Ryu Terauchi

Sequence‐specific gene silencing in murine muscle induced by electroporation‐mediated transfer of short interfering RNA

Post‐genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence‐specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation‐mediated transfer of short interfering RNA (siRNA).

Successful genetic transduction in vivo into synovium by means of electroporation

This present study aims at establishing a novel in vivo gene delivery system for intra-articular tissues. Plasmid DNA (pDNA) carrying the firefly luciferase or enhanced green fluorescent protein (EGFP) genes as markers was injected into a joint space and electric stimuli were given percutaneously with a pair of electrodes. Injection with naked pDNA alone did not induce any detectable level of luciferase activity, whereas electroporation at 25-500 V/0.7 cm resulted in a significant expression of the marker gene in the synovium. The expression level depended on the voltage, the optimum transfection being achieved at 150 V/0.7 cm. When the Epstein-Barr virus (EBV)-based plasmid vectors harboring the EBV nuclear antigen 1 (EBNA1) gene and oriP sequence were substituted for conventional pDNA, the transfection efficiency was increased approximately 5-10 times. Histological examination of the EGFP gene-transfected joints revealed that the marker gene was expressed in the synovial membrane while other intra-articular tissues such as articular cartilage were negative for the transgene product. Transgene-specific mRNA was demonstrated in synovium but not in other organs as estimated by RT-PCR analysis. The present results strongly suggest that in vivo electroporation is a quite simple, safe, and effective gene delivery method that could be applicable to gene therapy against articular diseases.

Hyperthermia for the treatment of articular cartilage with osteoarthritis

Osteoarthritis (OA) is one of the most frequent musculoskeletal disorders in the elderly population. OA is characterised by a gradual loss of extracellular matrix in the articular cartilage of joints. OA can only be managed by artificial joint replacement when joint destruction becomes severe. Therefore, it is preferable to administer conservative therapy that is easy, simple and effective in inhibiting OA progression at the early stage. Heat shock protein 70 (Hsp70) has a protective effect on the cartilage and inhibits the apoptosis of chondrocytes. Heat stimulation by microwave to the joints can increase Hsp70 expression in chondrocytes, and at the same time, Hsp70 expression partially enhances matrix metabolism of the cartilage. These findings suggest that hyperthermia can be positively applied to the treatment of OA. Hyperthermia is therefore expected to be an inexpensive and less-invasive conservative therapy for OA.

Silencing the expression of connexin 43 decreases inflammation and joint destruction in experimental arthritis.

The objective of the present study was to determine whether the expression of connexin 43 (Cx43) effected on inflammatory conditions in rat fibroblast‐like synoviocytes (FLS) and on rat model of rheumatoid arthritis (RA). The expression of Cx43 in rat FLS stimulated with lipopolysaccharide (LPS) was confirmed by real‐time reverse transcriptase polymerase chain reaction (RT‐PCR). The effects of small‐interfering RNA targeting Cx43 (siCx43) on pro‐inflammatory cytokines and chemokine were assessed by real‐time RT‐PCR and enzyme‐linked immunosorbent assay (ELISA). The therapeutic and side effects of siCx43 in a rat model of collagen‐induced arthritis (CIA) were examined by in vivo electroporation method. LPS markedly enhanced Cx43 gene expression in rat FLS, with transfection of siCx43 suppressing the over‐expression of pro‐inflammatory cytokines and the chemokine. Treatment of CIA rats with siCx43 significantly ameliorated paw swelling, and significantly reduced histological arthritis scores and radiographic scores. In histological appearance of rat ankle joints, siCx43 treatment significantly decreased the number of tartrate‐resistant acid phosphatase (TRAP)‐positive (osteoclast‐like) cells. These findings indicated that siCx43 had anti‐inflammatory effects in rat FLS and efficiently inhibited the development of CIA. Cx43 may play an important role in the pathophysiology of RA, and may be a potential target molecule for novel RA therapies.

HIF-1α-induced HSP70 regulates anabolic responses in articular chondrocytes under hypoxic conditions.

We assessed whether heat shock protein 70 (HSP70) is involved in hypoxia inducible factor 1 alpha (HIF‐1α)‐dependent anabolic pathways in articular chondrocytes under hypoxic conditions. Primary rabbit chondrocytes were cultured under normoxia (20% oxygen condition) or hypoxia (1% oxygen condition). Alternatively, cells cultured under normoxia were treated with CoCl2, which induces HIF‐1α, to simulate hypoxia, or transfected with siRNAs targeting HIF‐1α (si‐HIF‐1α) and HSP70 (si‐HSP70) under hypoxia. HSP70 expression was enhanced by the increased expression of HIF‐1α under hypoxia or simulated hypoxia, but not in the presence of si‐HIF‐1α. Hypoxia‐induced overexpression of ECM genes was significantly suppressed by si‐HIF‐1α or si‐HSP70. Cell viability positively correlated with hypoxia, but transfection with si‐HIF‐1α or si‐HSP70 abrogated the chondroprotective effects of hypoxia. Although LDH release from sodium nitroprusside‐treated cells and the proportion of TUNEL positive cells were decreased under hypoxia, transfection with si‐HIF‐1α or si‐HSP70 almost completely blocked these effects. These findings indicated that HIF‐1α‐induced HSP70 overexpression increased the expression levels of ECM genes and cell viability, and protected chondrocytes from apoptosis. HIF‐1α may regulate the anabolic effects of chondrocytes under hypoxic conditions by regulating HSP70 expression.

Antimicrobial megaprostheses supported with iodine

Deep infection associated with implants remains a serious complication of orthopedic surgery. We developed iodine coating for titanium implants. In this study, we performed a clinical trial of iodine-coated megaprostheses to evaluate its safety and antibacterial effects. Forty-seven patients with malignant bone tumor or pyogenic arthritis were treated using iodine-supported titanium megaprostheses between July 2008 and May 2013. The mean age was 53.6 years (range, 15–85 years). Twenty-six patients were males and 21 were females. The diagnoses included malignant bone tumor in 29 cases, infected total knee arthroplasty in 11 cases, chronic osteomyelitis due to pyogenic arthritis in six cases and loosening of total knee arthroplasty in one case. The iodine-supported implants used were 42 Kyocera Limb Salvage System and five KOBELCO K-MAX K-3. These megaprostheses were used to prevent infection in 21 patients, treat active infections in 26 patients. The mean follow-up period was 30.1 months (range, 8–50). Infection was prevented in 20 out of 21 patients. Only one patient had surgical site infection caused by Pseudomonas aeruginosa and was cured by intravenous administration of antibiotics alone without removal of the implant. In 26 treatment cases involving one- or two-stage revision surgery, infection subsided without any additional surgery. In all cases, there were no signs of infection at the time of the last follow-up. White blood cell and C-reactive protein levels returned to normal within four weeks after surgery. To confirm systemic effects of iodine, thyroid hormone levels in the blood were examined. Abnormalities of thyroid gland function were not detected. Loosening of the implants was not observed. Excellent bone ingrowth and ongrowth were found around iodine-supported megaprostheses. The iodine-supported titanium megaprostheses are highly effective and show promise for the prevention and treatment of infections in large bone defects. No cytotoxicity or adverse effects were detected with this treatment.

Combined microwave irradiation and intraarticular glutamine administration-induced HSP70 expression therapy prevents cartilage degradation in a rat osteoarthritis model.

The objective of the present study was to investigate the effects of heat stimulation and glutamine (Gln) on the expression of extracellular matrix genes and heat shock protein 70 (HSP70) in rat articular cartilage in vivo and to determine whether HSP70 expression achieved with a combination of microwave (MW) and Gln suppresses osteoarthritis (OA) progression in a rat OA model. Stimulation at 40 W was assumed to be appropriate in the present study, and the effects of heat treatment at this intensity were evaluated. Articular cartilage was collected at 8 h after heat stimulation and/or intraarticular Gln administration, and total RNA was extracted. The expression of HSP70, aggrecan, and type II collagen was quantified using real‐time RT‐PCR. Cartilage samples from the OA model were subjected to hematoxylin and eosin (HE) and safranin O staining. HSP70 and aggrecan expression was greatest in a group receiving both MW and Gln. In the rat OA model, the severity of OA was significantly milder in a group receiving MW and Gln than in the control group. HSP70, stimulated by the combination of MW heat and Gln, may be involved in the suppression of OA progression.

Radial magnetic resonance imaging and pathological findings of acetabular labrum in dysplastic hips

Radial MRI findings and pathological changes were comparatively examined in the acetabular labrum of 11 hips of 11 patients, who underwent total hip arthroplasty for osteoarthritis due to acetabular dysplasia. Diffuse high signal pattern on the radial MR images corresponded to histological degeneration of the labrum. High signal pattern which was equivalent to the synovial fluid, showed an intralabral tear. In the obscure areas of MR images, severe impairment of the labrum such as rupture, detachment, and displacement were found.

Sonoporation-mediated transduction of siRNA ameliorated experimental arthritis using 3 MHz pulsed ultrasound

The goal of this feasibility study was to examine whether sonoporation assisted transduction of siRNA could be used to ameliorate arthritis locally. If successful, such approach could provide an alternative treatment for the patients that have or gradually develop adverse response to chemical drugs. Tumor necrosis factor alpha (TNF-α) produced by synovial fibroblasts has an important role in the pathology of rheumatoid arthritis, inducing inflammation and bone destruction. In this study, we injected a mixture of microbubbles and siRNA targeting TNF-α (siTNF) into the articular joints of rats, and transduced siTNF into synovial tissue by exposure to a collimated ultrasound beam, applied through a probe 6mm in diameter with an input frequency of 3.0 MHz, an output intensity of 2.0 W/cm(2) (spatial average temporary peak; SATP), a pulse duty ratio of 50%, and a duration of 1 min. Sonoporation increased skin temperature from 26.8 °C to 27.3 °C, but there were no adverse effect such as burns. The mean level of TNF-α expression in siTNF-treated knee joints was 55% of those in controls. Delivery of siTNF into the knee joints every 3 days (i.e., 7, 10, 13, and 16 days after immunization) by in vivo sonoporation significantly reduced paw swelling on days 20-23 after immunization. Radiographic scores in the siTNF group were 56% of those in the CIA group and 61% of those in the siNeg group. Histological examination showed that the number of TNF-α positive cells was significantly lower in areas of pannus invasion into the ankle joints of siTNF- than of siNeg-treated rats. These results indicate that transduction of siTNF into articular synovium using sonoporation may be an effective local therapy for arthritis.

Hydrostatic pressure influences HIF-2 alpha expression in chondrocytes.

Hypoxia-inducible factor (HIF)-2α is considered to play a major role in the progression of osteoarthritis. Recently, it was reported that pressure amplitude influences HIF-2α expression in murine endothelial cells. We examined whether hydrostatic pressure is involved in expression of HIF-2α in articular chondrocytes. Chondrocytes were cultured and stimulated by inflammation or hydrostatic pressure of 0, 5, 10, or 50 MPa. After stimulation, heat shock protein (HSP) 70, HIF-2α, nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-13, MMP-3, and vascular endothelial growth factor (VEGF) gene expression were evaluated. The levels of all gene expression were increased by inflammatory stress. When chondrocytes were exposed to a hydrostatic pressure of 5 MPa, HIF-2α, MMP-13, and MMP-3 gene expression increased significantly although those of HSP70 and NF-κB were not significantly different from the control group. In contrast, HIF-2α gene expression did not increase under a hydrostatic pressure of 50 MPa although HSP70 and NF-κB expression increased significantly compared to control. We considered that hydrostatic pressure of 5 MPa could regulate HIF-2α independent of NF-κB, because the level of HIF-2α gene expression increased significantly without upregulation of NF-κB expression at 5 MPa. Hydrostatic pressure may influence cartilage degeneration, inducing MMP-13 and MMP-3 expression through HIF-2α.