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We showed earlier that activating autoantibodies against the angiotensin II type 1 (AT 1 ) receptor (AT1-AA) circulate in preeclamptic women. They may be involved in the pathogenesis of preeclampsia. Protein alignment suggests that the binding site for AT1-AAs is highly homologous to the capsid protein VP2 of parvovirus B19. We performed a prospective, nested, case-control study of 30 gestational age–matched women with preeclampsia and 30 normotensive pregnant women. We measured AT1-AA, soluble fms-like tyrosine kinase 1 (sFlt-1), and serum immunoglobulin G against parvovirus B19 proteins. AT1-AAs were present in 70% of preeclamptic patients and absent in 80% of controls. Prediction by AT1-AA was improved in late-onset preeclampsia. The discrimination for sFlt-1 was 96%. We did not find an interaction between sFlt-1 and AT1-AA. A human monoclonal immunoglobulin G antibody against parvovirus B19 VP2-protein showed a positive reaction in the AT1-AA bioassay, which could be blocked by an AT 1 receptor blocker, as well as by the epitope amino acid sequence. Immunoglobulin G against parvovirus B19 proteins was similarly distributed between preeclamptic patients and controls and had no significant importance. We detected significantly more AT1-AA in women with an immune response corresponding with parvovirus B19 infection corresponding with a distant viral infection associated with virus elimination. We concluded that AT1-AAs were common in patients with preeclampsia in a prospective case-control study, although sFlt-1 was a superior biomarker. AT1-AA may represent a better marker for late disease, whereas sFlt1 is a better marker for early onset disease.

Background— Vitronectin is a multifunctional protein with a multiple binding domain that interacts with a variety of plasma and cell proteins. Vitronectin binds multiple ligands, including the soluble vitronectin receptor. Abciximab binds equally well to soluble vitronectin receptor and glycoprotein IIb/IIIa, because both share the β 3 subunit. We tested whether vitronectin concentrations correlate with adverse outcomes in acute coronary syndrome patients. Methods and Results— Baseline serum samples (n=233) from a randomized, placebo-controlled trial of abciximab plus stenting (Evaluation of Platelet IIb/IIIa Inhibitor for Stenting EPISTENT) were retrospectively analyzed. We stratified vitronectin concentrations into the 3 lower quartiles (n=178; P =0.01) and 6 months (20.0% versus 6.2%; P =0.006). When baseline variables not predictive of major adverse cardiovascular event (eg, troponin positive, history of congestive heart failure, diabetes, history of hypertension, smoking status) were excluded from the multivariate model, only baseline vitronectin ≥49.7 μg/mL (at 30 days: OR, 3.23; 95% CI, 1.23, 8.49; at 6 months: OR, 3.36; 95% CI, 1.33, 8.52) and history of myocardial infarction (at 30 days: OR, 5.02; 95% CI, 1.41, 17.9; at 6 months: OR, 3.99; 95% CI, 1.28, 12.43) remained. No interaction occurred between abciximab and vitronectin. Conclusions— Our findings indicate that vitronectin may be an independent predictor of adverse cardiovascular outcomes following acute stenting.

Hypertensive disorders complicate pregnancy in more than 10 % worldwide.Generally several forms were differentiated from one another. Especiallypre-eclampsia, namely hypertension and proteinuria after the 20 th weekof gestation, is the most common cause of maternal and fetal mortalityand morbidity. Their aetiology is unknown. Because elective deliveryremains the only effective management approach, the main complicationis neonatal death due to prematurity. Now we do not have sufficientevidence to know when to begin antihypertensive treatment, how vigorouslyto treat, or whether to stop treatment in mild chronic hypertensionand hope that the hypotensive effect of normal pregnancy will beenough to control blood pressure. In several studies treatment ofhypertension in pregnancy resulted in an increased risk of growthrestriction in their infants. These results and the unknown long-termeffects on the infant of any treatment led to current recommendations totreat only blood pressure elevations with a potential acute riskfor the mother. Only less antihypertensive drugs are available whichcan be used in pregnancy. The only trial for treatment of hypertensionduring pregnancy with adequate infant follow-up was performed over30 years ago with alpha-methyldopa, the antihypertensive drug ofchoice in pregnancy as yet. Epidemiological data indicate that women with preeclampsia aremore likely to develop cardiovascular disease (e. g. myocardialinfarction, stroke) later in life, compared with normotensive pregnancy.Many risk factors are shared by cardiovascular diseases and preeclampsia,including endothelial dysfunction, obesity, hypertension, hyperglycemia,insulin resistance, and dyslipidemia. Therefore, it has been proposedthat the metabolic syndrome may be a possible underlying mechanism.A lifelong follow-up and counselling of women with a history ofpreeclampsia may be an opportunity for prevention of future disease.

Preeclampsia is the major cause of maternal and fetal morbidity and mortality, involving 15% to 20% of pregnancies in developed countries and even more in less developed parts of the world. Superficial placentation driven by immune maladaptation, with subsequently reduced concentrations of angiogenic growth factors and increased placental debris in the maternal circulation, are likely responsible. Recent advances suggest that antiangiogenic factors (soluble fms-like tyrosine receptor kinase and soluble endoglin), altered relaxin-mediated mechanisms leading to impaired nitric oxide production through asymmetrical dimethylarginine production, and activating antibodies directed at the angiotensin II type 1 receptor may be responsible. The field of preeclampsia research is enjoying a well-deserved blossoming of novel ideas and approaches. We hope the activity will lead to much earlier diagnostic capacities and novel prophylactic treatments. The prize will go to the affected women and their afflicted children. For the investigators in the area, such a prize would be welcome.

Background— Angiotensin (Ang) II–induced target-organ damage involves innate and acquired immunity. Mice deficient for the helix-loop-helix transcription factor inhibitor of differentiation (Id2 −/− ) lack Langerhans and splenic CD8a+ dendritic cells, have reduced natural killer cells, and have altered CD8 T-cell memory. We tested the hypothesis that an alteration in the number and quality of circulating blood cells caused by Id2 deletion would ameliorate Ang II–induced target-organ damage. Methods and Results— We used gene-deleted and transgenic mice. We conducted kidney and bone marrow transplants. In contrast to Ang II–infused Id2 +/− , Id2 −/− mice infused with Ang II remained normotensive and failed to develop albuminuria or renal damage. Bone marrow transplant of Id2 +/− bone marrow to Id2 −/− mice did not restore the blunted blood pressure response to Ang II. Transplantation of Id2 −/− kidneys to Id2 +/− mice also could not prevent Ang II–induced hypertension and renal damage. We verified the Ang II resistance in Id2 −/− mice in a model of local tissue Ang II production by crossing hypertensive mice transgenic for rat angiotensinogen with Id2 −/− or Id2 +/− mice. Angiotensinogen-transgenic Id2 +/− mice developed hypertension, albuminuria, and renal injury, whereas angiotensinogen-transgenic Id2 −/− mice did not. We also found that vascular smooth muscle cells from Id2 −/− mice showed an antisenescence phenotype. Conclusions— Our bone marrow and kidney transplant experiments suggest that alterations in circulating immune cells or Id2 in the kidney are not responsible for Ang II resistance. The present studies identify a previously undefined role for Id2 in the pathogenesis of Ang II–induced hypertension.

The recently cloned (pro)renin receptor [(P)RR] mediates renin-stimulated cellular effects by activating mitogen-activated protein kinases and promotes nonproteolytic prorenin activation. In vivo, (P)RR is said to be blocked with a peptide consisting of 10 amino acids from the prorenin prosegment called the “handle-region” peptide (HRP). We tested whether human prorenin and renin induce extracellular signal-regulated kinase (ERK) 1/2 activation and whether the direct renin inhibitor aliskiren or the HRP inhibits the receptor. We detected the (P)RR mRNA and protein in isolated human monocytes and in U937 monocytes. In U937 cells, we found that both human renin and prorenin induced a long-lasting ERK 1/2 phosphorylation despite angiotensin II type 1 and 2 receptor blockade. In contrast to angiotensin II-ERK signaling, renin and prorenin signaling did not involve the epidermal growth factor receptor. A mitogen-activated protein kinase kinase 1/2 inhibitor inhibited both renin and prorenin-induced ERK 1/2 phosphorylation. Neither aliskiren nor HRP inhibited binding of 125 I-renin or 125 I-prorenin to (P)RR. Aliskiren did not inhibit renin and prorenin-induced ERK 1/2 phosphorylation and kinase activity. Fluorescence-activated cell sorter analysis showed that, although fluorescein isothiocyanate-labeled HRP bound to U937 cells, HRP did not inhibit renin or prorenin-induced ERK 1/2 activation. In conclusion, prorenin and renin-induced ERK 1/2 activation are independent of angiotensin II. The signal transduction is different from that evoked by angiotensin II. Aliskiren has no (P)RR blocking effect and did not inhibit ERK 1/2 phosphorylation or kinase activity. Finally, we found no evidence that HRP affects renin or prorenin binding and signaling.

Maternal spiral artery remodeling is the consequence of controlled trophoblast invasive interaction with the maternal cellular environment and is fundamentally important for successful placentation. In preeclampsia, trophoblast invasion is shallow, remodeling is incomplete, and vessels develop an inflammatory appearance, termed “acute atherosis.” We noted that, in our preeclampsia, human renin-human angiotensinogen transgenic rat model, complement component 3 (C3), and tumor necrosis factor-α were upregulated and heavily expressed in atherotic uteroplacental vessels. We next used coculture involving human trophoblasts, rat vascular smooth muscle cells (VSMCs), and human VSMCs to observe VSMC-trophoblast regulatory interactions. Tumor necrosis factor-α induced complement C3 and interleukin-6 expression in VSMCs. We found that trophoblasts were able to reduce VSMC C3 and interleukin-6 expression after the VSMCs were stimulated with tumor necrosis factor-α. However, a direct VSMC-trophoblast cell-cell contact was necessary for this anti-inflammatory response. We also studied double-transgenic VSMCs that express inflammatory components and exhibit accelerated proliferation (“synthetic” phenotype). Trophoblasts could not downregulate C3 in these cells. We then examined uteroplacental tissues from preeclamptic and control patients. In control deciduas, only traces of C3 staining were observed, and vessels were thin walled without thrombus formation. In preeclampsia, the decidual vessels showed atherosis, thrombus formation, and C3 expression. Our data suggest that fetally derived trophoblasts require direct cell-cell contact with maternally derived VSMCs to downregulate VSMC C3 and interleukin-6 expression and to avoid atherosis. The findings also implicate C3 in the placental vasculopathy observed in preeclampsia.

We compared the effect n-3 polyunsaturated fatty acids (PUFAs) with direct renin inhibition on electrophysiological remodeling in angiotensin II–induced cardiac injury. We treated double-transgenic rats expressing the human renin and angiotensinogen genes (dTGRs) from week 4 to 7 with n-3 PUFA ethyl-esters (Omacor; 25-g/kg diet) or a direct renin inhibitor (aliskiren; 3 mg/kg per day). Sprague-Dawley rats were controls. We performed electrocardiographic, magnetocardiographic, and programmed electrical stimulation. Dietary n-3 PUFAs increased the cardiac content of eicosapentaenoic and docosahexaenoic acid. At week 7, mortality in dTGRs was 31%, whereas none of the n-3 PUFA- or aliskiren-treated dTGRs died. Systolic blood pressure was modestly reduced in n-3 PUFA-treated (180±3 mm Hg) compared with dTGRs (208±5 mm Hg). Aliskiren-treated dTGRs and Sprague-Dawley rats were normotensive (110±3 and 119±6 mm Hg, respectively). Both n-3 PUFA–treated and untreated dTGRs showed cardiac hypertrophy and increased atrial natriuretic peptide levels. Prolonged QRS and QT c intervals and increased T-wave dispersion in dTGRs were reduced by n-3 PUFAs or aliskiren. Both treatments reduced arrhythmia induction from 75% in dTGRs to 17% versus 0% in Sprague-Dawley rats. Macrophage infiltration and fibrosis were reduced by n-3 PUFAs and aliskiren. Connexin 43, a mediator of intermyocyte conduction, was redistributed to the lateral cell membranes in dTGRs. n-3 PUFAs and aliskiren restored normal localization to the intercalated disks. Thus, n-3 PUFAs and aliskiren improved electrical remodeling, arrhythmia induction, and connexin 43 expression, despite a 70-mm Hg difference in blood pressure and the development of cardiac hypertrophy.

Antibodies directed at receptors can block or stimulate them. Hallmark example of the latter action is Graves' disease where antibodies directed at the thyroid-stimulating hormone receptor exert an agonistic action. Recently, compelling evidence has been presented regarding agonistic antibodies directed against the alpha-adrenergic receptor, the beta-adrenergic receptors, the angiotensin II AT1 receptor, and the platelet-derived growth factor-alpha receptor. The antibodies could play a pathogenic role in various cardiovascular diseases, including hypertension, cardiomyopathy, pre-eclampsia, acute humoral rejection, and connective tissue disease. The mechanisms that result in the production of these antibodies are unclear, automated assays to determine their presence are beset with technical difficulties, and the therapeutic implications are uncertain. Nevertheless, the signaling phenomena resulting from these antibodies are well established and mechanistic studies are being intensively pursued. The discovery of agonistic antibodies may provide additional therapeutic avenues.

Mutations in the gene encoding dysferlin cause limb-girdle muscular dystrophy 2B (LGMD2B), a disorder that is believed to spare the heart. We observed dilated cardiomyopathy in two out of seven LGMD2B patients and cardiac abnormalities in three others. Cardiac biopsies showed that dysferlin was completely absent from the sarcolemma and appeared to be trapped within the cardiomyocytes. SJL/J mice (33-week-old) had diminished end-systolic pressure and reduced dP/dt; however, the hearts were histologically normal. Gene expression profiles of cardiac tissue were obtained and later confirmed by quantitative RT-PCR. Dysferlin-deficient and control mice had different gene expression patterns in terms of cardiomyocyte Z-disc and signal transduction proteins. CapZ, LIM-domain-binding protein 3 (LDB3, MLP), cypher (ZASP), desmin, and the cardiac ankyrin-repeated protein (CARP) were differentially expressed, compared to controls. Mechanical stress induced by the nonselective beta-adrenergic agonist isoproterenol (5 mg/kg body weight) given daily for 10 days resulted in reduced fractional shortening and increased cardiac fibrosis in SJL/J mice as compared to controls. Isoproterenol also caused metalloproteinase-2 upregulation in SJL/J mice. In A/J mice, the effect of isoproterenol injection was even more dramatic and lead to premature death as well as marked sarcolemmal injury as demonstrated by Evans blue dye penetration. Our data suggest that disturbances in dysferlin as well as Z-line proteins and transcription factors particularly under mechanical stress cause cardiomyopathy.

Objective Despite the success of antiproliferative therapies, restenosis remains a common problem after percutaneous transluminal coronary angioplasty (PTCA). Longer-term clinical results of brachytherapy (intracoronary radiation), the lack of long-term clinical results after implantation of drug eluting stents, and the occurrence of late thrombosis after both procedures leave room for skepticism. Neointimal proliferation is not substantially inhibited at late time points after brachytherapy, and late lumen loss with a “catch-up” proliferation can occur. We hypothesized that the transcription factors nuclear factor-{kappa}B (NF-κB) and activator protein-1 (AP-1) are involved in these processes. We addressed the role of these mediators in a porcine model of coronary restenosis. Methods Thirty-nine pigs underwent PTCA in two major coronary arteries. One of the two balloon-injured arteries was randomly assigned to receive immediate 20 Gy beta-irradiation (Brachy group) using a noncentered source train (90Sr/Y Beta-Cath, Novoste). Animals were sacrificed after 1 day, 14 days, or 28 days. Proliferating cells were labeled prior to euthanasia. Results At late time points, lumen area was significantly smaller and the inflammatory response was more pronounced in the Brachy group than in the PTCA group. These findings coincided with sustained activation of MMP-9 and transcription factors like NF-κB and AP-1. Initially, cell proliferation was reduced in the Brachy group; however, at late time points, differences between the two treatment groups were no longer significant. Conclusions Brachytherapy initially inhibits cell proliferation; however, cellular and molecular inflammatory processes (e.g. activation of NF−κB) are enhanced within the arterial wall. This proinflammatory side effect may be responsible for the observed delayed proliferation and the resulting lumen loss.

We studied the effects of extremely low-dose human renin inhibition (aliskiren) with low angiotensin II receptor blockade (losartan) in a novel double-transgenic rat model harbouring both human renin and angiotensinogen genes. We found that low-dose aliskiren and low-dose losartan effectively reduced mortality and target-organ damage with minimal, non-significant, effects on blood pressure (BP). Our data suggest that renin-angiotensin system (RAS) inhibition ameliorates target-organ damage in an Ang II-driven model of hypertension. Direct renin inhibition is equally efficacious in this regard. Our study does not fully answer the question of BP-lowering versus RAS inhibition. This question is important and was at least partially addressed with our low-dose model.

Activating angiotensin II type 1 autoantibodies (AT1-AAs) develop in women with preeclampsia and may contribute to the disorder. Insulin resistance and serum concentrations of the antiangiogenic soluble fms-like tyrosine kinase 1 (sFlt-1) are also increased in women with preeclampsia compared with normal pregnancy. sFlt-1 and insulin resistance decrease substantially after delivery; however, significant group differences persist postpartum. Women who have had preeclampsia are at increased cardiovascular risk later in life. We measured AT1-AAs in groups of women with previous preeclampsia (n=29) and previous normal pregnancies (n=35) 18±9 months after the first completed pregnancy. These women had had sFlt-1, insulin resistance homeostasis model assessment score, and related cardiovascular risk factors measured. Activating antibodies were detected by the chronotropic response of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists (losartan and prazosin). AT1-AAs were detected in 17.2% of women with previous preeclampsia versus 2.9% of women with previous uncomplicated pregnancies ( P

We investigated whether or not p38 mitogen-activated protein kinase inhibition ameliorates angiotensin II–induced target organ damage. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). dTGR, with or without p38 inhibitor (BIRB796; 30 mg/kg per day in the diet), and nontransgenic Sprague–Dawley rats were studied in 2 protocols. In protocol 1 (week 7), systolic blood pressure of untreated dTGRs was 204±4 mm Hg, but partially reduced after BIRB796 treatment (166±7 mm Hg), whereas Sprague–Dawley rats were normotensive. The cardiac hypertrophy index was unchanged in untreated and BIRB796-treated dTGRs. The β-myosin heavy chain expression of BIRB796-treated hearts was significantly lower in BIRB796 compared with dTGRs, indicating a delayed switch to the fetal isoform. BIRB796 treatment significantly reduced cardiac fibrosis, connective tissue growth factor, tumor necrosis factor-α, interleukin-6, and macrophage infiltration. Albuminuria was not reduced in BIRB796-treated dTGRs. Tubular and glomerular damage with tumor necrosis factor-α expression was unaltered, although serum creatinine and cystatin C were normalized. Renal macrophage infiltration, fibrosis, and vessel damage were reduced. In protocol 2 (week 8), we focused on mortality and arrhythmogenic electrical remodeling. Mortality of untreated dTGRs was 100% but was reduced to 10% in the BIRB796 group. Cardiac magnetic field mapping showed prolongation of depolarization and repolarization in untreated dTGRs compared with Sprague–Dawley rats with a partial reduction by BIRB796. Programmed electrical stimulation elicited ventricular tachycardias in 81% of untreated dTGRs but only in 48% of BIRB796-treated dTGRs. In conclusion, BIRB796 improved survival, target organ damage, and arrhythmogenic potential in angiotensin II–induced target organ damage.

Hypertension with left ventricular hypertrophy is a major cause of diastolic heart failure (DHF). Due to its high prevalence and high rate of mortality, DHF represents a major challenge in today's cardiovascular medicine; with limited therapeutic options. Soluble guanylate cyclase (sGC) stimulation is emerging as a promising treatment option in DHF, and is currently under investigation in preclinical and clinical studies. The present study investigates the effect of the sGC stimulator BAY 41-8543 in a transgenic rat model of hypertension-induced heart failure. We used 4 week-old male double transgenic rats expression both human renin and angiotensinogen genes (dTGRs). At 7 weeks of age, dTGRs exhibit striking cardiac hypertrophy with fibrosis and inflammation, ventricular arrhythmias and heart failure, which is accompanied with high mortality. We compared vehicle-treated dTGR (receiving 10% transcutol, 20% cremophor, 70% water) to those receiving 3 mg/kg/d BAY 41-8543, and vehicle-treated SD control rats (single oral dose per day for 3 weeks). We performed in vivo echocardiography, hemodynamic monitoring, cardiac electrophysiology studies and blood pressure measurements. Endothelial function was measured in isolated mesenteric arteries. Transcriptional analyses in cardiac tissue were performed using qRT-PCR and gene-microarray. Cardiac tissue was analyzed using histology. Treatment of dTGRs with BAY 41-8543 resulted in 100% survival at week 7, whereas only 24% of vehicle-treated dTGRs survived. Mean arterial pressure in dTGRs was significantly by BAY 41-8543 reduced (197 ± 11 mmHg vehicle vs 133 ± 4 mmHg BAY 41-8543). In addition, BAY 41-8543 significantly decreased in vivo total peripheral resistance and improved endothelium-dependent vasorelaxation of isolated mesenteric arteries. Furthermore BAY 41-8543 prevented fibrosis and inflammation of cardiac tissue. Echocardiography and invasive hemodynamic monitoring revealed BAY 41-8543 significantly increased ejection fraction and cardiac output in dTGR, whereas vehicle-treated had preserved systolic function but reduced diastolic function. In addition, diastolic compliance was significantly enhanced by BAY 41-8543, as shown by myocardial strain analysis and end-diastolic pressure volume relationship (EDPVR); indicative of an improved diastolic function. In vivo programmed electrical stimulation revealed a high ventricular tachycardia induction rate in vehicle-treated dTGRs (46%), which was significantly reduced in BAY 41-8543-treated dTGR (11%). Myocardial gene-microarray analysis showed a reversal of dysregulated genes in dTGR by BAY 41-8543 treatment. Our data demonstrate that BAY 41-8543 improves survival and cardiac performance in a transgenic rat model of hypertension-induced DHF. We postulate that treatment of DHF with sGC stimulators offers a novel therapeutic potential for humans.

Preeclampsia is associated with maternal perinatal morbidity and mortality and a high risk of premature birth and intrauterine growth restriction. The apelin system is a novel pleiotropic pathway with a potential for therapeutic targeting in preeclampsia. We have previously reported that total apelin content is lower in human preeclamptic chorionic villi. In this study, we determined whether (Pyr1)-apelin-13 improves hypertension, proteinuria, fetal characteristics, uteroplacental hemodynamics, and the autonomic function in preeclamptic rats (TgA, female transgenic for human angiotensinogen mated to male transgenic for human renin). (Pyr1)-apelin-13 (2 mg/kg/day) (n=7) or saline (n=5) was infused in TgA via osmotic minipumps starting at day 13 of gestation, when blood pressure begins to increase in these animals. Pregnant SD (n=6) rats were used as controls. At the 20th day of pregnancy, TgA rats had higher MAP (138±6 vs. 79±3 mmHg in SD, p

Objectives: Preeclamptic pregnancies feature placental anomalies. Villous trophoblast differentiation during placental development is regulated by feto-placental macrophages and disturbance of this well-balanced regulation can lead to pathological pregnancies. We hypothesized that Cluster of differentiation 74 (CD74) dysregulation of placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia. Methods and Results: We performed microarray analysis of placental tissue. CD74 was one of the most down-regulated (-2.5 fold) genes in placentas from preeclamptic women. We confirmed this finding in early onset ( Conclusions: We found that CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation led to altered macrophage activation towards a pro-inflammatory signature, a disturbed crosstalk with trophoblasts and an abnormal placental morphology.

Studies in our lab have previously shown that Vitamin D supplementation in the RUPP rat model of preeclampsia lowers blood pressure and reduces autoantibodies to the AT1 receptor (AT1-AA). Therefore, we sought to determine if the effects of Vitamin D supplementation to inhibit endothelial dysfunction and hypertension during pregnancy were mediated by AT1-AA reduction. We hypothesized that Vitamin D supplementation to AT1-AA-induced hypertensive pregnant rats would reduce anti-angiogenic factor soluble FMS-like tyrosine kinase-1 (sflt-1) and uterine artery resistance index (UARI) while improving blood pressure (MAP). Purified rat AT1-AA was infused (1:40) into Sprague-Dawley rats via miniosmotic pump from gestational day (GD) 12 to GD19. On GD14-18 we administered Vitamin D2 or D3 (VD2 or VD3) to AT1-AA rats (50ul/ml) by oral gavage. On GD18 indwelling carotid catheters were inserted and UARI assessed by Doppler sonography and MAP was measured on GD19. Consistent with previous studies, MAP was 119.0 mmHg in AT1-AA infused pregnant rats. MAP was unchanged with VD2 treatment at 121.7 mmHg (n=3), however, reduced to 109.3 mmHg (n=3) in AT1-AA+VD3 rats. Pup and placental weights were 1.79 and 0.46 g (n=3), respectively, in AT1-AA rats and were increased with VD2 treatment to 2.33 and 0.54 g (n=3) and to 2.39 and 0.56 g (n=3) in AT1-AA+VD3 rats. UARI was 0.577 (n=2) in AT1-AA rats but reduced with VD2 treatment to 0.491 (n=3) and VD3 to 0.452 (n=2). Plasma sflt-1, which is increased with AT1-AA infusion, was measured with ELISA and was >1050 pg/ml in AT1-AA rats (n=3) and greatly reduced in both AT1-AA+VD2 at 42.3 pg/ml (n=2) and AT1-AA+VD3 at 241.0 pg/ml (n=3). Our preliminary data demonstrate that Vitamin D supplementation improves uterine artery vascular resistance and sflt-1 and that these are potential mechanisms for improving fetal weight and hypertension that is induced by AT1-AA during pregnancy.

Objectives: Preeclampsia (PE) is one of the most-common, life-threatening pregnancy complication that affects maternal and offspring health, with short and long-term implications. We hypothesize that epigenetic abnormalities and/or dysregulation of imprinted genes together with genes located in their proximity during placentation affect expression pattern of a broad spectrum of genes leading to preeclampsia. These genes can induce life-long epigenetic changes in the genome of the mother following preeclamptic pregnancy. Methods and Results: Using large-scale bioinformatical analysis of human placenta gene expression in 25 preeclamptic patients and 23 controls we identified characteristic pattern for PE. It consists of three clusters of patients, suggesting that preeclampsia is a complex, heterologous disease. Moreover, the analysis identified distal-less homeobox 5 (DLX5), a maternally expressed gene, which is significantly upregulated in preeclamptic placenta (1.5 fold). We confirmed upregulation of DLX5 on mRNA level in the early onset (0.8 (n=6), indicating that LOI is involved in overexpression of DLX5 in PE. DLX5 is primarily known as a developmental transcription factor and is crucial for bone development. Although DLX5 is highly expressed in human placenta, its function and the target genes in trophoblasts as well as its role during placentation still remains unknown. Using Sleeping Beauty (SB) transposon system, we stably overexpressed DLX5 in SGHPL-4 trophoblast cell line that significantly reduced trophoblast proliferation as indicated by high throughput sampler cell count (654.2 ± 395.5 vs. 1709 ± 1119) and MTT colorimetric assay (A570: 0.154 ± 0.097 vs. 0.29 ± 0.099). Conclusions: We show that the imprinted transcription factor DLX5 is upregulated in preeclampsia and propose loss of imprinting as one underlying mechanism. Overexpression of DLX5 in first trimester trophoblasts reduces cell proliferation. Our data support the importance of dysregulated epigenetic mechanism as an important feature in preeclampsia.

Placental ischemia, a characteristic feature of preeclampsia, leads to impaired cerebral blood flow (CBF) autoregulation, cerebral edema, and increased blood-brain barrier (BBB) permeability; however, the placental factors that contribute to these cerebral abnormalities are not clear. Agonistic autoantibodies to the angiotensin II type 1 receptor (AT1-AA) are increased in preeclamptic patients as well as in a rat model of preeclampsia induced by placental ischemia. In this study, we tested the hypothesis that AT1-AA mediates placental ischemia-induced cerebrovascular abnormalities. To determine whether the AT1-AA contributes to impaired CBF autoregulation, we infused purified rat AT1-AA into normal pregnant rats from gestational day (GD) 12 to 19 via mini-osmotic pumps and measured CBF using laser Doppler flowmetry on GD 19. Autoregulatory index increased from 0.7 to 1.0±0.2 in the AT1-AA infused group over the range of 120 to 160 mmHg compared to pregnant controls (0.3 to 0.5±0.1 over the same range of pressures, p

Preeclamptic women produce agonistic autoantibodies to the Angiotensin II type 1 receptor (AT1-AA) and exhibit increased blood pressure (BP) and vascular sensitivity to angiotensin II (ANG II). Although, together AT1-AAs and ANGII increase the BP, renal artery resistant index, and vasoconstriction of renal afferent arterioles in pregnant rats; the renal hemodynamics in the presence of the AT1-AAs during pregnancy has not been examined. Thus the objective of this study was to examine the changes in the glomerular filtration rate (GFR) and renal blood flow (RBF) during pregnancy in the presence of AT1-AAs and/or ANGII. Methods: Pregnant Sprague Dawley rats were divided into 4 groups: Normal Pregnant (NP, n=6), Pregnant + ANG II (Preg + ANG II, n=6), Pregnant + AT1-AA (Preg + AT1-AA, n=8), and Pregnant + ANG II + AT1-AA (Preg + ANGII + AT1-AA, n= 6). On day 13 of pregnancy, rats were implanted with mini-pumps infusing ANG II (50 ng/kg/min) and/or AT1-AA (1:40 dilution). On day 19 of pregnancy, rats were subjected to terminal renal function surgeries using FITC labeled Inulin. During the surgery, the BP was recorded and a transonic flowmeter probe was placed on the left renal artery to measure RBF. Results: BP was elevated in all pregnant rats administered ANG II and/or the AT1-AA. Although GFR was reduced, it was not significant between Preg + ANG II and Preg + AT1-AA vs. NP rats (1.5 ±0.24, 1.60 ±0.17 vs. 1.90 ±0.16 ml/min). However, the GFR was further decreased in Preg + ANGII + AT1-AA rats (1.20 ±0.08). No difference was observed with the RBF between Preg + ANG II and Preg + AT1-AA vs NP rats (14.4 ±2.96, 14.4 ±1.48 vs. 15.4 ±1.75 ml/min). RBF was decreased in Preg + ANGII + AT1-AA vs NP rats (7.4 ±1.09 vs. 15.4 ±1.75 ml/min). No change in RVR between Preg + ANG II and Preg + AT1-AA vs. NP rats (9.7 ±2.69, 8.3 ±0.58 vs. 6.4 ±0.77). However, the RVR was drastically increased between Preg + ANGII + AT1-AA vs NP rats (18.4 ±2.91 vs. 6.4 ±0.77). Conclusion: Together ANG II and AT1-AA drastically decreases renal function by 37%, RBF by 50%, and caused a 3 fold increase in RVR vs NP rats. These data indicate the importance of AT1-AAs to drastically enhance ANG II induced renal vascular sensitivity and reduce renal function during preeclampsia. Research Supported by T32HL105324 and RO1HD067541

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with pro-inflammatory cytokines and decreased regulatory immune mechanisms such as Tregs, IL-10 and IL-4. We believe this decrease in immune regulatory mechanisms leads to an uncontrolled proinflammatory response which contributes to most of the pathophysiology associated with PE. The Reduced Uterine Perfusion Pressure, RUPP, rat model of induced placental ischemia exhibits similar characteristics as women with PE including high blood pressure, elevated pro-inflammatory cytokines and cells and decreased Tregs, IL-4 and IL-10. Therefore, we hypothesized that stimulating Tregs by administration of a superagonist (SA) would increase the Treg profile in the RUPP rats which could reduce pro-inflammatory cytokines and blood pressure. The RUPP procedure was performed at gestation day 14 (GD14); SA was administered intraperitoneally at GD15, GD18 carotid catheters inserted, and GD19 MAP and pup weight, serum and tissues were collected. MAP in NP rats was 91 +/- 3, 119.6 +/- 2 in RUPPs which was improved to 111 +/- 1 mmHg in RUPP+SA. Circulating FoxP3+ Treg cells were 6 +/- 1.7% in NP, 0.77% +/- 0.75 in RUPP rats but increased to 11% +/- 3 in RUPP+SA; IL-6 was 33.65+/- 3.4 in NP, 117.2 +/- 37.8 in RUPP, and 43.66+/- 6.1 pg/mL in RUPP+SA. Placental Pre-pro Endothelin-1 (PPET-1) was increased 44.42+/- 0.269 fold in RUPP compared to NP 1+/- 0.255, but was decreased to 18.78+/- 0.48 in RUPP+SA. These data suggest an important role for up-regulating Treg cells to enhance the immune regulatory interactions and lower the hypertension without causing further reduction in fetal weight in response to placental ischemia during pregnancy.

Increasing evidence suggests that the gut microbiota critically influence host health and immune homeostasis. Microbiome-host communication occurs via gut bacterial metabolites which are resorbed by the host and target various organs. Short-chain fatty acids (SCFA) are produced from bacterial fermentation, are highly abundant in the gut but can also be detected in the blood. Recently, the SCFA propionate has been shown to regulate T cell differentiation into effector and regulatory T cells in peripheral tissues. Since activation of the immune system is known to substantially contribute to hypertensive target organ damage and anti-inflammatory strategies have been shown to be beneficial in animal models, we hypothesized that treatment with propionate would be beneficial in angiotensin (AngII)-induced target organ damage. Male NMRI mice received AngII infusions for two weeks and propionate (P) or vehicle (C) in drinking water. To deplete endogenous SCFA production mice were fed a low-fibre diet. Body weight was similar among all groups. Propionate treatment significantly reduced albuminuria (C 1143 ± 193; P 302 ± 69 μg/d). Propionate significantly reduced cardiac hypertrophy as measured by heart-to-tibia ratio (C 10.1 ± 0.4; P 8.9 ± 0.4 mg/mm) and was confirmed by echocardiography. Propionate treatment significantly reduced interstitial (C 16.5 ± 0.8%; P 6.6 ± 0.2%) and perivascular cardiac fibrosis (C 1.5 ± 0.06; P 1.1 ± 0.03 μm/μm) as measured by fibronectin and collagen I immunofluorescence, respectively. In vivo cardiac electrophysiology studies showed a significantly reduced susceptibility to ventricular arrhythmias in propionate-treated mice (C 71 ± 14%; P 24 ± 16%), indicating the functional relevance of the improved cardiac morphology. Propionate reduced the expression of IL-17 in CD4+ T cells in spleen and lymph nodes as measured by flow cytometry. Our data indicate that propionate attenuates AngII-induced cardiac remodeling and reduces susceptibility to arrhythmias. The gut microbiome is a promising target for treatment of hypertensive heart disease.

Hypertension is present in about 10 % of all pregnancies. The frequency of chronic hypertension and that of gestational hypertension is increasing. The management of pregnant women with hypertension remains a significant, but controversial, public health problem. Although treatment of hypertension in pregnancy has shown to reduce maternal target organ damage, considerable debate remains concerning treatment. We review current evidence regarding treatment goals, the ideal treatment starting time, and which drugs are available for the treatment of hypertension in pregnancy.

The cytochrome P450 (CYP)-system regulates vascular functions, inflammation, and angiogenesis that are mechanistically important in preeclampsia.The aim of this study was to analyze the dysregulation of the Cytochrome P450 in the pathogenesis of preeclampsia.We performed microarray screening of placenta and decidua from 25 preeclamptic women and 23 controls. Results were confirmed by realtime RT-PCR, immunohistochemistry and Serum of patients were analyzed by HPLC tandem mass spectrometry. For functional testing we did cardiomyocyte contraction bioassay and myograph studies. The reduced uterine perfusion pressure (RUPP) rat model was proceed for interventional study.In microarray studies the CYP subfamily 2J polypeptide 2 (CYP2J2) was upregulated in preeclamptic decidual tissue (3.9 fold, p0.0001) and in preeclamptic placenta (1.55 fold, p0.001). RT-PCR confirmed the upregulation and immunohistochemistry, localized CYP2J2 in trophoblasts of villi and deciduas at week 12 and term. The CYP2J2 metabolites were analyzed by HPLC tandem mass spectrometry. 5,6- epoxyeicosatrienoic acids (EET), 14,15-EET, and the corresponding dihydroxyeicosatrienoic acids (DHET), were elevated in preeclamptic women compared to controls in the latter two-thirds of pregnancy and after delivery. Stimulation of the trophoblast-derived cell line SGHPL-4 with the preeclampsia-associated cytokine tumor necrosis factor-a enhanced CYP2J2 gene and protein expression. For functional testing, 5,6-EET increased the beating rate of neonatal cardiomyocytes in a bioassay and downregulated large-conductance calcium-activated potassium channel KCa 1.1 activity. In the RUPP rat model of preeclampsia, we observed elevated EET, DHET, and preeclamptic features that were ameliorated by the CYP inhibitor MsPPOH. Uterine arterial rings of rats also dilated in response to MsPPOH.Our data implicate CYP2J2 in the pathogenesis of preeclampsia and as a potential candidate for the disturbed uteroplacental remodeling, leading to hypertension and endothelial dysfunction.

INTRODUCTION: Vitamin D may ameliorate hypertension and kidney disease through genomic and extra-genomic pathways.OBJECTIVE: To investigate the impact of vitamin D in a transgenic rat model of angiotensin II-mediated hypertensive organ failure.METHODS: In 4-week-old age-matched rats overexpressing the human renin and angiotensinogen genes, group 1 (n=18) received vitamin D depleted chow; group 2 (n=15) standard chow and intraperitoneal paricalcitol at 800ng/kg thrice weekly; and group 3 (n=15) standard chow and vehicle injections. Blood pressure (tail cuff) and 24-h albuminuria were determined once weekly. After three weeks, animals were sacrificed. Heart tissue was examined for atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) by RT-PCR.RESULTS: The vitamin D depleted group had higher blood pressure at week 1 (mean difference 23.4mmHg, 95% CI 9.1-37.7) and tended to have higher blood pressure in weeks 2 and 3 (mean difference 14.3mmHg 95% CI -0.02-28.7 and 15.2mmHg 95% CI -1.5-33). The depletion group had higher heart-to-body weight ratio, and a trend towards higher ANP and BNP levels. The group receiving paricalcitol did not perform better. No differences were found between groups in mortality or proteinuria.CONCLUSION: Short-term vitamin D depletion aggravated hypertension and end-organ damage in a rat model of angiotensin II-induced hypertension. Short-term interventions with high-dose vitamin D analogues had no protective effect.

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.

Vitamin D (VD) deficiency is associated with preeclampsia (PE) and has been shown to regulate immune responses. We tested the hypothesis that VD2 and VD3 treatment ameliorates hypertension and or i...

The (pro)renin receptor (PRR) was originally thought to be important for regulating blood pressure via the renin-angiotensin system. However, it is now emerging that PRR has instead a generic role in cellular development. Here, we have specifically deleted PRR from T cells. T-cell-specific PRR-knockout mice had a significant decrease in thymic cellularity, corresponding with a 100-fold decrease in the number of CD4(+) and CD8(+) thymocytes, and a large increase in double-negative (DN) precursors. Gene expression analysis on sorted DN3 thymocytes indicated that PRR-deficient thymocytes have perturbations in key cellular pathways essential at the DN3 stage, including transcription and translation. Further characterization of DN T-cell progenitors leads us to propose that PRR deletion affects thymocyte survival and development at multiple stages; from DN3 through to DN4, double-positive, and single-positive CD4 and CD8. Our study thus identifies a new role for PRR in T-cell development.

Healthy placental development is essential for reproductive success; failure of the feto-maternal interface results in pre-eclampsia and intrauterine growth retardation. We found that grainyhead-like 2 (GRHL2), a CP2-type transcription factor, is highly expressed in chorionic trophoblast cells, including basal chorionic trophoblast (BCT) cells located at the chorioallantoic interface in murine placentas. Placentas from Grhl2-deficient mouse embryos displayed defects in BCT cell polarity and basement membrane integrity at the chorioallantoic interface, as well as a severe disruption of labyrinth branching morphogenesis. Selective Grhl2 inactivation only in epiblast-derived cells rescued all placental defects but phenocopied intraembryonic defects observed in global Grhl2 deficiency, implying the importance of Grhl2 activity in trophectoderm-derived cells. ChIP-seq identified 5282 GRHL2 binding sites in placental tissue. By integrating these data with placental gene expression profiles, we identified direct and indirect Grhl2 targets and found a marked enrichment of GRHL2 binding adjacent to genes downregulated in Grhl2−/− placentas, which encoded known regulators of placental development and epithelial morphogenesis. These genes included that encoding the serine protease inhibitor Kunitz type 1 (Spint1), which regulates BCT cell integrity and labyrinth formation. In human placenta, we found that human orthologs of murine GRHL2 and its targets displayed co-regulation and were expressed in trophoblast cells in a similar domain as in mouse placenta. Our data indicate that a conserved Grhl2-coordinated gene network controls trophoblast branching morphogenesis, thereby facilitating development of the site of feto-maternal exchange. This might have implications for syndromes related to placental dysfunction.

The mechanisms of deficient placentation in the first trimester remain poorly understood, although apoptosis, hypoxia, and oxidative stress have been implicated. High uterine artery Doppler resistance indexes (RIs) are predictive of placental complications of pregnancy, such as preeclampsia, fetal growth restriction, and stillbirth. We provide evidence that even in the first trimester, pregnancies with high uterine artery Doppler RI demonstrate alterations in placental gene and protein expression. Apoptosis was significantly higher in high RI placental tissue, as determined by Western blot analysis of cleaved poly (ADP-ribose) polymerase and caspase 3. Protein expression of the trophoblast survival factor insulin-like growth factor-2 was significantly lower. Both high and normal RI placentas showed evidence of hypoxia and oxidative stress with expression of hypoxia-inducible factors 1α and 2α, heat shock protein 70, presence of nitrotyrosine residues, and lipid peroxidation. We observed no exaggerated placental hypoxia or oxidative stress associated with high RI pregnancies. High RI placental tissue demonstrated an altered balance of antioxidant enzyme activity. Hypoxia and oxidative stress appear to be a physiological state in early pregnancy; our data did not support the hypothesis that they are associated with deficient placentation in the first trimester. Higher levels of apoptosis, reduced insulin-like growth factor-2 expression, and altered antioxidant defenses may contribute to abnormal placentation and the later development of pregnancy complications, such as preeclampsia, fetal growth restriction, and stillbirth.

The angiogenic factor ratio soluble Fms-kinase 1 (sFlt-1)/placental growth factor (PlGF) is a novel diagnostic tool for preeclampsia. We compared the efficacy of the KRYPTOR (BRAHMS) automated assays for sFlt-1 and PlGF with the Elecsys (Roche) assays in a routine clinical setting. Preeclamptic women (n = 39) were included shortly after the time of diagnosis. Normotensive control pregnancies were matched by gestational age (n = 76). The KRYPTOR assays performed comparably or superior to Elecsys (sFlt-1/PlGF area under the curve 0.746 versus 0.735; P = .09; for non-obese 0.820 versus 0.805, P = .047). For early-onset preeclampsia, KRYPTOR area under the curve increased to 0.929 with a 100% specificity for preeclampsia at cut-off 85 and an 88.9% sensitivity for preeclampsia at cut-off 33. For women with preeclampsia and preterm delivery or Hemolysis, Elevated Liver enzymes, Low Platelet count (HELLP) syndrome, the KRYPTOR sFlt-1/PlGF ratio was manifold increased (P < .01). The sFlt-1/PlGF ratio proved especially useful in early-onset preeclampsia, preeclampsia with preterm delivery or HELLP, and among non-obese women.

This chapter reviews physiological changes in volume and osmolality in normal pregnancy, as well as alterations in volume and related humoral systems associated with preeclampsia. There is a ~12 kg weight gain, associated with 30–50% increases in extracellular fluid, plasma, blood volume and total body water plus a resetting of the thresholds for vasopressin secretion and thirst, plasma osmolality averaging 10 mosmol below nonpregnant levels. There is a marked stimulation of the RAA system and other potent mineralocorticoids that accompany these changes, while opposing these salt-retaining influences are increases in GFR and a rise in the plasma concentration of several natriuretic hormones. There is ongoing debate concerning the cause and meaning of the increased plasma volume (does it represent “under-fill,” “over-fill,” or “normal-fill”?) and resolving this debate has implications for management of complications. Plasma volume decreases in preeclampsia (most marked in eclampsia), while the interstitial water may increase further (edema) or remain unchanged. Levels of all components of the RAA system are decreased compared to normal pregnancy. There is an evolving literature on the role of the dilating peptide Ang 1–7 in normal pregnancy and preeclampsia. Finally, current thoughts regarding treating preeclampsia with sodium loading or plasma volume expansion, a controversial area, were revisited in light of the recent discovery of genetic mutations leading to inefficient aldosterone production in preeclampsia. Contrarily, the possible presence of ouabain-like factors in animal models and women with preeclampsia was also noted.

Die Blockade des Renin-Angiotensin-Systems hat im Rahmen der medikamentosen Behandlung des Bluthochdrucks ihren Nutzen bewiesen. ACE-Hemmer und Angiotensinrezeptorblocker senken als Antihypertensiva der 1. Wahl effektiv den Blutdruck, verhindern die Progression bzw. bewirken die Regression von Endorganschaden und vermindern die kardiovaskulare Morbiditat und Mortalitat. Die neue Strategie der direkten Renininhibition scheint in diesem Zusammenhang ein Erfolg versprechender therapeutischer Ansatz zu sein, da auf diese Art und Weise der erste und geschwindigkeitsbestimmende Schritt des Renin-Angiotensin-Systems gehemmt wird. Im Sinn einer optimalen kardiovaskularen Protektion sollte bei Patienten mit arterieller Hypertonie ein „Niedrig-Reninaktivitat-“ und „Niedrig-Angiotensin-Zustand“ angestrebt werden. Auch aus diesem Grund ist das Konzept der direkten Renininhibition vielversprechend. Die bisher vorliegenden Daten weisen Aliskiren als ein potentes Antihypertensivum mit geringem Nebenwirkungsprofil aus. Seine Anwendung in der Hochdrucktherapie scheint sowohl in der Mono- als auch in der Kombinationstherapie mit anderen Antihypertensiva indiziert zu sein. Ob es in der Behandlung kardiovaskularer Endorganschaden und der Primar- oder Sekundarpravention kardiovaskularer Erkrankungen erfolgreich ist, wird sich herausstellen.

Immune mechanisms and circulating mediators may be important in the pathogenesis of preeclampsia. We review our findings on agonistic antibodies against the angiotensin II (Ang II) receptor (AT1-AA) and their possible role in the pathogenesis of this disorder. AT1-AA appear in the course of preeclampsia and are largely gone by 6 weeks after delivery. AT1-AA detection relies on a bioassay using spontaneously beating neonatal rat cardiomyocytes. Their specificity has been documented by other methods, including Western blotting, co-localization, and co-immunoprecipitation experiments. AT1-AA induce signaling in vascular cells and trophoblasts including transcription factor activation. The signaling results in tissue factor production and reactive oxygen species generation, both of which have been implicated in preeclampsia. The role of AT1-AA in preeclampsia and other severe hypertensive conditions has not yet been proved with certainty. However, we believe the findings are compelling and warrant further study.

Background In the recent years, a role of the immune system in Huntington’s disease (HD) is increasingly recognized. Here we investigate the presence of T cell activating auto-antibodies against angiotensin II type 1 receptors (AT1R) in all stages of the disease as compared to healthy controls and patients suffering from multiple sclerosis (MS) as a prototype neurologic autoimmune disease. Results As compared to controls, MS patients show higher titers of anti-AT1R antibodies, especially in individuals with active disease. In HD, anti-AT1R antibodies are more frequent than in healthy controls or even MS and occur in 37.9% of patients with relevant titers ≥ 20 U/ml. In a correlation analysis with clinical parameters, the presence of AT1R antibodies in the sera of HD individuals inversely correlated with the age of onset and positively with the disease burden score as well as with smoking and infection. Conclusions These data suggest a dysfunction of the adaptive immune system in HD which may be triggered by different stimuli including autoimmune responses, infection and possibly also smoking. peerReviewed

The pregnant female human angiotensinogen (hAGN) transgenic rat mated with the male human renin (hREN) transgenic rat is a model of preeclampsia (TgA) with increased blood pressure, proteinuria, and placenta alterations of edema and necrosis at late gestation. We studied vascular responses and the role of COX-derived prostanoids in the uterine artery (UA) at early gestation in this model. TgA UA showed lower stretch response, similar smooth muscle α-actin content, and lower collagen content compared with Sprague-Dawley (SD) UA. Vasodilation to acetylcholine was similar in SD and TgA UA (64 ± 8 vs. 75 ± 6% of relaxation, P > 0.05), with an acetylcholine-induced contraction in TgA UA that was abolished by preincubation with indomethacin (78 ± 6 vs. 83 ± 11%, P > 0.05). No differences in the contraction to phenylephrine were observed (159 ± 11 vs. 134 ± 12 %KMAX, P > 0.05), although in TgA UA this response was greatly affected by preincubation with indomethacin (179 ± 16 vs. 134 ± 9 %KMAX, P < 0.05, pD25.92 ± 0.08 vs. 5.85 ± 0.03, P < 0.05). Endothelium-independent vasodilation was lower in TgA UA (92 ± 2 vs. 74 ± 5% preconstricted tone, P < 0.05), and preincubation with indomethacin restored the response to normal values (90 ± 3 vs. 84 ± 3%). Immunostaining showed similar signals for α-actin, COX-2, and eNOS between groups ( P > 0.05). Plasma thromboxane levels were similar between groups. In summary, TgA UA displays functional alterations at early gestation before the preeclamptic phenotype is established. Inhibition of COX enzymes normalizes some of the functional defects in the TgA UA. An increased role for COX-derived prostanoids in this model of preeclampsia may contribute to the development of a hypertensive pregnancy.

The long-term relationship between 24-hour ambulatory blood pressure (ABP) and office BP in patients on therapy is not well documented. From a registry we included all patients in whom antihypertensive therapy needed to be uptitrated. Drug treatment included the direct renin inhibitor aliskiren or an angiotensin-converting enzyme inhibitor/angiotensin receptor blocker or drugs not blocking the renin–angiotensin system, alone or on top of an existing drug regimen. In all patients, office BP and 24-hour ABP were obtained at baseline and after 1 year with validated devices. In the study population of 2722 patients, there was a good correlation between the change in office BP and 24-hour ABP (systolic: r =0.39; P r =0.34; P

We used rats transgenic for the human angiotensinogen (hAogen) gene and the human renin (hRen) gene and crossed the strains to produce a model of preeclampsia in the dams. The female (n=9) hAogen × male hRen cross had severe (telemetry-measured) hypertension and albuminuria, which developed during the last trimester of pregnancy and subsided after delivery. The converse cross (n=9) and control (n=9) SD rats did not. We demonstrated that the female hAogen × male hRen cross had agonistic antibodies capable of activating the angiotensin (Ang) II AT1 receptor (AT1R-AA) and defined the epitope on the receptor’s second extracellular loop. The phenomenon also occurs in humans with preeclampsia. The rats displayed renal histology reminiscent of preeclampsia, including fibrin deposition confined to the glomeruli. The complement system was activated in glomeruli and IgG deposits were present that may represent AT1R-AA. Finally, we observed an atherosis-like lesion in the spiral arteries of the placental bed, which we called placental-bed arteriolosclerosis. Our model may be relevant to preeclampsia in humans.

Antibodies against HLA antigens cause refractory allograft rejection with vasculopathy in some, but not all, patients.We studied 33 kidney-transplant recipients who had refractory vascular rejection. Thirteen had donor-specific anti-HLA antibodies, whereas 20 did not. Malignant hypertension was present in 16 of the patients without anti-HLA antibodies, 4 of whom had seizures. The remaining 17 patients had no malignant hypertension. We hypothesized that activating antibodies targeting the angiotensin II type 1 (AT1) receptor might be involved.Activating IgG antibodies targeting the AT1 receptor were detected in serum from all 16 patients with malignant hypertension and without anti-HLA antibodies, but in no other patients. These receptor-activating antibodies are subclass IgG1 and IgG3 antibodies that bind to two different epitopes on the second extracellular loop of the AT1 receptor. Tissue factor expression was increased in renal-biopsy specimens from patients with these antibodies. In vitro stimulation of vascular cells with an AT1-receptor-activating antibody induced phosphorylation of ERK 1/2 kinase and increased the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor-kappaB. The AT1 antagonist losartan blocked agonistic AT1-receptor antibody-mediated effects, and passive antibody transfer induced vasculopathy and hypertension in a rat kidney-transplantation model.A non-HLA, AT1-receptor-mediated pathway may contribute to refractory vascular rejection, and affected patients might benefit from removal of AT1-receptor antibodies or from pharmacologic blockade of AT1 receptors.

Immune mechanisms have been shown to be important in the pathogenesis of preeclampsia. Here, we review our studies of agonistic antibodies against the AT1 receptor in the pathogenesis as well as a pathologic phenotype of this disorder, focusing on observations in our laboratory. We have demonstrated their specificity of the binding by Western blotting, co-localization, and co-immunoprecipitation experiments. AT1-AA induce signaling in vascular cells and trophoblasts including AP-1 and nuclear factor-kappaB (NF-kappaB) activation. The signaling results in tissue factor production and reactive oxygen species (ROS) generation, both of which have been implicated in preeclampsia. The role of AT1-AA in preeclampsia and other severe hypertensive conditions has not yet been proven with certainty. However, we believe the findings are compelling and warrant further study.

Preeclampsia remains a frequent and potentially dangerous complication of pregnancy. The cause remains largely unknown, but oxidative stress and a generalized inflammatory state are features of the maternal syndrome. The placenta appears to be the principal source of free radical synthesis but maternal leukocytes and the maternal endothelium are also likely contributors. Recent reports have suggested an important role for placental trophoblast NAD(P)H oxidase in free radical generation in preeclampsia. The antioxidant vitamin E is now known to have multiple actions in addition to prevention of lipid peroxidation (ie, inhibition of NAD(P)H oxidase activation and the inflammatory response). In view of the abnormally low plasma vitamin C concentrations in preeclampsia, a combination of vitamins C and E is a promising prophylactic strategy for prevention of preeclampsia. Several multicenter randomized clinical trials are now underway. The potential use of antioxidants and the recognized, albeit modest, benefit of low-dose aspirin prophylaxis have heightened the need for a reliable predictive test for preeclampsia. A combination test involving several relevant biomarkers is likely to provide the best predictive potential.

Background— In a double-transgenic human renin and human angiotensinogen rat model, we found that mineralocorticoid receptor (MR) blockade ameliorated angiotensin II (Ang II)–induced renal and cardiac damage. How Ang II and aldosterone (Ald) might interact is ill defined. Methods and Results— We investigated the effects of Ang II (10 −7 mol/L) and Ald (10 −7 mol/L) on extracellular signal–regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling in vascular smooth muscle cells (VSMCs) with Western blotting and confocal microscopy. Ang II induced ERK 1/2 and JNK phosphorylation by 2 minutes. Ald achieved the same at 10 minutes. Ang II+Ald had a potentiating effect by 2 minutes. Two oxygen radical scavengers and the epidermal growth factor receptor (EGFR) antagonist AG1478 reduced Ang II–, Ald-, and combination-induced ERK1/2 phosphorylation. Preincubating the cells with the MR blocker spironolactone (10 −6 mol/L) abolished Ang II–induced ROS generation, EGFR transactivation, and ERK1/2 phosphorylation. Conclusions— Ald potentiates Ang II–induced ERK-1/2 and JNK phosphorylation. Oxygen radicals, the MR, and the EGFR play a role in early signaling induced by Ang II and Ald in VSMCs. These in vitro data may help explain the effects of MR blockade on Ang II–induced end-organ damage in vivo.

Lipoic acid supplementation prevents angiotensin II–induced renal injury. Background Angiotensin II (Ang II)–induced renal injury is associated with perivascular inflammation, cell proliferation, and increased superoxide production in the vascular wall. We tested whether lipoic acid, an endogenous antioxidant, protects against the Ang II–induced inflammatory response and end-organ damage. Methods Light microscopy, immunohistochemistry, electrophoretic mobility shift assay, Northern blots, and high-pressure liquid chromatography (HPLC) were used in kidneys from double transgenic rats (dTGR) harboring human renin and angiotensinogen genes and normotensive Sprague Dawley (SD) rats. The effects of lipoic acid supplementation for three weeks were examined in dTGR and SD rats. Results Lipoic acid effectively prevented Ang II-induced glomerular and vascular damage in the kidneys and completely prevented the development of albuminuria. Ang II–induced leukocyte infiltration and cell proliferation in the kidney were attenuated. The redox-sensitive transcription factors nuclear factor (kappa) B (NF-κB) and activator protein-1 (AP-1) in the kidneys were increased in dTGR compared with SD, and were effectively reduced. Renal glutathione levels were much higher in dTGR than in SD, while the opposite was true for cysteine levels. These results suggested increased renal glutathione oxidation in dTGR, leading to cysteine shortage. Lipoic acid partly prevented renal cysteine depletion and increased hepatic cysteine and glutathione concentrations. This effect was accompanied by increased hepatic gamma-glutamylcysteine synthetase mRNA expression. Conclusion Our in vivo results suggest that lipoic acid protects against Ang II–induced renal injury through anti-inflammatory/antioxidative mechanisms. The effects are associated with decreased NF-κB and AP-1 activation, as well as improved thiol homeostasis.

Angiotensin (Ang) II promotes renal infiltration by immunocompetent cells in double-transgenic rats (dTGRs) harboring both human renin and angiotensinogen genes. To elucidate disease mechanisms, we investigated whether or not dexamethasone (DEXA) immunosuppression ameliorates renal damage. Untreated dTGRs developed hypertension, renal damage, and 50% mortality at 7 weeks. DEXA reduced albuminuria, renal fibrosis, vascular reactive oxygen stress, and prevented mortality, independent of blood pressure. In dTGR kidneys, p22phox immunostaining co-localized with macrophages and partially with T cells. dTGR dendritic cells expressed major histocompatibility complex II and CD86, indicating maturation. DEXA suppressed major histocompatibility complex II+, CD86+, dendritic, and T-cell infiltration. In additional experiments, we treated dTGRs with mycophenolate mofetil to inhibit T- and B-cell proliferation. Reno-protective actions of mycophenolate mofetil and its effect on dendritic and T cells were similar to those obtained with DEXA. We next investigated whether or not Ang II directly promotes dendritic cell maturation in vitro. Ang II did not alter CD80, CD83, and MHC II expression, but increased CCR7 expression and cell migration. To explore the role of tumor necrosis factor (TNF)-alpha on dendritic cell maturation in vivo, we treated dTGRs with the soluble TNF-alpha receptor etanercept. This treatment had no effect on blood pressure, but decreased albuminuria, nuclear factor-kappaB activation, and infiltration of all immunocompetent cells. These data suggest that immunosuppression prevents dendritic cell maturation and T-cell infiltration in a nonimmune model of Ang II-induced renal damage. Ang II induces dendritic migration directly, whereas in vivo TNF-alpha is involved in dendritic cell infiltration and maturation. Thus, Ang II may initiate events leading to innate and acquired immune response.