Kenneth E. Clark, Ph.D. - US grants

Hypertension in pregnancy is associated with elevated maternal and fetal mortality rates. Two major pathophysiological alterations which are known to occur in the pregnant hypertensive are elevated arterial blood pressure and chronic reduction in uterine blood flow. In previous studies from our laboratory we have investigated maternal and fetal effects of reduced uterine blood flow and have shown that decreased uterine perfusion can significantly alter fetal oxygenation. In the present proposal, we plan to utilize a renal hypertensive pregnant sheep model to evaluate hypertension induced alterations in uterine hemodynamic. In this study, the effects of renovascular hypertension on maternal and fetal cardiovascular, respiratory and biochemical parameters will be evaluated. Renal hypertension will be produced by unilateral nephrectomy and a 50% reduction in renal blood flow to the remaining kidney. Renal hypertension will be produced at 118 days gestation (term 145 days), eight days after maternal and fetal instrumentation (110 days gestation). Maternal arterial blood pressure changes will be measured from 90 days gestation to 142 days of gestation, at which time animals will be sacrificed for evaluation of fetal growth. During the period of instrumentation, maternal and fetal blood pressure, heart rate, blood gas values, oxygen content, pH, lactate, glucose and electrolytes will be determined. Because renal hypertension activates the renin angiotensin system, plasma renin activity and angiotensin II will also be measured. The ability of the altered renin-angiotensin system to modify prostaglandin levels will be investigated by measuring maternal arterial and uterine venous prostaglandin (PGD2, PGE2 and 6 keto PGF1Alpha) levels. In addition, uterine and femoral blood flow and their calculated vascular resistances will be determined. These measurements will allow us to determine if resistance changes occur in both vascular beds or if these changes are specific for the uterine vasculature. Finally throughout the experimental period (90-142 days gestation) systemic and local responses to vasoconstrictors (angiotensin II, norepinephrine and serotonin) will be evaluated to determine if renal hypertension alters vascular reactivity thus leading to increased vascular resistance. These studies will greatly improve our understanding of the effects of renal hypertension on the mother and fetus.

The present study is designed to evaluate the mechanism(s) responsible for fetal growth retardation in response to reduced uteroplacental blood flow. During the last four years we have been developing a model of intrauterine growth retardation (IUGR) using a pregnant sheep model in which uteroplacental blood flow has been mechanically reduced. Studies to data have shown a direct relationship (p less than or equal to 0.01) between uteroplacental blood flow and ponderal index, feta body weight, fetal heart and thymus weights and placental weight. The present study is designed to evaluate the mechanisms responsible for fetal growth retardation in response to reductions in uteroplacental blood flow. The first series of studies is designed to complete validation of the sheep model which has shown that graded reductions in uteroplacental blood flow result in graded decreases in fetal growth during the last five weeks of gestation. The second series of studies will evaluate the mechanism by which reduction in uteroplacental blood flow leads to decreased placental size. We will extensively investigate the mechanism by which umbilical blood flow is decreased and placental resistance is significantly increased in IUGR. Furthermore we plan to carefully evaluate changes which occur with internal and external Doppler ultrasound wave profiles (S/D ratios, pulsatile indices) in response to IUGR and vasoconstrictive agents. Changes in systolic and diastolic velocity ratios will be determined and compared using both implanted Doppler flow probes on the uterine and umbilical circulation and external spectral analysis Doppler waveform equipment. Studies are also designed to evaluate if autoregulation of blood blow occurs in the uterine and umbilical circulation. Further investigations are planned in regard to substrate and oxygen delivery and consumption in IUGR and control fetuses. These studies will include evaluation of delivery, extraction and consumption by the uterus, placenta, and fetus. Finally, the effect of increased uteroplacental perfusion (produced by release of the vascular occluder) on substrate delivery, fetal growth, umbilical vascular resistance and umbilical blood flow will be determined in IUGR animals after the vascular occluders have been returned to pre-occlusion values or after administration of oxygen to mother. These studies will result in significant insight into the mechanism of IUGR.

The present study is designed to evaluate the mechanism(s) responsible for increased systemic arterial pressure, reduced uteroplacental blood flow and proteinuria which occurs in response to maternal hypocalcemia in late term pregnant sheep. Pregnancy-induced hypertension (PIH) in women is known to be associated with similar changes in these parameters. The onset of PIH occurs in pregnant women when the maternal and fetal calcium demands are at their maximum. Additionally, recent studies have shown that reduction in calcium levels leads to a significant decrease in prostacyclin synthesis in vascular smooth muscle which has also been observed in PIH. The present proposal is designed to evaluate the maternal and fetal cardiovascular response to maternal hypocalcemia and to utilize this model to understand the underlying pathophysiology of PIH. Studies are planned which will evaluate systemic and uterine vascular responses to hypocalcemia and determine if changes occur in uterine and umbilical vascular resistance and reactivity in hypocalcemic animals. In these studies the effects of hypocalcemia and the resultant hypertension in fetal and placental growth will also be determined. Furthermore, we plan to carefully evaluate umbilical and uterine changes which occur with internal and external doppler ultrasound wave profiles (S/D ratios, pulsatility indices) response to maternal hypertension as well as in response to exogenously administered vasoconstrictors. Changes in systolic and diastolic velocity ratios will be determined and compared using both implanted doppler flow probes on the uterine and umbilical circulation and external continuous wave spectral analysis doppler equipment. These values will be related to fetal PaO2,O2 content, pH and lactate. Studies will also evaluate local uterine and umbilical responses to vasoconstrictors and determine if prostacyclin (PGI2, measured as 6 oxo PGF 1alpha) release in response to these vasoconstrictors is altered in hypertensive animals compared to controls. These studies will provide us with new and exciting information regarding this model of PIH.

Adequate uterine blood is required for normal growth and development of the fetus. Blood flowing to the uterus provides the blastocyst, embryo and fetus with all the necessary nutrients and oxygen which are required for growth and development. Since arterial blood pressure remains relatively constant during pregnancy, changes in uteroplacental blood flow are directly related to decreases in uterine vascular resistance. A significant portion of this change is due to the development of a new parallel circuit, the placenta. However, it is also thought that locally produced vasodilator agents are responsible for a significant portion of the increase in blood flow especially in late gestation. Recently, many cardiovascular scientists have turned their interest to the role of the endothelial cells in regulating blood flow in organs throughout the body. Endothelial cells produce several very potent vasodilators including endothelial derived relaxing factor (EDRF) and PGI(2). EDRF is thought to be the vasodilator nitric oxide, released during the conversion of L-arginine to L-citrulline, a reaction catalyzed by the calcium calmodulin dependent nitric oxide synthetase. This enzyme can be blocked by analogues of L-arginine (i.e., L-nitro arginine, L-mono-methyl arginine, etc.) an effect which is reversible by large doses of L-arginine but not D-arginine. The present application is based on exciting preliminary data (page 40) which demonstrates that estrogen induced increases in uterine blood flow are mediated by EDRF (NO) and that blockade of EDRF (NO) synthesis results in a 30% reduction in uteroplacental blood flow in late term sheep. Studies are outlined which will evaluate the role of EDRF in regulating systemic arterial blood pressure as well as uterine vascular resistance in normal pregnant and nonpregnant sheep and umbilical vascular resistance in the fetus. We will determine if nitric oxide synthetase (NOS) activity is increased in systemic and uterine vascular smooth muscle during pregnancy and determine if estrogen can induce NOS activity in the nonpregnant systemic and uterine vasculature. Studies are also planned which will determine if the nitric oxide breakdown product, nitrate, is elevated in the uterine and umbilical circulation in pregnancy and increases during gestation. We will also determine if estrogen administration increases systemic and uterine venous levels of plasma nitrate and cGMP. Finally, the ability of EDRF to modulate organ blood flow distribution as well as systemic and uterine vascular responses to endogenously occurring vasoconstrictors (norepinephrine, angiotensin II, serotonin and endothelin-1) in pregnant and nonpregnant sheep will be determined. These experiments should further clarify the role of endothelial derived relaxing factors in the regulation of systemic and uterine blood flow throughout pregnancy. Investigating their interactions with endogenous vasoconstrictors will increase our understanding of the physiological basis of vascular regulation and its pathologic deviation.

DESCRIPTION: (Adapted from the applicant's abstract) The objective of these experiments is to test the hypothesis that ET-1 plays an important role in regulating maternal systemic arterial blood pressure, systemic vascular resistance, uterine and renal hemodynamics, as well as renal permeability to proteins in nonpregnant and pregnant ewes. Aim 1 is to determine the dose of an ET-1 converting enzyme inhibitor required to block the conversion of big ET-1 to processed ET-1, and to determine if the endogenous production of ET-1 plays a role in modulating cardiovascular dynamics in pregnant and nonpregnant ewes. Aim 2 is to determine the systemic, uterine, and renal vascular responses to both local and intravenous infusion of ET-1. Aim 3 is to determine the mechanism by which the uterine artery becomes refractory to the vasoconstrictor actions of ET-1 during pregnancy. Aim 4 is to test the hypothesis that infusions of ET-1 results in hypertension, reduced uterine blood flow, hemoconcentration, and proteinuria in both pregnant and nonpregnant sheep.

Although the uterine vascular effects of estrogen have been studied for over 70 years, the mechanism by which estrogen produces vasodilation remains unclear. Our laboratory was the first to show that a significant component of the uterine response to estrogen in the nonpregnant sheep is mediated by the release of nitric oxide (NO). We and others have shown that estradiol-17beta increases the expression and activity of endothelial nitric oxide synthase (eNOS) in the uterine circulation. However, it is not currently clear how this occurs or if, in addition to eNOS, neuronal nitric oxide synthase (nNOS) or inducible nitric oxide synthase (iNOS) are also important in maintaining the sustained vasodilatory response seen after estrogen administration. Furthermore, it is not clear how estrogen modulates these NOS isoforms at the cellular and molecular level. Recently a new estrogen receptor, ERbeta has been isolated and emerging data suggest that this receptor may mediate a significant portion of the effects of estrogen in the vasculature. We hypothesize that uterine vasodilation produced by estradiol-17beta is mediated by specific interaction with both ERalpha and ERbeta, which subsequently activates eNOS (and potentially nNOS) via a nongenomic pathway, and iNOS via a genomic pathway, leading to increases in NO. The present application plans to evaluate the role of ER as a modulator of the NOS isoforms in the uterine circulation using a combination of physiologic and molecular endpoints. We will monitor the uterine hemodynamic responses to locally and systemically administered pharmacological antagonists that are selective for specific isoforms of NOS using a well-characterized ovine model. We intend to evaluate the expression of ERalpha and ERbeta in the ovine uterine vasculature and explore how estrogen alters eNOS, nNOS and NOS expression in endothelial cells and vascular smooth muscle. Finally we plan to determine if endogenous estrogen, acting through the uterine vascular NOS system, plays a critical role in increasing and maintaining uterine blood flow in late pregnancy. We believe that the information obtained in this revised application will provide new and important understanding into the mechanisms regulating vascular tone and hemodynamics in the uterine circulation in both the nonpregnant and pregnant animal.