Normal Pregnant Rats Research Articles

Endothelin-1, Oxidative Stress, and Endogenous Angiotensin II

Hypertension during preeclampsia is associated with increased maternal vascular sensitivity to angiotensin II (ANGII). This study was designed to determine mechanisms whereby agonistic autoantibodies to the ANGII type I receptor (AT1-AA) enhance blood pressure (mean arterial pressure [MAP]) and renal vascular sensitivity to ANGII during pregnancy. First, we examined MAP and renal artery resistance index in response to chronic administration of ANGII or AT1-AA or AT1-AA+ANGII in pregnant rats compared with control pregnant rats. To examine mechanisms of heightened sensitivity in response to AT1-AA during pregnancy, we examined the role of endogenous ANGII in AT1-AA–infused pregnant rats, and that of endothelin-1 and oxidative stress in AT1-AA+ANGII–treated rats. Chronic ANGII increased MAP from 95±2 in normal pregnant rats to 115±2 mm Hg; chronic AT1-AA increased MAP to 118±1 mm Hg in normal pregnant rats, which further increased to 123±2 mm Hg with AT1-AA+ANGII. Increasing ANGII from 10 −11 to 10 −8 decreased afferent arteriole diameter from 15% to 20% but sharply decreased afferent arteriole diameter to 60% in AT1-AA–pretreated vessels. Renal artery resistance index increased from 0.67 in normal pregnant rats to 0.70 with AT1-AA infusion, which was exacerbated to 0.74 in AT1-AA+ANGII–infused rats. AT1-AA–induced hypertension decreased with enalapril but was not attenuated. Both tissue endothelin-1 and reactive oxygen species increased with AT1-AA+ANGII compared with AT1-AA alone, and blockade of either of these pathways had significant effects on MAP or renal artery resistance index. These data support the hypothesis that AT1-AA, via activation of endothelin-1 and oxidative stress and interaction with endogenous ANGII, is an important mechanism whereby MAP and renal vascular responses are enhanced during preeclampsia.

Changes of calcium channel characteristics and its relationship with pancreatic islet β cell parasecretion in rat born small for gestational age

Method The animal model of small for gestation in rat was made under matemal calorie restriction by 50% from d1 of gestation until term, while the control groups named AGA rats, were obtained from normal pregnant rats. Both of SGA and AGA newborn rats were fed normally till 4 weeks and 8 weeks. Single islet b cell isolation and pancreatic tissue-slice technique were used to obtain b cells in neonatal rats and adult rats, respectively. Using whole cell patch clamp to record the characteristics of b cell voltage dependent calcium channel (such as current, reversal potential, I-V curve, activation curve, inactivation curve) in both SGA and AGA groups. Using capacitance measurement induced by a sequence of sine wave stimulus to reflect the insu lin secretion. In aw ord, study the tendency of calcium channel kinetics and secretion property by timing. Results The membrane capacitance of SGA was smaller than AGA rat after excluding the influence of whole cell membrane capacitance deviation both in neonatal rats, 4 weeks and 8 weeks (P 0.05). The same result was obtained in activation curve and inactivation curve of calcium channel, whose half activated voltage/ half inactivated voltage V1/2 and slope factor k between SGA and AGA both showed no significant difference (P>0.05) ; 2) adult rats: there was no statistical difference found in all average calcium current density (Id), including the total calcium, L-type and T-type. No significant difference was observed in the peak current density of I-V curve in SGA and AGA (P>0.05). However, the reversal potential in SGA group was much lower than control (P<0.05). The two group had the same shape of activation curve and inactivation curve of calcium channel, and 50% activated voltage between SGA and AGA had no significant difference, but the calcium channel of SGA was easily to be closed, the inactivated voltage in SGA group was much higher than control (P<0.01). Conclusion The effect of intrauterine malnourishment on pancreatic isletbcell last in postnatal life and the destroy of bcell tends to aggravate with time. Low calcium current density on b cell membrane devotes to the decrease of b cell secretion in SGA neonatal rat. However, the current of calcium channel returns to normal level in childhood. The kinetics of calcium channel is changed significantly,

Administration of Interleukin-17 Soluble Receptor C Suppresses T H 17 Cells, Oxidative Stress, and Hypertension in Response to Placental Ischemia During Pregnancy

Preeclampsia, new onset hypertension with proteinuria during pregnancy, is associated with chronic inflammation and placental oxidative stress (ROS). Chronic interleukin-17 (IL-17) increases blood pressure, autoantibodies (angiotensin II type I receptor [AT1-AA]), and ROS during pregnancy. The objective of this study was to determine whether T-helper 17 (TH17) suppression via IL-17 recombinant receptor C (IL-17RC) decreases pathophysiology associated with placental ischemia (reduced uterine perfusion pressure [RUPP]). On gestation day 14, miniosmotic pumps infusing 100 pg of IL-17RC per day were implanted into pregnant rats undergoing RUPP. On gestation day 18, carotid catheters were inserted. On gestation day 19, mean arterial pressure was recorded and TH17 cells, oxidative stress, and AT1-AA were measured and analyzed via 1-way ANOVA. Mean arterial pressure increased from 101 ± 2 mm Hg in normal pregnant rats (n = 19) to 120 ± 1 mm Hg in RUPP rats (n = 17) but decreased to 110 ± 2 mm Hg in RUPP+IL-17RC rats (n = 22). Pup weight decreased from 2.28 ± 0.2 g in normal pregnant rats to 1.96 ± 0.3 g in RUPP rats but was significantly increased to 2.01 ± 0.1 in RUPP+IL-17RC rats. TH17 cells were 1.77% in RUPP rats but decreased to 0.65% in RUPP+IL-17RC rats. Urinary isoprostanes were normalized in RUPP+IL-17RC rats (52 pg/µg) compared with 89 pg/µg in RUPP controls. Placental ROS was 652 relative light units in RUPP rats but decreased to 337 relative light units in RUPP+IL-17RC rats. AT1-AA was 17.27 ± 0.7 bpm in RUPP rats but decreased to 5.00 ± 0.5 bpm in RUPP+IL-17RC rats. With this study, we show that infusion of IL-17RC blunts TH17s, oxidative stress, AT1-AA, and hypertension in the RUPP model of preeclampsia, indicating that TH17 cells may play an important role in disease pathophysiology.

Abstract 279: Hypertension in Hemolysis, Elevated Liver Enzyme, Low Platelet Syndrome is Associated with Increased Endothelin-1

INTRODUCTION. HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) is a severe form of preeclampsia, a hypertensive disorder that occurs during pregnancy. We have shown that HELLP syndrome in both women and in an animal model causes hypertension and elevated TNF-α, IL-6, and agonistic autoantibodies to the angiotensin II receptor, all of which have been shown to activate the endothelin-1 system (ET-1). Therefore we tested the hypothesis that hypertension in response to HELLP syndrome is associated with ET-1 activation. METHODS. Plasma was collected from women with HELLP syndrome or pregnant women without maternal/fetal complications at the time of delivery. Endothelin (ET-1) was extracted from the plasma and measured with a Quantikine ET-1 enzyme linked immunosorbent assay kit. On gestational day (GD) 12 miniosmotic pumps infusing sEndoglin (7ug/kg/day) and sFlt-1 (4.7ug/kg/day) were implanted into normal pregnant rats to induce HELLP syndrome. On GD18 carotid catheters were inserted into HELLP and normal pregnant (NP) rats and mean arterial pressure (MAP) was recorded on GD19; serum, urine and tissues were collected for molecular analysis. RESULTS. Women with HELLP syndrome (n=4) had significantly more circulating ET-1 (3.35pg/mL) compared to normal pregnant women (0.75pg/mL; n=3; p=0.006) and increased MAP (120.8±6.2 mmHg HELLP vs. 87.33±6.67mmHg NP; p=0.015). In rats MAP increased from 91±2mmHg in NP rats (n=4) to 129.8±4.5mmHg in HELLP rats (n=4; p=0.0002). Preproendothelin mRNA expression was 3 fold higher in placentas and 1.3 fold higher in livers from HELLP rats compared to NP rats (p=0.02; p=0.05). In addition we measured ET-1 secretion from endothelial cells exposed to serum from NP and HELLP rats. ET-1 in response to NP serum was 4.03±.68pg/mg/mL and increased to 9.69±3.5pg/mg/mL with HELLP serum (p=0.04) after 24hrs. CONCLUSION. These data support the hypothesis that hypertension in both women and animals with HELLP syndrome is accompanied by increased ET-1 expression.

Abstract 272: Reduced Uterine Perfusion Pressure Stimulated T-helper 17 Cells Cause Hypertension during Pregnancy

Preeclampsia (PE), new onset hypertension with proteinuria during pregnancy, is associated with chronic inflammation and an imbalance among T-helper cell subtypes. Adoptive transfer of all CD4+ T cells from Reduced Uterine Perfusion Pressure (RUPP) rats into Normal Pregnant (NP) rats caused elevations in blood pressure and Angiotensin II type I receptor autoantibody (AT1-AA) and inflammatory cytokines. We have shown that chronic IL-17 infusion increases blood pressure and AT1-AA during pregnancy. Furthermore, we have shown that blockade of IL-17 decreases blood pressure and AT1-AA in the RUPP rat model of PE. The objective of this study was to determine a role for the elevated autoimmune associated T-helper 17 (T H 17) cells from RUPP rats to cause hypertension and chronic inflammation during pregnancy. CD4 + CD25 - T cells were isolated from RUPP rat spleens using biotinylated antibody. Isolated cells were then stimulated on CD3 and CD28 magnetic beads and cultured in T-helper media to differentiate cells into T H 17 cells. Differentiation of isolated cells into T H 17 cells was verified via flow cytometry. On day 12 of gestation, 1x10 6 T H 17 cells from RUPP rats were adoptively transferred into NP rats. Carotid catheters were inserted on gestation day18, and on day 19 blood pressure (MAP) was recorded, serum and plasma were collected. One-way ANOVA was used for statistical analysis. MAP increased from 99 ±2 mmHg in NP (n=8), to 128±4 mmHg in RUPP (n=9, P&lt;0.0001). Likewise, adoptive transfer of RUPP T H 17 cell into NP (NP + RUPP T H 17) significantly increased MAP to 111±3 mmHg (n=11, P&lt;0.001 compared to NP). AT1-AA significantly increased from 0.1±0.2 beats/min in NP to 15.6±0.7 beats/min in NP + RUPP T H 17 rats (p&lt;0.01). Plasma inflammatory cytokines were assessed via ELISA. IL-6 increased from 27.5±7.3 pg/mL in NP to 60.3±9.5 pg/mL in NP + RUPP T H 17 rats (p&lt;0.05); TNF-α increased from 11.4±9 pg/mL in NP to 19.9±6 pg/mL in NP + RUPP T H 17; IL-17increased from 0.49±0.32 pg/ml in NP to 5.46±5 pg/mL in NP +RUPP T H 17. In conclusion, RUPP T H 17 cells increased blood pressure, AT1-AA, and inflammatory cytokines when transferred to NP rats, indicating a role for autoimmune associated T H 17 cells, to cause much of the pathophysiology associated with PE.

Abstract 542: Progesterone Supplementation Attenuates Hypertension And AT1-AA in Response Tt Elevated IL-6 During Pregnancy

Preeclampsia is a multi-system disorder defined as new-onset hypertension with proteinuria developing after 20 weeks gestation. Preeclampsia remains a significant contributor to maternal morbidity and mortality, with great impact in perinatal morbidity. Research efforts throughout the world have focused on alternative therapies for the treatment of preeclampsia to the mainstay treatment of delivery of the fetus and placenta. 17-alpha-hydroxyprogesterone caproate (17-OHP) is a synthetic metabolite of progesterone used effectively for the prevention of recurrent preterm birth in singleton pregnancies. Previously we identified potential role for 17-OHP as an anti-inflammatory that suppressed inflammatory cytokines, hypertension and endothelin-1 in response to placental ischemia in the RUPP rat model of preeclampsia. In the current study we examined a role 17-OHP to blunt the pathophysiological effects to elevated IL-6 during pregnancy. IL-6 induced hypertension during pregnancy occurs with increased renin activity, autoantibodies to the angiotensin II type I receptor (AT1-AA) and reduced kidney function. This experiment was performed in the following groups of normal pregnant rats: NP (n=5); NP+17-OHP (n=6); NP+IL-6 (n=10); NP+IL-6+17-OHP (n=10). To test our hypothesis IL-6 (5ng/day) was infused via miniosmotic pump into normal pregnant rats beginning on day 14 of gestation and 17-OHP (3.32mg/kg) was diluted in normal saline and injected on day 18. Blood pressure (MAP) determination and serum collection was performed on day 19 of gestation. MAP in NP was 100+3mmHg which increased with IL-6 to 112+4mmHg, p &lt;0.05. Pregnant rats given 17-OHP alone had a MAP of 99+3mmHg and MAP only increased to 103+2mmHg in IL-6+17-OHP. AT1-AA was 1.2+0.5 bpm in NP rats and increased to 17+9 bpm with IL-6 infusion. Administration of 17-OHP significantly blunted AT1-AA to 4+0.8bpm in NP+IL-6+17-OHP. Importantly, this study illustrates that 17-OHP attenuates hypertension and AT1-AA in response to elevated IL-6 during pregnancy and could lend hope to a new potential therapeutic for preeclampsia.

Abstract 281: HO-1 Induction Increases ßENaC in Ischemic Placentas and Cultured Cytotrophoblasts

Preeclampsia is characterized by abnormal placentation that is thought to arise from inadequate cytotrophoblast migration and invasion of the spiral arteries. The resulting reduced perfusion of the utero-placental unit promotes the development of hypertension in preeclampsia. Beta Epithelial Na+ Channel (βENaC) protein, a mediator of cytotrophoblast migration in vitro, is decreased in placentas of preeclamptic patients. We have recently reported that hemeoxygenase-1 (HO-1) and its by-products reduce blood pressure in placental ischemic rats. Since HO and its byproducts can regulate ENaC expression, including βENaC, are important mediators of migration, the goal of this study was to test the hypothesis that HO-1 induction 1) stimulates βENaC expression and migration in cultured cytotrophoblasts and 2) restores βENaC expression in ischemic placentas. Cobalt protoporhyrin (CoPP, 1 - 10 μM, 48 hrs) stimulated HO-1 induction in cultured cytotrophoblasts (BEWO cells), upregulated βENaC expression, and increased spontaneous migration by 39.2 ± 6.0% (n=7, p&lt;0.001). To determine if HO-1 induction increased placental βENaC expression and prevented hypertension, CoPP was injected subcutaneously (5mg/kg) on gestational day 14 in timed pregnant Sprague-Dawley rats with placental ischemia. MAP was increased on gestational day 19 in placental ischemic rats (141 ± 4 vs.101 ± 1 mmHg, p&lt;0.001; n=4 per group). While HO-1 induction did not affect MAP in normal pregnant rats (101 ± 5 mmHg), MAP was significantly attenuated (p&lt;0.01) in placental ischemic rats treated with CoPP (117 ± 1 mmHg). Western blot analysis revealed a 62 ± 22% decrease in βENaC expression in ischemic placentas, which was restored by HO-1 induction (n= 4, p&lt;0.05). These findings suggest that HO-1 induction stimulates βENaC expression in cultured cytotrophoblasts and ischemic placentas. Future studies will determine if the beneficial effects of HO-1 occur through βENaC-mediated cytotrophoblast migration and spiral artery remodeling in ischemic placentas. Targeting the HO-1 pathway presents a potential therapeutic approach for improving placentation in preeclamptic women.

Abstract 270: Exercise During Pregnancy Restores Angiogenic Balance But Does Not Lower Blood Pressure in Rupp Hypertensive Rats

Although clinical studies suggest women continuing to exercise during pregnancy have a decreased incidence of preeclampsia, little is known regarding the effects of an exercise regimen started at the onset of pregnancy. Our laboratory has previously reported voluntary wheel running in rats for 6 weeks before and during (EBD) pregnancy increases circulating free vascular endothelial growth factor (VEGF), restores angiogenic potential, and attenuates placental ischemia induced hypertension. Our hypothesis was exercise during (ED) pregnancy without previous exercise training would decrease blood pressure and restore angiogenic potential in pregnant rats with reduced uterine perfusion pressure (RUPP)-induced hypertension. Rats were given free access to running wheels upon confirmation of pregnancy, and running distance was monitored weekly. RUPP (n = 10) or Sham (n= 10) surgery was performed on day 14 of gestation (term=21). Arterial pressure (AP) was measured via indwelling catheter in conscious rats and tissues were collected on day 19. Serum angiogenic potential was assessed with an endothelial tube formation assay using human umbilical vascular endothelial cells (HUVEC). RUPP increased AP compared to Sham (123±2 vs 105±3 mmHg; P&lt;0.05) and AP was not altered in the RUPP+ED (n=8; 117±4mmHg) or Sham+ED (n = 7; 98±3 mmHg). RUPP decreased free VEGF compared to Sham and Sham+ED rats (465.4 vs 842.9 vs 809.8 pg/ml; P&lt;0.05) and this was restored in the RUPP+ED (809.8 pg/ml). Exercise increased (P&lt;0.05) free VEGF in the RUPP but not the Sham rats. HUVEC tube length was decreased in RUPP compared to Sham and Sham+ED rats (61194 vs 74865 vs 79318 μm; P&lt;0.05) and this reduction was attenuated in the RUPP+ED (71552 μm) rats. RUPP, RUPP+ED and Sham+ED had decreased fetal weight compared to Sham (2.3 vs 2.0 vs. 2.2 vs 2.2 vs2.6g; P&lt;0.05). In contrast to our previous report that exercise before and during pregnancy increased free plasma VEGF in normal pregnant and sham rats and decreased AP and restored angiogenic balance in RUPP rats, the present data demonstrate that an exercise regimen started at pregnancy in rats that are exercise naïve at conception has both beneficial and detrimental effects on maternal-fetal outcomes in a gestation complicated by placental-ischemia.

Abstract 548: Reduced Urinary Angiotensinogen Excretion in Preeclampsia

Objective: Preeclampsia (preE) is a hypertensive disorder of pregnancy. We reported the suppression of circulatory renin-angiotensin system (RAS) in a rat model of preE. Urinary angiotensinogen has been considered as an indicator of intrarenal angiotensin status in hypertension. Little is known about the urinary angiotensinogen in preE. This study evaluates the level of urinary excretion of angiotensinogen in preE to assess the RAS status. Methods: Normal pregnant (n=57) and preE (n=32) patients were recruited from Scott &amp; White Hospital and had their blood drawn between 21 to 40 weeks of pregnancy. Criteria for diagnosis of preE included blood pressure &gt;140/90 mm Hg and proteinuria &gt;300 mg of protein/24h. Two groups of rats were used: normal pregnant (n=10) and preE rats (n=10) which were given weekly injections of desoxycorticosterone acetate and 0.9% saline to drink. Urinary angiotensinogen levels and the plasma AngII levels were assayed by ELISA. The kidney expression of (pro)renin receptor, AT 1 receptor, AT 2 receptor, and renin for the two groups of rats was measured by western blot. Results are expressed as means with SD and comparisons made using Student’s t test with p&lt;0.05 considered significant. Results: In preE patients, the mean urinary excretion of angiotensinogen (2.0 ± 1.1 ng/mg creatinine) differed (p&lt;0.05) from that in patients with normal pregnancy (2.7 ± 1.5 ng/mg creatinine). The urinary excretion of angiotensinogen (PreE: 1.5 ± 0.3, NP: 2. 3 ± 0.4 nmol/day) also differed (p&lt;0.05) in preE rats compared to NP. The plasma concentration of Ang II for preE rats (25 ± 3 fmol/ml) differed (p&lt;0.05) compared to NP rats (39 ± 5 fmol/ml). The kidney expression of (pro)renin receptor was downregulated (p=0.008), while the AT 1 receptor was upregulated (p=0.01) in preE rats compared to NP. However, the kidney expression of AT 2 receptor (1.0 vs 1.1) and renin (1.0 vs 1.4) were similar in NP and PDS rats (p=0.63 and p=0.12, respectively). Conclusions: We demonstrated that urinary excretion of angiotensinogen is reduced in both patients with preE and in a rat model of preE. The kidney expression of RAS components was either downregulated or remained unchanged in preE rats. This finding indicates that preE is an Ang-II independent form of systemic hypertension.

Hepatic bile acids and bile acid-related gene expression in pregnant and lactating rats

Background. Significant physiological changes occur during pregnancy and lactation. Intrahepatic cholestasis of pregnancy (ICP) is a liver disease closely related to disruption of bile acid homeostasis. The objective of this study was to examine the regulation of bile acid synthesis and transport in normal pregnant and lactating rats.Materials and Methods. Livers from timed pregnant SD rats were collected on gestational days (GD) 10, 14 and 19, and postnatal days (PND) 1, 7, 14 and 21. Total bile acids were determined by the enzymatic method, total RNA was isolated and subjected to real time RT-PCR analysis. Liver protein was extracted for western-blot analysis.Results. Under physiological conditions hepatic bile acids were not elevated during pregnancy but increased during lactation in rats. Bile acid synthesis rate-limiting enzyme Cyp7a1 was unchanged on gestational days, but increased on PND14 and 21 at mRNA and protein levels. Expression of Cyp8b1, Cyp27a1 and Cyp7b1 was also higher during lactation. The mRNA levels of small heterodimer partner (SHP) and protein levels of farnesoid X receptor (FXR) were increased during pregnancy and lactation. Bile acid transporters Ntcp, Bsep, Mrp3 and Mrp4 were lower at gestation, but increased during lactation. Hepatic Oatp transporters were decreased during pregnancy and lactation.Conclusion. Hepatic bile acid homeostasis is maintained during normal pregnancy in rats, probably through the FXR-SHP regulation. The expression of bile acid synthesis genes and liver bile acid accumulation were increased during lactation, together with increased expression of bile acid efflux transporter Bsep, Mrp3 and Mrp4.

Effects of magnesium sulfate on heart rate, blood pressure variability and baroreflex sensitivity in preeclamptic rats treated with L-NAME

Objective: Magnesium sulfate is used for the treatment of preeclampsia. This study aimed to evaluate effects of magnesium sulfate on heart rate variability (HRV), blood pressure variability (BPV) and baroreflex sensitivity (BRS) in pregnant rats treated with NG-nitro-.-arginine-methyl ester (L-NAME). Methods: Sprague–Dawley rats were randomly divided into four groups: non-pregnancy and three pregnant groups. From gestational day 2–12, normal pregnancy group (NOR) received sterile water through intravenous injection, LN was injected with L-NAME [25 mg/day] to induce preeclampsia, while LNM group was also treated with magnesium sulfate at 0.3 g/kg at gestational day 13. HRV, BPV and BRS were monitored, and endothelial functions were detected at gestational day 14. Results: Rats with treatment of L-NAME showed significantly increased blood pressure and endothelin-1 concentration and decreased plasma NO concentration; concomitant treatment with magnesium sulfate suppressed blood pressure. Also, rats treated with L-NAME and magnesium sulfate underwent lower heart rate variability (lower absolute and normalized LF) and higher blood pressure variability (lower normalized HF and LF, higher normalized VLF) compared with non-pregnant or normal pregnant rats, whereas normalized LF of HRV in rats co-administered magnesium sulfate returned to normal level. Additionally, rats treated with L-NAME underwent decreased BRS-SP, BRS-NE, BRS LF and HF, and rats with concomitant magnesium sulfate partially reversed the decline (p > 0.05) and had significantly lower BRS-SP, BRS-NE. Conclusion: Sympathetic and parasympathetic impairment exist in preeclamptic rats treated with L-NAME. Magnesium sulfate may ameliorate alterations in the autonomic nervous system noted in preeclampsia.

Euglycemic Hyperinsulinemia Increases Blood Pressure in Pregnant Rats Independent of Placental Antiangiogenic and Inflammatory Factors

Although pregnancies associated with hyperinsulinemia and altered placental angiogenic and inflammatory factors are at increased risk for developing preeclampsia, the effects of euglycemic hyperinsulinemia on placental factors and blood pressure regulation during pregnancy are unclear. We hypothesized that chronic hyperinsulinemia results in increased placental soluble fms-like tyrosine kinase 1(sFlt-1) and tumor necrosis factor α (TNF- α) levels and hypertension in pregnant rats. On gestational day (GD) 14, Sprague-Dawley rats were assigned as normal pregnant or pregnant + insulin. Insulin was infused subcutaneously by osmotic minipump for 5 days at a dose of 1.5 mU/kg/min. Those rats receiving insulin were supplemented with 20% glucose in drinking water to maintain euglycemia. On GD 19, mean arterial pressure (MAP) and heart rate (HR) were assessed in conscious rats by indwelling carotid catheters, followed by collections of blood, placentas, and fetuses. In addition to placental sFlt-1 and TNF-α levels, circulating insulin, glucose, leptin, cholesterol, triglyceride, and free fatty acid concentrations were measured. MAP was higher in pregnant + insulin vs. normal pregnant rats; however, HR was similar between groups. Although litter size and placental weight were comparable, fetuses from pregnant + insulin rats were heavier. Importantly, circulating insulin concentration was elevated in the pregnant + insulin group, with no change in glucose level. Moreover, circulating leptin, cholesterol, triglyceride, and free fatty acid concentrations were increased in the pregnant + insulin group. There were no differences in placental sFlt-1 and TNF-α concentrations between groups. In summary, sustained euglycemic hyperinsulinemia, comparable with insulin levels in preeclamptic women, can raise blood pressure in pregnancy independent of recognized placental factors associated with preeclampsia.

Antihypertensive effects of inducible nitric oxide synthase inhibition in experimental pre‐eclampsia

Upregulation of inducible nitric oxide synthase (iNOS) has been reported in both experimental and clinical hypertension. However, although pro-inflammatory cytokines that up-regulate iNOS contribute to pre-eclampsia, no previous study has tested the hypothesis that a selective iNOS inhibitor (1400 W) could exert antihypertensive effects associated with decreased iNOS expression and nitrosative stress in pre-eclampsia. This study examined the effects of 1400 W in the reduced uteroplacental perfusion pressure (RUPP) placental ischaemia animal model and in normal pregnant rats. Sham-operated and RUPP rats were treated with daily vehicle or 1 mg/kg/day N-[3-(Aminomethyl) benzyl] acetamidine (1400 W) subcutaneously for 5 days. Plasma 8-isoprostane levels, aortic reactive oxygen species (ROS) levels and nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ROS production were evaluated by ELISA, dihydroethidium fluorescence microscopy and lucigenin chemiluminescence respectively. Inducible nitric oxide synthase expression was assessed by western blotting analysis and aortic nitrotyrosine was evaluated by immunohistochemistry. Mean arterial blood pressure increased by ∼30 mmHg in RUPP rats, and 1400 W attenuated this increase by ∼50% (P < 0.05). While RUPP increased plasma 8-isoprostane levels, aortic ROS levels, and NADPH-dependent ROS production (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). Moreover, while RUPP increased iNOS expression and aortic nitrotyrosine levels (P < 0.05), treatment with 1400 W blunted these alterations (P < 0.05). These results clearly implicate iNOS in the hypertension associated with RUPP. Our findings may suggest that iNOS inhibitors could be clinically useful in the therapy of pre-eclampsia, especially in particular groups of patients genetically more prone to express higher levels of iNOS. This issue deserves further confirmation.

Cerebral White Matter CD4+ T cell Infiltration is Associated with Hypertension in a rat model of HELLP Syndrome

HELLP syndrome (hemolysis, elevated liver enzymes, low platelets) and preeclampsia (PE) (reduction in uterine perfusion pressure ‐RUPP), are hypertensive disorders that can occur during pregnancy with severe symptoms and consequences. The development of hypertension (HTN) may increase ischemia and inflammation in the maternal brain. We hypothesize that HTN in response to placental ischemia or HELLP increases CD4+ T cells expression in the maternal brain.On gestational day (GD) 12, sEndoglin (7ug/kg) and sFlt‐1 (4.7ug/kg) infusion began via miniosmotic pump into normal pregnant (NP) rats to induce HELLP. On GD14 RUPP rats had the lower abdominal aorta and ovarian arteries physically constricted. On GD18 carotid catheters were inserted (n=5/group), GD19 mean arterial pressure (MAP), plasma, tissue and maternal brains were collected.MAP increased from 96.2 ± 3.6 in NP rats to 127.8 ± 3.8 (p=.05) in RUPP rats; to 113.4±3.2mmHg in HELLP rats (p=.05). RUPP and HELLP rats had increased circulating CD4+ T cells compared to NP rats (p=.01, p=.05). CD4+ T cells along the corpus callosum in RUPP (136.1 OD) and HELLP rats (151.9 OD) were increased compared to NP (114.5 OD, p=.05, p=.05).These data indicate that CD4+ T cells are increased in both the circulation and the brain and may therefore be one potential link in the neuropathophysiology of HTN associated with PE and HELLP syndrome.

Tumor Necrosis Factor induces cerebral edema and increased cerebrovascular permeability in normal pregnant rats

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia deaths and preeclamptic patients commonly present with neurological symptoms. Our laboratory recently showed that placental ischemia, induced by reducing uterine perfusion pressure, closely mimics preeclamptic patients resulting in cerebral edema and increased inflammatory cytokines such as tumor necrosis factor (TNFα). In this study, we examined whether TNFα, infused via intraperitoneal implanted mini‐osmotic pumps (100ng/day from gestational day 14 to 19), contributes to cerebral edema and blood‐brain barrier (BBB) disruption in normal pregnant rats. Mean arterial pressure, measured via carotid catheters, was significantly increased in TNFα infused rats (112 vs. 107 mmHg; p=0.01). Brain water content, calculated as (wet weight ‐ dry weight)/ wet weight × 100, was also significantly increased in TNFα infused rats (78.2 vs. 77.9%; p=0.01). BBB permeability, assayed using the Evans blue extravasation method, was increased in the hippocampus of TNFα‐infused rats (p&lt;0.05) but was unchanged in the striatum, cerebellum, and anterior brain. Our results indicate that TNFα released from ischemic placentas, could mediate cerebral edema through increased BBB permeability. TNFα may be an important clinical target for preventing cerebrovascular complications associated with preeclampsia.Funding: NIH Grant HL108618

Effect of high‐fat diet (HFD) on resistance artery function in normal pregnant rats

Despite clinical evidence that obesity is the leading attributable risk for preeclampsia (PE) in developed countries, the mechanisms whereby metabolic factors increase the risk for PE are unclear. The purpose of this study was to examine the effects of HFD on resistance artery endothelial function in normal pregnant rats. Twelve‐week‐old Sprague‐Dawley rats were fed a normal diet (ND; 13% fat kcal; N=5–8) or HFD (40% fat kcal; N=9–11). At 21 weeks old, timed‐pregnant rats were generated. At gestational day 19, wire myography showed in third‐order mesenteric arteries that sensitivity (logEC50) of endothelial‐dependent relaxation to acetylcholine (ACh) was greater in HFD (−7.9 ± 0.3M) vs. ND (−6.7 ± 0.3M) (P&lt;0.05). In the HFD group, treatment of artery rings with the non‐selective nitric oxide synthase inhibitor L‐NAME reduced sensitivity to ACh (logEC50 of −6.6 ± 0.2M, P&lt;0.05 vs. untreated rings), whereas in the ND group, L‐NAME did not alter the ACh response (−6.6 ± 0.3M) vs. untreated rings. Endothelial‐independent relaxation to Na nitroprusside was similar (logEC50: ND: −6.5 ± 0.4M vs. HFD: −7.0 ± 0.2M). These data suggest that normal pregnancy protects against HFD‐induced endothelial dysfunction. However, this protective pathway may be targeted in PE by factors released from the ischemic placenta that are known to promote the hypertension and endothelial dysfunction of this disease. HL105324, HL51971

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

Effect of high‐fat diet (HFD) on blood pressure and placental levels of tumor necrosis factor (TNF)‐α and soluble fms‐like tyrosine kinase (sFlt)‐1 in pregnant rats

During preeclampsia (PE), placental ischemia increases circulating levels of inflammatory and anti‐angiogenic factors that ultimately leads to hypertension and proteinuria. While obesity is a leading risk factor for developing PE, the mechanisms whereby obesity increases this risk are unclear. Our aims were to examine the effects of HFD on cardiovascular and metabolic systems of normal pregnant rats. Twelve‐week‐old Sprague‐Dawley female rats were fed a normal diet (ND, 13% fat kcal; n=12) or HFD (40% fat kcal). After 9 weeks, rats receiving HFD were grouped in obese prone (OP, weight gain=78.6±2.2 g; n=10) and obese resistant (OR, wg=57.4±2.0 g; n=11) (vs. ND wg=53.6±3.2 g; P&lt;0.05). Rats were then allowed to breed, and mean arterial pressure (MAP) was measured by carotid catheters and tissues were harvested at gestational day 19. Body weight/litter size and visceral fat mass were similar in ND, OR and OP (32.0±2.6 vs. 32.7±2.6 vs. 30.0±1.4 g and 10.9±0.6 vs. 12.2±0.6 vs. 13.2±1.0 g; P&gt;;0.05). MAP was also similar in ND, OR and OP (112±7 vs. 108±3 vs. 110±4 mmHg; P&gt;;0.05). Placental levels of TNFα and sFlt1 were comparable between ND, OR and OP (1.0±0.1 vs. 1.5±0.2 vs. 1.2±0.1 ng/μg and 0.8±0.1 vs.0.9±0.1 vs. 0.9±0.1 pg/μg; P&gt;;0.05). In summary, while HFD increases body weight in OP virgin rats, HFD in OP pregnant rats had no effect on blood pressure regulation or placental levels of TNFα and sFlt1. Funding: HL51971 and HL105324.

Progesterone blunts vascular endothelial cell secretion of endothelin-1 in response to placental ischemia

Preeclampsia (PE) is associated with hypertension and elevated endothelin (ET-1), an indicator of endothelial cell activation and dysfunction. Reduction of uteroplacental perfusion (RUPP) in the pregnant rat model of PE is characterized by elevated mean arterial pressure, inflammatory cytokines, and activation of the ET-1 system. We aim to determine whether 17-alpha-hydroxyprogesterone caproate (17-OHPC) or progesterone suppresses these pathways. Plasma progesterone was purified from normal pregnant (NP) and PE patients and measured via enzyme-linked immunosorbent assay. Human umbilical vein endothelial cells were exposed to the sera with or without progesterone added and ET-1 wasmeasured. Pregnant rats underwent the RUPP procedure with or without intraperitoneal 17-OHPC. Mean arterial pressure was compared in RUPP vs NP rats. Human umbilical vein endothelial cells were exposed to NP or RUPP sera, with and without progesterone and ET-1 measured. Progesterone was significantly decreased in PE women compared with NP women. In response to human sera, ET-1 was elevated in PE women compared to NP women, and decreased with addition of progesterone. Mean arterial pressure was significantly elevated in RUPP vs NP rats but was attenuated by 17-OHPC. ET-1 secretion was stimulated significantly by RUPP compared to NP rat sera, but attenuated by progesterone. Circulating progesterone is significantly lower in PE women compared to controls. 17-OHPC attenuates hypertension in response to placental ischemia in RUPP rats. Progesterone blunts vascular ET-1 stimulated at cellular level by sera from PE women or RUPP rats. Decreased circulating progesterone is associated with stimulation of ET-1. 17-OHPC supplementation blunts hypertension and progesterone blunts endothelial cell ET-1 secretion in response to placental ischemia.

Pravastatin Attenuates Hypertension, Oxidative Stress, and Angiogenic Imbalance in Rat Model of Placental Ischemia-Induced Hypertension

Preeclampsia is a pregnancy-specific condition characterized by an imbalance of circulating angiogenic factors and new-onset hypertension. Although current treatment options are limited, recent studies suggest that pravastatin may improve angiogenic profile and reduce blood pressure in preeclampsia. We hypothesized pravastatin would restore angiogenic balance and reduce mean arterial pressure (MAP) in rats with reduced utero-placental perfusion pressure (RUPP)-induced hypertension. Pravastatin was administered intraperitoneally (1 mg/kg per day) in RUPP (RUPP+P) and normal pregnant rats (NP+P) from day 14 to 19 of pregnancy. On day 19, MAP was measured via catheter, conceptus data were recorded, and tissues collected. MAP was increased (P<0.05) in RUPP compared with NP dams, and pravastatin ameliorated this difference. Pravastatin attenuated decreased fetal weight and plasma vascular endothelial growth factor and the RUPP-induced increased soluble fms-like tyrosine kinase-1 when compared with NP dams. Pravastatin treatment did not improve angiogenic potential in RUPP serum and decreased (P<0.05) endothelial tube formation in NP rats. RUPP rats presented with indices of oxidative stress, such as increased placental catalase activity and plasma thiobarbituric acid reactive substances along with decreased plasma total antioxidant capacity compared with NP controls, and pravastatin attenuated these effects. MAP, fetal weight, plasma vascular endothelial growth factor, and plasma soluble fms-like tyrosine kinase-1 were unchanged in NP+P compared with NP controls. The present data indicate that treatment with pravastatin attenuates oxidative stress and lowers MAP in placental ischemia-induced hypertension, but may have negative effects on circulating angiogenic potential during pregnancy. Further studies are needed to determine whether there are long-term deleterious effects on maternal or fetal health after pravastatin treatment during pregnancy-induced hypertension or preeclampsia.