Carole M. Dodd

Identification of fibrocytes in postburn hypertrophic scar.

Fibrocytes are a unique leukocyte subpopulation implicated in wound healing. They are derived from peripheral blood mononuclear cells, display fibroblast‐like properties, and synthesize extracellular matrix macromolecules. This study investigated whether fibrocytes are present in healing burn wounds and whether the number of fibrocytes in tissue correlates with the degree of burn injury and the development of hypertrophic scar. Proteins extracted from cultured fibrocytes and nonadherent lymphocytes were found to be similar using two‐dimensional gel electrophoresis and quite distinct from those obtained from fibroblasts. However, one protein, identified as leukocyte‐specific protein 1 using mass spectrometric peptide mapping, was found in significantly larger amounts in fibrocytes than in lymphocytes but was undetectable in fibroblasts. Double immunostaining with antibodies to leukocyte‐specific protein‐1 and to the N‐terminal propeptide of type I collagen was performed on cryosections of hypertrophic scar, mature scar, and normal skin. Fibrocytes were seen in scar tissue as dual‐labeled spindle‐shaped cells but were absent from normal skin. Moreover, the number of fibrocytes was higher in hypertrophic than in mature scar tissue. We conclude that fibrocytes, which have been reported to be antigen‐presenting cells, are recruited to wounds following extensive burn injury and could potentially upregulate the inflammatory response and synthesize collagen and other matrix macromolecules, thus contributing to the development of hypertrophic scarring.

Deep dermal fibroblasts contribute to hypertrophic scarring

Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. The development of HTS involves numerous cell types and cytokines with dermal fibroblasts being a key cell. We have previously reported that the phenotype of fibroblasts isolated from HTS was altered compared to fibroblasts from normal skin. In this study, normal skin was horizontally sectioned into five layers using a dermatome from which fibroblasts were isolated and cultured. Cells from the deeper layers were observed to proliferate at a slow rate, but were morphologically larger. In ELISA and FACS assays, cells from the deeper layers produced more TGF-β1 and TGF-β1 producing cells were higher. In quantitative RT-PCR, the cells from the deeper layers had higher CTGF and HSP47 mRNA levels compared to those from superficial layers. In western blot, FACS and collagen gel assays, fibroblasts from the deeper layers produced more α-smooth muscle actin (α-SMA), had higher α-SMA positive cells and contracted collagen gels more. Fibroblasts from the deeper layers were also found to produce more collagen, but less collagenase by mass spectrometry and collagenase assay. Interestingly, cells from the deeper layers also produced more of the proteoglycan, versican, but less decorin. Taken together, these data strongly demonstrate that fibroblasts from the deeper layers of the dermis resemble HTS fibroblasts, suggesting that the deeper layer fibroblasts may be critical in the formation of HTS.

Immunoelectron Microscopic Localization of the Core Protein of Decorin Near the d and e Bands of Tendon Collagen Fibrils by Use of Monoclonal Antibodies

Two monoclonal antibodies, 6D6 and 7B1, previously shown to recognize different epitopes on different regions of the protein core of decorin were used to localize the protein core in relation to the positively stained bands in the D period of bovine tendon collagen fibrils. Peroxidase-antiperoxidase staining revealed that the antigen is associated with the surface of all fibrils and suggested that the axial distance between antigens is D-periodic. Immunoferritin labeling with each antibody produced a distribution of ferritin particles that showed that both epitopes of the protein core are localized near the d and e bands in the D period. The data indicate that the decorin protein core binding site(s) on tendon collagen fibrils is/are located near these bands, axially, within the D period.

Immunohistochemical localization of the proteoglycans decorin, biglycan and versican and transforming growth factor-β in human post-burn hypertrophic and mature scars

The distributions of the small proteoglycans, decorin and biglycan and the large proteoglycan, versican, in normal skin and post‐burn hypertrophic and mature scars, were compared using monoclonal and polyclonal antibodies to the core proteins. Biglycan and verscan were virtually undetectable in normal dermis but readily seen in hypertrophic scars. Staining for decorin was strong throughout the dermis in normal skin but restricted to the deep dermis and a narrow zone under the epidermis in hypertrophic scar—areas which did not stain for versican. Decorin was absent or reduced in the nodules in these specimens. In mature post‐burn scars, staining for all three proteoglycans demonstrated an intensity that was intermediate between that in normal dermis and that in the nodules of the hypertrophic scars. Transforming growth factor‐β was present in the nodules of hypertrophic scars but the deep dermis of these specimens stained most intensely for this cytokine which was also found in the dermis of mature scars but was not detectable in normal dermis. The apparent co‐distribution of decorin and transforming growth factor‐β suggests that this proteoglycan may play an active role in the resolution of the scars. Changes in proteoglycan type and distribution could possibly account, at least in part, for the derangement of collagen and the altered physical properties of hypertrophic scar tissue.

Crystal Structure of the Biglycan Dimer and Evidence That Dimerization Is Essential for Folding and Stability of Class I Small Leucine-rich Repeat Proteoglycans

Biglycan and decorin are two closely related proteoglycans whose protein cores contain leucine-rich repeats flanked by disulfides. We have previously shown that decorin is dimeric both in solution and in crystal structures. In this study we determined whether biglycan dimerizes and investigated the role of dimerization in the folding and stability of these proteoglycans. We used light scattering to show that biglycan is dimeric in solution and solved the crystal structure of the glycoprotein core of biglycan at 3.40-Å resolution. This structure reveals that biglycan dimerizes in the same way as decorin, i.e. by apposition of the concave inner surfaces of the leucine-rich repeat domains. We demonstrate that low concentrations of guanidinium chloride denature biglycan and decorin but that the denaturation is completely reversible following removal of the guanidinium chloride, as assessed by circular dichroism spectroscopy. Furthermore, the rate of refolding is dependent on protein concentration, demonstrating that it is not a unimolecular process. Upon heating, decorin shows a single structural transition at a Tm of 45-46 °C but refolds completely upon cooling to 25 °C. This property of decorin enabled us to show both by calorimetry and light scattering that dimer to monomer transition coincided with unfolding and monomer to dimer transition coincided with refolding; thus these processes are inextricably linked. We further conclude that folded monomeric biglycan or decorin cannot exist in solution. This implies novel interrelated functions for the parallel β sheet faces of these leucine-rich repeat proteoglycans, including dimerization and stabilization of protein folding.

A role for disulphide bridges in the protein core in the interaction of proteodermatan sulphate and collagen

Proteodermatan sulphate from bovine skin retarded precipitation of fibrils from solutions of purified acid-soluble bovine skin collagen. The isolated protein core was as effective as the intact proteoglycan. Thermal denaturation leading to almost complete loss of the native secondary structure, (determined by circular dichroism spectroscopy to consist of about 60% beta structure) did not diminish the effect unless accompanied by reduction of disulphides, of which there were shown to be three per molecule. The reduced and alkylated protein core was totally ineffective. Electron-microscopy revealed a D-periodic arrangement of glycosaminoglycan on the surfaces of collagen fibrils precipitated in the presence of proteodermatan sulphate. Dermatan sulphate (with attached small peptide) prepared from the proteoglycan, had no effect on the rate of fibrillogenesis and was apparently not bound to the fibrils.

Proteoglycans of the articular disc of the bovine temporomandibular joint. I: High molecular weight chondroitin sulphate proteoglycan

The low molecular weight proteoglycan fraction extracted from articular discs with 4 M guanidinium chloride was found to consist predominantly of an iduronate-rich dermatan sulphate proteoglycan, together with chondroitin sulphate-containing material. The dermatan sulphate proteoglycan was purified by ion-exchange and gel-filtration chromatography and its core protein isolated after digestion with chondroitinase ABC. The amino acid composition and pattern of cyanogen bromide peptides obtained from this core were closely similar to those of the protein core of bovine skin proteodermatan sulphate. Four monoclonal antibodies raised against bovine skin proteodermatan sulphate also reacted with the disc protein core and its cyanogen bromide peptides. Results of digestion with glycopeptidase F demonstrated the presence of three N-linked oligosaccharides. The combined size of these oligosaccharides appeared to be somewhat less than the size of those on skin proteodermatan sulphate. The glycosaminoglycan chain released by digestion with cathepsin C had a higher molecular weight than that from skin. These differences in glycosylated structures may be responsible for the different effects on collagen fibrillogenesis in vitro; whereas skin proteodermatan sulphate only reduced the rate of fibril growth, disc dermatan sulphate proteoglycan also increased the length of the lag-phase and the final opacity.

Light and X-ray Scattering Show Decorin to Be a Dimer in Solution

Decorin is a widely distributed member of the extracellular matrix small leucine-rich repeat glycoprotein/proteoglycan family. For investigation of its physical properties, decorin from two sources (young steer skin and a recombinant adenovirus) was used. The first sample was extracted into 7 m urea and purified, while the second was isolated from medium conditioned by 293A cells infected with adenovirus and purified without chaotropes. The only chemical differences detected between these materials were a slightly shorter glycosaminoglycan chain and the retention of the propeptide on the latter. Circular dichroism spectra of the two samples were virtually identical, showing a high proportion of β-sheet and β-turn and little α-helix. The protein cores were completely denatured in 2.25 m guanidine HCl (GdnHCl) but recovered their secondary structure on removal of chaotrope. Light scattering of material eluted from gel-filtration columns in Tris-buffered saline, pH 7.0, gave molecular mass values of 165 ± 1 kDa and 84.6 ± 4 kDa for intact decorin and the glycoprotein core produced by digestion with chondroitin ABC lyase, respectively. Intact recombinant prodecorin had a mass of 148 ± 18 kDa. These values, which are double those estimated from SDS gel electrophoresis or from the known sequences and compositions, were halved in 2.5 m GdnHCl. Data from solution x-ray scattering of intact decorin and its core in Tris-buffered saline are consistent with a dimeric particle whose protein component has a radius of gyration of 31.6 ± 0.4 Å, a maximum diameter of 98 ± 5 Å, and approximates two intertwined C shapes.

An immunohistochemical study of the localization of biglycan, decorin and large chondroitin-sulphate proteoglycan in adult rat temporomandibular joint disc.

To analyse regional variations in extracellular matrix components of adult rat temporomandibular joint discs, immunohistochemical techniques were used to examine the localization of two small dermatan-sulphate proteoglycans, biglycan and decorin, and a large chondroitin-sulphate proteoglycan. Staining for biglycan was intense in the posterior band, although it had a rather weak and even distribution throughout the disc. In contrast, staining for decorin was faint in the intermediate zone and the central part of the posterior band, moderate in the anterior and posterior attachments and most intense in the junction between the anterior band and attachment. The upper surface of the disc stained more intensely than the lower. Similarly, there was intense staining for large chondroitin-sulphate proteoglycan in the peripheral band, but both the anterior and the temporal parts of the posterior attachments were faintly stained. These results demonstrate marked regional differences in the expression of biglycan, decorin and large chondroitin-sulphate proteoglycan in the temporomandibular joint discs of adult rats. These variations probably reflect the different biomechanical environments caused by the complicated articulatory functions of the temporomandibular joint.

Production and characterization of monoclonal antibodies to bovine skin proteodermatan sulfate.

To study the molecular structure and function of bovine skin proteodermatan sulfate, on a determinant by determinant basis, several monoclonal antibodies to this molecule have been produced and characterized. Based on the results of a preliminary immunogenetic analysis of 4 inbred mouse strains, SJL/J (H-2s) mice were immunized for the fusions. Ten hybridomas were produced and the monoclonal antibodies from four of these were selected for further investigation. Employing an ELISA inhibition assay, none showed any detectable affinity for bovine collagen types I, II, III, or IV, bovine fibronectin or chondroitin or dermatan sulfate glycosaminoglycans. Each monoclonal antibody bound the chondroitinase ABC-derived protein core and none was significantly inhibited by proteinase digests of the intact molecule suggesting that the epitope of each contains a protein component. The results of competitive binding ELISA assays and immunoblots of the cyanogen bromide cleavage products of proteodermatan sulfate indicate that the 4 antibodies recognize at least 3 distinct antigenic determinants on this molecule. Immunohistochemical methods located the antigen in the dermis of bovine skin and revealed that a change in proteodermatan sulfate distribution occurs during skin development.