Masakazu Ishikawa

Dose-Dependent Contribution of CD34-Positive Cell Transplantation to Concurrent Vasculogenesis and Cardiomyogenesis for Functional Regenerative Recovery After Myocardial Infarction

Background— Multilineage developmental capacity of the CD34+ cells, especially into cardiomyocytes and smooth muscle cells (SMCs), is still controversial. In the present study we performed a series of experiments to prove our hypothesis that vasculogenesis and cardiomyogenesis after myocardial infarction (MI) may be dose-dependently enhanced after CD34+ cell transplantation. Methods and Results— Peripheral blood CD34+ cells were isolated from total mononuclear cells of patients with limb ischemia by apheresis after 5-day administration of granulocyte colony-stimulating factor. PBS and 1×103 (low), 1×105 (mid), or 5×105 (high) CD34+ cells were intramyocardially transplanted after ligation of the left anterior descending coronary artery of nude rats. Functional assessments with the use of echocardiography and a microtip conductance catheter at day 28 revealed dose-dependent preservation of left ventricular function by CD34+ cell transplantation. Necropsy examination disclosed dose-dependent augmentation of capillary density and dose-dependent inhibition of left ventricular fibrosis. Immunohistochemistry for human-specific brain natriuretic peptide demonstrated that human cardiomyocytes were dose-dependently observed in ischemic myocardium at day 28 (high, 2480±149; mid, 1860±141; low, 423±9; PBS, 0±0/mm2; P<0.05 for high versus mid and mid versus low). Immunostaining for smooth muscle actin and human leukocyte antigen or Ulex europaeus lectin type 1 also revealed dose-dependent vasculogenesis by endothelial cell and SMC development after CD34+ cell transplantation. Reverse transcriptase–polymerase chain reaction indicated that human-specific gene expression of cardiomyocyte (brain natriuretic peptide, cardiac troponin-I, myosin heavy chain, and Nkx 2.5), SMC (smooth muscle actin and sm22&agr;), and endothelial cell (CD31 and KDR) markers were dose-dependently augmented in MI tissue. Conclusions— Human CD34+ cell transplantation may have significant and dose-dependent potential for vasculogenesis and cardiomyogenesis with functional recovery from MI.

Expression of MicroRNA-146a in osteoarthritis cartilage.

OBJECTIVE A role of microRNA, which are approximately 22-nucleotide noncoding RNAs, has recently been recognized in human diseases. The objective of this study was to identify the expression pattern of microRNA-146a (miR-146a) in cartilage from patients with osteoarthritis (OA). METHODS The expression of miR-146a in cartilage from 15 patients with OA was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and by in situ hybridization. Induction of the expression of miR-146a by cultures of normal human articular chondrocytes following stimulation with interleukin-1beta (IL-1beta) was examined by quantitative RT-PCR. RESULTS All cartilage samples were divided into 3 groups according to a modification of the Mankin score (grade I = mild OA scored 0-5, grade II = moderate OA scored 6-10, and grade III = severe OA scored 11-14). In grade I OA cartilage samples, the expression of miR-146a and COL2A1 was significantly higher than that in the other groups (P < 0.05). In grades II and III OA cartilage, the expression of miR-146a and COL2A1 was decreased, whereas the expression of matrix metalloproteinase 13 (MMP-13) was elevated in grade II OA cartilage. These data showed that miR-146a is expressed intensely in cartilage with a low Mankin grade and that miR-146a expression decreases in parallel with the level of MMP-13 expression. Tissue section in situ hybridization of primary miR-146a (pri-miR-146a) revealed that pri-miR-146a was expressed in chondrocytes residing in all tissue layers, especially in the superficial layer, where it was intensely expressed. The expression of miR-146 was markedly elevated by IL-1beta stimulation in human chondrocytes in vitro. CONCLUSION This study shows that miR-146 is intensely expressed in low-grade OA cartilage and that its expression is induced by stimulation of IL-1beta. Thus, miR-146 might play a role in OA cartilage pathogenesis.

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.

Acceleration of muscle regeneration by local injection of muscle‐specific microRNAs in rat skeletal muscle injury model

MicroRNA (miRNA)s are a class of non‐coding RNAs that regulate gene expression post‐transcriptionally. Muscle‐specific miRNA, miRNA (miR)‐1, miR‐133 and miR‐206 play a crucial role in the regulation of muscle development and homeostasis. Muscle injuries are a common muscloskeletal disorder, and the most effective treatment has not been established yet. The purpose of this study was to demonstrate that a local injection of double‐stranded (ds) miR‐1, miR‐133 and 206 can accelerate muscle regeneration in a rat skeletal muscle injury model. After the laceration of the rat tibialis anterior muscle, ds miR‐1, 133 and 206 mixture mediated atelocollagen was injected into the injured site. The control group was injected with control siRNA. At 1 week after injury, an injection of miRNAs could enhance muscle regeneration morphologically and physiologically, and prevent fibrosis effectively compared to the control siRNA. Administration of exogenous miR‐1, 133 and 206 can induce expression of myogenic markers, MyoD1, myogenin and Pax7 in mRNA and expression in the protein level at 3 and 7 days after injury. The combination of miR‐1, 133 and 206 can promote myotube differentiation, and the expression of MyoD1, myogenin and Pax7 were up‐regulated in C2C12 cells in vitro. Local injection of miR‐1, 133 and 206 could be a novel therapeutic strategy in the treatment of skeletal muscle injury.

A Minimum 2-Year Follow-up After Selective Anteromedial or Posterolateral Bundle Anterior Cruciate Ligament Reconstruction

PURPOSE The purpose of this study was to investigate the 2-year follow-up clinical results of 45 patients who had undergone our anterior cruciate ligament (ACL) augmentation procedure using an autogenous semitendinosus tendon and the EndoButton CL (Smith & Nephew, Andover, MA). METHODS We followed up 45 patients for more than 2 years after their ACL augmentations. Of these, 37 were anteromedial (AM) bundle reconstructions and 8 were posterolateral (PL) bundle reconstructions. They were assessed using a KT-2000 knee arthrometer (MEDmetric, San Diego, CA) at 30 lb by joint position sense, Lysholm knee score, and magnetic resonance imaging (MRI). RESULTS The mean side-to-side difference of anterior displacement measured by the KT-2000 knee arthrometer at 30 degrees of knee flexion preoperatively was 3.3 +/- 2.4 mm, significantly improving to a mean of 0.5 +/- 2.7 mm 2 years after surgery. The preoperative joint position sense inaccuracy was 1.6 degrees +/- 1.8 degrees , which improved significantly to 0.3 degrees +/- 2.0 degrees after surgery. The median Lysholm knee score significantly improved from 74 (range, 44 to 95) to 100 points (range, 81 to 100) after surgery. In 20 of 29 patients who had the postoperative MRI examination, the augmented ACL resembled 1 bundle on the sagittal planes of the postoperative MRI. CONCLUSIONS Selective AM or PL bundle reconstruction showed improved joint stability, joint position sense, and Lysholm scores postoperatively. This procedure can be a treatment option for patients whose ACL remnants are left in certain conditions.

Induction of apoptosis in the synovium of mice with autoantibody-mediated arthritis by the intraarticular injection of double-stranded microRNA-15a

OBJECTIVE MicroRNA is a family of noncoding RNAs that exhibit tissue-specific or developmental stage-specific expression patterns and are associated with human diseases. MicroRNA-15a (miR-15a) is reported to induce cell apoptosis by negatively regulating the expression of Bcl-2, which suppresses the apoptotic processes. The purpose of this study was to investigate whether double-stranded miR-15a administered by intraarticular injection could be taken up by cells and could induce Bcl-2 dysfunction and cell apoptosis in the synovium of arthritic mice in vivo. METHODS Autoantibody-mediated arthritis was induced in male DBA/1J mice. In the experimental group, double-stranded miR-15a labeled with FAM-atelocollagen complex was injected into the knee joint. In the control group, control small interfering RNA-atelocollagen complex was injected into the knee joint. Synovial expression of miR-15a was analyzed by quantitative polymerase chain reaction, FAM by fluorescence microscopy, Bcl-2 by Western blotting, and Bcl-2 and caspase 3 by immunohistochemistry. RESULTS The expression of miR-15a in the synovium of the experimental group was significantly higher than that in the control group. Green fluorescence emission of FAM was observed in the synovium of the experimental group. Bcl-2 protein was down-regulated and the expression of caspase 3 was increased as compared with that in the control group. CONCLUSION These results indicate that the induction of cell apoptosis after intraarticular injection of double-stranded miR-15a occurs through inhibition of the translation of Bcl-2 protein in arthritic synovium.

A Novel Cell Delivery System Using Magnetically Labeled Mesenchymal Stem Cells and an External Magnetic Device for Clinical Cartilage Repair

PURPOSE The purpose of this study was to investigate whether it is possible to successfully accumulate magnetically labeled mesenchymal stem cells (MSCs), under the direction of an external magnetic force, to the desired portion of osteochondral defects of the patellae after intra-articular injection of the MSCs. METHODS MSCs were cultured from bone marrow and were labeled magnetically. Osteochondral defects were made in the center of rabbit and swine patellae, and magnetically labeled MSCs were injected into the knee joints either under the direction of an external magnetic force or with no magnetic force applied. In the rabbit model we evaluated the patellae macroscopically and histologically, and in the swine model we observed the patellae arthroscopically. RESULTS Accumulation of magnetically labeled MSCs to the osteochondral defect was shown macroscopically and histologically in the rabbit model and was shown by arthroscopic observation to be attached to the chondral defect in the swine model. CONCLUSIONS We showed the ability to deliver magnetically labeled MSCs to a desired place in the knee joint. CLINICAL RELEVANCE Our novel approach is applicable for human cartilage defects and may open a new era of repairing cartilage defects caused by osteoarthritis or trauma by use of a less invasive technique.

Responses of microRNAs 124a and 223 following spinal cord injury in mice.

Study design:We investigated microRNA (miRNA) expression after spinal cord injury (SCI) in mice.Objectives:The recent discovery of miRNAs suggests a novel regulatory control over gene expression during plant and animal development. MiRNAs are short noncoding RNAs that suppress the translation of target genes by binding to their mRNAs, and play a central role in gene regulation in health and disease. The purpose of this study was to examine miRNA expression after SCI.Setting:Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University.Methods:We examined the expression of miRNA (miR)-223 and miR-124a in a mouse model at 6 h, 12 h, 1 day, 3 days and 7 days after SCI using quantitative PCR. The miRNA expression was confirmed by in situ hybridization.Results:Quantitative PCR revealed two peaks of miR-223 expression at 6 and 12 h and 3 days after SCI. MiR-124a expression decreased significantly from 1 day to 7 days after SCI. In situ hybridization demonstrated the presence of miR-223 around the injured site. However, miR-124a, which was present in the normal spinal cord, was not observed at the injured site.Conclusion:Our results indicate a time-dependent expression pattern of miR-223 and miR-124a in a mouse model of SCI. In this study, the time course of miRNA-223 expression may be related to inflammatory responses after SCI, and the time course of decreased miR-124a expression may reflect cell death.

Acceleration of skeletal muscle regeneration in a rat skeletal muscle injury model by local injection of human peripheral blood-derived CD133-positive cells.

Muscle injuries in sport activities can pose challenging problems in traumatology and sports medicine. The best treatment for muscle injury has not been clearly established except for the conservative treatment that is routinely performed. We investigated the potential of human adult CD133+ cells to contribute to skeletal muscle regeneration in an athymic rat model. We tested whether CD133+ cells locally transplanted to the skeletal muscle lacerated models could (a) induce vasculogenesis/angiogenesis, (b) differentiate into endothelial and myogenic lineages, and (c) finally promote histological and functional skeletal myogenesis. Granulocyte colony stimulating factor‐mobilized peripheral blood (PB) CD133+ cells, PB mononuclear cells, or phosphate‐buffered saline was locally injected after creating a muscle laceration in the tibialis anterior muscle in athymic rats. After treatment, histological and functional skeletal myogenesis was observed significantly in the CD133+ group. The injected CD133+ cells differentiated into endothelial and myogenic lineages. Using real‐time polymerase chain reaction analysis, we found that the gene expressions related to microenvironment conduction for host angiogenesis, fibrosis, and myogenesis were ideally up/downregulated. Our results show that CD133+ cells have the potential to enhance the histological and functional recovery from skeletal muscle injury rather via indirect contribution to environment conduction for muscular regeneration. It would be relatively easy to purify this cell fraction from PB, which could be a feasible and attractive autologous candidate for skeletal muscle injuries in a clinical setting. These advantages could accelerate the progression of cell‐based therapies for skeletal muscle injuries from laboratory to clinical implementation. STEM CELLS 2009;27:949–960

Up-regulation of miR-210 by vascular endothelial growth factor in ex vivo expanded CD34+ cells enhances cell-mediated angiogenesis.

Ex vivo culture has been proposed as a means to augment and repair autologous cells in patients with chronic diseases, but the mechanisms governing improvement in cell function are not well understood. Although microRNAs (miRs) are increasingly appreciated as key regulators of cellular function, a role for these factors in CD34+ cell‐mediated angiogenesis has not been elucidated. Vascular endothelial growth factor (VEGF) was previously shown to induce expression of certain miRs associated with angiogenesis in endothelial cells and promote survival and number of vascular colony forming units of haematopoietic stem cells (HSCs). We sought to evaluate the role of VEGF in expansion and angiogenic function of CD34+ cells and to identify specific miRs associated with angiogenic properties of expanded cells. Umbilical cord blood CD34+ cells were effectively expanded (18‐ to 22‐fold) in culture medium containing stem cell factor (SCF), Flt‐3 ligand (Flt‐3), thrombopoietin (TPO) and interleukin‐6 (IL‐6) with (postEX/+VEGF) and without VEGF (postEX/noVEGF). Tube formation in matrigel assay and tissue perfusion/capillary density in mice ischaemic hindlimb were significantly improved by postEX/+VEGF cells compared with fresh CD34+ and postEX/noVEGF cells. MiR‐210 expression was significantly up‐regulated in postEX/+VEGF cells. MiR‐210 inhibitor abrogated and 210 mimic recapitulated the pro‐angiogenic effects by treatment of postEX/+VEGF and postEX/noVEGF cells respectively. Collectively, these observations highlight a critical role for VEGF in enhancing the angiogenic property of expanded cells, and identify miR‐210 as a potential therapeutic target to enhance CD34+ stem cell function for the treatment of ischaemic vascular disease.