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.     

Collagen XVIII and corneal reinnervation following keratectomy

The significance of collagen XVIII in the regulation of corneal reinnervation remains largely unknown. We used whole-mount immunoconfocal microscopy to localize collagen XVIII to the nerve basement membrane of wild-type (WT) mouse corneas. Transmission electron microscopy showed corneal nerve disorganization in collagen XVIII knockout mice (col18a1(-/-)). Antibody 2H3-specific neurofilament colocalized with collagens XVIII and IV and laminin-2 in WT mouse corneas, but did not colocalize with collagen IV and laminin-2 in col18a1(-/-) mouse corneas. Following keratectomy, col18a1(-/-) mice displayed decreased corneal neurite extension compared to WT mice. Our data indicate that collagen XVIII may play an important role in corneal reinnervation after wounding.     

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.     

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1)₃; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Importantly, type XV trimerbodies demonstrated substantially greater thermal and serum stability and resistance to protease digestion than type XVIII trimerbodies. In summary, the small size, high expression level, solubility and stability of the trimerization domain of type XV collagen make it the ideal choice for engineering homotrimeric antibodies for cancer detection and therapy.Key words: antibody engineering, multivalent antibody, collagen XVIII, collagen XV, tumor targeting     

TGFBIp mediates lymphatic sprouting in corneal lymphangiogenesis

Corneal lymphangiogenesis plays a key role in diverse pathological conditions of the eye. Here, we demonstrate that a versatile extracellular matrix protein, transforming growth factor‐β induced protein (TGFBIp), promotes lymphatic sprouting in corneal lymphangiogenesis. TGFBIp is highly up‐regulated in inflamed mouse corneas. Immunolocalization of TGFBIp is detected in infiltrating macrophages in inflamed mouse corneas. Subconjunctival injection of liposomal clodronate can significantly reduce macrophage infiltration in inflamed mouse cornea, and decrease the expression of TGFBIp and areas of corneal lymphangiogenesis and angiogenesis after corneal suture placement. In brief, these results indicate that the up‐regulation of TGFBIp in sutured cornea correlates with macrophage infiltration. Although TGFBIp alone cannot significantly stimulate corneal lymph vessel ingrowth in vivo, it can enhance the effect of vascular endothelial growth factor‐C in promoting corneal lymphangiogenesis. The in vitro results show that TGFBIp promotes migration, tube formation and adhesion of human lymphatic endothelial cells (HLECs), but it has no effect on HLECs' proliferation. We also find that the in vitro effect of TGFBIp is mediated by the integrin α5β1‐FAK pathway. Additionally, integrin α5β1 blockade can significantly inhibit lymphatic sprouting induced by TGFBIp. Taken together, these findings reveal a new molecular mechanism of lymphangiogenesis in which the TGFBIp‐integrin pathways plays a pivotal role in lymphatic sprouting.     

Expression of collagen XVIII and localization of its glycosaminoglycan attachment sites

Collagen XVIII is the only currently known collagen that carries heparan sulfate glycosaminoglycan side chains. The number and location of the glycosaminoglycan attachment sites in the core protein were determined by eukaryotic expression of full-length chick collagen XVIII and site-directed mutagenesis. Three Ser-Gly consensus sequences carrying glycosaminoglycan side chains were detected in the middle and N-terminal part of the core protein. One of the Ser-Gly consensus sequences carried a heparan sulfate side chain, and the remaining two had mixed chondroitin and heparan sulfate side chains; thus, recombinant collagen XVIII was a hybrid of heparan sulfate and chondroitin proteoglycan. In contrast, collagen XVIII from all chick tissues so far assayed have exclusively heparan sulfate side chains, indicating that the posttranslational modification of proteins expressed in vitro is not entirely identical to the processing that occurs in a living embryo. Incubating the various mutated collagen XVIIIs with retinal basement membranes showed that the heparan sulfate glycosaminoglycan side chains mediate the binding of collagen XVIII to basement membranes.     

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.     

Lack of collagen XVIII long isoforms affects kidney podocytes, whereas the short form is needed in the proximal tubular basement membrane

Collagen XVIII is characterized by three variant N termini, an interrupted collagenous domain, and a C-terminal antiangiogenic domain known as endostatin. We studied here the roles of this collagen type and its variant isoforms in the mouse kidney. Collagen XVIII appeared to be in a polarized orientation in the tubular basement membranes (BMs), the endostatin domain embedded in the BM, and the N terminus residing at the BM-fibrillar matrix interface. In the case of the glomerular BM (GBM), collagen XVIII was expressed in different isoforms depending on the side of the GBM. The orientation appeared polarized here, too, both the endothelial promoter 1-derived short variant of collagen XVIII and the epithelial promoter 2-derived longer variants having their C-terminal endostatin domains embedded in the BM and the N termini at the respective BM-cell interfaces. In addition to loosening of the proximal tubular BM structure, the Col18a1(-/-) mice showed effacement of the glomerular podocyte foot processes, and microindentation studies showed changes in the mechanical properties of the glomeruli, the Col18a1(-/-) glomeruli being ～30% softer than the wild-type. Analysis of promoter-specific knockouts (Col18a1(P1/P1) and Col18a1(P2/P2)) indicated that tubular BM loosening is due to a lack of the shortest isoform, whereas the glomerular podocyte effacement was due to a lack of the longer isoforms. We suggest that lack of collagen XVIII may also have disparate effects on kidney function in man, but considering the mild physiological findings in the mutant mice, such effects may manifest themselves only late in life or require other compounding molecular changes.     

Mode of action of 2-(diethylamino)-7-ethoxychromone on human platelets.

The in vitro effect of 2-(diethylamino)-7-ethoxychromone (RC39XVIII) on human platelet aggregation induced by several agonists and on thromboxane B2 formation, granule release and intracellular cAMP elevation has been studied. The chromosome-derivative exerts a dose-dependent inhibitory effect on aggregation produced by U46619, arachidonic acid, thrombin, collagen and ADP. RC39XVIII inhibits aggregation, TxB2 formation and granule release in parallel. Moreover the drug potentiates cAMP accumulation induced by iloprost and forskolin. The drug also inhibits soluble cAMP phosphodiesterase in a dose-dependent manner. No effect on adenylate cyclase activity measured in platelet membranes was evident.     

Improved stability of multivalent antibodies containing the human collagen XV trimerization domain

We recently described the in vitro and in vivo properties of an engineered homotrimeric antibody made by fusing the N-terminal trimerization region of collagen XVIII NC1 domain to the C-terminus of a scFv fragment [trimerbody (scFv-NC1)₃; 110 kDa]. Here, we demonstrated the utility of the N-terminal trimerization region of collagen XV NC1 domain in the engineering of trivalent antibodies. We constructed several scFv-based trimerbodies containing the human type XV trimerization domain and demonstrated that all the purified trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Importantly, type XV trimerbodies demonstrated substantially greater thermal and serum stability and resistance to protease digestion than type XVIII trimerbodies. In summary, the small size, high expression level, solubility and stability of the trimerization domain of type XV collagen make it the ideal choice for engineering homotrimeric antibodies for cancer detection and therapy.     

Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution

The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation–pain and swelling–by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process in vivo. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary in vivo approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75^NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities in vitro. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis in vivo. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.     

Tumor suppression by collagen XV is independent of the restin domain

Non-fibrillar collagen XV is a chondroitin sulfate modified glycoprotein that is associated with the basement membrane zone in many tissues. Its precise functions remain to be fully elucidated though it clearly plays a critical role in the structural integrity of the extracellular matrix. Loss of collagen XV from the basement membrane zone precedes invasion of a number of tumor types and we previously showed that collagen XV functions as a dose-dependent suppressor of tumorigenicity in cervical carcinoma cells. The carboxyl terminus of another non-fibrillar collagen (XVIII) is cleaved to produce endostatin, which has anti-angiogenic effects and thus may act as a tumor suppressor in vivo. Since collagen XV has structural similarity with collagen XVIII, its C-terminal restin domain could confer tumor suppressive functions on the molecule, though our previous data did not support this. We now show that expression of collagen XV enhances the adhesion of cervical carcinoma cells to collagen I in vitro as does the N-terminus and collagenous regions of collagen XV, but not the restin domain. Destruction of a cysteine residue in the collagenous region that is critical for intermolecular interactions of collagen XV abolished the enhanced adhesion to collagen I. Finally, we demonstrate that unlike full length collagen XV, expression of the restin domain alone does not suppress tumorigenicity of cervical carcinoma cells in vivo; hence, this process is dependent on functions and interactions of other parts of the protein.     

Epitope-defined monoclonal antibodies against multiplexin collagens demonstrate that type XV and XVIII collagens are expressed in specialized basement membranes

Type XV and type XVIII collagens are classified as part of multiplexin collagen superfamily and their C-terminal parts, endostatin and restin, respectively, have been shown to be anti-angiogenic in vivo and in vitro. The alpha1(XV) and alpha1(XVIII) collagen chains are reported to be localized mainly in the basement membrane zone, but their distributions in blood vessels and nonvascular tissues have yet to be thoroughly clarified. In the present study, we raised monoclonal antibodies against synthetic peptides of human alpha1(XV) and alpha1(XVIII) chains and used them for extensive investigation of the distribution of these chains. We came to the conclusion that nonvascular BMs contain mainly one of two types: subepithelial basement membranes that contained type XVIII in general, or skeletal and cardiac muscles that harbored mainly type XV. But basement membranes surrounding smooth muscle cells in vascular tissues contained one or both of them, depending on their locations. Interestingly, continuous capillaries contained both type XV and type XVIII collagens in their basement membranes; however, fenestrated or specialized capillaries such as glomeruli, liver sinusoids, lung alveoli, and splenic sinusoids expressed only type XVIII in their basement membranes, lacking type XV. This observation could imply that different functions of basement membranes in various tissues and organs use different mechanisms for the endogenous control of angiogenesis.     

Collagen XVIII,a basement membrane heparan sulfate proteoglycan,interacts with L-selectin and monocyte chemoattractant protein-1

Leukocyte infiltration during inflammation is mediated by the sequential actions of adhesion molecules and chemokines. By using a rat ureteral obstruction model, we showed previously that L-selectin plays an important role in leukocyte infiltration into the kidney. Here we report the purification, identification, and characterization of an L-selectin-binding heparan sulfate proteoglycan (HSPG) expressed in the rat kidney. Partial amino acid sequencing and Western blotting analyses showed that the L-selectin-binding HSPG is collagen XVIII, a basement membrane HSPG. The binding of L-selectin to isolated collagen XVIII was specifically inhibited by an anti-L-selectin monoclonal antibody, EDTA, treatment of the collagen XVIII with heparitinase or heparin but not by chemically desulfated heparin. A cell binding assay showed that the L-selectin-collagen XVIII interaction mediates cell adhesion. Interestingly, collagen XVIII also interacted with a chemokine, monocyte chemoattractant protein-1, and presented it to a monocytic cell line, THP-1, which enhanced the alpha(4)beta(1) integrin-mediated binding of the THP-1 cells to vascular cell adhesion molecule-1. Thus, collagen XVIII may provide a link between selectin-mediated cell adhesion and chemokine-induced cellular activation and accelerate the progression of leukocyte infiltration in renal inflammation.     

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.     

Vascular endothelial growth factor receptor-3 mediates induction of corneal alloimmunity

There are no studies so far linking molecular regulation of lymphangiogenesis and induction of adaptive immunity. Here, we show that blockade of vascular endothelial growth factor receptor-3 (VEGFR-3) signaling significantly suppresses corneal antigen-presenting (dendritic) cell trafficking to draining lymph nodes, induction of delayed-type hypersensitivity and rejection of corneal transplants. Regulating the function of VEGFR-3 may therefore be a mechanism for modulating adaptive immunity in the periphery.     

Lack of collagen XVIII/endostatin results in eye abnormalities

Mice lacking collagen XVIII and its proteolytically derived product endostatin show delayed regression of blood vessels in the vitreous along the surface of the retina after birth and lack of or abnormal outgrowth of retinal vessels. This suggests that collagen XVIII/endostatin is critical for normal blood vessel formation in the eye. All basement membranes in wild-type eyes, except Descemet's membrane, showed immunogold labeling with antibodies against collagen XVIII. Labeling at sites where collagen fibrils in the vitreous are connected with the inner limiting membrane and separation of the vitreal matrix from the inner limiting membrane in mutant mice indicate that collagen XVIII is important for anchoring vitreal collagen fibrils to the inner limiting membrane. The findings provide an explanation for high myopia, vitreoretinal degeneration and retinal detachment seen in patients with Knobloch syndrome caused by loss-of-function mutations in collagen XVIII.