血管新生是一種為原有的血管延伸成新的血管的生理現象。血管新生通常會發生像是在胚胎發育時期、傷口癒合和修復的現象。近幾年抑制血管新生為治療癌症研究的策略。Vasostatin為抗血管新生抑制者之一它是calreticulin 1-180 個胺基N端的部分，具有抑制腫瘤生長的功能。在先前的研究發現，Vasostatin48為Vasostatin180後面位置133-180的胺基酸片段，具有抑制脈絡膜血管新生雞絨毛膜血管新生的功能。但是目前在Vasosoatin48抑制血管新生的機制還未知。本研究主要是要探討Vasostatin在抑制血管新生中扮演的角色和VS48在內皮細胞膜上的接受器。 首先利用細胞表面結合的活性測試了解Vasoatatin是否會與細胞膜上面的接受器做結合。之後探討Vasostatin在抑制血管新生中的功能利用擴增、移動、管狀形成來測試。我們發現重組蛋白TRX-VS48會專一性與內皮細胞表面做結合，之後分離內皮細胞的膜蛋白與TRX-VS48相互作用利用親和力層析法將此結合蛋白質洗滌出來應用銀染做分析，出現在膠體上的可能為TRX-VS48在內皮細胞表面的接受器。在抑制血管新生的功能方面，發現TRX-VS48會抑制內皮細胞的擴增、轉移和管狀結構的形成。我們未來會繼續探討TRX-VS48在抗血管新生的機制。這些結果可以幫助我們未來將VS48應用在抑制血管新生和治癌相關的藥物上。

Angiogenesis is a physiological process involving the growth of new blood vessels from pre-existing vessels. Angiogenesis takes place in physiological situations, such as embryonic development, wound healing and reproduction. Endothelial cells, which constitute the vessels, become activated, detach, migrate, and proliferate to form new sprouts. Angiogenesis is essential for the growth and metastasis of cancer. Anti-angiogensis strategy has been investigated for cancer therapeutic in recent years. In present studies, Vasostatin48 (VS48) is the 133–180 N-terminal domain of calreticulin, inhibits Choroidal neovascularization (CNV) and CAM assay. However, the anti-angiogenic mechanism and receptor(s) of VS48 is still unclear. The specific aim of this study is to investigate anti-angiogenic function of VS48 and to identify the membrane receptor(s) of VS48. Cell surface binding assay was used to bind ability and to identify the plasma membrane receptor of VS48. The proliferation, migration, and tube formation assay was used to study the anti-angiogenic effect of VS48. Isolating the EA.hy962 endothelial cells plasma membrane proteins which interaction with TRX-VS48 by affinity chromatography. We found that Thioredoxin-vasostatin48 (TRX-VS48) specifically bound with the cell surface of endothelial cells. And the TRX-VS48 inhibited the proliferation, migration and suppressed the tube formation of endothelial cells. The EA.hy926 membrane proteins interacted with TRX-VS48 and to elute these fractions by affinity chromatography. These fractions used the silver stain and the bands are the candidate receptor(s) of TRX-VS48 on endothelial cells. We will investigate the anti-angiogenic mechanism of VS48 in the future. These results suggested that VS48 could be an anti-angiogenesis drug for treatment of anti-angiogenic and cancer

Abstract in Chinese………..……………………………….2
Abstract in English……………………………………………3
Introduction……………………………………………………5
Specific Aims…………………………………………………..12
Materials and Methods……………………………………….13
Results…………………………………………………………21
Purification of the recombinant proteins TRX, TRX-VS48, VS48 and VS132……………………………………………21
The TRX-VS48 and VS48 proteins can bind with endothelial cells specifically……………………………………………………21
The TRX-VS48 and VS48 can uptake in EA.hy926 cells………….22
The TRX-VS48 and VS48 inhibited the proliferation in EA.hy926 cells…………………………………………………………22
The VS48 inhibited the migration in EA.hy926 cells……………....22
The TRX-VS48 inhibited the tube formation in HUVEC cells........23
The stability of VS48 proteins storage at -80 ℃, -20 ℃, and 4 ℃ for 7 days and still remained to inhibition of the proliferation in EA.hy926cells………………………………………………23
The stability of VS48 proteins storage at -80 ℃, -20 ℃, and 4 ℃ for7 days and still remained to inhibition of the migration in EA.hy926 cells…………………………………………………………24
Identification of VS48 receptors by affinity chromatography.........24
Discussion…………………………………………………26
Figures and legends………………………………………31
References…………………………………………………43

1 A. Abdollahi, P. Hahnfeldt, C. Maercker, H. J. Grone, J. Debus, W. Ansorge, J. Folkman, L. Hlatky, and P. E. Huber, 'Endostatin's Antiangiogenic Signaling Network', Mol Cell, 13 (2004), 649-63.
2 Y. S. Bee, S. J. Sheu, Y. L. Ma, H. C. Lin, W. T. Weng, H. M. Kuo, H. C. Hsu, C. H. Tang, J. C. Liou, and M. H. Tai, 'Topical Application of Recombinant Calreticulin Peptide, Vasostatin 48, Alleviates Laser-Induced Choroidal Neovascularization in Rats', Mol Vis, 16 (2010), 756-67.
3 P. Carmeliet, 'Angiogenesis in Life, Disease and Medicine', Nature, 438 (2005), 932-6.
4 P. Carmeliet, and R. K. Jain, 'Molecular Mechanisms and Clinical Applications of Angiogenesis', Nature, 473 (2011), 298-307.
5 A. Carneiro, M. Falcao, A. Pirraco, P. Milheiro-Oliveira, F. Falcao-Reis, and R. Soares, 'Comparative Effects of Bevacizumab, Ranibizumab and Pegaptanib at Intravitreal Dose Range on Endothelial Cells', Exp Eye Res, 88 (2009), 522-7.
6 S. Frokjaer, and D. E. Otzen, 'Protein Drug Stability: A Formulation Challenge', Nat Rev Drug Discov, 4 (2005), 298-306.
7 A. Grothey, and C. Allegra, 'Antiangiogenesis Therapy in the Treatment of Metastatic Colorectal Cancer', Ther Adv Med Oncol, 4 (2012), 301-19.
8 M. K. Gupta, and R. Y. Qin, 'Mechanism and Its Regulation of Tumor-Induced Angiogenesis', World J Gastroenterol, 9 (2003), 1144-55.
9 D. Hanahan, and J. Folkman, 'Patterns and Emerging Mechanisms of the Angiogenic Switch During Tumorigenesis', Cell, 86 (1996), 353-64.
10 K. Javaherian, T. Y. Lee, R. M. Tjin Tham Sjin, G. E. Parris, and L. Hlatky, 'Two Endogenous Antiangiogenic Inhibitors, Endostatin and Angiostatin, Demonstrate Biphasic Curves in Their Antitumor Profiles', Dose Response, 9 (2011), 369-76.
11 R. Khurana, M. Simons, J. F. Martin, and I. C. Zachary, 'Role of Angiogenesis in Cardiovascular Disease: A Critical Appraisal', Circulation, 112 (2005), 1813-24.
12 L. Lu, F. Payvandi, L. Wu, L. H. Zhang, R. J. Hariri, H. W. Man, R. S. Chen, G. W. Muller, C. C. Hughes, D. I. Stirling, P. H. Schafer, and J. B. Bartlett, 'The Anti-Cancer Drug Lenalidomide Inhibits Angiogenesis and Metastasis Via Multiple Inhibitory Effects on Endothelial Cell Function in Normoxic and Hypoxic Conditions', Microvasc Res, 77 (2009), 78-86.
13 L. Ma, L. Luo, H. Qiao, X. Dong, S. Pan, H. Jiang, G. W. Krissansen, and X. Sun, 'Complete Eradication of Hepatocellular Carcinomas by Combined Vasostatin Gene Therapy and B7h3-Mediated Immunotherapy', J Hepatol, 46 (2007), 98-106.
14 F. Musumeci, M. Radi, C. Brullo, and S. Schenone, 'Vascular Endothelial Growth Factor (Vegf) Receptors: Drugs and New Inhibitors', J Med Chem (2012).
15 F. Nussenbaum, and I. M. Herman, 'Tumor Angiogenesis: Insights and Innovations', J Oncol, 2010 (2010), 132641.
16 N. M. Pandya, N. S. Dhalla, and D. D. Santani, 'Angiogenesis--a New Target for Future Therapy', Vascul Pharmacol, 44 (2006), 265-74.
17 L. Persano, M. Crescenzi, and S. Indraccolo, 'Anti-Angiogenic Gene Therapy of Cancer: Current Status and Future Prospects', Mol Aspects Med, 28 (2007), 87-114.
18 S. E. Pike, L. Yao, K. D. Jones, B. Cherney, E. Appella, K. Sakaguchi, H. Nakhasi, J. Teruya-Feldstein, P. Wirth, G. Gupta, and G. Tosato, 'Vasostatin, a Calreticulin Fragment, Inhibits Angiogenesis and Suppresses Tumor Growth', J Exp Med, 188 (1998), 2349-56.
19 S. E. Pike, L. Yao, J. Setsuda, K. D. Jones, B. Cherney, E. Appella, K. Sakaguchi, H. Nakhasi, C. D. Atreya, J. Teruya-Feldstein, P. Wirth, G. Gupta, and G. Tosato, 'Calreticulin and Calreticulin Fragments Are Endothelial Cell Inhibitors That Suppress Tumor Growth', Blood, 94 (1999), 2461-8.
20 M. Potente, H. Gerhardt, and P. Carmeliet, 'Basic and Therapeutic Aspects of Angiogenesis', Cell, 146 (2011), 873-87.
21 S. J. Sheu, Y. S. Bee, Y. L. Ma, G. S. Liu, H. C. Lin, T. L. Yeh, J. C. Liou, and M. H. Tai, 'Inhibition of Choroidal Neovascularization by Topical Application of Angiogenesis Inhibitor Vasostatin', Mol Vis, 15 (2009), 1897-905.
22 B. Sitohy, J. A. Nagy, and H. F. Dvorak, 'Anti-Vegf/Vegfr Therapy for Cancer: Reassessing the Target', Cancer Res, 72 (2012), 1909-14.
23 Q. M. Sun, L. Cao, L. Fang, C. Chen, J. Dai, L. L. Chen, and Z. C. Hua, 'Expression, Purification of Human Vasostatin120-180 in Escherichia Coli, and Its Anti-Angiogenic Characterization', Protein Expr Purif, 39 (2005), 288-95.
24 T. Tanaka, Y. Cao, J. Folkman, and H. A. Fine, 'Viral Vector-Targeted Antiangiogenic Gene Therapy Utilizing an Angiostatin Complementary DNA', Cancer Res, 58 (1998), 3362-9.
25 H. Toge, T. Inagaki, Y. Kojimoto, T. Shinka, and I. Hara, 'Angiogenesis in Renal Cell Carcinoma: The Role of Tumor-Associated Macrophages', Int J Urol, 16 (2009), 801-7.
26 W. Wang, 'Instability, Stabilization, and Formulation of Liquid Protein Pharmaceuticals', Int J Pharm, 185 (1999), 129-88.
27 S. M. Weis, and D. A. Cheresh, 'Tumor Angiogenesis: Molecular Pathways and Therapeutic Targets', Nat Med, 17 (2011), 1359-70.