The regulation of angiogenesis in pregnancy is tightly controlled; however, the molecular mechanism of sFlt-1 induction in pregnancy is unknown. Here we report that Ang II stimulates an increase in circulating sFlt-1 in pregnant mice, human placental villous explants, and trophoblasts but not from endothelial cells. Ang II induces sFlt-1 release via the AT1 receptor and calcineurin pathway. Furthermore, we demonstrate that Ang II–induced sFlt-1 reduces the ability of trophoblast-derived CM to promote angiogenesis. Taken together, our findings identify a previously unrecognized regulatory role for Ang II in the regulation of sFlt-1 expression during pregnancy.
A number of groups have shown that a complete renin/angiotensin system is present in the placenta of humans and other mammals.15,31
Analysis of pregnant mice revealed that renin gene expression is developmentally regulated during normal pregnancy, achieving maximal values near the end of gestation.15
Ang II is also increased in the maternal circulation during normal human pregnancy.32–34
Using pregnant mice, villous explants, and human trophoblasts, we provide in vivo and in vitro evidence for Ang II as a positive modulator of sFlt-1 synthesis and secretion via AT1
receptor activation. These results imply that the local renin angiotensin system is intact in placental villous explants and is capable of generating endogenous Ang II and activating AT1
receptors to induce sFlt-1 synthesis and secretion. Furthermore, we showed that FK506, a calcineurin specific inhibitor,24–25
inhibited Ang II–mediated sFlt-1 production in pregnant mice and by cultured human trophoblasts. Moreover, siRNA specific for calcineurin subunit α mRNA completely abolished the Ang II–induced sFlt-1 secretion. Thus, the results obtained from both in vivo and in vitro experiments using FK506 or siRNA specific for calcineurin subunit α suggest that calcineurin signaling is required for Ang II–mediated sFlt-1 induction in trophoblasts during pregnancy. In other experiments, we have shown that Ang II treatment of human trophoblasts results in the activation of nuclear factor of activated T cells (NFAT), a well-known transcription factor and substrate of calcineurin. Ang II–mediated activation of NFAT is blocked by both FK506 and cyclosporin A (see the online data supplement, available at http://circres.ahajournals.org
). Our earlier studies have shown that Ang II is capable of inducing NFAT nuclear translocalization and activation in human trophoblasts.16
Altogether, our results suggest that Ang II–induced synthesis and secretion of sFlt-1 involve AT1
receptor activation, calcineurin signaling, NFAT nuclear translocation, and the transcriptional activation of the Flt-1 gene.
Toward the end of gestation, continued placental vascular development is terminated, a process accomplished in part through the production of antiangiogenic factors. Multiple groups have demonstrated that sFlt-1, an antagonist to the action of VEGF and placental growth factor, is secreted from cytotrophoblasts in late pregnancy.6,8,19,26
The circulating levels of sFlt-1 are relatively low during the first trimester, begin to rise during the second trimester, and achieve relatively high levels during the third trimester. Thus, we propose that increased production of Ang II in the placenta late in the pregnancy stimulates production of antiangiogenic factors such as sFlt-1 to reduce continued placenta vascular development. We have provided the direct evidence that increased trophoblast sFlt-1 secretion by Ang II functions to antagonize angiogenesis as was earlier reported for preeclamptic CM.4
However, the biological significance of Ang II–mediated sFlt-1 secretion during pregnancy requires further investigation.
It is well known that serum concentrations of sFlt-1 are significantly higher in women with preeclampsia.35–39
However, Ang II levels are not elevated in women with preeclampsia. Thus, the following question remains: what factors contribute to the increased sFlt-1associated with preeclampsia? We40–42
have shown that autoantibodies present in the serum of women with preeclampsia are capable of activating AT1
receptors. In addition, these autoantibodies are able to induce the synthesis and secretion of plasminogen activator inhibitor-1, a feature that may also contribute to the shallow trophoblast invasion associated with preeclampsia.40
In this study, infusion of Ang II resulted in an increased arterial blood pressure and induced restricted fetal growth in pregnant mice (), which are 2 hallmark features of preeclampsia. We speculate that the AT1
receptor activating autoantibodies associated with preeclampsia may be responsible for the increased sFlt-1 observed in this disorder.