Print this pageVascular Smooth Muscle Cells Apoptosis induced by supercooling and re-warming
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Wai-ki Yiu, MBBS, MRCS1, John E Aruny, MD1, Stephen WK Cheng, MS, FRCS, FACS2, Bauer E Sumpio, MD, PhD1.
1Department of Vascular Surgery, Yale University School of Medicine, New Haven, CT, USA, 2Division of Vascular Surgery, Department of Surgery, University of Hong Kong Medical Centre, Hong Kong, Hong Kong Special Administrative Region of China.
Background: Cryoplasty is a technique that combines angioplasty with tissue cooling and has been introduced as a novel treatment for peripheral atherosclerotic disease with improved long-term patency. The mechanisms underlying this effect are not entirely clear. Vascular smooth muscle cells (SMC) play a critical role in neointimal hyperplasia, but the SMC responses to supercooling and/ or re-warming are still not well understood. The aim of this study was to determine SMC survival under temperature conditions similar to cryoplasty.
Material and methods: Bovine aortic SMC were cultured in medium supplemented with 10% fetal bovine serum. SMC were supercooled to -20oC for 0, 60, 120, 180 or 240 seconds using a custom-designed conduction cooling stage and then re-warmed to 37oC in an incubator for 0,12 or 24 hours. The 0 second cooling and 0 hour re-warming group served as control. The TUNEL assay was used to measure the degree of apoptosis. Activation of AKT, a cell signal protein involved in apoptosis, was assessed by western blot and quantified by densitometry. Results are given as mean + standard error of mean and analysed by ANOVA.
Results: Figure 1 shows that the % of apoptotic SMC progressively increased with increasing exposure to supercooling and subsequent re-warming.
Activation of Akt (phosphorylation, pAKT) was also observed (Figure 2) to be dependent on the supercooling and re-warming time. It was significantly peaked at 120s supercooling time by 2.03 fold after 24 hours re-warming (p<0.05).
Conclusion: The supercooling and re-warming processes induce significant effect on vascular smooth muscle cells survival. The mechanism of this effect appears to involve AKT activation. We hypothesize that SMC apoptosis in vivo after supercooling may reduce SMC proliferation and may explain the relatively low incidence of neointimal hyperplasia after cryoplasty. 
