Acute Lung Injury Edema Research Articles

BRD3308 suppresses macrophage oxidative stress and pyroptosis via upregulating acetylation of H3K27 in sepsis-induced acute lung injury

Abstract Background Sepsis-induced acute lung injury (ALI) leads to severe hypoxemia and respiratory failure, contributing to poor prognosis in septic patients. Endotoxin dissemination triggers oxidative stress and the release of inflammatory cytokines in macrophages, initiating diffuse alveolar damage. The role of epigenetic histone modifications in organ injury is increasingly recognized. The present study aimed to investigate the use of a histone modification inhibitor to alleviate sepsis-induced ALI, revealing a new strategy for improving sepsis patient survival. Methods In vivo models of ALI were established through the intraperitoneal injection of lipopolysaccharide and cecal ligation and puncture surgery. Furthermore, the disease process was simulated in vitro by stimulating Tamm-Horsfall protein-1 (THP-1) cells with lipopolysaccharide. Hematoxylin and eosin staining, blood gas analysis and pulmonary function tests were utilized to assess the extent of lung tissue damage. Western blot analysis, real-time polymerase chain reaction, enzyme-linked immunosorbent assay and immunofluorescence were used to measure the levels and distribution of the indicated indicators within cells and tissues. Reactive oxygen species and autophagic flux alterations were detected using specific probes. Results BRD3308, which is a inhibitor of histone deacetylase 3, improved lung tissue damage, inflammatory infiltration and edema in ALI by inhibiting Nod-like receptor protein3-mediated pyroptosis in macrophages. By upregulating autophagy, BRD3308 improved the disruption of redox balance in macrophages and reduced the accumulation of reactive oxygen species. Mechanistically, BRD3308 inhibited histone deacetylase 3 activity by binding to it and altering its conformation. Following histone deacetylase 3 inhibition, acetylation of H3K27 was significantly increased. Moreover, the increase in H3K27Ac led to the upregulation of autophagy-related gene 5, a key component of autophagosomes, thereby activating autophagy. Conclusions BRD3308 inhibits oxidative stress and pyroptosis in macrophages by modulating histone acetylation, thereby preventing sepsis-induced ALI. The present study provides a potential strategy and theoretical basis for the clinical treatment of sepsis-induced ALI.

EGR-1 Contributes to Pulmonary Edema by Regulating the Epithelial Sodium Channel in Lipopolysaccharide-Induced Acute Lung Injury

ABSTRACT Acute lung injury (ALI) is a common lung disease with increasing morbidity and mortality rates due to the lack of specific drugs. Impaired alveolar fluid clearance (AFC) is a primary pathological feature of ALI. Epithelial sodium channel (ENaC) is a primary determinant in regulating the transport of Na+ and the clearance of alveolar edema fluid. Therefore, ENaC is an important target for the development of drugs for ALI therapy. However, the role of ENaC in the progression of ALI remains unclear. Inhibition of early growth response factor (EGR-1) expression has been reported to induce a protective effect on ALI; therefore, we evaluated whether EGR-1 participates in the progression of ALI by regulating ENaC-α in alveolar epithelium. We investigated the potential mechanism of EGR-1-mediated regulation of ENaC in ALI. We investigated whether EGR-1 aggravates the pulmonary edema response in ALI by regulating ENaC. ALI mouse models were established by intrabronchial injection of lipopolysaccharides (LPS). Lentiviruses with EGR-1 knockdown were transfected into LPS-stimulated A549 cells. We found that EGR-1 expression was upregulated in the lung tissues of ALI mice and in LPS-induced A549 cells, and was negatively correlated with ENaC-α expression. Knockdown of EGR-1 increased ENaC-α expression and relieved cellular edema in ALI. Moreover, EGR-1 regulated ENaC-α expression at the transcriptional level, and correspondingly promoted pulmonary edema and aggravated ALI symptoms. In conclusion, our study demonstrated that EGR-1 could promote pulmonary edema by downregulating ENaC-α at the transcriptional level in ALI. Our study provides a new potential therapeutic strategy for treatment of ALI.

Calpain Promotes LPS-induced Lung Endothelial Barrier Dysfunction via Cleavage of Talin.

Acute lung injury (ALI) is characterized by lung vascular endothelial cell (EC) barrier compromise resulting in increased endothelial permeability and pulmonary edema. The infection of gram-negative bacteria that produce toxins like LPS is one of the major causes of ALI. LPS activates Toll-like receptor 4, leading to cytoskeleton reorganization, resulting in lung endothelial barrier disruption and pulmonary edema in ALI. However, the signaling pathways that lead to the cytoskeleton reorganization and lung microvascular EC barrier disruption remain largely unexplored. Here we show that LPS induces calpain activation and talin cleavage into head and rod domains and that inhibition of calpain attenuates talin cleavage, RhoA activation, and pulmonary EC barrier disruption in LPS-treated human lung microvascular ECs invitro and lung EC barrier disruption and pulmonary edema induced by LPS in ALI invivo. Moreover, overexpression of calpain causes talin cleavage and RhoA activation, myosin light chain (MLC) phosphorylation, and increases in actin stress fiber formation. Furthermore, knockdown of talin attenuates LPS-induced RhoA activation and MLC phosphorylation and increased stress fiber formation and mitigates LPS-induced lung microvascular endothelial barrier disruption. Additionally, overexpression of talin head and rod domains increases RhoA activation, MLC phosphorylation, and stress fiber formation and enhances lung endothelial barrier disruption. Finally, overexpression of cleavage-resistant talin mutant reduces LPS-induced increases in MLC phosphorylation in human lung microvascular ECs and attenuates LPS-induced lung microvascular endothelial barrier disruption. These results provide the first evidence that calpain mediates LPS-induced lung microvascular endothelial barrier disruption in ALI via cleavage of talin.

Ulinastatin alleviates pulmonary edema by reducing pulmonary permeability and stimulating alveolar fluid clearance in a rat model of acute lung injury.

Objective(s):Previous studies have shown that ulinastatin (UTI) alleviates pulmonary edema in acute lung injury (ALI) caused by lipopolysaccharide (LPS), although the mechanism behind this action is uncertain. This research aimed to identify the fundamental mechanism by which UTI alleviates pulmonary edema.Materials and Methods:We established a model of acute lung injury (ALI) in rats by using LPS as the inciting agent.The control, LPS, and LPS+UTI groups were each comprised of a specific number of randomly selected Wistar rats. We evaluated lung injury and determined pulmonary edema. The concentrations of TNF-α, IL-1β and IL-6 in BALF and the expression levels of α1Na, k-ATPase, β1Na, K-AtPase, α-ENaC, β-ENaC, γ-ENaC, Zonula occludens (ZO)-1, Occludin, Caludin-5, PI3K, Akt, TLR4, MyD88 and NF-ƘBwere identified in lung tissues.Results:The presence of UTI was associated with a reduction in lung pathological alterations, lung injury scores, the lung W/D ratio, and MPO activity, in addition to the improved gas exchange (P<0.01). Furthermore, UTI alleviated EB leakage and stimulated AFC (P<0.01). Importantly, UTI increased the expression of ZO-1, Occludin, Caludin-5, α1Na, K-ATPase, β1Na, K-AtPase, α-ENaC, β-ENaC, and γ-ENaC (P<0.01). Furthermore, UTI inhibited the inflammatory response, enhanced the expression of PI3K and Akt and hindered TLR4, MyD88, and NF-ƘB expression (P<0.01) in lung tissues.Conclusion: Our results demonstrated that UTI attenuated pulmonary edema by reducing pulmonary permeability and promoting AFC through inhibiting the inflammatory response, and the mechanism is related to promoting PI3K/Akt signaling pathways and suppressing TLR4/MyD88/NF-ƘB signaling pathways.

Inhibition of Inflammation and Regulation of AQPs/ENaCs/Na+-K+-ATPase Mediated Alveolar Fluid Transport by Total Flavonoids Extracted From Nervilia fordii in Lipopolysaccharide-induced Acute Lung Injury.

Aims: The occurrence of vascular permeability pulmonary edema in acute lung injury (ALI) is related to the imbalance of alveolar fluid transport. Regulating the active transport of alveolar fluid by aquaporins (AQPs), epithelial sodium channels (ENaCs), and Na+-K+-ATPase can effectively reduce the edema fluid in the alveolar cavity and protect against ALI. We evaluated the therapeutic effects of total flavonoids, extracted from Nervilia fordii (TFENF), and investigated its potential mechanisms of alveolar fluid transport in a rat ALI model. Materials and methods: A model of lipopolysaccharide (LPS, 5 mg/kg)-induced ALI was established in Sprague-Dawley (SD) rats through the arteriae dorsalis penis. SD rats were divided into six groups, including the vehicle, LPS model, TFENF (6 mg/kg, 12 mg/kg, 24 mg/kg), and dexamethasone group (DEX group, 5 mg/kg). The wet-to-dry (W/D) lung weight ratio, oxygenation index, and histopathological observation were used to evaluate the therapeutic effect of TFENF. The mRNA expression of AQPs, ENaCs, and pro-inflammatory cytokines was determined using real-time polymerase chain reaction, whereas protein expression was determined using immunohistochemistry. The Na + -K + -ATPase activity was assessed using enzyme-linked immunosorbent assay. Results: LPS significantly stimulated the production of inflammatory mediators including tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and disrupted the water transport balance in the alveolar cavity by inhibiting AQPs/ENaCs/Na + -K + -ATPase. Pretreatment with TFENF reduced the pathological damage and W/D ratio of the lungs and ameliorated the arterial blood oxygen partial pressure (PaO2) and oxygenation index. TFENF further decreased the mRNA level of TNF-α and IL-1β; increased the expression of AQP-1, AQP-5, αENaC, and βENaC; and increased Na + -K + -ATPase activity. Moreover, the regulation of AQPs, βENaC, and Na + -K + -ATPase and the inhibition of TNF-α and IL-1β by TFENF were found to be dose dependent. Conclusion: TFENF protects against LPS-induced ALI, at least in part, through the suppression of inflammatory cytokines and regulation of the active transport capacity of AQPs/ENaCs/Na + -K + -ATPase. These findings suggest the therapeutic potential of TFENF as phytomedicine to treat inflammation and pulmonary edema in ALI.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

Dysregulation of ion transport in the lung epithelium infected with SARS-CoV-2.

RBM3 Increases Cell Survival but Disrupts Tight Junction of Microvascular Endothelial Cells in Acute Lung Injury

BackgroundRNA-binding motif protein 3 (RBM3) is an important cold shock protein, which also responds to hypothermia or hypoxia. RBM3 is involved into multiple physiologic processes, such as promoting cell survival. However, its expression and function in acute lung injury (ALI) have not been reported. MethodsA mouse ALI model was established by lipopolysaccharides (LPS) treatment. The RBM3 and cold inducible RNA-binding protein mRNA levels were examined by RT-qPCR, and MMP9 mRNA stability was determined by actinomycin D assay. RBM3 and MMP9 mRNA was tested by RNA immunoprecipitation (RIP assay). RBM3 overexpression or silent stable cell lines were established using recombinant lentivirus and subsequently used for cell survival and tight junction measurements. ResultsIn this study, we found that RBM3, rather than cold inducible RNA-binding protein, was upregulated in lung tissue of ALI mice. RBM3 was increased in human pulmonary microvascular endothelial cells (HPMVECs) in response to LPS treatment, which is modulated by the NF-κB signaling pathway. Furthermore, RBM3 could reduce cell apoptosis induced by LPS, probably through suppressing p53 expression. Because increased permeability of HPMVECs leads to pulmonary edema in ALI, we subsequently examined the effect of RBM3 on cell tight junctions. Unexpectedly, RBM3 decreased the expression of tight junction protein zonula occludens-1 and increased cell permeability, and RBM3 overexpression increased MMP9 mRNA stability. Furthermore, RIP assay confirmed the interaction between RBM3 and MMP9 mRNA, possibly explaining the contribution of RBM3 to increase cell permeability. ConclusionsRBM3 seems to act as a "double-edged sword" in ALI, that RBM3 alleviates cell apoptosis but increases HPMVEC permeability in ALI.

Irisin alleviates pulmonary epithelial barrier dysfunction in sepsis-induced acute lung injury via activation of AMPK/SIRT1 pathways

ObjectiveAlveolar epithelial barrier dysfunction in response to inflammatory reaction contributes to pulmonary edema in acute lung injury(ALI).Irisin,a newly-found myokine,exerts the anti-inflammatory effects. This study aims to investigate the protective effects of irisin on lipopolysaccharide (LPS)-induced ALIin vivo and in vitro, and to explore its underlying mechanism. MethodsMale SD rats and A549 cells were divided into 4 groups: control group, LPS group, Irisin pretreated group, and Irisin/Compound C(a special inhibitor of AMPK)-treated group. The ALI model was established by intravenous injection of LPS in rats, and LPS challenge in A549 cells. Pulmonary specimens were harvested for microscopic examination of the pathological changes, and the expression of AMPK,SIRT1,NF-κB, p66Shc and caspase-3 in lung tissues. The pulmonary permeability were examined by wet/dry lung weight ratio(W/D) and lung permeability index(LPI). The apoptotic index, and the expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), monocyte chemoattractant activating protein-1 (MCP-1), tight junctions (occludin,ZO-1) were determined both in lung tissue and A549 cells. ResultsIrisin alleviated lung histological changes and decreased pulmonary microvascular permeability in LPS-induced rats. Irisin up-regulated the expression of occludin, ZO-1,AMPK,SIRT1, down-regulated the expression of TNF-α,IL-1β,MCP-1,NF-κB, p66Shc caspase-3, and decreased the apoptotic index in LPS-induced rats and A549 cells. All these protective effects of irisin could be reversed by Compound C. ConclusionIrisin improved LPS-induced alveolar epithelial barrier dysfunction via suppressing inflammation and apoptosis, and this protective effect might be mediated by activating AMPK/SIRT1 pathways.

New insights into the mechanisms of pulmonary edema in acute lung injury.

Appearance of alveolar protein-rich edema is an early event in the development of acute respiratory distress syndrome (ARDS). Alveolar edema in ARDS results from a significant increase in the permeability of the alveolar epithelial barrier, and represents one of the main factors that contribute to the hypoxemia in these patients. Damage of the alveolar epithelium is considered a major mechanism responsible for the increased pulmonary permeability, which results in edema fluid containing high concentrations of extravasated macromolecules in the alveoli. The breakdown of the alveolar-epithelial barrier is a consequence of multiple factors that include dysregulated inflammation, intense leukocyte infiltration, activation of pro-coagulant processes, cell death and mechanical stretch. The disruption of tight junction (TJ) complexes at the lateral contact of epithelial cells, the loss of contact between epithelial cells and extracellular matrix (ECM), and relevant changes in the communication between epithelial and immune cells, are deleterious alterations that mediate the disruption of the alveolar epithelial barrier and thereby the formation of lung edema in ARDS.

GLP-1 Analogue Liraglutide Enhances SP-A Expression in LPS-Induced Acute Lung Injury through the TTF-1 Signaling Pathway.

The reduction of pulmonary surfactant (PS) is essential for decreased pulmonary compliance and edema in acute lung injury (ALI). Thyroid transcription factor-1 (TTF-1) plays a major role in the regulation of surfactant protein-A (SP-A), the most abundant protein component of PS. Simultaneously, the glucagon-like peptide-1 (GLP-1) analogue can enhance SP-A expression in the lung. However, the underlying mechanism is still unknown. The purpose of this study was to explore whether liraglutide, a GLP-1 analogue, upregulates SP-A expression through the TTF-1 signaling pathway in ALI. In vivo, a murine model of ALI was induced by lipopolysaccharide (LPS). Pulmonary inflammation, edema, insulin level, ultrastructural changes in type II alveolar epithelial (ATII) cells, and SP-A and TTF-1 expression were analyzed. In vitro, rat ATII cells were obtained. SP-A and TTF-1 expression in cells was measured. ShRNA-TTF-1 transfection was performed to knock down TTF-1 expression. Our data showed that LPS-induced lung injury and increase in insulin level, and LPS-induced reduction of SP-A and TTF-1 expression in both the lung and cells, were significantly compromised by liraglutide. Furthermore, we also found that these effects of liraglutide were markedly blunted by shRNA-TTF-1. Taken together, our findings suggest that liraglutide enhances SP-A expression in ATII cells and attenuates pulmonary inflammation in LPS-induced ALI, most likely through the TTF-1 signaling pathway.

Gene expression of the concentration-sensitive sodium channel is suppressed in lipopolysaccharide-induced acute lung injury in mice

ABSTRACTPurpose: The concentration-sensitive sodium channel (NaC) is expressed in alveolar type II epithelial cells and pulmonary microvascular endothelial cells in mouse lungs. We recently reported that NaC contributes to amiloride-insensitive sodium transport in mouse lungs (Respiratory Physiology & Neurobiology, 2016). However, details regarding its physiological role in the lung remain unknown. To examine whether NaC is involved in alveolar fluid clearance during an acute lung injury (ALI), we analyzed the relationship between NaC gene expression in the lung and the development of pulmonary edema in lipopolysaccharide (LPS)-induced ALI mice. Methods: LPS-induced ALI mice were prepared by the intratracheal administration of LPS. Bronchoalveolar lavage (BAL) neutrophils and lung water content (LWCs) were used as a marker of ALI and pulmonary edema, respectively. NaC protein production in the lung was detected by immunoblotting and immunofluorescence. The gene expressions of NaC and the epithelial sodium channel (ENaC) of LPS-induced ALI mice were examined by quantitative RT-PCR over a time course of 14 days. Results: The BAL neutrophil count increased until day 2 after LPS administration and had nearly recovered by day 6. LWCs in LPS-induced mice gradually increased until day 8 and had recovered by day 14. The expression of the NaC protein in the lungs of LPS-induced mice dramatically decreased from day 2 to day 6, but recovered by day 8. The mRNA expression of NaC decreased in the lung, as well as those for α-, β-, and γ-ENaC during ALI. Thus, NaC expression is suppressed during the development stage of pulmonary edema and then recovers in the convalescent phase. Conclusion: Our results suggest that suppression of the gene expression of NaC is involved in the development of pulmonary edema in ALI.

Electrical impedance tomography (EIT) for quantification of pulmonary edema in acute lung injury

BackgroundAssessment of pulmonary edema is a key factor in monitoring and guidance of therapy in critically ill patients. To date, methods available at the bedside for estimating the physiologic correlate of pulmonary edema, extravascular lung water, often are unreliable or require invasive measurements. The aim of the present study was to develop a novel approach to reliably assess extravascular lung water by making use of the functional imaging capabilities of electrical impedance tomography.MethodsThirty domestic pigs were anesthetized and randomized to three different groups. Group 1 was a sham group with no lung injury. Group 2 had acute lung injury induced by saline lavage. Group 3 had vascular lung injury induced by intravenous injection of oleic acid. A novel, noninvasive technique using changes in thoracic electrical impedance with lateral body rotation was used to measure a new metric, the lung water ratioEIT, which reflects total extravascular lung water. The lung water ratioEIT was compared with postmortem gravimetric lung water analysis and transcardiopulmonary thermodilution measurements.ResultsA significant correlation was found between extravascular lung water as measured by postmortem gravimetric analysis and electrical impedance tomography (r = 0.80; p < 0.05). Significant changes after lung injury were found in groups 2 and 3 in extravascular lung water derived from transcardiopulmonary thermodilution as well as in measurements derived by lung water ratioEIT.ConclusionsExtravascular lung water could be determined noninvasively by assessing characteristic changes observed on electrical impedance tomograms during lateral body rotation. The novel lung water ratioEIT holds promise to become a noninvasive bedside measure of pulmonary edema.

Abstract 337: VE-Cadherin Mechanotransduction Regulates Lung Endothelial Contractility and Barrier Integrity

Dysfunctional regulation of endothelial adherens junctions is the direct underlying cause of vascular leak and pulmonary edema in acute lung injury. Indirect evidence suggests that cytoskeletal rearrangements are linked to adherens junction remodeling, but there is no clear demonstration that adherens junction proteins play an active role in the regulation of intracellular or extracellular mechanical tension. Here I investigate mechanotransduction (response to applied force) of VE-cadherin, the main structural protein at adherens junctions, and how this regulates global cell contractility and actin remodeling both locally and globally. Mechanical responses in human pulmonary artery endothelial cells were studied using magnetic twisting cytometry (MTC) experiments, in which shear stress was exerted on specific cell surface receptors by twisting magnetized beads bound to the cell surface. Remodeling of adherens junctions and F-actin in response to mechanical force was visualized by immunofluorescence. Confocal microscopy revealed force-actuated changes in both local and global cytoskeletal organization, as well as local rearrangements at both bead-cell and cell-cell junctions. This force-dependent remodeling correlated directly with the stiffening of VE-cadherin junctions with increasing applied stress. Treatment with cytoskeletal inhibitors significantly diminished this response. When a constant magnitude of shear stress was applied continuously over time, VE-cadherin junctions increased stiffness, suggesting active junction reinforcement. These data provide direct evidence that VE-cadherin complexes are tension sensors, and that associated mechanotransduction regulates global cell mechanics. Moreover, I present data showing that cadherin adhesions across the cell monolayer form a mechanically coupled network that regulates the endothelial barrier in response to force. This suggests a new mechanism regulating endothelial monolayer integrity.

The Fas/FasL pathway impairs the alveolar fluid clearance in mouse lungs

Alveolar epithelial damage is a critical event that leads to protein-rich edema in acute lung injury (ALI), but the mechanisms leading to epithelial damage are not completely understood. Cell death by necrosis and apoptosis occurs in alveolar epithelial cells in the lungs of patients with ALI. Fas activation induces apoptosis of alveolar epithelial cells, but its role in the formation of lung edema is unclear. The main goal of this study was to determine whether activation of the Fas/Fas ligand pathway in the lungs could alter the function of the lung epithelium, and the mechanisms involved. The results show that Fas activation alters the alveolar barrier integrity and impairs the ability of the lung alveolar epithelium to reabsorb fluid from the air spaces. This result was dependent on the presence of a normal Fas receptor and was not affected by inflammation induced by Fas activation. Alteration of the fluid transport properties of the alveolar epithelium was partially restored by β-adrenergic stimulation. Fas activation also caused apoptosis of alveolar endothelial cells, but this effect was less pronounced than the effect on the alveolar epithelium. Thus, activation of the Fas pathway impairs alveolar epithelial function in mouse lungs by mechanisms involving caspase-dependent apoptosis, suggesting that targeting apoptotic pathways could reduce the formation of lung edema in ALI.

TNF-Induced Death Signaling Triggers Alveolar Epithelial Dysfunction in Acute Lung Injury

The ability of the alveolar epithelium to prevent and resolve pulmonary edema is a crucial determinant of morbidity and mortality in acute lung injury (ALI). TNF has been implicated in ALI pathogenesis, but the precise mechanisms remain undetermined. We evaluated the role of TNF signaling in pulmonary edema formation in a clinically relevant mouse model of ALI induced by acid aspiration and investigated the effects of TNF p55 receptor deletion, caspase-8 inhibition, and alveolar macrophage depletion on alveolar epithelial function. We found that TNF plays a central role in the development of pulmonary edema in ALI through activation of p55-mediated death signaling, rather than through previously well-characterized p55-mediated proinflammatory signaling. Acid aspiration produced pulmonary edema with significant alveolar epithelial dysfunction, as determined by alveolar fluid clearance (AFC) and intra-alveolar levels of the receptor for advanced glycation end-products. The impairment of AFC was strongly correlated with lung caspase-8 activation, which was localized to type 1 alveolar epithelial cells by flow cytometric analysis. p55-deficient mice displayed markedly attenuated injury, with improved AFC and reduced caspase-8 activity but no differences in downstream cytokine/chemokine production and neutrophil recruitment. Caspase-8 inhibition significantly improved AFC and oxygenation, whereas depletion of alveolar macrophages attenuated epithelial dysfunction with reduced TNF production and caspase-8 activity. These results provide in vivo evidence for a novel role for TNF p55 receptor-mediated caspase-8 signaling, without substantial apoptotic cell death, in triggering alveolar epithelial dysfunction and determining the early pathophysiology of ALI. Blockade of TNF-induced death signaling may provide an effective early-phase strategy for ALI.

Epigallocatechin‐3‐Gallate inhibits the effects of TGF‐Beta and IL‐1B on ENaC expression in human alveolar epithelial cells.

Acute Lung Injury (ALI) is major clinical problem in the United States, affecting over 200,000 Americans a year. Increased mortality in ALI is attributed to pulmonary edema and decreased net fluid transport. Previous clinical studies from ALI patients have correlated the decrease net fluid transport with decreased expression of epithelium sodium channels (ENaC). Therefore drugs that enhance ENaC expression offer considerable therapeutic promise in ALI. Epigallocatechin‐3‐Gallate (EGCG), a green tea extract, has been shown to have potent anti‐inflammatory effects. However, its effect on ENaC expression in response to proinflammatory cytokines in ALI has not been shown. In this study, we investigated the hypothesis that EGCG rescues human alveolar epithelial cell (A549) ENaC expression following proinflammatory cytokine (IL‐1β and TGFβ) treatment. To test this, A549 cells were treated with TGF‐β and IL‐1β in the presence or absence of EGCG. Cell lysates as well as supernatants were collected for protein analysis. EGCG treatments resulted in increased expression of the alpha and beta subunits of ENaC in response to both TGF‐ β and IL‐ β. Further, EGCG treatments resulted in decreased cytokine secretion mediated through decreased NF‐kappaB and p‐38 MAP kinase activity in comparison to control. Our data suggests that EGCG up regulates ENaC, a major sodium channel involved in resolution of pulmonary edema in ALI.

MicroRNA 16 modulates epithelial sodium channel in human alveolar epithelial cells

Acute lung injury (ALI) is a devastating disease characterized by pulmonary edema. Removal of edema from the air spaces of lung is a critical function of the epithelial sodium channel (ENaC) in ALI. The molecular mechanisms behind resolution of pulmonary edema are incompletely understood. MicroRNA’s (miRNA) are crucial gene regulators and are dysregulated in various diseases including ALI. Recent studies suggest that microRNA-16 (miR-16) targets serotonin transporter (SERT) involved in the serotonin (5-HT) transmitter system. Alterations in serotonin levels have been reported in various pulmonary diseases. However, the role of miR-16 on its target SERT, and ENaC, a key ion channel involved in the resolution of pulmonary edema, have not been studied. In the present study, the expression patterns of miR-16, SERT, ENaC and serotonin were investigated in mice exposed to room air and hyperoxia. The effects of miR-16 overexpression on ENaC, SERT, TGF-β and Nedd4 in human alveolar epithelial cells were analyzed. miR-16 and ENaC were downregulated in mice exposed to hyperoxia. miR-16 downregulation in mouse lung was correlated with an increase in SERT expression and pulmonary edema. Overexpression of miR-16 in human alveolar epithelial cells (A549) suppressed SERT and increased ENaCβ levels when compared to control-vector transfected cells. In addition, miR-16 over expression suppressed TGFβ release, a critical inhibitor of ENaC. Interestingly Nedd4, a negative regulator of ENaC remained unaltered in miR-16 over expressed A549 cells when compared to controls. Taken together, our data suggests that miR-16 upregulates ENaC, a major sodium channel involved in resolution of pulmonary edema in ALI.

Derecruitment Test and Surfactant Therapy in Patients with Acute Lung Injury

Introduction. A recruitment maneuver (RM) may improve gas exchange in acute lung injury (ALI). The aim of our study was to assess the predictive value of a derecruitment test in relation to RM and to evaluate the efficacy of RM combined with surfactant instillation in patients with ALI. Materials and Methods. Thirteen adult mechanically ventilated patients with ALI were enrolled into a prospective pilot study. The patients received protective ventilation and underwent RM followed by a derecruitment test. After a repeat RM, bovine surfactant (surfactant group, n = 6) or vehicle only (conventional therapy group, n = 7) was instilled endobronchially. We registered respiratory and hemodynamic parameters, including extravascular lung water index (EVLWI). Results. The derecruitment test decreased the oxygenation in 62% of the patients. We found no significant correlation between the responses to the RM and to the derecruitment tests. The baseline EVLWI correlated with changes in SpO2 following the derecruitment test. The surfactant did not affect gas exchange and lung mechanics but increased EVLWI at 24 and 32 hrs. Conclusions. Our study demonstrated no predictive value of the derecruitment test regarding the effects of RM. Surfactant instillation was not superior to conventional therapy and might even promote pulmonary edema in ALI.

Caveolin-1 siRNA Increases the Pulmonary Microvascular and Alveolar Epithelial Permeability in Rats

Increased pulmonary microvascular and epithelial permeability are important contributors to pulmonary edema in acute lung injury. In this study, we used small interfering RNA (siRNA) to knock down caveolin-1 expression in rat lungs and to confirm the important role of caveolin-1 in regulating pulmonary edema. After pulmonary injection of siRNA against caveolin-1 messenger RNA incorporated in liposomes with three concentrations of 0.4, 0.8, and 1.2 mg/kg, the gene silencing rate and the effects of caveolin-1 siRNA on aquaporin (AQP)-1, AQP-5, and epithelial sodium channel (ENaC) were detected. For pulmonary permeability analysis, Evans blue fluorimetry, ratios of albumin concentrations between blood and bronchoalveolar lavage, and wet/dry weight ratios were measured. The impacts of caveolin-1 suppression on interendothelial junctions were evaluated by the performance of electron microscopy and the analysis of vascular endothelial (VE)-cadherin Western blot. Alveolar wall thickness analysis and chest fluoroscopy were performed to determine the pulmonary edema degree. After 72 hours of injection, the gene silencing rate of caveolin-1 siRNA is about 87%. AQP-1, AQP-5, ENaC-α, ENaC-β, ENaC-γ, and VE-cadherin protein levels were decreased by 63%, 66%, 80%, 90%, 89%, and 50%, respectively. Caveolin-1 siRNA also resulted in increasing microvascular and epithelial permeability and pulmonary edema. These data suggest that caveolin-1 plays an important part in regulating the pulmonary permeability by modifying AQP-1, AQP-5, ENaC, and VE-cadherin.

A Sigh of Relief About Treating Influenza in Individuals With Alcohol-Use Disorders?

The recent outbreak of so-called “swine flu,” or influenza caused by an H1N1 strain of the influenza A virus, has renewed the public fear (as well as within the medical community) about pandemic influenza. However, we must all remember that seasonal epidemic influenza has always been a serious public health concern. For example, an average of more than 41,000 per year died of influenza in the United States alone between 1979 and 2001 (Dushoff et al., 2006). Although annual vaccinations remain the foundation of public health efforts to limit the severity of seasonal epidemics, many unvaccinated individuals are either exposed to the virus or present with frank influenza illness and are treated with neuraminidase inhibitors such as oseltamivir (trade name Tamiflu) to reduce symptoms and/or transmission of the virus (Moscona, 2005). Although it has long been known that individuals with chronic alcohol-use disorders are at increased risk of a wide variety of community-acquired lung infections, there are no specific studies that have examined the role of alcohol-use disorders on the risks of acquiring and/or spreading influenza. However, studies in animal models have raised concerns; in particular, a study by Legge and colleagues showed that chronic alcohol consumption increased the mortality of influenza infection in a murine model (Meyerholz et al., 2008). In parallel, our group has shown that chronic alcohol ingestion increases the severity of acute edematous lung injury by impairing the normal epithelial barrier in the lung and placing an increased burden on the active transport of sodium and water from the airways to keep the airspaces dry (Guidot et al., 2000; Otis et al., 2008). In parallel, we also determined that the influenza virus interferes with this active fluid transport in the lung (Chen et al., 2004). Taken together, these experimental findings suggest that even otherwise healthy individuals with a chronic alcohol-use disorder could be at increased risk of acute edematous lung injury and death from influenza. Unfortunately, these subjects are also less likely to receive annual vaccinations, and therefore, their survival could depend on the ability of drugs such as oseltamivir to limit the acute illness. In this regard, the findings by Legge and colleagues (Langlois et al.) in the current issue provide some cautious optimism from their murine model that, at least in otherwise healthy subjects with underlying chronic alcohol use, oseltamivir might remain effective in limiting the severity of acute influenza illness. Specifically, mice given ethanol for 8 weeks in their drinking water and then inoculated with influenza virus had a 60% mortality when compared to a 15% mortality in control mice. In contrast, oseltamivir treatment markedly decreased morbidity and completely prevented any mortality in both control and ethanol-treated mice. Consistent with the elimination of mortality, oseltamivir decreased viral shedding and lung inflammation in both groups. Although one must obviously interpret these experimental findings cautiously, this study suggests that neuraminidase inhibitors retain their ability to limit the severity of acute influenza pneumonia even in the context of chronic alcohol ingestion. Although it appears for now that the extent and severity of the current H1N1 epidemic has been far less than initially feared, public health officials and clinicians can never drop their guard as epidemic seasonal influenza and intermittent (and unpredictable) pandemics are inevitable. For now, this new study suggests we can remain hopeful that antiviral drugs used to limit the spread of influenza in unvaccinated individuals will retain their efficacy even for those who suffer from chronic alcohol-use disorders.