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.     

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.     

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.     

Endostatin/collagen XVIII--an inhibitor of angiogenesis--is expressed in cartilage and fibrocartilage.

Aim of the study was to get a deeper insight in the mechanisms regulating avascularity of cartilaginious tissues. In the center of our interest was the expression of the anti-angiogenic fragment of collagen XVIII and its potency to inhibit angiogenesis. We observed a strong endostatin/collagen XVIII production in articular and fibrocartilage and an inhibitory potency concerning the VEGF-signalling pathway. INTRODUCTION: Cartilaginous tissue is mainly avascular and shows a limited intrinsic capacity for healing. Aim of this study was to investigate the expression of the antiangiogenic peptide endostatin/collagen XVIII in cartilage and fibrocartilage. RESULTS: In fetal epiphyseal cartilage of humans high endostatin/collagen XVIII levels could be detected by ELISA whereas significantly lower levels were found in articular cartilage of adults. In the fibrocartilaginous tissue of the menisci, there was no significant difference in the endostatin/collagen XVIII concentrations between samples of fetuses and adults. But in the menisci of adults, endostatin/collagen XVIII concentrations were higher in the internal avascular two thirds of the meniscus whereas in the fetal menisci higher endostatin/collagen XVIII concentrations were found in the external third. Endostatin/collagen XVIII immunostaining of rat articular cartilage shows that endostatin/collagen XVIII downregulation starts soon after birth. In fetal cartilage and fibrocartilage of rats and humans, endostatin/collagen XVIII could be immunostained in the extracellular matrix and in the pericellular matrix of endothelial cells, fibrochondrocytes and chondrocytes. In adult cells, weak endostatin/collagen XVIII immunostaining was restricted to the pericellular matrix of fibrochondrocytes and chondrocytes. The detection of endostatin/collagen XVIII could be verified by in situ hybridization. Chondrocytes in vitro released measurable amounts of endostatin/collagen XVIII into culture supernatants. Stimulation of chondrocytes with EGF, as an example of a growth factor, or dexamethasone had no influence on endostatin/collagen XVIII expression. Endostatin inhibited VEGF-induced phosphorylation of MAPK in chondrocytes. CONCLUSIONS: The spatial and temporal expression of endostatin/collagen XVIII in cartilaginous tissue and its potency regarding inactivation of VEGF signalling suggests that this antiangiogenic factor is important not only for the development but also for the maintenance of avascular zones in cartilage and fibrocartilage. EXPERIMENTAL PROCEDURES: We analyzed the spatial and temporal expression of endostatin/collagen XVIII--an endogenous angiogenesis inhibiting factor--in cartilage and fibrocartilage of humans and rats by immunohistochemical and biochemical (ELISA) methods and by in situ hybridization. To elucidate possible factors responsible for the induction or suppression of endostatin/collagen XVIII in cartilaginous tissues, chondrocytes (cell line C28/I2) were exposed to EGF and dexamethason. To study the possible interaction of endostatin/collagen XVIII with angiogenic factors, the immortalized human chondrocytes (C28/I2) have been incubated with VEGF and the phosphorylation of the MAPK Erk 1/2 (extracellular-regulated kinases), a known signal transduction pathway for VEGF has been determined under the influence of endostatin.     

Secreted cathepsin L generates endostatin from collagen XVIII

Endostatin, an inhibitor of angiogenesis and tumor growth, was identified originally in conditioned media of murine hemangioendothelioma (EOMA) cells. N-terminal amino acid sequencing demonstrated that it corresponds to a fragment of basement membrane collagen XVIII. Here we report that cathepsin L is secreted by EOMA cells and is responsible for the generation of endostatin with the predicted N-terminus, while metalloproteases produce larger fragments in a parallel processing pathway. Efficient endostatin generation requires a moderately acidic pH similar to the pericellular milieu of tumors. The secretion of cathepsin L by a tumor cell line of endothelial origin suggests that this cathepsin may play a role in angiogenesis. We propose that cleavage within collagen XVIII's protease-sensitive region evolved to regulate excessive proteolysis in conditions of induced angiogenesis.     

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.     

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.     

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.     

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.     

Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.     

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.     

Chronic NOS inhibition actuates endothelial-mesenchymal transformation

Chronic kidney diseases are accompanied by the accumulation of substances like asymmetric dimethylarginine, phenylacetic acid, homocysteine, and advanced glycation end products, known to either inhibit endothelial nitric oxide synthase (eNOS) or uncouple it, consequently limiting the amount of available nitric oxide (NO). Reduced bioavailability of NO induces endothelial dysfunction. An early loss of peritubular capillaries in tubulointerstitial fibrotic areas and injury to endothelial cells have been linked to progressive renal disease. Screening endothelial genes in cells treated with NOS inhibitors showed upregulation of collagen XVIII, a precursor of a potent antiangiogenic substance, endostatin. This finding was confirmed at the level of mRNA and protein expression. Tie-2 promoter-driven green fluorescent protein mice treated with nonhypertensinogenic doses of a NOS inhibitor exhibited upregulation of collagen XVIII/endostatin and rarefaction of capillary profiles. This was accompanied by the increased expression of transforming growth factor-beta and connective tissue growth factor in the kidney. Occasional endothelial cells expressed both the marker of endothelial lineage (green fluorescent protein) and mesenchymal marker (alpha-smooth muscle actin or calponin). In vitro studies of endothelial cells treated with asymmetric dimethylarginine showed decreased expression of eNOS and Flk-1 and enhanced expression of calponin and fibronectin, additional markers of smooth muscle and mesenchymal cells. These cells overexpressed transforming growth factor-beta and connective tissue growth factor, as well as endostatin. In conclusion, data presented here 1) ascribe to NO deficiency in endothelial cells the function of a profibrotic stimulus associated with the expression of an antiangiogenic fragment of collagen XVIII (endostatin) and 2) provide evidence of endothelial-mesenchymal transdifferentiation in the course of inhibition of NOS by a pathophysiologically important antagonist, asymmetric dimethylarginine. Both mechanisms may account for microvascular rarefaction.     

Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of 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.     

Autophagy and angiogenesis inhibition

Angiogenesis, the process by which new blood vessels are formed is critical for embryonic development and physiological functioning of normal tissues. Angiogenesis also plays a critical role in the pathology of many diseases including cancer, wherein the supply and demand for blood vessels determines the rate of cancer growth. A number of therapeutic strategies are being developed to inhibit pathological angiogenesis. Kringle domains of plasminogen such as kringle 5 (K5) and a proteolytic fragment of collagen type XVIII (endostatin) are well-characterized, potent angiogenesis inhibitors. These inhibitors activate different intracellular signaling pathways to induce apoptosis and inhibit cell proliferation. Recent studies from our group have shown that K5 and endostatin can also induce autophagy in addition to apoptosis in endothelial cells. A common feature of the two treatments was the upregulation of Beclin 1 levels leading to alterations in the Beclin 1-Bcl-2 complex. Angiogenesis inhibitor-induced autophagy in endothelial cells was independent of nutritional or hypoxic stress and initiated even in the presence of endothelial-specific survival factors such as vascular endothelial growth factor (VEGF). Interfering with the autophagic response by knocking down Beclin 1 levels dramatically increased apoptosis of endothelial cells. These findings identify the autophagic response as a novel target for enhancing the therapeutic efficacy of angiogenesis inhibitors.     

Hypoxic induction of endothelial cell growth factors in retinal cells: identification and characterization of vascular endothelial growth factor (VEGF) as the mitogen.

BACKGROUND: New vessel growth is often associated with ischemia, and hypoxic tissue has been identified as a potential source of angiogenic factors. In particular, ischemia is associated with the development of neovascularization in a number of ocular pathologies. For this reason, we have studied the induction of endothelial cell mitogens by hypoxia in retinal cells. MATERIALS AND METHODS: Human retinal pigment epithelium (hRPE) were grown under normoxic and hypoxic conditions and examined for the production of endothelial mitogens. Northern analysis, biosynthetic labeling and immunoprecipitation, and ELISA were used to assess the levels of vascular endothelial growth factor/vascular permeability factor (VEGF) and basic fibroblast growth factor (bFGF), two endothelial cell mitogens and potent angiogenic factors. Soluble receptors for VEGF were employed as competitive inhibitors to determine the contribution of the growth factor to the hypoxia-stimulated mitogen production. RESULTS: Following 6-24 hr of hypoxia, confluent and growing cultures of hRPE increase their levels of VEGF mRNA and protein synthesis. Biosynthetic labeling studies and RT-PCR analysis indicate that the cells secrete VEGF121 and VEGF165, the soluble forms of the angiogenic factor. In contrast, hRPE cultured under hypoxic conditions show reduced steady-state levels of basic fibroblast growth factor (bFGF) mRNA and decreased bFGF protein synthesis. Unlike VEGF, bFGF is not found in conditioned media of hRPE following 24 hr of hypoxia. Using a soluble high-affinity VEGF receptor as a competitive inhibitor of VEGF, we demonstrate that a VEGF-like activity is the sole hypoxia-inducible endothelial mitogen produced by cultured hRPE. CONCLUSIONS: From this comparison we conclude that hRPE do not respond to hypoxia with a general, nonspecific increase in the overall levels of growth factors, as is seen during cell wounding responses or serum stimulation. The physiological relevance of data from this in vitro model are affirmed by separate studies in an animal model of retinal ischemia-induced ocular neovascularization (1) in which retina-derived VEGF levels have been shown to correlate spatio-temporally with the onset of angiogenesis. Taken together, these data support the hypothesis that the induction of VEGF by hypoxia mediates the rapid, initial angiogenic response to retinal ischemia.