The possible role played by hypoxia and vascular endothelial growth factor (VEGF) in the regulation of follicular angiogenesis was studied in a three-dimensional fibrin gel model. Granulosa cells from follicles >5mm were subjected to normoxia (19% O2), partial (5% O2) or total (1% O2) hypoxia and their culture media were collected and used to stimulate porcine Aortic Endothelial Cells (AOC) included in the fibrin matrix. A suspension of AOC on microcarrier beads was pipetted in a fibrinogen solution (1 mg/ml PBS) before the addition of 1250 IU thrombine (250 microl) to catalize the gel formation. Granulosa cell conditioned media were tested in the presence or absence of VEGF Trap R1R2 (150 ng/ml), a potent VEGF inhibitor, that had its efficacy tested by adding VEGF (100 ng/ml) to AOC culture. Endothelial cell proliferation was measured at 48, 96, 144, 192 h by means of Scion Image Beta. A significant (p < 0.01) increase of AOC proliferation at each time of measurement was induced by culture media from granulosa cells subjected to partial (except at the end of the first 48 h) and total hypoxia compared to control and normoxia conditions, and by VEGF. VEGF Trap significantly (p < 0.01) inhibited the stimulatory effect of media conditioned by granulosa cells cultured in hypoxic conditions. These data suggest that hypoxia stimulates angiogenic activity of granulosa cells possibly by means of VEGF which could represent the main effector in promoting endothelial cell proliferation.
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