IS THE UPREGULATION OF VEGF BY hCG IN HUMAN LUTEAL CELLS DEPENDENT ON HIF-1ALPHA EXPRESSION?

Abstract

Angiogenesis is essential for normal growth and function of the corpus luteum. Several lines of research have demonstrated the importance of Vascular Endothelial Growth Factor (VEGF) in corpus luteum angiogenesis. Generally it is believed that hypoxia is the primary inducer of VEGF production in most tissues. Indeed, experiments using non-human primate cells suggested that hypoxia was a major regulator of luteal VEGF expression. However we have previously shown that VEGF is upregulated by human chorionic gonadotropin (hCG) in the human corpus luteum. As Hypoxia Inducible Factor-1alpha (HIF-1alpha) has been shown to regulate the expression of VEGF under non-hypoxic (i.e. ligand stimulated) conditions, as well as under hypoxic conditions, in cell lines, we hypothesised that the effect of hCG on luteal VEGF expression was mediated through HIF-1alpha. We therefore studied the effect of hCG on VEGF and HIF-1á expression in cultured luteinized granulosa cells (LGCs) in vitro and in human corpora lutea in vivo. LGCs were obtained from women undergoing oocyte recovering as part of an assisted conception program. Isolated LGCs were cultured under serum-free conditions, on Matrigel-coated plastic tissue culture plates, for one week and the culture medium was refreshed every two days. Cells were then treated with hCG alone (100 ng/ml) or hCG in the presence of synthetic inhibitory molecules before mRNA was collected. The expression of VEGF and HIF-1alpha mRNA was analyzed by Taqman quantitative real-time PCR. In vitro, hCG up-regulated both VEGF (P<0.0001) and HIF-1alpha (P<0.0001) mRNA transcripts in LGCs. In vivo, expression was investigated in corpora lutea from the late-luteal phase in the presence (n=5) or absence (n=5) of exogenous hCG concentrations similar to those seen in early pregnancy. Both VEGF (P=0.02) and HIF-1alpha (P=0.034) mRNA expression was upregulated by hCG during luteal rescue when compared to that in the late-luteal phase. To investigate whether the effect of hCG on HIF-1alpha/VEGF expression was under influence of progesterone, LGCs were stimulated with hCG in the presence or absence of the progesterone synthesis inhibitor aminoglutethamide (100 μM). The up-regulation of both HIF-1alpha and VEGF by hCG was independent of progesterone in vitro. As both the Raf/MEK/MAPK and the PI3 kinase/Akt signalling pathways have been shown to stimulate VEGF mRNA expression through the regulation of HIF-1alpha activity in a range of cell lines, LGCs were stimulated with hCG in the presence or absence of specific synthetic inhibitors of the Raf/MEK/MAPK (PD98059 (50 μM)) and PI3 kinase/Akt (LY294002 (50 μM)) pathways. PD98059 and LY294002 inhibited both VEGF and HIF-1alpha mRNA expression, whereas the mRNA expression of steroidogenic acute regulatory protein (StAR), which is regulated through a cAMP-dependent PKA pathway, was not affected. In summary, our results suggest that the expression of HIF- 1alpha is hormonally regulated in human luteal cells under normoxic conditions in vitro and in vivo and this may in turn regulate VEGF expression. (poster)