Takuya Niimoto

The inhibitory effect of microRNA-146a expression on bone destruction in collagen-induced arthritis.

OBJECTIVE MicroRNA, a class of noncoding RNA, play a role in human diseases. MicroRNA-146a (miR-146a) is a negative regulator of immune and inflammatory responses, and is strongly expressed in rheumatoid arthritis (RA) synovium and peripheral blood mononuclear cells (PBMCs). This study was undertaken to examine whether miR-146a expression inhibits osteoclastogenesis, and whether administration of miR-146a prevents joint destruction in mice with collagen-induced arthritis (CIA). METHODS PBMCs from healthy volunteers were isolated and seeded in culture plates. The following day, double-stranded miR-146a was transfected and cultured in the presence of macrophage colony-stimulating factor and either tumor necrosis factor α or RANKL. After 3 weeks, tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were counted. Three days after miR-146a culture, the expression of c-Jun, nuclear factor of activated T cells c1 (NF-ATc1), PU.1, and TRAP was evaluated by quantitative reverse transcriptase-polymerase chain reaction. After the onset of distinct arthritis in mice with CIA, double-stranded miR-146a or nonspecific double-stranded RNA was administered twice by intravenous injection. Radiographic and histologic examinations were performed at 4 weeks. RESULTS The number of TRAP-positive multinucleated cells in human PBMCs was significantly reduced by miR-146a in a dose-dependent manner. The expression of c-Jun, NF-ATc1, PU.1, and TRAP in PBMCs was significantly down-regulated by miR-146a. Administration of miR-146a prevented joint destruction in mice with CIA, although it did not completely ameliorate inflammation. CONCLUSION Our findings indicate that expression of miR-146a inhibits osteoclastogenesis and that administration of double-stranded miR-146a prevents joint destruction in arthritic mice. Administration of miR-146a has potential as a novel therapeutic target for bone destruction in RA.

MicroRNA-146a expresses in interleukin-17 producing T cells in rheumatoid arthritis patients

BackgroundInterleukin (IL)-17 is an important factor in rheumatoid arthritis (RA) pathogenesis. MicroRNA (miRNA)s are a family of non coding RNAs and associated with human diseases including RA. The purpose of this study is to identify the miRNAs in the differentiation of IL-17 producing cells, and analyze their expression pattern in the peripheral blood mononuclear cells (PBMC) and synovium from RA patients.MethodsIL-17 producing cells were expanded from CD4+T cell. MiRNA microarray was performed to identify the miRNAs in the differentiation of IL-17 producing cells. Quantitative polymerase chain reaction was performed to examine the expression patterns of the identified miRNAs in the PBMC and synovium from RA and osteoarthritis (OA) patients. Double staining combining in situ hybridization and immunohistochemistry of IL-17 was performed to analyze the expression pattern of identified miRNA in the synovium.ResultsSix miRNAs, let-7a, miR-26, miR-146a/b, miR-150, and miR-155 were significantly up regulated in the IL-17 producing T cells. The expression of miR-146a and IL-17 was higher than in PBMC in the patients with low score of Larsen grade and short disease duration. MiR-146a intensely expressed in RA synovium in comparison to OA. MiR-146a expressed intensely in the synovium with hyperplasia and high expression of IL-17 from the patients with high disease activity. Double staining revealed that miR-146a expressed in IL-17 expressing cells.ConclusionThese results indicated that miR-146a was associated with IL-17 expression in the PBMC and synovium in RA patients. There is the possibility that miR-146a participates in the IL-17 expression.

Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro

OBJECTIVE miRNAs, which are non-coding RNAs, play a role in the pathogenesis of disease including OA. miRNA (miR)-34a is induced by p53, subsequently leading to cell apoptosis, which is one of the major factors in the pathogenesis of OA. The purpose of this study is to investigate the effect of silencing miR-34a on IL-1β-induced chondrocyte apoptosis in a rat OA model in vitro. METHODS Locked nucleotide analogue (LNA)-modified miR-34a-specific anti-sense was transfected into rat chondrocyte monolayer culture. After that, IL-1β was added to the chondrocytes to create an OA model in vitro. The effect of silencing miR-34a on the prevention of chondrocyte apoptosis was analysed by assessment of the expression levels of Col2a1 and iNOS, also through assessment of cell viability and TUNEL staining. RESULTS The expression of miR-34a was significantly up-regulated by IL-1β. Silencing of miR-34a significantly prevented IL-1β-induced down-regulation of Col2a1, as well as IL-1β-induced up-regulation of iNOS. Finally, MiR-34a inhibitor could also reduce TUNEL-positive cells. CONCLUSION Silencing of miR-34a by LNA-modified anti-sense could effectively reduce rat chondrocyte apoptosis induced by IL-1β. This present study revealed that silencing of miR-34a might develop a novel intervention for OA treatment through the prevention of cartilage degradation.

Changes in microRNA expression in peripheral mononuclear cells according to the progression of osteoarthritis

MicroRNAs (miRNAs) are a family of non-coding RNAs that play an important role in human diseases, including osteoarthritis (OA). The objective of this study was to investigate the expression patterns of miRNAs in the peripheral blood mononuclear cells (PBMCs) of OA patients. PBMCs were isolated from 36 patients with OA, 6 RA patients, and 36 healthy controls. The expression patterns of miR-146a, 155, 181a, and 223 in PBMCs were analyzed using quantitative reverse transcription-polymerase chain reaction (qPCR). We investigated the expression patterns of the miRNAs in OA progression, and their relationships with the parameters of age, body mass index (BMI), the femorotibial angle (FTA), and serum keratan sulfate (KS). The relative expression levels of miR-146a, 155, 181a, and 223 in the OA patients were significantly higher than those found in healthy controls. In the early stages of OA, miR-146a and 223 expressions were significantly higher than they were at later stages. There was a significant correlation between the expression of miR-223 and KS. This study demonstrated that high expression levels of miR-146a, 155, 181a, and 223 in the PBMCs of OA patients might be related to the pathogenesis of OA. This evidence could lead to the elucidation of the mechanism underlying OA pathogenesis and hence to a novel therapeutic strategy for OA.

The Effect of Intra-articular Injection of MicroRNA-210 on Ligament Healing in a Rat Model

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.

The Transverse Ligament as a Landmark for Tibial Sagittal Insertions of the Anterior Cruciate Ligament: A Cadaveric Study

PURPOSE The purpose of this study was to determine the relation between the position of the transverse ligament, the anterior edge of the anterior cruciate ligament (ACL) tibial footprint, and the center of the ACL tibial insertion. We used arthroscopy for localization of the anatomic landmarks, followed by insertions of guide pins under direct visualization, and then the position of these guide pins was checked on plain lateral radiographs. METHODS The transverse ligament and the anterior aspect of the ACL tibial footprint were identified by arthroscopy in 20 unpaired cadaveric knees (10 left and 10 right). Guide pins were inserted with tibial ACL adapter drill guides under direct observation at the transverse ligament, the anterior aspect of the tibial footprint, and the center of tibial insertion of the ACL. Then, plain lateral radiographs of specimens were taken. The Amis and Jakob line was used to define the attachment of the ACL tibial insertion and the transverse ligament. A sagittal percentage of the location of the insertion point was determined and calculated from the anterior margin of the tibia in the anteroposterior direction. RESULTS The transverse ligament averaged 21.20% ± 4.1%, the anterior edge of the ACL tibial insertion averaged 21.60% ± 4.0%, and the center of the ACL tibial insertion averaged 40.30% ± 4.8%. There were similar percent variations between the transverse ligament and the anterior edge of the ACL tibial insertion, with no significant difference between them (P = .38). Intraobserver and interobserver reliability was high, with small standard errors of measurement. CONCLUSIONS This study shows that the transverse ligament coincides with the anterior edge of the ACL tibial footprint in the sagittal plane. CLINICAL RELEVANCE The transverse ligament can be considered as a new landmark for tibial tunnel positioning during anatomic ACL reconstruction.

Knee Articulated Distraction Arthroplasty for the Middle-aged Osteoarthritic Knee Joint

Objective We developed a knee distraction arthroplasty device that allows continuous joint movement. The objective of this article is to show the surgical procedure of knee distraction arthroplasty with a bone marrow-stimulating technique, for treatment of osteoarthritis of the knee and to evaluate the clinical results. Methods As we showed in Arthroscopy in 2007, we performed this distraction arthroplasty to 6 knees (in 6 patients, aged 42 to 63 y). Then we compared preoperative findings with postoperative ones. The fixation period for the distraction device ranged from 7 to 12 weeks and the follow-up period ranged from 24 months to 53 months (average 36 mo). Results The Japan Orthopaedic Association knee score, range of motion, and the values of the joint spaces were significantly improved in all cases at the latest follow-up (P<0.05). Visual analog pain scales were also significantly improved (P<0.05). Conclusions We conclude that treatment using this arthroplasty device in combination with a bone marrow stimulating method is effective for osteoarthritic knees in middle-aged patients.

Augmentation Procedure for Partial Rupture of the Anterior Cruciate Ligament

Arthroscopic examination before anterior cruciate ligament (ACL) reconstruction shows the presence of several types of ACL remnants within the intercondylar notch. It is thought that 10% to 20% of the ACL injury cases represent a partial rupture of the ACL. In these cases, although rupture of the anteromedial or posterolateral bundle can be seen, the other bundle is preserved with an attachment of the anatomical femoral origin. We considered it beneficial to perform the ACL augmentation procedure without sacrificing the remaining remnant in terms of proprioception, biomechanical functions, and vascularization of the graft. This study will present the ACL augmentation procedure for patients with partial ACL rupture. We believe that the described technique can be a treatment option for patients whose ACL remnants are left in certain conditions.