The in vitro effect on arterial wall function of serum from patients with pregnancy-induced hypertension

Tom Tulenko, Ph.D., Jan Schneider, M.D., Claro Floro, M.D., and Marita Sicilia

Philadelphia, Pennsylvania

The object of this study was to develop an in vitro model to identify the serum factor(s) responsible for the vascular changes that occur in pregnancy-induced hypertension (preeclampsia). Segments of rabbit carotid artery were perfused for 3 hours with nutrient medium to which serum from normal pregnant women or patients with preeclampsia was added. After perfusion, ring segments of the vessels were cut and mounted in muscle chambers for force measurement. Sensitivity of the vessels to constrictor agents and endothelium-mediated relaxation was studied. The results indicate that arteries exposed to serum from preeclamptic patients developed a 2.9-fold increase in sensitivity to angiotensin II, 1.6-fold increased sensitivity to norepinephrine, and no change of sensitivity to serotonin. These effects were not accompanied by changes in isometric force. Endothelium-mediated relaxation was not affected by serum from preeclamptic patients. These findings confirm clinical observations of increased angiotensin II sensitivity in preeclampsia. The system used may facilitate identification of factors that cause the difference between the sera of normal pregnant women and those with preeclampsia. (AM J OssTET GYNECOL 1987;156:817-23.)

Key words: Preeclampsia, pregnancy-induced hypertension, arterial function, arterial smooth muscle cells, endothelial cells, norepinephrine, angiotensin II, serotonin

The clinical manifestations and pathophysiologic ef­fects of pregnancy-induced hypertension (preeclamp­sia) are well known. However, studies of the causes of this disease have been limited by a series of obstacles. The most critical of these is the uniquely human nature of preeclampsia.' No similar condition exists in any other species. Although a variety of chemical and an­atomic attempts have been made to produce an animal model, none has been entirely satisfactory.2

• 3 Studies of

the human have yielded a wealth of data but have failed to reveal the causes of the disease. The altered pressor response in normal pregnancy and in preeclampsia has been extensively studied; however, no humoral differ­ence between the blood of normal women and that of preeclamptic women has been consistently demon­strated.4 Against this background, the present study was mounted to combine techniques of basic vascular phys­iology with those of clinical research. It was designed to assess and compare the effects of sera from normal pregnant women and women with preeclampsia on ar­terial wall vasoconstrictor potential in an in vitro ex-

From the Departments of Obstetrics and Gynecology and Physiology and Biochemistry, Medical College of Pennsylvania.

Supported in part by National Institutes of Health Grants HL-30496 and AG-04908 and by the W. W. Smith Trust.

Presented by invitation at the Fifth Annual Meeting of the American Gynecological and Obstetrical Society, Hot Springs, Virginia, Sep­tember 4-6, 1986.

Reprint requests:Jan Schneider, M.D., Department of Obstetrics and Gynecology, The Medical College of Pennsylvania, 3300 Henry Ave., Philadelphia, PA 19129.

periment with animal blood vessels. If successful this system could provide a major step in identifying dif­ferences between the blood of patients with preeclamp­sia and that of the normal pregnant woman.

Material and methods

The basic method used m this study involved the perfusion of rabbit carotid arteries in vitro with a nutrient perfusion medium to which serum from ei­ther preeclamptic women or normal pregnant control women was added. The perfusate in each experiment contained 70% nutrient medium and 30% serum. After perfusion, a series of physiologic and pharmacologic assessments was made to characterize vessel function. The differences between vessels perfused with sera from normal pregnant women and those of preeclamp­tic patients were compared.

Thirty milliliters of blood was drawn from 50 patients; in 26 preeclampsia was diagnosed by the traditional criteria of hypertension (blood pressure > 140/90 mm Hg) and proteinuria (3 + on dipstick or 300 mg/24 hours). The other 24 patients served as nor­mal pregnant control subjects. The patients were sim­ilar in age, gravidity and parity, and gestational age. The blood samples were drawn before any medications had been given. The blood was allowed to coagulate and the specimens were then centrifuged at 1000 x g for 20 minutes. The resulting serum samples were fro­zen at - 70° C until the perfusion studies could be started.

817

818 Tulenko et al.

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Water 95% air jackets 5% coa

Perfusate Bath Silastic reservoir reservoir lung

1-,Aolle• 5· -pump

PE tubing

Fig. 1. Diagram of the apparatus used to perfuse rabbit carotid arteries in vitro with a solution of nutrient medium and human serum.

Perfusion of vessels. Each experiment began with the perfusion of blood vessels. The separate perfusates of test and control sera were simultaneously circulated under identical conditions through 2 cm segments of the right or left carotid artery of the same rabbit. Each

perfusion was maintained for 3 hours. For each ex­periment a New Zealand White rabbit (2.5 to 3.0 kg) was anesthetized with intravenous pentobarbital so­dium (23 mg/kg) and the carotid arteries were removed by standard sterile procedures. In the dissection care was taken to avoid injury to the smooth muscle and endothelial cell layers of the arterial wall. The vessel segments were then mounted on cannulas stretched to their in vivo lengths and perfused in an apparatus that was thermoregulated at 37° C.

The perfusion apparatus (Fig. 1) essentially consisted of a sterilized miniature heart-lung machine. This sys­tem was originally devised in the laboratories of the Department of Physiology and Biochemistry of the Medical College of Pennsylvania to permit long-term metabolic and functional studies of the various cell lay­ers of blood vessels in vitro.5 In previous experiments blood vessels were maintained in a fully functional state for up to 36 hours. In each experiment pulsatile flow at 4 ml/min was provided by a Gilson MiniPulse roller pump that circulated both test and control media from their respective reservoirs through silicone rubber coils that served as an artificial lung, through a heat ex­changer, and on through the experimental vessels. The effluent perfusate returned to the reservoirs for recir­culation. A threaded displacement micrometer clamp located just distal to the vessel was adjusted to provide the appropriate resistance to maintain perfusion pres-

April 1987 Am J Obstet Gynecol

sure at 70 mm Hg systolic and 50 mm Hg diastolic. Pulsatile and mean pressure was monitored by an in­line arterial transducer just proximal to the vessel. The silicone rubber lung consisted of gas porous silicone rubber tubing coiled inside an Ehrlenmeyer flask, which was ventilated with a gas mixture of 95% room air and 5% carbon dioxide. The appropriate balance of gas flow, perfusate flow, and silicone rubber coil length provided for gas equilibration of the medium resulting in a Po2 of 100 torr, a Pco2 of 35 torr, and a pH of 7.35. Preliminary studies demonstrated that these values held constant for perfusion periods of up to 36 hours. This apparatus permitted two arteries to be perfused simultaneously with their respective media under identical hemodynamic conditions. The adven­titial surface of the vessels was maintained by bathing them in a crystalloid medium, which was also aerated and recirculated through the water-jacketed chamber bath. The perfusion medium to which the human se­rum samples were added contained sodium chloride, 119 mmol/L; potassium chloride, 4.6 mmol/L; calcium chloride, 1.6 mmol/L, potassium phosphate, mono­basic, 1.2 mmol/L, magnesium sulfate, 1.2 mmol/L; sodium bicarbonate, 22.6 mmol/L; dextrose, 5.0 mmol/L; calcium-sodium-ethylenediaminetetraacetic acid, 0.03 mmol/L; and was sterilized by filtration through a 0.45 µm filter (Millipore) before use. In each experiment, one carotid artery was perfused with the control medium while the other carotid from the same rabbit was simultaneously perfused with the test me­dium within the same chamber.

Arterial smooth muscle contractility. After a 3-hour perfusion period, 1 to 2 mm segments of the carotid arteries were cut to form rings. The arterial smooth muscle in these rings was tested for isometric force development. Each ring was suspended between stain­less steel wires in a 10 ml muscle chamber and stretched to its predetermined optimal resting length, which cor­related with a transmural pressure of 60 mm Hg. One wire was attached to a stationary base at the bottom of the chamber and the other to an isometric force dis­placement transducer (Grass FT .03). The mechanical activity was recorded on a six-channel Grass polygraph (Model 7D). After a 90-minute equilibration period in physiologic perfusion medium at 37° C with continual aeration, dose-response analyses were performed. The effects of cumulative additions of 10 µI of vasocon­strictor substances were studied. Experiments were performed with the use of angiotensin II, norepineph­rine, and serotonin. No vessel ring was exposed to more than two different vasoconstrictor agents in any ex­periment. The contractile effect induced by each of the pressor substances in increasing concentrations was thus measured.

The isometric force developed by the arterial smooth

Volume 156 Number 4

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Preeclampsia and arterial wall function 819

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Fig. 2. Dose-response relationship of angiotensin II and carotid arteries after their perfusion with either control or preeclamptic serum solution. Bars to the right represent the maximum force de­veloped to angiotensin II. See Table II for kinetic data. * = p < 0.05.

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Fig. 3. Dose-response relationship of norepinephrine and carotid arteries after their perfusion with either control or preeclamptic serum solution. Bars to the right represent the maximum force de­veloped to norepinephrine. See Table II for kinetic data. * = p < 0.05.

muscle to each agent was calculated and adjusted on the basis of cross-sectional area in order to correct for differences in ring size. The cross-sectional area was calculated from the wet weight and measured circum­ference of each ring. All force data were expressed as grams of tension per square centimeter of arterial wall. The agonist concentration eliciting half-maximal re­sponse (ED,0) for each experiment was taken as an in­dex of sensitivity to the agonist. This concentration was calculated from a log-logit transformation of the dose­response relationship expressed as the percent maximal response. The differences between the groups was tested by comparing the dose-response curves between each group by use of a test of parallelism followed by a two-way analysis of variance. The ED50 values for each experiment were averaged arithmetically and the dif­ferences between groups were further tested with Stu­dent's t test for unpaired data.

Endothelium-mediated vasodilation. Endothelial cells have recently been shown to have profound influ­ences on arterial tone.6 Therefore the endothelial cell­mediated vasodilator potential of these vessels was eval­uated. For these experiments acetylcholine and the cal­cium ionophore A23 l 87, both of which are known to cause endothelial cell-mediated relaxation, were stud­ied.7 The endothelial cells lining the arterial lumen were thereby analyzed for their ability to promote en­dothelial cell-mediated relaxation of the vessel wall. The method described by Furchgott and Zawadski6 was used. For this procedure the rings were contracted to

a steady state with a half-maximal concentration of se­rotonin followed by cumulative additions of either ace­tylcholine or A23187 to the chamber bath. The degree of relaxation caused by these agents was taken as an indication of the relative physiologic integrity of the endothelial cells.

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April 1987 Am J Obstet Gynecol

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Fig. 4. Dose-response relationship of serotonin and carotid arteries after their perfusion with either control or preeclamptic serum solution. Bars to the right represent the maximum force developed to serotonin. See Table II for kinetic data.

Table I. Effects of normal (control) and preeclamptic serum on the sensitivity of perfused carotid arteries

Sensitivity Condition (ED,0 X mol/L) Difference

Angiotensin II Control 3.04 ± 0.98 x 10-s

l (n = 7) Preeclamptic 1.07 ± 0.38 x 10-• 2.9x* (n = 6) Non perfused 1.16 ± 0.57 x 10-s (n = 16)

Norepinephrine Control 5.70 ± 1.35 x 10-7

) (n = 13) Preeclamptic 3.48 ± 0.76 x 10-7 l.6x* (n = 14) Non perfused 6.26 ± 1.21 x 10-7

(n = 3) Serotonin

Control 1.46 ± 0.17 x 10-7

) (n = 8) Preeclamptic 1.52 ± 0.19 x 10-7 I.Ox (NS) (n = 8) Non perfused 2.44 ± 0.41 x 10- 7

(n = 24)

Numbers in parentheses refer to the number of experi­ments. Experiments are reported as mean ± SEM.

*p < 0.05, control versus preeclampsia.

In all experiments of both vasoconstrictor and va­sodilator effects, duplicate rings from each artery stud­ied were cut and data were averaged between two rings from the same vessel. Each such average was reported as a single datum (n = 1). Additionally in all experi­ments two rings from each artery were cut before the perfusion and cold stored at 4° C during the time of perfusion. These were also then studied with the other vessels as nonperfused controls. In all statistical tests significance was accepted at p < 0.05. All values are reported as the mean ± SEM.

Results

Contractility. The sensitivity of the arteries to an­giotensin II was found to be 2.9 times greater in vessels perfused with serum from preeclamptic patients than in those perfused with serum from normal pregnant control subjects (Fig. 2). Similarly the sensitivity to nor­epinephrine was also found to be increased in blood vessels perfused with blood from preeclamptic patients when compared with those from normal control sub­jects. The 1.6-fold increase in sensitivity was less than that of angiotensin II (Fig. 3). The sensitivity of blood vessels to serotonin, however, was not altered and there was no significant difference between vessels perfused with serum of preeclamptic patients and normal control subjects (Fig. 4).

The maximum force-generating capacity of the ves­sels was unaffected by perfusion with either test or control sera. This would suggest that the increased con­tractile sensitivity evoked by serum from preeclamptic patients has its origin in the plasma membrane of the smooth muscle cell of the arterial wall. These findings are summarized in Table I.

Endothelial cell-mediated vasodilatation. The va­sodilator activity of the endothelial cells of the vessel wall was unaffected by their exposure to serum from preeclamptic patients when compared with control se­rum (Figs. 5 and 6). This was true in terms of both their sensitivity and their maximal relaxation capacity when exposed to either acetylcholine or A23187. The greater relaxation activity observed in the nonperfused vessels suggests that sera from either test or control patients equally impairs endothelial cell-mediated re­laxation.

Comment

Preeclampsia is considered to be mediated, at least in part, by a generalized increase in peripheral arterial tone. The present study tested the hypothesis that se-

Volume 156 Number 4

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Preeclampsia and arterial wall function 821

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Fig. 5. Endothelium-mediated relaxation stimulated by acetylcholine on carotid arteries after their perfusion with either control or preeclamptic serum solution.

rum from women with preeclampsia differs from that of normal pregnancy and contains a factor or factors that interact with the arterial wall to alter its vasocon­strictor function. The results of the study support this concept. It was demonstrated that perfusion of rabbit carotid arteries with serum from patients with pre­eclampsia increased their sensitivity to angiotensin II and to a lesser extent to norepinephrine but not to serotonin. These findings are directly consistent with human in vivo studies reported by Abdul-Karim and Assali" and by Gant et al.9

· 10 The alteration in sensitivity

to angiotensin II and norepinephrine as compared to serotonin suggests a selective effect in the arterial wall. The similarity of maximum force-generating capacity of the vessels to each agonist of both groups of arteries suggests that the cellular vasoconstrictor pathways sup­porting the contractile protein machinery in smooth muscle are not altered by preeclampsia. This implies that a cellular mechanism proximal to the intracellular vasoconstrictor pathways is altered. A likely site would be the smooth muscle cell membrane and its angio­tensin II and (or) norepinephrine receptor excitation­coupling mechanism.

The possible role of an endothelial lesion of the cells lining the arterial lumen in the etiology of preeclampsia needs to be considered. Furchgott and Zawadski6 were the first to demonstrate that the endothelium is capable of altering arterial smooth muscle tone by releasing an endothelial cell-derived relaxation factor. The release of endothelial cell-derived relaxation factor appears to be tonic, can be altered by various chemical and me­chanical interventions, and can alter sensitivity to cer­tain drugs in some vascular beds. 11

· 12 Endothelial dam­

age could therefore theoretically explain the elevated arterial pressure in preeclampsia as well as the in­creased capillary permeability, edema, proteinuria, vol-

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Fig. 6. Endothelium-mtediated relaxation stimulated by the calcium ionophore A23187 on carotid arteries after their per­fusion with either control or preeclamptic serum solution.

ume constriction, disseminated intravascular coagula­tion, and even the seizures of eclampsia. The results of the present study, however, do not support this theory. The findings noted above suggest that serum from pre­eclamptic patients has the same effect on endothelial function as does serum from normal pregnant women. It is evident, however, that more experiments must be conducted to verify this preliminary conclusion.

The data reported here were derived from an in vitro system that confirmed changes in arterial wall function

associated with preeclampsia. The selective enhancing effect of serum from preeclamptic patients on arterial angiotensin II receptor activity lends support to the proposal that in preeclampsia there is either an in­creased sensitivity of the vasculature to this substance

822 Tulenko et al.

or a failure of the vessels to become refractory to it. We believe that the in vitro system described will permit the isolation and characterization of the responsible serum factors. It may clarify the cause of these changes and whether they are due to a sensitizing substance in the serum of patients with preeclampsia or to the lack of a protective substance in women suffering from this disease.

REFERENCES

1. MacGillivray I. Preeclampsia. London: WB Saunders, 1983.

2. Hodgkinson CP, Hodari A, Bumpus FM. Experimental hypertensive disease in pregnancy. Obstet Gynecol 1967; 30:371.

3. Cavanagh D, Rao PS, Tung KK, Gaston L. Eclamptogenic toxemia: the development of an experimental model in the subhuman primate. AM J OBSTET GYNECOL 1974; 120: 183.

4. Lindheimer MD, Katz Al. Pathophysiology of preeclamp­sia. Ann Rev Med 1981;32:273.

5. Bialecki R, Tulenko T. Increased endothelial relaxation and smooth muscle contraction following cholesterol in­corporation into arterial wall membranes. Physiologist 1986;29:152.

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

7. Furchgott RF. Role of endothelium in responses of var­ious smooth muscle. Circ Res 1983;53:557-73.

8. Abdul-Karim R, Assali NS. Pressor response to angiotonin in pregnant and nonpregnant women. AM J 0BSTET Gv­NECOL 1961;82:246-51.

9. Gant NF, Daley GL, Chand S, Whalley PJ, MacDonald PC. A study of angiotensin II pressor response through­out primigravid pregnancy. J Clin Invest 1973;52: 2681-9.

l 0. Gant NF, Chand S, Whalley PJ, MacDonald PC. The na­ture of pressor responsiveness to angiotensin II in human pregnancy. Obstet Gynecol 1974;43:865.

11. Rubanyi GM, Lorenz RR, Vanhoutte PM. Bioassay of en­dothelium-derived relaxing factor(s): inactivation by cat­echolamines. Am J Physiol l985;249:H95-Hl0l.

12. Cocks TM, AngusJA. Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin. Na­ture l 983;305:627-30.

Editors' note: This manuscript was revised after these discussions were presented.

Discussion DR. NORMAN GANT, Dallas, Texas. The authors have

developed an in vitro model for assessing vascular re­sponsiveness to a variety of different pressor agents. This ingenious miniature heart-lung machine allows for the in vitro perpetuation of arterial strips for up to 36 hours in duration. What the authors set out to do, using this very clever system, was to develop a model for identifying possible serum factors that might be responsible for the vascular changes that occur in pa­tients with pregnancy-induced hypertension. They ob­served in this in vitro model that segments of rabbit carotid artery, when perfused with serum from patients with pregnancy-induced hypertension compared with serum of normotensive patients, exhibited an increased

April 1987 Am J Obstet Gynecol

sensitivity to angiotensin II and to norepinephrine. The increased sensitivity to angiotensin II was 2.9-fold over control and 1.6-fold over control with norepinephrine. There appeared to be no difference in sensitivity to serotonin. The authors concluded from their studies that the data they obtained were consistent with a view that there were changes in the arterial wall function associated with preeclampsia. This conclusion was based on the fact that the maximum force of generating capacity of the vessels to each of the agonists tested in the system was equal, and thus it appeared to the au­thors that the cellular vasoconstrictive pathways, which support contractile protein machinery in smooth mus­cle, are not altered by preeclampsia. Instead the defect most likely exists in the endothelium and (or) mem­brane of the contracting smooth muscle.

I congratulate the authors on this study. There is no doubt that the technique developed for use in this study will prove to be effective in future studies of pregnancy­induced hypertension. The research was carefully and meticulously done. The results were accurately re­ported. The interpretation of the results was exactly to the point. Results were succinct and not overstated. It is a true pleasure to read such a beautifully conducted and reported study. My only question is one that I have asked the authors before. Specifically, does the serum from the patients with pregnancy-induced hyperten­sion have a pressor substance, or is it more likely that the serum from the patients with pregnancy-induced hypertension is deficient in a factor that might render these vessels refractory to pressor agents?

DR. JOHN I. FISHBURNE, Oklahoma City, Oklahoma. This is a very interesting study using carotid arteries. Because of the differences between carotids and uter­ine vessels, I wonder if the work has been done with segments of uterine arteries.

DR. ROBERT GooouN, Denver, Colorado. Several ar­ticles have recently shown that women with toxemia often have an elevated serum digoxin-like substance that may be sodium-potassium-adenosine triphospha­tase related and affects arteries as in this study.

I wonder if the authors have measured the digoxin levels in these patients.

DR. WILLIAMJ. LEMAIRE, Miami, Florida. I wonder if the authors have examined serum from patients who are not pregnant and have hypertension.

DR. Guv M. HARBERT, Charlottesville, Virginia. I noticed the standard deviations and the differences were quite significant.

Have the authors attempted to make a correlation of the difference in response with the severity of toxemia in the patients from whom they obtained the serum? In other words, is this quantitated with severity?

DR. SCHNEIDER (Closing). Dr. Gant asked a most im­portant question that has puzzled many of us. Is pre­eclampsia due to a substance, the so-called "toxin," that causes preeclamptic patients to overreact to pressor substances or is it caused by the absence of a protective mechanism that makes normal pregnant women re­fractory to the effect of pressor substances such as an-

Volume 156 Number 4

giotensin. Perhaps I can attempt to answer this by ad­dressing the question asked by Dr. LeMaire. It seems evident to me now that we have performed the second experiment first. Our first experiment should have been to compare the effects of blood of healthy non­pregnant women with those of blood of normal preg­nant women. If the blood from nonpregnant women caused vessels to have the same increased reaction to angiotensin, then clearly the pressor response in pre­eclampsia must be due to the absence of a protective substance. I believe this to be the case. We plan to draw blood from patients at term and again 6 weeks post partum to see if they react differently. It is my postulate that the non pregnant response will be similar to that in preeclampsia.

Dr. Fishburne asked about the uterine artery. For technical reasons and the need for arterial segments long enough to be perfused in the apparatus, we have not been able to use rabbit uterine arteries. However,

Preeclampsia and arterial wall function 823

in another study, which perhaps is not for me to report since I am not personally involved in it, members of my department, in conjunction with Dr. Tulenko in physiology, are studying uterine arteries obtained at hysterectomy. These can be suspended and studied in the muscle chamber. The early data suggest that the uterine artery responds very differently than other ar­teries. This indicates again that not only are there spe­cies differences but in the same species there are vari­ations in the response of vessels from different ana­tomic sites.

Dr. Goodlin, we did not measure digoxin levels. Finally, to address Dr. Harbert's question about the

relationship of vessel response to the severity of pre­eclampsia, I confess that I cannot answer this excellent question. We have the clinical data and I could and should trace the relationship. I would like to think that the worse the preeclampsia, the greater the vessel re­sponse to angiotensin II. We will look into that.