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. 2012 Apr;64(4):1182-92.
doi: 10.1002/art.33444. Epub 2011 Oct 27.

Mechanical injury suppresses autophagy regulators and pharmacologic activation of autophagy results in chondroprotection (V体育官网入口)

Beatriz Caramés  1 , Noboru Taniguchi, Daisuke Seino, Francisco J Blanco, Darryl D'Lima, Martin Lotz
Affiliations

Mechanical injury suppresses autophagy regulators and pharmacologic activation of autophagy results in chondroprotection

Beatriz Caramés et al. Arthritis Rheum. 2012 Apr.

Abstract

Objective: Mechanical injury induces cell death in cartilage and triggers a remodeling process that ultimately can manifest as osteoarthritis VSports手机版. Autophagy is a process for turnover of intracellular organelles and macromolecules that protects cells during stress responses. This study was undertaken to determine changes in and functions of autophagy following mechanical injury to cartilage. .

Methods: Bovine and human cartilage explants were subjected to mechanical impact (40% strain for 500 msec). Cell viability, sulfated glycosaminoglycan (sGAG) release, and changes in the levels of the autophagy markers ULK1, beclin 1, and microtubule-associated protein 1 light chain 3 (LC3) were evaluated. Cartilage explants were treated with the mammalian target of rapamycin complex 1 (mTORC-1) inhibitor and the autophagy inducer rapamycin and tested for protective effects against mechanical injury. Explants were also treated with the cell death inducers nitric oxide and tumor necrosis factor α (TNFα) plus actinomycin D, and the proinflammatory cytokine interleukin-1α (IL-1α) V体育安卓版. .

Results: Mechanical injury induced cell death and loss of sGAG in a time-dependent manner. This was associated with significantly decreased ULK1, beclin 1, and LC3 expression in the cartilage superficial zone (P < 0. 05) 48 hours after injury. The levels of LC3-II were increased 24 hours after injury but decreased at 48 and 96 hours. Rapamycin enhanced expression of autophagy regulators and prevented cell death and sGAG loss in mechanically injured explants. Rapamycin also protected against cell death induced by sodium nitroprusside and TNFα plus actinomycin D and prevented sGAG loss induced by IL-1α V体育ios版. .

Conclusion: Our findings indicate that mechanical injury leads to suppression of autophagy, predominantly in the superficial zone where most of the cell death occurs VSports最新版本. Pharmacologic inhibition of mTORC-1, at least in part by enhancement of autophagy, prevents cell and matrix damage, suggesting a novel approach for chondroprotection. .

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Figures

Figure 1
Figure 1. Mechanical injury induces cell death and loss of glycosaminoglycans in bovine cartilage explants
Full-thickness cartilage explants (n=80 explants) were subjected to mechanical injury or placed in the loading machine but not loaded as control. The explants were analyzed by Live/Dead viability assay immediately after mechanical injury (0 hours) or following culture for 24, 48 or 96 hours. In addition, the effect of mechanical stress on cartilage extracellular matrix was evaluated by safranin O staining and by quantification of sulfated glycosaminoglycan (sGAG) release into supernatants. A, Percentage of viable cells in cartilage explants. Values are mean ± SD. ** = P < 0.001 vs control; * = P < 0.01 vs control. B, Percentage of viable cells in superficial zone (SZ) of cartilage explants. Values are mean ± SD. ** = P < 0.001 vs control; * = P < 0.05 vs control. C, Safranin O staining of control explants and after mechanical injury at 0, 24, 48 and 96 hours. Original magnification x40. D, Quantitative analysis of sGAG release into supernatants. Values are mean ± SD. * = P < 0.05 vs control; ** = P < 0.001 vs control. Values represent mean ± SD of five separate experiments each in duplicate.
Figure 2
Figure 2. ULK1, Beclin1 and LC3 expression is reduced in response to mechanical injury
Full-thickness cartilage explants (n=24) were subjected to mechanical injury or cultured without injury to evaluate the effect of mechanical injury on the autophagy pathway. A, Total protein from explants subjected to mechanical injury or cultured without injured at 24, 48 and 96 hours was analyzed by Western blotting using anti-LC3 and β-actin. B, Safranin O staining and immunohistochemical analysis of ULK1, Beclin1 and LC3 from explants subjected to mechanical injury or cultured without injury at 48 hours. Original magnification x40. C–E, Quantitative analysis of ULK1, Beclin1 and LC3 positive cells in superficial zone (SZ), mid zone (MZ) and deep zone (DZ). Values represent mean ± SD of four separate experiments each in duplicate, * = P < 0.05.
Figure 3
Figure 3. Rapamycin does not affect cell viability and increases expression of autophagy regulators
Full-thickness cartilage explants and bovine chondrocytes were employed to study the potential effect of rapamycin on cell viability by Live-Dead assay. Autophagy activation was assessed by immunohistochemistry. A, Results of Live/Dead viability assay at 48 hours. B, LC3 expression in explants in control conditions and after rapamycin treatment (Rapa; 1, 5 and 10 μM) at 48 hours. Original magnification X 40.
Figure 4
Figure 4. Autophagy induction by rapamycin protects against cell death in response to mechanical injury
Full-thickness cartilage explants (n=64 explants) were employed to study the potential effect of rapamycin on response to mechanical injury. Explants were subjected to mechanical injury, treated with rapamycin (Rapa; 1 μM) and analyzed by Live/Dead viability assay at 24, 48, 96 hours. A, Results of Live/Dead assay (original magnification X 40). B, Percentage of viable cells in bovine cartilage explants. Values are mean ± SD. * = P < 0.05 vs control; ** = P < 0.01 vs control. C, Percentage of viable cells in superficial zone (SZ). Values are mean ± SD. * = P < 0.05 vs control; ** = P < 0.001 vs control; & = P < 0.01 vs injury. Values represent mean ± SD of four separate experiments each in duplicate.
Figure 5
Figure 5. Rapamycin inhibits loss of glycosaminoglycans after mechanical injury
Full-thickness cartilage explants (n=64 explants) were used to study the effect of rapamycin on mechanical injury-induced sulfated glycosaminoglycan (sGAG) release into supernatants based on the dimethylmethylene blue method. Explants were subjected to mechanical injury, treated with rapamycin (Rapa; 1 μM) and analyzed at 24, 48, 96 hours. A, Safranin O staining of control explants and after mechanical injury at 48 hours. Original magnification x40. B, Quantitative analysis of sGAG release into supernatants. Values are mean ± SD. * = P < 0.05 vs injury; ** = P < 0.001 vs control. Values represent mean ± SD of four separate experiments each in duplicate.
Figure 6
Figure 6. Rapamycin protects against biochemically induced cell death and matrix degradation
Chondrocytes and cartilage were preincubated for 1 hour with rapamycin (Rapa; 1, 5, 10 μM ) and then stimulated by sodium nitroprusside (SNP; 2 mM) or TNFα (10 ng/ml) + Actinomycin D (ActD; 1 μg/ml) for 18 hours or IL-1α (10 ng/ml) for 72 hours. A, Quantitative analysis of cell death induced by SNP in chondrocytes at 18 hours was performed by propidium iodide staining. Values are mean ± SD. ** = P < 0.001 vs control; * = P < 0.01 vs SNP 2 mM. B, Quantitative analysis of cell death induced by TNFα + ActD was performed by Annexin-V staining. Values are mean ± SD. ** = P < 0.001 vs control; * = P < 0.01 vs TNFα + ActD. C, Morphological studies using DAPI staining in explants treated or not with TNFα + ActD and rapamycin for 18 h. D, Quantitative analysis of sGAG release into supernatants. Values are mean ± SD. ** = P < 0.001 vs control; * = P < 0.01 vs IL-1α at 72 hours. Values represent mean ± SD of three separate experiments each in duplicate.
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References

    1. Yang S, Kim J, Ryu JH, Oh H, Chun CH, Kim BJ, et al. Hypoxia-inducible factor-2alpha is a catabolic regulator of osteoarthritic cartilage destruction. Nat Med. 2010;16:687–93. - PubMed
    1. Patwari P, Cook MN, DiMicco MA, Blake SM, James IE, Kumar S, et al. Proteoglycan degradation after injurious compression of bovine and human articular cartilage in vitro: interaction with exogenous cytokines. Arthritis Rheum. 2003;48:1292–301. - PubMed
    1. Stevens AL, Wishnok JS, White FM, Grodzinsky AJ, Tannenbaum SR. Mechanical injury and cytokines cause loss of cartilage integrity and upregulate proteins associated with catabolism, immunity, inflammation, and repair. Mol Cell Proteomics. 2009;8:1475–89. - PMC - PubMed
    1. Sui Y, Lee JH, DiMicco MA, Vanderploeg EJ, Blake SM, Hung HH, et al. Mechanical injury potentiates proteoglycan catabolism induced by interleukin-6 with soluble interleukin-6 receptor and tumor necrosis factor alpha in immature bovine and adult human articular cartilage. Arthritis Rheum. 2009;60:2985–96. - "V体育官网入口" PubMed
    1. Taniguchi N, Caramés B, Ronfani L, Ulmer U, Komiya S, Bianchi ME, et al. Aging-related loss of the chromatin protein HMGB2 in articular cartilage is linked to reduced cellularity and osteoarthritis. Proc Natl Acad Sci U S A. 2009;106:1181–6. - PMC - PubMed

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