Blockade of endogenous angiotensin II type I receptor agonistic autoantibody activity improves mitochondrial reactive oxygen species and hypertension in a rat model of preeclampsia.

Preeclampsia (PE) is characterized by new-onset hypertension that usually occurs in the third trimester of pregnancy and is associated with oxidative stress and angiotensin II type 1 receptor agonistic autoantibodies (AT1-AAs). Inhibition of the AT1-AAs in the reduced uterine perfusion pressure (RUPP) rat, a model of PE, attenuates hypertension and many other characteristics of PE. We have previously shown that mitochondrial oxidative stress (mtROS) is a newly described PE characteristic exhibited by the RUPP rat that contributes to hypertension. However, the factors that cause mtROS in PE or RUPP are unknown. Thus, the objective of the current study is to use pharmacologic inhibition of AT1-AAs to examine their role in mtROS in the RUPP rat model of PE. AT1-AA inhibition in RUPP rats was achieved by administration of an epitope-binding peptide ('n7AAc'). Female Sprague-Dawley rats were divided into the following two groups: RUPP and RUPP + AT1-AA inhibition (RUPP + 'n7AAc'). On day 14 of gestation (GD), RUPP surgery was performed; 'n7AAc' peptide (2 µg/µL) was administered by miniosmotic pumps in a subset of RUPP rats; and on GD19, sera, placentas, and kidneys were collected. mitochondrial respiration and mtROS were measured in isolated mitochondria using the Oxygraph 2K and fluorescent microplate reader, respectively. Placental and renal mitochondrial respiration and mtROS were improved in RUPP + 'n7AAc' rats compared with RUPP controls. Moreover, endothelial cells (human umbilical vein endothelial cells) treated with RUPP + 'n7AAc' sera exhibited less mtROS compared with those treated with RUPP sera. Overall, our findings suggest that AT1-AA signaling is one stimulus of mtROS during PE.

Renal natural killer cell activation and mitochondrial oxidative stress; new mechanisms in AT1-AA mediated hypertensive pregnancy.

Women with preeclampsia (PE) have increased mean arterial pressure (MAP), natural killer (NK) cells, reactive oxygen species (ROS), and agonistic autoantibodies to the angiotensin II type 1 receptor (AT1-AA). AT1-AA's administered to pregnant rodents produces a well-accepted model of PE. However, the role of NK cells and mitochondrial reactive oxygen species (mtROS) in AT1-AA mediated hypertension during pregnancy is unknown. We hypothesize that AT1-AA induced model of PE will exhibit elevated MAP, NK cells, and mtROS; while inhibition of the AT1-AA binding to the AT1R would be preventative. Pregnant rats were divided into 4 groups: normal pregnant (NP) (n = 5), NP + AT1-AA inhibitory peptide (NP +'n7AAc') (n = 3), NP + AT1-AA infused (NP + AT1-AA) (n = 10), and NP + AT1-AA +'n7AAc' (n = 8). Day 13, rats were surgically implanted with mini-pumps infusing either AT1-AA or AT1-AA +'n7AAc'. Day 19, tissue and blood was collected. MAP was elevated in AT1-AA vs. NP (119 ±â€¯1 vs. 102 ±â€¯2 mmHg, p < 0.05) and this was prevented by 'n7AAc' (108 ±â€¯3). There was a 6 fold increase in renal activated NK cells in AT1-AA vs NP (1.2 ±â€¯0.4 vs. 0.2 ±â€¯0.1% Gated, p = 0.05) which returned to NP levels in AT1-AA +'n7AAc' (0.1 ±â€¯0.1% Gated). Renal mtROS (317 ±â€¯49 vs. 101 ±â€¯13% Fold, p < 0.05) was elevated with AT1-AA vs NP and was decreased in AT1-AA +'n7AAc' (128 ±â€¯16, p < 0.05). In conclusion, AT1-AA's increased MAP, NK cells, and mtROS which were attenuated by AT1-AA inhibition, thus highlighting new mechanisms of AT1-AA and the importance of drug therapy targeted to AT1-AAs in hypertensive pregnancies.

Natural killer cells contribute to mitochondrial dysfunction in response to placental ischemia in reduced uterine perfusion pressure rats.

Preeclampsia (PE) is characterized by new-onset hypertension during pregnancy and is associated with immune activation and placental oxidative stress. Mitochondrial dysfunction is a major source of oxidative stress and may play a role in the pathology of PE. We (Vaka VR, et al. Hypertension 72: 703-711, 2018. doi: 10.1161/HYPERTENSIONAHA.118.11290 .) have previously shown that placental ischemia is associated with mitochondrial oxidative stress in the reduced uterine perfusion pressure (RUPP) model of PE. Furthermore, we have also shown that placental ischemia induces natural killer (NK) cell activation in RUPP. Thus, we hypothesize that NK cell depletion could improve mitochondrial function associated with hypertension in the RUPP rat model of PE. Pregnant Sprague-Dawley rats were divided into three groups: normal pregnant (NP), RUPP, and RUPP+NK cell depletion rats (RUPP+NKD). On gestational day (GD)14, RUPP surgery was performed, and NK cells were depleted by administering anti-asialo GM1 antibodies (3.5 µg/100 µl ip) on GD15 and GD17. On GD19, mean arterial pressure (MAP) was measured, and placental mitochondria were isolated and used for mitochondrial assays. MAP was elevated in RUPP versus NP rats (119 ± 1 vs.104 ± 2 mmHg, P = 0.0004) and was normalized in RUPP+NKD rats (107 ± 2 mmHg, P = 0.002). Reduced complex IV activity and state 3 respiration rate were improved in RUPP+NKD rats. Human umbilical vein endothelial cells treated with RUPP+NKD serum restored respiration with reduced mitochondrial reactive oxygen species (ROS). The restored placental or endothelial mitochondrial function along with attenuated endothelial cell mitochondrial ROS with NK cell depletion indicate an important role of NK cells in mediating mitochondrial oxidative stress in the pathology of PE.

Role of Mitochondrial Dysfunction and Reactive Oxygen Species in Mediating Hypertension in the Reduced Uterine Perfusion Pressure Rat Model of Preeclampsia.

Placental ischemia is believed to be the initial event in the development of preeclampsia. Mitochondrial dysfunction is a cause of reactive oxygen species (ROS) generation and oxidative stress, however, there are not many studies examining the role of mitochondrial ROS in the pathology of preeclampsia. The purpose of this study was to not only examine the effect of placental ischemia on mitochondrial-mediated oxidative stress in reduced uterine perfusion pressure (RUPP) rat model of preeclampsia but to also examine the role of mitochondrial ROS in contributing to hypertension in response to placental ischemia. Female pregnant Sprague Dawley rats were used in this study. On gestational day 14, RUPP surgery was performed. On gestational day 19, blood pressure (mean arterial pressure) was measured, placentas and kidneys were collected from normal pregnant and RUPP rats and processed for mitochondrial respiration, ROS, and oxidative phosphorylation enzyme activities. Renal and placental complex activities, expressions and respiration rates were significantly reduced and mitochondrial ROS was increased in RUPP versus normal pregnant mitochondria. Mean arterial pressure was elevated in RUPP (n=6) compared with normal pregnant rats (n=5; 126±4 versus 103±4 mm Hg; P<0.05) and treatment with mitochondrial-specific antioxidants (MitoQ/MitoTEMPO) significantly reduced mean arterial pressure in RUPPs (n=5-10). Mitochondrial ROS was significantly elevated in endothelial cells incubated with RUPP serum compared from with normal pregnant rats, whereas serum from mito antioxidant-treated RUPP rats attenuated this response. Impaired mitochondrial function and vascular, placental, and renal mitochondrial ROS play an important role in hypertension and reduced fetal weight in response to placental ischemia during pregnancy.

AT1-AA (Angiotensin II Type 1 Receptor Agonistic Autoantibody) Blockade Prevents Preeclamptic Symptoms in Placental Ischemic Rats.

Women with preeclampsia produce AT1-AA (agonistic autoantibodies to the angiotensin II type 1 receptor), which stimulate reactive oxygen species, inflammatory factors, and hypertensive mechanisms (ET [endothelin] and sFlt-1 [soluble fms-like tyrosine kinase-1]) in rodent models of preeclampsia. The placental ischemic reduced uterine perfusion pressure (RUPP) rat model of preeclampsia exhibits many of these features. In this study, we examined the maternal outcomes of AT1-AA inhibition ('n7AAc') in RUPP rats. Blood pressure was higher in RUPP rats versus normal pregnant (NP) rats (123±2 versus 99±2 mm Hg, P<0.05), which was reduced in RUPP+'n7AAc' (105±3 versus 123±2 mm Hg, P<0.05 versus RUPP). Uterine artery resistant index was increased in RUPP versus NP rats (0.71±0.02 versus 0.49±0.02, P<0.05) and normalized in RUPP+'n7AAc' rats (0.55±0.03). Antiangiogenic factor sFlt-1 was elevated in RUPP versus NP rats (176±37 versus 77±15 pg/mL, P<0.05) but normalized in RUPP+'n7AAc' (86±9, P=0.05 versus RUPP). Plasma nitrate and nitrite were decreased (14±1 versus 20±1 µMNO3, P<0.05) and isoprostanes were elevated (20 117±6304 versus 2809±1375 pg/mL, P<0.05) in RUPP versus NP rats; and normalized in RUPP+'n7AAc' rats; (18±2 µMNO3; 4311±1 pg/mL). PPET-1 (preproendothelin-1) expression increased 4-fold in RUPP versus NP rats which were prevented with 'n7AAc'. Importantly, placental cytolytic natural killer cells were elevated in RUPP versus NP rats (8±2% versus 2±2% gated, P<0.05), which was prevented in RUPP+'n7AAc' total (3±1% gated, P<0.05) In conclusion, AT1-AA inhibition prevents the rise in maternal blood pressure and several pathophysiological factors associated with preeclampsia in RUPP rats and could be a potential therapy for preeclampsia.

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.

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.