Cissy Chenyi Zhou

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.

Autoantibody From Women With Preeclampsia Induces Soluble Fms-Like Tyrosine Kinase-1 Production via Angiotensin Type 1 Receptor and Calcineurin/Nuclear Factor of Activated T-Cells Signaling

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Recent evidence indicates that maternal endothelial dysfunction in preeclampsia results from increased soluble Fms-like tyrosine kinase-1 (sFlt-1), a circulating antiangiogenic protein. Factors responsible for excessive production of sFlt-1 in preeclampsia have not been identified. We tested the hypothesis that angiotensin II type 1 (AT1) receptor activating autoantibodies, which occur in women with preeclampsia, contribute to increased production of sFlt-1. IgG from women with preeclampsia stimulates the synthesis and secretion of sFlt-1 via AT1 receptor activation in pregnant mice, human placental villous explants, and human trophoblast cells. Using FK506 or short-interfering RNA targeted to the calcineurin catalytic subunit mRNA, we determined that calcineurin/nuclear factor of activated T-cells signaling functions downstream of the AT1 receptor to induce sFlt-1 synthesis and secretion by AT1-receptor activating autoantibodies. AT1-receptor activating autoantibody–induced sFlt-1 secretion resulted in inhibition of endothelial cell migration and capillary tube formation in vitro. Overall, our studies demonstrate that an autoantibody from women with preeclampsia induces sFlt-1 production via angiotensin receptor activation and downstream calcineurin/nuclear factor of activated T-cells signaling. These autoantibodies represent potentially important targets for diagnosis and therapeutic intervention.

Angiotensin II Induces Soluble fms-Like Tyrosine Kinase-1 Release via Calcineurin Signaling Pathway in Pregnancy

Maternal endothelial dysfunction in preeclampsia is associated with increased soluble fms-like tyrosine kinase-1 (sFlt-1), a circulating antagonist of vascular endothelial growth factor and placental growth factor. Angiotensin II (Ang II) is a potent vasoconstrictor that increases concomitant with sFlt-1 during pregnancy. Therefore, we speculated that Ang II may promote the expression of sFlt-1 in pregnancy. Here we report that infusion of Ang II significantly increases circulating levels of sFlt-1 in pregnant mice, thereby demonstrating that Ang II is a regulator of sFlt-1 secretion in vivo. Furthermore, Ang II stimulated sFlt-1 production in a dose- and time-dependent manner from human villous explants and cultured trophoblasts but not from endothelial cells, suggesting that trophoblasts are the primary source of sFlt-1 during pregnancy. As expected, Ang II–induced sFlt-1 secretion resulted in the inhibition of endothelial cell migration and in vitro tube formation. In vitro and in vivo studies with losartan, small interfering RNA specific for calcineurin and FK506 demonstrated that Ang II–mediated sFlt-1 release was via Ang II type 1 receptor activation and calcineurin signaling, respectively. These findings reveal a previously unrecognized regulatory role for Ang II on sFlt-1 expression in murine and human pregnancy and suggest that elevated sFlt-1 levels in preeclampsia may be caused by a dysregulation of the local renin/angiotensin system.

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.

Angiotensin Receptors, Autoimmunity, and Preeclampsia

Preeclampsia is a pregnancy-induced hypertensive disorder that causes substantial maternal and fetal morbidity and mortality. 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. Recent studies indicate that women with preeclampsia have autoantibodies that activate the angiotensin receptor, AT1, and that autoantibody-mediated receptor activation contributes to pathophysiology associated with preeclampsia. The research reviewed here raises the intriguing possibility that preeclampsia may be a pregnancy-induced autoimmune disease.

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.

Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice

Background Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth. Principal Findings By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus. Significance In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.