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Angiogenesis During Follicular Development in the Primate and its Inhibition by Treatment with Truncated Flt-1-Fc (Vascular Endothelial Growth Factor Trap_A40)¹

Medical Research Council (C.W., P.T.K.S., G.A.S., H.M.F.), Human Reproductive Sciences Unit, EH3 9ET Edinburgh, United Kingdom; and Regeneron Pharmaceuticals (S.J.W.), Tarrytown, New York 10591

Address all correspondence and requests for reprints to: Dr. C. Wulff, Medical Research Council, Human Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh EH3 9ET, United Kingdom. E-mail: c.wulff{at}hrsu.mrc.ac.uk

The aims of this study were to 1) quantify changes in angiogenesis during follicular growth in a primate model; 2) investigate the molecular regulation using in situ hybridization of vascular endothelial growth factor (VEGF), its receptor, Flt-1, the angiopoietins (Ang-1 and Ang-2), and their receptor, Tie-2; 3) elucidate the role of VEGF in follicular angiogenesis by blocking its action by treatment with a soluble truncated form of the Flt-1 receptor, (VEGF Trap_A40). Changes in angiogenesis were quantified using bromodeoxyuridine to obtain a proliferation index, and CD31 immunocytochemistry to visualize endothelial cell area. Percentage of proliferating endothelial cells was calculated by double labeling for bromodeoxyuridine and CD31. Vascularization was first observed in follicles containing four granulosa cell layers. A significant increase in proliferation in the thecal layer was observed from the early to late secondary stage, and dual staining showed that 25% of proliferating cells were of endothelial cell origin. VEGF messenger RNA (mRNA) was expressed in granulosa cells with an increase of grain density from late secondary to tertiary follicles. Ang-1 was weakly expressed in the theca of tertiary follicles. Ang-2 mRNA was not detected in any follicles. The mRNA for the Flt-1 and Tie-2 receptors was localized in endothelial cells of the theca. Unexpectedly, Tie-2 mRNA was also found in granulosa cells of early follicular stages and its translation was confirmed by immunocytochemistry. VEGF trap treatment for 3 days resulted in an 87% decrease of proliferation in the theca of secondary and tertiary follicles, a reduction in endothelial cell area and a marked decline in Flt-1 mRNA expression. Granulosa cell proliferation also decreased. These results show that onset and establishment of the follicle vasculature takes place early during follicular development. The ability of VEGF trap treatment to severely restrict follicular angiogenesis establishes that VEGF is the major regulator of this process in the primate ovary.

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