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Adarsha Koirala, Ann Marshak-Rothstein, Bruce R Ksander, Meredith S Gregory-Ksander; Regulation of vascular leakage by Fas Ligand: Distinguishing the role of soluble from membrane FasL in unique knock-in mice with laser-induced choroidal neovascularization. Invest. Ophthalmol. Vis. Sci. 2014;55(13):80.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies reported that membrane FasL (mFasL) on retinal pigment epithelial cells inhibited laser-induced CNV via apoptosis of choroidal endothelial cells. By contrast, soluble FasL (sFasL) promoted CNV by inducing infiltration of pro-angiogenic macrophages. However, these studies used a non-specific method to inhibit MMPs, the enzyme that cleaves mFasL to produce sFasL. By contrast, our current study uses a genetic approach to specifically inhibit cleavage of FasL via the ΔCS knock-in mutation that prevents cleavage of mFasL, thus completely preventing production of sFasL. We use this unique model to study the opposing roles of sFasL and mFasL in CNV leakage.
Laser-induced CNV was performed on three groups of C57BL/6 mice with distinct FasL phenotypes: (i) WT-B6.FasL+/+ mice (mFasL and/or sFasL) (ii) homozygous B6.FasLΔCS/ΔCS mice (only mFasL), (iii) heterozygous B6.FasLΔCS/+ mice (high mFasL and low sFasL). Laser-treated mice were analyzed for: vascular leakage by fluorescein angiography, CNV lesion size by optical coherence tomography, and vascular development by confocal microscopy of FITC dextran perfused and collagen IV stained retinal whole mounts. Sprouting and maturation of choroidal vessels were analyzed in choroidal explant matrigel cultures.
Contrary to previous predictions, B6.FasLΔCS/ΔCS mice (only mFasL) displayed no significant difference from WT-B6.FasL+/+ mice in either CNV lesion size or vascular leakage on days 3,5,8, or 14. Unexpectedly, B6.FasLΔCS/+ (high mFasL and low sFasL) displayed significantly reduced CNV lesions and vascular leakage at all time points. Surprisingly, reduced vascular leakage did not coincide with reduced angiogenesis, but rather an accelerated formation of fully perfused, non-leaky vessels. Moreover, choroidal explants from B6.FasLΔCS/+ mice displayed accelerated vessel growth with a highly reticular phenotype as compared to B6.FasL+/+ and B6.FasLΔCS/ΔCS mice.
Together these data demonstrate that mFasL alone does not inhibit lesion size or vascular leakage in laser-induced CNV. Moreover, sFasL is critical in preventing vascular leakage via promoting vessel maturation. We therefore propose that there is an optimal ratio of mFasL and sFasL required to prevent pathologic CNV.
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