Matrix Generate Angiostatin: Effects on Neovascularization Lynn A. Cornelius, Leslie C. Nehring, Elizabeth Harding, Mark Bolanowski, Howard G. Welgus, Dale K. Kobayashi, This information is current as Richard A. Pierce and Steven D. Shapiro of September 29, 2021. J Immunol 1998; 161:6845-6852; ; http://www.jimmunol.org/content/161/12/6845 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Matrix Metalloproteinases Generate Angiostatin: Effects on Neovascularization1

Lynn A. Cornelius,2* Leslie C. Nehring,* Elizabeth Harding,ሻ Mark Bolanowski,ሻ Howard G. Welgus,* Dale K. Kobayashi,¶ Richard A. Pierce,* and Steven D. Shapiro†‡§¶

Angiostatin, a cleavage product of plasminogen, has been shown to inhibit endothelial cell proliferation and metastatic tumor cell growth. Recently, the production of angiostatin has been correlated with tumor-associated macrophage production of elastolytic metalloproteinases in a murine model of Lewis lung cell carcinoma. In this report we demonstrate that purified murine and human matrix metalloproteinases generate biologically functional angiostatin from plasminogen. Macrophage (MMP-12 or MME) proved to be the most efficient angiostatin-producing MMP. MME was followed by gelatinases and then the stomelysins in catalytic efficiency; interstitial collagenases had little capacity to generate angiostatin. Both recombinant angiostatin and an- giostatin generated from recombinant MME-treated plasminogen inhibited human microvascular endothelial cell proliferation Downloaded from and differentiation in vitro. Finally, employing macrophages isolated from MME-deficient mice and their wild-type littermates, we demonstrate that MME is required for the generation of angiostatin that inhibits the proliferation of human microvascular endothelial cells. The Journal of Immunology, 1998, 161: 6845–6852.

t has long been recognized that the removal of certain pri- activity to generate angiostatin: urokinase and free sulfhydryl

mary tumors, in both experimental models (1–3) and clinical donors (6). http://www.jimmunol.org/ I practice (4), can be followed by the rapid growth of dormant Recently, however, Dong et al. (7) demonstrated that generation metastases. In searching for a factor that may mediate this re- of angiostatin in the LLC model was not caused by tumor cell sponse, O’Reilly recently isolated an inhibitor termed proteinases but, rather, was associated with the presence of mac- angiostatin, a fragment of plasminogen containing kringle regions rophages in the primary tumor. Furthermore, they found that an- 1–4 (3). Using a murine model of Lewis lung cell carcinoma giostatin activity, as measured by inhibition of endothelial cell (LLC),3 these investigators demonstrated that s.c. implanted LLC proliferation, was correlated with the presence of EDTA-inhibit- tumors generated angiostatin, which prevented the neovasculariza- able elastolytic activity, presumably the matrix metalloprotienase tion of lung metastases and maintained them in a dormant state. murine macrophage elastase (MME or MMP-12) (8–10). Along by guest on September 29, 2021 Upon removal of the primary tumor, presumably the source of these lines, a recent communication (11) described the angiostatin- angiostatin, lung metastases resumed angiogenesis and grew converting enzyme activities of MMP-7 (matrilysin) and MMP-9 rapidly. (gelatinase B, 92-kDa gelatinase). The biologic activity of the In addition to these findings, angiostatin was shown to be pro- MMP-generated products was not determined, however. duced in vitro upon exposure of plasminogen to pancreatic elas- MMPs represent a family of structurally related enzymes with tase; the source of endogenous proteolytic activity for the in vivo catalytic activity that is dependent upon coordination of zinc and generation of angiostatin in the carcinoma was not identified. In with catalysis that is specifically inhibited by the tissue inhibitors recent in vitro studies, human prostate carcinoma cells were found of metalloproteinases (TIMPs) (12, 13). Although MMPs possess to possess serine proteolytic capacity capable of converting plas- a broad capacity to degrade extracellular matrix components, these minogen to angiostatin (5). Further work identified two compo- proteinases are capable of cleaving nonmatrix that regu- nents present in carcinoma cells that provided sufficient protease late a variety of biologic processes. For example, MME has been ␣ shown to cleave 1-antitrypsin, releasing a 4-kDa fragment that is Divisions of *Dermatology and †Respiratory and Critical Care, Departments of ‡Med- chemotactic for neutrophils (14). MMPs have also been shown to icine, §Pediatrics, and ¶Cell Biology and Physiology, Washington University School possess the capacity to cleave latent TNF-␣, releasing the biolog- ࿣ of Medicine at Barnes-Jewish Hospital, St. Louis, MO 63110; and Monsanto/Searle ically active 17-kDa TNF-␣ from the surface of cells (15). In Co., St. Louis, MO 63141 this report we have studied the relative capacities of several Received for publication April 17, 1998. Accepted for publication August 31, 1998. MMPs to act upon plasminogen to generate angiostatin, and most The costs of publication of this article were defrayed in part by the payment of page importantly, we have investigated the effect of this product on charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. human endothelial cell proliferation and in an in vitro assay of 1 This work was supported by the National Institutes of Health (to L.A.C., H.G.W., angiogenesis. and S.D.S.), and a Monsanto/Searle-Washington University agreement (to L.A.C., H.G.W., and S.D.S.). 2 Address correspondence and reprint requests to Dr. Lynn A. Cornelius, Dermatol- Materials and Methods ogy Division, Barnes-Jewish Hospital, 216 S. Kingshighway, St. Louis, MO 63110. Reagents E-mail address: [email protected] 3 Purified MMPs were obtained from the following sources. Recombinant Abbreviations used in this paper: LLC, Lewis lung carcinoma; MME, murine mac- mouse (MME) (9) and human (HME) (10, 16) macrophage elastase and rophage elastase; MMP, matrix ; TIMP, tissue inhibitor of metal- loproteinases; HME, human macrophage elastase; K1–4, K1–3, K4, human - MMP-7 (17) were expressed and purified from Escherichia coli to homo- ogen kringle regions 1–4, 1–3, and 4; HPg, human plasminogen; MECs, human geneity in our laboratory. E. coli-derived enzymes were spontaneously ac- dermal microvascular endothelial cells; EBM, endothelial cell basal medium; bFGF, tive; that is, only their catalytic domains were expressed. Native MMP-1 basic fibroblast growth factor. (18) and MMP-9 (19) were purified in our laboratories (H.G.W. and

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00 6846 MATRIX METALLOPROTEINASES GENERATE ANGIOSTATIN

S.D.S.); MMP-3 was a gift from V. M. Baragi, Parke-Davis (Nutley, NJ) Human microvascular endothelial cell proliferation assay (17). MMP-8 and MMP-13 were provided by Monsanto-Searle (St. Louis, MO). Native enzymes were activated with p-aminophenylmercuric acetate MECs, isolated as described above, were maintained in culture and used at (10 mM) as described previously (19). Native TIMP was a gift from passages 2–5. MECs were plated onto gelatin-coated 24-well tissue culture ϫ 4 Dr. Carmichael (Synergen, Boulder, CO) (20). The bicinchoninic acid pro- plates at 1.25 10 cells/well and incubated overnight in EBM containing tein assay (Pierce, Rockford, IL) and a TIMP-1 inhibition assay were used 10% FCS, epidermal growth factor, hydrocortisone, cAMP, and antibiotics. to determine the total and active concentrations of MMPs, respectively The medium was then replaced with EBM containing 5% FCS, basic fi- (21). The latter method involved preincubation of known concentrations of broblast growth factor (bFGF; 1 ng/ml), and either buffer (300 mM Tris, 60 TIMP-1 with fixed amounts of active enzyme, followed by the addition of mM CaCl2, and 90 mM NaCl, pH 7.5), MME (240 nM), HPg (170 nM), Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-OEt. Hydrolysis of this thiopeptolide and MME-generated cleavage products of HPg (170 nM; 18-h incubation substrate (Bachem Bioscience, King of Prussia, PA) by metalloproteinases at 37°C) or recombinant HPg K1–4 (475 nM). Total protein synthesis was was determined as described previously (21). The accuracy and correspon- not affected (data not shown). The cells were trypsinized, pelleted, and dence of these two methods were confirmed further by reverse phase HPLC counted using a hemocytometer and trypan blue exclusion following 72-h amino acid analysis of the purified protein. MMPs were used at equimolar incubation at 37°C in 5% CO2. ϫ Ϫ7 Cell supernatants from peritoneal macrophages, harvested from mice concentrations (5 10 M) based on the TIMP inhibition assay. Ϫ/Ϫ Recombinant angiostatin (kringle regions 1–4, K1–4) was expressed in deficient in MME by targeted mutagenesis (MME ) (24) and their wild- type littermates (MMEϩ/ϩ), were also employed in an endothelial cell mammalian cell culture using the herpesvirus VP 16 trans-activator (22) ϩ/ϩ Ϫ/Ϫ and was then purified from conditioned medium by lysine-Sepharose chro- proliferation assay. Briefly, MME and MME mice received an i.p. matography. N-terminal sequence analysis of the first 15 amino acids con- injection of 1 ml of thioglycolate. Three days following the injection, peri- firmed the purity and presence of K1. toneal macrophages were harvested and plated into 24-well tissue culture ϫ 5 The hydroxymate MMP inhibitor (SC 44463) was a gift from D. Get- plates at 1 10 cells/well in serum-free EBM. After 5 days of culture at ␮ 37°C in 5% CO2, cells were treated with 50 g/ml of HPg or buffer control man (Monsanto-Searle). SC 44463 is a general MMP inhibitor (23). The Ki ϩ/ϩ for 72 h. Cell supernatants were harvested, and 100 ␮lofMME or Downloaded from for macrophage elastase is 0.67 nM and was determined using previously Ϫ/Ϫ described methods (23). MME supernatant was added to MECs plated onto 24-well tissue cul- ϫ 4 ␮ Ab to mouse macrophage elastase was generated in rabbits (24) and ture plates at 1.25 10 cells/well containing 200 l of EBM with 5% used at a 1/4000 dilution. Ab to human plasminogen was directed against FCS and bFGF (1 ng/ml). K1–3 (Enzyme Research Laboratories, South Bend, IN). Western blot analysis Glu-human plasminogen (HPg), pancreatic elastase, aprotinin, and p- aminophenylmercuric acetate were obtained from Sigma (St. Louis, MO). Cell supernatants harvested from buffer control and HPg-treated (50 ␮g/ ϩ/ϩ Ϫ/Ϫ

Glu-plasminogen was prepared from fresh-frozen plasma by affinity chro- ml) MME and MME macrophages were subjected to SDS-PAGE http://www.jimmunol.org/ matography on lysine-Sepharose, gel filtration, and ion exchange chroma- following addition of 25 mM DTT in protein running buffer at 100°C for tography. The purity is Ͼ98% Glu-plasminogen and Ͻ2% Lys-plasmino- 5 min. Proteins were transferred to a nylon membrane and blocked with 5% gen, as determined by acetic acid/urea PAGE. The purity of the HPg was casein in PBS overnight at 4°C. The membrane was then incubated with further verified in our laboratory by subjecting it to fast protein liquid rabbit anti-mouse IgG MME-specific Ab (1/4000 dilution) (9, 24) for 1 h chromatography and confirming a sole peak on elution that, by protein at 37°C, followed by goat anti-rabbit horseradish peroxidase (Amersham, electrophoresis, corresponded to HPg at 90 kDa. Arlington Heights, IL) and autoradiography. Control and HPg-treated MMEϩ/ϩ and MMEϪ/Ϫ macrophage supernatants were similarly subjected Cleavage of plasminogen by MMPs to Western analysis for angiostatin using a polyclonal rabbit anti-human plasminogen Ab (Enzyme Research Laboratories). Each active MMP was incubated with 10 ␮g of HPg (final concentration, ␮ 4 M) in buffer (300 mM Tris, 60 mM CaCl, and 90 mM NaCl, pH 7.5) Endothelial cell in vitro tube formation assay by guest on September 29, 2021 at 37°C for various times as indicated. The reaction was stopped by addi- tion of 25 mM DTT in protein running buffer (400 mM Tris (pH 7.4), 1.5% MECs were isolated and cultured as described above. Following trypsiniza- glycerol, 1 mg/ml bromphenol blue, and 2% SDS) at 100°C for 5 min, tion, the cells were plated onto two-chambered Tissue-Tek polystyrene followed by SDS-PAGE. In separate incubations, the serine proteinase in- slides (Nunc, Naperville, IL) coated with Matrigel (Collaborative Biomed- hibitor aprotinin (final concentration, 100 kallikrein inhibitor units/ml) or ical Products) diluted 1/1 with EBM containing buffer (300 mM Tris, 60 the MMP inhibitors SC 44463 (25 ␮M) and TIMP-1 (25 ␮M) were pre- mM CaCl, and 90 mM NaCl, pH 7.5), MME (288 nM), HPg (170 nM), incubated for1hat37°C with MME (5 ϫ 10Ϫ7 M) before the addition of MME/HPg product (170 nM), or K1–4 (475 nM). MECs plated on this 10 ␮g of HPg (final concentration, 4 ␮M) as described above. basement membrane matrix differentiate into “tubes” within 24 h of plating as described previously (25). The cells were plated in 500 ␮l of EBM with N-terminal amino acid sequence analysis of plasminogen 1% FCS containing the same additives, and the formation of tubes was cleavage products assessed at 24 h. To quantify tube formation, cells were fixed in 100% methanol at 4°C for 7 min, rinsed four times with PBS, and incubated Amino acid sequence analysis was performed on the major protein bands overnight at 20°C with rabbit polyclonal anti-human factor VIII-related Ag produced by purified MME cleavage of HPg. Ten micrograms of HPg was (von Willibrand factor; Dako, Glostrup, Denmark). The next day, the cells incubated with 250 ng of MME for 18 h at 37°C and subsequently resolved were counterstained with goat anti-rabbit IgG Cy3 (Jackson ImmunoRe- by 10–12% SDS-PAGE. Proteins were transferred to Problott membrane search Laboratories, West Grove, PA). Endothelial cells were then visual- (Applied Biosystems, Foster City, CA), visualized with 0.1% Coomassie ized using an Olympus microscope equipped with a fluorescence filter and blue, excised, and sequenced by automated Edman degradation using an Apochromat objectives linked to a Pentium 100-mHz computer containing Applied Biosystems 473 Sequenator. a frame-grabber board and Optimus Image Analysis software (Bothell, WA). Five separate random fields for each experimental condition were Isolation and culture of human microvascular endothelial cells visualized under low power, the color images were captured into the image (MECs) program Paxit (Midwest Information Systems, Chicago, IL), and analysis was performed using the Optimus program to quantify total endothelial cell MECs were isolated from neonatal foreskins by a method modified from area and total area encompassed by complete tubes with intact cell-cell that of Kubota et al. (25). Briefly, neonatal foreskins were incubated over- contacts, using the mean area of the five fields. Tube formation was quan- night with Dispase II (Collaborative Biomedical, Bedford, MA), the epi- tified as follows: total area encompassed by endothelial cell tubes/total dermis was removed, and then gentle pressure was exerted on the remain- endothelial cell surface area. Employing this analysis, efficient and com- ing dermis with the plunger of a tuberculin syringe, releasing vascular plete tube formation is represented by a larger number; inefficient, discon- fragments. The vascular fragments were then centrifuged, and the pellet tinuous tube formation yields a smaller number. was resuspended in endothelial cell basal medium (EBM; Clonetics, San Diego, CA) containing 10% FCS (Irvine Scientific, Irvine, CA), 10 ng/ml Results epidermal growth factor (Clonetics), 1 ␮g/ml hydrocortisone acetate (Sig- ma), 5 ϫ 10Ϫ5 M dibutyryl cAMP (Sigma), 2 ϫ 10Ϫ3 M glutamine (Ir- Human plasminogen is susceptible to cleavage by MMPs vine), and 100 U/ml penicillin and 100 ␮g/ml streptomycin (Sigma). Cells Pancreatic elastase (39 mM; Sigma) and all MMPs tested (final were cultured at 37°C in 5% CO2 on gelatin-coated tissue culture plastic. ϫ Ϫ7 Endothelial cell cultures were Ն99% pure as determined by factor VIII- concentration, 5 10 M), including mouse and human macro- related Ag staining (von Willibrand factor) and typical cobblestone cell phage elastase (MME and HME); MMP-3 (stromelysin); the ge- morphology. latinases MMP-2 and MMP-9; collagenases MMP-1, MMP-8, and The Journal of Immunology 6847

FIGURE 2. Time course of MME cleavage of human plasminogen. MME (final concentration, 5 ϫ 10Ϫ7 M) was incubated with 4 ␮M HPg (final concentration) for 0.5, 1, 2, 6, and 18 h at 37°C. The reaction mix- tures were stopped with SDS sample buffer containing DTT, and electro- phoresis was performed on a 10% SDS-polyacrylamide gel. Downloaded from

macrophage elastase (MME and HME) produced the most rapid and efficient cleavage of HPg to prominent 14-, 35-, and 38-kDa bands (arrows, Fig. 1A), molecular mass migrations consistent with angiostatin and other angiostatic HPg kringle regions as pre- viously described (3, 26). Catalytic efficacy was then followed by http://www.jimmunol.org/ MMP-9, MMP-3, and MMP-7. Therefore, macrophage elastase produced the most efficient cleavage of HPg to the 38-kDa protein product corresponding to angiostatin, followed by gelatinase and stromelysin, with the collagenases exhibiting weak, if any, an- giostatin-generating capacity. Separate experiments were then performed to examine the ef- fects of a inhibitor, aprotinin, as well as an MMP inhibitor, SC 44463, on this catalytic process (Fig. 1C). Aprotinin

did not inhibit MME cleavage of HPg (Fig. 1C), in contrast to by guest on September 29, 2021 recent reports of cleavage of HPg to angiostatin by a serine pro- tease in prostate tumor cells (5, 27). In contrast, the MMP inhib- itors SC 44463 (a hydroxymate) and TIMP-1 protein totally blocked MME cleavage of HPg (Fig. 1C). These data indicate that HPg processing to angiostatin is metalloproteinase dependent and that the generation of plasmin does not play a role in MMP-me- diated formation of angiostatin and other plasminogen fragments in this system. To more closely examine the MME cleavage of HPg, the catal- FIGURE 1. Cleavage of plasminogen by MMP. A, Pancreatic elastase (PE; 39 mM) and the MMPs (final concentration, 5 ϫ 10Ϫ7 M) mouse and ysis was examined over time (0.5–18 h). As shown in Fig. 2, MMP human macrophage elastase (MME and HME, respectively) were incu- cleavage of HPg began within 30 min and was complete by 18 h. bated with 4 ␮M HPg (final concentration) for 1 or 18 h at 37°C. The In fact, Ͼ50% generation of angiostatin by 1–2 h by MME at reaction mixtures were stopped with SDS sample buffer containing DTT submicromolar enzyme concentrations suggests potential physio- and subjected to electrophoresis on a 10% SDS-polyacrylamide gel. B, The logic relevance and perhaps greater catalytic potential than that of Ϫ MMPs (final concentration, 5 ϫ 10 7 M) stromelysin (MMP-3), 92-kDa serine such as pancreatic elastase. gelatinase (MMP-9), collagenase-2 (MMP-8), matrilysin (MMP-7), colla- genase-1 (MMP-1), and collagenase-3 (MMP-13) were incubated with 4 ␮ M HPg (final concentration) for the indicated times. C, MME was pre- MMP cleavage of plasminogen generates angiostatin (K1–4) incubated with aprotinin (Apr; 100 kallikrein inhibitor units/ml), a hy- and K4 droxymate MMP inhibitor (SC 44463; 25 ␮M), or TIMP (25 ␮M) for 1 h before the addition of HPg. For all panels, the arrows denote 38-, 35-, and To determine the sites of cleavage of HPg by MMPs, we incubated 14-kDa cleavage products. MME (1 ϫ 10Ϫ6 M) with 5 ␮g of HPg (2 ␮M) at 37°C for 18 h. The products were subjected to gel electrophoresis and transferred onto a Pro Blot nylon membrane, and the 14-, 35-, and 38-kDa protein bands produced by MME cleavage of HPg were excised MMP-13; and matrilysin (MMP-7), were incubated with 4 ␮M and subjected to N-terminal sequence analysis. The amino-termi- HPg (final concentration) for 1 or 18 h. As reported previously (3), nal analysis of both the 35- and the 38-kDa bands yielded an iden- pancreatic elastase cleaved HPg into smaller protein species (Fig. tical sequence of KVYLSECKTGN (Fig. 3B), representing the N- 1A). As shown in Fig. 1, A and B, each of the MMPs tested also terminus of K1. The molecular mass of the 38-kDa band cleaved HPg to several protein products with varying efficiencies corresponds to K1–4 (26). Since HPg is not glycosylated and both and in a time-dependent manner. Specifically, mouse and human the 35- and 38-kDa species have the same N-terminal sequence, 6848 MATRIX METALLOPROTEINASES GENERATE ANGIOSTATIN

FIGURE 3. MME cleavage of human plasminogen yields angiostatin and other kringle products. A, Amino acid sequence of human plasminogen kringle Downloaded from regions as described (see text), with the N terminus of K1 at amino acid 97 and the N terminus of K4 at amino acid 373. B, HPg (2 ␮M) (final concentration) was incubated with MME (1 ϫ 10Ϫ6 M) at 37°C for 18 h, the products were subjected to electrophoresis on a 10% reducing gel and electroblotted onto a Pro Blot nylon membrane. The 14-, 35-, and 38-kDa protein bands produced by MME cleavage of HPg were excised and subjected to N-terminal sequence analysis by automated Edman degradation. The amino-terminal sequence analysis of both the 35- and 38-kDa bands produced an identical amino acid sequence of KVYLSECKTGN, corresponding to the N terminus of K1. N-terminal sequence analysis of the 14-kDa band produced the sequence VVQD CYHGDGT, corresponding to the N terminus of K4. http://www.jimmunol.org/

C-terminal processing is likely for the 35-kDa product. Interest- and HPg-treated MMEϪ/Ϫ macrophages did not produce MME. ingly, in previous reports (26), K1–3 migrates at a molecular mass HPg-treated MMEϩ/ϩ and MMEϪ/Ϫ macrophage supernatants of 35 kDa. N-terminal amino acid sequence analysis of the 14-kDa were then subjected to Western analysis for angiostatin using a band produced the sequence VVQDCYHGDGT corresponding to polyclonal human plasminogen Ab directed against K1–3 (En- K4 of HPg (26). Thus, HPg cleavage by MME probably generates zyme Research Laboratories). Cleavage of plasminogen, in a time- K1–4 (38-kDa), K1–3 (35-kDa), and K4 (14-kDa) fragments. dependent manner, to the prominent 38-kDa protein band of an- by guest on September 29, 2021 giostatin was demonstrated by the MME-competent macrophages Basic FGF-stimulated endothelial cell proliferation is inhibited (Fig. 5B); this cleavage was inhibited by the hydroxymate MMP by MME-generated plasminogen products and recombinant inhibitor SC 44463 (data not shown). MME-deficient macrophages K1–4 were incapable of degradation of plasminogen to angiostatin (Fig. To determine whether MME-generated angiostatin and kringle re- 5B). In light of recent reports identifying urokinase activity in con- gions altered endothelial cell proliferation, primary MECs were junction with free sulfhydryl donors as a mechanism of angiostatin plated onto gelatin-coated 24-well tissue culture plates (Costar, generation in prostate carcinoma cells (6), we determined the pres- ϩ/ϩ Cambridge, MA) at 1.25 ϫ 104 cells/well containing 5% FCS and ence of macrophage-associated urokinase in our MME and Ϫ Ϫ 1 ng/ml bFGF. The cells were incubated with buffer (300 mM Tris, MME / macrophages by Western analysis. We found no differ- 60 mM CaCl, and 90 mM NaCl, pH 7.5), HPg (170 nM), MME ence in urokinase production between MME-competent and (240 nM), the MME cleavage products of HPg (170 nM), or K1–4 MME-deficient macrophages (data not shown). ϩ ϩ Ϫ Ϫ (475 nM). After 72 h, MECs were trypsinized, and cell counts MME / and MME / macrophage-conditioned media were were performed using trypan blue exclusion. As shown in Fig. 4A then used to determine their capacity to inhibit bFGF-stimulated (n ϭ 12), MME-generated HPg protein products inhibited bFGF- endothelial cell proliferation. The proliferation of MECs exposed induced endothelial cell proliferation by 58% ( p Ͻ 0.001); K1–4 to cell supernatant from HPg-treated MMEϩ/ϩ macrophages was inhibited proliferation by 44% ( p Ͻ 0.01). Of note, MME-gener- inhibited by 55% compared with that in control medium ( p Ͻ ated kringle regions did not inhibit human dermal fibroblast pro- 0.05; Fig. 6). MMEϪ/Ϫ macrophage-conditioned medium had no liferation (Fig. 4B), demonstrating the specificity of this effect for effect on endothelial cell proliferation even in the presence of HPg. endothelial cells.

MME is required for the generation of angiostatin and the MME-generated and recombinant angiostatin inhibit endothelial inhibition of bFGF-stimulated human endothelial cell cell tube formation in vitro proliferation To assess the effect of MME-generated kringle regions and recom- Mouse peritoneal macrophages harvested from MMEϩ/ϩ and binant angiostatin on endothelial cell differentiation, in vitro an- MMEϪ/Ϫ mice were incubated in the absence or the presence of giogenesis experiments were performed. MECs were plated on HPg (Sigma) for 48 h. Western analysis for MME showed a growth factor-reduced Matrigel in EBM with 1% FCS containing marked induction and activation of MME by the MMEϩ/ϩ mac- PBS (Fig. 7A), MMP buffer (300 mM Tris, 60 mM CaCl, and 90 rophages exposed to HPg as previously described (28) (Fig. 5A). mM NaCl, pH 7.5; Fig. 7C), recombinant K1–4 (475 nM; Fig. 7B), As expected, control macrophages did not produce active MME, HPg (170 nM; Fig. 7D), MME (288 nM; Fig. 7E), or the MME The Journal of Immunology 6849 Downloaded from http://www.jimmunol.org/

FIGURE 5. MME is required for the generation of angiostatin from plasminogen. A, Western analysis for MME in MMEϩ/ϩ and MMEϪ/Ϫ macrophage-conditioned medium. Peritoneal macrophages from MMEϪ/Ϫ mice and their wild-type littermates MMEϩ/ϩ were plated into 24-well FIGURE 4. Effects of kringle fragments on endothelial cell prolifera- tissue culture plates at 1 ϫ 105 cells/well and treated with either buffer tion. A, MME-generated kringle regions inhibit bFGF-induced prolifera- control or 50 ␮g/ml of HPg for 48 h. Cell supernatants were subjected to tion of human dermal microvascular endothelial cells. MECs were plated SDS-PAGE and Western analysis with polyclonal rabbit anti-mouse ϩ ϩ onto 24-well tissue culture plates at 1.25 ϫ 104 cells/well containing 5% MME-specific Ab. In MME / mice, exposure of HPg results in conver- FCS and 1 ng/ml bFGF. Cultures were incubated with MMP buffer control sion of pro-MME (54 kDa) to the active fully processed form (22 kDa) and by guest on September 29, 2021 Ϫ/Ϫ Ϫ/Ϫ (300 mM Tris, 60 mM CaCl2, and 90 mM NaCl, pH 7.5), HPg (170 nM), increased synthesis of MME. MME control and HPg-treated MME MME (240 nM), the MME cleavage products of HPg (170 nM), or recom- macrophages did not produce MME. B, Western analysis for angiostatin binant K1–4 (475 nM) for 72 h. Cell counts were performed, and the data generation by plasminogen-treated macrophages. Conditioned media from ϩ ϩ are shown as the increase in cell number (i.e., cell count (ϫ104) Ϫ cells MME / macrophages (lanes 1, 3, 5, and 7) demonstrate cleavage of plas- plated). Bars represent the SEM (n ϭ 12). B, MME-generated kringle minogen to a prominent 38-kDa protein band (arrow) in a time-dependent Ϫ Ϫ regions do not inhibit bFGF-induced proliferation of human dermal fibro- manner that is not present in MME / -conditioned media (lanes 2, 4, 6, blasts (HDF). HDFs were plated onto 12-well tissue culture plates at 1.5 ϫ and 8) or in HPg-exposed MECs (lane 9). The 90-kDa band is intact 105 cells/well containing 5% FCS and 1 ng/ml bFGF. Cultures were incu- plasminogen. bated with either MMP buffer control (300 mM Tris, 60 mM CaCl2, and 90 mM NaCl, pH 7.5) or the MME cleavage products of HPg (170 nM) for 72 h. Cell counts were performed, and the data are shown as cell number (cell count ϫ 105). Bars represent the SEM (n ϭ 3). Discussion The capacity of both primary and metastatic tumors to grow be- cleavage products of HPg (170 nM; Fig. 7F). The cells were ex- yond the limits of oxygen diffusion requires the establishment of a amined after 24 h of incubation on Matrigel, which induces in vitro neovasculature (29, 30). The isolation of angiostatin by O’Reilly et differentiation of the endothelial cells and subsequent formation of al. (3) demonstrated that tumors may limit metastatic growth by tube-like structures. As shown in Fig. 7, many of the tubes that generation of antiangiogenic agents. Dong et al. (7) further dem- were formed by the cells exposed to K1–4 (Fig. 7B) and MME- onstrated that production of antiangiogenic activity by tumors was generated kringle regions (Fig. 7F) were discontinuous compared associated with the production of host macrophage elastolytic en- with those formed in buffer (Fig. 7, A and C), HPg (Fig. 7D), or zyme(s). Additionally, Patterson et al. (11) demonstrated the in MME (Fig. 7E) alone. To quantify the degree of tube formation, vitro proteolytic activity of MMP-7 and MMP-9 to generate an- the cells were immunostained for factor VIII-related Ag (von Wil- giostatin from plasminogen. We have extended these findings to librand factor), and the total area encompassed by endothelial cell directly demonstrate that both mouse and human macrophage elas- tubes/total endothelial cell surface area was calculated. Exposure tase and to varying extents other human MMPs proteolytically to angiostatin (K1–4) resulted in a Ͼ50% decrease ( p Ͻ 0.01) in generate angiostatin that inhibits the proliferation of human mi- tube formation, reflecting the reduction in total tube area and hence crovascular endothelial cells. In murine macrophage-conditioned endothelial cell differentiation. Since Fig. 7F demonstrates similar medium, where MME accounts for most extracellular matrix-de- tube formation inhibition, a comparable reduction is implied fol- grading activities (24), MME is required for the production of lowing exposure to MME/HPg-generated kringle regions. angiostatin and the subsequent inhibition of endothelial cell 6850 MATRIX METALLOPROTEINASES GENERATE ANGIOSTATIN

proliferation. In fact, the HPg-MMP interaction is even more com- plex. Plasmin is capable of MMP activation (31). The mechanism of action of the antiproliferative and angiostatic effects of angiostatin remains unclear. As proposed by Cao et al. (26), endothelial cell proliferation may be blocked through the binding of kringle regions to a receptor that is up-regulated in or exclusive to proliferating endothelial cells. As also suggested by this group, angiostatin may be binding to an that is not only increased in response to an angiogenic endothelial cell mito- gen such as bFGF (32) but also important in the establishment of ␣ ␤ angiogensis, as is v 3 (33). Our own investigations (unpublished observations) as well as those previously reported (25) have es- FIGURE 6. Mouse macrophage-derived MME is required for the inhi- tablished that endothelial cell proliferation does not contribute to bition of bFGF-induced proliferation of human dermal MECs. One hun- ϩ ϩ Ϫ Ϫ the formation of cords or tubes when these cells are seeded on dred microliters of cell supernatants from MME / and MME / perito- neal macrophages exposed to 50 ␮g/ml of HPg or buffer were added to Matrigel. Therefore, our additional findings of the inhibition of MECs plated onto 24-well tissue culture plates at 1.25 ϫ 104 cells/well tube formation on Matrigel by angiostatin suggest that the in vivo containing 200 ␮l of EBM with 5% FCS and bFGF (1 ng/ml; n ϭ 6). effect of angiostatin may not solely be an inhibition of endothelial

MECs not exposed to macrophage-conditioned media served as controls cell proliferation but may also be an effect on a later stage of Downloaded from for proliferation. Cell counts were performed after 72 h using trypan blue angiogenesis that is represented by the in vitro tube-forming assay. exclusion. The proliferation of MECs exposed to cell supernatant from The identities of cell surface molecules that may mediate angiosta- HPg-treated MMEϩ/ϩ macrophages was inhibited by 55% compared with Ϫ Ϫ tin-endothelial cell binding remain under investigation. that in control media (n ϭ 3; p Ͻ 0.05). MME / macrophage-conditioned media had no effect on endothelial cell proliferation even in the presence The concept that MMPs generate angiostatin, potentially limit- of HPg. Bars represent the SEM. ing tumor neovascularization, is particularly intriguing given the http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. MME-generated kringle regions and recombinant angiostatin inhibit endothelial cell tube formation in vitro. MECs were plated on Matrigel in EBM containing 1% FCS and PBS (A; control), recombinant K1–4 (B; 475 nM), MMP buffer (C; 300 mM Tris, 60 mM CaCl, and 90 mM NaCl, pH 7.5), HPg (D; 170 nM), MME (E; 288 nM), or MME cleavage products of HPg (F; 170 nM). Endothelial cells formed complete tubes in control buffers (A and C), but cell-cell contacts were disrupted in recombinant angiostatin-treated cells (B) and also in cells treated with MME-generated kringle regions in a dose-responsive manner (F). Magnification: A and B, ϫ200; C–F, ϫ600. The Journal of Immunology 6851 fact that tumor-derived MMPs have been recognized as promoters References of tumor growth both by degrading matrix barriers and by enhanc- 1. Holmgren, L., M. O’Reilly, and J. Folkman. 1995. Dormancy of micrometastasis: ing angiogenesis (34, 35). Host-derived MMPs, such as macro- balanced proliferation and in the presence of angiogenesis suppression. phage-specific MME, may also contribute to the generation of Nat. Med. 1:149. 2. Gorelik, E. S., S. Segal, and M. Feldman. 1980. Growth of a local tumor exerts other antiangiogenic molecules from certain parent serum or ma- a specific inhibitory effect on progression of lung metastasis. Int. J. Cancer 21: trix molecules, such as or type XVIII collagen, 617. i.e., the antiangiogenic fragment of thrombospondin (36) or en- 3. O’Reilly, M. S., L. Holmgren, Y. Shing, C. Chen, R. A. Rosenthal, M. Moses, W. S. Lane, Y. Cao, E. H. Sage, and J. Folkman. 1994. A novel angiogenesis dostatin (37), respectively. Just as MMPs may be pro- or antian- inhibitor which mediates the suppression of metastasis by a Lewis lung carci- giogenic, macrophages are intimately involved in both initiating noma. Cell 79:315. 4. Sugarbaker, E. V., J. Thornewaite, and A. S. Ketchem. 1977. In Progress in and halting angiogenesis (7, 38, 39). It is well recognized that Cancer Research and Therapy. S. B. Day and W. P. L. Rogers, eds. Raven Press, macrophages release an array of angiogenic and angiostatic cyto- New York, p. 227. kine and growth factor secretory products (40). Mediators such as 5. Gately, S., P. Twardowski, S. Stack, M. Patrick, L. Boggio, D. L. Cundiff, H. W. Schnaper, L. Madison, O. Volpert, N. Bouck, et al. 1996. Human prostate bFGF, granulocyte-macrophage CSF, and IL-8 function as stimu- carcinoma cells express enzymatic activity that converts human plasminogen to lators of endothelial cell migration and mitosis. In contrast, inhib- the , angiostatin. Cancer Res. 56:4887. 6. Gately, S. G., P. Twardowski, M. S. Stack, D. L. Cundiff, D. Grella, itors of angiogenesis produced by macrophages include throm- F. J. Castellino, J. Enghild, H. C. Kwan, F. Lee, R. A. Kramer, et al. 1997. The bospondin and IFNs. As for most biologic systems, the mechanism of cancer-mediated conversion of plasminogen to the angiogenesis contribution of macrophages to angiogenesis must then depend inhibitor angiostatin. Proc. Natl. Acad. Sci. USA 94:10868. 7. Dong, Z., R. Kumar, X. Yang, and I. J. Fidler. 1997. Macrophage-derived met- upon the fine regulation and balance of proangiogenic and antian- alloelastase is responsible for the generation of angiostatin in Lewis lung carci- Downloaded from giogenic factors. This applies to physiologic angiogenesis such as noma. Cell 88:801. 8. Banda, M. J., and Z. Werb. 1981. Mouse macrophage elastase: purification and wound healing as well as to the pathologic angiogenesis observed characterization as a metalloproteinase. Biochem. J. 193:589. in tumor growth. 9. Shapiro, S. D., G. L. Griffin, D. J. Gilbert, N. A. Jenkins, N. G. Copeland, In light of our findings as well as those recently reported (6, 11, H. G. Welgus, R. M. Senior, and T. J. Ley. 1992. Molecular cloning, chromo- somal localization, and bacterial expression of a murine macrophage metalloela- 27), the biologic properties of MMPs, plasminogen, and angiosta- stase. J. Biol. Chem. 267:4664.

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