Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via Pl 3-kinase/Akt signaling pathways
Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via Pl 3-kinase/Akt signaling pathways
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DOI：
10.1111/j.1745-7254.2006.00335.x
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发表时间：
2006-06-01
影响因子：
8.2
通讯作者：
Zhou, Yu-Feng
中科院分区：
文献类型：
--
作者：
Fu, Jin-Rong;Liu, Wen-Li;Zhou, Yu-Feng
目的：研究Sonic hedgehog（shh）蛋白对骨髓内皮祖细胞（BM-EPC）增殖、迁移和血管内皮生长因子（VEGF）生成的影响，并探讨其可能的信号通路。研究方法：用MACS系统富集成年昆明小鼠骨髓Flk-1+细胞，然后将BM-EPC在明胶包被的培养皿中培养。采用MTT比色法检测shh N端肽对BM-EPC增殖的影响。使用改良的Boy den室技术测定细胞迁移。ELISA法和免疫荧光法检测VEGF的表达。采用选择性抑制剂或Western blot方法探讨PKC和PI 3 K信号通路的可能参与。结果如下：0.1 ~ 10 μ g/mL的内源性shh N端肽能促进BM-EPC的增殖、迁移和VEGF的产生，而抗shh抗体则能抑制其增殖、迁移和VEGF的产生。抗VEGF可部分抑制Shh介导的BM-EPC增殖和迁移。新分离的BM-EPC中磷酸化PI 3激酶的表达较低，加入外源性shh N端肽后磷酸化PI 3激酶的表达显著增加，而抗人/鼠shh N端肽抗体可减弱磷酸化PI 3激酶的表达。此外，PI 3-激酶抑制剂，而不是PKC抑制剂，显着抑制shh介导的增殖，迁移和VEGF的产生。结论：Shh蛋白可刺激骨髓来源的BM-EPC增殖、迁移和VEGF的产生，从而促进缺血组织的新生血管形成。这一结果也表明，PI 3-激酶/Akt信号通路参与了shh的血管生成作用。
Aim: To investigate the effects of Sonic hedgehog (shh) protein on bone marrow-derived endothelial progenitor cells (BM-EPC) proliferation, migration and vascular endothelial growth factor (VEGF) production, and the potential signaling pathways involved in these effects. Methods: Bone marrow-derived Flk-1+ cells were enriched using the MACS system from adult Kunming mice and then BM-EPC was cultured in gelatin-coated culture dishes. The effects of shh N-terminal pep-tide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boy den chamber technique. The production of VEGF was determined by ELISA and immunofluorescence analysis. The potential involvement of PKC and PI3K signaling pathways was explored using selective inhibitor or Western blot. Results: The proliferation, migration and VEGF production in BM-EPC could be promoted by endogenous shh N-terminal peptide at concentrations of 0.1 ug/mL to 10 ug/mL, and could be inhibited by anti-shh antibodies. Shh-mediated proliferation and migration in BM-EPC could be partly attenuated by anti-VEGF. Phospho-PI3 -kinase expression in newly separated BM-EPC was low, and it increased significantly when exogenous shh N-terminal peptide was added, but could be attenuated by anti-human/mouse shh N-terminal peptide antibody. Moreover, the inhibitor of the PI3-kinase, but not the inhibitor of the PKC, significantly inhibited the shh-mediated proliferation, migration and VEGF production. Conclusion: Shh protein can stimulate bone marrow-derived BM-EPC proliferation, migration and VEGF production, which may promote neovascularization to ischemic tissues. This results also suggests that the PI3-kinase/Akt signaling pathways are involved in the angiogenic effects of shh.