NC1 Domain Research Articles

NC1 domain of collagen α3(IV) derived from the basement membrane regulates Sertoli cell blood-testis barrier dynamics

The blood-testis barrier (BTB) is an important ultrastructure for spermatogenesis. Delay in BTB formation in neonatal rats or its irreversible damage in adult rats leads to meiotic arrest and failure of spermatogonial differentiation beyond type A. While hormones, such as testosterone and FSH, are crucial to BTB function, little is known if there is a local regulatory mechanism in the seminiferous epithelium that modulates BTB function. Herein, we report that collagen α3(IV) chain, a component of the basement membrane in the rat testis, could generate a noncollagenous (NC1) domain peptide [Colα3(IV) NC1] via limited proteolysis by matrix metalloproteinase-9 (MMP-9), and that the expression of MMP-9 was upregulated by TNFα. While recombinant Colα3(IV) NC1 protein produced in E. coli failed to perturb Sertoli cell tight junction (TJ)-permeability barrier function, possibly due to the lack of glycosylation, Colα3(IV) NC1 recombinant protein produced in mammalian cells and purified to apparent homogeneity by affinity chromatography was found to reversibly perturb the Sertoli cell TJ-barrier function. Interestingly, Colα3(IV) NC1 recombinant protein did not perturb the steady-state levels of several TJ- (e.g., occludin, CAR, JAM-A, ZO-1) and basal ectoplasmic specialization- (e.g., N-cadherin, α-catenin, β-catenin) proteins at the BTB but induced changes in protein localization and/or distribution at the Sertoli cell-cell interface in which these proteins moved from the cell surface into the cell cytosol, thereby destabilizing the TJ function. These findings illustrate the presence of a local regulatory axis known as the BTB-basement membrane axis that regulates BTB restructuring during spermatogenesis.

Exploratory biomarker analysis reveals a genetic epistatic interaction in interleukin 1 receptor antagonist (IL1RN) gene associated to cartilage volume growth measured by MRI in response to sprifermin therapy in patients with knee radiographic osteoarthritis

Exploratory biomarker analysis reveals a genetic epistatic interaction in interleukin 1 receptor antagonist (IL1RN) gene associated to cartilage volume growth measured by MRI in response to sprifermin therapy in patients with knee radiographic osteoarthritis

The Expression and Activity of Cathepsins D, H and K in Asthmatic Airways

Tumstatin is an anti-angiogenic collagen IV α3 fragment, levels of which are reduced in the airways of asthmatics. Its reduction may be due to the degradation by extracellular matrix (ECM) proteases. Cathepsins play a role in ECM remodelling, with cathepsin D, H and K (CTSD, CTSH and CTSK) being associated with lung diseases. CTSD modulates the NC1 domains of collagen molecules including tumstatin, while CTSH and CTSK are involved in ECM degradation. The role of these cathepsins in the regulation of tumstatin in the lung has not previously been examined. We demonstrated that CTSB, D, F, H, K, L and S mRNA was expressed in the airways. Quantification of immunohistochemistry showed that there is no difference in the global expression of CTSD, CTSH and CTSK between asthmatics and non-asthmatics. CTSD and CTSK, but not CTSH had the capacity to degrade tumstatin. No difference was observed in the activity of CTSD and H in bronchoalveolar lavage fluid of asthmatic and non-asthmatics, while CTSK was undetectable. This indicates that while CTSD possesses the potential to directly regulate tumstatin, and thus angiogenesis through this mechanism however, it is not likely to be involved in the dysregulation of tumstatin found in asthmatic airways.

A new anti-tumor strategy based on in vivo tumstatin overexpression after plasmid electrotransfer in muscle

The NC1 domains from the different α(IV) collagen chains were found to exert anti-tumorigenic and/or anti-angiogenic activities. A limitation to the therapeutic use of these matrikines is the large amount of purified recombinant proteins, in the milligram range in mice that should be administered daily throughout the experimental procedures. In the current study, we developed a new therapeutic approach based on tumstatin (NC1α3(IV)) overexpression in vivo in a mouse melanoma model. Gene electrotransfer of naked plasmid DNA (pDNA) is particularly attractive because of its simplicity, its lack of immune responsiveness and its safety. The pDNA electrotransfer in muscle mediates a substantial gene expression that lasts several months. A pVAX1© vector containing the tumstatin cDNA was injected into the legs of C57BL/6 mice and submitted to electrotranfer. Sera were collected at different times and tumstatin was quantified by ELISA. Tumstatin secretion reached a plateau at day 21 with an expression level of 12μg/mL. For testing the effects of tumstatin expression on tumor growth in vivo, B16F1 melanoma cells were subcutaneously injected in mice 7days after empty pVAX1© (Mock) or pVAX1©–tumstatin electrotransfer. Tumstatin expression triggered a large decrease in tumor growth and an increase in mouse survival. This new therapeutic approach seems promising to inhibit tumor progression in vivo.

The NC2 Domain of Type IX Collagen Determines the Chain Register of the Triple Helix

Precise mapping and unraveling the mechanism of interaction or degradation of a certain type of collagen triple helix requires the generation of short and stable collagenous fragments. This is a great challenge especially for hetero-trimeric collagens, where chain composition and register (stagger) are important factors. No system has been reported that can be efficiently used to generate a natural collagenous fragment with exact chain composition and desired chain register. The NC2 domain (only 35-50 residues) of FACIT collagens is a potent trimerization domain. In the case of type IX collagen it provides the efficient selection and hetero-trimerization of three distinct chains. The ability of the NC2 domain to determine the chain register of the triple helix is studied. We generated three possible sequence combinations (α1α1α2, α1α2α1, α2α1α1) of a type I collagen fragment (the binding region for the von Willebrand factor A3 domain) attached to the NC2 domain. In addition, two control combinations were produced that constitute homo-trimers of (α1)(3) or (α2)(3). For the hetero-trimeric constructs, α1α1α2 demonstrated a higher melting temperature than the other two. Binding experiments with the von Willebrand factor A3 domain revealed the homo-trimer of (α1)(3) as the strongest binding construct, whereas the homo-trimer of (α2)(3) showed no binding. For hetero-trimers, α1α1α2 was found to be the strongest binding construct. Differences in thermal stability and binding to the A3 domain unambiguously demonstrate that the NC2 domain of type IX collagen determines not only the chain composition but also the chain register of the adjacent triple helix.

Diagnosis and disease severity assessment of epidermolysis bullosa acquisita by ELISA for anti-type VII collagen autoantibodies: an Italian multicentre study

Epidermolysis bullosa acquisita (EBA) is a rare autoimmune mucocutaneous bullous disease caused by autoantibodies against type VII collagen, a component of anchoring fibrils that stabilizes dermoepidermal adherence. Type VII collagen is composed of a collagenous domain linked by the noncollagenous (NC)1 and NC2 domains. To assess the repeatability, sensitivity and specificity of a recently developed enzyme-linked immunosorbent assay (ELISA) for detection of anti-type VII collagen autoantibodies, and to ascertain whether they may be a marker of disease activity in EBA. Using this ELISA, which was able to recognize autoantibodies against the NC1 and NC2 epitopes of type VII collagen, we tested 14 EBA sera, 30 healthy control sera and 113 disease control sera. In the EBA sera group, 12 out of the 14 samples were positive in ELISA, with autoantibody titres varying from 7·2 to 127·9UmL(-1) (cutoff value <6), the sensitivity of the method being 86%. Among the controls, only two bullous pemphigoid sera tested positive, the specificity being 98·6%. A good correlation was found between EBA disease severity, expressed as autoimmune bullous skin disorder intensity score, and the serum levels of anti-collagen VII autoantibodies, measured by ELISA (n =14; r=0·965; P=0·0001). The intra- and interassay coefficients of variation of the ELISA method ranged from 6·3% to 18·3%. This NC1+NC2 ELISA can be a practical assay for the diagnosis of EBA. The correlation between autoantibody titres and disease severity suggests its usefulness as a marker of disease activity in EBA However, this should be confirmed by studies on larger series of patients.

NC1 Long and NC3 Short Splice Variants of Type XII Collagen Are Overexpressed during Corneal Scarring

To investigate type XII collagen expression in corneal scars in vivo. Type XII collagen protein expression was evaluated by immunohistochemistry in human corneal scars and in a mouse model of corneal scarring at several time points (from day 7 to day 210) after full-thickness excision. Alternative splice variants of the NC3 and NC1 domains of type XII collagen were investigated in the mouse wound-healing model using RT-PCR. Type XII collagen was overexpressed in human corneal scars in areas that were also positive for alpha-smooth muscle actin staining. In a mouse model of corneal wound injury we found that at 14 and 21 days postexcision, type XII collagen was largely concentrated in the subepithelial region of the cornea, especially in and near the wound bed. By 28 days postexcision, expression of type XII collagen decreased but remained higher than that in controls. NC3 short form is the main form expressed in the cornea during the wound-healing process. After injury, the NC1 long splice variant mRNA was the most highly overexpressed variant in the cornea, especially in the epithelium (×2.7, 3.72, and 5.57 at days 7, 14, and 21, respectively, P < 0.01 to 0.001 compared with uninjured samples). Corneal scars from a 7-month-old mouse revealed an overexpression of type XII collagen in the wound area similar to what we observed in human corneal scars. Type XII collagen is overexpressed in permanent human and mouse corneal scars and could represent a new target to treat corneal scarring.

L-arginine Mediated Renaturation Enhances Yield of Human, &amp;alpha;6 Type IV Collagen Non-collagenous Domain from Bacterial Inclusion Bodies

The anti-angiogenic, carboxy terminal non-collagenous domain (NC1) derived from human Collagen type IV alpha 6 chain, [α6(IV)NC1] or hexastatin, was earlier obtained using different recombinant methods of expression in bacterial systems. However, the effect of L-arginine mediated renaturation in enhancing the relative yields of this protein from bacterial inclusion bodies has not been evaluated. In the present study, direct stirring and on-column renaturation methods using L-arginine and different size exclusion chromatography matrices were applied for enhancing the solubility in purifying the recombinant α6(IV)NC1 from bacterial inclusion bodies. This methodology enabled purification of higher quantities of soluble protein from inclusion bodies, which inhibited endothelial cell proliferation, migration and tube formation. Thus, the scope for L-arginine mediated renaturation in obtaining higher yields of soluble, biologically active NC1 domain from bacterial inclusion bodies was evaluated.

Serum levels of anti‐type VII collagen antibodies detected by enzyme‐linked immunosorbent assay in patients with epidermolysis bullosa acquisita are correlated with the severity of skin lesions

Epidermolysis bullosa acquisita (EBA) is a chronic autoimmune subepidermal bullous disease characterized by circulating autoantibodies against type VII collagen. Detecting these autoantibodies is crucial for the diagnosis of this disease, and is also useful for measuring disease activity. Enzyme-linked immunosorbent assay (ELISA), a quantitative method to measure anti-type VII collagen antibody levels, is currently available to diagnose EBA. The aim of this study was to investigate the relationship of ELISA with overall clinical severity. Sera from patients with EBA (n = 30), bullous pemphigoid (n = 20), anti-laminin γ1 pemphigoid (n = 9) and healthy donors (n = 24) were tested using ELISA, using the recombinant non-collagenous 1 (NC1) and 2 (NC2) domains of type VII collagen. Relationships between clinical characteristics, indirect immunofluoroscence (IIF) titres and ELISA values were investigated. The sensitivity and specificity of the EBA ELISA were 96.7% and 98.1%, respectively. There was no significant difference between ELISA results for classic and inflammatory types. The severity of skin involvement was positively correlated with both ELISA value (r = 0.87, P < 0.01) and IIF titre (r = 0.59, P < 0.01). Time sequence analysis in four patients with EBA showed that ELISA values reflect disease activity better than IIF titres. Type VII collagen ELISA using the NC1 and NC2 domains is useful for diagnosing EBA and monitoring disease severity.

La lumière de la peau

Extracellular matrix proteins play a crucial role in influencing the invasion of trophoblast cells. However the role of collagens and collagen type IV (col-IV) in particular at the implantation site is not clear.Immunohistochemistry was used to determine the distribution of collagen types I, III, IV and VI in endometrium and decidua during the menstrual cycle and the first trimester of pregnancy. Expression of col-IV alpha chains during the reproductive cycle was determined by qPCR and protein localisation by immunohistochemistry. The structure of col-IV in placenta was examined using transmission electron microscopy. Finally, the expression of col-IV alpha chain NC1 domains and collagen receptors was localised by immunohistochemistry.Col-IV alpha chains were selectively up-regulated during the menstrual cycle and decidualisation. Primary extravillous trophoblast cells express collagen receptors and secrete col-IV in vitro and in vivo, resulting in the increased levels found in decidua basalis compared to decidua parietalis. A novel expression pattern of col-IV in the mesenchyme of placental villi, as a three-dimensional network, was found. NC1 domains of col-IV alpha chains are known to regulate tumour cell migration and the selective expression of these domains in decidua basalis compared to decidua parietalis was determined.Col-IV is expressed as novel forms in the placenta. These findings suggest that col-IV not only represents a structural protein providing tissue integrity but also influences the invasive behaviour of trophoblast cells at the implantation site.

Tetrastatin, the NC1 domain of the α4(IV) collagen chain: a novel potent anti-tumor matrikine.

BackgroundNC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far.Methodology/Principal FindingsWe demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (KD = 148±9.54 nM).Conclusion/SignificanceCollectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.

Genetic Susceptibility to Experimental Autoimmune Glomerulonephritis in the Wistar Kyoto Rat

In experimental autoimmune glomerulonephritis (EAG), a model of Goodpasture's disease, Wistar Kyoto (WKY) rats immunized with collagenase-solubilized glomerular basement membrane (GBM) or the recombinant NC1 domain of the α3 chain of type IV collagen [α3(IV)NC1] develop anti-GBM antibodies and focal necrotizing glomerulonephritis with crescent formation. However, Lewis (LEW) rats, which share the same major histocompatibility complex (MHC) haplotype, are resistant to EAG development. A genome-wide linkage analysis of backcrossed animals with EAG revealed a major quantitative trait locus (QTL) on rat chromosome 13 (LOD = 3.9) linked to the percentage of glomerular crescents. To investigate the role of this QTL in EAG induction, reciprocal congenic rats were generated (LEW.WCrgn1 congenic and WKY.LCrgn1 congenic), immunized with recombinant rat α3(IV)NC1, and assessed for EAG development. WKY.LCrgn1 rats showed a marked reduction in albuminuria, severity of crescentic nephritis, and number of glomerular macrophages compared with WKY controls. No reduction in antibody levels was observed. However, LEW.WCrgn1 rats were resistant to EAG development, as were LEW controls. Macrophage activation in vitro was assessed in parental and congenic rat bone marrow-derived macrophages (BMDMs). WKY.LCrgn1 BMDMs showed a significant reduction in Fc receptor-mediated oxidative burst, phagocytosis of opsonised polystyrene beads, and LPS-induced levels of MCP-1 secretion and iNOS mRNA expression compared with WKY rats. These results confirm the importance of Crgn1 on chromosome 13 in EAG susceptibility, mediated partly through differences in Fc receptor-mediated macrophage activation.

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.

Increased incidence of anti-GBM disease in Fcgamma receptor 2b deficient mice, but not mice with conditional deletion of Fcgr2b on either B cells or myeloid cells alone

Fcgamma receptor 2b (Fcgr2b) is the only inhibitory Fcgamma receptor in both humans and mice, and is implicated in both antibody production and effector responses to antibody complexes. Reduced function of Fcgr2b has previously been associated with anti-glomerular basement membrane antibody (anti-GBM) disease in mice. However, the mice used had 129 genetic elements flanking the deleted Fcgr2b gene, which are known to increase susceptibility to autoimmunity. In order to confirm a role for Fcgr2b in protection from anti-GBM disease, wild type (WT) mice, mice lacking Fcgr2b on a pure C57BL/6 background, or mice lacking Fcgr2b on a C57BL/6 background with 129 flanking sequences, were immunized with the recombinant NC1 domain of alpha 3 Type IV collagen. Twenty two weeks after immunization, there was a higher incidence of crescentic glomerulonephritis, macrophage infiltration and renal dysfunction in both groups of Fcgr2b−/− mice, indicating an important role of Fcgr2b in regulating the development of anti-GBM disease, on both genetic backgrounds. In order to determine the cellular origin of the Fcgr2b-associated effect, disease was induced in mice with deficiency of Fcgr2b on either B cells alone (CD19Cre), or a subset of myeloid cells (LysozymeMCre). Neither B cell nor myeloid specific knockout mice developed crescentic glomeruonephritis with higher incidence than WT mice indicating that Fcgr2b deficiency on either B cells or a subset of myeloid cells alone is not sufficient to increase susceptibility to anti-GBM disease, but that a combination of cell types, or deficiency of Fcgr2b in a different cell type, is also required.

Matrikines : une nouvelle stratégie thérapeutique anti-cancéreuse

Tumor microenvironment is a complex system composed of a largely altered extracellular matrix (ECM) with different cell types that determine the angiogenic response. Upon the influence of hypoxia, tumor cells secrete cytokines that activate stromal cells to produce proteases and angiogenic factors. The proteases degrade the stromal ECM and participate in the release of various ECM fragments, named matrikines or matricryptins, capable to control tumor invasion and metastasis dissemination. We will focus on the matrikines derived from the NC1 domains of the different constitutive chains of basement membrane-associated collagens and mainly collagen IV. The putative targets of the matrikine action are the proliferation and invasive properties of tumor or inflammatory cells, and the angiogenic and lymphangiogenic responses. For example, canstatin, tumstatin and tetrastatin, respectively derived from the NC1 domains of α2, α3 and α4 chains of collagenIV, inhibit in vivo tumor growth in various experimental cancer models. Their anti-cancer activity comprises an anti-proliferative effect on tumor cells and on endothelial cells by induction of cell apoptosis or cell cycle blockade and the induction of a loss of their migratory phenotype. Matrikines constitute a new family of potent anticancer agents that could be used under various therapeutic strategies: i) induction of their overexpression by cancer cells or by the host cells, ii) use of recombinant proteins or synthetic peptides or structural analogues designed from the structure of the active sequences. These matrikines could be used in combination with conventional chemotherapy or radiotherapy to limit tumor progression.

Detection of collagen VII autoantibodies to NC1 and NC2 domains of collagen VII by ELISA in suspected epidermolysis bullosa acquisita and bullous lupus erythematosus patients

Detection of collagen VII autoantibodies to NC1 and NC2 domains of collagen VII by ELISA in suspected epidermolysis bullosa acquisita and bullous lupus erythematosus patients

Type IX Collagen Interacts with Fibronectin Providing an Important Molecular Bridge in Articular Cartilage

Type IX collagen is covalently bound to the surface of type II collagen fibrils within the cartilage extracellular matrix. The N-terminal, globular noncollagenous domain (NC4) of the α1(IX) chain protrudes away from the surface of the fibrils into the surrounding matrix and is available for molecular interactions. To define these interactions, we used the NC4 domain in a yeast two-hybrid screen of a human chondrocyte cDNA library. 73% of the interacting clones encoded fibronectin. The interaction was confirmed using in vitro immunoprecipitation and was further characterized by surface plasmon resonance. Using whole and pepsin-derived preparations of type IX collagen, the interaction was shown to be specific for the NC4 domain with no interaction with the triple helical collagenous domains. The interaction was shown to be of high affinity with nanomolar K(d) values. Analysis of the fibronectin-interacting clones indicates that the constant domain is the likely site of interaction. Type IX collagen and fibronectin were shown to co-localize in cartilage. This novel interaction between the NC4 domain of type IX collagen and fibronectin may represent an in vivo interaction in cartilage that could contribute to the matrix integrity of the tissue.

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.

Stimulation of the PD-1/PDL-1 T-cell co-inhibitory pathway is effective in treatment of experimental autoimmune glomerulonephritis

Experimental autoimmune glomerulonephritis (EAG) can be induced in Wistar-Kyoto (WKY) rats by immunization with the recombinant NC1 domain of the alpha 3 chain of type IV collagen [α3(IV)NC1]. EAG is characterized by circulating and deposited anti-α3(IV)NC1 antibodies, accompanied by focal necrotizing glomerulonephritis with crescent formation. Programmed death-1 (PD-1) receptor is preferentially expressed on activated T cells and binds two known ligands present on antigen presenting cells, PDL-1 and PDL-2. Engagement of PD-1 by its ligands results in a negative regulatory effect, with inhibition of downstream cellular signalling events and diminished cellular proliferation. In order to investigate the role of the PD-1/PDL-1 co-inhibitory pathway in development of EAG, the in vivo effects of a stimulating PDL-1/Fc fusion protein were examined after the onset of disease. Stimulation of PD-1 led to a significant reduction in albuminuria, serum urea, serum creatinine, crescent formation and tubular damage compared with controls. There was also a reduction in numbers of glomerular macrophages, CD4+ T cells, CD8+ T cells and PD1+ cells compared with controls. No reduction was observed in levels of circulating or deposited antibodies. These results demonstrate that PDL-1/Fc fusion protein is effective in treatment of glomerulonephritis and confirm the importance of the PD-1/PDL-1 T-cell co-inhibitory pathway in development of EAG. Strategies designed to stimulate this pathway may provide a novel approach to treatment of human glomerulonephritis.

Recombinant canstatin inhibits angiopoietin‐1‐induced angiogenesis and lymphangiogenesis

We describe the effect of recombinant canstatin, the NC1 domain of the α2 chain of Type IV collagen, on suppression of angiogenesis and lymphangiogenesis both in vitro and in vivo. Recombinant canstatin produced from stably transformed Drosophila S2 cells reduced the expression of angiopoietin-1 in hypoxia mimetic agent, CoCl(2) -treated CT-26 cells. Recombinant canstatin inhibited proliferation, tube formation and migration of human angiopoietin-1 (rhAngpt-1)-treated human umbilical vein endothelial cells (HUVEC) and lymphatic endothelial cells (LEC). Recombinant canstatin suppressed the expression of Tie-2 and vascular endothelial growth factor-3 (VEGFR-3) transcripts in rhAngpt-1-treated HUVEC and LEC, respectively. The inhibitory effect of recombinant canstatin on tumor growth was also investigated using a heterotopic CT-26 colon carcinoma animal (BALB/c mice) model. Recombinant canstatin reduced the final volume and weight of tumors, and blood and lymphatic vessel densities of tumors, which were evaluated by CD-31 and LYVE-1 immunostaining. Immunohistochemical analysis showed that recombinant canstatin dramatically reduced the expression of angiopoietin-1 in CT-26 colon carcinoma-induced tumor, but not the expression of VEGF-C. Tie-2 and VEGFR-3 expressions were also reduced in recombinant canstatin-treated tumors. These results indicate that recombinant canstatin has anti-tumoral activities against CT-26 colon carcinoma cells. Recombinant canstatin reduces the expression of angiopoietin-1 in hypoxia-induced CT-26 cells and inhibits the angiogenic and lymphangiogenic signaling induced by angiopoietin-1. Recombinant canstatin probably inhibits angiogenesis and lymphangiogenesis via suppression of the integrin-dependent FAK signaling induced by angiopoietin-1/Tie-2 and/or VEGFR-3.