Collagen composition of normal and myxomatous human mitral heart valves.

The collagens were studied in 13 normal and 19 myxomatous human mitral valves. The collagens of the valve were completely solubilized by using a method consisting of guanidinium chloride extraction, limited pepsin digestions and CNBr cleavage of the residue. The normal valves contained 74% type I, 24% type III and 2% type V collagen. The type I and type III collagens had similar solubility patterns, although only type I collagen was detected in the guanidinium chloride extract. Type V collagen was only detected in the first pepsin extract. The type I and III collagens had higher contents of hydroxylysine than did the same collagens from age-matched dermis. The two-dimensional electrophoretic 'maps' of CNBr-cleavage peptides showed low recoveries of the C-terminal alpha 1(I) CB6 and alpha 1(III) CB9 peptides, which are involved in forming intermolecular cross-linkages. Most of the reducible cross-linkages were present in large-Mr peptide complexes, and these complexes were shown by labelling with 125I to include the tyrosine-containing alpha 1(I) CB6 peptide. The myxomatous valves contained 67% type I, 31% type III and 2% type V collagens. There was a significant increase in the concentration of each type of collagen, which consisted of a 9% increase of type I collagen, a 53% increase of type III collagen and a 25% increase of type V collagen. The contents of hydroxylysine in type I and III collagens and the electrophoretic 'maps' of the CNBr-cleavage peptides involved in cross-linkages did not differ significantly from the results obtained from the normal valves. The biochemical findings suggest that there is an increased production of collagen, in particular type III collagen, and glycosaminoglycan as well as a proliferation of cells as part of a repair process in the myxomatous valves.     

Fibril-forming collagens in lamprey

Five types of collagen with triple-helical regions approximately 300 nm in length were found in lamprey tissues which show characteristic D-periodic collagen fibrils. These collagens are members of the fibril forming family of this primitive vertebrate. Lamprey collagens were characterized with respect to solubility, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carboxylmethyl-cellulose chromatography, peptide digestion patterns, composition, susceptibility to vertebrate collagenase, thermal stability, and segment long spacing-banding pattern. Comparison with fibril-forming collagens in higher vertebrates (types I, II, III, V, and XI) identified three lamprey collagens as types II, V, and XI. Both lamprey dermis and major body wall collagens had properties similar to type I but not the typical heterotrimer composition. Dermis molecules had only alpha 1(I)-like chains, while body wall molecules had alpha 2(I)-like chains combined with chains resembling lamprey type II. Neither collagen exhibited the interchain disulfide linkages or solubility properties of type III. The conservation of fibril organization in type II/type XI tissues in contrast to the major developments in type I and type III tissues after the divergence of lamprey and higher vertebrates is consistent with these results. The presence of type II and type I-like molecules as major collagens and types V and XI as minor collagens in the lamprey, and the differential susceptibility of these molecules to vertebrate collagenase is analogous to the findings in higher vertebrates.     

Type V collagen. Quantitation in normal lungs and in lungs of rats with bleomycin-induced pulmonary fibrosis

Type V collagen was first isolated in 1976; there is still controversy as to how many molecular species of type V collagen exist. Although its structural and functional roles remain unclear, reports of changes in the relative amount of type V collagen from that present in normal tissue have been reported in such diverse pathologic conditions as atherosclerotic aortas, prolapsed mitral valves, and fibrotic lungs. Methods for quantitating type V collagen relative to other collagens have consisted of solubilizing the collagen with pepsin and then analyzing the ratios of the intact chains by gel electrophoresis or by column chromatography. In tissues in which only a small percentage of the total collagen can be solubilized by pepsin, such analyses may not accurately reflect changes in the total collagen present. In this report, a method for quantitating type V collagen relative to types I and III collagens based on CNBr peptide mapping is presented. CNBr solubilizes virtually all the collagen present in any tissue. The method is applied to a model of bleomycin-induced pulmonary fibrosis in rats. It was found that type I collagen increased relative to types III and V collagens, which seemed to remain at values comparable to those observed in lungs from control (normal) rats, both in terms of newly synthesized collagen (collagen synthesized by lung minces during 4 h in culture) and total unlabeled lung collagen (collagen synthesized during the life of the animal).     

Differences in the extent and heterogeneity of lysyl hydroxylation in embryonic chick cranial and long bone collagens

The hydroxylation of lysine in embryonic chick long bone and mandibular collagen was found to be approximately 3-fold greater than that of the collagens of adult animals. In contrast, no significant difference was found in extent of lysine hydroxylation of the collagens of frontal bones of embryos and postnatal animals. Both histochemical and biochemical evidence established that full thickness diaphyseal bone samples contained cartilage and, consequently, type II collagen which undoubtedly contributed to the higher hydroxylysine contents of young postnatal animals reported previously. DEAE ion exchange chromatography of the alpha 1(I) chains of lathyritic long bone and mandibular collagens isolated by carboxymethyl-cellulose ion exchange chromatography showed considerable heterogeneity, whereas the alpha 1(I) chains obtained from lathyritic frontal bone collagen did not. Three fractions of alpha 1(I) chains of long bones and mandibular collagen were isolated which differed significantly in their hydroxylysine contents. The relative proportion of the three peaks changed as a function of embryonic age and maturation: more of the alpha 1(I) chains with the highest hydroxylysine content was present in the collagen synthesized earliest during embryonic development. This is consistent with results which demonstrated that the collagens synthesized earliest during embryonic and postnatal development had the highest hydroxylysine contents.     

Involvement of HMGB1 and HMGB2 proteins in exogenous DNA integration reaction into the genome of HeLa S3 cells

Electrophoretic and Western blot studies were conducted on collagen fractions extracted from Sepia officinalis (cuttlefish) cartilage using a modified salt precipitation method developed for the isolation of vertebrate collagens. The antibodies used had been raised in rabbit against the following types of collagen: Sepia I-like; fish I; human I; chicken I, II, and IX; rat V; and calf IX and XI. The main finding was that various types of collagen are present in Sepia cartilage, as they are in vertebrate hyaline cartilage. However, the main component of Sepia cartilage is a heterochain collagen similar to vertebrate type I, and this is associated with minor forms similar to type V/XI and type IX. The cephalopod type I-like heterochain collagen can be considered a first step toward the evolutionary development of a collagen analogous to the typical collagen of vertebrate cartilage (type II homochain). The type V/XI collagen present in molluscs, and indeed all phyla from the Porifera upwards, may represent an ancestral collagen molecule conserved relatively unchanged throughout evolution. Type IX-like collagen seems to be essential for the formation of cartilaginous tissue.     

The effects of mechanical stability on the macromolecules of the connective tissue matrices produced during fracture healing. I. The collagens

Summary The distribution of types I, II, III, V and IX collagens in healing fractures of the rabbit tibia has been demonstrated by immunofluorescent techniques. It has also been shown that the mechanical stability of the healing fracture affects both the distribution and types of the collagens present.The initial fibrous matrix contains types III and V collagens; type I collagen was only located in this matrix if unfixed tissue was used. In mechanically stable fractures, cancellous bone forms over the entire periosteal surface by 5–7 days; type I collagen is laid down within the previous fibrous matrix. The trabeculae are heterogeneous in their collagen content. The cavities contain a matrix of types III and V collagens. Small nodules of cartilage may be present between 7 and 14 days; these contain types II and IX collagens.In mechanically unstable fractures, cancellous bone is initially formed away from the fracture gap. The fibrous tissue over the gap is replaced by cartilage; types II and IX collagens are laid down on the pre-existing fibrous matrix. The cartilage is replaced by endochondral ossification. At the ossification front, type I collagen is found around the chondrocyte lacunae of the spicules of cartilage. The new trabeculae contain a core of cartilage which is surrounded by a bone matrix of types I and V collagens.The fracture gaps are invaded by fibrous tissue, which contain types III and V collagens. This is later replaced by cancellous bone.     

Collagen defects in lethal perinatal osteogenesis imperfecta.

Quantitative and qualitative abnormalities of collagen were observed in tissues and fibroblast cultures from 17 consecutive cases of lethal perinatal osteogenesis imperfecta (OI). The content of type I collagen was reduced in OI dermis and bone and the content of type III collagen was also reduced in the dermis. Normal bone contained 99.3% type I and 0.7% type V collagen whereas OI bone contained a lower proportion of type I, a greater proportion of type V and a significant amount of type III collagen. The type III and V collagens appeared to be structurally normal. In contrast, abnormal type I collagen chains, which migrated slowly on electrophoresis, were observed in all babies with OI. Cultured fibroblasts from five babies produced a mixture of normal and abnormal type I collagens; the abnormal collagen was not secreted in two cases and was slowly secreted in the others. Fibroblasts from 12 babies produced only abnormal type I collagens and they were also secreted slowly. The slower electrophoretic migration of the abnormal chains was due to enzymic overmodification of the lysine residues. The distribution of the cyanogen bromide peptides containing the overmodified residues was used to localize the underlying structural abnormalities to three regions of the type I procollagen chains. These regions included the carboxy-propeptide of the pro alpha 1(I)-chain, the helical alpha 1(I) CB7 peptide and the helical alpha 1(I) CB8 and CB3 peptides. In one baby a basic charge mutation was observed in the alpha 1(I) CB7 peptide and in another baby a basic charge mutation was observed in the alpha 1(I) CB8 peptide. The primary defects in lethal perinatal OI appear to reside in the type I collagen chains. Type III and V collagens did not appear to compensate for the deficiency of type I collagen in the tissues.     

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.     

Metalloproteinases from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin.

Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.     

Electrophoretic and immunochemical study of collagens from Sepia officinalis cartilage

Electrophoretic and Western blot studies were conducted on collagen fractions extracted from Sepia officinalis (cuttlefish) cartilage using a modified salt precipitation method developed for the isolation of vertebrate collagens. The antibodies used had been raised in rabbit against the following types of collagen: Sepia I-like; fish I; human I; chicken I, II, and IX; rat V; and calf IX and XI. The main finding was that various types of collagen are present in Sepia cartilage, as they are in vertebrate hyaline cartilage. However, the main component of Sepia cartilage is a heterochain collagen similar to vertebrate type I, and this is associated with minor forms similar to type V/XI and type IX. The cephalopod type I-like heterochain collagen can be considered a first step toward the evolutionary development of a collagen analogous to the typical collagen of vertebrate cartilage (type II homochain). The type V/XI collagen present in molluscs, and indeed all phyla from the Porifera upwards, may represent an ancestral collagen molecule conserved relatively unchanged throughout evolution. Type IX-like collagen seems to be essential for the formation of cartilaginous tissue.     

Differential expression of fibrillar collagen genes during callus formation

An experimental fracture healing model in the rat tibio-fibular bone was employed to study the appearance of messenger RNAs for types I, II and III collagens during endochondral fracture repair. Total RNA was extracted from normal bone and from callus tissue at various time points. The total RNAs were analyzed in Northern hybridization for their contents of procollagen mRNAs using specific cDNA clones. The results show that during the first week of fracture repair type III collagen mRNA is increased to the greatest extent, followed by type II collagen mRNA during the second week. The 28-day callus resembles bone by containing mainly type I collagen mRNAs and very little type II or III collagen mRNA.     

The expression of the fibrillar collagen genes during fracture healing: heterogeneity of the matrices and differentiation of the osteoprogenitor cells

The cells that express the genes for the fibrillar collagens, types I, II, III and V, during callus development in rabbit tibial fractures healing under stable and unstable mechanical conditions were localized. The fibroblast-like cells in the initial fibrous matrix express types I, III and V collagen mRNAs. Osteoblasts, and osteocytes in the newly formed membranous bone under the periosteum, express the mRNAs for types I, III and V collagens, but osteocytes in the mature trabeculae express none of these mRNAs. Cartilage formation starts at 7 days in calluses forming under unstable mechanical conditions. The differentiating chondrocytes express both types I and II collagen mRNAs, but later they cease expression of type I collagen mRNA. Both types I and II collagens were located in the cartilaginous areas. The hypertrophic chondrocytes express neither type I, nor type II, collagen mRNA. Osteocalcin protein was located in the bone and in some cartilaginous regions. At 21 days, irrespective of the mechanical conditions, the callus consists of a layer of bone; only a few osteoblasts lining the cavities now express type I collagen mRNA.We suggest that osteoprogenitor cells in the periosteal tissue can differentiate into either osteoblasts or chondrocytes and that some cells may exhibit an intermediate phenotype between osteoblasts and chondrocytes for a short period. The finding that hypertrophic chondrocytes do not express type I collagen mRNA suggests that they do not transdifferentiate into osteoblasts during endochondral ossification in fracture callus.     

FBJ virus-induced osteosarcoma contains type I, type I trimer, type III as well as type V collagens

The FBJ osteosarcoma (a virus-induced osteosarcoma named after its discoverers, Finkel, Biskis, and Jinkins) contains an extensive extracellular matrix. Collagens were extracted by digestion with pepsin in dilute acetic acid from tumors grown in lathyritic mice and fractionated by differential salt precipitation, yielding five fractions. Fraction 1 (precipitated at acidic 0.7 M and neutral 2.0 M NaCl) gave rise mainly to alpha 1(III) chain on phosphocellulose column chromatography. The alpha 1(III) chain was identified by its typical behavior on interrupted electrophoresis and analysis of the CNBr-cleaved peptides. The alpha 1(III) chain of the FBJ tumor had a high content of hydroxylysine and neutral saccharide. Fraction 2 (precipitated at acidic 0.7 M and neutral 4.5 M NaCl) yielded alpha 1(I) and alpha 2(I) chains on the phosphocellulose column from which alpha 1(I) was eluted as a broad peak, conceivably reflecting a high content of hydroxylysine and neutral saccharide. Fraction 4 (precipitated at acidic 1.2 M and neutral 4.5 M NaCl) yielded type V collagen, which also featured an exceptionally high content of neutral saccharide (Yamagata, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 1208-1214). The proportions of type I, type I trimer, type III, and type V collagens extracted by pepsin digestion from FBJ tumor were calculated to be 33, 29, 26, and 12%, respectively. The FBJ tumor is free from invasion by blood vessels, shows no deposition of calcium, and thus has the appearance of cartilage. But type II collagen, a specific gene product of cartilage, could not be identified in any of the fractions analyzed. Contrary to its appearance, collagen type analyses indicate that FBJ osteosarcoma is literally induced from osteogenic cells.     

Altered ratio of collagen chains in bone of a patient with non-lethal osteogenesis imperfecta.

Bone from a patient with osteogenesis imperfecta contained type III collagen which was absent in control bone. The ratio of alpha 1(I)/alpha 2(I) in type I collagen of patient's bone was increased (2.9 vs. 2.3 +/- 0.2 in controls) and the ratio of dimers beta 11/beta 12/beta 22 was altered due to the increased beta 22 content. No abnormality was observed in collagen from the patient's skin. The altered composition of collagen in bone, but the normal composition in skin suggests that the disease in the patient is due to impaired regulation of the synthesis of collagens in bone, rather than by a mutation in one of the two type I collagen genes. Unlike in skin, all the type III collagen in patient's bone was pepsin-soluble indicating an inability of the bone to incorporate type III collagen into mature highly cross-linked extracellular matrix.     

Fibroblast behavior on gels of type I, III, and IV human placental collagens

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.     

Interaction of osteogenin, a heparin binding bone morphogenetic protein, with type IV collagen

Osteogenin, an extracellular matrix component of bone, is a heparin binding differentiation factor that initiates endochondral bone formation in rats when implanted subcutaneously with an insoluble collagenous matrix. We have examined the interaction of osteogenin with various extracellular matrix components including basement membranes. Osteogenin, purified from bovine bone, binds avidly to type IV collagen and to a lesser extent to both type I and IX collagens. Osteogenin binds equally well to both native and denatured type IV collagen. Both alpha 1 and alpha 2 chains of type IV collagen are recognized by osteogenin. Osteogenin binds to a collagen IV affinity column, and is eluted by 6.0 M urea with 1 M NaCl, pH 7.4, and the eluate contained the osteogenic activity as demonstrated in vivo. Binding of osteogenin to collagen IV is not influenced by either laminin or fibronectin. These results imply that osteogenin binding to extracellular matrix components including collagens I and IV and heparin may have physiological relevance, and such interactions may modulate its local action.     

Collagen heterogeneity within different growth regions of long bones of rachitic and nonrachitic chicks

The different anatomical regions involved in osteogenesis in the chick long bone have been examined for heterogeneities in collagen structure that might relate to the mechanism of ossification. Experimentally induced lathyrism was employed to enhance collagen solubility, and vitamin D deficiency to allow accumulation of osteoid, the precursor of bone matrix. The extractable lathyritic collagens of the cartilaginous and osseous regions of growing long bones from rachitic and non-rachitic chicks were examined for alpha-chain type and amino acid composition. In both groups of animals the growth plate and cartilaginous regions of the epiphysis gave collagen molecules of the constitution [alpha1(II)](3), whereas the ossifying regions contained [alpha1(I)](2) alpha2. The degree of hydroxylation of the lysine moieties was increased by approximately 50% in the alpha1(I)-chain and alpha2-chain of rachitic bone collagen. Since uncalcified osteoid is greatly enriched in rachitic bone, it is concluded that the collagen of osteoid has the configuration [alpha1(I)](2) alpha2, similar to that of bone matrix, but has an elevated hydroxylysine content. The possible relationship of this difference to the mechanism of calcification is discussed.     

Proteolytic inactivation of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin by oxidatively activated human neutrophil metalloproteinases.

Human neutrophils use the H2O2-myeloperoxidase-chloride system to generate chlorinated oxidants capable of activating metalloproteinase zymogens that hydrolyze not only native and denatured collagens, but also the serine proteinase inhibitor (serpin) alpha 1-proteinase inhibitor (alpha 1 PI). To identify the metalloenzyme that hydrolyzes and inactivates alpha 1 PI, neutrophil releasates were chromatographed over gelatin-Sepharose and divided into fractions containing either progelatinase or procollagenase. The gelatinase-containing fraction cleaved alpha 1 PI in a manner inhibitable by native type V, but not type I, collagen. Conversely, while the collagenase-containing fraction also cleaved alpha 1 PI, this activity was inhibited by type I, but not type V, collagen. Because type I and V collagens are competitive substrates for collagenase and gelatinase, respectively, each of the metalloproteinase zymogens were purified to apparent homogeneity and examined for alpha 1 PI-hydrolytic activities. Both purified gelatinase and collagenase inactivated alpha 1PI by hydrolyzing the serpin within its active-site loop at the Phe352-Leu353 and Pro357-Met358 bonds, albeit with distinct kinetic properties. Furthermore, purified collagenase, but not gelatinase, cleaved a second serpin, alpha 1-antichymotrypsin, by hydrolyzing the Ala362-Leu363 bond within its active-site loop. These data demonstrate that human neutrophils use chlorinated oxidants to activate collagenolytic metalloproteinases whose substrate specificities can be extended to members of the serpin superfamily.     

Changes in the distribution of fibrillar collagens in the collateral and cruciate ligaments of the rabbit knee joint during fetal and postnatal development

Summary The collateral ligaments can be clearly distinguished in the 25-day fetal rabbit knee joint. Types I and V collagens are present in the extracellular matrix between the cells of the lateral and medial collateral ligaments and this distribution persists until the rabbit is skeletally mature. From 8 months onwards type III collagen is also present, particularly around the cells. Type I collagen mRNA is expressed by the cells from the 25-day fetal to 8-month-old adult ligament. The ligament sheath is composed of types III and V collagens. The cruciate ligaments are present between the femur and tibia in the 20-day fetus. The matrix is composed of types I and V collagens from the 25-day fetus until at 12- to 14-weeks postnatal, type III collagen appears in the pericellular regions together with type V. At 8 months and 2 years, the amount of type III collagen has increased. All the cells express the mRNA for type I collagen at 12- to 14-weeks, but only isolated cells express this mRNA at 8 months. Thus, both the collateral and cruciate ligaments undergo changes in their complement of collagens during postnatal development and ageing. The implications of these complex interactions of different types of collagen are discussed in relation to healing and the surgical replacement of torn ligaments by tendons.