Comparative analysis of gene expression between cartilage and menisci in early-phase experimental osteoarthritis

Abstract

Purpose: Pathogenesis of knee osteoarthritis (OA) is still largely unknown but secondary OA gaining much interest in terms of pathogenesis and intervention. Primal initial trauma such as ligament tear, resulting in joint instability, unnatural articulation, causes eventual development of histopathologic abnormality as secondary OA. Some researchers stressed the importance of meniscal degeneration in OA development. Whether the association between meniscal and cartilage degeneration in OA is purely due to the mechanical property of the meniscus, or whether meniscal cells secrete enzymes and inflammatory mediators that contribute to degeneration of other joint tissues, are unclear. It would not be too much to say the integrity of the meniscus is vital in maintaining healthy articular cartilage and prevention of degenerative changes in the knee. Despite the clear link between meniscal deterioration and the development of OA, there have been relatively few studies investigating the molecular mechanisms of meniscal degeneration compared with cartilage. In this study, we analyzed the gene expression using microarray technique for the purpose of researching OA pathogenesis. We focus on evaluating molecular changes in very early phase of OA in ACLT in rats in order to further our understanding of the relationship between cartilage and meniscal degradation. Methods: OA was surgically induced in 10 week old male Wister rats by ACLT surgery. Articular cartilage from the femoral condyles and bilateral menisci were separately dissected of 6 ACLT and 6 sham operated rats. Each specimen composed of 3 knees was pooled for averaging and yielding enough RNA for subsequent analyses. Consequently, total of 8 RNA samples (2 ACLT and 2 sham femoral cartilage, 2 ACLT and 2 sham medial menisci) were extracted. Affymetrix® 3’GeneChips arrays were used to assess genome-wide changes in gene expression. Probe sets were filtered using the criterion of a minimum 1.5-fold change in differential gene expression between ACLT operated and sham operated in both cartilage and meniscus, respectively. Subsequently, the genes over 1.5-fond change were analyzed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) for cluster analysis (http://david.abcc.ncifcrf.gov/). Five rats in each group were underwent the same surgical protocol and sacrigficed 3 weeks after surgery for histological and immunohistochemical analysis. Results: Histologically, 3 weeks after ACLT operated rats showed slight fibrillation in bilateral surface of cartilage and menisci. The proteoglycan content appeared almost normal in ACLT operated rats since no loss of Safranin O staining was observed comparing to sham operated rats in both of cartilage and menisci. Of the 36,685 transcripts detectable by microarray, 641 (1.74%) and 651 (1.77%) transcripts in ACLT cartilage, 1439 (3.92%) and 1773 (4.83%) transcripts in ACLT menisci were up-regulated by more than 1.5 fold change comparing to sham operated, respectively. Cluster analysis using DAVID showed genes related to OA were found in ACLT menisci such as response to stimulus, angiogenesis, and apoptosis. Representative proteases including Adamts1,2,3,4, Mmp1,2,12,13,14,19, and extra cellular matrix genes including Versican(Vcan), lumican (Lum), syndecan 1 (Sdc1), and prostaglandin endoperoxide synthase2 (Ptgs2) were up-regulated in menisci but were not up-regulated in cartilage. Expression of type II collagen (Col2a1) was not affected in both cartilage and menisci in ACLT operated rats as well as sham operated rats. Immunohistochemical analysis revealed that MMP-13, Adamts-4 and type II collagen-related neoepitope (C2C) were present in bilateral menisci of ACLT operated rats, however no staining was found in cartilage. Conclusions: We evaluated molecular changes in very early phase of OA by microarray analysis in terms of the relationship between cartilage and meniscal degradation in surgically-induced OA models in rats. It has been unclear in the previous studies that meniscal and cartilage degeneration in OA is purely due to the mechanical property of the menisci, or cellular reaction of meniscal cells where secretion of enzymes or inflammatory mediators that contributes to degeneration of other joint tissues. Our results indicated that molecular changes occurred in menisci preceded those occurred in cartilage in very early phase of surgically-induced OA models. And this suggested the possibility of interventions targeting menisci in early phase of OA might be a new strategy to prevent OA development.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)