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A. J. Stempel, M. H. Davies, K. E. Hubert, M. R. Powers; Exogenous Soluble Fas Ligand Fails to Inhibit Retinal Neovascularization in a Model of Oxygen-Induced Retinopathy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):526. doi: https://doi.org/.
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Previous studies using Fas ligand (FasL) mutant mice in a model of oxygen-induced retinopathy (OIR) have suggested that Fas-FasL interactions help control the extent of retinal neovascularization (NV) (Davies et al, IOVS; 2003). The present study was performed to determine if intravitreal injection of soluble (s) FasL could enhance regression of retinal NV in the OIR model.
Postnatal day 7 (P7) C57BL/6 mice were exposed to 75% oxygen for 5 days and then recovered in room air. FACS analysis for Annexin-V was used to verify the ability of dimerized sFasL to induce apoptosis in Jerkat cells. On P16, one eye from each mouse was injected intravitreally with either 1.3µg (low dose) or 13.3µg (high dose) of dimerized sFasL in 1.5µl, while the contralateral eye was injected with the cross-linking antibody alone. Eyes were collected at P16.5 (low dose n=6, high dose n=6) and P17 (low dose n=6, high dose n=4) and NV was assessed in retinal sections by quantification of preretinal nuclei. Apoptotic cells were labeled using a standard TUNEL assay and quantified within the neovascular tufts and the neural retina. Retinal vessels were quantified in sections immunolabeled with an antibody to von Willebrand factor (vWF).
Jerkat cells stimulated with sFasL in the presence of a cross-linking antibody demonstrated a significant increase (~15-fold) in apoptosis as compared to controls. Quantification of retinal NV showed similar levels of NV at P16.5 and P17 in both sFasL injected and control injected eyes. TUNEL analysis revealed no significant decrease in the number of apoptotic cells located in the neovascular tufts of the sFasL injected eyes as compared to controls. Interestingly, at P17 the low dose injected eyes revealed a paradoxical reduction (~40%) in the number of apoptotic cells located in the inner retina eyes as compared to controls (p<0.001). TUNEL analysis of the neural retina revealed no significant differences between sFasL injections and controls. Immunostaining for vWF showed no difference in the intra-retinal vessels between sFasL injected and controls.
Intravitreal injection of sFasL was not sufficient to reduce the amount of NV at P16.5 and P17, the peak of disease in the OIR model. These data suggest that sFasL was unable to activate sufficient numbers of Fas receptors on neovascular tuft endothelial cells within the retinal microenvironment. To demonstrate a significant effect of Fas activation on vascular tuft regression, multiple injections or alternative doses of sFasL may be required.
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