探討氧化膽固醇促進人類臍帶靜脈內皮細胞血管新生之作用與機轉 Mechanism of oxysterol-induced angiogenesis on human umbilical vein endothelial cells
中文摘要
在動物及人體組織中含有大量形成細胞膜主要成分之膽固醇，膽固醇在體內(in vivo)轉化成膽 酸及荷爾蒙的過程會被氧化形成氧化性膽固醇；在活體外，當膽固醇受到加熱、空氣、光及一些 氧化劑時亦會被氧化形成氧化性膽固醇。氧化性膽固醇在動脈粥樣化(atherogenesis)、細胞毒 性(cytotoxicity)、致突變性(mutagenesis) and 致癌性(carcinogenesis)扮演很重要的角 色。許多文獻中都指出粥狀動脈硬化為氧化性膽固醇對平滑肌細胞與對內皮細胞的毒性傷害所導 致，氧化膽固醇亦會促進發炎反應和細胞壞死(necrosis)、增加發炎抑制及結腸癌等等。
我們發現在細胞存活率試驗(MTT assay)中，低濃度的氧化性膽固醇 7-keto-cholesterol (7-keto)和 Cholesterol-5 alpha, 6 alpha-epoxide (epoxide)會誘發人類臍帶靜脈內皮 細胞的增生，且在體外管柱形成試驗(tube formation)以及 wound healing 試驗中發現 7-keto 和 epoxide 這?種氧化性膽固醇?會誘發人?臍帶靜脈內皮細胞?血管組織的形成以及細 胞的增生與快速遷移。當觀察一些血管新生相關蛋白，如：mitogen-activated protein kinase (MAPK)之 extracellular signal-regulated kinase (ERK)、c-Jun N-terminal kinase (JNK)、endothelial nitric oxide synthase (eNOS)以及 adhension molecule，如：integrin 時發現7-keto 和 epoxide 誘發人?臍帶靜脈內皮細胞?血管組織的形成可能是經由 integrin alpha (v)和 beta 3 蛋白質?調控。除此之外，我們也發現了 7-keto 和 epoxide 可增加 MMP-2 蛋白活性。以上結果證實了，氧化膽固醇中的 7-keto 和 epoxide 有促血管新生作用，
這也是我們首次發現氧化膽固醇與血管新生之間的關係。
當進一步的研究7-keto 和 epoxide 誘發人類臍帶靜脈內皮細胞 integrinalpha (v)和 beta 3 蛋白質的表現是經由哪些訊息傳遞時，我們發現一些 kinase 的抑制劑(inhibitor)以及抗氧化 劑並不能抑制由7-keto 及 epoxide 所誘發之 integrin alpha (v)和 beta 3 蛋白質的表現。由 結果得知MAP kinase kinase (MEK) inhibitor PD98059 (PD, 10 μM)；JNK inhibitor SP600125 (SP, 10 μM)；p38 MAPK inhibitor SB203580 (SB, 10 μM)；PI3K inhibitor LY294002 (LY, 10 μM)；PKC alpha inhibitor G?6976 (G?, 10 nM)；PKC inhibitor rottlerin (rott, 10 μM)；nitric oxide synthase inhibitor L-NAME (NAME, 100 μM)；
antioxidant N-Acetyl Cysteine (NAC) (NAC, 1 mM)；antioxidant Tiron (Tiron, 1 mM)；
nuclear factor-kappa B (NF-kappa B) inhibitor PDTC (PDTC, 25 μM)；peroxisome proliferator activator receptor gamma(PPAR gamma) agonist rosiglitaze (rosi, 10 μ M)；Ca2+ antagonist nifedipine (nifedipine, 50 μM)並不會影響由 7-keto 和 epoxide 所誘發HUVECs integrin alpha (v)和 beta 3 蛋白質表現；故推測 7-keto 和 epoxide 誘發 HUVECs integrin alpha (v)和 beta 3 蛋白質表現非經由 MAPK、phosphatidylinositol 3-kinase (PI3K)、protein kinase C (PKC)等之訊息傳遞路徑。
未來將利用反轉錄-聚合酵素連鎖反應(reverse transcription- polymerase chain
reaction，RT-PCR)來研究 7-keto 和 epoxide 是否會誘發人類臍帶靜脈內皮細胞 integrin alpha (v)和 beta 3 mRNA 的表現，並更近一步的探討 7-keto 和 epoxide 誘發人類臍帶靜脈 內皮細胞integrin alpha (v)和 beta 3 蛋白質的表現是經由哪些過程傳遞訊息。
英文摘要
Sterols are abundant in human and animal tissues, and are essential for the formation and function of cellular membranes. Cholesterol may be oxidized, in part, to oxysterols both during its conversion to bile acids and hormones in vivo and also when exposed to heat, air, light, and oxidizing agents in vitro. Oxysterols play important roles in atherogenesis, cytotoxicity, mutagenesis, and carcinogenesis.
Much evidence indicates that oxysterols have been implicated in the initiation and progression of atherosclerosis due to their toxicity to arterial endothelium and smooth muscle cells. Oxysterols also promote tissue inflammation and necrosis, produce immunosuppression, and enhance colon carcinogenesis.
As an accidenial finding, oxysterol (7-keto and epoxide) in low concentration induced human umbilical vein endothelial cells proliferation. In the tube formation experiment, 7-keto and epoxide induced human umbilical vein endothelial cells tube formation. In the Western blot experiment, we speculated that the mechanisms might be related to integrin alpha v protein and beta 3 protein.
Then, we used the inhibitors and antioxidant to inhibition the 7-keto-induced integrin alpha (v) protein and beta3 protein expression and epoxide-induced integrin alpha(v)protein and beta3 protein expression. The inhibitors and
antioxidant including MAP kinase kinase (MEK) inhibitor PD98059 (PD, 10 μM)；JNK inhibitor SP600125 (SP, 10 μM)；p38 MAPK inhibitor SB203580 (SB, 10 μM)；PI3K inhibitor LY294002 (LY, 10 μM)；PKC alpha inhibitor G?6976 (G?, 10 nM)；PKC inhibitor rottlerin (rott, 10 μM)；nitric oxide synthase inhibitor L-NAME (NAME, 100 μM)；antioxidant N-Acetyl Cysteine (NAC) (NAC, 1 mM)；antioxidant Tiron (Tiron, 1 mM)；nuclear factor-kappa B (NF-kappa B) inhibitor PDTC (PDTC, 25 μM)；
peroxisome proliferator activator receptor gamma(PPAR gamma) agonist rosiglitaze (rosi, 10 μM)；Ca2+ antagonist nifedipine (nifedipine, 50 μM). According to Western blot analysis, we found that the inhibitors and antioxidant did not inhibit 7-keto-induced integrin alpha (v) protein and beta3 protein expression and epoxide-induced integrin alpha(v) protein and beta3 protein expression.
Further investigations are required to clarify the mechanisms of signal pathway on 7-keto-induced integrin (v) protein and beta3 protein expression and
epoxide-induced integrin alpha(v) protein and beta 3 protein expression. In the future, we will use RT-PCR (reverse transcription-polymerase chain reaction) to confirm that effect of 7-keto-induced integrin alpha(v)mRNA expression and beta 3
mRNA expression and epoxide-induced integrin alpha(v)mRNA and beta3 mRNA expression.
