血管新生作用（Angiogenesis）是指從舊有血管長出新血管的過程，其包含了內皮細胞的細胞間質分解、內皮細胞的移動與增生，最後形成血管。近來有不少證據顯示，血管新生是腫瘤生長與轉移所必須的，因此，抑制血管新生是治療癌症方法之一。血管靜止蛋白（Angiostatin）最初是從攜有Lewis lung cancer的老鼠血清及尿液中分離出的，是一種能抑制血管新生的內生性蛋白質，分子量為38KDa，是血纖維蛋白溶脢原（Plasminogen）經由酵素切割所釋出具有kringle結構的片段，而每個kringle結構則具有三個雙硫鍵。有不少研究報告顯示，不同的kringle片段具有不同的抑制血管新生能力。本實驗室為進一步探討血管靜止蛋白及其相關蛋白質中kringle domain的結構與其功能之間的關係，我們針對kringle 1、kringle 2、kringle 3、kringle 4中的三對雙硫鍵設計了不同長度的片段，分別為K185-548、K1106-548、K1134-548、K2164-548、K2167-548、K2188-548、K2216-548、K3250-548、K3257-548、K3278-548、K3306-548、K4355-548、K4359-548、K4380-548、K4408-548及K5443-548，以酵母菌系統表現蛋白質，經由lysine sepharose或Nickel column純化後，再利用牛肺動脈內皮細胞（CPAE）進行移動試驗及增生試驗檢測其抑制血管新生的能力：在移動試驗中發現上述所有蛋白質片段都可抑制細胞移動且雙硫鍵被破壞的蛋白質其抑制能力大部分比kringle結構完整的蛋白質高﹔在增生試驗中K5抑制效果最佳。再者，本實驗室曾發現：針對破壞雙硫鍵所設計出的K41-418在移動試驗及增生試驗中具較佳的抑制效果，為進一步研究在in vivo實驗中，K41-418是否也具抑制血管新生的潛力，我們利用反轉錄病毒分別將K41-418及控制組K1-4的基因送至攜有黑色素細胞瘤的小鼠腫瘤上，並觀察腫瘤大小之差異，結果發現施以K41-418處理的小鼠腫瘤體積比其他組小，故推論K41-418可在in vivo抑制腫瘤生長。

Angiogenesis is a multi-step process that includes basement membrane degradation, migration and proliferation of endothelial cell, and new lumen organization. It is generally accepted that tumor growth is angiogenesis -dependent. The inhibition of angiogenesis has been used as be effective strategy in cancer therapy. Angiostatin, a kringle-containing fragment of plasminogen, is an endogenous antiangiogenic agent and is initially isolated from urine and sera of mice bearing Lewis lung carcinoma. Many studies have demonstrated that individual and multiple kringle fragments of angiostatin have different inhibitory activities on angiogenesis. Each kringle contains the triple loop disulfide-linked structures; we want to understand the relationship between the function and its related fragments with disrupted kringle. We selected four specific cleavage sites in kringle 1, 2, 3 and 4, i.e. at residue 85, 106, 134 in kringle 1; 167, 188, 216 in kringle 2; 257, 278, 306 in kringle 3; and 359, 380, 408 in kringle 4, so that the kringle structure would be disrupted gradually. In addition, we also prepared four fragments with intact kringle, i.e. K2164-548, K3256-548, K4355-548 and K5443-548. We expressed these sixteen fragments of plasminogen using the Pichia pastoris expressing system and tested their antiangiogenic activity by using Calf Pulmonary Artery Endothelial Cell. In migration assay, we found that all fragments had inhibitory potential and most of the plasminogen fragments with disrupted disulfide bridge had higher inhibitory effect than these fragments with intact kringle structure. In proliferation assay, kringle 5 domain has the highest inhibitory activity. Besides, previous studies from our lab also showed that k41-418 had higher inhibitory activity in both migration assay and proliferation assay. To understand whether the protein also has the anti-angiogenesis activity in vivo, we used intratumor delivery of retrovirus vector expressing cDNA of k41-418 and K1-4 to C57BL/6 mice bearing B16F10 melanoma. We found that tumor sizes of mice treated with retrovirus expressing k41-418 were smaller than others. The result suggests that k41-418 can inhibit the tumor growth potentially in vivo.

1.Ausprunk DH. Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during tumor angiogenesis. Microvasc. Res. 14:53-65, 1977.
2.Bodine DM. Mcdonagh KT. Brandt SJ. Ney PA. Agricola B. Byrne E. Nienhuis AW. Development of a high-titer retrovirus producer cell line capable of gene transfer into rhesus monkey hematopoieic stem cells. Proc. Natl. Acad. Sci. USA. 87:3738-3742, 1990.
3.Cao R. Wu HL. Veitonmaki N. Linden P. Farnebo J. Shi GY. Cao Y. Suppression of angiogenesis and tumor growth by inhibitor K1-5 generated by plasmin-mediated proteolysis. Proc. Natl. Acad. Sci. USA. 96:5728-5733, 1999.
4.Cao Y. Ji RW. Davidson D. Schaller J. Marti D. Sohndel S. McCance SG. O'Reilly MS. Llinas M. Folkman J. Kringle domains of human angiostatin. Characterization of the anti-proliferative activity on endothelial cells. J. Biol. Chem. 271:29461-7, 1996.
5.Cao Y. Chen A. An SSA. Ji RW. Davidson D. Cao Y. Llinas M. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J. Biol. Chem. 272:22924-22928, 1997.
6.Carmeliet P. Jain RK. Angiogenesis in cancer and other diseases. Nature. 407:249-257, 2000.
7.Chang YS. Tomaso ED. Mcdonald DM. Jones R. Jain RK. Munn LL. Mosaic blood vessels in tumors: Frequency of cancer cells in contact with flowing blood. Proc. Natl. Acad. Sci. USA. 97:14608-14613, 2000.
8.Chang Y. Mochalkin I. Mccance SG. Cheng B. Tulinsky A. Castellino FJ. Structure and ligand binding determinants of the recombinant kringle 5 domain of human plasminogen. Biochemistry. 37:3258-3271, 1998.
9.Chavalis E. Dimmeler. Regulation of endothelial cell survival and apoptosis during angiogenesis.Arterioscler. Thromb. Vasc. Biol. 22:887-893, 2002.
10.Cornelius LA. Nehring LC. Harding E. Bolanowski M. Welgus HG. Kobayashi DK. Pierce RA. Shaprio SD. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immnol. 161: 6845-6852, 1998.
11.Denekamp J. Hobson B. Endothelial proliferation in normal and tumour blood vessels. Microvasc. Res. 27:388, 1984.
12.Folkman J. Shing Y. Angiogenesis. J. Biol. Chem. 267:10931-4, 1992.
13.Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N. Engl. J. Med. 333:1757-63, 1995.
14.Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med. 1:27-31, 1995.
15.Folkman J. Haudenschild C. Angiogenesis in vitro. Nature. 288:551-6, 1980.
16.Folkman J. Klagsbrun M. Angiogenic factors. Science. 235:442-7, 1987.
17.Folkman J. What is the evidence that tumors are angiogenesis dependent? J. Natl. Cancer Inst. 82:4-6, 1990.
18.Folkman J. Anti-angiogenesis: new concept for therapy of solid tumors. Ann. Surg. 175:409-16, 1972.
19.Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes memorial Award lecture. Cancer Res. 46:467-73, 1986.
20.Folkman J. What is the role of endothelial cells in angiogenesis? Lab. Invest. 51:601-4, 1984.
21.Fidler IJ. Ellis LM. The implications of angiogenesis for the biology and therapy of cancer metastasis. Cell. 79:185-8, 1994.
22.Gross JL. Moscatelli D. Rifkin DB. Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro. Proc. Natl. Acad. Sci. USA. 80:2623-7, 1983.
23.Gimbrone MA Jr. Leapman SB. Cotran RS. Folkman J. Tumor dormancy in vivo by prevention of neovascularization. J. Exp. Med. 136:261-76, 1972.
24.Gupta N. Nodzenski E. Khodarev NN. Yu J. Khorasani L. Beckett MA. Kufe DW. Weichselbaum RR. Angiostatin effects on endothelial cells mediated by ceramade and RhoA. EMBO reports. 2: 536-540, 2001.
25.Hanahan D. Signaling vascular morphogenesis and maintenance. Science. 277:48-50, 1997.
26.Hanahan D. Folkman J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 86:353-364, 1996.
27.Hochschwender SM. Laursen RA. The lysine binding sites of human plasminogen. Evidence for a critical tryptophan in the binding site of kringle 4. J. Biol. Chem. 256:11172-6, 1981.
28.Ji WR. Castellino FJ. Chang Y. Deford ME. Gray H. Villarreal X. Kondri ME. Marti DN. Llinas M. Schaller J. Kramer RA. Trail PA. Characterization of kringle domains of angiostatin as antagonists of endothelial cell migration, an important process in angiogenesis. FASEB Journal. 12:1731-8, 1998.
29.Ji WR. Barrientos LG. Llinas M. Gray H. Villarreal X. DeFord ME. Castellino FJ. Kramer RA. Trail PA. Selective inhibition by kringle 5 of human plasminogen on endothelial cell migration, an important process in angiogenesis. Biochem. & Biophy. Res. Commun. 247:414-9, 1998.
30.Naviaux RK. Costanzi E. Haas M. Verma IM. The pCL vector system: rapid production of helper-free, high-titer, recombinant retrovirus. J. Virol. 70:5701-5705,1996.
31.Montesano R. Vassalli JD. Baird A. Guillemin R. Orci L. Basic fibroblast growth factor induces angiogenesis in vitro. Proc. Natl. Acad. Sci. USA. 83:7297-301, 1986.
32.Moser TL. Stack MS. Asplin I. Enghild JJ. Hojrup P. Everitt L. Hubchak S.Schnaprt HW. Pizzo SV. Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc. Natl. Acad. Sci. USA. 96:2811-2816, 1999.
33.Moser TL. Stack MS. Wahl ML. Pizzo SV. The mechanism of action of angiostatin: can you teach an old dog new tricks? Thromb. Haemost. 87:394-401, 2002.
34.O'Reilly MS. Holmgren L. Shing Y. Chen C. Rosenthal RA. Moses M. Lane WS. Cao Y. Sage EH. Folkman J. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell. 79:315-28, 1994.
35.O'Reilly MS. Holmgren L. Chen C. Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nature Med. 2:689-92, 1996.
36.Petersen TE. Martzen MR. Ichinose A. Davie EW. Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system. J. Biol. Chem. 265:6104-11, 1990.
37.Poste G. Fidler IJ. The pathogenesis of cancer metastasis. Nature. 283:139-46, 1980.
38.Stathakis P. Lay AJ. Fitzgerald M. Schlieker C. Matthias LJ. Hogg PJ. Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 of plasmin. J. Biol. Chem. 274:8910-6, 1999.
39.Sim BK. O'Reilly MS. Liang H. Fortier AH. He W. Madsen JW. Lapcevich R. Nacy CA. A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Res. 57:1329-34, 1997.
40.Sim BK. MacDonald NJ. Gubish ER. Angiostatin and endostatin: endogenous inhibitors of tumor growth. Cancer and Metastasis Reviews. 19:181-190, 2000.
41.Soff GA. Angiostatin and angiostatin-related proteins. Cancer and Metastasis Reviews. 19:97-107, 2000.
42.Weidner N. Semple JP. Welch WR. Folkman J. Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. N. Engl. J. Med. 324:1-8, 1991.
43.Troyanovsky B. Levchenko T. Mansson G. Matvijenko O. Holmgren L. Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation. J. Cell Biol. 152: 1247-1254, 2001.
44.Yancopoulos GD. Davis S. Gale NW. Rudge JS. Wiegand SJ. Holash J. Vascular -specific growth factors and blood vessel formation. Nature. 407:242-248, 2000.
44.謝榮忠。血管靜止蛋白的產生與血纖維蛋白溶脢原代謝的關係。國立成功大學生
物化學研究所碩士論文，1997。
45.盧諭德。血管靜止蛋白及其相關蛋白的構造與功能之研究。國立成功大學生物化學研究所碩士論文，2000。
46.陳汶妤。血管靜止蛋白結構與功能。國立成功大學生物化學研究所碩士論文，2001。