Roxanna A. Irani

Angiotensin receptor agonistic autoantibodies induce pre-eclampsia in pregnant mice

Pre-eclampsia affects approximately 5% of pregnancies and remains a leading cause of maternal and neonatal mortality and morbidity in the United States and the world. The clinical hallmarks of this maternal disorder include hypertension, proteinuria, endothelial dysfunction and placental defects. Advanced-stage clinical symptoms include cerebral hemorrhage, renal failure and the HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome. An effective treatment of pre-eclampsia is unavailable owing to the poor understanding of the pathogenesis of the disease. Numerous recent studies have shown that women with pre-eclampsia possess autoantibodies, termed AT1-AAs, that bind and activate the angiotensin II receptor type 1a (AT1 receptor). We show here that key features of pre-eclampsia, including hypertension, proteinuria, glomerular endotheliosis (a classical renal lesion of pre-eclampsia), placental abnormalities and small fetus size appeared in pregnant mice after injection with either total IgG or affinity-purified AT1-AAs from women with pre-eclampsia. These features were prevented by co-injection with losartan, an AT1 receptor antagonist, or by an antibody neutralizing seven–amino-acid epitope peptide. Thus, our studies indicate that pre-eclampsia may be a pregnancy-induced autoimmune disease in which key features of the disease result from autoantibody-induced angiotensin receptor activation. This hypothesis has obvious implications regarding pre-eclampsia screening, diagnosis and therapy.

The Functional Role of the Renin-Angiotensin System in Pregnancy and Preeclampsia

During normal pregnancy, the renin-angiotensin system (RAS) plays a vitally important role in salt balance and subsequent well-being of mother and fetus. In this balance, one must consider not only the classical renal RAS but also that of the uteroplacental unit, where both maternal and fetal tissues contribute to the signaling cascade. Many studies have shown that in normal pregnancy there is an increase in almost all of the components of the RAS. In derangements of pregnancy this delicate equilibrium can become unbalanced. Preeclampsia is one such case. It is a disorder of pregnancy characterized by hypertension, proteinuria and placental abnormalities associated with shallow trophoblast invasion and impaired spiral artery remodeling. Despite being a leading cause of maternal death and a major contributor to maternal and perinatal morbidity, the mechanisms responsible for the pathogenesis of preeclampsia are poorly understood. Immunological mechanisms and the RAS have been long considered to be involved in the development of preeclampsia. Numerous recent studies demonstrate the presence of the angiotensin II type I receptor agonistic autoantibody (AT1-AA). This autoantibody can induce many key features of the disorder and upregulate molecules involved in the pathogenesis of preeclampsia. Here we review the functional role of the RAS during pregnancy and the impact of AT1-AA on preeclampsia.

Angiotensin Receptor Agonistic Autoantibody Is Highly Prevalent in Preeclampsia Correlation With Disease Severity

Preeclampsia (PE), a syndrome affecting 5% of pregnancies, characterized by hypertension and proteinuria, is a leading cause of maternal and fetal morbidity and mortality. The condition is often accompanied by the presence of a circulating maternal autoantibody, the angiotensin II type I receptor agonistic autoantibody (AT1-AA). However, the prevalence of AT1-AA in PE remains unknown, and the correlation of AT1-AA titers with the severity of the disease remains undetermined. We used a sensitive and high-throughput luciferase bioassay to detect AT1-AA levels in the serum of 30 normal, 37 preeclamptic (10 mild and 27 severe), and 23 gestational hypertensive individuals. Here we report that AT1-AA is highly prevalent in PE (≈95%). Next, by comparing the levels of AT1-AA among women with mild and severe PE, we found that the titer of AT1-AA is proportional to the severity of the disease. Intriguingly, among severe preeclamptic patients, we discovered that the titer of AT1-AA is significantly correlated with the clinical features of PE: systolic blood pressure (r=0.56), proteinuria (r=0.70), and soluble fms-like tyrosine kinase-1 level (r=0.71), respectively. Notably, only AT1-AA, and not soluble fms-like tyrosine kinase-1, levels are elevated in gestational hypertensive patients. These data serve as compelling clinical evidence that AT1-AA is highly prevalent in PE, and its titer is strongly correlated to the severity of the disease.

Angiotensin Receptor Agonistic Autoantibody–Mediated Tumor Necrosis Factor-α Induction Contributes to Increased Soluble Endoglin Production in Preeclampsia

Background— Preeclampsia is a prevalent life-threatening hypertensive disorder of pregnancy. The circulating antiangiogenic factor, soluble endoglin (sEng), is elevated in the blood circulation of women with preeclampsia and contributes to disease pathology; however, the underlying mechanisms responsible for its induction in preeclampsia are unknown. Methods and Results— Here, we discovered that a circulating autoantibody, the angiotensin receptor agonistic autoantibody (AT1-AA), stimulates sEng production via AT1 angiotensin receptor activation in pregnant mice but not in nonpregnant mice. We subsequently demonstrated that the placenta is a major source contributing to sEng induction in vivo and that AT1-AA–injected pregnant mice display impaired placental angiogenesis. Using drug screening, we identified tumor necrosis factor-&agr; as a circulating factor increased in the serum of autoantibody-injected pregnant mice contributing to AT1-AA–mediated sEng induction in human umbilical vascular endothelial cells. Subsequently, among all the drugs screened, we found that hemin, an inducer of heme oxygenase, functions as a break to control AT1-AA–mediated sEng induction by suppressing tumor necrosis factor-&agr; signaling in human umbilical vascular endothelial cells. Finally, we demonstrated that the AT1-AA–mediated decreased angiogenesis seen in human placenta villous explants was attenuated by tumor necrosis factor-&agr;–neutralizing antibodies, soluble tumor necrosis factor-&agr; receptors, and hemin by abolishing both sEng and soluble fms-like tyrosine kinase-1 induction. Conclusions— Our findings demonstrate that AT1-AA–mediated tumor necrosis factor-&agr; induction, by overcoming its negative regulator, heme oxygenase-1, is a key underlying mechanism responsible for impaired placental angiogenesis by inducing both sEng and soluble fms-like tyrosine kinase-1 secretion from human villous explants. Our results provide important new targets for diagnosis and therapeutic intervention in the management of preeclampsia.

Autoantibody-mediated IL-6-dependent endothelin-1 elevation underlies pathogenesis in a mouse model of preeclampsia.

Preeclampsia (PE) is a life-threatening hypertensive disorder of pregnancy. Elevated circulating endothelin-1 (ET-1) is associated with the disease. However the molecular basis of increased ET-1 production and its role in PE are unknown. This study aimed to investigate the causative factors, pathological role of elevated ET-1 production in PE, and the underlying mechanisms. In this study, we found that IgG from women with PE, in contrast to IgG from normotensive pregnant women, induced preproET-1 mRNA expression via angiotensin II type 1 receptor activation in kidneys and placentas in pregnant mice. The ET-A receptor-specific antagonist BQ123 significantly attenuated autoantibody-induced hypertension, proteinuria, and renal damage in pregnant mice, demonstrating that autoantibody-induced ET-1 production contributes to pathophysiology. Mechanistically, we discovered that IL-6 functioned downstream of TNF-α signaling, contributing to increased ET-1 production in pregnant mice. IL-6 blockade inhibited preeclamptic features in autoantibody-injected pregnant mice. Extending the data to human studies, we found that IL-6 was a key cytokine underlying ET-1 induction mediated by IgG from women with PE in human placental villous explants and that endothelial cells are a key source of ET-1. Overall, we provide human and mouse studies showing that angiotensin II type I receptor-agonistic autoantibody is a novel causative factor responsible for elevated ET-1 production and that increased TNF-α/IL-6 signaling is a key mechanism underlying increased ET-1 production and subsequent maternal features. Significantly, our findings revealed novel factors and signaling cascades involved in ET-1 production, subsequent disease symptom development, and possible therapeutic intervention in the management of PE.

The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia

Growth-restricted fetuses are at risk for a variety of lifelong medical conditions. Preeclampsia, a life-threatening hypertensive disorder of pregnancy, is associated with fetuses who suffer from intrauterine growth restriction (IUGR). Recently, emerging evidence indicates that preeclamptic women harbor AT1 receptor agonistic autoantibodies (AT1-AAs) that contribute to the disease features. However, the exact role of AT1-AAs in IUGR and the underlying mechanisms have not been identified. We report that these autoantibodies are present in the cord blood of women with preeclampsia and retain the ability to activate AT1 receptors. Using an autoantibody-induced animal model of preeclampsia, we show that AT1-AAs cross the mouse placenta, enter fetal circulation, and lead to small fetuses with organ growth retardation. AT1-AAs also induce apoptosis in the placentas of pregnant mice, human villous explants, and human trophoblast cells. Finally, autoantibody-induced IUGR and placental apoptosis are diminished by either losartan or an autoantibody-neutralizing peptide. Thus, these studies identify AT1-AA as a novel causative factor of preeclampsia-associated IUGR and offer two possible underlying mechanisms: a direct detrimental effect on fetal development by crossing the placenta and entering fetal circulation, and indirectly through AT1-AA–induced placental damage. Our findings highlight AT1-AAs as important therapeutic targets.

Autoantibody-mediated angiotensin receptor activation contributes to preeclampsia through tumor necrosis factor-alpha signaling.

Preeclampsia is a prevalent life-threatening hypertensive disorder of pregnancy for which the pathophysiology remains largely undefined. Recently, a circulating maternal autoantibody, the angiotensin II type I (AT1) receptor agonistic autoantibody (AA), has emerged as a contributor to disease features. Increased circulating maternal tumor necrosis factor &agr; (TNF-&agr;) is also associated with the disease; however, it is unknown whether this factor directly contributes to preeclamptic symptoms. Here we report that this autoantibody increases the proinflammatory cytokine TNF-&agr; in the circulation of AT1-AA–injected pregnant mice but not in nonpregnant mice. Coinjection of AT1-AA with a TNF-&agr; neutralizing antibody reduced cytokine availability in AT1-AA–injected pregnant mice. Moreover, TNF-&agr; blockade in AT1-AA–injected pregnant mice significantly attenuated the key features of preeclampsia. Autoantibody-induced hypertension was reduced from 131±4 to 110±4 mm Hg, and proteinuria was reduced from 212±25 to 155±23 &mgr;g of albumin per milligram of creatinine (both P<0.05). Injection of AT1-AA increased the serum levels of circulating soluble fms-like tyrosine kinase 1 and soluble endoglin (34.1±5.1, 2.4±0.3 ng/mL, respectively) and coinjection with the TNF-&agr; blocker significantly reduced their levels (21.7±3.4 and 1.2±0.4 ng/mL, respectively). Renal damage and placental abnormalities were also decreased by TNF-&agr; blockade. Lastly, the elevated circulating TNF-&agr; in preeclamptic patients is significantly correlated with the AT1-AA bioactivity in our patient cohort. Similarly, the autoantibody, through AT1 receptor–mediated TNF-&agr; induction, contributed to increased soluble fms-like tyrosine kinase 1, soluble endoglin secretion, and increased apoptosis in cultured human villous explants. Overall, AT1-AA is a novel candidate that induces TNF-&agr;, a cytokine that may play an important pathogenic role in preeclampsia.

Renin Angiotensin Signaling in Normal Pregnancy and Preeclampsia

Many reports indicate that there is an increase in almost all of the components of the renin-angiotensin system (RAS) during an uncomplicated pregnancy, but renin activity, angiotensin II, and aldosterone decrease in preeclampsia (PE) for reasons that are unclear. PE is a life-threatening disorder of late pregnancy characterized by hypertension, proteinuria, increased soluble fms-like tyrosine kinase-1, as well as renal and placental morphologic abnormalities. Although a leading cause of maternal and perinatal morbidity and mortality, the pathogenic mechanisms of PE remain largely undefined. Immunologic mechanisms and aberrations of the RAS have been long considered contributors to the disorder. Bridging these two concepts, numerous studies report the presence of the angiotensin II type I receptor agonistic autoantibody (AT(1)-AA) found circulating in preeclamptic women. This autoantibody induces many key features of the disorder through AT(1) receptor signaling, and has been implicated in the pathogenesis of PE. Here we review the functions of the RAS during normal pregnancy and PE, and highlight the role of AT(1)-AA in both animal models and in the human disorder.

Autoantibody-Mediated Angiotensin Receptor Activation Contributes to Preeclampsia Through Tumor Necrosis Factor-α Signaling

Preeclampsia is a prevalent life-threatening hypertensive disorder of pregnancy for which the pathophysiology remains largely undefined. Recently, a circulating maternal autoantibody, the angiotensin II type I (AT1) receptor agonistic autoantibody (AA), has emerged as a contributor to disease features. Increased circulating maternal tumor necrosis factor &agr; (TNF-&agr;) is also associated with the disease; however, it is unknown whether this factor directly contributes to preeclamptic symptoms. Here we report that this autoantibody increases the proinflammatory cytokine TNF-&agr; in the circulation of AT1-AA–injected pregnant mice but not in nonpregnant mice. Coinjection of AT1-AA with a TNF-&agr; neutralizing antibody reduced cytokine availability in AT1-AA–injected pregnant mice. Moreover, TNF-&agr; blockade in AT1-AA–injected pregnant mice significantly attenuated the key features of preeclampsia. Autoantibody-induced hypertension was reduced from 131±4 to 110±4 mm Hg, and proteinuria was reduced from 212±25 to 155±23 &mgr;g of albumin per milligram of creatinine (both P<0.05). Injection of AT1-AA increased the serum levels of circulating soluble fms-like tyrosine kinase 1 and soluble endoglin (34.1±5.1, 2.4±0.3 ng/mL, respectively) and coinjection with the TNF-&agr; blocker significantly reduced their levels (21.7±3.4 and 1.2±0.4 ng/mL, respectively). Renal damage and placental abnormalities were also decreased by TNF-&agr; blockade. Lastly, the elevated circulating TNF-&agr; in preeclamptic patients is significantly correlated with the AT1-AA bioactivity in our patient cohort. Similarly, the autoantibody, through AT1 receptor–mediated TNF-&agr; induction, contributed to increased soluble fms-like tyrosine kinase 1, soluble endoglin secretion, and increased apoptosis in cultured human villous explants. Overall, AT1-AA is a novel candidate that induces TNF-&agr;, a cytokine that may play an important pathogenic role in preeclampsia.

Autoantibody-Mediated Complement C3a Receptor Activation Contributes to the Pathogenesis of Preeclampsia

Preeclampsia (PE) is a prevalent life-threatening hypertensive disorder of pregnancy associated with increased complement activation. However, the causative factors and pathogenic role of increased complement activation in PE are largely unidentified. Here we report that a circulating maternal autoantibody, the angiotensin II type 1 receptor agonistic autoantibody, which emerged recently as a potential pathogenic contributor to PE, stimulates deposition of complement C3 in placentas and kidneys of pregnant mice via angiotensin II type 1 receptor activation. Next, we provide in vivo evidence that selectively interfering with C3a signaling by a complement C3a receptor–specific antagonist significantly reduces hypertension from 167±7 to 143±5 mm Hg and proteinuria from 223.5±7.5 to 78.8±14.0 &mgr;g of albumin per milligram creatinine (both P<0.05) in angiotensin II type 1 receptor agonistic autoantibody–injected pregnant mice. In addition, we demonstrated that complement C3a receptor antagonist significantly inhibited autoantibody-induced circulating soluble fms-like tyrosine kinase 1, a known antiangiogenic protein associated with PE, and reduced small placental size with impaired angiogenesis and intrauterine growth restriction. Similarly, in humans, we demonstrate that C3 deposition is significantly elevated in the placentas of preeclamptic patients compared with normotensive controls. Lastly, we show that complement C3a receptor activation is a key mechanism underlying autoantibody-induced soluble fms-like tyrosine kinase 1 secretion and decreased angiogenesis in cultured human villous explants. Overall, we provide mouse and human evidence that angiotensin II type 1 receptor agonistic autoantibody–mediated activation contributes to elevated C3 and that complement C3a receptor signaling is a key mechanism underlying the pathogenesis of the disease. These studies are the first to link angiotensin II type 1 receptor agonistic autoantibody with complement activation and to provide important new opportunities for therapeutic intervention in PE.