RESUMO
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been demonstrated to limit the host interferon response; however, the underlying mechanism remains unclear. Here, we found that SARS-CoV-2 infection upregulated the E3 ubiquitin ligase Huwe1, which in turn facilitated the degradation of the transcription factor Miz1. The degradation of Miz1 hampered interferon alpha and gamma responses, consequently fostering viral replication and impeding viral clearance. Conversely, silencing or inhibiting Huwe1 enhanced the interferon responses, effectively curbing viral replication. Consistently, overexpressing Miz1 augmented the interferon responses and limited viral replication, whereas silencing Miz1 had the opposite effect. Targeting Huwe1 or overexpressing Miz1 elicited transcriptomic alterations characterized by enriched functions associated with bolstered antiviral response and diminished virus replication. Further study revealed Miz1 exerted epigenetic control over the transcription of specific interferon signaling molecules, which acted as common upstream regulators responsible for the observed transcriptomic changes following Huwe1 or Miz1 targeting. These findings underscore the critical role of the Huwe1-Miz1 axis in governing the host antiviral response, with its dysregulation contributing to the impaired interferon response observed during COVID-19.
Assuntos
COVID-19 , Interferon-alfa , Interferon gama , SARS-CoV-2 , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Replicação Viral , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Humanos , SARS-CoV-2/fisiologia , Interferon gama/metabolismo , COVID-19/imunologia , COVID-19/virologia , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Interferon-alfa/metabolismo , Animais , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Inibidoras de STAT Ativados/genética , Proteólise , Células HEK293 , Chlorocebus aethiops , Fatores de Transcrição Kruppel-LikeRESUMO
For the first time, three acceptor-donor-acceptor (A-D-A)-type boranil fluorescent dyes, CSU-BF-R (R = H, CH3, and OCH3), featuring phenothiazine as the donor, were designed and synthesized. CSU-BF-R exhibited remarkable photophysical characteristics, including large Stokes shifts (>150 nm), high fluorescence quantum yields (up to 40%), long-wavelength emissions, and strong red solid-state fluorescence. Moreover, these CSU-BF-R fluorescent dyes were demonstrated to function as highly selective and sensitive ratiometric fluorescent probes for detecting hypochlorous acid (HClO). The preliminary biological applications of CSU-BF-OCH3 for sensing intracellular HClO in living cells and zebrafish were demonstrated. Therefore, CSU-BF-R possess the potential to further explore the physiological and pathological functions associated with HClO and provide valuable insights into the design of high-performance A-D-A-type fluorescent dyes.
Assuntos
Desenho de Fármacos , Corantes Fluorescentes , Ácido Hipocloroso , Peixe-Zebra , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Animais , Ácido Hipocloroso/análise , Ácido Hipocloroso/química , Humanos , Compostos de Anilina/química , Compostos de Anilina/síntese química , Estrutura Molecular , Imagem ÓpticaRESUMO
Given the issues of soil cracking, poor water retention during drought, and erosion damage caused by rainfall, we conducted an in-depth study on the water retention properties, cracking resistance, and scouring resistance of biogel-amended clay using evaporation cracking and scouring tests. The hydrophysical properties and cohesive aggregation mechanism of biogel-amended clay were explored, and the results showed that the incorporation of biogel improved the water retention, cracking resistance, and scour resistance of the clay samples. With an increase in the biogel content, the biogel mucous membrane inside the samples improved the cohesion between soil particles, reduced the generation and development of cracks, and improved the cracking resistance. There was no significant cracking of the samples after the biogel content reached 0.3%, which changed the migration of water in the sample, prevented water evaporation, and improved the water retention of the clay samples. Biofilm can change the migration of water in the sample, prevent some evaporation, and reduce the evaporation rate. To a certain extent, it can enhance the water retention capacity of the sample. Enhanced biofilm content significantly reduced scouring in the process of rainfall and runoff erosion of the sample, and biofilm content of 0.2% significantly reduced the surface of the specimen damaged by erosion. The hydrophysical properties of the composite-adhesive-amended clay samples were significantly improved compared with those of the single-bioadhesive-amended clay samples.
RESUMO
Drying-induced cracks and precipitation-induced erosion negatively impact the performance of soils in the context of extreme weather events. This study introduces two effective and sustainable materials, microbial biopolymer (MB) and palm fibers (PF), for cracking and erosion control in the sand-clay mixtures. A series of desiccation cracking tests, erosion tests, and SEM tests were conducted to evaluate the effectiveness of the treatment. The results showed that MB could significantly improve the resistance of the soil to cracking and scouring, and the improvement increased with increasing MB content. The optimum MB content was 0.15 % to achieve the maximum cracking and erosion resistance. For samples with varying sand contents, 0.15 % MB addition reduced the crack ratio, total crack length, and accumulative erosion ratio by 19.55 %-96.91 %, 4.22 %-99.58 %, and 57.88 %-89.53 %, respectively. In addition, PF positively affected the anti-crack and anti-erosion properties of the soil, and the application of 0.60 % PF had the best performance for both improvements. The cracks in the soils were mostly fine and shallow with the addition of 0.60 % PF, and therefore, the accumulative erosion ratio decreased by 44.18 %-62.76 % for samples with varying sand contents. Compared to the untreated soil, the degree of cracking and erosion was less due to the formation of a structure with more macropores and a sand skeleton in the treated samples with higher sand content. MB addition provides strong inter-particle bonding connections and a hydrophilic crust structure to improve the soils' resistance to cracking and erosion, while the fiber reinforcement effect benefits from interfacial friction and spatial restriction effects. This study provides mechanistic interpretations of desiccation cracking and erosion behavior in sand-clay mixtures under different treatments. It may guide the design of low-carbon technologies for geotechnical engineering applications.
Assuntos
Areia , Solo , Argila , Solo/química , BiopolímerosRESUMO
Aging is a major risk factor of high incidence and increased mortality of acute respiratory distress syndrome (ARDS). Here, we demonstrated that persistent lung injury and high mortality in aged mice after sepsis challenge were attributable to impaired endothelial regeneration and vascular repair. Genetic lineage tracing study showed that endothelial regeneration after sepsis-induced vascular injury was mediated by lung resident endothelial proliferation in young adult mice, whereas this intrinsic regenerative program was impaired in aged mice. Expression of forkhead box M1 (FoxM1), an important mediator of endothelial regeneration in young mice, was not induced in lungs of aged mice. Transgenic FOXM1 expression or in vivo endothelium-targeted nanoparticle delivery of the FOXM1 gene driven by an endothelial cell (EC)-specific promoter reactivated endothelial regeneration, normalized vascular repair and resolution of inflammation, and promoted survival in aged mice after sepsis challenge. In addition, treatment with the FDA-approved DNA demethylating agent decitabine was sufficient to reactivate FoxM1-dependent endothelial regeneration in aged mice, reverse aging-impaired resolution of inflammatory injury, and promote survival. Mechanistically, aging-induced Foxm1 promoter hypermethylation in mice, which could be inhibited by decitabine treatment, inhibited Foxm1 induction after sepsis challenge. In COVID-19 lung autopsy samples, FOXM1 was not induced in vascular ECs of elderly patients in their 80s, in contrast with middle-aged patients (aged 50 to 60 years). Thus, reactivation of FoxM1-mediated endothelial regeneration and vascular repair may represent a potential therapy for elderly patients with ARDS.
Assuntos
COVID-19 , Proteína Forkhead Box M1 , Lesão Pulmonar , Síndrome do Desconforto Respiratório , Sepse , Animais , Camundongos , Decitabina/farmacologia , Endotélio Vascular/fisiologia , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Pulmão/metabolismo , Lesão Pulmonar/genética , Camundongos Transgênicos , Regeneração/fisiologia , Sepse/metabolismoRESUMO
Inflammasome activation is of central importance for the process of generation of overwhelming inflammatory response and the pathogenesis of sepsis. The intrinsic molecular mechanism for controlling inflammasome activation is still poorly understood. Here we investigated the role of p120-catenin expression in macrophages in regulating nucleotide-binding oligomerization domain (NOD) and leucine-rich repeat (LRR)- and pyrin domain-containing proteins 3 (NLRP3) inflammasome activation. Depletion of p120-catenin in murine bone marrow-derived macrophages enhanced caspase-1 activation and secretion of active interleukin (IL)-1ß in response to ATP stimulation following LPS priming. Coimmunoprecipitation analysis showed that p120-catenin deletion promoted NLRP3 inflammasome activation by accelerating the assembly of the inflammasome complex comprised of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1. Depletion of p120-catenin also increased the production of mitochondrial reactive oxygen species. Pharmacological inhibition of mitochondrial reactive oxygen species nearly completely abolished NLRP3 inflammasome activation, caspase-1 activation, and the production of IL-1ß in p120-catenin-depleted macrophages. Furthermore, p120-catenin ablation significantly disrupted mitochondrial function, evidenced by decreased mitochondrial membrane potential and lower production of intracellular ATP. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of p120-catenin-deficient macrophages dramatically enhanced the accumulation of IL-1ß and IL-18 in bronchoalveolar lavage fluid. These results demonstrate that p120-catenin prevents NLRP3 inflammasome activation in macrophages by maintaining mitochondrial homeostasis and reducing the production of mitochondrial reactive oxygen species in response to endotoxin insult. Thus, inhibition of NLRP3 inflammasome activation by stabilization of p120-catenin expression in macrophages may be a novel strategy to prevent an uncontrolled inflammatory response in sepsis.
Assuntos
Inflamassomos , Sepse , Animais , Camundongos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , delta Catenina , Espécies Reativas de Oxigênio/metabolismo , Macrófagos/metabolismo , Caspase 1/metabolismo , Sepse/metabolismo , Trifosfato de Adenosina/metabolismo , Interleucina-1beta/metabolismoRESUMO
Clay is one of the important base materials in slope restoration. The adhesion of clay-rock interface plays a decisive role in the repairing effect on rock slopes. Fibers and polymers are widely used as a clay improvement method in rock slope repair. In this paper, the friction effect of sisal fiber and polyvinyl acetate (PVAc)-reinforced clay was studied through the design of an indoor rock-like interface sliding model test. Using modelled test results and scanning electron microscope (SEM) images, the reinforced clay was analyzed. The test results showed that the critical sliding angle and maximum static friction force of clay decreased with the increase of moisture content. An excess of fiber content and moisture content weakens the coupling effect of fiber-anchoring clay. Fiber content of 0.8% and PVAc content of 2% had the best effect on enhancing the sliding resistance of clay and provided good adhesion for dangerous interfaces of rock slope at 35° and 45°, respectively. PVAc formed a three-dimensional networked elastic membrane structure to improve the skid resistance and dynamic friction coefficient of the clay. The results provide an effective way for soil improvement and ecological restoration.
RESUMO
Considering that, in the context of the ecological restoration of a large number of exposed rock slopes, it is difficult for existing artificial soil to meet the requirements of mechanical properties and ecological construction at the same time, this paper investigates the stabilization benefits of polyvinyl acetate and attapulgite-treated clayey soil through a series of laboratory experiments. To study the effectiveness of polyvinyl acetate (PVA) and attapulgite as soil stabilizer, a triaxial strength test, an evaporation test and a vegetation growth test were carried out on improved soil with different amounts of PVA content (0, 1%, 2%, 3%, and 4%) and attapulgite replacement (0, 2%, 4%, 6%, and 8%). The results show that the single and composite materials of polyvinyl acetate and attapulgite can increase the peak deviator stress of the sample. The addition of polyvinyl acetate can improve the soil strength by increasing the cohesion of the sample; the addition of attapulgite improves the soil strength mainly by increasing the internal friction angle of the sample. The strength of the composite is greatly improved by increasing the cohesion and internal friction angle of the sample at the same time. The effect of adding materials increased significantly with increasing curing age. Moreover, polyvinyl acetate and attapulgite improve the soil water retention of the soil by improving the water-holding capacity, so that the soil can still ensure the good growth of vegetation under long-term drought conditions. The scanning electron microscopy (SEM) images indicated that the PVA and attapulgite of soil affect the strength characteristics of soil specimens by the reaction of PVA and water, which changes the structure of the soil and, by the interweaving of attapulgite soil particles, acts as the skeleton of the aggregate. Overall, PVA and attapulgite can effectively increase clayey soil stability by improving the cohesive force and internal friction angle of clayey soil.
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Mechanical ventilation is a life-sustaining therapy for patients with respiratory failure but can cause further lung damage known as ventilator-induced lung injury (VILI). However, the intrinsic molecular mechanisms underlying recovery of VILI remain unknown. Phagocytosis of apoptotic cells (also known as efferocytosis) is a key mechanism orchestrating successful resolution of inflammation. Here we show the positive regulation of macrophage Toll-like receptor (TLR) 4 in efferocytosis and resolution of VILI. Mice were depleted of alveolar macrophages and then subjected to injurious ventilation (tidal volume, 20 mL/kg) for 4 h. On day 1 after mechanical ventilation, Tlr4+/+ or Tlr4-/- bone marrow-derived macrophages (BMDMs) were intratracheally administered to alveolar macrophage-depleted mice. We observed that mice depleted of alveolar macrophages exhibited defective resolution of neutrophilic inflammation, exuded protein, lung edema, and lung tissue injury after ventilation, whereas these delayed responses were reversed by administration of Tlr4+/+ BMDMs. Importantly, these proresolving effects by Tlr4+/+ BMDMs were abolished in mice receiving Tlr4-/- BMDMs. The number of macrophages containing apoptotic cells or bodies in bronchoalveolar lavage fluid was much less in mice receiving Tlr4-/- BMDMs than that in those receiving Tlr4+/+ BMDMs. Macrophage TLR4 deletion facilitated a disintegrin and metalloprotease 17 maturation and enhanced Mer cleavage in response to mechanical ventilation. Heat shock protein 70 dramatically increased Mer tyrosine kinase surface expression, phagocytosis of apoptotic neutrophils, and rescued the inflammatory phenotype in alveolar macrophage-depleted mice receiving Tlr4+/+ BMDMs, but not Tlr4-/- BMDMs. Our results suggest that macrophage TLR4 promotes resolution of VILI via modulation of Mer-mediated efferocytosis.
Assuntos
Macrófagos Alveolares/metabolismo , Neutrófilos/imunologia , Fagocitose/fisiologia , Receptor 4 Toll-Like/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Proteína ADAM17/metabolismo , Animais , Apoptose/fisiologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , Células Cultivadas , Feminino , Proteínas de Choque Térmico HSP70/metabolismo , Pulmão/patologia , Macrófagos Alveolares/transplante , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Respiração Artificial/efeitos adversos , Transdução de Sinais , c-Mer Tirosina Quinase/metabolismoRESUMO
Ventilator-induced lung injury is associated with an increase in mortality in patients with respiratory dysfunction, although mechanical ventilation is an essential intervention implemented in the intensive care unit. Intrinsic molecular mechanisms for minimizing lung inflammatory injury during mechanical ventilation remain poorly defined. We hypothesize that Yes-associated protein (YAP) expression in endothelial cells protects the lung against ventilator-induced injury. Wild-type and endothelial-specific YAP-deficient mice were subjected to a low (7 mL/kg) or high (21 mL/kg) tidal volume (VT) ventilation for 4 h. Infiltration of inflammatory cells into the lung, vascular permeability, lung histopathology, and the levels of inflammatory cytokines were measured. Here, we showed that mechanical ventilation with high VT upregulated YAP protein expression in pulmonary endothelial cells. Endothelial-specific YAP knockout mice following high VT ventilation exhibited increased neutrophil counts and protein content in bronchoalveolar lavage fluid, Evans blue leakage, and histological lung injury compared with wild-type littermate controls. Deletion of YAP in endothelial cells exaggerated vascular endothelial (VE)-cadherin phosphorylation, downregulation of vascular endothelial protein tyrosine phosphatase (VE-PTP), and dissociation of VE-cadherin and catenins following mechanical ventilation. Importantly, exogenous expression of wild-type VE-PTP in the pulmonary vasculature rescued YAP ablation-induced increases in neutrophil counts and protein content in bronchoalveolar lavage fluid, vascular leakage, and histological lung injury as well as VE-cadherin phosphorylation and dissociation from catenins following ventilation. These data demonstrate that YAP expression in endothelial cells suppresses lung inflammatory response and edema formation by modulating VE-PTP-mediated VE-cadherin phosphorylation and thus plays a protective role in ventilator-induced lung injury.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Permeabilidade Capilar , Endotélio Vascular/metabolismo , Pulmão/metabolismo , Neutrófilos/imunologia , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controle , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Feminino , Pulmão/citologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Fosforilação , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Proteínas de Sinalização YAPAssuntos
Pulmão/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Alveolares/imunologia , Pneumonia/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Expressão Gênica/imunologia , Humanos , Pulmão/metabolismo , Pulmão/patologia , Ativação de Macrófagos/genética , Macrófagos Alveolares/classificação , Macrófagos Alveolares/metabolismo , Pneumonia/genética , Pneumonia/metabolismoRESUMO
BACKGROUND: Sevoflurane with its antiinflammatory properties has shown to decrease mortality in animal models of sepsis. However, the underlying mechanism of its beneficial effect in this inflammatory scenario remains poorly understood. Macrophages play an important role in the early stage of sepsis as they are tasked with eliminating invading microbes and also attracting other immune cells by the release of proinflammatory cytokines such as interleukin-1ß, interleukin-6, and tumor necrosis factor-α. Thus, the authors hypothesized that sevoflurane mitigates the proinflammatory response of macrophages, while maintaining their bactericidal properties. METHODS: Murine bone marrow-derived macrophages were stimulated in vitro with lipopolysaccharide in the presence and absence of 2% sevoflurane. Expression of cytokines and inducible NO synthase as well as uptake of fluorescently labeled Escherichia coli (E. coli) were measured. The in vivo endotoxemia model consisted of an intraperitoneal lipopolysaccharide injection after anesthesia with either ketamine and xylazine or 4% sevoflurane. Male mice (n = 6 per group) were observed for a total of 20 h. During the last 30 min fluorescently labeled E. coli were intraperitoneally injected. Peritoneal cells were extracted by peritoneal lavage and inducible NO synthase expression as well as E. coli uptake by peritoneal macrophages was determined using flow cytometry. RESULTS: In vitro, sevoflurane enhanced lipopolysaccharide-induced inducible NO synthase expression after 8 h by 466% and increased macrophage uptake of fluorescently labeled E. coli by 70% compared with vehicle-treated controls. Inhibiting inducible NO synthase expression pharmacologically abolished this increase in bacteria uptake. In vivo, inducible NO synthase expression was increased by 669% and phagocytosis of E. coli by 49% compared with the control group. CONCLUSIONS: Sevoflurane enhances phagocytosis of bacteria by lipopolysaccharide-challenged macrophages in vitro and in vivo via an inducible NO synthase-dependent mechanism. Thus, sevoflurane potentiates bactericidal and antiinflammatory host-defense mechanisms in endotoxemia.
Assuntos
Anti-Inflamatórios/farmacologia , Regulação Enzimológica da Expressão Gênica , Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo II/biossíntese , Fagocitose/fisiologia , Sevoflurano/farmacologia , Animais , Atividade Bactericida do Sangue/efeitos dos fármacos , Atividade Bactericida do Sangue/fisiologia , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/genética , Fagocitose/efeitos dos fármacos , Células RAW 264.7RESUMO
Endothelial barrier dysfunction is a central factor in the pathogenesis of persistent lung inflammation and protein-rich edema formation, the hallmarks of acute respiratory distress syndrome. However, little is known about the molecular mechanisms that are responsible for vascular repair and resolution of inflammatory injury after sepsis challenge. Herein, we show that hypoxia-inducible factor-1α (HIF-1α), expressed in endothelial cells (ECs), is the critical transcriptional factor mediating vascular repair and resolution of inflammatory lung injury. After sepsis challenge, HIF-1α but not HIF-2α expression was rapidly induced in lung vascular ECs, and mice with EC-restricted disruption of Hif1α (Hif1af/f/Tie2Cre+) exhibited defective vascular repair, persistent inflammation, and increased mortality in contrast with the wild-type littermates after polymicrobial sepsis or endotoxemia challenge. Hif1af/f/Tie2Cre+ lungs exhibited marked decrease of EC proliferation during recovery after sepsis challenge, which was associated with inhibited expression of forkhead box protein M1 (Foxm1), a reparative transcription factor. Therapeutic restoration of endothelial Foxm1 expression, via liposomal delivery of Foxm1 plasmid DNA to Hif1af/f/Tie2Cre+ mice, resulted in reactivation of the vascular repair program and improved survival. Together, our studies, for the first time, delineate the essential role of endothelial HIF-1α in driving the vascular repair program. Thus, therapeutic activation of HIF-1α-dependent vascular repair may represent a novel and effective therapy to treat inflammatory vascular diseases, such as sepsis and acute respiratory distress syndrome.
Assuntos
Células Endoteliais/metabolismo , Proteína Forkhead Box M1/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lesão Pulmonar/metabolismo , Pulmão/fisiologia , Regeneração , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células Endoteliais/patologia , Feminino , Proteína Forkhead Box M1/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Lesão Pulmonar/etiologia , Lesão Pulmonar/genética , Lesão Pulmonar/patologia , Masculino , Camundongos , Camundongos Transgênicos , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/genética , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Sepse/complicações , Sepse/genética , Sepse/metabolismo , Sepse/patologiaRESUMO
Calpain is an intracellular Ca2+-dependent non-lysosomal cysteine protease expressed ubiquitously in mammals. In endothelial cells, dysregulation of calpain has been shown to be involved in a wide variety of pathological conditions such as angiogenesis, vascular inflammation, and diabetes. Cell- or tissue-targeted in vivo delivery of small interfering RNA (siRNA) is a powerful research tool in the analysis of protein function and has been proposed as an attractive therapeutic modality that is applicable against a large number of human diseases including cancer. In this chapter we describe a method to knockdown calpain 1 in mouse pulmonary vascular endothelium using delivery of siRNA/cationic liposome complex. This technique results in a greater than 80% reduction in calpain 1 protein levels 48 h after a single i.v. injection of calpain 1 siRNA (0.5 mg siRNA/kg)/cationic liposome complex. We also describe confocal imaging to verify the loss of calpain 1 expression in pulmonary microvessel endothelial cells and application of this technique in the mouse model of ventilator-induced lung injury.
Assuntos
Calpaína/genética , Células Endoteliais/química , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/genética , Animais , Calpaína/antagonistas & inibidores , Células Endoteliais/metabolismo , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Humanos , Lipossomos/química , Pulmão/química , Pulmão/metabolismo , Camundongos , RNA Interferente Pequeno/químicaRESUMO
Uncontrolled inflammatory response during sepsis predominantly contributes to the development of multiorgan failure and lethality. However, the cellular and molecular mechanisms for excessive production and release of proinflammatory cytokines are not clearly defined. In this study, we show the crucial role of the GTPase Ras-related protein in brain (Rab)1a in regulating the nucleotide binding domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation and lung inflammatory injury. Expression of dominant negative Rab1 N124I plasmid in bone marrow-derived macrophages prevented the release of IL-1ß and IL-18, NLRP3 inflammasome activation, production of pro-IL-1ß and pro-IL-18, and attenuated TLR4 surface expression and NF-кB activation induced by bacterial LPS and ATP compared with control cells. In alveolar macrophage-depleted mice challenged with cecal ligation and puncture, pulmonary transplantation of Rab1a-inactivated macrophages by expression of Rab1 N124I plasmid dramatically reduced the release of IL-1ß and IL-18, neutrophil count in bronchoalveolar lavage fluid, and inflammatory lung injury. Rab1a activity was elevated in alveolar macrophages from septic patients and positively associated with severity of sepsis and respiratory dysfunction. Thus, inhibition of Rab1a activity in macrophages resulting in the suppression of NLRP3 inflammasome activation may be a promising target for the treatment of patients with sepsis.
Assuntos
Inflamassomos/metabolismo , Lesão Pulmonar/imunologia , Macrófagos Alveolares/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Pneumonia/imunologia , Sepse/imunologia , Proteínas rab1 de Ligação ao GTP/metabolismo , Animais , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Ativação de Macrófagos/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Neutrófilos/imunologia , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
Angiogenesis is initiated in response to a variety of external cues, including mechanical and biochemical stimuli; however, the underlying signaling mechanisms remain unclear. Here, we investigated the proangiogenic role of the endothelial mechanosensor Piezo1. Genetic deletion and pharmacological inhibition of Piezo1 reduced endothelial sprouting and lumen formation induced by wall shear stress and proangiogenic mediator sphingosine 1-phosphate, whereas Piezo1 activation by selective Piezo1 activator Yoda1 enhanced sprouting angiogenesis. Similarly to wall shear stress, sphingosine 1-phosphate functioned by activating the Ca2+ gating function of Piezo1, which in turn signaled the activation of the matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase during sprouting angiogenesis. Studies in mice in which Piezo1 was conditionally deleted in endothelial cells demonstrated the requisite role of sphingosine 1-phosphate-dependent activation of Piezo1 in mediating angiogenesis in vivo. These results taken together suggest that both mechanical and biochemical stimuli trigger Piezo1-mediated Ca2+ influx and thereby activate matrix metalloproteinase-2 and membrane type 1 matrix metalloproteinase and synergistically facilitate sprouting angiogenesis.
Assuntos
Canais Iônicos/deficiência , Metaloproteinase 14 da Matriz/metabolismo , Neovascularização Fisiológica/fisiologia , Transdução de Sinais/fisiologia , Animais , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Canais Iônicos/genética , Metaloproteinase 14 da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
RATIONALE: Microvascular inflammation and endothelial dysfunction secondary to unchecked activation of endothelium play a critical role in the pathophysiology of sepsis and organ failure. The intrinsic signaling mechanisms responsible for dampening excessive activation of endothelial cells are not completely understood. OBJECTIVE: To determine the central role of YAP (Yes-associated protein), the major transcriptional coactivator of the Hippo pathway, in modulating the strength and magnitude of endothelial activation and vascular inflammation. METHODS AND RESULTS: Endothelial-specific YAP knockout mice showed increased basal expression of E-selectin and ICAM (intercellular adhesion molecule)-1 in endothelial cells, a greater number of adherent neutrophils in postcapillary venules and increased neutrophil counts in bronchoalveolar lavage fluid. Lipopolysaccharide challenge of these mice augmented NF-κB (nuclear factor-κB) activation, expression of endothelial adhesion proteins, neutrophil and monocyte adhesion to cremaster muscle venules, transendothelial neutrophil migration, and lung inflammatory injury. Deletion of YAP in endothelial cells also markedly augmented the inflammatory response and cardiovascular dysfunction in a polymicrobial sepsis model induced by cecal ligation and puncture. YAP functioned by interacting with the E3 ubiquitin-protein ligase TLR (Toll-like receptor) signaling adaptor TRAF6 (tumor necrosis factor receptor-associated factor 6) to ubiquitinate TRAF6, and thus promoted TRAF6 degradation and modification resulting in inhibition of NF-κB activation. TRAF6 depletion in endothelial cells rescued the augmented inflammatory phenotype in mice with endothelial cell-specific deletion of YAP. CONCLUSIONS: YAP modulates the activation of endothelial cells and suppresses vascular inflammation through preventing TRAF6-mediated NF-κB activation and is hence essential for limiting the severity of sepsis-induced inflammation and organ failure.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Células Endoteliais/fisiologia , Endotélio Vascular/fisiopatologia , Fosfoproteínas/fisiologia , Fator 6 Associado a Receptor de TNF/metabolismo , Vasculite/etiologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Permeabilidade Capilar , Adesão Celular , Proteínas de Ciclo Celular , Selectina E/metabolismo , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Molécula 1 de Adesão Intercelular/metabolismo , Contagem de Leucócitos , Camundongos , Camundongos Knockout , Microvasos , Monócitos/fisiologia , NF-kappa B/metabolismo , Neutrófilos/citologia , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Sepse/complicações , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Vênulas/citologia , Proteínas de Sinalização YAPRESUMO
A patient's recovery from lung inflammatory injury or development of multi-system organ failure is determined by the host's ability to resolve inflammation and repair tissue damage, both of which require the clearance of apoptotic neutrophils by macrophages (efferocytosis). Here, we investigated the effects of isoflurane on macrophage efferocytosis and resolution of lung inflammatory injury. Treatment of murine bone marrow-derived macrophages (BMDMs) or alveolar macrophages with isoflurane dramatically enhanced phagocytosis of apoptotic neutrophils. Isoflurane significantly increased the surface expression of the receptor tyrosine kinase Mer in macrophages, but markedly decreased the levels of a soluble form of Mer protein in the medium. Isoflurane treatment also caused a decrease in a disintegrin and metalloproteinase 17 (ADAM17) on the cell surface and a concomitant increase in its cytoplasmic fraction. These responses induced by isoflurane were completely reversed by a pharmacological inhibitor or genetic deletion of AMP-activated protein kinase (AMPK). In a mouse model of lipopolysaccharide-induced lung injury, isoflurane accelerated the recovery of lung inflammation and injury that was coupled with an increase in the number of alveolar macrophages containing apoptotic bodies. In alveolar macrophage-depleted mice, administration of isoflurane-pretreated BMDMs facilitated resolution of lung inflammation following lipopolysaccharide challenge. Thus, isoflurane promoted resolution of lipopolysaccharide-induced lung inflammatory injury via enhancement of macrophage efferocytosis. Increased macrophage efferocytosis following isoflurane treatment correlates with upregulation of Mer surface expression through AMPK-mediated blockade of ADAM17 trafficking to the cell membrane.
Assuntos
Proteína ADAM17/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Apoptose , Isoflurano/farmacologia , Neutrófilos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Receptores Imunológicos/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP/genética , Lesão Pulmonar Aguda/induzido quimicamente , Animais , Células Cultivadas , Técnicas de Silenciamento de Genes , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/imunologiaRESUMO
Acute respiratory distress syndrome (ARDS) is characterized by acute hypoxemia respiratory failure, bilateral pulmonary infiltrates, and pulmonary edema of non-cardiac origin. Effective treatments for ARDS patients may arise from experimental studies with translational mouse models of this disease that aim to delineate the mechanisms underlying the disease pathogenesis. Mouse models of ARDS, however, can be limited by their rapid progression from injured to recovery state, which is in contrast to the course of ARDS in humans. Furthermore, current mouse models of ARDS do not recapitulate certain prominent aspects of the pathogenesis of ARDS in humans. In this study, we developed an improved endotoxemic mouse model of ARDS resembling many features of clinical ARDS including extended courses of injury and recovery as well as development of fibrosis following i.p. injection of lipopolysaccharide (LPS) to corn oil-preloaded mice. Compared with mice receiving LPS alone, those receiving corn oil and LPS exhibited extended course of lung injury and repair that occurred over a period of >2 weeks instead of 3-5days. Importantly, LPS challenge of corn oil-preloaded mice resulted in pulmonary fibrosis during the repair phase as often seen in ARDS patients. In summary, this simple novel mouse model of ARDS could represent a valuable experimental tool to elucidate mechanisms that regulate lung injury and repair in ARDS patients.