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Joanne A Matsubara, Miranda Tsuyuki, Jacob Ng, Erika Yuan Tian, Chao Zhu, Jing Z Cui, Sho Hiroyasu, Christopher T. Turner, Matthew R. Zeglinski, Trevor J McGill, Martha Neuringer, David J. Granville; Granzyme B Promotes Early Stages of AMD by Degrading Extracellular Matrix and Cell-Cell Contact Proteins in Bruch’s Membrane and RPE. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2285.
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Granzyme B (GzmB) is an extracellular serine protease found in tissues during aging and chronic inflammation, where it plays an important role in the abnormal degradation of the extracellular matrix (ECM). GzmB activity also reduces epithelial barrier function by cleaving cell-cell adhesion and basement membrane proteins in non-ocular systems. However, little is known of its action in eye disease. This study assesses ocular GzmB and its potential role in the reorganization of Bruch’s Membrane (BM) and breakdown of the blood-retinal barrier in genetically modified mice and non-human primates.
Knock-outs of Apolipoprotein-E (ApoE-KO), GzmB-KO and a combined ApoE+GzmB double KO (DKO) mice were kept on high fat diets for 30 weeks, euthanized and eyes processed for western blot (WB) and immunofluorescence (IF) in paraffin sections and wholemount preps. GzmB, intercellular junctional and ECM proteins were quantified in outer retina to assess BM remodeling and the degradation of the blood-retinal barrier. Image J analysis of confocal images were undertaken for IF intensity differences in eye tissue among GzmB-KO, Apo-E-KO, DKO and WT groups. A series of non-human primate tissues were also processed for GzmB IF.
GzmB immunolabeling was present in BM of WT and ApoE-KO mice and exhibited age-related increases. As GzmB is known to cleave intercellular junctional proteins, we analyzed GzmB’s presence in the intercellular space between RPE and determined its role in the structural degradation of the tight (ZO-1) and adherens (JAM-A, beta-catenin) junction proteins. WB demonstrated heightened levels of cleaved caspase-1 in ApoE-KO compared to GzmB-KO and DKO, suggesting a role for GzmB in inflammasome activation and proinflammation in outer retina. Non-human primate data demonstrate age-related increases in extracellular GzmB in BM and RPE, with spatial proximity to drusen deposits.
GzmB is a novel serine protease that is present in outer retina of the aged non-human primate and in the ApoE-KO model of accelerated aging characterized by early pathological features of AMD. In the ApoE-KO, GzmB levels are heightened and promote cleavage of ECM, remodeling of BM and the disruption of the RPE barrier function. These data suggest that inhibitors of extracellular GzmB activity may ameliorate changes in outer retina during the early stages of AMD.
This is a 2020 ARVO Annual Meeting abstract.
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