RESUMO
Hyaluronan (HA) retention inside the synovial cavity of joints serves diverse protective roles. We tested the hypothesis that HA retention is mediated by the network of extracellular matrix proteins in the synovial lining. Cannulated rabbit knee joints were infused with HA solution with or without pretreatment by chymopapain, a collagen-sparing protease. Trans-synovial fluid escape rate was measured and, after a period of trans-synovial filtration, samples of intra-articular fluid and subsynovial fluid were analysed for HA to assess its trans-synovial ultrafiltration. In control joints, HA ultrafiltration was confirmed by postfiltration increases in intra-articular HA concentration (259 +/- 17% of infused concentration) and reduced subsynovial concentration (30 +/- 8%; n = 11). The proportion of HA molecules reflected by the synovium was 57-75%. Chymopapain treatment increased the hydraulic permeability of the synovial lining approximately 13-fold, almost abolished the trans-synovial difference in HA concentration and reduced the HA reflected fraction to 3-7% (n = 6; P < 0.001, ANOVA). Structural studies confirmed that chymopapain treatment depleted the matrix of proteoglycans but preserved its collagen. The findings thus demonstrate that HA ultrafiltration and synovial hydraulic permeability are determined by the network of non-collagen, extracellular matrix proteins. This may be important clinically, since protease activity is raised in rheumatoid arthritis, as are HA and fluid escape.
Assuntos
Quimopapaína/farmacologia , Proteínas da Matriz Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Articulações/metabolismo , Líquido Sinovial/metabolismo , Animais , Galinhas , Quimopapaína/administração & dosagem , Dextranos , Filtração , Fluoresceínas , Corantes Fluorescentes , Injeções Intra-Arteriais , Articulações/efeitos dos fármacos , Articulações/ultraestrutura , Linfa/metabolismo , Coelhos , Líquido Sinovial/efeitos dos fármacos , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/ultraestruturaRESUMO
OBJECTIVE: Hyaluronan (HA) has a major role in regulating synovial fluid volume. This role depends on the synovium functioning as an ultrafilter that reflects HA during trans-synovial fluid drainage. Reflection boosts the HA concentration on the membrane surface, leading to osmotic retention of synovial fluid ("buffering"). In arthritic effusions, however, HA concentration and osmotic buffering are greatly reduced. We tested the hypothesis that reflection is reduced (escape increased) when the HA concentration falls below the molecular entanglement concentration (C*). METHODS: HA at 0.2 mg/ml (
Assuntos
Ácido Hialurônico/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Animais , Difusão , CoelhosRESUMO
Hyaluronan (HA) is central to joint function, contributing to synovial fluid retention, lubrication, matrix organisation and joint embryogenesis. HA synthesis by intimal synoviocytes is stimulated by stretch (SSHA), linking HA production to joint usage; but the signal transduction paths are unknown. Low passage rabbit synoviocytes (RS), cultured from micro dissected synovial intima, were subjected to 10min of 10% static stretch followed by 170-min relaxation, or to sustained stretch for 180min in a Flexcell 2000 apparatus. Medium HA content was analysed by a HA-binding assay. The roles of protein kinase C (PKC) isoforms, extracellular signal-regulated kinases (ERK1/2) and Ca(2+) signalling in SSHA were tested using kinase inhibitors, Ca(2+) chelators and Ca(2+) channel activators combined with Western blots for activated kinases. Stretch increased HA secretion by 57%, independently of stretch duration. PKCalpha translocated from cytosol to membrane and triggered the phosphorylation of ERK1/2. The PKC inhibitor bisindolylmaleimide (BIM) blocked both SSHA and ERK phosphorylation, as did Gö 6976, a specific inhibitor of Ca(2+)-dependent PKC. The Ca(2+) channel activator Bay K stimulated HA secretion and ERK phosphorylation. Extra- and intra-cellular Ca(2+) chelation by EGTA and BAPTA-AM (respectively) inhibited SSHA. SSHA was also blocked by the partially selective protein kinase A inhibitor, H-89. Connective tissue growth factor, CTGF, was not involved in SSHA. Thus, stimulation of synoviocyte HA secretion by static stretch is due at least in part the o activation of a Ca(2+) influx-dependent activation of the PKCalpha-MEK-ERK1/2 cascade. This is functionally important because it links joint lubrication to joint use.
Assuntos
Sinalização do Cálcio , Ácido Hialurônico/biossíntese , Sistema de Sinalização das MAP Quinases , Mecanotransdução Celular/fisiologia , Proteína Quinase C-alfa/metabolismo , Membrana Sinovial/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo , Proteínas Imediatamente Precoces/metabolismo , Proteínas Imediatamente Precoces/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mecanotransdução Celular/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Modelos Biológicos , Proteína Quinase C-alfa/antagonistas & inibidores , Coelhos , Proteínas Recombinantes/farmacologia , Estresse Mecânico , Membrana Sinovial/citologia , Membrana Sinovial/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta/farmacologiaRESUMO
OBJECTIVE: To investigate the role of adenosine in chondrocyte death in murine osteoarthritis (OA). METHODS: 5'-Nucleotidase (5'NT) generates adenosine. Enzyme activity was measured histochemically in normal murine and osteoarthritic STR/ort strain tibial cartilage. Adenosine-mediated cell death was investigated in MC615 chondrocyte cultures. Adenosine receptors (ARs) were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Cellular uptake of [(3)H] adenosine was measured with or without the inhibitor, nitrobenzylthioinosine (NBTI). Cell death was assessed by cell counting and DNA laddering following selective receptor stimulation, or after modulating adenosine metabolism with adenosine deaminase (ADA) or adenosine kinase (AK) inhibitors [erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and Iodotubericidin (Itub)], or with homocysteine (HC). Markers of apoptosis were assessed by Western blotting. Cell studies were validated by incubating normal murine knee joints in a medium containing adenosine and metabolic inhibitors. Apoptotic chondrocytes were identified with the TUNEL reaction. RESULTS: 5'NT activity in STR/ort tibial cartilage increased with development of OA, especially close to OA lesions. Adenosine induced MC615 cell death in the presence of ADA inhibition (100 microM EHNA), or 1mM HC, or both. Adenosine uptake, mediated by NBTI-sensitive adenosine transporters, was required for cell death. ARs were expressed (A2b>A2a>A1) but were not involved in mediating cell death. Cell death involved the activation of caspase-3 and DNA fragmentation and was prevented by inhibiting caspase activity. However, neither caspase-8 nor caspase-9 was detected. Adenosine+EHNA induced chondrocyte apoptosis in normal murine knee joints. CONCLUSION: Increased adenosine production may induce chondrocyte apoptosis and play a role in OA in STR/ort mice.
Assuntos
Adenosina/metabolismo , Morte Celular/fisiologia , Condrócitos/fisiologia , Osteoartrite/fisiopatologia , 5'-Nucleotidase/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Inibidores de Adenosina Desaminase , Adenosina Quinase/antagonistas & inibidores , Animais , Apoptose/fisiologia , Cartilagem Articular/metabolismo , Cartilagem Articular/fisiopatologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Fragmentação do DNA/fisiologia , Inibidores Enzimáticos/farmacologia , Homocisteína/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos , Receptores Purinérgicos P1/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tubercidina/análogos & derivados , Tubercidina/farmacologiaRESUMO
AIMS/HYPOTHESIS: We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-beta1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells. METHODS: THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/-THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA. RESULTS: In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF. CONCLUSIONS/INTERPRETATION: Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target.
Assuntos
Nefropatias Diabéticas/metabolismo , Mesângio Glomerular/metabolismo , Hiperglicemia/metabolismo , Proteínas Imediatamente Precoces/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Trombospondina 1/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adulto , Idoso , Western Blotting , Estudos de Casos e Controles , Fator de Crescimento do Tecido Conjuntivo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Fibronectinas/genética , Fibronectinas/metabolismo , Regulação da Expressão Gênica , Mesângio Glomerular/patologia , Glucose/farmacologia , Glucose/fisiologia , Produtos Finais de Glicação Avançada , Humanos , Proteínas Imediatamente Precoces/genética , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análise , RNA Mensageiro/genética , Albumina Sérica/farmacologia , Trombospondina 1/genética , Fator de Crescimento Transformador beta/genética , Albumina Sérica GlicadaRESUMO
Hyaluronan (HA) is an essential component of synovial interstitial matrix and synovial fluid, but the link between its production and joint use is unclear. HA secretion is enhanced by joint distension in vivo, but direct proof that synoviocytes exhibit mechanosensitive HA secretion is lacking. We tested this in vitro. Primary rabbit synoviocyte (PRS) cultures from microdissected synovial intima were subjected to 180 min of maintained 10% static stretch, or to 10 min of 10% static stretch followed by 170 min relaxation, in a Flexcell 2000 apparatus. Stretch stimulated HA secretion into the medium over 3 h by 57%. Notably, a short stretch (10 min) was as effective as sustained stretch. Actinomycin D and cycloheximide abolished stretch-stimulated HA secretion and also reduced basal HA secretion rate. RT-PCR showed that HAS2 was the major hyaluronan synthase expressed, but there was no increase in HAS2 mRNA (or other isoforms) in continuously stretched cells, and only a small increase (20%) at 180 min in cells stretched for the first 10-30 min. However HAS2 transcription increased 10-fold in response to TGF-beta1 and IL-1beta. Thus HA secretion by intimal synoviocytes is regulated by a mechanosensitive pathway which depends on transcription and de novo protein synthesis, possibly of HAS2, but also of other proteins involved in HA secretion.
Assuntos
Ácido Hialurônico/metabolismo , Mecanotransdução Celular/fisiologia , Líquido Sinovial/citologia , Líquido Sinovial/metabolismo , Animais , Células Cultivadas , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Regulação Enzimológica da Expressão Gênica/genética , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Ácido Hialurônico/genética , Fenótipo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Coelhos , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genéticaRESUMO
In joint fluid the polymer hyaluronan (HA) confers viscous lubrication and greatly attenuates trans-synovial fluid loss (outflow buffering). Outflow buffering arises from the molecular sieving (reflection) and concentration polarization of HA at the synovial membrane surface. Outflow buffering declines if HA chain length is reduced, as in arthritis, and this has been attributed to reduced HA reflection. This was tested directly in the present study. Infused solutions of HA of approximately 2200 kDa (HA2000, 0.2 mg ml(-1)) or approximately 500 kDa (HA500, 0.2 mg ml(-1)) or approximately 140 kDa (HA140, 0.2-4.0 mg ml(-1)) were filtered across the synovial lining of the knee joint cavity of anaesthetized rabbits at a constant rate, along with a freely permeating reference solute, 20 kDa fluorescein-dextran (FD20). After a priming period the femoral lymph was sampled over 3 h. Mixed intra-articular (i.a.) fluid and subsynovial fluid were sampled at the end. Fluids were analysed by gel exclusion chromatography. The trans-synovial concentration profile was found to depend on polymer size. The i.a. concentration of HA2000 increased substantially relative to infusate and the subsynovial and lymph concentrations fell substantially. For HA500 and HA140 the trans-synovial concentration gradients were less pronounced, and absent for FD. The reflected fractions for HA2000, HA500 and HA140 across the cavity-to-lymph barrier were 0.65 +/- 0.05 (n = 10), 0.43 +/- 0.09 (n = 3) and 0.19 +/- 0.05 (n = 7), respectively, at matched filtration rates (P < 0.0001, analysis of variance). Reflected fractions calculated from HA i.a. accumulation or subsynovial dilution showed the same trend. The results demonstrate size-selective molecular sieving by the synovial extracellular matrix, equivalent to steric exclusion from cylindrical pores of radius 33-59 nm. The findings underpin the concentration polarization-outflow buffering theory and indicate that reduced HA chain length in arthritis exacerbates lubricant loss from a joint.
Assuntos
Matriz Extracelular/química , Ácido Hialurônico/química , Ácido Hialurônico/isolamento & purificação , Articulação do Joelho/química , Líquido Sinovial/química , Membrana Sinovial/química , Ultrafiltração/métodos , Animais , Difusão , Conformação Molecular , Peso Molecular , Tamanho da Partícula , Coelhos , ViscosidadeRESUMO
OBJECTIVES: The aims were to assess the contribution of arg-gly-asp (RGD) mediated cell integrin-matrix bonds to interstitial hydraulic resistance and to fenestrated endothelial permeability in joints. Joint fluid is generated by filtration from fenestrated capillaries and drains through a fibronectin-rich synovial intercellular matrix. The role of parenchymal cell-matrix bonding in determining tissue hydraulic resistance is unknown. METHODS: The knee cavity of anesthetized rabbits was infused with saline or the competitive hexapeptide blocker GRGDTP, with or without added osmotic stress (600 mosm saline). Intra-articular pressure Pj, net trans-synovial drainage rate s, and the permeation of Evans blue-labeled albumin (EVA) from plasma into the joint cavity were measured. RESULTS: GRGDTP increased the hydraulic conductance of the synovial drainage pathway, ds/dPj, by 71% (p =.02, paired t test, n = 6 animals). Synovial plasma EVA clearance (control 7.1 +/- 0.8 microL h-1, mean +/- SEM, n = 15) was unaffected by GRGDTP (7.0 +/- 2.3 microL h(-1), n = 6) or hyperosmolarity (4.9 +/- 1.5 microL h(-1), n = 8) but was increased by GRGDTP and hyperosmolarity together (15.9 +/- 4.8 microL h(-1), n = 5) (p =.01, ANOVA). Changes in dPj/dt evoked by GRGDTP plus hyperosmolarity, but neither alone, demonstrated increased microvascular filtration into the joint cavity (p <.001, ANOVA), as did changes in fluid absorption from the infusion system at fixed Pj. CONCLUSIONS: RGD-mediated bonds between the parenchymal cells and interstitial polymers reduce the interstitial hydraulic conductance by 42%. This helps to retain the lubricating fluid inside a joint cavity. RGD-mediated bonds also support the macromolecular barrier function of fenestrated endothelium, but in vivo this is evident only in stressed endothelium (cf. in vitro).
Assuntos
Endotélio/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Soluções Hipertônicas/farmacologia , Articulação do Joelho/efeitos dos fármacos , Oligopeptídeos/farmacologia , Albuminas/farmacocinética , Animais , Corantes/farmacocinética , Endotélio/fisiologia , Azul Evans/farmacocinética , Matriz Extracelular/fisiologia , Soluções Isotônicas/farmacologia , Oligopeptídeos/antagonistas & inibidores , Concentração Osmolar , Permeabilidade , Pressão , Coelhos , Solução de Ringer , Líquido Sinovial/fisiologiaRESUMO
Hyaluronan (HA), a component of synovial fluid, buffers fluid loss from joints. To explain this, a quantitative theory for HA concentration polarisation at a partially sieving synovial lining was developed. The theory predicts a fall in HA reflected fraction R with increased filtration rate. To test this, knees of anaesthetised rabbits were infused with HA and fluorescein-dextran (FD) at constant trans-synovial filtration rates of 6-89 microl min(-1). Samples of femoral lymph, mixed intra-articular fluid and subsynovial fluid after >/= 3 h were analysed by high-performance liquid chromatography. R was calculated as (1 - downstream/upstream concentration), using [FD] to adjust for joint lymph dilution in femoral lymph. Intra-articular HA concentration after >/= 3 h, 0.47 +/- 0.02 mg ml(-1) (mean +/-s.e.m., n= 31), exceeded the infusate concentration, 0.20 mg ml(-1), while subsynovial and lymph [HA] were reduced relative to [FD]. The changes in [HA] demonstrated synovial molecular sieving of HA. R from cavity to lymph (R(lymph)) fell monotonically from 0.93 at 6 microl min(-1) to 0.14 at 89 microl min(-1) (P < 0.0001, regression analysis, n= 33). R values calculated from the intra-articular HA accumulation (R(asp)) or the low subsynovial concentrations (R(syn)) were similar negative functions of filtration rate. R for lymphatic capillary endothelium (R(endo)), calculated from lymph/subsynovial concentration ratios, was effectively zero (-0.03 +/- 0.18, n= 21), confirming that synovium, not initial lymphatic endothelium, is the reflection site. Logarithmic linearisation of the results evaluated the synovial HA reflection coefficient as 0.91. In conclusion, the existence of concentration polarisation during joint fluid drainage was supported by the demonstration of a negative relation between filtration rate and R(lymph), R(asp) and R(syn).
Assuntos
Ácido Hialurônico/metabolismo , Articulações/metabolismo , Linfa/metabolismo , Algoritmos , Animais , Capilares/metabolismo , Galinhas , Cromatografia Líquida de Alta Pressão , Células Endoteliais/metabolismo , Filtração , Ácido Hialurônico/química , Técnicas In Vitro , Articulações/química , Cinética , Modelos Lineares , Linfa/química , Coelhos , Líquido Sinovial/químicaRESUMO
OBJECTIVE: To determine whether chondrocyte apoptosis occurs during the progression of osteoarthritis (OA) in the STR/ort mouse model of OA. METHODS: Serial cryostat sections were cut (10 microns) through the knee joint of young and old male STR/ort mice and graded for the severity of OA lesions. Age- and sex-matched CBA mice were used as controls. Apoptotic chondrocytes were detected using the TUNEL assay. Ultrastructural changes were examined using electron microscopy (EM). Expression of biochemical markers associated with apoptosis (bax, bcl-2 and caspases-3, -8 & -9) was investigated using immunohistochemistry. RESULTS: TUNEL assays on histological sections of STR/ort knee joints showed that the number of TUNEL-positive chondrocytes in the tibial medial articular cartilage correlated with the severity of the OA damage. These cells were located close to the lesional area. Only very occasional TUNEL positive chondrocytes were detected in either morphologically normal STR/ort cartilage or in control CBA cartilage. Ultrastructural analysis of chondrocytes neighboring focal osteoarthritic lesions in STR/ort tibial cartilage revealed an abundance of abnormal cells exhibiting numerous morphological changes. These resembled, but in some cases differed, from changes reported in classical apoptosis. The changes include abnormal distribution of chromatin, cell shrinkage, membrane blebbing and deposition of cell remnants (apoptotic bodies) in the lacuna space. Despite the TUNEL and EM changes, immunohistochemistry failed to detect any changes in the ratio of bax to bcl-2 in tibial chondrocytes of STR/ort mice. Both bcl-2 and bax levels decreased with age in morphologically normal STR/ort and control CBA cartilage. None of the caspases tested for was detected in tibial chondrocytes of either strain. CONCLUSION: Chondrocyte cell death is correlated with the progression of OA in STR/ort mice and has many of the morphological characteristics of classical apoptosis. Absence of changes in bax to bcl-2 ratio in STR/ort chondrocytes indicate that the mitochondrial pathway of apoptosis is unlikely to be involved. Failure to detect caspases could be due to low levels of enzyme expression, expression within a very brief time period, or to a caspase-independent mechanism of cell death.
Assuntos
Apoptose/fisiologia , Cartilagem Articular/fisiopatologia , Condrócitos/fisiologia , Osteoartrite/fisiopatologia , Animais , Biomarcadores/análise , Cartilagem Articular/patologia , Caspase 8 , Caspase 9 , Caspases/análise , Modelos Animais de Doenças , Membro Posterior , Imuno-Histoquímica/métodos , Marcação In Situ das Extremidades Cortadas/métodos , Masculino , Camundongos , Camundongos Endogâmicos , Microscopia Eletrônica/métodos , Osteoartrite/patologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Tíbia , Proteína X Associada a bcl-2RESUMO
OBJECTIVE: To investigate the expression of a novel member of the mannose receptor family, Endo180 (also known as uPARAP), and the distribution of Endo180 ligand(s) in the articular cartilage and growth plate of normal CBA mice and STR/ort mice, a well characterized model of spontaneous osteoarthritis. DESIGN: A polyclonal anti-Endo180 antibody was used to determine receptor expression. The Endo180 extracellular domain fused to a human immunoglobulin Fc tail was used to detect ligand. RESULTS: Endo180 receptor was strongly expressed in chondrocytes both in vitro and throughout the articular cartilage of young CBA and STR/ort mice. Expression decreased in older animals. In STR/ort mice with osteoarthritic lesions, no upregulation of Endo180 was detected. In the developing growth plate, Endo180 was expressed strongly by the proliferating chondrocytes. In contrast, Endo180 ligand was detected most strongly in hypertrophic zone of the growth plate and only at low levels in articular cartilage. In cultured chondrocytes, Endo180 was localized on the cell surface and in intracellular vesicles. CONCLUSION: Constitutively recycling endocytic receptors function to internalize ligand from the extracellular milieu and the ability of Endo180 to bind both glycosylated ligands and collagens suggests a role in extracellular matrix remodeling. Expression of Endo180 in articular cartilage chondrocytes of young, but not old, mice and the reciprocal expression of Endo180 and its ligands in the growth plate suggest that this receptor is involved in cartilage development but not in cartilage homeostasis. In addition, our data indicates that Endo180 does not appear to play a role in the development or progression of murine osteoarthritis.
Assuntos
Cartilagem Articular/metabolismo , Osteoartrite/metabolismo , Receptores Mitogênicos/análise , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Cartilagem Articular/crescimento & desenvolvimento , Linhagem Celular , Condrócitos/metabolismo , Extremidades , Lâmina de Crescimento/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Microscopia de Fluorescência/métodosRESUMO
Synovial fluid hyaluronan (HA) profoundly buffers fluid loss from joints. This is attributed to the osmotic pressure of HA reflected by the joint lining. The aims were to quantify HA sieving during fluid drainage from joint to lymphatics and to compare the contributions of the synovial lining and lymphatic endothelium to sieving. HA (2100 kDa) and fluorescein-dextran (FD, 20 kDa) were infused under constant pressure into the knee cavity in anaesthetised rabbits. Samples were taken of femoral lymph and, after approximately 3-h transsynovial filtration, subsynovial fluid and mixed intra-articular fluid. [HA] and [FD] were analysed by HPLC and reflection was calculated as one minus transmitted fraction. Subsynovial and lymph [HA] were 16 and 12% of intra-articular [HA], which increased to 2.6 times infusate [HA] (P < 0.001, ANOVA, n = 19). [FD] was not significantly changed in infusate, aspirate, and subsynovial fluid but fell to 62% in femoral lymph due to dilution by skin/muscle lymph. The HA reflected fraction for the cavity-to-lymph barrier, R(lymph), was 0.54 +/- 0.03 (n = 82, mean +/- SEM), compared with 0.51 +/- 0.07 for cavity-to-subsynovium (R(syn), P > 0.05, Bonferroni) and 0.07 +/- 0.18 for subynovium-to-lymph (R(endo), P < 0.0001, Bonferroni). Lymphatic capillary endothelial reflection R(endo) was not significantly different from zero (one-sample t test). It is concluded that HA is partially sieved out of fluid leaving the joint cavity, and the sieve is the synovial lining interstitial matrix, not lymphatic capillary endothelium.
Assuntos
Endotélio Linfático/patologia , Ácido Hialurônico/farmacologia , Linfa/metabolismo , Vasos Linfáticos/patologia , Membrana Sinovial/patologia , Animais , Cromatografia Líquida de Alta Pressão , Dextranos/farmacologia , Endocitose , Fêmur/patologia , Fluoresceínas/farmacologia , Articulação do Joelho/patologia , Modelos Teóricos , Pressão Osmótica , Coelhos , Membrana Sinovial/efeitos dos fármacosRESUMO
Hyaluronan (HA) is important for joint cavitation, lubrication, volume regulation and synovial fluid drainage but little is known about the regulation of joint HA synthesis/secretion in vivo. We investigated whether HA secretion into joints in vivo can be regulated by protein kinase C (PKC). Secretion into the knee joint cavity of anaesthetised rabbits was measured over 6 h by washout and chromatography. Joints received intra-articular injections of Ringer vehicle (control) or an activator of classical PKC isoforms, phorbol-12-myristate-13-acetate (PMA), at 20-2000 ng ml(-1). The effects of PKC inhibition by bisindolylmaleimide (BIM) and protein synthesis inhibition by cycloheximide (CX) on basal and stimulated HA secretion were also studied. The endogenous HA mass, 181+/-8 microg (n=26, mean +/- S.E.M.), and basal secretion rate, 4.4+/-0.4 microg h(-1), indicated a turnover time of 41 h. Secretion rate showed a dose-dependent response to PMA (n=30), rising 5-fold to 21.7+/-5.0 microg h(-1) (n=5) at 2000 ng ml(-1) PMA (P<0.0001, one-way ANOVA). PMA-induced stimulation was partially suppressed by CX (HA secretion: 5.8+/-1.7 microg h(-1), n=8, P<0.01) and totally blocked by BIM (HA secretion: 3.2+/-0.6 microg h(-1), n=9, P<0.001). Basal HA secretion was unaffected by CX over 6 h (4.2+/-0.7 microg h(-1), n=8) but was reduced by 29 % by BIM (3.1+/-0.6 microg h(-1), n=10, P=0.03). It is concluded that: (1) PKC can stimulate HA secretion into joints in vivo through mechanisms involving protein synthesis de novo as well as phosphorylation; (2) basal HA secretion is only partially PKC dependent; and (3) hyaluronan synthase turnover time is >6 h in vivo, which is slower than in vitro (<2-3 h).
Assuntos
Ácido Hialurônico/metabolismo , Proteína Quinase C/fisiologia , Líquido Sinovial/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Articulações/patologia , Cinética , Maleimidas/farmacologia , Proteína Quinase C/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Coelhos , Sinovite/patologia , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
OBJECTIVE: To raise peptide antibodies recognizing the C-terminal amino acid sequence in the G1 domain of porcine aggrecan, generated by the action of either aggrecanase or neutral metalloproteinase(s), in rabbits and to use them to investigate the release of aggrecan from porcine articular cartilage. METHOD: An explant culture system was used to investigate the release of the G1 domain of aggrecan from porcine articular cartilage treated with retinoic acid or interleukin 1beta and to study how the activity of these agents is modified by the proteinase inhibitor, batimastat (BB94). RESULTS: Retinoic acid and interleukin 1beta induced both enzyme activities and the release of the G1 domain into the culture medium. Proteinase activity was significantly reduced when the tissue was incubated in the presence of BB94. The functional properties of the enzyme-generated G1 domain were studied using large-pore, agarose/polyacrylamide gel electrophoresis, and it was shown to interact with hyaluronan and link protein. CONCLUSIONS: The results show that there must be a mechanism for removing a functional G1 domain from aggrecan during tissue turnover using this culture system.
Assuntos
Cartilagem Articular/metabolismo , Proteínas da Matriz Extracelular , Ácido Hialurônico/metabolismo , Fenilalanina/análogos & derivados , Proteoglicanas/metabolismo , Agrecanas , Animais , Especificidade de Anticorpos , Cartilagem Articular/efeitos dos fármacos , Meios de Cultivo Condicionados , Técnicas de Cultura , Endopeptidases/farmacologia , Glicosaminoglicanos/metabolismo , Soros Imunes/imunologia , Interleucina-1/farmacologia , Lectinas Tipo C , Metaloendopeptidases/farmacologia , Fragmentos de Peptídeos/imunologia , Fenilalanina/farmacologia , Inibidores de Proteases/farmacologia , Proteoglicanas/imunologia , Coelhos , Suínos , Tiofenos/farmacologia , Tretinoína/farmacologiaRESUMO
AIMS: Off-line post-processing of colour tissue Doppler from digital loops may allow objective quantification of dobutamine stress echocardiography. We assessed the reproducibility of off-line measurements of regional myocardial velocities. METHODS AND RESULTS: Nine observers analysed 10 studies, each making 2400 observations. Coefficients of variation in basal segments from apical windows, at rest and maximal stress, were 9-14% and 11-18% for peak systolic velocity, 16-18% and 17-19% for time-to-peak systolic velocity, 9-17% and 18-24% for systolic velocity time integral, and 18-23% and 21-27% for systolic acceleration. Coefficients of variation for diastolic velocities in basal segments at rest were 11-40%. Coefficients of variation for peak systolic velocity were 10-24% at rest and 14-28% at peak in mid segments, and 19-53% and 29-69% in apical segments. From parasternal windows coefficients of variation for peak systolic velocity were 14-16% in basal posterior, and 19-29% in mid-anterior segments. High variability makes measurement unreliable in apical and basal anterior septal segments. The feasibility of obtaining traces was tested in 92 subjects, and >90% in all basal and mid segments apart from the anterior septum. CONCLUSION: Quantification of myocardial functional reserve by off-line analysis of colour tissue Doppler acquired during dobutamine stress is feasible and reproducible in 11 segments of the left ventricle. The most reliable measurements are systolic velocities of longitudinal motion in basal segments.
Assuntos
Ecocardiografia Doppler em Cores , Ecocardiografia sob Estresse , Processamento de Imagem Assistida por Computador , Isquemia Miocárdica/diagnóstico por imagem , Velocidade do Fluxo Sanguíneo , Estudos de Viabilidade , Humanos , Isquemia Miocárdica/fisiopatologia , Variações Dependentes do Observador , Reprodutibilidade dos TestesRESUMO
OBJECTIVES: Because fibroblast filamentous actin (F-actin) influences cutaneous interstitial matrix swelling pressure (5), we investigated whether F-actin in fibroblast-derived synoviocytes influences the hydraulic permeability of the trans-synovial interstitial pathway. The study also tested whether F-actin in fenestrated synovial endothelium contributes to the blood-joint barrier in vivo. METHODS: The clearance of Evans blue-albumin (EVA) from plasma into the knee joint cavity was determined in joint infused with F-actin disrupting cytochalasin D (1-200 microM), latrunculin B (100 microM) or vehicle in anesthetized rabbits. The hydraulic permeability of the lining was determined as the slope relating net trans-synovial flow Q(s) to intra-articular pressure P(j). Synovium was examined histologically after i.v. Monastral blue to assess endothelial leakiness. RESULTS: EVA permeation in vivo was increased up to 25-fold by cytochalasin (p = 0.0002, ANOVA), with an EC(50) of 23 microM (95% confidence limits 13-43 microM). Washout quickly reversed the increase. Latrunculin had a similar effect. F-actin disruption switched Q(s) from drainage (control) to filtration into the cavity at low P(j) in vivo and raised the conductance Q(s)/dP(j) by 2.13 (p = 0.001, ANOVA). Circulatory arrest abolished these effects. Monastral blue revealed numerous endothelial leaks. CONCLUSIONS: F-actin is crucial to the barrier function of fenestrated endothelium in situ. No significant effect of synoviocyte F-actin on matrix permeability was detected.
Assuntos
Actinas/fisiologia , Permeabilidade Capilar/fisiologia , Articulação do Joelho/irrigação sanguínea , Membrana Sinovial/fisiologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Citocalasina D/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/ultraestrutura , Azul Evans/farmacocinética , Espaço Extracelular , Articulação do Joelho/ultraestrutura , Microcirculação , Coelhos , Líquido Sinovial , Membrana Sinovial/citologia , Tiazóis/farmacologia , TiazolidinasRESUMO
OBJECTIVE: To study the temporal expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the STR/ort mouse model of osteoarthritis, using in situ hybridization with oligonucleotide probes and specific antisera for each protein. METHODS: In situ hybridization and immunolocalization experiments were performed on serial cryosections of knee joints from STR/ort and control CBA mice. The mRNA was localized using digoxygenin-labeled probes. RESULTS: MMP2, MMP3, MMP7, MMP9, MMP13, MT1-MMP and TIMP2 mRNA was detected in the tibial articular chondrocytes of STR/ort mice at all ages (12, 18, 24, 30 and 35 weeks). Levels were always higher than in age-matched CBA mice. Neither MMP8 nor TIMP1 mRNA was detected in murine cartilage. The location and distribution of each of the MMP mRNA transcripts varied within the tibial plateau. Immunolocalization consistently detected MMP3 and MT1-MMP in articular cartilage and MMP13 in calcified cartilage. Other proteases and their inhibitors were not detected in either of these cartilages but MMP2 and MMP9 were immunolocalized in bone marrow cells and growth cartilage respectively. CONCLUSION: Expression of all the detected MMPs and TIMP-2 is up-regulated in STR/ort mice at the mRNA level. However, failure to detect protein expression for MMPs 2, 7, 9, 13 and TIMPs 1 and 2 in murine chondrocytes by immunohistochemistry indicates that the changes in mRNA levels in STR/ort mice must be interpreted with caution.
Assuntos
Metaloproteinases da Matriz/genética , Osteoartrite/genética , Inibidores Teciduais de Metaloproteinases/genética , Animais , Cartilagem Articular , Condrócitos , Colagenases/genética , Imunofluorescência , Expressão Gênica , Hibridização In Situ , Metaloproteinase 13 da Matriz , Metaloproteinase 14 da Matriz , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 7 da Matriz/genética , Metaloproteinase 8 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/genética , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
The coupling of periarticular lymph flow to transsynovial flow from a joint cavity, i.e., fluid load, is essential to avoid periarticular edema, which is associated with arthritic morning stiffness. To study coupling in swollen joints, a new method, "inside-out" cannulation, which eliminates dead space, resistance and cutout, was used to collect lymph from fine femoral lymph trunks in anesthetised rabbits while the knee joint cavity was infused with Evans blue albumin (EVA) at controlled intraarticular pressure and transsynovial drainage rates. The amount of joint lymph in femoral lymph (volume fraction V(v)) was calculated by EVA analysis. Joint lymph flow and EVA clearance was 1.5 +/- 0.4 microl min(-1) (mean +/- SEM, n = 62) at mean trans-synovial flow, 23 microl min(-1), and increased with pressure. Volume fraction increased from 16% at 10 cmH(2)O to 43% at 41 cmH(2)O. The increase in lymph flow with pressure, 0.052 +/- 0.025 microl min(-1) cmH(2)O(-1) (n = 61) was much smaller than the increase in transsynovial flow (periarticular fluid load) with pressure, 0.71 +/- 0.14 microl min(-1) cmH(2)O(-1) (P < 0.001). Their ratio, the coupling coefficient, was only 0.06-0.11. Thus, although up to 43% of femoral lymph could stem from a single swollen joint, this drained away only a small fraction of the transsynovial filtrate. The study showed that joint lymphatic drainage is coupled to joint pressure and transsynovial flow; but the coupling is insufficient to prevent periarticular fluid accumulation under conditions of joint volume expansion and limb immobility. This may contribute to the periarticular edema of arthritis.
Assuntos
Articulação do Joelho/metabolismo , Albuminas/metabolismo , Animais , Artrite/patologia , Modelos Animais de Doenças , Linfa/metabolismo , Sistema Linfático/cirurgia , Microcirculação/fisiologia , Modelos Anatômicos , Pressão , Coelhos , Corantes de Rosanilina/farmacologia , Sinovectomia , Líquido Sinovial/fisiologia , Membrana Sinovial/irrigação sanguínea , Fatores de TempoRESUMO
The polysaccharide hyaluronan (HA) conserves synovial fluid by keeping outflow low and almost constant over a wide pressure range ('buffering'), but only at concentrations associated with polymer domain overlap. We therefore tested whether polymer interactions can cause buffering, using HA-chondroitin sulphate C (CSC) mixtures. Also, since it has been found that capillary filtration is insensitive to the Starling force interstitial osmotic pressure in frog mesenteries, this was assessed in synovium. Hyaluronan at non-buffering concentrations (0.50-0.75 mg ml(-1)) and/or 25 mg ml(-1) CSC (osmotic pressure 68 cmH(2)O) was infused into knees of anaesthetised rabbits in vivo. Viscometry and chromatography confirmed that HA interacts with CSC. Pressure (P(j)) versus trans-synovial flow (;Q(s)) relations were measured.;Q(s) was outwards for HA alone (1.2 +/- 0.9 microl min(-1) at 3 cmH(2)O, mean +/- S.E.M.; n = 6). CSC diffused into synovium and changed;Q(s) to filtration at low P(j) (-4.1 microl min(-1), 3 cmH(2)O, n = 5, P < 0.02, t test). Filtration ceased upon circulatory arrest (n = 3). At higher P(j), 0.75 mg ml(-1) HA plus CSC buffered;Q(s) to approximately 3 microl min(-1) over a wide range of P(j), with an outflow increase of only 0.04 +/- 0.02 microl min(-1) cmH(2)O(-1) (n = 4). With HA or CSC alone, buffering was absent (slopes 0.57 +/- 0.04 microl min(-1) cmH(2)O(-1) (n = 4) and 0.86 +/- 0.05 microl min(-1) cmH(2)O(-1) (n = 5), respectively). Therefore, polymer interactions can cause outflow buffering in joints. Also, interstitial osmotic pressure promoted filtration in fenestrated synovial capillaries, so the results for frog mesentery capillaries cannot be generalised. The difference is attributed to differences in pore ultrastructure.