Objective. A role of microRNA, which are ϳ22-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-1␤ (IL-1␤) 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-1␤ 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-1␤. Thus, miR-146 might play a role in OA cartilage pathogenesis.Osteoarthritis (OA) is a highly prevalent disease that is characterized by progressive degeneration of articular cartilage (1-4). Although little is known about the pathogenesis of OA, an imbalance between anabolic and catabolic factors that maintain the homeostasis of cartilage is thought to lead to cartilage degradation. While there is a delicate balance between anabolism and catabolism in the strict regulation of matrix turnover in normal cartilage, catabolism becomes dominant over anabolism in OA cartilage, leading to the degradation of cartilage. Several studies have demonstrated an interaction between anabolic factors, such as transforming growth factor ␤, and catabolic factors, such as matrix metalloproteinases (MMPs) and aggrecanases, in chondrocytes; however, the molecular mechanisms involved in OA remain unclear (5).MicroRNA (miRNA) are a family of ϳ22-nucleotide noncoding RNAs identified in organisms ranging from nematodes to humans (6-8). Many miRNA are evolutionarily conserved across phyla, regulating gene expression by posttranscriptional gene repression. The miRNA regulate gene expression by binding the 3Ј-untranslated region of...

Failures in fracture healing are mainly caused by a lack of vascularization. Adult human circulating CD34+ cells, an endothelial/hematopoietic progenitor-enriched cell population, have been reported to differentiate into osteoblasts in vitro; however, the therapeutic potential of CD34+ cells for fracture healing is still unclear. Therefore, we performed a series of experiments to test our hypothesis that functional fracture healing is supported by vasculogenesis and osteogenesis via regenerative plasticity of CD34+ cells. Peripheral blood CD34+ cells, isolated from total mononuclear cells of adult human volunteers, showed gene expression of osteocalcin in 4 of 20 freshly isolated cells by single cell reverse transcriptase-polymerase chain reaction analysis. Phosphate-buffered saline, mononuclear cells, or CD34+ cells were intravenously transplanted after producing nonhealing femoral fractures in nude rats. Reverse transcriptase-polymerase chain reaction and immunohistochemical staining at the peri-fracture site demonstrated molecular and histological expression of human-specific markers for endothelial cells and osteoblasts at week 2. Functional bone healing assessed by biomechanical as well as radiological and histological examinations was significantly enhanced by CD34+ cell transplantation compared with the other groups. Our data suggest circulating human CD34+ cells have therapeutic potential to promote an environment conducive to neovascularization and osteogenesis in damaged skeletal tissue, allowing the complete healing of fractures.

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 1×10 3 (low), 1×10 5 (mid), or 5×10 5 (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/mm 2 ; 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 sm22α), 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.

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.

BackgroundCell‐based therapies involving mononuclear cells (MNCs) have been developed for vascular regeneration to treat ischemic diseases; however, quality control of therapeutic MNCs has not been evaluated. We investigated the therapeutic potential of peripheral blood (PB) MNCs, operated by recently developed quality and quantity (QQ) culture of endothelial progenitor cells (EPCs).Methods and ResultsPBs were collected from healthy volunteers; peripheral blood mononuclear cells (PBMNCs) isolated from these PBs were subjected to QQ culture for 7 days with medium containing stem cell factor, thrombopoietin, Flt‐3 ligand, vascular endothelial growth factor, and interleukin‐6. The resulting cells (QQMNCs) in EPC colony‐forming assay generated significantly more definitive EPC colonies than PBMNCs. In flow cytometry, macrophages and helper T lymphocytes of QQMNCs became phenotypically polarized into angiogenic, anti‐inflammatory, and regenerative subsets: classical M1 to alternative M2; T helper (Th)1 to Th2; angiogenic or regulatory T‐cell expansion. Quantitative real‐time polymerase chain reaction (qRT‐PCR) assay revealed the predominant proangiogenic gene expressions in QQMNCs versus PBMNCs. Using murine ischemic hindlimb models, the efficacy of QQMNC intramuscular transplantation (Tx) was compared to that of PBMNCTx, cultured “early EPC” Tx (eEPCTx), and granulocyte colony‐stimulating factor mobilized CD34+ cell Tx (GmCD34Tx). Laser Doppler imaging revealed the blood perfusion recovery in ischemic hindlimbs after QQMNCTx superior to after PBMNCTx and eEPCTx, but also earlier than after GmCD34Tx. Histological evaluations and qRT‐PCR assays in ischemic hindlimbs demonstrated that QQMNCTx, similarly to GmCD34Tx, enhanced angiovasculogenesis and myogenesis, whereas it preponderantly inhibited inflammation and fibrosis versus PBMNCTx and eEPCTx.ConclusionsQQ culture potentiates the ability of PBMNCs to promote regeneration of injured tissue; considering the feasible cell preparation, QQ culture‐treated PBMNCs may provide a promising therapeutic option for ischemic diseases.Clinical Trial RegistrationURL: irb.med.u-tokai.ac.jp/d/2/monthly/2010.html; IRB No.: 10R‐020.URL: irb.med.u-tokai.ac.jp/d/2/monthly/201312.html; IRB No.: 13R228.

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.

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Quantitative and qualitative impairment of endothelial progenitor cells (EPCs) limits the efficacy of autologous cell therapy in patients with cardiovascular diseases. Here, we developed a serum-free quality and quantity control culture system for colony-forming EPCs to enhance their regenerative potential. A culture with serum-free medium containing stem cell factor, thrombopoietin, vascular endothelial growth factor, interleukin-6, and Flt-3 ligand was determined as optimal quality and quantity culture ( MEDICINE 2012;1:160 -171