Recapitulation of human pathophysiology and identification of forensic biomarkers in a translational model of chlorine inhalation injury.

Chlorine gas (Cl2) has been repeatedly used as a chemical weapon, first in World War I and most recently in Syria. Life-threatening Cl2 exposures frequently occur in domestic and occupational environments, and in transportation accidents. Modeling the human etiology of Cl2-induced acute lung injury (ALI), forensic biomarkers, and targeted countermeasures development have been hampered by inadequate large animal models. The objective of this study was to develop a translational model of Cl2-induced ALI in swine to understand toxico-pathophysiology and evaluate whether it is suitable for screening potential medical countermeasures and to identify biomarkers useful for forensic analysis. Specific pathogen-free Yorkshire swine (30-40 kg) of either sex were exposed to Cl2 (≤240 ppm for 1 h) or filtered air under anesthesia and controlled mechanical ventilation. Exposure to Cl2 resulted in severe hypoxia and hypoxemia, increased airway resistance and peak inspiratory pressure, and decreased dynamic lung compliance. Cl2 exposure resulted in increased total leucocyte and neutrophil counts in bronchoalveolar lavage fluid, vascular leakage, and pulmonary edema compared with the air-exposed group. The model recapitulated all three key histopathological features of human ALI, such as neutrophilic alveolitis, deposition of hyaline membranes, and formation of microthrombi. Free and lipid-bound 2-chlorofatty acids and chlorotyrosine-modified proteins (3-chloro-l-tyrosine and 3,5-dichloro-l-tyrosine) were detected in plasma and lung tissue after Cl2 exposure. In this study, we developed a translational swine model that recapitulates key features of human Cl2 inhalation injury and is suitable for testing medical countermeasures, and validated chlorinated fatty acids and protein adducts as biomarkers of Cl2 inhalation.NEW & NOTEWORTHY We established a swine model of chlorine gas-induced acute lung injury that exhibits several features of human acute lung injury and is suitable for screening potential medical countermeasures. We validated chlorinated fatty acids and protein adducts in plasma and lung samples as forensic biomarkers of chlorine inhalation.

Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Polymorphisms near the membrane bound O-acyltransferase domain containing 7 (MBOAT7) genes are associated with worsened nonalcoholic fatty liver (NASH), and nonalcoholic fatty liver disease (NAFLD)/NASH may decrease MBOAT7 expression independent of these polymorphisms. We hypothesized that enhancing MBOAT7 function would improve NASH. METHODS: Genomic and lipidomic databases were mined for MBOAT7 expression and hepatic phosphatidylinositol (PI) abundance in human NAFLD/NASH. Male C57BL6/J mice were fed either choline-deficient high-fat diet or Gubra Amylin NASH diet and subsequently infected with adeno-associated virus expressing MBOAT7 or control virus. NASH histological scoring and lipidomic analyses were performed to assess MBOAT7 activity, hepatic PI, and lysophosphatidylinositol (LPI) abundance. RESULTS: Human NAFLD/NASH decreases MBOAT7 expression and hepatic abundance of arachidonate-containing PI. Murine NASH models display subtle changes in MBOAT7 expression, but significantly decreased activity. After MBOAT7 overexpression, liver weights, triglycerides, and plasma alanine and aspartate transaminase were modestly improved by MBOAT7 overexpression, but NASH histology was not improved. Despite confirmation of increased activity with MBOAT7 overexpression, content of the main arachidonoylated PI species was not rescued by MBOAT7 although the abundance of many PI species was increased. Free arachidonic acid was elevated but the MBOAT7 substrate arachidonoyl-CoA was decreased in NASH livers compared to low-fat controls, likely due to the decreased expression of long-chain acyl-CoA synthetases. CONCLUSION: Results suggest decreased MBOAT7 activity plays a role in NASH, but MBOAT7 overexpression fails to measurably improve NASH pathology potentially due to the insufficient abundance of its arachidonoyl-CoA substrate.

Myeloperoxidase-derived hypochlorous acid targets human airway epithelial plasmalogens liberating protein modifying electrophilic 2-chlorofatty aldehydes.

Neutrophil and airway epithelial cell interactions are critical in the inflammatory response to viral infections including respiratory syncytial virus, Sendai virus, and SARS-CoV-2. Airway epithelial cell dysfunction during viral infections is likely mediated by the interaction of virus and recruited neutrophils at the airway epithelial barrier. Neutrophils are key early responders to viral infection. Neutrophil myeloperoxidase catalyzes the conversion of hydrogen peroxide to hypochlorous acid (HOCl). Previous studies have shown HOCl targets host neutrophil and endothelial cell plasmalogen lipids, resulting in the production of the chlorinated lipid, 2-chlorofatty aldehyde (2-ClFALD). We have previously shown that the oxidation product of 2-ClFALD, 2-chlorofatty acid (2-ClFA) is present in bronchoalveolar lavage fluid of Sendai virus-infected mice, which likely results from the attack of the epithelial plasmalogen by neutrophil-derived HOCl. Herein, we demonstrate small airway epithelial cells contain plasmalogens enriched with oleic acid at the sn-2 position unlike endothelial cells which contain arachidonic acid enrichment at the sn-2 position of plasmalogen. We also show neutrophil-derived HOCl targets epithelial cell plasmalogens to produce 2-ClFALD. Further, proteomics and over-representation analysis using the ω-alkyne analog of the 2-ClFALD molecular species, 2-chlorohexadecanal (2-ClHDyA) showed cell adhesion molecule binding and cell-cell junction enriched categories similar to that observed previously in endothelial cells. However, in contrast to endothelial cells, proteins in distinct metabolic pathways were enriched with 2-ClFALD modification, particularly pyruvate metabolism was enriched in epithelial cells and mitochondrial pyruvate respiration was reduced. Collectively, these studies demonstrate, for the first time, a novel plasmalogen molecular species distribution in airway epithelial cells that are targeted by myeloperoxidase-derived hypochlorous acid resulting in electrophilic 2-ClFALD, which potentially modifies epithelial physiology by modifying proteins.

Plasmalogen Loss in Sepsis and SARS-CoV-2 Infection.

Plasmalogens are plasma-borne antioxidant phospholipid species that provide protection as cellular lipid components during cellular oxidative stress. In this study we investigated plasma plasmalogen levels in human sepsis as well as in rodent models of infection. In humans, levels of multiple plasmenylethanolamine molecular species were decreased in septic patient plasma compared to control subject plasma as well as an age-aligned control subject cohort. Additionally, lysoplasmenylcholine levels were significantly decreased in septic patients compared to the control cohorts. In contrast, plasma diacyl phosphatidylethanolamine and phosphatidylcholine levels were elevated in septic patients. Lipid changes were also determined in rats subjected to cecal slurry sepsis. Plasma plasmenylcholine, plasmenylethanolamine, and lysoplasmenylcholine levels were decreased while diacyl phosphatidylethanolamine levels were increased in septic rats compared to control treated rats. Kidney levels of lysoplasmenylcholine as well as plasmenylethanolamine molecular species were decreased in septic rats. Interestingly, liver plasmenylcholine and plasmenylethanolamine levels were increased in septic rats. Since COVID-19 is associated with sepsis-like acute respiratory distress syndrome and oxidative stress, plasmalogen levels were also determined in a mouse model of COVID-19 (intranasal inoculation of K18 mice with SARS-CoV-2). 3 days following infection, lung infection was confirmed as well as cytokine expression in the lung. Multiple molecular species of lung plasmenylcholine and plasmenylethanolamine were decreased in infected mice. In contrast, the predominant lung phospholipid, dipalmitoyl phosphatidylcholine, was not decreased following SARS-CoV-2 infection. Additionally total plasmenylcholine levels were decreased in the plasma of SARS-CoV-2 infected mice. Collectively, these data demonstrate the loss of plasmalogens during both sepsis and SARS-CoV-2 infection. This study also indicates plasma plasmalogens should be considered in future studies as biomarkers of infection and as prognostic indicators for sepsis and COVID-19 outcomes.

Endothelial Cell Protein Targeting by Myeloperoxidase-Derived 2-Chlorofatty Aldehyde.

Neutrophils are important cellular mediators of injury and repair in diseases including ischemic heart disease, atherosclerosis, and sepsis. Myeloperoxidase-derived (MPO)-oxidants released from neutrophils are potential mediators of endothelial injury in disease. MPO-derived HOCl attacks plasmalogen phospholipid to liberate 2-chlorofatty aldehyde (2-ClFALD). Both 2-ClFALD and its oxidation product, 2-chlorofatty acid (2-ClFA), are electrophilic lipids, and both probably react with proteins through several mechanisms. In the present study, we investigate protein modification specifically by 2-ClFALD under non-reducing conditions (e.g., without stabilizing Schiff base bonds), which likely reflects nucleophilic targeting of the electrophilic chlorinated carbon. Protein modification by the ω-alkyne analog of 2-chlorohexadecanal (2-ClHDA), 2-ClHDyA, was compared to that with the ω-alkyne analog of 2-chlorohexadecanoic acid (2-ClHA), 2-ClHyA, in multiple cell lines, which demonstrated 2-ClFALD preferentially modifies proteins compared to 2-ClFA. The 2-ClHDyA modified proteins from EA.hy926 cells and human lung microvascular endothelial cells analyzed by shotgun proteomics and over-representation analysis included adherens junction, cell adhesion molecule binding, and cell substrate junction enrichment categories. It is possible that proteins in these groups may have roles in previously described 2-ClFALD-elicited endothelial barrier dysfunction.

Identification of novel neutrophil very long chain plasmalogen molecular species and their myeloperoxidase mediated oxidation products in human sepsis.

Plasmalogens are a class of phospholipids containing vinyl ether linked aliphatic groups at the sn-1 position. Plasmalogens are known to contain 16- and 18-carbon aliphatic groups at the sn-1 position. Here, we reveal that the human neutrophil plasmenylethanolamine pool uniquely includes molecular species with very long carbon chain (VLC) aliphatic groups, including 20-, 22- and 24-carbon vinyl ether linked aliphatic groups at the sn-1 position. We identified these novel VLC plasmalogen species by electrospray ionization mass spectrometry methods. VLC plasmalogens were only found in the neutrophil plasmenylethanolamine pool. During neutrophil activation, VLC plasmenylethanolamines undergo myeloperoxidase-dependent oxidation to produce VLC 2-chlorofatty aldehyde and its oxidation product, 2-chlorofatty acid (2-ClFA). Furthermore, plasma concentrations of VLC 2-ClFA are elevated in human sepsis. These studies demonstrate for the first time VLC plasmenylethanolamine molecular species, their myeloperoxidase-mediated chlorolipid products and the presence of these chlorolipids in human sepsis.

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.

Reprogramming lipid metabolism prevents effector T cell senescence and enhances tumor immunotherapy.

The functional state of T cells is a key determinant for effective antitumor immunity and immunotherapy. Cellular metabolism, including lipid metabolism, controls T cell differentiation, survival, and effector functions. Here, we report that development of T cell senescence driven by both malignant tumor cells and regulatory T cells is a general feature in cancers. Senescent T cells have active glucose metabolism but exhibit unbalanced lipid metabolism. This unbalanced lipid metabolism results in changes of expression of lipid metabolic enzymes, which, in turn, alters lipid species and accumulation of lipid droplets in T cells. Tumor cells and Treg cells drove elevated expression of group IVA phospholipase A2, which, in turn, was responsible for the altered lipid metabolism and senescence induction observed in T cells. Mitogen-activated protein kinase signaling and signal transducer and activator of transcription signaling coordinately control lipid metabolism and group IVA phospholipase A2 activity in responder T cells during T cell senescence. Inhibition of group IVA phospholipase A2 reprogrammed effector T cell lipid metabolism, prevented T cell senescence in vitro, and enhanced antitumor immunity and immunotherapy efficacy in mouse models of melanoma and breast cancer in vivo. Together, these findings identify mechanistic links between T cell senescence and regulation of lipid metabolism in the tumor microenvironment and provide a new target for tumor immunotherapy.

Oxidized Lipoproteins Promote Resistance to Cancer Immunotherapy Independent of Patient Obesity.

Antitumor immunity is impaired in obese mice. Mechanistic insight into this observation remains sparse and whether it is recapitulated in patients with cancer is unclear because clinical studies have produced conflicting and controversial findings. We addressed this by analyzing data from patients with a diverse array of cancer types. We found that survival after immunotherapy was not accurately predicted by body mass index or serum leptin concentrations. However, oxidized low-density lipoprotein (ox-LDL) in serum was identified as a suppressor of T-cell function and a driver of tumor cytoprotection mediated by heme oxygenase-1 (HO-1). Analysis of a human melanoma gene expression database showed a clear association between higher HMOX1 (HO-1) expression and reduced progression-free survival. Our in vivo experiments using mouse models of both melanoma and breast cancer revealed HO-1 as a mechanism of resistance to anti-PD1 immunotherapy but also exposed HO-1 as a vulnerability that could be exploited therapeutically using a small-molecule inhibitor. In conclusion, our clinical data have implicated serum ox-LDL as a mediator of therapeutic resistance in patients with cancer, operating as a double-edged sword that both suppressed T-cell immunity and simultaneously induced HO-1-mediated tumor cell protection. Our studies also highlight the therapeutic potential of targeting HO-1 during immunotherapy, encouraging further translational development of this combination approach.See article by Kuehm et al., p. 227.

Alterations in the Human Plasma Lipidome in Response to Tularemia Vaccination.

Tularemia is a highly infectious and contagious disease caused by the bacterium Francisella tularensis. To better understand human response to a live-attenuated tularemia vaccine and the biological pathways altered post-vaccination, healthy adults were vaccinated, and plasma was collected pre- and post-vaccination for longitudinal lipidomics studies. Using tandem mass spectrometry, we fully characterized individual lipid species within predominant lipid classes to identify changes in the plasma lipidome during the vaccine response. Separately, we targeted oxylipins, a subset of lipid mediators involved in inflammatory pathways. We identified 14 differentially abundant lipid species from eight lipid classes. These included 5-hydroxyeicosatetraenoic acid (5-HETE) which is indicative of lipoxygenase activity and, subsequently, inflammation. Results suggest that 5-HETE was metabolized to a dihydroxyeicosatrienoic acid (DHET) by day 7 post-vaccination, shedding light on the kinetics of the 5-HETE-mediated inflammatory response. In addition to 5-HETE and DHET, we observed pronounced changes in 34:1 phosphatidylinositol, anandamide, oleamide, ceramides, 16:1 cholesteryl ester, and other glycerophospholipids; several of these changes in abundance were correlated with serum cytokines and T cell activation. These data provide new insights into alterations in plasma lipidome post-tularemia vaccination, potentially identifying key mediators and pathways involved in vaccine response and efficacy.

In vitro oxidations of low-density lipoprotein and RAW 264.7 cells with lipophilic O(3P)-precursors.

A beneficial property of photogenerated reactive oxygen species (ROS) is the capability of oxidant generation within a specific location or organelle inside a cell. Dibenzothiophene S-oxide (DBTO), which is known to undergo a photodeoxygenation reaction to generate ground state atomic oxygen [O(3P)] upon irradiation, was functionalized to afford localization within the plasma membrane of cells. The photochemistry, as it relates to oxidant generation, was studied and demonstrated that the functionalized DBTO derivatives generated O(3P). Irradiation of these lipophilic O(3P)-precursors in the presence of LDL and within RAW 264.7 cells afforded several oxidized lipid products (oxLP) in the form of aldehydes. The generation of a 2-hexadecenal (2-HDEA) was markedly increased in irradiations where O(3P) was putatively produced. The substantial generation of 2-HDEA is not known to accompany the production of other ROS. These cellular irradiation experiments demonstrate the potential of inducing oxidation with O(3P) in cells.

Phosgene inhalation causes hemolysis and acute lung injury.

Phosgene (Carbonyl Chloride, COCl2) remains an important chemical intermediate in many industrial processes such as combustion of chlorinated hydrocarbons and synthesis of solvents (degreasers, cleaners). It is a sweet smelling gas, and therefore does not prompt escape by the victim upon exposure. Supplemental oxygen and ventilation are the only available management strategies. This study was aimed to delineate the pathogenesis and identify novel biomarkers of acute lung injury post exposure to COCl2 gas. Adult male and female C57BL/6 mice (20-25 g), exposed to COCl2 gas (10 or 20 ppm) for 10 min in environmental chambers, had a dose dependent reduction in PaO2 and an increase in PaCO2, 1 day post exposure. However, mortality increased only in mice exposed to 20 ppm of COCl2 for 10 min. Correspondingly, these mice (20 ppm) also had severe acute lung injury as indicated by an increase in lung wet to dry weight ratio, extravasation of plasma proteins and neutrophils into the bronchoalveolar lavage fluid, and an increase in total lung resistance. The increase in acute lung injury parameters in COCl2 (20 ppm, 10 min) exposed mice correlated with simultaneous increase in oxidation of red blood cells (RBC) membrane, RBC fragility, and plasma levels of cell-free heme. In addition, these mice had decreased plasmalogen levels (plasmenylethanolamine) and elevated levels of their breakdown product, polyunsaturated lysophosphatidylethanolamine, in the circulation suggesting damage to cellular plasma membranes. This study highlights the importance of free heme in the pathogenesis of COCl2 lung injury and identifies plasma membrane breakdown product as potential biomarkers of COCl2 toxicity.

Doubled lifespan and patient-like pathologies in progeria mice fed high-fat diet.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a devastating premature aging disease. Mouse models have been instrumental for understanding HGPS mechanisms and for testing therapies, which to date have had only marginal benefits in mice and patients. Barriers to developing effective therapies include the unknown etiology of progeria mice early death, seemingly unrelated to the reported atherosclerosis contributing to HGPS patient mortality, and mice not recapitulating the severity of human disease. Here, we show that progeria mice die from starvation and cachexia. Switching progeria mice approaching death from regular diet to high-fat diet (HFD) rescues early lethality and ameliorates morbidity. Critically, feeding the mice only HFD delays aging and nearly doubles lifespan, which is the greatest lifespan extension recorded in progeria mice. The extended lifespan allows for progeria mice to develop degenerative aging pathologies of a severity that emulates the human disease. We propose that starvation and cachexia greatly influence progeria phenotypes and that nutritional/nutraceutical strategies might help modulate disease progression. Importantly, progeria mice on HFD provide a more clinically relevant animal model to study mechanisms of HGPS pathology and to test therapies.

Disrupting cholesterol esterification by bitter melon suppresses triple-negative breast cancer cell growth.

Triple negative breast cancer (TNBC) is aggressive with a worse prognosis. We have recently shown that bitter melon extract (BME) treatment was more effective in inhibition of TNBC tumor growth in mouse models as compared to ER positive breast tumor growth. Aberrant dysregulation of lipid metabolism is associated with breast cancer progression, however, anti-cancer mechanism of BME linking lipid metabolism in breast cancer growth remains unexplored. Here, we observed that accumulation of esterified cholesterol was reduced in BME treated TNBC cell lines as compared to control cells. We next evaluated expression levels of acyl-CoA: cholesterol acyltransferase 1 (ACAT-1) in TNBC cells treated with BME. Our results demonstrated that BME treatment inhibited ACAT-1 expression in TNBC cells. Subsequently, we found that sterol regulatory element-binding proteins-1 and -2, and FASN was significantly reduced in BME treated TNBC cell lines. Low-density lipoprotein receptor was also downregulated in BME treated TNBC cells as compared to control cells. We further demonstrated that BME feeding reduced tumor growth in TNBC mammospheres implanted into NSG mice, and inhibits ACAT-1 expression. To our knowledge, this is the first report demonstrating BME suppresses TNBC cell growth through ACAT-1 inhibition, and have potential for additional therapeutic regimen against human breast cancer.

Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein.

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more α-BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br2 gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.

The chlorinated lipidome originating from myeloperoxidase-derived HOCl targeting plasmalogens: Metabolism, clearance, and biological properties.

Myeloperoxidase produces the two-electron oxidant HOCl, which targets plasmalogen phospholipids liberating 2-chlorofatty aldehyde. 2-Chlorofatty aldehyde has four known fates: 1) oxidation to 2-chlorofatty acid; 2) reduction to 2-chlorofatty alcohol; 3) Schiff base adduct formation with proteins and amines; and 4) reactivity with glutathione through nucleophilic attack of the α-chlorinated carbon. 2-Chlorofatty acid does not undergo conventional fatty acid ß-oxidation due to the presence of the α-chlorinated carbon; however, 2-chlorofatty acid does undergo sequential ω-oxidation and ß-oxidation from the ω-end, ultimately resulting in 2-chloroadipic acid urinary excretion. Recent studies have demonstrated that 2-chlorofatty acid clearance is increased by treatment with the PPAR-α agonist WY14643, which increases the enzymatic machinery responsible for hepatic ω-oxidation. Furthermore, 2-chlorofatty acid has been shown to be a PPAR-α agonist, and thus accelerates its own clearance. The roles of 2-chlorofatty aldehyde and 2-chlorofatty acid on leukocyte and endothelial function have been explored by several groups, suggesting that chlorinated lipids induce endothelial cell dysfunction, neutrophil chemotaxis, monocyte apoptosis, and alterations in vascular tone. Thus, the chlorinated lipidome, produced in response to leukocyte activation, is a potential biomarker and therapeutic target to modulate host response in inflammatory diseases.

2-Chlorofatty acids induce Weibel-Palade body mobilization.

Endothelial dysfunction is a hallmark of multiple inflammatory diseases. Leukocyte interactions with the endothelium have significant effects on vascular wall biology and pathophysiology. Myeloperoxidase (MPO)-derived oxidant products released from leukocytes are potential mediators of inflammation and endothelial dysfunction. 2-Chlorofatty acids (2-ClFAs) are produced as a result of MPO-derived HOCl targeting plasmalogen phospholipids. Chlorinated lipids have been shown to be associated with multiple inflammatory diseases, but their impact on surrounding endothelial cells has not been examined. This study tested the biological properties of the 2-ClFA molecular species 2-chlorohexadecanoic acid (2-ClHA) on endothelial cells. A synthetic alkyne analog of 2-ClHA, 2-chlorohexadec-15-ynoic acid (2-ClHyA), was used to examine the subcellular localization of 2-ClFA in human coronary artery endothelial cells. Click chemistry experiments revealed that 2-ClHyA localizes to Weibel-Palade bodies. 2-ClHA and 2-ClHyA promote the release of P-selectin, von Willebrand factor, and angiopoietin-2 from endothelial cells. Functionally, 2-ClHA and 2-ClHyA cause neutrophils to adhere to and platelets to aggregate on the endothelium, as well as increase permeability of the endothelial barrier which has been tied to the release of angiopoietin-2. These findings suggest that 2-ClFAs promote endothelial cell dysfunction, which may lead to broad implications in inflammation, thrombosis, and blood vessel stability.

Myeloperoxidase-derived 2-chlorofatty acids contribute to human sepsis mortality via acute respiratory distress syndrome.

Sepsis-associated acute respiratory distress syndrome (ARDS) is characterized by neutrophilic inflammation and poor survival. Since neutrophil myeloperoxidase (MPO) activity leads to increased plasma 2-chlorofatty acid (2-ClFA) levels, we hypothesized that plasma concentrations of 2-ClFAs would associate with ARDS and mortality in subjects with sepsis. In sequential consenting patients with sepsis, free 2-ClFA levels were significantly associated with ARDS, and with 30-day mortality, for each log increase in free 2-chlorostearic acid. Plasma MPO was not associated with either ARDS or 30-day mortality but was correlated with 2-ClFA levels. Addition of plasma 2-ClFA levels to the APACHE III score improved prediction for ARDS. Plasma 2-ClFA levels correlated with plasma levels of angiopoietin-2, E selectin, and soluble thrombomodulin. Endothelial cells treated with 2-ClFA responded with increased adhesion molecule surface expression, increased angiopoietin-2 release, and dose-dependent endothelial permeability. Our results suggest that 2-ClFAs derived from neutrophil MPO-catalyzed oxidation contribute to pulmonary endothelial injury and have prognostic utility in sepsis-associated ARDS.

Peroxisome proliferator-activated receptor-α accelerates α-chlorofatty acid catabolism.

α-Chlorofatty aldehydes are generated from myeloperoxidase-derived HOCl targeting plasmalogens, and are subsequently oxidized to α-chlorofatty acids (α-ClFAs). The catabolic pathway for α-ClFA is initiated by ω-oxidation. Here, we examine PPAR-α activation as a mechanism to increase α-ClFA catabolism. Pretreating both HepG2 cells and primary mouse hepatocytes with the PPAR-α agonist, pirinixic acid (Wy 14643), increased the production of α-chlorodicarboxylic acids (α-ClDCAs) in cells treated with exogenous α-ClFA. Additionally, α-ClDCA production in Wy 14643-pretreated wild-type mouse hepatocytes was accompanied by a reduction in cellular free α-ClFA. The dependence of PPAR-α-accelerated α-ClFA catabolism was further demonstrated by both impaired metabolism in mouse PPAR-α-/- hepatocytes and decreased clearance of plasma α-ClFA in PPAR-α-/- mice. Furthermore, Wy 14643 treatments decreased plasma 2-chlorohexadecanoic acid levels in wild-type mice. Additional studies showed that α-ClFA increases PPAR-α, PPAR-δ, and PPAR-γ activities, as well as mRNA expression of the PPAR-α target genes, CD36, CPT1a, Cyp4a10, and CIDEC. Collectively, these results indicate that PPAR-α accelerates important pathways for the clearance of α-ClFA, and α-ClFA may, in part, accelerate its catabolism by serving as a ligand for PPAR-α.

Platelet-Activating Factor Quantification Using Reversed Phase Liquid Chromatography and Selected Reaction Monitoring in Negative Ion Mode.

Platelet-activating factor (PAF) is a potent biologically active phospholipid that mediates human physiological and pathophysiologic responses. PAF levels increase transiently and are typically assessed by techniques with limitations related to expense, sensitivity, pre-analysis derivatization and interference with isobaric molecules. This study elucidates a facile, accurate liquid chromatography-mass spectrometry analytical method for PAF. In negative ion mode using electrospray ionization, collisionally-activated dissociation analysis showed a unique product ion for acetate adducts of PAF molecular species representing the loss of methyl acetate from the polar head group and loss of a part of the acetate group from the sn-2 position. This product ion was exploited for selected reaction monitoring of PAF molecular species following separation by reversed-phase liquid chromatography. Standard calibration responses were determined, and this method was able to detect as low as 100 fmol of PAF. Finally, PAF molecular species were quantified in human neutrophils and monocytes.