Concise Review: Stem/Progenitor Cell Proteoglycans Decorated with 7-D-4, 4-C-3, and 3-B-3(-) Chondroitin Sulfate Motifs Are Morphogenetic Markers of Tissue Development.

This study reviewed the occurrence of chondroitin sulfate (CS) motifs 4-C-3, 7-D-4, and 3-B-3(-), which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulfation motifs 7-D-4, 4-C-3, and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis. Stem Cells 2018;36:1475-1486.

Biodiversity of CS-proteoglycan sulphation motifs: chemical messenger recognition modules with roles in information transfer, control of cellular behaviour and tissue morphogenesis.

Chondroitin sulphate (CS) glycosaminoglycan chains on cell and extracellular matrix proteoglycans (PGs) can no longer be regarded as merely hydrodynamic space fillers. Overwhelming evidence over recent years indicates that sulphation motif sequences within the CS chain structure are a source of significant biological information to cells and their surrounding environment. CS sulphation motifs have been shown to interact with a wide variety of bioactive molecules, e.g. cytokines, growth factors, chemokines, morphogenetic proteins, enzymes and enzyme inhibitors, as well as structural components within the extracellular milieu. They are therefore capable of modulating a panoply of signalling pathways, thus controlling diverse cellular behaviours including proliferation, differentiation, migration and matrix synthesis. Consequently, through these motifs, CS PGs play significant roles in the maintenance of tissue homeostasis, morphogenesis, development, growth and disease. Here, we review (i) the biodiversity of CS PGs and their sulphation motif sequences and (ii) the current understanding of the signalling roles they play in regulating cellular behaviour during tissue development, growth, disease and repair.

Keratan sulfate, a complex glycosaminoglycan with unique functional capability.

From an evolutionary perspective keratan sulfate (KS) is the newest glycosaminoglycan (GAG) but the least understood. KS is a sophisticated molecule with a diverse structure, and unique functional roles continue to be uncovered for this GAG. The cornea is the richest tissue source of KS in the human body but the central and peripheral nervous systems also contain significant levels of KS and a diverse range of KS-proteoglycans with essential functional roles. KS also displays important cell regulatory properties in epithelial and mesenchymal tissues and in bone and in tumor development of diagnostic and prognostic utility. Corneal KS-I displays variable degrees of sulfation along the KS chain ranging from non-sulfated polylactosamine, mono-sulfated and disulfated disaccharide regions. Skeletal KS-II is almost completely sulfated consisting of disulfated disaccharides interrupted by occasional mono-sulfated N-acetyllactosamine residues. KS-III also contains highly sulfated KS disaccharides but differs from KS-I and KS-II through 2-O-mannose linkage to serine or threonine core protein residues on proteoglycans such as phosphacan and abakan in brain tissue. Historically, the major emphasis on the biology of KS has focused on its sulfated regions for good reason. The sulfation motifs on KS convey important molecular recognition information and direct cell behavior through a number of interactive proteins. Emerging evidence also suggest functional roles for the poly-N-acetyllactosamine regions of KS requiring further investigation. Thus further research is warranted to better understand the complexities of KS.

Mesenchymal-epithelial cell interactions and proteoglycan matrix composition in the presumptive stem cell niche of the rabbit corneal limbus.

PURPOSE: To investigate whether mesenchymal-epithelial cell interactions, similar to those described in the limbal stem cell niche in transplant-expired human eye bank corneas, exist in freshly enucleated rabbit eyes and to identify matrix molecules in the anterior limbal stroma that might have the potential to help maintain the stem cell niche. METHODS: Fresh limbal corneal tissue from adult Japanese white rabbits was obtained and examined in semithin resin sections with light microscopy, in ultrathin sections with transmission electron microscopy, and in three-dimensional (3D) reconstructions from data sets of up to 1,000 serial images from serial block face scanning electron microscopy. Immunofluorescence microscopy with five monoclonal antibodies was used to detect specific sulfation motifs on chondroitin sulfate glycosaminoglycans, previously identified in association with progenitor cells and their matrix in cartilage tissue. RESULTS: In the rabbit limbal cornea, while no palisades of Vogt were present, the basal epithelial cells stained differentially with Toluidine blue, and extended lobed protrusions proximally into the stoma, which were associated with interruptions of the basal lamina. Elongate processes of the mesenchymal cells in the superficial vascularized stroma formed direct contact with the basal lamina and basal epithelial cells. From a panel of antibodies that recognize native, sulfated chondroitin sulfate structures, one (6-C-3) gave a positive signal restricted to the region of the mesenchymal-epithelial cell associations. CONCLUSIONS: This study showed interactions between basal epithelial cells and subjacent mesenchymal cells in the rabbit corneal limbus, similar to those that have been observed in the human stem cell niche. A native sulfation epitope in chondroitin sulfate glycosaminoglycans exhibits a distribution specific to the connective tissue matrix of this putative stem/progenitor cell niche.

Sulfation of the bikunin chondroitin sulfate chain determines heavy chain·hyaluronan complex formation.

Inter-α-trypsin inhibitor (IαI) is a complex comprising two heavy chains (HCs) that are covalently bound by an ester bond to chondroitin sulfate (CS), which itself is attached to Ser-10 of bikunin. IαI is essential for the trans-esterification of HCs onto hyaluronan (HA). This process is important for the stabilization of HA-rich matrices during ovulation and some inflammatory processes. Bikunin has been isolated previously by anion exchange chromatography with a salt gradient up to 0.5 M NaCl and found to contain unsulfated and 4-sulfated CS disaccharides. In this study, bikunin-containing fractions in plasma and urine were separated by anion exchange chromatography with a salt gradient of 0.1-1.0 M NaCl, and fractions were analyzed for their reactivity with the 4-sulfated CS linkage region antibody (2B6). The fractions that reacted with the 2B6 antibody (0.5-0.8 M NaCl) were found to predominantly contain sulfated CS disaccharides, including disulfated disaccharides, whereas the fractions that did not react with this antibody (0.1-0.5 M NaCl) contained unsulfated and 4-sulfated CS disaccharides. IαI in the 0.5-0.8 M NaCl plasma fraction was able to promote the trans-esterification of HCs to HA in the presence of TSG-6, whereas the 0.1-0.5 M NaCl fraction had a much reduced ability to transfer HC proteins to HA, suggesting that the CS containing 4-sulfated linkage region structures and disulfated disaccharides are involved in the HC transfer. Furthermore, these data highlight that the structure of the CS attached to bikunin is important for the transfer of HC onto HA and emphasize a specific role of CS chain sulfation.

Transplantation of human umbilical mesenchymal stem cells cures the corneal defects of mucopolysaccharidosis VII mice.

Mucopolysaccharidosis (MPS) are a family of related disorders caused by a mutation in one of the lysosomal exoglycosidases which leads to the accumulation of glycosaminoglycans (GAGs). MPS VII, caused by a mutation in ß-glucuronidase, manifests hepatomegaly, skeletal dysplasia, short stature, corneal clouding, and developmental delay. Current treatment regimens for MPS are not effective for treating corneal clouding and impaired mental development. We hypothesized that human umbilical mesenchymal stem cells (UMSCs) transplanted into the corneal stroma could participate in the catabolism of GAGs providing a means of cell therapy for MPS. For such treatment, human UMSCs were intrastromally transplanted into corneas of MPS VII mice. UMSC transplantation restored the dendritic and hexagonal morphology of host keratocytes and endothelial cells, respectively, and in vivo confocal microscopy (HRT-II) revealed reduced corneal haze. Immunohistochemistry using antibodies against heparan sulfate and chondroitin sulfate chains as well as lysosomal-associated membrane protein 2 revealed a decrease in GAG content and both lysosomal number and size in the treated corneas. Labeling UMSC intracellular compartments prior to transplantation revealed the distribution of UMSC vesicles throughout the corneal stroma and endothelium. An in vitro coculture assay between skin fibroblasts isolated from MPS VII mice and UMSC demonstrated that neutral vesicles released by the UMSC are taken up by the fibroblasts and proceed to fuse with the acidic lysosomes. Therefore, transplanted UMSCs participate both in extracellular GAG turnover and enable host keratocytes to catabolize accumulated GAG products, suggesting that UMSC could be a novel alternative for treating corneal defects associated with MPS and other congenital metabolic disorders.

ADAMTS-4_v1 is a splice variant of ADAMTS-4 that is expressed as a protein in human synovium and cleaves aggrecan at the interglobular domain.

OBJECTIVE: We previously described a messenger RNA variant of ADAMTS4 (ADAMTS4_v1) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS-4, except that the C-terminal spacer domain would be different. The purpose of this study was to determine whether ADAMTS4_v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase. METHODS: Polyclonal antibodies were raised against unique C-terminal sequences of ADAMTS-4_v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG-tagged human ADAMTS4 was mutated to contain the different sequences of ADAMTS4_v1, and the resultant plasmid was used to transfect HEK 293 cells. ADAMTS-4_v1 produced by these cells was purified via the FLAG epitope, and the ability of this recombinant protein to cleave aggrecan, biglycan, and decorin was investigated. RESULTS: An antibody specific for ADAMTS-4_v1 was found to bind to the synovial membrane surface on cryosections, and the protein was detected in cell lysates from synovium obtained from OA patients. The recombinant ADAMTS-4_v1 demonstrated enzyme activity toward the target substrate in a commercial aggrecanase 1 enzyme-linked immunosorbent assay and was also found to cleave aggrecan at the pathologically important Glu(373↓374) Ala aggrecanase site. CONCLUSION: ADAMTS-4_v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase, and cleaves other proteoglycan substrates. This splice variant may be a major contributor to loss of aggrecan from the superficial zone of OA cartilage.

Viability, growth kinetics and stem cell markers of single and clustered cells in human intervertebral discs: implications for regenerative therapies.

PURPOSE: There is much interest in the development of a cellular therapy for the repair or regeneration of degenerate intervertebral discs (IVDs) utilising autologous cells, with some trials already underway. Clusters of cells are commonly found in degenerate IVDs and are formed via cell proliferation, possibly as a repair response. We investigated whether these clusters may be more suitable as a source of cells for biological repair than the single cells in the IVD. METHODS: Discs were obtained at surgery from 95 patients and used to assess the cell viability, growth kinetics and stem or progenitor cell markers in both the single and clustered cell populations. RESULTS: Sixty-nine percent (±15) of cells in disc tissue were viable. The clustered cell population consistently proliferated more slowly in monolayer than single cells, although this difference was only significant at P0-1 and P3-4. Both populations exhibited progenitor or notochordal cell markers [chondroitin sulphate epitopes (3B3(-), 7D4, 4C3 and 6C3), Notch-1, cytokeratin 8 and 19] via immunohistochemical examination; stem cell markers assessed with flow cytometry (CD73, 90 and 105 positivity) were similar to those seen on bone marrow-derived mesenchymal stem cells. CONCLUSIONS: These results confirm those of previous studies indicating that progenitor or stem cells reside in adult human intervertebral discs. However, although the cell clusters have arisen via proliferation, there appear to be no greater incidence of these progenitor cells within clusters compared to single cells. Rather, since they proliferate more slowly in vitro than the single cell population, it may be beneficial to avoid the use of clustered cells when sourcing autologous cells for regenerative therapies.

Biochemical composition and turnover of the extracellular matrix of the normal and degenerate intervertebral disc.

BACKGROUND: The intervertebral disc (IVD) is a complex cartilaginous structure which functions to resist biomechanical loads during spinal movement. It consists of the highly viscous cartilaginous nucleus pulposus, which is surrounded laterally by a thick outer ring of fibrous cartilage-the annulus fibrosus-and sandwiched inferiorly and superiorly by the cartilage end-plates. The main extracellular matrix molecules of the disc are collagens, proteoglycans, glycoproteins and elastin. The disc also contains appreciable amounts of water, matrix-degrading protease enzymes and their inhibitors, soluble signalling molecules and various metabolic breakdown products. METHODS: This review provides a comprehensive description of the biochemical composition of the extracellular matrix of the IVD and, specifically, the proteases involved in its molecular turnover. Quantitation of the turnover rates using racemization of aspartic acid as a molecular clock is also discussed. CONCLUSIONS: Molecular turnover rates of the major constituent matrix macromolecules of the IVD are found to be particularly slow, especially in the case of collagen. Over a normal human life span, this slow turnover may compromise the structural integrity of the IVD extracellular matrix essential for normal physiological functioning.

Expression of glycosaminoglycan epitopes during zebrafish skeletogenesis.

BACKGROUND: The zebrafish is an important developmental model. Surprisingly, there are few studies that describe the glycosaminoglycan composition of its extracellular matrix during skeletogenesis. Glycosaminoglycans on proteoglycans contribute to the material properties of musculo skeletal connective tissues, and are important in regulating signalling events during morphogenesis. Sulfation motifs within the chain structure of glycosaminoglycans on cell-associated and extracellular matrix proteoglycans allow them to bind and regulate the sequestration/presentation of bioactive signalling molecules important in musculo-skeletal development. RESULTS: We describe the spatio-temporal expression of different glycosaminoglycan moieties during zebrafish skeletogenesis with antibodies recognising (1) native sulfation motifs within chondroitin and keratan sulfate chains, and (2) enzyme-generated neoepitope sequences within the chain structure of chondroitin sulfate (i.e., 0-, 4-, and 6-sulfated isoforms) and heparan sulfate glycosaminoglycans. We show that all the glycosaminoglycan moieties investigated are expressed within the developing skeletal tissues of larval zebrafish. However, subtle changes in their patterns of spatio-temporal expression over the period examined suggest that their expression is tightly and dynamically controlled during development. CONCLUSIONS: The subtle differences observed in the domains of expression between different glycosaminoglycan moieties suggest differences in their functional roles during establishment of the primitive analogues of the skeleton.

The effect of beta-xylosides on the chondrogenic differentiation of mesenchymal stem cells.

Chondroitin/dermatan sulphate (CS/DS) sulphation motifs on cell and extracellular matrix proteoglycans (PGs) within stem/progenitor cell niches are involved in modulating cell phenotype during the development of many musculoskeletal connective tissues. Here, we investigate the importance of CS/DS chains and their motifs in the chondrogenic differentiation of bone marrow mesenchymal stem cells (bMSCs), using p-nitrophenyl xyloside (PNPX) as a competitive acceptor of CS/DS substitution on PGs. Comparison of cultures grown in control chondrogenic medium, with those grown in the presence of PNPX showed that PNPX delayed the onset of chondrogenesis, characterised by cell rounding and aggregation into spheroidal beads. PNPX reduced gene expression of SOX-9, aggrecan and collagen type II, and caused reduced levels of collagen type II protein. PNPX-treated cultures also showed delayed expression of a native CS/DS sulphation motif epitope recognised by antibody 6C3. This epitope appeared associated with a range of PGs, particularly biglycan, and its close association was lost after PNPX treatment. Overall our data show that perturbation of PG glycosylation with CS/DS GAGs using PNPX significantly delays the onset of chondrogenic differentiation of bMSCs, highlighting the importance of CS/DS during the initial stages of chondrogenesis. The delayed expression of the CS/DS sulphation motif recognised by 6C3 suggests that this motif, in particular, may have early involvement in chondrogenesis. The mechanism(s) by which CS/DS chains on PGs contribute to early chondrogenic events is unknown; however, they may be involved in morphogenetic signalling through the capture and cellular presentation of soluble bioactive molecules (e.g. growth factors).

The ovine newborn and human foetal intervertebral disc contain perlecan and aggrecan variably substituted with native 7D4 CS sulphation motif: spatiotemporal immunolocalisation and co-distribution with Notch-1 in the human foetal disc.

Composite agarose (1.2 %) polyacrylamide (0.6 %) gel electrophoresis was used to separate discrete populations of native aggrecan and perlecan in newborn to 10 year old ovine intervertebral discs (IVDs). Semi-dry immunoblotting using core-protein and glycosaminoglycan (GAG) side chain specific monoclonal antibodies in combination with chondroitin ABC lyase demonstrated intra-chain native 7-D-4 chondroitin sulphate (CS) sulphation motifs and variable proportions of non-reducing terminal Δ4,5-unsaturated uronate-N-acetylgalactosamine-4-sulphate [2B6(+)] and Δ4,5-unsaturated glucuronate-N-acetylgalactosamine-6-sulphate [3B3(+)] disaccharides. The relative abundance of 2-B-6(+) aggrecan increased with advancing age of the IVD samples while the converse was true for the 3-B-3(+) aggrecan population. Relative 7D4 levels in aggrecan and perlecan were highest in the newborn IVD and significantly lower in the older IVD and other cartilage samples. Quantitation of 7D4 proteoglycan by enzyme linked immunosorbent inhibition assay confirmed the newborn ovine nucleus pulposus (NP) and inner annulus fibrosus (AF) contained higher levels (1.2-1.32 µg 7-D-4-proteoglycan/mg tissue wet weight) than the 2 (0.35-0.42 µg/mg wet weight tissue) and 10 year old IVD samples (0.16-0.22 µg/mg tissue wet weight) with the outer AF zones consistently containing lower levels of 7-D-4 epitope in all cases (P < 0.001). Cell populations on the margins of the AF and cartilaginous vertebral rudiments in newborn ovine and human foetal IVD strongly expressed 7-D-4 CS epitope and perlecan, This was co-distributed with Notch-1 expression in human foetal IVDs consistent with the 7-D-4 CS sulphation motif representing a marker of tissue development expressed by disc progenitor cell populations.

Histopathology of chondronecrosis development in knee articular cartilage in a rat model of Kashin-Beck disease using T-2 toxin and selenium deficiency conditions.

The objective of this study is to observe pathogenic lesions of joint cartilages in rats fed with T-2 toxin under a selenium deficiency nutrition status in order to determine possible etiological factors causing Kashin-Beck disease (KBD). Sprague-Dawley rats were fed selenium-deficient or control diets for 4 weeks prior to their being exposed to T-2 toxin. Six dietary groups were formed and studied 4 weeks later, i.e., controls, selenium-deficient, low T-2 toxin, high T-2 toxin, selenium-deficient diet plus low T-2 toxin, and selenium-deficient diet plus high T-2 toxin. Selenium deficiencies were confirmed by the determination of glutathione peroxidase activity and selenium levels in serum. The morphology and pathology (chondronecrosis) of knee joint cartilage of experimental rats were observed using light microscopy and the expression of proteoglycans was determined by histochemical staining. Chondronecrosis in deep zone of articular cartilage of knee joints was seen in both the low and high T-2 toxin plus selenium-deficient diet groups, these chondronecrotic lesions being very similar to chondronecrosis observed in human KBD. However, the chondronecrosis observed in the rat epiphyseal growth plates of animals treated with T-2 toxin alone or T-2 toxin plus selenium-deficient diets were not similar to that found in human KBD. Our results indicate that the rat can be used as a suitable animal model for studying etiological factors contributing to the pathogenesis (chondronecrosis) observed in human KBD. However, those changes seen in epiphyseal growth plate differ from those seen in human KBD probably because of the absence of growth plate closure in the rat.

Chondroitin sulphate and heparan sulphate sulphation motifs and their proteoglycans are involved in articular cartilage formation during human foetal knee joint development.

Novel sulphation motifs within the glycosaminoglycan chain structure of chondroitin sulphate (CS) containing proteoglycans (PGs) are associated with sites of growth, differentiation and repair in many biological systems and there is compelling evidence that they function as molecular recognition sites that are involved in the binding, sequestration or presentation of soluble signalling molecules (e.g. morphogens, growth factors and cytokines). Here, using monoclonal antibodies 3B3(-), 4C3 and 7D4, we examine the distribution of native CS sulphation motifs within the developing connective tissues of the human foetal knee joint, both during and after joint cavitation. We show that the CS motifs have broad, overlapping distributions within the differentiating connective tissues before the joint has fully cavitated; however, after cavitation, they all localise very specifically to the presumptive articular cartilage tissue. Comparisons with the labelling patterns of heparan sulphate (HS), HS-PGs (perlecan, syndecan-4 and glypican-6) and FGF-2, molecules with known signalling roles in development, indicate that these also become localised to the future articular cartilage tissue after joint cavitation. Furthermore, they display interesting, overlapping distributions with the CS motifs, reflective of early tissue zonation. The overlapping expression patterns of these molecules at this site suggests they are involved, or co-participate, in early morphogenetic events underlying articular cartilage formation; thus having potential clinical relevance to mechanisms involved in its repair/regeneration. We propose that these CS sulphation motifs are involved in modulating the signalling gradients responsible for the cellular behaviours (proliferation, differentiation, matrix turnover) that shape the zonal tissue architecture present in mature articular cartilage.

Fell-Muir Lecture: chondroitin sulphate glycosaminoglycans: fun for some and confusion for others.

This review emphasizes the importance of glycobiology in nature and aims to highlight, simplify and summarize the multiple functions and structural complexities of the different oligosaccharide combinatorial domains that are found in chondroitin sulphate/dermatan sulphate (CS/DS) glycosaminoglycan (GAG) chains. For example, there are 1008 different pentasaccharide sequences possible within CS, DS or CS/DS hybrid GAG chains. These combinatorial possibilities provide numerous potential ligand-binding domains that are important for cell and extracellular matrix interactions as well as specific associations with cytokines, chemokines, morphogens and growth factors that regulate cellular differentiation and proliferation during tissue development, for example, morphogen gradient establishment. The review provides some details of the large and diverse number of different enzymes that are involved in CS/DS biosynthesis and attempts to explain how differences in their expression patterns in different cell types can lead to subtle but important differences in the GAG metabolism that influence cellular proliferation and differentiation in development as well as regeneration and repair in disease. Our laboratory was the first to generate and characterize monoclonal antibodies (mAb) that very specifically recognize different 'native' sulphation motif/epitopes in CS/DS GAG chains. These monoclonal antibodies have been used to identify very specific spatio-temporal expression patterns of CS/DS sulphation motifs that occur during tissue and organ development (in particular their association with stem/progenitor cell niches) and also their recapitulated expression in adult tissues with the onset of degenerative joint diseases. In summary, diversity in CS/DS sulphation motif expression is a very important necessity for animal life as we know it.

Proteoglycan metabolism, cell death and Kashin-Beck disease.

Kashin-Beck Disease (KBD) is an endemic, chronic and degenerative osteoarthropathy principally occurring in children. The characteristic pathological change of KBD is chondrocyte necrosis in hyaline articular cartilage. Proteoglycans are one of the major components in the extracellular matrix of articular cartilage, and disrupted proteoglycan metabolism and loss of proteoglycans in articular cartilage from KBD patients has been observed. In this mini-review, we discuss the close relationship between chondrocyte death including necrosis and loss of proteoglycan, and its potential mechanism during KBD onset and development, which may provide new clues for KBD research.

The effects of mycotoxins and selenium deficiency on tissue-engineered cartilage.

OBJECTIVE: To investigate the effects of 3 mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and T-2 toxin, in the presence and absence of selenium (Se) on the metabolism of tissue-engineered cartilage to mimic conditions found in Kashin-Beck disease (KBD) environments. MATERIALS AND METHODS: Chondrocytes were seeded onto bone matrix gelatin (BMG) to construct engineered cartilage. The 3 toxins were added to the culture media for 3 weeks followed by immunhistochemical analyses of collagens type II and X, aggrecan, matrix metalloproteinases 1 and 3 (MMP-1 and MMP-3), MMP inhibitors 1 and 3 (TIMP-1 and TIMP-3) and α(2) macroglobulin (α2M). RESULTS: Type II collagen was decreased while type X collagen was increased in response to DON, NIV and T-2 toxin. Aggrecan was reduced by all 3 mycotoxins. Compared with the control, the 3 toxins decreased the expression of α2M, TIMP-1 and TIMP-3, and increased the expression of MMP-1 and MMP-3. Se could partially inhibit the effects of DON, NIV and T-2 toxins. CONCLUSION: Under the low Se condition, the 3 mycotoxins produced procatabolic changes in cartilage resulting in the loss of aggrecan and type II collagen and promoted a hypertrophic phenotype of chondrocytes characterized by increasing type-X-collagen expression, enhancing the expression of MMPs, while weakening the TIMPs. Se could partially block the effects mentioned above. These results support the hypothesis that the combination of mycotoxin stress and Se deficiency would be the causative factors for KBD.

Articular cartilage glycosaminoglycans inhibit the adhesion of endothelial cells.

Articular cartilage undergoes severe loss of proteoglycan and its constituent glycosaminoglycans (GAGs) in osteoarthritis. We hypothesize that the low GAG content of osteoarthritic cartilage renders the tissue susceptible to pathological vascularization. This was investigated using an in vitro angiogenesis model assessing endothelial cell adhesion to GAG-depleted cartilage explants. Bovine cartilage explants were treated with hyaluronidase to deplete GAG content and then seeded with fluorescently tagged human endothelial cells (HMEC-1). HMEC-1 adherence was assessed after 4 hr and 7 days. The effect of hyaluronidase treatment on GAG content, chondrocyte viability, and biochemical composition of the extracellular matrix was also determined. Hyaluronidase treatment reduced the GAG content of cartilage explants by 78 ± 3% compared with that of controls (p < 0.0001). GAG depletion was associated with significantly more HMEC-1 adherence on both the surface (superficial zone) and the underside (deep zone) of the explants (both p < 0.0001). The latter provided a more favorable environment for extended culture of HMEC-1 compared with the articulating surface. Hyaluronidase treatment altered the immunostaining for chondroitin sulfate epitopes, but not for lubricin. Our results support the hypothesis that articular cartilage GAGs are antiadhesive to endothelial cells and suggest that chondroitin sulfate and/or hyaluronan are responsible. The loss of these GAGs in osteoarthritis may allow osteochondral angiogenesis resulting in disease progression.

A comparative evaluation of the small leucine-rich proteoglycans of pathological human intervertebral discs.

PURPOSE: Proteoglycans are important to the functioning of the intervertebral disc. In addition to aggrecan there are the small leucine-rich proteoglycans (SLRPs). These are less common but in other locations their functions include collagen organisation, sequestering growth factors and stimulating inflammation. We have performed a comparative analysis of the SLRP core protein species present in intervertebral discs with various pathologies. METHODS: Eighteen intervertebral discs from patients with scoliosis (n = 7, 19-53 years), degenerative disc disease (n = 6, 35-51 years) and herniations (n = 5, 33-58 years) were used in this study. Proteoglycans were dissociatively extracted from disc tissues and the SLRPs (biglycan, decorin, fibromodulin, keratocan and lumican) assessed by Western blotting following deglycosylation with chondroitinase ABC and keratanase. RESULTS: Intact SLRP core proteins and a number of core protein fragments were identified in most of the discs examined. Biglycan and fibromodulin were the most extensively fragmented. Keratocan generally occurred as two bands, one representing the intact core protein, the other a smaller fragment. The intact core protein of lumican was detected in all samples with fragmentation evident in only one of the older scoliotic discs. Decorin was less obvious in the disc samples and showed little fragmentation. CONCLUSION: In this cohort of pathological intervertebral discs, fragmentation of certain SLRP core proteins was common, indicating that some SLRPs are extensively processed during the pathological process. Identification of specific SLRP fragments which correlate with disc pathology may not only help understand their aetiopathogeneses, but also provide biomarkers which can be used to monitor disease progression or to identify particular disc disorders.