-X-C-motif) ligand 1 and 2 (CXCL1/2; 238- and 241-fold, respectively);P-selectin (Selp; 160-fold), CXCL10 (60-fold), FBJ osteosarcoma oncogene (FOS; 33-fold), E-selectin (Sele; 30-fold), chemokine (C-C-motif) ligand 2 (CCL2; 19-fold), peptidoglycan recognition protein 1 (PGLYRP1; 19-fold), CCL7 (13-fold) (table 2), early growth response 1 (EGR1; 20-fold), myelocytomatosis oncogene (MYC; 14-fold), zinc finger protein 36 (ZFP36; 11-fold) and runt related transcription factor 1 (RUNX1; 10-fold) (table 3). No genes were profoundly downregulated by CPB as compared to Sham. Of these 17 genes that are profoundly affected by CPB as compared to Sham, 13 were involved in the regulation of immune responses (tables 2 and 3) while the minority of affected genes was associated with other (non-immunological) processes (table 3).
Table 1. Genes involved in cytokine biosynthetic process and cytokine mediated signaling pathways GO: cytokine biosynthetic process and cytokine mediated signaling pathways
Acute day 1 day 5
Entrez ID
Symbol Definition CPB Sham p CPB Sham p CPB Sham p
24498 IL6 interleukin 6 283.4 0.8 .00 1.5 1.3 ns 0.4 2.0 ns 24494 IL1B interleukin 1 $ 19.4 2.6 .00 1.1 0.9 ns. 1.1 1.1 ns 310132 OSMR oncostatin M
receptor 17.4 4.1 .00 1.4 1.9 ns 1.6 1.7 ns
25325 IL10 interleukin 10 14.2 1.0 .00 1.0 1.0 ns 1.0 1.4 ns 24253 CEBPB CCAAT/enhancer
binding protein (C/EBP), $
9.3 2.3 .00 1.2 1.4 ns 1.2 1.2 ns
114203 SH2B2 SH2B adaptor protein 2
9.3 1.6 .00 0.5 1.3 ns 1.4 1.1 ns
24508 IRF1 interferon regulatory factor 1
6.2 1.5 .00 1.0 1.2 ns 1.1 0.9 ns
58954 KLF6 Kruppel-like factor 6
5.5 1.9 .00 1.2 1.3 ns 1.0 1.0 ns
310553 TLR2 toll-like receptor 2 3.4 1.1 .00 0.8 0.7 ns 0.9 0.8 ns 25625 TNFRSF1A tumor necrosis
factor receptor superfamily, member 1a
2.8 1.4 .00 0.9 1.3 ns 0.9 1.0 ns
301059 MYD88 myeloid differentiation primary response gene 88
2.2 1.0 .01 1.2
1.2 ns 1.2 1.0 ns
Table shows genes involved in cytokine biosynthetic processes and cytokine mediated signaling pathways, as determined by gene ontology analysis (cut-off P< 0.10) performed on all significant differences between CPB and Sham (ANOVA and post-hoc Tukey HSD; p < 0.05). Shown in the table is the fold-change compared to healthy control for CPB and Sham during the acute phase (60 minutes), 1 and 5 days following the procedure, as well as the p-value between CPB and Sham (ANOVA and post-hoc Tukey
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Discussion
Our analysis reveals 421 genes in rat kidney to be affected by CPB. The majority of changes was detected at a short interval after CPB, and normalized by 1 day following CPB. Geneontology analysis was performed to calculate which gene sets were significantly affected by CPB and demonstrated that these comprise mainly genes involved in immune responses (acute phase response) and metabolic processes (tables 2 – 4). The gp-130-cytokine receptor mediated signal transduction
Table 2. Genes involved in defense/immune response other than cytokine biosynthesis and signaling GO: defense/immune response other than cytokine biosynthesis and signaling
Acute day 1 day 5
Entrez ID
Symbol Definition CPB Sham p CPB Sham p CPB Sham p
114105 CXCL2 chemokine (C-X-Cmotif) ligand 2
241.3 2.5 .00 1.4 2.4 ns 2.9 0.7 ns
81503 CXCL1 chemokine (C-X-Cmotif) ligand 1
237.6 3.1 .00 1.1 1.1 ns 0.6 1.0 ns
25651 SELP selectin P 160.2 1.0 .01 0.5 0.9 ns 1.4 0.9 ns 245920 CXCL10 chemokine (C-
X-Cmotif) ligand 10
59.9 7.3 .00 1.0 1.9 ns 1.3 1.3 ns
314322 FOS FBJ osteosarcoma oncogene
33.3 1.3 .01 0.4 1.2 ns 1.6 1.6 ns
25544 SELE selectin E 29.7 1.5 .02 1.4 0.9 ns 1.9 1.2 ns
24770 CCL2 chemokine (C-Cmotif) ligand 2
19.3 2.1 .00 0.5 0.7 ns 0.9 0.5 ns
84387 PGLYRP1 peptidoglycan recognition protein 1
19.3 1.0 .01 0.7 1.6 ns 0.7 1.7 ns
287561 CCL7 chemokine (C-Cmotif) ligand 7
13.4 1.9 .00 0.8 0.9 ns 0.9 0.7 ns
25369 ADORA2A adenosine A2a receptor
6.1 2.1 .00 1.5 1.0 ns 1.4 1.2 ns
29187 CD69 CD69 molecule 4.9 1.1 .00 0.7 1.0 ns 0.4 0.6 ns 117029 CCR5 chemokine (C-
Cmotif) receptor 5
4.1 1.0 .01 1.3 1.2 ns 1.2 1.2 ns
60350 CD14 CD14 molecule 3.6 1.5 .00 1.2 1.0 ns 1.0 0.9 ns 171164 GBP2 guanylate
binding protein 2 3.3 0.8 .01 1.2 1.1 ns 1.1 0.7 ns
117540 PLSCR1 phospholipid scramblase 1
2.8 0.7 .01 1.1 1.3 ns 1.1 1.1 ns
246208 IFI47 Interferon-% inducible protein 47
2.6 1.0 .00 1.1 0.8 ns 0.8 0.8 ns
24165 ADA adenosine deaminase
0.4 1.5 .00 0.7 0.6 ns 0.7 0.7 ns
Table shows genes involved in defense/immune response other than cytokine biosynthesis and cytokine mediated signaling (which are shown in table 1), as determined by gene ontology analysis (cut-off P< 0.10) performed on all significant differences between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05). Shown in the table is the fold-change compared to healthy control for CPB and Sham during the acute phase (60 minutes), 1 and 5 days following the procedure, as well as the p-value between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05); a fold-change of 1.0 represents equal expression as observed in healthy control rats.
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that can be activated by (a.o.) IL6 and OSM was significantly upregulated following CPB. IL6 and the pathways that can be activated by IL6 are major players in the induction of the acute phase response. Downstream signaling leads to expression of chemokines, selectins and adhesion molecules in the kidney (fig. 4). Ultimately, this inflammatory response leads to influx of leukocytes (neutrophils, monocytes/macrophages and lymphocytes), and activation of coagulation cascades that lead to augmentation of the inflammatory response. Thus, CPB induces an acute phase response in the kidney that leads to the induction of a local inflammatory response with influx of leukocytes, which may play a role in the pathogenesis of renal dysfunction post-operatively.9;10
Although the etiology of renal dysfunction following CPB is complex, the design of our experiment allowed us to focus on the effect of the extracorporeal circulation on renal inflammation. Inflammation induced by CPB is postulated to be due to contact of leukocytes with the artificial surfaces of the machine, endotoxin released from the inflamed intestine, ischemia/reperfusion, hypothermia and surgical trauma during the procedure.9-12 In our model, hypothermia and surgical trauma were ruled out as factors influencing inflammation a priori, since animals were all kept normothermic during the procedure and the Sham procedure was included as a control for surgical trauma. Thus, our data suggests that the induction of a profound acute inflammatory response following CPB is primarily due to factors related to the use of extracorporal circulation.
Signal transduction pathways involved in the acute phase response induced by
CPB Interleukins play important signaling roles in inflammatory responses. In the
kidney, interleukin-1$ (IL1$) and IL6 are increased in expression in the acute phase (60 minutes) following CPB (table 1). IL1$ is 19-fold upregulated in CPB, while IL6 was almost 300-fold upregulated in CPB. Expression of IL6 might be induced by IL1$ via activation of MAP-kinases (ERK1/2 and p38),13 but also by Oncostatin M (OSM) that plays an important role in the acute phase response in the kidney.14 Both OSM and IL6 can activate the gp130-cytokine-receptor leading to the induction of expression of IL6, fibrinogen beta (FGB), OSMR and Serpine peptidase inhibitor 1 (Serpine 1)14 (fig. 4). Binding of IL6 to the gp130-receptor stimulates homodimerization that in turn leads to activation of downstream signalling pathways. Also heterodimerization of gp130 with OSMR, which shares extensive functional similarities with the gp130-cytokine receptor, leads to activation of downstream signal transduction pathways.15 In our analysis, OSMR was upregulated more than 17-fold by CPB (table 1). Transcription of IL6 stimulates a positive feedback mechanism, while negative feedback in the pathway is supplied by SOCS-3,16 which is upregulated more than 100-fold in the acute phase (60 minutes) following CPB. It has been shown that plasma levels of IL6 correlate with the presence of renal injury following CPB.17 Induction of expression of upstream activators and downstream signaling targets is consistent with activation of the gp130-cytokine receptor signal transduction pathway.
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Interleukin-6 (gp130-cytokine receptor) mediated signal transduction In the kidney, IL6 signals through the glycoprotein-130 (gp130)-cytokine-
receptor, downstream Janus Kinase/signal transducer and activator of transcription (JAK/STAT), and mitogen activated protein kinase (MAP-kinase) signal transduction pathways.16 Activation of this MAPK pathways is confirmed by our microarray, as the downstream transcription factors of MAPK-signaling Myc, Jun, Fos, and ATF-4 were upregulated by CPB (table 3 and Supplemental Digital Content 1, table), suggesting major involvement of MAPK-signaling in the kidney following CPB. Activation of MAP-kinases might occur via growth arrest and DNA-damage-inducible alpha and beta (GADD45A and –B) that are both upregulated in the acute phase (60 minutes) following CPB (Supplemental Digital Content 1, table) and can induce activation of mitogen activated protein kinase kinase kinase (MAP3K4). By activation of p38 and JNK, MAP3K4 can induce apoptosis of the cells.18 Another upstream activator of p38, dual specificity mitogen activated protein kinase kinase 6 (MAP2K6), was downregulated twice as much by CPB as by Sham (Supplemental Digital Content 1, table). Although MAP2K6 has been linked to p38, it does not cause activation of ERK.19 Downregulation of MAP2K6 might therefore be a counterregulatory pathway limiting p38-activation. This might shift the balance in MAPK-activation towards the ‘pro-survival’ MAP-kinase ERK.16 Our data are consistent with previous observations on gene expression changes in cardiac tissue following CPB, in which upregulation of Fos, Myc and Jun in patients20 and of mitogen activated protein kinase 1 (MAPK1) and mitogen activated protein kinase 3 (MAPK3) in rat.21 MAPK-pathways can be activated by several stimuli, including ischemia/reperfusion and circulating cytokines.16;22;23 Activation of different MAPK-signaling pathways determines cellular faith in terms of survival or apoptosis of the cells.24 MAP-kinases play an important role in signal transduction in inflammation by regulating the transcription of cytokines and/or growth factors16;22;23 (fig. 4). Taken together, our data suggest that a main pathway of local inflammation induction in kidney consists of activation of the gp130-cytokine receptor, activating downstream MAP-kinase signal transduction pathways that play a pivotal role in cell cycle regulation but also regulate the expression of cytokines.
Chemokines, selectins and adhesion molecules stimulate cellular influx into
the kidney Expression of chemoattractive cytokines (chemokines) can stimulate influx of
leukocytes into the kidney, thereby exaggerating the inflammatory response. In our analysis, C-X-C motif chemokine ligand 1 (CXCL1), CXCL2, C-C motif chemokine ligand 2 (CCL2), CCL7, and CXCL10 were grossly upregulated in the acute phase following CPB (table 2). While CXCL1 and CXCL2 stimulate attraction of neutrophils 25;26, CCL2 and CCL7 promote influx of monocytes/macrophages27;28 and CXCL10 stimulates influx of activated (i.e. CD69+-cells) T-lymphocytes.29 Local production of chemokines will stimulate influx of leukocytes into the kidney. Leukocyte influx is facilitated by the upregulation of E- and P-selectin, VCAM1 and ICAM1 in the acute phase (60 minutes) following CPB (table 2 and Supplemental Digital Content 1, table).
Table 3. Genes involved in regulation of cellular metabolic process (without direct immune function)
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GO: regulation of cellular metabolic process (without direct immune function)
acute day 1 day 5
Entrez ID
Symbol Definition CPB Sham p CPB Sham p CPB Sham p
24330 EGR1 early growth response 1
19.7 2.6 .00 0.5 1.2 ns 1.6 1.3 ns
24577 MYC myelocytomatosis oncogene
13.6 3.3 .00 1.2 1.8 ns 1.1 1.2 ns
79426 ZFP36 zinc finger protein 36
10.7 1.2 .00 1.2 1.2 ns 1.1 1.1 ns
50662 RUNX1 runt related transcription factor 1
10.1 2.2 .01 1.1 1.1 ns 2.0 1.3 ns
24517 JUNB jun B proto-oncogene
8.1 1.6 .00 0.9 1.4 ns 1.3 1.1 ns
59329 SNF1LK SNF1-like kinase 6.0 0.9 .01 1.1 1.2 ns 1.6 1.3 ns 304663 LYL1 lymphoblastic
leukemia derived sequence 1
0.2 1.0 .00 1.2 0.8 ns 1.3 1.0 ns
309452 NFKB2 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, p49/p100
4.6 1.3 .00 1.1 1.1 ns 1.0 1.1 ns
309175 CDC42EP2 CDC42 effector protein (Rho GTPase binding) 2
3.7 1.5 .01 1.2 1.2 ns 1.5 1.1 ns
63839 FHL2 four and a half LIM domains 2
0.3 0.7 .01 1.1 0.8 ns 0.9 0.9 ns
114121 CCNL1 cyclin L1 3.4 1.7 .00 1.3 1.1 ns 1.4 1.4 ns 306330 KLF2 Kruppel-like factor 2 0.3 0.6 .00 1.3 1.1 ns 0.9 1.1 ns 79431 BHLHE40 basic helix-loop-
helix family, member e40
3.3 1.7 .00 1.2 1.0 ns 1.1 1.0 ns
246760 MAFK v-maf musculoaponeurotic fibrosarcoma oncogene homolog K (avian)
2.9 1.7 .00 1.0 1.3 ns 1.1 1.2 ns
79255 ATF4 activating transcription factor 4 (tax-responsive enhancer element B67)
2.9 1.7 .00 0.9 1.1 ns 0.9 0.9 ns
114090 EGR2 early growth response 2
2.8 1.0 .00 1.0 0.9 ns 0.8 0.9 ns
302937 ANKS3 ankyrin repeat and sterile ! motif domain containing 3
0.4 0.8 .01 1.1 0.8 ns 0.8 0.8 ns
24516 JUN Jun oncogene 2.6 1.2 .00 0.8 1.0 ns 0.9 0.9 ns 29344 ZFP36L1 zinc finger protein
36, C3H type-like 1 2.3 1.0 .03 0.9 1.3 ns 1.0 1.1 ns
29618 BTG1 B-cell translocation gene 1, anti-proliferative
2.3 1.2 .00 1.0 1.0 ns 1.0 1.0 ns
311807 BMYC brain expressed myelocytomatosis oncogene
0.4 0.7 .00 0.9 0.9 ns 1.1 0.9 ns
288912 MRI1 methylthioribose-1-phosphate isomerase homolog
0.5 0.9 .01 0.9 0.8 ns 1.0 1.0 ns
286988 PNRC1 proline-rich nuclear receptor coactivator 1
2.1 1.2 .00 0.8 0.8 ns 0.8 0.8 ns
Table shows genes involved in regulation of cellular metabolic process (without direct immune function; these are shown in table 1 and 3), as determined by gene ontology analysis (cut-off P< 0.10) performed on all significant differences between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05). Shown in the table is the fold-change compared to healthy control for CPB and Sham during the acute phase (60 minutes), 1 and 5 days following the procedure, as well as the p-value between CPB and Sham (ANOVA and post-hoc Tukey HSD; P < 0.05); a fold-change of 1.0 represents equal expression as observed in healthy control rats.
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Increased expression of adhesion molecules has also been found in cardiac tissue of rats and in renal tissue from humans undergoing CPB.21;30 Taken together, our analysis provides evidence that local production of chemokines and increased expression of selectins and adhesion molecules following CPB stimulate cellular influx into the kidney, thereby amplifying the inflammatory response.
Neutrophil and monocyte influx Clinically, the number of circulating monocytes31 and neutrophils32 is increased
following surgery. Activation and influx of neutrophils and monocytes correlates with renal injury following CPB.31-34 The expression of CD14 (a marker of monocytes/macrophages) was significantly higher in the kidney following CPB (table 2). Activation of macrophages induces expression of CCL2, CCL7,35 CXCL1 and CXCL2,36;37 which is induced by CPB (table 2). While CCL2 and CCL7 provide a
Figure 4. Summary of major signal transduction pathways involved in renal injury following cardiopulmonary bypass. This figure shows schematically how activation of the interleukin-1-receptor (IL1R), gp130-cytokine receptor (interleukin-6-receptor or oncostatin-receptor; IL6R or OSMR) lead to activation of the mitogen activated protein kinases (MAP-kinases) extracellular signal-regulated kinase (ERK), Jun-kinase (JNK) and p38. Activation of JNK and p38 might result in apoptosis of the cell. Downstream signal transduction from the gp130-cytokine receptor can result in transcription of genes involved in the acute phase response in the kidney (fibrogen-$; FGB, OSMR; oncostatin M receptor, and Serpine 1), but also induces expression and activation of suppressor of cytokine signaling 3 (SOCS3) that provides a negative feedback loop by inhibiting the receptor. Expression of C-X-C chemokine ligand 1 and -2 (CXCL1 and -2) can be induced by downstream signal transduction of the IL1R in which MyD88 and the MAP-kinases ERK and p38 play important signaling roles. Further, activation of MAP-kinases is enhanced by growth arrest and DNA-damage inducible gene (GADD45) and MAP-kinase kinase kinase 4 (MAP3K4) and lead via activation of transcription factors (Myc, Fos, Jun, ATF2 and ATF4) to transcription of several genes that are involved in the inflammatory response and might contribute to the development of acute kidney injury following cardiopulmonary bypass (E-selectin, C-C motif chemokine ligand 2; CCL2, tumor necrosis factor alpha; TNF!, heme oxygenase; HMOX, and IL6).
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chemotactic signal for monocytes,27;28 CXCL1 and CXCL2 stimulate early recruitment of activated neutrophils into the tissue.36;37 Activated neutrophils increase their expression of CCRL2, which is a receptor for circulating chemokines and thus augments their sensitivity to chemokines.38 Expression of CCRL2 is significantly higher than Sham in the acute phase (60 minutes) following CPB (table 2); a finding that might represent influx of activated neutrophils into the kidney. CXCL1 and CXCL2 can be upregulated by ischemia/reperfusion,39 but also IL1$ is able to upregulate expression of CXCL1 and CXCL2 in which MyD88 and the MAP-kinases ERK1/2 and p38 play important signaling roles13;37 (fig. 4). Both the expression of IL1$ and MyD88 are increased in the acute phase (60 minutes) following CPB (table 1). Since CCL2, CCL7, CXCL1 and CXCL2 are mainly produced by macrophages; our array data support the view that the early activation of (resident) macrophages induces the production of chemokines, in turn causing an influx of monocytes/macrophages and neutrophils into the kidney.
Lymphocyte influx The number of circulating T-helper (Th-) cells, B-cells and natural killer cells
(NK-cells) is reduced following CPB.40;41 However, CPB induces the activation of both NK-cells40 and T-lymphocytes (i.e. CD69+-cells)42 in peripheral blood. Activated T-lymphocytes increase their expression of CD69.43 In our microarray, expression of CD69 increased about four fold in the acute phase (60 minutes) following CPB and thus likely represents an influx of activated T-cells in the kidney. Further, one of the chemokine receptors mainly expressed on T-cells and monocytes/macrophages, the C-C motif Chemokine Receptor 5 (CCR5),44 is upregulated significantly by CPB (table 2). A factor that plays a key role in the migration of lymphocytes from peripheral lymphoid organs is Sphingosine-1-phosphate (S1P).45 The expression of Sphk was more than eight fold increased by CPB, while this was only slightly upregulated following Sham (Supplemental Digital Content 1, table). We speculate that local production of S1P in the kidney stimulates influx of lymphocytes into the kidney. Further, influx of lymphocytes into the kidney is promoted by CXCL10.29 In our analysis, expression of CXCL10 was induced about 60-fold by CPB. Taken together, CPB induces activation of lymphocytes, while production of S1P and CXCL10 in the kidney causes influx of these activated cells.
Heme oxygenase 1 Heme oxygenase 1 (HMOX1) is the rate-limiting enzyme in the catabolism of
heme to form biliverdin, which is subsequently cleaved to free iron and carbon monoxide (CO).46 HMOX1 (Supplemental Digital Content 1, table) is upregulated almost 100-fold during the acute phase (60 minutes) following CPB, being ten times as much as following Sham. Ischemia induces expression of HMOX1, which can be mediated by activating transcription factor 4 (ATF4).47;48 Further, IL10 can induce expression of HMOX1 via p38 (fig. 4).49 Expression of ATF4 is increased almost three fold, while expression of IL10 was increased about 14-fold in the acute phase (60 minutes) following CPB (tables 4 and Supplemental Digital Content 1, table).
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HMOX1/CO are believed to regulate several (repair) processes to limit tissue injury: they stimulate the production of anti-inflammatory IL10 over pro-inflammatory IL6,50;51 induce the formation of FoxP3+ regulatory T-lymphocytes,50 and stimulate re-endothelialization after vascular injury by mobilizing progenitor cells.52 Induction of HMOX1 in the kidney seems to reflect direct cell injury as well as an adaptive response towards a cytoprotective state to limit renal injury, thereby protecting the kidneys from further ischemic or toxic damage.53
Characterization of the renal inflammatory response following CPB The sequence of events following CPB can be divided in events on a
molecular level (e.g. intracellular signaling pathways; summarized in fig. 4) as well as on a cellular level (e.g. influx of leukocytes). Taken together, our data suggest early activation of (resident) macrophages in the kidney following CPB that leads to local production of chemokines, thereby attracting other leukocytes into the kidney. Whereas production of CCL2 and CCL7 stimulates influx of monocytes, influx of neutrophils is promoted by CXCL1 and CXCL2, and production of CXCL10 and S1P stimulates lymphocyte infiltration. Upon activation of circulating cells, the expression of ligands for adhesion molecules is increased (CD11b, CCRL2 on neutrophils and CCR5 on monocytes and lymphocytes) that can bind the adhesion molecules expressed on endothelial cells on kidney vasculature (E-selectin, ICAM1, VCAM1) and facilitate transmigration of cells into the tissue. Signaling between leukocytes is mainly mediated by (chemoattractive) cytokines, while intercellular signaling can occur after these cytokines bind their receptors and activate the downstream signaling pathways. An important signaling pathway identified by our microarray is the gp130-cytokinereceptor mediated signaling pathway. This receptor can be activated by IL6 or OSM (grossly increased in our array) that leads via activation of MAP-kinase pathways to expression of adhesion molecules, (chemoattractive) cytokines and other molecules involved in the acute phase response.
Identification of pharmacological targets Resident macrophages might play a more important role than infiltrating cells
from the circulation in initiating the inflammatory response in the kidney. Our micoarray analysis shows early upregulation of chemokines that are mainly produced by (resident) macrophages rather than neutrophils or lymphocytes and stimulate influx of both monocytes/macrophages, neutrophils and lymphocytes. Since the application of leukocyte depleting filters during CPB is only partially effective in reducing the inflammatory response54;55 and does not affect clinical outcome following surgery,56 one may speculate that influx of circulating leukocytes into organs is of minor importance in the initiation of the local inflammatory response in organs following CPB. Conditional ablation of resident macrophages, an experimental technique that can be used to deplete the kidney of resident macrophages, may provide valuable data regarding the role of resident cells.57 Inhibition of signaling pathways, either directed at leukocytes (e.g. resident macrophages) or endothelial cells in the kidney, might reduce the inflammatory response in the kidney. As described above, signaling through the
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gp130-cytokine-receptor following activation of OSM or IL6 and downstream activation of MAP-kinases represents an important target in renal inflammation following CPB. Reducing the presence of the receptor-ligand, antagonizing the receptor or intervening in its signal transduction might limit renal inflammation following CPB. Identification of pharmacological agents that successfully reduce renal injury following CPB does not only provide more insight into the etiology of CPB-induced renal dysfunction and its influence on mortality, but might also improve survival following surgery.
Conclusion
Changes in gene expression of kidney occur early following CPB and represent mainly genes involved in inflammatory responses. The principle activation route consists of release of IL6 or OSM by resident macrophages, which in turn activate gp130-cytokine mediated signaling and downstream MAP-kinase pathways. Intervening with receptor activation or downstream signaling may represent novel therapeutic strategies to limit CPB-induced renal injury.
Reference List 1. Loef BG, Epema AH, Smilde TD, Henning RH, Ebels T, Navis G, Stegeman CA: Immediate
postoperative renal function deterioration in cardiac surgical patients predicts in-hospital mortality and long-term survival. J.Am.Soc.Nephrol. 2005; 16: 195-200
2. Karkouti K, Wijeysundera DN, Yau TM, Callum JL, Cheng DC, Crowther M, Dupuis JY, Fremes SE, Kent B, Laflamme C, Lamy A, Legare JF, Mazer CD, McCluskey SA, Rubens FD, Sawchuk C, Beattie WS: Acute kidney injury after cardiac surgery: focus on modifiable risk factors. Circulation 2009; 119: 495-502
3. Loef BG, Epema AH, Navis G, Ebels T, Stegeman CA: Postoperative renal dysfunction and preoperative left ventricular dysfunction predispose patients to increased long-term mortality after coronary artery bypass graft surgery. Br.J.Anaesth. 2009; 102: 749-55
4. Murphy GJ, Angelini GD: Side effects of cardiopulmonary bypass: what is the reality? J.Card Surg. 2004; 19: 481-8
5. Bapat V, Allen D, Young C, Roxburgh J, Ibrahim M: Survival and quality of life after cardiac surgery complicated by prolonged intensive care. J.Card Surg. 2005; 20: 212-7
6. Abu-Omar Y, Ratnatunga C: Cardiopulmonary bypass and renal injury. Perfusion 2006; 21: 209-13 7. Rosner MH, Okusa MD: Acute kidney injury associated with cardiac surgery. Clin.J.Am.Soc.Nephrol.
2006; 1: 19-32 8. Morariu AM, Loef BG, Aarts LP, Rietman GW, Rakhorst G, van OW, Epema AH: Dexamethasone:
benefit and prejudice for patients undergoing on-pump coronary artery bypass grafting: a study on myocardial, pulmonary, renal, intestinal, and hepatic injury. Chest 2005; 128: 2677-87
9. Caputo M, Yeatman M, Narayan P, Marchetto G, Ascione R, Reeves BC, Angelini GD: Effect of off-pump coronary surgery with right ventricular assist device on organ function and inflammatory response: a randomized controlled trial. Ann.Thorac.Surg. 2002; 74: 2088-95
10. Holmes JH, Connolly NC, Paull DL, Hill ME, Guyton SW, Ziegler SF, Hall RA: Magnitude of the inflammatory response to cardiopulmonary bypass and its relation to adverse clinical outcomes. Inflamm.Res. 2002; 51: 579-86
11. Asimakopoulos G: Systemic inflammation and cardiac surgery: an update. Perfusion 2001; 16: 353-60
12. Kourliouros A, Valencia O, Phillips SD, Collinson PO, van Besouw JP, Jahangiri M: Low cardiopulmonary bypass perfusion temperatures are associated with acute kidney injury following coronary artery bypass surgery. Eur.J.Cardiothorac.Surg. 2010; 37: 704-9
Gene expression profile in the kidney 75
!
13. Yang HT, Cohen P, Rousseau S: IL-1beta-stimulated activation of ERK1/2 and p38alpha MAPK mediates the transcriptional up-regulation of IL-6, IL-8 and GRO-alpha in HeLa cells. Cell Signal. 2008; 20: 375-80
14. Luyckx VA, Cairo LV, Compston CA, Phan WL, Mueller TF: Oncostatin M pathway plays a major role in the renal acute phase response. Am.J.Physiol Renal Physiol 2009; 296: F875-F883
15. Hermanns HM, Radtke S, Haan C, Schmitz-Van de Leur H, Tavernier J, Heinrich PC, Behrmann I: Contributions of leukemia inhibitory factor receptor and oncostatin M receptor to signal transduction in heterodimeric complexes with glycoprotein 130. J.Immunol. 1999; 163: 6651-8
16. Bouma HR, Ploeg RJ, Schuurs TA: Signal transduction pathways involved in brain death-induced renal injury. Am.J.Transplant. 2009; 9: 989-97
17. Gueret G, Lion F, Guriec N, Arvieux J, Dovergne A, Guennegan C, Bezon E, Baron R, Carre JL, Arvieux C: Acute renal dysfunction after cardiac surgery with cardiopulmonary bypass is associated with plasmatic IL6 increase. Cytokine 2009; 45: 92-8
18. Takekawa M, Saito H: A family of stress-inducible GADD45-like proteins mediate activation of the stress-responsive MTK1/MEKK4 MAPKKK. Cell 1998; 95: 521-30
19. Han J, Lee JD, Jiang Y, Li Z, Feng L, Ulevitch RJ: Characterization of the structure and function of a novel MAP kinase kinase (MKK6). J.Biol.Chem. 1996; 271: 2886-91
20. Ruel M, Bianchi C, Khan TA, Xu S, Liddicoat JR, Voisine P, Araujo E, Lyon H, Kohane IS, Libermann TA, Sellke FW: Gene expression profile after cardiopulmonary bypass and cardioplegic arrest. J.Thorac.Cardiovasc.Surg. 2003; 126: 1521-30
21. Podgoreanu MV, Michelotti GA, Sato Y, Smith MP, Lin S, Morris RW, Grocott HP, Mathew JP, Schwinn DA: Differential cardiac gene expression during cardiopulmonary bypass: ischemia-independent upregulation of proinflammatory genes. J.Thorac.Cardiovasc.Surg. 2005; 130: 330-9
22. di Mari JF, Davis R, Safirstein RL: MAPK activation determines renal epithelial cell survival during oxidative injury. Am.J.Physiol 1999; 277: F195-F203
23. Lawrence MC, Jivan A, Shao C, Duan L, Goad D, Zaganjor E, Osborne J, McGlynn K, Stippec S, Earnest S, Chen W, Cobb MH: The roles of MAPKs in disease. Cell Res. 2008; 18: 436-42
24. di Mari JF, Davis R, Safirstein RL: MAPK activation determines renal epithelial cell survival during oxidative injury. Am.J.Physiol 1999; 277: F195-F203
25. Li Q, Park PW, Wilson CL, Parks WC: Matrilysin shedding of syndecan-1 regulates chemokine mobilization and transepithelial efflux of neutrophils in acute lung injury. Cell 2002; 111: 635-46
26. Nieuwenhuis EE, MATSUMOTO T, Exley M, Schleipman RA, Glickman J, Bailey DT, Corazza N, Colgan SP, Onderdonk AB, Blumberg RS: CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung. Nat.Med. 2002; 8: 588-93
27. Mantovani A, Bottazzi B, Colotta F, Sozzani S, Ruco L: The origin and function of tumor-associated macrophages. Immunol.Today 1992; 13: 265-70
28. Corrigall VM, Arastu M, Khan S, Shah C, Fife M, Smeets T, Tak PP, Panayi GS: Functional IL-2 receptor beta (CD122) and gamma (CD132) chains are expressed by fibroblast-like synoviocytes: activation by IL-2 stimulates monocyte chemoattractant protein-1 production. J.Immunol. 2001; 166: 4141-7
29. Dufour JH, Dziejman M, Liu MT, Leung JH, Lane TE, Luster AD: IFN-gamma-inducible protein 10 (IP-10; CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J.Immunol. 2002; 168: 3195-204
30. Asimakopoulos G, Taylor KM: Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules. Ann.Thorac.Surg. 1998; 66: 2135-44
31. Wehlin L, Vedin J, Vaage J, Lundahl J: Peripheral blood monocyte activation during coronary artery bypass grafting with or without cardiopulmonary bypass. Scand.Cardiovasc.J. 2005; 39: 78-86
32. Rinder CS, Fontes M, Mathew JP, Rinder HM, Smith BR: Neutrophil CD11b upregulation during cardiopulmonary bypass is associated with postoperative renal injury. Ann.Thorac.Surg. 2003; 75: 899-905
33. Brix-Christensen V, Rheling M, Flo C, Ravn H, Hjortdal V, Marqversen J, Andersen N, Tonnesen E: Neutrophil and platelet dynamics at organ level after cardiopulmonary bypass: an in vivo study in neonatal pigs. APMIS 2004; 112: 133-40
34. Parikh CR, Mishra J, Thiessen-Philbrook H, Dursun B, Ma Q, Kelly C, Dent C, Devarajan P, Edelstein CL: Urinary IL-18 is an early predictive biomarker of acute kidney injury after cardiac surgery. Kidney Int. 2006; 70: 199-203
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!
35. Albright AV, Gonzalez-Scarano F: Microarray analysis of activated mixed glial (microglia) and monocyte-derived macrophage gene expression. J.Neuroimmunol. 2004; 157: 27-38
36. Wolpe SD, Sherry B, Juers D, Davatelis G, Yurt RW, Cerami A: Identification and characterization of macrophage inflammatory protein 2. Proc.Natl.Acad.Sci.U.S.A 1989; 86: 612-6
37. De FK, Henderson RB, Laschinger M, Hogg N: Neutrophil chemokines KC and macrophage-inflammatory protein-2 are newly synthesized by tissue macrophages using distinct TLR signaling pathways. J.Immunol. 2008; 180: 4308-15
38. Galligan CL, Matsuyama W, Matsukawa A, Mizuta H, Hodge DR, Howard OM, Yoshimura T: Up-regulated expression and activation of the orphan chemokine receptor, CCRL2, in rheumatoid arthritis. Arthritis Rheum. 2004; 50: 1806-14
39. Maheshwari A, Christensen RD, Calhoun DA, Dimmitt RA, Lacson A: Circulating CXC-chemokine concentrations in a murine intestinal ischemia-reperfusion model. Fetal Pediatr.Pathol. 2004; 23: 145-57
40. Akbas H, Erdal AC, Demiralp E, Alp M: Effects of coronary artery bypass grafting on cellular immunity with or without cardiopulmonary bypass: changes in lymphocytes subsets. Cardiovasc.Surg. 2002; 10: 586-9
41. Markewitz A, Lante W, Franke A, Marohl K, Kuhlmann WD, Weinhold C: Alterations of cell-mediated immunity following cardiac operations: clinical implications and open questions. Shock 2001; 16 Suppl 1: 10-5
42. Blacher C, Neumann J, Jung LA, Lucchese FA, Ribeiro JP: Off-pump coronary artery bypass grafting does not reduce lymphocyte activation. Int.J.Cardiol. 2005; 101: 473-9
43. Cambiaggi C, Scupoli MT, Cestari T, Gerosa F, Carra G, Tridente G, Accolla RS: Constitutive expression of CD69 in interspecies T-cell hybrids and locus assignment to human chromosome 12. Immunogenetics 1992; 36: 117-20
44. Rottman JB, Ganley KP, Williams K, Wu L, Mackay CR, Ringler DJ: Cellular localization of the chemokine receptor CCR5. Correlation to cellular targets of HIV-1 infection. Am.J.Pathol. 1997; 151: 1341-51
45. Schwab SR, Cyster JG: Finding a way out: lymphocyte egress from lymphoid organs. Nat.Immunol. 2007; 8: 1295-301
46. Guenegou A, Leynaert B, Benessiano J, Pin I, Demoly P, Neukirch F, Boczkowski J, Aubier M: Association of lung function decline with the heme oxygenase-1 gene promoter microsatellite polymorphism in a general population sample. Results from the European Community Respiratory Health Survey (ECRHS), France. J.Med.Genet. 2006; 43: e43
47. He CH, Gong P, Hu B, Stewart D, Choi ME, Choi AM, Alam J: Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation. J.Biol.Chem. 2001; 276: 20858-65
48. Fujita T, Toda K, Karimova A, Yan SF, Naka Y, Yet SF, Pinsky DJ: Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis. Nat.Med. 2001; 7: 598-604
49. Lee TS, Chau LY: Heme oxygenase-1 mediates the anti-inflammatory effect of interleukin-10 in mice. Nat.Med. 2002; 8: 240-6
50. Xia ZW, Xu LQ, Zhong WW, Wei JJ, Li NL, Shao J, Li YZ, Yu SC, Zhang ZL: Heme oxygenase-1 attenuates ovalbumin-induced airway inflammation by up-regulation of foxp3 T-regulatory cells, interleukin-10, and membrane-bound transforming growth factor- 1. Am.J.Pathol. 2007; 171: 1904-14
51. Kamimoto M, Mizuno S, Nakamura T: Reciprocal regulation of IL-6 and IL-10 balance by HGF via recruitment of heme oxygenase-1 in macrophages for attenuation of liver injury in a mouse model of endotoxemia. Int.J.Mol.Med. 2009; 24: 161-70
52. Lin HH, Chen YH, Yet SF, Chau LY: After vascular injury, heme oxygenase-1/carbon monoxide enhances re-endothelialization via promoting mobilization of circulating endothelial progenitor cells. J.Thromb.Haemost. 2009; 7: 1401-8
53. Zager RA: Uremia induces proximal tubular cytoresistance and heme oxygenase-1 expression in the absence of acute kidney injury. Am.J.Physiol Renal Physiol 2009; 296: F362-F368
54. Warren O, Wallace S, Massey R, Tunnicliffe C, Alexiou C, Powell J, Meisuria N, Darzi A, Athanasiou T: Does systemic leukocyte filtration affect perioperative hemorrhage in cardiac surgery? A systematic review and meta-analysis. ASAIO J. 2007; 53: 514-21
Gene expression profile in the kidney 77
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55. Lim HK, Anderson J, Leong JY, Pepe S, Salamonsen RF, Rosenfeldt FL: What is the role of leukocyte depletion in cardiac surgery? Heart Lung Circ. 2007; 16: 243-53
56. Warren OJ, Tunnicliffe CR, Massey RM, Wallace S, Smith AJ, Alcock EM, Darzi A, Vincent CA, Athanasiou T: Systemic leukofiltration does not attenuate pulmonary injury after cardiopulmonary bypass. ASAIO J. 2008; 54: 78-88
57. Duffield JS, Tipping PG, Kipari T, Cailhier JF, Clay S, Lang R, Bonventre JV, Hughes J: Conditional ablation of macrophages halts progression of crescentic glomerulonephritis. Am.J.Pathol. 2005; 167: 1207-19
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Supplemental Digital Content 1 Microarray annotations Group Microarray
number Rat numbers
Control 48716040004_I 77 + 78
Control 48716040004_L 79 + 80
Control 48716040005_E 81 + 82
Acute Sham 48716040004_D 18 + 19
Acute Sham 48716040005_C 20 + 21
Acute Sham 48716040005_G 16 + 17
1 day Sham 48716040004_E 42 + 61
1 day Sham 48716040005_H 34 + 36
1 day Sham 48716040005_J 39 + 41
1 day Sham 48716040005_K 37 + 38
5 day Sham 48716040004_B 70 + 73
5 day Sham 48716040004_H 68 + 69
5 day Sham 48716040005_D 74
Acute CPB 48716040004_A 12 + 14
Acute CPB 48716040004_K 15 + 43
Acute CPB 48716040005_B 5 + 7
Acute CPB 48716040005_L 9 + 10
1 day CPB 48716040004_C 32 + 35
1 day CPB 48716040004_F 25 + 26
1 day CPB 48716040004_G 27 + 31
1 day CPB 48716040004_J 63 + 64
5 day CPB 48716040005_A 2 + 67
5 day CPB 48716040005_F 71 + 72
5 day CPB 48716040005_I 75
Table shows annotation of microarray samples. In each group (n = 5 – 8 animals), RNA-samples were pooled to obtain n = 3 – 4 microarray samples. Samples were included in the different arrays as shown.
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Chapter 5
Protracted pulmonary inflammation during long-term recovery following
cardiopulmonary bypass in the rat
Iryna V. Samarska, Pieter A. van der Vorm, Hjalmar Bouma, Hendrik Buikema, Martin C. Houwertjes, Michel M.R.F. Struys,
Anne H. Epema, Robert H. Henning
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Abstract Background: Cardiopulmonary bypass (CPB) is associated with a multiple organ dysfunction syndrome involving disturbances in lung function. Pulmonary inflammation represents a main cause of postoperative lung dysfunction. The present study was intended to evaluate the time-dependent changes in markers of the pulmonary inflammation during a clinically relevant post-operative period. Materials and Methods: Male Wistar rats (n=78) were anesthetized with isoflurane (2,5-3%) and fentanyl/midazolam during CPB. Femoral and carotid artery and the right heart were cannulated and animals were randomly assigned to sham or CPB with normothermic extracorporeal circulation (ECC) for 60 min at a flow of 100-110 mL kg-1 min-1. Different markers of the endothelial activation, oxidative stress, pro- and anti-inflammatory cytokines were evaluated within 5 days recovery period by RT-PCR. Infiltration of the lung tissue with macrophages was evaluated by immunohistochemistry with anti-ED-1 staining. Results: After one hour of recovery, CPB rats showed increased expression of HO-1, E-selectin, ICAM-1, VCAM-1, IL6, IL-1beta (as compared with Control, p<0.05). At the second day of the recovery, CPB rats showed upregulation of SOD-1, TGFbeta-1 and IL1beta, which was higher in CPB group through the entire 5 days following CPB The amount of macrophages in lung tissue was relatively unchanged throughout the whole recovery period. Activity of neutrophil elastase in plasma was increased in CPB group after 1 hour of recovery (p<0.05). Conclusion: CPB evokes production of IL-6 and IL-1beta by residential macrophages of lung, which in turn activate neutrophils and endothelium. Activated neutrophils release neutrophil elastase, which participates in degradation of extra-cellular matrix components and contribute to acute lung tissue injury. Increased expression of TGFbeta1 is supposed to have immunosuppressive effects with modulatory influence on T-cell response. The prolonged upregulation of IL-1beta, TGFbeta1 and SOD-1 after 5 days of recovery period suggest ongoing inflammatory process in lungs that may contribute to post-CPB lung complications. Introduction
Cardiopulmonary bypass (CPB) plays an important role in thoracic surgery, including procedures such as coronary artery bypass grafting, valve surgery, heart-lung transplantation and pulmonary surgical interventions. However, CPB is also associated with the initiation of a systemic inflammatory response, which may affect function of vital organs including heart, kidney, lung, and liver.1-3 One of the severe clinical consequences of CPB includes postoperative lung dysfunction with interstitial pulmonary edema and subsequent abnormal gas exchange. Clinical data suggest that approximately 25% of the patients following an open-heart surgery exhibited signs of the pulmonary impairment during at least one week after. 4, 5 Lung damage following CPB may be explained by the specific character of during CPB, since lungs are more or less excluded from the circulation in this period. It is thought that the extracorporeal
Protracted inflammation in the lungs 81
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circulation causes the lung vascular injury, atelectasis, collapse of non-ventilated lungs, and massive or submassive pulmonary embolism.4, 5, 6 On the other hand, the CPB-related systemic inflammatory response plays a prominent role as well. Several triggers (surgical technique, effects of the anesthetic technique, extracorporeal circulation, metabolic changes, hypothermia, temporary cardiac dysfunction, contact of the blood with artificial surfaces, ischemia-reperfusion injury) may lead to the activation of the humoral and cellular immune system with subsequent release of the various mediators and factors that in turn cause multiple organ dysfunction syndrome. Several studies investigated lung injury after CPB and pulmonary inflammation was found to be the main cause.2, 7-10 The present study was intended to evaluate the time-dependent changes in markers of the pulmonary inflammation during a clinically relevant post-operative period (from 60 min up to 5 days of recovery). Material and Methods
The experimental protocol was approved by the Animal Ethics Committee of the University of Groningen. This study was performed in n=78 adult male Wistar rats with body weights of 507.4±31.3 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study.
Experimental groups Experimental animals were randomly allocated to one of four experimental
groups with different duration of the recovery period after the procedure, being 60 min and 1, 2 and 5 days. Each group was subdivided in two sub-groups: CPB and Sham. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the corresponding recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. In order to evaluate the normal vascular reactivity in rats without invasive interventions, an additional untreated control group was examined. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5-3%) only.
Experimental protocol The experimental protocol includes following parts: anesthesia, preparation,
extracorporeal circulation, a weaning and a recovery period. Anesthesia was induced with 2-3% isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.5±0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for blood pressure monitoring. The mean arterial pressure was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left
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carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (adapted from a Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.
Extracorporeal circulation Subsequently, extracorporeal circulation (ECC) was initiated in the CPB group
for 60 min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70), a neonatal membrane oxygenator (0140GM, Polystan, Vaerlose, Denmark) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of Haes 60 mg ml -1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). Animals were additionally heparinised (250 IU kg-1) after the start of ECC. During EEC, rats were anesthetized with intravenous fentanyl (125 "g kg-1), atracurium (0.5 mg kg-1), and midazolam (2 mg kg-1). During CBP, blood oxygen saturation was monitored continuously by a pulse oximeter. Targeted CBP flow was 100-110 mL kg-1 min-1, corresponding to 60-70% of normal cardiac output. Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the end of preparation period (15 min before start of ECC), twice during the ECC period (15 min after the start of it and 15 min before the its ending), and at 45 min after the end of ECC.
Weaning and recovery During the weaning period, ECC was terminated and mechanical ventilation
was initiated. Protamine (150 IU kg-1 i.v.) was administered in order to neutralize heparin after which cannules were removed and the wounds sutured. Following extubation, animals were first kept under isoflurane anesthesia (0.8-1.0%) for the first h of recovery in order to stabilize. The duration of the recovery period of various groups lasted 1 h, 1, 2 and 5 days after the end of the extracorporeal circulation. Sacrification was performed in the end of assigned protocol under isoflurane anesthesia (2.5-3%).
Real time PCR Total RNA was extracted from the lung using NucleoSpin RNAII (Macherey-
Nagel, Duren, Germany). First-Strand cDNA Synthesis from RNA (1ug) was performed by using Reverse Transcription reagents (Promega, Madison, WI, USA). The expression of SOD1, p22, HO-1, ICAM-1, E-selectin, VCAM-1, TNF-alpha, TGF beta 1, IL-6, IL1beta, FOXP3 (table 1) were analyzed by real-time PCR with Absolute QPCR SYBR Green reagents (Molecular Probes, Leiden, Netherlands) and CFX384 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The PCR protocol consisted of 15 min at 95ºC, followed by 40 cycles with heating to 95ºC and cooling to 60ºC for 1 minute. The amplification product was evaluated by melting curves analysis and agarose gel (1,5%) electrophoresis. Sequence-specific primers
Protracted inflammation in the lungs 83
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(table 1) were obtained from Biolegio (Nijmegen, the Netherlands). Cycle threshold (CT) values for genes analyzed were normalized to their CT value of GAPDH and presented as delta delta Ct (2-&&Ct).
Neutrophil elastase activity Neutrophil elastase activity was measured in plasma by using its specific
substrate N-methoxysuccinyl-Ala- Ala-Pro-Val p-nitroanilide.11 Briefly, 100ul plasma was incubated with 0.1M Tris–HCl buffer containing 0.5MNaCl and 1mM substrate at the temperature 37oC during 24 hours. This results in the formation of the product p-nitroanilide that was measured by spectrophotometry (Bio-Rad Laboratories, USA) at 405nm.
Immunohistochemistry The infiltration of the lung tissue with macrophages was evaluated by
immunohistochemistry. Fresh frozen lung sections were incubated with a monoclonal primary IgG antibody specific for the monocyte/macrophage antigen, ED-1 (Serotec, Oxford, UK). The immunostaining was quantitated by counting the number of ED-1 positive cells in 10 fields under dimensions 400x.
Data analysis Data are given as mean±SD and n refers to the number of animals in a
corresponding group. If the data passed normality test, then differences were evaluated using one-way ANOVA followed by Bonferroni test or with t-test (SPSS, Chicago, IL, USA). Unless the normality test was passed, data were analyzed by Kruskal-Wallis One Way Analysis of Variance on Ranks followed by Turkey test or Mann-Whitney Rank Sum Test (SigmaStat for SigmaPlot version 11, Systat Software Inc, San Jose, California). Differences were considered significant at P<0.05 (2-tailed).
Table 1. Primer pairs for RT-PCR
Gene Forward Reverse SOD1 AACATGGCGGTCCAGCGGAT GCCAAGCGGCTTCCAGCATT
p22 TGTTGCAGGAGTGCTCATCTGTCT AGGACAGCCCGGACGTAGTAATTT
HO-1 GTGCACATCCGTGCAGAGAA GAAGGCCATGTCCTGCTCTA
E-selectin CCATTCGGCCTCTTCAAGCTA TGCAGCTCACAGAGCCATTC
ICAM-1 CTGCCACCATCACTGTGT CTGACCTCGGAGACATTCTT
VCAM-1 TAAGTTACACAGCAGTCAAATGGA CACATACATAAATGCCGGAATCTT
IL-6 CCAGAAGACCAGAGCAGATT CACACTAGCAGGTCGTCATC
IL1beta CTGTGGCAGCTACCTATGTC CACACTAGCAGGTCGTCATC
TNF" CACGCTCTTCTGTCTACTGA GTACCACCAGTTGGTTGTCT
TGF beta 1 AGAGCTGCGCCTGCAGAG GAAGCCGGTTACCAAGGT
FOXP3 GCACAAGTGCTTTGTGCGAGT TGTCTGTGGTTGCAGACGTTGT
GAPDH AAGGTCGGTGTCAACGGATTT CAATGTCCACTTTGTCACAAGAGAA
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Results Hemodynamics and blood gas analysis during the protocol During the procedure, hemodynamic measurements (MAP) and blood gas
analysis were performed in order to monitor the animals. At baseline all parameters were in the normal range. Blood gas analysis was performed before start of ECC, twice during ECC and after weaning. During ECC, mean arterial blood pressure was significantly lower in the CPB groups compared with Sham (table 2). Also in the CPB group, a decreased pH, bicarbonate (HCO-
3), and Base excesses and increased levels of pCO2 and pO2 were found, which may represent moderate respiratory acidosis. Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with the priming solution. Intraoperative hyperglycemia was observed in both Sham and CPB groups during the first 2 hours. In the post-ECC period all parameters in CPB, including mean arterial blood pressure, returned to normal ranges and were similar in both Sham and CPB, except for hematocrit (table 2).
Expression of markers of the oxidative stress CPB is known to be associated with ischemia-reperfusion injury, hypoxia,
oxidative stress and increased ROS-production. Therefore the expression of SOD-1, p22 phox, HO-1 was evaluated by RT-PCR during long-term recovery (fig. 1). HO-1 was significantly increased in CPB after 1hour of recovery as compared with Control (P<0.05, t-test). Expression of SOD-1 was increased in CPB group at the second day of recovery and remained upregulated at 5th day of the postoperative period without a tendency for normalization. Expression of p22phox showe a tendency to increase in both CPB and Sham compared to Controls.
Thus, CPB evoked expression of HO-1, a marker of hypoxia injury, during short-term recovery and upregulated SOD-1, a marker of the oxidative stress, during later phases of the postoperative period.
Expression of markers of endothelial activation In order to evaluate the activation of the pulmonary endothelium, the relative
expression of E-selectin, ICAM-1, and VCAM-1 were studied by RT-PCR (fig.2). All three parameters were significantly upregulated in CPB after 1 hour of recovery as compared with Sham and/or Control. Interestingly, after 1 day of recovery, the values of all three markers returned to the baseline levels, even in the CPB group. Thus, CPB evoked an immediate endothelial activation which normalized within 24 h.
Protracted inflammation in the lungs 85
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Tabl
e 2.
Dat
a on
blo
od g
ases
ana
lysi
s, g
luco
se c
once
ntra
tion
and
mea
n ar
teri
al p
ress
ure
(M
AP
, fem
oral
art
ery)
in S
ham
and
CP
B g
roup
s du
ring
the
expe
rim
enta
l pro
toco
l P
aram
eter
s S
HA
M
CP
B
Bef
ore
EC
C
15 m
in E
CC
45
min
EC
C
Aft
er E
CC
B
efor
e E
CC
15
min
EC
C
45 m
in E
CC
A
fter
EC
C
MA
P, m
mH
g 10
2.4±
32.8
10
9.8±
12.9
10
3.5±
22.4
92
.0±1
8.2
84.1
±11.
6 56
.0*±
12.9
81
.5*±
25.6
80
.0±2
5.1
pH
7.43
±0.0
5 7.
45±0
.05
7.46
±0.0
7 7.
41±0
.06
7.42
±0.0
5 7.
40*±
0.07
7.
32 *
±0.0
8 7.
37±0
.05
pCO
2, k
Pa
4.74
±1.0
4.
51±0
.8
4.68
±1.0
5.
15±0
.9
4.77
±0.8
5.
1*±1
.1
5.72
*±1.
0 5.
22±0
.8
pO2,
kP
a 17
.4±4
.7
16.6
±4.0
17
.7±4
.2
17.0
±5.1
19
.6±7
.0
34.1
*±17
.1
36.0
*±18
.3
20.0
±10.
0
HC
O3,
mm
ol/l
23.0
±1.6
23
.3±1
.5
24.7
±1.5
24
.4±2
.2
22.6
±2.3
21
.5±3
.6
21.6
*±2.
4 22
.4*±
1.7
BE
, mm
ol/l
-0
.4±1
.0
0.5±
1.2
1.8±
1.5
0.5±
1.4
-0.6
±1.7
-1
.9*±
2.7
-3.6
*±3.
1 -2
.04±
1.9
Hct
c 0.
4±0.
02
0.
4±0.
03
0.
4±0.
03
0.
4±0.
03
0.4±
0.02
0.3*
±0.0
5
0.3*
±0.0
2
0.3*
±0.0
3
Glu
cose
, mm
ol/l
20.6
±5.4
15.2
±5.4
8.4±
4.3
6.
6±1.
0
22.9
±4.1
20.5
±5.1
11.6
±4.4
6.9±
3.0
Abb
revi
atio
ns: H
ctc,
hem
atoc
rit; C
PB
, car
diop
ulm
onar
y by
pass
. *
deno
tes
P<0
.05,
Sha
m v
s C
PB
, ind
epen
dent
t-te
st
86 Chapter 5
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Figure 1. Relative expression of markers of the oxidative stress and hypoxia (HO-1, SOD-1, p22phox) in Sham, CPB and Control groups within 5 days recovery period. * P<0.05, vs Control, one way ANOVA, Bonferroni test.
!
Protracted inflammation in the lungs 87
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Figure 2. Relative expression of markers of the endothelial activation (E-selectin, ICAM-1, VCAM-1) in Sham, CPB and Control groups within 5 days recovery period. * P<0.05, vs Control, Kruskal-Wallis One Way Analysis of Variance on Ranks, Turkey test; ** P<0.05, vs Control, One Way ANOVA, Bonferroni test; # P<0.05, vs 2days Sham, One way ANOVA, Bonferroni test
88 Chapter 5
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Neutrophil elastase activity CPB caused a marked increase in
the activity of neutrophil elastase in plasma after 1 hour recovery as compared with corresponding Sham group and Control (fig.3). After 1 day of recovery, neutrophil elastase acivity returned to baseline values. Thus, CPB induced an immediate increase in neutrophil elastase activity in plasma.
Expression of the pro and anti-inflammatory markers IL1beta, IL6 and TNF alpha expression were studied in lung tissue as pro-
inflammatory markers (fig. 4). mRNA levels of IL6 and IL1beta were significantly increased in CPB 1hour group as compared with Control and Sham. At 1 day of recovery, expression of IL6 was normalized, while IL 1beta remained upregulated in CPB groups up to 5th day of the recovery period. TNF alpha did not show pronounced changes in its expression and was even down-regulated at the earliest time point of recovery (1hour).
TGF beta 1 was evaluated as anti-inflammatory factor, as it is considered to be upregulated in SIRS.12 CPB induced a biphasic pattern in the expression of TGF beta: it was down regulated during the 1st day of the recovery period, followed by an upregulation ate the 5th day of the recovery period.
Together, these data show that CPB causes a profound upregulation of pro- and anti-inflammatory cytokines during recovery. During short-term recovery, CPB increased the expression of the pro-inflammatory cytokines IL-6 and IL-1beta and decreased the expression of anti-inflammatory cytokine TGF-beta. Expression of IL-6 was normalized at 1th day of the recovery period, while IL-1beta remained up-regulated during long-term recovery. Expression of TGF-beta was increased above normal value at 5th day of the recovery period.
Infiltration of lung with macrophages ED-1 staining was performed in order to evaluate the infiltration of lung tissue
with macrophages (fig. 6). Throughout the whole recovery period number of ED-1 positive cells remained almost on the same level in all groups. Thus, the amount of macrophages seems to be unaffected by the experimental procedure or CPB during 5 days recovery.
Figure 3. Neutrophil elastase activity in plasma in Sham, CPB and Control groups within 5 days recovery period. # P=0.051, vs Sham 1h, Mann-Whitney Rank Sum Test. * P<0.05, vs Control, Anova one way, Dunnet C test
!
Protracted inflammation in the lungs 89
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Figure 4. Relative expression of pro-inflammatory markers (Il-1beta, IL-6, TNF alpha) in Sham, CPB and Control groups within 5 days recovery period. * P<0.001, vs Control, Kruskal-Wallis One Way Analysis of Variance on Ranks, Turkey test. ** P<0.05, vs Control One way Anova with Bonferroni test # p<0.05, vs Sham 5days One way Anova with Bonferroni test.
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Figure 5. Relative expression of anti-inflammatory marker TGF beta 1 in Sham, CPB and Control groups within 5 days recovery period. * P<0.05, vs Control, One Way ANOVA, Bonferroni test. ** P<0.05, vs Control, Kruskal-Wallis One Way Analysis of Variance on Ranks, Turkey test
!
Figure 6. Infiltration of the lung with macrophages. ED-1 staining was performed in order to evaluate the infiltration of lung tissue with macrophages in Sham, CPB, and Control groups (resolution 400x). A: ED-1 staining in lung of Control group; B: negative control for anti-ED-1 antibody; C: Number of macrophages in the lung in all groups within the whole recovery period.
!
Protracted inflammation in the lungs 91
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Discussion The present study evaluates the time-dependent changes in markers of
pulmonary inflammation during a clinically relevant post-operative period (from 60 min up to 5 days of recovery). Several parameters were evaluated, including markers of hypoxia and oxidative stress (HO-1, SOD1, p22phox), markers of the endothelial activation (E-selectin, ICAM-1, and VCAM-1), pro- and anti-inflammatory markers (IL1 beta, IL-6, TNF alpha, and TGF beta), and activity of neutrophil elastase. Results show that CPB promoted endothelial activation and activation of neutrophils during short-term recovery period (1hour) and caused profound upregulation of specific pro- and anti-inflammatory cytokines during short and long-term recovery period.
Hypoxia and oxidative stress after CPB. CPB is known to be associated with hypoxia and oxidative stress injury, which
may contribute to acute lung injury after cardiac surgery.13, 14 In our study, CPB evoked significant upregulation of the hypoxia injury marker HO-1 during the first hour of recovery and a trend towards upregulation of the small NAD(P)H oxidase subunit p22phox. Similar results were shown previously in pigs after 2 hours CPB.13, 14 It is thought that ischemia-reperfusion injury may activate alveolar residential macrophages initiating NADPH-mediated ROS-production, which in turn cause alveolar tissue damage, and release of the proinflammatory cytokines.14 Interestingly, expression of antioxidant enzyme SOD1, that neutralizes superoxide radicals in cells, was increased only after the second day of recovery and remained upregulated during the entire recovery period without normalization. Such maintained activity of SOD1 in the CPB groups suggests ongoing pathological processes in lung tissue, probably due to increased inflammation, since acute CPB-mediated ischemia-reperfusion injury occurs immediately after the operation.15, 16
Endothelial and neutrophils activation. Our data show that CPB caused upregulation of E-selectin, which is a main
marker of the endothelium activation in response to inflammatory cytokine (TNF-! and IL-1$).17 Also CPB upregulated the cell adhesion molecules of the immunoglobulin superfamily, ICAM-1 and VCAM-1, that serve as ligands for leukocyte integrins.18-21 Further, CPB increased the plasma concentration of neutrophil elastase, which is a serine proteinase from the azurophilic granules of neutrophils, which is associated with acute lung injury.21 This enzyme is involved in several biological processes, such as degradation of extra-cellular matrix components and the modulation of the activity of some pro-inflammatory agents (TNF alpha, IL6, IL8) and anti-inflammatory mediators (TGF beta, TGF alpha, EGF).22
CPB is known to activate neutrophils and endothelial cells during acute phase reaction.23 The specific character of the lung functionality during CPB is represent extraordinary aspect since lungs are more or less excluded from the circulation in this period.3, 4 Clinical data suggest that approximately 25% of the patients following open heart surgery exhibited signs of the pulmonary impairment during at least one week after,3 while experimental studies in humans on endothelial activation marker
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expression covered mainly short term recovery period. Thus, elevated levels of circulating ICAM-1, VCAM-1, and E-selectin were shown during short term24 and 24h post-operative period.25 Our data showed that CPB caused endothelial activation during 1hour post-operative period with complete normalization till 1st day of recovery period. Similar pattern was found in our study for increased plasma level of neutrophil elastase as a marker of the neutrophils activation.
Pro and anti--inflammatory cytokines. The present study shows that CPB causes up-regulation of the pro-
inflammatory cytokines IL1beta and IL6 in lung tissue already after a short-term recovery period. Interestingly, CPB evoked short-lasting expression of IL6, which was completely normalized at 1day of recovery, while IL-1beta remained up-regulated during long-term recovery period, suggesting ongoing inflammation of the lung. CPB did not cause pronounced changes in TNF alpha expression. It is known that in lung TNF-alpha and IL-1beta are produced by resident or infiltrated macrophages in response to infective and non-infective agents and initiates the activation of neutrophils and cell adhesion molecules.26 IL-6 is a cytokine with various physiological function and several cells are responsible for its production, such as monocytes/macrophages, endotheliocytes, smooth muscle cells, fibroblasts.19, 26 Rapid up-regulation of IL-6 and IL-1beta, which was found in our study, might be as a result of its release from alveolar macrophages, which are abundantly distributed in lung tissue. The latter is supported by unchanged amounts of ED-1 positive cells (macrophages) in lung tissue during the whole recovery period, as found in this study.
Previously, short-time upregulation of these cytokines was shown after cardiac surgery and CPB 23, 27, 28 in different animal29 model of CPB and in human studies. We also evaluated long-term postoperative period. Interestingly, CPB evoked short-lasting expression of IL6, which was completely normalized up to 1day of recovery. IL-1beta remained up-regulated during long-term recovery, suggesting ongoing pulmonary inflammation.12, 30
TGF beta 1, which was evaluated as an anti-inflammatory factor, was found upregulated in SIRS.12, 31 TGF beta 1 has been shown to have a wide range of the immunosuppressive and immunomodulatory activity, inhibit superoxide anions production, and decrease the release of TNF-alpha.12, 31-33 In our study, CPB evoked an initial downregulation of TGF-beta during 1st day of the recovery period and a subsequent upregulation at the 5th day of recovery.
In summary, we suggest the following scheme of the CPB-mediated changes in lung: heart-lung machine-related triggers (i.e. ischemia-reperfusion injury, hypoxia, contact of blood with artificial surfaces or others) evoke the production of IL-6 and IL-1beta by residential macrophages of lung, which in turn activate neutrophils and endothelium. Activated neutrophils release neutrophil elastase, which participates in the degradation of extra-cellular matrix components and evoke acute lung tissue injury. At the same time, CPB increases expression of TGFbeta1, which has several immunosuppressive effects with a negative influence on T-cell response and may contribute to the CPB-evoked immunological disturbances. The prolonged upregulation
Protracted inflammation in the lungs 93
!
of IL-1beta, TGFbeta1 and SOD-1 after 5 days of recovery suggests ongoing pulmonary inflammation that may contribute to post-CPB lung complications.
Reference list 1. Salis S, Mazzanti VV, Merli G, Salvi L, Tedesco CC, Veglia F, Sisillo E: Cardiopulmonary bypass
duration is an independent predictor of morbidity and mortality after cardiac surgery. J Cardiothorac Vasc Anesth 2008; 22: 814-22
2. Yoshizumi K, Ishino K, Ugaki S, Ebishima H, Kotani Y, Kasahara S, Sano S: Effect of a miniaturized cardiopulmonary bypass system on the inflammatory response and cardiac function in neonatal piglets. Artif Organs 2009; 33: 941-6
3. Apostolakis EE, Koletsis EN, Baikoussis NG, Siminelakis SN, Papadopoulos GS: Strategies to prevent intraoperative lung injury during cardiopulmonary bypass. J Cardiothorac Surg 2010; 5: 1
4. Apostolakis E, Filos KS, Koletsis E, Dougenis D: Lung dysfunction following cardiopulmonary bypass. J Card Surg 2010; 25: 47-55
5. Sadaba JR, Greco E, Alvarez LA, Pulitani I, Juaristi A, Goiti JJ: The surgical option in the management of acute pulmonary embolism. J Card Surg 2008; 23: 729-32
6. Groeneveld AB, Jansen EK, Verheij J: Mechanisms of pulmonary dysfunction after on-pump and off-pump cardiac surgery: a prospective cohort study. J Cardiothorac Surg 2007; 2: 11
7. Goebel U, Siepe M, Mecklenburg A, Doenst T, Beyersdorf F, Loop T, Schlensak C: Reduced pulmonary inflammatory response during cardiopulmonary bypass: effects of combined pulmonary perfusion and carbon monoxide inhalation. Eur J Cardiothorac Surg 2008; 34: 1165-72
8. Risnes I, Wagner K, Ueland T, Mollnes T, Aukrust P, Svennevig J: Interleukin-6 may predict survival in extracorporeal membrane oxygenation treatment. Perfusion 2008; 23: 173-8
9. Yasser MA, Elmistekawy E, El-Serogy H: Effects of dexamethasone on pulmonary and renal functions in patients undergoing CABG with cardiopulmonary bypass. Semin Cardiothorac Vasc Anesth 2009; 13: 231-7
10. Zheng JH, Gao BT, Jiang ZM, Yu XQ, Xu ZW: Evaluation of Early Macrophage Activation and NF-kappaB Activity in Pulmonary Injury Caused by Deep Hypothermia Circulatory Arrest: An Experimental Study. Pediatr Cardiol 2009
11. Hagio T, Kishikawa K, Kawabata K, Tasaka S, Hashimoto S, Hasegawa N, Ishizaka A: Inhibition of neutrophil elastase reduces lung injury and bacterial count in hamsters. Pulm Pharmacol Ther 2008; 21: 884-91
12. Torre D, Tambini R, Aristodemo S, Gavazzeni G, Goglio A, Cantamessa C, Pugliese A, Biondi G: Anti-inflammatory response of IL-4, IL-10 and TGF-beta in patients with systemic inflammatory response syndrome. Mediators Inflamm 2000; 9: 193-5
13. Dodd-o JM, Welsh LE, Salazar JD, Walinsky PL, Peck EA, Shake JG, Caparrelli DJ, Ziegelstein RC, Zweier JL, Baumgartner WA, Pearse DB: Effect of NADPH oxidase inhibition on cardiopulmonary bypass-induced lung injury. Am J Physiol Heart Circ Physiol 2004; 287: H927-36
14. Shimoyama T, Tabuchi N, Kojima K, Akamatsu H, Arai H, Tanaka H, Sunamori M: Aprotinin attenuated ischemia-reperfusion injury in an isolated rat lung model after 18-hours preservation. Eur J Cardiothorac Surg 2005; 28: 581-7
15. Mumby S, Block R, Petros AJ, Gutteridge JM: Hydrogen peroxide and catalase are inversely related in adult patients undergoing cardiopulmonary bypass: implications for antioxidant protection. Redox Rep 1999; 4: 49-52
16. Davies SW, Duffy JP, Wickens DG, Underwood SM, Hill A, Alladine MF, Feneck RO, Dormandy TL, Walesby RK: Time-course of free radical activity during coronary artery operations with cardiopulmonary bypass. J Thorac Cardiovasc Surg 1993; 105: 979-87
17. Vallely MP, Bannon PG, Bayfield MS, Hughes CF, Kritharides L: Endothelial activation after coronary artery bypass surgery: comparison between on-pump and off-pump techniques. Heart Lung Circ 2010; 19: 445-52
18. Asimakopoulos G: Systemic inflammation and cardiac surgery: an update. Perfusion 2001; 16: 353-60
94 Chapter 5
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19. Asimakopoulos G, Taylor KM: Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules. Ann Thorac Surg 1998; 66: 2135-44
20. Elangbam CS, Qualls CW,Jr, Dahlgren RR: Cell adhesion molecules--update. Vet Pathol 1997; 34: 61-73
21. Moraes TJ, Zurawska JH, Downey GP: Neutrophil granule contents in the pathogenesis of lung injury. Curr Opin Hematol 2006; 13: 21-7
22. Lungarella G, Cavarra E, Lucattelli M, Martorana PA: The dual role of neutrophil elastase in lung destruction and repair. Int J Biochem Cell Biol 2008; 40: 1287-96
23. Paparella D, Yau TM, Young E: Cardiopulmonary bypass induced inflammation: pathophysiology and treatment. An update. Eur J Cardiothorac Surg 2002; 21: 232-44
24. Kalawski R, Bugajski P, Smielecki J, Wysocki H, Olszewski R, More R, Sheridan DJ, Siminiak T: Soluble adhesion molecules in reperfusion during coronary bypass grafting. Eur J Cardiothorac Surg 1998; 14: 290-5
25. Vallely MP, Bannon PG, Bayfield MS, Hughes CF, Kritharides L: Endothelial activation after coronary artery bypass surgery: comparison between on-pump and off-pump techniques. Heart Lung Circ 2010; 19: 445-52
26. Bhatia M, Moochhala S: Role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome. J Pathol 2004; 202: 145-56
27. Ng CS, Wan S, Yim AP, Arifi AA: Pulmonary dysfunction after cardiac surgery. Chest 2002; 121: 1269-77
28. Gilliland HE, Armstrong MA, McMurray TJ: Tumour necrosis factor as predictor for pulmonary dysfunction after cardiac surgery. Lancet 1998; 352: 1281-2
29. Bernard F, Romano A, Granel B: Regulatory T cells and systemic autoimmune diseases: systemic lupus erythematosus, rheumatoid arthritis, primary Sjogren's syndrome. Rev Med Interne 2010; 31: 116-27
30. Weber A, Wasiliew P, Kracht M: Interleukin-1beta (IL-1beta) processing pathway. Sci Signal 2010; 3: cm2
31. Sablotzki A, Welters I, Lehmann N, Menges T, Gorlach G, Dehne M, Hempelmann G: Plasma levels of immunoinhibitory cytokines interleukin-10 and transforming growth factor-beta in patients undergoing coronary artery bypass grafting. Eur J Cardiothorac Surg 1997; 11: 763-8
32. Markewitz A, Lante W, Franke A, Marohl K, Kuhlmann WD, Weinhold C: Alterations of cell-mediated immunity following cardiac operations: clinical implications and open questions. Shock 2001; 16 Suppl 1: 10-5
33. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009; 9: 447-5
!
!!!
Chapter 6
Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice
under isoflurane anesthesia1
Iryna V. Samarska, Matijs van Meurs, Hendrik Buikema, Martin C. Houwertjes, Francis M. Wulfert, Grietje Molema,
Anne H. Epema, Robert H. Henning
Anesthesiology 2009; 111:600 –8
1-This article is accompanied by an editorial in Anesthesiology: Does correcting the numbers improve long-term outcome? Robert D. Sanders, Mervyn Maze See supplement of this thesis
96 Chapter 6
!
Abstract Background: Hemorrhagic shock is associated with changes in vascular responsiveness that may lead to organ dysfunction and ultimately multiple organ dysfunction syndrome. Volatile anesthetics interfere with vasoresponsiveness, which may contribute to organ hypoperfusion. In this study, we examined the influence of adjunct nitrous oxide on the vascular responsiveness after short-term hemorrhagic shock under isoflurane anesthesia. Methods: Spontaneously breathing mice (n=31, 27.6 ± 0.31g) were anesthetized with isoflurane (1.4%) or with isoflurane (1.4%) and adjunct nitrous oxide (66%). Both groups were divided in Sham, Shock and Resuscitated groups. Vascular reactivity to phenylephrine and acetylcholine and expression of cyclooxygenases were studied in aorta. Results: In isoflurane-anesthetized groups, the contractile response to phenylephrine was increased in Shock as compared with the Sham and Resuscitated groups (Emax=3.2±0.4, 1.2±0.4 and 2.5±0.5 mN, respectively). Adjunct nitrous oxide increased phenylephrine contraction to a similar level in all three groups. In Sham-isoflurane group, acetylcholine caused a biphasic response: an initial relaxation followed by a contractile response sensitive to cyclooxygenases inhibition by indomethacine. The contractile response was abrogated in isoflurane-anesthetized groups that underwent shock. In all groups, adjunct nitrous oxide preserved the contractile phase. Shock induced a downregulation of cyclooxygenases-1, which was normalized by adjunct nitrous oxide. Conclusion: Adjunct nitrous oxide attenuates shock-induced changes in vascular reactivity and cyclooxygenases expression of mice under isoflurane anesthesia. This implies that vascular reactive properties during anesthesia in hemorrhagic shock conditions may be influenced by the choice of anesthetics. Introduction
Changed vascular reactivity in hemorrhagic shock (HS) may represent an important factor in the development of a multiple organ dysfunction syndrome. The mechanisms underlying this syndrome are not fully understood. However, early changes in vascular responsiveness may be of significance, as arterial hypotension and vascular leakage have been recognized as hallmarks of the post-shock state.1-3 Indeed, hypotension has been associated with altered vascular reactivity to different modulators of vascular tone.1 Several mechanisms have been proposed to cause post-shock vascular hyporeactivity including systemic and local release of nitric oxide, decreased plasma levels of vasopressin and changes in properties of vascular smooth muscle cells.2-5
Patients with hemorrhagic shock frequently receive anesthesia to facilitate diagnostic and therapeutic procedures. Many of the agents employed interfere with vasoresponsiveness and/or influence the therapeutic effectiveness of vasoactive drugs, and the development and outcome of hemorrhagic shock. Volatile anesthetics may evoke peripheral vasodilatation, myocardial depression, and lower sympathetic nervous system activity, subsequently leading to a decrease in blood pressure.6, 7
Anesthesia and vascular reactivity 97
!
Moreover, these agents may influence systemic secretion of vasoconstrictors such as vasopressin, angiotensin, and endothelin, further contributing to a reduction in blood pressure and deterioration of organ microcirculation.8, 9
Nitrous oxide, still widely used as an adjunct in various anesthetic techniques, has also been implied to influence hemodynamics. Adjunct nitrous oxide to sevoflurane (at 1.5 Minimum Alveolar Concentration [MAC]) or isoflurane (at 1.45 MAC) stabilizes several hemodynamic parameters of regional and systemic flow.10-12 The mechanisms through which nitrous oxide offsets the hemodynamic effects of volatile anesthetics are still not fully understood,13 although venoconstictor effects of nitrous oxide and sympathetic stimulation have been proposed as a possible explanation.12
The purpose of this study was to determine the change in vascular responsiveness obtained from mice after short-term hemorrhagic shock under isoflurane anesthesia, and to investigate the effects of adjunct nitrous oxide administration on the observed changes.
Materials and Methods
The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Groningen (Groningen, the Netherlands). This study was performed in 31 adult mice (27.6±0.31g; Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow) and drinking water throughout the study.
Experimental animals were assigned to two groups: A) a group of mice that were anesthetized with isoflurane (ISO,1.4%) in oxygen/air mixture (O2:33%), and B) a group of mice that were anesthetized with isoflurane (1.4%) and adjunct nitrous oxide (N2O, 66%) in oxygen (33%). Each group was divided in three experimental sub-groups (n = 5-6 per group). Sham animals were anesthetized and cannulated but not hemorrhaged, and kept under anesthesia for 90 min before being killed. Shock animals underwent anesthesia and hemorrhage and were sacrificed after 90 min of hemorrhagic shock. Resuscitated animals underwent anesthesia with hemorrhagic shock for 90 minutes, followed by resuscitation with 6% hydroxyethyl starch 130/0.4 (Voluven; Fresenius-Kabi, Bad Homburg, Germany). The resuscitation volume was twice the estimated volume of the blood withdrawn to induce hemorrhagic shock. Resuscitated mice were sacrificed 24 hours after induction of shock. All animals were breathing spontaneously throughout the protocol. A fixed-blood pressure model of hemorrhagic shock was used as described previously.14 Briefly, after induction of anesthesia the left femoral artery was cannulated with polyethylene tubing (internal diameter of 0.28 mm and an external diameter of 0.61 mm). Hemorrhagic shock was induced by blood withdrawal until mean arterial pressure (MAP) reached 30 mmHg; this blood pressure reduction was reached in approximately 11 minutes. An initial 0.5 ml of blood was withdrawn followed by additional portions of about 0.1 ml mean arterial pressure reached 30 mmHg. Total amount of blood withdrawn was estimated from the number of portions taken from the animal. Blood was collected in a heparinized 1-mL syringe to prevent clotting. Hypotension at 30 mmHg was maintained during 90 min with a continuous monitoring
98 Chapter 6
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of MAP. Small increments of blood were infused or withdrawn during the shock period to maintain MAP at 30 mmHg. In both Shock-groups, blood gas analysis was measured twice: just after femoral cannulation (t=0 min) and after the shock period immediately before killing (t=90 min). In both Resuscitated groups, blood gas analysis was performed just after femoral cannulation (t=0 min) and immediately before killing (t=24 hours). In Sham-groups, blood gas analysis was performed only immediately before sacrification (90 min) to avoid potential activation of endothelium and modification of vascular reactivity by withdrawal of blood at t=0. After killing, freshly isolated thoracic aorta was collected for in vitro vasomotor studies. Abdominal aorta was removed and snap-frozen in liquid nitrogen without pretreatment with any drug, and stored at -80oC until analysis.
Vasomotor responses After removal, the descending thoracic aorta was immediately placed into cold
physiological saline. Freshly isolated thoracic aortic rings (1.5 - 2 mm in length) were mounted on two 200µm stainless wires in the individual myograph baths wire myograph (Danish Myo Technology A/S, Aarhus, Denmark), containing 6ml of Kreb’s solution, warmed to 37°C and bubbled continuously with 95%O2/5%CO2 to maintain pH at 7.4. Length of aortic strips was assessed by microscopy. Aortic rings were equilibrated for 40 min until they were at a steady baseline. Then, rings were subjected twice to stimulation with potassium chloride (60 mM) in order to obtain reproducible contractile responses. Contraction was measured by obtaining concentration-response curves to phenylephrine (10 nM-100 "M). Endothelium-dependent relaxation was measured by obtaining concentration-response curves to acetylcholine (10 nM-300 "M) in rings precontracted with phenylephrine. The influence of vasoactive prostanoids on the response to acetylcholine was examined by incubating rings with the non-specific cyclooxygenase inhibitor indomethacine (10 "M) administered 20 min before application of phenylephrine. In the end of each experimental protocol, endothelium-independent relaxation was measured by applying the nitric oxide-donor sodium nitroprusside (0.1 mM).
Western blotting The methods used were described previously.15, 16 Briefly, after grinding, the
frozen aortas were placed in 300 "l of boiling 2% SDS followed by pounding by a polytron (Kinematica AG Littau, Switzerland). Then, samples were centrifuged (4000 rpm, 1 min) and boiled (95oC) for 5 min. After a second centrifugation (13000 rpm, 3 min), supernatant was collected and used for measurements. Protein concentration was determined by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Forty µg of total protein from each sample was separated on 4-20% Precise Protein Gels (Pierce, Rockford, IL) and transferred to nitrocellulose membranes. Anti-cyclooxygenase 1 antibody (ALEXIS Biochemicals, Lausen, Switzerland), anti-cyclooxygenase-2 antibody (BD Bioscience Pharmingen, San Diego, CA) and anti-$-actin (Sigma, St. Louis, MO) were used as a primary antibodies. Horseradish peroxidase-linked rabbit anti-mouse antibody was applied as a secondary antibody. Macrophage+IFNg/LPS (BD
Anesthesia and vascular reactivity 99
!
Bioscience Pharmingen) was used as a positive control for cyclooxygenase-2. The blots were analyzed using Super Signal assay (Pierce, Rockford, IL). $-actin served as a housekeeping protein. Cyclooxygenase-1 and cyclooxygenase-2 levels are expressed as ratios to $-actin protein levels.
Drugs Krebs solution was prepared freshly and of the following composition (mM):
120.4 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: Acetylcholine chloride, phenylephrine, indomethacine, sodium nitroprusside, NG-Monomethyl-L-arginine acetate salt. Stock solution (10 mM) for indomethacine, was prepared in 96% ethanol. All other drugs were dissolved in deionized water. NG-Monomethyl-L-arginine acetate salt was purchased form MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma. The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the organ baths.
Statistical analysis Data are present as mean ± SD and n refers to the number of animals in
corresponding group. The contractile response to phenylephrine was expressed in mN and relaxant responses to acetylcholine and sodium nitroprusside are expressed as a percentage of the preconstriction obtained with phenylephrine (0.1 mM). Concentration- response curves to phenylephrine and acetylcholine were characterized by Area Under the Curve, the maximal response (Emax), and the negative logarithm of the molar concentration that caused half-maximal response (EC50). Concentration-response curves of individual rings were plotted with SigmaPlot version 10.0 (Systat Software Inc, San Jose, CA). EC50 was calculated by Four Parameter Logistic Curve (SigmaPlot) for each ring. Statistical analysis was done with SPSS 16.0.2 for Windows (SPSS Inc., Chicago, IL). Differences between concentration-response curves were evaluated by Two-way Repeated-Measures ANOVA followed by Bonferroni test. Comparison of single parameters among multiple groups was done by One-Way ANOVA followed by Bonferroni test. Levene's test was used to confirm the homogeneity of variance. Independent sample t-test was used to compare the means of two groups where applicable. All tests were two-tailed and differences were considered significant at P<0.05. Results
Hemodynamic changes and routine lab parameters To monitor the animals during the procedure, hemodynamic measurements
and blood gas analyses were performed. After instrumentation at the start of the experimental protocol, hemodynamic parameters were comparable between the 3 experimental subgroups for a given type of anaesthesia. In mice undergoing blood withdrawal, shock was confirmed by a significant drop in blood pressure; MAP varied between 26 and 36 mmHg during the 90 min shock period followed by a near
100 Chapter 6
!
normalization to baseline values in resuscitated animals (fig. 1). The main difference between both types of anesthesia was that in Sham isoflurane animals MAP was slightly lower (~ 10 mmHg throughout the protocol) compared to Sham isoflurane/nitrous oxide animals receiving adjunct nitrous oxide (P<0.05). Blood gases, pCO2, Base excess, HCO3
-, SaO2, Hb, and Hct did not significantly differ between both Sham groups at all time points measured (table 1). The pH was increased in Shock isoflurane group compared to Sham isoflurane. Adjunct nitrous oxide resulted in a similar pH in Sham isoflurane/nitrous oxide and Shock isoflurane/nitrous oxide groups (table 1). During shock, pO2 was slightly increased in animals anesthetised with isoflurane only as compared to group with adjunct nitrous oxide (table 1). Other blood gas parameters were similar in both Shock groups.
Figure 1. Mean arterial pressure (MAP, femoral artery) of Sham, Shock and Resuscitated groups during the experimental procedure in animals anesthetized with isoflurane (ISO, panel A), or anesthesia with isoflurane and adjunct nitrous oxide (ISO + N2O, panel B). * denotes significantly different curves between Sham ISO and Sham ISO+N2O (P<0.05). ** P<0.05 Sham vs corresponding Shock and Resuscitated groups
!
Anesthesia and vascular reactivity 101
!
Tabl
e 1.
Art
eria
l blo
od g
as d
ata
at th
e di
ffer
ent t
ime
poin
ts o
f the
exp
erim
enta
l pro
toco
l
Par
amet
ers
Isof
lura
ne
Isof
lura
ne+N
itrou
s ox
ide
SH
AM
gro
up
Sho
ck g
roup
R
esus
cita
ted
grou
p S
HA
M g
roup
S
hock
gro
up
Res
usci
tate
d gr
oup
Num
ber o
f ani
mal
s 5
6 5
5 5
5 w
eigh
t, g
26.9
±2.5
27
.7±3
.3
26.2
±1.7
26
.6±3
.0
28.9
±0.9
29
.0±3
.2
pH
0 m
in
7.
32±0
.03
7.29
±0.0
4
7.37
±0.0
8 7.
34±0
.05
90 m
in
7.34
±0.0
1$ 7.
42±0
.03*
7.35
±0.0
5 7.
33±0
.01
24
h
7.40
±0.0
6
7.
37±0
.07
pCO
2, kP
a
0
min
4.5±
0.6
4.9±
0.4
3.
9±0.
4 4.
6±0.
5 90
min
3.
5±0.
8 2.
9±0.
7
3.4±
0.1
3.4±
0.9
24
h
2.8±
0.4
3.5±
0.1
pO2,
kPa
0
min
30.3
±1.4
*
31.9
±0.9
#
25.3
±1.7
25
.2±1
.9
90 m
in
33.1
±0.6
27
.8±1
.7
22
.7±9
.2
30.3
±3.3
24 h
27
.3±5
.0
39.2
±13.
7 B
E, m
mol
/l
0 m
in
-8
.3±1
.2
-8.3
±1.6
-7.3
±3.8
-6
.6±2
.2
90 m
in
-10.
5±2.
4 -9
.4±2
.2
-1
0.3±
2.5
-11.
5±4.
2
24 h
-1
1.9±
2.4
-9. 5
±3.0
H
CO
- 3, m
mol
/l
0 m
in
16
.6±1
.4
17.3
±0.1
16.4
±2.7
18
.0±1
.5
90 m
in
13.6
±2.7
13
.8±2
.6
13
.6±1
.6
13.0
±3.9
24 h
12
.1±1
.7
14.6
±2.1
S
aO2 ,
%
0 m
in
10
0.0±
0 10
0.0±
0
99.9
±0.2
10
0.0±
0 90
min
10
0.0±
0 10
0.0±
0
99.7
±0.5
10
0.0±
0
24 h
99
.9±0
.1
100.
0±0
Hb,
mm
ol/l
0 m
in
7.
9±0.
4 7.
9±0.
4
8.4±
0.3
8.6±
0.2
90 m
in
6.9±
0.9
4.6±
0.6
7.
3±0.
6 5.
6±1.
3
24 h
3.
1±0.
3
3.
6±0.
4 H
ct
0 m
in
39
.4±2
.1
39.5
±1.8
41.3
±1.5
42
.4±1
.1
90 m
in
34.7
±4.5
23
.0±2
.6
35
.9±2
.5
28.1
±6.4
24 h
15
.9±1
.3
18.4
±2.1
D
ata
on t
he v
alue
of
pH,
pCO
2, pO
2, B
E,
HC
O-3
, S
aO2,
Hb,
Hct
in g
roup
s of
mic
e an
esth
etiz
ed w
ith I
soflu
rane
sol
e an
d Is
oflu
rane
with
adj
unct
Nitr
ous
oxid
e. p
CO
3 ,
the
parth
ial a
rteria
l pre
ssur
e of
car
bon
diox
ide;
pO
2, th
e pa
rtial
arte
rial p
ress
ure
of o
xyge
n;
HC
O-3
, bi
carb
onat
e; B
E, B
ase
exce
ss; H
b, H
emog
lobi
n; H
ct, h
emat
ocrit
; SaO
2, th
e ox
ygen
sat
urat
ion.
*- P
<0.0
5 S
hock
Iso
flura
ne v
s S
hock
Is
oflu
rane
+Nitr
ous
oxid
e; $ -
P<0
.05
Sha
m I
soflu
rane
vs
Sho
ck I
soflu
rane
; #-
P<0
.05
Res
usci
tate
d Is
oflu
rane
vs
Res
usci
tate
d Is
oflu
rane
+Nitr
ous
oxid
e
102 Chapter 6
!
Tabl
e 2.
The
wid
th o
f aor
ta ri
ngs
in a
ll gr
oups
G
roup
s Is
oflu
rane
Is
oflu
rane
/Nitr
ous
oxid
e S
ham
1.
8 ±
0.3
1.7
± 0.
1
Sho
ck
1.7
± 0.
2 1.
8 ±
0.2
Res
usci
tate
d
1.7
± 0.
1 1.
7 ±
0.1
All
data
are
pre
sent
ed a
s m
ean
± S
D in
mm
.
Tabl
e 3.
Para
met
ers
of P
heny
leph
rine-
med
iate
d C
ontr
actio
n du
ring
Diff
eren
t Pha
ses
of E
xper
imen
tal S
hock
afte
r A
naes
thes
ia w
ith e
ither
Isof
lura
ne o
r Is
oflu
rane
with
Adj
unct
N
itrou
s O
xide
G
roup
s Is
oflu
rane
Is
oflu
rane
/Nitr
ous
oxid
e -lo
gEC
50
AU
C
Emax
, mN
-lo
gEC
50
AU
C
Emax
, mN
S
ham
-6
.9 ±
0.2
2.
1 ±
1.6
1.5
± 0.
41
-7.0
± 0
.1
4.2
± 1.
0*
2.19
± 0
.31
Sho
ck
-7.0
± 0
.3
7.
8 ±
2.8!
3
.24
± 0.
43!
-6
.9 ±
0.2
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Anesthesia and vascular reactivity 103
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Contractile reactivity Experiments in vitro were performed on freshly isolated aorta rings of similar
length (P=0.1) in all groups (table 2).Contractile responses of isolated aortic rings were obtained by constructing concentration-response curves to phenylephrine. The contractile response to phenylephrine in the Sham isoflurane/nitrous oxide group was enhanced (P < 0.05) as compared with the Sham isoflurane group (fig. 2). This enhancement was characterized by an increased Emax (P < 0.05) and an unchanged EC50 (P=0.2, table 3). The contractile response to phenylephrine in the Shock isoflurane and Resuscitated isoflurane groups was also enhanced as compared with the Sham isoflurane group (P < 0.05; fig. 2A). This enhancement was also characterized by an increased Emax (P < 0.05) and an unchanged EC50 (P=0.7, table 3). However, the contractile response to phenylephrine was identical between the Shock isoflurane and Shock isoflurane/nitrous oxide groups and between Resuscitated isoflurane and Resuscitated isoflurane/nitrous oxide groups (fig. 2); i.e. no significant differences were observed in the Emax and EC50 values between the corresponding two groups (table 3). These findings suggest that both shock and nitrous oxide increase contractility in a nonadditive manner.
Relaxation responses to acetylcholine Administration of acetylcholine to aortic rings from mice subjected to sham
conditions caused a biphasic response, characterized by a relaxation, which was
Figure 2. Contractile reactivity of aortic rings to phenylephrine of mice anesthetized with isoflurane (panel A) and isoflurane with adjunct nitrous oxide (panel B) in Sham, Shock and Resuscitated groups. Inserts show the responsiveness to phenylephrine presented as maximal response (Emax) in mN. ISO=isoflurane; N2O= Nitrous oxide. #P<0.05 Sham vs Shock Isoflurane. *P<0.05 Sham vs Shock Isoflurane
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maximal at 0.3 "M, followed by a contractile response at concentrations ' 0.3 "M (Sham groups in fig. 3, A and B). In Shock isoflurane and Resuscitated isoflurane mice, the contractile phase and hence the biphasic pattern in the concentration-response curve was fully suppressed (fig. 3A). In contrast, adjunct nitrous oxide preserved the biphasic pattern also after conditions of shock and resuscitation (fig. 3B). The latter is also reflected by the profound increase in acetylcholine concentration at which maximal relaxation was seen in Shock and Resuscitation groups that received anesthesia with isoflurane only (at 10 "M), as compared to groups with adjunct nitrous oxide (at 1 "M).
The biphasic pattern in the acetylcholine concentration-response curve was also abolished in the presence of indomethacine, an inhibitor of cyclooxygenase (fig. 3, C and D). Consequently, under these conditions concentration-response curves for acetylcholine did not differ between groups. These findings imply the involvement of contractile prostaglandins in the upward stroke at higher concentrations of acetylcholine. Collectively, these findings indicate that the biphasic pattern of the
Figure 3. Acetylcholine induced relaxation of aortic rings from Sham, Shock and Resuscitated groups anesthetized with isoflurane (A and C) or isoflurane with adjunct nitrous oxide (B and D), and in the absence of indomethacine (A and B) or presence of indomethacine (C and D). Concentration-response curves of Sham groups (closed circle), Shock groups (open circles) and Resuscitated groups (triangular down) are expressed as percentage of preconstriction value to phenylephrine (0.1mM) and are present as mean±SD. INDO=indomethacine; ISO= isoflurane; N2O= nitrous oxide.
Anesthesia and vascular reactivity 105
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response to acetylcholine after conditions of shock and resuscitation was maintained by adjunct nitrous oxide.
While groups differed in anesthetic regimes and the levels of phenylephrine-precontraction, the relaxant response to sodium nitroprusside was similar in all groups (table 4), suggesting that acetylcholine responses represent changes in endothelial cell function rather than altered vascular smooth muscle response to nitric oxide.
Vascular cyclooxygenase-1 and cyclooxygenase-2 expression Since short-term hemorrhagic shock differentially affected relaxation
responses to acetylcholine with both modes of anesthesia, we subsequently investigated vascular protein expression of cyclooxygenase-1 and cyclooxygenase-2 in segments collected from the unstimulated abdominal aorta. In mice anaesthetized with isoflurane only, vascular cyclooxygenase-1 expression decreased significantly after shock, followed by a recovery during resuscitation (fig. 4, A,B and C). In contrast, cyclooxygenase-1 expression was preserved in groups receiving adjunct nitrous oxide. A similar pattern was observed for vascular cyclooxygenase-2 expression, but changes did not reach statistical significance (fig. 4, A and B). Collectively, these findings suggest that adjunct nitrous oxide mitigates the shock-induced decrease in the expression of cyclooxygenases, particularly cyclooxygenase-1.
Discussion
We investigated the effects of nitrous oxide adjunct to anesthesia with isoflurane on vascular reactivity in an experimental model of shock in mice. Our data show that hemorrhagic shock in animals that received isoflurane during the procedure induced profound changes in the subsequent vascular reactivity of isolated thoracic rings, consisting of an increased contraction to phenylephrine and a loss of cyclooxygenase mediated contraction to acetylcholine. These functional changes were accompanied by a decrease in the vascular expression of cyclooxygenase-1. Secondly, adjunct nitrous oxide augmented phenylephrine-induced contractility of Sham mice, but no further increase was observed when these animals underwent shock. In addition, adjunct nitrous oxide protected from the loss of contractile response to acetylcholine during shock and preserved the vascular expression of cyclooxygenase-1. Collectively, these findings suggest that vascular reactivity during different phases of shock may be preserved when adjunct nitrous oxide is employed,
Table 4. Maximal relaxant response to sodium nitroprusside in all groups Maximal relaxation to SNP (%)
Isoflurane Isoflurane/Nitrous oxide Sham Shock Resuscitated Sham Shock Resuscitated
101.2±21.7 99.4±8.1 101.5±1.8 101.2±1.3 103.4±7.1 98.0±11.3 Data are expressed as a percentage of phenylephrine-mediated precontraction and were calculated from concentration response curves at 0.1 mM of sodium nitroprusside. No differences were found between groups (one-way ANOVA). SNP, sodium nitroprusside.
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which may involve (amongst others) a preservation of vascular cyclooxygenase-expression.
In this study, we investigated the effect of different anesthetic techniques applied during hemorrhagic shock in vivo on the vascular properties of aortic tissue that was harvested subsequently and examined in the absence of anesthetics. To our knowledge, the influence of anesthetics on vascular reactivity has not been explored in this way previously. Importantly, the changes observed in vascular reactivity in the shock group under isoflurane anesthesia imply that this procedure induces protracted changes in the vascular bed. Indeed, such view is in keeping with the observed downregulation of cyclooxygenase-1 in this group.
Systemic hypotension after isoflurane anesthesia has been reported previously,4, 12, 17 and its general depressant effects on hemodynamics are quite
Figure 4. Expressions level of COX enzymes in aortic tissue from Sham, Shock and Resuscitated groups in groups anesthetized with isoflurane (ISO), or anesthesia with isoflurane and adjunct nitrous oxide (ISO+N2O). Expression of COX-1 (A) and COX-2 (B) was assessed in Sham (n=5), Shock (n=5) and Resuscitated (n=5) groups. Data are normalized to the expression level of $-actin and presented in arbitrary units. Representative expressions pattern in Sham (n=5), Shock (n=5), Resuscitated (n=5) groups anesthetized with sole isoflurane with isoflurane and adjunct nitrous oxide (C). The difference between groups was borderline significant with One-Way ANOVA (P=0.06). However, independent t-test showed a significant reduction of COX-1 expression in Shock ISO versus Sham ISO (*P<0.05).
Anesthesia and vascular reactivity 107
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notorious indeed.10 It may represent an important unwanted side effect of anesthesia, particularly during conditions of shock in which tissue perfusion already is at threat. Investigation of the underlying mechanisms was not within the scope of this study, but previous studies reported the involvement of decrements in processes such as myofilament calcium-sensitivity, intracellular calcium concentration, or voltage-gated calcium influx,18-24 all of which might have contributed to decreased contractility after anesthesia. In the present study, decreased arterial pressure was observed together with decreased constriction to phenylephrine in isolated aortic ring preparation obtained from Sham mice anaesthetized with isoflurane, as compared to those receiving adjunct nitrous oxide. Recently, Pypendop et al showed that addition of 70% nitrous oxide to isoflurane anesthesia results in improved arterial and central venous pressures.12 In addition, a study in patients under sevoflurane anesthesia suggests that adjunct nitrous oxide normalizes hemodynamic parameters of regional and systemic flow.11 These findings suggest that hemodynamics may be better preserved during adjunct anesthesia with nitrous oxide compared to monoanesthesia with isoflurane. It should also be noted, however, that shock increased constriction to phenylephrine. It thus appears that contractile mechanisms become mobilized to maintain perfusion pressure during shock and overrule the effect of anesthesia. In keeping with that, contractility to phenylephrine was not further increased after shock with adjunct nitrous oxide, indicating that the effect of nitrous oxide was not additive.
Vascular responsiveness was also investigated by studying endothelial-mediated responses. As reported previously,25 administration of acetylcholine caused relaxation at low concentrations and contraction at higher concentrations thus generating a biphasic concentration-response curve. Preincubation with indomethacine fully abolished the normal upward stroke in the concentration-response curve, thus indicating the involvement of contractile prostaglandins derived from cyclooxygenase herein. Interestingly, the upward stroke in the concentration-response curve was also lost in aorta preparations of shock-mice anesthetized with isoflurane, but not in those receiving adjunct nitrous oxide. Such findings suggest that shock may alter the production of contractile prostaglandins, which may be preserved during anesthesia with adjunct nitrous oxide. In mouse aorta, endothelial denudation abolishes the acetylcholine-mediated contractile responses,25 implicating that contractile cyclooxygenases metabolites are derived from endothelial cells. In turn, this would implicate that the decreased cyclooxygenase-1 expression as observed following shock is caused by downregulation of the enzyme in endothelial cells. Previous studies suggest both isoforms of cyclooxygenase to be expressed in aorta endothelial and/or vascular smooth muscle cells,26, 27 and that both cyclooxygenase-1 and cyclooxygenase-2 may be involved in production of contractile prostaglandins. At present, there are few data on the influence of isoflurane on cyclooxygenase-protein expression. It has been reported that isoflurane produces cyclooxygenase-2 mediated anesthetic preconditioning, but it did not affect cyclooxygenase-1 and cyclooxygenase-2 protein expression,28, 29 Taken together, our results indicate that the loss of acetylcholine-induced contractile function in Shock-mice anesthetized solely with isoflurane, is due to decreased endothelial production of contractile prostaglandins
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caused by downregulation of endothelial cyclooxygenase-1. Moreover, adjunct nitrous oxide counteracts these changes most likely due to a preservation of normal endothelial function.
Alternatively, nitrous oxide-evoked hyperhomocysteinemia may offer an explanation for enhanced vascular reactivity to phenylephrine in Sham animals, because of an increase in asymmetric dimethylarginine, an endogenous inhibitor of nitric oxide synthase.30 Asymmetric dimethylarginine has been reported to increase arteriolar basal tone31 and participate in the maintenance of vasospasm.32 Previously, nitrous oxide anesthesia has been shown to enhance homocysteine concentration and impairment of endothelial function.33 However, acetylcholine evoked relaxation of groups with and without nitrous oxide was similar in the presence of indomethacine, i.e. upon blockade of prostaglandin synthesis. Consequently, nitric oxide mediated relaxation does not differ between both groups, making the involvement of hyperhomocysteinemia unlikely.
Furthermore, nitrous oxide was reported to stimulate the sympathetic nervous system, which induces systemic and local vasoconstriction. This effect possibly explains the increased MAP during anesthesia with nitrous oxide.34 Whether this phenomenon also explains the normalization of phenylephrine-mediated contraction in resuscitated animals from the nitrous oxide group remains to be established. Aortic rings were studied in the absence of anesthetics in the organ bath. Our study clearly demonstrates vasomotor changes and decreased expression of cyclooxygenase-1 in the post-shock period, which were prevented by the use of nitrous oxide during the shock period. In view of the absence of volatile anesthetics and nitrous oxide in the organ baths, these results likely reflect vascular changes present in the immediate post-shock period, e.g. at the post anesthesia care unit or intensive care unit. However, it is unknown how these results obtained in isolated arteries correspond to vasoreactivity in in vivo conditions. Further, isoflurane and isoflurane/nitrous oxide groups differed in depth of anesthesia, since equivalent concentrations of isoflurane were used. However, as the study aimed to evaluate the effect of adjunct nitrous oxide, we chose to use the same isoflurane concentration in all experimental groups. Also, because the blood volume withdrawn for the induction of hemorrhagic shock was not assessed, we cannot comment on possible intra-operative differences in circulating volume. Finally, as the number of experiments was about six, small differences in calculated parameters of acetylcholine and phenylephrine-mediated responsiveness, or of cyclooxygenase expression may have been unnoticed. Nowadays, despite intensive scientific discussion on the influence of adjunct nitrous oxide to volatile anesthesia,33-38 only few experimental data regarding this question are available. The present study shows positive effects of adjunct nitrous oxide on vasoresponsiveness after short-term hemorrhagic shock in mice, since this anesthetic agent normalized vascular reactivity after as observed under isoflurane anesthesia.
In summary, the findings of this study show that vascular reactivity after hemorrhagic shock is affected by the choice of general anesthesia. Short-term hemorrhagic shock under isoflurane anesthesia increased phenylephrine-mediated
Anesthesia and vascular reactivity 109
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contractile response and suppressed the acetylcholine-evoked contractile effect in thoracic mouse aorta, while adjunct nitrous oxide may attenuate changes in vascular reactivity caused by hemorrhagic shock and/or subsequent resuscitation. The results of this study also indicate that cyclooxygenase proteins participate in shock-dependent changes of vasoresponsiveness to acetylcholine. Our data suggest that the choice of the anesthetic regimen during emergency surgery for hemorrhagic shock may influence post-surgery vascular reactivity. !!Reference List 1. Keel M, Trentz O: Pathophysiology of polytrauma. Injury 2005; 36: 691-709 2. Landry DW, Oliver JA: The pathogenesis of vasodilatory shock. N Engl J Med 2001; 345: 588-95 3. Musser JB, Bentley TB, Griffith S, Sharma P, Karaian JE, Mongan PD: Hemorrhagic shock in swine:
Nitric oxide and potassium sensitive adenosine triphosphate channel activation. Anesthesiology 2004; 101: 399-408
4. Baumert JH, Hecker KE, Hein M, Reyle-Hahn SM, Horn NA, Rossaint R: Haemodynamic effects of haemorrhage during xenon anaesthesia in pigs. Br J Anaesth 2005; 94: 727-32
5. Xu J, Liu L: The role of calcium desensitization in vascular hyporeactivity and its regulation after hemorrhagic shock in the rat. Shock 2005; 23: 576-81
6. Park WK, Kim MH, Ahn DS, Chae JE, Jee YS, Chung N, Lynch C 3rd: Myocardial depressant effects of desflurane: Mechanical and electrophysiologic actions in vitro. Anesthesiology 2007; 106: 956-66
7. Akata T: General anesthetics and vascular smooth muscle: direct actions of general anesthetics on cellular mechanisms regulating vascular tone. Anesthesiology 2007; 106: 365-91
8. Akata T, Kanna T, Yoshino J, Takahashi S: Mechanisms of direct inhibitory action of isoflurane on vascular smooth muscle of mesenteric resistance arteries. Anesthesiology 2003; 99: 666-77
9. Izumi K, Akata T, Takahashi S: Role of endothelium in the action of isoflurane on vascular smooth muscle of isolated mesenteric resistance arteries. Anesthesiology 2001; 95: 990-8
10. Hopkins PM: Nitrous oxide: A unique drug of continuing importance for anaesthesia. Best Pract Res Clin Anaesthesiol 2005; 19: 381-9
11. Kaisti KK, Langsjo JW, Aalto S, Oikonen V, Sipila H, Teras M, Hinkka S, Metsahonkala L, Scheinin H: Effects of sevoflurane, propofol, and adjunct nitrous oxide on regional cerebral blood flow, oxygen consumption, and blood volume in humans. Anesthesiology 2003; 99: 603-13
12. Pypendop BH, Ilkiw JE, Imai A, Bolich JA: Hemodynamic effects of nitrous oxide in isoflurane-anesthetized cats. Am J Vet Res 2003; 64: 273-8
13. Sanders RD, Weimann J, Maze M. Biologic effects of nitrous oxide: A mechanistic and toxicologic review. Anesthesiology 2008;109:707-22.
14. van Meurs M, Wulfert FM, Knol AJ, De Haes A, Houwertjes M, Aarts LP, Molema G: Early organ-specific endothelial activation during hemorrhagic shock and resuscitation. Shock 2008; 29: 291-9
15. Darblade B, Pendaries C, Krust A, Dupont S, Fouque MJ, Rami J, Chambon P, Bayard F, Arnal JF: Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor. Circ Res 2002; 90: 413-9
16. Potenza MA, Botrugno OA, De Salvia MA, Lerro G, Nacci C, Marasciulo FL, Andriantsitohaina R, Mitolo-Chieppa D: Endothelial COX-1 and -2 differentially affect reactivity of MVB in portal hypertensive rats. Am J Physiol Gastrointest Liver Physiol 2002; 283: G587-94
17. Conzen PF, Vollmar B, Habazettl H, Frink EJ, Peter K, Messmer K: Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg 1992; 74: 79-88
18. Akata T: Cellular and molecular mechanisms regulating vascular tone. Part 2: regulatory mechanisms modulating Ca2+ mobilization and/or myofilament Ca2+ sensitivity in vascular smooth muscle cells. J Anesth 2007; 21: 232-42
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19. Stekiel TA, Contney SJ, Kokita N, Bosnjak ZJ, Kampine JP, Stekiel WJ: Mechanisms of isoflurane-mediated hyperpolarization of vascular smooth muscle in chronically hypertensive and normotensive conditions. Anesthesiology 2001; 94: 496-506
20. Stekiel TA, Kokita N, Yamazaki M, Bosnjak ZJ, Kampine JP, Stekiel WJ: Effect of isoflurane on in situ vascular smooth muscle transmembrane potential in spontaneous hypertension. Anesthesiology 1999; 91: 207-14
21. Kokita N, Stekiel TA, Yamazaki M, Bosnjak ZJ, Kampine JP, Stekiel WJ: Potassium channel-mediated hyperpolarization of mesenteric vascular smooth muscle by isoflurane. Anesthesiology 1999; 90: 779-88
22. Li T, Liu L, Xu J, Yang G, Ming J: Changes of Rho kinase activity after hemorrhagic shock and its role in shock-induced biphasic response of vascular reactivity and calcium sensitivity. Shock 2006; 26: 504-9
23. Li T, Liu L, Liu J, Ming J, Xu J, Yang G, Zhang Y: Mechanisms of Rho kinase regulation of vascular reactivity following hemorrhagic shock in rats. Shock 2008; 29: 65-70
24. Liu LM, Ward JA, Dubick MA: Hemorrhage-induced vascular hyporeactivity to norepinephrine in select vasculatures of rats and the roles of nitric oxide and endothelin. Shock 2003; 19: 208-14
25. Zhou Y, Varadharaj S, Zhao X, Parinandi N, Flavahan NA, Zweier JL: Acetylcholine causes endothelium-dependent contraction of mouse arteries. Am J Physiol Heart Circ Physiol 2005; 289: H1027-32
26. Li M, Kuo L, Stallone JN: Estrogen potentiates constrictor prostanoid function in female rat aorta by upregulation of cyclooxygenase-2 and thromboxane pathway expression. Am J Physiol Heart Circ Physiol 2008; 294:H2444-55
27. Kawka DW, Ouellet M, Hetu PO, Singer II, Riendeau D: Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium. Biochim Biophys Acta 2007; 1771: 45-54
28. Alcindor D, Krolikowski JG, Pagel PS, Warltier DC, Kersten JR: Cyclooxygenase-2 mediates ischemic, anesthetic, and pharmacologic preconditioning in vivo. Anesthesiology 2004; 100: 547-54
29. Tanaka K, Ludwig LM, Krolikowski JG, Alcindor D, Pratt PF, Kersten JR, Pagel PS, Warltier DC: Isoflurane produces delayed preconditioning against myocardial ischemia and reperfusion injury: role of cyclooxygenase-2. Anesthesiology 2004; 100: 525-31
30. Dayal S, Lentz SR: ADMA and hyperhomocysteinemia. Vasc Med 2005; 10:S27-33. 31. Toth J, Racz A, Kaminski PM, Wolin MS, Bagi Z, Koller A: Asymmetrical Dimethylarginine inhibits
shear stress-induced nitric oxide release and dilation and elicits superoxide-mediated increase in arteriolar tone. Hypertension 2007; 49:563-8
32. Pluta RM, Oldfield EH: Analysis of nitric oxide in cerebral vasospasm after aneursymal bleeding. Rev Recent Clin Trials 2007; 2:59-67.
33. Myles PS, Chan MT, Kaye DM, McIlroy DR, Lau CW, Symons JA, Chen S: Effect of nitrous oxide anesthesia on plasma homocysteine and endothelial function. Anesthesiology 2008;109:657-63
34. Jahn UR, Berendes E: Nitrous oxide--an outdated anaesthetic. Best Pract Res Clin Anaesthesiol 2005; 19: 391-7
35. Nagele P, Zeugswetter B, Wiener C, Burger H, Hüpfl M, Mittlböck M, Födinger M: Influence of methylenetetrahydrofolate reductase gene polymorphisms on homocysteine concentrations after nitrous oxide anesthesia. Anesthesiology 2008;109:36-43
36. Myles PS, Leslie K, Chan MT, Forbes A, Paech MJ, Peyton P, Silbert BS, Pascoe E; ENIGMA Trial Group: Avoidance of nitrous oxide for patients undergoing major surgery: a randomized controlled trial. Anesthesiology 2007;107:221-31
37. Preckel B, Bolten J: Pharmacology of modern volatile anaesthetics. Best Pract Res Clin Anaesthesiol 2005; 19: 331-48
38. Mashour GA, Forman SA, Campagna JA: Mechanisms of general anesthesia: From molecules to mind. Best Pract Res Clin Anaesthesiol 2005; 19: 349-64
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Chapter 7
S1P receptor modulation improves systemic vascular dysfunction after CPB in the rat independent of depletion
of lymphocytes: a comparison between FTY720 and SEW2871
Iryna V. Samarska, Hjalmar Bouma, Hendrik Buikema, Martin C. Houwertjes, Anne H. Epema, Robert H. Henning
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Abstract Background: Cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response syndrome and disturbances in endothelial function of systemic arteries. FTY720, a non-selective agonist for sphingosine 1-phosphate (S1P) receptors, evokes lymphopenia by sequestration of lymphocytes from circulation to secondary lymphoid organs. SEW2871 is a selective agonist of S1P1 receptors. We investigated whether FTY720 and SEW2871 improve vascular reactivity after CPB in the rat. Materials and Methods: Experiments were done in male Wistar rats (n=48). Anesthesia-induction consisted of isoflurane (2.5-3%), followed by fentanyl and midazolam during CPB. After catheterization of the left femoral and carotid artery and the right heart, normothermic extracorporeal circulation was instituted for 60 min. Following 1 day of recovery, constriction to phenylephrine (PE) and serotonin (SE) and relaxation to acetylcholine of small mesenteric artery segments was assessed in a wired myograph system. Relaxation was expressed as % of preconstriction and analyzed as the area under the concentration-response curve (AUC, arbitrary units). Results: Contractile responses were inhibited after both CPB and SHAM experimental procedures. In mesenteric artery, FTY720 normalized SE- and PE-mediated vascular reactivity after CPB. FTY720 also increased total relaxation to acetylcholine as compared with untreated CPB groups (AUC: 256.8±43.9 and 168.2± 28.4; respectively). SEW2871 produced vascular effects comparable with FTY720. Conclusions: FTY720 and SEW2871 improve vascular function in mesenteric after CPB. This pharmacological effect was mediated mainly through S1P1 receptors and did not require lymphopenia. S1P1 receptor agonism may provide a promising therapeutic intervention to prevent CPB-related vascular dysfunction. Introduction
Cardiopulmonary bypass (CPB) is a widely used technique invaluable to thoracic surgery. CPB is however also associated with several side-effects such as induction of a systemic inflammatory response syndrome, a multiple organ dysfunction syndrome and ischemia-reperfusion injury. Two main mechanisms of CPB-related complications have been investigated during last decade: systemic inflammatory events and vascular dysfunction with secondary tissue edema and/or tissue hypoperfusion, setting the stage for multiple organ dysfunction.1 In a previous study, we characterized the vascular changes in small systemic arteries during a 5 day follow-up post-CPB in rat. That was found that overall contractility was inhibited by experimental procedures most prominently at 1 day of the recovery period, suggesting CPB-related surgical procedures and/or extended anesthesia induce extensive and protracted changes in the contractility of small vessels.
Several anti-inflammatory pharmacological agents, including corticosteroids, aprotinin, antioxidants, complement inhibitors and phosphodiesterase inhibitors have been proposed to inhibit the CPB-related inflammatory processes and vascular
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dysfunction. However, none of them satisfactorily prevented pathological and clinical outcomes that are associated with extracorporeal circulation.2
FTY720 (Fingolimod) is a non-selective sphingosine 1-phosphate (S1P) receptor modulator and acts as an immunosuppressive agent. Treatment with FTY720 thus far has been studied for autoimmune disorders (multiple sclerosis, autoimmune neuritis, autoimmune glomerulonephritis)3-6 and in the setting of transplantation.7 One main mechanisms of its therapeutic activity is the induction of lymphopenia, through inhibition of the egress of lymphocytes from the secondary lymphoid organs to the peripheral blood. In addition, FTY720 exerts pharmacological effects, including vascular effects, via activation of different S1P receptors.8 FTY720 has been shown to prevent or decrease ischemia-reperfusion injury,9-15 enhance endothelial barrier and decrease vascular permeability,16-19 and improve vascular function through modulation of the type 1, 3, 4 and 5 S1P receptors.16;18;20
From the above we hypothesized that treatment with FTY720 may be a promising pharmacological strategy to prevent vascular dysfunction in CPB. To address this, we investigated the effect of FTY720 on systemic vascular dysfunction in a rat model of experimental CPB. Rats were pretreated with FTY720 and subjected to CPB-related anaesthetical and surgical procedures with or without extracorporeal circulation. 24-Hours following recovery, small mesenteric arteries were removed and studied for contractility and endothelial relaxant reactivity. Comparisons were made to control rats that underwent short anesthesia only. Additional comparisons were made to rats treated with the selective S1P1 receptor agonist SEW2871 to further detail the type of S1P receptor involved. Material and Methods
The experimental protocol was approved by the Animal Ethics Committee of the University of Groningen. This study was performed in n=48 adult male Wistar rats with body weights of 509.3±32.3 g (Harlan, Zeist, The Netherlands) housed under standard conditions with free access to food (standard rat chow; Hope Farms, Woerden, The Netherlands) and drinking water throughout the study.
Experimental groups Experimental animals were randomly allocated to one of three experimental
groups with different treatment: untreated, FTY720-pretreated and SEW2871-pretreated. Each group was subdivided in three sub-groups: Control, Sham and CPB. CPB animals were subjected to the full experimental protocol described below, including anesthesia, cannulation, extracorporeal circulation, weaning and the recovery period. Sham animals followed the same procedure except for the extracorporeal circulation, but maintained cannulated and mechanically ventilated. The duration of the recovery period in Sham and CPB groups was 24 hours, in the end of which animals were sacrificed. Control groups were employed in order to evaluate the normal vascular reactivity in rats without invasive interventions. Animals in this group were sacrificed under brief isoflurane anesthesia (2.5-3%) only.
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Experimental protocol The experimental protocol consisted of the following parts: anesthesia,
preparation, extracorporeal circulation, a weaning and a recovery period (see fig. 1). Three hour before the beginning of the protocol animals were injected with FTY720 (0.5mg/kg), SEW2871 (0.5mg/kg) or vehicle. Anesthesia was induced with 2-3% isoflurane in O2/air (1:1) before intubation and mechanical ventilation (Amsterdam Infant Ventilator; HoekLoos, Amsterdam, The Netherlands). Tidal volume was set to achieve normocapnia (verified by capnography and arterial blood gas analysis), with O2/air (1:2) at a ventilation rate of 50 min-1 (0.5 s inspiration time). Rectal temperature was kept at 37.5±0.5 °C, using an electrical heating pad. The left femoral artery was cannulated (26-gauge catheter) for blood pressure monitoring. The mean arterial pressure was kept between 70 and 100 mm Hg by adjusting the isoflurane concentration as necessary (typically between 2.0-2.5%). Immediately before insertion of the arterial line, 250 IU kg-1 heparin was administered. The left carotid artery was cannulated for arterial inflow using a 22-gauge catheter. A multi-orifice 4.5 French cannula (adapted from a Desilets-Hoffmann catheter, Cook Son, The Netherlands) was advanced into the right heart using the right common jugular vein for access. The tail vein was cannulated for the administration of intravenous anesthetics, heparin, and protamine sulfate.
Extracorporeal circulation Subsequently, extracorporeal circulation (ECC) was initiated in the CPB group
for 60 min. The set-up consisted of a glass venous reservoir, a peristaltic pump (Pericor® SF70), a neonatal membrane oxygenator (0140GM, Polystan, Vaerlose, Denmark) and a glass counter-flow heat exchanger with built-in bubble trap. All components were connected with polyethylene tubing (1.6 mm inner diameter). The venous reservoir and heat exchanger were sterilized prior to use. The circuit was primed with 15 ml of haes 60 mg ml -1 solution (Voluven®, Fresenius Kabi, Bad Homburg, Germany). Animals were additionally heparinized (250 IU kg-1) after the start of ECC. During EEC, rats were anesthetized with intravenous fentanyl (125 "g kg-1), atracurium (0.5 mg kg-1), and midazolam (2 mg kg-1). During CBP, blood oxygen
Figure 1. Scheme of the experimental protocol in Sham and CPB groups regarding the duration of recovery period. The experimental protocol consisted of the following parts: anesthesia, preparation, extracorporeal circulation, a weaning and a recovery period (see methods). Abbreviations: ECC, extracorporeal circulation
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S1P receptors’ agonists and vascular reactivity 115
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saturation was monitored continuously by a pulse oximeter. Targeted CBP flow was 100-110 mL kg-1 min-1, corresponding to 60-70% of normal cardiac output.
Weaning and recovery During the weaning period, ECC was terminated and mechanical ventilation
was initiated. Protamine (150 IU kg-1 i.v.) was administered in order to neutralize heparin after which cannulas were removed and the wounds sutured. Following extubation, animals were first kept under isoflurane anesthesia (0.8-1.0%) for the first hour of recovery in order to stabilize. The duration of the recovery period of various groups lasted 24 hours after the end of the extracorporeal circulation. Sacrifice was performed in the end of the recovery period under isoflurane anesthesia (2.5-3%).
Blood gas and cell analysis Samples for blood gas analysis (0.1 "l) were drawn at four time-points: at the
end of preparation period (15 min before start of ECC), twice during the ECC period (15 min after the start of ECC and 15 min before its ending), and at 45 min after the end of ECC. During the procedure, the amount of neutrophils, lymphocytes and monocytes was measured 4 times: just prior to injecting the compound (FTY720, SEW2871 or vehicle), 15 min after before the start of the ECC, 15 min after the end of the ECC, and at sacrification (24h after ECC). Blood gas analysis and cell count were performed at the Central laboratory of the University Medical Centre Groningen. MAP was monitored continuously during the entire protocol.
Vascular reactivity studies At sacrifice, all mesenteric loops were removed and placed into a cold
physiological saline solution. Several segments of third branch of the mesenteric superior artery were dissected, prepared as ring vessels preparations (1.8-2.0 mm in length) and mounted on two 40 µm stainless wires connected to force transducers in individual organ bath chambers for isometric tension recordings in a wire-myograph (Danish Myo Technology A/S, Denmark). The baths contained 6 ml of Kreb’s solution warmed to 37°C and bubbled continuously with 95%O2/5%CO2 at pH 7.4. Vessels were subjected to the standard normalization procedure21 and left to equilibrate for 40 min until they were at a steady baseline. In brief, the vessels were distended stepwise until effective pressure exceeded 100 mmHg (13.3 kPa). The internal circumference, IC100, was found from the Laplace’s equation and the experiments were performed at the IC1=0.9*IC100. Vascular segments were primed and checked for viability by two consecutive exposures to potassium chloride (60 mM).
The experimental protocol consisted of evaluation of contractile responses to potassium chloride (Emax to 60 mM) as a G-protein coupled receptor (GPCR)-independent contractile agent, and phenylephrine (PE; 10 nM to 100 "M) as a GPCR-dependent contractile agent. Endothelium-dependent relaxation to acetylcholine (ACh; 10 nM to 300 "M) was assessed in rings pre-constricted with PE. To assess the contribution of different endothelial mediators, ACh-induced relaxations were studied in the absence and presence of L-NMMA (0.1 mM; an inhibitor of NO-synthase) and/or
116 Chapter 7
!
indomethacine (1 "M; an inhibitor of cyclooxygenase) administered 20 min before application of PE [22]. Following the final concentration of ACh, a high concentration of the NO-donor sodium nitroprusside (SNP; 0.1 mM) was applied to assess maximal endothelium-independent relaxation.
Drugs The composition of Kreb’s solution was as follows (mM): 120.4 NaCl, 5.9 KCl,
2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 glucose; these chemicals were obtained from Merck (Darmstadt, Germany). The following drugs were used: acetylcholine chloride (ACh), phenylephrine (PE), indomethacine, SNP, NG-methyl-L-arginine (L-NMMA All other drugs were dissolved in deionized water. L-NMMA was purchased from MP Biomedicals (Illkirch, France). All other compounds were purchased from Sigma (St. Louis, Missouri, USA). The concentrations presented in the concentration-curve responses are expressed as a final molar concentration in the bath.
Data analysis Data are given as mean±SD and n refers to the number of animals in a
corresponding group. Contractile responses to KCl and PE are presented in mN and relaxations to ACh and SNP were calculated as a percentage of pre-constriction. For each concentration-response curve the following parameters were determined: 1) the Area Under the Curve (AUC) in arbitrary units (AU) (SigmaPlot, Systat Software, San Jose, CA, USA); 2) the maximal response (Emax); 3) the concentration at which half-maximal response was reached (EC50).Tthe difference in AUC for concentration-response curves to ACh in the absence or presence of an inhibitor was used as an estimate the contribution of different endothelial mediators [23]. The remainder of ACh induced relaxation resistant to the inhibition of both cyclooxygenase and NO-synthase was taken as an estimate of EDHF, as showed previously [24]. Differences were evaluated using one-way ANOVA combined with post-hoc Bonferroni test or with t-test (SPSS, Chicago, IL, USA). When data were not normally distributed, then nonparametric tests were used. Differences were considered significant at P<0.05 (2-tailed). The relationship between different relaxant pathways and total acetylcholine-mediated relaxation was evaluated with regression analysis and Spearman correlation. Results
Hemodynamics and blood gas analysis during the procedure Blood gas analysis was performed before start of ECC, twice during ECC and
after weaning. In untreated Sham and CPB groups at baseline all parameters were in the normal range. During ECC both in the untreated and treated CPB groups, a decreased pH, bicarbonate (HCO-
3), and Base excesses (BE) and increased levels of pCO2 were found, which may represent moderate respiratory acidosis (table 1). Hematocrit was significantly decreased in CPB groups as a consequence of hemodilution with the priming solution. In the post-ECC period, all parameters in CPB
S1P receptors’ agonists and vascular reactivity 117
!
groups returned to normal ranges and were similar for Sham and CPB groups, except for hematocrit (table 1).
!!!!!!!!!!!!!
%&!untreated rats, baseline MAP was similar between the groups. After onset of ECC, MAP dropped and remained significantly decreased until re-institution of normal circulation during which it recovered (fig. 2). In contrast, in pre-treated rats (all groups), MAP was significantly reduced already at baseline before the onset of ECC, and remained lower throughout the whole experimental procedure (fig. 2).!
Influence of FTY-720 and SEW-2871 on the amount of neutrophils, lymphocytes and monocytes in peripheral blood
Data on the amount of neutrophils, lymphocytes and monocytes are present in table 2. Since hemodilution can influence the results in CPB groups, all values are normalized to Hct value. At the start of recovery, the number of circulating neutrophils transiently increased in both Sham and CPB rats (table 2). Treatments did not affect the number of circulating neutrophils.
The amount of circulating lymphocytes did not significantly change in Sham rats, but was reduced in CPB rats at the end of the 24h recovery period (table 2). Treatment with FTY720 (0.5mg/kg i.p.) induced a pronounced lymphopenia, which was present already before onset of Sham/ECC (~4 hours after injection) and lasted until animals were sacrificed 24h thereafter. SEW2871 (0.5mg/kg) on the other hand, did not induce lymphopenia. Finally, the amount of monocytes did not significantly differ between groups throughout the study period. Thus, FTY720 caused pronounced lymphopenia, while SEW2871 did not show such effect.
Figure 2. Data on Mean arterial pressure (MAP, femoral artery) in Sham and CPB groups during the experimental protocol. Abbreviations: CPB, cardiopulmonary bypass. * indicates P<0.05, t-test; # indicates P<0.05, repeated measurements Anova, Bonferonni test !
118 Chapter 7
!
Alterations in vascular reactivity after CPB and the effect of treatments Differences in vascular contractility at the end of the 1-day study period were
observed particularly in Control versus Sham/CPB-groups, with no obvious additional effect of CPB compared to Sham. I.e. in mesenteric arteries both GPCR-independent and –dependent responses to KCl and PE, respectively, were significantly decreased in Sham/CPB as compared to Control (see fig. 3). Both treatments significantly increased and fully normalized responses to KCl - in Sham as well as in CPB rats - but seemed less effective in restoring contractility to PE. It should be noted, however, that both treatments also reduced GPCR-dependent responses to PE in control rats, while leaving GPCR-independent contraction to KCl unaffected (fig. 3).
Relaxant reactivity to ACh was similar in untreated control and Sham/CPB rats, thus suggesting that (systemic) endothelial function in this vessel type at this time-point post-CPB was (still) intact. Interestingly, both treatments enhanced total relaxation to ACh in all groups, including in control rats. Augmented endothelium-dependent reactivity was most pronounced in CPB rats treated with FTY720 and SEW2871, particularly due to increased contribution of EDHF in ACh-induced relaxation (fig. 4).
Thus, both treatments had tendency to enhance contractile and relaxant reactivity in mesenteric artery that became more pronounced in CPB groups.
S1P receptors’ agonists and vascular reactivity 119
!
Ta
ble 1
. Dat
a on
bloo
d ga
s ana
lysis
durin
g th
e exp
erim
enta
l pro
cedu
re in
all g
roup
s
SH
AM u
ntre
ated
Sham
FTY
720
Sham
SEW
2871
Be
fore
ECC
15
min
ECC
45
min
ECC
Af
ter E
CC
Befo
re E
CC
15 m
in E
CC
45 m
in E
CC
Afte
r ECC
Be
fore
ECC
15
min
ECC
45
min
ECC
Af
ter E
CC
pH
7.43
±0.0
5 7.
45±0
.05
7.46
±0.0
7 7.
41±0
.06
7.4±
0.06
7.
4±0.
07
7.4±
0.07
7.
36±0
.07
7.4±
0.06
7.
4±0.
05
7.37
±0.0
8 7.
35±0
.03
pCO 2
, kPa
4.
74±1
.0
4.51
±0.8
4.
68±1
.0
5.15
±0.9
4.
8±0.
8 4.
6±0.
8 5.
0±0.
8 5.
6±1.
1 4.
7±0.
9 4.
7±0.
8 5.
7±1.
3 5.
9±0.
7
HCO 3
,mm
ol/l
22.9
±1.5
23
.3±1
.5
24.7
±1.5
24
.4±2
.2
22.1
±0.7
22
.3±1
.2
23.8
±1.3
23
.8±1
.2
21.5
±2.0
21
.5±1
.7
23.9
±1.6
23
.9±1
.4
BE,m
mol
/l -0
.4±1
.0
0.5±
1.2
1.8±
1.5
0.5±
1.4
-1.8
±0.9
-0
.84±
1.3
0.05
±0.7
-1
.4±1
.1
-2.1
±1.6
-1
.9±1
.2
-0.7
±1.9
-1
.2±1
.1
Hctc
0.
4±0.
02
0.4±
0.03
0.
4±0.
03
0.4±
0.03
0.
39±0
.03
0.34
±0.0
3 0.
34±0
.06
0.34
±0.0
7 0.
4±0.
01
0.4±
0.06
0.
4±0.
02
0.4±
0.03
Gluc
ose,m
mol
/l 20
.6±5
.4
15.2
±5.4
8.
4±4.
3 6.
6±1.
0 19
.6±2
.4
16.4
±3.0
6.
9±1.
2 6.
9±0.
7 22
.7±3
.1
19.2
±4.4
7.
8±1.
4 8.
3±1.
2
CPB
untre
ated
CP
B FT
Y720
CP
B
SEW
2871
Be
fore
ECC
15
min
ECC
45
min
ECC
Af
ter E
CC
Befo
re E
CC
15 m
in E
CC
45 m
in E
CC
Afte
r ECC
Be
fore
ECC
15
min
ECC
45
min
ECC
Af
ter E
CC
pH
7.42
±0.0
5 7.
4*±0
.07
7.32
*±0.
08
7.
37±0
.05
7.
37±0
.07
7.
3#±0
.02
7.
3#±0
.04
7.33
±0.0
3 7.
35±0
.03
7.
3±0.
07
7.
3±0.
03
7.4±
0.08
pCO 2
, kPa
4.
77±0
.8
5.07
*±1.
1
5.72
*±1.
0
5.22
±0.8
5.0±
0.9
6.2#
±0.4
5
6.9#
±0.8
8 5.
8±0.
4 5.
4±0.
6 5.
7±0.
7 6.
6±0.
54
5.6±
1.2
HCO 3
,mm
ol/l
22.6
±2.3
21
.5±3
.6
21
.6*±
2.4
22.4
*±1.
7
21.3
±0.7
21.9
±0.5
6
22.3
±1.6
6
22.3
±1.5
22
.5±1
.0
21
.7±0
.9
23
.7±2
.5
23.7
±1.6
BE,m
mol
/l -0
.6±1
.7
-1.9
*±2.
7
-3.6
*±3.
1
-2.0
4±1.
9 -3
.2#±
0.7
-3
.6#±
0.5
-4
.2#±
1.7
-3.0
#±1.
4 -2
.3±0
.9
-3.2
±1.9
-2
.3±2
.1
1±2.
1
Hctc
0.
4±0.
02
0.3*
±0.0
5
0.3*
±0.0
2
0.3*
±0.0
3 0.
38±0
.03
0.2#
±0.0
2 0.
2#±0
.02
0.33
±0.0
2 0.
04±0
.07
0.22
±0.0
2 0.
28±0
.02
0.38
&±0.
02
Gluc
ose,m
mol
/l 22
.9±4
.1
20.5
±5.1
11
.6±4
.4
6.
9±3.
0
23.4
±1.9
22.0
±1.3
13.0
±2.3
7.
4±1.
4 24
.5±7
.4
23
.5±8
.5
18
.5±1
0.0
12.1
5&±3
.9
Ab
brev
iation
: CPB
, car
diopu
lmon
ary b
ypas
s. *P
<0.0
5, vs
Sha
m u
ntre
ated
, t-te
st; #
P<0
.05,
vs S
ham
FTY
720,
t-te
st; &
P<0
.05,
vs S
ham
SEW
2871
, t-te
st.
120 Chapter 7
!
Tabl
e 2.
Am
ount
of n
eutr
ophi
ls, l
ymph
ocyt
es a
nd m
onoc
ytes
in b
lood
of t
he e
xper
imen
tal a
nim
als
in a
ll gr
oups
. Va
lues
wer
e no
rmal
ized
to H
ct (1
0-9/l)
Sham
unt
reat
ed
CPB
unt
reat
ed
base
line
star
t EC
C
end
ECC
24
h re
cove
ry
base
line
star
t EC
C
end
ECC
24
h re
cove
ry
Neu
troph
ils
1.7±
0.4
1.7±
0.9
6.5a
±2.4
3.
1±1.
4 2.
4±1.
2 6.
5±2.
6 10
.3±5
.7a
6.6±
0.8
Lym
phoc
ytes
21
.1±6
.4
14.7
±2.6
18
.7±4
.7
15.0
±1.8
16
.1±7
.6
31.3
±16.
0 34
.9±5
.3
9.8±
3.6
b
Mon
ocyt
es
0.6±
0.5
0.3±
0.2
0.4±
0.5
0.75
±0.3
1.
2±0.
8 2.
3±2.
4 1.
8±1.
3 2.
3±2.
0
Sh
am F
TY 7
20
CPB
FTY
720
base
line
star
t EC
C
end
ECC
24
h re
cove
ry
base
line
star
t EC
C
end
ECC
24
h re
cove
ry
Neu
troph
ils
1.2±
0.1
4.2±
2.7
7.1±
0.9
c
4.6±
0.8
2.0±
1.5
7.8±
2.7
3.0
±0.7
5.
8±2.
9
Lym
phoc
ytes
23
.3±5
.4
5.9±
1.4
d 5.
1±1.
1 d
1.3
±0.7
d
19.0
±3.2
8.
3±2.
6 e
6.2±
3.8
e 2.
0±1.
0 e
Mon
ocyt
es
1.0±
0.3
0.6±
0.5
1.5±
0.6
0.9±
0.7
0.7±
0.4
0.7±
0.4
1.0±
0.6
0.7±
0.3
Sh
am S
EW28
71
CPB
SEW
2871
ba
selin
e st
art E
CC
en
d EC
C
24 h
reco
very
ba
selin
e st
art E
CC
en
d EC
C
24 h
reco
very
N
eutro
phils
2.
2±1.
4 2.
3±0.
9 9.
5±2.
2 f
5.2±
1.5
2.3±
1.7
4.8±
2.3
5.7±
1.7
6.3±
2.5
Lym
phoc
ytes
18
.8±2
.7
18.2
±7.5
23
.0±0
.65
12.8
±6.6
g
23.4
±3.2
40
.6±1
4.5
32.0
±7.
7 11
.4±5
.8 i
M
onoc
ytes
0.
7±0.
3 0.
5±0.
27
0.2±
0.1
0.5±
0.37
0.
5 ±0
.3
0.4
±0.2
1.
4±0.
9 0.
8 ±0
.4
Abb
revi
atio
n: C
PB=c
ardi
opul
mon
ary
bypa
ss;
base
line,
15
min
afte
r in
ject
ion
of t
he e
xper
imen
tal
com
poun
d of
the
veh
icle
; st
art
ECC
, 15
min
bef
ore
extra
corp
orea
l circ
ulat
ion;
end
EC
C, 1
5 m
in a
fter e
xtra
corp
orea
l circ
ulat
ion;
24h
reco
very
, sac
rific
atio
n, 2
4h la
ter t
he e
nd o
f the
ext
raco
rpor
eal c
ircul
atio
n. a
- P<
0.05
, vs
Sham
bas
elin
e, t-
test
; b -
P<0.
05, u
ntre
ated
CPB
24h
vs
all o
ther
CPB
unt
reat
ed g
roup
s, o
ne w
ay A
NO
VA, B
onfe
rroni
test
; c -
P<0.
05, v
s Sh
am
FTY7
20 b
asel
ine,
one
way
AN
OVA
on
rank
s, D
unn'
s M
etho
d; d
- P
<0.0
5, v
s Sh
am F
TY72
0 ba
selin
e, o
ne w
ay A
NO
VA, B
onfe
rroni
test
;e -
P<0
.05,
vs
CPB
FT
Y720
bas
elin
e, o
ne w
ay A
NO
VA, B
onfe
rroni
test
;f - P
<0.0
5, v
s Sh
am S
EW28
71 b
asel
ine,
one
way
AN
OVA
, Bon
ferro
ni te
st; g
- P<
0.05
, vs
Sham
SEW
2871
en
d EC
C, o
ne w
ay A
NO
VA, B
onfe
rroni
test
; i -
P<0.
05, C
PB S
EW28
71 2
4h v
s al
l oth
er C
PB28
71 g
roup
s, o
ne w
ay A
NO
VA, B
onfe
rroni
test
S1P receptors’ agonists and vascular reactivity 121
!
Discussion
The present work studied the acute pre-treatment effects of FTY720 and SEW2871, two different S1P receptor agonists, on systemic vascular reactivity in an experimental rat model of cardiopulmonary bypass. Isolated mesenteric artery preparations were studied 1-day post-CPB, hence at which time point contractility in untreated animals was generally depressed. The major finding of this study is that both FTY720 and SEW2871 both restored vascular contractility and augmented endothelial relaxant reactivity after CPB. Both compounds also evoked hypotension, yet differentially decreased the amount of lymphocytes in the peripheral blood. The observed effects are discussed with respect to the anti-inflammatory activity of S1P agonists and the modulation of the S1P receptors. It is concluded that the observed systemic vascular treatment effects were independent from lymphopenia but rather involved modulation of vascular S1P1 receptors.
Impaired contractility of mesenteric arteries to PE and KCl in the present study confirmed the development of systemic vascular dysfunction post-CPB in this model.
Figure 3. Contractile reactivity to phenylephrine and potassium chloride in all groups. A: maximal response to single dose of potassium chloride, mN; B: AUC values of the contractile responses to phenylephrine, mN. *- P<0.05, vs Control untreated, one way ANOVA, Bonferroni test; **- P<0.05, vs CPB untreated, one way ANOVA, Bonferroni test; #- P<0.05, vs Control, t-test; + -P<0.05 vs Sham untreated, one way ANOVA Bonferroni test
!
122 Chapter 7
!
Since both GPCR-dependent (PE) and –independent responses (KCl) were attenuated, this indicated a general depression of contractility. A Ca2+-desensitization process developing post-CPB might be implied, but further mechanistic investigation or qualification hereof was beyond the scope of the present study. Instead, because vascular dysfunction is believed to contribute to multiple organ dysfunction syndrome and related complications post-CPB, and therefore considered a relevant target of therapy, depressed mesenteric contractility was taken as an index (marker) to evaluate the potential of S1P receptor agonist treatment to modulate systemic vascular dysfunction in CPB.
Figure 4. Relaxant reactivity of mesenteric vessels to Acetylcholine in Control (A), Sham (B), and CPB (C) groups. Data are present as AUC values. ** - P=0.001 vs Control untreated, one way ANOVA, Bonferroni test. * P=0.003, vs CPB untreated, one way ANOVA, Bonferroni test.# P<0.05, vs CPB untreated, one way ANOVA, Bonferroni test.
S1P receptors’ agonists and vascular reactivity 123
!
Although S1P is of importance in the entire human body, it is a major regulator of vascular and immune systems. In this respect, the immunomodulatory effects of S1P agonists have been associated with the inhibition after S1P receptor activation of the egress of lymphocytes from secondary lymphoid organs to peripheral blood.8 Consistent herewith, FTY720 at the dose 0.5mg/kg in the present study induced a profound decrease in the amount of circulating lymphocytes (hence, but not in neutrophils or monocytes). Moreover, FTY720-induced lymphopenia was present at the time of onset of ECC (approximately 4 hour after the injection) and lasted for (at least) 24 hours thereafter (at which time-point vascular reactivity was assessed). In contrast, levels of circulating lymphocytes in animals treated SEW2871 at the same dose (0.5mg/kg) did not differ from (sham or CPB) rats that remained untreated. FTY720 is a potent non-selective S1P receptors agonist that causes the internalization of the receptors with their following degradation.25 SEW2871 is a highly selective S1P1 receptors agonist, that in contrast to FTY720 are capable only to internalize receptors, posses less efficacy and unable to downregulate S1P1 receptors.26;27
Despite the differential effects of FTY720 and SEW2871 on circulating lymphocytes in the present study, both compounds shared a similar treatment effect on vascular reactivity and blood pressure (see below). This suggests that the vascular treatment effects occurred independently from lymphopenia but rather involved (direct) modulation of vascular S1P receptors. Second to the above, therefore, S1P receptor agonists may exert direct vascular effects that may help to maintain vascular function. The five types of S1P receptors, S1P1-5, are widely distributed in different organs and tissues with S1P1-3 being the main receptors in the cardiovascular system. They are functionally involved in various processes, such as vascular permeability, endothelial proliferation, angiogenesis, and regulation of vascular tone and heart rate.19;28;29 The S1P receptor is a G-protein coupled receptor (GPCR), the type of G-protein coupled to it providing the diversity in downstream signaling pathways and responses.30 S1P receptors are not homogeneously expressed in the endothelial and smooth muscle cells derived from different vascular beds.29;30 Although it is generally considered that in endothelium the S1P1-receptor type is most abundantly expressed,30 both S1P1, -2 and -3 receptors participate in the maintenance of endothelial integrity and hence, the vascular barrier. Activation of S1P1 type receptor increases the endothelial integrity and thus decreases the vascular permeability, as found in studies with S1P1 agonists.17;19;30 Activation of S1P2 and S1P3 receptors on the other hand, leads to an increased vascular permeability. Activation of S1P2 and S1P3 receptors on smooth muscle cells causes vasoconstriction through activation of the phospholipase C, activation of myosine light chain kinase, and/or Rho-associated kinase-dependent inhibition of myosine light chain phosphatase.29-31 Several studies also showed the involvement of S1P1 receptors in regulation of contractile reactivity.32
In our study, impaired contractility was restored in Sham and CPB-rats pre-treated with FTY720 or SEW2871. This improvement mainly concerned the GRCP-independent responses to KCl, but was less eminent for GRCP-dependent responses to PE (except in SEW2871-treated CPB-rats). Of note, both treatments reduced
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contractility to PE, but not that to KCl, in control rats. Possibly, stimulation of the S1P receptor, which is also a G-protein coupled receptor, leads to a downregulation/desensitzation of G-protein signalling in general. This could explain the decreased response to PE, but not KCl, in treated control rats as well as in treated Sham and CPB rats.
Endothelium-dependent relaxation to ACh in untreated Sham and CPB rats was comparable to that in untreated controls. This indicates that endothelial function was still intact 1-day post-CPB but does not rule out attenuation of endothelial function in other vascular beds and/or at later stages post-CPB.chapter 2 Interestingly, FTY720 and SEW2871 both enhanced total relaxation to ACh in all groups, including in control rats. It has been shown previously that S1P1 and S1P3 receptors on endothelial cells may evoke the release of NO through activation of eNOS.12-15 On the other hand, NO is not the major mediator of endothelium-dependent relaxation in mesenteric artery. Augmented endothelium-dependent reactivity was most pronounced in CPB rats treated with FTY720 and SEW2871, due to an increased contribution of EDHF. Although the effect of S1P agonists on NO production has been described before, the effect of S1P-receptor activation in EDHF has not been reported previously. S1P was shown to activate large-conductance Ca(2+)-activated K(+) channel, but in G-protein independent manner.32 Thus, this effect requires further elucidation but that was not an aim of the present study. Nevertheless, because both experimental compounds exerted their effect in comparable values, mainly S1P1 receptor involvement may be implied in above vascular effects.
Finally, the results of our study showed that both experimental compounds decreased mean arterial blood pressure during the whole experimental procedure in both Sham and CPB-operated groups. Previously it has been shown that FTY720 increased blood pressure in human.29;32; 33 Probably, simultaneous application of S1P agonists and prolonged anesthetical intervention might produce hypotensive effects since both can modulate open probability of the potassium channels34;35 that are involved in the regulation of the heart rate and vascular tone. S1P1 receptors participate in the regulation of both relaxant and contractile reactivity of vascular beds that serve as balance of concurrent activity. The final effect depends on the most sensitive and most expressed pathway.31;32
In conclusion, our study for the first times demonstrated that agonists of the S1P receptors enhanced systemic vascular reactivity after CPB mainly due to modulation of the S1P1 subtype. However, this effect does not depend on the lymphopenia. This finding can initiate further research for the evaluation of such therapeutical agents in the prevention and/or treatment of the CPB-related complications. Reference List
1. Hirleman E, Larson DF: Cardiopulmonary bypass and edema: physiology and pathophysiology.
Perfusion 2008; 23: 311-22
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2. Apostolakis EE, Koletsis EN, Baikoussis NG, Siminelakis SN, Papadopoulos GS: Strategies to prevent intraoperative lung injury during cardiopulmonary bypass. J.Cardiothorac.Surg. 2010; 5: 1
3. Chun J, Hartung HP: Mechanism of Action of Oral Fingolimod (FTY720) in Multiple Sclerosis. Clin.Neuropharmacol. 2010;
4. Brinkmann V: FTY720 (fingolimod) in Multiple Sclerosis: therapeutic effects in the immune and the central nervous system. Br.J.Pharmacol. 2009; 158: 1173-82
5. Zhang Z, Zhang ZY, Fauser U, Schluesener HJ: FTY720 ameliorates experimental autoimmune neuritis by inhibition of lymphocyte and monocyte infiltration into peripheral nerves. Exp.Neurol. 2008; 210: 681-90
6. Zhang Z, Schluesener HJ: FTY720: a most promising immunosuppressant modulating immune cell functions. Mini.Rev.Med.Chem. 2007; 7: 845-50
7. Martini S, Peters H, Bohler T, Budde K: Current perspectives on FTY720. Expert.Opin.Investig.Drugs 2007; 16: 505-18
8. Mullershausen F, Zecri F, Cetin C, Billich A, Guerini D, Seuwen K: Persistent signaling induced by FTY720-phosphate is mediated by internalized S1P1 receptors. Nat.Chem.Biol. 2009; 5: 428-34
9. Hasegawa Y, Suzuki H, Sozen T, Rolland W, Zhang JH: Activation of sphingosine 1-phosphate receptor-1 by FTY720 is neuroprotective after ischemic stroke in rats. Stroke 2010; 41: 368-74
10. Egom EE, Ke Y, Musa H, Mohamed TM, Wang T, Cartwright E, Solaro RJ, Lei M: FTY720 prevents ischemia/reperfusion injury-associated arrhythmias in an ex vivo rat heart model via activation of Pak1/Akt signaling. J.Mol.Cell Cardiol. 2010; 48: 406-14
11. Lee KD, Chow WN, Sato-Bigbee C, Graf MR, Graham RS, Colello RJ, Young HF, Mathern BE: FTY720 reduces inflammation and promotes functional recovery after spinal cord injury. J.Neurotrauma 2009; 26: 2335-44
12. Delbridge MS, Shrestha BM, Raftery AT, El Nahas AM, Haylor JL: Reduction of ischemia-reperfusion injury in the rat kidney by FTY720, a synthetic derivative of sphingosine. Transplantation 2007; 84: 187-95
13. Kaudel CP, Frink M, Schmiddem U, Probst C, Bergmann S, Krettek C, Klempnauer J, van GM, Winkler M: FTY720 for treatment of ischemia-reperfusion injury following complete renal ischemia; impact on long-term survival and T-lymphocyte tissue infiltration. Transplant.Proc. 2007; 39: 499-502
14. Kaudel CP, Frink M, van GM, Schmiddem U, Probst C, Bergmann S, Krettek C, Klempnauer J, Winkler M: FTY720 application following isolated warm liver ischemia improves long-term survival and organ protection in a mouse model. Transplant.Proc. 2007; 39: 493-8
15. Kaudel CP, Schmiddem U, Frink M, Bergmann S, Pape HC, Krettek C, Klempnauer J, Winkler M: FTY720 for treatment of ischemia-reperfusion injury following complete renal ischemia in C57/BL6 mice. Transplant.Proc. 2006; 38: 679-81
16. Sarai K, Shikata K, Shikata Y, Omori K, Watanabe N, Sasaki M, Nishishita S, Wada J, Goda N, Kataoka N, Makino H: Endothelial barrier protection by FTY720 under hyperglycemic condition: involvement of focal adhesion kinase, small GTPases, and adherens junction proteins. Am.J.Physiol Cell Physiol 2009; 297: C945-C954
17. Camp SM, Bittman R, Chiang ET, Moreno-Vinasco L, Mirzapoiazova T, Sammani S, Lu X, Sun C, Harbeck M, Roe M, Natarajan V, Garcia JG, Dudek SM: Synthetic analogs of FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] differentially regulate pulmonary vascular permeability in vivo and in vitro. J.Pharmacol.Exp.Ther. 2009; 331: 54-64
18. Dudek SM, Camp SM, Chiang ET, Singleton PA, Usatyuk PV, Zhao Y, Natarajan V, Garcia JG: Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor. Cell Signal. 2007; 19: 1754-64
19. Brinkmann V, Cyster JG, Hla T: FTY720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function. Am.J.Transplant. 2004; 4: 1019-25
20. Tolle M, Levkau B, Keul P, Brinkmann V, Giebing G, Schonfelder G, Schafers M, von Wnuck LK, Jankowski J, Jankowski V, Chun J, Zidek W, van der GM: Immunomodulator FTY720 Induces eNOS-dependent arterial vasodilatation via the lysophospholipid receptor S1P3. Circ.Res. 2005; 96: 913-20
21. Xu Y, Henning RH, Lipsic E, van BA, van Gilst WH, Buikema H: Acetylcholine stimulated dilatation and stretch induced myogenic constriction in mesenteric artery of rats with chronic heart failure. Eur.J.Heart Fail. 2007; 9: 144-51
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22. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
23. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
24. Gschwend S, Pinto-Sietsma SJ, Buikema H, Pinto YM, van Gilst WH, Schulz A, de ZD, Kreutz R: Impaired coronary endothelial function in a rat model of spontaneous albuminuria. Kidney Int. 2002; 62: 181-91
25. Graler MH, Goetzl EJ: The immunosuppressant FTY720 down-regulates sphingosine 1-phosphate G-protein-coupled receptors. FASEB J. 2004; 18: 551-3
26. Graler MH: Targeting sphingosine 1-phosphate (S1P) levels and S1P receptor functions for therapeutic immune interventions. Cell Physiol Biochem. 2010; 26: 79-86
27. Huwiler A, Pfeilschifter J: New players on the center stage: sphingosine 1-phosphate and its receptors as drug targets. Biochem.Pharmacol. 2008; 75: 1893-900
28. Brinkmann V, Baumruker T: Pulmonary and vascular pharmacology of sphingosine 1-phosphate. Curr.Opin.Pharmacol. 2006; 6: 244-50
29. Brinkmann V: Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology. Pharmacol.Ther. 2007; 115: 84-105
30. Lucke S, Levkau B: Endothelial Functions of Sphingosine-1-phosphate. Cell Physiol Biochem. 2010; 26: 87-96
31. Yatomi Y: Sphingosine 1-phosphate in vascular biology: possible therapeutic strategies to control vascular diseases. Curr.Pharm.Des 2006; 12: 575-87
32. Igarashi J, Michel T: Sphingosine-1-phosphate and modulation of vascular tone. Cardiovasc.Res. 2009; 82: 212-20
33. Comi G, O'Connor P, Montalban X, Antel J, Radue EW, Karlsson G, Pohlmann H, Aradhye S, Kappos L: Phase II study of oral fingolimod (FTY720) in multiple sclerosis: 3-year results. Mult.Scler. 2010; 16: 197-207
34. Agnew NM, Pennefather SH, Russell GN: Isoflurane and coronary heart disease. Anaesthesia 2002; 57: 338-47
35. Koyrakh L, Roman MI, Brinkmann V, Wickman K: The heart rate decrease caused by acute FTY720 administration is mediated by the G protein-gated potassium channel I. Am.J.Transplant. 2005; 5: 529-36
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Chapter 8
Summary and future perspectives
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Cardiopulmonary bypass (CPB) is a widely used technique invaluable to thoracic surgery. However, CPB also associates with an (temporal) organ dysfunction and an increased incidence of postoperative clinical complications, not only in-hospital but also at the mid- and long-term thereafter.1 Changes in vascular function of macro- and microcirculatory beds represent a pivotal component in the development of organ dysfunction and are thought to result from systemic inflammation and ischemia-reperfusion injury following CPB.2 Hence, documenting the time-course and mechanisms of altered vascular reactivity post-CPB as well as its interrelationship with mediators of reperfusion injury and inflammation may provide better and/or new rationales for pharmacotherapeutic interventions to improve morbidity- and mortality outcome after CPB. The studies in the present thesis intended to explore this and help to fill the gap in the data on the role of altered vascular function in the pathogenesis of CPB. To this end, the main focus was put on (1) alterations in vascular reactivity after CPB in relation to (2) inflammatory processes induced in vital organs, and (3) the effect of pharmacological modulation of S1P-receptors as a therapeutic intervention herein. Employing a rat model of experimental CPB, vascular reactivity in the second and third chapter of this thesis was assessed at different time-points post-operatively during a clinically relevant recovery period (up to 5 days). Because anesthesia is part of CPB, but may have its own impact on vascular reactivity, this was further explored in the forth chapter in which the effect of different types of general anesthesia on vascular reactivity was tested in a mice model of hemorrhagic shock. In chapters five and six, the inflammatory response of vital organs (kidney and lung) to CPB was studied. Finally, the effect of pretreatment with two different S1P-receptor agonists on altered vascular reactivity after CPB was studied in chapter seven. Alterations in vascular reactivity after CPB
Influence of CPB-related anesthetic and surgical procedures Apart from extracorporeal circulation (ECC), CPB consists of intensive
anesthetic and surgical procedures, which by themselves represent a profound challenge to the subject’s condition. In chapters 2 and 3, time-course analysis of vascular reactivity was assessed (in vitro) in preparations of 3 different vessel types obtained from rats subjected to CPB-related procedures with or without ECC (i.e. rats from CPB and sham groups) and sacrificed at different time points (up to 5 days) after recovery hereof. Compared to animals studied after short anesthesia only (i.e. control rats), small mesenteric and coronary artery (but not aorta) contractility was depressed in sham rats the first day after recovery after which it normalized again. This early inhibition of small artery contractility concerned both receptor-dependent (phenylephrine or serotonin) and -independent (KCl) pathways. Such finding suggests a temporal decrease in general Ca2+ sensitivity post-CPB, possibly through modulation of the MLC kinase and/or MLC phosphatase activity.3
Volatile anesthetics are well known for their vasorelaxing effects4 and constitute a main candidate for the induction of changes in vascular contractility. In chapter 4, the effects of N2O adjunct to anesthesia with isoflurane (ISO) on vascular reactivity was investigated in a mouse model of hemorrhagic shock. Both arterial
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pressure and aortic contractility to phenylephrine were decreased in sham mice anaesthetized with ISO only, as compared to those receiving adjunct N2O. Systemic hypotension after ISO has been reported previously5-7 and may represent an unwanted side-effect of anesthesia. Volatile anesthetics including ISO have also been shown to inhibit contraction to angiotensin II, amongst others.4, 8 Collectively, the findings in chapter 6 suggest that vascular contractility during different phases of shock was preserved when adjunct N2O was employed. Moreover, changes in COX expression may represent the underlying mechanism of those effects of N2O. The results of this study fundamentally demonstrate that vascular contractility was modulated by the choice of general anesthesia. This may demand for a rational choice of intra-operative anesthesia to maintain vascular function post-operatively and avoid clinical complications after surgical intervention, including in CPB.
CPB-related anesthetic and surgical procedures not only affected vascular smooth cell function but also endothelial cell function. Additionally, as described in chapter 2, endothelium-dependent relaxation in small mesenteric arteries was significantly enhanced in sham rats at 2 days after recovery. Together with the above-mentioned findings, the results indicate that anesthetic and invasive procedures in CPB may induce profound changes in vascular function, even in the absence of ECC. The results suggest a pattern of temporal changes early post-CPB (i.e. 24-48h) both at the level of smooth muscle as well as endothelial cell function in small arteries. In contrast, however, aorta contractility and endothelium-dependent relaxation in sham rats remained relatively unaffected after early post-CPB (but increased EDHF in EF after 5 days). This seems to highlight that different vessel types may become differentially affected and underlines the differences along the vascular tree in susceptibility for alterations in vascular function following CPB.
Additional effects of extracorporeal circulation On top of the described effects of CPB-related anesthetic and surgical
procedures, ECC attenuated vascular relaxation in small mesenteric and coronary arteries (chapter2). This impairment was most outspoken at 2 days of recovery and involved a decrement of various different mediators of endothelial relaxation function. In mesenteric arteries, particularly, the contribution of EDHF was reduced, while in coronary arteries all endothelial mediators appeared suppressed by ECC.
The observation that ECC selectively affected (small artery) endothelial function constitutes an important finding. Others have found increased plasma TNF-! concentrations correlates to impaired endothelium dependent relaxation in rats after CPB9 and implied that (systemic) endothelial dysfunction results from the aggravated systemic inflammatory response that may be elicited by CPB.10; 11 ; 12 In chapter 3, we examined vascular expression of E-selectin, ICAM-1, and VCAM-1 and evaluated the correlation between these markers of endothelial cell activation and endothelial relaxation in rat aorta. Hence, E-selectin is a main marker of the endothelium activation in response to inflammatory cytokine (TNF-! and IL-1$)13 and the cell adhesion molecules of the immunoglobulin superfamily, ICAM-1 and VCAM-1, serve as ligands for leukocyte integrins.1 Substantial up-regulation of all these factors was found consistently in CPB and occasionally in Sham but only after 60 min of recovery from
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CPB. CPB also induced the up-regulation of TNF-! and TGF-$, which may modulate the expression of adhesion molecules, provide a chemotactic gradient for leukocytes and other cells participating in an inflammatory response,14 and increase expression of HO-1, an enzyme induced in response to oxidative stress and hypoxia. Again, however, these increases occurred only very acutely after CPB. Because aortic endothelium-dependent relaxation at that time-point of recovery did not differ between sham and CPB rats, it seems unlikely that endothelial relaxation (dys-)function was related with endothelial cell activation (which was present mainly in the CPB group). Alternatively, it seems that transient aortic inflammation acutely following CPB may have induced processes that lead to alterations in aortic reactivity at the long-term.
To summarise, vascular reactivity after CPB was greatly affected in different vessel types during long-term recovery period without trends for normalization. Sham-related procedures (anesthesia and surgery) seemed to evoke an initial decrease in general vascular contractility, whereas extracorporeal circulation (as a major constitute of CPB) additionally attenuated endothelial relaxant function (particularly in small vessels). Although ECC also prompted for a rapid systemic inflammatory response, endothelial cell activation alone did not explain the selective CPB-mediated impairment in endothelial function (see also fig. 1, A and B).
Changes in inflammatory processes in vital organs (kidney and lung) A high rate of postoperative morbidity in CPB is being associated with
(temporal) renal and pulmonary organ dysfunction. The systemic inflammatory response syndrome in CPB is thought to play a major role herein. In chapters 4 and 5, we evaluated the time-dependent changes in inflammatory marker expression after CPB in the lung and the kidney.
The observed expression patterns of different pro- and anti-inflammatory cytokines, endothelial activation markers, and markers of neutrophil activation indicated that CPB evoked an acute inflammatory response in both organs. Early activation of macrophages and profound release of the inflammatory cytokines (TNF-!, IL-6, IL-1$ and others) during the first hours of recovery has been shown previously both in lung15-18 and kidney.1, 19, 20 In our studies, macrophages infiltration in the lung was relatively high and remained elevated over time post-CPB in all groups, including in controls rats. However, expression of inflammatory cytokines was not increased in the latter. Such findings seem to dispute for a role solely of infiltrating macrophages in increased inflammatory cytokine production. Instead, macrophages already resident in organs may be importantly involved in the rapid up-regulation of inflammatory cytokines. Literature reporting on a poor correlation between inflammatory mRNA expression in organs with cytokine concentrations in the plasma may further support the importance of inflammatory processes locally in the kidney and the lung.21
Despite early similarities, the expressional patterns at later stages post-CPB differed between lung and kidney in our studies. In the kidney, all up-regulated markers were normalized by day 5 of the recovery period. In contrast, pulmonary expression of Il-1beta and TGFbeta1 remained increased during recovery, indicating the continuation of inflammatory processes in the lung. Such situation may be explained by the specific
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character of the lung functionality during CPB. Lungs are not only subjected to CPB-evoked release of cytokines initiating a prominent inflammatory response in this organ, but they are also more or less excluded from the circulation during the extracorporeal circulation period22, 23 that aggravates CPB-related pathological consequences.
To summarize, the inflammatory processes in kidney and lung in our study appeared early after CPB. Whereas all up-regulated markers in kidney normalised in due course thereafter, inflammatory processes in lung seem were maintained for at least 5 days thereafter with little trend for the normalization (fig.1C) Pharmacological modulation of S1P-receptors as a therapeutic intervention in CPB
Systemic inflammation has been implied to underlie changes in vascular function of macro- and microcirculatory beds. Altered vascular function is believed to represent a pivotal component in the development of organ dysfunction and clinical complications hereof.22, 23 Therefore, interventions to maintain vascular function in CPB may be aimed both at modulation of systemic inflammation and/or direct modulation of vascular function. Because sphingosine 1-phosphate (S1P) is a main regulator of immune as well as vascular systems, pharmacological modulation of S1P-receptors was considered a potential therapeutic intervention in CPB.24
Employing our experimental rat model of CPB, the acute pre-treatment effects of FTY720 and SEW2871, two different S1P-receptor agonists, on (systemic) vascular reactivity was investigated in chapter seven. Isolated mesenteric artery preparations were studied 1-day post-CPB, hence, at which time point contractility in untreated animals was generally depressed (see chapter 2). FTY720 and SEW2871 both restored contractility and augmented endothelial relaxant reactivity after CPB. Both treatments also evoked hypotension, yet differentially affected the amount of lymphocytes in the peripheral blood. I.e. circulating lymphocytes were decreased after FTY720, a non-selective S1P receptor modulator, but not after SEW2871, a selective agonist of the S1P1-receptor (see also fig. 2). Because both compounds shared a similar treatment effect on vascular reactivity and blood pressure, this suggests that the vascular treatment effects occurred independent from lymphopenia and inhibition of systemic inflammation but rather involved (direct) modulation of vascular S1P receptors.
The five types of S1P receptors, S1P1-5, are widely distributed in different organs and tissues with S1P1-3 being the main receptors in the cardiovascular system.25 S1P1 receptors participate in the regulation of both relaxant and contractile reactivity of vascular beds that serve as balance of concurrent activity.26, 27 Given the fact that the observed systemic vascular effects were also obtained with the selective agonist SEW2871, involvement of the vascular S1P1-receptor subtype herein is strongly implied. The findings of these studies may encourage further research for the evaluation of such therapeutic agents in the prevention and/or treatment of the CPB-related complications.
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Implications The studies in the first part of this thesis demonstrated that vascular reactivity
post-CPB was profoundly influenced in a vessel-type specific manner by CPB-related procedures (anesthesia and surgical intervention), and ECC in addition hereof. Per se anesthetic and surgical procedures in CPB evoked a pattern of temporal changes early post-CPB both at the level of smooth muscle (decreased contractility) as well as endothelial cell function (enhanced relaxation) in small (coronary and mesenteric) arteries. As depicted in figure 1A, this is in contrast to large conductance artery reactivity which was relatively unaffected early post-CPB but rather changed at the longer term (i.e. increased EDHF in endothelial function). ECC as a major constitute of CPB attenuated endothelial relaxant function in small arteries, but not in large vessel. The findings suggest differential sensitivity along the vascular tree for alterations in vascular function following CPB, and imply increased susceptibility of small arteries for impairment of endothelial function by ECC. These pathophysiological events may explain observations made in the clinic. The disturbances in microcirculatory beds of different organs can initiate secondary tissue edema and tissue hypoperfusion, thus founding a multiple organ dysfunction syndrome. Hemodynamic and microcirculatory abnormalities were shown to set a stage for the mesenteric complications,28, 29 acute lung injury and acute respiratory distress syndrome,15, 30 acute kidney injury 31, 32 after CPB.
As depicted in figure 1B, ECC also initiated a rapid systemic inflammatory response and vascular endothelial cell activation in the aorta, however, without a momentary impairment in the endothelial relaxation. The latter challenges the immediate translation of vascular inflammation into altered vascular reactivity. Rather, inflammation may act as an trigger of subsequent alterations in the expression or activity of components involved in vasomotor regulation, subsequently altering vascular reactivity after an initial lag time [2]. If so, the lag time may leave time for pharmacological modulation of vascular function to prevent microcirculatory disturbances.
Studies in the second part of this thesis showed that while ECC evoked an acute inflammatory processes in all organs studied, organs differed with respect of the duration of the response. As depicted in figure 1C, up-regulated markers in vascular and renal tissue normalised in due course, while the inflammatory processes in lung was maintained for at least 5 days post-CPB. The difference may be explained by the specifics of the lung perfusion during CPB, since lungs are deprived of the majority of their normal blood supply in this period, additionally to the exposition to all CPB-related pathogenetic factors that affect functionality of all organs.1, 20 Maintenance of the inflammatory processes in lung during long-term recovery can set the stage for the frequently occurring acute pulmonary complication after CPB, since approximately 25% of the patients following open heart surgery exhibited signs of pulmonary impairment for at least one week thereafter.1, 22, 23
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Figure 1. Schematic representation of the changes in vascular reactivity and inflammatory processes following cardiopulmonary bypass (CPB). (A) CPB evoked a differential pattern of temporal changes early post-CPB (i.e. 24-48h) in small (mesenteric and coronary) arteries vs. “long-term” changes in large conductance artery (aorta); (B) Rapid systemic inflammation and aorta endothelial cell activation after extracorporeal circulation (ECC) were not paralleled by acute changes in aorta endothelial relaxation function, but rather by changes at the “long-term”; (C) Up-regulated inflammatory markers in kidney and aorta normalised in due course after ECC, but were maintained elevated in the lung for at least 5 days after CPB.
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Figure 2. Effect of S1P-receptor agonism on circulating lymphocytes and vascular reactivity following CPB. (A) The amount of circulating lymphocytes was reduced after pre-treatment with the non-selective S1P-receptor agonist FTY720, but not with the selective S1P1-receptor agonist SEW2871. In contrast, both compounds similarly enhanced vascular (B) contractility and (C) endothelial relaxation function. This suggests that the enhanced vascular reactivity following pre-treatment was mediated via modulation vascular S1P1-receptors, independently from lymphopenia. * P<0.05, vs untreated group, one way ANOVA with Bonferroni test. # P<0.05, vs untreated group, t-test
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Studies in the last part of this thesis demonstrated that pre-treatment with an S1P-receptor agonist restored vascular reactivity post-CPB both in the absence and presence of concurrent reduction in circulating lymphocytes. As depicted in figure 2, lymphopenia was induced with FTY720 only, but not with SEW2871. However, both compounds shared a similar effect on vascular reactivity and blood pressure. The results imply positive vascular treatment effects independent from lymphopenia via direct modulation of vascular S1P1-receptors. Therefore agonists of S1P receptors can represent a new approach for the improvement of the functionality of microcirculatory beds and preventing of the organ dysfunction syndrome due to enhancing of the tissue perfusion. In conclusion, there are several major points disclosed to the research described in this thesis. First, CPB evokes profound abnormalities, which include different physiological processes; herein among others changes in vascular reactivity play a prominent role. Secondly, key components of physiology are not fully normalized after a prolonged recovery period, suggesting continuance of the pathological process. Thirdly, normalization of the microcirculation following CPB may be accomplished by modulation of S1P receptors. Despite recent progress in the understanding of the pathophysiological consequences of CPB, the remaining unsolved CPB-related problems point at a range of directions for future research. First, further investigation of the relative contribution of different factors to CPB pathogenesis is warranted. The contribution of the different components of the CPB-circuit to CPB-related complications needs exploration, as changes of the technical design of the CPB-circuit or the surgical protocol may represent a possible way for the prevention and/or treatment of the CPB-mediated medical problems.23, 33 Secondly, as shown in this thesis, anesthetic interventions may have profound impact on the physiology of vascular beds, however, our current knowledge in this respect is very limited. Thus, the choice of the optimal inhalation/intravenous anesthetics prevent multiple organ dysfunction syndrome, by improving hemodynamics, organ microcirculation and tissue perfusion.chapter 2, 3, and 6 of this
thesis; 34, 35 In addition, the influence of preoperatively existing chronic disease, such diabetes, heart, renal, and pulmonary disease, may influence the pathophysiology of CPB.36, 37. Further research in this direction provides better understanding of the underlying mechanisms and possible selective therapeutical modulations. Finally, evaluation of the efficacy of different therapeutic interventions to limit CPB-related complications remains an actual research topic. Further investigation of the S1P receptor modulation may be promising in prevention and/or treatment of the CPB-related complications. Reference List 1. Asimakopoulos G: Systemic inflammation and cardiac surgery: an update. Perfusion 2001; 16: 353
60 2. Verrier ED, Morgan EN: Endothelial response to cardiopulmonary bypass surgery. Ann Thorac Surg
1998; 66: S17,9; discussion S25-8
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3. Savineau JP, Marthan R: Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications. Fundam Clin Pharmacol 1997; 11: 289-99
4. Preckel B, Bolten J: Pharmacology of modern volatile anaesthetics. Best Pract Res Clin Anaesthesiol 2005; 19: 331-48
5. Baumert JH, Hecker KE, Hein M, Reyle-Hahn SM, Horn NA, Rossaint R: Haemodynamic effects of haemorrhage during xenon anaesthesia in pigs. Br J Anaesth 2005; 94: 727-32
6. Pypendop BH, Ilkiw JE, Imai A, Bolich JA: Hemodynamic effects of nitrous oxide in isoflurane-anesthetized cats. Am J Vet Res 2003; 64: 273-8
7. Conzen PF, Vollmar B, Habazettl H, Frink EJ, Peter K, Messmer K: Systemic and regional hemodynamics of isoflurane and sevoflurane in rats. Anesth Analg 1992; 74: 79-88
8. Qi F, Ogawa K, Tokinaga Y, Uematsu N, Minonishi T, Hatano Y: Volatile anesthetics inhibit angiotensin II-induced vascular contraction by modulating myosin light chain phosphatase inhibiting protein, CPI-17 and regulatory subunit, MYPT1 phosphorylation. Anesth Analg 2009; 109: 412-7
9. Doguet F, Litzler PY, Tamion F, Richard V, Hellot MF, Thuillez C, Tabley A, Bouchart F, Bessou JP: Changes in mesenteric vascular reactivity and inflammatory response after cardiopulmonary bypass in a rat model. Ann Thorac Surg 2004; 77: 2130,7; author reply 2137
10. Larmann J, Theilmeier G: Inflammatory response to cardiac surgery: cardiopulmonary bypass versus non-cardiopulmonary bypass surgery. Best Pract Res Clin Anaesthesiol 2004; 18: 425-38
11. Sistino JJ, Acsell JR: Systemic inflammatory response syndrome (SIRS) following emergency cardiopulmonary bypass: a case report and literature review. J Extra Corpor Technol 1999; 31: 37-43
12. Markewitz A, Lante W, Franke A, Marohl K, Kuhlmann WD, Weinhold C: Alterations of cell-mediated immunity following cardiac operations: clinical implications and open questions. Shock 2001; 16 Suppl 1: 10-5
13. Ulbrich H, Eriksson EE, Lindbom L: Leukocyte and endothelial cell adhesion molecules as targets for therapeutic interventions in inflammatory disease. Trends Pharmacol Sci 2003; 24: 640-7
14. Sanjabi S, Zenewicz LA, Kamanaka M, Flavell RA: Anti-inflammatory and pro-inflammatory roles of TGF-beta, IL-10, and IL-22 in immunity and autoimmunity. Curr Opin Pharmacol 2009; 9: 447-53
15. Fujii M, Miyagi Y, Bessho R, Nitta T, Ochi M, Shimizu K: Effect of a neutrophil elastase inhibitor on acute lung injury after cardiopulmonary bypass. Interact Cardiovasc Thorac Surg 2010; 10: 859-62
16. Zheng JH, Gao BT, Jiang ZM, Yu XQ, Xu ZW: Evaluation of Early Macrophage Activation and NF-kappaB Activity in Pulmonary Injury Caused by Deep Hypothermia Circulatory Arrest: An Experimental Study. Pediatr Cardiol 2009
17. Apostolakis E, Filos KS, Koletsis E, Dougenis D: Lung dysfunction following cardiopulmonary bypass. J Card Surg 2010; 25: 47-55
18. Apostolakis EE, Koletsis EN, Baikoussis NG, Siminelakis SN, Papadopoulos GS: Strategies to prevent intraoperative lung injury during cardiopulmonary bypass. J Cardiothorac Surg 2010; 5: 1
19. Gueret G, Lion F, Guriec N, Arvieux J, Dovergne A, Guennegan C, Bezon E, Baron R, Carre JL, Arvieux C: Acute renal dysfunction after cardiac surgery with cardiopulmonary bypass is associated with plasmatic IL6 increase. Cytokine 2009; 45: 92-8
20. Asimakopoulos G, Taylor KM: Effects of cardiopulmonary bypass on leukocyte and endothelial adhesion molecules. Ann Thorac Surg 1998; 66: 2135-44
21. Brix-Christensen V, Vestergaard C, Chew M, Johnsen CK, Andersen SK, Dreyer K, Hjortdal VE, Ravn HB, Tonnesen E: Plasma cytokines do not reflect expression of pro- and anti-inflammatory cytokine mRNA at organ level after cardiopulmonary bypass in neonatal pigs. Acta Anaesthesiol Scand 2003; 47: 525-31
22. Apostolakis E, Filos KS, Koletsis E, Dougenis D: Lung dysfunction following cardiopulmonary bypass. J Card Surg 2010; 25: 47-55
23. Apostolakis EE, Koletsis EN, Baikoussis NG, Siminelakis SN, Papadopoulos GS: Strategies to prevent intraoperative lung injury during cardiopulmonary bypass. J Cardiothorac Surg 2010; 5: 1
24. Huwiler A, Pfeilschifter J: New players on the center stage: sphingosine 1-phosphate and its receptors as drug targets. Biochem Pharmacol 2008; 75: 1893-900
25. Brinkmann V, Baumruker T: Pulmonary and vascular pharmacology of sphingosine 1-phosphate. Curr Opin Pharmacol 2006; 6: 244-50
Summary and future perspectives 137
!
26. Graler MH: Targeting sphingosine 1-phosphate (S1P) levels and S1P receptor functions for therapeutic immune interventions. Cell Physiol Biochem 2010; 26: 79-86
27. Lucke S, Levkau B: Endothelial functions of sphingosine-1-phosphate. Cell Physiol Biochem 2010; 26: 87-96
28. Abboud B, Daher R, Boujaoude J: Acute mesenteric ischemia after cardio-pulmonary bypass surgery. World J Gastroenterol 2008; 14: 5361-70
29. Andrasi TB, Buhmann V, Soos P, Juhasz-Nagy A, Szabo G: Mesenteric complications after hypothermic cardiopulmonary bypass with cardiac arrest: underlying mechanisms. Artif Organs 2002; 26: 943-6
30. Aghdaii N, Faritous SZ, Yazdanian F, Zade HR: Acute respiratory distress syndrome: rapid and significant response to volume-controlled inverse ratio ventilation--a case report. Middle East J Anesthesiol 2009; 20: 457-60
31. Picca S, Ricci Z, Picardo S: Acute kidney injury in an infant after cardiopulmonary bypass. Semin Nephrol 2008; 28: 470-6
32. Abu-Omar Y, Ratnatunga C: Cardiopulmonary bypass and renal injury. Perfusion 2006; 21: 209-13 33. Su XW, Undar A: Brain protection during pediatric cardiopulmonary bypass. Artif Organs 2010; 34:
E91-102 34. Piriou V, Mantz J, Goldfarb G, Kitakaze M, Chiari P, Paquin S, Cornu C, Lecharny JB, Aussage P,
Vicaut E, Pons A, Lehot JJ: Sevoflurane preconditioning at 1 MAC only provides limited protection in patients undergoing coronary artery bypass surgery: a randomized bi-centre trial. Br J Anaesth 2007; 99: 624-31
35. Pouzet B, Lecharny JB, Dehoux M, Paquin S, Kitakaze M, Mantz J, Menasche P: Is there a place for preconditioning during cardiac operations in humans? Ann Thorac Surg 2002; 73: 843-8
36. Ji Q, Mei Y, Wang X, Feng J, Cai J, Sun Y: Impact of diabetes mellitus on old patients undergoing coronary artery bypass grafting. Int Heart J 2009; 50: 693-700
37. Guo HW, Chang Q, Xu JP, Song YH, Sun HS, Hu SS: Coronary artery bypass grafting for Kawasaki disease. Chin Med J (Engl) 2010; 123: 1533-6
138 Chapter 8
!
!
!!!
Supplements
140 Supplement to Chapter 4
!
Sup
plem
enta
l Dig
ital C
onte
nt 2
Li
st o
f sig
nific
antly
diff
eren
t gen
es (4
21) b
etw
een
CP
B a
nd S
ham
. G
enes
ac
ute
day
1 da
y 5
ID
Sym
bol
Def
initi
on
CP
B
Sha
m
p C
PB
S
ham
p
CP
B
Sha
m
p 24
498
IL6
inte
rleuk
in 6
28
3.4
0.8
.00
1.5
1.3
ns
0.4
2.0
ns
1141
05
CX
CL2
ch
emok
ine
(C-X
-C m
otif)
liga
nd 2
24
1.3
2.5
.00
1.4
2.4
ns
2.9
0.7
ns
8150
3 C
XC
L1
chem
okin
e (C
-X-C
mot
if) li
gand
1
237.
6 3.
1 .0
0 1.
1 1.
1 ns
0.
6 1.
0 ns
25
651
SE
LP
sele
ctin
P
160.
2 1.
0 .0
1 0.
5 0.
9 ns
1.
4 0.
9 ns
89
829
SO
CS
3 su
ppre
ssor
of c
ytok
ine
sign
alin
g 3
114.
1 2.
0 .0
1 3.
0 2.
3 ns
2.
7 2.
1 ns
24
451
HM
OX
1 he
me
oxyg
enas
e (d
ecyc
ling)
1
90.2
9.
0 .0
0 1.
1 1.
2 ns
0.
7 0.
8 ns
30
4005
N
FKB
IZ
nucl
ear f
acto
r of k
appa
ligh
t pol
ypep
tide
gene
enh
ance
r in
B-c
ells
inhi
bito
r, ze
ta
89.2
2.
6 .0
0 1.
3 4.
0 ns
1.
3 3.
8 ns
3629
93
RN
D1
Rho
fam
ily G
TPas
e 1
60.0
1.
9 .0
0 0.
5 0.
9 ns
0.
4 0.
9 ns
24
5920
C
XC
L10
chem
okin
e (C
-X-C
mot
if) li
gand
10
59.9
7.
3 .0
0 1.
0 1.
9 ns
1.
3 1.
3 ns
36
0857
R
GS
16
regu
lato
r of G
-pro
tein
sig
nalin
g 16
59
.7
2.3
.01
0.6
2.9
ns
2.9
0.9
ns
2869
26
TFP
I2
tissu
e fa
ctor
pat
hway
inhi
bito
r 2
59.2
0.
5 .0
0 0.
6 0.
5 ns
0.
3 0.
3 ns
36
2850
A
NG
PTL
4 an
giop
oiet
in-li
ke 4
57
.8
9.9
.00
2.3
5.5
ns
1.8
1.6
ns
4980
87
STF
A2I
3 st
efin
A2-
like
3 44
.1
6.1
.00
1.0
1.0
ns
1.9
1.0
ns
2828
21
HA
S1
hyal
uron
an s
ynth
ase
1 40
.3
1.7
.00
2.2
1.5
ns
1.0
1.0
ns
3143
22
FOS
FB
J os
teos
arco
ma
onco
gene
33
.3
1.3
.01
0.4
1.2
ns
1.6
1.6
ns
2554
4 S
ELE
se
lect
in E
29
.7
1.5
.02
1.4
0.9
ns
1.9
1.2
ns
2461
7 S
ER
PIN
E1
serin
e (o
r cys
tein
e) p
eptid
ase
inhi
bito
r, cl
ade
E, m
embe
r 1
25.5
6.
1 .0
0 1.
2 1.
1 ns
0.
8 0.
8 ns
17
0496
LC
N2
lipoc
alin
2
19.9
1.
4 .0
2 6.
5 2.
4 ns
1.
1 0.
9 ns
24
330
EG
R1
early
gro
wth
resp
onse
1
19.7
2.
6 .0
0 0.
5 1.
2 ns
1.
6 1.
3 ns
24
494
IL1B
in
terle
ukin
1 b
eta
19.4
2.
6 .0
0 1.
1 0.
9 ns
1.
1 1.
1 ns
24
770
CC
L2
chem
okin
e (C
-C m
otif)
liga
nd 2
19
.3
2.1
.00
0.5
0.7
ns
0.9
0.5
ns
8438
7 P
GLY
RP
1 pe
ptid
ogly
can
reco
gniti
on p
rote
in 1
19
.3
1.0
.01
0.7
1.6
ns
0.7
1.7
ns
3163
27
AR
ID5A
A
T ric
h in
tera
ctiv
e do
mai
n 5A
(Mrf1
like
) 17
.9
1.7
.00
0.6
4.2
ns
2.3
1.7
ns
3101
32
OS
MR
on
cost
atin
M re
cept
or
17.4
4.
1 .0
0 1.
4 1.
9 ns
1.
6 1.
7 ns
29
9206
B
ATF
ba
sic
leuc
ine
zipp
er tr
ansc
riptio
n fa
ctor
, ATF
-like
16
.8
1.7
.00
1.1
1.2
ns
0.9
1.1
ns
7897
1 B
IRC
3 ba
culo
vira
l IA
P re
peat
-con
tain
ing
3 16
.3
2.1
.00
0.9
1.0
ns
1.1
1.0
ns
3652
45
SA
A4
seru
m a
myl
oid
A4,
con
stitu
tive
16.1
1.
3 .0
1 2.
1 9.
5 ns
1.
7 1.
3 ns
30
9527
C
H25
H
chol
este
rol 2
5-hy
drox
ylas
e 16
.1
2.7
.00
1.0
1.1
ns
1.0
0.8
ns
3023
78
LPA
R4
lyso
phos
phat
idic
aci
d re
cept
or 4
0.
1 0.
5 .0
1 0.
4 0.
6 ns
1.
2 0.
6 ns
29
619
BTG
2 B
TG fa
mily
, mem
ber 2
15
.1
1.8
.00
0.8
1.0
ns
1.1
0.9
ns
2879
74
KLH
L6
kelc
h-lik
e 6
0.1
0.9
.00
0.8
0.9
ns
0.9
0.9
ns
2568
5 IG
FBP
1 in
sulin
-like
gro
wth
fact
or b
indi
ng p
rote
in 1
14
.3
3.0
.00
1.2
1.7
ns
0.8
1.1
ns
2532
5 IL
10
inte
rleuk
in 1
0 14
.2
1.0
.00
1.0
1.0
ns
1.0
1.4
ns
5885
3 N
R4A
3 nu
clea
r rec
epto
r sub
fam
ily 4
, gro
up A
, mem
ber 3
14
.2
1.2
.00
1.0
1.3
ns
1.1
0.7
ns
2457
7 M
YC
m
yelo
cyto
mat
osis
onc
ogen
e 13
.6
3.3
.00
1.2
1.8
ns
1.1
1.2
ns
3628
48
PR
AM
1 P
ML-
RA
RA
regu
late
d ad
apto
r mol
ecul
e 1
13.5
1.
7 .0
2 2.
9 0.
6 ns
3.
1 1.
0 ns
Supplements 141
!
2875
61
CC
L7
chem
okin
e (C
-C m
otif)
liga
nd 7
13
.4
1.9
.00
0.8
0.9
ns
0.9
0.7
ns
1170
22
IL1R
2 in
terle
ukin
1 re
cept
or, t
ype
II 11
.7
1.1
.00
0.9
1.2
ns
0.3
0.4
ns
2956
71
RG
D13
0889
0 si
mila
r to
RIK
EN
cD
NA
493
0506
L13
0.1
0.6
.02
0.9
1.1
ns
1.1
1.0
ns
3063
51
GD
F1
grow
th d
iffer
entia
tion
fact
or 1
0.
1 0.
8 .0
1 1.
2 1.
0 ns
1.
5 1.
8 ns
69
0672
LO
C69
0672
si
mila
r to
Dis
cs la
rge
hom
olog
5 (P
lace
nta
and
pros
tate
DLG
) (D
iscs
larg
e pr
otei
n P
-dlg
) 0.
1 0.
7 .0
4 0.
4 0.
8 ns
0.
9 0.
4 ns
2924
61
RA
ET1
L re
tinoi
c ac
id e
arly
tran
scrip
t 1L
10.8
1.
0 .0
0 1.
0 4.
2 ns
1.
2 1.
0 ns
79
426
ZFP
36
zinc
fing
er p
rote
in 3
6 10
.7
1.2
.00
1.2
1.2
ns
1.1
1.1
ns
3628
61
SLC
41A
2 so
lute
car
rier f
amily
41,
mem
ber 2
10
.4
1.1
.02
1.6
1.4
ns
1.9
1.0
ns
3088
94
RG
D13
0984
7 si
mila
r to
pept
idyl
glyc
ine
alph
a-am
idat
ing
mon
ooxy
gena
se C
OO
H-te
rmin
al
inte
ract
or; p
eptid
ylgl
ycin
e al
pha-
amid
atin
g m
onoo
xyge
nase
CO
OH
-term
inal
in
tera
ctor
pro
tein
-1
0.1
0.8
.00
0.5
0.8
ns
1.1
1.0
ns
8531
1 P
LA1A
ph
osph
olip
ase
A1
mem
ber A
10
.2
1.8
.00
4.0
2.4
ns
1.2
1.1
ns
5066
2 R
UN
X1
runt
rela
ted
trans
crip
tion
fact
or 1
10
.1
2.2
.01
1.1
1.1
ns
2.0
1.3
ns
2543
3 H
BE
GF
hepa
rin-b
indi
ng E
GF-
like
grow
th fa
ctor
9.
8 1.
1 .0
1 0.
4 0.
6 ns
0.
7 0.
4 ns
11
6596
M
AP
3K8
mito
gen-
activ
ated
pro
tein
kin
ase
kina
se k
inas
e 8
9.5
1.1
.00
1.0
1.1
ns
0.9
0.9
ns
2869
29
CY
P3A
23/3
A1
cyto
chro
me
P45
0, fa
mily
3, s
ubfa
mily
a, p
olyp
eptid
e 23
/pol
ypep
tide
1 9.
4 1.
5 .0
2 1.
0 5.
6 ns
1.
0 1.
0 ns
25
066
PV
R
polio
viru
s re
cept
or
9.3
2.0
.01
1.0
1.4
ns
1.0
0.8
ns
2425
3 C
EB
PB
C
CA
AT/
enha
ncer
bin
ding
pro
tein
(C/E
BP
), be
ta
9.3
2.3
.00
1.2
1.4
ns
1.2
1.2
ns
1142
03
SH
2B2
SH
2B a
dapt
or p
rote
in 2
9.
3 1.
6 .0
0 0.
5 1.
3 ns
1.
4 1.
1 ns
31
5711
S
EM
A7A
se
ma
dom
ain,
imm
unog
lobu
lin d
omai
n (Ig
), an
d G
PI m
embr
ane
anch
or,
(sem
apho
rin) 7
A
9.2
2.5
.00
1.2
0.9
ns
1.0
1.0
ns
4449
83
AP
OLD
1 ap
olip
opro
tein
L d
omai
n co
ntai
ning
1
8.9
1.7
.00
1.1
1.4
ns
1.1
1.1
ns
2546
4 IC
AM
1 in
terc
ellu
lar a
dhes
ion
mol
ecul
e 1
8.9
2.0
.00
1.1
1.2
ns
0.9
1.0
ns
4986
82
RG
D15
6632
5 si
mila
r to
regu
lato
r of s
ex-li
mita
tion
cand
idat
e 16
0.
1 0.
9 .0
0 1.
7 1.
2 ns
1.
0 1.
3 ns
17
0897
S
PH
K1
sphi
ngos
ine
kina
se 1
8.
5 1.
4 .0
1 0.
8 1.
1 ns
1.
2 1.
1 ns
31
6241
N
FKB
IE
nucl
ear f
acto
r of k
appa
ligh
t pol
ypep
tide
gene
enh
ance
r in
B-c
ells
inhi
bito
r, ep
silo
n 4.
2 1.
0 n
s 0.
1 0.
8 0.
00
0.6
0.5
ns
5003
00
LOC
5003
00
sim
ilar t
o hy
poth
etic
al p
rote
in M
GC
6835
8.
3 0.
4 .0
0 1.
5 1.
1 ns
0.
8 1.
0 ns
29
527
PTG
S2
pros
tagl
andi
n-en
dope
roxi
de s
ynth
ase
2 8.
2 0.
8 .0
0 1.
0 0.
7 ns
0.
8 0.
8 ns
28
8529
G
JC3
gap
junc
tion
prot
ein,
gam
ma
3 8.
1 1.
9 .0
2 1.
2 1.
5 ns
1.
0 1.
0 ns
24
517
JUN
B
jun
B p
roto
-onc
ogen
e 8.
1 1.
6 .0
0 0.
9 1.
4 ns
1.
3 1.
1 ns
83
518
AP
LNR
ap
elin
rece
ptor
0.
1 0.
5 .0
0 0.
7 0.
8 ns
1.
0 1.
0 ns
28
7422
P
ER
1 pe
riod
hom
olog
1
7.8
1.6
.00
4.0
3.8
ns
2.6
3.0
ns
2898
01
UP
P1
urid
ine
phos
phor
ylas
e 1
7.7
3.2
.00
1.3
1.4
ns
0.9
0.9
ns
3676
20
MG
C11
6197
si
mila
r to
RIK
EN
cD
NA
170
0001
E04
0.
1 0.
6 .0
2 0.
7 0.
9 ns
1.
2 0.
8 ns
25
531
RA
B3A
R
AB
3A, m
embe
r RA
S o
ncog
ene
fam
ily
0.1
0.7
.01
1.1
1.2
ns
1.2
1.2
ns
1706
37
SA
MS
N1
SA
M d
omai
n, S
H3
dom
ain
and
nucl
ear l
ocal
izat
ion
sign
als,
1
7.5
1.2
.00
1.8
1.1
ns
1.0
0.9
ns
2536
1 V
CA
M1
vasc
ular
cel
l adh
esio
n m
olec
ule
1 7.
0 1.
4 .0
0 1.
1 1.
1 ns
1.
0 1.
0 ns
79
252
AD
AM
TS1
AD
AM
met
allo
pept
idas
e w
ith th
rom
bosp
ondi
n ty
pe 1
mot
if, 1
6.
8 1.
5 .0
2 1.
2 1.
7 ns
1.
2 1.
1 ns
84
586
FGL2
fib
rinog
en-li
ke 2
6.
7 2.
1 .0
0 1.
1 1.
0 ns
0.
7 0.
9 ns
29
194
CH
KA
ch
olin
e ki
nase
alp
ha
6.6
3.3
.00
1.1
1.1
ns
1.3
1.2
ns
4977
57
GU
CY
1A3
guan
ylat
e cy
clas
e 1,
sol
uble
, alp
ha 3
0.
2 0.
6 .0
0 1.
0 0.
9 ns
1.
0 0.
9 ns
142 Supplement to Chapter 4
!
3084
08
GP
R4
G p
rote
in-c
oupl
ed re
cept
or 4
6.
4 1.
6 .0
0 1.
5 1.
4 ns
1.
3 1.
2 ns
29
9626
G
AD
D45
B
grow
th a
rres
t and
DN
A-d
amag
e-in
duci
ble,
bet
a 6.
4 1.
2 .0
0 0.
8 0.
7 ns
0.
9 1.
1 ns
31
2309
ZN
F775
zi
nc fi
nger
pro
tein
775
0.
2 0.
7 .0
1 1.
2 0.
9 ns
1.
2 1.
4 ns
24
508
IRF1
in
terfe
ron
regu
lato
ry fa
ctor
1
6.2
1.5
.00
1.0
1.2
ns
1.1
0.9
ns
3108
77
TIFA
TR
AF-
inte
ract
ing
prot
ein
with
fork
head
-ass
ocia
ted
dom
ain
6.2
1.0
.00
1.0
0.9
ns
0.8
0.8
ns
1165
10
TIM
P1
TIM
P m
etal
lope
ptid
ase
inhi
bito
r 1
6.1
1.6
.02
2.7
1.7
ns
1.1
1.1
ns
2536
9 A
DO
RA
2A
aden
osin
e A
2a re
cept
or
6.1
2.1
.00
1.5
1.0
ns
1.4
1.2
ns
3146
19
SB
NO
2 st
raw
berr
y no
tch
hom
olog
2
6.1
1.4
.02
1.1
1.2
ns
1.2
1.2
ns
2924
4 G
CH
1 G
TP c
yclo
hydr
olas
e 1
6.1
0.7
.00
0.9
0.6
ns
0.5
0.5
ns
2549
3 N
FKB
IA
nucl
ear f
acto
r of k
appa
ligh
t pol
ypep
tide
gene
enh
ance
r in
B-c
ells
inhi
bito
r, al
pha
6.0
1.4
.00
1.2
1.2
ns
1.1
1.0
ns
3080
97
TAG
AP
T-
cell
activ
atio
n G
TPas
e ac
tivat
ing
prot
ein
6.0
1.0
.03
1.3
1.3
ns
1.0
1.0
ns
5932
9 S
NF1
LK
SN
F1-li
ke k
inas
e 6.
0 0.
9 .0
1 1.
1 1.
2 ns
1.
6 1.
3 ns
30
8451
IT
PK
C
inos
itol 1
,4,5
-tris
phos
phat
e 3-
kina
se C
5.
9 1.
1 .0
0 0.
9 0.
9 ns
1.
0 1.
1 ns
49
9839
R
GD
1564
664
sim
ilar t
o LO
C38
7763
pro
tein
5.
9 1.
3 .0
0 0.
7 0.
7 ns
0.
7 0.
7 ns
29
3538
H
MX
2 H
6 fa
mily
hom
eobo
x 2
0.2
1.2
.02
0.7
0.9
ns
0.6
0.9
ns
7896
5 C
SF1
co
lony
stim
ulat
ing
fact
or 1
(mac
roph
age)
5.
6 1.
3 .0
0 0.
9 1.
0 ns
1.
0 0.
9 ns
58
954
KLF
6 K
rupp
el-li
ke fa
ctor
6
5.5
1.9
.00
1.2
1.3
ns
1.0
1.0
ns
3096
96
BC
R
brea
kpoi
nt c
lust
er re
gion
5.
5 1.
1 .0
0 1.
0 1.
0 ns
1.
2 1.
2 ns
31
3806
R
NF1
9B
ring
finge
r pro
tein
19B
5.
4 1.
1 .0
0 1.
0 1.
0 ns
1.
3 0.
9 ns
64
195
MG
L1
mac
roph
age
gala
ctos
e N
-ace
tyl-g
alac
tosa
min
e sp
ecifi
c le
ctin
1
5.3
1.7
.00
1.8
1.2
ns
1.6
1.2
ns
3035
19
KR
T25
kera
tin 2
5 1.
0 1.
0 ns
3.
1 1.
0 ns
5.
2 1.
5 0.
02
3623
36
FAM
180A
fa
mily
with
seq
uenc
e si
mila
rity
180,
mem
ber A
0.
2 1.
0 .0
0 2.
1 1.
4 ns
1.
3 1.
4 ns
49
8276
LO
C49
8276
Fc
gam
ma
rece
ptor
II b
eta
5.1
1.7
.00
1.3
0.9
ns
1.5
1.1
ns
1710
71
PP
P1R
15A
pr
otei
n ph
osph
atas
e 1,
regu
lato
ry (i
nhib
itor)
sub
unit
15A
4.
9 1.
3 .0
0 0.
8 0.
9 ns
0.
9 1.
0 ns
29
187
CD
69
Cd6
9 m
olec
ule
4.9
1.1
.00
0.7
1.0
ns
0.4
0.6
ns
4943
44
IER
2 im
med
iate
ear
ly re
spon
se 2
4.
7 1.
3 .0
0 0.
8 0.
9 ns
1.
0 0.
9 ns
30
4663
LY
L1
lym
phob
last
ic le
ukem
ia d
eriv
ed s
eque
nce
1 0.
2 1.
0 .0
0 1.
2 0.
8 ns
1.
3 1.
0 ns
30
9452
N
FKB
2 nu
clea
r fac
tor o
f kap
pa li
ght p
olyp
eptid
e ge
ne e
nhan
cer i
n B
-cel
ls 2
, p4
9/p1
00
4.6
1.3
.00
1.1
1.1
ns
1.0
1.1
ns
5006
23
RG
D15
6370
1 si
mila
r to
BC
0682
81 p
rote
in
0.2
0.7
.00
0.9
0.9
ns
1.1
0.7
ns
2433
4 E
NO
2 en
olas
e 2,
gam
ma,
neu
rona
l 4.
6 1.
6 .0
0 1.
2 1.
2 ns
1.
6 1.
2 ns
36
2491
R
IPK
2 re
cept
or-in
tera
ctin
g se
rine-
thre
onin
e ki
nase
2
4.6
1.4
.00
1.3
1.2
ns
1.3
1.2
ns
4052
97
OLR
114
olfa
ctor
y re
cept
or 1
14
1.3
1.0
ns
1.0
1.0
ns
4.4
1.0
0.04
25
426
CY
P1B
1 cy
toch
rom
e P
450,
fam
ily 1
, sub
fam
ily b
, pol
ypep
tide
1 4.
4 1.
4 .0
0 0.
7 0.
8 ns
0.
5 0.
5 ns
30
7351
TU
BB
6 tu
bulin
, bet
a 6
4.4
1.5
.02
1.3
1.3
ns
1.1
0.8
ns
2491
4 LO
X
lysy
l oxi
dase
4.
3 1.
4 .0
0 1.
0 1.
0 ns
1.
0 0.
9 ns
29
7902
G
EM
G
TP b
indi
ng p
rote
in (g
ene
over
expr
esse
d in
ske
leta
l mus
cle)
4.
3 1.
7 .0
0 1.
6 1.
4 ns
0.
8 0.
9 ns
31
6019
C
CR
L2
chem
okin
e (C
-C m
otif)
rece
ptor
-like
2
4.3
0.9
.00
0.7
0.5
ns
0.5
0.9
ns
2909
11
RG
D15
6480
7 si
mila
r to
zinc
fing
er p
rote
in, s
ubfa
mily
1A
, 5
1.4
1.1
ns
4.3
1.0
0.01
1.
2 1.
0 ns
28
8239
D
SC
R6
Dow
n sy
ndro
me
criti
cal r
egio
n ho
mol
og 6
0.
2 0.
8 .0
0 0.
9 1.
0 ns
0.
9 1.
0 ns
17
1099
R
AS
D2
RA
SD
fam
ily, m
embe
r 2
0.2
0.9
.03
1.0
0.9
ns
0.9
1.0
ns
1408
60
SLC
O2B
1 so
lute
car
rier o
rgan
ic a
nion
tran
spor
ter f
amily
, mem
ber 2
b1
0.2
0.6
.00
1.1
0.9
ns
1.2
1.2
ns
Supplements 143
!
1170
29
CC
R5
chem
okin
e (C
-C m
otif)
rece
ptor
5
4.1
1.0
.01
1.3
1.2
ns
1.2
1.2
ns
4981
79
RG
D15
6348
2 si
mila
r to
hypo
thet
ical
pro
tein
FLJ
3866
3 0.
2 0.
6 .0
0 0.
9 0.
8 ns
1.
0 0.
9 ns
50
2018
R
GD
1563
728
sim
ilar t
o co
iled-
coil-
helix
-coi
led-
coil-
helix
dom
ain
cont
aini
ng 2
1.
2 1.
0 ns
4.
0 1.
0 0.
04
1.0
1.0
ns
3037
98
AR
VC
F ar
mad
illo
repe
at g
ene
dele
ted
in v
elo-
card
io-fa
cial
syn
drom
e 0.
3 0.
6 .0
0 0.
8 0.
7 ns
1.
0 1.
0 ns
36
3140
R
AS
SF1
R
as a
ssoc
iatio
n (R
alG
DS
/AF-
6) d
omai
n m
embe
r 1
4.0
1.5
.00
0.9
1.0
ns
0.8
1.0
ns
2940
07
PP
RC
1 pe
roxi
som
e pr
olife
rato
r-ac
tivat
ed re
cept
or g
amm
a, c
oact
ivat
or-r
elat
ed 1
4.
0 1.
8 .0
0 0.
9 1.
2 ns
1.
1 1.
2 ns
29
4235
IE
R3
imm
edia
te e
arly
resp
onse
3
3.9
1.0
.00
0.9
1.1
ns
1.2
1.1
ns
5026
57
RG
D15
6356
5 si
mila
r to
cyto
chro
me
c ox
idas
e su
buni
t IV
0.
7 0.
9 ns
0.
7 0.
7 ns
3.
9 0.
7 0.
00
3137
22
SP
SB
1 sp
lA/ry
anod
ine
rece
ptor
dom
ain
and
SO
CS
box
con
tain
ing
1 3.
9 1.
7 .0
0 1.
6 1.
5 ns
1.
5 1.
3 ns
49
7886
C
1QTN
F2
C1q
and
tum
or n
ecro
sis
fact
or re
late
d pr
otei
n 2
0.3
0.8
.00
0.9
1.0
ns
1.1
1.0
ns
2542
9 C
YP
7B1
cyto
chro
me
P45
0, fa
mily
7, s
ubfa
mily
b, p
olyp
eptid
e 1
3.8
0.8
.00
0.9
1.0
ns
1.0
0.9
ns
2882
57
DO
NS
ON
do
wns
tream
nei
ghbo
r of S
ON
3.
8 1.
3 .0
0 1.
5 1.
7 ns
1.
4 1.
4 ns
11
4856
D
US
P1
dual
spe
cific
ity p
hosp
hata
se 1
3.
8 1.
5 .0
0 0.
8 1.
0 ns
1.
1 0.
9 ns
30
7811
S
LC7A
6 so
lute
car
rier f
amily
7 (c
atio
nic
amin
o ac
id tr
ansp
orte
r, y+
sys
tem
), m
embe
r 6
3.7
1.3
.00
1.0
1.0
ns
1.0
0.8
ns
3091
75
CD
C42
EP
2 C
DC
42 e
ffect
or p
rote
in (R
ho G
TPas
e bi
ndin
g) 2
3.
7 1.
5 .0
1 1.
2 1.
2 ns
1.
5 1.
1 ns
24
423
GS
TM1
glut
athi
one
S-tr
ansf
eras
e m
u 1
1.5
0.9
ns
3.6
1.1
0.00
0.
9 0.
7 ns
60
350
CD
14
CD
14 m
olec
ule
3.6
1.5
.00
1.2
1.0
ns
1.0
0.9
ns
2991
45
RH
OJ
ras
hom
olog
gen
e fa
mily
, mem
ber J
3.
6 1.
5 .0
0 0.
9 0.
8 ns
1.
1 0.
9 ns
28
9443
LR
RC
8C
leuc
ine
rich
repe
at c
onta
inin
g 8
fam
ily, m
embe
r C
3.6
1.0
.04
1.0
0.9
ns
0.6
0.8
ns
2511
2 G
AD
D45
A
grow
th a
rres
t and
DN
A-d
amag
e-in
duci
ble,
alp
ha
3.6
1.3
.00
0.8
1.0
ns
1.0
1.0
ns
2417
3 A
DR
A1B
ad
rene
rgic
, alp
ha-1
B-,
rece
ptor
0.
3 0.
6 .0
0 0.
9 1.
0 ns
0.
8 1.
0 ns
29
1908
TO
X3
TOX
hig
h m
obili
ty g
roup
box
fam
ily m
embe
r 3
0.3
0.6
.01
0.7
0.9
ns
0.7
0.8
ns
3105
53
TLR
2 to
ll-lik
e re
cept
or 2
3.
4 1.
1 .0
0 0.
8 0.
7 ns
0.
9 0.
8 ns
36
4206
P
LEK
pl
ecks
trin
3.4
1.4
.00
1.3
1.1
ns
1.1
1.0
ns
6383
9 FH
L2
four
and
a h
alf L
IM d
omai
ns 2
0.
3 0.
7 .0
1 1.
1 0.
8 ns
0.
9 0.
9 ns
28
7129
TM
EM
204
trans
mem
bran
e pr
otei
n 20
4 0.
3 0.
8 .0
0 1.
1 1.
0 ns
1.
0 1.
1 ns
29
7508
N
AM
PT
nico
tinam
ide
phos
phor
ibos
yltra
nsfe
rase
3.
4 1.
3 .0
1 1.
2 1.
6 ns
1.
1 1.
2 ns
11
4121
C
CN
L1
cycl
in L
1 3.
4 1.
7 .0
0 1.
3 1.
1 ns
1.
4 1.
4 ns
36
1993
R
GD
1562
059
sim
ilar t
o R
IKE
N c
DN
A 1
1100
38F2
1 0.
3 0.
6 .0
1 0.
6 0.
7 ns
0.
7 1.
0 ns
30
6330
K
LF2
Kru
ppel
-like
fact
or 2
0.
3 0.
6 .0
0 1.
3 1.
1 ns
0.
9 1.
1 ns
25
7634
LH
X1
LIM
hom
eobo
x 1
0.3
0.8
.00
0.8
0.9
ns
0.9
1.0
ns
8152
0 M
AR
CK
SL1
M
AR
CK
S-li
ke 1
3.
3 1.
3 .0
1 1.
0 0.
8 ns
1.
5 0.
8 ns
84
398
CD
93
CD
93 m
olec
ule
3.3
1.2
.00
0.7
0.7
ns
1.0
0.9
ns
1711
64
GB
P2
guan
ylat
e bi
ndin
g pr
otei
n 2
3.3
0.8
.01
1.2
1.1
ns
1.1
0.7
ns
7943
1 B
HLH
E40
ba
sic
helix
-loop
-hel
ix fa
mily
, mem
ber e
40
3.3
1.7
.00
1.2
1.0
ns
1.1
1.0
ns
3054
23
C1Q
TNF7
C
1q a
nd tu
mor
nec
rosi
s fa
ctor
rela
ted
prot
ein
7 0.
3 0.
8 .0
0 1.
3 1.
1 ns
1.
1 1.
3 ns
29
3023
K
LHL2
5 ke
lch-
like
25
0.3
1.0
.04
1.1
0.6
ns
0.6
0.8
ns
7923
7 H
HE
X
hem
atop
oiet
ical
ly e
xpre
ssed
hom
eobo
x 0.
3 1.
2 .0
0 1.
0 1.
0 ns
1.
1 1.
0 ns
36
2564
B
EN
D5
BE
N d
omai
n co
ntai
ning
5
0.3
0.7
.00
0.7
0.8
ns
1.1
1.2
ns
3049
54
UA
P1L
2 U
DP
-N-a
ctey
lglu
cosa
min
e py
roph
osph
oryl
ase
1-lik
e 2
3.1
1.3
.00
0.7
1.0
ns
1.1
0.9
ns
3135
95
RG
D15
6307
2 si
mila
r to
hypo
thet
ical
pro
tein
FLJ
3898
4 0.
3 1.
1 .0
0 1.
2 1.
2 ns
1.
4 1.
2 ns
36
2703
W
DR
43
WD
repe
at d
omai
n 43
3.
1 1.
8 .0
0 1.
0 1.
2 ns
1.
0 1.
0 ns
144 Supplement to Chapter 4
!
3142
43
WD
R89
W
D re
peat
dom
ain
89
0.3
0.7
.00
0.9
0.9
ns
1.0
1.0
ns
2520
2 G
UC
Y1B
3 gu
anyl
ate
cycl
ase
1, s
olub
le, b
eta
3 0.
3 0.
7 .0
2 1.
0 0.
9 ns
0.
9 0.
8 ns
30
1416
C
OQ
10B
co
enzy
me
Q10
hom
olog
B
3.1
1.5
.00
1.0
1.2
ns
1.0
0.9
ns
2953
23
TRIM
45
tripa
rtite
mot
if-co
ntai
ning
45
0.3
0.6
.00
1.1
1.0
ns
1.2
1.0
ns
2469
7 P
TPN
1 pr
otei
n ty
rosi
ne p
hosp
hata
se, n
on-r
ecep
tor t
ype
1 3.
0 1.
3 .0
0 1.
0 1.
0 ns
1.
1 1.
2 ns
14
0942
D
DIT
4 D
NA
-dam
age-
indu
cibl
e tra
nscr
ipt 4
3.
0 1.
0 .0
4 1.
9 1.
2 ns
1.
1 1.
2 ns
31
3436
ZC
CH
C12
zi
nc fi
nger
, CC
HC
dom
ain
cont
aini
ng 1
2 3.
0 1.
3 .0
0 0.
6 1.
1 ns
1.
2 1.
3 ns
11
4495
M
AP
2K6
mito
gen-
activ
ated
pro
tein
kin
ase
kina
se 6
0.
3 0.
6 .0
0 1.
1 1.
1 ns
1.
0 1.
0 ns
29
9411
LO
C29
9411
si
mila
r to
Ig h
eavy
cha
in V
regi
on M
OP
C 2
1 pr
ecur
sor
1.0
1.3
ns
1.0
1.0
ns
3.0
1.3
0.00
65
161
LITA
F lip
opol
ysac
char
ide-
indu
ced
TNF
fact
or
3.0
1.5
.00
1.2
1.3
ns
1.1
1.1
ns
8458
3 R
GS
2 re
gula
tor o
f G-p
rote
in s
igna
ling
2 3.
0 1.
1 .0
0 1.
2 1.
3 ns
1.
1 1.
1 ns
24
6760
M
AFK
v-
maf
mus
culo
apon
euro
tic fi
bros
arco
ma
onco
gene
hom
olog
K (a
vian
) 2.
9 1.
7 .0
0 1.
0 1.
3 ns
1.
1 1.
2 ns
79
255
ATF
4 ac
tivat
ing
trans
crip
tion
fact
or 4
(tax
-res
pons
ive
enha
ncer
ele
men
t B67
) 2.
9 1.
7 .0
0 0.
9 1.
1 ns
0.
9 0.
9 ns
24
5963
E
GFL
7 E
GF-
like-
dom
ain,
mul
tiple
7
0.3
1.2
.01
0.8
0.9
ns
1.0
1.0
ns
3005
19
ES
AM
en
doth
elia
l cel
l adh
esio
n m
olec
ule
2.9
1.5
.00
1.0
1.0
ns
1.3
1.1
ns
3641
36
AA
SD
H
amin
oadi
pate
-sem
iald
ehyd
e de
hydr
ogen
ase
0.4
0.9
.00
1.3
0.9
ns
1.1
1.3
ns
3081
13
CN
KS
R3
Cnk
sr fa
mily
mem
ber 3
2.
8 1.
3 .0
1 0.
8 0.
9 ns
1.
1 1.
1 ns
11
7540
P
LSC
R1
phos
phol
ipid
scr
ambl
ase
1 2.
8 0.
7 .0
1 1.
1 1.
3 ns
1.
1 1.
1 ns
83
626
UG
CG
U
DP
-glu
cose
cer
amid
e gl
ucos
yltra
nsfe
rase
2.
8 0.
9 .0
0 0.
8 1.
0 ns
1.
0 1.
0 ns
25
472
KC
NJ8
po
tass
ium
inw
ardl
y-re
ctify
ing
chan
nel,
subf
amily
J, m
embe
r 8
2.8
1.1
.01
1.9
1.1
ns
1.2
1.1
ns
1140
90
EG
R2
early
gro
wth
resp
onse
2
2.8
1.0
.00
1.0
0.9
ns
0.8
0.9
ns
2562
5 TN
FRS
F1A
tu
mor
nec
rosi
s fa
ctor
rece
ptor
sup
erfa
mily
, mem
ber 1
a 2.
8 1.
4 .0
0 0.
9 1.
3 ns
0.
9 1.
0 ns
25
655
GJA
4 ga
p ju
nctio
n pr
otei
n, a
lpha
4
2.8
1.0
.03
1.1
0.8
ns
1.1
1.1
ns
2930
8 A
RL4
A
AD
P-r
ibos
ylat
ion
fact
or-li
ke 4
A
2.8
1.8
.00
1.2
1.3
ns
1.0
1.0
ns
1165
01
SLC
9A3R
2 so
lute
car
rier f
amily
9 (s
odiu
m/h
ydro
gen
exch
ange
r), m
embe
r 3 re
gula
tor 2
0.
4 0.
8 .0
1 1.
1 1.
0 ns
1.
0 1.
2 ns
17
0929
B
CL2
A1D
B
-cel
l leu
kem
ia/ly
mph
oma
2 re
late
d pr
otei
n A
1d
2.7
1.3
.00
1.0
1.0
ns
1.0
0.9
ns
3047
35
TME
M17
7 tra
nsm
embr
ane
prot
ein
177
0.4
0.9
.02
1.0
1.0
ns
1.0
1.0
ns
3063
38
AB
HD
8 ab
hydr
olas
e do
mai
n co
ntai
ning
8
0.4
0.8
.00
0.9
0.8
ns
0.8
0.8
ns
2906
48
PG
PE
P1
pyro
glut
amyl
-pep
tidas
e I
0.4
1.1
.01
1.0
1.0
ns
0.9
1.0
ns
3608
78
KLH
DC
9 ke
lch
dom
ain
cont
aini
ng 9
0.
4 0.
7 .0
0 0.
9 0.
9 ns
1.
1 1.
1 ns
30
2937
A
NK
S3
anky
rin re
peat
and
ste
rile
alph
a m
otif
dom
ain
cont
aini
ng 3
0.
4 0.
8 .0
1 1.
1 0.
8 ns
0.
8 0.
8 ns
50
672
ED
NR
B
endo
thel
in re
cept
or ty
pe B
2.
6 1.
8 .0
0 1.
0 1.
1 ns
1.
1 1.
3 ns
29
3511
ZN
F688
zi
nc fi
nger
pro
tein
688
0.
4 0.
7 .0
0 0.
8 0.
8 ns
0.
8 0.
9 ns
24
6311
P
DP
2 py
ruva
te d
ehyd
roge
nase
pho
spha
tase
isoe
nzym
e 2
0.4
0.8
.04
0.8
0.8
ns
0.9
0.9
ns
2936
69
CD
248
CD
248
mol
ecul
e, e
ndos
ialin
0.
4 0.
9 .0
0 1.
3 1.
1 ns
0.
9 1.
0 ns
24
516
JUN
Ju
n on
coge
ne
2.6
1.2
.00
0.8
1.0
ns
0.9
0.9
ns
2462
08
IFI4
7 in
terfe
ron
gam
ma
indu
cibl
e pr
otei
n 47
2.
6 1.
0 .0
0 1.
1 0.
8 ns
0.
8 0.
8 ns
31
4228
S
NA
PC
1 sm
all n
ucle
ar R
NA
act
ivat
ing
com
plex
, pol
ypep
tide
1 2.
6 1.
2 .0
0 0.
8 1.
1 ns
0.
9 1.
0 ns
49
7832
TR
PC
1 tra
nsie
nt re
cept
or p
oten
tial c
atio
n ch
anne
l, su
bfam
ily C
, mem
ber 1
2.
5 1.
1 .0
1 1.
0 0.
9 ns
1.
1 1.
2 ns
60
341
CA
MK
K1
calc
ium
/cal
mod
ulin
-dep
ende
nt p
rote
in k
inas
e ki
nase
1, a
lpha
0.
4 1.
0 .0
0 1.
1 1.
1 ns
1.
0 1.
1 ns
30
2642
S
AT1
sp
erm
idin
e/sp
erm
ine
N1-
acet
yl tr
ansf
eras
e 1
2.5
1.6
.00
1.1
1.3
ns
0.9
1.0
ns
4983
86
RG
D15
6104
2 si
mila
r to
RIK
EN
cD
NA
573
0509
K17
gen
e 0.
4 0.
8 .0
0 1.
1 1.
1 ns
1.
1 1.
1 ns
24
165
AD
A
aden
osin
e de
amin
ase
0.4
1.5
.00
0.7
0.6
ns
0.7
0.7
ns
Supplements 145
!
3141
26
BA
Z1A
br
omod
omai
n ad
jace
nt to
zin
c fin
ger d
omai
n, 1
A
2.5
1.4
.00
1.1
1.1
ns
0.9
0.9
ns
2945
18
SE
SN
1 se
strin
1
0.4
0.8
.00
0.9
0.9
ns
0.9
0.9
ns
3127
28
TSP
AN
9 te
trasp
anin
9
0.4
0.8
.01
1.0
0.7
ns
0.8
0.9
ns
3132
62
PA
PP
A
preg
nanc
y-as
soci
ated
pla
sma
prot
ein
A
2.5
1.1
.00
0.8
1.0
ns
1.1
1.1
ns
3612
26
CD
83
CD
83 m
olec
ule
2.5
1.2
.00
0.9
0.9
ns
0.7
0.9
ns
3151
19
MFN
G
MFN
G O
-fuco
sylp
eptid
e 3-
beta
-N-a
cety
lglu
cosa
min
yltra
nsfe
rase
0.
4 0.
8 .0
0 1.
5 1.
1 ns
1.
1 1.
0 ns
68
3385
LO
C68
8922
si
mila
r to
Insu
lin-in
duce
d ge
ne 1
pro
tein
(IN
SIG
-1) (
Insu
lin-in
duce
d gr
owth
re
spon
se p
rote
in C
L-6)
(Im
med
iate
-ear
ly p
rote
in C
L-6)
2.
5 1.
1 .0
1 1.
1 0.
8 ns
0.
8 0.
7 ns
3098
28
TSP
YL4
TS
PY
-like
4
0.4
0.7
.01
0.8
1.0
ns
1.0
1.0
ns
6502
6 R
WD
D3
RW
D d
omai
n co
ntai
ning
3
0.4
0.8
.00
1.0
0.9
ns
1.0
1.1
ns
3608
72
RC
SD
1 R
CS
D d
omai
n co
ntai
ning
1
0.4
0.7
.01
0.9
0.7
ns
0.9
0.9
ns
4982
72
UA
P1
UD
P-N
-act
eylg
luco
sam
ine
pyro
phos
phor
ylas
e 1
2.4
1.0
.01
0.9
0.8
ns
0.9
0.8
ns
2569
2 P
LAT
plas
min
ogen
act
ivat
or, t
issu
e 2.
4 1.
6 .0
0 0.
8 1.
0 ns
1.
1 0.
9 ns
36
2762
D
CA
F4
DD
B1
and
CU
L4 a
ssoc
iate
d fa
ctor
4
0.4
0.9
.01
0.8
0.9
ns
1.1
0.9
ns
2478
3 S
LC9A
2 so
lute
car
rier f
amily
9 (s
odiu
m/h
ydro
gen
exch
ange
r), m
embe
r 2
0.4
0.7
.01
0.7
1.0
ns
1.0
0.9
ns
6812
52
LOC
6812
52
sim
ilar t
o M
yris
toyl
ated
ala
nine
-ric
h C
-kin
ase
subs
trate
(MA
RC
KS
) (P
rote
in
kina
se C
sub
stra
te 8
0 kD
a pr
otei
n)
2.4
0.5
.02
1.0
1.0
ns
1.1
0.9
ns
2934
4 ZF
P36
L1
zinc
fing
er p
rote
in 3
6, C
3H ty
pe-li
ke 1
2.
3 1.
0 .0
3 0.
9 1.
3 ns
1.
0 1.
1 ns
30
1227
TR
EM
2 tri
gger
ing
rece
ptor
exp
ress
ed o
n m
yelo
id c
ells
2
0.7
0.6
ns
2.3
0.9
0.00
1.
7 1.
3 ns
29
618
BTG
1 B
-cel
l tra
nslo
catio
n ge
ne 1
, ant
i-pro
lifer
ativ
e 2.
3 1.
2 .0
0 1.
0 1.
0 ns
1.
0 1.
0 ns
31
1807
B
MY
C
brai
n ex
pres
sed
mye
locy
tom
atos
is o
ncog
ene
0.4
0.7
.00
0.9
0.9
ns
1.1
0.9
ns
3072
35
RG
D13
1191
0 si
mila
r to
hypo
thet
ical
p38
pro
tein
0.
4 0.
9 .0
4 0.
7 0.
7 ns
0.
8 0.
9 ns
36
5571
TY
SN
D1
tryps
in d
omai
n co
ntai
ning
1
0.4
0.8
.01
1.0
0.9
ns
1.0
1.0
ns
8533
2 P
RK
CD
BP
pr
otei
n ki
nase
C, d
elta
bin
ding
pro
tein
0.
4 0.
9 .0
0 1.
1 0.
8 ns
0.
9 0.
8 ns
30
4346
M
BLA
C1
met
allo
-bet
a-la
ctam
ase
dom
ain
cont
aini
ng 1
0.
4 0.
9 .0
1 0.
9 0.
8 ns
0.
9 0.
8 ns
50
0150
R
GD
1559
612
sim
ilar t
o tig
ger t
rans
posa
ble
elem
ent d
eriv
ed 2
0.
4 0.
8 .0
0 0.
8 0.
8 ns
0.
8 1.
0 ns
30
8336
ZN
F580
zi
nc fi
nger
pro
tein
580
0.
8 0.
5 ns
0.
4 1.
1 0.
04
1.0
0.9
ns
3099
18
ELL
2 el
onga
tion
fact
or R
NA
pol
ymer
ase
II 2
2.2
1.2
.00
1.0
1.1
ns
1.2
1.1
ns
5896
6 R
AM
P2
rece
ptor
(G p
rote
in-c
oupl
ed) a
ctiv
ity m
odify
ing
prot
ein
2 0.
5 1.
1 .0
0 1.
4 1.
1 ns
1.
1 1.
0 ns
63
997
SLC
29A
1 so
lute
car
rier f
amily
29
(nuc
leos
ide
trans
porte
rs),
mem
ber 1
0.
5 0.
8 .0
5 0.
8 0.
8 ns
0.
8 0.
9 ns
30
1059
M
YD
88
mye
loid
diff
eren
tiatio
n pr
imar
y re
spon
se g
ene
88
2.2
1.0
.01
1.2
1.2
ns
1.2
1.0
ns
3032
00
MA
P2K
3 m
itoge
n ac
tivat
ed p
rote
in k
inas
e ki
nase
3
2.2
1.3
.02
1.1
1.2
ns
1.0
1.1
ns
2889
12
MR
I1
met
hylth
iorib
ose-
1-ph
osph
ate
isom
eras
e ho
mol
og
0.5
0.9
.01
0.9
0.8
ns
1.0
1.0
ns
2871
48
RP
US
D1
RN
A p
seud
ourid
ylat
e sy
ntha
se d
omai
n 1
0.5
0.8
.01
0.8
0.8
ns
0.9
1.0
ns
2869
88
PN
RC
1 pr
olin
e-ric
h nu
clea
r rec
epto
r coa
ctiv
ator
1
2.1
1.2
.00
0.8
0.8
ns
0.8
0.8
ns
4992
14
CH
CH
D8
coile
d-co
il-he
lix-c
oile
d-co
il-he
lix d
omai
n 8
0.5
0.9
.03
1.1
1.0
ns
0.8
0.9
ns
2961
19
DTW
D1
DTW
dom
ain
cont
aini
ng 1
0.
5 0.
8 .0
0 0.
9 1.
0 ns
1.
1 1.
1 ns
30
7907
R
GD
1304
884
sim
ilar t
o R
IKE
N c
DN
A 6
4305
48M
08
0.5
1.0
.00
1.1
1.1
ns
1.1
1.1
ns
3135
60
CTP
S
CTP
syn
thas
e 2.
1 1.
0 .0
1 0.
8 0.
8 ns
0.
8 0.
8 ns
49
9630
R
GD
1565
059
sim
ilar t
o hy
poth
etic
al p
rote
in E
1303
11K
13
0.5
0.9
.00
1.3
1.1
ns
1.1
1.2
ns
3065
87
TCTA
T-
cell
leuk
emia
tran
sloc
atio
n al
tere
d ge
ne
0.5
0.8
.00
1.0
0.9
ns
0.8
0.9
ns
8438
6 S
LPI
secr
etor
y le
ukoc
yte
pept
idas
e in
hibi
tor
2.1
0.8
.01
1.1
0.7
ns
0.8
0.7
ns
6037
3 N
OP
58
nucl
eola
r pro
tein
NO
P58
2.
1 1.
2 .0
0 0.
8 1.
3 ns
1.
0 1.
0 ns
146 Supplement to Chapter 4
!
2553
7 R
OC
K2
Rho
-ass
ocia
ted
coile
d-co
il co
ntai
ning
pro
tein
kin
ase
2 2.
1 1.
3 .0
0 1.
1 1.
6 ns
1.
4 1.
6 ns
28
7063
N
MR
AL1
N
mrA
-like
fam
ily d
omai
n co
ntai
ning
1
0.5
0.7
.00
0.8
0.8
ns
0.9
1.1
ns
3108
11
PA
LMD
pa
lmde
lphi
n 0.
5 1.
0 .0
1 0.
9 1.
0 ns
1.
0 0.
9 ns
30
9109
TM
EM
80
trans
mem
bran
e pr
otei
n 80
0.
5 0.
9 .0
1 0.
9 0.
9 ns
0.
9 1.
0 ns
29
9207
R
GD
1310
769
sim
ilar t
o H
SP
C28
8 0.
5 0.
9 .0
0 1.
1 1.
1 ns
1.
1 1.
1 ns
30
5351
K
LHL5
ke
lch-
like
5
0.5
0.9
.00
1.0
0.9
ns
0.9
0.9
ns
3035
77
AC
BD
4 ac
yl-C
oenz
yme
A b
indi
ng d
omai
n co
ntai
ning
4
0.5
0.9
.04
0.9
0.8
ns
1.0
1.0
ns
5001
26
HO
XA
9 ho
meo
box
A9
0.5
0.9
.00
1.0
1.0
ns
0.9
1.0
ns
2919
75
RG
D13
0735
7 si
mila
r to
hypo
thet
ical
pro
tein
DK
FZp4
34A
1319
0.
5 0.
9 .0
0 1.
2 1.
1 ns
1.
1 1.
3 ns
29
1661
TM
CO
6 tra
nsm
embr
ane
and
coile
d-co
il do
mai
ns 6
0.
5 1.
0 .0
1 1.
3 1.
2 ns
0.
9 1.
3 ns
49
9085
P
ELI
1
pelli
no 1
2.
0 1.
3 .0
0 1.
0 1.
3 ns
1.
1 1.
1 ns
25
675
HM
GC
R
3-hy
drox
y-3-
met
hylg
luta
ryl-C
oenz
yme
A re
duct
ase
2.0
0.9
.00
1.0
0.9
ns
0.9
0.9
ns
3145
84
ZFP
563
zinc
fing
er p
rote
in 5
63
0.5
0.8
.00
1.1
1.1
ns
0.9
1.0
ns
3636
76
ZPB
P2
zona
pel
luci
da b
indi
ng p
rote
in 2
0.
5 0.
7 .0
0 1.
1 1.
0 ns
1.
2 1.
1 ns
58
840
MA
PK
6 m
itoge
n-ac
tivat
ed p
rote
in k
inas
e 6
1.9
1.1
.00
0.8
0.9
ns
1.1
1.0
ns
3036
12
PO
LG2
poly
mer
ase
(DN
A d
irect
ed),
gam
ma
2, a
cces
sory
sub
unit
0.5
1.1
.00
1.2
1.1
ns
1.0
1.1
ns
3604
61
RG
D13
0582
3 si
mila
r to
RIK
EN
cD
NA
061
0037
P05
0.
5 0.
9 .0
1 0.
9 1.
0 ns
1.
1 0.
9 ns
28
7427
TR
AP
PC
1 tra
ffick
ing
prot
ein
parti
cle
com
plex
1
0.5
0.9
.02
1.0
0.8
ns
0.8
0.9
ns
8434
8 C
XC
R7
chem
okin
e (C
-X-C
mot
if) re
cept
or 7
1.
9 0.
9 .0
0 0.
8 0.
8 ns
0.
8 0.
8 ns
26
6773
ZF
P70
9 zi
nc fi
nger
pro
tein
709
0.
5 1.
1 .0
0 1.
3 1.
1 ns
1.
3 1.
5 ns
28
2845
R
NF3
4 rin
g fin
ger p
rote
in 3
4 0.
5 0.
8 .0
0 1.
1 1.
1 ns
0.
9 1.
0 ns
29
6565
R
GD
1306
215
sim
ilar t
o hy
poth
etic
al p
rote
in M
GC
3683
1 0.
5 0.
9 .0
0 0.
9 0.
8 ns
0.
8 0.
7 ns
29
9639
A
NK
RD
24
anky
rin re
peat
dom
ain
24
0.5
0.9
.04
1.0
0.9
ns
0.8
1.0
ns
3606
47
ICA
M2
inte
rcel
lula
r adh
esio
n m
olec
ule
2 0.
5 1.
0 .0
0 1.
1 1.
0 ns
1.
1 1.
1 ns
36
0754
ZF
P35
8 zi
nc fi
nger
pro
tein
358
0.
5 1.
1 .0
0 1.
0 1.
0 ns
1.
0 1.
0 ns
49
7782
A
BC
B1A
A
TP-b
indi
ng c
asse
tte, s
ub-fa
mily
B (M
DR
/TA
P),
mem
ber 1
A
1.4
0.7
ns
0.5
1.0
0.00
1.
3 1.
0 ns
28
8651
G
TF2H
3 ge
nera
l tra
nscr
iptio
n fa
ctor
IIH
, pol
ypep
tide
3 0.
5 1.
0 .0
1 1.
0 1.
1 ns
0.
9 0.
9 ns
29
534
PX
MP
3 pe
roxi
som
al m
embr
ane
prot
ein
3 0.
5 0.
9 .0
5 0.
9 0.
9 ns
0.
9 0.
8 ns
49
7745
LS
S
lano
ster
ol s
ynth
ase
(2,3
-oxi
dosq
uale
ne-la
nost
erol
cyc
lase
) 1.
8 1.
0 .0
2 0.
9 1.
0 ns
1.
0 1.
0 ns
31
1723
S
OX
18
SR
Y (s
ex d
eter
min
ing
regi
on Y
)-bo
x 18
0.
5 1.
7 .0
1 1.
2 0.
9 ns
1.
1 1.
0 ns
83
614
PIA
S3
prot
ein
inhi
bito
r of a
ctiv
ated
STA
T, 3
0.
6 0.
8 .0
1 0.
9 0.
9 ns
1.
0 0.
9 ns
28
9150
C
EN
PL
cent
rom
ere
prot
ein
L 0.
6 0.
8 .0
0 1.
1 1.
0 ns
0.
9 0.
9 ns
30
9446
H
PS
6 H
erm
ansk
y-P
udla
k sy
ndro
me
6 0.
6 1.
2 .0
0 1.
0 1.
0 ns
1.
0 0.
9 ns
84
019
NA
E1
NE
DD
8 ac
tivat
ing
enzy
me
E1
subu
nit 1
0.
6 0.
9 .0
0 1.
0 0.
9 ns
1.
0 1.
0 ns
64
134
XY
LT2
xylo
syltr
ansf
eras
e II
0.6
0.8
.00
0.9
1.0
ns
1.0
1.1
ns
2976
27
MG
C94
282
sim
ilar t
o 59
3041
6I19
Rik
pro
tein
0.
6 0.
9 .0
0 1.
0 1.
1 ns
1.
1 1.
1 ns
36
5314
LI
PT2
lip
oyl(o
ctan
oyl)
trans
fera
se 2
(put
ativ
e)
0.6
1.0
.01
1.0
0.9
ns
0.9
0.9
ns
2882
33
WR
B
trypt
opha
n ric
h ba
sic
prot
ein
0.6
0.9
.01
1.0
0.9
ns
0.9
0.9
ns
3111
85
KB
TBD
4 ke
lch
repe
at a
nd B
TB (P
OZ)
dom
ain
cont
aini
ng 4
0.
6 1.
0 .0
2 0.
9 0.
9 ns
0.
9 0.
9 ns
36
2576
R
GD
1305
347
sim
ilar t
o R
IKE
N c
DN
A 2
6105
28J1
1 0.
6 0.
8 .0
0 0.
9 1.
0 ns
1.
0 1.
1 ns
36
2667
TH
AP
3 TH
AP
dom
ain
cont
aini
ng, a
popt
osis
ass
. pro
tein
3
0.6
0.9
.01
1.0
1.0
ns
1.0
1.1
ns
2998
28
XR
CC
6BP
1 X
RC
C6
bind
ing
prot
ein
1 0.
6 0.
8 .0
1 1.
0 0.
8 ns
0.
9 1.
0 ns
36
3022
ZF
P42
6L2
zinc
fing
er p
rote
in 4
26-li
ke 2
0.
6 1.
0 .0
1 1.
1 1.
2 ns
1.
0 1.
0 ns
Supplements 147
!
2537
4 A
LAD
am
inol
evul
inat
e, d
elta
-, de
hydr
atas
e 0.
6 1.
0 .0
1 1.
0 0.
9 ns
1.
0 1.
1 ns
30
7395
A
BLI
M3
actin
bin
ding
LIM
pro
tein
fam
ily, m
embe
r 3
0.7
0.7
ns
0.6
0.9
0.02
0.
9 0.
9 ns
29
1927
O
RC
6L
orig
in re
cogn
ition
com
plex
, sub
unit
6 lik
e 0.
6 0.
9 .0
0 1.
1 1.
1 ns
0.
9 0.
9 ns
36
2425
ZF
P63
7 zi
nc fi
nger
pro
tein
637
0.
6 0.
9 .0
2 1.
0 1.
0 ns
0.
9 1.
0 ns
30
4396
LO
C30
4396
si
mila
r to
hypo
thet
ical
pro
tein
DK
FZp4
34K
1815
0.
6 1.
0 .0
1 1.
0 1.
0 ns
1.
1 1.
0 ns
30
5341
R
HO
H
ras
hom
olog
gen
e fa
mily
, mem
ber H
1.
7 0.
9 .0
4 0.
8 1.
0 ns
1.
1 1.
1 ns
36
7874
R
GD
1563
579
sim
ilar t
o 60
S ri
boso
mal
pro
tein
L29
(P23
) 0.
6 1.
1 .0
3 0.
9 0.
9 ns
0.
7 0.
9 ns
28
9468
FA
M17
5A
fam
ily w
ith s
eque
nce
sim
ilarit
y 17
5, m
embe
r A
0.6
1.0
.00
1.0
1.0
ns
1.1
1.1
ns
3614
53
DE
AD
C1
deam
inas
e do
mai
n co
ntai
ning
1
0.6
1.1
.01
1.2
1.3
ns
0.9
1.0
ns
5006
27
RG
D15
6280
1 si
mila
r to
RN
A, U
tran
spor
ter 1
0.
6 0.
8 .0
0 0.
9 1.
0 ns
1.
0 1.
0 ns
36
2849
M
AR
CH
2 m
embr
ane-
asso
ciat
ed ri
ng fi
nger
(C3H
C4)
2
0.6
1.0
.02
1.0
0.9
ns
1.0
1.0
ns
2954
58
BD
H2
3-hy
drox
ybut
yrat
e de
hydr
ogen
ase,
type
2
0.6
0.9
.01
1.2
0.9
ns
0.9
1.0
ns
3089
93
SE
PH
S2
sele
noph
osph
ate
synt
heta
se 2
0.
6 0.
8 .0
0 1.
0 0.
8 ns
0.
9 0.
9 ns
49
9561
FA
M17
3B
fam
ily w
ith s
eque
nce
sim
ilarit
y 17
3, m
embe
r B
0.6
1.1
.02
1.3
1.0
ns
0.8
1.1
ns
2980
75
NC
BP
1 nu
clea
r cap
bin
ding
pro
tein
sub
unit
1, 8
0kD
a 0.
6 0.
9 .0
4 1.
0 1.
0 ns
0.
8 1.
0 ns
68
6765
LO
C68
6765
si
mila
r to
CG
1428
6-P
A
0.6
0.9
.01
1.0
0.9
ns
0.9
0.9
ns
1714
11
TME
M17
6B
trans
mem
bran
e pr
otei
n 17
6B
0.6
0.8
.00
1.2
1.0
ns
1.0
0.9
ns
5004
11
RG
D15
6494
0 si
mila
r to
RIK
EN
cD
NA
300
0004
N20
0.
6 1.
1 .0
0 1.
1 1.
3 ns
1.
0 1.
2 ns
49
9900
LO
C49
9900
si
mila
r to
Zinc
fing
er p
rote
in 1
33
0.6
1.1
.02
1.0
1.1
ns
0.9
1.0
ns
3119
52
GA
LNT1
1 U
DP
-N-a
cety
l-alp
ha-D
-gal
acto
sam
ine:
pol
ypep
tide
N-
acet
ylga
lact
osam
inyl
trans
fera
se 1
1 (G
alN
Ac-
T11)
0.
6 1.
0 .0
5 1.
2 1.
0 ns
1.
0 1.
1 ns
8448
0 B
NIP
3 B
CL2
/ade
novi
rus
E1B
inte
ract
ing
prot
ein
3 1.
7 1.
2 .0
0 1.
1 1.
0 ns
0.
9 1.
0 ns
50
0533
D
MA
P1
DN
A m
ethy
ltran
sfer
ase
1-as
soci
ated
pro
tein
1
0.6
0.8
.00
0.9
0.9
ns
0.9
0.9
ns
2558
6 A
LPL
alka
line
phos
phat
ase,
live
r/bon
e/ki
dney
0.
6 0.
8 .0
0 1.
0 0.
9 ns
0.
8 0.
8 ns
31
4788
R
GD
1307
947
sim
ilar t
o R
IKE
N c
DN
A C
4300
08C
19
0.6
0.9
.01
0.9
0.9
ns
0.9
0.9
ns
6422
6 M
LST8
M
TOR
ass
ocia
ted
prot
ein,
LS
T8 h
omol
og
0.6
0.9
.00
0.9
0.9
ns
1.0
1.1
ns
6451
3 P
AW
R
PR
KC
, apo
ptos
is, W
T1, r
egul
ator
1.
7 1.
1 .0
0 1.
0 1.
0 ns
1.
0 1.
0 ns
30
2890
C
PP
ED
1 ca
lcin
eurin
-like
pho
spho
este
rase
dom
ain
cont
aini
ng 1
0.
6 0.
8 .0
0 0.
9 0.
8 ns
0.
9 0.
9 ns
29
6758
A
RM
C10
ar
mad
illo
repe
at c
onta
inin
g 10
0.
6 0.
9 .0
0 0.
9 0.
9 ns
0.
9 1.
0 ns
36
2793
R
GD
1307
315
LOC
3627
93
0.6
1.0
.00
1.0
1.1
ns
1.0
1.1
ns
6828
93
LYR
M2
LYR
mot
if co
ntai
ning
2
0.6
0.8
.00
1.0
1.0
ns
0.9
0.9
ns
3615
42
U2A
F1L4
U
2 sm
all n
ucle
ar R
NA
aux
iliar
y fa
ctor
1-li
ke 4
0.
6 0.
9 .0
2 1.
0 0.
9 ns
0.
9 0.
9 ns
36
2789
ZF
YV
E21
zi
nc fi
nger
, FY
VE
dom
ain
cont
aini
ng 2
1 0.
6 0.
9 .0
0 1.
1 1.
0 ns
1.
0 1.
0 ns
31
6626
H
ES
6 ha
iry a
nd e
nhan
cer o
f spl
it 6
0.
6 0.
9 .0
0 1.
4 1.
1 ns
1.
2 1.
1 ns
28
7456
M
ED
11
med
iato
r com
plex
sub
unit
11
0.6
0.9
.01
1.1
1.0
ns
1.1
1.0
ns
3613
01
RG
D13
1057
1 si
mila
r to
hypo
thet
ical
pro
tein
0.
6 0.
8 .0
1 0.
7 0.
8 ns
0.
7 0.
7 ns
31
6008
C
CD
C51
co
iled-
coil
dom
ain
cont
aini
ng 5
1 0.
6 1.
0 .0
5 0.
9 1.
0 ns
1.
0 0.
9 ns
28
7383
B
9D1
B9
prot
ein
dom
ain
1 0.
6 0.
9 .0
0 0.
9 1.
1 ns
0.
9 0.
9 ns
84
427
GR
B7
grow
th fa
ctor
rece
ptor
bou
nd p
rote
in 7
0.
6 1.
2 .0
0 1.
0 1.
2 ns
1.
1 1.
3 ns
31
1902
R
BM
18
RN
A b
indi
ng m
otif
prot
ein
18
1.6
1.0
.01
0.8
0.9
ns
0.9
0.8
ns
6787
41
LOC
6787
41
sim
ilar t
o Zi
nc fi
nger
CC
CH
-type
dom
ain
cont
aini
ng p
rote
in 6
0.
6 1.
0 .0
1 1.
1 1.
0 ns
1.
0 1.
0 ns
64
364
PLL
P
plas
ma
mem
bran
e pr
oteo
lipid
(pla
smol
ipin
) 0.
6 0.
9 .0
3 1.
0 1.
1 ns
0.
9 0.
9 ns
36
0894
R
GD
1310
587
sim
ilar t
o hy
poth
etic
al p
rote
in F
LJ14
146
0.6
0.9
.00
1.1
1.1
ns
1.0
1.0
ns
148 Supplement to Chapter 4
!
3634
42
FUN
DC
1 FU
N14
dom
ain
cont
aini
ng 1
0.
6 0.
9 .0
3 0.
9 0.
9 ns
1.
0 0.
9 ns
36
1197
R
GD
1561
537
sim
ilar t
o pu
tativ
e re
pair
and
reco
mbi
natio
n he
licas
e R
AD
26L
0.6
0.9
.05
0.8
0.7
ns
0.7
0.7
ns
2903
22
INTS
9 in
tegr
ator
com
plex
sub
unit
9 0.
6 0.
9 .0
1 1.
0 0.
9 ns
1.
0 1.
0 ns
24
706
RA
RB
re
tinoi
c ac
id re
cept
or, b
eta
0.6
1.0
.04
0.9
0.8
ns
1.0
0.8
ns
3159
23
LYS
MD
3 Ly
sM, p
utat
ive
pept
idog
lyca
n-bi
ndin
g, d
omai
n co
ntai
ning
3
1.6
1.1
.01
0.9
1.2
ns
1.2
1.2
ns
3606
39
DC
AK
D
deph
osph
o-C
oA k
inas
e do
mai
n co
ntai
ning
0.
6 0.
9 .0
0 0.
9 1.
0 ns
1.
0 1.
0 ns
24
5974
C
OM
MD
5 C
OM
M d
omai
n co
ntai
ning
5
0.6
1.0
.00
1.0
0.9
ns
1.1
1.0
ns
3616
50
HIR
IP3
HIR
A in
tera
ctin
g pr
otei
n 3
0.6
1.0
.02
1.0
1.0
ns
1.0
1.0
ns
1711
59
ZHX
1 zi
nc fi
nger
s an
d ho
meo
boxe
s 1
0.6
0.8
.01
0.9
0.9
ns
0.9
0.9
ns
3651
79
ZNF5
24
zinc
fing
er p
rote
in 5
24
0.6
1.0
.00
1.1
1.1
ns
1.0
1.1
ns
3622
27
DTD
1 D
-tyro
syl-t
RN
A d
eacy
lase
1 h
omol
og
0.6
0.9
.01
0.9
0.9
ns
1.0
1.0
ns
2885
57
MO
SP
D3
mot
ile s
perm
dom
ain
cont
aini
ng 3
0.
6 1.
0 .0
0 1.
1 1.
0 ns
1.
0 1.
1 ns
30
4579
U
SP
30
ubiq
uitin
spe
cific
pep
tidas
e 30
0.
6 0.
9 .0
1 0.
9 1.
0 ns
1.
0 0.
9 ns
49
8957
K
LHL3
6 ke
lch-
like
36
0.6
1.0
.02
1.1
1.0
ns
1.0
1.1
ns
2424
4 C
ALM
3 ca
lmod
ulin
3
0.6
1.0
.00
1.3
1.0
ns
1.1
1.0
ns
2877
03
EIF
1 eu
kary
otic
tran
slat
ion
initi
atio
n fa
ctor
1
1.5
1.2
.00
1.0
1.1
ns
1.0
1.0
ns
4982
79
FCE
R1G
Fc
frag
men
t of I
gE, h
igh
affin
ity I,
rece
ptor
for;
gam
ma
poly
pept
ide
1.5
1.2
ns
1.5
1.0
0.00
1.
1 1.
0 ns
28
7436
E
IF4A
1 eu
kary
otic
tran
slat
ion
initi
atio
n fa
ctor
4A
1 1.
5 1.
1 .0
0 0.
9 1.
0 ns
1.
0 1.
0 ns
36
2304
O
RC
5L
orig
in re
cogn
ition
com
plex
, sub
unit
5-lik
e 0.
7 0.
8 .0
0 1.
0 1.
0 ns
1.
1 1.
0 ns
25
671
SM
AD
1 S
MA
D fa
mily
mem
ber 1
1.
5 1.
0 .0
0 0.
9 0.
8 ns
0.
8 0.
9 ns
25
670
IL15
in
terle
ukin
15
0.7
1.0
.00
1.3
1.3
ns
1.3
1.3
ns
2936
20
PTD
SS
2 ph
osph
atid
ylse
rine
synt
hase
2
0.7
0.9
.01
1.1
0.9
ns
0.9
0.9
ns
3061
82
IPO
5 im
porti
n 5
1.5
1.1
.02
0.9
1.1
ns
1.1
1.0
ns
4997
55
TME
M14
1 tra
nsm
embr
ane
prot
ein
141
0.7
0.9
.00
1.0
0.9
ns
1.0
1.0
ns
3008
49
FBX
O9
f-box
pro
tein
9
0.7
1.1
.05
0.9
1.0
ns
1.1
1.0
ns
3680
42
BTA
F1
BTA
F1 R
NA
pol
ymer
ase
II, B
-TFI
ID tr
ansc
riptio
n fa
ctor
-ass
ocia
ted,
(Mot
1 ho
mol
og, S
. cer
evis
iae)
1.
5 1.
2 .0
0 0.
8 1.
0 ns
0.
9 0.
9 ns
3014
32
PP
IL3
pept
idyl
prol
yl is
omer
ase
(cyc
loph
ilin)
-like
3
0.7
1.0
.04
0.9
1.0
ns
0.9
0.9
ns
2906
32
MR
PL3
4 m
itoch
ondr
ial r
ibos
omal
pro
tein
L34
0.
7 0.
9 .0
0 0.
9 1.
0 ns
1.
0 1.
0 ns
29
7432
A
BTB
1 an
kyrin
repe
at &
BTB
(PO
Z) d
omai
n co
ntai
ning
1
0.7
1.1
.00
1.1
1.2
ns
1.4
1.4
ns
3636
03
CN
OT8
C
CR
4-N
OT
trans
crip
tion
com
plex
, sub
unit
8 0.
7 1.
0 .0
0 0.
9 1.
0 ns
1.
0 1.
0 ns
36
1767
P
OLL
po
lym
eras
e (D
NA
dire
cted
), la
mbd
a 0.
7 1.
0 .0
0 1.
1 1.
1 ns
1.
2 1.
3 ns
36
2757
R
DH
11
retin
ol d
ehyd
roge
nase
11
(all-
trans
/9-c
is/1
1-ci
s)
0.7
0.9
.02
1.0
0.8
ns
0.9
0.9
ns
2996
12
RG
D13
5912
7 si
mila
r to
RIK
EN
cD
NA
231
0011
J03
0.7
1.0
.01
0.9
0.9
ns
1.0
0.9
ns
5009
65
RG
D15
6269
0 si
mila
r to
L-la
ctat
e de
hydr
ogen
ase
A c
hain
(LD
H-A
) 1.
5 1.
0 .0
3 1.
1 1.
1 ns
1.
2 1.
0 ns
17
0842
TO
B1
trans
duce
r of E
rbB
-2.1
0.
7 1.
0 .0
2 1.
1 1.
0 ns
1.
0 0.
9 ns
50
0899
M
FSD
3 m
ajor
faci
litat
or s
uper
fam
ily d
omai
n co
ntai
ning
3
0.7
0.9
.00
1.2
1.1
ns
1.2
1.2
ns
3152
87
AS
B8
anky
rin re
peat
SO
CS
box
-con
tain
ing
prot
ein
8 0.
7 0.
9 .0
0 0.
9 1.
1 ns
1.
0 1.
0 ns
29
4283
R
GL2
ra
l gua
nine
nuc
l. di
ssoc
iatio
n st
imul
ator
-like
2
0.7
1.1
.01
1.2
1.1
ns
1.2
1.1
ns
6877
99
LOC
6894
08
sim
ilar t
o H
2A h
isto
ne fa
mily
, mem
ber V
-1
0.7
0.9
.03
1.0
1.0
ns
0.9
1.0
ns
2950
50
EX
OS
C8
exos
ome
com
pone
nt 8
0.
7 1.
0 .0
0 1.
1 1.
1 ns
1.
1 1.
1 ns
36
2414
TA
DA
3L
trans
crip
tiona
l ada
ptor
3 (N
GG
1 ho
mol
og, y
east
)-lik
e 0.
7 1.
0 .0
3 1.
0 0.
9 ns
1.
0 1.
1 ns
58
918
CA
SP
9 ca
spas
e 9,
apo
ptos
is-r
elat
ed c
yste
ine
pept
idas
e 0.
7 1.
0 .0
4 1.
0 1.
0 ns
1.
1 1.
0 ns
Supplements 149
!
1409
08
CD
K5
cycl
in-d
epen
dent
kin
ase
5 0.
7 1.
0 .0
1 1.
0 1.
0 ns
1.
0 1.
0 ns
36
2484
P
LEK
HF2
pl
ecks
trin
hom
olog
y do
mai
n co
ntai
ning
, fam
ily F
(with
FY
VE
dom
ain)
m
embe
r 2
0.7
1.1
.03
0.9
0.9
ns
0.8
1.0
ns
9420
1 C
DK
4 cy
clin
-dep
ende
nt k
inas
e 4
0.7
1.1
.02
1.2
0.9
ns
1.0
0.9
ns
5026
03
SFR
S11
sp
licin
g fa
ctor
, arg
inin
e/se
rine-
rich
11
1.4
1.0
.03
1.0
1.0
ns
1.2
1.1
ns
2889
24
MO
RG
1 m
itoge
n-ac
tivat
ed p
rote
in k
inas
e or
gani
zer 1
0.
7 1.
0 .0
3 0.
9 1.
0 ns
1.
1 1.
0 ns
17
1565
S
TAM
BP
S
tam
bin
ding
pro
tein
0.
7 1.
0 .0
3 1.
1 1.
0 ns
1.
0 1.
1 ns
29
6851
P
ON
2 pa
raox
onas
e 2
0.7
0.9
.00
1.1
1.0
ns
1.0
0.9
ns
2916
70
CX
XC
5 C
XX
C fi
nger
5
0.7
1.0
.02
1.0
1.1
ns
1.0
1.0
ns
4989
09
CO
Q9
coen
zym
e Q
9 ho
mol
og
0.7
0.9
.03
1.0
0.9
ns
0.9
0.9
ns
3067
34
RM
I1
RM
I1, R
ecQ
med
iate
d ge
nom
e in
stab
ility
1, h
omol
og
0.7
1.0
.02
1.1
1.0
ns
1.1
1.2
ns
5672
5 C
RIP
T cy
stei
ne-r
ich
PD
Z-bi
ndin
g pr
otei
n 0.
7 1.
0 .0
2 0.
9 0.
9 ns
0.
9 1.
0 ns
28
8621
M
RP
S17
m
itoch
ondr
ial r
ibos
omal
pro
tein
S17
0.
7 0.
9 .0
1 1.
0 1.
1 ns
0.
9 1.
0 ns
29
5357
H
BX
IP
hepa
titis
B v
irus
x in
tera
ctin
g pr
otei
n 0.
7 1.
0 .0
1 1.
0 1.
0 ns
1.
1 1.
1 ns
28
8783
O
RM
DL2
O
RM
1-lik
e 2
0.7
0.9
.00
1.0
0.9
ns
1.0
0.9
ns
2980
95
PR
PF4
P
RP
4 pr
e-m
RN
A p
roce
ssin
g fa
ctor
4 h
omol
og
0.7
1.0
.04
1.0
0.9
ns
1.0
1.0
ns
3075
45
ELP
2 el
onga
tion
prot
ein
2 ho
mol
og
0.7
0.9
.00
1.0
0.9
ns
1.1
1.1
ns
3125
11
RG
D13
0674
6 si
mila
r to
Hyp
othe
tical
pro
tein
MG
C25
529
0.7
0.9
.00
1.2
1.3
ns
1.0
1.2
ns
4986
96
RG
D15
6368
4 si
mila
r to
hete
roge
neou
s nu
clea
r rib
onuc
leop
rote
in A
0 0.
7 0.
9 .0
2 1.
0 0.
8 ns
0.
9 0.
9 ns
36
1565
V
RK
3 va
ccin
ia re
late
d ki
nase
3
0.7
1.0
.01
1.1
1.1
ns
1.0
1.1
ns
8358
6 U
SF1
up
stre
am tr
ansc
riptio
n fa
ctor
1
0.7
1.0
.03
1.0
0.9
ns
1.0
1.0
ns
2918
13
CM
TM3
CK
LF-li
ke M
AR
VE
L tra
nsm
embr
ane
dom
ain
0.
7 1.
2 .0
0 1.
2 1.
1 ns
1.
3 1.
3 ns
81
859
SH
AR
PIN
S
HA
NK
-ass
ocia
ted
RH
dom
ain
inte
ract
or
0.7
1.1
.02
1.0
1.0
ns
1.1
1.0
ns
1144
89
DA
G1
dyst
rogl
ycan
1
0.7
0.9
.00
0.9
0.9
ns
1.0
0.9
ns
2899
92
FER
MT2
fe
rmiti
n fa
mily
hom
olog
2
1.4
1.0
.01
0.9
0.9
ns
0.9
0.9
ns
2974
55
LSM
3 LS
M3
hom
olog
, U6
smal
l nuc
lear
RN
A a
ssoc
iate
d 0.
7 0.
9 .0
3 1.
0 0.
9 ns
1.
0 1.
0 ns
30
5350
R
GD
1565
119
sim
ilar t
o M
itoch
ondr
ial c
arrie
r trip
le re
peat
1
0.7
0.9
.03
1.0
0.9
ns
1.0
1.0
ns
3605
47
SA
T2
sper
mid
ine/
sper
min
e N
1-ac
etyl
trans
fera
se fa
mily
mem
ber 2
0.
7 1.
0 .0
3 1.
1 0.
9 ns
1.
0 1.
0 ns
36
1888
S
FRS
12IP
1 S
FRS
12-in
tera
ctin
g pr
otei
n 1
0.7
0.9
.00
1.0
0.9
ns
1.0
1.0
ns
3158
04
RFX
7 re
gula
tory
fact
or X
, 7
0.7
1.0
.02
1.0
1.0
ns
1.0
1.0
ns
2928
08
PE
PD
pe
ptid
ase
D
0.7
1.0
.02
1.1
0.9
ns
0.9
0.8
ns
3114
14
ZC3H
8 zi
nc fi
nger
CC
CH
type
con
tain
ing
8 0.
7 1.
1 .0
0 1.
1 1.
2 ns
1.
2 1.
2 ns
50
0790
R
GD
1563
634
sim
ilar t
o R
3144
9_3
0.8
1.2
.01
0.9
0.9
ns
1.0
1.0
ns
2881
74
NIT
2 ni
trila
se fa
mily
, mem
ber 2
0.
8 1.
0 .0
3 1.
0 1.
0 ns
1.
1 1.
1 ns
85
261
MTE
RF
mito
chon
dria
l tra
nscr
iptio
n te
rmin
atio
n fa
ctor
0.
8 1.
0 .0
1 1.
0 1.
0 ns
1.
0 1.
1 ns
49
9185
M
RP
S11
m
itoch
ondr
ial r
ibos
omal
pro
tein
S11
0.
8 0.
9 .0
1 1.
1 1.
0 ns
1.
0 1.
0 ns
85
269
NM
E3
non-
met
asta
tic c
ells
3, p
rote
in e
xpre
ssed
in
0.8
1.0
.00
1.0
1.1
ns
1.2
1.2
ns
2923
06
RN
AS
ET2
rib
onuc
leas
e T2
0.
8 0.
9 .0
1 1.
0 0.
9 ns
1.
0 1.
0 ns
36
3252
FA
M13
4A
fam
ily w
ith s
eque
nce
sim
ilarit
y 13
4, m
embe
r A
0.8
1.1
.03
1.1
1.1
ns
1.0
1.0
ns
3630
68
CO
MM
D4
CO
MM
dom
ain
cont
aini
ng 4
0.
8 0.
9 .0
0 1.
2 1.
1 ns
1.
0 1.
0 ns
29
0997
U
IMC
1 ub
iqui
tin in
tera
ctio
n m
otif
cont
aini
ng 1
0.
8 1.
0 .0
0 1.
1 1.
2 ns
1.
2 1.
2 ns
29
2758
M
RP
S12
m
itoch
ondr
ial r
ibos
omal
pro
tein
S12
0.
8 0.
9 .0
0 1.
1 1.
1 ns
1.
1 1.
0 ns
49
9380
E
MX
2 em
pty
spira
cles
hom
eobo
x 2
0.8
1.1
.01
0.9
1.0
ns
1.1
1.1
ns
150 Supplement to Chapter 4
!
2910
44
NE
DD
9 ne
ural
pre
curs
or c
ell e
xpre
ssed
, dev
elop
men
tally
dow
n-re
gula
ted
9 0.
8 1.
1 .0
2 1.
1 1.
0 ns
1.
0 1.
1 ns
31
3018
R
GD
1303
271
sim
ilar t
o ch
rom
osom
e 1
open
read
ing
fram
e 17
2 0.
8 1.
0 .0
3 0.
9 1.
0 ns
1.
0 0.
9 ns
69
1947
E
IF3J
eu
kary
otic
tran
slat
ion
initi
atio
n fa
ctor
3, s
ubun
it J
1.3
1.0
.00
0.9
0.9
ns
0.9
0.9
ns
2985
52
TME
M50
A
trans
mem
bran
e pr
otei
n 50
A
0.8
1.0
.01
1.0
1.0
ns
1.1
1.0
ns
5882
1 ZR
AN
B2
zinc
fing
er, R
AN
-bin
ding
dom
ain
cont
aini
ng 2
0.
8 1.
0 .0
5 0.
9 1.
1 ns
1.
0 1.
0 ns
29
528
VA
MP
3 ve
sicl
e-as
soci
ated
mem
bran
e pr
otei
n 3
0.8
1.0
.00
1.2
1.1
ns
1.2
1.0
ns
3089
86
TME
M21
9 tra
nsm
embr
ane
prot
ein
219
0.8
0.9
.00
1.1
0.9
0.00
1.
0 1.
0 ns
29
4311
R
GD
7350
65
sim
ilar t
o G
I:133
8541
2-lik
e pr
otei
n sp
lice
form
I 0.
8 1.
0 .0
1 1.
0 1.
1 ns
1.
0 1.
0 ns
36
0389
ZF
P42
2 zi
nc fi
nger
pro
tein
422
0.
9 1.
1 .0
1 1.
1 1.
1 ns
1.
1 1.
1 ns
31
2903
TR
AM
1 tra
nslo
catio
n as
soci
ated
mem
bran
e pr
otei
n 1
0.9
1.0
.01
1.1
1.1
ns
1.0
1.1
ns
Tabl
e sh
ows
all g
enes
that
are
sig
nific
antly
diff
eren
t bet
wee
n C
PB
and
Sha
m (A
NO
VA
and
pos
t-hoc
Tuk
ey H
SD
; P <
0.0
5). S
how
n is
the
fold
-cha
nge
com
pare
d to
hea
lthy
cont
rol f
or
CP
B a
nd S
ham
dur
ing
the
acut
e ph
ase
(60
min
utes
), 1
and
5 da
ys fo
llow
ing
the
proc
edur
e, a
s w
ell a
s th
e p-
valu
e be
twee
n C
PB
and
Sha
m (A
NO
VA
and
pos
t-hoc
Tuk
ey H
SD
; P<
0.05
); a
fold
-cha
nge
of 1
.0 re
pres
ents
equ
al e
xpre
ssio
n as
obs
erve
d in
hea
lthy
cont
rol r
ats.
ID =
Ent
rez
ID; n
s =
non
sign
ifica
nt c
hang
e.
!
!!!
Supplement
Does correcting the numbers improve long-term outcome? This Editorial View accompanies the following article: Samarska IV, van Meurs M, Buikema H, Houwertjes MC, Wulfert FM, Molema G, Epema AH, Henning RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia.
Robert D. Sanders, B.Sc., M.B.B.S., F.R.C.A.,* Mervyn Maze, M.B., Ch.B., F.R.C.P., F.R.C.A., F.Med.Sci. *Department of Anaesthetics,
Pain Medicine, and Intensive Care, Imperial College London, United Kingdom. [email protected]
152 Supplement to Chapter 6
!
Advances in the understanding of anesthetic pharmacology and perioperative physiology, coupled with improved patient monitoring, have significantly contributed to improvements in quality of care and perioperative outcome.1–3 In this issue of Anesthesiology, Samarska et al. describe preclinical research that addresses the anesthetic modulatory effects on the physiologic adaptation to hemorrhagic shock; their data have led them to the conclusion that nitrous oxide promotes hemodynamic stability.4 In their studies, mice were exposed to anesthesia, either isoflurane (1.4%) in oxygen (33%) or isoflurane (1.4%) plus nitrous oxide (66%) in oxygen (33%), and underwent a sham procedure, hemorrhagic shock, or shock plus fluid resuscitation, during which time hemodynamic measurements were obtained. Thereafter, vascular responsiveness was assessed ex vivo in aortic rings. Isoflurane treatment attenuated the maximal aortic contractile responses to phenylephrine, corroborating earlier reports with volatile anesthetics.5 Shock, with or without resuscitation, mitigated the isoflurane-induced attenuation of phenylephrine responses, although the biphasic pattern of relaxation and then contraction with acetylcholine was altered. The ex vivo effects induced by in vivo isoflurane exposure were mitigated when supplemented with nitrous oxide. However, in the shock state the addition of nitrous oxide induced acidosis when compared with isoflurane, and further physiologic differences (such as oxygenation) confounds clear interpretation of the experimental findings. Even though animals were at different depths of anesthesia under these conditions, the authors attribute the pharmacologic properties of nitrous oxide for “normalizing” vasoreactivity, and speculate that nitrous oxide may induce increased perioperative hemodynamic stability. Samarska et al. also observed that nitrous oxide exposure was associated with a higher mean arterial blood pressure in the sham-treated animals despite the increased depth of anesthesia;4 this finding corroborates previous studies demonstrating the vasoconstrictive properties of nitrous oxide.6 Yet recent clinical studies have reported minor difference in blood pressure when comparing nitrous oxide or air as the carrier gas.6–9 Thus it is highly speculative that the modest increments (of the order of 10 mmHg) reported by Samarska et al. and others will exert a positive long-term clinical benefit. ! Placing Physiologic Normalization into Clinical Context
It is not possible to ascribe a benefit to the “normalization” achieved by the administration of nitrous oxide because there was no “nonanesthetized” control group, no correlation with postoperative hemodynamic changes, and no assessment of long-term outcome. Nonetheless, the authors’ interest in improving hemodynamic stability in the postoperative period is commendable, especially as there is a tendency to view improvement as simply an intraoperative endpoint within anesthesiology. Rather our discipline needs to focus on endpoints of anesthetic management that are important by virtue of the fact that patient outcome is affected. In this manner, physiologic variables should not be used as surrogate markers for long-term outcomes unless their association is tightly correlated.
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When Can Modifying Intraoperative Numbers Improve Long-term Outcome? Identification of patient comorbidities is critical to understanding risk
stratification of vulnerable patients and, therefore, their level of care. In addition, anesthesiologists need to determine the modifiable risk factors occurring in the perioperative period that may be manipulated to improve outcomes. A recent analysis started this process by evaluating the importance of intraoperative physiologic variables to determine long-term cardiovascular outcomes and death.10 Data from this large cohort study identified higher-risk patients as having two or more comorbidities: Age, obesity, emergency surgery, previous cardiac intervention, congestive cardiac failure, cerebrovascular disease, and hypertension. Patients with two or more risk factors who had an adverse cardiac event were more likely to have had intraoperative hypotension (mean arterial pressure less than 50 mmHg or decreased by 40%, lasting at least 10 min) among other modifiable risks. Similar to previous data for vascular surgical patients,11 those with three or more risk factors who sustained an adverse cardiac event were also more likely to have endured intraoperative tachycardia. Unfortunately the study was underpowered to ascertain an independent effect of the hemodynamic variables on adverse cardiac events; adequately powered studies are required to further investigate these findings and that of physiologic changes in the postoperative period,11 to determine long-term patient outcomes. Clearly it is important to “correct the numbers” intraoperatively, but how? Perhaps if tachycardia predisposes patients with three or more cardiovascular risk factors to adverse cardiac events, then these are the subjects who are most likely to benefit from perioperative $-blockade.11,
12 Exposing patients with fewer risk factors may merely increase their chance of hypotension and thus increase their cardiac and stroke risk.13 These findings also question the clinical significance of nitrous oxide induced improvement in intraoperative mean arterial blood pressure, suggesting that this effect will be too modest alone (approximately 10 mmHg) to alter cardiac risk. Whether postoperative hemodynamic parameters are improved after nitrous oxide exposure remains unknown.
Long-term Anesthetic Effects: Nitrous Oxide Case Study
The use of nitrous oxide for the maintenance of anesthesia exemplifies the need to focus on long-term outcomes. While the purported increased hemodynamic stability (“correcting the numbers”) with nitrous oxide has been regarded as good for cardiac risk, other factors may mitigate this benefit; for example, halogenated volatile anesthetics consistently demonstrate superior organ-protective effects as compared with nitrous oxide or intravenous agents in experimental studies.14–16 This may translate into improved tolerance to lower perfusion pressure or reduced oxygen supply with an anesthetic technique that is based solely on halogenated volatile anesthesia. Thus the addition of nitrous oxide, while “sparing” the volatile, may reduce this potential benefit accrued from the volatile anesthetic gas. Similarly, individual anesthetic effects on cellular metabolism could also be important.17 Nitrous oxide may also influence cardiac risk by increasing homocysteine levels.18, 19 Raised
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homocysteine levels predispose to higher cardiac risk in the community20 and in cardiac surgical patients21 via endothelial dysfunction and possible effects on coagulation.19 Putatively related to this increased perioperative myocardial ischemia, increased homocysteine levels have been noted with nitrous oxide-based anesthesia (however, long-term follow-up of these patients has not been conducted).18 To further ignite debate, nitrous oxide administration was recently associated with an increased number of delayed ischemic neurologic events in a post hoc subgroup analysis of the intraoperative hypothermia for aneurysm surgery trial.8 Again this may be secondary to raised homocysteine levels in the nitrous oxide group (although these were not measured). Critically though, the long-term outcomes between the nitrous oxide and no nitrous oxide groups were not different. Therefore, the use of nitrous oxide to improve hemodynamic stability based on the assumption that it will alter long-term patient outcomes may be flawed. It is therefore timely that the ENIGMA-II trial protocol has recently been published.22 ENIGMA-II is designed to ask whether nitrous oxide predisposes to adverse cardiac events based on its ability to modify homocysteine levels.
The trial has a solid scientific foundation,19, 22, 23 with proof of principal demonstrated in smaller clinical trials.18, 23 ENIGMA-II will be a 7,000-patient study designed to evaluate whether avoidance of nitrous oxide administration is associated with a 25% decrease risk of cardiac events or death (! =0.05; $=0.1). Of course it is possible that the study may find that the higher intraoperative mean arterial blood pressure induced by nitrous oxide may improve outcomes. Whatever the results of ENIGMA II, the critical approach here is to focus on long-term patient outcomes; outcomes that matter to the patient. Impact of Long-term Outcome Studies
Long-term outcomes studies are needed to define the optimal anesthetic management for the more than 234 million patients who undergo surgery each year.24 Going beyond the results that are based on cohorts of “average” patients, anesthesiologists will need to further personalize care for the individual patients, using careful clinical phenotyping that will be guided in the future by biomarkers that evolve from postgenomic research endeavors. Both our specialty and the welfare of our patients will benefit from rigorous translation of the evidence from well-conducted clinical research into practice. Our discipline’s research program has to focus on long-term outcomes to improve endpoints that both matter to the patient and improve the efficiency of healthcare resource use. Defining how to “correct the numbers” is a critical part of this approach; we need studies to define how these values should be modified. It is more than likely that anesthesiologists can continue to improve long-term patient outcomes, but we need the studies to demonstrate how.
Reference list 1. Holland R: Anaesthetic mortality in New South Wales. Br J Anaesth 1987; 59:834–41
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2. Lagasse RS: The right stuff: Veterans Affairs National Surgical Quality Improvement Project. Anesth Analg 2008; 107:1772–4
3. Mayfield JB: The impact of intraoperative monitoring on patient safety. Anesthesiol Clin 2006; 24:407–17
4. Samarska IV, van Meurs M, Buikema H, Houwertjes MC, Wulfert FM, Molema I, Epema AH, Hennings RH: Adjunct nitrous oxide normalizes vascular reactivity changes after hemorrhagic shock in mice under isoflurane anesthesia. Anesthesiology 2009; 111:600–8
5. Spiss CK, Smith CM, Tsujimoto G, Hoffman BB, Maze M: Prolonged hyporesponsiveness of vascular smooth muscle contraction after halothane anesthesia in rabbits. Anesth Analg 1985; 64:1–6
6. Inada T, Inada K, Kawachi S, Takubo K, Tai M, Yasugi H: Haemodynamic comparison of sevoflurane and isoflurane anaesthesia in surgical patients. Can J Anaesth 1997; 44:140–5
7. Fleischmann E, Lenhardt R, Kurz A, Herbst F, Fu¨lesdi B, Greif R, Sessler DI, Akc¸a O; Outcomes Research Group: Nitrous oxide and risk of surgical wound infection: A randomised trial. Lancet 2005; 366:1101–7
8. Pasternak JJ, McGregor DG, Lanier WL, Schroeder DR, Rusy DA, Hindman B, Clarke W, Torner J, Todd MM; IHAST Investigators: Effect of nitrous oxide use on long-term neurologic and neuropsychological outcome in patients who received temporary proximal artery occlusion during cerebral aneurysm clipping surgery. Anesthesiology 2009; 110:563–73
9. McKinney MS, Fee JP: Cardiovascular effects of 50% nitrous oxide in older adult patients anaesthetized with isoflurane or halothane. Br J Anaesth 1998; 80:169–73
10. Kheterpal S, O’Reilly M, Englesbe MJ, Rosenberg AL, Shanks AM, Zhang L, Rothman ED, Campbell DA, Tremper KK: Preoperative and intraoperative predictors of cardiac adverse events after general, vascular, and urological surgery. Anesthesiology 2009; 110:58–66
11. Feringa HH, Bax JJ, Boersma E, Kertai MD, Meij SH, Galal W, Schouten O, Thomson IR, Klootwijk P, van Sambeek MR, Klein J, Poldermans D: High-dose beta-blockers and tight heart rate control reduce myocardial ischemia and troponin T release in vascular surgery patients. Circulation 2006; 114:I344–9
12. Beattie WS, Wijeysundera DN, Karkouti K, McCluskey S, Tait G: Does tight heart rate control improve beta-blocker efficacy? An updated analysis of the noncardiac surgical randomized trials. Anesth Analg 2008; 106:1039–48
13. POISE Study Group; Devereaux PJ, Yang H, Yusuf S, Guyatt G, Leslie K, Villar JC, Xavier D, Chrolavicius S, Greenspan L, Pogue J, Pais P, Liu L, Xu S, Ma´laga G, Avezum A, Chan M, Montori VM, Jacka M, Choi P: Effects of extendedrelease metoprolol succinate in patients undergoing non-cardiac surgery (POISE trial): A randomised controlled trial. Lancet 2008; 371:1839–47
14. De Hert SG, Van der Linden PJ, Cromheecke S, Meeus R, Nelis A, Van Reeth V, ten Broecke PW, De Blier IG, Stockman BA, Rodrigus IE: Cardioprotective properties of sevoflurane in patients undergoing coronary surgery with cardiopulmonary bypass are related to the modalities of its administration. Anesthesiology 2004; 101:299–310
15. Sanders RD, Ma D, Maze M: Anaesthesia induced neuroprotection. Best Pract Res Clin Anaesthesiol 2005; 19:461–74
16. Weber NC, Toma O, Awan S, Fra¨ssdorf J, Preckel B, Schlack W: Effects of nitrous oxide on the rat heart in vivo: Another inhalational anesthetic that preconditions the heart? Anesthesiology 2005; 103:1174–82
17. Kaisti KK, La°ngsjo¨ JW, Aalto S, Oikonen V, Sipila¨ H, Tera¨s M, Hinkka S, Metsa¨honkala L, Scheinin H: Effects of sevoflurane, propofol, and adjunct nitrous oxide on regional cerebral blood flow, oxygen consumption, and blood volume in humans. Anesthesiology 2003; 99:603–13
18. Badner NH, Beattie WS, Freeman D, Spence JD: Nitrous oxide-induced increased homocysteine concentrations are associated with increased postoperative myocardial ischemia in patients undergoing carotid endarterectomy. Anesth Analg 2000; 91:1073–9
19. Myles PS, Chan MT, Kaye DM, McIlroy DR, Lau CW, Symons JA, Chen S: Effect of nitrous oxide anesthesia on plasma homocysteine and endothelial function. Anesthesiology 2008; 109:657–63
20. Nygard O, Nordrehaug JE, Refsum H, Ueland PM, Farstad M, Vollset SE: Plasma homocysteine levels and mortality in patients with coronary artery disease. N Engl J Med 1997; 337:230–6
21. Ranucci M, Ballotta A, Frigiola A, Boncilli A, Brozzi S, Costa E, Mehta RH: Pre-operative homocysteine levels and morbidity and mortality following cardiac surgery. Eur Heart J 2009; 30:995–1004
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22. Myles PS, Leslie K, Peyton P, Paech M, Forbes A, Chan MT, Sessler D, Devereaux PJ, Silbert BS, Jamrozik K, Beattie S, Badner N, Tomlinson J, Wallace S; ANZCA Trials Group: Nitrous oxide and perioperative cardiac morbidity (ENIGMA-II) Trial: Rationale and design. Am Heart J 2009; 157:488–94
23. Sanders RD, Weimann J, Maze M: Biologic effects of nitrous oxide: A mechanistic and toxicologic review. Anesthesiology 2008; 109:707–22
24. Weiser TG, Regenbogen SE, Thompson KD, Haynes AB, Lipsitz SR, Berry WR, Gawande AA: An estimation of the global volume of surgery: A modeling strategy based on available data. Lancet 2008; 372:139–44
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Samenvatting in het Nederlands
Cardiopulmonaire bypass (CPB) is een techniek waarbij een hart-longmachine de functie van het hart als circulatiepomp overneemt, waardoor openhartchirurgie mogelijk wordt gemaakt. CPB is daarmee van grote waarde voor de thoraxchirurgie gebleken. Het is echter ook een techniek waarbij er sprake is van een verhoogd optreden van postoperatieve klinische complicaties, zowel tijdens verblijf in het ziekenhuis als op midden en lange termijn daarna. CPB gaat gepaard met activering van ontstekingsprocessen en een meer of mindere mate van (tijdelijke) achteruitgang in de werking van verschillende organen. Het goed functioneren van organen is mede afhankelijk van het “gedrag” van bloedvaten (vaatfunctie) in deze organen en daarbuiten. Dit proefschrift richt zich op het beschrijven van het verloop van veranderingen in vaatfunctie na CPB in relatie tot de ontstekingsprocessen die hierbij optreden. Daarbij werd onderzocht of beïnvloeding van de vaatfunctie een mogelijk aangrijpingspunt van farmacotherapeutische interventie is (behandeling met geneesmiddelen).
In hoofdstuk 1 worden de huidige opvattingen besproken over de pathofysiologische veranderingen en de klinische gevolgen van CPB. Het is bekend dat het chirurgisch trauma, het contact van bloed met kunstmatige oppervlakken van de hart-long machine, en de afwijkende doorbloeding van organen tijdens CPB en het herstel van de normale doorbloeding na CPB (=ischemie/reperfusie) allen cellulaire en humorale afweermechanismen activeren. Deze processen spelen zowel op lokaal niveau in de organen als in de systemische circulatie. Ontstekingsprocessen en ischemie-reperfusie schade leidt tot de ontwikkeling van vaatdisfunctie. Hierbij speelt verhoogde productie van zuurstof radicalen (oxidatieve stress) - zoals bij ischemie-reperfusie - een belangrijke rol. Oxidatieve stress draagt bij aan het ontstaan van endotheeldisfunctie, d.w.z. het niet goed meer functioneren van de cellaag die de binnenbekleding van de vaatwand vormt en normaliter belangrijke processen reguleert t.b.v. van een gezonde structuur en functie van bloedvaten. Als gevolg van bovengenoemde veranderingen treden afwijkingen op van de bloedsomloop in grote en kleine bloedvaten, die – samen met weefseloedeem en secundaire hypoperfusie - aan de basis liggen van het zogenaamde meervoudige orgaan disfunctie syndroom in CPB.
Vervolgens is in hoofdstukken 2 en 3 eerst de veranderde reactiviteit van bloedvaten na CPB het onderwerp van onderzoek. Deze werd bestudeerd in een experimenteel model van CPB in de rat waarbij drie verschillende type bloedvaten (kleine coronair en mesenteriaal arteriën, en de aorta) werden bemeten op verschillende tijdstippen na CPB gedurende een klinische relevante periode van 5 dagen. Tijdens deze periode na CPB trad er een verandering in vaatreactiviteit op die kan worden omschreven als een algehele verschuiving naar minder contractie/meer (endotheel-) relaxatie, met daarbij een opvallend verschil in het tijdsverloop voor verschillende bloedvaten. De patronen in figuur 1A suggereren dat veranderingen in vaatfunctie in kleine bloedvaten op korte termijn na CPB (1-2 dagen) optreden en tijdelijk van aard zijn, terwijl veranderingen in grote bloedvaten pas later opkomen (5
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dagen na CPB) en mogelijk een meer blijvend karakter hebben. Deze veranderingen in vaatfunctie zijn grotendeels toe te schrijven aan chirurgische procedures bij CPB, zoals anesthesie en cannulaties.
Het overnemen van de bloedsomloop door de hartlongmachine (extracorporele circulatie [ECC]) lijkt hieraan nog specifiek een eigen effect toe te voegen m.b.t. de endotheelfunctie. Zo bleek de toename in endotheel-afhankelijke relaxatie in kleine mesenteriaal arteriën 2 dagen na CPB teniet te zijn gedaan door ECC. Dit effect viel samen met een verhoogd niveau van nitrotyrosine in dit vasculaire bed, wat aangeeft dat de oxidatieve stress verhoogd was bij ECC. Verder is opmerkelijk dat het effect van ECC op endotheelfunctie selectief aanwezig was bij kleine maar niet bij grote arteriën, hetgeen verder onderstreept hoe verschillende vaatbedden op eigen verschillende wijze reageren op CPB. Om veranderde vaatfunctie verder te onderzoeken in relatie tot ontstekingsreacties in CPB werd in hoofdstuk 3 de expressie van markers en mediatoren van ontsteking en endotheelcel-activatie in de aorta vaatwand bepaald. Het patroon in figuur 1B suggereert dat CPB gepaard gaat met acute ontstekingsreacties in de aorta die daarna echter ook snel weer normaliseren. Dit in tegenstelling tot de veranderingen in reactiviteit in de aorta, die zoals eerder besproken pas op latere tijdstippen (5 dagen) na CPB optraden. Het lijkt er dus mogelijk op dat tijdelijke ontsteking van de vaatwand acuut na CPB bepaalde processen in gang zet die zich pas op langere termijn vertalen naar functionele verandering in reactiviteit. Of dit ook het geval is in kleine bloedvaten, of dat hier ontstekingsreacties en veranderde vaatfunctie hand in hand gaan (en gelijktijdig optreden) zou nog verder onderzocht moeten worden.
In de hoofdstukken 4 en 5 van dit proefschrift werd vervolgens getracht de pathofysiologie van CPB-gerelateerde ontstekingsprocessen verder te karakteriseren. Hiertoe werd in het zelfde model als boven de expressie van ontstekingsmediatoren in longen en de nieren bepaald m.b.v. PCR, Western blot en micro-array analyse technieken. De expressiepatronen die daarbij naar voren komen van verschillende pro-en anti-inflammatoire cytokines, en van markers van endotheelcel- en neutrofiel-activatie, suggereren dat CPB een acute ontstekingsreactie opwekt in beide organen. Micro-array analyse van nierweefsel liet zien dat CPB hier de expressie beïnvloedde van 421 genen die voornamelijk betrokken zijn bij de regulatie van de afweermechanismen.
Daar waar acute en heftige ontstekingsreacties in nier en aorta van voorbijgaande aard waren en volledig normaliseerden op langere termijn na CPB, bleef de expressie van de markers van oxidatieve stress, endotheelcel- en neutrofiel-activatie, en van pro- en anti-inflammatoire markers in longweefsel substantieel verhoogd gedurende veel langere tijd. De verlengde verhoging in longweefsel van markers, zoals IL-1$, TGF-$1 en SOD-1 tot wel 5 dagen van herstel na CPB, suggereert een doorlopende ontstekingreactie in longweefsel, welke bij zou kunnen dragen aan longcomplicaties zoals waargenomen na hartchirurgie. CPB is ook een situatie waarin (tijdelijke) hypotensieve condities kunnen ontstaan, waarbij er op dat moment een lagere bloeddruk “heerst” dan eigenlijk wenselijk als het
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gaat om adequate orgaandoorbloeding. Immers, te weinig bloeddruk leidt tot te weinig bloeddoorstroming en daarmee tot orgaan-ischemie, -schade en –functieverlies (net als extreem het geval is in hemodynamische shock). De boven beschreven bevindingen in dit proefschrift over veranderde vaatfunctie in CPB – met een shift naar minder contractie/meer (endotheel-) relaxatie – passen bij dit beeld en geven een functionele verklaring. Omdat anesthesie – als onderdeel van chirurgische procedures bij CPB - zelf ook van invloed kan zijn op de vaattonus is in hoofdstuk 6 een klein uitstapje gemaakt naar het effect van verschillende vormen van anesthesie op reactiviteit van bloedvaten in een muis model van extreme hypotensie (hemorragische shock). De resultaten bevestigen in eerste instantie dat een gangbaar anestheticum zoals isofluraan, ingrijpende effecten heeft op reactiviteit van bloedvaten (- en daarmee orgaanfunctie). Tevens blijkt dat een geselecteerde combinatie met andere anesthetica (in dit geval distikstofoxide, N2O ofwel lachgas) deze beïnvloeding dramatisch kan beperken. Het fundamentele punt wat deze studie naar voren brengt is dat post-operatieve vaatreactiviteit kan worden beïnvloed/gestuurd door rationeel gebruik van intra-operatieve anesthesie, en daarmee wellicht ook de klinische gevolgen na een chirurgische ingreep.
Tot slot wordt in dit proefschrift de aandacht gericht op mogelijke therapeutische interventies (=ontwikkeling van geneesmiddelen) voor de preventie en/of behandeling van de complicaties die gerelateerd zijn aan CPB. De strategie die hierbij werd getest was om systemische immuunreacties die veranderde vaatreactiviteit zouden kunnen opwekken te onderdrukken en tegelijkertijd om vaatreactiviteit direct te beïnvloeden d.m.v. modulatie van de zogenaamde S1P-receptor, die bij beide processen betrokken is. In hoofdstuk 7 werden ratten kort voorafgaand aan CPB behandeld met FTY720, een niet-selectieve agonist (=stimulerende stof) die aangrijpt op verschillende subtypes S1P-receptoren, of met SEW2871, een selectieve agonist van de S1P1-subtype-receptor. In figuur 2 is een patroon te zien waarin beide stoffen eenzelfde effect hadden op vaatreactiveit maar een verschillend effect op de systemische immuunrespons. D.w.z. beide stoffen versterkten contractiele en relaxerende reactiviteit na CPB (figuur 2B en 2C), maar alleen FTY720 beïnvloede het aantal lymfocyten in het bloed (figuur 2A). Deze resultaten suggereren dat beide S1P-receptor agonisten de vaatreactiviteit na CPB direct versterkten door modulatie van de S1P1-receptoren op de endotheelcellen en gladde spiercellen in de vaatwand.
Samengevat, het onderzoek in dit proefschrift onderstreept dat zowel de chirurgische procedures als het overnemen van lichaamsfuncties door de hart-long machine bij CPB verschillende fysiologische processen sterk verandert. Het optreden van ontstekingsreacties en vaatfunctie zijn hiervan belangrijke uitingen. Verder blijkt dat belangrijke componenten zelfs tijdens een langere herstelperiode niet volledig normaliseren, hetgeen suggereert dat sommige processen langdurig verstoord zijn. Deze langdurige verstoring vormt wellicht een belangrijk onderdeel van de klinische complicaties die zowel op korte als langere termijn na CPB zijn beschreven. Om dit te vermijden is directe beïnvloeding van vaatreactiviteit m.b.v. S1P1 receptor agonisten voorafgaand aan CPB een potentieel interessante optie.
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!Figure 1. Schematic representation of the changes in vascular reactivity and inflammatory processes following cardiopulmonary bypass (CPB). (A) CPB evoked a differential pattern of temporal changes early post-CPB (i.e. 24-48h) in small (mesenteric and coronary) arteries vs. “long-term” changes in large conductance artery (aorta); (B) Rapid systemic inflammation and aorta endothelial cell activation after extracorporeal circulation (ECC) were not paralleled by acute changes in aorta endothelial relaxation function, but rather by changes at the “long-term”; (C) Up-regulated inflammatory markers in kidney and aorta normalised in due course after ECC, but were maintained elevated in the lung for at least 5 days after CPB.
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Figure 2. Effect of S1P-receptor agonism on circulating lymphocytes and vascular reactivity following CPB. (A) The amount of circulating lymphocytes was reduced after pre-treatment with the non-selective S1P-receptor agonist FTY720, but not with the selective S1P1-receptor agonist SEW2871. In contrast, both compounds similarly enhanced vascular (B) contractility and (C) endothelial relaxation function. This suggests that the enhanced vascular reactivity following pre-treatment was mediated via modulation vascular S1P1-receptors, independently from lymphopenia. * P<0.05, vs untreated group, one way ANOVA with Bonferroni test. # P<0.05, vs untreated group, t-test
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164 Ukrainian summary
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Ukrainian summary 165
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166 Ukrainian summary
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Acknowledgments
It is my pleasure to say that I enjoy working with all people from the Departments of Clinical Pharmacology and Anesthesiology whom I have been lucky to meet during these years. I will always keep the nice memories of this time in my hart.
First of all I would like to express my gratitude to my promotor Prof. Dr. Robert H. Henning, whose expertise, knowledge, assistance, fast work are always a template for his PhD-students. Dear Robert, it is always a pleasure to work with you because of your ability to encourage people, to make a nice work atmosphere, and, of course, to generate an enormous amount of the research ideas.
Prof. Dr. Michel M.R.F. Struys, my second promotor, thank you for your suggestions to improve our papers, the scientific support and for the constructive feedback.
Dr. Hendrik Buikema, my copromotor, thank you for your ideas, the excellent talent to present data and to write perfect articles, and for your work. I appreciate very much your supervision and learned a lot from you.
Dr. Anne H. Epema, my copromotor, thank you for your medical expertise in the developing of the experimental model, for sharing his knowledge and experience in the CPB research, and for the supervision and the encouragement.
During the past years I was lucky to work with Martin C. Houwertjes, who improved to the highest degree the experimental setup, performed the surgery perfectly, provided technical support of all experiments, and has done a lot of great work. Dear Martin, many thanks for your professionalism and for the excellent human qualities.
I would like to thank the members of the reading committee, Prof. Dr. Patrick Wouters, Prof. Dr. Ingrid Molema and Prof. Dr. Jozef G.R de Mey, for the revision and critical evaluation of this thesis.
Many nice people made my work in the Department enjoyable and convenient. AIO’s, postdoc’s, and the staff: Maggie, Roelien, Peter, Diana, Hjalmar, Talaei, Madhi, Yan, Sjoerd, Nadir, Hiddo, Maria, Bernadet, Hisko, Irma, Femke, Azuwerus, Marry, Maaike, Jan, Linda, Maria, Anouk, Pieter. Thank you all for the fun and support during theses years. Special thanks to the supporting staff of the Department: Ardy Kuperus, Alexandra Doeglas, Wessel Sloof, Adriaan van Doorn, Marja van der Ende. I appreciate very much everything you have done for me.
I would like to show appreciation to the academic staff of the Department of Clinical Pharmacology for the support and for the constructive, interesting meetings: Prof. Dr. Dick de Zeeuw, Dr. Bianca Brundel. Dr. Leo Deelman, Dr. Petra Denig, Dr. Richard van Dokkum, Dr. Pieter de Graeff, Prof. Dr. Floor Haaijer-Ruskamp,
168 Acknoledgments
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Dr. Frank Holtkamp, Dr. Frouke Hut, Dr. Cees de Langen, Dr. Peter Mol, Dr. Margie Monster-Simons, Dr. Itte de Waard.
I was also glad to collaborate and communicate with Matijs van Meurs, Francis M. Wulfert, Dr. Willem van Oeveren, Hubert Mungroop, Dr. Fellery de Lange, Yumei Wang.
Further, the work of the GUIDE team, Prof. Dr. Han Moshage, Riekje Banus, Maaike Bansema, Mathilde Pekelaer, and many others, is greatly appreciated.
I would like also to thank my friends Anna, Katya, Jullia, Yana, Natasha, Olya, Vladimir, Zhenya, Katya, Sasha, Zanna, Sergei, and many others for the friendship and support. It was a pleasure for me to meet you.
It is a pleasure to thank you, my paranimfen Maggie and Roelien, for your generous help and assistance.
Last but not at least, I would like to express my enormous gratitude to my family; my parents Tetiana and Vasyl, my husband Volodymyr, my brother Oleksii and my sister-in-law Ella, my small nephew Stella, a parents-in-law Georgii and Maria, also Iryna and Oleg, uncle Victor, aunt Ellena, cousins Maria, Iwan, Michail, and of course my small daughter Katja for the support during these years. Y1+12@> T*C* @ Z*)*, . C>0: 0>, <914 4[+*E@,G O*C <41= )+@E0*,>9G01<,G E* O*-. 9=514G, E*51,. @ )1DD>+J3.. O[ 4<>2D* 5[9@ D9: C>0: )+@C>+1C @ @D>*91C. Y1+121B O14*, <)*<@51, /,1 ,[ 9=5@-G @ )1DD>+J@4*>-G C>0: 4<> \,@ 21D[. M +*D*, /,1 C[ < ,151B 4C><,>, 0><C1,+: 0* 4<> @<)[,*0@:, 31,1+[> 4[)*9@ 0* 0*->C ).,@. T@9*: @ 9=5@C*: C1: D1/>0G3* 8*,=-*, < ,41@C )1:49>0@>C C1: J@E0G 0*)190@9*<G 1<15>00[C <C[<91C, : J@4. ,151B @ D9: ,>5:. Y1+12@> (9>-*, ]99* @ Q,>99*, <)*<@51 O*C E* ./*<,@>. M O*< 4<>H 1/>0G 9=59=.
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170 Notes
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