Key Points Fn-EDA+ promotes arterial thrombosis. Platelet TLR4 mediates the prothrombotic effect of cellular Fn.

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Background The fibronectin splicing variant containing extra domain A (Fn-EDA) is present in negligible amounts in the plasma of healthy humans, but markedly elevated in patients with comorbid conditions including diabetes and hypercholesterolemia, which are risk factors for stroke. It remains unknown, however, whether Fn-EDA worsens stroke outcomes in such conditions. We determined the role of Fn-EDA in stroke outcome in a model of hypercholesterolemia, the apolipoprotein E-deficient (Apoe−/−) mouse. Methods and Results In a transient cerebral ischemia/reperfusion injury model, Apoe−/− mice expressing Fn deficient in EDA (Fn-EDA−/−Apoe−/− mice) exhibited smaller infarcts and improved neurological outcomes at days 1 and 8 (P<0.05 vs. Apoe−/− mice). Concomitantly, intracerebral thrombosis (assessed by fibrin (ogen) deposition) and postischemic inflammation (phospho-NF-κB p65, phospho IKKα/β, IL1-β and TNFα) within lesions of Fn-EDA−/−Apoe−/− mice were markedly decreased (P<0.05 vs. Apoe−/− mice). In a FeCl3 injury-induced carotid artery thrombosis model, thrombus growth rate and the time to occlusion were prolonged in Fn-EDA−/−Apoe−/− mice (P<0.05 vs. Apoe−/− mice). Genetic ablation of TLR4 improved stroke outcome in Apoe−/− mice (P<0.05) but had no effect on stroke outcome in Fn-EDA−/−Apoe−/− mice. Bone marrow transplantation experiments revealed that non-hematopoietic cell-derived Fn-EDA exacerbates stroke through TLR4 expressed on hematopoietic cells. Infusion of a specific inhibitor of Fn-EDA into Apoe−/− mouse 15 minutes after reperfusion significantly improved stroke outcome. Conclusions Hypercholesterolemic mice deficient in Fn-EDA exhibit reduced cerebral thrombosis and less inflammatory response after ischemia/reperfusion injury. These findings suggest that targeting Fn-EDA could be an effective therapeutic strategy in stroke associated with hypercholesterolemia.

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Objective 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. Approach and Results 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), EDAfl/flApoe−/− mice (which constitutively express EDA+-FN) and control Apoe−/− mice fed a high-fat “Western” diet for 14 weeks. Irrespective of gender, EDAfl/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 vs. 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 EDAfl/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, TNFα and IL1β) 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. Conclusions 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.