Angiostatin Adonia E. Papathanassiu, Shawn J. Green*, Davida K. Grella and B. Kim Lee Sim EntreMed Inc., 9640 Medical Center Drive, Rockville, MD 20850, USA * corresponding author tel: 301-738-2494, fax: 301-217-9858, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.08005.
SUMMARY Angiostatin protein is a proteolytic fragment of plasminogen that has been shown to inhibit endothelial cell proliferation and migration in vitro and tumor angiogenesis in vivo.
BACKGROUND
suppressing growth factor-induced migration and proliferation of endothelial cells. Potent inhibition of angiogenesis was demonstrated with this form of angiostatin in the chick chorioallantoic membrane (CAM) assay as well as basic FGF-induced angiogenesis in the murine eye model. Systemic administration of angiostatin results in increased rates of apoptosis of endothelial cells as well as tumor cells in tumor models. This directly inhibits the growth of primary tumors and metastasis in murine models.
Discovery Angiostatin protein, an inhibitor of angiogenesis, was purified from the serum and urine of mice bearing Lewis lung carcinoma (LLC) tumors. N-terminal sequence analysis revealed that angiostatin is an internal fragment of plasminogen.
Structure Angiostatin contains the first three and/or four disulfide-linked domains of plasminogen referred to as kringle domains. Each kringle domain consists of approximately 80 amino acids arranged in a highly conserved, triple disulfide bond pattern which affords structural integrity. Although the X-ray crystal structure of angiostatin is not yet available, the NMR and crystal structures of the individual kringles are established.
Main activities and pathophysiological roles Angiostatin derived from elastase cleavage of human plasminogen was shown to inhibit angiogenesis by
GENE AND GENE REGULATION Angiostatin is the proteolytic product of plasminogen. The transcript of a gene encoding angiostatin has not been detected in various organs or tumors; however, plasminogen transcripts are found primarily in the liver.
Accession numbers Human plasminogen: XO5199
Chromosome location Human plasminogen: Chromosome 6q26±6q27
Cells and tissues that express the gene Human plasminogen is expressed primarily in liver. Other reports have shown local expression of plasminogen in the brain (Matsuoka et al., 1998) and in
810 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim the seminiferous tubules of the testis (Saksela and Vihko, 1986).
Important homologies Kringle domains analogous to those of plasminogen and angiostatin are found in variety of proteins including apolipoprotein(a), prothrombin, tissue-type and urokinase-type plasminogen activators, TrKrelated tyrosine kinases, and hepatocyte growth factor.
PROTEIN
Accession numbers The accession number for the parent protein of angiostatin, human plasminogen, is P00747.
Sequence
Posttranslational modifications As a proteolytic product of plasminogen, angiostatin is not subject to posttranslational modifications. Since human plasminogen exists in two forms that differ in glycosylation, human plasminogen-derived angiostatin also exists in two forms: one glycosylated at Asn289 and Thr346 and another one glycosylated at Thr346 and Ser249.
See Figure 1.
Description of protein Angiostatin was initially identified as a 38 kDa fragment of plasminogen which consists of five consecutive kringle domains followed by a serine protease domain. N-terminal sequence revealed that angiostatin begins at amino acid residue Val79 or Tyr80 of plasminogen. Many of the studies reporting on the potency of angiostatin use the form derived from elastase cleavage of plasminogen which frequently does not include kringle 4. Presently, the name angiostatin protein refers to a plasminogen fragment that includes either kringles 1 to 3 or kringles 1 to 4.
Discussion of crystal structure Presently, no X-ray crystal structure of angiostatin is available. NMR and X-ray crystallographic studies of individual kringle domains have shown that kringles 1, 2, 3, and 4 not only display a considerable sequence homology (48±50%), but also possess a remarkable structural similarity. In addition, kringles 1, 2, and 4 exhibit !-amino acid-binding capacity. A major difference between the various kringle domains is the presence of an exposed cationic cluster in the kringle 4 region that consists of two lysine pairs.
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce The origin of endogenous angiostatin is not known. Since tumor cells lack detectable amounts of mRNA for plasminogen or angiostatin, it has been proposed that these cells do not produce angiostatin directly but, rather, they express one or more proteases that cleave plasminogen to angiostatin. Table 1 and Table 2 summarize the production of angiostatin from plasminogen or plasmin in various cell-free and cell systems.
Eliciting and inhibitory stimuli including exogenous and endogenous modulators Modulators of angiostatin production involve mainly proteases, derived from tumor cells or cells found in
Figure 1 Amino acid sequence for human angiostatin protein. Human angiostatin protein: VYLSECKTGN GKNYRGTMSK TKNGITCQKW ILECEEECMH CSGENYDGKI SKTMSCLECQ DIPRCTTPPP SSGPTYQCLK GTGENYRGNV TNSQVRWEYC KIPSCDSSPV STEQLAPTAP NAGLTMNYCR NPDADKGPWC FTTDPSVRWE
SSTSPHRPRF AWDSQSPHAH AVTVSGHTCQ PELTPVVQDC YCCNLKKCSG
SPATHPSEGL GYIPSKFPNK HWSAQTPHTH YHGDGQSYRG TEA
EENYCRNPDN NLKKNYCRNP NRTPENFPCK TSSTTTTGKK
DPQGPWCYTT DRELRPWCFT NLDENYCRNP CQSWSSMTPH
DPEKRYDYCD TDPNKRWELC DGKRAPWCHT RHQKTPENYP
Angiostatin 811 Table 1 Enzymatic generation of human angiostatin protein from plasminogen in a cell-free system Enzyme/Reference
Site of cleavage
Form of angiostatin/Associated activity
Pancreatic elastase (O'Reilly et al., 1996)
38 kDa fragment of plasminogen In vitro activity: inhibition of endothelial cell proliferation In vivo activity: increased apoptotic index and inhibition of Lewis lung carcinoma (LLC), PC-3, Clone A (human colon carcinoma), T241 fibrosarcoma, MDA-MB (human breast cancer), reticulum cell sarcoma
Matrix metalloprotease 3 (Lijnen et al., 1998)
Hydrolysis of: Glu59±Asn60, Pro447±Val448, Pro544±Ser545
55 kDa fragment of plasminogen comprising kringles 1±4 Activity: not tested
Matrix metalloprotease 7 and 9 (Patterson and Sang, 1997)
Cleavage site of MMP-7: Pro466±Val467
40 kDa fragment of human plasminogen comprising kringles 1±4 Activity: not tested
Cleavage site of MMP-9: Pro465±Pro466 Plasminogen activators: uPA, tPA, or streptokinase in combination with a free sulfhydryl donor: N-acetyl-L-cysteine, D-penicillamine, captopril, L-cysteine, or reduced glutathione (Gately et al., 1996)
Conversion of plasminogen to plasmin. Autocatalytic cleavage of Lys77±Lys78 of plasmin in the presence of a free sulfhydryl donor
In vitro activity: inhibition of endothelial cell proliferation and migration In vivo activity: inhibition of cornea neovascularization
Table 2 Enzymatic generation of angiostatin protein from plasminogen in a cell system Enzyme/Reference
Form of angiostatin/Associated activity
PC-3 cell-derived serine proteinase (Gately et al., 1997)
50 kDa fragment of human plasminogen comprising of kringles 1±4 In vitro activity: inhibition of endothelial cell proliferation and tube formation In vivo activity: inhibition of cornea neovascularization
ASPC-1 cell-derived serine proteinase (O'Mahony et al., 1998)
48 kDa fragment of human plasminogen Activity: not tested
LLC-LM (low metastatic phenotype) (O'Reilly et al., 1994)
38 kDa fragment of human plasminogen isolated from murine urine In vitro activity: inhibition of endothelial cell proliferation In vivo activity: inhibition of CAM assay, suppression of metastatic growth in LLC-LM model
Macrophage-derived metalloelastase (Dong et al., 1997) CHO or HT1080 cell-derived `plasmin reductase' activity composed of two cofactors: a protein component and a low molecular weight molecule (Stathakis et al., 1997)
38 kDa fragment of human plasminogen In vitro activity: inhibition of endothelial cell proliferation Angiostatin-like fragment comprising of human kringles 1±4 with free sulfhydryl groups In vitro activity: inhibition of endothelial cell proliferation.
812 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim the tumor microenvironment, that are capable of cleaving plasminogen to a fragment with anti-angiogenic activity. These proteases include metalloelastase (MME) derived from macrophages infiltrating Lewis lung carcinoma (LLC) tumors, unidentified serine protease(s) derived from human prostate carcinoma (PC-3, DU-145, and LN-CaP), or human pancreatic carcinoma (ASPC-1) cells, and an unidentified `plasmin reductase' derived from Chinese hamster ovary (CHO) or HT1080 cells. Other enzymes implicated in the production of angiostatin from plasminogen include pancreatic elastase and various matrix metalloproteases.
RECEPTOR UTILIZATION The angiostatin receptor is not known.
IN VITRO ACTIVITIES
In vitro findings General in vitro observations are described in Table 3. The anti-angiogenic activity of angiostatin is Table 3 General in vitro observations with angiostatin protein Angiostatin induces
Angiostatin does not affect
Inhibition of endothelial cell proliferation, migration, and tube formation (O'Reilly et al., 1994; Barendz-Janson et al., 1998)
Growth of tumor cells (O'Reilly et al., 1994)
Upregulation of E-selectin expression in proliferating endothelial cells (Luo et al., 1998)
Expression of P-selectin and integrins avb3 and avb5 (Luo et al., 1998)
Regulatory molecules: Inhibitors and enhancers None known, although one study suggest that the presence of a free sulfhydryl group is essential for inhibition of endothelial cell proliferation (Gately et al., 1997).
Bioassays used The following bioassays were used for the in vitro experiments: Proliferation assay. Subconfluent cultures of endothelial or tumor cells are cultured with growth factor(s) in the presence or absence of an angiogenic inhibitor. The assay is terminated 72±96 hours later. Cell growth is determined by cell counting or DNA synthesis via thymidine or uridine incorporation. Wound migration assay. Confluent endothelial cell monolayers are wounded with a razor blade. Cells from the wound area migrate in the presence of growth factors. After a short incubation in the presence or absence of an angiogenic inhibitor, the cells are fixed and stained and the number of migrating cells are counted under a light microscope. The following bioassays were used for the in vivo experiments:
Downregulation of M-phase phosphoproteins in endothelial cells and disruption of G2/M transition (Griscelli et al., 1998)a Growth factor-stimulated Tyrosine phosphorylation Ras pathway of unidentified molecules with 250 kDa and 45 kDa molecular weights (Claesson-Welsh et al., 1998) a
summarized in Table 4. The anti-angiogenic activity of the individual kringles of angiostatin is summarized in Table 5 (Cao et al., 1996; Ji et al., 1998).
In contrast, a study published by Luo et al. (1998) suggests that angiostatin protein does not alter cell cycle progression and has little effect on the distribution of endothelial cells in G0/G1, S, and G2/M phases.
Chorioallantoic membrane (CAM). Disks containing various concentrations of an angiogenic inhibitor are placed on the chorioallantoic membrane of 6-day-old chicken embryos. After 48 hours of incubation, CAMs are checked for the presence of avascular zones in the periphery of the disk using a stereomicroscope. Mouse cornea micropocket assay. A pellet containing sucrose octasulfate, hydron, and 80±100 ng bFGF is placed into the cornea micropocket of a mouse and the cornea angiogenesis is evaluated by slit lamp microscopy. Primary tumor model. Mice or rats are implanted with a specific number of a certain tumor cell type. Systemic administration of an angiogenic inhibitor is initiated after the tumors have grown to a palpable size. Tumor size is assessed with a caliper and the tumor volume is determined.
Angiostatin 813 Table 4 The anti-angiogenic activity of angiostatin protein Source of angiostatin/Reference
Cells inhibited
Cells not inhibited
Murine; purified from serum or urine of tumor-bearing animals (O'Reilly et al., 1994)
Bovine capillary endothelial cells (BCE). Bovine aortic endothelial cells. Murine hemangioendothelioma cells (EOMA)
Lewis lung carcinoma (LLC) Mink lung epithelium 3T3 fibroblasts Bovine aortic smooth muscle Bovine retinal pigment epithelium MDK (canine renal epithelium) W138 (human fetal lung fibroblasts) EFN (murine fetal fibroblasts) LM (murine connective tissue)
Human; derived from proteolytic cleavage of plasminogen (Dong et al., 1997; Gately et al., 1997)
Bovine capillary endothelial (BCE) cells. Human umbilical vein endothelial cells (HUVEC). Bovine aortic endothelial cells
Lewis lung carcinoma (LLC) PC-3 (human prostate carcinoma) DU-145 (human prostate carcinoma) LN-CaP (human prostate carcinoma)
Human; derived from plasmin (Stathakis et al., 1997)
Human dermal microvascular endothelial cells
Bovine aortic endothelial Human umbilical vein endothelial (HUVEC) Chinese hamster ovary (CHO) HT1080
Human; recombinant (Sim et al., 1997)
Bovine capillary endothelial (BCE) cells
Lewis lung carcinoma (LLC)
Mouse; recombinant (Wu et al., 1997)
Bovine capillary endothelial (BCE) cells
Lewis lung carcinoma (LLC)
Table 5 Kringle localization of the antiproliferative and antimigratory activity of angiostatin protein Angiostatin domain
Inhibitory activity IC50 (nM) Proliferation
Migration
Kringle 1
320
> 1000
Kringle 2
±
Kringle 3
460
Kringle 4
±
1000
Domain combination
Observed effect
kringle 2±3
No inhibition of migration with kringle 2±3 in the presence of kringle 1
kringle 1
Inhibition of proliferation. Presence of kringle 2 did affect activity of kringle 1
kringle 3
Inhibition of migration, addition of kringle 3 did not increase the inhibition
kringle 1
Inhibition of proliferation. Presence of kringle 4 did affect activity of kringle 1
1000 500
kringle 1±3 Presence of kringle 1±3 resulted in a decrease of inhibition of migration Kringles 2±3
±
100
Kringles 1±3
70
> 1000
Kringles 1±4
135
50
Metastatic tumor model. Mice are implanted with tumor cells that exhibit a metastatic potential. The tumors are allowed to grow to a certain size before they are surgically removed. After tumor resection,
kringle 2
Reduced inhibition. Presence of kringles 2±3 decreased the activity of kringle 1
systemic treatment with an angiogenic inhibitor is initiated. At the end of the experiment, the number of pulmonary metastases and/or the lung weight of treated mice are compared to those of mice that
814 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim received a control treatment or that did not undergo tumor resection.
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles The studies performed thus far have explored the role of angiostatin only in the disease state.
Species differences Plasminogen and, therefore, angiostatin are highly conserved between species with a similarity index greater than 75%. Besides amino acid differences, posttranslational modifications, especially glycosylation, are known to differ between species.
Knockout mouse phenotypes Since angiostatin is the proteolytic fragment of plasminogen, the most relevant knockout mouse phenotype is the one that lacks plasminogen. Studies involving plasminogen-deficient mice showed that neither plasminogen nor plasmin are essential for embryonic development; however, the mice develop spontaneous fibrin deposits due to impaired thrombolytic potential, suffer retarded growth, and reduced fertility and survival (Lijnen, 1996).
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Role in experiments of nature and disease states Angiostatin has been shown to inhibit primary tumor growth and to suppress metastatic disease in murine models. The biological effects of angiostatin have been attributed to its ability to inhibit neovascularization. Inhibition of new blood vessel formation within a tumor with steady state proliferative rate results in lack of nutrients, accumulation of toxic metabolites, and induction of cell death. Therefore,
angiostatin treatment results in a higher apoptotic rate of tumor cells, while it leaves their proliferative rate unaffected (O'Reilly et al., 1996; Kirsch et al., 1998). Angiostatin suppresses neovascularization directly, by inhibiting proliferation, migration, and tube formation of endothelial cells and indirectly, by influencing the production of angiogenic molecules. For example, angiostatin treatment of C6 and 9L glioma tumors is associated with a 3-fold downregulation of VEGF mRNA expression. Angiostatin was also found to induce a 4-fold upregulation of bFGF mRNA in C6 tumors and to increase TGF expression in 9L tumors (Kirsch et al., 1998). Interestingly, the anti-angiogenic activity of angiostatin does not require an intact immune system, since angiostatin treatment leads to inhibition of primary tumors grown in mice lacking T lymphocytes (Kirsch et al., 1998).
IN THERAPY
Preclinical ± How does it affect disease models in animals? The in vivo biological properties of angiostatin are described in Table 6. The effect of angiostatin, derived from the proteolytic digest of plasminogen, on various primary tumor models is summarized on Table 7. The effect of angiostatin gene transfer on the growth of primary tumor models is outlined on Table 8. The combined effect of angiostatin and ionizing radiation on the growth of various primary tumor models is shown in Table 9 (Mauceri et al., 1998).
Pharmacokinetics There are no published pharmacokinetic studies for either recombinant or plasminogen-derived angiostatin. However, O'Reilly et al. (1994) estimated the half-life of endogenous, circulating angiostatin in tumor-bearing mice to be 2.5 days.
Toxicity Prolonged, systemic administration of angiostatin (50 mg/kg every 12 hours for 60 days) results in no apparent weight loss, lethargy, bleeding, hair loss, growth abnormalities, or other toxicity (O'Reilly et al., 1996). Angiostatin-treated animals do not suffer from anemia and have normal platelet counts
Table 6 In vivo activity of angiostatin protein in various animal disease models In vivo bioassay/Reference
Source of angiostatin
Treatment regimen
Result
CAM assay (O'Reilly et al., 1994)
Purified from serum of tumor-bearing mice
20±100 mg/embryo
Inhibition of blood vessel formation
Cornea micropocket assay (O'Reilly et al., 1994)
Proteolytic cleavage of human plasminogen
50 mg/kg twice/day
85% inhibition of neovascularization
Primary tumor models (see Table 7)
Proteolytic cleavage of human plasminogen
10±80 mg/kg
Partial or total inhibition of tumor growth
Lewis lung carcinoma spontaneous metastatic model (O'Reilly et al., 1994; Sim et al., 1997)
Proteolytic cleavage of human plasminogen, recombinant
0.6 mg/kg daily
Suppression of pulmonary metastases
Table 7 Effect of human angiostatin protein on primary tumor models Primary tumor model/Reference
Treatment regimen (mg/kg)
Effect on tumor growth
Lewis lung carcinoma (O'Reilly et al., 1994; Sim et al., 1997)
50 twice/day
87% inhibition
T241 fibrosarcoma (O'Reilly et al., 1994)
10 to 80
Suppression
PC-3 prostate carcinoma (O'Reilly et al., 1994)
50 twice/day
100% inhibition
Clone A colon carcinoma (O'Reilly et al., 1994)
50 twice/day
97% inhibition
MDA-MB breast carcinoma (O'Reilly et al., 1994)
50 twice/day
95% inhibition
M5076 reticulum cell sarcoma (O'Reilly et al., 1994)
50 twice/day
81% inhibition
Murine hemangioendothelioma (EOMA) (Lannutti et al., 1997)
40 twice/day
92% inhibition
Subcutaneous rat C6 glioma (Kirsch et al., 1998)
50 twice/day
74% inhibition
Intracranial rat C6 glioma (Kirsch et al., 1998)
50 twice/day
65% inhibition
Subcutaneous 9L glioma (Kirsch et al., 1998)
50 twice/day
69% inhibition
Intracranial 9L glioma (Kirsch et al., 1998)
50 twice/day
89% inhibition
Subcutaneous human U87 glioma (Kirsch et al., 1998)
50 twice/day
84% inhibition
Table 8 Effect of angiostatin protein gene transfer on primary tumor models Primary tumor model
Method of gene delivery
Effect
T241 fibrosarcoma (Cao et al., 1998)
Direct transfection into T241 fibrosarcoma cells with plasmid containing the angiostatin gene under the control of the cytomegalovirus promoter
77% inhibition of the transfected T241 fibrosarcoma
Rat C6 glioma (Griscelli et al., 1998)
Defective adenovirus expressing secretable kringles 1±3 from the cytomegalovirus promoter
80% inhibition of tumor growth
MDA-MB-231 breast carcinoma (Griscelli et al., 1998)
Defective adenovirus expressing secretable kringles 1±3 from the cytomegalovirus promoter
85% inhibition of tumor growth
Murine RT2 glioma (subrenal capsule assay, subdermal, intercerebral) (Tanaka et al., 1998)
Retroviral or adenoviral transduction
Suppression in the subrenal capsule assay Growth delay in the subdermal model Longer survival in the intercerebral model
Human U87MG glioma (subrenal capsule assay, intercerebral) (Tanaka et al., 1998)
Retroviral transduction
Suppression in the subrenal capsule assay Longer survival in the intercerebral model
816 Adonia E. Papathanassiu, Shawn J. Green, Davida K. Grella and B. Kim Lee Sim Table 9 Combined effect of angiostatin protein and ionizing radiation in various tumor models Primary tumor model
Angiostatin protein regimen
Radiation regimen
Effect
Lewis lung carcinoma
25 mg/kg daily
20 Gy on days 0 and 1
Tumor regression
D54 human glioma
25 mg/kg once
5 Gy per day/30 Gy total
Tumor regression
SQ-20B squamous cell carcinoma
25 mg/kg daily
5 Gy per day/50 Gy total
Tumor regression
PC-3 prostate adenocarcinoma
25 mg/kg daily
5 Gy per day/40 Gy total
Tumor regression
(Lannutti et al., 1997). Angiostatin does not affect tissue type plasminogen activator-induced whole blood clot lysis in vivo. Prolonged treatment with angiostatin does not result in development of drug resistance (Griscelli et al., 1998).
References Barendsz-Janson, A. F., Griffioen, A. W., Muller, A. D., van Dam-Mieras, M. C. E., and Hillen, H. F. P. (1998). In vitro tumor angiogenesis assays: Plasminogen lysine bindingsite 1 inhibits in vitro tumor-induced angiogenesis. J. Vasc. Res. 35, 109±114. Cao, Y., Ji, R.-W., Davidson, D., Schaller, J., Marti, D., Sohndel, S., McCane, S. G., O'Reilly, M. S., Llinas, M., and Folkman, J. (1996). Kringle domains of human Angiostatin1 Protein. J. Biol. Chem. 271, 29461±29467. Cao, Y., O'Reilly, M. S., Marshall, B., Flynn, E., Ji, R.-W., and Folkman, J. (1998). Expression of Angiostatin1 Protein cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormancy of metastases. J. Clin. Invest. 101, 1055±1063. Claesson-Welsh, L., Welsh, M., Ito, N., Anand-Apte, B., Soker, S., Zetter, B., O'Reilly, M., and Folkman, J. (1998). Angiostatin1 Protein induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc. Natl Acad. Sci. USA 95, 5579±5583. Dong, Z., Kumar, R., Yang, X., and Fiddler, I. J. (1997). Macrophage-derived metalloelastase is responsible for the generation of Angiostatin1 Protein in Lewis lung carcinoma. Cell 88, 801±810. Gately, Y., Twardowski, P., Stack, M. S., Patrick, M., Boggio, L., Cundiff, D. L., Schnaper, H. W., Madison, L., Volpert, O., Bouck, N., Enghild, J., Kwaan, H. C., and Soff, G.A, (1996). Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, Angiostatin1 Protein. Cancer Res. 56, 4887±4990. Gately, S., Twardowski, P., Stack, M. S., Cundiff, D. L., Grella, D., Castellino, F. J., Enghild, J., Kwaan, H. C., Lee, F., Kramer, R. A., Volpert, O., Bouck, N., and Soff, G. A. (1997). The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor Angiostatin1 Protein. Proc. Natl Acad. Sci. USA 94, 10868±10872. Griscelli, F., Li, H., Bennaceur-Griscelli, A., Soria, J., Opolon, P., Soria, C., Perricaudet, M., Yeh, P., and Lu, H. (1998). Angiostatin1 Protein gene transfer: Inhibition of tumor growth
in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest. Proc. Natl Acad. Sci. USA 95, 6367±6372. Ji, W. R., Castellino, F. J., Chang, Y., DeFord, M. E., Gray, H., Villareal, X., Eghtedarzadeh, K., Marti, D. N., Llinas, M., Schaller, J., Kramer, R. A., and Trail, P. A. (1998). Characterization of kringle domains of angiostatin as antagonists of endothelial cell migration, an important process in angiogenesis. FASEB J. 12, 1731±1738. Kirsch, M., Strasser, J., Allende, R., Bello, L., Zhang, J., and Black, P. McL. (1998). Angiostatin1 Protein suppresses malignant glioma growth in vivo. Cancer Res. 58, 4654±4659. Lannutti, B. J., Gately, S. T., Quevedo, M. E., Soff, G. A., and Paller, A. S. (1997). Human Angiostatin1 Protein inhibits murine hemangioendothelioma tumor growth. Cancer Res. 57, 5277±5280. Lijnen, H. R. (1996). Pathophysiology of the plasminogen/plasmin system. Int. J. Clin. Lab. Res. 26, 1±6. Lijnen, R. H., Ugwu, F., Bini, A., and Collen, D. (1998). Generation of an Angiostatin1 Protein-like fragment from plasminogen by stromelysin-1 (MMP-3). Biochemistry 37, 4699± 4702. Luo, J., Lin, J., Paranya, G., and Bischoff, J. (1998). Angiostatin1 Protein upregulates E-selectin in proliferating endothelial cells. Biochem. Biophys. Res. Commun. 245, 906±911. Matsuoka, Y., Kitamura, Y., and Taniguchi, T. (1998). Induction of plasminogen in rat hippocampal pyramidal neurons by kainic acid. Neurosci. Lett. 252, 119±122. Mauceri, H. J., Hanna, N. N., Beckett, M. A., Gorski, D. H., Staba, M.-J., Stellato, K. A., Bigelow, K., Heimann, R., Gately, S., Dhanaba, M., Soff, G. A., Sukhatme, V. P., Kufe, D. W., and Weichselbaum, R. R. (1998). Combined effects of Angiostatin1 Protein and ionizing radiation in antitumor therapy. Nature 394, 287±291. O'Mahony, C. A., Seidel, A., Albo, D., Chang, H., Tuszynski, G. P., and Berger, D. H. (1998). Angiostatin1 Protein generation by human pancreatic cancer. J. Surg. Res. 77, 55±58. O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994). Angiostatin1 Protein: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis Lung carcinoma. Cell 79, 315±328. O'Reilly, M. S., Holmgren, L., Chen, C., and Folkman, J. (1996). Angiostatin1 Protein induces and sustains dormancy of human primary tumors in mice. Nature Med. 2, 689±693. Patterson, B. C., and Sang, Q. X. A. (1997). Angiostatin1 Proteinconverting enzyme activities of human matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9). J. Biol. Chem. 272, 28823±28825.
Angiostatin 817 Saksela, O., and Vihko, K. K. (1986). Local synthesis of plasminogen by the seminiferous tubules of the testis. FEBS Lett. 204, 193±197. Sim, B. K. L., O'Reilly, M. S., Liang, H., Fortier, A. H., He, W., Madsen, J. W., Lapcevich, R., and Nacy, C. A. (1997). A recombinant human Angiostatin1 Protein protein inhibits experimental primary and metastatic cancer. Cancer Res. 57, 1329±1334. Stathakis, P., Fitzerald, M., Matthias, L. J., Chesterman, C. N., and Hogg, P. J. (1997). Generation of Angiostatin1 Protein by reduction and proteolysis of plasmin. J. Biol. Chem. 272, 20641± 20645. Tanaka, T., Cao, Y., Folkman, J., and Fine, H. A. (1998). Viral vector-targeted antiangiogenic gene therapy utilizing an
Angiostatin1 Protein complementary DNA. Cancer Res. 58, 3362±3369. Wu, Z., O'Reilly, M. S., Folkman, J., and Shing, Y. (1997). Suppression of tumor growth with recombinant murine growth. Biochem. Biophys. Res. Commun. 236, 651±654.
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