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1.
Free Radic Biol Med ; 90: 59-74, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26577177

ABSTRACT

Peripheral leukocytes aggravate brain damage by releasing cytotoxic mediators that compromise blood-brain barrier function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) that is formed via the myeloperoxidase-H2O2-chloride system. The reaction of HOCl with the endogenous plasmalogen pool of brain endothelial cells results in the generation of 2-chlorohexadecanal (2-ClHDA), a toxic, lipid-derived electrophile that induces blood-brain barrier dysfunction in vivo. Here, we synthesized an alkynyl-analog of 2-ClHDA, 2-chlorohexadec-15-yn-1-al (2-ClHDyA) to identify potential protein targets in the human brain endothelial cell line hCMEC/D3. Similar to 2-ClHDA, 2-ClHDyA administration reduced cell viability/metabolic activity, induced processing of pro-caspase-3 and PARP, and led to endothelial barrier dysfunction at low micromolar concentrations. Protein-2-ClHDyA adducts were fluorescently labeled with tetramethylrhodamine azide (N3-TAMRA) by 1,3-dipolar cycloaddition in situ, which unveiled a preferential accumulation of 2-ClHDyA adducts in mitochondria, the Golgi, endoplasmic reticulum, and endosomes. Thirty-three proteins that are subject to 2-ClHDyA-modification in hCMEC/D3 cells were identified by mass spectrometry. Identified proteins include cytoskeletal components that are central to tight junction patterning, metabolic enzymes, induction of the oxidative stress response, and electrophile damage to the caveolar/endosomal Rab machinery. A subset of the targets was validated by a combination of N3-TAMRA click chemistry and specific antibodies by fluorescence microscopy. This novel alkyne analog is a valuable chemical tool to identify cellular organelles and protein targets of 2-ClHDA-mediated damage in settings where myeloperoxidase-derived oxidants may play a disease-propagating role.


Subject(s)
Aldehydes/metabolism , Alkynes/metabolism , Brain/metabolism , Endothelial Cells/metabolism , Alkylation , Cells, Cultured , Female , Humans , Proteins/metabolism
2.
Biochem Pharmacol ; 93(4): 470-81, 2015 Feb 15.
Article in English | MEDLINE | ID: mdl-25576489

ABSTRACT

Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl(-) system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood-brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120min, decaying at a rate of 5.9×10(-3)min(-1). NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC-MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo.


Subject(s)
Aldehydes/metabolism , Blood-Brain Barrier/metabolism , Endothelium, Vascular/metabolism , Phloretin/metabolism , Plasmalogens/metabolism , Aldehydes/chemistry , Aldehydes/pharmacology , Animals , Blood-Brain Barrier/drug effects , Cells, Cultured , Endothelium, Vascular/drug effects , Male , Mice , Mice, Inbred C57BL , Phloretin/chemistry , Phloretin/pharmacology , Plasmalogens/chemistry , Plasmalogens/pharmacology , Sheep , Swine
3.
PLoS One ; 8(5): e64034, 2013.
Article in English | MEDLINE | ID: mdl-23691142

ABSTRACT

Peripheral leukocytes can exacerbate brain damage by release of cytotoxic mediators that disrupt blood-brain barrier (BBB) function. One of the oxidants released by activated leukocytes is hypochlorous acid (HOCl) formed via the myeloperoxidase (MPO)-H2O2-Cl(-) system. In the present study we examined the role of leukocyte activation, leukocyte-derived MPO and MPO-generated oxidants on BBB function in vitro and in vivo. In a mouse model of lipopolysaccharide (LPS)-induced systemic inflammation, neutrophils that had become adherent released MPO into the cerebrovasculature. In vivo, LPS-induced BBB dysfunction was significantly lower in MPO-deficient mice as compared to wild-type littermates. Both, fMLP-activated leukocytes and the MPO-H2O2-Cl(-) system inflicted barrier dysfunction of primary brain microvascular endothelial cells (BMVEC) that was partially rescued with the MPO inhibitor 4-aminobenzoic acid hydrazide. BMVEC treatment with the MPO-H2O2-Cl(-) system or activated neutrophils resulted in the formation of plasmalogen-derived chlorinated fatty aldehydes. 2-chlorohexadecanal (2-ClHDA) severely compromised BMVEC barrier function and induced morphological alterations in tight and adherens junctions. In situ perfusion of rat brain with 2-ClHDA increased BBB permeability in vivo. 2-ClHDA potently activated the MAPK cascade at physiological concentrations. An ERK1/2 and JNK antagonist (PD098059 and SP600125, respectively) protected against 2-ClHDA-induced barrier dysfunction in vitro. The current data provide evidence that interference with the MPO pathway could protect against BBB dysfunction under (neuro)inflammatory conditions.


Subject(s)
Blood-Brain Barrier/metabolism , Oxidants/metabolism , Peroxidase/metabolism , Aldehydes/pharmacology , Animals , Blood-Brain Barrier/drug effects , Endothelial Cells/cytology , Endothelial Cells/metabolism , Fatty Acids/biosynthesis , Humans , Lipopolysaccharides/pharmacology , Male , Mice , Microvessels/cytology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Mitogen-Activated Protein Kinase 8/metabolism , Mitogen-Activated Protein Kinase 9/metabolism , Neutrophils/drug effects , Neutrophils/metabolism , Peroxidase/deficiency , Plasmalogens/metabolism , Rats , Swine
4.
Neurosci Lett ; 539: 16-21, 2013 Feb 28.
Article in English | MEDLINE | ID: mdl-23347841

ABSTRACT

Aberrant oxysterol biosynthesis is implicated in the pathogenesis of neurodegenerative diseases. During the present study we have investigated the effects of exogenously added 25-hydroxycholesterol (25-HC) on transcription of cholesterol biosynthetic genes, sterol-regulatory element binding protein (SREBP) processing and cholesterol biosynthesis in the murine CATH.a neuronal cell line. A single i.p. injection of lipopolysaccharide resulted in robust induction of cholesterol 25-hydroxylase mRNA and protein levels in brains of treated mice. In vitro, 25-HC upregulated the transcription of ATP-binding cassette transporter A1 (ABCA1) and (to a lesser extent) apolipoprotein E (apoE) in CATH.a neurons. Cholesterol biosynthetic gene expression (squalene synthase, HMG-CoA synthase, HMG-CoA reductase, and SREBP2) was downregulated by 25-HC. 25-HC also significantly attenuated proteolytic processing of SREBP2. Finally, 25-HC downregulated cholesterol biosynthesis in CATH.a neurons. Our results demonstrate that 25-HC is a potent effector oxysterol of neuronal cholesterol homeostasis.


Subject(s)
Cholesterol/biosynthesis , Hydroxycholesterols/pharmacology , Neurons/drug effects , Animals , Cell Line , Cholesterol/genetics , Homeostasis , Hydroxycholesterols/metabolism , Lipopolysaccharides/pharmacology , Mice , Neurons/metabolism , RNA, Messenger/metabolism , Steroid Hydroxylases/genetics , Steroid Hydroxylases/metabolism , Sterol Regulatory Element Binding Proteins/genetics , Sterol Regulatory Element Binding Proteins/metabolism , Transcription, Genetic
5.
J Allergy Clin Immunol ; 131(2): 532-40.e1-2, 2013 Feb.
Article in English | MEDLINE | ID: mdl-22704539

ABSTRACT

BACKGROUND: Increased vascular permeability is a fundamental characteristic of inflammation. Substances that are released during inflammation, such as prostaglandin (PG) E(2), can counteract vascular leakage, thereby hampering tissue damage. OBJECTIVE: In this study we investigated the role of PGE(2) and its receptors in the barrier function of human pulmonary microvascular endothelial cells and in neutrophil trafficking. METHODS: Endothelial barrier function was determined based on electrical impedance measurements. Neutrophil recruitment was assessed based on adhesion and transendothelial migration. Morphologic alterations are shown by using immunofluorescence microscopy. RESULTS: We observed that activation of E-type prostanoid (EP) 4 receptor by PGE(2) or an EP4-selective agonist (ONO AE1-329) enhanced the barrier function of human microvascular lung endothelial cells. EP4 receptor activation prompted similar responses in pulmonary artery and coronary artery endothelial cells. These effects were reversed by an EP4 antagonist (ONO AE3-208), as well as by blocking actin polymerization with cytochalasin B. The EP4 receptor-induced increase in barrier function was independent of the classical cyclic AMP/protein kinase A signaling machinery, endothelial nitric oxide synthase, and Rac1. Most importantly, EP4 receptor stimulation showed potent anti-inflammatory activities by (1) facilitating wound healing of pulmonary microvascular endothelial monolayers, (2) preventing junctional and cytoskeletal reorganization of activated endothelial cells, and (3) impairing neutrophil adhesion to endothelial cells and transendothelial migration. The latter effects could be partially attributed to reduced E-selectin expression after EP4 receptor stimulation. CONCLUSION: These data indicate that EP4 agonists as anti-inflammatory agents represent a potential therapy for diseases with increased vascular permeability and neutrophil extravasation.


Subject(s)
Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Neutrophils/metabolism , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Actins/metabolism , Capillary Permeability/drug effects , Capillary Permeability/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Coronary Vessels/drug effects , Coronary Vessels/metabolism , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Cytochalasin B/pharmacology , Dinoprostone/metabolism , Endothelial Cells/drug effects , Endothelial Cells/immunology , Endothelium, Vascular/drug effects , Endothelium, Vascular/immunology , Humans , Lung/drug effects , Lung/metabolism , Methyl Ethers/pharmacology , Microvessels/drug effects , Microvessels/immunology , Microvessels/metabolism , Naphthalenes/pharmacology , Neutrophils/drug effects , Neutrophils/immunology , Nitric Oxide Synthase Type III/metabolism , Phenylbutyrates/pharmacology , Pulmonary Artery/drug effects , Pulmonary Artery/metabolism , Wound Healing/drug effects , Wound Healing/physiology , rac1 GTP-Binding Protein/metabolism
6.
Free Radic Biol Med ; 53(9): 1770-81, 2012 Nov 01.
Article in English | MEDLINE | ID: mdl-22982051

ABSTRACT

2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood-brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting.


Subject(s)
Aldehydes/pharmacology , Brain/blood supply , Endothelial Cells/metabolism , Microvessels/cytology , Phloretin/pharmacology , Aldehydes/metabolism , Animals , Apoptosis/drug effects , Capillary Permeability/drug effects , Cell Survival/drug effects , Cells, Cultured , Cytoprotection , Endothelial Cells/drug effects , Half-Life , Kinetics , Mitochondria/drug effects , Mitochondria/metabolism , Palmitic Acid/pharmacology , Phlorhizin/pharmacology , Primary Cell Culture , Swine
7.
Free Radic Biol Med ; 49(11): 1655-65, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-20807565

ABSTRACT

Plasmalogens, 1-O-alk-1'-enyl-2-acyl-sn-glycerophospholipids, are significant constituents of cellular membranes and are essential for normal brain development. Plasmalogens, which contain a vinyl ether bond at the sn-1 position, are preferential targets for hypochlorous acid (HOCl), generated by myeloperoxidase (MPO) from H(2)O(2) and chloride ions. Because MPO is implicated in neurodegeneration, this study pursued two aims: (i) to investigate the reactivity of mouse brain plasmalogens toward HOCl in vitro and (ii) to obtain in vivo evidence for MPO-mediated brain plasmalogen modification. Liquid chromatography coupled to hybrid linear ion trap-Fourier transform-ion cyclotron resonance mass spectrometry revealed plasmalogen modification in mouse brain lipid extracts at lower HOCl concentrations as observed for diacylphospholipids, resulting in the generation of 2-chloro fatty aldehydes and lysophospholipids. Lysophosphatidylethanolamine accumulation was transient, whereas lysophosphatidylcholine species containing saturated acyl residues remained stable. In vivo, a single, systemic endotoxin injection resulted in upregulation of cerebral MPO mRNA levels to a range comparable to that observed for tumor necrosis factor-α and cyclooxygenase-2. This inflammatory response was accompanied by a significant decrease in several brain plasmalogen species and concomitant in vivo generation of 2-chlorohexadecanal. The present findings demonstrate that activation of the MPO-H(2)O(2)-chloride system under neuroinflammatory conditions results in oxidative attack of the total cerebral plasmalogen pool. As this lipid class is indispensable for normal neuronal function, HOCl-mediated plasmalogen modification is likely to compromise normal synaptic transmission.


Subject(s)
Brain/drug effects , Hypochlorous Acid/pharmacology , Plasmalogens/chemistry , Plasmalogens/metabolism , Animals , Brain/metabolism , Brain Chemistry/drug effects , Cells, Cultured , Ethanolamines/analysis , Ethanolamines/metabolism , Inflammation Mediators/metabolism , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred C57BL , Oxidation-Reduction , Phospholipids/analysis , Phospholipids/metabolism , Phosphorylcholine/analysis , Phosphorylcholine/metabolism , Transcription, Genetic/drug effects
8.
Free Radic Biol Med ; 48(12): 1588-600, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20226853

ABSTRACT

Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCl), formed by MPO from H(2)O(2) and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCl, (ii) to define the impact of exogenously added SM and HOCl-modified SM (HOCl-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCl-SM. MALDI-TOF-MS analyses revealed that HOCl, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCl-SM but not SM induced the formation of reactive oxygen species. HOCl-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCl-SM. Our results demonstrate that HOCl modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO-H(2)O(2)-chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis.


Subject(s)
Apoptosis/drug effects , Hypochlorous Acid/pharmacology , Neurons/metabolism , Oxidants/pharmacology , Oxidative Stress/physiology , Sphingomyelins/metabolism , Animals , Apoptosis/physiology , Blotting, Western , Brain Chemistry , Cell Line , Dopamine/metabolism , Electrophoresis, Gel, Two-Dimensional , Fourier Analysis , Halogenation , Image Processing, Computer-Assisted , In Vitro Techniques , Lipids/chemistry , Membrane Potential, Mitochondrial , Mice , Mice, Inbred C57BL , Proteome/chemistry , Proteome/drug effects , Proteome/metabolism , Rats , Reactive Oxygen Species/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Sphingomyelins/chemistry
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