RESUMEN
The paucity of therapeutic strategies to reduce the severity of radiation-induced lung fibrosis (RILF), a life-threatening complication of intended or accidental ionizing radiation exposure, is a serious unmet need. We evaluated the contribution of eNAMPT (extracellular nicotinamide phosphoribosyltransferase), a damage-associated molecular pattern (DAMP) protein and TLR4 (Toll-like receptor 4) ligand, to the severity of whole-thorax lung irradiation (WTLI)-induced RILF. Wild-type (WT) and Nampt+/- heterozygous C57BL6 mice and nonhuman primates (NHPs, Macaca mulatta) were exposed to a single WTLI dose (9.8 or 10.7 Gy for NHPs, 20 Gy for mice). WT mice received IgG1 (control) or an eNAMPT-neutralizing polyclonal or monoclonal antibody (mAb) intraperitoneally 4 hours after WTLI and weekly thereafter. At 8-12 weeks after WTLI, NAMPT expression was assessed by immunohistochemistry, biochemistry, and plasma biomarker studies. RILF severity was determined by BAL protein/cells, hematoxylin and eosin, and trichrome blue staining and soluble collagen assays. RNA sequencing and bioinformatic analyses identified differentially expressed lung tissue genes/pathways. NAMPT lung tissue expression was increased in both WTLI-exposed WT mice and NHPs. Nampt+/- mice and eNAMPT polyclonal antibody/mAb-treated mice exhibited significantly attenuated WTLI-mediated lung fibrosis with reduced: 1) NAMPT and trichrome blue staining; 2) dysregulated lung tissue expression of smooth muscle actin, p-SMAD2/p-SMAD1/5/9, TGF-ß, TSP1 (thrombospondin-1), NOX4, IL-1ß, and NRF2; 3) plasma eNAMPT and IL-1ß concentrations; and 4) soluble collagen. Multiple WTLI-induced dysregulated differentially expressed lung tissue genes/pathways with known tissue fibrosis involvement were each rectified in mice receiving eNAMPT mAbs.The eNAMPT/TLR4 inflammatory network is essentially involved in radiation pathobiology, with eNAMPT neutralization an effective therapeutic strategy to reduce RILF severity.
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Lesión Pulmonar , Fibrosis Pulmonar , Alarminas/metabolismo , Animales , Anticuerpos Monoclonales , Citocinas/metabolismo , Pulmón/patología , Lesión Pulmonar/patología , Ratones , Ratones Endogámicos C57BL , Nicotinamida Fosforribosiltransferasa/genética , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/metabolismo , Tórax , Receptor Toll-Like 4/metabolismoRESUMEN
Increasing evidence suggests an important role for deubiquitinating enzymes (DUBs) in modulating a variety of biological functions and diseases. We previously identified the upregulation of the DUB ubiquitin carboxyl terminal hydrolase 1 (UCHL1) in murine ventilator-induced lung injury (VILI). However, the role of UCHL1 in modulating vascular permeability, a cardinal feature of acute lung injury (ALI) in general, remains unclear. We investigated the role of UCHL1 in pulmonary endothelial cell (EC) barrier function in vitro and in vivo and examined the effects of UCHL1 on VE-cadherin and claudin-5 regulation, important adherens and tight junctional components, respectively. Measurements of transendothelial electrical resistance confirmed decreased barrier enhancement induced by hepatocyte growth factor (HGF) and increased thrombin-induced permeability in both UCHL1-silenced ECs and in ECs pretreated with LDN-57444 (LDN), a pharmacological UCHL1 inhibitor. In addition, UCHL1 knockdown (siRNA) was associated with decreased expression of VE-cadherin and claudin-5, whereas silencing of the transcription factor FoxO1 restored claudin-5 levels. Finally, UCHL1 inhibition in vivo via LDN was associated with increased VILI in a murine model. These findings support a prominent functional role of UCHL1 in regulating lung vascular permeability via alterations in adherens and tight junctions and implicate UCHL1 as an important mediator of ALI.
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Permeabilidad Capilar , Endotelio Vascular/patología , Ubiquitina Tiolesterasa/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/patología , Animales , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Técnicas In Vitro , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Oximas/farmacología , Transducción de Señal , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Ubiquitina Tiolesterasa/genética , Ubiquitinación , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismoRESUMEN
RATIONALE: The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. METHODS: Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo. RESULTS: Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. CONCLUSIONS: These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.
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Lesión Pulmonar Aguda , COVID-19 , Animales , Anticuerpos Monoclonales , Humanos , Ratones , Ratones Endogámicos C57BL , SARS-CoV-2RESUMEN
RATIONALE: The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically relevant inflammatory stimuli and promoter SNPs in nmMLCK promoter regulation. METHODS: Full-length or serially deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein-DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2'-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was utilized for nmMLCK validation. RESULTS: Lung EC levels of nmMLCK were significantly increased in LPS-challenged mice and LPS, tumor necrosis factor-α (TNF-α), 18% cyclic stretch (CS) and 5-Aza each significantly up-regulated EC nmMYLK promoter activities. EC exposure to FG-4592, a prolyl hydroxylase inhibitor that increases hypoxia-inducible factor (HIF) expression, increased nmMYLK promoter activity, confirmed by HIF1α/HIF2α silencing. nmMYLK promoter deletion studies identified distal inhibitory and proximal enhancing promoter regions as well as mechanical stretch-, LPS- and TNFα-inducible regions. Insertion of ARDS-associated SNPs (rs2700408, rs11714297) significantly increased nmMYLK promoter activity via increased transcription binding (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (ISX), respectively). Finally, the MYLK rs78755744 SNP (-261G/A), residing within a nmMYLK CpG island, significantly attenuated 5-Aza-induced promoter activity. CONCLUSION: These findings indicate nmMYLK transcriptional regulation by clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.
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Lesión Pulmonar Aguda/inducido químicamente , Epigénesis Genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Animales , Células Cultivadas , Decitabina , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Lesión Pulmonar/inducido químicamente , Masculino , Ratones Endogámicos C57BL , Quinasa de Cadena Ligera de Miosina/genética , Neumonía , Polimorfismo de Nucleótido Simple , Síndrome de Dificultad Respiratoria/genética , Estrés Mecánico , Factor de Necrosis Tumoral alfaRESUMEN
NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE-dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of MYLK, which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.
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Factor 2 Relacionado con NF-E2/genética , Proteína de Replicación A/genética , Proteínas Represoras/genética , Transcripción Genética/genética , Activación Transcripcional/genética , Células A549 , Animales , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Genoma/genética , Humanos , Ratones , Regiones Promotoras Genéticas/genética , Elementos de Respuesta/genéticaRESUMEN
We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterial hypertension (PAH) and now examine NAMPT regulation and extracellular NAMPT's (eNAMPT's) role in PAH vascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-ß1 [transforming growth factor-ß1], and hypoxia). Endothelial-to-mesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPT-neutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2α (hypoxia-inducible factor-2α) also potently regulated NAMPT promoter activity, and HIF-2α binding sites were identified between -628 bp and -328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2α-dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-κB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Citocinas/genética , Hipertensión Pulmonar/genética , Nicotinamida Fosforribosiltransferasa/genética , Factores de Transcripción SOX/genética , Transcripción Genética/genética , Remodelación Vascular/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica/genética , Humanos , Masculino , RatasRESUMEN
RATIONALE: Genetic factors are involved in acute respiratory distress syndrome (ARDS) susceptibility. Identification of novel candidate genes associated with increased risk and severity will improve our understanding of ARDS pathophysiology and enhance efforts to develop novel preventive and therapeutic approaches. OBJECTIVES: To identify genetic susceptibility targets for ARDS. METHODS: A genome-wide association study was performed on 232 African American patients with ARDS and 162 at-risk control subjects. The Identify Candidate Causal SNPs and Pathways platform was used to infer the association of known gene sets with the top prioritized intragenic SNPs. Preclinical validation of SELPLG (selectin P ligand gene) was performed using mouse models of LPS- and ventilator-induced lung injury. Exonic variation within SELPLG distinguishing patients with ARDS from sepsis control subjects was confirmed in an independent cohort. MEASUREMENTS AND MAIN RESULTS: Pathway prioritization analysis identified a nonsynonymous coding SNP (rs2228315) within SELPLG, encoding P-selectin glycoprotein ligand 1, to be associated with increased susceptibility. In an independent cohort, two exonic SELPLG SNPs were significantly associated with ARDS susceptibility. Additional support for SELPLG as an ARDS candidate gene was derived from preclinical ARDS models where SELPLG gene expression in lung tissues was significantly increased in both ventilator-induced (twofold increase) and LPS-induced (5.7-fold increase) murine lung injury models compared with controls. Furthermore, Selplg-/- mice exhibited significantly reduced LPS-induced inflammatory lung injury compared with wild-type C57/B6 mice. Finally, an antibody that neutralizes P-selectin glycoprotein ligand 1 significantly attenuated LPS-induced lung inflammation. CONCLUSIONS: These findings identify SELPLG as a novel ARDS susceptibility gene among individuals of European and African descent.
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Negro o Afroamericano/genética , Estudio de Asociación del Genoma Completo , Genotipo , Síndrome de Dificultad Respiratoria/genética , Síndrome de Dificultad Respiratoria/fisiopatología , Selectinas/genética , Población Blanca/genética , Adulto , Anciano , Estudios de Cohortes , Femenino , Predisposición Genética a la Enfermedad , Humanos , Masculino , Persona de Mediana Edad , Síndrome de Dificultad Respiratoria/epidemiología , Factores de Riesgo , Estados Unidos/epidemiologíaRESUMEN
Acute respiratory distress syndrome (ARDS) is a devastating critical illness disproportionately affecting the elderly population, with both higher incidence and mortality. The integrity of the lung endothelial cell (EC) monolayer is critical for preservation of lung function. However, mechanisms mediating EC barrier regulation in the context of aging remain unclear. We assessed the severity of acute lung injury (ALI) in young (2 mo) and aged (18 mo) mice using a two-hit preclinical model. Compared with young cohorts, aged mice exhibited increased ALI severity, with greater vascular permeability characterized by elevated albumin influx and levels of bronchoalveolar lavage (BAL) cells (neutrophils) and protein. Aged/injured mice also demonstrated elevated levels of reactive oxygen species (ROS) in the BAL, which was associated with upregulation of the ROS-generating enzyme, Nox4. We evaluated the role of aging in human lung EC barrier regulation utilizing a cellular model of replicative senescence. Senescent EC populations were defined by increases in ß-galactosidase activity and p16 levels. In response to lipopolysaccharide (LPS) challenge, senescent ECs demonstrate exacerbated permeability responses compared with control "young" ECs. LPS challenge led to a rapid induction of Nox4 expression in both control and senescent ECs, which was posttranslationally mediated via the proteasome/ubiquitin system. However, senescent ECs demonstrated deficient Nox4 ubiquitination, resulting in sustained expression of Nox4 and alterations in cellular redox homeostasis. Pharmacological inhibition of Nox4 in senescent ECs reduced LPS-induced alterations in permeability. These studies provide insight into the roles of Nox4/senescence in EC barrier responses and offer a mechanistic link to the increased incidence and mortality of ARDS associated with aging.
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Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Envejecimiento/metabolismo , NADPH Oxidasas/metabolismo , Ubiquitinación , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Susceptibilidad a Enfermedades , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , NADPH Oxidasa 4 , Oxidación-Reducción/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Acute lung injury (ALI) results from infectious challenges and from pathologic lung distention produced by excessive tidal volume delivered during mechanical ventilation (ventilator-induced lung injury [VILI]) and is characterized by extensive alveolar and vascular dysfunction. Identification of novel ALI therapies is hampered by the lack of effective ALI/VILI biomarkers. We explored endothelial cell (EC)-derived microparticles (EMPs) (0.1-1 µm) as potentially important markers and potential mediators of lung vascular injury in preclinical models of ALI and VILI. We characterized EMPs (annexin V and CD31 immunoreactivity) produced from human lung ECs exposed to physiologic or pathologic mechanical stress (5 or 18% cyclic stretch [CS]) or to endotoxin (LPS). EC exposure to 18% CS or to LPS resulted in increased EMP shedding compared with static cells (â¼ 4-fold and â¼ 2.5-fold increases, respectively). Proteomic analysis revealed unique 18% CS-derived (n = 10) and LPS-derived EMP proteins (n = 43). VILI-challenged mice (40 ml/kg, 4 h) exhibited increased plasma and bronchoalveolar lavage CD62E (E-selectin)-positive MPs compared with control mice. Finally, mice receiving intratracheal instillation of 18% CS-derived EMPs displayed significant lung inflammation and injury. These findings indicate that ALI/VILI-producing stimuli induce significant shedding of distinct EMP populations that may serve as potential ALI biomarkers and contribute to the severity of lung injury.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Micropartículas Derivadas de Células/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Endotoxinas/farmacología , Estrés Mecánico , Lesión Pulmonar Inducida por Ventilación Mecánica/tratamiento farmacológico , Lesión Pulmonar Aguda/metabolismo , Lesión Pulmonar Aguda/patología , Animales , Micropartículas Derivadas de Células/patología , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Humanos , Lipopolisacáridos/farmacología , Masculino , Ratones Endogámicos C57BL , Neumonía/inducido químicamente , Neumonía/metabolismo , Neumonía/patología , Lesión Pulmonar Inducida por Ventilación Mecánica/metabolismo , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
Acute lung injury/acute respiratory distress syndrome (ALI/ARDS), an illness characterized by life-threatening vascular leak, is a significant cause of morbidity and mortality in critically ill patients. Recent preclinical studies and clinical observations have suggested a potential role for the chemotherapeutic agent imatinib in restoring vascular integrity. Our prior work demonstrates differential effects of imatinib in mouse models of ALI, namely attenuation of LPS-induced lung injury but exacerbation of ventilator-induced lung injury (VILI). Because of the critical role of mechanical ventilation in the care of patients with ARDS, in the present study we pursued an assessment of the effectiveness of imatinib in a "two-hit" model of ALI caused by combined LPS and VILI. Imatinib significantly decreased bronchoalveolar lavage protein, total cells, neutrophils, and TNF-α levels in mice exposed to LPS plus VILI, indicating that it attenuates ALI in this clinically relevant model. In subsequent experiments focusing on its protective role in LPS-induced lung injury, imatinib attenuated ALI when given 4 h after LPS, suggesting potential therapeutic effectiveness when given after the onset of injury. Mechanistic studies in mouse lung tissue and human lung endothelial cells revealed that imatinib inhibits LPS-induced NF-κB expression and activation. Overall, these results further characterize the therapeutic potential of imatinib against inflammatory vascular leak.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Mesilato de Imatinib/uso terapéutico , Inflamación/tratamiento farmacológico , Pulmón/irrigación sanguínea , Pulmón/patología , Lesión Pulmonar Aguda/complicaciones , Lesión Pulmonar Aguda/patología , Animales , Líquido del Lavado Bronquioalveolar , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Humanos , Mesilato de Imatinib/farmacología , Inflamación/complicaciones , Inflamación/patología , Lipopolisacáridos , Pulmón/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Sustancias Protectoras/farmacología , Sustancias Protectoras/uso terapéutico , Arteria Pulmonar/patología , Respiración Artificial/efectos adversos , Factor de Necrosis Tumoral alfa/biosíntesis , Lesión Pulmonar Inducida por Ventilación Mecánica/complicaciones , Lesión Pulmonar Inducida por Ventilación Mecánica/tratamiento farmacológico , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
We previously reported protective effects of GADD45a (growth arrest and DNA damage-inducible gene 45 alpha) in murine ventilator-induced lung injury (VILI) via effects on Akt-mediated endothelial cell signaling. In the present study we investigated the role of GADD45a in separate murine models of radiation- and bleomycin-induced lung injury. Initial studies of wild-type mice subjected to single-dose thoracic radiation (10 Gy) confirmed a significant increase in lung GADD45a expression within 24 h and persistent at 6 wk. Mice deficient in GADD45a (GADD45a(-/-)) demonstrated increased susceptibility to radiation-induced lung injury (RILI, 10 Gy) evidenced by increased bronchoalveolar lavage (BAL) fluid total cell counts, protein and albumin levels, and levels of inflammatory cytokines compared with RILI-challenged wild-type animals at 2 and 4 wk. Furthermore, GADD45a(-/-) mice had decreased total and phosphorylated lung Akt levels both at baseline and 6 wk after RILI challenge relative to wild-type mice while increased RILI susceptibility was observed in both Akt(+/-) mice and mice treated with an Akt inhibitor beginning 1 wk prior to irradiation. Additionally, overexpression of a constitutively active Akt1 transgene reversed RILI-susceptibility in GADD45a(-/-) mice. In separate studies, lung fibrotic changes 2 wk after treatment with bleomycin (0.25 U/kg IT) was significantly increased in GADD45a(-/-) mice compared with wild-type mice assessed by lung collagen content and histology. These data implicate GADD45a as an important modulator of lung inflammatory responses across different injury models and highlight GADD45a-mediated signaling as a novel target in inflammatory lung injury clinically.
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Bleomicina/efectos adversos , Proteínas de Ciclo Celular/metabolismo , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/metabolismo , Pulmón/metabolismo , Proteínas Nucleares/metabolismo , Animales , Colágeno/metabolismo , Modelos Animales de Enfermedad , Femenino , Inflamación/inducido químicamente , Inflamación/metabolismo , Pulmón/efectos de los fármacos , Pulmón/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Radiación , Transducción de Señal/fisiologíaRESUMEN
RATIONALE: Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis. OBJECTIVES: We aimed to examine whether SphKs and their product, S1P, play a role in the development of pulmonary arterial hypertension (PAH). METHODS: SphK1(-/-), SphK2(-/-), and S1P lyase heterozygous (Sgpl1(+/-)) mice, a pharmacologic SphK inhibitor (SKI2), and a S1P receptor 2 (S1PR2) antagonist (JTE013) were used in rodent models of hypoxia-mediated pulmonary hypertension (HPH). S1P levels in lung tissues from patients with PAH and pulmonary arteries (PAs) from rodent models of HPH were measured. MEASUREMENTS AND MAIN RESULTS: mRNA and protein levels of SphK1, but not SphK2, were significantly increased in the lungs and isolated PA smooth muscle cells (PASMCs) from patients with PAH, and in lungs of experimental rodent models of HPH. S1P levels were increased in lungs of patients with PAH and PAs from rodent models of HPH. Unlike SphK2(-/-) mice, SphK1(-/-) mice were protected against HPH, whereas Sgpl1(+/-) mice were more susceptible to HPH. Pharmacologic SphK1 and S1PR2 inhibition prevented the development of HPH in rodent models of HPH. Overexpression of SphK1 and stimulation with S1P potentially via ligation of S1PR2 promoted PASMC proliferation in vitro, whereas SphK1 deficiency inhibited PASMC proliferation. CONCLUSIONS: The SphK1/S1P axis is a novel pathway in PAH that promotes PASMC proliferation, a major contributor to pulmonary vascular remodeling. Our results suggest that this pathway is a potential therapeutic target in PAH.
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Hipertensión Pulmonar/etiología , Lisofosfolípidos/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Esfingosina/análogos & derivados , Animales , Humanos , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Esfingosina/fisiología , Técnicas de Cultivo de TejidosRESUMEN
Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from -2,428 to -2,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (-948G/T) and rs7789066 (-2,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.
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Citocinas/genética , Células Endoteliales/fisiología , Nicotinamida Fosforribosiltransferasa/genética , Síndrome de Dificultad Respiratoria/genética , Factor de Transcripción STAT5/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Regiones no Traducidas 5'/genética , Lesión Pulmonar Aguda/etiología , Lesión Pulmonar Aguda/genética , Lesión Pulmonar Aguda/metabolismo , Animales , Células Cultivadas , Citocinas/fisiología , Metilación de ADN/fisiología , Modelos Animales de Enfermedad , Células Endoteliales/citología , Epigénesis Genética/genética , Regulación de la Expresión Génica/fisiología , Variación Genética/genética , Humanos , Masculino , Ratones Endogámicos C57BL , Nicotinamida Fosforribosiltransferasa/fisiología , Regiones Promotoras Genéticas/fisiología , Arteria Pulmonar/citología , ARN Interferente Pequeño/genética , Respiración Artificial/efectos adversos , Síndrome de Dificultad Respiratoria/etiología , Síndrome de Dificultad Respiratoria/metabolismo , Estrés MecánicoRESUMEN
We previously identified the intracellular nicotinamide phosphoribosyltransferase (iNAMPT, aka pre-B-cell colony enhancing factor) as a candidate gene promoting acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI) with circulating nicotinamide phosphoribosyltransferase potently inducing NF-κB signaling in lung endothelium. iNAMPT also synthesizes intracellular nicotinamide adenine dinucleotide (iNAD) in response to extracellular oxidative stress, contributing to the inhibition of apoptosis via ill-defined mechanisms. We now further define the role of iNAMPT activity in the pathogenesis of ARDS/VILI using the selective iNAMPT inhibitor FK-866. C57/B6 mice were exposed to VILI (40 ml/kg, 4 h) or LPS (1.5 mg/kg, 18 h) after osmotic pump delivery of FK-866 (100 mg/kg/d, intraperitoneally). Assessment of total bronchoalveolar lavage (BAL) protein, polymorphonuclear neutrophil (PMN) levels, cytokine levels (TNF-α, IL-6, IL-1α), lung iNAD levels, and injury scores revealed that FK-866-mediated iNAMPT inhibition successfully reduced lung tissue iNAD levels, BAL injury indices, inflammatory cell infiltration, and lung injury scores in LPS- and VILI-exposed mice. FK-866 further increased lung PMN apoptosis, as reflected by caspase-3 activation in BAL PMNs. These findings support iNAMPT inhibition via FK-866 as a novel therapeutic agent for ARDS via enhanced apoptosis in inflammatory PMNs.
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Acrilamidas/farmacología , Antiinflamatorios/farmacología , Citocinas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Pulmón/efectos de los fármacos , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Piperidinas/farmacología , Neumonía/tratamiento farmacológico , Síndrome de Dificultad Respiratoria/tratamiento farmacológico , Lesión Pulmonar Inducida por Ventilación Mecánica/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Líquido del Lavado Bronquioalveolar/inmunología , Caspasa 3/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Pulmón/enzimología , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , NAD/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/inmunología , Nicotinamida Fosforribosiltransferasa/metabolismo , Neumonía/enzimología , Neumonía/inmunología , Neumonía/patología , Síndrome de Dificultad Respiratoria/enzimología , Síndrome de Dificultad Respiratoria/inmunología , Síndrome de Dificultad Respiratoria/patología , Lesión Pulmonar Inducida por Ventilación Mecánica/enzimología , Lesión Pulmonar Inducida por Ventilación Mecánica/inmunología , Lesión Pulmonar Inducida por Ventilación Mecánica/patologíaRESUMEN
Myosin light chain kinase (MLCK; gene code, MYLK) is a multifunctional enzyme involved in isoform-specific nonmuscle (nm) and smooth muscle contraction, inflammation, and vascular permeability, processes directly relevant to asthma pathobiology. In this report, we highlight the contribution of the nm isoform (nmMLCK) to asthma susceptibility and severity, supported by studies in two lines of transgenic mice with knocking out nmMLCK or selectively overexpressing nmMLCK in endothelium. These mice were sensitized to exhibit ovalbumin-mediated allergic inflammation. Genetically engineered mice with targeted nmMLCK deletion (nmMLCK(-/-)) exhibited significant reductions in lung inflammation and airway hyperresponsiveness. Conversely, mice with overexpressed nmMLCK in endothelium (nmMLCK(ec/ec)) exhibited elevated susceptibility and severity in asthmatic inflammation. In addition, reduction of nmMLCK expression in pulmonary endothelium by small interfering RNA results in reduced asthmatic inflammation in wild-type mice. These pathophysiological assessments demonstrate the positive contribution of nmMLCK to asthmatic inflammation, and a clear correlation of the level of nmMLCK with the degree of experimental allergic inflammation. This study confirms MYLK as an asthma candidate gene, and verifies nmMLCK as a novel molecular target in asthmatic pathobiology.
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Asma/enzimología , Quinasa de Cadena Ligera de Miosina/metabolismo , Neumonía/enzimología , Animales , Asma/genética , Asma/metabolismo , Citocinas/genética , Citocinas/metabolismo , Células Endoteliales/metabolismo , Endotelio/metabolismo , Leucocitos/metabolismo , Pulmón/enzimología , Pulmón/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Quinasa de Cadena Ligera de Miosina/genética , Neumonía/genética , Neumonía/metabolismoRESUMEN
OBJECTIVE: Effective therapies are needed to reverse the increased vascular permeability that characterizes acute inflammatory diseases such as acute lung injury. FTY720 is a pharmaceutical analog of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate. Because both FTY720 and sphingosine 1-phosphate have properties that may limit their usefulness in patients with acute lung injury, alternative compounds are needed for therapeutic use. The objective of this study is to characterize the effects of FTY720 (S)-phosphonate, a novel analog of FTY720-phosphate, on variables of pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo. SETTING: University-affiliated research institute. SUBJECTS: Cultured human pulmonary endothelial cells; C57BL/6 mice. INTERVENTIONS: Endothelial cells were stimulated with sphingosine 1-phosphate receptor 1 agonists to determine effects on sphingosine 1-phosphate receptor 1 expression. Acute lung injury was induced in C57BL/6 mice with bleomycin to assess effects of sphingosine 1-phosphate receptor 1 agonists. MEASUREMENTS AND MAIN RESULTS: FTY720 (S)-phosphonate potently increases human pulmonary endothelial cell barrier function in vitro as measured by transendothelial electrical resistance. Reduction of sphingosine 1-phosphate receptor 1 with small interference RNA significantly attenuates this transendothelial electrical resistance elevation. FTY720 (S)-phosphonate maintains endothelial sphingosine 1-phosphate receptor 1 protein expression in contrast to greater than 50% reduction after incubation with sphingosine 1-phosphate, FTY720, or other sphingosine 1-phosphate receptor 1 agonists. FTY720 (S)-phosphonate does not induce ß-arrestin recruitment, sphingosine 1-phosphate receptor 1 ubiquitination, and proteosomal degradation that occur after other agonists. Intraperitoneal administration of FTY720 (S)-phosphonate every other day for 1 week in normal or bleomycin-injured mice maintains significantly higher lung sphingosine 1-phosphate receptor 1 expression compared with FTY720. FTY720 fails to protect against bleomycin-induced acute lung injury in mice, while FTY720 (S)-phosphonate significantly decreases lung leak and inflammation. CONCLUSION: FTY720 (S)-phosphonate is a promising barrier-promoting agent that effectively maintains sphingosine 1-phosphate receptor 1 levels and improves outcomes in the bleomycin model of acute lung injury.
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Lesión Pulmonar Aguda/tratamiento farmacológico , Arrestinas/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Glicoles de Propileno/farmacología , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/análogos & derivados , Lesión Pulmonar Aguda/fisiopatología , Animales , Arrestinas/efectos de los fármacos , Bleomicina/farmacología , Western Blotting , Células Cultivadas , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Clorhidrato de Fingolimod , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Ratones Endogámicos C57BL , Distribución Aleatoria , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Lisoesfingolípidos/efectos de los fármacos , Sensibilidad y Especificidad , Esfingosina/farmacología , beta-ArrestinasRESUMEN
Thyroid hormone (TH) metabolism, mediated by deiodinase types 1, 2, and 3 (D1, D2, and D3) is profoundly affected by acute illness. We examined the role of TH metabolism during ventilator-induced lung injury (VILI) in mice. Mice exposed to VILI recapitulated the serum TH findings of acute illness, namely a decrease in 3,5,3'-triiodothyronine (T(3)) and thyroid-stimulating hormone and an increase in reverse T(3). Both D2 immunoreactivity and D2 enzymatic activity were increased significantly. D1 and D3 activity did not change. Using D2 knockout (D2KO) mice, we determined whether the increase in D2 was an adaptive response. Although similar changes in serum TH levels were observed in D2KO and WT mice, D2KO mice exhibited greater susceptibility to VILI than WT mice, as evidenced by poorer alveoli integrity and quantified by lung chemokine and cytokine mRNA induction. These data suggest that an increase in lung D2 is protective against VILI. Similar findings of increased inflammatory markers were found in hypothyroid WT mice exposed to VILI compared with euthyroid mice, indicating that the lungs were functionally hypothyroid. Treatment of D2KO mice with T(3) reversed many of the lung chemokine and cytokine profiles seen in response to VILI, demonstrating a role for T(3) in the treatment of lung injury. We conclude that TH metabolism in the lung is linked to the response to inflammatory injury and speculate that D2 exerts its protective effect by making more TH available to the injured lung tissue.
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Lesión Pulmonar Aguda/enzimología , Yoduro Peroxidasa/metabolismo , Pulmón/enzimología , Lesión Pulmonar Inducida por Ventilación Mecánica/enzimología , Lesión Pulmonar Aguda/sangre , Lesión Pulmonar Aguda/genética , Animales , Quimiocinas/genética , Citocinas/genética , Activación Enzimática/fisiología , Expresión Génica/efectos de los fármacos , Genotipo , Inmunohistoquímica , Yoduro Peroxidasa/genética , Pulmón/metabolismo , Pulmón/patología , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad de la Especie , Tirotropina/sangre , Tiroxina/sangre , Triyodotironina/sangre , Triyodotironina/farmacología , Lesión Pulmonar Inducida por Ventilación Mecánica/sangre , Lesión Pulmonar Inducida por Ventilación Mecánica/genética , Yodotironina Deyodinasa Tipo IIRESUMEN
RATIONALE: Cortactin, an actin-binding cytoskeletal protein, plays a crucial role in maintaining endothelial cell (EC) barrier integrity and regulating vascular permeability. The gene encoding cortactin, CTTN, is implicated in various lung inflammatory disorders. Despite this, the transcriptional regulation of CTTN by inflammatory stimuli and promoter SNPs remains unexplored. METHODS: We transfected human lung ECs with a full-length CTTN promoters linked to a luciferase reporter to measure promoter activity. SNP-containing CTTN promoter was created via site-directed mutagenesis. Transfected ECs were exposed to LPS (PAMP), TNF-α (cytokine), cyclic stretch (CS), FG-4592 (HIF-inducer), NRF2 (anti-oxidant modulator), FTY-(S)-phosphate (endothelial barrier enhancer) and 5'-Aza (demethylation inducer). Immunohistochemistry was used to assess cortactin expression in mouse lungs exposed to LPS. RESULTS: LPS, TNF-α, and 18%CS significantly increased CTTN promoter activities in a time-dependent manner (p<0.05). The variant rs34612166 (-212T/C) markedly enhanced LPS- and 18%CS- induced CTTN promoter activities (p<0.05). FG-4592 significantly boosted CTTN promoter activities (p<0.01), which were partially inhibited by HIF1a (KC7F2) and HIF2a (PT2385) inhibitors (p<0.05). NRF2 activator Bixin increased CTTN promoter activities, whereas NRF2 inhibitor Brusatol reduced them (p<0.05). 5'-Aza increased CTTN promoter activities by 2.9-fold (p<0.05). NF-kB response element mutations significantly reduced CTTN promoter activities response to LPS and TNF-α. FTY-(S)-phosphate significantly increased CTTN promoter activities in 24hrs. In vivo, cortactin levels were significantly elevated in inflammatory mouse lungs exposed to LPS for 18hrs. CONCLUSION: CTTN transcriptional is significantly influenced by inflammatory factors and promoter variants. Cortactin, essential in mitigating inflammatory edema, presents a promising therapeutic target to alleviate severe inflammatory disorders.
RESUMEN
Rationale: Circadian systems drive the expression of multiple genes in nearly all cells and coordinate cellular-, tissue-, and system-level processes that are critical to innate immunity regulation. Objective: We examined the effects of circadian rhythm disorganization, produced by light shift exposure, on innate immunity-mediated inflammatory lung responses including vascular permeability and gene expression in a C57BL/6J murine model of inflammatory lung injury. Methods: A total of 32 C57BL/6J mice were assigned to circadian phase shifting (CPS) with intratracheal phosphate-buffered saline (PBS), CPS with intratracheal lipopolysaccharide (LPS), control (normal lighting) condition with intratracheal PBS, and control condition with intratracheal LPS. Bronchoalveolar lavage (BAL) protein, cell counts, tissue immunostaining, and differentially expressed genes (DEGs) were measured in lung tissues at 2 and 10 weeks. Measurements and results: In mice exposed to both CPS and intratracheal LPS, both BAL protein and cell counts were increased at both 2 and 10 weeks compared to mice exposed to LPS alone. Multiple DEGs were identified in CPS-LPS-exposed lung tissues compared to LPS alone and were involved in transcriptional pathways associated with circadian rhythm disruption, regulation of lung permeability, inflammation with Rap1 signaling, and regulation of actin cytoskeleton. The most dysregulated pathways included myosin light chain kinase, MAP kinase, profilin 2, fibroblast growth factor receptor, integrin b4, and p21-activated kinase. Conclusion: Circadian rhythm disruption results in exacerbated immune response and dysregulated expression of cytoskeletal genes involved in the regulation of epithelial and vascular barrier integrity-the mechanistic underpinnings of acute lung injury. Further studies need to explore circadian disorganization as a druggable target.
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Lesión Pulmonar Aguda , Lipopolisacáridos , Ratones , Animales , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Pulmón , Expresión GénicaRESUMEN
Myocardial infarction (MI) triggers adverse ventricular remodeling (VR), cardiac fibrosis, and subsequent heart failure. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is postulated to play a significant role in VR processing via activation of the TLR4 inflammatory pathway. We hypothesized that an eNAMPT specific monoclonal antibody (mAb) could target and neutralize overexpressed eNAMPT post-MI and attenuate chronic cardiac inflammation and fibrosis. We investigated humanized ALT-100 and ALT-300 mAb with high eNAMPT-neutralizing capacity in an infarct rat model to test our hypothesis. ALT-300 was 99mTc-labeled to generate 99mTc-ALT-300 for imaging myocardial eNAMPT expression at 2 hours, 1 week, and 4 weeks post-IRI. The eNAMPT-neutralizing ALT-100 mAb (0.4 mg/kg) or saline was administered intraperitoneally at 1 hour and 24 hours post-reperfusion and twice a week for 4 weeks. Cardiac function changes were determined by echocardiography at 3 days and 4 weeks post-IRI. 99mTc-ALT-300 uptake was initially localized to the ischemic area at risk (IAR) of the left ventricle (LV) and subsequently extended to adjacent non-ischemic areas 2 hours to 4 weeks post-IRI. Radioactive uptake (%ID/g) of 99mTc-ALT-300 in the IAR increased from 1 week to 4 weeks (0.54 ± 0.16 vs. 0.78 ± 0.13, P < 0.01). Rats receiving ALT-100 mAb exhibited significantly improved myocardial histopathology and cardiac function at 4 weeks, with a significant reduction in the collagen volume fraction (%LV) compared to controls (21.5 ± 6.1% vs. 29.5 ± 9.9%, P < 0.05). Neutralization of the eNAMPT/TLR4 inflammatory cascade is a promising therapeutic strategy for MI by reducing chronic inflammation, fibrosis, and preserving cardiac function.