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Uppal, Manjosh

University of British Columbia

Age-related macular degeneration (AMD) is a multifactorial disease which is characterized by drusen deposits, retinal pigmented epithelium (RPE) atrophy and, in a subset of cases, the formation of choroidal neovascularization lesions. Granzyme B (GzmB) is a serine protease traditionally found within natural killer (NK) cells and promotes apoptosis in target cells. Recently, GzmB has been discovered extracellularly and is implicated in chronic inflammatory disorders such as multiple sclerosis (MS), rheumatoid arthritis (RA) and impaired wound healing. GzmB is present within the outer retina and choroid and is upregulated in human donor eyes with wet AMD. This study hypothesizes that mast cells contain GzmB and release it upon degranulation, contributing to a pro-angiogenic response. In order to test this hypothesis, mast cells were first characterized in the mouse choroid tissue using toluidine blue. Peripheral areas of the mouse choroid/sclera were found to contain more mast cells than the central area. Mast cells were then further explored using immunofluorescence (IF) and found to contain and release GzmB when activated. Choroidal sprouting assays (CSA) using ex-vivo RPE/choroidal/scleral tissue from mice exposed to 48/80, a mast cell activator, confirmed that mast cell degranulation increased angiogenesis. Increases in TGF- β and VEGF-A were observed in the supernatants, illustrating the pro-inflammatory and pro-angiogenic effect of mast cell activation. Application of ketotifen fumarate (KF), a mast cell stabilizer, resulted in dramatically reduced sprouting in the CSA, providing further evidence for the pro-angiogenic impact of mast cell degranulation. In order to investigate which mast cell component(s) contribute to the angiogenic response, a GzmB inhibitor, VTI-1002, was applied and resulted in decreased sprouting in the CSA. Using GzmB-KO animals compared to controls in the CSA, it was found that the global knock-out for GzmB increased choroidal sprouting. These findings support the hypothesis that extracellular GzmB plays a pathogenic role in wet AMD. This study is the first to explore the pathogenic implication of extracellular GzmB in choroidal neovascularization.

Text; Still Image

2024-04-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/84390

Experimental Medicine

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/