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Oxidative stress has been reported to play an important role in the pathogenesis of skin fibrosis in systemic sclerosis (SSc). We previously identified that botulinum toxin (BTX) injection suppresses pressure ulcer formation in a cutaneous ischemia-reperfusion injury mouse model by regulation of oxidative stress. However, the therapeutic possibility of BTX administration for preventing skin fibrosis in SSc is unclear. The objective of this study was to investigate the effect of BTX-B on skin fibrosis in a murine model of SSc and determine the underlying mechanism. We found that BTX-B injection significantly reduced dermal thickness and inflammatory cell infiltration in bleomycin-induced skin fibrosis lesion in mice. We also identified that the oxidative stress signal detected through bioluminescence in OKD48 mice after bleomycin injection in the skin was significantly decreased by BTX-B. Additionally, mRNA levels of oxidative stress associated factors (NOX2, HO-1, Trx2) were significantly decreased by BTX-B. Apoptotic cells in the lesional skin of bleomycin-treated mice were significantly reduced by BTX-B. Oxidant-induced intracellular accumulation of reactive oxygen species in SSc fibroblasts was also inhibited by BTX-B. In conclusion, BTX-B might improve bleomycin-induced skin fibrosis via the suppression of oxidative stress and inflammatory cells in the skin. BTX-B injection may have a therapeutic effect on skin fibrosis in SSc.

Homocysteine is a sulfhydryl-containing amino acid that is derived from dietary methionine, and there has been increasing evidence that elevated plasma homocysteine levels are associated with increased risk of central and peripheral vascular disorders, including carotid, coronary and peripheral arterial diseases, and Raynaud's phenomenon. Recently, associations of plasma homocysteine levels with autoimmune diseases such as systemic lupus erythematodes and systemic sclerosis have been reported. However, no study analyzed the association between plasma homocysteine levels and dermatomyositis (DM). The objective of this study was to examine plasma homocysteine levels and their clinical associations in patients with DM. Plasma homocysteine levels in 28 Japanese patients with DM and 22 healthy controls were examined. We found that the plasma homocysteine levels in DM patients were significantly higher than those in healthy individuals (15.8 ± 1.1 vs 8.5 ± 0.5 µmol/L, P < 0.01). Presence of mechanic's hand, complication of interstitial lung disease (ILD), high serum Krebs von den Lungen-6 (KL-6), surfactant protein-D and creatine kinase levels, and anti-aminoacyl-tRNA synthetase (ARS) antibody (Ab) positivity were significantly more prevalent among DM patients with elevated plasma homocysteine levels. The plasma homocysteine levels in DM patients with mechanic's hand, ILD and anti-ARS Ab were significantly higher than those in DM without those features. Furthermore, the plasma homocysteine levels were positively correlated with serum KL-6 levels. These results suggest that the pathogenesis of elevated plasma homocysteine levels may be associated with ILD in DM patients, especially with anti-ARS Ab, and further examination is required.

Background There is growing evidence that vasculopathy-induced hypoxia and oxidative stress enhance the process of fibrosis in systemic sclerosis (SSc). Kaempferol is a natural flavonoid widely found in various vegetables and fruits, and has been reported to have excellent antioxidant activity. Objective Objective was to elucidate the effect of kaempferol on skin fibrosis and the mechanism of the inhibitory regulation of fibrosis by kaempferol. Methods We assessed the effect of intraperitoneally administered kaempferol on bleomycin-induced dermal fibrosis in mice. The effect of kaempferol on oxidative stress in bleomycin-treated mice and SSc fibroblasts was assessed in vivo and in vitro. Results We identified that kaempferol injection significantly inhibited bleomycin-induced dermal fibrosis in mice. The number of αSMA+ myofibroblasts, CD3+ T-cells, and CD68+ macrophages in lesional skin was significantly decreased by kaempferol injections. Kaempferol administration also significantly suppressed the bleomycin-induced oxidative stress signal in OKD48 mice. Additionally, mRNA levels of oxidative stress-associated factors, such as HO-1 and NOX2, as well as inflammatory and pro-fibrotic cytokines, including IL-6, TGF-β and TNFα in sclerotic skin were significantly decreased by kaempferol. Kaempferol also reduced bleomycin-induced TUNEL+ apoptotic cells in the lesional skin of bleomycin-treated mice. Furthermore, the oxidant-induced intracellular accumulation of reactive oxygen species (ROS) in SSc fibroblasts was inhibited by kaempferol treatment. In addition, the oxidant-induced apoptosis of SSc fibroblasts was decreased by kaempferol in vitro. Conclusion Kaempferol might improve bleomycin-induced fibrosis by reducing oxidative stress, inflammation, and oxidative cellular damage. Administration of kaempferol might be an alternative treatment for skin fibrosis in SSc.

Tissue injury/hypoxia and oxidative stress induced-extracellular adenosine triphosphate (ATP) can act as damage-associated molecular pattern molecules, which initiate inflammatory response. Our objective was to elucidate the role of extracellular ATP in skin fibrosis in systemic sclerosis (SSc). We identified that hypoxia enhanced ATP release and that extracellular ATP enhanced IL-6 production more significantly in SSc fibroblasts than in normal fibroblasts. There were no significant differences of P2X and P2Y receptor expression levels between normal and SSc fibroblasts. Nonselective P2 receptor antagonist and selective P2Y2 receptor antagonists, kaempferol and AR-C118925XX, significantly inhibited ATP-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. ATP-induced IL-6 production was significantly inhibited by p38 inhibitors, SB203580, and doramapimod. Collagen type I production in SSc fibroblasts by ATP-induced IL-6/IL-6 receptor trans-signaling was inhibited by kaempferol and SB203580. The amount of ATP in bleomycin-treated skin was increased, and administration of AR-C118925XX significantly inhibited bleomycin-induced dermal fibrosis in mice. These results suggest that vasculopathy-induced hypoxia and oxidative stress might enhance ATP release in the dermis in SSc and that extracellular ATP-induced phosphorylation of p38 via P2Y2 receptor might enhance IL-6 and collagen type I production in SSc fibroblasts. P2Y2 receptor antagonist therapy could be a treatment for skin sclerosis in patients with SSc.

Background Systemic sclerosis (SSc) is a connective tissue disorder characterized by the development of fibrosis in the skin and internal organs. Increasing evidence suggests that mesenchymal stem cells (MSCs) can be used to a treatment for fibrotic diseases. Recent studies have demonstrated that some of the biological effects of MSCs are due to the secretion of exosomes. However, the precise mechanisms underlying MSCs-derived exosomes in skin fibrosis are not well understood. Objective We aimed to elucidate the effect of MSCs-derived exosomes on skin fibrosis in SSc and the mechanism underlying their inhibitory action on fibrosis. Methods Exosome was collected from MSCs by ultracentrifugation method. We examined the suppressive effect of MSCs-derived exosome on skin fibrosis in bleomycin-induced SSc mouse model. Skin samples from the injected site were collected for further examination, and micro-RNA analysis of MSCs-derived exosome was performed. Results Injection of MSCs-derived exosomes significantly inhibited bleomycin-induced dermal fibrosis in mice. MSCs-derived exosomes significantly reduced the amount of collagen and the number of α-SMA+ myofibroblasts and CD68+ macrophages in lesional skin. They also reduced the expression of type I collagen and TGF-β receptor 1 in fibroblasts in vitro. Moreover, micro-RNA analysis revealed that several microRNAs in MSCs-derived exosomes have antifibrotic potential. We confirmed that overexpression of miR-196b-5p in fibroblasts significantly suppressed collagen type I alpha 2 expression. Conclusion This study demonstrated that inhibition of collagen type I expression by miR-196b-5p in exosomes might be one of the mechanisms by which MSCs suppress skin fibrosis in an SSc mouse model.

Background Psoriasis is a multifactorial disease arises from a complex interaction of genetics, immune system, and environmental aspects. IL-23/Th17 immune axis has been considered as a primary modulator in psoriasis. In addition, several findings imply that nervous system may take a part in the pathogenesis of psoriasis, suggesting that nervous system, through its neuropeptide, may interact with immune system and lead to the formation of psoriasis. Objective We aimed to ascertain the role of neuropeptides secreted from neurons in the pathogenesis of psoriasis in vivo. Methods The release of neuropeptide was inhibited by injecting Botulinum toxin B (BTX-B) on Imiquimod (IMQ)-induced psoriasis-like dermatitis mice model. Quantification of skin dermatitis, infiltrating inflammatory cells, and the production of cytokines at the lesional skin area were performed by PSI score, immunostaining, and real-time PCR. We also tested the effect of selective CGRP antagonist (CGRP8−37) on psoriasis-like dermatitis in IMQ-treated mice. Results BTX-B injection significantly suppressed PSI score and reduced the number of CD4+ T cells, CD11c+ dendritic cells, and the production of IL-17A/F in the lesional skin. The expressions of PGP9.5+ nerve fibers and neuropeptides (SP, CGRP) were also significantly reduced following BTX-B injection. Additionally, CGRP antagonist also suppressed the development of IMQ-induced psoriasis-like dermatitis in mice. Conclusion The suppression of neuropeptide secretion in the skin by BTX injection might inhibit nerve elongation, the infiltration of immune cells, as well as IL-17 production, resulting in the improvement of psoriasis. Neuropeptide inhibitor could also be applied to the treatment of psoriasis.

Objective Several studies have demonstrated that the secreted glycoprotein and integrin ligand milk fat globule-associated protein with epidermal growth factor- and factor VIII-like domains (MFG-E8) negatively regulates fibrosis in the liver, lungs, and respiratory tract. However, the mechanisms and roles of MFG-E8 in skin fibrosis in systemic sclerosis (SSc) have not been characterized. We undertook this study to elucidate the role of MFG-E8 in skin fibrosis in SSc. Methods We assessed expression of MFG-E8 in the skin and serum in SSc patients. We examined the effect of recombinant MFG-E8 (rMFG-E8) on latent transforming growth factor β (TGFβ)-induced gene/protein expression in SSc fibroblasts. We examined the effects of deficiency or administration of MFG-E8 on fibrosis mouse models. Results We demonstrated that MFG-E8 expression around dermal blood vessels and the serum MFG-E8 level in SSc patients (n = 7 and n = 44, respectively) were lower than those in healthy individuals (n = 6 and n = 28, respectively). Treatment with rMFG-E8 significantly inhibited latent TGFβ-induced expression of type I collagen, α-smooth muscle actin, and CCN2 in SSc fibroblasts (n = 3-8), which suggested that MFG-E8 inhibited activation of latent TGFβ as well as TGFβ signaling via binding to αv integrin. In a mouse model of bleomycin-induced fibrosis (n = 5-8) and in a TSK mouse model (a genetic model of SSc) (n = 5-10), deficient expression of MFG-E8 significantly enhanced both pulmonary and skin fibrosis, and administration of rMFG-E8 significantly inhibited bleomycin-induced dermal fibrosis. Conclusion These results suggest that vasculopathy-induced dysfunction of pericytes and endothelial cells, the main cells secreting MFG-E8, may be associated with the decreased expression of MFG-E8 in SSc and that the deficient inhibitory regulation of latent TGFβ-induced skin fibrosis by MFG-E8 may be involved in the pathogenesis of SSc and may be a therapeutic target for fibrosis in SSc patients.

Dermatomyositis (DM) patients are known to have various infectious complications, such as cytomegalovirus (CMV) reactivation, due to immune dysfunction caused by DM itself and immunosuppressants used for treatment. Although CMV reactivation has been known to be a major cause of mortality in immunocompromised hosts, there has not been sufficient study of CMV reactivation in DM patients. The objective of this study was to examine the frequency of CMV reactivation in DM patients and to investigate risk factors potentially associated with development of CMV reactivation. We analyzed 52 Japanese DM patients, and CMV reactivation was observed in 21 (40.4%). The mean duration from the initiation of prednisolone (PSL) to the diagnosis of CMV reactivation was 6.1 ± 0.5 weeks. The total amount of oral PSL before the diagnosis of CMV reactivation was 2000.3 ± 169.3 mg. Using a univariate analysis, we found that the prevalence of interstitial lung disease and the frequency of diabetes mellitus complications in DM patients with CMV reactivation was significantly higher than that in DM patients without CMV. We identified that low lymphocytes (

Ischemia-reperfusion (I/R) is associated with various pathogenic conditions, and there has been increasing evidence that cutaneous I/R injury is associated with the pathogenesis of pressure ulcers (PUs), especially at the early stage presenting as non-blanchable erythema. Several studies demonstrated that oxidative stress is a key player in I/R injury, and the inhibition of oxidative stress may be capable of protecting tissue damage after I/R injury in various organs including skin. Dimethyl fumarate (DMF) approved by the Food and Drug Administration is Nrf2 activator, and recent studies revealed the antioxidative and anti-inflammatory effects of DMF on I/R injury in animal models. Our objective was to assess the effects of oral administration of DMF on the development of PUs after cutaneous I/R injury in mice. We found that DMF administration significantly decreased the size of PUs after cutaneous I/R. Cutaneous I/R-induced oxidative stress was also significantly inhibited by DMF in OKD48 mice, in which oxidative stress can be visually assessed. In addition, DMF treatment decreased hypoxic area, the numbers of apoptotic cells, and vascular loss in I/R area. DMF treatment suppressed the infiltration of MPO+ neutrophils and the production of proinflammatory cytokines in I/R site after cutaneous I/R injury. in vitro experiments, DMF treatment suppressed the production of reactive oxygen species in pericyte-like cells. These results suggest that DMF treatment might prevent the formation of PUs induced by cutaneous I/R injury via suppressing oxidative stress and subsequent inflammation. DMF treatment during the early phase of decubitus ulcers might protect against further progression.

Patients with systemic sclerosis (SSc) develop various vasculopathy-induced vascular disorders such as Raynaud's phenomenon, abnormal nail-fold capillaries, persistent digital ischemia, digital ulcers (DU), and sometimes develop renal crisis and pulmonary artery hypertension (PAH), affecting prognosis. Earlobe crease (ELC), also known as Frank's sign, is a wrinkle extending from the tragus to the outer border of the earlobe and is generally recognized as the sign of cardiovascular events. However, no previous study analyzed the association between ELC and SSc. In this study, we examined the prevalence and clinical characteristics of ELC in SSc patients. We analyzed 145 Japanese SSc patients and found that the prevalence of ELC in SSc patients was 23.4% (43/145), similar to that previously reported in the general population without SSc. Using univariate analysis, we found that SSc patients with ELC were characterized by old age, high incidence of DU, ILD and PAH, and high complication of coronary artery diseases (CAD) compared with SSc patients without ELC. In multivariate analysis, ELC was significantly associated with old age and incidence of DU. PAH tended to correlate with ELC without statistical significance. These results suggest that ELC may be associated with vascular disorders in SSc patients. This is the first report concerning the prevalence and clinical characteristics of ELC in patients with SSc. ELC is very easy to detect in clinical practice and helps physicians to identify SSc patients at risk of developing vascular disorders such as DU and PAH.

Background We previously identified that botulinum toxin A (BTX-A) suppressed pressure ulcer (PU) formation after cutaneous ischemia-reperfusion (I/R) injury; however, regulation of cutaneous I/R-induced oxidative and endoplasmic reticulum (ER) stress by BTX-B was not investigated. Additionally, the efficacy of BTX-B injection has never been examined. Objective Objective was to assess the effects of BTX-B on the formation of PU by cutaneous I/R injury, and the regulation of oxidative and ER stress in I/R injury by BTX-B. Methods BTX-B was subcutaneously injected into I/R area, and wound size, vascular damage, hypoxic area, and apoptotic cells in I/R area were analyzed. We evaluated the extent of oxidative and ER stress in I/R area by using OKD48 mice and ERAI mice, respectively, which enabled evaluating oxidative and ER stress through bioluminescence detection. Results BTX-B injection significantly suppressed the formation of PU by cutaneous I/R injury. Cutaneous I/R-induced vascular damage, hypoxic area, and number of oxidative-damaged cells and apoptotic cells were suppressed by BTX-B injection. BTX-B administration significantly inhibited I/R-induced oxidative stress signal in OKD48 mice. BTX-B reduced the I/R-induced oxidative stress-associated factors. BTX-B significantly inhibited the oxidant-induced reactive oxygen species and apoptosis of endothelial cells and fibroblasts. BTX-B significantly inhibited I/R-induced ER stress signal in ERAI mice. Cutaneous I/R injury-induced ER stress-response factors and GRP78/BiP and CHOP-positive cells in I/R area were significantly decreased by BTX-B injection. Conclusion BTX-B injection might have protective effects against PU formation after cutaneous I/R injury by reducing vascular damage, hypoxia-induced oxidative and ER stress, and apoptosis.

Several studies have demonstrated potential roles for apelin/APJ signaling in the regulation of oxidative stress associated with ischemia-reperfusion (I/R) injury in several organs. Objective was to assess the role of apelin/APJ signaling in the development of pressure ulcers (PUs) formation after cutaneous I/R injury in mice. We identified that cutaneous I/R injury increased the expression of apelin in the skin at I/R site. Administration of apelin significantly inhibited the formation of PUs. The reductions of blood vessels, hypoxic area and apoptosis in I/R site were inhibited by apelin injection. Oxidative stress signals in OKD48 mice and the expressions of oxidative stress related genes in the skin were suppressed by apelin injection. H2O2-induced intracellular ROS and apoptosis in endothelial cells and fibroblasts were suppressed by apelin in vitro. Furthermore, MM07, biased agonist of APJ, also significantly suppressed the development of PUs after cutaneous I/R, and the inhibitory effect of MM07 on PUs formation was higher than that in apelin. We conclude that apelin/APJ signaling may inhibit cutaneous I/R injury-induced PUs formation by protecting the reduction of vascularity and tissue damage via suppression of oxidative stress. Exogenous application of apelin or MM07 might have therapeutic potentials against the development of PUs.

Tissue injury/hypoxia and oxidative stress induced-extracellular adenosine triphosphate (ATP) can act as damage-associated molecular pattern molecules, which initiate inflammatory response. Our objective was to elucidate the role of extracellular ATP in skin fibrosis in systemic sclerosis (SSc). We identified that hypoxia enhanced ATP release and that extracellular ATP enhanced IL-6 production more significantly in SSc fibroblasts than in normal fibroblasts. There were no significant differences of P2X and P2Y receptor expression levels between normal and SSc fibroblasts. Nonselective P2 receptor antagonist and selective P2Y

Cutaneous ischemia-reperfusion (I/R) injury is associated with the early pathogenesis of cutaneous pressure ulcers (PUs). The objective of this study was to investigate the effect of mesenchymal stem cells (MSCs) injection on the formation of PUs after I/R injury and determine the underlying mechanisms. We found that the subcutaneous injection of MSCs into areas of I/R injured skin significantly suppressed the formation of PUs. I/R-induced vascular damage, hypoxia, oxidative DNA damage, and apoptosis were decreased by MSCs injection. Oxidative stress signals detected after I/R in OKD48 (Keap1-dependent oxidative stress detector, No-48-luciferase) mice were decreased by the injection of MSCs. In cultured fibroblasts, MSCs-conditioned medium significantly inhibited oxidant-induced reactive oxygen species (ROS) generation and apoptosis. Furthermore, endoplasmic reticulum (ER) stress signals detected after I/R in ERAI (ER stress-activated indicator) mice were also decreased by the injection of MSCs. These results suggest that the injection of MSCs might protect against the development of PUs after cutaneous I/R injury by reducing vascular damage, oxidative cellular damage, oxidative stress, ER stress, and apoptosis.