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Abstract

Pterygium is a chronic ocular surface disease characterized by fibrovascular growth of the conjunctiva, with increasing evidence indicating that elevated immune cell populations and protease activity contribute to its pathogenesis. Granzyme B (GzmB), a serine protease classically known for its intracellular cytotoxic roles, has received growing attention for its extracellular involvement in inducing inflammation, extracellular matrix (ECM) remodeling, neovascularization, and fibrosis in various inflammatory conditions. Previous research has demonstrated elevated GzmB expression in pterygium tissues and identified mast cells as a source. Based on this, I hypothesize that immune cell-derived GzmB is secreted extracellularly in the conjunctiva and contributes to the molecular mechanisms underlying pterygium development. This thesis further characterizes GzmB⁺ mast cell alterations in pterygium, identifies an additional cellular source of GzmB in the conjunctiva, and investigates the functional effects of extracellular GzmB on human conjunctival epithelial cells (HCEC) in vitro. Age matched human postmortem conjunctival and pterygium tissues were analyzed using immunofluorescence staining and confocal microscopy to quantify mast cells and macrophages and to assess GzmB expression and release. HCEC were cultured and treated with increasing concentrations of exogenous GzmB to evaluate effects on cell viability, migration, and morphology. Pterygium tissues exhibited a significant increase in mast cell density and degranulation compared to healthy conjunctiva. Mast cells accounted for the majority of GzmB⁺ cells and released GzmB extracellularly through degranulation. Co-localization of CD68 and GzmB identified macrophages as an additional potential source of GzmB in the conjunctiva. In vitro, prolonged exposure to GzmB reduced HCEC viability at high doses, altered migratory capacity in a dose-dependent manner, and induced morphological changes consistent with epithelial mesenchymal transition (EMT) in cell culture studies. These findings highlight a potential pathogenic role for GzmB in the conjunctiva, impairing epithelial cell survival, altering migratory behaviour, and inducing morphological changes. Increased GzmB⁺ mast cell density and degranulation in pterygium drive extracellular GzmB accumulation, supporting GzmB inhibition and mast cell stabilization as potential therapeutic strategies to halt pterygium development or recurrence.