The role of collagen-derived proteolytic fragments in angiogenesis.

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 (NC1) 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 XVIII/NC1 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.     

Endostatins derived from collagens XV and XVIII differ in structural and binding properties, tissue distribution and anti-angiogenic activity

Endostatin is a fragment of the C-terminal domain NC1 of collagen XVIII that inhibits angiogenesis and tumor growth. We report the characterization of a collagen XV endostatin analogue and its parent NC1 domain, obtained by recombinant expression in mammalian cells. Both NC1 domains contain a trimerization domain, a hinge region that is more sensitive to proteolysis in collagen XVIII and the endostatin domain. Unlike endostatin-XVIII, endostatin-XV does not bind zinc or heparin, which is explained by the crystal structure of endostatin-XV. The collagen XV and XVIII fragments inhibited chorioallantoic membrane angiogenesis induced by basic fibroblast growth factor (FGF-2) or vascular endothelial growth factor (VEGF), but there are striking differences depending on which cytokine is used and whether free endostatins or NC1 domains are applied. The collagen XV and XVIII fragments showed a similar binding repertoire for extracellular matrix proteins. Differences were found in the immunohistological localization in vessel walls and basement membrane zones. Together, these data indentify endostatin-XV as an angiogenesis inhibitor, which differs from endostatin-XVIII in several important functional details.     

Collagen XVIII/endostatin structure and functional role in angiogenesis

The angiogenesis inhibitor endostatin is a 20 kDA C-terminal fragment of collagen XVIII, a proteoglycan/collagen found in vessel walls and basement membranes. The endostatin fragment was originally identified in conditioned media from a murine endothelial tumor cell line. Endostatin inhibits endothelial cell migration in vitro and appears to be highly effective in murine in vivo studies. The molecular mechanisms behind the inhibition of angiogenesis have not yet been elucidated. Studies of the crystal structure of endostatin have shown a compact globular fold, with one face particularly rich in arginine residues acting as a heparin-binding epitope. It was initially suggested that zinc binding was essential for the antiangiogenic mechanism but later studies indicate that zinc has a structural rather than a functional role in endostatin. The generation of endostatin or endostatin-like collagen XVIII fragments is catalyzed by proteolytic enzymes, including cathepsin L and matrix metalloproteases, that cleave peptide bonds within the protease-sensitive hinge region of the C-terminal domain. The processing of collagen XVIII to endostatin may represent a local control mechanism for the regulation of angiogenesis.     

Endostatin inhibits human tongue carcinoma cell invasion and intravasation and blocks the activation of matrix metalloprotease-2, -9, and -13

Endostatin, a 20-kDa collagen XVIII fragment, inhibits angiogenesis and tumor growth in vivo, but the mechanisms are still unclear. Matrix metalloproteases (MMPs), a family of extracellular and membrane-associated endopeptidases, collectively digest almost all extracellular matrix and basement membrane components, and thus play an important role in tumor progression. We studied the effects of recombinant human endostatin on human MMP-2, -9, -8, and -13. We found that endostatin inhibited the activation and catalytic activity of pro-MMP-9 and -13 as well as recombinant pro-MMP-2. It prevented the fragmentation of pro-MMP-2 that was associated with reduction of catalytic activity. Endostatin had no effect on MMP-8 as shown by collagenase activity assays. An in vitro migration assay and an in vivo chicken chorioallantoic membrane intravasation assay with the human tongue squamous cell carcinoma cell line HSC-3 revealed the biphasic nature of endostatin; low endostatin concentrations inhibited intravasation and migration of these cells in a dose-dependent manner, but at increased concentrations, the inhibitory effect was far less efficient. The results show that endostatin blocks the activation and activities of certain tumor-associated pro-MMPs, such as pro-MMP-2, -9, and -13, which may explain, at least in part, the antitumor effect of endostatin. Our results also suggest that endostatin inhibits tumor progression by directly affecting the tumor cells and not just acting via endothelial cells and blockage of angiogenesis.     

Cloning, expression, and in vitro activity of human endostatin.

Endostatin, a 20 kDa C-terminal fragment of collagen XVIII, is a specific inhibitor of endothelial cell proliferation and angiogenesis. In the present study, we have expressed human endostatin in a yeast expression system (10 mg/L). The recombinant protein was expressed in a soluble form and purified to homogeneity. It specifically inhibited the proliferation and migration of endothelial cells. In addition, we report for the first time that endostatin caused G1 arrest of endothelial cells. Also, we show that endostatin treatment resulted in apoptosis of HUVE and HMVE cells and that all of these effects do not occur in nonendothelial cells. Collectively, these findings demonstrate the expression of a biologically active form of human endostatin in yeast and provide important mechanistic insight into endostatin action on endothelial cells.     

Cleavage of the matricellular protein SPARC by matrix metalloproteinase 3 produces polypeptides that influence angiogenesis

SPARC, a matricellular protein that affects cellular adhesion and proliferation, is produced in remodeling tissue and in pathologies involving fibrosis and angiogenesis. In this study we have asked whether peptides generated from cleavage of SPARC in the extracellular milieu can regulate angiogenesis. Matrix metalloproteinase (MMP)-3, but not MMP-1 or 9, showed significant activity toward SPARC. Limited digestion of recombinant human (rhu)SPARC with purified catalytic domain of rhuMMP-3 produced three major fragments, which were sequenced after purification by HPLC. Three synthetic peptides (Z-1, Z-2, and Z-3) representing motifs from each fragment were tested in distinct assays of angiogenesis. Peptide Z-1 (3.9 kDa, containing a Cu2+-binding sequence KHGK) exhibited a biphasic effect on [3H]thymidine incorporation by cultured endothelial cells and stimulated vascular growth in the chick chorioallantoic membrane (CAM). In contrast, peptides Z-2 (6.1 kDa, containing Ca2+-binding EF hand-1) and Z-3 (2.2 kDa, containing neither Cu2+-binding motifs nor EF hands), inhibited cell proliferation in a concentration-dependent manner and exhibited no effects on vessel growth in the CAM. Reciprocal results were obtained in a migration assay in native collagen gels: peptide Z-1 was ineffective over a range of concentrations, whereas Z-2 or Z-3 stimulated cell migration. Therefore, proteolysis of SPARC by MMP-3 produced peptides that regulate endothelial cell proliferation and/or migration in vitro in a mutually exclusive manner. One of these peptides containing KHGK also demonstrated a concentration-dependent effect on angiogenesis.     

人内皮抑素在毕赤酵母中的表达、纯化与生物功能研究

内皮抑素（Endostatin）是近年来新发现的一种内源性新生血管生成（Angiogenesis）抑制因子,通过抑制新血管生成而抑制肿瘤的形成和转移且不会引起耐药性,具有极高的临床应用前景。巴斯德毕赤酵母（Pichia pastoris)具有表达率高、产物可分泌、可对高等真核生物蛋白正确进行翻译后加工、遗传稳定、发酵工艺成熟等优点被用来进行重组人Endostatin的表达。本研究用PCR的方法从人胎肝cDNA文库中扩增出人Endostatin的cDNA,测序正确后转入毕赤巴斯德甲醇酵母,并获得了高效可溶型表达,用肝素亲和层析的方法进行纯化,纯化后产物经SDSPAGE薄层扫描分析纯度达987％以上,质谱测定分子量为2043kD与理论值一致,蛋白质N端序列测定结果为SPPAHTHRDFQPVLH与天然序列一致。生物活性检测证明可抑制鸡胚尿囊绒毛膜（CAM）的新生血管生成（Angiogenesis),并可抑制血管内皮细胞的增殖。因此用酵母表达系统可以得到具有生物活性的内皮抑素,经纯化后可用于进一步的生物功能和作用机理试验。     

Functional characterization of neostatins, the MMP-derived, enzymatic cleavage products of type XVIII collagen

Several anti-angiogenic factors are derived from proteolytic processing of large molecules including endostatin from type XVIII collagen and angiostatin from plasminogen. In previous studies we showed that neostatin-7, the C-terminal 28kDa endostatin-spanning proteolytic fragment, is generated from the proteolytic action of matrix metalloproteinase matrilysin (MMP)-7 on type XVIII collagen. Now, we report a second member of the neostatin family of proteins, neostatin-14. Given the small quantities of neostatin-7 and -14 generated by the breakdown of naturally occurring collagen XVIII (using MMP-7 and -14, respectively), we used two other approaches to characterize the anti-angiogenic properties of these molecules: murine recombinant neostatin in vitro, and gene therapy. We demonstrate that murine recombinant neostatin-7 inhibits calf pulmonary artery endothelial cell proliferation and that microinjection of neostatin-7 and neostatin-14 naked DNA into the corneal stroma of mice results in significant reduction of basic fibroblast growth factor-induced corneal neovascularization. These results provide supportive evidence of the possible anti-angiogenic effect of neostatins.     

Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin

Based on a homology search with endostatin, the C-terminus 185 aa of collagen XVIII, we report the cloning, expression, and antiangiogenic activity of a 22 kDa human collagen XV fragment, that we have named restin. Restin was expressed in the prokaryotic pET expression system. We have shown that restin inhibits the migration of endothelial cells in vitro but has no effect on the proliferation of these cells. A polyclonal antibody raised against endostatin cross-reacted with restin. Systemic administration of restin suppressed the growth of tumors in a xenograft renal carcinoma model.     

NC1 domain of human type VIII collagen (alpha 1) inhibits bovine aortic endothelial cell proliferation and causes cell apoptosis.

Endostatin, a natural angiogenesis inhibitor, had been identified for years. It opened a new approach for cancer therapy. Sequence analysis revealed that endostatin is the NC1 domain (non-triple-helical domain) of collagen XVIII. In this report, the cDNA of NC1 domain of type VIII collagen (alpha 1) was cloned and expressed as soluble form in Escherichia coli. The recombinant protein was purified with Ni-NTA agarose column and named as vastatin. It inhibited the proliferation of bovine aortic endothelial (BAE) cell stimulated by basic fibroblast growth factor (bFGF) in a dose-dependent manner. The ED(50) of vastatin was 0.6 microg/ml, while the ED(50) of endostatin was 0.5 microg/ml. Treatment of BAE cell with vastatin caused G(0)-G(1) arrest and cell apoptosis. It is interesting that sequence analysis showed that there was only about 12% amino acid sequence homology between vastatin and endostatin. The structure-function relationship of these angiogenesis molecules remains to be elucidated.     

Isolation and characterization of the circulating form of human endostatin

Recently, fragments of extracellular proteins, including endostatin, were defined as a novel group of angiogenesis inhibitors. In this study, human plasma equivalent hemofiltrate was used as a source for the purification of high molecular weight peptides (10–20 kDa), and the isolation and identification of circulating human endostatin are described. The purification of this C-terminal fragment of collagen α1(XVIII) was guided by MALDI-MS and the exact molecular mass determined by ESI-MS was found to be 18 494 Da. N-terminal sequencing revealed the identity of this putative angiogenesis inhibitor and its close relation to mouse endostatin. The cysteine residues 1–3 and 2–4 in the molecule are linked by disulfide bridges. In vitro biological characterization of the native protein demonstrated no anti-proliferative activity on different endothelial cell types. These data indicate that human endostatin, which is a putative angiogenesis inhibitor, is present in the circulation.     

Sequence and embryonic expression of collagen XVIII NC1 domain (endostatin) in the zebrafish

Endostatin, located in the NC1 domain of the collagen XVIII, is believed to inhibit the migration and proliferation of endothelial cells (Fed. Am. Soc. Exp. Biol. J. 15 (2001) 1044) and to play a role in axon guidance in Caenorhabditis elegans (J. Cell Biol. 152 (2001) 1219). Zebrafish is an attractive vertebrate model to determine the role of endostatin and the entire molecule of collagen XVIII during vertebrate development. Therefore, we have investigated the expression pattern of COL18A1 in zebrafish embryos from the segmentation to the hatching period stages.     

Endostatin inhibits adhesion of endothelial cells to collagen I via alpha(2)beta(1) integrin, a possible cause of prevention of chondrosarcoma growth

Endostatin derived from collagen XVIII is a potent endogenous anti-angiogenic factor that induces regression of various tumors of epithelial origin. Endostatin has been shown to inhibit endothelial cell functions, however, its effect remains controversial. We first attempted here to apply the inhibitory effect of recombinant human endostatin on chondrosarcomas, which originate from the mesenchyme, in nude mice. Endostatin induced reduction of chondrosarcoma growth and tumor angiogenesis in vivo. However, endostatin showed no effect on the proliferation and migration of chondrosarcoma cells in vitro. Next, we investigated the interactions between endostatin and endothelial cells in detail. Endostatin inhibited the migration on and attachment to collagen I but did not affect the proliferation of endothelial cells. Although the migration of endothelial cells was stimulated by angiogenic factors such as basic fibroblast growth factor and vascular endothelial growth factor, endostatin showed similar inhibitory effects on it in the presence and absence of the stimulants. Moreover, the inhibitory effect against endothelial cell attachment to collagen I was attenuated or modulated in the presence of neutralizing antibodies of alpha(2), alpha(5)beta(1), and alpha(V)beta(3) integrins but not that of alpha(1) integrin. Our results suggest that endostatin might suppress the alpha(2)beta(1) integrin function of endothelial cells via alpha(5)beta(1) or alpha(V)beta(3) integrin. We propose here that endostatin might be effective for anti-angiogenic therapy for human chondrosarcomas through the suppression of alpha(2)beta(1) integrin functions in endothelial cells.     

Structure, function and tissue forms of the C-terminal globular domain of collagen XVIII containing the angiogenesis inhibitor endostatin.

The C-terminal domain NC1 of mouse collagen XVIII (38 kDa) and the shorter mouse and human endostatins (22 kDa) were prepared in recombinant form from transfected mammalian cells. The NC1 domain aggregated non-covalently into a globular trimer which was partially cleaved by endogenous proteolysis into several monomers (25-32 kDa) related to endostatin. Endostatins were obtained in a highly soluble, monomeric form and showed a single N-terminal sequence which, together with other data, indicated a compact folding. Endostatins and NC1 showed a comparable binding activity for the microfibrillar fibulin-1 and fibulin-2, and for heparin. Domain NC1, however, was a distinctly stronger ligand than endostatin for sulfatides and the basement membrane proteins laminin-1 and perlecan. Immunological assays demonstrated endostatin epitopes on several tissue components (22-38 kDa) and in serum (120-300 ng/ml), the latter representing the smaller variants. The data indicated that the NC1 domain consists of an N-terminal association region (approximately 50 residues), a central protease-sensitive hinge region (approximately 70 residues) and a C-terminal stable endostatin domain (approximately 180 residues). They also demonstrated that proteolytic release of endostatin can occur through several pathways, which may lead to a switch from a matrix-associated to a more soluble endocrine form.     

Crystal structure of the angiogenesis inhibitor endostatin at 1.5 A resolution.

A number of extracellular proteins contain cryptic inhibitors of angiogenesis. Endostatin is a 20 kDa C-terminal proteolytic fragment of collagen XVIII that potently inhibits endothelial cell proliferation and angiogenesis. Therapy of experimental cancer with endostatin leads to tumour dormancy and does not induce resistance. We have expressed recombinant mouse endostatin and determined its crystal structure at 1.5 A resolution. The structure reveals a compact fold distantly related to the C-type lectin carbohydrate recognition domain and the hyaluronan-binding Link module. The high affinity of endostatin for heparin is explained by the presence of an extensive basic patch formed by 11 arginine residues. Endostatin may inhibit angiogenesis by binding to the heparan sulphate proteoglycans involved in growth factor signalling.     

The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.     

Cysteine cathepsins S and L modulate anti-angiogenic activities of human endostatin

Human endostatin, a potent anti-angiogenic protein, is generated by release of the C terminus of collagen XVIII. Here, we propose that cysteine cathepsins are involved in both the liberation and activation of bioactive endostatin fragments, thus regulating their anti-angiogenic properties. Cathepsins B, S, and L efficiently cleaved in vitro FRET peptides that encompass the hinge region corresponding to the N terminus of endostatin. However, in human umbilical vein endothelial cell-based assays, silencing of cathepsins S and L, but not cathepsin B, impaired the generation of the ～22-kDa endostatin species. Moreover, cathepsins L and S released two peptides from endostatin with increased angiostatic properties and both encompassing the NGR sequence, a vasculature homing motif. The G10T peptide (residues 1455-1464: collagen XVIII numbering) displayed compelling anti-proliferative (EC(50) = 0.23 nm) and proapoptotic properties. G10T inhibited aminopeptidase N (APN/CD13) and reduced tube formation of endothelial cells in a manner similar to bestatin. Combination of G10T with bestatin resulted in no further increase in anti-angiogenic activity. Taken together, these data suggest that endostatin-derived peptides may represent novel molecular links between cathepsins and APN/CD13 in the regulation of angiogenesis.     

Novel glycosylated forms of human plasma endostatin and circulating endostatin-related fragments of collagen XV.

Circulating elongated forms of the angiogenesis inhibitor and potential anti-cancer drug endostatin were isolated from human blood filtrate. Immunoreactive endostatin was identified by a polyclonal rabbit antiserum raised against an N-terminal epitope of the polypeptide and purified by consecutive chromatographic steps and immunoblotting. N- and C-terminal sequence analyses of the isolated molecules revealed different forms of endostatin starting with V(117)HLRPAR. lacking the last and final three residues of the noncollagenous domain 1 (NC-1) of collagen XVIII, respectively. These polypetides are found to be O-glycosylated at T(125) (residue 9) with a glycan structure of the mucin type consisting of galactose N-acetylgalactosamine and N-acetylneuraminic acid residues. Carbohydrate analyses were performed via the semiquantitative HPLC-electrospray ionization mass spectrometry (ESMS) technique after exoglycosidase hydrolysis. Circulating endostatins are present as sialoglycoprotein (22 000 and 21 841 Da +/- 0.02%) and asialoglycoprotein structures (21 710 and 21 549 Da +/- 0.02%), while the two completely deglycosylated forms are obtained only after enzymatic incubation. The described glycosylated endostatins may represent intermediates in the proteolytic pathway of the NC-1 domain of collagen XVIII resulting in bioactive endostatins. Furthermore, immunoreactive endostatin-related C-terminal fragments of human collagen XV are found in the hemofiltrate. These polypeptides exhibit the N-terminal sequences P(66)HLLPPP. and Y(81)EKPALH. of the collagen XV NC-1 domain. ESMS and immunoblotting analyses reveal three glycosylated polypeptides with a molecular mass ranging from 16 to 21 kDa. Due to the high degree of homology between collagen XV and collagen XVIII as well as their analoqous proteolytic processing, functional similarities of collagen XVIII- and XV-related fragments should be revealed in future experiments.     

Hypoxia-induced increase of endostatin in murine aorta and lung

In the lung, hypoxia induces pulmonary hypertension caused by vasoconstriction and vascular remodeling. Additionally, hypoxia is an inducer of angiogenesis, which is assumed to counteract pulmonary hypertension. We asked whether the anti-angiogenic factor endostatin—a cleavage product of collagen XVIII—participates in the vascular alterations induced by hypoxia. By employing Western blotting of tissue extracts of murine brain, liver and heart an endostatin fragment of 22 kDa was detectable, whereas in lung and aorta additional bands of 24 and 26 kDa were found. The amount of these larger fragments was increased in tissues obtained from mice housed for 4 days or 3 weeks at hypobaric hypoxia. By immunohistochemistry endostatin was detected in association with elastic fibers and in close neighborhood to smooth muscle cells of intrapulmonary vessels and the aorta. In the lung, the activity of matrix metalloproteinases (MMP) known to generate endostatin by cleavage of collagen XVIII was increased (MMP-2) and decreased (proMMP-9), respectively, by hypoxia. Elevated amounts of endostatin within the aortic wall of mice exposed to hypobaric hypoxia may stabilize the vascular wall by inhibition of microvascular sprouting. The surprising finding of increased endostatin in the lung presumably contributes to the development of pulmonary hypertension by reduction of angiogenesis.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

人内皮抑素基因的克隆与序列分析

目的 :克隆与鉴定人内皮抑素基因功能区片段。方法 :采用反转录聚合酶链反应 (RT PCR)的方法 ,从人胎肝总RNA中扩增人内皮抑素基因 ,将其克隆入 pGEM T载体 ,命名为pT hES ,并应用A377自动序列分析仪进行序列分析。结果 :成功克隆了人内皮抑素基因 ,经序列分析表明 ,所克隆的基因序列正确。这为利用基因工程技术生产重组蛋白奠定了基础。