Sample Set Annotation Tool: Hypoxia induced HIF-1/HIF-2 activity alters trophoblast transcriptional regulation and promotes invasion

Study Design
Assay
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Purpose

Reduced or absent cytotrophoblast invasion of the maternal uterine spiral arteries is a common clinical finding in studies of pregnancies complicated by preeclampsia, suggesting that the mechanisms behind 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, in vitro, 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 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-1 and HIF-2 to evaluate whether HIFs are differentially expressed and localised across the period of development characterised by hypoxia (6-8 weeks) and maternal blood perfusion (10-12 weeks). Eighty-eight genes were differentially expressed between cells cultured in 1% oxygen (where HIF-1 was localised to the nucleus) and 5% oxygen (where HIF-1 was cytoplasmic). 65% of the genes were predicted to contain HIF-1?:ARNT transcription factor binding sites. Increased nuclear localisation of HIF-1? was seen in extravillous cytotrophoblasts in early first trimester compared with late, while cellular expression of HIF-2? in the villous stroma was higher in late first trimester. While HIFs and their downstream targets are clearly induced in trophoblasts during early placental development, and in vitro hypoxic conditions, the mechanism and pathways by which invasion is increased under hypoxic conditions is not clear from the gene expression profile. Further insight beyond the transcription level is required to fully understand this complex phenomenon.

Hypothesis

Experimental Design

In this study we sought to elucidate the role of hypoxia-inducible genes in HTR8/SVneo invasiveness by performing global gene expression analysis under conditions where HIF-1? was predicted to be active (1% O2) and inactive (5% O2, physiological normoxia). Ambient oxygen standard culture conditions (20% O2) were also included to evaluate the effect of using 5% oxygen rather than 20% as a representation of normoxia. Particular attention was paid to the role of HIFs, which mediate the effects of hypoxia on the cell, as a key regulator of gene expression. Differential HIF-1 localization in HTR8/SVneo was confirmed under our culture conditions, and in first trimester placenta sections of different gestational ages to confirm that HIF is highly expressed in trophoblasts during different stages of differentiation. Microarray experiments were performed with and without growth factor reduced Matrigel; cells cultured on Matrigel representing an extravillous, endovascular-like phenotype of HTR8/SVneo (Highet et al., 2012) while HTR8/Svneo cultured on plastic are believed to be a phenotype resembling cells of the proximal region of cytotrophoblast columns (Kilburn et al., 2000).

Experimental Variables

Controls

Methods

Additional Information

Expected Samples:

18 Samples Loaded: 18

Species

Human (Homo sapiens)

Sample Source

HTR8/SVneo cells

Disease

Treatment Protocol

Growth Protocol

Extraction Protocol

Storage Conditions

Sample Set Spreadsheet

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Sample ID	!Sample Title	Group	Cell line	treatment	media
GSM1591506	One.matri.1	Matrigel-One percent oxygen	HTR8/SVneo	One percent oxygen	Matrigel
GSM1591507	One.matri.2	Matrigel-One percent oxygen	HTR8/SVneo	One percent oxygen	Matrigel
GSM1591508	One.matri.3	Matrigel-One percent oxygen	HTR8/SVneo	One percent oxygen	Matrigel
GSM1591509	Five.matri.2	Matrigel-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Matrigel
GSM1591510	Five.matri.3	Matrigel-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Matrigel

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sampleset4000024_sampleannotations_group.csv Sample Set Spreadsheet
sampleset4000024_sampleannotations_group.csv Sample Set Spreadsheet

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Sample ID	!Sample Title	Group	Cell line	Treatment	Media
GSM1591506	One.matri.1	Matrigel-One percent oxygen	HTR8/SVneo	One percent oxygen	Matrigel
GSM1591507	One.matri.2	Matrigel-One percent oxygen	HTR8/SVneo	One percent oxygen	Matrigel
GSM1591508	One.matri.3	Matrigel-One percent oxygen	HTR8/SVneo	One percent oxygen	Matrigel
GSM1591509	Five.matri.2	Matrigel-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Matrigel
GSM1591510	Five.matri.3	Matrigel-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Matrigel
GSM1591511	Twenty.matri.1	Matrigel-Twenty percent oxygen	HTR8/SVneo	Twenty percent oxygen	Matrigel
GSM1591512	Twenty.matri.2	Matrigel-Twenty percent oxygen	HTR8/SVneo	Twenty percent oxygen	Matrigel
GSM1591513	Twenty.matri.3	Matrigel-Twenty percent oxygen	HTR8/SVneo	Twenty percent oxygen	Matrigel
GSM1591514	One.plasti.1	Plastic-One percent oxygen	HTR8/SVneo	One percent oxygen	Plastic
GSM1591515	One.plasti.3	Plastic-One percent oxygen	HTR8/SVneo	One percent oxygen	Plastic
GSM1591516	One.plasti.4	Plastic-One percent oxygen	HTR8/SVneo	One percent oxygen	Plastic
GSM1591517	Five.plasti.1	Plastic-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Plastic
GSM1591518	Five.plasti.2	Plastic-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Plastic
GSM1591519	Five.plasti.3	Plastic-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Plastic
GSM1591520	Five.plasti.4	Plastic-Five percent oxygen	HTR8/SVneo	Five percent oxygen	Plastic
GSM1591521	Twenty.plasti.2	Plastic-Twenty percent oxygen	HTR8/SVneo	Twenty percent oxygen	Plastic
GSM1591522	Twenty.plasti.3	Plastic-Twenty percent oxygen	HTR8/SVneo	Twenty percent oxygen	Plastic
GSM1591523	Twenty.plasti.4	Plastic-Twenty percent oxygen	HTR8/SVneo	Twenty percent oxygen	Plastic

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