Granzyme B degrades extracellular matrix and promotes choroidal neovascularization in an ex-vivo microvascular angiogenesis model

Abstract

Purpose : Granzyme B (GzmB) is a cytotoxic serine protease that advances aging/age-related diseases and chronic inflammation via pathological extracellular matrix (ECM) degradation and increased vascular permeability. Extracellular GzmB is increased in the aging outer retina and in the choroid of donor eyes with neovascular AMD (nAMD) and with choroidal neovascularization (CNV), but its role in CNV is not known. This study determines the effect of exogenous GzmB using an ex vivo model of microvascular angiogenesis.

Methods : Ex vivo choroid sprouting assay (CSA) using peripheral retinal pigment epithelium (RPE)/Bruch’s membrane (BrM)/choroid/sclera tissue explants from 3-month-old C57BL/6 mice was established and cultured for 10 days. Explants were stimulated with either exogenous GzmB (50nM) or PBS on Days 4, 6, 8 and 10 of culture. Western blot was performed to assess the expression and cleavage of ECM proteins, decorin and fibronectin in the CSA supernatant. Images of CSA explants were captured, vascular sprouting was analysed and quantified by a standardized SWIFT-Choroid macro based on ImageJ software. CSA relative vascular sprouting area (mm²) and western blot relative band intensity data were statistically evaluated.

Results : Relative vascular sprouting area was significantly (p<0.05; n ≥11 per group) increased in choroid explants stimulated with exogenous GzmB compared with controls. Western blot also reveals significantly increased cleavage of fibronectin (p=0.001; n=3 per group) and expression of decorin (p<0.05; n=4 per group) in GzmB-stimulated choroid explants compared with controls. ECM remodelling induced by GzmB cleavage of important ECM proteins could diminish outer retinal barrier function, promote vascular leakage and inflammation, and dysregulated angiogenesis in CNV.

Conclusions : GzmB cleavage of ECM proteins including decorin and fibronectin may contribute to CNV development in nAMD via an ECM remodelling/pro-angiogenesis pathway. Further studies are needed to investigate the possibility that pharmacological inhibition of extracellular GzmB could mitigate CNV in nAMD.

This is a 2021 ARVO Annual Meeting abstract.