Wang Feng-Sheng, Wang Ching-Jen, Chen Yeung-Jen, Chang Per-Rong, Huang Yu-Ting, Sun Yi-Chih, Huang Hueng-Chen, Yang Ya-Ju, Yang Kuender D
Department of Medical Research, Chang Gung Memorial Hospital, Kaohsiung 833, Taiwan.
J Biol Chem. 2004 Mar 12;279(11):10331-7. doi: 10.1074/jbc.M308013200. Epub 2003 Dec 16.
Vascular endothelial growth factor (VEGF) released by osteoblasts plays an important role in angiogenesis and endochondral ossification during bone formation. In animal studies, we have reported that shock waves (SW) can promote osteogenic differentiation of mesenchymal stem cells through superoxide-mediated signal transduction (Wang, F. S., Wang, C. J., Sheen-Chen, S. M., Kuo, Y. R., Chen, R. F., and Yang, K. D. (2002) J. Biol. Chem. 277, 10931-10937) and vascularization of the bone-tendon junction. Here, we found that SW elevation of VEGF-A expression in human osteoblasts to be mediated by Ras-induced superoxide and ERK-dependent HIF-1alpha activation. SW treatment (0.16 mJ/mm(2), 1 Hz, 500 impulses) rapidly activated Ras protein (15 min) and Rac1 protein (30 min) and increased superoxide production in 30 min and VEGF mRNA expression in 6 h. Early scavenging of superoxide, but not nitric oxide, peroxide hydrogen, or prostaglandin E(2), reduced SW-augmented VEGF-A levels. Inhibition of superoxide production by diphenyliodonium, an NADPH oxidase inhibitor, was found to suppress VEGF-A expression. Transfection of osteoblasts with a dominant negative (S17N) Ras mutant abrogated the SW enhancement of Rac1 activation, superoxide synthesis, and VEGF expression. Further studies demonstrated that SW significantly promoted ERK activation in 1 h and HIF-1alpha phosphorylation and HIF-1alpha binding to VEGF promoter in 3 h. In support of the observation that superoxide mediated the SW-induced ERK activation and HIF-1alpha transactivation, we further demonstrated that scavenging of superoxide by superoxide dismutase and inhibition of ERK activity by PD98059 decreased HIF-1alpha activation and VEGF-A levels. Moreover, culture medium harvested from SW-treated osteoblasts increased vessel number of chick chorioallantoic membrane. Superoxide dismutase pretreatment and anti-VEGF-A antibody neutralization reduced the promoting effect of conditioned medium on angiogenesis. Thus, modulation of redox reaction by SW may have some positive effect on angiogenesis during bone regeneration.
成骨细胞释放的血管内皮生长因子（VEGF）在骨形成过程中的血管生成和软骨内骨化中起重要作用。在动物研究中，我们曾报道冲击波（SW）可通过超氧化物介导的信号转导促进间充质干细胞的成骨分化（Wang, F. S., Wang, C. J., Sheen-Chen, S. M., Kuo, Y. R., Chen, R. F., and Yang, K. D. (2002) J. Biol. Chem. 277, 10931-10937）以及骨-肌腱连接处的血管化。在此，我们发现SW提高人成骨细胞中VEGF-A表达是由Ras诱导的超氧化物和ERK依赖的HIF-1α激活所介导。SW处理（0.16 mJ/mm(2)，1 Hz，500次脉冲）迅速激活Ras蛋白（15分钟）和Rac1蛋白（30分钟），并在30分钟内增加超氧化物生成，在6小时内增加VEGF mRNA表达。早期清除超氧化物，但不是一氧化氮、过氧化氢或前列腺素E(2)，可降低SW增强的VEGF-A水平。发现NADPH氧化酶抑制剂二苯基碘鎓抑制超氧化物生成可抑制VEGF-A表达。用显性负性（S17N）Ras突变体转染成骨细胞可消除SW对Rac1激活、超氧化物合成和VEGF表达的增强作用。进一步研究表明，SW在1小时内显著促进ERK激活，在3小时内促进HIF-1α磷酸化以及HIF-1α与VEGF启动子的结合。为支持超氧化物介导SW诱导的ERK激活和HIF-1α反式激活这一观察结果，我们进一步证明超氧化物歧化酶清除超氧化物以及PD98059抑制ERK活性可降低HIF-1α激活和VEGF-A水平。此外，从SW处理的成骨细胞收获的培养基增加了鸡胚绒毛尿囊膜的血管数量。超氧化物歧化酶预处理和抗VEGF-A抗体中和降低了条件培养基对血管生成的促进作用。因此，SW对氧化还原反应的调节可能对骨再生过程中的血管生成有一些积极作用。
