Impaired Alveolar Fluid Clearance Research Articles

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.

Insulin reverses impaired alveolar fluid clearance in ARDS by inhibiting LPS-induced autophagy and inflammatory.

Until now, acute respiratory distress syndrome (ARDS) has been a difficult clinical condition with a high mortality and morbidity rate, and is characterized by a build-up of alveolar fluid and impaired clearance. The underlying mechanism is not yet fully understood and no effective medications available. Autophagy activation is associated with ARDS caused by different pathogenic factors. It represents a new direction of prevention and treatment of ARDS to restrain autophagy to a reasonable level through pharmacological and molecular genetic methods. Na, K-ATPase is the main gradient driver of pulmonary water clearance in ARDS and could be degraded by the autophagy-lysosome pathway to affect its abundance and enzyme activity. As a normal growth hormone in human body, insulin has been widely used in clinical for a long time. To investigate the association of insulin with Na, K-ATPase, autophagy and inflammatory markers in LPS-treated C57BL/6 mice by survival assessment, proteomic analysis, histologic examination, inflammatory cell counting, myeloperoxidase, TNF-α and IL-1β activity analysis etc. This was also verified on mouse alveolar epithelial type II (AT II) and A549 cells by transmission electron microscopy. We found that insulin restored the expression of Na, K-ATPase, inhibited the activation of autophagy and reduced the release of inflammatory factors caused by alveolar epithelial damage. The regulation mechanism of insulin on Na, K-ATPase by inhibiting autophagy function may provide new drug targets for the treatment of ARDS.

Upregulation of alveolar fluid clearance is not sufficient for Na+,K+-ATPase β subunit-mediated gene therapy of LPS-induced acute lung injury in mice

Acute Lung Injury/Acute Respiratory Distress Syndrome (ALI/ARDS) is characterized by diffuse alveolar damage and significant edema accumulation, which is associated with impaired alveolar fluid clearance (AFC) and alveolar‐capillary barrier disruption, leading to acute respiratory failure. Our previous data showed that electroporation‐mediated gene delivery of the Na+, K+-ATPase β1 subunit not only increased AFC, but also restored alveolar barrier function through upregulation of tight junction proteins, leading to treatment of LPS‐induced ALI in mice. More importantly, our recent publication showed that gene delivery of MRCKα, the downstream effector of β1 subunit-mediated signaling towards upregulation of adhesive junctions and epithelial and endothelial barrier integrity, also provided therapeutic potential for ARDS treatment in vivo but without necessarily accelerating AFC, indicating that for ARDS treatment, improving alveolar capillary barrier function may be of more benefit than improving fluid clearance. In the present study, we investigated the therapeutical potential of β2 and β3 subunits, the other two β isoforms of Na+, K+-ATPase, for LPS‐induced ALI. We found that gene transfer of either the β1, β2, or β3 subunits significantly increased AFC compared to the basal level in naïve animals and each gave similar increased AFC to each other. However, unlike that of the β1 subunit, gene transfer of the β2 or β3 subunit into pre-injured animal lungs failed to show the beneficial effects of attenuated histological damage, neutrophil infiltration, overall lung edema, or increased lung permeability, indicating that β2 or β3 gene delivery could not treat LPS induced lung injury. Further, while β1 gene transfer increased levels of key tight junction proteins in the lungs of injured mice, that of either the β2 or β3 subunit had no effect on levels of tight junction proteins. Taken together, this strongly suggests that restoration of alveolar-capillary barrier function alone may be of equal or even more benefit than improving AFC for ALI/ARDS treatment.

Mechanisms of impaired alveolar fluid clearance.

Impaired alveolar fluid clearance (AFC) is an important cause of alveolar edema fluid accumulation in patients with acute respiratory distress syndrome (ARDS). Alveolar edema leads to insufficient gas exchange and worse clinical outcomes. Thus, it is important to understand the pathophysiology of impaired AFC in order to develop new therapies for ARDS. Over the last few decades, multiple experimental studies have been done to understand the molecular, cellular, and physiological mechanisms that regulate AFC in the normal and the injured lung. This review provides a review of AFC in the normal lung, focuses on the mechanisms of impaired AFC, and then outlines the regulation of AFC. Finally, we summarize ongoing challenges and possible future research that may offer promising therapies for ARDS.

Dichotomous Role of Tumor Necrosis Factor in Pulmonary Barrier Function and Alveolar Fluid Clearance.

Alveolar-capillary leak is a hallmark of the acute respiratory distress syndrome (ARDS), a potentially lethal complication of severe sepsis, trauma and pneumonia, including COVID-19. Apart from barrier dysfunction, ARDS is characterized by hyper-inflammation and impaired alveolar fluid clearance (AFC), which foster the development of pulmonary permeability edema and hamper gas exchange. Tumor Necrosis Factor (TNF) is an evolutionarily conserved pleiotropic cytokine, involved in host immune defense against pathogens and cancer. TNF exists in both membrane-bound and soluble form and its mainly -but not exclusively- pro-inflammatory and cytolytic actions are mediated by partially overlapping TNFR1 and TNFR2 binding sites situated at the interface between neighboring subunits in the homo-trimer. Whereas TNFR1 signaling can mediate hyper-inflammation and impaired barrier function and AFC in the lungs, ligand stimulation of TNFR2 can protect from ventilation-induced lung injury. Spatially distinct from the TNFR binding sites, TNF harbors within its structure a lectin-like domain that rather protects lung function in ARDS. The lectin-like domain of TNF -mimicked by the 17 residue TIP peptide- represents a physiological mediator of alveolar-capillary barrier protection. and increases AFC in both hydrostatic and permeability pulmonary edema animal models. The TIP peptide directly activates the epithelial sodium channel (ENaC) -a key mediator of fluid and blood pressure control- upon binding to its α subunit, which is also a part of the non-selective cation channel (NSC). Activity of the lectin-like domain of TNF is preserved in complexes between TNF and its soluble TNFRs and can be physiologically relevant in pneumonia. Antibody- and soluble TNFR-based therapeutic strategies show considerable success in diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease, but their chronic use can increase susceptibility to infection. Since the lectin-like domain of TNF does not interfere with TNF’s anti-bacterial actions, while exerting protective actions in the alveolar-capillary compartments, it is currently evaluated in clinical trials in ARDS and COVID-19. A more comprehensive knowledge of the precise role of the TNFR binding sites versus the lectin-like domain of TNF in lung injury, tissue hypoxia, repair and remodeling may foster the development of novel therapeutics for ARDS.

Pro-Resolving Mediator Resolvin E1 Restores Alveolar Fluid Clearance in Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) is a life-threatening condition characterized by increased permeability of the alveolar-capillary barrier and impaired alveolar fluid clearance. Resolvin E1 (RvE1) is a specialized pro-resolving mediator derived endogenously from omega-3-polyunsaturated fatty acids. RvE1 (10 μg/kg i.v.) was injected to rats 6 h post-lipopolysaccharide (LPS) (14 mg/kg) induction. After another 3 h, alveolar fluid clearance was measured in live rats (n = 8-9). The primary Type II alveolar epithelial cell was isolated and treated by LPS (1 μg/mL) with or without RvE1 (250 nM). The expression of epithelial sodium channel (ENaC), Na+/K+-ATPase (NKA), AKT, serum- and glucocorticoid-induced kinase 1 (SGK1), and Nedd4-2 were detected. RvE1 improved survival rate (30% vs. 70%, P = 0.048), increased the clearance of alveolar fluid (13.34% vs. 18.73%, P < 0.001), reduced lung wet-dry weight ratio (5.01 vs. 4.63, P < 0.001), mitigated lung injury scores (13.38 vs. 7.0, P < 0.05) and inflammation in LPS-induced ARDS in rats. RvE1 upregulated alveolar ENaC and NKA expression in vivo and in vitro. In addition, RvE1 significantly increased the expression of phosphorylated AKT, SGK1, and phosphorylated Nedd4-2 in LPS-stimulated primary alveolar type II cells. The effects of RvE1 were abrogated by blocking phosphatidylinositide3'-kinase (PI3K) and SGK1 with LY294002 and GSK650394, respectively. In summary, RvE1 upregulated ENaC and NKA expression by activating PI3K/AKT/SGK1 pathway to promote alveolar fluid clearance, suggesting that RvE1 may be a potentially effective drug for ARDS treatment.

The roles of sodium-potassium-chloride cotransporter isoform-1 in acute lung injury.

Acute lung injury (ALI) is often characterized by severe lung inflammation and pulmonary edema with poor gas exchange and hypoxemia. Alveolar inflammation and water flooding are, in fact, notable features of ALI pathogenesis. The sodium-potassium-chloride co-transporter isoform 1 (NKCC1), localized at the basolateral surface of the lung epithelium, drives water transport via back transport of Na+ and Cl− to the alveolar air space. NKCC1, therefore, is crucial in regulating alveolar fluid. Increased expression of NKCC1 results in increased alveolar fluid secretion and impaired alveolar fluid clearance. During ALI, the with no lysine kinase (WNK), oxidative stress responsive kinase 1 (OSR1), and STE20/SPS1-related proline/alanine-rich kinase (SPAK) pathways are activated, which upregulates NKCC1 expression. Proinflammatory cytokines also enhance the expression of NKCC1 via c-Jun N-terminal kinase-and p38-dependent pathways. NKCC1 activation also increases the expression of proinflammatory cytokines via cell rupture and activation of macrophages. Increased proinflammatory cytokines, in turn, recruit inflammatory cells to the site of injury and cause further lung damage. Animals with high expression of NKCC1 show more severe lung injury with presentations of more severe pulmonary edema and microvascular permeability, higher expression of proinflammatory cytokines, and greater neutrophilic infiltration. In contrast, animals with low expression of NKCC1 or those treated with NKCC1 inhibitors show less severe lung injury with milder levels of presentations of ALI. These reports collectively highlight a novel role of NKCC1 in ALI pathogenesis. Manipulation of NKCC1 expression levels could, therefore, represent novel modalities for effective ALI treatment.

Hypercapnia Induces Inositol-Requiring Enzyme 1α-Driven Endoplasmic Reticulum-associated Degradation of the Na,K-ATPase β-Subunit.

Acute respiratory distress syndrome is often associated with elevated levels of CO2 (hypercapnia) and impaired alveolar fluid clearance. Misfolding of the Na,K-ATPase (NKA), a key molecule involved in both alveolar epithelial barrier tightness and resolution of alveolar edema, in the endoplasmic reticulum (ER) may decrease plasma membrane abundance of the transporter. Here, we investigated how hypercapnia affects the NKA β-subunit (NKA-β) in the ER. Exposing murine precision-cut lung slices and human alveolar epithelial A549 cells to elevated CO2 levels led to a rapid decrease of NKA-β abundance in the ER and at the cell surface. Knockdown of ER mannosidase α class 1B member 1 and ER degradation-enhancing α-mannosidase like protein 1 by siRNA or treatment with the mannosidase α class 1B member 1 inhibitor kifunensine rescued loss of NKA-β in the ER, suggesting ER-associated degradation (ERAD) of the enzyme. Furthermore, hypercapnia activated the unfolded protein response by promoting phosphorylation of inositol-requiring enzyme 1α (IRE1α), and treatment with an siRNA against IRE1α prevented the decrease of NKA-β in the ER. Of note, the hypercapnia-induced phosphorylation of IRE1α was triggered by a Ca2+-dependent mechanism. In addition, inhibition of the inositol trisphosphate receptor decreased phosphorylation levels of IRE1α in precision-cut lung slices and A549 cells, suggesting that Ca2+ efflux from the ER might be responsible for IRE1α activation and ERAD of NKA-β. In conclusion, here we provide evidence that hypercapnia attenuates maturation of the regulatory subunit of NKA by activating IRE1α and promoting ERAD, which may contribute to impaired alveolar epithelial integrity in patients with acute respiratory distress syndrome and hypercapnia.

Gene Delivery of MRCKα Repairs the Alveolar‐Capillary Barrier and Rescues the Experimental Lipopolysaccharide‐Induced Acute Lung Injury

Rationale Acute Respiratory Distress Syndrome (ARDS) is a devastating condition which is characterized by the pulmonary accumulation of protein-rich edema fluids and insufficient gas exchange, leading to acute hypoxemic respiratory failure. During ARDS, the net fluid clearance is undermined not only due to the impaired alveolar fluid clearance (AFC) which osmotically drives water following the ion gradient out of alveoli, but also the disrupted alveolar-capillary barrier. The previous data from our lab indicated that electroporation-mediated gene delivery of the β1 subunit of the Na+, K+-ATPase into mouse lungs treats LPS induced acute lung injury (ALI), showing significantly decreased lung edema, and improved overall outcome of lung injury. Overexpression of β1 increased AFC in healthy and injured murine lungs, and more importantly, gene delivery of β1 restored pulmonary barrier function, demonstrated by upregulated tight junction (TJ) proteins, and decreased lung permeability, total protein and cellularity in BAL fluid. MRCKα (CDC42 binding protein kinase alpha) was identified by our lab as an interacting partner of the β1 subunit through mass spectrometry. MRCKα is a Rho GTPase effector kinase, regulating diverse cell behaviors, including junction formation. Our previous in vitro data indicate that MRCKα mediated β1’s upregulation of TJ proteins and epithelial barrier integrity. Overexpression of MRCKα alone sufficiently increased the trans-epithelial electrical resistance (TEER) in cell culture. Thus, it is intriguing to investigate whether MRCKα has the therapeutic potential to treat LPS induced ALI by restoring pulmonary barrier function in vivo. This study tested whether electroporation-mediated gene transfer of MRCKα could upregulate TJ protein and barrier function and treat LPS induced ALI in mice. Methods ALI was induced by LPS intratracheal administration and 24 hours later, plasmids encoding MRCKα, β1- Na+, K+-ATPase, or nothing (control) were delivered individually or in combination to mouse lungs by oropharyngeal aspiration and electroporation. Two days after gene delivery, various endpoint assays were performed to evaluate lung edema, permeability, inflammation and histological injury. In addition, AFC was performed on mice with MRCKα overexpression to evaluate the fluid clearance. Results Gene transfer of MRCKα alone did not enhance AFC, but alone or in combination with β1- Na+, K+-ATPase attenuated LPS-increased Wet/Dry ratio, lung leakage, and BAL cellularity and protein concentration, restored TJ protein expression, and improved overall outcome of lung injury. Conclusions Gene delivery ofMRCKα repair alveolar capillary barrier integrity without promoting ion transport, and could benefit the pre-injured lungs by reducing pulmonary edema, restoring lung barrier function and reducing inflammation. Moreover, the data also suggest that improving barrier function alone may be of equal or even more benefit than improving alveolar fluid clearance in order to treat ALI/ARDS.

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.

Sodium-coupled neutral amino acid transporter SNAT2 counteracts cardiogenic pulmonary edema by driving alveolar fluid clearance.

The constant transport of ions across the alveolar epithelial barrier regulates alveolar fluid homeostasis. Dysregulation or inhibition of Na+ transport causes fluid accumulation in the distal airspaces resulting in impaired gas exchange and respiratory failure. Previous studies have primarily focused on the critical role of amiloride-sensitive epithelial sodium channel (ENaC) in alveolar fluid clearance (AFC), yet activation of ENaC failed to attenuate pulmonary edema in clinical trials. Since 40% of AFC is amiloride-insensitive, Na+ channels/transporters other than ENaC such as Na+-coupled neutral amino acid transporters (SNATs) may provide novel therapeutic targets. Here, we identified a key role for SNAT2 (SLC38A2) in AFC and pulmonary edema resolution. In isolated perfused mouse and rat lungs, pharmacological inhibition of SNATs by HgCl2 and α-methylaminoisobutyric acid (MeAIB) impaired AFC. Quantitative RT-PCR identified SNAT2 as the highest expressed System A transporter in pulmonary epithelial cells. Pharmacological inhibition or siRNA-mediated knockdown of SNAT2 reduced transport of l-alanine across pulmonary epithelial cells. Homozygous Slc38a2-/- mice were subviable and died shortly after birth with severe cyanosis. Isolated lungs of Slc38a2+/- mice developed higher wet-to-dry weight ratios (W/D) as compared to wild type (WT) in response to hydrostatic stress. Similarly, W/D ratios were increased in Slc38a2+/- mice as compared to controls in an acid-induced lung injury model. Our results identify SNAT2 as a functional transporter for Na+ and neutral amino acids in pulmonary epithelial cells with a relevant role in AFC and the resolution of lung edema. Activation of SNAT2 may provide a new therapeutic strategy to counteract and/or reverse pulmonary edema.

Bone marrow mesenchymal stem cell-conditioned medium facilitates fluid resolution via miR-214-activating epithelial sodium channels.

Acute lung injury (ALI) is featured with severe lung edema at the early exudative phase, resulting from the imbalance of alveolar fluid turnover and clearance. Mesenchymal stem cells (MSCs) belong to multipotent stem cells, which have shown potential therapeutic effects during ALI. Of note, MSC‐conditioned medium (MSC‐CM) improved alveolar fluid clearance (AFC) in vivo, whereas the involvement of miRNAs is seldom known. We thus aim to explore the roles of miR‐214 in facilitating MSC‐CM mediated fluid resolution of impaired AFC. In this study, AFC was increased significantly by intratracheally administrated MSC‐CM in lipopolysaccharide‐treated mice. MSC‐CM augmented amiloride‐sensitive currents in intact H441 monolayers, and increased α‐epithelial sodium channel (α‐ENaC) expression level in H441 and mouse alveolar type 2 epithelial cells. Meanwhile, MSC‐CM increased the expression of miR‐214, which may participate in regulating ENaC expression and function. Our results suggested that MSC‐CM enhanced AFC in ALI mice in vivo through a novel mechanism, involving miR‐214 regulation of ENaC.

Adenosine A2B receptor activation stimulates alveolar fluid clearance through alveolar epithelial sodium channel via cAMP pathway in endotoxin-induced lung injury.

Clinical studies have established that the capacity of removing excess fluid from alveoli is impaired in most patients with acute respiratory distress syndrome. Impaired alveolar fluid clearance (AFC) correlates with poor outcomes. Adenosine A2B receptor (A2BAR) has the lowest affinity with adenosine among four adenosine receptors. It is documented that A2BAR can activate adenylyl cyclase (AC) resulting in elevated cAMP. Based on the understanding that cAMP is a key regulator of epithelial sodium channel (ENaC), which is the limited step in sodium transport, we hypothesized that A2BAR signaling may affect AFC in acute lung injury (ALI) through regulating ENaC via cAMP, thus attenuating pulmonary edema. To address this, we utilized pharmacological approaches to determine the role of A2BAR in AFC in rats with endotoxin-induced lung injury and further focused on the mechanisms in vitro. We observed elevated pulmonary A2BAR level in rats with ALI and the similar upregulation in alveolar epithelial cells exposed to LPS. A2BAR stimulation significantly attenuated pulmonary edema during ALI, an effect that was associated with enhanced AFC and increased ENaC expression. The regulatory effects of A2BAR on ENaC-α expression were further verified in cultured alveolar epithelial type II (ATII) cells. More importantly, activation of A2BAR dramatically increased amiloride-sensitive Na+ currents in ATII cells. Moreover, we observed that A2BAR activation stimulated cAMP accumulation, whereas the cAMP inhibitor abolished the regulatory effect of A2BAR on ENaC-α expression, suggesting that A2BAR activation regulates ENaC-α expression via cAMP-dependent mechanism. Together, these findings suggest that signaling through alveolar epithelial A2BAR promotes alveolar fluid balance during endotoxin-induced ALI by regulating ENaC via cAMP pathway, raising the hopes for treatment of pulmonary edema due to ALI.

Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the β-Subunit of the Enzyme in Alveolar Epithelial Cells.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO2 levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO2 concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus. These effects were associated with a marked reduction of the plasma membrane (PM) abundance of the NKA-α/β complex as well as a decreased total and ouabain-sensitive ATPase activity. Furthermore, our study revealed that the ER-retained NKA-β subunits were only partially assembled with NKA α-subunits, which suggests that hypercapnia modifies the ER folding environment. Moreover, we observed that elevated CO2 levels decreased intracellular ATP production and increased ER protein and, particularly, NKA-β oxidation. Treatment with α-ketoglutaric acid (α-KG), which is a metabolite that has been shown to increase ATP levels and rescue mitochondrial function in hypercapnia-exposed cells, attenuated the deleterious effects of elevated CO2 concentrations and restored NKA PM abundance and function. Taken together, our findings provide new insights into the regulation of NKA in alveolar epithelial cells by elevated CO2 levels, which may lead to the development of new therapeutic approaches for patients with ARDS and hypercapnia.

In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells.

Acute respiratory distress syndrome (ARDS) is a syndrome of diffuse alveolar injury with impaired alveolar fluid clearance and severe inflammation. The use of halogenated agents, such as sevoflurane or isoflurane, for the sedation of intensive care unit (ICU) patients can improve gas exchange, reduce alveolar edema, and attenuate inflammation during ARDS. However, data on the use of inhaled agents for continuous sedation in the ICU to treat or prevent lung damage is lacking. To study the effects of halogenated agents on alveolar epithelial cells under "physiologic" conditions, we describe an easy system to culture cells at the air-liquid interface and expose them to halogenated agents to provide precise controlled "air" fractions and "medium" concentrations for these agents. We developed a sealed air-tight chamber in which plates with human alveolar epithelial immortalized cells could be exposed to a precise, controlled fraction of sevoflurane or isoflurane using a continuous gas flow provided by an anesthetic machine circuit. Cells were exposed to 4% of sevoflurane and 1% of isoflurane for 24 hours. Gas mass spectrometry was performed to determine the concentration of halogenated agents dissolved in the medium. After the first hour, the concentrations of sevoflurane and isoflurane in the medium were 251 mg/L and 25 mg/L, respectively. The curves representing the concentrations of both sevoflurane and isoflurane dissolved in the medium showed similar courses over time, with a plateau reached at one hour after exposure. This protocol was specifically designed to reach precise and controlled concentrations of sevoflurane or isoflurane in vitro to improve our understanding of mechanisms involved in epithelial lung injury during ARDS and to test novel therapies for the syndrome.

Therapeutic Implications of Human Umbilical Cord Mesenchymal Stromal Cells in Attenuating Influenza A(H5N1) Virus-Associated Acute Lung Injury.

Highly pathogenic avian influenza viruses can cause severe forms of acute lung injury (ALI) in humans, where pulmonary flooding leads to respiratory failure. The therapeutic benefits of bone marrow mesenchymal stromal cells (MSCs) have been demonstrated in a model of ALI due to influenza A(H5N1) virus. However, clinical translation is impractical and limited by a decline in efficacy as the age of the donor increases. Umbilical cord MSCs (UC-MSCs) are easier to obtain by comparison, and their primitive source may offer more-potent therapeutic effects. Here we investigate the therapeutic efficacy of UC-MSCs on the mechanisms of pulmonary edema formation and alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs were also tested in a mouse model of influenza ALI. We found that UC-MSCs were effective in restoring impaired alveolar fluid clearance and protein permeability of A(H5N1)-infected human alveolar epithelial cells. UC-MSCs consistently outperformed bone marrow MSCs, partly because of greater growth factor secretion of angiopoietin 1 and hepatocyte growth factor. Conditioned UC-MSC medium and UC-MSC exosomes were also able to recapitulate these effects. However, UC-MSCs only slightly improved survival of A(H5N1)-infected mice. Our results suggest that UC-MSCs are effective in restoring alveolar fluid clearance and protein permeability in A(H5N1)-associated ALI and confer functional in addition to practical advantages over conventional bone marrow MSCs.

Inhaled AP301 for treatment of pulmonary edema in mechanically ventilated patients with acute respiratory distress syndrome: a phase IIa randomized placebo-controlled trial

BackgroundHigh-permeability pulmonary edema is a hallmark of acute respiratory distress syndrome (ARDS) and is frequently accompanied by impaired alveolar fluid clearance (AFC). AP301 enhances AFC by activating epithelial sodium channels (ENaCs) on alveolar epithelial cells, and we investigated its effect on extravascular lung water index (EVLWI) in mechanically ventilated patients with ARDS.MethodsForty adult mechanically ventilated patients with ARDS were included in a randomized, double-blind, placebo-controlled trial for proof of concept. Patients were treated with inhaled AP301 (n = 20) or placebo (0.9% NaCl; n = 20) twice daily for 7 days. EVLWI was measured by thermodilution (PiCCO®), and treatment groups were compared using the nonparametric Mann–Whitney U test.ResultsAP301 inhalation was well tolerated. No differences in mean baseline-adjusted change in EVLWI from screening to day 7 were found between the AP301 and placebo group (p = 0.196). There was no difference in the PaO2/FiO2 ratio, ventilation pressures, Murray lung injury score, or 28-day mortality between the treatment groups. An exploratory subgroup analysis according to severity of illness showed reductions in EVLWI (p = 0.04) and ventilation pressures (p < 0.05) over 7 days in patients with initial sequential organ failure assessment (SOFA) scores ≥11 inhaling AP301 versus placebo, but not in patients with SOFA scores ≤10.ConclusionsThere was no difference in mean baseline-adjusted EVLWI between the AP301 and placebo group. An exploratory post-hoc subgroup analysis indicated reduced EVLWI in patients with SOFA scores ≥11 receiving AP301. These results suggest further confirmation in future clinical trials of inhaled AP301 for treatment of pulmonary edema in patients with ARDS.Trial registrationThe study was prospectively registered at clinicaltrials.gov, NCT01627613. Registered 20 June 2012.

Inflammatory Responses Regulating Alveolar Ion Transport during Pulmonary Infections

The respiratory epithelium is lined by a tightly balanced fluid layer that allows normal O2 and CO2 exchange and maintains surface tension and host defense. To maintain alveolar fluid homeostasis, both the integrity of the alveolar–capillary barrier and the expression of epithelial ion channels and pumps are necessary to establish a vectorial ion gradient. However, during pulmonary infection, auto- and/or paracrine-acting mediators induce pathophysiological changes of the alveolar–capillary barrier, altered expression of epithelial Na,K-ATPase and of epithelial ion channels including epithelial sodium channel and cystic fibrosis membrane conductance regulator, leading to the accumulation of edema and impaired alveolar fluid clearance. These mediators include classical pro-inflammatory cytokines such as TGF-β, TNF-α, interferons, or IL-1β that are released upon bacterial challenge with Streptococcus pneumoniae, Klebsiella pneumoniae, or Mycoplasma pneumoniae as well as in viral infection with influenza A virus, pathogenic coronaviruses, or respiratory syncytial virus. Moreover, the pro-apoptotic mediator TNF-related apoptosis-inducing ligand, extracellular nucleotides, or reactive oxygen species impair epithelial ion channel expression and function. Interestingly, during bacterial infection, alterations of ion transport function may serve as an additional feedback loop on the respiratory inflammatory profile, further aggravating disease progression. These changes lead to edema formation and impair edema clearance which results in suboptimal gas exchange causing hypoxemia and hypercapnia. Recent preclinical studies suggest that modulation of the alveolar–capillary fluid homeostasis could represent novel therapeutic approaches to improve outcomes in infection-induced lung injury.

150 - Molecular Target(S) Responsible for the Alveolar Tight Junction Proteins Disruption Under Hypoxia: Prophylactic Role of Curcumin in Refurbishing the Alveolar Barrier Integrity Under Hypoxia?

Pulmonary edema (PE) is attributed to ruptured alveolar-epithelial barrier integrity together with impaired alveolar fluid clearance (AFC). Disruption of tight junction (TJ) integrity can occur from loss of TJ protein expression and/or disorganization. TJ determine the barrier properties of cell-cell contact existing between two neighbouring cells and regulate paracellular permeability. The TJ disruption has been related to multiple signal transduction pathways; understanding such mechanisms in hypoxia induced disruption of epithelial and endothelial barrier functions is likely to provide insight into the pathogenesis of various inflammatory diseases, and may form a basis for the design of treatment strategies for different diseases involving barrier dysfunction. The present study was undertaken to determine whether differential expression of tight junction protein(s) is a mechanism for regulation of hypoxia-induced pulmonary epithelial permeability both in-vitro and in-vivo. For in-vitro studies, Human lung adenocarcinoma cell line (A549) was exposed to 3% O2 for different time periods viz. 1 h, 3h, 6h, 12h, 24h and 48 h. For in-vivo studies male Sprague Dawley rats were used as an animal model and exposed them to 25000 ft at 25°C for 6 hrs. Simultaneously, to rule out the fact that inflammation causes impaired tight junction proteins integrity leading to pulmonary edema, A549 cells were pre-treated with 10 µM curcumin and the rats were pre-treated with curcumin (50 mg/kg body weight) 1 h prior to 6-h hypoxia. Hypoxia exposure resulted into increased RBC, WBC, Hb, monocytes, lymphocytes and HCT. Blood gas analysis revealed that PaO2 mmHg, PaCO2 mmHg and SaO2 (%) were significantly (P≤0.001) altered compared to control. Hypoxia induced a progressive time dependent decrease in TJ proteins ZO-1, Claudin-4, and JAM-C proteins expression over control. Further, hypoxic exposure also increased the NFkB, TNF-α, Claudin -5 and Occludin protein expression levels with time as compared to control. However prior treatment with curcumin both in-vitro and in-vivo showed significant changes in tight junction proteins integrity and attenuated NFkB activity with reduced expression of its regulatory genes in lung tissue, serum and BALF. Further we confirmed by pre-treatment with NFkB inhibitor MG132 or siRNA mediated knock down of p65 significantly abrogated (p≤0.001) hypoxia induced NFkB expression followed by significant reduction in (p ≤0.05 ) in dextran FITC efflux in to the lungs. Similarly, HIF-1α reporter gene studies further, confirmed that curcumin stabilized the lung HIF-1α levels under hypoxia. Immunohistochemistry and immunofluorescence observations supports the notion that curcumin restored the brain ZO-1, JAMC, claudin 4 and claudin 5 levels (p ≤ 0.001) under hypoxia. These results indicate that curcumin is a potent drug in amelioration of pulmonary edema as it effectively attenuated inflammation with enhanced alveolar fluid clearance via maintaining the tight junction proteins integrity under hypoxia.

SOCS-1 rescues IL-1β-mediated suppression of epithelial sodium channel in mouse lung epithelial cells via ASK-1.

BackgroundAcute lung injury (ALI) is characterized by alveolar damage, increased levels of pro-inflammatory cytokines and impaired alveolar fluid clearance. Recently, we showed that the deletion of Apoptosis signal-regulating kinase 1 (ASK1) protects against hyperoxia-induced acute lung injury (HALI) by suppressing IL-1β and TNF-α. Previously, our data revealed that the suppressor of cytokine signaling-1 (SOCS-1) overexpression restores alveolar fluid clearance in HALI by inhibiting ASK-1 and suppressing IL-1β levels. Furthermore, IL-1β is known to inhibit the expression of epithelial sodium channel α-subunit (ENaC) via a p38 MAPK signaling pathway.ObjectiveTo determine whether SOCS-1 overexpression in MLE-12 cells would protect against IL-1β-mediated depletion of αENaC by suppressing ASK-1 expression.MethodsWe co-transfected MLE-12 cells with SOCS-1 overexpressing plasmid with or without IL-1β in the presence or absence of sodium channel inhibitor, amiloride. We measured potential difference, transepithelial current, resistance, and sodium uptake levels across MLE-12 cells. We studied the effect of ASK-1 depletion, as well as ASK-1 and SOCS-1 overexpression on αENaC expression.ResultsSOCS-1 overexpression sufficiently restored transepithelial current and resistance in MLE-12 cells treated with either IL-1β or amiloride. The αENaC mRNA levels and sodium transport were increased in SOCS-1 overexpressing MLE-12 cells exposed to IL-1β. Depletion of ASK-1 in MLE-12 cells increased αENaC mRNA levels. Interestingly, SOCS-1 overexpression restored αENaC expression in MLE-12 cells in the presence of ASK-1 overexpression.ConclusionCollectively, these findings suggest that SOCS-1 may exert its protective effect by rescuing αENaC expression via suppression of ASK-1.