Monoclonal antibodies to type V collagen for immunohistological examination of new tissue deposition associated with biomaterial implants

We developed a panel of highly specific monoclonal antibodies (MAb) against dog Type V collagen. Each antibody showed differential reactivities towards Type V collagen from other species. All the antibodies were highly reactive in conventional ELISA, as well as with electroblots of collagen after polyacrylamide gel electrophoresis using non-denaturing conditions. The MAb were shown to be suitable for the immunohistological detection of Type V collagen in tissue sections, although this normally required pre-treatment of sections with 50 mM acetic acid. In particular, the antibodies were shown to be useful for examining samples of a collagen-based biomaterial, a vascular prosthesis, after explant from evaluation in an animal model. This showed that Type V collagen was most prominent in regions of new tissue formation within the neointima, close to the inner surface of the prosthesis. The broad spectrum of differential reactivities allows the antibodies to be used for a wide range of experimental models. These MAb therefore provide a novel approach for the evaluation of biomaterial performance, particularly for collagen-based implants.     

Abnormal structure of type II collagen in a patients with funnel chest

The electrophoretical analysis and CNBr-peptide mapping of the collagens, isolated from the costal cartilage of 30 patients with non-classified and syndromal forms of pex excavatum (funnel chest) (27 patients) and pex carinatum (3 patients) was carried out. In case of one patient with the nonclassified form of funnel chest the electrophoretical mobility of CB 9.7-peptide was found to be decreased. The electrophoretical mobilities of other peptides are not markedly changed. The data obtained allow one to suggest the mutation causing the defect in the region about 160 amino acid residues distant from the C-end of alpha 1 (II) chain of type II collagen of the patient.     

Inhibition of cell adhesion by type V collagen.

Human umbilical vein endothelial cells grew well in dishes coated with collagen types I, II, III, or IV. However, the same cells tended to detach themselves from dishes coated with type V collagen, and cell proliferation in these dishes was inhibited. Such anti-adhesive activity was partially retained by heat-denatured type V collagen or by its alpha 1 chain, but not by its alpha 2 chain. Several other cell types did not adhere to the type V collagen substratum even in the presence of 10% serum. The cell types strongly inhibited from adhering by type V collagen included Swiss mouse 3T3 cells and their MSV-transformants, BALB/c 3T3 cells and their methylcholanthrene-transformants, NIH 3T3 cells and their ras-transformants, BHK cells, CHO-9 cells, CHO-K1 cells, and mouse melanoma B16-F10 cells. Using Swiss mouse 3T3, we studied the effects of type V collagen on cell adhesion to fibronectin in serum-free medium. When the culture dishes were coated with a mixture of fibronectin with various concentrations of type V collagen, the adhesion of the cells was inhibited depending on the concentration of type V collagen. The inhibition of cell adhesion by type V collagen was competitively overcome by increased concentrations of fibronectin. The activity that interferes with the effects of fibronectin was retained mainly by the alpha 1 chain of heat-denatured type V collagen.     

Subepithelial type II collagen deposition during embryonic chick limb development

Summary Type II collagen is a major component of hyaline cartilage but recent studies have demonstrated the presence of this protein in a variety of interfaces that separate epithelia from mesenchyme, particularly in early stages of embryonic chick development. In the present study an immunohistochemical analysis of the distribution of type II collagen was performed on closely staged wing buds of early chick embryo. This report describes how using two different monoclonal antibodies against type II collagen and the peroxidase or fluorescence staining technique reveals that deposition of type II collagen at the ectoderm-mesenchyme interface occurs in the proximal part of the limb coincidentally with the appearance of this protein in the proximal core region, where chondrogenesis begins (stage 25). Then the staining in the subepithelial region spreads distallly with time, following the progression of the formation of cartilage rudiments. At about 7 days of development type II collagen is present under the apical ectoderm ridge and surrounds completely the wing bud underneath the epithelium. At the same time, another antibody directed against the cartilage-specific proteoglycan core protein only stains the chondrogenic central core of the limb and not the subepithelium. Although type II collagen and cartilage-specific proteoglycan are closely associated in the cartilage, the observations presented here suggest that the deposition of these proteins can be regulated independently during limb formation. The role of type II collagen at the epithelium-mesenchyme interface during limb formation is still to be determined.     

Development of a functional skin matrix requires deposition of collagen V heterotrimers

Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the alpha2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the alpha1(V)(2)alpha2(V) heterotrimer with the result that the alpha1(V) homotrimer is the predominant species deposited into the matrix. Second, the alpha1(V) homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the alpha1(V) collagen gene that leads to additional deposition of alpha1(V) homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.     

Neoplastic association of enhanced type V collagen production in rat fibrosarcoma

Collagens present in the connective tissues of the extracellular matrix of fibrosarcoma were isolated and characterized. The fibrosarcoma was induced in rats by the administration of 3-methylcholanthrene. The results obtained were compared with normal muscle. An excess amount of type V collagen was found to be produced by the fibrosarcoma tissue compared to the normal muscle. Type V collagen from fibrosarcoma was characterized on the basis of solubility behavior in sodium chloride solutions, electrophoretic mobility on SDS-polyacrylamide gels, elution pattern of phosphocellulose chromatography and amino acid composition.     

Biosynthesis and regulation of type V collagen in diploid human fibroblasts

The biosynthesis of type V collagen and its regulation were studied using diploid human gingival fibroblasts. Cells were metabolically labeled with radioactive amino acids and labeled proteins were subjected to limited pepsin digestion, DEAE-cellulose chromatography at 15 degrees C, and polyacrylamide gel electrophoresis. Proteins eluted from DEAE-cellulose columns by 0.25 M NaCl contained a collagen species which was resistant to mammalian collagenase and had alpha chains with hydroxylysine/lysine ratios and CNBr peptide patterns similar to alpha 1(V) and alpha 2(V). Procollagen(V) fractions obtained by DEAE-cellulose chromatography and immunoprecipitates of type V collagen antibody contained polypeptides with Mr = 239,000, 219,000, 198,000, 174,000, 157,000, and 132,000. By comparing the CNBr peptide maps of these proteins with those of standard alpha 1(V) and alpha 2(V) chains, the first three polypeptides were shown to be related to alpha 1(V) and the others to alpha 2(V). It was concluded that the gingival fibroblasts synthesize type V collagen, that the pro alpha 1(V) and the pro alpha 2(V) chains have Mr = 239,000 and 174,000, respectively, and that the alpha 1(V) and alpha 2(V) chains laid in the form of fibrils have Mr = 198,000 and 132,000, respectively. A detectable amount of type V collagen was synthesized only at high cell density, and it was associated with the cell layer. The amount and proportion of type V synthesized were increased when the cells were labeled in the presence of serum, and the increase was accompanied by a decrease in type III. This effect was dependent on serum concentration. Serum obtained from platelet-poor plasma failed to elicit this effect, and it was restored by the addition of platelet-derived growth factor. Platelet-derived growth factor was effective in medium with and without platelet-poor serum. Thus, it appears that platelet-derived growth factor may be an important regulatory factor in the synthesis of types V and III collagens.     

Abnormal growth plate function in pigs carrying a dominant mutation in type X collagen

We have identified a naturally occurring, dominant mutation that causes dwarfism in domestic pigs (Sus scrofa). With a positional candidate gene approach, the dwarf phenotype was shown to be a result of a single amino acid change, G590R, in the α1(X) chain of type X collagen. Type X collagen is a homotrimer of α1(X) chains encoded by the COL10A1 gene, which is expressed in hypertrophic chondrocytes during the process of endochondral ossification. An amino acid substitution at the equivalent position in human type X collagen, G595E, has previously been shown to cause Schmid metaphyseal chondrodysplasia (SMCD), which is a relatively mild skeletal disorder associated with dwarfism and growth plate abnormality. Consistent with the clinical phenotype of SMCD patients, radiological and histological examination of the dwarf pigs revealed metaphyseal chondrodysplasia in the long bones. Yeast-based, two-hybrid protein interaction studies and in vitro assembly experiments demonstrated that the amino acid substitution interfered with the ability of the mutated collagen molecules to engage in trimerization. This work establishes that the chondrodysplastic dwarf pigs by genetic, biochemical, radiological and histological criteria provide a valid animal model of SMCD. Received: 25 May 2000 / Accepted: 25 July 2000     

Primary structure of the heparin-binding site of type V collagen

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.     

The enhancement of muscle trichinosis in rabbits after immunization with heterologous serum proteins

The enhancement of muscle trichinosis in rabbits after immunization with heterologous serum proteins. International Journal for Parasitology4, 193–196. Rabbits were immunized with electrophoretically-purified rat serum albumin, β-globulin or γ-globulin in Freund's Complete Adjuvant and then inoculated with Trichinella spiralis larvae. Muscle parasitism was greatly enhanced in all the treated animals; this, plus the high levels of anti-rat and low levels of antiparasite antibody, indicates that antigenic competition can increase parasite numbers in trichinosis. Antigenic competition may play a role in the establishment of natural infections by T. spiralis by deflecting a protective immunological response by the host.     

Distribution and synthesis of type V collagen in the rat mammary gland

In the 100-day-old virgin and lactating rat mammary glands, type V collagen is mainly present in the interstitial connective tissue and in association with blood vessels. It is not present in the basement membrane region surrounding the ducts in mature virgin glands but is present in this region in neonatal and lactating glands. Ultrastructural localization of type V collagen reveals that it is mainly located on the basal surface (i.e., the surface in contact with the basement membrane) of epithelial but not myoepithelial cells. In addition, type V collagen is located on some interstitial collagen fibers and on a large number of granules that are in close proximity to the basal surface of both epithelial and myoepithelial cells. Immunofluorescence and biochemical studies indicate that several clonal mammary fibroblastic cell lines synthesize type V collagen in vitro. In some cell lines, type V collagen is secreted as an extensive fibrillar meshwork on the surface of the cells, whereas in other cell lines, it is secreted beneath the cells around their periphery. A number of mammary epithelial and myoepithelial-like cells, however, do not synthesize type V collagen in vitro.     

Intercellular accumulation of type V collagen fibrils in accordance with cell aggregation

We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization.     

Abnormal type I collagen metabolism by cultured fibroblasts in lethal perinatal osteogenesis imperfecta.

Cultured skin fibroblasts from seven consecutive cases of lethal perinatal osteogenesis imperfecta (OI) expressed defects of type I collagen metabolism. The secretion of [14C]proline-labelled collagen by the OI cells was specifically reduced (51-79% of control), and collagen degradation was increased to twice that of control cells in five cases and increased by approx. 30% in the other two cases. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that four of the OI cell lines produced two forms of type I collagen consisting of both normally and slowly migrating forms of the alpha 1(I)- and alpha 2(I)-chains. In the other three OI cell lines only the 'slow' alpha (I)'- and alpha 2(I)'-chains were detected. In both groups inhibition of the post-translational modifications of proline and lysine resulted in the production of a single species of type I collagen with normal electrophoretic migration. Proline hydroxylation was normal, but the hydroxylysine contents of alpha 1(I)'- and alpha 2(I)'-chains purified by h.p.l.c. were greater than in control alpha-chains. The glucosylgalactosylhydroxylysine content was increased approx. 3-fold while the galactosylhydroxylysine content was only slightly increased in the alpha 1(I)'-chains relative to control alpha 1(I)-chains. Peptide mapping of the CNBr-cleavage peptides provided evidence that the increased post-translational modifications were distributed throughout the alpha 1(I)'- and alpha 2(I)'-chains. It is postulated that the greater modification of these chains was due to structural defects of the alpha-chains leading to delayed helix formation. The abnormal charge heterogeneity observed in the alpha 1 CB8 peptide of one patient may reflect such a structural defect in the type I collagen molecule.     

FBJ virus-induced osteosarcoma has type V collagen consisting of A, B and C-like chains in addition to type I collagen

A study was carried out on collagen chains of FBJ virus-induced osteosarcoma. Collagens were extracted from pepsin-digested tissues and fractionated by differential salt precipitation. An acidic 0.7 M NaCl precipitate contained type I, type I trimer and/or type III collagens. Collagen fractions precipitated at acidic 1.2 M NaCl showed features characteristic of type V collagen consisting of three chains (mol. weights of which were 120K, 110K and 100K daltons). None of these chains, however, was identical to any of the B, C or A chains reported by Sage et al. in 1979 (1), judging from amino acid composition, cyanogen bromide cleavage and phosphocellulose chromatography data.     

Repetitive immunization breaks tolerance to type XVII collagen and leads to bullous pemphigoid in mice

Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease of the elderly associated with considerable morbidity and mortality. As unspecific immunosuppressants are still the mainstay of BP therapy, several animal models, based on the passive transfer of autoantibodies or immune cells, have been developed to obtain a better understanding of the pathogenesis of BP and evaluate novel therapeutic interventions. We describe in this study an experimental model inducing BP by immunization of immunocompetent mice with a recombinant form of the immunodominant 15th noncollagenous domain of murine BP180 (type XVII collagen). The homologous noncollagenous 16A domain of human BP180 has previously been identified as an immunodominant region in human BP. Immunization of female SJL/J mice with the murine peptide led to clinical disease within 14 wk in 56% of mice. In contrast, none of the other strains developed blisters despite the presence of autoantibodies. The clinical disease manifested for at least 8 wk without further manipulation. This novel immunization-induced model reflects key immunopathological characteristics of human BP, including binding of complement-fixing autoantibodies along the dermal-epidermal junction, elevated total IgE serum levels, and infiltration of skin lesions with eosinophilic granulocytes. The use of immunocompetent mice and the induction of sustained clinical disease not requiring additional interventions make this immunization-induced mouse model most suitable to further explore the pathogenesis of BP and novel therapeutic interventions for this and other autoantibody-mediated diseases.     

Molecular assembly, secretion, and matrix deposition of type VI collagen

Monoclonal antibodies reactive with the tissue form of type VI collagen were used to isolate the type VI collagen polypeptides from cultured fibroblasts and muscle cells. Two [35S]methionine-labeled polypeptides of 260 and 140 kD were found intracellularly, in the medium, and in the extracellular matrix of metabolically labeled cells. These polypeptides were disulfide cross-linked into very large complexes. The 260- and 140-kD polypeptides were intimately associated and could not be separated from each other by reduction without denaturation. In the absence of ascorbic acid, both polypeptides accumulated inside the cell, and their amounts in the medium and in the matrix were decreased. These results suggest that both the 260- and the 140-kD polypeptides are integral parts of the type VI collagen molecule. Examination of type VI collagen isolated from the intracellular pool by electron microscopy after rotary shadowing revealed structures corresponding to different stages of assembly of type VI collagen. Based on these images, a sequence for the intracellular assembly of type VI collagen could be discerned. Type VI collagen monomers are approximately 125 nm long and are composed of two globules separated by a thin strand. The monomers assemble into dimers and tetramers by lateral association. Only tetramers were present in culture media, whereas both tetramers and multimers were found in extracellular matrix extracts. The multimers appeared to have assembled from tetramers by end-to-end association into filaments that had prominent knobs and a periodicity of approximately 110 nm. These results show that, unlike other collagens, type VI collagen is assembled into tetramers before it is secreted from the cells, and they also suggest an extracellular aggregation mechanism that appears to be unique to this collagen.     

Insulin binds to type V collagen with retention of mitogenic activity

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.