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Background: Both latent class analysis (LCA) assignment based upon a panel of plasma biomarkers and interleukin-18 (IL-18) plasma level have been shown to predict prognosis and treatment response in Acute Respiratory Distress Syndrome (ARDS). Interleukin-18 is a measure of inflammasome activation and plays a distinct role in inflammation that is not captured by the biomarkers used in LCA assignments. We hypothesized that elevated IL-18 would provide additive prognostic and therapeutic information to previously published LCA assignments in ARDS, identifying additional “high-risk” patients not captured by LCA who could be eligible for inclusion in future precision medicine-focused trials. Methods: IL-18 and a panel of protein markers used for LCA had been previously measured in plasma from 683/745 patients in the Statins for Acutely Injured Lungs from Sepsis (SAILS) and 511/540 patients in the Hydroxymethylglutaryl-CoA reductase inhibition with simvastatin in Acute lung injury to Reduce Pulmonary dysfunction (HARP-2) trials. We tested the association between high IL-18 (>800 pg/mL) and LCA class assignment using McNemar’s test and evaluated the association of each subgrouping as well as treatment with 60-day mortality using Fisher’s exact test. We assessed 60-day mortality in each combination (high/low IL-18, hypo-/hyper-inflammatory LCA class, and treatment/placebo) using Kaplan-Meier survival analysis. We evaluated the correlation between the log2 transformed IL-18 level and LCA biomarkers using Pearson’s correlation coefficient. Results: 33% of patients in SAILS and HARP-2 were discordant by IL-18 level and LCA class. Elevated IL-18 identified a high-risk group of individuals previously classified as hypo-inflammatory by LCA in both SAILS (OR 3.3, 95% CI 1.8-6.1, p of 0.17-0.47. Conclusions: High Plasma IL-18 level provides additional prognostic information to LCA sub-phenotypes in two large ARDS cohorts.

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.

ObjectiveA 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.DesignRetrospective analysis of randomized controlled clinical trial.SettingMulticenter North American clinical trial, the ARDS Network Statins for Acutely Injured Lungs from Sepsis.PatientsSix hundred eighty-three subjects with infection-related acute respiratory distress syndrome, representing 92% of the original trial population.InterventionsRandom assignment of rosuvastatin or placebo for up to 28 days or 3 days after ICU discharge.Measurements and main resultsWe 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.ConclusionsElevated 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.

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.

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.

One mechanism utilized by cells to maintain signaling pathways is to regulate the levels of specific signal transduction proteins. The compound geldanamycin (GA) specifically interacts with heat shock protein 90 (hsp90) complexes and has been widely utilized to study the role of hsp90 in modulating the function of signaling proteins. In this study, we used GA to demonstrate that levels of heterotrimeric Galpha subunits can be regulated through interactions with hsp90. In a dose-dependent manner, GA significantly reduced the steady state levels of endogenous Galpha(o) expression in two cell lines (PC12 and GH3) and had a similar effect on Galpha(o) transiently expressed in COS cells. Galpha(o) synthesis and degradation was studied in PC12 cells and in transiently transfected COS cells. (35)S labeling followed by immunoprecipitation demonstrated no effect of GA on the rate of Galpha(o) synthesis, but GA accelerated degradation of Galpha(o) in both PC12 cells and COS cells. The use of inhibitors, including lactacystin (a proteosome-specific inhibitor), suggests that Galpha(o) is predominantly degraded through the proteosome pathway. In vitro translated (35)S-labeled Galpha(o) could be detected in hsp90 immunoprecipitates, and this interaction did not require N-terminal myristoylation. Taken together, these results suggest that heterotrimeric Galpha(o) subunits are protected from degradation by interaction with hsp90 and that the interaction of Galpha subunits with heat shock proteins may be a general mechanism for regulating Galpha levels in the cell.

Expression of the von Willebrand factor (vWF) gene is restricted to the endothelial and megakaryocyte lineages. Within the endothelium, expression of vWF varies between different vascular beds. We have previously shown that the human vWF promoter spanning a region between −2182 (relative to the start site of transcription) and the end of the first intron contains information for environmentally responsive, vascular bed-specific expression in the heart, skeletal muscle, and brain. In the present study, we cloned the mouse vWF (mvWF) promoter and studied its function in cultured endothelial cells and transgenic mice. In transient transfection assays, the mvWF gene was found to be regulated by distinct mechanisms in different endothelial cell subtypes. In independent lines of transgenic mice, an mvWF promoter fragment containing DNA sequences between −2645 and the end of the first intron directed endothelial cell-specific expression in the microvascular beds of the heart, brain, and skeletal muscle as well as the endothelial lining of the aorta. In 1 line of mice, reporter gene activity was also detected in bone marrow megakaryocytes. Taken together, these findings suggest that both the mouse and human vWF promoters are regulated by vascular bed-specific mechanisms.

Extracellular superoxide dismutase (EC-SOD) is the major extracellular antioxidant enzyme. We have determined the primary structure of mouse EC-SOD by characterization of complementary DNA (cDNA) clones and by amino-acid sequence analysis of purified protein. cDNA sequence analysis indicates that mouse EC-SOD is synthesized as a 251-amino-acid precursor protein with a predicted molecular weight of 27,400 D. Amino-terminal micro sequence analysis of purified mature mouse lung EC-SOD demonstrated the sequence to begin with SSFDLADRLDPV-. These results indicate that EC-SOD as initially synthesized contains a 24-amino-acid precursor peptide, and that the mature protein is 227 amino acids in length. Computer algorithms that predict the most likely site of cotranslational signal peptidase cleavage suggest that processing will occur between amino acids 18 and 19 or 20 and 21, which implies that EC-SOD may be initially synthesized as a pre-pro-protein. Like human EC-SOD, mature mouse EC-SOD is glycosylated. The full-length mouse EC-SOD cDNA is 1,834 base pairs long and is 82% (79% for protein) identical to rat EC-SOD, but only 60% (60% for protein) identical to human EC-SOD. The mouse EC-SOD gene locus (Sod3) was mapped by interspecific backcross haplotype analysis as being 0.9 +/- 0.9 centimorgans distal to the Qdpr locus on mouse Chromosome 5, a position suggesting that the human homologue of EC-SOD will map close to the human QDPR locus (4p15.3). Of nine tissues examined by Northern blot analysis, those of the kidney and lung are by far the major tissues that express EC-SOD messenger RNA. Using in situ hybridization in the mouse lung, we demonstrate EC-SOD gene expression to be highly localized to alveolar Type II epithelial cells. These data suggest that alveolar Type II cells play a central role in mediating EC-SOD antioxidant function in the lung.

Extracellular acidosis (EA) regulates Heme Oxygenase-1 (HO-1) expression in vascular smooth muscle cells via transcriptional and posttranscriptional mechanisms but the signaling pathways involved are not known. We examined the role of Mitogen-Activated Protein Kinase (MAPK) pathways in HO-1 regulation by EA. Primary rat aortic smooth muscle cells were exposed to EA or physiologic pH. Levels of the total and phosphorylated forms of p38, extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and HO-1 protein were assessed by Western analysis and HO-1 mRNA levels were assessed by quantitative PCR. Inhibition of p38 MAPK was achieved with the chemical inhibitor SB203580, or adenoviral infection of a dominant-negative form of p38alpha. Phospho p38 MAPK activity was evaluated with an in vitro kinase activity assay. Binding of Activator Protein-1 (AP-1), a known target of MAPK pathways, was assessed by Electromobility shift assay (EMSA). EA induced phosphorylation of p38 MAPK in a biphasic manner while total p38 was unchanged. EA did not alter levels of phospho ERK 1/2 and phospho JNK 1/2. There was increased phospho p38 MAPK activity in the setting of EA which preceded the induction of HO-1. Inhibition of phospho p38 activity with either SB20358 or a dominant negative p38alpha oligonucleotide abrogated the induction of HO-1 by EA. Increased specific binding of AP-1 in the setting of EA was shown by EMSA. Increased phospho p38 activity precedes and likely mediates HO-1 induction by EA. Increased AP-1 binding may underlie the transcriptional regulation of HO-1 by EA.

The early growth response (Egr)-1 transcription factor serves to couple changes in the extracellular environment to alterations in gene expression. An understanding of the mechanisms that underlie Egr-1 gene regulation should provide important insights into how environmental signals are transduced by endothelial cells. The aim of the present study was to determine whether epidermal growth factor (EGF) induces Egr-1 expression in endothelial cells. In ECV304 cells, Egr-1 mRNA and protein levels were increased in response to EGF. In stable transfection assays, the 1,200-bp promoter of the mouse Egr-1 gene contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent pathway. The endogenous Egr-1 gene was similarly responsive to EGF in primary human umbilical vein endothelial cells, human coronary artery endothelial cells, and rat fat pad endothelial cells, but not in bovine aortic endothelial cells, calf pulmonary artery endothelial cells, or PY-4-1 endothelial cells. Together, these results suggest that the Egr-1 gene is responsive to EGF in a subset of endothelial cells.

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.