17 beta-estradiol increases VEGF receptor-2 and promotes DNA synthesis in retinal microvascular endothelial cells

PURPOSE. Estrogen is known to promote angiogenesis in gonads. The presence of estrogen receptors in the vascular endothelium of organs other than gona ds has been reported. The goal of this study was to determine whether estro gen promotes the proliferation of retinal microvascular endothelial cells a nd to explore the mechanism of it. METHODS. DNA was quantitated using primary cultures of bovine retinal endot helial cells that were incubated with different doses of 17 beta-estradiol (E-2), VEGF, or both. Thr changes in expression level of VEGF and VEGF rece ptor-2 (VEGFR2) were measured using northern blot analysis after treatment with E-2. The presence of estrogen receptors in the endothelial cells was s tudied by immunohistochemistry and western blot analysis. RESULTS. 17 beta-Estradiol (E-2) increased the DNA level in bovine retinal capillary endothelial cells (BRECs) by 177% at 1 nM (P < 0.05) and 150% at 10 nM (P < 0.05) by comparison with unstimulated BREC. One hundred nanomole tamoxifen completely blocked the E-2-induced DNA synthesis in BRECs. Ten n anomole E-2 augmented vascular endothelial growth factor (VEGF)-induced DNA synthesis in BRECs significantly (160%, P < 0.01). Ten nanomole E-2 also i ncreased VEGF mRNA expression, which peaked after 24 hours (6.7 times, P < 0.05), and VEGF receptor-2 (VEGFR2) mRNA expression, which peaked after 9 h ours (2.4 times, P < 0.05). The mRNA expression level of VEGFR2 peaked with 10 nM E-2 (P < 0.05) and that of VEGF reached maximum with 1 nM E-2 (15 ti mes, P < 0.001), VEGFR2 and VEGF proteins increased in parallel with their mRNA levels. Immunocytochemistry showed estrogen receptor expression in BRE Cs, and western blot analysis indicated the presence of a 67-kDa protein th at was compatible with the estrogen receptor. CONCLUSIONS. These findings suggest that E-2 may stimulate BREC growth by t he receptor-mediated pathway and that E-2 may augment the VEGF-dependent an giogenesis partly through the upregulation of VEGFR2.