The role of LRG1 in the regulation of angiogenesis in Experimental Autoimmune Uveoretinitis.

Abstract

Purpose: Leucine-rich alpha-2 glycopro­­tein 1 (LRG1) stimulates angiogenesis via modulation of the TβRII receptor complex. LRG1 binds to TβRII in association with Endoglin, inducing TGFβ mediated phosphorylation of ALK1 and subsequent Smad1/5/8 transcription factor activation, causing a pro-angiogenic signalling switch. LRG1 depletion reduces angiogenesis in models of acute ocular pathology. However, in Experimental Autoimmune Uveoretinitis (EAU), where altered immune regulation and ocular inflammation results in early blood retinal barrier (BRB) breakdown and late retinal angiogenesis, the role of LRG1 is unknown. We hypothesised that LRG1 regulates BRB function and angiogenesis in acute and chronic disease respectively.

Methods: Female C57BL/6 Lrg1 knockout (KO) and wild type (WT) mice aged 6-8 weeks were immunized to induce EAU using RBP-3 1-20 peptide. Fluorescein angiography (FA) and topical endoscopic fundus imaging (TEFI) were used to track disease and vascular permeability. Disease matched mice were sacrificed on day 18 (n=7), 25 (n=12) and 80 (n=7-9) post immunisation (p.i.). Infiltrating leukocytes were quantified by flow cytometry and day 80 retinas were flat mounted and stained for collagen IV and CD11b, for quantification of retinal neovascular membrane (RNVM) by confocal microscopy.

Results: At day 15 and 17 p.i. FA revealed reduced fluorescein leakage in KO mice, despite equal numbers of infiltrating retinal leukocytes as controls. At peak EAU (day 25 p.i.), KO mice had significantly fewer retinal-infiltrating leukocytes than WT controls (p<0.01), and by day 80 p.i, numbers in both genotypes were equivalent. Whilst the number of RNVM remained unchanged in KO mice, there was a significant reduction in RNVM volume (p <0.05).

Conclusions: In EAU, LRG1 depletion results in reduced early vascular leakage and reduced inflammatory infiltrate at peak disease. The significant reduction of RNVM volume in KO mice infers an altered angiogenic phenotype in chronic EAU.<br /> <br /> References: Wang et al. 2013. LRG1 promotes angiogenesis by modulating endothelial TGF-β signalling. Nature 499(7458), pp. 306-311