Hemopexin reverses activation of lung eIF2α and decreases mitochondrial injury in chlorine-exposed mice.

We assessed the mechanisms by which nonencapsulated heme, released in the plasma of mice after exposure to chlorine (Cl2) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult male and female C57BL/6 mice to Cl2 (500 ppm for 30 min), returned them to room air, and injected them intramuscularly with either human hemopexin (hHPX; 5 µg/g BW in 50-µL saline) or vehicle at 1 h post-exposure. Upon return to room air, Cl2-exposed mice, injected with vehicle, developed respiratory acidosis, increased concentrations of plasma proteins in the alveolar space, lung mitochondrial DNA injury, increased levels of free plasma heme, and major alterations of their lung proteome. hHPX injection mice mitigated the onset and development of lung and mitochondrial injury and the increase of plasma heme, reversed the Cl2-induced changes in 83 of 237 proteins in the lung proteome at 24 h post-exposure, and improved survival at 15 days post-exposure. Systems biology analysis of the lung global proteomics data showed that hHPX reversed changes in a number of key pathways including elF2 signaling, verified by Western blotting measurements. Recombinant human hemopexin, generated in tobacco plants, injected at 1 h post-Cl2 exposure, was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl2 results in acute lung injury and the therapeutic effects of hemopexin.NEW & NOTEWORTHY Herein, we demonstrate that exposure of mice to chlorine gas causes significant changes in the lung proteome 24 h post-exposure. Systems biology analysis of the proteomic data is consistent with damage to mitochondria and activation of eIF2, the master regulator of transcription and protein translation. Post-exposure injection of hemopexin, which scavenges free heme, attenuated mtDNA injury, eIF2α phosphorylation, decreased lung injury, and increased survival.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

BACKGROUND: Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical, host-derived regulators of the microbiota. However, mechanisms that support microbiota homeostasis in response to inflammatory stimuli, such as supraphysiologic oxygen, remain unclear. RESULTS: We show that supraphysiologic oxygen exposure to neonatal mice, or direct exposure of intestinal organoids to supraphysiologic oxygen, suppresses the intestinal expression of AMPs and alters intestinal microbiota composition. Oral supplementation of the prototypical AMP lysozyme to hyperoxia-exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. CONCLUSIONS: Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury in newborns. Together, these data support that intestinal AMPs modulate lung injury and repair. Video Abstract.

Hemopexin Reverses Activation of Lung eIF2a and Decreases Mitochondrial Injury in Chlorine Exposed Mice.

We assessed the mechanisms by which non-encapsulated heme, released in the plasma of mice post exposure to chlorine (Cl 2 ) gas, resulted in the initiation and propagation of acute lung injury. We exposed adult C57BL/6 male and female to Cl 2 (500 ppm for 30 min) in environmental chambers and returned them to room air and injected them intramuscularly with a single dose of human hemopexin (hHPX; 5 µg/ g BW), the most efficient scavenger of heme, 30-60 min post exposure. Concentrations of hHPX in plasma of air and Cl 2 exposed mice were 9081±900 vs. 1879± 293 at 6 h and 2966±463 vs. 1555±250 at 50 h post injection (ng/ml; X±1 SEM=3; p<0.01). Cl 2 exposed mice developed progressive acute lung injury post exposure characterized by increased concentrations of plasma heme, marked inflammatory response, respiratory acidosis and increased concentrations of plasma proteins in the alveolar space. Injection of hHPX decreased the onset of acute lung injury at 24 h post exposure; mean survival, for the saline and hHPX groups were 40 vs. 80% (P<0.001) at 15 d post exposure. Non-supervised global proteomics analysis of mouse lungs at 24 h post exposure, revealed the upregulation of 92 and downregulation of 145 lung proteins. Injection of hHPX at one h post exposure moderated the Cl 2 induced changes in eighty-three of these 237 lung proteins. System biology analysis of the global proteomics data showed that hHPX reversed changes in mitochondrial dysfunction and elF2 and integrin signaling. Western blot analysis of lung tissue showed significant increase of phosphorylated elF2 at 24 h post exposure in vehicle treated mice but normal levels in those injected with hHPX. Similarly, RT-PCR analysis of lung tissue showed that hHPX reversed the onset of mtDNA lesions. A form of recombinant human hemopexin generated in tobacco plants was equally effective in reversing acute lung and mtDNA injury. The results of this study offer new insights as to the mechanisms by which exposure to Cl 2 results in acute lung injury and to the therapeutic effects of hemopexin.

The fungal intestinal microbiota predict the development of bronchopulmonary dysplasia in very low birthweight newborns.

RATIONALE: Bronchopulmonary dysplasia (BPD) is the most common morbidity affecting very preterm infants. Gut fungal and bacterial microbial communities contribute to multiple lung diseases and may influence BPD pathogenesis. METHODS: We performed a prospective, observational cohort study comparing the multikingdom fecal microbiota of 144 preterm infants with or without moderate to severe BPD by sequencing the bacterial 16S and fungal ITS2 ribosomal RNA gene. To address the potential causative relationship between gut dysbiosis and BPD, we used fecal microbiota transplant in an antibiotic-pseudohumanized mouse model. Comparisons were made using RNA sequencing, confocal microscopy, lung morphometry, and oscillometry. RESULTS: We analyzed 102 fecal microbiome samples collected during the second week of life. Infants who later developed BPD showed an obvious fungal dysbiosis as compared to infants without BPD (NoBPD, p = 0.0398, permutational multivariate ANOVA). Instead of fungal communities dominated by Candida and Saccharomyces, the microbiota of infants who developed BPD were characterized by a greater diversity of rarer fungi in less interconnected community architectures. On successful colonization, the gut microbiota from infants with BPD augmented lung injury in the offspring of recipient animals. We identified alterations in the murine intestinal microbiome and transcriptome associated with augmented lung injury. CONCLUSIONS: The gut fungal microbiome of infants who will develop BPD is dysbiotic and may contribute to disease pathogenesis.

Mitochondrial DNA Variations Modulate Alveolar Epithelial Mitochondrial Function and Oxidative Stress in Newborn Mice Exposed to Hyperoxia.

Oxidative stress is an important contributor to bronchopulmonary dysplasia (BPD), a form of chronic lung disease that is the most common morbidity in very preterm infants. Mitochondrial functional differences due to inherited and acquired mutations influence the pathogenesis of disorders in which oxidative stress plays a critical role. We previously showed using mitochondrial-nuclear exchange (MNX) mice that mitochondrial DNA (mtDNA) variations modulate hyperoxia-induced lung injury severity in a model of BPD. In this study, we studied the effects of mtDNA variations on mitochondrial function including mitophagy in alveolar epithelial cells (AT2) from MNX mice. We also investigated oxidant and inflammatory stress as well as transcriptomic profiles in lung tissue in mice and expression of proteins such as PINK1, Parkin and SIRT3 in infants with BPD. Our results indicate that AT2 from mice with C57 mtDNA had decreased mitochondrial bioenergetic function and inner membrane potential, increased mitochondrial membrane permeability and were exposed to higher levels of oxidant stress during hyperoxia compared to AT2 from mice with C3H mtDNA. Lungs from hyperoxia-exposed mice with C57 mtDNA also had higher levels of pro-inflammatory cytokines compared to lungs from mice with C3H mtDNA. We also noted changes in KEGG pathways related to inflammation, PPAR and glutamatergic signaling, and mitophagy in mice with certain mito-nuclear combinations but not others. Mitophagy was decreased by hyperoxia in all mice strains, but to a greater degree in AT2 and neonatal mice lung fibroblasts from hyperoxia-exposed mice with C57 mtDNA compared to C3H mtDNA. Finally, mtDNA haplogroups vary with ethnicity, and Black infants with BPD had lower levels of PINK1, Parkin and SIRT3 expression in HUVEC at birth and tracheal aspirates at 28 days of life when compared to White infants with BPD. These results indicate that predisposition to neonatal lung injury may be modulated by variations in mtDNA and mito-nuclear interactions need to be investigated to discover novel pathogenic mechanisms for BPD.

Antimicrobial peptides modulate lung injury by altering the intestinal microbiota.

Mammalian mucosal barriers secrete antimicrobial peptides (AMPs) as critical host-derived regulators of the microbiota. However, mechanisms that support homeostasis of the microbiota in response to inflammatory stimuli such as supraphysiologic oxygen remain unclear. Here, we show that neonatal mice breathing supraphysiologic oxygen or direct exposure of intestinal organoids to supraphysiologic oxygen suppress the intestinal expression of AMPs and alters the composition of the intestinal microbiota. Oral supplementation of the prototypical AMP lysozyme to hyperoxia exposed neonatal mice reduced hyperoxia-induced alterations in their microbiota and was associated with decreased lung injury. Our results identify a gut-lung axis driven by intestinal AMP expression and mediated by the intestinal microbiota that is linked to lung injury. Together, these data support that intestinal AMPs modulate lung injury and repair. In Brief: Using a combination of murine models and organoids, Abdelgawad and Nicola et al. find that suppression of antimicrobial peptide release by the neonatal intestine in response to supra-physiological oxygen influences the progression of lung injury likely via modulation of the ileal microbiota. Highlights: Supraphysiologic oxygen exposure alters intestinal antimicrobial peptides (AMPs).Intestinal AMP expression has an inverse relationship with the severity of lung injury.AMP-driven alterations in the intestinal microbiota form a gut-lung axis that modulates lung injury.AMPs may mediate a gut-lung axis that modulates lung injury.

Platelet Activating Factor Activity Modulates Hyperoxic Neonatal Lung Injury Severity.

Hyperoxia-induced inflammation contributes significantly to developmental lung injury and bronchopulmonary dysplasia (BPD) in preterm infants. Platelet activating factor (PAF) is known to be a major driver of inflammation in lung diseases such as asthma and pulmonary fibrosis, but its role in BPD has not been previously investigated. Therefore, to determine whether PAF signaling independently modulates neonatal hyperoxic lung injury and BPD pathogenesis, lung structure was assessed in 14 day-old C57BL/6 wild-type (WT) and PAF receptor knockout (PTAFR KO) mice that were exposed to 21% (normoxia) or 85% O 2 (hyperoxia) from postnatal day 4. Lung morphometry showed that PTAFR KO mice had attenuated hyperoxia-induced alveolar simplification when compared to WT mice. Functional analysis of gene expression data from hyperoxia-exposed vs. normoxia-exposed lungs of WT and PTAFR KO showed that the most upregulated pathways were the hypercytokinemia/hyperchemokinemia pathway in WT mice, NAD signaling pathway in PTAFR KO mice, and agranulocyte adhesion and diapedesis as well as other pro-fibrotic pathways such as tumor microenvironment and oncostatin-M signaling in both mice strains, indicating that PAF signaling may contribute to inflammation but may not be a significant mediator of fibrotic processes during hyperoxic neonatal lung injury. Gene expression analysis also indicated increased expression of pro-inflammatory genes such as CXCL1, CCL2 and IL-6 in the lungs of hyperoxia-exposed WT mice and metabolic regulators such as HMGCS2 and SIRT3 in the lungs of PTAFR KO mice, suggesting that PAF signaling may modulate BPD risk through changes in pulmonary inflammation and/or metabolic reprogramming in preterm infants.

The SARS-CoV-2 spike S1 protein induces global proteomic changes in ATII-like rat L2 cells that are attenuated by hyaluronan.

The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants, COVID-19 has led to 651,918,402 confirmed cases and 6,656,601 deaths worldwide (as of December 27, 2022; https://covid19.who.int/). Despite advances in our understanding of COVID-19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in the proteome of alveolar type II (ATII)-like rat L2 cells that lack ACE2 receptors. Systems biology analysis revealed that the S1-induced proteomics changes were associated with three significant network hubs: E2F1, CREB1/RelA, and ROCK2/RhoA. We also found that pretreatment of L2 cells with high molecular weight hyaluronan (HMW-HA) greatly attenuated the S1 effects on the proteome. Western blotting analysis and cell cycle measurements confirmed the S1 upregulation of E2F1 and ROCK2/RhoA in L2 cells and the protective effects of HMW-HA. Taken as a whole, our studies revealed profound and novel biological changes that contribute to our current understanding of both S1 and hyaluronan biology. These data show that the S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV-2-induced cell injury.

Mesenchymal stem cell bioenergetics and apoptosis are associated with risk for bronchopulmonary dysplasia in extremely low birth weight infants.

Oxidant stress contributes significantly to the pathogenesis of bronchopulmonary dysplasia (BPD) in extremely low birth weight (ELBW) infants. Mitochondrial function regulates oxidant stress responses as well as pluripotency and regenerative ability of mesenchymal stem cells (MSCs) which are critical mediators of lung development. This study was conducted to test whether differences in endogenous MSC mitochondrial bioenergetics, proliferation and survival are associated with BPD risk in ELBW infants. Umbilical cord-derived MSCs of ELBW infants who later died or developed moderate/severe BPD had lower oxygen consumption and aconitase activity but higher extracellular acidification-indicative of mitochondrial dysfunction and increased oxidant stress-when compared to MSCs from infants who survived with no/mild BPD. Hyperoxia-exposed MSCs from infants who died or developed moderate/severe BPD also had lower PINK1 expression but higher TOM20 expression and numbers of mitochondria/cell, indicating that these cells had decreased mitophagy. Finally, these MSCs were also noted to proliferate at lower rates but undergo more apoptosis in cell cultures when compared to MSCs from infants who survived with no/mild BPD. These results indicate that mitochondrial bioenergetic dysfunction and mitophagy deficit induced by oxidant stress may lead to depletion of the endogenous MSC pool and subsequent disruption of lung development in ELBW infants at increased risk for BPD.

The SARS-CoV-2 Spike S1 Protein Induces Global Proteomic Changes in ATII-Like Rat L2 Cells that are Attenuated by Hyaluronan.

The COVID-19 pandemic continues to impose a major impact on global health and economy since its identification in early 2020, causing significant morbidity and mortality worldwide. Caused by the SARS-CoV-2 virus, along with a growing number of variants that have been characterized to date, COVID-19 has led to 571,198,904 confirmed cases, and 6,387,863 deaths worldwide (as of July 15 th , 2022). Despite tremendous advances in our understanding of COVID19 pathogenesis, the precise mechanism by which SARS-CoV2 causes epithelial injury is incompletely understood. In this current study, robust application of global-discovery proteomics applications combined with systems biology analysis identified highly significant induced changes by the Spike S1 protein of SARS-CoV-2 in an ATII-like Rat L2 cells that include three significant network hubs: E2F1, CREB1/ RelA, and ROCK2/ RhoA. Separately, we found that pre-treatment with High Molecular Weight Hyaluronan (HMW-HA), greatly attenuated the S1 effects. Immuno-targeted studies carried out on E2F1 and Rock2/ RhoA induction and kinase-mediated activation, in addition to cell cycle measurements, validated these observations. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with standard immuno-targeted and cell cycle measurements revealed profound and novel biological changes that contribute to our current understanding of both Spike S1 and Hyaluronan biology. This data shows that the Spike S1 protein may contribute to epithelial injury induced by SARS-CoV-2. In addition, our work supports the potential benefit of HMW-HA in ameliorating SARS CoV2 induced cell injury.

Halogen-Induced Chemical Injury to the Mammalian Cardiopulmonary Systems.

The halogens chlorine (Cl2) and bromine (Br2) are highly reactive oxidizing elements with widespread industrial applications and a history of development and use as chemical weapons. When inhaled, depending on the dose and duration of exposure, they cause acute and chronic injury to both the lungs and systemic organs that may result in the development of chronic changes (such as fibrosis) and death from cardiopulmonary failure. A number of conditions, such as viral infections, coexposure to other toxic gases, and pregnancy increase susceptibility to halogens significantly. Herein we review their danger to public health, their mechanisms of action, and the development of pharmacological agents that when administered post-exposure decrease morbidity and mortality.

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.

Chlorine inhalation induces acute chest syndrome in humanized sickle cell mouse model and ameliorated by postexposure hemopexin.

Triggering factors of Acute Chest Syndrome (ACS) is a leading cause of death in patients with Sickle Cell Disease (SCD) and targeted therapies are limited. Chlorine (Cl2) inhalation happens frequently, but its role as a potential trigger of ACS has not been determined. In this study, we hypothesized that Cl2 exposure resembling that in the vicinity of industrial accidents induces acute hemolysis with acute lung injury, reminiscent of ACS in humanized SCD mice. When exposed to Cl2 (500 ppm for 30 min), 64% of SCD mice succumbed within 6 h while none of the control mice expressing normal human hemoglobin died (p<0.01). Surviving SCD mice had evidence of acute hemolysis, respiratory acidosis, acute lung injury, and high concentrations of chlorinated palmitic and stearic acids (p<0.05) in their plasmas and RBCs compared to controls. Treatment with a single intraperitoneal dose of human hemopexin 30 min after Cl2 inhalation reduced mortality to around 15% (p<0.01) with reduced hemolysis (decreased RBCs fragility (p<0.001) and returned plasma heme to normal levels (p<0.0001)), improved oxygenation (p<0.0001) and reduced acute lung injury scores (p<0.0001). RBCs from SCD mice had significant levels of carbonylation (which predisposes RBCs to hemolysis) 6 h post-Cl2 exposure which were absent in RBCs of mice treated with hemopexin. To understand the mechanisms leading to carbonylation, we incubated RBCs from SCD mice with chlorinated lipids and identified sickling and increased hemolysis compared to RBCs obtained from control mice and treated similarly. Our study indicates that Cl2 inhalation induces ACS in SCD mice via induction of acute hemolysis, and that post exposure administration of hemopexin reduces mortality and lung injury. Our data suggest that SCD patients are vulnerable in Cl2 exposure incidents and that hemopexin is a potential therapeutic agent.

Noninvasive Assessment of Right Ventricle Function and Pulmonary Artery Pressure Using Transthoracic Echocardiography in Women With Pre-Eclampsia: An Exploratory Study.

Background and objective Pre-eclampsia (PEC) is associated with the release of anti-angiogenic factors that are incriminated in raising systemic and pulmonary vascular resistance (PVR). Compared to the left heart and systemic circulation, much less attention has been paid to the right heart and pulmonary circulation in patients with PEC. We used transthoracic echocardiography (TTE) to estimate pulmonary artery (PA) pressure and right ventricular (RV) function in women with PEC. Materials and methods We conducted a case-control study at a tertiary care academic center. Ten early PEC (<34-week gestation) and nine late PEC (≥34-week gestation) patients with 11 early and 10 late gestational age-matched controls were enrolled. Two-dimensional TTE was performed on all patients. The estimated mean PA pressure (eMPAP) was calculated based on PA acceleration time (PAAT). PVR was estimated from eMPAP and RV cardiac output (RV CO). RV myocardial performance index (RV MPI), tricuspid annular plane systolic excursion (TAPSE), tissue tricuspid annular displacement (TTAD), and lateral tricuspid annular tissue peak systolic velocity (S') were measured. Results Compared to early controls, in early PEC, the eMPAP and estimated PVR (ePVR) were elevated, PAAT was reduced, RV MPI was increased, TTAD was reduced, and TAPSE and TV S' were unchanged. Compared to late controls, in late PEC, the eMPAP and ePVR were elevated, PAAT was reduced, and RV MPI was increased, while TAPSE, TTAD, and TV S' were unchanged. Conclusions In a sample of women with PEC, early PEC was found to be associated with increased eMPAP and ePVR and subclinical decrement of RV function as assessed by TTE. TTE may be a useful noninvasive screening tool for early detection of pulmonary hypertension and RV dysfunction in PEC. An adequately powered longitudinal study is needed to determine the implications of these findings on long-term outcomes.

Halogen gas exposure: toxic effects on the parturient.

The elemental halogens include chlorine, bromine, and phosgene. Halogen gas can be directly weaponized and employed in warfare or terrorism. Industrial stockpiles or halogen transport can provide targets for terrorist attack as well as an origin for accidental release creating a risk for potential mass-casualty incidents. Pregnant and post-partum women represent a substantial and vulnerable subset of the population who may be at particular risk during an attack or accidental exposure. We review the effects of halogen exposure on the parturient with a focus on bromine toxicity. Bromine is the most extensively studied agent in the context of pregnancy and to-date murine models form the basis for the majority of current knowledge. Pregnancy potentiates the acute lung injury after halogen exposure. In addition, halogen exposure precipitates a preeclamptic-like syndrome in mice. This phenotype is characterized by systemic and pulmonary hypertension, endothelial dysfunction, decreased cardiac output, placental injury and fetal growth restriction. This constellation contributes to increased maternal and fetal mortality observed after bromine exposure. Angiogenic imbalance is noted with overexpression of the soluble fms-like tyrosine kinase-1 (sFlt-1) form of the vascular endothelial growth factor receptor 1 reminiscent of human preeclampsia. Additional research is needed to further explore the effect of halogen gas exposure in pregnancy and to develop therapeutic interventions to mitigate risk to this unique population.

Halogen exposure injury in the developing lung.

Owing to a high-volume industrial usage of the halogens chlorine (Cl2 ) and bromine (Br2 ), they are stored and transported in abundance, creating a risk for accidental or malicious release to human populations. Despite extensive efforts to understand the mechanisms of toxicity upon halogen exposure and to develop specific treatments that could be used to treat exposed individuals or large populations, until recently, there has been little to no effort to determine whether there are specific features and or the mechanisms of halogen exposure injury in newborns or children. We established a model of neonatal halogen exposure and published our initial findings. In this review, we aim to contrast and compare the findings in neonatal mice exposed to Br2 with the findings published on adult mice exposed to Br2 and the neonatal murine models of bronchopulmonary dysplasia. Despite remarkable similarities across these models in overall alveolar architecture, there are distinct functional and apparent mechanistic differences that are characteristic of each model. Understanding the mechanistic and functional features that are characteristic of the injury process in neonatal mice exposed to halogens will allow us to develop countermeasures that are appropriate for, and effective in, this unique population.

Hyaluronan and halogen-induced airway hyperresponsiveness and lung injury.

Chlorine (Cl2 ) and bromine (Br2 ) are produced in large quantities throughout the world and used in the industry and the sanitation of water. These halogens can pose a significant threat to public health when released into the atmosphere during transportation and industrial accidents, or as acts of terrorism. In this review, we discuss the evidence showing that the activity of Cl2 and Br2 , and of products formed by their interaction with biomolecules, fragment high-molecular-weight hyaluronan (HMW-HA), a key component of the interstitial space and present in epithelial cells, to form proinflammatory, low-molecular-weight hyaluronan fragments that increase intracellular calcium (Ca2+ ) and activate RAS homolog family member A (RhoA) in airway smooth muscle and epithelial and microvascular cells. These changes result in airway hyperresponsiveness (AHR) to methacholine and increase epithelial and microvascular permeability. The increase in intracellular Ca2+ is the result of the activation of the calcium-sensing receptor by Cl2 , Br2 , and their by-products. Posthalogen administration of a commercially available form of HMW-HA to mice and to airway cells in vitro reverses the increase of Ca2+ and the activation of RhoA, and restores AHR to near-normal levels of airway function. These data have established the potential of HMW-HA to be a countermeasure against Cl2 and Br2 toxicity.

Vascular Endothelial Growth Factor-121 Administration Mitigates Halogen Inhalation-Induced Pulmonary Injury and Fetal Growth Restriction in Pregnant Mice.

Background Circulating levels of sFLT-1 (soluble fms-like tyrosine kinase 1), the extracellular domain of vascular endothelial growth factor (VEGF) receptor 1, and its ratio to levels of placental growth factor are markers of the occurrence and severity of preeclampsia. Methods and Results C57BL/6 pregnant mice on embryonic day 14.5 (E14.5), male, and non-pregnant female mice were exposed to air or to Br2 at 600 ppm for 30 minutes and were treated with vehicle or with VEGF-121 (100 µg/kg, subcutaneously) daily, starting 48 hours post-exposure. Plasma, bronchoalveolar lavage fluid, lungs, fetuses, and placentas were collected 120 hours post-exposure. In Br2-exposed pregnant mice, there was a time-dependent and significant increase in plasma levels of sFLT-1 which correlated with increases in mouse lung wet/dry weights and bronchoalveolar lavage fluid protein content. Supplementation of exogenous VEGF-121 improved survival and weight gain, reduced lung wet/dry weights, decreased bronchoalveolar lavage fluid protein levels, enhanced placental development, and improved fetal growth in pregnant mice exposed to Br2. Exogenous VEGF-121 administration had no effect in non-pregnant mice. Conclusions These results implicate inhibition of VEGF signaling driven by sFLT-1 overexpression as a mechanism of pregnancy-specific injury leading to lung edema, maternal mortality, and fetal growth restriction after bromine gas exposure.

Effects of hyperoxia on alveolar and pulmonary vascular development in germ-free mice.

Airway microbial dysbiosis is associated with subsequent bronchopulmonary dysplasia (BPD) development in very preterm infants. However, the relationship of airway microbiome in normal pulmonary development has not been defined. To better understand the role of the airway microbiome, we compared normal and abnormal alveolar and pulmonary vascular development in mice with or without a microbiome. We hypothesized that the lungs of germ-free (GF) mice would have an exaggerated phenotypic response to hyperoxia compared with non-germ-free (NGF) mice. With the use of a novel gnotobiotic hyperoxia chamber, GF and NGF mice were exposed to either normoxia or hyperoxia. Alveolar morphometry, pulmonary mechanics, echocardiograms, inflammatory markers, and measures of pulmonary hypertension were studied. GF and NGF mice in normoxia showed no difference, whereas GF mice in hyperoxia showed protected lung structure and mechanics and decreased markers of inflammation compared with NGF mice. We speculate that an increase in abundance of pathogenic bacteria in NGF mice may play a role in BPD pathogenesis by regulating the proinflammatory signaling and neutrophilic inflammation in lungs. Manipulation of the airway microbiome may be a potential therapeutic intervention in BPD and other lung diseases.

Mitochondrial DNA variation modulates alveolar development in newborn mice exposed to hyperoxia.

Hyperoxia-induced oxidant stress contributes to the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. Mitochondrial functional differences due to mitochondrial DNA (mtDNA) variations are important modifiers of oxidant stress responses. The objective of this study was to determine whether mtDNA variation independently modifies lung development and mechanical dysfunction in newborn mice exposed to hyperoxia. Newborn C57BL6 wild type (C57n/C57mt, C57WT) and C3H/HeN wild type (C3Hn/C3Hmt, C3HWT) mice and novel Mitochondrial-nuclear eXchange (MNX) strains with nuclear DNA (nDNA) from their parent strain and mtDNA from the other-C57MNX (C57n/C3Hmt) and C3HMNX (C3Hn/C57mt)-were exposed to 21% or 85% O2 from birth to postnatal day 14 (P14). Lung mechanics and histopathology were examined on P15. Neonatal mouse lung fibroblast (NMLF) bioenergetics and mitochondrial superoxide (O2-) generation were measured. Pulmonary resistance and mitochondrial O2- generation were increased while alveolarization, compliance, and NMLF basal and maximal oxygen consumption rate were decreased in hyperoxia-exposed C57WT mice (C57n/C57mt) versus C57MNX mice (C57n/C3Hmt) and in hyperoxia-exposed C3HMNX mice (C3Hn/C57mt) versus C3HWT (C3Hn/C3Hmt) mice. Our study suggests that neonatal C57 mtDNA-carrying strains have increased hyperoxia-induced hypoalveolarization, pulmonary mechanical dysfunction, and mitochondrial bioenergetic and redox dysfunction versus C3H mtDNA strains. Therefore, mtDNA haplogroup variation-induced differences in mitochondrial function could modify neonatal alveolar development and BPD susceptibility.