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

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-1α:ARNT transcription factor binding sites. Increased nuclear localisation of HIF-1α was seen in extravillous cytotrophoblasts in early first trimester compared with late, while cellular expression of HIF-2α 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. 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-1α 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).

子痫前期合并妊娠的相关研究中，细胞滋养层细胞侵袭母体子宫螺旋动脉的能力减弱或缺失是常见临床发现，提示此类细胞的侵袭机制发生紊乱。胎盘最初在1%~2%氧浓度的低氧环境中发育，直至妊娠第10周之后。在此期间，氧浓度对滋养层细胞活性具有显著调控作用；体外实验表明，缺氧诱导因子（hypoxia inducible factors, HIFs）被认为是调控孕早期滋养层细胞行为的关键因子之一。本研究采用全基因组基因表达微阵列技术，旨在鉴定缺氧条件下（此时HIF-1处于激活状态）孕早期滋养层细胞系HTR8/SVneo的侵袭相关信号通路。此外，我们使用抗HIF-1与抗HIF-2抗体对不同孕周的孕早期胎盘样本进行标记，以评估在以低氧为特征的阶段（孕6~8周）及母体血液灌注阶段（孕10~12周），HIFs的表达与定位是否存在差异。在氧浓度为1%（此时HIF-1定位于细胞核）与5%（此时HIF-1定位于细胞质）的环境中培养的细胞之间，共有88个基因存在差异表达。其中65%的基因被预测含有HIF-1α:ARNT转录因子结合位点。孕早期早期的绒毛外细胞滋养层细胞中，HIF-1α的核定位水平高于孕早期晚期；而绒毛间质中HIF-2α的细胞表达水平在孕早期晚期更高。尽管在胎盘发育早期以及体外低氧环境中，滋养层细胞内的HIFs及其下游靶标均被显著诱导，但通过基因表达谱仍无法明确低氧条件下细胞侵袭能力增强的具体机制与通路。要充分理解这一复杂现象，还需获取转录水平之外的更多研究证据。本研究旨在通过在HIF-1α被预测为激活状态（1% O₂）与失活状态（5% O₂，生理性常氧）的条件下开展全基因组基因表达分析，阐明缺氧诱导基因在HTR8/SVneo细胞侵袭中的作用。本研究同时设置了常规大气氧培养条件（20% O₂），以评估以5%氧而非20%氧作为常氧代表所产生的影响。本研究重点关注了介导低氧细胞效应、作为基因表达关键调控因子的HIFs。我们在本次培养条件下以及不同孕周的孕早期胎盘切片中均证实了HTR8/SVneo细胞中HIF-1定位的差异，以验证HIFs在滋养层细胞分化的不同阶段均呈高表达。本研究分别在添加与不添加生长因子消减型基质胶（growth factor reduced Matrigel）的条件下开展微阵列实验：在基质胶上培养的HTR8/SVneo细胞呈现绒毛外、血管内皮样表型（Highet等，2012），而在塑料培养板上培养的HTR8/SVneo细胞则被认为更接近细胞滋养层柱近端区域的细胞表型（Kilburn等，2000）。