Microarray analysis reveals age-related differences in gene expression during the development of osteoarthritis in mice

Abstract

Objective: To better understand the contribution of age to the development of osteoarthritis (OA).

Methods: Surgical destabilization of the medial meniscus (DMM) was used to model OA in 12-week-old and 12-month-old male C57BL/6 mice. OA severity was evaluated histologically. RNA used for microarray and real-time polymerase chain reaction analysis was isolated from joint tissue collected from the medial side of the joint, including cartilage, meniscus, subchondral bone, and the joint capsule with synovium. Computational analysis was used to identify patterns of gene expression, and immunohistochemistry was used to evaluate tissue distribution of selected proteins.

Results: OA was more severe in older mice than in young mice. Only 55 genes showed a similar expression with DMM-induced OA in the 2 age groups, while 493 genes showed differential expression, the majority having increased expression in older mice. Functional categories for similarly expressed genes included extracellular matrix- and cell adhesion-related genes; differentially expressed genes included those related to muscle structure and development and immune response genes. Comparison of expression in sham-operated control joints revealed an age-related decrease in matrix gene expression and an increase in immune and defense response gene expression. Interleukin-33 was present in multiple joint tissue cells, while CCL21 was more localized to chondrocytes and meniscal cells. Periostin was found in the extracellular matrix of cartilage and meniscus.

Conclusion: Age affects both the basal pattern of gene expression in joint tissues and the response to surgically induced OA. Examining tissue from the joint beyond only cartilage revealed novel genes and proteins that would be important to consider in OA.

Figure 1

Flowchart of filtering process. As described in the Methods, the 45,101 probe sets on the Affymetrix arrays were filtered, in a step-wise process, to identify genes that responded similarly or differently to DMM treatment between younger and older mice. Step 1 identified all genes with a signal that was significantly detected on the chip over the background. Step 2 identified significantly expressed or repressed genes in DMM joints, with respect to sham, in 2 or more replicate pools. Step 3 identified genes that either responded in a similar fashion between young and older mice, or had a significantly different response. These genes were then assigned to an expression pattern in Step 4for further analysis.

Figure 2

Histological evaluation of osteoarthritis in medial knee joint compartments of 12-week-old and 12-month-old mice. Younger and older mice underwent DMM or sham surgery and OA severity was evaluated after 8 weeks. Sections were stained with H&E or Safranin-O. A) 12-month-old DMM, B) 12-month-old sham, C) 12-week-old DMM, D) 12-week-old sham. Bar = 100 microns; insert is a higher magnification (20×) image of a central area of the medial tibial plateau of the H&E-stained section. E) Articular cartilage structure (ACS) scores were calculated on a 0–12 scale for the medial and lateral sides of the joint. Controls are the contralateral (non-operated) limb. Results are mean ± SEM presented for the medial plus lateral tibial plateaus. p < 0.05 by ANOVA.

Figure 3

Heat maps of the genes identified in each expression pattern. Each group summarized in Step 4 of Figure 1 is represented here as a heat map with a summary of annotation terms found to be significantly over represented in each pattern by DAVID. Columns represent replicate arrays of Young mice (left 3 columns) and Older mice (right 3 columns) with each row representing a single gene. Blue=down regulation, Red=up regulation and White=no change in expression as compared to sham.

Figure 4

Immunohistochemical staining for selected proteins in mouse knee joints. Strong positive staining of chondrocytes in articular cartilage (arrows) and meniscus (arrowhead) for A) IL-33 in a young DMM mouse and B) CCL21 in an old sham mouse; C) positive staining for periostin in articular cartilage matrix (arrows) in a young DMM mouse and D) periosteum (arrows)in a young sham mouse; Bar = 50μm.