Role of Myeloperoxidase in ROS Generation and Inflammation Response on Prostate Epithelial Cells.

Myeloperoxidase (MPO) has been reported in prostate tissue, and considering its pro-oxidant properties, this location might be linked to prostate pathology. The possibility that the glandular prostatic tissue might be the source of MPO and its potential inflammatory effects must be tested. Human prostate material was obtained from prostate biopsies and radical prostatectomies. Immunohistochemistry was performed using MPO-specific human antibody. In situ hybridization using MPO-specific probes and laser-assisted microdissection for quantitative real-time RT-PCR were performed to observe whether MPO is being produced in prostate tissue. Mass spectrometry on prostate biopsies was used to detect products of MPO activity in nucleic acids (DNA/RNA). MPO contribution to intracellular accumulation of ROS and interleukin-8 in prostatic epithelial cells was monitored in vitro. Immunohistochemistry confirmed cellular localization of MPO in epithelial cells of the prostate. The staining varied from light to high intensity. In situ hybridization did not address the presence of mRNA coding for MPO. No MPO-specific modifications on nucleic acids were detected. Mox-LDL was a major factor inducing ROS and cytokines production in prostatic epithelial cells. We did not demonstrate that MPO was synthetized by prostatic epithelial cells. However, in vitro experiments showed the ability of MPO to potentiate the ROS production and inflammation on prostate epithelial cells. Results do not allow us to demonstrate a role of MPO in prostate to date but further studies are mandatory to focus on the potential impact of MPO in the development of prostatic diseases.

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 LOQ's 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.

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].

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.

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.

The presence of modified nucleosides in extracellular fluids leads to the specific incorporation of 5-chlorocytidine into RNA and modulates the transcription and translation.

Myeloperoxidase (MPO) is able to promote several kinds of damage and is involved in mechanisms leading to various diseases such as atherosclerosis or cancers. An example of these damages is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity. Since 5-chlorocytidine has been recently shown in healthy donor plasmas, this study aimed at discovering if these circulating modified nucleosides could be incorporated into RNA and DNA and if their presence impacts the ability of enzymes involved in the incorporation, transcription, and translation processes. Experimentations, which were carried out in vitro with endothelial and prostatic cells, showed a large penetration of all chloronucleosides but an exclusive incorporation of 5-chlorocytidine into RNA. However, no incorporation into DNA was observed. This specific incorporation is accompanied by an important reduction of translation yield. Although, in vitro, DNA polymerase processed in the presence of chloronucleosides but more slowly than in control conditions, ribonucleotide reductase could not reduce chloronucleotides prior to the replication. This reduction seems to be a limiting step, protecting DNA from chloronucleoside incorporation. This study shows the capacity of transcription enzyme to specifically incorporate 5-chlorocytidine into RNA and the loss of capacity-complete or partial-of different enzymes, involved in replication, transcription or translation, in the presence of chloronucleosides. Questions remain about the long-term impact of such specific incorporation in the RNA and such decrease of protein production on the cell viability and function.

Validation of a sensitive LC/MSMS method for chloronucleoside analysis in biological matrixes and its applications.

Myeloperoxidase promotes several kinds of damage and is involved in the development of various diseases (as atherosclerosis and cancers). An example of these damage is the chlorination of nucleic acids, which is considered as a specific marker of the MPO activity on those acids. This study aimed to develop and validate a method to analyze oxidized and MPO-specific chlorinated nucleosides in biological matrixes (cells, tissues and plasma). Although a lot of methods to quantify oxidized or chlorinated nucleosides have already been established, none of them took into account all these derivatives together. The new method used a Triple Quadrupole mass spectrometer fitted with a Jet Stream electrospray ionization source. This approach has two advantages compared with existing LC/MSMS analyses: it includes MPO-induced modifications in a unique analysis and obtains a better sensitivity. Our optimized method reached LOQs of 1.50pg and 1.42pg respectively for oxoG and oxo(d)G, being 4 times more sensitive than previous methods, and LOQs of 1.39pg, 1.30pg and 63.4 fg respectively for 5-chlorocytidine, 5-chloro-2'-deoxycytidine and 8-chloroguanosine. Developed method is also 25 times more sensitive for chloroguanosine than the best existing method. Nevertheless, this method is not specific enough for 8-chloro-(2'-deoxy)adenosine analysis. Examples of applications demonstrate the interest of this validated method. Indeed analysis of plasma from healthy donors highlighted exclusively the presence of 5-chlorocytidine (1.0±0.2nM) whereas analysis of treated endothelial cells by HOCl showed chlorination of guanosine and cytidine in cytoplasmic pools and chlorination of (deoxy)cytidine in DNA and RNA. In conclusion, this study shows that 5-chloro-2'-deoxycytidine, 5-chlorocytidine and 8-chloroguanosine are good markers allowing us to detect the MPO activity in biological fluids. The robust, specific and sensitive developed method enables future studies on MPO implications in human diseases.

Phosphatidylethanolamine Is a Key Regulator of Membrane Fluidity in Eukaryotic Cells.

Adequate membrane fluidity is required for a variety of key cellular processes and in particular for proper function of membrane proteins. In most eukaryotic cells, membrane fluidity is known to be regulated by fatty acid desaturation and cholesterol, although some cells, such as insect cells, are almost devoid of sterol synthesis. We show here that insect and mammalian cells present similar microviscosity at their respective physiological temperature. To investigate how both sterols and phospholipids control fluidity homeostasis, we quantified the lipidic composition of insect SF9 and mammalian HEK 293T cells under normal or sterol-modified condition. As expected, insect cells show minimal sterols compared with mammalian cells. A major difference is also observed in phospholipid content as the ratio of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) is inverted (4 times higher in SF9 cells). In vitro studies in liposomes confirm that both cholesterol and PE can increase rigidity of the bilayer, suggesting that both can be used by cells to maintain membrane fluidity. We then show that exogenously increasing the cholesterol amount in SF9 membranes leads to a significant decrease in PE:PC ratio whereas decreasing cholesterol in HEK 293T cells using statin treatment leads to an increase in the PE:PC ratio. In all cases, the membrane fluidity is maintained, indicating that both cell types combine regulation by sterols and phospholipids to control proper membrane fluidity.

Advancement in stationary phase for peptide separation helps in protein identification: application to atheroma plaque proteomics using nano-chip liquid chromatography and mass spectrometry.

In the last decades, proteomics has largely progressed. Mass spectrometry and liquid chromatography (LC) are generally used in proteomics. These techniques enable proper separation of peptides and good identification and/or quantification of them. Later, nano-scaled liquid chromatography, improvements of mass spectrometry resolution and sensitivity brought huge advancements. Enhancements in chemistry of chromatographic columns also brought interesting results. In the present work, the potency of identification of proteins by different nano-chip columns was studied and compared with classical LC column. The present study was applied to cardiovascular field where proteomics has shown to be highly helpful in research of new biomarkers. Protein extracts from atheroma plaques were used and proteomics data were compared. Results show that fewer spectra were acquired by the mass spectrometer when nano-chip columns were used instead of the classical ones. However, approximately 40% more unique peptides were identified by the recently optimized chip named Polaris-HR-chip-3C18 column, and 20% more proteins were identified. This fact leads to the identification of more low-abundance proteins. Many of them are involved in atheroma plaque development such as apolipoproteins, ceruloplasmin, etc. In conclusion, present data shows that recent developments of nanoLC column chemistry and dimensions enabled the improved detection and identification of low-abundance proteins in atheroma plaques. Several of them are of major interest in the field of cardiovascular disease.

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.

4-Bromo-2-(piperidin-1-yl)thiazol-5-yl-phenyl methanone (12b) inhibits Na+/K(+)-ATPase and Ras oncogene activity in cancer cells.

The in vitro growth inhibitory activity of 26 thiazoles (including 4-halogeno-2,5-disubtituted-1,3-thiazoles) and 5 thienothiazoles was assessed on a panel of 6 human cancer cell lines, including glioma cell lines. (4-Chloro-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12a) and (4-bromo-2-(piperidin-1-yl)thiazol-5-yl)(phenyl)methanone (12b) displayed ~10 times greater in vitro growth inhibitory activity than perillyl alcohol (POH), which therapeutically benefits glioma patients through the inhibition of both alpha-1 Na(+)/K(+)-ATPase (NAK) and Ras oncogene activity. The in vitro cytostatic activities (as revealed by quantitative videomicroscopy) displayed by 12a and 12b were independent of the intrinsic resistance to pro-apoptotic stimuli associated with cancer cells. Compounds 12a and 12b displayed relatively similar inhibitory activities on purified guinea pig brain preparations that mainly express NAK alpha-2 and alpha-3 subunits, whereas only compound 12b was efficacious against purified guinea pig kidney preparations that mainly express the NAK alpha-1 subunit, which is also expressed in gliomas, melanomas and non-small-cell lung cancers NSCLCs.

Vasorelaxant and antihypertensive effects of methanolic extracts from Hymenocardia acida Tul.

ETHNOPHARMACOLOGICAL RELEVANCE: In Congolese traditional medicine, decoctions of Hymenocardia acida root bark (HaRB) and trunk bark (HaTrB) are used for the treatment of conditions assumed to be hypertension. In this work, we propose to study the vasorelaxant effect of HaRB and HaTrB methanolic extracts on isolated rat thoracic aorta, to characterize the group of molecules responsible for the observed vasorelaxant activity, to evaluate the in vitro antioxidant activity of these extracts and to determine the antihypertensive activity of the HaRB extract on spontaneously hypertensive rats (SHR). MATERIALS AND METHODS: The vasorelaxant effect of the HaRB and HaTrB methanolic extracts was studied on endothelium-intact aortic rings pre-contracted with phenylephrine (PE, 1µM). The mechanism of this vasorelaxant effect was investigated on endothelium-denuded vessels and on endothelium-intact aortic rings in the presence of three inhibitors: l-N(G)-nitroarginine methyl ester (100µM), indomethacin (10µM) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10µM). To determine the nature of the compounds responsible for the vasorelaxant activity, we carried out a fractionation of the extracts and a thiolysis of the most active fraction followed by a liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) analysis. The extracts antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) colorimetric assay. In vivo anti-hypertensive activity of the HaRB extract was conducted on SHR. RESULTS: HaRB and HaTrB methanolic extracts produced a concentration-dependent vasorelaxation on intact aortic rings pre-contracted with PE (1µM). The vasorelaxant responses obtained were 95.3±1.5% (5µg/ml) and 100.6±3.0% (1µg/ml), respectively. The effect was markedly attenuated by removal of endothelium or pretreatment of aortic rings with all inhibitors except indomethacin. The LC/ESI-MS analysis of the thiolysis products indicated that the fraction which caused the most important vasorelaxation (97.9±2.5% at 3µg/ml) was a mixture of procyanidins and prodelphinidins, with a predominance of procyanidins. Both extracts and all fractions from HaRB extract showed a DPPH scavenging activity, ranging from 0.4 to 0.8 quercetin-equivalents. The HaRB methanolic extract reduced the systolic blood pressure in SHR (from 214±3mmHg to 194±4mmHg) after a 5-week treatment. CONCLUSIONS: The methanolic extracts of Hymenocardia acida root and trunk bark have vasorelaxant activity. The vasorelaxant effect observed is endothelium-dependent and seems mainly mediated through the NO-cGMP pathway. The COX pathway is not involved. The vasorelaxant activity appears to be due to polymeric procyanidins and prodelphinidins. These extracts also have an antioxidant effect. The extract of Hymenocardia acida root bark shows a significant but weak antihypertensive activity in SHR.

Simultaneous measurement of protein-bound 3-chlorotyrosine and homocitrulline by LC-MS/MS after hydrolysis assisted by microwave: application to the study of myeloperoxidase activity during hemodialysis.

A high degree of uremia is common in patients with end-stage renal disease and has been linked to the development of chronic inflammation and cardiovascular diseases. In conditions where transplantation is not possible, uremia can be reduced by hemodialysis although the repeated interventions have been implicated in loss of renal function, partially as a result of chronic inflammation and/or oxidative stress processes. In this context, it has been suggested that myeloperoxidase (MPO) can contribute to the oxidative stress during hemodialysis and to the cardiovascular risk. Protein damages due to MPO activity have never been assessed during hemodialysis although two of its reaction products, 3-chlorotyrosine and homocitrulline, are of interest. Indeed, the first one is a specific product of MPO activity and the formation of the second one could be catalyzed by MPO. In order to analyze these products in plasma proteins, a total hydrolysis method followed by liquid chromatography mass spectrometry analysis was developed. Different conditions of hydrolysis were tested and the optimized procedure was assessed for complete hydrolysis and artifactual chlorination. Finally, the method was used for analyzing 3-chlorotyrosine and homocitrulline in plasma proteins during a hemodialysis session in fifteen patients and data were related to measurements of MPO concentration and activity. Both increases in MPO activity and protein-bound 3-chlorotyrosine were observed, highlighting the involvement of MPO in oxidative stress during hemodialysis and further demonstrating the link between hemodialysis and cardiovascular diseases.

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.