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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 vascular endothelial growth factor (VEGF) expression in the articular cartilage. (A) Representative images of immunostaining for VEGF (green). Scale bar = 20 μm. (B) Quantification of VEGF-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for VEGF in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. *P <0.05, **P <0.01, ***P <0.001.
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Fig3: The effect of rapamycin on vascular endothelial growth factor (VEGF) expression in the articular cartilage. (A) Representative images of immunostaining for VEGF (green). Scale bar = 20 μm. (B) Quantification of VEGF-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for VEGF in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. *P <0.05, **P <0.01, ***P <0.001.

Mentions: As it has been reported that the expression of VEGF is related to the development of OA changes [16], VEGF expression was examined with immunohistochemistry and qPCR (Figure 3). The number of VEGF-positive cells in the articular cartilage was significantly lower in the rapamycin group compared with the DMSO group at 8 and 12 week post DMM injury (at 8 weeks DMSO 143.8 ± 29.6, rapamycin 21.2 ± 8.4, P <0.001 for DMSO versus rapamcyin; at 12 weeks DMSO 275.5 ± 56.9, rapamycin 96.0 ± 27.8, P <0.001 for DMSO versus rapamcyin). The number of VEGF-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 3A, B). Similarly, the expression of VEGF by qPCR revealed a significant increase in the DMSO-treated mice at 8 weeks after DMM surgery compared to sham knees (Figure 3C, P <0.001 relative to sham). Rapamycin treatment decreased VEGF expression when compared to the DMSO-treated mice at the 8 and 12 week time points post DMM injury (Figure 3C, P <0.001 at 8 and 12 weeks), suggesting that the local intra-articular injection of rapamycin reduced articular cartilage damage, at least in part, through a reduction in VEGF expression.Figure 3


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 vascular endothelial growth factor (VEGF) expression in the articular cartilage. (A) Representative images of immunostaining for VEGF (green). Scale bar = 20 μm. (B) Quantification of VEGF-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for VEGF in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. *P <0.05, **P <0.01, ***P <0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4269094&req=5

Fig3: The effect of rapamycin on vascular endothelial growth factor (VEGF) expression in the articular cartilage. (A) Representative images of immunostaining for VEGF (green). Scale bar = 20 μm. (B) Quantification of VEGF-positive cells in the cartilage was calculated. (C) Graph indicating quantitative RT-PCR for VEGF in dimethyl sulphoxide (DMSO)- and rapamycin-treated mice at 8 and 12 weeks after destabilization of the medial meniscus (DMM) or sham surgery. *P <0.05, **P <0.01, ***P <0.001.
Mentions: As it has been reported that the expression of VEGF is related to the development of OA changes [16], VEGF expression was examined with immunohistochemistry and qPCR (Figure 3). The number of VEGF-positive cells in the articular cartilage was significantly lower in the rapamycin group compared with the DMSO group at 8 and 12 week post DMM injury (at 8 weeks DMSO 143.8 ± 29.6, rapamycin 21.2 ± 8.4, P <0.001 for DMSO versus rapamcyin; at 12 weeks DMSO 275.5 ± 56.9, rapamycin 96.0 ± 27.8, P <0.001 for DMSO versus rapamcyin). The number of VEGF-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 3A, B). Similarly, the expression of VEGF by qPCR revealed a significant increase in the DMSO-treated mice at 8 weeks after DMM surgery compared to sham knees (Figure 3C, P <0.001 relative to sham). Rapamycin treatment decreased VEGF expression when compared to the DMSO-treated mice at the 8 and 12 week time points post DMM injury (Figure 3C, P <0.001 at 8 and 12 weeks), suggesting that the local intra-articular injection of rapamycin reduced articular cartilage damage, at least in part, through a reduction in VEGF expression.Figure 3

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