Pulmonary toxicity following inhalation exposure to VX in anesthetized rats: Possible roles for compromised immunity and oxidative stress-induced lung injury.

The nerve agent VX is one of the most deadly threat agents available in weapons stockpiles for intentional release. While mostly considered a percutaneous toxicant, it can be fatal when aerosolized. The objective of this study was to investigate toxic responses in the lung up to two weeks following a single 10-minute exposure to inhaled VX. Anesthetized rats were exposed singly and only once to VX. The nebulization rate in this system was 0.2-0.3 ml per minute with the delivery of a consistent particle size of 2.1 µm. Following exposure, all rats were removed from the ventilator and allowed to recover in the glovebox for 10-15 minutes. Results showed that inhaled VX altered several respiratory parameters and caused increased lung resistance up to 6 h post-exposure (PE). There was a trending increase in SOD and xanthine oxidoreductase (XOR) activities, both of which are indicative of oxidative stress. Based on increased lung tissue p38 signaling, MAP kinase expression was activated after VX exposure. IL-6 expression was also increased at 6 h post-inhalation for the 31.6 mg/m3 exposed group. Innate survival response mechanisms in rats may be present due to increased lung tissue mRNA AChE expression 6 h after exposure. Immunohistochemistry showed reduced staining for surfactant D and increased expression of iNOS, indicating that the activation of •NO precursor pathways. Bronchoalveloar lavage fluid (BALF) results from 1 h to 2 weeks PE show that inflammatory cells are highly active as evidenced by the increased production of cytokines and chemokines. This is the first study linking VX-induced lung injury to a possible innate survival amplification of AChE and possibly compromised immune function. These results could supplement medical treatment strategies with regard to therapeutic approaches against VX inhalational challenge.

Acute pulmonary toxicity following inhalation exposure to aerosolized VX in anesthetized rats.

This study evaluated acute toxicity and pulmonary injury in rats at 3, 6 and 24 h after an inhalation exposure to aerosolized O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX). Anesthetized male Sprague-Dawley rats (250-300 g) were incubated with a glass endotracheal tube and exposed to saline or VX (171, 343 and 514 mg×min/m³ or 0.2, 0.5 and 0.8 LCt50, respectively) for 10 min. VX was delivered by a small animal ventilator at a volume of 2.5 ml × 70 breaths/minute. All VX-exposed animals experienced a significant loss in percentage body weight at 3, 6, and 24 h post-exposure. In comparison to controls, animals exposed to 514 mg×min/m³ of VX had significant increases in bronchoalveolar lavage (BAL) protein concentrations at 6 and 24 h post-exposure. Blood acetylcholinesterase (AChE) activity was inhibited dose dependently at each of the times points for all VX-exposed groups. AChE activity in lung homogenates was significantly inhibited in all VX-exposed groups at each time point. All VX-exposed animals assessed at 20 min and 3, 6 and 24 h post-exposure showed increases in lung resistance, which was prominent at 20 min and 3 h post-exposure. Histopathologic evaluation of lung tissue of the 514 mg×min/m³ VX-exposed animals at 3, 6 and 24 h indicated morphological changes, including perivascular inflammation, alveolar exudate and histiocytosis, alveolar septal inflammation and edema, alveolar epithelial necrosis, and bronchiolar inflammatory infiltrates, in comparison to controls. These results suggest that aerosolization of the highly toxic, persistent chemical warfare nerve agent VX results in acute pulmonary toxicity and lung injury in rats.

Determination of LCt(50)s in anesthetized rats exposed to aerosolized nerve agents.

Nerve agents pose a threat to the respiratory tract with exposure that could result in acute compromised lung performance and death. The determination of toxicity by inhalation is important for the rational development of timely therapeutic countermeasures. This study was designed to deliver aerosolized dilute nerve agents in a dose-response manner to investigate the extent of lethality of nerve agents: soman, sarin, VX and VR. Male rats (240-270 g) were anesthetized intramuscularly with 10 mg/kg xylazine and 90 mg/kg ketamine. Following anesthesia, rats were intubated with a glass endotracheal tube (ET) and placed in a glove box. The ET was connected to a closed circuit nebulizer system (Aeroneb, Aerogen, Inc.) that delivered a particle size of < 2.0 µm and was in series between the ventilator and the ET. Nerve agents were delivered by a small animal ventilator set for a volume of 2.5 mL × 60-80 breaths/min. VX or VR were nebulized and delivered in concentrations ranging from 6.25-800 µg/kg over a 10-min exposure time period. Sarin (GB) or soman (GD), 6.5-1250 µg/kg, were delivered in a similar manner. Lethality by inhalation occurred either during the 10-min exposure period or less than 15 min after the cessation of exposure. Survivors were euthanized at 24 h postexposure. LCt(50) estimates (± 95% confidence intervals [CIs]) were obtained from the sequential stage-wise experiments using the probit analysis. Probit analysis revealed that the LD(50) for VX was 110.7 µg/kg (CI: 73.5-166.7), VR 64.2 µg/kg (CI: 42.1-97.8); soman (GD), 167 µg/kg (CI: 90-310), and sarin (GB), 154 µg/kg (CI: 98-242), respectively. Although VR is a structural isomer of VX, the compounds appear to be markedly different in terms of toxicity when delivered by aerosol. These relationships were converted to actual 10 min LCt(50) equivalents: VX = 632.2, VR = 367, GD = 954.3 and GB = 880 mg·min/m(3). Validation of exposure was verified by the determination of blood levels of acetylcholinesterase (AChE) across doses for the agent VR.

Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung.

AIM: To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling. METHODS: Male C57BL/J6 (wild-type, WT) or eNOS knockout mice (eNOS(-/-)) were exposed to mechanical ventilation (MV) with low (LV(T), 7 mL/kg) and high tidal volume (HV(T), 20 mL/kg) for 0-4 h. A subset of WT mice was administered the specific inhibitors of PI3K (100 nmol/L Wortmannin [Wort], ip) or of p38 MAPK (SB203580, 2 mg/kg, ip) 1 h before MV. Cultured type II alveolar epithelial cells C10 were exposed to 18% cyclic stretch for 2 h with or without 20 nmol/L Wort pretreatment. At the end of the experiment, the capillary leakage in vivo was assessed by extravasation of Evans blue dye (EBD), wet/dry weight ratio and lung lavage protein concentration. The lung tissue and cell lysate were also collected for protein and histological review. RESULTS: MV decreased PI3K/Akt phosphorylation and eNOS expression but increased phospho-p38 MAPK expression along with a lung leakage of EBD. Inhibitions of phospho-Akt by Wort worsen the lung edema, whereas inhibition of p38 MAPK kinase restored activation of Akt together with alleviated capillary leakage. eNOS(-/-) mice showed an exacerbated lung edema and injury. The stretched C10 cells demonstrated that Wort diminished the activation of Akt, but potentiated phosphorylation of MAPK p38. CONCLUSION: Our results indicate that PI-3K/Akt/eNOS pathway has significant protective effects in VALI by preventing capillary leakage, and that there is a cross-talk between PI3K/Akt and p38 MAPK pathways in vascular barrier dysfunction resulting from VALI.

Mitogen activated protein kinase activated protein kinase 2 regulates actin polymerization and vascular leak in ventilator associated lung injury.

Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.

Alveolar cell apoptosis is dependent on p38 MAP kinase-mediated activation of xanthine oxidoreductase in ventilator-induced lung injury.

Signaling via p38 MAP kinase has been implicated in the mechanotransduction associated with mechanical stress and ventilator-induced lung injury (VILI). However, the critical downstream mediators of alveolar injury remain incompletely defined. We provide evidence that high-tidal volume mechanical ventilation (HVt MV) rapidly activates caspases within the lung, resulting in increased alveolar cell apoptosis. Antagonism of MV-induced p38 MAP kinase activity with SB-203580 suppresses both MV-induced caspase activity and alveolar apoptosis, placing p38 MAP kinase upstream of MV-induced caspase activation and programmed cell death. The reactive oxygen species (ROS)-producing enzyme xanthine oxidoreductase (XOR) is activated in a p38 MAP kinase-dependent manner following HVt MV. Allopurinol, a XOR inhibitor, also suppresses HVt MV-induced apoptosis, implicating HVt MV-induced ROS in the induction of alveolar cell apoptosis. Finally, systemic administration of the pan-caspase inhibitor, z-VAD-fmk, but not its inactive peptidyl analog, z-FA-fmk, blocks ventilator-induced apoptosis of alveolar cells and alveolar-capillary leak, indicating that caspase-dependent cell death is necessary for VILI-associated barrier dysfunction in vivo.

Regression of chronic hypoxic pulmonary hypertension by simvastatin.

The 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor, simvastatin, has been shown to attenuate chronic hypoxic pulmonary hypertension (CHPH). Here, we assess whether simvastatin is capable of inducing regression of established CHPH and explore potential mechanisms of statin effect. Rats (n = 8 in each group) were exposed to chronic hypoxia (10% Fi(O(2))) for 2 or 4 wk. Simvastatin treatment (20 mg.kg(-1).day(-1)) commenced after 2 wk of hypoxia, at which time CHPH was fully established, reduced mean pulmonary artery pressure (19 +/- 0.5 vs. 27 +/- 0.9 mmHg; P < 0.001), the ratio of right ventricular free wall to left ventricular plus septal weight (0.41 +/- 0.03 vs. 0.54 +/- 0.03; P < 0.001), and medial thickening of small pulmonary arteries (13 +/- 0.4 vs. 16 +/- 0.4%; P < 0.01) compared with 4-wk hypoxic controls. Supplementation with mevalonate (50 mg.kg(-1).day(-1)) prevented the attenuation of CHPH induced by simvastatin during 2 wk of hypoxia. Because statins are known to inhibit Rho-kinase (ROCK), we determined expression of ROCK-1 and -2 in whole lung by Western blot and ROCK activity by phosphorylation of the myosin-binding subunit of myosin phosphatase. Expression of both ROCK-1 and -2 were markedly diminished in simvastatin-treated animals during normoxia and hypoxia (2- and 4-wk) exposure (P < 0.01). ROCK activity was increased threefold under hypoxic conditions and normalized with simvastatin treatment (P < 0.001). We conclude that simvastatin attenuates and induces regression of established CHPH through inhibition of HMG-CoA reductase. Inhibition of ROCK expression and activity may be an important mechanism of statin effect.

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways.

Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.

Inducible nitric oxide synthase contributes to ventilator-induced lung injury.

RATIONALE: Inducible nitric oxide synthase (iNOS) has been implicated in the development of acute lung injury. Recent studies indicate a role for mechanical stress in iNOS and endothelial NOS (eNOS) regulation. OBJECTIVES: This study investigated changes in lung NOS expression and activity in a mouse model of ventilator-induced lung injury. METHODS: C57BL/6J (wild-type [WT]) and iNOS-deficient (iNOS(-/-)) mice received spontaneous ventilation (control) or mechanical ventilation (MV; VT of 7 and 20 ml/kg) for 2 hours, after which NOS gene expression and activity were determined and pulmonary capillary leakage assessed by the Evans blue albumin assay. RESULTS: iNOS mRNA and protein expression was absent in iNOS(-/-) mice, minimal in WT control mice, but significantly upregulated in response to 2 hours of MV. In contrast, eNOS protein was decreased in WT mice, and nonsignificantly increased in iNOS(-/-) mice, as compared with control animals. iNOS and eNOS activities followed similar patterns in WT and iNOS(-/-) mice. MV caused acute lung injury as suggested by cell infiltration and nitrotyrosine accumulation in the lung, and a significant increase in bronchoalveolar lavage cell count in WT mice, findings that were reduced in iNOS(-/-) mice. Finally, Evans blue albumin accumulation in lungs of WT mice was significant (50 vs. 15% increase in iNOS(-/-) mice compared with control animals) in response to MV and was prevented by treatment of the animals with the iNOS inhibitor aminoguanidine. CONCLUSION: Taken together, our results indicate that iNOS gene expression and activity are significantly upregulated and contribute to lung edema in ventilator-induced lung injury.

Sphingosine 1-phosphate reduces vascular leak in murine and canine models of acute lung injury.

Excessive mechanical stress is a key component of ventilator-associated lung injury, resulting in profound vascular leak and an intense inflammatory response. To extend our in vitro observations concerning the barrier-protective effects of the lipid growth factor sphingosine 1-phosphate (Sph 1-P), we assessed the ability of Sph 1-P to prevent regional pulmonary edema accumulation in clinically relevant rodent and canine models of acute lung injury induced by combined intrabronchial endotoxin administration and high tidal volume mechanical ventilation. Intravenously delivered Sph 1-P significantly attenuated both alveolar and vascular barrier dysfunction while significantly reducing shunt formation associated with lung injury. Whole lung computed tomographic image analysis demonstrated the capability of Sph 1-P to abrogate significantly the accumulation of extravascular lung water evoked by 6-hour exposure to endotoxin. Axial density profiles and vertical density gradients localized the Sph 1-P response to transitional zones between aerated and consolidated lung regions. Together, these results indicate that Sph 1-P represents a novel therapeutic intervention for the prevention of pulmonary edema related to inflammatory injury and increased vascular permeability.

Dependency of cortical functional hyperemia to forepaw stimulation on epoxygenase and nitric oxide synthase activities in rats.

Individual inhibition of nitric oxide (NO) synthase and cytochrome P450 (CYP) epoxygenase activity attenuates cortical functional hyperemia evoked by whisker stimulation. The objectives of the present study were to determine (1) if administration of epoxygenase inhibitors attenuates cortical functional hyperemia by using a different modality of sensory activation (i.e., electrical stimulation of the rat forepaw), (2) if epoxygenase inhibition has an additive effect with NO synthase inhibition on the flow response, and (3) the cellular localization of the epoxygenase CYP2C11 in cerebral cortex. In six groups of anesthetized rats, the cortical surface was superfused for 90 minutes with (1) vehicle; (2) 1-mmol/L Nomega-nitro-L-arginine (L-NNA), to inhibit NO synthase activity; (3) 20-micromol/L N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), a substrate inhibitor of P450 epoxygenase; (4) MS-PPOH plus L-NNA; (5) 20-micromol/L miconazole, a reversible inhibitor at the heme site of P450 epoxygenase; and (6) miconazole plus L-NNA. The percent increases in laser-Doppler perfusion over primary sensory cortex during 20-second forepaw stimulation were reduced by 44% to 64% in all drug-treated groups. The addition of L-NNA to MS-PPOH produced no additional reduction (64%) compared with MS-PPOH alone (64%) or L-NNA alone (60%). The addition of L-NNA to miconazole also produced no additional reduction in the flow response. In situ hybridization of CYP2C11 mRNA showed localization in astrocytes, including those adjacent to blood vessels. Thus, activity of both epoxygenase, presumably localized in astrocytes, and NO synthase is required for generating a complete cortical hyperemic response evoked by electrical forepaw stimulation. The lack of additional blood flow attenuation with the combination of the NO synthase and the distinct epoxygenase inhibitors suggests that the signaling pathways do not act in a simple parallel fashion and that other mediators may be involved in coupling cortical blood flow to neuronal activation.

Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury.

Our prior in vitro studies indicate that sphingosine 1-phosphate (S1P), a phospholipid angiogenic factor, produces endothelial cell barrier enhancement through ligation of endothelial differentiation gene family receptors. We hypothesized that S1P may reduce the vascular leak associated with acute lung injury and found that S1P infusion produced a rapid and significant reduction in lung weight gain (more than 50%) in the isolated perfused murine lung. The effect of S1P was next assessed in a murine model of LPS-mediated microvascular permeability and inflammation with marked increases in parameters of lung injury at both 6 and 24 hours after intratracheal LPS. Each parameter assessed was significantly reduced by intravenous S1P (1 microM final) and in selected experiments by the S1P analogue FTY720 (0.1 mg/kg, intraperitoneally) delivered 1 hour after LPS. S1P produced an approximately 40-50% reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage protein content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Consistent with systemic barrier enhancement, S1P significantly decreased Evans blue dye albumin extravasation and myeloperoxidase content in renal tissues of LPS-treated mice. These studies indicate that S1P significantly decreases pulmonary/renal vascular leakage and inflammation in a murine model of LPS-mediated acute lung injury and may represent a novel therapeutic strategy for vascular barrier dysfunction.

Wall stiffness suppresses Akt/eNOS and cytoprotection in pulse-perfused endothelium.

Increased steady shear stress stimulates nitric oxide synthase (eNOS) in part by Akt-dependent phosphorylation. Arteries in vivo are exposed to pulse perfusion (PP) combining phasic shear with stretch. In compliant vessels, enhancing PP lowers vascular tone by stimulating eNOS; whereas in aged, stiff arteries, flow-mediated dilation declines and PP is a prominent risk factor. Here, we tested the hypothesis that reduced wall distensibility alters PP-induced eNOS/Akt mechano-signaling. Bovine aortic endothelial cells cultured within distensible tubes were exposed to physiological nonreversing steady or PP (7 dynes/cm(2) mean shear, pulse pressure 0 or 90 mm Hgx2 hours) in a custom servo-system. In compliant tubes, PP doubled Akt phosphorylation above nonpulsatile flow levels, whereas P-Akt declined to static levels from PP in stiffer tubes. eNOS phosphorylation (S-1179) similarly increased with PP in compliant tubes but was nearly undetectable with increased PP in stiffer tubes. After PP, brief exposure of cells to ultraviolet irradiation (oxidant stress) and subsequent culture revealed cytoprotection in compliant tubes but diffuse cytotoxicity and cell detachment in stiffer tubes. NOS inhibition by L-NAME converted compliant-tube post-UV behavior to that of stiffer tubes. These data provide novel evidence that wall compliance can directionally mediate endothelial Akt/eNOS phosphorylation and mechano-signaling. This may help explain increased vascular risks resulting from artery stiffening.

Suppression of cortical functional hyperemia to vibrissal stimulation in the rat by epoxygenase inhibitors.

Application of glutamate to glial cell cultures stimulates the formation and release of epoxyeicosatrienoic acids (EETs) from arachidonic acid by cytochome P-450 epoxygenases. Epoxygenase inhibitors reduce the cerebral vasodilator response to glutamate and N-methyl-D-aspartate. We tested the hypothesis that epoxygenase inhibitors reduce the somatosensory cortical blood flow response to whisker activation. In chloralose-anesthetized rats, percent changes in cortical perfusion over whisker barrel cortex were measured by laser-Doppler flowmetry during whisker stimulation. Two pharmacologically distinct inhibitors were superfused subdurally: 1) N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), an epoxygenase substrate inhibitor; and 2) miconazole, a reversible cytochrome P-450 inhibitor acting on the heme moiety. Superfusion with 5 micromol/l MS-PPOH decreased the hyperemic response to whisker stimulation by 28% (from 25 +/- 9 to 18 +/- 7%, means +/- SD, n = 8). With 20 micromol/l MS-PPOH superfusion, the response was decreased by 69% (from 28 +/- 9% to 9 +/- 4%, n = 8). Superfusion with 20 micromol/l miconazole decreased the flow response by 67% (from 31 +/- 6% to 10 +/- 3%, n = 8). Subsequent superfusion with vehicle restored the response to 26 +/- 11%. Indomethacin did not prevent MS-PPOH inhibition of the flow response, suggesting that EET-related vasodilation was not dependent solely on cyclooxygenase metabolism of 5,6-EET. Neither MS-PPOH nor miconazole changed baseline flow, reduced the blood flow response to an adenosine A(2) agonist, or decreased somatosensory evoked potentials. The marked reduction of the cortical flow response to whisker stimulation with two different types of epoxygenase inhibitors indicates that EETs play an important role in the physiological coupling of blood flow to neural activation.