Download PDF
Thromb Haemost 2015; 114(01): 173-185
DOI: 10.1160/TH14-10-0880
Stroke, Systemic or Venous Thromboembolism
Schattauer GmbH

NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium

Kefeng Zhai
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
2   Institute of Pharmaceutical Biotechnology, Suzhou University, Suzhou, P. R. China
,
Youmei Tang
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
,
Yuanyuan Zhang
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
,
Fang Li
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
,
Yan Wang
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
3   Department of Internal Medicine, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, Conneticut, USA
,
Zhengyu Cao
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
,
Jun Yu
3   Department of Internal Medicine, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, Conneticut, USA
4   Center of Translation Medicine, Jiangxi University of Traditional Chinese Medicine, Nanchang, Jiangxi, P. R. China
,
Junping Kou
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
3   Department of Internal Medicine, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, Conneticut, USA
,
Boyang Yu
1   State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, Nanjing, P. R. China
› Author Affiliations Financial support: The present research was supported by the National Natural Science Foundation of China (No. 81274131), 2011’ Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education, a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, and the Project Program of the State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. JKGZ201107).
Further Information

Publication History

Received: 24 October 2014

Accepted after major revision: 19 February 2015

Publication Date:
22 November 2017 (online)

    Permissions and Reprints

Summary

Non-muscle myosin heavy chain IIA (NMMHC IIA) has been shown to be involved in thrombus formation and inflammatory microparticle release in endothelial cells. However, the role of NMMHC IIA in regulating the expression of tissue factor (TF) and deep venous thrombosis remains to be elucidated. In the present study, endothelial cells were stimulated with tumour necrosis factor-α (TNF-α) to induce TF expression. Pretreatment with the NMMHC II inhibitor blebbistatin suppressed the mRNA and protein expressions as well as the procoagulant activity of TF in a dose-dependent manner. Blebbistatin enhanced Akt and GSK3β phosphorylation and inhibited NF-κB p65 nuclear translocation and IκBα degradation. These observations were similar to the effect of CHIR99021, a GSK3β inhibitor. TF downregulation by blebbistatin was antagonised by the PI3K inhibitor, wortmannin. Furthermore, siRNA knockdown of NMMHC IIA but not IIB or IIC, inhibited TF expression, activated Akt/GSK3β and suppressed NF-κB signalling pathways, whereas the overexpression of NMMHC IIA increased TF expression. The binding of NMMHC IIA and TNF receptor 2 mediated signal internalisation in TNF-α-stimulated endothelial cells. Importantly, blebbistatin decreased endothelium NMMHC IIA and TF expression, deactivated GSK3β by inducing its phosphorylation, suppressed p65 nuclear translocation, and inhibited thrombus formation in a mouse deep venous thrombosis model. Our findings provide solid evidence that inhibition of NMMHC II most likely NMMHC IIA impedes TF expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium both in vitro and in vivo. NMMHC IIA might be a potential novel target for the treatment of thrombotic disorders.

Keywords

NMMHC IIA - venous thrombosis - tissue factor - endothelium - signalling transduction

 

  • References

  • 1 Dasari TW, Pappy R, Hennebry TA. Pharmacomechanical thrombolysis of acute and chronic symptomatic deep vein thrombosis: a systematic review of literature. Angiology 2012; 63: 138-145.
    CrossrefPubMedGoogle Scholar
  • 2 Mackman N. New insights into the mechanisms of venous thrombosis. J Clin Invest 2012; 122: 2331-2336.
    CrossrefPubMedGoogle Scholar
  • 3 Schulz C, Engelmann B, Massberg S. Crossroads of coagulation and innate immunity: the case of deep vein thrombosis. J Thromb Haemost 2013; 11: 233-241.
    CrossrefPubMedGoogle Scholar
  • 4 Reitsma PH, Versteeg HH, Middeldorp S. Mechanistic view of risk factors for venous thromboembolism. Arterioscler Thromb Vasc Biol 2012; 32: 563-568.
    CrossrefPubMedGoogle Scholar
  • 5 Manly DA, Boles J, Mackman N. Role of tissue factor in venous thrombosis. Annu Rev Physiol 2011; 73: 515-525.
    CrossrefPubMedGoogle Scholar
  • 6 Watson SP. Platelet activation by extracellular matrix proteins in haemostasis and thrombosis. Curr Pharm Des 2009; 15: 1358-1372.
    CrossrefPubMedGoogle Scholar
  • 7 Eto M, Kouroedov A, Cosentino F. et al. Glycogen synthase kinase-3 mediates endothelial cell activation by tumor necrosis factor-alpha. Circulation 2005; 112: 1316-1322.
    CrossrefPubMedGoogle Scholar
  • 8 Zhang W, Wang J, Wang H. et al. Acadesine inhibits tissue factor induction and thrombus formation by activating the phosphoinositide 3-kinase/Akt signalling pathway. Arterioscler Thromb Vasc Biol 2010; 30: 1000-1006.
    CrossrefPubMedGoogle Scholar
  • 9 Li YD, Ye BQ, Zheng SX. et al. NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis. J Biol Chem 2009; 284: 4473-4483.
    CrossrefPubMedGoogle Scholar
  • 10 Johnson GJ, Leis LA, Bach RR. Tissue factor activity of blood mononuclear cells is increased after total knee arthroplasty. Thromb Haemost 2009; 102: 728-734.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 11 Ettelaie C, Collier ME, Mei MP. et al. Enhanced binding of tissue factor-micro-particles to collagen-IV and fibronectin leads to increased tissue factor activity in vitro. Thromb Haemost 2013; 109: 61-71.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 12 Schabbauer G, Tencati M, Pedersen B. et al. PI3K–Akt pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler Thromb Vasc Biol 2004; 24: 1963-1969.
    CrossrefPubMedGoogle Scholar
  • 13 Guha M, Mackman N. The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signalling pathways and expression of inflammatory mediators in human monocytic cells. J Biol Chem 2002; 277: 32124-32132.
    CrossrefPubMedGoogle Scholar
  • 14 Kim I, Oh JL, Ryu YS. et al. Angiopoietin-1 negatively regulates expression and activity of tissue factor in endothelial cells. FASEB J 2002; 16: 126-128.
    CrossrefPubMedGoogle Scholar
  • 15 Zhang WJ, Wei H, Hagen T. et al. Alpha-lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signalling pathway. Proc Natl Acad Sci USA 2007; 104: 4077-4082.
    CrossrefPubMedGoogle Scholar
  • 16 Yu M, Ge C, Zeng W. et al. Retinoic acid promotes proliferation of chicken primordial germ cells via activation of PI3K/Akt-mediated NF-kappaB signalling cascade. Cell Biol Int 2012; 36: 705-712.
    CrossrefPubMedGoogle Scholar
  • 17 Calaminus SD, Auger JM, McCarty OJ. et al. MyosinIIa contractility is required for maintenance of platelet structure during spreading on collagen and contributes to thrombus stability. J Thromb Haemost 2007; 05: 2136-2145.
    CrossrefPubMedGoogle Scholar
  • 18 Liu Z, van Grunsven LA, Van Rossen E. et al. Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells. Br J Pharmacol 2010; 159: 304-315.
    CrossrefPubMedGoogle Scholar
  • 19 Maravillas-Montero JL, Santos-Argumedo L. The myosin family: unconventional roles of actin-dependent molecular motors in immune cells. J Leukoc Biol 2012; 91: 35-46.
    CrossrefPubMedGoogle Scholar
  • 20 Lv Y, Lu S, Lu T. et al. Homology model of nonmuscle myosin heavy chain IIA and binding mode analysis with its inhibitor blebbistatin. J Mol Model 2013; 19: 1801-1810.
    CrossrefPubMedGoogle Scholar
  • 21 Clark K, Middelbeek J, Dorovkov MV. et al. The alpha-kinases TRPM6 and TRPM7, but not eEF-2 kinase, phosphorylate the assembly domain of myosin IIA, IIB and IIC. FEBS lett 2008; 582: 2993-2997.
    CrossrefPubMedGoogle Scholar
  • 22 Léon C, Hechler B, Aleil B. et al. Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion. Blood 2007; 110: 3183-3191.
    CrossrefPubMedGoogle Scholar
  • 23 Ono A, Westein E, Hsiao S. et al. Identification of a fibrin-independent platelet contractile mechanism regulating primary hemostasis and thrombus growth. Blood 2008; 112: 90-99.
    CrossrefPubMedGoogle Scholar
  • 24 Chandrasekharan UM, Dechert L, Davidson UI. et al. Release of Nonmuscle Myosin II from the Cytosolic Domain of Tumor Necrosis Factor Receptor 2 Is Required for Target Gene Expression. Sci Signal 2013; 06: ra60.
    PubMedGoogle Scholar
  • 25 Betapudi V, Lominadze G, Hsi L. et al. Anti-beta2GPI antibodies stimulate endothelial cell microparticle release via a nonmuscle myosin II motor protein-dependent pathway. Blood 2013; 122: 3808-3817.
    CrossrefPubMedGoogle Scholar
  • 26 Sun Q, Chen L, Gao M. et al. Ruscogenin inhibits lipopolysaccharide-induced acute lung injury in mice: involvement of tissue factor, inducible NO synthase and nuclear factor (NF)-kappaB. Int immunopharmacol 2012; 12: 88-93.
    CrossrefPubMedGoogle Scholar
  • 27 Gullberg M, Andersson A-C. Visualization and quantification of protein-protein interactions in cells and tissues. Nat Methods 2010; 07: 641-647.
    PubMedGoogle Scholar
  • 28 Diaz JA, Obi AT, Myers Jr. DD. et al. Critical review of mouse models of venous thrombosis. Arterioscler Thromb Vasc Biol 2012; 32: 556-562.
    CrossrefPubMedGoogle Scholar
  • 29 Wrobleski SK, Farris DM, Diaz JA. et al. Mouse complete stasis model of inferior vena cava thrombosis. J Vis Exp 2011; 2738.
    PubMedGoogle Scholar
  • 30 Straight AF, Cheung A, Limouze J. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science 2003; 299: 1743-1747.
    CrossrefPubMedGoogle Scholar
  • 31 Kovacs M, Toth J, Hetenyi C. et al. Mechanism of blebbistatin inhibition of myosin II. J Biol Chem 2004; 279: 35557-35563.
    CrossrefPubMedGoogle Scholar
  • 32 Di Santo A, Amore C, Dell’Elba G. et al. Glycogen synthase kinase-3 negatively regulates tissue factor expression in monocytes interacting with activated platelets. J Thromb Haemost 2011; 09: 1029-1039.
    CrossrefPubMedGoogle Scholar
  • 33 Breitenstein A, Camici GG, Tanner FC. Tissue factor: beyond coagulation in the cardiovascular system. Clin Sci 2010; 118: 159-172.
    PubMedGoogle Scholar
  • 34 Eisenreich A, Malz R, Pepke W. et al. Role of the phosphatidylinositol 3-kinase/ protein kinase B pathway in regulating alternative splicing of tissue factor mRNA in human endothelial cells. Circ J 2009; 73: 1746-1752.
    CrossrefPubMedGoogle Scholar
  • 35 Risbud MV, Fertala J, Vresilovic EJ. et al. Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signalling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal. Spine 2005; 30: 882-889.
    CrossrefPubMedGoogle Scholar
  • 36 Witkowski M, Weithauser A, Rauch U. Tissue Factor-a Link Between Vascular Procoagulability and Inflammation. Exp Clin Cardiol 2014; 20: 1-7.
    PubMedGoogle Scholar
  • 37 Sorensen BB, Rao LV, Tornehave D. et al. Antiapoptotic effect of coagulation factor VIIa. Blood 2003; 102: 1708-1715.
    CrossrefPubMedGoogle Scholar
  • 38 Meng F, Liu L, Chin PC. et al. Akt is a downstream target of NF-kappa B. J Biol Chem 2002; 277: 29674-29680.
    CrossrefPubMedGoogle Scholar
  • 39 Molor-Erdene P, Okajima K, Isobe H. et al. Inhibition of lipopolysaccharide-induced tissue factor expression in monocytes by urinary trypsin inhibitor in vitro and in vivo. Thromb Haemost 2005; 94: 136-145.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 40 Martin M, Rehani K, Jope RS. et al. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol 2005; 06: 777-784.
    PubMedGoogle Scholar
  • 41 Chandrasekharan UM, Siemionow M, Unsal M. et al. Tumor necrosis factor alpha (TNF-alpha) receptor-II is required for TNF-alpha-induced leukocyte-endothelial interaction in vivo. Blood 2007; 109: 1938-1944.
    CrossrefPubMedGoogle Scholar
  • 42 Chandrasekharan UM. Decreased atherosclerosis in mice deficient in tumor necrosis factor-alpha receptor-II (p75). Arterioscler Thromb Vasc Biol 2007; 27: e16-17.
    CrossrefPubMedGoogle Scholar
  • 43 Pircher J, Merkle M, Wornle M. et al. Prothrombotic effects of tumor necrosis factor alpha in vivo are amplified by the absence of TNF-alpha receptor subtype 1 and require TNF-alpha receptor subtype 2. Arthritis Res Ther 2012; 14: R225.
    CrossrefPubMedGoogle Scholar
  • 44 Diaz JA, Alvarado CM, Wrobleski SK. et al. The electrolytic inferior vena cava model (EIM) to study thrombogenesis and thrombus resolution with continuous blood flow in the mouse. Thromb Haemost 2013; 109: 1158-1169.
    Article in Thieme ConnectPubMedGoogle Scholar
  • 45 Zhou J, May L, Liao P. et al. Inferior vena cava ligation rapidly induces tissue factor expression and venous thrombosis in rats. Arterioscler Thromb Vasc Biol 2009; 29: 863-869.
    CrossrefPubMedGoogle Scholar
  • 46 Mikulich A, Kavaliauskiene S, Juzenas P. Blebbistatin, a myosin inhibitor, is phototoxic to human cancer cells under exposure to blue light. Biochim Biophys Acta 2012; 1820: 870-877.
    CrossrefPubMedGoogle Scholar
  • 47 Si J, Ge Y, Zhuang SG. et al. Inhibiting nonmuscle myosin II impedes inflammatory infiltration and ameliorates progressive renal disease. Lab Invest 2010; 90: 448-458.
    CrossrefPubMedGoogle Scholar
  • 48 Swift LM, Asfour H, Posnack NG. et al. Properties of blebbistatin for cardiac optical mapping and other imaging applications. Eur J Physiol 2012; 464: 503-512.
    CrossrefPubMedGoogle Scholar
  • 49 Kolega J. Phototoxicity and photoinactivation of blebbistatin in UV and visible light. Biochem Biophys Res Commun 2004; 320: 1020-1025.
    CrossrefPubMedGoogle Scholar