The Effects of Freezing versus Supercooling on the Vascular Smooth Muscle Cell

Cryoplasty, a technique combining conventional angioplasty with the delivery of cold thermal energy, has emerged as a treatment for peripheral vascular disease with improved late results, achieving greater long term patency rates compared to conventional angioplasty. Vascular smooth muscle cells (SMC) are known to play a significant role in restenosis and neointimal hyperplasia but the mechanisms were not well understood. Our previous studies suggest supercooling induces SMC apoptosis. This study compares the effects of freezing versus supercooling on the SMC. METHOD: Bovine aortic SMC were either frozen or supercooled at approximately 0°C and −10°C for 0, 30 or 60 seconds using a cooling stage and then re‐warmed to 37°C in an incubator for 24 hours. TUNEL assay was used to identify apoptotic cells. RESULTS: Up to 29.5% of cells in apoptosis was observed when SMC's were frozen. SMC supercooling yielded rates reaching only 14.3% (Fig. 1). Rates of apoptosis also increased with increasing frozen time (1.1% with 0 second frozen time vs. 29.5 % after 60 seconds frozen time) (Fig. 1). An increasing number of detached cells were observed with increasing frozen time (38.6% after 60 seconds frozen time) (Fig. 2). CONCLUSION: Freezing induces SMC apoptosis. This was more pronounced then with supercooling alone. The rate of apoptosis increases with longer SMC frozen time. Furthermore, longer frozen times result in an increasing number of dead cells including those that have completed apoptosis, contributing to an even higher apoptosis rate. Further studies into the physics and pathophysiology of freezing and its apoptotic pathway may elucidate the mechanism behind the long term patency rates associated with cryoplasty. 12