Plasma exosomes from children with juvenile dermatomyositis are taken up by human aortic endothelial cells and are associated with altered gene expression in those cells

BACKGROUND: The pathology of juvenile dermatomyositis (JDM) is characterized by prominent vessel wall and perivascular inflammation. This feature of the disease has remained unexplained and under-investigated. We have hypothesized that plasma exosomes, which play an important role in inter-cellular communication, may play a role in the vascular injury associated with JDM. OBJECTIVE: To characterize the circulating exosomes of children with JDM and determine whether the small RNA cargoes within those exosomes are capable of altering transcriptional programs within endothelial cells. DESIGN/METHODS: We purified exosomes from plasma samples of children with active, untreated JDM (n = 6) and healthy controls (n = 9). We characterized the small RNA cargoes in JDM and control exosomes by RNA sequencing using the Illumina HiSeq 2500 platform. We then incubated isolated exosomes from healthy controls and children with JDM with cultured human aortic endothelial cells (HAEC) for 24 h. Fluorescence microscopy was used to confirm that both control and JDM exosomes were taken up by HAEC. RNA was then purified from HAEC that had been incubated with either control or JDM exosomes and sequenced on the Illumina platform. Differential expression of mRNAs from HAEC incubated with control or JDM exosomes was ascertained using standard computational methods. Finally, we assessed the degree to which differential gene expression in HAEC could be attributed to the different small RNA cargoes in JDM vs control exosomes using conventional and novel analytic methods. RESULTS: We identified 10 small RNA molecules that showed differential abundance when we compared JDM and healthy control exosomes. Fluorescence microscopy of labeled exosomes confirmed that both JDM and control exosomes were taken up by HAEC. Differential gene expression analysis revealed 59 genes that showed differential expression between HAEC incubated with JDM exosomes vs HAEC incubated with exosomes from controls. Statistical analysis of gene expression data demonstrated that multiple miRNAs exerted transcriptional control on multiple genes with HAEC. CONCLUSIONS: Plasma exosomes from children with active, untreated JDM are taken up by HAEC and are associated with alterations in gene expression in those cells. These findings provide new insight into potential mechanisms leading to the targeting of vascular tissue by the immune system in JDM

Water-soluble fullerene (C60) inhibits the osteoclast differentiation and bone destruction in arthritis

Kazuo Yudoh1, Rie Karasawa1, Kayo Masuko2, Tomohiro Kato21Department of Frontier Medicine, 2Department of Biochemistry, Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, JapanAbstract: Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the receptor activator NFκB (RANK) signal pathway required for osteoclast differentiation. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against the RANK-induced osteoclastogenesis and osteoclastic bone destruction in arthritis, both in vitro and in vivo. The effects of C60 on the RANK-induced osteoclastogenesis and osteoclastic bone resorption were examined in vitro. Adjuvant-induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 1.0 µM. The left ankle joint was injected intra-articularly with water-soluble C60 (20 µl) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 µl) once weekly for eight weeks. Ankle joint tissues were prepared for histologic analysis. C60 significantly inhibited the responses of osteoclast precursor cells to RANK ligand, including osteoclast differentiation and osteoclastic bone resorption in vitro. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced the number of osteoclasts and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of joint destruction with time. These findings indicate that C60 downregulates the RANK-induced osteoclast differentiation and is a potential therapeutic agent for inhibition of osteoclastic bone destruction in arthritis.Keywords: fullerene, osteoclast, bone resorption arthritis, antioxidan

Water-soluble fullerene (c60) inhibits the development of arthritis in the rat model of arthritis

Kazuo Yudoh1, Rie Karasawa1, Kayo Masuko2, Tomohiro Kato2 1Institute of Medical Science, 2Department of Biochemistry, St. Marianna University School of Medicine, Kawasaki, JapanAbstract: Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the development of inflammation and osteoclastogenesis, suggesting the implication of oxygen free radicals in the pathogenesis of arthritis. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against synovitis in arthritis, both in vitro and in vivo. In the presence or absence of C60 (0.1, 1.0, 10.0 µM), human synovial fibroblasts, synovial infiltrating lymphocytes or macrophages were incubated with tumor necrosis factor-α (TNF-α) (10.0 ng/mL), and the production of proinflammatory cytokines by the individual cells were analyzed. C60 significantly suppressed the TNF-α-induced production of proinflammatory cytokines in synovial fibroblasts, synovial infiltrating lymphocytes and macrophages in vitro. Adjuvant induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 10.0 µM. The left ankle joint was injected intraarticularly with water-soluble C60 (20 µl) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 µl), once weekly for eight weeks. Ankle joint tissues were prepared for histological analysis. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced synovitis and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of synovitis and joint destruction with time. These findings indicate that C60 is a potential therapeutic agent for inhibition of arthritis.Keywords: fullerene, inflammation, arthritis, synovitis, bone resorptio