Gene expression in phenotypically homogeneous chondrocytes from different articular cartilage layers of equine osteoarthritic and control joints: method validation and gene array analysis
Date
2007
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Abstract
Osteoarthritis remains a common and debilitating disease in horses, despite advances in diagnosis and treatment. Cartilage is commonly considered to play a central role in the pathophysiology of osteoarthritis. The investigation of differences in gene expression in cells from osteoarthritic and control cartilage is expected to yield genes possibly playing a role in the pathophysiology of osteoarthritis, representing new targets for treatment of the disease. The goals of this investigation were to develop a methodology to isolate RNA from phenotypically homogeneous cells of various cartilage layers for gene array analysis and to determine differentially expressed genes in these cells in osteoarthritic and control cartilage. A methodology to isolate phenotypically homogeneous chondrocytes from frozen sections of adult equine articular cartilage was developed using laser capture microdissection, RNA isolation, amplification and qrt-PCR. Expression levels of candidate genes were compared to those in conventionally isolated RNA from paired cartilage samples. The methodology was adequate to produce sufficient amounts of RNA for gene array analysis. Gene expression was found to be altered, but in a consistent fashion. The validated methodology, followed by gene array analysis was utilized to compare expression patterns in chondrocytes from tangential and radial layers of experimentally induced osteoarthritic and control cartilage. 154 genes were differentially expressed between tangential and radial cartilage layers and 17 genes were differentially expressed between osteoarthritic and control cartilage. The gene expression pattern of the tangential layer reflected support of cell proliferation, suppression of apoptosis and several genes involved in cell-matrix interactions or inflammatory processes. In contrast, the gene expression pattern of the radial layer was dominated by genes supporting the synthesis of proteins and proteoglycans. The gene expression pattern from osteoarthritic cartilage suggested an active response to oxidative stress, activation of the NF-κB pathway, decreased anti-apoptotic ability and downregulation of proteoglycan synthesis and glycolysis. This study was the first to determine gene expression patterns between two different layers of articular cartilage, improving our understanding of cartilage homeostasis in health and disease.
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Subject
articular cartilage
chondrocytes
equine osteoarthritis
molecular biology