Cellular fibronectin containing extra domain A (EDA⁺-FN) is abundant in the arteries of patients with atherosclerosis. Several in vitro studies suggest that EDA⁺-FN interacts with Toll-like receptor 4 (TLR4). We tested the hypothesis that EDA⁺-FN exacerbates atherosclerosis through TLR4 in a clinically relevant model of atherosclerosis, the apolipoprotein E–deficient (Apoe^−/−) mouse.

The extent of atherosclerosis was evaluated in whole aortae and cross sections of the aortic sinus in male and female EDA^−/−Apoe^−/− mice (which lack EDA⁺-FN), EDA^fl/flApoe^−/− mice (which constitutively express EDA⁺-FN), and control Apoe^−/− mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA^fl/flApoe^−/− mice exhibited a 2-fold increase in atherosclerotic lesions (aorta and aortic sinus) and macrophage content within plaques, whereas EDA^−/−Apoe^−/− mice exhibited reduced atherosclerotic lesions (P<0.05 versus Apoe^−/−, n=10–12 mice/group), although cholesterol and triglyceride levels and circulating leukocytes were similar. Genetic ablation of TLR4 partially reversed atherosclerosis exacerbation in EDA^fl/flApoe^−/− mice (P<0.05) but had no effect on atherosclerotic lesions in EDA^−/−Apoe^−/− mice. Purified cellular FN, which contains EDA, potentiated dose-dependent NFκB-mediated inflammation (increased phospho-NFκB p65/NFκB p65, tumor necrosis factor-α, and interleukin-1β) in bone marrow–derived macrophages from EDA^−/−Apoe^−/− mice but not from EDA^−/−TLR4^−/−Apoe^−/− mice. Finally, using immunohistochemistry, we provide evidence for the first time that EDA⁺-FN colocalizes with macrophage TLR4 in murine aortic lesions and human coronary artery atherosclerotic plaques.

Our findings reveal that TLR4 signaling contributes to EDA⁺-FN–mediated exacerbation of atherosclerosis. We suggest that EDA⁺-FN could be a therapeutic target in atherosclerosis.