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AIMS: Study of the role of mitochondria-generated reactive oxygen species (mtROS) and mitochondrial polarization in mitochondrial fragmentation at the initial stages of myogenesis. MAIN METHODS: Mitochondrial morphology, Drp1 protein phosphorylation, mitochondrial electron transport chain components content, mtROS and mitochondrial lipid peroxidation levels, and mitochondrial polarization were evaluated on days 1 and 2 of human MB135 myoblasts differentiation. A mitochondria-targeted antioxidant SkQ1 was used to elucidate the effect of mtROS on mitochondria. KEY FINDINGS: In immortalized human MB135 myoblasts, mitochondrial fragmentation began on day 1 of differentiation before the myoblast fusion. This fragmentation was preceded by dephosphorylation of p-Drp1 (Ser-637). On day 2, an increase in the content of some mitochondrial proteins was observed, indicating mitochondrial biogenesis stimulation. Furthermore, we found that myogenic differentiation, even on day 1, was accompanied both by an increased production of mtROS, and lipid peroxidation of the inner mitochondrial membrane. SkQ1 blocked these effects and partially reduced the level of mitochondrial fragmentation, but did not affect the dephosphorylation of p-Drp1 (Ser-637). Importantly, mitochondrial fragmentation at early stages of MB135 differentiation was not accompanied by depolarization, as an important stimulus for mitochondrial fragmentation. SIGNIFICANCE: Mitochondrial fragmentation during early myogenic differentiation depends on mtROS production rather than mitochondrial depolarization. SkQ1 only partially inhibited mitochondrial fragmentation, without significant effects on mitophagy or early myogenic differentiation.
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Many muscular pathologies are associated with oxidative stress and elevated levels of the tumor necrosis factor (TNF) that cause muscle protein catabolism and impair myogenesis. Myogenesis defects caused by TNF are mediated in part by reactive oxygen species (ROS), including those produced by mitochondria (mitoROS), but the mechanism of their pathological action is not fully understood. We hypothesized that mitoROS act by triggering and enhancing mitophagy, an important tool for remodelling the mitochondrial reticulum during myogenesis. We used three recently developed probes-MitoTracker Orange CM-H2TMRos, mito-QC, and MitoCLox-to study myogenesis in human myoblasts. Induction of myogenesis resulted in a significant increase in mitoROS generation and phospholipid peroxidation in the inner mitochondrial membrane, as well as mitophagy enhancement. Treatment of myoblasts with TNF 24 h before induction of myogenesis resulted in a significant decrease in the myoblast fusion index and myosin heavy chain (MYH2) synthesis. TNF increased the levels of mitoROS, phospholipid peroxidation in the inner mitochondrial membrane and mitophagy at an early stage of differentiation. Trolox and SkQ1 antioxidants partially restored TNF-impaired myogenesis. The general autophagy inducers rapamycin and AICAR, which also stimulate mitophagy, completely blocked myogenesis. The autophagy suppression by the ULK1 inhibitor SBI-0206965 partially restored myogenesis impaired by TNF. Thus, suppression of myogenesis by TNF is associated with a mitoROS-dependent increase in general autophagy and mitophagy.
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Mitochondria-targeted antioxidants have become promising candidates for the therapy of various pathologies. The mitochondria-targeted antioxidant SkQ1, which is a derivative of plastoquinone, has been successfully used in preclinical studies for the treatment of cardiovascular and renal diseases, and has demonstrated anti-inflammatory activity in a number of inflammatory disease models. The present work aimed to investigate the therapeutic potential of SkQ1 and C12TPP, the analog of SkQ1 lacking the antioxidant quinone moiety, in the prevention of sodium dextran sulfate (DSS) experimental colitis and impairment of the barrier function of the intestinal epithelium in mice. DSS-treated animals exhibited weight loss, bloody stool, dysfunction of the intestinal epithelium barrier (which was observed using FITC-dextran permeability), reduced colon length, and histopathological changes in the colon mucosa. SkQ1 prevented the development of clinical and histological changes in DSS-treated mice. SkQ1 also reduced mRNA expression of pro-inflammatory molecules TNF, IL-6, IL-1ß, and ICAM-1 in the proximal colon compared with DSS-treated animals. SkQ1 prevented DSS-induced tight junction disassembly in Caco-2 cells. Pretreatment of mice by C12TPP did not protect against DSS-induced colitis. Furthermore, C12TPP did not prevent DSS-induced tight junction disassembly in Caco-2 cells. Our results suggest that SkQ1 may be a promising therapeutic agent for the treatment of inflammatory bowel diseases, in particular ulcerative colitis.
Assuntos
Antioxidantes , Colite , Humanos , Camundongos , Animais , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Células CACO-2 , Modelos Animais de Doenças , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Mucosa Intestinal/patologia , Mitocôndrias/patologiaRESUMO
Heterogeneous and homogeneous-heterogeneous assays for the quantitation of hsa-miR-141-3p (miRNA-141) were constructed. Both microplate assays were based on the use of the isothermal circular strand-displacement polymerization reaction (ICSDPR), which was carried out in heterogeneous and homogeneous media, respectively. In addition, a streptavidin-polyperoxidase conjugate and enhanced chemiluminescence were used to increase the assay's sensitivity. A comparison of the developed assays showed that the sensitivity of the heterogeneous assay was higher than that of the homogeneous-heterogeneous assay. The detection limit values of the heterogeneous and homogeneous-heterogeneous assays were 51 fM and 10 pM, respectively. The amplification index for the ICSDPR used in the heterogeneous assay of miRNA-141 was 100. Using miRNAs of the miRNA-200 family, the high specificity of the assay was demonstrated. MiRNA-141 in human cultured cells was determined by the heterogeneous ICSDPR-assisted assay with chemiluminescence detection. To assess the purification yield of miRNAs from cellular lysates, the heterogeneous assay of miRNA-39 developed on the same platform was used. The intracellular content of miRNA-141 in Caco-2, HepG2, MCF-7 and HeLa was shown to be 3400, 1400, 1300 and 470 copies per cell, respectively.
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MicroRNAs , Técnicas de Amplificação de Ácido Nucleico , Células CACO-2 , Humanos , Limite de Detecção , MicroRNAs/genética , Polimerização , EstreptavidinaRESUMO
Muscles of patients with facioscapulohumeral dystrophy (FSHD) are characterized by sporadic DUX4 expression and oxidative stress which is at least partially induced by DUX4 protein. Nevertheless, targeting oxidative stress with antioxidants has a limited impact on FSHD patients, and the exact role of oxidative stress in the pathology of FSHD, as well as its interplay with the DUX4 expression, remain unclear. Here we set up a screen for genes that are upregulated by DUX4 via oxidative stress with the aim to target these genes rather than the oxidative stress itself. Immortalized human myoblasts expressing DUX4 (MB135-DUX4) have an increased level of reactive oxygen species (ROS) and exhibit differentiation defects which can be reduced by treating the cells with classic (Tempol) or mitochondria-targeted antioxidants (SkQ1). The transcriptome analysis of antioxidant-treated MB135 and MB135-DUX4 myoblasts allowed us to identify 200 genes with expression deregulated by DUX4 but normalized upon antioxidant treatment. Several of these genes, including PITX1, have been already associated with FSHD and/or muscle differentiation. We confirmed that PITX1 was indeed deregulated in MB135-DUX4 cells and primary FSHD myoblasts and revealed a redox component in PITX1 regulation. PITX1 silencing partially reversed the differentiation defects of MB135-DUX4 myoblasts. Our approach can be used to identify and target redox-dependent genes involved in human diseases.
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Distrofia Muscular Facioescapuloumeral , Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Humanos , Mioblastos/metabolismo , Estresse OxidativoRESUMO
A sensitive and specific heterogeneous assay for quantitation of cel-miRNA-39-3p (miRNA-39) was constructed. To improve the assay sensitivity an amplification strategy based on the use of isothermal circular strand-displacement polymerization reaction (ICSDPR), polyperoxidase conjugated with streptavidin and enhanced chemiluminescence was used. The detection limit of the proposed assay was 4 × 10-13 M. The coefficient of variation (CV) for quantitation of miRNA-39 within the working range was below 8%. The study of cross-reactivity of different miRNAs including miRNA-39 demonstrated high specificity of the proposed assay. Comparison of the calibration curves of miRNA-39 dissolved in the buffer and the lysate of MCF-7 cells (prepared by lysis of the cells with phenol/guanidine thiocyanate mixture and purified using silica membrane spin column) has demonstrated a negligible matrix effect. The proposed assay makes it possible to estimate the yield of purification of miRNAs from cells, which is necessary for the quantitative calculation of the intracellular content of miRNAs measured with the isothermal assay coupled with ICSDPR.
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Medições Luminescentes/métodos , MicroRNAs/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Medições Luminescentes/normas , Células MCF-7 , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/normas , Peroxidase/metabolismo , Sensibilidade e EspecificidadeRESUMO
Leukotriene synthesis in neutrophils is critical for host survival during infection. In particular, leukotriene B4 (LTB4) is a powerful neutrophil chemoattractant that plays a crucial role in neutrophil swarming. In this work, we demonstrated that preincubation of human neutrophils with Salmonella typhimurium strongly stimulated LTB4 production induced by the bacterial chemoattractant, peptide N-formyl-L-methionyl-L-leucyl-l-phenylalanine (fMLP), while the reverse sequence of additions was ineffective. Preincubation with bacterial lipopolysaccharide or yeast polysaccharide zymosan particles gives weaker effect on fMLP-induced LTB4 production. Activation of 5-lipoxygenase (5-LOX), a key enzyme in leukotrienes biosynthesis, depends on rise of cytosolic concentration of Ca2+ and on translocation of the enzyme to the nuclear membrane. Both processes were stimulated by S. typhimurium. With an increase in the bacteria:neutrophil ratio, the transformation of LTB4 to ω-OH-LTB4 was suppressed, which further supported increased concentration of LTB4. These data indicate that in neutrophils gathered around bacterial clusters, LTB4 production is stimulated and at the same time its transformation is suppressed, which promotes neutrophil swarming and elimination of pathogens simultaneously.
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A new mitochondria-targeted probe MitoCLox was designed as a starting compound for a series of probes sensitive to cardiolipin (CL) peroxidation. Fluorescence microscopy reported selective accumulation of MitoCLox in mitochondria of diverse living cell cultures and its oxidation under stress conditions, particularly those known to cause a selective cardiolipin oxidation. Ratiometric fluorescence measurements using flow cytometry showed a remarkable dependence of the MitoCLox dynamic range on the oxidation of the sample. Specifically, MitoCLox oxidation was induced by low doses of hydrogen peroxide or organic hydroperoxide. The mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenyl-phosphonium (SkQ1), which was shown earlier to selectively protect cardiolipin from oxidation, prevented hydrogen peroxide-induced MitoCLox oxidation in the cells. Concurrent tracing of MitoCLox oxidation and membrane potential changes in response to hydrogen peroxide addition showed that the oxidation of MitoCLox started without a delay and was complete during the first hour, whereas the membrane potential started to decay after 40 minutes of incubation. Hence, MitoCLox could be used for splitting the cell response to oxidative stress into separate steps. Application of MitoCLox revealed heterogeneity of the mitochondrial population; in living endothelial cells, a fraction of small, rounded mitochondria with an increased level of lipid peroxidation were detected near the nucleus. In addition, the MitoCLox staining revealed a specific fraction of cells with an increased level of oxidized lipids also in the culture of human myoblasts. The fraction of such cells increased in high-density cultures. These specific conditions correspond to the initiation of spontaneous myogenesis in vitro, which indicates that oxidation may precede the onset of myogenic differentiation. These data point to a possible participation of oxidized CL in cell signalling and differentiation.
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Peroxidação de Lipídeos/fisiologia , Microscopia de Fluorescência/métodos , Mitocôndrias/metabolismo , Animais , Cardiolipinas/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Desenvolvimento Muscular/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Plastoquinona/análogos & derivados , Plastoquinona/farmacologiaRESUMO
Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca2+ concentration, therefore, the use of Ca2+ ionophore A23187 can be considered a semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by NADPH oxidase, however, NETosis induced by Ca2+ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS). Using the mitochondria-targeted antioxidant SkQ1 and specific inhibitors of NADPH oxidase, we showed that both sources of ROS, mitochondria and NADPH oxidase, are involved in NETosis induced by A23187 in human neutrophils. In support of the critical role of mtROS, SkQ1-sensitive NETosis was demonstrated to be induced by A23187 in neutrophils from patients with chronic granulomatous disease (CGD). We assume that Ca2+-triggered mtROS production contributes to NETosis either directly (CGD neutrophils) or by stimulating NADPH oxidase. The opening of the mitochondrial permeability transition pore (mPTP) in neutrophils treated by A23187 was revealed using the electron transmission microscopy as a swelling of the mitochondrial matrix. Using specific inhibitors, we demonstrated that the mPTP is involved in mtROS production, NETosis, and the oxidative burst induced by A23187.
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Armadilhas Extracelulares/metabolismo , Doença Granulomatosa Crônica/patologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , NADPH Oxidase 2/metabolismo , Neutrófilos/metabolismo , Explosão Respiratória/fisiologia , Adolescente , Calcimicina/farmacologia , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Células Cultivadas , Criança , Transporte de Elétrons , Sequestradores de Radicais Livres/farmacologia , Doença Granulomatosa Crônica/sangue , Voluntários Saudáveis , Humanos , Mutação com Perda de Função , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial/ultraestrutura , Poro de Transição de Permeabilidade Mitocondrial , NADPH Oxidase 2/antagonistas & inibidores , NADPH Oxidase 2/genética , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/ultraestrutura , Oxirredução/efeitos dos fármacos , Plastoquinona/análogos & derivados , Plastoquinona/farmacologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Explosão Respiratória/efeitos dos fármacosRESUMO
Oxidative stress is widely recognized as an important factor in the delayed wound healing in diabetes. However, the role of mitochondrial reactive oxygen species in this process is unknown. It was assumed that mitochondrial reactive oxygen species are involved in many wound-healing processes in both diabetic humans and animals. We have applied the mitochondria-targeted antioxidant 10-(6'-plastoquinonyl)decyltriphenylphosphonium (SkQ1) to explore the role of mitochondrial reactive oxygen species in the wound healing of genetically diabetic mice. Healing of full-thickness excisional dermal wounds in diabetic C57BL/KsJ-db-/db- mice was significantly enhanced after long-term (12 weeks) administration of SkQ1. SkQ1 accelerated wound closure and stimulated epithelization, granulation tissue formation, and vascularization. On the 7th day after wounding, SkQ1 treatment increased the number of α-smooth muscle actin-positive cells (myofibroblasts), reduced the number of neutrophils, and increased macrophage infiltration. SkQ1 lowered lipid peroxidation level but did not change the level of the circulatory IL-6 and TNF. SkQ1 pretreatment also stimulated cell migration in a scratch-wound assay in vitro under hyperglycemic condition. Thus, a mitochondria-targeted antioxidant normalized both inflammatory and regenerative phases of wound healing in diabetic mice. Our results pointed to nearly all the major steps of wound healing as the target of excessive mitochondrial reactive oxygen species production in type II diabetes.
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Derme/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Mitocôndrias/metabolismo , Plastoquinona/análogos & derivados , Cicatrização/efeitos dos fármacos , Animais , Derme/patologia , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Estresse Oxidativo/efeitos dos fármacos , Plastoquinona/farmacologiaRESUMO
Activation of neutrophils is accompanied by the oxidative burst, exocytosis of various granule types (degranulation) and a delay in spontaneous apoptosis. The major source of reactive oxygen species (ROS) in human neutrophils is NADPH oxidase (NOX2), however, other sources of ROS also exist. Although the function of ROS is mainly defensive, they can also play a regulatory role in cell signaling. However, the contribution of various sources of ROS in these processes is not clear. We investigated a possible role of mitochondria-derived ROS (mtROS) in the regulation of neutrophil activation induced by chemoattractant fMLP in vitro. Using the mitochondria-targeted antioxidant SkQ1, we demonstrated that mtROS are implicated in the oxidative burst caused by NOX2 activation as well as in the exocytosis of primary (azurophil) and secondary (specific) granules. Scavenging of mtROS with SkQ1 slightly accelerated spontaneous apoptosis and significantly stimulated apoptosis of fMLP-activated neutrophils. These data indicate that mtROS play a critical role in signal transduction that mediates the major neutrophil functional responses in the process of activation.
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Fatores Quimiotáticos/farmacologia , Grânulos Citoplasmáticos/metabolismo , Mitocôndrias/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antioxidantes/farmacologia , Apoptose , Células Cultivadas , Exocitose , Humanos , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Estresse Oxidativo , Plastoquinona/análogos & derivados , Plastoquinona/farmacologiaRESUMO
Mitochondrial dysfunctions occur in many diseases linked to the systemic inflammatory response syndrome (SIRS). Mild uncoupling of oxidative phosphorylation is known to rescue model animals from pathologies related to mitochondrial dysfunctions and overproduction of reactive oxygen species (ROS). To study the potential of SIRS therapy by uncoupling, we tested protonophore dinitrophenol (DNP) and a free fatty acid (FFA) anion carrier, lipophilic cation dodecyltriphenylphosphonium (C12TPP) in mice and in vitro models of SIRS. DNP and C12TPP prevented the body temperature drop and lethality in mice injected with high doses of a SIRS inducer, tumor necrosis factor (TNF). The mitochondria-targeted antioxidant plastoquinonyl decyltriphenylphosphonium (SkQ1) which also catalyzes FFA-dependent uncoupling revealed similar protective effects and downregulated expression of the NFκB-regulated genes (VCAM1, ICAM1, MCP1, and IL-6) involved in the inflammatory response of endothelium in aortas of the TNF-treated mice. In vitro mild uncoupling rescued from TNF-induced endothelial permeability, disassembly of cell contacts and VE-cadherin cleavage by the matrix metalloprotease 9 (ÐÐÐ 9). The uncouplers prevented TNF-induced expression of MMP9 via inhibition of NFκB signaling. Water-soluble antioxidant Trolox also prevented TNF-induced activation and permeability of endothelium in vitro via inhibition of NFκB signaling, suggesting that the protective action of the uncouplers is linked to their antioxidant potential.
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Permeabilidade Capilar/efeitos dos fármacos , Endotélio Vascular/metabolismo , Compostos Heterocíclicos/farmacologia , Compostos Organofosforados/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Desacopladores/farmacologia , Animais , Antioxidantes/farmacologia , Cromanos/farmacologia , Endotélio Vascular/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Síndrome de Resposta Inflamatória Sistêmica/patologiaRESUMO
Systemic inflammatory response syndrome (SIRS) development is accompanied by mitochondrial dysfunction and excessive ROS production. Mitochondrial dysfunctions also occur in many SIRS-related diseases and may be critical for their pathogenesis; therefore, a use of mitochondria-targeted drugs is a promising trend in SIRS research and therapy. Here, we review recent studies concerning the application of the mitochondria-targeted antioxidants and uncouplers of oxidative phosphorylation in animal models of SIRS and related diseases. We propose that a new class of uncouplers of oxidative phosphorylation, lipophilic cations could be a base for a new generation of drugs for SIRS treatment. J. Cell. Physiol. 232: 904-912, 2017. © 2016 Wiley Periodicals, Inc.
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Antioxidantes/farmacologia , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Síndrome de Resposta Inflamatória Sistêmica/metabolismo , Animais , Antioxidantes/química , Antioxidantes/uso terapêutico , Ensaios Clínicos como Assunto , Humanos , Mitocôndrias/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Síndrome de Resposta Inflamatória Sistêmica/tratamento farmacológico , Síndrome de Resposta Inflamatória Sistêmica/patologiaRESUMO
The process of skin wound healing is delayed or impaired in aging animals. To investigate the possible role of mitochondrial reactive oxygen species (mtROS) in cutaneous wound healing of aged mice, we have applied the mitochondria-targeted antioxidant SkQ1. The SkQ1 treatment resulted in accelerated resolution of the inflammatory phase, formation of granulation tissue, vascularization and epithelization of the wounds. The wounds of SkQ1-treated mice contained increased amount of myofibroblasts which produce extracellular matrix proteins and growth factors mediating granulation tissue formation. This effect resembled SkQ1-induced differentiation of fibroblasts to myofibroblast, observed earlierin vitro. The Transforming Growth Factor beta (TGFb) produced by SkQ1-treated fibroblasts was found to stimulated motility of endothelial cells in vitro, an effect which may underlie pro-angiogenic action of SkQ1 in the wounds. In vitro experiments showed that SkQ1 prevented decomposition of VE-cadherin containing contacts and following increase in permeability of endothelial cells monolayer, induced by pro-inflammatory cytokine TNF. Prevention of excessive reaction of endothelium to the pro-inflammatory cytokine(s) might account for anti-inflammatory effect of SkQ1. Our findings point to an important role of mtROS in pathogenesis of age-related chronic wounds.
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Antioxidantes/farmacologia , Mitocôndrias/efeitos dos fármacos , Plastoquinona/análogos & derivados , Cicatrização/efeitos dos fármacos , Envelhecimento , Animais , Caderinas/metabolismo , Movimento Celular/efeitos dos fármacos , Citocinas/metabolismo , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Proteínas da Matriz Extracelular/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Miofibroblastos/metabolismo , Plastoquinona/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Pele/lesões , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Vascular aging is accompanied by increases in circulatory proinflammatory cytokines leading to inflammatory endothelial response implicated in early atherogenesis. To study the possible role of mitochondria-derived reactive oxygen species (ROS) in this phenomenon, we applied the effective mitochondria-targeted antioxidant SkQ1, the conjugate of plastoquinone with dodecyltriphenylphosphonium. Eight months treatment of (CBAxC57BL/6) F1 mice with SkQ1 did not prevent age-related elevation of the major proinflammatory cytokines TNF and IL-6 in serum, but completely abrogated the increase in adhesion molecule ICAM1 expression in aortas of 24-month-old animals. In endothelial cell culture, SkQ1 also attenuated TNF-induced increase in ICAM1, VCAM, and E-selectin expression and secretion of IL-6 and IL-8, and prevented neutrophil adhesion to the endothelial monolayer. Using specific inhibitors to transcription factor NF-κB and stress-kinases p38 and JNK, we demonstrated that TNF-induced ICAM1 expression depends mainly on NF-κB activity and, to a lesser extent, on p38. SkQ1 had no effect on p38 phosphorylation (activation) but significantly reduced NF-κB activation by inhibiting phosphorylation and proteolytic cleavage of the inhibitory subunit IκBα. The data indicate an important role of mitochondrial reactive oxygen species in regulation of the NF-κB pathway and corresponding age-related inflammatory activation of endothelium.
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Envelhecimento/metabolismo , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antioxidantes/farmacologia , Aorta/efeitos dos fármacos , Aorta/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Endotélio Vascular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/sangue , Camundongos , Mitocôndrias/efeitos dos fármacos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Plastoquinona/análogos & derivados , Plastoquinona/farmacologia , Fator de Necrose Tumoral alfa/sangueRESUMO
Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.
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Infecções por Cardiovirus/virologia , Efeito Citopatogênico Viral , Regulação para Baixo , Interações Hospedeiro-Patógeno , Mengovirus/fisiologia , Replicação Viral , Infecções por Cardiovirus/imunologia , Infecções por Cardiovirus/patologia , Células HeLa , Humanos , Mengovirus/genética , Mengovirus/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
The goal of this study was to investigate the possible role of reactive oxygen species (ROS) in signaling, in modulation of the cytoskeleton, and in differentiation of fibroblasts. For this purpose, we have applied a novel mitochondria-targeted antioxidant: plastoquinone conjugated with decyltriphenylphosphonium (SkQ1). This antioxidant at nanomolar concentration prevented ROS accumulation and cell death induced by H(2)O(2) in fibroblasts. We found that scavenging of ROS produced by mitochondria activated the Rho/ROCK/LIMK signaling pathway that was followed by phosphorylation of cofilin and stabilization of actin stress fibers. The mitochondria-targeted antioxidant induced differentiation of human subcutaneous fibroblasts to myofibroblasts as revealed by expression of fibronectin isoform (EDA-FN) and smooth muscle actin (α-SMA). This effect was shown to be mediated by transforming growth factor ß1 (TGFß1), which was activated by matrix metalloprotease 9 (MMP9) in the culture medium. Scavenging of ROS stimulated secretion of MMP9 rather than its processing. The same effect was achieved by the nontargeted antioxidant Trolox at higher concentration, but the thiol antioxidant N-acetylcysteine (NAC) inhibited MMP activity and was not able to induce myofibroblast differentiation. The myofibroblast phenotype was supported due to autocrine TGFß1-dependent stimulation after removal of SkQ1. It is concluded that ROS scavenging in mitochondria induces TGFß1-dependent myofibroblast differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Actinas/metabolismo , Actinas/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Cromanos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/farmacologia , Mitocôndrias/metabolismo , Músculo Liso/metabolismo , Fosforilação , Plastoquinona/análogos & derivados , Plastoquinona/metabolismo , Plastoquinona/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismoRESUMO
The present state of the art in studies on the mechanisms of antioxidant activities of mitochondria-targeted cationic plastoquinone derivatives (SkQs) is reviewed. Our experiments showed that these compounds can operate as antioxidants in two quite different ways, i.e. (i) by preventing peroxidation of cardiolipin [Antonenko et al., Biochemistry (Moscow) 73 (2008) 1273-1287] and (ii) by fatty acid cycling resulting in mild uncoupling that inhibits the formation of reactive oxygen species (ROS) in mitochondrial State 4 [Severin et al. Proc. Natl. Acad. Sci. USA 107 (2009), 663-668]. The quinol and cationic moieties of SkQ are involved in cases (i) and (ii), respectively. In case (i) SkQH2 interrupts propagation of chain reactions involved in peroxidation of unsaturated fatty acid residues in cardiolipin, the formed SkQ- being reduced back to SkQH2 by heme bH of complex III in an antimycin-sensitive way. Molecular dynamics simulation showed that there are two stable conformations of SkQ1 with the quinol residue localized near peroxyl radicals at C9 or C13 of the linoleate residue in cardiolipin. In mechanism (ii), fatty acid cycling mediated by the cationic SkQ moiety is involved. It consists of (a) transmembrane movement of the fatty acid anion/SkQ cation pair and (b) back flows of free SkQ cation and protonated fatty acid. The cycling results in a protonophorous effect that was demonstrated in planar phospholipid membranes and liposomes. In mitochondria, the cycling gives rise to mild uncoupling, thereby decreasing membrane potential and ROS generation coupled to reverse electron transport in the respiratory chain. In yeast cells, dodecyltriphenylphosphonium (capital ES, Cyrillic12TPP), the cationic part of SkQ1, induces uncoupling that is mitochondria-targeted since capital ES, Cyrillic12TPP is specifically accumulated in mitochondria and increases the H+ conductance of their inner membrane. The conductance of the outer cell membrane is not affected by capital ES, Cyrillic12TPP.
Assuntos
Antioxidantes/farmacologia , Cardiolipinas/metabolismo , Ácidos Graxos/metabolismo , Plastoquinona/análogos & derivados , Animais , Antioxidantes/química , Cardiolipinas/química , Desenho de Fármacos , Humanos , Técnicas In Vitro , Cinética , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Biológicos , Simulação de Dinâmica Molecular , Oxirredução , Plastoquinona/química , Plastoquinona/farmacologia , RatosRESUMO
Energy catastrophe, when mitochondria hydrolyze glycolytic ATP instead of producing respiratory ATP, has been modeled. In highly glycolyzing HeLa cells, 30-50% of the population survived after inhibition of respiration and uncoupling of oxidative phosphorylation for 2-4 days. The survival was accompanied by selective elimination of mitochondria. This type of mitoptosis includes (i) fission of mitochondrial filaments, (ii) clustering of the resulting roundish mitochondria in the perinuclear area, (iii) occlusion of mitochondrial clusters by a membrane (formation of a "mitoptotic body"), (iv) decomposition of mitochondria inside this body to small membrane vesicles, (v) protrusion of the body from the cell, and (vi) disruption of the body boundary membrane. Autophagy was not involved in this mitoptotic program. Increased production of reactive oxygen species (ROS) was necessary for execution of the program, since antioxidants prevent mitoptosis and kill the cells treated with the mitochondrial poisons as if a ROS-linked mitoptosis serves for protection of the cells under conditions of severe mitochondrial stress. It is suggested that exocytosis of mitoptotic bodies may be involved in maturation of reticulocytes and lens fiber cells.
Assuntos
Mitocôndrias/fisiologia , Apoptose , Membrana Celular/fisiologia , Sobrevivência Celular , Citosol/fisiologia , Metabolismo Energético , Células HeLa , Humanos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Fosforilação Oxidativa , Espécies Reativas de Oxigênio/metabolismoRESUMO
Sulfatides - sulfated derivatives of galactocerebroside - are endogenous ligands for P- and L-selectins and are able to induce intracellular signaling in neutrophils through a L-selectin dependent pathway. Sulfatides are implicated in a variety of physiological functions and have been found to suppress the synthesis of 5-lipoxygenase (5-LO) metabolites and impede 5-LO translocation to the nuclear envelope in adherent human polymorphonuclear leukocytes (PMNs) [Sud'ina, G. F., Brock, T. G., Pushkareva, M. A., Galkina, S. I., Turutin, D. V., Peters-Golden, M., et al. (2001). Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase. The Biochemical Journal, 359, 621-629]. In this study we investigated the mechanism of the leukotriene (LT) synthesis inhibition by sulfatides. Sulfatides neither attenuated the ionophore-induced rise in [Ca(2+)](i) nor promoted PKA activation. We demonstrated that sulfatides directly inhibited 5-LO enzyme activity in a cell-free assay. BODIPY-labeled sulfatides were able to rapidly penetrate into the cells. Sulfatides induced rearrangement and redistribution of cytoskeletal components in adherent PMNs. The lipid incorporation as well as sulfatide-induced inhibition of LT synthesis were abolished by cytochalasin D, an inhibitor of actin polymerization and endocytosis. Importantly, sulfatides caused a prominent intracellular cholesterol redistribution, increasing its abundance at the uropod region. On the basis of these data, we suggest that increased cholesterol accumulation in cell compartments represents a novel mechanism by which sulfatides abrogate 5-LO translocation and activation.