BACKGROUND: It is known from clinical and experimental studies that the healing potential of the anterior cruciate ligament (ACL) is extremely poor and that early phases of ligament healing require an augmented blood supply. MicroRNA (miRNA) is a type of small, noncoding RNA that negatively regulates gene expression, and miRNA (miR)-210 is reported to be crucial for cell response to hypoxia, vascular endothelial growth factor (VEGF)-driven endothelial cell migration, and formation of capillary-like structures. PURPOSE: The purpose of this study was to examine the effect of intra-articular injection of miRNA miR-210 on acceleration of ACL healing. STUDY DESIGN: Controlled laboratory study. METHODS: Two experiments were performed in this study. The ACLs of 12-week-old male LEW/CrlCrlj rats were partially transected. First, the temporal expression change of miR-210 after ACL injury was analyzed using real-time polymerase chain reaction (PCR) on day zero, and 1, 2, and 4 weeks after injury (n = 5 at each time point). Next, intra-articular injection of double-stranded (ds) miR-210 with atelocollagen was performed soon after injury. The control group was injected with control small interfering RNA (siRNA). Four weeks after injection, biomechanical and histological assessments of samples stained with H&E as well as Masson trichrome, and immunohistochemistry for VEGF, fibroblast growth factor 2 (FGF2), isolectin B4, and collagen type I, were performed. Real-time PCR analysis was also performed for quantitative evaluation of miR-210, VEGF-A, and collagen type I. RESULTS: Real-time PCR analysis revealed that miR-210 expression was decreased soon after injury but gradually increased thereafter. Histological analysis confirmed that the transected area was covered with healing tissue in the miR-210 group but remained devoid of any tissue in the control group 4 weeks after injury. Biomechanical analysis confirmed the improvement of biomechanical properties in the miR-210 group; the ultimate failure loads 4 weeks after injection were 30.5 ± 3.1 N in the miR-210 group and 22.8 ± 3.1 N in the control group (P < .05). Real-time PCR analysis showed that endogenous miR-210, VEGF, and collagen type I were highly expressed compared with controls, and immunohistochemistry for VEGF, FGF2, isolectin B4, and collagen type I showed that VEGF and FGF2 were highly upregulated, and there were abundant blood vessels and fibrotic deposition in the miR-210 group. CONCLUSION: Injection of ds miR-210 was effective in promoting the healing of partially torn ACLs through enhancement of angiogenesis via upregulation of VEGF and FGF2. CLINICAL RELEVANCE: It might represent a potential therapeutic approach for treatment of ACL injury.
BACKGROUND: It is known from clinical and experimental studies that the healing potential of the anterior cruciate ligament (ACL) is extremely poor and that early phases of ligament healing require an augmented blood supply. MicroRNA (miRNA) is a type of small, noncoding RNA that negatively regulates gene expression, and miRNA (miR)-210 is reported to be crucial for cell response to hypoxia, vascular endothelial growth factor (VEGF)-driven endothelial cell migration, and formation of capillary-like structures. PURPOSE: The purpose of this study was to examine the effect of intra-articular injection of miRNA miR-210 on acceleration of ACL healing. STUDY DESIGN: Controlled laboratory study. METHODS: Two experiments were performed in this study. The ACLs of 12-week-old male LEW/CrlCrlj rats were partially transected. First, the temporal expression change of miR-210 after ACL injury was analyzed using real-time polymerase chain reaction (PCR) on day zero, and 1, 2, and 4 weeks after injury (n = 5 at each time point). Next, intra-articular injection of double-stranded (ds) miR-210 with atelocollagen was performed soon after injury. The control group was injected with control small interfering RNA (siRNA). Four weeks after injection, biomechanical and histological assessments of samples stained with H&E as well as Masson trichrome, and immunohistochemistry for VEGF, fibroblast growth factor 2 (FGF2), isolectin B4, and collagen type I, were performed. Real-time PCR analysis was also performed for quantitative evaluation of miR-210, VEGF-A, and collagen type I. RESULTS: Real-time PCR analysis revealed that miR-210 expression was decreased soon after injury but gradually increased thereafter. Histological analysis confirmed that the transected area was covered with healing tissue in the miR-210 group but remained devoid of any tissue in the control group 4 weeks after injury. Biomechanical analysis confirmed the improvement of biomechanical properties in the miR-210 group; the ultimate failure loads 4 weeks after injection were 30.5 ± 3.1 N in the miR-210 group and 22.8 ± 3.1 N in the control group (P < .05). Real-time PCR analysis showed that endogenous miR-210, VEGF, and collagen type I were highly expressed compared with controls, and immunohistochemistry for VEGF, FGF2, isolectin B4, and collagen type I showed that VEGF and FGF2 were highly upregulated, and there were abundant blood vessels and fibrotic deposition in the miR-210 group. CONCLUSION: Injection of dsmiR-210 was effective in promoting the healing of partially torn ACLs through enhancement of angiogenesis via upregulation of VEGF and FGF2. CLINICAL RELEVANCE: It might represent a potential therapeutic approach for treatment of ACL injury.
Authors: Antonio Marmotti; Roberto Rossi; Filippo Castoldi; Eliana Roveda; Gianni Michielon; Giuseppe M Peretti Journal: Biomed Res Int Date: 2015-05-05 Impact factor: 3.411