Alveolar Fluid Clearance Research Articles

Thyroid Function Modulates Lung Fluid and Alveolar Viscoelasticity in Mechanically Ventilated Rat

BackgroundMechanical ventilation (MV) is life saving; yet it may induce severe lung injury and lead to multisystem organ failure and death. Thyroid hormones (THs) promote alveolar fluid clearance and alleviates hypoxia-induced lung injury. Given that the mechanism involved in hypoxia-induced lung injury is different from that of ventilator-induced lung injury, we examined the effects of thyroid function on lung extravascular fluid (LF), aquaporin 5 (AQP 5) expression, and alveolar viscoelasticity (AVE) in mechanically ventilated rat. MethodsHypothyroid (hypo) and hyperthyroid (hyper) animals were generated by administration of metimazole and L-thyroxine, respectively. Lung injury was induced by high-tidal volume MV. The LF was estimated by lung wet weight-to-dry weight ratio assessment. Expression of AQP 5 was evaluated by western blotting and in situ immunohistochemistry. The AVE was judged by elastic lung pressure/volume curve recording. ResultsInjurious MV significantly reduced lung AQP 5 expression and altered LF and AVE in a thyroid function–dependent manner. Regardless of animals' ventilation mode, hyper state caused significant reductions in LF and lung AQP 5 protein. It also improved AVE irrespective of animals' ventilation mode. The effects of hypo condition on LF, AQP 5 expression, and AVE were in contrast to that of hyper state. ConclusionsThese data indicate that thyroid function has profound effects on LF, AQP 5, and AVE in mechanically ventilated lungs. Given that the effects of thyroidal status were as prominent as that of injurious MV, we suggest that thyroid function should be considered when patients are to be subjected to MV.

Lipoxin A4 alleviates lung injury in sepsis rats through p38/MAPK signaling pathway.

The aim of the study was to explore the effect of lipoxin A4 (LXA4) on lung injury in sepsis rats through the p38/mitogen-activated protein kinase (MAPK) signaling pathway. Sprague-Dawley rats were used for the study. The rat model of sepsis-induced acute lung injury was established via cecal ligation (Sepsis group, n=20). LXA4 (0.1 mg/kg) was injected at 6 h after modeling (Treatment group, n=20), and a The Control group (n=20) was also set up. The 7-day survival rate was 100% in The Control group, and LXA4 raised the survival rate of rats in the Sepsis group from 40% to 60% (P<0.01). Alveolar fluid clearance (AFC) significantly declined and the wet/dry weight (W/D) ratio of lung tissues rose remarkably in the Sepsis group compared with those in the Control group, while LXA4 restored AFC and reduced the W/D ratio of lung tissues (P<0.05), suggesting that LXA4 treatment reduces lung fluids and partially enhances AFC, thus lowering the W/D ratio of lung. The total cell count, polymorphonuclear neutrophils (PMN) percentage and concentration of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 in bronchoalveolar lavage fluid (BALF) were obviously increased in the Sepsis group compared with those in the Control group, while they were markedly decreased in the Treatment group (P<0.05). The activity of myeloperoxidase (MPO) in lung tissue homogenate was evidently higher in the Sepsis group than that in The Control group, while it was notably lower in the Treatment group than that in the Sepsis group after LXA4 treatment (P<0.05). Moreover, it was observed microscopically that the morphology of lung tissues was intact in the Control group. Finally, the results of Western blotting manifested that the p-p38/ MAPK protein expression was remarkably increased in the Sepsis group, indicating the activation of the p38/MAPK pathway, while it was remarkably decreased in the Treatment group, indicating the inhibited activity of the pathway (P<0.05). LXA4 has an anti-inflammatory effect on sepsis rats with lung injury, and such effect is related to the p38/MAPK signaling pathway.

Acute respiratory distress syndrome subphenotypes and therapy responsive traits among preclinical models: protocol for a systematic review and meta-analysis

BackgroundSubphenotypes were recently reported within clinical acute respiratory distress syndrome (ARDS), with distinct outcomes and therapeutic responses. Experimental models have long been used to mimic features of ARDS pathophysiology, but the presence of distinct subphenotypes among preclinical ARDS remains unknown. This review will investigate whether: 1) subphenotypes can be identified among preclinical ARDS models; 2) such subphenotypes can identify some responsive traits.MethodsWe will include comparative preclinical (in vivo and ex vivo) ARDS studies published between 2009 and 2019 in which pre-specified therapies were assessed (interleukin (IL)-10, IL-2, stem cells, beta-agonists, corticosteroids, fibroblast growth factors, modulators of the receptor for advanced glycation end-products pathway, anticoagulants, and halogenated agents) and outcomes compared to a control condition. The primary outcome will be a composite of the four key features of preclinical ARDS as per the American Thoracic Society consensus conference (histologic evidence of lung injury, altered alveolar-capillary barrier, lung inflammatory response, and physiological dysfunction). Secondary outcomes will include the single components of the primary composite outcome, net alveolar fluid clearance, and death. MEDLINE, Embase, and Cochrane databases will be searched electronically and data from eligible studies will be extracted, pooled, and analyzed using random-effects models. Individual study reporting will be assessed according to the Animal Research: Reporting of In Vivo Experiments guidelines. Meta-regressions will be performed to identify subphenotypes prior to comparing outcomes across subphenotypes and treatment effects.DiscussionThis study will inform on the presence and underlying pathophysiological features of subphenotypes among preclinical models of ARDS and should help to determine whether sufficient evidence exists to perform preclinical trials of subphenotype-targeted therapies, prior to potential clinical translation.Systematic review registrationPROSPERO (ID: CRD42019157236).

Impact of Bacterial Toxins in the Lungs.

Bacterial toxins play a key role in the pathogenesis of lung disease. Based on their structural and functional properties, they employ various strategies to modulate lung barrier function and to impair host defense in order to promote infection. Although in general, these toxins target common cellular signaling pathways and host compartments, toxin- and cell-specific effects have also been reported. Toxins can affect resident pulmonary cells involved in alveolar fluid clearance (AFC) and barrier function through impairing vectorial Na+ transport and through cytoskeletal collapse, as such, destroying cell-cell adhesions. The resulting loss of alveolar-capillary barrier integrity and fluid clearance capacity will induce capillary leak and foster edema formation, which will in turn impair gas exchange and endanger the survival of the host. Toxins modulate or neutralize protective host cell mechanisms of both the innate and adaptive immunity response during chronic infection. In particular, toxins can either recruit or kill central players of the lung’s innate immune responses to pathogenic attacks, i.e., alveolar macrophages (AMs) and neutrophils. Pulmonary disorders resulting from these toxin actions include, e.g., acute lung injury (ALI), the acute respiratory syndrome (ARDS), and severe pneumonia. When acute infection converts to persistence, i.e., colonization and chronic infection, lung diseases, such as bronchitis, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) can arise. The aim of this review is to discuss the impact of bacterial toxins in the lungs and the resulting outcomes for pathogenesis, their roles in promoting bacterial dissemination, and bacterial survival in disease progression.

Maresin Conjugates in Tissue Regeneration 1 improves alveolar fluid clearance by up-regulating alveolar ENaC, Na, K-ATPase in lipopolysaccharide-induced acute lung injury.

Maresin Conjugates in Tissue Regeneration 1 (MCTR1) is a newly identified macrophage‐derived sulfido‐conjugated mediator that stimulates the resolution of inflammation. This study assessed the role of MCTR1 in alveolar fluid clearance (AFC) in a rat model of acute lung injury (ALI) induced by lipopolysaccharide (LPS). Rats were intravenously injected with MCTR1 at a dose of 200 ng/rat, 8 hours after administration of 14 mg/kg LPS. The level of AFC was then determined in live rats. Primary rat ATII (Alveolar Type II) epithelial cells were also treated with MCTR1 (100 nmol/L) in a culture medium containing LPS for 8 hours. MCTR1 treatment improved AFC (18.85 ± 2.07 vs 10.11 ± 1.08, P < .0001) and ameliorated ALI in rats. MCTR1 also significantly promoted AFC by up‐regulating epithelial sodium channel (ENaC) and Na+‐K+‐adenosine triphosphatase (Na, K‐ATPase) expressions in vivo. MCTR1 also activated Na, K‐ATPase and elevated phosphorylated‐Akt (P‐Akt) by up‐regulating the expression of phosphorylated Nedd4‐2 (P‐Nedd4‐2) in vivo and in vitro. However, BOC‐2 (ALX inhibitor), KH7 (cAMP inhibitor) and LY294002 (PI3K inhibitor) abrogated the improved AFC induced by MCTR1. Based on the findings of this study, MCTR1 may be a novel therapeutic approach to improve reabsorption of pulmonary oedema during ALI/acute respiratory distress syndrome (ARDS).

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.

Alveolar Type 2 Epithelial Cells as Potential Therapeutics for Acute Lung Injury/Acute Respiratory Distress Syndrome.

Acute lung injury/acute respiratory distress syndrome is a common clinical illness with high morbidity and mortality, which is still one of the medical problems urgently needed to be solved. Alveolar type 2 epithelial cells are an important component of lung epithelial cells and as a kind of stem cells, they can proliferate and differentiate into alveolar type 1 epithelial cells, thus contributing to lung epithelial repairment. In addition, they synthesize and secrete all components of the surfactant that regulates alveolar surface tension in the lungs. Moreover, alveolar type 2 epithelial cells play an active role in enhancing alveolar fluid clearance and reducing lung inflammation. In recent years, as more advanced approaches appear in the field of stem and progenitor cells in the lung, many preclinical studies have shown that the cell therapy of alveolar type 2 epithelial cells has great potential effects for acute lung injury/acute respiratory distress syndrome. We reviewed the recent progress on the mechanisms of alveolar type 2 epithelial cells involved in the damaged lung repairment, aiming to explore the possible therapeutic targets in acute lung injury/acute respiratory distress syndrome.

Curcumin prophylaxis refurbishes alveolar epithelial barrier integrity and alveolar fluid clearance under hypoxia

We have studied the prophylactic efficacy of curcumin to ameliorate the impairment of tight junction protein integrity and fluid clearance in lungs of rats under hypoxia. A549 cells wereexposed to 3 % O2 for 1 h, 3 h, 6 h, 12 h, 24 h and 48 h and rats were exposed to 7620 m for 6 h. NF-κB, Hif-1α and their related genes, tight junction protein (TJ) (ZO-1, JAM-C, claudin-4 and claudin-5, claudin-18) expressions were determined in A549 cells and lungs of rats by western blotting, ELISA and their activity by reporter gene assay, siRNAp65 knock out. Tissue specific localization of tight junction protein was determined by immunohistochemistry and immunoflorescence. Further transmission electron microscopy (TEM) was used to visualize the TJ structures between pulmonary epithelial cells. Blood gas and hematological parameters were also assessed. Later we checked, whether prior treatment with curcumin can restore the altered alveolar epithelial barrier integrity that is compromised through inflammatory mediators under hypoxia, A549 cells were pre-treated (1 h) with 10 μM curcumin and rats with 50 mg curcumin/kg BW and exposed to hypoxia. Curcumin pre-treatment both in vitro and in vivo showed significant changes in TJ protein integrity, attenuated NF-κB activity with reduced expression of its regulatory genes in lung tissues, serum and bronchoalveolar lavage fluid (BALF) along with stabilized HIF-1α levels under hypoxia. NF-κB inhibitors MG132, SN50 or siRNA mediated p65 knock down significantly reduced the dextran FITC influx into the lungs. The present study indicates that, curcumin prophylaxis augments alveolar epithelial barrier integrity and alveolar fluid clearance under hypoxia.

Adaptive Support Ventilation Attenuates Ventilator Induced Lung Injury: Human and Animal Study.

Adaptive support ventilation (ASV) is a closed-loop ventilation, which can make automatic adjustments in tidal volume (VT) and respiratory rate based on the minimal work of breathing. The purpose of this research was to study whether ASV can provide a protective ventilation pattern to decrease the risk of ventilator-induced lung injury in patients of acute respiratory distress syndrome (ARDS). In the clinical study, 15 ARDS patients were randomly allocated to an ASV group or a pressure-control ventilation (PCV) group. There was no significant difference in the mortality rate and respiratory parameters between these two groups, suggesting the feasible use of ASV in ARDS. In animal experiments of 18 piglets, the ASV group had a lower alveolar strain compared with the volume-control ventilation (VCV) group. The ASV group exhibited less lung injury and greater alveolar fluid clearance compared with the VCV group. Tissue analysis showed lower expression of matrix metalloproteinase 9 and higher expression of claudin-4 and occludin in the ASV group than in the VCV group. In conclusion, the ASV mode is capable of providing ventilation pattern fitting into the lung-protecting strategy; this study suggests that ASV mode may effectively reduce the risk or severity of ventilator-associated lung injury in animal models.

Efficacy of Quercetin as a potent sensitizer of β2-AR in combating the impairment of fluid clearance in lungs of rats under hypoxia

BackgroundHypoxia reportedly increases free radical generation in the body, causing oxidative stress and inhibiting β2-AR signaling. The present study correlates the prophylactic potential of quercetin and salbutamol in ameliorating fluid clearing capacity of lungs by re-sensitizing β2-AR signaling under hypoxia. MethodsMale SD rats supplemented orally with quercetin (50 mg/Kg BW), and salbutamol (2 mg/Kg BW) were exposed to hypobaric hypoxia at 7620 m for 6 h. Western blotting and ELISA quantitated NFĸB and related genes and GPCR pathway proteins. The binding affinities of drugs with receptor were determined by SPR spectroscopy and further confirmed insilico. ResultsQuercetin and salbutamol pre-treatment significantly up-regulated the expressions of β2-AR, GPR-1, GPR-10, GCSα, cAMP content, and down-regulated GRK-2, β-arrestin, ROS, NFκB (p < 0.001), thus, enhancing alveolar fluid clearance (AFC). SPR and insilico findings revealed a higher binding affinity of β2-AR with quercetin over salbutamol. ConclusionResults indicated quercetin to be a better prophylactic that augmented AFC in rats exposed to hypoxia by attenuating inflammation and stimulating β2-AR.

Current understanding of the therapeutic benefits of mesenchymal stem cells in acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) is a multifaceted lung disorder in which no specific therapeutic intervention is able to effectively improve clinical outcomes. Despite an improved understanding of molecular mechanisms and advances in supportive care strategies, ARDS remains associated with high mortality, and survivors usually face long-term morbidity. In recent years, preclinical studies have provided mounting evidence of the potential of mesenchymal stem cell (MSC)-based therapies in lung diseases and critical illnesses. In several models of ARDS, MSCs have been demonstrated to induce anti-inflammatory and anti-apoptotic effects, improve epithelial and endothelial cell recovery, and enhance microbial and alveolar fluid clearance, thus resulting in improved lung and distal organ function and survival. Early-stage clinical trials have also demonstrated the safety of MSC administration in patients with ARDS, but further, large-scale investigations are required to assess the safety and efficacy profile of these therapies. In this review, we summarize the main mechanisms whereby MSCs have been shown to exert therapeutic effects in experimental ARDS. We also highlight questions that need to be further elucidated and barriers that must be overcome in order to efficiently translate MSC research into clinical practice.

Novel insights into the role of LRRC8A in ameliorating alveolar fluid clearance in LPS induced acute lung injury

Leucine-rich repeat-containing 8A (LRRC8A) protein was recently identified as an essential component of volume-regulated anion channel which plays a central role in maintaining cell volume. The aim of this study was to elucidate the role of LRRC8A in alveolar fluid clearance (AFC) and the effect of inflammatory cytokines on LRRC8A and the underlying mechanism. Lipopolysaccharide (LPS) was used to generate a rat acute lung injury model. The results showed that the concentrations of IL-1β, TNF-α and IL-6 in bronchoalveolar lavage fluid increased significantly, but the expression of LRRC8A in the lung tissue decreased dramatically in the acute lung injury group followed by a decline in the AFC rate. Additionally, LRRC8A knockdown reduced AFC in normal rats. However, specific overexpression of LRRC8A in the lung could increase AFC. Furthermore, we observed the effects of LPS, IL-1β, TNF-α and IL-6 on the LRRC8A current in alveolar type II (ATII) cells, and IL-1β showed the greatest inhibition among them, which was involved in phospho-p38 activation. Overall, LRRC8A plays an essential role in the progression of AFC in LPS-induced acute lung injury, and chronic treatment with IL-1β or TNF-α could inhibit the function of LRRC8A in ATII cells by targeting phospho-p38. All of the findings suggested that LRRC8A could be a new partner in AFC and a potential target for the treatment of acute lung injury.

Lipoxin A4 ameliorates alveolar fluid clearance disturbance in lipopolysaccharide-induced lung injury via aquaporin 5 and MAPK signaling pathway.

A characteristic of acute lung injury (ALI) is the inflammatory damage of alveolar fluid transport. Lipoxins are endogenous lipids involving in the resolution of inflammation. It is found that lipoxin A4 (LXA4) has the distinct properties to improve the anti-edema and pro-resolution function in inflammation. Since aquaporins (AQPs) have essential roles in the integrity of barrier function during fluid transport, especially AQP5 in the maintaining of the epithelium permeability, the current study is aimed to evaluate the potential role of LXA4 in regulating alveolar fluid clearance (AFC) during fluid transport and the corresponding change of AQP5 in the lung. ALI was induced by the lipopolysaccharide (LPS) intraperitoneal injection, and LXA4 treatment was given 8 hours after LPS administration. We investigated changes in the capacity of AFC, pro-inflammatory cytokine concentrations in bronchoalveolar lavage fluid (BALF) and the severity of ALI. Then AQP5 expression in lung tissue and potential regulatory pathways in LPS-induced ALI was explored. LXA4 treatment was found to inhibit AFC capacity, inflammatory cytokine release, partially, alleviate ALI severity, and restored AQP5 expression partially. Additionally, we found that LXA4 played a protective role by the inhibition of the phosphorylation of p38 and JNK. In summary, our results suggest that LXA4 plays a protective role in lipopolysaccharide-induced ALI by restoring AFC capacity and upregulating AQP5 expression and inhibiting the phosphorylation of p38 and JNK. These findings suggest potential new mechanism of LXA4 as anti-inflammation therapy for the impairment of alveolar fluid transport in ALI.

Cardiogenic pulmonary edema: mechanisms and treatment - an intensivist's view.

This review summarizes current understanding of the pathophysiology of cardiogenic pulmonary edema, its causes and treatment. The pathobiology and classification of pulmonary edema is more complex than the hydrostatic vs. permeability dichotomy of the past. Mechanisms of alveolar fluid clearance and factors that affect the clearance rate are under intensive study to find therapeutic strategies. Patients need early stabilization of oxygenation and ventilation, preferably with high-flow nasal cannula oxygen or noninvasive ventilation whereas the diagnostic cause is quickly sought with echocardiography and other testing. Treatments must be initiated early, whereas evaluation still is occurring and requires multimodality intervention. The general treatment of cardiogenic pulmonary edema includes diuretics, possibly morphine and often nitrates. The appropriate use of newer approaches - such as, nesiritide, high-dose vasodilators, milrinone, and vasopressin receptor antagonists - needs larger clinical trials.

Inhibition of the Receptor for Advanced Glycation End-Products in Acute Respiratory Distress Syndrome: A Randomised Laboratory Trial in Piglets

The receptor for advanced glycation end-products (RAGE) modulates the pathogenesis of acute respiratory distress syndrome (ARDS). RAGE inhibition attenuated lung injury and restored alveolar fluid clearance (AFC) in a mouse model of ARDS. However, clinical translation will require assessment of this strategy in larger animals. Forty-eight anaesthetised Landrace piglets were randomised into a control group and three treatment groups. Animals allocated to treatment groups underwent orotracheal instillation of hydrochloric acid (i) alone; (ii) in combination with intravenous administration of a RAGE antagonist peptide (RAP), or (iii) recombinant soluble (s)RAGE. The primary outcome was net AFC at 4 h. Arterial oxygenation was assessed hourly and alveolar-capillary permeability, alveolar inflammation and lung histology were assessed at 4 h. Treatment with either RAP or sRAGE improved net AFC (median [interquartile range], 21.2 [18.8–21.7] and 19.5 [17.1–21.5] %/h, respectively, versus 12.6 [3.2–18.8] %/h in injured, untreated controls), oxygenation and decreased alveolar inflammation and histological evidence of tissue injury after ARDS. These findings suggest that RAGE inhibition restored AFC and attenuated lung injury in a piglet model of acid-induced ARDS.

Ursodeoxycholic acid stimulates alveolar fluid clearance in LPS-induced pulmonary edema via ALX/cAMP/PI3K pathway.

This study aims to examine the impact of ursodeoxycholic acid (UDCA) on pulmonary edema and explore the underlying molecular mechanisms. The effects of UDCA on pulmonary edema were assessed through hematoxylin and eosin (H&E) staining, lung dry/wet (W/D) ratio, TNF-α/IL-1β levels of bronchoalveolar lavage fluid (BALF), protein expression of epithelial sodium channel (ENaC), and Na+ /K+ -ATPase. Besides, the detailed mechanisms were explored in primary rat alveolar type (AT) II epithelial cells by determining the effects of BOC-2 (ALX [lipoxin A4 receptor] inhibitor), Rp-cAMP (cAMP inhibitor), LY294002 (PI3K inhibitor), and H89 (PKA inhibitor) on the therapeutic effects of UDCA against lipopolysaccharide (LPS)-induced changes. Histological examination suggested that LPS-induced lung injury was obviously attenuated by UDCA. BALF TNF-α/IL-1β levels and lung W/D ratios were decreased by UDCA in LPS model rats. UDCA stimulated alveolar fluid clearance (AFC) though the upregulation of ENaC and Na+ /K+ -ATPase. BOC-2, Rp-cAMP, and LY294002 largely suppressed the therapeutic effects of UDCA. Significant attenuation of pulmonary edema and lung inflammation was revealed in LPS-challenged rats after the UDCA treatment. The therapeutic efficacy of UDCA against LPS was mainly achieved through the ALX/cAMP/PI3K pathway. Our results suggested that UDCA might be a potential drug for the treatment of pulmonary edema induced by LPS.

Sodium‐coupled neutral amino acid transporter SNAT2 is critical for alveolar fluid transport and resolution of pulmonary edema

ObjectiveIn the intact lung, constant movement of Na+ and Cl− across the epithelial barrier regulates alveolar liquid volume and maintains a balance between fluid secretion and clearance, which is critical for normal lung function. Dysregulation or inhibition of Na+ transport can lead to accumulation of fluid in the airspace resulting in impaired gas exchange and respiratory failure. Previous studies have largely focused on the critical role of the amiloride‐sensitive epithelial sodium channel (ENaC) in alveolar fluid transport, yet activation of ENaC failed to reverse edema formation in clinical trials. Since 40–50% of alveolar fluid clearance is amiloride‐insensitive, sodium channels other than ENaC such as sodium‐coupled neutral amino acid transporter (SNAT, Slc38a2) 2 may provide for new potential therapeutic targets. SNAT2 co‐transports a neutral amino acid along with Na+ and may promote alveolar fluid clearance by mediating epithelial Na+ uptake. Here, we studied the role of SNAT2 in a murine model of lung injury, in isolated perfused lungs (IPL) and in a pulmonary epithelial cell culture System.MethodsFor functional in vitro analyses, L‐alanine transport across NCI‐H441 cells was analyzed by ELISA, with cells cultured in the presence or absence of amino acids, and following treatment with HgCl2, a SNAT inhibitor, or siRNA (control or siSNAT2).In IPL of slc38a2+/− and wildtype mice, edema formation was induced by hydrostatic stress (7 cm H2O) and fluid transport was assessed in the absence or presence of the SNAT2 inhibitor methylaminoisobutyric acid (MeAIB) or SNAT2 substrate L‐alanine in the alveolar instillate. Edema formation was assessed after 30 min of perfusion and ventilation by measurement of wet‐to‐dry lung weight ratio (W/D).For in vivo measurements of edema formation and resolution, acid (HCl) or saline was instilled intratracheally in slc38a2+/− and wildtype mice. After 2 h of mechanical ventilation, lungs were collected for W/D measurement.ResultsIn vitro, L‐alanine transport was significantly decreased in H441 cells treated with HgCl2 or SNAT2‐siRNA as compared to respective controls.In vivo, SNAT2 knockout (slc38a2−/−) mice were found to be sublethal and newborn pups typically succumbed to cyanotic dyspnea.In IPL, lungs of slc38a2+/− showed elevated W/D ratio as compared to wildtype lungs in response to hydrostatic stress and after treatment with the SNAT2 inhibitor MeAIB.Similarly, in HCl‐induced ALI, W/D ratio was increased in slc38a2+/− mice.ConclusionIn its function as Na+ transporter, we propose a crucial role for SNAT2 in alveolar fluid transport. Our results indicate that SNAT2 is functional relevant for alveolar fluid absorption and may contribute to the resolution of pulmonary edema. Hence, activation of SNAT2 may, provide a new therapeutic strategy to counteract and/or reverse formation of pulmonary edema.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Progesterone receptor isoform B expression in pulmonary neuroendocrine cells decreases cell proliferation

The progesterone receptor (PR) has been reported to play important roles in lung development and function, such as alveolarization, alveolar fluid clearance (AFC) and upper airway dilator muscle activity. In the lung, pulmonary neuroendocrine cells (PNECs) are important in the etiology and progression of lung neuroendocrine tumors (NETs). Women with lung NETs had significantly better survival rates than men, suggesting that sex steroids and their receptors, such as the PR, could be involved in the progression of lung NETs. The PR exists as two major isoforms, PRA and PRB. How the expression of different PR isoforms affects proliferation and the development of lung NETs is not well understood. To determine the role of the PR isoforms in PNECs, we constructed H727 lung NET cell models expressing PRB, PRA, Green Fluorescence Protein (GFP) (control). The expression of PRB significantly inhibited H727 cell proliferation better than that of PRA in the absence of progestin. The expression of the unrelated protein, GFP, had little to no effect on H727 cell proliferation. To better understand the role of the PR isoform in PNECs, we examined PR isoform expression in PNECs in lung tissues. A monoclonal antibody specific to the N-terminus of PRB (250H11 mAb) was developed to specifically recognize PRB, while a monoclonal antibody specific to a common N-terminus epitope present in both PRA and PRB (1294 mAb) was used to detect both PRA and PRB. Using these PR and PRB-specific antibodies, we demonstrated that PR (PRA&PRB) and PRB were expressed in the PNECs of the normal fetal and adult lung, with significantly higher PR expression in the fetal lung. Interestingly, PRB expression in the normal lung was associated with lower cell proliferation than PR expression, suggesting a distinct role of PRB in the PNECs. A better understanding of the molecular mechanism of PR and PR isoform signaling in lung NET cells may help in developing novel therapeutic strategies that will benefit lung NET patients in the future.

RETRACTED ARTICLE: Overexpressing p130/E2F4 in mesenchymal stem cells facilitates the repair of injured alveolar epithelial cells in LPS-induced ARDS mice

BackgroundLow differentiation rates of mesenchymal stem cells (MSCs) limit their therapeutic effects on patients in clinical studies. Our previous study demonstrated that overexpressing p130 or E2F4 affected the multipotential differentiation of MSCs, and the underlying mechanism was attributed to the regulation of the G1 phase. Improving the efficiency of MSC differentiation into epithelial cells is considered to be a new method. Therefore, this study was conducted to evaluate the effects of overexpressing p130 or E2F4 in MSCs on improving re-epithelization in lipopolysaccharide (LPS)-induced ARDS animals.MethodsMouse MSCs (mMSCs) stably transfected with p130 and E2F4 were transplanted intratracheally into LPS-induced ARDS mice. After 7 and 14 days, the mice were sacrificed, and the histopathology of the lungs was assessed by haematoxylin-eosin staining and lung injury scoring. Homing and differentiation of mMSCs were analysed by labelling and tracking mMSCs with NIR815 dye and immunofluorescent staining. Surfactant proteins A and C and occludin in the lungs were assessed by western blot. Permeability was evaluated by analysing the protein concentration of BALF using ELISA. Alveolar fluid clearance was assessed by absorbance measurements of BALF. Lung fibrosis was assessed by Masson’s trichrome staining and Ashcroft scoring.ResultsThe engraftment of mMSCs overexpressing p130 or E2F4 led to attenuated histopathological impairment of the lung tissue, and the lung injury scores of the LPS+mBM-MSC-p130 and LPS+mBM-MSC-E2F4 groups were also decreased (p < 0.05). Overexpression of p130 or E2F4 also increased the retention of mMSCs in the lung (p < 0.05), increased differentiation into type II alveolar epithelial cells (p < 0.05), and improved alveolar epithelial permeability (p < 0.05). Additionally, mMSCs overexpressing p130 or E2F4 inhibited lung fibrosis according to the deposition of collagen and the fibrosis score in the lungs (p < 0.05).ConclusionOverexpressing p130 or E2F4 in mMSCs could further improve the injured structure and function of epithelial cells in the lungs of ARDS mice as a result of improved differentiation of mMSCs into epithelial cells.