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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 食品暨應用生物科技學系 > 依資料類型分類 > 碩博士論文 >  2-去氧葡萄糖可藉由弱化人類臍靜脈內皮細胞中的血管內皮生長因子受器-2及整合素β1所調控的訊息傳遞路徑而抑制血管新生作用

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/153194

標題: 2-去氧葡萄糖可藉由弱化人類臍靜脈內皮細胞中的血管內皮生長因子受器-2及整合素β1所調控的訊息傳遞路徑而抑制血管新生作用
2-Deoxyglucose inhibits angiogenesis through attenuating vascular endothelial growth factor receptor 2- and integrin β1-mediated signaling pathway in human umbilical vascular endothelial cells
作者: 莊宜臻
Chuang, I-Chen
Contributors: 胡淼琳
Miao-Lin Hu
食品暨應用生物科技學系所
關鍵字: 血管新生;2-去氧葡萄糖;人類臍靜脈內皮細胞;整合素β1;蛋白質糖基化作用;血管內皮生長因子受器-2
angiogenesis;2-deoxyglucose;human umbilical vascular endothelial cells;integrin β1;N-linked glycosylation;vascular endothelial growth factor receptor-2
日期: 2013
Issue Date: 2013-11-18 14:15:14 (UTC+8)
Publisher: 食品暨應用生物科技學系所
摘要: 血管新生為在既有的血管中衍生出新血管的過程,在許多生理及病理過程中扮演重要的角色。血管內皮生長因子受器-2 (vascular endothelial growth factor receptor-2, VEGFR2)為存在於血管內皮細胞上的一種專一性酪胺酸激酶受器,與血管內皮細胞的增生、侵襲及移行能力有關。整合素β1 (integrin β1)為表現在細胞膜上的細胞黏附分子,對於內皮細胞的移行、增生以及分化為不可缺少的要件。2-去氧葡萄糖(2-deoxyglucose, 2-DG)為人工合成非代謝性葡萄糖類似物,它能與葡萄糖及甘露糖競爭,分別干擾糖解作用與蛋白質糖基化作用。研究指出,2-DG可藉由干擾蛋白質糖基化作用,導致內質網產生非摺疊蛋白反應(unfolded protein response),促使人類臍靜脈內皮細胞(human umbilical vascular endothelial cells, HUVECs)凋亡,而抑制血管新生。然而,2-DG的抗血管新生機制,包含是否可經由弱化VEGFR-2及整合素β1所調控的訊息傳遞路徑,而抑制血管內皮細胞降解基底膜、移行和分化能力,進而抑制血管新生,目前仍不清楚。因此,本研究利用大鼠動脈環與HUVECs細胞為試驗模式,探討2-DG對於大鼠動脈環新血管生成的作用,並利用HUVECs釐清其相關分子機制。結果顯示,大鼠動脈環與2-DG (0.05-1 mM)培養7天,可顯著抑制新血管的生成,其最佳濃度為1 mM,抑制率為40% (P < 0.05)。細胞試驗部分,以2-DG (0.1-1 mM)與HUVECs預培養24小時,可顯著抑制其脈管生成與細胞侵襲、移行及黏附能力,並且具有濃度效應,其最佳濃度為1 mM,抑制率分別為50%、58%、55%以及26% (P < 0.05)。此外,2-DG (1 mM)可顯著抑制VEGFR-2 mRNA與蛋白質表現量(同時蛋白分子量有向低分子量移動的趨勢)以及抑制整合素β1的蛋白質表現。分子機制部份,2-DG可顯著抑制VEGFR-2及整合素β1所調控的分子,包括: (1)黏著斑激酶(focal adhesion kinase, FAK)的磷酸化作用,而抑制磷脂肌醇激酶(phosphatidylinositide 3-kinases, PI3K)的蛋白質表現與絲胺酸激酶(Akt)的磷酸化作用,進而抑制Rho small GTPase (Rho及Rac)的蛋白質表現;(2) p38的磷酸化作用;(3)基質金屬蛋白酶-2 (matrix metalloproteinase-2, MMP-2) mRNA、蛋白質表現量與活性,以及(4)抑制基質金屬蛋白酶內生性抑制劑-2 (tissue inhibitor of metalloproteinase-2, TIMP-2)蛋白表現量。此外,甘露糖(2 mM)可顯著回復2-DG所抑制的脈管生成能力、MMP-2活性以及VEGFR-2與整合素β1的蛋白質表現。最後以免疫螢光染色法觀察2-DG對於VEGFR2在HUVECs中分布的影響,結果顯示2-DG (1 mM)與HUVECs預培養24小時後,會造成VEGFR2累積在細胞內質網上,而甘露糖(2 mM)可明顯逆轉VEGFR2的累積。以上結果指出,2-DG具有抑制血管新生的能力,其機制包含了弱化VEGFR2及整合素β1所調控的訊息路徑以及干擾蛋白質糖基化作用。
Angiogenesis, the process of vascular growth by sprouting of pre-existing vessels, plays an important role in many physiological and pathological processes. Vascular endothelial growth factor receptor-2 (VEGFR2) is a specific tyrosine kinase receptor and is involved in cell proliferation, invasion and migration in vascular endothelial cells. Intergrin β1, a cell adhesion molecule that is expressed on the endothelial cell surface, plays an important role in cell migration, proliferation, differentiation and survival of endothelium. 2-Deoxyglucose (2-DG), a synthetic non-metabolizable analogue of glucose, can interfere with glycolysis and N-linked glycosylation by competition with glucose and mannose metabolism, respectively. The interference of 2-DG with N-linked glycosylation has been shown to induce unfolded protein response in endoplasmic reticulum resulting in promotion of apoptosis and inhibition of angiogenesis in human umbilical vascular endothelial cells (HUVECs). However, the anti-angiogenic mechanisms of 2-DG are not well-investigated, it is unclear whether the anti-angiogenic activity of 2-DG is related to VEGFR2- and integrin β1-mediated pathways involved in extracellular matrix (ECM) degradation, cell migration and differentiation in vascular endothelial cells. In this study, we employed an ex vivo rat aortic rings assay to substantiate the anti-angiogenic action of 2-DG and investigated the molecular mechanism underlying such actions in HUVECs. Results reveal that rat aortic rings treated with 2-DG (0.05-1 mM) for 7 d resulted in a significant inhibition of new blood formation, with an inhibition of 40% (P < 0.05) at 1 mM of 2-DG. 2-DG (0.1-1 mM) also significantly inhibited tube formation, invasion, migration and adhesion of HUVECs in a concentration-dependent manner at 24 h of incubation, with an inhibition of 50%, 58%, 55% and 26% (P < 0.05) at 1 mM 2-DG, respectively. In addition, 2-DG significantly inhibited mRNA and protein expression of VEGFR2 as well as protein expression of integrin β1. Meanwhile, VEGFR2 tended to shift to low molecular weight position by 2-DG treatment. Mechanistically, 2-DG significantly inhibited VEGFR2- and integrin β1-mediated signaling pathways, such as: (1) phosphorylation of focal adhesion kinase (FAK) followed by attenuated PI3K-Akt axis, leading to inhibited protein expression of Rho small GTPase (Rho and Rac1); (2) phosphorylation of p38; (3) mRNA, protein expression and activity of matrix metalloproteinase-2 (MMP-2) and (4) protein expression of tissue inhibitor of metalloproteinase-2 (TIMP-2). In addition, mannose (2 mM) reversed the effects of 2-DG on tube formation, MMP-2 avtivity as well as on VEGFR2 and integrin β1 protein expression in HUVECs. We then employed immunofluorescence assay to illustrate the effects of 2-DG on VEGFR2 distribution in HUVECs. Results reveal that pre-incubation of HUVECs with 2-DG (1 mM) for 24 h clearly induced accumulation of VEGFR2 on endoplasmic reticulum, whereas co-incubation with mannose (2 mM) significantly reversed such an accumulation. In conclusion, the present study demonstrats that 2-DG has anti-angiogenic activity which involves attenuating of VEGFR2- and integrin β1-mediated signaling pathways as well as interference of N-linked glycosylation.
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