SOX gene expression in human osteoarthritic cartilage.

Objective: While the developmental role of the SOX transcription factors in fetal chondrocyte differentiation is well documented, much less is known about the expression of SOX family members in normal and osteoarthritic adult cartilage. Therefore, the aim of the present study was to present a thorough analysis of SOX gene expression in normal and osteoarthritic human adult cartilage.

Methods: RNA from normal and osteoarthritic knee cartilage from human adults was analyzed by gene expression profiling using GeneChip technology (Affymetrix) and quantitative real time PCR.

Mechanisms of disease: role of chondrocytes in the pathogenesis of osteoarthritis--structure, chaos and senescence.

The extracellular matrix of articular cartilage is the primary target of osteoarthritic cartilage degradation. However, cartilage cells have a pivotal role during osteoarthritis, as they are mainly responsible for the anabolic-catabolic balance required for matrix maintenance and tissue function. In addition to the severe changes in the extracellular matrix, the cells also display abnormalities during osteoarthritic cartilage degeneration, such as inappropriate activation of anabolic and catabolic activities, and alterations in cell number through processes like proliferation and (apoptotic) cell death.

Phenotyping of chondrocytes in vivo and in vitro using cDNA array technology.

The cDNA array technology is a powerful tool to analyze a high number of genes in parallel. We investigated whether large-scale gene expression analysis allows clustering and identification of cellular phenotypes of chondrocytes in different in vivo and in vitro conditions. In 100% of cases, clustering analysis distinguished between in vivo and in vitro samples, suggesting fundamental differences in chondrocytes in situ and in vitro regardless of the culture conditions or disease status.

Large-scale gene expression profiling reveals major pathogenetic pathways of cartilage degeneration in osteoarthritis.

Objective: Despite many research efforts in recent decades, the major pathogenetic mechanisms of osteoarthritis (OA), including gene alterations occurring during OA cartilage degeneration, are poorly understood, and there is no disease-modifying treatment approach. The present study was therefore initiated in order to identify differentially expressed disease-related genes and potential therapeutic targets.

Methods: This investigation consisted of a large gene expression profiling study performed based on 78 normal and disease samples, using a custom-made complementary DNA array covering >4,000 genes.

Gene expression profiling of serum- and interleukin-1 beta-stimulated primary human adult articular chondrocytes--a molecular analysis based on chondrocytes isolated from one donor.

In order to understand the cellular disease mechanisms of osteoarthritic cartilage degeneration it is of primary importance to understand both the anabolic and the catabolic processes going on in parallel in the diseased tissue. In this study, we have applied cDNA-array technology (Clontech) to study gene expression patterns of primary human normal adult articular chondrocytes isolated from one donor cultured under anabolic (serum) and catabolic (IL-1beta) conditions. Significant differences between the different in vitro cultures tested were detected.

Analysis of differential gene expression in healthy and osteoarthritic cartilage and isolated chondrocytes by microarray analysis.

The regulation of chondrocytes in osteoarthritic cartilage and the expression of specific gene products by these cells during early-onset and late-stage osteoarthritis are not well characterized. With the introduction of cDNA array technology, the measurement of thousands of different genes in one small tissue sample can be carried out. Interpretation of gene expression analyses in articular cartilage is aided by the fact that this tissue contains only one cell type in both normal and diseased conditions.

Quantification of mRNA expression levels in articular chondrocytes with PCR technologies.

Unlike any other technology in molecular biology, the polymerase chain reaction (PCR) has changed the technological armamentarium of molecular scientists working on cartilage, in terms of outstanding sensitivity and accuracy. Four approaches to determine mRNA expression levels by PCR amplification of specific cDNA sequences are currently in use and are discussed in this chapter: conventional PCR with end-point determination, conventional PCR in the logarithmic amplification phase, conventional PCR using internal competitive DNA fragments, and real-time PCR as offered by TaqMan technology and others. The determination of mRNA expression levels by real-time quantitative PCR appears to be the most reliable method for accurate determination of gene expression levels within cartilage and cultured chondrocytes, as in other tissues and cell types.

Down-regulation of the GTPase RhoB might be involved in the pre-apoptotic phenotype of osteoarthritic chondrocytes.

Anabolic activity, phenotypic alterations, and in particular survival of the chondrocytes are essential for the maintenance of proper articular cartilage and appears to fail during osteoarthritic cartilage degeneration. In this study, we investigated the presence and expression of RhoB in adult human articular cartilage and its regulation in osteoarthritic cartilage as well as in chondrocytes in vitro. RhoB belongs to the family of small GTPases, which are thought to be involved in a large range of activities important for eukaryotic cells.

SOX9 expression does not correlate with type II collagen expression in adult articular chondrocytes.

Anabolic activity is a crucial activity of articular chondrocytes and its failure is one major reason of osteoarthritic cartilage degeneration. The intracellular factors responsible for the increase or decrease of anabolic activity of articular chondrocytes remain largely unknown. A recent candidate, the transcription factor SOX9, has elicited much interest as it is suggested to be a central factor in chondrocytic differentiation during development, including collagen type II (COL2A1) expression, the major anabolic gene product of chondrocytes.

Quantification of expression levels of cellular differentiation markers does not support a general shift in the cellular phenotype of osteoarthritic chondrocytes.

Many studies have shown increased anabolic activity in osteoarthritic cartilage and have suggested changes in the cellular phenotypes of articular chondrocytes. Most of these studies relied on non-quantitative technologies, which did not allow the estimation of the relative importance of the different differentiation phenomena. In the present study, we developed and used quantitative PCR assays for collagen types I, II(total), IIA, III, and X as marker genes indicating cellular synthetic activity (collagen type II) as well as differentiation pattern of chondrocytes (collagen types I, IIA, III, and X) and quantified these genes in normal, early degenerative, and late stage osteoarthritic cartilage in parallel.

Bone morphogenetic protein-mediating receptor-associated Smads as well as common Smad are expressed in human articular chondrocytes but not up-regulated or down-regulated in osteoarthritic cartilage.

Bone morphogenetic proteins (BMPs) are supposed to be important for cartilage matrix anabolism. In this study, we investigated whether the intracellular mediators of BMP activity, Smads 1, 4, 5, and 8, are expressed in normal human articular chondrocytes in vivo and in vitro and whether alterations in expression and distribution pattern are found in osteoarthritic cartilage or in vitro after stimulation with interleukin (IL)-1, because down-regulation of these mediators could be responsible for the decrease of anabolic activity in osteoarthritic cartilage. RNA was isolated from normal and osteoarthritic human knee cartilage and analyzed by (quantitative) polymerase chain reaction (PCR) technology.

Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro.

Objective: Osteoarthritic (OA) cartilage destruction depends on collagen- and aggrecan-degrading proteases such as collagenases (MMP-1 and MMP-13), stromelysin (MMP-3), MMP-14, as well as the so-called aggrecanases (ADAM-TS4 and ADAM-TS5). In this study, we tried to clarify whether these proteases are expressed in vivo in human normal and OA cartilage (and whether they are up-regulated or down-regulated during the disease process) and in interleukin-1beta (IL-1beta)-stimulated chondrocytes in vitro.

Methods: Quantitative polymerase chain reaction assays were developed and performed on RNA isolated directly from normal and degenerative cartilage tissue as well as from primary human articular chondrocytes cultured with and without IL-1beta.