Tan Wenbin, Jia Wangcun, Sun Victor, Mihm Martin C, Nelson J Stuart
Department of Surgery, Beckman Laser Institute and Medical Clinic, University of California, Irvine, Irvine, California 92617, USA.
Lasers Surg Med. 2012 Dec;44(10):796-804. doi: 10.1002/lsm.22101. Epub 2012 Dec 4.
Pulsed dye laser (PDL) is the most effective treatment for port wine stain (PWS) birthmarks. However, regeneration and revascularization of photocoagulated blood vessels may result in poor therapeutic outcome. We have recently shown that rapamycin (RPM), an angiogenesis inhibitor, can reduce the regeneration and revascularization of photocoagulated blood vessels. Herein, we attempt to further elucidate the molecular pathophysiology on the inhibition of the regeneration and revascularization of photocoagulated blood vessels by topical RPM in an animal model.
Two separate skin areas on each hamster were irradiated by PDL. After PDL exposure, topical RPM was applied daily to one of the randomly selected test sites. PDL, PDL + RPM and normal skin test sites were biopsied on day 3 after PDL exposure. The total ribonucleic acid (RNA) and protein were extracted from biopsied skin samples and quantified. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot were subsequently performed to quantify the mRNA and protein levels of hypoxia-inducible factor-1alpha (HIF-1α), vascular endothelial growth factor (VEGF) and ribosomal protein S6 kinase (S6). The phosphorylation levels of S6 and AKT were also evaluated by immunoblot.
The mRNA and protein levels of HIF-1α, VEGF, and S6 significantly increased after PDL exposure as compared to the normal hamster skin. Topical application of 1% RPM suppressed the PDL-induced increase in mRNA and protein levels of those genes on day 3 post-PDL exposure. The phosphorylation levels of S6 and AKT increased after PDL exposure but the increases were suppressed by the topical application of RPM.
The increase in expression of HIF-1α, VEGF, and S6 after PDL-exposure suggests that angiogenesis pathways play very active roles in the process of skin blood vessel regeneration and revascularization. Topical application of 1% RPM can suppress the angiogenesis pathways and, therefore, reduce the regeneration and revascularization of photocoagulated blood vessels.
脉冲染料激光（PDL）是治疗葡萄酒色斑（PWS）胎记最有效的方法。然而，光凝血管的再生和再血管化可能导致治疗效果不佳。我们最近发现，血管生成抑制剂雷帕霉素（RPM）可以减少光凝血管的再生和再血管化。在此，我们试图在动物模型中进一步阐明局部应用RPM抑制光凝血管再生和再血管化的分子病理生理学机制。
对每只仓鼠的两个不同皮肤区域进行PDL照射。PDL照射后，每天对随机选择的一个测试部位局部应用RPM。在PDL照射后第3天，对PDL、PDL + RPM和正常皮肤测试部位进行活检。从活检的皮肤样本中提取总核糖核酸（RNA）和蛋白质并进行定量。随后进行实时逆转录-聚合酶链反应（RT-PCR）和免疫印迹，以定量缺氧诱导因子-1α（HIF-1α）、血管内皮生长因子（VEGF）和核糖体蛋白S6激酶（S6）的mRNA和蛋白质水平。还通过免疫印迹评估S6和AKT的磷酸化水平。
与正常仓鼠皮肤相比，PDL照射后HIF-1α、VEGF和S6的mRNA和蛋白质水平显著升高。局部应用1% RPM可在PDL照射后第3天抑制PDL诱导的这些基因的mRNA和蛋白质水平升高。PDL照射后S6和AKT的磷酸化水平升高，但局部应用RPM可抑制这种升高。
PDL照射后HIF-1α、VEGF和S6表达的增加表明血管生成途径在皮肤血管再生和再血管化过程中发挥着非常活跃的作用。局部应用1% RPM可抑制血管生成途径，从而减少光凝血管的再生和再血管化。
