Hypoxia induced HIF-1/HIF-2 activity alters trophoblast transcriptional regulation across gestation

Abstract

Background: Delivery of oxygen to placental and fetal tissues changes across gestation with a significant increase once maternal blood flow into the placenta is established in late first trimester. Prior to 10 weeks of gestation, fetal and placental development takes place in a physiologically hypoxic environment of 1-2% oxygen tension. Subsequently, the oxygen concentration rises to a level considered normoxic (5-8%) for other tissues following dislocation of extravillous trophoblast cells that have invaded into and initially occluded the maternal spiral arterioles where they remodel the maternal decidual vasculature. Hypoxia inducible factors (HIFs) are transcription factors which respond to changes in oxygen tension and are active in a low oxygen environment. Therefore, oxygen, hypoxia and HIFs play a crucial role in placentation and inadequate trophoblast invasion which when impaired has been associated with pregnancy pathologies such as preeclampsia (PE). The aims of this study were to examine (1) the gene expression profile of first trimester trophoblast cells cultured in different oxygen concentrations and (2) the differential expression of hypoxia responsive genes in the placenta of early and late first trimester, term uncomplicated and term complicated pregnancy. Methods: HTR8/SVneo trophoblast cells were cultured in 1% (hypoxia), 5% (normoxia) and 20% oxygen (standard culture conditions) for 6 hours. Localisation of HIF-1α and HIF-2α protein in these cells was performed using immunofluorescence. Microarray analyses were undertaken to determine the differential gene expression profile of HTR8/SVneo cells in response to oxygen. Differential expression of selected genes (IGFBP3, IGFBP5, MMP1, VEGFA, P4HA1, P4HA2 and ANGPTL4) was validated by qPCR in independent samples. HIF-1α and HIF-2α protein were localised by immunohistochemistry in early (6-8 weeks of gestation) and late (10-12 weeks of gestation) first trimester, and term, placenta tissue samples. Expression of selected genes in placenta samples of early (n=11) and late (n=7) first trimester, term uncomplicated (n=10), term complicated by gestational hypertension (n=8) and term complicated by PE (n=12) pregnancy was also quantified by qPCR. Results: 290 genes were differentially expressed in HTR8/SVneo cells treated with 1% compared to 5% oxygen. HIF-1α was identified as the top upstream regulator of 41 genes using Ingenuity Pathway Analysis. qPCR validation showed that the expression of IGFBP3 (P<0.0001), VEGFA (P<0.0001), P4HA1 (P=0.0023), P4HA2 (P=0.0009) and ANGPTL4 (P=0.0001) are significantly increased in 1% oxygen compared to 5% oxygen. These genes were predicted to contain HIF-1 transcription factor binding sites. Immunofluorescence showed that HIF-1α is highly expressed in the nucleus of 1% oxygen treated trophoblasts compared to 5% oxygen whereas expression of HIF-2α remained unchanged by oxygen treatment. HIF-1α and HIF-2α were localised to the nucleus and cytoplasm of syncytiotrophoblast, cytotrophoblast and villous stroma of early and late first trimester placenta with an increased expression in the nucleus in early first trimester. Subtle but significantly lower expression of HIF-1α (-4% P<0.013) and higher expression of HIF-2α (+57% P<0.001) was observed in late first trimester placenta compared to early first trimester placenta. qPCR showed that the expression of IGFBP3 (P<0.0001), IGFBP5 (P<0.0001), VEGFA (P=0.0195) and ANGPTL4 (P<0.0001) was significantly increased in term control compared to first trimester placenta. No difference was observed in the expression of any genes between term control vs complicated pregnancy. Conclusion: HIF translocation to the nucleus was confirmed in trophoblasts under low oxygen tension. Genome wide assessment of trophoblast cells in vitro identified a large number of differentially expressed genes in different oxygen atmospheres. Of the seven selected genes of interest, IGFBP3, VEGFA and ANGPTL4 are known to be involved in trophoblast function and P4HA1 and P4HA2 have known roles in cellular proliferation and migration. Together our findings provide additional molecular evidence that expression of HIFs and hypoxia responsive genes is altered with the change of oxygen tension which may mediate known altered trophoblast activity in low oxygen tension.