Direct comparison of endothelial cell and smooth muscle cell response to supercooling and rewarming

Abstract

Background: Cryoplasty combines mechanical dilatation with the delivery of hypothermia to atherosclerotic plaques. The response of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) to supercooling and subsequent rewarming is still not clear. This study investigated the differential effects of vascular cell survival and proliferation in an in vitro model simulating cryoplasty. Methods: Bovine aortic ECs and SMCs were cultured separately with medium supplemented with 10% fetal bovine serum. The samples were supercooled to -10°C for 0, 60, or 120 seconds on a cooling stage and then rewarmed in an incubator at 37°C for 0, 6, 12, or 24 hours. Terminal deoxynucleotide transferase-mediated deoxy uridine triphosphate nick-end labeling (TUNEL) and 5′-bromo-2′-deoxyuridine incorporation were used to measure the degree of apoptosis and proliferation respectively. Activation of protein kinase B (AKT), P70 S6 kinase, and P44/42 mitogen-activated protein kinase (MAPK) were assessed by Western blot and quantified using densitometry. Results are given as mean ± standard error of mean and analyzed by analysis of variance. Results: SMC and EC apoptosis were significantly increased with increasing supercooling and rewarming time, with a higher rate in SMCs. SMC apoptosis was maximal at 60 seconds cooling, followed by 24 hours rewarming (17.05% ± 0.44%), whereas maximal EC apoptosis was after 120 seconds cooling, followed by 24 hours rewarming (4.21% ± 0.22%, P < .05). Higher AKT activation was observed in ECs, with a maximum obtained of 3.34-fold at 120 seconds cooling with 24 hours rewarming (P < .05); only modest activation was found in SMCs. ECs had a decreased proliferation with cooling and rewarming time, and although SMCs maintained their low proliferative rate, ECs still had a higher overall proliferation rate that was statistically significant at 60 and 120 seconds cooling without rewarming compared with noncooling and nonrewarming (P < .05). Both p70S6 kinase and p44/42 MAPK activities decreased in SMCs, with significant drop at 60 seconds cooling, followed by 12 hours rewarming (P < .05). However, ECs showed a significant rise of P70 S6 kinase activity at 60 seconds cooling with 12 hours rewarming by 1.62-fold and P44/42 MAPK at 120 seconds cooling with 24 hours rewarming by 1.74-fold (P < .05). Conclusion: The higher apoptosis and lower proliferation of SMCs compared with ECs demonstrate the different effects of supercooling and rewarming on different vascular cell types. This information may be important in helping to understand the mechanism by which cryoplasty of atherosclerotic lesions may result in less restenosis. © 2007 The Society for Vascular Surgery.