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Effect of transfection strategy on growth factor overexpression by articular chondrocytes.

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.

SUBMITTER: Shi S

PROVIDER: S-EPMC4133934 | biostudies-other | 2010 Jan

REPOSITORIES: biostudies-other

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Effect of transfection strategy on growth factor overexpression by articular chondrocytes.

Shi Shuiliang S Mercer Scott S Trippel Stephen B SB

Journal of orthopaedic research : official publication of the Orthopaedic Research Society 20100101 1

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-ass ...[more]

PMID: 19637273

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Project description: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, up-regulation 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.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.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.FGFR1 is the major mediator with the degenerative potential in the presence of FGF-2 in human adult articular chondrocytes. FGFR1 activation by FGF-2 promotes catabolism and impedes anabolism. Disruption of the balance between FGFR1 and FGFR3 signaling ratio may contribute to the pathophysiology of OA.

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Effect of transfection strategy on growth factor overexpression by articular chondrocytes.

Publications

Effect of transfection strategy on growth factor overexpression by articular chondrocytes.

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