AICAR decreases acute lung injury by phosphorylating AMPK and upregulating heme oxygenase-1.

AIM: We investigated the mechanisms by which N1-(ß-d-ribofuranosyl)-5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of AMP-activated protein kinase (AMPK), decreases lung injury and mortality when administered to mice post exposure to bromine gas (Br2). METHODS: We exposed male C57BL/6 mice and heme oxygenase-1 (HO-1)-deficient (HO-1-/-) and corresponding wild-type (WT) littermate mice to Br2 (600 ppm for 45 or 30 min, respectively) in environmental chambers and returned them to room air. AICAR was administered 6 h post exposure (10 mg·kg-1, intraperitoneal). We assessed survival, indices of lung injury, high mobility group box 1 (HMGB1) in the plasma, HO-1 levels in lung tissues and phosphorylation of AMPK and its upstream liver kinase B1 (LKB1). Rat alveolar type II epithelial (L2) cells and human club-like epithelial (H441) cells were also exposed to Br2 (100 ppm for 10 min). After 24 h we measured apoptosis and necrosis, AMPK and LKB1 phosphorylation, and HO-1 expression. RESULTS: There was a marked downregulation of phosphorylated AMPK and LKB1 in lung tissues and in L2 and H441 cells post exposure. AICAR increased survival in C57BL/6 but not in HO-1-/- mice. In WT mice, AICAR decreased lung injury and restored phosphorylated AMPK and phosphorylated LKB1 to control levels and increased HO-1 levels in both lung tissues and cells exposed to Br2. Treatment of L2 and H441 cells with small interfering RNAs against nuclear factor erythroid 2-related factor 2 or HO-1 abrogated the protective effects of AICAR. CONCLUSIONS: Our data indicate that the primary mechanism for the protective action of AICAR in toxic gas injury is the upregulation of lung HO-1 levels.

Upregulation of airway smooth muscle calcium-sensing receptor by low-molecular-weight hyaluronan.

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20-25 g) exposed to either bromine (Br2) or Cl2 (600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2 or Cl2 exposure, produced higher AHR compared with Br2 or Cl2 alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2 resulted in membrane potential depolarization (Vm Air: 62 ± 3 mV; 3 h post Br2:-45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2 and nifedipine-induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2 reversed the effects of Br2 and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vm depolarization, [Ca2+]i increase, and Ca-SR expression to a similar extent as exposure to Br2 and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure.

Bromine (Br2) is an organohalide found in nature and is integral to many manufacturing processes. Br2 is toxic to living organisms, and high concentrations can prove fatal. To meet industrial demand, large amounts of purified Br2 are produced, transported, and stored worldwide, providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br2 exposure, we have surveyed the lung proteomes of C57BL/6 male mice and human lung-derived microvascular endothelial cells (HMECs) at 24 h following exposure to Br2 in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 h after Br2 exposure. These experiments revealed significant increases in the filtration coefficient (Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br2 and Br-lipid-treated HMECs exhibited differential levels of zona occludens-1 that were found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadherin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br2 toxicity.

Instillation of hyaluronan reverses acid instillation injury to the mammalian blood gas barrier.

Acid (HCl) aspiration during anesthesia may lead to acute lung injury. There is no effective therapy. We hypothesized that HCl instilled intratracheally in C57BL/6 mice results in the formation of low-molecular weight hyaluronan (L-HA), which activates RhoA and Rho kinase (ROCK), causing airway hyperresponsiveness (AHR) and increased permeability. Furthermore, instillation of high-molecular weight hyaluronan (H-HA; Yabro) will reverse lung injury. We instilled HCl in C57BL/6 wild-type (WT), myeloperoxidase gene-deficient (MPO-/-) mice, and CD44 gene-deficient (CD44-/-) mice. WT mice were also instilled intranasally with H-HA (Yabro) at 1 and 23 h post-HCl. All measurements were performed at 1, 5, or 24 h post-HCl. Instillation of HCl in WT but not in CD44-/- resulted in increased inflammation, AHR, lung injury, and L-HA in the bronchoalveolar lavage fluid (BALF) 24 h post-HCl; L-HA levels and lung injury were significantly lower in HCl-instilled MPO-/- mice. Isolated perfused lungs of HCl instilled WT but not of CD44-/- mice had elevated values of the filtration coefficient ( Kf). Addition of L-HA on the apical surface of human primary bronchial epithelial cell monolayer decreased barrier resistance ( RT). H-HA significantly mitigated inflammation, AHR, and pulmonary vascular leakage at 24 h after HCl instillation and mitigated the increase of Kf and RT, as well as ROCK2 phosphorylation. Increased H- and L-HA levels were found in the BALF of mechanically ventilated patients but not in healthy volunteers. HCl instillation-induced lung injury is mediated by the L-HA-CD44-RhoA-ROCK2 signaling pathway, and H-HA is a potential novel therapeutic agent for acid aspiration-induced lung injury.

N-cadherin coordinates AMP kinase-mediated lung vascular repair.

Injury to the pulmonary circulation compromises endothelial barrier function and increases lung edema. Resolution of lung damage involves restoring barrier integrity, a process requiring reestablishment of endothelial cell-cell adhesions. However, mechanisms underlying repair in lung endothelium are poorly understood. In pulmonary microvascular endothelium, AMP kinase α1 (AMPKα1) stimulation enhances recovery of the endothelial barrier after LPS-induced vascular damage. AMPKα1 colocalizes to a discrete membrane compartment with the adhesion protein neuronal cadherin (N-cadherin). This study sought to determine N-cadherin's role in the repair process. Short-hairpin RNA against full-length N-cadherin or a C-terminally truncated N-cadherin, designed to disrupt the cadherin's interactions with intracellular proteins, were expressed in lung endothelium. Disruption of N-cadherin's intracellular domain caused translocation of AMPK away from the membrane and attenuated AMPK-mediated restoration of barrier function in LPS-treated endothelium. AMPK activity measurements indicated that lower basal AMPK activity in cells expressing the truncated N-cadherin compared with controls. Moreover, the AMPK stimulator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) failed to increase AMPK activity in cells expressing the modified N-cadherin, indicating uncoupling of a functional association between AMPK and the cadherin. Isolated lung studies confirmed a physiologic role for this pathway in vivo. AMPK activation reversed LPS-induced increase in permeability, whereas N-cadherin inhibition hindered AMPK-mediated repair. Thus N-cadherin coordinates the vascular protective actions of AMPK through a functional link with the kinase. This study provides insight into intrinsic repair mechanisms in the lung and supports AMPK stimulation as a modality for treating vascular disease.

Metformin-stimulated AMPK-α1 promotes microvascular repair in acute lung injury.

Acute lung injury secondary to sepsis is a leading cause of mortality in sepsis-related death. Present therapies are not effective in reversing endothelial cell dysfunction, which plays a key role in increased vascular permeability and compromised lung function. AMP-activated protein kinase (AMPK) is a molecular sensor important for detection and mediation of cellular adaptations to vascular disruptive stimuli. In this study, we sought to determine the role of AMPK in resolving increased endothelial permeability in the sepsis-injured lung. AMPK function was determined in vivo using a rat model of endotoxin-induced lung injury, ex vivo using the isolated lung, and in vitro using cultured rat pulmonary microvascular endothelial cells (PMVECs). AMPK stimulation using N1-(α-d-ribofuranosyl)-5-aminoimidizole-4-carboxamide or metformin decreased the LPS-induced increase in permeability, as determined by filtration coefficient (Kf) measurements, and resolved edema as indicated by decreased wet-to-dry ratios. The role of AMPK in the endothelial response to LPS was determined by shRNA designed to decrease expression of the AMPK-α1 isoform in capillary endothelial cells. Permeability, wounding, and barrier resistance assays using PMVECs identified AMPK-α1 as the molecule responsible for the beneficial effects of AMPK in the lung. Our findings provide novel evidence for AMPK-α1 as a vascular repair mechanism important in the pulmonary response to sepsis and identify a role for metformin treatment in the management of capillary injury.

Influenza A-induced cystic fibrosis transmembrane conductance regulator dysfunction increases susceptibility to Streptococcus pneumoniae.

Influenza A virus (IAV) infection is commonly complicated by secondary bacterial infections that lead to increased morbidity and mortality. Our recent work demonstrates that IAV disrupts airway homeostasis, leading to airway pathophysiology resembling cystic fibrosis disease through diminished cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we use human airway organotypic cultures to investigate how IAV alters the airway microenvironment to increase susceptibility to secondary infection with Streptococcus pneumoniae (Spn). We observed that IAV-induced CFTR dysfunction and airway surface liquid acidification is central to increasing susceptibility to Spn. Additionally, we observed that IAV induced profound transcriptional changes in the airway epithelium and proteomic changes in the airway surface liquid in both CFTR-dependent and -independent manners. These changes correspond to multiple diminished host defense pathways and altered airway epithelial function. Collectively, these findings highlight both the importance of CFTR function during infectious challenge and demonstrate a central role for the lung epithelium in secondary bacterial infections following IAV.

TRPV4 channels augment macrophage activation and ventilator-induced lung injury.

We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4(-/-) and TRPV4(+/+) mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (K(f)) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH(2)O PIP did not significantly increase in lungs from TRPV4(-/-) mice but increased >2.2-fold in TRPV4(+/+) lungs, TRPV4(+/+) lungs instilled with TRPV4(-/-) macrophages, and TRPV4(-/-) lungs instilled with TRPV4(+/+) macrophages after ventilation with 35 cmH(2)O PIP. Activation of TRPV4 with 4-alpha-phorbol didecanoate (4alphaPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4(+/+) macrophages compared with TRPV4(-/-) macrophages after 4alphaPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4(+/+) lungs compared with low PIP ventilated TRPV4(+/+) or high PIP ventilated TRPV4(-/-) lungs. Thus TRPV4(+/+) macrophages restored susceptibility of TRPV4(-/-) lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. K(f) increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.

Exogenous surfactant instillation attenuates inflammatory response to acid-induced lung injury in rat.

The present study was performed to investigate the role of exogenous surfactant on hydrochloric acid (HCL) - induced lung injury in rats. Six-week-old male Sprague-Dawley rats were anesthetized by intraperitoneal injection of pentobarbital sodium (40mg/kg) and HCL (0.1N, 2mL/kg) or normal saline (NS, 2mL/kg) was instilled into the trachea. Thirty minutes after HCL instillation, surfactant at a dose of 60mg (=2mL)/body or NS (2mL) was instilled into the rat lungs. Animals in another experimental group were also treated with the same dose of surfactant supplement 2hours after the first administration. Bronchoalveolar lavage fluid (BALF) was obtained 5hours after HCL instillation. In BALF, increases in total nuclear cell counts, neutrophil counts, optical density at 412nm as an indicator of pulmonary hemorrhage, neutrophil elastase activity, concentrations of albumin and cytokine-induced neutrophil chemoattractant (CINC) induced by HCL instillation were significantly attenuated by surfactant treatment. The wet-to-dry weight (W/D) ratio in the lung and partial oxygen tension (P(O2)) were also estimated; surfactant treatment significantly attenuated the W/D ratio and improved deteriorated P(O2) induced by HCL. Additional surfactant supplementation did not show further beneficial effects on HCL-induced lung injury compared with a single treatment. These results suggest that surfactant shows an anti-inflammatory effect on acid lung injury in rats but the beneficial effects may be dose limited.

Comparison of acid-induced inflammatory responses in the rat lung during high frequency oscillatory and conventional mechanical ventilation.

BACKGROUND: The present study was performed to compare the effects of high frequency oscillatory ventilation (HFOV) with conventional mechanical ventilation (CMV) on pulmonary inflammatory responses in a rat acid-induced lung injury model. METHODS: Anesthetized rats were instilled intratracheally with HCl (0.1 N, 2 mL/kg) and then randomly divided into three mechanical ventilation settings: HFOV (an oscillatory frequency of 15 Hz, mean airway pressure (MAP) of 9 cmH(2)O), CMV at tidal volume of 12 and 6 mL/kg for 5 h. RESULTS: After HCl instillation, HFOV significantly attenuated the increases in neutrophil infiltration and TNF-α concentration in bronchoalveolar lavage fluid compared with the CMV groups. During HFOV, there was an inhibition of an increase in TNF-α mRNA expression and a decrease in SP-A mRNA expression induced by acid instillation. CONCLUSION: This animal study demonstrates that HFOV is a suitable form of mechanical ventilation to prevent inflammatory responses in acid-induced lung injury.

Ca2+ entry via alpha1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium.

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca(2+) influx in response to diverse environmental stimuli. However, it is not clear whether Ca(2+) influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the alpha(1G) T-type Ca(2+) channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca(2+), immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K(+) perfusate induces Ca(2+) entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca(2+) entry in septal endothelium evoked by the selective TRPV4 activator 4alpha-phorbol-12,13-didecanoate (4alpha-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of alpha(1G) abolishes depolarization-induced Ca(2+) entry and surface expression of P-selectin but does not prevent 4alpha-PDD-activated Ca(2+) entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4alpha-PDD-activated Ca(2+) entry and the increase in permeability, while not impacting depolarization-induced Ca(2+) entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca(2+) entry through alpha(1G) and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

High vascular pressure-induced lung injury requires P450 epoxygenase-dependent activation of TRPV4.

High vascular pressure targets the lung septal network, causing acute lung injury. While calcium entry in septal endothelium has been implicated, the channel involved is not known. This study tested the hypothesis that the vanilloid transient receptor potential channel, TRPV4, is a critical participant in the permeability response to high vascular pressure. Isolated lungs from TRPV4(+/+) or TRPV4(-/-) mice were studied at baseline or during high pressure challenge. Permeability was assessed via the filtration coefficient. Endothelial calcium transients were assessed using epifluorescence microscopy of the lung subpleural network. Light microscopy and point counting were used to determine the alveolar fluid volume fraction, a measure of alveolar flooding. Baseline permeability, calcium intensity, and alveolar flooding were no different in TRPV4(+/+) versus TRPV4(-/-) lungs. In TRPV4(+/+) lungs, the high pressure-induced permeability response was significantly attenuated by low calcium perfusate, the TRPV antagonist ruthenium red, the phospholipase A(2) inhibitor methyl arachidonyl fluorophosphonate, or the P450 epoxygenase inhibitor propargyloxyphenyl hexanoic acid. Similarly, the high pressure-induced calcium transient in TRPV4(+/+) lungs was attenuated with ruthenium red or the epoxygenase inhibitor. High vascular pressure increased the alveolar fluid volume fraction compared with control. In lungs from TRPV4(-/-) mice, permeability, calcium intensity, and alveolar fluid volume fraction were not increased. These data support a role for P450-derived epoxyeicosatrienoic acid-dependent regulation of calcium entry via TRPV4 in the permeability response to high vascular pressure.

Functional coupling of TRPV4, IK, and SK channels contributes to Ca(2+)-dependent endothelial injury in rodent lung.

Our previous work has shown that the increased lung endothelial permeability response to 14,15-epoxyeicosatrienoic acid (14,15-EET) in rat lung requires Ca(2+) entry via vanilloid type-4 transient receptor potential (TRPV4) channels. Recent studies suggest that activation of TRPV4 channels in systemic vascular endothelium prolongs agonist-induced hyperpolarization and amplifies Ca(2+) entry by activating Ca(2+)-activated K(+) (KCa) channels, resulting in vessel relaxation. Activation of endothelial KCa channels thus has potential to increase the electrochemical driving force for Ca(2+) influx via TRPV4 channels and to amplify permeability responses to TRPV4 activation in lung. To examine this hypothesis, we used Western blot analysis, electrophysiological recordings, and isolated-lung permeability measurements to document expression of TRPV4 and KCa channels and the potential for functional coupling. The results show that rat pulmonary microvascular endothelial cells express TRPV4 and 3 KCa channels of different conductances: large (BK), intermediate (IK), and small (SK3). However, TRPV4 channel activity modulates the IK and SK3, but not the BK, channel current density. Furthermore, the TRPV4-mediated permeability response to 14,15-EET in mouse lung is significantly attenuated by pharmacologic blockade of IK and SK3, but not BK, channels. Collectively, this functional coupling suggests that endothelial TRPV4 channels in rodent lung likely form signaling microdomains with IK and SK3 channels and that the integrated response dictates the extent of lung endothelial injury caused by 14,15-EET.

JTE-607, a cytokine release blocker, attenuates acid aspiration-induced lung injury in rats.

The present study was designed to clarify the effects of (-)-ethyl N-[3,5-dichloro-2-hydroxy-4-[2-(4-methyl-piperazin-1-yl)ethoxy]benzoyl]-l-phenylalaninate dihydrochloride (JTE-607), a novel multiple cytokine inhibitor, on hydrochloric acid (HCl) aspiration lung injury in rats. HCl (0.1 N, 2 ml kg(-1)) was instilled into male Sprague-Dawley rats that were pretreated with or without JTE-607 (30 or 75 mg kg(-1) h(-1)). As a control, normal saline (2 ml kg(-1)) was instilled in rats. All the animals were anesthetized with intraperitoneally injected pentobarbital sodium (40 mg kg(-1)). Bronchoalveolar lavage was performed 5 h (h) after HCl or normal saline instillation. In bronchoalveolar lavage fluid, the increases in total nuclear cell counts, neutrophil counts, optical density at 412 nm as an indication of pulmonary hemorrhage, concentrations of albumin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant induced by HCl instillation were significantly reduced by JTE-607 pretreatment. The level of expression of tumor necrosis factor-alpha and interleukin-6 mRNA in lung tissue was analyzed. The mean expression level of tumor necrosis factor-alpha and interleukin-6 mRNA in the JTE-607 group was lower than that in the HCl and NS groups. The wet-to-dry weight ratio was also determined, and JTE-607 at the dose of 75 mg kg(-1) h(-1) significantly attenuated the increased wet-to-dry weight ratio induced by HCl. These results suggest that JTE-607 can inhibit the production of inflammatory cytokines such as tumor necrosis factor-alpha, interleukin-6 and cytokine-induced neutrophil chemoattractant and attenuate acid-induced lung injury in rats. This agent might be therapeutically useful for lung injury.

Effects of granulocyte colony-stimulating factor (G-CSF) and neutrophil elastase inhibitor (ONO-5046) on acid-induced lung injury in rats.

It has been suggested that neutrophils play an important role in acid-aspirated lung injury. We examined the effects of the high dose of granulocyte-colony stimulating factor (G-CSF), which is capable of increasing peripheral neutrophils, and a specific neutrophil elastase inhibitor (ONO-5046) on acid lung injury in rats. Animals were anesthetized and normal saline (NS, 2 mL kg(-1)) or hydrochloric acid (HCl, 0.1 N 2 mL kg(-1)) was then instilled into trachea. Thirty minutes before HCl instillation, G-CSF (150 microg kg(-1)) was injected subcutaneously or ONO-5046 (10 mg kg(-1) h(-1)) was infused continuously into the right jugular vein. Animals were ventilated during the experiments. Five hours after HCl or NS instillation, bronchoalveolar lavage fluid (BALF) and lung tissue samples were obtained. Total nuclear cell count, absorbance, albumin, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, cytokine-induced neutrophil chemoattractant (CINC), neutrophil elastase in BALF, wet-to-dry (W/D) ratio were measured. HCl aspiration markedly increased these values in BALF and W/D ratio. Both ONO-5046 and G-CSF attenuated the parameters increased by acid-induced lung injury in rats. The data suggests that neutrophils play an important role in acid-induced lung injury. However, high-dose G-CSF does not exacerbate acid-aspirated lung injury in rats, although this agent causes an increase in peripheral neutrophils.

An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs.

We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K(f)) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH(2)O PIP at both 35 degrees C and 40 degrees C. Ventilation with 35 cmH(2)O PIP increased K(f) by 2.2-fold at 35 degrees C and 3.3-fold at 40 degrees C compared with baseline, but K(f) increased significantly with time at 40 degrees C with 9 cmH(2)O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in K(f). In TRPV4(-/-) knockout mice, the high PIP ventilation protocol did not increase K(f) at either temperature. We have also found that lung distention caused Ca(2+) entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4(-/-) and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4(-/-) lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation.

Effects of nitric oxide synthase inhibitor on acid aspiration-induced lung injury in rats.

The current study was designed to determine the effects of nitric oxide synthase (NOS) in the development of acid aspiration-induced lung injury in rats. Hydrochloric acid (HCl, 0.1 N; 2 ml/kg) or normal saline (NS, 2 ml/kg) was instilled into the lung of anesthetized, ventilated Sprague-Dawley rats. NG-monomethyl-L-arginine (L-NMMA, 20 mg kg(-1)) and a selective inducible nitric oxide synthase (iNOS) inhibitor, ONO-1714 (0.1 and 0.3 mg kg(-1)), were used to block NOS. Bronchoalveolar lavage fluid (BALF) and wet and dry measurements of lung (W/D) were obtained 5h after HCl or NS instillation. Unlike the control group, rats instilled with HCl showed significant increases in total nuclear cell counts (NCC), neutrophil counts, concentrations of albumin, tumor necrosis factor-alpha (TNF-alpha), interleukine-6 (IL-6) and nitrites/nitrates (NO(x)) in BALF. These parameters were associated with the significantly increased W/D in the HCl group compared with the NS group. ONO-1714 (0.1 mg kg(-1)) significantly prevented the increases in all these parameters. Its inhibitory effects were superior to those of L-NMMA and 0.3 mg kg(-1) of ONO-1714. NOS plays an important role in the pathogenesis of acid aspiration-induced lung injury. Furthermore, selective iNOS inhibition at the optimal dose was most effective in improving lung injury induced by acid aspiration in rats.

Activated protein C attenuates acid-aspiration lung injury in rats.

Acid aspiration causes direct lung damage and secondary inflammatory response involving several cytokines and accumulation of neutrophils. Activated protein C (APC) exhibits antithrombotic and anti-inflammatory properties. We examined the effect and mechanism of pre-treatment APC on acid-aspirated lung injury in rats. Anesthetized rats were instilled intratracheally with normal saline (NS, 2 ml kg(-1)) or hydrochloric acid (HCl, 0.1 N, 2 ml kg(-1)). Thirty minutes before HCl instillation, APC (200 U kg(-1) h(-1)) was infused continuously into the right jugular vein. Animals were ventilated during the experiments. Five hours after HCl or NS instillation, bronchoalveolar lavage fluid (BALF) and lung tissue samples were obtained. Total and differential cell count, absorbance, albumin concentration, concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and cytokine-induced neutrophil chemoattractant (CINC) in BALF, wet and dry weight (W/D) ratio were measured. Platelet count and fibrin degradation product (FDP) in peripheral blood were also measured. HCl instillation markedly increased these values in BALF as well as W/D ratio. APC attenuated the parameters increased by HCl-induced lung injury in rats. However, HCl instillation and APC treatment did not cause significant changes in platelet count and FDP compared with the control. We conclude that APC treatment protected the rats against HCl-induced lung injury and that this action seemed to be due to the anti-inflammatory properties of this protein rather than its anti-coagulant effects.