title: NMMHC IIA inhibition impedes tissue factor expression and venous thrombosis via Akt/GSK3β-NF-κB signalling pathways in the endothelium.
abstract: 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.
authors.forename: Kefeng
authors.initials: K
authors.lastname: Zhai
authors.forename: Youmei
authors.initials: Y
authors.lastname: Tang
authors.forename: Yuanyuan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Zhengyu
authors.initials: Z
authors.lastname: Cao
authors.affiliation: Dr. Jun Yu, Department of Internal Medicine, Section of Cardiovascular Medicine, Yale University School of Medicine, New Haven, CT 06519, USA, Tel.: +1 203 7372869, Fax: +1 203 7372290, E-mail: jun.yu@yale.edu.
authors.forename: Jun
authors.initials: J
authors.lastname: Yu
authors.affiliation: Dr. Junping Kou, State Key Laboratory of Natural Products, Jiangsu Key Laboratory of TCM Evaluation and Translational Research, Department of Complex Prescription of TCM, China Pharmaceutical University, 639 Longmian Road, Nanjing, 211198, P. R. China, Tel./Fax: +86 25 86185158, E-mail: junpingkou@cpu.edu.cn.
authors.forename: Junping
authors.initials: J
authors.lastname: Kou
authors.forename: Boyang
authors.initials: B
authors.lastname: Yu
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects* / Q000201:enzymology; D005343:Fibrinolytic Agents / Q000494:pharmacology*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071679:Glycogen Synthase Kinase 3 beta; D057809:HEK293 Cells; D006576:Heterocyclic Compounds, 4 or More Rings / Q000494:pharmacology*; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism; D008810:Mice, Inbred C57BL; D020409:Molecular Motor Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D024507:Nonmuscle Myosin Type IIA / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034622:RNA Interference; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013925:Thromboplastin / Q000378:metabolism*; D051996:Transcription Factor RelA / Q000378:metabolism; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D020246:Venous Thrombosis / Q000378:metabolism / Q000517:prevention & control*
chemical_list: D005343:Fibrinolytic Agents; D006576:Heterocyclic Compounds, 4 or More Rings; D020672:I-kappa B Proteins; C103814:MYH9 protein, human; D020409:Molecular Motor Proteins; C525088:Myh9 protein, mouse; D016328:NF-kappa B; D047428:Protein Kinase Inhibitors; C498143:RELA protein, human; D047889:Receptors, Tumor Necrosis Factor, Type II; C415902:TNFRSF1B protein, human; D051996:Transcription Factor RelA; D014409:Tumor Necrosis Factor-alpha; C472645:blebbistatin; D013925:Thromboplastin; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D024507:Nonmuscle Myosin Type IIA; D018995:Myosin Heavy Chains
keywords: NMMHC IIA; endothelium; signalling transduction; tissue factor; venous thrombosis