Differential survival responses of vascular smooth muscle cells (SMC) and endothelial cells (EC) towards different cycles of supercooling and re-warming

Abstract

BACKGROUND: Cryoplasty combines mechanical dilation with supercooling of the vessel used in the treatment of peripheral arterial disease. The mechanism of action is not well defined. This study investigates the optimal supercooling and re-warming cycles by studying the survival responses of SMC and EC. ATERIAL AND METHODS: Bovine aortic SMC and EC were cultured separately in 6 well plates with medium supplemented with 10% fetal bovine serum. In the 1 cycle treatment group, the cells were supercooled for 60 s to -10°C and then re-warmed rapidly in a water bath at 37°C for another 60 s. The samples were then put into an incubator at 37°C for 0, 6, 12 and 24 h. Two cycles treatment was done by supercooling and re-warming the cells twice. TUNEL assay and immunohistochemistry (IHC) and western blot (WB) were used to measure apoptosis and Akt activation. Results are given as mean +/- standard error of mean and analysed by ANOVA. RESULTS: Both EC and SMC showed increasing apoptosis with re-warming time and number of cycle treatment with significantly higher rate in SMC (P < 0.05). Akt activation decreased in SMC (P < 0.05) but significantly peaked at 6 h by threefold in EC (P < 0.05). CONCLUSION: The higher apoptotic rate with longer re-warming times and repeated treatment cycle in SMC, together with lower Akt activation may explain why cryoplasty can produce lower restenosis rate. This information will be useful in determining optimal treatment regimen for cryoplasty.