Evaluation of chondrocyte cell-associated matrix metabolism by flow cytometry

Wang, Lai; Almqvist, Karl; Broddelez, C; Veys, Eric; Verbruggen, G.

doi:10.1053/joca.2001.0412

Cited by 17 publications

(18 citation statements)

References 37 publications

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“…Monolayer cultures were trypsinized, resuspended in complete media, then mixed 1:1 with 0.8% low melt agarose (Life Technologies, Rockville, MD) in DMEM at 37°C (before gelling), giving a final concentration of 1 × 10 4 cells/ml suspended in 0.4% low melt agarose. Eight milliliter were pipeted into 10‐cm tissue culture plates that were previously coated with 8 ml of 0.7% low melt agarose in DMEM, 10% FBS (Robson et al, 2000; Wang et al, 2001) which yielded a total of 16 ml of agarose‐media to 8 × 10 4 cells. The agarose was allowed to gel at room temperature, and then placed in a 37°C, 5% CO 2 humidified incubator for 3–4 weeks to allow cell cluster growth to reach quantifiable size and number.…”

Section: Methodsmentioning

confidence: 99%

Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis

Quintavalla¹,

Kumar²,

Daouti³

et al. 2005

Journal Cellular Physiology

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Understanding altered gene expression in osteoarthritic cartilage can lead to new targets for drug intervention. We established a functional assay based on chondrocyte cluster formation, a phenotype associated with osteoarthritis (OA), to screen an OA cartilage gene library. Previous reports have demonstrated that normal chondrocytes grown in suspension culture maintain their chondrocytic phenotype, however, certain growth factors such as basic fibroblast growth factor (bFGF) will induce the cells to proliferate in tight clusters similar to those seen in osteoarthritic cartilage. In this study we validate that overexpression of bFGF by retrovirally transduced normal chondrocytes would similarly induce the proliferation of tight cell clusters. We then used this approach as a basis to set up a functional screen where an entire OA cartilage cDNA library was tranduced into normal chondrocytes to search for other genes that would also induce cluster formation. Seven potential genes were isolated from the OA gene library, including BPOZ, IL-17 receptor C, NADH ubiquinone oxidoreductase, COMP, Soluble carrier 16 (MCT 3), C1r, and bFGF itself. None of the identified genes were upregulated by bFGF, however, all of them upregulated the expression of bFGF suggesting a common pathway. Although cluster formation is not considered to be destructive in OA cartilage, it is consistent with the disease and could yield answers to the altered phenotype. Further studies are needed to elucidate how these genes are linked to the disease state.

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Section: Methodsmentioning

confidence: 99%

Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis

Quintavalla¹,

Kumar²,

Daouti³

et al. 2005

Journal Cellular Physiology

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“…Cartilage ECM is formed and maintained by chondrocytes (12)(13)(14)(15). Although the mechanisms of ECM turnover and homeostasis have not been studied in great detail, research in several laboratories has substantiated the importance of cartilage-specific collagens and the large aggregating proteoglycan aggrecan in the maintenance of cartilage integrity and homeostasis (12)(13)(14)(15). Extensive research has also confirmed the importance of the sex-determining region Y-type high mobility group box (SOX) family of high mobility group transcription factors (L-SOX5, SOX6, and SOX9) in the expression of cartilage-specific proteins (13).…”

mentioning

confidence: 99%

WISP3‐dependent regulation of type II collagen and aggrecan production in chondrocytes

Sen¹,

Cheng²,

Goldring³

et al. 2004

Arthritis & Rheumatism

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Objective. WISP3 (Wnt-1-inducible secreted protein 3) is a member of the CCN (connective tissue growth factor, cysteine-rich 61, nephroblastoma overexpressed) family of connective tissue growth factors. WISP3 mutations have been linked to progressive pseudorheumatoid dysplasia (PPRD). The present study was conducted to investigate whether WISP3 is responsible for the expression of cartilage-specific molecules.Methods. WISP3 expression in human cartilage was assessed by immunostaining with anti-WISP3 antibody. The effect of WISP3 on chondrocyte-specific gene regulation was determined by transfecting human chondrocyte lines C-28/I2 and T/C-28a2 with a WISP3 expression vector. Alterations in WISP3-mediated messenger RNA and protein expression of cartilage-specific molecules were assessed by reverse transcriptasepolymerase chain reaction and immunoblotting.Results. Immunohistochemistry experiments demonstrated that WISP3 protein is expressed in the midzone chondrocytes of normal adult articular cartilage, in chondrocyte clusters of osteoarthritic cartilage, and in the zone of proliferating chondrocytes of fetal growth cartilage. Human chondrocyte lines C-28/I2 and T/C-28a2 transfected with a WISP3 expression vector produced increased amounts of the cartilage-specific matrix molecules type II collagen and aggrecan, in part via activation of the sex-determining region Y-type high mobility group box (SOX) family of transcription factors. In contrast, a mutant WISP3, previously found to be associated with PPRD, had impaired effects on cartilage-specific gene expression.Conclusion. Our experimental results suggest that WISP3 supports cartilage integrity by regulating the expression of type II collagen and aggrecan, and mutations linked with PPRD can compromise this function and produce cartilage loss.

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“…The MFI of the cell samples was then analyzed without changing any of the instrument settings. The reproducibility and reliability of this procedure have been demonstrated previously (36, 38).…”

Section: Methodsmentioning

confidence: 58%

“…Cells were gated on forward and side scatter to exclude dead cells, debris, and aggregates. Propidium iodide was additionally used to exclude dead cells when the epitopes outside the cells (i.e., IGFRI, IL‐1RI, and IL‐1RII) and the ECM molecules were analyzed (36, 38). The mean fluorescence intensity (MFI) of the positive cell population, which is due to the binding of the conjugated antibodies to the specific antigen, was used to quantify the presence of IGFRI, IL‐1RI, and IL‐1RII on the plasma membrane, the ECM molecules in the CAM, and the accumulation of IGF‐1, IL‐1α, and IL‐1β inside the cells.…”

Section: Methodsmentioning

confidence: 99%

Insulin‐like growth factor 1–induced interleukin‐1 receptor II overrides the activity of interleukin‐1 and controls the homeostasis of the extracellular matrix of cartilage

Wang¹,

Elewaut²,

Veys³

et al. 2003

Arthritis & Rheumatism

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Objective. We examined the effect of the insulinlike growth factor 1 (IGF-1)/IGF receptor I (IGFRI) autocrine/paracrine anabolic pathway on the extracellular matrix (ECM) of human chondrocytes and the mechanism by which IGF-1 reverses the catabolic effects of interleukin-1 (IL-1).Methods. Phenotypically stable human articular cartilage cells were obtained from normal cartilage and maintained in culture in alginate beads for 1 week to reach equilibrium of accumulated cell-associated matrix (CAM) compounds. Levels of CAM components aggrecan and type II collagen ( Conclusion. These findings reveal a new paradigm by which IGF-1 influences chondrocyte metabolism, by reversing the IL-1-mediated catabolic pathway through up-regulation of its decoy receptor.Homeostasis of the extracellular matrix (ECM) of articular cartilage is dependent on the responses of articular cartilage cells to autocrine and paracrine anabolic and catabolic pathways. The most relevant growth factors and cytokines known to be involved in cartilage metabolism are produced by the chondrocytes themselves (1,2) Synthesis and accumulation of the ECM is regulated by locally produced growth factors, such as the insulin-like growth factors (IGFs) and transforming growth factor ␤. A large body of experimental data has substantiated the importance of IGFs 1 and 2 as promoters of growth and matrix synthesis by articular cartilage cells. Both IGFs enhance aggrecan synthesis by human articular cartilage cells cultured in serum-free medium (3,4). The two forms of IGF, however, differentially affect chondrocyte metabolism. It has been suggested that in cartilage, IGF-1 is the regulator of growth and differentiation, whereas IGF-2 may be an important regulator of glucose metabolism (5). IGF-1 has been shown to enhance the synthesis of aggrecan and type II collagen (CII) by chondrocytes (6-9) and to directly decrease both basal and cytokine-stimulated degradation of proteoglycan in cartilage (10). Although it has been proposed that the action of IGF-1 is predominantly as an endocrine factor, whereas IGF-2 interacts Supported by an FWO grant (3G013201). Studies were performed in collaboration with the

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Evaluation of chondrocyte cell-associated matrix metabolism by flow cytometry

Abstract: Flow cytometry offers an efficient tool to study the metabolism of the chondrocyte CAM. The MFI has been used as a parameter to quantify the ECM molecules in the CAM.

Cited by 17 publications

References 37 publications

Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis

Chondrocyte cluster formation in agarose cultures as a functional assay to identify genes expressed in osteoarthritis

WISP3‐dependent regulation of type II collagen and aggrecan production in chondrocytes

Insulin‐like growth factor 1–induced interleukin‐1 receptor II overrides the activity of interleukin‐1 and controls the homeostasis of the extracellular matrix of cartilage

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