Hiroyuki Ishitobi

Mesenchymal‐stem‐cell‐derived exosomes accelerate skeletal muscle regeneration

Mesenchymal stem cell (MSC) transplantation is used for treatment of many diseases. The paracrine role of MSCs in tissue regeneration is attracting particular attention. We investigate the role of MSC exosomes in skeletal muscle regeneration. MSC exosomes promote myogenesis and angiogenesis in vitro, and muscle regeneration in an in vivo model of muscle injury. Although MSC exosomes had low concentrations of muscle‐repair‐related cytokines, a number of repair‐related miRNAs were identified. This study suggests that the MSC‐derived exosomes promote muscle regeneration by enhancing myogenesis and angiogenesis, which is at least in part mediated by miRNAs such as miR‐494.

Exosome-formed synthetic microRNA-143 is transferred to osteosarcoma cells and inhibits their migration

MicroRNAs (miRNAs) have emerged as potential anticancer agents, but their clinical application is limited by the lack of an effective delivery system to tumors. Exosomes are small vesicles that play important roles in intercellular communication. Here, we show that synthetic miR-143 introduced into cells is released enveloped in exosomes and that the secreted exosome-formed miR-143 is transferred to osteosarcoma cells. The delivery of exosome-formed miR-143 significantly reduced the migration of osteosarcoma cells. The delivery efficiency of exosome-formed miR-143 was less than that achieved with lipofection, but the migratory potential of osteosarcoma cells was similarly inhibited after both strategies. Our results suggest that exosomes can deliver synthetic miR-143 and are a potentially efficient and functional delivery system.

Exosomes from IL-1β stimulated synovial fibroblasts induce osteoarthritic changes in articular chondrocytes

IntroductionOsteoarthritis (OA) is a whole joint disease, and characterized by progressive degradation of articular cartilage, synovial hyperplasia, bone remodeling and angiogenesis in various joint tissues. Exosomes are a type of microvesicles (MVs) that may play a role in tissue-tissue and cell-cell communication in homeostasis and diseases. We hypothesized that exosomes function in a novel regulatory network that contributes to OA pathogenesis and examined the function of exosomes in communication among joint tissue cells.MethodsHuman synovial fibroblasts (SFB) and articular chondrocytes were obtained from normal knee joints. Exosomes isolated from conditioned medium of SFB were analyzed for size, numbers, markers and function. Normal articular chondrocytes were treated with exosomes from SFB, and Interleukin-1β (IL-1β) stimulated SFB. OA-related genes expression was quantified using real-time PCR. To analyze exosome effects on cartilage tissue, we performed glycosaminoglycan release assay. Angiogenic activity of these exosomes was tested in migration and tube formation assays. Cytokines and miRNAs in exosomes were analyzed by Bio-Plex multiplex assay and NanoString analysis.ResultsExosomes from IL-1β stimulated SFB significantly up-regulated MMP-13 and ADAMTS-5 expression in articular chondrocytes, and down-regulated COL2A1 and ACAN compared with SFB derived exosomes. Migration and tube formation activity were significantly higher in human umbilical vein endothelial cells (HUVECs) treated with the exosomes from IL-1β stimulated SFB, which also induced significantly more proteoglycan release from cartilage explants. Inflammatory cytokines, IL-6, MMP-3 and VEGF in exosomes were only detectable at low level. IL-1β, TNFα MMP-9 and MMP-13 were not detectable in exosomes. NanoString analysis showed that levels of 50 miRNAs were differentially expressed in exosomes from IL-1β stimulated SFB compared to non-stimulated SFB.ConclusionsExosomes from IL-1β stimulated SFB induce OA-like changes both in vitro and in ex vivo models. Exosomes represent a novel mechanism by which pathogenic signals are communicated among different cell types in OA-affected joints.

Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

Paracrine signaling by bone‐marrow‐derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. Although the production of regulatory cytokines by MSC transplantation is a critical modulator of tissue regeneration, we focused on exosomes, which are extracellular vesicles that contain proteins and nucleic acids, as a novel additional modulator of cell‐to‐cell communication and tissue regeneration. To address this, we used radiologic imaging, histological examination, and immunohistochemical analysis to evaluate the role of exosomes isolated from MSC‐conditioned medium (CM) in the healing process in a femur fracture model of CD9−/− mice, a strain that is known to produce reduced levels of exosomes. We found that the bone union rate in CD9−/− mice was significantly lower than wild‐type mice because of the retardation of callus formation. The retardation of fracture healing in CD9−/− mice was rescued by the injection of exosomes, but this was not the case after the injection of exosomes‐free conditioned medium (CM‐Exo). The levels of the bone repair‐related cytokines, monocyte chemotactic protein‐1 (MCP‐1), MCP‐3, and stromal cell‐derived factor‐1 in exosomes were low compared with levels in CM and CM‐Exo, suggesting that bone repair may be in part mediated by other exosome components, such as microRNAs. These results suggest that exosomes in CM facilitate the acceleration of fracture healing, and we conclude that exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process.

Bach1 deficiency reduces severity of osteoarthritis through upregulation of heme oxygenase-1.

IntroductionBTB and CNC homology 1 (Bach1) is a transcriptional repressor of Heme oxygenase-1 (HO-1), which is cytoprotective through its antioxidant effects. The objective of this study was to define the role of Bach1 in cartilage homeostasis and osteoarthritis (OA) development using in vitro models and Bach1-/- mice.MethodsHO-1 expression in Bach1-/- mice was analyzed by real-time PCR, immunohistochemistry and immunoblotting. Knee joints from Bach1-/- and wild-type mice with age-related OA and surgically-induced OA were evaluated by OA scoring systems. Levels of autophagy proteins and superoxide dismutase 2 (SOD2) were determined by immunohistochemistry. The relationship between HO-1 and the protective effects for OA was determined in chondrocytes treated with small interfering RNA (siRNA) targeting HO-1 gene.ResultsHO-1 expression decreased with aging in articular cartilages and menisci of mouse knees. Bach1-/- mice showed reduced severity of age-related OA and surgically-induced OA compared with wild-type mice. Microtubule-associated protein 1 light chain 3 (LC3), autophagy marker, and SOD2 were increased in articular cartilage of Bach1-/- mice compared with wild-type mice. Interleukin-1β (IL-1β) induced a significant increase in Adamts-5 in wild-type chondrocytes but not in Bach1-/- chondrocytes. The expression of SOD2 and the suppression of apoptosis in Bach1-/- chondrocytes were mediated by HO-1.ConclusionsBach1 deficiency reduces the severity of OA-like changes. This may be due to maintenance of cartilage homeostasis and joint health by antioxidant effects through HO-1 and downregulation of extracellular matrix degrading enzymes. These results suggest that inactivation of Bach1 is a novel target and signaling pathway in OA prevention.

Carnosic acid protects starvation-induced SH-SY5Y cell death through Erk1/2 and Akt pathways, autophagy, and FoxO3a

Abstract Carnosic acid (CA) is recognized as a unique neuroprotective compound in the herb rosemary, since it induces expression of antioxidant enzymes including heme oxygenase-1 (HO-1), γ-glutamylcysteine synthase (γ-GCS), and glutathione S-transferase (GST) via activation of nuclear factor erythroid 2-related factor 2 (Nrf2), which is a nuclear transcription factor. In this study, we examined the cytoprotective effects of CA against starvation. We found that CA protected starvation-induced SH-SY5Y cell death by activating Akt and extracellular signal-regulated kinase 1/2 (Erk1/2). Interestingly, CA induced moderate autophagy and dephosphorylation of a transcriptional factor, the forkhead box protein O3a (FoxO3a). These effects of CA play an important role in cytoprotection.

Carnosic acid attenuates cartilage degeneration through induction of heme oxygenase-1 in human articular chondrocytes

Abstract Osteoarthritis (OA) is common age‐associated disease, and associated with joint pain, mobility limitations and compromised overall quality of life. OA treatment is currently limited to pain management and joint arthroplasty at end stage disease. Oxidative damage to cartilage extracellular matrix and cells is an important mechanism in joint aging and OA pathogenesis. Evidence from in vitro and in vivo models of OA suggests that pharmaceuticals and natural compounds with antioxidant properties reduce expression of mediators of OA pathogenesis and OA severity in animal models. Among the signaling pathways that control cellular protective mechanisms against oxygen radical damage is heme oxygenase‐1 (HO‐1). We recently report HO‐1 reduced OA severity in a mouse model. This led to the hypothesis that compounds that increase HO‐1 expression have therapeutic potential in OA. Carnosic acid (CA), a natural diterpene with oxidant activity, is prevents cartilage degeneration though induction of HO‐1. CA induced HO‐1 and miR‐140 expression in human articular chondrocytes, and cartilage degeneration was attenuated by CA treatment. Induced HO‐1 by CA was in part associated with downregulation via miR‐140 binding to 3’UTR of BTB and CNC homology 1 (BACH1). These findings suggest that CA attenuates cartilage degradation through HO‐1 upregulation and has potential as a supplement for OA prevention.