- 學位論文
陰電性低密度脂蛋白引發血管內皮細胞活化在心血管疾病所扮演的角色
Role of Electronegative LDL-induced Endothelial Activation in Cardiovascular Diseases
摘要
完整的血管內皮細胞能預防心血管疾病與血栓的發生，L5是人體內自然產生的陰電性低密度脂蛋白，目前已知L5濃度在心血管高風險族群體內升高，並且能引起血管內皮細胞凋亡，因此L5被認為是一個心血管疾病的危險因子，然而仍然需要更多證據支持L5的致病機轉，因此我們針對L5對內皮細胞功能的影響提出一系列的討論。 根據高醫人體試驗委員會審核通過的程序收案並分離LDL，利用內建陰離子交換樹脂的快速蛋白質層析儀，以多個鹽梯度的步驟分離LDL成L1–L5共五個低密度脂蛋白次群。我們發現L5能透過活化內皮細胞釋出發炎媒介物，間接引起心肌凋亡。此外L5改變血管內皮細胞表面黏著分子表現，增加血管內皮表現P-selectin與CX3CL1，增加血小板與單核球黏附至內皮細胞，這可能與早發性動脈粥樣硬化有關。L5能降低內皮細胞抗血栓形成的功能，促使血管內皮細胞表現tissue factor，增加血小板的活化與凝集，L5注射小鼠尾部凝血時間明顯縮短，因此L5可能幫助動脈粥樣硬化血栓形成。實驗動物模式 (db/db) 證實，陰電性低密度脂蛋白與心血管的老化有關，導致心血管疾病發生風險提高。 急性心肌梗塞病人與紅斑性狼瘡病人體內低密度脂蛋白膽固醇的濃度及其他脂質參數都沒有明顯變化，然而分離自急性心肌梗塞病人的L5能增加血小板活化，L5刺激血管內皮細胞活化表現P-selectin，結果增加血小板黏附至內皮細胞的機率。而分離自紅斑性狼瘡病人的L5能刺激血管內皮表現CX3CL-1，引發CX3CL1-CX3CR1調控的內皮細胞與單核球相互作用，因此L5藉由引發血管內皮細胞活化，吸引免疫細胞黏著至血管內皮引起發炎反應。 為了探討L5本質，我們深入探討L5的化學組成，借助於質譜儀得分析，結果顯示紅斑性狼瘡病人的L5與LDL含有豐富的lysophosphatidylcholines (LPC) 與platelet-activating factor (PAF)。高脂血症患者體內的L5具有高度sphingomyelinase (SMase) 活性，質譜分析結果顯示，高脂血症患者體內的L5含有大量ceramide。上述這些脂質成分都具有生物活性，皆可以引發內皮細胞的活化。然而不同來源的L5脂質分佈不盡相同，這部分值得我們再深入研究。總之，透過對L5致病機轉的瞭解，或許未來可以改變心血管疾病的評估指標與治療標的，並發展出新的醫療技術與抗體藥物。
並列摘要
Healthy blood vessel endothelial cells can function properly to prevent cardiovascular diseases and thrombosis. Previous studies have shown that L5 is naturally occurred electronegative low-density lipoprotein (LDL). Elevated plasma level in patients with cardiovascular diseases, L5 induces endothelial apoptosis in vivo and in vitro. Therefore, L5 has confirmed the atherogenic property and can be an important risk factor for cardiovascular diseases. However, detail mechanisms are still not clear and need further investigations. According to agreement of human Institutional Review Board (IRB) committee, we followed the instructions to recruit patients and healthy controls. Blood samples were collected and plasma samples were undergoing serial ultra-centrifugations. LDL were isolated and purified for further analysis. A fast protein liquid chromatography (FPLC) equipped with anion-exchange column was used to divide LDL into five subfractions, L1-L5, with increasing electronegativity. We found that L5 stimulates endothelial cells release inflammatory cytokines, in turns to induce the apoptosis of cardiomyocytes. In addition, L5 changes the surface markers of endothelial, including P-selectin and CX3CL1, to increase the interaction or attachment of platelets and monocytes. These findings indicate that L5 may contribute to inflammation. L5 defects the anti-thrombotic function of endothelium, increases the expression of tissue factor, and enhances the platelet activation and aggregation. Together, L5 induces the formation of atherothrombosis. Animal db/db mice study has also shown low-does L5 induces the endothelial senesces and increases the risks of cardiovascular diseases. In patients with ST-segment elevation myocardial infraction (STEMI) and systemic lupus erythematosus (SLE), traditional lipid profiles have no significantly difference. However, L5 was highly elevated in STEMI patients. L5 from STEMI patients induces platelet activation, attachment and aggregation. Tail-vein L5-injection caused the shortening of coagulation time testing. The L5-induced platelet activation was highly correlated to overexpression of P-selectin and activation of GPIIb/IIIa, which were important to thrombotic events. On the other hand, L5 from SLE patients induces the CX3CL1-CX3CR1 signaling between endothelial cells and monocytes, which was the fundamental stage of atherosclerosis. In order to investigate the underlying property of L5, we further analyze the chemical compositions of lipid moiety by parallel data acquisition liquid chromatography mass spectrometry (LC/MSE) and matrix-assisted laser desorption/ionization-time of fly (MALDI-TOF) mass spectrometry. Results show L5 from SLE patients has high concentration of lysophosphatidylcholines (LPC) and platelet-activating factor (PAF). In contrast, L5 from hyperlipidemia patients has increased ceramide in addition to LPC and PAF. These lipid contents are bioactive materials for activating endothelial cells. In summary, through understanding the detail mechanisms of L5 in endothelial cells, we may develop new therapeutic compounds or antibodies in the future.
參考文獻
延伸閱讀
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