We report here that IgG from women with preeclampsia stimulates sFlt-1 synthesis and secretion by pregnant mice, human placental villous explants, and human trophoblast cells through AT1 receptor activation and that the increased production of sFlt-1 results in the inhibition of endothelial cell migration and capillary tube formation. Notably, losartan or an antibody-blocking epitope peptide prevented the excessive sFlt-1 secretion, thereby highlighting therapeutic possibilities for the treatment of preeclampsia. In addition, we have shown that NFAT, a transcription factor and a well-known calcineurin substrate, is required for antibody-mediated transcriptional enhancement of the FLT-1 gene. In summary, our studies indicate that autoantibodies functioning as Ang II are capable of activating AT1 receptors and contribute to excess sFlt-1 secretion in preeclampsia.
The level of sFlt-1 in the maternal circulation increases significantly during the third trimester.16,43,44
We have shown recently that Ang II stimulates increased production of sFlt-1 by human placental villous explants, human trophoblast cells, and in pregnant mice.25
We speculate that the increase in circulating sFlt-1 that occurs during the third trimester of a normal pregnancy is because of the increase in circulating Ang II that occurs at this time.20–22,45
Preeclampsia is characterized by an earlier and more extensive increase in the concentration of circulating sFlt-1.16–18
Factors accounting for the precocious and excessive accumulation of sFlt-1 associated with preeclampsia have not been identified previously. Ang II levels are not elevated in preeclampsia over that occurring in a normal pregnancy,20,22,26,45,46
and, therefore, other factors may contribute to the elevated levels of sFlt-1 observed in women with preeclampsia. We hypothesize that AT1-AA present in women with preeclampsia contributes to the increased production of sFlt-1 observed in these women. These autoantibodies are detected at as early as 18 to 20 weeks of gestation in women with impaired placental development,47
thereby placing their appearance within a time-frame congruent with subsequent increases in circulating sFlt-1.16
Here, we showed that IgG from women with preeclampsia activates AT1
receptors and thereby stimulates increased sFlt-1 secretion from human placental explants and cultured human trophoblast cells. We also showed that Ang II and AT1-AA can function additively to induce increased sFlt-1 secretion through additional AT1
receptor activation. We suggest that Ang II is a key regulator of sFlt-1 synthesis and secretion during normal pregnancy25
and that the excessive accumulation of sFlt-1 observed in women with preeclampsia is because of additional activation of AT1
receptors mediated by AT1-AA.
The following lines of evidence indicate that autoantibody-induced sFlt-1 secretion results from transcriptional activation of the FLT-1 gene. First, we have shown here that autoantibody-induced sFlt-1 secretion is accompanied by a corresponding increase in sFlt-1 mRNA abundance. Second, a transcriptional control mechanism is suggested by our finding that antibody-mediated AT1 receptor activation is accompanied by downstream activation of the calcineurin/NFAT signaling pathway. Calcineurin is a calcium/calmodulin-dependent phosphatase that dephosphorylates multiple residues within the regulatory domain of the transcription factor NFAT, leading to its nuclear translocation and the activation of target genes. Additional evidence for the importance of autoantibody-induced NFAT activation comes from experiments using Chinese hamster ovary cells stably transformed with an NFAT-luciferase reporter gene. We found that the NFAT-luciferase reporter gene was activated by IgG from women with preeclampsia and that the activation was blocked by the calcineurin inhibitor FK506. Finally, a role for NFAT in the transcriptional regulation of the FLT-1 gene is consistent with the presence of 4 consensus NFAT binding sites in the 5′ flanking region of the gene within 550 bp of the transcription start site (GenBank accession No. D64016). Overall, our findings suggest that NFAT, functioning downstream of calcineurin, may directly regulate FLT-1 transcriptional activation after autoantibody-mediated AT1 receptor activation.
In addition to AT1-AA, other factors may contribute to the increased sFlt-1 secretion associated with preeclampsia. Preeclampsia is often associated with poorly developed placentas characterized by shallow trophoblast invasion, deficient spiral artery remodeling, and reduced perfusion. These placental abnormalities result in a hypoxic environment for the placenta that is believed to contribute to increased sFlt-1 production in preeclampsia.14,48–50
It has been suggested that hypoxia-induced upregulation of sFlt-1 expression is through the presence of hypoxia-inducible factor-1α
response elements in the sFLT-1
Reduced oxygen tension is also associated with increased production of placental cytokines, including tumor necrosis factor-α
, which is elevated in the maternal circulation of women with preeclampsia.42,52
Recent studies show that tumor necrosis factor-α
, in a concentration-dependent manner, stimulates the release of sFlt-1 from placental villous explants.42
Complement activation, especially C5a, is associated with increased sFlt-1 production.53
Negative regulators of sFlt-1 production have also been identified. The carbon monoxide produced by the heme oxygenase reaction serves as a negative regulator of sFlt-1 production.54
Thus, multiple factors influence sFlt-1 production by the placenta. Stepan et al55
have recently examined the relationship between AT1-AA and elevated sFlt-1 in pregnant women. They found that most of the preeclamptic patients were characterized by high sFlt-1 levels and the presence of AT1-AA. However, discordance between AT1-AA and sFlt-1 was observed in a population of patients characterized by reduced uterine perfusion and no other pregnancy complications. In these cases, AT1-AA was frequently present, whereas sFlt-1 was not usually elevated. They also identified an occasional preeclamptic patient with elevated sFlt-1 who lacked AT1-AA. Thus, in addition to AT1-AA, other factors influence sFlt-1 production and may account for those cases identified by Stepan et al55
in which there is discordance between sFlt-1 and AT1-AA in pregnant women.
Important recent work by Walther et al47
shows that AT-AA can be detected before 20 weeks in women with impaired uterine perfusion detected by Doppler sonography. The fact that AT1-AA can be detected many weeks before the symptoms of preeclampsia has significant implications regarding early diagnosis and therapeutic intervention. If maternal circulating AT1-AA contributes to the pathophysiology of preeclampsia, the removal of these autoantibodies from women with preeclampsia may provide therapeutic benefit. Another possible therapeutic approach is to block the action of the autoantibody. AT1-AAs typically recognize a 7-amino acid sequence present on the second extracellular loop of the AT1
receptor. Available evidence indicates that a 7-amino acid peptide, corresponding with this epitope, blocks antibody-induced stimulation of the AT1
Our current studies show that this 7-amino acid peptide can neutralize AT1-AA and thereby prevent autoantibody-induced sFlt-1 production in placental explants and cultured trophoblast cells. Thus, the use of epitope peptide therapy to block the action of angiotensin receptor–activating autoantibodies has the potential of being a safe and effective treatment of preeclampsia.
In 1999, Wallukat et al27
reported their remarkable findings that sera from women with preeclampsia contain autoantibodies that react with the AT1
receptor in a stimulatory fashion. In recent years, these important findings have been extended in numerous ways showing that these autoantibodies activate AT1
receptors on cardiac myocytes, trophoblast cells, endothelial cells, mesangial cells, vascular smooth muscle cells, and Chinese hamster ovary cells.56,57
Here we reported that these autoantibodies stimulate increased production of sFlt-1 by human trophoblast cells, human villous explants, and in pregnant mice and in this way may contribute to the production of a major soluble factor released from the placenta that contributes to the maternal syndrome. Altogether, the studies show that the AT1-AAs activate AT1
receptors on a variety of cell types and provoke biological responses that are relevant to the pathophysiology of preeclampsia. These findings provide a new way of thinking about the abnormalities associated with preeclampsia and raise the possibility that preeclampsia may be an autoimmune disease. Thus, removing AT1-AA or inhibiting AT1
receptor activation by 7-amino acid antibody-blocking epitope peptide may be a potential safe and effective therapeutic treatment for preeclampsia.