Endocrine Abstracts

Several studies have demonstrated that atherosclerosis is associated with low serum levels of testosterone and replacement therapy reduces myocardial ischaemia in men. Vascular smooth muscle cell (VSMC) apoptosis reduces plaque stability. The role of testosterone if any, in this process is unknown. Testosterone has been demonstrated act via both the classic androgen receptor and by calcium channel antagonism. Calcium channel blockers have been shown to induce VSMC apoptosis. We have compared the effects of different testosterone preparations on apoptosis in the VSMC cell line A7r5.

A7r5 cells were plated at 150000 cells per well in DMEM supplemented with 10% foetal bovine serum (FBS), and left to adhere overnight. Media was then replaced with phenol red-free DMEM supplemented with 2% FBS containing either testosterone, testosterone-3-carboxymethyloxime or testosterone-3-carboxymethyloxime-BSA (all 10^-9, 10^-7 or 10^-5M) alone or in combination with the appropriate control; BSA (3.45x10^-4M) or ethanol (0.1%). Separately, cells were also pre-incubated with flutamide (10^-5M) or ethanol control (0.05%) 2-hours before addition of testosterone (10^-5) or ethanol vehicle (0.05%). All experiments were incubated for 24-hours at 37degC. Adherent cells were trysinised, combined with floating cells via centrifugation, washed in PBS and resuspended in annexin-binding buffer. Cells were then dual stained with FITC-conjugated annexin V and propidium iodide, and analysed via flow cytometry.

Compared to appropriate vehicle, neither the percentage of apoptotic nor late apoptotic/necrotic cells were affected by either testosterone-3-carboxymethyloxime: 2.95plus/minus0.40% and 4.86plus/minus0.69% (10^-9M), 3.01plus/minus0.43% and 3.88plus/minus0.29% (10^-7M), and 3.47plus/minus0.50% and 4.88plus/minus0.87% (10^-5M) respectively; testosterone-3-carboxymethyloxime-BSA: 3.48plus/minus0.43% and 4.61plus/minus0.77% (10^-9M), 3.07plus/minus0.35% and 3.56plus/minus0.39% (10^-7M), and 3.62plus/minus0.42% and 4.07plus/minus0.73% (10^-5M) respectively; testosterone (10^-5M): 3.24plus/minus0.47% and 7.95plus/minus0.63% respectively; or flutamide (10^-5M) plus testosterone (10^-5M): 2.46plus/minus0.31 and 10.46plus/minus1.09 respectively. All p>0.05.

None of the testosterone preparations tested showed any significant effects on A7r5 apoptosis, therefore replacement therapy is unlikely to promote plaque instability via inducing VSMC death.