Abstract 522: Shotgun Proteomics Implicates Matrix Proteolysis, Cell Adhesion and Cell Death in Urokinase-Accelerated Vascular Disease

Abstract

Introduction: Elevated activity of urokinase plasminogen activator (uPA) in human arteries associates with atherosclerosis, aneurysms, and plaque rupture. However, the underlying mechanisms remain unknown. ApoE null mice with macrophage-specific uPA overexpression (SR-uPA mice) have elevated vascular uPA activity, accelerated atherosclerosis, dilated aortas, and histologic features of plaque rupture. Thus, SR-uPA mice may be a useful model of uPA-accelerated human vascular disease.

Hypothesis: We hypothesized that mechanisms of uPA-accelerated vascular disease would be revealed by shotgun proteomics of aortas of SR-uPA and nontransgenic (ntg) ApoE null mice. We focused our studies on extracellular matrix (ECM) because previous studies implicate uPA in alteration of matrix-associated proteins.

Methods: Using aortas, we developed a method for generating tissue extracts (TE) that selectively isolate ECM proteins. We extracted proteins from aortic arches of SR-uPA and ntg ApoE null mice. We also collected conditioned medium (CM) from their explanted aortas.

Results: Mass spectrometry-based proteomics demonstrated that aortic TE were greatly enriched in ECM proteins as determined by Gene Ontology (GO) analysis. These TE contained all major and several minor vascular collagens, and many proteoglycans. CM contained a similar number of ECM proteins as TE; however, TE were enriched in collagens, proteoglycans, elastin, and laminins. Using stringent dual statistical criteria and a FDR of 5%, 107 of 1125 proteins were differentially present in SR-uPA aorta. MMP-2, -3, and -10 were increased; laminins, collagen I, fibronectin, and elastin were decreased. CM analysis revealed 1161 proteins; 57 were differentially present in SR-uPA. NuMa1, nuclear lamins, histones, actin, and myosins were increased; PAI-1, Plg, and A2m were decreased. Unbiased GO analysis helped identify proteolysis, ECM, cell adhesion, and cell death as processes that differed significantly between SR-uPA and ntg aortas.

Conclusions: Elevated aortic uPA activity alters abundance of a limited number of proteins and affects specific biological processes, implicating ECM proteolysis, altered cell adhesion, and cell death in the pathogenesis of uPA-mediated vascular disease.