Haptoglobin在動脈硬化中所扮演之功能角色

Abstract

冠狀動脈及其他心血管疾病(包括心肌梗塞)是造成西方工業化國家人口死亡的主要原因。在台灣心血管疾病雖名列死亡疾病前三、四名，但在民國八十九年內因血管疾病造成死亡的人數竟高達三萬五千人次；即使利用降低膽固醇藥物(hypocholesterolemic drugs)來進行治療。以此種危險性觀之，心血管疾病仍是引起死亡的主要殺手。冠狀動脈粥狀化之病理研究中，最為人注意及採信的病理觀點為低密度脂蛋白修飾假說(LDL-modification hypothesis)，其主要觀點為冠狀動脈斑(atherosclerotic plaques)的形成是由於巨噬細胞(macrophages) 在動脈管壁內吞噬氧化修飾之低密度脂蛋白(oxidatively modified LDL)，導致平滑肌細胞的遷移及增生作用(smooth muscle cell migration and proliferation)。其中，值得注意的是了解氧化作用所受的緊迫、平滑肌細胞在intima的轉移及增生作用之各項生化角色相對地變得十分重要。

冠狀動脈斑部位之各種發炎現象逐漸地被認為引起動脈粥狀化之主要決定因素，因此影響此種發炎現象的分子便成為研究的重要對象。近來我們研究發現haptoglobin(Hp)(一種急性蛋白在受感染及發炎反應Hp之蛋白量會大量產生)，在冠狀動脈病變處大量累積此蛋白分子，並證明Hp蛋白是由巨噬細胞(macrophages)所產生，而並非平滑肌細胞所產生。更加興奮的是我們在研究環境中第一個報導平滑肌細胞在不同濃度之氧化修飾後低密度脂蛋白中可表現出Hp mRNA。我們更證明Hp蛋白之抗氧化效力比probucol更強大。雖然冠狀動脈粥狀化之病理機制持續被研究證明，但並無任何研究指出Hp蛋白可能參與保護並防止冠狀動脈粥狀化之形成。

本計劃的初始目標在測試Hp表現型(Hp1-1, 2-2, 2-1)與心血管疾病有關之假說，及研究平滑肌細胞與巨噬細胞在氧化之LDL刺激後表現出Hp蛋白的機制。長期目標為於探討Hp在冠狀動脈粥狀化所扮演的功能性角色。本三年計劃之主要目標如下：

1. 利用單株抗體研發新型及快速偵測Hp表現型(Hp1-1, 2-2, 2-1)，並且測試Hp表現型與台灣心血管疾病病人有相關性的假說。 (第一至第三年)

2. 研究平滑肌細胞及巨噬細胞在氧化之LDL(oxidized LDL)及其他脂蛋白(VLDL, LDL, and HDL)刺激後Hp mRNA的表現。 (第一至第二年)

3. 研究巨噬細胞及平滑肌細胞中Hp之抗氧化角色，並利用Hp sense及antisense 之 cDNA轉殖入巨噬細胞及平滑肌細胞，來測試Hp抗氧化能力與Hp表現型之相關性。 (第一至第二年)

4. 利用餵食膽固醇的兔子及Hp基因轉殖小鼠，研究Hp在形成冠狀動脈粥狀化所扮演之功能角色。 (第二至第三年)

Coronary artery and other vascular diseases (including myocardial infarction, MI) are the leading cause of death in the Western industrialized countries. The mortality of the diseases are the top third-fourth in Taiwan, the number of death reached more than 35,000 in year 2001 and has since become the leading killer, despite the overall improvement in mortality of the patients treated with hypocholesterolemic drugs. One concept that has received much recent attention for the pathogenesis of atherosclerosis is the LDL-modification hypothesis, which postulates that atherosclerotic plaque resulted from the uptake of oxidized LDL by macrophages in the arterial wall followed by smooth muscle (SMC) migration and proliferation. In this respect, an understanding of the biochemical events, which impact on the oxidation stress SMC proliferation and its migration into the intima deserves to receive much attention. Inflammatory phenomena at sites of atherosclerotic plaques are increasingly thought to be major determinants of the progression and clinical outcome of atherosclerosis disease. Therefore, attention is being paid to systemic markers that may reflect the inflammatory activity in the plaques. Recently we found that haptoglobin (Hp), an acute phase protein elevated sharply during the infection and inflammation, was substantially accumulated in atherosclerotic lesions. Subsequently, we demonstrated that Hp was endogenously synthesized in macrophages, but not in SMC. Interestingly, for the first time we showed that SMC can express Hp mRNA in the presence of oxidized LDL with a dose-dependent manner. We also demonstrated in vitro that Hp is a highly potent antioxidant superior to probucol. Nevertheless, while the pathogenesis of atherosclerosis has been continuously explored, the Hp molecule that may protect against the progression of atherosclerosis has never been reported.

The initial goal of this proposal is to test the hypothesis whether or not the phenotypes of Hp (Hp 1-1, 2-2, and 2-1) are related to the patients with coronary artery disease and to explore the mechanisms by which oxidized LDL stimulates the expression of Hp in SMC and macrophages. Utimately, the long-term goal is to focus on the functional role of Hp in atherogenesis. The specific aims in the next 3 years are to:

1. Develop a new and fast approach for human Hp phenotping (Hp 1-1, 2-2, and 2-1) using monoclonal antibodies, and to test the hypothesis whether or not Hp phenotypes are correlated to the patients with coronary artery disease in Taiwan (Years 1-3).

2. Study the Hp mRNA expression in macrophages and SMC in the presence of oxidized LDL and lipoproteins VLDL, LDL, and HDL (Years 1-2).

3. Test the hypothesis that Hp may exert an antioxidant role in macrophages and SMC transfected with sense and antisense Hp cDNA; to define the antioxidant activity of Hp phenotypes in vitro (Years 1-2).

4. Investigate the functional role of Hp in the progression of atherosclerosis using cholesterol-fed rabbits and antiatherogenic effect (if any) using Hp transgenic mice (Years 2-3).