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Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis.

Takayama K, Kawakami Y, Kobayashi M, Greco N, Cummins JH, Matsushita T, Kuroda R, Kurosaka M, Fu FH, Huard J - Arthritis Res. Ther. (2014)

Bottom Line: VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice.Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.

Methods: Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).

Results: Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

Conclusion: These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

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The effect of rapamycin on phospho- mammalian target of rapamycin (p-mTOR) and light chain 3 (LC3) expression. (A) Representative images of immunostaining for p-mTOR (green) and LC3 (green). Scale bar = 20 μm. (B) Quantification of p-mTOR-positive cells was calculated. Rapamycin treatment suppressed p-mTOR expression in the rapamycin-treated mice compared to those treated with dimethyl sulphoxid (DMSO) at both the 8- and 12-week time points (at 8 weeks DMSO 108.5 ± 13.3, rapamycin 43.0 ± 8.9 mm; at 12 weeks DMSO 273.8 ± 27.0, rapamycin 76.5 ± 14.7 mm). (C) Quantification of LC3-positive cells was calculated. **P <0.01, ***P <0.001. DMM, destabilization of the medial meniscus.
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Fig2: The effect of rapamycin on phospho- mammalian target of rapamycin (p-mTOR) and light chain 3 (LC3) expression. (A) Representative images of immunostaining for p-mTOR (green) and LC3 (green). Scale bar = 20 μm. (B) Quantification of p-mTOR-positive cells was calculated. Rapamycin treatment suppressed p-mTOR expression in the rapamycin-treated mice compared to those treated with dimethyl sulphoxid (DMSO) at both the 8- and 12-week time points (at 8 weeks DMSO 108.5 ± 13.3, rapamycin 43.0 ± 8.9 mm; at 12 weeks DMSO 273.8 ± 27.0, rapamycin 76.5 ± 14.7 mm). (C) Quantification of LC3-positive cells was calculated. **P <0.01, ***P <0.001. DMM, destabilization of the medial meniscus.

Mentions: To determine whether the local intra-articular injection of rapamycin modulates mTOR and autophagy, the effects that rapamycin had on phospho-mTOR (p-mTOR) and LC3 expressions were examined. The expression of p-mTOR was increased in DMSO-treated mice at 8 and 12 weeks post DMM surgery compared to DMSO-treated mice undergoing the sham knee operation. Rapamycin treatment suppressed p-mTOR expression in these mice compared to those treated with DMSO at both the 8- and 12-week time points post DMM injury. The expression of p-mTOR in the rapamycin-treated mice was significantly increased at 12 weeks compared to 8 weeks after DMM injury (at 8 weeks DMSO 108.5 ± 13.3, rapamycin 43.0 ± 8.9; at 12 weeks DMSO 273.8 ± 27.0, rapamycin 76.5 ± 14.7) (Figure 2A, B). In contrast, there were more LC3-positive cells in the articular cartilage maintaining proteoglycan staining but fewer in the degenerated articular cartilage of the DMSO-treated mice after DMM surgery. An increase in LC3-positive cells was observed in the rapamycin-treated mice compared to those treated with DMSO at 8 and 12 weeks post DMM surgery. The number of LC3-positive cells in the rapamycin-treated mice decreased significantly at 12 weeks compared to 8 weeks post DMM injury (at 8 weeks DMSO 88.2 ± 13.1, rapamycin 236.0 ± 56.3; at 12 weeks DMSO 46.2 ± 12.8, rapamycin 106.3 ± 27.1) (Figure 2A, C).Figure 2


Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis.

Takayama K, Kawakami Y, Kobayashi M, Greco N, Cummins JH, Matsushita T, Kuroda R, Kurosaka M, Fu FH, Huard J - Arthritis Res. Ther. (2014)

The effect of rapamycin on phospho- mammalian target of rapamycin (p-mTOR) and light chain 3 (LC3) expression. (A) Representative images of immunostaining for p-mTOR (green) and LC3 (green). Scale bar = 20 μm. (B) Quantification of p-mTOR-positive cells was calculated. Rapamycin treatment suppressed p-mTOR expression in the rapamycin-treated mice compared to those treated with dimethyl sulphoxid (DMSO) at both the 8- and 12-week time points (at 8 weeks DMSO 108.5 ± 13.3, rapamycin 43.0 ± 8.9 mm; at 12 weeks DMSO 273.8 ± 27.0, rapamycin 76.5 ± 14.7 mm). (C) Quantification of LC3-positive cells was calculated. **P <0.01, ***P <0.001. DMM, destabilization of the medial meniscus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig2: The effect of rapamycin on phospho- mammalian target of rapamycin (p-mTOR) and light chain 3 (LC3) expression. (A) Representative images of immunostaining for p-mTOR (green) and LC3 (green). Scale bar = 20 μm. (B) Quantification of p-mTOR-positive cells was calculated. Rapamycin treatment suppressed p-mTOR expression in the rapamycin-treated mice compared to those treated with dimethyl sulphoxid (DMSO) at both the 8- and 12-week time points (at 8 weeks DMSO 108.5 ± 13.3, rapamycin 43.0 ± 8.9 mm; at 12 weeks DMSO 273.8 ± 27.0, rapamycin 76.5 ± 14.7 mm). (C) Quantification of LC3-positive cells was calculated. **P <0.01, ***P <0.001. DMM, destabilization of the medial meniscus.
Mentions: To determine whether the local intra-articular injection of rapamycin modulates mTOR and autophagy, the effects that rapamycin had on phospho-mTOR (p-mTOR) and LC3 expressions were examined. The expression of p-mTOR was increased in DMSO-treated mice at 8 and 12 weeks post DMM surgery compared to DMSO-treated mice undergoing the sham knee operation. Rapamycin treatment suppressed p-mTOR expression in these mice compared to those treated with DMSO at both the 8- and 12-week time points post DMM injury. The expression of p-mTOR in the rapamycin-treated mice was significantly increased at 12 weeks compared to 8 weeks after DMM injury (at 8 weeks DMSO 108.5 ± 13.3, rapamycin 43.0 ± 8.9; at 12 weeks DMSO 273.8 ± 27.0, rapamycin 76.5 ± 14.7) (Figure 2A, B). In contrast, there were more LC3-positive cells in the articular cartilage maintaining proteoglycan staining but fewer in the degenerated articular cartilage of the DMSO-treated mice after DMM surgery. An increase in LC3-positive cells was observed in the rapamycin-treated mice compared to those treated with DMSO at 8 and 12 weeks post DMM surgery. The number of LC3-positive cells in the rapamycin-treated mice decreased significantly at 12 weeks compared to 8 weeks post DMM injury (at 8 weeks DMSO 88.2 ± 13.1, rapamycin 236.0 ± 56.3; at 12 weeks DMSO 46.2 ± 12.8, rapamycin 106.3 ± 27.1) (Figure 2A, C).Figure 2

Bottom Line: VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice.Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.

Methods: Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).

Results: Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

Conclusion: These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.

Show MeSH
Related in: MedlinePlus