Tara B. Hendry-Hofer

Treatment with the catalytic metalloporphyrin AEOL 10150 reduces inflammation and oxidative stress due to inhalation of the sulfur mustard analog 2-chloroethyl ethyl sulfide

Sulfur mustard (bis-2-(chloroethyl) sulfide; SM) is a highly reactive vesicating and alkylating chemical warfare agent. A SM analog, 2-chloroethyl ethyl sulfide (CEES), has been utilized to elucidate mechanisms of toxicity and as a screen for therapeutics. Previous studies with SM and CEES have demonstrated a role for oxidative stress as well as decreased injury with antioxidant treatment. We tested whether posttreatment with the metalloporphyrin catalytic antioxidant AEOL 10150 would improve outcome in CEES-induced lung injury. Anesthetized rats inhaled 5% CEES for 15 min via a nose-only inhalation system. At 1 and 9 h after CEES exposure, rats were given AEOL 10150 (5 mg/kg, sc). At 18 h post-CEES exposure BALF lactate dehydrogenase activity, protein, IgM, red blood cells, and neutrophils were elevated but were decreased by AEOL 10150 treatment. Lung myeloperoxidase activity was increased after CEES inhalation and was ameliorated by AEOL 10150. The lung oxidative stress markers 8-OHdG and 4-HNE were elevated after CEES exposure and significantly decreased by AEOL 10150 treatment. These findings demonstrate that CEES inhalation increased lung injury, inflammation, and oxidative stress, and AEOL 10150 was an effective rescue agent. Further investigation utilizing catalytic antioxidants as treatment for SM inhalation injury is warranted.

Airway Obstruction Due to Bronchial Vascular Injury after Sulfur Mustard Analog Inhalation

RATIONALE Sulfur mustard (SM) is a frequently used chemical warfare agent, even in modern history. SM inhalation causes significant respiratory tract injury, with early complications due to airway obstructive bronchial casts, akin to those seen after smoke inhalation and in single-ventricle physiology. This process with SM is poorly understood because animal models are unavailable. OBJECTIVES To develop a rat inhalation model for airway obstruction with the SM analog 2-chloroethyl ethyl sulfide (CEES), and to investigate the pathogenesis of bronchial cast formation. METHODS Adult rats were exposed to 0, 5, or 7.5% CEES in ethanol via nose-only aerosol inhalation (15 min). Airway microdissection and confocal microscopy were used to assess cast formation (4 and 18 h after exposure). Bronchoalveolar lavage fluid (BALF) retrieval and intravascular dye injection were done to evaluate vascular permeability. MEASUREMENTS AND MAIN RESULTS Bronchial casts, composed of abundant fibrin and lacking mucus, occluded dependent lobar bronchi within 18 hours of CEES exposure. BALF contained elevated concentrations of IgM, protein, and fibrin. Accumulation of fibrin-rich fluid in peribronchovascular regions (4 h) preceded cast formation. Monastral blue dye leakage identified bronchial vessels as the site of leakage. CONCLUSIONS After CEES inhalation, increased permeability from damaged bronchial vessels underlying damaged airway epithelium leads to the appearance of plasma proteins in both peribronchovascular regions and BALF. The subsequent formation of fibrin-rich casts within the airways then leads to airways obstruction, causing significant morbidity and mortality acutely after exposure.

Tissue Plasminogen Activator Prevents Mortality from Sulfur Mustard Analog–Induced Airway Obstruction

Sulfur mustard (SM) inhalation causes the rare but life-threatening disorder of plastic bronchitis, characterized by bronchial cast formation, resulting in severe airway obstruction that can lead to respiratory failure and death. Mortality in those requiring intubation is greater than 80%. To date, no antidote exists for SM toxicity. In addition, therapies for plastic bronchitis are solely anecdotal, due to lack of systematic research available to assess drug efficacy in improving mortality and/or morbidity. Adult rats exposed to SM analog were treated with intratracheal tissue plasminogen activator (tPA) (0.15-0.7 mg/kg, 5.5 and 6.5 h), compared with controls (no treatment, isoflurane, and placebo). Respiratory distress and pulse oximetry were assessed (for 12 or 48 h), and arterial blood gases were obtained at study termination (12 h). Microdissection of fixed lungs was done to assess airway obstruction by casts. Optimal intratracheal tPA treatment (0.7 mg/kg) completely eliminated mortality (0% at 48 h), and greatly improved morbidity in this nearly uniformly fatal disease model (90-100% mortality at 48 h). tPA normalized plastic bronchitis-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress (i.e., clinical scores) while decreasing airway fibrin casts. Intratracheal tPA diminished airway-obstructive fibrin-containing casts while improving clinical respiratory distress, pulmonary gas exchange, tissue oxygenation, and oxygen utilization in our model of severe chemically induced plastic bronchitis. Most importantly, mortality, which was associated with hypoxemia and clinical respiratory distress, was eliminated.

Airway tissue plasminogen activator prevents acute mortality due to lethal sulfur mustard inhalation

RATIONALE Sulfur mustard (SM) is a chemical weapon stockpiled today in volatile regions of the world. SM inhalation causes a life-threatening airway injury characterized by airway obstruction from fibrin casts, which can lead to respiratory failure and death. Mortality in those requiring intubation is more than 80%. No therapy exists to prevent mortality after SM exposure. Our previous work using the less toxic analog of SM, 2-chloroethyl ethyl sulfide, identified tissue plasminogen activator (tPA) an effective rescue therapy for airway cast obstruction (Veress, L. A., Hendry-Hofer, T. B., Loader, J. E., Rioux, J. S., Garlick, R. B., and White, C. W. (2013). Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction. Am. J. Respir. Cell Mol. Biol. 48, 439-447). It is not known if exposure to neat SM vapor, the primary agent used in chemical warfare, will also cause death due to airway casts, and if tPA could be used to improve outcome. METHODS Adult rats were exposed to SM, and when oxygen saturation reached less than 85% (median: 6.5 h), intratracheal tPA or placebo was given under isoflurane anesthesia every 4 h for 48 h. Oxygen saturation, clinical distress, and arterial blood gases were assessed. Microdissection was done to assess airway obstruction by casts. RESULTS Intratracheal tPA treatment eliminated mortality (0% at 48 h) and greatly improved morbidity after lethal SM inhalation (100% death in controls). tPA normalized SM-associated hypoxemia, hypercarbia, and lactic acidosis, and improved respiratory distress. Moreover, tPA treatment resulted in greatly diminished airway casts, preventing respiratory failure from airway obstruction. CONCLUSIONS tPA given via airway more than 6 h after exposure prevented death from lethal SM inhalation, and normalized oxygenation and ventilation defects, thereby rescuing from respiratory distress and failure. Intra-airway tPA should be considered as a life-saving rescue therapy after a significant SM inhalation exposure incident.

Airway tissue factor-dependent coagulation activity in response to sulfur mustard analog 2-chloroethyl ethyl sulfide

Acute lung injury is a principal cause of morbidity and mortality in response to mustard gas (SM) inhalation. Obstructive, fibrin-containing airway casts have recently been reported in a rat inhalation model employing the SM analog 2-chloroethyl ethyl sulfide (CEES). The present study was designed to identify the mechanism(s) causing activation of the coagulation cascade after CEES-induced airway injury. Here we report that CEES inhalation elevates tissue factor (TF) activity and numbers of detached epithelial cells present in lavage fluid (BALF) from rats after exposure (18 h). In vitro studies using 16HBE cells, or with rat BALF, indicated that detached epithelial cells could convert factor X (FX) to the active form FXa when incubated with factor VII and could elicit rapid clotting of plasma. In addition, immunocytochemical analysis demonstrated elevated cell surface (TF) expression on CEES-exposed 16HBE cells as a function of time. However, total cell TF expression did not increase. Since membrane surfaces bearing TF are important determinants of clot initiation, anticoagulants directed against these entities were tested for ability to limit plasma clotting or FX activation capacity of BALF or culture media. Addition of tifacogin, a TF pathway inhibitor, effectively blocked either activity, demonstrating that the procoagulant actions of CEES were TF pathway dependent. Lactadherin, a protein capable of competing with clotting factors for phospholipid-binding sites, was partially effective in limiting these procoagulant actions. These findings indicate that TF pathway inhibition could be an effective strategy to prevent airway obstruction after SM or CEES inhalation.

Differential Regulation of Pulmonary Vascular Cell Growth by Hypoxia-Inducible Transcription Factor–1α and Hypoxia-Inducible Transcription Factor–2α

Hypoxia-inducible transcription factors HIF-1α and HIF-2α can contribute to pulmonary hypertension and vascular remodeling, but their mechanisms remain unknown. This study investigated the role of HIF-1α and HIF-2α in pulmonary artery endothelial and smooth muscle cells. The exposure of human pulmonary artery endothelial cells (HPAECs) to hypoxia (10% O₂ or 5% O₂) increased proliferation over 48 hours, compared with cells during normoxia (21% O₂). The adenovirus-mediated overexpression of HIF-2α that is transcriptionally active during normoxia (mutHIF-2α) increased HPAEC proliferation, whereas the overexpression of HIF-1α, which is transcriptionally active during normoxia (mutHIF-1α), exerted no effect. The knockdown of HIF-2α decreased proliferation during both hypoxia and normoxia. Both HIFs increased migration toward fibrinogen, used as a chemoattractant. In an angiogenesis tube formation assay, mutHIF-2α-transduced cells demonstrated increased tube formation, compared with the mutHIF-1α-transduced cells. In addition, the tubes formed in HIF-2α-transduced cells were more enduring than those in the other groups. In human pulmonary artery smooth muscle cells (HPASMCs), chronic exposure to hypoxia increased proliferation, compared with cells during normoxia. For HPASMCs transduced with adenoviral HIFs, HIF-1α increased proliferation, whereas HIF-2α exerted no such effect. Thus, HIF-1α and HIF-2α exert differential effects in isolated cells of the human pulmonary vasculature. This study demonstrates that HIF-2α plays a predominant role in the endothelial growth pertinent to the remodeling process. In contrast, HIF-1α appears to play a major role in pulmonary smooth muscle growth. The selective targeting of each HIF in specific target cells may more effectively counteract hypoxic pulmonary hypertension and vascular remodeling.

Tissue factor pathway inhibitor prevents airway obstruction, respiratory failure and death due to sulfur mustard analog inhalation

UNLABELLED Sulfur mustard (SM) inhalation causes airway injury, with enhanced vascular permeability, coagulation, and airway obstruction. The objective of this study was to determine whether recombinant tissue factor pathway inhibitor (TFPI) could inhibit this pathogenic sequence. METHODS Rats were exposed to the SM analog 2-chloroethyl ethyl sulfide (CEES) via nose-only aerosol inhalation. One hour later, TFPI (1.5mg/kg) in vehicle, or vehicle alone, was instilled into the trachea. Arterial O2 saturation was monitored using pulse oximetry. Twelve hours after exposure, animals were euthanized and bronchoalveolar lavage fluid (BALF) and plasma were analyzed for prothrombin, thrombin-antithrombin complex (TAT), active plasminogen activator inhibitor-1 (PAI-1) levels, and fluid fibrinolytic capacity. Lung steady-state PAI-1 mRNA was measured by RT-PCR analysis. Airway-capillary leak was estimated by BALF protein and IgM, and by pleural fluid measurement. In additional animals, airway cast formation was assessed by microdissection and immunohistochemical detection of airway fibrin. RESULTS Airway obstruction in the form of fibrin-containing casts was evident in central conducting airways of rats receiving CEES. TFPI decreased cast formation, and limited severe hypoxemia. Findings of reduced prothrombin consumption, and lower TAT complexes in BALF, demonstrated that TFPI acted to limit thrombin activation in airways. TFPI, however, did not appreciably affect CEES-induced airway protein leak, PAI-1 mRNA induction, or inhibition of the fibrinolytic activity present in airway surface liquid. CONCLUSIONS Intratracheal administration of TFPI limits airway obstruction, improves gas exchange, and prevents mortality in rats with sulfur mustard-analog-induced acute lung injury.

Sarcoendoplasmic Reticulum Ca2+ ATPase. A Critical Target in Chlorine Inhalation–Induced Cardiotoxicity

Autopsy specimens from human victims or experimental animals that die due to acute chlorine gas exposure present features of cardiovascular pathology. We demonstrate acute chlorine inhalation-induced reduction in heart rate and oxygen saturation in rats. Chlorine inhalation elevated chlorine reactants, such as chlorotyrosine and chloramine, in blood plasma. Using heart tissue and primary cardiomyocytes, we demonstrated that acute high-concentration chlorine exposure in vivo (500 ppm for 30 min) caused decreased total ATP content and loss of sarcoendoplasmic reticulum calcium ATPase (SERCA) activity. Loss of SERCA activity was attributed to chlorination of tyrosine residues and oxidation of an important cysteine residue, cysteine-674, in SERCA, as demonstrated by immunoblots and mass spectrometry. Using cardiomyocytes, we found that chlorine-induced cell death and damage to SERCA could be decreased by thiocyanate, an important biological antioxidant, and by genetic SERCA2 overexpression. We also investigated a U.S. Food and Drug Administration-approved drug, ranolazine, used in treatment of cardiac diseases, and previously shown to stabilize SERCA in animal models of ischemia-reperfusion. Pretreatment with ranolazine or istaroxime, another SERCA activator, prevented chlorine-induced cardiomyocyte death. Further investigation of responsible mechanisms showed that ranolazine- and istaroxime-treated cells preserved mitochondrial membrane potential and ATP after chlorine exposure. Thus, these studies demonstrate a novel critical target for chlorine in the heart and identify potentially useful therapies to mitigate toxicity of acute chlorine exposure.

Tissue Factor Signals Airway Epithelial Basal Cell Survival via Coagulation and Protease-Activated Receptor Isoforms 1 and 2

Tissue factor (TF) initiates the extrinsic coagulation cascade and is a high-affinity receptor for coagulation factor VII. TF also participates in protease-activated receptor (PAR)1 and PAR2 activation. Human epithelial basal cells were previously purified on the basis of TF expression. The purpose of this study was to determine if tracheobronchial epithelial basal cell-associated TF drives coagulation and/or activates PARs to promote basal cell functions. We used human tracheobronchial tissues to isolate human airway epithelial cells using specific cell surface markers by flow cytometry and studied TF expression by immunostaining. TF-dependent fibrin network formation was observed by confocal and scanning electron microscopy. TF knockdown was done using short hairpin RNA, and TF mRNA was measured using quantitative RT-PCR. We found that 97 ± 5% of first-passage human tracheobronchial epithelial cells were basal cells, and 100% of these basal cells expressed TF. Basal cell-associated TF was active, but TF activity was dependent on added extrinsic coagulation cascade factors. TF inhibition caused basal cell apoptosis and necrosis. This was due to two parallel but interdependent TF-regulated processes: failure to generate a basal cell-associated fibrin network and suboptimal PAR1 and PAR2 activity. The data indicate that membrane surface TF mediates airway epithelial basal cell attachment, which maintains cell survival and mitotic potential. The implications of these findings are discussed in the context of basal cell-associated TF activity in normal and injured tissues and of the potential for repair of airway epithelium in lung disease.

Intratracheal heparin improves plastic bronchitis due to sulfur mustard analog

Background: Inhalation of sulfur mustard (SM) and SM analog, 2‐chloroethyl ethyl sulfide (CEES), cause fibrinous cast formation that occludes the conducting airways, similar to children with Fontan physiology‐induced plastic bronchitis. These airway casts cause significant mortality and morbidity, including hypoxemia and respiratory distress. Our hypothesis was that intratracheal heparin, a highly cost effective and easily preserved rescue therapy, could reverse morbidity and mortality induced by bronchial cast formation. Methods: Sprague‐Dawley rats were exposed to 7.5% CEES via nose‐only aerosol inhalation to produce extensive cast formation and mortality. The rats were distributed into three groups: non‐treated, phosphate‐buffered saline (PBS)‐treated, and heparin‐treated groups. Morbidity was assessed with oxygen saturations and clinical distress. Blood and bronchoalveolar lavage fluid (BALF) were obtained for analysis, and lungs were fixed for airway microdissection to quantify the extent of airway cast formation. Results: Heparin, given intratracheally, improved survival (100%) when compared to non‐treated (75%) and PBS‐treated (90%) controls. Heparin‐treated rats also had improved oxygen saturations, clinical distress and airway cast scores. Heparin‐treated rats had increased thrombin clotting times, factor Xa inhibition and activated partial thromboplastin times, indicating systemic absorption of heparin. There were also increased red blood cells (RBCs) in the BALF in 2/6 heparin‐treated rats compared to PBS‐treated control rats. Conclusions: Intratracheal heparin 1 hr after CEES inhalation improved survival, oxygenation, airway obstruction, and clinical distress. There was systemic absorption of heparin in rats treated intratracheally. Some rats had increased RBCs in BALF, suggesting a potential for intrapulmonary bleeding if used chronically after SM inhalation. Pediatr Pulmonol. 2015; 50:118–126.