Abstract 5309: Perforin-Independent Granzyme B-Mediated Extracellular Matrix Protein Cleavage Contributes to Abdominal Aortic Aneurysm
Background: Granzyme B (GrB) is a pro-apoptotic serine protease present in atherosclerotic human vessels, where its expression corresponds to increased disease severity and plaque instability. In addition to its perforin-dependent cytotoxic role, GrB accumulates extracellularly and contributes to the degradation of extracellular matrix (ECM) proteins.
Hypothesis: GrB contributes to abdominal aortic aneurysm (AAA) through the cleavage of medial and/or adventitial ECM proteins.
Methods: Human AAA and thoracic aortic aneurysm (TAA) samples were assessed for the presence of granzymes, immune cell markers, and ECM. Apolipoprotein E (apoE)−/− × GrB−/− double knockout (GDKO) and apoE−/− × perforin−/− DKO (PDKO) mice were generated to assess the functional intracellular and extracellular roles of GrB, respectively, in AAA. To induce AAA, mice were implanted with osmotic mini pumps containing angiotensin II (AII, 1000 ng/kg/min) or saline for 28 days. To further assess extracellular GrB activity, a novel serpin inhibitor of GrB was injected 3h prior to pump implantation. Survival, degree of AAA, ECM composition and GrB localization were evaluated. In vitro protease assays were performed to identify novel extracellular GrB substrates that may contribute to AAA.
Results: While GrB was not present in healthy human aorta, GrB was abundantly expressed in the vessel wall and thrombus of aneurysmal tissue. AII-treated GDKO mice but not PDKO mice had increased 28-day survival (92.86%; n=14 vs. 56.25%, n=16) when compared to apoE-KO mice (53.33%; n=15). While AAA rupture was observed frequently in apoE-KO (46.7%; n=15) and PDKO (43.3%; n=16) mice, rupture was rarely observed in GDKO (7.1%; n=14) mice. Reduced degradation of medial fibrillin-1 and adventitial decorin and collagen was observed in the blood vessels of GDKO and serpin-treated mice versus apoE-KO mice. GrB localized to macrophages, mast cells and areas of medial disruption and adventitial weakening. Protease assays demonstrated GrB-cleavage of fibrillin-1 and decorin along with other ECM components.
Conclusions: GrB contributes to the loss of vessel wall integrity and AAA through the cleavage of medial and adventitial ECM. Inhibition of GrB attenuates medial disruption and aortic rupture in mice.