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Global Gene Expression Analysis Identifies Age-Related Differences in Knee Joint Transcriptome During the Development of Post-Traumatic Osteoarthritis in Mice

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Global Gene Expression Analysis Identifies Age-Related Differences in Knee Joint Transcriptome During the Development of Post-Traumatic Osteoarthritis in Mice

Aimy Sebastian et al. Int J Mol Sci.

Abstract

Aging and injury are two major risk factors for osteoarthritis (OA). Yet, very little is known about how aging and injury interact and contribute to OA pathogenesis. In the present study, we examined age- and injury-related molecular changes in mouse knee joints that could contribute to OA. Using RNA-seq, first we profiled the knee joint transcriptome of 10-week-old, 62-week-old, and 95-week-old mice and found that the expression of several inflammatory-response related genes increased as a result of aging, whereas the expression of several genes involved in cartilage metabolism decreased with age. To determine how aging impacts post-traumatic arthritis (PTOA) development, the right knee joints of 10-week-old and 62-week-old mice were injured using a non-invasive tibial compression injury model and injury-induced structural and molecular changes were assessed. At six-week post-injury, 62-week-old mice displayed significantly more cartilage degeneration and osteophyte formation compared with young mice. Although both age groups elicited similar transcriptional responses to injury, 62-week-old mice had higher activation of inflammatory cytokines than 10-week-old mice, whereas cartilage/bone metabolism genes had higher expression in 10-week-old mice, suggesting that the differential expression of these genes might contribute to the differences in PTOA severity observed between these age groups.

Keywords: PTOA; RNA-seq; aging; cartilage degeneration; chondrocytes; gene expression; osteoarthritis; scRNA-seq.

Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Age-related changes in the knee joint gene expression. Genes up- (A) and down-regulated (B) in 62-week-old (62W) mice and 95-week-old (95W) mice compared with 10-week-old (10W) mice and in 95-week-old mice compared with 62-week-old mice. (C) Inflammatory response-related genes up-regulated in both 62-week-old and 95-week-old compared with 10-week-old mice (top 30 genes). (D) Cartilage development-associated genes down-regulated in both 62-week-old and 95-week-old compared with 10-week-old mice. (E) Examples of inflammatory response genes showing progressive increase with age. (F) Key cartilage development-associated genes showing an age-related decrease in expression.
Figure 2
Figure 2
Characterization of post-traumatic osteoarthritis (PTOA)-associated structural changes in 10-week-old and 62-week-old mice. (A) Histological evaluation of uninjured contralateral joints and injured joints at six-week post-injury using Safranin-O and Fast Green staining which stains cartilage in red and surrounding tissue in green (5× magnification). Scale bars: 200 µm. (B) Osteoarthritis Research Society International (OARSI) scoring of histological sections of injured and uninjured contralateral joints at six-week post-injury. (C) Osteophyte volume at six-week post-injury. (D) Epiphyseal trabecular bone volume fraction (BV/TV) of the distal femur was quantified using µCT and analyzed between injured and uninjured contralateral joints at six-week post-injury. 10W: 10-week-old; 62W: 62-week-old. * p < 0.05, ** p < 0.01, *** p < 0.001.
Figure 3
Figure 3
Age related differences in anterior cruciate ligament (ACL) injury-induced gene expression changes at six-week post-injury. (A) Number of genes differentially expressed in response to injury in 10-week-old and 62-week-old mice at six-week post-injury. (B) Overlap between genes up-regulated in 10-week-old and 62-week-old mice compared with respective uninjured controls and genes up-regulated in injured joints of 10-week-old compared with injured joints of 62-week-old mice. (C) Injury-induced regulators of cartilage and bone development/metabolism showing highest expression in injured joints of 10-week-old-mice. 10W_I: injured joints of 10-week-old; 10W_U: uninjured joints of 10-week-old; 62W_I: injured joints of 62-week-old; 62W_U: uninjured joints of 62-week-old.
Figure 4
Figure 4
Injury-induced transcriptional changes at early post-injury timepoints. Number of genes up- and down-regulated in injured knee joints of 62-week-old (A) and 10-week-old (B) mice at one-day (1D), one-week (1W), and two weeks (2W) post-injury compared with uninjured contralateral joints. (C) Key biological processes associated with genes up-regulated in 62-week-old mice and 10-week-old mice at early timepoints. (D) Inflammatory cytokines up-regulated at one-day post-injury in 62-week-old mice. (E) Matrix degrading enzymes up-regulated in 62-week-old mice after injury. Majority of these had highest expression at 1–2 weeks post-injury. 10W: 10-week-old; 62W: 62-week-old; 1DPI: one-day post-injury; 1WPI: one-week post-injury; 2WPI: two weeks post-injury.
Figure 5
Figure 5
Chad (A) and Plod2 (B) gene expression at six-week post injury in 10-week-old and 62-week-old mice. Chad (C) and Plod2 (D) protein expression in 10-week-old and 62-week-old mice Green: protein staining. Blue: DAPI staining marking the nucleus. (40× magnification).
Figure 6
Figure 6
(A) Table showing Cytl1 fold down-regulation in injured joints compared with uninjured controls at various post-injury timepoints, in both 10-week-old and 62-week-old mice. (B) Cytl1 protein expression at six-week post injury in 10-week-old and 62-week-old mice. Green: protein staining. Blue: DAPI staining marking the nucleus. (40× magnification) (C) tSNE plot of mouse cartilage cells identified using scRNA-seq. Each color represents a distinct cell type/subtype. Chondrocyte subtypes identified in mouse cartilage based on the expression of chondrocyte markers Col2a1, Acan, and Sox9 are shown in black oval. (D) Expression of chondrocyte markers Sox9 and Col2a1 in scRNA-seq data. (E) Cytl1 expression is restricted to a chondrocyte subtype that also express high levels of Bmp2, Wif1, and Prg4 at high levels. Ns: not significant.

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