Low testosterone levels in men are associated with increased atherosclerosis, and testosterone replacement therapy has been shown to reduce myocardial ischemia in men with coronary artery disease. Vascular smooth muscle cell (VSMC) apoptosis is proposed to reduce plaque stability, but the role of testosterone in this process is unknown. Testosterone causes vasodilatation via calcium channel antagonism and the calcium channel antagonist nifedipine induces VSMC apoptosis. We have compared the actions of these two agents on A7r5 VSMC apoptosis.
A7r5 cells were plated at 150000 cells per well in 6-well plates in DMEM supplemented with 10% foetal bovine serum (FBS), and left to adhere overnight. Media was replaced with phenol red-free DMEM supplemented with 2% FBS, containing either testosterone (10-9, 10-7 or 10-5M) or ethanol control (0.1%), or nifedipine (10-6, 10-5 or 10-4M) or DMSO control (0.1%), for 24 or 48-hours. Adherent cells were removed with trypsin-EDTA, combined with floating cells via centrifugation at 1000rpm for 5-minutes, washed in PBS and resuspended in annexin-binding buffer. Cells were then dual stained with FITC-conjugated annexin V and propidium iodide, and analysed by flow-cytometry.
Compared to ethanol control, testosterone (10-9, 10-7 and 10-5M) significantly decreased the number of early apoptotic cells over 24-hours; 1.84plus/minus0.17%, 1.75plus/minus0.12%, 1.55plus/minus0.12% and 1.45plus/minus0.10% respectively (p=0.03), but not over 48-hours. Compared to DMSO control, nifedipine (10-6, 10-5 and 10-4M) increased the number of early apoptotic cells over 24-hours; 4.23plus/minus0.25%, 4.84plus/minus0.41%, 5.56plus/minus0.34% and 7.59plus/minus0.60% respectively (p<0.001). This effect was enhanced after 48-hours; 3.62plus/minus0.32%, 5.02plus/minus0.25%, 6.30plus/minus0.92% and 33.1plus/minus2.40% respectively (p<0.001).
Nifedipine induces, whereas testosterone protects against, A7r5 VSMC apoptosis, indicating different mechanisms of action. Testosterone replacement therapy may improve atherosclerotic plaque stability via this action.
22 - 24 Mar 2004
British Endocrine Societies