Two-pore potassium channel TREK-1 (K2P2.1) regulates NLRP3 inflammasome activity in macrophages.

Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages (AMs) and bone marrow-derived macrophages (BMDMs) from wild-type (wt) and TREK-1-/- mice, we measured responses to inflammasome priming [using lipopolysaccharide (LPS)] and activation (LPS + ATP). We measured IL-1ß, caspase-1, and NLRP3 via ELISA and Western blot. A membrane-permeable potassium indicator was used to measure potassium efflux during ATP exposure, and a fluorescence-based assay was used to assess changes in membrane potential. Inflammasome activation induced by LPS + ATP increased IL-1ß secretion in wt AMs, whereas activation was significantly reduced in TREK-1-/- AMs. Priming of BMDMs using LPS was not affected by either genetic deficiency or pharmacological inhibition of TREK-1 with Spadin. Cleavage of caspase-1 following LPS + ATP treatment was significantly reduced in TREK-1-/- BMDMs. The intracellular potassium concentration in LPS-primed wt BMDMs was significantly lower compared with TREK-1-/- BMDMs or wt BMDMs treated with Spadin. Conversely, activation of TREK-1 with BL1249 caused a decrease in intracellular potassium in wt BMDMs. Treatment of LPS-primed BMDMs with ATP caused a rapid reduction in intracellular potassium levels, with the largest change observed in TREK-1-/- BMDMs. Intracellular K+ changes were associated with changes in the plasma membrane potential (Em), as evidenced by a more depolarized Em in TREK-1-/- BMDMs compared with wt, and Em hyperpolarization upon TREK-1 channel opening with BL1249. These results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.NEW & NOTEWORTHY Because of the importance of potassium efflux in inflammasome activation, we investigated the role of the two-pore potassium (K2P) channel TREK-1 in macrophage inflammasome activity. Using primary alveolar macrophages and bone marrow-derived macrophages from wild-type and TREK-1-/- mice, we measured responses to inflammasome priming (using LPS) and activation (LPS + ATP). Our results suggest that TREK-1 is an important regulator of NLRP3 inflammasome activation in macrophages.

Endothelial Glycocalyx Degradation Patterns in Sepsis-Associated Pediatric Acute Respiratory Distress Syndrome: A Single Center Retrospective Observational Study.

BACKGROUND: Sepsis-associated destruction of the pulmonary microvascular endothelial glycocalyx (EGCX) creates a vulnerable endothelial surface, contributing to the development of acute respiratory distress syndrome (ARDS). Constituents of the EGCX shed into circulation, glycosaminoglycans and proteoglycans, may serve as biomarkers of endothelial dysfunction. We sought to define the patterns of plasma EGCX degradation products in children with sepsis-associated pediatric ARDS (PARDS), and test their association with clinical outcomes. METHODS: We retrospectively analyzed a prospective cohort (2018-2020) of children (≥1 month to <18 years of age) receiving invasive mechanical ventilation for acute respiratory failure for ≥72 h. Children with and without sepsis-associated PARDS were selected from the parent cohort and compared. Blood was collected at time of enrollment. Plasma glycosaminoglycan disaccharide class (heparan sulfate, chondroitin sulfate, and hyaluronan) and sulfation subtypes (heparan sulfate and chondroitin sulfate) were quantified using liquid chromatography tandem mass spectrometry. Plasma proteoglycans (syndecan-1) were measured through an immunoassay. RESULTS: Among the 39 mechanically ventilated children (29 with and 10 without sepsis-associated PARDS), sepsis-associated PARDS patients demonstrated higher levels of heparan sulfate (median 639 ng/mL [interquartile range, IQR 421-902] vs 311 [IQR 228-461]) and syndecan-1 (median 146 ng/mL [IQR 32-315] vs 8 [IQR 8-50]), both p = 0.01. Heparan sulfate subtype analysis demonstrated greater proportions of N-sulfated disaccharide levels among children with sepsis-associated PARDS (p = 0.01). Increasing N-sulfated disaccharide levels by quartile were associated with severe PARDS (n = 9/29) with the highest quartile including >60% of the severe PARDS patients (test for trend, p = 0.04). Higher total heparan sulfate and N-sulfated disaccharide levels were independently associated with fewer 28-day ventilator-free days in children with sepsis-associated PARDS (all p < 0.05). CONCLUSIONS: Children with sepsis-associated PARDS exhibited higher plasma levels of heparan sulfate disaccharides and syndecan-1, suggesting that EGCX degradation biomarkers may provide insights into endothelial dysfunction and PARDS pathobiology.

Activation of TREK-1 (K2P2.1) potassium channels protects against influenza A-induced lung injury.

Influenza-A virus (IAV) infects yearly an estimated one billion people worldwide, resulting in 300,000-650,000 deaths. Preventive vaccination programs and antiviral medications represent the mainstay of therapy, but with unacceptably high morbidity and mortality rates, new targeted therapeutic approaches are urgently needed. Since inflammatory processes are commonly associated with measurable changes in the cell membrane potential (Em), we investigated whether Em hyperpolarization via TREK-1 (K2P2.1) K+ channel activation can protect against influenza-A virus (IAV)-induced pneumonia. We infected mice with IAV, which after 5 days caused 10-15% weight loss and a decrease in spontaneous activity, representing a clinically relevant infection. We then started a 3-day intratracheal treatment course with the novel TREK-1 activating compounds BL1249 or ML335. We confirmed TREK-1 activation with both compounds in untreated and IAV-infected primary human alveolar epithelial cells (HAECs) using high-throughput fluorescent imaging plate reader (FLIPR) assays. In mice, TREK-1 activation with BL1249 and ML335 counteracted IAV-induced histological lung injury and decrease in lung compliance and improved BAL fluid total protein levels, cell counts, and inflammatory IL-6, IP-10/CXCL-10, MIP-1α, and TNF-α levels. To determine whether these anti-inflammatory effects were mediated by activation of alveolar epithelial TREK-1 channels, we studied the effects of BL1249 and ML335 in IAV-infected HAEC, and found that TREK-1 activation decreased IAV-induced inflammatory IL-6, IP-10/CXCL10, and CCL-2 secretion. Dissection of TREK-1 downstream signaling pathways and construction of protein-protein interaction (PPI) networks revealed NF-κB1 and retinoic acid-inducible gene-1 (RIG-1) cascades as the most likely targets for TREK-1 protection. Therefore, TREK-1 activation may represent a novel therapeutic approach against IAV-induced lung injury.

Cell saver blood transfusions may be associated with a decrease in inflammation and improved outcome measures in pediatric cardiac surgery patients.

OBJECTIVE: Cardiopulmonary bypass (CPB) is a requisite for correction of congenital heart disease by open-heart surgery and induces a systemic inflammatory response that can lead to complications such as acute lung injury and acute kidney injury. In addition, blood transfusions are commonly required for this type of surgery, and they may further exacerbate this inflammatory response and increase morbidity and mortality. We hypothesized that, in contrast to red blood cells, intraoperative cell saver (CS) blood transfusions attenuate the post-CPB proinflammatory cytokine response. METHODS: Serum cytokine concentrations of IL-10, IL-1RA, IL-6, IL-8, and TNF-α were measured at four time points (preoperatively and postoperatively on postoperative days 0, 1, and 2). RESULTS: Anti-inflammatory IL-10 levels were significantly lower in the CS group on POD 0 than in the control group (mean 1083.2 pg/mL vs 2080.2 pg/mL, 95%CI 357.4-1636.6, p = .0026). Of the clinical parameters measured, mean BUN and creatinine levels on POD 2 were significantly lower in the CS group (13.79 vs 21.88, p = .004 and 0.45 vs 0.55, p = .055, respectively). In addition, the duration of milrinone use decreased by 80% in the CS group (0.20, 95%CI 0.04, 0.94; p = .048), the median time to extubation in hours was significantly lower in the CS group (3.5 vs 6.5; 95%CI -38.00, -0.50; p = .026), and hospital length of stay was decreased by 60% in the CS group (p = .003). CONCLUSIONS: CS transfusions in children may lower postoperative anti-inflammatory IL-10 levels, possibly due to an overall decrease in proinflammatory state, and may be associated with improvements in renal and pulmonary functions.

Activation of Endothelial Large Conductance Potassium Channels Protects against TNF-α-Induced Inflammation.

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.

Beyond the Alveolar Epithelium: Plasma Soluble Receptor for Advanced Glycation End Products Is Associated With Oxygenation Impairment, Mortality, and Extrapulmonary Organ Failure in Children With Acute Respiratory Distress Syndrome.

OBJECTIVES: Soluble receptor for advanced glycation end products is a known plasma marker of alveolar epithelial injury. However, RAGE is also expressed on cell types beyond the lung, and its activation leads to up-regulation of pro-inflammatory mediators. We sought to examine the relationship between plasma soluble receptor for advanced glycation end products and primary pulmonary dysfunction, extrapulmonary organ dysfunction, and mortality in pediatric acute respiratory distress syndrome patients at two early time points following acute respiratory distress syndrome diagnosis and compare these results to plasma surfactant protein-D, a marker of pure alveolar epithelial injury. DESIGN: Prospective observational study. SETTING: Five academic PICUs. PATIENTS: Two hundred fifty-eight pediatric patients 30 days to 18 years old meeting Berlin Criteria for acute respiratory distress syndrome. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Plasma was collected for soluble receptor for advanced glycation end products and surfactant protein-D measurements within 24 hours (day 1) and 48 to 72 hours (day 3) after acute respiratory distress syndrome diagnosis. Similar to surfactant protein-D, plasma soluble receptor for advanced glycation end products was associated with a higher oxygenation index (p < 0.01) and worse lung injury score (p < 0.001) at the time of acute respiratory distress syndrome diagnosis. However, unlike surfactant protein-D, plasma soluble receptor for advanced glycation end products was associated with worse extrapulmonary Pediatric Logistic Organ Dysfunction score during ICU stay (day 3; p < 0.01) and positively correlated with plasma levels of interleukin-6 (p < 0.01), tumor necrosis factor-α (p < 0.01), and angiopoietin-2 (p < 0.01). Among children with indirect lung injury, plasma soluble receptor for advanced glycation end products was associated with mortality independent of age, sex, race, cancer/bone marrow transplant, and Pediatric Risk of Mortality score (day 3; odds ratio, 3.14; 95% CI, 1.46-6.75; p < 0.01). CONCLUSIONS: Unlike surfactant protein-D, which is primarily localized to the alveolar epithelium plasma soluble receptor for advanced glycation end products is systemically expressed and correlates with markers of inflammation, extrapulmonary multiple organ dysfunction, and death in pediatric acute respiratory distress syndrome with indirect lung injury. This suggests that unlike surfactant protein-D, soluble receptor for advanced glycation end products is a multifaceted marker of alveolar injury and increased inflammation and that receptor for advanced glycation end products activation may contribute to the pathogenesis of multiple organ failure among children with indirect acute respiratory distress syndrome.

BK Channels Regulate LPS-induced CCL-2 Release from Human Pulmonary Endothelial Cells.

We recently established a role for the stretch-activated two-pore-domain K+ (K2P) channel TREK-1 (K2P2.1) in inflammatory cytokine secretion using models of hyperoxia-, mechanical stretch-, and TNF-α-induced acute lung injury. We have now discovered the expression of large conductance, Ca2+-activated K+ (BK) channels in human pulmonary microvascular endothelial cells and primary human alveolar epithelial cells using semiquantitative real-time PCR, IP and Western blot, and investigated their role in inflammatory cytokine secretion using an LPS-induced acute lung injury model. As expected, LPS induced IL-6 and CCL-2 secretion from pulmonary endothelial and epithelial cells. BK activation with NS1619 decreased LPS-induced CCL-2 but not IL-6 secretion from endothelial cells and had no effect on epithelial cells, although fluorometric assays revealed that BK activation hyperpolarized the plasma membrane potential (Em) of both cell types. Interestingly, BK inhibition (Paxilline) did not alter cytokine secretion or the Em in either cell type. Furthermore, LPS treatment by itself did not affect the Em or intracellular Ca2+ concentrations. Therefore, we propose BK channel activation as a novel targeted approach to counteract LPS-induced CCL-2 secretion from endothelial cells. This protective effect appears to occur via Em hyperpolarization but independent of intracellular Ca2+ concentrations.

TREK-1 protects the heart against ischemia-reperfusion-induced injury and from adverse remodeling after myocardial infarction.

The TWIK-related K+ channel (TREK-1) is a two-pore-domain potassium channel that produces background leaky potassium currents. TREK-1 has a protective role against ischemia-induced neuronal damage. TREK-1 is also expressed in the heart, but its role in myocardial ischemia-reperfusion (IR)-induced injury has not been examined. In the current study, we used a TREK-1 knockout (KO) mouse model to show that TREK-1 has a critical role in the cardiac I/R-induced injury and during remodeling after myocardial infarction (MI). At baseline, TREK-1 KO mice had similar blood pressure and heart rate as the wild-type (WT) mice. However, the lack of TREK-1 was associated with increased susceptibility to ischemic injury and compromised functional recovery following ex vivo I/R-induced injury. TREK-1 deficiency increased infarct size following permanent coronary artery ligation, resulting in greater systolic dysfunction than the WT counterpart. Electrocardiographic (ECG) analysis revealed QT interval prolongation in TREK-1 KO mice, but normal heart rate (HR). Acutely isolated TREK-1 KO cardiomyocytes exhibited prolonged Ca2+ transient duration associated with action potential duration (APD) prolongation. Our data suggest that TREK-1 has a protective effect against I/R-induced injury and influences the post-MI remodeling processes by regulating membrane potential and maintaining intracellular Ca2+ homeostasis. These data suggest that TREK-1 activation could be an effective strategy to provide cardioprotection against ischemia-induced damage.

Apoptosis signal-regulating kinase-1 promotes inflammasome priming in macrophages.

We previously showed that mice deficient in apoptosis signal-regulating kinase-1 (ASK1) were partially protected against ventilator-induced lung injury. Because ASK1 can promote both cell death and inflammation, we hypothesized that ASK1 activation regulates inflammasome-mediated inflammation. Mice deficient in ASK1 expression (ASK1-/-) exhibited significantly less inflammation and lung injury (as measured by neutrophil infiltration, IL-6, and IL-1ß) in response to treatment with inhaled lipopolysaccharide (LPS) compared with wild-type (WT) mice. To determine whether this proinflammatory response was mediated by ASK1, we investigated inflammasome-mediated responses to LPS in primary macrophages and bone marrow-derived macrophages (BMDMs) from WT and ASK1-/- mice, as well as the mouse alveolar macrophage cell line MH-S. Cells were treated with LPS alone for priming or LPS followed by ATP for activation. When macrophages were stimulated with LPS followed by ATP to activate the inflammasome, we found a significant increase in secreted IL-1ß from WT cells compared with ASK1-deficient cells. LPS priming stimulated an increase in NOD-like receptor 3 (NLRP3) and pro-IL-1ß in WT BMDMs, but expression of NLRP3 was significantly decreased in ASK1-/- BMDMs. Subsequent ATP treatment stimulated an increase in cleaved caspase-1 and IL-1ß in WT BMDMs compared with ASK1-/- BMDMs. Similarly, treatment of MH-S cells with LPS + ATP caused an increase in both cleaved caspase-1 and IL-1ß that was diminished by the ASK-1 inhibitor NQDI1. These results demonstrate, for the first time, that ASK1 promotes inflammasome priming.

Hyperoxia treatment of TREK-1/TREK-2/TRAAK-deficient mice is associated with a reduction in surfactant proteins.

We previously proposed a role for the two-pore domain potassium (K2P) channel TREK-1 in hyperoxia (HO)-induced lung injury. To determine whether redundancy among the three TREK isoforms (TREK-1, TREK-2, and TRAAK) could protect from HO-induced injury, we now examined the effect of deletion of all three TREK isoforms in a clinically relevant scenario of prolonged HO exposure and mechanical ventilation (MV). We exposed WT and TREK-1/TREK-2/TRAAK-deficient [triple knockout (KO)] mice to either room air, 72-h HO, MV [high and low tidal volume (TV)], or a combination of HO + MV and measured quasistatic lung compliance, bronchoalveolar lavage (BAL) protein concentration, histologic lung injury scores (LIS), cellular apoptosis, and cytokine levels. We determined surfactant gene and protein expression and attempted to prevent HO-induced lung injury by prophylactically administering an exogenous surfactant (Curosurf). HO treatment increased lung injury in triple KO but not WT mice, including an elevated LIS, BAL protein concentration, and markers of apoptosis, decreased lung compliance, and a more proinflammatory cytokine phenotype. MV alone had no effect on lung injury markers. Exposure to HO + MV (low TV) further decreased lung compliance in triple KO but not WT mice, and HO + MV (high TV) was lethal for triple KO mice. In triple KO mice, the HO-induced lung injury was associated with decreased surfactant protein (SP) A and SPC but not SPB and SPD expression. However, these changes could not be explained by alterations in the transcription factors nuclear factor-1 (NF-1), NKX2.1/thyroid transcription factor-1 (TTF-1) or c-jun, or lamellar body levels. Prophylactic Curosurf administration did not improve lung injury scores or compliance in triple KO mice.

The role of stretch-activated ion channels in acute respiratory distress syndrome: finally a new target?

Mechanical ventilation (MV) and oxygen therapy (hyperoxia; HO) comprise the cornerstones of life-saving interventions for patients with acute respiratory distress syndrome (ARDS). Unfortunately, the side effects of MV and HO include exacerbation of lung injury by barotrauma, volutrauma, and propagation of lung inflammation. Despite significant improvements in ventilator technologies and a heightened awareness of oxygen toxicity, besides low tidal volume ventilation few if any medical interventions have improved ARDS outcomes over the past two decades. We are lacking a comprehensive understanding of mechanotransduction processes in the healthy lung and know little about the interactions between simultaneously activated stretch-, HO-, and cytokine-induced signaling cascades in ARDS. Nevertheless, as we are unraveling these mechanisms we are gathering increasing evidence for the importance of stretch-activated ion channels (SACs) in the activation of lung-resident and inflammatory cells. In addition to the discovery of new SAC families in the lung, e.g., two-pore domain potassium channels, we are increasingly assigning mechanosensing properties to already known Na(+), Ca(2+), K(+), and Cl(-) channels. Better insights into the mechanotransduction mechanisms of SACs will improve our understanding of the pathways leading to ventilator-induced lung injury and lead to much needed novel therapeutic approaches against ARDS by specifically targeting SACs. This review 1) summarizes the reasons why the time has come to seriously consider SACs as new therapeutic targets against ARDS, 2) critically analyzes the physiological and experimental factors that currently limit our knowledge about SACs, and 3) outlines the most important questions future research studies need to address.

Chronic hypersensitivity pneumonitis caused by Saccharopolyspora rectivirgula is not associated with a switch to a Th2 response.

Hypersensitivity pneumonitis (HP) is an immune-mediated interstitial lung disease that develops following repeated exposure to inhaled environmental antigens. The disease results in alveolitis and granuloma formation and may progress to a chronic form associated with fibrosis; a greater understanding of the immunopathogenic mechanisms leading to chronic HP is needed. We used the Saccharopolyspora rectivirgula (SR) mouse model of HP to determine the extent to which a switch to a Th2-type immune response is associated with chronic HP. Exposure of wild-type (WT) and tlr2/9(-/-) mice to SR for 14 wk resulted in neutrophilic and lymphocytic alveolitis that was not dependent on Toll-like receptors (TLRs) 2 and 9. Long-term exposure of WT mice to SR resulted in a significant increase in collagen deposition, protein leakage, and IL-1α accompanied by a decrease in quasistatic compliance and total lung capacity compared with unexposed mice. This was associated with an increase in IL-17 but not IL-4 production or recruitment of Th2 cells. tlr2/9(-/-) mice exhibited an increase in protein leakage but less IL-1α and collagen deposition in the lungs compared with WT mice, yet they still displayed a decrease in quasistatic compliance, although total lung capacity was not affected. These mice exhibited an increase in both IL-13 and IL-17, which suggests that IL-13 may ameliorate some of the lung damage caused by long-term SR exposure. Our results suggest that lung pathology following long-term SR exposure in WT mice is associated with the IL-17 response and that TLRs 2 and 9 may inhibit the development of the IL-13/Th2 response.

Regulation of inflammatory biomarkers by intravenous methylprednisolone in pediatric ARDS patients: Results from a double-blind, placebo-controlled randomized pilot trial.

OBJECTIVE: A double-blind, randomized controlled trial showed that low-dose glucocorticoid therapy in pediatric ARDS patients is feasible and may improve both ventilation and oxygenation indices in these patients. However, the molecular mechanisms underlying potential changes in outcomes remain unclear. Based on these clinical findings, this study was designed to examine the effects of intravenous methylprednisolone on circulating inflammatory biomarkers in pediatric ARDS patients. DESIGN: Double-blind, placebo-controlled randomized trial with blood collection on study entry and day 7. SETTING: Tertiary care children's hospital. PATIENTS: Children (0-18years) with ARDS undergoing mechanical ventilation. INTERVENTIONS: 35 children were randomized within 72h of mechanical ventilation. The glucocorticoid group received methylprednisolone 2mg/kg loading dose followed by 1mg/kg/day continuous infusion from days 1 to 7. Both groups were ventilated following the ARDSnet recommendations. WBC and differential cell counts, plasma cytokines and CRP levels, and coagulation parameters were analyzed on days 0 and 7. RESULTS: At study entry, the placebo group had higher IL-15 and basophil levels. On day 7, in comparison to study entry, the placebo group had lower IL-1α, IFN-γ and IL-10 levels. The glucocorticoid group had lower INF-α, IL-6, IL-10, MCP-1, G-CSF and GM-CSF levels, and higher IL-17α levels on day 7 in comparison to study entry. Total and differential cell counts remained unchanged within the placebo group between days 0 and 7, whereas in the glucocorticoid group total WBC and platelets counts were increased on day 7. Pearson's correlation studies within the placebo and glucocorticoid groups revealed positive and negative correlations between cytokine levels, cell counts, coagulation parameters and relevant clinical parameters of disease severity identified in our previous study. Multiple regression models identified several cytokines as predictors for alterations in clinical parameters of disease severity. CONCLUSION: This pilot study shows the feasibility of simultaneously measuring multiple inflammatory cytokines, cell counts and coagulation parameters in pediatric ARDS patients. We report statistical models that may be useful for future, larger trials to predict ARDS severity and outcomes.

Pulmonary epithelial barrier function: some new players and mechanisms.

The pulmonary epithelium serves as a barrier to prevent access of the inspired luminal contents to the subepithelium. In addition, the epithelium dictates the initial responses of the lung to both infectious and noninfectious stimuli. One mechanism by which the epithelium does this is by coordinating transport of diffusible molecules across the epithelial barrier, both through the cell and between cells. In this review, we will discuss a few emerging paradigms of permeability changes through altered ion transport and paracellular regulation by which the epithelium gates its response to potentially detrimental luminal stimuli. This review is a summary of talks presented during a symposium in Experimental Biology geared toward novel and less recognized methods of epithelial barrier regulation. First, we will discuss mechanisms of dynamic regulation of cell-cell contacts in the context of repetitive exposure to inhaled infectious and noninfectious insults. In the second section, we will briefly discuss mechanisms of transcellular ion homeostasis specifically focused on the role of claudins and paracellular ion-channel regulation in chronic barrier dysfunction. In the next section, we will address transcellular ion transport and highlight the role of Trek-1 in epithelial responses to lung injury. In the final section, we will outline the role of epithelial growth receptor in barrier regulation in baseline, acute lung injury, and airway disease. We will then end with a summary of mechanisms of epithelial control as well as discuss emerging paradigms of the epithelium role in shifting between a structural element that maintains tight cell-cell adhesion to a cell that initiates and participates in immune responses.

Inverse Relationship between Cardio-Ankle Vascular Index and Body Mass Index in Healthy Children.

OBJECTIVE: To establish reference scores for cardio-ankle vascular index (CAVI), a noninvasive measure of vascular function, which reflects the stiffness of arteries, in healthy children, to test for racial and ethnic differences, and to compare CAVI scores between overweight and normal weight children. STUDY DESIGN: Subjects included 292 children aged 10-18 years: 100 non-Hispanic whites, 89 non-Hispanic blacks, and 103 Hispanics. Subjects were grouped as normal weight (body mass index [BMI] <85th percentile for age) and overweight (BMI >85th percentile for age). Blood pressure (BP) and CAVI scores were measured in all subjects. RESULTS: After controlling for age, sex, and BMI, normal weight black males had a higher CAVI score (indicating stiffer arteries) in comparison with Hispanic males and white males (5.53 ± 0.15 vs 5.13 ± 0.15 vs 5.02 ± 0.15, P = .04). BMI had an inverse association on the CAVI score (r = -0.335, P < .0001). In multivariable analysis, BMI and average CAVI scores were significant predictors of each other (R(2) = 0.37, P < .0001, R(2) = 0.21, P < .0001). There was no significant correlation between CAVI scores and resting BP values, confirming that CAVI scores were independent of concurrent BP values. CONCLUSIONS: Significant differences in vascular function exist among ethnic groups of children. Overweight children had lower CAVI scores, suggestive of vascular adaptation to obesity in early life. CAVI, by providing a noninvasive measure of vascular health, may help identify children at increased risk for cardiovascular disease.

Double-blind, placebo-controlled pilot randomized trial of methylprednisolone infusion in pediatric acute respiratory distress syndrome.

OBJECTIVE: Low-dose methylprednisolone therapy in adults with early acute respiratory distress syndrome reduces systemic inflammation, duration of mechanical ventilation, and ICU length of stay. We report a pilot randomized trial of glucocorticoid treatment in early pediatric acute respiratory distress syndrome. DESIGN: Double-blind, placebo-controlled randomized clinical trial. SETTING: Le Bonheur Children's Hospital, Memphis, TN. PATIENTS: Children (0-18 yr) with acute respiratory distress syndrome undergoing mechanical ventilation. INTERVENTIONS: Patients were randomly assigned to steroid or placebo groups within 72 hours of intubation. IV methylprednisolone administered as loading dose (2 mg/kg) and continuous infusions (1 mg/kg/d) on days 1-7 and then tapered over days 8-14. Both groups were ventilated according to the Acute Respiratory Distress Syndrome Network protocol modified for children. Daily surveillance was performed for adverse effects. MEASUREMENTS AND MAIN RESULTS: Thirty-five patients were randomized to the steroid (n = 17, no death) and placebo groups (n = 18, two deaths). No differences occurred in length of mechanical ventilation, ICU stay, hospital stay, or mortality between the two groups. At baseline, higher plateau pressures (p = 0.006) and lower Pediatric Logistic Organ Dysfunction scores (p = 0.04) occurred in the steroid group; other characteristics were similar. Despite higher plateau pressures on days 1 (p = 0.006) and 2 (p = 0.025) due to poorer lung compliance in the steroid group, they had lower PaCO2 values on days 2 (p = 0.009) and 3 (p = 0.014), higher pH values on day 2 (p = 0.018), and higher PaO2/FIO2 ratios on days 8 (p = 0.047) and 9 (p = 0.002) compared with the placebo group. Fewer patients in the steroid group required treatment for postextubation stridor (p = 0.04) or supplemental oxygen at ICU transfer (p = 0.012). Steroid therapy was not associated with detectable adverse effects. CONCLUSION: This study demonstrates the feasibility of administering low-dose glucocorticoid therapy and measuring clinically relevant outcomes in pediatric acute respiratory distress syndrome. Changes in oxygenation and/or ventilation are consistent with early acute respiratory distress syndrome pathophysiology and results of similar clinical trials in adults. We propose and design a larger randomized trial to define the role of glucocorticoid therapy in pediatric acute respiratory distress syndrome.

Deficiency of the two-pore-domain potassium channel TREK-1 promotes hyperoxia-induced lung injury.

OBJECTIVES: We previously reported the expression of the two-pore-domain K channel TREK-1 in lung epithelial cells and proposed a role for this channel in the regulation of alveolar epithelial cytokine secretion. In this study, we focused on investigating the role of TREK-1 in vivo in the development of hyperoxia-induced lung injury. DESIGN: Laboratory animal experiments. SETTING: University research laboratory. SUBJECTS: Wild-type and TREK-1-deficient mice. INTERVENTIONS: Mice were anesthetized and exposed to 1) room air, no mechanical ventilation, 2) 95% hyperoxia for 24 hours, and 3) 95% hyperoxia for 24 hours followed by mechanical ventilation for 4 hours. MEASUREMENTS AND MAIN RESULTS: Hyperoxia exposure accentuated lung injury in TREK-1-deficient mice but not controls, resulting in increase in lung injury scores, bronchoalveolar lavage fluid cell numbers, and cellular apoptosis and a decrease in quasi-static lung compliance. Exposure to a combination of hyperoxia and injurious mechanical ventilation resulted in further morphological lung damage and increased lung injury scores and bronchoalveolar lavage fluid cell numbers in control but not TREK-1-deficient mice. At baseline and after hyperoxia exposure, bronchoalveolar lavage cytokine levels were unchanged in TREK-1-deficient mice compared with controls. Exposure to hyperoxia and mechanical ventilation resulted in an increase in bronchoalveolar lavage interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α levels in both mouse types, but the increase in interleukin-6 and monocyte chemotactic protein-1 levels was less prominent in TREK-1-deficient mice than in controls. Lung tissue macrophage inflammatory protein-2, keratinocyte-derived cytokine, and interleukin-1ß gene expression was not altered by hyperoxia in TREK-1-deficient mice compared with controls. Furthermore, we show for the first time TREK-1 expression on alveolar macrophages and unimpaired tumor necrosis factor-α secretion from TREK-1-deficient macrophages. CONCLUSIONS: TREK-1 deficiency resulted in increased sensitivity of lungs to hyperoxia, but this effect is less prominent if overwhelming injury is induced by the combination of hyperoxia and injurious mechanical ventilation. TREK-1 may constitute a new potential target for the development of novel treatment strategies against hyperoxia-induced lung injury.

Regulation of interleukin-6 secretion by the two-pore-domain potassium channel Trek-1 in alveolar epithelial cells.

We recently proposed a role for the two-pore-domain K(+) (K2P) channel Trek-1 in the regulation of cytokine release from mouse alveolar epithelial cells (AECs) by demonstrating decreased interleukin-6 (IL-6) secretion from Trek-1-deficient cells, but the underlying mechanisms remained unknown. This study was designed to investigate the mechanisms by which Trek-1 decreases IL-6 secretion. We hypothesized that Trek-1 regulates tumor necrosis factor-α (TNF-α)-induced IL-6 release via NF-κB-, p38-, and PKC-dependent pathways. We found that Trek-1 deficiency decreased IL-6 secretion from mouse and human AECs at both transcriptional and translational levels. While NF-κB/p65 phosphorylation was unchanged, p38 phosphorylation was decreased in Trek-1-deficient cells, and pharmacological inhibition of p38 decreased IL-6 secretion in control but not Trek-1-deficient cells. Similarly, pharmacological inhibition of PKC also decreased IL-6 release, and we found decreased phosphorylation of the isoforms PKC/PKDµ (Ser(744/748)), PKCθ, PKCδ, PKCα/ßII, and PKCζ/λ, but not PKC/PKDµ (Ser(916)) in Trek-1-deficient AECs. Phosphorylation of PKCθ, a Ca(2+)-independent isoform, was intact in control cells but impaired in Trek-1-deficient cells. Furthermore, TNF-α did not elevate the intracellular Ca(2+) concentration in control or Trek-1-deficient cells, and removal of extracellular Ca(2+) did not impair IL-6 release. In summary, we report the expression of Trek-1 in human AECs and propose that Trek-1 deficiency may alter both IL-6 translation and transcription in AECs without affecting Ca(2+) signaling. The results of this study identify Trek-1 as a new potential target for the development of novel treatment strategies against acute lung injury.

Insulin-like growth factor-I stimulates differentiation of ATII cells to ATI-like cells through activation of Wnt5a.

Alveolar type II (ATII) epithelial cells play a crucial role in the repair and remodeling of the lung following injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo and into an ATI-like phenotype in vitro. While previous reports indicate that the differentiation of ATII cells into ATI cells is a complex biological process, the underlying mechanism responsible for differentiation is not fully understood. To investigate factors involved in this differentiation in culture, we used a PCR array and identified several genes that were either up- or downregulated in ATI-like cells (day 6 in culture) compared with day 2 ATII cells. Insulin-like growth factor-I (IGF-I) mRNA was increased nearly eightfold. We found that IGF-I was increased in the culture media of ATI-like cells and demonstrated a significant role in the differentiation process. Treatment of ATII cells with recombinant IGF-I accelerated the differentiation process, and this effect was abrogated by the IGF-I receptor blocker PQ401. We found that Wnt5a, a member of the Wnt-Frizzled pathway, was activated during IGF-I-mediated differentiation. Both protein kinase C and ß-catenin were transiently activated during transdifferentiation. Knocking down Wnt5a using small-interfering RNA abrogated the differentiation process as indicated by changes in the expression of an ATII cell marker (prosurfactant protein-C). Treatment of wounded cells with either IGF-I or Wnt5a stimulated wound closure. These results suggest that IGF-I promotes differentiation of ATII to ATI cells through the activation of a noncanonical Wnt pathway.

Protection from acute lung injury by a peptide designed to inhibit the voltage-gated proton channel.

There are no targeted medical therapies for Acute Lung Injury (ALI) or its most severe form acute respiratory distress syndrome (ARDS). Infections are the most common cause of ALI/ARDS and these disorders present clinically with alveolar inflammation and barrier dysfunction due to the influx of neutrophils and inflammatory mediator secretion. We designed the C6 peptide to inhibit voltage-gated proton channels (Hv1) and demonstrated that it suppressed the release of reactive oxygen species (ROS) and proteases from neutrophils in vitro. We now show that intravenous C6 counteracts bacterial lipopolysaccharide (LPS)-induced ALI in mice, and suppresses the accumulation of neutrophils, ROS, and proinflammatory cytokines in bronchoalveolar lavage fluid. Confirming the salutary effects of C6 are via Hv1, genetic deletion of the channel similarly protects mice from LPS-induced ALI. This report reveals that Hv1 is a key regulator of ALI, that Hv1 is a druggable target, and that C6 is a viable agent to treat ALI/ARDS.