Differential survival responses of vascular smooth muscle cells and endothelial cells towards different cycles of supercooling and rewarming in a model simulating cryoplasty

Abstract

BACKGROUND: Restenosis, in which the interactions of Smooth muscle cells (SMC) and endothelial cells (EC) play a significant role, is a major complication in treating peripheral arterial disease. Though recent studies have shown that cryoplasty may produce less restenosis but its underlying mechanism is still not well understood. This study investigates the survival responses of SMC and EC after different cycle treatments of supercooling and re-warming in an in vitro model simulating cryoplasty. MATERIAL 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 seconds to -10 degree C and then re-warmed rapidly in a water bath at 37degree C for another 60 seconds. The samples were then put into an incubator at 37 degree C for 0, 6, 12 and 24 hours. 2 cycles treatment was done by supercooling and re-warming the cells twice. TUNEL assay, 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 apoptotic rates with increasing re-warming time and no. of cycle treatment, but significantly higher in SMC (p<0.05), corresponding to its decreasing Akt activation with prolonging re-warming time and cycles of treatment (p<0.05). On the other hand, EC showed significant higher Akt activation, as assessed by IHC and WB, at 6 and 12 hours re-warming time compared with SMC (p<0.001). It was significantly peaked at 6 hours by 3 fold (p<0.05) and then decreased with re-warming time. CONCLUSION: The higher apoptotic rate and lower Akt activation with increasing re-warming times and treatment cycles in SMC, together with the relative lower apoptosis in EC may explain why cryoplasty can produce lower restenosis, where the integrity of EC and the lower SMC survival are the important factors in reducing restenosis. This information will also be useful in determining optimal cycle regimen for cryoplasty.