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Extracellular granzyme B and pathophysiological implications Boivin, Wendy Anne

Abstract

Granzyme B (GzmB) is a serine protease that contributes to immune-mediated elimination of cells by initiating a tightly-regulated form of death known as apoptosis. However, during inflammation, GzmB leaks out and accumulates in the extracellular space, retains its activity, and proficiently cleaves extracellular matrix (ECM) proteins. I therefore hypothesized that extracellular GzmB is capable of cleaving novel ECM substrates, contributing to dysregulated ECM integrity and function in disease. In the present dissertation I identified eleven novel extracellular GzmB substrates. Further investigations revealed that GzmB-mediated proteoglycan cleavage was implicated in the dysregulation of active transforming growth factorbeta (TGF-β) sequestration and bioavailability. GzmB cleavage sites were identified in biglycan and betaglycan and active TGF-β was shown to be released from decorin, biglycan and betaglycan. The pathophysiological role of my findings were further investigated and validated using animal models of disesase in which inflammation and elevated GzmB are observed. Evidence of fibrillin-1 and decorin cleavage were observed in atherosclerosis, abdominal aortic aneurysm and in skin aging pathogenesis. I also assessed the activity of GzmB in advanced atherosclerosis using perforin/apolipoprotein E- double knockout (Perf/apoE-DKO) and granzyme B/apolipoprotein E-double knockout (GzmB/apoE-DKO) mice. Interestingly, unlike our aneurysm findings whereby only GzmB/apoE-DKO mice were protected, both Perf/apoEDKO and GzmB/apoE-DKO mice were protected from atherosclerosis compared to apoE-KO controls, suggesting the intracellular Perf-dependent activities of granzymes are also important in the pathogenesis of atherosclerosis. In summary, GzmB is a protease that functions both intracellularly and extracellularly in disease. My findings suggest that the use of Perf knockout mice alone to study the role of GzmB in disease should be re-evaluated given the increasing evidence in both animal models and in human disease showing elevated GzmB in bodily fluids is associated with inflammation and age.

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