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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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Proposed schematic model showing how rapamycin modulates osteoarthritis (OA) in our animal model. There are three potential mechanisms by which rapamycin has a beneficial effect on OA (autophagy, vascular endothelial growth factor (VEGF), and the Akt pathway). mTOR, mammalian target of rapamycin; MMP13, matrix metallopeptidase 13; COL10A1, collagen, type X alpha 1.
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Fig6: Proposed schematic model showing how rapamycin modulates osteoarthritis (OA) in our animal model. There are three potential mechanisms by which rapamycin has a beneficial effect on OA (autophagy, vascular endothelial growth factor (VEGF), and the Akt pathway). mTOR, mammalian target of rapamycin; MMP13, matrix metallopeptidase 13; COL10A1, collagen, type X alpha 1.

Mentions: Recent studies have demonstrated that mTOR inhibition by rapamycin triggers a negative feedback loop, resulting in the activation of Akt signaling [17,18]. In adult articular cartilage, phosphoinositide 3 (PI-3) kinase-Akt signaling promotes matrix synthesis as well as the survival of chondrocytes. Activation of Akt in human articular chondrocytes significantly increase proteoglycan synthesis and type II collagen expression [19-21] (Figure 6). Another important function of the mTOR signaling pathway is the regulation of autophagy, a process in which the cell degrades damaged or excess cellular components, ranging from individual proteins and protein aggregates to whole organelles, through the use of the cell lysosomal machinery. A previous in vitro study indicated that autophagy activation by 10 μM rapamycin regulated a change in the expression of OA-related genes through the modulation of apoptosis and reactive oxygen species (ROS) in human chondrocytes [8]. Similarly, during the development of OA, autophagy increased as an adaptive response to protect the cells from various stresses, while in severely damaged articular cartilage, autophagy was reduced [8]. The present study demonstrated that LC3-positive cells resided in healthy articular cartilage maintaining proteoglycan staining, and LC3-expressing cells decreased in degenerating articular cartilage after DMM surgery. Thus, it is plausible that articular cartilage degradation after surgical induction of OA is partially due to insufficient autophagy. In support of this hypothesis, our results also indicate that the beneficial effect of local intra-articular rapamycin treatment on OA-induced cartilage damage correlates with an increase in LC3-expressing cells (Figure 6).Figure 6


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)

Proposed schematic model showing how rapamycin modulates osteoarthritis (OA) in our animal model. There are three potential mechanisms by which rapamycin has a beneficial effect on OA (autophagy, vascular endothelial growth factor (VEGF), and the Akt pathway). mTOR, mammalian target of rapamycin; MMP13, matrix metallopeptidase 13; COL10A1, collagen, type X alpha 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Proposed schematic model showing how rapamycin modulates osteoarthritis (OA) in our animal model. There are three potential mechanisms by which rapamycin has a beneficial effect on OA (autophagy, vascular endothelial growth factor (VEGF), and the Akt pathway). mTOR, mammalian target of rapamycin; MMP13, matrix metallopeptidase 13; COL10A1, collagen, type X alpha 1.
Mentions: Recent studies have demonstrated that mTOR inhibition by rapamycin triggers a negative feedback loop, resulting in the activation of Akt signaling [17,18]. In adult articular cartilage, phosphoinositide 3 (PI-3) kinase-Akt signaling promotes matrix synthesis as well as the survival of chondrocytes. Activation of Akt in human articular chondrocytes significantly increase proteoglycan synthesis and type II collagen expression [19-21] (Figure 6). Another important function of the mTOR signaling pathway is the regulation of autophagy, a process in which the cell degrades damaged or excess cellular components, ranging from individual proteins and protein aggregates to whole organelles, through the use of the cell lysosomal machinery. A previous in vitro study indicated that autophagy activation by 10 μM rapamycin regulated a change in the expression of OA-related genes through the modulation of apoptosis and reactive oxygen species (ROS) in human chondrocytes [8]. Similarly, during the development of OA, autophagy increased as an adaptive response to protect the cells from various stresses, while in severely damaged articular cartilage, autophagy was reduced [8]. The present study demonstrated that LC3-positive cells resided in healthy articular cartilage maintaining proteoglycan staining, and LC3-expressing cells decreased in degenerating articular cartilage after DMM surgery. Thus, it is plausible that articular cartilage degradation after surgical induction of OA is partially due to insufficient autophagy. In support of this hypothesis, our results also indicate that the beneficial effect of local intra-articular rapamycin treatment on OA-induced cartilage damage correlates with an increase in LC3-expressing cells (Figure 6).Figure 6

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