臺灣博碩士論文加值系統

全身性的膽固醇代謝失調是導致動脈粥狀硬化及脂肪肝發生的主因之一。體內膽固醇的運送包括兩個主要階段：將腸道吸收的膽固醇送往周邊組織作運用而多餘的膽固醇則透過脂蛋白送回肝臟作進一步代謝。當血液中的膽固醇含量過高時，便容易堆積在血管壁的巨噬細胞中或是累積在肝臟細胞中，導致動脈粥狀硬化或是脂肪肝，而目前已知有許多細胞激素參與並調控此病理機轉。半胱胺酸蛋白61，屬於細胞外基質蛋白，在胚胎發育的血管新生中扮演很重要的角色。研究亦發現，在動脈粥狀硬化的病變處，半胱胺酸蛋白61表現量明顯增加，然而其在全身性的膽固醇代謝及相關疾病中所扮演的角色仍非常不明確。首先，我們發現半胱胺酸蛋白61大量表現於載脂蛋白E基因剔除小鼠的主動脈的粥樣硬化病變處，且主要是位於巨噬細胞衍生之泡沫細胞的位置。而細胞實驗中，給予氧化的低密度脂蛋白則顯著的提升半胱胺酸蛋白61在巨噬細胞中的表現量，且半胱胺酸蛋白61加劇了氧化低密度脂蛋白所造成的脂質堆積現象。主要是因為半胱胺酸蛋白61令載脂蛋白A及高密度脂蛋白專一的細胞膽固醇排除功能失調，且是基於三磷酸腺苷結合盒轉運體A1和G1及肝異受體alpha的蛋白表現量受到調降的影響所致。而在過量表現半胱胺酸蛋白61於巨噬細胞中亦得到相似的結果，膽固醇排除及三磷酸腺苷結合盒轉運體A1和G1與肝異受體alpha的表現量接下降。除此之外，腹腔注射半胱胺酸蛋白61於載脂蛋白E基因剔除小鼠，惡化其高血脂、全身性發炎反應及動脈粥狀硬化的生成。半胱胺酸蛋白61亦降低其膽固醇排除的能力及主動脈中相關蛋白質表現。值得注意的是，半胱胺酸蛋白61加劇肝臟的脂質堆積其調控肝臟與膽固醇代謝相關脂蛋白表現，包括：肝異受體alpha、三磷酸腺苷結合盒轉運體G5和G8、膽固醇7alpha-羥化酶、低密度脂蛋白受體和前蛋白轉化酶枯草溶菌素9。總結來說，我們的研究發現，半胱胺酸蛋白61惡化粥狀動脈硬化及脂肪肝的生成透過促進泡沫細胞形成及損害肝臟細胞之膽固醇代謝的能力。

Deregulation of cholesterol metabolism plays a critical role in pathogenesis of atherosclerosis or liver steatosis. Cholesterol delivers to peripheral tissues for usage and remains cholesterol transports back to liver for clearance are two key events in cholesterol homeostasis. When the circulating levels of cholesterol are overloaded, lipid will accumulate in macrophage foam cells or in liver, leading to atherosclerosis or liver steatosis. Several cytokines are known to participate in deregulation of cholesterol metabolism in atherosclerosis or liver steatosis. Cysteine-rich protein 61 (CCN1), an extracellular matrix cytokine, plays an important role in embryonic angiogenesis. Additionally, CCN1 can be detected in lesion during the development of atherosclerosis. However, the role of CCN1 in regulation of cholesterol metabolism and related metabolic diseases is largely unknown. Our results demonstrated that the protein level of CCN1 was increased in atherosclerotic aortas and the predominant expression of CCN1 was found in foamy macrophage of atherosclerotic lesions of apolipoprotein E–deficient (apoE−/−) mice. In macrophages, treatment with oxidized low-density lipoprotein (oxLDL) increased the CCN1 expression and CCN1 exacerbated oxLDL-induced lipid accumulation. Furthermore, CCN1 impaired the apolipoprotein AI- and high-density lipoprotein-dependent cholesterol efflux by down-regelation the expression of ATP-binding cassette transporter A1 (ABCA1) and ABCG1 and liver X receptor alpha(LXR alpha). Similarly, overexpression of CCN1 in macrophage decreased cholesterol efflux and the expression of ABCA1 and ABCG1. Moreover, administration with CCN1 in apoE−/− mice worsened hyperlipidemia, systemic inflammation and atherosclerosis. CCN1 also decreased the capacity of cholesterol efflux and down-regulated protein expression of ABCA1 and ABCG1 in aortas. Notably, CCN1 exacerbated lipid accumulation in liver and down-regulated the expression of cholesterol clearance-related proteins, including ABCG5, ABCG8, LXR alpha cholesterol 7 alpha-hydrolase, LDL receptor and proprotein convertase subtilisin/kexin type 9. In conclusion, our findings suggest that CCN1 exacerbates atherosclerosis and liver steatosis by promoting macrophage foam cell formation and impairing hepatic cholesterol metabolism.

致謝 VII
Abbreviations VIII
Abstract X
摘要 XII
Introduction 1
1.1 CCN family 1
1.2 CCN1/CYR61 2
1.3 Biological function of CCN1 3
1.4 CCN1 in pathologies 4
2.1 Atherosclerosis 4
2.2 Lipoproteins 5
2.3 Progression of early-stage of atherosclerosis 6
2.4 The homeostasis of cholesterol metabolism in macrophages 8
2.5 Atherosclerotic lesion progression 10
2.6 The role of liver in reverse cholesterol transport system 11
Objective 13
Materials and methods 14
1. Reagents 14
2. Mice 15
3. Histological and immunohistochemical examination 15
4. Cell culture 16
5. Preparation of tissue and cell lysates 17
6. Western blot analysis 17
7. Preparation of oxLDL 18
8. Dil-oxLDL binding assay 18
9. Cholesterol efflux assay 19
10. Reverse transcription polymerase chain reaction (RT-PCR) 20
11. Transient Transfection 21
12. Cholesterol and triglyceride measurement 22
13. Serum lipid profile analysis 22
14. Measurement of inflammatory cytokines 22
15.Statistical analysis 23
Results 24
1. Expression of CCN1 is increased in atherosclerotic lesions of apoE−/− mice. 24
2. oxLDL increases CCN1 expression and CCN1 exacerbates oxLDL-induced lipid accumulation in macrophages. 24
3. CCN1 does not affect oxLDL internalization and protein expression of CD36, SR-A, LAL and ACAT-1. 25
4. CCN1 decreases apoAI- and HDL-dependent cholesterol efflux and related protein expression in macrophages. 25
5. CCN1 provokes down-regulation of LXRa , ABCA1 and ABCG1 in macrophages. 26
6. Overexpression of CCN1 in macrophages decreases cholesterol efflux efficiency and related protein expression. 27
7. CCN1 exacerbates atherosclerosis, hyperlipidemia and inflammation in apoE−/− mice. 27
8. CCN1 decreases cholesterol efflux efficiency and RCT-related protein expression in aortas of apoE−/− mice. 28
9. CCN1 deregulates cholesterol metabolism and related protein expression in liver of apoE−/− mice. 29
10. Effects of CCN1 on body weight, fat weight and fat morphology in apoE−/− mice. 30
11. CCN1 increases the levels of smooth muscle cell in fibrous cap of atherosclerotic lesions in apoE−/− mice. 30
Discussion 31
References 40

Figures 58
Figure 1. Expression of CCN1 is increased in atherosclerotic lesions of apoE−/− mice. 58
Figure 2. Expression of CCN1 is increased in atherosclerotic lesions of apoE−/− mice. 59
Figure 3. OxLDL induces CCN1 expression in macrophages. 60
Figure 4. CCN1 exacerbates oxLDL-induced lipid accumulation in macrophages. 61
Figure 5. CCN1 does not affect oxLDL internalization. 62
Figure 6. CCN1 does not affect the protein level of scavenger receptors in macrophages. 63
Figure 7. CCN1 does not affect cholesterol storage-related protein level in macrophages 64
Figure 8. CCN1 reduces apoAI- and HDL-dependent cholesterol efflux in macrophages. 65
Figure 9. CCN1 decreases cholesterol efflux related protein expression in macrophages. 66
Figure 10. CCN1 reduces mRNA level of ABCA1 and ABCG1 in macrophages. 67
Figure 11. CCN1 decreases LXRa protein expression and its activity in macrophages. 68
Figure 12. Overexpression of CCN1 in macrophages decreases cholesterol efflux related protein expression. 69
Figure 13. Overexpression of CCN1 in macrophages decreases apoAI- and HDL-dependent cholesterol efflux efficiency. 70
Figure 14. CCN1 exacerbates atherosclerotic lesion size in apoE−/− mice. 71
Figure 15. CCN1 worsens dyslipidemia in apoE−/− mice. 72
Figure 16. CCN1 elevates CETP activity and protein expression in apoE−/− mice. 73
Figure 17. CCN1 aggravates inflammation in apoE−/− mice. 74
Figure 18. CCN1 reduces cholesterol efflux efficiency in apoE−/− mice. 75
Figure 19. CCN1 decreases RCT-related protein expression in aortas of apoE−/− mice. 76
Figure 20. CCN1 worsens cholesterol metabolism in liver of apoE−/− mice. 77
Figure 21. CCN1 disrupts cholesterol metabolism related protein expression in liver of apoE−/− mice. 78
Figure 22. Effects of CCN1 on body weight, fat weight and fat morphology in apoE−/− mice. 79
Figure 23. CCN1 increases the levels of smooth muscle cell in fibrous cap of atherosclerotic lesions in apoE−/− mice. 80
Figure 24. A proposed model with the role of CCN1 in systemic cholesterol homeostasis in apoE−/− mice. 81

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