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Role of extracellular granzyme B (GzmB) in the disruption of the outer blood-retina barrier (oBRB) and remodeling of Bruch’s membrane (BM)

University of British Columbia

Age-related macular degeneration (AMD) is a poorly understood chronic inflammatory eye disease, which is characterized by the atrophy of retinal pigment epithelium (RPE) and the breakdown of Bruch’s membrane (BM), often leading to the formation of choroidal neovascularization (CNV), a hallmark of the exudative form of AMD. Currently, the effective treatments are anti-vascular endothelial growth factor (VEGF) therapies targeting CNV growth. Few studies have been conducted to explore the functions of Granzyme B (GzmB) in AMD. GzmB is a serine protease, stored in the granules of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), and also expressed by different immune and non-immune cells. It was initially believed to be involved in immune-targeted cell death, and recently many studies have revealed an additional role and an extracellular function in several chronic inflammatory disorders, such as asthma, multiple sclerosis (MS), and rheumatoid arthritis (RA). Whether extracellular GzmB activity is involved in the pathogenesis of AMD, a chronic inflammatory disease of the eye, is still not known. This present study hypothesized that extracellular GzmB may affect the function of the outer blood-retina barrier (oBRB) and BM remodeling by cleaving potential substrates in the outer retina. In order to test this hypothesis, this study used immunohistochemistry (IHC) to identify increased numbers of GzmB+ choroidal cells in aged and CNV human ocular tissues, most of which were confirmed to be choroidal mast cells. RPE cells were also found to be another source of GzmB in the outer retina. This study then used cell culture methods and immunocytochemistry (ICC) and western blot to show that exogenous GzmB cleaves RPE cell-cell adhesion proteins (ZO-1, JAM-A, occludin) and RPE-derived extracellular matrix (ECM) substrates (fibronectin, laminin), leading to damaged RPE barrier function and the degradation of ECM components in BM in vitro. These results support the hypothesis that extracellular GzmB may play a role in disrupting the oBRB function and BM remodeling. This study is the first to explore such pathophysiological implications of extracellular GzmB activity in AMD.

Text

2020-04-14

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/74001

Neuroscience

University of British Columbia

UBCV

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