A Novel Mucidosphaerium sp. Downregulates Inflammatory Gene Expression in Skin and Articular Cells.

CONTEXT: Hot-spring therapy is occasionally used for the treatment of inflammatory diseases. Microorganisms might contribute to the anti-inflammatory functions seen in thermal mud therapies. Natural microorganisms, derived from traditional spa resorts, could be useful as a preventive strategy for alternative medical applications. OBJECTIVE: The aim of the study was to find effective microalgae from prominent hot springs to use for the treatment of inflammatory diseases. DESIGN: The research team performed an in-vitro study. Microalgae, derived from Beppu hot springs, were isolated and homogeneously cultured. SETTING: The study took place at the Saravio Central Institute at Saravio Cosmetics in Oita, Japan and the Department of Bioscience and Biotechnology in the Graduate School of Agriculture at Shinshu University in Nagano, Japan. INTERVENTION: For identification, the 18S ribosomal RNA genes of microalgae were investigated by DNA sequencing and homology search, together with microscopic observation. OUTCOME MEASURES: To examine the pharmacological activities of the algal extracts, real-time polymerase chain reactions were performed, using either primary dermal fibroblasts (DFs), dermal papilla cells (DPCs), or fibroblast-like synoviocytes (FLSs). To test the antioxidant activity, both the oxygen radical absorbance capacity and the generation of intracellular reactive oxygen species (ROS) were evaluated. RESULTS: A novel strain of green algae, Mucidosphaerium sp., was isolated from a Beppu hot spring. The algal extract downregulated gene-expression levels of pro-inflammatory cytokines, such as interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor- alpha (TNF-α), in various primary cells pre-exposed to IL-1ß. The protein level of the risk factors was concomitantly reduced. In addition, the algal extract suppressed the IL-1ß-induced upregulation of cyclooxygenase-2, nerve growth factor, and matrix metalloproteinase-1 (MMP-1) and MMP-3 in DFs. It also inhibited that of MMP-1, -3, and -9 in FLSs. Moreover, the extract inhibited total MMP protease activities. The microalgae decreased the intracellular reactive oxygen species (ROS) level in FLSs with an antioxidant activity of 178.3 ± 0.9 µmol of trolox equivalent/g. CONCLUSIONS: The present study showed that the novel Mucidosphaerium sp., derived from a Beppu hot spring, suppressed inflammatory reactions in both cutaneous and articular cells, partly due to its antioxidative properties. The novel algal strain may be a useful tool as an alternative medicine for skin and joint inflammatory disorders.

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

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.

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.

Molecular basis for Flk1 expression in hemato-cardiovascular progenitors in the mouse.

The mouse Flk1 gene is expressed in various mesodermal progenitor cells of developing embryos. Recent studies have shown that Flk1 expression marks multipotent mesodermal progenitors, giving rise to various hemato-cardiovascular cell lineages during development. Flk1 expression also marks hemato-cardiovascular cell lineages in differentiating embryonic stem (ES) cells, which may be used in transplantation decisions to treat cardiovascular diseases. Despite its developmental and clinical importance in cardiovascular tissues, the transcriptional regulatory system of Flk1 has remained unclear. Here, we report a novel enhancer of the mouse Flk1 gene directing early mesodermal expression during development as well as ES differentiation. The enhancer enriches various mesodermal progenitors, such as primitive erythropoietic progenitors, hemangioblast (BL-CFC) and cardiovascular progenitors (CV-CFC). The enhancer is activated by Bmp, Wnt and Fgf, and it contains Gata-, Cdx-, Tcf/Lef-, ER71/Etv2- and Fox-binding sites, some of which are bound specifically by each of these transcription factors. As these transcription factors are known to act under the control of the Bmp, Wnt and Fgf families, early Flk1 expression may be induced by cooperative interactions between Gata, Tcf/Lef, Cdx and ER71/Etv2 under the control of Bmp, Wnt and Fgf signaling. The enhancer is required for early Flk1 expression and for hemangioblast development during ES differentiation.

Genome-wide identification of endothelial cell-enriched genes in the mouse embryo.

The early blood vessels of the embryo and yolk sac in mammals develop by aggregation of de novo-forming angioblasts into a primitive vascular plexus, which then undergoes a complex remodeling process. Angiogenesis is also important for disease progression in the adult. However, the precise molecular mechanism of vascular development remains unclear. It is therefore of great interest to determine which genes are specifically expressed in developing endothelial cells (ECs). Here, we used Flk1-deficient mouse embryos, which lack ECs, to perform a genome-wide survey for genes related to vascular development. We identified 184 genes that are highly enriched in developing ECs. The human orthologs of most of these genes were also expressed in HUVECs, and small interfering RNA knockdown experiments on 22 human orthologs showed that 6 of these genes play a role in tube formation by HUVECs. In addition, we created Arhgef15 knockout and RhoJ knockout mice by a gene-targeting method and found that Arhgef15 and RhoJ were important for neonatal retinal vascularization. Thus, the genes identified in our survey show high expression in ECs; further analysis of these genes should facilitate our understanding of the molecular mechanisms of vascular development in the mouse.

Human IgA-binding peptides selected from random peptide libraries: affinity maturation and application in IgA purification.

Phage display system is a powerful tool to design specific ligands for target molecules. Here, we used disulfide-constrained random peptide libraries constructed with the T7 phage display system to isolate peptides specific to human IgA. The binding clones (A1-A4) isolated by biopanning exhibited clear specificity to human IgA, but the synthetic peptide derived from the A2 clone exhibited a low specificity/affinity (K(d) = 1.3 µm). Therefore, we tried to improve the peptide using a partial randomized phage display library and mutational studies on the synthetic peptides. The designed Opt-1 peptide exhibited a 39-fold higher affinity (K(d) = 33 nm) than the A2 peptide. An Opt-1 peptide-conjugated column was used to purify IgA from human plasma. However, the recovered IgA fraction was contaminated with other proteins, indicating nonspecific binding. To design a peptide with increased binding specificity, we examined the structural features of Opt-1 and the Opt-1-IgA complex using all-atom molecular dynamics simulations with explicit water. The simulation results revealed that the Opt-1 peptide displayed partial helicity in the N-terminal region and possessed a hydrophobic cluster that played a significant role in tight binding with IgA-Fc. However, these hydrophobic residues of Opt-1 may contribute to nonspecific binding with other proteins. To increase binding specificity, we introduced several mutations in the hydrophobic residues of Opt-1. The resultant Opt-3 peptide exhibited high specificity and high binding affinity for IgA, leading to successful isolation of IgA without contamination.

Study of normal and pathological blood vessel morphogenesis in Flt1-tdsRed BAC Tg mice.

Blood vessel development and network patterning are controlled by several signaling molecules, including VEGF, FGF, TGF-ß, and Ang-1,2. Among these, the role of VEGF-A signaling in vessel morphogenesis is best understood. The biological activity of VEGF-A depends on its reaction with specific receptors Flt1 and Flk1. Roles of VEGF-A signaling in endothelial cell proliferation, migration, survival, vascular permeability, and induction of tip cell filopodia have been reported. In this study, we have generated Flt1-tdsRed BAC transgenic (Tg) mice to monitor Flt1 gene expression during vascular development. We show that tdsRed fluorescence is observed within blood vessels of adult mice and embryos, indicative of retinal angiogenesis and tumor angiogenesis. Flt1 expression recapitulated by Flt1-tdsRed BAC Tg mice overlapped well with Flk1, while Flt1 was expressed more abundantly in endothelial cells of large blood vessels such as dorsal aorta and presumptive stalk cells in retina, providing a unique model to study blood vessel development.

Senescence-accelerated mice prone 8 (SAMP8) in male as a spontaneous osteoarthritis model.

BACKGROUND: Animal models of spontaneous osteoarthritis (OA) are sparse and not well characterized. The purpose of the present study is to examine OA-related changes and mechanisms in senescence-accelerated mouse prone 8 (SAMP8) that displays a phenotype of accelerated aging.  METHODS: Knees of male SAMP8 and SAM-resistant 1 (SAMR1) mice as control from 6 to 33 weeks of age were evaluated by histological grading systems for joint tissues (cartilage, meniscus, synovium, and subchondral bone), and µCT analysis. Gene expression patterns in articular cartilage were analyzed by real-time PCR. Immunohistochemistry was performed for OA-related factors, senescence markers, and apoptosis. RESULTS: Starting at 14 weeks of age, SAMP8 exhibited mild OA-like changes such as proteoglycan loss and cartilage fibrillation. From 18 to 33 weeks of age, SAMP8 progressed to partial or full-thickness defects with exposure of subchondral bone on the medial tibia and exhibited synovitis. Histological scoring indicated significantly more severe OA in SAMP8 compared with SAMR1 from 14 weeks [median (interquartile range): SAMR1: 0.89 (0.56-1.81) vs SAMP8: 1.78 (1.35-4.62)] to 33 weeks of age [SAMR1: 1.67 (1.61-1.04) vs SAMP8: 13.03 (12.26-13.57)]. Subchondral bone sclerosis in the medial tibia, bone mineral density (BMD) loss of femoral metaphysis, and meniscus degeneration occurred much earlier than the onset of cartilage degeneration in SAMP8 at 14 weeks of age. CONCLUSIONS: SAMP8 are a spontaneous OA model that is useful for investigating the pathogenesis of primary OA and evaluating therapeutic interventions.

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

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.

The role of tetraspanin CD9 in osteoarthritis using three different mouse models.

Although osteoarthritis (OA) is the most prevalent aging-related joint disease, the understanding of mechanisms of OA pathogenesis remains limited. Key features include the progressive degradation of articular cartilage, synovial hyperplasia, and angiogenesis in joint tissues. CD9, a member of the tetraspanin family, is localized in the cell membranes and partly in the endosomes of all mammalian cell types. CD9 is associated with inflammation and angiogenesis through cell adhesion, migration, and signal transduction. This study examined the role of CD9 in OA development in three different mouse models: an aging model, a surgical model and antigen-induced arthritis (AIA) model, using CD9 deficient mice. Our study showed that CD9 deficiency reduced the severity of hallmarks of OA including cartilage degradation and soft tissue inflammation in aged mice. In the AIA model, cartilage damage and inflammation were also reduced in CD9-/- mice. This was in contrast to the surgical OA model where disease severity was similar in wild-type and CD9-/- mice. Col2a1 and Aggrecan expression was increased in chondrocytes of CD9-/- mice compared with those of wild-type mice. Our results indicate that the suppression of cartilage degradation in CD9-/- could be in part related to an increase in the expression of the two main cartilage extracellular matrix proteins aggrecan and type II collagen.

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. SIGNIFICANCE: This work focuses on exosomes, which are extracellular vesicles, as a novel additional modulator of cell-to-cell communication. This study evaluated the role of exosomes isolated from mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) in the fracture-healing process of CD9-/- mice, a strain that is known to produce reduced levels of exosomes. Retardation of fracture healing in CD9-/- mice was rescued by the injection of MSC exosomes, but this was not the case after the injection of exosome-free CM. This study finds that MSC exosomes are a novel factor of MSC paracrine signaling, with an important role in the tissue repair process.

Tough Physical Double-Network Hydrogels Based on Amphiphilic Triblock Copolymers.

A series of physical double-network hydrogels is synthesized based on an amphiphilic triblock copolymer. The gel, which contains strong hydrophobic domains and sacrificial dynamic bonds of hydrogen bonds, is stiff and tough, and even stiffens in concentrated saline solution. Furthermore, due to its supramolecular structure, the gel features improved self-healing and self-recovery abilities.

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

INTRODUCTION: BTB 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. METHODS: HO-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. RESULTS: HO-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-1ß (IL-1ß) 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. CONCLUSIONS: Bach1 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.

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.

Asymmetric synthesis of α-chiral hydroxyalkylphosphines by a catalytic enantioselective reduction of acylphosphines.

Enantioselective reduction of acylphosphines, after precomplexation with borane, proceeded smoothly in the presence of a chiral oxazaborolidine catalyst and catecholborane. α-Hydroxyalkylphosphine products were obtained as phosphine-borane complexes in good yield and enantioselectivity. One of the products of the enantioselective reduction was successfully applied as an optically active phosphine ligand for asymmetric catalysis after suitable derivatization.

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

INTRODUCTION: Osteoarthritis (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. METHODS: Human 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-1ß (IL-1ß) 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. RESULTS: Exosomes from IL-1ß 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-1ß 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-1ß, 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-1ß stimulated SFB compared to non-stimulated SFB. CONCLUSIONS: Exosomes from IL-1ß 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.

Delayed cutaneous wound healing in Fam129b/Minerva-deficient mice.

A recent integrative analysis using a phosphoproteomic approach identified FAM129B, also known as MINERVA, as a downstream effector of the MAP kinase pathway in human melanoma cells. FAM129B protein, which is a member of a small family of proteins, was also found to suppress TNFα/cycloheximide-induced apoptosis in HeLa cells. To investigate the physiological functions of Fam129b in vivo, we generated gene-targeted Fam129b-mutant mice in which, the amino terminal coding exon was replaced by lacZ. We found that homozygous mutant mice are viable and fertile and that Fam129b is considerably expressed in most of the epidermal keratinocytes of both embryonic and adult mice. Although no gross defect was observed in the skin of the Fam129b-deficient mice, wound healing subsequent to skin puncturing was significantly delayed. Furthermore, overexpression of Fam129b promoted HaCaT cell motility in an N-terminal pleckstrin homology domain-dependent manner, but not proliferation. Microarray analysis of the Fam129b transfectant exhibited substantial upregulation of several genes related to wound repair and cell motility. These results suggest that expression of Fam129b in epidermal keratinocytes accompanied by alteration of wound healing-related gene expression is necessary for regulation of cell motility and thereby, contributes to the appropriate wound healing process.

Flk1-GFP BAC Tg mice: an animal model for the study of blood vessel development.

The mouse Flk1 (also called Kdr or Vegf-r2) gene encodes a receptor for VEGF-A. Flk1 is expressed in endothelial cells of the developing embryo. Recent studies have shown that Flk1 is expressed by multi-potent mesodermal progenitors, which give rise to various hematopoietic and cardiovascular cell lineages during development, and in differentiating ES cells, which may be used for cell transplantation therapy to treat cardiovascular diseases. Given its developmental and clinical importance in cardiovascular tissues, an animal model of Flk1 activity would be very useful. Here, we report the generation of Flk1-GFP BAC transgenic mice for monitoring Flk1 gene expression during development. We show that GFP expression in these mice serves as a surrogate marker for developing endothelial cells. Immunohistochemical analysis showed that the regions of expression of GFP and endogenous FLK1 largely overlap. Uniform GFP expression was observed in most endothelial cells at 8.5 dpc and thereafter. Flk1-GFP BAC transgenic mice should be useful for the study of both vascular development and pathological angiogenesis.

Runx1 is involved in primitive erythropoiesis in the mouse.

Targeted disruption of the Runx1/ AML1 gene in mice has demonstrated that it is required for the emergence of definitive hematopoietic cells but that it is not essential for the formation of primitive erythrocytes. These findings led to the conclusion that Runx1 is a stage-specific transcription factor acting only during definitive hematopoiesis. However, the zebrafish and Xenopus homologs of Runx1 have been shown to play roles in primitive hematopoiesis, suggesting that mouse Runx1 might also be involved in the development of primitive lineages. In this study, we show that primitive erythrocytes in Runx1(-/-) mice display abnormal morphology and reduced expression of Ter119, Erythroid Kruppel-like factor (EKLF, KLF1), and GATA-1. These results suggest that mouse Runx1 plays a role in the development of both primitive and definitive hematopoietic cells.

New broad-spectrum parenteral cephalosporins exhibiting potent activity against both methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Part 2: Synthesis and structure-activity relationships in the S-3578 series.

Among the prepared novel cephalosporin derivatives related to S-3578, a series of 7beta-[2-(5-amino-1,2,4-thiadiazol-3-yl)-2-(Z)-ethoxyiminoacetamido]-3-[1-(aminoalkyl)-1H-pyrazolo[4,3-b]pyridinium-4-yl]methyl-3-cephem-4-carboxylate showed potent activity against both MRSA and Pseudomonas aeruginosa, and displayed good water solubility.