Kathy Cockrell

Role of Endothelin in Mediating Tumor Necrosis Factor-Induced Hypertension in Pregnant Rats

Hypertension during preeclampsia is associated with an increase in plasma levels of tumor necrosis factor (TNF)-α, a cytokine known to contribute to endothelial dysfunction. Recently, our laboratory reported that a 2-fold increase in plasma TNF-α produces hypertension in pregnant rats. Endothelin is also elevated in preeclampsia and endothelin synthesis is enhanced by TNF-α. The purpose of this study was to determine the role of endothlelin in mediating TNF-α–induced hypertension in pregnant rats. To achieve this goal, TNF-α (50 ng/d for 5 days) was infused into control pregnant rats and pregnant rats treated with an endothelin receptor A antagonist, ABT 627 (5 mg/kg per day for 5 days). At day 19 of gestation, arterial pressure was measured and aorta, kidneys, and placentas were harvested. Infusion of TNF-α into pregnant rats increased plasma concentration of TNF-α (13.5±0.8 to 28.0±3.7 pg/mL) and arterial pressure (101±2 to 122±1 mm Hg). The increase in arterial pressure was associated with an increase in preproendothelin mRNA expression in placenta, aorta, and kidneys measured by real-time polymerase chain reaction (PCR). Pretreatment with the endothelin receptor A antagonist completely abolished the blood pressure response to TNF-α in pregnant rats (105±1 versus 97±2 mm Hg). In sharp contrast, the ETA receptor antagonist had no effect on arterial pressure in normal pregnant rats (97±2 versus 101±2 mm Hg). Moreover, chronic infusion of TNF-α had no significant effect on arterial pressure or renal preproendothelin levels in virgin rats. These results suggest an important role for endothelin in mediating TNF-α–induced hypertension in pregnant rats.

Hypertension Produced by Reductions in Uterine Perfusion in the Pregnant Rat: Role of Tumor Necrosis Factor-α

Inflammatory cytokines such as tumor necrosis factor (TNF)-&agr; are elevated in preeclamptic women and are thought to be an important link between placental ischemia and endothelial dysfunction. The purpose of this study was to determine the role of TNF in mediating hypertension in response to chronic reductions in uterine perfusion (RUPPs) in pregnant rats. Arterial pressure was significantly higher in RUPP rats (138±1 mm Hg) than in pregnant rats (107±1 mm Hg). Serum TNF-&agr; levels in the RUPP rats were 17±4 pg/mL compared with 8±1 pg/mL in normal pregnant rats. To determine the long-term effects of a 2- to 3-fold elevation in plasma TNF-&agr; on renal and systemic hemodynamics in pregnant rats, we infused TNF-&agr; for 5 days at a rate of 50 ng/d during days 14 to 19 of gestation in pregnant rats. Serum levels were 7±2 pg/mL in the control pregnant rats and 14±2 pg/mL in the TNF-&agr;–treated pregnant rats. Mean arterial pressure was higher in the TNF-&agr;–treated pregnant rats (123±3 mm Hg) compared with pregnant controls (96±3 mm Hg) at day 19 of gestation. TNF-&agr; increased renal vascular resistance in pregnant rats by 182%. Renal plasma flow was 5.4±1.2 mL/min in the TNF-&agr;–treated group and 9.2±1.6 mL/min in the control group. Glomerular filtration rate was 1.7±0.4 mL/min in the TNF-&agr;–treated group and 2.6±0.4 mL/min in the control group. In summary, these data suggest that TNF-&agr; may play an important role in mediating the increased arterial pressure in response to chronic RUPPs in pregnant rats.

Hypertension in Response to Autoantibodies to the Angiotensin II Type I Receptor (AT1-AA) in Pregnant Rats: Role of Endothelin-1

Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) and endothelin -1 (ET-1) are suggested to be important links between placental ischemia and hypertension during preeclampsia. Activation of the angiotensin II type 1 receptor (AT1R) increases endothelial cell production of ET-1; however, the importance of ET-1 in response to AT1-AA–mediated AT1 R activation during preeclampsia is unknown. Furthermore, the role of AT1-AA–mediated increases in blood pressure during pregnancy remains unclear. The objective of this study was to test the hypothesis that AT1-AA, increased to levels observed in preeclamptic women and placental ischemic rats, increases mean arterial pressure (MAP) by activation of the ET-1 system. Chronic infusion of purified rat AT1-AA into normal pregnant (NP) rats for 7 days increased AT1-AA from 0.68±0.5 to 10.88±1.1 chronotropic units (P<0.001). The increased AT1-AA increased MAP from 99±1 to 119±2 mm Hg (P<0.001). The hypertension was associated with significant increases in renal cortices (11-fold) and placental (4-fold) ET-1. To determine whether ET-1 mediates AT1-AA–induced hypertension, pregnant rats infused with AT1-AA and NP rats were treated with an ETA receptor antagonist. MAP was 100±1 mm Hg in AT1-AA+ETA antagonist-treated rats versus 98±2 mm Hg in ETA antagonist-treated rats. Collectively, these data support the hypothesis that one potential pathway whereby AT1-AAs increase blood pressure during pregnancy is by an ET-1–dependent mechanism.

Tumor necrosis factor–α–induced hypertension in pregnant rats results in decreased renal neuronal nitric oxide synthase expression

BACKGROUND Preeclampsia is associated with increases in plasma levels of tumor necrosis factor-alpha (TNF-alpha), a cytokine known to contribute to endothelial dysfunction. We recently reported that a twofold elevation in plasma TNF-alpha produces significant reductions in renal function and hypertension in pregnant rats. The purpose of this study was to determine the role of the nitric oxide (NO) system in TNF-alpha-induced hypertension in pregnant rats. METHODS Tumor necrosis factor-alpha (50 ng/day) was chronically infused starting at day 14 of gestation. Mean arterial pressure, 24-h urinary nitrite/nitrate excretion, and renal nitric oxide synthase (NOS) protein expression by Western blot analysis was measured at day 19 of gestation. RESULTS A twofold increase in plasma TNF-alpha levels in pregnant rats resulted in a significant increase in arterial pressure (97 +/- 3.6 v 116 +/- 2.1 mm Hg, pregnant versus TNF-alpha pregnant, respectively, P < .05), but no significant change in urinary nitrite/nitrate excretion (22.0 +/- 1.9 v 20.8 +/- 2.5 micromol/24 h, pregnant versus TNF-alpha pregnant, respectively), a measure of whole body NO production. As abnormalities in renal production of NO would not be reflected in the measure of whole body NO production, changes in renal NOS protein levels were determined. The protein expression of both neuronal (nNOS) and inducible (iNOS) nitric oxide synthase were significantly decreased in the medulla of TNF-alpha pregnant rats (nNOS: 10.6 +/- 0.7 v 8.2 +/- 0.8 densitometric units, P < .05; and iNOS: 19.2 +/- 0.9 v 15.4 +/- 0.8 densitometric units, P < .05, pregnant versus TNF-alpha pregnant, respectively). CONCLUSION The hypertension associated with a chronic twofold increase in TNF-alpha in pregnant rats is associated with significant decreases in renal nNOS and iNOS protein production.

Reduced uterine perfusion pressure (RUPP) model for studying cardiovascular-renal dysfunction in response to placental ischemia.

Despite being one of the leading causes of maternal death and a major contributor of maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are unknown. The initiating event in preeclampsia has been postulated to be reduced uteroplacental perfusion. Placental ischemia/hypoxia is thought to lead to widespread activation/dysfunction of the maternal vascular endothelium, vasoconstriction and hypertension. Experimental induction of chronic uteroplacental ischemia appears to be the most promising animal model to study potential mechanisms of preeclampsia since reductions in uteroplacental blood flow in a variety of animal models lead to a hypertensive state that closely resembles preeclampsia in women. This chapter details the methods we use in our laboratory to produce the reduced uterine perfusion pressure (RUPP) model in the pregnant rat.

Endothelin Type A Receptor Blockade Attenuates the Hypertension in Response to Chronic Reductions in Uterine Perfusion Pressure

A chronic reduction in uterine perfusion pressure in pregnant rats is associated with a significant elevation in mean arterial pressure (MAP) and reduction in kidney function. The purpose of this study was to examine the role of endothelin in mediating the hypertension in response to chronic reductions in uterine perfusion pressure in conscious, chronically instrumented, pregnant rats. MAP in pregnant rats with chronic reductions in uterine perfusion pressure (123.0±1.8 mm Hg) was significantly higher than that in control pregnant rats (101.3±4.0 mm Hg). Renal expression of preproendothelin mRNA as determined by ribonuclease protection assay was also significantly elevated in the medulla (>45%, P <0.05) and in the cortex (>22%, P <0.05) of the pregnant rats with chronic reductions in uterine perfusion pressure compared with control pregnant rats. Chronic administration of the selective endothelin type A receptor antagonist (ABT-627, 5 mg/kg per day for 10 days) markedly attenuated the increase in MAP observed in the pregnant rats with chronic reductions in uterine perfusion pressure (103.3±5.6 mm Hg, plus endothelin antagonist;P <0.05). However, endothelin type A receptor blockade had no significant effect on blood pressure in the normal pregnant animals (96.0±2.7 mm Hg, plus endothelin antagonist). These findings suggest that endothelin plays a major role in mediating the hypertension produced by chronic reductions in uterine perfusion pressure in pregnant rats.

Role of Endothelin in Mediating Soluble fms-Like Tyrosine Kinase 1–Induced Hypertension in Pregnant Rats

Although soluble fms-like tyrosine kinase 1 (sFlt-1), an antagonist of vascular endothelial growth factor and placental growth factor, has been implicated in the pathogenesis of hypertension during preeclampsia, the mechanisms whereby enhanced sFlt-1 production leads to hypertension remain unclear. Both sFlt-1 and endothelin 1 productions are elevated in women with preeclampsia and in placental ischemic animal models of preeclampsia; however, the importance of endothelin 1 and sFlt-1 interactions in the control of blood pressure during pregnancy is unknown. The purpose of this study was to determine the role of endothelin 1 in mediating sFlt-1–induced hypertension in pregnant rats. To achieve this goal, sFlt-1 (3.7 &mgr;g/kg per day for 6 days) was infused into normal pregnant rats and pregnant rats treated with a selective endothelin type A receptor antagonist, ABT 627 (5 mg/kg per day for 6 days). Plasma concentration of sFlt-1 increased from 735±34 pg/mL in normal pregnant rats to 2498±645 pg/mL (P<0.05) with infusion of sFlt-1. Arterial pressure increased from 100±1 mm Hg in normal pregnant rats to 122±3 mm Hg (P<0.05) in sFlt-1–infused rats. Chronic increases in plasma sFlt-1 in normal pregnant rats increased preproendothelin mRNA expression in the renal cortices by ≈3-fold. In addition, chronic endothelin type A receptor blockade completely abolished the blood pressure response to sFlt-1 in pregnant rats (104±3 versus 100±1 mm Hg; P<0.05), whereas the endothelin A receptor antagonist had no effect on arterial pressure in NP rats (105±2 versus 100±1 mm Hg). In conclusion, this study demonstrates that endothelin 1, via endothelin type A receptor activation, plays an important role in mediating the hypertension in response to excess sFlt-1 during pregnancy.

Hypertension in Response to Chronic Reductions in Uterine Perfusion in Pregnant Rats: Effect of Tumor Necrosis Factor-α Blockade

Reductions in uterine perfusion pressure (RUPP) in pregnant rats is associated with increased tumor necrosis factor-&agr; (TNF-&agr;). This study was designed to determine the role of endogenous TNF-&agr; in mediating changes in arterial pressure and endothelin-1 (ET-1) in RUPP rats. To achieve this goal we examined the effect of RUPP in the presence and absence of a TNF-&agr;–soluble receptor, etanerecept (0.4 mg/kg). Mean arterial pressure increased from 102±1 mm Hg in normal pregnant (NP) rats to 134±3 mm Hg (P<0.05) in RUPP rats. Serum TNF-&agr; increased to 40±7.6 pg/mL in RUPP rats (n=24) versus 14.8±3.3 pg/mL (n=16; P<0.05) in NP rats. Administration of etanerecept decreased TNF-&agr; in RUPP rats (n=20) to 17.2±3 pg/mL and mean arterial pressure to 118±2 mm Hg (P<0.05). Tissue ET-1 decreased in etanerecept-treated RUPP rats compared with control RUPP rats. The direct effect of TNF-&agr; blockade on endothelial activation in response to placental ischemia was examined in human umbilical vein endothelial cells. ET-1 secreted from human umbilical vein endothelial cells treated with RUPP serum was 59.2+16 pg/mg and decreased when etanerecept was added to the medium with RUPP serum (7.60±0.77 pg/mg), as well as in response to serum from etanerecept-treated RUPP rats (7.30±0.55 pg/mg; P<0.001). ET-1 secreted from human umbilical vein endothelial cells was 15.6±2 pg/mg when treated with NP serum. These data support the hypothesis that endogenous TNF-&agr; is an important stimulus for ET-1 in response to placental ischemia and is important in mediating endothelial cell activation and hypertension during pregnancy.

Induction of Heme Oxygenase 1 Attenuates Placental Ischemia–Induced Hypertension

Recent in vitro studies have reported that heme oxygenase 1 (HO-1) downregulates the angiostatic protein soluble fms-like tyrosine kinase 1 from placental villous explants and that the HO-1 metabolites CO and bilirubin negatively regulate endothelin 1 and reactive oxygen species. Although soluble fms-like tyrosine kinase 1, endothelin 1, and reactive oxygen species have been implicated in the pathophysiology of hypertension during preeclampsia and in response to placental ischemia in pregnant rats, it is unknown whether chronic induction of HO-1 alters the hypertensive response to placental ischemia. The present study examined the hypothesis that HO-1 induction in a rat model of placental ischemia would beneficially affect blood pressure, angiogenic balance, superoxide, and endothelin 1 production in the ischemic placenta. To achieve this goal we examined the effects of cobalt protoporphyrin, an HO-1 inducer, in the reduced uterine perfusion pressure (RUPP) placental ischemia model and in normal pregnant rats. In response to RUPP treatment, mean arterial pressure increases 29 mm Hg (136±7 versus 106±5 mm Hg), which is significantly attenuated by cobalt protoporphyrin (118±5 mm Hg). Although RUPP treatment causes placental soluble fms-like tyrosine kinase 1/vascular endothelial growth factor ratios to alter significantly to an angiostatic balance (1.00±0.10 versus 1.27±0.20), treatment with cobalt protoporphyrin causes a significant shift in the ratio to an angiogenic balance (0.68±0.10). Placental superoxide increased in RUPP (952.5±278.8 versus 243.9±70.5 relative light units/min per milligram) but was significantly attenuated by HO-1 induction (482.7±117.4 relative light units/min per milligram). Also, the preproendothelin message was significantly increased in RUPP, which was prevented by cobalt protoporphyrin. These data indicate that HO-1, or its metabolites, is a potential therapeutic for the treatment of preeclampsia.

Enhanced Endothelin Synthesis by Endothelial Cells Exposed to Sera From Pregnant Rats With Decreased Uterine Perfusion

The initiating event in preeclampsia is thought be to reduced uteroplacental perfusion. Although we have reported previously that chronic reductions in uterine perfusion pressure (RUPP) in pregnant rats results in hypertension and enhanced endothelin production, the factors linking placental ischemia and endothelial cell activation remain unclear. The purpose of this study was to determine the role of angiotensin II type-1 (AT1) receptor activation on endothelin production induced by serum from pregnant rats exposed to reductions in uterine perfusion. To achieve this goal, human umbilical vein endothelial cells were exposed to sera collected from RUPP rats or normal pregnant rats. Arterial pressure was significantly higher in RUPP rats (135±2 mm Hg) than in pregnant rats (106±1 mm Hg). Six hours after exposure to RUPP serum (n=17), cell media endothelin concentration was 18.4±2.7 pg/mL as compared with 9.22±1.3 pg/mL from cells exposed to serum from normal pregnant rats (n=9). Eighteen hours after exposure to RUPP serum (n=7), endothelin concentration was 30.5±3.8 pg/mL as compared with 12.8±5.3 pg/mL from cells exposed to normal pregnant rat serum (n=6). In contrast, serum from RUPP rats did not increase endothelin production in human umbilical vein endothelial cells pretreated with an AT1 receptor antagonist, losartan (15 &mgr;mol/L). Eighteen hours after exposure to RUPP serum and losartan (n=14), endothelin concentration was 21.3±2.2 pg/mL as compared with 16.4±3.3 pg/mL from cells exposed to normal pregnant rat serum and losartan (n=10). These data indicate that serum from pregnant rats exposed to reductions in uterine perfusion enhances endothelin production by endothelial cells via by AT1 receptor activation.