Leucine-rich Alpha-2 Glycoprotein 1 (Lrg1) Contributes To The Development Of Ocular Neovascularisation

Abstract

Purpose: : Aberrant angiogenesis is the hallmark of many blinding eye diseases including retinopathy of prematurity, proliferative diabetic retinopathy, age-related wet macular degeneration (AMD) and macular telangiectasia. The formation of new vessels is a complex and tightly regulated process involving multiple intercellular pro- and anti-angiogenic molecules. We have previously demonstrated that leucine rich α-2-glycoprotein-1 (Lrg1) is involved in the fine-tuning of angiogenesis and drives this delicate process in a pro-angiogenic direction. The aim of this study was to gain new insight into the molecular mechanisms of Lrg1 signalling and its interaction with other signalling pathways involved in angiogenesis, as well as to evaluate its potential as a therapeutic target.

Methods: : Co-immunoprecipitation and GST-pulldown assay were used to identify Lrg1 binding proteins. The interaction between Lrg1 and the TGFβ signalling pathway in angiogenesis was studied in vitro (proliferation assay and organotypic angiogenesis assay), ex vivo (metatarsal assay and aortic ring assay) and in vivo. The therapeutic potential of a Lrg1 neutralizing antibody was studied in vitro (Matrigel assay) and in vivo (mouse model of wet AMD).

Results: : We observed that Lrg1 interacts with TGFβ1 and its receptors in vitro. Recombinant human Lrg1 (rhLrg1) promotes TGFβ1 stimulated Smad1/5 phosphorylation and subsequent endothelial cell (EC) proliferation. rhLrg1 and TGFβ1 promote vessel outgrowth from metatarsal and aortic ring explants, which could be attenuated by polyclonal antibody against full length human Lrg1 protein. Lrg1 neutralizing antibody completely blocks HUVEC tube formation in Matrigel and inhibits laser induced choroidal neovascularization.

Conclusions: : This study demonstrates that Lrg1 plays an important role in angiogenesis through regulating the TGFβ signalling pathway and is a potentially promising therapeutic target.

Keywords: choroid: neovascularization • neovascularization • retinal neovascularization