Florence Reye

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100.

Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.

Design, synthesis, and structure-activity relationship studies of novel 3-alkylindole derivatives as selective and highly potent myeloperoxidase inhibitors.

Due to its production of potent antimicrobial oxidants including hypochlorous acid, human myeloperoxidase (MPO) plays a critical role in innate immunity and inflammatory diseases. Thus MPO is an attractive target in drug design. (Aminoalkyl)fluoroindole derivatives were detected to be very potent MPO inhibitors; however, they also promote inhibition of the serotonin reuptake transporter (SERT) at the same concentration range. Via structure-based drug design, a new series of MPO inhibitors derived from 3-alkylindole were synthesized and their effects were assessed on MPO-mediated taurine chlorination and low-density lipoprotein oxidation as well as on inhibition of SERT. The fluoroindole compound with three carbons in the side chain and one amide group exhibited a selectivity index of 35 (Ki/IC50) with high inhibition of MPO activity (IC50 = 18 nM), whereas its effect on SERT was in the micromolar range. Structure-function relationships, mechanism of action, and safety of the molecule are discussed.

Optimization of apolipoprotein-B-100 sequence coverage by liquid chromatography-tandem mass spectrometry for the future study of its posttranslational modifications.

Proteomic applications have been increasingly used to study posttranslational modifications of proteins (PTMs). For the purpose of identifying and localizing specific but unknown PTMs on huge proteins, improving their sequence coverage is fundamental. Using liquid chromatography coupled to mass spectrometry (LC-MS/MS), peptide mapping of the native apolipoprotein-B-100 was performed to further document the effects of oxidation. Apolipoprotein-B-100 is the main protein of low-density lipoprotein particles and its oxidation could play a role in atherogenesis. Because it is one of the largest human proteins, the sequence recovery rate of apolipoprotein-B-100 only reached 1% when conventional analysis parameters were used. The different steps of the peptide mapping process-from protein treatment to data analysis-were therefore reappraised and optimized. These optimizations allowed a protein sequence recovery rate of 79%, a rate which has never been achieved previously for such a large human protein. The key points for improving peptide mapping were optimization of the data analysis software; peptide separation by LC; sample preparation; and MS acquisition. The new protocol has allowed us to increase by a factor of 4 the detection of modified peptides in apolipoprotein-B-100. This approach could easily be transferred to any study of PTMs using LC-MS/MS.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

Data on myeloperoxidase-oxidized low-density lipoproteins stimulation of cells to induce release of resolvin-D1

This article present data related to the publication entitled “Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells” (Dufour et al., 2018). The supporting materials include results obtained by Mox-LDLs stimulated macrophages and investigation performed on scavenger receptors. Linear regressions (RvD1 vs age of mice and RvD1 vs CL-Tyr/Tyr) and Data related to validation were also presented. The interpretation of these data and further extensive insights can be found in Dufour et al. (2018) [1].

Native and myeloperoxidase-oxidized low-density lipoproteins act in synergy to induce release of resolvin-D1 from endothelial cells

BACKGROUND AND AIMS Oxidation of native low-density lipoproteins (LDLs-nat) plays an important role in the development of atherosclerosis. A major player in LDL-nat oxidation is myeloperoxidase (MPO), a heme enzyme present in azurophil granules of neutrophils and monocytes. MPO produces oxidized LDLs called Mox-LDLs, which cause a pro-inflammatory response in human microvascular endothelial cells (HMEC), monocyte/macrophage activation and formation of foam cells. Resolvin D1 (RvD1) is a compound derived from the metabolism of the polyunsaturated fatty acid DHA, which promotes resolution of inflammation at the ng/ml level. METHODS In the present study, we used liquid chromatography-mass spectrometry (LC-MS/MS) to investigate the synthesis of RvD1 and its precursors - 17(S)-hydroxy docosahexaenoic acid (17S-HDHA) and docosahexaenoic acid (DHA) - by HMEC, in the presence of several concentrations of Mox-LDLs, copper-oxidized-LDLs (Ox-LDLs), and native LDLs or in mouse plasma. The LC-MS/MS method has been validated and applied to cell supernatants and plasma to measure production of RvD1 and its precursors in several conditions. RESULTS Mox-LDLs played a significant role in the synthesis of RvD1 and 17S-HDHA from DHA compared to Ox-LDLs. Moreover, Mox-LDLs and LDLs-nat acted in synergy to produce RvD1. In addition, different correlations were found between RvD1 and M1 macrophages, age of mice or Cl-Tyr/Tyr ratio. CONCLUSIONS These results suggest that although Mox-LDLs are known to be pro-inflammatory and deleterious in the context of atherosclerosis, they are also able to induce a pro-resolution effect by induction of RvD1 from HMEC. Finally, our data also suggest that HMEC can produce RvD1 on their own.

High Wattage E-cigarettes Induce Tissue Hypoxia and Lower Airway Injury: A Randomized Trial.

More than 5% of adults in the United States are current electronic cigarette (e-cigarette) users (1). The liquid vehicles that are vaporized in e-cigarettes are propylene glycol and glycerol. Regular e-cigarette users buy last-generation devices delivering a high energy level to low coil resistance (subohm vaping). High-energy settings are used to increase heat and vapor production, to enhance the throat and nicotine hits, while nicotine concentration in the e-liquid is reduced (2, 3). E-cigarettes with high output wattage increase the quantity of e-liquid consumed per puff and produce volatile carbonyls by thermal degradation (3). Daily exposures to large amounts of high-temperature propylene glycol/glycerol aerosol may present a hazard to health. The preliminary results presented here are a part of a larger in-depth, placebo-controlled, randomized, crossover, and single-blinded study on the cardiorespiratory effects of e-cigarettes. We present here the main hypothesis tested, namely, that acute vaporization of a propylene glycol and glycerol mix (50:50), under intense use conditions, alters lung and skin microvascular functions via an oxidative stress pathway (ClinicalTrials.gov identifier: NCT03036644).

Validation of a LC/MSMS method for simultaneous quantification of 9 nucleotides in biological matrices

Nucleotides play a role in inflammation processes: cAMP and cGMP in the endothelial barrier function, ADP in platelet aggregation, ATP and UTP in vasodilatation and/or vasoconstriction of blood vessels, UDP in macrophages activation. The aim of this study is to develop and validate a LC/MS-MS method able to quantify simultaneously nine nucleotides (AMP, cAMP, ADP, ATP, GMP, cGMP, UMP, UDP and UTP) in biological matrixes (cells and plasma). The method we developed, has lower LOQs than others and has the main advantage to quantify all nucleotides within one single injection in less than 10 min. The measured nucleotides concentrations obtained with this method are similar to those obtained with assay kits commercially available. Analysis of plasma and red blood cells from healthy donors permits to estimate the physiological concentration of those nucleotides in human plasma and red blood cells, such information being poorly available in the literature. Furthermore, the protocol presented in this paper allowed us to observe that AMP, ADP, ATP concentrations are modified in human red blood cells and plasma after a venous stasis of 4 min compared to physiological blood circulation. Therefore, this specific method enables future studies on nucleotides implications in chronic inflammatory diseases but also in other pathologies where nucleotides are implicated in.

Differential Effects of E-Cigarette on Microvascular Endothelial Function, Arterial Stiffness and Oxidative Stress: A Randomized Crossover Trial.

Propylene glycol and glycerol are electronic cigarettes vehicles allowing liquid vaporization and nicotine transport. The respective effects of these different constituents on the cardiovascular system are unknown. We assessed the differential effects of vehicles (propylene glycol and glycerol) and nicotine on microcirculatory function, arterial stiffness, hemodynamic parameters and oxidative stress. Twenty-five tobacco smokers were exposed to vaping with and without nicotine, and sham vaping, in a randomized, single blind, 3-period crossover design study. Neither sham-vaping nor vaping in the absence of nicotine resulted in modifications of cardiovascular parameters or oxidative stress. In contrast, vaping with nicotine: 1) impaired acetylcholine mediated vasodilation (mean ± standard error mean) (area under curve, perfusion unit (PU), 3385 ± 27PU to 2271 ± 27PU, p < 0.0001); 2) increased indices of arterial stiffness, namely augmentation index corrected for heart rhythm (−3.5 ± 1.5% to 1.9 ± 2.3%; p = 0.013) and pulse wave velocity (4.9 ± 0.1 m.s−1 to 5.3 ± 0.1 m.s−1; p < 0.0001); 3) increased systolic and diastolic blood pressures as well as heart rate (all p < 0.0001) and finally; 4) raised plasma myeloperoxidase (median [interquartile range]) (13.6 ng.ml−1 [10–17.7] to 18.9 ng.ml−1 [12.2–54.4], p = 0.005). Our findings demonstrated that high temperature e-cigarette vehicle vaporization does not alter micro- and macro-vascular function, and oxidative stress, and that these effects are solely attributable to nicotine.