Autophagy activation by rapamycin reduces severity of experimental osteoarthritis - PubMed (original) (raw)

Autophagy activation by rapamycin reduces severity of experimental osteoarthritis

Beatriz Caramés et al. Ann Rheum Dis. 2012 Apr.

Abstract

Objectives: Osteoarthritis is associated with cell death and extracellular matrix degradation in articular cartilage. Autophagy is an essential cellular homeostasis mechanism that was found to be deficient in ageing and osteoarthritic cartilage. This study determined whether pharmacological inhibition of the mammalian target of rapamycin (mTOR), a key inhibitor of autophagy, has disease-modifying activity in experimental osteoarthritis.

Methods: Experimental osteoarthritis was induced by transection of the medial meniscotibial ligament and the medial collateral ligament in 2-month-old C57Bl/6 mice (n=36). Rapamycin (1 mg/kg weight/day) (n=18 mice) or dimethyl sulphoxide vehicle control (n=18 mice) was administered intraperitoneally for 10 weeks. Histopathological changes in articular cartilage and synovium were examined by using semiquantitative scoring systems. Rapamycin effects on mTOR signalling, autophagy, cartilage homeostasis and inflammation were analysed by immunohistochemistry and immunofluorescence staining.

Results: Rapamycin affected the mTOR signalling pathway in mouse knee joints as indicated by the inhibition of ribosomal protein S6 phosphorylation, a target of mTOR and activation of LC3, a main marker of autophagy. The severity of cartilage degradation was significantly (p<0.01) reduced in the rapamycin-treated group compared with the control group and this was associated with a significant (p<0.05) decrease in synovitis. Rapamycin treatment also maintained cartilage cellularity and decreased ADAMTS-5 and interleukin-1β expression in articular cartilage.

Conclusions: These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental osteoarthritis. Pharmacological activation of autophagy may be an effective therapeutic approach for osteoarthritis.

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Figures

Figure 1

Figure 1. Systemic administration of rapamycin modulates mTOR signaling and autophagy in mouse knee joints

A, Knee joints from C57Bl/6J mice were collected 10 weeks after knee destabilization or and treatment with rapamycin (rapa) or vehicle (n=3/each). Contralateral knees were used as a control. Sections were analyzed by immunohistochemistry for phosphorylation of ribosomal protein rpS6. Magnification: ×10 and ×40. B, Knee joints from 2 months old C57Bl/6J mice after OA surgery treated with vehicle (n=4) or rapa (n=4) were analyzed by immunofluorescence for LC3. Magnification: 10× and 100×.

Figure 2

Figure 2. Rapamycin reduces severity of experimental OA

Two months old C57Bl/6J mice were subjected to OA surgery in right the knee. The left knee was not subjected to surgery and used as control. Two separate experiments were performed. Each experiment included 18 mice, with 9 receiving rapamycin (rapa) and 9 receiving vehicle. A, Knee joints were analyzed by staining with Safranin O. Magnification:10x, 40x. B–C, Histological scores for the two separate experiments. Values are mean ± SD. * = P < 0.01 versus control-vehicle and control-rapa; ** = P < 0.01 versus OA-vehicle. D, Histological scores after combining both experiments (n= 36) showed a significant decrease in OA scores after rapa treatment. Values are the mean ± SD. * = P < 0.01 versus OA-vehicle.

Figure 3

Figure 3. Rapamycin prevents reduction in cartilage cellularity during experimental OA

A, Knee joint sections from C57Bl/6J mice with or without OA surgery under treatment with rapamycin (rapa) or vehicle (n=6 each) were stained with Hematoxylin Eosin (H&E). Magnification: 10× and 40×. B, Quantitative analysis of cell number showed a significant increase of cellularity after rapa treatment compared to vehicle treatment. Values are the mean ± SD. * = P < 0.001 versus OA-vehicle.

Figure 4

Figure 4. Rapamycin reduces ADAMTS-5 expression. A

Knee joints from C57Bl/6J mice were collected 10 weeks after OA surgery and treatment with rapamycin (rapa) or vehicle (n=5 each). Sections were analyzed by immunohistochemistry for ADAMTS-5. Magnification: 10× and 40×. B, Quantitative analysis of ADAMTS-5 positive cells. Total cell number in three fields and ADAMTS-5 positive cells were counted and the percentage of ADAMTS-5 positive cells was calculated. Results show a significant decrease in ADAMTS-5 after rapa treatment compared to vehicle. Values are mean ± SD. * = P < 0.05 versus OA-vehicle.

Figure 5

Figure 5. Effect of Rapamycin on inflammation in experimental OA

A, Synovium from mice after experimental OA and vehicle (N= 16) or rapamycin (rapa, N= 11) treatment was analyzed by staining with Safranin O. Magnification: 40×. B, The histological score for inflammation was significantly decreased after rapa treatment. Values are the mean ± SD. * = P < 0.05 versus OA-vehicle. C, Synovium sections from vehicle or rapa treated mice (N=5 each) were analyzed by immunohistochemistry for IL-1β. Magnification: 40×.

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