Article |
Address correspondence to Peter C. Brooks, Departments of Radiation Oncology and Cell Biology, Kaplan Cancer Center, Rusk Building Room 806, New York University School of Medicine, 400 East 34th St., New York, NY 10016. Tel.: (212) 263-3021. Fax: (212) 263-3018. E-mail: peter.brooks{at}med.nyu.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: angiogenesis; ECM; cryptic sites; tumor; migration
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A major component of the vascular basement membrane is collagen type IV (Hudson et al., 1993). The most widely expressed form is composed of two 1(IV) chains and one
2(IV) chain and is found in the basement membrane of virtually all blood vessels. Interestingly, the triple helical nature of collagen is thought to regulate integrin-mediated cellular interactions (Messent et al., 1998; Emsley et al., 2000). In fact, proteolytic cleavage and denaturation can convert triple helical collagen type I from a ß1 integrindirected ECM ligand to an
vß3-dependent ligand (Davis, 1992; Montgomery et al., 1994). This shift in integrin-mediated interactions may represent an important regulatory mechanism to activate distinct signal transduction pathways necessary for invasive cellular behavior. However, no direct evidence is available to indicate that interaction of denatured collagen with endothelial cells is a functionally important step in angiogenesis.
Here, we provide evidence that proteolytic cleavage of collagen type IV can expose a cryptic site which is normally hidden within its triple helical structure. This cryptic site was shown to be specifically exposed within the subendothelial basement membrane of angiogenic blood vessels, whereas little if any was detected in association with quiescent vessels. Importantly, our studies provide evidence that proteolytic exposure of this cryptic site within collagen IV plays a functional role in angiogenesis. In fact, a Mab HUIV26 directed to this cryptic site potently inhibited angiogenesis and tumor growth in multiple animal models. Together, these studies suggest a novel mechanism by which proteolytic remodeling of the ECM exposes cryptic protein sequences that promote novel integrinligand interactions required for angiogenesis in vivo.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Exposure of the HUIV26 cryptic site within the subendothelial basement membranes of angiogenic blood vessels
We assessed whether the HUIV26 cryptic epitope could be exposed within the basal lamina of blood vessels in vivo. Unfixed biopsy sections from normal human skin were incubated with either activated or proMMP-2, HT1080 tumorconditioned medium, or control buffer. The tissues were costained with Mab HUIV26 (green) and a polyclonal antibody directed to factor VIIIrelated antigen (red), a known marker of blood vessels. As shown in Fig. 2 A, blood vessels (red) from normal human skin were readily detected. Little if any of the cryptic HUIV26 epitope (green) was detected within the vascular basement membranes or the surrounding interstitial matrix from tissues treated with either inactive proMMP-2 or control buffer (Fig. 2 A, top). In contrast, tissues treated with either proteolytically active MMP-2 (Fig. 2 A, bottom left) or HT1080 tumorconditioned medium (Fig. 2 A, bottom right) demonstrated exposure of the HUIV26 cryptic epitope, as indicated by colocalization (yellow) due to overlap of the exposed HUIV26 epitope (green) and factor VIIIrelated antigen (red). Together, these findings provide further evidence that the HUIV26 cryptic sites could be exposed by proteolytic activity in a physiological tissue.
|
|
|
|
|
Mab HUIV26 inhibits human endothelial cell adhesion and migration on denatured, but not triple helical, collagen IV
It is possible that exposure of the HUIV26 cryptic epitope may contribute to angiogenesis in part by regulating endothelial cellintegrin interactions. To examine this possibility, we evaluated the effects of Mab HUIV26 on human endothelial cell adhesion to either triple helical or denatured human collagen IV. HUVECs were allowed to attach to immobilized triple helical or denatured collagen IV in the presence or absence of Mab HUIV26 or isotype-matched control antibody (50 µg/ml). As shown if Fig. 6 A, HUVECs readily attached to both triple helical and denatured collagen IV. In contrast, HUVEC adhesion to denatured collagen IV was inhibited by 60% in the presence of Mab HUIV26, while having little if any effect on adhesion to triple helical collagen IV. An isotype-matched control antibody had no effect of cell adhesion to either triple helical or denatured collagen IV.
|
Mab HUIV26 inhibits purified integrin vß3 binding to denatured collagen IV
We sought to determine whether an integrin receptor was involved in mediating cellular interactions with the HUIV26 cryptic epitope. To facilitate these studies, microtiter wells were coated with either triple helical or denatured collagen IV. The wells were incubated with purified integrin receptors, including 1ß1,
2ß1,
vß3, and
5ß1. After incubation, bound integrins were detected with antiintegrin-specific antibodies. As shown in Fig. 7 A, the collagen-binding integrins
1ß1 and
2ß1 bound to triple helical collagen in a dose-responsive manner, whereas integrins
vß3 and
5ß1 showed little if any interaction. After denaturation,
1ß1 binding was lost; however, denatured collagen IV acquired the capacity to bind to integrin
vß3 (Fig. 7 B). Moreover, denatured collagen IV retained its ability to bind to
2ß1, whereas the control fibronectin receptor integrin
5ß1 failed to interact (Fig. 7 B). These findings suggest that denaturation of the triple helical structure of collagen IV can shift integrin binding specificity from that of a ß1 dependency to both ß1 and
vß3. To determine whether integrin
2ß1 or integrin
vß3 interacts with the HUIV26 cryptic epitope, similar receptor binding assays were performed in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. As shown in Fig. 7 C, Mab HUIV26 failed to block the ability of purified integrin
2ß1 to bind to denatured collagen IV, while inhibiting integrin
vß3 binding by >70%. Together, these findings suggest that integrin
vß3 may function as a receptor for the HUIV26 cryptic epitope.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies have indicated that proteolytic enzymes, such as members of the MMP family, play an important role in angiogenesis (Hiraoka et al., 1998; Stetler-Stevenson, 1999; Werb et al., 1999). In fact, mice deficient in MMP-2 or MMP-9 exhibit reduced angiogenesis in vivo (Itoh et al., 1998; Vu et al., 1998). Moreover, our recent studies suggest that MMP-9deficient mice exhibit reduced exposure of the HUIV26 sites within the retina during hypoxia-induced retinal neovascularization in vivo (unpublished data). Together, these findings suggest that proteolysis of collagen, as well as perhaps other ECM proteins, is of critical importance in angiogenesis. Although soluble fragments of collagen have been detected in the circulation, little if any direct evidence is available that proteolyzed matrixassociated forms of collagen IV exist within the subendothelial basement membrane or that they play a functional role in angiogenesis (Jukkola et al., 1997). Here, we describe the use of a unique Mab that specifically binds to proteolyzed and denatured collagen IV, but does not react with triple helical collagen IV. This cryptic HUIV26 epitope was shown to be specifically exposed within the subendothelial basement membrane of angiogenic and tumor-associated blood vessels, but not within the basement membrane of normal vessels. The high degree of specificity for angiogenic and tumor vessels is likely due to the slow turnover of members of the collagen family in healthy tissues as compared with the rapid remodeling that likely occurs during angiogenesis.
Recent evidence suggests that cellular interactions with proteolyzed forms of ECM molecules such as osteopontin, and laminin may result in altered cellular behavior including changes in cell motility (Senger and Perruzzi, 1996; Giannelli et al., 1997; Davis et al., 2000). Thus, proteolytic cleavage of ECM proteins, together with cell surface receptor binding events, may represent a previously unappreciated mechanism to transmit cryptic regulatory signals that are required for angiogenesis. Consistent with this hypothesis, we provide evidence that a Mab directed to the matrix-immobilized HUIV26 cryptic site potently inhibits angiogenesis in multiple animal models. Importantly, angiogenesis was inhibited irregardless of the cytokine used or the animal species in which these assays were conducted. Moreover, systemic administration of Mab HUIV26 also potently inhibited the growth of several tumor types of distinct histological origin. Interestingly, the exposure of the HUIV26 epitope was associated with a loss of 1ß1 binding and a gain in
vß3 binding, whereas
2ß1-mediated interactions were unaffected. This shift in integrin binding may initiate a unique signaling cascade required for angiogenesis in vivo.
Importantly, recent studies have indicated that MMP-mediated cleavage of laminin 5 can expose a cryptic epitope which potentiates tumor cell motility in vitro (Giannelli et al., 1997). Moreover, proteolytic cleavage of fibronectin and osteopontin also enhance cellular migration in vitro (Bowersox and Sorgenete, 1982; Senger and Perruzzi, 1996). However, little is known concerning the roles that these proteolyzed ECM proteins may have on pathological processes in vivo. Here, we provide evidence for the first time that proteolytic exposure of the HUIV26 cryptic site is required for angiogenesis and tumor growth in vivo. Moreover, our results suggest that proteolytic remodeling is not solely a mechanism to destroy physical barriers that obstruct vascular cell migration, but can expose cryptic sites that are essential for the angiogenic process. In fact, our systematic search for cryptic sites in other ECM proteins have resulted in the generation of several distinct Mab directed to different cryptic epitopes which potently inhibit angiogenesis and tumor growth (unpublished data). Thus, an in depth knowledge of the roles these cryptic sites play in angiogenesis is critical to our understanding of blood vessel formation. Together, our findings indicate that targeting matrix-immobilized cryptic sites within ECM molecules may be a highly specific and powerful new approach for the treatment of neoplastic diseases.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells and cell culture
Human melanoma cell line M21 was a gift from Dr. David Cheresh (Scripps Research Institute, La Jolla, CA). CS1 hamster melanoma cells were provided by Dr. C. Damsky (University of California, San Francisco, CA). Human Fibrosarcoma cell line HT1080 was obtained from the American Type Culture Collection. Cell lines were maintained in RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, and Pen-Strep. HUVECs were obtained from Clonectics Corp. and were maintained in M199 medium containing 20% FBS, 100 µg/ml gentamicin, 4 mM L-glutamine, 0.9 mg/ml heparin, and 30 µg/ml ECGS (Upstate Biotechnology).
Solid phase ELISA
Nontissue culturetreated 96-well ELISA plates were coated (50 µl/well) with ECM proteins (25 µg/ml in PBS) for 18 h at 4°C. Plates were blocked with 100 µl/well of 1.0% BSA in PBS for 1 h at 37°C. Purified Mab HUIV26 (1.0 µg/ml) was diluted in 1.0% BSA in PBS (100 µl/well). Plates were incubated for 1 h at 37°C and washed three times with PBS. Goat antimouse peroxidaseconjugated IgG was added and allowed to incubate for 1 h at 37°C. The plates were washed three times with PBS and ELISA substrate (OPD) was added and the OD was measured with an ELISA plate reader at a wavelength of 490 nm. All measurements were corrected for nonspecific binding to BSA and reactivity of secondary antibody.
For proteolyzed collagen IV ELISAs, microtiter plates were coated as described above with collagen IV. Concentrated (20x) HUVEC serum-freeconditioned medium (100 µl/well) with or without EDTA (50 mM) or aprotinin (10 µg/ml) was incubated for 1, 6, and 24 h at 37°C. At each time point, the wells were washed five times with PBS/EDTA and blocked with 1.0% BSA. No significant loss of total bound collagen IV was noted between experimental conditions, as detected by control incubations with polyclonal antibodies directed to collagen IV. Detection of Mab HUIV26 immunoreactivity was performed as described above.
Immunofluorescence analysis of tissue sections
Human and chick tissues were embedded in OCT and snap frozen in liquid nitrogen (Brooks et al., 1996). In brief, 4-um sections of normal human skin, retina, and chick CAM, or human malignant melanoma or retina from patients with diabetic retinopathy were fixed by incubation for 30 s in 50% methanol/50% acetone. Tissue were blocked by incubation with 2.5% BSA in PBS followed by incubation with primary antibodies HUIV26 (100 µg/ml), anti-MMP-2 (50 µg/ml), or polyclonal antifactor VIII (1:100 dilution) in 1.0% BSA in PBS for 2 h at 37°C. In control experiments, tissues were incubated with secondary antibodies only. Tissue were washed five times in PBS for 5 min each followed by incubation with FITC- and rhodamine-conjugated secondary antibodies (1:400 dilution in 1.0% BSA in PBS) for 1 h at 37°C. In experiments in which the tissues were proteolyzed before staining, unfixed frozen sections were incubated with either control buffer alone (50 mM Tris, 200 mM NaCl, 10 mM CaCl2, pH 7.5), activated or Pro MMP-2 (1.0 µg/ml), or concentrated (20x) serumfree HT1080conditioned medium for 2 h at 37°C. The tissues were then washed extensively five times with PBS. Costaining with primary antibodies was carried out as described above. Photomicrographs were taken at either low (200x) or high power (630x).
Quantitation of HUIV26-positive tumor blood vessels
To assess the relative percentage of HUIV26-positive tumor blood vessels, costain analysis was performed on frozen sections of human melanoma tumor biopsies. In brief, 4.0-um tissue sections were cut from frozen blocks of human malignant melanoma tumors. The tissues were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIIIrelated antigen. 10 sections were analyzed for each of 5 distinct tumors. For each tumor, the percentage of HUIV26-positive vessels were estimated by determining the number of tumor vessels that costained for both HUIV26- and factor VIIIrelated antigen, as compared with the vessels that only stained positive for factor VIIIrelated antigen. These observations were conducted using low power magnification (200x).
Gelatin zymography and dot blot analysis
Angiogenesis was induced within the CAMs of 10-d-old chick embryos by placing a filter disc saturated with bFGF (25 µl) at 1.0 µg/ml (Brooks et al., 1998). Tissue directly beneath the filter discs were harvested at 2, 24, 48, and 72 h after addition of the bFGF. CAM tissues were homogenized in lysis buffer containing 1.0% TX-100, 50 mM Tris, 300 mM NaCl, pH 7.5. 20 µg of total CAM lysate were electrophoresed through a 10% SDS-PAGE gel polymerized with 0.2% gelatin (Brooks et al., 1996). Gels were washed three times for 1 h each with 2.5% TX-100 and incubated for 16 h at 37°C in collagenase buffer containing 50 mM TRIS, 200 mM NaCl, and 10 mM CaCl2, pH 7.5. Gelatinolytic activity was visualized by staining with 0.5% Coomassie blue. Gelatinolytic bands were confirmed to be pro and active MMP-2 by Western blot analysis with anti-MMP-2specific Mab.
In dot blot analysis, 10 µg of total protein was spotted (10 µl/spot) on nitrocellulose paper. Blots were incubated in 10% milk diluted in TBS-T to block nonspecific binding and incubated with either anticollagen IV polyclonal antibody or Mab HUIV26 (1.0 µg/ml). Blots were next washed and incubated with peroxidase-labeled secondary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence according to the manufacture's instructions.
Rat corneal micropocket angiogenesis assay
The rat corneal micropocket angiogenesis assay was performed essentially as described (Koch et al., 1995; Dipietro et al., 1998). In brief, hydron pellets (Polyhydroxyethyl methacrylate; Interferon Sciences) were prepared containing 1.2 µl of saline, 1.2 µl of bFGF, either Mab HUIV26 or control Ab (50 µg), and 12 µl of 12% hydron in ethanol. Mixtures were applied to a 1.5-mm diameter Teflon rod (Dupont). The hydron pellets were dried in a laminar flow hood. Pockets were cut in the corneal stroma of F344 female rats, 1.5-mm from the limbus and the Hydron pellets implanted (Koch et al., 1995; Dipietro et al., 1998). Corneas were routinely examined by slit-lamp biomicroscopy for up to 5 d. Corneas were photographed on day 5 after implantation. Rats were anesthetized and perfused with saline followed by colloidal carbon to enhance visualization of blood vessels. The corneas were dissected, fixed in 4% paraformaldehyde, and mounted on glass slides in 50% glycerol/50% gelatin solution. Corneal neovascularization was quantified by measuring the area of neovascularization from the limbus to the pellet. The area of neovascularization was acquired with Image Pro Plus 3.0 software (Media Cybernetics). Experiments were conducted two to three times with five to seven eyes per condition.
Chick embryo tumor growth assays
Single cell suspensions of CS1 melanoma (5 x 106 per embryo) or HT1080 fibrosarcoma (4 x 105 per embryo) were applied in a total volume of 40 µl of RPMI to the CAMs of 10-d-old embryos (Brooks et al., 1998). 24 h later, the embryos received a single intravenous injection of purified Mabs HUIV26 or control Mabs (100 µg per embryo). Tumors were grown for 7 d, then resected and wet weights were determined. Experiments were performed three to four times with five to ten embryos per condition.
SCID mouse tumor growth assay
Subconfluent human M21 melanoma cells were harvested, washed, and resuspended in sterile PBS (20 x 106 per ml). SCID mice were injected subcutaneously with 100 µl of M21 human melanoma cell (2 x 106) suspension. 3 d after tumor cell injection, mice were either untreated or treated i.p. (100 µg/ mouse) with either Mab HUIV26 or an isotype-matched control antibody. The mice were treated daily for 24 d. Tumor size was measured with calipers and the volume was estimated using the formula V = L2 x W/2, where V is equal to the volume, L is equal to the length, and W is equal to the width. Experiments were completed three times with similar results
Cell adhesion assays
Human collagen type IV (triple helical or denatured) was immobilized (25 µg/ml) on 48-well nontissue culturetreated plates. Wells were washed and incubated with 1% BSA in PBS for 1 h at 37°C. Subconfluent HUVECs were harvested, washed, and resuspended in adhesion buffer containing RPMI 1640, 1 mM MgCl2, 0.2 mM MnCl2, and 0.5% BSA. HUVECs (105) were resuspended in 200 µl of the adhesion buffer in the presence or absence of Mab HUIV26 or control antibodies (50 µg/ml) and were added to each well and allowed to attach for 30 min at 37°C. The nonattached cells were removed and the attached cells were stained for 10 min with crystal violet as described (Petitclerc et al., 1999). The wells were washed three times with PBS and cell-associated crystal violet was eluted by addition of 100 µl of 10% acetic acid. Cell adhesion was quantified by measuring the optical density of eluted crystal violet at a wavelength of 600 nm.
Cell migration assays
Transwells membranes (8.0-µm pore size) were coated with human collagen type IV (triple helical or denatured) for 16 h at 4°C. Next, 600 µl of migration buffer (RPMI 1640, 1 mM MgCl2, 0.2 mM MnCl2, and 0.5% BSA) was added to the lower chamber. HUVECs (105) were resuspended in migration buffer in the presence or absence of Mab HUIV26 or control antibodies (50 µg/ml) and added to upper chamber and allowed to migrate for 5 h at 37°C. Cells remaining on the top of the membrane were removed and cells that had migrated to the underside were fixed and stained with crystal violet. The membranes were washed and the cell-associated crystal violet was eluted with 10% acetic acid. Cell migration was quantified by measuring the optical density of eluted crystal violet at a wavelength of 600 nm.
Purified integrin receptor binding assays
ELISA plates (96 well) were coated with either 25 µg/ml of denatured or triple helical collagen IV for 18 h at 4°C. Plates were washed three times with 200 µl of PBS and blocked with 100 µl/well of 1.0% BSA in PBS for 1 h at 37°C. Purified human integrin receptors (1ß1,
2ß1,
5ß1, and
vß3) were diluted in binding buffer containing 20 mM Tris, 150 mM NaCl, 1 mM MgCl2, 0.2 mM MnCl2, 0.5% BSA, pH 7.5. Integrins (0.54.0 µg/ml) were allowed to bind for 1 h at 37°C. Next, the plates were washed three times with binding buffer and incubated with antiintegrin specific Mabs. The plates were washed three times with PBS and incubated with goat antimouse peroxidaseconjugated IgG for 1 h at 37°C. The plates were washed three times with PBS and ELISA substrate (OPD) was added and the OD was measured with an ELISA plate reader at a wavelength of 490 nm. All measurements were corrected for nonspecific binding to BSA and for reactivity with secondary antibody.
Statistical analysis
Statistical analysis was performed using Student's t test. P values <0.05 were considered significant.
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
P.C. Brooks was supported by grants CA74132 and CA086140, and G.E. Davis was supported by grant HL 59971 from the National Institutes of Health (NCH/NCI).
Submitted: 26 March 2001
Revised: 6 July 2001
Accepted: 13 July 2001
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Blood, C.H., and B.R. Zetter. 1990. Functional interactions with the vasculature: angiogenesis and tumor metastasis. Biochim. Biophys. Acta. 1032:89118.[Medline]
Bowersox, J.C., and N. Sorgenete. 1982. Chemotaxis of aortic endothelial cells in response to fibronectin. Cancer Res. 42:25472551.[Abstract]
Brooks, P.C., A.M.P. Montgomery, M. Rosenfeld, R.A. Reisfeld, T. Hu, G. Klier, and D.A. Cheresh. 1994. Integrin vß3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell. 79:11571164.[Medline]
Brooks, P.C., S. Stromblad, L.C. Sanders, T.L. von Schalscha, R.T. Aimes, W.G. Stetler-Stevenson, J.P. Quigley, and D.A. Cheresh. 1996. Localization of matrix metalloproteinase MMP-2 to the surface of invasive cells by interaction with integrin. Cell. 85:683693.[Medline]
Brooks, P.C., A.M.P. Montgomery, and D.A. Cheresh. 1998. Use of the 10 day old chick embryo model for studying angiogenesis. A.R. Howlett, editor. Humana Press Inc., Totowa, NJ. 257269.
Davis, G.E. 1992. Affinity of integrins for damaged extracellular matrix: vß3 binds to denatured collagen type I through RGD sites. Biochem. Biophys. Res. Commun. 182:10251031.[Medline]
Davis, G.E., K.J. Bayless, M.J. Davis, and G.A. Meinirger. 2000. Regulation of tissue injury responses by the exposure of matricryptic sites within extracellular matrix molecules. Am. J. Pathol. 156:14891498.
Dipietro, L.A., M. Burdick, Q.E. Low, S.L. Kunkel, and R.M. Strieter. 1998. MIP-1 as a critical macrophage chemoattractant in murine wound repair. J. Clin. Invest. 101:16931698.
Dogic, D., B. Eckes, and M. Aumailley. 1999. Extracellular matrix, integrins and focal adhesions. Curr. Top. Pathol. 93:7583.[Medline]
Emsley, J., G. Knight, R.W. Frarndale, M.J. Barnes, and R.C. Liddington. 2000. Structural basis of collagen recognition by integrin 2ß1. Cell. 100:4756.
Friedlander, M., C.L. Theesefeld, M. Sugita, M. Fruttiger, M.A. Thomas, S. Change, and D.A. Cheresh. 1996. Involvement of integrins vß3 and
vß5 in ocular neovascular diseases. Proc. Natl. Acad. Sci. USA. 93:97649769.
Giannelli, G., J. Falk-Marzillier, O. Schiraldi, W.G. Stetler-Stevenson, and V. Quaranta. 1997. Induction of cell migration by matrix metalloproteinase-2 cleavage of laminin-5. Science. 277:225228.
Hanahan, D., and J. Folkman. 1996. J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 86:353364.[Medline]
Herbst, T.J., J.B. McCarthy, E.C. Tsilbary, and L.T. Furcht. 1998. Differential effects of laminin, intact type IV collagen, and specific domains of type IV collagen on endothelial cell adhesion and migration. J. Cell Biol. 106:13651373.[Abstract]
Hiraoka, N., E. Allen, I.J. Apel, M.R. Gyetko, and S.J. Weiss. 1998. Matrix metalloproteinases regulate neovascularization by acting as pericellular fibrinolysins. Cell. 95:365377.[Medline]
Hudson, B.G., S.T. Reeders, and K.J. Tryggvason. 1993. Type IV collagen: structure, gene organization, and role in human diseases. J. Biol. Chem. 268:2603326036.
Itoh, T., M. Tanioka, H. Yoshida, T. Yoshioka, H. Nishimoto, and S. Itohara. 1998. Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res. 58:10481051.[Abstract]
Jukkola, A., R. Taktela, E. Tholix, K. Vuorinen, G. Blanco, L. Risteli, and J. Risteli. 1997. Aggressive breast cancer leads to discrepant serum levels of the type I procollagen propeptides PINP and PICP. Cancer Res. 57:55175520.[Abstract]
Koch, A.E., M.M. Halloran, C.J. Haskell, S.R. Manisha, P.J. Polverini. 1995. Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1. Nature. 376:517519.[Medline]
Liotta, L.A., P.A. Steeg, and W.G. Stetler-Stevenson. 1991. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell. 64:327336.[Medline]
Messent, A.J., D.S. Tuckwell, V. Knauper, M.J. Humphries, G. Murphy, and J. Gavrilovic. 1998. Effects of collagenase-cleavage of type I collagen on 2ß1 integrin mediated cell adhesion. J. Cell Sci. 111:11271135.
Montgomery, A.M.P., R.A. Reisfeld, and D.A. Cheresh. 1994. Integrin vß3 rescues melanoma cells from apoptosis in a three-dimensional dermal collagen. Proc. Natl. Acad. Sci. USA. 91:88568860.[Abstract]
O'Reilly, M.S., M.S. Holmgren, L. Shing, Y. Chen, R.A. Rosenthal, M. Moszes, W.W. Lane, Y. Cao, E.H. Sage, and J. Folkman. 1994. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by lewis lung carcinoma. Cell. 79:315328.[Medline]
Petitclerc, E., S. Stromblad, T.L. von Schalsacha, F. Mitjans, J. Piutats, A.M.P. Montgomery, D.A. Cheresh, and P.C. Brooks. 1999. Integrin vß3 promotes M21 melanoma growth in human skin by regulating tumor cell survival. Cancer Res. 59:27242730.
Rak, J., J. Filmus, G. Finkenzeller, S. Grugel, D. Marme, and R.S. Kerbel. 1995. Oncogenes as inducers of tumor angiogenesis. Cancer Metastasis Rev. 14:263277.[Medline]
Risau, W., and V. Lemmon. 1988. Changes in vascular extracellular matrix during embryonic vasculogenesis and angiogenesis. Dev. Biol. 125:441450.[Medline]
Schnaper, W.H., D.S. Grant, W.G. Stetler-Stevenson, R. Fridaman, G. D'razi, A.N. Murphy, R.E. Bird, M. Hoythya, T.R. Fuerst, D.L. French, J.P. Quigley, and H.K. Kleinman. 1993. Type IV collagenases and TIMPs modulate endothelial cell morphogenesis in vitro. J. Cell. Physiol. 156:235246.[Medline]
Senger, D.R., and C.A. Perruzzi. 1996. Cell migration promoted by a potent RGDS-containing thrombin-cleavage fragment of osteopontin. Biochim. Biophys. Acta. 1314:1324.[Medline]
Smith, J.W., and D.A. Cheresh. 1988. The Arg-Gly-Asp binding domain of the vitronectin receptor. J. Biol. Chem. 263:1872618731.
Stetler-Stevenson, W.G. 1999. Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention. J. Clin. Invest. 103:12371241.
Suri, C., P.F. Jones, S. Patan, S. Bartunkova, P.C. Maisonpierre, S. Davis, T.N. Sato, and G.D. Yancopoulos. 1996. Requisite role of Angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell. 87:11711180.[Medline]
Timpl, R. 1996. Macromolecular organization of basement membranes. Curr. Opin. Cell Biol. 8:618624.[Medline]
Timpl, R., and J.C. Brown. 1995. Supramolecular assembly of basement membranes. Bioessays. 18:123132.
Tsai, L.-H. 1998. Stuck on the ECM. Trends Cell Biol. 8:192295.
Vu, T., M.J. Shipley, G. Bergers, J.E. Berger, J.A. Helms, D. Hanahan, S.D. Shapiro, R.M. Senior, and Z. Werb. 1998. MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell. 93:411422.[Medline]
Weidner, N., J.P. Semple, W.R. Welch, and J. Folkman. 1991. Tumor angiogenesis and metastasis, correlation in invasive breast carcinoma. N. Engl. J. Med. 324:18.[Abstract]
Weidner, N., J. Folkman, F. Pozza, P. Bevilacqua, E.N. Allred, D.H. Moore, S. Meli, and G. Gasparini. 1992. Tumor angiogenesis: a new significant and independent prognostic indicator in early-stage breast carcinoma. J. Natl. Cancer Inst. 84:18751887.[Abstract]
Werb, Z., T. Vu, J.L. Rinkenberger, and L.M. Coussens. 1999. Matrix-degrading proteases and angiogenesis during development and tumor formation. APMIS. 107:1118.[Medline]
Xu, J., D. Rodriguez, J.J. Kim, and P.C. Brooks. 2000. Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization. Hybridoma. 19:375385.[Medline]
Related Article