Oral Presentations - Pathology and Laboratory Medicine - University ...

Graduate StudentAbstract # 18Wendy A. Boivin 1,2 , Marlo Shackleford 2 , Hongyan Zhao 2 , and David J. Granville 1,21Department of Pathology and Laboratory Medicine, Faculty of Medicine, Univeristy ofBritish Columbia. 2 Providence Heart + Lung Institute, St. Paul’s HospitalWendy Boivingranzyme b cleaves proteoglycans and releasessequestered tgf-β from extracellular matrix:implications in atherosclerosis and plaque instabilityBackround/ObjectivesGranzyme B (GrB) is a cytotoxic serine protease that contributes to chronic inflammation and disease throughimmune cell-mediated apoptosis and through the degradation of extracellular matrix (ECM). GrB resides in humanatherosclerotic plaques and preliminary data in the apolipoproteinE-knockout model of atherosclerosis suggests thatGrB localizes to the rupture-prone shoulder regions of advanced atherosclerotic plaques. We hypothesize that GrBdegrades proteoglycans located in the cap region of atherosclerotic plaques and promotes plaque instability throughthe release of cytokines and promotion of inflammation.MethodsFor ECM cleavage assays, GrB, with and without the inhibitor 3,4-dichloroisocoumarin (DCI), was incubatedfor 4 and 24h, at room temperature, with decorin, biglycan or betaglycan and visualized by ponceau staining.Cleavage fragments were subjected to Edman degradation for cleavage site identification. As transforming growthfactor-beta (TGF-beta) is sequestered by the aforementioned proteoglycans, GrB was incubated with TGF-betabound proteoglycans to determine if GrB cleavage results in the release of sequestered TGF-beta. Cytokine releasewas assessed in supernatants using Western blotting. To determine if the TGF-beta released by GrB was active,supernatants from the above release assay were incubated on human coronary artery smooth muscle cells (HCASMC)and Erk activation was examined by Western blotting.ResultsGrB cleaved biglycan, betaglycan and decorin, at both time points and cleavage was evident at GrB concentrations aslow as 50 nM. This proteolysis was GrB-dependent, as cleavage was inhibited by the GrB inhibitor DCI and not bythe inhibitor solvent control DMSO. Edman sequencing analysis determined GrB cleavage sites in decorin, biglycanand betaglycan with P1 residues of either aspartic acid or glutamic acid, consistent with the P1 specificity of theenzyme. In cytokine release assays, TGF-beta was released from decorin, biglycan, and betaglycan in a GrB-dependentmanner after 24h incubation. TGF-beta was not released in the absence of GrB or when GrB was inhibited by DCI,thus there was no non-specific dissociation of TGF-beta from decorin, biglycan or betaglycan. In addition, the TGFbetareleased by GrB remained active and induced Erk phosphorylation in HCASMC after 24h of incubation.ConclusionGrB cleaves decorin, biglycan, and betaglycan resulting in the release of sequestered active TGF-beta. This may haveimplications in vivo, where cleavage of these structural proteins and a subsequent GrB-dependent increase of TGFbetabioavailability may promote plaque instability and atherosclerosis progression.28 2 0 1 0 * O r a l P r e s e n t a t i o n s