動脈硬化斑塊之特徵：型態學、代謝學及臨床表現之相關研究

Abstract

研究背景： 心血管疾病在國人的罹病率、死亡率、造成的醫療照護支出及社會成本逐年快速增加，付出之醫療及社會相關成本超過惡性腫瘤的比重。研究顯示，不穩定性斑塊產生裂隙、突然破裂及後續之血栓形成，是造成急性心肌梗塞、猝死及缺血性腦中風等病變的主要原因。因此在症狀及併發症發生前，對高危險病群早期診斷及積極治療，極為重要；而心血管疾病之臨床表現、分子影像及相關的基礎之研究及其整合，對於該疾病發生之早期偵測及預後評估都有莫大的助益。 動脈硬化疾病的發生是由致病性脂蛋白等引起的急性發炎；白血球受到多種趨化激素吸引，加上多種細胞黏著分子合成或被活化，將白血球與內皮細胞繫鏈在一起，使白血球變形且內皮細胞的間隙打開，讓白血球進入組織形成浸潤。另一方面，基質金屬蛋白酶 (matrix metalloproteinases, MMPs) 會分解細胞間質，使斑塊纖維蓋弱化，產生裂隙、潰瘍、崩解，造成動脈硬化加劇。 偵測「不穩定性斑塊」(vulnerable plaques) 是臨床診斷的一大挑戰。不穩定性斑塊主要組織學變化包括發炎細胞 (單核球、巨噬細胞及部分T 細胞) 的浸潤，斑塊多具有薄蓋及巨大脂肪核心，內皮裸露，血小板凝集，出現裂隙或是造成嚴重管腔阻塞。近年來超音波、電腦斷層 (computed tomography, CT) 及磁振造影 (magnetic resonance imaging, MRI) 在斑塊結構分析有不少進步，不過多以解剖變化為主，對於發炎偵測及斑塊破裂跟併發症的預測仍未臻理想。核醫影像利用細胞代謝功能的變化來造影，其中氟-18去氧葡萄糖（18F-fluorodeoxyglucose, 18F-FDG）會蓄積於發炎部位，與發炎細胞的代謝活性成正相關。過去小動物實驗及臨床研究觀察到血管炎或動脈硬化時，18F-FDG在血管壁的蓄積量會增加，與預後有關，且在消炎或降血脂藥物治療後活性會減少，顯示18F-FDG為一有潛力的同位素示蹤劑。早期的正子電腦斷層影像 (positron emission tomography, PET) 解析力較差，新近科技發展迅速，當代的正子電腦斷層掃描儀 (PET scanner) 配有CT，可得清晰的解剖及代謝功能的融合影像，可做全身性掃描，並對發炎部位也可以作定性及半定量分析，可能是研究動脈硬化機轉及治療效果最具潛力的造影工具。 由於炎症反應跟動脈硬化及不穩定性斑塊形成關係密切，曾被研究過的血液指標很多，但何者最為重要卻未有定論。牽涉白血球和內皮細胞交互作用的分子中，VCAM-1是匯聚巨噬細胞的重要黏著因子之ㄧ，且早在斑塊形成之前就已經出現。另一方面，根據組織學及動脈硬化動物模型的研究顯示，MMP-1可被多種趨化激素及生長激素誘發，集中於富含巨噬細胞的區域，是動脈硬化斑塊主要成分之ㄧ，也可能是具有潛力的不穩定斑塊生化指標。過去曾有少數報告指出MMP-1與急性冠心症或是冠狀動脈複雜性病灶有關，不過，迄今仍欠缺大型的研究來證實MMP-1在臨床上的應用價值。對於這些血液指摽的臨床價值及其調控機轉仍有待進一步研究。 巨噬細胞為動脈硬化斑塊中的主要細胞，與斑塊的不穩定有密切的關係，在斑塊裡的分布也符合18F-FDG示蹤劑及MMP-1在斑塊中聚集的位置。過去，動脈硬化的研究大多利用血管細胞株 (例如內皮細胞或平滑肌細胞株) 進行，並不能完全反應斑塊真正的變化，而研究巨噬細胞的訊息傳遞及基因表現機轉，應有助於吾人對動脈硬化斑塊的了解。由於流行病學研究已知糖尿病為動脈硬化疾病的重要危險因子，新近糖尿病這項危險因子對心血管的危險性更被視為等同於冠心症。當血糖升高會增加細胞的氧化壓力並誘發全身的發炎反應，可加速動脈硬化，進一步造成各種心血管併發症。我們利用THP-1細胞 (monoblastic leukemic cell line) 的分化過程模擬發炎過程，建立實驗室動脈斑塊研究的細胞模式，研究高糖環境的影響及巨噬細胞的調控機轉，是有其必要性。 研究目的： 1. 探討18F-FDG PET/CT在頸動脈硬化斑塊造影的可行性，並分析18F-FDG PET/CT動脈斑塊造影，與血液中生化代謝指標、臨床預後 (如急性缺血性腦中風發生率、經導管支架置入治療產生栓塞併發症及支架再狹窄等) 的關連性。 2. 研究週邊各項血液指標 (如hs-CRP、MMP-1及VCAM-1等) 預測頸動脈狹窄、冠狀動脈心臟病或換心後植體冠狀動脈血管病變等的可行性及其價值。 3. 建立實驗室巨噬細胞高糖環境模式，研究： (a) 高糖環境是否會使分化後的巨噬細胞MMP-1表現量增加； (b) 降血糖藥物是否可以抑制MMP-1； (c) MMP-1是否經由nuclear factor-kB (NF-kB)或 Jun N-terminal kinase (JNK) 的途徑進行調控，以及MMP-1與HIF-1α的相關性； (d) 建立MMP-1與代謝症候群、糖尿病及不穩定動脈斑塊間之關聯性。 研究方法： 1. 在25位換心者導管檢查時經冠狀靜脈竇採血，分析心肌切片及冠狀動脈攝影所見，探討冠狀竇的E-selectin、ICAM-1及 VCAM-1濃度與急性排斥及植體冠狀血管病變的關連性。 2. 我們首先在25位頸動脈狹窄患者與22位接受健康檢查的受試者做18F-FDG PET/CT全身動脈造影，研究動脈斑塊的性質與周邊血液的總白血球數量、hs-CRP及MMP-1的相關性；進一步比較37位頸動脈狹窄患者與84位對照組在周邊血液指標的差異性。利用血管內支架置入造成動脈硬化斑塊破裂，探索支架置入術前後，動脈斑塊受擠壓時周邊血液中MMP-1短時間的變化。 3. 比較217位重度冠狀動脈狹窄患者 (二條或以上冠狀動脈或其主要分枝具有50%或以上管徑狹窄) 與81位對照組在周邊血液指標 (包括MMP-1、hs-CRP 及adiponectin) 的差異性，探討這些血液指標作為評估動脈硬化疾病的臨床價值。 4. 在THP-1細胞株的研究，利用THP-1經phorbol 12-myristate 13-acetate (PMA) 誘發分化模擬急性炎症反應研究MMP-1表現量的變化，探討高糖環境的影響，進一步比較糖尿病藥物 (glimepiride、metformin及BRL 49653) 在高糖環境下MMP-1表現量在分化的THP-1細胞株的變化。最後，利用低氧誘導因子1α (Hypoxia-inducible factor-1α, HIF-1α) 的增強劑或抑制劑，研究MMP-1跟HIF-1α之間的關連性。並利用NF-kB及JNK抑制劑的有無，研究HIF-1α及MMP-1是否均經由NF-kB及JNK途徑進行調控。 結果： 1. 25位換心者共接受過40次的心肌切片及冠狀動脈攝影，在冠狀靜脈竇的細胞黏著分子濃度與心肌切片急性排斥分級 (International Society for Heart and Lung Transplantation grade, ISHLT grade) 間並無顯著關聯性。其中六位換心者發生植體血管病變。我們的結果顯示是否發生植體血管病變，與冠心病的危險因子、術前疾病、人類白血球組織抗原或急性排斥次數等均無明顯相關性；僅VCAM-1在發生植體血管病變者明顯升高 (1015.9 ± 457.3 vs. 594.8 ±233.0 ng/mL, P = 0.005)。在接受過兩次或以上檢查的12 位受試者，共有三位的VCAM-1值明顯的隨換心後時間而升高，其中兩位最終出現植體血管病變。 2. 正子影像研究顯示，18F-FDG 會在動脈硬化斑塊蓄積，未必與血管壁的鈣化程度成正相關性；頸動脈硬化斑塊的18F-FDG 蓄積量與周邊血液的MMP-1成正相關，顯示斑塊內18F-FDG 的蓄積跟MMP-1 成份有關。支架置入後MMP-1 會立即升高，表示頸動脈斑塊被擠壓時將大量MMP-1 釋出，佐證MMP-1 為動脈硬化斑塊裡的主要成分之一。 3. 在多條冠狀動脈阻塞患者，其周邊血液中的MMP-1 及hs-CRP 均明顯地較對照組為高，但與adiponectin 無關；多變項分析顯示MMP-1 甚至比hs-CRP 更具預測價值，顯示MMP-1 可作為動脈硬化疾病的周邊血液生化標記，。 4. THP-1 細胞被PMA 誘發分化時MMP-1 基因表現量會顯著增加，高糖狀態時更為明顯。無論是glimepiride，metformin 及BRL 49653 (rosiglitazone, PPAR γagonist) 都可抑制高糖狀態時MMP-1 的表現，其中以 BRL 49653 抑制效果最為明顯。NF-κB 抑制劑 (IMD-0354) 及JNK 抑制劑 (SP-600125) 均可抑制MMP-1 的表現，尤其是NF-κB 抑制劑有顯著的劑量效應。THP-1 經PMA 分化時，HIF-1α 蛋白的量會增加，而NF-κB 抑制劑可減少HIF-1α 蛋白的量，顯示HIF-1α 也可能經由NF-κB 的途徑調控。我們進一步發現，MMP-1 基因表現量也受HIF-1α 促進及抑制劑的調控，暗示HIF-1α 可能是MMP-1 的上游調控因子之ㄧ。 結論： 本論文顯示冠狀靜脈竇血液中 VCAM-1 的升高與植體發生動脈硬化有關，具有預測心臟移植後換心人發生冠狀動脈病變的價值。本研究亦顯示18F-FDGPET/CT 在動脈斑塊影像的應用價值，進一步證明MMP-1 是動脈硬化斑塊中重要的成分，並與18F-FDG 的蓄積量成正相關性。在THP-1 細胞模式下，MMP-1的基因表現量在巨噬細胞分化時會增加，在高糖時尤其明顯，並受到NF-κB/JNK途徑調控。同時，本研究發現HIF-1α 可能與MMP-1 有關，顯示MMP-1 在動脈硬化斑塊的不穩性上扮演重要的角色。

Background: Rupture of atherosclerotic plaques and subsequent formation of thrombi are currently recognized as the major cause of morbidity and mortality in ischemic stroke and cardiovascular diseases, including acute coronary syndrome. Several imaging modalities have shown promises as potential surrogate markers for atherosclerosis. They may help clinicians and investigators to directly visualize atherosclerotic plaque burden, refine cardiovascular risk assessments, and allow serial monitoring of disease activity once interventions have been initiated. The proposed major criteria for defining vulnerable plaques, based on the autopsy studies, include active inflammation (monocyte/macrophage and sometimes T-cell infiltration), thin cap with large lipid core, endothelial denudation with superficial platelet aggregation, fissured plaque and severe luminal stenosis. Conventional imaging of atherosclerosis is based on the degree of luminal stenosis and morphologic characteristics of atheromas, including X-ray contrast angiography, computed tomography (CT) and magnetic resonance (MR) imaging. Nuclear imaging also has the ability to non-invasively image pathophysiological process of atherosclerosis. Recent studies have shown that 18F-fluorodeoxyglucose (18F-FDG) accumulates in macrophage-rich plaques and the inflammatory activity of individual plaques could be detected and measured by using positron emission tomography (PET). In addition, hybrid PET/CT scanners could detect calcification and provide better localization of systemic atherosclerotic plaques, thus may allow to screen, diagnose or monitor treatment response in patients with atherosclerosis. Atherosclerosis is a complex and multifactorial disorder, and a specific profile of biomarkers may help identify subjects at risk for progressive atherosclerosis or plaque rupture. Inflammation plays a significant role in the pathogenesis and progression of atherosclerosis. Numerous systemic serological markers have been reported to provide additional information about the risk of developing cardiovascular disease and reflect the overall burden of atherosclerosis. However, many of these are not specific to systemic atherosclerosis or plaque vulnerability, and the clinical significance remains to be elucidated. Levels of cellular adhesion molecules (such as E-selectin, ICAM-1, VCAM-1) may reflect extent of expression and/or shedding of molecules on endothelial surface that are influenced by pro-inflammatory cytokines. Recent data have suggested that macrophages and vascular smooth muscle cells are important component of vulnerable plaques, and are the sources of matrix metalloproteinases (MMPs) production. Several immunochemical findings have showed that MMP-1 is abundant in the macrophage-rich shoulder regions of atherosclerotic plaques and is robustly induced by cytokines and growth factor. However, scant studies have reported that MMP-1 is associated with acute coronary syndrome and the presence of complex coronary lesions. The significance of circulating levels of MMP-1 remains to be evaluated in larger-scale clinical studies. Macrophages are important for intracellular lipid accumulation and foam cell formation in the process of atherosclerosis. MMPs secretion by macrophages is believed to play a key role in the matrix degradation that underlies atherosclerotic plaque instability. Diabetes is a major risk factor for atherosclerosis, thus we investigate the regulatory mechanism of MMP-1 in THP-1 differentiated macrophages under high glucose media. Purposes: The aims of this study are to evaluate the feasibility of 18F-FDG PET/CT as a non-invasive imaging modality in the detection of systemic atherosclerosis, and correlation between characteristics of atherosclerotic plaques by using 18F-FDG PET/CT and circulating biomarkers among subjects with or without carotid stenosis. We further evaluate the role of cellular adhesion molecules in patients with transplant vasculopathy and MMP-1 in patients with significant atherosclerosis. We establish an in-vitro system of THP-1 monoblastic leukemic cell-line to explore MMP-1 and hypoxia-inducible factor-1α (HIF-1α) expression and regulation signal pathways including nuclear factor-kB (NF-kB) or c-Jun N-terminal kinase (JNK) pathways, especially in high glucose conditions. We also evaluate the relationship between hypoxia-inducible factor-1α (HIF-1α) and MMP-1. Research Designs and Results: 1. We analyzed ICAM-1, VCAM-1 and E-selectin levels from the coronary sinus of 25 cardiac allograft recipients, correlated with the degree of acute rejection detected in endomyocardial biopsy specimens and the presence of transplant vasculopathy assessed with coronary angiography. We found that VCAM-1 significantly increased in patients with transplant vasculopathy compared with those without transplant vasculopathy, whereas E-selectin and ICAM-1 did not. 2.We examined 25 patients with significant carotid stenosis (≥ 50%) and 22 controls using 18F-FDG PET/CT. 18F-FDG arterial uptake, as well as calcifications, was significantly higher in extensive distributions in patients with established carotid stenosis; but their distribution was not consistently overlapping. The values of circulating MMP-1 and leukocyte counts were significantly higher in patients with carotid stenosis (all P < 0.05). In addition, subjects with higher 18F-FDG uptake (SUVmax > 2.0) on target lesions had higher baseline and post-stenting MMP-1 levels (all P < 0.05). We further measured hs-CRP and MMP-1 in 37 patients with significant carotid stenosis and 84 controls. We also confirmed that patients with carotid stenosis exhibited significant higher MMP-1 as compared with controls, but no difference in hs-CRP. Moreover, MMP-1 elevated immediately after stenting (n = 30). In multivariate analyses, MMP-1 was negatively correlated with statin and angiotensin converting enzyme inhibitor/angiotensin-II receptor blocker use in controls. 3. MMP-1, hs-CRP and adiponectin were measured in 217 subjects with angiographically documented multivessel coronary artery disease (CAD, two or three-vessel disease by luminal stenosis ≥ 50%) and 81 controls. MMP-1 and hs-CRP were notably higher in patients with CAD; while adiponectin was not significantly different between two groups. Levels of hs-CRP positively correlated with body mass index and left ventricular dysfunction (R2 = 0.16, P < 0.0001); while adiponectin was significantly associated with age, gender, and levels of cholesterol and triglyceride (R2 = 0.09, P < 0.0001). On the contrary, MMP-1 was not associated with any clinical cardiovascular risk factors, and still an independent predictor (OR = 1.49, P < 0.0001) of multivessel CAD after the adjustment of clinical risk factors and hs-CRP. 4. We have established an in-vitro THP-1 macrophage cell model. THP-1 treated with PMA may mimic inflammatory stimulation. High glucose concentration could augment PMA-stimulated MMP-1 expression in THP-1. MMP-1 mRNA expression is through cytokines/inflammatory process, via NF-κB and JNK pathways, especially NF-κB. Of glimepiride, metformin and BRL-49653 (rosiglitazone, PPAR γ agonist), BRL-49653 notably attenuates PMA-stimulated MMP-1 expression in THP-1 in high glucose concentration. We have demonstrated that PMA could upregulate HIF-1α which is suppressed by NF-κB inhibitor. HIF-1α inducers could upregulate MMP-1 while HIF-1α inhibitor could attenuate MMP-1, suggesting MMP-1 could be regulated by HIF-1α. Conclusions: This study has demonstrated that an increased coronary sinus level of sVCAM-1 is a reliable marker in assessing cardiac transplant vasculopathy. Our study has also shown that 18F-FDG PET/CT imaging could be used as an adjunct to the clinical management of high-risk atherosclerosis and an in vivo tool to study plaque biology. Elevated MMP-1 could predict the presence of advanced atherosclerosis. Higher levels and rapid surge after stenting in patients with carotid stenosis support MMP-1 is an important composition of plaques. Our investigation provides a link between 18F FDG uptake and MMP-1. In THP-1 cell model, increased transcription of macrophage MMP-1 under high glucose conditions provides a mechanism for accelerated atherosclerosis in diabetes. MMP-1 expression is regulated via NF-κB and JNK pathways, as well as HIF-1α., thus providing a molecular basis for regulation of MMP-1 in differentiated THP-1 cells.