Limits...
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.

Show MeSH

Related in: MedlinePlus

The effect of rapamycin on matrix metallopeptidase 13 (MMP13) expression in articular cartilage. (A) Representative images of immunostaining for MMP13 (green). Scale bar = 20 μm. (B) Quantification of MMP13-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for MMP13 in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice 8 and 12 weeks after destabilization of the medial meniscus (DMM) injury. ***P <0.001, **P <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4269094&req=5

Fig5: The effect of rapamycin on matrix metallopeptidase 13 (MMP13) expression in articular cartilage. (A) Representative images of immunostaining for MMP13 (green). Scale bar = 20 μm. (B) Quantification of MMP13-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for MMP13 in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice 8 and 12 weeks after destabilization of the medial meniscus (DMM) injury. ***P <0.001, **P <0.01.

Mentions: To investigate the mechanism behind rapamycin’s ability to prevent experimental OA, subsequent experimentation focused on chondrocyte hypertrophy and consequently the expression of COL10A1 and MMP13 (hypertrophic chondrocyte markers) was examined by immunohistochemistry and qPCR (Figures 4, 5). The number of CoL10A1-positive cells in the articular cartilage was significantly lower in the rapamycin group when compared to the DMSO group at 8 and 12 weeks post DMM injury (at 8 weeks DMSO 88.2 ± 13.1, rapamycin 236.0 ± 56.3, P <0.001 for DMSO versus rapamcyin; at 12 weeks DMSO 46.2 ± 12.8, rapamycin 106.3 ± 27.1, P <0.001 for DMSO versus rapamcyin). The number of CoL10A1-positive cells in the rapamycin-treated mice increased at 12 weeks compared to 8 weeks post DMM injury (P <0.001 for DMSO at 8 weeks versus 12 weeks) (Figure 4A, B). Similarly the expression of COL10A1, as measured by qPCR, was increased in the DMSO-treated mice at 8 and 12 weeks after DMM surgery compared to the sham knees (Figure 4C: P = 0.006 at 8 weeks, P <0.001 at 12 weeks, respectively). Also intra-articular injection of rapamycin significantly decreased the expression of COL10A1 compared to the DMSO-treated mice at 8 and 12 weeks after DMM surgery (Figure 4C: P = 0.001at 8 weeks, P <0.001 at 12 weeks, respectively).Figure 4


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 matrix metallopeptidase 13 (MMP13) expression in articular cartilage. (A) Representative images of immunostaining for MMP13 (green). Scale bar = 20 μm. (B) Quantification of MMP13-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for MMP13 in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice 8 and 12 weeks after destabilization of the medial meniscus (DMM) injury. ***P <0.001, **P <0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269094&req=5

Fig5: The effect of rapamycin on matrix metallopeptidase 13 (MMP13) expression in articular cartilage. (A) Representative images of immunostaining for MMP13 (green). Scale bar = 20 μm. (B) Quantification of MMP13-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for MMP13 in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice 8 and 12 weeks after destabilization of the medial meniscus (DMM) injury. ***P <0.001, **P <0.01.
Mentions: To investigate the mechanism behind rapamycin’s ability to prevent experimental OA, subsequent experimentation focused on chondrocyte hypertrophy and consequently the expression of COL10A1 and MMP13 (hypertrophic chondrocyte markers) was examined by immunohistochemistry and qPCR (Figures 4, 5). The number of CoL10A1-positive cells in the articular cartilage was significantly lower in the rapamycin group when compared to the DMSO group at 8 and 12 weeks post DMM injury (at 8 weeks DMSO 88.2 ± 13.1, rapamycin 236.0 ± 56.3, P <0.001 for DMSO versus rapamcyin; at 12 weeks DMSO 46.2 ± 12.8, rapamycin 106.3 ± 27.1, P <0.001 for DMSO versus rapamcyin). The number of CoL10A1-positive cells in the rapamycin-treated mice increased at 12 weeks compared to 8 weeks post DMM injury (P <0.001 for DMSO at 8 weeks versus 12 weeks) (Figure 4A, B). Similarly the expression of COL10A1, as measured by qPCR, was increased in the DMSO-treated mice at 8 and 12 weeks after DMM surgery compared to the sham knees (Figure 4C: P = 0.006 at 8 weeks, P <0.001 at 12 weeks, respectively). Also intra-articular injection of rapamycin significantly decreased the expression of COL10A1 compared to the DMSO-treated mice at 8 and 12 weeks after DMM surgery (Figure 4C: P = 0.001at 8 weeks, P <0.001 at 12 weeks, respectively).Figure 4

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