Abstract 5488: Granzyme B Contributes to Extracellular Matrix Degradation and Advanced Atherosclerotic Plaque Formation
Introduction: Granzyme B (GrB) is a cytotoxic serine protease that acts in chronic inflammation and disease through immune cell-mediated apoptosis and putatively through the degradation of extracellular matrix (ECM). Elevated tissue and plasma levels of GrB are associated with advanced, unstable atherosclerotic plaques.
Hypothesis: GrB promotes atherosclerosis and the loss of arterial and skin integrity through extracellular matrix degradation in apolipoprotein E-knockout (ApoE-KO) mice.
Methods: ApoE-KO, GrB-KO, and GrB/apoE-double KO (DKO) mice were fed a high fat diet for 30 weeks and assessed for atherosclerotic lesion morphology, apoptosis, immune cell infiltration and ECM composition. Skin sections were examined by histology and electron microscopy for evidence of ECM degradation, aging and pathogenesis. As we have found that GrB localizes to elastic fibres, elastin binding/cleavage assays as well as ECM screening assays were performed.
Results: ApoE-KO mice on high fat diet developed advanced atherosclerosis, medial thinning and age-associated skin pathologies. GrB deficiency in ApoE/GrB-DKO mice significantly reduced the advanced atherosclerosis, medial thinning of vessels, loss of skin elasticity, and arterial and skin aging that are characteristic of apoE-KO mice. Although ApoE-KO mice were euthanized at 30 weeks for humane purposes, preliminary studies indicate that ApoE/GrB-DKO mice are normal and healthy at 60 weeks, suggesting that GrB promotes aging. GrB deficiency did not affect blood lipid and cholesterol levels in the ApoE-KO mice. In blood vessels, GrB strongly co-localized to the elastic lamellae suggesting it could have ECM cleavage potential. Furthermore, GrB activity assays suggested that GrB exhibited weak elastolytic activity but also efficiently cleaved microfibril proteins such as fibrillin-1, fibrillin-2, fibulin-2 and fibronectin, which have important structural and functional properties in normal vessels and skin. Immunostaining for these proteins supported these findings and suggested that ECM degradation by GrB is involved in these age related pathologies.
Conclusion: GrB contributes to the age-related loss of skin and arterial elasticity through the proteolytic cleavage of ECM.