Hypertensive Pregnant Rats Research Articles

Prostaglandins, but not sympathetic nervous system, modulate Renin/Prorrenin receptor expression during normal pregnancy and pregnancy‐induced hypertension in rats

Pregnancy is associated with blunted response to contractile agents as angiotensin II (Ang II), catecholamines and prostaglandins (PG's). In contrast, their responses are increased in pregnancy‐induced hypertension (PIH). The renin‐angiotensin system (RAS) has become more complex with new components with cardiovascular functions. Renin/Prorenin receptor (RPR) is one of these components. The aim of this work was to elucidate if sympathetic nervous system (SNS) and/or PG's modulate RPR expression in normal and pregnancy‐induced hypertensive pregnant rats. We used a subrenal aorctic coarctation (SRAC) model. Using immunoblot, RT‐PCR and immunohistochemistry, we determined RPR expression in aorta and kidney from normal and PIH animals. Similar groups were treated with Prazosine (P, 2mg/Kg), Metoprolol (M, 5 mg/Kg), Indomethacine (I, 3 mg/Kg) or Celecoxib (C, 10 mg/Kg) during the third week of pregnancy. Neither P, M nor Cox inhibitors altered blood pressure. In normal pregnancy, RPR increased its expression significantly. RPR expression during PIH decreased even lower than control levels. Adrenergic blockers did not affect RPR expression significantly. I and C, in contrast, normalized RPR expression during non‐complicated pregnancy and increased it to higher levels during PIH. These results suggest that PG's, but no SNS, modulates RPR expression. Supported by SIP‐IPN 20120122 and 20120929 grants

Necrotic trophoblast debris increases blood pressure during pregnancy

Preeclampsia is a major disease of human pregnancy characterised by hypertension and proteinuria. These signs are preceded by systemic maternal endothelial dysfunction. Hypertension in preeclampsia appears to be triggered by a placental factor, which leads to endothelial activation/dysfunction. One potential placental trigger for preeclampsia is necrotic trophoblast cell debris shed from the placenta into maternal blood. The larger trophoblast debris is trapped in the maternal pulmonary vessels and is hypothesised to be cleared by endothelial cells. Phagocytosis of necrotic but not apoptotic trophoblast debris by endothelial cells leads to their activation in vitro. We hypothesised that intravenous injection of necrotic trophoblast debris would induce hypertension in pregnant rats. Virgin female Wistar rats were surgically implanted with telemetry devices to monitor arterial blood pressure and chronic intravenous catheters to allow delivery of necrotic trophoblast debris. After recovery, the rats were mated and, from day 6 of gestation until parturition, they were given five consecutive daily injections per week of 5×106 necrotic Jeg-3 cells per kilo bodyweight. Control rats received vehicle injections. The normalised mean arterial blood pressure of rats receiving injections of necrotic trophoblast debris was higher than control rats during the third week of gestation, while mean arterial blood pressure decreased less from the pre-pregnancy baseline compared to control rats. These results suggest that necrotic trophoblast debris has a hypertensive effect which manifests in late gestation in Wistar rats and supports the theory that necrotic trophoblast debris may trigger the symptoms of preeclampsia.

Endothelial dysfunction in gestational hypertension induced by catechol‐O‐methyltransferase inhibition

The present study evaluated whether catechol-O-methyltransferase inhibition in pregnant rats results in increased blood pressure and vascular endothelial dysfunction as a consequence of decreased nitric oxide bioavailability. Pregnant Sprague-Dawley rats were given entacapone (a catechol-O-methyltransferase inhibitor) by gavage from the 10th to the 20th day of pregnancy. Blood pressure was measured by plethysmography in the tail artery. Vascular endothelial function and NO release were assessed both in the absence and in the presence of tempol. Systolic blood pressure increased significantly in pregnant rats treated with entacapone compared with untreated pregnant rats on days 14 (143 ± 4 versus 122 ± 3 mmHg) and 19 of gestation (129 ± 4 versus 115 ± 5 mmHg). Both conductance (aortic rings) and resistance vessels (mesenteric small arterial vessels) from entacapone-treated pregnant rats showed diminished relaxation in response to acetylcholine compared with vessels from vehicle-treated pregnant and virgin rats. In mesenteric arterioles, this endothelial dysfunction was abolished in the presence of l-NAME, indicating that it was caused by reduced NO availability, and it also improved in the presence of tempol, suggesting increased oxidative stress in hypertensive pregnant rats. Endothelial release of nitric oxide induced by calcium ionophore (A23187) was significantly greater in aortas from vehicle-treated pregnant rats than in aortas from pregnant rats given entacapone. This endothelial dysfunction seen in hypertensive rats was prevented by addition of tempol. The present study provides evidence that catechol-O-methyltransferase inhibition in pregnant rats produces arterial hypertension and endothelial dysfunction due to reduced nitric oxide bioavailability.

Sildenafil Administration Attenuates Placental Ischemia and sFlt‐1 Induced Hypertension in Pregnant Rats

Preeclampsia is a complication of pregnancy which is marked by hypertension, proteinuria, and maternal endothelial dysfunction. A central factor in the etiology of the disease is the development of placental hypoxia/ischemia, which results in release of pathogenic soluble factors. There is currently no effective treatment for preeclampsia, but one proposed therapeutic agent is the phosphodiesterase‐5 (PDE‐5) inhibitor sildenafil, as PDE‐5 is enriched in the uterus, and its antagonism could improve uteroplacental perfusion. Here we report in two rodent models that administration of oral sildenafil is effective in attenuating hypertension during gestation. In the RUPP placental ischemia model, arterial pressure is increased significantly when compared to control animals (132±3 vs 100±2 mmHg, p<0.05). Administration of sildenafil (45mg/kg/day) in drinking water had no effect on blood pressure in control rats, but decreased pressure in RUPP rats (115±1mmHg, p<0.05). Similarly, in the sFlt‐1 infusion model of hypertension, pressure is increased significantly over control rats (116±2 vs 99±2mmHg, p<0.05). In these rats sildenafil administration essentially normalized blood pressure (103±2, p<0.05 vs sFlt‐1 infused alone). These data suggest that administration of sildenafil might prove an effective therapeutic in the management of hypertension during preeclampsia.

Hypertension and Pregnancy are accompanied by differential changes in GABAA and NMDA receptor subunit expression in the hypothalamic Paraventricular Nucleus

Sympathoexcitation is characteristic of cardiovascular disease and pregnancy. Sympathoexcitation emanates, in part, from the hypothalamic paraventricular nucleus (PVN). Hypertensive rats show that the normal balance of GABAergic inhibition and glutamatergic excitation is altered. We hypothesise that this alteration is caused by changes in the subunit composition of the GABAA and NMDA receptors in the PVN. Brains from female Wistars, female spontaneously hypertensive rats (SHR) and pregnant Wistar rats (E19) were subjected to either immunoblotting of PVN micropunches or immunohistochemistry for NMDA and GABAA receptor subunits. Expression of NMDA subunits GluN1, ‐2A and ‐2B was found in all subnuclei of the PVN. A significant increase in expression of the GluN2A subunit was observed in the PVN of SHR (P<0.05 n=3). Furthermore, a significant reduction in GABAA α1 was observed in the PVN of SHR and pregnant animals (P<0.05, n=3). A significant reduction in expression of the GABAA α5 subunits was observed in the PVN of SHR animals (P<0.001 n=3).We hypothesise that the sympathoexcitation observed in pregnancy is driven by a withdrawal in phasic GABAergic inhibition. Conversely, we believe that in SHR, the sympathoexcitation is driven by a decrease in both phasic (α1) and tonic (α5) GABAA‐mediated inhibition, as well as an increase in NMDA‐driven excitation.Work supported by the BBSRC

774: AT1-AA enhances renal sensitivity and blood pressure in response to ANGII during pregnancy

pressure in response to ANGII during pregnancy Justin Brewer, Ruisheng Liu, Kedra Wallace, Florian Herse, Janae Moseley, Ralf Dechend, Gerd Walluk, James Martin, Babbette LaMarca University of Mississippi Medical Center, Maternal Fetal Medicine, Jackson, MS, University of Mississippi Medical Center, Department of Physiology, Jackson, MS, University of Mississippi Medical Center, Obstetrics and Gynecology, Jackson, MS, HELIOS Clinic, Max-DelbruckCenter for Molecular Medicine, Berlin, Germany OBJECTIVE: Agonistic autoantibodies to the angiotensin II (ANGII) type I receptor (AT1-AA) are proposed to be important links between hypertension and increased maternal vascular sensitivity to ANGII during preeclampsia. This study was designed to determine effects of AT1-AA to enhance blood pressure (MAP) and renal vascular sensitivity to ANGII. STUDY DESIGN: First, we examined acute administration of ANGII on MAP in AT1-AA induced hypertensive pregnant rats. ANGII (100ng/ kg/min) was administered via jugular infusion to normal pregnant (NP) (n 6) controls and AT1-AA (1:50, n 10) infused pregnant rats. We examined the direct effects of ANGII on renal vasoconstriction in the presence and absence of the AT1-AA on isolated afferent arterioles (Af-Art) from NP rats. A dose response of increased constriction of the Af-Art with ANGII alone (10-11-10-8) or increasing AT1-AA alone (1:200, 1:150, and 1:100) was established. We subsequently measured Af-Art constriction to ANGII (10-11, 10-10, 10-9) with nonconstrictor dose of AT1-AA (1:200). Second, we examined chronic ANGII on MAP and renal artery resistive index (RI) in AT1-AA induced hypertensive pregnant rats. Power Doppler velocimetry was performed, bilaterally at two levels, using Vevo 770 unit with 30 Hz transducer and insonating angle 30. Statistical analysis was performed using STATA. RESULTS: MAP increased 30mmHg with acute ANGII in NP rats but markedly increased 47mmHg with ANGII in AT1-AA infused rats. Increasing ANGII from (10-11-10-8) decreased Af-Art diameter 1520% but sharply decreased Af-Art diameter 80% in AT1-AA pretreated vessels. MAP was 95 / 1 mmHg in NP and increased to 116 / 3 mmHg with chronic ANGII, to 120 / 3 mmHg with AT1-AA and was 112 / 3 mmHg, with ANGII AT1-AA (P 0.05). RI in the renal arteries was 0.67 in NP, 0.70 in AT1-AA infused pregnant rats, and was 0.74 (P 0.002) in ANGII AT1-AA pregnant rats. CONCLUSION: These data support the hypothesis that AT1-AA may be an important mechanism whereby MAP and renal vascular responses are greatly enhanced during preeclampsia.

L-Arginine supplementation abolishes the blood pressure and endothelin response to chronic increases in plasma sFlt-1 in pregnant rats

While soluble fms-like tyrosine kinase-1 (sFlt-1) and endothelin-1 (ET-1) have been implicated in the pathogenesis of preeclampsia (PE), the mechanisms whereby increased sFlt-1 leads to enhanced ET-1 production and hypertension remain unclear. It is well documented that nitric oxide (NO) production is reduced in PE; however, whether a reduction in NO synthesis plays a role in increasing ET-1 and blood pressure in response to chronic increases in plasma sFlt-1 remains unclear. The purpose of this study was to determine the role of reduced NO synthesis in the increase in blood pressure and ET-1 in response to sFlt-1 in pregnant rats. sFlt-1 was infused into normal pregnant (NP) Sprague-Dawley rats (3.7 μg·kg(-1)·day(-1) for 6 days beginning on day 13 of gestation) treated with the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 mg/l for 4 days) or supplemented with 2% L-Arg (in drinking water for 6 days beginning on day 15 of gestation). Infusion of sFlt-1 into NP rats significantly elevated mean arterial pressure compared with control NP rats: 116 ± 2 vs. 103 ± 1 mmHg (P < 0.05). NO synthase inhibition had no effect on the blood pressure response in sFlt-1 hypertensive pregnant rats (121 ± 3 vs. 116 ± 2 mmHg), while it significantly increased mean arterial pressure in NP rats (128 ± 4 mmHg, P < 0.05). In addition, NO production was reduced ∼70% in isolated glomeruli from sFlt-1 hypertensive pregnant rats compared with NP rats (P < 0.05). Furthermore, prepro-ET-1 in the renal cortex was increased ∼3.5-fold in sFlt-1 hypertensive pregnant rats compared with NP rats. Supplementation with L-Arg decreased the sFlt-1 hypertension (109 ± 3 mmHg, P < 0.05) but had no effect on the blood pressure response in NP rats (109 ± 3 mmHg) and abolished the enhanced sFlt-1-induced renal cortical prepro-ET expression. In conclusion, a reduction in NO synthesis may play an important role in the enhanced ET-1 production in response to sFlt-1 hypertension in pregnant rats.

Induction of heme oxygenase-1 attenuates sFlt-1-induced hypertension in pregnant rats

Preeclampsia (PE) is one of the leading causes of fetal and maternal morbidity, affecting 5-10% of all pregnancies, and lacks an effective treatment. The exact etiology of the disorder is unclear, but placental ischemia has been shown to be a central causative agent. In response to placental ischemia, the antiangiogenic protein fms-like tyrosine kinase-1 (sFlt-1), a VEGF antagonist, and reactive oxygen species are secreted, leading to the maternal syndrome. One promising therapeutic approach to treat PE is through manipulation of the heme oxygenase-1 (HO-1) protein. It has been previously reported that HO-1 and carbon monoxide downregulate sFlt-1 production in vitro, and we have recently shown that HO-1 induction significantly attenuates placental ischemia-induced hypertension, partially through normalization of the sFlt-1-to-VEGF ratio in the placenta. The purpose of this study was to determine whether HO-1 induction would have beneficial effects independently of sFlt-1 suppression. To that end, pregnant rats were continuously infused with recombinant sFlt-1 from gestational days 14-19, and circulating sFlt-1 increased approximately twofold, similar to rats with experimentally induced placental ischemia. In response, mean arterial pressure increased 17 mmHg, which was completely normalized by HO-1 induction. Unbound circulating VEGF was decreased ∼17% in response to sFlt-1 infusion but was increased ∼50% in response to HO-1 induction. Finally, endothelial function was improved as measured by reductions in vascular expression of preproendothelin mRNA. In conclusion, manipulation of HO-1 presents an intriguing therapeutic approach to the treatment of PE.

Hypertension in Response to AT1-AA: Role of Reactive Oxygen Species in Pregnancy-Induced Hypertension

Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) and reactive oxygen species (ROS) are implicated in the pathophysiology of preeclampsia. The objective of this study was to determine the role of AT1-AA to stimulate placental oxidative stress in vivo and role ROS in mediating hypertension in response to AT1-AA during pregnancy. To achieve these goals, blood pressure (mean arterial pressure (MAP)) and ROS were analyzed in AT1-AA-induced hypertensive pregnant rats in the presence and absence of a superoxide dismutase mimetic, tempol. Rat AT1-AA (1:50) and tempol (30 mg/kg/day) were administered to pregnant rats beginning on day 12 of gestation. On day 19, MAP was analyzed and tissues collected for ROS analysis via lucigenin chemiluminescence. MAP increased from 101 ± 2 normal pregnant (NP) rats to 116 ± 2 mm Hg in chronic AT1-AA infused rats (P = 0.002). Placental basal and NADPH oxidase stimulated ROS was 29 ± 6 and 92 ± 10 relative light units (RLUs) in NP rats. These levels increased to 159 ± 29 (P < 0.0001) and 287 ± 60 RLUs (P < 0.006) in AT1-AA infused rats. MAP in AT1-AA + tempol rats was 109 ± 2 mm Hg, no difference than tempol-treated controls (109 ± 3 mm Hg). Administration of tempol decreased basal and NADPH-stimulated placental ROS in AT1-AA-treated rats (121 ± 13; 262 ± 21 RLUs). Basal and NADPH-stimulated ROS in tempol-treated controls were 69 ± 24; 141 ± 33 RLUs. This study indicates that AT1-AA's contribute to placental oxidative stress; one mechanism whereby the AT1-AA mediates hypertension during pregnancy.

The Effect of Immune Factors, Tumor Necrosis Factor- , and Agonistic Autoantibodies to the Angiotensin II Type I Receptor on Soluble fms-Like Tyrosine-1 and Soluble Endoglin Production in Response to Hypertension During Pregnancy

Preeclampsia is considered a disease of immunological origin associated with abnormalities in inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and activated lymphocytes secreting autoantibodies to the angiotensin II receptor (AT1-AA). Recent studies have also demonstrated that an imbalance of angiogenic factors, soluble fms-like tyrosine kinase (sFlt-1), and sEndoglin, exists in preeclampsia; however, the mechanisms that initiate their overproduction are unclear. To determine the role of immune regulation of these factors, circulating and placental sFlt-1 and/or sEndoglin was examined from pregnant rats chronically treated with TNF-alpha or AT1-AA. On day 19 of gestation blood pressure was analyzed and serum and tissues were collected. Placental villous explants were excised and cultured on matrigel coated inserts for 24 h and sFlt-1 and sEndoglin was measured from media. In response to TNF-alpha-induced hypertension, sFlt-1 increased from 180 +/- 5 to 2,907 +/- 412 pg/ml. sFlt-1 was also increased from cultured placental explants of TNF-alpha induced hypertensive pregnant rats (n = 12) (2,544 +/- 1,132 pg/ml) vs. explants from normal pregnant (NP) rats (n = 12) (2,189 +/- 586 pg/ml) where as sEng was undetectable. Circulating sFlt-1 increased from 245 +/- 38 to 3,920 +/- 798 pg/ml in response to AT1-AA induced hypertension. sFlt-1 levels were higher (3,400 +/- 350 vs. 2,480 +/- 900 pg/ml) in placental explants from AT1-AA infused rats (n = 12) than NP rats (n = 7). In addition, sEndoglin increased from 30 +/- 2.7 to 44 +/- 3.3 pg/ml (P < 0.047) in AT1-AA infused rats but was undetectable in the media of the placental explants. These data suggest that immune factors may serve as an important stimulus for both sFlt-1 and sEndoglin production in response to placental ischemia.

L‐Arginine supplementation abolishes the blood pressure and endothelin response to chronic increases in plasma sFlt‐1 in pregnant rats

While soluble fms‐like tyrosine kinase (sFlt‐1) and endothelin (ET‐1) have been implicated in the pathogenesis of preeclampsia (PE), the mechanisms whereby increased sFlt‐1 leads to enhanced ET‐1 production and hypertension remain unclear. NO production is reduced in PE, however, whether a reduction in NO synthesis plays a role in increasing ET‐1 and blood pressure in response to chronic increases in plasma sFlt‐1 remains unclear. The purpose of this study was to determine the role of reduced NO synthesis is mediating the increase in blood pressure and ET‐1 in response to sFlt‐1 in pregnant rats. To achieve this goal, sFlt‐1 was infused into NP rats (3.7μg/kg/d for 6 days) and pregnant rats supplemented with 2% L‐arginine (in drinking water for 6 days). Infusion of sFlt‐1 into NP rats significantly elevated MAP (117±2 mmHg, p&lt;0.05) versus NP (103±1 mmHg). sFlt‐1 hypertensive pregnant rats had ~3.5 fold increase in preproendothelin‐1 (PPET‐1) in the renal cortex when compared to NP rats. Supplementation with L‐arginine decreased the sFlt‐1 hypertension (108±3 mmHg, p&lt;0.05) while having no effect on the blood pressure response in NP rats (108±2 mmHg), and abolished the enhanced sFlt‐1 induced PPET‐1 expression. In conclusion, a reduction in nitric oxide synthesis may play an important role in the enhanced ET‐1 production in response to sFlt‐1 hypertension in pregnant rats. NIH grant HL51971

Hypertension in response to agonistic autoantibodies to the angiotensin II type I receptor (AT1‐AA): role of reactive oxygen species (ROS)

AT1‐AA and ROS are implicated in hypertension in preeclamptic women. Administration of purified rat AT1‐AA significantly increases mean arterial pressure (MAP) and placental ROS in normal pregnant rats. The objective of this study was to determine the role of ROS in mediating AT1‐AA induced hypertension during pregnancy.To achieve this goal, MAP and ROS were analyzed in AT1‐AA induced hypertensive pregnant rats in the presence and absence of a superoxide dismutase mimetic, Tempol. Rat AT1‐AA (1:50) was infused intraperitoneally via osmotic minipumps and Tempol (30mg/kg/day)was delivered in drinking water of pregnant rats beginning on day 12 of gestation. On day 19 MAP was analyzed and placentas were collected for ROS analysis via lucigenin chemiluminescence technique.MAP was 101 + 2 in NP rats and 116 + 2 mmHg AT1‐AA rats (p=0.002). Placental basal and NADPH oxidase stimulated ROS was 29 +/− 6 and 92 +/− 10 RLUs in NP rats. These levels increased to 159 +/− 29 (P&lt;0.01) and 287 +/− 60 RLUs (P&lt;0.001) in AT1‐AA rats. MAP in AT1‐AA + Tempol rats was 109 +/−3 vs 109+/−2 mmHg in Tempol treated controls. Administration of Tempol decreased ROS in AT1‐AA treated rats (121+/−13; 262 +/−21 RLUs) compared to Tempol treated controls (69 +/−24; 141 +/− 33 RLUs)AT1‐AA induced hypertension is associated with placental placenta oxidative stress and is attenuated with administration of an SOD mimetic

Interleukin-10 reduces inflammation, endothelial dysfunction, and blood pressure in hypertensive pregnant rats

Hypertensive disorders of pregnancy are characterized by systemic and placental inflammation; however, treatment for these conditions has remained elusive. We tested whether administration of the anti-inflammatory cytokine interleukin-10 (IL-10) during pregnancy would attenuate the hypertension, endothelial dysfunction, proteinuria, and inflammation seen in pregnant DOCA/saline-treated (PDS) rats. Normal pregnant (NP) rats and PDS were given daily intraperitoneal injections of recombinant IL-10 from gestational day 13 until death on day 20. Systolic blood pressure, aortic endothelium-dependent relaxation responses, and urinary protein excretion were measured on days 13 and 20 of gestation. Fetal number and development, plasma endothelin-1 levels, serum and placental levels of IFNgamma and IL-10, and aortic and placental levels of platelet endothelial cell adhesion molecule (PECAM) were assessed on gestational day 20. Systolic blood pressure, aortic endothelial dysfunction, and urinary protein excretion were significantly increased at gestational day 13 in PDS rats. However, all of these were restored to NP levels following IL-10 treatment in PDS rats. IL-10 treatment also significantly increased the number of pups per litter in PDS rats and did not further affect fetal development. The beneficial effects of IL-10 in PDS rats were likely mediated by the decreased plasma levels of endothelin-1, decreased levels of circulating and placental IFNgamma, as well as decreased aortic and placental expression of PECAM. These data demonstrate that exogenous IL-10 can normalize blood pressure and endothelial function in pregnancy-induced hypertensive rats and may be beneficial in women with hypertensive disorders of pregnancy.

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 microg/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 approximately 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.

Immunosuppression Improves Blood Pressure and Endothelial Function in a Rat Model of Pregnancy-Induced Hypertension

Hypertensive disorders of pregnancy, including preeclampsia (PE), affect approximately 7-10% of pregnancies in the US. Clinical and experimental studies strongly suggest that the maternal immune system plays a role in the development of these disorders; however, few therapeutic options exist aside from delivery. Using a deoxycorticosterone acetate (DOCA)/salt-low renin rat model, which exhibits hypertension, proteinuria, endothelial dysfunction, and intrauterine growth restriction (IUGR), we measured serum cytokine levels as an indication of immune system activation. In addition, we suppressed the immune system with either azathioprine (Aza) or mycophenolate mofetil (MMF) during the second half of pregnancy to determine whether the these symptoms could be ameliorated. Our results demonstrate that serum T helper-1 (Th1)-type inflammatory cytokines interleukin (IL)-2, IL-12, interferon-gamma (IFNgamma), and RANTES were significantly elevated in hypertensive pregnant rats while the Th2-type cytokine IL-4 was elevated in normal pregnant animals. Either Aza or MMF significantly attenuated the hypertension, proteinuria, and endothelial dysfunction as well as the increased proinflammatory Th1 cytokine profile in pregnant rats treated with DOCA/salt, and had no effect on these parameters in normal pregnant rats. These data strongly suggest that maternal immune system activation plays a role in the development of pregnancy-induced hypertension (PIH).

Maternal Adaptation in Pregnant Hypertensive Rats: Improvement of Vascular and Inflammatory Variables and Oxidative Damage in the Kidney

Mechanisms of normalization of blood pressure in spontaneously hypertensive rats (SHR) during pregnancy were investigated. We hypothesized that at the end of pregnancy (20th day), the modified renal renin-angiotensin system (RAS) plays a pivotal role in this effect associated with reduced inflammation and oxidative damage. We measured blood pressure and heart rate (HR) using a noninvasive tail-cuff method in conscious SHR and Wistar Kyoto rats (WKY). Nonpregnant (-NP) or pregnant (-P) SHR and WKY were used to compare the changes of angiotensin II (ANG II) type 1 (AT1) and type 2 (AT2) receptor expression in the kidney. Renal modification of proinflammatory enzyme expression, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) and their transcription factor nuclear factor-kappaB (NF-kappaB) were also evaluated. Renal malonyldialdehyde (MDA) content and protein nitrotyrosylation, as indicators of oxidative stress, were assessed. Moreover monocyte chemotactic protein-1 (MCP-1) mRNA was determined. Our findings indicate that the significant reduction of blood pressure induced by pregnancy in the SHR strain could be related to reduced AT1 and increased AT2 expression. We also saw a significant decline in renal NF-kappaB, COX-2, iNOS, and macrophage infiltration, as well as the fall in oxidative stress indicators. The increased proinflammatory and oxidative variables, seen in SHR, are strongly ameliorated by pregnancy. In pregnant SHR animals, the adaptive and compensative changes of RAS and inflammation in the kidney seem to contribute to the reduction of blood pressure near term.

IL‐6 induced hypertension in pregnant rats is associated with agonistic autoantibodies to the angiotensin II type I receptor

Increases in interleukin 6 (IL‐6) and agonistic autoantibodies to the angiotensin II type 1 receptor (AT1‐AA) are proposed to be important links between placental ischemia and hypertension in preeclampsia. We reported that hypertension in response to placental ischemia in pregnant rats is associated with elevated IL‐6 and AT1‐AA. However, whether IL‐6 stimulates AT1‐AA production is unknown. Therefore, the purpose of this study was to determine whether IL‐6 excess in pregnant rats leads to production of AT1‐AA and whether AT1 R activation plays a role in mediating IL‐6 induced hypertension. To achieve this goal, IL‐6 (5ng/day) was infused into NP rats and mean arterial pressure (MAP) and AT1‐AA were analyzed. Serum IL‐6 increased with chronic infusion from 63 ±5 pg/ml to 230 ±54 pg/ml was associated with an increase in MAP from 102 ± 2 to 118 ± 4 mm Hg. Furthermore, AT1‐AA increased markedly with chronic IL‐6 (0.7 ± 0.3 bpm NP vs 14.1 ± 1.4 bpm). To determine the importance of AT1 R activation in mediating hypertension in IL‐6 treated rats, we measured MAP in the presence and absence of an AT1R antagonist, losartan, L. Blood pressure responses to IL‐6 were abolished in losartan pretreated rats (85± 4 in NP+L vs 85±3 mmHg in IL‐6 + L). Collectively, these data indicate that IL‐6 is an important stimulus of AT1‐AA and that activation of the AT1 receptor appears to mediate hypertension associated with IL‐6 during pregnancy.

Soluble fms‐like tyrosine‐1 (sFlt‐1) is enhanced in response to chronic tumor necrosis factor‐ alpha excess during pregnancy

Recent studies have shown that an imbalance of pro‐angiogenic factors and the anti‐angiogenic factor, sFlt‐1, exists in preeclampsia. While increases in sFlt‐1 are thought to play an important role in the pathogenesis of hypertension during preeclampsia, the mechanisms that lead to increased production are unclear. Although preeclampsia in women and placental ischemia induced hypertension in pregnant rats is associated with enhanced production of inflammatory cytokines such as tumor necrosis factor‐alpha (TNF‐ a), the role of TNF‐a in the regulation of sFlt‐1 production is unknown. Therefore, the purpose of this study was to determine the effect of chronic TNF‐a excess on plasma sFlt‐1 levels in pregnant rats. Chronic infusion of TNF‐a into normal pregnant (NP) rats significantly increased arterial pressure from 101±2 to 125±1 mmHg. Associated with the hypertension were significant increases in circulating sFlt‐1 (180±5 NP vs 2907±412 pg/ml TNF‐a treated). Moreover, sFlt‐1 production was elevated in cultured placental explants of TNF‐a induced hypertensive pregnant rats (2544±1132 pg/ml) vs NP rats (2189±586 pg/ml). In summary, we report circulating and placental sFlt‐1 to be elevated in response to chronic TNF‐α excess during pregnancy. These data support a potential role for inflammatory cytokines, such as TNF‐α, as an important stimulus for sFlt‐l production during preeclampsia. NIH grant HL51971.

Vascular Endothelial Growth Factor Improves Renal and Endothelial Function, and Normalizes Blood Pressure in Hypertensive Pregnant Rats.

We have recently shown that chronic VEGF administration during the last trimester of pregnancy partially decreases the hypertension associated with placental ischemia. While we have shown that placental ischemia results in endothelial dysfunction, whether VEGF can restore endothelial cell function remains unknown. We hypothesized that restoration of VEGF levels to that seen in normal pregnant rats would further attenuate the hypertension, normalize glomerular filtration rate (GFR), and reverse the endothelial dysfunction associated with placental ischemia. VEGF (180ug/kg/day) was administered for five days via osmotic mini‐pump placed intraperitoneal. Arterial pressure (AP) and tissues were obtained on day 19 of pregnancy from RUPP+VEGF, RUPP and normal pregnant (NP) dams. Endothelial function was assessed by acetylcholine (ACh) induced relaxation of U‐46619 preconstricted carotid artery rings via wire myography. AP (mm Hg) was increased in the RUPP (131±3) compared to the NP (102±1) and infusion of VEGF resolved the hypertension (105±5). GFR (ml/min), decreased in the RUPP dams (1.5±0.3) returned to NP levels (3.1±0.5) by VEGF treatment (3.1±0.4). Relaxation to ACh was inhibited in the RUPP dams compared to the NP but was enhanced by the VEGF treatment (P&lt;0.05). These data demonstrate that VEGF restores GFR and endothelial function, and reduces high blood pressure associated with placental ischemia.

Monoclonal antibody to an endogenous bufadienolide, marinobufagenin, reverses preeclampsia-induced Na/K-ATPase inhibition and lowers blood pressure in NaCl-sensitive hypertension

Levels of marinobufagenin (MBG), an endogenous bufadienolide Na/K-ATPase (NKA) inhibitor, increase in preeclampsia and in NaCl-sensitive hypertension. We tested a 3E9 monoclonal anti-MBG antibody (mAb) for the ability to lower blood pressure (BP) in NaCl-sensitive hypertension and to reverse the preeclampsia-induced inhibition of erythrocyte NKA. Measurements of MBG were performed via immunoassay based on 4G4 anti-MBG mAb. In hypertensive Dahl-S rats, intraperitoneal administration of 50 microg/kg 3E9 mAb lowered BP by 32 mmHg and activated the Na/K-pump in the thoracic aorta by 51%. NaCl supplementation of pregnant rats (n = 16) produced a 37 mmHg increase in BP, a 3.5-fold rise in MBG excretion, and a 25% inhibition of the Na/K-pump in the thoracic aorta, compared with pregnant rats on a normal NaCl intake. In eight pregnant hypertensive rats, 3E9 mAb reduced the BP (21 mmHg) and restored the vascular Na/K-pump. In 14 patients with preeclampsia (mean BP, 126 +/- 3 mmHg; 26.9 +/- 1.4 years; gestational age, 37 +/- 0.8 weeks), plasma MBG was increased three-fold and erythrocyte NKA was inhibited compared with that of 12 normotensive pregnant women (mean BP, 71 +/- 3 mmHg) (1.5 +/- 0.1 vs. 3.1 +/- 0.2 micromol Pi/ml/h, respectively; P < 0.01). Ex-vivo 3E9 mAb restored NKA activity in erythrocytes from patients with preeclampsia. As compared with 3E9 mAb, Digibind, an affinity-purified antidigoxin antibody, was less active with respect to lowering BP in both hypertensive models and to restoration of NKA from erythrocytes from patients with preeclampsia. Anti-MBG mAbs may be a useful tool in studies of MBG in vitro and in vivo and may offer treatment of preeclampsia.