Volker Homuth

Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor.

Immune mechanisms and the renin-angiotensin system are implicated in preeclampsia. We investigated 25 preeclamptic patients and compared them with 12 normotensive pregnant women and 10 pregnant patients with essential hypertension. Antibodies were detected by the chronotropic responses to AT1 receptor-mediated stimulation of cultured neonatal rat cardiomyocytes coupled with receptor-specific antagonists. Immunoglobulin from all preeclamptic patients stimulated the AT1 receptor, whereas immunoglobulin from controls had no effect. The increased autoimmune activity decreased after delivery. Affinity-column purification and anti-human IgG and IgM antibody exposure implicated an IgG antibody directed at the AT1 receptor. Peptides corresponding to sites on the AT1 receptors second extracellular loop abolished the stimulatory effect. Western blotting with purified patient IgG and a commercially obtained AT1 receptor antibody produced bands of identical molecular weight. Furthermore, confocal microscopy of vascular smooth muscle cells showed colocalization of purified patient IgG and AT1 receptor antibody. The protein kinase C (PKC) inhibitor calphostin C prevented the stimulatory effect. Our results suggest that preeclamptic patients develop stimulatory autoantibodies against the second extracellular AT1 receptor loop. The effect appears to be PKC-mediated. These novel autoantibodies may participate in the angiotensin II-induced vascular lesions in these patients.

AT1 Receptor Agonistic Antibodies From Preeclamptic Patients Stimulate NADPH Oxidase

Background—We recently identified agonistic autoantibodies directed against the angiotensin AT1 receptor (AT1-AA) in the plasma of preeclamptic women. To elucidate their role further, we studied the effects of AT1-AA on reactive oxygen species (ROS), NADPH oxidase expression, and nuclear factor-&kgr;B (NF-&kgr;B) activation. Methods and Results—We investigated human vascular smooth muscle cells (VSMC) and trophoblasts, as well as placentas. AT1-AA were isolated from sera of preeclamptic women. Angiotensin II (Ang II) and AT1-AA increased ROS production and the NADPH oxidase components, p22, p47, and p67 phox in Western blotting. We next tested if AT1-AA lead to NF-&kgr;B activation in VSMC and trophoblasts. AT1-AA activated NF-&kgr;B. Inhibitor-&kgr;B&agr; (I-&kgr;B&agr;) expression was reduced in response to AT1-AA. AT1 receptor blockade with losartan, diphenylene iodonium, tiron, and antisense against p22 phox all reduced ROS production and NF-&kgr;B activation. VSMC from p47phox−/− mice showed markedly reduced ROS generation and NF-&kgr;B activation in response to Ang II and AT1-AA. The p22, p47, and p67 phox expression in placentas from preeclamptic patients was increased, compared with normal placentas. Furthermore, NF-&kgr;B was activated and I-&kgr;B&agr; reduced in placentas from preeclamptic women. Conclusions—NADPH oxidase is potentially an important source of ROS that may upregulate NF-&kgr;B in preeclampsia. We suggest that AT1-AA through activation of NADPH oxidase could contribute to ROS production and inflammatory responses in preeclampsia.

AT1 Receptor Agonistic Antibodies From Preeclamptic Patients Cause Vascular Cells to Express Tissue Factor

BACKGROUND We recently described autoantibodies (angiotensin-1 receptor autoantibodies, AT(1)-AA) directed at the AT(1) receptor in the serum of preeclamptic patients, whose placentas are commonly infarcted and express tissue factor (TF). Mechanisms of how AT(1)-AA might contribute to preeclampsia are unknown. We tested the hypothesis that AT(1)-AA cause vascular smooth muscle cells (VSMC) to express TF. METHODS AND RESULTS IgG from preeclamptic patients containing AT(1)-AA was purified with anti-human IgG columns. AT(1)-AA were separated from the IgG by ammonium sulfate precipitation. We transfected Chinese hamster ovary cells overexpressing the AT(1) receptor with TF promoter constructs coupled to a luciferase reporter gene. VSMC were obtained from human coronary arteries. Extracellular signal-related kinase activation was detected by an in-gel kinase assay. AP-1 activation was determined by electromobility shift assay. TF was measured by ELISA and detected by immunohistochemistry. Placentas from preeclamptic women stained strongly for TF, whereas control placentas showed far less staining. We proved AT(1)-AA specificity by coimmunoprecipitating the AT(1) receptor with AT(1)-AA but not with nonspecific IgG. Angiotensin (Ang) II and AT(1)-AA both activated extracellular signal-related kinase, AP-1, and the TF promoter transfected VSMC and Chinese hamster ovary cells, but only when the AP-1 binding site was present. We then demonstrated TF expression in VSMC exposed to either Ang II or AT(1)-AA. All these effects were blocked by losartan. Nonspecific IgG or IgG from nonpreeclamptic pregnant women had a negligible effect. CONCLUSIONS We conclude that AT(1)-AA and Ang II both stimulate the AT(1) receptor and initiate a signaling cascade resulting in TF expression. These results show an action of AT(1)-AA on human cells that could contribute to the pathogenesis of preeclampsia.

New Aspects in the Pathophysiology of Preeclampsia

Preeclampsia, the de novo occurrence of hypertension and proteinuria after the 20th week of gestation, continues to exert an inordinate toll on mothers and children alike. Recent clinical trials, new physiologic insights, and novel observations on pathogenesis have altered the thinking about preeclampsia. The mechanisms surrounding relaxin and its effects on the circulation and on matrix metalloproteinases have been elucidated. The growth factors receptor, fms-like tyrosine kinase 1, has been shown to exist in a soluble form that is able to inactivate vascular endothelial-derived growth factor and human placental growth factor. Compelling evidence has been brought forth suggesting that fms-like tyrosine kinase 1 is a circulating factor that can cause preeclampsia. Preeclamptic women have high circulating levels of asymmetric dimethyl arginine that could account for the generalized endothelial dysfunction observed in preeclampsia. Preeclamptic women also produce novel autoantibodies that may serve to activate angiotensin receptors. These new observations raise the possibility that the treatment of preeclamptic women will soon be improved.

α1-Adrenergic Receptor Antibodies in Patients With Primary Hypertension

Autoimmune mechanisms have been proposed to play a role in the pathogenesis of primary (essential) hypertension. Autoantibodies against the α 1 -adrenergic receptor have been described in patients with malignant and secondary hypertension. To investigate the incidence of autoantibodies against the α 1 -adrenoceptor in patients with primary hypertension, we examined the immunoglobulin fractions of sera from 54 patients with primary hypertension and 26 normotensive control subjects for the presence of autoantibodies against the α 1 -adrenoceptor. Sera from 24 patients (44%) and 3 subjects (12%) were positive. An epitope analysis of 16 autoantibody-positive immunoglobulin fractions revealed that in two thirds of the cases, the antibodies were directed against the first extracellular loop of the α 1 -adrenoceptor and in one third, against the second. The autoantibodies had a positive chronotropic effect on isolated neonatal rat cardiomyocytes, an effect that was blocked by α 1 -adrenergic antagonists. Since the functional characteristics of the autoantibodies showed no desensitization phenomena, they may play a role in elevating peripheral vascular resistance and promoting cardiac hypertrophy in patients with primary hypertension.

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

Background Agonistic autoantibodies directed at the α1-adrenergic receptor (α1-AAB) have been described in patients with hypertension. We implied earlier that α1-AAB might have a mechanistic role and could represent a therapeutic target. Methodology/Principal Findings To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had α1-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α1-adrenergic receptor antibodies (α1-AB). Patient α1-AAB and rabbit α1-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human α1A-adrenergic receptor were incubated with patient α1-AAB and rabbit α1-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient α1-AAB and rabbit α1-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca2+ in cardiomyocytes and induce mesentery artery segment contraction. Conclusions/Significance Patient α1-AAB and rabbit α1-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α1-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

Agonistic Antibodies Directed at the Angiotensin II, AT1 Receptor in Preeclampsia

Immune mechanisms and circulating mediators may be important in the pathogenesis of preeclampsia. We review our findings on agonistic antibodies against the angiotensin II (Aug 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.

Endothelial-cell permeability and protein kinase C in pre-eclampsia

BACKGROUND Oedema and vascular leakage play a part in the pathogenesis of pre-eclampsia. We tested the hypothesis that serum from pre-eclamptic patients increases endothelial-cell permeability and examined possible signal-transduction pathways. METHODS We studied eight patients with pre-eclampsia, eight normotensive pregnant women, eight non-pregnant women, five pregnant patients with pre-existing hypertension, and four hypertensive non-pregnant women. Cultured human umbilical-vein endothelial-cell monolayers were used and permeability was measured by albumin flux. The part played by protein kinase C (PKC) signalling was examined by down-regulation with phorbol ester and with the inhibitors Goe 6976 and staurosporine. PKC isoforms were assessed by western blot and confocal microscopy. Antisense oligodesoxynucleotides (ODN) were used to test for specific PKC isoforms. FINDINGS Serum from pre-eclamptic women increased endothelial permeability significantly (by 100%, p<0.01). The change in permeability decreased rapidly after delivery. Serum from normotensive pregnant women and non-pregnant women had no effect. Permeability was not influenced by serum from patients with essential hypertension or pregnant patients with pre-existing hypertension. Serum from pre-eclamptic patients induced a translocation of PKC isoforms alpha and epsilon within the cells. Goe 6976 and staurosporine (10(-8) mol/L) inhibited the increase in permeability induced by serum from pre-eclamptic patients. Down-regulation of PKC alpha and, to a lesser extent, PKC epsilon by antisense ODN also inhibited the pre-eclampsia-induced permeability increase. INTERPRETATION Serum from pre-eclamptic patients contains a factor or factors that increase endothelial-cell permeability. The effect of pre-eclamptic serum may be mediated by PKC alpha and epsilon.

Agonistic AT1 receptor autoantibodies and monocyte stimulation in hypertensive patients

BACKGROUND Agonistic AT(1) receptor autoantibodies (AT(1)-AA) have been described in hypertensive and preeclamptic patients. Furthermore, monocytes are activated in hypertensive patients. We investigated and compared the ability of angiotensin II (Ang II) and AT(1)-AA to stimulate monocytes from hypertensive and normotensive persons. The adhesiveness of the monocytes to endothelial cell layers, tissue factor expression, and chemiluminescence were determined. METHODS Blood samples were obtained from 17 patients with essential hypertension and from 20 normotensive subjects. Peripheral blood monocytes were isolated by Dynabeads and used in adhesion experiments. Adherence assays, Western blotting, and reactive oxygen species release by chemiluminescence were done. RESULTS Monocyte adhesion to human aortic or umbilical vein endothelial cell layers was significantly higher after stimulation with AT(1)-AA, compared to Ang II or no stimulation. The effect was blocked with tissue factor antibody or epitope peptide preincubation. Eposartan was partially effective in blocking the effects. Western blotting after AT(1)-AA or Ang II stimulation showed that the monocytes expressed tissue factor. The AT(1)-AA and Ang II induced significantly higher chemiluminescence in monocytes from hypertensive than control subjects. Endothelial cells, on the other hand, showed much less chemiluminescence. CONCLUSIONS These data show that monocytes can be stimulated by AT(1)-AA and Ang II to adhere, produce tissue factor, and probably reactive oxygen species. They underscore the importance of monocyte activation in hypertensive patients. The relevance of AT(1)-AA in hypertension will require further studies.

HELLP syndrome in the 18th week of gestation in association with elevated angiotensin AT1-receptor autoantibodies

We present a 24-year-old woman with a twin pregnancy who was with a typical HELLP syndrome at the 18th week of pregnancy. One fetus was dead, while the other was severely growth retarded. Our patient had agonistic autoantibodies directed at the angiotensin AT(1)-receptor. Termination of the pregnancy proved necessary. This report is the first to our knowledge associating HELLP syndrome with angiotensin AT(1)-receptor antibodies. Since the antibodies may have a pathogenic significance, their removal could permit the prolongation of pregnancy in preeclamptic and HELLP syndrome patients.