Over–Expression of FASL in RPE Layer Reduces CNV Lesions

Abstract

Abstract: : Purpose: Choroidal neovascularization (CNV) is a sight threatening complication of age–related macular degeneration. We aimed to rule out the role of Fas–FasL in a mice model of laser–induced choroidal neovacsularization and to define whether FasL over–expression in RPE can inhibit choroidal vessel growth through a Fas–FasL–mediated apoptosis in this model. Methods: A construct consisting of a tissue–specific murine retinal pigment epithelium promoter (RPE65 promoter) coupled to murine FasL cDNA with was introduced into the genome of C57BL/6J mice. RT–PCR, immunoblot and immunohistochemistry were used to examine the FasL transgene expression in RPE. Laser photocoagulation was used to induce CNV in wild type C57BL/6J and FasL over–expressing transgenic mice. CNV responses were compared by determination of fluorescein angiographic leakage at day 0 and 14 after laser injury. The development of CNV was assessed on RPE–choroidal flatmounts after FITC–dextran perfusion and CD31–labeling as well as in paraffin cross sections and the size of CNV areas was quantitated accordingly. In situ DNA end labeling was used to assess apoptosis. The expression of active form of caspase–8 was investigated by Western blot. Results: Transgenic FasL mRNA and protein were highly expressed in RPE of FasL mice before and after induction of laser photocoagulation. In contrast, FasL was only weakly expressed in RPE layer of wild type C57BL/6J mice. Two to three weeks afterlaser injury, there were significantly fewer lesions exhibited pathological fluorescein leakage in FasL transgenic mice, as well as, significantly greater number of lesions without fluorescein leakage in comparison to wild type mice. The area of leakage (mm2)decreased significantly in FasL transgenic mice in comparison to control mice. Histologically, on paraffin sections, there were ruptures of Bruch's membrane and CNV formation in transgenic as well as control eyes, 2 to 3 weeks after laser photocoagulation. However, the severity and sizes of CNV lesions were reduced in mice over–expressing FasL. The expression of other antiangiogenic factors such as PEDF was not changed in FasL transgenic mice. Conclusions: Over–expression of FasL in RPE layer reduces CNV after laser photocoagulation. Our results suggest the importance of FasL–Fas pathway in controlling pathological choroidal neovascularization.

Keywords: age-related macular degeneration • choroid: neovascularization • apoptosis/cell death