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.     

Cathepsin B regulates the intrinsic angiogenic threshold of endothelial cells

The lysosomal protease cathepsin B has been implicated in a variety of pathologies including pancreatitis, tumor angiogenesis, and neuronal diseases. We used a tube formation assay to investigate the role of cathepsin B in angiogenesis. When cultured between two layers of collagen I, primary endothelial cells formed tubes in response to exogenously added VEGF. Overexpressing cathepsin B reduced the VEGF-dependent tube response, whereas pharmacologically or molecularly suppressing cathepsin B eliminated the dependence on exogenous VEGF. However, tube formation still required VEGF receptor activity, which suggested that endothelial cells generated VEGF. Indeed, VEGF mRNA and protein was detectable in cells treated with cathepsin B inhibitor, which correlated with a rise in the level of HIF-1alpha. In addition to boosting the level of proangiogenic factors, blocking cathepsin B activity reduced the amount of the antiangiogenic protein endostatin. Thus endothelial cells have the intrinsic capacity to generate pro- and antiangiogenic agents. These observations complement and expand our appreciation of how endothelial cell-derived proteases regulate angiogenesis.     

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.     

Generation of biologically active endostatin fragments from human collagen XVIII by distinct matrix metalloproteases

Endostatin, a potent inhibitor of endothelial cell proliferation, migration, angiogenesis and tumor growth, is proteolytically cleaved from the C-terminal noncollagenous NC1 domain of type XVIII collagen. We investigated the endostatin formation from human collagen XVIII by several MMPs in vitro. The generation of endostatin fragments differing in molecular size (24-30 kDa) and in N-terminal sequences was identified in the cases of MMP-3, -7, -9, -13 and -20. The cleavage sites were located in the protease-sensitive hinge region between the trimerization and endostatin domains of NC1. MMP-1, -2, -8 and -12 did not show any significant activity against the C-terminus of collagen XVIII. The anti-proliferative effect of the 20-kDa endostatin, three longer endostatin-containing fragments generated in vitro by distinct MMPs and the entire NC1 domain, on bFGF-stimulated human umbilical vein endothelial cells was established. The anti-migratory potential of some of these fragments was also studied. In addition, production of endostatin fragments between 24-30 kDa by human hepatoblastoma cells was shown to be due to MMP action on type XVIII collagen. Our results indicate that certain, especially cancer-related, MMP family members can generate biologically active endostatin-containing polypeptides from collagen XVIII and thus, by releasing endostatin fragments, may participate in the inhibition of endothelial cell proliferation, migration and angiogenesis.     

Amyloid endostatin induces endothelial cell detachment by stimulation of the plasminogen activation system

Endostatin is a fragment of collagen XVIII that acts as an inhibitor of tumor angiogenesis and tumor growth. Anti-tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study, we have investigated the cellular effects of insoluble endostatin. We previously found that insoluble endostatin shows all the hallmarks of amyloid aggregates and potently stimulates tissue plasminogen activator-mediated formation of the serine protease plasmin. We here show that amyloid endostatin induces plasminogen activation by endothelial cells, resulting in vitronectin degradation and plasmin-dependent endothelial cell detachment. Endostatin-mediated stimulation of plasminogen activation, vitronectin degradation, and endothelial cell detachment is inhibited by carboxypeptidase B, indicating an essential role for carboxyl-terminal lysines. Our results suggest that amyloid endostatin may inhibit angiogenesis and tumor growth by stimulating the fibrinolytic system.     

Inhibitory effect of full-length human endostatin on in vitro angiogenesis.

Endostatin, a C-terminal product of collagen XVIII, is a very powerful angiogenesis inhibitor. In vivo experiments in mice indicate that endostatin dramatically reduces tumor mass without causing the onset of any resistance to the treatment. Recently, a 12-aa shorter human endostatin has been purified from plasma, but is ineffective in in vitro angiogenesis assays. Here we report that the full-length human recombinant endostatin has a potent inhibitory activity in in vitro angiogenesis assays. Two powerful angiogenic factors were used to stimulate endothelial cells: FGF-2 and VEGF-165. Endostatin prevented cell growth both in the basal condition and after stimulation with FGF-2 or VEGF-165. Migration of microvascular endothelial cells toward FGF-2 or VEGF-165 was impaired, both when cells were pretreated with the inhibitor and when endostatin was added together with the growth factors. Furthermore, experiments of inhibition of proliferation performed on nonmicroendothelial cells showed that endostatin was ineffective. This study indicates that human endostatin is a potent angiogenesis inhibitor and suggests its use in human anticancer therapy.     

Cathepsin S controls angiogenesis and tumor growth via matrix-derived angiogenic factors

The cysteine protease cathepsin S is highly expressed in malignant tissues. By using a mouse model of multistage murine pancreatic islet cell carcinogenesis in which cysteine cathepsin activity has been functionally implicated, we demonstrated that selective cathepsin S deficiency impaired angiogenesis and tumor cell proliferation, thereby impairing angiogenic islet formation and the growth of solid tumors, whereas the absence of its endogenous inhibitor cystatin C resulted in opposite phenotypes. Although mitogenic vascular endothelial growth factor, transforming growth factor-beta1, and the anti-angiogenic endostatin levels in either serum or carcinoma tissue extracts did not change in cathepsin S- or cystatin C-null mice, tumor tissue basic fibroblast growth factor and serum type 1 insulin growth factor levels were higher in cystatin C-null mice, and serum type 1 insulin growth factor levels were also increased in cathepsin S-null mice. Furthermore, cathepsin S affected the production of type IV collagen-derived anti-angiogenic peptides and the generation of bioactive pro-angiogenic gamma2 fragments from laminin-5, revealing a functional role for cathepsin S in angiogenesis and neoplastic progression.     

Efficient secretion of human endostatin in the yeast, Pichia pastoris

Endostatin is a 20 kDa COOH-terminal fragment of collagen XVIII that inhibits angiogenesis and tumor growth. The cDNA coding for human endostatin in human fetal liver has been cloned into the secreting expression organism Pichia pastoris, and the high level expression of human endostatin has been achieved (about 200 mg of endostatin in 1 l of culture). The recombinant human endostatin was purified to homogeneity by heparin-affinity column, and showed antiproliferative effect on rat brain micro-vascular endothelia cells.     

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.     

Endostatin's heparan sulfate-binding site is essential for inhibition of angiogenesis and enhances in situ binding to capillary-like structures in bone explants

The functional role of endostatin's affinity for heparan sulfates was addressed using an ex vivo bone angiogenesis model. Capillary-like sprouts showed prominent expression of collagen XVIII/endostatin. Outgrowth of endothelial cells was not altered in the absence of collagen XVIII but inhibited by the addition of recombinant endostatin. Mutant non-heparan sulfate binding endostatin and the collagen XV endostatin homologue were ineffective. The ability of mutant endostatin to bind to capillary structures was reduced when compared to endostatin. Endostatin-XV completely failed to bind to endothelial cells. Our data indicate that endostatin's angiostatic function is heparan sulfate-dependent, and that in situ-binding of endostatin to endothelial cells is increased by heparan sulfates.     

内皮抑素与肿瘤治疗

内皮抑素(endostatin)是近年来发现的,天然产生的新血管生成抑制因子。它在体内由胶原ⅩⅧ经酶解产生,可以抑制新血管生成,在肿瘤动物模型中显示出显著的抑瘤活动。通过抑制肿瘤相关的新血管的形成来治疗肿瘤,是目前出现的新疗法,内皮抑素是这种疗法中很有前景的侯选药物之一。在美国已经开始了用内皮抑素治疗肿瘤的Ⅰ期临床试验,关于它的临床前基础实验也在广泛开展。本文综述了内皮抑素在肿瘤治疗中的应用基础研究 。     

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.     

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.     

The collagen type XVIII endostatin domain is co-localized with perlecan in basement membranes in vivo.

The C-terminal globular endostatin domain of collagen type XVIII is anti-angiogenic in a variety of experimental tumor models, and clinical trials to test it as an anti-tumor agent are already under way. In contrast, many of its cell biological properties are still unknown. We systematically localized the mRNA of collagen type XVIII with the help of in situ hybridization (ISH) and detected it in epithelial and mesenchymal cells of almost all organ systems throughout mouse development. Light and electron microscopic immunohistochemistry (IHC) revealed that the endostatin domain is a widespread component of almost all epithelial basement membranes in all major developing organs, and in all basement membranes of capillaries and blood vessels. Furthermore, quantitative immunogold double labeling demonstrated a co-localization of 50% of the detected endostatin domain together with perlecan in basement membranes in vivo. We conclude that the endostatin domain of collagen type XVIII plays a role, even in early stages of mouse development, other than regulating angiogenesis. In the adult, the endostatin domain could well be involved in connecting collagen type XVIII to the basement membrane scaffolds. At least in part, perlecan appears to be an adaptor molecule for the endostatin domain in basement membranes in vivo.     

A key angiogenic role of monocyte chemoattractant protein-1 in hemangioendothelioma proliferation

Angiomatous lesions are common in infants and children. Hemangioendotheliomas (HE) represent one type of these lesions. Endothelial cell proliferation and the development of vascular/blood cell-filled spaces are inherent in the growth of HE. Therefore, understanding mechanisms that regulate the proliferation of these lesions should provide key insight into mechanisms regulating angiogenesis. A murine model was used to test the significance of monocyte chemoattractant protein (MCP)-1 in HE proliferation. EOMA cells, a cell line derived from a spontaneously arising murine HE, generate these lesions with 100% efficiency when injected subcutaneously into syngeneic mice. MCP-1 produced by EOMA cells recruit macrophages, which were shown to induce angiogenic behavior in EOMA cells by stimulating transwell migration and inducing sprout formation on type I collagen gels. When EOMA cells were injected into MCP-1^–/– mice, only 50% of the mice developed tumors, presumably because the low levels of MCP-1 expressed by the injected EOMA cells were enough to overcome any host deficits of this chemokine. When EOMA cells were coinjected with a neutralizing antibody to MCP-1, tumors failed to develop in any of the treated mice, including syngeneic 129P3, C57Bl/6 (wild type), and MCP-1^–/–. These results present the first evidence that MCP-1 is required for HE proliferation and may promote the growth of these lesions by stimulating angiogenic behavior of endothelial cells. This study has produced the first in vivo evidence of a complete response for any neoplasm, specifically a vascular proliferative lesion, to anti-MCP-1 therapy in animals with intact immune systems. endothelium; vascular; macrophage; redox; angiogenesis     

Protein kinase B inhibits endostatin-induced apoptosis in HUVECs

Endostatin is a tumor-derived angiogenesis inhibitor, and the endogenous 20 kDa carboxyl-terminal fragment of collagen XVIII. In addition to inhibiting angiogenesis,endostatin inhibits tumor growth and the induction of apoptosis in several endothelial cell types. However, the mechanisms that regulate endostatin-induced apoptotic cell death are unclear. Here, we investigated apoptotic cell death and the underlying regulatory mechanisms elicited of endostatin in human umbilical vein endothelial cells (HUVECs). Endostatin was found to induce typical apoptotic features, such as, chromatin condensation and DNA fragmentation in these cells. Thus, as the phosphoinositide 3-OH kinase (PI3K)/protein kinase B (PKB) signaling pathway has been shown to prevent apoptosis in various cell types, we investigated whether this pathway could protect cells against endostatin induced apoptosis. It was found that the inhibition of PI3K/PKB significantly increased endostatin-induced apoptosis, and that endostatininduced cell death is physiologically linked to PKB-mediated cell survival through caspase-8.     

Knockdown of endothelial NOS by lentivirus-mediated short hairpin RNA in hemangioendothelioma cells increases proliferation and tumor formation

Endothelial nitric oxide synthase (ecNOS) derived nitric oxide (NO) is a key contributor to the angiogenic process. By augmenting angiogenesis NO could potentially promote tumor progression. The object of this study was to determine how knockdown of ecNOS affects endothelial NO production and the angiogenic response in endothelial cells. EOMA cells derived from a spontaneously arising murine hemangioendothelioma were genetically manipulated to stably express siRNA targeting ecNOS. Knockdown of ecNOS in different stably transfected EOMA cell lines was demonstrated by quantitative RT-PCR, Western blot and ecNOS specific ELISA. An EOMA cell line with near complete knockdown of ecNOS exhibited dramatically altered morphology and changes in the expression of mRNAs encoding proteins involved in angiogenesis. This cell line exhibited a 4-fold increase in proliferation in vitro, altered tube formation in matrigel and formed tumors in mice more rapidly than the parental cells. In contrast, a cell line in which ecNOS protein levels were reduced only 5-fold did not show changes in proliferation rate, tube formation or tumor growth. These results suggest that ecNOS derived nitric oxide reduces the growth of hemangioendothelioma derived tumors, and underscore the importance of careful consideration of the tumor type when selecting modulation of nitric oxide signaling as a treatment strategy.     

人血管内皮抑制素在毕赤酵母中的表达纯化及活性测定

血管内皮抑制素是胶原ⅩⅧ C-末端的一个片段,是一个有效的血管生成抑制因子。本文克隆了人血管内皮抑制素的基因,并用毕赤酵母进行表达,表达量为50.5 mg/L。发酵液上清经SP Sepharose Fast Flow凝胶一步层析,产物纯度达到98%以上,纯化收率达到95%以上。毕赤酵母分泌表达的人血管内皮抑制素具有免疫活性;能明显抑制鸡胚尿囊膜新生血管的生长;并且能特异性地抑制bFGF刺激的人微血管内皮细的迁移,达到抑制效果为50%时所需的蛋白浓度（IC50）为0.4μg/ml。本研究为应用人血管内皮抑制素治疗肿瘤奠定了初步实验基础。