RESUMO
Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells (ECs). Recent studies have identified several microRNA (miRNA) families that either promote or inhibit angiogenesis. To date, the nature and functional importance of miRNAs in Ang-1-induced angiogenesis are unknown. Microarray screening of known miRNAs in human umbilical vein endothelial cells (HUVECs) revealed that the expressions of miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p significantly decrease following exposure to Ang-1 for 24 h. Exposure to the angiogenesis factors angiopoietin-2 (Ang-2), vascular endothelial growth factor, fibroblast growth factor 2, and transforming growth factor ß also inhibits miR-103b expression, but exerts varying effects on the other miRNAs. By overexpressing miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p with selective mimics, we demonstrated that the pro-survival effects of Ang-1 are eliminated, Caspase-3 activity increases, and cell migration, proliferation, and capillary-like tube formation decreases. Conversely, transfection with selective miRNA inhibitors increases cell survival, inhibits Caspase-3 activity, and stimulates migration, proliferation and tube formation. miRNet miRNA-target gene network analyses revealed that miR-103, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p directly interact with 47, 95, 165, 108, 49, and 16 gene targets, respectively. Since many of these genes are positive regulators of angiogenic processes, we conclude that these miRNAs function as anti-angiogenic miRNAs and that their downregulation may be essential for Ang-1-induced angiogenesis to occur.
Assuntos
MicroRNAs/fisiologia , Neovascularização Fisiológica , Angiopoietina-1/farmacologia , Ciclo Celular , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , HumanosRESUMO
Activation of muscle progenitor cell myogenesis and endothelial cell angiogenesis is critical for the recovery of skeletal muscle from injury. Angiopoietin-1 (Ang-1), a ligand of Tie-2 receptors, enhances angiogenesis and skeletal muscle satellite cell survival; however, its role in skeletal muscle regeneration after injury is unknown. We assessed the effects of Ang-1 on fiber regeneration, myogenesis, and angiogenesis in injured skeletal muscle (tibialis anterior, TA) in mice. We also assessed endogenous Ang-1 levels and localization in intact and injured TA muscles. TA fiber injury was triggered by cardiotoxin injection. Endogenous Ang-1 mRNA levels immediately decreased in response to cardiotoxin then increased during the 2 wk. Ang-1 protein was expressed in satellite cells, both in noninjured and recovering TA muscles. Positive Ang-1 staining was present in blood vessels but not in nerve fibers. Four days after the initiation of injury, injection of adenoviral Ang-1 into injured muscles resulted in significant increases in in situ TA muscle contractility, muscle fiber regeneration, and capillary density. In cultured human skeletal myoblasts, recombinant Ang-1 protein increased survival, proliferation, migration, and differentiation into myotubes. The latter effect was associated with significant upregulation of the expression of the myogenic regulatory factors MyoD and Myogenin and certain genes involved in cell cycle regulation. We conclude that Ang-1 strongly enhances skeletal muscle regeneration in response to fiber injury and that this effect is mediated through induction of the myogenesis program in muscle progenitor cells and the angiogenesis program in endothelial cells.
Assuntos
Angiopoietina-1/metabolismo , Terapia Genética/métodos , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Doenças Musculares/terapia , Regeneração , Adenoviridae/genética , Adulto , Angiopoietina-1/genética , Angiopoietina-2/genética , Angiopoietina-2/metabolismo , Animais , Cardiotoxinas , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica , Vetores Genéticos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/induzido quimicamente , Doenças Musculares/genética , Doenças Musculares/patologia , Doenças Musculares/fisiopatologia , Mioblastos/metabolismo , Mioblastos/patologia , Necrose , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Acute lung injury during bacterial infection is associated with neutrophilic inflammation, epithelial cell apoptosis, and disruption of the alveolar-capillary barrier. TLR4 is required for lung injury in animals exposed to bacterial LPS and initiates proinflammatory responses in part via the transcription factor NF-κB. Ligation of TLR4 also initiates a proapoptotic response by activating IFN-ß and STAT1-dependent genes. We recently demonstrated that mammalian target of rapamycin (mTOR), a key controller of cell growth and survival, can physically interact with STAT1 and suppress the induction of STAT1-dependent apoptosis genes. We therefore hypothesized that the mTOR inhibitor rapamycin would increase LPS-induced apoptosis and lung injury in vivo. Rapamycin increased lung injury and cellular apoptosis in C57BL/6J mice exposed to intratracheal LPS for 24 h. Rapamycin also augmented STAT1 activation, and the induction of STAT1-dependent genes that mediate cellular apoptosis (i.e., Fas, caspase-3). LPS-induced lung injury was attenuated in STAT1 knockout mice. In addition, LPS and IFN-ß-induced apoptosis was absent in cultured cells lacking STAT1, and, unlike in wild-type cells, a permissive effect of rapamycin was not observed. In contrast to its effect on STAT1, rapamycin inhibited NF-κB activation in vivo and reduced selected markers of inflammation (i.e., neutrophils in the bronchoalveolar lavage fluid, TNF-α). Therefore, although it inhibits NF-κB and neutrophilic inflammation, rapamycin augments LPS-induced lung injury and apoptosis in a mechanism that involves STAT1 and the induction of STAT1-dependent apoptosis genes.
Assuntos
Lesão Pulmonar Aguda/imunologia , Apoptose/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Serina-Treonina Quinases TOR/imunologia , Receptor 4 Toll-Like/imunologia , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Antibacterianos/farmacologia , Apoptose/genética , Apoptose/imunologia , Lavagem Broncoalveolar , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , NF-kappa B/genética , NF-kappa B/imunologia , NF-kappa B/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
OBJECTIVE: Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion. METHODS AND RESULTS: Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs. CONCLUSIONS: Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation.
Assuntos
Angiopoietina-1/farmacologia , Células Endoteliais/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/fisiologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/fisiologia , Histona Desacetilases/metabolismo , Humanos , Quinase I-kappa B/genética , Leucócitos/fisiologia , NF-kappa B/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/fisiologia , Canais de Cátion TRPP/metabolismo , Células U937 , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
This study uses a relational work design perspective to explore substitutes for leadership behaviors that promote team meaningfulness and performance. We propose that team task interdependence, a structural feature facilitating interaction among team members, can be a substitute for the contributions of empowering leadership. Data were collected from 47 R&D and technology implementation teams across three organizations in a cross-sectional field study. The results revealed that high task interdependence attenuated the contributions of empowering leadership concerning team meaningfulness and, indirectly, to team performance. These findings highlight that the importance of leaders as generators of team meaningfulness is contingent on team relational work design.
RESUMO
Mental retardation (MR) occurs in 2 to 3 % of the general population and is still not therapeutically addressed. Milder forms of MR result from deficient synaptogenesis and/or impaired synaptic plasticity during childhood. These alterations would result from disequilibrium in signalling pathways regulating the balance between long term potentiation (LTP) and long term depression (LTD) in certain neurons such as hippocampus neurons. To provide mentally retarded children with increased cognitive abilities, novel experimental approaches are currently being developed to characterize signalling status associated with MR and to identify therapeutic targets that would restore lost equilibrium. Several studies also highlighted the major role played by molecular switches like kinases, phosphatases, small G proteins and their regulators in the coordination and integration of signalling pathways associated with synaptic plasticity. These proteins may therefore constitute promising therapeutic targets for a number of cognitive deficiencies.
Assuntos
Deficiência Intelectual/tratamento farmacológico , Nootrópicos/uso terapêutico , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/fisiologia , Cognição/fisiologia , Dendritos/ultraestrutura , Modelos Animais de Doenças , Desenho de Fármacos , GTP Fosfo-Hidrolases/fisiologia , Hipocampo/patologia , Humanos , Deficiência Intelectual/epidemiologia , Deficiência Intelectual/patologia , Deficiência Intelectual/fisiopatologia , Modelos Neurológicos , Família Multigênica , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Nootrópicos/farmacologia , Fosfoproteínas Fosfatases/fisiologia , Fosforilação/efeitos dos fármacos , Proteínas Quinases/fisiologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
BACKGROUND: Angiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors. OBJECTIVE: The main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response. METHODS: In silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos). RESULTS: Ang-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively. CONCLUSION: We conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.
RESUMO
Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes angiogenesis. It has been established that regulatory loops exist between angiogenic growth factors and distinct pro or anti-angiogenic miRNAs, but the nature and the roles of Ang-1-regulated miRNAs remain unclear. In this study, we assessed the role of miR-640 in Ang-1-induced angiogenesis in human umbilical vein endothelial cells (HUVECs). Exposure to Ang-1 (300 ng/mL) from 6 to 72 h significantly decreased expression of mature miR-640, a response that was mediated by Tie-2 receptors and was also observed in response to Ang-2, the vascular endothelial growth factor, and transforming growth factor ß. Increasing miR-640 levels using a mimic inhibited Ang-1-induced cell migration and capillary-like tube formation whereas inhibition of miR-640 enhanced these responses. Pull down assays of biotinylated miR-640 revealed that miR-640 directly targets Zinc Finger Protein 91 (ZFP91), an atypical E3-ubiquitin ligase. Ang-1 exposure induced ZFP91 expression through down-regulation of miR-640. Silencing of ZFP91 significantly inhibited Ang-1-induced cell migration and tube formation. We conclude that Ang-1 upregulates ZFP91 expression through transcriptional down-regulation of miR-640 and that ZFP91 plays important roles in the promotion of Ang-1-induced endothelial cell migration and differentiation.
Assuntos
MicroRNAs/metabolismo , Neovascularização Fisiológica , Ubiquitina-Proteína Ligases/genética , Angiopoietina-1/metabolismo , Diferenciação Celular , Movimento Celular , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , MicroRNAs/genética , Receptor TIE-2/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND AND AIMS: MicroRNA (miR)-146 is a key regulator of inflammation, endothelial activation and atherosclerosis. This study sought to define its potential role for the modulation of ischemia-induced neovascularization in atherosclerotic conditions. METHODS: Next generation sequencing and qRT-PCR analyses were used to compare microRNA expression in the ischemic muscles of hypercholesterolemic ApoE-deficient (ApoE-/-) mice vs. wild type mice, and in HUVECs exposed or not to oxLDL. Neovascularization was investigated in a mouse model of hindlimb ischemia and the functional activities of HUVECs and pro-angiogenic cells (PACs) were assessed in vitro. RESULTS: We found that miR-146b (but not miR-146a) is significantly reduced in the ischemic muscles of ApoE-/- mice, and in HUVECs exposed to oxLDL. Inhibition of miR-146b reduces angiogenesis in vitro, whereas forced expression of miR-146b rescues oxLDL-mediated impairment of endothelial cell proliferation and tube formation. Mechanistically, miR146b directly targets tumor necrosis factor-alpha (TNFa) Receptor Associated Factor 6 (TRAF6) to inhibit inflammation. We found that hypercholesterolemia and oxLDL exposure are associated with higher levels of TRAF6, and increased expression of TNFa. However, forced expression of miR-146b in high cholesterol conditions reduces the expression of these inflammatory factors. In vivo, intramuscular injection of miR-146b mimic reduces ischemic damages and restores blood flow recuperation and capillary density in the ischemic muscles of ApoE-/- mice. Treatment with miR-146b also increases the number and functional activities of pro-angiogenic cells (PACs). CONCLUSIONS: Hypercholesterolemia is associated with reduced expression of miR-146b, which increases TRAF6-dependent inflammation and is associated with poor neovascularization in response to ischemia. Forced expression of miR-146b using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in atherosclerotic conditions.
Assuntos
Hipercolesterolemia/metabolismo , Inflamação/metabolismo , MicroRNAs/genética , Neovascularização Patológica/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Animais , Velocidade do Fluxo Sanguíneo , Movimento Celular , Proliferação de Células , Membro Posterior/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Isquemia/fisiopatologia , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Knockout para ApoE , MicroRNAs/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Análise de Sequência de RNA , Células THP-1RESUMO
Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells. Several studies have linked reactive oxygen species (ROS) to Ang-1 signaling and distinct angiogenic responses, but the molecular sources of these ROS have never been clearly identified. In this study, we have identified source-specific contributions of ROS to Ang-1/Tie 2 signaling and angiogenic responses in human umbilical vein endothelial cells (HUVECs), specifically the differential contributions of mitochondrial ROS (mtROS) and ROS from two isoforms of NADPH oxidase (NOX2, NOX4). We demonstrate that: 1) Ang-1 induces significant increases in mtROS production under normal conditions but does not when cells are pre-incubated with mitochondrial antioxidants; 2) Ang-1 induces rapid Tie-2-dependent increases in cytosolic ROS production but does not when NOX2 and NOX4 are knocked down; 3) Ang-1 induces simultaneous increases in phosphorylation of AKT, ERK1/2, p38, and SAPK/JNK proteins within a few minutes of exposure, but this response is strongly and selectively attenuated when NOX2 and NOX4 are knocked down or cells are pre-treated with mitochondrial antioxidants; 4) Ang-1 exerts a strong effect on HUVEC survival in serum-deprived medium and enhances cell migration and capillary tube formation, but the survival response is inhibited by NOX2 knockdown and the migration and tube formation responses are entirely absent with NOX4 knockdown or pre-treatment with mitochondrial antioxidants. We conclude that Ang-1 triggers NOX2, NOX4, and the mitochondria to release ROS and that ROS derived from these sources play distinct roles in the regulation of the Ang-1/Tie 2 signaling pathway and pro-angiogenic responses.
Assuntos
Indutores da Angiogênese/farmacologia , Angiopoietina-1/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , NADPH Oxidases/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Caspase 3/metabolismo , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Glicoproteínas de Membrana/genética , Mitocôndrias/enzimologia , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/genética , Interferência de RNA , Receptor TIE-2/agonistas , Receptor TIE-2/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
PURPOSE: The purpose of this study is to present a retrospective study on clients with Acquired Brain Injury (ABI) enrolled in a tele-motion-rehabilitation service program for two or more months. METHODS: Data from 82 clients (46 males; 74 with ABI), aged 22-85 years, are reported. The Kinect-based CogniMotion System (ReAbility Online, Gertner Institute, Tel Hashomer, Israel) provided services that included 30-min biweekly sessions. Participants were evaluated prior to and 2 months following the commencement of service with clinical assessments that measured movements and function of the weaker upper extremity and cognitive abilities. RESULTS: Clients enrolled in the service had intact or mild cognitive impairment, mild-moderate motor impairment but little use of their weak upper extremity for daily activities. They were satisfied with the service and reported high levels of system usability. Post-intervention clinical assessments were performed on about half of the participants after 2 months; significant improvements in active movements of the weak upper extremity, shoulder flexion range of motion and in the Trail Making Test were found (p < 0.05). CONCLUSIONS: The service appears to be feasible for people with ABI and effective in important clinical outcomes related to improvements in upper extremity function. Implications for Rehabilitation Tele-rehabilitation provided with Microsoft Kinect 3D sensor virtual reality tracking system is feasible for people with Acquired Brain Injury. People with Acquired Brain Injury in the chronic stage were satisfied with the tele-rehabilitation service and perceived it as beneficial to improve their motor and cognitive abilities The CogniMotion System service appears to be effective in important clinical outcomes related to improvements in upper extremity function.
Assuntos
Lesões Encefálicas/reabilitação , Paresia/reabilitação , Satisfação Pessoal , Telerreabilitação/métodos , Extremidade Superior/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Grupos Focais , Serviços de Assistência Domiciliar , Humanos , Israel , Masculino , Pessoa de Meia-Idade , Amplitude de Movimento Articular , Análise de Regressão , Estudos Retrospectivos , Terapia de Exposição à Realidade Virtual , Adulto JovemRESUMO
AIMS: Bacterial lipopolysaccharides (LPS) induce innate immune inflammatory responses in endothelial cells by activating toll-like receptor 4 (TLR4) signalling. Here, we investigate the effects of angiopoietin-1 (Ang-1) on LPS-induced TLR4 signalling and the role of the miR-146 family of micro RNAs in the effects of Ang-1 on TRL4 signalling. METHODS AND RESULTS: Leucocyte adhesion to human umbilical vein endothelial cells (HUVECs) was detected using fluorescence microscopy. Adhesion molecule, pro-inflammatory cytokine, miR-146a, and miR-146b-5p expressions in HUVECs were quantified using real-time PCR. TLR4 signalling protein levels were measured using immunoblotting. Exposure of HUVECs to LPS for 4-6 h induces robust inflammatory responses, including enhanced leucocyte adhesion, up-regulation of adhesion molecule expression (VCAM1, ICAM1, E-SELECTIN), enhanced cytokine production (TNFα, IL1ß, IL6, and IL8), and increased NFκB luciferase reporter activity. Addition of Ang-1 to the culture medium for 24 h prior to LPS exposure significantly attenuates these responses. Prolonged Ang-1 exposure significantly decreases IRAK1 and TRAF6 protein levels but has no effect on TLR4, MYD88, IRAK4, or TAK1 expressions. Ang-1 triggers significant up-regulation of miR-146b-5p levels but has no effect on miR-146a or miR-146b-3p expressions. Transfection of HUVECs with a miR-146b-5p mimic significantly attenuates LPS-induced inflammatory responses and IRAK1 and TRAF6 expressions. In HUVECs transfected with a miR-146b-5p inhibitor, Ang-1 has no effect on LPS-induced inflammatory responses or IRAK1 and TRAF6 expressions. CONCLUSION: Ang-1 disrupts TLR4 signalling, resulting in inhibition of LPS-induced inflammatory responses in endothelial cells. This inhibition occurs through selective targeting of IRAK1 and TRAF6 proteins by miR-146b-5p.
Assuntos
Angiopoietina-1/farmacologia , Anti-Inflamatórios/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Inflamação/prevenção & controle , MicroRNAs/metabolismo , Receptor 4 Toll-Like/antagonistas & inibidores , Adesão Celular/efeitos dos fármacos , Técnicas de Cocultura , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Transdução de Sinais/efeitos dos fármacos , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fatores de Tempo , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo , Transfecção , Células U937RESUMO
OBJECTIVE: To evaluate the effects of physiological levels of mitochondrial-derived reactive oxygen species (ROS) on skeletal muscle autophagy, a proteolytic pathway designed to regulate contractile and myofilament homeostasis and to recycle long-lived proteins and damaged organelles. RESULTS: Basal levels of autophagy and autophagy triggered by 1.5 to 4 h of acute nutrient deprivation, rapamycin treatment, or leucine deprivation were measured in differentiated C2C12 myotubes using long-lived protein degradation assays, LC3B lipidation, autophagy-related gene expression, and electron microscopy. Preincubation with the general antioxidants tempol (superoxide dismutase mimic) and N-acetyl cysteine (NAC) or the mitochondria-specific antioxidants mito-tempol and SS31 significantly decreased the rates of long-lived protein degradation and LC3B flux and blocked the induction of autophagy-related gene expression. Mitochondrial ROS levels significantly increased in response to acute nutrient deprivation and rapamycin treatment. Mito-tempol and tempol blocked this response. Antioxidants decreased AMP-activated protein kinase (AMPK) phosphorylation by 40% and significantly increased protein kinase B (AKT) phosphorylation, but exerted no effects on mTORC1-dependent ULK1 phosphorylation on Ser(555). NAC significantly decreased basal LC3B autophagic flux in skeletal muscles of mice. INNOVATION: We report for the first time that endogenous ROS promote skeletal muscle autophagy at the basal level and in response to acute nutrient starvation and mTORC1 inhibition. We also report for the first time that mitochondrial-derived ROS promote skeletal muscle autophagy and that this effect is mediated, in part, through regulation of autophagosome initiation and AKT inhibition. CONCLUSION: Mitochondrial-derived ROS promote skeletal muscle autophagy and this effect is mediated, in part, through activation of AMPK and inhibition of AKT.
Assuntos
Autofagia/genética , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases Ativadas por AMP , Animais , Antioxidantes/farmacologia , Autofagia/efeitos dos fármacos , Linhagem Celular , Óxidos N-Cíclicos/farmacologia , Alimentos , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Transdução de Sinais , Sirolimo/farmacologia , Marcadores de SpinRESUMO
Cell shape changes are crucial for metazoan development. During Caenorhabditis elegans embryogenesis, epidermal cell shape changes transform ovoid embryos into vermiform larvae. This process is divided into two phases: early and late elongation. Early elongation involves the contraction of filamentous actin bundles by phosphorylated non-muscle myosin in a subset of epidermal (hypodermal) cells. The genes controlling early elongation are associated with two parallel pathways. The first one involves the rho-1/RHOA-specific effector let-502/Rho-kinase and mel-11/myosin phosphatase regulatory subunit. The second pathway involves the CDC42/RAC-specific effector pak-1. Late elongation is driven by mechanotransduction in ventral and dorsal hypodermal cells in response to body-wall muscle contractions, and involves the CDC42/RAC-specific Guanine-nucleotide Exchange Factor (GEF) pix-1, the GTPase ced-10/RAC and pak-1. In this study, pix-1 is shown to control early elongation in parallel with let-502/mel-11, as previously shown for pak-1. We show that pix-1, pak-1 and let-502 control the rate of elongation, and the antero-posterior morphology of the embryos. In particular, pix-1 and pak-1 are shown to control head, but not tail width, while let-502 controls both head and tail width. This suggests that let-502 function is required throughout the antero-posterior axis of the embryo during early elongation, while pix-1/pak-1 function may be mostly required in the anterior part of the embryo. Supporting this hypothesis we show that low pix-1 expression level in the dorsal-posterior hypodermal cells is required to ensure high elongation rate during early elongation.
Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/genética , Proteínas de Transporte/fisiologia , Fosfatase de Miosina-de-Cadeia-Leve/fisiologia , Quinases Associadas a rho/fisiologia , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Mecanotransdução Celular/genética , Mutação , Fenótipo , Fosforilação , Transdução de SinaisRESUMO
The role of angiogenesis factors in skeletal muscle dysfunction in patients with chronic obstructive pulmonary disease (COPD) is unknown. The first objective of this study was to assess various pro- and antiangiogenic factor and receptor expressions in the vastus lateralis muscles of control subjects and COPD patients. Preliminary inquiries revealed that angiopoietin-2 (ANGPT2) is overexpressed in limb muscles of COPD patients. ANGPT2 promotes skeletal satellite cell survival and differentiation. Factors that are involved in regulating muscle ANGPT2 production are unknown. The second objective of this study was to evaluate how oxidants and proinflammatory cytokines influence muscle-derived ANGPT2 expression. Angiogenic gene expressions in human vastus lateralis biopsies were quantified with low-density real-time PCR arrays. ANGPT2 mRNA expressions in cultured skeletal myoblasts were quantified in response to proinflammatory cytokine and H2O2 exposure. Ten proangiogenesis genes, including ANGPT2, were significantly upregulated in the vastus lateralis muscles of COPD patients. ANGPT2 mRNA levels correlated negatively with forced expiratory volume in 1 s and positively with muscle wasting. Immunoblotting confirmed that ANGPT2 protein levels were significantly greater in muscles of COPD patients compared with control subjects. ANGPT2 expression was induced by interferon-γ and -ß and by hydrogen peroxide, but not by tumor necrosis factor. We conclude that upregulation of ANGPT2 expression in vastus lateralis muscles of COPD patients is likely due to oxidative stress and represents a positive adaptive response aimed at facilitating myogenesis and angiogenesis.
Assuntos
Angiopoietina-2/fisiologia , Músculo Esquelético/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Idoso , Proteínas Angiogênicas/genética , Proteínas Angiogênicas/fisiologia , Angiopoietina-2/genética , Estudos de Casos e Controles , Citocinas/metabolismo , Diafragma/fisiopatologia , Feminino , Humanos , Masculino , Desenvolvimento Muscular/genética , Neovascularização Fisiológica/genética , Estresse Oxidativo , Doença Pulmonar Obstrutiva Crônica/genética , Músculo Quadríceps/fisiopatologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
BACKGROUND: The symptoms of numerous diseases result from genetic mutations that disrupt the homeostasis maintained by the appropriate integration of signaling gene activities. The relationships between signaling genes suggest avenues through which homeostasis can be restored and disease symptoms subsequently reduced. Specifically, disease symptoms caused by loss-of-function mutations in a particular gene may be reduced by concomitant perturbations in genes with antagonistic activities. METHODOLOGY/PRINCIPAL FINDINGS: Here we use network-neighborhood analyses to predict genetic interactions in Caenorhabditis elegans towards mapping antagonisms and synergisms between genes in an animal model. Most of the predicted interactions are novel, and the experimental validation establishes that our approach provides a gain in accuracy compared to previous efforts. In particular, we identified genetic interactors of gdi-1, the orthologue of GDI1, a gene associated with mental retardation in human. Interestingly, some gdi-1 interactors have human orthologues with known neurological functions, and upon validation of the interactions in mammalian systems, these orthologues would be potential therapeutic targets for GDI1-associated neurological disorders. We also observed the conservation of a gdi-1 interaction between different cellular systems in C. elegans, suggesting the involvement of GDI1 in human muscle degeneration. CONCLUSIONS/SIGNIFICANCE: We developed a novel predictor of genetic interactions that may have the ability to significantly streamline the identification of therapeutic targets for monogenic disorders involving genes conserved between human and C. elegans.