Increased Lung Vascular Permeability Research Articles

Protectin DX Relieve Hyperoxia-induced Lung Injury by Protecting Pulmonary Endothelial Glycocalyx.

Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in premature infants with limited treatments and poor prognosis. Damaged endothelial glycocalyx leads to vascular permeability, lung edema and inflammation. However, whether hyperoxia increases neonatal pulmonary microvascular permeability by degrading the endothelial glycocalyx remains unknown. Newborn mice were maintained in 60-70% O2 for 7 days. Protectin DX (PDX), an endogenous lipid mediator, was injected intraperitoneally on postnatal d 0, 2, 4 and 6. Lung samples and bronchoalveolar lavage fluid were taken at the end of the study. Primary human umbilical vein endothelial cells (HUVECs) were cultured in 80%O2. Hyperoxia exposure for 7 days led to neonatal mice alveolar simplification with less radial alveolar count (RAC), mean linear intercept (MLI) and mean alveolar diameter (MAD) compared to the control group. Hyperoxia exposure increased lung vascular permeability with more fluid and proteins and inflammatory factors, including TNF-α and IL-1β, in bronchoalveolar lavage fluid while reducing the heparan sulfate (HS), the most abundant component of the endothelial glycocalyx, in the pulmonary endothelial cells. PDX relieve these changes. PDX attenuated hyperoxia-induced high expression of heparanase (HPA), the endoglycosidase that shed endothelial glycocalyx, p-P65, P65, and low expression of SIRT1. BOC-2 and EX527 abolished the affection of PDX both in vivo and intro. In summary, our findings indicate that PDX treatment relieves hyperoxia-induced alveolar simplification, vascular leakage and lung inflammation by attenuating pulmonary endothelial glycocalyx injury via the SIRT1/NF-κB/ HPA pathway.

Fluoxetine reduces organ injury and improves motor function after traumatic brain injury in mice.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality in trauma patients worldwide. Brain injury is associated with significant inflammation, both within the brain and in the peripheral organs. This inflammatory response in TBI leads to a secondary injury, worsening the effects of the original brain injury. Serotonin is also linked to inflammation in the intestine and inflammatory bowel disease, but its role in the gut-brain axis is not known. We hypothesized that using fluoxetine to block serotonin reuptake would reduce organ inflammation and improve outcomes after TBI. C57/B6 mice were given a severe TBI using a controlled cortical impact. To measure intestinal permeability, a piece of terminal ileum was resected, the lumen was filled with 4-kDa fluorescein isothiocyanate (FITC)-dextran, and the ends were tied. The intestinal segment was submerged in buffer and fluorescence in the buffer measured over time. To measure lung permeability, 70-kDa FITC-dextran is injected retro-orbitally. Thirty minutes later, the left lung was homogenized and the fluorescence was measured. To measure performance on the rota-rod, mice were placed on a spinning rod, and the time to fall off was measured. Those treated with fluoxetine received a single dose of 5 mg/kg via intraperitoneal injection immediately after injury. Traumatic brain injury was associated with an increase in intestinal permeability to FITC-dextran, increased lung vascular permeability, and worse performance on the rota-rod. Fluoxetine significantly reduced lung and intestinal permeability after TBI and improved performance on the rota-rod after TBI. Use of fluoxetine has the potential to reduce lung injury and improve motor coordination in severe TBI patients. Further study will be needed to elucidate the mechanism behind this effect.

A cortactin CTTN coding SNP contributes to lung vascular permeability and inflammatory disease severity in African descent subjects

The cortactin gene (CTTN), encoding an actin-binding protein critically involved in cytoskeletal dynamics and endothelial cell (EC) barrier integrity, contains single nucleotide polymorphisms (SNPs) associated with severe asthma in Black patients. As loss of lung EC integrity is a major driver of mortality in the Acute Respiratory Distress Syndrome (ARDS), sepsis, and the acute chest syndrome (ACS), we speculated CTTN SNPs that alter EC barrier function will associate with clinical outcomes from these types of conditions in Black patients. In case-control studies, evaluation of a nonsynonymous CTTN coding SNP Ser484Asn (rs56162978, G/A) in a severe sepsis cohort (725 Black subjects) revealed significant association with increased risk of sepsis mortality. In a separate cohort of sickle cell disease (SCD) subjects with and without ACS (177 SCD Black subjects), significantly increased risk of ACS and increased ACS severity (need for mechanical ventilation) was observed in carriers of the A allele. Human lung EC expressing the cortactin S484N transgene exhibited: (i) delayed EC barrier recovery following thrombin-induced permeability; (ii) reduced levels of critical Tyr486 cortactin phosphorylation; (iii) inhibited binding to the cytoskeletal regulator, nmMLCK; and (iv) attenuated EC barrier-promoting lamellipodia dynamics and biophysical responses. ARDS-challenged Cttn+/- heterozygous mice exhibited increased lung vascular permeability (compared to wild-type mice) which was significantly attenuated by IV delivery of liposomes encargoed with CTTN WT transgene but not by CTTN S484N transgene. In summary, these studies suggest that the CTTN S484N coding SNP contributes to severity of inflammatory injury in Black patients, potentially via delayed vascular barrier restoration.

Endothelial-Specific Loss of Sphingosine-1-Phosphate Receptor 1 Increases Vascular Permeability and Exacerbates Bleomycin-induced Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Evidence for reprogramming of monocytes into reparative alveolar macrophages in vivo by targeting PDE4b.

Increased lung vascular permeability and neutrophilic inflammation are hallmarks of acute lung injury. Alveolar macrophages (AMϕ), the predominant sentinel cell type in the airspace, die in massive numbers while fending off pathogens. Recent studies indicate that the AMϕ pool is replenished by airspace-recruited monocytes, but the mechanisms instructing the conversion of recruited monocytes into reparative AMϕ remain elusive. Cyclic AMP (cAMP) is a vascular barrier protective and immunosuppressive second messenger in the lung. Here, we subjected mice expressing GFP under the control of the Lysozyme-M promoter (LysM-GFP mice) to the LPS model of rapidly resolving lung injury to address the impact of mechanisms determining cAMP levels in AMϕ and regulation of mobilization of the reparative AMϕ-pool. RNA-seq analysis of flow-sorted Mϕ identified phosphodiesterase 4b (PDE4b) as the top LPS-responsive cAMP-regulating gene. We observed that PDE4b expression markedly increased at the time of peak injury (4 h) and then decreased to below the basal level during the resolution phase (24 h). Activation of transcription factor NFATc2 was required for the transcription of PDE4b in Mϕ. Inhibition of PDE4 activity at the time of peak injury, using intratracheal rolipram, increased cAMP levels, augmented the reparative AMϕ pool, and resolved lung injury. This response was not seen following conditional depletion of monocytes, thus establishing airspace-recruited PDE4b-sensitive monocytes as the source of reparative AMϕ. Interestingly, adoptive transfer of rolipram-educated AMϕ into injured mice resolved lung edema. We propose suppression of PDE4b as an effective approach to promote reparative AMϕ generation from monocytes for lung repair.

Circulating BMP9 Protects the Pulmonary Endothelium during Inflammation-induced Lung Injury in Mice.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis.Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge.Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice.Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS–induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001).Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

Abstract 13667: Programming to S1PR1 + Endothelial Cell Population Promotes the Restoration of Vascular Integrity in vivo

Introduction: Increased endothelial permeability and failure to repair is the hallmark of several vascular diseases including acute lung injury (ALI). However, little is known about the intrinsic pathways that activate the endothelial cell (EC) regenerative programs facilitating thereby tissue repair. Studies have invoked a crucial role of sphingosine-1-phosphate (S1P) in resolving endothelial hyperpermeability through activation of the G-protein coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1). Hypothesis: We postulate that S1PR1 + EC serve as an endogenous means to prevent endothelial injury. Methods: Studies were made using EC-S1PR1 null mice and S1PR1-GFP reporter mice to trace the generation and characteristics of S1PR1 + EC by exploiting immuno-histochemical analysis and FACS. RNA-seq analysis was performed to identify the genetic signature of S1PR1 + EC. Combination of genetic and pharmacological strategies were included for mechanistic study. Transplantation of S1PR1 + EC and edema measurement was performed in EC-S1PR1 null mice. Results: We observed in a mouse model of endotoxemia that LPS via generation of S1P induced the programming of S1PR1 lo EC to S1PR1 + EC, comprising 80% of lung EC. Their generation preceded the vascular repair phase and these cells were required for reestablishing the endothelial barrier function. Thus, conditional deletion of S1PR1 in EC spontaneously increased lung vascular permeability. RNA-seq analysis of S1PR1 + EC showed enrichment of genes regulating S1P synthesis and transport, sphingosine kinase 1 (SPHK1) and SPNS2, respectively, as well as transcription factors EGR1 and STAT3. EGR1 and STAT3 were essential for transcribing SPHK1 and SPNS2, respectively to increase S1P concentration that served to amplify S1PR1 + EC transition. Transplantation of S1PR1 + EC into injured lung vasculature of EC-S1PR1 -/- mice restored endothelial integrity. Conclusions: Findings illustrate that generation of a specialized S1PR1 + EC population has the potential to activate key endothelial regenerative program mediating vascular endothelial repair raising the possibility of activating this pathway to restore vascular homeostasis in inflammatory lung injury.

Macrophage TLR4 and PAR2 Signaling: Role in Regulating Vascular Inflammatory Injury and Repair.

Macrophages play a central role in dictating the tissue response to infection and orchestrating subsequent repair of the damage. In this context, macrophages residing in the lungs continuously sense and discriminate among a wide range of insults to initiate the immune responses important to host-defense. Inflammatory tissue injury also leads to activation of proteases, and thereby the coagulation pathway, to optimize injury and repair post-infection. However, long-lasting inflammatory triggers from macrophages can impair the lung's ability to recover from severe injury, leading to increased lung vascular permeability and neutrophilic injury, hallmarks of Acute Lung Injury (ALI). In this review, we discuss the roles of toll-like receptor 4 (TLR4) and protease activating receptor 2 (PAR2) expressed on the macrophage cell-surface in regulating lung vascular inflammatory signaling.

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.

Alveolar Stretch Activation of Endothelial Piezo1 Protects Adherens Junctions and Lung Vascular Barrier.

Disruption of alveolar-capillary barriers is a major complication of high-volume mechanical ventilation referred to as "ventilator-induced lung injury." The stretching force in alveoli is transmitted to endothelial cells, increasing the tension on underlying endothelial plasma membrane. The mechanosensor Piezo1, a plasma membrane cation channel, was inducibly deleted in endothelial cells of mice (Piezo1iEC-/-), which allowed us to study its role in regulating the endothelial barrier response to alveolar stretch. We observed significant increase in lung vascular permeability in Piezo1iEC-/- mice as compared with control Piezo1fl/fl mice in response to high-volume mechanical ventilation. We also observed that human lung endothelial monolayers depleted of Piezo1 and exposed to cyclic stretch had increased permeability. We identified the calcium-dependent cysteine protease calpain as a downstream target of Piezo1. Furthermore, we showed that calpain maintained stability of the endothelial barrier in response to mechanical stretch by cleaving Src kinase, which was responsible for disassembling endothelial adherens junctions. Pharmacological activation of calpain caused Src cleavage and thereby its inactivation, and it restored the disrupted lung endothelial barrier seen in Piezo1iEC-/- mice undergoing high-volume mechanical ventilation. Our data demonstrate that downregulation of Piezo1 signaling in endothelium is a critical factor in the pathogenesis of ventilator-induced lung injury, and thus augmenting Piezo1 expression or pharmacologically activating Piezo1 signaling may be an effective therapeutic strategy.

MiR-144-mediated Inhibition of ROCK1 Protects against LPS-induced Lung Endothelial Hyperpermeability.

Dysfunctional endothelial cell (EC) barrier and increased lung vascular permeability is a cardinal feature of acute lung injury and sepsis that may result in a pathophysiological condition characterized by alveolar flooding, pulmonary edema, and subsequent hypoxemia. In lung ECs, activation of Rho-associated kinase-1 (ROCK1) phosphorylates myosin light chain (MLC)-associated phosphatase at its inhibitory site, which favors phosphorylation of MLC, stress fiber formation, and hyperpermeability during acute lung injury. The role of microRNA-144 (miR-144) has been well investigated in many human diseases, including cardiac ischemia/reperfusion-induced injury, lung cancer, and lung viral infection; however, its role in pulmonary EC barrier regulation remains obscure. Here, we investigated the miR-144-mediated mechanism in the protection of endothelial barrier function in an LPS-induced lung injury model. By using transendothelial electrical resistance and transwell permeability assay to examine in vitro permeability and immunofluorescence microscopy to determine barrier integrity, we showed that ectopic expression of miR-144 effectively blocked lung EC barrier disruption and hyperpermeability in response to proinflammatory agents. Furthermore, using a gain-and-loss-of-function strategy, overexpression of miR-144 significantly decreased ROCK1 expression. Concomitantly, miR-144 inhibits ROCK1-mediated phosphorylation of MLC phosphataseThr853 and thus phosphorylation of MLCThr18/Ser19 to counteract stress fiber formation in LPS-activated EC. Finally, in LPS-challenged mice, intranasal delivery of miR-144 mimic via liposomes attenuated endotoxemia-induced increases in lung wet/dry ratio, vascular permeability, and inflammation. In conclusion, these data suggest that miR-144-attenuated activation of inflammatory ROCK1/MLC pathway in vascular ECs is a promising therapeutic strategy to counter inflammatory lung injury.

Upregulated miR‐144‐3p Attenuates LPS‐induced Acute Lung Injury via Inhibition of ROCK1 Signaling Pathway

Dysfunctional endothelial cell (EC) barrier and increased lung vascular permeability is a cardinal feature of Acute Lung Injury (ALI) and sepsis that may results in a pathophysiological condition characterized by alveolar flooding and pulmonary edema and subsequent hypoxemia. In lung ECs, activation of Rho associated kinase‐1 (ROCK1) phosphorylates myosin light chain (MLC), stress fiber formation and hyper‐permeability during ALI. The role of MicroRNA‐144 (miR‐144) has been well investigated in many human diseases including cardiac ischaemia/reperfusion‐induced injury, lung cancer and lung viral infection, however, its role in pulmonary EC barrier regulation remains obscure. Here, we investigated miR‐144 mediated mechanism in the protection of endothelial barrier function in bacterial wall lipopolysaccharide‐(LPS) induced lung injury model. By using transwell endothelial permeability assay to examine in vitro permeability and immunofluorescence microscopy to determine barrier integrity, we showed that ectopic expression of miR‐144 effectively blocked human lung EC barrier disruption and hyper‐permeability in response to LPS. Furthermore, using a gain and loss of function strategy, overexpression of miR‐144 significantly decreased ROCK1 expression. Concomitantly miR‐144 inhibits ROCK1 mediated phosphorylation of MLC phosphatase and thus phosphorylation of MLC to counteract actin stress fibers formation in LPS activated human lung EC. Finally, in mice challenged with LPS, in vivo delivery of miR‐144 mimic via liposomes attenuated endotoxemia‐induced increased in lung wet‐to‐dry ratio, vascular permeability and inflammatory cytokines. In conclusion, our data suggest miR‐144 attenuates activation of the inflammatory ROCK1/MLC pathway in vascular EC is a promising therapeutic strategy to counter inflammatory lung injury.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cigarette smoke alters lung vascular permeability and endothelial barrier function (2017 Grover Conference Series).

Smoking of tobacco products continues to be widespread, despite recent progress in decreasing use. Both in the United States and worldwide, cigarette smoking is a major cause of morbidity and mortality. Growing evidence indicates that acute respiratory distress syndrome (ARDS) is among the consequences of cigarette smoking. Based on the topic from the 2017 Grover Conference, we review evidence that cigarette smoking increases lung vascular permeability using both acute and longer exposures of mice to cigarette smoke (CS). We also review studies indicating that CS extract disrupts cultured lung endothelial cell barrier function through effects on focal adhesion contacts, adherens junctions, actin cytoskeleton, and microtubules. Among the potentially injurious components of CS, the reactive aldehyde, acrolein, similarly increases lung vascular permeability and disrupts barrier function. We speculate that inhibition of aldehyde-induced lung vascular permeability may prevent CS-induced lung injury.

Differential Expression of Aquaporins in Experimental Models of Acute Lung Injury.

The mammalian lung expresses at least three aquaporin (AQP) water channels whose precise role in lung injury or inflammation is still controversial. Three murine models of lung inflammation and corresponding controls were used to evaluate the expression of Aqp1, Aqp4, Aqp5 and Aqp9: lipopolysaccharide (LPS)-induced lung injury; HCl-induced lung injury; and ventilation-induced lung injury (VILI). All models yielded increased lung vascular permeability, and inflammatory cell infiltration in the broncho-alveolar lavage fluid; VILI additionally produced altered lung mechanics. Lung expression of Aqp4 decreased in the models that targeted primarily the alveolar epithelium, i.e. acid aspiration and mechanical ventilation, while Aqp5 expression decreased in the model that appeared to target both the capillary endothelium and alveolar epithelium, i.e. LPS. Participation of aquaporins in the acute inflammatory process depends on localization and the type of lung injury.

Cell-Free Hemoglobin-mediated Increases in Vascular Permeability. A Novel Mechanism of Primary Graft Dysfunction and a New Therapeutic Target.

Cell-free hemoglobin (CFH) is a potent oxidant associated with poor clinical outcomes in a variety of clinical settings. Recent studies suggest that acetaminophen (APAP), a specific hemoprotein reductant, can abrogate CFH-mediated oxidative injury and organ dysfunction. Preoperative plasma CFH levels are independently associated with primary graft dysfunction (PGD) after lung transplant ( 1 ). Our objectives were to determine whether CFH would increase lung vascular permeability in the isolated perfused human lung and whether APAP would limit these effects. Human lungs declined for transplant were inflated and perfused with Dulbecco's modified Eagle medium/5% albumin at a pulmonary artery pressure of 8-12 mm Hg. After steady state was achieved, CFH (100 mg/dl) was added to the perfusate ± APAP (15 μg/ml). Lung permeability was measured by continuous monitoring of lung weight gain and by extravasation of Evans blue dye-labeled albumin from the vasculature into bronchoalveolar lavage. To test the mechanism of increased permeability, human pulmonary microvascular endothelial cells were exposed to CFH (0.5 mg/ml) ± APAP (160 μM) for 24 hours and permeability was assessed by electrical cell-substrate impedance sensing. In the isolated perfused human lung, CFH increased lung permeability over 2 hours compared with control lungs (12% vs. 2% weight gain from baseline, P = 0.03). Increased vascular permeability was confirmed by a 4.8-fold increase in Evans blue dye-labeled albumin in the airspace compared with control lungs. Pretreatment with APAP prevented lung weight gain (P = 0.06 vs. CFH). In human pulmonary microvascular endothelial cells, CFH increased monolayer permeability (P = 0.03 vs. control), and this was attenuated by APAP (P = 0.045 vs. CFH). Circulating CFH increases vascular permeability in the isolated perfused human lung and paracellular permeability in lung microvascular endothelial cells. These effects may explain the association of plasma CFH levels with PGD. The hemoprotein reductant APAP attenuates the effects of CFH and merits further exploration as a potential therapy for PGD prevention.

Endothelial cell signaling and ventilator-induced lung injury: molecular mechanisms, genomic analyses, and therapeutic targets

Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS). Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The pathobiology of VILI and ARDS shares many inflammatory features including increases in lung vascular permeability due to loss of endothelial cell barrier integrity resulting in alveolar flooding. While there have been advances in the understanding of certain elements of VILI and ARDS pathobiology, such as defining the importance of lung inflammatory leukocyte infiltration and highly induced cytokine expression, a deep understanding of the initiating and regulatory pathways involved in these inflammatory responses remains poorly understood. Prevailing evidence indicates that loss of endothelial barrier function plays a primary role in the development of VILI and ARDS. Thus this review will focus on the latest knowledge related to 1) the key role of the endothelium in the pathogenesis of VILI; 2) the transcription factors that relay the effects of excessive mechanical stress in the endothelium; 3) the mechanical stress-induced posttranslational modifications that influence key signaling pathways involved in VILI responses in the endothelium; 4) the genetic and epigenetic regulation of key target genes in the endothelium that are involved in VILI responses; and 5) the need for novel therapeutic strategies for VILI that can preserve endothelial barrier function.

Hyperglycemia-Mediated Oxidative Stress Increases Pulmonary Vascular Permeability.

Hyperglycemia in diabetes mellitus is associated with endothelial dysfunction as evidenced by increased oxidative stress and vascular permeability. Whether impaired glucose control in metabolic syndrome impacts pulmonary vascular permeability is unknown. We hypothesized that in metabolic syndrome, hyperglycemia increases lung vascular permeability through superoxide. Lung capillary Kf and vascular superoxide were measured in the isolated lungs of LZ and OZ rats. OZ were subjected to 4 weeks of metformin treatment (300 mg/kg/day orally) to improve insulin sensitivity. In a separate experiment, lung vascular permeability and vascular superoxide were measured in LZ exposed to acute hyperglycemia (30 mM). As compared to LZ, OZ had impaired glucose and insulin tolerance and elevated vascular superoxide which was associated with an elevated lung Kf. Chronic metformin treatment in OZ improved glucose control and insulin sensitivity which was associated with decreased vascular oxidative stress and lung Kf. Acute hyperglycemia in isolated lungs from LZ increased lung Kf, which was blocked with the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin (3 mM). Apocynin also decreased baseline Kf in OZ. These data suggest that hyperglycemia in metabolic syndrome exacerbates lung vascular permeability through increases in vascular superoxide, possibly through NADPH oxidase.

Effects of Acute and Chronic Hyperglycemia on Lung Capillary Permeability

Hyperglycemia is correlated with increased vascular oxidative stress and increased vascular permeability. The role of acute and chronic hyperglycemia in affecting lung endothelial permeability is unclear in a type 2 diabetes setting. Lung capillary filtration coefficient (Kf) was measured in the isolated lung of lean Zucker (LZ) and obese Zucker rats (OZ). We hypothesized obesity increases lung vascular permeability through chronic hyperglycemia. OZ had impaired glucose and insulin tolerance which was associated with elevated lung Kf (19.4 ± 3 ml/mmHg/g/min) as compared to LZ (11.4 ± 2 ml/mmHg/g/min). In a separate experiment, OZ were subjected to 4 weeks of Metformin treatment (300 mg/kg/day orally) to improve insulin sensitivity. Metformin treatment improved oral glucose tolerance, insulin sensitivity, and vascular oxidative stress, and this was associated with improved baseline lung Kf (11.5 ± 1 ml/mmHg/g/min) as compared to OZ control animals. To examine the role of acute hyperglycemia in lung permeability, lungs from LZ and OZ were subjected to 30 minutes of an elevated glucose concentration (550 mg/dl). Acute hyperglycemia had no effect in OZ but increased lung Kf in LZ, which was ameliorated with acute antioxidant treatment (apocynin, 3mM) added to the lung perfusion solution. These data suggest that the chronic hyperglycemia in obesity can exacerbate lung vascular permeability, possibly through oxidative stress, and may blunt any further alterations in Kf during acute episodes hyperglycemia. This warrants further investigation into the use of antioxidants in the treatment of diabetic and acute hyperglycemic-induced lung hyperpermeability.

Regulation of Radixin Gene Expression in Pulmonary Endothelium by LPS and Cyclic Stretch

Increased lung vascular permeability is a cardinal feature of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We previously have shown that the ezrin, radixin, and moesin (ERM) proteins differentially participate in agonists-induced endothelial cell (EC) barrier enhancement and hyperpermeability. In the present study we explore the regulation of radixin gene expression in lung inflammation by disease-associated genetic variants in the promoter and intronic regions of the RDX. A combination of genomic wide association study (GWAS), in silico analysis, and transfection of RDX promoter luciferase reporter were utilized. We analyzed RDX expression with Genomatix and Haploview for gene regulation and allele linkage disequilibrium (LD), respectively. Inflammatory stimuli LPS and 18% cyclic stretch (a model of mechanical lung injury) each time-dependently modulate both the protein expression of radixin and phosphorylation of ERMs. A GWAS study of African Americans identified one promoter (rs1...

HIF2α signaling inhibits adherens junctional disruption in acute lung injury.

Vascular endothelial barrier dysfunction underlies diseases such as acute respiratory distress syndrome (ARDS), characterized by edema and inflammatory cell infiltration. The transcription factor HIF2α is highly expressed in vascular endothelial cells (ECs) and may regulate endothelial barrier function. Here, we analyzed promoter sequences of genes encoding proteins that regulate adherens junction (AJ) integrity and determined that vascular endothelial protein tyrosine phosphatase (VE-PTP) is a HIF2α target. HIF2α-induced VE-PTP expression enhanced dephosphorylation of VE-cadherin, which reduced VE-cadherin endocytosis and thereby augmented AJ integrity and endothelial barrier function. Mice harboring an EC-specific deletion of Hif2a exhibited decreased VE-PTP expression and increased VE-cadherin phosphorylation, resulting in defective AJs. Mice lacking HIF2α in ECs had increased lung vascular permeability and water content, both of which were further exacerbated by endotoxin-mediated injury. Treatment of these mice with Fg4497, a prolyl hydroxylase domain 2 (PHD2) inhibitor, activated HIF2α-mediated transcription in a hypoxia-independent manner. HIF2α activation increased VE-PTP expression, decreased VE-cadherin phosphorylation, promoted AJ integrity, and prevented the loss of endothelial barrier function. These findings demonstrate that HIF2α enhances endothelial barrier integrity, in part through VE-PTP expression and the resultant VE-cadherin dephosphorylation-mediated assembly of AJs. Moreover, activation of HIF2α/VE-PTP signaling via PHD2 inhibition has the potential to prevent the formation of leaky vessels and edema in inflammatory diseases such as ARDS.