Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies

Girkontaitė, Irutė; Frischholz, Svenja; Lammi, Pirkko; Wagner, Klaus; Swoboda, B.; Aigner, Thomas; K, von der Mark

doi:10.1016/s0945-053x(96)90114-6

Cited by 197 publications

(154 citation statements)

References 34 publications

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“…We found that many genes (e.g., Ltbp2, Phex, and Reln) were regulated similarly in both degrading cartilage and differentiating chondrocytes, positioning mechanisms leading to hypertrophic differentiation of chondrocytes in early OA. This finding is supported by the results of other studies, which have led investigators to propose that recapitulation of chondrocyte differentiation to hypertrophy may be responsible for cartilage loss in human OA (12,13,46,63).…”

Section: Discussionsupporting

confidence: 79%

Global analyses of gene expression in early experimental osteoarthritis

Appleton¹,

Pitelka²,

Henry³

et al. 2007

Arthritis & Rheumatism

222

224

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Objective. To analyze genome-wide changes in chondrocyte gene expression in a surgically induced model of early osteoarthritis (OA) in rats, to assess the similarity of this model to human OA, and to identify genes and mechanisms leading to OA pathogenesis.Methods. OA was surgically induced in 5 rats by anterior cruciate ligament transection and partial medial meniscectomy. Sham surgery was performed in 5 additional animals, which were used as controls. Both groups underwent 4 weeks of forced mobilization, 3 times per week. RNA was extracted directly from articular chondrocytes in the OA (operated), contralateral, and sham-operated knees. Affymetrix GeneChip expression arrays were used to assess genome-wide changes in gene expression. Expression patterns of selected dysregulated genes, including Col2a1, Mmp13, Adamts5, Ctsc, Ptges, and Cxcr4, were validated by real-time polymerase chain reaction, immunofluorescence, or immunohistochemistry 2, 4, and 8 weeks after surgery.Results. After normalization, comparison of OA and sham-operated samples showed 1,619 differentially expressed probe sets with changes in their levels of expression >1.5-fold, 722 with changes >2-fold, 135 with changes >4-fold, and 20 with changes of 8-fold. Dysregulated genes known to be involved in human OA included Mmp13, Adamts5, and Ptgs2, among others. Several dysregulated genes (e.g., Reln, Phex, and Ltbp2) had been identified in our earlier microarray study of hypertrophic chondrocyte differentiation. Other genes involved in cytokine and chemokine signaling, including Cxcr4 and Ccl2, were identified. Changes in gene expression were also observed in the contralateral knee, validating the sham operation as the appropriate control.Conclusion. Our results demonstrate that the animal model mimics gene expression changes seen in human OA, supporting the relevance of newly identified genes and pathways to early human OA. We propose new avenues for OA pathogenesis research and potential targets for novel OA treatments, including cathepsins and cytokine, chemokine, and growth factor signaling pathways, in addition to factors controlling the progression of chondrocyte differentiation.

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Section: Discussionsupporting

confidence: 79%

Global analyses of gene expression in early experimental osteoarthritis

Appleton¹,

Pitelka²,

Henry³

et al. 2007

Arthritis & Rheumatism

222

224

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“…It has also been 416 STOKES ET AL shown that chondrocytes within the deep zones of OA cartilage can express type X collagen, indicating that they may be undergoing hypertrophy (8,9,72,73). Others have observed the synthesis by OA chondrocytes of aggrecan molecules that resemble the structure of aggrecan molecules in fetal chondrocytes (11).…”

Section: Discussionmentioning

confidence: 99%

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

Stokes¹,

Liu²,

Coimbra³

et al. 2002

Arthritis & Rheumatism

152

106

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Objective. To study the changes in patterns of gene expression exhibited by human chondrocytes as they dedifferentiate into fibroblastic cells in culture in order to better understand the mechanisms that control this process and its relationship to the phenotypic changes that occur in chondrocytes during the development of osteoarthritis (OA).Methods. Human fetal epiphyseal chondrocytes (HFCs) were cultured either on poly-(2-hydroxyethyl methacrylate)-coated plates (differentiated HFC cultures) or in plastic tissue culture flasks as monolayers (dedifferentiated HFC cultures). Following 11 days of culture under either condition, poly(A؉) RNA was isolated from the two cell populations and subjected to a gene expression analysis using a microarray containing ϳ5,000 known human genes and ϳ3,000 expressed sequence tags (ESTs).Results. A >2-fold difference in the expression of 62 known genes and 6 ESTs was observed between the two cell types. The differences in expression of several of the genes detected by the microarray hybridization were confirmed by Northern analyses. Two transcription factor genes, TWIST and HIF-1␣, and a cellular adhesion protein gene, cadherin 11, were markedly regulated in response to differentiation and dedifferentiation. Expression of these genes was also detected in adult normal and OA cartilage and chondrocytes. Analysis of the gene expression profile of HFCs revealed a complex pattern of gene expression, including many genes not yet reported to be expressed by chondrocytes.Conclusion. Chondrocytes in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one of expression of chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. Changes in gene expression associated with the process of dedifferentiation of HFCs in vitro were observed in a wide variety of genes, including genes encoding extracellular matrix proteins, transcription factors, and growth factors. At least 3 of the genes that were regulated in response to dedifferentiation were also found to be expressed in adult normal and OA articular cartilage and chondrocytes.

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“…Alternatively, cells were incubated with unconjugated antibodies specific for Fgfr3, Igf2r, Pthr1, or collagen X, (26) washed, and labeled with corresponding conjugated secondary antibodies (Molecular Probes, Carlsbad, CA) in 50 mL PBS-5% FCS on ice for 15 minutes. For detection of intracellular collagen X, cells were fixed in 1% paraformaldehyde (PFA)-PBS for 10 minutes and permeabilized in 0.2% Triton-X-100-PBS for 5 minutes on ice prior to staining.…”

Section: Immunomagnetic Cell Selectionmentioning

confidence: 99%

“…For this reason, we established a new staining method to detect intracellular collagen X. Once growth plate-derived cells were fixed and permeabilized, a monoclonal antibody directed against collagen X (26) was used to quantify collagen X-containing The ratio of proliferative versus prehypertrophic/hypertrophic cell numbers for 12-and 13-day-old mice is given with standard deviation (n ! 3).…”

Section: Assessment Of the Growth Plate Composition During Developmentmentioning

confidence: 99%

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

Belluoccio¹,

Etich²,

Rosenbaum³

et al. 2010

Journal of Bone and Mineral Research

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Axial growth of long bones occurs through a coordinated process of growth plate chondrocyte proliferation and differentiation. This maturation of chondrocytes is reflected in a zonal change in gene expression and cell morphology from resting to proliferative, prehypertrophic, and hypertrophic chondrocytes of the growth plate followed by ossification. A major experimental limitation in understanding growth plate biology and pathophysiology is the lack of a robust technique to isolate cells from the different zones, particularly from small animals. Here, we report on a new strategy for separating distinct chondrocyte populations from mouse growth plates. By transcriptome profiling of microdissected zones of growth plates, we identified novel, zone-specific cell surface markers and used these for flow cytometry and immunomagnetic cell separation to quantify, enrich, and characterize chondrocytes populations with respect to their differentiation status. This approach provides a novel platform to study cartilage development and characterize mouse growth plate chondrocytes to reveal unique cellular phenotypes of the distinct subpopulations within the growth plate. ß

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Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies

Cited by 197 publications

References 34 publications

Global analyses of gene expression in early experimental osteoarthritis

Global analyses of gene expression in early experimental osteoarthritis

Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis

Sorting of growth plate chondrocytes allows the isolation and characterization of cells of a defined differentiation status

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