Cissy Chenyi Zhou

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 (AT(1)) 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 AT(1) 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 AT(1) receptor to induce sFlt-1 synthesis and secretion by AT(1)-receptor activating autoantibodies. AT(1)-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.

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.

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 (AT(1)) receptor agonistic autoantibody (AA), has emerged as a contributor to disease features. Increased circulating maternal tumor necrosis factor alpha (TNF-alpha) 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-alpha in the circulation of AT(1)-AA-injected pregnant mice but not in nonpregnant mice. Coinjection of AT(1)-AA with a TNF-alpha neutralizing antibody reduced cytokine availability in AT(1)-AA-injected pregnant mice. Moreover, TNF-alpha blockade in AT(1)-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 microg of albumin per milligram of creatinine (both P<0.05). Injection of AT(1)-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-alpha 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-alpha blockade. Lastly, the elevated circulating TNF-alpha in preeclamptic patients is significantly correlated with the AT(1)-AA bioactivity in our patient cohort. Similarly, the autoantibody, through AT(1) receptor-mediated TNF-alpha induction, contributed to increased soluble fms-like tyrosine kinase 1, soluble endoglin secretion, and increased apoptosis in cultured human villous explants. Overall, AT(1)-AA is a novel candidate that induces TNF-alpha, a cytokine that may play an important pathogenic role in 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.