芴促進人類臍靜脈內皮細胞血管新生作用機制之探討

Abstract

多環芳香烴為普遍存在的環境汙染物，主要來源包括工廠製程排放的氣體、汽機車排放廢氣、抽煙、不當的食物烹煮等，之前文獻也指出多環芳香烴具致癌性、免疫毒性以及與心血管疾病相關。血管新生指新的血管生成，是從現存分布的血管內皮所形成，在多種生理與病理過程中扮演重要的角色，例如傷口癒合、胚胎發育、女性月經週期、糖尿病所引起的視網膜病變、腫瘤生長、轉移以及各種慢性發炎反應等。本實驗室之前發現多環芳香烴會促進血管內皮細胞內eNOS的蛋白表現，並且增加eNOS的活性和一氧化氮的合成量，同時這些多環芳香烴亦會影響細胞內鈣離子之衡定以及誘導細胞黏附分子的表現。而許多文獻也指出一氧化氮、鈣離子濃度的變化及黏附因分子的表現在血管新生中扮演一種媒介的角色，所以在此我們選取具三苯環的芴來探討多環芳香烴對於血管新生方面的影響。 血管新生為一複雜的過程，包括胞外基質分解、細胞黏附、細胞增生、細胞移行以及細胞型態分化成管狀結構。我們以細胞黏附、細胞移行與管狀形成三部份實驗來評估芴對血管新生方面的影響。結果發現芴會促進細胞黏附、移行以及管狀形成作用。此外，芴會藉由促進整合素αvβ3 mRNA的表現，來增加整合素αvβ3蛋白表現。為了觀察整合素αvβ3是否與芴引發細胞黏附、移行以及管狀形成作用有直接關聯，而外加抗整合素αvβ3抗體(LM609)，發現其可抑制芴引發細胞黏附、移行以及管狀形成作用增加的情形，表示整合素αvβ3有決定性的角色。進一步探討芴對於血管新生作用的機制，實驗結果顯示加入PI3K抑制劑LY294002與抗氧化劑NAC皆可降低整合素αvβ3蛋白的表現量，並且抑制芴引發細胞黏附、移行以及管狀形成作用。綜合以上結果，發現芴可藉由增加整合素αvβ3蛋白的表現來促進血管新生的作用，而ROS與PI3K/Akt途徑參與其中。

Polycyclic aromatic hydrocarbons (PAHs) are common environmental contaminants which have been found in the exhausts of industrial manufacture and motor vehicles, tobacco smoke, and contaminated food and water due to improper cooking. In previous studies, PAHs are potently carcinogenic and immunotoxic, and they have also been suggested to be involved in cardiovascular dysfunction. Angiogenesis, the formation of new blood vessels from pre-existing endothelium, plays a critical role in a variety of physiological and pathological conditions including wound healing, embryonic development, female reproductive cycle, diabetic retinopathy, tumor growth, metastasis and various chronic inflammation disorders. We recently found that PAHs induced eNOS protein expression in HUVECs, and increased eNOS protein activity and NO production. They also affected the homeostasis of intracellular calcium concentration and induced cell adhesion molecules expression. In addition, NO, calcium and adhesion molecules mediate the processes of angiogenesis. Thus, we chose fluorene, one of PAHs which contain three bezene rings, to study the effect of PAHs on angiogenesis in HUVECs. The process of angiogenesis is complex and involves several discrete steps, such as extracellular matrix degradation, cell adhesion, proliferation, migration, and morphological differentiation of endothelial cells to form tubes. In this study, we found that fluorene induced angiogenic effects by cell adhesion, cell migration, and tube formation assays. In addition, we also found that fluorene increased integrin αvβ3 protein expression by transcription. The angiogenic effect induced by fluorene may be via the expression of integrin αvβ3 because cell adhesion, migration, and tube formation were inhibited by anti-integrin αvβ3 antibody (LM609) treatment. Fluorene elicited angiogenic effects in in vitro angiogenesis assays, and incubation of HUVECs with fluorene induced integrin αvβ3 expression; these effects were inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002) and antioxidant (NAC). These findings suggested that fluorene induced vascular endothelial cell adhesion, migration, and tube formation via increasing integrin αvβ3 expression, and ROS and PI3K/Akt signaling pathway involved in angiogenesis activated by fluorene were also established.