Src blockage by siRNA inhibits VEGF-induced vascular hyperpemeability and osteoclast activity - an in vitro mechanism study for preventing destructive repair of osteonecrosis.
INTRODUCTION
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.
['Cao|Hui-Juan|HJ|', 'Zheng|Li-Zhen|LZ|', 'Wang|Nan|N|', 'Wang|Lin-Ying|LY|', 'Li|Ye|Y|', 'Li|Dan|D|', 'Lai|Yu-Xiao|YX|', 'Wang|Xin-Luan|XL|', 'Qin|Ling|L|']
D000818:Animals D002199:Capillary Permeability D002454:Cell Differentiation D048430:Cell Shape D004730:Endothelium, Vascular D051418:Focal Adhesion Kinase 2 D055785:Gene Knockdown Techniques D061307:Human Umbilical Vein Endothelial Cells D006801:Humans D008297:Male D008810:Mice, Inbred C57BL D010010:Osteoclasts D010020:Osteonecrosis D010766:Phosphorylation D050721:Proto-Oncogene Proteins c-cbl D016392:Proto-Oncogene Proteins pp60(c-src) D034741:RNA, Small Interfering D015398:Signal Transduction D014162:Transfection D042461:Vascular Endothelial Growth Factor A D014945:Wound Healing
Osteoclasts activity Destructive repair Osteonecrosis Vascular permeability VEGF c-Src