Mark W. Cunningham

Preeclampsia: long-term consequences for vascular health

Preeclampsia (PE) is a pregnancy-specific syndrome and one of the leading causes of preterm birth, neonatal and maternal morbidity and mortality. This disease is characterized by new onset hypertension usually in the third trimester of pregnancy and is sometimes associated with proteinuria, although proteinuria is not a requirement for the diagnosis of PE. In developing countries, women have a higher risk of death due to PE than more affluent countries and one of the most frequent causes of death is high blood pressure and stroke. Although PE only affects approximately 2%–8% of pregnancies worldwide it is associated with severe complications such as eclampsia, hemorrhagic stroke, hemolysis, elevated liver enzymes and low platelets (HELLP syndrome), renal failure and pulmonary edema. Importantly, there is no “cure” for the disease except for early delivery of the baby and placenta, leaving PE a health care risk for babies born from PE moms. In addition, PE is linked to the development of cardiovascular disease and stroke in women after reproductive age, leaving PE a risk factor for long-term health in women. This review will highlight factors implicated in the pathophysiology of PE that may contribute to long-term effects in women with preeclamptic pregnancies.

Identifying immune mechanisms mediating the hypertension during preeclampsia.

Preeclampsia (PE) is a pregnancy-associated disorder that affects 5-8% of pregnancies and is a major cause of maternal, fetal, and neonatal morbidity and mortality. Hallmark characteristics of PE are new onset hypertension after 20 wk gestation with or without proteinuria, chronic immune activation, fetal growth restriction, and maternal endothelial dysfunction. However, the pathophysiological mechanisms that lead to the development of PE are poorly understood. Recent data from studies of both clinical and animal models demonstrate an imbalance in the subpopulations of CD4+ T cells and a role for these cells as mediators of inflammation and hypertension during pregnancy. Specifically, it has been proposed that the imbalance between two CD4+ T cell subtypes, regulatory T cells (Tregs) and T-helper 17 cells (Th17s), is involved in the pathophysiology of PE. Studies from our laboratory highlighting how this imbalance contributes to vasoactive factors, endothelial dysfunction, and hypertension during pregnancy will be discussed in this review. Therefore, the purpose of this review is to highlight hypertensive mechanisms stimulated by inflammatory factors in response to placental ischemia, thereby elucidating a role.

Vitamin D supplementation improves pathophysiology in a rat model of preeclampsia

Deficiency of vitamin D (VD) is associated with preeclampsia (PE), a hypertensive disorder of pregnancy characterized by proinflammatory immune activation. We sought to determine whether VD supplementation would reduce the pathophysiology and hypertension associated with the reduced uterine perfusion pressure (RUPP) rat model of PE. Normal pregnant (NP) and RUPP rats were supplemented with VD2 or VD3 (270 IU and 15 IU/day, respectively) on gestation days 14-18 and mean arterial pressures (MAPs) measured on day 19. MAP increased in RUPP to 123 ± 2 mmHg compared with 102 ± 3 mmHg in NP and decreased to 113 ± 3 mmHg with VD2 and 115 ± 3 mmHg with VD3 in RUPP rats. Circulating CD4+ T cells increased in RUPP to 7.90 ± 1.36% lymphocytes compared with 2.04 ± 0.67% in NP but was lowered to 0.90 ± 0.19% with VD2 and 4.26 ± 1.55% with VD3 in RUPP rats. AT1-AA, measured by chronotropic assay, decreased from 19.5 ± 0.4 bpm in RUPPs to 8.3 ± 0.5 bpm with VD2 and to 15.4 ± 0.7 bpm with VD3. Renal cortex endothelin-1 (ET-1) expression was increased in RUPP rats (11.6 ± 2.1-fold change from NP) and decreased with both VD2 (3.3 ± 1.1-fold) and VD3 (3.1 ± 0.6-fold) supplementation in RUPP rats. Plasma-soluble FMS-like tyrosine kinase-1 (sFlt-1) was also reduced to 74.2 ± 6.6 pg/ml in VD2-treated and 91.0 ± 16.1 pg/ml in VD3-treated RUPP rats compared with 132.7 ± 19.9 pg/ml in RUPP rats. VD treatment reduced CD4+ T cells, AT1-AA, ET-1, sFlt-1, and blood pressure in the RUPP rat model of PE and could be an avenue to improve treatment of hypertension in response to placental ischemia.

Placental Ischemia and Resultant Phenotype in Animal Models of Preeclampsia.

Preeclampsia is new onset (or worsening of preexisting) hypertension that occurs during pregnancy. It is accompanied by chronic inflammation, intrauterine growth restriction, elevated anti-angiogenic factors, and can occur with or without proteinuria. Although the exact etiology is unknown, it is thought that preeclampsia begins early in gestation with reduced uterine spiral artery remodeling leading to decreased vasculogenesis of the placenta as the pregnancy progresses. Soluble factors, stimulated by the ischemic placenta, shower the maternal vascular endothelium and are thought to cause endothelial dysfunction and to contribute to the development of hypertension during pregnancy. Due to the difficulty in studying such soluble factors in pregnant women, various animal models have been designed. Studies from these models have contributed to a better understanding of how factors released in response to placental ischemia may lead to increased blood pressure and reduced fetal weight during pregnancy. This review will highlight various animal models and the major findings indicating the importance of placental ischemia to lead to the pathophysiology observed in preeclamptic patients.

Serelaxin improves the pathophysiology of placental ischemia in the reduced uterine perfusion pressure rat model of preeclampsia

Preeclampsia is a hypertensive disorder of pregnancy that has limited therapeutic options. In healthy pregnancy, relaxin plays an important vasodilatory role to maintain vascular compliance; however, currently, there is no preclinical evidence to support the use of relaxin during preeclampsia. Therefore, the goal of this study was to test the hypothesis that recombinant human relaxin-2 (Serelaxin, Novartis; RLX) could reduce mean arterial pressure (MAP) and improve uterine artery resistance index (UARI) and nitric oxide bioavailability, and/or decrease prepro-endothelin-1 (PPET-1), soluble fms-like tyrosine kinase-1 (sFlt-1), and TNF-α) in the reduced uterine perfusion pressure (RUPP) model of preeclampsia. On day 14 of gestation (GD14), pregnant rats were assigned to normal pregnant (NP), RUPP, RUPP+RLX, or NP+RLX groups. Treated rats received RLX at 0.4 μg/h or RLX2 4 μg/h RLX via minipump implanted on GD14. On GD18, carotid arterial catheters were inserted, and on GD19, MAP and tissues were collected. MAP was increased in RUPP rats compared with NP but was lowered with either dose of RLX. UARI and sFlt-1 were significantly improved in both treated RUPP groups. Total circulating nitrate-nitrite improved and placental PPET-1 and TNF-α were significantly decreased with the higher dose of RLX. Renal cortex PPET-1 was reduced with both doses of RLX. In conclusion, Serelaxin improved blood pressure, sFlt-1, TNF-α, UARI, and nitric oxide bioavailability and PPET-1 in a rat model of preeclampsia, thereby suggesting a potential therapeutic role for RLX in maintaining maternal health and prolonging pregnancy in the face of placental ischemia.

Agonistic Autoantibodies to the Angiotensin II Type 1 Receptor Enhance Angiotensin II-Induced Renal Vascular Sensitivity and Reduce Renal Function During Pregnancy.

Preeclamptic women produce agonistic autoantibodies to the angiotensin II type 1 receptor (AT1-AA) and exhibit increased blood pressure (mean arterial pressure), vascular sensitivity to angiotensin II (ANG II), and display a decrease in renal function. The objective of this study was to examine the renal hemodynamic changes during pregnancy in the presence of AT1-AAs with or without a slow pressor dose of ANG II. In this study, mean arterial pressure was elevated in all pregnant rats treated with ANG II with or without AT1-AA. Glomerular filtration rate was reduced from 1.90±0.16 mL/min in normal pregnant (NP) to 1.20±0.08 in ANG II+AT1-AA rats. Renal blood flow was decreased in ANG II+AT1-AA versus NP rats to 7.4±1.09 versus 15.4±1.75 mL/min. Renal vascular resistance was drastically increased between ANG II+AT1-AA versus NP rats (18.4±2.91 versus 6.4±0.77 mm Hg/mL per minute). Isoprostane excretion was increased by 3.5-fold in ANG II+AT1-AA versus NP (1160±321 versus 323±52 pg/mL). In conclusion, ANG II and AT1-AA together significantly decrease glomerular filtration rate by 37% and renal blood flow by 50% and caused a 3-fold increase in renal vascular resistance and isoprostane levels versus NP rats. These data indicate the importance of AT1-AAs to enhance ANG II–induced renal vasoconstriction and reduce renal function as mechanisms to cause hypertension as observed during preeclampsia.

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.

Natural killer cells mediate pathophysiology in response to reduced uterine perfusion pressure

Preeclampsia is associated with hypertension, small-for-gestational-age babies, and increased cytolytic natural killer (NK) cells. The specific role of cytolytic NK cells in the pathophysiology of preeclampsia has not been clearly defined. We hypothesized that Reduced Uterine Perfusion Pressure (RUPP) stimulates proliferation and cytolytic activation of NK cells, and that reducing NK cells in RUPP would prevent hypertension, intrauterine growth restriction, and inflammation in response to placental ischemia. RUPP was induced on gestation day (GD) 14 in pregnant rats. NK cells were depleted by i.p. administration of anti-asialo GM1 antibody on GDs 15 and 17. Placental and circulating NK cells were quantified via flow cytometry, mean arterial pressure (MAP), fetal weights, and cytokines were measured on GD 19. Total placental NK cells were 7.4 ± 2% of gated cells in normal pregnant (NP; n=10) and 16.5 ± 3% of gated cells in RUPP (n=10) rats. Furthermore, cytolytic placental NK cells also increased in RUPP. Depletion of NK cells in RUPP (RUPP + anti-ASGM1) significantly improved MAP and fetal weights. MAP was 108 ± 2 mmHg in NP, 125 ± 2 mmHg in RUPP, and 112 ± 2 mmHg in RUPP + anti-ASGM1 (n=12). Fetal weight was 2.32 ± 0.05 in NP, 1.8 ± 0.04g in RUPP, and increased to 2.0 ± 0.04g in RUPP + anti-ASGM1. Placental interferon-γ (IFN-γ) was 40.4 ± 5.2 pg/mg in NP, 72.17 ± 3.2 pg/mg in RUPP, and 44.0 ± 6.5 pg/mg in RUPP + anti-ASGM1 (P<0.05). Placental tumor necrosis factor-α (TNF-α) was 17.9 ± 1.7 pg/mg in NP, 23.9 ± 2.2 pg/mg in RUPP, and 12.9 ± 2.3 pg/mg in RUPP + anti-ASGM1 (P<0.05). Depletion of NK cells significantly lowered MAP, intrauterine growth restriction, and inflammation in RUPP rats indicating that cytolytic NK cells are important in preeclampsia pathophysiology.

Proliferation of endogenous regulatory T cells improve the pathophysiology associated with placental ischaemia of pregnancy

Preeclampsia (PE) is associated with inflammation and decreased Treg cells and IL‐10. The reduced uterine perfusion pressure (RUPP) rat model of PE exhibits these characteristics, and we hypothesized that induction of endogenous Tregs by a specific stimulus (CD28 superagonistic monoclonal antibody) would reduce inflammation, vasoactive factors, and hypertension in RUPP rats.

Vitamin D supplementation reduces some AT1-AA induced downstream targets implicated in preeclampsia including hypertension

Autoantibodies to the ANG II type I receptor (AT1-AA) are associated with preeclampsia (PE). We found that vitamin D supplementation reduced AT1-AA and blood pressure (MAP) in the RUPP rat model of PE. However, it was undetermined whether the decrease in AT1-AA was the mechanism whereby vitamin D lowered MAP or if it were through factors downstream of AT1-AA. Uterine artery resistance index, placental ET-1, and soluble FMS-like tyrosine kinase-1 are increased with AT1-AA-induced hypertension and are considered markers of PE in pregnant women. Therefore, we hypothesized that vitamin D would reduce PE factors during AT1-AA-induced hypertension and could lower blood pressure in a model of hypertension during pregnancy without PE features. Either ANG II (50 ng·kg-1·day) or AT1-AA (1:40) was infused from gestational day (GD) 12-19. vitamin D2 (VD2, 270 IU/day) or vitamin D3 (VD3, 15 IU/day) was administered orally from GD14-GD18. MAP (mmHg) increased in AT1-AA (121 ± 4) and ANG II (113 ± 1)-infused pregnant rats compared with normal pregnant rats (NP) (101 ± 2) but was lower in AT1-AA+VD2 (105 ± 2), AT1-AA+VD3 (109 ± 2), ANG II+VD2 (104 ± 4), and ANG II+VD3 (104 ± 3). VD2 and/or VD3 improved PE features associated with AT1-AA during pregnancy, while ANG II did not induce such features, supporting the hypothesis that AT1-AA induces PE features during pregnancy, and these are improved with vitamin D. In this study, we demonstrate that vitamin D improved many factors associated with PE and reduced blood pressure in a hypertensive model without PE features, indicating that vitamin D could be beneficial for various hypertensive disorders of pregnancy.