Fibronectin extra domain A stabilises atherosclerotic plaques in apolipoprotein E and in LDL-receptor-deficient mice

Summary

The primary transcript of fibronectin undergoes alternative splicing in the cassette-type EDA and EDB exons and in the IIICs segment to generate different protein isoforms. Human carotid atherosclerotic plaques with a more stable phenotype are enriched with EDA containing fibronectin (FN-EDA). The aim of this study was to investigate the role of EDA containing fibronectin during atherogenesis. Mice constitutively expressing or lacking the EDA domain of fibronectin (EDA^+/+ or EDA^-/-) were crossed with ApoE^-/- or LDL-R^-/- mice and fed with a western type diet for 12 weeks. Lack of FN-EDA resulted in reduced atherosclerosis and in a plaque phenotype characterised by decreased calponin positive VSMC’s (-15 %) and increased macrophages (+20 %). This was paralleled by increased MMP2, MMP9, and reduced TIMP2, collagen 1A1, 1A2 and 3A1 gene expression compared to that of wild-type and EDA^+/+ mice. In vitro, VSMCs and macrophages isolated from EDA^-/- mice showed increased MMPs expression and activity compared to wild-type or EDA^+/+ mice. Albumin-Cre recombinase/ EDA^+/+/ApoE^-/- mice, which produce EDA containing FN only in peripheral tissues, presented an extension, a composition and a gene expression pattern in the atherosclerotic lesions similar to that of controls. The inclusion of EDA in FN results in larger atherosclerotic plaques compared to mice lacking EDA but with a more favourable phenotype in two animals models of atherosclerosis. This effect depends on the EDA-containing fibronectin produced by cells in the vasculature but not in the liver. These observations set the stage for investigating the properties of circulating EDA containing FN in improving plaque stability.

Keywords

Atherosclerosis - extracellular matrix - lipoproteins

• References

• 1 White ES, Baralle FE, Muro AF. New insights into form and function of fibronectin splice variants. J Pathol 2008; 216: 1-14.
• 2 Arslan F, Smeets MB, Riem Vis PW. et al. Lack of fibronectin-EDA promotes survival and prevents adverse remodelling and heart function deterioration after myocardial infarction. Circulation Res 2011; 108: 582-592.
• 3 Tersteeg C, Roest M, Mak-Nienhuis EM. et al. A fibronectin-fibrinogen-tropoelastin coating reduces smooth muscle cell growth but improves endothelial cell function. J Cell Mol Med 2012; 16: 2117-2126.
• 4 van Keulen JK, de Kleijn DP, Nijhuis MM. et al. Levels of extra domain A containing fibronectin in human atherosclerotic plaques are associated with a stable plaque phenotype. Atherosclerosis 2007; 195: e83-91.
• 5 Paloschi V, Kurtovic S, Folkersen L. et al. Impaired splicing of fibronectin is associated with thoracic aortic aneurysm formation in patients with bicuspid aortic valve. Arterioscl Thromb Vasc Biol 2011; 31: 691-697.
• 6 Babaev VR, Porro F, Linton MF. et al. Absence of regulated splicing of fibronectin EDA exon reduces atherosclerosis in mice. Atherosclerosis 2008; 197: 534-540.
• 7 Tan MH, Sun Z, Opitz SL. et al. Deletion of the alternatively spliced fibronectin EIIIA domain in mice reduces atherosclerosis. Blood 2004; 104: 11-18.
• 8 Rohwedder I, Montanez E, Beckmann K. et al. Plasma fibronectin deficiency impedes atherosclerosis progression and fibrous cap formation. EMBO Mol Med 2012; 04: 564-576.
• 9 Muro AF, Chauhan AK, Gajovic S. et al. Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan. J Cell Biol 2003; 162: 149-160.
• 10 Chauhan AK, Iaconcig A, Baralle FE. et al. Alternative splicing of fibronectin: a mouse model demonstrates the identity of in vitro and in vivo systems and the processing autonomy of regulated exons in adult mice. Gene 2004; 324: 55-63.
• 11 Ammirati E, Cianflone D, Vecchio V. et al. Effector Memory T cells Are Associated With Atherosclerosis in Humans and Animal Models. J Am Heart Assoc 2012; 01: 27-41.
• 12 Norata GD, Venu VK, Callegari E. et al. Effect of Tie-2 conditional deletion of BDNF on atherosclerosis in the ApoE null mutant mouse. Biochim Biophys Acta 2012; 1822: 927-935.
• 13 Tibolla G, Norata GD, Meda C. et al. Increased atherosclerosis and vascular inflammation in APP transgenic mice with apolipoprotein E deficiency. Atherosclerosis 2010; 210: 78-87.
• 14 Norata GD, Marchesi P, Pulakazhi Venu VK. et al. Deficiency of the long pentraxin PTX3 promotes vascular inflammation and atherosclerosis. Circulation 2009; 120: 699-708.
• 15 Norata GD, Cattaneo P, Poletti A. et al. The androgen derivative 5alpha-androstane-3beta,17beta-diol inhibits tumor necrosis factor alpha and lipopolysaccharide induced inflammatory response in human endothelial cells and in mice aorta. Atherosclerosis 2010; 212: 100-106.
• 16 Norata GD, Raselli S, Grigore L. et al. Small dense LDL and VLDL predict common carotid artery IMT and elicit an inflammatory response in peripheral blood mononuclear and endothelial cells. Atherosclerosis 2009; 206: 556-562.
• 17 Sala F, Aranda JF, Rotllan N. et al. MiR-143/145 deficiency attenuates the progression of atherosclerosis in Ldlr-/-mice. Thromb Haemost 2014; 112: 796-802.
• 18 Bellosta S, Via D, Canavesi M. et al. HMG-CoA reductase inhibitors reduce MMP-9 secretion by macrophages. Arterioscler Thromb Vasc Biol 1998; 18: 1671-1678.
• 19 Rensen SS, Doevendans PA, van Eys GJ. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Netherl Heart J 2007; 15: 100-108.
• 20 Tamkun JW, Hynes RO. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem 1983; 258: 4641-4647.
• 21 Moretti FA, Chauhan AK, Iaconcig A. et al. A major fraction of fibronectin present in the extracellular matrix of tissues is plasma-derived. J Biol Chem 2007; 282: 28057-28062.
• 22 Engvall E, Ruoslahti E, Miller EJ. Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J Exp Med 1978; 147: 1584-1595.
• 23 Gildner CD, Lerner AL, Hocking DC. Fibronectin matrix polymerization increases tensile strength of model tissue. Am J Physiol Heart Circul Physiol 2004; 287: H46-53.
• 24 Clark RA. Fibronectin in the skin. J Invest Dermatol 1983; 81: 475-479.
• 25 Jarnagin WR, Rockey DC, Koteliansky VE. et al. Expression of variant fibronec-tins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis. J Cell Biol 1994; 127: 2037-2048.
• 26 Serini G, Bochaton-Piallat ML, Ropraz P. et al. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J Cell Biol 1998; 142: 873-881.
• 27 Abe Y, Bui-Thanh NA, Ballantyne CM. et al. Extra domain A and type III connecting segment of fibronectin in assembly and cleavage. Biochem Biophys Res Commun 2005; 338: 1640-1647.
• 28 Manabe R, Ohe N, Maeda T. et al. Modulation of cell-adhesive activity of fibronectin by the alternatively spliced EDA segment. J Cell Biol 1997; 139: 295-307.
• 29 Minami T, Kuwahara K, Nakagawa Y. et al. Reciprocal expression of MRTF-A and myocardin is crucial for pathological vascular remodelling in mice. EMBO J 2012; 31: 4428-4440.
• 30 Howard EW, Crider BJ, Updike DL. et al. MMP-2 expression by fibroblasts is suppressed by the myofibroblast phenotype. Exp Cell Res 2012; 318: 1542-1553.
• 31 Hesselvik JF. Plasma fibronectin levels in sepsis: influencing factors. Crit Care Med 1987; 15: 1092-1097.