March K L, Patton B L, Wilensky R L, Hathaway D R
Krannert Institute of Cardiology, Indianapolis, Ind. 46202-4800.
Circulation. 1993 Jan;87(1):184-91. doi: 10.1161/01.cir.87.1.184.
Smooth muscle cell proliferation plays a major role in the genesis of restenosis after angioplasty or vascular injury. Although the effects of arterial exposure to high-energy radiation sources such as laser have been investigated in detail, the effects on vascular cells of low-intensity radiant energy in combination with photoactive agents have not been extensively characterized. Psoralens are photoactive agents that are known to be well tolerated when used in conjunction with local exposure to ultraviolet light in the A band (UVA) for the treatment of various dermatologic proliferative disorders.
We have investigated the effects of psoralen/UVA (PUVA) exposure on the proliferation of bovine aortic smooth muscle cells. Proliferation and viability were assessed over a 14-day period by trypan blue exclusion counts. Cell cycle effects were evaluated by thymidine incorporation and flow cytometry with DNA quantitation after addition of serum or platelet-derived growth factor B-chain (PDGF-BB) to subconfluent cells synchronized by serum withdrawal. No effect was observed after exposure to 8-methoxypsoralen (8-MOP) at concentrations up to 10 microM or UVA irradiation at energies up to 2.5 J/cm2. Longwave ultraviolet light and 8-MOP were found to behave synergistically as potent inhibitors of DNA synthesis in bovine aortic smooth muscle cells with the EC50 in combination ranging from 7 microM at 0.35 J/cm2 to 0.2 microM at 2.1 J/cm2. Similar antiproliferative effects were obtained by an inverse variation of dose and energy delivered. After serum stimulation, inhibition of DNA synthesis was found with either an immediate or delayed (16-hour) application of PUVA. This effect was independent of subsequent 8-MOP washout. Flow cytometry of cells treated with PUVA at several times after serum stimulation demonstrated for each time point a block in further cell cycle progression for cells in all phases of the cell cycle. Evaluation of [125I]-labeled PDGF and epidermal growth factor (EGF) binding revealed no effect of PUVA on the apparent number or affinity of PDGF binding sites present but did reveal a dose-dependent inhibition by PUVA of EGF binding. This inhibition of EGF binding occurred increasingly at higher PUVA doses than the cell cycle inhibition and accordingly did not appear to represent a critical mechanism for the antiproliferative effect. Cell counting after a single exposure to PUVA (1 microM, 1.5 J/cm2) revealed complete stasis of cell proliferation over a 28-day period without recurrent exposure. No increase in trypan-positive cells was noted over this period.
PUVA treatment represents a novel method for locally inhibiting proliferation of vascular smooth muscle cells without producing cytolysis.
平滑肌细胞增殖在血管成形术或血管损伤后再狭窄的发生过程中起主要作用。尽管已经详细研究了动脉暴露于高能辐射源（如激光）的影响，但低强度辐射能与光活性剂联合对血管细胞的影响尚未得到广泛描述。补骨脂素是一类光活性剂，已知其与局部暴露于A波段紫外线（UVA）联合用于治疗各种皮肤增殖性疾病时耐受性良好。
我们研究了补骨脂素/UVA（PUVA）照射对牛主动脉平滑肌细胞增殖的影响。通过台盼蓝排斥计数在14天内评估细胞增殖和活力。在对通过血清饥饿同步化的亚汇合细胞添加血清或血小板衍生生长因子B链（PDGF-BB）后，通过胸苷掺入和流式细胞术结合DNA定量来评估细胞周期效应。在浓度高达10μM的8-甲氧基补骨脂素（8-MOP）或能量高达2.5J/cm²的UVA照射后未观察到影响。发现长波紫外线和8-MOP协同作用，作为牛主动脉平滑肌细胞中DNA合成的有效抑制剂，联合使用时的半数有效浓度（EC50）范围从0.35J/cm²时的7μM到2.1J/cm²时的0.2μM。通过剂量和传递能量的反向变化获得了类似的抗增殖作用。血清刺激后，立即或延迟（16小时）应用PUVA均可抑制DNA合成。这种作用与随后的8-MOP洗脱无关。在血清刺激后不同时间用PUVA处理的细胞的流式细胞术显示，在每个时间点，细胞周期各阶段的细胞进一步的细胞周期进展均受阻。对[125I]标记的PDGF和表皮生长因子（EGF）结合的评估显示，PUVA对存在的PDGF结合位点的表观数量或亲和力没有影响，但确实显示PUVA对EGF结合有剂量依赖性抑制作用。与细胞周期抑制相比，这种对EGF结合的抑制在更高的PUVA剂量下越来越明显，因此似乎不代表抗增殖作用的关键机制。单次暴露于PUVA（1μM，1.5J/cm²）后的细胞计数显示，在28天内细胞增殖完全停滞，无需再次暴露。在此期间未观察到台盼蓝阳性细胞增加。
PUVA治疗是一种局部抑制血管平滑肌细胞增殖而不产生细胞溶解的新方法。
