RESUMO
OBJECTIVE: Dialysis-related amyloidosis (DRA) is a severe complication of maintenance hemodialysis (HD). Given the predominant deposition of beta(2)-microglobulin (beta2m) fibrils on articular cartilage in early DRA, we investigated the significance of beta2m and its relationship to distinct cartilage biomarkers in early DRA diagnosis in HD patients. Furthermore, we assessed the effects of beta2m on articular chondrocytes in vitro. METHODS: Serum samples from 133 patients were collected before and after HD. Type II collagen cleavage product (C2C), procollagen II c-propeptide (CPII), aggrecan chondroitin sulfate 846 epitope (CS-486) and cartilage oligomeric matrix protein (COMP) levels were determined by enzyme-linked immunosorbent assay. Primary bovine articular chondrocytes were cultured as monolayers and incubated with beta2m at 1.5mg/l and 20mg/l. Cartilage glucosaminoglycan synthesis was measured by [(35)S]sulfate incorporation. mRNA expression of interleukin (IL)-1beta, matrix metalloproteinases (MMPs)-3 and -9 was measured by reverse-transcriptase polymerase chain reaction (RT-PCR). RESULTS: Incubation with beta2m at 20mg/l significantly decreased matrix biosynthesis. PCR analysis revealed an increase of IL-1beta, as well as MMPs-3 and -9 on the mRNA level. C2C/CPII, CS-486 and COMP levels were increased only in a subset of patients without a significant correlation with beta2m concentrations. A subgroup analysis elucidated an increase in type II collagen degradation during the first years of HD, as shown by the elevation of C2C/CPII ratio. CONCLUSION: beta2m exerted anti-anabolic effects on articular chondrocytes in vitro and might be involved in cartilage degradation in HD patients. beta2m serum levels, however, did not reflect cartilage degradation in DRA. The assessment of C2C/CPII, CS-486 or COMP concentrations apparently has minor relevance in DRA diagnosis in HD patients. However, the increased type II collagen breakdown within 5 years after HD onset possibly mirrors the early stages of DRA. Thus, the C2C/CPII ratio could be employed in longitudinal studies, since it may reflect a risk for DRA related arthropathy development in a subset of patients.
Assuntos
Amiloidose/etiologia , Cartilagem Articular/metabolismo , Colágeno Tipo II/metabolismo , Diálise Renal/efeitos adversos , Microglobulina beta-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Amiloidose/sangue , Animais , Biomarcadores/metabolismo , Doenças das Cartilagens , Estudos de Casos e Controles , Bovinos , Células Cultivadas , Condrócitos/metabolismo , Humanos , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
OBJECTIVE: To assess the presence of Toll-like receptors (TLRs) 1-9 in human articular cartilage, and to investigate the effects of lipopolysaccharide (LPS)-induced activation of TLR-4 on biosynthetic activity and matrix production by human articular chondrocytes. METHODS: TLRs 1-9 were assessed in human articular cartilage by reverse transcription-polymerase chain reaction (RT-PCR); TLR-4 was also analyzed by Western blotting and immunohistochemistry. Articular chondrocytes were isolated from human donors and from wild-type or TLR-4(-/-) mice. Chondrocyte monolayer cultures were incubated with interleukin-1beta (IL-1beta) and LPS in the absence or presence of bone morphogenetic protein 7 (BMP-7) and IL-1 receptor antagonist (IL-1Ra). Neosynthesis of sulfated glycosaminoglycans (sGAG) was measured by (35)S-sulfate incorporation. Endogenous gene expression of cartilage markers as well as IL-1beta was examined using RT-PCR. The involvement of p38 kinase or p44/42 kinase (ERK-1/2) in LPS-mediated TLR-4 signaling was investigated by immunoblotting, RT-PCR, and sGAG synthesis. RESULTS: TLRs 1-9 were found on the messenger RNA (mRNA) level in human articular chondrocytes. The presence of TLR-4 was also observed on the protein level. In murine and human articular chondrocytes, but not in chondrocytes derived from TLR-4(-/-) mice, stimulation with LPS resulted in a decrease in total proteoglycan synthesis. IL-1beta mRNA expression was increased by TLR-4 activation, whereas expression of aggrecan and type II collagen was significantly decreased. The presence of BMP-7 and IL-1Ra antagonized the anti-anabolic effects of LPS. Blocking of p38, but not ERK-1/2, resulted in inhibition of both LPS-mediated IL-1beta gene expression and the negative effects of LPS on matrix biosynthesis. CONCLUSION: These data demonstrate the presence of TLRs in human articular cartilage. The suppressive effects of LPS on cartilage biosynthetic activity are dependent on the presence of TLR-4, are governed, at least in part, by an up-regulation of IL-1beta, and are mediated by p38 kinase. These in vitro data indicate an anti-anabolic effect of TLR-4 in articular chondrocytes that may hamper cartilage repair in various joint diseases.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Condrócitos/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Lipopolissacarídeos/farmacologia , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Proteína Morfogenética Óssea 7 , Proteína de Matriz Oligomérica de Cartilagem , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Proteínas da Matriz Extracelular/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Glicoproteínas/genética , Humanos , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1beta/genética , Interleucina-1beta/fisiologia , Proteínas Matrilinas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor 4 Toll-Like/genética , Receptores Toll-Like/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
OBJECTIVE: To investigate the effects of a pulsed electromagnetic field (EMF) on articular cartilage matrix biosynthesis with regard to age and cartilage damage using a matrix depleted cartilage explant model. METHODS: Cartilage explants were obtained from metacarpophalangeal joints of calves and adult cows. After depletion of the extracellular matrix by trypsin digestion, samples were maintained in serum-free basal medium with and without the addition of interleukin 1beta (IL1beta). Half the samples were subjected to an EMF for 24 minutes daily; the other half were left untreated. Undigested and untreated explants served as negative controls. After 7 days, biosynthesis of matrix macromolecules was assessed by [35S]sulphate incorporation and values were normalised to hydroxyproline content. RESULTS: The EMF increased matrix macromolecule synthesis in undigested, untreated explants (p<0.009). In matrix depleted samples the EMF had no stimulatory effect on proteoglycan biosynthesis. IL1beta significantly decreased the de novo synthesis of matrix macromolecules (p<0.00004) in young and adult samples, but an EMF partly counteracted this inhibitory effect in cartilage samples from young, but not old animals. CONCLUSION: EMF promoted matrix macromolecule biosynthesis in intact tissue explants but had no stimulatory effect on damaged articular cartilage. The supressive effects of IL1beta were partially counteracted by EMF exposure, exclusively in cartilage derived from young animals. An EMF has age dependent chondroprotective but not structure modifying properties when cartilage integrity is compromised.
Assuntos
Envelhecimento/fisiologia , Cartilagem Articular/metabolismo , Campos Eletromagnéticos , Proteínas da Matriz Extracelular/biossíntese , Matriz Extracelular/metabolismo , Animais , Cartilagem Articular/citologia , Bovinos , Feminino , Interleucina-1/farmacologia , Articulação Metacarpofalângica , Técnicas de Cultura de TecidosRESUMO
OBJECTIVE: To investigate effects of cartilage derived morphogenetic protein-1 and -2 (CDMP-1, CDMP-2), bone morphogenetic protein (BMP)-7 and BMP-6 on metabolism of ligament fibroblasts and their osteogenic or chondrogenic differentiation potential. METHODS: Ligament fibroblasts were obtained from 3 month old calves, plated as monolayers or micromass cultures, and incubated with or without CDMP-1, CDMP-2, BMP-7, and BMP-6. Expression of the indicated growth factors was assessed by RT-PCR and western immunoblotting. The presence of their respective type I and II receptors, and lineage related markers, was investigated in stimulated and unstimulated cells by RT-PCR and northern blotting. Biosynthesis of matrix proteoglycans was assessed by [(35)S]sulphate incorporation in monolayers. Alcian blue and toluidine blue staining was done in micromass cultures. RESULTS: CDMP-1, CDMP-2, BMP-7, and BMP-6 were detected on mRNA and on the protein level. Type I and II receptors were endogenously expressed in unstimulated ligament fibroblasts. The growth factors significantly stimulated total proteoglycan synthesis as assessed by [(35)S]sulphate incorporation. Toluidine blue staining showed cartilage-specific metachromasia in the growth factor treated micromass cultures. Transcription analysis of stimulated ligament fibroblasts demonstrated coexpression of chondrocyte markers but no up regulation of osteogenic markers. CONCLUSION: CDMP-1, CDMP-2, BMP-7, and BMP-6 and their receptors were expressed in ligament tissue. These growth factors induced matrix synthesis in fibroblasts derived from bovine ligament. The preferential expression of cartilage markers in vitro suggests that CDMP-1, CDMP-2, BMP-7, and BMP-6 have the potential to induce differentiation towards a chondrogenic phenotype in ligament fibroblasts. Thus, fibroblasts from ligaments may serve as a source for chondrogenesis and tissue repair.