下肢動脈疾病的功能性評估、影像學分析與相關動脈硬化因子的研究

Abstract

研究背景： 隨著台灣社會年齡層的老化，國民心血管疾病相關死亡佔比居高不下。由衛生福利部的統計年報發現，除了冠狀動脈疾病與腦血管疾病之外，周邊血管疾病的盛行率也有逐年上升的趨勢。在下肢動脈疾病（PAD）中，接近九成的患者往往沒有明顯症狀或是症狀不典型，常常會被輕忽，以致於患者求助就醫師時常常已經是下肢潰瘍壞疽，而面臨被截肢的命運。從對生活品質的影響來看，下肢動脈疾病對生活品質的影響並不亞於其他心血管疾病，對於行走能力的影響則有過之而無不及。針對下肢動脈疾病高危險群的篩檢，踝肱動脈壓比（ABI）仍然是公認的首選。核磁共振血管攝影、電腦斷層血管攝影、以及動脈導管血管攝影因為價格、顯影劑使用、輻射暴露與侵入性等問題，主要應用在動脈血管成形術前後的治療計畫與治療追蹤。都普勒超音波檢查號費時間較長、也受到檢查者因素的部分影響。然而，這些檢查工具都侷限於血流與壓力變化的分析，而缺少對終端組織器官（如皮膚、肌肉）的評估。因此，我們計畫利用不同的研究工具，針對下肢的皮膚與肌肉作功能性的評估，同時合併冠狀動脈疾病研究中常用的鈣化指數分析。希望透過這些研究設計，增加我們對下肢動脈疾病的了解，提升目前的評估方式，進而改善下肢動脈疾病的預後，降低下肢動脈疾病對於病患與社會的影響。 研究目的： 為拓展我們對下肢動脈疾病的認知，我們在臨床上設計了三個不同的研究，利用三種不同的研究工具（紅外線熱影像、近紅外線光譜與多排螺旋電腦斷層），分別對下肢動脈疾病中三個受影響組織（皮膚、肌肉與血管），進行影像與功能性的研究，研究的設計包括橫斷面的分析、介入治療、以及長期的預後追蹤。我們希望這些研究工具能提供更完整的下肢動脈疾病的臨床評估，對未來下肢動脈疾病的治療產生影響。在基礎研究中，我們分析脂肪細胞脂肪酸結合蛋白A-FABP與下肢動脈疾病的關聯性，並研究下肢動脈疾病藥物cilostazol的調控機轉。 研究方法： 1. 針對下肢動脈疾病的高風險族群（已證實有粥狀動脈硬化性疾病患者、70歲以上老年人、年齡超過50歲同時合併有的糖尿病或抽菸者、以及末期腎病患者）進行運動前後的下肢紅外線熱影像（Infrared thermography）分析。運動的方式為六分鐘行走測試，運動測試前先進行踝肱動脈壓比與分段血壓的測量，並且完成行走損傷問卷、七日日常活動問卷、血管生活品質問卷等三份下肢動脈疾病相關問卷。 2. 收錄30至70歲合併高血壓與至少一種其他心血管疾病風險的患者，接受12週olmesartan (10-40mg/day)的治療。治療前後以運動心電圖機進行最大運動耐受力測試心肺功能，測試前先進行雷射都普勒超音波（Laser Doppler Flowmetry）分析下肢血管內皮依賴與非血管內皮依賴微血流變化，運動中以近紅外線光譜（Near-infrared Spectroscopy, NIRS）分析肌肉組織血液灌流（以組織氧合度回升50%的時間SatT50代表）以及氧氣擷取能力（以去氧血紅素最大變化量△deoxyHb代表）。同時分析治療前後血壓變化、血清內皮細胞功能指標（ICAM-1、VCAM-1、PAI-1）以及微量蛋白尿。 3. 回溯分析82位因為下肢動脈疾病（Fontaine Ⅱ–Ⅳ）在台大醫院接受多排螺旋下肢電腦斷層檢查並追蹤治療的患者。下肢動脈鈣化計分上緣啟始在降主動脈及髂總動脈的交界處，下緣為腳踝處；鈣化分數則參照Agatston等學者所定定義的Agatston score計算方式。研究的主要終點為死亡，次要終點為因下肢動脈疾病而截肢。 4. 分析脂肪細胞脂肪酸結合蛋白A-FABP與下肢動脈疾病的關聯性。建立THP-1細胞研究模型，在PMA誘導分化後，加入ox-LDL模擬粥狀動脈硬化發生過程，分析A-FABP與其他與泡沫細胞（foam cell）形成有關的蛋白表現（MCP-1、ABCA-1、CD36），同時研究下肢動脈疾病治療藥物cilostazol的調控角色與可能機轉。 研究結果： 1. 51位下肢動脈高危險群患者有20位被診斷有下肢動脈疾病。PAD患者有較短的六分鐘行走距離以及較低的下肢動脈疾病相關問卷分數。休息狀態的下肢體表溫度在兩組之間沒有差別，運動後一分鐘體表溫度的改變在PAD患者有略低的趨勢。然而比較運動前後的下肢體表溫度，可以發現非PAD患者的溫度在運動後通常會上升，而非PAD患者的下肢體表溫度則持平甚至下降。這樣變化的幅度及方向與下肢動脈疾病的嚴重度、行走距離以及日常活動能力相關。 2. 12週olmesartan治療後，30位高血壓受試者收縮壓平均下降22 mmHg，舒張壓平均下降 10 mmHg。在治療前的檢測中，我們發現年HbA1c、微量蛋白尿與NIRS指標SatT50及△deoxyHb呈現輕度正相關。治療後△deoxyHb有增加的趨勢，SatT50也有縮短的傾向，但都沒有達到統計學的意義。此外，研究發現SatT50改善的程度與糖尿病與否有明顯相關性，但內皮細胞依賴的血管擴張程度治療前後的改變量與SatT50的改善程度並無關聯。此子研究因機器取得問題而提前終止。 3. 研究結果發現高齡患者、糖尿病、高脂血症，以及末期腎病伴隨著較高的下肢動脈鈣化分數。下肢動脈鈣化為均勻對稱分布，各分段間的鈣化分數有高度的相關性。鈣化分數與下肢動脈的臨床症狀嚴重度（Fontaine stage）相關，同時也是是預測下肢動脈疾病患者截肢與死亡的獨立危險因子。 4. 下肢動脈疾病患者有較高的血清A-FABP濃度。在THP-1細胞模式中加入ox-LDL模擬粥狀動脈硬化，同樣發現A-FABP表現量上升，而cilostazol可以抑制這樣的反應。Cilostazol同時影響了數個與泡沫細胞形成相關的基因表現，包括ABCA-1、CD36、MCP-1等。 結論 本研究跳脫原有下肢動脈疾病分析的框架，廣泛分析下肢動脈阻塞對下肢各個組織可能造成的影響。從這些檢查工具得到的多個特殊且獨立的指標，與下肢動脈疾病的嚴重度及預後相關，可以應用於日後下肢動脈疾病的篩檢與追蹤。我們也證實A-FABP同樣在下肢動脈疾病扮演重要的角色，而下肢動脈疾病治療藥物cilostazol可以調控其表現。

Background: Peripheral artery disease (PAD) affects 15%-20% of persons older than 70 years of age, though its prevalence is probably even greater if we include asymptomatic persons. Apart from claudication, patients with PAD may experience many problems, such as ischemic rest pain, digital ulceration and gangrene changes, repeated hospitalizations, revascularizations, and finally limb loss. These lead to limited physical activity, poor quality of life, and high rate of depression. Moreover, various epidemiologic studies have shown that up to 50% of patients with PAD also have symptoms of coronary and cerebrovascular disease. They also have higher rate of cardiovascular and all-cause mortality compared with patients without PAD. Several techniques are currently being used to aid low extremity PAD diagnosis and severity assessment. These include ankle-brachial index, Duplex ultrasound, computed tomography angiography, magnetic resonance angiography, and catheter-based angiography. Unlike the multidimensional approachs in patients with clinical suspicion of CAD, these techniques focus on lower extremity arteries only, and provide limited information about stenotic severity, blood flow, and pressure gradients. Purposes: The aims of our study were to evaluate patients with PAD in multiple dimensions. We focused on skin temperature, muscular oxygenation, and arterial calcification, which were analyzed by infrared thermography, near-infrared spectroscopy (NIRS), and multidetector computed tomography (MDCT), respectively in three separate studies. Finally, the laboratory study analyzed the regulating roles of cilostazol, medication for PAD treatment, on adipocyte fatty acid-binding protein (A-FABP) and other foam cell formation-associated gene expression in THP-1 cell model. Research Designs and Results: 1. Fifty one subjects at high risk for lower extremity PAD were recruited from cardiovascular clinics. Patients with end-stage renal disease (ESRD) were also enrolled. The cutaneous temperatures of lower extremity before and after exercise were measured by a digital infrared thermal image system. The exercise was a six-minute walk test. The ankle-brachial index and three PAD-associated questionnaires were analyzed before examination. In this study, we demonstrated that there was no difference in rest temperature between PAD and non-PAD patients. However, the exercise-induced temperature changes were correlated with PAD severity, walking capacity, and daily physical activity. 2. Thirsty hypertensive subjects with at least another cardiovascular risk factor were enrolled and treated with 12-week olmesartan. Muscular microcirculation (estimated by half-time of tissue saturation recovery SatT50) and capacity of oxygenation extraction (estimated by deoxy-hemoglobin changes △deoxyHb) were analyzed by NIRS. The blood pressure, serum endothelial biomarkers (ICAM-1、VCAM-1、PAI-1), and above-mentioned examinations were performed before and after treatment. There were mild positive correlations between hemoglobin A1c, microalbuminuria, and NIRS derived parameters (SatT50 and △deoxyHb) at baseline. After 12-week olmesartan treatment, there was a trend of lower SatT50 and higher △deoxyHb. However, there was no significant change in endothelial function, serum biomarker levels, exercise duration, and maximal oxygen uptake. 3. Eighty two symptomatic PAD patients (Fontaine stage II-IV) with MDCT of lower extremity artery images in NTUH were analyzed retrospectively. The scoring of calcification started at the junction of descending aorta and common iliac artery, and ended at the ankle. The calcium score (CS) for each segments of interest was determined and expressed as Agatston score. The primary and secondary endpoints were all-cause mortality and amputation. Our study demonstrated that lower extremity arterial CS, which was higher in patients with old age, diabetes, hyperlipidemia, and ESRD, was independently associated with amputation and all-cause mortality in patients with symptomatic PAD. 4. In a small clinical study, we demonstrated that the levels of A-FABP were significantly higher in patients with PAD than normal controls. In THP-1 cell model, we analyzed the role of cilostazol, medication for PAD treatment, in regulating foam cell formation-associated gene expression. We demonstrated that cilostazol suppresses the upregulation of A-FABP induced by ox-LDL. The expression of CD36, ABCA-1, and MCP-1 were also modulated by cilostazol. Conclusions: In conclusion, our study demonstrated that the multidimensional assessment (skin, muscle, and artery) provided objective, functional, and outcome-predicting information of PAD. We believe that these techniques and parameters will contribute to PAD diasnosis in the future. Besides, we have explored the regulatory mechanism of cilostazol on A-FABP and other foam-cell formation associated gene expression. These results partially explained the anti-atherosclerotic and metabolic modulating effects of cilostazol observed in clinical practice.