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1.
Am J Pathol ; 189(7): 1423-1434, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31051168

RESUMO

Preserving the mature articular cartilage of joints is a critical focus in the prevention and treatment of osteoarthritis. We determined whether the genetic inactivation of high-temperature requirement A1 (HtrA1) can significantly attenuate the degradation of articular or condylar cartilage. Two types of mouse models of osteoarthritis were used, a spontaneous mutant mouse model [type XI collagen-haploinsufficient (Col11a1+/-) mice] and two post-traumatic mouse models [destabilization of the medial meniscus (DMM) on the knee and a partial discectomy (PDE) on the temporomandibular joint]. Three different groups of mice were generated: i) HtrA1 was genetically deleted from Col11a1+/- mice (HtrA1-/-;Col11a1+/-), ii) HtrA1-deficient mice (HtrA1-/-) were subjected to DMM, and iii) HtrA1-/- mice were subjected to PDE. Knee and temporomandibular joints from the mice were characterized for evidence of cartilage degeneration. The degradation of articular or condylar cartilage was significantly delayed in HtrA1-/-;Col11a1+/- mice and HtrA1-/- mice after DMM or PDE. The amount of collagen type VI was significantly higher in the articular cartilage in HtrA1-/-;Col11a1+/- mice, compared with that in Col11a1+/- mice. The genetic removal of HtrA1 may delay the degradation of articular or condylar cartilage in mice.


Assuntos
Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Osteoartrite/enzimologia , Animais , Cartilagem Articular/enzimologia , Cartilagem Articular/patologia , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Modelos Animais de Doenças , Serina Peptidase 1 de Requerimento de Alta Temperatura A/genética , Articulação do Joelho/enzimologia , Articulação do Joelho/patologia , Côndilo Mandibular/enzimologia , Côndilo Mandibular/patologia , Camundongos , Camundongos Knockout , Osteoartrite/genética , Osteoartrite/patologia
2.
Acta Orthop Traumatol Turc ; 53(2): 140-144, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30655094

RESUMO

PURPOSE: The aim of this study is to investigate which ADAMTS genes play a major role in the development of primary hip osteoarthritis, by comparing the tissue and blood samples in patients with hip osteoarthritis and a control group. MATERIAL AND METHODS: Human articular cartilage was obtained from femoral heads of 15 patients with end stage osteoarthritis undergoing total hip replacement. As the control group, the cartilages was obtained from femoral heads of 15 patients, who did not have osteoarthritis or degenerative changes in hip joint, undergoing hip replacement following the fracture of the femoral neck. After the cartilage samples were taken from the resection materials, the DNA polymorphisms in the patients' cartilage samples were tested by Polymerase Chain Reaction (PCR), the serum levels of aggrecanase genes were analyzed with Enzyme-Linked ImmunoSorbent Assay (ELISA). RESULTS: The level of ADAMTS5 and ADAMTS9 genes were found significantly lower as a result of ELISA analysis degenerative arthritis group than the control group (p < 0,05). ADAMTS 1, 4, 8, 15 were similar between the two groups in ELISA analysis (p > 0,05). As a result of quantitative real time RT-PCR analysis, the level of ADAMTS8 mRNA increased 3.5 fold in hip degenerative arthritis group when compared with femoral neck fractures group. ADAMTS1, ADAMTS4 and ADAMTS5 expression levels in hip degenerative arthritis group were decreased 2.5, 2 and 2.5 fold, respectively. ADAMTS9, 15 were found to be similar between two groups. CONCLUSON: As a result of this study on hip osteoarthritis, the ADAMTS8 levels was found to be significantly higher in the end stage of hip osteoarthritis. Unlike similar studies on knee osteoarthritis, ADAMTS1,4,5 levels were found to be lower.


Assuntos
Proteínas ADAMTS/genética , Proteína ADAMTS1/genética , Cartilagem Articular , Endopeptidases , Osteoartrite do Quadril , Proteínas ADAMTS/análise , Idoso , Artroplastia de Quadril/métodos , Cartilagem Articular/enzimologia , Cartilagem Articular/patologia , Correlação de Dados , Endopeptidases/sangue , Endopeptidases/genética , Feminino , Fraturas do Colo Femoral/genética , Fraturas do Colo Femoral/patologia , Fraturas do Colo Femoral/cirurgia , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/sangue , Osteoartrite do Quadril/genética , Osteoartrite do Quadril/patologia , Osteoartrite do Quadril/cirurgia
3.
Ann Rheum Dis ; 78(3): 421-428, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30610061

RESUMO

OBJECTIVE: Osteoarthritis (OA) appears to be associated with various metabolic disorders, but the potential contribution of amino acid metabolism to OA pathogenesis has not been clearly elucidated. Here, we explored whether alterations in the amino acid metabolism of chondrocytes could regulate OA pathogenesis. METHODS: Expression profiles of amino acid metabolism-regulating genes in primary-culture passage 0 mouse chondrocytes were examined by microarray analysis, and selected genes were further characterised in mouse OA chondrocytes and OA cartilage of human and mouse models. Experimental OA in mice was induced by destabilisation of the medial meniscus (DMM) or intra-articular (IA) injection of adenoviruses expressing catabolic regulators. The functional consequences of arginase II (Arg-II) were examined in Arg2-/- mice and those subjected to IA injection of an adenovirus encoding Arg-II (Ad-Arg-II). RESULTS: The gene encoding Arg-II, an arginine-metabolising enzyme, was specifically upregulated in chondrocytes under various pathological conditions and in OA cartilage from human patients with OA and various mouse models. Adenovirus-mediated overexpression of Arg-II in mouse joint tissues caused OA pathogenesis, whereas genetic ablation of Arg2 in mice (Arg2 -/-) abolished all manifestations of DMM-induced OA. Mechanistically, Arg-II appears to cause OA cartilage destruction at least partly by upregulating the expression of matrix-degrading enzymes (matrix metalloproteinase 3 [MMP3] and MMP13) in chondrocytes via the nuclear factor (NF)-κB pathway. CONCLUSIONS: Our results indicate that Arg-II is a crucial regulator of OA pathogenesis in mice. Although chondrocytes of human and mouse do not identically, but similarly, respond to Arg-II, our results suggest that Arg-II could be a therapeutic target of OA pathogenesis.


Assuntos
Arginase/fisiologia , Artrite Experimental/enzimologia , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Osteoartrite/enzimologia , Animais , Artrite Experimental/induzido quimicamente , Modelos Animais de Doenças , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Osteoartrite/induzido quimicamente , Regulação para Cima
4.
J Orthop Res ; 37(2): 490-502, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30457172

RESUMO

The etiology of joint tissue degeneration following rotator cuff tear remains unclear. Thus, the purpose of this study was to understand the timeline of protease activity in the soft tissues of the shoulder (tendon, muscle, and cartilage) that may lead to down-stream degeneration following rotator cuff tear. A well-established rat model involving suprascapular nerve denervation and supraspinatus/infraspinatus tendon transection was employed. Histological staining and/or micro-computed tomography (µCT) were used to observe structural damage in the supraspinatus tendon and muscle, humeral head cartilage, and subchondral bone. Multiplex gelatin zymography was utilized to assess protease activity in the supraspinatus tendon and muscle, and humeral head cartilage. Zymography analysis demonstrated that cathepsins were upregulated in the first week in all tissues, while MMP-2 maintained prolonged activity in supraspinatus tendon between 1 and 3 weeks and increased only at 3 weeks in supraspinatus muscle. In supraspinatus tendon, increased cathepsin L and MMP-2 activity in the first week was concurrent with matrix disorganization and infiltration of inflammatory cells. In contrast, significant upregulation of cathepsin L and K activity in supraspinatus muscle and humeral head cartilage did not correspond to any visible tissue damage at 1 week. However, focal defects developed in half of all animals' humeral head cartilage by 12 weeks (volume: 0.12 ± 0.09 mm3 ). This work provides a more comprehensive understanding of biochemical changes to joint tissue over time following rotator cuff tear. Overall, this provides insight into potential therapeutic targets and will better inform ideal intervention times and treatments for each tissue. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:490-502, 2019.


Assuntos
Catepsinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Lesões do Manguito Rotador/enzimologia , Manguito Rotador/enzimologia , Articulação do Ombro/enzimologia , Animais , Osso Esponjoso/diagnóstico por imagem , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/enzimologia , Masculino , Ratos Sprague-Dawley , Manguito Rotador/patologia , Lesões do Manguito Rotador/diagnóstico por imagem , Lesões do Manguito Rotador/patologia , Articulação do Ombro/diagnóstico por imagem , Fatores de Tempo , Microtomografia por Raio-X
5.
J Cell Physiol ; 234(6): 9156-9167, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30311192

RESUMO

Mechanical stress plays a key role in regulating cartilage degradation in osteoarthritis (OA). The aim of this study was to evaluate the effects and mechanisms of mechanical stress on articular cartilage. A total of 80 male Sprague-Dawley rats were randomly divided into eight groups (n = 10 for each group): control group (CG), OA group (OAG), and CG or OAG subjected to low-, moderate-, or high-intensity treadmill exercise (CL, CM, CH, OAL, OAM, and OAH, respectively). Chondrocytes were obtained from the knee joints of rats; they were cultured on Bioflex 6-well culture plates and subjected to different durations of cyclic tensile strain (CTS) with or without exposure to interleukin-1ß (IL-1ß). The results of the histological score, immunohistochemistry, enzyme-linked immunosorbent assay, and western-blot analyses indicated that there were no differences between CM and CG, but OAM showed therapeutic effects compared with OAG. However, CH and OAH experienced more cartilage damage than CG and OAG, respectively. CTS had no therapeutic effects on collagen II of normal chondrocytes, which is consistent with findings after treadmill exercise. However, CTS for 4 hr could alleviate the chondrocyte damage induced by IL-1ß by activating AMP-activated protein kinase (AMPK) phosphorylation and suppressing nuclear translocation of nuclear factor (NF)-κB p65. Our findings indicate that mechanical stress had no therapeutic effects on normal articular cartilage and chondrocytes; mechanical stress only caused damage with excessive stimulation. Still, moderate biomechanical stress could reduce sensitization to the inflammatory response of articular cartilage and chondrocytes through the AMPK/NF-κB signaling pathway.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Artrite Experimental/prevenção & controle , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Terapia por Exercício , Fator de Transcrição RelA/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Artrite Experimental/enzimologia , Artrite Experimental/patologia , Artrite Experimental/fisiopatologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Cartilagem Articular/fisiopatologia , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/patologia , Interleucina-1beta/farmacologia , Masculino , Fosforilação , Ratos Sprague-Dawley , Corrida , Transdução de Sinais , Estresse Mecânico
6.
Arthritis Rheumatol ; 71(4): 571-582, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30379418

RESUMO

OBJECTIVE: Cartilage destruction in osteoarthritis (OA) is mediated mainly by matrix metalloproteinases (MMPs) and ADAMTS. The therapeutic candidature of targeting aggrecanases has not yet been defined in joints in which spontaneous OA arises from genetic susceptibility, as in the case of the STR/Ort mouse, without a traumatic or load-induced etiology. In addition, we do not know the long-term effect of aggrecanase inhibition on bone. We undertook this study to assess the potential aggrecanase selectivity of a variant of tissue inhibitor of metalloproteinases 3 (TIMP-3), called [-1A]TIMP-3, on spontaneous OA development and bone formation in STR/Ort mice. METHODS: Using the background of STR/Ort mice, which develop spontaneous OA, we generated transgenic mice that overexpress [-1A]TIMP-3, either ubiquitously or conditionally in chondrocytes. [-1A]TIMP-3 has an extra alanine at the N-terminus that selectively inhibits ADAMTS but not MMPs. We analyzed a range of OA-related measures in all mice at age 40 weeks. RESULTS: Mice expressing high levels of [-1A]TIMP-3 were protected against development of OA, while those expressing low levels were not. Interestingly, we also found that high levels of [-1A]TIMP-3 transgene overexpression resulted in increased bone mass, particularly in females. This regulation of bone mass was at least partly direct, as adult mouse primary osteoblasts infected with [-1A]TIMP-3 in vitro showed elevated rates of mineralization. CONCLUSION: The results provide evidence that [-1A]TIMP-3-mediated inhibition of aggrecanases can protect against cartilage degradation in a naturally occurring mouse model of OA, and they highlight a novel role that aggrecanase inhibition may play in increased bone mass.


Assuntos
Densidade Óssea/genética , Cartilagem Articular/enzimologia , Endopeptidases/metabolismo , Osteoartrite/enzimologia , Inibidores de Proteases/metabolismo , Animais , Condrócitos/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Osteoartrite/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo
7.
Arch Oral Biol ; 97: 238-244, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30412863

RESUMO

OBJECTIVES: The structure of the mandibular condylar cartilage (MCC) is regulated by dynamic and multifactorial processes. The aim of this study was to examine the effects of altered dietary loading, estrogen level, and aging on the structure of the condylar cartilage and the expressions of matrix metalloproteinase (MMP) -3 and MMP-8 of rat MCC. METHODS: In this study, Crl:CD (SD) female rats were randomly divided into 3 groups according to dietary hardness: hard diet (diet board), normal diet (pellet), and soft diet (powder). In each group, the rats were further divided into 2 subgroups by ovariectomy at the age of 7 weeks. The rats were sacrificed at 5- and 14-month-old. Histomorphometric analysis of the MCC thickness was performed after toluidine blue staining. Immunochemical staining was done for MMP-3 and MMP-8. A linear mixed model was used to assess the effects of dietary loading, estrogen level, and aging. RESULTS: Increased dietary loading was the main factor to increase the MMP-3 expression and the anterior and central thickness of the MCC. Lack of estrogen was the main factor associated with decreased MMP-8. Aging was associated with the thickness changes of the whole condylar cartilage and the reduced expression of MMP-8. CONCLUSION: The condylar cartilage structure and metabolism of the female rats are sensitive to dietary loading changes, estrogen level as well as aging. The proper balance of these factors seems to be essential for the maintenance of the condylar cartilage.


Assuntos
Cartilagem Articular/enzimologia , Dieta , Estrogênios/metabolismo , Côndilo Mandibular/enzimologia , Metaloproteinase 3 da Matriz/metabolismo , Metaloproteinase 8 da Matriz/metabolismo , Animais , Feminino , Ovariectomia , Distribuição Aleatória , Ratos , Coloração e Rotulagem
8.
Osteoarthritis Cartilage ; 27(1): 99-105, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30240939

RESUMO

OBJECTIVE: Animal studies suggest that S100A8/S100A9 may be involved in the pathogenesis of osteoarthritis (OA); however, there has been no clinical study examining the associations between serum S100A8/S100A9 and knee symptoms, joint structures and cartilage degradation enzymes in knee OA patients so far. Therefore, this study was designed to investigate the cross-sectional associations between serum levels of S100A8/S100A9 and the outcomes in patients with knee OA. DESIGN: A total of 141 subjects with clinical knee OA were included. Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) score was used to assess joint symptoms. Magnetic resonance imaging (MRI) was used to measure knee structural abnormalities including cartilage defects. Knee radiography was used to assess joint space narrowing (JSN), osteophytes and the radiographic severity of OA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum levels of S100A8/S100A9, matrix metalloproteinase (MMP)-3, MMP10 and MMP13. RESULTS: In multivariable analyses, serum S100A8/S100A9 were positively associated with total WOMAC score (ß: 0.111 per 10 ng/ml, P = 0.021), WOMAC weight-bearing pain (ß: 0.015 per 10 ng/ml, P = 0.043) and WOMAC physical dysfunction (ß: 0.091 per 10 ng/ml, P = 0.010), and had positive associations with total cartilage defects and cartilage defects at lateral femoral, lateral tibial and medial femoral sites (ORs: 1.006-1.008 per 10 ng/ml, all P < 0.05) and serum levels of MMP3 (ß: 0.002 per 10 ng/ml, P = 0.032) in patients with clinical knee OA. CONCLUSIONS: Serum levels of S100A8/S100A9 were positively associated with increased knee symptoms, cartilage defects and serum cartilage degradation enzymes in patients with knee OA, suggesting that S100A8/S100A9 may have a role to play in knee OA. Future longitudinal studies are required to confirm these findings.


Assuntos
Calgranulina A/sangue , Calgranulina B/sangue , Cartilagem Articular/enzimologia , Osteoartrite do Joelho/sangue , Adulto , Idoso , Biomarcadores/sangue , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Estudos Transversais , Feminino , Humanos , Articulação do Joelho/diagnóstico por imagem , Imagem por Ressonância Magnética/métodos , Masculino , Metaloproteinase 10 da Matriz/sangue , Metaloproteinase 13 da Matriz/sangue , Metaloproteinase 3 da Matriz/sangue , Pessoa de Meia-Idade , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/patologia , Radiografia , Índice de Gravidade de Doença
9.
PLoS One ; 13(9): e0203944, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30222787

RESUMO

Recent studies have shown that superoxide dismutase 1 (SOD1), SOD2, and SOD3 are significantly decreased in human osteoarthritic cartilage. SOD activity is a marker that can be used to comprehensively evaluate the enzymatic capacities of SOD1, SOD2, and SOD3; however, the trend of SOD activity in end-stage osteoarthritic tissues remains unknown. In the present study, we found that SOD activity in end-stage osteoarthritic synovium of the knee was significantly lower than that in control synovium without the influence of age. The SOD activity was significantly lower in the end-stage knee osteoarthritic cartilage than in the control, but a weak negative correlation was observed between aging and SOD activity. However, SOD activity in end-stage hip osteoarthritic cartilage was significantly lower than that in control cartilage without the influence of aging. The relationship between osteoarthritis and SOD activity was stronger than the relationship between aging and SOD activity. These results indicate that direct regulation of SOD activity in joint tissues may lead to suppression of osteoarthritis progression.


Assuntos
Cartilagem Articular/enzimologia , Osteoartrite do Quadril/enzimologia , Osteoartrite do Joelho/enzimologia , Superóxido Dismutase/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Malondialdeído/metabolismo , Pessoa de Meia-Idade , Espécies Reativas de Oxigênio/metabolismo , Membrana Sinovial/enzimologia , Adulto Jovem
10.
J Biol Chem ; 293(31): 12259-12270, 2018 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-29929979

RESUMO

Certain dysregulated chondrocyte metabolic adaptive responses such as decreased activity of the master regulator of energy metabolism AMP-activated protein kinase (AMPK) promote osteoarthritis (OA). Metabolism intersects with epigenetic and transcriptional responses. Hence, we studied chondrocyte ATP-citrate lyase (ACLY), which generates acetyl-CoA from mitochondrial-derived citrate, and modulates acetylation of histones and transcription factors. We assessed ACLY in normal and OA human knee chondrocytes and cartilages by Western blotting and immunohistochemistry, and quantified acetyl-CoA fluorometrically. We examined histone and transcription factor lysine acetylation by Western blotting, and assessed histone H3K9 and H3K27 occupancy of iNOS, MMP3, and MMP13 promoters by chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR). We analyzed iNOS, MMP3, MMP13, aggrecan (ACAN), and Col2a1 gene expression by RT-qPCR. Glucose availability regulated ACLY expression and function, nucleocytosolic acetyl-CoA, and histone acetylation. Human knee OA chondrocytes exhibited increased ACLY activation (assessed by Ser-455 phosphorylation), associated with increased H3K9 and H3K27 acetylation. Inhibition of ACLY attenuated IL-1ß-induced transcription of iNOS, MMP3, and MMP13 by suppressing acetylation of p65 NF-κB, H3K9, and H3K27, blunted release of NO, MMP3, and MMP13, and also reduced SOX9 acetylation that promoted SOX9 nuclear translocation, leading to increased aggrecan and Col2a1 mRNA expression. ACLY is a novel player involved in regulation of cartilage matrix metabolism. Increased ACLY activity in OA chondrocytes increased nucleocytosolic acetyl-CoA, leading to increased matrix catabolism via dysregulated histone and transcription factor acetylation. Pharmacologic ACLY inhibition in OA chondrocytes globally reverses these changes and stimulates matrix gene expression and AMPK activation, supporting translational investigation in OA.


Assuntos
ATP Citrato (pro-S)-Liase/metabolismo , Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Matriz Extracelular/enzimologia , Osteoartrite do Joelho/enzimologia , ATP Citrato (pro-S)-Liase/genética , Acetilcoenzima A/metabolismo , Acetilação , Agrecanas/genética , Agrecanas/metabolismo , Cartilagem Articular/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Histonas/metabolismo , Humanos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/metabolismo
11.
Cell Death Dis ; 9(6): 699, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899528

RESUMO

Osteoarthritis (OA) is the most common form of arthritis involving major structural changes of peripheral joints and local or systemic inflammation and in lack of therapeutic approaches because of complexity of underlying molecular basis. Our previous work showed that HS6ST2, an enzyme involved in the transfer of sulfate, is downregulated in cartilage tissues of OA patients compared with normal donors, but little is known about its regulatory mechanism. In this study, we demonstrated that the expression of HS6ST2 was lower in OA-damaged cartilage than smooth cartilage from the same patient. In chondrocytes, HS6ST2 could be targeted by miR-23b-3p, which was higher expressed in OA-damaged cartilage. Under TNF-α stimulation, the expression of HS6ST2 was found inversely correlated with the expression of miR-23b-3p. Downregulation of HS6ST2 regulated by overexpression of miR-23b-3p and siRNAs against HS6ST2 could enhance the protein level of MMP13 and aggravate the matrix degradation in chondrocytes. Increased expression of MMP13 depended on activity of p38 MAPK rather than total p38 MAPK level and was abrogated by HS6ST2 overexpression. Together, the results indicated that downregulated HS6ST2 targeted by miR-23b-3p promotes matrix degradation by activating p38 MAPK in chondrocytes and OA cartilage.


Assuntos
Regulação para Baixo , Matriz Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases , MicroRNAs/metabolismo , Osteoartrite/enzimologia , Osteoartrite/genética , Sulfotransferases/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Idoso , Cartilagem Articular/enzimologia , Cartilagem Articular/patologia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Feminino , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Metaloproteinase 13 da Matriz/metabolismo , MicroRNAs/genética , Fosforilação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sulfotransferases/genética , Fator de Necrose Tumoral alfa/farmacologia
12.
Mol Med Rep ; 18(1): 541-549, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29749508

RESUMO

The aim of the present study was to investigate the role of microRNA (miR)­27a­3p in osteoarthritis (OA). Reverse transcription­quantitative polymerase chain reaction and western blotting were performed to determine the expression of miR­27a­3p and aggrecanase­2 (ADAMTS5) in cartilage tissues from patients with OA and healthy controls, and also in interleukin (IL)­1ß­treated primary human chondrocytes. Primary human chondrocytes were transfected with miR­27a­3p. A luciferase reporter assay was used to validate the direct contact between miR­27a­3p and its putative binding site in the 3'­untranslated region ADAMTS5 mRNA. Furthermore, the effects of IL­1ß­induced activation of mitogen­activated protein kinase (MAPK) and nuclear factor (NF)­κB on miR­27a­3p were evaluated using specific inhibitors. The results revealed that the level of miR­27a­3p was reduced in OA cartilage tissues compared with those of normal controls. In addition, decreased miR­27a­3p and increased ADAMTS5 expression was observed in a time­ and dose­dependent manner in chondrocytes treated with IL­1ß. Furthermore, overexpression of miR­27a­3p suppressed the expression of ADAMTS5 in human chondrocytes induced by IL­1ß. miR­27a­3p overexpression also decreased the luciferase activity of the wild­type ADAMTS5 reporter plasmid. Mutation of the miR­27a­3p binding site in the 3'­untranslated region of ADAMTS5 mRNA abolished the miR­27a­3p­mediated repression of reporter activity. Furthermore, the use of specific inhibitors demonstrated that IL­1ß may regulate miR­27a­3p expression via NF­κB and MAPK signaling pathways in chondrocytes. The present study concluded that miR­27a­3p was downregulated in human OA and was suppressed by IL­1ß, and functions as a crucial regulator of ADAMTS5 in OA chondrocytes. In addition, IL­1ß­mediated suppression of miR­27a­3p activity may occur via the MAPK and NF­κB pathways. The present study may provide a novel strategy for clinical treatment of OA caused by upregulation of miR­27a­3p.


Assuntos
Cartilagem Articular/metabolismo , Interleucina-1beta/metabolismo , MicroRNAs/genética , Osteoartrite do Joelho/metabolismo , Transdução de Sinais , Proteína ADAMTS5/genética , Adolescente , Adulto , Idoso , Cartilagem Articular/enzimologia , Células Cultivadas , Criança , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Osteoartrite do Joelho/enzimologia , Osteoartrite do Joelho/etiologia
13.
Osteoarthritis Cartilage ; 26(8): 1110-1117, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29803826

RESUMO

OBJECTIVES: The chondrocytes' pericellular matrix acts as a mechanosensor by sequestering growth factors that are bound to heparan sulfate (HS) proteoglycans. Heparanase is the sole mammalian enzyme with HS degrading endoglycosidase activity. Here, we aimed to ascertain whether heparanase plays a role in modulating the anabolic or catabolic responses of human articular chondrocytes. METHODS: Primary chondrocytes were incubated with pro-heparanase and catabolic and anabolic gene expression was analyzed by quantitative polymerase chain reaction (PCR). MMP13 enzymatic activity in the culture medium was measured with a specific fluorescent assay. Extracellular regulated kinase (ERK) phosphorylation was evaluated by Western blot. Human osteoarthritis (OA) cartilage was assessed for heparanase expression by reverse-transcriptase PCR, by Western blot and by a heparanase enzymatic activity assay. RESULTS: Cultured chondrocytes rapidly associated with and activated pro-heparanase. Heparanase induced the catabolic genes MMP13 and ADAMTS4 and the secretion of active MMP13, and down-regulated the anabolic genes ACAN and COL2A1. PG545, a HS-mimetic, inhibited the effects of heparanase. Heparanase expression and enzymatic activity were demonstrated in adult human osteoarthritic cartilage. Heparanase induced ERK phosphorylation in cultured chondrocytes and this could be inhibited by PG545, by fibroblast growth factor 2 (FGF2) neutralizing antibodies and by a FGF-receptor inhibitor. CONCLUSIONS: Heparanase is active in osteoarthritic cartilage and induces catabolic responses in primary human chondrocytes. This response is due, at least in part, to the release of soluble growth factors such as FGF2.


Assuntos
Cartilagem Articular/enzimologia , Condrócitos/enzimologia , Glucuronidase/metabolismo , Osteoartrite/enzimologia , Adulto , Western Blotting , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Humanos , Metaloproteinase 13 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Biomed Pharmacother ; 103: 346-354, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29669300

RESUMO

INTRODUCTION: Rheumatoid arthritis (RA) represents the most commonly occurring inflammatory type of arthritis and is a major cause of disability. Reports have placed emphasis on the potential of, granzyme B (GZMB) as a potentially valuable prognostic marker in early RA, the mechanism of which still remains largely unclear. Thus, the aim of the current study was to investigate the effects GZMB gene silencing influences synovial tissue hyperplasia and articular cartilage tissue injury of RA through the regulation of the MAPK signaling pathway. METHODS: Following the successful establishment of the collagen-induced animal model of RA in rats, a five-grade scoring method was applied to evaluate the swelling degree measurement of the rats for model identification. The various rat responses to GZMB shRNA and U-46619 (activator of the MAPK signaling pathway) were subsequently detected. The general status of rats was observed and recorded, with their weight and ankle diameter kept accurate record of. ELISA was employed to detect the levels of inflammatory cytokines, while RT-qPCR and Western blotting techniques were applied to determine the expressions of GZMB and pathway-related genes and proteins. RESULTS: GZMB gene silencing was observed to aid in the maintenance of rat weight increases, while acting to reduce the degree of ankle swelling, while hypertrophy of the synovial tissue and the injury of the articular cartilage tissue were not obvious. GZMB gene silencing was shown to decrease inflammatory cytokine levels, as well as decreased bcl-2, Cyclin D1, VEGF and bFGF while increasing caspase 3. Notably, GZMB gene silencing suppressed the activation of the MAPK signaling pathway by reducing the phosphorylation extent of ERK and MEK. CONCLUSION: Taken together, the key findings of the present study ultimately suggest that GZMB gene silencing acts to inhibit MAPK signaling pathway through regulating the expressions of inflammatory factors, factors correlated with apoptosis (bcl-2 and caspase), as well as factors associated with angiogenesis (VEGF and bFGF), thus relieving synovial tissue hyperplasia and articular cartilage tissue injury brought about by RA. The GZMB gene could well be a new therapeutic target for RA treatment.


Assuntos
Artrite Experimental/enzimologia , Cartilagem Articular/enzimologia , Granzimas/metabolismo , Hiperplasia/enzimologia , Sistema de Sinalização das MAP Quinases/fisiologia , Membrana Sinovial/enzimologia , Animais , Artrite Experimental/genética , Artrite Experimental/patologia , Cartilagem Articular/lesões , Inativação Gênica/fisiologia , Granzimas/antagonistas & inibidores , Granzimas/genética , Hiperplasia/genética , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Masculino , Ratos , Ratos Wistar , Membrana Sinovial/patologia
15.
Nutrients ; 10(4)2018 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-29641501

RESUMO

Osteoarthritis (OA) is an age-related degenerative joint disease characterized by high oxidative stress, chondrocyte death and cartilage damage. Zinc has been implicated in the antioxidant capacity of the cell, and its deficiency might inhibit chondrocyte proliferation. The present study examined the potential of zinc as a preventive supplement against OA using the in vitro chondrosarcoma cell line SW1353 and an in vivo Wistar rat model to mimic OA progress induced by monosodium iodoacetate (MIA). The results demonstrated that, in SW1353 cells, 5 µM MIA exposure increased oxidative stress and decreased the expression of GPx1 and Mn-SOD but still increased GSH levels and HO-1 expression and enhanced the expression of interleukin (IL)-10, IL-1ß, and matrix metalloproteinase (MMP)-13. Zinc addition could block these changes. Besides, the expression of Nrf2 and phosphorylated (p)-Akt was dramatically increased, implicating the p-Akt/Nrf2 pathway in the effects of zinc on MIA-treated cells. A rat model achieved similar results as those of cell culture, and 1.6 mg/kg/day of zinc supplementation is sufficient to prevent OA progress, while 8.0 mg/kg/day of zinc supplementation does not have a better effect. These findings indicate that zinc supplementation exerts a preventive effect with respect to MIA-induced OA progress.


Assuntos
Antioxidantes/farmacologia , Cartilagem Articular/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Interleucinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Osteoartrite/prevenção & controle , Sulfato de Zinco/farmacologia , Animais , Antioxidantes/metabolismo , Cartilagem Articular/enzimologia , Cartilagem Articular/patologia , Linhagem Celular Tumoral , Condrócitos/enzimologia , Condrócitos/patologia , Citoproteção , Heme Oxigenase-1/metabolismo , Humanos , Masculino , Osteoartrite/enzimologia , Osteoartrite/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos
16.
Ann Rheum Dis ; 77(6): 935-943, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29555825

RESUMO

OBJECTIVES: To investigate the role of tyrosine kinase Fyn in the development of osteoarthritis (OA) and the underlying mechanisms, and to define whether targeting Fyn could prevent OA in mice. METHODS: Cartilage samples from normal and aged mice were analysed with proteome-wide screening. Fyn expression was examined with immunofluorescence in human and age-dependent or experimental mouse OA cartilage samples. Experimental OA in Fyn-knockout mice was induced by destabilisation of the medial meniscus. Primary cultured mouse chondrocytes were treated with proinflammatory cytokine interleukin-1ß. The inhibitor of Src kinase family, AZD0530 (saracatinib), and inhibitor of Fyn, PP1, were used to treat experimental OA in mice. RESULTS: Fyn expression was markedly upregulated in human OA cartilage and in cartilage from aged mice and those with post-traumatic OA. Fyn accumulates in articular chondrocytes and interacts directly with and phosphorylates ß-catenin at Tyr142, which stabilises ß-catenin and promotes its nuclear translocation. The deletion of Fyn effectively delayed the development of post-traumatic and age-dependent OA in mice. Fyn inhibitors AZD0530 and PP1 significantly attenuated OA progression by blocking the ß-catenin pathway and reducing the levels of extracellular matrix catabolic enzymes in the articular cartilage. CONCLUSIONS: Fyn accumulates and activates ß-catenin signalling in chondrocytes, accelerating the degradation of the articular cartilage and OA development. Targeting Fyn is a novel and potentially therapeutic approach to the treatment of OA.


Assuntos
Artrite Experimental/enzimologia , Osteoartrite/enzimologia , Proteínas Proto-Oncogênicas c-fyn/fisiologia , beta Catenina/metabolismo , Envelhecimento/metabolismo , Animais , Artrite Experimental/prevenção & controle , Benzodioxóis/farmacologia , Benzodioxóis/uso terapêutico , Cartilagem Articular/enzimologia , Células Cultivadas , Condrócitos/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Técnicas de Inativação de Genes , Humanos , Camundongos Knockout , Terapia de Alvo Molecular/métodos , Osteoartrite/prevenção & controle , Proteínas Proto-Oncogênicas c-fyn/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fyn/deficiência , Proteínas Proto-Oncogênicas c-fyn/genética , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
17.
J Bone Miner Res ; 33(5): 945-958, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29314205

RESUMO

Cdc42, a member of Rho family small guanosine triphosphatases (GTPases), is critical for cartilage development. We investigated the roles of Cdc42 in osteoarthritis and explored the potential mechanism underlying Cdc42-mediated articular cartilage degeneration and subchondral bone deterioration. Cdc42 is highly expressed in both articular cartilage and subchondral bone in a mouse osteoarthritis model with surgical destabilization of the medial meniscus (DMM) in the knee joints. Specifically, genetic disruption of Cdc42, knockdown of Cdc42 expression, or inhibition of Cdc42 activity robustly attenuates the DMM-induced destruction, hypertrophy, high expression of matrix metallopeptidase-13 and collagen X, and activation of Stat3 in articular cartilages. Notably, genetic disruption of Cdc42, knockdown of Cdc42 expression or inhibition of Cdc42 activity significantly restored the increased numbers of mesenchymal stem cells, osteoprogenitors, osteoblasts, osteoclasts, and neovascularized vessels, the increased bone mass, and the activated Erk1/2, Smad1/5 and Smad2 in subchondral bone of DMM-operated mice. Mechanistically, Cdc42 mediates interleukin-1ß-induced interleukin-6 production and subsequent Jak/Stat3 activation to regulate chondrocytic inflammation, and also lies upstream of Erk/Smads to regulate subchondral bone remodeling during transform growth factor-ß1 signaling. Cdc42 is apparently required for both articular cartilage degeneration and subchondral bone deterioration of osteoarthritis, thus, interventions targeting Cdc42 have potential in osteoarthritic therapy. © 2018 American Society for Bone and Mineral Research.


Assuntos
Osso e Ossos/enzimologia , Cartilagem Articular/enzimologia , Articulação do Joelho/enzimologia , Osteoartrite do Joelho/enzimologia , Proteína cdc42 de Ligação ao GTP/biossíntese , Animais , Osso e Ossos/patologia , Cartilagem Articular/patologia , Colágeno Tipo X/genética , Colágeno Tipo X/metabolismo , Regulação Enzimológica da Expressão Gênica , Interleucina-6/genética , Interleucina-6/metabolismo , Articulação do Joelho/patologia , Masculino , Metaloproteinase 3 da Matriz/genética , Metaloproteinase 3 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteoartrite do Joelho/genética , Osteoartrite do Joelho/patologia , Osteoblastos/enzimologia , Osteoblastos/patologia , Osteoclastos/enzimologia , Osteoclastos/patologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1 , Proteína cdc42 de Ligação ao GTP/genética
18.
Biomacromolecules ; 19(1): 94-102, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29211452

RESUMO

We investigated the effects of different oxygen tension (21% and 2.5% O2) on the chondrogenesis of different cell systems cultured in pH-degradable PVA hydrogels, including human articular chondrocytes (hACs), human mesenchymal stem cells (hMSCs), and their cocultures with a hAC/hMSC ratio of 20/80. These hydrogels were prepared with vinyl ether acrylate-functionalized PVA (PVA-VEA) and thiolated PVA-VEA (PVA-VEA-SH) via Michael-type addition reaction. The rheology tests determined the gelation of the hydrogels was controlled within 2-7 min, dependent on the polymer concentrations. The different cell systems were cultured in the hydrogel scaffolds for 5 weeks, and the safranin O and GAG assay showed that hypoxia (2.5% O2) greatly promoted the cartilage matrix production with an order of hAC > hAC/hMSC > hMSC. The real time quantitative PCR (RT-PCR) revealed that the hMSC group exhibited the highest hypertrophic marker gene expression (COL10A1, ALPL, MMP13) as well as the dedifferentiated marker gene expression (COL1A1) under normoxia conditions (21% O2), while these expressions were greatly inhibited by coculturing with a 20% amount of hACs and significantly further repressed under hypoxia conditions, which was comparative to the sole hAC group. The enzyme-linked immunosorbent assay (ELISA) also showed that coculture of hMSC/hAC greatly reduced the catabolic gene expression of MMP1 and MMP3 compared with the hMSC group. It is obvious that the hypoxia conditions promoted the chondrogenesis of hMSC by adding a small amount of hACs, and also effectively inhibited their hypotrophy. We are convinced that coculture of hAC/hMSC using in situ forming hydrogel scaffolds is a promising approach to producing cell source for cartilage engineering without the huge needs of primary chondrocyte harvest and expansion.


Assuntos
Hipóxia Celular , Condrócitos/citologia , Condrogênese , Hidrogéis/química , Células-Tronco Mesenquimais/citologia , Tecidos Suporte , Fosfatase Alcalina/metabolismo , Materiais Biocompatíveis/química , Cartilagem Articular/citologia , Cartilagem Articular/enzimologia , Cartilagem Articular/metabolismo , Condrócitos/enzimologia , Condrócitos/metabolismo , Técnicas de Cocultura , Colágeno/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Glicosaminoglicanos/metabolismo , Humanos , Metaloproteinase 13 da Matriz/genética , Células-Tronco Mesenquimais/enzimologia , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Fenazinas/metabolismo , Álcool de Polivinil/química
19.
Sci Rep ; 7(1): 7028, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28765635

RESUMO

The catabolism of hyaluronan in articular cartilage remains unclear. The aims of this study were to investigate the effects of hyaluronidase 2 (Hyal2) knockdown in articular cartilage on the development of osteoarthritis (OA) using genetic manipulated mice. Destabilization of the medial meniscus (DMM) model of Col2a promoter specific conditional Hyal2 knockout (Hyal -/- ) mice was established and examined. Age related and DMM induced alterations of articular cartilage of knee joint were evaluated with modified Mankin score and immunohistochemical staining of MMP-13, ADAMTS-5, KIAA11199, and biotinylated- hyaluronan binding protein staining in addition to histomorphometrical analyses. Effects of Hyal2 suppression were also analyzed using explant culture of an IL-1α induced articular cartilage degradation model. The amount and size of hyaluronan in articular cartilage were higher in Hyal2 -/- mice. Hyal2 -/- mice exhibited aggravated cartilage degradation in age-related and DMM induced mice. MMP-13 and ADAMTS-5 positive chondrocytes were significantly higher in Hyal2 -/- mice. Articular cartilage was more degraded in explant cultures obtained from Hyal2 -/- mice. Knockdown of Hyal2 in articular cartilage induced OA development and progression possibly mediated by an imbalance of HA metabolism. This suggests that Hyal2 knockdown exhibits mucopolysaccharidosis-like OA change in articular cartilage similar to Hyal1 knockdown.


Assuntos
Cartilagem Articular/enzimologia , Técnicas de Silenciamento de Genes , Hialuronoglucosaminidase/metabolismo , Osteoartrite/patologia , Proteína ADAMTS5/análise , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Histocitoquímica , Hialuronoglucosaminidase/genética , Imuno-Histoquímica , Articulação do Joelho/patologia , Metaloproteinase 13 da Matriz/análise , Menisco/patologia , Camundongos , Índice de Gravidade de Doença
20.
Eur Rev Med Pharmacol Sci ; 21(10): 2329-2337, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28617559

RESUMO

OBJECTIVE: Rheumatoid arthritis (RA) is one systemic auto-immune disorder featured as chronic synovitis and can destruct joint cartilage. Fibroblast-like synoviocyte (FLS) secretes various factors affecting chondrocyte matrix and degradation. This study thus investigated the effect of interleukin-17A (IL-17A) on FLS and osteoclast. MATERIALS AND METHODS: Type II collagen-induced arthritis (CIA) rats were assigned to CIA model, CIA + IgG1 isotype, and CIA + Anti-Rat IL-17A groups. Tissue volume and arthritis index (AI) evaluated arthritis condition. ELISA and flow cytometry measured IL-17A content and Th17 cell percentage in joint cavity fluid. Matrix metallopeptidase 13 (MMP-13) and collagen type II alpha 1 (COL2A1) expression in synovial tissues were compared. FLS-osteoclast co-culture system was treated with IL-17A + IgG1 Isotype or CIA + Anti-Rat IL-17A. MMP-13 and COL2A1 expression were compared. RESULTS: CIA model rats had significantly higher IL-17A and Th17 cell ratio in joint cavity fluid. Injection of Anti-Rat IL-17A decreased AI and tissue volume in model rats, decreased MMP-13 while increased COL2A1 expression in synovial or cartilage tissues. IL-17A treatment remarkably up-regulated MMP-13 mRNA or protein expression in chondrocytes. Anti-IL-17A weakened effects of IL-17A on FLS or chondrocytes. CONCLUSIONS: IL-17A inhibits COL2A1 mRNA and protein expression of chondrocyte in the co-culture system via inducing MMP-13 expression in FLS, thus enhancing collagen degradation and playing a role in RA-related cartilage injury.


Assuntos
Artrite Experimental/imunologia , Cartilagem Articular/imunologia , Interleucina-17/antagonistas & inibidores , Metaloproteinase 13 da Matriz/metabolismo , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/enzimologia , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/enzimologia , Técnicas de Cocultura , Imunoglobulina G/administração & dosagem , Imunoglobulina G/uso terapêutico , Inflamação , Masculino , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Osteoclastos/imunologia , Ratos Wistar , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/enzimologia , Sinoviócitos/imunologia , Células Th17/imunologia
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