Gail Otulakowski

Lung-derived soluble mediators are pathogenic in ventilator-induced lung injury.

Ventilator-induced lung injury (VILI) due to high tidal volume (V(T)) is associated with increased levels of circulating factors that may contribute to, or be markers of, injury. This study investigated if exclusively lung-derived circulating factors produced during high V(T) ventilation can cause or worsen VILI. In isolated perfused mouse lungs, recirculation of perfusate worsened injury (compliance impairment, microvascular permeability, edema) induced by high V(T). Perfusate collected from lungs ventilated with high V(T) and used to perfuse lungs ventilated with low V(T) caused similar compliance impairment and permeability and caused a dose-dependent decrease in transepithelial electrical resistance (TER) across rat distal lung epithelial monolayers. Circulating soluble factors derived from the isolated lung thus contributed to VILI and had deleterious effects on the lung epithelial barrier. These data demonstrate transferability of an injury initially caused exclusively by mechanical ventilation and provides novel evidence for the biotrauma hypothesis in VILI. Mediators of the TER decrease were heat-sensitive, transferable via Folch extraction, and (following ultrafiltration, 3 kDa) comprised both smaller and larger molecules. Although several classes of candidate mediators, including protein cytokines (e.g., tumor necrosis factor-α, interleukin-6, macrophage inflammation protein-1α) and lipids (e.g., eicosanoids, ceramides, sphingolipids), have been implicated in VILI, only prostanoids accumulated in the perfusate in a pattern consistent with a pathogenic role, yet cyclooxygenase inhibition did not protect against injury. Although no single class of factor appears solely responsible for the decrease in barrier function, the current data implicate lipid-soluble protein-bound molecules as not just markers but pathogenic mediators in VILI.

Early Growth Response-1 Worsens Ventilator-induced Lung Injury by Up-Regulating Prostanoid Synthesis

RATIONALE Ventilator-induced lung injury (VILI) is common and serious and may be mediated in part by prostanoids. We have demonstrated increased expression of the early growth response-1 (Egr1) gene by injurious ventilation, but whether-or how-such up-regulation contributes to injury is unknown. OBJECTIVES We sought to define the role of Egr1 in the pathogenesis of VILI. METHODS An in vivo murine model of VILI was used, and Egr1(+/+) (wild-type) and Egr1(-/-) mice were studied; the effects of prostaglandin E receptor subtype 1 (EP1) inhibition were assessed. MEASUREMENTS AND MAIN RESULTS Injurious ventilation caused lung injury in wild-type mice, but less so in Egr1(-/-) mice. The injury was associated with expression of EGR1 protein, which was localized to type II cells and macrophages and was concentrated in nuclear extracts. There was a concomitant increase in expression of phosphorylated p44/p42 mitogen-activated protein kinases. The prostaglandin E synthase (mPGES-1) gene has multiple EGR1 binding sites on its promoter, and induction of mPGES-1 mRNA (as well as the prostanoid product, PGE2) by injurious ventilation was highly dependent on the presence of the Egr1 gene. PGE2 mediates many lung effects via EP1 receptors, and EP1 blockade (with ONO-8713) lessened lung injury. CONCLUSIONS This is the first demonstration of a mechanism whereby expression of a novel gene (Egr1) can contribute to VILI via a prostanoid-mediated pathway.

Mechanical ventilation induces neutrophil extracellular trap formation.

Background:Mechanical ventilation can injure the lung and induce a proinflammatory state; such ventilator-induced lung injury (VILI) is associated with neutrophil influx. Neutrophils release DNA and granular proteins as cytotoxic neutrophil extracellular traps (NETs). The authors hypothesized that NETs were produced in a VILI model and may contribute to injury. Methods:In a two-hit lipopolysaccharide/VILI mouse model with and without intratracheal deoxyribonuclease (DNase) treatment or blockade of known inducers of NET formation (NETosis), the authors assessed compliance, bronchoalveolar lavage fluid protein, markers of NETs (citrullinated histone-3 and DNA), and markers of inflammation. Results:Although lipopolysaccharide recruited neutrophils to airways, the addition of high tidal mechanical ventilation was required for significant induction of NETs markers (e.g., bronchoalveolar lavage fluid DNA: 0.4 ± 0.07 µg/ml [mean ± SEM], P < 0.05 vs. all others, n = 10 per group). High tidal volume mechanical ventilation increased airway high-mobility group box 1 protein (0.91 ± 0.138 vs. 0.60 ± 0.095) and interleukin-1&bgr; in lipopolysaccharide-treated mice (22.4 ± 0.87 vs. 17.0 ± 0.50 pg/ml, P < 0.001) and tended to increase monocyte chemoattractant protein-1 and interleukin-6. Intratracheal DNase treatment reduced NET markers (bronchoalveolar lavage fluid DNA: 0.23 ± 0.038 vs. 0.88 ± 0.135 µg/ml, P < 0.001; citrullinated histone-3: 443 ± 170 vs. 1,824 ± 403, P < 0.01, n = 8 to 10) and attenuated the loss of static compliance (0.9 ± 0.14 vs. 1.58 ± 0.17 ml/mmHg, P < 0.01, n = 19 to 20) without significantly impacting other measures of injury. Blockade of high-mobility group box 1 (with glycyrrhizin) or interleukin-1&bgr; (with anakinra) did not prevent NETosis or protect against injury. Conclusions:NETosis was induced in VILI, and DNase treatment eliminated NETs. In contrast to experimental transfusion-related acute lung injury, NETs do not play a major pathogenic role in the current model of VILI.

Hypercapnia attenuates ventilator‐induced lung injury via a disintegrin and metalloprotease‐17

Hypercapnia is common in mechanically ventilated patients with lung injury; while CO2 can ameliorate experimental lung injury, it can also cause harm. Because hypercapnia can protect against ventilator‐induced lung injury (VILI), understanding its impact on key signalling pathways may provide insight into the mechanisms of VILI. We show that hypercapnia blocks stretch‐mediated activation of p44/42 mitogen‐activated protein kinase (MAPK) signalling in alveolar epithelial cells; this occurs through inhibition of sheddase (i.e. the metalloprotease, ADAM17), which releases ligands that bind to the epidermal growth factor receptor. In vivo pharmacological blockade of ADAM17 reduces downstream MAPK activation and attenuates VILI in a two‐hit mouse model. Thus, hypercapnia uncovered a novel ADAM17‐dependent mechanism of VILI, and this represents a potential therapeutic target.

Global and Gene-Specific Translational Regulation in Rat Lung Development

During the peripartum period, the lung must respond to dramatic changes in circulating hormones, nutritional factors, and physiologic signals during its transition to becoming the organ of gas exchange. Protein synthesis consumes a significant proportion of metabolic resources and is inhibited by many environmental stresses. We hypothesized that translational control mechanisms play a role in the perinatal lung. Immunoblots of late-gestation (Fetal Day [FD] 17-22) rat lung extracts revealed gradual decreases in phosphorylated forms of the mammalian target of rapamycin effectors, eukaryotic initiation factor (eIF) 4E-binding protein, p70 S6 kinase, and ribosomal protein S6, followed by sharp increases on Postnatal Day 1 (P1). Immunohistochemistry showed phospho-S6 staining was most prominent in epithelial cells of the large and small airways. m(7)GTP-sepharose pulldown experiments showed a decrease in association of translation initiation factor, eIF4E, with its inhibitor, eIF4E-binding protein, and a concomitant increase in eIF4E association with eIF4G immediately after birth, and polysome profiles confirmed a decrease in abundance of large polysomes between FD19 and FD22, which was reversed on P1. Microarray analysis of polysomal versus total RNA from FD19, FD22, and P1 lungs was used to identify specific genes, the association of which with large polysomes changed either pre- or postnatally. RT-PCR and Northern blotting were used to confirm translational changes in selected candidate genes, including a prenatal increase in IL-18 and a postnatal decrease in regulatory subunit 2 of protein phosphatase 1. Translational regulation of IL-18 and protein phosphatase 1 regulatory (inhibitor) subunit 2 is gene-specific, as these changes contrast with the corresponding global changes in polysome abundance.

Adverse Heart–Lung Interactions in Ventilator-induced Lung Injury

Rationale: In the original 1974 in vivo study of ventilator‐induced lung injury, Webb and Tierney reported that high Vt with zero positive end‐expiratory pressure caused overwhelming lung injury, subsequently shown by others to be due to lung shear stress. Objectives: To reproduce the lung injury and edema examined in the Webb and Tierney study and to investigate the underlying mechanism thereof. Methods: Sprague‐Dawley rats weighing approximately 400 g received mechanical ventilation for 60 minutes according to the protocol of Webb and Tierney (airway pressures of 14/0, 30/0, 45/10, 45/0 cm H2O). Additional series of experiments (20 min in duration to ensure all animals survived) were studied to assess permeability (n = 4 per group), echocardiography (n = 4 per group), and right and left ventricular pressure (n = 5 and n = 4 per group, respectively). Measurements and Main Results: The original Webb and Tierney results were replicated in terms of lung/body weight ratio (45/0 > 45/10 ≈ 30/0 ≈ 14/0; P < 0.05) and histology. In 45/0, pulmonary edema was overt and rapid, with survival less than 30 minutes. In 45/0 (but not 45/10), there was an increase in microvascular permeability, cyclical abolition of preload, and progressive dilation of the right ventricle. Although left ventricular end‐diastolic pressure decreased in 45/10, it increased in 45/0. Conclusions: In a classic model of ventilator‐induced lung injury, high peak pressure (and zero positive end‐expiratory pressure) causes respiratory swings (obliteration during inspiration) in right ventricular filling and pulmonary perfusion, ultimately resulting in right ventricular failure and dilation. Pulmonary edema was due to increased permeability, which was augmented by a modest (approximately 40%) increase in hydrostatic pressure. The lung injury and acute cor pulmonale is likely due to pulmonary microvascular injury, the mechanism of which is uncertain, but which may be due to cyclic interruption and exaggeration of pulmonary blood flow.

Hydrogen Sulfide in Lung Injury: Therapeutic Hope from a Toxic Gas?

COULD a toxic gas treat injured lungs in critically ill patients? Maybe. In this issue of the journal, Faller et al. provide evidence that hydrogen sulfide is beneficial in an animal model, raising the question: might it help patients? To understand the importance of these findings, we need to understand the context. Acute respiratory distress syndrome is common, often lethal, and consumes enormous resources. Mechanical ventilation is the mainstay of supportive therapy, allowing adequate oxygenation and assisting with the increased work of breathing. Although such mechanical ventilation is acutely lifesaving, it has an attributable mortality and morbidity caused by lung injury that is initiated by cyclic distension of lung tissue. This problem, ventilator-associated lung injury, has been the subject of immense progress for the past 40 yr. Since the original in vivo model was described by Webb and Tierney in 1976, there have been several landmark mechanistic insights. One of the first such insights was the recognition of the obligatory role of neutrophils in ventilator-induced lung injury, confirming an inflammatory basis for the condition, and one of the most recent major insights describes some of the fundamental cell signaling events that propagate many forms of acute lung injury. Clinical practice has also evolved. That high tidal volumes should be avoided was recognized in several case series from the 1980s that described improved outcomes with lowered tidal volumes in premature neonates and in adults with status asthmaticus or lung injury. Ten years ago, an important prospective trial confirmed that tidal volume makes a critical difference in patients with acute respiratory distress syndrome: mortality was less in patients randomized to lower versus higher tidal volumes. Since then, clinical trial groups have expended extensive effort in testing approaches to treatment—beyond lowering tidal volumes—that the clinician could apply to populations of patients with acute respiratory distress syndrome and expect to improve outcome. Such approaches (e.g., prone positioning, increased positive end-expiratory pressure, surfactant, or inhaled nitric oxide) have certainly advanced our knowledge in the field, but they have not provided the clinician with new therapies that will necessarily improve their patients’ outcome. Whether progress in clinical trials seems swift or stalled, future advances will always depend on discovering new mechanisms that are amenable to clinical testing. Such insights might come from physiologic or molecular studies, and although molecular mechanisms are most distant from the minds of the bedside clinician, they might ultimately offer the brightest hope for testable “candidate” therapies. Faller et al. offer early signs of one such candidate. Hydrogen sulfide, a potent toxin, is a gaseous mediator that has created great excitement as a therapy for preserving organ function—and life—during suspended animation in in vivo models. Faller et al. hypothesized that because ventilator-induced lung injury involved inflammatory and apoptotic pathways, suspending such processes might decrease the injurious effects of ventilation with high tidal volume. In this important study—the first of its kind in the field— low concentrations of inhaled hydrogen sulfide (80 ppm) attenuated the key indices of lung injury in the anesthetized in vivo mouse. The injury was induced solely by high tidal volume without the confounding effects of previous injury, and in addition to reducing the histologic evidence of lung injury, hydrogen sulfide inhibited several inflammatory pathways and decreased neutrophil activation, apoptosis, and heme-oxygenase expression. A separate series of experiments confirmed that although both hydrogen sulfide and hypothermia protect against ventilator-induced lung injury and hydrogen sulfide does induce hypothermia, the protective effects of hydrogen sulfide were demonstrable when hypothermia was already induced. Thus, hydrogen sulfide was protective over and above the protective effects of hypothermia. This is potentially important because hypothermia from a therapy such as hydrogen sulfide might not result in larger animals (e.g., humans), and in situations where hypothermia exists, hydrogen sulfide might be expected to have additional benefit. Although the biologic effects of hydrogen sulfide have been described in a variety of in vivo models, the molecular mechanisms by which it elicits these responses implicate a

Hypercapnia: clinical relevance and mechanisms of action.

Purpose of reviewMultiple clinical and laboratory studies have been conducted to illustrate the effects of hypercapnia in a range of injuries, and to understand the mechanisms underlying these effects. The aim of this review is to highlight and interpret information obtained from these recent reports and discuss how they may inform the clinical context. Recent findingsIn the last decade, several important articles have addressed key elements of how carbon dioxide interacts in critical illness states. Among them the most important insights relate to how hypercapnia affects critical illness and include the effects and mechanisms of carbon dioxide in pulmonary hypertension, infection, inflammation, diaphragm dysfunction, and cerebral ischemia. In addition, we discuss molecular insights that apply to multiple aspects of critical illness. SummaryExperiments involving hypercapnia have covered a wide range of illness models with varying degrees of success. It is becoming evident that deliberate hypercapnia in the clinical setting should seldom be used, except wherever necessitated to avoid ventilator-associated lung injury. A more complete understanding of the molecular mechanisms must be established.

Dissociation of inflammatory mediators and function: experimental lung injury in nonpulmonary sepsis.

Background:Sepsis is a common indication for mechanical ventilation, which, with higher tidal volume, can cause ventilator-associated lung injury. Inflammatory mediators in the plasma or bronchoalveolar fluid are sometimes proposed as biomarkers in ICU patients. Objective:To test the hypothesis that “priming” with subthreshold sepsis in a clinically relevant model would worsen lung function, increase ventilator-induced mediator production, and differentially impact systemic vs. pulmonary mediator levels. The model used was cecal ligation and perforation modified so that alone it caused lung inflammatory responses but not injury. Methods and Main Results:Anesthetized mice were randomized to cecal ligation and perforation (vs. sham) with or without dexamethasone and 6 hrs later further randomized to: 1) sham, nonventilated, saline; 2) cecal ligation and perforation, nonventilated, saline; 3) cecal ligation and perforation, nonventilated, dexamethasone; 4) sham, high tidal volume, saline; 5) sham, high tidal volume, dexamethasone; 6) cecal ligation and perforation, high tidal volume, saline; or 7) cecal ligation and perforation, high tidal volume, dexamethasone. Mediators associated with sepsis and lung injury (cytokines: interleukin-6, tumor necrosis factor-&agr;; chemokine: keratinocyte stimulating factor) were measured in the plasma and the bronchoalveolar lavage, and lung function (compliance, oxygenation, alveolar protein leak) assessed. High tidal volume and cecal ligation and perforation increased individual bronchoalveolar lavage and plasma mediators; high tidal volume but not cecal ligation and perforation impaired lung function. Priming of high tidal volume by cecal ligation and perforation intensified plasma and bronchoalveolar lavage mediators; the plasma (but not the bronchoalveolar lavage) mediators were inhibited by dexamethasone pretreatment. Conclusions:Mediator—but not functional—responses to high tidal volume are augmented by subthreshold sepsis priming. There is important discordance among systemic and pulmonary mediators, physiologic function, and response to corticosteroids; thus, mediator levels may be incomplete surrogates for measures of lung injury or response to therapy in the context of systemic sepsis.

Cyclooxygenase inhibition in ventilator-induced lung injury.

BACKGROUND:We tested the hypothesis that inhibition of cyclooxygenase (COX) attenuates in vivo ventilator-induced lung injury (VILI) in a prospective, randomized laboratory investigation in a university-affiliated laboratory. Adult male rats were anesthetized and randomized with or without nonselective COX inhibition (ibuprofen) and were subjected to injurious mechanical ventilation (positive end-expiratory pressure = 0; peak inspiratory pressure = 21 mm Hg). METHODS:We investigated the profile of VILI (respiratory mechanics, cytokines, eicosanoids), expression of COX enzymes, and activation of nuclear factor (NF)-&kgr;B in ibuprofen- versus vehicle-treated animals. Injurious ventilation caused lung injury (i.e., decrement in compliance, tissue edema, and elevated inflammatory cytokines, eicosanoids, and COX-2). RESULTS:Pretreatment with ibuprofen that effectively inhibited eicosanoid synthesis and COX-2 activity increased survival and attenuated lung edema and decrement in respiratory mechanics. Ibuprofen had no modulatory effect on ventilator-induced activation of NF-&kgr;B or inflammatory cytokines (tumor necrosis factor-&agr;, interleukin [IL]-1&bgr;, IL-6, GRO/KC [growth-related oncogene/keratinocyte chemoattractant]). COX activity seems important in the pathogenesis of VILI in the in vivo rat. Inhibition of COX provides significant protection (i.e., survival, pulmonary function) in VILI, but without affecting levels of important mediators (tumor necrosis factor-&agr;, IL-1&bgr;, IL-6, GRO/KC) or activation of NF-&kgr;B. CONCLUSIONS:These data confirm that nonselective COX inhibition provides partial protection against VILI and that the NF-&kgr;B signaling pathway is not exclusively eicosanoid dependent. Studies of COX inhibition in ventilator-associated lung injury might benefit from multimodal targeting that includes a comprehensive focus on inflammatory cytokines and NF-&kgr;B.