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Joanne A Matsubara, Yuan Tian, Jing Z Cui, Sijia Cao, Sho Hiroyasu, Chris Turner, Yue Shen, David J. Granville; Potential Role of Extracellular Granzyme B in the Pathogenesis of Age-related Macular Degeneration (AMD). Invest. Ophthalmol. Vis. Sci. 2019;60(9):1234.
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Granzyme B (GzmB) is a serine protease that has been shown to accumulate in the extracellular milieu in conditions associated with age and chronic inflammation. In the extracellular space 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. However, little is known of its action in eye disease. This current study assessed ocular GzmB and its potential role in reorganizing/degrading Bruch’s Membrane (BM) during the development and progression of AMD.
Postmortem human eyes and rodent models of AMD were studied. Young (<55), old (>65 yrs), and diseased (numerous soft drusen, geographic atrophy GA, or choroidal neovascularization CNV) donor eyes were processed for GzmB immunohistochemistry (IHC). GzmB labeling in RPE and choroid was quantified per choroidal area (mm2), or per mm length BM. Double labeling for CD 68 (macrophages) or toluidine blue (mast cells) was quantified. Eyes from mouse models of AMD were processed for GzmB IHC. The intensity of GzmB positive (+) staining was quantified by Image J software and statistical tests by SPSS software.
There was a significant increase in GzmB+ cells with aging in human choroidal tissues (older: 61.13 ± 3.83 vs younger: 22.77 ± 4.93, p<0.05). There was a significant increase in GzmB+ choroidal cells in donor eyes with CNV compared to those with numerous soft drusen or GA (4.67 ± 0.56 vs 2.93 ± 0.50 or 1.76 ± 0.27, p<0.05). GzmB and CD68 double-labeled cells were found in choroid of normal and CNV eyes. Numerous double-labeled mast cells were found in the human choroid. RPE cells in human eyes also express GzmB, but levels were moderate compared to choroidal cells. In mouse RPE, GzmB IHC in 18 month old mice was significantly higher than those in 3 month old mice (2.38 ± 0.31 vs 1.88 ± 0.13, p<0.05 chromogenic analysis and 19.65 ± 1.57 vs 12.64 ± 0.69, p<0.05 immunofluorescence analysis).
Aging is associated with increased GzmB in human and mouse eyes. Macrophages and mast cells in human choroid express GzmB. GzmB choroidal IHC is significantly increased in CNV, but not GA or age matched control eyes. RPE cells also express GzmB in their basal compartment. GzmB cleavage of ECM proteins (fibronectin, laminin, etc) in BM may contribute to the development of AMD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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