The Effect And Mechanism Of Estrogen Receptor Ligands On DHT-Induced Endothelial Cells Growth

Objective:1. To examine the effect of estrogen receptor(ER)-ligands and androgen on cell proliferation and gene expression in human aortic endothelial cells (HAECs).2. To investigate the differential modulation of DHT actions by ER-ligands.Method:1. To examine the effect of ER-ligands, HAECs were cultured in various doses of17alpha-estradiol (αE2),17beta-estradiol (βE2), diethylstilbestrol (DES), tamoxifen and genistein for48hours, at the end of the experiment, MTS cell proliferation assay was used to determine viable cell numbers, qRT-PCR was used to analyze the expression of vascular endothelial growth factor (VEGF) and cyclin A mRNA, MSD assay was used to evaluate the secretion of VEGF in medium. To determine the role of estrogen receptor alpha (ERa) and estrogen receptor beta (ERβ) in cell proliferation, we first examined the expression of ERa and ERβ using RT-PCR and Western blot, then cultured HAECs in various doses of ERa agonist PPT and ERβ agonist DPN for48hour, at the end of the experiment, analyzed the viable cell numbers by MTS assay.2. HAECs were cultured in various doses of dihydrotestosterone (DHT) for0hour,24hours or48hours, viable cell numbers were determined to optimize DHT treatment condition for HAECs. The expression of AR, VEGF, Cyclin A and Cyclin D were analyzed by qRT-PCR, Western blot and MSD assay in the optimized condition. To explore the role of androgen receptor (AR) in DHT-induced HAECs growth, AR was knocked-down by specific AR siRNA, then HAECs were treated with DHT lOnM for48hours, at the end the experiment, viable cell numbers were determined.3. To investigate the modulation of DHT actions by ER-ligands, the corresponding doses of various ER-ligands with DHT10nM were used to treat HAECs for48hours, DHT10nM serves as a positive control, viable cell numbers were determined at the end of the experiments. Further, ERa agonist PPT and ER(3agonist DPN and ER antagonist ICI182780(ICI) were used to evaluate the function of ER subtypes in modulation of DHT actions by ER-ligands.Results:1. We found that viable cell numbers were significantly increased by approximately24%and31%, respectively, at aE210nmol/L and aE2100nmol/L, while various doses ofβE2, DES, tamoxifen and genistein treatment had no significant effect. The expression of cyclin A mRNA was significantly up-regulated at αE2100nmol/L but not0E2100nmol/L. Neither aE2.100nmol/L nor βE2100nmol/L had significant effect on VEGF expression. The viable cell numbers were increased at PPT treatment, specifically significantly increased by24%at PPT1nmol/L DPN treatment had no significant effect.2. To determine the effect of DHT on HAECs proliferation, HAECs were treated at various doses of DHT in different time periods. The viable cell numbers were significantly increased in a dose and time-dependent manner. The expression of AR, VEGF, cyclin A and cyclin D mRNA was significantly up-regulated in a dose-dependent way for48hours. AR protein was significantly increased at DHT10nmol/L. The secretion of VEGF was significantly increased at DHT5nmol/L and10nmol/L When transfected by specific AR siRNA200nmol/L, AR was silenced effectively(>90%)。After transfection, HAECs were treated with DHT10nmol/L, the viable cell numbers were significantly decreased by AR siRNA (P<0.01)3. The viable cell numbers induced by DHT10nmol/L were not decreased by co-administration with αE2(10nmol/L and100nmol/L)、 genistein (1μmol/L) and PPT(0.05nmol/L, O.lnmol/L,1nmol/L). While treated with various doses of βE2, DES, tamoxifen, DPN and ICI combined with DHT10nmol/L, the viable cell numbers were decreased significantly comparing with DHT10nmol/L.Conclusion: 1. The effect of various ER-ligands on HAECs proliferation is different. αE2and PPT induces HAECs growth, but βE2, DES, tamoxifen, genistein, ICI182780and DPN have no effect on HAECs proliferation.2. DHT acting via androgen receptor induces HAECs growth, and up-regulates the expression of AR、VEGF、Cyclin A and Cyclin D.3. The DHT-induced HAECs growth is modulated by ER-ligands differentially. aE2, genistein and PPT have no effect on DHT induction of HAECs growth, while βE2, DES, tamoxifen, ICI182780and DPN inhibit DHT-induced HAECs proliferation.