Atherosclerotic plaque smooth muscle cells (SMCs) are sensitive to apoptotic stimuli, increasing plaque instability. Testosterone replacement therapy is beneficial in men with coronary artery disease, although its effect upon plaque stability is unknown. We have utilised the rat A7r5 vascular SMC line to investigate the effects of testosterone on apoptosis induced by serum deprivation or the calcium channel antagonist nifedipine.
Cells were plated at 150000 cells per well in 6-well plates in DMEM supplemented with 10% foetal bovine serum (FBS), and left overnight to adhere. In serum-free experiments, media was replaced with serum-free DMEM, containing ethanol (0.1%) or testosterone (10-9, 10-7 or 10-5M) for 48-hours. In nifedipine experiments, media was replaced with DMEM supplemented with 2% FBS containing ethanol (0.1%) or testosterone (10-7M) for 30-minutes. Nifedipine (10-5 or 10-4M) was then added 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) did not affect the degree of early apoptosis induced by serum deprivation; 2.42plus/minus0.93%, 2.30plus/minus0.83%, 2.09plus/minus0.58%, 1.96plus/minus0.34% respectively (p>0.1). Following incubation with ethanol, the degree of early apoptosis induced by nifedipine (10-5 and 10-4M) was 3.96plus/minus0.41% and 6.30plus/minus0.61% (24-hours) and 4.95plus/minus0.21% and 13.4plus/minus2.16% (48-hours) respectively. Incubation with testosterone (10-7M) had no additional effect; 4.15plus/minus0.17% and 7.60plus/minus4.18% (24-hours) and 4.26plus/minus0.12% and 11.5plus/minus1.59% (48-hours) respectively (all p>0.1).
Testosterone does not affect apoptosis induced by either serum deprivation or nifedipine in A7r5 vascular SMCs, therefore, replacement therapy is unlikely to promote atherosclerotic plaque instability by further inducing VSMC apoptosis.
22 - 24 Mar 2004
British Endocrine Societies