Gene expression analyses of subchondral bone in early experimental osteoarthritis by microarray

Abstract

Osteoarthritis (OA) is a degenerative joint disease that affects both cartilage and bone. A better understanding of the early molecular changes in subchondral bone may help elucidate the pathogenesis of OA. We used microarray technology to investigate the time course of molecular changes in the subchondral bone in the early stages of experimental osteoarthritis in a rat model. We identified 2,234 differentially expressed (DE) genes at 1 week, 1,944 at 2 weeks and 1,517 at 4 weeks post-surgery. Further analyses of the dysregulated genes indicated that the events underlying subchondral bone remodeling occurred sequentially and in a time-dependent manner at the gene expression level. Some of the identified dysregulated genes that were identified have suspected roles in bone development or remodeling; these genes include Alp, Igf1, Tgf β1, Postn, Mmp3, Tnfsf11, Acp5, Bmp5, Aspn and Ihh. The differences in the expression of these genes were confirmed by real-time PCR, and the results indicated that our microarray data accurately reflected gene expression patterns characteristic of early OA. To validate the results of our microarray analysis at the protein level, immunohistochemistry staining was used to investigate the expression of Mmp3 and Aspn protein in tissue sections. These analyses indicate that Mmp3 protein expression completely matched the results of both the microarray and real-time PCR analyses; however, Aspn protein expression was not observed to differ at any time. In summary, our study demonstrated a simple method of separation of subchondral bone sample from the knee joint of rat, which can effectively avoid bone RNA degradation. These findings also revealed the gene expression profiles of subchondral bone in the rat OA model at multiple time points post-surgery and identified important DE genes with known or suspected roles in bone development or remodeling. These genes may be novel diagnostic markers or therapeutic targets for OA.

Figure 1. Macroscopic analysis of femoral condyles in a rat model of osteoarthritis.

No detectable macroscopic surface changes were observed in the femoral condyle (A) of E-group rats at 1 week post-surgery. At 2 weeks post-surgery, the local medial femoral condyle of the distal femur exhibited a slightly rough articular surface (B, arrow). Significant roughness of the articular surface was observed both on the medial and lateral femoral condyle (C, arrow) at 4 weeks post-surgery. The knee joints (D, E, F) of the S-group exhibited a normal articular surface at all time points following the surgery.

Figure 2. Histologic analysis of cartilage degradation induced by medial meniscectomy and medial collateral ligament (MCL) transection, evaluated by section staining with safranin-O and rapid green.

The sections were stained with safranin-O (red stain) for glycosaminoglycans, rapid green for bone and fibrous tissue (green stain), and counterstained with Mayer's hematoxylin for nuclei (blue). A slight decrease in glycosaminoglycan staining at 1 week post-surgery is shown (A, arrow). A loss of superficial cartilage and focal fibrillation of the articular surface at 2 weeks post-surgery was seen (B, arrow). A focal loss of chondrocytes and exposure of subchondral bone wear observed at 4 weeks post-surgery (C, arrow). A healthy articular surface was observed throughout the study (D, E, F). Each of the above images was captured from representative locations from sections of femur condyles and is shown at the same magnification. The scale bar represents 200 µm.

Figure 3. Integrated analyses of the DE genes in the subchondral bone of E-Group versus S-group samples.

The number of DE genes at each time point is shown in A. DE genes were classified according to their differential expression levels with a minimum of 2-fold, 3-fold, 4-fold and 6-fold differences (B). Venn diagram depicting the overlap of dysregulated genes at three time points post-surgery (C). The expression patterns of 112 genes that were differentially expressed at all three time points are shown in D. Dendrogram of the unsupervised hierarchical clustering analysis of the E-Group and S-group samples at each time point is shown in E. The clustering was performed based on DE genes of the E-group versus the S-group at each time point. Euclidean distances were used to measure the similarities between the expression profiles of the samples.

Figure 4. The distribution of the DE genes at each post-surgical time point is described based on the three gene ontology categories and the numbers of DE genes in the first four gene functional classifications of each gene ontology category.

A. The distribution of DE genes was similar among the three gene ontology categories at each post-surgical time point. B. The functional classifications of the dysregulated genes involved in the biological processes category and the numbers of dysregulated genes in the first four functional classifications at each post-surgical time point. C. The functional classifications of the dysregulated genes involved in the cellular component category and the numbers of dysregulated genes in the first four functional classifications at each post-surgical time point. D. The functional classifications of the dysregulated genes involved in the molecular function category and the numbers of dysregulated genes in the first four functional classifications at each post-surgical time point.

Figure 5. Gene expression patterns of Alp, Igf1, Tgf-β1 and Postn (genes involved in osteoblasts differentiation and function), Mmp3, Tnfsf11 and Acp5 (genes involved in osteoclasts differentiation and function), and Bmp5, Aspn, Ihh (known OA-related genes), evaluated by real-time polymerase chain reaction (PCR).

The expression profiles of each gene matched the tested probes at a minimum of two time points. A similar degree of variability was observed at other time points in the real-time PCR results without statistically significant differences between groups. The values are the mean and SEM of the gene expression levels in 5 animals (separate from the animals used for microarray analyses), as determined by ΔCt analysis, normalized to GAPDH expression, and relative to the expression levels of sham-operated controls. * p<0.05, ** p<0.01 (versus sham-operated controls).

Figure 6. Evaluation of the expression levels of Mmp3 in the subchondral bone of the E-Group and the S-Group using immunohistochemistry staining.

The antibody against Mmp3 was used to assess the spatial and temporal expression of the protein by colorimetric detection (brown precipitate). The mononuclear cells of both groups appeared positive for the same levels of Mmp3 at 1, 2 and 4 weeks post-surgery. The polynuclear giant cells in the subchondral bone of the E-Group expressed Mmp3 in their cytoplasm 1 and 2 weeks post-surgery, but not at 4 weeks, wheras those of the S-Group appeared negative at all three time points post-surgery. Specifically, obvious differences were discovered in the E-Group between 1 week and 2 weeks post-surgery, including stronger positive signals and more positive cells at 1 week compared with 2 weeks post-surgery. All sections were counterstained with hematoxylin (blue stain). The scale bars for 10X, 40X and Cell-view are 200 µm, 50 µm, and 50 µm respectively.