| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 91 === Angiogenesis, the sprouting of new capillaries from established vessels, occurs physiologically during tissue growth, wound healing, and female reproductive cycle. Because tumor growth and metastasis are also angiogenesis-dependent, anti-angiogenesis is a strategy for cancer therapy. Angiostatin, the first four kringles of plasminogen, is an endogenous inhibitor of angiogenesis. Pervious studies in our laboratory found two plasminogen fragments, K420-436 and K420-448, which exhibited more remarkable anti-proliferative and anti-migratory activities than K420-461 (angiostatin). The purpose of this study was to further elucidate the mechanism of how they inhibit angiogenesis. Plasminogen fragments were expressed in Pichia pastoris and purified by anion exchange (DEAE) and lysine sepharose. Molecular weights of these proteins were larger than predict ones due to glycosylation and most of these saccharides attached to plasminogen fragments were mannose. The receptors for these proteins were studied in attempt to realize their action. ATP synthase, which was believed to be a receptor for angiostatin on the surface of human umbilical vein endothelial cells (HUVECs), did not exist on the calf pulmonary artery endothelial cell (CPAE) surface. Because the morphological changes, cell rounded and detached, were observed after treatment, integrins were suspected to be receptors for plasminogen fragments. The expression of a1, a2, b1 and avb3 integrins on CPAE cells was examined and all of them were constitutively expressed except a2. Integrin avb3, which had a high affinity for RGD (Arg-Gly-Asp) sequence-containing proteins, was a fundamental protein in angiogenesis so it was further studied whether they bound to plasminogen fragments. Cells pre-incubated with anti-avb3 antibody or RGD peptides could not adhere to immobilized plasminogen fragments anymore. The results indicated plasminogen fragments were bound to CPAE cells through avb3 in an RGD-dependent manner despite that there is no RGD sequence in plasminogen fragments. Following treatment with plasminogen fragments, CPAE cells underwent apoptosis (anoikis) but not non-endothelial cells (A549) and the apoptotic effects of K420-436 and K420-448 were much higher than K420-461. The apoptosis of CPAE cells could completely be abolished by pre-treatment with a broad caspase inhibitor (zVAD-FMK) indicating caspases played a fundamental role in the plasminogen fragments- induced apoptosis. In the apoptotic pathway, caspase 9 and caspase 3 were greatly activated and cytosolic levels of cytochrome c increased.
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