Granzyme B deficiency attenuates subretinal fibrosis in neovascular age-related macular degeneration

Abstract

Purpose : >50% of patients with neovascular age-related macular degeneration (nAMD) eventually develop subretinal fibrotic lesions that expand over time and exacerbate vision loss. There are no treatment options for subretinal fibrosis, as its pathophysiology remains elusive. Granzyme B (GzmB) is a serine protease that is elevated in postmortem human eyes with nAMD, and recent evidence suggests that it contributes to key features of subretinal fibrosis. We investigated the role of GzmB in subretinal fibrosis by using a two-stage laser-induced mouse model of subretinal fibrosis.

Methods : Subretinal fibrotic lesions were induced in younger (3-6 month) and older (7-13 month) C57BL/6J (wild type; WT) and GzmB deficient (GzmB KO) mice. At Day 7 post second laser, eyes were processed for immunofluorescence (IF) in flatmounts or paraffin cross-sections. Fibrosis markers, collagen-1 (Col1) and α- smooth muscle actin (α-SMA), were used in flatmounts to quantify the size of fibrotic lesions and confirm the presence of myofibroblasts, respectively. GzmB substrates, pro-fibrotic thrombospondin-1 (TSP-1) and anti-fibrotic decorin (DCN), within lesions were quantified in cross-sections to determine possible mechanisms for the anti-fibrotic effects of GzmB deficiency. The presence and degranulation of mast cells within lesions were quantified in flatmounts by using toluidine blue and tryptase immunolabeling. Confocal images were analyzed by Image J to quantify lesion size and IF intensity.

Results : GzmB KO mice displayed much smaller Col1+ lesions (p<0.05; n =17-18 lesions from 10 animals). While lesions in both WT and GzmB KO mice contained myofibroblasts, α-SMA+ lesions in GzmB KO mice had less TSP-1 immunolabeling but more DCN immunolabeling, compared to α-SMA+ lesions in WT mice. Additionally, lesions in GzmB KO mice had significantly less tryptase immunolabeling, compared to lesions in WT mice (p<0.05; n =6-7 lesions from 3-4 animals), and toluidine blue staining revealed that there were fewer mast cells within lesions in GzmB KO mice.

Conclusions : Our previous studies have shown GzmB is involved in choroidal neovascularization of nAMD. Our recent data reveal GzmB is also involved in subretinal fibrosis secondary to nAMD, and GzmB deficiency attenuates subretinal fibrosis possibly by preserving DCN and reducing TSP-1 within fibrotic lesions and delaying the accumulation of mast cells within fibrotic lesions.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.