Coordinated augmentation of NFAT and NOD signaling mediates proliferative VSMC phenotype switch under hyperinsulinemia

  • Abhijit Shiny
    Corresponding author. Department of Cell and Molecular Biology, Madras Diabetes Research Foundation Gopalapuram, Chennai 600 086, India.
    Department of Cell and Molecular Biology, Madras Diabetes Research Foundation, Dr. Mohan's Diabetes Specialities Centre Gopalapuram, Chennai, India
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  • Bhaskaran Regin
    Department of Cell and Molecular Biology, Madras Diabetes Research Foundation, Dr. Mohan's Diabetes Specialities Centre Gopalapuram, Chennai, India
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  • Viswanathan Mohan
    Department of Cell and Molecular Biology, Madras Diabetes Research Foundation, Dr. Mohan's Diabetes Specialities Centre Gopalapuram, Chennai, India
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  • Muthuswamy Balasubramanyam
    Department of Cell and Molecular Biology, Madras Diabetes Research Foundation, Dr. Mohan's Diabetes Specialities Centre Gopalapuram, Chennai, India
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      • Atherosclerosis is characterized by endothelial dysfunction, smooth muscle cell proliferation and inflammation.
      • Our study shows the role of transcription factor, NFATc1 in hyperinsulinemia induced proliferation and migration of VSMCs.
      • This study advocates the coordinated augmentation of NFATc1 and NOD signaling in the proliferation and migration of VSMCs.
      • In patients with type 2 diabetes, expression of NFATc1 and NOD1/2 showed positive correlation with insulin resistance.



      Although hyperglycemia has been demonstrated to play a significant role in the vascular disease associated with type 2 diabetes, the mechanisms underlying hyperinsulinemia mediated vascular dysfunction are not well understood. We have analyzed whether hyperinsulinemia could activate NFAT (Nuclear factor of activated T cells) signaling and thereby influence vascular smooth muscle cell (VSMC) migration and proliferation, a major event in the progression of atherosclerosis.

      Methods and results

      Human aortic VSMCs upon chronic insulin treatment exhibited increased expression of NFATc1 both at the mRNA and protein levels. The mechanistic role of NFAT in VSMC migration and proliferation was examined using 11R-VIVIT, a cell permeable NFAT specific inhibitor, where it reduced the insulin effect on VSMC, which was further substantiated by over expression or silencing of NFATc1gene (p < 0.05). This study also report for the first time the role of NFAT in NOD (Nucleotide oligomerization domain) mediated innate immune signaling and its significance in insulin effect on VSMCs. mRNA expression of NOD was up regulated when cells were treated with insulin or ligands whereas pretreatment with 11R-VIVIT reversed this effect (p < 0.05). Our study uphold the clinical significance as we observed an increased mRNA expression of NFATc1 in monocytes isolated from patients with type 2 diabetes which correlated positively with insulin resistance and glycemic load (p < 0.05).


      This study suggests that targeted NFAT inhibition can be an effective strategy to coordinately quench insulin induced proliferative and inflammatory responses along with innate immunity alterations in vascular smooth muscle cells, which underlie atherosclerosis.


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        • Randriamboavonjy V.
        • Schrader J.
        • Busse R.
        • Fleming I.
        Insulin induces the release of vasodilator compounds from platelets by nitric oxide-G Kinase-VAMP-3-dependent pathway.
        J. Exp. Med. 2004; 199: 347-356
        • Anfossi G.
        • Russo I.
        • Doronzo G.
        • Trovati M.
        Contribution of insulin resistance to vascular dysfunction.
        Arch. Physiol. Biochem. 2009; 115: 199-217
        • Wang C.C.
        • Goalstone M.L.
        • Draznin B.
        Molecular mechanisms of insulin resistance that impact cardiovascular biology.
        Diabetes. 2004; 53: 2735-2740
        • Singh R.B.
        • Mengi S.A.
        • Xu Y.J.
        • Arneja A.S.
        • Dhalla N.S.
        Pathogenesis of atherosclerosis-A multifactorial process.
        Exp. Clin. Cardiol. 2002; 7: 40-53
        • Abhijit S.
        • Bhaskaran R.
        • Narayanasamy A.
        • Chakroborty A.
        • Manickam N.
        • Dixit M.
        • Mohan V.
        • Balasubramanyam M.
        Hyperinsulinemia-induced vascular smooth muscle cell (VSMC) migration and proliferation is mediated by converging mechanisms of mitochondrial dysfunction and oxidative stress.
        Mol. Cell. Biochem. 2013; 373: 95-105
        • Orr A.W.
        • Hastings N.E.
        • Blackman B.R.
        • Wamhoff B.R.
        Complex regulation and function of the inflammatory smooth muscle cell phenotype in atherosclerosis.
        J. Vasc. Res. 2010; 47: 168-180
        • Vallejo J.G.
        Role of Toll-like receptors in cardiovascular diseases.
        Clin. Sci. 2011; 121: 1-10
        • Lee G.L.
        • Chang Y.W.
        • Wu J.Y.
        • Wu M.L.
        • Wu K.K.
        • Yet S.F.
        • Kuo C.C.
        TLR 2 induces vascular smooth muscle cell migration through cAMP response element-binding protein-mediated interleukin-6 production.
        Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2751-2760
        • Delbridge L.M.
        • O'Riordan M.X.
        Innate recognition of intracellular bacteria.
        Curr. Opin. Immunol. 2007; 19: 10-16
        • Bracey N.A.
        • Beck P.L.
        • Muruve D.A.
        • Hirota S.A.
        • Guo J.
        • Jabagi H.
        • Wright J.R.
        • Macdonald J.A.
        • Lees Miller J.P.
        • Roach D.
        • Semeniuk L.M.
        • Duff H.J.
        The Nlrp3 inflammasome promotes myocardial dysfunction in structural cardiomyopathy through interleukin-1beta.
        Exp. Physiol. 2013; 98: 462-472
        • Sun J.
        • Ding Y.
        NOD2 agonist promotes the production of inflammatory cytokines in VSMC in synergy with TLR2 and TLR4 agonists.
        Sci. World J. 2012; 2012: 607157
        • Shiny A.
        • Regin B.
        • Balachandar V.
        • Gokulakrishnan K.
        • Mohan V.
        • Babu S.
        • Balasubramanyam M.
        Convergence of innate immunity and insulin resistance as evidenced by increased nucleotide oligomerization domain (NOD) expression and signaling in monocytes from patients with type 2 diabetes.
        Cytokine. 2013; 64: 564-770
        • Horsley V.
        • Pavlath G.K.
        NFAT:ubiquitous regulator of cell differentiation and adaptation.
        J. Cell. Biol. 2002; 4: 771-774
        • Wu H.
        • Peisley A.
        • Graef I.A.
        • Crabtree G.R.
        NFAT signaling and the invention of vertebrates.
        Trends Cell. Biol. 2007; 17: 251-260
        • Rao A.
        • Luo C.
        • Hogan P.G.
        Transcription factors of the NFAT family: regulation and function.
        Annu. Rev. Immunol. 1997; 15: 707-747
        • Minematsu H.
        • Shin M.J.
        • Celil Aydemir A.B.
        • Kim K.O.
        • Nizami S.A.
        • Chung G.J.
        • Lee F.Y.
        Nuclear presence of nuclear factor of activated T cells (NFAT) c3 and c4 is required for toll-like receptor-activated innate inflammatory response of monocytes/macrophages.
        Cell. Signal. 2011; 23: 1785-1793
        • Nilsson J.
        • Nilsson L.M.
        • Chen Y.-W.
        • Molkentin J.D.
        • Erlinge D.
        • Gomez M.F.
        High glucose activates nuclear factor of activated t cells in native vascular smooth muscle.
        Arterioscler. Thromb. Vasc. Biol. 2006; 26: 794-800
        • Nilsson L.M.
        • Sun Z.W.
        • Nilsson J.
        • Nordstrom I.
        • Chen Y.W.
        • Molkentin J.D.
        • Wide-Swensson D.
        • Hellstrand P.
        • Lydrup M.L.
        • Gomez M.F.
        Novel blocker of NFAT activation inhibits IL-6 production in human myometrial arteries and reduces vascular smooth muscle cell proliferation.
        Am. J. Physiol. Cell. Physiol. 2007; 292: C1167-C1178
        • Deepa M.
        • Pradeepa R.
        • Rema M.
        • Mohan A.
        • Deepa R.
        • Shanthirani S.
        • Mohan V.
        The Chennai urban rural epidemiology study (CURES)–study design and methodology (urban component) (CURES-I).
        J. Assoc. Phys. India. 2003; 51: 863-870
        • Deepa R.
        • Shanthirani C.S.
        • Premalatha G.
        • Sastry N.G.
        • Mohan V.
        Prevalence of insulin resistance syndrome in a selected south Indian population–the Chennai urban population study-7 (CUPS-7).
        Indian J. Med. Res. 2002; 115: 118-127
        • Yang T.T.
        • Suk H.Y.
        • Yang X.
        • Olabisi O.
        • Yu R.Y.
        • Durand J.
        • Jelicks L.A.
        • Kim JY,Scherer P.E.
        • Wang Y.
        • Feng Y.
        • Rossetti L.
        • Graef I.A.
        • Crabtree G.R.
        • Chow C.W.
        Role of transcription factor NFAT in glucose and insulin homeostasis.
        Mol. Cell. Biol. 2006; 26: 7372-7387
        • Lipskaia L.
        • del Monte F.
        • Capiod T.
        • Yacoubi S.
        • Hadri L.
        • Hours M.
        • Hajjar R.J.
        • Lompré A.M.
        Sarco/endoplasmic reticulum Ca2+-ATPase gene transfer reduces vascular smooth muscle cell proliferation and neointima formation in the rat.
        Circ. Res. 2005; 97: 488-495
        • Pang X.
        • Sun N.L.
        Calcineurin-NFAT signaling is involved in phenylephrine-induced vascular smooth muscle cell proliferation.
        Acta Pharmacol. Sin. 2009; 30: 537-544
        • Liu Z.
        • Zhang C.
        • Dronadula N.
        • Li Q.
        • Rao G.N.
        Blockade of nuclear factor of activated T cells activation signaling suppresses balloon injury-induced neointima formation in a rat carotid artery model.
        J. Biol. Chem. 2005; 280: 14700-14708
        • Zhu J.
        • McKeon F.
        Nucleocytoplasmic shuttling and the control of NF-AT signaling.
        Cell. Mol. Life Sci. 2000; 57: 411-420
        • Galkina E.
        • Ley K.
        Immune and inflammatory mechanisms of atherosclerosis.
        Annu. Rev. Immunol. 2009; 27: 165-197
        • Jager J.
        • Gremeaux T.
        • Cormont M.
        • Le Marchand-Brustel Y.
        • Tanti J.F.
        Interleukin-1beta-induced insulin resistance in adipocytes through downregulation of insulin receptor substrate-1 expression.
        Endocrinol. 2007; 148: 241-251
        • Larsen C.M.
        • Faulenbach M.
        • Vaag A.
        • Volund A.
        • Ehses J.A.
        • Seifert B.
        • Mandrup-Poulsen T.
        • Donath M.Y.
        Interleukin-1-receptor antagonist in type 2 diabetes mellitus.
        N. Engl. J. Med. 2007; 356: 1517-1526
        • Mandrup-Poulsen T.
        • Pickersgill L.
        • Donath M.Y.
        Blockade of interleukin 1 in type 1 diabetes mellitus.
        Nat. Rev. Endocrinol. 2010; 6: 158-166
        • Qi X.
        • Li S.
        • Li J.
        The prognostic value of IL-8 for cardiac events and restenosis in patients with coronary heart diseases after percutaenous coronary intervention.
        Jpn. Heart J. 2003; 44: 623-632
        • Westwell-Roper C.
        • Nackiewicz D.
        • Dan M.
        • Ehses J.A.
        Toll-like receptors and NLRP3 as central regulators of pancreatic islet inflammation in type 2 diabetes.
        Immunol. Cell. Biol. 2014; 92: 314-323
        • Wen C.
        • Yang X.
        • Yan Z.
        • Zhao M.
        • Yue X.
        • Cheng X.
        • Zheng Z.
        • Guan K.
        • Dou J.
        • Xu T.
        • Zhang Y.
        • Song T.
        • Wei C.
        • Zhong H.
        Nalp3 inflammasome is activated and required for vascular smooth muscle cell calcification.
        Int. J. Cardiol. 2013; 168: 2242-2247
        • Yuan H.
        • Zelkha S.
        • Burkatovskaya M.
        • Gupte R.
        • Leeman S.E.
        • Amar S.
        Pivotal role of NOD2 in inflammatory processes affecting atherosclerosis and periodontal bone loss.
        Proc. Natl. Acad. Sci. 2013; 110: E5059-E5068
        • Liu H.Q.
        • Zhang X.Y.
        • Edfeldt K.
        • Nijhuis M.O.
        • Idborg H.
        • Bäck M.
        • Roy J.
        • Hedin U.
        • Jakobsson P.J.
        • Laman J.D.
        • de Kleijn D.P.
        • Pasterkamp G.
        • Hansson G.K.
        • Yan Z.Q.
        NOD2-mediated innate immune signaling regulates the eicosanoids in atherosclerosis.
        Arterioscler. Thromb. Vasc. Biol. 2013; 33: 2193-2201
        • Zhang S.
        • Zhang S.
        • Hu L.
        • Zhai L.
        • Xue R.
        • Ye J.
        • Chen L.
        • Cheng G.
        • Mruk J.
        • Kunapuli S.P.
        • Ding Z.
        Nucleotide-binding oligomerization domain 2 receptor is expressed in platelets and enhances platelet activation and thrombosis.
        Circulation. 2015; 131: 1160-1170
        • Jauliac S.
        • et al.
        The role of NFAT transcription factors in integrin-mediated carcinoma invasion.
        Nat. Cell. Biol. 2002; 4: 540-544
        • Medyouf H.
        • et al.
        Targeting calcineurin activation as a therapeutic strategy for T-cell acute lymphoblastic leukemia.
        Nat. Med. 2007; 13: 736-741