The Generation of Endostatin Is Mediated by Elastase1

[CANCER RESEARCH 59, 6052–6056, December 15, 1999] Advances in Brief

Wei Wen, Marsha A. Moses, Dmitri Wiederschain, Jack L. Arbiser, and Judah Folkman2 Laboratory for Surgical Research, Department of Surgery, The Children’s Hospital, Boston, Massachusetts 02115 [W. W., M. A. M., D. W., J. F.]; Departments of Surgery [M. A. M., J. F.] and Cellular Biology [J. F.], Harvard Medical School, Boston, Massachusetts 02115; and Department of Dermatology, Emory University School of Medicine, Atlanta, Georgia 30322 [J. L. A.]

Abstract function as an EPE by specifically cleaving recombinant NC1 at the Ala-His linkage and releasing endostatin. In addition, we show Endostatin, a potent inhibitor of angiogenesis and tumor growth, is a that at least two steps, a metal-dependent early step and an elas- COOH-terminal fragment of collagen XVIII derived through cleavage of tase-dependent final step, were involved in the generation of an Ala-His linkage by an as yet unidentified endostatin-processing enzyme. Endostatin was originally isolated from the conditioned medium of endostatin from collagen XVIII. hemangioendothelioma (EOMA) cells. By investigating the processing of collagen XVIII to endostatin by EOMA cells, we show here that the Materials and Methods generation of endostatin can be mediated by an elastase activity. We also show that several members of the elastase family can act as an endostatin- Materials. Aprotinin, bestatin, chymostatin, E-64, leupeptin, pepstatin, processing enzyme by specifically cleaving the Ala-His linkage and releas- soybean trypsin inhibitor, TLCK, and TPCK were purchased from Boehringer ing endostatin from a precursor molecule. We further suggest that the Mannheim. AEBSF, elastatinal, elastase inhibitor, pAPMSF, porcine pancre- generation of endostatin from collagen XVIII is at least a two-step process, atic elastase, and human neutrophil elastase were obtained from Calbiochem. involving a metal-dependent early step and an elastase activity-dependent Cell Culture. EOMA cells were cultured as described previously (1). Cells final step. were maintained in DMEM supplemented with 10% heat-inactivated calf serum and 1% glutamine-penicillin-streptomycin. Cells were passaged by Introduction trypsinization (Trypsin/EDTA; Life Technologies, Inc.), followed by dilution (100–200-fold) in DMEM medium containing 10% calf serum. Angiogenesis inhibitors, which inhibit angiogenesis specifically Expression and Purification of rNC1 Protein. The cDNA encoding NC1 and selectively, have been shown to suppress tumor growth in mice was amplified from a mouse ␣1 (XVIII) cDNA clone mc3b (kindly provided without toxicity or drug resistance. Many of the known angiogenesis by N. Fukai and B. Olsen, Harvard Medical School, Boston, MA) by PCR and inhibitors are fragments of larger molecules. For instance, endostatin, was cloned into the EcoRI and XbaI sites of the expression vector pSecTagA (Invitrogen). The vector contains a secretion signal at the NH -terminus and a potent angiogenesis inhibitor, is a fragment of collagen XVIII (1), 2 myc and His-tags at the COOH-terminus to facilitate purification and detec- and angiostatin is a fragment of plasminogen (2). Elucidation of the tion. mechanisms that control the release of angiogenesis inhibitors from The rNC1 expression vector was introduced into 293T human epithelial their parental molecules will have a profound influence on our un- kidney cells using LipofectAMINE (Life Technologies, Inc.). Transfected cells derstanding of angiogenesis and may also lead to potential therapeutic were selected with Zeocin (400 ␮g/ml) in DMEM with 10% calf serum, applications. expanded, and thereafter maintained in medium containing Zeocin. After The generation of angiostatin has been shown to involve a reaching confluence, NC1-expressing cells were washed with PBS and variety of pathways that appear to be different in different model changed into serum-free medium (Opti-MEM, supplemented with insulin- systems (3–9). However, the mechanism by which endostatin is transferrin-selenium). After a 48 h incubation, the medium was collected and ␮ generated from collagen XVIII has not been elucidated. Collagen filtered through a 0.45 m filter. His-tagged NC1 protein was purified using a XVIII consists of an NH -terminal noncollagenous domain, a TALON affinity column (Clontech) according to the manufacturer’s instruc- 2 tions. The purified protein was dialyzed against PBS and stored at Ϫ80°C in series of collagen-like domains, and a COOH-terminal NC13 do- aliquots of 50 ␮l. main (Fig. 1A; Ref. 1). Endostatin, originally isolated from the Western Blot Analysis. Proteins were separated on a 12.5% SDS-PAGE, medium of hemangioendothelioma (EOMA) cells, is generated electrotransferred onto a PVDF membrane, and probed with a rabbit polyclonal from collagen XVIII through the cleavage of an Ala-His linkage by antibody against endostatin, followed by an horseradish peroxidase-conjugated an unidentified EPE. The present study was undertaken to identify sheep antirabbit antibody (Amersham). Three different rabbit antibodies were the enzymes involved in the generation of endostatin. In this study, used for these experiments. The antibody raised against recombinant mouse we report that members of the elastase family of enzymes can endostatin was used routinely, whereas two other antibodies raised either against recombinant human endostatin or a peptide (GLSGTFRAFLSSR- LQDLYSIVRRADRGSVC) derived from mouse endostatin were used to Received 10/4/99; accepted 10/29/99. The costs of publication of this article were defrayed in part by the payment of page verify the results. After washing the PVDF membrane extensively, endostatin- charges. This article must therefore be hereby marked advertisement in accordance with related proteins were visualized using ECL Western blotting detection reagents 18 U.S.C. Section 1734 solely to indicate this fact. (Amersham). 1 This study was supported by Grant R01 CA64481 from the NIH (to J. F.), a grant to Generation of Endostatin from rNC1. rNC1 (100 ng) was incubated with Children’s Hospital from EntreMed, Inc. (Rockville, MD), Grant RPG-97-013 from the ␮ American Cancer Society (to M. A. M.), and by Grants KO8 AR 02096-01 and RO3 AR CM from EOMA cells (30 l, collected 3 days after cells were plated) at 37°C 44947-03 from NIAMS (to J. L. A.). W. W. is supported by the Susan G. Komen Breast for the indicated time either in the absence or presence of protease inhibitors. Cancer Foundation. Cleavage products were examined by Western analysis. 2 To whom requests for reprints should be addressed, at Children’s Hospital, Hun- Detection of Endostatin in Conditioned Medium of EOMA Cells. CM newell 103, 300 Longwood Avenue, Boston, MA 02115. ␮ 3 The abbreviations used are: NC1, noncollageneous domain 1 of collagen XVIII; (3 ml) was incubated with 30 l of heparin Sepharose beads (Pharmacia rNC1, recombinant NC1; CM, conditioned medium; EPE, endostatin-processing en- Biotech, Inc.) on a rotating shaker for1hat4°Ctoconcentrate endostatin. zyme; HNE, human neutrophil elastase; PPE, porcine pancreatic elastase; MMP, After washing three times with 10 mM Tris (pH 7.0), the beads were boiled in matrix metalloprotease; PVDF, polyvinylidene difluoride; AEBSF, 4-(2-aminoethyl)- 20 ␮l of SDS sample buffer. Endostatin was then detected by Western blotting. benzenesulfonyl fluoride; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; TLCK, N-␣-p-tosyl-L-lysine chloromethyl ketone; pAPMSF, (4-amidinophenyl)- Purification of an EPE. An extract of porcine pancreas (Life Technol- methanesulfonyl fluoride. ogies, Inc.) was chosen as the source of purification, because it was found 6052

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1999 American Association for Cancer Research. PROCESSING OF ENDOSTATIN FROM COLLAGEN XVIII to contain an EPE activity and was readily available in larger quantities Results than EOMA CM. The extract was first dialyzed against 10 mM Tris (pH Generation of Endostatin by EOMA Cells May Involve Several 7.0) and then applied to a Hitrap Q Sepharose column (5 ml) (Pharmacia Biotech, Inc.) preequilibrated with the same buffer. Proteins were eluted Proteolytic Activities. To investigate the processing of endostatin, through a stepwise profile with 0.2, 0.3, 0.4, and 2 M NaCl at a flow rate CM from EOMA cells, from which endostatin was first identified, of 1 ml/min using fast protein liquid chromatography (Pharmacia). Each was used for study. For controls, CM from several other collagen fraction was screened for its ability to cleave rNC1 to endostatin. Fractions XVIII-producing cell lines was also examined. The presence of en- with cleavage activity, which eluted at 0.2 M NaCl, were pooled and applied dostatin was determined by Western blot analysis using antibodies to a Superose 12 gel filtration column (Pharmacia) using fast protein liquid specific for endostatin as described in “Materials and Methods.” As chromatography. Active fractions were then fractionated on a SynChropak expected, endostatin was detected in the CM of EOMA cells (Fig. 1B). RP-4 column using high performance liquid chromatography. The activity It was not, however, present in the medium of a cell line that does not eluted at a concentration of 50–53% acetonitrile in 0.1% trifluoroacetic produce collagen XVIII (CCL188), nor was it detected in the media of acid. several cell lines that produce collagen XVIII (BAE, BCE, and Protein Sequencing. After SDS-PAGE, proteins were blotted to a PVDF membrane. Coomassie blue-stained proteins were excised from the membrane BASMC). Endostatin was also found, albeit at a lower level than in and applied directly to a Perkin Elmer/Applied Biosystems Division model the CM of EOMA cells, in the medium of a mouse capillary endo- 477A protein sequencer, with an on-line model 120A PHP-amino acid ana- thelial cell line SVR (transformed with SV40 large T and H-ras) but lyzer. Protein sequencing was conducted by the HHMI/Harvard Medical was absent in the medium of its parental cell line MS1 (expressing School Biopolymers Facilities. only SV40 large T; Fig. 1B; Ref. 10).

Fig. 1. Generation of endostatin by EOMA cells involves an elastase activity. A, Schematic repre- sentation of collagen XVIII and endostatin. Open squares, collagen-like domains. Arrow, cleavage site of endostatin. Es, endostatin. B and C, generation of endostatin by EOMA cells. Cells were plated, grown to near confluency, and refed with serum-free medium with (C) or without (B) various protease inhibitors. CM (3 ml) of EOMA (B and C) and other cells (B) were collected 24 h later. En- dostatin was enriched by incubation with heparin beads and subsequently detected by Western blotting with an antibody against endostatin. BAE, bovine aortic endothelial cells; BCE, bovine capillary endothelial cells; BSMC, bovine smooth muscle cells; CCL188, human colon CCL 188 carcinoma cell line; MS1, mouse capillary endothelial cells immortalized with SV40 large T; SVR, MS1 transformed with H-ras. D and E, conversion of endostatin from rNC1 by CM of EOMA cells. Purified rNC1 (100 ng) was incubated with CM (30 ␮l, collected 3 days after cell were plated) for the indicated time (16 h in E) in the absence (D)or presence (E) of protease inhibitors. Cleavage products were detected by Western blotting with an antibody against endostatin.

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Table 1 Effects of protease inhibitors on the generation of endostatin Inhibitory effect on generation of endostatina

EOMA cellsb (from endogenous CM of EOMA cellsc Purified EPEd Inhibitor (concentration) Class of protease inhibited collagen XVIII) (from rNC1) (from rNC1) Leupeptin (50 ␮g/ml) Serine proteases ϪϪϪ Aprotinin (2 ␮g/ml) Serine proteases ϪϪϪ SBTIe (1 mg/ml) Serine proteases ϪϪϪ AEBSF (1 mM) Serine proteases ND ϩϩ TLCK (100 ␮M) Serine proteases ϪϪϪ TPCK (100 ␮M) Serine proteases ND ϪϪ pAPMSF (100 ␮M) Serine proteases ϪϪϪ Chymostatin (100 ␮M) Chymotrypsin Ϫ ND Ϫ Elastase inhibitor (100 ␮M) Elastase ϩϩϩ Elastatinal (100 ␮M) Elastase ϩϩϩ E64 (10 ␮M) Cysteine proteases ϪϪϪ Pepstatin (1 ␮M) Aspartic proteases ϪϪϪ Bestatin (10 ␮M) Aminopeptidases ϪϪϪ EDTA (1 mM) Metalloproteases ND ϪϪ 1,10-Phenanthroline (10 ␮M) Metalloproteases ϩϪϪ a Ϫ, not inhibited; ϩ, inhibited; ND, not determined. b CM of EOMA cells was collected 24 h after incubation with cells in the presence of various protease inhibitors as described in Fig. 1C. c rNC1 was incubated with CM of EOMA cells plus protease inhibitors as described in Fig. 1E. d rNC1 was incubated with the purified EPE in the presence of protease inhibitors as described in Fig. 2C. e SBTI, soybean trypsin inhibitor.

In addition to endostatin, several other endostatin-related peptides of rNC1 to endostatin by the CM of EOMA cells, the effect of were detected in the CM of EOMA cells. One of the peptides migrates elastatinal was tested (Fig. 1E and Table 1). As expected, this ϳ at Mr 32,000 and therefore is likely to be NC1. The presence of inhibitor significantly blocked the cleavage of rNC1. AEBSF, a these intermediate-sized fragments indicates that the generation of general serine protease inhibitor, also blocked the cleavage of endostatin from collagen XVIII by EOMA cells might involve several rNC1, but some other serine protease inhibitors that are more proteolytic activities or several steps. specific to trypsin or chymotrypsin had little effect on the cleav- Generation of Endostatin by EOMA Cells Is Inhibited by an age. These results suggest that an elastase-like serine protease is Elastase Inhibitor and by a Matrix Metalloprotease Inhibitor. involved in the cleavage of rNC1 to endostatin. To examine the proteolytic activity involved in the generation of Elastases Can Directly Cleave rNC1 to Endostatin. We next endostatin by EOMA cells, a panel of class-specific protease asked whether an elastase-like protease directly cleaves NC1 to en- inhibitors was tested. Generation of endostatin in the CM of dostatin or whether such a protease triggers a cascade of proteolytic EOMA cells was significantly impaired in the presence of elasta- processes, leading to the release of endostatin. To address this ques- tinal, a specific inhibitor of elastases, but not by several other tion, a protease that is capable of directly cleaving rNC1 to endostatin classes of protease inhibitors (Fig. 1C and Table 1). The elastase at the site of Ala-His (EPE) was isolated as described in “Materials inhibitor, however, did not inhibit the generation of the cleavage and Methods” and identified to be a member of the elastase family product that corresponds to NC1 (Fig. 1C). A metal chelator, (Fig. 2A). We chose an extract of porcine pancreas for the purification, 1,10-phenanthroline, on the other hand, blocked the generation of because we found that the presence of residual amounts (0.5% v/v) of both NC1 and endostatin, implying that a metal-dependent activity TC trypsin (an extract of porcine pancreas) greatly enhanced the was required for the generation of a precursor of endostatin, e.g., accumulation of endostatin in the CM of EOMA cells, and that this NC1. A MMP might be responsible for such a metal-dependent extract was capable of cleaving rNC1 to endostatin. The purified activity, because 1,10-phenanthroline can specifically inhibit enzyme was able to cleave rNC1 to a Mr 20,000 peptide, the size of MMP activity. These results suggest that the proteolytic activities endostatin. NH2-terminal sequencing of this cleavage product showed mediating the generation of endostatin might involve both an that it begins with amino acids HTHQDFQP, identical to that of elastase activity and a MMP activity. endostatin (1).

Elastase Activity Is Involved in the Cleavage of rNC1 to En- NH2-terminal sequencing of the purified EPE (Fig. 2B) revealed dostatin by the CM of EOMA Cells. The above results suggest that that it is identical to hypocalcemic factor (11), except at position NC1 is likely to be a precursor of endostatin and that an elastase 20, where a clear signal was not detected. Hypocalcemic factor activity likely mediates the generation of endostatin from NC1 by belongs to the type IIIB elastase family (11, 12) and has been EOMA cells. To further analyze the second step in the process of shown to decrease ionized calcium levels in the blood stream and endostatin generation, we next used rNC1 as a substrate to test to inhibit bone resorption (11). Cleavage of rNC1 to endostatin by whether the CM of EOMA cells is capable of cleaving rNC1 to the purified EPE was abolished by specific inhibitors of elastase, endostatin, and whether an elastase activity is involved in the cleav- elastatinal, and elastase inhibitor, as well as a general serine age. His-tagged rNC1 was expressed in 293T cells as a secreted protease inhibitor AEBSF, but not by several other protease inhib- protein and was purified using a TALON metal affinity column as itors (Fig. 2C and Table 1). This result was consistent with the described in “Materials and Methods.” Purified rNC1 protein was profile of inhibitors seen in the generation of endostatin by EOMA incubated with the CM of EOMA cells, and cleavage products were cells (Fig. 1, C and E; Table 1), suggesting that the purified EPE analyzed by Western blotting. As illustrated in Fig. 1D, the CM was may represent the activity present in the CM of EOMA cells. capable of cleaving rNC1 to endostatin in a time-dependent manner. Taken together, our results demonstrate that an elastase-like pro- The cleavage was first observed 1 h after incubation and was complete tease can act as an EPE by directly cleaving at the Ala-His site. within 16 h. We also tested the cleavage of rNC1 by representatives of two To determine whether an elastase was involved in the cleavage major classes of elastase, porcine pancreatic elastase (PPE) and hu- 6054

Fig. 2. An elastase-like protease can directly release endostatin from rNC1. A, SDS-PAGE gel of a purified EPE. Fractions eluted from an RP-4 column were resolved on an SDS-PAGE gel and stained with Coomassie blue 250. Fraction 23 contains the EPE activity. B, the NH2-terminal sequence of the purified EPE is aligned with several members of the elastase family. Nonconserved amino acids are underlined; amino acids that were determined with low confidence are shown in lower case; X, an undetermined amino acid. C, rNC1 was incubated with the purified EPE in the presence of inhibitors for elastase or other proteases. D, rNC1 was incubated with PPE and HNE in the presence or absence of elastase inhibitor, elastatinal, for 16 h. Cleavage products were analyzed for the presence of endostatin by Western blotting and were also NH2-terminal sequenced. man neutrophil elastase (HNE) (Fig. 2D). Both elastases were capable collagen XVIII is not a suitable substrate for elastase and that the of directly converting rNC1 to a peptide with a size similar to that of generation of endostatin may involve more than one step. For in- endostatin. The cleavage activities were also blocked by elastatinal. stance, native collagen XVIII may need to be initially modified or

NH2-terminal sequencing of the cleavage products showed that the processed, allowing it to be readily cleaved by an elastase in a main cleavage site is the Ala-His linkage. Cleavage after alanine is consistent with the substrate preference of elastase (13).

Discussion In this report, we describe for the first time a mechanism for the processing of the angiogenesis inhibitor endostatin from collagen XVIII. We show that the generation of endostatin by EOMA cells can be prevented by either a metal chelator or an elastase inhibitor and suggest that the processing of collagen XVIII to endostatin involves at least two steps, a metal-dependent early step and an elastase activity- dependent final step (Fig. 3). In addition, we identify an elastase-like protease as an EPE, which can specifically cleave rNC1 at the Ala-His site to release a peptide with an identical size and NH2-terminal sequence (HTH) to that of the endostatin protein isolated originally (1). The suggestion of a two-step process for the generation of endostatin and the involvement of a metal-dependent activity in the early step Fig. 3. A model for the generation of endostatin from collagen XVIII. At least two steps are involved in the generation of endostatin (Es) from collagen XVIII, a metal- are supported by the following observations: dependent early step and an elastase activity-dependent final step. 1,10-Phenanthroline, a (a) Several other collagen XVIII-producing cells could not generate metal chelator, inhibits the generation of endostatin by preventing the generation of a precursor of endostatin. Elastases (hypocalcemic factor, PPE, and HNE) are capable of endostatin, even when large amounts of the purified EPE were sup- cleaving a precursor at the Ala-His site and releasing endostatin. Elastatinal, a specific plemented into the cultures (not shown). One possibility is that native elastase inhibitor, blocks the generation of endostatin at this final step. 6055

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1999 American Association for Cancer Research. PROCESSING OF ENDOSTATIN FROM COLLAGEN XVIII subsequent step. EOMA cells, as well as SVR cells, appear to be involves at least two steps and that the final step can be mediated by capable of this initial modification or processing. an elastase activity. Elucidation of the regulatory machinery that (b) Elastase inhibitors blocked the generation of endostatin in the controls the release of angiogenesis inhibitors from their precursors culture of EOMA cells with a concomitant accumulation of NC1 (Fig. will provide a better understanding of the process of angiogenesis and 1C), indicating that NC1 is likely an intermediate product of collagen may also lead to potential therapeutic applications. XVIII processing. Acknowledgments (c) A metal chelator, although not inhibiting the cleavage of rNC1 to endostatin (Fig. 1E), impaired the production of both NC1 and We thank Dr. Michael O’Reilly for EOMA cells; Drs. Thomas Boehm and endostatin by EOMA cells (Fig. 1C), suggesting that a metal-depen- Kashi Javaherian for generously providing antibodies against endostatin; Dr. dent activity (e.g., MMP activity) is required for generating a precur- Naomi Fukai and Bjo¨rn Olsen for mc3b cDNA clones; Dr. Yuen Shing for sor of endostatin (e.g., NC1). Finally, another cell line SVR that also helpful advice on protein purification; Catherine Butterfield for help with produces endostatin was shown to have an elevated level of MMP tissue culture; and Drs. Deborah Freedman, David Resnick, Thomas Boehm, (10), consistent with a potential role of MMP in the generation of an and Steven Pirie-Shepherd for very thoughtful discussions and critical reading endostatin precursor. of the manuscript. Elastases are a group of proteases that can cleave elastin, an important References connective tissue protein. They have also been demonstrated to process a 1. O’Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., number of molecules from their precursors, including tumor necrosis Birkhead, J. R., Olsen, B. R., and Folkman, J. Endostatin: an endogenous inhibitor of factor ␣ (14) and interleukin-1␤ (15). Here we show that an elastase is angiogenesis and tumor growth. Cell, 88: 277–285, 1997. 2. O’Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., also involved in the generation of a potent angiogenesis inhibitor, en- Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. Angiostatin: a novel angiogenesis dostatin. Our observation that several members of the elastase family can inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma [see convert NC1 to endostatin implies that there may not be a unique elastase comments]. Cell, 79: 315–328, 1994. 3. O’Reilly, M. S., Wiederschain, D., Stetler-Stevenson, W. G., Folkman, J., and Moses, for the processing of endostatin and that different elastases may be M. A. Regulaton of angiostatin production by matrix metalloproteinase-2 in a model involved in its generation in different tissues. of concomitant resistance. J. Biol. Chem., 274: 29568–29571, 1999. Endostatin-like molecules ranging from M 22,000–38,000 with 4. Dong, Z., Kumar, R., Yang, X., and Fidler, I. J. Macrophage-derived metalloelastase r is responsible for the generation of angiostatin in Lewis lung carcinoma. Cell, 88: distinct NH2-terminal sequences have been found in the circulation 801–810, 1997. and in many tissues (16, 17), but the physiological roles of these 5. 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, endostatin-related proteins have not yet been established. Whether G. A. The mechanism of cancer-mediated conversion of plasminogen to the angio- functional endostatin can be produced in vivo and whether elastases genesis inhibitor angiostatin. Proc. Natl. Acad. Sci. USA, 94: 10868–10872, 1997. are involved in its generation remain to be determined. It is very 6. Stathakis, P., Lay, A. J., Fitzgerald, M., Schlieker, C., Matthias, L. J., and Hogg, P. J. Angiostatin formation involves disulfide bond reduction and proteolysis in kringle 5 plausible, however, that endostatin can be generated by elastases in of plasmin. J. Biol. Chem., 274: 8910–8916, 1999. vivo, given that elastases are produced in a highly regulated manner by 7. Lijnen, H. R., Ugwu, F., Bini, A., and Collen, D. 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A., Fernandez, C. A., Ghiso, N., Cao, Y., Klauber, N., found that although many proteases including thrombin, plasmin, Frank, D., Brownlee, M., Flynn, E., Parangi, S., Byers, H. R., and Folkman, J. chymotrypsin, and corneal extracts containing MMPs did not generate Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc. Natl. Acad. Sci. USA, 94: 861–866, 1997. endostatin, cathepsin B and cathepsin L could convert rNC1 into a 11. Izbicka, E., Yoneda, T., Takaoka, Y., Horn, D., Williams, P., and Mundy, G. R. fragment with a size similar to that of endostatin. We did not further Identification of a novel bone/calcium metabolism-regulating factor in porcine pan- characterize the cleavage of rNC1 by cathepsins. However, at the time creas. J. Biol. Chem., 271: 23230–23234, 1996. 12. Tani, T., Ohsumi, J., Mita, K., and Takiguchi, Y. 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Wei Wen, Marsha A. Moses, Dmitri Wiederschain, et al.

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