隨者生活水平的提升、飲食習慣的改變，高尿酸血症正在成為一個重要且常見的醫學問題，其流行率和相關的合併症在過去幾十年中急劇攀升。近年來研究顯示，體內尿酸含量增高會增加罹患心血管疾病的風險，甚至提升心血管疾病的死亡風險。在臨床上，尿酸濃度過高易產生腎臟及血管方面的疾病，而痛風為最常見疾病之一，過高的尿酸會在體內形成尿酸鈉鹽堆積，尿酸鈉鹽結晶因沉積於關節、軟骨、肌腱等或軟組織中而造成一種發炎性疾病；高尿酸( Hyperuricemia ) 環境亦容易造成血管與內皮內的發炎，促使內皮細胞一氧化碳( Nitric Oxide )含量降低、平滑肌細胞增生，進一步產生動脈血管粥狀硬化，但高尿酸血症引發動脈硬化與增加心血管風險的原因仍不是很清楚。
內皮前驅細胞（endothelial progenitor cells, EPC）為1997年由日本學者Dr. Asahara發現，其同時具有幹細胞及內皮細胞的特性，其功能可以修復內皮細胞以及血管新生的作用。近年來對於內皮前驅細胞已有明確的定義及相關分離方式，如此有助於更加了解 EPC 參與血管新生所扮演的角色；內源性血管新生可促使腫瘤的發展、傷口癒合、嚴重下肢缺血及心肌缺血。由Takahashi T等人也提出內源性刺激，如組織缺血，與外源性細胞因子治療，如granulocyte macrophage-colony stimulating factor（GM-CSF），皆可導致內皮前驅細胞產生遷移行為的發生和促使缺血組織內產生血管新生的進行。由骨髓分離出的內皮前驅細胞，可透過內源性組織缺血的誘導以及經由外源性的細胞因子（cytokine），可使內皮前驅細胞從而具有遷移的能力。由於尿酸對於內皮細胞、內皮前驅細胞與缺氧組織血管新生的影響和其作用機轉尚未清楚。因此本研究藉由體外試驗研究高濃度尿酸對內皮前驅細胞的影響及其機制探討，高尿酸環境下是否對血管內皮產生傷害，並阻礙缺氧組織誘發血管新生的預後情形；藉由體內試驗觀察高尿酸血症老鼠在下肢缺血後誘發血管新生及血流恢復之程度，以了解高尿酸血症與缺氧組織中血管新生的相關性。
在本研究中，我們發現高濃度尿酸的刺激下，內皮細胞內的氧化壓力及發炎因子會大量表現；同時對內皮前驅細胞之一氧化氮產量、細胞遷移與管狀成形能力皆有抑制作用，氧化壓力及細胞凋亡的程度則有提高的現象。實驗亦發現高濃度尿酸會活化內皮細胞 NF-κB、內皮前驅細胞 eNOS 和 JNK 的表現，分別加入抗氧化劑、一氧化氮補充劑與拮抗劑後，可改善高濃度尿酸對細胞所引發的傷害；動物實驗發現高尿酸模式下的小鼠，經由下肢缺血創傷後，會降低血流的恢復能力；高尿酸鼠再搭配尿酸飲食(嚴重高尿酸鼠) 之血流恢復力又比高尿酸鼠更差，相較於對照組則為更差。藉由釐清高濃度尿酸環境對於血管內皮細胞、內皮前驅細胞與組織缺氧過程對血管新生的影響，這些研究結果對未來臨床醫師在心血管疾病控制與治療上有更進一步的了解。

In the past decades, hyperuricemia is becoming a critical medical problem, and its prevalence and related comorbidities have dramatically increased worldwide. Uric acid (UA) is the final breakdown product of purine metabolism, degraded by urate oxidase to allantoin and freely excreted in the urine. Uric acid metabolism of abnormalities may cause hyperuricemia, gout, and hyperuricemia-related morbidity. Clinical studies have demonstrated that hyperuricemia may be associated with cardiovascular disease, independent of traditional risk factors. Furthermore, elevated serum uric acid levels in the circulation are associated with endothelial dysfunction, induced systemic inflammation, increased oxidative stress, and enhanced risk of cardiovascular mortality.
Endothelial progenitor cells (EPCs) are special sub-type progenitor cells with the ability to differentiate into mature endothelial cells and contribute to reendothelialization and neovascularization. Accumulating evidence indicates that neovascularization in adults is not solely the result of angiogenesis but may also involve bone marrow (BM)-derived EPCs in the process of vasculogenesis. These circulating EPCs can be mobilized endogenously in response to tissue ischemia or exogenously by cytokine stimulation. However, the effects of chronic hyperuricemia on cultured endothelial cell and ischemia-induced neovascularization, EPC mobilization and EPC angiogenic functions remain unclear. In this study, we aimed to investigate the impact of high concentration of uric acid on endothelial cell and EPCs functions, and further tested the hypothesis that chronic hyperuricemia can inhibit ischemia-induced neovascularization and attenuate blood flow recovery in a mouse model of hindlimb ischemia.
Incubation human umbilical vein endothelial cells (HUVEC) with high concentrations of uric acid medium significantly suppressed cell proliferation and elevated reactive oxygen species (ROS) production. High uric acid concentrations promoted cellular inflammation in cultured HUVEC through regulated the NF-κB pathway. Moreover, high uric acid levels significantly suppressed EPC proliferation, and down-regulated phosphorylation of eNOS and Akt. High concentrations of uric acid also promoted cellular apoptosis and enhanced oxidative stress, as well as attenuated nitric oxide (NO) production and EPC functions. On the other hand, administration of NAC (antioxidant), SNP (NO donor) and JNK inhibitors reversed high concentration uric acid-impaired EPC functions. Chronic hyperuricemia mice had significantly diminished ischemia-induced tissue reperfusion, EPC mobilization, and impaired neovascularization in the ischemic hindlimbs compared with the control mice. Our result provided that chronic hyperuricemia impaired endothelial and its function thought oxidative stress and diminished blood flow recovery and EPC mobilization in response to tissue ischemia.

論文電子檔著作權授權書 i
論文審定同意書 ii
誌謝 Acknowledgements iii
目錄… iv
English Abstract viii
Chinese Abstract x
List of Abbreviations xii
1. Introduction 01
1.1 Hyperuricemia and cardiovascular disease 01
1.2 The clinical relationship between uric acid and endothelial function 01
1.3 Role of endothelial and endothelial progenitor cells in cardiovascular
diseases 02
1.4 Angiogenesis and Endothelial progenitor cells 03
1.5 Aim and hypothesis of the study 03
2. Materials and methods 05
2.1 In vitro study 05
2.1.1 Reagents 05
2.1.2 Cell culture 05
2.1.3 EPC characterization 06
2.1.4 Cell survival 06
2.1.5 EPC tube formation assay 07
2.1.6 EPC migration 07
2.1.7 EPC senescence 08
2.1.8 Nitric oxide (NO) assay 08
2.1.9 Reactive oxygen species (ROS) production assay 08
2.1.10 Western blotting 09
2.2 In vivo experiments 09
2.2.1 Animals 09
2.2.2 Hyperuricemia model 09
2.2.3 Mouse hindlimb ischemic model 10
2.2.4 Measurement of serum levels of uric acid, creatinine, blood urea nitrogen, Glutamic Oxaloacetic Transaminase and Glutamic Pyruvic Transaminase 10
2.2.5 Reactive oxygen species measurement in mice 11
2.2.6 Nitric oxide measurement in mice 11
2.2.7 EPC mobilization in the hyperuricemia hindlimb ischemic model 11
2.3. Histology and Immunohistochemistry… 12
2.3.1 Measurement of capillary density in the ischemic leg 12
2.4. Statistical analysis 12
3. Results 13
3.1 Uric acid elevated inflammation of endothelial cell 13
3.1.1 Effects of uric acid on Human umbilical vein endothelial cells (HUVEC) survival 13
3.1.2 High-level uric acid stimulates HUVEC reactive oxygen species production 13
3.1.3 High-level uric acid inactivates HUVEC SirT1 proteins but activates IL-1β and Tissue Factor 14
3.1.4 Antioxidant reverses the high-level uric acid-induced TNFα, IL-1β and high-level uric acid-induced attenuated SirT1 15
3.1.5 Uric acid promotes cell phosphorylation of p65, Tissue Fact and IL-1β 15
3.1.6 High-level uric acid promoter phosphorylation of p65 pathway through regulate activation of SirT1 expression 16
3.1.7 SirT1 activator restores uric acid-induced Tissue Factor, TNFα and IL-1β expression 17
3.2 Hyperuricemia effects on endothelial progenitor cells and function 18
3.2.1 Characterization of human EPC 18
3.2.2 Effects of uric acid on EPC survival and apoptosis 18
3.2.3 High-level uric acid reduce EPC phosphorylation of eNOS and Akt 19
3.2.4 High-level uric acid reduced nitric oxide (NO) production on EPC 19
3.2.5 High-level uric acid stimulates EPC reactive oxygen species (ROS) production 20
3.2.6 High-level uric acid activates by JNK pathway on EPC 20
3.2.7 High-level uric acid activates EPC caspase 3 & 9 proteins 21
3.2.8 High-levels of uric acid facilitate cellular senescence and activation of p16 expression and suppress activation of SirT1 expression 22
3.2.9 High-levels of uric acid reduce EPC migration and tube formation 23
3.2.10 Antioxidant, JNK inhibitor, and NO donor reverse the high-level uric acid- attenuated of late EPCs survival 23
3.3 Chronic hyperuricemia impairs blood flow recovery in the
ischemic hindlimb 24
3.3.1 C57BL/6J mice-induced chronic hyperuricemia model and have no
accompanied with the side effect 24
3.3.2 Chronic hyperuricemia impairs ischemia-induced blood flow recovery and new vessels formation 24
3.3.3 High-level uric acid reduces endothelial progenitor cell mobilization 25
3.3.4 Allopurinol rescues chronic hyperuricemia impairs ischemia-induced blood flow recovery 26
3.3.5 Allopurinol improves chronic hyperuricemia-induced reactive oxygen
species production 26
3.3.6 Allopurinol improves chronic hyperuricemia-suppressed nitric oxide
production ischemia-induced blood flow recovery 27
4. Discussion 28
5. Conclusions 34
6. Perspectives 35
7. References 36
8. Figures and Table 43
9. Publication 74

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