Abstract 458: Alternatively-Spliced Extra Domain A of Fibronectin Exacerbates Atherosclerosis via a Toll-Like Receptor 4- Dependent Mechanism

Abstract

Rationale Fibronectin containing the alternatively-spliced extra domain A (EDA + -FN) is absent in healthy arteries, but expressed in the atherosclerotic arteries of humans and mice. The mechanistic role of EDA + -FN in the pathophysiology of atherosclerosis remains unclear. Recent studies by us and others suggest that EDA + -FN is a ligand for toll-like receptor 4 (TLR4). Hypothesis EDA + -FN exacerbates atherosclerosis via TLR4 pathway in ApoE -/- mice. Model and Methods We generated EDA +/+ /ApoE -/- (constitutively over express EDA + -FN) and EDA -/- /ApoE -/- mice (EDA-deleted allele of FN and express FN lacking EDA). ApoE -/- mice was used as control. To elucidate the role of TLR4 in EDA + -FN-mediated atherosclerosis, we generated EDA +/+ /TLR4 -/- /ApoE -/- and EDA -/- /TLR4 -/- /ApoE -/- mice. Male mice (n=12-14/group) were fed a high-fat Western diet (21% fat and 0.2% cholesterol) beginning at 6 weeks until they were sacrificed at 5 months of age (i.e., 14 weeks on high-fat Western diet). We compared the extent of atherosclerosis in whole aortae, stained with Oil Red O and en face lesion area measured by morphometry, and in the cross section area of the aortic sinus using the Verhoeff-Van Gieson stain. Macrophage infiltration was quantified by immunohistochemistry. Results We report that atherosclerotic plaque formation in the aortae and aortic sinus of EDA +/+ /ApoE -/- mice was increased by 2-fold, concomitant with significant increased macrophage infiltration (% of total plaque area) ( P <0.001 vs.ApoE -/- mice, ANOVA). Deletion of the EDA exon of FN gene in ApoE -/- mice significantly reduced atherosclerotic plaque formation (aorta and aortic sinus) and macrophage infiltration ( P <0.01 vs. ApoE -/- mice, ANOVA). Total cholesterol and triglycerides levels were similar among groups. TLR4 deficiency in EDA +/+ /ApoE -/- mice significantly reduced atherosclerotic plaque formation in the aorta and aortic sinus, concomitant with decreased macrophage infiltration in aortic sinus ( P <0.05 vs. EDA +/+ /ApoE -/- mice, Two way ANOVA), whereas TLR4 deficiency in EDA -/- /ApoE -/- mice had no effect compared to EDA -/- /ApoE -/- mice. Conclusion These findings reveal a mechanistic role of endogenous EDA + -FN in modulating atherosclerosis via a TLR4-dependent pathway.