Abstract 458: Alternatively-Spliced Extra Domain A of Fibronectin Exacerbates Atherosclerosis via a Toll-Like Receptor 4- Dependent Mechanism
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.
- © 2013 by American Heart Association, Inc.