RESUMEN
UNLABELLED: : Familial osteochondritis dissecans (FOCD) is an inherited skeletal defect characterized by the development of large cartilage lesions in multiple joints, short stature, and early onset of severe osteoarthritis. It is associated with a heterozygous mutation in the ACAN gene, resulting in a Val-Met replacement in the C-type lectin domain of aggrecan. To understand the cellular pathogenesis of this condition, we studied the chondrogenic differentiation of patient bone marrow mesenchymal stromal cells (BM-MSCs). We also looked at cartilage derived from induced pluripotent stem cells (iPSCs) generated from patient fibroblasts. Our results revealed several characteristics of the differentiated chondrocytes that help to explain the disease phenotype and susceptibility to cartilage injury. First, patient chondrogenic pellets had poor structural integrity but were rich in glycosaminoglycan. Second, it was evident that large amounts of aggrecan accumulated within the endoplasmic reticulum of chondrocytes differentiated from both BM-MSCs and iPSCs. In turn, there was a marked absence of aggrecan in the extracellular matrix. Third, it was evident that matrix synthesis and assembly were globally dysregulated. These results highlight some of the abnormal aspects of chondrogenesis in these patient cells and help to explain the underlying cellular pathology. The results suggest that FOCD is a chondrocyte aggrecanosis with associated matrix dysregulation. The work provides a new in vitro model of osteoarthritis and cartilage degeneration based on the use of iPSCs and highlights how insights into disease phenotype and pathogenesis can be uncovered by studying differentiation of patient stem cells. SIGNIFICANCE: The isolation and study of patient stem cells and the development of methods for the generation of iPSCs have opened up exciting opportunities in understanding causes and exploring new treatments for major diseases. This technology was used to unravel the cellular phenotype in a severe form of inherited osteoarthritis, termed familial osteochondritis dissecans. The phenotypic abnormalities that give rise to cartilage lesions in these patients were able to be described via the generation of chondrocytes from bone marrow-derived mesenchymal stromal cells and iPSCs, illustrating the extraordinary value of these approaches in disease modeling.
Asunto(s)
Condrocitos/patología , Estrés del Retículo Endoplásmico/fisiología , Matriz Extracelular/patología , Osteocondritis Disecante/congénito , Adulto , Agrecanos/genética , Animales , Cartílago/metabolismo , Técnicas de Cultivo de Célula/métodos , Condrocitos/metabolismo , Condrogénesis/fisiología , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Masculino , Espectrometría de Masas , Células Madre Mesenquimatosas/citología , Ratones , Microscopía Electrónica de Transmisión , Persona de Mediana Edad , Osteocondritis Disecante/genética , Osteocondritis Disecante/metabolismo , Osteocondritis Disecante/patología , FenotipoRESUMEN
cyclicCHAD is a peptide representing the α2ß1 integrin binding sequence of the matrix protein chondroadherin (CHAD), which in our hands proved effective at counteracting bone loss in ovariectomised mice by inhibiting osteoclastogenesis. Given that bone metastases are characterised by exacerbated osteoclast activity as well, we tested this therapy in mice intracardiacally injected with the osteotropic human breast cancer cell line MDA-MB-231. Treatment with cyclicCHAD significantly decreased cachexia and incidence of bone metastases, and induced a trend of reduction of visceral metastasis volume, while in orthotopically injected mice cyclicCHAD reduced tumour volume. In vitro studies showed its ability to impair tumour cell motility and invasion, suggesting a direct effect not only on osteoclasts but also on the tumour cell phenotype. Interestingly, when administered together with a suboptimal, poorly effective, dose of doxorubicin (DXR), cyclicCHAD improved survival and reduced visceral metastases volume to a level similar to that of the optimal dose of DXR alone. Taken together, these preclinical data suggest that cyclicCHAD is a new inhibitor of bone metastases, with an appreciable direct effect also on tumour growth and a synergistic activity in combination with low dose chemotherapy, underscoring an important translational impact.
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Neoplasias Óseas/tratamiento farmacológico , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de la Matriz Extracelular/metabolismo , Integrina alfa2beta1/metabolismo , Animales , Neoplasias Óseas/patología , Neoplasias Óseas/secundario , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/patología , Neoplasias de la Mama/patología , Caquexia/tratamiento farmacológico , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Proteínas de la Matriz Extracelular/administración & dosificación , Femenino , Humanos , Ratones , Osteoclastos/efectos de los fármacos , Osteoclastos/patología , Estructura Terciaria de ProteínaRESUMEN
The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease.
Asunto(s)
Cartílago Articular/metabolismo , Matriz Extracelular/química , Proteínas Matrilinas/aislamiento & purificación , Proteómica/métodos , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Espectrometría de Masas , Tenascina/metabolismoRESUMEN
Chondroadherin (CHAD), a class IV small leucine rich proteoglycan/protein (SLRP), was hypothesized to play important roles in regulating chondrocyte signaling and cartilage homeostasis. However, its roles in cartilage development and function are not well understood, and no major osteoarthritis-like phenotype was found in the murine model with CHAD genetically deleted (CHAD(-/-)). In this study, we used atomic force microscopy (AFM)-based nanoindentation to quantify the effects of CHAD deletion on changes in the biomechanical function of murine cartilage. In comparison to wild-type (WT) mice, CHAD-deletion resulted in a significant ≈70-80% reduction in the indentation modulus, Eind, of the superficial zone knee cartilage of 11 weeks, 4 months and 1 year old animals. This mechanical phenotype correlates well with observed increases in the heterogeneity collagen fibril diameters in the surface zone. The results suggest that CHAD mainly plays a major role in regulating the formation of the collagen fibrillar network during the early skeletal development. In contrast, CHAD-deletion had no appreciable effects on the indentation mechanics of middle/deep zone cartilage, likely due to the dominating role of aggrecan in the middle/deep zone. The presence of significant rate dependence of the indentation stiffness in both WT and CHAD(-/-) knee cartilage suggested the importance of both fluid flow induced poroelasticity and intrinsic viscoelasticity in murine cartilage biomechanical properties. Furthermore, the marked differences in the nanomechanical behavior of WT versus CHAD(-/-) cartilage contrasted sharply with the relative absence of overt differences in histological appearance. These observations highlight the sensitivity of nanomechanical tools in evaluating structural and mechanical phenotypes in transgenic mice.
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Cartílago Articular/fisiología , Cartílago Articular/ultraestructura , Proteínas de la Matriz Extracelular/deficiencia , Fenotipo , Animales , Fenómenos Biomecánicos , Cartílago Articular/metabolismo , Colágeno/metabolismo , Elasticidad , Ratones , Microscopía de Fuerza Atómica , Microscopía Electrónica de RastreoRESUMEN
To identify patients at risk for progressive joint damage, there is a need for early diagnostic tools to detect molecular events leading to cartilage destruction. Isolation and characterization of distinct cartilage oligomeric matrix protein (COMP) fragments derived from cartilage and released into synovial fluid will allow discrimination between different pathological conditions and monitoring of disease progression. Early detection of disease and processes in the tissue as well as an understanding of the pathologic mechanisms will also open the way for novel treatment strategies. Disease-specific COMP fragments were isolated by affinity chromatography of synovial fluids from patients with rheumatoid arthritis, osteoarthritis, or acute trauma. Enriched COMP fragments were separated by SDSPAGE followed by in-gel digestion and mass spectrometric identification and characterization.Using the enzymes trypsin, chymotrypsin, and Asp-N for the digestions, an extensive analysis of the enriched fragments could be accomplished. Twelve different neoepitopes were identified and characterized within the enriched COMP fragments. For one of the neoepitopes, Ser77, an inhibition ELISA was developed. This ELISA quantifies COMP fragments clearly distinguishable from total COMP. Furthermore, fragments containing the neoepitope Ser77 were released into the culture medium of cytokine (TNF-α and IL-6/soluble IL-6 receptor)-stimulated human cartilage explants. The identified neoepitopes provide a complement to the currently available commercial assays for cartilage markers. Through neoepitope assays, tools to pinpoint disease progression, evaluation methods for therapy, and means to elucidate disease mechanisms will be provided.
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Proteína de la Matriz Oligomérica del Cartílago , Cromatografía de Afinidad , Epítopos , Artropatías/metabolismo , Espectrometría de Masas , Líquido Sinovial , Adulto , Proteína de la Matriz Oligomérica del Cartílago/química , Proteína de la Matriz Oligomérica del Cartílago/aislamiento & purificación , Proteína de la Matriz Oligomérica del Cartílago/metabolismo , Células Cultivadas , Epítopos/química , Epítopos/aislamiento & purificación , Epítopos/metabolismo , Humanos , Interleucina-6/metabolismo , Artropatías/patología , Receptores de Interleucina-6/metabolismo , Líquido Sinovial/química , Líquido Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Presently, there are no established treatments to prevent, stop or even retard back pain arising from disc degeneration. Previous studies have shown that Link N can act as a growth factor and stimulate the synthesis of proteoglycans and collagens, in IVD. However, the sequences in Link N involved in modulating cellular activity are not well understood. To determine if disc cells can proteolytically process Link N, human disc cells were exposed to native Link N over a 48 h period and mass spectrometric analysis revealed that a peptide spanning residues 1-8 was generated in the presence of AF cells but not NP cells. Link N 1-8 significantly induced proteoglycan production in the presence of IL-1ß NP and AF cells, confirming that the biological effect is maintained in the first 8 amino acids of the peptide and indicating that the effect is sustained in an inflammatory environment. Thus Link-N 1-8 could be a promising candidate for biologically induced disc repair, and the identification of such a stable specific peptide may facilitate the design of compounds to promote disc repair and provide alternatives to surgical intervention for early stage disc degeneration.
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Factores Biológicos/metabolismo , Factores Biológicos/farmacología , Disco Intervertebral/efectos de los fármacos , Disco Intervertebral/metabolismo , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Adolescente , Adulto , Secuencia de Aminoácidos , Animales , Bovinos , Células Cultivadas , Colágeno/metabolismo , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/metabolismo , Degeneración del Disco Intervertebral/patología , Vértebras Lumbares/patología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteoglicanos/metabolismo , Regeneración/efectos de los fármacos , Vértebras Torácicas/patología , Adulto JovenRESUMEN
Chondroadherin (CHAD) is a leucine-rich protein promoting cell attachment through binding to integrin α2 ß1 and syndecans. We observed that CHAD mRNA and protein were lower in bone biopsies of 50-year-old to 65-year-old osteoporotic women and in bone samples of ovariectomized mice versus gender/age-matched controls, suggesting a role in bone metabolism. By the means of an internal cyclic peptide (cyclicCHAD), we observed that its integrin binding sequence impaired preosteoclast migration through a nitric oxide synthase 2-dependent mechanism, decreasing osteoclastogenesis and bone resorption in a concentration-dependent fashion, whereas it had no effect on osteoblasts. Consistently, cyclicCHAD reduced transcription of two nitric oxide downstream genes, migfilin and vasp, involved in cell motility. Furthermore, the nitric oxide donor, S-nitroso-N-acetyl-D,L-penicillamine, stimulated preosteoclast migration and prevented the inhibitory effect of cyclicCHAD. Conversely, the nitric oxide synthase 2 (NOS2) inhibitor, N5-(1-iminoethyl)-l-ornithine, decreased both preosteoclast migration and differentiation, confirming a role of the nitric oxide pathway in the mechanism of action triggered by cyclicCHAD. In vivo, administration of cyclicCHAD was well tolerated and increased bone volume in healthy mice, with no adverse effect. In ovariectomized mice cyclicCHAD improved bone mass by both a preventive and a curative treatment protocol, with an effect in line with that of the bisphosphonate alendronate, that was mimicked by the NOS2 inhibitor [L-N6-(1-Iminoethyl)-lysine.2 dihydrochloride]. In both mouse models, cyclicCHAD reduced osteoclast and bone resorption without affecting osteoblast parameters and bone formation. In conclusion, CHAD is a novel regulator of bone metabolism that, through its integrin binding domain, inhibits preosteoclast motility and bone resorption, with a potential translational impact for the treatment of osteoporosis.
Asunto(s)
Resorción Ósea/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/farmacología , Osteoclastos , Anciano , Animales , Western Blotting , Conservadores de la Densidad Ósea/farmacología , Diferenciación Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Proteínas de la Matriz Extracelular/química , Femenino , Perfilación de la Expresión Génica , Humanos , Ratones , Persona de Mediana Edad , Ovariectomía , Péptidos/farmacologíaRESUMEN
During inflammatory processes the extracellular matrix (ECM) is extensively remodeled, and many of the constituent components are released as proteolytically cleaved fragments. These degradative processes are better documented for inflammatory joint diseases than tendinopathy even though the pathogenesis has many similarities. The aims of this study were to investigate the proteomic composition of injured tendons during early and late disease stages to identify disease-specific cleavage patterns of the ECM protein cartilage oligomeric matrix protein (COMP). In addition to characterizing fragments released in naturally occurring disease, we hypothesized that stimulation of tendon explants with proinflammatory mediators in vitro would induce fragments of COMP analogous to natural disease. Therefore, normal tendon explants were stimulated with IL-1ß and prostaglandin E2, and their effects on the release of COMP and its cleavage patterns were characterized. Analyses of injured tendons identified an altered proteomic composition of the ECM at all stages post injury, showing protein fragments that were specific to disease stage. IL-1ß enhanced the proteolytic cleavage and release of COMP from tendon explants, whereas PGE2 had no catabolic effect. Of the cleavage fragments identified in early stage tendon disease, two fragments were generated by an IL-1-mediated mechanism. These fragments provide a platform for the development of neo-epitope assays specific to injury stage for tendon disease.
Asunto(s)
Proteína de la Matriz Oligomérica del Cartílago/metabolismo , Matriz Extracelular/metabolismo , Proteómica/métodos , Tendinopatía/metabolismo , Tendinopatía/patología , Tendones/metabolismo , Tendones/patología , Secuencia de Aminoácidos , Animales , Western Blotting , Proteína de la Matriz Oligomérica del Cartílago/química , Cromatografía Liquida , Medios de Cultivo , Dinoprostona/farmacología , Caballos , Humanos , Interleucina-1beta/farmacología , Espectrometría de Masas , Datos de Secuencia Molecular , Tendones/efectos de los fármacos , Supervivencia Tisular/efectos de los fármacosRESUMEN
Chondroadherin, a leucine rich repeat extracellular matrix protein with functions in cell to matrix interactions, binds cells via their α2ß1 integrin as well as via cell surface proteoglycans, providing for different sets of signals to the cell. Additionally, the protein acts as an anchor to the matrix by binding tightly to collagens type I and II as well as type VI. We generated mice with inactivated chondroadherin gene to provide integrated studies of the role of the protein. The null mice presented distinct phenotypes with affected cartilage as well as bone. At 3-6 weeks of age the epiphyseal growth plate was widened most pronounced in the proliferative zone. The proteome of the femoral head articular cartilage at 4 months of age showed some distinct differences, with increased deposition of cartilage intermediate layer protein 1 and fibronectin in the chondroadherin deficient mice, more pronounced in the female. Other proteins show decreased levels in the deficient mice, particularly pronounced for matrilin-1, thrombospondin-1 and notably the members of the α1-antitrypsin family of proteinase inhibitors as well as for a member of the bone morphogenetic protein growth factor family. Thus, cartilage homeostasis is distinctly altered. The bone phenotype was expressed in several ways. The number of bone sialoprotein mRNA expressing cells in the proximal tibial metaphysic was decreased and the osteoid surface was increased possibly indicating a change in mineral metabolism. Micro-CT revealed lower cortical thickness and increased structure model index, i.e. the amount of plates and rods composing the bone trabeculas. The structural changes were paralleled by loss of function, where the null mice showed lower femoral neck failure load and tibial strength during mechanical testing at 4 months of age. The skeletal phenotype points at a role for chondroadherin in both bone and cartilage homeostasis, however, without leading to altered longitudinal growth.
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Huesos/patología , Proteínas de la Matriz Extracelular/deficiencia , Animales , Fenómenos Biomecánicos , Huesos/diagnóstico por imagen , Huesos/fisiopatología , Huesos/ultraestructura , Cartílago/diagnóstico por imagen , Cartílago/metabolismo , Cartílago/patología , Cartílago/fisiopatología , Epífisis/diagnóstico por imagen , Epífisis/patología , Epífisis/fisiopatología , Proteínas de la Matriz Extracelular/metabolismo , Fémur/metabolismo , Fémur/patología , Fémur/fisiopatología , Silenciador del Gen , Placa de Crecimiento/diagnóstico por imagen , Placa de Crecimiento/patología , Placa de Crecimiento/fisiopatología , Sialoproteína de Unión a Integrina/genética , Sialoproteína de Unión a Integrina/metabolismo , Ratones , Osteopontina/metabolismo , Fenotipo , Proteoma/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Microtomografía por Rayos XRESUMEN
INTRODUCTION: Complexes between cartilage oligomeric matrix protein (COMP) and the complement activation product C3b have been found in the circulation of patients with rheumatoid arthritis and systemic lupus erythematosus. In systemic sclerosis (SSc) COMP expression in the skin is upregulated both in lesional and non-lesional skin, which is also reflected in an increased amount of circulating COMP. We investigated the presence of COMP-C3b complexes in serum and skin biopsies of patients with SSc. METHODS: The presence of COMP and COMP-C3b complexes in the serum of 80 patients with limited cutaneous SSc (lcSSc, n = 40) and diffuse cutaneous SSc (dcSSc, n = 40) and 97 healthy controls was measured by ELISA and correlated to different clinical parameters. Samples were collected both at baseline and after three to five years to assess longitudinal changes in COMP-C3b complex levels. Furthermore, skin biopsies from seven patients with dcSSc and three healthy controls were analyzed for expression of COMP and deposition of C3b and IgG. RESULTS: Serum levels of COMP-C3b were found to be elevated in both dcSSc and lcSSc compared to healthy controls and decreased at the second measurement in patients on immunosuppressive therapy. No co-localization of COMP and C3b was found in the skin biopsies, indicating that the COMP-C3b complexes are formed upon release of COMP into the circulation. CONCLUSION: COMP-C3b complexes are found in the serum of patients with SSc. The lack of co-localization between COMP and C3b in the skin suggests that COMP does not drive complement activation in the skin in SSc.
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Proteína de la Matriz Oligomérica del Cartílago/sangre , Activación de Complemento/fisiología , Complemento C3b/metabolismo , Esclerodermia Sistémica/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Proteína de la Matriz Oligomérica del Cartílago/análisis , Complemento C3b/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Microscopía Fluorescente , Persona de Mediana Edad , Piel/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: To investigate biomarker patterns in rheumatoid arthritis (RA) with extraarticular manifestations. METHODS: Cartilage oligomeric matrix protein (COMP), COMP-C3b, and soluble terminal complement complexes (sTCC) were measured by ELISA. RESULTS: COMP-C3b levels were higher in patients with RA than in healthy controls and lower in extraarticular RA (ExRA) than in RA controls. In patients with ExRA, sTCC levels were higher than in RA controls, and correlated inversely with serum COMP-C3b levels in the ExRA group. CONCLUSION: Patients with ExRA had lower levels of COMP-C3b. This may be a consequence of complement consumption or a lower potential for COMP from these patients to activate complement.
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Artritis Reumatoide/inmunología , Proteína de la Matriz Oligomérica del Cartílago/inmunología , Complemento C3b/inmunología , Índice de Severidad de la Enfermedad , Adulto , Anciano , Artritis Reumatoide/metabolismo , Biomarcadores/metabolismo , Proteína de la Matriz Oligomérica del Cartílago/sangre , Cartílago Articular/inmunología , Cartílago Articular/metabolismo , Complemento C3b/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Líquido Sinovial/inmunología , Líquido Sinovial/metabolismoRESUMEN
BACKGROUND: Currently available biomarkers for the early tissue process leading to joint damage in rheumatoid arthritis are insufficient and lack prognostic accuracy, possibly a result of variable activity of the disease over time. This study represents a novel approach to detect an altered activity of the disease process detected as increasing serum-COMP levels over a short time and whether this would correlate with joint damage progression over the first 5 years of disease. METHODS: In all, 349 patients from the Swedish BARFOT early RA study were examined. Serum-COMP was analysed by ELISA at diagnosis and after 3 months. Based on changes in serum-COMP levels, three subgroups of patients were defined: those with unchanged levels (change ≤ 20%) (N=142), decreasing levels (> 20%) (N=173) and increasing levels (> 20%) (N=34). Radiographs of hands and feet were obtained at inclusion, after 1, 2 and 5 years and scored according to Sharp van der Heijde (SHS). Radiographic progression was defined as increase in SHS by ≥5.8. RESULTS: The group of patients with increasing COMP levels showed higher median change in total SHS and erosion scores at 1, 2 and 5 year follow-up compared with the groups with stable or decreasing COMP levels. Furthermore, the odds ratio of radiographic progression was 2.8 (95% CI 1.26-6.38) for patients with increasing COMP levels vs. patients with unchanged levels.The group of patients with increasing COMP levels had higher ESR at inclusion but there were no baseline differences between the groups for age, gender, disease duration, disease activity (DAS28), function (HAQ), CRP, nor presence of rheumatoid factor or anti-CCP. Importantly, neither did changes over the 3-month period in DAS28, HAQ, ESR nor CRP differ between the groups and these variables did not correlate to joint damage progression. CONCLUSION: Increasing serum-COMP levels between diagnosis and the subsequent 3 months in patients with early RA represents a novel indicator of an activated destructive process in the joint and is a promising tool to identify patients with significant joint damage progression during a 5-year period.
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Artritis Reumatoide/diagnóstico , Biomarcadores/sangre , Proteínas de la Matriz Extracelular/sangre , Glicoproteínas/sangre , Articulaciones/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/sangre , Artritis Reumatoide/fisiopatología , Artrografía , Proteína de la Matriz Oligomérica del Cartílago , Progresión de la Enfermedad , Diagnóstico Precoz , Femenino , Humanos , Articulaciones/fisiopatología , Masculino , Proteínas Matrilinas , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
Chondroadherin, a member of the leucine-rich repeat family, has previously been demonstrated to be fragmented in some juveniles with idiopathic scoliosis. This observation led us to investigate adults with disc degeneration. Immunoblotting analysis demonstrated that non-degenerate discs from three different age groups show no chondroadherin fragmentation. Furthermore, the chondroadherin fragments in adult degenerate disc and the juvenile scoliotic disc were compared via immunoblot analysis and appeared to have a similar size. We then investigated whether or not chondroadherin fragmentation increases with the severity of disc degeneration. Three different samples with different severities were chosen from the same disc, and chondroadherin fragmentation was found to be more abundant with increasing severity of degeneration. This observation led us to the creation of a neoepitope antibody to the cleavage site observed. We then observed that the cleavage site in adult degenerate discs and juvenile scoliotic discs was identical as confirmed by the neoepitope antibody. Consequently, investigation of the protease capable of cleaving chondroadherin at this site was necessary. In vitro digests of disc tissue demonstrated that ADAMTS-4 and -5; cathepsins K, B, and L; and MMP-3, -7, -12, and -13 were incapable of cleavage of chondroadherin at this site and that HTRA1 was indeed the only protease capable. Furthermore, increased protein levels of the processed form of HTRA1 were demonstrated in degenerate disc tissues via immunoblotting. The results suggest that chondroadherin fragmentation can be used as a biomarker to distinguish the processes of disc degeneration from normal aging.
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Envejecimiento , Proteínas de la Matriz Extracelular/metabolismo , Degeneración del Disco Intervertebral/enzimología , Serina Endopeptidasas/metabolismo , Adolescente , Factores de Edad , Sitios de Unión , Diagnóstico Diferencial , Matriz Extracelular/metabolismo , Serina Peptidasa A1 que Requiere Temperaturas Altas , Humanos , Inflamación , Disco Intervertebral/metabolismo , Disco Intervertebral/patología , Persona de Mediana Edad , Péptido Hidrolasas/metabolismoRESUMEN
(hbd) PRELP is a peptide corresponding to the N-terminal heparin binding domain of the matrix protein proline/arginine-rich end leucine-rich repeat protein (PRELP). (hbd) PRELP inhibits osteoclastogenesis entering pre-fusion osteoclasts through a chondroitin sulfate- and annexin 2-dependent mechanism and reducing the nuclear factor-κB transcription factor activity. In this work, we hypothesized that (hbd) PRELP could have a pharmacological relevance, counteracting bone loss in a variety of in vivo models of bone diseases induced by exacerbated osteoclast activity. In healthy mice, we demonstrated that the peptide targeted the bone and increased trabecular bone mass over basal level. In mice treated with retinoic acid to induce an acute increase of osteoclast formation, the peptide consistently antagonized osteoclastogenesis and prevented the increase of the serum levels of the osteoclast-specific marker tartrate-resistant acid phosphatase. In ovariectomized mice, in which osteoclast activity was chronically enhanced by estrogen deficiency, (hbd) PRELP counteracted exacerbated osteoclast activity and bone loss. In mice carrying osteolytic bone metastases, in which osteoclastogenesis and bone resorption were enhanced by tumor cell-derived factors, (hbd) PRELP reduced the incidence of osteolytic lesions, both preventively and curatively, with mechanisms involving impaired tumor cell homing to bone and tumor growth in the bone microenvironment. Interestingly, in tumor-bearing mice, (hbd) PRELP also inhibited breast tumor growth in orthotopic sites and development of metastatic disease in visceral organs, reducing cachexia and improving survival especially when administered preventively. (hbd) PRELP was retained in the tumor tissue and appeared to affect tumor growth by interacting with the microenvironment rather than by directly affecting the tumor cells. Because safety studies and high-dose treatments revealed no adverse effects, (hbd) PRELP could be employed as a novel biological agent to combat experimentally induced bone loss and breast cancer metastases, with a potential translational impact.
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Resorción Ósea/tratamiento farmacológico , Resorción Ósea/patología , Proteínas de la Matriz Extracelular/farmacología , Proteínas de la Matriz Extracelular/uso terapéutico , Glicoproteínas/farmacología , Glicoproteínas/uso terapéutico , Osteoclastos/patología , Animales , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/secundario , Resorción Ósea/complicaciones , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteínas de la Matriz Extracelular/efectos adversos , Proteínas de la Matriz Extracelular/química , Femenino , Glicoproteínas/efectos adversos , Glicoproteínas/química , Humanos , Neoplasias Mamarias Experimentales/complicaciones , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Osteoporosis/complicaciones , Osteoporosis/tratamiento farmacológico , Osteoporosis/patología , Unión Proteica/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Excessive complement activation contributes to joint diseases such as rheumatoid arthritis and osteoarthritis during which cartilage proteins are fragmented and released into the synovial fluid. Some of these proteins and fragments activate complement, which may sustain inflammation. The G3 domain of large cartilage proteoglycan aggrecan interacts with other extracellular matrix proteins, fibulins and tenascins, via its C-type lectin domain (CLD) and has important functions in matrix organization. Fragments containing G3 domain are released during normal aggrecan turnover, but increasingly so in disease. We now show that the aggrecan CLD part of the G3 domain activates the classical and to a lesser extent the alternative pathway of complement, via binding of C1q and C3, respectively. The complement control protein (CCP) domain adjacent to the CLD showed no effect on complement initiation. The binding of C1q to G3 depended on ionic interactions and was decreased in D2267N mutant G3. However, the observed complement activation was attenuated due to binding of complement inhibitor factor H to CLD and CCP domains. This was most apparent at the level of deposition of terminal complement components. Taken together our observations indicate aggrecan CLD as one factor involved in the sustained inflammation of the joint.
Asunto(s)
Agrecanos/inmunología , Activación de Complemento/inmunología , Proteínas del Sistema Complemento/inmunología , Dominios y Motivos de Interacción de Proteínas/inmunología , Agrecanos/química , Agrecanos/metabolismo , Animales , Cartílago/química , Cartílago/inmunología , Bovinos , Línea Celular , Complemento C1q/inmunología , Complemento C1q/metabolismo , Factor H de Complemento/metabolismo , Vía Alternativa del Complemento/inmunología , Vía Clásica del Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Humanos , Modelos Biológicos , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genéticaRESUMEN
Skin fibrosis is characterized by activated fibroblasts and an altered architecture of the extracellular matrix. Excessive deposition of extracellular matrix proteins and altered cytokine levels in the dermal collagen matrix are common to several pathological situations such as localized scleroderma and systemic sclerosis, keloids, dermatosclerosis associated with venous ulcers and the fibroproliferative tissue surrounding invasively growing tumors. Which factors contribute to altered organization of dermal collagen matrix in skin fibrosis is not well understood. We recently demonstrated that cartilage oligomeric matrix protein (COMP) functions as organizer of the dermal collagen I network in healthy human skin (Agarwal et al., 2012). Here we show that COMP deposition is enhanced in the dermis in various fibrotic conditions. COMP levels were significantly increased in fibrotic lesions derived from patients with localized scleroderma, in wound tissue and exudates of patients with venous leg ulcers and in the fibrotic stroma of biopsies from patients with basal cell carcinoma. We postulate enhanced deposition of COMP as one of the common factors altering the supramolecular architecture of collagen matrix in fibrotic skin pathologies. Interestingly, COMP remained nearly undetectable in normally healing wounds where myofibroblasts transiently accumulate in the granulation tissue. We conclude that COMP expression is restricted to a fibroblast differentiation state not identical to myofibroblasts which is induced by TGFß and biomechanical forces.
Asunto(s)
Carcinoma Basocelular/metabolismo , Proteína de la Matriz Oligomérica del Cartílago/metabolismo , Dermis/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Úlcera de la Pierna/metabolismo , Esclerodermia Localizada/metabolismo , Neoplasias Cutáneas/metabolismo , Anciano , Carcinoma Basocelular/genética , Carcinoma Basocelular/patología , Proteína de la Matriz Oligomérica del Cartílago/genética , Estudios de Casos y Controles , Diferenciación Celular , Proliferación Celular , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Dermis/patología , Matriz Extracelular/genética , Matriz Extracelular/patología , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica , Humanos , Úlcera de la Pierna/genética , Úlcera de la Pierna/patología , Esclerodermia Localizada/genética , Esclerodermia Localizada/patología , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Cicatrización de Heridas/fisiologíaRESUMEN
Chondroadherin, a leucine-rich repeat family member, contains a very C-terminal sequence CKFPTKRSKKAGRH(359), now shown to bind to heparin with a K(D) of 13 µm. This observation led us to investigate whether chondroadherin interacts via this C-terminal heparin-binding domain with glycosaminoglycan chains of proteoglycans at the cell surface. Cells were shown to bind this heparin-binding peptide in FACS analysis, and the interaction was shown to be with glycosaminoglycans because it was abolished when sulfation was inhibited by chlorate treatment of the cells. In separate experiments, heparin and heparan sulfate inhibited the peptide interaction in a dose-dependent manner. Using a human chondrosarcoma and a murine osteoblast cell line, heparan sulfate proteoglycans were identified as the cell surface receptors involved in the binding. Different binding syndecans were identified in the two different cell lines, indicating that the same protein core of a proteoglycan may have structural and functional differences in the attached heparan sulfate chains. Upon binding to coated peptide, cells spread, demonstrating engagement of the cytoskeleton, but no focal adhesion complex was formed. The number of cells adhering via their ß(1) integrin receptor to collagen type II or chondroadherin was profoundly and rapidly enhanced by the addition of the heparin-binding peptide. The peptide added to the cells caused ERK phosphorylation, showing that it triggered intracellular signaling. The results show that heparan sulfate chains differ between various members of the proteoglycan families on a given cell, but also differ between the same proteoglycan on different cells with a potential for differential regulation of cellular activities.
Asunto(s)
Proteínas de la Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calorimetría , Línea Celular , Cromatografía de Afinidad , Cartilla de ADN , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Humanos , Ratones , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
This is a descriptive study of tendon pathology with different structural appearances of repair tissue correlated to immunolocalization of cartilage oligomeric matrix protein (COMP) and type I and III collagens and expression of COMP mRNA. The material consists of nine tendons from seven horses (5-25 years old; mean age of 10 years) with clinical tendinopathy and three normal tendons from horses (3, 3, and 13 years old) euthanized for non-orthopedic reasons. The injured tendons displayed different repair-tissue appearances with organized and disorganized fibroblastic regions as well as areas of necrosis. The normal tendons presented distinct immunoreactivity for COMP and expression of COMP mRNA and type I collagen in the normal aligned fiber structures, but no immunolabeling of type III collagen. However, immunoreactivity for type III collagen was present in the endotenon surrounding the fiber bundles, where no expression of COMP could be seen. Immunostaining for type I and III collagens was present in all of the pathologic regions indicating repair tissue. Interestingly, the granulation tissues showed immunostaining for COMP and expression of COMP mRNA, indicating a role for COMP in repair and remodeling of the tendon after fiber degeneration and rupture. The present results suggest that not only type III collagen but also COMP is involved in the repair and remodeling processes of the tendon.
Asunto(s)
Colágeno Tipo II/metabolismo , Colágeno Tipo I/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Glicoproteínas/metabolismo , Enfermedades de los Caballos/metabolismo , Traumatismos de los Tendones/veterinaria , Tendones/metabolismo , Animales , Colágeno Tipo I/análisis , Colágeno Tipo I/genética , Colágeno Tipo II/análisis , Colágeno Tipo II/genética , Proteínas de la Matriz Extracelular/análisis , Expresión Génica , Glicoproteínas/análisis , Enfermedades de los Caballos/patología , Caballos , Proteínas Matrilinas , ARN Mensajero/metabolismo , Traumatismos de los Tendones/metabolismo , Traumatismos de los Tendones/patologíaRESUMEN
Rheumatoid arthritis (RA) is a highly disabling disease affecting all structures of the joint. Understanding the pathology behind the development of RA is essential for developing targeted therapeutic strategies as well as for developing novel markers to predict disease onset. Several molecules normally hidden within the cartilage tissue are exposed to complement components in the synovial fluid upon cartilage breakdown. Some of these have been shown to activate complement and toll-like receptors, which may enhance an already existing inflammatory response, thereby worsening the course of disease. Other cartilage-resident molecules have in contrast shown to possess complement-inhibitory properties. Knowledge about mechanisms behind pathological complement activation in the joints will hopefully lead to methods which allow us to distinguish patients with pathological complement activation from those where other inflammatory pathways are predominant. This will help to elucidate which patients will benefit from complement inhibitory therapies, which are thought to aid a specific subset of patients or patients at a certain stage of disease. Future challenges are to target the complement inhibition specifically to the joints to minimize systemic complement blockade.
Asunto(s)
Artritis Reumatoide/inmunología , Activación de Complemento , Proteínas del Sistema Complemento/metabolismo , Matriz Extracelular/metabolismo , Artropatías/inmunología , Animales , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Cartílago/inmunología , Cartílago/patología , Proteína de la Matriz Oligomérica del Cartílago , Proteínas de la Matriz Extracelular/inmunología , Glicoproteínas/inmunología , Humanos , Inflamación/inmunología , Artropatías/metabolismo , Proteínas Repetidas Ricas en Leucina , Proteínas Matrilinas , Proteínas/inmunología , Proteínas/metabolismo , Líquido Sinovial/inmunología , Líquido Sinovial/metabolismo , Receptores Toll-Like/inmunologíaRESUMEN
The tensile and scaffolding properties of skin rely on the complex extracellular matrix (ECM) that surrounds cells, vasculature, nerves, and adnexus structures and supports the epidermis. In the skin, collagen I fibrils are the major structural component of the dermal ECM, decorated by proteoglycans and by fibril-associated collagens with interrupted triple helices such as collagens XII and XIV. Here we show that the cartilage oligomeric matrix protein (COMP), an abundant component of cartilage ECM, is expressed in healthy human skin. COMP expression is detected in the dermal compartment of skin and in cultured fibroblasts, whereas epidermis and HaCaT cells are negative. In addition to binding collagen I, COMP binds to collagens XII and XIV via their C-terminal collagenous domains. All three proteins codistribute in a characteristic narrow zone in the superficial papillary dermis of healthy human skin. Ultrastructural analysis by immunogold labeling confirmed colocalization and further revealed the presence of COMP along with collagens XII and XIV in anchoring plaques. On the basis of these observations, we postulate that COMP functions as an adapter protein in human skin, similar to its function in cartilage ECM, by organizing collagen I fibrils into a suprastructure, mainly in the vicinity of anchoring plaques that stabilize the cohesion between the upper dermis and the basement membrane zone.