Our results clearly indicate that persistent reperfusion occurred in photocoagulated blood vessels in this animal model treated by laser only ( and ). However, topical RPM inhibits reperfusion of such vessels in the DWC model ( and ). Reperfusion can be caused by mechanical and biological mechanisms such as flow restoration in incompletely photocoagulated blood vessels, angiogenesis and neovasculogenesis [26
]. Because vessel coagulation was essentially complete in this study, it is hypothesized that angiogenesis/neovasculogenesis is responsible for vessel reperfusion. Shutdown of major branches in the window induced a severely hypoxic microenvironment which can cause overexpression of hypoxia-inducible factor-1 alpha (HIF-1α) which in turn promoted the secretion of angiogenesis-stimulating factors [34
] such as platelet-derived growth factor [35
] and VEGF [36
]. Therefore, inhibition of the mTOR–HIF-1α–VEGF pathway by RPM is expected to play a major role in preventing vascular reperfusion after laser irradiation. Moreover, the antiangiogenic effect of RPM is also due to a direct antiproliferative response on VEGF-stimulated endothelial cells through inhibition of the PI3K–p70S6 kinase pathway [14
]. The exact underlying mechanism deserves further study to optimize a combined laser and RPM approach to improve PWS therapeutic outcome.
The nearly 100% reperfusion rate () in the laser only group suggests a lower therapeutic efficacy than that is achieved by laser treatment of PWS. The discrepancy might be related to the length of the irradiated blood vessel segment (2 mm in this study as compared to 7–10 mm for PDL). Although further study is needed to determine if a lower reperfusion rate can be achieved when the segment irradiated is longer, it is reasonable to assume that reperfusion would occur and topical RPM would still be required.
The DWC model is one of the few in vivo animal models which permits serial imaging and application of topical agents. Although hamster skin is thinner and contains some elements (e.g., subdermal muscle) not seen in human skin, the ultrastructure of the post-capillary venules within rodent skin is comparable to those in humans because the alteration of PWS vessels is believed to be caused by the progressive ectasia due to a congenital absence of perivascular nerve tissue [38
]. The major difference between the hamster skin and non-scalp PWS human skin is that there are numerous hair follicles in hamster skin. Fur regrowth after depilation may alter the window’s microenvironment because VEGF expression in hair dermal papilla cells (specialized mesenchymal cells in the hair follicle) was previously reported [39
]. We noted that fur in the window grew much slower as compared to other shaved and depilated skin when topical RPM was applied. Also of concern with the DWC model is that the direct contact between subdermis and glass window can induce an inflammatory response which may affect the window’s microenvironment. This concern can be alleviated by coating the glass window with a thin layer of biocompatible material.
The advantage of topical application is that RPM could be delivered to the dermis while avoiding significant systemic drug absorption and associated side effects [41
]. Two different topical RPM formulae were tested in this study. In both formulae, a skin penetration enhancer (PET™) was used. The results indicate that RPM can be effectively delivered through the stratum corneum using this enhancer. In the development of the first formula, emphasis was give to penetration enhancement. Thus, ingredients such as castor oil were used and irritation of laser-irradiated skin resulted. Much milder ingredients were carefully selected for the second formula, and thus, no skin irritation was observed while the delivery of RPM was maintained. Therefore, the second RPM formula appears promising for use in clinical trials on PWS patients.
The period of 14 days to apply RPM onto the DWC was determined by the fact that laser-only blood vessels fully reperfused within such a time frame. How long after laser irradiation should RPM be applied to PWS skin to obtain an optimal antiangiogenic effect? Future work should focus on assessing the flow dynamics of the PWS vasculature following laser irradiation as well as after the combination therapy. Shortly after laser irradiation, perfusion in PWS skin is reduced significantly [42
], which may cause local hypoxia and stimulate secretion of angiogenesis-stimulating factors. Perfusion in PWS skin could be monitored with LSI for an extended period of time. The suggested point to stop RPM application might be the moment when perfusion reaches its plateau.