In this study, we have provided both in vivo mouse evidence and in vitro human studies that AT1-AA contributes to impaired placental angiogenesis via AT1 receptor activation. Mechanistically, we have identified for the first time that the induction of sEng and sFlt1 by AT1-AA is mediated through TNF-α signaling that is negatively regulated by HO-1. Overall, both mouse and human studies reported here provide strong evidence that AT1-AA-mediated TNF-α induction is an underlying mechanism for increased secretion of antiangiogenic factors and suggest that these signaling pathways contribute to impaired placenta angiogenesis in PE and novel therapeutic possibilities for the disease ().
Working model of AT1-AA-mediated TNF-α induction in impaired placental angiogenesis in preeclampsia
Although the molecular basis for sEng induction in PE is poorly understood , hypoxia-inducible factor-1(HIF-1), TGF-β and TNF-α are reported involved in regulating the endoglin gene expression and the release of sEng from the placenta.17, 18
We have shown here that IgG harbored by women with PE induce TNF-α production via AT1
R activation which in turn stimulates sEng production and its release into the maternal circulation from placenta but not kidney. These results indicate that AT1
-AA is the causative factor responsible for sEng induction via TNF-α signaling and the placenta is a major organ contributing to increased sEng secretion. Moreover, we provide the compelling evidence that hemin, a well-known inducer of heme oxygenase-1 (HO-1), attenuated AT1
-AA-induced impaired human placental angiogenesis by blocking increased secretion of both sFlt1 and sEng from human placenta villous explants. Thus, our studies provide the first evidence that HO-1 is a key intracellular molecule to control AT1
-AA-induced dysregualted placental angiogenesis by inhibiting TNF-a-mediated sFlt-1 and sEng induction. HO-1 is an inducible, endoplamic reticulum bound enzyme that catalyzes the nicotinamide adenosine dinucleotide phosphate-cytochrome P450 reductase-dependent oxidation of heme to biliverdin in a 3-step process that liberates carbon monoxide (CO) and Fe2+
. Thus, our current findings are strongly supported by the earlier studies showing that HO-1 activity is protective against oxidant injury and inflammatory responses.19
More importantly, it is known that women with PE have significantly decreased CO concentrations in their exhaled breath associated with a decreased expression of HO activity in their placentas.20
Overall, we have provided both mouse and human evidence that AT1
-AA-mediated TNF-α induction contributes to sEng and sFlt-1 induction and subsequent impaired placenta angiogenesis by overcoming HO-1 signaling.
TNF-α is a potent pro-inflammatory cytokine found at increased levels in the plasma of preeclamptic women.21–23
Notably, recent studies demonstrate that there are significant increases of soluble TNF-α receptors in the plasma of preeclamtpic patients.24, 25
Although soluble TNF-α receptor is likely to bind with circulating TNF-α and decreases its availability as a ligand, we have provided both human and mouse evidence that AT1
-AA-mediated TNF-α induction is responsible for both sEng and sFlt-1 induction and contributes to decreased placental angiogenesis, suggesting that increased TNF-α may contribute to pathogenesis of PE. These implication is strongly supported by recent studies demonstrating that TNFα is an important factor contributing to pathology seen an experimental model of preeclampsia in rats based on reduced uterine perfusion pressure.26, 27
More importantly, chronic perfusion of TNF-α at a similar level as those seen in the plasma of the preeclamptic women into pregnant rats leads to hypertension and proteinuria.26
Although our studies present here and others21, 22, 26
provide a strong evidence of pathoglogical role of TNF-α in PE, the relative importance of increased soluble TNF-α receptors in PE remain to be determined.
While there is general agreement that plasma levels of TNF-α are higher in women with PE 28–30
, conflicting reports have appeared regarding the placental contribution to increased levels of TNF-α in women with preeclampsia.16, 31
Two earliest reports28, 30
provided evidence for increased TNF-α in placentas from women with preeclampsia. Two subsequent reports 16, 31
did not find any significant difference in TNF-α levels between placentas from women with preeclampsia compared to placentas from normotensive pregnant women. Because TNF-α is synthesized and released from the placenta the amount of TNF-α remaining in placental tissue may not accurately reflect the amount of TNF-α produced and released by the placenta. The abundance of TNF-α mRNA will more likely reflect the potential for TNF-α protein production. Data presented here show that autoantibody mediated induction of sEng only occurs in pregnant animals and that TNF-α is a critical signaling intermediate. We see a significant increase in TNF-α mRNA in the placentas of pregnant mice injected with IgG from women with preeclampsia. The increase is prevented by co-injection with losartan or the 7-aa epitope peptide, indicating that the increase is due to AT1
-AA mediated AT1
receptor activation. Thus, in our AT1
-AA induced model of preeclampsia in mice, and the data from human placenta villous explants, suggest that the placenta is a major contributor to the resulting increase in circulating TNF-α.
In conclusion, the work reported here is the first to link AT1-AA with the inflammatory system to regulate placental-derived factors in both human and murine pregnancy and suggest that TNF-α is likely an important mediator of autoantibody-induced pathophysiology associated with PE resulting from increased production of sEng and sFlt1. Therefore the use of TNF-α neutralizing antibodies, soluble forms of TNF-α receptors or inducers of Ho-1 to blunt the effects of elevated TNF-α may be useful in the treatment of PE.
Preeclampsia (PE) is a life-threatening hypertensive complication of pregnancy that is a leading cause of maternal and neonatal mortality and morbidity in the United States and the world. It is highly prevalent and affects ~7% of first pregnancies. Available strategies used to manage PE are poor and currently limited to the delivery of the baby and placenta, secondary to the lack of fundamental understanding of the etiology and pathophysiology of the disorder. PE accounts for over 80,000 premature births each year in the US (approximately 15% of total premature births) and over $4 billion in medical cost. Recent studies demonstrate that two antiangiogenic factors, sFlt-1 and sEng, are elevated in the sera of preeclamptic women and contribute to pathophysiology of PE. However, the causative factors and molecular mechanisms responsible for their induction remain unknown. In this study, we have identified that a circulating maternal autoantibody, the angiotensin receptor agonistic autoantibody (AT1-AA), recently emerged as a prominent component in the pathogenesis of the disease, stimulates both sFlt-1 and sEng production via AT1 receptor activation and subsequent impaired placental angiogenesis in both pregnant mice and human villous explants. We also discovered that AT1-AA-mediated TNF-α induction, by overcoming its negative regulator, heme oxygenase-1 (HO-1), is a key underlying mechanism responsible for impaired placenta angiogenesis by inducing both sEng and sFlt-1 secretion from human villous explants. Thus, these studies are the first to link AT1-AA with the inflammatory system to regulate placental derived factors and provide important new targets for diagnosis and therapeutic intervention in the management of PE.