Smooth muscle cell–specific fibronectin-EDA mediates phenotypic switching and neointimal hyperplasia
Manish Jain, Nirav Dhanesha, Prakash Doddapattar, Mehul R. Chorawala, Manasa K. Nayak, Anne Cornelissen, Liang Guo, Aloke V. Finn, Steven R. Lentz, Anil K. Chauhan
Manish Jain, Nirav Dhanesha, Prakash Doddapattar, Mehul R. Chorawala, Manasa K. Nayak, Anne Cornelissen, Liang Guo, Aloke V. Finn, Steven R. Lentz, Anil K. Chauhan
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Research Article Vascular biology

Smooth muscle cell–specific fibronectin-EDA mediates phenotypic switching and neointimal hyperplasia

Abstract

Fibronectin–splice variant containing extra domain A (Fn-EDA) is associated with smooth muscle cells (SMCs) following vascular injury. The role of SMC-derived Fn-EDA in SMC phenotypic switching or its implication in neointimal hyperplasia remains unclear. Herein, using human coronary artery sections with a bare metal stent, we demonstrate the expression of Fn-EDA in the vicinity of SMC-rich neointima and peri-strut areas. In mice, Fn-EDA colocalizes with SMCs in the neointima of injured carotid arteries and promotes neointima formation in the comorbid condition of hyperlipidemia by potentiating SMC proliferation and migration. No sex-based differences were observed. Mechanistic studies suggested that Fn-EDA mediates integrin- and TLR4-dependent proliferation and migration through activation of FAK/Src and Akt1/mTOR signaling, respectively. Specific deletion of Fn-EDA in SMCs, but not in endothelial cells, reduced intimal hyperplasia and suppressed the SMC synthetic phenotype concomitant with decreased Akt1/mTOR signaling. Targeting Fn-EDA in human aortic SMCs suppressed the synthetic phenotype and downregulated Akt1/mTOR signaling. These results reveal that SMC-derived Fn-EDA potentiates phenotypic switching in human and mouse aortic SMCs and neointimal hyperplasia in the mouse. We suggest that targeting Fn-EDA could be explored as a potential therapeutic strategy to reduce neointimal hyperplasia.

Authors

Manish Jain, Nirav Dhanesha, Prakash Doddapattar, Mehul R. Chorawala, Manasa K. Nayak, Anne Cornelissen, Liang Guo, Aloke V. Finn, Steven R. Lentz, Anil K. Chauhan

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Figure 4

Exogenous cFn-EDA potentiates Akt1 signaling and promotes phenotypic switching.

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Exogenous cFn-EDA potentiates Akt1 signaling and promotes phenotypic swi...
Quiescent aortic SMCs from Fn-EDA–/– Apoe–/– mice were stimulated with cellular Fn containing EDA (cFn-EDA; 0–50 μg/mL) for 24 hours. (A) Duplicate samples were run in the same gels, with the membrane cut in half, and then probed for total and phosphorylated proteins separately. Representative Western blots and densitometric analysis showing the dose-dependent effect of cFn-EDA on Akt signaling components (n = 4 per group). (B) ELISA quantification of TNF-α and IL-1β in supernatant medium from cFn-EDA (20 μg/mL) treated SMCs (n = 4 per group). (C) Quiescent aortic SMCs from Fn-EDA–/– Apoe–/– mice were stimulated with either cFn-EDA (20 μg/mL) or pFn lacking EDA (20 μg/mL). The left panels show representative immunostaining images for the contractile proteins SM22α (green) and SM-MHC (green), and the synthetic proteins vimentin (red) and osteopontin (red). Scale bars: 25 μm. The right panel shows quantification of the immunostaining for SM22α, SM-MHC, vimentin, and osteopontin (n = 4 per group). (D) Representative immunoblots and densitometric analysis of SM22α, SM-MHC, vimentin, and osteopontin in aortic SMCs from Fn-EDA–/– Apoe–/– mice stimulated with either cFn-EDA (20 μg/mL) or pFn lacking EDA (20 μg/mL) (n = 4 per group). Blots for SM22α and osteopontin are from the same biological sample, and blots for SM-MHC and vimentin are from another biological sample. Values are represented as mean ± SEM. Statistical analysis: (A and B) 1-way ANOVA with Bonferroni’s post hoc test; (C and D) unpaired Student’s t test. *P < 0.05 vs. cFn (0 μg/mL).