Publications by authors named "Helga Reicher"

23 Publications

  • Page 1 of 1

Lysophosphatidic Acid Induces Aerobic Glycolysis, Lipogenesis, and Increased Amino Acid Uptake in BV-2 Microglia.

Int J Mol Sci 2021 Feb 17;22(4). Epub 2021 Feb 17.

Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, Austria.

Lysophosphatidic acid (LPA) species are a family of bioactive lipids that transmit signals via six cognate G protein-coupled receptors, which are required for brain development and function of the nervous system. LPA affects the function of all cell types in the brain and can display beneficial or detrimental effects on microglia function. During earlier studies we reported that LPA treatment of microglia induces polarization towards a neurotoxic phenotype. In the present study we investigated whether these alterations are accompanied by the induction of a specific immunometabolic phenotype in LPA-treated BV-2 microglia. In response to LPA (1 µM) we observed slightly decreased mitochondrial respiration, increased lactate secretion and reduced ATP/ADP ratios indicating a switch towards aerobic glycolysis. Pathway analyses demonstrated induction of the Akt-mTOR-Hif1α axis under normoxic conditions. LPA treatment resulted in dephosphorylation of AMP-activated kinase, de-repression of acetyl-CoA-carboxylase and increased fatty acid content in the phospholipid and triacylglycerol fraction of BV-2 microglia lipid extracts, indicating de novo lipogenesis. LPA led to increased intracellular amino acid content at one or more time points. Finally, we observed LPA-dependent generation of reactive oxygen species (ROS), phosphorylation of nuclear factor erythroid 2-related factor 2 (Nrf2), upregulated protein expression of the Nrf2 target regulatory subunit of glutamate-cysteine ligase and increased glutathione synthesis. Our observations suggest that LPA, as a bioactive lipid, induces subtle alterations of the immunometabolic program in BV-2 microglia.
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http://dx.doi.org/10.3390/ijms22041968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7923140PMC
February 2021

Myeloperoxidase-Derived 2-Chlorohexadecanal Is Generated in Mouse Heart during Endotoxemia and Induces Modification of Distinct Cardiomyocyte Protein Subsets In Vitro.

Int J Mol Sci 2020 Dec 3;21(23). Epub 2020 Dec 3.

Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, 8010 Graz, Austria.

Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.
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http://dx.doi.org/10.3390/ijms21239235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730634PMC
December 2020

MAPK signaling determines lysophosphatidic acid (LPA)-induced inflammation in microglia.

J Neuroinflammation 2020 Apr 23;17(1):127. Epub 2020 Apr 23.

Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Neue Stiftingtalstrasse 6/6, 8010, Graz, Austria.

Background: In the extracellular environment, lysophosphatidic acid (LPA) species are generated via autotaxin (ATX)-mediated hydrolysis of lysophospholipid precursors. Members of the LPA family are potent lipid mediators transmitting signals via six different G protein-coupled LPA receptors (LPAR1-6). The LPA signaling axis is indispensable for brain development and function of the nervous system; however, during damage of the central nervous system, LPA levels can increase and aberrant signaling events counteract brain function. Here, we investigated regulation of the ATX/LPA/LPAR axis in response to lipopolysaccharide-induced systemic inflammation in mice and potential neurotoxic polarization programs in LPA-activated primary murine microglia.

Methods: In vivo, LPAR1-6 expression was established by qPCR in whole murine brain homogenates and in FACS-sorted microglia. ELISAs were used to quantitate LPA concentrations in the brain and cyto-/chemokine secretion from primary microglia in vitro. Transcription factor phosphorylation was analyzed by immunoblotting, and plasma membrane markers were analyzed by flow cytometry. We used MAPK inhibitors to study signal integration by the JNK, p38, and ERK1/2 branches in response to LPA-mediated activation of primary microglia.

Results: Under acute and chronic inflammatory conditions, we observed a significant increase in LPA concentrations and differential regulation of LPAR, ATX (encoded by ENPP2), and cytosolic phospholipase A2 (encoded by PLA2G4A) gene expression in the brain and FACS-sorted microglia. During pathway analyses in vitro, the use of specific MAPK antagonists (SP600125, SB203580, and PD98059) revealed that JNK and p38 inhibition most efficiently attenuated LPA-induced phosphorylation of proinflammatory transcription factors (STAT1 and -3, p65, and c-Jun) and secretion of IL-6 and TNFα. All three inhibitors decreased LPA-mediated secretion of IL-1β, CXCL10, CXCL2, and CCL5. The plasma membrane marker CD40 was solely inhibited by SP600125 while all three inhibitors affected expression of CD86 and CD206. All MAPK antagonists reduced intracellular COX-2 and Arg1 as well as ROS and NO formation, and neurotoxicity of microglia-conditioned media.

Conclusion: In the present study, we show that systemic inflammation induces aberrant ATX/LPA/LPAR homeostasis in the murine brain. LPA-mediated polarization of primary microglia via MAPK-dependent pathways induces features reminiscent of a neurotoxic phenotype.
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http://dx.doi.org/10.1186/s12974-020-01809-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7178949PMC
April 2020

Myeloperoxidase and Septic Conditions Disrupt Sphingolipid Homeostasis in Murine Brain Capillaries In Vivo and Immortalized Human Brain Endothelial Cells In Vitro.

Int J Mol Sci 2020 Feb 9;21(3). Epub 2020 Feb 9.

Division of Molecular Biology and Biochemistry, Gottfried Schatz Research Center, Medical University of Graz, Graz 8010, Austria.

During inflammation, activated leukocytes release cytotoxic mediators that compromise blood-brain barrier (BBB) function. Under inflammatory conditions, myeloperoxidase (MPO) is critically involved in inflicting BBB damage. We used genetic and pharmacological approaches to investigate whether MPO induces aberrant lipid homeostasis at the BBB in a murine endotoxemia model. To corroborate findings in a human system we studied the impact of sera from sepsis and non-sepsis patients on brain endothelial cells (hCMEC/D3). In response to endotoxin, the fatty acid, ceramide, and sphingomyelin content of isolated mouse brain capillaries dropped and barrier dysfunction occurred. In mice, genetic deficiency or pharmacological inhibition of MPO abolished these alterations. Studies in metabolic cages revealed increased physical activity and less pronounced sickness behavior of MPO compared to wild-type mice in response to sepsis. In hCMEC/D3 cells, exogenous tumor necrosis factor α (TNFα) potently regulated gene expression of pro-inflammatory cytokines and a set of genes involved in sphingolipid (SL) homeostasis. Notably, treatment of hCMEC/D3 cells with sera from septic patients reduced cellular ceramide concentrations and induced barrier and mitochondrial dysfunction. In summary, our in vivo and in vitro data revealed that inflammatory mediators including MPO, TNFα induce dysfunctional SL homeostasis in brain endothelial cells. Genetic and pharmacological inhibition of MPO attenuated endotoxin-induced alterations in SL homeostasis in vivo, highlighting the potential role of MPO as drug target to treat inflammation-induced brain dysfunction.
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http://dx.doi.org/10.3390/ijms21031143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037060PMC
February 2020

Hepatocyte-specific lysosomal acid lipase deficiency protects mice from diet-induced obesity but promotes hepatic inflammation.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 04 9;1864(4):500-511. Epub 2019 Jan 9.

Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria. Electronic address:

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.
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http://dx.doi.org/10.1016/j.bbalip.2019.01.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372077PMC
April 2019

2-Chlorohexadecanoic acid induces ER stress and mitochondrial dysfunction in brain microvascular endothelial cells.

Redox Biol 2018 05 5;15:441-451. Epub 2018 Jan 5.

Gottfried Schatz Research Center for Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz, Austria; BioTechMed Graz, Austria. Electronic address:

Peripheral leukocytes induce blood-brain barrier (BBB) dysfunction through the release of cytotoxic mediators. These include hypochlorous acid (HOCl) that is formed via the myeloperoxidase-HO-chloride system of activated phagocytes. HOCl targets the endogenous pool of ether phospholipids (plasmalogens) generating chlorinated inflammatory mediators like e.g. 2-chlorohexadecanal and its conversion product 2-chlorohexadecanoic acid (2-ClHA). In the cerebrovasculature these compounds inflict damage to brain microvascular endothelial cells (BMVEC) that form the morphological basis of the BBB. To follow subcellular trafficking of 2-ClHA we synthesized a 'clickable' alkyne derivative (2-ClHyA) that phenocopied the biological activity of the parent compound. Confocal and superresolution structured illumination microscopy revealed accumulation of 2-ClHyA in the endoplasmic reticulum (ER) and mitochondria of human BMVEC (hCMEC/D3 cell line). 2-ClHA and its alkyne analogue interfered with protein palmitoylation, induced ER-stress markers, reduced the ER ATP content, and activated transcription and secretion of interleukin (IL)-6 as well as IL-8. 2-ClHA disrupted the mitochondrial membrane potential and induced procaspase-3 and PARP cleavage. The protein kinase R-like ER kinase (PERK) inhibitor GSK2606414 suppressed 2-ClHA-mediated activating transcription factor 4 synthesis and IL-6/8 secretion, but showed no effect on endothelial barrier dysfunction and cleavage of procaspase-3. Our data indicate that 2-ClHA induces potent lipotoxic responses in brain endothelial cells and could have implications in inflammation-induced BBB dysfunction.
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http://dx.doi.org/10.1016/j.redox.2018.01.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5975063PMC
May 2018

Dipeptidyl peptidase-4 independent cardiac dysfunction links saxagliptin to heart failure.

Biochem Pharmacol 2017 12 30;145:64-80. Epub 2017 Aug 30.

Department of Internal Medicine, Division of Cardiology, Medical University of Graz, Austria.

Saxagliptin treatment has been associated with increased rate of hospitalization for heart failure in type 2 diabetic patients, though the underlying mechanism(s) remain elusive. To address this, we assessed the effects of saxagliptin on human atrial trabeculae, guinea pig hearts and cardiomyocytes. We found that the primary target of saxagliptin, dipeptidyl peptidase-4, is absent in cardiomyocytes, yet saxagliptin internalized into cardiomyocytes and impaired cardiac contractility via inhibition of the Ca/calmodulin-dependent protein kinase II-phospholamban-sarcoplasmic reticulum Ca-ATPase 2a axis and Na-Ca exchanger function in Ca extrusion. This resulted in reduced sarcoplasmic reticulum Ca content, diastolic Ca overload, systolic dysfunction and impaired contractile force. Furthermore, saxagliptin reduced protein kinase C-mediated delayed rectifier K current that prolonged action potential duration and consequently QTc interval. Importantly, saxagliptin aggravated pre-existing cardiac dysfunction induced by ischemia/reperfusion injury. In conclusion, our novel results provide mechanisms for the off-target deleterious effects of saxagliptin on cardiac function and support the outcome of SAVOR-TIMI 53 trial that linked saxagliptin with the risk of heart failure.
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http://dx.doi.org/10.1016/j.bcp.2017.08.021DOI Listing
December 2017

Lysosomal lipid hydrolysis provides substrates for lipid mediator synthesis in murine macrophages.

Oncotarget 2017 Jun;8(25):40037-40051

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.

Degradation of lysosomal lipids requires lysosomal acid lipase (LAL), the only intracellular lipase known to be active at acidic pH. We found LAL to be expressed in murine immune cells with highest mRNA expression in macrophages and neutrophils. Furthermore, we observed that loss of LAL in mice caused lipid accumulation in white blood cells in the peripheral circulation, which increased in response to an acute inflammatory stimulus. Lal-deficient (-/-) macrophages accumulate neutral lipids, mainly cholesteryl esters, within lysosomes. The cholesteryl ester fraction is particularly enriched in the PUFAs 18:2 and 20:4, important precursor molecules for lipid mediator synthesis. To investigate whether loss of LAL activity affects the generation of lipid mediators and to eliminate potential systemic effects from other cells and tissues involved in the pronounced phenotype of Lal-/- mice, we treated macrophages from Wt mice with the LAL-specific inhibitor LAListat-2. Acute inhibition of LAL resulted in reduced release of 18:2- and 20:4-derived mediators from macrophages, indicating that lipid hydrolysis by LAL is an important source for lipid mediator synthesis in macrophages. We conclude that lysosomes should be considered as organelles that provide precursor molecules for lipid mediators such as eicosanoids.
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http://dx.doi.org/10.18632/oncotarget.16673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522325PMC
June 2017

1-Oleyl-lysophosphatidic acid (LPA) promotes polarization of BV-2 and primary murine microglia towards an M1-like phenotype.

J Neuroinflammation 2016 08 26;13(1):205. Epub 2016 Aug 26.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Harrachgasse 21, 8010, Graz, Austria.

Background: Microglia, the immunocompetent cells of the CNS, rapidly respond to brain injury and disease by altering their morphology and phenotype to adopt an activated state. Microglia can exist broadly between two different states, namely the classical (M1) and the alternative (M2) phenotype. The first is characterized by the production of pro-inflammatory cytokines/chemokines and reactive oxygen and/or nitrogen species. In contrast, alternatively activated microglia are typified by an anti-inflammatory phenotype supporting wound healing and debris clearance. The objective of the present study was to determine the outcome of lysophosphatidic acid (LPA)-mediated signaling events on microglia polarization.

Methods: LPA receptor expression and cyto-/chemokine mRNA levels in BV-2 and primary murine microglia (PMM) were determined by qPCR. M1/M2 marker expression was analyzed by Western blotting, immunofluorescence microscopy, or flow cytometry. Cyto-/chemokine secretion was quantitated by ELISA.

Results: BV-2 cells express LPA receptor 2 (LPA2), 3, 5, and 6, whereas PMM express LPA1, 2, 4, 5, and 6. We show that LPA treatment of BV-2 and PMM leads to a shift towards a pro-inflammatory M1-like phenotype. LPA treatment increased CD40 and CD86 (M1 markers) and reduced CD206 (M2 marker) expression. LPA increased inducible nitric oxide synthase (iNOS) and COX-2 levels (both M1), while the M2 marker Arginase-1 was suppressed in BV-2 cells. Immunofluorescence studies (iNOS, COX-2, Arginase-1, and RELMα) extended these findings to PMM. Upregulation of M1 markers in BV-2 and PMM was accompanied by increased cyto-/chemokine transcription and secretion (IL-1β, TNFα, IL-6, CCL5, and CXCL2). The pharmacological LPA5 antagonist TCLPA5 blunted most of these pro-inflammatory responses.

Conclusions: LPA drives BV-2 and PMM towards a pro-inflammatory M1-like phenotype. Suppression by TCLPA5 indicates that the LPA/LPA5 signaling axis could represent a potential pharmacological target to interfere with microglia polarization in disease.
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http://dx.doi.org/10.1186/s12974-016-0701-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5002165PMC
August 2016

Assessment of electrophile damage in a human brain endothelial cell line utilizing a clickable alkyne analog of 2-chlorohexadecanal.

Free Radic Biol Med 2016 Jan 11;90:59-74. Epub 2015 Nov 11.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria; BioTechMed Graz, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.11.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392177PMC
January 2016

Interference with distinct steps of sphingolipid synthesis and signaling attenuates proliferation of U87MG glioma cells.

Biochem Pharmacol 2015 Jul 19;96(2):119-30. Epub 2015 May 19.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria; BioTechMed Graz, Austria. Electronic address:

Glioblastoma is the most common malignant brain tumor, which, despite combined radio- and chemotherapy, recurs and is invariably fatal for affected patients. Members of the sphingolipid (SL) family are potent effectors of glioma cell proliferation. In particular sphingosine-1-phosphate (S1P) and the corresponding G protein-coupled S1P receptors transmit proliferative signals to glioma cells. To investigate the contribution to glioma cell proliferation we inhibited the first step of de novo SL synthesis in p53(wt) and p53(mut) glioma cells, and interfered with S1P signaling specifically in p53(wt) U87MG cells. Subunit silencing (RNAi) or pharmacological antagonism (using myriocin) of serine palmitoyltransferase (SPT; catalyzing the first committed step of SL biosynthesis) reduced proliferation of p53(wt) but not p53(mut) GBM cells. In U87MG cells these observations were accompanied by decreased ceramide, sphingomyelin, and S1P content. Inhibition of SPT upregulated p53 and p21 expression and induced an increase in early and late apoptotic U87MG cells. Exogenously added S1P (complexed to physiological carriers) increased U87MG proliferation. In line, silencing of individual members of the S1P receptor family decreased U87MG proliferation. Silencing and pharmacological inhibition of the ATP-dependent cassette transporter A1 (ABCA1) that facilitates S1P efflux in astrocytes attenuated U87MG growth. Glyburide-mediated inhibition of ABCA1 resulted in intracellular accumulation of S1P raising the possibility that ABCA1 promotes S1P efflux in U87MG glioma cells thereby contributing to inside-out signaling. Our findings indicate that de novo SL synthesis, S1P receptor-mediated signaling, and ABCA1-mediated S1P efflux could provide pharmacological targets to interfere with glioma cell proliferation.
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http://dx.doi.org/10.1016/j.bcp.2015.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490581PMC
July 2015

Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin.

Biochem Pharmacol 2015 Feb 6;93(4):470-81. Epub 2015 Jan 6.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bcp.2014.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321883PMC
February 2015

Endocytosis and intracellular processing of BODIPY-sphingomyelin by murine CATH.a neurons.

Biochim Biophys Acta 2013 Dec 22;1831(12):1665-78. Epub 2013 Aug 22.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz, Austria.

Neuronal sphingolipids (SL) play important roles during axonal extension, neurotrophic receptor signaling and neurotransmitter release. Many of these signaling pathways depend on the presence of specialized membrane microdomains termed lipid rafts. Sphingomyelin (SM), one of the main raft constituents, can be formed de novo or supplied from exogenous sources. The present study aimed to characterize fluorescently-labeled SL turnover in a murine neuronal cell line (CATH.a). Our results demonstrate that at 4°C exogenously added BODIPY-SM accumulates exclusively at the plasma membrane. Treatment of cells with bacterial sphingomyelinase (SMase) and back-exchange experiments revealed that 55-67% of BODIPY-SM resides in the outer leaflet of the plasma membrane. Endocytosis of BODIPY-SM occurs via caveolae with part of internalized BODIPY-fluorescence ending up in the Golgi and the ER. Following endocytosis BODIPY-SM undergoes hydrolysis, a reaction substantially faster than BODIPY-SM synthesis from BODIPY-ceramide. RNAi demonstrated that both, acid (a)SMase and neutral (n)SMases contribute to BODIPY-SM hydrolysis. Finally, high-density lipoprotein (HDL)-associated BODIPY-SM was efficiently taken up by CATH.a cells. Our findings indicate that endocytosis of exogenous SM occurs almost exclusively via caveolin-dependent pathways, that both, a- and nSMases equally contribute to neuronal SM turnover and that HDL-like particles might represent physiological SM carriers/donors in the brain.
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http://dx.doi.org/10.1016/j.bbalip.2013.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3807659PMC
December 2013

Myeloperoxidase-derived oxidants induce blood-brain barrier dysfunction in vitro and in vivo.

PLoS One 2013 14;8(5):e64034. Epub 2013 May 14.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0064034PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3653856PMC
December 2013

Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro.

Free Radic Biol Med 2012 Nov 25;53(9):1770-81. Epub 2012 Aug 25.

Institute of Molecular Biology and Biochemistry, University Children's Hospital, Medical University of Graz, Austria.

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2012.08.575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485557PMC
November 2012

Alterations in lipid metabolism mediate inflammation, fibrosis, and proliferation in a mouse model of chronic cholestatic liver injury.

Gastroenterology 2012 Jan 14;142(1):140-151.e12. Epub 2011 Oct 14.

Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Medical University of Graz, Graz, Austria.

Background & Aims: The liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice.

Methods: We used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer.

Results: Alterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice.

Conclusions: Alterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.
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http://dx.doi.org/10.1053/j.gastro.2011.09.051DOI Listing
January 2012

Sequential synthesis and methylation of phosphatidylethanolamine promote lipid droplet biosynthesis and stability in tissue culture and in vivo.

J Biol Chem 2011 May 22;286(19):17338-50. Epub 2011 Mar 22.

Department of Molecular Biology and Biochemistry, Center for Molecular Medicine, Medical University of Graz, A-8010 Graz, Austria.

Triacylglycerols are stored in eukaryotic cells within lipid droplets (LD). The LD core is enwrapped by a phospholipid monolayer with phosphatidylcholine (PC), the major phospholipid, and phosphatidylethanolamine (PE), a minor component. We demonstrate that the onset of LD formation is characterized by a change in cellular PC, PE, and phosphatidylserine (PS). With induction of differentiation of 3T3-L1 fibroblasts into adipocytes, the cellular PC/PE ratio decreased concomitant with LD formation, with the most pronounced decline between confluency and day 5. The mRNA for PS synthase-1 (forms PS from PC) and PS decarboxylase (forms PE from PS) increased after day 5. Activity and protein of PE N-methyltransferase (PEMT), which produces PC by methylation of PE, are absent in 3T3-L1 fibroblasts but were induced at day 5. High fat challenge induced PEMT expression in mouse adipose tissue. PE, produced via PS decarboxylase, was the preferred substrate for methylation to PC. A PEMT-GFP fusion protein decorated the periphery of LD. PEMT knockdown in 3T3-L1 adipocytes correlated with increased basal triacylglycerol hydrolysis. Pemt(-/-) mice developed desensitization against adenosine-mediated inhibition of basal hydrolysis in adipose tissue, and adipocyte hypotrophy was observed in Pemt(-/-) animals on a high fat diet. Knock-out of PEMT in adipose tissue down-regulated PS synthase-1 mRNA, suggesting coordination between PE supply and converting pathways during LD biosynthesis. We conclude that two consecutive processes not previously related to LD biogenesis, (i) PE production via PS and (ii) PE conversion via PEMT, are implicated in LD formation and stability.
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http://dx.doi.org/10.1074/jbc.M111.234534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089575PMC
May 2011

Mouse brain plasmalogens are targets for hypochlorous acid-mediated modification in vitro and in vivo.

Free Radic Biol Med 2010 Dec 31;49(11):1655-65. Epub 2010 Aug 31.

Institute of Molecular Biology and Biochemistry, Center for Molecular Medicine, Medical University of Graz, Graz, Austria.

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4061399PMC
December 2010

Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12 neurons in vitro.

Free Radic Biol Med 2010 Jun 11;48(12):1588-600. Epub 2010 Mar 11.

Institute of Molecular Biology and Biochemistry, Medical University of Graz, 8010 Graz, Austria.

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.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.02.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4061462PMC
June 2010

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture.

Proteomics 2010 Jan;10(1):141-58

Institute of Molecular Biology and Biochemistry, Medical University of Graz, Austria.

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein-coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA(1), LPA(2), and LPA(3) on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal-regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA-induced global alterations in protein expression patterns a 2-D DIGE/LC-ESI-MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.
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http://dx.doi.org/10.1002/pmic.200900195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060044PMC
January 2010

Apolipoprotein A-I coating of protamine-oligonucleotide nanoparticles increases particle uptake and transcytosis in an in vitro model of the blood-brain barrier.

J Control Release 2007 Feb 28;117(3):301-11. Epub 2006 Nov 28.

Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, Harrachgasse 21, 8010 Graz, Austria.

Drug delivery to the brain is severely restricted by formation of tight junctions between adjacent brain capillary endothelial cells (BCEC). In the present study we have evaluated the effects of protamine-oligonucleotide nanoparticles (proticles) on the functional properties of primary porcine BCEC and characterized uptake and transcytosis of proticles by these cells. Proticles had no adverse effects on BCEC properties relevant to blood-brain barrier (BBB) function. Transcytosis of (125)I-labeled proticles across polarized BCEC cultures occurred in a time- and concentration-dependent manner. As apolipoproteins were suggested to enhance cellular proticle uptake, proticle coating was performed with apoA-I, the major apolipoprotein component of high density lipoproteins. Adsorption of apoA-I on the surface of proticles resulted in significantly improved uptake and transcytosis properties as compared to uncoated proticles. ApoA-I coating enhanced proticle delivery to astrocytes in an in vitro model of the BBB almost twofold. Blocking of scavenger receptor class B, type I (the prime receptor for high density lipoprotein/apoA-I that is expressed on BCEC) reduced transcytosis of apoA-I-coated proticles to levels observed for uncoated proticles. Our data indicate that apoA-I-coating of proticles could be a feasible targeting technology to improve delivery across the BBB.
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http://dx.doi.org/10.1016/j.jconrel.2006.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861216PMC
February 2007

Scavenger receptor class B, type I mediates uptake of lipoprotein-associated phosphatidylcholine by primary porcine cerebrovascular endothelial cells.

Neurosci Lett 2004 Sep;368(1):11-4

Institute of Molecular Biology and Biochemistry, Medical University Graz, Harrachgasse 21, A-8010 Graz, Austria.

We previously reported that scavenger receptor class B, type I (SR-BI) mediates uptake of lipoprotein-associated cholesteryl ester and Vitamin E by porcine brain capillary endothelial cells (pBCECs). In the present study we investigated whether SR-BI is capable of mediating phosphatidylcholine (PC) uptake by pBCECs from low- and high density lipoproteins. SR-BI-overexpressing CHO cells and pBCECs showed significantly enhanced uptake rates of PC from both lipoprotein classes. In addition, preincubation of pBCECs in the presence of both lipoprotein species resulted in a significant increase of the cellular fatty acid content, particularly linoleic and arachidonic acid. Our results suggest that uptake of lipoprotein-associated PC by the cerebrovasculature via SR-BI could generate a pool of lipids containing polyunsaturated fatty acids available for transport into deeper regions of the brain.
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http://dx.doi.org/10.1016/j.neulet.2004.04.097DOI Listing
September 2004

Effects of lipoprotein lipase on uptake and transcytosis of low density lipoprotein (LDL) and LDL-associated alpha-tocopherol in a porcine in vitro blood-brain barrier model.

J Biol Chem 2002 Aug 24;277(32):28537-44. Epub 2002 May 24.

Institute of Medical Biochemistry and Molecular Biology, University Graz, Harrachgasse 21, Graz 8010, Austria.

During the present study the contribution of lipoprotein lipase (LPL) to low density lipoprotein (LDL) holoparticle and LDL-lipid (alpha-tocopherol (alphaTocH)) turnover in primary porcine brain capillary endothelial cells (BCECs) was investigated. The addition of increasing LPL concentrations to BCECs resulted in up to 11-fold higher LDL holoparticle cell association. LPL contributed to LDL holoparticle turnover, an effect that was substantially increased in response to LDL-receptor up-regulation. The addition of LPL increased selective uptake of LDL-associated alphaTocH in BCECs up to 5-fold. LPL-dependent selective alphaTocH uptake was unaffected by the lipase inhibitor tetrahydrolipstatin but was substantially inhibited in cells where proteoglycan sulfation was inhibited by treatment with NaClO(3). Thus, selective uptake of LDL-associated alphaTocH requires interaction of LPL with heparan-sulfate proteoglycans. Although high level adenoviral overexpression of scavenger receptor BI (SR-BI) in BCECs resulted in a 2-fold increase of selective LDL-alphaTocH uptake, SR-BI did not act in a cooperative manner with LPL. Although the addition of LPL to BCEC Transwell cultures significantly increased LDL holoparticle cell association and selective uptake of LDL-associated alphaTocH, holoparticle transcytosis across this porcine blood-brain barrier (BBB) model was unaffected by the presence of LPL. An important observation during transcytosis experiments was a substantial alphaTocH depletion of LDL particles that were resecreted into the basolateral compartment. The relevance of LPL-dependent alphaTocH uptake across the BBB was confirmed in LPL-deficient mice. The absence of LPL resulted in significantly lower cerebral alphaTocH concentrations than observed in control animals.
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http://dx.doi.org/10.1074/jbc.M203989200DOI Listing
August 2002