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Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model.

Kawanishi Y, Nakasa T, Shoji T, Hamanishi M, Shimizu R, Kamei N, Usman MA, Ochi M - Arthritis Res. Ther. (2014)

Bottom Line: In gene expression analysis of the meniscus, the expression of miR-210, Collagen type 2 alpha 1 (Col2a1), Vascular endothelial growth factor (VEGF), and Fibroblast growth factor-2 (FGF2) in the miR-210 group was significantly higher than that in the control.In vitro, miR-210 upregulated Col2a1 expression in the meniscus cells and VEGF and FGF2 expression in the synovial cells.An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The important functions of the meniscus are shock absorption, passive stabilization and load transmission of the knee. Because of the avascularity of two-thirds of the meniscal center region, the treatment of tears in this area is hard. Recently, microRNAs have been proven to play an important role in the pathogenesis of diseases. We focused on microRNA (miR)-210, which plays a wide spectrum of roles comprising mitochondrial metabolism, angiogenesis, DNA repair and cell survival. This study aimed to investigate the effect of intra-articular injection of synthetic miR-210 on the injured meniscus in the avascular zone.

Methods: The middle segments of the medial meniscus of Spraque Dawley rats were incised longitudinally with a scalpel. An intra-articular injection of double-stranded (ds) miR-210 (for control group using control dsRNA) with atelocollagen was administered immediately after injury. Four weeks and 12 weeks after the injection, we conducted a histologic evaluation, immunohistochemical evaluation and Real-time PCR analysis. In vitro, the inner meniscus and synovial cells were isolated from rat knee joint, and were transfected with ds miR-210 or control dsRNA. Real-time PCR and immunohistochemical evaluations were performed.

Results: Twenty-four hours after the injection, FAM (Fluorescein amidite) labeled miR-210 was observed in the cells around the injured site. Four weeks after the injection, the injured site of the miR-210 group was filled with repaired tissue while that of the control was not repaired. In gene expression analysis of the meniscus, the expression of miR-210, Collagen type 2 alpha 1 (Col2a1), Vascular endothelial growth factor (VEGF), and Fibroblast growth factor-2 (FGF2) in the miR-210 group was significantly higher than that in the control. At 12 weeks, the intra-articular injection of miR-210 had healed the injured site of the meniscus and had prevented articular cartilage degeneration. In vitro, miR-210 upregulated Col2a1 expression in the meniscus cells and VEGF and FGF2 expression in the synovial cells.

Conclusions: An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells.

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Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (*P < 0.05, N.S. (no significant difference), n = 6 for each group). (B) Immunohistochemical analysis indicates that VEGF and FGF2 were highly expressed in the miR-210 group (bar = 100 μm). These results demonstrated that miR-210 could enhance the expression of VEGF and FGF2 expression in synovial cells. ds, double stranded.
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Fig6: Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (*P < 0.05, N.S. (no significant difference), n = 6 for each group). (B) Immunohistochemical analysis indicates that VEGF and FGF2 were highly expressed in the miR-210 group (bar = 100 μm). These results demonstrated that miR-210 could enhance the expression of VEGF and FGF2 expression in synovial cells. ds, double stranded.

Mentions: Real-time PCR revealed that Col2a1 expression was significantly upregulated in the inner meniscus cells, and immunocytochemistry also demonstrated that miR-210 could enhance the expression of collagen type 2 in the meniscus cells (P < 0.05, n = 6 for each group) (Figure 5A,B). On the other hand, the expression of VEGF and FGF2 was significantly upregulated in the synovial cells (P < 0.05, n = 6 for each group) (Figure 6A). Immunocytochemistry indicated that VEGF and FGF2 expression were enhanced in the synovial cells (Figure 6B).Figure 5


Intra-articular injection of synthetic microRNA-210 accelerates avascular meniscal healing in rat medial meniscal injured model.

Kawanishi Y, Nakasa T, Shoji T, Hamanishi M, Shimizu R, Kamei N, Usman MA, Ochi M - Arthritis Res. Ther. (2014)

Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (*P < 0.05, N.S. (no significant difference), n = 6 for each group). (B) Immunohistochemical analysis indicates that VEGF and FGF2 were highly expressed in the miR-210 group (bar = 100 μm). These results demonstrated that miR-210 could enhance the expression of VEGF and FGF2 expression in synovial cells. ds, double stranded.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4265493&req=5

Fig6: Gene expression analyses in synovial cells after overexpression of miR-210. (A) Real-time PCR analysis of collagen type 1 alpha 1 (Col1a1), collagen type 2 alpha 1 (Col2a1), vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) at 7 days after in vitro transfection of synovial cells. Expression of Col2a1 was not detected in both groups. Expression of VEGF and FGF2 was significantly higher than in the control group (*P < 0.05, N.S. (no significant difference), n = 6 for each group). (B) Immunohistochemical analysis indicates that VEGF and FGF2 were highly expressed in the miR-210 group (bar = 100 μm). These results demonstrated that miR-210 could enhance the expression of VEGF and FGF2 expression in synovial cells. ds, double stranded.
Mentions: Real-time PCR revealed that Col2a1 expression was significantly upregulated in the inner meniscus cells, and immunocytochemistry also demonstrated that miR-210 could enhance the expression of collagen type 2 in the meniscus cells (P < 0.05, n = 6 for each group) (Figure 5A,B). On the other hand, the expression of VEGF and FGF2 was significantly upregulated in the synovial cells (P < 0.05, n = 6 for each group) (Figure 6A). Immunocytochemistry indicated that VEGF and FGF2 expression were enhanced in the synovial cells (Figure 6B).Figure 5

Bottom Line: In gene expression analysis of the meniscus, the expression of miR-210, Collagen type 2 alpha 1 (Col2a1), Vascular endothelial growth factor (VEGF), and Fibroblast growth factor-2 (FGF2) in the miR-210 group was significantly higher than that in the control.In vitro, miR-210 upregulated Col2a1 expression in the meniscus cells and VEGF and FGF2 expression in the synovial cells.An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells.

View Article: PubMed Central - PubMed

ABSTRACT

Introduction: The important functions of the meniscus are shock absorption, passive stabilization and load transmission of the knee. Because of the avascularity of two-thirds of the meniscal center region, the treatment of tears in this area is hard. Recently, microRNAs have been proven to play an important role in the pathogenesis of diseases. We focused on microRNA (miR)-210, which plays a wide spectrum of roles comprising mitochondrial metabolism, angiogenesis, DNA repair and cell survival. This study aimed to investigate the effect of intra-articular injection of synthetic miR-210 on the injured meniscus in the avascular zone.

Methods: The middle segments of the medial meniscus of Spraque Dawley rats were incised longitudinally with a scalpel. An intra-articular injection of double-stranded (ds) miR-210 (for control group using control dsRNA) with atelocollagen was administered immediately after injury. Four weeks and 12 weeks after the injection, we conducted a histologic evaluation, immunohistochemical evaluation and Real-time PCR analysis. In vitro, the inner meniscus and synovial cells were isolated from rat knee joint, and were transfected with ds miR-210 or control dsRNA. Real-time PCR and immunohistochemical evaluations were performed.

Results: Twenty-four hours after the injection, FAM (Fluorescein amidite) labeled miR-210 was observed in the cells around the injured site. Four weeks after the injection, the injured site of the miR-210 group was filled with repaired tissue while that of the control was not repaired. In gene expression analysis of the meniscus, the expression of miR-210, Collagen type 2 alpha 1 (Col2a1), Vascular endothelial growth factor (VEGF), and Fibroblast growth factor-2 (FGF2) in the miR-210 group was significantly higher than that in the control. At 12 weeks, the intra-articular injection of miR-210 had healed the injured site of the meniscus and had prevented articular cartilage degeneration. In vitro, miR-210 upregulated Col2a1 expression in the meniscus cells and VEGF and FGF2 expression in the synovial cells.

Conclusions: An intra-articular injection of ds miR-210 was effective in the healing of the damaged white zone meniscus through promotion of the collagen type 2 production from meniscus cells and through upregulated of VEGF and FGF2 from synovial cells.

Show MeSH
Related in: MedlinePlus