Daniel Herbage

A differential scanning calorimetry analysis of the age-related changes in the thermal stability of rat skin collagen

Differential scanning calorimetry (DSC) has been applied to the study of connective tissue to evaluate the denaturation process of collagen. We have applied this technique to the study of the ageing of rat skin. We have tried to correlate the variations of the parameters measured by DSC and the modifications of collagen crosslinks with ageing. The thermograms obtained are composed of one main peak located between two shoulders. The relative size of each peak varies with time: the first peak diminishes regularly from 2 to 20 months whilst, at the same time, the third peak increases; the recovery temperature increases with age (+ 16 degrees C between 2 and 20 months); the total denaturation enthalpy does not vary: the main value obtained is 5.9 X 10(-2) J/mg collagen. On the other hand, the assay of reducible collagen crosslinks in rat skin, over the same age range, shows a decrease of heat-labile aldimine crosslink (essentially hydroxylysinonorleucine). These results and the study of thermograms obtained with altered rat skin (animals treated with beta-aminopropionitrile or skin reduced with NaBH4) allow us to conclude that heat-labile and heat-stable crosslinks account for a collagen thermal stabilization which can explain the delay of denaturation characterized by the third peak of DSC thermograms.

Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis.

The aim of this study was to characterize the cellular phenotypes of articular cartilage and meniscus in rabbits with experimentally induced osteoarthritis (OA), by histological and molecular biological techniques. OA was induced by severing the anterior cruciate ligament of the knee and rabbits were killed 2, 4 or 9 weeks following surgery. Our histological observations show a progressive destruction of extracellular matrix in both tissues. To determine whether these morphological changes could be related to alterations in the regulation of gene expression for a subset of relevant molecules, levels of mRNA for proteinases and one inhibitor (MMP-1, -3 and -13, aggrecanase-1 and -2 and TIMP-1), matrix molecules and one chaperone (type II and X collagens, aggrecan, osteonectin, betaig-h3 and BiP) were assessed by reverse transcription-polymerase chain reaction. Our results indicate that for most markers expression profiles were similar in both tissues. In particular, matrix protein gene expression remained stable or varied little during progression of OA, suggesting a poor repair capacity of the tissues. MMP gene expression increased rapidly whereas aggrecanase gene expression remained stable. These findings suggest that differential regulation of mRNA levels of MMP-1, -3 and -13 on the one hand and aggrecanase-1 and -2 on the other, occurs during OA.

Biochemical properties and immunolocalization of minor collagens in foetal calf cartilage

Type II collagen is the major collagen of cartilage. However, new collagenous chains have been described in different cartilaginous tissues, recently. The la, 2a and 3a chains were extracted from human and bovine hyaline cartilage [1]. Other minor collagenous components were extracted from neonatal pig and human cartilage noted M, CFI, CF2 [2-4], from bovine nasal cartilage and human intervertebral disc noted CPS1, CPS2 [5,6] and from chicken sternal cartilage noted HMW, LMW [7,8] and M 1, M 2 [9]. Here, we report the partial characterization of 3 collagenous fractions obtained after limited pepsin treatment of foetal calf cartilage and isolated according to their solubility properties. The 1.2 M NaC1 fraction contains the la, 2t~ and 3a chains. The 2.0 M NaC1 and 3.0 M NaCl fractions contain at least 7 collagenous chains, which are related to the disulfide-bonded new chains enumerated above, but show some major differences, Antibodies against the 1.2 M and 2.0 M NaC1 fractions were raised in rabbits and their specificity tested by radioimmunoprecipitation. The localization of the corresponding collagenous chains was achieved by indirect immunofluorescence in epiphyseal proper and growth cartilage from foetal calf cartilage. 2. MATERIALS AND METHODS

Immunofluorescent localization of collagen types I and III, and of fibronectin during feather morphogenesis in the chick embryo.

Abstract Collagen types I and III were purified from the skin of 3- or 7-week chickens and fibronectin from human serum. Antibodies were raised in rabbits and used in indirect immunofluorescence on frozen sections of 5- to 16-day chick embryo feather-forming skin. Prior to the formation of dense feather-forming dermis, anticollagen fluorescence was confined to a thin underlining of the dermal-epidermal junction (DEJ), while antifibronectin label was retained on loosely dispersed material in the predermal mesenchyme. Dense feather-forming dermis was characterized by loosening of the anti-collagen label along the DEJ, by its spreading throughout the thickness of dermis, and by an overall densification of antifibronectin label. Feather formation coincided with a decrease of anti-collagen and an increase of antifibronectin label density in the dermal feather condensations and in the core of outgrowing feather buds. By contrast, density of anti-collagen-labeled material was highest and anti-fibronectin-labeled material was lowest in interplumar and glabrous skin. In slanting feather buds and feather filaments, distribution of anti-collagen-labeled material exhibited a type-specific cranial-caudal asymmetry. The microheterogeneous distribution of extracellular matrix components might constitute part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of cutaneous appendages.

Localization of the Expression of Type I, II, III Collagen, and Aggrecan Core Protein Genes in Developing Human Articular Cartilage

The expression of mRNAs for collagen types I, II, III and for aggrecan core protein was studied in developing human femoral cartilage by in situ hybridization, with special attention given to the cartilage covered by the perichondrium and to the articular surface. In parallel, the synthesis of the related proteins was monitored by immunohistochemistry. The cells metabolically active for type I and type III collagen expression were identified by hybridization using [32P]-labeled cDNA clones coding for human alpha 1(I) and alpha 1(III), respectively. Type II collagen and core protein mRNAs were detected by hybridization with specific [32P]-labeled oligonucleotide probes. In the femoral heads of one 22-week old fetus and of one newborn, our in situ hybridization and immunohistochemical analysis revealed that chondrocytes located immediately subjacent to the perichondrium produced collagen types I, II, III as well as aggrecan; whereas only type II collagen and aggrecan gene expression was detected deeper in the cartilage covered by the perichondrium. This observation supports the hypothesis that the inner cell layers of perichondrium are chondrogenic, with a transient state where cells express all the markers studied here. At the articular surface different patterns of expression were observed at the two developmental stages. After 22 weeks of fetal development only collagen types I and III were expressed by the surface zone cells while in the newborn cartilage, these cells expressed all the molecules studied (collagen types I, II, III and cartilage proteoglycan). At both ages the underlying cartilage cells expressed only the cartilage-specific molecules (type II collagen and aggrecan). Thus a progressive transformation of cartilaginous matrix occurs with time from the deep cartilage up to the surface by addition of new components, i.e. aggrecan and type II collagen. These results supplemented by an immunofluorescence analysis on 20-, 26- and 38-week old fetal femoral heads suggest that expression of collagen and aggrecan in the cartilage covered by the perichondrium and in the cartilage at the articular surface are subject to different regulatory mechanisms during development. Furthermore, the appearance of hybridizable core protein and type II collagen mRNAs at the articular surface, closely followed by the appearance of the proteins for which they code, indicates that core protein and type II collagen expression is regulated primarily at the transcriptional level in this region. Finally, the similar topography observed for the expression of these two proteins suggests that the genes for these two major constituents of cartilage matrix are coordinately regulated during growth of articular cartilage.

Type IX collagen is a potent inducer of PGE2 and interleukin 1 production by human monocyte macrophages

Type IX collagen and its collagenous fragments are potent stimulatory agents on human blood mononuclear cells for the production of prostaglandin E2 and interleukin 1/mononuclear cell factor. Type IX collagen is 2–4‐fold more potent that type I and II and 1α‐, 2α‐ and 3α‐collagens. This property may be important in the destructive process of cartilage in inflammatory diseases.

Collagen: A Family of Proteins with many Facets

Publisher Summary Many of the recently identified molecules are very different from the originally described type I collagen molecule. The generally accepted current definition of a collagen is that it is a structural protein of the extracellular matrix that contains one or more domains having the conformation of a triple helix. This definition implies that collagen molecules are multidomain proteins and that some domain(s) are involved in the multimolecular aggregates forming the architecture of the extracellular matrix. The triple-helical domains of collagens are thus the main characteristic of this family of proteins to which they confer unique properties of aggregation. This chapter provides a brief overview of thecollagen family of proteins and discusses that particular protein conformation, how it is formed, and how triple-helical domains can interact to form multimolecular aggregates. The nontriple-helical domains of molecules containing triple helices of the collagen type could be of any nature, and they are indeed highly diverse in structure and function. The chapter discusses the domains separately.

Immunofluorescent localization of collagen types I, III, IV, fibronectin and laminin during morphogenesis of scales and scaleless skin in the chick embryo

SummaryCollagen types I and III were purified from the skin of 3-or 7-week-old chickens, collagen type IV from bovine skin or EHS mouse tumour, fibronectin from human serum, and laminin from EHS mouse tumour. Antibodies were produced in rabbits or sheep, and used in indirect immunofluorescence on frozen sections of 9-to 16-day-old normal or mutant (scaleless) chick-embryo foot skin. In normal scale-forming skin and inscaleless skin, the distribution of anti-laminin and anti-type IV collagen label was uniform along the dermal-epidermal junction and showed no stage-related variations, except for fluorescent granules located in the dermis of early scale rudiments. By contrast, in normal scale-forming skin, the density of anti-types I and III label decreased in the dermis within scale rudiments, whereas it gradually increased in interscale skin. Conversely, anti-fibronectin label accumulated at a higher density within scale rudiments than in interscale skin. In the dermis of thescaleless mutant, anti-types I and III label and antifibronectin label were distributed evenly: the density of anti-collagen label increased with age, while that of antifibronectin decreased and almost completely vanished in 16-day-old skin, except around blood vessels. The microheterogeneous distribution of some extracellular matrix components, namely interstitial collagen types I and III and fibronectin, is interpreted as part of the morphogenetic message that the dermis is known to transmit to the epidermis during the formation of scales. The even distribution of these components in mutantscaleless skin is in agreement with this view. Basement membrane constituents laminin and type-IV collagen do not appear to be part of the dermal morphogenetic message.

Biochemical characterization of the cuticle collagen of the nematode Caenorhabditis elegans.

Proteins of purified cuticles from adults of the small free-living nematode Caenorhabditis elegans are solubilized by reduction in the presence of a strong denaturing agent and then carboxymethylated. As in the large parasitic nematode Ascaris lumbricoïdes, these soluble proteins appeared to be collagens by their amino acid compositions. C. elegans cuticle collagen is separated into seven major components with different apparent molecular weights by molecular sieve chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The two main components, which together account for more than 64% of the total cuticle collagen, were extracted from gel after electrophoresis and analyzed. They differ in their amino acid compositions and would seem to represent genetically distinct collagen chains. The results presented lead to the hypothesis of the presence in this collagen of at least two different chains.

A recurrent Vα17 / Vβ10 TCR-expressing T cell clone is involved in the pathogenicity of collagen-induced arthritis in DBA / 1 mice

Collagen‐induced arthritis (CIA) is an experimental model that mimics clinical and histological features of rheumatoid arthritis. In this disease, a crucial role in initiating the pathological changes has been assigned to T lymphocytes expressing the Th1 phenotype. Aiming at identifying type II collagen (CII)‐specific T cells involved in CIA, T cell clones were generated in vitro from the lymph nodes (LN) of CII‐immunized DBA / 1 mice. In three independent experiments, we repeatedly isolated CD4+ Th1 clones recognizing the immunodominant epitope in the CB11 fragment of bovine CII and expressing a unique α βTCR produced by the rearrangement of Vα17 / Jα20 and Vβ10 / Dβ1.1 / Jβ2.5 gene segments. By reverse transcriptase‐PCR, we demonstrated the presence of mRNA transcripts specific for the β complementary‐determining region 3 of this clonotype in the LN of the majority (73 %) of mice with CIA whereas it was never detected in control animals. When transferred to CII‐immunized DBA / 1 mice, this recurrent Th1 clone augmented the incidence, aggravated significantly the clinical signs of CIA and greatly enhanced the anti‐CII antibody response. Altogether, these results provide evidence that a CD4+ Th1 clone belonging to the public arm of the response toward the immunodominant epitope of CII is involved in the cascade of events leading to CIA.