Abstract 509: Fibronectin Containing Extra Domain a Modulates TLR4-Dependent Smooth Muscle Cell Proliferation and Neointimal Hyperplasia in Apolipoprotein E-Deficient Mice

Abstract

Background: Fibronectin-splice variant containing extra domain A (Fn-EDA), an endogenous ligand of toll-like-receptor 4 (TLR4), is a characteristic feature of proliferating vascular smooth muscle cells (VSMCs) that contributes to intimal thickening and atherosclerosis. Very little is known about the role of Fn-EDA in VSMC proliferation or its clinical implication in neointima formation after vascular injury in the comorbid condition of hyperlipidemia. Methods: To explore the mechanism and therapeutic potential of targeting Fn-EDA in vascular diseases, we used carotid wire-injury model to induce neointimal hyperplasia (28 days) in hyperlipidemic Apoe -/- , Fn-EDA -/- Apoe -/- , TLR4 -/- Apoe -/- , Fn-EDA -/- TLR4 -/- Apoe -/- and SMC- specific Fn-EDA -/- mice (Fn-EDA fl/fl αSMACre + Apoe -/- ) (male and female; 10 weeks old). Furthermore, VSMCs were isolated from different genotypes and stimulated with PDGF to dissect the mechanisms. Results: Using immunostaining, we found increased expression of Fn-EDA in human atheroma, injured carotid artery and proliferating VSMCs of Apoe -/- mice (P<0.05 vs. control). Irrespective of gender, genetic deletion of Fn-EDA in Apoe -/- mice reduced neointimal area after vascular injury (P<0.05 vs. Apoe -/- ). Aortic VSMCs isolated from Fn-EDA -/- Apoe -/- mice exhibited reduced cell proliferation (BrdU assay, G1/S phase transition) and migration (wound healing assay) that was concomitant with elevated pAKT2 and pFOXO4 expression and reduction in pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion levels (P<0.05 vs. Apoe -/- ). Genetic ablation of TLR4 in Apoe -/- mice reduced neointimal hyperplasia concomitant with decreased VSMCs proliferation and migration (P<0.05 vs. Apoe -/- ), but had no additive effect in Fn-EDA -/- Apoe -/- mice. Purified cellular Fn, which contains EDA, attenuated pAKT2 and pFOXO4 and potentiated pAKT1, pmTOR, pNFκB, TNF-α and IL-1β secretion in SMCs from Fn-EDA -/- Apoe -/- mice (P<0.05 vs. plasma Fn that lacks Fn-EDA) but not from Fn-EDA -/- TLR4 -/- Apoe -/- mice. Finally, SMC-specific Fn-EDA -/- mice (Fn-EDA fl/fl αSMACre + Apoe -/- ) exhibited reduced neointimal hyperplasia. Conclusion: Fn-EDA modulates TLR4-dependent VSMCs proliferation and intimal hyperplasia after vascular injury in the comorbid condition of hyperlipidemia.