Mechanical ventilation enhances extrapulmonary sepsis-induced lung injury: role of WISP1-αvß5 integrin pathway in TLR4-mediated inflammation and injury.

BACKGROUND: High tidal volume ventilation of healthy lungs or exacerbation of existing acute lung injury (ALI) by more moderate mechanical ventilation (MTV) produces ventilator-induced lung injury. It is less clear whether extrapulmonary sepsis sensitizes the lung to MTV. METHODS: We used a two-hit model of cecal ligation and puncture (CLP) followed 12 h later by MTV (10 ml/kg; 6 h) to determine whether otherwise noninjurious MTV enhances CLP-induced ALI by contrasting wildtype and TLR4-/- mice with respect to: alveolar-capillary permeability, histopathology and intrapulmonary levels of WNT-inducible secreted protein 1 (WISP1) and integrin ß5; plasma levels of cytokines and chemokines (TNF-α, IL-6, MIP-2, MCP-1) and intrapulmonary neutrophil infiltration; and other inflammatory signaling via intrapulmonary activation of JNK, p38 and ERK. A separate cohort of mice was pretreated with intratracheal neutralizing antibodies to WISP1, integrin ß5 or IgG as control and the presented phenotyping repeated in a two-hit model; there were 10 mice per group in these first three experiments. Also, isolated peritoneal macrophages (PM) from wildtype and TLR4-/-, MyD88-/- and TRIF-/- mice were used to identify a WISP1-TLR4-integrin ß5 pathway; and the requisite role of integrin ß5 in WISP1-induced cytokine and chemokine production in LPS-primed PM was examined by siRNA treatment. RESULTS: MTV, that in itself did not cause ALI, exacerbated increases in alveolar-capillary permeability, histopathologic scoring and indices of pulmonary inflammation in mice that previously underwent CLP; the effects of this two-hit model were abrogated in TLR4-/- mice. Attendant with these findings was a significant increase in intrapulmonary WISP1 and integrin ß5 in the two-hit model. Anti-WISP1 or anti-integrin ß5 antibodies partially inhibited the two-hit phenotype. In PM, activation of TLR4 led to an increase in integrin ß5 expression that was MyD88 and NF-κB dependent. Recombinant WISP1 increased LPS-induced cytokine release in PM that was inhibited by silencing either TLR4 or integrin ß5. CONCLUSIONS: These data show for the first time that otherwise noninjurious mechanical ventilation can exacerbate ALI due to extrapulmonary sepsis underscoring a potential interactive contribution of common events (sepsis and mechanical ventilation) in critical care, and that a WISP1-TLR4-integrin ß5 pathway contributes to this phenomenon.

Biosynthesis of oxidized lipid mediators via lipoprotein-associated phospholipase A2 hydrolysis of extracellular cardiolipin induces endothelial toxicity.

We (66) have previously described an NSAID-insensitive intramitochondrial biosynthetic pathway involving oxidation of the polyunsaturated mitochondrial phospholipid, cardiolipin (CL), followed by hydrolysis [by calcium-independent mitochondrial calcium-independent phospholipase A2-γ (iPLA2γ)] of oxidized CL (CLox), leading to the formation of lysoCL and oxygenated octadecadienoic metabolites. We now describe a model system utilizing oxidative lipidomics/mass spectrometry and bioassays on cultured bovine pulmonary artery endothelial cells (BPAECs) to assess the impact of CLox that we show, in vivo, can be released to the extracellular space and may be hydrolyzed by lipoprotein-associated PLA2 (Lp-PLA2). Chemically oxidized liposomes containing bovine heart CL produced multiple oxygenated species. Addition of Lp-PLA2 hydrolyzed CLox and produced (oxygenated) monolysoCL and dilysoCL and oxidized octadecadienoic metabolites including 9- and 13-hydroxyoctadecadienoic (HODE) acids. CLox caused BPAEC necrosis that was exacerbated by Lp-PLA2 Lower doses of nonlethal CLox increased permeability of BPAEC monolayers. This effect was exacerbated by Lp-PLA2 and partially mimicked by authentic monolysoCL or 9- or 13-HODE. Control mice plasma contained virtually no detectable CLox; in contrast, 4 h after Pseudomonas aeruginosa (P. aeruginosa) infection, 34 ± 8 mol% (n = 6; P < 0.02) of circulating CL was oxidized. In addition, molar percentage of monolysoCL increased twofold after P. aeruginosa in a subgroup analyzed for these changes. Collectively, these studies suggest an important role for 1) oxidation of CL in proinflammatory environments and 2) possible hydrolysis of CLox in extracellular spaces producing lysoCL and oxidized octadecadienoic acid metabolites that may lead to impairment of pulmonary endothelial barrier function and necrosis.

Protein-tyrosine phosphatase 4A3 (PTP4A3) promotes vascular endothelial growth factor signaling and enables endothelial cell motility.

Protein-tyrosine phosphatase 4A3 (PTP4A3) is highly expressed in multiple human cancers and is hypothesized to have a critical, albeit poorly defined, role in the formation of experimental tumors in mice. PTP4A3 is broadly expressed in many tissues so the cellular basis of its etiological contributions to carcinogenesis may involve both tumor and stromal cells. In particular, PTP4A3 is expressed in the tumor vasculature and has been proposed to be a direct target of vascular endothelial growth factor (VEGF) signaling in endothelial cells. We now provide the first in vivo experimental evidence that PTP4A3 participates in VEGF signaling and contributes to the process of pathological angiogenesis. Colon tumor tissue isolated from Ptp4a3-null mice revealed reduced tumor microvessel density compared with wild type controls. Additionally, vascular cells derived from Ptp4a3-null tissues exhibited decreased invasiveness in an ex vivo wound healing assay. When primary endothelial cells were isolated and cultured in vitro, Ptp4a3-null cells displayed greatly reduced migration compared with wild type cells. Exposure to VEGF led to an increase in Src phosphorylation in wild type endothelial cells, a response that was completely ablated in Ptp4a3-null cells. In loss-of-function studies, reduced VEGF-mediated migration was also observed when human endothelial cells were treated with a small molecule inhibitor of PTP4A3. VEGF-mediated in vivo vascular permeability was significantly attenuated in PTP4A3-deficient mice. These findings strongly support a role for PTP4A3 as an important contributor to endothelial cell function and as a multimodal target for cancer therapy and mitigating VEGF-regulated angiogenesis.

Orai1 determines calcium selectivity of an endogenous TRPC heterotetramer channel.

RATIONALE: Canonical transient receptor potential 4 (TRPC4) contributes to the molecular composition of a channel encoding for a calcium selective store-operated current, I(SOC), whereas Orai1 critically comprises a channel encoding for the highly selective calcium release activated calcium current, I(CRAC). However, Orai1 may interact with TRPC proteins and influence their activation and permeation characteristics. Endothelium expresses both TRPC4 and Orai1, and it remains unclear as to whether Orai1 interacts with TRPC4 and contributes to calcium permeation through the TPRC4 channel. OBJECTIVE: We tested the hypothesis that Orai1 interacts with TRPC4 and contributes to the channel's selective calcium permeation important for endothelial barrier function. METHODS AND RESULTS: A novel method to purify the endogenous TRPC4 channel and probe for functional interactions was developed, using TRPC4 binding to protein 4.1 as bait. Isolated channel complexes were conjugated to anti-TRPC protein antibodies labeled with cy3-cy5 pairs. Förster Resonance Energy Transfer among labeled subunits revealed the endogenous protein alignment. One TRPC1 and at least 2 TRPC4 subunits constituted the endogenous channel (TRPC1/4). Orai1 interacted with TRPC4. Conditional Orai1 knockdown reduced the probability for TRPC1/4 channel activation and converted it from a calcium-selective to a nonselective channel, an effect that was rescued on Orai1 reexpression. Loss of Orai1 improved endothelial cell barrier function. CONCLUSION: Orai1 interacts with TRPC4 in the endogenous channel complex, where it controls TRPC1/4 activation and channel permeation characteristics, including calcium selectivity, important for control of endothelial cell barrier function.

Zinc deficiency enhances sensitivity to influenza A associated bacterial pneumonia in mice.

Although zinc deficiency (secondary to malnutrition) has long been considered an important contributor to morbidity and mortality of infectious disease (e.g. diarrhea disorders), epidemiologic data (including randomized controlled trials with supplemental zinc) for such a role in lower respiratory tract infection are somewhat ambiguous. In the current study, we provide the first preclinical evidence demonstrating that although diet-induced acute zinc deficiency (Zn-D: ~50% decrease) did not worsen infection induced by either influenza A (H1N1) or methicillin-resistant staph aureus (MRSA), Zn-D mice were sensitive to the injurious effects of superinfection of H1N1 with MRSA. Although the mechanism underlying the sensitivity of ZnD mice to combined H1N1/MRSA infection is unclear, it was noteworthy that this combination exacerbated lung injury as shown by lung epithelial injury markers (increased BAL protein) and decreased genes related to epithelial integrity in Zn-D mice (surfactant protein C and secretoglobins family 1A member 1). As bacterial pneumonia accounts for 25%-50% of morbidity and mortality from influenza A infection, zinc deficiency may be an important pathology component of respiratory tract infections.

Probing the structural basis of Zn2+ regulation of the epithelial Na+ channel.

Extracellular Zn(2+) activates the epithelial Na(+) channel (ENaC) by relieving Na(+) self-inhibition. However, a biphasic Zn(2+) dose response was observed, suggesting that Zn(2+) has dual effects on the channel (i.e. activating and inhibitory). To investigate the structural basis for this biphasic effect of Zn(2+), we examined the effects of mutating the 10 extracellular His residues of mouse γENaC. Four mutations within the finger subdomain (γH193A, γH200A, γH202A, and γH239A) significantly reduced the maximal Zn(2+) activation of the channel. Whereas γH193A, γH200A, and γH202A reduced the apparent affinity of the Zn(2+) activating site, γH239A diminished Na(+) self-inhibition and thus concealed the activating effects of Zn(2+). Mutation of a His residue within the palm subdomain (γH88A) abolished the low-affinity Zn(2+) inhibitory effect. Based on structural homology with acid-sensing ion channel 1, γAsp(516) was predicted to be in close proximity to γHis(88). Ala substitution of the residue (γD516A) blunted the inhibitory effect of Zn(2+). Our results suggest that external Zn(2+) regulates ENaC activity by binding to multiple extracellular sites within the γ-subunit, including (i) a high-affinity stimulatory site within the finger subdomain involving His(193), His(200), and His(202) and (ii) a low-affinity Zn(2+) inhibitory site within the palm subdomain that includes His(88) and Asp(516).

Metallothionein-induced zinc partitioning exacerbates hyperoxic acute lung injury.

Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.

WNT1-inducible signaling pathway protein 1 contributes to ventilator-induced lung injury.

Although strides have been made to reduce ventilator-induced lung injury (VILI), critically ill patients can vary in sensitivity to VILI, suggesting gene-environment interactions could contribute to individual susceptibility. This study sought to uncover candidate genes associated with VILI using a genome-wide approach followed by functional analysis of the leading candidate in mice. Alveolar-capillary permeability after high tidal volume (HTV) ventilation was measured in 23 mouse strains, and haplotype association mapping was performed. A locus was identified on chromosome 15 that contained ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (Asap1), adenylate cyclase 8 (Adcy8), WNT1-inducible signaling pathway protein 1 (Wisp1), and N-myc downstream regulated 1 (Ndrg1). Information from published studies guided initial assessment to Wisp1. After HTV, lung WISP1 protein increased in sensitive A/J mice, but was unchanged in resistant CBA/J mice. Anti-WISP1 antibody decreased HTV-induced alveolar-capillary permeability in sensitive A/J mice, and recombinant WISP1 protein increased HTV-induced alveolar-capillary permeability in resistant CBA/J mice. HTV-induced WISP1 coimmunoprecipitated with glycosylated Toll-like receptor (TLR) 4 in A/J lung homogenates. After HTV, WISP1 increased in strain-matched control lungs, but was unchanged in TLR4 gene-targeted lungs. In peritoneal macrophages from strain-matched mice, WISP1 augmented LPS-induced TNF release that was inhibited in macrophages from TLR4 or CD14 antigen gene-targeted mice, and was attenuated in macrophages from myeloid differentiation primary response gene 88 gene-targeted or TLR adaptor molecule 1 mutant mice. These findings support a role for WISP1 as an endogenous signal that acts through TLR4 signaling to increase alveolar-capillary permeability in VILI.

A critical role for increased labile zinc in reducing sensitivity of cultured sheep pulmonary artery endothelial cells to LPS-induced apoptosis.

We previously noted an important signaling role for decreased labile intracellular zinc ([ Zn ] (i)) in LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) (Tang ZL, Wasserloos KJ, Liu X, Stitt MS, Reynolds IJ, Pitt BR, St Croix CM. Mol Cell Biochem 234-235: 211-217, 2002; Thambiayya K, Wasserloos KJ, Huang Z, Kagan VE, St Croix CM, Pitt BR. Am J Physiol Lung Cell Mol Physiol 300: L624-632, 2011). In the present study, we used small interfering RNA (siRNA) to important contributors of zinc homeostasis [ SLC39A14 or Zrt/Irt-like protein 14 (ZIP14), a zinc importer; metallothionein (MT), a zinc binding protein ] to define molecular pathways by which extracellular zinc or nitric oxide (NO) increase labile [ Zn ] (i) [ e.g., zinc-sensitive fluorophore (FluoZin-3) detectable and/or chelatable by N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine ] and reduce the sensitivity of SPAEC to LPS. Addition of 10 µM zinc to serum-free medium of SPAEC increased [ Zn ] (i) and abolished LPS-induced apoptosis (e.g., increased annexin V binding). The increase in [ Zn ] (i) and the protective effect of extracellular zinc were sensitive to reduction in ZIP14 expression (by siRNA), but not affected by collectively knocking down major isoforms of sheep MT (sMT-Ia, -Ib, -Ic, and -II). Pretreatment of wild-type SPAEC with 250 µM of the NO donor S-nitroso-N-acetylpenicillamine (SNAP) increased labile zinc in a relatively similar fashion to addition of extracellular zinc and reduced sensitivity of SPAEC to LPS-induced apoptosis (e.g., caspase-3/7 activation) in a N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine-sensitive fashion. The antiapoptotic effects of SNAP were insensitive to siRNA knockdown of ZIP14, but were abolished (along with SNAP-induced increase in [ Zn ] (i)) when SPAEC were pretreated with siRNA to sheep MT. Zinc was able to directly inhibit recombinant caspase-3 activity in an in vitro assay. Collectively, these data show that increases in labile [ Zn ] (i) are an important component of ZIP14- or NO-mediated resistance to LPS-induced apoptosis. Cytoprotection via ZIP14 appeared to be secondary to transcellular movement of extracellular zinc, whereas NO-mediated protection was secondary to S-nitrosation of MT and redistribution of [ Zn ] (i).

Iron-induced remodeling in cultured rat pulmonary artery endothelial cells.

Although iron is known to be a component of the pathogenesis and/or maintenance of acute lung injury (ALI) in experimental animals and human subjects, the majority of these studies have focused on disturbances in iron homeostasis in the airways resulting from exposure to noxious gases and particles. Considerably less is known about the effect of increased plasma levels of redox-reactive non-transferrin bound iron (NTBI) and its impact on pulmonary endothelium. Plasma levels of NTBI can increase under various pathophysiological conditions, including those associated with ALI, and multiple mechanisms are in place to affect the [Fe(2+)]/[Fe(3+)] redox steady state. It is well accepted, however, that intracellular transport of NTBI occurs after reduction of [Fe(3+)] to [Fe(2+)] (and is mediated by divalent metal transporters). Accordingly, as an experimental model to investigate mechanisms mediating vascular effects of redox reactive iron, rat pulmonary artery endothelial cells (RPAECs) were subjected to pulse treatment (10 min) with [Fe(2+)] nitriloacetate (30 µM) in the presence of pyrithione, an iron ionophore, to acutely increase intracellular labile pool of iron. Cellular iron influx and cell shape profile were monitored with time-lapse imaging techniques. Exposure of RPAECs to [Fe(2+)] resulted in: (i) an increase in intracellular iron as detected by the iron sensitive fluorophore, PhenGreen; (ii) depletion of cell glutathione; and (iii) nuclear translocation of stress-response transcriptional factors Nrf2 and NFkB (p65). The resulting iron-induced cell alterations were characterized by cell polarization and formation of membrane cuplike and microvilli-like projections abundant with ICAM-1, caveolin-1, and F-actin. The iron-induced re-arrangements in cytoskeleton, alterations in focal cell-cell interactions, and cell buckling were accompanied by decrease in electrical resistance of RPAEC monolayer. These effects were partially eliminated in the presence of N,N'-bis (2-hydroxybenzyl) ethylenediamine-N,N'-diacetic acid, an iron chelator, and Y27632, a Rho-kinase inhibitor. Thus acute increases in labile iron in cultured pulmonary endothelium result in structural remodeling (and a proinflammatory phenotype) that occurs via post-transcriptional mechanisms regulated in a redox sensitive fashion.

LPS-induced decrease in intracellular labile zinc, [Zn]i, contributes to apoptosis in cultured sheep pulmonary artery endothelial cells.

A role in signal transduction for a vanishingly small labile pool of intracellular zinc ([Zn](i)) has been inferred by the sensitivity of various physiological pathways to zinc chelators such as N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and/or associations with changes in nonprotein-bound zinc-sensitive fluorophores. Although we (44) reported that LPS-induced apoptosis in cultured sheep pulmonary artery endothelial cells (SPAEC) was exacerbated by TPEN, 1) we did not detect acute (30 min) changes in [Zn](i), and 2) it is unclear from other reports whether LPS increases or decreases [Zn](i) and whether elevations or decreases in [Zn](i) are associated with cell death and/or apoptosis. In the present study, we used both chemical (FluoZin-3 via live cell epifluorescence microscopy and fluorescence-activated cell sorting) and genetic (luciferase activity of a chimeric reporter encoding zinc-sensitive metal-response element and changes in steady-state mRNA of zinc importer, SLC39A14 or ZIP14) techniques to show that LPS caused a delayed time-dependent (2-4 h) decrease in [Zn](i) in SPAEC. A contributory role of decreases in [Zn](i) in LPS-induced apoptosis (as determined by caspase-3/7 activation, annexin-V binding, and cytochrome c release) in SPAECs was revealed by mimicking the effect of LPS with the zinc chelator, TPEN, and inhibiting LPS- (or TPEN)-induced apoptosis with exogenous zinc. Collectively, these are the first data demonstrating a signaling role for decrease in [Zn](i) in pulmonary endothelial cells and suggest that endogenous levels of labile zinc may affect sensitivity of pulmonary endothelium to the important and complex proapoptotic stimulus of LPS.

A role for zinc in regulating hypoxia-induced contractile events in pulmonary endothelium.

We previously reported that zinc thiolate signaling contributes to hypoxic contraction of small, nonmuscularized arteries of the lung. The present studies were designed to investigate mechanisms by which hypoxia-released zinc induces contraction in isolated pulmonary endothelial cells and to delineate the signaling pathways involved in zinc-mediated changes in the actin cytoskeleton. We used fluorescence-based imaging to show that hypoxia induced time-dependent increases in actin stress fibers that were reversed by the zinc chelator, N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN). We further showed that hypoxia-induced phosphorylation of the contractile protein myosin light chain (MLC) and assembly of actin stress fibers were each TPEN sensitive. Hypoxia and zinc-induced inhibition of MLC phosphatase (MLCP) were independent of the regulatory subunit (MYPT1) of MLCP, and therefore hypoxia-released zinc likely inhibits MLCP at its catalytic (PP1) subunit. Inhibition of PKC by Ro-31-8220 and a dominant-negative construct of PKC-ε attenuated hypoxia-induced contraction of isolated pulmonary endothelial cells. Furthermore, zinc-induced phosphorylation of MLC (secondary to inhibition of MLCP) was PKC dependent, and hypoxia-released zinc promoted the phosphorylation of the PKC substrate, CPI-17. Collectively, these data suggest a link between hypoxia, elevations in labile zinc, and activation of PKC, which in turn acts through CPI-17 to inhibit MLCP activity and promote MLC phosphorylation, ultimately inducing stress fiber formation and endothelial cell contraction.

Cytoprotective effects of albumin, nitrosated or reduced, in cultured rat pulmonary vascular cells.

S-nitrosoalbumin (SNO-Alb) has been shown to be an efficacious cytoprotective molecule in acute lung injury, as well as ischemia-reperfusion injury in heart and skeletal muscle. Nonetheless, limited information is available on the cellular mechanism of such protection. Accordingly, we investigated the protective effects of SNO-Alb [ and its denitrosated congener, reduced albumin (SH-Alb) ] on tert-butyl hydroperoxide (tBH)-mediated cytotoxicity in cultured rat pulmonary microvascular endothelial cells (RPMEC), as well as hydrogen sulfide (H(2)S)-mediated cytotoxicity in rat pulmonary artery smooth muscle cells (RPASMC). We noted that tBH caused a concentration-dependent necrosis in RPMEC, and pretreatment of RPMEC with SNO-Alb dose-dependently decreased the sensitivity of these cells to tBH. A component of SNO-Alb cytoprotection was sensitive to N(G)-nitro-L-arginine methyl ester and was associated with activation of endothelial nitric oxide synthase (eNOS), phenomena that could be reproduced with pretreatment with SH-Alb. Exogenous H(2)S caused concentration-dependent apoptosis in RPASMC due to activation of ERK1/2 and p38, as well as downregulation of Bcl-2. Pretreatment with SNO-Alb reduced H(2)S-mediated apoptosis in a concentration-dependent manner that was associated with SNO-Alb-mediated inhibition of activation of ERK1/2 and p38. Pretreatment with SNO-Alb reduced toxicity of 1 mM sodium hydrosulfide in an N(G)-nitro-L-arginine methyl ester-sensitive fashion in RPASMC that expressed gp60 and neuronal NOS and was capable of transporting fluorescently labeled SH-Alb. Therefore, SNO-Alb is cytoprotective against models of oxidant-induced necrosis (tBH) and inhibitors of cellular respiration and apoptosis (H(2)S) in both pulmonary endothelium and smooth muscle, respectively, and a component of such protection can be attributed to a SH-Alb-mediated activation of constitutive NOS.

Acute, sublethal cyanide poisoning in mice is ameliorated by nitrite alone: complications arising from concomitant administration of nitrite and thiosulfate as an antidotal combination.

Sodium nitrite alone is shown to ameliorate sublethal cyanide toxicity in mice when given from ∼1 h before until 20 min after the toxic dose as demonstrated by the recovery of righting ability. An optimum dose (12 mg/kg) was determined to significantly relieve cyanide toxicity (5.0 mg/kg) when administered to mice intraperitoneally. Nitrite so administered was shown to rapidly produce NO in the bloodsteam as judged by the dose-dependent appearance of EPR signals attributable to nitrosylhemoglobin and methemoglobin. It is argued that antagonism of cyanide inhibition of cytochrome c oxidase by NO is the crucial antidotal activity rather than the methemoglobin-forming action of nitrite. Concomitant addition of sodium thiosulfate to nitrite-treated blood resulted in the detection of sulfidomethemoblobin by EPR spectroscopy. Sulfide is a product of thiosulfate hydrolysis and, like cyanide, is known to be a potent inhibitor of cytochrome c oxidase, the effects of the two inhibitors being essentially additive under standard assay conditions rather than dominated by either one. The findings afford a plausible explanation for an observed detrimental effect in mice associated with the use of the standard nitrite-thiosulfate combination therapy at sublethal levels of cyanide intoxication.

Mechanical Ventilation with Moderate Tidal Volume Exacerbates Extrapulmonary Sepsis-Induced Lung Injury via IL33-WISP1 Signaling Pathway.

ABSTRACT: IL-33 and WNT1-inducible secreted protein (WISP1) play central roles in acute lung injury (ALI) induced by mechanical ventilation with moderate tidal volume (MTV) in the setting of sepsis. Here, we sought to determine the inter-relationship between IL-33 and WISP1 and the associated signaling pathways in this process.We used a two-hit model of cecal ligation puncture (CLP) followed by MTV ventilation (4 h 10 mL/kg) in wild-type, IL-33-/- or ST2-/- mice or wild-type mice treated with intratracheal antibodies to WISP1. Macrophages (Raw 264.7 and alveolar macrophages from wild-type or ST2-/- mice) were used to identify specific signaling components.CLP + MTV resulted in ALI that was partially sensitive to genetic ablation of IL-33 or ST2 or antibody neutralization of WISP1. Genetic ablation of IL-33 or ST2 significantly prevented ALI after CLP + MTV and reduced levels of WISP1 in the circulation and bronchoalveolar lung fluid. rIL-33 increased WISP1 in alveolar macrophages in an ST2, PI3K/AKT, and ERK dependent manner. This WISP1 upregulation and WNT ß-catenin activation were sensitive to inhibition of the ß-catenin/TCF/CBP/P300 nuclear pathway.We show that IL-33 drives WISP1 upregulation and ALI during MTV in CLP sepsis. The identification of this relationship and the associated signaling pathways reveals a number of possible therapeutic targets to prevent ALI in ventilated sepsis patients.

The HIV protease inhibitor Saquinavir attenuates sepsis-induced acute lung injury and promotes M2 macrophage polarization via targeting matrix metalloproteinase-9.

Imbalance of macrophage polarization plays an indispensable role in acute lung injury (ALI), which is considered as a promising target. Matrix metalloproteinase-9 (MMP-9) is expressed in the macrophage, and has a pivotal role in secreting inflammatory cytokines. We reported that saquinavir (SQV), a first-generation human immunodeficiency virus-protease inhibitor, restricted exaggerated inflammatory response. However, whether MMP-9 could regulate macrophage polarization and inhibit by SQV is still unknown. We focused on the important role of macrophage polarization in CLP (cecal ligation puncture)-mediated ALI and determined the ability of SQV to maintain M2 over M1 phenotype partially through the inhibition of MMP-9. We also performed a limited clinical study to determine if MMP-9 is a biomarker of sepsis. Lipopolysaccharide (LPS) increased MMP-9 expression and recombinant MMP-9 (rMMP-9) exacerbated LPS-mediated M1 switching. Small interfering RNA to MMP-9 inhibited LPS-mediated M1 phenotype and SQV inhibition of this switching was reversed with rMMP-9, suggesting an important role for MMP-9 in mediating LPS-induced M1 phenotype. MMP-9 messenger RNA levels in peripheral blood mononuclear cells of these 14 patients correlated with their clinical assessment. There was a significant dose-dependent decrease in mortality and ALI after CLP with SQV. SQV significantly inhibited LPS-mediated M1 phenotype and increased M2 phenotype in cultured RAW 264.7 and primary murine bone marrow-derived macrophages as well as lung macrophages from CLP-treated mice. This study supports an important role for MMP-9 in macrophage phenotypic switching and suggests that SQV-mediated inhibition of MMP-9 may be involved in suppressing ALI during systemic sepsis.

Mechanical Ventilation Exacerbates Poly (I:C) Induced Acute Lung Injury: Central Role for Caspase-11 and Gut-Lung Axis.

Background: The mechanisms by which moderate tidal volume ventilation (MTV) exacerbates preexisting lung injury are unclear. We hypothesized that systemic endotoxemia via the gut-lung axis would lead to non-canonical and canonical inflammasome activation and pyroptosis in a two-hit model involving polyinosinic-polycytidylic acid (Poly(I:C)), a synthetic analog of dsRNA and MTV and that this would associate with acute lung injury (ALI). Methods: Anesthetized mice were administered Poly(I:C) intratracheally and then 6 h later, they were mechanically ventilated for 4 h with otherwise non-injurious MTV (10ml/kg). Changes in intestinal and alveolar capillary permeability were measured. Further documentation of ALI was assessed by evans blue albumin permeability, protein and IL-1 family concentration in bronchoalveolar lavage fluid (BALF) or plasma, and histopathology in cohorts of wildtype (WT), whole body genetically ablated caspase-11 (caspase-11-/-), caspase-1/caspase-11 double knockout (caspase-1/11-/-), gasdermin D (GSDMD)-/-, nucleotide-binding domain leucine-rich repeat-containing protein 3 (NLRP3)-/- and advanced glycosylation end product-specific receptor (RAGE) -/- mice. Results: Non-injurious MTV exacerbated the mild lung injury associated with Poly(I:C) administration. This included the disruption of alveolar-capillary barrier and increased levels of interleukin (IL)-6, high mobility group proteins 1 (HMGB-1), IL-1ß in BALF and IL-18 in plasma. Combined (Poly(I:C)-MTV) injury was associated with increase in gastrointestinal permeability and endotoxin in plasma and BALF. Poly(I:C)-MTV injury was sensitive to caspase-11 deletion with no further contribution of caspase-1 except for maturation and release of IL-18 (that itself was sensitive to deletion of NLRP3). Combined injury led to large increases in caspase-1 and caspase-11. Genetic ablation of GSDMD attenuated alveolar-capillary disruption and release of cytokines in combined injury model. Conclusions: The previously noted exacerbation of mild Poly(I:C)-induced ALI by otherwise non-injurious MTV is associated with an increase in gut permeability resulting in systemic endotoxemia. The gut-lung axis resulted in activation of pulmonary non-canonical (cytosolic mediated caspase-11 activation) and canonical (caspase-1) inflammasome (NLRP3) mediated ALI in this two-hit model resulting in GSDMD sensitive alveolar capillary barrier disruption, pyroptosis (alveolar macrophages) and cytokine maturation and release (IL-1ß; IL-18). Pharmacologic strategies aimed at disrupting communication between gut and lung, inhibition of inflammasomes or GSDMD in pyroptosis may be useful in ALI.

S1P2 receptor-dependent Rho-kinase activation mediates vasoconstriction in the murine pulmonary circulation induced by sphingosine 1-phosphate.

Vasoactive properties of sphingosine 1-phosphate (S1P) have been demonstrated by many investigators to vary in systemic vascular beds. These variations appear to reflect differential S1P receptor expression in the vasculature of these tissues. Although S1P has been demonstrated to enhance endothelial barrier function, induce airway hyperresponsiveness, and modulate immune responses in the lung, the pulmonary vasomotor effects of S1P remain poorly defined. In the present study, we sought to define the vasoregulatory effects of S1P in the pulmonary vasculature and to elucidate the underlying mechanisms operative in effecting the response in the intact lung. S1P (10 microM) increased pulmonary vascular resistance (PVR) by 36% in the isolated perfused mouse lung. S1P-induced vasoconstriction was reduced by 64% by concomitant administration of the Rho-kinase inhibitor Y27632 (10 microM). Similarly, the S1P response was attenuated by >50% after S1P(2) receptor antagonism (JTE-013; 10 microM) and in S1P(2) receptor null mice. In contrast, S1P(3) receptor antagonism (VPC23019; 10 microM) had no effect on the contractile response to S1P. Furthermore, we confirmed the role of Rho-kinase as an important regulator of basal vasomotor tone in the isolated perfused mouse lung. These results suggest that S1P is capable of altering pulmonary vascular tone in vivo and may play an important role in the modulation of pulmonary vascular tone both in the normal lung and under pathological conditions.

Oxidative lipidomics of hyperoxic acute lung injury: mass spectrometric characterization of cardiolipin and phosphatidylserine peroxidation.

Reactive oxygen species have been shown to play a significant role in hyperoxia-induced acute lung injury, in part, by inducing apoptosis of pulmonary endothelium. However, the signaling roles of phospholipid oxidation products in pulmonary endothelial apoptosis have not been studied. Using an oxidative lipidomics approach, we identified individual molecular species of phospholipids involved in the apoptosis-associated peroxidation process in a hyperoxic lung. C57BL/6 mice were killed 72 h after exposure to hyperoxia (100% oxygen). We found that hyperoxia-induced apoptosis (documented by activation of caspase-3 and -7 and histochemical terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of pulmonary endothelium) was accompanied by nonrandom oxidation of pulmonary lipids. Two anionic phospholipids, mitochondria-specific cardiolipin (CL) and extramitochondrial phosphatidylserine (PS), were the two major oxidized phospholipids in hyperoxic lung. Using electrospray ionization mass spectrometry, we identified several oxygenation products in CL and PS. Quantitative assessments revealed a significant decrease of CL and PS molecular species containing C(18:2), C(20:4), C(22:5), and C(22:6) fatty acids. Similarly, exposure of mouse pulmonary endothelial cells (MLEC) to hyperoxia (95% oxygen; 72 h) resulted in activation of caspase-3 and -7 and significantly decreased the content of CL molecular species containing C(18:2) and C(20:4) as well as PS molecular species containing C(22:5) and C(22:6). Oxygenated molecular species were found in the same two anionic phospholipids, CL and PS, in MLEC exposed to hyperoxia. Treatment of MLEC with a mitochondria-targeted radical scavenger, a conjugate of hemi-gramicidin S with nitroxide, XJB-5-131, resulted in significantly lower oxidation of both CL and PS and a decrease in hyperoxia-induced changes in caspase-3 and -7 activation. We speculate that cytochrome c driven oxidation of CL and PS is associated with the signaling role of these oxygenated species participating in the execution of apoptosis and clearance of pulmonary endothelial cells, thus contributing to hyperoxic lung injury.

Mass-spectrometry based oxidative lipidomics and lipid imaging: applications in traumatic brain injury.

Lipids, particularly phospholipids, are fundamental to CNS tissue architecture and function. Endogenous polyunsaturated fatty acid chains of phospholipids possess cis-double bonds each separated by one methylene group. These phospholipids are very susceptible to free-radical attack and oxidative modifications. A combination of analytical methods including different versions of chromatography and mass spectrometry allows detailed information to be obtained on the content and distribution of lipids and their oxidation products thus constituting the newly emerging field of oxidative lipidomics. It is becoming evident that specific oxidative modifications of lipids are critical to a number of cellular functions, disease states and responses to oxidative stresses. Oxidative lipidomics is beginning to provide new mechanistic insights into traumatic brain injury which may have significant translational potential for development of therapies in acute CNS insults. In particular, selective oxidation of a mitochondria-specific phospholipid, cardiolipin, has been associated with the initiation and progression of apoptosis in injured neurons thus indicating new drug discovery targets. Furthermore, imaging mass-spectrometry represents an exciting new opportunity for correlating maps of lipid profiles and their oxidation products with structure and neuropathology. This review is focused on these most recent advancements in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury.