隨著世界老化人口的快速增加，與老化相關之疾病：癌症、腎臟和心血管等疾病相關問題也應更加重視之。其中老化對於心血管以動脈血管的鈣化最為顯著，通常伴隨著血管順硬性降低與血管壁的硬化增厚，此病理表現同樣好發於慢性腎臟病和糖尿病。目前已知老化患者體內會累積糖化終產物 (Advanced glycation end-porducts, AGEs) 且先前研究指出AGEs會透過和其受體 (RAGE) 結合或與其他蛋白交叉連結作用造成心血管或其他病變。Aminoguanidine (AG) 為目前研究中最常使用之AGEs 抑制劑，並已於動物實驗中證實其減緩視網膜、腎臟和神經病變。Alagebrium chloride (ALT-711) 為AGEs與其他蛋白交互之阻斷劑，且目前已應用於臨床試驗中，並能明顯降低50歲受試者之血壓且增加其心血觀之彈性。因此，本研究欲探討AGEs在細胞和動物實驗中，對於血管平滑肌之鈣化之角色，且在給予AG和ALT-711是否能得到回復。本研究於兩種細胞(A7r5和RASMC)中同樣使用無機磷和氯化鈣作誘導平滑肌細胞鈣化，以及添加AGEs觀察其對於鈣化的影響，細胞實驗結果顯示AGEs能使平滑肌細胞鈣化的情況更顯著，並再添加其阻斷劑ALT-711後於茜紅素染色結果及西方墨點法結果顯示能顯著回復血管鈣化指標蛋白 (RunX2和BMP-2)之表現。另外，在動物實驗的部分以100 mg/kg/body weight 的AG 和 1 mg/kg/body weight 的ALT-711餵食小鼠，實驗分成14週與8週，8週的小鼠只給予AG。犧牲後取其主動脈並進行蘇木素-伊紅染色、免疫組織染色 (RunX2, BMP-2和AGE)、茜紅素染色 (鈣沉積)及均質化後進行西方墨點法分析鈣化相關蛋白表現，並檢測血清AGE、鈣、磷濃度及尿液鈣和磷之濃度。動物實驗結果顯示於免疫組織染色107週的組別RunX2及茜紅素染色鈣化表現顯著增加，而在給予AG的組別能得到回復，但BMP-2和AGE表現無顯著差異，而骨質相關係數檢測數值呈現同樣趨勢。血清鈣離子濃度於兩個實驗中都呈現同樣上升趨勢而磷濃度無顯著差異；血清AGE濃度僅於8週實驗中檢測，結果顯示AG於14週和53週組別能降低其濃度。然而鈣化相關蛋白表現在給予AG後呈現相似下降趨勢但無顯著差異。綜合以上結果，AGE會促進血管平滑肌細胞之鈣化，但其與老化之間的相關性於動物實驗中尚未能得到有力證據，但處理AG相對於ALT-711於動物實驗中，更具有減緩老化所影響之血管鈣化之趨勢。然而，關於相關蛋白訊號傳遞路徑仍需進一步探討之。

Population of aging around the world is rapidly increasing which is the main risk factor of prevalent diseases such as cancer, kidney diseases and cardiovascular diseases, etc. Calcification of the arterial media is a hallmark of vascular ageing with the characteristics of vascular compliance reduction and vessel wall stiffening enhancement. It is known that advanced glycation end-products (AGEs) accumulated during the aging process and previous studies also indicated that AGEs accelerates cardiovascular diseases and other disorders through two pathway either bind with its receptor (RAGE) or cross-linking with proteins. Aminoguanidine (AG), the most extensively used of the AGEs inhibitors and has been shown to attenuate retinopathy, nephropathy and neuropathy in animal studies. Alagebrium chloride (ALT-711), an novel AGEs crosslink breaker which was already been used in clinical trial with the ability of reducing blood pressure and enhancing the flexibility of vessels in patients over 50 years old. Therefore, the aim of the present study is to investigate the role of AGEs in vascular calcification in vitro and in vivo, and the treatment of AG and ALT-711. We simulate the vascular calcification by Na2HPO4• 2H2O and CaCl2 for 6 days and added with ALT-711 as its inhibitor. The result of in vitro study from alizarin red staining and western blot analysis, showed that AGEs promotes the calcification which was induced by inorganic phosphate and calcium in two types of smooth muscle cell (A7r5 and RASMC); and ALT-711 could alleviate the calcified condition by reducing the expression of RunX2 and BMP-2. As for in vivo study, we perform two animal experiment, 14 week of AG (100 mg/g per body weight) and ALT-711 (1 mg/g per body weight) and 8 week of AG (100 mg/g per body weight) treatment only. Aortas were extracted for hematoxylin-eosin (H&E) stain, IHC stain of RunX2, BMP-2, and AGE, and alizarin red staining for calcium deposition. The expression of calcification-related protein were analyzed by means of western blot. The concentration of serum and urinary calcium and phosphorus and serum AGEs were measured as well. The result of our in vivo study showed that 107 week-old group presented a significant increased amount of calcification in alizarin red staining and the expression of RunX2 but opposite in BMP-2. The result of bone-related value decreased significantly in 107 week-old group. The level of serum calcium elevated in 107 week-old group but no significant changes in phosphate. On the other hand, treated with AG reduced serum AGE level, but the expression of calcification related protein decreased but no significant difference. Taken together, these results indicated that AGEs promotes the calcification in smooth muscle cell and the better effect of AG than ALT-711 on ameliorating the results of aging in our animal model. However, the signaling pathway of reversing vascular calcification should be elucidated, and the evidence of animal experiment in protein level also needed to be analyzed in the future.

口試委員會審定書 ........................................................................................................................ I
致謝 .............................................................................................................................................. II
中文摘要 ..................................................................................................................................... IV
Abstract ...................................................................................................................................... VI
PART 1：Introduction ............................................................................................................... 1
1.1 Aging ................................................................................................................................... 1
1.2 Vascular calcification .......................................................................................................... 2
1.3 Smooth muscle cell ............................................................................................................. 3
1.4 Advanced glycation end-products (AGEs).......................................................................... 4
1.5 Aminoguanidine (AG)......................................................................................................... 6
1.6 Alagebrium chloride (ALT-711) ......................................................................................... 7
PART 2：Aim .............................................................................................................................. 8
PART 3：Materials and Methods ............................................................................................. 9
3.1 Cell culture ........................................................................................................................... 9
3.1.1 A7r5 cell line ................................................................................................................ 9
3.1.2 Primary rat aortic smooth muscle cell (RASMC) ........................................................ 9
3.2 In Vitro Calcification of A7r5 and RASMC ...................................................................... 10
3.3 Preparation of AGEs .......................................................................................................... 10
3.4 Alizarin Red Staining......................................................................................................... 11
3.4.1 Cell staining................................................................................................................ 11
3.4.2 Aorta staining ............................................................................................................. 11
3.5 Measurement of Cell viability ........................................................................................... 12
3.6 Animals .............................................................................................................................. 12
3.6.1 Aged model and treatment with aminoguanidine (AG) and alagebrium chloride for
(ALT-711) for 14 weeks ..................................................................................................... 12
3.6.2 Treatment with aminoguanidine (AG) for 8 weeks. ................................................... 13
3.7 Histological Assessment .................................................................................................... 13
3.8 Micro CT ........................................................................................................................... 14
3.9 Serum AGEs level analysis ............................................................................................ 14
3.10 Protein extraction and Western Blot Analysis ............................................................... 15
3.11 Statistical Analysis ......................................................................................................... 17
PART 4：Results ...................................................................................................................... 18
4.1 Effects of AGEs and ALT-711 on Na 2 HPO 4 • 2H 2 O-induced calcification in vitro-A7r5
cell. 18
4.1.1 Na 2 HPO 4 • 2H 2 O induced A7r5 calcification. ............................................................. 18
4.1.2 AGEs promoted the Na 2 HPO 4 • 2H 2 O induced-calcification via RAGE in A7r5. ...... 19
4.1.3 ALT-711 reversed the AGEs-promoted Na 2 HPO 4 • 2H 2 O induced-A7r5 calcification
limitedly. ............................................................................................................................. 20
4.2 Effects of AGEs and ALT-711 on Na 2 HPO 4 • 2H 2 O-induced calcification in vitro-
RASMC primary cell. ............................................................................................................. 21
4.2.1 Na 2 HPO 4 • 2H 2 O, CaCl 2 and AGEs induced RASMC calcification in a lower
concentration compared with A7r5. .................................................................................... 21
4.2.2 ALT-711 can reverse the AGEs-promoted Na 2 HPO 4 • 2H 2 O induced-RASMC
calcification. ........................................................................................................................ 22
4.3 Effects of AG and ALT-711 on aortic calcification in vivo. ............................................. 23
4.3.1 AG inhibits calcification not ALT-711 in vivo. ......................................................... 23
4.3.2 Effects of AG and ALT-711 on bone, calcium and phosphorus metabolism in vivo. 24
4.4 Effects of AG on aortic calcification in vivo. .................................................................... 26
4.4.1 No effects of AG on the body weight, food and water consumption in vivo. ............ 26
4.4.2 AG reduced serum AGEs level in vivo. ..................................................................... 26
4.4.3 Effects of AG on calcium and phosphorus metabolism in vivo. ................................ 26
4.4.4 Effects of AG treatment on protein expression of calcification-related proteins in
vivo. ..................................................................................................................................... 27
PART 5：Discussion ................................................................................................................. 28
PART 6：Conclusion ................................................................................................................ 35
PART 7：Figures and legends ................................................................................................. 36
Figure 1. Na2HPO4•2H2O induced A7r5 cell calcification. ................................................... 37
Figure 2. Effect of Na2HPO4•2H2O on cell viability. ............................................................. 38
Figure 3. Effect of Na2HPO4•2H2O on the expression of BMP signaling molecules in
response to smooth muscle calcification in vitro. ................................................................... 39
Figure 4.The effect of AGEs in Na2HPO4•2H2O induced calcification. ................................ 41
Figure 5. The combination treatment of Na2HPO4•2H2O and AGEs accelerated calcification
through AGE receptor and BMP signaling pathway. .............................................................. 43
Figure 6. The effects of ALT-711 on AGEs accelerated phosphate-calcium calcification in
A7r5. 44
Figure 7. Effect of Na2HPO4•2H2O, CaCl2, AGEs and ALT-711 on calcification ................ 46
Figure 8. Effects of ALT-711 in protein expression of BMP signaling pathway associated
protein. .................................................................................................................................... 47
Figure 9. The effects of the combination treatment of Na 2 HPO 4 • 2H 2 O, CaCl 2 , AGEs and
ALT-711 on cell morphology. ................................................................................................ 48
Figure 10. The effects of the combination treatment of Na 2 HPO 4 • 2H 2 O, CaCl 2 , AGEs and
ALT-711 on calcification. ....................................................................................................... 51
Figure 11. The effects of the combination treatment of Na 2 HPO 4 • 2H 2 O, CaCl 2 , AGEs and
ALT-711 on cell viability. ....................................................................................................... 52
Figure 12. The effects of the combination treatment of Na 2 HPO 4 • 2H 2 O, CaCl 2 , AGEs and
ALT-711 with lower concentration on cell viability. .............................................................. 53
Figure 13. The effects of the combination treatment of Na 2 HPO 4 • 2H 2 O, CaCl 2 , AGEs and
ALT-711 with lower concentration on calcification. .............................................................. 54
Figure 14. The expression of the BMP signaling pathway associated protein in combination
treatment of Na2HPO4•2H2O, CaCl2, AGEs and ALT-711 with lower concentration. .......... 56
Figure 15. The effects of the combination treatment of Na2HPO4•2H2O, CaCl2, AGEs and
ALT-711 with final concentration on cell morphology and calcification. .............................. 58
Figure 16. The effects of AG and ALT-711 on aorta in vivo. ................................................. 60
Figure 17. The effects of AG and ALT-711 on aorta calcification in vivo. ............................ 62
Figure 18. The immunohistochemical changes of RunX2 expression in aortas of mice with
AG and ALT-711 treatment. ................................................................................................... 64
Figure 19. The immunohistochemical changes of BMP-2 expression in aortas of mice with
AG and ALT-711 treatment. ................................................................................................... 66
Figure 20. The immunohistochemical changes of AGE expression in aortas of mice with AG
and ALT-711 treatment. .......................................................................................................... 68
Figure 21. Effects of AG on mice bones. ................................................................................ 70
Figure 22. Effects of AG and ALT-711 treatment on urinary and serum measurement of
calcium and phosphorus concentration in vivo........................................................................ 71
Figure 23. Effects of AG on the body weight, food and water consumption in vivo. ............. 72
Figure 24. Effects of AGs on mice aortas. .............................................................................. 73
Figure 25. Effects of AG on serum AGE concentration in vivo. ............................................. 74
Figure 26. Effects of AG treatment on urinary and serum measurement of calcium and
concentration in vivo. .............................................................................................................. 76
Figure. 27 Effects of AG on protein expression of calcification-related proteins in vivo. ...... 77
Figure 28. Effects of AG treatment on protein expression of calcification-related proteins in
vivo. 78
PART 8：References ................................................................................................................ 79

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