J. Univers1, D. J. Mountain1, B. M. Freeman1, R. T. Fisher1, S. S. Kirkpatrick1, F. A. Klein1, M. B. Freeman1, O. H. Grandas1 1University Of Tennessee Graduate School Of Medicine,Department Of Surgery,Knoxville, TN, USA
Introduction: Androgen deficiency (AD) is associated with increased risk of vascular disease, though the underlying mechanisms remain unclear. We have previously demonstrated testosterone (TST) and dihydrotestosterone (DHT) inhibit vascular smooth muscle cell (VSMC) migration and proliferation in a dose dependent manner in vitro. Furthermore, we have shown that AD increases intimal hyperplasia (IH) in vivo in a rodent model of vascular injury, while physiological TST replacement attenuated this effect. Urotensin II (UTS) is a potent vasoconstrictor and can stimulate cellular proliferation. Activation of the UTS/UTS receptor mechanism has been shown to exacerbate vascular pathologies. Here we investigated the role of UTS in AD-induced IH.
Methods: Three groups of aged orchiectomized (AO) male rats underwent TST supplementation via controlled release pellet (0.5-5 mg). Young and aged intact (YI, AI) and AO placebo (Plac) groups served as controls. All groups underwent balloon angioplasty of the left common carotid following 14d TST therapy. Carotid tissue was collected 14d post-injury, and stained for UTS quantification. Human male VSMCs were treated with TST or DHT (0-3000nM) for 24h then subjected to qPCR for UTS/UTS receptor expression or PCNA/Ki67 proliferation marker analyses. Cells were stained with Phalloidin-FITC for visualization of cytoskeletal organization.
Results:UTS receptor staining was low in injured vessels of YI, AI, and Plac controls but was significantly upregulated in all AO groups receiving TST supplementation, irrespective of dose (Fig1A/B). UTS peptide in the tissue was not affected by TST exposure. In vitro exposure to DHT increased the expression of UTS receptor in VSMCs in a dose-dependent manner, with 3000nM increasing expression by 55±13% vs. 0nM; n=3; P<0.05. However, this did not correlate with any change in proliferative markers PCNA or Ki67. Phalloidin staining of filamentous-actin revealed that DHT induced cytoskeletal organization in a dose–dependent manner (Fig1C).
Conclusion:AD alone does not affect the UTS/UTS receptor mechanism. However TST and DHT increase the expression of UTS receptor, in vivo and in vitro, respectively. This regulation has no effect on proliferative markers but does correlate to a shift in filamentous-actin organization. Analyses of this organization in the presence of UTS receptor agonist/antagonists, and its downstream effect on vasotone, are ongoing. Future studies will examine the potential for exogenous TST therapy to exacerbate dysfunctional vasoconstriction via the upregulation of UTS receptor. These studies are needed in determining if TST replacement in AD men should be evaluated for attenuation of vascular pathogenesis.
