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The role of collagen-derived proteolytic fragments in angiogenesis
Matrix Biology 20 Ž2001. 337᎐345
Mini review
The role of collagen-derived proteolytic fragments in angiogenesis Alexander G. MarnerosU , Bjorn R. Olsen Department of Cell Biology, Har¨ ard Medical School, 240 Longwood A¨ enue, Boston, MA 02115, USA Accepted 7 June 2001
Abstract Basement membrane molecules and fragments derived from them are regulators of biological activities such as cell growth, differentiation and migration. This review describes proteolytically derived fragments from the non-collagenous ŽNC1. domain at the C-terminus of the basement membrane collagens type IV, XV and XVIII, which have been implicated as regulators of angiogenesis. Endostatin is an endogenous collagen XVIIIrNC1 derivative, inhibiting endothelial cell proliferation and migration in vitro and tumor-growth in vivo. A homologous NC1 domain fragment of type XV collagen has anti-angiogenic activity as well. Furthermore, NC1 domain fragments of the most abundant basement membrane collagen, type IV collagen, have been shown to inhibit induced vessel growth. 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Angiogenesis; Collagen XVIII; Collagen XV; Collagen IV; Endostatin; Basement membrane
1. Introduction In a process termed vasculogenesis, the primary vascular plexus in the embryo is formed by angioblasts Žundifferentiated precursor cells. that differentiate into endothelial cells. In angiogenesis, this network expands and remodels into a more complex vascular network, either by sprouting of capillaries from pre-existing vessels, or by splitting of vessels. In sprouting angiogenesis, the perivascular extracellular matrix is proteolytically degraded, endothelial cells proliferate and migrate, and new vessels are formed in response to growth and chemotactic factors, such as VEGF or FGF-2. Alternatively, new vessels are formed through splitting of pre-existing vessels by U
Corresponding author. Tel.: q1-617-432-1874; fax: q1-617432-0638. E-mail address: alexander y [email protected] ŽA.G. Marneros..
transcapillary posts of extracellular matrix, a process called non-sprouting angiogenesis or intussusception Žreviewed by Risau, 1997; Carmeliet, 2000.. In the adult, new vessels form mainly through angiogenesis, although vasculogenesis can also occur through mobilized bone marrow angioblasts. Physiological vessel growth in the adult occurs in placenta formation and during the ovarian cycle. Pathological angiogenesis, often induced by inflammation, is a hallmark of tumor formation, arteriosclerosis and various other pathological conditions. Although embryonic and adult angiogenesis have distinct mechanisms, they are mainly regulated by the same factors. For example, VEGF and its receptors are essential for embryonic blood vessel formation, as demonstrated by the lethality of VEGFrVEGFR null mutant mice ŽCarmeliet et al., 1996; Shalaby et al., 1995; Fong et al., 1995.. Similarly, inhibition of VEGF signaling results in reduced angiogenesis in many different tumors. This association between reduced angiogenesis
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and reduced tumor growth, has led to much interest in inhibitors of angiogenesis. Interestingly, various angiogenesis inhibitors have been found to be proteolytically derived fragments of basement membrane collagens type IV, type XV and type XVIII. 2. Type XVIII collagen Non-fibrillar collagens have interruptions within the collagenous Gly-X-Y motif, creating potential regions of flexibility and allowing them to form polymers that are different from the fibrils formed by fibrillar collagens type I, II, III, VrXI. A recently defined subfamily of non-fibrillar collagens, the multiplexins, is comprised of type XV and type XVIII collagens ŽOh et al., 1994a., and characterized by multiple interruptions in the central triple-helical domain and the presence of a unique non-triple-helical domain at the C-terminus ŽNC1.. Type XVIII collagen contains 10 collagenous triple-helical ŽCOL. domains, separated by 11 noncollagenous ŽNC. regions ŽOh et al., 1994b.. Three distinct variants of this collagen have been described in mice, but only two variants Ža short and a long form. in humans ŽSaarela et al., 1997.. The differences are in the N-terminal NC11-domain, as a result of transcription from two different promoters ŽRehn and Pihlajaniemi, 1995.. Each of the variants shows a characteristic expression pattern, with the highest levels of RNA in liver, kidney and lung. Immunohistochemical studies show type XVIII collagen to be a component of vascular and epithelial basement membranes ŽMuragaki et al., 1995; Saarela et al., 1998.. Interestingly, the human short variant localizes to most vascular and epithelial basement membranes, whereas the long variant is highly and almost exclusively expressed in the liver, suggesting a distinct liver-specific function. Besides the typical features of a collagen, such as sensitivity to bacterial collagenase digestion, type XVIII collagen also has properties of a heparan sulfate proteoglycan ŽHSPG., and contains long heparitinase-sensitive carbohydrate chains. Thus, this molecule belongs to the group of basement membrane HSPGs ŽHalfter et al., 1998., in addition to being a collagen. Only little is known about the physiological role of type XVIII collagen. Col18a1-deficient mice are viable and reproduce normally. Although type XVIII collagen is a component of basement membranes, these mice demonstrate no significant abnormality in basement membrane assembly Žour own unpublished results .. A truncating mutation in the COL18A1 gene affecting the short form has recently been found in a family with autosomal recessive Knobloch syndrome ŽSertie et al., 2000.. This rare disorder is character-
Fig. 1. The NC1 domain of collagen XVIII. This domain consists of three segments: an N-terminal trimerization region, implicated in the self-assembly of type XVIII collagen homotrimers; a central protease-sensitive hinge region; and a 20-kDa C-terminal domain, termed endostatin.
ized by various ocular abnormalities, such as high myopia, macular and vitreoretinal degeneration with retinal detachment, and occipital encephalocele. Inactivating mutations affecting both the short and the long variants of type XVIII collagen ŽPassos-Bueno, personal communication. have also been identified in Knobloch syndrome patients. Thus, this disorder represents the effects of type XVIII collagen deficiency in humans, and implies a role of this collagen in the development or maintenance of the vitreoretinal structure in the human eye. The amino acid sequences of the human and mouse ␣1ŽXVIII. collagen chains show a high degree of homology, particularly in their C-terminal non-triple helical domain ŽNC1.. This domain consists of three segments: an N-terminal association region Ž; 50 residues., implicated in the self-assembly of type XVIII collagen homotrimers; a central protease-sensitive hinge region Ž; 70 residues.; and a 20-kDa C-terminal domain Ž; 180 residues., termed endostatin ŽFig. 1. ŽSasaki et al., 1998..
3. Endostatin Endostatin was originally purified from the conditioned media of a murine hemangioendothelioma cell line ŽEOMA. as a specific inhibitor of endothelial cell proliferation in vitro and a potent angiogenesis inhibitor in vivo ŽO’Reilly et al., 1997.. In the initial study, soluble recombinant endostatin, made in the baculovirus system, specifically inhibited the proliferation of endothelial cells in a dose-dependent fashion,
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whereas the proliferation of non-endothelial cell lines was not inhibited. Systemic administration of recombinant mouse endostatin, made as an insoluble precipitate in E. coli, suppressed the growth of Lewis lung carcinoma metastases with no evidence of any toxicity or resistance, as well as the growth of primary tumors growing in syngeneic mice, such as the Lewis lung carcinoma, T241 fibrosarcoma, EOMA hemangioendothelioma and B16F10 melanoma. The antiangiogenic and anti-tumor activity of endostatin has subsequently been demonstrated in a variety of different tumors, e.g. renal or mammary carcinomas, leading to the initiation of human clinical trials. Several strategies for a continuous and local administration of endostatin in sufficient doses have already been described, such as the use of microencapsulated producer cells ŽRead et al., 2001; Joki et al., 2001. or gene transfer approaches ŽSauter et al., 2000.. Physiological serum levels of monomeric endostatin indicate a biological role for the proteolytic processing of collagen XVIII. The basement membrane location of type XVIII collagen suggests a local regulatory role of endostatin in vessel growth. Additionally, circulating endostatin might participate in regulation of angiogenesis, since its concentration in serum Ž100᎐300 ngrml. is similar to the concentrations that efficiently inhibited endothelial cell proliferation in vitro ŽO’Reilly et al., 1997; Sasaki et al., 1998.. Endostatin appears not to be a rate-limiting regulator of vessel growth, however, since Col18a1rendostatindeficient mice display no major vascular abnormalities. Preliminary data also suggest no altered growth of primary tumors in these mice compared to wild-type mice, as well as no significantly altered angiogenic response in these tumors ŽMarneros and Fukai, unpublished data.. Additionally, patients with Knobloch syndrome have not been reported to show increased frequency of vascular abnormalities. Some insight into the physiological role of endostatin was recently provided through studies in Caenorhabditis elegans. The C. elegans type XVIII collagen homologue, cle-1, is found in basement membranes, but particularly at high levels in the nervous system. Notably, deletion of the cle-1 NC1rendostatin domain leads to multiple cell migration and axon guidance defects, suggesting a promigratory activity of the NC1 domain in C. elegans ŽAckley et al., 2001.. The NC1 domain of type XVIII collagen has the ability to oligomerize via its association domain, whereas endostatin remains monomeric. Interestingly, NC1 trimers could rescue the defects of the C. elegans NC1-deletion mutants, whereas monomeric endostatin expression in the wild-type background phenocopied the NC1-deletion mutants, acting as an inhibitor of migration. Such an oligomerization-dependent pro-migratory activity of the NC1
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domain of type XVIII collagen and its inhibition by monomeric endostatin has also been demonstrated in cell culture experiments for the human form ŽKuo et al., 2001.. The anti-migratory activity of endostatin was previously shown in migration assays with human umbilical vein endothelial cells ŽHUVECs. ŽYamaguchi et al., 1999.. Interestingly, the pro-migratory activity of the NC1 domain oligomers was not specific for endothelial cells, but was also observed with various other nonendothelial cells. This activity strictly required the presence of the extracellular matrix, and of rac, cdc42 and the MAP kinase pathway. The pro-migratory activity of the NC1 domain on endothelial and non-endothelial cells is in contrast to the anti-migratory effect of monomeric endostatin on only endothelial cells. Cell culture experiments demonstrated that endothelial cell adhesion on immobilized endostatin is mediated by ␣ 5- and ␣ v-integrins ŽRehn et al., 2001.. Additionally, it was shown that immobilized endostatin promotes and soluble endostatin inhibits endothelial cell migration in an integrin-dependent and specific manner. These integrins are expressed on the surface of proliferating endothelial cells and mediate interactions with the extracellular matrix. They are thought to be important for the anchorage-dependent proliferation of endothelial cells, since antagonists inhibit tumor angiogenesis in animal models ŽKim et al., 2000.. Such integrin antagonists have been shown to induce apoptosis of the proliferative angiogenic endothelial cells, without affecting pre-existing quiescent blood vessels ŽBrooks et al., 1994.. To further investigate the role of endostatin in cell adhesion and migration, the binding of several extracellular matrix proteins to immobilized mouse trimeric NC1 and monomeric endostatin was assessed in solid-phase assays ŽSasaki et al., 1998.. The NC1 trimers strongly bound to perlecan and to laminin-1 complexed to nidogen-1, but the interaction with monomeric endostatin was approximately 100-fold weaker. The laminin-1-nidogen-1 complex promotes cell attachment and has been implicated in the regulation of angiogenesis ŽChakravarti et al., 1990., showing concentration-dependent effects on formation of microvessels in vitro ŽNicosia et al., 1994.. Thus, it is possible that the type XVIII collagen NC1 domain interferes with basement membrane-cell interactions resulting in a pro-migratory activity. The anti-migratory effects of monomeric endostatin are likely to be the result of a different mechanism. Physiologic proteolysis in the hinge region of NC1 converts endostatin from a trimerized form into a monomeric form. These data suggest that the in vivo proteolytic generation of endostatin might be part of an autoregulatory feedback loop, which antagonizes
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collagen XVIIIrNC1 activity. In vitro studies identified cathepsin L as an enzyme capable of generating endostatin, whereas matrix metalloproteases ŽMMPs. produce larger fragments in a parallel processing pathway ŽFelbor et al., 2000.. In vivo, proteolytic processing of type XVIII collagen generates both NC1 trimers and endostatin monomers ŽSasaki et al., 1998; Wen et al., 1999., since both types of fragment are found in tissues and serum ŽStandker et al., 1997; John et al., 1999.. A role for endostatin in an autoregulatory feedback loop in conditions of induced angiogenesis is supported by the observations that cathepsins that can generate endostatin are produced by both endothelial cells and tumor cells, and that such proteases are activated by a moderately acidic pH similar to what is found in the pericellular milieu of tumors. The central protease-sensitive hinge region of the NC1 domain is sensitive to many different proteases ŽFerreras et al., 2000., e.g. cathepsins, MMPs or pancreatic serine elastase, which generate distinct endostatin containing fragments with varying efficiency. Incubation of human endostatin with these proteases showed that cathepsins L and D efficiently degraded endostatin, whereas MMPs showed no such activity. These data imply that not only the generation of endostatin, but also its degradation, is dependent on the activities of pericellular proteases, pointing to an additional mechanism for regulating local levels of endostatin. Some data indicate that endostatin is a protease inhibitor ŽKim et al., 2000b.. Activation of proMMP-2 is blocked by endostatin, and the catalytic activity of MMP-2 is inhibited. This is an interesting finding,
since MMPs are produced by endothelial cells and proteolytically degrade the perivascular extracellular matrix during sprouting angiogenesis. Thus, locally generated endostatin at sites of induced angiogenesis may directly inhibit MMP activity. In the original report describing the identification of endostatin ŽO’Reilly et al., 1997., the apoptosis-rate of tumor cells in endostatin treated tumor-bearing mice was seven-fold higher than in untreated mice. The authors concluded that a high apoptosis rate in endostatin treated tumors reflected the inhibition of angiogenesis. Subsequently, others demonstrated that endostatin can induce apoptosis in endothelial cells ŽDhanabal et al., 1999., in a process which seems to be dependent on tyrosine phosphorylation of the adaptor protein Shb ŽDixelius et al., 2000.. The crystal structure of mouse endostatin reveals a compact fold with a heparin-binding basic patch formed by 11 arginine residues ŽFig. 2A. ŽHohenester et al., 1998.. The heparin-binding ability of endostatin was reported to be required for the induction of apoptosis in cultured endothelial cells ŽDhanabal et al., 1999., and for its inhibition of FGF-2 Žbut not VEGF. induced angiogenesis in a chick chorioallantoic membrane ŽCAM. assay ŽSasaki et al., 1999.. The identification of glypicans, being cell surface HSPGs, as low-affinity endostatin receptors ŽKarumanchi et al., 2001. support a critical role of the heparin-binding site for endostatin actions, although heparin-binding does not appear to be important for inhibition of VEGF-induced endothelial cell migration ŽYamaguchi et al., 1999.. The intracellular signaling events in response to
Fig. 2. Ža. Crystal structure of murine endostatin. Žb. Crystal structure of the murine endostatin-like fragment of type XV collagen. The crystal structure of the endostatin-like collagen XV fragment shows a very similar overall fold compared to the crystal structure of endostatin, but contains no heparin-binding site. Structures were obtained from the protein data bank wPDB Ids: 1DY1 and 1DY2x.
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cell binding of endostatin are only partially understood. Endostatin’s anti-angiogenic activity is likely to involve intracellular signaling pathways that antagonize pro-angiogenic actions of VEGF or FGF-2, since no cross-competition for receptor binding between VEGF or FGF-2 and endostatin was observed ŽKarumanchi et al., 2001.. Some findings suggest that the effects of endostatin on endothelial cells in vitro are dependent on the cell culture conditions ŽShichiri and Hirata, 2001.. Apoptosis or growth inhibition of endostatin treated endothelial cells was observed under reduced serum conditions, under serum-supplemented conditions no apoptosis was detected. In experiments where migration was potently inhibited, endostatin was found to rapidly down-regulate the expression of a broad variety of genes.
4. Type XV collagen Type XV collagen is structurally homologous to type XVIII collagen; the two collagens form the multiplexin family of non-fibrillar collagens. The genomic structure of their genes reveals a considerable conservation in exon᎐intron organization, indicating a common ancestor. Type XV collagen contains several interruptions in the triple-helical sequence of the central collagenous domain, a large non-collagenous globular domain at the N-terminus and a smaller C-terminal non-collagenous domain ŽNC1. ŽMyers et al., 1992; Kivirikko et al., 1994; Oh et al., 1994a,b; Muragaki et al., 1994; Hagg ¨ et al., 1997a.. Type XVIII collagen is a heparan sulfate proteoglycan; in contrast, type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan ŽLi et al., 2000.. Type XV collagen shows a widespread tissue distribution, with high expression in heart, skeletal muscle and placenta ŽKivirikko et al., 1995.. This collagen is synthesized by fibroblasts, endothelial cells, myoblasts and some epithelial cells. It localizes to vascular, neuronal, mesenchymal and some epithelial basement membrane zones ŽMyers et al., 1996., but also to other locations, such as to the fibrillar collagen matrix of the papillary dermis. The basement membrane location is not ubiquitous, but in immunohistochemical studies strong positive staining was found particularly at basement membranes of capillaries and skeletal muscle cells ŽHagg ¨ et al., 1997b.. Consistent with this localization are the findings observed in Col15a1-deficient mice ŽEklund et al., 2001.. These mice, which are viable and reproduce normally, showed a mild but progressive degeneration of skeletal muscle cells, and a susceptibility to exercise-induced muscle damage. Additionally, the mice were reported to have abnormal microvessels in heart and skeletal muscle, whereas the development of the over-
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all vasculature appeared normal. These findings suggest that type XV collagen is required for the structural integrity of skeletal muscle cells and capillaries.
5. NC1 domain of type XV collagen Type XV collagen contains in its NC1 domain an endostatin-like region, showing approximately 60% sequence identity with the endostatin domain of type XVIII collagen. Based on the anti-angiogenic activity of endostatin, derived from collagen XVIII, such an activity was investigated also for fragments of the NC1 domain of type XV collagen. The endostatin-like region in collagen XV was produced as a recombinant protein and tested for anti-angiogenic activity ŽRamchandran et al., 1999.. It showed no anti-proliferative effects on several types of endothelial cells. A dose-dependent inhibition of migration, in response to FGF-2, was observed with endothelial cells, but not with non-endothelial cells. The authors conducted in vivo tumor assays using a renal cell carcinoma xenograft model with systemic administration of the recombinant human type XV collagen fragment. This treatment resulted in a reduced increase in tumor volume over time, but produced no tumor regression, and demonstrated a less potent anti-tumor effect than treatment with endostatin derived from type XVIII collagen. In contrast to the type XVIII collagen NC1 domain, the recombinant type XV collagen NC1 domain has no pro-migratory activity, as a complete lack of inhibition of HUVEC tube formation on Matrigel was observed ŽKuo et al., 2001.. In solid-phase assays, type XV collagen NC1 domain binds significantly weaker to nidogen-1, the laminin-1-nidogen-1 complex and perlecan, than type XVIII collagen NC1 domain ŽSasaki et al., 2000.. Similar weak binding affinities for these extracellular matrix proteins were shown for the endostatin-like fragment of type XV collagen as well. Further structural and functional characterization of the NC1 domain of type XV collagen demonstrated that it also contains a trimerization domain, a hinge region that is less sensitive to proteolysis than in collagen XVIII, and an endostatin-like domain at the C-terminus ŽSasaki et al., 2000.. The crystal structure of the endostatin-like collagen XV fragment shows a very similar overall fold compared to the crystal structure of endostatin, but contains no heparin-binding site ŽFig. 2B.. The in vitro and in vivo anti-angiogenic activities of the endostatin-like fragment of collagen XV have been demonstrated using recombinant protein. Immunoreactive endostatin-related C-terminal fragments of human collagen XV have been isolated from
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human blood filtrate, supporting the view that this proteolytic fragment or the processing of type XV collagen may have a physiological role ŽJohn et al., 1999..
type IV collagen and its inhibition with type IV collagen peptides has been demonstrated for several cells ŽAumailley and Timpl, 1986; Tsilibary et al., 1990.. The data indicate that type IV collagen has an important role in promoting the adhesion and motility of various cell types.
6. Type IV collagen Type IV collagen forms a network structure in basement membranes, providing a scaffold that incorporates several other basement membrane components and regulates the interaction with adhering cells. Six distinct genes encode for the six type IV collagen chains ␣1᎐␣6 ŽIV.. Their genomic location shows a pair-wise head-to-head arrangement with a bi-directional promoter, mapping to three different chromosomes Žreviewed by Olsen and Ninomiya, 1999; Kuhn, 1995.. The chains of this predominant collagenous component of basement membranes contain a cysteine-rich Ž7S. domain at the N-terminus, a central triple-helical collagenous domain and a C-terminal non-collagenous ŽNC1. domain. Type IV collagen molecules form a network structure through covalently cross-linked and laterally associated 7S domains, end-to-end interactions of their NC1 domains, and lateral associations of their central collagenous domain. The ␣1ŽIV. and ␣ 2ŽIV. chains are the predominant forms of this collagen in most basement membranes. The ␣ 3᎐␣6ŽIV. chains seem to have distinct functional properties, since they are found in specialized basement membranes. Of particular interest is the composition of the kidney glomerular basement membrane, in which ␣ 3᎐␣ 5ŽIV. chains replace ␣1ŽIV. and ␣ 2ŽIV. as development proceeds. The functional importance of these chains are underscored through mutations in the ␣ 3᎐␣ 5ŽIV. chains in Alport syndrome, resulting in structural changes of the glomerular basement membrane with the clinical manifestation of glomerulonephritis. The ␣6ŽIV. chain shows a distinct localization in epidermal basement membranes, in Bowman’s capsule and renal distal tubules, and around adipocytes and smooth muscle cells. Type IV collagen plays a role in the interaction of basement membranes with cells. This interaction can be either direct or mediated through laminin, which shows low affinity binding to type IV collagen. The affinity is increased through nidogen, which binds strongly to laminin, and contains binding sites for type IV collagen and cells as well. Additionally, type IV collagen is able to bind heparin and heparan sulfate proteoglycans. The w ␣1ŽIV.x2 ␣ 2ŽIV. trimers contain in their triple-helical domain a cell-binding site with the recognition site for the two integrin receptors ␣ 1  1 and ␣ 2  1 ŽVandenberg et al., 1991.. Cell binding to
7. NC1 domain of type IV collagen The NC1 domain of type IV collagen is thought to be essential for the oligomerization of the ␣-chains. Several experiments, however, have pointed to functional roles beyond the assembly of basement membrane networks. A peptide derived from the NC1 domain of the ␣1ŽIV. chain was shown to promote the adhesion of bovine aortic endothelial cells ŽTsilibary et al., 1990.. Recombinant ␣1ŽIV.NC1 domain inhibited VEGF-induced proliferation and migration of endothelial cells in vitro, and neovascularization in a Matrigel plug assay in mice ŽColorado et al., 2000.. Thus, recombinant NC1 domains of type IV collagen have been further investigated as inhibitors of angiogenesis and regulators of endothelial cell behavior. Kefalides and co-workers demonstrated that a peptide of the NC1 domain of ␣ 3ŽIV. Žresidue sequence 185᎐203. inhibited the activation of leucocytes ŽMonboisse et al., 1994.. This peptide also promoted cell adhesion of human melanoma cells, and inhibited their proliferation as well ŽHan et al., 1997.. A triplet sequence Ž ᎐SNS᎐ at residues 189᎐191., which is unique to the ␣ 3ŽIV. chain, was essential for this activity. The receptors for the ␣ 3ŽIV.185᎐203 peptide were identified as CD47rintegrin-associated protein and the integrin ␣ v  3 ŽShahan et al., 1999.. These proteins were shown to regulate tumor cell chem otaxis to type IV collagen and the ␣ 3ŽIV.185᎐203 peptide through a calcium-dependent mechanism ŽShahan et al., 2000.. Based on these studies, recombinant ␣ 3ŽIV. NC1 domain was assessed for potential anti-angiogenic activity. The ␣ 3ŽIV. NC1 domain inhibited the proliferation of capillary endothelial cells, induced endothelial cell apoptosis and reduced tumor growth in vivo ŽMaeshima et al., 2000.. These activities were localized to residues 54᎐132 of the ␣ 3ŽIV. NC1 domain ŽMaeshima et al., 2001.. Using specific function-blocking anti-integrin antibodies, it has been shown that the attachment of endothelial cells to immobilized recombinant ␣ 2ŽIV. NC1 is dependent largely on integrin ␣ v  3 , partially also on ␣ v  5 and ␣ 3  1. This NC1 domain does not contain a RGD site recognized by ␣ v  3 , suggesting a novel binding site ŽPetitclerc et al., 2000.. Endothelial cell adhesion to the ␣ 3ŽIV. and ␣6ŽIV. NC1 domains
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were shown to depend only on ␣ v  3 , but not on ␣ v  5 or ␣ 3 1 integrins. These findings suggest that distinct ␣ ŽIV. NC1 domains bind to endothelial cells via ␣ v and  1 integrins, independently of the ␣ 1  1 and ␣ 2  1 integrin binding sites in the central triple-helical region of type IV collagen. In previous studies, function-blocking antibodies directed to integrins that bind type IV collagen inhibited angiogenesis in vitro and in vivo ŽDavis and Camarillo, 1996.. Integrin ␣ 1-blocking antibodies inhibited cell attachment of microvascular endothelial cells to type IV collagen and laminin-1 ŽSenger et al., 1997.. Systemic administration of recombinant ␣ 2ŽIV., ␣ 3ŽIV. and ␣6ŽIV. NC1 domains potently inhibited VEGF- and FGF-2-induced angiogenesis ŽCAM assay. and tumor growth in vivo Žchick embryo tumor growth assay., but no inhibition was observed with ␣1ŽIV., ␣4ŽIV. and ␣ 5ŽIV. NC1 domains ŽPetitclerc et al., 2000.. In further in vitro studies with a recombinant ␣ 2ŽIV. NC1 domain, endothelial cell tube formation, migration and proliferation were inhibited in a dose-dependent manner and endothelial cell apoptosis was induced ŽKamphaus et al., 2000.. Sensitivity of endothelial cells to apoptotic signals was dependent on growth factor stimulation, indicating that ␣ 2ŽIV. NC1 domain selectively targets angiogenic and not pre-formed quiescent endothelium. This recombinant peptide inhibited the growth of human prostate adenocarcinoma cells in SCID or athymic Žnurnu. mice, without inhibiting the proliferation of tumor cells in vitro. Notably, soluble NC1 domain and derivatives have been shown to block type IV collagen matrix assembly by binding along the length of the central region of type IV collagen, and thereby disrupting lateral associations of individual type IV collagen molecules ŽTsilibary and Charonis, 1986; Tsilibary et al., 1988, 1990.. Alternatively, soluble NC1 domains might disrupt the association of triple helical type IV collagen molecules at the C-terminus. In conclusion, it is possible that the anti-angiogenic activity of the recombinant ␣ ŽIV. NC1 domains may not only result through interfering with endothelial cell integrin signaling, but in part be the result of a disruption of type IV collagen matrix assembly. Additionally, an inhibition of the activity of proteases, such as MMP-2 and MMP-3, has been implicated ŽNetzer et al., 1998., similar to the MMP inhibitory activity of endostatin.
8. Conclusions The identified anti-angiogenic fragments of type IV, XV and XVIII collagens are derived from their NC1 domains, underscoring a role of these domains
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as regulators of cell migration and proliferation. Since the pro-migratory activity of the type XVIII collagen NC1 domain on endothelial and non-endothelial cells was strictly dependent on extracellular matrix, it is tempting to speculate that this pro-migratory activity results partially from the high-affinity binding of this domain to extracellular matrix components implicated in cell adhesion and migration, such as laminin-1 or nidogen-1, thus interfering with cell adhesion to the basement membrane. Such pro-migratory activity is lacking for the NC1 domain of type XV collagen, which binds significantly weaker to the laminin-1nidogen-1 complex in comparison to the NC1 domain of type XVIII collagen. Monomeric endostatin, which shows weak binding to laminin-1-nidogen-1 complex, inhibited the migration of endothelial cells, but not of non-endothelial cells ŽKuo et al., 2001; Shichiri and Hirata, 2001.. Thus, its anti-migratory activity might be mediated by endothelial cell-specific molecules and pathways that are different from those that are mediating the pro-migratory activity of type XVIII collagen NC1 domain on endothelial and non-endothelial cells. Endostatin binds with low affinity to endothelial cells through ␣ v- and ␣ 5-integrins and glypicans, as well as to an as yet unidentified high-affinity receptor. Type IV collagen NC1 domains have also been shown to bind to ␣ v-integrins, which mediate interactions of proliferating endothelial cells with the extracellular matrix. Thus, the different collagen-derived proteolytic fragments described here might regulate cellextracellular matrix interactions via closely related mechanisms, and affect angiogenesis in similar ways. References Ackley, B.D., Crew, J.R., Elamaa, H., Pihlajaniemi, T., Kuo, C.J., Kramer, J.M., 2001. The NC1rendostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance. J. Cell Biol. 152, 1219᎐1232. Aumailley, M., Timpl, R., 1986. Attachment of cells to basement membrane collagen type IV. J. Cell Biol. 103, 1569᎐1575. Brooks, P.C., Montgomery, A.M., Rosenfeld, M. et al., 1994. Integrin alpha V beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157᎐1164. Carmeliet, P., 2000. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389᎐395. Carmeliet, P., Ferreira, V., Breier, G. et al., 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435᎐439. Chakravarti, S., Tam, M.F., Chung, A.E., 1990. The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions. J. Biol. Chem. 265, 10597᎐10603. Colorado, P.C., Torre, A., Kamphaus, G. et al., 2000. Anti-angiogenic cues from vascular basement membrane collagen. Cancer Res. 60, 2520᎐2526. Davis, G.E., Camarillo, C.W., 1996. An alpha2 beta1 integrin-dependent pinocytic mechanism involving intracellular vacuole formation and coalescence regulates capillary lumen and tube
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human tissue and their production by muscle cells and fibroblasts. Am. J. Pathol. 147, 1500᎐1509. Kuhn, K., 1995. Basement membrane Žtype IV. collagen. Matrix Biol. 14, 439᎐445. Kuo, C.J., LaMontagne Jr., K.R., Garcia-Cardena, G. et al., 2001. Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1rendostatin domain. J. Cell Biol. 152, 1233᎐1246. Li, D., Clark, C.C., Myers, J.C., 2000. Basement membrane zone type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan in human tissues and cultured cells. J. Biol. Chem. 275, 22339᎐22347. Maeshima, Y., Colorado, P.C., Torre, A. et al., 2000. Distinct antitumor properties of a type IV collagen domain derived from basement membrane. J. Biol. Chem. 275, 21340᎐21348. Maeshima, Y., Manfredi, M., Reimer, C. et al., 2001. Identification of the anti-angiogenic site within vascular basement membranederived tumstatin. J. Biol. Chem. 276, 15240᎐15248. Monboisse, J.C., Garnotel, R., Bellon, G. et al., 1994. The alpha 3 chain of type IV collagen prevents activation of human polymorphonuclear leukocytes. J. Biol. Chem. 269, 25475᎐25482. Muragaki, Y., Abe, N., Ninomiya, Y., Olsen, B.R., Ooshima, A., 1994. The human alpha 1ŽXV. collagen chain contains a large amino-terminal non-triple helical domain with a tandem repeat structure and homology to alpha 1ŽXVIII. collagen. J. Biol. Chem. 269, 4042᎐4046. Muragaki, Y., Timmons, S., Griffith, C.M., Oh, S.P., Fadel, B., Quertermous, T., Olsen, B.R., 1995. Mouse Col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones. Proc. Natl. Acad. Sci. USA 92, 8763᎐8767. Myers, J.C., Dion, A.S., Abraham, V., Amenta, P.S., 1996. Type XV collagen exhibits a widespread distribution in human tissues but a distinct localization in basement membrane zones. Cell Tissue Res. 286, 493᎐505. Myers, J.C., Kivirikko, S., Gordon, M.K., Dion, A.S., Pihlajaniemi, T., 1992. Identification of a previously unknown human collagen chain, alpha 1ŽXV., characterized by extensive interruptions in the triple-helical region. Proc. Natl. Acad. Sci. USA 89, 10144᎐10148. Netzer, K.O., Suzuki, K., Itoh, Y., Hudson, B.G., Khalifah, R.G., 1998. Comparative analysis of the noncollagenous NC1 domain of type IV collagen: identification of structural features important for assembly, function, and pathogenesis. Protein Sci. 7, 1340᎐1351. Nicosia, R.F., Bonanno, E., Smith, M., Yurchenco, P., 1994. Modulation of angiogenesis in vitro by laminin-entactin complex. Dev. Biol. 164, 197᎐206. O’Reilly, M.S., Boehm, T., Shing, Y. et al., 1997. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88, 277᎐285. Oh, S.P., Kamagata, Y., Muragaki, Y., Timmons, S., Ooshima, A., Olsen, B.R., 1994aa. Isolation and sequencing of cDNAs for proteins with multiple domains of Gly-Xaa-Yaa repeats identify a distinct family of collagenous proteins. Proc. Natl. Acad. Sci. USA 91, 4229᎐4233. Oh, S.P., Warman, M.L., Seldin, M.F. et al., 1994bb. Cloning of cDNA and genomic DNA encoding human type XVIII collagen and localization of the alpha 1ŽXVIII. collagen gene to mouse chromosome 10 and human chromosome 21. Genomics 19, 494᎐499. Olsen, B.R, Ninomiya, Y., 1999. Collagens. In: Kreis, T., Vale, R. ŽEds.., Guidebook to the Extracellular Matrix, Anchor, and Adhesion Proteins, 2nd edition Oxford University Press, Oxford, UK, pp. 380᎐408. Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J. et al., 2000. New functions for non-collagenous domains of human collagen type
A.G. Marneros, B.R. Olsen r Matrix Biology 20 (2001) 337᎐345 IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo. J. Biol. Chem. 275, 8051᎐8061. Ramchandran, R., Dhanabal, M., Volk, R. et al., 1999. Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin. Biochem. Biophys. Res. Commun. 255, 735᎐739. Read, T.A., Sorensen, D.R., Mahesparan, R. et al., 2001. Local endostatin treatment of gliomas administered by microencapsulated producer cells. Natl. Biotechnol. 19, 29᎐34. Rehn, M., Pihlajaniemi, T., 1995. Identification of three N-terminal ends of type XVIII collagen and tissue-specific differences in the expression of the corresponding transcripts. J. Biol. Chem. 270, 4705᎐4711. Rehn, M., Veikkola, T., Kukk-Valdre, E. et al., 2001. Interaction of endostatin with integrins implicated in angiogenesis. Proc. Natl. Acad. Sci. USA 98 Ž3., 1024᎐1029. Risau, W., 1997. Mechanisms of angiogenesis. Nature 386, 671᎐674. Saarela, J., Ylikarppa, ¨ ¨ R., Rehn, M., Purmonen, S., Pihlajaniemi, T., 1997. Complete primary structure of two variant forms of human type XVIII collagen and tissue-specific differences in the expression of the corresponding transcripts. Matrix Biol. 16, 319᎐328. Saarela, J., Rehn, M., Oikarinen, A., Autio-Harmainen, H., Pihlajaniemi, T., 1998. The short and long forms of type XVIII collagen show clear tissue specificities in their expression and location in basement membrane zones in humans. Am. J. Pathol. 153, 611᎐626. Sasaki, T., Fukai, N., Mann, K., Gohring, W., Olsen, B.R., Timpl, R., 1998. Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin. Embo J. 17, 4249᎐4256. Sasaki, T., Larsson, H., Kreuger, J. et al., 1999. Structural basis and potential role of heparinrheparan sulfate binding to the angiogenesis inhibitor endostatin. Embo J. 18, 6240᎐6248. Sasaki, T., Larsson, H., Tisi, D., Claesson-Welsh, L., Hohenester, E., Timpl, R., 2000. Endostatins derived from collagens XV and XVIII differ in structural and binding properties, tissue distribution and anti-angiogenic activity. J. Mol. Biol. 301, 1179᎐1190. Sauter, B.V., Martinet, O., Zhang, W.J., Mandeli, J., Woo, S.L., 2000. Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases. Proc. Natl. Acad. Sci. USA 97, 4802᎐4807. Senger, D.R., Claffey, K.P., Benes, J.E., Perruzzi, C.A., Sergiou, A.P., Detmar, M., 1997. Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and
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Mini review
The role of collagen-derived proteolytic fragments in angiogenesis Alexander G. MarnerosU , Bjorn R. Olsen Department of Cell Biology, Har¨ ard Medical School, 240 Longwood A¨ enue, Boston, MA 02115, USA Accepted 7 June 2001
Abstract Basement membrane molecules and fragments derived from them are regulators of biological activities such as cell growth, differentiation and migration. This review describes proteolytically derived fragments from the non-collagenous ŽNC1. domain at the C-terminus of the basement membrane collagens type IV, XV and XVIII, which have been implicated as regulators of angiogenesis. Endostatin is an endogenous collagen XVIIIrNC1 derivative, inhibiting endothelial cell proliferation and migration in vitro and tumor-growth in vivo. A homologous NC1 domain fragment of type XV collagen has anti-angiogenic activity as well. Furthermore, NC1 domain fragments of the most abundant basement membrane collagen, type IV collagen, have been shown to inhibit induced vessel growth. 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. Keywords: Angiogenesis; Collagen XVIII; Collagen XV; Collagen IV; Endostatin; Basement membrane
1. Introduction In a process termed vasculogenesis, the primary vascular plexus in the embryo is formed by angioblasts Žundifferentiated precursor cells. that differentiate into endothelial cells. In angiogenesis, this network expands and remodels into a more complex vascular network, either by sprouting of capillaries from pre-existing vessels, or by splitting of vessels. In sprouting angiogenesis, the perivascular extracellular matrix is proteolytically degraded, endothelial cells proliferate and migrate, and new vessels are formed in response to growth and chemotactic factors, such as VEGF or FGF-2. Alternatively, new vessels are formed through splitting of pre-existing vessels by U
Corresponding author. Tel.: q1-617-432-1874; fax: q1-617432-0638. E-mail address: alexander y [email protected] ŽA.G. Marneros..
transcapillary posts of extracellular matrix, a process called non-sprouting angiogenesis or intussusception Žreviewed by Risau, 1997; Carmeliet, 2000.. In the adult, new vessels form mainly through angiogenesis, although vasculogenesis can also occur through mobilized bone marrow angioblasts. Physiological vessel growth in the adult occurs in placenta formation and during the ovarian cycle. Pathological angiogenesis, often induced by inflammation, is a hallmark of tumor formation, arteriosclerosis and various other pathological conditions. Although embryonic and adult angiogenesis have distinct mechanisms, they are mainly regulated by the same factors. For example, VEGF and its receptors are essential for embryonic blood vessel formation, as demonstrated by the lethality of VEGFrVEGFR null mutant mice ŽCarmeliet et al., 1996; Shalaby et al., 1995; Fong et al., 1995.. Similarly, inhibition of VEGF signaling results in reduced angiogenesis in many different tumors. This association between reduced angiogenesis
0945-053Xr01r$ - see front matter 䊚 2001 Elsevier Science B.V.rInternational Society of Matrix Biology. All rights reserved. PII: S 0 9 4 5 - 0 5 3 X Ž 0 1 . 0 0 1 5 1 - 2
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and reduced tumor growth, has led to much interest in inhibitors of angiogenesis. Interestingly, various angiogenesis inhibitors have been found to be proteolytically derived fragments of basement membrane collagens type IV, type XV and type XVIII. 2. Type XVIII collagen Non-fibrillar collagens have interruptions within the collagenous Gly-X-Y motif, creating potential regions of flexibility and allowing them to form polymers that are different from the fibrils formed by fibrillar collagens type I, II, III, VrXI. A recently defined subfamily of non-fibrillar collagens, the multiplexins, is comprised of type XV and type XVIII collagens ŽOh et al., 1994a., and characterized by multiple interruptions in the central triple-helical domain and the presence of a unique non-triple-helical domain at the C-terminus ŽNC1.. Type XVIII collagen contains 10 collagenous triple-helical ŽCOL. domains, separated by 11 noncollagenous ŽNC. regions ŽOh et al., 1994b.. Three distinct variants of this collagen have been described in mice, but only two variants Ža short and a long form. in humans ŽSaarela et al., 1997.. The differences are in the N-terminal NC11-domain, as a result of transcription from two different promoters ŽRehn and Pihlajaniemi, 1995.. Each of the variants shows a characteristic expression pattern, with the highest levels of RNA in liver, kidney and lung. Immunohistochemical studies show type XVIII collagen to be a component of vascular and epithelial basement membranes ŽMuragaki et al., 1995; Saarela et al., 1998.. Interestingly, the human short variant localizes to most vascular and epithelial basement membranes, whereas the long variant is highly and almost exclusively expressed in the liver, suggesting a distinct liver-specific function. Besides the typical features of a collagen, such as sensitivity to bacterial collagenase digestion, type XVIII collagen also has properties of a heparan sulfate proteoglycan ŽHSPG., and contains long heparitinase-sensitive carbohydrate chains. Thus, this molecule belongs to the group of basement membrane HSPGs ŽHalfter et al., 1998., in addition to being a collagen. Only little is known about the physiological role of type XVIII collagen. Col18a1-deficient mice are viable and reproduce normally. Although type XVIII collagen is a component of basement membranes, these mice demonstrate no significant abnormality in basement membrane assembly Žour own unpublished results .. A truncating mutation in the COL18A1 gene affecting the short form has recently been found in a family with autosomal recessive Knobloch syndrome ŽSertie et al., 2000.. This rare disorder is character-
Fig. 1. The NC1 domain of collagen XVIII. This domain consists of three segments: an N-terminal trimerization region, implicated in the self-assembly of type XVIII collagen homotrimers; a central protease-sensitive hinge region; and a 20-kDa C-terminal domain, termed endostatin.
ized by various ocular abnormalities, such as high myopia, macular and vitreoretinal degeneration with retinal detachment, and occipital encephalocele. Inactivating mutations affecting both the short and the long variants of type XVIII collagen ŽPassos-Bueno, personal communication. have also been identified in Knobloch syndrome patients. Thus, this disorder represents the effects of type XVIII collagen deficiency in humans, and implies a role of this collagen in the development or maintenance of the vitreoretinal structure in the human eye. The amino acid sequences of the human and mouse ␣1ŽXVIII. collagen chains show a high degree of homology, particularly in their C-terminal non-triple helical domain ŽNC1.. This domain consists of three segments: an N-terminal association region Ž; 50 residues., implicated in the self-assembly of type XVIII collagen homotrimers; a central protease-sensitive hinge region Ž; 70 residues.; and a 20-kDa C-terminal domain Ž; 180 residues., termed endostatin ŽFig. 1. ŽSasaki et al., 1998..
3. Endostatin Endostatin was originally purified from the conditioned media of a murine hemangioendothelioma cell line ŽEOMA. as a specific inhibitor of endothelial cell proliferation in vitro and a potent angiogenesis inhibitor in vivo ŽO’Reilly et al., 1997.. In the initial study, soluble recombinant endostatin, made in the baculovirus system, specifically inhibited the proliferation of endothelial cells in a dose-dependent fashion,
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whereas the proliferation of non-endothelial cell lines was not inhibited. Systemic administration of recombinant mouse endostatin, made as an insoluble precipitate in E. coli, suppressed the growth of Lewis lung carcinoma metastases with no evidence of any toxicity or resistance, as well as the growth of primary tumors growing in syngeneic mice, such as the Lewis lung carcinoma, T241 fibrosarcoma, EOMA hemangioendothelioma and B16F10 melanoma. The antiangiogenic and anti-tumor activity of endostatin has subsequently been demonstrated in a variety of different tumors, e.g. renal or mammary carcinomas, leading to the initiation of human clinical trials. Several strategies for a continuous and local administration of endostatin in sufficient doses have already been described, such as the use of microencapsulated producer cells ŽRead et al., 2001; Joki et al., 2001. or gene transfer approaches ŽSauter et al., 2000.. Physiological serum levels of monomeric endostatin indicate a biological role for the proteolytic processing of collagen XVIII. The basement membrane location of type XVIII collagen suggests a local regulatory role of endostatin in vessel growth. Additionally, circulating endostatin might participate in regulation of angiogenesis, since its concentration in serum Ž100᎐300 ngrml. is similar to the concentrations that efficiently inhibited endothelial cell proliferation in vitro ŽO’Reilly et al., 1997; Sasaki et al., 1998.. Endostatin appears not to be a rate-limiting regulator of vessel growth, however, since Col18a1rendostatindeficient mice display no major vascular abnormalities. Preliminary data also suggest no altered growth of primary tumors in these mice compared to wild-type mice, as well as no significantly altered angiogenic response in these tumors ŽMarneros and Fukai, unpublished data.. Additionally, patients with Knobloch syndrome have not been reported to show increased frequency of vascular abnormalities. Some insight into the physiological role of endostatin was recently provided through studies in Caenorhabditis elegans. The C. elegans type XVIII collagen homologue, cle-1, is found in basement membranes, but particularly at high levels in the nervous system. Notably, deletion of the cle-1 NC1rendostatin domain leads to multiple cell migration and axon guidance defects, suggesting a promigratory activity of the NC1 domain in C. elegans ŽAckley et al., 2001.. The NC1 domain of type XVIII collagen has the ability to oligomerize via its association domain, whereas endostatin remains monomeric. Interestingly, NC1 trimers could rescue the defects of the C. elegans NC1-deletion mutants, whereas monomeric endostatin expression in the wild-type background phenocopied the NC1-deletion mutants, acting as an inhibitor of migration. Such an oligomerization-dependent pro-migratory activity of the NC1
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domain of type XVIII collagen and its inhibition by monomeric endostatin has also been demonstrated in cell culture experiments for the human form ŽKuo et al., 2001.. The anti-migratory activity of endostatin was previously shown in migration assays with human umbilical vein endothelial cells ŽHUVECs. ŽYamaguchi et al., 1999.. Interestingly, the pro-migratory activity of the NC1 domain oligomers was not specific for endothelial cells, but was also observed with various other nonendothelial cells. This activity strictly required the presence of the extracellular matrix, and of rac, cdc42 and the MAP kinase pathway. The pro-migratory activity of the NC1 domain on endothelial and non-endothelial cells is in contrast to the anti-migratory effect of monomeric endostatin on only endothelial cells. Cell culture experiments demonstrated that endothelial cell adhesion on immobilized endostatin is mediated by ␣ 5- and ␣ v-integrins ŽRehn et al., 2001.. Additionally, it was shown that immobilized endostatin promotes and soluble endostatin inhibits endothelial cell migration in an integrin-dependent and specific manner. These integrins are expressed on the surface of proliferating endothelial cells and mediate interactions with the extracellular matrix. They are thought to be important for the anchorage-dependent proliferation of endothelial cells, since antagonists inhibit tumor angiogenesis in animal models ŽKim et al., 2000.. Such integrin antagonists have been shown to induce apoptosis of the proliferative angiogenic endothelial cells, without affecting pre-existing quiescent blood vessels ŽBrooks et al., 1994.. To further investigate the role of endostatin in cell adhesion and migration, the binding of several extracellular matrix proteins to immobilized mouse trimeric NC1 and monomeric endostatin was assessed in solid-phase assays ŽSasaki et al., 1998.. The NC1 trimers strongly bound to perlecan and to laminin-1 complexed to nidogen-1, but the interaction with monomeric endostatin was approximately 100-fold weaker. The laminin-1-nidogen-1 complex promotes cell attachment and has been implicated in the regulation of angiogenesis ŽChakravarti et al., 1990., showing concentration-dependent effects on formation of microvessels in vitro ŽNicosia et al., 1994.. Thus, it is possible that the type XVIII collagen NC1 domain interferes with basement membrane-cell interactions resulting in a pro-migratory activity. The anti-migratory effects of monomeric endostatin are likely to be the result of a different mechanism. Physiologic proteolysis in the hinge region of NC1 converts endostatin from a trimerized form into a monomeric form. These data suggest that the in vivo proteolytic generation of endostatin might be part of an autoregulatory feedback loop, which antagonizes
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collagen XVIIIrNC1 activity. In vitro studies identified cathepsin L as an enzyme capable of generating endostatin, whereas matrix metalloproteases ŽMMPs. produce larger fragments in a parallel processing pathway ŽFelbor et al., 2000.. In vivo, proteolytic processing of type XVIII collagen generates both NC1 trimers and endostatin monomers ŽSasaki et al., 1998; Wen et al., 1999., since both types of fragment are found in tissues and serum ŽStandker et al., 1997; John et al., 1999.. A role for endostatin in an autoregulatory feedback loop in conditions of induced angiogenesis is supported by the observations that cathepsins that can generate endostatin are produced by both endothelial cells and tumor cells, and that such proteases are activated by a moderately acidic pH similar to what is found in the pericellular milieu of tumors. The central protease-sensitive hinge region of the NC1 domain is sensitive to many different proteases ŽFerreras et al., 2000., e.g. cathepsins, MMPs or pancreatic serine elastase, which generate distinct endostatin containing fragments with varying efficiency. Incubation of human endostatin with these proteases showed that cathepsins L and D efficiently degraded endostatin, whereas MMPs showed no such activity. These data imply that not only the generation of endostatin, but also its degradation, is dependent on the activities of pericellular proteases, pointing to an additional mechanism for regulating local levels of endostatin. Some data indicate that endostatin is a protease inhibitor ŽKim et al., 2000b.. Activation of proMMP-2 is blocked by endostatin, and the catalytic activity of MMP-2 is inhibited. This is an interesting finding,
since MMPs are produced by endothelial cells and proteolytically degrade the perivascular extracellular matrix during sprouting angiogenesis. Thus, locally generated endostatin at sites of induced angiogenesis may directly inhibit MMP activity. In the original report describing the identification of endostatin ŽO’Reilly et al., 1997., the apoptosis-rate of tumor cells in endostatin treated tumor-bearing mice was seven-fold higher than in untreated mice. The authors concluded that a high apoptosis rate in endostatin treated tumors reflected the inhibition of angiogenesis. Subsequently, others demonstrated that endostatin can induce apoptosis in endothelial cells ŽDhanabal et al., 1999., in a process which seems to be dependent on tyrosine phosphorylation of the adaptor protein Shb ŽDixelius et al., 2000.. The crystal structure of mouse endostatin reveals a compact fold with a heparin-binding basic patch formed by 11 arginine residues ŽFig. 2A. ŽHohenester et al., 1998.. The heparin-binding ability of endostatin was reported to be required for the induction of apoptosis in cultured endothelial cells ŽDhanabal et al., 1999., and for its inhibition of FGF-2 Žbut not VEGF. induced angiogenesis in a chick chorioallantoic membrane ŽCAM. assay ŽSasaki et al., 1999.. The identification of glypicans, being cell surface HSPGs, as low-affinity endostatin receptors ŽKarumanchi et al., 2001. support a critical role of the heparin-binding site for endostatin actions, although heparin-binding does not appear to be important for inhibition of VEGF-induced endothelial cell migration ŽYamaguchi et al., 1999.. The intracellular signaling events in response to
Fig. 2. Ža. Crystal structure of murine endostatin. Žb. Crystal structure of the murine endostatin-like fragment of type XV collagen. The crystal structure of the endostatin-like collagen XV fragment shows a very similar overall fold compared to the crystal structure of endostatin, but contains no heparin-binding site. Structures were obtained from the protein data bank wPDB Ids: 1DY1 and 1DY2x.
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cell binding of endostatin are only partially understood. Endostatin’s anti-angiogenic activity is likely to involve intracellular signaling pathways that antagonize pro-angiogenic actions of VEGF or FGF-2, since no cross-competition for receptor binding between VEGF or FGF-2 and endostatin was observed ŽKarumanchi et al., 2001.. Some findings suggest that the effects of endostatin on endothelial cells in vitro are dependent on the cell culture conditions ŽShichiri and Hirata, 2001.. Apoptosis or growth inhibition of endostatin treated endothelial cells was observed under reduced serum conditions, under serum-supplemented conditions no apoptosis was detected. In experiments where migration was potently inhibited, endostatin was found to rapidly down-regulate the expression of a broad variety of genes.
4. Type XV collagen Type XV collagen is structurally homologous to type XVIII collagen; the two collagens form the multiplexin family of non-fibrillar collagens. The genomic structure of their genes reveals a considerable conservation in exon᎐intron organization, indicating a common ancestor. Type XV collagen contains several interruptions in the triple-helical sequence of the central collagenous domain, a large non-collagenous globular domain at the N-terminus and a smaller C-terminal non-collagenous domain ŽNC1. ŽMyers et al., 1992; Kivirikko et al., 1994; Oh et al., 1994a,b; Muragaki et al., 1994; Hagg ¨ et al., 1997a.. Type XVIII collagen is a heparan sulfate proteoglycan; in contrast, type XV collagen is a disulfide-bonded chondroitin sulfate proteoglycan ŽLi et al., 2000.. Type XV collagen shows a widespread tissue distribution, with high expression in heart, skeletal muscle and placenta ŽKivirikko et al., 1995.. This collagen is synthesized by fibroblasts, endothelial cells, myoblasts and some epithelial cells. It localizes to vascular, neuronal, mesenchymal and some epithelial basement membrane zones ŽMyers et al., 1996., but also to other locations, such as to the fibrillar collagen matrix of the papillary dermis. The basement membrane location is not ubiquitous, but in immunohistochemical studies strong positive staining was found particularly at basement membranes of capillaries and skeletal muscle cells ŽHagg ¨ et al., 1997b.. Consistent with this localization are the findings observed in Col15a1-deficient mice ŽEklund et al., 2001.. These mice, which are viable and reproduce normally, showed a mild but progressive degeneration of skeletal muscle cells, and a susceptibility to exercise-induced muscle damage. Additionally, the mice were reported to have abnormal microvessels in heart and skeletal muscle, whereas the development of the over-
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all vasculature appeared normal. These findings suggest that type XV collagen is required for the structural integrity of skeletal muscle cells and capillaries.
5. NC1 domain of type XV collagen Type XV collagen contains in its NC1 domain an endostatin-like region, showing approximately 60% sequence identity with the endostatin domain of type XVIII collagen. Based on the anti-angiogenic activity of endostatin, derived from collagen XVIII, such an activity was investigated also for fragments of the NC1 domain of type XV collagen. The endostatin-like region in collagen XV was produced as a recombinant protein and tested for anti-angiogenic activity ŽRamchandran et al., 1999.. It showed no anti-proliferative effects on several types of endothelial cells. A dose-dependent inhibition of migration, in response to FGF-2, was observed with endothelial cells, but not with non-endothelial cells. The authors conducted in vivo tumor assays using a renal cell carcinoma xenograft model with systemic administration of the recombinant human type XV collagen fragment. This treatment resulted in a reduced increase in tumor volume over time, but produced no tumor regression, and demonstrated a less potent anti-tumor effect than treatment with endostatin derived from type XVIII collagen. In contrast to the type XVIII collagen NC1 domain, the recombinant type XV collagen NC1 domain has no pro-migratory activity, as a complete lack of inhibition of HUVEC tube formation on Matrigel was observed ŽKuo et al., 2001.. In solid-phase assays, type XV collagen NC1 domain binds significantly weaker to nidogen-1, the laminin-1-nidogen-1 complex and perlecan, than type XVIII collagen NC1 domain ŽSasaki et al., 2000.. Similar weak binding affinities for these extracellular matrix proteins were shown for the endostatin-like fragment of type XV collagen as well. Further structural and functional characterization of the NC1 domain of type XV collagen demonstrated that it also contains a trimerization domain, a hinge region that is less sensitive to proteolysis than in collagen XVIII, and an endostatin-like domain at the C-terminus ŽSasaki et al., 2000.. The crystal structure of the endostatin-like collagen XV fragment shows a very similar overall fold compared to the crystal structure of endostatin, but contains no heparin-binding site ŽFig. 2B.. The in vitro and in vivo anti-angiogenic activities of the endostatin-like fragment of collagen XV have been demonstrated using recombinant protein. Immunoreactive endostatin-related C-terminal fragments of human collagen XV have been isolated from
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human blood filtrate, supporting the view that this proteolytic fragment or the processing of type XV collagen may have a physiological role ŽJohn et al., 1999..
type IV collagen and its inhibition with type IV collagen peptides has been demonstrated for several cells ŽAumailley and Timpl, 1986; Tsilibary et al., 1990.. The data indicate that type IV collagen has an important role in promoting the adhesion and motility of various cell types.
6. Type IV collagen Type IV collagen forms a network structure in basement membranes, providing a scaffold that incorporates several other basement membrane components and regulates the interaction with adhering cells. Six distinct genes encode for the six type IV collagen chains ␣1᎐␣6 ŽIV.. Their genomic location shows a pair-wise head-to-head arrangement with a bi-directional promoter, mapping to three different chromosomes Žreviewed by Olsen and Ninomiya, 1999; Kuhn, 1995.. The chains of this predominant collagenous component of basement membranes contain a cysteine-rich Ž7S. domain at the N-terminus, a central triple-helical collagenous domain and a C-terminal non-collagenous ŽNC1. domain. Type IV collagen molecules form a network structure through covalently cross-linked and laterally associated 7S domains, end-to-end interactions of their NC1 domains, and lateral associations of their central collagenous domain. The ␣1ŽIV. and ␣ 2ŽIV. chains are the predominant forms of this collagen in most basement membranes. The ␣ 3᎐␣6ŽIV. chains seem to have distinct functional properties, since they are found in specialized basement membranes. Of particular interest is the composition of the kidney glomerular basement membrane, in which ␣ 3᎐␣ 5ŽIV. chains replace ␣1ŽIV. and ␣ 2ŽIV. as development proceeds. The functional importance of these chains are underscored through mutations in the ␣ 3᎐␣ 5ŽIV. chains in Alport syndrome, resulting in structural changes of the glomerular basement membrane with the clinical manifestation of glomerulonephritis. The ␣6ŽIV. chain shows a distinct localization in epidermal basement membranes, in Bowman’s capsule and renal distal tubules, and around adipocytes and smooth muscle cells. Type IV collagen plays a role in the interaction of basement membranes with cells. This interaction can be either direct or mediated through laminin, which shows low affinity binding to type IV collagen. The affinity is increased through nidogen, which binds strongly to laminin, and contains binding sites for type IV collagen and cells as well. Additionally, type IV collagen is able to bind heparin and heparan sulfate proteoglycans. The w ␣1ŽIV.x2 ␣ 2ŽIV. trimers contain in their triple-helical domain a cell-binding site with the recognition site for the two integrin receptors ␣ 1  1 and ␣ 2  1 ŽVandenberg et al., 1991.. Cell binding to
7. NC1 domain of type IV collagen The NC1 domain of type IV collagen is thought to be essential for the oligomerization of the ␣-chains. Several experiments, however, have pointed to functional roles beyond the assembly of basement membrane networks. A peptide derived from the NC1 domain of the ␣1ŽIV. chain was shown to promote the adhesion of bovine aortic endothelial cells ŽTsilibary et al., 1990.. Recombinant ␣1ŽIV.NC1 domain inhibited VEGF-induced proliferation and migration of endothelial cells in vitro, and neovascularization in a Matrigel plug assay in mice ŽColorado et al., 2000.. Thus, recombinant NC1 domains of type IV collagen have been further investigated as inhibitors of angiogenesis and regulators of endothelial cell behavior. Kefalides and co-workers demonstrated that a peptide of the NC1 domain of ␣ 3ŽIV. Žresidue sequence 185᎐203. inhibited the activation of leucocytes ŽMonboisse et al., 1994.. This peptide also promoted cell adhesion of human melanoma cells, and inhibited their proliferation as well ŽHan et al., 1997.. A triplet sequence Ž ᎐SNS᎐ at residues 189᎐191., which is unique to the ␣ 3ŽIV. chain, was essential for this activity. The receptors for the ␣ 3ŽIV.185᎐203 peptide were identified as CD47rintegrin-associated protein and the integrin ␣ v  3 ŽShahan et al., 1999.. These proteins were shown to regulate tumor cell chem otaxis to type IV collagen and the ␣ 3ŽIV.185᎐203 peptide through a calcium-dependent mechanism ŽShahan et al., 2000.. Based on these studies, recombinant ␣ 3ŽIV. NC1 domain was assessed for potential anti-angiogenic activity. The ␣ 3ŽIV. NC1 domain inhibited the proliferation of capillary endothelial cells, induced endothelial cell apoptosis and reduced tumor growth in vivo ŽMaeshima et al., 2000.. These activities were localized to residues 54᎐132 of the ␣ 3ŽIV. NC1 domain ŽMaeshima et al., 2001.. Using specific function-blocking anti-integrin antibodies, it has been shown that the attachment of endothelial cells to immobilized recombinant ␣ 2ŽIV. NC1 is dependent largely on integrin ␣ v  3 , partially also on ␣ v  5 and ␣ 3  1. This NC1 domain does not contain a RGD site recognized by ␣ v  3 , suggesting a novel binding site ŽPetitclerc et al., 2000.. Endothelial cell adhesion to the ␣ 3ŽIV. and ␣6ŽIV. NC1 domains
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were shown to depend only on ␣ v  3 , but not on ␣ v  5 or ␣ 3 1 integrins. These findings suggest that distinct ␣ ŽIV. NC1 domains bind to endothelial cells via ␣ v and  1 integrins, independently of the ␣ 1  1 and ␣ 2  1 integrin binding sites in the central triple-helical region of type IV collagen. In previous studies, function-blocking antibodies directed to integrins that bind type IV collagen inhibited angiogenesis in vitro and in vivo ŽDavis and Camarillo, 1996.. Integrin ␣ 1-blocking antibodies inhibited cell attachment of microvascular endothelial cells to type IV collagen and laminin-1 ŽSenger et al., 1997.. Systemic administration of recombinant ␣ 2ŽIV., ␣ 3ŽIV. and ␣6ŽIV. NC1 domains potently inhibited VEGF- and FGF-2-induced angiogenesis ŽCAM assay. and tumor growth in vivo Žchick embryo tumor growth assay., but no inhibition was observed with ␣1ŽIV., ␣4ŽIV. and ␣ 5ŽIV. NC1 domains ŽPetitclerc et al., 2000.. In further in vitro studies with a recombinant ␣ 2ŽIV. NC1 domain, endothelial cell tube formation, migration and proliferation were inhibited in a dose-dependent manner and endothelial cell apoptosis was induced ŽKamphaus et al., 2000.. Sensitivity of endothelial cells to apoptotic signals was dependent on growth factor stimulation, indicating that ␣ 2ŽIV. NC1 domain selectively targets angiogenic and not pre-formed quiescent endothelium. This recombinant peptide inhibited the growth of human prostate adenocarcinoma cells in SCID or athymic Žnurnu. mice, without inhibiting the proliferation of tumor cells in vitro. Notably, soluble NC1 domain and derivatives have been shown to block type IV collagen matrix assembly by binding along the length of the central region of type IV collagen, and thereby disrupting lateral associations of individual type IV collagen molecules ŽTsilibary and Charonis, 1986; Tsilibary et al., 1988, 1990.. Alternatively, soluble NC1 domains might disrupt the association of triple helical type IV collagen molecules at the C-terminus. In conclusion, it is possible that the anti-angiogenic activity of the recombinant ␣ ŽIV. NC1 domains may not only result through interfering with endothelial cell integrin signaling, but in part be the result of a disruption of type IV collagen matrix assembly. Additionally, an inhibition of the activity of proteases, such as MMP-2 and MMP-3, has been implicated ŽNetzer et al., 1998., similar to the MMP inhibitory activity of endostatin.
8. Conclusions The identified anti-angiogenic fragments of type IV, XV and XVIII collagens are derived from their NC1 domains, underscoring a role of these domains
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as regulators of cell migration and proliferation. Since the pro-migratory activity of the type XVIII collagen NC1 domain on endothelial and non-endothelial cells was strictly dependent on extracellular matrix, it is tempting to speculate that this pro-migratory activity results partially from the high-affinity binding of this domain to extracellular matrix components implicated in cell adhesion and migration, such as laminin-1 or nidogen-1, thus interfering with cell adhesion to the basement membrane. Such pro-migratory activity is lacking for the NC1 domain of type XV collagen, which binds significantly weaker to the laminin-1nidogen-1 complex in comparison to the NC1 domain of type XVIII collagen. Monomeric endostatin, which shows weak binding to laminin-1-nidogen-1 complex, inhibited the migration of endothelial cells, but not of non-endothelial cells ŽKuo et al., 2001; Shichiri and Hirata, 2001.. Thus, its anti-migratory activity might be mediated by endothelial cell-specific molecules and pathways that are different from those that are mediating the pro-migratory activity of type XVIII collagen NC1 domain on endothelial and non-endothelial cells. Endostatin binds with low affinity to endothelial cells through ␣ v- and ␣ 5-integrins and glypicans, as well as to an as yet unidentified high-affinity receptor. Type IV collagen NC1 domains have also been shown to bind to ␣ v-integrins, which mediate interactions of proliferating endothelial cells with the extracellular matrix. Thus, the different collagen-derived proteolytic fragments described here might regulate cellextracellular matrix interactions via closely related mechanisms, and affect angiogenesis in similar ways. References Ackley, B.D., Crew, J.R., Elamaa, H., Pihlajaniemi, T., Kuo, C.J., Kramer, J.M., 2001. The NC1rendostatin domain of Caenorhabditis elegans type XVIII collagen affects cell migration and axon guidance. J. Cell Biol. 152, 1219᎐1232. Aumailley, M., Timpl, R., 1986. Attachment of cells to basement membrane collagen type IV. J. Cell Biol. 103, 1569᎐1575. Brooks, P.C., Montgomery, A.M., Rosenfeld, M. et al., 1994. Integrin alpha V beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 79, 1157᎐1164. Carmeliet, P., 2000. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. 6, 389᎐395. Carmeliet, P., Ferreira, V., Breier, G. et al., 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380, 435᎐439. Chakravarti, S., Tam, M.F., Chung, A.E., 1990. The basement membrane glycoprotein entactin promotes cell attachment and binds calcium ions. J. Biol. Chem. 265, 10597᎐10603. Colorado, P.C., Torre, A., Kamphaus, G. et al., 2000. Anti-angiogenic cues from vascular basement membrane collagen. Cancer Res. 60, 2520᎐2526. Davis, G.E., Camarillo, C.W., 1996. An alpha2 beta1 integrin-dependent pinocytic mechanism involving intracellular vacuole formation and coalescence regulates capillary lumen and tube
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