Sonic hedgehog protein promotes bone marrow-derived endothelial progenitor cell proliferation, migration and VEGF production via PI 3-kinase/Akt signaling pathways.
OBJECTIVE
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 peptide on BM-EPC proliferation were evaluated using the MTT colorimetric assay. Cell migration was assayed using a modified Boyden 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 microg/mL to 10 microg/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.


CONCLUSIONS
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.
Fu
Jin-Rong
JR
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Liu
Wen-Li
WL
Zhou
Jian-Feng
JF
Sun
Han-Ying
HY
Xu
Hui-Zhen
HZ
Luo
Li
L
Zhang
Heng
H
Zhou
Yu-Feng
YF
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D053823:Hedgehog Proteins / Q000494:pharmacology*; D051379:Mice; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D053823:Hedgehog Proteins; D042461:Vascular Endothelial Growth Factor A; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt