Osteoarthritis (OA) is unusual in young adults but occurs quite commonly in older adults, such that symptomatic OA affects 10–20% of people age >50 years (1). In addition to age, joint injury is a common risk factor, especially for knee OA, with a pooled odds ratio of 3.86 (95% confidence interval 2.6–5.7) for knee OA after joint injury (2). Importantly, aging and joint injury interact. People who experienced a meniscal injury after age 30 years developed radiographic evidence of OA 3 times faster than those who had a similar injury between ages 17 and 30 years (3). The mechanisms by which aging contributes to the development of OA and the ways in which age and joint injury interact are incompletely understood and are the subject of the present work.
Several studies have analyzed gene expression microarray data to discover the genes and pathways that are regulated at the transcriptional level during the development of OA. Studies of human OA have used cartilage removed at the time of joint replacement surgery, which represents end-stage disease, or from autopsies for studies of early lesions (4–7). Gene expression has also been evaluated in animal models of OA, including the rat anterior cruciate ligament transection (ACLT) and meniscal tear models (8, 9). A limitation to these studies is that gene expression was evaluated only in 1 tissue, most often the articular cartilage. It is well accepted that OA is a process that involves the joint as an organ rather than the articular cartilage alone. Microarray studies have been performed using RNA isolated from OA subchondral bone (10) and synovium (11, 12), but, like the cartilage studies, the analysis was limited to those selected tissues.
In the present study, we evaluated and compared gene expression in knee joint tissues from younger and older adult mice after the induction of OA by destabilization of the medial meniscus (DMM). The DMM model is a postinjury model described by Glasson et al (13–15) that has become popular because it involves the meniscus, which is commonly involved in human OA, and because the histologic lesions within the affected joint are similar to those observed in human OA. Most commonly, young male mice (129/SvEv or C57BL/6 strains) in the age range of 8–12 weeks are used in this model. Mice are considered to be skeletally mature at age ∼10 weeks, which is the age generally recommended for studies of surgically induced OA in this species (16). However, a 10-week-old C57BL/6 mouse corresponds approximately to a teenaged human, while a 12-month-old mouse would represent a 40–50-year-old human (17).
Therefore, to study the effects of age on the development of posttraumatic OA, we measured OA severity histologically and analyzed gene expression by microarray in joints from 12-week-old and 12-month-old mice, which we will refer to as young and older adult mice, respectively. RNA was isolated from joint tissue that was removed from the medial (affected) side of the joint, including cartilage, subchondral bone, meniscus, and the joint capsule with synovium, in order to study the joint as an organ. We identified significant histologic differences in OA severity between younger and older mice as well as differences in gene expression that included genes not previously identified in OA that might play an important role in the disease process.
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- MATERIALS AND METHODS
- AUTHOR CONTRIBUTIONS
- Supporting Information
Similar to findings in humans, we found that age contributes to the development of both spontaneous and injury-induced OA in mice. Mild OA-like pathology was present in the unoperated knees of the 12-month-old mice, suggesting that mice at this age are in the early stages of developing naturally occurring OA, similar to what has been described in pathologic studies of human knees at the equivalent age of ∼40 years (25). The older mice also exhibited more severe histologic OA after a joint injury that destabilized the medial meniscus, which is also consistent with studies on the development of OA after joint injury in humans (3) and supports the use of this model to study age-related differences. More severe OA lesions were also noted in the contralateral (unoperated) knee of older DMM-operated mice relative to the contralateral (unoperated) knee of older sham-operated mice, which could have been due to either gait changes or systemic factors released from the DMM-operated joint, suggesting that contralateral joints should not be used as controls in mouse models of surgically induced OA.
Annotations of genes expressed at lower levels in the older sham-operated control animals included extracellular matrix, metabolic processes, and tissue development, with a predominance of cartilage extracellular matrix genes including those for types II, IX, and XI collagen, aggrecan, COMP, and link protein, as well as for the transcription factor SOX9 and the growth factor TGFβ2. These findings are consistent with those of studies demonstrating an age-related decline in matrix production when chondrocytes are stimulated with anabolic factors (for review, see ref.26) and of a study showing reduced immunostaining for TGFβ2 in the articular cartilage of older mice (27). We also noted a decrease in expression of the gene for HMGB-2, consistent with a previous study in mice that showed that decreased levels may contribute to chondrocyte death in the superficial zone (28). Genes expressed at higher levels in older sham-operated controls included chemokine and HLA genes and had annotations for immune and defense response. Expression of the gene for CXCR2, which serves as an IL-8 receptor and has been found to play an important role in promoting cell senescence (29), was also increased in the sham-operated joints of older mice.
In the joints with surgically induced OA, more genes were significantly up-regulated in older mice (n = 421) compared to younger mice (n = 72), potentially indicating a more active disease process. Genes with increased expression in OA joints of older mice included extracellular matrix genes, such as those for aggrecan and type II collagen, the expression of which decreased with age in the sham-operated controls. DAVID analysis and IPA of the relatively smaller set of genes that were up-regulated in younger (but not older) mice with OA identified significant annotations for immune response genes and B cell signaling, while these annotations were found in genes expressed in the sham-operated joints of older mice. Genes with muscle-related annotations and genes involved in calcium signaling were more often down-regulated in the younger mice with OA, while these genes were up-regulated in DMM-operated joints of older mice. Because we did not include muscle in the tissue used for RNA isolation, muscle-related genes are most likely being expressed by other tissues including cartilage, as previous studies have noted chondrocyte expression of genes such as that for α-smooth muscle actin (30).
A unique aspect of the present study was that the RNA isolated for microarray analysis was extracted from the multiple tissues that make up the joint rather than from a single tissue. Although this approach might be less sensitive in detecting genes that were up- or down-regulated in a single tissue and might limit the ability to determine which particular tissue contributed to expression of a specific gene, it has the advantage of allowing discovery of genes that are more globally involved in the OA process. Despite the potential limitations, the genes for aggrecan, link protein, and type II collagen, primarily expressed in articular cartilage and in the inner zone of the meniscus, were detected on the arrays as being up-regulated in DMM-operated joints of older mice, even though cartilage and meniscus loss was greater in these mice than in the younger mice.
Periostin was up-regulated in DMM-operated joints of older but not younger mice. Periostin is a vitamin K–dependent (Gla-containing) protein produced by osteoblasts and also found in the periosteum (31). We confirmed its presence in periosteum and osteoblasts and in addition found that it was present in the cartilage matrix and in low numbers of chondrocytes. Periostin appears to play a role in diverse processes including tooth development (32), cancer metastasis (33), and tissue repair after injury, such as repair of heart tissue after a myocardial infarction (34). Likewise, secreted Frizzled-related protein 2, which can augment Wnt3a signaling (35) and whose gene expression was increased in DMM-operated joints from older mice, has also been shown to be involved in myocardial repair (36). The increased expression of these and other genes involved in extracellular matrix formation and tissue repair indicates an active repair response in the knee joints of the older mice.
IL-33 was found to be up-regulated in the DMM-operated joints of younger but not older mice, although its expression was higher in sham-operated joints of older mice than in sham-operated joints of younger mice. IL-33 is a member of the IL-1 superfamily and is thought to serve as an alarmin in a number of inflammatory diseases, including RA (for review, see ref.37), but, to our knowledge, it has not been reported to be up-regulated in OA. In RA, IL-33 was found in synovial tissue; however, it can also be expressed by osteoblasts, where it is thought to inhibit bone resorption (38). We found that although IL-33 is fairly widely distributed in joint tissues, there was strong and consistent positive immunostaining for IL-33 in articular and growth plate chondrocytes and meniscal cells, suggesting that it may function in these tissues as well.
The 55 genes that were similarly expressed in DMM-operated knees relative to sham-operated knees of younger and older mice were involved in extracellular matrix remodeling and included up-regulated genes involved in matrix degradation, such as those for MMP-2, MMP-3, and HtrA serine peptidase 1. Although not as extensively studied as the MMPs, several serine proteases, including HtrA serine peptidase 1, have been shown to be increased in human OA cartilage (39). HtrA serine peptidase 1 has been implicated in degradation of fibronectin (40) and aggrecan (21) and was also found by others to be increased in the cartilage of mice 8 weeks after DMM surgery (41). In addition, HtrA serine peptidase 1 was a prominent protease found in a proteomic analysis of human OA cartilage (42). CCL21 is a novel chemokine gene found to have increased expression in DMM-operated joints of both young and old mice; it was found by immunostaining to be localized to chondrocytes and meniscal cells and the growth plate matrix. CCL21 is a ligand for CCR7, the gene for which was also increased in DMM-operated joints of young mice and in sham-operated control joints of older mice. A recent study demonstrated elevated levels of CCL21 in both RA and OA synovial fluid, with higher levels in RA synovial fluid, compared to those in normal controls (43).
In a recently reported cartilage microarray study that used the rat meniscal tear model of OA, Wei et al (9) integrated their findings with those of previous microarray studies of differential gene expression in the rat ACLT model (8) and in human OA cartilage (5) and generated a list of 20 OA genes that were in common in the human study and at least 1 rat model. We searched for those 20 genes in our list of 548 genes that were differentially expressed in either younger or older DMM-operated mice and in the list of 861 genes differentially regulated by age in sham-operated joints of young and older mice. We found 7 genes in common in the list of genes differentially expressed in DMM-operated mice (COL3A1, COL6A2, lumican, MMP3, NDRG2, PCOLCE, and TIMP1); 6 were differentially regulated in the older DMM-operated mice and 5 in the young DMM-operated mice. We also found 4 genes in the list of genes in sham-operated mice that were differentially regulated with age in the same direction as differential regulation in the OA list (LTBP2 [the gene for latent TGFβ binding protein 2], NDRG2, SERPINA1, and TIMP1). Given the importance of TGFβ in joint tissues, the increase in LTBP2 expression in OA and with age may be particularly important.
We also compared our list of genes differentially expressed in DMM-operated mice with a list of 150 genes reported by Hopwood et al to be differentially expressed in human OA subchondral bone (10), and we found 4 genes in common (CCR2, crystallin alpha B, synuclein alpha, and tyrosyl–transfer RNA [tRNA] synthetase). Of these, the expression patterns matched for DMM-operated joints of young but not old mice for crystallin alpha B and tyrosyl-tRNA synthetase (down-regulated) and synuclein alpha (up-regulated). However, we found more genes (n = 10) in common in a comparison of the bone list with the list of genes differentially expressed in sham-operated young versus old mice, with 8 genes having the same expression pattern (crystallin alpha B, guanine nucleotide binding protein alpha Z, glycoprotein V, lymphotoxin beta, matrix extracellular phosphoglycoprotein with ASARM motif, RAB27B, selectin P, and tubulin, beta 1).
Finally, we made a similar comparison between the mouse joint microarray gene lists and a list of 260 genes found to be ≥2-fold differentially expressed between inflamed and uninflamed biopsy specimens of human synovial tissue obtained from subjects undergoing meniscectomy for meniscal injuries (12). A total of 30 genes were present in both the synovial gene list and the mouse joint lists, with expression of 8 genes regulated in the same direction in synovium and the lists of genes differentially regulated in DMM-operated and sham-operated mice (see Supplementary Table 2, available on the Arthritis & Rheumatism Web site at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1529-0131). One of those was the gene for CCL21, which our immunohistochemical staining showed to be present in articular cartilage, suggesting that this gene is up-regulated in more than one tissue in joints with meniscal damage. The comparison of genes regulated in the same direction between the sham-operated joints of old–young mice and the human synovial samples showed 20 genes in common, demonstrating that a significant number of genes that are up-regulated with age in the joint are also found in inflamed synovia from meniscal injuries. This included the gene for IL-7R, which our group had previously shown to be expressed by human chondrocytes (44).
In summary, the analysis of gene expression in joint tissues in a meniscal injury model of OA found OA-related genes that had been previously reported in other animal models and in human OA, but also revealed novel genes and pathways that could be important in the OA process. The results also demonstrated clear age-related transcriptional differences in both the sham-operated control joints and the DMM-operated joints. These findings demonstrate the importance of age when considering the results of animal model studies of OA. Most studies that have used transgenic mice to study the role of specific genes in OA have used animals at an age similar to that in our younger group. Genes and pathways important in the OA process may be missed if only young animals are used in such studies. Age at onset of joint injury clearly affects the way in which the cells within joint tissues respond. The older animals exhibited a very active response to joint injury, including the up-regulation of matrix genes, chemokines, and matrix-degrading enzymes, consistent with the concept that OA is not a degenerative disease but rather a condition that activates remodeling of joint tissues. Further studies of the differences in the transcriptional response between joints of younger and older mice should help elucidate the mechanisms underlying the contribution of age to the development of OA.