Articular Chondrocytes

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Martin Lotz - One of the best experts on this subject based on the ideXlab platform.

  • exosomes from il 1β stimulated synovial fibroblasts induce osteoarthritic changes in Articular Chondrocytes
    Arthritis Research & Therapy, 2014
    Co-Authors: Tomohiro Kato, Martin Lotz, Shigeru Miyaki, Hiroyuki Ishitobi, Yoshihiro Nakamura, Tomoyuki Nakasa, Mitsuo Ochi
    Abstract:

    Osteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of Articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells. Human synovial fibroblasts (SFB) and Articular Chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal Articular Chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis. Exosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in Articular Chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB. Exosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.

  • chemotaxis of human Articular Chondrocytes and mesenchymal stem cells
    Journal of Orthopaedic Research, 2008
    Co-Authors: Yasunori Mishima, Martin Lotz
    Abstract:

    Migration of Chondrocytes and mesenchymal stem cells (MSCs) may be important in cartilage development, tissue response to injury, and in tissue engineering. This study analyzed growth factors and cytokines for their ability to induce migration of human Articular Chondrocytes and bone marrow-derived mesenchymal stem cells in Boyden chamber assays.In human Articular Chondrocytes serum induced dose- and time-dependent increases in cell migration. Among a series of growth factors and cytokines tested only PDGF induced a significant increase in cell migration. The PDGF isoforms AB and BB were more potent than AA. There was an aging-related decline in the ability of Chondrocytes to migrate in response to serum and PDGF. Human bone marrow MSC showed significant chemotaxis responses to several factors, including FBS, PDGF, VEGF, IGF-1, IL-8, BMP-4, and BMP-7. In summary, these results demonstrate that directed cell migration is inducible in human Articular Chondrocytes and MSC. PDGF is the most potent factor analyzed, and may be useful to promote tissue integration during cartilage repair or tissue engineering. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1407–1412, 2008

  • il 18 is produced by Articular Chondrocytes and induces proinflammatory and catabolic responses
    Journal of Immunology, 1999
    Co-Authors: Tsaiwei Olee, Sanshiro Hashimoto, Jacqueline Quach, Martin Lotz
    Abstract:

    IL-18, a cytokine originally identified as IFN-γ-inducing factor, is a member of the IL-1 family of proteins. Because IL-1α and IL-1β are important mediators in the pathogenesis of arthritis, the present study addresses the expression of IL-18 and its role in regulating in Articular Chondrocytes. IL-18 mRNA was induced by IL-1β in Chondrocytes. Chondrocytes produced the IL-18 precursor and in response to IL-1 stimulation secreted the mature form of IL-18. Studies on IL-18 effects on Chondrocytes showed that it inhibits TGF-β-induced proliferation and enhances nitric oxide production. IL-18 stimulated the expression of several genes in normal human Articular Chondrocytes including inducible nitric oxide synthase, inducible cyclooxygenase, IL-6, and stromelysin. Gene expression was associated with the synthesis of the corresponding proteins. Treatment of normal human Articular cartilage with IL-18 increased the release of glycosaminoglycans. These finding identify IL-18 as a cytokine that regulates chondrocyte responses and contributes to cartilage degradation.

  • Integrin expression by human Articular Chondrocytes.
    Arthritis & Rheumatism, 1994
    Co-Authors: Virgil L. Woods, Paul J. Schreck, Dirk S. Gesink, Hector O. Pacheco, David Amiel, Wayne H. Akeson, Martin Lotz
    Abstract:

    Objective. To perform a comprehensive analysis of the integrin forms expressed by normal human Articular Chondrocytes. Methods. Cartilage sections and collagenase-released Chondrocytes were probed with a comprehensive panel of integrin isoform–specific monoclonal antibodies (MAb), using in situ immunohistochemistry techniques, indirect immunofluorescence and flow cytometry, and immunoprecipitation/sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Results. Chondrocytes in cartilage sections reacted with MAb specific for the α5, αv, and β1 integrin subunits and the αvβ3 and αvβ5 heterodimers. They also reacted with a polyclonal antibody specific for the intracytoplasmic portion of the α1 subunit. MAb specific for the αv subunit reacted more strongly with Chondrocytes near the Articular surface than with those in deeper layers of cartilage, and the αvβ3-specific MAb reacted exclusively with Chondrocytes within the most superficial 30 μm of cartilage. Flow cytometric analysis and SDS-PAGE analysis of immunoprecipitates prepared from extracts of cell-surface radioiodinated Chondrocytes confirmed the above observations, and additionally revealed the presence of the α3β1 integrin. Conclusion. Normal human Articular Chondrocytes prominently display substantial quantities of the α1β1, α5β1, and αvβ5 integrin heterodimers, as well as lesser quantities of the α3β1 and αvβ3 heterodimers, The αv subunit–containing integrins are detected more readily on the more superficial Chondrocytes than on Chondrocytes deep within cartilage. These observations provide the basis for analysis of the role of chondrocyte integrins in cartilage homeostasis and in the pathogenesis of joint diseases.

  • hiv 1 transactivator protein tat induces proliferation and tgf beta expression in human Articular Chondrocytes
    Journal of Cell Biology, 1994
    Co-Authors: Martin Lotz, I Clarklewis
    Abstract:

    The human immunodeficiency virus-1 (HIV-1) protein Tat binds to cell surface antigens and can regulate cellular responses. Tat has similar immunosuppressive effects as transforming growth factor-beta (TGF beta) and both inhibit lymphocyte proliferation. TGF beta is expressed by primary human Articular Chondrocytes and is their most potent growth factor. The present study analyzed the interactions of TGF beta and HIV Tat in the regulation of human Articular Chondrocytes. Synthetic or recombinant full-length Tat (1-86) induced chondrocyte proliferation and this was of similar magnitude as the response to TGF beta. Tat peptides that did not contain the RGD motif had similar chondrocyte stimulatory activity as full-length Tat. Among a series of Tat peptides, peptide 38-62 which contains the basic domain was the only one active, suggesting that this region is responsible for the effects on chondrocyte proliferation. Full-length Tat and peptide 38-62 synergized with TGF beta and induced proliferative responses that were greater than those obtained with any combination of the known chondrocyte growth factors. Further characterization of the interactions between Tat and TGF beta showed that Tat increased synthesis and TGF beta activity and TGF beta 1 mRNA levels. The stimulatory effects of Tat and peptide 38-62 on chondrocyte proliferation were reduced by neutralizing antibodies to TGF beta and by TGF beta antisense oligonucleotides. These results identify a virally encoded protein and a synthetic peptide derived from it as novel and potent chondrocyte growth stimuli which act at least in part through the induction of TGF beta.

Di Chen - One of the best experts on this subject based on the ideXlab platform.

  • fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2 induced catabolic activities in human Articular Chondrocytes
    Arthritis Research & Therapy, 2011
    Co-Authors: Di Chen, Katalin Mikecz, Simon M Cool, Andre J Van Wijnen, Gillian Murphy, Heejeong Im
    Abstract:

    Introduction: Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human Articular Chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, upregulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA. Methods: Primary human Articular Chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity. Results: Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human Articular Chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic Chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal Chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human Articular Chondrocytes.

  • inhibition of β catenin signaling in Articular Chondrocytes results in Articular cartilage destruction
    Arthritis & Rheumatism, 2008
    Co-Authors: Mo Chen, Michael J Zuscik, Qiuqian Wu, Yong Jun Wang, Randy N Rosier, Regis J Okeefe, Di Chen
    Abstract:

    Osteoarthritis (OA) is a degenerative joint disease. Numerous genetic and environmental factors have been proposed as contributors to the development of OA. The progression of the disease is slow and eventually results in degeneration and loss of the Articular cartilage in various joints, including fingers, knees, hips, and spine. The disease process leads to limitation of joint movement, joint deformity, joint stiffness, inflammation, and severe pain. The mechanism of OA pathogenesis remains undefined. OA is mediated by a dynamic interplay between the Articular Chondrocytes, their matrix, and synovial cells. Articular Chondrocytes, the cell type present in Articular cartilage, are responsible for producing and maintaining the extracellular matrix (ECM), which supports the appropriate biomechanical function of the tissue. Phenotypic changes seen in OA are often accompanied by ECM degradation, which eventually results in cartilage destruction (1). Synovial fibroblasts are mesenchyme-derived cells that form a thin lining of synovial tissue surrounding the fibrous capsule of the joint. The physiologic roles of synovial tissue are to produce synovial fluid that lubricates the joints and to supply nutrients to the Articular Chondrocytes. Articular cartilage plays a crucial role in joint function during adulthood, and destruction of Articular cartilage has severe consequences. While most cartilages are replaced by bone during development, only a few cartilages are permanent, including Articular cartilage. Articular Chondrocytes, which function to maintain the matrix, are unique among growth plate Chondrocytes in that they exhibit very limited mitotic activity, have a slow rate of matrix synthesis and degradation, and do not progress fully to the terminally differentiated phenotype displayed by cells in the growth plate (2). Very little is known regarding the mechanisms involved in establishing and maintaining this Articular chondrocyte phenotype. The function of Articular Chondrocytes is regulated by a variety of growth factors, including Wnt/β-catenin signaling molecules, which play a critical role in chondrocyte proliferation, differentiation, and apoptosis. Beta-catenin is a key molecule in the canonical Wnt signaling pathway and plays a critical role in multiple steps during chondrocyte formation and maturation (3,4). Inhibitor of β-catenin and T cell factor (ICAT) is an 82-amino-acid small molecule (5) whose crystal structure reveals binding capacity to the armadillo repeats of β-catenin. This binding disrupts the complex formation of β-catenin with T cell factor (TCF)/lymphoid enhancer factor (LEF) (6,7) and thus leads to inhibition of signaling in this pathway. To investigate the role of β-catenin signaling in the maintenance of Articular cartilage function under physiologic and pathophysiologic conditions, we generated Col2a1-ICAT–transgenic mice in which the ICAT transgene was targeted specifically to Chondrocytes using the Col2a1 promoter. We found that the ICAT transgene was highly expressed in Articular Chondrocytes in 6-month-old transgenic mice, leading to inhibition of β-catenin signaling in transgenic mouse Chondrocytes. Associated with this, severe Articular cartilage destruction was observed in Col2a1-ICAT–transgenic mice. Articular chondrocyte apoptosis was significantly increased, providing novel evidence regarding the contribution of β-catenin signaling to the maintenance of normal Articular cartilage function.

  • Tamoxifen-Inducible Cre-Recombination in Articular Chondrocytes of Adult Col2a1-CreERT2 Transgenic Mice
    Osteoarthritis and Cartilage, 2007
    Co-Authors: Mo Chen, Alexander C. Lichtler, Regis J. O'keefe, Di Chen
    Abstract:

    Summary Objective To determine the specificity and efficiency of the tamoxifen (TM)-induced Cre-recombination in Articular Chondrocytes of adult Col2a1-CreER T2 transgenic mice. Methods Col2a1-CreER T2 transgenic mice were bred with Rosa26 reporter mice. Two-week-old Col2a1-CreER T2 ;R26R mice were administered TM for 5 days and were sacrificed 1 and 6 months after TM induction. X-Gal staining was performed. Results Efficient Cre-recombination is achieved in adult Articular Chondrocytes 1 and 6 months after TM induction. Conclusion Our findings demonstrate that the Col2a1-CreER T2 transgenic mouse model is a valuable tool to target genes specifically expressed in Articular Chondrocytes in a temporally controlled manner in adult mice.

Heejeong Im - One of the best experts on this subject based on the ideXlab platform.

  • fibroblast growth factor receptor 1 is principally responsible for fibroblast growth factor 2 induced catabolic activities in human Articular Chondrocytes
    Arthritis Research & Therapy, 2011
    Co-Authors: Di Chen, Katalin Mikecz, Simon M Cool, Andre J Van Wijnen, Gillian Murphy, Heejeong Im
    Abstract:

    Introduction: Cartilage degeneration driven by catabolic stimuli is a critical pathophysiological process in osteoarthritis (OA). We have defined fibroblast growth factor 2 (FGF-2) as a degenerative mediator in adult human Articular Chondrocytes. Biological effects mediated by FGF-2 include inhibition of proteoglycan production, upregulation of matrix metalloproteinase-13 (MMP-13), and stimulation of other catabolic factors. In this study, we identified the specific receptor responsible for the catabolic functions of FGF-2, and established a pathophysiological connection between the FGF-2 receptor and OA. Methods: Primary human Articular Chondrocytes were cultured in monolayer (24 hours) or alginate beads (21 days), and stimulated with FGF-2 or FGF18, in the presence or absence of FGFR1 (FGF receptor 1) inhibitor. Proteoglycan accumulation and chondrocyte proliferation were assessed by dimethylmethylene blue (DMMB) assay and DNA assay, respectively. Expression of FGFRs (FGFR1 to FGFR4) was assessed by flow cytometry, immunoblotting, and quantitative real-time PCR (qPCR). The distinctive roles of FGFR1 and FGFR3 after stimulation with FGF-2 were evaluated using either pharmacological inhibitors or FGFR small interfering RNA (siRNA). Luciferase reporter gene assays were used to quantify the effects of FGF-2 and FGFR1 inhibitor on MMP-13 promoter activity. Results: Chondrocyte proliferation was significantly enhanced in the presence of FGF-2 stimulation, which was inhibited by the pharmacological inhibitor of FGFR1. Proteoglycan accumulation was reduced by 50% in the presence of FGF-2, and this reduction was successfully rescued by FGFR1 inhibitor. FGFR1 inhibitors also fully reversed the up-regulation of MMP-13 expression and promoter activity stimulated by FGF-2. Blockade of FGFR1 signaling by either chemical inhibitors or siRNA targeting FGFR1 rather than FGFR3 abrogated the up-regulation of matrix metalloproteinases 13 (MMP-13) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif 5 (ADAMTS5), as well as down-regulation of aggrecan after FGF-2 stimulation. Flow cytometry, qPCR and immunoblotting analyses suggested that FGFR1 and FGFR3 were the major FGFR isoforms expressed in human Articular Chondrocytes. FGFR1 was activated more potently than FGFR3 upon FGF-2 stimulation. In osteoarthritic Chondrocytes, FGFR3 was significantly down regulated (P < 0.05) with a concomitant increase in the FGFR1 to FGFR3 expression ratio (P < 0.05), compared to normal Chondrocytes. Our results also demonstrate that FGFR3 was negatively regulated by FGF-2 at the transcriptional level through the FGFR1-ERK (extracellular signal-regulated kinase) signaling pathway in human Articular Chondrocytes.

Stephen B Trippel - One of the best experts on this subject based on the ideXlab platform.

  • Role of Sox9 in Growth Factor Regulation of Articular Chondrocytes
    Journal of Cellular Biochemistry, 2015
    Co-Authors: Congrong Wang, George J. Eckert, Anthony J. Acton, Stephen B Trippel
    Abstract:

    Chondrogenic polypeptide growth factors influence Articular chondrocyte functions that are required for Articular cartilage repair. Sox9 is a transcription factor that regulates chondrogenesis, but its role in the growth factor regulation of chondrocyte proliferation and matrix synthesis is poorly understood. We tested the hypotheses that selected chondrogenic growth factors regulate sox9 gene expression and protein production by adult Articular Chondrocytes and that sox9 modulates the actions of these growth factors. To test these hypotheses, we delivered insulin-like growth factor-I (IGF-I), fibroblast growth factor-2 (FGF-2), bone morphogenetic protein-2 (BMP-2) and/or bone morphogenetic protein-7 (BMP-7), or their respective transgenes to adult bovine Articular Chondrocytes, and measured changes in sox9 gene expression and protein production. We then knocked down sox9 gene expression with sox9 siRNA, and measured changes in the expression of the genes encoding aggrecan and types I and II collagen, and in the production of glycosaminoglycan, collagen and DNA. We found that FGF-2 or the combination of IGF-I, BMP-2, and BMP-7 increased sox9 gene expression and protein production and that sox9 knockdown modulated growth factor actions in a complex fashion that differed both with growth factors and with chondrocyte function. The data suggest that sox9 mediates the stimulation of matrix production by the combined growth factors and the stimulation of chondrocyte proliferation by FGF-2. The mitogenic effect of the combined growth factors and the catabolic effect of FGF-2 appear to involve sox9-independent mechanisms. Control of these molecular mechanisms may contribute to the treatment of cartilage damage. J. Cell. Biochem. 116: 1391–1400, 2015. © 2015 Wiley Periodicals, Inc.

  • Endogenous versus Exogenous Growth Factor Regulation of Articular Chondrocytes
    Journal of Orthopaedic Research, 2013
    Co-Authors: Albert Chan, George J. Eckert, Scott Mercer, Stephen B Trippel
    Abstract:

    Anabolic growth factors that regulate the function of Articular Chondrocytes are candidates for Articular cartilage repair. Such factors may be delivered by pharmacotherapy in the form of exogenous proteins, or by gene therapy as endogenous proteins. It is unknown whether delivery method influences growth factor effectiveness in regulating Articular chondrocyte reparative functions. We treated adult bovine Articular Chondrocytes with exogenous recombinant insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-β1), or with the genes encoding these growth factors for endogenous production. Treatment effects were measured as change in chondrocyte DNA content, glycosaminoglycan production, and aggrecan gene expression. We found that IGF-I stimulated chondrocyte biosynthesis similarly when delivered by either exogenous or endogenous means. In contrast, exogenous TGF-s1 stimulated these reparative functions, while endogenous TGF-s1 had little effect. Endogenous TGF-s1 became more bioactive following activation of the transgene protein product. These data indicate that effective mechanisms of growth factor delivery for Articular cartilage repair may differ for different growth factors. In the case of IGF-I, gene therapy or protein therapy appear to be viable options. In contrast, TGF-s1 gene therapy may be constrained by a limited ability of Chondrocytes to convert latent complexes to an active form.

  • Growth factor transgenes interactively regulate Articular Chondrocytes
    Journal of Cellular Biochemistry, 2013
    Co-Authors: Scott Mercer, George J. Eckert, Stephen B Trippel
    Abstract:

    Adult Articular Chondrocytes lack an effective repair response to correct damage from injury or osteoarthritis. Polypeptide growth factors that stimulate Articular chondrocyte proliferation and cartilage matrix synthesis may augment this response. Gene transfer is a promising approach to delivering such factors. No single growth factor gene is likely to optimize these cell functions, but multiple growth factor gene transfer remains unexplored. We tested the hypothesis that multiple growth factor gene transfer selectively modulates Articular chondrocyte proliferation and matrix synthesis. We tested the hypothesis by delivering combinations of the transgenes encoding insulin-like growth factor I (IGF-I), fibroblast growth factor-2 (FGF-2), transforming growth factor beta1 (TGF-β1), bone morphogenetic protein-2 (BMP-2) and bone morphogenetic protien-7 (BMP-7) to Articular Chondrocytes and measured changes in the production of DNA, glycosaminoglycan and collagen. The transgenes differentially regulated all these chondrocyte functions. In concert, the transgenes interacted to generate widely divergent responses from the cells. These interactions ranged from inhibitory to synergistic. The transgene pair encoding IGF-I and FGF-2 maximized cell proliferation. The three-transgene group encoding IGF-I, BMP-2 and BMP-7 maximized matrix production and also optimized the balance between cell proliferation and matrix production. These data demonstrate a potentially tunable approach to Articular chondrocyte regulation and suggest that certain growth factor gene combinations have potential value for cell-based Articular cartilage repair.

  • Effect of transfection strategy on growth factor overexpression by Articular Chondrocytes
    Journal of Orthopaedic Research, 2009
    Co-Authors: Scott Mercer, Stephen B Trippel
    Abstract:

    Articular cartilage damage remains an unsolved problem in orthopaedics. Insulin-like growth factor I (IGF-I) and fibroblast growth factor-2 (FGF-2) are anabolic and mitogenic for Articular Chondrocytes, and are candidates for the application of gene therapy to Articular cartilage repair. We tested the hypothesis that the production of IGF-I and FGF-2 can be augmented by modulating vector designs and delivery methods used for gene transfer to Articular Chondrocytes. We developed a novel adeno-associated virus (AAV)-based plasmid (pAAV) to overexpress IGF-I and FGF-2 cDNAs in adult bovine Articular Chondrocytes. We found that the pAAV-based vectors generated significantly more growth factor than pcDNA vectors carrying the same cDNAs. Chondrocytes cotransfected with both IGF-I and FGF-2 cDNAs in two separate pAAV plasmids produced significantly more IGF-I and FGF-2 than cells transfected by a single pAAV plasmid carrying both cDNAs in a dicistronic cassette. These data indicate that pAAV vectors are more effective than pcDNA vectors for transfer of IGF-I and FGF-2 genes to Articular Chondrocytes. They further suggest that cotransfection may be an effective strategy for multiple gene transfer to these cells. These findings may be important in applying growth factor gene transfer to cell-based Articular cartilage gene therapy.

  • efficient lipid mediated gene transfer to Articular Chondrocytes
    Gene Therapy, 2000
    Co-Authors: Henning Madry, Stephen B Trippel
    Abstract:

    We examined nonviral, lipid-mediated gene transfer methods as potential tools for efficient transfection of Articular Chondrocytes. Transfection conditions were determined for primary cultures of normal human Articular, osteoarthritic human Articular and normal bovine Articular Chondrocytes using a lacZ reporter gene construct with the commercially available cationic liposomes Cellfectin, DMRIE-C, LipofectAmine, Lipofectin, LipoTaxi, TransFast and the lipid-based reagent FuGENE 6. Optimized conditions were then evaluated in an ex vivo model of chondrocyte transplantation. FuGENE 6 transfection produced the maximum levels of transgene expression. Transfection efficiency was cell type specific and affected by DNA concentration, lipid/DNA ratio and the presence of hyaluronidase, a matrix-degrading enzyme. Analysis of X-gal staining demonstrated an efficiency of 41.0% in normal bovine Articular Chondrocytes, 20.7% in normal human Articular Chondrocytes and 7.8% in osteoarthritic human Chondrocytes. Transfected Chondrocytes were found to successfully populate the Articular cartilage surface in explant cultures. Transplanted genetically modified Chondrocytes adhered to the Articular cartilage and continued to produce β-galactosidase for 2 weeks. This evaluation and optimization of lipid-based gene transfer into Articular Chondrocytes may serve as a useful tool in studies of genes involved in Articular cartilage damage and repair and as a potential delivery method for therapeutic genes.

M.c. Lopes - One of the best experts on this subject based on the ideXlab platform.

  • Hydrogen peroxide mediates interleukin-1β-induced AP-1 activation in Articular Chondrocytes: Implications for the regulation of iNOS expression
    Cell Biology and Toxicology, 2003
    Co-Authors: A Ferreira Mendes, M.m. Caramona, A.p. Carvalho, M.c. Lopes
    Abstract:

    The pro-inflammatory cytokine interleukin-1β (IL-1) induces Articular Chondrocytes to produce reactive oxygen species (ROS), including hydrogen peroxide (H_2O_2), which mediate some IL-1-induced responses. This study aimed at elucidating the role of ROS, pArticularly H_2O_2, in mediating IL-1-induced activation of the transcription factor activator protein-1 (AP-1) in primary cultures of Articular Chondrocytes. AP-1 may function either as an inducer or as a repressor of the inducible nitric oxide synthase (iNOS) gene promoter. Since we observed that AP-1 is not required for iNOS expression in Chondrocytes, we also investigated whether it is a repressor of this gene. The results of electrophoretic mobility shift assays showed that both IL-1 and H_2O_2 activated AP-1 and that inhibition of IL-1-induced ROS production abrogated AP-1 activation. The AP-1 complexes, induced by either IL-1 or H_2O_2, contained c-Fos/c-Jun and c-Fos/JunD heterodimers, but IL-1 activated AP-1 with a kinetics slower than that observed with H_2O_2. Pre-activation of AP-1, before stimulation of the cells with IL-1, did not inhibit iNOS mRNA and protein synthesis, relative to cells treated with IL-1 alone. These results indicate that H_2O_2 is a major mediator of IL-1-induced AP-1 activation in Articular Chondrocytes and that inhibition of ROS production is an effective strategy to block this IL-1-induced response. This study also identifies c-Fos/c-Jun and c-Fos/JunD heterodimers as the AP-1 transcription factors induced by IL-1, which, although not involved in the transcriptional regulation of the iNOS gene, may be important for the regulation of other genes also relevant in arthritic diseases, namely the collagenase-1 and IL-8 genes.