Reduced Uterine Perfusion Pressure Research Articles

THE IMPORTANCE OF B2 CELLS IN THE HYPERTENSION AND PATHOPHYSIOLOGY OF PLACENTAL ISCHEMIA DURING PREGNANCY

Objective: Preeclampsia (PE), is characterized by placental ischemia, inflammation, hypertension and IUGR. Antibodies activating the angiotensin II type I receptor (AT1-AA) activate cytolytic Natural Killer cells (NK) and cause clinical features of PE. The Reduced uterine perfusion pressure (RUPP) pregnant rat model of PE has elevated AT1-AA, NK cells and blood pressure. B cell depletion or AT1-AA epitope binding protein (‘n7AAc’) improved hypertension and AT1-AA activity and NK cells in RUPP rats. One unanswered question is the importance of B2 cells vs B1 cells in the production of the AT1-AA and thus the pathology of PE. Design and method: To test this hypothesis, splenic B Cells and B2 cells were isolated from RUPP rats on Gestation Day (GD) 19 and transferred into NP rats on GD12. Blood pressure (MAP) and cNKs were measured by flow cytometry on GD19. To test the role of AT1-AA in the hypertension, ‘n7AAc’ was administered to a group of recipient rats on GD14. Results: MAP increased in NP+RUPP B cells to 117 ± 1 mmHg, n = 10, and to 111 mmHg ± 3, n = 8, in NP+RUPP B2 cells, compared to NP rats, n = 10 (p < 0.001). ‘n7AAc’ attenuated hypertension in response to total RUPP B cells (99 mmHg ± 6, n = 5)(p < 0.01) and in response to RUPP B2 s (100 mmHg ± 5, p = 0.08). Circulating and placental cNKs were elevated with total RUPP B cells and RUPP B2 cells compared to NP. Circulating cNKs were elevated in NP+RUPPBcells (6 ± 2.5, n = 5)(p < 0.01) and in NP+RUPP B2 cells (8 ± 3.33, n = 4)(p < 0.01) compared to NP (0.67 ± 0.33, n = 10). Placental cNKs were elevated in NP+RUPPBcells (2.32 ± 0.73, n = 6)(p < 0.01) and in NP+RUPP B2 cells (1.33 ± 0.24, n = 3)(p < 0.01) compared to NP (0.2250 ± 0.133, n = 10). Conclusions: We conclude that RUPP total B Cells and RUPP B2 cells cause hypertension and NK cell activation via secretion of the AT1-AA during pregnancy. However, our studies do not rule out the importance of B1 cells in hypertension and AT1-AA production during PE or RUPP. Future studies from our laboratory will further examine B1 cells in the pathology of PE.

Protective role of SIRT1-mediated Sonic Hedgehog signaling pathway in the preeclampsia rat models.

To explore the role of silent information regulator 1 (SIRT1)-mediated Sonic Hedgehog (SHH) pathway in reduced uterine perfusion pressure (RUPP) model of preeclampsia (PE) in rats. The pregnant rats were divided into sham, RUPP, RUPP + rSIRT1 (recombinant SIRT1 protein), RUPP + rSHH (recombinant SHH protein), and RUPP + rSIRT1+ Cy (cyclopamine, an SHH pathway inhibitor) groups, followed by the determination of mean arterial pressure (MAP) and pregnancy outcome. The gene or protein expression was determined by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction (qRT-PCR), or Western blotting. RUPP rats showed increases MAP with the lower levels of vascular endothelial growth factor (VEGF) and nitrite and nitrate (NOx), as well as the higher levels of soluble FMS-like tyrosine kinase-1 (sFlt-1), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in maternal plasma, which was attenuated after rSIRT1 or rSHH treatment. Besides, the improvement in the pregnancy outcome was seen in the rats from the RUPP + rSIRT1/rSHH groups as compared with the RUPP group. However, the therapeutic effect of rSIRT1 was reversed by cyclopamine. Placenta tissues of RUPP rats manifested the down-regulations of SIRT1, Patched-1 (PTCH1), and GLI family zinc finger 2 (GLI2), which were up-regulated in the RUPP + rSIRT1 group. SIRT1 was down-regulated while SHH pathway was inhibited in the placental tissue of PE rats. SIRT1 improved the blood pressure, angiogenic imbalance, inflammation, and pregnancy outcome in PE rats via SHH pathway, supporting its potential use for the treatment of PE.

Soluble guanylate cyclase stimulation in late gestation does not mitigate asymmetric intrauterine growth restriction or cardiovascular risk induced by placental ischemia in the rat.

Stimulation of soluble guanylate cyclase (sGC) improves fetal growth at gestational day 20 in the reduced uterine perfusion pressure (RUPP) rat model of placental ischemia suggesting a role for sGC in the etiology of intrauterine growth restriction (IUGR). This study tested the hypothesis that stimulation of sGC until birth attenuates asymmetric IUGR mitigating increased cardiovascular risk in offspring. Sham or RUPP surgery was performed at gestational day 14 (G14); vehicle or the sGC stimulator Riociguat (10 mg/kg/day sc) was administered G14 until birth. Birth weight was reduced in offspring from RUPP [intrauterine growth restricted (IUGR)], sGC RUPP (sGC IUGR), and sGC Sham (sGC Control) compared with Sham (Control). Crown circumference was maintained, but abdominal circumference was reduced in IUGR and sGC IUGR compared with Control indicative of asymmetrical growth. Gestational length was prolonged in sGC RUPP, and survival at birth was reduced in sGC IUGR. Probability of survival to postnatal day 2 was also significantly reduced in IUGR and sGC IUGR versus Control and in sGC IUGR versus IUGR. At 4 mo of age, blood pressure was increased in male IUGR and sGC IUGR but not male sGC Control born with symmetrical IUGR. Global longitudinal strain was increased and stroke volume was decreased in male IUGR and sGC IUGR compared with Control. Thus late gestational stimulation of sGC does not mitigate asymmetric IUGR or increased cardiovascular risk in male sGC IUGR. Furthermore, late gestational stimulation of sGC is associated with symmetrical growth restriction in sGC Control implicating contraindications in normal pregnancy.NEW & NOTEWORTHY The importance of the soluble guanylate cyclase-cGMP pathway in a rat model of placental ischemia differs during critical windows of development, implicating other factors may be critical mediators of impaired fetal growth in the final stages of gestation. Moreover, increased blood pressure at 4 mo of age in male intrauterine growth restriction offspring is associated with impaired cardiac function including an increase in global longitudinal strain in conjunction with a decrease in stroke volume, ejection fraction, and cardiac output.

Impact of hyperleptinemia during placental ischemia-induced hypertension in pregnant rats.

The prevalence of preeclampsia and obesity have increased. Although obesity is a major risk factor for preeclampsia, the mechanisms linking these morbidities are poorly understood. Circulating leptin levels increase in proportion to fat mass. Infusion of this adipokine elicits hypertension in nonpregnant rats, but less is known about how hyperleptinemia impacts blood pressure during placental ischemia, an initiating event in the pathophysiology of hypertension in preeclampsia. We tested the hypothesis that hyperleptinemia during reduced uterine perfusion pressure (RUPP) exaggerates placental ischemia-induced hypertension. On gestational day (GD) 14, Sprague-Dawley rats were implanted with osmotic mini-pumps delivering recombinant rat leptin (1 µg/kg/min iv) or vehicle concurrently with the RUPP procedure to induce placental ischemia or Sham. On GD 19, plasma leptin was elevated in Sham + Leptin and RUPP + Leptin. Leptin infusion did not significantly impact mean arterial pressure (MAP) in Sham. MAP was increased in RUPP + Vehicle vs. Sham + Vehicle. In contrast to our hypothesis, placental ischemia-induced hypertension was attenuated by leptin infusion. To examine potential mechanisms for attenuation of RUPP-induced hypertension during hyperleptinemia, endothelial-dependent vasorelaxation to acetylcholine was similar between Sham and RUPP; however, endothelial-independent vasorelaxation to the nitric oxide (NO)-donor, sodium nitroprusside, was increased in Sham and RUPP. These findings suggest that NO/cyclic guanosine monophosphate (cGMP) signaling was increased in the presence of hyperleptinemia. Plasma cGMP was elevated in Sham and RUPP hyperleptinemic groups compared with vehicle groups but plasma and vascular NO metabolites were reduced. These data suggest that hyperleptinemia during placental ischemia attenuates hypertension by compensatory increases in NO/cGMP signaling.NEW & NOTEWORTHY Ours is the first study to examine the impact of hyperleptinemia on the development of placental ischemia-induced hypertension using an experimental animal model.

Effect of edaravone on pregnant mice and their developing fetuses subjected to placental ischemia

Growing evidence indicates that reduced uterine perfusion pressure (RUPP) triggers the cascade of events leading to preeclampsia. Edaravone is a powerful free radical scavenger used for the treatment of ischemia/reperfusion diseases due to its anti-oxidative stress and anti-inflammatory properties. Here we investigate the effect of edaravone (3 mg/kg) on different maternal and fetal outcomes of RUPP-induced placental ischemia mice model. RUPP surgery was performed on gestation day (GD) 13 followed by edaravone injection from GD14 to GD18, sacrifice day. The results showed that edaravone injection significantly decreased the maternal blood pressure (113.2 ± 2.3 mmHg) compared with RUPP group (131.5 ± 1.9 mmHg). Edaravone increased fetal survival rate (75.4%) compared with RUPP group (54.4%), increased fetal length, weights, and feto-placental ratio (7.2 and 5.7 for RUPP and RUPP-Edaravone groups, respectively) compared with RUPP group. In addition, RUPP resulted in many fetal morphological abnormalities as well as severe delayed ossification, however edaravone decreased the morphological abnormalities and increased the ossification of the fetal endoskeleton. Edaravone improved the histopathological structure of the maternal kidney and heart as well as decreased the elevated blood urea and creatinine levels (31.5 ± 0.15 mg/dl (RUPP), 25.6 ± 0.1 mg/dl (RUPP+edaravone) for urea and 5.4 ± 0.1 mg/dl (RUPP), 3.5 ± 0.1 mg/dl (RUPP+edaravone) for creatinine) and decreased cleaved caspase-3 expression in the maternal kidney. In conclusion, this study demonstrated that our RUPP mice model recapitulated preeclampsia symptoms and edaravone injection ameliorated most of these abnormalities suggesting its effectiveness and potential application in preeclampsia treatment regimes.

Progesterone-induced blocking factor improves blood pressure, inflammation, and pup weight in response to reduced uterine perfusion pressure (RUPP).

Preeclampsia (PE) is characterized by new-onset hypertension in association with elevated natural killer (NK) cells and inflammatory cytokines, which are likely culprits for decreased fetal weight during PE pregnancies. As progesterone increases during normal pregnancy, it stimulates progesterone-induced blocking factor (PIBF). PIBF has been shown to decrease inflammation and cytolytic NK cells, both of which are increased during PE. We hypothesized that PIBF reduces inflammation as a mechanism to improve hypertension in the preclinical reduced uterine perfusion pressure (RUPP) rat model of PE. PIBF (2.0 µg/mL) was administered intraperitoneally on gestational day 15 to either RUPP or normal pregnant (NP) rats. On day 18, carotid catheters were inserted. Mean arterial blood pressure (MAP) and samples were collected on day 19. MAP in NP rats (n = 11) was 100 ± 2 mmHg and 105 ± 3 mmHg in NP+PIBF rats (n = 8) and 122 ± 1 mmHg in RUPP rats (n = 10), which improved to 110 ± 2 mmHg in RUPP+PIBF rats (n = 11), P < 0.05. Pup weight was 2.4 ± 0.1 g in NP, 2.5 ± 0.1 g in NP+PIBF, 1.9 ± 0.1 g in RUPP, and improved to 2.1 ± 0.1 g in RUPP+PIBF rats. Circulating and placental cytolytic NK cells, IL-17, and IL-6 were significantly reduced while IL-4 and T helper (TH) 2 cells were significantly increased in RUPP rats after PIBF administration. Importantly, vasoactive pathways preproendothelin-1, nitric oxide, and soluble fms-Like tyrosine Kinase-1 (sFlt-1) were normalized in RUPP+PIBF rats compared with RUPP rats, P < 0.05. Our findings suggest that PIBF normalized IL-4/TH2 cells, which was associated with improved inflammation, fetal growth restriction, and blood pressure in the RUPP rat model of PE.

784 Evaluation of hyaluronic acid as a possible biomarker in rat models of preeclampsia

Endothelial injury and hypertension are principal features of preeclampsia (PE), suggesting that plasma endothelial glycocalyx fragments are potential biomarkers. We aimed to: 1) compare the newer ‘selective reduced uterine perfusion pressure’ (sRUPP) model and the traditional RUPP model in their ability to simulate PE; and 2) determine whether plasma hyaluronic acid (HA), a major endothelial glycocalyx component, differs in the two models as compared to sham rats. Thirty-two timed-pregnant Sprague-Dawley rats underwent sham surgery (n=9), sRUPP procedure (n=10), or RUPP procedure (n=13) on gestational day 14. On day 18, each rat underwent carotid artery cannulation, and conscious mean arterial pressure (MAP; PowerLab, ADInstruments, CO) was measured on day 19, followed by plasma and tissue collection. Pup survival rate was calculated based on the number of pups on day 19 vs. day 14. Plasma HA on day 19 was quantified by Quantikine ELISA (R&D Systems, MN). Statistical significance was determined by one-way ANOVA or Kruskal-Wallis test, with post-hoc analyses as appropriate. Both the sRUPP and RUPP rats had significant yet comparable pup resorption (p<0.001) and blunted maternal weight gain (p<0.001) vs. sham rats, suggesting underlying placental ischemia. The RUPP rats showed significantly higher MAP than sham rats (p<0.01); this increase was approximately twice as large as that of the sRUPP rats. There was a trend towards increased plasma HA concentration in the RUPP animals, though it did not differ significantly across all three groups. Our preliminary data suggest that the rat RUPP model is superior to the sRUPP model in modeling blood pressure changes of PE; the latter may represent a milder disease phenotype. Plasma HA may not be a reliable marker in either model. Given the current evidence for endothelial glycocalyx damage occurring in human PE, future studies will investigate the role of other endothelial glycocalyx markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Investigation of interleukin-2-mediated changes in blood pressure, fetal growth restriction, and innate immune activation in normal pregnant rats and in a preclinical rat model of preeclampsia

Two important clinical features of preeclampsia (PE) are hypertension and fetal growth restriction. The reduced uterine perfusion pressure (RUPP) preclinical rat model of PE exhibits both of these features. Moreover, RUPP and PE women have elevated vasoconstrictor peptide endothelin-1 (ET-1) and inflammation. Interleukin-2 (IL-2) is a cytokine that regulates NK cell activity and is elevated in miscarriage, PE, and RUPP rats. The objective of this study was to examine a role for IL-2 in NK cell activation, fetal growth restriction, and hypertension during pregnancy by either infusion of IL-2 or blockade of IL-2 (basiliximab) in normal pregnant (NP) and RUPP rats. On gestational day 14, NP and RUPP rats received low (LD), middle (MD), or high dose (HD) IL-2 (0.05, 0.10, or 0.20 ng/ml) IP or basiliximab (0.07 mg per rat) by IV infusion. On day 19, blood pressure (MAP), pup weights, and blood were collected. Basiliximab had no effect on blood pressure, however, significantly lowered NK cells and may have worsened overall fetal survival in RUPP rats. However, IL-2 LD (102 ± 4 mmHg) and IL-2 HD (105 ± 6 mmHg) significantly lowered blood pressure, ET-1, and activated NK cells compared to control RUPPs (124 ± 3 mmHg, p < 0.05). Importantly, IL-2 in RUPP rats significantly reduced fetal weight and survival. These data indicate that although maternal benefits may have occurred with low dose IL-2 infusion, negative effects were seen in the fetus. Moreover, inhibition of IL-2 signaling did not have favorable outcome for the mother or fetus.

Adoptive transfer of placental ischemia-stimulated natural killer cells causes a preeclampsia-like phenotype in pregnant rats.

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many characteristics of preeclampsia including maternal hypertension, intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). While we have previously shown a 5-fold higher cytotoxicity of RUPP NKs versus normal pregnant NKs, their role in RUPP pathophysiology remains unclear. In this study, we tested the hypotheses that (1) adoptive transfer of RUPP-stimulated NKs will induce maternal hypertension and IUGR in normal pregnant control (Sham) rats and (2) adoptive transfer of Sham NKs will attenuate maternal hypertension and IUGR in RUPP rats. On gestation day (GD)14, vehicle or 5×106 RUPP NKs were infused i.v. into a subset of Sham rats (Sham+RUPP NK), and vehicle or 5×106 Sham NKs were infused i.v. into a subset of RUPP rats (RUPP+Sham NK; n=12/group). On GD18, Uterine Artery Resistance Index (UARI) was measured. On GD19, mean arterial pressure (MAP) was measured, animals were sacrificed, and blood and tissues were collected for analysis. Adoptive transfer of RUPP NKs into Sham rats resulted in elevated NK activation, UARI, placental oxidative stress, and preproendothelin expression as well as reduced circulating nitrate/nitrite. This led to maternal hypertension and IUGR. RUPP recipients of Sham NKs demonstrated normalized NK activation, sFlt-1, circulating and placental VEGF, and UARI, which led to improved maternal blood pressure and normal fetal growth. These data suggest a direct role for cNKs in causing preeclampsia pathophysiology and a role for normal NKs to improve maternal outcomes and IUGR during late gestation.

CD4+ T cells cause renal and placental mitochondrial oxidative stress as mechanisms of hypertension in response to placental ischemia.

The reduced uterine perfusion pressure (RUPP) rat model and normal pregnant (NP) rat recipients of RUPP CD4+ T cells recapitulate many characteristics of preeclampsia such as hypertension and oxidative stress. We have shown an important hypertensive role for natural killer (NK) cells to cause mitochondrial dysfunction in RUPP rats; however, the role for RUPP CD4+ T cells to stimulate NK cells is unknown. Therefore, we hypothesized that RUPP-induced CD4+ T cells activate NK cells to cause mitochondrial dysfunction/reactive oxygen species (ROS) as mechanisms of hypertension during pregnancy. We tested our hypothesis by adoptive transfer of RUPP CD4+T cells into NP rats or by inhibiting the activation of RUPP CD4+T cells with Orencia (abatacept) and examining hypertension, NK cells, and mitochondrial function. RUPP was performed on gestation day (GD) 14, and splenic CD4+ T cells were isolated on GD 19 and injected into NP rats on GD 13. In a separate group of rats, Orencia was infused and the RUPP procedure was performed. Mean arterial pressure and placental and renal mitochondrial ROS increased in RUPP (n = 7, P < 0.05) and NP+RUPP CD4+ T-cell recipients (n = 13, P < 0.05) compared with control NP (n = 7) and NP+NP CD4+ T-cell recipients (n = 5) but was reduced with Orencia (n = 13, P < 0.05). Placental and renal respiration was reduced in RUPP (n = 6, P < 0.05) and NP+RUPP CD4+ T-cell recipients (n = 6, state 3 P < 0.05) compared with NP (n = 5) and NP+NP CD4+ T-cell recipients (n = 5) but improved with Orencia (n = 9, n = 8 P < 0.05). These data indicate that CD4+ T cells, independent of NK cells, cause mitochondrial dysfunction/ROS contributing to hypertension in response to placental ischemia during pregnancy.

Stimulation of soluble guanylate cyclase diminishes intrauterine growth restriction in a rat model of placental ischemia.

Placental ischemia in preeclampsia (PE) results in hypertension and intrauterine growth restriction (IUGR). Stimulation of soluble guanylate cyclase (sGC) reduces blood pressure in the clinically relevant reduced uterine perfusion pressure (RUPP) rat model of PE, implicating involvement in RUPP-induced hypertension. However, the contribution of sGC in the development of IUGR in PE is not known. Thus, this study demonstrated the efficacy of Riociguat, an sGC stimulator, in IUGR reversion in the RUPP rat model of PE, and tested the hypothesis that improvement in fetal weight occurs in association with improvement in placental perfusion, placental morphology, and placental nutrient transport protein expression. Sham or RUPP surgery was performed at gestational day 14 (G14) with administration of vehicle (Sham or RUPP) or the sGC stimulator (Riociguat, 10 mg/kg/day sc; sGC-treated) until G20. Fetal weight was reduced (P = 0.004) at G20 in RUPP but not in sGC-treated RUPP compared with Sham, the control group. At G20, uterine artery resistance index (UARI) was increased (P = 0.010) in RUPP, indicating poor placental perfusion; proportional junctional zone surface area was elevated (P = 0.035), indicating impaired placental development. These effects were ameliorated in sGC-treated RUPP. Placental protein expression of nutrient transporter heart fatty acid-binding protein (hFABP) was increased (P = 0.008) in RUPP but not in sGC-treated RUPP, suggesting a compensatory mechanism to maintain normal neurodevelopment. Yet, UARI (P < 0.001), proportional junctional zone surface area (P = 0.013), and placental hFABP protein expression (P = 0.008) were increased in sGC-treated Sham, suggesting a potential adverse effect of Riociguat. Collectively, these results suggest sGC contributes to IUGR in PE.

Angiotensin II type 1 receptor autoantibody blockade improves cerebral blood flow autoregulation and hypertension in a preclinical model of preeclampsia

ABSTRACT Introduction:Women with preeclampsia (PE) and reduced uterine perfusion pressure (RUPP) pre-clinical rat model of PE have elevated angiotensin II type 1 receptor agonistic autoantibodies (AT1-AA) and cerebrovascular dysfunction. Methods:Sprague Dawley rats had RUPP surgery with/without AT1-AA inhibitor (‘n7AAc’144 μg/day) osmotic minipumps. Mean arterial pressure (MAP), CBF autoregulation, blood brain barrier (BBB) permeability, cerebral edema, oxidative stress, and eNOS were assessed. Results:‘n7AAc’ improved MAP, restored CBF autoregulation, prevented cerebral edema, elevated oxidative stress, and increased phosphorylated eNOS protein in RUPP rats. Conclusion:Inhibiting the AT1-AA in placental ischemic rats prevents hypertension, cerebrovascular dysfunction, and improves cerebral metabolic function.

Abstract P213: Placental Ischemia-Stimulated Natural Killer Cells Contribute To Hypertension, Vascular Dysfunction, And Intrauterine Growth Restriction In Pregnant Rats

The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia mimics many characteristics of preeclampsia (PE) such as hypertension (HTN), intrauterine growth restriction (IUGR), and increased cytolytic natural killer cells (cNKs). We have previously shown that natural killer (NK) cell depletion in RUPP rats improves PE pathophysiology and that RUPP NKs have a 5-fold increase in cytotoxicity vs normal pregnant NKs. In this study, we tested the hypotheses that (1) RUPP stimulated NKs play a direct role in causing HTN and IUGR in pregnant rats and (2) normal pregnant control (Sham) NKs attenuate HTN and IUGR in RUPP rats. NKs were isolated from the placentas of Sham and RUPP rats on gestation day (GD) 19. On GD14, vehicle or 5x10 6 RUPP NKs were infused i.v. into a subset of Sham rats, and vehicle or 5x10 6 Sham NKs were infused i.v. into a subset of RUPP rats. On GD18, Uterine Artery Resistance Index (UARI) was measured via Doppler Ultrasound; GD19, cNKs were quantified via flow cytometry and MAP, fetal weight, and blood were acquired. Plasma VEGF and sFlt-1 were measured via ELISA. Placental cNKs (% gated) increased from 3±1% in Sham to 19±5% in RUPP and 21±4% in Sham+RUPP NK (p&lt;0.05 vs Sham), and decreased to 3±1% in RUPP+Sham NK (p&lt;0.05 vs RUPP). Circulating cNKs also followed this trend. MAP increased from 102±1 mmHg in Sham to 130±2 mmHg in RUPP and 121±2 mmHg in Sham+RUPP NK (p&lt;0.05 vs Sham), and was blunted to 113±1 mmHg in RUPP+Sham NK (p&lt;0.05 vs RUPP). Fetal weight decreased from 2.4±0.04 g in Sham to 2.1±0.07 g in RUPP and 2.1±0.03 g in Sham+RUPP NK (p&lt;0.05 vs Sham), and this was normalized to 2.3±0.03 g in RUPP+Sham NK (p&lt;0.05 vs RUPP). UARI increased from 0.56±0.05 in Sham to 0.75±0.06 in RUPP and 0.76±0.05 in Sham+RUPP NK (p&lt;0.05 vs Sham), and decreased to 0.64±0.05 in RUPP+Sham NK (p&lt;0.05 vs RUPP). Circulating sFlt-1 increased from 76±15 pg/mL in Sham to 1391±424 pg/mL in RUPP (p&lt;0.05 vs Sham), 780±256 pg/mL in Sham+RUPP NK, and decreased to 67±8 pg/mL in RUPP+Sham NK (p&lt;0.05 vs RUPP). Furthermore, circulating VEGF decreased in RUPP and Sham+RUPP NK compared to Sham (p&lt;0.05), and increased in RUPP+Sham NK (p&lt;0.05 vs RUPP). These data demonstrate a direct role for cNKs to mediate vascular dysfunction in PE and for normal NKs to promote positive maternal and fetal outcomes.

Abstract P225: Rupp Rat Model Cd4+ T Cells Activate Nk Cells And Cause Mitochondrial Oxidative Stress And Hypertension In Normal Pregnant Rats

Preeclampsia (PE) is new onset hypertension during pregnancy and is associated with elevated inflammatory response such as CD4+ T cells, NK cells, and cytokines. We have previously shown women with PE exhibit increases in circulating and placental CD4+T cells and placental mitochondrial (mt) dysfunction/ROS compared to normal pregnant (NP) women. The Reduced Uterine Perfusion Pressure (RUPP) rat model produces many characteristics of PE such as hypertension, increases in CD4+ cells, increases in renal and placental NK cells, and mt dysfunction/ROS. We have previously demonstrated that RUPP CD4+T cells cause hypertension in NP rats, however the role of RUPP CD4+ T cells in stimulating NK cells to cause mt dysfunction/ROS are not elucidated. Therefore, we examined the effect of adoptive transfer of RUPP CD4+ T cells to activate NK cells in NP rats. Splenic CD4+ T cells were isolated from RUPP rats, cultured, and injected into NP rats on GD 13. On GD19, MAP values and blood/tissue samples were collected from both RUPP CD4+ T cell recipients and NP controls. Mitochondrial respiration and mtROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader, respectively. A student’s t-test was used for statistical analysis. On GD19, MAP increased to 110±2 mmHg (n=13) in RUPP CD4+ T cell recipients compared to control NP rats 102±2 mmHg (n=7, p&lt;0.05). Circulating cytolytic NK cells increased to 3±0.6% in RUPP CD4+ T cell recipients (n=8) compared to NP controls 0.3±0.2% (n=7, p&lt;0.05). Placental state 3 (209.3±31.3 vs 422.7 ±83.3 pmol/sec/mg, p&lt;0.05) and maximal (152.1±46.2 vs 229.7±58.9 pmol/sec/mg) and renal state 3 (133.4 ±21.4 vs 289.8±43.4 pmol/sec/mg, p&lt;0.05) and maximal (61.8±18 vs 242.4±27.7 pmol/sec/mg, p&lt;0.05) respiration rates, indicative of ATP production and electron transport chain efficacy respectively, were reduced with RUPP CD4+ T cells (n=6; n=9) compared to NP (n=5; n=5). Collectively, the data indicate that the adoptive transfer of RUPP CD4+ T cells stimulates cytolytic NK cells and placental and renal mitochondrial dysfunction/ROS during pregnancy as important mechanisms of hypertension in the pathophysiology of preeclampsia. Keywords: Preeclamspia, Hypertension, Oxidative stress

Abstract P208: Imaging The Longitudinal Reduction In Placental Ischemia In Response To Therapeutic Intervention For Preeclampsia

Preeclampsia is believed to be induced by abnormal vascular remodeling and the resulting placental ischemia during early development. Sildenafil, a phosphodiesterase-5 inhibitor, reduces the maternal symptoms of preeclampsia by promoting angiogenesis and enhancing NO-mediated vasodilation. However, the effect of sildenafil on in vivo placental function has not been demonstrated. We performed longitudinal spectral photoacoustic (sPA) imaging of placental therapeutic response to sildenafil in the reduced uterine perfusion pressure (RUPP) model of preeclampsia. Sprague Dawley rats were administered sildenafil (S) via drinking water beginning on gestational day (GD) 11. Imaging was performed on GD14, 16, and 18 with the RUPP procedure implemented after the first imaging session. sPA images of oxygenation show that the placental ischemia induced by the RUPP surgery (RUPP, n=8, 48%; NP, n=8, 54%), was effectively eliminated by treatment with sildenafil (RUPP+S, n=8, 53%, p&lt;0.05). In addition to improved placental oxygenation, sildenafil was also found to reduce mean arterial pressure in RUPP animals by GD18 (RUPP, 110 mmHg; RUPP+S, 98 mmHg; p&lt;0.05), consistent with prior reports. Our studies demonstrate that sPA imaging can detect changes in placental oxygenation which could be used to indicate in vivo placental therapeutic response. Figure 1: Placental hypoxia in the RUPP is improved by treatment with sildenafil on GD16. B-mode US images (a, c) of anatomy were used to manually select the placenta (p) and average placental oxygenation was calculated. A custom red-blue oxygenation colormap was then overlaid for visualization (b, d). Scale bars = 3mm.

Abstract P211: B Cells Contribute To Hypertension And Cytolytic Nk Cell Activation In Response To Placental Ischemia In Pregnancy

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with inflammation and B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1-AA). Two recognized subsets of B Cells are B1 and B2 B Cells. B2 B Cells are involved in adaptive immune responses and produce specific antibodies. B1 B Cells produce natural antibodies and are implicated in the pathophysiology of PE through AT1-AA. The Reduced Uterine Perfusion Pressure (RUPP) rat model of placental ischemia recapitulates many characteristics of PE. We have previously shown that either B cell depletion or AT1-AA epitope binding protein (7AA) in RUPP rats improved hypertension. It is currently unknown which B cell subsets make the AT1AA in PE. This information would add to the design of new therapies targeting one B cell type for depletion. Therefore, we hypothesize that B cells and specifically B2 cells cause hypertension and NK cell activation during pregnancy through production of the AT1-AA. To test this hypothesis, total splenic B Cells and B2 cells were isolated from RUPP rats on Gestation Day (GD)19 RUPP and adoptively transferred into NP rats on GD12, blood pressure (MAP), placental and circulating cNKs were measured by flow cytometry on GD19. To test the role of AT1-AA in the hypertension, 7AA was administered to a group of recipient rats on GD14. MAP increased in NP+RUPP B cells to 116 ± 3 mmHg, n=11, and to 112 mmHg ± 2, n=12, in NP+RUPP B2 cells, compared to NP rats(p&lt;0.001). Administration of 7AA attenuated hypertension in response to total RUPP B cells (99 mmHg ± 6, n=5)(p&lt;0.01) and in response to RUPP B2s (98 mmHg ± 4, n=6) (p&lt;0.05). Circulating cNKs were elevated in NP+RUPPBcells (6 ± 2.5, n=5)(p&lt;0.01) and in NP+RUPP B2 cells (4.8 ± 1.6, n=11)(p&lt;0.01) compared to NP (0.275 ± 0.2, n=11). Placental cNKs were elevated in NP+RUPPBcells (2.32 ± 0.73, n=6)(p&lt;0.01) and in NP+RUPP B2 cells (1.01 ± 0.2, n=11)(p&lt;0.01) compared to NP (0.225 ± 0.2, n=11). In conclusion total B Cells and B2 B cells activated in response to placental ischemia cause hypertension via secretion of the AT1-AA and NK cell activation during pregnancy and play an important role in PE.

Abstract P215: Hyperglycemic Rats Have Increased Fetal Demise But Not Hypertension During Pregnancy

While obesity is a major cause for pregnancy complications including preeclampsia and fetal demise, it is not exactly clear the precise obesity-related metabolic factors that promote these adverse outcomes. Epidemiological studies have pointed toward hyperglycemia as one such factor. Therefore, we tested the hypothesis that hyperglycemic rats have hypertension and fetal demise during pregnancy. For this purpose, we utilized the type II diabetic model, the Goto-Kakizaki (GK) rat (N=16), compared to normoglycemic Wistar Hannover (WH) rats (N=9), which were maintained on Envigo 8640 standard chow. The GK rat allows for assessment of hyperglycemia on pregnancy without confounding obesity. Maternal fasting glucose levels were significantly greater (P&lt;0.05) in GK (97±8 mg/dL) vs. WH (72±9 mg/dL) rats by gestational day 19. Body weight was lower (P&lt;0.05) in GK (248±4 g) versus WH (289±4 g) pregnant rats, whereas perirenal fat (1.56±0.07 g vs. 1.38±0.07 g, P&gt;0.05) and circulating levels of the adipokine, leptin (1.6±0.2 ng/mL vs. 2.2±0.3 ng/mL, P&gt;0.05) were similar between GK and WH pregnant groups, respectively. Endothelial-dependent relaxation to acetylcholine (sensitivity as logEC50: -5.2±0.3 M vs -5.2±0.4 M) and endothelial-independent relaxation to the nitric oxide-donor sodium nitroprusside (logEC50: -7.2±0.2 M vs. -7.5±0.1 M) were similar (P&gt;0.05) in uterine arteries isolated from GK and WH rats, respectively. It was then determined if reduced uterine perfusion pressure (RUPP)-induced placental ischemia, a significant contributor to the development of preeclampsia, promoted greater maternal hypertension in GK rats. RUPP was conducted on gestational day 14 and blood pressure assessed on day 19. RUPP produced hypertension to a similar extent (P&gt;0.05) in GK (116±5 mmHg vs. Sham 102±5 mmHg) and WH (124±4 mmHg vs. Sham 100±2 mmHg) groups. Blood pressure was similar under Sham conditions. Fetal demise was already greater in Sham GK vs. Sham WH pregnant rats (% absorptions: 13±2 vs. 2±2, P&lt;0.05) but increased similarly following RUPP in GK (61±11 %) and WH (65±5 %) pregnant rats. In conclusion, these data suggest that high glucose levels promote fetal demise during pregnancy but do not exaggerate the outcomes of placental ischemia-induced hypertension.

Abstract 2: Increased Complement Regulator CD55 Is Associated With A Reduction In Pancreatic Beta Cell Area In Islets Of Rat Offspring Following Chronic Placental Ischemia-induced Hypertension

Placental insufficiency can result in gestational hypertension and/or intrauterine growth restriction (IUGR) in the offspring. Gestational hypertension with or without low birth weight is associated with increased risk for Type 2 diabetes in the offspring. Pancreatic β cell mass, set very early in life, can influence whether an individual develops Type 2 diabetes. Our previous studies demonstrated a significant reduction in β cell area in embryonic day 19 and postnatal day (PD)13 female offspring of rats subjected to chronic placental ischemia-induced hypertension. Previous studies in human islets demonstrated important roles for complement C3 and activation product C3a in determining β cell area, with increased C3a generation associated with prevention of islet cell atrophy. Thus, we hypothesized that increases in self-proteins that limit complement system activation, i.e. complement regulators, are associated with reductions in β cell area in the offspring following placental insufficiency. The Reduced Uterine Perfusion Pressure (RUPP) model was used to induce placental insufficiency in the dam by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation to mechanically reduce placental perfusion. This results in hypertension in the dam and IUGR in the offspring. Islets from the pancreas of (PD)13 female offspring of RUPP and Sham dams were isolated by collagenase perfusion and hand picking. The transcript message for complement regulators CD55 and Crry (regulators of C3 activation) and CD59 (terminal pathway regulator) was determined using qRT-PCR. Complement regulator CD55 was significantly increased in female RUPP offspring (2.01 + 0.29 fold) compared to female Sham offspring (1.00 + 0.13 fold change; p &lt; 0.05; 6-13 pups from 4-7 dams; mixed model ANOVA including litter effect), whereas the Crry increase of 1.38 + 0.20 fold in female RUPP vs Sham offspring was not significant (p = 0.22). No significant change was observed in message for CD59 or C3. Thus, these data suggest increased islet complement regulator CD55 may limit complement activation in islets of PD13 offspring following placental ischemia and thereby contribute to reduction of β cell area and long term susceptibility for Type 2 diabetes.

Abstract P207: Mitochondrial Dysfunction And Natural Killer Cell Activation Stimulated By Il-17 Signaling From Th17 Cells In Response To Placental Ischemia During Pregnancy; Sarah Fitzgerald, Evangeline Deer, Owen Herrock, Tarek Ibrahim, Lorena Amaral, Denise Cornelius And Babbette Lamarca; 1 Department Of Pharmacology, University Of Mississippi Medical Center.

Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension associated with placental ischemia (PI), mitochondrial (mt) dysfunction, and an imbalance in T helper (TH) and Natural Killer (NK) cells during pregnancy. The reduced uterine perfusion pressure (RUPP) model is an established model for PE. We have previously shown an important role for IL-17 in hypertension and activation of NK cells in the RUPP rat. We don’t know the role for IL-17 or TH17 cells in contributing to NK cell mediated mt dysfunction and ROS associated with PI. Therefore, we hypothesize that hypertension in response to PI stimulated TH17 cells causes mt ROS mediated through IL-17 signaling to NK cells. On gestation day 12 (GD12) RUPP-induced TH17s (splenic CD4+/CD25- cells) were adoptively transferred (Ad-T) into normal pregnant (NP) rats. Recombinant mouse IL-17 receptor C (IL-17RC) (100 pg/day) was administered from GD14-19 via osmotic mini-pump. On GD19, samples were collected and mean arterial pressure (MAP) was measured. Mt respiration and mt ROS data was measured in isolated mt from renal and placental tissues using the Oxygraph 2K and fluorescent microplate reader, respectively. A one-way ANOVA with Bonferroni post hoc test was used for statistical analysis. MAP was increased in Ad-T rats (112 ± 0.72mmHg, n=14) compared to NP (92 ± 3.03mmHg, n=14) (p&lt;0.05), and was lowered with IL-17RC (97.2± 2.14 mmHg, n=13). Circulating activated NK cells were significantly increased with Ad-T (3.326± 0.76 %gated, n=9) (p&lt;0.0001) compared to NP (0.05± 0.05 %, n=11), and were attenuated with IL-17RC (0.42± 0.4 %, n=5) (p&lt;0.01). Placental activated NK cells were significantly increased with Ad-T (2.5± 1.01 %, n=4) (p&lt;0.05) compared to NP (0.03± 0.03 %, n=8) and were attenuated with IL-17RC (0.21± 0.12 %, n=4) (p&lt;0.05). Renal mtROS increased in Ad-T (312.4 ± 44.7%, n=9) compared to NP (191.9 ± 20.5%, n=5). Placental mtROS significantly increased in Ad-T (312.5 ± 23.6%, n=9) (P&lt;0.0005) compared to NP controls (134 ± 9.6%, n=5) (p&lt;0.0001), and was decreased by administration of IL-17RC (174.5± 42.5 %, n=5) (p&lt;0.0001). These data demonstrate that IL-17 signaling plays an important role in NK cell activation and tissue mt function in response to TH17 cells stimulated during PE.

Quercetin attenuates reduced uterine perfusion pressure -induced hypertension in pregnant rats through regulation of endothelin-1 and endothelin-1 type A receptor

BackgroundQuercetin was reported to be crucial for a broad range of activities, including attenuating inflammation, platelet aggregation, capillary permeability, and lipid peroxidation. However, the effect of quercetin in hypertension during pregnancy, was not fully understood.MethodsThe model of hypertension in pregnancy was established in rats by reduced uterine perfusion pressure (RUPP). Quercetin was administrated by gavage. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured using the CODA 6 BP system. Plasma concentrations of Endothelin-1 (ET-1), soluble fms-like tyrosine kinase-1 (sFlt-1), and vascular endothelial growth factor (VEGF) were detected using enzyme-linked immunosorbent assay kits. The mRNA and protein levels of ET-1 and endothelin-1 type A receptor (ETAR) were determined by RT-PCR and Western blotting. The ETAR antagonist BQ-123 was performed by osmotic minipumps.ResultsIn RUPP induced rats, quercetin treatment decreased SBP and DBP, fetal resorptions percentage, plasma ET-1 and sFlt-1 concentrations, ET-1 and ETAR levels, but increased fetal body weight and VEGF expression. BQ-123 administration attenuated SBP and DBP, suppressed fatal resorptions percentage, and increased fetal body weight of RUPP rats.ConclusionQuercetin attenuates RUPP induced hypertension in pregnant rats through the regulation of ET-1 and ETAR.