title: Src blockage by siRNA inhibits VEGF-induced vascular hyperpemeability and osteoclast activity - an in vitro mechanism study for preventing destructive repair of osteonecrosis.
abstract: BACKGROUND
  Destructive repair is the pathological feature of ONFH characterized with the elevated vascular permeability and persistent bone resorption, which is associated with higher VEGF expression, activated c-Src, and vascular leakage. Activated c-Src also participates in mediating endothelial permeability and osteoclasts activity. However, the molecular mechanism of the VEGF and c-Src contributing to the destructive repair process remains unknown. The purpose of this study is to delineate the role of VEGF and c-Src in triggering destructive repair of osteonecrosis in vitro, as well as to elucidate if VEGF mediating vascular permeability and osteoclastic bone resorption are Src dependent.
  
  
  METHODS
  We employed pharmacological VEGF to induce higher endothelial permeability and osteoclasts activity for simulating related pathological features of destructive repair in vitro. Src specific pp60(c-src)siRNA was used for determining the contribution of VEGF and Src to destructive repair. The primary endothelial cells and osteoclasts were treated with 50ng/ml VEGF and/or transfected with the pp60(c-src)siRNA, while equivalent PBS and non-targeting siRNA were treated in the control groups.
  
  
  RESULTS
  VEGF enhanced Src bioactivity through promoting dephosphorylation of Src at Y527 and phosphorylation of Src at Y416. Meanwhile, Src specific pp60(c-src)siRNA significantly reduced Src expression in both cells. VEGF destroyed the junctional integrity of endothelial cells resulting in higher endothelial permeability. However, Src blockade significantly relieved VEGF induced actin stress and inhibited caveolae and VVOs formation, meanwhile further stabilized the complex β-catenin/VE-cadherin/Flk-1 through decreasing phosphorylation of VE-cadherin, ultimately decreasing VEGF-mediating higher vascular permeability. In addition, VEGF promoted osteoclasts formation and function without affecting the adhesion activity and cytoskeleton. We further found that Src blockade significantly impaired cytoskeleton resulting in a lower adhesion activity through down-regulation of phosphorylation of Src, Pyk2 and Cbl, and ultimately inhibited osteoclasts formation and function.
  
  
  CONCLUSIONS
  These findings provide a new insight into VEGF and c-Src mode of reaction in triggering destructive repair of osteonecrosis and further indicate that VEGF mediating vascular permeability and osteoclasts activity are Src-dependent. Blockade of Src may have great potential as an effective therapy targeting destructive repair in osteonecrosis.
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China. Electronic address: hj.cao@siat.ac.cn.
authors.forename: Hui-Juan
authors.initials: HJ
authors.lastname: Cao
authors.affiliation: Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong SAR, PR China. Electronic address: zhenglz@ort.cuhk.edu.hk.
authors.forename: Li-Zhen
authors.initials: LZ
authors.lastname: Zheng
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China. Electronic address: wangnan@siat.ac.cn.
authors.forename: Nan
authors.initials: N
authors.lastname: Wang
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China; Nano Science and Technology Institute, The University of Science and Technology of China, Suzhou, PR China. Electronic address: wangliny@siat.ac.cn.
authors.forename: Lin-Ying
authors.initials: LY
authors.lastname: Wang
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China. Electronic address: ye.li1@siat.ac.cn.
authors.forename: Ye
authors.initials: Y
authors.lastname: Li
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China. Electronic address: danli@siat.ac.cn.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China. Electronic address: yx.lai@siat.ac.cn.
authors.forename: Yu-Xiao
authors.initials: YX
authors.lastname: Lai
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong SAR, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China. Electronic address: xl.wang@siat.ac.cn.
authors.forename: Xin-Luan
authors.initials: XL
authors.lastname: Wang
authors.affiliation: Translational Medicine R&D Center, Institute of Biomedical and Health Engineering, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, PR China; Musculoskeletal Research Laboratory, Department of Orthopaedics & Traumatology, The Chinese University of Hong Kong, Hong Kong SAR, PR China; Shenzhen Bioactive Materials Engineering Lab for Medicine, Shenzhen, PR China. Electronic address: lingqin@cuhk.edu.hk.
authors.forename: Ling
authors.initials: L
authors.lastname: Qin
mesh_terms: D000818:Animals; D002199:Capillary Permeability / Q000187:drug effects*; D002454:Cell Differentiation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism; D055785:Gene Knockdown Techniques; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010020:Osteonecrosis / Q000473:pathology / Q000503:physiopathology* / Q000517:prevention & control*; D010766:Phosphorylation / Q000187:drug effects; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology*; D014945:Wound Healing / Q000187:drug effects
chemical_list: D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D050721:Proto-Oncogene Proteins c-cbl; D051418:Focal Adhesion Kinase 2; D016392:Proto-Oncogene Proteins pp60(c-src); C496053:Ptk2b protein, mouse
keywords: Destructive repair; Osteoclasts activity; Osteonecrosis; VEGF; Vascular permeability; c-Src