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OBJECTIVES: Interleukin-18 (IL-18) plasma level and latent class analysis (LCA) have separately been shown to predict prognosis and treatment response in acute respiratory distress syndrome (ARDS). IL-18 is a measure of inflammasome activation, a pathway potentially distinct from inflammation captured by biomarkers defining previously published LCA classes. We hypothesized that elevated IL-18 would identify distinct "high-risk" patients not captured by prior LCA classifications. DESIGN: Statins for acutely injured lungs from sepsis (SAILS) and hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in acute lung injury to reduce pulmonary dysfunction trial (HARP-2) are two large randomized, controlled trials in ARDS in which both LCA assignments and IL-18 levels were shown to predict mortality. We first evaluated the overlap between high IL-18 levels (≥ 800 pg/mL) with prior LCA class assignments using McNemar's test and then tested the correlation between IL-18 and LCA biomarkers using Pearson's exact test on log-2 transformed values. Our primary analysis was the association of IL-18 level with 60-day mortality in the hypoinflammatory LCA class, which was assessed using the Fisher exact test and Cox proportional hazards modeling adjusting for age, Acute Physiology and Chronic Health Evaluation score, and gender. Secondary analyses included the association of IL-18 and LCA with mortality within each IL-18/LCA subgroup. SETTING: Secondary analysis of two multicenter, randomized controlled clinical trials of ARDS patients. SUBJECTS: Six hundred eighty-three patients in SAILS and 511 patients in HARP-2. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We found that 33% of patients in SAILS and HARP-2 were discordant by IL-18 level and LCA class. We further found that IL-18 level was only modestly correlated (0.17-0.47) with cytokines used in the LCA assignment. A substantial subset of individuals classified as hypoinflammatory by LCA (14% of SAILS and 43% of HARP-2) were classified as high risk by elevated IL-18. These individuals were at high risk for mortality in both SAILS (42% 60-d mortality, odds ratio [OR] 3.3; 95% CI, 1.8-6.1; p < 0.001) and HARP-2 (27% 60-d mortality, OR 2.1; 95% CI, 1.2-3.8; p = 0.009). CONCLUSIONS: Plasma IL-18 level provides important additional prognostic information to LCA subphenotypes defined largely by traditional inflammatory biomarkers in two large ARDS cohorts.
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Interleucina-18 , Síndrome do Desconforto Respiratório , Humanos , Análise de Classes Latentes , Estudos Retrospectivos , Citocinas , Ensaios Clínicos Controlados Aleatórios como Assunto , Síndrome do Desconforto Respiratório/terapia , Biomarcadores , Interleucina-8RESUMO
BACKGROUND: Two acute respiratory distress syndrome (ARDS) trials showed no benefit for statin therapy, though secondary analyses suggest inflammatory subphenotypes may have a differential response to simvastatin. Statin medications decrease cholesterol levels, and low cholesterol has been associated with increased mortality in critical illness. We hypothesized that patients with ARDS and sepsis with low cholesterol could be harmed by statins. METHODS: Secondary analysis of patients with ARDS and sepsis from two multicenter trials. We measured total cholesterol from frozen plasma samples obtained at enrollment in Statins for Acutely Injured Lungs from Sepsis (SAILS) and Simvastatin in the Acute Respiratory Distress Syndrome (HARP-2) trials, which randomized subjects with ARDS to rosuvastatin versus placebo and simvastatin versus placebo, respectively, for up to 28 days. We compared the lowest cholesterol quartile (< 69 mg/dL in SAILS, < 44 mg/dL in HARP-2) versus all other quartiles for association with 60-day mortality and medication effect. Fisher's exact test, logistic regression, and Cox Proportional Hazards were used to assess mortality. RESULTS: There were 678 subjects with cholesterol measured in SAILS and 509 subjects in HARP-2, of whom 384 had sepsis. Median cholesterol at enrollment was 97 mg/dL in both SAILS and HARP-2. Low cholesterol was associated with higher APACHE III and shock prevalence in SAILS, and higher Sequential Organ Failure Assessment score and vasopressor use in HARP-2. Importantly, the effect of statins differed in these trials. In SAILS, patients with low cholesterol who received rosuvastatin were more likely to die (odds ratio (OR) 2.23, 95% confidence interval (95% CI) 1.06-4.77, p = 0.02; interaction p = 0.02). In contrast, in HARP-2, low cholesterol patients had lower mortality if randomized to simvastatin, though this did not reach statistical significance in the smaller cohort (OR 0.44, 95% CI 0.17-1.07, p = 0.06; interaction p = 0.22). CONCLUSIONS: Cholesterol levels are low in two cohorts with sepsis-related ARDS, and those in the lowest cholesterol quartile are sicker. Despite the very low levels of cholesterol, simvastatin therapy seems safe and may reduce mortality in this group, though rosuvastatin was associated with harm.
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Inibidores de Hidroximetilglutaril-CoA Redutases , Síndrome do Desconforto Respiratório , Sepse , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Rosuvastatina Cálcica/farmacologia , Rosuvastatina Cálcica/uso terapêutico , Sinvastatina/farmacologia , Sinvastatina/uso terapêutico , Síndrome do Desconforto Respiratório/terapia , Sepse/complicaçõesRESUMO
Vascular dysfunction is a hallmark of chronic diseases in elderly. The contribution of the vasculature to lung repair and fibrosis is not fully understood. Here, we performed an epigenetic and transcriptional analysis of lung endothelial cells (ECs) from young and aged mice during the resolution or progression of bleomycin-induced lung fibrosis. We identified the transcription factor ETS-related gene (ERG) as putative orchestrator of lung capillary homeostasis and repair, and whose function is dysregulated in aging. ERG dysregulation is associated with reduced chromatin accessibility and maladaptive transcriptional responses to injury. Loss of endothelial ERG enhances paracrine fibroblast activation in vitro, and impairs lung fibrosis resolution in young mice in vivo. scRNA-seq of ERG deficient mouse lungs reveales transcriptional and fibrogenic abnormalities resembling those associated with aging and human lung fibrosis, including reduced number of general capillary (gCap) ECs. Our findings demonstrate that lung endothelial chromatin remodeling deteriorates with aging leading to abnormal transcription, vascular dysrepair, and persistent fibrosis following injury.
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Fibrose Pulmonar , Idoso , Envelhecimento/genética , Animais , Bleomicina , Células Endoteliais/metabolismo , Fibrose , Humanos , Pulmão/patologia , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Transdução de Sinais , Regulador Transcricional ERG/genética , Regulador Transcricional ERG/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by myofibroblast accumulation and progressive lung scarring. To identify transcriptional gene programs driving persistent lung fibrosis in aging, we performed RNA-Seq on lung fibroblasts isolated from young and aged mice during the early resolution phase after bleomycin injury. We discovered that, relative to injured young fibroblasts, injured aged fibroblasts exhibited a profibrotic state characterized by elevated expression of genes implicated in inflammation, matrix remodeling, and cell survival. We identified the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and its target nuclear factor of activated T cells-1 (NFATc1) as putative drivers of the sustained profibrotic gene signatures in injured aged fibroblasts. PIM1 and NFATc1 transcripts were enriched in a pathogenic fibroblast population recently discovered in IPF lungs, and their protein expression was abundant in fibroblastic foci. Overexpression of PIM1 in normal human lung fibroblasts potentiated their fibrogenic activation, and this effect was attenuated by NFATc1 inhibition. Pharmacological inhibition of PIM1 attenuated IPF fibroblast activation and sensitized them to apoptotic stimuli. Interruption of PIM1 signaling in IPF lung explants ex vivo inhibited prosurvival gene expression and collagen secretion, suggesting that targeting this pathway may represent a therapeutic strategy to block IPF progression.
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Fibroblastos , Fibrose Pulmonar Idiopática , Envelhecimento/genética , Animais , Bleomicina/toxicidade , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , CamundongosRESUMO
OBJECTIVE: A high plasma level of inflammasome mediator interleukin-18 was associated with mortality in observational acute respiratory distress syndrome cohorts. Statin exposure increases both inflammasome activation and lung injury in mouse models. We tested whether randomization to statin therapy correlated with increased interleukin-18 in the ARDS Network Statins for Acutely Injured Lungs from Sepsis trial. DESIGN: Retrospective analysis of randomized controlled clinical trial. SETTING: Multicenter North American clinical trial, the ARDS Network Statins for Acutely Injured Lungs from Sepsis. PATIENTS: Six hundred eighty-three subjects with infection-related acute respiratory distress syndrome, representing 92% of the original trial population. INTERVENTIONS: Random assignment of rosuvastatin or placebo for up to 28 days or 3 days after ICU discharge. MEASUREMENTS AND MAIN RESULTS: We measured plasma interleukin-18 levels in all Statins for Acutely Injured Lungs from Sepsis patients with sample available at day 0 (baseline, n = 683) and day 3 (after randomization, n = 588). We tested the association among interleukin-18 level at baseline, rising interleukin-18, and the impact of statin therapy on 60-day mortality, adjusting for severity of illness. Baseline plasma interleukin-18 level greater than or equal to 800 pg/mL was highly associated with 60-day mortality, with a hazard of death of 2.3 (95% CI, 1.7-3.1). Rising plasma interleukin-18 was also associated with increased mortality. For each unit increase in log2 (interleukin-18) at day 3 compared with baseline, the hazard of death increased by 2.3 (95% CI, 1.5-3.5). Subjects randomized to statin were significantly more likely to experience a rise in plasma interleukin-18 levels. Subjects with acute kidney injury, shock, low baseline interleukin-18, and those not receiving systemic corticosteroids were more likely to experience rising interleukin-18. Randomization to statin therapy was associated with rising in interleukin-18 in all of those subsets, however. CONCLUSIONS: Elevated baseline plasma interleukin-18 was associated with higher mortality in sepsis-induced acute respiratory distress syndrome. A rise in plasma interleukin-18 was also associated with increased mortality and was more common in subjects randomized to statin therapy in this clinical trial.
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Interleucina-18/sangue , Alvéolos Pulmonares/fisiopatologia , Síndrome do Desconforto Respiratório/imunologia , Síndrome do Desconforto Respiratório/mortalidade , Lesão Pulmonar Aguda/imunologia , Adulto , Feminino , Humanos , Unidades de Terapia Intensiva , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Sepse/sangue , Sepse/mortalidadeRESUMO
Mechanical ventilation is necessary to support patients with acute lung injury, but also exacerbates injury through mechanical stress-activated signaling pathways. We show that stretch applied to cultured human cells, and to mouse lungs in vivo, induces robust expression of metallothionein, a potent antioxidant and cytoprotective molecule critical for cellular zinc homeostasis. Furthermore, genetic deficiency of murine metallothionein genes exacerbated lung injury caused by high tidal volume mechanical ventilation, identifying an adaptive role for these genes in limiting lung injury. Stretch induction of metallothionein required zinc and the zinc-binding transcription factor MTF1. We further show that mouse dietary zinc deficiency potentiates ventilator-induced lung injury, and that plasma zinc levels are significantly reduced in human patients who go on to develop acute respiratory distress syndrome (ARDS) compared with healthy and non-ARDS intensive care unit (ICU) controls, as well as with other ICU patients without ARDS. Taken together, our findings identify a potentially novel adaptive response of the lung to stretch and a critical role for zinc in defining the lung's tolerance for mechanical ventilation. These results demonstrate that failure of stretch-adaptive responses play an important role in exacerbating mechanical ventilator-induced lung injury, and identify zinc and metallothionein as targets for lung-protective interventions in patients requiring mechanical ventilation.
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BACKGROUND: Isoflurane may be protective in preclinical models of lung injury, but its use in patients with lung injury remains controversial and the mechanism of its protective effects remains unclear. The authors hypothesized that this protection is mediated at the level of alveolar tight junctions and investigated the possibility in a two-hit model of lung injury that mirrors human acute respiratory distress syndrome. METHODS: Wild-type mice were treated with isoflurane 1 h after exposure to nebulized endotoxin (n = 8) or saline control (n = 9) and then allowed to recover for 24 h before mechanical ventilation (MV; tidal volume, 15 ml/kg, 2 h) producing ventilator-induced lung injury. Mouse lung epithelial cells were similarly treated with isoflurane 1 h after exposure to lipopolysaccharide. Cells were cyclically stretched the following day to mirror the MV protocol used in vivo. RESULTS: Mice treated with isoflurane following exposure to inhaled endotoxin and before MV exhibited significantly less physiologic lung dysfunction. These effects appeared to be mediated by decreased vascular leak, but not altered inflammatory indices. Mouse lung epithelial cells treated with lipopolysaccharide and cyclic stretch and lungs harvested from mice after treatment with lipopolysaccharide and MV had decreased levels of a key tight junction protein (i.e., zona occludens 1) that was rescued by isoflurane treatment. CONCLUSIONS: Isoflurane rescued lung injury induced by a two-hit model of endotoxin exposure followed by MV by maintaining the integrity of the alveolar-capillary barrier possibly by modulating the expression of a key tight junction protein.
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Lesão Pulmonar Aguda/tratamento farmacológico , Lesão Pulmonar Aguda/metabolismo , Anestésicos Inalatórios/administração & dosagem , Isoflurano/administração & dosagem , Junções Íntimas/metabolismo , Lesão Pulmonar Aguda/patologia , Animais , Linhagem Celular Transformada , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Junções Íntimas/efeitos dos fármacosRESUMO
The pcp2/L7 gene is characterized by its very cell type-specific expression restricted to cerebellar Purkinje cells and retinal bipolar neurons. Although remarkable progress as to the biochemical properties of the encoded protein has been made, knowledge on its physiological functions remains sparse. While characterizing a pcp2-driven transgenic strain, we observed the presence of a longer, so far unknown, pcp2 transcript. Different from another recently discovered splice variant, ret-pcp2, expression of this novel transcript is observed in bipolar as well as cerebellar Purkinje cells of mid-postnatal mice. The protein encoded by our novel variant appears to be less efficient in binding to Gα subunits compared to the original L7/pcp2 protein and it is also less inhibitory with respect to GTPγ binding. Its expression in the eye appears to be independent from eye opening in postnatal mice.
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Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Retina/crescimento & desenvolvimento , Retina/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Neuropeptídeos/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Células de Purkinje/metabolismo , Células Bipolares da Retina/metabolismo , Regulação para Cima , Visão Ocular , Percepção VisualRESUMO
Purkinje cell protein-2 (Pcp2 or L7) is highly expressed in cerebellar Purkinje cells and retinal bipolar neurons and interacts with the Galpha(i/o) family of G-proteins. Although the expression pattern of Pcp2 in the developing central nervous system suggests a role in differentiation, its function remains unknown. We established Tet-off inducible expression of Pcp2 in PC12 cells (rat pheochromocytoma cells) to determine whether Pcp2 regulates neuronal differentiation. Utilizing a polyclonal antibody, Pcp2 was localized in the cell body and throughout neurites of differentiated PC12 cells, similar to its localization in cerebellar Purkinje cells. Pcp2 expression in PC12 cells stimulated process formation (5-fold) and NGF (nerve growth factor)-stimulated neurite length (2-fold). Under basal conditions, Pcp2-PC12 cells demonstrated a 5-fold increase in Ras activation relative to non-induced PC12 cells and there was no change in extracellular-signal-regulated kinase 1/2 activity with Pcp2 expression. However, Pcp2 induction led to a >3-fold increase in basal p38 MAPK (mitogen-activated protein kinase) activity and the addition of NGF significantly stimulated both Ras and p38 MAPK in Pcp2-PC12 cells relative to the controls. Pretreatment of Pcp2-PC12 cells with the p38-specific inhibitor SB203580 blocked both the increased neurite formation and NGF-stimulated neurite growth. Pertussis toxin treatment had no effect on neurite growth in control cells, but completely blocked Pcp2-mediated increased neurite growth. Transient transfection of the beta-adrenergic receptor kinase C-terminus to prevent signalling through Gbetagamma in Pcp2-PC12 cells also inhibited the Pcp2-induced phenotype and reduced the Pcp2-stimulated Ras activation. Taken together, these findings demonstrate that Pcp2 induces differentiation in PC12 cells, in part through Gbetagamma-mediated Ras and p38 MAPK activation and suggest the potential for similar signalling mechanisms in Purkinje cells.