RESUMO
BACKGROUND: Ischemia- reperfusion (I/R) injury-induced oxidative stress is a key factor in the pathogenesis of pressure ulcer formation. Ferroptosis is an iron-dependent programmed cell death that connects oxidative stress and inflammation in various diseases. Recent studies revealed the protective effect of inhibition of ferroptosis in I/R injury. However, the role of ferroptosis in cutaneous I/R injury remains elusive. OBJECTIVE: To assess the role of ferroptosis in the progression of cutaneous I/R injury. METHODS: Cutaneous I/R injury experiments and histopathological studies were performed in wild-type mice with or without exposure to volatile ferroptosis inhibitor, TEMPO (2,2,6,6-Tetramethylpiperidine-1-oxyl). The suppressive effects of TEMPO on ferroptosis inducing cell death and oxidative stress were examined in vitro. RESULTS: Inhibition of ferroptosis with TEMPO significantly reduced ulcer formation after cutaneous I/R injury. Fluctuated ferroptosis markers, such as GPX4, ACSL4, and 4-HNE expression in the I/R skin site, were reversed by TEMPO treatment. Inhibition of ferroptosis reduced apoptosis, CD3+ infiltrating lymphocytes, and improved vascularity in the I/R skin site. Inhibition of ferroptosis also suppressed the enhancement of Nrf2 activation. In vitro, ferroptosis and the activation of ferroptosis-related gene expression by RSL3 stimulation were markedly ameliorated by TEMPO treatment in mouse fibroblasts. Inhibiting ferroptosis also suppressed the elevation of the mRNA levels of NOX2 and HO-1 caused by ferroptosis. CONCLUSION: Cutaneous I/R injury-induced ferroptosis likely promotes cell death, vascular loss, infiltration of inflammatory cells, and oxidative stress. The inhibition of ferroptosis with TEMPO might have potential clinical application as novel therapeutic agent for cutaneous I/R injury.
RESUMO
Ischemia-reperfusion (I/R) injury is a key player in the pathogeneses of pressure ulcer formation. Our previous work demonstrated that inducing the transcription factor SOX2 promotes cutaneous wound healing through EGFR signaling pathway enhancement. However, its protective effect on cutaneous I/R injury was not well-characterized. We aimed to assess the role of SOX2 in cutaneous I/R injury and the tissue-protective effect of SOX2 induction in keratinocytes (KCs) in cutaneous I/R injury. SOX2 was transiently expressed in KCs after cutaneous I/R injury. Ulcer formation was significantly suppressed in KC-specific SOX2-overexpressing mice. SOX2 in skin KCs significantly suppressed the infiltrating inflammatory cells, apoptotic cells, vascular damage, and hypoxic areas in cutaneous I/R injury. Oxidative stress-induced mRNA levels of inflammatory cytokine expression were suppressed, and antioxidant stress factors and amphiregulin were elevated by SOX2 induction in skin KCs. Recombinant amphiregulin administration suppressed pressure ulcer development after cutaneous I/R injury in mice and suppressed oxidative stress-induced ROS production and apoptosis in vitro. These findings support that SOX2 in KCs might regulate cutaneous I/R injury through amphiregulin production, resulting in oxidative stress suppression. Recombinant amphiregulin can be a potential therapeutic agent for cutaneous I/R injury.
Assuntos
Úlcera por Pressão , Traumatismo por Reperfusão , Animais , Camundongos , Anfirregulina/genética , Anfirregulina/metabolismo , Apoptose , Queratinócitos/metabolismo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/metabolismo , Pele/metabolismoRESUMO
BACKGROUND: Transient receptor potential vanilloid 4 (TRPV4), a cation ion channel, is expressed in different cells, and it regulates the development of different diseases. We recently found a high TRPV4 expression in the wounded skin area. However, the role of TRPV4 in cutaneous wound healing is unknown. OBJECTIVE: To investigate the role of TRPV4 in cutaneous wound healing in a mouse model. METHODS: Skin wound healing experiment and histopathological studies were performed between WT and TRPV4 KO mice. The effect of TRPV4 antagonist and agonist on cell migration, proliferation, and differentiation were examined in vitro. RESULTS: TRPV4 expression was enhanced in wounded area in the skin. TRPV4 KO mice had impaired cutaneous wound healing compared with the WT mice. Further, they had significantly suppressed re-epithelialization and formation of granulation tissue, amount of collagen deposition, and number of α-SMA-positive myofibroblasts in skin wounds. qPCR revealed that the KO mice had decreased mRNA expression of COL1A1 and ACTA2 in skin wounds. In vitro, treatment with selective TRPV4 antagonist suppressed migrating capacity, scratch stimulation enhanced the expression of phospho-ERK in keratinocytes, and TGF-ß stimulation enhanced the mRNA expression of COL1A1 and ACTA2 in fibroblasts. Selective TRPV4 agonist suppressed cell migration in keratinocytes, and did not enhance proliferation and migration, but promoted differentiation in fibroblasts. CONCLUSION: TRPV4 mediates keratinocytes and fibroblasts migration and increases collagen deposition in the wound area, thereby promoting cutaneous wound healing.
Assuntos
Canais de Cátion TRPV , Cicatrização , Animais , Camundongos , Movimento Celular/genética , Movimento Celular/fisiologia , Colágeno/metabolismo , Modelos Animais de Doenças , Fibroblastos/metabolismo , Queratinócitos/metabolismo , RNA Mensageiro/metabolismo , Pele/patologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
BACKGROUND: Atopic dermatitis is a common skin disease caused by genetic susceptibility, environmental factors, immune response, and skin barrier dysfunction. Kaempferol is a natural flavonoid widely found in tea, vegetables, and fruits and has been reported to have excellent anti-inflammation activity. However, the therapeutic effect of kaempferol on atopic dermatitis is unclear. OBJECTIVE: This study aimed to elucidate the effect of kaempferol on skin inflammation in atopic dermatitis. METHODS: The suppressive effect of kaempferol administration on skin inflammation was examined using MC903-induced atopic dermatitis-like skin inflammation mouse model. Quantification of skin dermatitis and transepidermal water loss was performed. A histopathological study was performed to examine thymic stromal lymphopoietin expression, cornified envelope proteins such as filaggrin, loricrin, and involucrin, and the numbers of infiltrating inflammatory cells, including lymphocytes, macrophages, and mast cells in the dermatitis area. The expressions of IL-4 and IL-13 were investigated by qPCR and flow cytometry analysis using skin tissues. The expression of HO-1 was investigated by western blot and qPCR. RESULTS: Kaempferol therapy significantly suppressed MC903-induced dermatitis, TEWL, TSLP, and HO-1 expression, and infiltration of inflammatory cells. Kaempferol therapy improved the decreased expressions of filaggrin, loricrin, and involucrin in MC903-induced dermatitis skin site. The expressions of IL-4, and IL-13 were partially decreased in kaempferol-treated mice. CONCLUSION: Kaempferol might improve MC903-induced dermatitis via suppression of type 2 inflammation and improvement of barrier dysfunction by inhibition of TSLP expression and oxidative stress. Kaempferol might have the potential to be a new treatment for atopic dermatitis.
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Dermatite Atópica , Camundongos , Animais , Dermatite Atópica/induzido quimicamente , Dermatite Atópica/tratamento farmacológico , Proteínas Filagrinas , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Quempferóis/farmacologia , Quempferóis/uso terapêutico , Citocinas/metabolismo , Pele/patologia , Inflamação/metabolismo , Estresse OxidativoRESUMO
TRPV4 is a calcium ion channel that is widely expressed in various cells. It is also involved in physiological and pathological processes. However, the role of TRPV4 in psoriasis remains unknown. We aimed to investigate the role of TRPV4 in psoriasis using human psoriasis skin samples and an imiquimod-induced psoriasis-like mouse model. Keratinocytes in human psoriasis skin had high TRPV4 expression. Trpv4-knockout mice had less severe dermatitis than wild-type mice in the imiquimod-induced mouse model. Knockout mice had significantly reduced epidermal thickness and a low number of infiltrated CD3+ T cells and CD68+ macrophages on the basis of histopathological studies and decreased mRNA expression of Il17a, Il17f, and Il23, as detected through qPCR. Furthermore, knockout mice had a significantly low expression of neuropeptides and the neuron marker PGP9.5. Adenosine triphosphate release was significantly suppressed by TRPV4 knockdown in both human and mouse keratinocytes in vitro. Finally, treatment with TRPV4 antagonist was significantly effective in preventing the progression of psoriasis-like dermatitis. In conclusion, TRPV4 mediates the expression of keratinocyte-derived adenosine triphosphate and increases the secretion of neuropeptides, resulting in the activation and amplification of IL-23/Th17 responses. Hence, TRPV4 can serve as a novel therapeutic target in psoriasis.
Assuntos
Dermatite , Neuropeptídeos , Psoríase , Humanos , Animais , Camundongos , Imiquimode/farmacologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Trifosfato de Adenosina/metabolismo , Camundongos Knockout , Queratinócitos/metabolismo , Psoríase/induzido quimicamente , Psoríase/genética , Psoríase/tratamento farmacológico , Pele/metabolismo , Dermatite/patologia , Neuropeptídeos/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos BALB CRESUMO
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-ß 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.
Assuntos
Exossomos/transplante , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Escleroderma Sistêmico/terapia , Pele/patologia , Animais , Bleomicina/administração & dosagem , Bleomicina/toxicidade , Células Cultivadas , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Exossomos/metabolismo , Feminino , Fibroblastos/metabolismo , Fibrose , Humanos , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/patologia , Pele/citologia , Pele/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
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.
Assuntos
Escleroderma Sistêmico , Dermatopatias , Animais , Bleomicina , Modelos Animais de Doenças , Fibroblastos/patologia , Fibrose , Camundongos , Estresse Oxidativo , Escleroderma Sistêmico/patologia , Pele/patologia , Dermatopatias/patologiaRESUMO
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.
Assuntos
Toxinas Botulínicas Tipo A/administração & dosagem , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Neuroimunomodulação/efeitos dos fármacos , Psoríase/tratamento farmacológico , Substância P/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Imiquimode/administração & dosagem , Imiquimode/imunologia , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos BALB C , Psoríase/induzido quimicamente , Psoríase/diagnóstico , Psoríase/imunologia , Índice de Gravidade de Doença , Pele/efeitos dos fármacos , Pele/imunologia , Pele/inervação , Pele/patologia , Substância P/imunologiaRESUMO
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.
Assuntos
Fumarato de Dimetilo/farmacologia , Úlcera por Pressão/etiologia , Úlcera por Pressão/prevenção & controle , Traumatismo por Reperfusão/complicações , Administração Oral , Animais , Fumarato de Dimetilo/administração & dosagem , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Estresse OxidativoRESUMO
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.
Assuntos
Receptores de Apelina/metabolismo , Apelina/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais , Pele/irrigação sanguínea , Pele/metabolismo , Úlcera/etiologia , Úlcera/metabolismo , Animais , Apelina/genética , Receptores de Apelina/genética , Apoptose/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Expressão Gênica , Camundongos , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Úlcera/patologiaRESUMO
OBJECTIVE: Although studies have shown inconsistent results in terms of prevalence of eating disorders, the Eating Attitudes Test (EAT-26) was used to screen students for abnormal eating behaviors. The results of the self-reported EAT-26 and body frame, as well as the efficacy of using self-administered questionnaires (SAQs) were examined to detect eating disorders in new college students. RESULTS: An anonymous questionnaire (EAT-26) was provided to 7738 new students; 4552 (58.8%) responders were included in the final analysis. Semi-structured interviews were conducted for 131 (1.7%) students. Among them, 6 students showed a high EAT-26 score, but were not diagnosed with an eating disorder based on the Structured Clinical Interview for DSM-IV Axis I Disorders (SCID-I). Three students were diagnosed with an eating disorder using SCID-I, but their EAT-26 scores were below the threshold. From these results, in a non-clinical population, findings on EAT-26 do not agree with those on SCID-I in terms of the diagnosis of eating disorders, and this battery is not appropriate for detecting eating disorders.
Assuntos
Testes Anônimos/métodos , Escala de Avaliação Comportamental/estatística & dados numéricos , Comportamento Alimentar/psicologia , Transtornos da Alimentação e da Ingestão de Alimentos/diagnóstico , Adolescente , Adulto , Índice de Massa Corporal , Transtornos da Alimentação e da Ingestão de Alimentos/epidemiologia , Transtornos da Alimentação e da Ingestão de Alimentos/psicologia , Feminino , Humanos , Japão/epidemiologia , Masculino , Prevalência , Autorrelato , Estudantes , Inquéritos e QuestionáriosRESUMO
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 ß (TGFß)-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 TGFß-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 TGFß as well as TGFß 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 TGFß-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.
Assuntos
Antígenos de Superfície/metabolismo , Fibroblastos/metabolismo , Integrina alfaV/metabolismo , Proteínas do Leite/metabolismo , Escleroderma Sistêmico/metabolismo , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Antígenos de Superfície/genética , Antígenos de Superfície/farmacologia , Bleomicina/toxicidade , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibrose , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Leite/genética , Proteínas do Leite/farmacologia , Escleroderma Sistêmico/induzido quimicamente , Escleroderma Sistêmico/patologia , Pele/efeitos dos fármacos , Pele/patologiaRESUMO
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.
Assuntos
Receptores Purinérgicos P2Y2/biossíntese , Escleroderma Sistêmico/complicações , Pele/patologia , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Immunoblotting , Interleucina-6/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Transdução de Sinais , Pele/metabolismoRESUMO
OBJECTIVE: Apelin/APJ signaling has been determined to regulate cardiac and arterial fibrosis and to be involved in the pathogenesis of pulmonary arterial hypertension. Our objective was to elucidate the role of apelin in skin fibrosis in systemic sclerosis (SSc). METHODS: Expression of apelin/APJ in normal and SSc fibroblasts was compared. Effects of small interfering RNA depletion and the addition of apelin in fibroblasts were analyzed. The effect of apelin injections on bleomycin-induced dermal fibrosis in mice was investigated. We analyzed the effects of the biased agonist of APJ, MM07, on skin fibrosis in vitro and in vivo. RESULTS: The expression of apelin in SSc fibroblasts was significantly lower than that in normal fibroblasts. Serum apelin levels were negatively correlated with the modified Rodnan skin thickness score in SSc patients. Stimulation with transforming growth factor ß1 (TGFß1) inhibited apelin expression in fibroblasts, suggesting that activation of TGFß1 signaling in SSc might be responsible for reduced apelin expression in SSc fibroblasts. Small interfering RNA depletion of apelin from fibroblasts significantly enhanced fibrosis-related gene expression, and treatment with apelin protein significantly inhibited TGFß1 signaling in fibroblasts. Administration of apelin significantly inhibited bleomycin-induced dermal fibrosis in mice. We demonstrated that MM07 had greater potential than apelin to inhibit fibrosis in vivo and in vitro. CONCLUSION: Collectively, TGFß1 signaling and apelin signaling may counteract each other in the fibrotic process of SSc. Inhibitory regulation of TGFß1-induced skin fibrosis by apelin/APJ signaling may be involved in the pathogenesis of SSc and could be a therapeutic target for fibrosis in SSc patients.
Assuntos
Receptores de Apelina/metabolismo , Apelina/metabolismo , Escleroderma Sistêmico/patologia , Dermatopatias/patologia , Pele/patologia , Adulto , Animais , Bleomicina , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Fibrose , Humanos , Masculino , Camundongos , Transdução de Sinais , Pele/citologia , Dermatopatias/induzido quimicamenteRESUMO
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.
Assuntos
Toxinas Botulínicas Tipo A/uso terapêutico , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Úlcera por Pressão/tratamento farmacológico , Traumatismo por Reperfusão/complicações , Animais , Apoptose/efeitos dos fármacos , Toxinas Botulínicas Tipo A/farmacologia , Modelos Animais de Doenças , Chaperona BiP do Retículo Endoplasmático , Fibroblastos , Células Endoteliais da Veia Umbilical Humana , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Oxidantes/farmacologia , Úlcera por Pressão/etiologia , Úlcera por Pressão/patologia , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/patologia , Pele/irrigação sanguínea , Pele/efeitos dos fármacos , Pele/patologia , Resultado do TratamentoRESUMO
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.
Assuntos
Estresse do Retículo Endoplasmático , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Estresse Oxidativo , Úlcera por Pressão/prevenção & controle , Substâncias Protetoras , Traumatismo por Reperfusão/complicações , Animais , Apoptose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , Úlcera por Pressão/etiologia , Úlcera por Pressão/metabolismo , Úlcera por Pressão/patologia , Espécies Reativas de Oxigênio , Dermatopatias/complicaçõesRESUMO
BACKGROUND: Diabetic wounds are intractable due to complex factors, such as the inhibition of angiogenesis, dysfunction of phagocytosis by macrophages and abnormal inflammatory responses. It is recognized that mesenchymal stem cells (MSCs) promote wound healing in diabetic mice. We previously demonstrated that MSCs produce large amounts of MFG-E8. OBJECT: The objective was to ascertain the role of MSCs-derived MFG-E8 in murine diabetic wounds. METHODS: MFG-E8 WT/KO MSCs or rMFG-E8 were subcutaneously injected around the wound in diabetic db/db mice, and wound areas were analyzed. Quantification of angiogenesis, infiltrating inflammatory cells, apoptotic cells at the wound area was performed by immunofluorescence staining and real-time PCR. Phagocytosis assay was performed using peritoneal macrophages from WT or db/db mice. RESULTS: MFG-E8 expression in granulation tissue in diabetic mice was significantly reduced compared with that in non-diabetic mice. We next examined the effect of subcutaneous injection of MFG-E8 WT/KO MSCs around the wound. Diabetic wound healing was significantly accelerated by the injection of MSCs. Diabetic wound healing in MFG-E8 KO MSCs-injected wounds was significantly delayed compared to that in WT MSCs-injected wounds. The numbers of CD31+ EC and NG2+ pericytes, as well as M2 macrophages in wounds in KO MSCs-injected mice were significantly decreased. MFG-E8 WT MSCs treatment suppressed the number of apoptotic cells and TNF-α+ cells in wounds. In an in vitro assay, MFG-E8 WT MSCs-conditioned medium enhanced phagocytosis of apoptotic cells by peritoneal macrophages from diabetic mice. CONCLUSION: MSCs-derived MFG-E8 might accelerate diabetic wound healing by promoting angiogenesis, the clearance of apoptotic cells, and the infiltration of M2 macrophages, and by suppressing inflammatory cytokines in wound area.
Assuntos
Antígenos de Superfície/metabolismo , Complicações do Diabetes/cirurgia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Proteínas do Leite/metabolismo , Úlcera Cutânea/cirurgia , Pele/metabolismo , Cicatrização , Animais , Antígenos de Superfície/genética , Apoptose , Células Cultivadas , Citocinas/metabolismo , Complicações do Diabetes/genética , Complicações do Diabetes/metabolismo , Complicações do Diabetes/patologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Mediadores da Inflamação/metabolismo , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas do Leite/genética , Neovascularização Fisiológica , Comunicação Parácrina , Pericitos/metabolismo , Pericitos/patologia , Fagocitose , Fenótipo , Transdução de Sinais , Pele/patologia , Úlcera Cutânea/genética , Úlcera Cutânea/metabolismo , Úlcera Cutânea/patologia , Fatores de TempoRESUMO
Raynaud's phenomenon is frequently observed in systemic sclerosis (SSc) patients, and cold- or stress-induced norepinephrine (NE) has been speculated to be associated with vasoconstriction. Objective was to elucidate the role of NE in fibrosis in SSc. IL-6 is a potent stimulator of collagen production in fibroblasts. NE enhanced IL-6 production and proliferation more significantly in SSc fibroblasts than in normal fibroblasts. Furthermore, the production of IL-6 and phosphorylation of p38 in SSc fibroblasts was enhanced by adrenergic receptor (AR)ß agonist, isoproterenol, but not ARα agonist, oxymetazoline. ARß blocker, propranolol, inhibited NE-induced IL-6 production and phosphorylation of p38 in SSc fibroblasts. NE-induced IL-6 was significantly inhibited by p38 inhibitor, SB203580, suggesting that NE-induced phosphorylation of p38 via ARß enhances IL-6 production in SSc fibroblasts. NE-induced phosphorylation of ERK1/2 via ARα inhibited IL-6 production in SSc fibroblasts. Combined treatment with NE and endothelin-1 resulted in an additive increase in IL-6 production in SSc fibroblasts. NE-induced IL-6/IL-6 receptor trans-signaling increased the production of collagen type I in SSc fibroblasts, and both propranolol and SB203580 inhibited NE-induced collagen production. These results suggest that cold exposure and/or emotional stress-induced NE might contribute to the skin fibrosis via potentiation of IL-6 production from fibroblasts in SSc.
Assuntos
Fibroblastos/metabolismo , Norepinefrina/efeitos adversos , Escleroderma Sistêmico/metabolismo , Feminino , Fibroblastos/patologia , Fibrose , Humanos , Interleucina-6/biossíntese , Masculino , Norepinefrina/farmacologia , Fosforilação/efeitos dos fármacos , Escleroderma Sistêmico/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Atypical progeroid syndrome (APS), including atypical Werner syndrome (AWS), is a disorder of premature ageing caused by mutation of the lamin A gene, the same causal gene involved in Hutchinson-Gilford syndrome (HGS). We previously reported the first Japanese case of APS/AWS with a LMNA mutation (p.D300N). Recently, it has been reported that UVA induced abnormal truncated form of lamin A, called progerin, as well as HGS-like abnormal nuclear structures in normal human fibroblasts, being more frequent in the elderly, suggesting that lamin A may be involved in the regulation of photoageing. The objective of this study was to elucidate the sensitivity to cell damage induced by oxidative stress or UVA in fibroblasts from APS/AWS patient. Using immunofluorescence staining and flow cytometry analysis, the amount of early apoptotic cells and degree of intra-cellular reactive oxygen species (ROS) generation were higher in H2 02 - or UVA-treated APS/AWS fibroblasts than in normal fibroblasts, suggesting that repeated UV exposure may induce premature ageing of the skin in APS/AWS patients and that protecting against sunlight is possibly important for delaying the emergence of APS/AWS symptoms. In addition, we demonstrated that H2 O2 -, or UVA-induced apoptosis and necrosis in normal and APS/AWS fibroblasts were enhanced by farnesyltransferase inhibitor (FTI) treatment, indicating that FTI might not be useful for treating our APS/AWS patient.
Assuntos
Lamina Tipo A/genética , Mutação de Sentido Incorreto , Síndrome de Werner/genética , Síndrome de Werner/patologia , Substituição de Aminoácidos , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Farnesiltranstransferase/antagonistas & inibidores , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Humanos , Peróxido de Hidrogênio/toxicidade , Necrose , Estresse Oxidativo , Quinolonas/farmacologia , Pele/metabolismo , Pele/patologia , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Síndrome de Werner/metabolismoRESUMO
Secretion of the powerful angiogenic factor MFG-E8 by pericytes can bypass the therapeutic effects of anti-VEGF therapy, but the mechanisms by which MFG-E8 acts are not fully understood. In this study, we investigated how this factor acts to promote the growth of melanomas that express it. We found that mouse bone marrow-derived mesenchymal stromal cells (MSC) expressed a substantial amount of MFG-E8. To assess its expression from this cell type, we implanted melanoma cells and MSC derived from wild type (WT) or MFG-E8 deficient [knockout (KO)] into mice and monitored tumor growth. Tumor growth and M2 macrophages were each attenuated in subjects coimplanted with KO-MSC compared with WT-MSC. In both xenograft tumors and clinical specimens of melanoma, we found that MFG-E8 expression was heightened near blood vessels where MSC could be found. Through in vitro assays, we confirmed that WT-MSC-conditioned medium was more potent at inducing M2 macrophage polarization, compared with KO-MSC-conditioned medium. VEGF and ET-1 expression in KO-MSC was significantly lower than in WT-MSC, correlating in vivo with reduced tumor growth and numbers of pericytes and M2 macrophages within tumors. Overall, our results suggested that MFG-E8 acts at two levels, by increasing VEGF and ET-1 expression in MSC and by enhancing M2 polarization of macrophages, to increase tumor angiogenesis. Cancer Res; 76(14); 4283-92. ©2016 AACR.