RESUMEN
Recent advances in genomics resources and tools are facilitating quantitative trait locus mapping. We developed a crossbreed pedigree for mapping quantitative trait loci for hip dysplasia in dogs by crossing dysplastic Labrador Retrievers and normal Greyhounds. We show that one advantage to using a crossbreed pedigree is the increased marker informativeness in the backcross/F2 population relative to the founder populations. We also discuss three factors that affect the detection power in the context of this crossbreed pedigree: being able to detect and correct genotyping errors, increasing marker density for chromosomes with a sparse coverage, and adding individuals to the mapping population as soon as they become available.
Asunto(s)
Alelos , Displasia Pélvica Canina/genética , Repeticiones de Microsatélite/genética , Polimorfismo Genético/genética , Animales , Mapeo Cromosómico/métodos , Perros , Femenino , Hibridación Genética , Masculino , Linaje , Reproducibilidad de los Resultados , Proyectos de Investigación , Estadística como Asunto/métodosRESUMEN
The Affymetrix canine GeneChip with 23,836 probe sets was used to look for cartilage genes that are significantly altered in response to mechanical impact. The model using canine articular cartilage explants loaded in vitro has been described previously (Chen et al., J Orthop Res 19:703-711, 2001). It is our hypothesis that genes that are activated or repressed in articular cartilage after impact injury initiate cartilage degeneration, leading to osteoarthritis in dogs. Gene expression of known cartilage genes was generally consistent with cartilage biology. A total of 528 genes were significantly (P < .01) up- or down- regulated in response to mechanical damage. After applying the strict Bonferroni correction, 172 remained significantly affected. One of these genes, MIG-6/gene 33, was chosen for verification by real- time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). A 3.8- fold increase in expression was confirmed, consistent with the microarray chip data. Deficiencies in the current annotation of the canine chip are discussed. Gene expression studies with the Affymetrix canine GeneChip are potentially valuable, but await more complete annotation.
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Cartílago Articular/química , Cartílago Articular/lesiones , Perros/genética , Regulación de la Expresión Génica/genética , Animales , Análisis por Conglomerados , Perros/lesiones , Regulación hacia Abajo/genética , Femenino , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Regulación hacia Arriba/genéticaRESUMEN
An outcrossed canine pedigree was developed for quantitative trait locus (QTL) mapping of hip dysplasia by breeding dysplastic Labrador retrievers to trait-free greyhounds. Measured susceptibility traits included age at onset of femoral capital chondroepiphyseal ossification (OSS), maximum hip distraction (laxity) index (DI), and the dorsolateral subluxation (DLS) score. The pedigrees consisted of 147 dogs representing four generations. For 59 dogs genotyped with 65 microsatellites, the median heterozygosity and polymorphic information content (PIC) values of the F(1) generation were 0.82 and 0.68, respectively. Seventy-seven percent of microsatellites had a PIC greater than 0.59 in the F(1)s. Ninety-six percent of alleles showed Mendelian inheritance. Based on marker informativeness, approximately 350 randomly selected markers would be required for genome-wide screening to obtain an average interval between informative markers of 10 cM. Heritability was estimated as 0.43, 0.5, and 0.61 for OSS, DI, and the DLS score, respectively. Biometric estimates of the mean (+/- variance) effective number of segregating QTLs was 1.2 (+/- 0.05), 0.8 (+/- 0.02), and 1.0 (+/- 0.03) for OSS, DI, and the DLS score, respectively. The distributions of simulated backcross trait data suggested that the loci controlling these traits acted additively and that the DI may be controlled by a major locus. When combined with previous power and quantitative genetic analyses, these estimates indicate that this pedigree is informative for QTL mapping of hip dysplasia traits.
Asunto(s)
Perros/genética , Predisposición Genética a la Enfermedad , Displasia Pélvica Canina/genética , Repeticiones de Microsatélite , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , Femenino , Masculino , Linaje , Sitios de Carácter CuantitativoRESUMEN
OBJECTIVE: Small proteoglycans (PGs) may accumulate in late stage osteoarthritis even as aggrecan is lost. It is not clear what role transforming growth factor (TGF) beta has in this accumulation. Our goal was to investigate the ability of TGF beta 1 to modulate the synthesis and accumulation of decorin, biglycan, and fibromodulin in cartilage explants cultured under conditions in which aggrecan synthesis remains relatively constant. DESIGN: Articular cartilage was cultured in the presence or absence of 4 ng/ml TGF beta 1 for up to 16 days. Material extracted from cartilage was assayed for 35SO(4)-large and small PGs and for total endogenous decorin, biglycan and fibromodulin. RESULTS: The synthesis of 35SO(4)-small PGs increased during the 16 days in culture in response to TGF beta 1, but declined in control cultures. The difference in 35SO(4)-decorin between TGF beta 1 and control samples reached nine-fold after 16 days, while the difference in total endogenous decorin was less than 1.5-fold. 35SO(4)-decorin, which was present in TGF beta 1-treated cultures had an identical core protein, but a longer glycosaminoglycan chain than that of decorin in control cultures. No significant differences in endogenous biglycan were detected, but accumulation of fibromodulin in TGF beta 1 explants exceeded fibromodulin in controls, on average, by 3.8-fold. Fibromodulin was present in cartilage in both keratan sulfate- and non-sulfated oligosaccharide-substituted forms. CONCLUSIONS: The accumulation of each of the three small PGs was affected to a different extent in response to TGF beta 1. Of the three, fibromodulin content was most rapidly augmented in response to TGF beta 1.
Asunto(s)
Proteínas Portadoras/metabolismo , Cartílago Articular/efectos de los fármacos , Proteínas de la Matriz Extracelular , Osteoartritis/metabolismo , Proteoglicanos/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Animales , Autorradiografía , Biglicano , Western Blotting/métodos , Proteínas Portadoras/análisis , Proteínas Portadoras/biosíntesis , Cartílago Articular/metabolismo , Células Cultivadas , Decorina , Perros , Electroforesis en Gel de Poliacrilamida/métodos , Fibromodulina , Glicosilación , Mediciones Luminiscentes , Proteoglicanos/análisis , Proteoglicanos/biosíntesisRESUMEN
OBJECTIVE: To investigate the topographical variations among the morphological and physical properties of articular cartilage over a canine humeral head. DESIGN: Nine side-by-side specimens from a canine humeral head were used in the combined polarized light microscopy (PLM) and microscopic magnetic resonance imaging (muMRI). Sixteen images were acquired from each specimen in muMRI. Subsequently, ten to fourteen histological slices were prepared at the location of the muMRI slice from each specimen. Four optical images were acquired from each histological slice. Using these images, the images of the T(2) relaxation, the optical retardation, and the collagen-fiber orientation were constructed in two dimensions. RESULTS: Along the medial/lateral direction of the humeral head, we have observed a number of topographical variations. These include the total thickness of the cartilage tissue, the thickness of the individual histological zones, the depth of the minimum optical retardation in the tissue, and the depth of the maximum T(2) relaxation in the tissue. We found that the depth of the minimum retardation from PLM occurs at the geometric middle point of the transitional zone in histology, and that the depth of the maximum T(2) relaxation from muMRI is closely correlated with the minimum retardation depth in PLM. In addition, although the thickness of the total tissue increases approximately by the same amount on both the lateral and medial sides of the joint, a slight asymmetry exists between the measurements from the medial and lateral halves of the humerus. CONCLUSIONS: Cartilage from different sites over a single joint could have different properties, possibly the consequence of the variation in load-bearing status. Because of these topographical variations, one must keep in mind that a different sampling site within a relatively small area of the same joint may significantly influence the results of the study.
Asunto(s)
Cartílago Articular/anatomía & histología , Perros/anatomía & histología , Húmero/anatomía & histología , Articulación del Hombro/anatomía & histología , Animales , Femenino , Imagen por Resonancia Magnética/métodos , Microscopía de PolarizaciónRESUMEN
OBJECTIVE: To compare the accuracy of the extended-hip radiographic (EHR) score, the distraction index (DI), and the dorsolateral subluxation (DLS) score for identifying hip dysplasia in dogs at 8 months of age. DESIGN: Cohort study ANIMALS: 129 Labrador Retrievers, Greyhounds, and Labrador Retriever-Greyhound crossbreds. PROCEDURE: Radiography was performed when dogs were 8 months of age. Dogs were euthanatized at 8 to 36 months of age; hip dysplasia was diagnosed at the time of necropsy on the basis of results of a gross examination of the articular cartilage of the hip joints for signs of osteoarthritis. RESULTS: The EHR score, DI, and DLS score at 8 months of age were all significantly correlated with degree of cartilage degeneration at necropsy. Sensitivity and specificity of using EHR score at 8 months of age to diagnose hip dysplasia (scores > 3 were considered abnormal) were 38 and 96%, respectively; sensitivity and specificity of using DI (values > 0.7 were considered abnormal) were 50 and 89%; and sensitivity and specificity of using DLS score (scores < 55% were considered abnormal) were 83 and 84%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that specificities of the 3 methods for diagnosing hip dysplasia in dogs at 8 months of age were similar. However, the DLS score had higher sensitivity, indicating that there were fewer false-negative results.
Asunto(s)
Enfermedades de los Perros/diagnóstico por imagen , Displasia Pélvica Canina/diagnóstico por imagen , Articulación de la Cadera/diagnóstico por imagen , Osteoartritis/veterinaria , Animales , Estudios de Cohortes , Enfermedades de los Perros/diagnóstico , Enfermedades de los Perros/patología , Perros , Reacciones Falso Negativas , Femenino , Displasia Pélvica Canina/diagnóstico , Displasia Pélvica Canina/patología , Articulación de la Cadera/patología , Masculino , Oportunidad Relativa , Osteoartritis/diagnóstico , Osteoartritis/diagnóstico por imagen , Osteoartritis/patología , Radiografía , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Soporte de PesoRESUMEN
OBJECTIVE: To determine whether dorsolateral subluxation (DLS) scores in young dogs could be used to reliably predict which dogs would develop evidence of hip osteoarthritis and whether DLS scores measured at various ages correlated with each other. ANIMALS: 129 Labrador Retrievers, Greyhounds, and Labrador Retriever-Greyhound crossbreds. PROCEDURES: DLS scores were measured on radiographs taken at 4, 8, and 12 months of age and at necropsy (8 to 36 months of age). At necropsy, the hip joints were examined macroscopically and a score assigned for degree of cartilage degeneration. RESULTS: DLS scores at 4 (n = 35, r(s) = -0.62), 8 (n = 106, r(s) = -0.54), and 12 (n = 15, r(s) = -0.87) months of age were significantly correlated with cartilage degeneration scores, and DLS scores at 8 months of age were significantly correlated with scores obtained at the time of necropsy (n = 39, r(s) = 0.87). The DLS scores at 4 months of age were significantly different from scores at 8 months of age, but scores did not differ significantly thereafter. Likelihood ratios for cartilage lesions for low (< 45%), intermediate (> or = 45 but < or = 55%), and high (> 55%) DLS scores at 8 months of age were 8.0, 2.6, and 0.2, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that DLS score at 8 months of age was a reasonable, albeit imperfect, predictor of the condition of the hip joint cartilage at necropsy. Thus, the DLS method might be useful for early identification of dogs with hip dysplasia.
Asunto(s)
Cartílago Articular/patología , Enfermedades de los Perros/patología , Osteoartritis de la Cadera/veterinaria , Pelvis/diagnóstico por imagen , Animales , Enfermedades de los Perros/diagnóstico por imagen , Perros , Osteoartritis de la Cadera/diagnóstico por imagen , Osteoartritis de la Cadera/patología , Valor Predictivo de las Pruebas , Radiografía , Reproducibilidad de los Resultados , Estadísticas no ParamétricasRESUMEN
Experimental evidence suggests that recommended dosages of some corticosteroids used clinically as antiinflammatory agents for treating arthropathies damage articular cartilage, but low dosages may be chondroprotective. The purpose of this study was to evaluate how different concentrations of methylprednisolone affect chondrocyte function and viability. Articular cartilage and chondrocytes were obtained from young adult horses, 1.5-3.5 years of age. Corticosteroid-induced changes in collagen expression were studied at the transcriptional level by Northern blot analyses and at the translational level by measuring [3H]-proline incorporation into [3H]-hydroxyproline. Fibronectin mRNA splicing patterns were evaluated with ribonuclease protection assays. Cytotoxicity was studied using erythrosin B dye exclusion. Steady-state levels of type II procollagen mRNA decreased without concurrent changes in type I procollagen expression as the medium methylprednisolone concentrations were increased from 1 x 10(1) to 1 x 10(8) pg/ml, dropping below 10% of control values by 1 x 10(5) pg/ml. Cytotoxicity occurred as methylprednisolone levels were increased further from 1 x 10(8) to 1 x 10(9) pg/ml. Changes in total collagen (protein) synthesis were less pronounced, but also demonstrated significant suppression between 1 x 10(4) and 1 x 10(8) pg/ml. Corticosteroid-induced changes in fibronectin isoform levels were evaluated in articular cartilage samples without in vitro culture. The cartilage-specific (V + C)(-) isoform was suppressed in both normal and inflamed joints by a single intraarticular injection (0.1 mg/kg) of methylprednisolone. Combined, these data indicate that methylprednisolone suppresses matrix protein markers of chondrocytic differentiation. Decreased and altered chondrocyte expression of matrix proteins likely contributes to the pathogenesis of corticosteroid-induced cartilage degeneration.
Asunto(s)
Antiinflamatorios/farmacología , Cartílago Articular/citología , Condrocitos/citología , Condrocitos/efectos de los fármacos , Hemisuccinato de Metilprednisolona/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Expresión Génica/efectos de los fármacos , Caballos , Fenotipo , Procolágeno/genética , ARN Mensajero/análisisRESUMEN
A decrease in chondrocyte numbers is one characteristic of osteoarthritic cartilage. This decrease may be the result of apoptosis or other forms of cell death induced by mechanical damage. Furthermore, cell death may contribute to the structural and metabolic changes found in osteoarthritic cartilage. Therefore, we investigated cell viability and the mode of cell death in cartilage subjected to an increasing severity of impact loads expected to cause compositional damage and osteoarthritic-like metabolic alterations. Canine cartilage explants were subjected to cyclic indentation impacts of 5 megapascals at 0.3 Hz for 0, 2, 20, and 120 min and then kept in culture for 2, 4, 48, and 144 h. Cell death was assessed by the TUNEL assay and by uptake of propidium iodide. Viable cells were detected by the ability to metabolize fluorescein diacetate. Nuclear morphology and ultrastructure of the cell were examined using Hoechst 33342 fluorescent staining and transmission electron microscopy (TEM). As controls for necrosis and apoptosis, cartilage was, respectively, frozen and thawed or incubated with mitomycin-C, an apoptosis inducer. In cartilage that had been loaded for 2 h, 32% of the chondrocytes in the loaded core took up propidium iodide within 2 h after loading. Most of these were in the middle to superficial zones and reflected leaky cell membranes usually characteristic of necrosis. Less than 1% of these chondrocytes were positive in the TUNEL assay after 4 h. After additional culture for 2 days, however, the proportion of chondrocytes which were positive in the TUNEL assay reached 73%. A dose dependent response to duration of loading was detected with the TUNEL assay at this time. The TUNEL assay was not specific for apoptosis since 92% of chondrocytes in freeze/thawed cartilage were TUNEL positive. However, some cells with apoptotic bodies and chromatin condensation characteristic of apoptosis were found in the transition zone between necrotic and normal chondrocytes, but not in the superficial and upper zones, in impact damaged cartilage. We concluded that in this study, necrosis occurred first, followed by apoptosis.
Asunto(s)
Apoptosis , Cartílago Articular/lesiones , Cartílago Articular/patología , Condrocitos/patología , Animales , Núcleo Celular/ultraestructura , Supervivencia Celular , Células Cultivadas , Condrocitos/ultraestructura , Perros , Etiquetado Corte-Fin in Situ , Microscopía Electrónica , NecrosisRESUMEN
OBJECTIVE: To establish the correlation between the non-invasive imaging by magnetic resonance microscopy (microMRI) and the histological imaging by polarized light microscopy (PLM) accurately, quantitatively, at the highest possible MRI resolution (13.7 microm), and based on the same piece of tissue (articular cartilage from canine shoulder joint). DESIGN: In microMRI experiments, the laminar appearance (the magic angle effect) of the proton intensity images and the anisotropic characteristics of the T(2)relaxation images were analysed. In PLM experiments, the images of the optical retardation and collagen-fibre orientation in cartilage were constructed in two dimensions. RESULTS: The T(2)profile has a distinctly asymmetric bell-shaped curve and three featured zones. The retardation profile has a non-zero minimum at the middle of the transitional zone of the tissue. The angle profile has a smooth variation across the transitional zone. These facts suggest that the collagen fibres in the transitional zone are not entirely random but have a residual order. In addition, the peak of the T(2)profile coincides with the minimum of the retardation profile, both represent the most isotropic region of the tissue. A hyperbolic tangent function was found to best describe the transition of the collagen fibres in cartilage. A set of criteria was developed for each technique to define the features in the quantitative measurements. CONCLUSIONS: The criteria offer, for the first time, a set of quantitative and objective means to subdivide the tissue thickness into the zones in histology and in MRI. It is shown that the microMRI zones based on the T(2)characteristics are statistically equivalent to the histological zones based on the collagen fibre orientation (t-probabilities of 0.730, 0.973, 0.647, 0.850 for the superficial, transitional, radial zones and the total thickness).
Asunto(s)
Cartílago Articular/citología , Colágeno/ultraestructura , Animales , Birrefringencia , Perros , Femenino , Miembro Anterior , Articulaciones , Imagen por Resonancia Magnética/métodos , Imagen por Resonancia Magnética/normas , Microscopía de Polarización/métodos , Microscopía de Polarización/normasRESUMEN
UNLABELLED: Recently, in vitro cartilage studies have shown that impact loading can produce structural damage and osteoarthritis-like changes, including tissue swelling, collagen denaturation, and cell death. OBJECTIVE: This study was to determine whether a signal for cell death moves through the cartilage matrix, resulting in the spread of cell death over time from impacted to unimpacted regions. DESIGN: Cyclic impacts were applied to the 2 mm core of 4 mm cartilage discs. Post-impact culturing extended for 3, 6 or 21 days and occurred in one of two ways. In one, discs were cultured intact. In the second, cores were removed immediately after cessation of impact and cores and rings cultured separately. Cells in apoptosis and later stage necrosis were monitored using the TUNEL assay. RESULTS: The extent of cell death in impacted samples increased with increased duration of post-impact culturing. At the early time, the majority of cell death was located in the regions of direct impact whereas after extended culture, the extent of cell death was similar in the surrounding unimpacted regions and in the impacted core region. However, the physical separation of the impacted core from the surrounding, non-impacted ring regions immediately after impact, and prior to independent culture, kept the level of cell death in the surrounding ring close to control levels, even after 21 days of incubation. DISCUSSION: These findings indicate that soluble intercellular signalling is involved in the spreading of cell death through the cartilage matrix, and that its effects can be prevented by physical isolation of the surrounding ring from the impacted core.
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Cartílago Articular/citología , Comunicación Celular/fisiología , Análisis de Varianza , Animales , Cartílago Articular/fisiología , Técnicas de Cultivo de Célula/métodos , Muerte Celular/fisiología , Perros , Matriz Extracelular/fisiología , Glicosaminoglicanos/metabolismo , Etiquetado Corte-Fin in Situ , Microscopía Fluorescente , Óxido Nítrico/metabolismo , Espectrofotometría , Factores de Tiempo , Soporte de Peso/fisiologíaRESUMEN
Numerous in vitro culture models have been developed for the investigation of chondrocyte and cartilage biology. In this study, we investigated the stability of the chondrocytic phenotype in monolayer, aggregate, pellet, and explant culture models and assessed the effects of recombinant human bone morphogenetic protein 2 (rhBMP-2) and serum supplementation on the phenotype in each model. Phenotypic effects were assessed by analyses of procollagen type II, aggrecan, (V + C)- fibronectin, and procollagen type I messenger RNA expression. In monolayer cultures, we noted a characteristic loss of procollagen type II and induction of procollagen type I expression. The aggregate and pellet culture models supported matrix protein gene expression profiles more reflective of in vivo levels. In explant cultures, expression of matrix protein genes was consistently depressed. Treatment with rhBMP-2 significantly increased the expression of procollagen type II and aggrecan in monolayer cultures; however, other models showed comparatively little response. Similarly, serum supplementation significantly down-regulated procollagen type II and aggrecan expression in monolayer cultures but had less effect on gene expression in the other models. Serum supplementation increased procollagen type I expression in monolayer and aggregate cultures. These results suggest that the influence of exogenous BMP-2 and serum on expression of chondrocyte-specific matrix protein genes is influenced by aspects of substrate attachments, cellular morphology, and/or cytoskeletal organization. Finally, the analyses of fibronectin expression suggest that V and C region alternative splicing in chondrocytes is linked to the establishment of a three-dimensional multicellular complex.
Asunto(s)
Sangre , Proteínas Morfogenéticas Óseas/farmacología , Cartílago Articular/citología , Condrocitos/citología , Proteínas de la Matriz Extracelular , Factor de Crecimiento Transformador beta , Agrecanos , Animales , Secuencia de Bases , Proteína Morfogenética Ósea 2 , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Cartilla de ADN , Fibronectinas/genética , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Caballos , Humanos , Lectinas Tipo C , Modelos Biológicos , Fenotipo , Procolágeno/genética , Procolágeno/metabolismo , Proteoglicanos/genética , Proteoglicanos/metabolismo , ARN Mensajero/genética , Proteínas Recombinantes/farmacologíaRESUMEN
OBJECTIVE: To determine which characteristics of cartilage lesion pathology are detected in dogs at high risk to develop osteoarthritis prior to diagnosis by standard radiographs or macroscopic cartilage abnormality on necropsy. METHODS: Fourteen disease-free dogs were assigned to risk groups based on hip distraction index. For seven dogs, three dimensional images of hip joints from computed tomography were available. At necropsy, ligamentum capitis femoris volumes were measured and articular cartilage was harvested and analyzed for percent water, swelling, glycosaminoglycan, and fibronectin. Comparisons were made with nine dogs with macroscopic cartilage lesions (OA group). RESULTS: Ligament volumes were greater in the high distraction index group (P=0.000). Water content was elevated in the lesion area in both low and high risk dogs (P=0.000); no additional increase was noted in the high risk group. Glycosaminoglycan content was slightly elevated in the surrounding area in both groups (P< 0.02) but loss was noted histologically in the lesion area of the high risk group. Fibronectin was increased in the lesion area and in the high risk group (P=0.000). The magnitude of this increase was greatest in the lesion area (P=0.000) in explants. Computed tomography indicated dorsal acetabular rim impingement on the lesion area in high risk dogs. CONCLUSIONS: Water content and swelling suggest matrix structure is weaker at the site of lesion predilection in all dogs regardless of risk status. Computed tomography imaging is consistent with site specific initiation of lesions by mechanical factors.
Asunto(s)
Cartílago Articular/metabolismo , Enfermedades de los Perros/diagnóstico por imagen , Articulación de la Cadera/diagnóstico por imagen , Osteoartritis de la Cadera/veterinaria , Animales , Agua Corporal/metabolismo , Cartílago Articular/patología , Técnicas de Cultivo , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/patología , Perros , Cabeza Femoral/diagnóstico por imagen , Fibronectinas/análisis , Glicosaminoglicanos/análisis , Displasia Pélvica Canina/complicaciones , Displasia Pélvica Canina/diagnóstico por imagen , Displasia Pélvica Canina/patología , Osteoartritis de la Cadera/diagnóstico por imagen , Osteoartritis de la Cadera/metabolismo , Factores de Riesgo , Tomografía Computarizada por Rayos XRESUMEN
Fibronectin is an extracellular-matrix glycoprotein encoded by a single gene, but with significant protein heterogeneity introduced through alternative RNA splicing and post-translational modifications. The (V+C)(-) splice variant, in which nucleotides encoding protein segments III-15 and I-10 are deleted along with the entire variable region, is unique in that expression is restricted to cartilaginous tissues. All known fibronectin splice variants retain the two C-terminal cysteine residues essential for dimerization, but cellular and/or structural constraints appear to influence homo- and heterodimerization patterns. Dimerization patterns of the (V+C)(-) isoform were studied under native conditions within canine articular cartilage and experimentally in COS-7, NIH-3T3 and CHO-K1 cell cultures. In all systems, (V+C)(-) fibronectin secretion was predominantly in a homodimeric configuration. Lower levels of (V+C)(-) monomers were also present. Heterodimers of (V+C)(-) with V(+),C(+) (V120) isoforms were not detected. Heterodimers of (V+C)(-) with V(-),C(+) (V0) subunits were detected only at low levels. Functional properties may differ significantly among monomers, homodimers and heterodimers. The unique dimerization pattern of (V+C)(-) fibronectin is consistent with this isoform having specialized functional properties in situ that are important for either the structural organization and biomechanical properties of cartilage matrix or regulation of a chondrocytic phenotype.
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Cartílago Articular/metabolismo , Fibronectinas/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Línea Celular , Dimerización , Perros , Fibronectinas/química , Isoformas de Proteínas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Canine hip dysplasia (CHD) is a prevalent, debilitating, polygenic disease characterized by hip subluxation and laxity which results in osteoarthritis. We are developing an informative pedigree for linkage analysis of CHD. The seven greyhound founders had excellent hip conformation with high dorsolateral subluxation scores (percentage of femoral head covered by the dorsal acetabulum in a weight-bearing position) of 66 +/- 4% (mean +/- SD averaged over both hips) and low hip distraction (laxity) indices of 0.14 +/- 0.08. Nine greyhounds bred on site had radiographic evidence of ossification in the capital femoral chondroepiphysis at 7.7 +/- 0.9 days of age. At 8 months of age they had a mean distraction index of 0.24 +/- 0.08 and dorsolateral subluxation score of 76 +/- 1%. Of the four dysplastic Labrador retriever founders, three had mean age at onset of capital femoral chondroeplphyseal ossification of 20 +/- 7 days of age n = 3) and a mean distraction index of 0.46 +/- 0.1 accompanied by hip osteoarthritis. Thirty-four F1s had mean onset of capital femoral ossification (10.7 +/- 4.0 days of age) and mean dorsolateral subluxation scores (61 +/- 12%) similar to the greyhound founders, but distraction indices (0.42 +/- 0.2) more similar to the Labrador retriever founders. One F1 had CHD radiographically but none of 20 F1s had osteoarthritis at necropsy at 10 months of age. These data suggested that maximum passive laxity (as measured by the distraction index) and normal osseous conformation (as indicated by a high dorsolateral subluxation score) were both dominant traits and were controlled by separate quantitative trait loci (QTL). Forty-three back-crosses between F1s with the highest hip laxity and greyhound founders had mean onset of capital femoral ossification at 9.9 +/- 2.6 days of age. Of 10 dogs in the backcross generation that have reached 8 months of age, 2 had palpable subluxation without marked CHD radiographically. The mean distraction index of these dogs was 0.36 +/- 0.16 and the dorsolateral subluxation score was 65 +/- 5%. Although dogs in the backcross generation that were three-quarter greyhound had a broad range of hip laxity, a protective effect of the greyhound QTLs for good osseous conformation has mitigated thus far against subluxation and CHD.
Asunto(s)
Ligamiento Genético , Displasia Pélvica Canina/genética , Animales , Perros , Femenino , Displasia Pélvica Canina/diagnóstico por imagen , Masculino , Linaje , Fenotipo , Carácter Cuantitativo Heredable , RadiografíaRESUMEN
The first objective of this study was to determine if the cumulative effects of impact or smoothly arising compression would damage the matrix of articular cartilage. Canine cartilage explants were subjected to repeated impacts or to smoothly arising compressions of as much as 20 MPa at 0.3 Hz for as long as 120 minutes. An increase in the water content of the loaded core compared with the surrounding ring was considered indicative of matrix damage. The results showed that damage to cartilage required repeated impacts with a peak stress of at least 2.5 MPa and a stress rate of at least 30 MPa/sec for 2 minutes or longer. This suggested that impact damage is cumulative and stress-rate dependent. The second objective was to identify biosynthetic and compositional changes in impact-damaged cartilage over a period of time after loading. Accordingly, canine cartilage explants were subjected to repetitive impacts of 5 MPa at 0.3 Hz for 2, 20, and 120 minutes. The loaded explants were then cultured for as long as 10 days. The increase in water content (1.9-3.8%) in the core region relative to the surrounding ring persisted during the 10-day culture. A significant increase in fibronectin synthesis (22-47%) was found in the core region of impact-damaged cartilage. Proteoglycan synthesis was increased by 41-104%. An increase in denatured collagens (11-70%) in the loaded cores substantiated damage to the collagen network. Denatured collagens stained with COL2-3/4m monoclonal antibody were consistent with the compositional findings and were mainly located near the articular surface and in the deep zone. These changes were consistent with early osteoarthritis and suggested the induction of the initial stages of osteoarthritis in the impact-damaged cartilage.
Asunto(s)
Cartílago/patología , Animales , Cartílago/química , Cartílago/metabolismo , Colágeno/análisis , Perros , Fibronectinas/biosíntesis , Glicosaminoglicanos/biosíntesis , Inmunohistoquímica , Osteoartritis/etiología , Estrés Mecánico , Factores de Tiempo , Agua/análisisRESUMEN
This study examined fetal chondrocyte proliferation and function following exposure to transforming growth factor-beta and insulin-like growth factor-I. Fetal equine articular chondrocytes of the early third-trimester were isolated and cultured in monolayer conditions, then exposed to 0, 1, 5, or 10 ng/ml transforming growth factor-beta or 0, 10, 50, or 100 ng/ml insulin-like growth factor-I for 48 hours. Proliferative responses were assessed by cell counts and [3H]thymidine uptake into precipitable DNA. Differentiated chondrocyte metabolic activity was determined by sulfated glycosaminoglycan quantitation, 35[SO4] incorporation into precipitable glycosaminoglycan, and proteoglycan molecular sizing by CL-2B column chromatography. Morphological changes seen on phase-contrast microscopy included a larger proportion of rounded cells in monolayer cultures supplemented with insulin-like growth factor-I and cytotoxic changes in cells treated with transforming growth factor-beta. Both insulin-like growth factor-I and transforming growth factor-beta resulted in significant elevations of [3H]thymidine uptake; however, cell numbers did not rise sufficiently over the 48-hour culture period to reach significant levels. Maximum mitogenic responses were evident at 50 and 100 ng/ml insulin-like growth factor-I and 5 ng/ml transforming growth factor-beta. The production of proteoglycan was also enhanced (435%) by exposure to 50 ng/ml insulin-like growth factor-I, and an increased proportion of larger proteoglycan monomer species was evident in cultures treated with 50 and 100 ng/ml insulin-like growth factor-I. A similar dose-response was also evident in cultures treated with transforming growth factor-beta (maximal 164% increase with 5 ng/ml), although the presence of serum in the culture medium altered the pattern of enhanced proteoglycan synthesis to favor the lower concentration of 1 ng/ml (191%). Additionally, larger proteoglycan molecules were synthesized in response to high concentrations of transforming growth factor-beta in serum-free cultures. Significant biochemical changes resulted from the addition of transforming growth factor-beta to fetal chondrocyte cultures; however, monolayer cultures that were treated with transforming growth factor-beta and supplemented with serum began to develop cellular toxicity, including nuclear pyknosis and cytoplasmic fragmentation. Degenerative cellular changes were not evident in cultures treated with insulin-like growth factor-I, and significant differentiated metabolic activity resulted from the presence of insulin-like growth factor-I in the culture medium. These data suggest that the responses of fetal chondrocytes to insulin-like growth factor-I and transforming growth factor-beta were enhanced compared with the responses of chondrocytes derived from postnatal animals and that these metabolically active cells can be primed by endogenous or exogenous growth factors to provide enhanced articular function and repair.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Condrocitos/citología , Factor I del Crecimiento Similar a la Insulina/farmacología , Factor de Crecimiento Transformador beta/farmacología , Animales , Cartílago/citología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , División Celular/fisiología , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Relación Dosis-Respuesta a Droga , Equidae , Feto/citología , Glicosaminoglicanos/biosíntesis , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Peso Molecular , Proteoglicanos/biosíntesis , Proteoglicanos/química , Proteoglicanos/metabolismo , Radioisótopos de AzufreRESUMEN
Fibronectin is encoded by a single gene, but heterogeneity is introduced by alternative splicing of the pre-mRNA. An unique splice variant, designated (V+C)-, which deletes nucleotides encoding the V, III-15 and I-10 segments, has been identified in articular cartilage. In this study, a ribonuclease protection assay was used to quantitate expression of the (V+C)- isoform in eight canine cartilaginous tissues and in chondrocytes cultured as monolayers or in alginate beads. The (V+C)- fibronectin isoform was detected in all cartilaginous tissues examined, ranging from a low of 11% of steady-state fibronectin mRNA in the nucleus pulposus to 71% in the rib. An age dependent increase, from 18% in the epiphyseal cartilage of a newborn to 54% in the articular cartilage of dogs over 10 months of age, was observed. The ubiquitous presence of this isoform in cartilaginous tissues and its absence in all non-cartilaginous tissues examined to date is consistent with a very strong association of the (V+C)- fibronectin isoform with the cartilaginous phenotype. Results from a ribonuclease protection assay using a probe extending into the V region from III-14 were combined with the quantitative information about (V+C)- fibronection expression to develop an over-all profile of splicing within the V region in cartilage. Monolayer culture of articular chondrocytes altered fibronectin splicing patterns. The (V+C)- isoform was rapidly lost and ED-A(+) fibronectin was induced. Three-dimensional culture in alginate beads prevented induction of ED-A(+) fibronection, but failed to sustain expression of the (V+C)- isoform. Thus, some matrix component or structure, lost in cell culture, may be essential to maintain expression of the (V+C)- isoform. The possible relationship of changing patterns of fibronectin isoforms in cultured chondrocytes to maintenance of the differentiated phenotype is discussed.
Asunto(s)
Cartílago/metabolismo , Matriz Extracelular/metabolismo , Fibronectinas/biosíntesis , Isoformas de Proteínas/biosíntesis , Empalme Alternativo , Animales , Cartílago/citología , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Perros , Fibronectinas/genética , Isoformas de Proteínas/genéticaRESUMEN
To understand the origin of the laminated appearance of cartilage in MRI (the magic angle effect), microscopic MRI (mu MRI) experiments were performed at 14-microns pixel resolution on normal canine articular cartilage from the shoulder joints. Two-dimensional images of the spin-spin relaxation time (T2) of the cartilage-bone plug at two angles (0 degree and 55 degrees) were calculated quantitatively. A distinct T2 anisotropy was observed as a function of the cartilage tissue depth. The surface and the deep regions exhibit strong orientational dependence of T2, whereas the upper-middle region exhibits little orientational dependence of T2. These three mu MRI regions correspond approximately to the three histologic zones in cartilage tissue. The results from the bulk T2 measurements agreed with these mu MRI results. Our studies show that the laminated appearance of cartilage in MRI is caused by T2 anisotropy of the tissue. We further suggest that the molecular origin of the T2 anisotropy is the nuclear dipolar interaction. The structure of the cartilage tissue indicates that the collagen meshwork defines this T2 anisotropy. The results show that the T2 anisotropy provides an indirect but sensitive indicator for the orientation of macromolecular structures in cartilage. The clinical implications of this anisotropy are discussed.
Asunto(s)
Membrana Basal/anatomía & histología , Cartílago Articular/anatomía & histología , Imagen por Resonancia Magnética , Animales , Técnicas de Cultivo , Modelos Animales de Enfermedad , Perros , Valores de Referencia , Sensibilidad y Especificidad , Articulación del HombroRESUMEN
OBJECTIVE: To determine whether onset of mineralization of the femoral and proximal tibial epiphyses and age at closure of the femoral and acetabular triradiate growth plates was different for Labrador Retrievers that were radiographically normal or that had canine hip dysplasia (CHD). DESIGN: Cohort study. ANIMALS: 36 Labrador Retriever puppies. PROCEDURE: Puppies were radiographed every other day from the time they were 8 to 10 days old until ossification of the femoral heads was apparent. Radiographs were then obtained weekly until puppies were 1 month old and then monthly until puppies were 8 to 12 months old. Age at which mineralization was first observed in the proximal and distal femoral and proximal tibial epiphyses and at which the femoral capital, triradiate acetabular, and distal femoral growth plates were no longer radiographically visible were recorded. Fifteen dogs were euthanatized and necropsied to determine whether dogs had CHD. RESULTS: There were 26 radiographically normal left and right hip joints and 10 dysplastic left and right hip joints. Onset of mineralization of the proximal femoral epiphyses and of the right proximal tibial epiphysis was significantly later in dysplastic than in radiographically normal puppies. The left femoral capital growth plates closed significantly later in dysplastic than in radiographically normal joints, but other differences in growth plate closure were not detected. CLINICAL IMPLICATIONS: Endochondral ossification may be abnormal in dogs with CHD. The disease appears to affect multiple joints, even though it is most evident clinically in the hip joint.