Abstract 639: Deletion of Alternatively Spliced Extra Domain A of Cellular Fibronectin Stabilizes Advanced Rupture-Prone Plaques in Hyperlipidemic Mice

Abstract

Background: The most important clinical manifestation of atherosclerosis is rupture of advanced plaques. The fibronectin containing alternatively spliced extra domain A (Fn-EDA) is abundant in extracellular matrix around macrophages and endothelial cells in advanced human plaques. In vitro studies suggest that Fn-EDA is a ligand for TLR4 and upregulates MMP9, which has been shown to enhance elastin degradation and promote plaque rupture in advanced plaques of hyperlipidemic apolipoprotein E-deficient (Apoe -/- ) mice. The mechanistic role of Fn-EDA in advanced plaques remains unknown. Hypothesis: Fn-EDA/TLR4 signaling in activated macrophages contributes to plaque vulnerability by upregulating MMP9 during advanced atherosclerosis. Methods and Results: We compared atherosclerotic plaques in the brachiocephalic artery (a model artery for study of plaque vulnerability) of female Fn- EDA -/- Apoe -/- and Apoe -/- mice at 50 weeks of age (44 weeks on high-fat Western diet). Fn- EDA -/- Apoe -/- mice exhibited decreased plaque size and characteristics of stable plaques, with decreased necrosis, reduced influx of activated macrophages, increased SMC staining and greater collagen content compared to Apoe -/- mice (P<0.05, n=11-13 mice/group), although cholesterol and triglyceride levels, and circulating leukocyte counts were similar. Purified cellular FN, which contains EDA, potentiated the NFκB-mediated inflammatory pathway (increased phospho-NFκB p65/ NFκB p65, TNFα and IL1β) and MMP9 protein expression (gelatin zymography) in a dose-dependent manner in purified macrophages from Fn-EDA -/- Apoe -/- mice but not from Fn-EDA -/- TLR4 -/- Apoe -/- mice. Using immunohistochemistry, we demonstrated colocalization of macrophage TLR4 and Fn-EDA within advanced atherosclerotic plaques in murine brachiocephalic arteries and human coronary arteries. Genetic deletion of TLR4 in Apoe -/- mice stabilized advanced plaques, and parameters of plaque stability were comparable between TLR4 -/- Apoe -/- and Fn-EDA -/- TLR4 -/- Apoe -/- mice. Conclusions: These findings suggest that Fn-EDA/TLR4 signaling in macrophages is a key mechanism that upregulates MMP9, and thereby promotes plaque vulnerability in advanced atherosclerosis.