transforming-growth-factor-beta and Osteoarthritis--Hip

The study of primary hip OA is continuing to redefine what was once considered a stagnant pathology as one of dynamic change, occurring over a long period of time involving the many composite tissue types of the joint including the bone. Examination of the inverse relationships evident between OA and fracture cohorts, including individuals with osteoporosis (OP), indicates an imbalance in formation and resorption in the bony component of both pathologies. This review contains an overview of primary OA followed by an assessment of differential gene expression and altered cellular characteristics identified in the bony compartments of primary hip OA, with a focus on the wingless mouse mammary tumor virus integration (Wnt) and TGF-β signalling pathways. The studies reviewed here suggest that OA is a systemic disease involving the bone and validate the assessment of molecular changes to further investigate this complex disease.

Topics: Animals; Biomarkers; Bone Remodeling; Cell Proliferation; Gene Expression; Hip Fractures; Humans; Mice; Osteoarthritis, Hip; Osteoblasts; Rats; Risk Factors; Signal Transduction; Spinal Fractures; Transforming Growth Factor beta; Wnt Proteins

Femoroacetabular impingement (FAI) is an important trigger of hip osteoarthritis (OA). Epigenetic changes in DNA methyltransferase 3B (DNMT3B) attenuate catabolic gene expression in cartilage hemostasis. This study aimed to examine the articular chondrocyte catabolic state and DNMT3B and 4-aminobutyrate aminotransferase promoter (ABAT) expression during OA progression in FAI. Cartilage samples were collected from the impingement zone of 12 patients with cam FAI (early-FAI) and 12 patients with advanced OA secondary to cam FAI (late-FAI-OA). Five healthy samples were procured from cadavers (ND: nondiseased). Explants were cultured under unstimulated conditions, catabolic stimulus (IL1β), or anabolic stimulus (TGFβ). Histology was performed with safranin-O/fast-green staining. Gene expression was analyzed via qPCR for GAPDH, DNMT3B, ABAT, MMP-13, COL10A1. Methylation specific PCR assessed methylation status at the ABAT promoter. Cartilage samples in early-FAI and late-FAI-OA showed a histological OA phenotype and increased catabolic marker expression (MMP13/COL10A1, ND vs. early-FAI, p = 0.004/p < 0.001, ND vs. late-FAI-OA, p < 0.001/p < 0.001). RT-PCR confirmed DNMT3B underexpression (ND vs. early-FAI, p < 0.001, early-FAI vs. late-FAI-OA, p = 0.016) and ABAT overexpression (ND vs. early-FAI, p < 0.001, early vs. late-FAI-OA, p = 0.035) with advanced disease. End-stage disease showed ABAT promoter hypomethylation. IL1β stimulus accentuated ABAT promoter hypomethylation and led to further ABAT and catabolic marker overexpression in early-FAI and late-FAI-OA while TGFβ normalized these alterations in gene expression. Catabolic and epigenetic molecule expression suggested less catabolism in early-stage disease. Sustained inflammation induced ABAT promoter hypo-methylation causing a catabolic phenotype. Suppression of ABAT by methylation control could be a new target for therapeutic intervention to prevent OA progression in hip FAI.

Topics: Cartilage, Articular; Disease Progression; Epigenesis, Genetic; Femoracetabular Impingement; Hip Joint; Humans; Osteoarthritis, Hip; Transforming Growth Factor beta

When serially passaged in standard monolayer culture to expand cell number, articular chondrocytes lose their phenotype. This results in the formation of fibrocartilage when they are used clinically, thus limiting their use for cartilage repair therapies. Identifying a way to redifferentiate these cells in vitro is critical if they are to be used successfully. Transforming growth factor beta (TGFβ) family members are known to be crucial for regulating differentiation of fetal limb mesenchymal cells and mesenchymal stromal cells to chondrocytes. As passaged chondrocytes acquire a progenitor-like phenotype, the hypothesis of this study was that TGFβ supplementation will stimulate chondrocyte redifferentiation in vitro in serum-free three-dimensional (3D) culture.. Human articular chondrocytes were serially passaged twice (P2) in monolayer culture. P2 cells were then placed in high-density (3D) culture on top of membranes (Millipore) and cultured for up to 6 weeks in chemically defined serum-free redifferentiation media (SFRM) in the presence or absence of TGFβ. The tissues were evaluated histologically, biochemically, by immunohistochemical staining, and biomechanically.. Passaged human chondrocytes cultured in SFRM supplemented with 10 ng/mL TGFβ3 consistently formed a continuous layer of articular-like cartilage tissue rich in collagen type 2 and aggrecan and lacking collagen type 1 and X in the absence of a scaffold. The tissue developed a superficial zone characterized by expression of lubricin and clusterin with horizontally aligned collagen fibers.. This study suggests that passaged human chondrocytes can be used to bioengineer a continuous layer of articular cartilage-like tissue in vitro scaffold free. Further study is required to evaluate their ability to repair cartilage defects in vivo.

Topics: Cells, Cultured; Chondrocytes; Humans; Hyaline Cartilage; Osteoarthritis, Hip; Transforming Growth Factor beta

Articular cartilage is well studied in osteoarthritis (OA). However, the role of supporting structures, such as the acetabular labrum, a sealing structure surrounding the hip joint, has been investigated much less. We recently showed that fibrochondrocytic labrum cells are metabolically active. This study was undertaken to investigate hip OA–associated changes in human acetabular labrum cells.. Microarray analysis was performed to compare OA labrum cells to healthy labrum cells cultured in a 3-dimensional alginate bead system. Data were analyzed by cluster analysis using gene set enrichment analysis software and by gene list analysis using PANTHER gene family tools. Selected candidates were validated by quantitative polymerase chain reaction analysis on labrum and cartilage samples and by immunohistochemistry. The functional impacts of the genes identified were investigated by in vitro stimulation experiments in labrum cells.. Pathway analysis revealed increased cytokine and chemokine signaling in OA labrum cells, whereas reduced extracellular matrix interactions and transforming growth factor β signaling were observed. Several genes were significantly differentially expressed in OA compared to healthy labrum. We specifically focused on 3 small leucine-rich repeat proteins (SLRPs), osteomodulin, osteoglycin, and asporin, that appeared to be distinctly regulated in OA labrum compared to OA cartilage. SLRPs were strongly down-regulated in OA labrum but up-regulated in OA articular chondrocytes. Moreover, in vitro stimulation with osteomodulin increased aggrecan expression in OA labrum cells.. OA labrum fibrochondrocytes have several features similar to OA chondrocytes. However, SLRP expression seems to be differentially influenced by degeneration in OA labrum compared to cartilage, suggesting a specific role for this supporting structure in OA. The functional impact of SLRPs on labrum cells makes them interesting targets for further studies in hip OA.

Topics: Acetabulum; Adolescent; Adult; Aged; Cartilage, Articular; Cells, Cultured; Down-Regulation; Extracellular Matrix; Extracellular Matrix Proteins; Female; Gene Expression Regulation; Humans; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Ligaments, Articular; Male; Middle Aged; Osteoarthritis, Hip; Proteoglycans; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Young Adult

Osteoarthritis is a disease characterized by an increase in the production of reactive oxygen species (ROS) in afflicted joints. Excess iron, due to its role in the production of ROS and crystal deposition in the joints, is implicated in the disease progression of osteoarthritis. Ferritin is a major regulator of the bioavailability of iron, and its functions are determined largely by the combination of H- and L-subunits present in its outer protein shell. The purpose of the study was to investigate the expression of the H- and L-subunits of ferritin in bone marrow macrophages of osteoarthritis patients. The cytokine profiles were assessed as cytokines play an important role in the expression of the ferritin subunits. The H-subunit of ferritin in the bone marrow macrophages was significantly higher (P value = 0.035) in the osteoarthritis patients compared with the controls (107.84; 69.25-167.94 counts/μm(2); n = 7 versus 71.07; 58.56-86.26 counts/μm(2); n = 19). A marginally significant increase (P value = 0.059) was shown for the expression of the L-subunit in the osteoarthritis patients compared with the controls (133.03; 104.04-170.10 counts/μm(2); n = 7 versus 104.23; 91.53-118.70 counts/μm(2); n = 19). The osteoarthritis and control groups had comparable C-reactive protein, as well as proinflammatory and anti-inflammatory cytokine concentrations. The major exception was for transforming growth factor-β (TGF-β), which was higher (P value = 0.014) in the plasma of the osteoarthritis patients (16.69; 13.09-21.28 ng/mL; n = 7 versus 8.60; 6.34-11.67 ng/mL; n = 19). Up-regulation of the ferritin subunits decreases the levels of bioavailable iron and provides protection against the unwarranted production of ROS and crystal deposition. A role for TGF-β in the up-regulation of the expression of the H-subunit, and possibly the L-subunit, of ferritin is postulated in osteoarthritis.

Topics: Apoferritins; C-Reactive Protein; Case-Control Studies; Cytokines; Humans; Macrophages; Osteoarthritis, Hip; Reactive Oxygen Species; Transforming Growth Factor beta; Up-Regulation

Decreased levels of transforming growth factor beta (TGFbeta) have been related to the failure of cartilage repair in experimental models of osteoarthritis. This study aimed to examine this aspect of osteoarthritis in human cartilage.. Cartilage samples were obtained from 11 patients with hip osteoarthritis and 11 patients with femoral neck fracture who were undergoing total hip replacement. Gene expression of the three TGFbeta isoforms, collagen type II (COL2A1) and aggrecan (AGC1) was analysed by reverse transcription quantitative PCR and immunohistochemistry.. Expression of the three TGFbeta isoforms was increased in osteoarthritis cartilage. The upregulation was more marked for the TGFbeta3 isoform (2.3-fold) than for TGFbeta1 (1.6-fold) or TGFbeta2 (1.7-fold). The messenger RNA levels of TGFbeta1 and TGFbeta2 were strongly correlated in osteoarthritis cartilage (r(s) = 0.83, p = 0.002), but levels of TGFbeta3 were uncorrelated with any of the two other TGFbeta isoforms. Immunohistochemistry showed an extension of immunoreactivity for the three TGFbeta isoforms to more chondrocytes and to deeper cartilage layers in the more severe osteoarthritis lesions. No correlation of TGFbeta isoforms with COL2A1 or AGC1 expression levels was found.. The three isoforms of TGFbeta were differentially upregulated in late osteoarthritis in relation to an increased percentage of TGFbeta-positive chondrocytes. These results indicate that cartilage damage progresses in spite of the TGFbeta stimulus for cartilage anabolism and that other causes of the failure to cope with the increased cartilage catabolism of osteoarthritis should be investigated.

Topics: Cartilage, Articular; Case-Control Studies; Cytoplasm; Gene Expression; Humans; Immunohistochemistry; Osteoarthritis, Hip; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Statistics, Nonparametric; Transforming Growth Factor beta; Up-Regulation

The proenzyme of matrix metalloproteinase 7 (proMMP-7), which can degrade various extracellular matrix (ECM) and non-ECM molecules after being activated, is overexpressed in osteoarthritic (OA) articular cartilage, but the process of its activation in the cartilage remains unknown. The present study was undertaken to investigate the expression of tetraspanin CD151 in OA cartilage and its involvement in proMMP-7 activation.. The expression of CD151 in articular cartilage was examined by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunohistochemistry, in situ hybridization, and immunoblotting. Chondrocytes were used to study the interaction between CD151 and proMMP-7, and activation of proMMP-7.. RT-PCR revealed expression of CD151 messenger RNA in all OA cartilage samples, but in only 30% of normal control cartilage samples. Immunohistochemistry and in situ hybridization findings indicated that CD151 was coexpressed with proMMP-7 in chondrocytes, mainly in the superficial and transitional zones of OA cartilage. CD151 immunoreactivity directly correlated with the Mankin score (r = 0.757, P < 0.0001 [n = 30]) and the degree of chondrocyte cloning (r = 0.83, P < 0.0001 [n = 30]) in the cartilage samples. Complexes CD151 and proMMP-7 and their colocalization on the cell membranes were demonstrated by immunoprecipitation and double fluorescence immunostaining of the OA chondrocytes. In situ zymography indicated that chondrocytes exhibit pericellular proteolytic activity, which was abolished by treatment with MMP inhibitors, anti-MMP-7 antibody, or anti-CD151 antibody.. These data demonstrate that CD151 is overexpressed in OA cartilage and suggest that CD151 plays a role in the pericellular activation of proMMP-7, leading to cartilage destruction and/or chondrocyte cloning.

Topics: Aged; Aged, 80 and over; Antigens, CD; Cartilage; Cells, Cultured; Chondrocytes; Gene Expression Regulation; Humans; Interleukin-1alpha; Matrix Metalloproteinase 7; Middle Aged; Osteoarthritis, Hip; Osteoarthritis, Knee; RNA, Messenger; Tetraspanin 24; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A

Osteoarthritis (OA) is characterized by erosion of cartilage and formation of osteophytes. Since transforming growth factor beta (TGF-beta) is known to be involved in chondrogenesis and osteogenesis, we studied by immunochemistry the expression of TGF-beta isoform types 1, 2, and 3 and their receptor types I and II in slightly and strongly altered areas of human OA cartilage and in osteophytes.. Specimens were collected from femoral heads at the time of hip arthroplasty, selecting osteophytic regions and areas of slight or severe degradation according to the Mankin score. Cryostat sections were prepared and submitted to immunohistochemistry using appropriate antibodies to TGF-beta(1-3) and TGF-beta receptors I and II.. TGF-beta1 expression was shown to be depressed in strongly degraded cartilage, compared to normal and slightly altered areas. TGF-beta2 was barely detectable in all samples studied. In osteophytes, a marked overexpression of TGF-beta1 and -beta3 was observed. An important decrease in TGF-beta receptor II was found in fibrillated cartilage areas.. The three major isoforms of TGF-beta are expressed in human OA cartilage, albeit the TGF-beta2 level is very low. Their expression patterns and the ratio of receptors I and II varies according to the degree of OA severity. The decrease in TGF-beta1 production and marked downregulation of receptor II in fibrillated cartilage may lead to reduced chondrocyte responsiveness to TGF-beta and contribute to the irreversibility of the disease. Overexpression of TGF-beta1 and -beta3 in osteophytes suggests that the two isoforms are involved in the formation of these structures.

Topics: Aged; Aged, 80 and over; Arthroplasty, Replacement, Hip; Cartilage, Articular; Femur Head; Humans; Immunohistochemistry; Osteoarthritis, Hip; Receptors, Transforming Growth Factor beta; Transforming Growth Factor beta

Osteoarthritis is the most common form of human arthritis. We investigated the potential role of asporin, an extracellular matrix component expressed abundantly in the articular cartilage of individuals with osteoarthritis, in the pathogenesis of osteoarthritis. Here we report a significant association between a polymorphism in the aspartic acid (D) repeat of the gene encoding asporin (ASPN) and osteoarthritis. In two independent populations of individuals with knee osteoarthritis, the D14 allele of ASPN is over-represented relative to the common D13 allele, and its frequency increases with disease severity. The D14 allele is also over-represented in individuals with hip osteoarthritis. Asporin suppresses TGF-beta-mediated expression of the genes aggrecan (AGC1) and type II collagen (COL2A1) and reduced proteoglycan accumulation in an in vitro model of chondrogenesis. The effect on TGF-beta activity is allele-specific, with the D14 allele resulting in greater inhibition than other alleles. In vitro binding assays showed a direct interaction between asporin and TGF-beta. Taken together, these findings provide another functional link between extracellular matrix proteins, TGF-beta activity and disease, suggesting new therapeutic strategies for osteoarthritis.

Topics: Aggrecans; Aspartic Acid; Carrier Proteins; Chondrogenesis; Chromosome Mapping; Disease Susceptibility; Extracellular Matrix Proteins; Glycoproteins; Humans; In Vitro Techniques; Lectins, C-Type; Minisatellite Repeats; Molecular Sequence Data; Osteoarthritis; Osteoarthritis, Hip; Osteoarthritis, Knee; Polymorphism, Genetic; Proteoglycans; Transforming Growth Factor beta

Recently it has been shown that an inactivating mutation in the TGFb-SMAD3 signaling pathway, which increases the conversion of osteoblasts to osteocytes, is accompanied by bone loss combined with increased osteocyte density. We hypothesized that increased matrix TGFb, known to occur in osteoarthritis, might cause the reverse of these effects in man. Because coxarthrosis (cOA) is associated with a reduced risk of femoral neck fracture, whole cross-section femoral neck biopsies were obtained from 11 patients with femoral neck fracture, 14 patients with cOA, and 22 age-and sex-matched controls. Lacunar density (Lc x mm2), osteocyte density (Ot x mm2), and cancellous wall width (Cn x W x Wi), were compared between cases of coxarthrosis, femoral neck fracture (FNF) and controls. In cOA, Lc.mm2 was reduced by 24% (P <0.001) while in FNF it was increased by 20% (P <0.001). Cn x W x Wi was increased in cOA by 22% (P <0.05) and in FNF was reduced by 27% (P <0.001). Lc x mm2 was inversely related to percentage cancellous bone area (adj. r2 = 0.373; P <0.01) and wall widths, r2 = 0.382, P <0.001. The reduction in osteocyte lacunar density coupled with increased wall width is consistent with a model of cOA effects on bone in which increased levels of matrix TGFb might prolong the effective lifespan or work rate of the osteoblast and delay its incorporation into the matrix as an osteocyte. One possible approach to strengthening bone in osteoporosis might be to enhance the effective lifespan of the osteoblast by modulating TGFb-related pathway activity in its local environment.

Topics: Aged; Aged, 80 and over; Bone Matrix; Bone Resorption; DNA-Binding Proteins; Female; Femoral Neck Fractures; Femur Neck; Humans; Image Processing, Computer-Assisted; Male; Middle Aged; Osteoarthritis, Hip; Osteocytes; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

Bone- and cartilage-derived morphogenetic proteins (BMPs and CDMPs), which are TGFbeta superfamily members, are growth and differentiation factors that have been recently isolated, cloned and biologically characterized. They are important regulators of key events in the processes of bone formation during embryogenesis, postnatal growth, remodelling and regeneration of the skeleton. In the present study, we used immunohistochemical methods to investigate the distribution of BMP-2, -3, -5, -6, -7 and CDMP-1, -2, -3 in human osteophytes (abnormal bony outgrowths) isolated from osteoarthritic hip and knee joints from patients undergoing total joint replacement surgery. All osteophytes consisted of three different areas of active bone formation: (1) endochondral bone formation within cartilage residues; (2) intramembranous bone formation within the fibrous tissue cover and (3) bone formation within bone marrow spaces. The immunohistochemistry of certain BMPs and CDMPs in each of these three different bone formation sites was determined. The results indicate that each BMP has a distinct pattern of distribution. Immunoreactivity for BMP-2 was observed in fibrous tissue matrix as well as in osteoblasts; BMP-3 was mainly present in osteoblasts; BMP-6 was restricted to young osteocytes and bone matrix; BMP-7 was observed in hypertrophic chondrocytes, osteoblasts and young osteocytes of both endochondral and intramembranous bone formation sites. CDMP-1, -2 and -3 were strongly expressed in all cartilage cells. Surprisingly, BMP-3 and -6 were found in osteoclasts at the sites of bone resorption. Since a similar distribution pattern of bone morphogenetic proteins was observed during embryonal bone development, it is suggested that osteophyte formation is regulated by the same molecular mechanism as normal bone during embryogenesis.

Topics: Aged; Aged, 80 and over; Biomarkers; Bone and Bones; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 3; Bone Morphogenetic Protein 5; Bone Morphogenetic Protein 6; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Bone Regeneration; Femur Head; Growth Differentiation Factor 5; Humans; Immunohistochemistry; Middle Aged; Osteoarthritis; Osteoarthritis, Hip; Osteoarthritis, Knee; Tibia; Transforming Growth Factor beta

To investigate the relationships between serum and urinary molecular markers (MM) used to monitor osteoarthritis.. Forty osteoarthritis patients had blood and urine collected at baseline and 1, 3, 6 and 12 months later. Specimens from 20 controls were obtained twice at a one month interval. The concentration of 14 different markers was determined at each time point and the data were analyzed by statistical methodology.. The markers could be divided by the method of principal components analysis into five clusters of related markers: inflammation markers (C-reactive protein, tumor necrosis receptor type I and tumor necrosis receptor type II, interleukin 6, eosinophilic cationic protein), bone markers (bone sialoprotein, hydroxylysyl pyridinoline, lysyl pyridinoline), putative markers of cartilage anabolism (carboxypropeptide of type II procollagen, hyaluronan, epitope 846) and catabolism (keratan sulfate, cartilage oligomeric matrix protein), and transforming growth factor beta. Three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters discriminated osteoarthritis patients from controls. Inflammation was not a confounding factor in measurement, but a recognizable distinguishing factor in osteoarthritis.. The markers separated into rational groups on the basis of their covariance, a finding with independent biochemical support. The covariance of markers from the same cluster suggests the use of a representative marker from the cluster to reflect changes in osteoarthritis. If multiple markers are being measured within a single cluster, then the use of a weighted cluster 'factor' may be preferable to the separate use of individual markers.

Topics: Amino Acids; Biomarkers; Blood Proteins; C-Reactive Protein; Carboxypeptidases; Case-Control Studies; Epitopes; Extracellular Matrix Proteins; Female; Humans; Hyaluronic Acid; Interleukin-6; Keratan Sulfate; Male; Middle Aged; Osteoarthritis; Osteoarthritis, Hip; Osteoarthritis, Knee; Procollagen; Receptors, Tumor Necrosis Factor; Sialoglycoproteins; Transforming Growth Factor beta

Osteoarthritis is characterized by marked osteophyte formation consisting of new cartilage and bone. Because several growth factors are known to be involved in chondrogenesis and osteogenesis, the expression of transforming growth factor-beta 1 and basic fibroblast growth factor in the osteophytes of the human femoral heads in osteoarthritis were examined. Transforming growth factor-beta 1 messenger ribonucleic acid was detected in the osteophytes by reverse transcription-polymerase chain reaction. All of the nine examined osteophytes expressed transforming growth factor-beta 1 messenger ribonucleic acid, whereas one of four osteoarthritic femoral heads and none of four osteonecrotic femoral heads expressed transforming growth factor-beta 1 messenger ribonucleic acid. The extent of transforming growth factor-beta 1 messenger ribonucleic acid expression varied among the osteophytes. Transforming growth factor-beta 1 or basic fibroblast growth factor was analyzed in osteophytes immunohistochemically. Transforming growth factor-beta 1 was localized in the superficial cells in the osteophyte cartilage, but it was scarcely detected in the superficial cells in the degenerative articular cartilage. Basic fibroblast growth factor was detected in the cells in the whole layer of osteophyte cartilage and in the articular cartilage. There was a difference in the localization, which suggests the different roles of transforming growth factor-beta 1 and basic fibroblast growth factor in bone and cartilage metabolism in osteophyte formation.

Topics: Fibroblast Growth Factor 2; Gene Expression; Humans; Immunohistochemistry; Osteoarthritis, Hip; RNA, Messenger; Transforming Growth Factor beta