血管新生作用在腫瘤生長與轉移的過程中扮演很重要的角色。在人類血纖維蛋白溶原中含有五個相似的片段，稱之為kringle。抗血管新生素(angiostatin)具有抑制血管新生的作用，其主要是由人類血纖維蛋白溶原中前四個相似片段所組成，由過去的研究中發現，kringle 1-5和kringle 5蛋白抑制血管新生與腫瘤生長的作用，比抗血管新生素來的明顯。為了進一步提高kringle片段抗血管新生的能力，我們利用突變的方式在人類血纖維蛋白溶原中五個相似的片段上，除去兩個糖化位置(Asn-289和Thr-346)與增強第五個片段上(Leu-532)離氨酸吸附的能力，再以酵母菌(Pichia pastoris)發酵與純化系統製作出七種突變蛋白(K1-5N289A, K1-5T346A, K1-5L532R, K1-5N289A/T346A, K1-5N289A/L532R, K1-5T346A/L532R, K1-5N289A/T346A/L532R)。由實驗的結果發現，由人類血纖維蛋白溶原所切下來的K1-5片段與利用酵母菌純化而得到的K1-5蛋白，其對於抑制內皮細胞的生長具有相同的能力。各種突變蛋白都具有抑制內皮細胞增生與促進細胞凋亡的作用，其中以K1-5N289A/T346A/L532R的效果最為顯著。另外，我們發現內皮細胞主要是透過胚胎著床分子alpha v beta 3的調控，吸附到K1-5N289A/T346A/L532R突變蛋白，而且吸附的作用比K1-5蛋白還強。更進一步我們由動物實驗的結果得知，K1-5N289A/T346A/L532R突變蛋白對於bFGF所刺激的血管新生作用具有明顯的抑制效果，且腫瘤生長時所引起的血管新生也能被K1-5N289A/T346A/L532R突變蛋白有效的抑制。除此之外，K1-5N289A/T346A/L532R在血液中的半衰期也比K1-5長。這可能是因為K1-5本身容易被asialoglycoprotein receptor所辨識而內吞到HepG2細胞內，並且造成下游ERK的活化。由以上的結果證明，改變糖化與離氨酸結合的能力可以增強K1-5蛋白與內皮細胞上胚胎著床分子alpha v beta 3的吸附能力，並且增強K1-5蛋白抗血管新生的作用。
另一方面，最近的報告指出，利用抗血管新生素可以有效的抑制動脈粥狀硬化的斑塊形成與前期發炎反應。血管內皮細胞的活化在動脈粥狀硬化的生成與血管斑塊的穩定度有舉足輕重的影響。為了進一步探討K1-5在動脈粥狀硬化中所扮演的角色，我們利用自發性動脈硬化小鼠與頸動脈結紮小鼠來進行實驗。在自發性動脈硬化小鼠實驗中，小鼠接受持續性皮下注射K1-5蛋白，可以有效的抑制小鼠主動脈血管斑塊的形成。而在頸動脈結紮小鼠實驗中，同樣觀察到K1-5蛋白可以有效的抑制血管栓塞。另外，我們利用腫瘤壞死因子刺激人類臍帶內皮細胞，使其表現出細胞間黏著分子-1與血管細胞黏著分子-1，模擬動脈粥狀硬化的前期發炎反應。並以前處理的方式給予K1-5蛋白，結果發現K1-5蛋白可以有效抑制前期發炎反應中細胞間黏著分子-1與血管細胞黏著分子-1的表現，其機制可能是透過抑制下游NF-kB活化與降低細胞內ROS的產生。經由動物實驗和細胞實驗的結果，我們推測K1-5蛋白可能藉由抑制動脈硬化前期發炎反應中細胞間黏著分子-1與血管細胞黏著分子-1的表現，進而降低動脈粥狀硬化的程度。

Angiogenesis plays a primary role in tumor growth and metastasis. Angiostatin, a proteolytic fragment containing the first four kringle domains of human plasminogen, can inhibit angiogenesis. The anti-angiogenic activities of kringle 1-5 (K1-5) and kringle 5 fragments of plasminogen are greater than angiostatin in inhibiting angiogenesis and angiogenesis-dependent tumor growth. To further optimize kringle fragment anti-angiogenic activities, mutations were created at the potential glycosylation sites Asn-289 and Thr-346 and the Lys binding site, Leu-532, at kringle 5, including K1-5N289A (replacing Asn by Ala at residue 289), K1-5T346A, K1-5L532R, K1-5N289A/T346A, K1-5T346A/L532R, K1-5N289A/L532R, and K1-5N289A/T346A/L532R. Wild-type and mutant K1-5 proteins were expressed successfully by the Pichia pastoris expression system. Native K1-5 from proteolytic cleavage and wild-type K1-5 have similar activity in inhibiting basic fibroblast growth factor-induced endothelial cell proliferation. Among these mutated proteins, K1-5N289A/T346A/L532R exhibited the greatest effect on inhibiting endothelial cell proliferation and inducing endothelial cell apoptosis. Integrin alpha v beta 3-mediated adhesion of K1-5N289A/T346A/L532R to endothelial cells was more greatly enhanced when compared to wild type K1-5. Furthermore, K1-5N289A/T346A/L532R was most potent in inhibiting basic fibroblast growth factor-induced angiogenesis in Matrigel assay in vivo. Angiogenesis-dependent tumor growth was inhibited by systemically injected K1-5N289A/T346A/L532R into mice. Pharmacokinetic experiments showed that K1-5N289A/T346A/L532R had a longer half-life compared with wild-type K1-5 following intraperitoneal administration to mice. Moreover, wild-type K1-5 was more easily internalized by HepG2 cells than the K1-5 protein variants. Wild-type K1-5 induced extracellular signal-regulated kinase and asialoglycoprotein receptor phosphorylation more easily in HepG2 cells. These results demonstrate that mutation of human plasminogen K1-5 prolongs its half-life and enhances the interaction with integrin alpha v beta 3 resulting in improved anti-angiogenic action.
On the other hand, activation of vascular endothelial cells plays an important role in atherogenesis and plaque instability. Recent research demonstrated that late stage inhibition of plaque angiogenesis by angiostatin reduces macrophage accumulation and slows the progression of advanced atherosclerosis. K1-5 is a variant of angiostatin that contain the first five kringle domains of plasminogen. To investigate whether K1-5 has an inhibitory effect on early-stage atherosclerosis, the apolipoprotein E-deficient mice model and a carotid artery ligation model were used. The areas of the lesion in the aortas of apolipoprotein E-deficient mice that received K1-5 injections were notably decreased, and formation of carotid neointima in the C57BL/6 mice was decreased by treatment with K1-5. Expression of TNF-alpha-induced intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 was inhibited by K1-5 treatment, possibly via down-regulation of translocation of nuclear factor-kB and expression of reactive oxygen species. K1-5 reduced atherosclerosis and neointima formation in mice, possibly through inhibition of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in endothelial cells.

1 Carmeliet P, Collen D. Molecular basis of angiogenesis. Role of VEGF and VE-cadherin. Ann N Y Acad Sci. 2000; 902: 249-62.
2 Folkman J, Shing Y. Angiogenesis. J Biol Chem. 1992; 267: 10931-4.
3 Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med. 1995; 333: 1757-63.
4 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. 1994; 79: 315-28.
5 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. 1996; 271: 29461-7.
6 Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971; 285: 1182-6.
7 Gately S, Twardowski P, Stack MS, Patrick M, Boggio L, Cundiff DL, Schnaper HW, Madison L, Volpert O, Bouck N, Enghild J, Kwaan HC, Soff GA. Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin. Cancer Res. 1996; 56: 4887-90.
8 Gately S, Twardowski P, Stack MS, Cundiff DL, Grella D, Castellino FJ, Enghild J, Kwaan HC, Lee F, Kramer RA, Volpert O, Bouck N, Soff GA. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proc Natl Acad Sci U S A. 1997; 94: 10868-72.
9 Falcone DJ, Khan KM, Layne T, Fernandes L. Macrophage formation of angiostatin during inflammation. A byproduct of the activation of plasminogen. J Biol Chem. 1998; 273: 31480-5.
10 O'Mahony CA, Seidel A, Albo D, Chang H, Tuszynski GP, Berger DH. Angiostatin generation by human pancreatic cancer. J Surg Res. 1998; 77: 55-8.
11 Stathakis P, Fitzgerald M, Matthias LJ, Chesterman CN, Hogg PJ. Generation of angiostatin by reduction and proteolysis of plasmin. Catalysis by a plasmin reductase secreted by cultured cells. J Biol Chem. 1997; 272: 20641-5.
12 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. 1999; 274: 8910-6.
13 Dong Z, Kumar R, Yang X, Fidler IJ. Macrophage-derived metalloelastase is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell. 1997; 88: 801-10.
14 Patterson BC, Sang QA. Angiostatin-converting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J Biol Chem. 1997; 272: 28823-5.
15 Cornelius LA, Nehring LC, Harding E, Bolanowski M, Welgus HG, Kobayashi DK, Pierce RA, Shapiro SD. Matrix metalloproteinases generate angiostatin: effects on neovascularization. J Immunol. 1998; 161: 6845-52.
16 Lijnen HR, Ugwu F, Bini A, Collen D. Generation of an angiostatin-like fragment from plasminogen by stromelysin-1 (MMP-3). Biochemistry. 1998; 37: 4699-702.
17 Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995; 1: 27-31.
18 O'Reilly MS, Holmgren L, Chen C, Folkman J. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med. 1996; 2: 689-92.
19 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 J. 1998; 12: 1731-8.
20 Cao R, Wu HL, Veitonmaki N, Linden P, Farnebo J, Shi GY, Cao Y. Suppression of angiogenesis and tumor growth by the inhibitor K1-5 generated by plasmin-mediated proteolysis. Proc Natl Acad Sci U S A. 1999; 96: 5728-33.
21 Li F, Yang J, Liu X, He P, Ji S, Wang J, Han J, Chen N, Yao L. Human glioma cell BT325 expresses a proteinase that converts human plasminogen to kringle 1-5-containing fragments. Biochem Biophys Res Commun. 2000; 278: 821-5.
22 Westphal JR, Van't Hullenaar R, Geurts-Moespot A, Sweep FC, Verheijen JH, Bussemakers MM, Askaa J, Clemmensen I, Eggermont AA, Ruiter DJ, De Waal RM. Angiostatin generation by human tumor cell lines: involvement of plasminogen activators. Int J Cancer. 2000; 86: 760-7.
23 Scapini P, Nesi L, Morini M, Tanghetti E, Belleri M, Noonan D, Presta M, Albini A, Cassatella MA. Generation of biologically active angiostatin kringle 1-3 by activated human neutrophils. J Immunol. 2002; 168: 5798-804.
24 Hortin GL, Gibson BL, Fok KF. Alpha 2-antiplasmin's carboxy-terminal lysine residue is a major site of interaction with plasmin. Biochem Biophys Res Commun. 1988; 155: 591-6.
25 Hortin GL, Trimpe BL, Fok KF. Plasmin's peptide-binding specificity: characterization of ligand sites in alpha 2-antiplasmin. Thromb Res. 1989; 54: 621-32.
26 Miles LA, Fless GM, Scanu AM, Baynham P, Sebald MT, Skocir P, Curtiss LK, Levin EG, Hoover-Plow JL, Plow EF. Interaction of Lp(a) with plasminogen binding sites on cells. Thromb Haemost. 1995; 73: 458-65.
27 Christensen U. C-terminal lysine residues of fibrinogen fragments essential for binding to plasminogen. FEBS Lett. 1985; 182: 43-6.
28 Tran-Thang C, Kruithof EK, Atkinson J, Bachmann F. High-affinity binding sites for human Glu-plasminogen unveiled by limited plasmic degradation of human fibrin. Eur J Biochem. 1986; 160: 599-604.
29 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. 1998; 37: 3258-71.
30 McCance SG, Menhart N, Castellino FJ. Amino acid residues of the kringle-4 and kringle-5 domains of human plasminogen that stabilize their interactions with omega-amino acid ligands. J Biol Chem. 1994; 269: 32405-10.
31 Marti D, Schaller J, Ochensberger B, Rickli EE. Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for omega-aminocarboxylic acids. Eur J Biochem. 1994; 219: 455-62.
32 Pirie-Shepherd SR. Role of carbohydrate on angiostatin in the treatment of cancer. J Lab Clin Med. 1999; 134: 553-60.
33 Hayes ML, Castellino JF. Carbohydrate of the human plasminogen variants. I. Carbohydrate composition, glycopeptide isolation, and characterization. J Biol Chem. 1979; 254: 8768-71.
34 Davidson DJ, Castellino FJ. Oligosaccharide structures present on asparagine-289 of recombinant human plasminogen expressed in a Chinese hamster ovary cell line. Biochemistry. 1991; 30: 625-33.
35 Pirie-Shepherd SR, Stevens RD, Andon NL, Enghild JJ, Pizzo SV. Evidence for a novel O-linked sialylated trisaccharide on Ser-248 of human plasminogen 2. J Biol Chem. 1997; 272: 7408-11.
36 Stockert RJ. The asialoglycoprotein receptor: relationships between structure, function, and expression. Physiol Rev. 1995; 75: 591-609.
37 Fallon RJ. Tyrosine phosphorylation of the asialoglycoprotein receptor. J Biol Chem. 1990; 265: 3401-6.
38 Geffen I, Fuhrer C, Spiess M. Endocytosis by the asialoglycoprotein receptor is independent of cytoplasmic serine residues. Proc Natl Acad Sci U S A. 1991; 88: 8425-9.
39 Bonvin C, Guillon A, van Bemmelen MX, Gerwins P, Johnson GL, Widmann C. Role of the amino-terminal domains of MEKKs in the activation of NF kappa B and MAPK pathways and in the regulation of cell proliferation and apoptosis. Cell Signal. 2002; 14: 123-31.
40 Ding Q, Wang Q, Evers BM. Alterations of MAPK activities associated with intestinal cell differentiation. Biochem Biophys Res Commun. 2001; 284: 282-8.
41 Kyosseva SV, Harris EN, Weigel PH. The hyaluronan receptor for endocytosis mediates hyaluronan-dependent signal transduction via extracellular signal-regulated kinases. J Biol Chem. 2008; 283: 15047-55.
42 Ross R. Atherosclerosis--an inflammatory disease. N Engl J Med. 1999; 340: 115-26.
43 Libby P. Inflammation in atherosclerosis. Nature. 2002; 420: 868-74.
44 Gotto AM, Jr. Role of C-reactive protein in coronary risk reduction: focus on primary prevention. Am J Cardiol. 2007; 99: 718-25.
45 Deanfield JE, Halcox JP, Rabelink TJ. Endothelial function and dysfunction: testing and clinical relevance. Circulation. 2007; 115: 1285-95.
46 Simionescu M. Implications of early structural-functional changes in the endothelium for vascular disease. Arterioscler Thromb Vasc Biol. 2007; 27: 266-74.
47 Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med. 2005; 352: 1685-95.
48 Eriksson EE, Xie X, Werr J, Thoren P, Lindbom L. Importance of primary capture and L-selectin-dependent secondary capture in leukocyte accumulation in inflammation and atherosclerosis in vivo. J Exp Med. 2001; 194: 205-18.
49 Calabro P, Samudio I, Safe SH, Willerson JT, Yeh ET. Inhibition of tumor-necrosis-factor-alpha induced endothelial cell activation by a new class of PPAR-gamma agonists. An in vitro study showing receptor-independent effects. J Vasc Res. 2005; 42: 509-16.
50 Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease. Arterioscler Thromb Vasc Biol. 2005; 25: 29-38.
51 Moore KJ, Freeman MW. Scavenger receptors in atherosclerosis: beyond lipid uptake. Arterioscler Thromb Vasc Biol. 2006; 26: 1702-11.
52 Fan J, Watanabe T. Inflammatory reactions in the pathogenesis of atherosclerosis. J Atheroscler Thromb. 2003; 10: 63-71.
53 Kraiss LW, Kirkman TR, Kohler TR, Zierler B, Clowes AW. Shear stress regulates smooth muscle proliferation and neointimal thickening in porous polytetrafluoroethylene grafts. Arterioscler Thromb. 1991; 11: 1844-52.
54 Gibbons GH, Dzau VJ. The emerging concept of vascular remodeling. N Engl J Med. 1994; 330: 1431-8.
55 Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest. 1994; 94: 2493-503.
56 Li YH, Hsieh CY, Wang DL, Chung HC, Liu SL, Chao TH, Shi GY, Wu HL. Remodeling of carotid arteries is associated with increased expression of thrombomodulin in a mouse transverse aortic constriction model. Thromb Haemost. 2007; 97: 658-64.
57 Niculescu F, Rus H. The role of complement activation in atherosclerosis. Immunol Res. 2004; 30: 73-80.
58 Liu SL, Li YH, Shi GY, Chen YH, Huang CW, Hong JS, Wu HL. A novel inhibitory effect of naloxone on macrophage activation and atherosclerosis formation in mice. J Am Coll Cardiol. 2006; 48: 1871-9.
59 Paigen B, Morrow A, Brandon C, Mitchell D, Holmes P. Variation in susceptibility to atherosclerosis among inbred strains of mice. Atherosclerosis. 1985; 57: 65-73.
60 Piedrahita JA, Zhang SH, Hagaman JR, Oliver PM, Maeda N. Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells. Proc Natl Acad Sci U S A. 1992; 89: 4471-5.
61 Jawien J, Nastalek P, Korbut R. Mouse models of experimental atherosclerosis. J Physiol Pharmacol. 2004; 55: 503-17.
62 Nakashima Y, Plump AS, Raines EW, Breslow JL, Ross R. ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree. Arterioscler Thromb. 1994; 14: 133-40.
63 Plump AS, Smith JD, Hayek T, Aalto-Setala K, Walsh A, Verstuyft JG, Rubin EM, Breslow JL. Severe hypercholesterolemia and atherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. Cell. 1992; 71: 343-53.
64 Berk BC, Korshunov VA. Genetic determinants of vascular remodelling. Can J Cardiol. 2006; 22 Suppl B: 6B-11B.
65 Lindner V, Fingerle J, Reidy MA. Mouse model of arterial injury. Circ Res. 1993; 73: 792-6.
66 Cercek B, Yamashita M, Dimayuga P, Zhu J, Fishbein MC, Kaul S, Shah PK, Nilsson J, Regnstrom J. Nuclear factor-kappaB activity and arterial response to balloon injury. Atherosclerosis. 1997; 131: 59-66.
67 Chyu KY, Dimayuga P, Zhu J, Nilsson J, Kaul S, Shah PK, Cercek B. Decreased neointimal thickening after arterial wall injury in inducible nitric oxide synthase knockout mice. Circ Res. 1999; 85: 1192-8.
68 Golino P, Ambrosio G, Pascucci I, Ragni M, Russolillo E, Chiariello M. Experimental carotid stenosis and endothelial injury in the rabbit: an in vivo model to study intravascular platelet aggregation. Thromb Haemost. 1992; 67: 302-5.
69 Burchenal JE, Deible CR, Deglau TE, Russell AJ, Beckman EJ, Wagner WR. Polyethylene glycol diisocyanate decreases platelet deposition after balloon injury of rabbit femoral arteries. J Thromb Thrombolysis. 2002; 13: 27-33.
70 Ueno H, Kanellakis P, Agrotis A, Bobik A. Blood flow regulates the development of vascular hypertrophy, smooth muscle cell proliferation, and endothelial cell nitric oxide synthase in hypertension. Hypertension. 2000; 36: 89-96.
71 Korshunov VA, Berk BC. Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening. Arterioscler Thromb Vasc Biol. 2003; 23: 2185-91.
72 Hatada EN, Krappmann D, Scheidereit C. NF-kappaB and the innate immune response. Curr Opin Immunol. 2000; 12: 52-8.
73 Baeuerle PA, Baltimore D. NF-kappa B: ten years after. Cell. 1996; 87: 13-20.
74 Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med. 1997; 336: 1066-71.
75 Chen KH, Reece LM, Leary JF. Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction. Free Radic Biol Med. 1999; 27: 100-9.
76 Garcia-Ruiz C, Colell A, Morales A, Kaplowitz N, Fernandez-Checa JC. Role of oxidative stress generated from the mitochondrial electron transport chain and mitochondrial glutathione status in loss of mitochondrial function and activation of transcription factor nuclear factor-kappa B: studies with isolated mitochondria and rat hepatocytes. Mol Pharmacol. 1995; 48: 825-34.
77 Schulze-Osthoff K, Bakker AC, Vanhaesebroeck B, Beyaert R, Jacob WA, Fiers W. Cytotoxic activity of tumor necrosis factor is mediated by early damage of mitochondrial functions. Evidence for the involvement of mitochondrial radical generation. J Biol Chem. 1992; 267: 5317-23.
78 Beyaert R, Fiers W. Molecular mechanisms of tumor necrosis factor-induced cytotoxicity. What we do understand and what we do not. FEBS Lett. 1994; 340: 9-16.
79 Schulze-Osthoff K, Beyaert R, Vandevoorde V, Haegeman G, Fiers W. Depletion of the mitochondrial electron transport abrogates the cytotoxic and gene-inductive effects of TNF. EMBO J. 1993; 12: 3095-104.
80 Goossens V, Grooten J, De Vos K, Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci U S A. 1995; 92: 8115-9.
81 Dawson TL, Gores GJ, Nieminen AL, Herman B, Lemasters JJ. Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am J Physiol. 1993; 264: C961-7.
82 Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J. 1995; 9: 899-909.
83 Gamble JR, Harlan JM, Klebanoff SJ, Vadas MA. Stimulation of the adherence of neutrophils to umbilical vein endothelium by human recombinant tumor necrosis factor. Proc Natl Acad Sci U S A. 1985; 82: 8667-71.
84 Moulton KS, Vakili K, Zurakowski D, Soliman M, Butterfield C, Sylvin E, Lo KM, Gillies S, Javaherian K, Folkman J. Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci U S A. 2003; 100: 4736-41.
85 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970; 170: 1095-6.
86 Wu HL, Chang BI, Wu DH, Chang LC, Gong CC, Lou KL, Shi GY. Interaction of plasminogen and fibrin in plasminogen activation. J Biol Chem. 1990; 265: 19658-64.
87 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227: 680-5.
88 Shi GY, Hau JS, Wang SJ, Wu IS, Chang BI, Lin MT, Chow YH, Chang WC, Wing LY, Jen CJ, et al. Plasmin and the regulation of tissue-type plasminogen activator biosynthesis in human endothelial cells. J Biol Chem. 1992; 267: 19363-8.
89 Passaniti A, Taylor RM, Pili R, Guo Y, Long PV, Haney JA, Pauly RR, Grant DS, Martin GR. A simple, quantitative method for assessing angiogenesis and antiangiogenic agents using reconstituted basement membrane, heparin, and fibroblast growth factor. Lab Invest. 1992; 67: 519-28.
90 Cao Y, O'Reilly MS, Marshall B, Flynn E, Ji RW, Folkman J. Expression of angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormancy of metastases. J Clin Invest. 1998; 101: 1055-63.
91 Zhang XP, Kamata T, Yokoyama K, Puzon-McLaughlin W, Takada Y. Specific interaction of the recombinant disintegrin-like domain of MDC-15 (metargidin, ADAM-15) with integrin alphavbeta3. J Biol Chem. 1998; 273: 7345-50.
92 Knedla A, Riepl B, Lefevre S, Kistella S, Grifka J, Straub RH, Gay S, Scholmerich J, Muller-Ladner U, Neumann E. The therapeutic use of osmotic minipumps in the severe combined immunodeficiency (SCID) mouse model for rheumatoid arthritis. Ann Rheum Dis. 2009; 68: 124-9.
93 Kumar A, Lindner V. Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arterioscler Thromb Vasc Biol. 1997; 17: 2238-44.
94 Chang PC, Chang YJ, Wu HL, Chang CW, Lin CI, Wang WC, Shi GY. Mutation of human plasminogen kringle 1-5 enhances anti-angiogenic action via increased interaction with integrin alpha(v)beta(3). Thromb Haemost. 2008; 99: 729-38.
95 Monroe RS, Huber BE. The major form of the murine asialoglycoprotein receptor: cDNA sequence and expression in liver, testis and epididymis. Gene. 1994; 148: 237-44.
96 Ashwell G, Harford J. Carbohydrate-specific receptors of the liver. Annu Rev Biochem. 1982; 51: 531-54.
97 Tarui T, Miles LA, Takada Y. Specific interaction of angiostatin with integrin alpha(v)beta(3) in endothelial cells. J Biol Chem. 2001; 276: 39562-8.
98 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. 2001; 152: 1247-54.
99 Veitonmaki N, Cao R, Wu LH, Moser TL, Li B, Pizzo SV, Zhivotovsky B, Cao Y. Endothelial cell surface ATP synthase-triggered caspase-apoptotic pathway is essential for k1-5-induced antiangiogenesis. Cancer Res. 2004; 64: 3679-86.
100 Chen YH, Wu HL, Li C, Huang YH, Chiang CW, Wu MP, Wu LW. Anti-angiogenesis mediated by angiostatin K1-3, K1-4 and K1-4.5. Involvement of p53, FasL, AKT and mRNA deregulation. Thromb Haemost. 2006; 95: 668-77.
101 Rudd PM, Woods RJ, Wormald MR, Opdenakker G, Downing AK, Campbell ID, Dwek RA. The effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator. Biochim Biophys Acta. 1995; 1248: 1-10.
102 Tarui T, Akakura N, Majumdar M, Andronicos N, Takagi J, Mazar AP, Bdeir K, Kuo A, Yarovoi SV, Cines DB, Takada Y. Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation. Thromb Haemost. 2006; 95: 524-34.
103 Rajan S, Ye J, Bai S, Huang F, Guo YL. NF-kappaB, but not p38 MAP kinase, is required for TNF-alpha-induced expression of cell adhesion molecules in endothelial cells. J Cell Biochem. 2008; 105: 477-86.
104 Blankenberg S, Barbaux S, Tiret L. Adhesion molecules and atherosclerosis. Atherosclerosis. 2003; 170: 191-203.
105 Chavakis T, Athanasopoulos A, Rhee JS, Orlova V, Schmidt-Woll T, Bierhaus A, May AE, Celik I, Nawroth PP, Preissner KT. Angiostatin is a novel anti-inflammatory factor by inhibiting leukocyte recruitment. Blood. 2005; 105: 1036-43.
106 Raskopf E, Gerceker S, Vogt A, Standop J, Sauerbruch T, Schmitz V. Plasminogen fragment K1-3 inhibits expression of adhesion molecules and experimental HCC recurrence in the liver. Int J Colorectal Dis. 2009. 10.1007/s00384-009-0652-z.
107 Sparrow CP, Olszewski J. Cellular oxidative modification of low density lipoprotein does not require lipoxygenases. Proc Natl Acad Sci U S A. 1992; 89: 128-31.
108 Morel DW, DiCorleto PE, Chisolm GM. Endothelial and smooth muscle cells alter low density lipoprotein in vitro by free radical oxidation. Arteriosclerosis. 1984; 4: 357-64.
109 Marui N, Offermann MK, Swerlick R, Kunsch C, Rosen CA, Ahmad M, Alexander RW, Medford RM. Vascular cell adhesion molecule-1 (VCAM-1) gene transcription and expression are regulated through an antioxidant-sensitive mechanism in human vascular endothelial cells. J Clin Invest. 1993; 92: 1866-74.
110 Walter JJ, Sane DC. Angiostatin binds to smooth muscle cells in the coronary artery and inhibits smooth muscle cell proliferation and migration In vitro. Arterioscler Thromb Vasc Biol. 1999; 19: 2041-8.