Normal Pregnant Research Articles

Abstract P173: Serum Soluble and Placental (Pro)Renin Receptor in Normal and Preeclamptic Pregnancy

Objective: Preeclampsia (preE), a syndrome of hypertension, proteinuria and edema, has many elusive triggers. The renin-angiotensin system (RAS) has been implicated in preE pathogenesis. Recently, we have demonstrated that (pro)renin levels are increased in preE patients and that levels of (pro)renin and (pro)renin receptor ((P)RR) are elevated in a rat model of preE. It has also been demonstrated that high circulating levels of soluble (P)RR at delivery is associated with preE. We evaluated the placental expression of (P)RR in preE patients as well as in a nonhuman primate. We also evaluated the serum levels of soluble (P)RR in preE patients and in a rat model of preE. Study Design: (1) Blood and placental samples were collected from 15 normal pregnant (NP) and 15 preE consenting patients in an IRB approved prospective study. (2) We used NP rats ( n=10) and pregnant rats receiving weekly injections of desoxycorticosterone acetate and whose drinking water was replaced with 0.9% saline (preE, n=10). (3) The placental samples from owl monkey (both early and term, NP, n= 2) were collected. The (P)RR expression was measured both by western blotting (WB) and Immunohistochemistry (IHC) with anti-ATP6IP2 (detect (P)RR). The extracellular-signal-regulated kinase ½ (ERK ½) expression was evaluated by WB. The levels of serum soluble (P)RR were measured by a commercially available ELISA kit. Results: The placental expression of (P)RR was higher (p<0.05) in preE compared to NP patients. The ERK1/2 expression was higher (p<0.05) in preE placenta compared to NP. The soluble (P)RR levels were higher in preE (preE patients: 29.2 ± 4.5; PDS rats: 16.9 ± 1.9 ng/mL) compared to NP (NP human: 19.3 ± 4.2; NP rats: 10.4 ± 3.7 ng/mL). The early placenta of owl monkey expressed higher (P)RR compared to term. Conclusions: These data suggest that increased expression of (P)RR and ERK1/2 in preE placenta is related to the occurrence of preE. These data also reconfirmed that high level of circulatory soluble (P)RR is associated with preE. The higher expression of (P)RR in early pregnancy compared to term placenta in owl monkey suggests that the (P)RR is important for normal placental development. The expression of (P)RR in nonhuman primates reveals the approach of future studies on owl monkey preE model.

Blockade of CD40 ligand for intercellular communication reduces hypertension, placental oxidative stress, and AT1-AA in response to adoptive transfer of CD4+ T lymphocytes from RUPP rats.

Preeclampsia (PE) is associated with altered immune activation during pregnancy. We have previously shown that adoptive transfer of CD4(+) T cells from the reduced uterine perfusion pressure (RUPP) rat model of PE increases blood pressure, oxidative stress (ROS), and inflammation in normal pregnant recipient rats. The objective of this study was to determine if blockade of communication via the CD40-CD40 ligand (CD40L) interaction between placental ischemia-induced CD4(+) T cells with endogenous normal pregnant (NP) cells would improve pathophysiology that was previously observed in NP recipient rats of RUPP CD4(+) T cells. Splenic CD4(+) T lymphocytes were magnetically separated, incubated with 2.5 μg/ml anti-CD40 ligand (αCD40L) overnight, and transferred into NP rats on day 12 of gestation (NP+RUPP CD4(+) T+anti-CD40L). On day 19 of gestation, blood pressure (MAP), blood, and tissues were collected. MAP was 99 ± 2 in NP (n = 13), 116 ± 4 in NP+RUPP CD4(+) T cells (n = 7; P < 0.01); MAP only increased to 104 ± 2 in NP+RUPP CD4(+) T cells+CD40L (n = 24) (P < 0.05 vs. NP+RUPP CD4(+) T cells). Mechanisms of hypertension in response to RUPP CD4(+) T cells include endothelin-1 (ET-1), ROS, and angiotensin II type I receptor (AT1-AA) were analyzed. Inhibition of CD40L binding reduced placental ET-1 to 2.3-fold above NP rats and normalized placental ROS from 318.6 ± 89 in NP+RUPP CD4(+) T cells (P < 0.05) to 118.7 ± 24 in NP+RUPP CD4(+) T+anti-CD40L (P < 0.05). AT1-AA was also normalized with inhibition of CD40L. These data suggest that placental ischemia-induced T-cell communication via the CD40L is one important mechanism leading to much of the pathophysiology of PE.

An increased population of regulatory T cells improves the pathophysiology of placental ischemia in a rat model of preeclampsia.

The reduced uterine perfusion pressure (RUPP) rat model of preeclampsia exhibits much of the pathology characterizing this disease, such as hypertension, inflammation, suppressed regulatory T cells (TRegs), reactive oxygen species (ROS), and autoantibodies to the ANG II type I receptor (AT1-AA) during pregnancy. The objective of this study was to determine whether supplementation of normal pregnant (NP) TRegs into RUPP rats would attenuate the pathophysiology associated with preeclampsia during pregnancy. CD4(+)/CD25(+) T cells were isolated from spleens of NP and RUPP rats, cultured, and injected into gestation day (GD) 12 normal pregnant rats that underwent the RUPP procedure on GD 14. On GD 1, mean arterial pressure (MAP) was recorded, and blood and tissues were collected for analysis. One-way ANOVA was used for statistical analysis. MAP increased from 99 ± 2 mmHg in NP (n = 12) to 127 ± 2 mmHg in RUPP (n = 21) but decreased to 118 ± 2 mmHg in RUPP+NP TRegs (n = 17). Circulating IL-6 and IL-10 were not significantly changed, while circulating TNF-α and IL-17 were significantly decreased after supplementation of TRegs. Placental and renal ROS were 339 ± 58.7 and 603 ± 88.1 RLU·min(-1)·mg(-1) in RUPP and significantly decreased to 178 ± 27.8 and 171 ± 55.6 RLU·min(-1)·mg(-1), respectively, in RUPP+NP TRegs; AT1-AA was 17.81 ± 1.1 beats per minute (bpm) in RUPP but was attenuated to 0.50 ± 0.3 bpm with NP TRegs. This study demonstrates that NP TRegs can significantly improve inflammatory mediators, such as IL-17, TNF-α, and AT1-AA, which have been shown to increase blood pressure during pregnancy.

Early Administration of 17‐Hydroxyprogesterone Caproate to Reduced Uterine Perfusion Pressure (RUPP) Rat Model of Preeclampsia Improves Inflammation, Uterine artery Vasoconstriction and Blood Pressure During Pregnancy

Preeclampsia (PE) is characterized by new onset hypertension and increased uterine artery resistance (UARI), elevated TNF-α, sFlt-1, and decreased of nitric oxide (NO) bioavailability during pregnancy. 17-hydroxyprogesterone caproate (17-OHPC) is approved for cessation of preterm labor in pregnancies not complicated by PE. Therefore, we hypothesized that 17-OHPC administered on day 15 of gestation into RUPP rats could reduce blood pressure (MAP), TNF-α, sFlt-1, UARI, and increase pup weight and NO bioavailability. Carotid catheters were inserted on day 18. MAP, blood and tissues were collected on day 19. MAP in normal pregnant (NP) rats (n=9) was 92 +1.35, 126 +1.98 in RUPP rats (n=17) and 111+1.54 mmHg in RUPP+17-OHPC (n=13), p <0.05. Pup weight was 2.31+0.12 in NP (n=13), 1.88+ 0.05 in RUPP rats (n=24), which improved to 2.01+0.07 grams in RUPP+17-OHPC (n=15), p <0.05. UARI was 0.79+0.03 in RUPP rats (n=4) and 0.59+0.04 in RUPP+17-OHPC (n=5), p<0.05. Plasma levels of TNFα, sFlt1 were 64.0+14.1, 385.9+141 in RUPP rats (n=7), which were blunted to 17.8+9.6, 110.2+11.1 pg/mL in RUPP+17OHPC (n=5), p<0.05. Plasma NOx was 10.82+2.3 in RUPP rats (n=13) but was improved to 25.5+5.2 µM in RUPP+17-OHPC (n=5), p<0.05. In conclusion, early administration of 17-OHPC improves TNF-α, sFtl-1, UARI, hypertension, pup weight and nitric oxide bioavailability in response to placental ischemia and should be considered for addition to the management of severe preterm PE. NIH RO1HD067541, T32HL105324

Serelaxin Improves Blood Pressure and Uterine Artery Resistance in the Reduced Uterine Perfusion Pressure (RUPP) Rat Model of Preeclampsia

Preeclampsia (PE), new-onset hypertension during pregnancy, is associated with systemic and renal vasoconstriction, as well as fetal growth restriction. Relaxin, vasodilator in humans, plays an important role in maintaining vascular compliance during normal pregnancy; however, there is currently no preclinical evidence to support the use of relaxin during PE. Therefore, we hypothesized that Serelaxin (recombinant human relaxin-2) subcutaneously administered via miniosmotic pump (4 ug/h) days 14-19 of gestation into RUPP rats could improve vascular compliance as a mechanism of reducing uterine artery resistance index (UARI) and improve fetal growth and mean arterial pressure (MAP). Carotid catheters were inserted on day 18. MAP, blood and tissues were collected on day 19. MAP in normal pregnant (NP) rats (n=5) was 106+5, 128+3 in RUPP rats (n=10) and 110+3 mmHg in RUPP+Serelaxin (n=10), p<0.05. Pup weight was reduced with RUPP from 2.3±0.1 in NP (n=5) to 1.9±0.1 g (n=10, p<0.05), and was unchanged in RUPP+Serelaxin (1.9.0±0.1 g, n=10). UARI was 0.66+0.01 in RUPP rats (n=6), but was improved to 0.59+0.02 in RUPP+Serelaxin (n=4), p<0.05. Total nitrate-nitrite concentration was 16.0+2.4 in RUPP rats (n=9), which increased to 25.4+2.5 µM in RUPP+Serelaxin (n=6), p<0.05. In conclusion, Serelaxin improves MAP, UARI and nitric oxide bioavailability. These data suggest an important role for relaxin in maintaining normal blood pressure and vascular compliance during pregnancy which would be helpful to maintain maternal health and prolong pregnancy in the face of placental ischemia.

Placental Ischemia‐Induced TH17 Cells Mediate the Pathophysiology Associated with Preeclampsia

Preeclampsia (PE) is associated with hypertension, inflammation, intrauterine growth restriction (IUGR), increased CD4+ THelper 17 cells (TH17s) and agonistic auto‐antibodies to the AT1 receptor (AT1‐AA). The objective of this study was to determine a role for TH17s in mediating pathophysiology associated with PE using the reduced uterine perfusion pressure (RUPP) rat model of PE. On gestation day 12 (GD12) RUPP‐induced TH17s were injected into normal pregnant (NP) rats; On GD19 blood pressure (MAP) was recorded, and blood and tissues were collected. One‐way ANOVA was used for statistical analysis. MAP increased from 99±2 in NP (n=8) to 128±4 in RUPP (n=9) and to 111±3 mmHg in NP+TH17 (n=11; p&lt;0.05). Placental and renal ROS increased from 238 and 411 RLU, respectively, in NP to 328 and 603 RLU in RUPP and to 339 and 833 RLU in NP+TH17 (p&lt;0.05). Importantly pup weight (gm) decreased significantly from 2.19 in NP to 1.91 in RUPP, and to 1.9 in NP+TH17 (p&lt;0.05). Adoptive transfer of TH17s also increased inflammation: IL‐6 increased significantly from 27.5 pg/mL in NP to 62.2 pg/mL in RUPP and to 75.9 pg/mL in NP+TH17 (p&lt;0.5); IL‐17 increased from 0.5 pg/mL in NP to 1.29 pg/mL in RUPP and increased to 5.5 pg/mL in NP+TH17 (p&gt;0.05). AT1‐AA significantly increased from 0.1beats/min in NP to 16.82beats/min in RUPP and to 15.6beats/min in NP+TH17. TH17s induced hypertension, ROS, IUGR, inflammation, and AT1‐AA in pregnant rats, demonstrating the importance of TH17s to mediate the pathophysiology associated with PE and their potential as a new therapeutic target for treatment of PE.NIH grants RO1HD067541 and T32HL105324

Alterations in Placental Eicosanoid Production Contribute to Impaired Vascular Remodeling in Preeclampsia

Preeclampsia (PreE) results from an impaired vascular remodeling of the placental spiral arteries. Recent data suggests cytochromeP450 (CYP450) vasoactive eicosanoids in the etiology of the disease. Therefore, we investigated whether alterations in the profile of arachidonic acid (AA) metabolites of the CYP450 pathway contribute to the impaired vascular remodeling in preeclampsia. To this end, we isolated microsomes from placental vessels of normal pregnant (NP) and PreE women and CYP450 enzyme activity was determined by HPLC/mass spectrometry. In addition, the proliferative actions of 20‐HETE, a major CYP450 metabolite were investigated in an in vivo model of trophoblast syncytialisation. Placental vascular microsomes from PreE women displayed a significant increase in 20‐HETE production relative to NP women (0.192±0.04 vs 0.119±0.06 pg/min/mg, p&lt;0.05). 15‐HETE (74±8 vs 43±11 pmol/min/mg), 12‐HETE (70±20 vs 27±7 pmol/min/mg, p&lt;0.05), and total HETE production (287±61 vs 170±44 pmol/min/mg) were also increased in PreE compared to NP. Incubation of BeWo cells with 20‐HETE (10nM; 100nM; 1uM) displayed an increase in cell proliferation, while inhibition of 20‐HETE with 17‐ODYA (10uM) and HET0016 (1nM) displayed a 10% and 75% inhibition. Thus, increases in 20‐HETE production promote, while inhibition of 20‐HETE reduces proliferation of trophoblasts in vitro and may contribute to the altered trophoblast invasion and vascular remodeling in PreE.

Regulation of soluble fms-like tyrosine kinase-1 production in response to placental ischemia/hypoxia: role of angiotensin II.

While soluble fms-like tyrosine-1 (sFlt-1) is implicated in the pathogenesis of hypertension during preeclampsia, the mechanisms leading to the enhanced sFlt-1 production remain unclear. A recent report suggests exogenous angiotensin II (ANGII) stimulates sFlt-1 production in pregnant rats, however, the role of endogenous ANGII in mediating the placental production of sFlt-1 in response to placental ischemia remains unknown. Therefore, the purpose of this study was to determine the role of endogenous ANGII in mediating the placental production of sFlt-1 in response to placental ischemia in pregnant Sprague–Dawley rats. To this end we compared sFlt-1 and ANGII levels from placental explants collected from normal pregnant (NP) and Reduced Uterine Perfusion Pressure (RUPP) rats. sFlt-1 (3271 ± 264 vs. 2228 ± 324 pg/mL, P < 0.05) and ANGII levels (43.2 ± 2.8 vs. 26.7 ± 1.9 pg/mL, P < 0.05) were higher in placental explants from RUPP rats versus NP rats. Administration of Losartan, an angiotensin type 1 (AT1) receptor antagonist, (10 mg/day for 5 days) to RUPP rats significantly reduced plasma levels of sFlt-1 (1432 ± 255 pg/mL, P < 0.05) when compared with untreated control rats (3431 ± 454 pg/mL). In addition, RUPP-induced hypertension was significantly reduced (113 ± 2 mmHg vs. 139 ± 2 mmHg, P < 0.05). In conclusion, placental sFlt-1 and ANGII production are significantly elevated in response to placental ischemia in pregnant rats. In addition, AT1 receptor activation, by endogenous ANGII, appears to play an important role in mediating the placental production of sFlt-1 in response to placental ischemia in pregnant rats.

Evaluation of Maternal Thyroid Hormone Status and Atherogenic Potency in Pre Eclampsia in Kolar District- A South West of India

Aim: To determine maternal thyroid hormone status and lipid profile in preeclampsia. Study Design: A prospective case control study consist of two groups such as group 1 normotensive non-preeclamptic group and group 2 as cases with clinically diagnosed preeclampsia Place and Duration of Study: Department of Obstetrics and Gynaecology, RL Jalappa Hospital and Research Centre kolar, between January 2012 and June 2013. Methodology: A total number of 100 pregnant patients were enrolled in the present study. Amongst, normotensive and preeclamptic pregnant women Group 1 (n=50) as controls (n=50). Group-2 (n=50) were preeclampsia cases. Five ml of blood samples were collected from each normal pregnant and preeclampsia patients. Thyroid hormone levels and lipid parameters were estimated using spectrophotometric method. Statistical analysis carried out by using Mean ± SD, and ‘t’ test. Original Research Article Aparna et al.; BJMMR, 10(4): 1-8, 2015; Article no.BJMMR.19452 2 Results: The Mean ± SD values of T3 ng/ml (1.56±0.38), T4 (1.37±0.36) mcg/dl, TSH (2.45±1.23) mcIU/ml in normal pregnants and T3 ng/ml (1.37±0.36), T4 (9.87±2.83) mcg/dl, TSH (6.15±5.51) mcIU/ml in preeclampsia cases were presented. Similarly Mean ± SD values of total cholesterol (181.62±44.33) mg/dl, HDL cholesterol (49.44±11.54) mg/dl, Triglycerides (198.10±49.84) mg/dl, LDL cholesterol (104.90±29.58) mg/dl, and Total cholesterol/HDL cholesterol ratio (3.67) were from control group and total cholesterol (222.60±70.07) mg/dl, HDL cholesterol (45.92±11.81), Triglycerides (278.66±93.46) mg/dl, LDL cholesterol (116.52±49.26) mg/dl, and cholesterol and HDL cholesterol ratio (4.84) in preeclampsia were presented respectively. Conclusion: Pre eclampsia patients have higher levels of TSH and lower levels of T3 and T4 in comparison to normotensive pregnant women. This changes in the thyroid hormones did not correlate with the severity of preeclampsia between mild and severe pre eclampsia groups. Dyslipidemia was observed in pre eclampsia group than in the normotensive group indicates the possible atherogenic potential. This association may be useful in understanding the pathologic processes of preeclampsia.

262-POS

Objectives Preeclampsia (PreE) is thought to originate from an impaired vascular remodeling of the placental spiral arteries. Recent data suggests a possible role of cytochromeP450 (CYP450) vasoactive eicosanoids in the etiology of the disease. We tested the hypothesis that alterations in the profile of arachidonic acid (AA) metabolites of the CYP450 pathway may contribute to the impaired vascular remodeling in preeclampsia. Methods Proliferation of BeWo cells, an in vivo model of trophoblast syncytialisation, was measured in the presence of 20-HETE, the major CYP450 metabolite, and 17-ODYA, a mechanism-based suicide substrate CYP450 inhibitor, in order to determine the proliferative actions of 20-HETE. In addition, microsomes were isolated from placental vessels of normal pregnant (NP) and PreE women and CYP450 enzyme activity was determined by HPLC/mass spectrometry. Results Incubation of BeWo cells with 10 nM, 100 nM, and 1 μ M of 20-HETE displayed a dose-dependent increase in cell proliferation with an approximate 40% increase at 1 μ M, while inhibition of 20-HETE with 17-ODYA (10 μ M) displayed a 40% decrease in BeWo cell proliferation. Placental vascular microsomes isolated from PreE women displayed a significant increase in 20-HETE production relative to NP women. 15-HETE, 12-HETE, and total HETE production were also increased in PreE compared to NP. Conclusions Increases in 20-HETE production promote, while inhibition of 20-HETE reduces proliferation of trophoblasts in vitro. Increases in the production of an endogenous inhibitor of 20-HETE, such as15-HETE, may contribute to the reduced trophoblast invasion and impaired vascular remodeling seen in PreE. Disclosures N. Lee: None. S. Spencer: None. B.B. LaMarca: None. S. Murphy: None.

274-POS

Objectives Preeclampsia (preE), a syndrome of hypertension and proteinuria in pregnancy, is thought to be initiated with alterations of placental function. Hypoxia and oxidative stress can lead to placental apoptotic signaling, which pass the placental barrier and leave persistent defect in the circulation of the offspring that may predispose to a pathological response later in life. We assessed apoptotic signaling in placentas and umbilical cords from patients with or without preE. We also evaluated the pregnancy outcomes. Methods In this study, we recruited 20 normal pregnant (NP) and 20 preE consenting patients in an IRB approved prospective study. Inclusion criteria of preE patients include blood pressure >140/90 and proteinuria >300 mg of protein/24 h urine. Samples of placenta and umbilical cord were collected after deliveries. Pro-apoptotic Bcl-2-associated X protein (Bax), anti-apoptotic Bcl-2 protein, caspase 9 and pro-inflammatory protein cyclooxygenase-2 (Cox-2) expression were assayed both by western blot and immunohistochemistry. The p38 MAPK phosphorylation was evaluated by western blot. Comparisons were performed using ANOVA with Duncan’s post-hoc test. Results The ratio of Bax/Bcl-2 expression (Placenta: 1.2 fold, Cord: 1.5 fold), Cox-2 (Placenta: 0.8 fold, Cord: 2.5 fold), Caspase 9 (Placenta: 1.5 fold, Cord: 1.8 fold), and p38 phosphorylation (Placenta: 1.5 fold, Cord: 1.7 fold) were up-regulated (p Conclusions Apoptotic signaling is augmented in preE which alters the intrauterine environment by modulating the pattern of hormonal signals and activating the detrimental cellular signaling that has been transported to the fetus. Disclosures M.M. Co: None. D. Leonard: None. L. Perger: None. V.P. Govande: None. M.R. Beeram: None. R.O. Jones: None. S.R. Allen: None. T.J. Kuehl: None. M.N. Uddin: None.

526: Alterations in placental eicosanoids contribute to impaired vascular remodeling in preeclampsia

526: Alterations in placental eicosanoids contribute to impaired vascular remodeling in preeclampsia

Abstract 027: T cell-Dependent B cell Activation Plays a Role in Mediating Hypertension and Pathophysiology in Response to CD4+ T cells from Reduced Uterine Perfusion Pregnant Rats

Preeclampsia, (PE) newly developed hypertension during pregnancy, is associated with altered immune activation. We have previously shown that adoptive transfer of CD4+T cells from a rat model of PE, the RUPP Rat, increases blood pressure, endothelin (ET) expression, oxidative stress (ROS), inflammation, and agonist autoantibodies to the AT1 receptor in NP recipients, all of which have been shown to play a role in hypertension during pregnancy. The objective of this study was to determine if blockade of lymphocyte crosstalk via the CD40-CD40 ligand interaction between RUPP CD4+ T cells with endogenous B cells in NP recipient rats would improve pathophysiology observed previously with RUPP CD4+ T cells. To do so splenic CD4+ T lymphocytes were magnetically separated, incubated with 2.5 μg/mL anti-CD40 ligand (αCD40L) overnight and transferred into Normal Pregnant (NP) rats on day 12 of gestation (NP+ αCD40L RUPP T cells). On day 19 of gestation, blood pressure (MAP), blood, and tissues were collected from all groups of rats. MAP was 99+/-1.2 in NP (n=18), 120+/-2.3 mmHg in control RUPP rats (n=19;P&lt;0.001); 121+/-1.2 in NP+RUPP T cells(n=5;P&lt;0.001); but only increased to 103 +/-1.7 in NP + αCD40L RUPP T cells (n=21) (P&lt;0.001 vs NP+RUPP T cells). Placental ROS increased from 160.4 +/- 31 in NP to 380.5 +/- 60.7 in RUPP (p&lt;0.05) and 318.6 +/-89 in NP+RUPP T cells. CD40 ligand binding reduced placental ROS to 118.7 +/-24 in NP + αCD40L RUPP T cells (p&lt;0.05). Plasma IL-6 increased from 38.71+/-8 in NP to 92.94+/-15 in RUPP (p&lt;0.05) and 107.4+/-26 in NP+RUPP T cells and only increased to 50.1+/-3.6 pg/ml in NP + αCD40L RUPP T cells. TNF-alpha increased from 10.9+/-5 in NP to 79+/-15 in RUPP (p&lt;0.05) and 23+/-17 in NP+RUPP T cells and was only 8+/-3 pg/ml in NP + αCD40L RUPP T cells. Plasma sFLT-1 increased from 79+/-14 in NP to 172+/-34 in RUPP (p&lt;0.05) and 107+/-9 in NP+RUPP T cells and was only 74+/-21 pg/ml in NP + αCD40L RUPP T cells. These studies indicate that placental ischemic induced T cell communication with B cells is one important mechanism of PE that leads to much of the pathophysiology of the disease.

Abstract 416: Hypertension During Pregnancy is Associated with Cerebral Damage in an Animal Model of severe preeclampsia

HELLP (hemolysis, elevated liver enzymes, low platelet) syndrome is a severe form of preeclampsia that is accompanied by hypertension, headaches, visual disturbances and cerebral infarctions during pregnancy. HELLP is associated with hypertension and elevations in TNF-α and IL-6, both of which have been shown to activate the endothelin-1 system (ET-1). The objective of the current study was to determine if hypertension in an animal model of HELLP is associated with cerebral impairment. On gestational day (GD) 12 miniosmotic pumps infusing sEndoglin and sFlt-1 (7 and 4.7ug/kg/day) were implanted into normal pregnant (NP) rats to induce HELLP. On GD18 carotid catheters were inserted into HELLP and NP rats and mean arterial pressure (MAP) was recorded on GD19 followed by brain collection. Myogenic tone was calculated from the middle cerebral artery; brain water content was measured to determine cerebral edema. Blood brain barrier (BBB) permeability was determined by exposing cerebral vessels from non-pregnant rats intraluminally to plasma from NP or HELLP rats. MAP increased from 92±2.6mmHg in NP rats (n=10) to 120.6±3.6mmHg in HELLP rats (n=11; p&lt;0.01). HELLP rats had significant cerebral edema compared to NP rats (p=0.04). Myogenic tone in HELLP rats was significantly impaired (p&lt;0.05) at pressures greater than or equal to 80mmHg compared to NP rats (n=2/group). Plasma from HELLP rats (n=6) significantly increased BBB permeability at 100mmHg compared to plasma from NP rats (n=5). Administration of an endothelin A (ET A ) receptor antagonist (ABT-627, 5mg/kg on GD12-19), significantly decreased MAP in HELLP rats compared to untreated HELLP rats (97±5.3mmHg, n=5, p&lt;0.01) and decreased cerebral edema to levels comparable to NP rats. Data from this study demonstrates that high MAP in HELLP rats is accompanied by cerebral edema, increased BBB permeability and impaired myogenic tone. Any of which could contribute to the neurologic complications that plague women with HELLP. Furthermore, as elevated ET-1 can damage the BBB and lead to cerebral damage we administered an ET A receptor antagonist which significantly decreased MAP and cerebral edema; future studies will determine if these changes are accompanied by improvement in myogenic tone and BBB permeability.

Abstract 294: CD4 + T cells Isolated from Preeclamptic Women Produce a Preeclampsia-Like Phenotype in Pregnant Nude-Athymic Rats

Preeclampsia (PE) is the development of hypertension, with proteinuria, during the second half of pregnancy and is associated with an altered immune function, resulting in a chronic state of inflammation, which is believed to play an important role to increase vasoconstrictors such as Endothelin (ET-1) and autoantibodies as well as much of the pathophysiology associated with PE. It has been previously shown that women with PE have an altered CD4 + T cell population compared to normal pregnant women. In addition, Ana Zenclussen’s lab has shown that adoptive transfer of activated T helper 1 like splenocytes increased blood pressure, fetal rejection and cytokines secreted from uterine cells when transferred to normal pregnant mice. However, mechanisms causing the increased blood pressure and renal abnormalities were not further investigated. The purpose of this study was to determine if CD4 + T cells isolated from placentas of PE patients have altered consequences during pregnancy compared to those collected from normal pregnant (NP) women that may further define their role in causing the pathophysiology of PE. CD4 + T cells were isolated from placentas of PE and NP women, using magnetic anti-CD4 antibodies, cultured in TexMACS medium (Miltenyi Biotec) with IL-2 at 37°C in 5% CO 2 , and injected intraperitoneally into first timed pregnant, nude-athymic rats on day 12 of gestation. On day 18 carotid catheters were implanted and on day 19 MAP was measured and blood and tissues were collected. MAP was significantly increased from 125±3 mmHg in rats injected with NP CD4 + T cells to 140±9 mmHg in rats injected with PE CD4 + T cells. Circulating inflammatory cytokines IL-6 and TNF-α were measured and found to be increased in rats exposed to PE vs NP CD4 + T cells (TNF-α- PE=142.4 pg/mL, NP=79.4 pg/mL; IL-6 - PE=311.6 pg/mL, NP=277.8 pg/mL). Renal cortical ET-1 mRNA expression was found to be increased 4.5 fold in rats exposed to PE CD4+ T cells compared to those exposed to NP CD4+ T cells (p=0.06). These data suggest an important role for placental CD4 + T cells stimulated in women with PE to cause hypertension and inflammation and renal abnormalities during the course of this disease.

Abstract 580: Apoptotic Signaling Is Augmented In Preeclampsia: An Adverse Impact On The Offspring

Objective: Preeclampsia (preE), a syndrome of hypertension and proteinuria in pregnancy, is thought to be initiated with alterations of placental function. Hypoxia and oxidative stress can lead to placental apoptotic signaling, which pass the placental barrier and leave persistent defect in the circulation of the offspring that may predispose to a pathological response later in life. We assessed apoptotic signaling in placentas and umbilical cords from patients with or without preE. We also evaluated the pregnancy outcomes. Methods: In this study, we recruited 20 normal pregnant (NP) and 20 preE consenting patients in an IRB approved prospective study from Scott &amp; White hospital, Temple, Texas. Inclusion criteria for determination of preE patients include blood pressure &gt;140/90 and presence of proteinuria &gt;300 mg of protein/24h urine. Samples of placenta and umbilical cord were collected from those subjects after deliveries. Apoptotic and stress signaling proteins; pro-apoptotic Bcl-2-associated X protein (Bax), anti-apoptotic Bcl-2 protein, caspase 9 and pro-inflammatory protein cyclooxygenase-2 (Cox-2) expression were assayed both by western blot and immunohistochemistry. The p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation was evaluated by western blot. Comparisons were performed using ANOVA with Duncan’s post-hoc test. Results: The ratio of Bax/Bcl-2 expression (Placenta: 1.2 fold, Cord: 1.5 fold), Cox-2 (Placenta: 0.8 fold, Cord: 2.5 fold), Caspase 9 (Placenta: 1.5 fold, Cord: 1.8 fold), and p38 MAPK phosphorylation (Placenta: 1.5 fold, Cord: 1.7 fold) were up-regulated (p&lt;0.05) in placental tissues and umbilical cords of preE compared to NP patients. We did follow up examination of the babies for both groups of patients to assess the pregnancy outcome. Average hospital stay for PreE babies were significantly longer than those of NP babies (3.4 vs 6.1). There were no complications has been reported for the NP babies. However, all of preE babies had multiple medical complications. Conclusions: Apoptotic signaling is augmented in preE which alters the intrauterine environment by modulating the pattern of hormonal signals and activating the detrimental cellular signaling that has been transported to the fetus.

Abstract 093: Progesterone Supplementation Improves Blood Pressure And Uterine Artery Resistance In Response To Placental Ischemia During Pregnancy

Preeclampsia (PE), new onset hypertension at 20 weeks of gestation, is characterized by increased uterine artery resistance index (UARI), chronic immune activation and decreased of vasodilators such as nitric oxide (NO). Despite being one of the leading causes of death in pregnant women, currently there is no effective treatment for PE except for early delivery of the fetus. We have demonstrated that PE women have significantly lower circulating progesterone than normal pregnant (NP). 17-alpha-hydroxyprogesterone caproate (17-OHPC) is a synthetic metabolite of progesterone used for the prevention of recurrent preterm birth and although the mechanism is not understood, is suspected to have vasodilatory and anti-inflammatory effects. Therefore, we hypothesized that progesterone (17-OHPC) supplementation could reduce blood pressure (MAP), pro-inflammatory cytokines, CD4+ T cells, UARI, as well as increase NO bioavailability in a hypertensive rat model of PE. To address this question 17-OHPC (3.32mg/kg) was intraperitoneally administered on day 18 of gestation into Reduced Uterine Perfusion Pressure (RUPP) rats and carotid catheters were inserted. MAP, blood and tissues were collected on day 19. MAP in NP rats (n=13) was 92±2; 123±2 in RUPP (n=18), and 116 ±1 mmHg in RUPP+17-OHPC (n=10), p &lt;0.05. UARI was 0.78±0.03 in RUPP (n=4) and 0.63±0.038 in RUPP+17-OHPC (n=8), p&lt;0.05. Circulating CD4+ T cells were 1.19±1.0% of gated cells in NP (n=7), which increased to 8.52±2.4% in RUPP rats (n=10) but was significantly reduced to 2.72±0.87% (n=14) in RUPP + 17 O-HPC, p &lt;0.05. Total circulating nitrate/nitrite was 26.34 ±3.5 μM in NP (n=12); 14.58±3.1 in RUPP rats (n=8) and increased to 26.69±1.62 in RUPP+17-OHPC (n=7), p &lt;0.05. Aortic eNOS expression was 0.65±0.11 A.U in NP (n=4), which decreased to 0.33±0.01 in RUPP rats (n=4) but increased to 0.57±0.01 in RUPP+17-OHPC (n=5), p &lt;0.05. Levels of TNF-alpha were 65.84±17.7 pg/ml in RUPP rats (n=5) but were blunted to 17.24±3.9 in RUPP+17-OHPC (n=8), p &lt;0.05. In conclusion, 17-OHPC supplementation improves inflammation, UARI, hypertension, and nitric oxide bioavailability in response to placental ischemia during pregnancy and should therefore be considered further for addition to the clinical management of PE

Abstract 292: Vitamin D Supplementation Reduces CD4+ T Cells, Blood Pressure and Autoantibodies to the AT1 Receptor (AT1-AA) in a Rat Model of Preeclampsia

Preeclampsia (PE) is characterized by the development of hypertension, endothelial dysfunction and chronic immune activation after 20 weeks of gestation. PE women exhibit elevated CD4 + T cells, proinflammatory cytokines and agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA). Vitamin D deficiency is associated with increased prevalence of hypertension and chronic inflammatory diseases both in the presence and absence of pregnancy. We utilize the Reduced Uterine Perfusion Pressure (RUPP) model of PE in the pregnant rat which exhibits hypertension, endothelial dysfunction as well as increased CD4 + T cells, inflammatory cytokines and AT1-AA. Therefore, we tested the hypothesis that administration of exogenous Vitamin D2 (VD2) and Vitamin D3 (VD3) would ameliorate hypertension and or chronic inflammation associated with placental ischemia in the RUPP rat. For these studies, the RUPP procedure was performed on gestation day 14 (GD14) and normal pregnant (NP) and RUPP rats were treated daily with 50uM VD2 or VD3 via oral gavage on GD14-18. Carotid catheters were inserted on GD18 and blood pressure (MAP), blood and tissues were collected on GD19. Flow cytometry was used to evaluate CD4 + T cells in control RUPP rats and RUPP rats treated with Vitamin D. On GD19, MAP was increased in RUPP rats to 123 ± 3.5 compared to 108.8 ± 4.3 mmHg in NP rats (n=5-6, p&lt;0.05). Supplementation with either VD2 or VD3 lowered MAP to 113 ± 6.9 or 115.4 ± 2.3 mmHg, respectively (n=4-5). CD4 + T cells in RUPP compared to NP rats increased from 2.80 ± 0.95 to 8.49 ± 1.52 percent (n=8-11, p&lt;0.05). Circulating CD4 + T cells fell to 1.22 ± 0.41 percent with VD2 and to 4.59 ± 3.67 percent with VD3 (n=2-3). AT1-AA levels, measured via chronotropic response in cardiomyocytes, were 19.50 ± 0.44 beats/min in RUPP rats and decreased to 15.35 ± 0.69 with VD3, and further to 8.33 ± 0.51 with VD2 supplementation (n=5, p&lt;0.05). Taken together, these data indicate that pharmaceutical Vitamin D supplementation reduced blood pressure and markers of chronic inflammation in a rat model of PE, thereby suggesting a role for Vitamin D supplementation to improve hypertension and chronic inflammation in response to diseases associated with placental ischemia, such as PE.

Oxidative stress signaling in response to CD4+T cells stimulated via placental ischemia play an important role in hypertension during preeclampsia (860.14)

Preeclampsia (PE) is associated with hypertension, oxidative stress, and chronic inflammation. The RUPP rat model of PE and normal pregnant (NP) recipient rats of RUPP CD4+T cells exhibit much of the disease pathology. Reactive oxygen species (ROS) could be released from monocytes or neutropils, however the objective of this study was to determine if RUPP CD4 T cells are a cellular source of ROS and hypertension (MAP) and during pregnancy. CD4+Tcells from RUPP rats were injected into NP recipients on gestational day (GD)13; GD19 MAP, urine and tissues were analyzed. Prior to injection RUPP CD4+ T cells secreted more myleoperoxidase compared to NP (116.9+15.2 vs. 48.5+8.3 ng/mg/mL; p=0.003). Placental NADPH oxidase mediated ROS in RUPP rats was 830.6+68, 726.2+43.73 in NP + RUPP CD4+ T cells and 420.7+51.4 RLU/min/mg in NP rats; p=0.009 and p=0.004, respectively. MAP was 100+2 in NP rats, 126+4 in RUPP rats (p=0.0001) and 118+4mmHg in NP+RUPP CD4+ T cell rats (p=0.002) which was attenuated with apocynin; 111+2 in RUPP + apocynin; 102+3 mmHg in NP + RUPP CD4+ T cells (p=0.01). These data suggest that CD4+Tcells are an important source of ROS mediating hypertension in response to placental ischemia.Grant Funding Source: NIH NICHD; APS STRIDE

Normal pregnant T regs inhibit pathophysiology associated with hypertension in response to reduced uterine perfusion pressure in pregnant rats (860.18)

The RUPP rat model of preeclampsia (PE) exhibits much of the pathology characterizing this disease such as hypertension, inflammation, suppressed regulatory Tcells (Tregs), oxidative stress (ROS), and AT1‐AA during pregnancy. The objective of this study was to determine if supplementation of normal pregnant (NP) Tregs into RUPP rats would attenuate the pathophysiology associated with preeclampsia. CD4+/CD25+ T cells were isolated from spleens of NP rats and differentiated into Tregs in culture. On day 12 of gestation NP Tregs were injected into pregnant rats undergoing RUPP; day18 carotid catheters were inserted and day19 blood pressure (MAP) was recorded, blood and tissues were analyzed. MAP increased from 100±2 mmHg in NP (n=17) to 125±2 mmHg in RUPP (n=22) but was 117±3 mmHg in RUPP+NPTregs (n=18; p&lt;0.05). Importantly, pup weight was 1.91±0.06 in RUPPs but increased to 2.06 ±0.09 in RUPP + NPTregs (p&gt;0.05). Inflammatory markers in RUPPs decreased with NP Tregs; placental and renal ROS was 339 and 603 RLU in RUPP but was 178 and 171 RLU in RUPP+Tregs (p&lt;0.05); TNF‐α was 20.9 pg/mL in RUPP and 12.59 pg/mL in RUPP+Tregs rats (p&gt;0.5); AT1‐AA was 17.27 bpm in RUPP but was attenuated with NP Tregs to 0.50 bpm (p&gt;.0001). We conclude that suppressed Tregs may play an important role in the inflammation and hypertension associated with PE.Grant Funding Source: Supported by NIH grants RO1HD067541 and T32HL105324