蛇毒蛋白Echispydin對血管平滑肌細胞增生與移行之抑制作用及機轉之探討

The inhibitory effects and action mechanism of Echispydin on proliferation and migration of vascular smooth muscle cells

張博雁(Po-Yen Chang)

指導教授 ： 黃德富

國立臺灣大學/醫學院/藥理學研究所/碩士(2014年)

https://doi.org/10.6342/NTU.2014.00465

摘要

心導管冠狀動脈血管氣球擴張術 (percutaneous coronary intervention，PTCA) 後所造成的血管再狹窄 (restenosis) 與血小板活化產生血栓以及血管平滑肌細胞異常地過度生長與移行有關。血小板衍生因子 (platelet-derived growth factor，PDGF) 在這過程中被認為是一個重要的刺激因子。Echispydin是一種從來自 Echis pyramidium 的蛇毒蛋白中純化出的dimeric disintegrin, 在本研究中證實在體外可濃度相關地抑制由collagen，thrombin以及U46619所以引發的人類血小板凝集。此外，藉由受體結合位的競爭實驗中發現 Echispydin 與 GPIIb/IIIa 拮抗劑 7E3 在血小板上有類似的結合部位而與10E5在不同的部位，並且在大鼠平滑肌細胞中可拮抗 integrin β3 的 mAb F11有競爭結合位的現象，因此證明 Echispydin 不只作用在血小板 GPIIb/IIIa 上，也會結合到老鼠平滑肌細胞上的αvβ3 integrin。我們進而觀察到 Echispydin 在老鼠平滑肌細胞上可濃度相關地抑制 PDGF 與 10% FBS 所引起的血管平滑肌細胞增生與移行，且在PDGF的組別中抑制效果明顯大於10% FBS的組別，也藉由乳酸脫氫酶釋放實驗 (LDH release assay) 確認 Echispydin 的這些作用並非來自於它的細胞毒性。此外，我們利用PI染色來分析 Echispydin 對細胞週期分布的影響，觀察到在 PDGF 組隨著 Echispydin 劑量上升，有輕微的 sub-G1 累積並且有 G1/S phase transition 停止的現象。由於在 PDGF 組別中發現 Echispydin 造成明顯的細胞型態改變與細胞脫離，所以我們進一步研究 Echispydin 對於 PDGF 所引起之 F-actin 形成之影響，發現 Echispydin 會抑制 PDGF 所引起之 F-actin 的形成也發現 Echispydin 會抑制平滑肌細胞貼附到 fibrinogen 與 fibronectin 上。接著我們研究 Echispydin 對於 PDGF 所引起的訊號傳遞之影響，結果顯示 Echispydin 會抑制 PDGF 引起的 PDGFRβ、PLCγ1、FAK、c-Src 與 MAPKs (p38、ERK1/2、JNK1/2) 在大鼠主動脈平滑肌細胞的磷酸化，但不影響 PI3K 和 Akt 的磷酸化。此外，Echispydin 也會抑制 PDGF 刺激後和細胞週期相關的 Cyclin D1/2 以及抑制和細胞骨架相關的 small GTPase, Rho的活化。綜合以上結果我們認為 Echispydin 抑制 PDGFRβ 下游的訊息傳導的原因是藉由結合到 αvβ3 integrin 上而抑制了 PDGFRβ 與 αvβ3 integrin 間的 crosstalk。基於 Echispydin 具有抗血小板凝集與抑制平滑肌細胞生長與移行的能力，我們認為其有治療動脈損傷後血管再狹窄的療效潛力。

關鍵字

Disintegrin ； 血管平滑肌細胞 ； 血小板衍生因子 ； αvβ3 integrin ； 血管再狹窄

並列摘要

Restenosis caused by percutaneous coronary intervention (PTCA) is related to thrombosis induced by platelet activation and abnormal growth and migration of vascular smooth muscle cells. Platelet-derived growth factor (PDGF) is considered as an essential stimulation factor in this process. Echispydin is a dimeric disintegrin purified from the snake venom of Echis pyramidium and in this study, it concentration-dependently inhibited human platelet aggregation induced by collagen, thrombin, or U46619 in vitro. According to the binding analysis, Echispydin and 7E3 may bind to a common epitope which is different from that of 10E5 on platelet, and Echispydin decreased the binding of mAb F11 raised against β3 integrin on rat arotic smooth muscle cell, confirming that Echispydin not only binds to GPIIb/IIIa on platelet but also binds to integrin αvβ3 on rat arotic smooth muscle cell. Echispydin inhibited PDGF and 10% FBS induced proliferation and migration of smooth muscle cell in a concentration-dependent manner and the inhibitory effect of Echispydin on PDGF induced condition was more pronounced than 10% FBS induced condition. We also confirmed that these effects are not due to its cytotoxicity by LDH release assay. Moreover, we analyzed the effect of Echispydin on cell cycle progression by PI staining and the results indicated that Echispydin treated cells under PDGF stimulation exhibited a slightly increase of sub-G1 phase and an arrest of G1/S phase transition in a concentration-dependent manner. Since we observed that Echispydin caused significant morphological change and cell detachment on smooth muscle cell under PDGF stimulation, so we further investigated the effect of Echispydin on PDGF-induced F-actin formation, and results showed that Echispydin inhibited PDGF-induced the formation of F-actin. Additionally, it also inhibited smooth muscle cell adhesion to fibrinogen and fibronectin. Echispydin inhibited PDGF-induced phosphorylation of PDGFRβ, PLCγ1, FAK, c-Src and MAPKs (p38, ERK1/2, JNK1/2) in rat aortic smooth muscle cells, but did not affect the phosphorylation of PI3K and Akt. Furthermore, Echispydin also inhibited cell cycle related molecules, Cyclin D1/2 and inhibited cytoskeleton-related small GTPase, Rho activation after PDGF stimulation. These data suggest that Echispydin inhibited PDGFRβ downstream signaling by binding to αvβ3 integrin and then inhibited crosstalk between PDGFRβ and αvβ3 integrin. Therefore, we consider Echispydin as a potent reagent for the prevention of restenosis based on its anti-platelet activity and inhibitory effects on proliferation and migration of smooth muscle cell.

並列關鍵字

Disintegrin ； Vascular smooth muscle cell ； PDGF ； αvβ3 integrin ； Restenosis

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