Evidence of human thrombomodulin domain as a novel angiogenic factor.
BACKGROUND
Thrombomodulin is an anticoagulant, endothelial-cell-membrane glycoprotein. A recombinant thrombomodulin domain containing 6 epidermal growth factor-like structures exhibits mitogenic activity. This study explored the novel angiogenic effects of the recombinant domain using in vitro and in vivo models.


RESULTS
Human recombinant thrombomodulin containing 6 epidermal growth factor-like structures (TMD2) and TMD2 plus a serine and threonine-rich domain (TMD23) were prepared using the Pichia pastoris expression system. Combined with purified TMD2 or TMD23, thrombin effectively activated protein C. TMD23 had higher activity than TMD2 in stimulating DNA synthesis in cultured human umbilical vein endothelial cells. Additionally, TMD23 stimulated chemotactic motility and capillarylike tube formation in human umbilical vein endothelial cells, an effect mediated through phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase and the phosphatidylinositol-3 kinase/Akt/endothelial nitric oxide synthase pathway. TMD23 also stimulated endothelial cell expression of matrix metalloproteinases and plasminogen activators, which mediated extracellular proteolysis, leading to endothelial cell invasion and migration during angiogenesis. Furthermore, TMD23-containing implants in rat cornea induced ingrowth of new blood vessels from the limbus. With the murine angiogenesis assay, TMD23 not only induced neovascularization coinjected with Matrigel and heparin but also enhanced angiogenesis in Matrigel containing melanoma A2058 cells in nude mice.


CONCLUSIONS
The recombinant thrombomodulin domain TMD23 enhanced the angiogenic response in vitro and in vivo, suggesting that thrombomodulin fragments may play a role in the formation of new vessels. These findings may provide a new therapeutic option for treating ischemic diseases.
Shi
Chung-Sheng
CS
Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Republic of China.
Shi
Guey-Yueh
GY
Chang
Yi-Sheng
YS
Han
Huai-Song
HS
Kuo
Cheng-Hsiang
CH
Liu
Che
C
Huang
Huey-Chun
HC
Chang
Yu-Jia
YJ
Chen
Pin-Shern
PS
Wu
Hua-Lin
HL
D043925:Angiogenesis Inducing Agents / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D003315:Cornea / Q000098:blood supply; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D004815:Epidermal Growth Factor; D006801:Humans; D051379:Mice; D009389:Neovascularization, Pathologic / Q000139:chemically induced; D018919:Neovascularization, Physiologic / Q000187:drug effects*; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D011486:Protein C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology*; D018180:Thrombomodulin / Q000737:chemistry / Q000627:therapeutic use*
D043925:Angiogenesis Inducing Agents; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D004815:Epidermal Growth Factor