Earlier studies indicated a role for reactive oxygen species (ROS) in host defense against Pseudomonas aeruginosa infection. However, the role of nicotinamide adenine dinucleotide phosphate–reduced (NADPH) oxidase (NOX) proteins and the mechanism of activation for NADPH oxidase in P. aeruginosa infection are not well-defined. Here, we investigated the role of NOX2 and NOX4 proteins in P. aeruginosa infection, ROS generation, and endothelial barrier function in murine lungs and in human lung microvascular endothelial cells (HLMVECs). Airway instillation of P. aeruginosa strain 103 (PA103) significantly increased ROS concentrations in bronchial alveolar lavage (BAL) fluid, along with the expression of NOX2 and NOX4, but not NOX1 and NOX3, in lung tissue. In addition, PA103-infected HLMVECs revealed elevated concentrations of ROS, NOX2, and NOX4. In murine lungs and HLMVECs, PA103 induced the NF-κB pathway, and its inhibition blocked PA103-dependent NOX2 and NOX4 expression. Barrier function analysis showed that heat-killed PA103 induced endothelial permeability in a dose-dependent manner, which was attenuated by treatment with small interfering (si)RNA specific for NOX4, but not NOX2. Furthermore, the knockdown of NOX4, but not NOX2, with siRNA reduced PA103-mediated apoptosis in HLMVECs. In vivo, the down-regulation of NOX4 with NOX4 siRNA attenuated PA103-induced lung vascular permeability. The deletion of NOX2 in mice exerted no effect on permeability, but offered significant resistance to P. aeruginosa–induced lung inflammation. These data show that P. aeruginosa lung infection up-regulates NOX2 and NOX4 expression and ROS generation, which play distinct roles in regulating lung inflammation, apoptosis, and permeability.
Pseudomonas aeruginosa; NADPH oxidase; reactive oxygen species; apoptosis; vascular permeability
The inflamed lung exhibits oxidative and nitrative modifications of multiple target proteins, potentially reflecting disease severity and progression. We identified sphingosine-1–phosphate receptor–3 (S1PR3), a critical signaling molecule mediating cell proliferation and vascular permeability, as a nitrated plasma protein in mice with acute lung injury (ALI). We explored S1PR3 as a potential biomarker in murine and human ALI. In vivo nitrated and total S1PR3 concentrations were determined by immunoprecipitation and microarray studies in mice, and by ELISA in human plasma. In vitro nitrated S1PR3 concentrations were evaluated in human lung vascular endothelial cells (ECs) or within microparticles shed from ECs after exposure to barrier-disrupting agonists (LPS, low-molecular-weight hyaluronan, and thrombin). The effects of S1PR3-containing microparticles on EC barrier function were assessed by transendothelial electrical resistance (TER). Nitrated S1PR3 was identified in the plasma of murine ALI and in humans with severe sepsis-induced ALI. Elevated total S1PR3 plasma concentrations (> 251 pg/ml) were linked to sepsis and ALI mortality. In vitro EC exposure to barrier-disrupting agents induced S1PR3 nitration and the shedding of S1PR3-containing microparticles, which significantly reduced TER, consistent with increased permeability. These changes were attenuated by reduced S1PR3 expression (small interfering RNAs). These results suggest that microparticles containing nitrated S1PR3 shed into the circulation during inflammatory lung states, and represent a novel ALI biomarker linked to disease severity and outcome.
acute lung injury; sphingosine-1–phosphate receptor–3; microparticles; nitration; biomarker
Exposure to particulate air pollution is associated with increased cardiopulmonary morbidity and mortality, although the pathogenic mechanisms are poorly understood. We previously demonstrated that particulate matter (PM) exposure triggers massive oxidative stress in vascular endothelial cells (ECs), resulting in the loss of EC integrity and lung vascular hyperpermeability. We investigated the protective role of hydrogen sulfide (H2S), an endogenous gaseous molecule present in the circulation, on PM-induced human lung EC barrier disruption and pulmonary inflammation. Alterations in EC monolayer permeability, as reflected by transendothelial electrical resistance (TER), the generation of reactive oxygen species (ROS), and murine pulmonary inflammatory responses, were studied after exposures to PM and NaSH, an H2S donor. Similar to N-acetyl cysteine (5 mM), NaSH (10 μM) significantly scavenged PM-induced EC ROS and inhibited the oxidative activation of p38 mitogen-activated protein kinase. Concurrent with these events, NaSH (10 μM) activated Akt, which helps maintain endothelial integrity. Both of these pathways contribute to the protective effect of H2S against PM-induced endothelial barrier dysfunction. Furthermore, NaSH (20 mg/kg) reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in a murine model of PM-induced lung inflammation. These data suggest a potentially protective role for H2S in PM-induced inflammatory lung injury and vascular hyperpermeability.
particulate matter; hydrogen sulfide; endothelial permeability; Akt
A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL+/− mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.
intracellular sphingosine-1-phosphate; sphingosine-1-phosphate lyase; IL-6; transendothelial resistance; acute lung injury
Novel therapies are desperately needed for radiation-induced lung injury (RILI), which, despite aggressive corticosteroid therapy, remains a potentially fatal and dose-limiting complication of thoracic radiotherapy. We assessed the utility of simvastatin, an anti-inflammatory and lung barrier–protective agent, in a dose- and time-dependent murine model of RILI (18–(25 Gy). Simvastatin reduced multiple RILI indices, including vascular leak, leukocyte infiltration, and histological evidence of oxidative stress, while reversing RILI-associated dysregulated gene expression, including p53, nuclear factor–erythroid-2–related factor, and sphingolipid metabolic pathway genes. To identify key regulators of simvastatin-mediated RILI protection, we integrated whole-lung gene expression data obtained from radiated and simvastatin-treated mice with protein–protein interaction network analysis (single-network analysis of proteins). Topological analysis of the gene product interaction network identified eight top-prioritized genes (Ccna2a, Cdc2, fcer1 g, Syk, Vav3, Mmp9, Itgam, Cd44) as regulatory nodes within an activated RILI network. These studies identify the involvement of specific genes and gene networks in RILI pathobiology, and confirm that statins represent a novel strategy to limit RILI.
radiation pneumonitis; lung vascular permeability; simvastatin; gene dysregulation; protein–protein interaction
The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1–phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR1, we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR1) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar–capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR1, intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar–capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR1 regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR1 inverse agonist, SB-649146, or S1PR1+/− mice exhibited reduced S1P/SEW-2871–mediated barrier protection after challenge with LPS. In contrast, S1PR2−/− knockout mice as well as mice with reduced S1PR3 expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR1 receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR1 agonist concentration, and S1PR1 expression in target tissues.
SEW-2871; LPS; SB-649146; S1P; lung edema
Epidemiologic studies have linked exposure to airborne pollutant particulate matter (PM) with increased cardiopulmonary mortality and morbidity. The mechanisms of PM-mediated lung pathophysiology, however, remain unknown. We tested the hypothesis that PM, via enhanced oxidative stress, disrupts lung endothelial cell (EC) barrier integrity, thereby enhancing organ dysfunction. Using PM collected from Ft. McHenry Tunnel (Baltimore, MD), we assessed PM-mediated changes in transendothelial electrical resistance (TER) (a highly sensitive measure of barrier function), reactive oxygen species (ROS) generation, and p38 mitogen-activated protein kinase (MAPK) activation in human pulmonary artery EC. PM induced significant dose (10–100 μg/ml)- and time (0–10 h)-dependent EC barrier disruption reflected by reduced TER values. Exposure of human lung EC to PM resulted in significant ROS generation, which was directly involved in PM-mediated EC barrier dysfunction, as N-acetyl-cysteine (NAC, 5 mM) pretreatment abolished both ROS production and barrier disruption induced by PM. Furthermore, PM induced p38 MAPK activation and HSP27 phosphorylation, events that were both attenuated by NAC. In addition, PM-induced EC barrier disruption was partially prevented by the p38 MAP kinase inhibitor SB203580 (10 μM) as well as by reduced expression of either p38 MAPK β or HSP27 (siRNA). These results demonstrate that PM induces ROS generation in human lung endothelium, resulting in oxidative stress–mediated EC barrier disruption via p38 MAPK- and HSP27-dependent pathways. These findings support a novel mechanism for PM-induced lung dysfunction and adverse cardiopulmonary outcomes.
endothelial permeability; HSP27; particulate matter; p38 MAP kinase; ROS
The functional significance of the expression of cystic fibrosis transmembrane regulator (CFTR) on endothelial cells has not yet been elucidated. Since CFTR has been implicated in the regulation of intracellular sphingolipid levels, which are important regulators of endothelial cell apoptosis in response to various insults, we investigated the role of CFTR in the apoptotic responses of lung endothelial cells. CFTR was detected as a functional chloride channel in primary lung endothelial cells isolated from both pulmonary arteries (human or mouse) and bronchial arteries (sheep). Both specific CFTR inhibition with 2-(phenylamino) benzoic acid diphenylamine-2-carboxylic acid, 5-[(4-carboxyphenyl)methylene]-2-thioxo-3-[(3-trifluoromethyl)phenyl-4-thiazolidinone (CFTRinh-172), or 5-nitro-2-(3-phenylpropylamino)benzoic acid and CFTR knockdown significantly attenuated endothelial cell apoptosis induced by staurosporine or H2O2. CFTRinh-172 treatment prevented the increases in the ceramide:sphingosine-1 phosphate ratio induced by H2O2 in lung endothelial cells. Replenishing endogenous ceramides via sphingomyelinase supplementation restored the susceptibility of CFTR-inhibited lung endothelial cells to H2O2-induced apoptosis. Similarly, the anti-apoptotic phenotype of CFTR-inhibited cells was reversed by lowering the intracellular pH, and was reproduced by alkalinization before H2O2 challenge. TUNEL staining and active caspase-3 immunohistochemistry indicated that cellular apoptosis was decreased in lung explants from patients with cystic fibrosis compared with those with smoking-induced chronic obstructive lung disease, especially in the alveolar tissue and vascular endothelium. In conclusion, CFTR function is required for stress-induced apoptosis in lung endothelial cells by maintaining adequate intracellular acidification and ceramide activation. These results may have implications in the pathogenesis of cystic fibrosis, where aberrant endothelial cell death may dysregulate lung vascular homeostasis, contributing to abnormal angiogenesis and chronic inflammation.
cystic fibrosis; vascular; sphingolipids; cell death; pulmonary artery; bronchial artery; oxidative stress
We examined the mechanism by which lysophosphatidylcholine (LPC) regulates β2-integrin–mediated adhesion of eosiniophils. Eosinophils were isolated from blood of mildly atopic volunteers by negative immunomagnetic selection. β2-integrin–dependent adhesion of eosinophils to plated bovine serum albumin (BSA) was measured by residual eosinophil peroxidase activity. LPC caused maximal adhesion of eosinophils to plated BSA at 4 μM. Lysophosphatidylinositol, which has a similar molecular shape, mimicked the effect of LPC on eosinophil adhesion, while neither lysophosphatidylserine nor lysophosphatidylethanolamine had any effect. Phosphatidylethanolamine, a lipid that has a molecular orientation that is the inverse of LPC, blocked eosinophil adhesion caused by LPC. Unlike platelet-activating factor, a G-protein–coupled receptor agonist, LPC did not cause Ca2+-store depletion, but caused increased Ca2+ influx upon addition of Ca2+ to extracellular medium. This influx was not inhibited by U73122, a phospholipase C inhibitor, demonstrating independence from the G protein–activated phospholipase C pathway. Ca2+ influx was inhibited by either preincubation of phosphotidylethanolamine or La3+, a broad spectrum blocker of cation channels. LPC induced up-regulation of the active conformation of CD11b, which was blocked by preincubation with phosphatidylethanolamine. These data suggest that LPC causes a non–store-operated Ca2+ influx into eosinophils, which subsequently activates CD11b/CD18 to promote eosinophil adhesion.
lysophosphatidylcholine; eosinophils; adhesion; calcium