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研究生：	陳儒逸 Chen, Ju-Yi
論文名稱：	動脈硬度與肥胖的研究 Study on Arterial Stiffness in Obesity
指導教授：	林立人 Lin, Li-Jen 蔡曜聲 Tsai, Yau-Sheng
學位類別：	博士 Doctor
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2013
畢業學年度：	101
語文別：	英文
論文頁數：	122
中文關鍵詞：	肥胖、動脈硬度、賴胺醯氧化酶、糖尿病、非酒精性脂肪肝
外文關鍵詞：	Obesity, Arterial Stiffness, Lysyl Oxidase, Diabetes, Nonalcoholic Fatty Liver Disease
相關次數：	點閱：125 下載：0
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肥胖是全球性的一種流病。而且越來越多的研究發現過重或肥胖有逐漸增加的趨勢，這與飲食習慣及缺乏運動有很大的關聯。大量的肉類及飽和脂肪酸攝取，沒有規律且適度的有氧運動，都非常容易產生過重或肥胖。臨床上，肥胖與非常多疾病有關聯，例如：高血壓、糖尿病、脂肪肝、代謝症候群、動脈硬化、腦血管疾病、腎臟病、心臟病、及心衰竭。肥胖也容易增加全身性動脈硬度。過多的脂肪堆積、或中心腹部脂肪不當堆積也已被很多研究證實與增加動脈硬度相關，而且年齡分布橫跨年輕到年老。但肥胖與增加動脈硬度的關聯性鮮少被研究，特別是因果關聯相關的研究。因此存在很多未知。
我們知道血管結構蛋白，例如彈力蛋白、膠原蛋白等，以及血管平滑肌細胞都與動脈硬度息息相關。彈力蛋白及膠原蛋白的質與量的異常都可能對動脈硬度產生可觀的影響。而影響彈力蛋白及膠原蛋白的成熟最重要的酵素是賴胺醯氧化酶(LOX)。鮮少研究提及賴胺醯氧化酶、氧化壓力，及發炎細胞與動脈硬度的關聯，特別是與肥胖的關係。
首先，在第二章，我們將探討在原發性高血壓族群中，新發生的糖尿病與中心動脈壓力指數的關係。我們發現中央收縮壓及中心動脈波反彈指數的增加，在原發性高血壓族群中，與新發生的糖尿病有密切相關。在第三章，在非酒精性脂肪肝族群中，我們將揭露中心型肥胖及全身性發炎增加對動脈硬度的影響。我們證實：中心型肥胖是決定動脈硬度是否增加及高敏感度反應蛋白之重要因子。而且，彈性指數(CI)在臨床上，應是一個有用的早期偵測動脈硬度的動脈硬度指數。在第四章，我們進一步研究在糖尿病前期族群中，異常的血糖恆定對動脈硬化的影響。我們發現，相較於非糖尿病族群，糖尿病前期族群有較低的彈性指數(CI)。我們亦發現，在糖尿病前期族群中，增加胰島素抗性與動脈硬度息息相關。最後在第五章，我們將探討肥胖與動脈硬度的獨立相關性，並在人體及動物模式印證。在肥胖鼠動物模式中探討肥胖引起動脈硬度增加的機制。我們發現不論在肥胖鼠或人，動脈硬度都有明顯增加。不穩定的彈力蛋白及蛋白裂解酶活性，都與肥胖鼠動脈硬度明顯增加有關。用賴胺醯氧化酶(LOX)抑制物β-aminopropionitrile (BAPN)治療的老鼠，造成賴胺醯氧化酶(LOX)活性的下降，就足以造成動脈波傳導速率增加及彈力蛋白的斷裂，進而引起動脈硬度明顯增加。而發炎及氧化壓力增加的動脈旁脂肪組織可能是導致上述現象的始作傭者。回歸人類，我們也印證肥胖的人有較低的血中賴胺醯氧化酶(LOX)濃度。
本論文的結論是：我們揭露了動脈硬度增加在肥胖及肥胖相關疾病的臨床重要性。我們的研究更明確提示出賴胺醯氧化酶(LOX)與彈力蛋白的不穩定對動脈硬度增加的明顯影響。

Obesity is a global epidemic. Obesity has been shown to be an important factor associated with cardiovascular mortality, cerebrovascular morbidity, and all-cause mortality in longitudinal studies. Obesity is also well associated with hypertension, diabetes mellitus (DM), and nonalcoholic fatty liver disease (NAFLD). Obesity might also exert adverse effects on the vascular system by increasing arterial stiffness, thus predisposing the individual to premature vascular aging. Excess abdominal visceral fat and larger waist circumference have been identified as important risk factors for accelerated arterial stiffness. Increasing arterial stiffness is believed to play an important role in the development of atherosclerotic cardiovascular diseases, as well as cerebrovascular diseases, and renal disease in different subjects.
Pathophysiology that links obesity to arterial stiffness is still largely unknown. The key cross-linking enzyme for elastin and collagen maturation is lysyl oxidase (LOX). The impaired qualities and decreased quantities of elastin and collagen, reactive oxygen species, and vascular inflammation have also been shown to contribute to arterial stiffness. The exact role of LOX, oxidative stress and inflammation in promoting vascular stiffness in obesity, however, remains unclear.
My first clinical study in chapter 2 is focus on the association of central aortic pressures indices with development of DM in essential hypertension. We found that increased central systolic blood pressure and pressure wave reflection index were associated with future development of new DM in essential hypertension. My second clinical study in chapter 3 is to elucidate the effects of increased systemic inflammation and central obesity on arterial stiffness in patients with NAFLD. We demonstrated that central obesity was an important determinant both for increased arterial stiffness and high-sensitive C reactive protein in NAFLD patients. Compliance index (CI) may be a clinically more useful parameter for arterial stiffness in early assessment of arteriosclerosis in patients with NAFLD. My third clinical study in chapter 4 is to show the effects of deranged glucose homeostasis on peripheral arterial stiffness index in patients with pre-DM. We demonstrated that pre-DM patients have a lower CI, a novel peripheral arterial parameter for arterial stiffness than non-DM patients. Increased insulin resistance may be associated with increased arterial stiffness in pre-DM patients. Finally, in chapter 5, we would dissect the possible mechanisms by which obesity causes the aortic stiffness in obese animal model and to see the relation between LOX and arterial stiffness in human. Our results demonstrated that obesity resulted in increased aortic stiffness in both humans and mice. Both destabilized elastic fiber networks and increased elastolytic activity in the aortas of obese mice contributed to increased aortic stiffness. The downregulation of LOX is sufficient to cause elastin fragmentation and increased pulse wave velocity in wild-type lean mice. Proinflammatory and pro-oxidative adipose tissue surrounding the aorta provide an explanation for LOX attenuation in the aortas of obese mice. Moreover, obese humans have stiffer arteries and lower serum LOX levels.
In conclusion, our studies highlight the importance of the clinical observation of increased arterial stiffness in obesity and obesity related diseases. More important, our study points out potential avenues for exploration of LOX and elastic fiber crosslinking that could illuminate the mechanism behind this clinical observation.

Contents
English abstract………………………………………………………………………i
Chinese abstract……………………………………………………………………..iii
Acknowledgement………………………………………………………………….....v
Abbreviations………………………………………………………………………..vii
List of Figures………………………………………………………………………. ix
List of Tables……………………………….…………………………………........... x
Chapter 1. Introduction……………………………………………………………...1
Chapter 2. Association of Central Aortic Pressures Indices with Development of Diabetes Mellitus in Essential Hypertension……………………………………......5
1 Introduction……………………………………………………………..…......5
2 Methods……………………………………………………………………....7
2.1 Study population……………………………………………………….7
2.2 Measurement of insulin resistance……………………………………8
2.3 Measurement of carotid-to-radial pulse wave velocity and central aortic pressure indices………………………………………………..8
2.4 Statistical analysis…………………………………………………......9
3 Results…………………………………………………………………….....10
3.1 Baseline characteristics………………………………………………10
3.2 Predictors of new DM……………………………………………........10
4 Discussion…………………………………………………………………...11
4.1 Predictors for new DM in hypertension………………………………11
4.2 Central blood pressures, pressure wave reflection indices and DM…..11
4.3 Study limitations……………………………………………………..13
5 Figures and Tables…........…………………………………………….……...15

Chapter 3. Effects of Increased Systemic Inflammation and Central Obesity on Arterial Stiffness in Patients with Nonalcoholic Fatty Liver Disease………..…. .19
1 Introduction…………………………………………………………….…….....19
2 Methods……………………………………………………………….……..21
2.1 Study population…………………………………………………….21
2.2 Clinical and laboratory evaluation……………………………………22
2.3 Abdominal ultrasonography…………………………………………22
2.4 Arterial stiffness measurement……………………………………….23
2.5 Measurement of hs-CRP and adiponectin……………………………..24
2.6 Measurement of insulin and free fatty acid………………………… .25
2.7 Statistical analysis…………………………………………………….25
3 Results……………………………………………………………………….. 26
3.1 Clinical characteristics of each patient group……………………. . . ……26
3.2 The impact of systemic inflammation and central obesity on arterial stiffness in patients with NAFLD……………………………….... . . 26
3.3 The independent predictors of high hs-CRP (>median value) and low CI (<median value) in patients with NAFLD………………………28
4 Discussion………………………………………………………………...….30
4.1 The impact of NAFLD progression on different arterial stiffness indices……………………………………………………………….30
4.2 The impact of central obesity on systemic inflammation, insulin resistance, and arterial stiffness in NAFLD patients……………….. ..32
4.3 Limitations…………………………………………………….……... ...33
5 Figures and Tables…......…………………………………………….……...35

Chapter 4. Effects of Deranged Glucose Homeostasis On Peripheral Arterial Stiffness Index In Patients With Pre-Diabetes Mellitus………………….……. …...41
1 Introduction………………………………………………………………….41
2 Methods……………………………………………………………………. . .43
2.1 Study population……………………………………………………. ..43
2.2 Clinical and laboratory evaluation…………………………………...44
2.3 Measurement of hsCRP, adiponectin, and insulin…………………. …44
2.4 Arterial stiffness measurement………………………………...……….45
2.5 Definitions of metabolic syndrome…………………………………...46
2.6 Statistical analysis……………………………………………………..46
3 Results…………………………………………………………………………..47
3.1 Clinical characteristics between each group……………………....…..47
3.2 The independent determinants of SI and CI with regard to different glucose homeostasis indices in non-DM and pre-DM patients……….48
4 Discussion……………………………………………………………………...50
4.1 Main findings…………………………………………………………..50
4.2 The association between glucose homeostasis indices and different arterial stiffness indices in pre-DM……………………………………50
4.3 Possible mechanisms of increased arterial stiffness in pre-DM…..…….51
4.4 Limitations………………………………………………………...…....53
5 Figures and Tables…......………………………………………………..….54

Chapter 5. Increased Aortic Stiffness and Attenuated Lysyl Oxidase Activity in Obesity…………………………………………………………………………….……59
1 Introduction……………………………………………………………….….59
2 Methods………………………………………………………………….……61
2.1 Animals……………………………………………………………..…61
2.2 PWV measurement in mice………………………………………….61
2.3 AFM…………………………………………………………………61
2.4 Pressure myography…………………………………………………62
2.5 Histological examination and elastic fiber stain…………………….62
2.6 Immunohistochemical and immunofluorescence staining…………..63
2.7 Immunoblot analysis………………………………………………...63
2.8 LOX enzyme activity………………………………….………….….64
2.9 Desmosine analysis…………………………………….……………64
2.10 Elastolytic Activity…………………………………………………65
2.11 Cell culture…………………………………………….….…………..65
2.12 RNA analysis……………………………………………………….65
2.13 Human participants………………………………………………...66
2.14 Arterial stiffness measurement in humans………………………….66
2.15 Data analysis……………………………………………………….67
3 Results…………………………………………………………………….....68
3.1 Higher aortic PWV and stiffness in obese mice……………………...68
3.2 Structural proteins and maturation enzyme in the ob/ob aorta………68
3.3 PVAT and aorta geometry in ob/ob mice…………………………….69
3.4 Higher inflammatory cytokine expression in the PVAT and macrophage infiltrate in the aortas of ob/ob mice…………………..69
3.5 Higher oxidative stress in the aortas and PVAT of ob/ob mice……..70
3.6 Factors attenuating LOX activity……..…………………………..….71
3.7 Effect of LOX inhibition on aortic stiffness in lean and obese mice...71
3.8 Arterial stiffening and lower LOX levels in obese humans…………..72
4 Discussion………………………………………………………….………….73
5 Figures and Tables…......………………………………….......…………….79

Chapter 6. Conclusions……………………………………………………….……....96
References…………………………………………………………………………...…….98
Associated publications during PhD periods….………………………………....…120
                                    

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