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Preeclampsia: Evidence for impaired shear stress–mediated nitric oxide release in uterine circulation
Preeclampsia: Evidence for impaired shear stress–mediated nitric oxide release in uterine circulation Karolina R. Kublickiene, MD, PhD,a Bo Lindblom, MD, PhD,b Kerstin Krüger, MD,a and Henry Nisell, MD, PhDa Huddinge and Uppsala, Sweden OBJECTIVE: We sought to compare flow-mediated dilatation and myogenic and norepinephrine-induced tone in myometrial resistance arteries from women with preeclampsia and healthy pregnant women and to evaluate the role that nitric oxide may play in these responses. STUDY DESIGN: Arteries (approximately 200µm, at 50 mm Hg) were dissected from myometrial biopsy specimens from women undergoing emergency cesarean delivery because of preeclampsia (n = 6) and from healthy control subjects undergoing planned cesarean delivery (n = 9). Responses to intraluminal flow, pressure, and a constrictor agonist (norepinephrine, 10-6 mol/L) were studied in the absence and presence of the nitric oxide synthase inhibitor Nωnitro-L-arginine (10–4 mol/L). Myogenic and norepinephrine-induced tone were calculated after the determination of artery diameter in the absence of extracellular calcium and in the presence of papaverine (10–4 mol/L). RESULTS: An increase in intraluminal flow led to dilatation of isolated myometrial arteries from healthy gravid women, whereas flow-mediated dilatation was absent in arteries from gravid patients with preeclampsia (increase in diameter at maximum flow rate of 204 µL/min, 28% ± 5% in healthy gravid patients vs –15% ± 6% in gravid women with preeclampsia; analysis of variance, P < .05). Addition of Nω-nitro-L-arginine had no significant effect on flow-mediated responses in arteries from women with preeclampsia, whereas flowmediated dilatation was abolished after addition of Nω-nitro-L-arginine in arteries from healthy gravid women (increase in diameter at a maximum flow rate of 204 µL/min, 28% ± 5% control vs –9% ± 5% Nω-nitro-L-arginine; analysis of variance, P < .05). Arteries from women with preeclampsia developed pressure-induced myogenic and norepinephrine-induced tone, similar to that obtained in arteries from healthy gravid women. In arteries from gravid women with preeclampsia, inhibition of nitric oxide synthase enhanced myogenic-induced tone (25% ± 4% control vs 35% ± 5% Nω-nitro-L-arginine; P < .05) and norepinephrine-induced tone (36% ± 4% control vs 46% ± 6% Nω-nitro-L-arginine; P < .05), as in arteries from healthy gravid women. CONCLUSIONS: Nitric oxide may participate in modulation of pressure- and norepinephrine-induced tone even in preeclampsia, but the shear stress–mediated release of nitric oxide is absent. Failure of shear stress–mediated dilation in myometrial arteries from gravid women with preeclampsia might contribute to the impaired uteroplacental blood flow in this disease. (Am J Obstet Gynecol 2000;183:160-6.)
Key words: Preeclampsia, arteries, tone, shear stress, nitric oxide
Endothelial dysfunction is considered an important pathogenic feature of preeclampsia, confirmed by alterations in a variety of biochemical markers1 and more recently by the demonstration of impaired vasodilatory response of resistance vessels to endothelium-dependent agonists such as acetylcholine and bradykinin in vitro.2-6 From the Department of Clinical Science, Section for Obstetrics and Gynaecology, Karolinska Institute, Huddinge University Hospital,a and the Department of Women’s and Children’s Health, Section for Obstetrics and Gynaecology, Uppsala University.b Received for publication March 3, 1999; revised December 22, 1999; accepted January 14, 2000. Reprint requests: Karolina-Rasa Kublickiene, MD, PhD, Department of Obstetrics and Gynaecology, Karolinska Institute, Huddinge University Hospital, 14186 Huddinge, Sweden. E-mail Karolina.Kublickiene@ kfcmail.hs.sll.se. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/105820 doi:10.1067/mob.2000.105820
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The belief that endothelial dysfunction would be linked to impaired nitric oxide activity in preeclampsia still remains controversial. Thus the role played by nitric oxide in endothelium-dependent agonist-mediated responses is far from settled.3-6 Moreover, contrasting data have been presented concerning the levels of stable nitric oxide metabolites in preeclampsia.7-10 Thus the relevance of these methods for assessing nitric oxide function in preeclampsia might be questioned. It is increasingly accepted that the predominant physiologic stimulus of endothelial nitric oxide synthesis is shear stress generated by blood flow.11 We have recently demonstrated that flow-induced shear stress is a physiologic modulator of vascular tone in isolated myometrial arteries from normal pregnant women and that nitric oxide mediates this response12 but vasodilatory prostanoids do not. Accordingly, studies in isolated myometrial
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arteries from women with preeclampsia by use of a physiologic stimulus for nitric oxide release might contribute to our understanding of the part that nitric oxide may play in preeclampsia and particularly in the uterine circulation. This is a particularly relevant circulation to investigate in view of the indication from Doppler waveform analysis of the uterine artery that resistance is increased in this circulation from an early stage in preeclamptic pregnancies.13 This study was therefore undertaken to compare shear stress–mediated vasodilatation in myometrial resistance arteries from women with preeclampsia and healthy gravid women. In addition, the role of nitric oxide in the modulation of pressure-induced myogenic and norepinephrine-induced tone has been determined in myometrial arteries from women with preeclampsia and compared with that in healthy gravid women. These studies have been carried out in isolated myometrial arteries mounted on a pressure arteriograph. Methods Subjects. This study was approved by the Ethical Committee of Huddinge University Hospital, and all women gave their informed consent to participate. Informed consent was obtained from 6 nulliparous women with preeclampsia, with a median age of 27 years (range, 2236), and a median gestational age of 36 weeks (range, 3139) who were undergoing cesarean delivery because of worsening of the preeclamptic state. Preeclampsia was defined as blood pressure ≥140/90 mm Hg and proteinuria exceeding >300 mg/24 h in the absence of urinary tract infection after 20 weeks’ gestation in women with previously normal blood pressure and without proteinuria. Any women with a history of chronic hypertension, kidney disease, or diabetes mellitus were excluded from the study. Women with preeclampsia who had received antihypertensive agents were also excluded. The control group included 9 healthy pregnant women (5 nulliparous) with a median age of 28 years (range, 18-34) and a median gestational age of 39 weeks (range, 37-41) undergoing elective cesarean delivery because of breech presentation (n = 5) or previous cesarean delivery (n = 4). These women were part of a control group that participated in a recent study demonstrating the effects of both nitric oxide and endothelin 1 on myometrial resistance artery tone under pressurized and perfused conditions.14 The biopsy specimen was obtained immediately after delivery from the upper edge of the transverse incision in the lower uterine segment. Experimental setup. Intramyometrial small arteries (internal diameter ~200 µm at 50 mm Hg intraluminal pressure and lengths of ~2.5 to 3 mm) were immediately dissected from the biopsy specimens, and the surrounding myometrium, connective tissue, and adventitia were carefully removed. The myometrial arteries were then
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mounted in a pressure arteriograph (Living Systems Instrumentation Inc, Burlington, Vt) as previously described.12, 14 The vessels were orientated in the in vivo direction of flow on a pair of opposing glass microcannulas previously matched for flow resistance. The organ bath was perfused (7 mL/min) with physiologic sodium chloride solution (sodium chloride, 119 mmol/L; potassium chloride, 4.7 mmol/L; calcium chloride, 2.5 mmol/L; magnesium sulfate, 1.17 mmol/L; sodium bicarbonate, 25 mmol/L; sodium phosphate, monobasic, 1.18 mmol/L; ethylenediaminetetraacetic acid, 0.026 mmol/L; and glucose 5.5 mmol/L; pH 7.4; 37°C, gassed with 5% carbon dioxide in oxygen). A servocontrolled pump maintained the required intraluminal pressure, and the internal diameter of the artery was recorded continuously with a video dimension analyser. “In-line” pressure transducers monitored the proximal and distal pressures on each side of the specimen, enabling calculation of the mean intraluminal pressure. Each artery was equilibrated for 45 minutes while being pressurized to 50 mm Hg of intraluminal pressure. Viability was confirmed by exposure to extraluminal norepinephrine (10–6 mol/L) in potassium-substituted physiologic sodium chloride solution (64 mmol/ L) and endothelial function by relaxation induced by acetylcholine (10–6 mol/L). Arteries failing to maintain pressure, to demonstrate complete occlusion of the lumen in response to norepinephrine, or to show relaxation in response to acetylcholine were excluded from the study. Altered acetylcholine-mediated dilatation of preeclamptic vessels was not an exclusion criterion because impaired dilatation to endothelium-dependent agonists has been reported in preeclampsia.5, 6 Effects of nitric oxide synthase inhibition on pressure-induced tone, norepinephrine-induced tone, and flow-mediated responses. After the equilibration period, the intraluminal pressure was increased to 80 mm Hg for 30 minutes, and then the inner diameter was recorded. This pressure was chosen because it approximates the in vivo pressure at the proximal part of these arteries. Norepinephrine (10–6 mol/L) was then added to the superfusate for 30 minutes, and inner diameter was recorded before flow was started. Intraluminal flow was initiated with a flow pump. Flow was increased at 5-minute intervals (from 0 to 204 µL/min), and the inner diameter was recorded at the end of each flow step. During flow establishment at different flow rates, the proximal and distal pressure gradients were controlled by changing the distal pressure to keep the intraluminal pressure constant, that is, 80 mm Hg. After achievement of the first flow-response curve at increasing flow rates, intraluminal flow was stopped and intraluminal pressure was kept at 80 mm Hg. Inhibition of nitric oxide synthase was then induced by addition of Nω-nitro-L-arginine (10–3 mol/L) for 30 minutes in the absence of flow and norepinephrine. After the inner diameter was recorded, the arteries were then constricted
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with norepinephrine (10–6 mol/L) again in the continued presence of Nω-nitro-L-arginine for 30 minutes and the flow protocol was repeated. Finally, while pressure was maintained at 80 mm Hg, the arteries were incubated in calcium-free solution with ethylene glycol-bis (β-aminoethyl ether)N,N,N´,N´-tetraacetic acid (EGTA) (1 mmol/L plus papaverine 0.1 mmol/L) for 30 minutes, and the increase in inner diameter was recorded. Calculation of wall shear stress and myogenic and norepinephrine-induced tone. Wall shear stress (dyne/cm2) was calculated as follows: WSS = 4 × ρ × Q × 109/π × r3 where WSS is wall shear stress, ρ is viscosity in poise (dyne s/cm2) at 37°C, Q is flow rate (µL/s), and r is artery radius (µm). Viscosity of the physiologic sodium chloride solution was 0.7 cP. The factor of 109 in the equation is to correct for the use of both microliters per second for flow and micrometers for arterial radius (1 µL is equivalent to 109 µm3). The difference in inner diameter of the arteries when pressurized at 80 mm Hg before and after equilibration in calcium-free physiologic sodium chloride solution (with EGTA) provides an estimate of myogenic tone. This was calculated as the percentage decrease of the internal diameter of arteries in calcium-free physiologic sodium chloride solution, from the following equation: Myogenic tone (%) = (id2 – id1)/id2 × 100 where id2 is internal diameter in calcium-free physiologic sodium chloride solution (with EGTA) and id1 is internal diameter when pressurized in physiologic sodium chloride solution at 80 mm Hg. Similarly, myogenic tone after the addition of norepinephrine is calculated as follows: Myogenic tone after norepinephrine addition (%) = (id2 – id3)/id2 × 100 where id3 is internal diameter after the addition of norepinephrine. For simplicity, hereafter this is referred to as norepinephrine-induced tone. Statistical analysis. Values in figures are given as mean ± SEM. The flow responses in the absence and presence of Nω-nitro-L-arginine at different flow steps between the groups were compared by multivariate analysis of variance for repeated measurements (StatSoft, Inc, Tulsa, Okla). Post hoc comparisons between groups at different flow steps were performed with the Scheffé test (adjusted for multiple comparisons). The effects of flow steps within 1 group were evaluated by 1-way analysis of variance for repeated measurements (eg, flow-mediated dilatation in physiologic sodium chloride solution in arteries from healthy pregnant women). The 2 groups were compared with regard to the effect of Nω-nitro-L-arginine on myogenic and norepinephrine-induced tone by 2–way analysis of variance. Significance was assumed if P < .05.
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Results The arteries from women with preeclampsia (n = 6) and control subjects (n = 9) had similar diameters when equilibrated in physiologic sodium chloride solution alone at 50 mm Hg (212 ± 24 vs 215 ± 20 µm, respectively). Flow-mediated responses. Myometrial arteries from women with preeclampsia failed to dilate in response to increasing intraluminal flow (Fig 1). In fact, these arteries in physiologic sodium chloride solution constricted significantly (analysis of variance, F = 4.7; P = .004) in response to increasing intraluminal flow (eg, percentage change in preconstricted inner diameter, –15% ± 6% at the maximum flow rate of 204 µL/min; Fig 1). After incubation with Nω-nitro-L-arginine, these arteries responded similarly (–12% ± 4% at the maximum flow rate of 204 µL/min; Fig 1). In contrast, the myometrial arteries from healthy pregnant women demonstrated a substantial relaxation in response to flow (analysis of variance, F = 32; P = .0008; Fig 1), which was most pronounced at a flow rate of 130 µL/min (percentage change in preconstricted inner diameter, 30% ± 5%) and was maintained at this level up to the maximum flow rate of 204 µL/min. In the presence of Nω-nitroL-arginine, these arteries responded significantly differently (multivariate analysis of variance, F = 33.4; P = .0001), because increasing intraluminal flow led to a small but significant (analysis of variance, F = 3.28; P = .02) flow-induced constriction (eg, percentage change in preconstricted inner diameter, –9% ± 5% at a flow rate of 204 µL/min). The relationship between wall shear stress (calculated in each artery with the internal diameter immediately before the change in intraluminal flow) and the percentage of dilatation in arteries from both patient groups, in the absence and presence of Nω-nitro-L-arginine, is presented in Fig 2. The range of shear stress values achieved in arteries from women with preeclampsia was considerably greater than that in arteries from healthy pregnant women (6 ± 1 to 89 ± 24 dyne/cm2 vs 7 ± 2 to 29 ± 7 dyne/cm2, respectively), implying that the arteries from women with preeclampsia were less sensitive to shear stress because no vasodilatation was observed. In the presence of Nω-nitro-L-arginine, shear stress values ranged from 14 ± 5 to 151 ± 58 dyne/cm2 in the preeclampsia group compared with 17 ± 6 to 221 ± 108 dyne/cm2 in the normotensive control group. Within these increased ranges of values for shear stress, no vasodilatation was observed (Fig 2). Pressure-induced myogenic and norepinephrineinduced tone. When pressurized to 80 mm Hg in physiologic sodium chloride solution alone, the arteries developed myogenic tone, which was similar in both groups (25% ± 4% in the preeclampsia group vs 24% ± 4% in the control group). After incubation with Nω-nitro-L-arginine, myogenic tone increased both in arteries from
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Fig 1. Percentage of flow-mediated vasodilatation in myometrial arteries from women with preeclampsia (solid line) and normal pregnant women (dashed line) in physiologic sodium chloride solution (filled circles and triangles, respectively) and in Nω-nitro-L-arginine (open circles and triangles, respectively). Data were analyzed by multivariate analysis of variance for repeated measurements and presented as mean ± SEM. Asterisks, Post hoc comparisons between 2 groups in physiologic sodium chloride solution at different flow steps (P < .001).
Fig 2. Relationship between wall shear stress (WSS) and percentage of dilatation (change in diameter) in myometrial arteries from women with preeclampsia (solid line) and normal pregnant women (dashed line), in physiologic sodium chloride solution (filled circles and triangles, respectively) and in Nω-nitro-L-arginine (open circles and triangles, respectively).
women with preeclampsia (35% ± 5% in Nω-nitro-L-arginine vs 25% ± 4% in physiologic sodium chloride solution; n = 6; Fig 3) and in arteries from healthy pregnant women (33% ± 4% in Nω-nitro-L-arginine vs 24% ± 4% in physiologic sodium chloride solution, n = 9; Fig 3). The 2way analysis of variance did not demonstrate any difference between the groups (F = 0.08; P = .78). However, there was a significant (F = 15.6; P = .002) effect of Nωnitro-L-arginine on pressure-induced tone in arteries from both groups. Norepinephrine-induced tone in isolated myometrial arteries did not differ significantly between the groups (36% ± 4% in the preeclampsia group vs 41% ± 5% in the
control group). In myometrial arteries from women with preeclampsia, inhibition of nitric oxide synthase increased norepinephrine-induced tone (46% ± 7% in Nωnitro-L-arginine vs 36% ± 4% in physiologic sodium chloride solution; Fig 3). A similar effect was observed in arteries obtained from healthy pregnant women (49% ± 6% in Nω-nitro-L-arginine vs 41% ± 5% in physiologic sodium chloride solution; Fig 3). As for pressure-induced tone, 2-way analysis of variance did not demonstrate any difference in norepinephrine-induced tone between the groups (F = 0.25; P = .63). However, there was a significant (F = 10.2; P = .007) effect of Nω-nitro-L-arginine on norepinephrine-induced tone in arteries from both groups.
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Fig 3. Percentage of pressure-induced myogenic tone (crosshatched bars) and norepinephrine-induced tone(solid bars) in isolated myometrial arteries from healthy pregnant women and women with preeclampsia (PE), in physiologic sodium chloride solution (PSS) and Nω-nitro-L-arginine (NOARG). Asterisks, Effect of Nω-nitro-L-arginine on pressure and norepinephrine-induced tone in arteries from both groups (2-way analysis of variance) (P < .01).
Comment In this study and in a recent report,12 we have demonstrated that shear stress is a potent stimulus for nitric oxide–mediated vasodilatation to occur in isolated small myometrial arteries from healthy pregnant women. However, in arteries from women with preeclampsia, wall shear stress fails to induce vasodilatation; indeed, these arteries demonstrate vasoconstriction in response to flowinduced shear stress. Our findings are thus in agreement with the absence of flow-mediated vasodilatation reported in the resistance vasculature of the cutaneous circulation from women with preeclampsia15 and suggest a generalized impairment in flow-mediated vasodilatation. The inhibition of nitric oxide synthase in our study failed to alter this response, indicating the absence of any flowmediated nitric oxide synthesis in these arteries in preeclampsia. The absence of nitric oxide–mediated relaxation in response to shear stress could therefore contribute to the vasoconstriction and the increase in vascular resistance in preeclampsia observed not only in the uterine circulation16 but also in other maternal vascular beds, such as cutaneous circulation.15 The flow-mediated vasoconstriction in the myometrial arteries from women with preeclampsia in this study might partly be explained by shear stress–induced release of endothelin 1 in the uterine circulation, which may be triggered or up-regulated during preeclampsia. We have recently demonstrated in isolated myometrial arteries from healthy pregnant women that shear stress is a potent stimulus not only for nitric oxide but also for endothelin 1 release, the response of which adds to the effect of nitric oxide synthase inhibition per se in causing vasoconstriction.14 Future studies with the endothelinconverting enzyme inhibitor and endothelin receptor an-
tagonists may define the role of endothelin 1 in shear stress–mediated vasoconstriction in preeclampsia. In vivo studies have shown vascular responses to norepinephrine to be enhanced in pregnant women with preeclampsia, as compared with healthy pregnant women.17 We found responses to adrenergic stimulation in pressurized myometrial resistance arteries to be similar in the 2 groups of gravid women. This is in agreement with earlier reports demonstrating a similar response to norepinephrine in isolated arteries from the omentum18 or subcutaneous fat,3, 15 but it is in contrast with reports in which sensitivity to norepinephrine was found to be increased in isolated wire-mounted myometrial19 and omental20 arteries from women with preeclampsia. It is possible that the discrepancies in results are attributable to differences in experimental setup, that is, wire-mounted versus pressurized arteries in our study. We found that norepinephrine-induced tone, as well as pressure-induced myogenic tone, was significantly enhanced after nitric oxide synthase inhibition in myometrial arteries from women with preeclampsia. A possible modulatory role of nitric oxide in adrenergic stimulation has been suggested in isolated omental arteries from women with preeclampsia, although this was reported to be manifest only at a much higher level of vessel wall tension than it was in the arteries from healthy pregnant women.20 Our observations imply that basal release of nitric oxide is unaffected in preeclampsia and that nitric oxide is, at least to some extent, involved in the modulation of myometrial resistance artery tone even in this disorder. At first glance this might seem incompatible with the absence of nitric oxide–mediated flow-induced vasodilatation demonstrated in the preeclampsia group and is worth comment. In fact, it has been demonstrated that nitric oxide synthase
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activity in cultured endothelial cells was significantly greater after exposure to plasma from women with preeclampsia than after exposure of cultured endothelial cells to plasma from pregnant women with normal blood pressure.21 This, however, does not preclude posttranslational modification of nitric oxide synthase activity in preeclampsia, and the physiologic importance of this in vitro observation remains to be proven. It is tempting to speculate that preeclampsia might affect elements of the shear stress transduction pathway. For example, preeclampsia could impair shear stress signal transduction through the endothelial cell cytoskeleton by altering F actin alignment, by influencing the β integrins in focal adhesion sites, the tyrosine kinases, or the mitogen-activated protein kinases, or by altering G protein expression.22-25 In fact, in a recent report Pascoal et al4 evaluated the vasodilatory response to acetylcholine and bradykinin in isolated omental resistance arteries from women with preeclampsia and found that acetylcholine-mediated vasodilatation was impaired but that bradykinin-mediated vasodilatation was not (both findings were independent of nitric oxide or prostanoid release). The authors suggested that preeclampsia might be associated with impaired signal transduction emanating from specific endothelial receptors (in their case the muscarinic acetylcholine receptor) at the level of specific G protein–mediated signal activation rather than a general disturbance in endothelial receptor function. An impaired signal transduction pathway coupled to Gprotein activation has also been suggested in a study by Koller and Huang26 in which impaired shear stress–mediated vasodilatation was observed in isolated skeletal resistance arteries from animals with hypertension. If this holds true, our observations might imply that preeclampsia is associated with an impairment or down-regulation of signal transduction elements involved in the initiation of shear stress–mediated response and thus nitric oxide release rather than reduced nitric oxide synthesis per se. We cannot, however, exclude that the impaired sensitivity to wall shear stress is caused by a reduced sensitivity of the smooth muscle to nitric oxide in women with preeclampsia because this was not evaluated in this study. Nevertheless, there are several studies from other laboratories in which endothelium-independent relaxation in resistance arteries from subcutaneous fat,2, 3 omentum,4 and myometrium5 of women with preeclampsia and healthy pregnant women showed no difference in response to sodium nitroprusside. In summary, we have shown that preeclampsia is associated with a failure of shear stress–mediated nitric oxide release in vitro, although the basal production remains unaltered. We believe that the proposed perturbation in the shear stress–mediated signal transduction leading to impaired vasodilatation in the presence
of an enhanced myogenic response6 will increase vascular resistance in the uterine circulation during preeclampsia. REFERENCES
1. Roberts JM, Taylor RN, Goldfien A. Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia. Am J Hypertens 1991;4:700-8. 2. McCarthy AL, Woolfson RG, Raju SK, Poston L. Abnormal endothelial cell function of resistance arteries from women with preeclampsia. Am J Obstet Gynecol 1993;168:1323-30. 3. Knock GA, Poston L. Bradykinin-mediated relaxation of isolated maternal resistance arteries in normal pregnancy and preeclampsia. Am J Obstet Gynecol 1996;175:1668-74. 4. Pascoal IF, Lindheimer MD, Nalbantian-Brandt C, Umans JG. Preeclampsia selectively impairs endothelium-dependent relaxation and leads to oscillatory activity in small omental arteries. J Clin Invest 1998;101:464-70. 5. Ashworth JR, Warren AY, Baker PN, Johnson IR. Loss of endothelium-dependent relaxation in myometrial resistance arteries in pre-eclampsia. Br J Obstet Gynaecol 1997;104:1152-8. 6. Kublickiene KR, Grunewald C, Lindblom B, Nisell H. Myogenic and endothelial properties in myometrial resistance arteries from women with preeclampsia. Hypertens Preg 1998;17:271-81. 7. Seligman SP, Buyon JP, Clancy RM, Young BK, Abramson SB. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944-8. 8. Davidge ST, Stranko CP, Roberts JM. Urine but not plasma nitric oxide metabolites are decreased in women with preeclampsia. Am J Obstet Gynecol 1996;174:1008-13. 9. Silver RK, Kupferminc MJ, Russell TL, Adler L, Mullen TA, Caplan MS. Evaluation of nitric oxide as a mediator of severe preeclampsia. Am J Obstet Gynecol 1996;175:1013-7. 10. Cameron IT, Van Paperndorp CL, Palmer RMJ, Smith SK, Moncada S. Relationship between nitric oxide synthesis and increase in systolic blood pressure in women with hypertension in pregnancy. Hypertens Preg 1993;12:85-92. 11. Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, et al. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res 1995;76:536-43. 12. Kublickiene KR, Cockell AP, Nisell H, Poston L. Role of nitric oxide in the regulation of vascular tone in pressurized and perfused resistance myometrial arteries from term pregnant women. Am J Obstet Gynecol 1997;177:1263-9. 13. Bower S, Schuchter K, Campbell S. Doppler ultasound screening as part of routine antenatal scanning: prediction of preeclampsia and intrauterine growth retardation. Br J Obstet Gynaecol 1993;100:989-94. 14. Kublickiene KR, Nisell H, Poston L, Krüger K, Lindblom B. Modulation of vascular tone by nitric oxide and endothelin 1 in myometrial resistance arteries from pregnant women at term. Am J Obstet Gynecol 2000;182:87-93. 15. Cockell AP, Poston L. Flow mediated nitric oxide synthesis is enhanced in normal pregnancy but reduced in preeclampsia. Hypertension 1997;30:247-51. 16. Lunell NO, Nylund L, Lewander R, Sarby B. Uteroplacental blood flow in pre-eclampsia: measurements with Indium-113m and a computerlinked gamma camera. Clin Exp Hypertens Preg 1982;1:105-17. 17. Nisell H, Hjemdahl P, Linde B. Cardiovascular responses to circulating catecholamines in normal pregnancy and in pregnancy-induced hypertension. Clin Physiol 1985;5:479-93. 18. Aalkjaer C, Danielsen H, Johannesen P, Pedersen EB, Rasmussen A, Mulvany MJ. Abnormal vascular function and morphology in pre-eclampsia: a study of isolated resistance vessels. Clin Sci 1985;69:477-82. 19. Allen J, Forman A, Maigaard S, Jespersen LT, Andersson KE. Effects of endogenous vasoconstrictors on maternal intramyometrial and fetal stem villous arteries in pre-eclampsia. J Hypertens 1989;7:529-36. 20. Belfort MA, Saade GR, Suresh M, Kramer W, Vedernikov YP. Ef-
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fects of selected vasoconstrictor agonists on isolated omental artery from premenopausal nonpregnant women and from normal and preeclamptic pregnant women. Am J Obstet Gynecol 1996;174:687-93. 21. Baker PN, Davidge ST, Roberts JM. Plasma from women with preeclampsia increases endothelial cell nitric oxide production. Hypertension 1995;26:244-8. 22. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 1995;75:519-60. 23. Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R. Intracellular pH and tyrosine phosphorylation but not calcium deter-
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mines shear stress-induced nitric oxide production in native endothelial cells. Circ Res 1996:78;750-8. 24. Muller JM, Chilian WM, Davis MJ. Integrin signaling transduces shear stress–dependent vasodilation of coronary arterioles. Circ Res 1997;80:320-6. 25. Takahashi M, Ishida T, Traub O, Corson M, Berk BC. Mechanotransduction in endothelial cells: temporal signaling events in response to shear stress. J Vasc Res 1997;34:212-9. 26. Koller A, Huang A. Impaired nitric oxide–mediated flowinduced dilatation in arterioles of spontaneous hypertensive rats. Circ Res 1994;74:416-21.
Key words: Preeclampsia, arteries, tone, shear stress, nitric oxide
Endothelial dysfunction is considered an important pathogenic feature of preeclampsia, confirmed by alterations in a variety of biochemical markers1 and more recently by the demonstration of impaired vasodilatory response of resistance vessels to endothelium-dependent agonists such as acetylcholine and bradykinin in vitro.2-6 From the Department of Clinical Science, Section for Obstetrics and Gynaecology, Karolinska Institute, Huddinge University Hospital,a and the Department of Women’s and Children’s Health, Section for Obstetrics and Gynaecology, Uppsala University.b Received for publication March 3, 1999; revised December 22, 1999; accepted January 14, 2000. Reprint requests: Karolina-Rasa Kublickiene, MD, PhD, Department of Obstetrics and Gynaecology, Karolinska Institute, Huddinge University Hospital, 14186 Huddinge, Sweden. E-mail Karolina.Kublickiene@ kfcmail.hs.sll.se. Copyright © 2000 by Mosby, Inc. 0002-9378/2000 $12.00 + 0 6/1/105820 doi:10.1067/mob.2000.105820
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The belief that endothelial dysfunction would be linked to impaired nitric oxide activity in preeclampsia still remains controversial. Thus the role played by nitric oxide in endothelium-dependent agonist-mediated responses is far from settled.3-6 Moreover, contrasting data have been presented concerning the levels of stable nitric oxide metabolites in preeclampsia.7-10 Thus the relevance of these methods for assessing nitric oxide function in preeclampsia might be questioned. It is increasingly accepted that the predominant physiologic stimulus of endothelial nitric oxide synthesis is shear stress generated by blood flow.11 We have recently demonstrated that flow-induced shear stress is a physiologic modulator of vascular tone in isolated myometrial arteries from normal pregnant women and that nitric oxide mediates this response12 but vasodilatory prostanoids do not. Accordingly, studies in isolated myometrial
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arteries from women with preeclampsia by use of a physiologic stimulus for nitric oxide release might contribute to our understanding of the part that nitric oxide may play in preeclampsia and particularly in the uterine circulation. This is a particularly relevant circulation to investigate in view of the indication from Doppler waveform analysis of the uterine artery that resistance is increased in this circulation from an early stage in preeclamptic pregnancies.13 This study was therefore undertaken to compare shear stress–mediated vasodilatation in myometrial resistance arteries from women with preeclampsia and healthy gravid women. In addition, the role of nitric oxide in the modulation of pressure-induced myogenic and norepinephrine-induced tone has been determined in myometrial arteries from women with preeclampsia and compared with that in healthy gravid women. These studies have been carried out in isolated myometrial arteries mounted on a pressure arteriograph. Methods Subjects. This study was approved by the Ethical Committee of Huddinge University Hospital, and all women gave their informed consent to participate. Informed consent was obtained from 6 nulliparous women with preeclampsia, with a median age of 27 years (range, 2236), and a median gestational age of 36 weeks (range, 3139) who were undergoing cesarean delivery because of worsening of the preeclamptic state. Preeclampsia was defined as blood pressure ≥140/90 mm Hg and proteinuria exceeding >300 mg/24 h in the absence of urinary tract infection after 20 weeks’ gestation in women with previously normal blood pressure and without proteinuria. Any women with a history of chronic hypertension, kidney disease, or diabetes mellitus were excluded from the study. Women with preeclampsia who had received antihypertensive agents were also excluded. The control group included 9 healthy pregnant women (5 nulliparous) with a median age of 28 years (range, 18-34) and a median gestational age of 39 weeks (range, 37-41) undergoing elective cesarean delivery because of breech presentation (n = 5) or previous cesarean delivery (n = 4). These women were part of a control group that participated in a recent study demonstrating the effects of both nitric oxide and endothelin 1 on myometrial resistance artery tone under pressurized and perfused conditions.14 The biopsy specimen was obtained immediately after delivery from the upper edge of the transverse incision in the lower uterine segment. Experimental setup. Intramyometrial small arteries (internal diameter ~200 µm at 50 mm Hg intraluminal pressure and lengths of ~2.5 to 3 mm) were immediately dissected from the biopsy specimens, and the surrounding myometrium, connective tissue, and adventitia were carefully removed. The myometrial arteries were then
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mounted in a pressure arteriograph (Living Systems Instrumentation Inc, Burlington, Vt) as previously described.12, 14 The vessels were orientated in the in vivo direction of flow on a pair of opposing glass microcannulas previously matched for flow resistance. The organ bath was perfused (7 mL/min) with physiologic sodium chloride solution (sodium chloride, 119 mmol/L; potassium chloride, 4.7 mmol/L; calcium chloride, 2.5 mmol/L; magnesium sulfate, 1.17 mmol/L; sodium bicarbonate, 25 mmol/L; sodium phosphate, monobasic, 1.18 mmol/L; ethylenediaminetetraacetic acid, 0.026 mmol/L; and glucose 5.5 mmol/L; pH 7.4; 37°C, gassed with 5% carbon dioxide in oxygen). A servocontrolled pump maintained the required intraluminal pressure, and the internal diameter of the artery was recorded continuously with a video dimension analyser. “In-line” pressure transducers monitored the proximal and distal pressures on each side of the specimen, enabling calculation of the mean intraluminal pressure. Each artery was equilibrated for 45 minutes while being pressurized to 50 mm Hg of intraluminal pressure. Viability was confirmed by exposure to extraluminal norepinephrine (10–6 mol/L) in potassium-substituted physiologic sodium chloride solution (64 mmol/ L) and endothelial function by relaxation induced by acetylcholine (10–6 mol/L). Arteries failing to maintain pressure, to demonstrate complete occlusion of the lumen in response to norepinephrine, or to show relaxation in response to acetylcholine were excluded from the study. Altered acetylcholine-mediated dilatation of preeclamptic vessels was not an exclusion criterion because impaired dilatation to endothelium-dependent agonists has been reported in preeclampsia.5, 6 Effects of nitric oxide synthase inhibition on pressure-induced tone, norepinephrine-induced tone, and flow-mediated responses. After the equilibration period, the intraluminal pressure was increased to 80 mm Hg for 30 minutes, and then the inner diameter was recorded. This pressure was chosen because it approximates the in vivo pressure at the proximal part of these arteries. Norepinephrine (10–6 mol/L) was then added to the superfusate for 30 minutes, and inner diameter was recorded before flow was started. Intraluminal flow was initiated with a flow pump. Flow was increased at 5-minute intervals (from 0 to 204 µL/min), and the inner diameter was recorded at the end of each flow step. During flow establishment at different flow rates, the proximal and distal pressure gradients were controlled by changing the distal pressure to keep the intraluminal pressure constant, that is, 80 mm Hg. After achievement of the first flow-response curve at increasing flow rates, intraluminal flow was stopped and intraluminal pressure was kept at 80 mm Hg. Inhibition of nitric oxide synthase was then induced by addition of Nω-nitro-L-arginine (10–3 mol/L) for 30 minutes in the absence of flow and norepinephrine. After the inner diameter was recorded, the arteries were then constricted
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with norepinephrine (10–6 mol/L) again in the continued presence of Nω-nitro-L-arginine for 30 minutes and the flow protocol was repeated. Finally, while pressure was maintained at 80 mm Hg, the arteries were incubated in calcium-free solution with ethylene glycol-bis (β-aminoethyl ether)N,N,N´,N´-tetraacetic acid (EGTA) (1 mmol/L plus papaverine 0.1 mmol/L) for 30 minutes, and the increase in inner diameter was recorded. Calculation of wall shear stress and myogenic and norepinephrine-induced tone. Wall shear stress (dyne/cm2) was calculated as follows: WSS = 4 × ρ × Q × 109/π × r3 where WSS is wall shear stress, ρ is viscosity in poise (dyne s/cm2) at 37°C, Q is flow rate (µL/s), and r is artery radius (µm). Viscosity of the physiologic sodium chloride solution was 0.7 cP. The factor of 109 in the equation is to correct for the use of both microliters per second for flow and micrometers for arterial radius (1 µL is equivalent to 109 µm3). The difference in inner diameter of the arteries when pressurized at 80 mm Hg before and after equilibration in calcium-free physiologic sodium chloride solution (with EGTA) provides an estimate of myogenic tone. This was calculated as the percentage decrease of the internal diameter of arteries in calcium-free physiologic sodium chloride solution, from the following equation: Myogenic tone (%) = (id2 – id1)/id2 × 100 where id2 is internal diameter in calcium-free physiologic sodium chloride solution (with EGTA) and id1 is internal diameter when pressurized in physiologic sodium chloride solution at 80 mm Hg. Similarly, myogenic tone after the addition of norepinephrine is calculated as follows: Myogenic tone after norepinephrine addition (%) = (id2 – id3)/id2 × 100 where id3 is internal diameter after the addition of norepinephrine. For simplicity, hereafter this is referred to as norepinephrine-induced tone. Statistical analysis. Values in figures are given as mean ± SEM. The flow responses in the absence and presence of Nω-nitro-L-arginine at different flow steps between the groups were compared by multivariate analysis of variance for repeated measurements (StatSoft, Inc, Tulsa, Okla). Post hoc comparisons between groups at different flow steps were performed with the Scheffé test (adjusted for multiple comparisons). The effects of flow steps within 1 group were evaluated by 1-way analysis of variance for repeated measurements (eg, flow-mediated dilatation in physiologic sodium chloride solution in arteries from healthy pregnant women). The 2 groups were compared with regard to the effect of Nω-nitro-L-arginine on myogenic and norepinephrine-induced tone by 2–way analysis of variance. Significance was assumed if P < .05.
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Results The arteries from women with preeclampsia (n = 6) and control subjects (n = 9) had similar diameters when equilibrated in physiologic sodium chloride solution alone at 50 mm Hg (212 ± 24 vs 215 ± 20 µm, respectively). Flow-mediated responses. Myometrial arteries from women with preeclampsia failed to dilate in response to increasing intraluminal flow (Fig 1). In fact, these arteries in physiologic sodium chloride solution constricted significantly (analysis of variance, F = 4.7; P = .004) in response to increasing intraluminal flow (eg, percentage change in preconstricted inner diameter, –15% ± 6% at the maximum flow rate of 204 µL/min; Fig 1). After incubation with Nω-nitro-L-arginine, these arteries responded similarly (–12% ± 4% at the maximum flow rate of 204 µL/min; Fig 1). In contrast, the myometrial arteries from healthy pregnant women demonstrated a substantial relaxation in response to flow (analysis of variance, F = 32; P = .0008; Fig 1), which was most pronounced at a flow rate of 130 µL/min (percentage change in preconstricted inner diameter, 30% ± 5%) and was maintained at this level up to the maximum flow rate of 204 µL/min. In the presence of Nω-nitroL-arginine, these arteries responded significantly differently (multivariate analysis of variance, F = 33.4; P = .0001), because increasing intraluminal flow led to a small but significant (analysis of variance, F = 3.28; P = .02) flow-induced constriction (eg, percentage change in preconstricted inner diameter, –9% ± 5% at a flow rate of 204 µL/min). The relationship between wall shear stress (calculated in each artery with the internal diameter immediately before the change in intraluminal flow) and the percentage of dilatation in arteries from both patient groups, in the absence and presence of Nω-nitro-L-arginine, is presented in Fig 2. The range of shear stress values achieved in arteries from women with preeclampsia was considerably greater than that in arteries from healthy pregnant women (6 ± 1 to 89 ± 24 dyne/cm2 vs 7 ± 2 to 29 ± 7 dyne/cm2, respectively), implying that the arteries from women with preeclampsia were less sensitive to shear stress because no vasodilatation was observed. In the presence of Nω-nitro-L-arginine, shear stress values ranged from 14 ± 5 to 151 ± 58 dyne/cm2 in the preeclampsia group compared with 17 ± 6 to 221 ± 108 dyne/cm2 in the normotensive control group. Within these increased ranges of values for shear stress, no vasodilatation was observed (Fig 2). Pressure-induced myogenic and norepinephrineinduced tone. When pressurized to 80 mm Hg in physiologic sodium chloride solution alone, the arteries developed myogenic tone, which was similar in both groups (25% ± 4% in the preeclampsia group vs 24% ± 4% in the control group). After incubation with Nω-nitro-L-arginine, myogenic tone increased both in arteries from
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Fig 1. Percentage of flow-mediated vasodilatation in myometrial arteries from women with preeclampsia (solid line) and normal pregnant women (dashed line) in physiologic sodium chloride solution (filled circles and triangles, respectively) and in Nω-nitro-L-arginine (open circles and triangles, respectively). Data were analyzed by multivariate analysis of variance for repeated measurements and presented as mean ± SEM. Asterisks, Post hoc comparisons between 2 groups in physiologic sodium chloride solution at different flow steps (P < .001).
Fig 2. Relationship between wall shear stress (WSS) and percentage of dilatation (change in diameter) in myometrial arteries from women with preeclampsia (solid line) and normal pregnant women (dashed line), in physiologic sodium chloride solution (filled circles and triangles, respectively) and in Nω-nitro-L-arginine (open circles and triangles, respectively).
women with preeclampsia (35% ± 5% in Nω-nitro-L-arginine vs 25% ± 4% in physiologic sodium chloride solution; n = 6; Fig 3) and in arteries from healthy pregnant women (33% ± 4% in Nω-nitro-L-arginine vs 24% ± 4% in physiologic sodium chloride solution, n = 9; Fig 3). The 2way analysis of variance did not demonstrate any difference between the groups (F = 0.08; P = .78). However, there was a significant (F = 15.6; P = .002) effect of Nωnitro-L-arginine on pressure-induced tone in arteries from both groups. Norepinephrine-induced tone in isolated myometrial arteries did not differ significantly between the groups (36% ± 4% in the preeclampsia group vs 41% ± 5% in the
control group). In myometrial arteries from women with preeclampsia, inhibition of nitric oxide synthase increased norepinephrine-induced tone (46% ± 7% in Nωnitro-L-arginine vs 36% ± 4% in physiologic sodium chloride solution; Fig 3). A similar effect was observed in arteries obtained from healthy pregnant women (49% ± 6% in Nω-nitro-L-arginine vs 41% ± 5% in physiologic sodium chloride solution; Fig 3). As for pressure-induced tone, 2-way analysis of variance did not demonstrate any difference in norepinephrine-induced tone between the groups (F = 0.25; P = .63). However, there was a significant (F = 10.2; P = .007) effect of Nω-nitro-L-arginine on norepinephrine-induced tone in arteries from both groups.
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Fig 3. Percentage of pressure-induced myogenic tone (crosshatched bars) and norepinephrine-induced tone(solid bars) in isolated myometrial arteries from healthy pregnant women and women with preeclampsia (PE), in physiologic sodium chloride solution (PSS) and Nω-nitro-L-arginine (NOARG). Asterisks, Effect of Nω-nitro-L-arginine on pressure and norepinephrine-induced tone in arteries from both groups (2-way analysis of variance) (P < .01).
Comment In this study and in a recent report,12 we have demonstrated that shear stress is a potent stimulus for nitric oxide–mediated vasodilatation to occur in isolated small myometrial arteries from healthy pregnant women. However, in arteries from women with preeclampsia, wall shear stress fails to induce vasodilatation; indeed, these arteries demonstrate vasoconstriction in response to flowinduced shear stress. Our findings are thus in agreement with the absence of flow-mediated vasodilatation reported in the resistance vasculature of the cutaneous circulation from women with preeclampsia15 and suggest a generalized impairment in flow-mediated vasodilatation. The inhibition of nitric oxide synthase in our study failed to alter this response, indicating the absence of any flowmediated nitric oxide synthesis in these arteries in preeclampsia. The absence of nitric oxide–mediated relaxation in response to shear stress could therefore contribute to the vasoconstriction and the increase in vascular resistance in preeclampsia observed not only in the uterine circulation16 but also in other maternal vascular beds, such as cutaneous circulation.15 The flow-mediated vasoconstriction in the myometrial arteries from women with preeclampsia in this study might partly be explained by shear stress–induced release of endothelin 1 in the uterine circulation, which may be triggered or up-regulated during preeclampsia. We have recently demonstrated in isolated myometrial arteries from healthy pregnant women that shear stress is a potent stimulus not only for nitric oxide but also for endothelin 1 release, the response of which adds to the effect of nitric oxide synthase inhibition per se in causing vasoconstriction.14 Future studies with the endothelinconverting enzyme inhibitor and endothelin receptor an-
tagonists may define the role of endothelin 1 in shear stress–mediated vasoconstriction in preeclampsia. In vivo studies have shown vascular responses to norepinephrine to be enhanced in pregnant women with preeclampsia, as compared with healthy pregnant women.17 We found responses to adrenergic stimulation in pressurized myometrial resistance arteries to be similar in the 2 groups of gravid women. This is in agreement with earlier reports demonstrating a similar response to norepinephrine in isolated arteries from the omentum18 or subcutaneous fat,3, 15 but it is in contrast with reports in which sensitivity to norepinephrine was found to be increased in isolated wire-mounted myometrial19 and omental20 arteries from women with preeclampsia. It is possible that the discrepancies in results are attributable to differences in experimental setup, that is, wire-mounted versus pressurized arteries in our study. We found that norepinephrine-induced tone, as well as pressure-induced myogenic tone, was significantly enhanced after nitric oxide synthase inhibition in myometrial arteries from women with preeclampsia. A possible modulatory role of nitric oxide in adrenergic stimulation has been suggested in isolated omental arteries from women with preeclampsia, although this was reported to be manifest only at a much higher level of vessel wall tension than it was in the arteries from healthy pregnant women.20 Our observations imply that basal release of nitric oxide is unaffected in preeclampsia and that nitric oxide is, at least to some extent, involved in the modulation of myometrial resistance artery tone even in this disorder. At first glance this might seem incompatible with the absence of nitric oxide–mediated flow-induced vasodilatation demonstrated in the preeclampsia group and is worth comment. In fact, it has been demonstrated that nitric oxide synthase
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activity in cultured endothelial cells was significantly greater after exposure to plasma from women with preeclampsia than after exposure of cultured endothelial cells to plasma from pregnant women with normal blood pressure.21 This, however, does not preclude posttranslational modification of nitric oxide synthase activity in preeclampsia, and the physiologic importance of this in vitro observation remains to be proven. It is tempting to speculate that preeclampsia might affect elements of the shear stress transduction pathway. For example, preeclampsia could impair shear stress signal transduction through the endothelial cell cytoskeleton by altering F actin alignment, by influencing the β integrins in focal adhesion sites, the tyrosine kinases, or the mitogen-activated protein kinases, or by altering G protein expression.22-25 In fact, in a recent report Pascoal et al4 evaluated the vasodilatory response to acetylcholine and bradykinin in isolated omental resistance arteries from women with preeclampsia and found that acetylcholine-mediated vasodilatation was impaired but that bradykinin-mediated vasodilatation was not (both findings were independent of nitric oxide or prostanoid release). The authors suggested that preeclampsia might be associated with impaired signal transduction emanating from specific endothelial receptors (in their case the muscarinic acetylcholine receptor) at the level of specific G protein–mediated signal activation rather than a general disturbance in endothelial receptor function. An impaired signal transduction pathway coupled to Gprotein activation has also been suggested in a study by Koller and Huang26 in which impaired shear stress–mediated vasodilatation was observed in isolated skeletal resistance arteries from animals with hypertension. If this holds true, our observations might imply that preeclampsia is associated with an impairment or down-regulation of signal transduction elements involved in the initiation of shear stress–mediated response and thus nitric oxide release rather than reduced nitric oxide synthesis per se. We cannot, however, exclude that the impaired sensitivity to wall shear stress is caused by a reduced sensitivity of the smooth muscle to nitric oxide in women with preeclampsia because this was not evaluated in this study. Nevertheless, there are several studies from other laboratories in which endothelium-independent relaxation in resistance arteries from subcutaneous fat,2, 3 omentum,4 and myometrium5 of women with preeclampsia and healthy pregnant women showed no difference in response to sodium nitroprusside. In summary, we have shown that preeclampsia is associated with a failure of shear stress–mediated nitric oxide release in vitro, although the basal production remains unaltered. We believe that the proposed perturbation in the shear stress–mediated signal transduction leading to impaired vasodilatation in the presence
of an enhanced myogenic response6 will increase vascular resistance in the uterine circulation during preeclampsia. REFERENCES
1. Roberts JM, Taylor RN, Goldfien A. Clinical and biochemical evidence of endothelial cell dysfunction in the pregnancy syndrome preeclampsia. Am J Hypertens 1991;4:700-8. 2. McCarthy AL, Woolfson RG, Raju SK, Poston L. Abnormal endothelial cell function of resistance arteries from women with preeclampsia. Am J Obstet Gynecol 1993;168:1323-30. 3. Knock GA, Poston L. Bradykinin-mediated relaxation of isolated maternal resistance arteries in normal pregnancy and preeclampsia. Am J Obstet Gynecol 1996;175:1668-74. 4. Pascoal IF, Lindheimer MD, Nalbantian-Brandt C, Umans JG. Preeclampsia selectively impairs endothelium-dependent relaxation and leads to oscillatory activity in small omental arteries. J Clin Invest 1998;101:464-70. 5. Ashworth JR, Warren AY, Baker PN, Johnson IR. Loss of endothelium-dependent relaxation in myometrial resistance arteries in pre-eclampsia. Br J Obstet Gynaecol 1997;104:1152-8. 6. Kublickiene KR, Grunewald C, Lindblom B, Nisell H. Myogenic and endothelial properties in myometrial resistance arteries from women with preeclampsia. Hypertens Preg 1998;17:271-81. 7. Seligman SP, Buyon JP, Clancy RM, Young BK, Abramson SB. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944-8. 8. Davidge ST, Stranko CP, Roberts JM. Urine but not plasma nitric oxide metabolites are decreased in women with preeclampsia. Am J Obstet Gynecol 1996;174:1008-13. 9. Silver RK, Kupferminc MJ, Russell TL, Adler L, Mullen TA, Caplan MS. Evaluation of nitric oxide as a mediator of severe preeclampsia. Am J Obstet Gynecol 1996;175:1013-7. 10. Cameron IT, Van Paperndorp CL, Palmer RMJ, Smith SK, Moncada S. Relationship between nitric oxide synthesis and increase in systolic blood pressure in women with hypertension in pregnancy. Hypertens Preg 1993;12:85-92. 11. Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, et al. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res 1995;76:536-43. 12. Kublickiene KR, Cockell AP, Nisell H, Poston L. Role of nitric oxide in the regulation of vascular tone in pressurized and perfused resistance myometrial arteries from term pregnant women. Am J Obstet Gynecol 1997;177:1263-9. 13. Bower S, Schuchter K, Campbell S. Doppler ultasound screening as part of routine antenatal scanning: prediction of preeclampsia and intrauterine growth retardation. Br J Obstet Gynaecol 1993;100:989-94. 14. Kublickiene KR, Nisell H, Poston L, Krüger K, Lindblom B. Modulation of vascular tone by nitric oxide and endothelin 1 in myometrial resistance arteries from pregnant women at term. Am J Obstet Gynecol 2000;182:87-93. 15. Cockell AP, Poston L. Flow mediated nitric oxide synthesis is enhanced in normal pregnancy but reduced in preeclampsia. Hypertension 1997;30:247-51. 16. Lunell NO, Nylund L, Lewander R, Sarby B. Uteroplacental blood flow in pre-eclampsia: measurements with Indium-113m and a computerlinked gamma camera. Clin Exp Hypertens Preg 1982;1:105-17. 17. Nisell H, Hjemdahl P, Linde B. Cardiovascular responses to circulating catecholamines in normal pregnancy and in pregnancy-induced hypertension. Clin Physiol 1985;5:479-93. 18. Aalkjaer C, Danielsen H, Johannesen P, Pedersen EB, Rasmussen A, Mulvany MJ. Abnormal vascular function and morphology in pre-eclampsia: a study of isolated resistance vessels. Clin Sci 1985;69:477-82. 19. Allen J, Forman A, Maigaard S, Jespersen LT, Andersson KE. Effects of endogenous vasoconstrictors on maternal intramyometrial and fetal stem villous arteries in pre-eclampsia. J Hypertens 1989;7:529-36. 20. Belfort MA, Saade GR, Suresh M, Kramer W, Vedernikov YP. Ef-
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fects of selected vasoconstrictor agonists on isolated omental artery from premenopausal nonpregnant women and from normal and preeclamptic pregnant women. Am J Obstet Gynecol 1996;174:687-93. 21. Baker PN, Davidge ST, Roberts JM. Plasma from women with preeclampsia increases endothelial cell nitric oxide production. Hypertension 1995;26:244-8. 22. Davies PF. Flow-mediated endothelial mechanotransduction. Physiol Rev 1995;75:519-60. 23. Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R. Intracellular pH and tyrosine phosphorylation but not calcium deter-
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mines shear stress-induced nitric oxide production in native endothelial cells. Circ Res 1996:78;750-8. 24. Muller JM, Chilian WM, Davis MJ. Integrin signaling transduces shear stress–dependent vasodilation of coronary arterioles. Circ Res 1997;80:320-6. 25. Takahashi M, Ishida T, Traub O, Corson M, Berk BC. Mechanotransduction in endothelial cells: temporal signaling events in response to shear stress. J Vasc Res 1997;34:212-9. 26. Koller A, Huang A. Impaired nitric oxide–mediated flowinduced dilatation in arterioles of spontaneous hypertensive rats. Circ Res 1994;74:416-21.