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1.
Methods Mol Biol ; 2598: 141-156, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36355290

RESUMO

Metabolism has long been recognized as a critical physiological process necessary to maintain homeostasis in all types of cells including the chondrocytes of articular cartilage. Alterations in metabolism in disease and metabolic adaptation to physiological stimuli such as mechanical loading are increasingly recognized as important for understanding musculoskeletal systems such as synovial joints. Metabolomics is an emerging technique that allows quantitative measurement of thousands of small molecule metabolites that serve as both products and reactants to myriad reactions of cellular biochemistry. This protocol describes procedures to perform metabolomic profiling on chondrocytes and other tissues and fluids within the synovial joint.


Assuntos
Cartilagem Articular , Osteoartrite , Humanos , Condrócitos/metabolismo , Osteoartrite/metabolismo , Cartilagem Articular/metabolismo , Metabolômica , Homeostase
2.
Methods Mol Biol ; 2598: 157-176, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36355291

RESUMO

Primary cilia regulate and coordinate a variety of cell signaling pathways important in chondrocyte physiology and cartilage development, health, and disease. Despite this, the chondrocyte primary cilium and its associated role in cartilage biology remains poorly understood. Key to elucidating primary cilia structure and function in chondrocytes is the ability to visualize this unique structure. Here we describe materials and methods for immunofluorescence labeling, microscopy, and measurement of chondrocyte primary cilia.


Assuntos
Cartilagem Articular , Condrócitos , Condrócitos/metabolismo , Cartilagem Articular/metabolismo , Cílios/metabolismo , Transdução de Sinais
3.
Methods Mol Biol ; 2598: 217-225, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36355295

RESUMO

Proteins from hyaline or articular cartilage can be isolated and purified using a series of chemical extraction steps and various identification techniques including mass spectrometry and immunoblotting. The isolation and purification of proteins from cartilage will facilitate the study of specific proteins and multimeric complexes of cartilage proteins to better understand their functions in normal healthy cartilage as well as pathological conditions of cartilage. Cartilage tissue engineering efforts rely on the comprehensive understanding of the composition of cartilage and the function of each of the protein constituents.


Assuntos
Cartilagem Articular , Cartilagem Hialina , Cartilagem Hialina/metabolismo , Cartilagem Articular/metabolismo , Proteoglicanas/metabolismo , Colágeno/metabolismo
4.
J Adv Res ; 41: 63-75, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36328754

RESUMO

INTRODUCTIONS: Excessive mechanical stress is closely associated with cell death in various conditions. Exposure of chondrocytes to excessive mechanical loading leads to a catabolic response as well as exaggerated cell death. Ferroptosis is a recently identified form of cell death during cell aging and degeneration. However, it's potential association with mechanical stress remains to be illustrated. OBJECTIVES: To identify whether excessive mechanical stress can cause ferroptosis. To explore the role of mechanical overloading in chondrocyte ferroptosis. METHODS: Chondrocytes were collected from loading and unloading zones of cartilage in patients with osteoarthritis (OA), and the ferroptosis phenotype was analyzed through transmission electron microscope and microarray. Moreover, the relationship between ferroptosis and OA was analyzed by GPX4-conditional knockout (Col2a1-CreERT: GPX4flox/flox) mice OA model and chondrocytes cultured with high strain mechanical stress. Furthermore, the role of Piezo1 ion channel in chondrocyte ferroptosis and OA development was explored by using its inhibitor (GsMTx4) and agonist (Yoda1). Additionally, chondrocyte was cultured in calcium-free medium with mechanical stress, and ferroptosis phenotype was tested. RESULTS: Human cartilage and mouse chondrocyte experiments revealed that mechanical overloading can induce GPX4-associated ferroptosis. Conditional knockout of GPX4 in cartilage aggravated experimental OA process, while additional treatment with ferroptosis suppressor protein (FSP-1) and coenzyme Q10 (CoQ10) abated OA development in GPX4-CKO mice. In mouse OA model and chondrocyte experiments, inhibition of Piezo1 channel activity increased GPX4 expression, attenuated ferroptosis phenotype and reduced the severity of osteoarthritis. Additionally, high strain mechanical stress induced ferroptosis damage in chondrocyte was largely abolished by blocking calcium influx through calcium-free medium. CONCLUSIONS: Our findings show that mechanical overloading induces ferroptosis through Piezo1 activation and subsequent calcium influx in chondrocytes, which might provide a potential target for OA treatment.


Assuntos
Cartilagem Articular , Ferroptose , Osteoartrite , Animais , Humanos , Camundongos , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Modelos Animais de Doenças , Canais Iônicos/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Estresse Mecânico
5.
Biochem Biophys Res Commun ; 636(Pt 1): 25-32, 2022 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-36332479

RESUMO

This study aimed to evaluate the efficacy of intra-articular delivery of peripheral blood derived mesenchymal stromal cells (PB-MSCs) on the progression of trauma-induced osteoarthritis (OA) in mice. Adult male C57BL/6J mice subjected to destabilization of the medial meniscus surgeries (DMM) were randomly divided into four groups: sham surgery group; vehicle control group (treated with saline), PBMSC-treated group, or adipose tissue derived MSCs (AD-MSC)-treated group (n = 4 per group). PB-MSCs and AD-MSCs were harvested and cultured following previously established protocols, and pre-labeled with BrdU for 48 h before transplantation. PB-MSCs or AD-MSCs (5 × 105 cells/mouse; passage 3-5) were intra-articular injected into the right knee joints thrice post-surgery. The mice were euthanized at 8 weeks post-surgery and knee joint samples were collected for micro-CT and histological examinations. PB-MSCs administration significantly reduced subchondral bone volume comparing to the vehicle control group. Safranin O staining showed that PB-MSCs treatment ameliorated degeneration of articular cartilage, which was comparable to AD-MSCs treatment. The expression of catabolic marker MMP13 was significantly reduced in articular cartilage of the PB-MSCs treated group comparing to that of the vehicle control group. Co-expression of BrdU and Sox9 indicated that injected PB-MSCs differentiated in chondrocytes in situ, along with reduced levels of IL-6 within peripheral sera of PB-MSCs- and AD-MSCs-treated mice. Therefore, administration of PB-MSCs or AD-MSCs attenuated trauma-induced OA progression through inhibiting cartilage degradation and inflammation. PB-MSCs are ideal cell source for treating cartilage-associated diseases.


Assuntos
Cartilagem Articular , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Osteoartrite , Animais , Masculino , Camundongos , Bromodesoxiuridina , Cartilagem Articular/metabolismo , Modelos Animais de Doenças , Injeções Intra-Articulares , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Osteoartrite/patologia
6.
BMC Biol ; 20(1): 253, 2022 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-36352408

RESUMO

BACKGROUND: Without the availability of disease-modifying drugs, there is an unmet therapeutic need for osteoarthritic patients. During osteoarthritis, the homeostasis of articular chondrocytes is dysregulated and a phenotypical transition called hypertrophy occurs, leading to cartilage degeneration. Targeting this phenotypic transition has emerged as a potential therapeutic strategy. Chondrocyte phenotype maintenance and switch are controlled by an intricate network of intracellular factors, each influenced by a myriad of feedback mechanisms, making it challenging to intuitively predict treatment outcomes, while in silico modeling can help unravel that complexity. In this study, we aim to develop a virtual articular chondrocyte to guide experiments in order to rationalize the identification of potential drug targets via screening of combination therapies through computational modeling and simulations. RESULTS: We developed a signal transduction network model using knowledge-based and data-driven (machine learning) modeling technologies. The in silico high-throughput screening of (pairwise) perturbations operated with that network model highlighted conditions potentially affecting the hypertrophic switch. A selection of promising combinations was further tested in a murine cell line and primary human chondrocytes, which notably highlighted a previously unreported synergistic effect between the protein kinase A and the fibroblast growth factor receptor 1. CONCLUSIONS: Here, we provide a virtual articular chondrocyte in the form of a signal transduction interactive knowledge base and of an executable computational model. Our in silico-in vitro strategy opens new routes for developing osteoarthritis targeting therapies by refining the early stages of drug target discovery.


Assuntos
Cartilagem Articular , Osteoartrite , Humanos , Camundongos , Animais , Cartilagem Articular/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/genética , Osteoartrite/metabolismo , Condrócitos/metabolismo , Hipertrofia/metabolismo , Transdução de Sinais
7.
Biomaterials ; 291: 121870, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36343609

RESUMO

Treating the concomitant inflammation in the process of injury and repair, and simultaneously promoting cartilage regeneration is very important for the repair of articular cartilage (AC) defects. Nevertheless, this remains a massive challenge. To address this issue, a collagen membrane-based modified citrus pectin (MCP) delivery system (MCP-C) was developed in this study by targeting galectin-3 (Gal-3), an upstream proinflammatory factor. As expected, MCP shows anti-inflammatory effects; it downregulates the expressions of IL-1ß, MMP13, Gal-3, and COL1A2, inhibits the degenerative effects of Gal-3 on chondrocytes in vitro, and protects chondrocytes from degeneration and death in vivo. Unexpectedly, MCP promotes the proliferation of chondrocytes, upregulates the expression of COL2A1 and SOX9 in the chondrocytes in vitro, and enhances the repair of AC defect in rabbit knee, especially MCP500-C with a complete release of the loading amount of approximately 500 µg/cm2 in a day. Mechanistically, MCP upregulates the expressions of multiple endogenous growth factors for chondrogenesis via the transcriptome sequencing of MCP-treated chondrocytes, and downregulates the expressions of various inflammatory factors. These findings demonstrate that locally delivered MCP can simultaneously modulate both regenerative and inflammatory responses, and can enhance the repair of AC defects.


Assuntos
Cartilagem Articular , Animais , Coelhos , Cartilagem Articular/metabolismo , Galectina 3/metabolismo , Condrócitos/metabolismo , Regeneração , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/metabolismo
8.
Int J Mol Sci ; 23(22)2022 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-36430655

RESUMO

Osteoarthritis (OA) is a complex disorder of diarthrodial joints caused by multiple risk factors and is characterized by articular cartilage destruction as well as changes in other articular tissues. Semaphorin 3A (Sema3A), known to be a chemo-repellent for sensory nerve fibers, has recently been implicated in cartilage OA pathophysiology. We demonstrated that the expression of SEMA3A and its receptor neuropilin-1 (NRP1) are synchronously upregulated in chondrocytes isolated from knee cartilage of OA patients compared to non-OA control chondrocytes. In addition, we observed that during in vitro passaging of OA chondrocytes, the Nrp-1 level increases, whereas the Sema3A level decreases. In this study, we aimed to uncover how Sema3A-Nrp-1 signaling affects metabolism and viability of OA chondrocytes via siRNA-mediated inhibition of Nrp-1 expression. We observed a decreased proliferation rate and an increase in adhesion and senescence after Nrp-1 silencing. Moreover, MMP13 gene expression was reduced by approximately 75% in NRP1 knockdown OA chondrocytes, whereas MMP13 expression was induced by Sema3A treatment in control (nt siRNA) OA chondrocytes, accompanied by an impaired AKT phosphorylation. These findings suggest a potential catabolic function of Sema3A signaling in OA chondrocytes by inducing MMP13 expression and by compromising pro-survival AKT activation. We propose that targeting the Sema3A-Nrp-1 signaling axis might be an opportunity to interfere with OA pathogenesis and progression.


Assuntos
Cartilagem Articular , Osteoartrite , Humanos , Condrócitos/metabolismo , Metaloproteinase 13 da Matriz/genética , Metaloproteinase 13 da Matriz/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Cartilagem Articular/metabolismo , Osteoartrite/metabolismo
9.
Medicine (Baltimore) ; 101(45): e31725, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36397317

RESUMO

Growing evidence indicates that lncRNAs are involved in the progression of several diseases, including osteoarthritis (OA). However, the role of the lncRNA PVT1 in OA is still unclear. The present study was aimed at exploring the impact of PVT1 on OA progression, along with potential underlying mechanisms. PVT1 expression levels in articular cartilage tissue of OA patients and non-OA patients were evaluated. To assess the proliferation and apoptosis of chondrocytes subject to treatment, PVT1, miR-497, and AKT3 were either knocked down or upregulated in IL-1ß-induced chondrocytes. The variables detected were changes in levels of AKT3 and extracellular matrix (ECM)-related factors (including aggrecan, collagen Type II, and MMP-9). Elevated PVT1 levels were found in cartilage tissue of OA patients and IL-1ß-induced chondrocytes. It was also observed that PVT1 knockdown and miR-497 upregulation led to enhanced cell proliferation and suppressed apoptosis. In addition, a decrease in aggrecan and collagen type II levels and an increase in MMP-9 levels were observed in IL-1ß-induced chondrocytes. A dual luciferase reporter assay was performed to identify the factors that interacted with miR-497, PVT1, and AKT3. It was observed through rescue experiments that enhancing AKT3 expression or knocking down miR-497 could reverse the impacts of PVT1 knockdown in IL-1ß-induced chondrocytes. An upregulation of PVT1 is observed in OA patients. On the other hand, PVT1 knockdown can decrease the effects of IL-1ß on the proliferation, apoptosis, and expression of ECM-related proteins of chondrocytes through the regulation of the miR-497/AKT3 axis. PVT1 levels are elevated in the cartilage tissue of OA patients and IL-1ß-induced chondrocytes. PVT1 knockdown alleviates the effects of IL-1ß treatment on the proliferation and apoptosis of chondrocytes and ECM degradation in chondrocytes by regulating the miR-497/AKT3 axis.


Assuntos
Cartilagem Articular , MicroRNAs , Osteoartrite , RNA Longo não Codificante , Humanos , Agrecanas/genética , Agrecanas/metabolismo , Cartilagem Articular/metabolismo , Colágeno Tipo II , Metaloproteinase 9 da Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo
10.
Acta Biomater ; 153: 243-259, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36374749

RESUMO

There are no drugs or treatment methods known to prevent the development of post-traumatic osteoarthritis (PTOA), a type of osteoarthritis (OA) that is triggered by traumatic joint injuries and accounts for ∼12% of the nearly 600 million OA cases worldwide. Lack of effective drug delivery techniques remains a major challenge in developing clinically effective treatments, but cationic delivery carriers can help overcome this challenge. Scaling up treatments that are effective in in vitro models to achieve success in preclinical in vivo models and clinical trials is also a challenging problem in the field. Here we use a cationic green fluorescent protein (GFP) as a carrier to deliver Insulin-Like Growth Factor 1 (IGF-1), a drug considered as a potential therapeutic for PTOA. GFP-IGF-1 conjugates were first synthesized as fusion proteins with different polypeptide linkers, and their transport properties were characterized in human cartilage explants. In vitro experimental data were used to develop a predictive mathematical transport model that was validated using an independent in vitro experimental data set. The model was used to predict the transport of these fusion proteins upon intra-articular injection into human knee joints. The predictions included results for the rate and extent of fusion protein penetration into cartilage, and the maximum levels of fusion proteins that would escape into systemic circulation through the joint capsule. Together, our transport measurements and model set the stage for translation of such explant culture studies to in vivo preclinical studies and potentially clinical application. STATEMENT OF SIGNIFICANCE: The lack of blood supply in cartilage and rapid clearance of drugs injected into human knees presents a major challenge in developing clinically effective treatments for osteoarthritis. Cationic delivery carriers can target negatively charged cartilage and help overcome this problem. Scaling up treatments that are effective in vitro to achieve success in vivo is also challenging. Here, we use a cationic green fluorescent protein (GFP) to deliver Insulin-Like Growth Factor-1 (IGF-1) into cartilage. Experiments measuring transport of GFP-IGF-1 fusion proteins in human cartilage explants were used to develop and validate a mathematical model to predict fusion protein transport upon injection into human knee joints. This work translates such explant culture studies to in vivo preclinical studies and potentially clinical application.


Assuntos
Cartilagem Articular , Fator de Crescimento Insulin-Like I , Osteoartrite , Humanos , Cartilagem Articular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Articulação do Joelho , Osteoartrite/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Sistemas de Liberação de Medicamentos
11.
Cells ; 11(22)2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36429007

RESUMO

The problem of isolating high-quality total RNA from intervertebral discs has no recognized solution yet. This is due to the extremely low content of live cells in the samples and the voluminous intercellular matrix. A variety of published protocols focused on isolating RNA from articular cartilage have recommended the use of expensive equipment, enzymatic matrix cleavage, or cell culture. In our study, we used a combination of the traditional QIAzol protocol (Qiagen, Germany) and RNEasy column purification (Qiagen, Germany) to obtain high-quality RNA from post-surgical intervertebral disc fragments. Only a mortar and a pestle were used for grinding, making our method particularly accessible. The isolated RNA with a RIN of ~7 is suitable for studying the expression profile of chondrocytes in situ. RNA-seq analysis of three samples demonstrated cell type ratios to be mostly relevant to intervertebral disc tissues, with over 70% of the chondrocytes of the three subtypes having an admixture of blood-related cells.


Assuntos
Cartilagem Articular , Disco Intervertebral , Humanos , RNA-Seq , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , RNA/metabolismo
12.
Biochem Biophys Res Commun ; 636(Pt 1): 64-74, 2022 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-36332484

RESUMO

Gap junctional intercellular communication (GJIC) is indispensable for the maintenance of physiological balance in articular cartilage. Transforming growth factor-ß3 (TGF-ß3), an important growth factor of TGF-ß superfamily, is well recognized to play a unique regulatory role in cartilage development and diseases. However, the role of TGF-ß3 in GJIC in adult chondrocytes remains elusive. This work aims to investigate the effect of TGF-ß3 on gap-junction mediated intercellular communication in chondrocytes. We first showed that TGF-ß3 could enhance the synaptic connections between chondrocytes by scanning electron microscopy (SEM) and promote the cell-to-cell communication in living chondrocytes by scrape loading/dye transfer assay. We then confirmed that TGF-ß3 enhanced cell-to-cell communication via up-regulation of connexin 43 (Cx43). We next found that TGF-ß3-enhanced GJIC required the participation of TGF-beta type I receptor ALK5 and depended on the activation of p-Smad3 signalling. Finally, through inhibitor experiments of SB525334 and SIS3, we demonstrated that TGF-ß3-induced functional GJIC in chondrocytes via the axis of ALK5/p-Smad3 signalling. Taking together, these results demonstrate a strong correlation between TGF-ß3 and GJIC in chondrocytes, which provides a new perspective on the importance of TGF-ß3 on cartilage physiology and pathobiology.


Assuntos
Cartilagem Articular , Condrócitos , Condrócitos/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Fator de Crescimento Transformador beta3/farmacologia , Fator de Crescimento Transformador beta3/metabolismo , Comunicação Celular , Cartilagem Articular/metabolismo , Junções Comunicantes/metabolismo
13.
Nat Commun ; 13(1): 7295, 2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36435829

RESUMO

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.


Assuntos
Cartilagem Articular , Fenômenos Fisiológicos Musculoesqueléticos , Cartilagem Articular/metabolismo , Proteoglicanas/metabolismo , Condrócitos/metabolismo , Regulação da Expressão Gênica
14.
Front Immunol ; 13: 930511, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36325344

RESUMO

Osteoarthritis (OA) reduces the quality of life as a result of the pain caused by continuous joint destruction. Inactivated Lactobacillus (LA-1) ameliorated osteoarthritis and protected cartilage by modulating inflammation. In this study, we evaluated the mechanism by which live LA-1 ameliorated OA. To investigate the effect of live LA-1 on OA progression, we administered LA-1 into monosodium iodoacetate (MIA)-induced OA animals. The pain threshold, cartilage damage, and inflammation of the joint synovial membrane were improved by live LA-1. Furthermore, the analysis of intestinal tissues and feces in the disease model has been shown to affect the systems of the intestinal system and improve the microbiome environment. Interestingly, inflammation of the intestinal tissue was reduced, and the intestinal microbiome was altered by live LA-1. Live LA-1 administration led to an increase in the level of Faecalibacterium which is a short-chain fatty acid (SCFA) butyrate-producing bacteria. The daily supply of butyrate, a bacterial SCFA, showed a tendency to decrease necroptosis, a type of abnormal cell death, by inducing autophagy and reversing impaired autophagy by the inflammatory environment. These results suggest that OA is modulated by changes in the gut microbiome, suggesting that activation of autophagy can reduce aberrant cell death. In summary, live LA-1 or butyrate ameliorates OA progression by modulating the gut environment and autophagic flux. Our findings suggest the regulation of the gut microenvironment as a therapeutic target for OA.


Assuntos
Cartilagem Articular , Osteoartrite , Animais , Condrócitos/metabolismo , Cartilagem Articular/metabolismo , Butiratos/metabolismo , Lactobacillus , Qualidade de Vida , Modelos Animais de Doenças , Osteoartrite/metabolismo , Inflamação/metabolismo , Autofagia , Morte Celular
15.
PLoS One ; 17(11): e0277495, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36367882

RESUMO

Osteoarthritis (OA) is a common disorder and a major cause of disability in the elderly population. WNT16 has been suggested to play important roles in joint formation, bone homeostasis and OA development, but the mechanism of action is not clear. Transgenic mice lacking Wnt16 expression (Wnt16-/-) have a more severe experimental OA than control mice. In addition, Wnt16-/- mice have a reduced cortical thickness and develop spontaneous fractures. Herein, we have used Cre-Wnt16flox/flox mice in which Wnt16 can be conditionally ablated at any age through tamoxifen-inducible Cre-mediated recombination. Wnt16 deletion was induced in 7-week-old mice to study if the Cre-Wnt16flox/flox mice have a more severe OA phenotype after destabilizing the medial meniscus (DMM surgery) than littermate controls with normal Wnt16 expression (Wnt16flox/flox). WNT16 deletion was confirmed in articular cartilage and cortical bone in Cre-Wnt16flox/flox mice, shown by immunohistochemistry and reduced cortical bone area compared to Wnt16flox/flox mice. After DMM surgery, there was no difference in OA severity in the articular cartilage in the knee joint between the Cre-Wnt16flox/flox and Wnt16flox/flox mice in neither female nor male mice. In addition, there was no difference in osteophyte size in the DMM-operated tibia between the genotypes. In conclusion, inactivation of Wnt16 in adult mice do not result in a more severe OA phenotype after DMM surgery. Thus, presence of WNT16 in adult mice does not have an impact on experimental OA development. Taken together, our results from Cre-Wnt16flox/flox mice and previous results from Wnt16-/- mice suggest that WNT16 is crucial during synovial joint establishment leading to limited joint degradation also later in life, after onset of OA. This may be important when developing new therapeutics for OA treatment.


Assuntos
Cartilagem Articular , Osteoartrite , Osteófito , Idoso , Camundongos , Masculino , Feminino , Humanos , Animais , Osteoartrite/genética , Osteoartrite/metabolismo , Cartilagem Articular/metabolismo , Meniscos Tibiais/cirurgia , Meniscos Tibiais/metabolismo , Camundongos Transgênicos , Modelos Animais de Doenças , Proteínas Wnt/metabolismo
16.
Nat Biomed Eng ; 6(10): 1105-1117, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36229661

RESUMO

Treatments for osteoarthritis would benefit from the enhanced visualization of injured articular cartilage and from the targeted delivery of disease-modifying drugs to it. Here, by using ex vivo human osteoarthritic cartilage and live rats and minipigs with induced osteoarthritis, we report the application of collagen-binding peptides, identified via phage display, that are home to osteoarthritic cartilage and that can be detected via magnetic resonance imaging when conjugated with a superparamagnetic iron oxide. Compared with the use of peptides with a scrambled sequence, hyaluronic acid conjugated with the collagen-binding peptides displayed enhanced retention in osteoarthritic cartilage and better lubricated human osteoarthritic tissue ex vivo. Mesenchymal stromal cells encapsulated in the modified hyaluronic acid and injected intra-articularly in rats showed enhanced homing to osteoarthritic tissue and improved its regeneration. Molecular docking revealed WXPXW as the consensus motif that binds to the α1 chain of collagen type XII. Peptides that specifically bind to osteoarthritic tissue may aid the diagnosis and treatment of osteoarthritic joints.


Assuntos
Cartilagem Articular , Osteoartrite , Animais , Humanos , Ratos , Suínos , Cartilagem Articular/diagnóstico por imagem , Cartilagem Articular/metabolismo , Ácido Hialurônico/metabolismo , Lubrificação , Colágeno Tipo XII/metabolismo , Simulação de Acoplamento Molecular , Porco Miniatura , Osteoartrite/metabolismo , Regeneração , Peptídeos/metabolismo
17.
Biochem Biophys Res Commun ; 635: 77-83, 2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-36257195

RESUMO

Articular cartilage defects remain the most common and challenging joint disease. Cartilage lacks the self-healing capacity after injury due to its avascularity. Recently, stem cell-based therapy has been applied for cartilage regeneration. However, the critical target for stem cells during chondrogenesis remains unclear. We first reported that LDL receptor-related protein 3 (LRP3) expression was markedly increased during chondrogenesis in stem cells. Furthermore, LRP3 was an effective chondrogenic stimulator, as confirmed by knockdown and overexpression experiments and RNA sequencing. In addition, inhibition of LRP3 suppressed proliferation and induced apoptosis. Therefore, our study first defined a new chondrogenic stimulator, LRP3, with detailed clarification, which provided a novel target for stem cell-based cartilage regeneration.


Assuntos
Cartilagem Articular , Células-Tronco Mesenquimais , Condrogênese/genética , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Células-Tronco , Cartilagem Articular/metabolismo , Apoptose , Proliferação de Células , Receptores de LDL/metabolismo
18.
Biochem Biophys Res Commun ; 635: 267-276, 2022 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-36308906

RESUMO

Osteoarthritis, a prevalent orthopedic disease, can affect the elderly and causes impairment. The degradation and aberrant homeostasis of cartilage extracellular matrix figure pivotally in the progression of osteoarthritis. Thioredoxin systems plays a role in a wide range of biological processes, including cell proliferation, apoptosis, and oxidative stress. The present study aimed to investigate the unique function and underlying pathophysiological mechanism of TXNRD1 in chondrocytes. An upregulated expression of TXNRD1 was observed in the articular cartilage of osteoarthritis patients compared with normal articular cartilage. Furthermore, in vitro experiments showed that the expression of TXNRD1 was also abnormally increased in IL-1ß-induced primary mouse chondrocytes. Silencing TXNRD1 using siRNA in chondrocytes could effectively inhibit the expression of ADAMTS5 and MMP13, and enhance the expression of COL2A1 and SOX9. The same was true for auranofin, an inhibitor of TXNRD1. This phenomenon indicated that inhibition of TXNRD1 attenuated il-1ß-induced metabolic imbalance of extracellular matrix (ECM) and the progression of chondrocyte osteoarthritis. Further mechanism analysis revealed that the activation of Nrf2 signaling pathway and the expression of heme oxygenase-1 (HO-1) were increased upon TXNRD1 inhibition. Furthermore, auranofin was found to attenuate DMM-induced osteoarthritis progression in vivo. Therefore, the pharmacological downregulation of TXNRD1 may provide an effective novel therapy for OA.


Assuntos
Cartilagem Articular , Osteoartrite , Tiorredoxina Redutase 1 , Animais , Camundongos , Auranofina/farmacologia , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Interleucina-1beta/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Osteoartrite/metabolismo , Tiorredoxina Redutase 1/genética
19.
Food Funct ; 13(22): 11825-11839, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36314362

RESUMO

The imbalance of intestinal flora would induce immune inflammation. Cedrol (CE), found from ginger by our group earlier, has been proven to play an excellent role in ameliorating rheumatoid arthritis (RA) via acting on JAK3, MAPK, and NF-κB. However, there have been no studies on CE ameliorating RA through the regulation of the micro-environment. In this study, the adjuvant arthritis model (AIA) is established to evaluate the weight, arthritis score, paw swelling, bone destruction, immune organ index, inflammatory cell infiltration, cartilage erosion, and metabolic enzymes of kidneys in AIA rats after CE intervention. The results indicated CE could alleviate paw swelling, reduce arthritis score, decrease the secretion of TNF-α, IL-6, and IL-1ß in serum in a dose-dependent manner, and inhibit the immune organ index of the spleen while having no significant effect on metabolic enzymes of the kidney. In addition, pathological sections of ankle and knee joints suggested CE might significantly prevent inflammatory cell infiltration, synovial hyperplasia, and joint degeneration and protect articular cartilage. Then, for the first time, 16S rRNA gene was applied to analyze the regulatory effect of CE on intestinal flora. CE could effectively improve the uniformity, diversity, and richness of intestinal flora, reduce the number of pathogenic bacteria, and increase the proportion of beneficial bacteria, and it significantly inhibited the abundance of Prevotella in RA rats, which was 12.43 times smaller than that in methotrexate. The distribution and excretion of CE in vivo were detected by GC-MS. It was found that CE would massively accumulate in the gastrointestinal tract after oral administration, which is then mainly excreted through feces. Interestingly, the research suggested that CE, which plays a role in the dynamic regulation of the intestinal micro-environment, could be used as a potential component to prevent RA.


Assuntos
Artrite Experimental , Artrite Reumatoide , Cartilagem Articular , Gengibre , Ratos , Animais , RNA Ribossômico 16S , Ratos Sprague-Dawley , Artrite Reumatoide/tratamento farmacológico , Artrite Experimental/metabolismo , Edema , Cartilagem Articular/metabolismo
20.
Exp Mol Med ; 54(10): 1727-1740, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36224344

RESUMO

Osteoarthritis, characterized by articular cartilage degradation, is the leading cause of chronic disability in older adults. Studies have indicated that circular RNAs are crucial regulators of chondrocyte development and are involved in the progression of osteoarthritis. In this study, we investigated the function and mechanism of a circular RNA and its potential for osteoarthritis therapy. The expression levels of circCREBBP, screened by circular RNA sequencing during chondrogenic differentiation in adipose tissue-derived stem cells, and TGFß2 were significantly increased in the cartilage of patients with osteoarthritis and IL-1ß-induced chondrocytes. circCREBBP knockdown increased anabolism in the extracellular matrix and inhibited chondrocyte degeneration, whereas circCREBBP overexpression led to the opposite effects. Luciferase reporter assays, rescue experiments, RNA immunoprecipitation, and RNA pulldown assays confirmed that circCREBBP upregulated TGFß2 expression by sponging miR-1208, resulting in significantly enhanced phosphorylation of Smad1/5 in chondrocytes. Moreover, intra-articular injection of adeno-associated virus-sh-circCrebbp alleviated osteoarthritis in a mouse model of destabilization of the medial meniscus. Our findings reveal a critical role for circCREBBP in the progression of osteoarthritis and provide a potential target for osteoarthritis therapy.


Assuntos
Cartilagem Articular , MicroRNAs , Osteoartrite , Animais , Camundongos , Apoptose , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Proteína de Ligação a CREB/metabolismo , Interleucina-1beta/metabolismo , MicroRNAs/genética , Osteoartrite/genética , Osteoartrite/metabolismo , RNA Circular/genética , Proteína Smad1/metabolismo , Proteína Smad1/farmacologia
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