Inhibition of Myeloperoxidase Pro-Fibrotic Effect by Noscapine in Equine Endometrium.

Myeloperoxidase is an enzyme released by neutrophils when neutrophil extracellular traps (NETs) are formed. Besides myeloperoxidase activity against pathogens, it was also linked to many diseases, including inflammatory and fibrotic ones. Endometrosis is a fibrotic disease of the mare endometrium, with a large impact on their fertility, where myeloperoxidase was shown to induce fibrosis. Noscapine is an alkaloid with a low toxicity, that has been studied as an anti-cancer drug, and more recently as an anti-fibrotic molecule. This work aims to evaluate noscapine inhibition of collagen type 1 (COL1) induced by myeloperoxidase in equine endometrial explants from follicular and mid-luteal phases, at 24 and 48 h of treatment. The transcription of collagen type 1 alpha 2 chain (COL1A2), and COL1 protein relative abundance were evaluated by qPCR and Western blot, respectively. The treatment with myeloperoxidase increased COL1A2 mRNA transcription and COL1 protein, whereas noscapine was able to reduce this effect with respect to COL1A2 mRNA transcription, in a time/estrous cycle phase-dependent manner (in explants from the follicular phase, at 24 h of treatment). Our study indicates that noscapine is a promising drug to be considered as an anti-fibrotic molecule to prevent endometrosis development, making noscapine a strong candidate to be applied in future endometrosis therapies.

Therapeutic inhibition of MPO stabilizes pre-existing high risk atherosclerotic plaque.

Currently there are no established therapies to treat high-risk patients with unstable atherosclerotic lesions that are prone to rupture and can result in thrombosis, abrupt arterial occlusion, and a precipitous infarction. Rather than being stenotic, rupture-prone non-occlusive plaques are commonly enriched with inflammatory cells and have a thin fibrous cap. We reported previously that inhibition of the pro-inflammatory enzyme myeloperoxidase (MPO) with the suicide inhibitor AZM198 prevents formation of unstable plaque in the Tandem Stenosis (TS) mouse model of plaque instability. However, in our previous study AZM198 was administered to animals before unstable plaque was present and hence it did not test the significant unmet clinical need present in high-risk patients with vulnerable atherosclerosis. In the present study we therefore asked whether pharmacological inhibition of MPO with AZM198 can stabilize pre-existing unstable lesions in an interventional setting using the mouse model of plaque instability. In vivo molecular magnetic resonance imaging of arterial MPO activity using bis-5-hydroxytryptamide-DTPA-Gd and histological analyses revealed that arterial MPO activity was elevated one week after TS surgery, prior to the presence of unstable lesions observed two weeks after TS surgery. Animals with pre-existing unstable plaque were treated with AZM198 for one or five weeks. Both short- and long-term intervention effectively inhibited arterial MPO activity and increased fibrous cap thickness, indicative of a more stable plaque phenotype. Plaque stabilization was observed without AZM198 affecting the arterial content of Ly6B.2+- and CD68+-cells and MPO protein. These findings demonstrate that inhibition of arterial MPO activity converts unstable into stable atherosclerotic lesions in a preclinical model of plaque instability and highlight the potential therapeutic potency of MPO inhibition for the management of high-risk patients and the development of novel protective strategies against cardiovascular diseases.

Inhibiting cardiac myeloperoxidase alleviates the relaxation defect in hypertrophic cardiomyocytes.

AIMS: Hypertrophic cardiomyopathy (HCM) is characterized by cardiomyocyte hypertrophy and disarray, and myocardial stiffness due to interstitial fibrosis, which result in impaired left ventricular filling and diastolic dysfunction. The latter manifests as exercise intolerance, angina, and dyspnoea. There is currently no specific treatment for improving diastolic function in HCM. Here, we investigated whether myeloperoxidase (MPO) is expressed in cardiomyocytes and provides a novel therapeutic target for alleviating diastolic dysfunction in HCM. METHODS AND RESULTS: Human cardiomyocytes derived from control-induced pluripotent stem cells (iPSC-CMs) were shown to express MPO, with MPO levels being increased in iPSC-CMs generated from two HCM patients harbouring sarcomeric mutations in the MYBPC3 and MYH7 genes. The presence of cardiomyocyte MPO was associated with higher chlorination and peroxidation activity, increased levels of 3-chlorotyrosine-modified cardiac myosin binding protein-C (MYBPC3), attenuated phosphorylation of MYBPC3 at Ser-282, perturbed calcium signalling, and impaired cardiomyocyte relaxation. Interestingly, treatment with the MPO inhibitor, AZD5904, reduced 3-chlorotyrosine-modified MYBPC3 levels, restored MYBPC3 phosphorylation, and alleviated the calcium signalling and relaxation defects. Finally, we found that MPO protein was expressed in healthy adult murine and human cardiomyocytes, and MPO levels were increased in diseased hearts with left ventricular hypertrophy. CONCLUSION: This study demonstrates that MPO inhibition alleviates the relaxation defect in hypertrophic iPSC-CMs through MYBPC3 phosphorylation. These findings highlight cardiomyocyte MPO as a novel therapeutic target for improving myocardial relaxation associated with HCM, a treatment strategy which can be readily investigated in the clinical setting, given that MPO inhibitors are already available for clinical testing.

Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1ß) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Polyamine-Conjugated Nitroxides Are Efficacious Inhibitors of Oxidative Reactions Catalyzed by Endothelial-Localized Myeloperoxidase.

The heme enzyme myeloperoxidase (MPO) is a key mediator of endothelial dysfunction and a therapeutic target in cardiovascular disease. During inflammation, MPO released by circulating leukocytes is internalized by endothelial cells and transcytosed into the subendothelial extracellular matrix of diseased vessels. At this site, MPO mediates endothelial dysfunction by catalytically consuming nitric oxide (NO) and producing reactive oxidants, hypochlorous acid (HOCl) and the nitrogen dioxide radical (•NO2). Accordingly, there is interest in developing MPO inhibitors that effectively target endothelial-localized MPO. Here we studied a series of piperidine nitroxides conjugated to polyamine moieties as novel endothelial-targeted MPO inhibitors. Electron paramagnetic resonance analysis of cell lysates showed that polyamine conjugated nitroxides were efficiently internalized into endothelial cells in a heparan sulfate dependent manner. Nitroxides effectively inhibited the consumption of MPO's substrate hydrogen peroxide (H2O2) and formation of HOCl catalyzed by endothelial-localized MPO, with their efficacy dependent on both nitroxide and conjugated-polyamine structure. Nitroxides also differentially inhibited protein nitration catalyzed by both purified and endothelial-localized MPO, which was dependent on •NO2 scavenging rather than MPO inhibition. Finally, nitroxides uniformly inhibited the catalytic consumption of NO by MPO in human plasma. These studies show for the first time that nitroxides effectively inhibit local oxidative reactions catalyzed by endothelial-localized MPO. Novel polyamine-conjugated nitroxides, ethylenediamine-TEMPO and putrescine-TEMPO, emerged as efficacious nitroxides uniquely exhibiting high endothelial cell uptake and efficient inhibition of MPO-catalyzed HOCl production, protein nitration, and NO oxidation. Polyamine-conjugated nitroxides represent a versatile class of antioxidant drugs capable of targeting endothelial-localized MPO during vascular inflammation.

Small molecule and macrocyclic pyrazole derived inhibitors of myeloperoxidase (MPO).

Myeloperoxidase (MPO), a critical enzyme in antimicrobial host-defense, has been implicated in chronic inflammatory diseases such as coronary artery disease. The design and evaluation of MPO inhibitors for the treatment of cardiovascular disease are reported herein. Starting with the MPO and triazolopyridine 3 crystal structure, novel inhibitors were designed incorporating a substituted pyrazole, which allowed for substituents to interact with hydrophobic and hydrophilic patches in the active site. SAR exploration of the substituted pyrazoles led to piperidine 17, which inhibited HOCl production from activated neutrophils with an IC50 value of 2.4 µM and had selectivity against thyroid peroxidase (TPO). Optimization of alkylation chemistry on the pyrazole nitrogen facilitated the preparation of many analogs, including macrocycles designed to bridge two hydrophobic regions of the active site. Multiple macrocyclization strategies were pursued to prepare analogs that optimally bound to the active site, leading to potent macrocyclic MPO inhibitors with TPO selectivity, such as compound 30.

Microbial-derived indoles inhibit neutrophil myeloperoxidase to diminish bystander tissue damage.

During episodes of acute inflammation, polymorphonuclear leukocytes (PMNs) are actively recruited to sites of inflammation or injury where they provide anti-microbial and wound-healing functions. One enzyme crucial for fulfilling these functions is myeloperoxidase (MPO), which generates hypochlorous acid from Cl- and hydrogen peroxide. The potential exists, however, that uncontrolled the extracellular generation of hypochlorous acid by MPO can cause bystander tissue damage and inhibit the healing response. Previous work suggests that the microbiota-derived tryptophan metabolites 1H-indole and related molecules ("indoles") are protective during intestinal inflammation, although their precise mechanism of action is unclear. In the present work, we serendipitously discovered that indoles are potent and selective inhibitors of MPO. Using both primary human PMNs and recombinant human MPO in a cell-free system, we revealed that indoles inhibit MPO at physiologic concentrations. Particularly, indoles block the chlorinating activity of MPO, a reliable marker for MPO-associated tissue damage, as measured by coulometric-coupled HPLC. Further, we observed direct interaction between indoles and MPO using the established biochemical techniques microscale thermophoresis and STD-NMR. Utilizing a murine colitis model, we demonstrate that indoles inhibit bystander tissue damage, reflected in decreased colon 3-chlorotyrosine and pro-inflammatory chemokine expression in vivo. Taken together, these results identify microbiota-derived indoles that acts as endogenous immunomodulatory compounds through their actions on MPO, suggesting a symbiotic association between the gut microbiota and host innate immune system. Such findings offer exciting new targets for future pharmacological intervention.

Chalcones and their B-aryl analogues as myeloperoxidase inhibitors: In silico, in vitro and ex vivo investigations.

In the present study, a series of chalcones and their B-aryl analogues were prepared and evaluate as inhibitors of myeloperoxidase (MPO) chlorinating activity, using in vitro and ex vivo assays. Among these, B-thiophenyl chalcone (analogue 9) demonstrated inhibition of in vitro and ex vivo MPO chlorinating activity, exhibiting IC50 value of 0.53 and 19.2 µM, respectively. Potent ex vivo MPO inhibitors 5, 8 and 9 were not toxic to human neutrophils at 50 µM, as well as displayed weak 2,2-diphenyl-1-pycrylhydrazyl radical (DPPH•) and hypochlorous acid (HOCl) scavenger abilities. Docking simulations indicated binding mode of MPO inhibitors, evidencing hydrogen bonds between the amino group at 4'position (ring A) of chalcones with Gln91, Asp94, and Hys95 MPO residues. In this regard, the efficacy and low toxicity promoted aminochalcones and arylic analogues to the rank of hit compounds in the search for new non-steroidal anti-inflammatory compounds.

Suppression of L-arginine-induced acute necrotizing pancreatitis in rats by metformin associated with the inhibition of myeloperoxidase and activation of interleukin-10 / Supresión de la pancreatitis necrotizante aguda inducida por L-arginina en ratas por metformina asociada con la inhibición de mieloperoxidasa y activación de interleucina-10

SUMMARY: Acute pancreatitis is a frequent life-threatening inflammatory disease of the pancreas characterized by severe abdominal pain that lasts for days to weeks. We sought to determine whether the antidiabetic and anti-inflammatory drug, metformin can substantially protect against acute pancreatitis in an animal model of L-arginine-induced acute pancreatitis, and whether this is associated with the augmentation of the anti-inflammatory cytokine interleukin-10 (IL-10) and inhibition of the enzyme that promotes tissue damage, myeloperoxidase (MPO). Rats were either injected with two doses of the amino acid L-arginine (2.5 gm/kg; i.p., at one-hour intervals) before being sacrificed after 48 hours (model group) or were pretreated with metformin (50 mg/kg) daily for two weeks prior to L- arginine injections and continued receiving metformin until the end of the experiment (protective group). Using microscopic examination of the pancreas and blood chemistry, we observed that L-arginine induced acute pancreatic injury. This is demonstrated by an enlarged pancreas with patchy areas of haemorrhage, vacuolated cytoplasm and pyknotic nuclei in the acini, disorganized lobular architecture with infiltration of inflammatory cells within the interlobular connective tissue (CT) septa, and the presence of congested blood vessels that were substantially ameliorated by metformin. Metformin also significantly (p<0.05) inhibited L-arginine-induced MPO, lactate dehydrogenase (LDH), and the inflammatory biomarker tumor necrosis factor alpha (TNF-α). Whereas, metformin significantly (p<0.05) increased IL-10 levels that were inhibited by pancreatitis induction. We further demonstrated a significant (p<0.001) correlation between the scoring of the degree of pancreatic lobules damage tissue damage and the blood levels of TNF-α, IL-10, LDH, and MPO. Thus, metformin effectively protects against L-arginine-induced acute pancreatitis, which is associated with the inhibition of MPO and augmentation of IL-10.

RESUMEN: La pancreatitis aguda es una enfermedad inflamatoria del páncreas que amenaza la vida y se caracteriza por un dolor abdominal intenso que dura de días a semanas. Buscamos determinar si la metformina, fármaco antidiabético y antiinflamatorio, puede proteger contra la pancreatitis aguda en un modelo animal de pancreatitis aguda inducida por L-arginina. Además se estudió la asociación con el aumento de la citocina antiinflamatoria interleucina-10. (IL-10) e inhibición de la enzima que promueve el daño tisular, mieloperoxidasa (MPO). Las ratas se inyectaron con dos dosis del aminoácido L-arginina (2,5 g / kg; ip, a intervalos de una hora) antes de ser sacrificadas des- pués de 48 horas (grupo modelo) o se pre trataron con metformina (50 mg / kg) durante dos semanas antes del tratamiento de L- arginina y continuaron recibiendo metformina hasta el final del experimento (grupo protector). Mediante el examen microscópico del páncreas y la química sanguínea, se observó que la L- arginina inducía una lesión pancreática aguda. Se observó un aumento significativo de tamaño del páncreas con áreas hemorrágicas, citoplasma vacuolado y núcleos picnóticos en los acinos, arquitectura desorganizada con infiltración de células inflamatorias dentro de los tabiques del tejido conjuntivo interlobulillar (TC) y la presencia de vasos sanguíneos congestionados mejorados por metformina. Se observó que la metformina inhibió significativamente (p <0,05) la MPO inducida por L- arginina, la lactato deshidrogenasa (LDH) y el factor de necrosis tumoral alfa (TNF-α). Además, demostramos una correlación significativa (p <0,001) entre la puntuación del grado de daño tisular de los lóbulos pancreáticos y los niveles sanguíneos de TNF-α, IL-10, LDH y MPO. Por tanto, la metformina protege eficazmente contra la pancreatitis aguda inducida por L-arginina, que se asocia con la inhibición de MPO y el aumento de IL-10.

Artificial intelligence based identification of the functional role of hirudin in diabetic erectile dysfunction treatment.

Diabetic erectile dysfunction (DED) hugely affected the patients' sexual life quality. However, there are no satisfactory therapeutic methods and intervention targets for this subtype of erectile dysfunction (ED). Inspired by the clinical practice of traditional Chinese medicine (TCM), we found that hirudin, the main active ingredient in the leech, could ameliorate the ED symptoms of the DED mouse model. To further reveal the underlying mechanism of hirudin, we designed a novel strategy to discover potential targets based on the diagnostic system of TCM, and found that myeloperoxidase (MPO) was a promising target of hirudin. Hirudin directly interacts with MPO and inhibits its activity, thus further decreases the content of oxidized low-density lipoprotein (ox-LDL) in serum. Our results demonstrated that the hirudin could ameliorate the symptoms of DED, and revealed the underlying mechanism of hirudin in regulating the activity of MPO.

Hemocyte extracellular traps of Manila clam Ruditapes philippinarum: Production characteristics and antibacterial effects.

Extracellular traps (ETs) have been found to be an important strategy of mammals to immobilize and kill invading microorganisms. In the present study, we observed the formation of ETs in the hemocytes of marine mollusks Ruditapes philippinarum in response to challenge from bacteria Vibrio anguillarum, and examined the potential factors and signaling pathways underling this process. We detected an increase of reactive oxygen species (ROS) and myeloperoxidase (MPO) production during ETosis, accompanied by significantly up-regulated expression of ROS-related and MPO genes. The suppression of ETs structures by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (diphenyleneiodonium chloride, DPI) and MPO inhibitor (aminobenzoic acid hydrazide, ABAH) further confirmed the essential roles ROS and MPO played in ETosis. Furthermore, ET production could be inhibited by phosphotidylinsitol-3-kinase (PI3K) inhibitor (LY294002) and extracellular regulated protein kinase (ERK) inhibitor (U0126), suggesting the idea that both the PI3K and ERK pathways were suggested to function during ETosis. In addition, the ETosis process was accompanied by enhancement of glycolysis-related enzymatic activities, e.g., pyruvate kinase (PK) and hexokinase (HK), and over-expression of the glycolysis-related genes, e.g., PK, HK and glucose transport protein (GLUT), indicating high involvement of glycolysis in the ETosis process. Furthermore, our scanning electron microscopy (SEM) observation and antibacterial activities test successfully showed the patterns how clam ETs entrapped and killed the invading V. anguillarum. Taken together, our results revealed that ETosis with bactericidal effect increased ROS, MPO and glycolysis level and carried out in a ROS-, MPO-, PI3K-ERK-dependent manner.

Blanching effects of radio frequency heating on enzyme inactivation, physiochemical properties of green peas (Pisum sativum L.) and the underlying mechanism in relation to cellular microstructure.

Fresh green peas require blanching to terminate enzymatic reaction induced quality deterioration before frozen storage. Radio frequency (RF) heating is a novel way of dry blanching for fruits and vegetables with high processing efficiency. In this study, blanching effects of RF heating on relative activities of lipoxygenase (LOX) and peroxidase (POD), physiochemical properties as well as cellular morphology changes of green peas were investigated. Results showed relative activities of pea LOX and POD reduced to 0.90 ± 0.78% and 1.10 ± 0.71%, respectively at 85 °C by RF heating with an electrode gap 105 mm. Weight loss, color, texture and electrolyte leakage of peas changed significantly with increasing temperature (60-85 °C). Ascorbic acid, chlorophyll and mineral contents had different loss after RF processing and long term heating at 115 mm exacerbated the loss of nutrients. Microstructure features showed the deconstruction of pea cell well and starch granule gelatinization.

The effect of ultrasonic on reducing anti-browning minimum effective concentration of purslane extract on fresh-cut potato slices during storage.

Enzymatic browning is one of the major difficulties for the preservation and commercial value of fresh-cut products. To research more healthy and inexpensive anti-browning methods, we investigated the effect of ultrasonic coupling purslane extract on the browning resistance of fresh-cut potato during 8d storage at 4 °C. Firstly, the optimal ultrasonic time (10 min) was obtained. Then, the results showed that the combined application with lower purslane extract concentration (0.02%, w/w) could achieve a better anti-browning effect than the optimal concentration of alone purslane extract (0.05%, w/w). The combined application not only significantly inhibited the key enzyme activities of polyphenol oxidase (PPO) and peroxidase (POD), but also effectively reduced the damage to cell membrane, maintained its integrity and permeability. Meanwhile, it also improved antioxidant capacity during storage. Overall, the ultrasonic cavitation combined with purslane extract would be a promising method for fresh-cut industry.

Early Clinical Experience With AZD4831, A Novel Myeloperoxidase Inhibitor, Developed for Patients With Heart Failure With Preserved Ejection Fraction.

We evaluated safety, tolerability, pharmacokinetics (PKs), and pharmacodynamics of AZD4831, a novel oral myeloperoxidase (MPO) inhibitor, in a randomized, single-blind, placebo-controlled study, following once-daily multiple ascending dosing to steady-state in healthy subjects. Target engagement was measured as specific MPO activity in plasma following ex vivo zymosan stimulation of whole blood. Except for generalized maculopapular rash in 4 of 13 subjects receiving the 2 highest doses, 15 and 45 mg AZD4831, no clinically relevant safety and tolerability findings were observed. AZD4831 was rapidly absorbed and plasma concentrations declined slowly with an elimination half-life of ~ 60 hours. A dose/concentration-effect relationship between MPO inhibition vs. AZD4831 exposure was established with > 50% MPO inhibition in plasma at concentrations in the low nanomolar range. Steady-state levels were achieved within 10 days. Taken together, the PK profile, the sustained dose/concentration-dependent MPO inhibition, and available clinical data support further clinical development of AZD4831 in patients with heart failure with preserved ejection fraction.

Nitroxides Mitigate Neutrophil-Mediated Damage to the Myocardium after Experimental Myocardial Infarction in Rats.

Reperfusion therapy increases survival post-acute myocardial infarction (AMI) while also stimulating secondary oxidant production and immune cell infiltration. Neutrophils accumulate within infarcted myocardium within 24 h post-AMI and release myeloperoxidase (MPO) that catalyses hypochlorous acid (HOCl) production while increasing oxidative stress and inflammation, thereby enhancing ventricular remodelling. Nitroxides inhibit MPO-mediated HOCl production, potentially ameliorating neutrophil-mediated damage. Aim: Assess the cardioprotective ability of nitroxide 4-methoxyTEMPO (4MetT) within the setting of AMI. Methods: Male Wistar rats were separated into 3 groups: SHAM, AMI/R, and AMI/R + 4MetT (15 mg/kg at surgery via oral gavage) and subjected to left descending coronary artery ligation for 30 min to generate an AMI, followed by reperfusion. One cohort of rats were sacrificed at 24 h post-reperfusion and another 28 days post-surgery (with 4MetT (15 mg/kg) administration twice daily). Results: 3-chlorotyrosine, a HOCl-specific damage marker, decreased within the heart of animals in the AMI/R + 4-MetT group 24 h post-AMI, indicating the drug inhibited MPO activity; however, there was no evident difference in either infarct size or myocardial scar size between the groups. Concurrently, MPO, NfκB, TNFα, and the oxidation marker malondialdehyde increased within the hearts, with 4-MetT only demonstrating a trend in decreasing MPO and TNF levels. Notably, 4MetT provided a significant improvement in cardiac function 28 days post-AMI, as assessed by echocardiography, indicating potential for 4-MetT as a treatment option, although the precise mechanism of action of the compound remains unclear.

Discovery and structure activity relationships of 7-benzyl triazolopyridines as stable, selective, and reversible inhibitors of myeloperoxidase.

Myeloperoxidase (MPO) is a heme peroxidase found in neutrophils, monocytes and macrophages that efficiently catalyzes the oxidation of endogenous chloride into hypochlorous acid for antimicrobial activity. Chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. Triazolopyrimidine 5 is a reversible MPO inhibitor; however it suffers from poor stability in acid, and is an irreversible inhibitor of the DNA repair protein methyl guanine methyl transferase (MGMT). Structure-based drug design was employed to discover benzyl triazolopyridines with improved MPO potency, as well as acid stability, no reactivity with MGMT, and selectivity against thyroid peroxidase (TPO). Structure-activity relationships, a crystal structure of the MPO-inhibitor complex, and acute in vivo pharmacodynamic data are described herein.

Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.

Loss-of-Function Myeloperoxidase Mutations Are Associated with Increased Neutrophil Counts and Pustular Skin Disease.

The identification of disease alleles underlying human autoinflammatory diseases can provide important insights into the mechanisms that maintain neutrophil homeostasis. Here, we focused our attention on generalized pustular psoriasis (GPP), a potentially life-threatening disorder presenting with cutaneous and systemic neutrophilia. Following the whole-exome sequencing of 19 unrelated affected individuals, we identified a subject harboring a homozygous splice-site mutation (c.2031-2A>C) in MPO. This encodes myeloperoxidase, an essential component of neutrophil azurophil granules. MPO screening in conditions phenotypically related to GPP uncovered further disease alleles in one subject with acral pustular psoriasis (c.2031-2A>C;c.2031-2A>C) and in two individuals with acute generalized exanthematous pustulosis (c.1705C>T;c.2031-2A>C and c.1552_1565del;c.1552_1565del). A subsequent analysis of UK Biobank data demonstrated that the c.2031-2A>C and c.1705C>T (p.Arg569Trp) disease alleles were also associated with increased neutrophil abundance in the general population (p = 5.1 × 10-6 and p = 3.6 × 10-5, respectively). The same applied to three further deleterious variants that had been genotyped in the cohort, with two alleles (c.995C>T [p.Ala332Val] and c.752T>C [p.Met251Thr]) yielding p values < 10-10. Finally, treatment of healthy neutrophils with an MPO inhibitor (4-Aminobenzoic acid hydrazide) increased cell viability and delayed apoptosis, highlighting a mechanism whereby MPO mutations affect granulocyte numbers. These findings identify MPO as a genetic determinant of pustular skin disease and neutrophil abundance. Given the recent interest in the development of MPO antagonists for the treatment of neurodegenerative disease, our results also suggest that the pro-inflammatory effects of these agents should be closely monitored.

The MPO system participates actively in the formation of an oxidative environment produced by neutrophils and activates the antioxidant mechanism of Naegleria fowleri.

Naegleria fowleri produces a fatal disease called primary amebic meningoencephalitis (PAM), which is characterized by an extensive inflammatory reaction in the CNS. It is known that the immune response is orchestrated mainly by neutrophils, which activate several defense mechanisms in the host, including phagocytosis, the release of different enzymes such as myeloperoxidase (MPO), and the production of neutrophil extracellular traps. However, the mechanisms by which amoebas evade the neutrophil response are still unknown. In this study, we analyzed the ability of N. fowleri to respond to the stress exerted by MPO. Interestingly, after the interaction of trophozoites with neutrophils, the amoeba viability was not altered; however, ultrastructural changes were observed. To analyze the influence of MPO against N. fowleri and its participation in free radical production, we evaluated its enzymatic activity, expression, and localization with and without the specific 4-aminobenzoic acid hydrazide inhibitor. The production of oxidizing molecules is the principal mechanism used by neutrophils to eliminate pathogens. In this context, we demonstrated an increase in the production of NO, superoxide anion, and reactive oxygen species; in addition, the overexpression of several antioxidant enzymes present in the trophozoites was quantified. The findings strongly suggest that N. fowleri possesses antioxidant machinery that is activated in response to an oxidative environment, allowing it to evade the neutrophil-mediated immune response, which may contribute to the establishment of PAM.

A patent review of myeloperoxidase inhibitors for treating chronic inflammatory syndromes (focus on cardiovascular diseases, 2013-2019).

INTRODUCTION: Myeloperoxidase (MPO) is an immune enzyme found in neutrophils and macrophages. It produces the highly oxidative compound HOCl from H2O2 and Cl- ions inside the phagosome of the neutrophil. Leakage of the enzyme outside the cell causes oxidative damages for the biomolecules promoting many inflammatory diseases such as atherosclerosis. Thus, there is a real interest to develop potent inhibitors of MPO as non-steroidal anti-inflammatory agents. This review highlights the several published MPO inhibitors, their activity, and the challenges met during the development of these compounds. AREAS COVERED: This article covers the patent literature published on MPO inhibitors from 2013 to 2019, as well as the potential use of these compounds as therapeutics for inflammatory syndromes, especially that plays an important role in the initiation and progression of atherosclerosis. EXPERT OPINION: To date, many MPO inhibitors with different structures have been studied, many of which have prominent inhibitory activities. Furthermore, the specificity of these drugs offers hope for the targeted therapy of inflammatory syndromes. Although many data have proved that MPO can contribute to several chronic inflammatory syndromes, the usefulness of MPO inhibitors in preventing and treating inflammatory disorders is still under investigation.