Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

Introduction

Similar to matrix metalloproteinases, glycosidases also play a major role in cartilage degradation. Carbohydrate cleavage products, generated by these latter enzymes, are released from degrading cartilage during arthritis. Some of the cleavage products (such as hyaluronate oligosaccharides) have been shown to bind to Toll-like receptors and provide endogenous danger signals, while others (like N-acetyl glucosamine) are reported to have chondroprotective functions. In the current study for the first time we systematically investigated the expression of glycosidases within the joints.

Methods

Expressions of β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, sperm adhesion molecule 1 and klotho genes were measured in synovial fibroblasts and synovial membrane samples of patients with rheumatoid arthritis and osteoarthritis by real-time PCR. β-D-Glucuronidase, β-D-glucosaminidase and β-D-galactosaminidase activities were characterized using chromogenic or fluorogenic substrates. Synovial fibroblast-derived microvesicles were also tested for glycosidase activity.

Results

According to our data, β-D-hexosaminidase, β-D-glucuronidase, hyaluronidase, and klotho are expressed in the synovial membrane. Hexosaminidase is the major glycosidase expressed within the joints, and it is primarily produced by synovial fibroblasts. HexA subunit gene, one of the two genes encoding for the alpha or the beta chains of hexosaminidase, was characterized by the strongest gene expression. It was followed by the expression of HexB subunit gene and the β-D-glucuronidase gene, while the expression of hyaluronidase-1 gene and the klotho gene was rather low in both synovial fibroblasts and synovial membrane samples. Tumor growth factor-β1 profoundly downregulated glycosidase expression in both rheumatoid arthritis and osteoarthritis derived synovial fibroblasts. In addition, expression of cartilage-degrading glycosidases was moderately downregulated by proinflammatory cytokines including TNFα, IL-1β and IL-17.

Conclusions

According to our present data, glycosidases expressed by synovial membranes and synovial fibroblasts are under negative regulation by some locally expressed cytokines both in rheumatoid arthritis and osteoarthritis. This does not exclude the possibility that these enzymes may contribute significantly to cartilage degradation in both joint diseases if acting in collaboration with the differentially upregulated proteases to deplete cartilage in glycosaminoglycans.     

Mesenchymal progenitor cell markers in human articular cartilage: normal distribution and changes in osteoarthritis

Introduction  

Recent findings suggest that articular cartilage contains mesenchymal progenitor cells. The aim of this study was to examine the distribution of stem cell markers (Notch-1, Stro-1 and VCAM-1) and of molecules that modulate progenitor differentiation (Notch-1 and Sox9) in normal adult human articular cartilage and in osteoarthritis (OA) cartilage.     

Mesenchymal progenitor cells in adult human articular cartilage

The transmembrane receptor Notch-1 regulates cell fate and differentiation and was suggested to identify a cell type with progenitor characteristics in newborn bovine articular cartilage. We show that Notch-1 is expressed on > 70% of BM-MSC in early passage monolayer culture. We also demonstrate that normal articular cartilage contains Notch-1+ cells and that the frequency is increased in OA. Most Notch-1+ cells in OA cartilage are located in the clusters of proliferating cells. These findings indicate that multipotential mesenchymal progenitor cells are present in articular cartilage from adult humans and that their frequency is increased in OA. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.     

Type III collagen in normal human articular cartilage

Summary Type III collagen in normal human articular cartilage has been detected biochemically and its location in a diffuse area around the chondrocytes demonstrated by immunofluorescence. It can be found pericellularly throughout the depth of the cartilage and is evident in specimens ranging in age from 17 to 81 years.     

Identification of latexin by a proteomic analysis in rat normal articular cartilage

Background  

Osteoarthritis (OA) is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS) were used. Interestingly, latexin (LXN) was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage.     

Glycosyltransferase activities in chondrocytes from osteoarthritic and normal human articular cartilage

Six glycosyltransferases (mannosyl-, glucosyl-, N-acetyl-glucosaminyl-, galactosyl-, sialyl- and fucosyltransferases) are studied and characterized for their optimal conditions and their relations with interfering reactions (glycosyl-nucleotide pyrophosphatases, glycosidases and proteinases) in chondrocytes from osteoarthritic and normal human articular cartilage. Osteoarthritis induces increased activities for five glycosyl-transferases. The observed modifications are not explained by alterations in physico-chemical parameters of the enzymes or by intervention of glycosyl-nucleotide pyrophosphatases, glycosidases or proteolytic enzymes.     

The distribution of serum albumin in human normal and degenerate articular cartilage

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymatic methods. With naturally occuring cartilage large a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partitiion coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

The distribution of serum albumin in human normal and degenerate articular cartilage.

A method for studying the distribution of a high molecular weight solute (serum albumin) between physiological saline and human articular cartilage is described. Samples of normal and fibrillated articular cartilage from both femoral condyles and femoral heads have been studied. Limited studies have also been performed where the glycosaminoglycan content of normal cartilage has been reduced by chemical or enzymic methods. With naturally occurring cartilage a wide range of partition coefficients (0.3 to less than 0.002) was obtained. The partition coefficients are very dependent upon proteoglycan concentration, with the partition coefficient decreasing with increasing fixed charge density. An attempt is made to interpret the observed partitioning in terms of the steric exclusion by the proteoglycans.     

Identification of link proteins in canine synovial cell cultures and canine articular cartilage

Link proteins are glycoproteins in cartilage that are involved in the stabilization of aggregates of proteoglycans and hyaluronic acid. We have identified link proteins in synovial cell cultures form normal canine synovium using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunofluorescence, and immunolocation with specific antibodies by electrophoretic transfer. We have also found evidence for the synthesis of link proteins in these cultures by fluorography of radiolabeled synovial cell extracts. We have identified a 70,000 mol-wt protein in canine synovial cell culture extracts that has antigenic cross-reactivity with the 48,000-mol-wt link protein. Three link proteins were identified in normal canine articular cartilage. These results indicate that link proteins are more widely distributed in connective tissues than previously recognized and may have biological functions other than aggregate stabilization.     

Synthesis of fibronectin in normal and osteoarthritic articular cartilage

The content and the biosynthesis of fibronectin was examined in disease-free articular cartilage and in articular cartilage from osteoarthritic canine joints. Fibronectin content was increased in extracts of cartilage from osteoarthritic joints. Incubation of cartilage in vitro with [³H]phenylalanine and subsequent isolation of [³H]fibronectin from a gelatin affinity column and characterization by SDS-polyacrylamide gel electrophoresis and by immunoprecipitation indicated that disease-free and osteoarthritic cartilage explants synthesized fibronectin. About 50% of the [³H]fibronectin was recovered in the incubation medium. The osteoarthritic cartilage synthesized and accumulated up to 5-fold more [³H]fibronectin than disease-free cartilage.     

Relative percentage and zonal distribution of mesenchymal progenitor cells in human osteoarthritic and normal cartilage

Introduction  

Mesenchymal stem cells (MSC) are highly attractive for use in cartilage regeneration. To date, MSC are usually recruited from subchondral bone marrow using microfracture. Recent data suggest that isolated cells from adult human articular cartilage, which express the combination of the cell-surface markers CD105 and CD166, are multi-potent mesenchymal progenitor cells (MPC) with characteristics similar to MSC. MPC within the cartilage matrix, the target of tissue regeneration, may provide the basis for in situ regeneration of focal cartilage defects. However, there is only limited information concerning the presence/abundance of CD105⁺/CD166⁺ MPC in human articular cartilage. The present study therefore assessed the relative percentage and particularly the zonal distribution of cartilage MPC using the markers CD105/CD166.     

Aerobic glycolysis: a study of human articular cartilage

Cartilage generally is one of those tissues that exhibit aerobic glycolysis. In a previous study on rat epiphyseal cartilage it had been suggested that this phenomenon is related to potentially excessive production of pyruvate and acetyl coenzyme A, the latter derived from fatty acid oxidation and inhibiting pyruvate dehydrogenase activity. The present study has shown that, in human articular cartilage, the contribution from fatty acid oxidation is too small to account for this phenomenon although the total potential production of pyruvate could still be in excess of the requirements for acetyl coenzyme A for the Krebs' cycle. Of greater relevance may be the apparent correlations that have been found between the activities of lactate and glyceraldehyde 3-phosphate dehydrogenases (r = 0 X 82: 0.01 greater than p greater than 0.001) and between those of lactate and glucose 6-phosphate dehydrogenases (r = 0.92; p less than 0.001).     

Degradome expression profiling in human articular cartilage

Introduction

The molecular mechanisms underlying cartilage destruction in osteoarthritis are poorly understood. Proteolysis is a key feature in the turnover and degradation of cartilage extracellular matrix where the focus of research has been on the metzincin family of metalloproteinases. However, there is strong evidence to indicate important roles for other catalytic classes of proteases, with both extracellular and intracellular activities. The aim of this study was to profile the expression of the majority of protease genes in all catalytic classes in normal human cartilage and that from patients with osteoarthritis (OA) using a quantitative method.

Methods

Human cartilage was obtained from femoral heads at joint replacement for either osteoarthritis or following fracture to the neck of femur (NOF). Total RNA was purified, and expression of genes assayed using Taqman^® low-density array quantitative RT-PCR.

Results

A total of 538 protease genes were profiled, of which 431 were expressed in cartilage. A total of 179 genes were differentially expressed in OA versus NOF cartilage: eight aspartic proteases, 44 cysteine proteases, 76 metalloproteases, 46 serine proteases and five threonine proteases. Wilcoxon ranking as well as the LogitBoost-NR machine learning approach were used to assign significance to each gene, with the most highly ranked genes broadly similar using each method.

Conclusions

This study is the most complete quantitative analysis of protease gene expression in cartilage to date. The data help give direction to future research on the specific function(s) of individual proteases or protease families in cartilage and may help to refine anti-proteolytic strategies in OA.     

Effect of hyaluronic acid on human chondrocyte cell lines from articular cartilage

The effects of hyaluronic acid (HA) derivative on the proliferation and metabolism of human chondrocytes were examined. Cells were obtained from cartilage from metatarsal phalangeal joints of 20 adult humans (aged 22-63) and from femoral knee condyles of 10 subjects (aged 22-77). Chondrocytes isolated by collagenase/Dnase digestion were cultured with addition of different doses of HA for 4 weeks. Morphological studies demonstrated that HA enhanced the adhesion of cells to substrate; HA-treated chondrocytes proliferated better than chondrocytes cultured in HA-free medium. This study shows that HA improves in vitro substrate adhesion ability and proliferative activity of human cartilage cells and that the response to the treatment varies on an individual basis.     

Abnormalities of DNA in human osteoarthritic articular cartilage

The normal amount of DNA in human diploid nuclei was determined by the use of the Feulgen reaction measured by microdensitometry. The DNA-content of nuclei in normal human articular cartilage was determined in nuclei of zones 3 and 4 of cartilage of the femoral head removed from osteoporotic fractured necks of femur. Analysis of the results indicated that a degree of synthesis of DNA occurred even in these zones of very elderly persons. Results on these zones in the articular cartilage of osteoarthritic joints indicated that different populations occurred. In some there was DNA-synthesis related to tetraploidy; in others, the DNA was very stable to acid hydrolysis with no sign of biosynthetic activity; in the last group, which contained erosions of the superficial zones, the DNA was unstable to hydrolysis.     

Electron microscopy of human articular cartilage]

Scanning and transmission microscopy of the articular cartilage was performed in femoral condyles of persons at the age of 30-50 years. It was demonstrated that hyaline cartilage is covered with a protective fibrillar layer consisting of tightly pressed collagenous fibrillae with an underlying layer of fibroblastic cells. In the intracellular substance of the hyaline cartilage fibrillar structures form a complex reticular web with vertical arrangement of the main collagenous fasiculi. In the superficial layer of the hyaline cartilage the collagenous fibrillae and their fasciculi form arcade-like structures. Lacunar chondrocytes have a rough villose surface, cellular secrete is discharged as round granules through cytoplasmic membrane. Ultrastructural changes in chondrocytes are observed simultaneously with their degenerative-dystrophic changes.     

The proteoglycans of bovine nasal cartilage and human articular cartilage: a sedimentation equilibrium analysis

Cartilage proteoglycan was isolated from bovine nasal septum and fractionated according to buoyant density after dissociative CsCl density gradient centrifugation. Gel-exclusion chromatography showed that hyaluronic acid was present in fractions of density lower than 1.69 g/mL. The molecular weight, assessed by sedimentation equilibrium analysis, of the proteoglycan present in the fractions with density > 1.69 g/mL, which appeared chromatographically homogeneous and constituted 54% of the preparation, ranged from 1.0 to 2.6 × 10⁶ for v = 0.55 cm³ g^?1. Carbodiimide-induced modification of the carboxyl groups by methylamine resulted in a reduction of the molecular weight to 0.74 – 1.25 × 10⁶. An analogous reduction in molecular weight was obtained after equilibration of this proteoglycan fraction with hyaluronic acid oligomers containing five disaccharide units. Since both procedures are known to cause inhibition of the interaction between proteoglycans and hyaluronic acid, it is suggested that this lower molecular-weight range represents the true degree of polydispersity of the sub-units of hyaline cartilage proteoglycan constituting this fraction, while the higher values obtained for the intact proteoglycan are the result of the presence of hyaluronic acid in the sample. The molecular-weight range of the whole proteoglycan subunit preparation, assessed after carboxyl group modification, was 0.5–1.2 × 10⁶. Apparently normal and abnormal cartilage was excised from single human osteoarthrosic femoral heads. Proteoglycans extracted by 4M guanidine hydrochloride were isolated after dissociative density gradient centrifugation and subjected to carboxyl group modification. Preparations from normal tissue exhibited molecular-weight averages ranging from 5 to 9 × 10⁵. A molecular-weight reduction was observed with proteoglycans isolated from abnormal areas.