Joanne Aiko Matsubara

Purinergic signaling via ATP-activated P2X receptors has been studied in age-related macular degeneration (AMD), where retinal pigment epithelium (RPE) cells release ATP and contribute to inflammation and apoptosis. This study focuses on the less-explored P2X4 receptor alongside the well-studied P2X7 receptor in cultured RPE cells, human post-mortem eyes, and mouse eyes to investigate their potential roles in AMD. Immunocytochemistry indicated expression of P2X4 and P2X7 receptors in cultured ARPE-19 cells (cell surface and intracellular), particularly when permeabilized with Triton X-100. Immunohistochemistry on human post-mortem eyes showed that the P2X4 receptor subtype was the most immunoreactive in the RPE of eyes with soft drusen among the five donor groups. In contrast, the P2X7 receptor subtype was the least immunoreactive in the RPE of eyes with soft drusen among the five donor groups. In support of this, RT-qPCR revealed the presence of P2X4 and P2X7 mRNA in ARPE-19 cells, with P2X4 mRNA showing greater upregulation than P2X7 mRNA. Double immunolabeling using Fab fragments in human post-mortem eyes demonstrated co-localization of P2X4 and P2X7 receptors, particularly within and around choroidal cell nuclei. Spectral unmixing for RPE autofluorescence demonstrated co-localization of P2X4 and P2X7 receptors in the RPE of human post-mortem eyes, particularly within and around RPE cell nuclei. Immunohistochemistry on mouse eyes showed P2X4 and P2X7 immunolabeling in the RPE, choroid, and neuroretina. These findings suggest that P2X4 and P2X7 receptors are co-localized in human and mouse retina, but their expression profiles vary with age, donor group/mouse strain, and retinal degeneration. P2X4 receptors potentially play a role in RPE cells, warranting further investigation into their function and interaction with P2X7 receptors in AMD.

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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.

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Glaucoma is a neurodegenerative eye disease characterized by elevated intraocular pressure (IOP) in the majority of cases, followed by retinal ganglion cell (RGC) loss. Neuroinflammation is an important factor contributing to RGC death during glaucoma pathogenesis. Evidence suggests that NLRP3 (nucleotide-binding oligomerization domain (NOD)-like receptor protein 3) inflammasome activity is associated with IOP-induced RGC death. To investigate whether NLRP3 inflammasome is activated by IOP elevation, the DBA/2J mouse model of glaucoma with age-related IOP elevation was studied. Female mice were sacrificed at 3 or 9 months of age. IOP was measured before sacrifice. NLRP3 inflammasome related proteins and RGC loss were quantified in eye tissues. To further assess whether NLRP3 inflammasome activation contributes to RGC death, mice were treated with 8 weeks of daily oral administration of 30 mg/kg MCC950, a specific small-molecule drug that prevents NLRP3 inflammasome activation.In the 9-month DBA/2J mice, IOP was significantly increased compared to 3-month mice. Concomitantly, protein levels of NLRP3 and cleaved caspase-1 were significantly higher in 9- month DBA/2J. The plasma level of circulating MCC950 (7650±775.3 ng/ml) after 8 weeks of drug administration was above the efficacy threshold (>1000 ng/ml). The MCC950 treated mice retained a higher number of surviving RGCs, exhibited lower levels of NLRP3 expression and caspase-1 cleavage compared to age-matched controls. In this study, we discovered MCC950 treatment inhibits the age-related increase of NLRP3 inflammasome activity and ameliorates RGC loss, suggesting that NLRP3 inflammasome activation is associated with RGC death in this mouse model.

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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.

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