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1.
Clin Exp Rheumatol ; 37 Suppl 120(5): 40-47, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31621575

RESUMO

MicroRNAs are small double-stranded RNAs, which negatively regulate gene expression and have been shown to have key roles in both chondrocyte development and cartilage homeostasis with age. Deletion of all microRNAs in chondrocytes leads to skeletal growth defects in mice, whilst deletion of specific microRNAs, e.g. miR-140, leads to premature articular cartilage degradation and increased susceptibility to posttraumatic osteoarthritis. Studies comparing microRNA expression in normal human articular cartilage compared to osteoarthritic cartilage show differential expression, but varying sample groups make interpretation difficult. MicroRNAs have been proposed as circulating biomarkers of osteoarthritis, but again, this differs amongst patient cohorts. Many micro-RNAs have been shown to have roles in chondrocyte phenotype via signalling pathways, apoptosis, autophagy and senescence. Modulating microRNAs in the joint has been shown to reduce osteoarthritis in animal models and translating this to man as a novel therapeutic strategy will be key.


Assuntos
Autofagia , Cartilagem Articular , MicroRNAs , Osteoartrite , Animais , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Condrócitos/metabolismo , Condrócitos/patologia , Humanos , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/fisiologia , Osteoartrite/genética , Osteoartrite/metabolismo
2.
Cell Physiol Biochem ; 53(4): 623-637, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31550089

RESUMO

BACKGROUND/AIMS: In articular cartilage, chondrocytes are the predominant cell type. A long-term stay in space can lead to bone loss and cartilage breakdown. Due to the poor regenerative capacity of cartilage, this may impair the crewmembers' mobility and influence mission activities. Beside microgravity other factors such as cosmic radiation and vibration might be important for cartilage degeneration. Vibration at different frequencies showed various effects on cartilage in vivo, but knowledge about its impact on chondrocytes in vitro is sparse. METHODS: Human chondrocytes were exposed to a vibration device, simulating the vibration profile occurring during parabolic flights, for 24 h (VIB) and compared to static controls. Phase-contrast microscopy, immunofluorescence, F-actin and TUNEL staining as well as quantitative real-time PCR were performed to examine effects on morphology, cell viability and shape as well as gene expression. The results were compared to earlier studies using semantic analyses. RESULTS: No morphological changes or cytoskeletal alterations were observed in VIB and no apoptotic cells were found. A reorganization and increase in fibronectin were detected in VIB samples by immunofluorescence technique. PXN, VCL, ANXA1, ANXA2, BAX, and BCL2 revealed differential regulations. CONCLUSION: Long-term VIB did not damage human chondrocytes in vitro. The reduction of ANXA2, and up-regulation of ANXA1, PXN and VCL mRNAs suggest that long-term vibration might even positively influence cultured chondrocytes.


Assuntos
Condrócitos/metabolismo , Vibração , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Anexina A2/genética , Anexina A2/metabolismo , Linhagem Celular , Condrócitos/citologia , Condrócitos/patologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Redes Reguladoras de Genes , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Vimentina/genética , Vimentina/metabolismo
3.
Braz J Med Biol Res ; 52(9): e8525, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31411316

RESUMO

Many compounds of ginsenosides show anti-inflammatory properties. However, their anti-inflammatory effects in intervertebral chondrocytes in the presence of inflammatory factors have never been shown. Increased levels of pro-inflammatory cytokines are generally associated with the degradation and death of chondrocytes; therefore, finding an effective and nontoxic substance that attenuates the inflammation is worthwhile. In this study, chondrocytes were isolated from the nucleus pulposus tissues, and the cells were treated with ginsenoside compounds and IL-1ß, alone and in combination. Cell viability and death rate were assessed by CCK-8 and flow cytometry methods, respectively. PCR, western blot, and immunoprecipitation assays were performed to determine the mRNA and protein expression, and the interactions between proteins, respectively. Monomeric component of ginsenoside Rd had no toxicity at the tested range of concentrations. Furthermore, Rd suppressed the inflammatory response of chondrocytes to interleukin (IL)-1ß by suppressing the increase in IL-1ß, tumor necrosis factor (TNF)-α, IL-6, COX-2, and inducible nitric oxide synthase (iNOS) expression, and retarding IL-1ß-induced degradation of chondrocytes by improving cell proliferation characteristics and expression of aggrecan and COL2A1. These protective effects of Rd were associated with ubiquitination of IL-1 receptor accessory protein (IL1RAP), blocking the stimulation of IL-1ß to NF-κB. Bioinformatics analysis showed that NEDD4, CBL, CBLB, CBLC, and ITCH most likely target IL1RAP. Rd increased intracellular ITCH level and the amount of ITCH attaching to IL1RAP. Thus, IL1RAP ubiquitination promoted by Rd is likely to occur by up-regulation of ITCH. In summary, Rd inhibited IL-1ß-induced inflammation and degradation of intervertebral disc chondrocytes by increasing IL1RAP ubiquitination.


Assuntos
Condrócitos/efeitos dos fármacos , Ginsenosídeos/farmacologia , Proteína Acessória do Receptor de Interleucina-1/metabolismo , Interleucina-1beta/efeitos dos fármacos , Degeneração do Disco Intervertebral/metabolismo , Adulto , Idoso , Agrecanas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Feminino , Ginsenosídeos/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Dor Lombar/metabolismo , Masculino , Pessoa de Meia-Idade , Óxido Nítrico Sintase/metabolismo , Núcleo Pulposo/citologia , Núcleo Pulposo/efeitos dos fármacos , Núcleo Pulposo/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinação
4.
Cell Prolif ; 52(5): e12666, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31407423

RESUMO

OBJECTIVES: Cartilaginous tissue degradation occurs because of the lack of survival of chondrocytes. Here, we ascertained whether bakuchiol (BAK) has the capability of activating chondrocyte proliferation. MATERIALS AND METHODS: The effect of BAK on the proliferation of rat chondrocytes at a concentration of 10 and 20 µmol/L was investigated. The molecular mechanisms involving target binding and signalling pathways were elucidated by RNA-sequencing, qPCR, molecular docking and Western blotting. Matrigel mixed with bakuchiol was implanted locally into rat knee articular cartilage defects to verify the activation of chondrocytes due to bakuchiol in vivo. RESULTS: Bakuchiol implantation resulted in the activation of rat chondrocyte proliferation in a dose-dependent manner. RNA-sequencing revealed 107 differentially expressed genes (DEGs) with 75 that were up-regulated and 32 that were down-regulated, indicating increased activation of the PI3K-Akt and cell cycle pathways. Activation of the phosphorylation of Akt, ERK1/2 and their inhibitors blocked the proliferative effect of bakuchiol treatment, confirming its direct involvement in these signal transduction pathways. Molecular docking and siRNA silencing revealed that estrogen receptor-α (ERα) was the target of bakuchiol in terms of its cell proliferative effect via PI3K activation. Two weeks after implantation of bakuchiol, the appearance and physiological structure of the articular cartilage was more integrated with abundant chondrocytes and cartilage matrix compared to that of the control. CONCLUSIONS: Bakuchiol demonstrated significant bioactivity towards chondrocyte proliferation via the PI3K-Akt and ERK1/2 pathways mediated by estrogen receptor activation and exhibited enhanced promotion of the remodelling of injured cartilage.


Assuntos
Cartilagem Articular/fisiologia , Proliferação de Células/efeitos dos fármacos , Fenóis/farmacologia , Receptores Estrogênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/patologia , Condrócitos/citologia , Condrócitos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Regeneração/efeitos dos fármacos
5.
Cell Physiol Biochem ; 53(1): 172-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31264811

RESUMO

BACKGROUND/AIMS: MicroRNAs (miRs) are transcribed as stem-loop precursors harboring two different miRs on either side of the structure. Both miRs can modulate levels of cellular transcripts based on sequence complementarity between the miR and the mRNA target. The miR of the current study, miR-675, is encoded in the H19 gene with high expression in fetal/placental tissues but low levels in most adult tissues except for skeletal muscle and articular cartilage. miR-675 has a supportive role in expression of the major collagen component of articular cartilage (COL2A1) but it is unknown which arm contributes to this effect. Objectives: To determine the active arm of miR-675 in human articular chondrocytes. To evaluate effects of overexpression of both arms of miR-675 on MMP1 and MMP13, two enzymes involved in breakdown of COL2A1. To investigate whether abundance of both arms of miR-675 is dynamic. METHODS: miR-arm activity was determined by association with the AGO2 complex using immunoprecipitation with an AGO2 specific antibody. miR overexpression and inhibition was used to identify indirect downstream effects on two targets of the Matrix-Metalloprotease family, MMP1 and MMP13. Data was evaluated by qPCR and enzymatic activity assays. Early passage human articular chondrocytes (up to passage 2) obtained from cartilage from both healthy and osteoarthritis affected tissue were used. To evaluate miR-675 levels in a different model, myotube differentiation was employed. RESULTS: We show that both arms of miR-675 have opposing effects on MMP1 and MMP13; however only one arm, miR-675-3' is active in human articular chondrocytes. We demonstrate that during myotube differentiation, high expression of both arms of miR-675 is observed as well as an increase in expression of MMP1. CONCLUSION: We show that both arms of miR-675 result in opposing effects on two downstream molecules MMP1 and MMP13. We propose that miR abundance may arise as response to direct target transcript levels and are thus dynamic to meet the requirements of the cellular environment.


Assuntos
Condrócitos/metabolismo , Regulação da Expressão Gênica , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 1 da Matriz/genética , MicroRNAs/genética , Osteoartrite/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Criança , Condrócitos/citologia , Condrócitos/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Regulação para Cima , Adulto Jovem
6.
Can J Vet Res ; 83(3): 206-217, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31308593

RESUMO

The use of dietary supplements as an alternative treatment for joint-related pathologies such as osteoarthritis (OA) is increasing. However, there is little scientific evidence to support the intended use. The aim of this study was to evaluate the anti-inflammatory effects of creatine- and amino acid-based supplements in primary cultured canine chondrocytes (CnCs) as an in-vitro model of OA and compare the effects to more commonly used agents, such as the non-steroidal anti-inflammatory drug (NSAID), carprofen, and the joint supplement, glucosamine (GS). CnCs were stimulated with interleukin-1ß (IL-1ß) and the subsequent release of prostaglandin E2 (PGE2) and tumor necrosis factor alpha (TNFα) was measured using an enzyme-linked immunosorbent assay (ELISA). Changes in oxylipins were also assessed using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). All compounds examined were able to significantly reduce the release of PGE2 and TNFα and were associated with reductions in cyclooxygenase-2 (COX-2) expression and nuclear factor-kappaB (NF-κB) phosphorylation. The creatine- and amino acids-based supplements also altered the profile of oxylipins produced. All compounds examined were less effective at reducing the release of PGE2 than carprofen. Carprofen significantly increased release of TNFα from CnCs, however, while the other agents reduced TNFα release. This study suggests that creatine- and amino acid-based supplements may have a beneficial role in preventing inflammation within the joint and that further studies are warranted.


Assuntos
Condrócitos/efeitos dos fármacos , Suplementos Nutricionais , Doenças do Cão/tratamento farmacológico , Inflamação/veterinária , Osteoartrite/veterinária , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Carbazóis/farmacologia , Sobrevivência Celular , Células Cultivadas , Condrócitos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Cães , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Osteoartrite/tratamento farmacológico
7.
Life Sci ; 232: 116625, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276691

RESUMO

AIMS: The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is critical for cartilage regeneration. Tissues constructed from BMSCs through cartilage tissue engineering still exhibit some histological, morphological, and biomechanical differences from normal cartilage tissues. Cyclic tensile strain (CTS) can increase chondrogenic gene expression and reduce hypertrophic gene expression in chondrocytes. miR-365 has been identified as a mechanoresponsive microRNA and is an important regulator of both chondrocyte hypertrophy and differentiation. Therefore, we hypothesized that CTS may promote the chondrogenesis of BMSCs by upregulating the expression of miR-365. METHODS: BMSCs were subjected to CTS to investigate the effects and mechanism on chondrogenesis. An Agilent miRNA microarray was used to profile miRNAs in the CTS-treated BMSCs and 3D-cultured control BMSCs. miR-365 was shown to interact with HDAC4 mRNA through a luciferase reporter assay. An animal cartilage defect model was constructed and different groups of BMSCs were implanted to investigate their in vivo effect. KEY FINDINGS: CTS promoted BMSC chondrogenesis. miR-365 was significantly upregulated in CTS-treated cells and played an important role in CTS-mediated chondrogenesis. Luciferase assays showed that HDAC4 is a direct target of miR-365. An in vivo study showed that CTS treatment and miR-365 overexpression could promote cartilage regeneration from BMSCs. SIGNIFICANCE: CTS can promote the expression of miR-365, a crucial mechanosensitive microRNA involved in the chondrogenesis of BMSCs, which directly inhibits the expression of HDAC4, in turn, enhancing the chondrogenesis of BMSCs.


Assuntos
Condrogênese/genética , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/genética , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Condrócitos/metabolismo , Condrogênese/fisiologia , MicroRNAs/metabolismo , Ratos , Transdução de Sinais , Resistência à Tração/fisiologia , Engenharia Tecidual
8.
Toxicol Lett ; 314: 18-26, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31299270

RESUMO

Epidemiological investigations indicate that effects related to prenatal adverse environments on the organs of the offspring could continue to adulthood. This study intends to confirm that prenatal nicotine exposure (PNE) increases the susceptibility of osteoarthritis (OA) in the male offspring, and to explore the potential intrauterine programming mechanism. During pregnancy, rats were divided into a PNE group and a control group. After birth, rats were given a high-fat diet for 6 months and long-distance running for 6 weeks. The rats were euthanized at 18 months after birth (PM18) and on gestational day 20 (GD20), respectively. Knee joints were collected for histochemistry, immunohistochemistry, and quantitative polymerase chain reaction (qPCR) assays. Histological analyses and the Mankin's score showed increased cartilage destruction and accelerated OA progression in adult offspring from the PNE group. Immunohistochemistry results showed decreased expression of transforming growth factor beta (TGFß) signaling pathway. Furthermore, the expression of apoptosis factors (caspase-3 and caspase-8), inflammatory factors [interleukin (IL)-1, IL-6] and matrix degradation enzymes [matrix metalloproteinase (MMP)-3, MMP-13] were also significantly increased. Traced back to the intrauterine period, it was found that the number of chondrocytes and the contents of Col2A1 and aggrecan in the matrix in the PNE group were decreased. And, the expression of the TGFß signaling pathway was inhibited. These results suggested that PNE enhanced the susceptibility of OA in male elderly offspring rats by down-regulating TGFß signaling, which increased articular cartilage local inflammation, matrix degradation, and cell apoptosis. This study confirmed the developmental origin of OA, and clarified the congenital and the living environment impact on the occurrence and development of OA. Our findings provide a theoretical and experimental basis for OA early prevention.


Assuntos
Articulações/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Osteoartrite/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Fatores Etários , Agrecanas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Condrogênese/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Feminino , Idade Gestacional , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Articulações/patologia , Masculino , Exposição Materna , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Gravidez , Ratos Wistar , Fatores de Risco , Fatores Sexuais
9.
Artif Cells Nanomed Biotechnol ; 47(1): 2139-2145, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31146598

RESUMO

Osteoarthritis (OA) is a common joint disease for which a safe and reliable treatment has yet to be developed. Here, we demonstrated the potential benefit of treatment with paeonol, a derivative of Paeonia suffruticosa, in the treatment and prevention of OA. Chondrogenic cell line ATDC5 cells were cultured with IL-1ß and the effects of paeonol were assessed through qRT-PCR, western blot analysis, MTT, ELISA, and NF-κB luciferase reporter gene assay. Our findings demonstrate a novel ability of paeonol to inhibit numerous factors of OA, including expressions of IL-6, TNF-α, NOX2, PTGS2, NUCB2/nesfatin-1, ICAM-1, VCAM-1, MMP-3/13, degradation of type II collagen, and NF-κB activation through the rescue of IκBα. Additionally, we assessed the effects of paeonol on cell viability to confirm its safety. These findings implicate a valuable potential role of paeonol in the treatment and prevention of OA.


Assuntos
Acetofenonas/farmacologia , Condrócitos/efeitos dos fármacos , Colágeno Tipo II/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Proteólise/efeitos dos fármacos , Acetofenonas/uso terapêutico , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Ciclo-Oxigenase 2/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 3 da Matriz/metabolismo , NADPH Oxidase 2/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Osteoartrite/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Molécula 1 de Adesão de Célula Vascular/metabolismo
10.
Mol Cell Biochem ; 459(1-2): 205-214, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31227976

RESUMO

Osteoarthritis (OA) is characterized by degradation of articular cartilage. MiRNAs are involved in the regulation of chondrogenesis and OA. We aimed to investigate effects and mechanisms of miR-19b-3p in regulating chondrocytes viability, cartilage degradation and inflammatory response. Primary chondrocytes were isolated from cartilages in control subjects and patients with OA. Murine ATDC5 cells were pre-conditioned with IL-1ß in vitro. Expressions and interaction of miR-19b-3p with G protein-coupled receptor kinase 6 (GRK6), and their effects on inflammation, chondrocytes viability and cartilage degradation were determined after miR-19b-3p mimic or GRK6 siRNA transfection. MiR-19b-3p was significantly decreased in OA chondrocytes and IL-1ß-stimulated ATDC5 cells, in paralleled with the elevated type-II-collagen, aggrecan, MMP13 and GRK6 expression. MiR-19b-3p mimic dramatically increased the viability of chondrocytes and suppressed cell apoptosis. It also increased type-II-collagen, aggrecan expression and glycosaminoglycan (sGAG) content, and decreased the expression of MMP-1 and MMP-13 that controlled by IL-1ß. Overexpression of miR-19b-3p inhibited the production of IL-6 and IL-8 in ATDC5 cells. However, the protective effects of miR-19b-3p mimic on IL-1ß induced cell death; IL-8 production and sGAG decrease were greatly discounted by GRK6 lentiviral vectors. Luciferase reporter assay confirmed that GRK6 gene was a direct target ofmiR-19b-3p. GRK6 siRNA transfection antagonized the IL-1ß-induced chondrocytes injury, extracellular matrix degradation and inflammatory response. MiR-19b-3p mimic and GRK6 siRNA showed comparable inhibitory effect on IL-1ß-provoked NF-κB as reflected by the expression of p-p65. NF-κB translocation inhibition with PS1154 reversed the effects of IL-1ß on IL-8 and sGAG. Collectively, miR-19b-3p attenuated OA by targeting GRK6-NF-κB pathway.


Assuntos
Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Quinases de Receptores Acoplados a Proteína G/metabolismo , Interleucina-1beta/metabolismo , MicroRNAs/metabolismo , Osteoartrite do Joelho/metabolismo , Idoso , Linhagem Celular , Condrócitos/patologia , Matriz Extracelular/patologia , Feminino , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/patologia , Transdução de Sinais
11.
Cell Mol Life Sci ; 76(23): 4795-4809, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31201465

RESUMO

Fibrillin microfibrils are ubiquitous elements of extracellular matrix assemblies that play crucial roles in regulating the bioavailability of growth factors of the transforming growth factor beta superfamily. Recently, several "a disintegrin and metalloproteinase with thrombospondin motifs" (ADAMTS) proteins were shown to regulate fibrillin microfibril function. Among them, ADAMTS17 is the causative gene of Weill-Marchesani syndrome (WMS) and Weill-Marchesani-like syndrome, of which common symptoms are ectopia lentis and short stature. ADAMTS17 has also been linked to height variation in humans; however, the molecular mechanisms whereby ADAMTS17 regulates skeletal growth remain unknown. Here, we generated Adamts17-/- mice to examine the role of Adamts17 in skeletogenesis. Adamts17-/- mice recapitulated WMS, showing shorter long bones, brachydactyly, and thick skin. The hypertrophic zone of the growth plate in Adamts17-/- mice was shortened, with enhanced fibrillin-2 deposition, suggesting increased incorporation of fibrillin-2 into microfibrils. Comprehensive gene expression analysis of growth plates using laser microdissection and RNA sequencing indicated alteration of the bone morphogenetic protein (BMP) signaling pathway after Adamts17 knockout. Consistent with this, phospho-Smad1 levels were downregulated in the hypertrophic zone of the growth plate and in Adamts17-/- primary chondrocytes. Delayed terminal differentiation of Adamts17-/- chondrocytes, observed both in primary chondrocyte and primordial metatarsal cultures, and was prevented by BMP treatment. Our data indicated that Adamts17 is involved in skeletal formation by modulating BMP-Smad1/5/8 pathway, possibly through inhibiting the incorporation of fibrillin-2 into microfibrils. Our findings will contribute to further understanding of disease mechanisms and will facilitate the development of therapeutic interventions for WMS.


Assuntos
Proteínas ADAMTS/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Transdução de Sinais , Proteínas ADAMTS/deficiência , Animais , Proteínas Morfogenéticas Ósseas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Fibrilina-2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microfibrilas/metabolismo , Músculo Esquelético/patologia , Pele/fisiopatologia , Proteína Smad1/metabolismo , Proteína Smad5/metabolismo , Proteína Smad8/metabolismo , Síndrome de Weill-Marchesani/metabolismo , Síndrome de Weill-Marchesani/patologia , Síndrome de Weill-Marchesani/veterinária
12.
Nat Commun ; 10(1): 2434, 2019 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-31164647

RESUMO

To date, genome-wide association studies have implicated at least 35 loci in osteoarthritis but, due to linkage disequilibrium, the specific variants underlying these associations and the mechanisms by which they contribute to disease risk have yet to be pinpointed. Here, we functionally test 1,605 single nucleotide variants associated with osteoarthritis for regulatory activity using a massively parallel reporter assay. We identify six single nucleotide polymorphisms (SNPs) with differential regulatory activity between the major and minor alleles. We show that the most significant SNP, rs4730222, exhibits differential nuclear protein binding in electrophoretic mobility shift assays and drives increased expression of an alternative isoform of HBP1 in a heterozygote chondrosarcoma cell line, in a CRISPR-edited osteosarcoma cell line, and in chondrocytes derived from osteoarthritis patients. This study provides a framework for prioritization of GWAS variants and highlights a role of HBP1 and Wnt signaling in osteoarthritis pathogenesis.


Assuntos
Condrócitos/metabolismo , Redes Reguladoras de Genes , Proteínas de Grupo de Alta Mobilidade/genética , Osteoartrite/genética , Proteínas Repressoras/genética , Alelos , Cartilagem Articular/citologia , Linhagem Celular Tumoral , Ensaio de Desvio de Mobilidade Eletroforética , Técnicas de Introdução de Genes , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Haplótipos , Proteínas de Grupo de Alta Mobilidade/metabolismo , Humanos , Desequilíbrio de Ligação , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Isoformas de Proteínas , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt
13.
Artif Cells Nanomed Biotechnol ; 47(1): 2612-2617, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31237151

RESUMO

Aging-related osteoarthritis (OA) is the most common type of arthritis. Chondrocyte senescence has been linked with the pathogenesis of OA. Here, we examined the expression of GPR39 in chondrocytes and its modulatory effect on IL-1ß-induced cellular senescence. We show that GPR39 is moderately expressed in human chondrocytes and its expression is repressed by the pro-inflammatory cytokine IL-1ß. The GPR39 agonist TC-G 1008 mitigates IL-1ß-induced chondrocyte senescence. Mechanistically, we show that TC-G 1008 mitigates IL-1ß-induced cell cycle arrest at G1 phase by suppressing the expression of p53, p21, PAI-1, and K382 acetylation of p53. Moreover, we show that TC-G 1008 treatment restores IL-1ß-induced inhibition of SIRT1 and the silencing of SIRT1 abolishes the function of TC-G 1008 on p53 acetylation and senescence, suggesting that the function of GPR39 signaling is mediated by SIRT1 in chondrocytes. Altogether, our findings implicate that the activation of GPR39 signaling ameliorates IL-1ß-induced chondrocyte senescence and the GPR39 agonist TC-G 1008 could have the potential to modulate aging-associated OA.


Assuntos
Senescência Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/efeitos dos fármacos , Interleucina-1beta/farmacologia , Pirimidinas/farmacologia , Receptores Acoplados a Proteínas-G/agonistas , Sulfonamidas/farmacologia , Acetilação/efeitos dos fármacos , Linhagem Celular , Condrócitos/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo
14.
Cell Prolif ; 52(4): e12637, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31168899

RESUMO

OBJECTIVES: Chondrocyte proliferation and differentiation are crucial for endochondral ossification, but their regulatory mechanism remains unclear. The present study aimed to determine the physiological function of TGFß1 signalling in the proliferation and differentiation of antler chondrocytes and explore its relationship with Notch, Shh signalling and Foxa. MATERIALS AND METHODS: Immunofluorescence, Western blot, MTS assay, flow cytometry, RNA interference and real-time PCR were used to analyse the function and regulatory mechanisms of TGFß1 signalling in antler chondrocyte proliferation and differentiation. RESULTS: TGFß1, TGFBR1 and TGFBR2 were highly expressed in antler cartilage. TGFß1 promoted chondrocyte proliferation, increased the proportion of S-phase cells and induced the expression of hypertrophic chondrocyte markers Col X, Runx2 and Alpl. However, this induction was weakened by TGFß receptor inhibitor SB431542 and Smad3 inhibitor SIS3. Simultaneously, TGFß1 activated Notch and Shh signalling whose blockage attenuated the above effects of rTGFß1, whereas addition of rShh rescued the defects in chondrocyte proliferation and differentiation elicited by SB431542 and SIS3. Further analysis revealed that inhibition of Notch signalling impeded TGFß1 activation of the Shh pathway. Knockdown of Foxa1, Foxa2 and Foxa3 abrogated the effects of TGFß1 on chondrocyte differentiation. Notch and Shh signalling mediated the regulation of Foxa transcription factors by TGFß1. CONCLUSIONS: TGFß1 signalling could induce the proliferation and differentiation of antler chondrocytes through Notch-Shh-Foxa pathway.


Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Animais , Chifres de Veado , Benzamidas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Dioxóis/farmacologia , Proteínas Hedgehog/metabolismo , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Isoquinolinas/farmacologia , Piridinas/farmacologia , Pirróis/farmacologia , Receptores Notch/metabolismo , Fase S/efeitos dos fármacos , Fase S/fisiologia , Transdução de Sinais/efeitos dos fármacos
15.
Cell Prolif ; 52(4): e12587, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31206838

RESUMO

OBJECTIVES: Cellular aggregates are readily applicable in cell-based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated. MATERIALS AND METHODS: The aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow-derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion-related molecules was analysed. RESULTS: It was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin ß1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery. CONCLUSIONS: Cellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large-scale production of cellular aggregates for cell-based therapy, such as cartilage regeneration.


Assuntos
Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Animais , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Bovinos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica/fisiologia , Células-Tronco Mesenquimais/metabolismo , Coelhos
16.
Artif Cells Nanomed Biotechnol ; 47(1): 1971-1977, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31155960

RESUMO

Objective: Cyclin D1 was an important molecular involved in the pathological process of osteoarthritis (OA). The purpose of this study was to identify the effect and potential mechanism of Cyclin D1 for the proliferation and apoptosis of OA chondrocytes. Methods: We used polymerase chain reaction (PCR) method to identify the expression levels of Cyclin D1 and down-stream Wnt/ß-catenin pathway-related genes in OA chondrocytes according to the grade of OA. Small interfering RNA (siRNA) or overexpression of Cyclin D1 were used to identify the role of Cyclin D1 in cell proliferation and apoptosis. Next, we used XAV-939 to inhibit the Wnt/ß-catenin pathway and explore the relevant mechanism. Results: Cyclin D1 was significantly decreased with OA grade (p < .05). After siCyclin D1 transfection, the expression level of WNT3 and nuclear ß-catenin were significantly increased, while Wnt10a and total ß-catenin were not obviously changed. Co-cultured with XAV-939 and siCyclin D1 abolished the effects of siCyclin D1 on proliferation and apoptosis of OA chondrocytes (p < .05). Conclusions: Cyclin D1 regulated chondrocyte proliferation and apoptosis through Wnt3/ß-catenin instead of Wnt10a/ß-catenin signalling pathway.


Assuntos
Apoptose , Condrócitos/patologia , Ciclina D1/metabolismo , Osteoartrite/metabolismo , Osteoartrite/patologia , Via de Sinalização Wnt , Proteína Wnt3/metabolismo , Estudos de Casos e Controles , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Condrócitos/metabolismo , Ciclina D1/genética , Regulação da Expressão Gênica , Humanos , Osteoartrite/genética
17.
Biomed Res Int ; 2019: 4328219, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31179322

RESUMO

High molecular weight hyaluronan (H-HA) has a pivotal role in the maintenance of normal functions of synovial fluid and structure of the articular joint, but it has been shown that its concentration is reduced in patients affected by degenerative cartilage diseases, such as osteoarthritis (OA). The aim of this study was to investigate the anti-inflammatory effects and properties of hybrid cooperative complexes based on high and low molecular weight hyaluronan (HCC) compared to H-HA on human primary cells derived by pathological joints. In addition, the rheological behavior of HCC was evaluated in order to define their potential as viscosupplement gel in degenerated joints. The experiments were performed using an in vitro model of OA based on human chondrocytes and synoviocytes isolated from degenerated joints of patients hospitalized for surgical replacement. In order to assess the anti-inflammatory effects of HCC, we evaluated NF-kB, COMP-2, IL-6, and IL-8 as specific markers at the transcriptional and/or protein level. Moreover, the proliferative properties of HCC were assessed using time lapse video microscopy. We showed that chondrocytes and synoviocytes clearly presented an altered cytokine profile compatible with a severe ongoing inflammation status. H-HA and, above all, HCC significantly reduced levels of the specific biomarkers evaluated and improved cartilage healing. The rheological profile indicated HCC suitability for intra-articular injection in joint diseases. HCC viscoelastic properties and the protective/anti-inflammatory effect on human chondrocytes and synoviocytes suggest the novel HCC-based gels as a valid support for OA management.


Assuntos
Proliferação de Células/efeitos dos fármacos , Condrócitos/metabolismo , Ácido Hialurônico , Modelos Biológicos , Osteoartrite/tratamento farmacológico , Sinoviócitos/metabolismo , Condrócitos/patologia , Géis , Humanos , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Peso Molecular , Osteoartrite/metabolismo , Osteoartrite/patologia , Sinoviócitos/patologia
18.
Int J Mol Sci ; 20(11)2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31146351

RESUMO

Evaluating cell migration after cell-based treatment is important for several disorders, including osteoarthritis (OA), as it might influence the clinical outcome. This research explores migrating expanded-adipose stromal cells (ASCs) and adipose niches after enzymatic and mechanical processes. Bilateral anterior cruciate ligament transection induced a mild grade of OA at eight weeks in adult male New Zealand rabbits. ASCs, enzymatic stromal vascular fraction (SVF), and micro fragmented adipose tissue (MFAT) were intra-articularly injected in the knee joint. Assessments of cell viability and expression of specific markers, including CD-163 wound-healing macrophages, were done. Cell migration was explored through labelling with PKH26 dye at 7 and 30 days alongside co-localization analyses for CD-146. All cells showed good viability and high percentages of CD-90 and CD-146. CD-163 was significantly higher in MFAT compared to SVF. Distinct migratory potential and time-dependent effects were observed among cell-based treatments. At day 7, both ASCs and SVF migrated towards synovium, whereas for MFAT versus cartilage, a different migration pattern was noticed at day 30. The long-term distinct cell migration of ASCs, SVF, and MFAT open interesting clinical insights on their potential use for OA treatment. Moreover, the highest expression of CD-163 in MFAT, rather than SVF, might have an important role in directly mediating cartilage tissue repair responses.


Assuntos
Adipócitos/transplante , Osteoartrite do Joelho/terapia , Regeneração , Transplante de Células-Tronco/métodos , Adipócitos/citologia , Adipócitos/fisiologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Movimento Celular , Células Cultivadas , Condrócitos/citologia , Condrócitos/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Cultura Primária de Células/métodos , Coelhos
19.
Cell Mol Life Sci ; 76(20): 3939-3952, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31201464

RESUMO

Articular cartilage is formed at the end of epiphyses in the synovial joint cavity and permanently contributes to the smooth movement of synovial joints. Most skeletal elements develop from transient cartilage by a biological process known as endochondral ossification. Accumulating evidence indicates that articular and growth plate cartilage are derived from different cell sources and that different molecules and signaling pathways regulate these two kinds of cartilage. As the first sign of joint development, the interzone emerges at the presumptive joint site within a pre-cartilage tissue. After that, joint cavitation occurs in the center of the interzone, and the cells in the interzone and its surroundings gradually form articular cartilage and the synovial joint. During joint development, the interzone cells continuously migrate out to the epiphyseal cartilage and the surrounding cells influx into the joint region. These complicated phenomena are regulated by various molecules and signaling pathways, including GDF5, Wnt, IHH, PTHrP, BMP, TGF-ß, and FGF. Here, we summarize current literature and discuss the molecular mechanisms underlying joint formation and articular development.


Assuntos
Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Condrogênese/genética , Regulação da Expressão Gênica , Cápsula Articular/metabolismo , Via de Sinalização Wnt , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/crescimento & desenvolvimento , Diferenciação Celular , Linhagem da Célula/genética , Movimento Celular , Condrócitos/citologia , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 5 de Diferenciação de Crescimento/genética , Fator 5 de Diferenciação de Crescimento/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Cápsula Articular/citologia , Cápsula Articular/crescimento & desenvolvimento , Osteogênese/genética , Proteína Relacionada ao Hormônio Paratireóideo/genética , Proteína Relacionada ao Hormônio Paratireóideo/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
20.
Nat Commun ; 10(1): 2876, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253842

RESUMO

Osteoarthritis (OA) is a common, painful disease. Currently OA is incurable, and its etiology largely unknown, partly due to limited understanding of OA as a whole-joint disease. Here we report that two homologous microRNAs, miR-204 and miR-211, maintain joint homeostasis to suppress OA pathogenesis. Specific knockout of miR-204/-211 in mesenchymal progenitor cells (MPCs) results in Runx2 accumulation in multi-type joint cells, causing whole-joint degeneration. Specifically, miR-204/-211 loss-of-function induces matrix-degrading proteases in articular chondrocytes and synoviocytes, stimulating articular cartilage destruction. Moreover, miR-204/-211 ablation enhances NGF expression in a Runx2-dependent manner, and thus hyper-activates Akt signaling and MPC proliferation, underlying multiplex non-cartilaginous OA conditions including synovial hyperplasia, osteophyte outgrowth and subchondral sclerosis. Importantly, miR-204/-211-deficiency-induced OA is largely rescued by Runx2 insufficiency, confirming the miR-204/-211-Runx2 axis. Further, intraarticular administration of miR-204-expressing adeno-associated virus significantly decelerates OA progression. Collectively, miR-204/-211 are essential in maintaining healthy homeostasis of mesenchymal joint cells to counteract OA pathogenesis.


Assuntos
Células-Tronco Mesenquimais/fisiologia , MicroRNAs/metabolismo , Osteoartrite/metabolismo , Animais , Condrócitos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Camundongos , Camundongos Knockout , MicroRNAs/genética , Osteoartrite/etiologia , Osteoartrite/patologia , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Sinoviócitos/metabolismo
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