| Summary: | 博士 === 國立成功大學 === 基礎醫學研究所 === 94 === Thrombomodulin (TM), a thrombin receptor on endothelial cell surface, inhibits the procoagulant functions of thrombin and acts as a protein cofactor in thrombin-catalyzed activation of protein C. TM converts pro-coagulant thrombin into an anticoagulant and plays a central role in protein C anticoagulant pathways. Recent studies indicate that TM not only functions as an anticoagulant factor by modulating thrombin’s function. Owing to TM’s widely expression patterns on various cell types, TM may also participate in other physiological functions. In the present study, the DNA fragments of TM domain were subcloned into the Pichia pastoris expression system, and the TM domain (TMD) proteins from ferments were further purified by affinity nickel-chelating column chromatography. Previous study has shown that a recombinant TM domain containing six epidermal growth factor-like structures exhibits mitogenic activity. I explored the novel angiogenic effects of TMD proteins by using in vitro and in vivo models. When thrombin was combined with purified TMD123, TMD23, TMD2, and TMD2/EGF456, respectively, the complex could activate protein C. TMD23 had higher activity than the other TMD proteins in stimulation of DNA synthesis and migration in cultured human umbilical vein endothelial cells (HUVECs). These results suggest that TMD23 is the most potent region of TM for activation of HUVECs. Besides, TMD23 enhanced chemotactic motility and capillary-like tube formation in HUVECs, which was through an effect mediated by 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 the expression of matrix metalloproteinase-3 and inhibited plasminogen activator activity. These steps mediated extracellular proteolysis, which leads to endothelial-cell’s invasion and migration during angiogenesis. In murine angiogenic assays, TMD23 induced neovascularization in Matrigel and enhanced angiogenesis in Matrigel containing melanoma A2058 cells in severe combined immune deficiency mice. Furthermore, in Matrigel assay, TMD23 was a more potent angiogenic activator than TMD123 and TMD1. On the other hand, TMD1 diminished TMD23-induced angiogenic response and HUVEC migration. The results revealed that the biological function of TMD23 was modulated by TMD1 (lectin-like domain). In conclusion, TM not only acts as a natural anticoagulant, but also participates in regulation of angiogenesis by an auto-regulatory manner. TMD23 promoted the angiogenic activities in vitro and in vivo, suggesting that TM fragments may play a role in the formation of new vessels. These findings may provide a new therapeutic option for treating ischemia diseases.
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