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www.arquivosonline.com.br Sociedade Brasileira de Cardiologia • ISSN-0066-782X • Volume 103, Nº 1, July 2014

EditorialRegarding Hypertension Treatment are we following Worldwide

Tendencies?

Original ArticlesEffect of Pitavastatin on Vascular Reactivity in Hypercholesterolemic

Rabbits

Mortality and Embolic Potential of Cardiac Tumors

Impact of Different Obesity Assessment Methods after Acute Coronary

Syndromes

Resistance Exercise Restores Endothelial Function and Reduces Blood

Pressure in Type 1 Diabetic Rats

Evaluation of Sexual Dimorphism in the Efficacy and Safety of

Simvastatin/Atorvastatin Therapy in a Southern Brazilian Cohort

Obesity does not Lead to Imbalance Between Myocardial

Phospholamban Phosphorylation and Dephosphorylation

Functional Vascular Study in Hypertensive Subjects with Type 2

Diabetes Using Losartan or Amlodipine

Resistance Training After Myocardial Infarction in Rats: Its Role on

Cardiac and Autonomic Function

High-Sensitivity C-Reactive Protein as a Predictor of Cardiovascular

Events after ST-Elevation Myocardial Infarction

Review ArticleLipoprotein (a): Structure, Pathophysiology and Clinical Implications

Eletronic Pages

Anatomopathological SessionCase 3/2014 - 81-Year-Old Patient Hospitalized for Decompensated

Heart Failure

Case ReportBioresorbable Vascular Scaffold Use in a Case of In-stent

Restenosis

ViewpointValvular Heart Team

ImageInsights of Optical Coherence Tomography in Renal Artery

Fibromuscular Dysplasia in a Patient with Spontaneous Coronary

Artery Dissection

Figure 2 – A. The prevalence of phosphorylation occurs when PKA is activated while it phosphorylates I-1, thus preventing PLB dephosphorylation. B. The prevalence of dephosphorylation occurs when PKA is not activated. There is no PLB and I-1 phoshporylation, therefore PP-1 maintains its active state. cAMP: 3’5’ cyclic adenosine monophosphate; I-1: inhibitory protein 1; P: phosphate; PKA: protein kinase A; pPLB: phosphorylated phospholamban; PLB: dephosphorylated phospholamban; PP-1: phosphatase-1; Serca2a: Ca2+ pump. Page: 43

Arquivos Brasileiros de Cardiologia - Volume 103, Nº 1, July 2014

REVISTA DA SOCIEDADE BRASILEIRA DE CARDIOLOGIA - Publicada desde 1948

Contents

Editorial

When Caring For Hypertension, Are We In Line With Worldwide Trends?Paulo César B. Veiga Jardim.........................................................................................................................................................................page 1

Original Articles

Atherosclerosis/Endothelium/Vascular

Effect of Pitavastatin on Vascular Reactivity in Hypercholesterolemic RabbitsEros Antonio de Almeida, Michiko Regina Ozaki.........................................................................................................................................................................page 4

Heart Surgery - Adults

Mortality and Embolic Potential of Cardiac TumorsRicardo Ribeiro Dias, Fábio Fernandes, Félix José Alvarez Ramires, Charles Mady, Cícero Piva Albuquerque, Fábio Biscegli Jatene.......................................................................................................................................................................page 13

Acute Coronary Artery Disease

Impact of Different Obesity Assessment Methods after Acute Coronary SyndromesCaroline N. M. Nunes, Marcos F. Minicucci, Elaine Farah, Daniéliso Fusco, Paula S. Azevedo, Sergio A. R. Paiva, Leonardo A. M. Zornoff.......................................................................................................................................................................page 19

Exercising

Resistance Exercise Restores Endothelial Function and Reduces Blood Pressure in Type 1 Diabetic RatsMarcelo Mendonça Mota, Tharciano Luiz Teixeira Braga da Silva, Milene Tavares Fontes, André Sales Barreto, João Eliakim dos Santos Araújo, Antônio Cesar Cabral de Oliveira, Rogério Brandão Wichi, Márcio Roberto Viana Santos.......................................................................................................................................................................page 25

Pharmacology/Toxicology

Evaluation of Sexual Dimorphism in the Efficacy and Safety of Simvastatin/Atorvastatin Therapy in a Southern Brazilian CohortLisiane Smiderle, Luciana O. Lima, Mara Helena Hutz, Cézar Roberto Van der Sand, Luiz Carlos Van der Sand, Maria Elvira Wagner Ferreira, Renan Canibal Pires, Silvana Almeida, Marilu Fiegenbaum.......................................................................................................................................................................page 33


Genetics/Molecular Biology

Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and DephosphorylationPaula Paccielli Freire; Carlos Augusto Barnabe Alves; Adriana Fernandes de Deus; Ana Paula Lima Leopoldo; André Soares Leopoldo; Danielle Cristina Tomaz da Silva; Loreta Casquel de Tomasi; Dijon Henrique Salomé Campos; Antonio Carlos Cicogna.......................................................................................................................................................................page 41

Systemic Hypertension

Functional Vascular Study in Hypertensive Subjects with Type 2 Diabetes Using Losartan or AmlodipineCesar Romaro Pozzobon, Ronaldo A. O. C. Gismondi, Ricardo Bedirian, Marcia Cristina Ladeira, Mario Fritsch Neves, Wille Oigman.......................................................................................................................................................................page 51

Ischemia/Myocardial Infarction

Resistance Training After Myocardial Infarction in Rats: Its Role on Cardiac and Autonomic FunctionCamilla Figueiredo Grans, Daniele Jardim Feriani, Marcos Elias Vergilino Abssamra, Leandro Yanase Rocha, Nicolle Martins Carrozzi, Cristiano Mostarda, Diego Mendrot Figueroa, Kátia De Angelis, Maria Cláudia Irigoyen, Bruno Rodrigues.......................................................................................................................................................................page 60

High-Sensitivity C-Reactive Protein as a Predictor of Cardiovascular Events after ST-Elevation Myocardial InfarctionDaniel Rios Pinto Ribeiro, Adriane Monserrat Ramos, Pedro Lima Vieira, Eduardo Menti, Odemir Luiz Bordin Jr., Priscilla Azambuja Lopes de Souza, Alexandre Schaan de Quadros, Vera Lúcia Portal.......................................................................................................................................................................page 69

Review Article

Lipoprotein (a): Structure, Pathophysiology and Clinical ImplicationsRaul Cavalcante Maranhão, Priscila Oliveira Carvalho, Celia Cassaro Strunz, Fulvio Pileggi.......................................................................................................................................................................page 76


Arquivos Brasileiros de Cardiologia - Eletronic Pages

Anatomopathological Session

Case 3/2014 - 81-Year-Old Patient Hospitalized for Decompensated Heart FailureBruna Affonso Madaloso e Paulo Sampaio Gutierrez....................................................................................................................................................................page e1

Case Report

Bioresorbable Vascular Scaffold Use in a Case of In-stent RestenosisJulia Cadrin-Tourigny, Liang Dong, Akiko Maehara, Erick Schampaert, Philippe Genereux..................................................................................................................................................................page e11

Viewpoint

Valvular Heart TeamMax Grinberg..................................................................................................................................................................page e15

Clinical Update

Insights of Optical Coherence Tomography in Renal Artery Fibromuscular Dysplasia in a Patient with Spontaneous Coronary Artery DissectionTeresa Bastante e Fernando Alfonso..................................................................................................................................................................page e18

* Indicate manuscripts only in the electronic version. To view them, visit: http://www.arquivosonline.com.br/2014/english/10301/edicaoatual.asp

Editorial BoardBrazilAguinaldo Figueiredo de Freitas Junior (GO)Alfredo José Mansur (SP)Aloir Queiroz de Araújo Sobrinho (ES)Amanda G. M. R. Sousa (SP)Ana Clara Tude Rodrigues (SP)André Labrunie (PR)Andrei Sposito (SP)Angelo A. V. de Paola (SP)Antonio Augusto Barbosa Lopes (SP) Antonio Carlos C. Carvalho (SP) Antônio Carlos Palandri Chagas (SP) Antonio Carlos Pereira Barretto (SP) Antonio Cláudio L. Nobrega (RJ) Antonio de Padua Mansur (SP)Ari Timerman (SP)Armenio Costa Guimarães (BA)Ayrton Pires Brandão (RJ)Beatriz Matsubara (SP)Brivaldo Markman Filho (PE)Bruno Caramelli (SP)Carisi A. Polanczyk (RS)Carlos Eduardo Rochitte (SP)Carlos Eduardo Suaide Silva (SP) Carlos Vicente Serrano Júnior (SP) Celso Amodeo (SP)Charles Mady (SP)Claudio Gil Soares de Araujo (RJ) Cláudio Tinoco Mesquita (RJ)Cleonice Carvalho C. Mota (MG)Clerio Francisco de Azevedo Filho (RJ)Dalton Bertolim Précoma (PR)Dário C. Sobral Filho (PE)Décio Mion Junior (SP)Denilson Campos de Albuquerque (RJ) Djair Brindeiro Filho (PE)Domingo M. Braile (SP)Edmar Atik (SP)Emilio Hideyuki Moriguchi (RS)

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Editorial

Regarding Hypertension Treatment are we following Worldwide Tendencies?Paulo César B. Veiga JardimPrograma de Pós-Graduação em Ciências da Saúde da Faculdade de Medicina da Universidade Federal de Goiás, Goiania, Goiás – Brazil

Mailing Address: Paulo César B. Veiga Jardim •Rua 115-F n 135, Setor Sul. Postal Code 74085-300, Goiania, GO – BrazilE-mail: [email protected], [email protected]

KeywordsHypertension/trends; Antihypertensive drugs /diagnostic

use; Drug Combinations.

DOI: 10.5935/abc.20140111

Between July 2012 and March 2014, the Department of Arterial Hypertension of the Brazilian Society of Cardiology prepared and published four documents addressing some specific aspects on hypertension care1-4. Similarly, between July 2013 and January 2014, major international institutions also produced documents updating their guidelines regarding hypertension5-9. Some of the Brazilian documents were published before the international ones, while others were published concurrently.

The first Brazilian document, published in 2012, addressed resistant arterial hypertension1, and extensive review of the subject was performed based on evidence available at the time. In addition to definition, care with diagnosis and possible causes, there was a concern regarding that most cases are, in fact, pseudo-resistant cases, mainly caused by lack of adherence, while highlighting the importance of possible secondary hypertension. The document finally proposed treatment that included the classic and known - but almost always overlooked – nonpharmacological treatment and an objective indication of pharmacological treatment, which takes into account the use of full doses of drugs that inhibit the renin-angiotensin-aldosterone system (ACEI or ARB), of a calcium channel antagonist and long-acting, of a thiazide diuretic and, as a fourth drug, proposed the use of spironolactone. The following was the indication of a beta-blocker with vasodilating action or a drug with central action.

When comparing the Brazilian document with the guidelines published in 2013 and 2014 by ESH/ESC5, JNC-8 6, ASH / ISH7 and CHEP8, no significant differences were observed; however, it was verified that the ESH/ESC and the CHEP documents did not have specific recommendations for resistant hypertension, suggesting only the conventional algorithm. The JNC-8 and ASH/ ISH documents propose as a fourth drug the free choice between aldosterone antagonist, beta-blockers or drug with central action. In fact, as described in the Brazilian document, there is no definitive evidence on the fourth drug to be used, and a true Brazilian ongoing multicenter study10, which is at the final stages of implementation, should partially answer this question, at least regarding this aspect.

The second document was published in mid-2013, dealing specifically with the care of hypertensive diabetic patients2, and was also published before the international documents. On this subject there are major differences between the several guidelines (Table 1), although they are actually small details that will ultimately have a minor effect on the final result.

The Brazilian guidelines define a target BP of around 130 x 80 mmHg, a value that was also adopted by CHEP8 in their document published in early 2014. The ESH/ESC5 document defined a BP target of 140 x 80-85. The American Diabetes Association (ADA)9 defined a target BP < 140 x 80 mmHg, while JNC-8 6 and ASH/ISH7 defined a target BP < 140 x 90 mmHg for this group. The preferred drugs were also defined, with ACE inhibitors or ARBs being mandatory for diabetics with kidney dysfunction, whereas any one of four classes (ACEIs, ARBs, diuretics, calcium channel antagonists) were also defined for those without kidney dysfunction. When in combination, ARBs or ACE inhibitors with calcium channel antagonists showed to be advantageous, although ACE inhibitors or ARBs associated with diuretics can also be used.

As complementary drugs, beta-blockers and drugs with central action are part of the associations. JNC-8 and CHEP make the same recommendations as the Brazilian document does for drug use. The initial use of ACEI or ARB for all diabetics is recommended by ESH/ASH, ASH/ISH and ADA, and, regarding the drug association, the recommendations are similar to the others. Additionally, the Brazilian document established targets for glycemic control (HbA1c < 7%), similar to that proposed by the ESH/ESC and the ADA, while the JNC-8, ASH/ ISH and CHEP did not address this issue. Targets for blood lipid control (LDL < 100 mg/dL for those age < 40 years without CVD and < 70 mg/dL for those aged > 40 years and with CVD) have also been defined.

Similarly, ESH/ESC and ADA established the same values for LDL-CT for patients with associated CVD (LDL-CT < 70 mg/dL), whereas target LDL-CT < 115 mg/dL was established for diabetics with moderate to high risk by ESH / ESC and the ADA established LDL-TC < 100 mg/dL for those with low risk. The JNC-8, ASH / ISH and CHEP documents did not address these aspects, either.

The third issue revised by DHA / SBC is associated with pre-hypertension, white-coat hypertension and masked hypertension, and appeared in ABC in early 20143. Conceptual, diagnostic, prognostic, and, finally, conduct aspects were discussed. In all cases, recommendations were made regarding an accurate diagnosis, special attention to the identified cases and conduct; all of them received indication for lifestyle changes with frequent monitoring. With regard to drug therapy, the same therapeutic regimen used in common hypertensive patients was indicated for those with masked hypertension.

1

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Jardim PCBVHypertension Treatment, Tendencies Worldwide

Arq Bras Cardiol. 2014; 103(1):1-3

Table 1 – Comparison between documents for patients with type 2 diabetes

Document Target BP Target HbA1C Target LDL-CT Initial drug

DHA/SBC2 130 x 80 < 7% < 100 without CVD ACEI/ARB

< 70 > 40 years CCA/DIUR

< 70 with CVD

ESH/ESC5 140 x 80-85 < 7% < 115 mod/high CVR ACEI/ARB

< 70 with CVD

JNC-86 < 140 x 90 – – ACEI/ARB

CCA/DIUR

ASH/ISH7 <140 x 90 – – ACEI/ARB

CHEP8 < 130 x 80 – – ACEI/ARB

CCA/DIUR

ADA9 < 140 x 80 < 7% < 100 low CVR ACEI/ARB

< 70 with CVD

ACC: calcium channel antagonist; ADA: American Diabetes Association; ASH: American Society of Hypertension; ARB: angiotensin receptor blocker II; CHEP: Canadian Hypertension Education Program; DHA-SBC: Department of Arterial Hypertension of SBC; DIUR: diuretics; ESC: European Society of Cardiology; ESH: European Society of Hypertension; HbA1C: glycated hemoglobin; ACEI: angiotensin-converting enzyme inhibitor; ISH: International Society of Hypertension; JNC-8: 8 Joint National Committee; LDL-CT: LDL cholesterol; BP: blood pressure; CVR: cardiovascular risk; CVD: cardiovascular disease.

For individuals with white-coat hypertension, careful monitoring was indicated without the use of drugs, while leaving open the possibility of careful drug management for individuals with very high risk or target-organ injury. As for patients with prehypertension, according to the existing evidence, they did not receive an indication for drug treatment, while leaving open the possibility of this indication in selected cases only, in patients at high cardiovascular risk, with diabetes or established renal injury, always dependent on individualized medical decision. Additionally, in these cases, a large study being carried out in Brazil aims to answer the question of whether or not antihypertensive drugs should be used at low doses in this group of patients11.

Again, the DHA document is absolutely consistent with the ESH/ESC one. The ASH / ISH document makes a passing reference to prehypertension and also indicates the non-pharmacological treatment for these cases. Moreover, it does not make any comments about white-coat hypertension and masked hypertension. The JNC-8 and the CHEP do not mention this issue in their documents.

Finally, the last DHA publication describes a matter of considerable interest, which is the combination of drugs4.

This document is absolutely in line with all the others and, moreover, gives recommendations on the use of associations, clearly, didactically and based on all available evidence. This type of information will certainly facilitate medical practices, allowing better treatment options.

Therefore, one can see that the Brazilian scientific community has been consistently working, using the existing information in an up-to-date manner and aligned with other international organizations that deal with this issue. There are small differences in documents related to diabetic patients and they are present in all guidelines, demonstrating that it is not different information, but the lack of definitive information that ultimately leads to this diversity of options.

Finally, it is worth a reflection, emphasizing what is defined in several publications in their closing remarks. All of them seek to provide support to good medical practice, based on the set of the best currently available scientific information. However, no guideline can replace clinical judgment and careful medical management targeted for each specific type of patient, based on the principles of science and bioethics.

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Jardim PCBVHypertension Treatment, Tendencies Worldwide


1. Alessi A, Brandão AA, Coca A, Cordeiro AC, Nogueira AR, Diógenes de Magalhães F, et al; Departamento de Hipertensão Arterial da Sociedade Brasileira de Cardiologia I. Posicionamento Brasileiro sobre hipertensão arterial resistente. Arq Bras Cardiol. 2012;99(1):576-85. Erratum in Arq Bras Cardiol. 2013;100(3):304.

2. Alessi A, Bonfim AV, Brandão AA, Feitosa A, Amodeo C, Alves CR, et al; Departamento de Hipertensão Arterial da Sociedade Brasileira de Cardiologia I Posicionamento Brasileiro em hipertensão arterial e diabetes mellitus. Arq Bras Cardiol. 2013;100(6):491-501.

3. Alessi A, Brandão AA, de Paiva AM, da Rocha Nogueira A, Feitosa A, de Campos Gonzaga C, et al; Departamento de Hipertensão Arterial da Sociedade Brasileira de Cardiologia. I Posicionamento Brasileiro sobre pré-hipertensão, hipertensão do avental branco e hipertensão mascarada: diagnóstico e conduta. Arq Bras Cardiol. 2014;102(2):110-8.

4. Póvoa R, Barroso WS, Brandão AA, Jardim PC, Barroso O, Passarelli O Jr, et al; Departamento de Hipertensão Arterial da Sociedade Brasileira de Cardiologia. I Posicionamento Brasileiro sobre combinação de fármacos anti-hipertensivos. Arq Bras Cardiol. 2014;102(3):203-10.

5. Mancia G, Fagard R, Narkiewicz K, Redón J, Zanchetti A, Bohm M, et al; Task Force Members. 2013 ESH/ESC Guidelines for the management of arterial hypertension The Task Force for the management of arterial hypertension of the European Society of Hypertension (ESH) and of the European Society of Cardiology (ESC). J Hypertens. 2013;31(7):1281-357.

6. James PA, Oparil S, Carter BL, Cushman WC, Dennison-Himmelfarb C, Handler J, et al. 2014 Evidence-based guideline for the management of high blood pressure in adults report from the panel members appointed to the Eighth Joint National Committee (JNC 8). JAMA. 2014;311(5):507-20. Erratum in JAMA. 2014;311(17):1809.

7. Weber MA, Schiffrin EL, White WB, Mann S, Lindholm LH, Kenerson JG, Flack JM, et al. Clinical practice guidelines for the management of hypertension in the community a statement by the American Society of Hypertension and the International Society of Hypertension. J Hypertens. 2014;32:3-15.

8. Dasgupta K, Quinn RR, Zarnke KB, Rabi DM, Ravani P, Daskalopoulou SS, et al. The 2014 Canadian Hypertension Education Program. Canadian Hypertension Education Program recommendations for blood pressure measurement, diagnosis, assessment of risk, prevention, and treatment of hypertension. Can J Cardiol. 2014;30(5):485-501.

9. American Diabetes Association. Standards of Medical Care in Diabetes 2014. Diabetes Care. 2014;37 Suppl 1:S14-80.

10. Krieger EM, Drager LF, Giorgi DM, Krieger JE, Pereira AC, Barreto-Filho JA, et al; ReHOT Investigators. Resistant hypertension optimal treatment trial: a randomized controlled trial. The ReHOT Investigators. Clin Cardiol. 2014;37(1):1-6.

11. Fuchs FD, Fuchs SC, Moreira LB, Gus M, Nóbrega AC, Poli-de- Figueiredo CE, et al. Prevention of hypertension in patients with pre-hypertension: protocol for the PREVER-prevent trial. Trials. 2011;12:65.

References

3

Original Article

Effect of Pitavastatin on Vascular Reactivity in Hypercholesterolemic RabbitsEros Antonio de Almeida and Michiko Regina OzakiUniversidade Estadual de Campinas, Campinas, SP - Brazil

Mailing Address: Eros Antonio de Almeida •Rua Professor Jorge Nogueira Ferraz, XX Jardim Chapadão. Postal Code 13070-120, Campinas, SP – BrazilE-mail: [email protected]; [email protected] received January 06, 2014; revised manuscript February 04, 2014; accepted February 19, 2014.

DOI: 10.5935/abc.20140090

Abstract

Background: Pitavastatin is the newest statin available in Brazil and likely the one with fewer side effects. Thus, pitavastatin was evaluated in hypercholesterolemic rabbits in relation to its action on vascular reactivity.

Objective: To assess the lowest dose of pitavastatin necessary to reduce plasma lipids, cholesterol and tissue lipid peroxidation, as well as endothelial function in hypercholesterolemic rabbits.

Methods: Thirty rabbits divided into six groups (n = 5): G1 - standard chow diet; G2 - hypercholesterolemic diet for 30 days; G3 - hypercholesterolemic diet and after the 16th day, diet supplemented with pitavastatin (0.1 mg); G4 - hypercholesterolemic diet supplemented with pitavastatin (0.25 mg); G5 - hypercholesterolemic diet supplemented with pitavastatin (0.5 mg); G6 - hypercholesterolemic diet supplemented with pitavastatin (1.0 mg). After 30 days, total cholesterol, HDL, triglycerides, glucose, creatine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT) were measured and LDL was calculated. In-depth anesthesia was performed with sodium thiopental and aortic segments were removed to study endothelial function, cholesterol and tissue lipid peroxidation. The significance level for statistical tests was 5%.

Results: Total cholesterol and LDL were significantly elevated in relation to G1. HDL was significantly reduced in G4, G5 and G6 when compared to G2. Triglycerides, CK, AST, ALT, cholesterol and tissue lipid peroxidation showed no statistical difference between G2 and G3-G6. Significantly endothelial dysfunction reversion was observed in G5 and G6 when compared to G2.

Conclusion: Pitavastatin starting at a 0.5 mg dose was effective in reverting endothelial dysfunction in hypercholesterolemic rabbits. (Arq Bras Cardiol. 2014; 103(1):4-12)

Keywords: Hydroxymethylglutaryl - CoA Reductase Inhibitors; Endothelial Dysfunction; Rabbits; Hypercholesterolemia.

Introduction Inhibitors of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme

A reductase (HMG-CoA), the statins, are potent inhibitors of cholesterol biosynthesis in the liver by blocking the conversion to mevalonate1. Clinical studies with simvastatin (4S) and pravastatin (WOSCOPS) demonstrated that statins act by decreasing the concentration of cholesterol in the blood, decreasing the incidence of myocardial infarction and its mortality2,3.

Aiming at correlating decreases in mortality with the atherosclerotic plaque size, studies were carried out with different commercial products of statins. Aware of changes in lipid profile, the studies MARS4 and REGRESS5 showed that there were stabilization and regression of atherosclerosis as assessed by coronary angiography.

Ribeiro Jorge et al6 in 1994 suggested that statins might have an antioxidant action when they observed that hypercholesterolemic rabbits showed improvement of endothelial dysfunction that was disproportionate to lipid reduction, when treated with pravastatin. This observation was once again seen in 19977, when the rapid reversal of hypercholesterolemia with statins was studied in the same animal model. These actions, in addition to lowering cholesterol known as pleiotropic effects, refer to endothelial function protection, anti-inflammatory and anti-thrombotic action and stabilization of atherosclerotic plaque, among others8-14.

Pitavastatin is the latest available statin in the market, also known as nisvastatin and itavastatin. It was developed in 2003 in Japan, and approved in 2009 by the U.S. Food and Drug Administration of the United States of America, being the seventh statin to be developed and commercialized15,16.

It is a synthetic and lipophilic statin, of which pharmacokinetics and pharmacodynamics have distinct properties compared with other statins and can offer greater pleiotropic effects in relation to endothelial function, inflammation, oxidative stress and antithrombosis. It is minimally metabolized in the liver and primarily metabolized by enzymes CYP2C9 and CYP2C8, showing bioavailability of 80% of the administered dose17,18. The low affinity of

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Almeida & OzakiPitavastatin and rabbits


pitavastatin to CYP3A4 reduces interactions with other drugs metabolized by this enzyme and can decrease toxic manifestations19 -21.

To date, no clinical or experimental study on the pitavastatin was found in the Brazilian literature and thus, the aim of this study was to verify the action of this statin, particularly in decreasing endothelial dysfunction in experimental hypercholesterolemia, as well as defining its lowest effective dose for this purpose.

MethodsThe experimental protocol was approved by the Ethics

Committee for Animal Experimentation (EAEC)-IB-UNICAMP, under n. 2528-1.

The animals were fed 100g a day of standard chow with the following composition (g/100): Proteins, 16.00; Carbohydrates, 45.00; Fibers, 20.00; Fat, 5.00 and Ash, 14.00. We studied 30 New Zealand male rabbits initially weighing from 2.0 to 2.5 kg, which were divided into six groups. In the group considered as control in relation to hypercholesterolemic ones (G1), the rabbits were sacrificed after one month of standard diet and adaptation to the biothery. The other 25 rabbits received a hypercholesterolemic diet, containing standard chow supplemented with 0.5% cholesterol and 10% coconut oil. With the exception of the hypercholesterolemic control group (G2), the other groups were treated with pitavastatin (Kowa Company, Nagoya, Japan) dur ing the las t 15 days, by gavage, at doses of 0.1 mg/animal/day (G3), 0 25 mg/animal/day (G4), 0.5 mg/animal/day (G5) and 1 mg/animal/day (G6).

Biochemical analysisTotal plasma cholesterol, HDL-cholesterol (high-density

lipoproteins), triglycerides, glucose, AST (aspartate aminotransferase), ALT (alanine aminotransferase) and creatine kinase were measured using enzymatic kits (Laborclin, Bioliquid, Pinhais, PR, Brazil), and the reading was performed by spectrophotometry (Thermo Spectronic, Genesys 10 uv, Rochester, NY, USA) with a wavelength of 500 nm. LDL was calculated using Friedewald formula.

Tissue Cholesterol At the end of the experiment, the animals were sacrificed

and the thoracic aorta was removed. Tissue cholesterol was measured in segments according to the method of Naito and David22. In brief, the specimens were dried and homogenized at 4° C in 5 mL of Tris HCl buffer, pH 7.4, plus 0.01 NaNO3. Total lipids were extracted and homogenized in 10 vol of chloroform-methanol. The extracted total cholesterol was measured by enzymatic kits.

Tissue lipid peroxidationOne segment of the thoracic aorta was homogenized

with trichloroacetic acid (1 g tissue + 10 vol 20% TCA). After centrifugation, 0.67% thiobarbituric acid volume was added and the mixture was heated at 100 ° C for 20 minutes.

The concentration of malondialdehyde was calculated from the absorbance of 532 nm using extinction coefficient of 1.49 x 10-5 expressed as nmoL/mg tissue x 10-7 23.

Endothelial function Endothelial function was measured in the thoracic aorta

segments of approximately 5 mm, with intact endothelium, suspended in a recipient with a capacity of 10 mL in Krebs-Henseleit solution at 37° and pH 7.4 and heated to 37º C. The solution was continuously aerated with a carbogen mixture containing 95% oxygen and 5% carbonic gas. The segments were mounted on two metal hooks attached to a support in the container and the force transducer (Narco, 40 Narcotrace, Texas, USA). Then they were left to equilibrate for 60 minutes with replacement of the Krebs Henseleit solution every 20 minutes. The segments were stretched to a basal tension of 1 g. All segments of the aorta were contracted with NE (10-7M) and, after stabilization, ACh was added cumulatively (10-8 to 10-5 M)6,24 and relaxation was verified.

Statistical Analysis The SAS for Windows (Statistical Analysis System) software,

version 9.2 (SAS Institute Inc., 2002-2008, Cary, NC, USA) was used in the statistical analysis.

ANOVA with rank transformation was used to compare treatment groups through the collected variables, followed by Tukey test, to locate the differences. When comparing endothelial function to locate the differences in concentrations between the groups, the contrast profile test was used. The significance level for statistical tests was 5%.

ResultsThe results of means and standard deviations of the

different parameters studied are shown in Table 1. Figure 1, depicting total cholesterol in the end of the

experiment, showed that there was a decrease in total cholesterol in groups G5 (25.8% reduction) and G6 (25.7%), where the rabbits were treated with 0.5 and 1.0 mg of pitavastatin, when compared to the hypercholesterolemic group G2, with statistically significant difference.

Figure 2, depicting LDL, showed that there was a decrease in G5 (20.07%) and G6 (26.62%), with no statistically significant difference when compared to G2.

Figure 3, depicting HDL, showed that a decrease occurred in G3 (40.88%), G5 (56.68%) and G6 (56.53%) when compared to G2, with a statistically significant difference.

Figure 4, depicting triglycerides, showed that a decrease occurred in G3 (44.62%), G4 (33.53%), G5 (52.05%) and G6 (45.56%), but with no statistically significant difference when compared to G2.

Figure 5, depicting tissue cholesterol, showed a decrease in groups G5 (28.2%) and G6 (20.09%), but with no statistically significant difference when compared to G2.

Figure 6, depicting lipid peroxidation, showed a decrease in G5 (26.25%) and G6 (31.25%), with no statistically significant difference compared to G2.

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900

700

600

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100

0G1

Total Cholesterol

(mg/dL)

G2 G3 G4 G5 G6

800

Figure 1 – Total cholesterol in all groups expressed as mean and standard deviation. * p < 0.05 in relation to G2.

Table 1 – Results of groups G1 to G6 with means and standard deviations

G1 G2 G3 G4 G5 G6

Col (mg/dL) 63.6 ± 4.3 753.9 ± 32.0 650.5 ± 212.5 705.3 ± 164.0 559.6 ± 203.6 527.6 ± 100.9

HDL (mg/dL) 16.5 ± 3.7 61.4 ± 8.8 *36.3 ± 15.0 *25.2 ± 10.9 *26.6 ± 12.5 *26.7 ± 5.7

LDL (mg/dL) 26.6 ± 3.9 650.0 ± 33.3 599.7 ± 196.3 663.0 ± 161.1 519.6 ± 202.3 477.7 ± 103.4

Trig (mg/dL) 104.2 ± 16.2 212.5 ± 99.9 117.7 ± 32.9 141.2 ± 36.5 101.9 ± 22.5 115.7 ± 15.0

Gli (mg/dL) 115.8 ± 19.2 127.3 ± 28.9 114.6 ± 25.5 123.5 ± 21.6 119.8 ± 23.7 94.7 ± 26.2

Col tec (mg/g) 21.6 ± 4.9 28.7 ± 4.8 22.8 ± 2.0 29.1 ± 7.8 20.4 ± 5.6 22.7 ± 4.5

Perox (ng/mg de prot) 5.1 ± 0.6 8.0 ± 1.9 5.7 ± 1.6 5.3 ± 1.4 5.9 ± 1.2 5.5 ± 1.4

Rel Máx (%) 93.2 ± 6.7 60.2 ± 12.64 62.3 ± 12.1 61.3 ± 11.7 *80.40 ± 5.1 *79.8 ± 12.0

Cknac (U/l) 236.1 ± 79.9 354.0 ± 62.3 243.8 ± 89.0 200.5 ± 88.7 336.1 ± 135.2 298.3 ± 118.6

AST (U/l) 35.7 ± 15.4 25.9 ± 8.3 50.0 ± 24.5 22.7 ± 8.3 30.0 ± 9.1 34.7 ± 10.8

ALT (U/l) 20.3 ± 11.2 25.1 ± 6.7 37.2 ± 18.6 36.1 ± 13.8 *18.3 ± 6.1 30.1 ± 6.8

Col: total cholesterol; HDL: high-density lipoprotein; LDL: low-density lipoprotein; TG: triglycerides; Glu: glucose; Tissue Col: tissue cholesterol; Perox: peroxidation tissue (ng / mg protein); Rel Max (%): endothelial function; Cknac: creatine phosphokinase; AST: aspartate aminotransferase; ALT: alanine aminotransferase. * p < 0.05 in relation to G2.

Figure 7 shows an improvement in endothelial function in G5 and G6 in relation to group G2, which was statistically significant.

Regarding glucose, creatine kinase, AST and ALT, there were no statistically significant alterations between the groups (Table 1).

DiscussionSevera l d i f ferent s ta t ins are ava i lab le in the

pharmaceutical market, acting through the inhibition of 3-Hydroxy-3-Methylglutaryl Coenzyme A (HMG-CoA) reductase, which makes them members of a group of a specific class of drugs, all with the precise indication of hypercholesterolemia reduction. Molecular pharmacokinetic

and pharmacodynamic modifications have been performed to differentiate the statins, almost always seeking the more effective blocking of HMGCoA reductase and thus, better control of plasma lipids, but also for the purpose of drug individualization, in addition its generic quality.

Pitavastatin is the newest statin in the market, starting in 2003 in Japan and currently available in Brazil25. To the best of our knowledge, this constitutes the first experimental study available in the Brazilian literature, to date, which evaluated pitavastatin action on plasma and tissue lipids, lipid peroxidation and vascular reactivity, seeking to identify the lowest dose at which it can be effective in controlling these parameters. Moreover, clinical studies have not been published in the national literature addressing the different aspects involving pitavastatin.

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Figure 2 – LDL-col in all groups expressed as mean and standard deviation.* p < 0.05 in relation to G2.

700

600

500

400

300

200

100

0G1

LDL-col (mg/dL)

G2 G3 G4 G5 G6

800

The addition of fat to the standard chow fed to the rabbits has been the most widely used model for induction of experimental hypercholesterolemia and it was effective in this study. The results shown in Table 1 showed that group G2 showed significantly higher elevations in serum lipids than those in G1, fed the standard chow. The same occurred with the tissue parameters, with elevations in total cholesterol, lipid peroxidation and reduced endothelial function in aortic segments.

The pitavastatin dose proposed for human use ranges from 1 to 2 mg/day, with a maximum dose of 4 mg/day, whereas in experimental studies, a dose < 1 mg/kg/day has been used, with no reports of groups of animals receiving increasingly higher doses, as in the present study19,26,27. The pitavastatin doses used in this study, although lower than the lowest used in humans,

are high for rabbits, considering the differences between the species, especially weight. However, they are necessary to achieve the effect of cholesterol reduction in these animals, which was observed only with a minimum dose 0.5 mg/animal. Differences in metabolism between species may probably explain why high doses are not toxic or lethal to some of them. Not only were the doses different regarding their action on lipids, but also their time of use. In the present study, only 15 days of drug use were sufficient for the lipid-lowering action of pitavastatin to occur, while other studies used at least 12 weeks19.

These data are similar to those observed in studies using other statins, when doses are optimized for a same total cholesterol percentage reduction28. A clinical study has demonstrated that pitavastatin improves peripheral microvasculature function verified through reactive hyperemia measured by arterial tonometry

G1

HDL-col (mg/dL)

G2 G3 G4 G5 G6

70

60

50

40

30

20

10

0

Figure 3 – HDL-col in all groups expressed as mean and standard deviation. * p < 0.05 in relation to G2.

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G1

Triglycerides (mg/dL)

G2 G3 G4 G5 G6

240220200180160140120100

80604020

0

Figure 4 – Triglycerides in all groups expressed as mean and standard deviation.

in hypercholesterolemic individuals with coronary artery disease, only two hours after oral administration of the drug, demonstrating the promptness of drug action in improving endothelial function, regardless of its action on plasma cholesterol29.

The results shown in Table 1 and Figure 7 demonstrated that pitavastatin was effective in improving vascular reactivity, as there was a significant improvement in endothelial dysfunction in the treated groups when compared to the hypercholesterolemic group. However, this effect occurred only with doses ≥ 0.5 mg, meaning that lower doses are unable to exercise the same effect during the time period of the experiment. Similar results in endothelial dysfunction reversal by pitavastatin have been reported in other experimental studies19 and in humans29 without great differences from those observed when other statins were assessed28,30-32.

The improvement in endothelial dysfunction cannot be determined only by LDL reduction, although this reduction has occurred, as shown in Table 1 and Figure 2, because the absolute values still remained much higher than in the G1 group, not hypercholesterolemic. However, the percentage of relaxation was very close to that of G1. Oxidative stress involving LDL in hypercholesterolemia has been held responsible for the endothelial dysfunction observed in these situations and was one of the goals of this study. Even without full control of hypercholesterolemia, one can achieve reversal of endothelial dysfunction by reducing oxidative stress. This effect has been produced by statins and occurs with pitavastatin, as observed in the results, as there was a decrease in tissue lipid peroxidation in relation to G2, in the treated groups.

Figure 5 – Tissue cholesterol in all groups expressed as mean and standard deviation.

G1 G2 G3 G4 G5 G6

40

35

30

25

20

15

10

5

0

Tissu

e cho

lester

ol (m

g/g)

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G1 G2 G3 G4 G5 G6

10

9

8

7

6

5

4

3

2

1

0

Tissue peroxidation (ng/mg tissue X 10-7.0)

Figure 6 – Tissue peroxidation in all groups expressed as mean and standard deviation.

Figure 7 – Endothelial function in all groups expressed as mean and standard deviation.*p < 0.05 in relation to G2.

Closely related to both situations, an improvement in endothelial dysfunction and decreased lipid oxidation is the reduction in tissue cholesterol, as reported in Table 1 and Figure 5, as well as in previous studies28,30-32. However the reduction in tissue cholesterol and lipid peroxidation occur similarly in all treated groups, unlike endothelial dysfunction reversal, which was observed only in the groups where animals received higher doses of pitavastatin (G5 and G6).

A literature study that used the same experimental model26 aimed to evaluate the effect of pitavastatin and probucol on atherosclerosis progression by studying oxidative stress. For that purpose, superoxide dismutase and the expression of peroxisome proliferator-activated receptors (PPARs) were used as parameters. The authors observed that, at a dose of 0.05 mg / kg / day, pitavastatin was effective in reducing oxidative stress without any action on serum

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cholesterol levels. Thus, it appears that pitavastatin acts on other mechanisms, which could explain the results obtained in this study, justifying the reduction in lipid peroxidation, without altering plasma cholesterol levels.

Thus, endothelial dysfunction reversal may depend on other factors in addition to oxidative ones, requiring larger doses of pitavastatin for it to occur.

One of the remarkable features of pitavastatin, observed from the clinical point of view, is its action in increasing HDL, especially in individuals in which it is reduced33,34. The main mechanism by which this statin is better than others in increasing HDL levels is the capacity to increase the expression of ApoA-1 gene, through the activation of PPARs35, the largest intra- and extracellular regulator of fatty acid metabolism, increasing its secretion.

In the present study, there was a reduction of HDL, accompanying a decrease in total cholesterol and LDL levels. Pitavastatin did not determine the increase or prevented the decrease in HDL, as observed in clinical studies. In another experimental study in ovariectomized hypercholesterolemic rabbits19, the authors found no significant changes in HDL and triglycerides. These findings regarding HDL have been observed with other statins in the literature30,32, and they were not aimed at specifying the mechanisms by which these animals exhibit such behavior. Differences in lipid metabolism between species that justify such results should exist and should be the objectives of future researches in order to better understand this phenomenon.

The results observed in relation to triglycerides (Table 1 and Figure 4), showing significant decrease in the treated groups, demonstrate the efficacy of pitavastatin in this sense, as observed in other experimental studies19,27, which would make this statin the choice to treat individuals with hypercholesterolemia, as well as those with hypertriglyceridemia, especially diabetic ones.

The actions of statins, in addition to those dependent on the reduction in LDL and cholesterol, are known in the literature12,14. Such effects, known as pleiotropic ones, have been generally beneficial by reducing lipid oxidation and reversing endothelial dysfunction, as demonstrated in the present study, in addition to blocking inflammatory processes, among others, resulting in the interruption of atherosclerosis progression and, consequently, of clinical events. However, lately it has been observed that these pleiotropic effects may also be deleterious to the body, especially in relation to glucose metabolism35.

Although clinical studies have shown controversial results regarding the adverse events of statins in inducing diabetes as, in the WOSCOPS3 study, pravastatin prevented diabetes onset, and in the JUPITER study36, rosuvastatin induced it, experimental evidence consistently demonstrate that statins may impair glucose homeostasis37. In the present study, there was no change in blood glucose levels of treated groups when compared to controls.

Although experimental studies involving pitavastatin and adverse events related to glucose metabolism have not been performed, clinical studies comparing it to other statins have shown that the onset of diabetes in users has been significantly lower, especially regarding atorvastatin and rosuvastatin, and comparable to pravastatin38. Furthermore, this event has occurred when higher doses of statins were used, perhaps justifying the results of this study, in which lower doses were used. The same result occurred in relation to liver and creatine kinase enzymes, which showed no change in the groups treated with pitavastatin when compared to controls, demonstrating that the doses used were safe (Table 1). This fact has been reported in the literature39.

ConclusionPitavastatin was effective in reducing plasma lipids, lipid

peroxidation and tissue cholesterol, reversing endothelial dysfunction in hypercholesterolemic rabbits, starting with a 0.5 mg dose.

Author contributionsConception and design of the research and Analysis and

interpretation of the data: Almeida EA, Ozaki MR; Acquisition of data and Writing of the manuscript: Ozaki MR; Obtaining financing and Critical revision of the manuscript for intellectual content: Almeida EA.

Potential Conflict of InterestNo potential conflict of interest relevant to this article was

reported.

Sources of FundingThis study was funded by FAEPEX (Fundo de apoio ao

ensino, pesquisa e extensão – UNICAMP).

Study AssociationThis study is not associated with any thesis or dissertation work.

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37. Kostapanos MS, Liamis GL, Milionis HJ, Elisaf MS. Do statins beneficially or adversely affect glucose homeostasis? Curr Vasc Pharmacol. 2010;8(5):612-31.

38. Navarese EP, Buffon A, Andreotti F, Kozinski M, Welton N, Fabiszak T, et al. Meta-analysis of impact of different types and doses of statins on new-onset diabetes mellitus. Am J Cardiol. 2013;111(8):1123-30.

39. Kitahara M, Kanaki T, Toyoda K, Miyakoshi C, Tanaka S, Tamaki T, et al. NK-104, a newly developed HMG-CoA reductase inhibitor, suppresses neointimal thickening by inhibiting smooth muscle cell growth and fibronectin production in ballon-injured rabbit carotid artery. Jpn J Pharmacol. 1998;77(2):117-28.

12

Original Article

Mortality and Embolic Potential of Cardiac TumorsRicardo Ribeiro Dias, Fábio Fernandes, Félix José Alvarez Ramires, Charles Mady, Cícero Piva Albuquerque, Fábio Biscegli JateneInstituto do Coração (InCor) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, São Paulo, SP - Brazil

Mailing Address: Ricardo Ribeiro Dias •Instituto do Coração da Faculdade de Medicina da Universidade de São Paulo - Ricardo Ribeiro Dias – Av. Dr. Enéas de Carvalho Aguiar, 44 2ºandar sala 13, Cerqueira César. Postal Code 05403-000, São Paulo, SP - BrazilE-mail: [email protected]; [email protected] received September 13, 20/13; revised manuscript February 24, 2014; accepted March 14, 2014.

DOI: 10.5935/abc.20140096

Abstract

Background: Cardiac tumors are rare, mostly benign with high embolic potential.

Objectives: To correlate the histological type of cardiac masses with their embolic potential, implantation site and long term follow up in patients undergoing surgery.

Methods: Between January 1986 and December 2011, we retrospectively analyzed 185 consecutive patients who underwent excision of intracardiac mass (119 females, mean age 48 ± 20 years). In 145 patients, the left atrium was the origin site. 72% were asymptomatic and prior embolization was often observed (19.8%). The diagnosis was established by echocardiography, magnetic resonance and histological examination.

Results: Most tumors were located in the left side of the heart. Myxoma was the most common (72.6%), followed by fibromas (6.9%), thrombi (6.4%) and sarcomas (6.4%). Ranging from 0.6cm to 15cm (mean 4.6 ± 2.5cm) 37 (19.8%) patients had prior embolization, stroke 10.2%, coronary 4.8%, peripheral 4.3% 5.4% of hospital death, with a predominance of malignant tumors (40% p < 0.0001). The histological type was a predictor of mortality (rhabdomyomas and sarcomas p = 0.002) and embolic event (sarcoma, lipoma and fibroelastoma p = 0.006), but not recurrence. Tumor size, atrial fibrillation, cavity and valve impairment were not associated with the embolic event. During follow-up (mean 80±63 months), there were 2 deaths (1.1%) and two recurrences 1 and 11 years after the operation, to the same cavity.

Conclusion: Most tumors were located in the left side of the heart. The histological type was predictor of death and preoperative embolic event, while the implantation site carries no relation with mortality or to embolic event. (Arq Bras Cardiol. 2014; 103(1):13-18)

Keywords: Heart Neoplasms / mortality; Heart n/ surgery; Embolism / complications.

IntroductionPrimary cardiac tumors are uncommon and often

asymptomatic with an incidence ranging from 0.0017% to 0.28%1. The differential diagnosis of cardiac masses includes vegetations, thrombi, and tumors. It may involve the endocardium, myocardium or epicardium. Secondary involvement of the heart by extra-cardiac tumors is rare. Benign tumors constitute 80% of primary cardiac neoplasms and myxomas are the prevalent type2. Angiosarcoma is the most frequent malignant cardiac tumor and is characterized by rapid growth, local invasion and distant metastasis3.

The clinical findings of cardiac tumors vary. They may mimic either cardiac or systemic diseases, and, therefore, should be

considered in the differential diagnosis of distinct heart diseases. In addition, as surgical therapy is curative in most cases, early diagnosis is required to prevent complications. In patients with primary cardiac neoplasm, tumors location and cells type are important in determining clinical presentation, therapy options, and outcomes4,5.

The aim of this study is to correlate the histological type of cardiac masses with their embolic potential, implantation site and long term follow up in patients undergoing surgery.

MethodsBetween January 1986 and December 2011, we retrospectively

analyzed 185 consecutive patients (118 females - 64%), with age ranging from 1 month to 86 years (mean age 48 ± 20 years), who were operated on at our Institution for primary cardiac mass.

The diagnosis was established by echocardiography, magnetic resonance and histological examination.

Patients’ characteristics, as well as demographic distribution, are listed in tables one to three. The most frequent primary cardiac tumors origin site was the left atrium (Table 1), most of the patients were asymptomatic (72%) and when with symptoms, dyspnea was the most common clinical presentation. Embolization was often observed (19.8%) especially to the central nervous system (Table 2).

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Table 1 – Classification of 185 patients with intracardiac tumors according to implantation site

Origin Site Nº of patients (%)

Left atrium 143 (77.3%)

Right atrium 23 (12.4%)

Left ventricle 15 (8.1%)

Right ventricle 4 (2.2%)

Table 2 – Preoperative clinical profile of 185 patients with cardiac tumors

Symptom Cardiac Nº of patients

Dyspnea 52 (28%)

Atrial fibrilation 50 (26.9%)

Palpitation 17 (9.1%)

Syncope 18 (9.7%)

Embolic

CNS 19 (10.2%)

AMI 9 (4.8%)

Peripheral 9 (4.8%)

Systemic

Fever 9 (4.8%)

Anemia 7 (3.8%)

Weight loss 3 (1.6%)

Arthralgia 1 (0.5%)

CNS: Central nervous system; AMI: Acute myocardial infarction.

Table 3 – Institutional experience with cardiac tumors resection

Type of tumor Nº of patients (%) Male/Female Mean age (years) Size (mean) (cm)

Myxoma 135 (72.6%) 45 / 90 52 ± 16 1-12.5 (4.7 ± 2.1)

Fibroma 13 (7%) 8 / 5 38 ± 37 0.6 – 11 (3.9)

Thrombus 11 (5.9%) 3 / 8 35 ± 22 1.8 – 5.7 (2.9)

Sarcoma 7 (3.8%) 3 / 4 55 2.5 – 6.4 (4.6)

Papillary Fibroelastoma 4 (2.2%) 2 / 2 46 0.6 – 1.8 (1)

Lipoma 4 (2.2%) 2 / 2 45 1.5 – 8.3 (4.7)

Rhabdomyoma 4 (2.2%) 1 / 3 0.03 2.2 – 10 (4.3)

Angissarcoma 3 (1.6%) 1 / 2 40 2.5 – 3 (2.7)

Osteossarcoma 2 (1.1%) 0 / 2 13.5 4.5 – 5.5

Hemangioma 1 (0.5%) 1 / 0 67 2.5

Meningioma 1 (0.5%) 1 / 0 19 9

despite high-dose inotropic support due to surgical resection and reconstruction of involved cardiac cavity. The third patient was a 12 year-old young girl who died of unknown cause during recovery of a 2,5cm left atrial thrombus and ductus

Complete patients’ follow-up was collected by chart review and patients’ phone interview to assess late functional status.

Statistical AnalysisAll data were initially descriptively analyzed. Analysis of

the minimum and maximum values, calculation of means, standard deviations and median were done for quantitative variables. For qualitative variables were calculated absolute and relative frequencies. The continuous variable studied was tumor size, the others were categorical (recurrence, embolism, origin site, histological type, atrial fibrillation, valve disease, death and hospital death).

For comparison of means of two groups (variable tumor size) it was used the Student t test (normal data distribution). The test of proportions of the categorical variables were performed using the chi-square or Fisher’s exact test (when expected frequencies less than 5%). Long-term cumulative evaluations were assessed by means of Kaplan-Meier analysis and curves were compared with the log-rank test. A p value less than 0.05 was considered statistically significant. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) version 16.0 for Windows (IBM Corporation Armonk, New York).

This study was approved by our Institutional Ethics Committee / Review Board, which waived the requirement for informed patient consent due to the retrospective design of this study.

ResultsOne hundred seventy four patients (93.6%) had benign

cardiac tumors. Their preferred implantation site was the left side of the heart (88%). 78% were in the left atrium, 8% in the left ventricle, 13% in the right atrium and 2% in the right ventricle. Myxoma was the most common histological type (72.6%), followed by fibroma (7%) and intracardiac thrombus (6.5%); the remaining types are listed in table 3.

Hospital mortality was 5.4%: four for patients with benign tumors (2.3%) and six malign (50%). Two infants with rhabdomyoma developed untreated low output syndrome

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arteriosus resection, and necropsy was not allowed. The fourth was a 42-year-old woman with coronary embolization prior to her myxoma resection, who died of untreated low output syndrome after surgery. The six patients with malignant tumors died early during the postoperative period due to rapidly disease progression (54.5%).

When comparing hospital mortality with cells type, more deaths related to malign tumors (p = 0.002) were observed. The histological type was predictor of mortality (rhabdomyomas, sarcomas and angiosarcomas; p < 0.001) (Figure 1) but not of recurrence (p = 0.182). There was no relation between implantation site and mortality (p = 0.346) (Figure 2).

Preoperative thromboembolic event occurred in 37 patients (19.8%) (Table 2), mainly to the central nervous system (10.2%), but also to coronary arteries (4.8%), to lower limbs (4.3%) and to pulmonary territory (0.5%). The tumor size (p = 0.132), pre-operatory atrial fibrillation (p = 0.206) and implantation site (p = 0.121) were not associated with embolic event. Nevertheless, the histological type was predictor of embolic event (osteosarcoma, angiosarcoma, papillary fibroelastoma and lipoma; p = 0.006).

During follow up (mean 87 ± 51.9 months) there were two patients with prior myxoma resection who died 60 and 27 months after surgery of unknown cause and cerebrovascular accident respectively. Actuarial survival was 100%, 98.8% and 98.8% at 1, 5 and 10 years respectively. There were two recurrences to the same cavity; one left atrial mixoma recurrence 11.2 years after the surgical resection and a right atrial osteossarcoma 11 months after the operation. Both were operated on and had an eventful

recovery. One neurological embolic event was observed at 27 months follow up but not related to intracardiac mass.

DiscussionIn this study it was observed that the histological type was

a predictor of mortality and embolic events.

Malignant tumors (sarcomas) were predictors of hospital mortality. Angiosarcoma is the most common malignant tumor of the heart and is characterized by rapid growth, local invasion, and distant metastasis with reserved prognosis. Patients with non metastatic cardiac sarcoma amenable to complete resection experienced improved survival6.

However, the high overall rates of disease progression and mortality, highlight the need for more effective local and systemic treatments that may be used in conjunction with surgery to improve patient outcomes 7. Some authors recommend, in order to reducing mortality, multimodal therapy (surgical resection, radiofrequency ablation, or radiation treatment). They achieved reasonable survival for patients with resected cardiac sarcomas (mean 47 months). Patients with local tumor recurrence or metastatic disease may still benefit from aggressive treatment8.

Elbardissi et al5 in 48 years of experience in 323 patients with primary cardiac tumors found that patients with malignant tumors have a dismal prognosis and despite aggressive adjuvant chemotherapy after resection, the median survival rates of these patients were less than 1 year.

We observed a decrease in mortality over time. In the period between 1980 and 1998 we had 16% of mortality9. At that time,

Figure 1 – The tumors’ histological type was related with higher mortality.

10.9

0.80.7

0.6

0.5

0.4

0.30.2

0.40

0 24 48 72 96 120 144

Months

Survi

val

Benign Malign

168

p < 0.001

192 216 240 264 288

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Arq Bras Cardiol. 2014; 103(1):13-18

the great majority of patients were diagnosed belatedly and, with the advent of routine use of additional methods, such as echo complemented with MRI, the diagnosis can be performed early and avoid complications. In the subsequent period up to 2004, the mortality dropped to 6%10. In this study, the hospital mortality of 5.4% is still considered high, although mainly due to the malignant tumors aggressive behavior.

As it has been written, the majority of cardiac tumors are benign and cardiac myxomas are the most frequent ones (over 70%). Myxoma is the most prevalent cardiac tumor in the adult population, predominating in the female sex, with a gelatinous and transparent gross appearance. Myxoma is usually a single tumor, preferably located in the left atrium and inserted close to the oval fossa. They are considered benign tumors, even though they may behave in a malignant way with local relapse, invasion of the thoracic wall, and embolism11.

The clinical presentation of cardiac tumors is usually divided into cardiac, embolic and systemic manifestations, such as fever, cachexia, arthralgia, Raynaud’s phenomenon, rash, and anemia, which are attributed to the production of certain cytokines, such as interleukin-612, and symptoms related to metastasis. In our series, systemic manifestations were present in 10.9% of the patients, and were reported as fever (4.8%), chronic anemia (4%), weight loss (1.6%) and arthralgia (0.5%).

Despite the fact that 72% of our patients were asymptomatic, cardiac manifestations may be related to tumor’s size, because as they grow, they can progressively obstruct cardiac chambers mimicking valve disease. So, dyspnea may be followed by others symptoms of heart failure, as happened in our series.

In order to avoid complications such as intracavity obstruction or embolic events, the treatment of choice for primary cardiac tumors is resection13.

Primary cardiac tumors should be considered as a possible diagnosis for all patients with embolization, although in our casuist, we had 12 patients (6.4%) with thrombus mimicking primary cardiac tumors. Cardiac imaging has difficulty in categorizing intracardiac mass and some patients may have their mass classified as thrombus after pathological evaluation. Two of these patients were subsequently diagnosed as having antiphospholipid syndrome. There are several cases in literature of atrial and ventricular thrombus related to antiphospholipid syndrome mimicking primary cardiac tumors14. Another recent study4 found a prevalence of cardiac thrombus in 13 of 84 (15.4%) patients with cardiac tumors. They showed thrombotic masses of variable age, occasionally with features of organization and typical concentric fibrinous layers. One case showed evidence of early peripheral calcification. None of the cases contained areas suggestive of residual myxoma or other-type neoplasm.

Most patients, in whom tumor was diagnosed and later confirmed thrombus by pathology, belonged to our initial series in which the diagnosis was established only by echocardiography. We have currently used, to all cardiac masses, transthoracic echo at first, followed by transesophageal echo and resonance to confirm the diagnosis.

Recurrence develops in about 3% of tumors, although the rate is higher with familial cardiac myxomas. Our study showed tumor recurrence in two cases. One of them was a myxoma 11 years after initial surgical treatment. It was observed that the cumulative myxoma recurrence was as high as 13%, and has

Figure 2 – The tumors’ implantation site did not interfere with survival (RA: right atrium; LA: left atrium; RV: right ventricle; LV: left ventricle).

1

0.9

0.8

0.70.6

0.50.4

0.30.2

0.10

0 24 48 72 96 120 144 168 192 216 240 264 288

Months

RA LA LVRV

Survi

val p = 0.346

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Arq Bras Cardiol. 2014; 103(1):13-18

increased steadily up to 4 years after which there was a low hazard of tumor recurrence. There was no association between the method of surgical excision and the incidence of tumor recurrence. Patients who experienced a myxoma recurrence were younger than those who did not (mean 42 years versus 57 years). The site of recurrence was at the location of the original tumor in 81% of cases. These authors recommended, based on this finding, all patients (especially younger patients) who undergo tumor resection should be followed closely with echocardiography semiannually for 4 years after resection5.

ConclusionOur analysis shows that most tumors were located in the

left side of the heart, most of intracardiac mass is benign and myxoma is the most common histological type. And also the histological type was a predictor of death and preoperative embolic event while the implantation site carries no relation to mortality or to embolic event.

Author contributionsConception and design of the research and Acquisition of

data: Dias RR; Analysis and interpretation of the data: Dias RR, Fernandes F, Ramires FJA; Writing of the manuscript: Dias RR, Fernandes F; Critical revision of the manuscript for intellectual content: Mady C, Albuquerque CP, Jatene FB.


reported.

Sources of FundingThere were no external funding sources for this study.


1. Silverman NA. Primary cardiac tumors. Ann Surg. 1980;191(2):127-38.

2. Garatti A, Nano G, Canziani A, Gagliardotto P, Mossuto E, Frigiola A, et al. Surgical excision of cardiac myxomas: twenty years experience at a single institution. Ann Thorac Surg. 2012;93(3):825-31.

3. Roberts WC. Primary and secondary neoplasm of the heart. Am J Cardiol. 1997;80(5):671-82.

4. Strecker T, Rösch J, Weyand M, Agaimy A. Primary and metastatic cardiac tumors: imaging characteristics, surgical treatment, and histopathological spectrum: a 10-year-experience at a German heart center. Cardiovasc Pathol. 2012;21(5):436-43.

5. Elbardissi AW, Dearani JA, Daly RC, Mullany CJ, Orszulak TA, Puga FJ, et al. Survival after resection of primary cardiac tumors: a 48-year experience. Circulation. 2008;118:(14 Suppl):S7-15.

6. Look Hong NJ, Pandalai PK, Hornick JL, Shekar PS, Harmon DC, Chen YL, et al. Cardiac angiosarcoma management and outcomes: 2 0 - y e a r s i n g l e - i n s t i t u t i o n e x p e r i e n c e . A n n S u r g O n c o l . 2012;19(8):2707-15.

7. Truong PT, Jones SO, Martens B, Alexander C, Paquette M, Joe H, et al. Treatment and outcomes in adult patients with primary cardiac sarcoma: the British Columbia Cancer Agency experience. Ann Surg Oncol. 2009;16(12):3358-65.

8. Bakaeen FG, Jaroszewski DE, Rice DC, Walsh GL, Vaporciyan AA, Swisher SS, et al. Outcomes after surgical resection of cardiac sarcoma in the multimodality treatment era. J Thorac Cardiovasc Surg. 2009;137(6):1454-60.

9. Fernandes F, Soufen HN, Ianni BM, Arteaga E, Ramires FJ, Mady C. Primary neoplasms of the heart. Clinical and histological presentation of 50 cases. Arq Bras Cardiol. 2001;76(3):231-7.

10. Dias RR, Stolf NA, Malbouisson LM, Fernandes F, Ramirez FJ, Mady C, et al. Morbidity and embolic potential of left atrial cardiac tumors. Thorac Cardiovasc Surg. 2006;54(6):400-3.

11. Bjessmo S, Ivert T. Cardiac myxoma: 40 years’ experience in 63 patients. Ann Thorac Surg. 1997;63(3):697-700.

12. Elbardissi AW, Dearani JA, Daly RC, Mullany CJ, Orszulak TA, Puga FJ, et al. Embolic potential of cardiac tumors and outcome after resection: a case-control study. Stroke. 2009;40(1):156-62.

13. Cianciulli TF, Saccheri MC, Lax JA, Neme RO, Sevillano JF, Maiori ME, et al. Left ventricular thrombus mimicking primary cardiac tumor in a patient with primary antiphospholipid syndrome and recurrent systemic embolism. Cardiol J. 2009;16(6):560-3.

14. Cianciulli TF, Saccheri MC, Redruello HJ, Cosarinsky LA, Celano L, Trila CS, et al. Right atrial thrombus mimicking myxoma with pulmonary embolism in a patient with systemic lupus erythematosus and secondary antiphospholipid syndrome. Tex Heart Inst J. 2008;35(4):454-7.

References

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Original Article

Impact of Different Obesity Assessment Methods after Acute Coronary SyndromesCaroline N. M. Nunes, Marcos F. Minicucci, Elaine Farah, Daniéliso Fusco, Paula S. Azevedo, Sergio A. R. Paiva, Leonardo A. M. ZornoffFaculdade de Medicina de Botucatu, Botucatu, SP - Brazil

Mailing Address: Leonardo A. M. Zornoff •Faculdade de Medicina de Botucatu - Departamento de Clínica Médica, Rubião Jr., Botucatu. Postal Code 18618-970. São Paulo, SP - BrazilE-mail: [email protected]; [email protected] received December 04, 2013; revised manuscript February 13, 2014; accepted February 18, 2014.

DOI: 10.5935/abc.20140073

Abstract

Background: Abdominal obesity is an important cardiovascular risk factor. Therefore, identifying the best method for measuring waist circumference (WC) is a priority.

Objective: To evaluate the eight methods of measuring WC in patients with acute coronary syndrome (ACS) as a predictor of cardiovascular complications during hospitalization.

Methods: Prospective study of patients with ACS. The measurement of WC was performed by eight known methods: midpoint between the last rib and the iliac crest (1), point of minimum circumference (2); immediately above the iliac crest (3), umbilicus (4), one inch above the umbilicus (5), one centimeter above the umbilicus (6), smallest rib and (7) the point of greatest circumference around the waist (8). Complications included: angina, arrhythmia, heart failure, cardiogenic shock, hypotension, pericarditis and death. Logistic regression tests were used for predictive factors.

Results: A total of 55 patients were evaluated. During the hospitalization period, which corresponded on average to seven days, 37 (67%) patients had complications, with the exception of death, which was not observed in any of the cases. Of these complications, the only one that was associated with WC was angina, and with every cm of WC increase, the risk for angina increased from 7.5 to 9.9%, depending on the measurement site. It is noteworthy the fact that there was no difference between the different methods of measuring WC as a predictor of angina.

Conclusion: The eight methods of measuring WC are also predictors of recurrent angina after acute coronary syndromes. (Arq Bras Cardiol. 2014; 103(1):19-24)

Keywords: Evaluation; Acute Coronary Syndrome; Abdominal Circumference.

IntroductionObesity is currently considered an epidemic in

many countries and is one of the main health problems of contemporary society, being associated with high prevalence and incidence of cardiovascular disease1. Cardiovascular disease, in turn, is the leading cause of death and disability worldwide. Despite the decrease in the proportion of deaths from cardiovascular disease in developed countries in recent decades, rates have grown enormously in low- and middle-income ones2.

Complications associated with overweight and obesity are related mainly to adipose tissue deposition, which leads to excess adiposity or body fat. However, the way in which fat is distributed throughout the body can be more important than total body fat in determining cardiovascular risk. Thus, recent

evidence suggests that, although excess fat is associated with cardiovascular risk, the accumulation of intra-abdominal adipose tissue, specifically, is characterized by more severe cardiovascular risk3-7.

There are several indirect methods to precisely estimate the total amount of body fat, as well as its distribution, to establish the diagnosis of obesity. Comparison of anthropometric measures with diagnostic imaging tests, such as magnetic resonance imaging and computed tomography, shows that waist circumference (WC) was the anthropometric variable that showed the best correlation with visceral adipose tissue8.

However, in a recent literature review9, experts detected eight different documented methods of measuring the WC: the midpoint between the last rib and the iliac crest (1), at the point of minimum circumference (2); immediately above the iliac crest (3), at the umbilicus (4), one inch above the umbilicus (5), one centimeter inch above the umbilicus (6), at the smallest rib (7) and the point of greatest circumference around the waist (8). This variability in WC measurement may hinder the use of this measurement as a marker of cardiovascular risk.

Thus, the aim of this study was to evaluate the eight measurement sites of WC in hospitalized patients with acute coronary syndrome and determine which methods are predictors of cardiovascular complications during hospitalization.

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Arq Bras Cardiol. 2014; 103(1):19-24

Table 1 – Presence of complications

Variables n (%)

Angina 8 (16.3)

Arrhythmia 5 (10.2)

Congestive heart failure 18 (36.7)

Cardiogenic shock 3 (6.10)

Hypotension 14 (28.6)

Pericarditis 1 (2.00)

Death 0 (0.00)

MethodsThis is a prospective, observational study carried out in

the Coronary Care Unit of our institution. Patients with acute coronary syndrome, characterized by acute myocardial infarction with ST-segment elevation (STEMI), acute myocardial infarction without ST-segment elevation (NSTEMI) or unstable angina (UA)10, 11 between August 2012 and April 2013, were included in the study.

The diagnosis of STEMI was made by the presence of ST-segment elevation in at least two leads that represent the same region, larger than 2 mm in men and 1.5 mm in women in leads V1-V3; or greater than 1 mm in other leads10. The diagnosis of NSTEMI was based on elevated marker of myocardial injury (troponin). Unstable angina was diagnosed by the presence of: recent angina with intensity of at least II of CCS; angina at rest and prolonged; angina after acute myocardial infarction (AMI); or accelerated angina, according to previous definitions11. The study protocol was approved by the Ethics Committee of our institution and patients were enrolled after signing a free and informed consent form.

Regarding the clinical profile, data were obtained from the clinical history and physical examination on admission. The variables analyzed were: age, gender, ethnicity, heart rate, blood pressure and duration of chest pain, the symptom onset to the time of initial evaluation in the emergency room. Regarding laboratory data, it included peak total creatine phosphokinase (CPK) isoenzyme MB (CK-MB) and troponin. The investigated risk factors were patient’s personal and family history, smoking status and presence of hypertension, diabetes mellitus, dyslipidemia, family history of premature atherosclerosis and obesity.

The assessed treatments were platelet antiaggregants, anticoagulants, angiotensin-converting enzyme (ACE) inhibitors, beta-blockers, calcium-channel blockers, nitrates, positive inotropic agents, diuretics and reperfusion therapy.

The presence of complications was evaluated during hospital stay and the following variables were considered: angina, characterized by chest pain with angina characteristics and/or acute and dynamic ischemic changes at ECG (T-wave inversion or ST segment elevation/depression); arrhythmias (ventricular fibrillation, ventricular tachycardia, sinus bradycardia, AV block of at least second degree, atrial tachyarrhythmias); cardiogenic shock defined by systemic hypotension (systolic blood pressure ≤ 80 mmHg), signs of hypoperfusion, such as cold extremities, oliguria and dyspnea caused by pulmonary congestion; heart failure, characterized by clinical or radiological pulmonary congestion requiring intravenous diuretics; arterial hypotension, when the systolic blood pressure is ≤ 80 mmHg; pericarditis (confirmed by echocardiography) and death. These and other definitions were similar to those of previous studies11-15.

WC measurements were performed by the eight methods described previously. Regarding the statistical analysis, continuous variables were tested for normality; when the variables were tested, the mean values and standard deviations were calculated for the studied groups. In the case of normal distribution, Student's t test was used to compare

variables. For nonparametric variables, median values and interquartile ranges were calculated and Mann-Whitney test was used to compare the groups.

Existing associations between the independent variables and complications were analyzed by means of uni- and multivariate logistic regression analyses. Data analysis was performed with the statistical package SigmaPlot v. 12.0. The level of significance was set at 5% for all tests.

ResultsA total of 55 patients, mean age of 62 ± 12 years, 42 (76%)

males, were assessed. During the hospitalization period, which corresponded on average to seven days, 37 (67%) patients had at least one complication observed, except death, which was not observed in none of the cases, as shown in Table 1.

Of the variables analyzed, including clinical profiles, risk factors and medications used by patients, only diastolic blood pressure showed a positive association with the occurrence of angina (Tables 2 and 3).

Considering the complications, angina was the only variable that, after being adjusted for gender, age and infarct size, showed a positive association (p <0.05) with abdominal obesity, regardless of the method used for WC measurement, as shown in Table 4. Additionally, we observe that for every centimeter of waist circumference increase, the risk for angina increased from 7.5 to 9.9%, depending on the measurement site, as shown in Table 5.

DiscussionThe aim of this study was to investigate the impact of

eight sites for the measurement of waist circumference in predicting complications after acute coronary syndromes during patient hospitalization. The results suggest that all sites used for WC measurement are associated with the incidence of angina after acute cardiac events, with no significant differences among them.

The first aspect to be considered refers to the fact that some studies suggest that body fat distribution, as assessed by WC and waist-hip ratio (WHR), may be more relevant than BMI as a cardiovascular risk factor16. This topic is controversial, as the study that evaluated WC as a predictor of 30-day evolution in patients with acute coronary

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Table 2 – Demographic, clinical and laboratory data

VariablesAngina

p valueNo (n = 47) Yes (n = 8)

Demographic data - - -

Age (years) 64.4 ± 11.1 60.5 ± 14.1 0.385

Time of hospitalization (days) 6.00 (4.00 – 8.00) 7.00 (6.30 – 8.00) 0.355

Time of precordial pain (minutes) 240.0(30.00 – 1440) 135.0 (75.00 – 1155) 0.924

Male gender, n (%) 35.0 (74.5) 7.00 (87.0) 0.664

Diagnosis - - -

NSTEMI, n (%) 13 (27.7) 2 (25.0) 0.918

Inferior AMI, n (%) 13 (27.7) 3 (37.5) 0.918

Anterior AMI, n (%) 8 (17.0) 1 (12.5) 0.918

HRUA, n (%) 10 (21.3) 1 (12.5) 0.918

MRUA, n (%) 3 (6.40) 1 (12.5) 0.918

Raça - - -

Caucasian, n (%) 34 (72.3) 6 (75.0) 0.949

African descendant, n (%) 12 (25.5) 2 (25.0) 0.949

Asian, n (%) 1 (2.2) 0 (0) 0.949

Family history - - -

Smoking, n (%) Yes 40 (85.1) 5 (62.5) 0.149

SAH, n (%) Yes 23 (48.9) 4 (50.0) 1.000

DM, n (%) Yes 18 (38.3) 4 (50.0) 0.700

DLP, n (%) Yes 13 (27.7) 3 (37.5) 0.678

Obesity, n (%) Yes 23 (48.9) 7 (87.5) 0.059

Personal history - - -

Smoking, n (%) Yes 14 (29.8) 1 (12.5) 0,423

SAH, n (%) Yes 34 (72.3) 8 (100.0) 0,176

DM, n (%) Yes 17 (36.2) 5 (62.5) 0,244

DLP, n (%) Yes 43 (91.5) 7 (87.5) 0,559

Obesity, n (%) Yes 20 (42.5) 5 (62.5) 0,446

Clinical data - - -

HR (bpm) 71,0 (63,0 – 83,0) 75.5 (65.2 – 92.0) 0.310

SBP (mmHg) 120 (102 – 136) 115 (105 – 133) 0.943

DBP (mmHg) 66,0 (60,0 – 80,0) 78.5 (70.0 – 88.5) 0.012

Laboratory data - - -

CPK (U/L) 333,0 (109,0 – 2678) 320.0 (88.2 – 2764) 0.711

CK-MB (U/L) 60,0 (160-235) 38.5 (15.0 – 282) 0.711

Troponin (U/L) 0,90 (0,20 – 4,00) 1.80 (0.0 0– 8.30) 0.886

NSTEMI: non-ST segment elevation acute myocardial infarction; HRUA: high-risk unstable angina; MRUA: moderate-risk unstable angina; SAH: systemic arterial hypertension; DM: diabetes mellitus; DLP dyslipidemia; HR: heart rate; SBP: systolic blood pressure; DBP: diastolic blood pressure; CPK: Total creatine phosphokinase; CK-MB: MB isoform.

syndrome (ACS) in reference hospital for the treatment of cardiovascular disease, found no association between WC and major cardiovascular events17.

On the other hand, in other studies, WC was the measure most associated with risk factors and death from cardiovascular disease18-20. Additionally, in an analysis of 6,560 patients with

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Table 3 – Drug therapy

VariablesAngina

p valueNo (n = 47) Yes (n = 8)

Reperfusion therapy (angioplasty), n (%) 10 (21.3) 1 (12.5) 1.000

ASA and clopidogrel therapy, n (%) 46 (97.9) 8 (100) 1.000

Enoxaparin therapy, n (%) 1 (100) 49 (90.7) 1.000

ACEI, n (%) 3 (60.0) 32 (64.0) 1.000

Beta-blockers, n (%) 38 (80.8) 8 (100) 0.327

Calcium-channel blockers, n (%) 7 (14.9) 2 (25.0) 0.604

Nitrates, n (%) 10 (21.7) 3 (33.3) 0.428

Positive inotropics, n (%) 9 (19.1) 1 (12.5) 1.000

Diuretics, n (%) 30 (63.8) 3 (37.5) 0.244

ACEI: angiotensin-converting enzyme inhibitors.

ACS, the disproportion between body mass index and WC (indicative of central obesity) increased the probability of cardiovascular death, myocardial infarction and recurrent ischemia21. Similarly, the WC, but not BMI, was a predictor of the remodeling process after anterior-wall acute myocardial infarction7. Therefore, identifying the best method of WC measurement may have important clinical implications.

The main finding of our study was that there was no difference between the WC measurement procedure and complications during hospital stay in patients hospitalized with ACS. In this sense, the WC, regardless of the method used, could predict recurrent angina during hospitalization. For each centimeter of WC increase, the patients in our study had, on average, a nine-fold higher chance to have angina. Similarly,

Table 4 – Association of different sites for waist circumference measurement and the incidence of angina

WC measurement method (cm) Angina (Yes) n = 8 Angina (No) n = 47 p

Greatest circumference 114.5 ± 16.70 99.80 ± 14.47 0.012

Smallest rib 106.5 (100.5 - 117.0) 93.00 (85.00 - 104.0) 0.011

One centimeter above the umbilicus 113.1 ± 16.10 97.80 ± 14.50 0.009

One inch above the umbilicus 113.3 ± 17. 90 96.80 ± 14.30 0.006

Umbilicus 114.4 ± 18.70 98.60 ± 14.10 0.007

Above iliac crest 110.2 ± 17.80 98.10 ± 13.40 0.029

Minimum circumference 107.6 ± 12.70 94.30 ± 13.00 0.010

Midpoint 113.1 ± 16.40 97.70 ± 13.70 0.006

WC: waist circumference.

Table 5 – Logistic regression for predicting angina adjusted for gender, age and peak CPK

Variables OR (%) 95%CI p value

Greatest circumference 1.081 1.016 - 1.151 0.015

Smallest rib 1.095 1.017 - 1.179 0.016

One centimeter above the umbilicus 1.088 1.018 - 1.164 0.013

One inch above the umbilicus 1.090 1.020 - 1.166 0.011

Umbilicus 1.086 1.018 - 1.158 0.012

Above iliac crest 1.075 1.010 - 1.144 0.023

Minimum circumference 1.096 1.016 - 1.182 0.017

Midpoint 1.099 1.022 - 1.182 0.011

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1. James PT, Leach R, Kalamara E, Shayeghi M. The worldwide obesity epidemic. Obes Res. 2001;9 Suppl 4:228S-33S.

2. Yusuf S, Hawken S, Ounpuu S, Bautista L, Franzosi MG, Commerford P, et al. Obesity and the risk of myocardial infarction in 27,000 participants from 52 countries: a case-control study. Lancet. 2005;366(9497):1640-9.

3. Despre´s JP, Moorjani S, Lupien PJ, Tremblay A, Nadeau A, Bouchard C. Regional distribution of body fat, plasma li)oproteins, and cardiovascular disease. Arteriosclerosis. 1990;10(4):497-511.

4. Kelley DE, Thaete FL, Troost F, Huwe T, Goodpaster BH. Subdivisions of subcutaneous abdominal adipose tissue and insulin resistance. Am J Physiol Endocrinol Metab. 2000;278(5):E941-8.

5. Carmienke S, Freitag MH, Pischon T, Schlattmann P, Fankhaenel T, Goebel H, et al. General and abdominal obesity parameters and their combination in relation to mortality: a systematic review and meta-regression analysis. Eur J Clin Nutr. 2013;67(6):573-85.

6. Tchernof A, Després J-P. Pathophysiology of human visceral obesity: an update. Physiol Rev. 2013;93(1):359-404.

7. Lee MJ, Wu Y, Fried SK. Adipose tissue heterogeneity: implication of depot differences in adipose tissue for obesity complications. Mol Aspects Med. 2013;34(1):1-11.

8. Pouliot MC, Després JP, Lemieux S, Moorjani S, Bouchard C, Tremblay A, et al. Waist circumference and abdominal sagittal diameter: best simple anthropometric indexes of abdominal visceral adipose tissue accumulation and related cardiovascular risk in men and woman. Am J Cardiol. 1994;73(7):460-8.

9. Cornier MA, Després JP, Davis N, Grossniklaus DA, Klein S, Lamarche B, et al. Assessing Adiposity: a Scientific Statement from the American Heart Association. Circulation. 2011;124(18):1996-2019.

10. O’Gara PT, Kushner FG, Ascheim DD, Casey DE Jr, Chung MK, de Lemos JA, et al. 2013 ACCF/AHA guideline for the management of ST-elevation myocardial infarction: executive summary: a report of the American College of Cardiology

References

none of the methods of WC measurement was associated with the other evaluated complications. Therefore, one can deduce that any of the eight available methods for WC assessment could be incorporated into clinical practice.

Another relevant aspect is related to the mechanisms involved in the cardiovascular risk associated with abdominal obesity. Although the mechanisms are complex and not completely understood, several hypotheses have been formulated. For instance, the visceral adipose tissue has greater capacity to secrete components of the renin-angiotensin-aldosterone system. Similarly, abdominal obesity would more adversely modulate the release / inhibition of substances secreted by adipose tissue (adipokines), which can regulate blood pressure, insulin sensitivity, energy homeostasis, immune response, oxidative stress and inflammatory response5-7. Therefore, these mechanisms alone or concomitant, could explain the association found in our study between waist circumference and recurrent ischemia.

Some issues should be considered when interpreting our results. Firstly, the clinical, demographic and laboratory data, as well as drug therapy used for the treatment of patients at the hospital discharge showed no association with the presence of angina in the studied population. Thus, in the absence of better markers, our result further highlights the importance of using different sites for WC measurement when predicting the risk of angina in individuals at risk of cardiovascular complications.

A second characteristic that expands the importance of abdominal obesity assessment is that the measure of the WC is considered a reliable, easy to use and low-cost anthropometric indicator. Additionally, the WC may have other important applications, considering it is used to predict the risk of early onset of certain diseases such as diabetes mellitus and other cardiovascular diseases, as well as to provide useful information to identify populations at risk even before obesity is identified through body mass index22,23.

Finally, some limitations should be taken into account when interpreting the results. Our study assessed a

low number of patients and included patients from a single center. Despite these limitations, we believe our study raises two important hypotheses to be confirmed in large clinical trials: first, this study suggests no significant differences between the different methods of WC assessment; and, second, our data suggest that WC measurement may be a clinically relevant marker for predicting the risk of angina after acute coronary syndromes and may, in this scenario, be incorporated as a marker of cardiovascular risk by health professionals.

ConclusionFor these reasons, we conclude that the eight methods

used for waist circumference measurement are associated with the presence of angina after acute coronary events, with no significant differences between them.

Author contributionsConception and design of the research: Azevedo PS,

Paiva SAR, Zornoff LAM; Acquisition of data: Nunes CNM, Minicucci MF, Farah E, Fusco D; Analysis and interpretation of the data: Nunes CNM, Minicucci MF, Farah E, Fusco D, Zornoff LAM; Statistical analysis: Minicucci MF, Paiva SAR; Writing of the manuscript: Nunes CNM, Minicucci MF, Azevedo PS, Paiva SAR, Zornoff LAM; Critical revision of the manuscript for intellectual content: Azevedo PS, Zornoff LAM.


reported.



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Foundation/American Heart Association Task Force on Practice Guidelines: developed in collaboration with the American College of Emergency Physicians and Society for Cardiovascular Angiography and Interventions. Circulation. 2013;127(4): e362-425. Erratum in Circulation. 2013;128(25):e481.

11. Hamm CW, Bassand JP, Agewall S, Bax J, Boersma E, Bueno H,et al. ESC guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation. The Task Force for the management of acute coronary syndromes (ACS) in patients presenting without persistent ST-segment elevation of the European Society of Cardiology (ESC). Eur Heart J. 2011;32(23):2999-3054.

12. Cogni AL, Farah E, Minicucci MF, Azevedo PS, Okoshi K, Matsubara BB, et al. Waist circumference, but not body mass index, is a predictor of ventricular remodeling. Nutrition. 2013;9(1):122-6.

13. Farah E, Cogni AL, Minicucci MF, Azevedo PS, Okoshi K, Matsubara BB, et al. Prevalence and predictors of ventricular remodeling after anterior myocardial infarction in the era of modern medical therapy. Med Sci Monit. 2012;18(5):CR276-81.

14. Azevedo PS, Cogni AL, Farah E, Minicucci MF, Okoshi K, Matsubara BB, et al. Predictors of right ventricle dysfunction after anterior myocardial infarction. Can J Cardiol. 2012;28(4):438-42.

15. Farah E, Fusco DR, Okumoto PR, Minicucci MF, Azevedo PS, Matsubara BB, et al. Impact of ventricular geometric pattern on cardiac remodeling after myocardial infarction. Arq Bras Cardiol. 2013;100(6):518-23.

16. Pitanga FJ, Lessa I. Indicadores antropométricos de obesidade como instrumento de triagem para risco coronariano elevado em adultos na cidade de Salvador - Bahia. Arq Bras Cardiol. 2005;85(1):26-31.

17. Souza PA, Fayh AP, Portal VL. Circunferencia abdominal como preditor de evolução em 30 dias na síndrome coronariana aguda. Arq Bras Cardiol. 2011;96(5):399-404.

18. Schneider HJ, Glaesmer H, Klotsche J, Böhler S, Lehnert H, Zeiher AM, et al. Accuracy of anthropometric indicators of obesity to predict cardiovascular risk. J Clin Endocrinol Metab. 2007;92(2):589-94.

19. Rezende FA, Rosado LE, Ribeiro RL, Vidigal FC, Vasques AJ, Bonard IS, et al. Indice de massa corporal e circunferencia abdominal: associação com fatores de risco cardiovascular. Arq Bras Cardiol. 2006;87(6):728-34.

20. Hauner H, Bramlage P, Lösch C, Steinhagen-Thiessen E, Schunkert H, Wasem J, et al. Prevalence of obesity in primary care using different anthropometric measures: results of the German Metabolic and Cardiovascular Risk Project (GEMCAS). BMC Public Health. 2008;8:282.

21. Kadakia MB, Fox CS, Scirica BM, Murphy SA, Bonaca MP, Morrow DA. Central obesity and cardiovascular outcomes in patients with acute coronary syndrome: observations from the MERLIN-TIMI 36 trial. Heart. 2011;97(21):1782-7.

22. Pouliot M, Després J, Lemieux S, Moorjani S, Bouchard C, Tremblay A, et al. Waist circumference and abdominal sagittal diameter: Best simple anthropometric indexes of abdominal visceral adipose tissue accumulation and related cardiovascular risk in men and women. Am J Cardiol. 1994; 73(7):460-8.

23. Zhu S, Heshka S, Wang Z, Shen W, Allison D, Ross R, et al. Combination of BMI and waist circumference for identifying cardiovascular risk factor in whites. Obes Res. 2004;12(4):633-45.

24

Original Article

Resistance Exercise Restores Endothelial Function and Reduces Blood Pressure in Type 1 Diabetic RatsMarcelo Mendonça Mota1, Tharciano Luiz Teixeira Braga da Silva1, Milene Tavares Fontes1, André Sales Barreto1, João Eliakim dos Santos Araújo1, Antônio Cesar Cabral de Oliveira2, Rogério Brandão Wichi2, Márcio Roberto Viana Santos1

Departamento de Fisiologia - Universidade Federal de Sergipe (UFS)1; Departamento de Educação Física - UFS2, São Cristóvão, SE − Brazil

Mailing Address: Marcio Roberto Viana Santos •Departamento de Fisiologia, Universidade Federal de Sergipe, Avenida Marechal Rondon, s/n, Rosa Elze. Postal Code 49100-000, São Cristóvão, SE − BrazilE-mail: [email protected], [email protected] Manuscript received June 29, 2013; revised manuscript November 19, 2013; accepted November 28, 2013.

DOI: 10.5935/abc.20140087

Abstract

Background: Resistance exercise effects on cardiovascular parameters are not consistent.

Objectives: The effects of resistance exercise on changes in blood glucose, blood pressure and vascular reactivity were evaluated in diabetic rats.

Methods: Wistar rats were divided into three groups: control group (n = 8); sedentary diabetic (n = 8); and trained diabetic (n = 8). Resistance exercise was carried out in a squat device for rats and consisted of three sets of ten repetitions with an intensity of 50%, three times per week, for eight weeks. Changes in vascular reactivity were evaluated in superior mesenteric artery rings.

Results: A significant reduction in the maximum response of acetylcholine-induced relaxation was observed in the sedentary diabetic group (78.1 ± 2%) and an increase in the trained diabetic group (95 ± 3%) without changing potency. In the presence of NG-nitro-L-arginine methyl ester, the acetylcholine-induced relaxation was significantly reduced in the control and trained diabetic groups, but not in the sedentary diabetic group. Furthermore, a significant increase (p < 0.05) in mean arterial blood pressure was observed in the sedentary diabetic group (104.9 ± 5 to 126.7 ± 5 mmHg) as compared to that in the control group. However, the trained diabetic group showed a significant decrease (p < 0.05) in the mean arterial blood pressure levels (126.7 ± 5 to 105.1 ± 4 mmHg) as compared to the sedentary diabetic group.

Conclusions: Resistance exercise could restore endothelial function and prevent an increase in arterial blood pressure in type 1 diabetic rats. (Arq Bras Cardiol. 2014; 103(1):25-32)

Keywords: Rats; Exercise; Physical endurance; Endothelium, vascular / physiology; Arterial Pressure / physiology; Diabetes.

Introduction Diabetes mellitus is a heterogeneous group of metabolic

disorders that have in common hyperglycemia associated with secondary cardiovascular system complications1,2. Increased blood glucose levels are associated with in vivo and in vitro endothelial dysfunction3,4. Endothelial dysfunction is a systemic phenomenon related to an unbalance in the endothelial production of mediators that regulate vascular tone; it contributes partially to increase arterial blood pressure levels5. The endothelial dysfunction in type 1 diabetes mellitus can be considered an early marker of cardiovascular disease6.

Several factors, such as hyperlipidemia, insulin resistance, hyperglycemia and hypertension, can explain the endothelial dysfunction in type 1 diabetes mellitus7. Resistance exercise

has been reported to contribute to prevent/treat pathologies that affect the metabolism and cardiovascular function8-10. Resistance exercise has proved to have an important therapeutic potential by promoting skeletal muscle mass gain, increased insulin sensitivity and blood glucose reduction in diabetic rats8,11. Aerobic exercise also have those effects11,12.

Some studies have suggested that aerobic exercise is effective to treat endothelial dysfunction in diabetes13-15. However, little is known about the chronic effects of resistance exercise on the arterial blood pressure and endothelial function of type 1 diabetic rats. We raised the hypothesis that long-term resistance exercise can minimize the deleterious effects on the cardiovascular system and on the metabolic control of type 1 diabetes mellitus-induced animals. Thus, this study aimed at assessing the chronic effects of resistance exercise on blood glucose changes, vascular reactivity and arterial blood pressure of diabetic rats.

Methods

Animals and experimental delineation Male Wistar rats (Rattus norvegicus), aged 3 months,

weighing 250-300 g, were used in all experiments. They were

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kept under controlled temperature (22 ± 1 °C) and 12-hour light-dark cycles, with free access to water and food specific for rodents (Labina, Purina®). All procedures described in this study were approved by the Committee on Ethics and Animal Research of the Universidade Federal de Sergipe (UFS) (protocol number 01/2008). The animals were divided into the following three groups of eight animals each: control group (C); diabetic sedentary (DS); and diabetic trained (DT). In groups C and DS, the animals were kept in their cages and not exposed to exercise, while group DT animals underwent eight weeks of resistance exercise.

DrugsThe following drugs were used: acetylcholine chloride

(ACh); L-phenylephrine (Phe); NG-nitro-L-arginine methyl ester (L-NAME); alloxan (SIGMA®, USA); and sodium thiopental (Thiopentax, Cristália, Itapira, SP, Brazil).

Induction of diabetes and blood glucose measurement Experimental diabetes was induced as described by Da

Silva Costa et al16. After a 24-hour fasting period, diabetes was induced by use of a single intravenous dose of alloxan (40 mg/kg - penile vein), two weeks before starting the exercise protocol. The animals with blood glucose level ≥ 200 mg/dL were selected as diabetic. Blood glucose level was measured one week after alloxan administration by using reagent test strips (ACCU-CHEK Advantage II, Roche®, São Paulo, SP, Brazil) coupled to a portable digital glucometer (ACCU-CHEK Advantage II, Roche®, São Paulo, SP, Brazil).

Exercise protocolResistance exercise was performed in a squat device

according to the model by Tamaki et al17. After one week of adaptation, DT animals underwent training with three sets of ten repetitions, with 60-second rest intervals and intensity of 50% of the load established through the one-repetition maximum (1RM) test, three times a week. For determining the maximum strength, successive loads were added to the equipment, and the animals were electrically stimulated to repeat the exercise. Between load increments, 5-minutes rests were observed to allow the musculature to recover. The maximum load for each animal was the maximum amount of weight allowing the complete movement. Training loads were readjusted every two weeks by using a new 1RM test18. The parameters of electrical stimulation were those described by Cássia Cypriano Ervati Pinter et al19. Electrical stimulation (20 V, 0.3-second duration, 3-second interval)20 with electrodes (Valu Trode, Model CF3200, Axelgaard, Fallbrook, CA, USA) fixed to the animals’ tails and connected to an electrical stimulator (BIOSET, Physiotonus Four, Model 3050, Rio Claro, SP, Brazil) was used to make the animals performed their exercise sets.

Surgical procedure and direct recording of mean arterial blood pressure

For this procedure, the animals were anesthetized with sodium thiopental (45 mg/kg, intraperitoneal), and a polyethylene catheter (PE-10/50, Intramedic, Becton

Dickinson and Company, Sparks, MD, USA) with heparin saline (1:20 v/v) was implanted through an inguinal incision in the left femoral artery to record arterial blood pressure. After insertion and fixation, the catheter was exteriorized in the animal’s posterior cervical region (scapulae) and the incision, sutured. After finishing the surgical procedures, all animals received a single intramuscular dose (0.2 g/kg) of oxytetracycline hydrochloride (long-lasting antibiotic) and oral sodium diclofenac (10 mg/kg/day). Then, the animals were put into individual cages, where they remained for a minimum period of 24 hours (postoperative recovery).

After the animals recovered and were moving spontaneously, the catheter was coupled to a pressure transducer (Edwards Lifescience®, Irvine, CA, USA), and 30 minutes after the signal stabilized, a 5-minute recording was performed. After recording the mean arterial blood pressure for 24 hours, the animals were anesthetized and prepared for the vascular reactivity experiments.

Vascular reactivity of the superior mesenteric arteryThe tissue was prepared as described by Araujo et al10.

Endothelial function was assessed via the ability of 1 µM of ACh to induce relaxation in more than 75% of the previously contracted superior mesenteric artery rings with 1 µM of FEN21.

The vascular reactivity changes were assessed by using concentration-response curves for ACh (10-9-10-4 M), a non-selective muscarinic agonist. To assess the nitric oxide (NO) participation in ACh-induced relaxation, the curves for that agent were also obtained in the presence of L-NAME (100 µM), a nitric oxide synthase (NOS) inhibitor.

Statistical analysesInitially, all data underwent the Kolmogorov-Smirnov test to

determine whether the probability distributions were parametric or non-parametric. All data had normal distribution. The values were expressed as mean ± standard error of the mean (SEM). When necessary, Student t test for independent samples and repeated measures or two-way analysis of variance (ANOVA), followed by the Bonferroni post-test, were used to assess the significance of the differences between the means. Pearson’s correlation was used to determine the association between ACh-induced relaxation and blood glucose levels. The significance level adopted was p < 0.05. The GraphPad Prism statistical software, version 3.02 (GraphPad Software, San Diego, CA, USA), was used for all procedures.

Results

Maximum strengthAt the beginning of the experiment, the strength levels

were similar in all groups (C: 956.3 ± 63.3 g, n = 8; DS: 1,022.2 ± 32.3 g, n = 8; and DT: 945.4 ± 108.7 g, n=8). After eight weeks, the strength levels of animals in groups C and DS showed no statistically significant difference (1,032.2 ± 44.0 and 1,030.5 ± 61.2 g, respectively). In addition, the resistance exercise promoted an increase in strength levels from 945.4 ± 108.7 g to 1,327.3 ± 98.7 g (p < 0.01).

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Blood glucose levelsFigure 1 shows the effect of resistance exercise on blood

glucose levels. Alloxan induced an increase (p < 0.001) in blood glucose concentration in both experimental groups. In addition, resistance exercise caused a reduction (p < 0.05) in blood glucose levels after eight weeks (Figure 1).

Endothelium-dependent relaxationFigure 2 shows ACh induced relaxation, depending on its

concentration, in isolated rings of the superior mesenteric artery, with intact endothelium in all groups. Neither diabetes, nor resistance exercise interfered with arterial sensitivity, considering that the concentration from which the agonist produces 50% of maximum response (potency - pD2) remained unaltered. However, in group DS, diabetes reduced (p < 0.001) the maximum response (Rmax) as compared to that in group C. That was reversed (p < 0.01) in DT animals. In addition, a strong negative correlation was observed between ACh-induced relaxation and blood glucose levels in groups DS (r = -0.9710; p = 0.001, n = 8) and DT (r = -0.9874; p = 0.001, n = 8) (Figure 3).

Endothelium-dependent relaxation in the presence of L-NAMETable 1 shows that L-NAME could reduce (p < 0.001)

pD2 and Rmax (p < 0.01 and p < 0.001) of ACh-induced relaxations in groups C and DT, respectively. That reduction was not observed in group DS. The presence of L-NAME significantly increased (p < 0.001) the pD2 of ACh-induced relaxations of group DS as compared to group C, without modifying the Rmax. However, L-NAME significantly reduced (p < 0.001) the pD2 and Rmax (p < 0.01) of the ACh-induced relaxations of group DT as compared to group DS.

Mean arterial blood pressureDiabetes induction with alloxan increased (p < 0.05)

mean arterial blood pressure in group DS. Inversely, resistance exercise reduced (p < 0.05) arterial blood pressure levels of group DT animals (Figure 4).

DiscussionAccording to the results, resistance exercise reduces blood

glucose levels, restores endothelial function and decreases arterial blood pressure of type 1 diabetic animals after eight weeks of training.

Resistance exercise is characterized by intermittent movements and a predominantly anaerobic metabolic pathway22,23. In this study, resistance exercise was performed in a squat device for rats, and proved to be effective in mimicking the beneficial cardiovascular effects found in humans practicing that type of exercise19,20. Tail electrical stimulation was required for the animals to perform the squat movement. The electrical stimulation parameters used in this study have been reported to promote no cardiovascular system changes20. Based on the literature, we suggest that the effects observed in trained diabetic animals are directly related to resistance exercise.

The maximum strength test was used as an indicator of training efficacy. From that perspective, we observed that trained diabetic animals gained muscle strength after eight weeks. This indicates that the training protocol could promote chronic adjustments resulting from resistance exercise. The importance of the therapeutic potential of that type of exercise has been recently emphasized9. Resistance exercise has proved to have a beneficial effect on insulin action improvement, muscle mass gain, fatty mass reduction, blood glucose control and arterial blood pressure reduction in individuals with diabetes24-26.

Experimental and clinical evidence has shown that metabolic disorders, mainly the chronic increase in blood glucose levels, are strictly related to cardiovascular complications of diabetes mellitus7,26. To better understand the metabolic and cardiovascular complications originating from diabetes, several experimental models of diabetes induced in rats have been widely used by different research groups27. Alloxan has been reported to cause the destruction of a large number of beta-pancreatic cells, hindering insulin production27,28. The experimental model of alloxan-induced diabetes is of type 1, with symptoms similar to those found in humans, such as weight loss, polyuria, polydipsia, polyphagia, glycosuria, ketonuria, increased production of oxygen reactive species, decreased blood insulin levels and increased blood glucose levels27-29.

The increased blood glucose levels of diabetic animals observed at the beginning of the study were reduced after eight weeks of training. Similarly, Farrell et al8 have shown that treatment with resistance exercise, by the end of eight weeks, reduced blood glucose levels of animals with type 1 diabetes. Studies have shown that muscle contraction during physical exercise stimulates the translocation of glucose transporter type 4 (GLUT4), independently of insulin action, resulting in an increase in peripheral glucose uptake30,31. Thus, a possible explanation for blood glucose level reduction in trained animals in this study could relate to a greater activation in the signaling pathways involved in glucose transportation regardless of insulin action, because our animals had insulin deficiency or lack of insulin production, since ours was a type 1 diabetes mellitus model8,28,32.

According to Gross et al33, increased blood glucose levels cause damages, dysfunctions and even failure of several organs, involving severe micro- and macrovascular changes. In some cases, restoration of normal blood glucose levels reverts cell damages. In others, however, such damages are irreversible, making blood glucose control a physiological parameter of essential importance to prevent the severe chronic complications of diabetes30-33. Studies have shown that diabetes mellitus causes changes in endothelium-dependent relaxation of different vascular beds, promoting endothelial dysfunction34,35. Endothelial dysfunction is considered a biomarker of cardiovascular risk, and the importance of the endothelium in maintaining vascular health is a consensus in the literature36.

The group DS animals showed loss of vascular function. However, eight weeks of resistance exercise could restore the vascular function of diabetic animals. That can be due

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Figure 1 – Variation of blood glucose levels of rats at the beginning (0) and by the end (8) of eight weeks of training: control group (C); diabetic sedentary group (DS); and diabetic trained group (DT). Data are expressed as mean ± standard error of the mean. The statistical differences were determined by repeated measures analysis of variance followed by Bonferroni post-test. *** p < 0.001 vs. C 0; †† p < 0.01 vs. DS 0; and # p < 0.05 vs. DT 0.

450 C

*** ***

††

#

DSDT

400350300

Gluc

ose (

mg/dL

)

250200150100

500

0 0 08 8Weeks

8

Figure 2 – Concentration-response curves for acetylcholine (ACh: 10-9 – 10 -4 M) in isolated superior mesenteric artery rings, with intact endothelium and pre-contracted with L-phenylephrine (1 µM). The rings were obtained from rats of the groups control (C), diabetic sedentary (DS) and diabetic trained (DT). Data are expressed as mean ± standard error of the mean. The statistical differences were determined by two-way analysis of variance followed by Bonferroni post-test. ** p < 0.01 and *** p <0.001 vs. C; ## p < 0.01 vs. DS.

0

25

50

% of

relax

ation

75

100

125

9

CDSDT

8 7–Log [ACh] M

***

##

**

6 5 4

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Figure 3 – Correlation between blood glucose levels and the percentage of maximum response of acetylcholine-induced relaxations in superior mesenteric artery rings of the groups diabetic sedentary (A) and diabetic trained (B).

100

90

80

Rmax

(%)

70

60

200

DSDT

250 300 350

Glucose (mg/dL)

400 450 500 550

Table 1 – Potency (pD2) and maximum response (Rmax) obtained from concentration-response curves for acetylcholine before and after pretreatment with NG-nitro-L-arginine methyl ester (L-NAME)

Groups Condition pD2 Rmax

C- L-NAME 7.0 ± 0.0 99.0 ± 2.7

+ L-NAME 5.5 ± 0.1* 70.0 ± 6.2**

DS- L-NAME 7.2 ± 0.0 78.0 ± 1.8

+ L-NAME 6.8 ± 0.1† 72.4 ± 3.1

DT- L-NAME 7.0 ± 0.1 95.0 ± 3.5

+ L-NAME 4.9 ± 0.2* # 50.0 ± 3.6* ##

The superior mesenteric artery rings were obtained from rats of the groups control (C), diabetic sedentary (DS), and diabetic trained (DT). The experiments were performed in the absence of L-NAME (- L-NAME) or presence of 100 µM of L-NAME (+ L-NAME). Data are expressed as mean ± standard error of the mean. The statistical differences were determined by Student t test for independent samples (intragroup) or analysis of variance followed by Bonferroni post-test (intergroup). *p <0.001 or **p <0.01 for - L-NAME vs. + L-NAME values; † p<0.001 vs. C and # p<0.001 or ## p<0.01 vs. DS.

to the reduction in blood glucose levels observed in group DT animals. Our results evidenced that the ACh-induced relaxation had a strong inverse correlation with blood glucose levels. The group DS animals had an increase in blood glucose levels and an important loss in endothelial function, however, the reduction in blood glucose levels is associated with the vascular function restoration of group DT animals. Such results emphasize the findings of several other studies, which indicate resistance exercise as a possible tool to treat and/or prevent diseases with vascular function loss, such as hypertension and diabetes8,10,19,20,38.

Diabetes mellitus has been reported to reduce the endothelial production of vasoactive substances responsible for regulating the vascular tone, such as NO and prostaglandins1. To investigate the participation of NO in endothelium-dependent relaxations, concentration-response curves for ACh were obtained in the presence of L-NAME. Under that experimental condition, L-NAME antagonized the ACh-induced relaxation in animals of groups C and DT, but did not change the relaxation in group DS animals, characterizing a reduction in the participation of one of the major endothelium-derived relaxing factors in group DS

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Figure 4 – Mean arterial blood pressure of rats of the groups control (C), diabetic sedentary (DS), and diabetic trained (DT) after eight weeks of resistance exercise. Data are expressed as mean ± standard error of the mean. The statistical differences were determined by repeated measures analysis of variance followed by Bonferroni post-test. * p < 0.05 DS vs. C and # p < 0.05 DT vs. DS.

150

125

100

MAP

(mmH

g)

75

50

25

0

C DS DT

#

*

animals. Those findings are in accordance with the results by Chen et al39, who have reported that ACh-induced Rmax was also reduced in the presence of L-NAME in animals undergoing aerobic exercise for eight weeks.

Group DT animals had a higher percentage of relaxation inhibition in the presence of L-NAME as compared with healthy animals. That phenomenon might have resulted from a possible increase in NO-dependent relaxation caused by resistance exercise. The increase in relaxations observed in this study corroborates other findings, in which there was an increase in aerobic-exercise-mediated NO production in the experimental type 1 diabetes model14.

Studies with humans with type 1 diabetes have also shown that aerobic exercise improved endothelial function in vascular beds not directly involved during exercise13. In this study, the effects observed in the arteries of animals exercising also suggest a possible generalized vascular effect, because the artery analyzed was far from the tissues more active during exercise. That generalized vascular effect has also been observed by Faria et al38, and one single session of resistance exercise increased the NO-dependent relaxation in the caudal artery, reducing arterial blood pressure in spontaneously hypertensive rats.

In addition, in our study, resistance exercise reduced mean arterial blood pressure in DT animals, proving to be effective to treat endothelial dysfunction related to increased blood glucose levels. Recent data from our laboratory have shown that L-NAME-induced hypertensive animals also showed a

reduction in arterial blood pressure levels after four weeks of resistance exercise10. The mechanisms responsible for the decrease in arterial blood pressure of animals undergoing resistance exercise can be the reduction in peripheral vascular resistance and the increase in systemic vascular conductance39.

Thus, our results suggest that low-intensity resistance exercise induces metabolic and cardiovascular responses similar to those observed in studies submitting diabetic animals to aerobic exercise14,15. Although those modalities of exercise have different characteristics, such as the energetic pathway and movement execution, both promote beneficial cardiometabolic effects that help to treat both types of diabetes mellitus11,12.

Thus, this study indicates that the model of resistance exercise used could reduce blood glucose levels, restore endothelial function and reduce arterial blood pressure in diabetic animals. Finally, resistance exercise might cause beneficial vascular and metabolic adjustments for the treatment of type 1 diabetes mellitus dysfunctions in an experimental model40.

AcknowledgementsWe thank the Brazilian Board of Scientific and

Technological Development (CNPq), the Coordination for the Improvement of Higher-Level-Education Personnel (CAPES) and the Foundation for Support to Technological Research and Innovation of the State of Sergipe (FAPITEC-SE) for the financial support.

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Author contributionsConception and design of the research: Mota MM,

Silva TLTB, Barreto AS, Santos MRV; Acquisition of data: Mota MM, Silva TLTB, Fontes MT, Araújo JES; Analysis and interpretation of the data: Mota MM, Silva TLTB, Fontes MT, Oliveira ACC; Statistical analysis: Mota MM, Silva TLTB, Barreto AS; Obtaining financing: Santos MRV; Writing of the manuscript: Mota MM, Silva TLTB, Wichi RB; Critical revision of the manuscript for intellectual content: Mota MM, Silva TLTB, Oliveira ACC, Wichi RB, Santos MRV.

Potential Conflict of InterestNo potential conflict of interest relevant to this article

was reported.

Sources of FundingThis study was funded by CNPq, CAPES e FAPITEC/SE.

Study AssociationThis article is part of the thesis of master submitted by Marcelo

Mendonça Mota, from Programa de Pos-Graduação em Ciencias da Saúde da Universidade Federal de Sergipe (UFS).

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14. Chakraphan D, Sridulyakul P, Thipakorn B, Bunnag S, Huxley VH, Patumraj S. Attenuation of endothelial dysfunction by exercise training in STZ-induced diabetic rats. Clin Hemorheol Microcirc. 2005;32(3):217-26.

15. Mota MM, Silva TL, Fontes MT, Barreto AS, Oliveira AC, Santos MR. Treinamento aerobio previne alterações na vasodilatação dependente do endotélio em ratos diabéticos. Rev educ fis UEM. 2013;24(3).

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20. Barauna VG, Junior ML, Costa Rosa LF, Casarini DE, Krieger JE, De Oliveira EM. Cardiovascular adaptations in rats submitted to a resistance-training model. Clin Exp Pharmacol Physiol. 2005;32(4):249-54.

21. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980;288(5789):373-6.

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24. Castaneda C, Layne JE; Munoz-Orians L, Gordon PL, Walsmith J, Foldvari M, et al. A randomized controlled trial of resistance exercise training to improve glycemic control in older adults with type 2 diabetes. Diabetes Care. 2002;25(12):2335-41.

25. Dunstan DW, Daly RM, Owen N, Jolley D, de Courten M, Shaw J, et al. High-intensity resistance training improves glycemic control in older patients with type 2 diabetes. Diabetes Care. 2002;25(10):1729-36.

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Evaluation of Sexual Dimorphism in the Efficacy and Safety of Simvastatin/Atorvastatin Therapy in a Southern Brazilian CohortLisiane Smiderle1, Luciana O. Lima2, Mara Helena Hutz2, Cézar Roberto Van der Sand3, Luiz Carlos Van der Sand3, Maria Elvira Wagner Ferreira3, Renan Canibal Pires3, Silvana Almeida1, Marilu Fiegenbaum1

Universidade Federal de Ciências da Saúde de Porto Alegre1; Universidade Federal do Rio Grande do Sul2; Centro de Diagnóstico Cardiológico3, Porto Alegre, RS - Brazil

Mailing Address: Marilu Fiegenbaum •Rua Sarmento Leite 245/403, Centro Historico. Postal Code: 90050-170, Porto Alegre, RS – BrazilEmail: [email protected]; [email protected] received December 9, 2013; revised April 2, 2014; accepted April 17, 2014.

DOI: 10.5935/abc.20140085

Abstract

Background: Dyslipidemia is the primary risk factor for cardiovascular disease, and statins have been effective in controlling lipid levels. Sex differences in the pharmacokinetics and pharmacodynamics of statins contribute to interindividual variations in drug efficacy and toxicity.

Objective: To evaluate the presence of sexual dimorphism in the efficacy and safety of simvastatin/atorvastatin treatment.

Methods: Lipid levels of 495 patients (331 women and 164 men) were measured at baseline and after 6 ± 3 months of simvastatin/atorvastatin treatment to assess the efficacy and safety profiles of both drugs.

Results: Women had higher baseline levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) compared with men (p < 0.0001). After treatment, women exhibited a greater decrease in plasma TC and LDL-C levels compared with men. After adjustment for covariates, baseline levels of TC and LDL-C influenced more than 30% of the efficacy of lipid-lowering therapy (p < 0.001), regardless of sex. Myalgia [with or without changes in creatine phosphokinase (CPK) levels] occurred more frequently in women (25.9%; p = 0.002), whereas an increase in CPK and/or abnormal liver function was more frequent in in men (17.9%; p = 0.017).

Conclusions: Our results show that baseline TC and LDL-C levels are the main predictors of simvastatin/atorvastatin therapy efficacy, regardless of sex. In addition, they suggest the presence of sexual dimorphism in the safety of simvastatin/atorvastatin. The effect of sex differences on receptors, transporter proteins, and gene expression pathways needs to be better evaluated and characterized to confirm these observations. (Arq Bras Cardiol. 2014; 103(1):33-40)

Keywords: Simvastatin; Atorvastatin; Sexual Dimorphism; Lipids.

IntroductionDyslipidemia has been established as the primary risk

factor for cardiovascular disease (CVD)1. Statins are a class of lipid-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase, a key enzyme in the intracellular synthesis of cholesterol. Statins promote an increase in low-density lipoprotein cholesterol (LDL-C) receptors in hepatocytes, an increase in the removal of LDL-C particles from blood, and a decrease in total cholesterol (TC) and LDL-C levels2.

In addition to their cardioprotective effects, statins also exhibit many pleiotropic effects, including anti-inflammatory

and antioxidant properties3. Although statins are well tolerated by patients and have a good safety profile, some patients develop adverse drug reactions (ADRs) or do not show the desired pharmacological efficacy4. Simultaneous drug use with statins may increase the risk of ADRs due to drug–drug interactions5.

Drug response may vary according to sexual dimorphism6. Differences in pharmacokinetics and pharmacodynamics between sexes contribute to interindividual variations in drug efficacy and toxicity7. Endogenous hormonal factors differ between men and women, and the hormonal effects and quantities change with age6,8. The incidence of CVD is lower in women during their reproductive period than in men of the same age9. The sex hormone estrogen (17β-estradiol) may contribute to the decreased incidence of cardiac diseases in females10. However, women in their postmenopausal period are more likely to develop CVD compared with men8.

A recently published meta-analysis shows that the benefits of statins in primary and secondary CVD prevention did not differ between sexes11. However, of the 18 studies analyzed, only seven were related to the use of simvastatin/atorvastatin.

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Moreover, this study did not provide data on sex differences in the safety and efficacy of statins12. Therefore, we highlight the importance of studies providing efficacy and safety data for lipid-lowering therapies, with focus on the role of sexual dimorphism and interaction with co-medications.

This study aimed to determine the effects of sexual dimorphism and interaction with co-medications on the efficacy and safety of simvastatin/atorvastatin therapy in a southern Brazilian cohort of European descent.

Methods

PatientsThis open prospective cohort study included patients with

hypercholesterolemia who were receiving lipid-lowering therapy with simvastatin/atorvastatin. The patients were of European descent, as ascertained by skin color and morphological characteristics, lived in Porto Alegre, Brazil, and were collected for convenience. The sample size was estimated by the standard deviation in LDL-C levels and the expected difference between men and women after considering the following values: power of 80%, significance level of 5%, difference between men and women of 5 percentage points, and a standard deviation of 18 mg/dL. Considering these data, the estimated sample size initially comprised 205 men and 205 women. Our initial screening included 658 patients. The exclusion criteria for this study were as follows: age < 20 years, triglyceride concentration ≥ 400 mg/dL, altered thyroid stimulating hormone levels, impaired hepatic or renal function, unstable or uncontrolled disease that influences lipid metabolism, and previous therapy with other lipid-lowering drugs. After application of these exclusion criteria, 495 patients were considered eligible (simvastatin/atorvastatin therapy use). Physical examination, clinical data, and clinical laboratory data were obtained by the physician. Biochemical evaluation was performed prior to statin (simvastatin/atorvastatin) therapy initiation and after 6 months of treatment for the evaluation of therapeutic efficacy. Statin therapy and dose administered were determined by the physician on the basis of clinical characteristics. The patients received other medications, including calcium channel blockers, diuretics, and antithrombotic agents, and did not alter their medication regimens throughout the study.

To assess the lipid-lowering efficacy, lipid levels were measured at baseline and after 6 ± 3 months of treatment. Totally, 162 patients who received treatment for at least a year (35 ± 22 months) without developing ADRs were designated as a control group. Patients who exhibited an ADR, regardless of the duration of statin treatment, were designated as the case group. ADRs were diagnosed by the physician when patients presented one or more events of myalgia with or without an increase in creatine phosphokinase (CPK) levels and impairment in liver function concomitant with statin therapy. Myalgia was defined as muscle pain with normal or increased serum CPK levels and changes in liver enzyme levels as confirmed by increased

serum levels of alanine aminotransferase or aspartate aminotransferase. The presence of ADRs was evaluated on an average of every 3 months. Patients were not included in the ADR group when other unrelated conditions caused muscle pain, such as exercise-induced myalgia, arthritis, and viral myalgia.

This study was approved by the Federal University of Health Sciences of Porto Alegre Ethics Committee. All subjects who agreed to participate in this study gave their informed consent. Previous studies have already analyzed part of this sample13-17. Financial support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq - Brazil), REUNI/UFCSPA scholarship program, PROAP-CAPES, PRONEX-FAPERGS/CNPq, and PRONEN-FAPERGS/CNPq.

Biochemical analyses and benchmarks Serum levels of TC), high-density lipoprotein cholesterol

(HDL-C), and triglycerides (TGs) were determined from peripheral blood obtained after 12 h of fasting using standard methods with commercial kits. LDL-C levels were assessed by Friedewald et al18. The percentage of patients with normalized lipid levels after therapy was assessed according to the following National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III) guidelines19 for primary prevention of CVD: TC < 200 mg/dL, LDL-C < 100 mg/dL, HDL-C > 60 mg/dL, and TG < 150 mg/dL.

Statistical analysisStatistical analyses were performed using SPSS® software

version 18.0 (Chicago, IL, USA). TG levels were ln-transformed to eliminate skewness in data, and untransformed values are shown. Analyses were conducted using the whole sample and stratified by sex. We created a standardized statin dosage variable to avoid differences in lipid-lowering efficacy. According to the findings of Kivistö et al20, the daily doses of simvastatin were transformed to equivalent doses of atorvastatin at a ratio of 2:1. The mean percentage change in plasma lipid levels was obtained from the difference in pre-and post-treatment lipid levels, multiplied by 100, and divided by the pre-treatment level for each parameter. To analyze the association between sexual dimorphism and lipid-lowering treatment, the mean percentage change in plasma lipid levels was compared using the general linear model Type III sum of squares. Models were adjusted for age, smoking status, baseline lipid levels, prior CVD, controlled hypothyroidism, and antithrombotic use. Linear regression analysis was performed to assess differences in the dependence of variables on lipid-lowering efficacy. Continuous variables are presented as means ± standard deviations. A p-value of <0.05 was considered statistically significant. Student’s t-test was performed to assess differences between continuous variables. Categorical variables were compared using chi-square tests (a two-sided p-value of < 0.05 was considered statistically significant) with Yates’s correction. When appropriate, adjusted residual values (cell-by-cell analyses) and the power of the tests were assessed by WINPEPI 21.

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Results

Clinical characteristics of patientsWe investigated 495 European descendants from southern

Brazil who used simvastatin/atorvastatin lipid-lowering therapy. This population-based study comprised 331 (66.9%) women and 164 (33.1%) men; 85.1% participants were simvastatin users and 14.9% participants were atorvastatin users. The average treatment duration for women and men was approximately 6.4 ± 3.4 months and 6.1 ± 2.9 months (p = 0.357), respectively, with no difference between sexes. The standard dose of statins did not differ between women (10.3 ± 4.7 mg) and men (10.2 ± 4.3 mg; p = 0.840). Patients were aged between 25 and 82 years (61 ± 11 years). The mean age of the female patients (62.3 ± 10.7 years) was higher than that of the male patients (59.9 ± 11.1 years; p = 0.021). The number of smokers was greater among men than among women (p = 0.008), while hypothyroidism was more frequent in women (p < 0.0001). The clinical and demographic characteristics of the sample are described in Table 1. In our study, women had higher mean baseline levels of TC, LDL-C, and HDL-C compared with men (p < 0.0001; Table 2).

Efficacy and safety of the lipid-lowering therapy and sexual dimorphism

During treatment, 96.5% patients achieved standard LDL-C levels, while 73.8% and 72.6% patients achieved the desired TC and TG levels, respectively. Analysis of HDL-C levels revealed that 85.5% men and 51.9% women achieved the recommended levels. A similar percentage of men and women exhibited normalized TC and LDL-C levels: 88.9% and 86.8%, respectively, for men and 88.5% and 87.2%, respectively, for women. Table 3 describes the mean percentage change in lipid levels with respect to sex. After 6 months of follow-up, women exhibited a greater decrease in plasma TC (−27.32 ± 12.51 vs. −24.57 ± 12.08; p = 0.028) and LDL-C (−37.61 ± 1 8.34 vs. −33.49 ± 7.38; p = 0.014) levels compared with men. After adjusting for covariates, baseline TC and LDL-C levels were the primary predictors of simvastatin/atorvastatin efficacy. After adjustment for covariates, baseline levels of TC and LDL-C influenced 35.8% and 36.3% of the efficacy of lipid-lowering therapy (p < 0.001), regardless of sex (Table 4).

When assessing the safety of simvastatin/atorvastatin therapy, we observed that 74 (14.9%) patients in the total sample developed ADRs. Myalgia (with or without an increase in CPK levels) occurred more frequently in women (25.9%; p = 0.002) than in men, whereas increased CPK levels and/or abnormal liver function developed more frequently in men (17.9%) than in women (p = 0.017; Table 5).

Interactions with co-medicationsWe analyzed the effects of the lipid-lowering therapies in

the presence of other medications, including beta-blockers, antithrombotic agents, levothyroxines, diuretics, angiotensin

enzyme converting inhibitors, calcium-channel blockers, vasodilators, nitrates, benzodiazepines, cytochrome (CYP) inhibitors, CYP substrates, and CYP inducers. Women who used calcium-channel blockers, diuretics, and antithrombotic agents exhibited a greater frequency of ADRs (p = 0.014, p = 0.014, p = 0.002; respectively).

The concomitant use of drugs affecting the lipid-lowering metabolic pathway, such as CYP3A4 inducers, substrates, and inhibitors, was assessed and showed no influence on the safety and efficacy of simvastatin/atorvastatin therapy. Likewise, analysis of other co-medications showed no influence on the simvastatin/atorvastatin therapy endpoint in this patient group.

DiscussionThe objective of this study was to determine the effects of

sexual dimorphism and interactions with co-medications on the efficacy and safety of simvastatin/atorvastatin therapy. High levels of TC, LDL-C, and TGs and low levels of HDL-C are significant predictors of atherosclerosis and CVD in all populations22.

Analysis of the baseline lipid profile revealed that women had higher mean levels of TC and LDL-C compared with men. In addition, the average age was greater in women than in men in our study. The majority of women studied were post-menopausal and hormone-deficient. This condition leads to a worse lipid profile due to a decrease in estrogen production, resulting in downregulation of LDL receptors in the liver and, subsequently, decreased clearance of LDL-C from the serum23.

During statin therapy, there is a decrease in LDL-C levels due to the hepatic inhibition of cholesterol synthesis, which leads to upregulation of LDL receptors and a clearance of LDL from the circulatory system2. Previous studies have shown that the percentage of men who achieve normal TC and LDL-C levels during statin therapy is significantly higher than that of women24-25. However, in our study, a similar percentage of men and women exhibited normalized TC and LDL-C levels.

3-hydroxy-3-methylglutaryl-coenzime A reductase is primarily expressed in hepatocytes, and the efficacy of statins is dependent on the local concentration of these drugs in the liver. In addition, individual differences in pharmacokinetics and pharmacodynamics contribute to interindividual variations that characterize drug responses7. An efficacy analysis of statin therapy revealed that the decrease in plasma TC and LDL-C levels was greater in females than in males. After adjustment for covariates, we observed that baseline TC and LDL-C levels were statistically significant covariates. Although several studies in the literature have shown differences in drug response between men and women, approximately 35%–40% of the efficacy of simvastatin/atorvastatin therapy was related to baseline lipid levels in each patient, regardless of sex. Statin therapy is more effective in subjects with higher TC and LDL-C levels. Sexual dimorphism is defined as differences in characteristics between men and women. Sexual dimorphism in humans is also associated with the prevalence, severity, and development of many common diseases such as autoimmune diseases26, asthma27, and cardiovascular disease28, which may be due to differences

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Table 1 – Patient characteristics

Characteristics All Women Men pa

Number 495 331 (66.9%) 164 (33.1%)

Age (years) 61.5 ± 10.9 62.3 ± 10.7 59.9 ± 11.1 0.021

Statin Use (%) 0.229

Simvastatin 85.1 86.8 81.8

Atorvastatin 14.9 13.2 18.2

Treatment time (months) 6.3 ± 3.2 6.4 ± 3.4 6.1 ± 2.9 0.357

Standard dose (mg) 10.2 ± 4.6 10.3 ± 4.7 10.2 ± 4.3 0.840

Postmenopausal (%) 76.0

Hormone therapy use (%) 14.1

Smoking (%) 0.020

Past 12.1 8.9 18.2

Current 8.7 8.1 9.8

Never 78.5 82.8 71.2

Prior CVD (%) 32.5 28.8 40.0 0.014

CVD Family history (%) 17.7 16.7 19.7 0.578

Glucose (mg/dL) 100.2 ± 23.9 99.8 ± 26.1 100.9 ± 19.3 0.658

Diabetes (%) 18.7 17.4 21.2 0.465

Hypertension (%) 71.3 71.3 71.2 1.000

Controlled hypothyroidism (%) 15.1 20.5 4.5 < 0.001

Concomitant therapy use (%)

calcium channel blockers 17.9 16.3 21.3 0.165

Diuretics 39.5 42.0 34.7 0.126

Antithrombotic 26.6 23.8 32.3 0.039

CYP3A4 substrates 17.6 16.6 19.5 0.362

CYP3A4 inducers 1.6 1.5 1.8 0.719

CYP3A4 inhibitors 21.6 20.2 24.4 0.228

Values for age, treatment duration, standard dose, and glucose levels are expressed as means ± standard deviations; ap-values represent differences between women and men; CVD: Cardiovascular disease.

Table 2 – Pretreatment lipid levels according to sex

All (n = 495) Women (n = 331) Men (n = 164) pa

TC (mg/dL) 247.3 ± 39.9 254.1 ± 39.5 234.2 ± 37.5 < 0.0001

LDL-C(mg/dL) 164.4 ± 35.6 169.1 ± 34.9 154.9 ± 36.3 < 0.0001

HDL-C (mg/dL) 51.1 ± 12.8 54.0 ± 13.0 46.2 ± 11.0 < 0.0001

TG (mg/dL)b 157.7 ± 78.6 154.2 ± 68.3 164.8 ± 96.3 0.384

Values are means ± standard deviations; TC: total cholesterol; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; TG: triglycerides; aStudent’s t-test for differences between woman and men; bTG values were ln-transformed for comparisons.

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Table 3 – Mean percentage changes in lipid levels according to sex

All (n = 495) Women (n = 331) Men (n = 164) pa pb

TC (%) -26.40 ± 12.42 -27.32 ± 12.51 -24.57 ± 12.08 0.028 0.406

LDL-C (%) -36.41 ± 17.93 -37.90 ± 17.82 -33.41 ± 17.84 0.014 0.179

HDL-C (%) 3.68 ± 21.95 2.90 ± 21.11 5.25 ± 23.55 0.291 0.713

TGs (%) -11.95 ± 32.14 -12.56 ± 31.56 -10.72 ± 33.36 0.572 0.328

Values are means ± standard deviations;TC: total cholesterol; LDL-C: low-density lipoprotein cholesterol; HDL-C: high-density lipoprotein cholesterol; TGs: triglycerides;aStudent’s t-test for comparison between woman and men; b covariates included in the model: age, smoking status, baseline lipid levels, prior CVD, controlled hypothyroidism, and antithrombotic use.

Table 4 – Linear regression analysis: factors associated with percentage change in TC and LDL-C levels

Covariates Regression coefficient ± standard error Partial R2 × 100 p

TC

Constant 1.742 ± 5.594 0.756

Age 0.009 ± 0.057 0.8 0.868

Gender 1.073 ± 1.290 3.8 0.406

Smoking 0.697 ± 0.817 3.9 0.394

Previous CVD -0.276 ± 1.318 -0.9 0.834

Baseline TC levels -0.117 ± 0.015 -35.8 < 0.001

Controlled hypothyroidism 1.324 ± 1.648 3.6 0.422

Antitrombothic use -1.305 ± 1.393 -4.2 0.350

LDL-C

Constant 0.988 ± 7.164 0.890

Age -0.100 ± 0.083 -5.5 0.228

Sex 2.521 ± 1.872 6.2 0.179

Smoking 0.833 ± 1.209 3.2 0.491

Previous CVD -1.729 ± 1.930 -4.1 0.371

Baseline LDL-C -0.192 ± 0.024 -36.3 < 0.001

Controlled hypothyroidism 1.380 ± 2.411 2.6 0.567

Antitrombothic use -1.433 ± 2.029 -3.2 0.481

TC: total cholesterol; LDL-C: low-density lipoprotein cholesterol; CVD: cardiovascular disease.

Table 5 – Adverse Drug Reactions (ADRs) according to sex

All (n = 236) Women (n = 158) Men (n = 78) p

Control 162 (68.6%) 105 (66.5%) 57a (73.1%)

Myalgia (regardless of changes in CPK levels) 48 (20.3%) 41 (25.9%) 7b (9.0%) 0.002

Increased CPK and/or abnormal liver function 26 (11.1%) 12 (7.6%) 14c (17.9%)

CPK: creatine phosphokinase; aAdjusted residual = 1.03, p = 0.302; bAdjusted residual = -3.05, p = 0.002; cAdjusted residual = 2.39, p = 0.017.

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in gene regulation between men and women. Previous studies have shown that sexual dimorphism affects mRNA expression29-30. Pilote et al22 suggested the possibility that the evolution of sex differences in risk profiles can be partially attributed to sex hormones or their receptors. Previous studies have identified controversial data on differences in statin-induced responses between women and men6,11-12,31-32. Kostis et al11 performed a meta-analysis to evaluate the effectiveness of statins in decreasing cardiovascular events in men and women. This study evaluated 18 randomized clinical trials of statin involving 141,235 participants in order to analyze sex-specific differences. They observed that the benefits of statins were similar between sexes, regardless of the type of control, baseline risk, or type of endpoint in primary and secondary prevention. Mosca12 evaluated that study and stated that Kostis et al11 did not provide data on statin efficacy with regard to sex-specific differences and safety. In addition, of the 18 trials evaluated by Kostis et al11, only seven involved the use of simvastatin/atorvastatin. Therefore, we emphasize the importance of conducting cohort studies with a follow-up analysis to assess the efficacy of simvastatin/atorvastatin therapy.

Statin use can be associated with ADRs. In this study, we observed that myalgia (with or without an increase in CPK levels) occurred more frequently in women (25.9%; p = 0.002) than in men, whereas increased CPK levels and/or abnormal liver function was more commonly observed in men than in women (17.9%; p = 0.017). Studies have shown a difference in pharmacokinetics of up to 40% between men and women in terms of pharmacological response; furthermore, women have been shown to be at risk of clinically relevant ADRs33. Females have a higher percentage body fat compared with males, which can affect the drug distribution volume. A larger distribution volume will decrease the maximum concentration of the drug and increase its half-life and efficacy33. Genetic, physiological, and environmental effects can affect enzyme activity. Previous studies have shown that genetic polymorphisms promote variations in the expression of metabolizing enzymes, such as genes in the CYP 450 family, and are related to the efficacy and toxicity of statin therapy34.

We also observed that women consuming calcium channel blockers, diuretics, and antithrombotic co-medications had a higher prevalence of ADRs compared with men. Although the pathogenesis of statin-related toxicity has been explained by multiple hypotheses, there is no clear consensus and uniform theory that can explain these effects35.

Simvastatin and atorvastatin are metabolized by CYP 450 3A4 (simvastatin acid is also metabolized by CYP2C8). Exposure to CYP3A4 inhibitor drugs concomitantly with statins can lead to ADRs. Calcium channel blockers are potent CYP3A4 inhibitors at clinically relevant doses36. Kornstein et al37 observed that the tolerability of antidepressant therapies is also sex-dependent. Other co-medications that were analyzed showed no influence on the simvastatin/atorvastatin therapy endpoint in this group of patients. However, an interaction between age and sex has also been demonstrated for a variety of CYP3A4 substrates33. The risk

of ADR occurrence is 1.5–1.7-fold greater in women than in men38. Female sex, advanced age, presence of comorbidities, and use of concomitant medications were described as risk factors for statin-induced ADRs39. The Cholesterol Treatment Trialists’ (CTT) Collaborators40 published a systematic review of statin trials, suggesting strong evidence that the benefits of statin outweigh any possible serious ADRs. However, ADRs occurred in 14.9% patients in this study. Assessment of the cause of ADRs is important for optimizing the management of these subjects and the efficacy of lipid-lowering therapy.

The main strength of our study is that it is focused on the characteristics of patients receiving simvastatin/atorvastatin therapy as well as efficacy and safety profiles in a southern Brazilian cohort.

This study also has some limitations. It was an observational study with no placebo control. The sample was not equally distributed in terms of the baseline lipid profile, proportion of men and women, and age, and there was no age and sex matching. Some patients had incomplete data on hormonal status (women), hormone therapy use, genetic dyslipidemia, and lifestyle (diet and physical exercise). All patients were advised to begin physical activity and diet control, but monitoring and evaluation of these changes were not part of this work. Simvastatin/atorvastatin-induced ADRs were determined according to the physician’s criteria and do not represent the incidence of ADRs in this population. In order to increase the power of the analysis, we collected patients who developed ADRs at some point during lipid-lowering therapy. Although the final sample size was different from the initially calculated sample size, our investigation displayed a power of 82% to detect differences of a five-percentage point decrease in LDL-C levels between men and women. We minimized the possibility of type II errors by using adjustment variables with covariates that could influence the final response.

ConclusionsIn summary, we observed that simvastatin/atorvastatin

therapy use was more effective in patients with higher TC and LDL-C levels, regardless of sex, and that ADRs were more frequent in women than in men. In the literature, we found a wide range of pharmacogenetic, pharmacokinetic, and pharmacodynamic statin-related studies. However, the role of sex differences in receptors, transporters proteins, and gene expression pathways needs to be better evaluated and characterized.

Author contributionsConception and design of the research: Smiderle L,

Almeida S, Hutz MH, Van der Sand CR, Van der Sand LC, Ferreira MEW, Pires RC, Fiegenbaum M; Acquisition of data: Smiderle L, Lima LO, Van der Sand CR, Van der Sand LC, Ferreira MEW, Pires RC; Analysis and interpretation of the data and Statistical analysis: Smiderle L, Almeida S, Fiegenbaum M; Obtaining financing: Almeida S, Fiegenbaum M; Writing of the manuscript: Smiderle L; Critical revision of the manuscript for intellectual content: Almeida S, Hutz MH, Fiegenbaum M.

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16. Sortica VA, Fiegenbaum M, Lima LO, Van der Sand CR, Van der Sand LC, Ferreira ME, et al. SLCO1B1 gene variability influences lipid-lowering efficacy on simvastatin therapy in Southern Brazilians. Clin Chem Lab Med. 2012;50(3):441-8.

17. Lima LO, Almeida S, Hutz MH, Fiegenbaum M. PPARA, RXRA, NR1I2 and NR1I3 gene polymorphisms and lipid and lipoprotein levels in a Southern Brazilian population. Mol Biol Rep. 2013;40(2):1241-7.

18. Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem. 1972;18(6):499-502.

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References

Potential Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Sources of Funding

This study was funded by CNPq, PROAP-CAPES, PRONEX-FAPERGS/CNPq, Bolsa REUNI USCSPA, DECIT/SCTIE-MS.

Study AssociationThis article is part of the thesis of Doctoral submitted by

Lisiane Smiderle, from Universidade Federal de Ciencias da Saúde de Porto Alegre.

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Obesity does not Lead to Imbalance Between Myocardial Phospholamban Phosphorylation and Dephosphorylation Paula Paccielli Freire1; Carlos Augusto Barnabe Alves1; Adriana Fernandes de Deus1; Ana Paula Lima Leopoldo2; André Soares Leopoldo2; Danielle Cristina Tomaz da Silva1; Loreta Casquel de Tomasi1; Dijon Henrique Salomé Campos1; Antonio Carlos Cicogna1

Departamento de Clínica Médica - Faculdade de Medicina de Botucatu - Universidade Estadual Paulista1, Botucatu, SP; Centro de Educação Física e Desportos - Universidade Federal do Espírito Santo2, Vitória, ES - Brazil

Mailing Address: Paula Paccielli Freire •Rua Joaquim Francisco de Barros, Bairro Alto. Postal Code 18600-380, Botucatu, SP – BrazilE-mail: [email protected]; [email protected] Manuscript received November 26, 2013; revised manuscript January 27, 2014; accepted February 18, 2014

DOI: 10.5935/abc.20140083

AbstractBackground: The activation of the beta-adrenergic system promotes G protein stimulation that, via cyclic adenosine monophosphate (cAMP), alters the structure of protein kinase A (PKA) and leads to phospholamban (PLB) phosphorylation. This protein participates in the system that controls intracellular calcium in muscle cells, and it is the primary regulator of sarcoplasmic reticulum calcium pump activity. In obesity, the beta-adrenergic system is activated by the influence of increased leptin, therefore, resulting in higher myocardial phospholamban phosphorylation via cAMP-PKA.

Objective: To investigate the involvement of proteins which regulate the degree of PLB phosphorylation due to beta-adrenergic activation in obesity. In the present study, we hypothesized that there is an imbalance between phospholamban phosphorylation and dephosphorylation, with prevalence of protein phosphorylation.

Methods: Male Wistar rats were randomly distributed into two groups: control (n = 14), fed with normocaloric diet; and obese (n = 13), fed with a cycle of four unsaturated high-fat diets. Obesity was determined by the adiposity index, and protein expressions of phosphatase 1 (PP-1), PKA, PLB, phosphorylated phospholamban at serine16 (PPLB-Ser16) were assessed by Western blot.

Results: Obesity caused glucose intolerance, hyperinsulinemia, hypertriglyceridemia, hyperleptinemia and did not alter the protein expression of PKA, PP-1, PLB, PPLB-Ser16.

Conclusion: Obesity does not promote an imbalance between myocardial PLB phosphorylation and dephosphorylation via beta-adrenergic system. (Arq Bras Cardiol. 2014; 103(1):41-50)

Keywords: Obesity; Phosphorylation; Rats; Leptin; Adyposity.

Introduction The beta-adrenergic system (BAS) modulates cardiac

performance via beta receptor, G protein, adenylyl cyclase, and cyclic adenosine monophosphate (cAMP). The cAMP alters protein kinase A (PKA), thus releasing the catalytic subunit and activating the phosphorylation of myocardial proteins1,2, which are involved in calcium (Ca2+) transport - Figure 1.

Phospholamban (PLB) participates in the control of intracellular calcium in the myocardium; it is the protein that regulates the activity of Ca2+ pump of the sarcoplasmic reticulum (SERCA2a)1,3-5; the dephosphorylated PLB forms

the complex PLB-SERCA2a, which inhibits the pump and does not allow the transfer of the cytosolic Ca2+ to the sarcoplasmic reticulum; phosphorylation uncouples the complex PLB-SERCA2a, therefore increasing the calcium recapture by SERCA2a3.

The connection PLB-SERCA2a is controlled by cycles of phosphorylation and dephosphorylation, by the action of PKA and phosphatase 1 (PP-1), respectively. The prevalence of PLB phosphorylation, site of serine 16, occurs with the activation of PKA. Simultaneously, phosphorylates the inhibitory protein (I-1), thus forming the complex I-1/PP-1 and preventing PLB dephosphorylation caused by PP-1. Dephosphorylation is prevalent when PKA is deactivated, therefore there is no PLB and I-1 phosphorylation; when phosphate is not added to I-1, the formation of I-1/PP-1 is not possible, which allows PP-1 to dephosphorylate PLB, in its active state6-8 (Figure 2).

Obesity - excessive fat tissue in relation to lean mass9 - produces adipokines, which interfere in biological processes, including the activation of BAS by leptin4,10. The BAS stimulation phosphorylates the myocardial PLB via cAMP-PKA. There are no studies analyzing the balance between PLB phosphorylation

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Arq Bras Cardiol. 2014; 103(1):41-50

Figure 1 – The activation of the beta-adrenergic system, by means of the beta receptor, leads to the stimulation of the G protein, via alpha subunit, thus activating AC and promoting the transformation of ATP into cAMP. The latter alters the conformation of PKA, releasing and stimulating the PKA catalytic subunit, which triggers the phosphorylation of different proteins involved in calcium transport. AC: adenyl cyclase; cAMP: 3’. 5’ cyclic adenosine monophosphate; ATP: adenosine triphosphate; PKA: protein kinase A.

Beta-adrenergic receptor

G Protein

Cytosol

ATP

ACas

as

β

β

γcAMP

Sarcolemma

PKAR R

C

C

C

Regulatory subunit

Activated catalytic subunit

Myocardial protein phosphorylation

Calcium transport changes

Catalytic subunit

C

and dephosphorylation via BAS in obesity. Relling et al11 used obese rats for 12 weeks and showed increased PLB expression and decreased phosphorylated PLB (pPLB). Lima-Leopoldo12 verified decreased pPLB via cAMP in serine 16 in obese rats for 15 weeks. These authors did not evaluate kinase and phosphatase proteins in the animals.

The inexistence of papers analyzing PLB activation and deactivation in obesity induced the investigation concerning the involvement of proteins that regulate PLB phosphorylation via BAS. The hypothesis of this study is that obesity promotes the imbalance between phospholamban phosphorylation and dephosphorylation, with prevalent phosphorylation.

Methods

Animals and experimental protocolTwenty-seven male 30-day old Wistar rats were used,

coming from the bioterium of the Medical Clinic Department at the Medical School of Botucatu (SP) — Unesp, under the following conditions: individual polypropylene cages

with chrome wire tops covered with sterilized pine wood shaving; room temperature of 24ºC and 12-hour light cycles. All of the procedures were conducted according to the Guide for the Care and Use of Laboratory Animals13, being afterwards approved by the Committee on Animal Research and Ethics of the Medical School of Botucatu (Unesp, Botucatu), protocol number 765.

Animals were randomized into two groups: control (C) and obese (Ob). Animals in C (n = 15) were fed with a normocaloric diet, RC Focus 1765, Agroceres ®, Rio Claro, São Paulo, Brazil (22% protein, 42.7% carbohydrate, 4% fat, 9% minerals, 8% fibers, 12% humidity, 1.5% calcium, 0.8% phosphorus); animals in group Ob received a cycle of four hyperlipidic diets Agroceres®,

Rio Claro, São Paulo, Brazil (20% protein, 26;4% carbohydrate, 20% fat, 10% minerals, 9% fibers, 12.5% humidity, 1.4% calcium, 0.7 phosphorus), which were rotating for a 15-week period. The profile analysis of fatty acids in the diet showed that unsaturated ones correspond to 80%, and saturated ones, to 20%. The food intake of animals was measured daily, and the intake of water, ad libitum. Animals were weighed weekly, with the digital scale Mettler®, model Spider 2. After 15 weeks of treatment, all

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Figure 2 - A. The prevalence of phosphorylation occurs when PKA is activated while it phosphorylates I-1, thus preventing PLB dephosphorylation. B. The prevalence of dephosphorylation occurs when PKA is not activated. There is no PLB and I-1 phoshporylation, therefore PP-1 maintains its active state. cAMP: 3’5’ cyclic adenosine monophosphate; I-1: inhibitory protein 1; P: phosphate; PKA: protein kinase A; pPLB: phosphorylated phospholamban; PLB: dephosphorylated phospholamban; PP-1: phosphatase-1; Serca2a: Ca2+ pump.

A BPrevalence of Phosphorylation

Sarcoplasmic Reticulum

Sarcoplasmic Reticulum

Ca2

Ca2+

Serca2a Serca2a

pPLB PLB

P

PPI-1 I-1PP-1 PP-1

+

cAMP

Active PKA

Inactive PKA

Prevalence of Dephosphorylation

of the animals were anesthetized with pentobarbital sodium (50 mg /kg/ip; Cristália® Produtos Químicos Farmaceuticos Ltda., Itapira, São Paulo, Brazil) and euthanized by decapitation.

Constitution of control and obese groupsIn the biological testing, especially in experimental trials,

even at similar laboratory conditions, the response homogeneity is not certain. In this sense, rats submitted to standard and hyperlipidic diets may present characteristics in common, in higher or lower scales, such as adiposity index. A study published previously14 showed that this fact may lead to classification errors, that is, animals submitted to standard diets could be classified as control, when in fact they exhibit aspects of obese animals, and vice-versa. Therefore, it is necessary to establish criteria to separate the animals in two distinct groups, according to the adiposity index. With that purpose, a 95% confidence interval (CI) was established for the average adiposity level in control and obese rats. The adopted separation point (SP) stood between the mean and the upper limit point in group C and the lower limit point of group Ob; considering that point, animals with adiposity index higher than the SP were excluded from group C, and those with adiposity index lower than the SP were excluded from group Ob.

Nutritional profile of the animalsIn order to assess if obesity had altered the nutritional

profile, food consumption was analyzed, as well as caloric intake, dietary efficiency, body mass, body fat and adiposity index. Food intake was daily calculated from individual leftovers. Caloric intake was calculated by the following formula: weekly food consumption multiplied by the energetic value of each diet (g x kcal). With the objective of analyzing the capacity of converting the consumed food energy into body weight, dietary efficiency was calculated by dividing the total body weight gain of the animals (g) by the total energy intake (kcal).

Characterization of obesityThe characterization of obesity, at the end of the

15-week period, was established by the adiposity index. The deposits of epididymal, retroperitoneal and visceral fat in the animals were dissected in order to quantify body fat. The adiposity index was measured by the sum of fat deposits normalized by final body weight multiplied by 100. This method allows a consistent analysis of body fat deposits15.

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Comorbidities associated with obesitySince obesity can lead to cardiovascular, metabolic

and hormonal comorbidities, such as systemic arterial hypertension, glucose intolerance, systemic resistance to insulin, dyslipidemia, hyperglycemia, hyperinsulinemia, and hyperleptinemia16,17, the following variables were analyzed:

a) Systemic blood pressureBlood pressure was assessed by measuring the

systolic arterial pressure (SAP). SAP was measured by plethysmography, using the electronic sphygmomanometer, Narco Bio-System®, model 709-0610 (International Biomedical, Austin, TX, United States). The rats were previously warmed, at a temperature of 40ºC for five minutes, in a wooden box (50 × 40 cm), covered with sterilized wood shaving, with the objective of producing the vasodilatation of the caudal artery. Afterwards, the cuff was connected to a pulse transducer placed around the animal’s tail and insufflated to 200 mmHg; then, it was uninsufflated. The arterial pulses were recorded with a Gould RS 3200 polygraph (Gould Instrumenta Valley View, Ohio, United States).

b) Glucose tolerance testAnimals were submitted to a six-hour fasting period.

Blood collection in the caudal artery was conducted in basal condition and after the intraperitoneal administration of 25% glucose (Sigma®-Aldrich, Saint Louis, MO, United States), equivalent to 2.0 g/kg . Blood samples were collected in moments 0 (basal condition), at 15, 30, 60, 90 and 120 minutes. The ACCU-CHEK GO glucose monitor kit (Roche Diagnostic Brazil Ltda., São Paulo, Brazil) was used to measure the glycemic index.

c) Hormonal profile: insulin and serum leptinThe serum concentrations of these hormones were

determined by the ELISA method, by using specific kits (Linco Research Inc, St. Louis, MO, United States). A microplate reader was used for the analysis (Spectra MAX 190, Molecular Devics, Sunnyvale, CA, United States).

d) Glycemic and lipid profileThe lipid and glycemic profiles were assessed by analyzing

serum glucose, triacylglycerol, total cholesterol, high and low density lipoprotein and non-esterified fatty acids (NEFA). Animals fasted for 12 to 15 hours, and they were anesthetized with pentobarbital sodium (50 mg/kg/IP, Cristália® Produtos Químicos Farmaceuticos Ltda., Itapira, São Paulo, Brazil) and euthanized. Afterwards, blood samples were collected in heparinized Falcon tubes, which were centrifuged (3,000 rpm; 10 minutes; Eppendorf® Centrifuge 5804-R, Hamburg, Germany) and stored at −80ºC. Concentrations of serum glucose, triacylglycerol, total cholesterol, and high and low density lipoprotein were determined with specific kits (CELM, Barueri, São Paulo, Brazil) and analyzed by the automated colorimetric enzymatic method (Technicon, RA-XTTM System,

Global Medical Instrumentation, Minnesota, United States). NEFA levels were determined with the method by Johnson & Peters18, using a colorimetric kit (WAKO NEFA-C, Wako Pure Chemical Industries, Osaka, Japan).

Characterization of cardiac remodelingSince obesity can lead to cardiac remodeling, it was studied

by the structural post mortem evaluation and by analyzing the expression of kinase and phosphatase proteins, which regulate the level of PLB phosphorylation resulting from the beta-adrenergic activation of the myocardium.

a) Cardiac structural analysisAnimals were submitted to fasting from 12 to 15 hours,

being afterwards anesthetized with pentobarbital sodium (50 mg/kg/ip; Cristália® Produtos Químicos Farmaceuticos Ltda., Itapira, São Paulo, Brazil) and euthanized by decapitation. The heart of the animals was removed and dissected, and the following determinations were made: total weight of the heart, of the left and right ventricles, and the atrium, and their respective relations with body weight and tibial length at the time of euthanasia. These analyses may indicate the presence of cardiac remodeling at atrial and ventricular levels.

b) Protein expression analysisThe protein expression of total PLB, pPLB (ser-16), PKA and

PP-1 was conducted by the Western Blot technique.

The Western Blot technique

a) Protein extractionFragments of the left ventricle were rapidly frozen in liquid

nitrogen and stored in a freezer at −80°C. The frozen sample was homogenized in a Polytron device (Ika Ultra TurraxTM T25 Basic, Wilmington, United States) with hypotonic lysis buffer (potassium phosphate 50 mM pH 7.0, sucrose 0.3 M, DTT 0.5 mM, EDTA 1 mM pH 8.0, PMSF 0.3 mM, NaF 10 mM and phosphatase inhibitor). The process was performed three times for 10 seconds at 4ºC, with 20-second intervals. The product of homogenization was centrifuged (Eppendorf 5804R, Hamburg, Germany) at 12.000 rpm for 20 minutes at 4ºC, and the supernatant was transferred to Eppendorf tubes and stored in a freezer at −80oC. The protein concentration was analyzed by the Bradford method19, using the curves in the BSA Protein Standard (Bio-Rad, Hercules, CA, United States) as a pattern.

The protein samples were diluted in a Laemmli buffer (Tris-HCL 240mM, SDS, 0.8%, 40% glicerol, 0.02% bromophenol blue and 200 mM beta-mercaptoethanol) and separated by electrophoresis using the Mini-Protean 3 Electrophoresis Cell system (Bio-Rad, Hercules, CA, United States). Electrophoresis was conducted with biphasic stacking (Tris -HCL 240mM pH 6.8, 30% polyacrylamide, APS and Temed) and resolution gel (Tris-HCL 240mM pH 8.8, 30% polyacrylamide, APS and Temed), with concentrations of 6% to 12%, depending on the molecular mass of the analyzed protein. In the first gel well, one molecular mass pattern was applied,

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with the Kaleidoscope Prestained Standards (Bio-Rad, Hercules, CA, United States), in order to identify the size of the bands. Electrophoresis was made at 120 V (Power Pac HC 3.0A, Bio-Rad, Hercules, CA, United States), for approximately three hours, with loading buffer (Tris 0.25M, glycine 192 mM and 1% SDS). Afterwards, proteins were transferred to a nitrocellulose membrane in a Mini-Trans Blot system (Bio-Rad, Hercules, CA, United States), by using the transfer buffer (Tris 25 mM, glycine 192 mM, 20% methanol and 0.1% SDS). Membranes were washed twice with a TBS buffer (Tris-HCl 20mM pH 7.6 and NaCl 137mM). The non-specific binding sites of the primary antibody to the membrane were blocked by incubation, with a 0.5% skimmed milk powder solution dissolved in a TBS-T buffer, pH 7.4 (Tris-HCl 20mM, NaCl 137mM and 0.1% Tween 20 detergent) for 120 minutes at room temperature under constant agitation. Afterwards, the membrane was washed three times in TBS-T buffer (Tris 1M pH2.8, NaCl 5M and Tween 20) and incubated with the primary antibody diluted in the blocking solution, under constant agitation for 12 hours. After the incubation with the primary antibody, the membrane was washed three times in TBS-T buffer and incubated with the secondary antibody in a blocking solution for two hours under constant agitation. In order to remove the excessive secondary antibody, the membrane was washed three times in TBS-T buffer. Finally, immunodetection was performed by the chemiluminescence method, according to the manufacturer’s instructions (Enhancer Chemi-Luminescence, Amersham Biosciences, NJ, United States). The nitrocellulose membranes were exposed to radiographic films X-Omat AR (Eastman Kodak Co., United States), in the periods standardized for each of the analyzed proteins.

b) Antibodies• PLB mouse IgG (Thermo Scientific, Golden, CO, United

States, MA3-922). Used concentration: 1:5,000.• Phospho-Phospholamban (Ser16), rabbit IgG (Badrilla,

Leeds, West Yorkshire, United Kingdom, A010-12). Used concentration: 1:5,000.

• PKA rabbit IgG (Abcam Inc, MA, United States, AB71764). Used concentration: 1:500.

• PP1 rabbit IgG (Abcam Inc, MA, United States, AB16446). Used concentration: 1:1,000.

• β-Actin, rabbit IgG1 (Santa Cruz Biotechnology Inc, Santa Cruz, CA, United States, SC81178). Used concentration: 1:1,000.Quantitave blot analyses were conducted with the

software Scion Image (Scion Corporation, Frederick, Maryland, United States), which is a free software available at: http://www.scioncorp.com/

Statistical analysisAll of the variables were submitted to the test of normality

Kolmogorov-Smirnov. The nutritional profile, the comorbidities associated with obesity, the anatomical data and the cardiac protein expression were analyzed by the Student’s t-test

for independent samples. The glucose tolerance test was examined by the analysis of variance (ANOVA) for the model of repeated measures in two independent groups, being complemented by the Bonferroni test20. The Sigma Plot 3.5 for Windows was used for statistical analyses (Systat Software Inc., San Jose, CA, United States). Data were presented as mean ± standard-deviation. The 5% significance level was considered for all of the variables.

Results

Composition of control and obese groupsAfter the criterion established to compose the

experimental groups was applied, 27 animals remained in the study and constituted the control (C, n = 14) and the obese group (Ob, n = 13).

Nutritional profileTable 1 shows the nutritional profile of animals in C and

Ob. Final body weight, weight gain, deposits of epididymal, retroperitoneal and visceral fat, total body fat and adiposity index were higher in the obese group in relation to the control group. Animals in the Ob consumed less food than those in the C group. There was no difference between both groups with regard to caloric intake.

Analysis of comorbidities

a) Hormonal profile and systolic arterial pressure Figure 3 illustrates the result of serum insulin values (A) and

leptin (B); obesity leads to increasing concentrations of these hormones. The result of the final systolic arterial pressure (C) did not present any significant differences between groups.

b) Glucose tolerance testFigure 4 shows the results of the glucose tolerance test

performed in groups C and Ob. Glycemic levels were similar at the baseline between groups. After the intraperitoneal administration of glucose, glycemia was high in the Ob group and in moments 15, 30, 60 and 90 in comparison to group C.

c) Glycemic and lipid profileTable 2 shows the serum biochemical analyses of animals

in groups C and Ob. The plasma concentrations of glucose, cholesterol, HDL and NEFA were not different between treatments; the triglyceride concentration was significantly higher in Ob than in C.

Cardiac remodeling

a) Macroscopic structure of the heartTable 3 shows the post mortem macroscopic structure of

the heart of rats in C and Ob. After 15 weeks of obesity, there was a significant difference concerning the weight of the atria.

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Figure 3 – Serum insulin (A) and leptin levels (B) in control (n = 8) and obese animals (n = 8). Systolic arterial pressure (C) of control (n = 14) and obese animals (n = 13). Data expressed as mean ± standard-deviation. Student’s t-test for independente samples, *p <0,05 × C

A

5 8 150

100

50

0

6

4

2

0

* *43210

Control

Insuli

n (ng

/dL)

Lepti

n (ng

/dL)

SAP

(mmH

g)

Control ControlObese Obese Obese

B C

Table 1 - Nutritional profile

VariablesGroups

C (n = 14) Ob (n = 13)

IBW (g) 290 ± 13 308 ± 23*

FBW (g) 445 ± 39 486 ± 45*

Weight gain (g) 300 ± 16 342 ± 16*

Food intake (g/day) 26.0 ± 2.1 22.0 ± 2.4*

Caloric intake (kcal/day) 76.7 ± 6.2 80.3 ± 8.7

Dietary efficiency (%) 2.05 ± 0.30 2.33 ± 0.25*

Epididymal (g) 8.4 ± 1.7 14.2 ± 4.4*

Retroperitoneal (g) 7.3 ± 1.9 14.4 ± 4.7*

Visceral (g) 4.80 ± 1.20 8.10 ± 1.80*

Total body fat (g) 20.5 ± 4.1 36.7 ± 7.1*

Adiposity index (%) 4.61 ± 0.85 7.55 ± 1.36*

C: control; Ob: obese; FBW: final body weight; IBW: initial body weight. Data expressed as mean ± standard deviation. Student’s t-test for independent samples, * p <0.05 × C.

b) Protein expression analysisAccording to Figure 5, we did not observe significant

differences in protein expressions of PLB (A), pPLB Ser-16 (B), PKA (C) and PP-1 (D) between the control and obese groups.

DiscussionThe main finding in this study was that obesity induced

by an unsaturated high-fat diet did not lead to changes in the balance between phosphorylation and dephosphorylation in the heart; the behaviors of kinase and phosphatase proteins were similar in both analyzed groups.

The diet-induced obesity is similar to that found in the human population, and it has been used to reproduce possible molecular, structural, metabolic, and functional changes in different organs of the human body21. The high calorie content of the diet used in this experiment, which was enough to promote obesity among rats, was a result of the high content of unsaturated fats. In this study, results showed that the adiposity index was significantly higher among obese rats (control = 4.61 ± 0.85; obese = 7.55 ± 1.36; p < 0.005)

in relation to the ones in the control group. This result is in accordance with studies (conducted with rodents) that classify obesity using this index22.

Obesity has been characterized by several comorbidities, such as glucose intolerance, insulin resistance, systemic arterial hypertension, dyslipidemia, hyperinsulinemia and hyperleptinemia11,23,24. In this study, obese animals presented the following comorbidities: glucose intolerance, hyperinsulinemia, hypertriglyceridemia, and hyperleptinemia. Glucose intolerance associated with increasing serum insulin showed that animals were resistant to the action of insulin. The increasing levels of insulin in obese rats were not able to maintain the homeostasis of carbohydrates facing the supplementation of this substrate in obese animals. The increasing levels of triglycerides in obese rats can be a consequence of the high capture of triglycerides in the form of chylomicrons and/or the decreasing absorption of triglycerides by peripheral tissues25. The increased leptin levels were caused by larger fat deposits, since there is correlation between the levels of leptin and the fat tissue14,26. Since leptin is a hormone that derives from the fat tissue, it participates

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Table 2 – Glycemic and lipid profile

VariablesGroups

C (n = 14) Ob (n = 13)

Glucose (mg/dL) 125 ± 16 138 ± 14

Triglycerides (mg/dL) 43.3 ± 10.3 82,1 ± 15,7*

Cholesterol (mg/dL) 62.4 ± 11.5 67.5 ± 18.0

HDL (mg/dL) 23.5 ± 3.0 26.6 ± 5.8

NEFA (mmol/L) 0.42 ± 0.10 0.43 ± 0.10

HDL: high-density lipoprotein; NEFA: non-esterified fatty acids. Data expressed as mean ± standard deviation. Student’s t-test for independent samples, * p <0.05 × C

Figure 4 – Glucose tolerance test in control (n = 14) and obese animals (n = 13). Data expressed as mean standard-deviation. Analysis of variance (ANOVA) for the model of repeated measures in independent groups, complemented by the Bonferroni test. * p <0.05 × C.

350

300

250

200

Glyc

emia

(mg/d

L)

150

100

50

0basal 15 min 30 min 60 min 90 min 120 min

Control

* *

*

Obese

in energy balance, thus regulating the food intake and the oxidation of lipids27,28. The reduced food intake by obese rats suggests that the increasing levels of leptin were effective for appetite control. Data concerning comorbidities observed in this study are in accordance with other studies that induced obesity experimentally14,17,29,30.

The most important observation in this study was that diets induced by unsaturated fat did not change the pPLB-ser16 expression, and proteins in charge of balancing phosphorylation and dephosphorylation, PKA and PP-1, respectively. Since the beta-adrenergic via is in charge of phosphorylation in the site of serum-16 of the PLB, we can infer that this system was not

sufficiently stimulated to lead to changes in the myocardial PLB phosphorylation or that another system was opposed to such an activation. The behavior of pPLB-ser16 in this study is not in accordance with a previous study conducted in our laboratory12, in which decreased PLB was found in its phosphorylated state in serum 16 among obese rats treated with the same diet used in this study. We could not find an explanation for these different results; such a divergence could be related to the adiposity index in obese animals, which was 16% higher in the study conducted by Lima-Leopoldo12. No studies in literature analyzed the relationship between proteins that interfere in myocardial PLB phosphorylation and dephosphorylation in obese rats submitted to a hyperlipidic diet.

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Figure 5 – Expressions of PLB (A), pPLB-ser16 (B), PKA (C) and PP-1 (D) normalyzed by beta-actin. PKA: protein kinase A; PLB: dephosphorylated phospholamban; pPLB-ser16: phosphorylated phospholamban in serin-16; PP-1: phosphatase-1. Control (n = 6) and obese (n = 6). Data are expressed as mean ± standard-deviation. Student’s t-test * p <0.05 × C.

1.5

1.5 1.5

1.5A

C D

B

1.0

1.0 1.0

1.0

0.5

0.5 0.5

0.5

0.0

0.0 0.0

Control

Control Control

Control

Control

Obese

Obese Obese

Obese

Obese

A - PLB β-actin

pPLB-ser16 β-actin

PKA β-actin

PP-1 β-actin

B -

C -

D -

0.0

PLB/

β-ac

tinPK

A/β-

actin

PP-1

/β-a

ctin

pPLB

-ser1

6/ β-

actin

Table 3 – Macroscopic structure of the heart and tibia

VariablesGroups

C (n = 14) Ob (n = 13)

Tibia (cm) 4.30 ± 0.07 4.30 ± 0.10

LV (g) 0.79 ± 0.06 0.85 ± 0.09

RV (g) 0.23 ± 0.02 0.25 ± 0.03

AT (g) 0.09 ± 0.01 0.10 ± 0.01*

Heart total (g) 1.11 ± 0.09 1.20 ± 0.14

LF/tibia (g/cm) 0.18 ± 0.01 0.19 ± 0.02

RV/tibia (g/cm) 0.050 ± 0.005 0.060 ± 0.008

AT/tibia (g/cm) 0.020 ± 0.002 0.020 ± 0.003

Heart/tibia (g/cm) 0.30 ± 0.02 0.30 ± 0.02

C: control; Ob: obese; AT: atrial mass; RV: right ventricle mass; LV: left ventricle mass; AT/tibia: AT to tibia length ratio; RV/tibia: RV to tibia length ratio; LV/tibia: LV to tibia length ratio; Data expressed in mean ± standard-deviation. Student’s t-test for independent samples. *p< 0.05 vs C

ConclusionThe initial hypothesis of this study was not confirmed.

Obesity does not promote imbalance between myocardial PLB phosphorylation and dephosphorylation by the beta-adrenergic via.

Author contributionsConception and design of the research: Freire PP,

Lima-Leopoldo AP, Leopoldo AS, Silva DCT, Campos DHS, Cicogna AC; Acquisition of data: Freire PP, Alves CAB,

Deus AF, Campos DHS; Analysis and interpretation of the data: Freire PP, Alves CAB, Deus AF, Lima-Leopoldo AP, Leopoldo AS, Silva DCT, Tomasi LC, Campos DHS, Cicogna AC; Statistical analysis: Freire PP, Alves CAB, Lima-Leopoldo AP, Leopoldo AS, Silva DCT, Tomasi LC, Campos DHS, Cicogna AC; Obtaining financing: Freire PP, Lima-Leopoldo AP, Leopoldo AS, Cicogna AC; Writing of the manuscript: Freire PP, Tomasi LC, Cicogna AC; Critical revision of the manuscript for intellectual content: Freire PP, Alves CAB, Deus AF, Lima-Leopoldo AP, Leopoldo AS, Silva DCT, Tomasi LC, Cicogna AC.

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13. Guide for the Care and Use of Laboratory Animals. 8th ed. Washington, DC: The National Academies Press; c2010.

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References



Sources of Funding

This study was funded by FAPESP.

Study Association

This article is part of the end-of-the-course essay of Paula Paccielli Freire from Universidade Estadual Paulista (UNESP) – Faculdade de Medicina de Botucatu (FMB).

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Original Article

Functional Vascular Study in Hypertensive Subjects with Type 2 Diabetes Using Losartan or AmlodipineCesar Romaro Pozzobon, Ronaldo A. O. C. Gismondi, Ricardo Bedirian, Marcia Cristina Ladeira, Mario Fritsch Neves, Wille OigmanUniversidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ - Brazil

Mailing Address: Cesar Romaro Pozzobon •Rua Oman, 61 apt.º 102, Barra da Tijuca. Postal Code 22620-190, Rio de Janeiro, RJ – BrazilE-mail: [email protected]; [email protected] received September 18, 2013; revised manuscript January 13, 2014; accepted March 14, 2014.

DOI: 10.5935/abc.20140089

Abstract

Background: Antihypertensive drugs are used to control blood pressure (BP) and reduce macro- and microvascular complications in hypertensive patients with diabetes.

Objectives: The present study aimed to compare the functional vascular changes in hypertensive patients with type 2 diabetes mellitus after 6 weeks of treatment with amlodipine or losartan.

Methods: Patients with a previous diagnosis of hypertension and type 2 diabetes mellitus were randomly divided into 2 groups and evaluated after 6 weeks of treatment with amlodipine (5 mg/day) or losartan (100 mg/day). Patient evaluation included BP measurement, ambulatory BP monitoring, and assessment of vascular parameters using applanation tonometry, pulse wave velocity (PWV), and flow-mediated dilation (FMD) of the brachial artery.

Results: A total of 42 patients were evaluated (21 in each group), with a predominance of women (71%) in both groups. The mean age of the patients in both groups was similar (amlodipine group: 54.9 ± 4.5 years; losartan group: 54.0 ± 6.9 years), with no significant difference in the mean BP [amlodipine group: 145 ± 14 mmHg (systolic) and 84 ± 8 mmHg (diastolic); losartan group: 153 ± 19 mmHg (systolic) and 90 ± 9 mmHg (diastolic)]. The augmentation index (30% ± 9% and 36% ± 8%, p = 0.025) and augmentation pressure (16 ± 6 mmHg and 20 ± 8 mmHg, p = 0.045) were lower in the amlodipine group when compared with the losartan group. PWV and FMD were similar in both groups.

Conclusions: Hypertensive patients with type 2 diabetes mellitus treated with amlodipine exhibited an improved pattern of pulse wave reflection in comparison with those treated with losartan. However, the use of losartan may be associated with independent vascular reactivity to the pressor effect. (Arq Bras Cardiol. 2014; 103(1):60-68)

Keywords: Hypertension / complications; Diabetes Mellitus, Type 2 / complications; Atherosclerosis; Endothelium / physiopathology; Losartan / therapeutic, use; Amlodipine / therapeutic, use.

IntroductionSystemic arterial hypertension (SAH) and type 2 diabetes

mellitus (T2DM) are often associated1. SAH induces vascular damage by promoting endothelial dysfunction and atherosclerosis. Early treatment of hypertension is particularly important in patients with diabetes to prevent cardiovascular disease (CVD) and to minimize the progression of kidney disease and diabetic retinopathy2.

Arterial stiffness has been recognized as a cardiovascular risk marker3. Patients with both SAH and T2DM exhibit increased arterial stiffness compared with those with either diabetes or hypertension4. Increased arterial stiffness is an important

and independent risk factor associated with early mortality and assumes greater importance in clinical prognosis when compared with other known cardiovascular risk factors such as age, gender, smoking history, and dyslipidemia5. The gold standard for assessment of arterial stiffness is pulse wave velocity (PWV)6. An important parameter used to estimate arterial compliance is the augmentation index (AIx), which can be obtained using applanation tonometry7.

SAH, when associated with atherosclerosis and endothelial dysfunction, constitutes a risk factor that significantly increases cardiovascular morbidity and mortality8. Flow-mediated dilatation (FMD) of the brachial artery is a noninvasive method used to assess endothelial function. Using FMD, previous studies have indicated improved endothelial function in patients with hypertension, coronary artery disease, and heart failure who were treated with angiotensin-converting enzyme inhibitors (ACEIs)9,10 and in patients with diabetes treated with losartan11. The effects of amlodipine on endothelial function were evaluated in subjects with risk factors for coronary artery disease. Although, the subjects showed improvement in the parameters evaluated with FMD, this improvement was not significant when compared with the placebo group12.

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Pozzobon et al.Vascular study in diabetic hypertensives

Arq Bras Cardiol. 2014; 103(1):60-68

The present study aimed to compare the functional vascular changes in hypertensive patients with T2DM after 6 weeks of use of a calcium channel antagonist (CCA; amlodipine) or an angiotensin receptor blocker (ARB; losartan).

Methods

Study samplePatients with SAH and T2DM were selected for this study

during clinical follow-up at the Pedro Ernesto University Hospital [Hospital Universitário Pedro Ernesto (HUPE)], State University of Rio de Janeiro. Patients of both sexes aged between 40 and 70 years who were diagnosed with SAH and T2DM, without changes in dietary treatment or oral medication usage in the last 4 weeks, were included in the study. The main exclusion criteria were signs of secondary hypertension, decompensated diabetes mellitus [fasting glucose levels of > 300 mg/dL or glycated hemoglobin (HbA1c) levels of > 7%], need for insulin therapy, and chronic renal disease with an estimated glomerular filtration rate (GFR) of < 30 mL/min. The study was approved by the Research Ethics Committee of HUPE (protocol No. 20406/2012), and all participants read and signed the informed consent form.

After initial clinical and laboratory evaluation, the patients were randomized into 2 groups for treatment with either amlodipine (5 mg/day) or losartan (100 mg/day). After 6 weeks, a cross-sectional study involving analysis of clinical and laboratory data and performance of vascular tests was conducted.

Clinical evaluationTo determine blood pressure (BP), the patients remained

seated for 30 min and refrained from using tobacco or caffeine. BP was evaluated using a semi-automatic calibrated device, model HEM-705CP (Omron Healthcare Inc., Illinois, USA), with the cuff adjusted for arm circumference. Three measurements were obtained in each upper limb, and the respective mean value was calculated. The highest mean value was used in data analysis.

With regard to anthropometric measurements, a precision scale (Filizola) with a maximum capacity of 180 kg was used to determine weight, and a stadiometer was used to measure height. The body mass index (BMI) was calculated by dividing the weight (in kilograms) by the squared height (in meters). Waist circumference was measured at the midpoint between the last rib and the iliac crest, and hip circumference was measured at the point of largest circumference in the gluteal region. The waist–hip ratio (WHR) was determined by dividing the values obtained for the respective circumferences.

Laboratory testsFor performance of biochemical tests and quantification of

HbA1c levels using turbidimetry (BioSystems), venous blood samples were collected after fasting period of 10–12 h. GFR was estimated using the Modification of Diet in Renal Disease (MDRD) formula: GFRMDRD = 186 (serum creatinine)1.154 × (age)0.203 × [0.742 (if female) or 1.212 (if black)]13.

Serum lipids were analyzed using a colorimetric method (Bioclin). When the triglyceride level was 400 mg/dL, low-density lipoprotein (LDL) cholesterol was calculated using the Friedewald formula: LDL cholesterol = [total cholesterol − high-density lipoprotein (HDL) cholesterol] − (triglycerides/5). For quantitation of C-reactive protein (CRP), turbidimetry (BioSystems) was used, and the possibility of evaluating patients with acute infectious or inflammatory processes developed in recent weeks was excluded.

For evaluation of microalbuminuria, the urine albumin-to-creatinine ratio (UACR) was obtained from urine samples collected in the morning. Urine creatinine was quantitated using a colorimetric method, and albuminuria was determined using a nephelometric method.

Ambulatory blood pressure monitoring (ABPM)ABPM was performed using the SpaceLabs 90207 device

(SpaceLabs Inc., Redmond, WA, USA) scheduled to start between 8 and 9 am, with a minimum duration of 24 h. BP was measured every 20 min during the waking period (6 am–11 pm) and every 30 min during the sleeping period (11 pm–6 am). The test was considered satisfactory when at least 70% of the BP readings were valid, with a minimum of 16 readings during the waking period, 8 readings in the sleeping period, and a period < 2 h without BP measurements.

Vascular tests

Determination of the central aortic pressurePulse waves of the radial artery were obtained using an

applanation tonometer (model SPC-301–Millar Instruments, Houston, Texas, USA), calibrated according to the brachial artery pressure. Pulse wave analysis was performed to obtain central arterial pressures and other hemodynamic parameters using the SphygmoCor system (Atcor, United States)14. Aortic pressure waves were subjected to several analyses to identify the time between the beginning and the first and second peaks of the arterial pulse wave during systole. The pressure difference between the first component and the maximum pressure at systole [pressure increase (PI)] was identified as the reflected pressure wave during systole. AIx was defined as the ratio between PI and central pulse pressure (PP); it was expressed as a percentage [AIx = (PI/PP) × 100] and was subsequently adjusted for a heart rate of 75 bpm (AIx@75).

Pulse wave velocityPulse waves were obtained via the transcutaneous

route using a COMPLIOR SP device (Artech Medical), with transducers placed in the regions of the right carotid artery, right radial artery (peripheral PWV), and right femoral artery (central PWV)15. Two measurements were made during the same consultation, and when differences were > 10%, a third measurement was made. The mean of the 2 measurements was used for analysis. The measure was corrected for the mean arterial pressure (MAP) by using the formula PWV-N = (PWV/MAP) × 100.

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Flow-mediated dilation of the brachial arteryThis technique was performed using a 2-dimensional

ultrasound device with color and spectral Doppler (Vivid-3, GE, United States) and a linear transducer with a frequency of 10 MHz. The patients were placed in supine position with the right arm slightly stretched. After the brachial artery was located, the transducer was placed on the anteromedial side of the right upper limb, perpendicular to the arm axis and 5–10 cm above the antecubital fold in the region of the brachial artery. The basal brachial artery diameter (BBAD) and post-occlusion brachial artery diameter (POBAD) were measured manually between the lumen-intima interfaces at end-diastole. After BBAD measurement, the site of contact of the probe on the skin was marked to allow POBAD measurement at the same site. Flow occlusion was maintained for 5 min using an arm cuff, allowing the recording of a BP of 60 mmHg above the systolic arterial pressure. POBAD was measured at 30, 60, and 90 s after blood flow release16. The tests were performed by the same examiner who was unaware of the data recorded. FMD was calculated as the percentage increase in POBAD compared with baseline values using the following formula: FMD (%) = (POBAD − BBAD/BBAD) × 100.

Statistical AnalysisTo calculate the sample size, the Sample Power module

of the SPSS® software version 18.0 was used. By adopting a significance criterion (alpha) of 0.05, a minimum sample size of 16 patients was proposed for each group. After analyzing the changes in FMD of the brachial artery and based on the results of other clinical studies, the statistical significance power was determined as 80.7% and the accuracy was ± 1.44 points, using a 95% confidence interval11,12.

Statistical analyses were performed using SPSS® software version 18.0. The results were expressed as the mean ± standard deviation or as absolute and percentage values. The continuous variables of each group were compared using unpaired Student’s t test between groups with a 95% confidence interval. A p-value of < 0.05 was considered significant.

ResultsInitially, 120 hypertensive patients with diabetes who met

the inclusion criteria were selected. Among these, 73 (61%) met the exclusion criteria and were withdrawn from the study. Accordingly, 47 subjects were included in the study and were randomly divided into the losartan or amlodipine group. Because of 5 follow-up interruptions during the evaluation period, only 42 patients completed the study (21 in each group) (Figure 1).

The groups were evaluated and compared according to their clinical and epidemiological profiles, presence of cardiovascular risk factors, and prior use of medications. All patients were sedentary. A comparison between the 2 groups indicated no significant difference between the variables analyzed (Table 1).

The initial BP values were calculated in each group for the determination of systolic blood pressure (SBP) and diastolic blood pressure (DBP). Mean SBP and DBP were 147 ± 11 mmHg and 85 ± 9 mmHg, respectively, in the losartan group and 147 ± 21 mmHg and 84 ± 8 mmHg, respectively, in the amlodipine group.

Table 2 shows the laboratory profiles, estimated GFRs using MDRD, and UACR. Mean microalbuminuria was lower in the amlodipine group when compared with the losartan group; however, the difference was not significant. In the losartan group, 33% of patients presented with microalbuminuria, with a mean value of 81.3 mg/g creatinine; in the amlodipine group, 24% of patients presented with microalbuminuria, with a mean value of 57.3 mg/g creatinine.

After 6 weeks of treatment, the losartan and amlodipine groups no significant differences in SBP (153 ± 19 mmHg and 145 ± 14 mmHg, respectively, p = 0.127), and DBP (90 ± 9 mmHg and 84 ± 8 mmHg, respectively, p = 0.063), although the values were slightly lower in the amlodipine group. The data obtained with ABPM after treatment with losartan or amlodipine revealed no significant differences between the 2 groups, and similar results were obtained during the waking and sleeping periods (Table 3).

The PWV results revealed no significant differences between the 2 treatment groups. In contrast, applanation tonometry data indicated that the mean augmentation pressure was significantly higher in the losartan group in comparison with the amlodipine group (20 ± 8 mmHg and 16 ± 6 mmHg, respectively, p = 0.045). Similarly, a significantly higher mean AIx was observed in the losartan group when compared with the amlodipine group (36% ± 8% and 30% ± 9%, respectively, p = 0.025, Table 4). Increased endothelial function was observed in the losartan group using FMD (8.4% ± 4.6% and 7.5% ± 3.0%, respectively p = 0.431); however, the difference was not significant between the 2 groups (Figure 2).

DiscussionThis cross-sectional study aimed to evaluate cardiovascular risk

markers, including arterial stiffness and endothelial dysfunction, in a group of hypertensive patients with T2DM after 6 weeks of treatment with an ARB or a CCA, which are important antihypertensive drugs currently used in the treatment of such patients. Diabetes mellitus increases the risk of cardiovascular morbidity and mortality in patients with hypertension by 2- to 3-fold, and a decline in BP can decrease the rate of cardiovascular events and renal impairment in these patients17. However, it is unclear whether all classes of antihypertensive drugs play a similar role in the treatment of hypertensive patients with diabetes and can lead to alterations in cardiovascular risk markers.

A comparison between the 2 groups at the beginning of the study indicated similarities in their clinical and epidemiological profiles, presence of cardiovascular risk factors, and previous use of medications. Furthermore, the initial antihypertensive treatment performed was similar between the 2 groups, eliminating the difference in treatment as a confounding factor in the interpretation of the results.

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Figure 1 – Patient selection flowchart. ANL: amlodipine; T2DM: type 2 diabetes mellitus; PMH: past medical history; CRF: chronic renal failure; LOS: losartan; DBP: diastolic blood pressure; SBP: systolic blood pressure.

Table 1 – Profile of each group evaluated and comparison between the variables analyzed

Variables Losartan group (n = 21) Amlodipine group (n = 21) p-value

Age (years) 54.0 ± 6.9 54.9 ± 4.5 0.619

Female, n (%) 15 (71.4) 15 (71.4) 1.000

Black, n (%) 4 (19.0) 3 (14.3) 0.679

BMI, kg/m2 30.4 ± 3.5 29.8 ± 4.0 0.636

Smoking, n (%) 5 (23.8) 6 (28.6) 0.939

Dyslipidemia, n (%) 13 (61.9) 12 (57.1) 0.753

Statin use, n (%) 8 (38.1) 6 (28.6) 0.513

ASA use, n (%) 7 (33.3) 7 (33.3) 1.000

Metformin use, n (%) 18 (85.7) 18 (85.7) 1.000

Sulfonylurea use, n (%) 5 (23.8) 6 (28.6) 0.726

Data are expressed as mean ± standard deviation or as percentages, as indicated. ASA: acetylsalicylic acid; BMI: body mass index. The p-values were calculated using Student’s t test for continuous variables and the chi-square test for categorical variables.

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Table 3 – Data obtained with ambulatory blood pressure monitoring


SBP in 24 h, mmHg 136 ± 14 137 ± 14 0.947

DBP in 24 h, mmHg 81 ± 11 82 ± 9 0.892

MAP in 24 h, mmHg 100 ± 12 101 ± 10 0.888

SBP during the waking period, mmHg 139 ± 15 139 ± 14 0.888

DBP during the waking period, mmHg 84 ± 12 85 ± 10 0.787

MAP during the waking period, mmHg 103 ± 12 104 ± 10 0.836

SBP during the sleeping period, mmHg 132 ± 15 131 ± 16 0.846

DBP during the sleeping period, mmHg 75 ± 12 75 ± 9 0.942

MAP during the sleeping period, mmHg 95 ± 13 94 ± 10 0.893

Data are expressed as mean ± standard deviation. DBP: diastolic blood pressure; SBP: systolic blood pressure; MAP: mean arterial pressure. The p-values were calculated using Student’s t test.

Table 4 – Values obtained with pulse wave velocity and applanation tonometry


CR-PWV, m/s 9.9 ± 1.1 9.5 ± 1.4 0.347

CF-PWV, m/s 10.4 ± 2.2 10.6 ± 2.7 0.880

NCF-PWV, m/s 9.5 ± 1.8 9.8 ± 2.5 0.595

ASBP, mmHg 144 ± 19 136 ± 12 0.108

ADBP, mmHg 90 ± 10 84 ± 10 0.100

PI, mmHg 20 ± 8 16 ± 6 0.045

AIx, % 36 ± 8 30 ± 9 0.025

APP, mmHg 53 ± 16 49 ± 11 0.386

AIx@75, % 32 ± 7 28 ± 7 0.050

Data are expressed as mean ± standard deviation. AIx: augmentation index; AP: augmentation pressure; AIx@75: augmentation index corrected for a heart rate of 75 bpm; ADBP: aortic diastolic blood pressure; ASBP: aortic systolic blood pressure; APP: aortic pulse pressure; CF-PWV: carotid–femoral pulse wave velocity; NCF-PWV: normalized carotid–femoral pulse wave velocity; CR-PWV: carotid–radial pulse wave velocity. The p-values were calculated using Student’s t test.

Table 2 – Laboratory profile of the study population


Creatinine, mg/dL 0.7 ± 0.2 0.7 ± 0.2 0.577

Potassium, mEq/L 4.3 ± 0.4 4.3 ± 0.5 0.684

Blood glucose, mg/dL 111.7 ± 43.0 122.0 ± 47.8 0.467

HbA1c, % total Hb 6.2 ± 0.5 6.4 ± 0.5 0.270

Total cholesterol, mg/dL 196.4 ± 35.6 191.9 ± 30.0 0.662

HDL cholesterol, mg/dL 55.1 ± 19.5 55.0 ± 12.1 0.985

LDL cholesterol, mg/dL 115.9 ± 40.1 111.6 ± 27.1 0.585

Triglycerides, mg/dL 127.3 ± 48.0 127.0 ± 56.4 0.984

CRP, mg/dL 0.3 ± 0.3 0.5 ± 0.5 0.393

GRFMDRD, mL/min/1, 73 m2 91.9 ± 21.4 100.0 ± 34.6 0.368

UACR, mg/g creatinine 34.6 ± 40.1 24.8 ± 25.9 0.352

Data are expressed as mean ± standard deviation. Hb: hemoglobin; HbA1c: glycated hemoglobin; HDL: high-density lipoprotein; LDL: low-density lipoprotein; CRP: C-reactive protein; MDRD: Modification of Diet in Renal Disease; UACR: urinary albumin-to-creatinine ratio; GFR: glomerular filtration rate. The p-values were calculated using Student’s t test.

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Figure 2 – Distribution of values of flow-mediated dilation (FMD) of the brachial artery in the amlodipine (ANL) and losartan (LOS) group; p = 0.431 using Student’s t test.

Glucose and lipid metabolism, serum potassium levels, and microalbuminuria were also evaluated, although assessment of these parameters was not the primary aim of this study. In the study by Otero et al18, improved glycaemia and HbA1c levels were reported in patients treated with manidipine when compared with those treated with enalapril. In contrast, Derosa et al19 found no difference in blood glucose levels between patients who received telmisartan or nifedipine, whereas Nishida et al20 observed better HbA1c levels in patients who received ARBs when compared with those who received CCAs. In another study, Giordano et al21 obtained similar results for blood glucose and HbA1c levels in patients on captopril or nifedipine. In the present study, the mean blood glucose levels were higher in the amlodipine group than in the losartan group; however, the difference was not significant. In contrast, HbA1c levels were similar between the 2 groups. Moreover, no difference was found between the 2 groups regarding the use of hypoglycemic agents. In view of these findings, little can be inferred about the influence of ARBs or CCAs on glycemic control.

In the present study, we found no significant difference in mean serum potassium levels between the losartan and amlodipine groups. These results are in contrast with those obtained by Nishida et al20, who reported higher levels in patients using ARBs than in those using CCAs. With regard to lipid metabolism, the levels of total cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides were similar between the 2 groups.

In the study conducted by Derosa et al19, lower values of total cholesterol and LDL cholesterol were found in patients treated with telmisartan compared with those treated with nifedipine. These results are similar to those obtained by Nishida et al20, who compared patients using ARBs and those using CCAs, thereby suggesting that ARBs may have improved benefits on lipid metabolism in comparison with CCAs.

Microalbuminuria was determined by calculating UACR. Mean microalbuminuria was lower in the amlodipine group in comparison with the losartan group; however, the difference was not significant. The profile of the evaluated study group indicated a decreased risk of nephropathy. Because of its cross-sectional nature, the present study did not evaluate the effects of each drug on the variables analyzed, including microalbuminuria. In contrast, Yasuda et al22 conducted a prospective study comparing patients on losartan or amlodipine and found lower microalbuminuria in the losartan group. The authors of the IDNT study23 evaluated patients with SAH and nephropathy secondary to T2DM; as primary outcomes, they observed a lower risk of renal function impairment in patients who received irbesartan when compared with those who received amlodipine or placebo. However, there was no significant difference in the risk of all-cause death, even in subjects with renal function impairment. Moreover, the RENAAL study24 evaluated the effects of losartan on renal and cardiovascular outcomes in patients with T2DM and nephropathy compared with the placebo group and found a decrease in the progression of renal disease in the

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losartan group. However, no decrease in nephropathy- and CVD-related mortality was observed. In contrast, Yilmaz et al25 compared the use of valsartan, amlodipine, or a combination of these drugs in patients with hypertension and T2DM and observed decreased microalbuminuria in the 3 patient groups. These findings suggest that although CCAs do not have the same effects as ARBs on kidney hemodynamics, CCAs may have an important role in reducing microalbuminuria in patients with hypertension and diabetes, which can be explained by their antihypertensive efficacy.

After 6 weeks of treatment with losartan or amlodipine, lower mean BP levels were observed in the amlodipine group in comparison with the losartan group, although the differences were not significant. The Valsartan Antihypertensive Long-term Use Evaluation (VALUE) trial26 compared patients with hypertension who were treated with valsartan or amlodipine and revealed an increased control of BP in patients treated with amlodipine. Two years later, Zanchetti et al27 performed further analysis of the VALUE trial data according to subgroups and indicated improved BP control in the group treated with amlodipine compared with the group treated with valsartan; furthermore, this reduction was more pronounced in women. Phillips et al28 compared patients with hypertension who were treated with amlodipine or losartan and found a decrease in BP in both groups; however, this decrease was greater in the group receiving amlodipine.

Previous studies have evaluated patients with hypertension and diabetes treated using CCAs and compared them with those using ARBs or ACEIs. Derosa et al19 compared the use of telmisartan or amlodipine, revealing similarities between the 2 drugs in the ability to decrease BP; no significant difference was found between the treatment groups. Yasuda et al22 studied the effects of losartan and amlodipine on BP in patients with hypertension and diabetes and reported that both drugs were capable of reducing BP levels; no significant difference was found between these 2 groups. Similarly, Miyashita et al29 compared the effects of olmesartan and amlodipine on BP and found similar results between the 2 drugs.

In the present study, the mean BP values obtained with ABPM were similar in both groups, consistent with the results reported by Yasuda et al22 when comparing the effects of losartan and amlodipine on BP. Ishimitsu et al30 studied the effects of losartan in hypertensive patients without diabetes in comparison with patients receiving amlodipine and reported that both drugs were able to decrease BP over 24 h; however, the effects of amlodipine were greater than those of losartan. Otero et al18 used ABPM to evaluate BP in patients with hypertension and diabetes treated with manidipine or enalapril and found no differences between these 2 drug classes.

SAH is associated with the pathogenesis of arterial stiffness and is a manifestation of decreased arterial compliance31. Decrease in mean BP levels, and consequently in the pressure distension on blood vessels, is the greatest beneficial effect common to all classes of antihypertensive drugs31. In this context, growing evidence indicates the beneficial effects of CCAs and other drugs that interfere with the renin–angiotensin–aldosterone system (RAAS) for the management of important hemodynamic parameters31. After conducting a meta-analysis comparing ACEIs and ARBs with CCAs, diuretics, and beta

blockers, Boutouyrie et al32 reported the improved ability of ACEIs and ARBs in decreasing PWV and AIx. Kim et al33 evaluated 98 patients with hypertension and T2DM after 12 weeks of use of valsartan and found a decrease in aortic pulse pressure (APP), AIx, and PWV. Similarly, Asmar et al34 studied a group of 28 patients with hypertension and T2DM who were treated with telmisartan and found a decrease in PWV and AIx in these patients compared with the placebo group.

Kita et al35 evaluated 29 patients hypertension treated with benidipine for 1 year and found a decrease in PWV in these patients. Previous studies have compared the effects of drugs that affect RAAS with CCAs. Rajzer et al36 evaluated 180 patients with hypertension who were randomized into 3 groups according to the drug administered [quinapril (20 mg/day), amlodipine (10 mg/day), and losartan (100 mg/day)] and reported that all 3 drugs were able to decrease BP. However, only the quinapril group exhibited a significant decrease in PWV. Moreover, Ichihara et al37 compared the effects of amlodipine with those of valsartan on PWV. The study monitored 100 patients with hypertension for 1 year and indicated a similar decrease in PWV in the 2 groups.

In the present study, applanation tonometry results revealed lower mean values of systolic and diastolic central aortic pressure, AIx, PI, and APP in the amlodipine group when compared with the losartan group. These findings suggest an improved pattern of pulse wave reflection in the amlodipine group. The results obtained with ABPM indicated no differences in the final BP in both groups, which corroborates these important hemodynamic findings. The average BP values over 24 h indicate an improved control of BP in hypertensive subjects. The lowest casual BP observed in the amlodipine group appears to reflect a more pronounced acute effect of the CCA in comparison with the ARB. With regard to PWV, the results were similar in both groups and indicated no significant differences in arterial stiffness.

In the present, FMD was used to assess endothelial dysfunction. Some previous studies have also reported the effect of antihypertensive drugs on FMD. Cheetham et al11 compared the efficacy of losartan (50 mg/day) for 4 weeks with that of placebo in 12 hypertensive patients with T2DM. The results indicated a significant increase in FMD in the losartan group. Clarkson et al12 compared the efficacy of amlodipine (5 mg/day) with that of placebo in 91 patients with hypertensive. A significant increase in FMD was observed in both groups. However, no significant differences between the groups could indicate the superiority of one intervention over the other. Another study conducted by Anderson et al38 evaluated the effects of 3 classes of drugs on FMD in 80 patients who were randomized into 4 groups according to the treatment received: enalapril (10 mg/day), quinapril (20 mg/day), losartan (50 mg/day), and amlodipine (5 mg/day). After 8 weeks, only quinapril resulted in a significant increase in FMD. Yilmaz et al25 compared the effect of amlodipine (10 mg/day), valsartan (160 mg/day), or a combination of these 2 drugs on FMD. They observed that all treatment regimens effectively increased FMD, and the largest increase was observed in the group treated with the drug combination. In the present study, the mean percentage increase in FMD was higher in the losartan group compared with the amlodipine group. However, this difference was not significant, thereby preventing further conclusions in the evaluation of this method.

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ConclusionsIn hypertensive patients with T2DM with no evidence of

advanced nephropathy, treatment with amlodipine at an average dose resulted in similar BP levels in a 24-h period when compared with treatment with losartan at the maximum dose. Assessment of functional vascular alterations revealed a more favorable pattern of pulse wave reflection in the amlodipine group when compared with the losartan group. The decreased casual BP levels in patients treated with amlodipine, although not significant, may have clinical importance. However, the other functional vascular parameters evaluated were similar in both groups, which may indicate the occurrence of vascular effects associated with the use of losartan, regardless of its antihypertensive effect.

Author contributionsConception and design of the research and Analysis

and interpretation of the data: Pozzobon CR, Neves MF, Oigman W; Acquisition of data: Pozzobon CR, Gismondi

RAOC, Bedirian R, Ladeira MC, Neves MF; Statistical analysis: Pozzobon CR, Neves MF; Writing of the manuscript: Pozzobon CR; Critical revision of the manuscript for intellectual content: Pozzobon CR, Gismondi RAOC, Bedirian R, Neves MF, Oigman W.


reported.


Study AssociationThis article is part of the thesis of master submitted by

Cesar Romaro Pozzobon, from Universidade do Estado do Rio de Janeiro.

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30. Ishimitsu T, Minami J, Yoshii M, Suzuki T, Inada H, Ohta S, et al. Comparison of the effects of amlodipine and losartan on 24-hour ambulatory blood pressure in hypertensive patients. Clin Exp Hypertens. 2002;24(1-2):41-50.

31. Milan A, Tosello F, Fabbri A, Vairo A, Leone D, Chiarlo M, et al. Arterial stiffness: from physiology to clinical implications. High Blood Press Cardiovasc Prev. 2011;18(1):1-12.

32. Boutouyrie P, Lacolley P, Briet M, Regnault V, Stanton A, Laurent S, et al. Pharmacological modulation of arterial stiffness. Drugs. 2011;71(13):1689-701.

33. Kim JH, Oh SJ, Lee JM, Hong EG, Yu JM, Han KA, et al. The effect of an angiotensin receptor blocker on arterial stiffness in type 2 diabetes mellitus patients with hypertension. Diabetes Metab J. 2011; 35(3):236-42.

34. Asmar R, Gosse P, Topouchian J, N’Tela G, Dudley A, Shepherd GL. Effects of telmisartan on arterial stiffness in Type 2 diabetes patients with essential hypertension. J Renin Angiotensin Aldosterone Syst. 2002;3(3):176-80.

35. Kita T, Suzuki Y, Eto T, Kitamura K. Long-term anti-hypertensive therapy with benidipine improves arterial stiffness over blood pressure lowering. Hypertens Res. 2005;28(12):959-64.

36. Rajzer M, Klocek M, Kawecka-Jaszcz K. Effect of amlodipine, quinapril, and losartan on pulse wave velocity and plasma collagen markers in patients with mild-to-moderate arterial hypertension. Am J Hypertens. 2003;16(6):439-44.

37. Ichihara A, Kaneshiro Y, Takemitsu T, Sakoda M. Effects of amlodipine and valsartan on vascular damage and ambulatory blood pressure in untreated hypertensive patients. J Hum Hypertens. 2006;20(10):787-94.

38. Anderson TJ, Elstein E, Haber H, Charbonneau F. Comparative study of ACE-inhibition, angiotensin II antagonism, and calcium channel blockade on flow-mediated vasodilation in patients with coronary disease (BANFF study). J Am Coll Cardiol. 2000;35(1):60-6.

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Resistance Training After Myocardial Infarction in Rats: Its Role on Cardiac and Autonomic FunctionCamilla Figueiredo Grans1, Daniele Jardim Feriani1, Marcos Elias Vergilino Abssamra1, Leandro Yanase Rocha1, Nicolle Martins Carrozzi1, Cristiano Mostarda2, Diego Mendrot Figueroa3, Kátia De Angelis4, Maria Cláudia Irigoyen3, Bruno Rodrigues1

Laboratório do Movimento Humano, Universidade São Judas Tadeu (USJT)1, São Paulo, SP; Departamento de Educação Física, Universidade Federal do Maranhão (UFMA)2, São Luís, MA; Laboratório de Hipertensão Experimental, Instituto do Coração (InCor), Faculdade de Medicina, Universidade de São Paulo (USP)3, São Paulo, SP; Laboratório de Fisiologia Translacional, Universidade Nove de Julho (Uninove)4, São Paulo, SP – Brazil

Mailing address: Bruno Rodrigues •Laboratorio do Movimento Humano, Universidade São Judas Tadeu. Rua Taquari, 546, Mooca. Postal Code: 03166-000, São Paulo, SP – BrazilE-mail: [email protected]; [email protected] received December 03, 2013; revised manuscript February 10, 2014; accepted February 24, 2014.

DOI: 10.5935/abc.20140093

Abstract

Background: Although resistance exercise training is part of cardiovascular rehabilitation programs, little is known about its role on the cardiac and autonomic function after myocardial infarction.

Objective: To evaluate the effects of resistance exercise training, started early after myocardial infarction, on cardiac function, hemodynamic profile, and autonomic modulation in rats.

Methods: Male Wistar rats were divided into four groups: sedentary control, trained control, sedentary infarcted and trained infarcted rats. Each group with n = 9 rats. The animals underwent maximum load test and echocardiography at the beginning and at the end of the resistance exercise training (in an adapted ladder, 40% to 60% of the maximum load test, 3 months, 5 days/week). At the end, hemodynamic, baroreflex sensitivity and autonomic modulation assessments were made.

Results: The maximum load test increased in groups trained control (+32%) and trained infarcted (+46%) in relation to groups sedentary control and sedentary infarcted. Although no change occurred regarding the myocardial infarction size and systolic function, the E/A ratio (-23%), myocardial performance index (-39%) and systolic blood pressure (+6%) improved with resistance exercise training in group trained infarcted. Concomitantly, the training provided additional benefits in the high frequency bands of the pulse interval (+45%), as well as in the low frequency band of systolic blood pressure (-46%) in rats from group trained infarcted in relation to group sedentary infarcted.

Conclusion: Resistance exercise training alone may be an important and safe tool in the management of patients after myocardial infarction, considering that it does not lead to significant changes in the ventricular function, reduces the global cardiac stress, and significantly improves the vascular and cardiac autonomic modulation in infarcted rats. (Arq Bras Cardiol. 2014; 103(1):51-59)

Keywords: Myocardial Infarction; Rehabilitation; Resistance Training; Exercise; Ventricular Function; Autonomic Nervous System; Rats.

IntroductionMyocardial infarction (MI) is one of the most prevalent

cardiovascular diseases worldwide, leading to high morbidity and mortality rates1. MI triggers a ventricular remodeling process characterized by progressive left ventricular (LV) dilatation, rearrangement of the ventricular wall structure, increase in the remaining muscle mass, and decrease in cardiac function2. The cardiovascular autonomic imbalance following MI is a key element in the pathophysiology of heart

failure (HF) and is accompanied by abnormalities in the reflex cardiorespiratory control3,4.

Since the classical study conducted by Sullivan et al5 in late 1980, evidences have accumulated regarding the beneficial effects of aerobic exercise training (ET), which is considered a fundamental intervention in preventive cardiology6-8. Additionally, the moment to start ET after MI seems to be an important variable as regards the benefits observed6. Experimentally, our group has consistently demonstrated that aerobic ET started early after MI is able to reduce the infarct size, improve LV function, increase peripheral blood flow, and promote positive adjustments in the autonomic nervous system of infarcted rats9-12.

Not long ago, resistance training (RT) was being traditionally discouraged for patients after MI or with HF, because of concerns about compromising the LV function. However, in recent years, RT has been recommended for patients with HF, based on the logic that this form of training may be more effective in reverting skeletal muscle atrophy and in improving the quality of life of these individuals13,14. In fact, RT seems to

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attenuate the muscle mass reduction15 and improve strength16 and resistance17, as well as the maximal oxygen consumption18 of patients with HF. However, the cardiac and autonomic effects of RT started early after MI are not yet fully understood. Thus, the objective of the present study was to assess the effects of low/moderate-intensity RT started early after MI on ventricular remodeling and function, as well as on the hemodynamic profile and cardiovascular autonomic control of rats undergoing myocardial ischemia.

Methods

AnimalsMale Wistar rats (250g to 300 g) from the Animal Shelter of

Sao Judas Tadeu University were used. The animals were kept in groups, in an environment with temperature between 22oC and 24oC) and light controlled in 12-hour cycles (light/dark). Water and food were supplied ad libitum, and the diet had normal protein content. The present study was in accordance with the ethical principles of animal experimentation of the Brazilian College of Animal Experimentation (Colégio Brasileiro de Experimentação Animal – COBEA) and the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Washington D.C., 1996); it was approved by the Ethics Commission on the Use of Animals of São Judas Tadeu University (CEUA-USJT: 008/2013).

The animals were randomly divided into four groups (n = 9, each): sedentary control (SC); trained control (TC); sedentary infarcted (SI); and trained infarcted (TI). After MI or sham surgery, the animals underwent echocardiographic assessment and maximum load test (MLT), and either the RT protocol or follow-up was started. At the end of 3 months, echocardiographic study, MLT, and catheterization of the femoral arteries and veins for direct blood pressure (BP) and heart rate recording were performed.

Induction of myocardial infarctionGroups SI and TI were anesthetized (ketamine 80 mg/kg,

and xylazine 12 mg/kg, i.p.) and underwent MI by surgical occlusion of the left anterior descending coronary artery, as described elsewhere12,13. In sum, left thoracotomy was performed, the third intercostal space was dissected, and the heart was exposed. The left descending coronary artery was occluded using nylon 6-0 suture at approximately 1 mm of the left auricle. The thorax was closed using nylon 4-0 suture and the animals were kept under artificial ventilation until recovery. Groups SC and TC underwent the same procedure, except for myocardial ischemia, which was not performed (sham).

Echocardiographic assessmentEchocardiographic assessment was performed by an

observer blind to the groups which the animals had been assigned to, and followed the guidelines of the American Society of Echocardiography. The animals underwent two echocardiographic assessments: the first, 2 days after MI or sham surgery (baseline assessment); and the second, three months after RT or follow-up (final assessment), according to methodology described elsewhere12,13.

The rats were anesthetized (ketamine 80 mg/kg, and xylazine 12 mg/kg, i.p.) and the images were obtained using a 10 to 14-MHz linear transducer in a Sequoia 512 device (ACUSON Corporation, Mountain View, CA) for the assessment of the following parameters: (1) morphometric: LV mass, LV diastolic diameter (LVDD) and relative wall thickness (RWT); (2) systolic function: ejection fraction (EF) and velocity of circumferential fiber shortening (VCF); (3) diastolic function: isovolumic relaxation time (IVRT) and E/A wave ratio; and (4) overall function: myocardial performance index (MPI).

Maximum load test and resistance trainingAll groups underwent a MLT protocol and RT, performed

in a ladder adapted for rats, featuring 54 vertical steps 0.5 cm apart from each other. The animals were gradually adapted to climbing for 5 consecutive days before the MLT. The test consisted of an initial load of 75% of body weight, which was progressively increased with an additional 15% of body weight in subsequent climbings, as previously described by our group19. MLT was performed 5 days after MI or sham surgery (baseline assessment), 45 days after training or follow-up for load adjustment (data not presented), and at the end of 3 months of protocol (final assessment).

The RT protocol was performed for 3 months, 5 days a week, 15 climbings per session, with a 1-minute rest between each climbing, at low/moderate intensity (40% to 60% of the maximum load)19, as recommended for patients with cardiovascular disease14.

Hemodynamic assessmentsOne day after the final MLT, two catheters containing

0.06 mL of saline solution were implanted in the femoral artery and vein of the anesthetized animals (ketamine 80 mg/kg, and xylazine 12 mg/kg, i.p.). The next day, the arterial catheter was connected to a pressure transducer (Blood Pressure XDCR; Kent Scientific, Torrington, CT) and the BP and pulse interval (PI) signals were recorded for 30 minutes with the animals awaken, as previously described12,13,20.

After the baseline recording, sequential injections (0.1 mL) of up-titrated doses of phenylephrine (0.25 to 32 mg/kg) and sodium nitroprusside (0.05 to 1.6 mg/kg) were administered, inducing responses of increased or decreased mean BP (MBP), ranging from 5 to 40 mmHg. Baroreflex sensitivity was expressed as bradycardic response (BR) and tachycardic response (TR), in beats per minute by millimeter of mercury, as described elsewhere12,13,20.

Cardiovascular autonomic modulationThe overall PI and systolic BP (SBP) variability in the

time domain was assessed using standard deviation (SD) of time series. PI and SBP variations were also assessed in the frequency domain, using autoregressive modeling of the spectral analysis. The theoretical and analytical procedures for autoregressive modeling of the oscillatory components are described elsewhere20,21. In sum, the PI and SBP series, derived from each recording, were divided into 300-beat segments, with 50% overlapping. The spectra of each segment were

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calculated using the Levinson-Durbin algorithm; the order of the model chosen was in agreement with Akaike`s criterion, with oscillatory components quantified in low frequency (LF: 0.2 to 0.6 Hz) and high frequency (HF: 0.6 to 3.0 Hz).

Tissue weighingAfter cardiovascular assessments, the animals were

euthanized by decapitation, and the soleus and gastrocnemius muscles, as well as the retroperitoneal white adipose tissue were immediately removed and weighed.

Statistical analysis The statistical analysis was carried out using the Statistical

Package for the Social Sciences (SPSS), version 20.0 for Windows (Chicago, USA). Data are presented as mean ± standard error of the mean. After using the Kolmogorov-Smirnov test to confirm that all continuous variables were normally distributed, the statistical differences between the groups were obtained using two-way ANOVA followed by Bonferroni post-test. Statistical differences between data assessed throughout time were obtained using ANOVA for repeated measures, with assessed group factor, followed by Bonferroni test. The significance level was set at p < 0.05.

Results

Physical capacity and body weightAt baseline, the infarcted animals (SI and TI) showed a

reduction of MLT in comparison to non-infarcted animals (SC and TC). After the RT or follow-up period, the trained groups (TC and TI) showed increased MLT values in relation to their baseline assessments and in comparison to the respective controls (SC and SI) (Figure 1). However, group TI remained with reduced MLT values in relation to TC at the end of the study. In MLT, there was interaction between the assessment moments and the experimental groups (F = 72.402; p < 0.001).

At baseline, body weight was similar between the groups studied (~277 ± 7 g); however, at the end, the experimental groups showed increased body weight in relation to baseline (SC: 490 ± 10; TC: 454 ± 12; SI: 478 ± 13; TI: 465 ± 18 g). As regards the retroperitoneal white adipose tissue weight, groups TC (4.5 ± 0.6g) and TI (5.9 ± 0.5 g) showed reduced values in relation to groups SC (8.1 ± 1.1g) and SI (7.1 ± 0.8 g), respectively. The weight of the soleus and gastrocnemius muscles, which was reduced in group SI (0.17 ± 0.02 and 1.00 ± 0.09 g) in relation to SC (0.28 ± 0.02 and 1.37 ± 0.05 g), increased with RT, as observed in groups TC (0.41 ± 0.03 and 1.59 ± 0.04 g) and TI (0.30 ± 0.02 and 1.26 ± 0.01g) in relation to their sedentary peers.

Morphometry and ventricular functionThe echocardiographic variables related to morphometry

and ventricular function are presented in Table 1.The LV infarct size, which was similar among the infarcted

groups (ST and TI) in the baseline echocardiographic

assessment, was not modified by the RT period. LV mass and RWT were similar among the groups in the baseline assessment. At the end of the study, the trained groups (TC and TI) showed increased LV mass in relation to the baseline assessments, as well as in comparison to their respective controls (SC and SI). In relation to RWT, group TC showed elevation, and group SI showed reduction of this variable, when compared to SC. However, RT was efficient in preventing this reduction in group TI in relation to group SI. The end-LV diastolic diameter, which was similar among the groups in the beginning of the protocol, was increased at the end of the study in groups SI and TI, in relation to SC (Table 1). In the morphometric variables, there were interactions between the assessment moments and the experimental groups regarding the LV mass assessments (F = 19.805; p < 0.001) and RWT (F = 0.0296; p < 0.001).

As regards the LV systolic function variables, groups SI and TI showed reduction in EF and shortening velocity in the baseline and final echocardiographic assessments in relation to groups SC and TC. The E/A ratio, initially increased in animals of groups SI and TI, improved with RT in IT animals when compared to SI animals as well as in relation to their baseline assessment. In the final assessment, this diastolic function parameter was increased in groups TC and SI in relation to SC, as well as in group SI in relation to TC. On the other hand, IVRT did not change with MI or RT (Table 1).

The global LV function assessment, performed by MPI, was similar among the groups in the beginning of the study. However, at the end of the study, this variable increased in group SI in comparison to SC. We should point out that the RT period improved MPI in groups TC and TI in relation to their respective controls (SC and SI). However, group TI remained with increased MPI in relation to TC. Interactions between the assessment moments and the experimental groups were observed in the cardiac function variables: E/A ratio (F = 2248.060; p < 0.001) and MPI (F = 23.293; p < 0.001).

Hemodynamic assessmentsGroup SI showed reduction in SBP in relation to group SC.

Although differences between TI and SC were not observed, group TI showed decreased SBP in relation to SC. As regards SBP, there was interaction between RT and MI (F = 13.068; P = 0.001). Additionally, no differences were observed in the parameters related to diastolic BP (DBP), MBP and HR among the groups assessed, as shown in Table 2.

TR to blood pressure drops after injection of sodium nitroprusside was reduced in animals of groups SI and TI in relation to those of groups SC and TC. BR to blood pressure increases after up-titrated doses of phenylephrine was reduced in animals of group SI in relation to those of group SC. RT induced BR increase in group TC rats in comparison to group SC. We should point out that no differences were observed in BR between groups TI and SC; however, group TI showed reduced BR in relation to TC (Table 2). Interaction between RT and MI was observed in BR (F = 12.087; p = 0.001).

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Table 1 – Baseline and final echocardiographic assessments of morphometry and cardiac function in groups sedentary control (SC), trained control (TC), sedentary infarcted (SI), and trained infarcted (TI)

Variables/groups SC TC SI TI

LV mass (g)Baseline 1.07 ± 0.02 1.10 ± 0.04 1.15 ± 0.04 1.11 ± 0.05

Final 1.10 ± 0.05 1.77 ± 0.09#* 1.29 ± 0.05#* 1.71 ± 0.08#*†

RWTBaseline 0.39 ± 0.04 0.43 ± 0.02 0.37 ± 0.02 0.44 ± 0.05

Final 0.40 ± 0.01 0.52 ± 0.03* 0.28 ± 0.02* 0.48 ± 0.04†

LVDD (cm)Baseline 0.65 ± 0.01 0.63 ± 0.01 0.80 ± 0.01 0.82 ± 0.02

Final 0.73 ± 0.01 0.74 ± 0.02 0.92 ± 0.04* 0.87 ± 0.05*

Systolic function

EF (%)Baseline 74 ± 3 72 ± 6 46 ± 4*‡ 44 ± 3*‡

Final 71 ± 1 68 ± 2 43 ± 4*‡ 40 ± 2*‡

VCF (circ/seg 10-4)Baseline 51 ± 4 49 ± 3 30 ± 5*‡ 35 ± 2*‡

Final 46 ± 1 45 ± 5 32 ± 2*‡ 33 ± 3*‡

Diastolic function

IVRT (ms)Baseline 30 ± 2 31 ± 1 28 ± 1 30 ± 2

Final 31 ± 3 32 ± 1 30 ± 1 32 ± 3

E/A Baseline 1.57 ± 0.11 1.61 ± 0.22 2.74 ± 0.12* 2.77 ± 0.21*

Final 1.61 ± 0.12 1.87 ± 0.04* 2.69 ± 0.05*‡ 2.07 ± 0.10#*†

Overall function

MPIBaseline 0.44 ± 0.03 0.46 ± 0.03 0.45 ± 0.04 0.46 ± 0.02

Final 0.37 ± 0.03 0.19 ± 0.01* 0.54 ± 0.04* 0.33 ± 0.02†‡

Values express mean ± standard error of the mean. # p < 0.05 vs. baseline assessment; *p < 0.05 vs. SC; † p < 0.05 vs. SI; ‡ p < 0.05 vs. TC.LV: left ventricle; RWT: relative wall thickness; LVDD: left ventricular diastolic diameter; EF: ejection fraction; VCF: velocity of circumferential fiber shortening; IVRT: isovolumic relaxation time; E/A: E and A waves ratio; MPI: myocardial performance index.

Figure 1 – Values of the maximum load test in groups sedentary control (SC), trained control (TC), sedentary infarcted (SI), and trained infarcted (TI). # p < 0.05 vs. baseline assessment; * p < 0.05 vs. SC; ‡ p < 0.05 vs. TC; † p < 0.05 vs. SI.

Gram

s

Baseline

SCTCSITI

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Cardiovascular autonomic modulationData regarding PI and SBP variability in the time and frequency

domains are shown in Table 3. Group SI animals showed a reduction of the standard deviation of PI (SD-PI), of PI variance (PI-var), and of the root mean square of successive RR-interval differences (RMSSD) in relation to SC animals. There was additional PI-var reduction in groups SI and TI also in relation to group TC. After the RT period, further reduction of PI-var was prevented in group TI in relation to group SI; also, RMSSD became normal in group TI, since it showed values similar to those of group SC. There was interaction between RT and MI in the PI-var variable (F = 12.106; p = 0.001). As regards PI-var, no changes were observed in the experimental groups.

MI induced changes in the cardiac autonomic modulation of the animals studied, namely a reduction in LF (Figure 2A) and HF (Figure 2B) bands of PI, as well as in the autonomic balance (LF/HF) in group SI in relation to SC. We should point out that 3 months of RT were efficient in preventing these changes, as observed in group TI. However, both the LF range and the HF band remained reduced in group TI in relation to TC. On the other hand, the LF band of SBP, which was increased in group SI, became normal in group TI (Figure 2C). There was interaction between RT and MI in the absolute values of LF band of PI (F = 11.041; p = 0.02).

Corroborating the data regarding baroreflex sensitivity, as assessed by responses to vasoactive drugs, the alpha index of the LF band of SBP, which was reduced in group SI, did not change with RT.

DiscussionThe present study was conducted to test the hypothesis

that low/moderate intensity dynamic RT could bring benefits to the cardiac function and improve the autonomic control of circulation in infarcted rats. The main findings of this study point to the fact that RT promoted ventricular morphometric changes in infarcted animals, which are not associated with changes in cardiac function. However, although the baroreflex sensitivity and the alpha index had not improved, RT was efficient in preventing further impairment of the cardiovascular autonomic modulation in animals undergoing MI.

RT has been accepted as the main component of an encompassing exercise program both for apparently healthy individuals and for those with cardiovascular disease. In this sense, the guidelines stress the importance of incorporating RT for an optimal exercise prescription for patients with heart diseases, with the purpose of improving muscular strength, physical capacity and the quality of life14-18.

Corroborating clinical data, in the present study, RT increased MLT values in infarcted animals in relation to sedentary animals, thus suggesting improvement of muscular strength in these animals. In addition, the reduction in the retroperitoneal adipose tissue and the weight increase of the soleus and gastrocnemius muscles in trained infarcted rates suggest a positive body composition change in these animals. Our findings in previous studies had already demonstrated positive adaptations to RT in body composition and in the increase in muscular strength in diabetic ovariectomized rats19 and healthy rats22.

Despite its known benefits on the quality of life, muscular strength, and body composition, RT had been traditionally discouraged for patients with HF23 due to concerns that it could lead to impairment of the LV function and have a potential adverse effect on cardiac remodeling, especially resulting from increased afterload. However, when RT was performed at a low/moderate intensity by patients with HF, the hemodynamic responses did not exceed the levels reached during a standard exercise test24, and adverse cardiac remodeling was not observed in patients after a RT period15.

In the present study, although the MI size and LVDD had not changed with RT in infarcted animals, the LV mass increased and RWT became normal in these animals, and these changes could suggest a positive cardiac remodeling. However, when variables related to the ventricular function were assessed, positive adaptation in EF, VCF, and IVRT were not observed in trained infarcted animals. Unlike these findings, our group has previously demonstrated that aerobic RT was able to reduce the MI size, LVDD, and improve the systolic and diastolic function of infarcted rats10,13. It is possible that a greater training volume, as well as the hemodynamic overload triggered by the aerobic training, had been responsible for these adaptations.

Table 2 – Hemodynamic assessments in groups sedentary control (SC), trained control (TC), sedentary infarcted (SI), and trained infarcted (TI)

Variables/groups SC TC SI TI

Hemodynamics

SBP (mmHg) 125 ± 4 130 ± 3 113 ± 2* 121 ± 4†

DBP (mmHg) 85 ± 2 87 ± 3 85 ± 4 88 ± 5

MBP (mmHg) 98 ± 5 101 ± 4 94 ± 4 99 ± 3

HR (bpm) 327 ± 8 334 ± 10 351 ± 12 355 ± 14

Baroreflex sensitivity

TR (bpm/mmHg) 3.5 ± 0.1 4.4 ± 0.6 1.8 ± 0.1*† 2.2 ± 0.3*†

BR (bpm/mmHg) -2.4 ± 0.09 -4.3 ± 0.31* -1.3 ± 0.05*† -2.1 ± 0.21†

Values express mean ± standard error of the mean. * p < 0.05 vs. SC; † p < 0.05 vs. TC.SBP: systolic blood pressure; DBP: diastolic blood pressure; MBP: mean blood pressure; HR: heart rate; TR: tachycardic response; BR: bradycardic response.

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Table 3 – Pulse interval and systolic blood pressure variability, in the time and frequency domains, in groups sedentary control (SC), trained control (TC), sedentary infarcted (SI), and trained infarcted (TI)

Variables/groups CS CT IS IT

PI variance

SD-PI (bpm) 11.5 ± 0.7 9.4 ± 1.3 5.3 ± 0.4* 8.1 ± 0.4*†

PI-var (ms2) 136.4 ± 16.1 140.0 ± 15.3 35.7 ± 7.0*‡ 64.4 ± 4.0*†‡

RMSSD (ms) 6.3 ± 0.4 8.4 ± 0.8* 4.6 ± 0.2*‡ 6.0 ± 0.2

HF/LF 0.41 ± 0.02 0.43 ± 0.05 0.19 ± 0.03* 0.28 ± 0.07

SBP variance

SBP-var (mmHg2) 27.5 ± 6.0 23.2 ± 3.9 19.7 ± 2.2 26.9 ± 4.4

HF (mmHg2) 1.9 ± 0.2 1.7 ± 0.2 1.3 ± 0.2 1.8 ± 0.3

α index (LF, ms/mmHg) 1.13 ± 0.09 1.33 ± 0.32 0.62 ± 0.24* 0.76 ± 0.06*

Values express mean ± standard error of the mean. * p < 0.05 vs. SC; † p < 0.05 vs. SI; ‡ p < 0.05 vs. TC.PI: pulse interval; SD-PI: standard deviation of the pulse interval; PI-var: pulse interval variance; RMSSD: root mean square of successive RR interval differences; LF: low frequency band; HF: high frequency range; SBP: systolic blood pressure; SBP-var: systolic blood pressure variance; HF: high frequency.

On the other hand, the E/A ratio and MPI, an index that represents the overall cardiac stress, improved with RT in infarcted animals, thus suggesting some favorable cardiac adaptation to dynamic RT in these animals. Corroborating this hypothesis, group SI animals showed a SBP reduction in comparison to those of group SC. However, after the RT period, this variable became normal in trained animals. Recognizing that SBP reflects the cardiac work capacity, as suggested by Yu and McNeill25, we can hypothesize that the SBP reduction observed in the present study may be related to a reduction in ventricular performance in the sedentary group, thus becoming normal after the RT period.

Using a RT equipment different from the one used in the present study, Pinter et al26 demonstrated that a 8-week RT promoted reduction in BP and HR, improvement in papillary muscle contractility and increase in cardiac myosin ATPase activity in healthy rats. On the other hand, Barauna et al22 suggest that RT leads to the development of concentric cardiac hypertrophy without changing the ventricular function or cavity in healthy rats. Thus, the disagreement between the findings related to the cardiac function may have resulted from the choice of the RT model, as well as from the presence of ventricular dysfunction triggered by MI in the animals.

Evidences from the literature suggest that aerobic RT performed during MI recovery provided increased HR variability, which is an important index of the autonomic function and predictive of mortality27. In addition, La Rovere et al8 showed that aerobic RT after MI may favorably modify the long-term survival and that this benefit is probably related to improvement of the baroreflex sensitivity and, consequently, of the autonomic imbalance after training in these infarcted individuals. In agreement with findings in humans, our group recently demonstrated that a 3-month aerobic training was able to improve HR variability, autonomic modulation, and baroreflex sensitivity in rats after MI10,13, thus increasing the survival of trained animals.

In fact, most of the studies point to aerobic RT as an important tool for the management of autonomic dysfunction in patients after MI; however, the effects of RT on the cardiac autonomic variables remain poorly examined. In clinical and experimental studies with CI, increases in the LF band of HR variability have been shown to be linked to the degree of sympathoexcitation, as assessed by direct measures of the sympathetic nervous activity or of plasma norepinephrine28,29. However, in advanced stages of the disease, the opposite is also true, i.e., the LF band of HRV almost disappears because of increased sympathetic activity30,31. Reduction of this component has been associated with a poor prognosis in patients with heart failure, since La Rovere et al32 showed that decreased LF band is an independent predictor of cardiovascular mortality in these individuals.

In the present study, although the baroreflex sensitivity and alpha index did not change, RT promoted positive adaptations in SD-PI and RMSSD, increased the LF and HF bands of PI, and normalized the LF/HF balance. In addition, group TI showed a reduction in the LF range of SBP in relation to group SI. Therefore, we can suggest that RT triggered a reduction in the cardiac and vascular sympathetic modulation, and promoted an increase in the parasympathetic modulation, thus improving, in turn, the cardiac autonomic balance of infarcted rats undergoing 3-month RT. The failure to change the baroreflex sensitivity and the alpha index of SBP suggests that RT may have led to important adaptations in the cardiovascular control centers, which could explain a better cardiac and vascular autonomic control in trained animals. However, further studies investigating the effects of RT on the central control of circulation are necessary to better explain these evidences.

ConclusionsIn conclusion, the findings of the present study suggest that low/

moderate intensity resistance exercise training may be an important and safe therapeutic tool after myocardial infarction, considering

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Figure 2 – Absolute values of low (A) and high frequency (B) bands of pulse interval variability, and of low frequency band (C) of systolic blood pressure in the experimental groups. * p < 0.05 vs. group sedentary control (SC); ‡ p < 0.05 vs. group trained control (TC); † p < 0.05 vs. Group sedentary infarcted (SI), Trained infarcted (TI).

LF P

I (m

s2 )HF

PI (

ms2 )

LF S

BP (m

mHg

2 )

SC

SC

SC

TC

TC

TC

SI

SI

SI

TI

TI

TI

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1. Go AS, Mozaffarian D, Roger VL, Benjamin EJ, Berry JD, Borden WB, et al; American Heart Association Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics-2013 update: a report from the American Heart Association. Circulation. 2013;127(1):e6-e245. Erratum in: Circulation. 2013;127(23):e841.

2. Yousef ZR, Redwood SR, Marber MS. Postinfarction left ventricular remodelling: where are the theories and trials leading us? Heart. 2000;83(1):76-80.

3. La Rovere MT, Bigger JT Jr, Marcus FI, Mortara A, Schwartz PJ. Baroreflex sensitivity and heart-rate variability in prediction of total cardiac mortality after myocardial infarction. ATRAMI (Autonomic Tone and Reflexes After Myocardial Infarction) Investigators. Lancet. 1998;351(9101):478-84.

4. Negrao CE, Middlekauff HR. Adaptations in autonomic function during exercise training in heart failure. Heart Fail Rev. 2008;13(1):51-60.

5. Sullivan MJ, Higginbotham MB, Cobb FR. Exercise training in patients with chronic heart failure delays ventilatory anaerobic threshold and improves submaximal exercise performance. Circulation. 1989;79(2):324-9.

6. Haykowsky M, Scott J, Esch B, Schopflocher D, Myers J, Paterson I. A meta-analysis of the effects of exercise training on left ventricular remodeling following myocardial infarction: start early and go longer for greatest exercise benefits on remodeling. Trials. 2011;12:92.

7. Vona M, Codeluppi GM, Iannino T, Ferrari E, Bogousslavsky J, von Segesser LK. Effects of different types of exercise training followed by detraining on endothelium-dependent dilation in patients with recent myocardial infarction. Circulation. 2009;119(12):1601-8.

8. La Rovere MT, Bersano C, Gnemmi M, Specchia G, Schwartz PJ. Exercise-induced increase in baroreflex sensitivity predicts improved prognosis after myocardial infarction. Circulation. 2002;106(8):945-9.

9. Flores LJ, Figueroa D, Sanches IC, Jorge L, Irigoyen MC, Rodrigues B, et al. Effects of exercise training on autonomic dysfunction management in an experimental model of menopause and myocardial infarction. Menopause. 2010;17(4):712-7.

10. Jorge L, Rodrigues B, Rosa KT, Malfitano C, Loureiro TC, Medeiros A, et al. Cardiac and peripheral adjustments induced by early exercise training intervention were associated with autonomic improvement in infarcted rats: role in functional capacity and mortality. Eur Heart J. 2011;32(7):904-12.

12. Rodrigues B, Jorge L, Mostarda CT, Rosa KT, Medeiros A, Malfitano C, et al. Aerobic exercise training delays cardiac dysfunction and improves autonomic control of circulation in diabetic rats undergoing myocardial infarction. J Card Fail. 2012;18(9):734-44.

13. Barboza CA, Rocha LY, Mostarda CT, Figueroa D, Caperuto EC, De Angelis K, et al. Ventricular and autonomic benefits of exercise training persist after detraining in infarcted rats. Eur J Appl Physiol. 2013;113(5):1137-46.

14. Williams MA, Haskell WL, Ades PA, Amsterdam EA, Bittner V, Franklin BA, et al; American Heart Association Council on Clinical Cardiology; American Heart Association Council on Nutrition, Physical Activity, and Metabolism. Resistance exercise in individuals with and without cardiovascular disease: 2007 update: a scientific statement from the American Heart Association Council on Clinical Cardiology and Council on Nutrition, Physical Activity, and Metabolism. Circulation. 2007;116(5):572-84.

15. Pu CT, Johnson MT, Forman DE, Hausdorff JM, Roubenoff R, Foldvari M, et al. Randomized trial of progressive resistance training to counteract the myopathy of chronic heart failure. J Appl Physiol (1985). 2001;90(6):2341-50.

16. Levinger I, Bronks R, Cody DV, Linton I, Davie A. Resistance training for chronic heart failure patients on beta blocker medications. Int J Cardiol. 2005;102(3):493-9.

17. Mandic S, Myers J, Selig SE, Levinger I. Resistance versus aerobic exercise training in chronic heart failure. Curr Heart Fail Rep. 2012;9(1):57-64.

18. Selig SE, Carey MF, Menzies DG, Patterson J, Geerling RH, Williams AD, et al. Moderate-intensity resistance exercise training in patients with chronic heart failure improves strength, endurance, heart rate variability, and forearm blood flow. J Card Fail. 2004;10(1):21-30.

19. Sanches IC, Conti FF, Sartori M, Irigoyen MC, De Angelis K. Standardization of resistance exercise training: effects in diabetic ovariectomized rats. Int J Sports Med. 2014 Apr;35(4):323-9.

20. Mostarda C, Rodrigues B, Vane M, Moreira ED, Rosa KT, Moraes-Silva IC, et al. Autonomic impairment after myocardial infarction: role in cardiac remodeling and mortality. Clin Exp Pharmacol Physiol. 2010;37(4):447-52.

21. Malliani A, Pagani M. Spectral analysis of cardiovascular variabilities in the assessment of sympathetic cardiac regulation in heart failure. Pharmacol Res. 1991;24 Suppl 1:43-53.

References

that it does not lead to considerable changes in ventricular function, reduces the overall cardiac stress, and significantly improves the cardiac and vascular autonomic modulation.

AcknowledgementsTo Professor Dr. Claudia Borim, for her technical support

in the statistical analyses.This study was financially supported by Fundação de

Amparo à Pesquisa do Estado de São Paulo (FAPESP), protocol 2013/14788-9. CFG had a Masters scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). MCI, KDA, and BR had financial support from Conselho National de Pesquisa e Desenvolvimento (CNPq-BPQ).

Author contributionsConception and design of the research: Irigoyen MC,

Rodrigues B; Acquisition of data: Grans CF, Feriani DJ, Abssamra MEV, Rocha LY, Carrozzi NM, Mostarda C, Figueroa

DM; Analysis and interpretation of the data: Grans CF, Feriani DJ, Abssamra MEV, Rocha LY, Carrozzi NM, Mostarda C, Figueroa DM, Angelis KD, Rodrigues B; Statistical analysis: Grans CF, Feriani DJ, Abssamra MEV, Rocha LY, Carrozzi NM, Rodrigues B; Obtaining financing: Rodrigues B; Writing of the manuscript: Grans CF, Angelis KD, Irigoyen MC, Rodrigues B; Critical revision of the manuscript for intellectual content: Angelis KD, Irigoyen MC, Rodrigues B.

Potential Conflict of InterestNo potential conflict of interest relevant to this article

was reported.

Sources of FundingThis study was funded by CAPES and FAPESP.

Study AssociationThis article is part of the thesis of master submitted by

Camila F. Grans, from Universidade São Judas Tadeu (USJT).

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22. Barauna VG, Rosa KT, Irigoyen MC, de Oliveira EM. Effects of resistance training on ventricular function and hypertrophy in a rat model. Clin Med Res. 2007;5(2):114-20.

23. Harrington D, Coats AJ. Skeletal muscle abnormalities and evidence for their role in symptom generation in chronic heart failure. Eur Heart J. 1997;18(12):1865-72.

24. McKelvie RS, McCartney N, Tomlinson C, Bauer R, MacDougall JD. Comparison of hemodynamic responses to cycling and resistance exercise in congestive heart failure secondary to ischemic cardiomyopathy. Am J Cardiol. 1995;76(12):977-9.

25. Yu Z, McNeill JH. Blood pressure and heart rate response to vasoactive agents in conscious diabetic rats. Can J Physiol Pharmacol. 1992;70(12):1542-8.

26. de Cássia Cypriano Ervati Pinter R, Padilha AS, de Oliveira EM, Vassallo DV, de Fúcio Lizardo JH. Cardiovascular adaptive responses in rats submitted to moderate resistance training. Eur J Appl Physiol. 2008;103(5):605-13.

27. Kukielka M, Holycross BJ, Billman GE. Endurance exercise training reduces cardiac sodium/calcium exchanger expression in animals susceptible to ventricular fibrillation. Front Physiol. 201;2:3.

28. Kienzle MG, Ferguson DW, Birkett CL, Myers GA, Berg WJ, Mariano DJ. Clinical, hemodynamic and sympathetic neural correlates of heart rate variability in congestive heart failure. Am J Cardiol. 1992;69(8):761-7.

29. Ishise H, Asanoi H, Ishizaka S, Joho S, Kameyama T, Umeno K, et al. Time course of sympathovagal imbalance and left ventricular dysfunction in conscious dogs with heart failure. J Appl Physiol (1985). 1998;84(4):1234-41.

30. Van de Borne P, Montano N, Pagani M, Oren R, Somers VK. Absence of low-frequency variability of sympathetic nerve activity in severe heart failure. Circulation. 1997;95(6):1449-54.

31. Galinier M, Pathak A, Fourcade J, Androdias C, Curnier D, Varnous S, et al. Depressed low frequency power of heart rate variability as an independent predictor of sudden death in chronic heart failure. Eur Heart J. 2000;21(6):475-82.

32. La Rovere MT, Pinna GD, Maestri R, Mortara A, Capomolla S, Febo O, et al. Short-term heart rate variability strongly predicts sudden cardiac death in chronic heart failure patients. Circulation. 2003;107(4):565-70.

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High-Sensitivity C-Reactive Protein as a Predictor of Cardiovascular Events after ST-Elevation Myocardial InfarctionDaniel Rios Pinto Ribeiro, Adriane Monserrat Ramos, Pedro Lima Vieira, Eduardo Menti, Odemir Luiz Bordin Jr., Priscilla Azambuja Lopes de Souza, Alexandre Schaan de Quadros, Vera Lúcia PortalPrograma de Pós-Graduação em Ciências da Saúde: Cardiologia - Instituto de Cardiologia/Fundação Universitária de Cardiologia, Porto Alegre, RS – Brazil

Mailing address: Vera Lúcia Portal •Avenida Princesa Isabel, 370, Santana. Postal Code 90620-000, Porto Alegre, RS – Brazil.E-mail: [email protected] received October 16, 2013; revised manuscript received January 29, 2014; accepted January 31, 2014.

DOI: 10.5935/abc.20140086

Abstract

Background: The association between high-sensitivity C-reactive protein and recurrent major adverse cardiovascular events (MACE) in patients with ST-elevation myocardial infarction who undergo primary percutaneous coronary intervention remains controversial.

Objective: To investigate the potential association between high-sensitivity C-reactive protein and an increased risk of MACE such as death, heart failure, reinfarction, and new revascularization in patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention.

Methods: This prospective cohort study included 300 individuals aged >18 years who were diagnosed with ST-elevation myocardial infarction and underwent primary percutaneous coronary intervention at a tertiary health center. An instrument evaluating clinical variables and the Thrombolysis in Myocardial Infarction (TIMI) and Global Registry of Acute Coronary Events (GRACE) risk scores was used. High-sensitivity C-reactive protein was determined by nephelometry. The patients were followed-up during hospitalization and up to 30 days after infarction for the occurrence of MACE. Student’s t, Mann–Whitney, chi-square, and logistic regression tests were used for statistical analyses. P values of ≤0.05 were considered statistically significant.

Results: The mean age was 59.76 years, and 69.3% of patients were male. No statistically significant association was observed between high-sensitivity C-reactive protein and recurrent MACE (p = 0.11). However, high-sensitivity C-reactive protein was independently associated with 30-day mortality when adjusted for TIMI [odds ratio (OR), 1.27; 95% confidence interval (CI), 1.07–1.51; p = 0.005] and GRACE (OR, 1.26; 95% CI, 1.06–1.49; p = 0.007) risk scores.

Conclusion: Although high-sensitivity C-reactive protein was not predictive of combined major cardiovascular events within 30 days after ST-elevation myocardial infarction in patients who underwent primary angioplasty and stent implantation, it was an independent predictor of 30-day mortality. (Arq Bras Cardiol. 2014; 103(1):69-75)

Keywords: Protein C; Myocardial Infarction / mortality; Electrocardiography; Diagnosis; Prognosis.

IntroductionCoronary artery disease (CAD) is a major cause of

mortality worldwide. It accounted for 7 million deaths in the year of 2011, which corresponds to 11.2% of the overall mortality during that period1. Within the clinical spectrum of CAD, ST-elevation myocardial infarction (STEMI) accounts for 29%–47% cases of acute coronary syndrome (ACS)2,3. STEMI results from the rupture of an atherosclerotic plaque with superimposed coronary thrombosis in approximately 75% patients4,5.

Inflammatory responses play a key role in plaque rupture6,7. The usefulness of inflammatory markers as indicators of hidden atherosclerosis, in the improvement of risk algorithms8-12, and as predictors of the risk of recurrent events and death during ACS13-20 has been investigated. Among these markers, high-sensitivity C-reactive protein (hs-CRP) is the most extensively studied21.

When it comes to the prognosis of STEMI, there is a series of conflicting results with regard to hs-CRP22-27. Selection biases and heterogeneity of the reperfusion modalities are some of the limitations of the different studies. In the setting of primary percutaneous coronary intervention (pPCI), the data available are controversial and scarce, particularly with regard to short-term events28. Moreover, risk scores with a strong prognostic ability, particularly Thrombolysis in Myocardial Infarction (TIMI) and Global Registry of Acute Coronary Events (GRACE) scores, have been broadly used with the aim of early risk stratification29,30. However, these scores do not include inflammatory markers. This study aimed to assess the association of hs-CRP with a composite endpoint including

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Arq Bras Cardiol. 2014; 103(1):69-75

death, reinfarction, new revascularization, and heart failure, that occurred within 30 days of the index event in patients with STEMI who underwent pPCI and stenting.

Material and Methods

SampleThe study sample was selected from patients diagnosed with

STEMI according to the World Health Organization criteria who were admitted to a tertiary care hospital of interventional cardiology between 2002 and 2010. The inclusion criteria were as follows: age ≥ 18 years, both sexes, indication of pPCI and stenting, and ability to fast for at least 12 h. The exclusion criteria were as follows: history of malignancy, presence of human immunodeficiency virus (HIV) infection, presence of inflammatory disease, duration of >24 h between infarction and hospital admission, use of corticosteroid therapy, and current or recent use of nonsteroidal anti-inflammatory drugs (NSAIDs; within the last month).

MethodsThis was a prospective cohort study in which each patient was

first approached right after admission to the emergency room. Patients meeting the inclusion criteria, but not the exclusion criteria, were invited to participate in the study and were asked for written informed consent. After receiving consent, a complete history was taken, followed by physical examination. Patients were assessed with respect to population data (age, sex, race), the presence of risk factors for ischemic heart disease (hypertension, diabetes mellitus, dyslipidemia, cigarette smoking, family history of CAD, and obesity), and medications used. Patients with systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg, those using anti-hypertensive medication, and those with a previous diagnosis of hypertension were considered hypertensive. Patients with diabetes were defined as those with a previous diagnosis of diabetes mellitus, those consuming hypoglycemic drugs, or those with fasting blood glucose ≥ 126 mg/dL before admission. Dyslipidemia was defined as serum low-density lipoprotein cholesterol (LDL-C) > 130 mg/dL, high-density lipoprotein cholestrol (HDL-C) < 40 mg/dL, triglycerides > 150 mg/dL, or a combination of these. Smokers were considered as those who currently smoked on a regular basis or those who had quit for <1 year. A positive family history was defined as a diagnosis of CAD or other atherosclerotic disease in a male first-degree relative aged < 55 years or a female first-degree relative aged < 65 years. Obesity was diagnosed on the basis of a body mass index of ≥30 kg/m2. Clinical and electrocardiographic characteristics of the infarct, TIMI and GRACE risk scores, and type of hospital care were recorded. Then, a blood sample was drawn for hs-CRP determination. The sample was collected after a 12-h fast, but no later than within the first 24 h of infarct onset. Hs-CRP was determined using the nephelometry method (Dade Behring BNTM II, Liederbach, Germany). From hospital admission to discharge, all patients were followed-up by the research team, and the occurrence of clinical complications such as arrhythmias, heart failure, reinfarction, percutaneous reintervention, coronary artery bypass grafting, and death were recorded.

The primary endpoint of the study was the combination of major adverse cardiovascular events (MACE) including death, heart failure, reinfarction, or new revascularization (whether percutaneous or surgical) within the first 30 days after the index event. Death was considered as that from all causes. The heart failure endpoint was investigated on the basis of clinical parameters suggestive of pulmonary congestion and/or signs of low cardiac output. Reinfarction was defined as angina or anginal equivalent accompanied by a new ST-segment elevation in leads consistent with those of the territory of the artery affected in the index event. New revascularization was characterized by the need for percutaneous or surgical intervention motivated by instability of the clinical picture (elective revascularization were not considered endpoints).

Thirty days after infarction, the patients were assessed in the ambulatory care section of the Institute of Cardiology so that the occurrence of any endpoint of interest could be identified.

Ethical considerationsThe research project was approved by the Research

Ethics Committee of the Institute of Cardiology/University Foundation of Cardiology, under protocol number 4406.09. All patients provided written informed consent.

Statistical analysisSample size calculation: Assuming a MACE prevalence

rate of 23% (checked in a preliminary analysis of the database), with an error margin of 5% and a 95% confidence interval (95% CI), the required sample size was calculated to be at least 273 patients. Categorical variables were described as proportions, while quantitative variables were described as means and standard deviations or medians and interquartile ranges. For quantitative variables, Student’s t-test or the Mann–Whitney test was used to evaluate comparisons between groups. The chi-square test was used to evaluate comparisons between categorical variables. Multiple logistic regression multivariate analysis was conducted with MACE as a dependent variable. The results of multivariate analyses were expressed as odds ratios (ORs). The significance level was set at p ≤ 0.05, and analyses were conducted using Statistical Package for the Social Sciences (SPSS) software version 19.

ResultsA total of 300 patients with a mean age of 59 ± 11

years were evaluated. Most patients were male (69.3%) and Caucasian (89.8%). The most prevalent risk factor for ischemic heart disease was systemic hypertension, found in 62.2% patients. Previous conditions and procedures, as well as the medications used prior to hospitalization, are shown in Table 1.

The mean TIMI and GRACE risk scores in the sample cohort were 3.57 (standard deviation, 2.40) and 143.07 (standard deviation, 35.35), respectively. The percentage of patients assigned to Killip classification groups I, II, III, and IV were 83, 12.3, 2, and 2.7%, respectively.

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Table 1 – Baseline patient characteristics

Mean age (years) 59.76 (± 11)

Male sex (%) 69.3

Caucasian race (%) 89.8

Risk factors (%)

SAH 62.2

Cigarette smoking 48.0

FH 44.3

Dyslipidemia 39.9

Diabetes mellitus 21.6

Obesity 18.2

Most frequent previous conditions and procedures (%)

AMI 14.5

Peripheral vascular disease 7.4

Gastrointestinal disease 6.8

Ischemic stroke 5.7

COPD 3.4

PCI 11.8

CABG 3.4

Medications more frequently used before the index AMI (%)

ACEI 27.0

Antiplatelet drugs 25.0

Beta-blockers 22.0

Diuretics 16.0

Statins 11.0

SAH: systemic arterial hypertension, FH: family history of cardiovascular disease, AMI: acute myocardial infarction, COPD: chronic obstructive pulmonary disease, PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; ACEI, angiotensin converting-enzyme inhibitors

Table 2 – Incidence of the most frequent endpoints 30 days after ST-elevation AMI

Endpoints N Incidence (%)

Heart failure 96 32.2

Death 16 5.3

New AMI 10 3.3

CABG 3 1.1

New PCI 1 0.4

MACE 104 34.7

AMI: acute myocardial infarction; CABG: coronary artery bypass grafting; PCI: percutaneous coronary intervention; MACE: major adverse cardiovascular events (death + heart failure + new PCI + CABG + new AMI).

Table 3 – High-sensitivity C-reactive protein (Hs-CRP) levels and major adverse cardiovascular events (MACE) 30 days after ST-elevation acute myocardial infarction

MACE* hs-CRP** (mg/L) p value

Yes 8.0 (3.7-23.5) 0.112

No 6.4 (3.1-17.2) 0.112

*Deaths + heart failure + new percutaneous coronary intervention + coronary artery bypass grafting + new acute myocardial infarction**values expressed as medians and interquartile ranges

The patients were divided into two groups on the basis of the year of enrollment: group 1 (137 individuals hospitalized between 2002 and 2006) and group 2 (163 patients hospitalized between 2007 and 2010). No significant difference was observed in the in-hospital use of acetylsalicylic acid, thienopyridine, angiotensin-converting enzyme inhibitors (ACEI), beta-blockers, or statins between groups.

During the first 30 days of post-infarction follow-up, 16 deaths occurred, corresponding to an overall mortality of 5.3%. In addition, 96 patients with heart failure (32.2%), one PCI procedure (0.4%), three coronary artery bypass graftings (1.1%), and ten patients with new AMI (3.3%) were identified. Recurrent MACE were observed in 104 patients (34.7%; Table 2).

When the patients were compared with respect to the occurrence of MACE within the first 30 days after the index event, we observed a median hs-CRP of 8.0 mg/L (range, 3.7–23.5 mg/L) in the group presenting with the endpoint and 6.4 mg/L (range, 3.1–17.2 mg/L) in the remaining (p = 0.11; Table 3). Among those who developed heart failure, the

median hs-CRP was 8.0 mg/L (range, 3.7–26.0 mg/L), while the median hs-CRP was 6.4 mg/L (range, 3.1–15.5 mg/L) in the group without heart failure (p = 0.057). When hs-CRP was assessed in relation to death, there was a significant association (p = 0.05; Figure 1). The causes of death and the respective hs-CRPs in each patient are shown in Table 4.

Multivariate analysis of hs-CRP in relation to all-cause mortality, after adjusting the markers for TIMI risk score (Table 5), showed an OR of 1.28 (95% CI, 1.08–1.52; p = 0.005). The OR of the TIMI score extracted from the logistic regression equation was 1.32 (95% CI, 1.09–1.59; p = 0.004) in relation to the same endpoint. On the other hand, when hs-CRP and GRACE risk scores were included in logistic regression, an independent association was found between hs-CRP and 30-day mortality (OR, 1.26; 95% CI, 1.07–1.50; p = 0.007). The OR corresponding to the GRACE score obtained from the same analysis was 1.02 (95% CI, 1.01–1.03; p = 0.002).

DiscussionThe present study evaluated the prognostic role of hs-CRP

with regard to the occurrence of 30-day recurrent MACE in patients with AMI treated with pPCI. No statistically significant association between this marker and the composite endpoint was observed. However, hs-CRP was significantly higher in patients who died within 30 days of the index event, and this association remained even after adjustment for TIMI and GRACE risk scores.

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Table 4 – Causes of death and respective hs-CRP levels

Cases Death cause(s) hs-CRP (mg/L)

1 Cardiogenic and septic shock 4.1

2 Cardiogenic shock 7.6






8 CHF and pneumonia 48.8


10 Cardiogenic shock; contrast-induced AKI 3.4

11 CHF 49.9




15 Ventricular fibrillation 22

16 Undetermined 4.2

Hs-CRP: high-sensitivity C-reactive protein; CHF: congestive heart failure; AKI: acute kidney injury.

The nature of our findings is based on the hypothesis that, in individuals with AMI, the elevation of this biomarker is more attributable to the inflammatory response resulting from myocardial lesions than to vascular inflammation, unlike in individuals with unstable angina, in whom the elevation of

Figure 1 – Bivariate analysis of high-sensitivity C-reactive protein (hs-CRP) in relation to 30-day mortality after ST-elevation acute myocardial infarction (p = 0.05)

CRP levels is associated with plaque instability and recurrent infarctions. Therefore, in patients with ACS and ST-elevation, elevated CRP levels have been related to mortality and heart failure, but not to recurrent infarctions24.

There is controversy surrounding the prognostic role of CRP in patients with STEMI. In an analysis of approximately 1000 patients with a mean follow-up of 23 months, Suleiman et al.22 verified an association between CRP and higher rates of overall mortality and heart failure after hospital discharge. Foussas et al23 found higher mortality rates within 30 days after STEMI in patients with CRP levels ≥ 5 mg/L, while the TIMI scores were the same in both groups. However, this study was initiated before the use of clopidrogel was widespread, and it did not provide information on the percentage of patients undergoing mechanical reperfusion23.

Whether the results of studies point to an association between CRP and relevant clinical endpoints24 or oppose that hypothesis25,27,31, most available evidence is based on patients receiving thrombolytic drugs. The retrospective design and failure to use the high-sensitivity method for determining CRP are also important limitations25.

In the context of STEMI treated with pPCI, the role of CRP as a prognostic marker has remained unclear. This is attributed to the paucity of evidence in this field and the disparity of information and methodological flaws present in the different publications. Tomoda and Aoki32 evaluated 234 patients with STEMI who underwent pPCI plus stenting and showed that a CRP level of ≥0.3 mg/dL was an independent predictor of a composite endpoint comprising in-hospital coronary reocclusion, reinfarction, target-vessel revascularization, and death. Limitations of this study were a retrospective design, failure to use contemporary technologies of percutaneous intervention, failure to use

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the high-sensitivity method to determine CRP, and failure to exclude individuals with inflammatory diseases or using antiinflamatory drugs.

In an Italian cohort comprising 758 patients, hs-CRP was associated with short-term mortality, long-term death, AMI and target-vessel revascularization33. This was also a retrospective study and was associated with a high rate of patient exclusion from the cohort (22% of the initial sample) because of the lack of CRP data on admission .

Two other studies showed an association between CRP and short-term MACE34,35. However, these studies were limited by a small sample size of 230 and 146 patients, respectively, and failure to explicitly list potential exclusion criteria, such as the use of NSAIDs and presence of inflammatory diseases. Kruk et al28 also reported that hs-CRP was predictive of in-hospital death without mentioning the exclusion criteria in their article.

Unlike the studies previously mentioned, and still in the context of PCI, Ohlmann et al36 did not show a prognostic relationship among CRP, infarct size, and mortality in a multivariate analysis. A small sample size (87 patients) and the fact that CRP was not determined using the high-sensitivity method may have influenced the results36. In turn, Damman et al37 did not include CRP in the score for the prediction of mortality in a very recent study with a significant number of patients (n = 1034) because the marker was not proven to be associated with mortality when adjusted for TIMI score variables. We should point out that, once again, concomitant inflammatory conditions were not considered and individuals in cardiogenic shock were excluded.

Our study plays an important role in the debate regarding prognosis and patient stratification because it shows an independent association between hs-CRP and a hard endpoint such as mortality. As previously mentioned, the association of CRP with clinically relevant endpoints in the setting of acute ischemic syndromes with ST-elevation has not yet reached a consensus in the literature. This was a prospective study that excluded clinical situations accompanied by an inflammatory response and the use of medications that interfere in this response, and it was conducted with all the contemporary therapeutic armamentarium38. The adjustment of hs-CRP to TIMI and GRACE risk scores29,30 provides our results with even greater consistency.

Table 5 – Predictors of death within 30 days after ST-elevation AMI (multivariate analysis)

Odds ratio (95%CI) p value

hsCRP adjusted for TIMI score 1.28 (1.08-1.52) 0.005

hsCRP adjusted for GRACE score 1.26 (1.07-1.50) 0.007

AMI: acute myocardial infarction; 95% CI: 95% confidence interval; hs-CRP: high-sensitivity C-reactive protein; TIMI: Thrombolysis in Myocardial Infarction; GRACE: Global Registry of Acute Coronary Events.

This study also has some limitations. First was its observational design. Second, it failed to establish a cut-off point for CRP level in relation to mortality. Third, it failed to use the C statistic to evaluate the prognostic accuracy of CRP in the context of STEMI because of the low frequency of this endpoint. Another limitation was the lack of hemodynamic data corresponding to coronary anatomy, which is justified by the fact that this was a fundamentally clinical study.

ConclusionsAlthough hs-CRP was not predictive of composite MACE

within the first 30 days after acute ST-elevation myocardial infarction in patients who underwent primary angioplasty and stenting, this marker proved to be an independent predictor of all-cause mortality.

Author contributionsConception and design of the research: Ribeiro DRP,

Ramos AM, Quadros AS, Portal VL; Acquisition of data: Ribeiro DRP, Ramos AM, Vieira PL, Menti E, Bordin Jr. OL, Souza PAL; Analysis and interpretation of the data, Writing of the manuscript and Critical revision of the manuscript for intellectual content: Ribeiro DRP, Quadros AS, Portal VL; Statistical analysis: Ribeiro DRP, Portal VL.



Sources of Funding

There were no external funding sources for this study.

Study Association

This article is part of the thesis of master submitted by Daniel Rios Pinto Ribeiro, from Programa de pos-graduação de Ciencias da Saúde – Cardiologia da Fundação Universitária de Cardiologia.

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8. Ridker PM, Buring JE, Rifai N, Cook NR. Development and validation of improved algorithms for the assessment of global cardiovascular risk in women: the Reynolds Risk Score. JAMA. 2007;297(6):611-9. Erratum in JAMA. 2007;297(13):1433.

9. Zethelius B, Berglund L, Sundström J, Ingelsson E, Basu S, Larsson A, et al. Use of multiple biomarkers to improve the prediction of death from cardiovascular causes. N Engl J Med. 2008;358(20):2107-16.

10. Ridker PM, Rifai N, Clearfield M, Downs JR, Weis SE, Miles JS, et al; Air Force/Texas Coronary Atherosclerosis Prevention Study Investigators. Measurement of C-reactive protein for the targeting of statin therapy in the primary prevention of acute coronary events. N Engl J Med. 2001;344(26):1959-65.

11. Blake GJ, Ridker PM, Kuntz KM. Projected life-expectancy gains with statin therapy for individuals with elevated C-reactive protein levels. J Am Coll Cardiol. 2002;40(1):49-55.

12. Ridker PM, Danielson E, Fonseca FA, Genest J, Gotto AM Jr, Kastelein JJ, et al; JUPITER Study Group. Rosuvastatin to prevent vascular events in men and women with elevated C-reactive protein. N Engl J Med. 2008;359(21):2195 207.

13. Morrow DA, de Lemos JA, Sabatine MS, Wiviott SD, Blazing MA, Shui A, et al. Clinical relevance of C-reactive protein during follow-up of patients with acute coronary syndromes in the Aggrastat-to-Zocor Trial. Circulation. 2006;114(4):281-8.

14. Liuzzo G, Biasucci LM, Gallimore JR, Grillo RL, Rebuzzi AG, Pepys MB, et al. The prognostic value of C-reactive protein and serum amyloid a protein in severe unstable angina. N Engl J Med. 1994;331(7):417-24.

15. Morrow DA, Rifai N, Antman EM, Weiner DL, McCabe CH, Cannon CP, et al. C-reactive protein is a potent predictor of mortality independently of and in combination with troponin T in acute coronary syndromes: a TIMI 11A substudy. Thrombolysis in Myocardial Infarction. J Am Coll Cardiol. 1998;31(7):1460-5.

16. Toss H, Lindahl B, Siegbahn A, Wallentin L. Prognostic influence of increased fibrinogen and C-reactive protein levels in unstable coronary artery disease. FRISC Study Group. Fragmin during Instability in Coronary Artery Disease. Circulation. 1997;96(12):4204-10.

17. Lindahl B, Toss H, Siegbahn A, Venge P, Wallentin L. Markers of myocardial damage and inflammation in relation to long-term mortality in unstable coronary artery disease. FRISC Study Group. Fragmin during Instability in Coronary Artery Disease. N Engl J Med. 2000;343(16):1139-47.

18. Heeschen C, Hamm CW, Bruemmer J, Simoons ML. Predictive value of C-reactive protein and troponin T in patients with unstable angina: a comparative analysis. CAPTURE Investigators. Chimeric c7E3 AntiPlatelet Therapy in Unstable angina REfractory to standard treatment trial. J Am Coll Cardiol. 2000;35(6):1535-42.

19. Biasucci LM, Liuzzo G, Grillo RL, Caligiuri G, Rebuzzi AG, Buffon A, et al. Elevated levels of C-reactive protein at discharge in patients with unstable angina predict recurrent instability. Circulation. 1999;99(7):855-60.

20. James SK, Armstrong P, Barnathan E, Califf R, Lindahl B, Siegbahn A, et al; GUSTO-IV-ACS Investigators. Troponin and C-reactive protein have different relations to subsequent mortality and myocardial infarction after acute coronary syndrome: a GUSTO-IV substudy. J Am Coll Cardiol. 2003;41(6):916-24.

21. Pepys MB, Hirschfield GM. C-reactive protein: a critical update. J Clin Invest. 2003;111(12):1805-12.

22. Suleiman M, Khatib R, Agmon Y, Mahamid R, Boulos M, Kapeliovich M, et al. Early Inflammation and Risk of Long-Term Development of Heart Failure and Mortality in Survivors of Acute Myocardial Infarction: Predictive Role of C-Reactive Protein. J Am Coll Cardiol. 2006;47(5):962-8.

23. Foussas SG, Zairis MN, Lyras AG, Patsourakos NG, Tsirimpis VG, Katsaros K, et al. Early prognostic usefulness of C-reactive protein added to the Thrombolysis In Myocardial Infarction Risk Score in acute coronary syndromes. Am J Cardiol. 2005;96(4):533-7.

24. Suleiman M, Aronson D, Reisner SA, Kapeliovich MR, Markiewicz W, Levy Y, et al. Admission C-reactive protein levels and 30-day mortality in patients with acute myocardial infarction. Am J Med. 2003;115(9):695-701.

25. Nikfardjam M, Müllner M, Schreiber W, Oschatz E, Exner M, Domanovits H, et al. The association between C-reactive protein on admission and mortality in patients with acute myocardial infarction. J Intern Med. 2000; 247(3):341-5.

26. Zebrack JS, Anderson JL, Maycock CA, Horne BD, Bair TL, Muhlestein JB, et al; Intermountain Heart Collaborative (IHC) Study Group. Usefulness of high-sensitivity C-reactive protein in predicting long-term risk of death or acute myocardial infarction in patients with unstable or stable angina pectoris or acute myocardial infarction. Am J Cardiol. 2002;89(2):145-9.

27. Mega JL, Morrow DA, De Lemos JA, Sabatine MS, Murphy SA, Rifai N, et al. B-type natriuretic peptide at presentation and prognosis in patients with ST-segment elevation myocardial infarction: An ENTIRE–TIMI-23 substudy. J Am Coll Cardiol. 2004;44(2):335-9.

28. Kruk M, Przyłuski J, Kalińczuk Ł, Pregowski J, Deptuch T, Kadziela J, et al; ANIN Myocardial Infarction Registry Group. Association of Non-Specific Inflammatory Activation With Early Mortality in Patients With ST-Elevation Acute Coronary Syndrome Treated With Primary Angioplasty. Circ J. 2008;72(2):205-11.

29. Morrow DA, Antman EM, Charlesworth A, Cairns R, Murphy SA, de Lemos JA, et al. TIMI risk score for ST-elevation myocardial infarction: a convenient, bedside, clinical score for risk assessment at presentation: an In TIME trial substudy. Circulation. 2000;102(17):2031-7.

30. Granger CB, Goldberg RJ, Dabbous O, Pieper KS, Eagle KA, Cannon CP, et al; Global Registry of Acute Coronary Events Investigators. Predictors of Hospital Mortality in the global registry of acute coronary events. Arch Intern Med. 2003;163(19):2345-53.

31. Steg PG, Ravaud P, Tedgui A, Puel J, Moyse D, Curaudeau E, et al; ELISCOR Investigators. Predischarge C-reactive protein and 1-year outcome after acute coronary syndromes. Am J Med. 2006;119(8):684-92.

32. Tomoda H, Aoki N. Prognostic value of C-reactive protein levels within six hours after the onset of acute myocardial infarction. Am Heart J. 2000;140(2):324-8.

33. Ortolani P, Marzocchi A, Marrozzini C, Palmerini T, Saia F, Taglieri N, et al. Predictive value of high sensitivity C-reactive protein in patients with ST-elevation myocardial infarction treated with percutaneous coronary intervention. Eur Heart J. 2008;29(10):1241-9.

References

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34. Magadle R, Hertz I, Merlon H, Weiner P, Mohammedi I, Robert D. The relation between preprocedural C-reactive protein levels and early and late complications in patients with acute myocardial infarction undergoing interventional coronary angioplasty. Clin Cardiol. 2004;27(3):163-8.

35. Yip HK, Hang CL, Fang CY, Hsieh YK, Yang CH, Hung WC, et al. Level of high-sensitivity C-reactive protein is predictive of 30-day outcomes in patients with acute myocardial infarction undergoing primary coronary intervention. Chest. 2005;127(3):803-8.

36. Ohlmann P, Jaquemin L, Morel O, El Behlgiti R, Faure A, Michotey MO, et al. Prognostic value of C-reactive protein and cardiac troponin I in primary

percutaneous interventions for ST-elevation myocardial infarction. Am Heart J. 2006;152(6):1161-7.

37. Damman P, Beijk MA, Kuijt WJ, Verouden NJ, van Geloven N, Henriques JP, et al. Multiple biomarkers at admission significantly improve the prediction of mortality in patients undergoing primary percutaneous coronary intervention for acute ST-segment elevation myocardial infarction. J Am Coll Cardiol. 2011;57(1):29-36.

38. Boden WE, Eagle K, Granger CB. Reperfusion strategies in acute ST-segment elevation myocardial infarction: a comprehensive review of contemporary management options. J Am Coll Cardiol. 2007;50(10):917-29.

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Lipoprotein (a): Structure, Pathophysiology and Clinical ImplicationsRaul Cavalcante Maranhão, Priscila Oliveira Carvalho, Celia Cassaro Strunz, Fulvio PileggiInstituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (USP), São Paulo, SP – Brazil

KeywordsLipoprotein (a) / metabolism; Lipoproteins, LDL /

ultrastructure; Risk Factors; Cardiovascular Diseases.

Mailing Address: Raul Cavalcante Maranhão •Avenida Dr. Enéas de Carvalho Aguiar, 44, bloco 2, 1º subsolo, sala 21 – Cerqueira César. Postal Code 05403-000, São Paulo, SP – BrazilE-mail: [email protected], [email protected] Manuscript received June 21, 2013; revised manuscript September 25, 2013; accepted October 01, 2013.

DOI: 10.5935/abc.20140101

AbstractThe chemical structure of lipoprotein (a) is similar to

that of LDL, from which it differs due to the presence of apolipoprotein (a) bound to apo B100 via one disulfide bridge. Lipoprotein (a) is synthesized in the liver and its plasma concentration, which can be determined by use of monoclonal antibody-based methods, ranges from < 1 mg to > 1,000 mg/dL. Lipoprotein (a) levels over 20-30 mg/dL are associated with a two-fold risk of developing coronary artery disease. Usually, black subjects have higher lipoprotein (a) levels that, differently from Caucasians and Orientals, are not related to coronary artery disease. However, the risk of black subjects must be considered. Sex and age have little influence on lipoprotein (a) levels. Lipoprotein (a) homology with plasminogen might lead to interference with the fibrinolytic cascade, accounting for an atherogenic mechanism of that lipoprotein. Nevertheless, direct deposition of lipoprotein (a) on arterial wall is also a possible mechanism, lipoprotein (a) being more prone to oxidation than LDL. Most prospective studies have confirmed lipoprotein (a) as a predisposing factor to atherosclerosis. Statin treatment does not lower lipoprotein (a) levels, differently from niacin and ezetimibe, which tend to reduce lipoprotein (a), although confirmation of ezetimibe effects is pending. The reduction in lipoprotein (a) concentrations has not been demonstrated to reduce the risk for coronary artery disease. Whenever higher lipoprotein (a) concentrations are found, and in the absence of more effective and well-tolerated drugs, a more strict and vigorous control of the other coronary artery disease risk factors should be sought.

Lipoprotein (a) and apolipoprotein (a) structuresThe particle of lipoprotein (a), Lp(a), first detected by Berg in

19631, is a spherical macromolecular complex with a diameter of approximately 25 nm, and density ranging from 1.05 to 1.12 g/mL. The Lp(a) structure is similar to that of low-density lipoprotein (LDL), regarding size and lipid composition of the particles and the presence of apolipoprotein B100 (apo B100). The major structural difference between both is that, in

addition to apo B, Lp(a) has a second protein, apolipoprotein (a) [apo(a)], bound to apo B100 via noncovalent interactions and one single disulfide bridge. The presence of apo(a) determines the differences in density and electrophoretic mobility between LDL and Lp(a), and the molecular weight of that glycoprotein varies widely from 400 to 700 kDa. For the purpose of comparison, the molecular weight of apo B100 is approximately 550 kDa, and those of apo A1 and apo Cs are approximately 7 and 29 kDa, respectively2.

A fundamental discovery was that apo(a) is markedly similar to plasminogen, one of the proteins of the fibrinolytic system. Apo(a) comprises a domain of inactive protease or serine-protease, whose amino acid sequence coincides with that of plasminogen in 94%. In addition, there are 2 other domains constituted by tridimensional heavy-chain structures, highly glycosylated, known as kringles3.

The serine-protease domain of apo(a) shows replacement of the serine amino acid by arginine in the activation site equivalent to that of plasminogen. That hinders the conversion of Lp(a) into active protease by tissue plasminogen activator (t-PA), urokinase or streptokinase, such as with plasminogen3.

Of the kringle domains of apo(a), one is similar to the kringle V (KV) of plasminogen, with only 9% replacement of amino acids. The other, kringle IV (KIV), which is present only once in the plasminogen structure, has 10 different types in apo(a) (KIV types 1 to 10). Only KIV type 2 occurs repeatedly in the apo(a) sequence, coinciding with about 84% of the amino acid sequence of KIV in plasminogen. Thus, KV is a single copy, while KIV repeats 10 to 40 times in the apo(a) structure. The number of KIV repetitions is genetically determined, ranging from 12 to 51 times, resulting in 34 different apo(a) isoforms3,4.

Using electrophoresis and immunoblotting, the following 6 different alleles for Lp(a) were identified: Lp(a)F; Lp(a)B; Lp(a)S1; Lp(a)S2; Lp(a)S3; and Lp(a)S4. The letters F, S and B relate to apo(a) mobility as compared to that of apo B100, standing for “fast” (fast mobility), “slow” (slow mobility), and mobility similar to that of apo B100, respectively. The isoform is determinant to Lp(a) plasma concentration, because it represents a limiting factor in Lp(a) synthesis. Smaller proteins are secreted more efficiently than those of higher molecular weight. Isoforms with fewer KIV type 2 repetitions, that is, smaller sequences of apo(a), tend to determine higher Lp(a) concentrations and to increase atherothrombogenic activity4. Thus, there is a strong inverse correlation between the molecular weight of apo(a) isoforms and the plasma concentration of Lp(a).

The existence of a seventh allele, called ‘null’ [Lp(a)0], which would lead to absence of the lipoprotein in plasma, has not been confirmed; by using more sensitive methods to detect Lp(a), none totally negative individuals have been observed. In addition, no subject with more than 2 alleles for apo(a) exists4.

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The presence of apo B100 in Lp(a) makes that lipoprotein co-precipitate with LDL in the assays currently used to separate lipoproteins by using the chemical precipitation method. This interferes with LDL values calculated with the Friedewald formula5. Thus, if the Lp(a) concentration in a patient is high, LDL-cholesterol calculation with that formula is not accurate without corrections that consider the Lp(a) concentration.

Methodology to determine Lp(a)The most common method to quantify Lp(a) consists in

determining the apo(a) concentration by using monoclonal anti-apo(a) antibodies. The first commercial kits measured Lp(a) by use of radioimmunoassay or radial immunodiffusion6. Currently, enzyme immunoassay (ELISA) and methods based on nephelometry or turbidimetry are more often used7. The wide variation in apo(a) molecular weight makes the ratio between mass and molar concentration vary between individuals. When the method to determine Lp(a) involves antibodies that react with the apo(a) kringle region, which has high individual variability, differences in reaction not related to molar concentration might occur, explaining the differences in normal Lp(a) plasma levels in different population samples. In that context, there are difficulties in standardizing the methodology to determine Lp(a) to allow a more accurate comparison between different studies. So far, new methods to determine Lp(a) are being developed.

Lp(a) synthesis and metabolismDespite the structural similarities between Lp(a) and

LDL, Lp(a) synthesis and metabolism, which have not been completely clarified, are totally independent from LDL synthesis and metabolism. In vitro studies have shown that apo(a) synthesis takes place in hepatocytes, and its association with apo B100 should occur on cell surface8. Thus, the liver has been described as the major site of Lp(a) synthesis. There is no coordination between the synthesis pathways of apo(a) and of apo B100, as there is no coordination between the synthesis of Lp(a) and of plasminogen, its structural analogue.

Similarly to LDL, Lp(a) does not derive from the catabolism of another lipoprotein9. In individuals with elevated triglyceridemia, Lp(a) is reduced, probably due to an increase in the plasma lipoprotein clearance10. However, when VLDL lipolysis was stimulated by heparin inoculation during catheterization in patients with normal lipid levels, there was a reduction in triglyceride levels, with no change in Lp(a) concentration. This confirms that Lp(a) levels are not related to the lipoprotein lipase activity11.

The way Lp(a) cellular uptake occurs has not been well established. Several studies have shown that Lp(a) binds to specific LDL receptors, although with less affinity. Two possible explanations for that difference in affinity are: (1) some Lp(a) domains near the domain of LDL-receptor binding would be covered by apo(a); or (2) apo(a) would not bind to apo B100 in the receptor binding site, causing changes in the apo B100 binding region. However, it is worth noting that, when apo(a) is dissociated from Lp(a) by cleavage of disulfide bridges, the binding capacity of the lipoprotein increases, becoming equivalent to that of LDL12.

There is evidence that the LDL receptor might not be so important in Lp(a) plasma removal. Large clinical studies have reported that statins have no effect on Lp(a) concentrations. Because statins induce superexpression of LDL receptors, greater Lp(a) plasma removal and consequent lower Lp(a) plasma levels would be expected if the receptor was essential for that process. Other receptors, such as asialoglycoprotein receptors, megalin receptors, and macrophage scavenger receptors, can also be involved in Lp(a) uptake13. The capacity of macrophages to uptake Lp(a) is important, because the excessive uptake of lipoproteins by macrophages, with their subsequent transformation into foam cells, is the major mechanism of atherogenesis.

Other studies have shown elevated Lp(a) plasma levels in patients with heterozygous familial hypercholesterolemia, known to have deficiency of LDL receptors. Considering that such increase is a direct consequence of a defect in the receptor that interacts with the apo B100 of Lp(a), the genetic defect in apo B100 would be expected to cause that same situation, similarly to that with LDL. However, that condition could not be confirmed, because the Lp(a) plasma levels were not affected by apo B100 mutation. In addition, only a small fraction of Lp(a) binds to hepatoma cells via LDL receptor, and the major part of lipoproteins associates with those cells via another cellular mechanism14. Thus, although the LDL receptor acts upon Lp(a) removal, its role in that process is limited.

The experiences carried out so far have not evidenced a physiological function for Lp(a) in lipid transportation or metabolism regulation. Up to now, Lp(a) remains conceptually only a “pathogenic lipoprotein”. In individuals with residual Lp(a) concentrations, neither organic deficiencies nor predisposition to any disease have been reported15.

Apo(a) genetic and ethnic aspects In men, the gene encoding the apo(a) protein, the LPA,

was cloned and sequenced for the first time in 1987, showing homology with up to 70% of the human plasminogen gene. The LPA gene is located in the same cluster of the plasminogen gene, in the long arm of chromosome 6, in the 6q2.6-2.7 region3. The LPA gene is characterized by 10 different variants present in the KIV domain and by multiple repetitions, ranging from 2 to 43, in the KIV type 2 domain3,4.

Because of that impressive genetic variability of apo(a) and the involvement of other genes related to Lp(a) synthesis and metabolism, that lipoprotein plasma levels can vary more than 1,000 times between individuals of the same population16. The LPA gene might be responsible for 91% of the variation in Lp(a) concentration. Of that variation, 69% are due to the number of KIV type 2 repetitions, and 22% to other factors17.

The allele frequency varies even more according to ethnicity, indicating that the racial factor has an important influence on Lp(a) levels. Such levels have a non-Gaussian distribution in white and Oriental individuals, being similar in those 2 populations. In the Sub-Saharan population and Afro-Americans, that distribution is Gaussian, and Lp(a) levels are more elevated, reaching means up to 2 to 3 times those of the Caucasian or Oriental population18.

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In a study with several ethnic groups, Lp(a) polymorphism has influenced in 17% to 77% of the variation in Lp(a) concentrations19. Regarding the variation in Lp(a) levels, 80% resulted from the number of kringles (KIV/KV ratio)20. In more than 7,000 individuals, divided into non-Hispanic whites, non-Hispanic blacks, and Mexican Americans, 19 polymorphisms were analyzed. Of the 19 polymorphisms, 15 were associated with Lp(a) levels in at least one of the subpopulations, 6 in at least 2 subpopulations, and none in all 3 subpopulations21. Those data are consistent with data from other studies that have shown little or no effect of other factors, such as gender and age, on Lp(a) concentrations7. The genetic factor is the major responsible for that variation.

Lp(a) pathophysiology Plasma concentrations of Lp(a) have a hereditary character,

with large interindividual variation, being not altered by environmental factors, and tending to remain constant throughout life. In the general population, Lp(a) concentrations can range from < 1 mg/dL to > 1,000 mg/dL.

Increases in Lp(a) levels can be transient in the presence of inflammatory processes or tissue damages, such as those occurring with other acute phase proteins (haptoglobin, alpha-1-antitripsin, and C-reactive protein)22. This can follow an episode of acute myocardial infarction, in which Lp(a) levels increase considerably in the first 24 hours, returning to baseline values in approximately 30 days23.

Lp(a) levels are increased in chronic inflammatory disease, such as rheumatoid arthritis24, systemic lupus erythematosus25, and acquired immunodeficiency syndrome26, and under some conditions, such as after heart transplantation27, chronic renal failure28, and pulmonary arterial hypertension29. On the other hand, liver diseases and abusive use of steroid hormones decrease Lp(a) levels28.

The relationship between Lp(a) and diabetes mellitus has not been well established. Regarding type 1 diabetes mellitus, some studies have reported higher Lp(a) levels30, which have not been confirmed by other studies31. Conflicting results have also been reported for type 2 diabetes mellitus. In a sub-study carried out from the San Antonio Heart Study, diabetic men and women showed no difference in Lp(a) concentrations when compared with non-diabetic individuals32. On the other hand, a prospective study carried out with 26,746 North-American women has shown a higher incidence of type 2 diabetes mellitus among those with lower Lp(a) levels33.

Several mechanisms of Lp(a) participation in atherogenesis have been proposed. One of them consists in the direct deposition of that lipoprotein on arterial wall, similarly to that which happens with LDL and oxidized LDL. The fact that Lp(a) is more likely to undergo oxidation than LDL itself might facilitate uptake by macrophages via scavenger receptors13. That is the most universal mechanism of atherogenesis, in which macrophages ‘indulge themselves’ in the cholesterol from LDL, and eventually from Lp(a), transforming themselves into foam cells, precursors of atherosclerosis. Another pro-atherogenic mechanism of Lp(a) would relate to the inverse correlation between that lipoprotein levels and vascular reactivity, in which case the increase in Lp(a) plasma levels would induce endothelial dysfunction34.

The influence of Lp(a) levels on carotid intima-media thickness is still controversial. While Kotani and Sakane35 have found an inverse association in the Japanese population, no relationship between that thickness and Lp(a) levels has been found in Spaniards by Calmarza et al36.

Other authors have found a positive association of Lp(a) gene polymorphisms and that lipoprotein levels with the incidence of ischemic cerebral vascular accident of large vessels, peripheral arterial disease, and abdominal aorta aneurysm. Association with the number of obstructed coronary arteries was observed, but not with carotid intima-media thickness. In addition, patients with coronary artery disease (CAD) and those polymorphisms are more susceptible to atherosclerotic manifestations outside the coronary tree37.

Associations between Lp(a) and inflammatory cytokines, such as tumor necrosis factor alpha (TNF-a), transforming growth factor beta (TGF-β), interleukine 6 (IL-6), and monocyte chemoattractant protein (MCP-1), have been reported38,39. Thus, the participation of Lp(a) in atherogenesis could be multifaceted. In addition to a reduction in fibrinolysis, it would involve platelet aggregation, induction of the expression of adhesion molecules, vascular remodeling via changes in the proliferative and migratory capacity of endothelial cells and resident smooth muscle cells, oxidative modification and formation of foam cells.

It is worth noting that the apo(a) gene has multiple elements of IL-6 response, and in vitro studies have demonstrated that the expression of that gene is increased by IL-6, leading to the accumulation of Lp(a) particles39. The Lipid Analytic Cologne (LIANCO) Study has found an association between the IL-6 polymorphism 74G/C and elevated Lp(a) levels (≥ 60 mg/dL)40.

Along with the discovery of homology between apo(a) and plasminogen, a mechanism linking thrombogenesis and atherogenesis with plasma lipoproteins via Lp(a) has caused great excitement in the scientific field. The hypothesis is as follows: Lp(a) would interfere with the fibrinolytic system, suggesting that Lp(a) competes with plasminogen for binding sites of endothelial cells, inhibiting fibrinolysis and promoting intravascular thrombosis41. In that scenario, Lp(a) would be a link between atherogenesis and thrombogenesis, explaining the redoubled interest in that possible mechanism.

An interesting question has been raised by Edelberg et al42, who have reported that Lp(a) interferes in vitro with the thrombolytic action of t-PA. However, Santos Filho et al43 have tested the hypothesis in patients undergoing post-acute myocardial infarction thrombolysis with rt-PA, and have observed no difference in the restenosis frequency of those with high Lp(a) levels.

In patients with cardiovascular disease, the possibility of accumulating Lp(a) in the postprandial period, due to competition between Lp(a) and remnants of chylomicrons generated by absorption of fat from the diet, has been studied. However, that possibility has been ruled out by the evidence that Lp(a) levels have not changed in those patients after a fatty meal44.

Lp(a) as a risk factor for atherosclerosisCross-sectional studies performed so far have widely

confirmed the association between Lp(a) levels and the risk for developing CAD, regardless of other risk factors. Kostner

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et al45 have estimated that risk as being 2.3 times higher in patients with Lp(a) levels over 50 mg/dL, while Riches and Porter38 have calculated that risk as twice greater for Lp(a) levels over 20 mg/dL. The relationship between Lp(a) and CAD and cerebral infarction has been confirmed by Murai et al46 in the Japanese population and by Rhoads et al47 in Japanese descendants in Hawaii. The latter study has reported that, in individuals under the age of 60 years with Lp(a) levels over 30 mg/dL, the risk was 2.5 times greater and decreased as age increased, dropping to 1.6 in the age group from 60 to 69 years, and to 1.2 in the age group over 70 years. In the Brazilian population of São Paulo, Maranhão et al48 have reported a risk of developing CAD 2.3 times greater when Lp(a) levels were over 25 mg/dL.

Several prospective studies have been published, and, contrary to the cross-sectional studies, they have not been so assertive in identifying Lp(a) as an independent risk factor. Their results are conflicting, ranging from strong positive associations to complete lack of association between Lp(a) and cardiovascular diseases. However, most prospective studies have supported the hypothesis that Lp(a) is really an independent risk factor for cardiovascular disease.

In one of the first studies, carried out in Boston, United States of America, with almost 15,000 men (age range, 40 to 84 years), no prevalence of high Lp(a) levels was identified in those who would subsequently develop acute myocardial infarction49. In the prospective study conducted in Quebec, Canada, for 5 years, with 2,000 men (age range, 47 to 76 years), Lp(a) has not appeared as an independent risk factor for cardiac events, although high Lp(a) levels have apparently exacerbated the potency as risk factors of both hypercholesterolemia and low HDL-cholesterol concentration50.

Lp(a) has been identified as an independent risk factor in a population of 6,000 Koreans with CAD, in which patients with high Lp(a) levels had worse disease course51. A meta-analysis encompassing 27 prospective studies and involving approximately 5,500 individuals has shown a clear independent association between Lp(a) and CAD, although 9 of those studies included individuals with preexisting disease52.

In addition to CAD, Lp(a) can be a risk factor for atherosclerosis in other arterial beds, such as in ischemic cerebral disease, in which the risk appears with a Lp(a) cutoff point of 30 mg/dL53.

In a North-American prospective study with approximately 14,000 participants, Caucasian women and Afrodescendant men and women with high Lp(a) have shown a higher incidence of ischemic cerebral disease over a 13-year follow-up. Caucasian men, however, have not shown an increased risk associated with high Lp(a) levels54.

Smolders et al55, reviewing 31 cross-sectional and prospective studies involving approximately 50,000 individuals, have suggested that high Lp(a) levels can be associated with the risk for ischemic cerebral vascular accident. A cohort study involving 2,365 individuals with CAD, 284 with ischemic cerebral vascular accident and 596 with peripheral arterial disease has shown an association of increased Lp(a) levels with

future events of arterial diseases, but not with ischemic cerebral disease. It is worth noting that such association was independent of LDL-cholesterol levels56.

Atherogenesis is a common causal factor of abdominal aortic aneurysm, while thoracic aortic aneurysm results from aortic dissection and is not associated with atherosclerosis. Lp(a) levels seem more elevated in abdominal aneurysm than in thoracic aneurysm, which is in accordance with the concept of the association between lipoprotein and atherogenesis57.

An important aspect relates to extremely high Lp(a) levels, whether they can represent a more significant risk factor. A Danish prospective study involving more than 9,000 individuals over a 10-year follow-up has shown that extremely high Lp(a) levels (≥ 120 mg/dL) increased 3 to 4 times the risk for CAD58.

In a meta-analysis of 40 prospective studies with 58,000 participants, a 2-fold increase in the risk for developing CAD and cerebral vascular accident has been found in individuals with smaller apo(a) isoforms, regardless of the Lp(a) concentration and the classical risk factors59.

Another important aspect is the relationship that Lp(a) might have with sex. Although most studies have shown no difference between sexes in Lp(a) concentrations, more elevated lipoprotein levels seem to be more significant risk factors in the female sex than in the male sex60. The last Atherosclerosis Risk in Communities (ARIC) Study, assessing Lp(a) as a risk factor, has found a difference between sexes in that lipoprotein plasma concentration, which was higher in women, both Caucasian and black61. Knoflach et al62, assessing risk factors for atherosclerosis in young women, have shown that Lp(a) levels related to the carotid intima-media thickness, while the classical risk factors had no influence on that parameter. In postmenopausal women, elevated Lp(a) and triglyceride levels were predictive of the presence of CAD63.

In black individuals, the mean Lp(a) concentrations are markedly high, 2 to 3 times greater than in Caucasian and Oriental individuals18. In older studies, with a more limited statistical power, Lp(a) levels have been assumed as non-predictive of cardiovascular disease in black individuals. However, a recent study with almost 3,500 Afro-Americans has reported a higher incidence of cardiovascular diseases and events when comparing between the highest and lowest Lp(a) concentrations61.

Table 1 lists several cross-sectional and prospective studies assessing Lp(a) levels as a risk factor for atherosclerotic vascular diseases.

Effects of drugs on Lp(a) concentrationTraditional lipid-lowering therapies, such as statins or fibrates,

do not consistently result in a reduction in Lp(a) concentrations. The use of atorvastatin at the dose of 20 mg/day for 24 weeks has resulted in both lack of effect on Lp(a) levels64 and a decrease in that lipoprotein levels in hypercholesterolemic individuals with no disease65. In a double-blind study with placebo, using doses of 10 or 40 mg/day for 12 weeks, the Lp(a) concentration has significantly decreased66. Of lovastatin, simvastatin and gemfibrozil, the latter has shown greater efficacy in reducing Lp(a)67.

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Ezetimibe reduces Lp(a) levels in as much as 29%68. However, ezetimibe is most often used in association with simvastatin, which has no additive effect to that of ezetimibe in regard to Lp(a).

Another compound widely used in the treatment of dyslipidemia, niacin, effectively reduces Lp(a) levels when administered at high doses. Patients receiving 2 g/day and 4 g/day of niacin have shown a 25% and 38% reduction in Lp(a) levels,

respectively. At lower doses (1 g/day), niacin has not shown that effectiveness69. Etofibrate, a hybrid drug that combines niacin and clofibrate, at the dose of 1 g/day reduces Lp(a) levels by 26% in type IIb dyslipidemic patients70. Patients with type IIa and IIb hyperlipidemia, undergoing treatment with neomycin, have reduced their Lp(a) levels by 24%, while the neomycin-niacin association has resulted in a 45% reduction. That effect is obtained with high doses of both drugs71.

Table 1 – Studies assessing lipoprotein (a), Lp(a), as a risk factor for atherosclerotic vascular disease: coronary artery disease (CAD), acute myocardial infarction (AMI), and ischemic cerebral vascular accident (ICVA)

Study type / duration Population Lp(a) cutoff point (mg/dL)

Independent risk or association Atherosclerotic manifestation Reference

Cross-sectional 183 men > 50 2.3 times higher AMI Kostner et al45

Cross-sectional 426 Japanese: 268 men and 158 women > 17 Positive CAD and ICVA Murai et al46

Cross-sectional 711 Japanese men in Hawaii > 302.5 times: < 60 years1.6 time: 60-69 years

1.2 time: > 70 years of ageAMI Rhoads et al47

Cross-sectional 162 Brazilians: 112 men and 50 women ≥ 25 2.3 times higher CAD Maranhão et al48

Prospective (5 years) 15,000 North-American men 30 Negative AMI Stampfer et al49

Prospective (5 years) 2,000 Canadian men 30 Negative CAD Cantin et al50

Meta-analysis (10 years) 27 prospective studies, 5,500 individuals of both sexes 20-100 Positive CAD Danesh et al52

Retrospective (1997-1999) 182 Brazilian postmenopausal women 2 to 3 times higher Obstructive CAD Sposito et al63

Prospective 346 men and 184 women of European descent 2 times higher CAD

Lp(a) 2 times higher in women Frohlich et al60

Prospective (13.5 years) 14,000 Caucasians and Afrodescendants of both sexes 30 Positive ICVA, except for Caucasian men Ohira et al54

Meta-analysis (1966-2006)31 cross-sectional and

prospective studies, 50,000 individuals of both sexes

≥ 30 Positive ICVA Smolders et al55

Meta-analysis (1966-2008) 2,000 individuals of both sexes Positive Abdominal aortic aneurysm Takagi et al57

Cross-sectional 205 young women (18 to 22 years of age) ≥ 30 Positive Carotid intima-media thickness Knoflach et al62

Prospective730 Caucasian, black and

Hispanic individuals of both sexes

≥ 30 PositiveICVA

Higher incidence in men and black individuals

Boden-Albala et al53

Meta-analysis 58,000 individuals of both sexes

smaller apo(a) isoforms 2 times higher

CAD and ICVAIndividuals with smaller apo(a)

isoforms had higher riskErqou et al59

Prospective 6,000 Koreans of both sexes ≥ 20.1 1.8 time higher CAD Worse disease course Kwon et al51

Prospective 2,000 Europeans of the United Kingdom of both sexes ≥ 25 Positive

Future events of arterial diseases (coronary and peripheral),

but not ICVAGurdasani et al56

Prospective (20 years)3,467 Afro-Americans and 9,851 Caucasians of both

sexes

≤ 10 and > 10≤ 20 and > 20≤ 30 and > 30

Positive

Higher number of cardiovascular events in women, higher incidence

when comparing between the highest and lowest Lp(a)

concentrations

Virani et al61

Prospective (10 years) 9,000 Danish individuals of both sexes ≥ 120 3-4 times higher CAD Kamstrup et al58

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Extended-release (ER) niacin has reduced Lp(a) levels in diabetic patients with dyslipidemia. Both ER niacin and conventional niacin at high doses are good drugs to treat dyslipidemia, because, in addition to reducing LDL-cholesterol levels, they increase HDL-cholesterol levels and decrease Lp(a) levels72. However, high doses of that drug can be associated with some adverse effects, such as migraine, flushing, diarrhea, vomiting, tachycardia, and liver toxicity. The administration of aspirin 30 minutes prior to niacin can relieve some of those effects. Japanese patients with elevated Lp(a) levels (> 300 mg/L) have shown a 20% reduction in Lp(a) levels with low doses of aspirin (81 mg/day)73. Women with high Lp(a) levels and an apo(a) polymorphic allele seem to have benefited more from the treatment with aspirin than those who lack that allele74.

In addition, LDL apheresis has been able to reduce Lp(a) concentration in more than 50% of patients with familial hypercholesterolemia75.

In hormone replacement, for both men and women76, as well as in hypothyroidism77, Lp(a) concentrations seem to decrease. Even considering the beneficial effects of estrogen therapy on Lp(a) and other plasma lipids, it is worth noting the controversies on hormone replacement regarding the increased risk for certain malignant neoplasias and thromboembolic accidents.

Other agents that might reduce Lp(a) levels are as follows: L-carnitine; a combination of L-lysine and ascorbate; thyromimetics; CETP inhibitors; anti-proprotein convertase subtilisin/kexin type 9 (anti-PCSK-9) monoclonal antibodies; protein responsible for degrading LDL receptor; and anti-tocilizumab antibody, that can block IL-6 signaling and is still in an experimental phase75.

Mipomersen, approved by Food and Drug Administration (FDA) to be used in homozygous familial hypercholesterolemia in January 2013, might be a promise to decrease Lp(a) levels75. Mipomersen is an antisense oligonucleotide that acts on messenger RNA, inhibiting apoB synthesis by the liver, reducing the concentration of lipoproteins that contain that apolipoprotein. That drug can reduce both LDL-cholesterol and Lp(a) levels; however, the safety of its use has not been established78.

Methotrexate, an immunosuppressive and anti-inflammatory drug used in the treatment of rheumatoid arthritis, has also reduced Lp(a) levels79.

So far, there is no specific therapy to decrease Lp(a) levels. New therapeutic agents that can more effectively reduce the concentration of that lipoprotein, which has a high pro-atherogenic potential, being thus a risk factor for cardiovascular disease, are still being sought.

Final considerationsAlmost half a century after the discovery of Lp(a) by Berg,

there is little doubt whether Lp(a) is an independent risk factor for cardiovascular disease. However, the mechanisms linking Lp(a) to atherogenesis are still unclear. In extreme cases, LDL apheresis is recommended80, but studies proving that the therapeutic decrease of Lp(a) reduces the number of events still lack.

In daily clinical practice and in the absence of well-tolerated drugs that effectively decrease Lp(a) concentrations, levels over 25-30 mg/dL should lead to a more strict control of the other risk factors for CAD.

Author contributionsConception and design of the research: Maranhão RC;

Acquisition of data: Carvalho PO; Writing of the manuscript: Maranhão RC, Carvalho PO, Strunz CC; Critical revision of the manuscript for intellectual content: Maranhão RC, Carvalho PO, Strunz CC, Pileggi F.


reported.



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Case 3/2014 - 81-Year-Old Patient Hospitalized for Decompensated Heart FailureBruna Affonso Madaloso and Paulo Sampaio GutierrezInstituto do Coração (InCor) HC-FMUSP, São Paulo, SP - Brazil

Mailing Address: Vera Demarchi Aiello •Avenida Dr. Enéas de Carvalho Aguiar, 44, subsolo, bloco I, Cerqueira César.Postal Code 05403-000, São Paulo, SP – BrazilE-mail: [email protected], [email protected]

KeywordsAmyloidosis / diagnosis; Heart Failure / complications;

Multiple Myeloma; Pulmonary Embolism.

Section editor: Alfredo José Mansur ([email protected])

Associate editors: Desidério Favarato ([email protected])

Vera Demarchi Aiello ([email protected])

DOI: 10.5935/abc.20140102

JJSA is an 81-year-old male patient hospitalized for decompensated heart failure. At 71 years of age, he experienced progressive dyspnea that lasted for 1 month and an episode of palpitation that lasted for 1 day and arrived at at the emergency department. He gave history of undergoing cardioversion in the past. He was then referred to the outpatient clinic of the InCor in October 1995. He also reported that he had long-term pulmonary arterial hypertension and had undergone surgery for the removal of an intracranial hematoma in the past.

Physical examination, during the first consultation (October 30, 1995), revealed the following: heart rate of 76 beats per minute (bpm), systemic blood pressure of 120/80 mmHg, normal findings on chest auscultation. However, cardiac auscultation revealed normal heart sounds, regular rhythm, and a systolic murmur (1+/4+) in the aortic area. Abdominal examination did not reveal any abnormalities and the patient did not have pedal edema.

ECG (October 30, 1995) showed sinus rhythm with atrial extrasystoles, heart rate of 100 bpm, and signs of right atrial overload (small QRS complex in lead V1 and normal in lead V2) as shown in Figure 1.

Four days later, he sought emergency medical assistance because the dyspnea had worsened. Physical examination (November 3, 1995) showed the patient was in a stable condition, had tachypnea (respiratory rate of 24 breaths per minute), heart rate of 80 bpm, and systemic arterial pressure of 90/70 mmHg. Chest auscultation revealed decreased breath sounds at the base of the right lung and crepitations at the base of the left lung. Cardiac auscultation revealed a regular rhythm with the heart sounds were muffled in both systolic and diastolic phases. There were no murmurs or pericardial rub. The liver was palpated 3 cm below the right costal margin and there

was swelling of the right calf. Chest X-ray revealed right atrial opacification; ECG showed no changes.

The results of the laboratory tests were as follows: hemoglobin 16.9 g/dL, hematocrit 39%, leucocytes 5,500/mm³ (60% neutrophils, 33% lymphocytes, and 7% monocytes), platelets 244,000/mm³, sodium 145 mEq/L, potassium 4.3 mEq/L, urea 39 mg/dL, creatinine 1.7 mg/dL, and blood glucose 97 mg/dL. Room-air pulse oximetry showed pH 7.34, pO2 66 mmHg, pCO2 33 mmHg, oxygen saturation 94%, bicarbonate 22 mEq/L, base excess −8 mEq/L.

Pulmonary perfusion/ventilation scintigraphy (November 3, 1995) showed hypoperfusion in the right upper lobe, anterior basal and lateral basal segments of the right lower lobe, lateral basal segment of the left lower lobe, and anterior segment of the left upper lobe. Ventilation was normal except in the base of the right lung, where it was decreased. The examination indicated a parenchymatous pathology in the base of the right lung and thromboembolism in the other pulmonary regions (Figure 2).

The patient received oxygen supplementation via nasal catheter (1 L/min), intravenous heparin (1.000 U/h), 40 mg of oral furosemide, and 37.5 mg of oral captopril daily.

On the third day of hospitalization the patient showed tachycardia, and ECG revealed atrial flutter with atrioventricular block 2:1 (Figure 3). The patient underwent cardioversion and sinus rhythm was maintained (Figure 4) with amiodarone (600 mg daily).

The dyspnea ameliorated and the patient remained hemodynamically stable. He was discharged on the seventh day of hospitalization and was prescribed warfarin (5 mg), captopril (38.5 mg), furosemide (40 mg), and digoxin (0.25 mg) daily.

Echocardiographic evaluation (June 25, 1996) revealed the following measurements: aorta 33 mm, left atrium 52 mm, right ventricle 30 mm, left ventricle (diastole/systole) 64/55 mm, and ejection fraction 36%. There was thickening of the aortic valve without stenosis, moderate tricuspid insufficiency, diffuse hypokinesis of both ventricles, and the systolic pressure of the right ventricle was estimated as 77 mmHg, with signs of pulmonary arterial hypertension (pulmonary valve corrected with absent A wave). Serological tests for Chagas disease was negative.

Echocardiographic evaluation performed later (February 10, 1999) revealed the following measurements: aorta 34 mm, left atrium 47 mm, right ventricle 33 mm, left ventricle (diastole/systole) 64/51 mm, and ejection fraction 49%. There was thickening of the aortic valve with without stenosis, moderate tricuspid insufficiency, diffuse hypokinesis of both ventricles, and the systolic pressure of the right ventricle was estimated as 79 mmHg, with signs of pulmonary arterial hypertension (pulmonary valve corrected with absent A wave).

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Madaloso & Gutierrez81-year-old patient with decompensated heart failure

Arq Bras Cardiol. 2014; 103(1):e1-e10

Figure 1 – Electrocardiogram showing sinus rhythm and right atrial overload.

The patient always experienced dyspnea on moderate exertion and there were several episodes of atrial flutter with subsequent 2:1 atrioventricular conduction, and the control of coagulation was irregular.

Laboratory tests performed in 2004 showed the following results: TSH 9.54 µU/mL, total cholesterol 112 mg/dL, HDL-C 47 mg/dL, LDL-C 53 mg/dL, triglycerides 52 mg/dL, creatinine 1.1 mg/dL, and glucose 87 mg/dL.

From 1999 onwards, the patient’s daily medication consisted of spironolactone (25 mg), enalapril (40 mg), furosemide (40 mg), carvedilol (12.5 mg), digoxin (0.25 mg), and warfarin (5 mg).

An X-ray obtained on May 10, 2005, revealed right pleural thickening with opacification of the right costophrenic angle and the lower third of the right hemithorax.

A laboratory evaluation performed in January 2005 showed that creatinine had increased to 1.9 mg/dL. An analysis performed later (February 2006) showed an even higher level of 2.3 mg/dL, with urea of 66 mg/dL.

In February 2006, the laboratory tests results were as follows: total cholesterol 84 mg/dL, triglycerides 44 mg/dL, potassium 4.8 mEq/L, sodium 144 mEq/L, creatinine 2.3 mg/dL, urea 66 mg/dL, glucose 78 mg/dL, and PT-INR 1.6. The urinalysis results were as follows: density 1.01, pH 5.0, protein 0.6 g/L, free hemoglobin +++, leucocytes 56,000/mL, and erythrocytes 30,000/mL.

The patient was in a stable condition until March 3rd, 2006, when he sought medical assistance because of worsening dyspnea worsening and pedal edema (present for 10 days). He reported having stopped his medication approximately 1 month before.

Physical examination (March 3, 2006) revealed anasarca, heart rate of 80 bpm, systemic arterial pressure of 90/70 mmHg, lungs with decreased breath sounds in the base of the left lung. Cardiac auscultation showed irregular rhythm, muffled heart sounds, and a systolic murmur (++/4+) in the tricuspid area. There was abdominal swelling without tenderness, and bowel movements were present. Scrotal and pedal edema were present (++++/4+).

ECG (March 3, 2006) revealed an irregular rhythm without a visible P wave, frequent ventricular extrasystoles, low-voltage QRS complexes in the frontal plane and in V4 to V6 leads, and left bundle branch block (Figure 5).

The laboratory tests performed on March 3, 2006 showed the following results: erythrocytes 4.4 million/mm³, hematocrit 43%, hemoglobin 14.4 g%, leukocytes 2,900/mm³ (neutrophils 56%, eosinophils 2%, lymphocytes 32%, monocytes 16%), platelets 70,000/mm³, PT-INR 1.8, magnesium, 1.9 mEq/L, calcium 4.76 mEq/L, creatinine 3.3 mg/dL, urea 124 mg/dL, potassium 4.8 mEq/L, sodium 140 mEq/L, and glucose 88 mg/dL.

The patient received 40 mg of furosemide intravenously and was prescribed 120 mg of furosemide, 37.5 mg of captopril, and 0.5 mg of digoxin daily.

The patient was diagnosed with congestive cardiac failure and malnutrition, thrombocytopenia, lymphopenia, and pneumonia. He was further prescribed 2 g of ceftriaxone as antibiotic prophylaxis.

The patient remained hypotensive and was administered intravenous dobutamine.

The laboratory test results on the second day of hospitalization (March 4, 2006) revealed the following: erythrocytes 3.7 million/mm3, hemoglobin 12.7 g/dL, hematocrit 36%, MCV 97 µm³, leukocytes 2,300/mm³ (neutrophils 60%, eosinophils 2%, lymphocytes 25%, and monocytes 13%), platelets 54,000/mm³, urea 127 mg/dL, creatinine 3.3 mg/dL, potassium 4.8 mEq/L, and sodium 145 mEq/L. In the afternoon that day, the patient suffered a cardiorespiratory arrest and died despite the attempts for cardiopulmonary resuscitation.

Clinical aspectsAn 81-year-old man hospitalized for worsening dyspnea.

The patient reported having systemic arterial hypertension for 10 years, with a history of electrical cardioversion for the treatment of cardiac arrhythmia and having been hospitalized in the past for decompensated heart failure.

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Figure 2 – Pulmonary perfusion/ventilation scintigraphy showing hypoperfusion in the right upper lobe, anterior basal and lateral basal segments of the right lower lobe, lateral basal segment of the left lower lobe, and anterior segment of the left upper lobe, and decreased ventilation in the base of the right lung.

During the penultimate hospitalization he had tachypnea and hypoxemia with the swelling of the right calf. Chest X-ray revealed opacification of the right lung base. On the basis of this data, the most probable diagnosis was pulmonary thromboembolism1.

The clinical diagnosis was confirmed by perfusion/ventilation scintigraphy, which showed hypoperfusion

with preserved ventilation. The complementary test most currently used is angiotomography of the pulmonary arteries, which is an alternative less invasive than the gold standard technique of pulmonary angiography. Pulmonary perfusion/ventilation scintigraphy has an important role as it diagnoses 30%–50% cases, although it may have a high rate of inconclusive results1.

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Figure 3 – Electrocardiogram showing atrial flutter with a 2:1 atrioventricular block.

Figure 4 – Electrocardiogram showing sinus rhythm and lateral inactive area.

Ecocardiography revealed systolic dysfunction (left ventricular ejection fraction of 36%), enlarged left atrium (47 mm), and increased pulmonary artery pressure, a complication of pulmonary thromboembolism that is observed in up to 26% of cases1.

Support therapy and anticoagulation treatment were initiated and maintained after hospital discharge.

With time, the patient improved but remained oligosymptomatic and showed progressive renal failure. He showed new cardiac decompensation after having interrupted the use of medications and was hospitalized for treatment. On admission, leukopenia, lymphopenia, and thrombocytopenia were observed. On admission, the chest X-ray showed pleural effusion and a pulmonary consolidation.

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Figure 5 – Electrocardiogram: ventricular extrasystoles, atrial fibrillation, left bundle branch block.

The most probable diagnosis for this elderly patient, who had acute cardiac decompensation, was hypotensive, and whose chest X-ray showed consolidation, would be pneumonia and antibiotic therapy should be initiated. However, a diagnosis of myeloproliferative disorder should be considered because the patient had leukopenia, lymphopenia, and thrombocytopenia. The pulmonary thromboembolism, due to tumor embolism, may present itself as a consolidation in the chest X-ray1.

Therefore, multiple myeloma is a possible diagnosis, because plasmocyte mutations are unique, with neoplastic proliferation of plasmocytes and production of monoclonal immunoglobulin that results in several alterations, such as cytopenia and renal failure.

Renal failure occurs in 20% patients with multiple myeloma. Renal damage is caused by the immunoglobulin light chains produced by the neoplasm. The early mortality rate is 30% among patients with myeloma who progress with renal failure and is mainly associated with sepsis2.

Circulating immunoglobulins in multiple myeloma increase serum viscosity and allow the formation of thrombi and the occurrence of embolism. Therefore, it is reasonable to think that the patient may have had deep vein thrombosis followed by pulmonary thromboembolism, which can be an early manifestation of multiple myeloma3.

Cardiac involvement can also occur, usually resulting in recurrent high-output decompensated cardiac failure4. Decompensated heart failure, in patients with multiple myeloma, can be divided into 2 subgroups: low-output failure, which is more common, and high-output failure5.

High-output decompensated cardiac failure usually occurs in patients with with preserved left ventricular contractility or

underlying structural heart disease, which leads to contractile impairment because of structural deterioration5.

The cause of the high-output failure in multiple myeloma remains only partially understood and has been associated with several factors. These include increased splenic inflow in patients who have splenomegaly with a splenic inflow increase of 55% (in these cases, the spleen functions almost like a arteriovenous fistula), anemia, osteoclastic lesions caused by the disease, which produce substances that lead to high-heart output state6.

Amyloidosis should be considered as a differential diagnosis or associated with multiple myeloma, because it can coexist in 10% cases7. Most frequently, multiple myeloma occurs first and amyloidosis develops later. Some authors have reported cases in which amyloidosis was diagnosed first; however, these cases tend to be more serious and have lower survival of approximately 1-2 years7.

Amyloidosis is an uncommon disease and is therefore often not taken into consideration during diagnosis. It usually presents as restrictive cardiomyopathy, with systolic function preserved until its last stage. Biatrial dilation is also present, which leads to an increase in the incidence of thromboembolic events8,9.

Therefore, secondary causes should be investigated in elderly patients with dilated cardiomyopathy, progressive worsening of renal function, and thromboembolic phenomena. Clinical changes primarily treated as conditions separate from the underlying disease may have a common origin of monoclonal gammopathy, which may present as amyloidosis and multiple myeloma. (Dra. Bruna Affonso Madaloso)

Diagnostic hypotheses: pulmonary thromboembolism, cardiac failure attributed to cardiac amyloidosis, multiple myeloma. (Dra. Bruna Affonso Madaloso)

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Figure 6 – (A) Histological section of the myocardium stained with Congo red, showing amyloid deposits in small vessels and the interstitium. (B) Same area observed under fluorescence microscopy where the amyloid deposition is shown. Objective magnification: 10×.

AutopsyAt autopsy, the heart was heavy and enlarged; however,

the chambers appeared normal. Microscopic examination showed that the primary disease was amyloidosis. This condition affected the myocardium (Figure 6) and the pericardium (Figure 7). In particular, obstructions were observed in the vascular tree of both coronary arteries, leading to the formation of areas of myocardial fibrosis. In addition, obstructions of the vessels supplying the pulmonary artery, and the aorta (vessels of the vasa vasorum) and of renal vessels were noted.

To i n v e s t i g a t e a m y l o i d o s i s , w e p e r f o r m e d immunohistochemical analysis of immunoglobulin light chains. Kappa light-chain staining was negative and lambda light-chain staining was positive (Figure 8), indicating that the patient probably had multiple myeloma. Supporting this was the fact that he had pancytopenia and systemic signs compatible with neoplasia. In the bone marrow sample obtained at autopsy (rib, Figure 9), there was no plasmacytic proliferation or amyloid deposition; however, we cannot completely rule out the possibility that the disease was present in other locations.

The patient was hypertensive and had benign nephrosclerosis and myocardiocyte hypertrophy. In addition, he had chronic obstructive pulmonary disease. As a consequence, the patient had cardiac failure. Secondarily, he had thrombosis in the right atrium. Thus, pulmonary thromboembolism was the causative factor of death (Figure 10). (Dr. Paulo Sampaio Gutierrez)

Primary diagnosis: cardiovascular amyloidosis (AL type) (deposition of lambda light chain).

Secondary diagnosis: systemic arterial hypertension.Causa mortis: pulmonary thromboembolism. (Dr. Paulo

Sampaio Gutierrez)

CommentThe course of progression of cardiac failure suggests that

the primary reason may not be amyloidosis. Possibly, the initial oligosymptomatic phase was caused by systemic arterial hypertension with hypertensive myocardiopathy. Amyloidosis had a major role in the worsening of the patient’s condition and in the final outcome. Amyloid deposition was very significant in vessel walls.

In most cases, cardiovascular amyloidosis is usually diagnosed at autopsy10. A few years ago, we reported that <50% cases were diagnosed before patients die11. In particular, cardiac failure occurred in elderly patients and in patients with other diseases, as was the case with the present patient11. Other difficulties include the absence of low-voltage QRS complexes and the existence of systolic dysfunction. Hence, amyloidosis is suspected in cases of unusual clinical and physiopathological patterns, which normally involve younger patients; however, it occurs mostly in elderly patients who have comorbid conditions.

More than 20 proteins with anomalous conformation are known to form amyloid deposits. In the heart, the most common proteins are transthyretin (especially in senile cardiovascular amyloidosis) and light-chain amyloidosis (AL amyloidosis)12. AL amyloidosis appears to be more frequent; however, the number of cases of senile cardiovascular amyloidosis is increasing as a result of the aging of the population13. The prognosis of AL amyloidosis is worse than that of senile cardiovascular amyloidosis14. In the present case, lambda light-chain staining showed that the disease was AL amyloidosis, despite the patient’s advanced age.

Gammopathies leading to AL amyloidosis and multiple myeloma are plasmocyte dyscrasias, but the relationship between these conditions is not direct. Only 10%-15% patients with myeloma exhibit amyloid deposition. The reverse, i.e., the number of patients with amyloidosis who present

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Figure 7 – Histological section of the epicardium showing amyloid deposit indicated by arrows. Hematoxylin and eosin staining. Objective magnification: 40×.

Figure 8 – Histological section adjacent to that of Figure 6, with immunoperoxidase labeling for lambda light chain, showing positivity in the same areas of the amyloid deposition. Objective magnification: 10×.

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Figure 9 – Histological section of the bone marrow showing absence of plasmacytic proliferation. Hematoxilin and eosin staining; objective magnification: 20×.

myeloma, has been less analyzed. A study conducted with 46 patients with amyloidosis in several organs (83% with cardiac involvement) showed that 57% of them met the criteria for myeloma15. In the case of our patient, because amyloidosis was not clinically suspected, tests for multiple myeloma were not conducted. The bone marrow sample analyzed during autopsy did not exhibit plasmocyte proliferation; however, we cannot completely rule out the possibility that the disease was present in other locations.

Although amyloid deposition in the pericardium has been described16, it is uncommon.

Pulmonary thromboembolism is the most frequent cause of death without diagnosis. In a survey conducted in our hospital17, it accounted for 34% of the cases in which there were discrepancies between diagnoses and the autopsy findings.

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Figure 10 – Thromboembolism of the central pulmonary artery: gross examination (A) and microscopy (B). Hematoxylin and eosin staining. Objective magnification: 5×.

1. Torbicki A1, Perrier A, Konstantinides S, Agnelli G, Galiè N, Pruszczyk P, et al; ESC Committee for Practice Guidelines (CPG). Guidelines on the diagnosis and management of acute pulmonary embolism: the Task Force for the Diagnosis and Management of Acute Pulmonary Embolism of the European Society of Cardiology (ESC). Eur Heart J. 2008;29(18):2276-315.

2. Goldschimdt H, Lannert H, Bommer J, Ho AD. Multiple myeloma and renal failure. Nephrol Dial Transplant. 2000;15(3):301-4.

3. International Myeloma Working Group. Criteria for the classfication of monoclonal gammopathies, multiple myeloma and related disorders: a report of the International Myeloma Working Group. Br J Haematol. 2003;121(5):749-57.

4. Robin J, Fintel B, Pikovskaya O, Davidson C, Cilley J, Flaherty J. Multiple myeloma presenting with high-output heart failure and improving with anti-angiogenesis therapy: two cases report and a review of the literature. J Med Case Rep. 2008;2:229.

5. Mehta PA, Dubrey SW. High output heart failure. QJM. 2009;102(4):235-41.

6. Kosinski D, Roush K, Fraker TD Jr, Grubb BP. High cardiac output state in patients with multiple myeloma: case report and review of the literature. Clin Cardiol. 1994;17(12):678-80.

7. Rajkumar SV, Gertz MA, Kyle RA. Primary systemic amyloidosis with delayed progression to multiple myeloma. Cancer. 1998;82(8):1501-5.

8. Dubrey SW, Cha K, Anderson J, Chamarthi B, Reisinger J, Skinner M, et al. The clinical features of immunoglobulin light-chain (AL) amyloidosis with heart involvement. QJM. 1998;91(2):141-57.

9. Rahman JE, Helou EF, Gelzer-Bell R, Thompson RE, Kuo C, Rodriguez ER, et al. Noninvasive diagnosis of biopsy-proven cardiac amyloidosis. J Am Coll Cardiol. 2004;43(3):410-5.

10. Esplin BL, Gertz MA. Current trends in diagnosis and management of cardiac amyloidosis. Curr Probl Cardiol. 2013;38(2):53-96.

References

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11. Gutierrez PS, Fernandes F, Mady C, Higuchi ML. Características clínicas, eletrocardiográficas e ecocardiográficas na amiloidose cardíaca significativa detectada apenas à necrópsia: comparação com casos diagnosticados em vida. Arq Bras Cardiol. 2008;90(3):191-6.

12. Guan J, Mishra S, Falk RH, Liao R. Current perspectives on cardiac amyloidosis. Am J Physiol Heart Circ Physiol. 2012;302(3):H544-52.

13. Dungu JN, Anderson LJ, Whelan CJ, Hawkins PN. Cardiac transthyretin amyloidosis. Heart. 2012;98(21):1546-54.

14. Ruberg FL, Berk JL. Transthyretin (TTR) cardiac amyloidosis. Circulation. 2012;126(10):1286-300.

15. Dinner S, Witteles W, Witteles R, Lam A, Arai S, Lafayette R, et al. The prognostic value of diagnosing concurrent multiple myeloma in immunog lobul in l i gh t cha in amylo idos i s . Br J Haemato l . 2013;161(3):367-72.

16. Daubert JP, Gaede J, Cohen HJ. A fatal case of constrictive pericarditis due to a marked, selective pericardial accumulation of amyloid. Am J Med. 1993;94(3):335-40.

17. Saad R, Yamada AT, Pereira da Rosa FH, Gutierrez PS, Mansur AJ. Comparison between clinical and autopsy diagnoses in a cardiology hospital. Heart. 2007;93(11):1414-9.

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Case Report

Bioresorbable Vascular Scaffold Use in a Case of In-stent RestenosisJulia Cadrin-Tourigny1, Liang Dong2, Akiko Maehara2, Erick Schampaert1, Philippe Genereux1,2

Hôpital du Sacré-Coeur de Montréal1, Québec, Canada; Columbia University Medical Center and the Cardiovascular Research Foundation2, New York, New York - USA

Mailing Address: Philippe Genereux •111 E, 59th street 12th floor. Postal Code 10023, New York, NY - USAE-mail: [email protected] received 18/10/13; revised manuscript received 12/11/13; accepted 13/11/13.

KeywordsStents; Coronary Artery Disease; Cardiac Catheterization;

Bioresorbable Vascular Scaffold

DOI: 10.5935/abc.20140098

Abbreviation listBMS – Bare metal stentBVS – Bioresorbable vascular scaffoldDES – Drug-eluting stentISR – In-stent restenosisLAD – Left anterior descendingOCT – Optical coherence tomographyPCI – Percutaneous coronary intervention

IntroductionBioresorbable vascular scaffolds (BVS) demonstrate

favourable outcomes in patients with stable coronary disease with simple de novo coronary lesions1,2 and are considered the “fourth revolution” in percutaneous coronary intervention (PCI) technology. BVS represent a promising alternative to drug-eluting stents (DES) while offering the same advantages. We present the first case of in-stent restenosis (ISR) successfully treated with an everolimus-eluting BVS (ABSORB; Abbott Vascular, Santa Clara, CA) and discuss its potential advantages in such lesion.

Case ReportA 72-year-old male presented with a 3-week history of

relapsing Canadian Cardiovascular Society class 3/4 angina. He had undergone PCI of the proximal left anterior descending (LAD) 4 years prior using a 2.75 x 18 mm sirolimus-eluting stent (Cypher Cordis, Miami, FL). His medical regimen included aspirin and a statin. Resting ECG and cardiac biomarkers were normal. Given the severity of his symptoms and his past medical history, it was decided to proceed directly to coronary angiography.

The coronary angiogram showed a severe 95% ISR lesion of the proximal LAD with thrombus and multiple areas of contrast staining outside the stent contour, compatible with coronary micro-aneurysms (Figure 1A). Thromboaspiration revealed white

thrombus. Optical coherence tomography (OCT) was compatible with a localized hypersensitivity reaction to first-generation DES (Figure 1B). After consensus with the referring physician, an ABSORB everolimus-eluting BVS 3.0 x 18 mm was deployed (Figure 2A). Repeated OCT showed good scaffold apposition and coverage of the micro-aneurysms (Figure 2B). The patient was discharged the following day on dual antiplatelet therapy with aspirin and ticagrelor. The patient was asymptomatic at 6 months clinical follow-up.

DiscussionTo the best of our knowledge, this is the first report

describing the use of a BVS in a case of ISR and late acquired malapposition secondary to first-generation DES. The ABSORB BVS consists of resorbable polymers containing the antiproliferative drug everolimus. Prospective studies of BVS showed favourable outcomes in simple, de novo coronary lesions1,2, with long-term positive vessel remodelling, late lumen enlargement, complete resorption of the vascular scaffold, and progressive filling of the previously occupied space with proteoglycans at follow-up3. However, these studies specifically excluded ISR lesions.

In the current report, a local delayed hypersensitivity vasculitis caused by a sirolimus-eluting stent was suspected to be the causal mechanism leading to late acquired stent malapposition, restenosis, with superimposed very late thrombosis4. Typical angiographic findings present in our patient, including positive remodelling and micro-aneurysms of the mid-segment of the stent. This hypersensitivity reaction has been attributed to the polymer coating of sirolimus-eluting stents, which, importantly, differs from the one used in the ABSORB BVS4.

In this particular case, the use of BVS had several advantages: avoidance of accumulation of multiple layers of metallic stent, avoidance of re-exposure to the same “allergic antigen” and most importantly, hypothetically, the healing process of BVS characterized by late lumen enlargement and replacement of the platform by proteoglycans, resulting in filling of the micro-aneurysms and exclusion of the nidus involved in the genesis of neo-thrombi. Angiographic follow-up with OCT imaging at 2 years or beyond will potentially validate the latest hypothesis.

ConclusionBVS are a promising addition to the PCI arsenal in managing

complex coronary lesions. This case report demonstrates novel use of this technology to treat an ISR lesion of DES characterized by late acquired malapposition.

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Case Report

Cadrin-Tourigny et al.BVS in the treatment of DES restenosis


Figure 1 – Baseline Angiogram and Optical Coherence Tomography(A) Baseline angiogram showing a severe restenotic lesion of the proximal left anterior descending artery with 2 micro-aneurysms (asterisk), with contrast staining outside the previously implanted drug-eluting stent suggesting late-acquired malapposition secondary to hypersensitivity vasculitis. (B) Optical coherence tomography performed after initial thrombectomy and balloon dilatation angioplasty showing typical late acquired malapposition features with “sunflower” appearance (top right panel) and 2 small pockets representing micro-aneurysms (asterisk; lower panel).

A

B

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Case Report



Figure 2 – Post PCI Angiogram and Optical Coherence Tomography(A) Final coronary angiography showing exclusion of the 2 micro-aneurysms present at baseline. (B) Final optical coherence tomography showing good BVS apposition with covering of the 2 micro-aneurysms and thrombus entrapment.

A

B

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Case Report



1. Ormiston JA, Serruys PW, Regar E, Dudek D, Thuesen L, Webster MW, et al. A bioabsorbable everolimus-eluting coronary stent system for patients with single de-novo coronary artery lesions (ABSORB): a prospective open-label trial. Lancet. 2008;371(9616):899-907.

2. Serruys PW, Onuma Y, Ormiston JA, de Bruyne B, Regar E, Dudek D, et al. Evaluation of the second generation of a bioresorbable everolimus drug-eluting vascular scaffold for treatment of de novo coronary artery stenosis: six-month clinical and imaging outcomes. Circulation. 2010;122(22):2301-12.

3. Serruys PW, Ormiston JA, Onuma Y, Regar E, Gonzalo N, Garcia-Garcia HM, et al. A bioabsorbable everolimus-eluting coronary stent system (ABSORB): 2-year outcomes and results from multiple imaging methods. Lancet. 2009;373(9667):897-910.

4. Virmani R, Guagliumi G, Farb A, Musumeci G, Grieco N, Motta T, et al. Localized hypersensitivity and late coronary thrombosis secondary to a sirolimus-eluting stent: should we be cautious? Circulation. 2004;109(6):701-5.

References

Author contributionsCadrin-Tourigny J and Dong L, conception and design of the

research, acquision of data, analysis and interpretation of the data, statistical analysis, writing of the manuscript and critical revision of the manuscript for intellectual content; Maehara A, conception and design of the research, acquision of data, analysis and interpretation of the data, obtaining financing, writing of the manuscript, critical revision of the manuscript for intellectual content; Schampaert E and Genereux P, conception and design of the research, acquision of data, analysis and interpretation of the data, writing of the manuscript, critical revision of the manuscript for intellectual content.


The author Erick Schampaert has conflict with Abbott Vascular Consultant and Philippe Genereux has conflict with Abbott Vascular Speaker Fee.

Sources of Funding


Study Association

This study is not associated with any thesis or dissertation work.

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Viewpoint

Valvular Heart Team Max GrinbergInCor – HCFMUSP, São Paulo, SP - Brazil

“An idealist is a person who helps other people to be prosperous.”

Henry Ford (1863 − 1947)

The morality of medical practice is based on beneficence. The benefit is applicable to an organ, however, it is not enough. The safety of the procedure to the patient’s life is a concern1.

The sense of the usefulness of a therapeutic method is well validated by effect extent class I/IIa in a guideline, but he/she can be harmed when the unexpected wins predictability. The high complexity collects unquestionable medical indications that exhibit poor individual prognosis due to severe comorbidities2.

In this comparison between illness and patient, the contraindication illustrates that the major limitations of physician are the limits of medical science. Fortunately, we live in a moment of endless technological innovations. Readily globalized, they expand the skill bounderies and, at the same time, they give vitality to the classic, for the ongoing development is imperative in medical science.

A new horizon requires interdisciplinarity, which, in turn, demands interpersonal communication. In the interests of the best clinical practice for the patient, the propaedeutics communicates with clinical and scientific evidence that is understood as the clinical reasoning under the tension of the imminence of conduct, listening to the values and preferences of the patient. The efficient communication — to talk, to hear oneself (am I being objective?), to listen and to hear oneself hearing (am I connected?) — energizes the trespassing through the essential ethical tolls: benefit of method, patient safety and human character of medical science3.

In recent millennium transition, the routine of “discussion of the case” included the gap between the availability of medical science and medical care to the elderly with aortic valve stenosis ineligible for conventional surgical treatment. However, already in the early years of the 21st century, the discomfort of “if we can’t do-good, at least let us not do harm” was reduced by the prospects of support to this subgroup of patients by means of transcateter implant of a bioprosthesis.

Due to the initial results, the sentence has changed, following the change of the paradigm, to “we can do-good, but we have to take care of its potential harm”. The systematized research4 proved the positive impact on the “hard” outcome pro-life and raised the likelihood of the certainty of benefit to B level. However, the best survival curve does not invalidate the uncertainties of complications related to innovation, a base of the modern concept of iatrogenesis5.

Bedside conflicts arise between using an alternative method to conventional surgery to correct aortic valve hemodynamics and, at the same time, envision strong objections to satisfy the purpose both of survival and quality of life. They direct the physician to establish the applicable proportions of science and humanism, strongly advised by soliloquy, with the ontological component of ethics.

The “I do-good to the patient because it crops out from being who I am and not just because I read the code of ethics” makes prudence and zeal flow in indicating/non-indicating/contraindicating depending on the symbolism of the Hippocratic oath. The resulting ordering of adjustments to the benefit ensures value for deliberation.

It is worth remembering that, about 20 years ago, this moral plumbing occurred in establishing balloon catheter of mitral valvuloplasty and encouraged a close and progressive knowledge feedback and skill on the patient with mitral stenosis. Physician, surgeon, interventional cardiologist and echocardiographist got together and converted the field of the then innovation into an efficient relationship benefit/security that made it a rare AI recommendation in guideline of valvular heart disease. Currently, the transcateter implant of bioprosthetic in aortic position causes similar mobilization6. The search for answers to the questions brought about by the new therapeutic heritage recommends to appreciate it further than just a procedure. It is preferable to see it as a program comparable to a transplant7.

The commitment of a collective of experts as far as the learning curve of this change of therapeutic standard in patients with valvular heart disease is concerned neither plastic nor native tissue replacement — is better structured in the formation of an interdisciplinary team to valvular heart disease. The team set-up replaces the one of disciplinary workgroup enough for sustainable routines by the coldness of reports and the monologue opinions juxtaposed.

The interdisciplinary team for valvular heart disease links space and time. These dimensions make the refinement of movements and countermovements easier and to support excellence. Indication/non indication/contraindication may, therefore, be appropriately customized to the symptomatic elderly with aortic valve stenosis, respecting the enormous heterogeneity of the real world twinned by right to dignity.

Mailing Address: Max Grinberg •Instituto do Coração HC FMUSP, Rua Manoel Antonio Pinto, 4 ap. 21A, Paraisópolis. Postal Code 05663-020, São Paulo, SP - Brazil E-mail: [email protected]; [email protected] received December 16, 2013; revised manuscript March 14, 2014; accepted March 14, 2014.

KeywordsAortic Valve Stenosis; Cardiac Catheterization; Heart Valve

Prosthesis Implantation / methods; Bioprosthesis; Bioethics.

DOI: 10.5935/abc.20140099

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Viewpoint

Max GrinbergValvular Team


Transparency in interdisciplinary coordination of complementary expertises in patients with valvular heart disease is the raw material for the construction of a platform of clarity of tasks to be fulfilled, limits to be respected and performance levels to be achieved. Interdisciplinary meanings for the professional proficiency make the team an opinion maker.

The interdisciplinary team for valvular heart disease confers nationality. The hierarchy between methods with flexibility in its borders, the tone for subjectivities (body weakness) and inaccurate objectivities (surgical risk scores), critical analysis on personal and literature results and the modulation to the sociocultural and economic develop the necessary fine tuning with Brazilian realities.

The interdisciplinary team for valvular heart disease is greater than the sum of their participants, which does not happen with the working group. The expansion lies in the attitude of each member — sometimes as a disclosure, other as a receiver. I teach, you learn, he improves, we progress, is the conjugation intended from a system connected to attain the highest level of mutual understanding on cost-risk-effectiveness about the trinomial aortic valve stenosis –non valvular cardiac abnormalities−extra cardiac comorbidities.

The interdisciplinary team to valvular heart disease thrusts the clippings out of interest the vertical provisions verticalized by the hyperspecialization turns to a horizontal solidarity position between one another, useful for the needs, preferences ad values of the Brazilian elderly

The in terd i sc ip l inary team for va lvu lar hear t disease highlights the value of current cardiology as it extracts information from the three imaging giants — ultrasonography, CT scan and MRI — and introduces it into the hammer of decision making present in the calloused hands of sovereign clinic, powerful surgical clinic and the skillful interventionist cardiology.

In short, the interdisciplinary team for valvular heart disease builds a strong interdisciplinarity. When exchanging not only methods but also concepts, the transdisciplinarity8 becomes closer by using rigor together with fundamental concepts, the opening to the unknown and the tolerance toward the gaps of medical science based on evidence about practices that cannot be disproved by personal experience. The hybrid room is the emblem.

It is known that languages are neither static nor closed. Loan words occur as the result of dominance over a particular segment of society. In this context, the niche of present medical science is influenced by the supremacy of English language literature. TAVI is an anglicism that was quietly incorporated. Just as we should not insist on a Brazilian acronym by repositioning letters — ITVA or IVAT —, It seems reasonable to

us to adopt the globalized (and synthetic) name Heart Team to express an interdisciplinary team specialized in cardiology.

The concept of Heart Team was revived less than a decade ago as a methodological imperative deriving from the SYNTAX study9. The name gained notoriety for its contribution to the discipline communication and has migrated from research to assistance fields. The Heart Team acquired high organizational value in valvular heart disease, being understood that its absence is an absolute contraindication to the bioprosthetic transcateter implant in aortic position10.

I propose that the Heart Team expression that (in)vests the shirt on in reliable relationship in a interdisciplinary network and acquires scientific capital facilitator of complex deliberations before the symptomatic elderly suffering from aortic valve stenosis to be termed as Heart Valve Team (VHT).

VHT specificity includes: a) bioprosthesis management techniques and improvement ; b) contributions from imaging methods; c) safety by reducing adversity; d) early and late results, including participation in records; e) propensity to the use transcateter implant under minor surgical risk.

It is appropriate to emphasize that the VHT should not be viewed with an expiration date to be set by taking innovation for granted. The VHT means aggregation in favour for excellence in the attention given to grey areas experienced by patients with valvular heart disease with dubious issues according to valvular and/or non valvular cardiac and /or extra-cardiac complexities.

Final ly, the VHT does not reinvent the wheel. VHT rediscovers the union and the gathering of people that give vitality to the analysis of uncertainties, the overcoming of adversity and the extent of the benefit.

Author contributionsConception and design of the research, Acquisition of

data, Writing of the manuscript and Critical revision of the manuscript for intellectual content: Grinberg M.


reported.



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Max GrinbergValvular Team


1. Singer SJ, Vogus TJ. Safety climate research: taking stock and looking forward. BMJ Qual Saf. 2013;22(1):1-4.

2. Atwater BD, Daí D, Allen-Lapointe NM, Al-Khatib SM, Zimmer LO, Sanders GD, et al. Is heart failure guideline adherence being underestimated? The impact of therapeutic contraindications. Am Heart J. 2012;164(5):750-5.

3. Grinberg M, Tarasoutchi F, Sampaio RO. Roteiro para resolução de valvopatia (Resolva). Arq Bras Cardiol. 2011;97(4):e86-90.

4. Leon MB, Smith CR, Mack M, Miller DC, Moses JW, Svensson LG, et al; PARTNER Trial Investigators. Transcatheter aortic-valve implantation for aortic stenosis in patients who cannot undergo surgery. N Engl J Med. 2010;363(17):1597-607.

5. Ligi I, Millet V, Sartor C, Jouve E, Tardieu S, Sambuc R, Simeoni U. Iatrogenic events in neonates: beneficial effects of prevention strategies and continuous monitoring. Pediatrics. 2010;126(6):e1461-8.

6. Webb JG, Pasupati S, Humphries K, Thompson C, Altwegg L, Moss R, et al. Percutaneous transarterial aortic valve replacement in selected high-risk patients with aortic stenosis. Circulation. 2007;116(7):755-63.

7. Mack MJ, Holmes Jr DR. Rational dispersion for the introduction of transcatheter valve therapy. JAMA. 2011;306(19):2149-50.

8. Sommermann A. Inter ou transdisciplinaridade? Da fragmentação disciplinar ao novo diálogo entre os saberes. São Paulo: Paulus; 2006.

9. Nallamothu BK, Cohen DJ. No ‘’I’’ in heart team: incentivizing multidisciplinary care in cardiovascular medicine. Circ Cardiovasc Qual Outcomes. 2012;5(3):410-3.

10. Vahanian A, Alfieri O, Andreotti F, Antunes MJ, Barón-Esquivias G, Baumgartner H, et al; Joint Task Force on the Management of Valvular Heart Disease of the European Society of Cardiology (ESC); European Association for Cardio-Thoracic Surgery (EACTS). Guidelines on the management of valvular heart disease (version 2012). Eur Heart J. 2012;33(19):2451-96.

References

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Insights of Optical Coherence Tomography in Renal Artery Fibromuscular Dysplasia in a Patient with Spontaneous Coronary Artery DissectionTeresa Bastante and Fernando AlfonsoHospital Universitario La Princesa, Madrid - Spain

Mailing Address: Teresa Bastante •Universitario de La Princesa, Teresa Bastante – C. Diego de Leon, 62. Postal Code 28006, Madrid - EspanhaE-mail: [email protected] received March 18, 2014; revised April 4, 2014; accepted April 4, 2014.

KeywordsOptical Ccoherence Tomography.

DOI: 10.5935/abc.20140100

A 60-year-old woman was admitted for an acute coronary syndrome. Coronary angiography showed a spontaneous coronary artery dissection (SCAD) in the left anterior descending coronary artery (A). A typical image of fibromuscular dysplasia (FMD) was also observed in the right renal artery (B). Optical coherence tomography (OCT) (C) revealed alternating areas of thickening and thinning of the medial layer, corresponding to the typical image of "string of beads" readily identified in the longitudinal reconstruction of the OCT and also in angiography.

A very high prevalence of FMD in non-coronary arteries has been recently reported in patients with DCE. Our findings suggest that OCT may provide unique diagnostic clues in these challenging patients.

Author contributionsConception and design of the research, Acquisition of data,

Analysis and interpretation of the data and Critical revision of the manuscript for intellectual content: Bastante T, Alfonso F; Writing of the manuscript: Bastante T.



Sources of Funding


Study Association

This study is not associated with any thesis or dissertation work.

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Image

Bastante & AlfonsoOCT in renal artery fibromusular dysplasia


Figure 1 – Panel A: Coronary angiography showing a linear filling defect in left anterior descending coronary artery, corresponding with the SCAD (arrows). Panel B: Renal artery angiography disclosing the typical image of FMD (arrows). Panel C: OCT of renal the artery depicting the characteristic areas of thickening (asterisks) and thinning (arrows) of the middle layer.

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