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Pharmacological inhibition of RhoA signaling prevents connective tissue growth factor induction in endothelial cells exposed to non-uniform shear stress

      Abstract

      Shear stress changes play an important role in atheroma formation. This study focussed on atherogenic protein expression under non-uniform shear stress and the pharmacological modulation of shear-related endothelial dysfunction. Bifurcating flow-through cell culture slides were used to expose HUVECs to steady laminar or non-uniform shear stress for 18 h at 10 dyn/cm2. Protein expression was determined by immunofluorescence, and quantified using MetaVue software.
      Laminar shear stress resulted in cell alignment, reduced F-actin fibers, and significant induction of endothelial nitric oxide synthase expression. Under non-uniform shear stress at bifurcations, minor upregulation of adhesion molecules was observed. Connective tissue growth factor (CTGF) was significantly downregulated by laminar shear stress and induced in cells exposed to non-uniform shear stress. CTGF upregulation by non-uniform shear stress was RhoA-dependent, because it was almost completely inhibited in cells transfected with dominant negative RhoA-N19, and when cells were treated with 1 μmol/L simvastatin during flow. Pre-incubation of HUVECs with inhibitors of Rho-associated kinase before exposure to flow significantly suppressed the CTGF induction in regions of non-uniform shear stress.
      In conclusion, non-uniform shear stress-dependent CTGF expression requires active RhoA and can be prevented pharmacologically. Interference with shear stress-induced protein expression may inhibit endothelial dysfunction in athero-prone vessel regions.

      Keywords

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      References

        • Cunningham K.S.
        • Gotlieb A.I.
        The role of shear stress in the pathogenesis of atherosclerosis.
        Lab Invest. 2005; 85: 9-23
        • Cheng C.
        • Tempel D.
        • van Haperen R.
        • et al.
        Atherosclerotic lesion size and vulnerability are determined by patterns of fluid shear stress.
        Circulation. 2006; 113: 2744-2753
        • Carbajal J.M.
        • Schaeffer R.C. Jr,
        RhoA inactivation enhances endothelial barrier function.
        Am J Physiol. 1999; 277: 955-964
        • Mallat Z.
        • Gojova A.
        • Sauzeau V.
        • et al.
        Rho-associated protein kinase contributes to early atherosclerotic lesion formation in mice.
        Circ Res. 2003; 93: 884-888
        • Vicari R.M.
        • Chaitman B.
        • Keefe D.
        • et al.
        • Fasudil Study Group
        Efficacy and safety of fasudil in patients with stable angina: a double-blind, placebo-controlled, phase 2 trial.
        J Am Coll Cardiol. 2005; 46: 1803-1811
        • Mason J.C.
        Statins and their role in vascular protection.
        Clin Sci (Lond). 2003; 105: 251-266
        • McCormick S.M.
        • Eskin S.G.
        • McIntire L.V.
        • et al.
        DNA microarray reveals changes in gene expression of shear stressed human umbilical vein endothelial cells.
        Proc Natl Acad Sci USA. 2001; 98: 8955-8960
        • Garcia-Cardena G.
        • Comander J.I.
        • Blackman B.R.
        • Anderson K.R.
        • Gimbrone M.A.
        Mechanosensitive endothelial gene expression profiles: scripts for the role of hemodynamics in atherogenesis?.
        Ann NY Acad Sci. 2001; 947: 1-6
        • Yoshisue H.
        • Suzuki K.
        • Kawabata A.
        • et al.
        Large scale isolation of non-uniform shear stress-responsive genes from cultured human endothelial cells through the preparation of a subtracted cDNA library.
        Atherosclerosis. 2002; 162: 323-334
        • Hajra L.
        • Evans A.I.
        • Chen M.
        • Hyduk S.J.
        • Collins T.
        • Cybulsky M.I.
        The NF-kappa B signal transduction pathway in aortic endothelial cells is primed for activation in regions predisposed to atherosclerotic lesion formation.
        Proc Natl Acad Sci USA. 2000; 97: 9052-9057
        • Passerini A.G.
        • Polacek D.C.
        • Shi C.
        • et al.
        Coexisting proinflammatory and antioxidative endothelial transcription profiles in a disturbed flow region of the adult porcine aorta.
        Proc Natl Acad Sci USA. 2004; 101: 2482-2487
        • Graness A.
        • Cicha I.
        • Goppelt-Struebe M.
        Contribution of Src-FAK signaling to the induction of connective tissue growth factor in renal fibroblasts.
        Kidney Int. 2006; 69: 1341-1349
        • Chaqour B.
        • Goppelt-Struebe M.
        Mechanical regulation of the Cyr61/CCN1 and CTGF/CCN2 proteins.
        FEBS J. 2006; 273: 3639-3649
        • Oemar B.S.
        • Werner A.
        • Garnier J.M.
        • et al.
        Human connective tissue growth factor is expressed in advanced atherosclerotic lesions.
        Circulation. 1997; 95: 831-839
        • Cicha I.
        • Yilmaz A.
        • Klein M.
        • et al.
        Connective tissue growth factor is overexpressed in complicated atherosclerotic plaques and induces mononuclear cell chemotaxis in vitro.
        Arterioscler Thromb Vasc Biol. 2005; 25: 1008-1013
        • Dai G.
        • Kaazempur-Mofrad M.R.
        • Natarajan S.
        • et al.
        Distinct endothelial phenotypes evoked by arterial waveforms derived from atherosclerosis-susceptible and -resistant regions of human vasculature.
        Proc Natl Acad Sci USA. 2004; 101: 14871-14876
        • Jakobisiak M.
        • Bruno S.
        • Skierski J.S.
        • Darzynkiewicz Z.
        Cell cycle-specific effects of lovastatin.
        Proc Natl Acad Sci USA. 1991; 88: 3628-3632
        • Essig M.
        • Vrtovsnik F.
        • Nguyen G.
        • Sraer J.D.
        • Friedlander G.
        Lovastatin modulates in vivo and in vitro the plasminogen activator/plasmin system of rat proximal tubular cells: role of geranylgeranylation and Rho proteins.
        J Am Soc Nephrol. 1998; 9: 1377-1388
        • Raedler U.
        • Zantl R.
        Simulation von Blutgefäßen in Zellkultur-Biochips.
        Laborwelt. 2005; 1 ([in German]): 34-36
        • Resnick N.
        • Yahav H.
        • Shay-Salit A.
        • et al.
        Fluid shear stress and the vascular endohelium: for better and for worse.
        Prog Biophys Mol Biol. 2003; 81: 177-199
        • Eberlein M.
        • Heusinger-Ribeiro J.
        • Goppelt-Struebe M.
        Rho-dependent inhibition of the induction of connective tissue growth factor (CTGF) by HMG CoA reductase inhibitors (statins).
        Br J Pharmacol. 2001; 133: 1172-1180
        • Muehlich S.
        • Schneider N.
        • Hinkmann F.
        • Garlichs C.D.
        • Goppelt-Struebe M.
        Induction of connective tissue growth factor (CTGF) in human endothelial cells by lysophosphatidic acid, sphingosine-1-phosphate, and platelets.
        Atherosclerosis. 2004; 175: 261-268
        • Brunton V.G.
        • MacPherson I.R.
        • Frame M.C.
        Cell adhesion receptors, tyrosine kinases and actin modulators: a complex three-way circuitry.
        Biochim Biophys Acta. 2004; 1692: 121-144
        • Fan W.-H.
        • Karnovsky M.J.
        Increased MMP-2 expression in connective tissue growth factor over-expression vascular smooth muscle cells.
        J Biol Chem. 2002; 277: 9800-9805
        • Ott C.
        • Iwanciw D.
        • Graness A.
        • Giehl K.
        • Goppelt-Struebe M.
        Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton.
        J Biol Chem. 2003; 278: 44305-44311
        • Hahn A.
        • Heusinger-Ribeiro J.
        • Lanz T.
        • Zenkel S.
        • Goppelt-Struebe M.
        Induction of connective tissue growth factor by activation of heptahelical receptors: modulation by rho proteins and the actin cytoskeleton.
        J Biol Chem. 2000; 275: 37429-37435
        • Wojciak-Stothard B.
        • Potempa S.
        • Eichholtz T.
        • Ridley A.J.
        Rho and Rac but not Cdc42 regulate endothelial cell permeability.
        J Cell Sci. 2001; 114: 1343-1355
        • McCue S.
        • Dajnowiec D.
        • Xu F.
        • Zhang M.
        • Jackson M.R.
        • Langille B.L.
        Shear stress regulates forward and reverse planar cell polarity of vascular endothelium in vivo and in vitro.
        Circ Res. 2006; 98: 939-946
        • Mehrotra M.
        • Saegusa M.
        • Wadhwa S.
        • Voznesensky O.
        • Peterson D.
        • Pilbeam C.
        Fluid flow induces Rankl expression in primary murine calvarial osteoblasts.
        J Cell Biochem. 2006; 98: 1271-1283
        • Heusinger-Ribeiro J.
        • Eberlein M.
        • Wahab N.
        • Goppelt-Struebe M.
        Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP.
        J Am Soc Nephrol. 2001; 12: 1853-1861
        • Kothapalli D.
        • Hayashi N.
        • Grotendorst G.R.
        Inhibition of TGF-β-stimulated CTGF gene expression and anchorage-independent growth by cAMP identifies a CTGF-dependent restriction point in the cycle.
        FASEB J. 1998; 12: 1151-1161
        • Bo Y.C.
        Shear stress stimulates phosphorylation of protein kinase A substrate proteins including endothelial nitric oxide synthase in endothelial cells.
        Exp Mol Med. 2006; 38: 63-71
        • Laufs U.
        • Liao J.K.
        Post-transcriptional regulation of endothelial nitric oxide synthase mRNA stability by Rho GTPase.
        J Biol Chem. 1998; 273: 24266-24271
        • Hernandez-Perera O.
        • Perez-Sala D.
        • Soria E.
        • Lamas S.
        Involvement of Rho GTPases in the transcriptional inhibition of preproendothelin-1 gene expression by simvastatin in vascular endothelial cells.
        Circ Res. 2000; 87: 616-622
        • Shiu Y.T.
        • Li S.
        • Yuan S.
        • Wang Y.
        • Nguyen P.
        • Chien S.
        Shear stress-induced c-fos activation is mediated by Rho in a calcium-dependent manner.
        Biochem Biophys Res Commun. 2003; 303: 548-555