Efficient lipid-mediated gene transfer to articular chondrocytes

Madry, Henning; Trippel, Stephen B.

doi:10.1038/sj.gt.3301086

Cited by 144 publications

(137 citation statements)

References 34 publications

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“…Cationic liposomes and other lipid-based systems have the advantages of being easy and safe to prepare and do not restrict the size of the DNA that can be delivered [11]. When liposomes have been used for in vivo transfection of chondrocytes in normal rat knees, gene expression occurred in the superficial and middle zones and persisted for at least 3 weeks [19,27]. Therefore, in gene therapy related to human tissues or cells, nonviral, lipidmediated gene transfer may be a safer method.…”

Section: Discussionmentioning

confidence: 99%

“…Another transgenic study compared the efficiencies of different commercially available cationic liposomes including Cellfectin, DMRIE-C, LipofectAmine, Lipofectin, LipoTaxi, TransFast, and the lipid-based reagent FuGENE 6 [19]. 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.…”

Section: Discussionmentioning

confidence: 99%

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Treating Human Meniscal Fibrochondrocytes with hIGF-1 Gene by Liposome

Zhang

Leng

Wang

et al. 2009

Clin Orthop Relat Res

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The menisci are intraarticular fibrocartilaginous structures essential to the normal function of the knee that lack the ability to self-repair. Human meniscal fibrochondrocytes may respond to beneficial genes like human insulin growth factor-1 (hIGF-1) and the meniscal cell may be a feasible donor for gene therapy. To explore this possibility, we amplified the hIGF-1 gene sequence in full length and cloned it into a bicistronic plasmid. This gene was then transfected into cultured human meniscal fibrochondrocytes by the liposome FuGene 6. Green fluorescence was expressed in part of the cells 6 hours after transfection and increased gradually, with a peak concentration of the hIGF-1 in the supernatants to 22.68 ng/mL 56 hours after transfection. Phenotypes of some cells changed and the proliferation accelerated after transfection. Flow cytometry analysis demonstrated upregulation of cell numbers in the G2 and S stages after hIGF-1 gene introduction. We conclude the hIGF-1 gene can be transfected into the human meniscal cell efficiently by liposome and it causes accelerated proliferation and differentiation. Within 10 days after transfection, the cytokine appears to be secreted into supernatants with the bioactivity and promotes the proliferation of the NIH 3T3 cell line.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Treating Human Meniscal Fibrochondrocytes with hIGF-1 Gene by Liposome

Zhang

Leng

Wang

et al. 2009

Clin Orthop Relat Res

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“…The activity of ␤-gal was detected by X-Gal staining to determine the transduction efficiencies (24,25,32,34). Transgene expression was also monitored by indirect immunostaining (25).…”

Section: Methodsmentioning

confidence: 99%

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

Cucchiarini¹,

Thurn²,

Weimer³

et al. 2007

Arthritis & Rheumatism

137

220

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Objective. Human osteoarthritis (OA) is characterized by a pathologic shift in articular cartilage homeostasis toward the progressive loss of extracellular matrix (ECM). The purpose of this study was to investigate the ability of rAAV-mediated SOX9 overexpression to restore major ECM components in human OA articular cartilage.Methods. We monitored the synthesis and content of proteoglycans and type II collagen in 3-dimensional cultures of human normal and OA articular chondrocytes and in explant cultures of human normal and OA articular cartilage following direct application of a recombinant adeno-associated virus (rAAV) SOX9 vector in vitro and in situ. We also analyzed the effects of this treatment on cell proliferation in these systems.Results. Following SOX9 gene transfer, expression levels of proteoglycans and type II collagen increased over time in normal and OA articular chondrocytes in vitro. In situ, overexpression of SOX9 in normal and OA articular cartilage stimulated proteoglycan and type II collagen synthesis in a dose-dependent manner. These effects were not associated with changes in chondrocyte proliferation. Notably, expression of the 2 principal matrix components could be restored in OA articular cartilage to levels similar to those in normal cartilage.Conclusion. These data support the concept of using direct, rAAV-mediated transfer of chondrogenic genes to articular cartilage for the treatment of OA in humans.Osteoarthritis (OA) is a progressive disease that affects diarthrodial joints and is mainly characterized by a gradual deterioration of the articular cartilage. A disturbed balance in cartilage metabolism is thought to play an important role in the pathogenesis of OA and to be a key factor in determining its progression. Over the course of OA, the cartilage loses major components of its extracellular matrix (ECM), such as proteoglycans and type II collagen (1). Proinflammatory cytokines, such as interleukin 1 (IL-1) and tumor necrosis factor ␣, produced locally by the inflamed synovium likely contribute to the pathophysiology of OA (2). Articular chondrocytes are the focus of OA because of pathologic changes in their gene expression pattern (3), the loss of their capacity to synthesize cartilage-specific matrix molecules, and their increased production of matrixdegrading enzymes (4).Despite various therapeutic options, including systemic nonsteroidal antiinflammatory drugs, local corticosteroids, physical therapy, regular exercise, use of orthopedic appliances, or with the advent of disease-and structure-modifying drugs, the management of OA remains an unresolved problem, especially for patients who are too young to undergo endoprosthetic total joint replacement. The difficulty in treating OA is largely due to its slow and irreversible progression and to the limited intrinsic ability of the cartilage to reequilibrate its natural components. Application of therapeutic genes to OA cartilage may offer potent alternatives for reestab- Most of the current approaches to restoring the physiolog...

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“…Recircularized pcDNA 3.1 plasmid was used for mock transfection controls. Tranfections were performed with FuGENE transfection reagent (Roche Diagnostics), according to the manufacturer's instructions, using previously described modifications (36). FuGENE-DNA complexes were applied to the chondrocytes for 4 hours, at which time fresh medium (with or without IGF-1 [100 ng/ml]) was applied, and the transfection was allowed to continue for an additional 44 hours to achieve a total transfection time of 48 hours.…”

Section: Reagentsmentioning

confidence: 99%

Hsp90 mediates insulin‐like growth factor 1 and interleukin‐1β signaling in an age‐dependent manner in equine articular chondrocytes

Boehm¹,

Seth²,

Mayr³

et al. 2007

Arthritis & Rheumatism

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Objective. Many metabolic processes in chondrocytes thought to contribute to age-related changes in the extracellular matrix are influenced by known roles of Hsp90. Age-related decreases in the level of Hsp90 have been documented in numerous cell types and could contribute to cartilage degeneration. The aim of this study was to investigate the roles of age and Hsp90 in insulin-like growth factor 1 (IGF-1) and interleukin-1␤ (IL-1␤) signaling in chondrocytes.Methods. Levels of Hsp90 messenger RNA (mRNA) and protein, with respect to age, were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. The Hsp90 inhibitor geldanamycin (50 nM, 100 nM, or 500 nM) was used to assess age-related responses to Hsp90 with concurrent IGF-1 or IL-1␤ stimulation of chondrocytes. Quantitative real-time PCR was used to measure COL2A1 and matrix metalloproteinase 13 (MMP13) gene expression; Western blot analysis was performed to determine the phosphorylation status of p42/44 and Akt/protein kinase B.Results. The effects of Hsp90 inhibition with geldanamycin were concentration dependent. Inhibition of Hsp90 with 100 nM or 500 nM geldanamycin blocked IGF-1-induced cell proliferation, Akt and p42/44 activation, and COL2A1 expression. Basal and IL-1␤-induced up-regulation of MMP13 mRNA was blocked by all concentrations of geldanamycin tested. Gain-offunction assays with Hsp90 resulted in increased expression of MMP13 mRNA.Conclusion. These results suggest that Hsp90 is involved in opposing signaling pathways of cartilage homeostasis, and that catabolic responses are more sensitive to Hsp90 inhibition than are anabolic responses. Further studies are needed to determine the role of Hsp90 inhibition in osteoarthritis in order to assess its potential as a therapeutic target.

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Efficient lipid-mediated gene transfer to articular chondrocytes

Cited by 144 publications

References 34 publications

Treating Human Meniscal Fibrochondrocytes with hIGF-1 Gene by Liposome

Treating Human Meniscal Fibrochondrocytes with hIGF-1 Gene by Liposome

Restoration of the extracellular matrix in human osteoarthritic articular cartilage by overexpression of the transcription factor SOX9

Hsp90 mediates insulin‐like growth factor 1 and interleukin‐1β signaling in an age‐dependent manner in equine articular chondrocytes

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