Minor Collagen Research Articles

Gene expression patterns in the bone tissue of women with fibrous dysplasia

Fibrous dysplasia is an isolated skeletal disorder caused by a somatic activating mutation of GNAS gene with abnormal unmineralized matrix overproduction and extensive undifferentiated bone cell accumulation in the fibro-osseous lesions. The aim of our investigation was to identify genes that are differently expressed in fibrous versus non-fibrous human bone and to describe the relationships between these genes using multivariate data analysis. Six bone tissue samples from female patients with fibrous dysplastia (FD) and seven bone tissue samples from women without FD (non-FD) were examined. The expression differences of selected 118 genes were analyzed by the TaqMan probe-based quantitative real-time RT-PCR system. The Mann-Whitney U-test indicated marked differences in the expression of 22 genes between FD and non-FD individuals. Nine genes were upregulated in FD women compared to non-FD ones and 18 genes showed a downregulated pattern. These altered genes code for minor collagen molecules, extracellular matrix digesting enzymes, transcription factors, adhesion molecules, growth factors, pro-inflammatory cytokines, and lipid metabolism-affected substrates. Canonical variates analysis demonstrated that FD and non-FD bone tissues can be distinguished by the multiple expression profile analysis of numerous genes controlled via a G-protein coupled pathway and BMP cascade as well as genes coding for extracellular matrix composing molecules. The remarkable changed gene expression profile observed in the fibrous dysplastic human bone tissue may provide further insight into the pathogenetic process of fibrous degeneration of bone.

Sp7/Osterix up-regulates the mouse pro-α3(V) collagen gene ( Col5a3) during the osteoblast differentiation

Type V collagen is a quantitatively minor collagen, but acts as critical regulator of fibril formation in the extracellular matrix. The purpose of this study is to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Sp7/Osterix is a transcription factor specifically expressed by osteoblasts and is important for osteoblast differentiation. The overexpression of Sp7/Osterix significantly increased the promoter activity and the endogenous mRNA level of the Col5a3 gene in osteoblastic cells. Conversely, a reduction of Sp7/Osterix by siRNA treatment decreased the promoter activity and the endogenous mRNA level of the Col5a3 gene. A CHIP assay confirmed that Sp7/Osterix interacted with the Col5a3 core promoter in vivo at the Sp1 binding site. The data from the experiments using the osteoblast differentiation model and the co-overexpression of Sp7/Osterix with Sp1 suggest that Sp7/Osterix promotes the expression of the collagen gene, Col5a3, and thereby playing a role in bone formation.

Pulmonary exposures to Sepiolite nanoclay particulates in rats: Resolution following multinucleate giant cell formation

Sepiolite is a magnesium silicate-containing nanoclay mineral and is utilized as a nanofiller for nanocomposite applications. We postulated that lung exposures to Sepiolite clay samples could produce sustained effects. Accordingly, the pulmonary and extrapulmonary systemic impacts in rats of intratracheally instilled Sepiolite nanoclay samples were compared with quartz or ultrafine (uf) titanium dioxide particle-types at doses of 1 mg/kg or 5 mg/kg. All particulates were well characterized, and dedicated groups were evaluated by bronchoalveolar lavage, lung cell proliferation, macrophage functional assays and full body histopathology at selected times postexposure (pe). Bronchoalveolar lavage results demonstrated that quartz particles produced persistent, dose-dependent lung inflammatory responses measured from 24 h through 3 months pe. Exposures to uf TiO 2 particles or Sepiolite samples produced transient neutrophilic responses at 24-h pe; however, unlike the other particle-types, Sepiolite exposures produced macrophage-agglomerates or multinucleate giant cells at 1 week, 5 weeks and 3 months pe. In vitro alveolar macrophage functional studies demonstrated that mononuclear cells recovered from quartz but not Sepiolite or uf TiO 2-exposed rats were deficient in their chemotactic capacities. Moreover, lung parenchymal cell proliferation rates were increased in rats exposed to quartz but not Sepiolite or uf TiO 2 particles. Histopathological evaluation of lung tissues revealed that pulmonary exposures to Sepiolite nanoclay or quartz samples produced inflammation in centriacinar regions at 24-h pe but the effects decreased in severity over time for Sepiolite and increased for quartz-exposed rats. The quartz-induced lesions were progressive and were characterized at 3 months by acinar foamy alveolar macrophage accumulation and septal thickening due to inflammation, alveolar Type II cell hyperplasia and collagen deposition. In the Sepiolite nanoclay group, the finding of multinucleated giant cell accumulation associated with minor collagen deposition in acinar regions was rarely observed. Exposures to ultrafine TiO 2 produced minimal effects characterized by the occurrence of phagocytic macrophages in alveolar ducts. Full body histopathology studies were conducted at 24 h and 3 months post particle exposures. Histopathological evaluations revealed minor particle accumulations in some mediastinal or thoracic lymph nodes. However, it is noteworthy that no extrapulmonary target organ effects were observed in any of the particle-exposed groups at 3 months postexposure.

PREPARATION AND CHARACTERIZATION OF PEPSIN-SOLUBILIZED TYPE I COLLAGEN FROM THE SCALES OF SNAKEHEAD (OPHIOCEPHALUS ARGUS)

ABSTRACT Pepsin-solubilized collagen prepared from the scales of snakehead (Ophiocephalus argus) was separated into two fractions, major and minor, by NaCl precipitation. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), amino acid composition, and secondary structure showed that the major collagen was typical type I collagen; in contrast, the minor collagen might be classified as type V collagen from the SDS-PAGE patterns and precipitation properties by NaCl. A sharp decrease in solubility of type I collagen was observed at the NaCl concentration of 40 g/L. The maximum and the minimum solubilities of collagen were observed at pH 3 and 8, respectively. Peptide maps of type I collagen digested by trypsin and V8 protease were different from those of calfskin and fish skin collagens. The imino acid content of type I collagen was lower than those of mammalian collagens and so did its denaturation temperature that was 30.3C obtained by viscosity measurement. PRACTICAL APPLICATIONS Collagen has been widely utilized as a material for foods, cosmetics, and pharmaceuticals. However, the use of collagen-derived products from land animals (e.g., bovine and pig) has been called into question because of foot-and-mouth disease crisis etc. Aquatic animal offals, which are readily available and inexpensive, seem to be safe sources for extraction of collagen. This work reports on preparation and characterization of collagen from snakehead scales, which will have potential in supplementing the skins and bones of land animals as an important collagen resource for use in functional food, biomedical, and cosmetic industries.

Caso para diagnóstico?

Mulher branca de 30 anos de idade há 15 anos apresentando lesões com depressões e herniações anulares, atróficas, bem circunscritas em pele previamente normal, predominando na região cervical e tronco; sem história familiar. Exames laboratoriais sem alterações e histopatológico com infiltrado inflamatório discreto na derme. Coloração com orceína evidenciou fibras elásticas fragmentadas e encurtadas. Após correlação anatomoclínica confirmou-se o diagnóstico de anetodermia de Schweninger-Buzzi.

Caso para diagnóstico?

Os autores relatam caso clínico de paciente do sexo masculino, diabético do tipo II insulinodependente, que apresentava lesões eritêmato-acastanhadas no abdômen e nas coxas, com ligeira atrofia assintomática. O exame histopatológico das lesões revelou a combinação de angiopatia e alterações leves do colágeno, compatíveis com o diagnóstico de dermopatia diabética.

The NC1 domain of type XIX collagen inhibits in vivo melanoma growth

Type XIX collagen is a minor collagen that localizes to basement membrane zones, together with types IV, XV, and XVIII collagens. Because several NC1 COOH-terminal domains of other chains from basement membrane collagens were reported to exhibit antitumor activity, we decided to study the effects of the NC1(XIX) collagen domain on tumor progression using an experimental in vivo model of mouse melanoma. We observed a 70% reduction in tumor volume in NC1(XIX)-treated mice compared with the corresponding controls. Histologic examination of the tumors showed a strong decrease in tumor vascularization in treated mice. In vitro, NC1(XIX) inhibited the migrating capacity of tumor cells and their capacity to invade Matrigel. It also inhibited the capacity of human microvascular endothelial cells to form pseudotubes in Matrigel. This effect was accompanied by a strong inhibition of membrane type-1 matrix metalloproteinase (matrix metalloproteinase-14) and vascular endothelial growth factor expression. Collectively, our data indicate that the NC1 domain of type XIX collagen exerts antitumor activity. This effect is mediated by a strong inhibition of the invasive capacities of tumor cells and antiangiogenic effects. NC1(XIX) should now be considered as a new member of the basement membrane collagen-derived matrikine family with antitumor and antiangiogenic activity.

Missense and silent mutations in COL2A1 result in Stickler syndrome but via different molecular mechanisms

Stickler syndrome due to mutations in COL2A1 is usually the result of premature termination codons and nonsense mediated decay resulting in haploinsufficiency of type II collagen. Here we present two missense mutations and one apparently silent mutation that each result in Stickler syndrome, but via different molecular mechanisms. One alters the translation initiating ATG codon. The second mutation is a unique glycine substitution in the minor collagen helix of the procollagen. To our knowledge a glycine substitution has not previously been reported in this region of fibrillar procollagens. The third mutation appears to be a silent change altering a GGC codon to GGT both for glycine, but use of a splicing reporter assay demonstrates that it results in missplicing and a shift in the reading frame.

Quantifying the contributions of structure to annulus fibrosus mechanical function using a nonlinear, anisotropic, hyperelastic model

The annulus fibrosus of the intervertebral disc is comprised of concentric lamella of oriented collagen fibers embedded in a hydrated proteoglycan matrix with smaller amounts of minor collagens, elastin, and small proteoglycans. Its structure and composition enable the disc to withstand complex loads and result in inhomogeneous, anisotropic, and nonlinear mechanical behaviors. The specific contributions of the annulus fibrosus constituent structures to mechanical function remain unclear. Therefore, the objective of this study was to use a structurally motivated, anisotropic, nonlinear strain energy model of annulus fibrosus to determine the relative contributions of its structural components to tissue mechanical behavior. A nonlinear, orthotropic hyperelastic model was developed for the annulus fibrosus. Terms to describe fibers, matrix, and interactions between annulus fibrosus structures (shear and normal to the fiber directions) were explicitly included. The contributions of these structures were analyzed by including or removing terms and determining the effect on the fit to multidimensional experimental data. Correlation between experimental and model-predicted stress, a Bland-Altman analysis of bias and standard deviation of residuals, and the contribution of structural terms to overall tissue stress were calculated. Both shear and normal interaction terms were necessary to accurately model multidimensional behavior. Inclusion of shear interactions more accurately described annulus fibrosus nonlinearity. Fiber stretch and shear interactions dominated contributions to circumferential direction stress, while normal and shear interactions dominated axial stress. The results suggest that interactions between fibers and matrix, perhaps facilitated by crosslinks, elastin, or minor collagens, augment traditional (i.e., fiber-uncrimping) models of nonlinearity.

TGF‐β1 generates a specific multicomponent extracellular matrix in human coronary SMC

Transforming growth factor (TGF-beta(1)) is postulated to play an important role in maintaining the structure and function of arterial tissue and protection against development of arteriosclerosis. The TGF-beta(1)-induced production of a stable extra-cellular matrix-rich plaque phenotype is suggested to be part of the protection against a switch to an unstable rupture-prone arteriosclerotic plaque. This study addresses the question of whether the expression profile and the type of extra-cellular matrix (ECM) generated by TGF-beta(1) stimulation have the structural feature of a fibril-rich stable matrix. Seventeen genes codings for ECM components of human coronary smooth muscle cells (SMCs) after a 24-h stimulation by TGF-beta(1) have been analyzed. Real-time RT-PCR was used to quantify the mRNA of genes under investigation. It was found that after TGF-beta(1) stimulation (a) the up-regulation of COL1A1-specific mRNA was associated with increased [(3)H]proline incorporation into the alpha-1 and -2 chains of collagen type I, (b) the up-regulation of biglycan- and syndecan-1-specific mRNA corresponded to an increased [(35)S]sulphate and [4,5-(3)H]leucine incorporation into the biglycan molecule and to an increase of syndecan-1 protein, (c) the up-regulated FGF-2 gene accounted predominantly for the ECM-bound subfraction of FGF-2-protein and (d) fibronectin and thrombospondin exhibited a significantly higher mRNA level. In contrast collagen XIV, a minor collagen type, and the proteoglycan decorin were down-regulated. The down-regulated decorin changed its structure by elongation and reduced GlcA to IdoA epimerization of the dermatan sulphate side-chain as judged by [(35)S]sulphate metabolic labelling experiments. No significant changes in response to TGF-beta(1) were observed for the collagen types III, VI and XVI, for versican, perlecan and the syndecans-2 and -4. It was concluded from the data that the TGF-beta(1)-induced formation of a highly specific multicomponent extra-cellular matrix on coronary arterial SMCs could provide in vivo mechanical strength to the neointima in arteriosclerotic lesions and to the fibrous cap overlying the lipid core.

Murine Model of the Ehlers-Danlos Syndrome: col5a1 HAPLOINSUFFICIENCY DISRUPTS COLLAGEN FIBRIL ASSEMBLY AT MULTIPLE STAGES

The most commonly identified mutations causing Ehlers-Danlos syndrome (EDS) classic type result in haploinsufficiency of proalpha1(V) chains of type V collagen, a quantitatively minor collagen that co-assembles with type I collagen as heterotypic fibrils. To determine the role(s) of type I/V collagen interactions in fibrillogenesis and elucidate the mechanism whereby half-reduction of type V collagen causes abnormal connective tissue biogenesis observed in EDS, we analyzed mice heterozygous for a targeted inactivating mutation in col5a1 that caused 50% reduction in col5a1 mRNA and collagen V. Comparable with EDS patients, they had decreased aortic stiffness and tensile strength and hyperextensible skin with decreased tensile strength of both normal and wounded skin. In dermis, 50% fewer fibrils were assembled with two subpopulations: relatively normal fibrils with periodic immunoreactivity for collagen V where type I/V interactions regulate nucleation of fibril assembly and abnormal fibrils, lacking collagen V, generated by unregulated sequestration of type I collagen. The presence of the aberrant fibril subpopulation disrupts the normal linear and lateral growth mediated by fibril fusion. Therefore, abnormal fibril nucleation and dysfunctional fibril growth with potential disruption of cell-directed fibril organization leads to the connective tissue dysfunction associated with EDS.

Collagen XXVIII, a Novel von Willebrand Factor A Domain-containing Protein with Many Imperfections in the Collagenous Domain

Here we describe a novel collagen belonging to the class of von Willebrand factor A (VWA) domain-containing proteins. This novel protein was identified by screening the EST data base and was subsequently recombinantly expressed and characterized as an authentic tissue component. The COL28A1 gene on human chromosome 7p21.3 and on mouse chromosome 6A1 encodes a novel protein that structurally resembles the beaded filament-forming collagens. The collagenous domain contains several very short interruptions arranged in a repeat pattern. As shown for other novel minor collagens, the expression of collagen XXVIII protein in mouse is very restricted. In addition to small amounts in skin and calvaria, the major signals were in dorsal root ganglia and peripheral nerves. By immunoelectron microscopy, collagen XXVIII was detected in the sciatic nerve, at the basement membrane of certain Schwann cells surrounding the nerve fibers. Even though the protein is present in the adult sciatic nerve, collagen XXVIII mRNA was only detected in sciatic nerve of newborn mice, indicating that the protein persists for an extended period after synthesis.

Purification and characterization of molecular species of collagen in the skin of skate ( Raja kenojei)

Two types, one major and the other minor, of collagen were obtained from pepsin-solubilized collagen of the skate ( Raja kenojei) skin. The major collagen was identified as Type I collagen because of a similar characteristic to those of Type I collagens from the skate muscle and cartilage. On the other hand, the SDS–PAGE pattern and peptide maps of the minor collagen were different from those of Type V and XI collagens of the skate muscle and cartilage, respectively. However, the minor collagen was similar, in amino acid composition, to Type V collagen from the skate muscle. These results suggest the existence of a molecular subspecies of Type V collagen in skate skin which is different from the muscle Type V collagen in primary structure or subunit composition.

Cloning and expression of type III collagen in normal and injured tendons of horses

To clone the 5' end of type III collagen and describe its pattern of mRNA and protein expression in normal and healing tendons in horses. 14 healthy adult horses. The tensile region of collagenase-injured superficial digital flexor tendons was harvested at intervals from 1 to 24 weeks after injury. Total RNA was reverse-transcribed into cDNA for cloning and sequencing of type III collagen. Equine-specific nucleic acid probes were developed and used for northern blot analysis and in situ hybridization. Type III collagen protein and cyanogen bromide-cleaved collagen peptides were assessedby gel electrophresis. Type III collagen mRNA expression and protein content increased immediately after injury and remained increased. Type III collagen was localized to the endotenon in normal tendon and in injured tendon at 1 week. At 8 and 24 weeks, expression became more widely distributed throughout the tendon parenchyma. Injured tendon contained 6 times more type I than type III collagen mRNA. Quantities of type III collagen protein were maximal in the first 4 weeks after injury (approx 33%) and then began to decrease. Type III collagen expression is increased initially in endotenon and subsequently in parenchyma of healing tendon; however, type III remains the minor collagen throughout the healing process. The role of type III collagen in tendon healing is not fully elucidated.

Detection of Mature Collagen in Human Dental Enamel

Mature dental enamel is the most mineralized of all mammalian tissues and considered to be free of collagen. Hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) are two nonreducible cross-links of mature collagen. Hydroxyproline (Hyp) is an amino acid that is believed to be indicative of the presence of collagen. We set out to assess the concentrations of Hyp, HP, and LP in dental enamel and dentin (control) to clarify whether there was minor collagen content in dental enamel. We studied 17.53 g of enamel and 22.12 g of dentin gained from 120 extracted human teeth. Enamel and dentin (control) were separated with a diamond dental drill under microscopic control by wasting a margin of enamel (Ca. 2 mm) at the dentin-enamel border. Collagen alpha-chains were analyzed by Sodium dodecylsulfate-polyacrylamide gel (SDS-PAGE) after decalcification and collagen extraction. Concentrations of HP and LP where measured by using high-performance liquid chromatography (HPLC). Hyp was analyzed by a spectrophotometric method. The pooled probe of enamel contained 0.23 mug/g of Hyp. This concentration was 49 times lower than that in dentin. Concentrations of HP and LP in enamel were 0.07 nmol/g and 0.02 nmol/g, respectively being 605.57 (HP) and 251.50 (LP) times lower in enamel as compared to dentin. Collagen type I was found in enamel; collagen types I and V were found in dentin samples. In reports of many studies and textbooks, collagen is considered to be completely absorbed in the course of the mineralization and maturation of dental enamel. We show that this is not the case. However, the concentration of collagen in enamel was considerably lower as compared to that in dentin.

Identification and characterization of molecular species of collagen in ordinary muscle and skin of the Japanese flounder Paralichthys olivaceus

Pepsin-solubilized collagens prepared from ordinary muscle and skin tissues of the Japanese flounder (Paralichthys olivaceus) were separated into two fractions, major and minor, by ammonium sulfate precipitation. Collagens in these fractions were further purified by cation-exchange column chromatography. The results of SDS–PAGE, peptide mapping and amino acid analysis of collagen in both fractions suggested that the purified major and minor collagens might be classified as types I and V collagens, respectively. Each type of collagen was fundamentally similar, among the ordinary muscle and the skin, in amino acid composition and peptide maps.

Collagen XXIV, a Vertebrate Fibrillar Collagen with Structural Features of Invertebrate Collagens: SELECTIVE EXPRESSION IN DEVELOPING CORNEA AND BONE*

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar alpha1(V), alpha1(XI), and alpha2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the alpha1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains alpha1(V) and alpha1(XI). However, a short imperfection in the triple helix makes alpha1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.

Characterization of molecular species of collagen in muscles of Japanese amberjack, Seriola quinqueradiata

Pepsin-solubilized collagens prepared from the muscle tissues (ordinary and dark muscles) of Japanese amberjack were separated into two fractions, major and minor, by ammonium sulphate precipitation. Collagens in these fractions were further purified by cation-exchange column chromatography. The results of SDS-PAGE, peptide mapping, and amino acid analysis suggested that the purified major and minor collagens might be classified as type I and V collagens, respectively. Each type of collagen was fundamentally similar, among the ordinary and dark muscles, in amino acid compositions and peptide maps.

Comparison of collagen types of arm and mantle muscles of the common octopus ( Octopus vulgaris)

Properties of muscle collagens of arm and mantle of the common octopus, Octopus vulgaris, were compared with regard to molecular species and thermal behaviour. The major collagens were isolated from pepsin-solubilized collagen preparations of these tissues by salt precipitation and were shown, by peptide mapping, to be essentially identical to each other in the triple helical domain. Alpha and beta subunits of the major collagen of the arm, in intact form (not solubilized by pepsin digestion), on electrophoresis, moved more slowly than those of the mantle. The major collagen of the arm showed higher hot-water solubility and lower thermal stability than that from the mantle muscle. These results suggested that there were some structural and functional differences between the major collagens from these tissues although they were genetically identical molecular species. In addition, immunoblot analysis revealed the existence of a minor collagen type, which corresponded to Type SQ-II collagen in decapod molluscs, in both arm and mantle muscles.

Molecular species of collagen in pectoral fin cartilage of skate ( Raja kenojei)

Soluble collagen was prepared from the pectoral fin cartilage of skate ( Raja kenojei) by limited pepsin digestion. It was fractionated into three fractions by differential salt precipitation. All collagen fractions were further purified by phosphocellulose column chromatography and were characterized with respect to solubility, mobility on SDS–PAGE, peptide map, and amino acid composition. The resultant data indicate the distribution of three molecular species of collagen, corresponding to Type I and II as major collagens and Type XI as a minor collagen, respectively.