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Gene expression profiles in the rat streptococcal cell wall-induced arthritis model identified using microarray analysis.

ABSTRACT: Experimental arthritis models are considered valuable tools for delineating mechanisms of inflammation and autoimmune phenomena. Use of microarray-based methods represents a new and challenging approach that allows molecular dissection of complex autoimmune diseases such as arthritis. In order to characterize the temporal gene expression profile in joints from the reactivation model of streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats, total RNA was extracted from ankle joints from naive, SCW injected, or phosphate buffered saline injected animals (time course study) and gene expression was analyzed using Affymetrix oligonucleotide microarray technology (RAE230A). After normalization and statistical analysis of data, 631 differentially expressed genes were sorted into clusters based on their levels and kinetics of expression using Spotfire profile search and K-mean cluster analysis. Microarray-based data for a subset of genes were validated using real-time PCR TaqMan analysis. Analysis of the microarray data identified 631 genes (441 upregulated and 190 downregulated) that were differentially expressed (Delta > 1.8, P < 0.01), showing specific levels and patterns of gene expression. The genes exhibiting the highest fold increase in expression on days -13.8, -13, or 3 were involved in chemotaxis, inflammatory response, cell adhesion and extracellular matrix remodelling. Transcriptome analysis identified 10 upregulated genes (Delta > 5), which have not previously been associated with arthritis pathology and are located in genomic regions associated with autoimmune disease. The majority of the downregulated genes were associated with metabolism, transport and regulation of muscle development. In conclusion, the present study describes the temporal expression of multiple disease-associated genes with potential pathophysiological roles in the reactivation model of SCW-induced arthritis in Lewis (LEW/N) rat. These findings improve our understanding of the molecular events that underlie the pathology in this animal model, which is potentially a valuable comparator to human rheumatoid arthritis (RA).

SUBMITTER: Rioja I

PROVIDER: S-EPMC1064886 | biostudies-literature | 2005

REPOSITORIES: biostudies-literature

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Gene expression profiles in the rat streptococcal cell wall-induced arthritis model identified using microarray analysis.

Rioja Inmaculada I Clayton Chris L CL Graham Simon J SJ Life Paul F PF Dickson Marion C MC

Arthritis research & therapy 20041119 1

Experimental arthritis models are considered valuable tools for delineating mechanisms of inflammation and autoimmune phenomena. Use of microarray-based methods represents a new and challenging approach that allows molecular dissection of complex autoimmune diseases such as arthritis. In order to characterize the temporal gene expression profile in joints from the reactivation model of streptococcal cell wall (SCW)-induced arthritis in Lewis (LEW/N) rats, total RNA was extracted from ankle joint ...[more]

PMID: 15642130

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Project description:DNA microarray is a powerful tool in biomedical research. However, transcriptomic profiling using DNA microarray is subject to many variations including biological variability. To evaluate the different sources of variation in mRNA gene expression profiles, gene expression profiles were monitored using the Affymetrix RatTox U34 arrays in cultured primary hepatocytes derived from six rats over a 26 hour period at 6 time points (0 h, 2h, 5h, 8h, 14 h and 26 h) with two replicate arrays at each time point for each animal. In addition, the impact of sample size on the variability of differentially expressed gene lists and the consistency of biological responses were also investigated. Excellent intra-animal reproducibility was obtained at all time points with 0 out of 370 present probe sets across all time points showing significant difference between the 2 replicate arrays (3-way ANOVA, p <or= 0.0001). However, large inter-animal biological variation in mRNA expression profiles was observed with 337 out of 370 present probe sets showing significant differences among 6 animals (3-way ANOVA, p <or= 0.05). Principal Component Analysis (PCA) revealed that time effect (PC1) in this data set accounted for 47.4% of total variance indicating the dynamics of transcriptomics. The second and third largest effects came from animal difference, which accounted for 16.9% (PC2 and PC3) of the total variance. The reproducibility of gene lists and their functional classification was declined considerably when the sample size was decreased. Overall, our results strongly support that there is significant inter-animal variability in the time-course gene expression profiles, which is a confounding factor that must be carefully evaluated to correctly interpret microarray gene expression studies. The consistency of the gene lists and their biological functional classification are also sensitive to sample size with the reproducibility decreasing considerably under small sample size.

Project description:Rheumatoid arthritis (RA) is a common systemic autoimmune disease mainly involving the formation of a synovial pannus, for which no effective treatment is available. In order to study the molecular biological mechanisms underlying the inhibition of RA synovial pannus by triptolide, differentially expressed genes in synovial tissues from an adjuvant arthritis (AA) rat model with and without triptolide treatment were detected in an mRNA microarray profile produced by Agilent Technologies and verified by reverse transcription-quantitative polymerase chain reaction analysis (RT-qPCR). An AA model was established by subcutaneously injecting 0.1 ml Freund's complete adjuvant daily for 18 days and scored by arthritis index assessment. Subsequently, triptolide (0.4 mg/kg) or an equivalent amount of saline was administered daily for 14 days. At the end of the experiment, synovial tissues were obtained from the ankle joints of the rats' hind legs. Total RNA was extracted and purified, and microarray hybridization was used to obtain the gene expression profile for RA with and without triptolide treatment. A total of 48 genes were identified to be differentially expressed between the treatment and model groups, including 32 upregulated and 16 downregulated genes. The possible signaling pathways associated with the effect of triptolide were investigated by Gene Ontology and pathway analysis, revealing that the phosphoinositide-3 kinase (PI3K)/AKT signaling pathway has a key role in the proliferation and apoptosis of synovial cells in RA joints. Reverse transcription-quantitative polymerase chain reaction analysis was applied to confirm the aberrant expression of key mRNAs and revealed that vascular endothelial growth factor (VEGF) A and C1q and tumor necrosis factor related protein 3 (C1QTNF3) were downregulated in the treatment group compared with the model group (P<0.05). In conclusion, triptolide may exert its effects against RA via the PI3K/AKT pathway and has an inhibitory effect on the expression of VEGFA and C1QTNF3, thus are potentially associated with the occurrence and development of RA.

Dataset Information

Gene expression profiles in the rat streptococcal cell wall-induced arthritis model identified using microarray analysis.

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Gene expression profiles in the rat streptococcal cell wall-induced arthritis model identified using microarray analysis.

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