key: cord-0034528-aa3914bl
authors: Tang, Tao; Zhang, Ge; Guo, Baosheng; Hung, Leung-kim; Qin, Ling
title: Role of Src in VEGF mediating the activation of rabbit osteoclasts in vitro
date: 2010-10-15
journal: Bone
DOI: 10.1016/j.bone.2010.09.261
sha: 020fbcc7ca09802e2b4ee820ff8aef3b989b8fbc
doc_id: 34528
cord_uid: aa3914bl

nan

Introduction: Pulsed administration of steroids, which was frequently applied for the therapy of serious infectious diseases such as Severe Acute Respiratory Syndrome [1] and Acquired Immune Deficiency Syndrome [2] , often results in osteonecrosis. It was reported that the blockade of Src in mice may induce a defect in osteoclastic bone resorption leading to osteopetrosis [3] . Our previous studies also showed that persistent vascular hyperpermeability with extensive edema occurred during bone destruction within steroid-associated osteonecrotic lesions in a rabbit model [4] , which were simultaneously accompanied with Src phosphorylation [5] . Accordingly, the hypothesis was that Src might play an important role in VEGF induced bone resorption in rabbit osteoclasts. Furthermore, a rabbit Src siRNA with high knock down efficiency have been identified by authors, which would facilitate the test of the hypothesis. Objective: To examine the effect of the silence of Src by siRNA on VEGF mediated bone resorption in rabbit osteoclasts. Materials and methods: VEGF was diluted in PBS. Lipofectamine™ 2000 (Invitrogen) was used as vehicle for siRNA delivery. Twentyfour hours before the treatment of 50 ng/ml VEGF [6] , the rabbit Src siRNA si-1 with vehicle (RNAi-VEGF Group, n = 4) or Non-sense siRNA with vehicle (NC-VEGF Group, n = 4) or vehicle only (VC-VEGF Group, n = 4) was treated for rabbit osteoclasts in vitro. In parallel, 24 h before the treatment of same volume of PBS, the rabbit Src siRNA si-1 with vehicle (RNAi Group, n = 4) or Non-sense siRNA with vehicle (NC Group, n = 4) or vehicle only (VC Group, n = 4) was treated for rabbit osteoclasts in vitro. And then, 48 h after transfection, cells were collected. The mRNA expression level of Src and TRAP were quantified by real-time polymerase chain reaction (PCR), which was to evaluate the knock down efficiency of the Src siRNA and the bone resorption, respectively. Results: The rabbit Src siRNAs significantly reduced the rabbit Src mRNA expression in RNAi-VEGF and RNAi Group. For the bone resorption marker, the mRNA expression of TRAP in NC-VEGF and VC-VEGF Group was significantly higher than that in NC and VC Group. The TRAP mRNA expression in NC and VC Group was significantly higher than that in RNAi-VEGF and RNAi Group. There was no significant difference between NC-VEGF and VC-VEGF Group, no significant difference between NC and VC Group and no significant difference between RNAi-VEGF and RNAi Group on TRAP mRNA expression level. Conclusion: Src could play an important role in VEGF mediating the activation of rabbit osteoclasts in vitro. Introduction: Our previous studies documented that persistent vascular hyperpermeability with extensive edema occurred during bone destruction within steroid-associated osteonecrotic lesions in a rabbit model [1] , which were simultaneously accompanied with Src phosphorylation [2] . It was also reported that VEGF-mediating vascular permeability, which results in the disruption of the complex of Flk, VE-cadherin, and beta-catenin, requires participation of Src phosphorylation [3] [4] . However, the role of Src in VEGF-mediating vascular permeability in the rabbit osteonecrosis model was still not clear. In the present study, authors applied Src siRNA for silencing Src to study its role in VEGF-mediating vascular permeability in the rabbit vascular endothelial cells in vitro. Objective: To examine the effect of the silence of Src by siRNA on vascular hyperpermeability mediated by VEGF in rabbit vascular endothelial cells. Materials and methods: VEGF was diluted in PBS. Lipofectamine™ 2000 (Invitrogen) was used as vehicle for siRNA delivery. Twenty-four hours before the treatment of 50 ng/ml VEGF [5] , the rabbit Src siRNA si-1 with vehicle (RNAi-VEGF Group, n = 4) or Non-sense siRNA with vehicle (NC-VEGF Group, n = 4) or vehicle only (VC-VEGF Group, n = 4) was treated for rabbit osteoclasts in vitro. In parallel, 24 h before the treatment of same volume of PBS, the rabbit Src siRNA si-1 with vehicle (RNAi Group, n = 4) or Non-sense siRNA with vehicle (NC Group, n = 4) or vehicle only (VC Group, n = 4) was treated for rabbit osteoclasts in vitro. And then, 48 h after transfection cells were collected. The mRNA expression level of Src was quantified by real-time polymerase chain reaction (PCR) to evaluate the knockdown efficiency of the Src siRNA. Immunoprecipitation (IP) of Flk and immunoblotting (IB) of Flk, VE-cadherin, and beta-catenin were performed to detect the complex of Flk, VE-cadherin and beta-catenin for analysis of the vascular permeability in the rabbit vascular endothelial cells [4] . Results: The rabbit Src siRNAs significantly reduced the rabbit Src mRNA expression in RNAi-VEGF and RNAi Group. For the evaluation of the vascular permeability, the complex of Flk, VE-cadherin and beta-catenin in NC and VC Group was higher than that in RNAi-VEGF and RNAi Group. The complex of Flk, VE-cadherin and beta-catenin in RNAi-VEGF and RNAi Group was higher than that in NC-VEGF and VC-VEGF Group. There was no difference between NC-VEGF and VC-VEGF Group, no difference between NC and VC Group and no difference between RNAi-VEGF and RNAi Group on the level of RNAi-VEGF and RNAi Group. Conclusion: SRC could play an important role in VEGF mediating vascular permeability in rabbit endothelial cell in vitro.

Development of a standard treatment protocol for severe acute respiratory syndrome

Osteonecrosis in HIV: a case-control study

Rescue of osteoclast function by transgenic expression of kinase-deficient Src in src−/−mutant mice

Continuous occurrence of both insufficient neovascularization and elevated vascular permeability in rabbit proximal femur during inadequate repair of steroid-associated osteonecrotic lesions

A novel semisynthesized small molecule icaritin reduces incidence of steroid-associated osteonecrosis with inhibition of both thrombosis and lipiddeposition in a dose-dependent manner

VEGF enhancement of osteoclast survival and bone resorption involves VEGF receptor-2 signaling and beta3-integrin

Authors would like to thank our collaborators, Wang Xinluan, Xie Xinhui and Zheng Lizhen.

Authors would like to thank our collaborators, Wang Xinluan, Xie Xinhui and Zheng Lizhen.

Abstracts / Bone 47 (2010) S385-S458 S430