Fibronectin Splicing Variants Containing Extra Domain A Promote Atherosclerosis in Mice Through Toll-Like Receptor 4

Arterioscler Thromb Vasc Biol. 2015 Nov;35(11):2391-400. doi: 10.1161/ATVBAHA.115.306474. Epub 2015 Oct 1.

Abstract

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), EDA(fl/fl)Apoe(-/-) mice (which constitutively express EDA(+)-FN), and control Apoe(-/-) mice fed a high-fat Western diet for 14 weeks. Irrespective of sex, EDA(fl/fl)Apoe(-/-) 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 versus 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 EDA(fl/fl)Apoe(-/-) 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, tumor necrosis factor-α, and interleukin-1β) 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.

Keywords: TLR4; apolipoprotein E; atherosclerosis; cellular fibronectin EDA; macrophages.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Aorta / metabolism*
  • Aorta / pathology
  • Aortic Diseases / genetics
  • Aortic Diseases / immunology
  • Aortic Diseases / metabolism*
  • Aortic Diseases / pathology
  • Aortic Diseases / prevention & control
  • Apolipoproteins E / genetics
  • Apolipoproteins E / metabolism
  • Atherosclerosis / genetics
  • Atherosclerosis / immunology
  • Atherosclerosis / metabolism*
  • Atherosclerosis / pathology
  • Atherosclerosis / prevention & control
  • Coronary Artery Disease / metabolism
  • Coronary Artery Disease / pathology
  • Coronary Vessels / metabolism
  • Coronary Vessels / pathology
  • Diet, High-Fat
  • Disease Models, Animal
  • Female
  • Fibronectins / deficiency
  • Fibronectins / genetics
  • Fibronectins / metabolism*
  • Humans
  • Lipoproteins, LDL / metabolism
  • Macrophages / immunology
  • Macrophages / metabolism*
  • Male
  • Mice, Inbred C57BL
  • Mice, Knockout
  • NF-kappa B / metabolism
  • Plaque, Atherosclerotic
  • Protein Isoforms
  • Signal Transduction*
  • Toll-Like Receptor 4 / deficiency
  • Toll-Like Receptor 4 / genetics
  • Toll-Like Receptor 4 / metabolism*

Substances

  • Apolipoproteins E
  • Fibronectins
  • Lipoproteins, LDL
  • NF-kappa B
  • Protein Isoforms
  • TLR4 protein, human
  • Tlr4 protein, mouse
  • Toll-Like Receptor 4
  • extra domain A fibronectin, mouse