Yiu Wai-ki, Cheng Stephen W K, Sumpio Bauer E
Department of Vascular Surgery, Yale University School of Medicine, New Haven, CT 06520-8062, USA.
J Vasc Surg. 2007 Sep;46(3):557-564. doi: 10.1016/j.jvs.2007.04.072.
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.
Bovine aortic ECs and SMCs were cultured separately with medium supplemented with 10% fetal bovine serum. The samples were supercooled to -10 degrees C for 0, 60, or 120 seconds on a cooling stage and then rewarmed in an incubator at 37 degrees 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.
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).
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.
冷冻球囊血管成形术将机械扩张与向动脉粥样硬化斑块输送低温相结合。血管平滑肌细胞（SMC）和内皮细胞（EC）对过冷及随后复温的反应仍不清楚。本研究在模拟冷冻球囊血管成形术的体外模型中，研究了血管细胞存活和增殖的差异效应。
牛主动脉内皮细胞和平滑肌细胞分别在补充有10%胎牛血清的培养基中培养。样本在冷却台上过冷至-10℃ 0、60或120秒，然后在37℃培养箱中复温0、6、12或24小时。分别采用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记法（TUNEL）和5'-溴-2'-脱氧尿苷掺入法测量凋亡程度和增殖情况。通过蛋白质印迹法评估蛋白激酶B（AKT）、P70 S6激酶和P44/42丝裂原活化蛋白激酶（MAPK）的激活情况，并使用光密度测定法定量。结果以平均值±平均标准误差表示，并通过方差分析进行分析。
随着过冷和复温时间的增加，SMC和EC凋亡显著增加，SMC的凋亡率更高。SMC凋亡在冷却60秒时达到最大值，随后复温24小时（17.05%±0.44%），而EC最大凋亡发生在冷却120秒后，随后复温24小时（4.21%±0.22%，P<.05）。在EC中观察到更高的AKT激活，在冷却120秒并复温24小时时达到最大值3.34倍（P<.05）；在SMC中仅发现适度激活。随着冷却和复温时间的延长，EC的增殖减少，尽管SMC维持其低增殖率，但与未冷却和未复温相比，在冷却60秒和120秒且未复温时，EC的总体增殖率仍更高，具有统计学意义（P<.05）。SMC中p70S6激酶和p44/42 MAPK活性均降低，在冷却60秒，随后复温12小时时显著下降（P<.05）。然而，在冷却60秒并复温12小时时，EC的P70 S6激酶活性显著升高1.62倍，在冷却120秒并复温24小时时，P44/42 MAPK活性升高1.74倍（P<.05）。
与EC相比，SMC更高的凋亡率和更低的增殖率表明过冷和复温对不同血管细胞类型有不同影响。这些信息可能有助于理解动脉粥样硬化病变冷冻球囊血管成形术导致再狭窄较少的机制。
