Effects of estradiol on cultured retinal endothelial cells is partially repressed by NGF signaling blocking

Abstract

Abstract: : Purpose: Receptors on endothelial cell promise the cell a multi–regulatory target of several signal pathways. Estrogen–estrogen receptor (E–ER) complex regulates many genes involved in cell survival and growth. It was reported recently that in cells outside the reproduction system and nervous system, NGF signal is also regulated by Estrogen. This study is to verify the estradiol–NGF regulatory cascade in retinal endothelial cells. Methods: Retina–choroid vascular endothelial cell line RF/6A from rhesus was cultured in M199 medium contain 20% FBS. mRNA of ER, NGF and its TrkA and P75NTR receptors was detected with RT–PCR. The cells were incubated with NGF, VEGF, antibodies against these factors, and specific TrkA inhibitor K252a(100nM) separately or in different combination. MTT based cell counting assay was used to study the viability of the cells. The apoptosis was evaluated with FACS as well. Would healing assay combined with image analysis were carried out to study the mass migration of the cell. The distance of migration was calculated and calibrated in free software NIH Image. AngioMatrix assay was used to study the tubogenesis effects of estradiol. Results: We amplified specific fragments of cDNA of ER, NGF and NGF receptor TrkA using RT–PCR. 10nM–1uM estradiol stimulates the proliferation and increases the viability of RF/6A in a dosage dependent manner. In the wound healing based migration assay, we found the endothelial cells react in a similar fashion when treated with the reagents. The increscent effects of estradiol was partially blocked by NGF neutralized antibody and K252a. BDNF as a control has similar effects on the viablity and migration of RF/6A, but was not blocked by TrkA inhibitor K252a. Apoptosis rate is also lower in estradiol treated cells. Tubogenesis was increased when treated with estradiol, but not with NGF. This effect of estradiol is not blocked by K252 as well. Conclusions: NGF, first identified as the survival factor of nerve system, now also an activator of retinal endothelial cell, is under the regulation of estrogen. The proliferation and migration, but not the tubogenesis of retinal endothelial cells is regulated by this regulatory cascade.

Keywords: retinal neovascularization • signal transduction • growth factors/growth factor receptors