812 TESTOSERONE MODULATES VASCULAR SMOOTH MUSCLE CELL MEDIATED MATRIX METALLOPROTEINASE EXPRESSION AND FUNCTION, IN VITRO

Abstract

INTRODUCTION AND OBJECTIVES: A strong association between serum testosterone levels and cardiovascular disease has been established. However, this relationship is complex, and the exact mechanism by which testosterone affects the vasculature system remains to be elucidated. Intimal hyperplasia (IH), the general response of a vessel to injury, is a well-established hallmark of atherosclerosis. An important component of this pathway involves matrix metalloproteinases (MMPs), implicated in vascular remodeling due to their ability to degrade components of the extracellular matrix. Previous studies have demonstrated a significant reduction of IH in the presence of testosterone, and our group has shown a positive correlation between female sex hormones and MMP activity. Therefore, we hypothesize testosterone reduces IH through modulation of MMP expression and function in male vascular smooth muscle cells (VSMCs). METHODS: Male human aortic VSMCs were treated with low to high physiological concentrations of testosterone (TST; 0.3nM-3 M) or Dihydrotestosterone (DHT; 0.3nM-3 M) for 24hr. Control cells were incubated with delivery vehicle only. Total RNA was isolated and subjected to quantitative polymerase chain reaction (qPCR). Cell lysates were collected and subjected to Western blot analysis. MMP activities were measured in conditioned media using in-gel protease analysis. A modified MTT proliferation assay was used to investigate the role of TST and DHT on VSMC proliferation. Finally, VSMC migration was evaluated using a modified collagen type IV coated Boyden chamber assay. RESULTS: qPCR revealed no significant change in the gene expression of MMPs. Western blot analysis and in-gel zymography demonstrated a significant change (P 0.05) in MMP-2 protein levels and enzymatic activity at higher physiological TST and DHT concentrations. Androgen stimulation at high physiological levels inhibited VSMC proliferation and migration through a collagen type IV lattice. CONCLUSIONS: Dysfunctional remodeling underlies the pathogenesis of major vascular diseases, such as atherosclerosis. A key group of enzymes involved in these processes are MMPs. Testosterone affects the MMP pathway in a concentration-dependent manner. With increasing physiological levels of testosterone, MMP activity and VSMC migration are significantly inhibited, in vitro. Our data suggests the risk of developing IH may decrease with higher physiological testosterone concentrations via the downregulation of MMP activity.