Lung Endothelial Permeability Research Articles

PLD2 deficiency alleviates endothelial glycocalyx degradation in LPS-induced ARDS/ALI

- Acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a life-threatening condition marked by severe lung inflammation and increased lung endothelial barrier permeability. Endothelial glycocalyx deterioration is the primary factor of vascular permeability changes in ARDS/ALI. Although previous studies have shown that phospholipase D2 (PLD2) is closely related to the onset and progression of ARDS/ALI, its role and mechanism in the damage of endothelial cell glycocalyx remains unclear. We used LPS-induced ARDS/ALI mice (in vivo) and LPS-stimulated injury models of EA.hy926 endothelial cells (in vitro). We employed C57BL/6 mice, including wild-type and PLD2 knockout (PLD2−/−) mice, to establish the ARDS/ALI model. We applied immunofluorescence and ELISA to examine changes in syndecan-1 (SDC-1), matrix metalloproteinase-9 (MMP9), inflammatory cytokines (TNF-α, IL-6, and IL-1β) levels and the effect of external factors, such as phosphatidic acid (PA), 1-butanol (a PLD inhibitor), on SDC-1 and MMP9 expression levels. We found that PLD2 deficiency inhibits SDC-1 degradation and MMP9 expression in LPS-induced ARDS/ALI. Externally added PA decreases SDC-1 levels and increases MMP9 in endothelial cells, hence underlining PA's role in SDC-1 degradation. Additionally, PLD2 deficiency decreases the production of inflammatory cytokines (TNF-α, IL-6, and IL-1β) in LPS-induced ARDS/ALI. In summary, these findings suggest that PLD2 deficiency plays a role in inhibiting the inflammatory process and protecting against endothelial glycocalyx injury in LPS-induced ARDS/ALI.

Etanercept’s Protective Effect Against Lung Inflammation Caused by Polymicrobial Sepsis Induced by CLP in Male Rats

Inflammation and oxidative stress are critical factors leading to sepsis-induced severe lung injury (ALI). This study investigates the regulatory effects of Etanercept (ENT) in an animal model of sepsis-induced ALI. We established a cecal ligation and puncture (CLP) model for ALI in adult rats under anesthesia. Sepsis amplifies pathological lung tissue damage, lung endothelial permeability, serum inflammatory factor production, and the number of inflammatory cells in bronchoalveolar lavage fluid. Furthermore, it enhances the expression of phosphorylated NF-\(\kappa\)B p65, reduces lung superoxide dismutase due to CLP, and elevates lung malondialdehyde levels due to LPS-induced inflammatory factor production. This cascade of events exacerbates the expression of several genes, including TNF-\(\alpha\) and cytokines. LPS-induced inflammatory factor production also enhances the formation of reactive oxygen species in mitochondria. In summary, our current research provides evidence that ENT acts as a novel regulator of inflammation in sepsis-induced ALI in rats. The control of oxidative stress and the modulation of the inflammatory response are two key underlying mechanisms for the effects of ENT.

Abstract 15352: Lung Endothelial Transport in Glypican 1 Knockout Mice

Introduction: Pulmonary edema is a life-threatening condition that can result from the acute increase in hemodynamic forces in the lung vasculature. The mechanisms whereby pulmonary edema develops are unresolved but increased lung endothelial permeability may be important. The endothelial glycocalyx is a surface layer known to mediate the effects of mechanical forces in the lung vasculature by eNOS-dependent mechanisms. Among the components of the glycocalyx, glypican 1 (Gpc1) is considered a potential mechano-transducer due to its physical interaction with the cellular membrane. Herein we sought to investigate the mechanisms whereby Gpc1 regulates pulmonary permeability. Hypothesis: We hypothesize that Gpc1 regulates endothelial transport by eNOS-dependent mechanisms. Methods: Mouse lung endothelial cells (MLEC) isolated from male and female wild-type (WT) and Gpc1 knockout mice (Gpc1KO, 6-8 weeks old) were used. Endothelial transport was assessed by dextran permeability in vitro, lung barrier stability was assessed by TEER, eNOS pathway was assessed by immunoblotting. To assess the role of eNOS to Gpc1 effects on endothelial transport, MLEC were exposed to an eNOS decoy peptide. Results: Gpc1KO MLEC formed a tighter barrier in vitro when compared to WT MLEC (3.18±0.11 [Gpc1KO] vs 2.34±0.11 [WT], normalized TEER, n=3/group). This was preserved during high pressure conditions. High pressure increased transcellular but not paracellular transport in both WT and Gpc1KO MLEC (1.8 fold in WT and 1.2 fold in Gpc1KO, n=6/group). Gpc1KO showed decreased eNOS expression (0.3 fold, n=4/group ) and activity during (1.947±0.32 [WT] vs 1.316±0.13 [Gpc1KO], n=4/group). The eNOS decoy peptide increased transcellular transport only in Gpc1KO MLEC (3 fold, n=5/group). Conclusions: In conclusion, these data suggest that intracellular transport may be the main mechanism for pressure-induced changes in endothelial permeability in MLEC . The mechanisms whereby Gpc1 regulates transcellular transport seem to be associated with eNOS signaling. This may be important for the management of acute changes in pressure seen in clinical settings when NO donors are used as vasodilators to decrease blood pressure.

Hyaluronic acid restored protein permeability across injured human lung microvascular endothelial cells.

Lung endothelial permeability is a key pathological feature of acute respiratory distress syndrome. Hyaluronic acid (HA), a major component of the glycocalyx layer on the endothelium, is generated by HA synthase (HAS) during inflammation and injury and is critical for repair. We hypothesized that administration of exogenous high molecular weight (HMW) HA would restore protein permeability across human lung microvascular endothelial cells (HLMVEC) injured by an inflammatory insult via upregulation of HAS by binding to CD44. A transwell coculture system was used to study the effects of HA on protein permeability across HLMVEC injured by cytomix, a mixture of IL‐1β, TNFα, and IFNγ, with or without HMW or low molecular weight (LMW) HA. Coincubation with HMW HA, but not LMW HA, improved protein permeability following injury at 24 h. Fluorescence microscopy demonstrated that exogenous HMW HA partially prevented the increase in “actin stress fiber” formation. HMW HA also increased the synthesis of HAS2 mRNA expression and intracellular HMW HA levels in HLMVEC following injury. Pretreatment with an anti‐CD44 antibody or 4‐methylumbelliferone, a HAS inhibitor, blocked the therapeutic effects. In conclusion, exogenous HMW HA restored protein permeability across HLMVEC injured by an inflammatory insult in part through upregulation of HAS2.

Evaluation of Dynamic Contrast-Enhanced MRI Measures of Lung Congestion and Endothelial Permeability in Heart Failure: A Prospective Method Validation Study.

BackgroundMethods for accurate quantification of lung fluid in heart failure (HF) are needed. Dynamic contrast‐enhanced (DCE)‐MRI may be an appropriate modality.PurposeDCE‐MRI evaluation of fraction of fluid volume in the interstitial lung space (v e ) and vascular permeability (K trans).Study TypeProspective, single‐center method validation.PopulationSeventeen evaluable healthy volunteers (HVs), 12 participants with HF, and 3 with acute decompensated HF (ADHF).Field Strength/Sequence T 1 mapping (spoiled gradient echo variable flip angle acquisition) followed by dynamic series (three‐dimensional spoiled gradient‐recalled echo acquisitions [constant echo time, repetition time, and flip angle at 1.5 T]).AssessmentThree whole‐chest scans were acquired: baseline (Session 1), 1‐week later (Session 2), following exercise (Session 3). Extended Tofts model quantified v e and K trans (voxel‐wise basis); total lung median measures were extracted and fitted via repeat measure analysis of variance (ANOVA) model. Patient tolerability of the scanning protocol was assessed.Statistical TestsThis was constructed as an experimental medicine study. Primary endpoints: K trans and v e at baseline (HV vs. HF), change in K trans and v e following exercise, and following lung congestion resolution (ADHF). K trans and v e were fitted separately using ANOVA. Secondary endpoint: repeatability, that is, within‐participant variability in v e and K trans between sessions (coefficient of variation estimated via mixed effects model).ResultsThere was no significant difference in mean K trans between HF and HV (P ≤ 0.17): 0.2216 minutes−1 and 0.2353 minutes−1 (Session 1), 0.2044 minutes−1 and 0.2567 minutes−1 (Session 2), 0.1841 minutes−1 and 0.2108 minutes−1 (Session 3), respectively. v e was greater in the HF group (all scans, P ≤ 0.02). Results were repeatable between Sessions 1 and 2; mean values for HF and HV were 0.4946 and 0.3346 (Session 1), 0.4353 and 0.3205 (Session 2), respectively. There was minimal difference in K trans or v e between scans for participants with ADHF (small population precluded significance testing). Scanning was well tolerated.Data ConclusionWhile no differences were detected in K trans, v e was greater in chronic HF patients vs. HV, augmented beyond plasma and intracellular volume. DCE‐MRI is a valuable diagnostic and physiologic tool to evaluate changes in fluid volume in the interstitial lung space associated with symptomatic HF.Level of Evidence2Technical Efficacy Stage2

Functional Transcriptomic Studies of Immune Responses and Endotoxin Tolerance in Early Human Sepsis.

Limited studies have functionally evaluated the heterogeneity in early ex vivo immune responses during sepsis. Our aim was to characterize early sepsis ex vivo functional immune response heterogeneity by studying whole blood endotoxin responses and derive a transcriptional metric of ex vivo endotoxin response. Blood collected within 24 h of hospital presentation from 40 septic patients was divided into two fractions and incubated with media (unstimulated) or endotoxin. Supernatants and cells were isolated, and responses measured using: supernatant cytokines, lung endothelial permeability after supernatant exposure, and RNA expression. A transcriptomic signature was derived in unstimulated cells to predict the ex vivo endotoxin response. The signature was tested in a separate cohort of 191 septic patients to evaluate for association with clinical outcome. Plasma biomarkers were quantified to measure in vivo host inflammation. Ex vivo response to endotoxin varied and was unrelated to immunosuppression, white blood cell count, or the causative pathogen. Thirty-five percent of patients demonstrated a minimal response to endotoxin, suggesting early immunosuppression. High ex vivo cytokine production by stimulated blood cells correlated with increased in vitro pulmonary endothelial cell permeability and was associated with attenuated in vivo host inflammation. A four-gene signature of endotoxin response detectable without the need for a functional assay was identified. When tested in a separate cohort of septic patients, its expression was inversely associated with hospital mortality. An attenuated ex vivo endotoxin response in early sepsis is associated with greater host in vivo inflammation and a worse clinical outcome.

Essential role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, ROS generation and lung endothelial barrier loss

We have shown that both reactive oxygen species (ROS) and paxillin tyrosine phosphorylation regulate LPS-induced human lung endothelial permeability. Mitochondrial ROS (mtROS) is known to increase endothelial cell (EC) permeability which requires dynamic change in mitochondrial morphology, events that are likely to be regulated by paxillin. Here, we investigated the role of paxillin and its tyrosine phosphorylation in regulating LPS-induced mitochondrial dynamics, mtROS production and human lung microvascular EC (HLMVEC) dysfunction. LPS, in a time-dependent manner, induced higher levels of ROS generation in the mitochondria compared to cytoplasm or nucleus. Down-regulation of paxillin expression with siRNA or ecto-expression of paxillin Y31F or Y118F mutant plasmids attenuated LPS-induced mtROS in HLMVECs. Pre-treatment with MitoTEMPO, a scavenger of mtROS, attenuated LPS-induced mtROS, endothelial permeability and VE-cadherin phosphorylation. Further, LPS-induced mitochondrial fission in HLMVECs was attenuated by both a paxillin siRNA, and paxillin Y31F/Y118F mutant. LPS stimulated phosphorylation of dynamin-related protein (DRP1) at S616, which was also attenuated by paxillin siRNA, and paxillinY31/Y118 mutants. Inhibition of DRP1 phosphorylation by P110 attenuated LPS-induced mtROS and endothelial permeability. LPS challenge of HLMVECs enhanced interaction between paxillin, ERK, and DRP1, and inhibition of ERK1/2 activation with PD98059 blocked mitochondrial fission. Taken together, these results suggest a key role for paxillin tyrosine phosphorylation in LPS-induced mitochondrial fission, mtROS generation and EC barrier dysfunction.

Human mesenchymal stromal cells small extracellular vesicles attenuate sepsis-induced acute lung injury in a mouse model: the role of oxidative stress and the mitogen-activated protein kinase/nuclear factor kappa B pathway

Background aimsAcute lung injury (ALI) secondary to sepsis is a complex disease associated with high morbidity and mortality. Mesenchymal stem cells (MSCs) and their conditioned medium have been demonstrated to reduce alveolar inflammation, improve lung endothelial barrier permeability and modulate oxidative stress in vivo and in vitro. Recently, MSCs have been found to release small extracellular vesicles (sEVs) that can deliver functionally active biomolecules into recipient cells. The authors’ study was designed to determine whether sEVs released by MSCs would be effective in sepsis-induced ALI mice and to identify the potential mechanisms. MethodsA total of 6 h after cercal ligation and puncture, the mice received saline, sEV-depleted conditioned medium (sEVD-CM) or MSC sEVs via the tail vein. ResultsThe administration of MSC sEVs improved pulmonary microvascular permeability and inhibited both histopathological changes and the infiltration of polymorphonuclear neutrophils into lung tissues. In addition, the activities of antioxidant enzymes were significantly increased in the group treated with sEVs compared with the saline and sEVD-CM groups, whereas lipid peroxidation was significantly decreased. Furthermore, sEVs were found to possibly inhibit phosphorylation of the mitogen-activated protein kinase/nuclear factor kappa B (MAPK/NF-κB) pathway and degradation of IκB but increase the activities of nuclear factor erythroid 2-related factor 2 and heme oxygenase 1. ConclusionsThese findings suggest that one of the effective therapeutic mechanisms of sEVs against sepsis-induced ALI may be associated with upregulation of anti-oxidative enzymes and inhibition of MAPK/NF-κB activation.

UCHL1, a deubiquitinating enzyme, regulates lung endothelial cell permeability in vitro and in vivo.

Increasing evidence suggests an important role for deubiquitinating enzymes (DUBs) in modulating a variety of biological functions and diseases. We previously identified the upregulation of the DUB ubiquitin carboxyl terminal hydrolase 1 (UCHL1) in murine ventilator-induced lung injury (VILI). However, the role of UCHL1 in modulating vascular permeability, a cardinal feature of acute lung injury (ALI) in general, remains unclear. We investigated the role of UCHL1 in pulmonary endothelial cell (EC) barrier function in vitro and in vivo and examined the effects of UCHL1 on VE-cadherin and claudin-5 regulation, important adherens and tight junctional components, respectively. Measurements of transendothelial electrical resistance confirmed decreased barrier enhancement induced by hepatocyte growth factor (HGF) and increased thrombin-induced permeability in both UCHL1-silenced ECs and in ECs pretreated with LDN-57444 (LDN), a pharmacological UCHL1 inhibitor. In addition, UCHL1 knockdown (siRNA) was associated with decreased expression of VE-cadherin and claudin-5, whereas silencing of the transcription factor FoxO1 restored claudin-5 levels. Finally, UCHL1 inhibition in vivo via LDN was associated with increased VILI in a murine model. These findings support a prominent functional role of UCHL1 in regulating lung vascular permeability via alterations in adherens and tight junctions and implicate UCHL1 as an important mediator of ALI.

MicroRNA-451 increases vascular permeability and suppresses angiogenesis in pulmonary burn injury in a rat model

Burns are common traumas that cause systemic symptoms by increasing vascular permeability. To investigate the role of miRNA-451 and to clarify the underlying mechanism of the burn process. We established a heat-induced third-degree burn with acute lung injury (ALI) model in rats. Hematoxylin and eosin (H&E) staining and in situ hybridization were performed. Overexpressed miRNA-451 in human umbilical vascular endothelial cells (HUVEC) were carried out. The migration and proliferation of HUVEC cells were examined. The H&E staining showed that the burn injury caused by heat went through the dermis and damaged deep tissues. Meanwhile, the heat also induced acute lung injury, characterized by inflammatory exudation in the alveoli and significant enlargement of the alveolar septum. In situ hybridization showed that the expression of miRNA-451 increased in the lung endothelial cells. We overexpressed miRNA-451 in human umbilical vascular endothelial cells (HUVEC) and the results showed that miRNA-451 inhibited the migration and proliferation of HUVEC cells, increased HUVEC cell permeability, inhibited cell adhesion, and induced cell apoptosis. Furthermore, the expression of occludin and ZO-1, 2 key protein molecules in forming tight junction between cells, decreased, and the proteins dispersed in the cytoplasm of HUVEC cells. MiRNA-451 was upregulated in the lung endothelial cells of the rat model, and contributed to increase lung endothelial cell permeability. It suppresses angiogenesis of lung endothelial cells, indicating their potential as a target in the treatment of burn injuries.

Unfolded protein response in the COVID-19 context

The unfolded protein response (UPR) maintains cellular homeostasis by regulating key elements of cellular growth and defense. Recent evidence suggests that this mechanism affects the vascular barrier function, by modulating lung endothelial permeability. Dysregulation of this barrier contributes in the irreversible outcomes of the SARS-CoV-2 - inflicted acute respiratory distress syndrome (ARDS). Thus, it is highly probable that the targeted activation of those UPR components in charge of repairing the destructed lung endothelium of the COVID-19 patients, may deliver a promising therapeutic possibility for those subjected to the devastating outcomes of the ongoing pandemic.

PV1 in Caveolae Controls Lung Endothelial Permeability

Caveolae are prominent plasmalemmal invaginations in endothelial cells, especially in the lung vasculature, which comprises a vast surface area. PV1 (plasmalemmal vesicle-associated protein-1), a 60-kD glycoprotein expressed in endothelial cells, is essential for generating spoke-like diaphragmatic structures that span the neck region of endothelial caveolae. However, their role in caveolae-mediated uptake and endothelial-barrier function is unknown. Here, we generated mice with endothelial cell-specific deletion of PV1 through tamoxifen-induced Cdh5.Cre.ERT2 (endothelial-specific vascular cadherin.Cre.estrogen receptor 2)-mediated excision of the floxed PV1 allele. We observed that loss of PV1 specifically in endothelial cells increased lung vascular permeability of fluid and protein, indicating that PV1 is required for maintenance of lung vascular-barrier integrity. Endothelial-specific PV1 deletion also increased caveolae-mediated uptake of tracer albumin compared with controls, promoted Au-albumin accumulation in the bulb of caveolae, and induced caveolar swelling. In addition, we observed the progressive loss of plasma proteins from the circulation and reduced arterial pressure resulting from transudation of water and protein as well as edema formation in multiple tissues, including lungs. These changes seen after endothelial-specific PV1 deletion occurred in the absence of disruption of endothelial junctions. We demonstrated that exposure of wild-type mice to endotoxin, which is known to cause acute lung injury and increase protein permeability, also significantly reduced PV1 protein expression. We conclude that the key function of PV1 is to regulate lung endothelial permeability through its ability to restrict the entry of plasma proteins such as albumin into caveolae and their transport through the endothelial barrier.

Inhibition of ERRα Aggravates Sepsis-Induced Acute Lung Injury in Rats via Provoking Inflammation and Oxidative Stress.

Inflammation and oxidative stress are critical pathologies that contribute to sepsis-induced acute lung injury (ALI). This study investigated the regulatory role of estrogen-related receptor alpha (ERRα) in an experimental model of sepsis-induced ALI. In vivo, a cecal ligation and puncture- (CLP-) induced ALI model was established in anesthetized rats. Animals were then randomly assigned to receive an intraperitoneal injection of vehicle or ERRα inverse agonist (XCT-790, 2.5 mg/kg). Administration of XCT-790 significantly aggravated a sepsis-induced increase in pathological damage of lung tissues, lung endothelial permeability, myeloperoxidase (MPO) activity in lung tissues, production of serum inflammatory factors, and inflammatory cell accumulation in bronchoalveolar lavage fluid. In addition, XCT-790 treatment exacerbated a CLP-induced decrease in lung superoxide dismutase and an increase in lung malondialdehyde levels. In vitro, the exposure of rat pulmonary microvascular endothelial cells (PMVECs) to lipopolysaccharide (LPS) resulted in increased endothelial permeability and reduced expression of tight junction protein ZO-1, Occludin, JAM-A, and adherens junction protein VE-cadherin, which were further deteriorated by knockdown of ERRα. In addition, LPS-triggered inflammatory factor production and increase in the expression of phosphorylated IκBα and NF-κB p65 were also exacerbated by silencing ERRα gene. Meanwhile, knockdown of ERRα dramatically promoted LPS-activated mitochondrial reactive oxygen species production and LPS-induced downregulation of Sirt3 protein levels in rat PMVECs. Taken together, our present study provides evidences that ERRα functions as a novel negative modulator of sepsis-induced ALI in rats. The underlying mechanisms responsible for ERRα-elicited effects are largely dependent on the regulation of inflammatory response and oxidative stress.

Loss of AC10 impairs neutrophil recruitment into the lung following Pseudomonas aeruginosa infection

Pseudomonas aeruginosa is a gram‐negative, opportunistic bacteria and a common cause of nosocomial pneumonia in critically ill patients that can progress to acute respiratory distress syndrome (ARDS). These patients have impaired gas exchange leading to hypoxia, altered blood pH and elevated bicarbonate. Bicarbonate, delivered into the cell via sodium bicarbonate cotransporters (NBCs), activates adenylyl cyclase 10 (AC10) to increase intracellular cAMP. Indeed, bicarbonate activation of AC10 is required for LPS‐induced lung endothelial permeability. Neutrophil recruitment into the airspaces plays a central role in the progression of ARDS. Recently, AC10 has been implicated in transendothelial emigration of neutrophils from the blood into the underlying tissue via CD99, a transmembrane protein critical for neutrophil recruitment. We sought to determine whether AC10 is necessary for neutrophil recruitment into the airspaces following P. aeruginosa intratracheal inoculation. Increasing doses of bacteria were introduced directly into the lung of wild type and AC10 and NBCn2 knockout mice. Bronchoalveolar lavage fluid (BALF) was recovered 24 hours later and examined for recruitment of polymorphonuclear cells (PMNs) into the airspaces. The number of PMNs recruited into the airspace following Pseudomonas inoculation increased with bacterial number and reached a maximum at a dose of 1E5 bacteria after which 100% of cells in the BALF were PMNs. Thus, doses upto 1E5 bacteria were used to examine the role of AC10 and NBCn2 in PMN recruitment into the airspace between wild type and knockout animals. Our data reveal a decreased number of PMNs in the BALF of both AC10 and NBCn2 knockout female mice; however, no difference in the number of PMNs recruited into the airspace was detected between male wild type and knockout animals. In addition, we confirmed the expression of CD99 in pulmonary microvascular endothelial cells and whole lung from both rat and mouse. Thus, CD99 is expressed in pulmonary endothelial cells and female mice utilize AC10 and NBCn2 to facilitate neutrophil recruitment into the airspace following a model of P. aeruginosa pneumonia.Support or Funding InformationNIH R01HL121513

ISSUE HIGHLIGHTS

IUBMB LifeVolume 72, Issue 4 p. 523-523 ISSUE HIGHLIGHTSFree Access ISSUE HIGHLIGHTS First published: 11 March 2020 https://doi.org/10.1002/iub.2243AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat 706 Association between dietary inflammatory index and risk of cardiovascular disease in the Mashhad stroke and heart atherosclerotic disorder study population Zahra Asadi, Mahdiyeh Yaghooti-Khorasani, Hamideh Ghazizadeh, Fatemeh Sadabadi, Ehsan Mosa-Farkhany, Susan Darroudi, Niloofar Shabani, Atiyeh Kamel-khodabandeh, Afsane Bahrami, Mohammad-Sadegh Khorrami-Mohebbseraj, Sahar Heidari-Bakavoli, Alireza Heidari-Bakavoli, Habibollah Esmaily, Mohsen Moohebati, Mohammad R. Oladi, Nitin Shivappa, James R. Hébert, Gordon A. Ferns, Majid Ghayour-Mobarhan Thedietary inflammatory index (DII®) as a novel index for evaluating the inflammatory potential of an individual's diet calculated based on the pro/anti-inflammatory macro/micro-nutrients of diet. In theMASHADcohort study population,weused a food frequency questionnaire to provide datafor calculating DII®.This cohort study showedthat thedietary intake of vegetable, fruitsand legume were higher among those with anti-inflammatory diet; while, higher dietary intake of carbohydrate beverages and processedmeat were observed in subjects with pro-inflammatory diet.The percentage ofCVDevent in subjects with pro andanti-inflammatorydietwere2.5% and 2.8%,respectivelybut werenot significant in adultsmiddle-agedof theIranianpopulation. Itmay be because the study period is partially short and there are various lifestyle factors such as diet among different ethnicities of Iranian people. Thus, future studies should be conducted in different geographic locations in theworld to confirmthese findings. 716 Neonicotinoid trapping by the FA1 site of human serum albumin Loris Leboffe, Alessandra di Masi, Viviana Trezza, Andrea Pasquadibisceglie, Gabriele Macari, Fabio Polticelli, Paolo Ascenzi Neonicotinoids are a widely used class of insecticides.Although they display a high specificity for insects, their use has been recently debated since it has been hypothesized that they may have adverse ecological effects and potential risks to mammals and even humans. Due to their hydrophobicnature,neonicotinoidsneedspecificcarrierstoallowtheirdistributioninbodyfluids. Human serum albumin (HSA), the most abundant plasma protein, is a key carrier of endogenous and exogenous compounds. In particular, HSA is essential for neonicotinoid transport and distribution to tissues and organs, including the liver where they are metabolized. 801 Regulation of lung endothelial permeability by NEK kinases Nektarios Barabutis Lung hyperpermeability is the cause and consequence of the acute respiratory distress syndrome(ARDS).Tocounteract thelethaloutcomesof this respiratorydisorder,weinvestigate the molecular cascades which regulate the pulmonary endothelial barrier function. It was recentlyrevealedthatP53inductionbytheunfoldedproteinresponseinducersgrowthhormone releasing hormone antagonists and heat shock protein 90 inhibitors; enhances endothelial barrier function. Since NEK kinases have been shown to regulate the activities of P53, it is anticipated that the stochasticmanipulation of one ormoremember(s) of this family of kinases; may deliver a promising therapeutic approach towards ARDS. REFERENCES 1Asadi, Z., Yaghooti-Khorasani, M., Ghazizadeh, H., Sadabadi, F., Mosa-Farkhany, E., et al. (2020) Association between dietary inflammatory index and risk of cardiovascular disease in the Mashhad stroke and heart atherosclerotic disorder study population. IUBMB Life 72, 706– 715. 2Leboffe, L., di Masi, A., Trezza, V., Pasquadibisceglie, A., Macari, G., et al. (2020) Neonicotinoid trapping by the FA1 site of human serum albumin. IUBMB Life. 72, 716– 723. 3Barabutis, N. (2020) Regulation of lung endothelial permeability by NEK kinases. IUBMB Life. 72, 801– 804. Volume72, Issue4Special Issue: Molecular Mechanisms of Neuronal Trafficking and Synapse Remodeling From Focused Meeting MNS 2019April 2020Pages 523-523 ReferencesRelatedInformation

Regulation of lung endothelial permeability by NEK kinases.

Dysregulation of lung endothelial barrier function may lead to lethal outcomes, as demonstrated in the case of the acute respiratory distress syndrome (ARDS). p53 participates in the regulation of the lung endothelial barrier, and it has been associated both in vivo and in vitro with protective effects against the LPS-induced hyperpermeability. Family members of the never in mitosis A-related kinases (NEKs) are crucial mediators of fundamental cellular processes, including mitosis, and have been shown to posttranslationally modify p53. Since such modifications affect p53 stability and activity, it is highly probable that NEK kinases are also regulators of lung endothelial permeability. Thus, they may serve as possible therapeutic targets for treatment of pathologies associated with endothelial barrier dysfunction.

Contrasting effects of stored allogeneic red blood cells and their supernatants on permeability and inflammatory responses in human pulmonary endothelial cells.

Transfusion of red blood cells (RBCs) is a common life-saving clinical practice in severely anemic or hemorrhagic patients; however, it may result in serious pathological complications such as transfusion-related acute lung injury. The factors mediating the deleterious effects of RBC transfusion remain unclear. In this study, we tested the effects of washed long-term (RBC-O; >28 days) versus short-term (RBC-F; <14 days) stored RBCs and their supernatants on lung endothelial (EC) permeability under control and inflammatory conditions. RBCs enhanced basal EC barrier function as evidenced by an increase in transendothelial electrical resistance and decrease in permeability for macromolecules. RBCs also attenuated EC hyperpermeability and suppressed secretion of EC adhesion molecule ICAM-1 and proinflammatory cytokine IL-8 in response to LPS or TNF-α. In both settings, RBC-F had slightly higher barrier protective effects as compared with RBC-O. In contrast, supernatants from both RBC-F and RBC-O disrupted the EC barrier. The early phase of EC permeability response caused by RBC supernatants was partially suppressed by antioxidant N-acetyl cysteine and inhibitor of Src kinase family PP2, while addition of heme blocker and inhibition of NOD-like receptor family pyrin domain containing protein 3 (NLRP3), stress MAP kinases, receptor for advanced glycation end-products (RAGE), or Toll-like receptor-4 (TLR4) signaling were without effect. Morphological analysis revealed that RBC supernatants increased LPS- and TNF-α-induced breakdown of intercellular junctions and formation of paracellular gaps. RBC supernatants augmented LPS- and TNF-α-induced EC inflammation reflected by increased production of IL-6, IL-8, and soluble ICAM-1. These findings demonstrate the deleterious effects of RBC supernatants on EC function, which may have a major impact in pathological consequences associated with RBC transfusion.

Chemokine (C-X-C motif) receptor 4 regulates lung endothelial barrier permeability during resuscitation from hemorrhagic shock

Chemokine (C-X-C motif) receptor 4 (CXCR4) agonists have been shown to protect lung endothelial barrier function in vitro. In vivo effects of CXCR4 modulation on lung endothelial permeability are unknown. Here we tested the effects of the CXCR4 agonist ubiquitin and the antagonist AMD3100 on lung vascular permeability and cytokine concentrations in a rat hemorrhage model. Animals were hemorrhaged (mean arterial blood pressure 30 mmHg for 30 min), treated with vehicle, ubiquitin (0.7 and 3.5 µmol/kg) or AMD3100 (3.5 µmol/kg), and resuscitated with crystalloids. Evans blue extravasation was employed to quantify lung vascular permeability. Ubiquitin dose-dependently reduced Evans blue extravasation into the lung. AMD3100 increased Evans blue extravasation. With AMD3100, TNFalpha levels in lung homogenates were increased; while TNFalpha levels were lower with ubiquitin, these differences did not reach statistical significance. Our findings suggest that CXCR4 regulates lung vascular permeability and further point towards CXCR4 as a drug target to confer lung protection during resuscitation from traumatic-hemorrhagic shock.

Pressure-dependent NOS activation contributes to endothelial hyperpermeability in a model of acute heart failure

Aims: Acute increases in left ventricular end diastolic pressure (LVEDP) can induce pulmonary edema (PE). The mechanism(s) for this rapid onset edema may involve more than just increased fluid filtration. Lung endothelial cell permeability is regulated by pressure-dependent activation of nitric oxide synthase (NOS). Herein, we demonstrate that pressure-dependent NOS activation contributes to vascular failure and PE in a model of acute heart failure (AHF) caused by hypertension.Methods and results: Male Sprague–Dawley rats were anesthetized and mechanically ventilated. Acute hypertension was induced by norepinephrine (NE) infusion and resulted in an increase in LVEDP and pulmonary artery pressure (Ppa) that were associated with a rapid fall in PaO2, and increases in lung wet/dry ratio and injury scores. Heart failure (HF) lungs showed increased nitrotyrosine content and ROS levels. L-NAME pretreatment mitigated the development of PE and reduced lung ROS concentrations to sham levels. Apocynin (Apo) pretreatment inhibited PE. Addition of tetrahydrobiopterin (BH4) to AHF rats lung lysates and pretreatment of AHF rats with folic acid (FA) prevented ROS production indicating endothelial NOS (eNOS) uncoupling.Conclusion: Pressure-dependent NOS activation leads to acute endothelial hyperpermeability and rapid PE by an increase in NO and ROS in a model of AHF. Acute increases in pulmonary vascular pressure, without NOS activation, was insufficient to cause significant PE. These results suggest a clinically relevant role of endothelial mechanotransduction in the pathogenesis of AHF and further highlights the concept of active barrier failure in AHF. Therapies targetting the prevention or reversal of endothelial hyperpermeability may be a novel therapeutic strategy in AHF.

Connexin 40 regulates lung endothelial permeability in acute lung injury via the ROCK1-MYPT1- MLC20 pathway.

Increased pulmonary vascular permeability is a hallmark of acute lung injury (ALI). Connexin 40 (Cx40) is a gap junctional protein abundantly present in the lung microvascular endothelium. Yet, the role of Cx40 in the regulation of lung vascular permeability and its underlying mechanisms are unclear. Here, we tested the hypothesis that Cx40 participates in regulation of lung endothelial permeability via a mechanism involving a Rho-associated protein kinase (ROCK) dependent regulation of myosin light chain (MLC). In murine models of intratracheal acid- or LPS-induced lung injury, genetic deficiency of Cx40 attenuated key features of ALI including vascular barrier failure. In human pulmonary microvascular endothelial cells (PMVECs), thrombin-induced loss of transendothelial electrical resistance was attenuated by a Cx40-inhibiting mimetic peptide (40GAP27), Cx40-specific shRNA, or ROCK inhibitor Y27632. In isolated perfused mouse lungs, platelet-activating factor-induced lung weight gain was abrogated by gap junction blocker carbenoxolone, 40GAP27, Y27632, or genetic deficiency of Cx40. Phosphorylation of MLC20 increased drastically in both LPS-treated PMVECs and HCl-treated mouse lungs. Expression of ROCK1 was increased in both LPS-treated PMVECs and HCl-treated mouse lungs, and paralleled by phosphorylation of MLC20. Coimmunoprecipitation experiments revealed protein-protein interaction between ROCK1 and Cx40. LPS-induced upregulation of ROCK1 and phosphorylation of MLC20 were blocked by knockdown of Cx40. LPS caused phosphorylation of myosin phosphatase targeting subunit 1, which could be abrogated by Y27632 or Cx40-shRNA. Our findings reveal a role of Cx40 in regulation of ROCK1 and MLC20 that contributes critically to lung vascular barrier failure in ALI.