Abstract 328: Capillary Formation and Autologous Regulation of Androgen Receptor Expression in Endothelial Progenitor Cells by DHT: Potential Implications for EPC-Mediated Cardiovascular Repair in Men

Abstract

Therapeutic use of Endothelial Progenitor Cells (EPCs) is known to repair and protect against cardiovascular damage. Since androgens are associated with cardiovascular protection in men, we investigated the mechanism(s) by which androgens (dihydrotestosterone; DHT) can modulate androgen receptor (AR) dependent EPC activity. Tube/capillary formation and androgen receptor expression by cultured EPCs was assessed using 2D-cultures/co-cultures, western blotting and RT-PCR. Treatment with DHT (100nM) induced capillary/tube formation by EPCs (≈179±3%; p<.05 vs control) and these effects were attenuated by AR antagonist flutamide and AR siRNA. DHT (10-100nM) up-regulated AR protein expression and this effect was abrogated by AR antagonist flutamide (1μM), AR silencing siRNA and cycloheximide (protein synthesis inhibitor). Compared to AR protein, DHT did not induce AR mRNA expression and DHT-induced AR expression was not blocked by the transcription inhibitor actinomycin-D (10ng/ml). Treatment with proteasome inhibitor MG132 (100nM) mimicked the effects of DHT on AR protein expression, moreover, when combined with DHT, the stimulatory effects on AR expression were additive. To ascertain the role of protein stabilization in mediating AR up-regulation in EPCs, the effects of MG132 and DHT on Raf-1, which is known to undergo proteasomal degradation, were assessed. Treatment with MG132, but not DHT, increased Raf-1 expression in EPCs. Moreover, the stimulatory effects of DHT on capillary formation were enhanced in presence of MG132. These findings suggest that DHT up-regulates AR expression and function in EPCs via protein stabilization, however, participation of other pathways cannot be ruled out. In EPCs, DHT induces capillary formation via AR and regulates AR expression in an autologous fashion. Since, androgens induce endothelial growth, and most patients receiving EPCs for cardiovascular repair are older, the low endogenous levels of androgens would decrease the potential of stem cell mediated tissue repair. More importantly, pretreatment of patients or priming of EPCs with androgens/DHT, as well as co-administration of DHT with EPCs may potentiate ARs and EPC mediate cardiovascular tissue repair in men.