Synergistic effect of cool/thaw cycles on vascular cells in an in vitro model of cryoplasty

PURPOSE: Cryoplasty combines mechanical dilation with supercooling of the vessel and has shown encouraging preliminary results in the management of atherosclerotic lesions. However, the mechanisms of action and the optimum inflation regimen are still not well established. This study investigates the effects of single and dual supercooling and rewarming cycles on the survival responses of smooth muscle cells (SMCs) and endothelial cells (ECs). MATERIALS AND METHODS: Bovine aortic SMCs and ECs were cultured separately in six-well plates with medium supplemented with 10% fetal bovine serum. In the one-cycle treatment group, the cells were supercooled for 60 seconds to -10 degrees C and then rewarmed rapidly in a water bath at 37 degrees C for another 60 seconds. Two-cycle treatment was done by supercooling and rewarming the cells twice. The samples were then put into an incubator at 37 degrees C for 0, 6, 12, and 24 hours. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was used to measure apoptosis and phospho-Akt immunohistochemistry and immunoblot analysis were employed to assess activation of Akt. RESULTS: A significant increase of apoptotic cells was observed in the two-cycle procedure versus the one-cycle procedure. In both groups, there were more apoptotic SMCs than ECs. Akt activation was higher in ECs by a factor of three compared with SMCs (P <.05). CONCLUSIONS: The higher apoptotic rate and the absence of Akt activation of SMCs versus controls in both treatment groups may imply the potential of a lower restenosis rate, especially after two cycles of supercooling and rewarming. Further in vivo and clinical investigations are needed to confirm results of the in vitro testing.