Hypoxia induced HIF-1/HIF-2 activity alters trophoblast transcriptional regulation and promotes invasion

• We aimed to uncover the mechanism by which low oxygen regulates trophoblast invasion.
• Hypoxia increased invasion and HIF activity in a cytotrophoblast cell line.
• Transcription of a limited set of hypoxia responsive genes was increased.
• Future work could uncover new roles for the identified factors in trophoblast invasion.

Reduced or absent cytotrophoblast invasion of the maternal uterine spiral arterioles is a common clinical finding in studies of pregnancies complicated by preeclampsia, suggesting that the mechanisms mediating invasion of these cells is perturbed. The placenta initially develops in a low oxygen environment of 1–2% oxygen until after the 10th week of pregnancy. During this time oxygen concentration exerts a major influence over trophoblast activity and, hypoxia inducible factors are proposed to be one of many key regulators of first trimester trophoblast behaviour. We used a global gene expression microarray approach to identify signalling pathways and hypoxia-responsive genes of interest involved in invasion of the first trimester trophoblast cell line HTR8/SVneo under hypoxic conditions where HIF-1 was active. Additionally, first trimester placental samples from different gestational age groups were labelled with anti HIF-1α and HIF-2α to evaluate whether HIFs are differentially expressed and localised across two periods of placental development: (1) early first trimester characterised by hypoxia (6–8 weeks) and (2) late first trimester after initiation of maternal blood flow into the placenta (10–12 weeks).Invasion of HTR8/SVneo was assessed in real-time and was significantly increased in 1% compared with 5% and 21% oxygen and did not differ between 5% and 21% oxygen treatments. Eighty-eight genes were differentially expressed between cells cultured in 1% oxygen (where HIF-1α protein was localised to the nucleus) and 5% oxygen (where HIF-1α was mainly cytoplasmic). 65% of the genes were predicted to contain HIF-1α:HIF-1β transcription factor binding sites. While HIF-2α staining intensity in trophoblasts of late first trimester placenta was higher than early first trimester (+57%) the percentage of positively stained trophoblast nuclei did not differ between the two time points.There was no difference in the expression level of any of the hypoxia responsive genes of interest, IGFBP3, P4HA1, P4HA2, ANGPTL4 and MMP1 between early and late first trimester placenta. While HIF-1α and its downstream targets are clearly induced in HTR8/SVneo during in vitro hypoxic conditions, it appears that hypoxia inducible factors and genes are not altered throughout the first 7–12 weeks of placental development, during which the onset of maternal blood flow to the intervillous space takes place.