Recombinant mouse canstatin inhibits chicken embryo chorioallantoic membrane angiogenesis and endothelial cell proliferation.
Human canstatin, a 24 kD fragment of the alpha2 chain of type IV collagen, has been proved to be one of the most effective inhibitors of angiogenesis and tumor growth. To investigate in vivo antiangiogenesis activity and in vitro effects on endothelial cell proliferation of recombinant mouse canstatin, the cDNA of mouse canstatin was introduced into an expression vector pQE40 to construct a prokaryotic expression vector pQE-mCan. The recombinant mouse canstatin efficiently expressed in E. coli M15 after IPTG induction was monitored by SDS-PAGE and by Western blotting with an anti-hexahistidine tag antibody. The expressed mouse canstatin, mainly as inclusion bodies, accounted for approximately 35% of the total bacterial proteins. The inclusion bodies were washed, lysed and purified by the nickel affinity chromatography to a purity of approximately 93%. The refolded mouse canstatin was tested on the chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. In addition, recombinant mouse canstatin potently inhibited endothelial cell proliferation with no inhibition on non-endothelial cells. Taken together, these findings demonstrate that the recombinant mouse canstatin effectively inhibited angiogenesis of the chicken embryo in a dose-dependent manner and specially suppressed in vitro the proliferation of human umbilical vein endothelial cells.
Hou
Wei-Hong
WH
Laboratory for Cell Biology, Zhengzhou University, Zhengzhou 450052, China.
Wang
Tian-Yun
TY
Yuan
Bao-Mei
BM
Chai
Yu-Rong
YR
Jia
Yan-Long
YL
Tian
Fang
F
Wang
Jian-Min
JM
Xue
Le-Xun
LX
D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002642:Chick Embryo; D049033:Chorioallantoic Membrane / Q000098:blood supply* / Q000187:drug effects; D024141:Collagen Type IV / Q000494:pharmacology*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010446:Peptide Fragments / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology*
D020533:Angiogenesis Inhibitors; C502625:COL4A2 protein, human; C502624:Col4a2 protein, mouse; D024141:Collagen Type IV; D010446:Peptide Fragments; D011994:Recombinant Proteins