Abstract 18817: Granzyme B-Mediated Proteoglycan Cleavage Results in the Release of Matrix-Sequestered TGF-β: Relevance to Aortic Aneurysm Pathogenesis
Background: Granzyme B (GZMB) is a pro-apoptotic serine protease that has been traditionally viewed to function solely in immune-mediated apoptosis wherein perforin facilitates its entry into cells. However, during aging and inflammation, GZMB accumulates extracellularly, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. We have previously shown in mice that GZMB contributes to aortic aneurysm progression through a perforin-independent mechanism involving the cleavage of ECM.
Hypothesis: GZMB contributes to aneurysm pathogenesis by cleaving TGF-β-sequestering proteoglycans (PG) resulting in the release of active TGF-β.
Methods: Using a cell-free ECM cleavage assay, decorin, biglycan and betaglycan were identified as GZMB substrates. As TGF-β is sequestered by the aforementioned PG, GZMB was incubated with TGF-β-bound PG to determine whether GZMB cleavage could induce TGF-β release. Supernatants from TGF-β release assays were incubated with smooth muscle cells (SMC) and ERK phosphorylation was assessed. The effect of GZMB inhibition (i.v. serpina3n) on PG cleavage was also investigated in a mouse model (Angiotensin II) of aortic aneurysm.
Results: GZMB released TGF-β from all PG substrates and its activity was maintained as indicated by the induction of Erk-1 phosphorylation in SMC. Cleavage sites were identified and corroborated with anticipated cleavage sites for GZMB. Inhibiting GZMB during aneurysm induction in mice was associated with lower adventitial decorin degradation, while increased SMAD3 phosphorylation was observed in the media and adventitia. Consistent with the key role of decorin in regulating collagen spacing and organization, GZMB inhibition was associated with improved adventitial collagen spacing and organization.
Conclusion: PG cleavage by GZMB can release active TGF-B. Increased GZMB activity in vivo during aneurysm formation may therefore lead to dysregulated release of ECM-sequestered cytokines in addition to contributing to the loss of ECM and structural integrity.
- © 2010 by American Heart Association, Inc.