RESUMEN
This study demonstrates the structure-activity relationship of Col-003, a potent collagen-heat-shock protein 47 (Hsp47) interaction inhibitor. Col-003 analogues were successfully synthesized by Pd(0)-catalyzed cross-coupling reactions of 5-bromosalicylaldehyde derivatives with alkyl-metal species, and the inhibitory activities of the synthetic analogues were evaluated using surface plasmon resonance analysis (BIAcore). We succeeded in discovering two potent inhibitors that showed 85 and 81% inhibition at a concentration of 1.9 µM against the collagen-Hsp47 interaction. This indicates that elongation of an alkyl linker between two aromatic rings could considerably improve inhibitory activity due to the adjustment of a pendant phenyl moiety to an appropriate position, in addition to the hydrophobic interaction with an alkyl linker moiety.
Asunto(s)
Aldehídos/química , Colágeno/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Aldehídos/síntesis química , Aldehídos/farmacología , Animales , Catálisis , Colágeno/antagonistas & inhibidores , Proteínas del Choque Térmico HSP47/antagonistas & inhibidores , Paladio/química , Mapas de Interacción de Proteínas/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Resonancia por Plasmón de SuperficieRESUMEN
Molecular chaperones perform pivotal roles in proteostasis by engaging in protein-protein interactions (PPIs). The collagen-specific molecular chaperone Hsp47 (heat shock protein 47) interacts with procollagen in the endoplasmic reticulum (ER) and plays crucial roles in collagen synthesis. PPIs between Hsp47 and collagen could offer a therapeutic target for fibrosis, which is characterized by abnormal collagen accumulation in the extracellular matrix of fibrotic organs. Herein, we established a bioluminescence resonance energy transfer (BRET) system for assessing Hsp47-collagen interaction dynamics within the ER. After optimization and validation of the method, we could demonstrate inhibition of the interaction between Hsp47 and collagen by a small molecule (Col003) in the ER. Using the BRET system, we also found that Hsp47 interacts not only with the Gly-Pro-Arg motif but also weakly with Gly-Pro-Hyp motifs of triple-helical collagen in cells. Moreover, we found that the serpin loop of Hsp47 (SerpinH1) contributes to its binding to collagen. We propose that the method developed here can provide valuable information on PPIs between Hsp47 and collagen and on the effects of PPI inhibitors important for the management of fibrotic disorders.
Asunto(s)
Colágeno/metabolismo , Proteínas del Choque Térmico HSP47/metabolismo , Sitios de Unión , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Colágeno/química , Retículo Endoplásmico/metabolismo , Células HEK293 , Proteínas del Choque Térmico HSP47/antagonistas & inhibidores , Proteínas del Choque Térmico HSP47/química , Humanos , Unión ProteicaRESUMEN
Glucocorticoids and hypoxia is considered to promote osteocyte apoptosis and necrosis, which are observed in glucocorticoid-associated osteonecrosis and osteoporosis. Heme oxygenase-1 (HO-1) induced by hemin is reported to have cytoprotective effects in ischemic diseases. The objective of this study was to evaluate the effect of HO-1 on osteocyte death caused by glucocorticoids and hypoxia. We confirmed that hemin induced HO-1 expression in MLO-Y4 mouse osteocytes. MLO-Y4 was cultured with dexamethasone (Dex) under hypoxia (DH group). Furthermore, these cells were cultured with hemin (DH-h group) or hemin and zinc protoporphyrin IX (an HO-1 inhibitor) (DH-h-PP group). The rates of apoptosis and necrosis of these groups were analyzed by flow cytometry and compared with cells cultured under normal condition. Both apoptosis and necrosis increased in the DH group. Hemin administration significantly reduced cell death caused by glucocorticoids and hypoxia in the DH-h group, and its effect was attenuated by the HO-1 inhibitor in DH-h-PP group. Capase-3 activity significantly decreased in the DH-h group. This implied that the cell death inhibition effect due to hemin is mediated by HO-1 and caspase-3. HO-1 induction may be useful in the treatment of glucocorticoid-associated osteonecrosis and osteoporosis.
Asunto(s)
Apoptosis , Hemo-Oxigenasa 1/metabolismo , Osteocitos/patología , Animales , Glucocorticoides/farmacología , Hemo-Oxigenasa 1/fisiología , Hipoxia , Ratones , Osteocitos/enzimología , Osteocitos/metabolismo , Osteoporosis/enzimología , Osteoporosis/metabolismo , Osteoporosis/fisiopatologíaRESUMEN
This study aimed to evaluate femoral perfusion using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) for two weeks after the simultaneous initiation of electrical stimulation (ES) and steroid treatment in a steroid-induced osteonecrosis (ON) model. A single dose of methylprednisolone was injected into 14 rabbits. Seven rabbits underwent ES (ES group), and seven rabbits did not (control group). DCE-MRI was performed before steroid administration and 1, 5, 10, and 14 days after steroid administration. Regions of interest were set in the bilateral proximal femora. The enhancement ratio, initial slope, and area under the curve were analyzed. These parameters were evaluated after steroid administration in each group and between the two groups, and the ratios of ON in both groups were compared. In the control group, the minimum values of all parameters decreased significantly after steroid administration (P < 0.05), but in the ES group, the parameters did not decrease. In the ES group, all parameter values were significantly increased on the 10th and 14th days (P < 0.05). All parameter values in the ES group were significantly higher than those in the control group on the 14th day (P < 0.05). In the control group, ON was detected in three of five rabbits (in three of ten femora). In the ES group, ON was not detected. These results suggest that increased femoral blood flow elicited by ES may be related to ON prevention after steroid administration.
Asunto(s)
Medios de Contraste , Estimulación Eléctrica , Fémur/irrigación sanguínea , Imagen por Resonancia Magnética , Osteonecrosis/fisiopatología , Flujo Sanguíneo Regional , Esteroides/efectos adversos , Animales , Modelos Animales de Enfermedad , Fémur/diagnóstico por imagen , Fémur/efectos de los fármacos , Fémur/patología , Masculino , Osteonecrosis/inducido químicamente , Osteonecrosis/diagnóstico por imagen , Conejos , Flujo Sanguíneo Regional/efectos de los fármacos , Factores de TiempoRESUMEN
BACKGROUND: Steroid (glucocorticoid)-induced osteonecrosis of the femoral head (ONFH) in young adults has been a challenging disorder due to frequent incidence of collapse of the femoral head and resulting dysfunction of the hip joint and impairing quality of life. In Japan, the working group on ONFH in the Specific Disease Investigation Committee under auspices of the Japanese Ministry of Health, Labor and Welfare was founded in 1975, clinical and related basic research on ONFH have been continued for more than 40 years. EPIDEMIOLOGY AND CLINICAL COURSE: A national epidemiologic survey in 2004 estimated that 2200 new patients per year would be diagnosed with ONFH in Japan. ONFH was associated with steroid intake (51%), heavy alcohol intake (31%), both (3%), and neither (15%). The male-to-female ratio was 5:4, and the peak decades of age at definitive diagnosis were the 40s in male patients and the 30s in females. MRI studies revealed that ONFH would have occurred in early phase after start of steroid administration and no expansion of necrotic lesion within the femoral head in spite of continued steroid use. To standardize ONFH diagnosis and treatment strategy, the Committee established validated diagnostic criteria, a radiological staging system, and type categorization. TREATMENT OPTIONS: Most symptomatic patients with collapse of the femoral head require various surgical procedures. Joint preserving surgery, such as transtrochanteric rotational osteotomy and curved varus osteotomy, should be the treatment choice for young patients with healthy areas without severe collapse of the femoral head. CLINICAL AND RELATED BASIC RESEARCH: Clinical and basic research has been performed to determine the pathogenesis of steroid-induced ONFH. Low hepatic CYP3A activity has been reported to significantly contribute to the risk of steroid-induced ONFH. Several gene polymorphisms related to steroid metabolism were shown to be associated with the occurrence of ONFH.
Asunto(s)
Necrosis de la Cabeza Femoral , Glucocorticoides/efectos adversos , Necrosis de la Cabeza Femoral/epidemiología , Necrosis de la Cabeza Femoral/patología , Necrosis de la Cabeza Femoral/terapia , Humanos , Japón/epidemiología , InvestigaciónAsunto(s)
Alcoholismo/complicaciones , Artroplastia de Reemplazo de Cadera/métodos , Necrosis de la Cabeza Femoral/etiología , Necrosis de la Cabeza Femoral/cirugía , Adulto , Artralgia/diagnóstico , Artralgia/etiología , Biopsia con Aguja , Femenino , Necrosis de la Cabeza Femoral/diagnóstico por imagen , Estudios de Seguimiento , Articulación de la Cadera , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética/métodos , Masculino , Dimensión del Dolor , Cuidados Posoperatorios/métodos , Rango del Movimiento Articular/fisiología , Índice de Severidad de la Enfermedad , Factores de Tiempo , Tomografía Computarizada por Rayos X/métodos , Resultado del TratamientoRESUMEN
BACKGROUND: Differentiated thyroid cancer (DTC) is relatively rare and can metastasize to both the lungs and bones. The great majority of bone metastases occur in red marrow regions where blood flow is high. Only one patient has been described with direct DTC metastasis to the subchondral bone of the femoral head. CASE PRESENTATION: The patient was a 68-year-old Japanese female who had presented with left hip joint pain at age 63 years. At age 51 years, she had been diagnosed with DTC and underwent partial excision. X-rays showed partial femoral head collapse, suggesting osteoarthritis or idiopathic necrosis of the left femoral head. Three years later, a (131) I whole-body scan showed accumulation in the left femoral head, resulting in a diagnosis of DTC metastasis to the left femoral head. Bipolar hip arthroplasty was performed. Examination of the excised femoral head resulted in a final diagnosis of metastasis of follicular thyroid cancer, which was limited histopathologically to the subchondral bone of the femoral head. CONCLUSION: Tumor metastasis to the subchondral bone of the femoral head is exceedingly rare. Overall survival of patients with bone metastasis is improved by complete resection. Differential diagnosis of patients with a previous history of DTC who present with femoral head collapse should include bone metastasis of DTC.
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Adenocarcinoma Folicular/secundario , Neoplasias Femorales/secundario , Cabeza Femoral/patología , Neoplasias de la Tiroides/patología , Anciano , Femenino , Cabeza Femoral/diagnóstico por imagen , Humanos , RadiografíaRESUMEN
This study was designed to evaluate femoral perfusion after pulsed electromagnetic field (PEMF) stimulation in a steroid-induced osteonecrosis rabbit model by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Steroid-induced osteonecrosis was produced by single intramuscular injection of methylprednisolone in 15 rabbits. Eight rabbits underwent PEMF stimulation (PEMF group) and seven did not (control group). DCE-MRI was performed before PEMF stimulation, immediately before steroid administration, and 1, 5, 10, and 14 days after steroid administration. Regions of interest were set in the bilateral proximal femora. Enhancement ratio (ER), initial slope (IS), and area under the curve (AUC) were analyzed. ER, IS, and AUC in the control group significantly decreased after steroid administration compared with before administration (P<0.05). In PEMF group, IS significantly decreased; however, ER and AUC showed no significant differences after steroid administration compared with before. ER and IS in PEMF group were higher than in control group until 10th day, and AUC was higher until 5th day after steroid administration (P<0.05). PEMF stimulation restrains the decrease in blood flow after steroid administration.
Asunto(s)
Terapia por Estimulación Eléctrica/métodos , Fémur/irrigación sanguínea , Fémur/fisiopatología , Osteonecrosis/fisiopatología , Osteonecrosis/terapia , Animales , Área Bajo la Curva , Modelos Animales de Enfermedad , Imagen por Resonancia Magnética , Masculino , Metilprednisolona , Conejos , Flujo Sanguíneo Regional , Factores de Tiempo , Resultado del TratamientoRESUMEN
PURPOSE: To evaluate perfusion during the early phase after steroid administration in vivo using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with a high magnetic field MRI system. The main pathogenesis of steroid-induced osteonecrosis is considered to be ischemia. MATERIALS AND METHODS: A single dose of methylprednisolone (MPSL) was injected into nine rabbits. DCE-MRI was performed for these rabbits before MPSL administration and 1, 5, 10, and 14 days after administration. Time-signal intensity curves were created for each femur based on the signal intensity to evaluate perfusion. Enhancement ratio (ER), initial slope (IS), and area under the curve (AUC) were calculated and the value before MPSL administration and the minimal value after administration were compared statistically. RESULTS: ER, IS, and AUC values after MPSL administration significantly decreased (P < 0.05, P < 0.01, and P < 0.01, respectively). All of them decreased by the 5th day in 56% of the femora and by the 14th day in 83%, and some femora even showed a decrease from the 1st day. CONCLUSION: In this study, decreased perfusion in the femora after steroid administration was proven. Additionally, we could show that it occurred from the early days after steroid administration.
Asunto(s)
Velocidad del Flujo Sanguíneo , Arteria Femoral/fisiopatología , Angiografía por Resonancia Magnética/métodos , Metilprednisolona , Osteonecrosis/inducido químicamente , Osteonecrosis/fisiopatología , Algoritmos , Animales , Medios de Contraste , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Masculino , Osteonecrosis/patología , Conejos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EsteroidesRESUMEN
BACKGROUND AND PURPOSE: It has been suggested that avascular osteonecrosis (AVN) of the femoral head occurs early after systemic steroid administration. The purpose of this study was to investigate the risks regarding development of AVN at a very early stage after renal transplantation. METHODS: The presence or absence of AVN was determined by MRI at 4 weeks, at 6-12 weeks, at 24 weeks, and at 12 months after renal transplantation in 286 patients (183 males) with a mean age of 39 (16-65) years. The relationship between AVN and age, sex, absence or presence of acute rejection (AR), type of transplanted kidney (living or cadaveric), type of immune suppressor, and total dose of orally administered steroids given in the 2-week period after transplantation was investigated. RESULTS: There were no statistically significant correlations between the development of AVN and age, sex, absence or presence of AR, type of transplanted kidney, or type of immune suppressor. A significant dose-response relationship was found between development of AVN and the total dose of steroid administered in the first 2 weeks after surgery. INTERPRETATION: We found a relationship between AVN development and steroid dose in the early postoperative period, and we also showed a dose-response relationship.
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Corticoesteroides/efectos adversos , Necrosis de la Cabeza Femoral/diagnóstico , Necrosis de la Cabeza Femoral/epidemiología , Trasplante de Riñón , Periodo Posoperatorio , Administración Intravenosa , Administración Oral , Adolescente , Corticoesteroides/administración & dosificación , Adulto , Anciano , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Necrosis de la Cabeza Femoral/patología , Humanos , Incidencia , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Factores de Tiempo , Adulto JovenRESUMEN
The goal of this feasibility study was to examine whether sonoporation assisted transduction of siRNA could be used to ameliorate arthritis locally. If successful, such approach could provide an alternative treatment for the patients that have or gradually develop adverse response to chemical drugs. Tumor necrosis factor alpha (TNF-α) produced by synovial fibroblasts has an important role in the pathology of rheumatoid arthritis, inducing inflammation and bone destruction. In this study, we injected a mixture of microbubbles and siRNA targeting TNF-α (siTNF) into the articular joints of rats, and transduced siTNF into synovial tissue by exposure to a collimated ultrasound beam, applied through a probe 6mm in diameter with an input frequency of 3.0 MHz, an output intensity of 2.0 W/cm(2) (spatial average temporary peak; SATP), a pulse duty ratio of 50%, and a duration of 1 min. Sonoporation increased skin temperature from 26.8 °C to 27.3 °C, but there were no adverse effect such as burns. The mean level of TNF-α expression in siTNF-treated knee joints was 55% of those in controls. Delivery of siTNF into the knee joints every 3 days (i.e., 7, 10, 13, and 16 days after immunization) by in vivo sonoporation significantly reduced paw swelling on days 20-23 after immunization. Radiographic scores in the siTNF group were 56% of those in the CIA group and 61% of those in the siNeg group. Histological examination showed that the number of TNF-α positive cells was significantly lower in areas of pannus invasion into the ankle joints of siTNF- than of siNeg-treated rats. These results indicate that transduction of siTNF into articular synovium using sonoporation may be an effective local therapy for arthritis.
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Artritis/genética , Artritis/terapia , Electroporación/métodos , Terapia Genética/métodos , ARN Interferente Pequeño/administración & dosificación , Sonicación/métodos , Transfección/métodos , Animales , Artritis/patología , Silenciador del Gen , Masculino , Ratas , Resultado del TratamientoRESUMEN
A heterozygous patient with dysfibrinogenemia with slight bleeding and no thrombotic complications was diagnosed with fibrinogen Kyoto VI (K-VI). To elucidate the genetic mutation(s) and characterize the variant protein, we performed the following experiments and compared with identical and similar variants that have already been reported. The proposita's PCR-amplified DNA was analyzed by sequencing and her purified plasma fibrinogen underwent SDS-PAGE followed by immunoblotting, fibrin polymerization, and scanning electron microscopic observation of fibrin clot and fibers. Sequence analyses showed that K-VI fibrinogen substituted W (TGG) for terminal codon (TAG), resulting in 12 amino acid elongation 462-473 (WSPIRRFLLFCM) in the Bß-chain. Protein analyses indicated that the presence of some albumin-binding variant fibrinogens and a dimeric molecule of variant fibrinogens reduced fibrin polymerization, with a thinner fiber and aberrant fibrin network. These results are almost the same as for the identical variant of Magdeburg, however, different from the similar variant of Osaka VI [12 amino acid elongation 462-473 (KSPIRRFLLFCM) in the Bß-chain] in the presence of variant forms and clot structure. We speculate the side-chain difference at 462 residues, W in K-VI, K in Osaka VI, and/or the difference in the presence of disulfide bridged forms of variant fibrinogens, led to the notable difference in the fibrin bundle network. Although a strong evolutional and structural association between Bß-chain and γ-chain molecules is established, the corresponding recombinant 15 residue elongation variants of the fibrinogen γ-chain showed reduced assembly and secretion.
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Afibrinogenemia/genética , Fibrinógeno/genética , Afibrinogenemia/sangre , Aminoácidos , Electroforesis en Gel de Poliacrilamida , Femenino , Fibrinógeno/metabolismo , Humanos , Microscopía Electrónica de Rastreo , Persona de Mediana EdadRESUMEN
We evaluated the preoperative errors in the pelvic tilt of 249 hips before total hip arthroplasty using fluoroscopic imaging while the patients were in the lateral decubitus position. The mean absolute value errors of the pelvic tilt were 2.94° (SD, 2.92°), 2.49° (SD, 2.68°), and 5.92° (SD, 5.20°) in the coronal, transverse, and sagittal planes, respectively. Such preoperative errors in the pelvic tilt contribute to malpositioning of the acetabular component, as is frequently observed on postoperative radiographs. We reduced the incidence of malpositioning by correcting the errors in the pelvic tilt through repositioning of the operating table using fluoroscopic imaging before surgery. The new technique using fluoroscopic imaging described in this article can be performed within a short time without a navigation system.
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Acetábulo/cirugía , Artroplastia de Reemplazo de Cadera , Fluoroscopía , Prótesis de Cadera , Posicionamiento del Paciente , Pelvis/diagnóstico por imagen , Acetábulo/diagnóstico por imagen , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Cuidados PreoperatoriosRESUMEN
We investigated whether N-acetylcysteine (NAC), a precursor of glutathione, could protect rabbit articular chondrocytes against nitric oxide (NO)-induced apoptosis and could prevent cartilage destruction in an experimental model of osteoarthritis (OA) in rats. Isolated chondrocytes were treated with various concentrations of NAC (0-2 mM). Apoptosis was induced by 0.75 mM sodium nitroprusside (SNP) dehydrate, which produces NO. Cell viability was assessed by MTT assay, while apoptosis was evaluated by Hoechst 33342 and TUNEL staining. Intracellular reactive oxygen species (ROS) and glutathione levels were measured, and expression of p53 and caspase-3 were determined by Western blotting. To determine whether intraarticular injection of NAC prevents cartilage destruction in vivo, cartilage samples of an OA model were subjected to H&E, Safranin O, and TUNEL staining. NAC prevented NO-induced apoptosis, ROS overproduction, p53 up-regulation, and caspase-3 activation. The protective effects of NAC were significantly blocked by buthionine sulfoximine, a glutathione synthetase inhibitor, indicating that the apoptosis-preventing activity of NAC was mediated by glutathione. Using a rat model of experimentally induced OA, we found that NAC also significantly prevented cartilage destruction and chondrocyte apoptosis in vivo. These results indicate that NAC inhibits NO-induced apoptosis of chondrocytes through glutathione in vitro, and inhibits chondrocyte apoptosis and articular cartilage degeneration in vivo.
Asunto(s)
Acetilcisteína/administración & dosificación , Apoptosis/efectos de los fármacos , Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Depuradores de Radicales Libres/administración & dosificación , Osteoartritis/prevención & control , Animales , Cartílago Articular/citología , Cartílago Articular/metabolismo , Caspasa 3/metabolismo , Condrocitos/metabolismo , Modelos Animales de Enfermedad , Glutatión/efectos de los fármacos , Glutatión/metabolismo , Inyecciones Intraarticulares , Masculino , Óxido Nítrico/efectos adversos , Osteoartritis/inducido químicamente , Osteoartritis/fisiopatología , Conejos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. METHODS: Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. RESULTS: Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. CONCLUSIONS: The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.
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Proteínas de la Matriz Extracelular/genética , Osteoartritis de la Rodilla/genética , Osteoblastos/metabolismo , Osteofito/genética , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta1/genética , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Células Cultivadas , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Furanos , Perfilación de la Expresión Génica , Humanos , Masculino , Osteoartritis de la Rodilla/metabolismo , Osteofito/metabolismo , Índice de Severidad de la Enfermedad , Tiofenos , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta3/genética , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
BACKGROUND: The purpose of this study was to investigate the influence of hydrostatic pressure (HP) on the gene expression of cartilage matrix, cytokines, and apoptosis-associated factors in chondrocytes in which the cartilage was in extracellular matrix (ECM)-rich or ECM-poor condition. METHODS: Chondrocytes were isolated from rabbit joints and cultured in alginate beads. Immediately after embedding (0W group) or after 2 weeks culture (2W group), the amounts of glycosaminoglycan (GAG) in the alginate beads were quantified. Both groups were exposed to continuous HP of 10 or 50 MPa for 12 h. The expression of inflammatory cytokines, proteases, and apoptosis-related factors were examined by reverse transcription-polymerase chain reaction (RT-PCR). The expression of proteoglycan core protein (PG) and collagen type II were quantified by real-time RT-PCR. RESULTS: All of the GAG components in alginate beads markedly increased in the 2W group. The expression of PG and collagen type II increased after exposure to 10 MPa in both groups. In the 0W group, these levels decreased after exposure to 50 MPa of HP. The expression of interleukins IL-6 and IL-8 increased after exposure to HP in the 0W group. HP at 50 MPa induced mRNA expression of ADAMTS-5 in the 0W group but not in the 2W group. The expression of Fas increased after exposure to HP in the 0W group. CONCLUSIONS: These findings suggested that nonphysiological, excessive HP on chondrocytes with the ECM in poor condition reduced matrix gene expression and increased expression of the genes associated with apoptosis and catabolism of the cartilage matrix. These results might therefore be associated with the pathogenesis of osteoarthritis.
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Apoptosis/genética , Condrocitos/metabolismo , Citocinas/genética , Matriz Extracelular/fisiología , Regulación de la Expresión Génica/fisiología , Presión Hidrostática , Animales , Cartílago Articular/metabolismo , Células Cultivadas , Citocinas/metabolismo , ConejosRESUMEN
To clarify the significance of the osteophytes that appear during the progression of osteoarthritis (OA), we investigated the expression of inflammatory cytokines and proteases in osteoblasts from osteophytes. We also examined the influence of mechanical stress loading on osteoblasts on the expression of inflammatory cytokines and proteases. Osteoblasts were isolated from osteophytes in 19 patients diagnosed with knee OA and from subchondral bone in 4 patients diagnosed with femoral neck fracture. Messenger RNA expression and protein production of inflammatory cytokines and proteases were analyzed using real-time RT-PCR and ELISA, respectively. To examine the effects of mechanical loading, continuous hydrostatic pressure was applied to the osteoblasts. We determined the mRNA expression and protein production of IL-6, IL-8, and MMP-13, which are involved in the progression of OA, were increased in the osteophytes. Additionally, when OA pathological conditions were simulated by applying a nonphysiological mechanical stress load, the gene expression of IL-6 and IL-8 increased. Our results suggested that nonphysiological mechanical stress may induce the expression of biological factors in the osteophytes and is involved in OA progression. By controlling the expression of these genes in the osteophytes, the progression of cartilage degeneration in OA may be reduced, suggesting a new treatment strategy for OA.
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Interleucina-6/metabolismo , Interleucina-8/metabolismo , Metaloproteinasa 13 de la Matriz/metabolismo , Osteoartritis/metabolismo , Osteoblastos/metabolismo , Osteofito/patología , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Línea Celular , Células Cultivadas , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Humanos , Presión Hidrostática , Interleucina-6/genética , Interleucina-6/farmacología , Interleucina-8/genética , Masculino , Metaloproteinasa 13 de la Matriz/genética , Persona de Mediana Edad , Osteoartritis/patología , Osteoblastos/efectos de los fármacos , Osteocalcina/genética , Osteofito/metabolismo , Receptores de Interleucina-6 , Estrés Mecánico , Soporte de Peso/fisiologíaRESUMEN
RNA interference (RNAi) provides a powerful means of sequence-specific gene silencing. Several studies show that RNAi may provide promising strategies to treat human diseases by suppressing disease responsible genes in vivo. In locomotor diseases, the progression of collagen-induced arthritis (CIA) is suppressed by tumor necrosis factor-alpha (TNF-alpha)-specific small interfering RNA (siRNA) delivered into the joint. The aim of this study, is to compare the effects of intraarticularly administered siRNAs targeting TNF-alpha, interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and receptor activator of NF-kappaB ligand (RANKL) on CIA in rats. We confirmed that the silencing effects of siRNA duplexes specific for rat TNF-alpha, IL-1beta, IL-6 and RANKL in vitro. Each siRNA was also delivered into the knee joint of CIA rats by the in vivo electroporation method 7, 10, 13 and 16 days after immunization with collagen. Local delivery of TNF-alpha or IL-1beta-specific siRNA ameliorated CIA in rats effectively at the gross morphological, radiographical and histological evaluations. Our results suggested that TNF-alpha and IL-1beta were the cytokines to be targeted in the joint for the treatment of rheumatoid arthritis. The in vivo siRNA transfection method may be useful for selection of target molecules to be silenced for treatment of joint diseases.
Asunto(s)
Antirreumáticos , Artritis Experimental/terapia , Citocinas/antagonistas & inhibidores , Interferencia de ARN , Animales , Articulación del Tobillo/patología , Artritis Experimental/diagnóstico por imagen , Artritis Experimental/patología , Colágeno , Citocinas/genética , Pie/diagnóstico por imagen , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Interleucina-6/antagonistas & inhibidores , Interleucina-6/genética , Ligando RANK/antagonistas & inhibidores , Ligando RANK/genética , ARN Interferente Pequeño , Radiografía , Ratas , Líquido Sinovial/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: The aim of this study was to clarify the significance of subchondral bone in the pathology of osteoarthritis (OA) by investigating the expression of inflammatory cytokines, proteases, and receptor activator of NF-kappaB ligand (RANKL)/receptor activator of NF-kappaB (RANK)/osteoprotegerin (OPG) involved in cartilage degeneration. METHODS: Subchondral bone was obtained from 19 patients diagnosed with knee OA and 4 patients diagnosed with femoral neck fracture. Subchondral bone osteoblasts (SBOs) were isolated, and total RNA was extracted. Messenger RNA expression of inflammatory cytokines, proteases, and RANKL/RANK/OPG were analyzed using a real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Real-time RT-PCR showed that mRNA expressions of interleukin-6 (IL-6), matrix metalloproteinase-13 (MMP-13), and RANKL were significantly enhanced in OA SBOs compared to SBOs without OA. The expressions of these genes was greater in patients with severe cartilage damage than in those with mild cartilage damage. A high correlation between mRNA expression of IL-6 and that of MMP-13 was found in OA SBOs. CONCLUSION: The increases in IL-6, MMP-13, and RANKL expression in OA SBOs suggest that in subchondral bone OA progression involves abnormal osseous tissue remodeling, which induces mechanical property changes. Cartilage degeneration in OA may also be due, at least in part, to IL-6 and MMP-13 produced by SBOs. Comprehensive research on these pathological features may lead to the development of more effective therapies for OA by administration of molecules that affect bone remodeling and metabolism.