RESUMO
OBJECTIVES: Arthritis is a common clinical manifestation of hereditary hemochromatosis (HH), and HH is one of a handful of conditions linked to calcium pyrophosphate deposition (CPPD) in joints. The connection between these two types of arthritis has not yet been fully elucidated. In light of new pathogenic pathways recently implicated in CPPD involving bone, we reviewed the literature on the etiology of hemochromatosis arthropathy (HHA) seeking shared pathogenic mechanisms. RESULTS: Clinical observations reinforce striking similarities between HHA and CPPD even in the absence of CPP crystals. They share a similar joint distribution, low grade synovial inflammation, and generalized bone loss. Excess iron damages chondrocytes and bone cells in vitro. While direct effects of iron on cartilage are not consistently seen in animal models of HH, there is decreased osteoblast alkaline phosphatase activity, and increased osteoclastogenesis. These abnormalities are also seen in CPPD. Joint repair processes may also be impaired in both CPPD and HHA. CONCLUSIONS: Possible shared pathogenic pathways relate more to bone and abnormal damage/repair mechanisms than direct damage to articular cartilage. While additional work is necessary to fully understand the pathogenesis of arthritis in HH and to firmly establish causal links with CPPD, this review provides some plausible hypotheses explaining the overlap of these two forms of arthritis.
Assuntos
Calcinose , Cartilagem Articular , Condrocalcinose , Hemocromatose , Artropatias , Animais , Pirofosfato de Cálcio , Cartilagem Articular/patologia , Condrocalcinose/patologia , Hemocromatose/complicações , Hemocromatose/genética , Hemocromatose/metabolismo , Humanos , Ferro/metabolismo , Artropatias/complicaçõesRESUMO
OBJECTIVE: The gene TNFRSF11B encodes for osteoprotegerin (OPG) and was recently identified as the CCAL1 locus associated with familial calcium pyrophosphate deposition disease (CPDD). While the CCAL1 OPG mutation (OPG-XL) was originally believed to be a gain-of-function mutation, loss of OPG activity causes arthritis-associated osteolysis in mice, which is likely related to excess subchondral osteoclast formation and/or activity. The purpose of the present study was to further explore the effect of OPG-XL in osteoclastogenesis. METHODS: The effects of recombinant OPG-XL and wild-type (WT) OPG were determined in monoculture and coculture models of RANKL-induced osteoclastogenesis. The effects of OPG-XL on osteoclast survival as well as on TRAIL-induced apoptosis were determined using standard in vitro assays and compared to WT OPG. The ability of OPG-XL and WT OPG to bind to osteoblasts was measured with enzyme-linked immunosorbent assay and flow cytometry using the osteoblastic MC3T3-E1 cell line. RESULTS: OPG-XL was less effective than WT OPG at blocking RANKL-induced osteoclastogenesis in monoculture and coculture models. Osteoclast survival and inhibition of TRAIL-induced apoptosis were similar in the presence of OPG-XL and WT OPG. Compared to WT OPG, considerably less OPG-XL bound to cells. CONCLUSION: These findings indicate that OPG-XL is a loss-of-function mutation as it relates to RANKL-mediated osteoclastogenesis, and thus may permit increased osteoclast numbers and heightened bone turnover. Further studies are necessary to demonstrate how this mutation contributes to arthritis in individuals carrying this mutation.
Assuntos
Condrocalcinose/genética , Mutação com Perda de Função/genética , Osteogênese/genética , Osteoprotegerina/genética , Animais , Remodelação Óssea/genética , Linhagem Celular , Técnicas de Cocultura , Modelos Animais de Doenças , CamundongosRESUMO
PURPOSE: Although there are general methodologies for lean implementation in manufacturing companies, a specific methodology for the implementation of lean healthcare in hospitals has not been addressed by the literature. Addressing this gap, the purpose of this paper is to develop a practice-driven methodology for implementing lean in hospital operations. DESIGN/METHODOLOGY/APPROACH: Three case studies were conducted to collect evidence on the lean implementation process in Brazilian hospitals. From empirical evidence and literature, the implementation methodology was proposed and submitted to critical assessment by experts from the field. FINDINGS: The process of lean implementation was very similar in all cases, triggered by strategic planning and operationalized by continuous improvement projects. On the other hand, in all cases, the lean implementation teams had to deal with employees' resistance. These findings were valuable inputs to the development of the implementation methodology. After refinement, it was proposed a feasible, useful and user-friendly methodology. RESEARCH LIMITATIONS/IMPLICATIONS: The proposed methodology was raised from the practice through case study research. However, the proposed methodology was not fully applied, and the associated performance measures were not elaborated in this paper. Therefore, more case studies and applications will be necessary to generalize the findings. PRACTICAL IMPLICATIONS: The methodology provides practical guidelines that support lean implementation in hospital operations. Although it demands adaptations for each specific hospital setting, this initial step may encourage hospital managers to start the lean journey. ORIGINALITY/VALUE: This study addressed the gap in the literature regarding the lack of methodologies for implementing lean healthcare in hospital operations. The methodology synthesizes the knowledge, principles and tools of lean thinking that can be applied in hospital operations.
Assuntos
Eficiência Organizacional/normas , Hospitais/normas , Melhoria de Qualidade , Gestão da Qualidade Total/métodos , Brasil , Estudos de Casos OrganizacionaisRESUMO
The protein product of the progressive ankylosis gene, known as ANK, is a 492-amino acid multi-pass transmembrane protein. This protein is critical for the regulation of pyrophosphate, and gain of function ANK mutations is associated with calcium pyrophosphate deposition disease. Much about the structure, function, and regulation of ANK remain unstudied. This review of the current literature examines recent contributions to our understanding of ANK. We focus on new work on the function, binding partners, and regulators of ANK. A more complete understanding of this important protein may help to identify future therapeutic targets for the treatment of calcium pyrophosphate deposition disease.
Assuntos
Condrocalcinose/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Condrocalcinose/genética , Humanos , Mutação , Proteínas de Transporte de Fosfato/genética , Conformação ProteicaRESUMO
Chondrocyte-derived extracellular organelles known as articular cartilage vesicles (ACVs) participate in non-classical protein secretion, intercellular communication, and pathologic calcification. Factors affecting ACV formation and release remain poorly characterized; although in some cell types, the generation of extracellular vesicles is associated with up-regulation of autophagy. We sought to determine the role of autophagy in ACV production by primary articular chondrocytes. Using an innovative dynamic model with a light scatter nanoparticle counting apparatus, we determined the effects of autophagy modulators on ACV number and content in conditioned medium from normal adult porcine and human osteoarthritic chondrocytes. Healthy articular chondrocytes release ACVs into conditioned medium and show significant levels of ongoing autophagy. Rapamycin, which promotes autophagy, increased ACV numbers in a dose- and time-dependent manner associated with increased levels of autophagy markers and autophagosome formation. These effects were suppressed by pharmacologic autophagy inhibitors and short interfering RNA for ATG5. Caspase-3 inhibition and a Rho/ROCK inhibitor prevented rapamycin-induced increases in ACV number. Osteoarthritic chondrocytes, which are deficient in autophagy, did not increase ACV number in response to rapamycin. SMER28, which induces autophagy via an mTOR-independent mechanism, also increased ACV number. ACVs induced under all conditions had similar ecto-enzyme specific activities and types of RNA, and all ACVs contained LC3, an autophagosome-resident protein. These findings identify autophagy as a critical participant in ACV formation, and augment our understanding of ACVs in cartilage disease and repair.
Assuntos
Autofagia , Cartilagem Articular/citologia , Condrócitos/citologia , Organelas/metabolismo , Osteoartrite/patologia , Fagossomos/fisiologia , Adulto , Animais , Apoptose , Transporte Biológico , Western Blotting , Cartilagem Articular/metabolismo , Caspase 3/metabolismo , Proliferação de Células , Células Cultivadas , Condrócitos/metabolismo , Citometria de Fluxo , Humanos , Imunossupressores/farmacologia , Pessoa de Meia-Idade , Osteoartrite/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sirolimo/farmacologia , Suínos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
INTRODUCTION: Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process. METHODS: We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release. RESULTS: eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels. CONCLUSIONS: These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.
Assuntos
Trifosfato de Adenosina/metabolismo , Condrócitos/metabolismo , Espaço Extracelular/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Animais , Anquilose/genética , Anquilose/metabolismo , Anquilose/patologia , Transporte Biológico/efeitos dos fármacos , Cálcio/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Leucina/análogos & derivados , Leucina/farmacologia , Proteínas de Transporte de Fosfato/antagonistas & inibidores , Proteínas de Transporte de Fosfato/genética , Probenecid/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X4/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Sulfonamidas/farmacologia , Suínos , Canais de Cátion TRPV/agonistasRESUMO
RATIONALE: Diabetic tendinopathy is characterized by increased stiffness, thickness, and excess calcification of affected tendons. We investigated the hypothesis that proteoglycans (PGs), as key regulators of tendon structure and calcification, are altered in diabetic tendons. METHODS: Adult porcine patellar tendons were incubated in iso-osmolar media with high or normal glucose levels for 2 weeks. The PG fraction was isolated and analyzed. Protein and mRNA levels of five PGs were measured. PG production was assessed in primary tenocyte monolayers by (35)S-sulfate labeling in high and normal glucose conditions with and without exposure to advanced glycation end-products (AGEs). Levels of transforming growth factor ß, which commonly mediates some effects of hyperglycemia, were also measured and the effects of free radical scavengers on (35)S-sulfate incorporation were determined. RESULTS: PG levels were significantly decreased in tendons exposed to high glucose media compared with tendons in iso-osmolar control media. Relative quantities of individual PGs were unchanged by exposure to hyperglycemia and mRNAs for PGs were variably affected. High glucose media decreased PG production by tenocytes as measured by (35)S-sulfate labeling, whereas AGE-modified type I collagen and free radical scavengers had no effects. Hyperglycemic conditions increased levels of transforming growth factor ß1 in an AGE-independent manner. CONCLUSIONS: Hyperglycemia produces a reduction in PG levels related to decreased synthesis or sulfation of glycosaminoglycans, which may contribute to the tendon pathology observed clinically in diabetes.
Assuntos
Complicações do Diabetes/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Hiperglicemia/metabolismo , Peptidoglicano/metabolismo , Tendinopatia/metabolismo , Tendões/metabolismo , Animais , Colágeno Tipo I/metabolismo , Complicações do Diabetes/patologia , Hiperglicemia/patologia , Técnicas de Cultura de Órgãos , Suínos , Tendinopatia/patologia , Tendões/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: Articular cartilage vesicles (ACVs) are extracellular organelles found in normal articular cartilage. While they were initially defined by their ability to generate pathologic calcium crystals in cartilage of osteoarthritis (OA) patients, they can also alter the phenotype of normal chondrocytes through the transfer of RNA and protein. The purpose of this study was to analyze the proteome of ACVs from normal and OA human cartilage. METHODS: ACVs were isolated from cartilage samples from 10 normal controls and 10 OA patients. We identified the ACV proteomes using in-gel trypsin digestion, nanospray liquid chromatography tandem mass spectrometry analysis of tryptic peptides, followed by searching an appropriate subset of the Uniprot database. We further differentiated between normal and OA ACVs by Holm-Sidak analysis for multiple comparison testing. RESULTS: More than 1,700 proteins were identified in ACVs. Approximately 170 proteins satisfied our stringent criteria of having >1 representative peptide per protein present, and a false discovery rate of ≤5%. These proteins included extracellular matrix components, phospholipid binding proteins, enzymes, and cytoskeletal components, including actin. While few proteins were seen exclusively in normal or OA ACVs, immunoglobulins and complement components were present only in OA ACVs. Compared to normal ACVs, OA ACVs displayed decreases in matrix proteoglycans and increases in transforming growth factor ß-induced protein ßig-H3, DEL-1, vitronectin, and serine protease HtrA1 (P < 0.01). CONCLUSION: These findings lend support to the concept of ACVs as physiologic structures in articular cartilage. Changes in OA ACVs are largely quantitative and reflect an altered matrix and the presence of inflammation, rather than revealing fundamental changes in composition.
Assuntos
Cartilagem Articular/metabolismo , Osteoartrite do Joelho/metabolismo , Vesículas Transportadoras/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Cromatografia Líquida de Alta Pressão , Humanos , Microquímica , Nanotecnologia , Osteoartrite do Joelho/patologia , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Vesículas Transportadoras/química , Vesículas Transportadoras/patologiaRESUMO
Small membrane-bound extracellular organelles known as articular cartilage matrix vesicles (ACVs) participate in pathologic mineralization in osteoarthritic articular cartilage. ACVs are also present in normal cartilage, although they have no known functions other than mineralization. Recently, RNA was identified in extracellular vesicles derived from mast cells, suggesting that such vesicles might carry coding information from cell to cell. We found that ACVs from normal porcine and human articular cartilage and primary chondrocyte conditioned media contained 1 microg RNA/80 microg ACV protein. No DNA could be detected. RT-PCR of ACV RNA demonstrated the presence of full length mRNAs for factor XIIIA, type II transglutaminase, collagen II, aggrecan, ANKH and GAPDH. RNA in intact ACVs was resistant to RNase, despite the fact that ACV preparations contained measurable levels of active RNases. Significantly, radiolabeled RNA in ACVs could be transferred to unlabeled chondrocytes by co-incubation and produced changes in levels of chondrocyte enzymes and proteins. The demonstration that ACVs contain mRNAs suggests that they may function to shuttle genetic information between articular cells and indicate novel functions for these structures in articular cartilage.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Transporte de RNA , RNA Mensageiro/metabolismo , Agrecanas/genética , Animais , Fator XIIIa/genética , Proteínas de Ligação ao GTP/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Humanos , Proteínas de Transporte de Fosfato/genética , Proteína 2 Glutamina gama-Glutamiltransferase , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Ribonucleases/química , Ribonucleases/metabolismo , Suínos , Transglutaminases/genética , Vesículas Transportadoras/metabolismoRESUMO
OBJECTIVE: Pathologic mineralization is common in osteoarthritic (OA) cartilage and may be mediated by extracellular organelles known as articular cartilage vesicles (ACVs). Paradoxically, ACVs isolated from OA human cartilage mineralize poorly in vitro compared with those isolated from normal porcine cartilage. We recently showed that collagens regulate ACV mineralization. We sought to determine differences between collagens and collagen receptors on human and porcine ACVs as a potential explanation of their different mineralization behaviors. METHODS: ACVs were enzymatically released from old and young human and porcine hyaline articular cartilage. Western blotting was used to determine the presence of types I, II, VI, and X collagen and various collagen receptors on ACVs. Type II collagen was quantified by enzyme-linked immunosorbent assay. Biomineralization was assessed by measuring the uptake of (45)Ca by isolated ACVs in agarose gels and by ACVs in situ in freeze-thawed cartilage. RESULTS: As previously shown, isolated human ACVs mineralized poorly in response to ATP compared with porcine ACVs, but human and porcine ACVs mineralized similarly in situ in freeze-thawed cartilage. Type II collagen levels were 100-fold higher in isolated human ACVs than in porcine ACVs. Type II collagen in human ACVs was of high molecular weight. Transglutaminase-crosslinked type II collagen showed increased resistance to collagenase, suggesting a possible explanation for residual collagen on human ACVs. Expression of other collagens and collagen receptors was similar on human and porcine ACVs. CONCLUSION: Higher levels of type II collagen in human ACV preparations, perhaps mediated by increased transglutaminase crosslinking, may contribute to the decreased mineralization observed in isolated human ACVs in vitro.
Assuntos
Fosfatos de Cálcio/metabolismo , Pirofosfato de Cálcio/metabolismo , Cartilagem Articular/metabolismo , Colágeno Tipo II/metabolismo , Organelas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Cartilagem Articular/patologia , Humanos , Organelas/patologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Receptores de Colágeno/metabolismo , Suínos , Transglutaminases/metabolismo , Adulto JovemRESUMO
OBJECTIVES: Tendon abnormalities, such as increased stiffness, thickness, and excess calcification, occur commonly in patients with diabetes mellitus and cause considerable disability. These changes are frequently attributed to increased cross-linking of extracellular matrix components by advanced glycation end-products (AGEs). However, cellular effects of AGEs, such as increased activity of the cross-linking transglutaminase (Tgase) enzymes, could also contribute to altered tissue biomechanics and calcification in diabetic tendons. We determined the effect of AGE-modified protein on tenocyte Tgase activity. RESEARCH DESIGN AND METHODS: Primary porcine tenocytes were exposed to N- carboxymethyl-lysine (CML)-modified type I collagen in high or normal glucose media. Protein and mRNA levels of the Tgase enzymes and Tgase activity levels were measured, as were markers of apoptosis. We also determined the effect of antioxidants on CML-collagen mediated Tgase activity. RESULTS: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes 2.3- to 5.6-fold over unmodified collagen controls in both normal and high glucose media, without altering enzyme protein levels. Anti-oxidant treatment reduced the effect of CML-collagen on Tgase activity. Deoxyribonucleic acid laddering and annexin V protein levels were not altered by CML-collagen exposure. CONCLUSIONS: Carboxymethyl-lysine-collagen increased Tgase activity in tenocytes, likely posttranslationally. Increased levels of Tgase-mediated cross-links may contribute to the excess calcification and biomechanical pathology seen in diabetic tendons.
Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Tendões/enzimologia , Transglutaminases/metabolismo , Animais , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lisina/análogos & derivados , Lisina/farmacologia , RNA Mensageiro/metabolismo , Joelho de Quadrúpedes/metabolismo , Suínos , Tendões/efeitos dos fármacos , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismo , Transglutaminases/genéticaRESUMO
OBJECTIVE: Basic calcium phosphate (BCP) crystals are common components of osteoarthritis (OA) synovial fluid. Progress in understanding the role of these bioactive particles in clinical OA has been hampered by difficulties in their identification. Tetracyclines stain calcium phosphate mineral in bone. The aim of this study was to investigate whether tetracycline staining might be an additional or alternative method for identifying BCP crystals in synovial fluid. METHODS: A drop of oxytetracycline was mixed with a drop of fluid containing synthetic or native BCP, calcium pyrophosphate dihydrate (CPPD), or monosodium urate (MSU) crystals and placed on a microscope slide. Stained and unstained crystals were examined by light microscopy, with and without a portable broad-spectrum ultraviolet (UV) pen light. A small set of characterized synovial fluid samples were compared by staining with alizarin red S and oxytetracycline. Synthetic BCP crystals in synovial fluid were quantified fluorimetrically using oxytetracycline. RESULTS: After oxytetracycline staining, synthetic and native BCP crystals appeared as fluorescent amorphous aggregates under UV light. Oxytetracycline did not stain CPPD or MSU crystals or other particulates. Oxytetracycline staining had fewer false-positive test results than did alizarin red S staining and could provide estimates of the quantities of synthetic BCP crystals in synovial fluid. CONCLUSION: With further validation, oxytetracycline staining may prove to be a useful adjunct or alternative to currently available methods for identifying BCP crystals in synovial fluid.
Assuntos
Pirofosfato de Cálcio/análise , Oxitetraciclina , Líquido Sinovial/química , Animais , Estudos de Viabilidade , Histocitoquímica/métodos , Humanos , Microscopia Ultravioleta/instrumentação , Sus scrofaRESUMO
OBJECTIVE: Calcium pyrophosphate dihydrate (CPPD) and basic calcium phosphate (BCP) crystals occur in up to 60% of osteoarthritic joints and predict an increased severity of arthritis. Articular cartilage vesicles (ACVs) generate CPPD crystals in the presence of ATP and BCP crystals with added beta-glycerophosphate. While ACVs are present in normal articular cartilage, they mineralize primarily in cartilage from osteoarthritic joints. The aim of this study was to explore the hypothesis that ACV mineralization is regulated by components of the surrounding extracellular matrix. METHODS: Porcine ACVs were embedded in agarose gels containing type II and/or type I collagen and/or proteoglycans. Mineralization was measured as (45)Ca accumulation stimulated by ATP or beta-glycerophosphate and reflects both nucleation and growth. Synthetic CPPD and BCP crystals were embedded in similar gels to isolate the effect of matrix components on crystal growth. RESULTS: After establishing baseline responsiveness of ACVs to ATP and beta-glycerophosphate in agarose gels, we examined the ability of ATP and beta-glycerophosphate to stimulate mineral formation in gels containing various matrix components. Type II collagen suppressed the ability of ATP to stimulate mineralization, while a combination of type II plus type I collagen increased the effect of ATP and beta-glycerophosphate on mineralization. Type I collagen affected ACV mineralization in a dose-responsive manner. Neither type of collagen significantly affected crystal growth or levels of mineralization-regulating enzymes. Proteoglycans suppressed mineral formation by ACVs in gels containing both type I and type II collagen. CONCLUSION: Cartilage matrix changes that occur with osteoarthritis, such as increased quantities of type I collagen and reduced proteoglycan levels, may promote ACV mineralization.
Assuntos
Cartilagem Articular/metabolismo , Condrocalcinose/metabolismo , Colágeno Tipo I/metabolismo , Matriz Extracelular/metabolismo , Articulação do Joelho/metabolismo , Animais , Western Blotting , Cartilagem Articular/efeitos dos fármacos , Técnicas de Cultura de Células , Células Cultivadas , Condrocalcinose/tratamento farmacológico , Colágeno Tipo I/farmacologia , Colágeno Tipo II/metabolismo , Colágeno Tipo II/farmacologia , Matriz Extracelular/efeitos dos fármacos , Microscopia Eletrônica , Proteoglicanas/metabolismo , Proteoglicanas/farmacologia , Índice de Gravidade de Doença , SuínosRESUMO
Calcific tendonitis is a common clinical condition associated with high rates of tendon rupture, prolonged symptoms, and poor response to therapy. Little is known about the pathogenesis of calcifications in tendons and consequently few effective therapies are available. We hypothesized that tendon calcification, like pathologic calcification in other sites, was generated by extracellular organelles known as matrix vesicles and that isolated matrix vesicles would constitute the basis for a useful model of this process. Tendon matrix vesicles were isolated from adult porcine patellar tendons using enzymatic digestion and differential centrifugation. Vesicle morphology was examined with electron microscopy. Levels of calcium, phosphate, pyrophosphate, ATP, and mineralization-associated enzymes were measured and compared with articular cartilage vesicles from porcine articular cartilage. Vesicles were embedded in agarose gels with or without type I collagen or dermatan sulfate and incubated in calcifying salt solution trace labeled with (45)calcium. (45)Calcium in the vesicle fraction was measured after 5-7 days. The type of mineral formed was determined by micro-x-ray diffraction. Matrix vesicles isolated from adult porcine tendon were similar morphologically to those obtained from articular cartilage. They contained mineralization-related enzymes and formed hydroxyapatite mineral in vitro. Mineralization was suppressed by levamisole and modulated by extracellular matrix components. Matrix vesicles isolated from tendons mineralize in vitro. This model may aid in the study of the pathogenesis of calcific tendonitis as well as serve as a means to identify effective therapies for this common disorder.
Assuntos
Calcinose/patologia , Tendinopatia/patologia , Animais , Cartilagem Articular/ultraestrutura , Colágeno Tipo I/farmacologia , Dermatan Sulfato/farmacologia , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/fisiologia , Glicerofosfatos/farmacologia , Levamisol/farmacologia , Microscopia Eletrônica , Ligamento Patelar/efeitos dos fármacos , Ligamento Patelar/patologia , Ligamento Patelar/ultraestrutura , Sus scrofaRESUMO
Calcium pyrophosphate dihydrate (CPPD) crystals are commonly found in osteoarthritic joint tissues, where they predict severe disease. Unlike other types of calcium phosphate crystals, CPPD crystals form almost exclusively in the pericellular matrix of damaged articular cartilage, suggesting a key role for the extracellular matrix milieu in their development. Osteopontin is a matricellular protein found in increased quantities in the pericellular matrix of osteoarthritic cartilage. Osteopontin modulates the formation of calcium-containing crystals in many settings. We show here that osteopontin stimulates ATP-induced CPPD crystal formation by chondrocytes in vitro. This effect is augmented by osteopontin's incorporation into extracellular matrix by transglutaminase enzymes, is only modestly affected by its phosphorylation state, and is inhibited by integrin blockers. Surprisingly, osteopontin stimulates transglutaminase activity in cultured chondrocytes in a dose-responsive manner. As elevated levels of transglutaminase activity promote extracellular matrix changes that permit CPPD crystal formation, this is one possible mechanism of action. We demonstrate the presence of osteopontin in the pericellular matrix of chondrocytes adjacent to CPPD deposits and near active transglutaminases. Thus, osteopontin may play an important role in facilitating CPPD crystal formation in articular cartilage.
Assuntos
Calcinose/metabolismo , Pirofosfato de Cálcio/metabolismo , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Osteopontina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cadaverina/análogos & derivados , Cadaverina/farmacologia , Cistamina/farmacologia , Difosfatos/metabolismo , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/metabolismo , Imuno-Histoquímica , Sus scrofa , Trombina/metabolismo , Transglutaminases/antagonistas & inibidoresRESUMO
OBJECTIVE: Trappins are small serine protease inhibitors bound to extracellular matrix (ECM) through the actions of transglutaminase (TGase) enzymes. Trappin-2 is present in many tissues and is upregulated at sites of injury. In osteoarthritis (OA), serine proteases contribute to articular cartilage destruction, and TGase activity is increased. Yet little is known about matrix-bound serine protease inhibitors or TGase substrates in articular cartilage. Our purpose was to determine if trappin-2 was present in OA cartilage and synovial fluid (SF). METHODS: OA knee articular cartilage and SF were assayed for trappin-2 protein by Western blotting, ELISA, and immunohistochemistry. Trappin-2 mRNA was detected with RT-PCR. The ECM components bound to trappin-2 were identified by 2-D gel electrophoresis and peptide fingerprinting. RESULTS: Trappin-2 was detectable in OA articular cartilage extracts, cultured chondrocytes, conditioned media, and SF by Western blotting. OA cartilage protein extracts contained significantly higher quantities of trappin-2 than normal cartilage protein extracts (22.98 +/- 1.28 ng/mg wet weight vs 14.97 +/- 1.92 ng/mg wet weight; p < 0.01). RT-PCR confirmed the presence of trappin-2 mRNA in OA chondrocytes. Immunohistochemical studies of OA cartilage revealed trappin-2 protein in chondrocytes. Peptide mapping of trappin-2 binding partners showed that fibromodulin was bound to trappin-2 in cartilage. CONCLUSION: We confirmed the presence of trappin-2 in OA cartilage and SF. Elevated levels of TGase activity in OA cartilage may increase levels of this serine protease inhibitor in response to injury.
Assuntos
Cartilagem Articular/metabolismo , Elastase de Leucócito/antagonistas & inibidores , Osteoartrite/metabolismo , Precursores de Proteínas/metabolismo , Líquido Sinovial/metabolismo , Adolescente , Adulto , Cartilagem Articular/patologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Meios de Cultivo Condicionados/química , Elafina , Humanos , Técnicas Imunoenzimáticas , Osteoartrite/patologia , Precursores de Proteínas/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Líquido Sinovial/citologiaRESUMO
Calcium pyrophosphate dihydrate (CPPD) crystals are common components of osteoarthritic joints and correlate with a poor prognosis. Transglutaminase (Tgase) enzymes have been implicated in pathologic mineralization in cartilage; yet, definitive studies linking Tgase activity to CPPD crystal formation in osteoarthritic articular cartilage are lacking. We measured in-vivo Tgase activity in osteoarthritic and normal human cartilage, and explored the effect of Tgase inhibitors on CPPD crystal formation by normal chondrocytes. Osteoarthritic articular cartilage from was obtained from specimens discarded at the time of knee replacement surgery. Normal adult cartilage samples from a tissue bank were used as controls. Tgase-specific isopeptide (epsilon-(gamma-glutamyl) lysine) bonds were measured in cartilage extracts by HPLC. Tgase-specific crosslinks were localized in osteoarthritic cartilage by immunohistochemistry. The effect of Tgase inhibition was determined in an in-vitro model of CPPD crystal formation. Tgase-specific crosslink levels were 1.55 +/- 0.3 picomoles/ng protein in normal human adult articular cartilage and 4.74 +/- 0.7 picomoles/ng protein in osteoarthritic human cartilage (p less than 0.001). Immunostaining confirmed the presence of Tgase crosslinks in the pericellular matrix of chondrocytes at potential sites of CPPD crystal formation. Tgase inhibitors suppressed CPPD crystal formation by porcine chondrocytes. These findings support a role for Tgase in CPPD crystal formation in aging or degenerated cartilage.
Assuntos
Osteoartrite/metabolismo , Transglutaminases/fisiologia , Animais , Pirofosfato de Cálcio/química , Cartilagem/metabolismo , Sobrevivência Celular , Células Cultivadas , Condrócitos/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Imuno-Histoquímica , Suínos , Sais de Tetrazólio/farmacologia , Tiazóis/farmacologia , Transglutaminases/metabolismoRESUMO
PURPOSE OF REVIEW: Calcium pyrophosphate dihydrate crystals are common components of synovial fluids from degenerated joints and often accompany unusually severe cartilage destruction. Progress in understanding why and how calcium pyrophosphate dihydrate crystals form in articular cartilage has been hampered by the scarcity of good models in which to study this phenomenon. RECENT FINDINGS: In this review, the author discusses various models of calcium pyrophosphate dihydrate crystal formation from early work with solutions and gels to more recent models using cells, tissues, and cell fractions. Each of these systems has advantages and disadvantages. SUMMARY: Current models of calcium pyrophosphate dihydrate crystal formation are less than ideal for studying the major factors involved in calcium pyrophosphate dihydrate crystal formation. Borrowing from the transplantation literature, solid matrices that support cell growth and allow for easy manipulation of matrix components hold some promise for better models in the future.
Assuntos
Pirofosfato de Cálcio/metabolismo , Cartilagem Articular/fisiopatologia , Animais , Células Cultivadas , Cristalização , Matriz Extracelular/fisiologia , Humanos , Modelos Animais , Técnicas de Cultura de ÓrgãosRESUMO
Transglutaminases are a family of enzymes that catalyze the formation of epsilon-(gamma-glutamyl)lysine isopeptide bonds in proteins, an activity that has been implicated in the pathogenesis of cartilage matrix mineralization in degenerative arthritis. Type II transglutaminase and thrombin-activatable factor XIII have been identified in articular cartilage. Thrombin, a coagulation protease, is found in pathological synovial fluids, and is known to stimulate transglutaminase activity in non-articular tissues. We investigated the effects of thrombin on transglutaminase activity in porcine articular chondrocytes. Direct addition of thrombin to chondrocyte lysates resulted in increased transglutaminase activity due to proteolytic conversion of factor XIII to XIIIa. Thrombin-treated chondrocyte cultures (0.001 to 2.0 U/ml) also showed increased transglutaminase activity. Thrombin treatment of chondrocyte cultures increased transglutaminase activity as early as 15 minutes after addition, an effect that we attributed to factor XIII activation. Additional stimulatory effects of thrombin were observed in cultured chondrocytes at 4 and 24 hours. A thrombin receptor agonist peptide (TRAP) which activates the PAR1 thrombin receptor mimicked these later effects. Thrombin treatment of chondrocyte cultures increased factor XIII mRNA and protein levels, without affecting levels of type II transglutaminase. Thus, thrombin stimulates transglutaminase activity in articular cartilage by directly cleaving factor XIII and by receptor-mediated up-regulation of factor XIII synthesis. Such increases in potential transglutaminase activity may facilitate pathological matrix calcification in degenerative arthritis.