45 results on '"Afonyushkin T"'
Search Results
2. Malondialdehyde-specific IGM antibodies define residual atherothrombotic risk
- Author
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Koller, T., primary, Afonyushkin, T., additional, Oszvar-Kozma, M., additional, Taqi, S., additional, Hoke, M., additional, and Binder, C., additional
- Published
- 2023
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3. Natural IgM attenuate formation of neutrophil extracellular traps induced by oxidation-specific epitopes in vitro and in vivo
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Ondracek, A, primary, Afonyushkin, T, additional, Taqi, S, additional, Aszlan, A, additional, Koller, T, additional, Hofbauer, T M, additional, Ozsvar-Kozma, M, additional, Sharma, S, additional, Scherz, T, additional, Beitzke, D, additional, Testori, C, additional, Lang, I M, additional, and Binder, C J, additional
- Published
- 2022
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- View/download PDF
4. Natural igm recognizing oxidation-specific epitopes block netosis induced by extracellular vesicles
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Ondracek, A.S., primary, Afonyushkin, T., additional, Taqi, S., additional, Hofbauer, T.M., additional, Kozma, M. Ozsvar, additional, Sharma, S., additional, Scherz, T., additional, Beitzke, D., additional, Testori, C., additional, Lang, I.M., additional, and Binder, C.J., additional
- Published
- 2022
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5. High-throughput screening assay identifies drugs that modulate the release of vasculo-inflammatory extracellular vesicles enriched in mitochondrial content
- Author
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Zisser, L., primary, Afonyushkin, T., additional, Puhm, F., additional, Ringler, A., additional, Kubicek, S., additional, Hacker, M., additional, and Binder, C.J., additional
- Published
- 2022
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- View/download PDF
6. Oxidized low‐density lipoprotein in inflammation‐driven thrombosis
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Obermayer, G., Afonyushkin, T., and Binder, C. J.
- Published
- 2018
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7. Pharmacological insight into the anti-inflammatory activity of sesquiterpene lactones from Neurolaena lobata (L.) R.Br. ex Cass
- Author
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McKinnon, R., Binder, M., Zupko, I., Afonyushkin, T., Lajter, I., Vasas, A., de Martin, R., Unger, C., Dolznig, H., Diaz, R., Frisch, R., Passreiter, C.M., Krupitza, G., Hohmann, J., Kopp, B., and Bochkov, V.N.
- Subjects
Lactones -- Health aspects ,Inflammation -- Care and treatment ,Materia medica, Vegetable -- Health aspects ,Compositae -- Health aspects ,Plant extracts -- Health aspects ,Biological sciences ,Health ,Science and technology - Abstract
ABSTRACT Purpose: Neurolaena lobata is a Caribbean medicinal plant used for the treatment of several conditions including inflammation. Recent data regarding potent anti-inflammatory activity of the plant and isolated sesquiterpene [...]
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- 2014
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8. P531Mitochondria of parental cells control the capacity of monocyte-derived microvesicles to activate endothelial cells
- Author
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Puhm, F, primary, Afonyushkin, T A, additional, Obermayer, G O, additional, Resch, U R, additional, Melki, I M, additional, Wagner, G W, additional, Mackman, N M, additional, Jilma, B J, additional, Boilard, E B, additional, and Binder, CJB, additional
- Published
- 2018
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9. The generation of malondialdehyde-positive microvesicles
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Puhm, F., primary, Afonyushkin, T., additional, and Binder, C., additional
- Published
- 2016
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10. 271 OXIDIZED PHOSPHOLIPIDS (OXPLS) INDUCE PRODUCTION OF STEM CELL FACTOR (SCF) AND ACTIVATION OF ITS RECEPTOR C-KIT IN ENDOTHELIAL CELLS
- Author
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Afonyushkin, T., primary, Oskolkova, O.V., additional, and Bochkov, V.N., additional
- Published
- 2011
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11. The inflammatory mediator oncostatin M induces stromal derived factor‐1 in human adult cardiac cells
- Author
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Hohensinner, P. J., primary, Kaun, C., additional, Rychli, K., additional, Niessner, A., additional, Pfaffenberger, S., additional, Rega, G., additional, Furnkranz, A., additional, Uhrin, P., additional, Zaujec, J., additional, Afonyushkin, T., additional, Bochkov, V. N., additional, Maurer, G., additional, Huber, K., additional, and Wojta, J., additional
- Published
- 2008
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12. Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB
- Author
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Afonyushkin, T., primary
- Published
- 2005
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13. The inflammatory mediator oncostatin M induces stromal derived factor-1 in human adult cardiac cells.
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Hohensinner, P. J., Kaun, C., Rychli, K., Niessner, A., Pfaffenberger, S., Rega, G., Furnkranz, A., Uhrin, P., Zaujec, J., Afonyushkin, T., Bochkov, V. N., Maurer, G., Huber, K., and Wojta, J.
- Subjects
INFLAMMATORY mediators ,CHEMOKINES ,HEART cells ,STEM cells ,LIGANDS (Biochemistry) ,LABORATORY mice - Abstract
Stromal derived factor 1 (SDF-1) is a CXC chemokine important in the homing process of stem cells to injured tissue. It has been implicated in healing and tissue repair. Growing evidence suggests that the glycoprotein-130 (gp130) ligand family is involved in repair processes in the heart. The aim of our study was to determine whether gp130 ligands could affect SDF-1 expression in cardiac cells. Human adult cardiac myocytes (HACMs) and fibroblasts (HACFs) were treated with gp130 ligands. Protein and mRNA levels of SDF-1 were determined using ELISA and RT-PCR, respectively, mRNA levels of SDF-1 were determined in human and mouse heart samples by RT-PCR. HACMs and HACFs constitutively express SDF-1, which was significantly up-regulated by the gp130 ligand oncostatin M (OSM). This effect was counteracted by a p38 inhibitor and to a lesser extent by a PI3K inhibitor, mRNA expression of SDF-1 in hearts of mice injected with OSM increased significantly. Levels of OSM and SDF-1 mRNA correlated significantly in human failing hearts. Our data, showing that OSM induces SDF-1 protein secretion in human cardiac cells in vitro and murine hearts in vivo, suggest that OSM via the induction of SDF-1 might play a key role in repair and tissue regeneration. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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14. Malondialdehyde-specific natural IgM inhibit NETosis triggered by culprit site-derived extracellular vesicles from myocardial infarction patients.
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Ondracek AS, Afonyushkin T, Aszlan A, Taqi S, Koller T, Artner T, Porsch F, Resch U, Sharma S, Scherz T, Spittler A, Haertinger M, Hofbauer TM, Ozsvar-Kozma M, Seidl V, Beitzke D, Krueger M, Testori C, Lang IM, and Binder CJ
- Published
- 2024
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15. Low Concentrations of Oxidized Phospholipids Increase Stress Tolerance of Endothelial Cells.
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Mauerhofer C, Afonyushkin T, Oskolkova OV, Hellauer K, Gesslbauer B, Schmerda J, Ke Y, Zimmer A, Birukova AA, Birukov KG, and Bochkov V
- Abstract
Oxidized phospholipids (OxPLs) are generated by enzymatic or autooxidation of esterified polyunsaturated fatty acids (PUFAs) residues. OxPLs are present in circulation and atherosclerotic plaques where they are thought to induce predominantly proinflammatory and toxic changes in endothelial (ECs) and other cell types. Unexpectedly, we found that low concentrations of OxPLs were not toxic but protected ECs from stress induced by serum deprivation or cytostatic drugs. The protective effect was observed in ECs obtained from different vessels and was monitored using a variety of readouts based on different biological and chemical principles. Analysis of the structure−activity relationship identified oxidized or missing fatty acid residue (OxPLs or Lyso-PLs, respectively) as a prerequisite for the protective action of a PL. Protective OxPLs or Lyso-PLs acquired detergent-like properties and formed in solution aggregates <10 nm in diameter (likely micelles), which were in striking contrast with large aggregates (>1000 nm, likely multilayer liposomes) produced by nonoxidized precursor PLs. Because surfactants, OxPLs, and Lyso-PLs are known to extract membrane cholesterol, we tested if this effect might trigger the protection of endothelial cells. The protective action of OxPLs and Lyso-PLs was inhibited by cotreatment with cholesterol and mimicked by cholesterol-binding beta-cyclodextrin but not inactive α-cyclodextrin. Wide-scale mRNA expression analysis in four types of ECs showed the induction of genes encoding for heat shock proteins (HSPs) and secreted prosurvival peptides and proteins. Inducers of HSPs, chemical chaperones, and pure prosurvival factors mimicked the protective action of OxPLs/Lyso-PLs. We hypothesize that oxidation changes the physicochemical properties of PLs, thus promoting membrane cholesterol redistribution or extraction leading to the expression of intra- and extracellular prosurvival factors.
- Published
- 2022
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16. Association of Lipoproteins with Neutrophil Extracellular Traps in Patients with Abdominal Aortic Aneurysm.
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Brandau A, Ibrahim N, Klopf J, Hayden H, Ozsvar-Kozma M, Afonyushkin T, Bleichert S, Fuchs L, Watzinger V, Nairz V, Manville E, Kessler V, Stangl H, Eilenberg W, Neumayer C, and Brostjan C
- Abstract
Neutrophil extracellular traps (NETs) are DNA-protein structures released by neutrophils in response to various stimuli, including oxidized, low-density lipoprotein (oxLDL). Accumulating evidence suggests a role for NETs in the pathogenesis of abdominal aortic aneurysm (AAA). In this study, we investigated the potential association of lipoprotein particles and NETs in AAA in comparison to non-AAA control groups. The concentrations of neutrophil myeloperoxidase (MPO), the NET parameters citrullinated histone H3 (citH3) and circulating cell-free DNA (cfDNA), as well as of blood lipids were determined in plasma or serum of patients with AAA ( n = 40), peripheral artery occlusive disease (PAD; n = 40) and healthy donors ( n = 29). A sandwich ELISA detecting oxidized phosphatidylcholine in association with apolipoprotein B-100 (oxPL/apoB) was applied to measure oxidized phospholipids in circulation. The effect of lipoparticles on NET formation was tested using a DNA release assay with isolated human neutrophils. Plasma MPO, citH3 and cfDNA levels were significantly increased in AAA patients in comparison to healthy donors and PAD patients. Plasma concentrations of citH3 positively correlated with serum oxPL/apoB in AAA patients. In functional in vitro assays, the addition of oxLDL induced NET formation in pre-stimulated neutrophils. In conclusion, our data suggest a promoting role of oxLDL on NET formation in AAA patients.
- Published
- 2022
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17. APRIL limits atherosclerosis by binding to heparan sulfate proteoglycans.
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Tsiantoulas D, Eslami M, Obermayer G, Clement M, Smeets D, Mayer FJ, Kiss MG, Enders L, Weißer J, Göderle L, Lambert J, Frommlet F, Mueller A, Hendrikx T, Ozsvar-Kozma M, Porsch F, Willen L, Afonyushkin T, Murphy JE, Fogelstrand P, Donzé O, Pasterkamp G, Hoke M, Kubicek S, Jørgensen HF, Danchin N, Simon T, Scharnagl H, März W, Borén J, Hess H, Mallat Z, Schneider P, and Binder CJ
- Subjects
- Animals, B-Cell Maturation Antigen metabolism, Binding Sites, Cardiovascular Diseases blood, Cardiovascular Diseases mortality, Female, Humans, Male, Mice, Mice, Inbred C57BL, Protein Binding, Transmembrane Activator and CAML Interactor Protein metabolism, Tumor Necrosis Factor Ligand Superfamily Member 13 blood, Tumor Necrosis Factor Ligand Superfamily Member 13 deficiency, Atherosclerosis metabolism, Atherosclerosis prevention & control, Heparan Sulfate Proteoglycans metabolism, Tumor Necrosis Factor Ligand Superfamily Member 13 metabolism
- Abstract
Atherosclerotic cardiovascular disease causes heart attacks and strokes, which are the leading causes of mortality worldwide
1 . The formation of atherosclerotic plaques is initiated when low-density lipoproteins bind to heparan-sulfate proteoglycans (HSPGs)2 and become trapped in the subendothelial space of large and medium size arteries, which leads to chronic inflammation and remodelling of the artery wall2 . A proliferation-inducing ligand (APRIL) is a cytokine that binds to HSPGs3 , but the physiology of this interaction is largely unknown. Here we show that genetic ablation or antibody-mediated depletion of APRIL aggravates atherosclerosis in mice. Mechanistically, we demonstrate that APRIL confers atheroprotection by binding to heparan sulfate chains of heparan-sulfate proteoglycan 2 (HSPG2), which limits the retention of low-density lipoproteins, accumulation of macrophages and formation of necrotic cores. Indeed, antibody-mediated depletion of APRIL in mice expressing heparan sulfate-deficient HSPG2 had no effect on the development of atherosclerosis. Treatment with a specific anti-APRIL antibody that promotes the binding of APRIL to HSPGs reduced experimental atherosclerosis. Furthermore, the serum levels of a form of human APRIL protein that binds to HSPGs, which we termed non-canonical APRIL (nc-APRIL), are associated independently of traditional risk factors with long-term cardiovascular mortality in patients with atherosclerosis. Our data reveal properties of APRIL that have broad pathophysiological implications for vascular homeostasis., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)- Published
- 2021
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18. Extracellular vesicles are associated with C-reactive protein in sepsis.
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Fendl B, Weiss R, Eichhorn T, Linsberger I, Afonyushkin T, Puhm F, Binder CJ, Fischer MB, and Weber V
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- Case-Control Studies, Cells, Cultured, Humans, Inflammation metabolism, Sepsis metabolism, C-Reactive Protein metabolism, Extracellular Vesicles metabolism, Inflammation diagnosis, Monocytes metabolism, Sepsis diagnosis
- Abstract
There is increasing evidence that C-reactive protein (CRP) can mediate inflammatory reactions following the transformation of functionally inert pentameric CRP (pCRP) into its structural isoform pCRP* and into monomeric CRP (mCRP). This conversion can occur on the membranes of apoptotic or activated cells or on extracellular vesicles (EVs) shed from the cell surface. Here, we characterized the association of CRP with EVs in plasma from sepsis patients using flow cytometry, and found highly elevated levels of total EV counts and CRP
+ EVs as compared to healthy individuals. We further assessed the ability of PentraSorb CRP, an extracorporeal device for the adsorption of CRP, to deplete free CRP and CRP+ EVs. Treatment of septic plasma with the adsorbent in vitro resulted in almost complete removal of both, free CRP and CRP+ EVs, while total EV counts remained largely unaffected, indicating the detachment of CRP from the EV surface. EVs from septic plasma elicited a release of interleukin-8 from cultured human monocytes, which was significantly reduced by adsorbent treatment prior to EV isolation. Our findings provide evidence that CRP+ EVs exhibit pro-inflammatory characteristics and can contribute to the spreading of inflammation throughout the circulation on top of their pro-coagulant activity.- Published
- 2021
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19. Natural IgM antibodies inhibit microvesicle-driven coagulation and thrombosis.
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Obermayer G, Afonyushkin T, Göderle L, Puhm F, Schrottmaier W, Taqi S, Schwameis M, Ay C, Pabinger I, Jilma B, Assinger A, Mackman N, and Binder CJ
- Subjects
- Animals, Blood Platelets cytology, Blood Platelets metabolism, Humans, Immunoglobulin M analysis, Mice, Inbred C57BL, Thrombosis blood, Mice, Blood Coagulation, Cell-Derived Microparticles metabolism, Immunoglobulin M metabolism, Thrombosis metabolism
- Abstract
Thrombosis and its associated complications are a major cause of morbidity and mortality worldwide. Microvesicles (MVs), a class of extracellular vesicles, are increasingly recognized as mediators of coagulation and biomarkers of thrombotic risk. Thus, identifying factors targeting MV-driven coagulation may help in the development of novel antithrombotic treatments. We have previously identified a subset of circulating MVs that is characterized by the presence of oxidation-specific epitopes and bound by natural immunoglobulin M (IgM) antibodies targeting these structures. This study investigated whether natural IgM antibodies, which are known to have important anti-inflammatory housekeeping functions, inhibit the procoagulatory properties of MVs. We found that the extent of plasma coagulation is inversely associated with the levels of both free and MV-bound endogenous IgM. Moreover, the oxidation epitope-specific natural IgM antibody LR04, which recognizes malondialdehyde adducts, reduced MV-dependent plasmatic coagulation and whole blood clotting without affecting thrombocyte aggregation. Intravenous injection of LR04 protected mice from MV-induced pulmonary thrombosis. Of note, LR04 competed the binding of coagulation factor X/Xa to MVs, providing a mechanistic explanation for its anticoagulatory effect. Thus, our data identify natural IgM antibodies as hitherto unknown modulators of MV-induced coagulation in vitro and in vivo and their prognostic and therapeutic potential in the management of thrombosis., (© 2021 by The American Society of Hematology.)
- Published
- 2021
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20. Mitochondria Are a Subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells.
- Author
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Puhm F, Afonyushkin T, Resch U, Obermayer G, Rohde M, Penz T, Schuster M, Wagner G, Rendeiro AF, Melki I, Kaun C, Wojta J, Bock C, Jilma B, Mackman N, Boilard E, and Binder CJ
- Subjects
- Adult, Cells, Cultured, Endothelial Cells drug effects, Endothelial Cells immunology, Extracellular Vesicles drug effects, Extracellular Vesicles immunology, Human Umbilical Vein Endothelial Cells drug effects, Human Umbilical Vein Endothelial Cells immunology, Human Umbilical Vein Endothelial Cells metabolism, Humans, Lipopolysaccharides toxicity, Male, Mitochondria drug effects, Mitochondria immunology, Monocytes drug effects, Monocytes immunology, Young Adult, Endothelial Cells metabolism, Extracellular Vesicles metabolism, Interferon Type I biosynthesis, Mitochondria metabolism, Monocytes metabolism, Tumor Necrosis Factor-alpha biosynthesis
- Abstract
Rationale: Extracellular vesicles, including microvesicles, are increasingly recognized as important mediators in cardiovascular disease. The cargo and surface proteins they carry are considered to define their biological activity, including their inflammatory properties. Monocyte to endothelial cell signaling is a prerequisite for the propagation of inflammatory responses. However, the contribution of microvesicles in this process is poorly understood., Objective: To elucidate the mechanisms by which microvesicles derived from activated monocytic cells exert inflammatory effects on endothelial cells., Methods and Results: LPS (lipopolysaccharide)-stimulated monocytic cells release free mitochondria and microvesicles with mitochondrial content as demonstrated by flow cytometry, quantitative polymerase chain reaction, Western Blot, and transmission electron microscopy. Using RNAseq analysis and quantitative reverse transcription-polymerase chain reaction, we demonstrated that both mitochondria directly isolated from and microvesicles released by LPS-activated monocytic cells, as well as circulating microvesicles isolated from volunteers receiving low-dose LPS-injections, induce type I IFN (interferon), and TNF (tumor necrosis factor) responses in endothelial cells. Depletion of free mitochondria significantly reduced the ability of these microvesicles to induce type I IFN and TNF-dependent genes. We identified mitochondria-associated TNFα and RNA from stressed mitochondria as major inducers of these responses. Finally, we demonstrated that the proinflammatory potential of microvesicles and directly isolated mitochondria were drastically reduced when they were derived from monocytic cells with nonrespiring mitochondria or monocytic cells cultured in the presence of pyruvate or the mitochondrial reactive oxygen species scavenger MitoTEMPO., Conclusions: Mitochondria and mitochondria embedded in microvesicles constitute a major subset of extracellular vesicles released by activated monocytes, and their proinflammatory activity on endothelial cells is determined by the activation status of their parental cells. Thus, mitochondria may represent critical intercellular mediators in cardiovascular disease and other inflammatory settings associated with type I IFN and TNF signaling.
- Published
- 2019
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21. Oxidized phospholipids stimulate production of stem cell factor via NRF2-dependent mechanisms.
- Author
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Afonyushkin T, Oskolkova OV, and Bochkov VN
- Subjects
- Animals, Aorta pathology, Apolipoproteins E deficiency, Human Umbilical Vein Endothelial Cells metabolism, Human Umbilical Vein Endothelial Cells pathology, Humans, Male, Mice, Mice, Knockout, ApoE, NF-E2-Related Factor 2 genetics, Oxidation-Reduction, Phosphatidylcholines genetics, Stem Cell Factor genetics, Aorta metabolism, Gene Expression Regulation, NF-E2-Related Factor 2 metabolism, Phosphatidylcholines metabolism, Stem Cell Factor biosynthesis
- Abstract
Receptor tyrosine kinase c-Kit and its ligand stem cell factor (SCF) regulate resident vascular wall cells and recruit circulating progenitors. We tested whether SCF may be induced by oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) known to accumulate in atherosclerotic vessels. Gene expression analysis demonstrated OxPAPC-induced upregulation of SCF mRNA and protein in different types of endothelial cells (ECs). Elevated levels of SCF mRNA were observed in aortas of ApoE
-/- knockout mice. ECs produced biologically active SCF because conditioned medium from OxPAPC-treated cells stimulated activation (phosphorylation) of c-Kit in naïve ECs. Induction of SCF by OxPAPC was inhibited by knocking down transcription factor NRF2. Inhibition or stimulation of NRF2 by pharmacological or molecular tools induced corresponding changes in SCF expression. Finally, we observed decreased levels of SCF mRNA in aortas of NRF2 knockout mice. We characterize OxPLs as a novel pathology-associated stimulus inducing expression of SCF in endothelial cells. Furthermore, our data point to transcription factor NRF2 as a major mediator of OxPL-induced upregulation of SCF. This mechanism may represent one of the facets of pleiotropic action of NRF2 in vascular wall.- Published
- 2018
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22. Extracellular Vesicles Act as Messengers of Macrophages Sensing Atherogenic Stimuli.
- Author
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Afonyushkin T and Binder CJ
- Subjects
- Atherosclerosis, Cell Movement, Humans, Macrophages, Extracellular Vesicles, MicroRNAs
- Published
- 2018
- Full Text
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23. Anti-Inflammatory Effects of OxPAPC Involve Endothelial Cell-Mediated Generation of LXA4.
- Author
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Ke Y, Zebda N, Oskolkova O, Afonyushkin T, Berdyshev E, Tian Y, Meng F, Sarich N, Bochkov VN, Wang JM, Birukova AA, and Birukov KG
- Subjects
- Acute Lung Injury prevention & control, Animals, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Cells, Cultured, Humans, Lipoxins pharmacology, Lipoxins therapeutic use, Mice, Mice, Inbred C57BL, Mice, Knockout, Phosphatidylcholines pharmacology, Treatment Outcome, Acute Lung Injury metabolism, Anti-Inflammatory Agents, Non-Steroidal therapeutic use, Endothelial Cells drug effects, Endothelial Cells metabolism, Lipoxins metabolism, Phosphatidylcholines therapeutic use
- Abstract
Rationale: Oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) generates a group of bioactive oxidized phospholipid products with a broad range of biological activities. Barrier-enhancing and anti-inflammatory effects of OxPAPC on pulmonary endothelial cells are critical for prevention of acute lung injury caused by bacterial pathogens or excessive mechanical ventilation. Anti-inflammatory properties of OxPAPC are associated with its antagonistic effects on Toll-like receptors and suppression of RhoA GTPase signaling., Objective: Because OxPAPC exhibits long-lasting anti-inflammatory and lung-protective effects even after single administration in vivo, we tested the hypothesis that these effects may be mediated by additional mechanisms, such as OxPAPC-dependent production of anti-inflammatory and proresolving lipid mediator, lipoxin A4 (LXA4)., Methods and Results: Mass spectrometry and ELISA assays detected significant accumulation of LXA4 in the lungs of OxPAPC-treated mice and in conditioned medium of OxPAPC-exposed pulmonary endothelial cells. Administration of LXA4 reproduced anti-inflammatory effect of OxPAPC against tumor necrosis factor-α in vitro and in the animal model of lipopolysaccharide-induced lung injury. The potent barrier-protective and anti-inflammatory effects of OxPAPC against tumor necrosis factor-α and lipopolysaccharide challenge were suppressed in human pulmonary endothelial cells with small interfering RNA-induced knockdown of LXA4 formyl peptide receptor-2 (FPR2/ALX) and in mFPR2
-/- (mouse formyl peptide receptor 2) mice lacking the mouse homolog of human FPR2/ALX., Conclusions: This is the first demonstration that inflammation- and injury-associated phospholipid oxidation triggers production of anti-inflammatory and proresolution molecules, such as LXA4. This lipid mediator switch represents a novel mechanism of OxPAPC-assisted recovery of inflamed lung endothelium., (© 2017 American Heart Association, Inc.)- Published
- 2017
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24. Alterations of lung microbiota in a mouse model of LPS-induced lung injury.
- Author
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Poroyko V, Meng F, Meliton A, Afonyushkin T, Ulanov A, Semenyuk E, Latif O, Tesic V, Birukova AA, and Birukov KG
- Subjects
- Animals, Base Sequence, Bronchoalveolar Lavage Fluid microbiology, Brucellaceae genetics, Brucellaceae isolation & purification, DNA, Bacterial genetics, Disease Models, Animal, Lipopolysaccharides toxicity, Mice, Mice, Inbred C57BL, RNA, Ribosomal, 16S genetics, Sequence Analysis, DNA, Xanthomonadaceae genetics, Xanthomonadaceae isolation & purification, Lung microbiology, Lung Injury microbiology, Microbiota drug effects, Respiratory Distress Syndrome microbiology
- Abstract
Acute lung injury (ALI) and the more severe acute respiratory distress syndrome are common responses to a variety of infectious and noninfectious insults. We used a mouse model of ALI induced by intratracheal administration of sterile bacterial wall lipopolysaccharide (LPS) to investigate the changes in innate lung microbiota and study microbial community reaction to lung inflammation and barrier dysfunction induced by endotoxin insult. One group of C57BL/6J mice received LPS via intratracheal injection (n = 6), and another received sterile water (n = 7). Bronchoalveolar lavage (BAL) was performed at 72 h after treatment. Bacterial DNA was extracted and used for qPCR and 16S rRNA gene-tag (V3-V4) sequencing (Illumina). The bacterial load in BAL from ALI mice was increased fivefold (P = 0.03). The community complexity remained unchanged (Simpson index, P = 0.7); the Shannon diversity index indicated the increase of community evenness in response to ALI (P = 0.07). Principal coordinate analysis and analysis of similarity (ANOSIM) test (P = 0.005) revealed a significant difference between microbiota of control and ALI groups. Bacteria from families Xanthomonadaceae and Brucellaceae increased their abundance in the ALI group as determined by Metastats test (P < 0.02). In concordance with the 16s-tag data, Stenotrohomonas maltophilia (Xanthomonadaceae) and Ochrobactrum anthropi (Brucellaceae) were isolated from lungs of mice from both groups. Metabolic profiling of BAL detected the presence of bacterial substrates suitable for both isolates. Additionally, microbiota from LPS-treated mice intensified IL-6-induced lung inflammation in naive mice. We conclude that the morbid transformation of ALI microbiota was attributed to the set of inborn opportunistic pathogens thriving in the environment of inflamed lung, rather than the external infectious agents., (Copyright © 2015 the American Physiological Society.)
- Published
- 2015
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25. Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies.
- Author
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Tsiantoulas D, Perkmann T, Afonyushkin T, Mangold A, Prohaska TA, Papac-Milicevic N, Millischer V, Bartel C, Hörkkö S, Boulanger CM, Tsimikas S, Fischer MB, Witztum JL, Lang IM, and Binder CJ
- Subjects
- Acute Coronary Syndrome immunology, Acute Coronary Syndrome metabolism, Adult, Female, Humans, Male, Malondialdehyde metabolism, Oxidation-Reduction, Cell-Derived Microparticles immunology, Cell-Derived Microparticles metabolism, Epitopes immunology, Immunoglobulin M immunology
- Abstract
Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA(+) MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE(+) MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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26. MicroRNA miR-320a modulates induction of HO-1, GCLM and OKL38 by oxidized phospholipids in endothelial cells.
- Author
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Schrottmaier WC, Oskolkova OV, Schabbauer G, and Afonyushkin T
- Subjects
- Animals, Apolipoproteins E genetics, Apoptosis Regulatory Proteins, Enzyme-Linked Immunosorbent Assay, Female, Glutamate-Cysteine Ligase genetics, Human Umbilical Vein Endothelial Cells, Humans, Male, Mice, Mice, Inbred C57BL, MicroRNAs genetics, Oligonucleotide Array Sequence Analysis, Oligonucleotides chemistry, Oxidative Stress, Oxygen chemistry, RNA, Messenger metabolism, Sulfoxides, Transfection, Glutamate-Cysteine Ligase metabolism, Heme Oxygenase-1 metabolism, Isothiocyanates chemistry, MicroRNAs metabolism, NF-E2-Related Factor 2 metabolism, Phospholipids chemistry, Proteins metabolism
- Abstract
Objective: Oxidized phospholipids (OxPLs), which are highly abundant in atherosclerotic lesions, are known to induce electrophilic stress response (ESR). ESR induces cytoprotective genes via the NF-E2-related factor 2 (NRF2) transcription factor. In order to get further insight into the mechanisms of ESR, we studied the role of microRNA (miR)-320a in induction of NRF2-dependent genes by OxPLs., Methods: Microarray profiling and qRT-PCR methods were used for measurements of mRNA and miRNA levels. miR-320a levels were changed by transfection with synthetic oligonucleotides. Protein analysis was performed by Western blotting. The functional activity of NRF2 was measured by DNA-binding ELISA., Results: Oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) induced miR-320a in endothelial cells. Induction of HO-1, OKL38 and GCLM mRNAs by OxPAPC and sulforaphane was attenuated upon knockdown of miR-320a. In contrast, transfection of ECs with miR-320a mimic oligonucleotide potentiated the effects of OxPAPC and sulforaphane on induction of HO-1, OKL38 and GCLM mRNAs. OxPAPC-induced p38 activation, levels of NRF2 protein and its ability to bind to consensus NRF2 DNA binding site were elevated in ECs transfected with miR-320a mimic. miR-320a positively regulated induction of VEGF mRNA by OxPAPC. Levels of miR-320a and HO-1 and OKL38 mRNAs were elevated in aortas of ApoE knockout mice fed with high fat diet. Manipulation of miR-320a level in ECs did not affect ability of OxPAPC to induce IL-8, COX-2 and MCP-1., Conclusion: miR-320a plays important role in induction of expression of HO-1, GCLM and OKL38 upon ESR induced either by OxPAPC or sulforaphane. These observations propose a general role of miR-320a in control of ESR induced by different electrophilic agents., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2014
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27. Retinal pigment epithelium cells produce VEGF in response to oxidized phospholipids through mechanisms involving ATF4 and protein kinase CK2.
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Pollreisz A, Afonyushkin T, Oskolkova OV, Gruber F, Bochkov VN, and Schmidt-Erfurth U
- Subjects
- Activating Transcription Factor 4 biosynthesis, Blotting, Western, Casein Kinase II biosynthesis, Cells, Cultured, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Humans, Macular Degeneration genetics, Macular Degeneration metabolism, Macular Degeneration pathology, Mass Spectrometry, Oxidation-Reduction, Real-Time Polymerase Chain Reaction, Retinal Pigment Epithelium pathology, Vascular Endothelial Growth Factor A biosynthesis, Activating Transcription Factor 4 genetics, Casein Kinase II genetics, Phospholipids metabolism, RNA, Messenger genetics, Retinal Pigment Epithelium metabolism, Up-Regulation, Vascular Endothelial Growth Factor A genetics
- Abstract
Oxidized phospholipids (OxPLs) are pleiotropic lipid mediators known to induce proangiogenic and proinflammatory cellular effects that are increasingly recognized to be involved in a number of physiologic and pathologic processes in the retina. Immunohistochemical studies have detected OxPLs in retinal structures, such as retinal pigment epithelium (RPE) or photoreceptor cells. This study analyzed whether OxPLs could play a role in upregulation of VEGF, which is a cause of pathological neovascularization characteristic of eye diseases such as age-related macular degeneration. We confirmed accumulation of OxPLs in the eye using reversed-phase liquid chromatography coupled to mass spectrometry. Multiple species of oxidized phosphatidylcholines (OxPCs) were detected in human vitreous, including biologically active fragmented species POVPC, PGPC, PONPC and PAzPC. In in vitro experiments human fetal RPE and primary RPE cells were stimulated with OxPLs. Primary RPE cells were transfected with small interfering RNAs targeting ATF4. mRNA levels of VEGF in fetal and primary RPE cells were determined by real-time quantitative PCR. VEGF protein concentrations were measured in culture medium by ELISA. We found that OxPCs and other classes of OxPLs upregulated the expression of VEGF in fetal and primary RPE cells, which critically depended on ATF4. In addition, upregulation of VEGF in primary RPE cells was blocked by a chemical inhibitor of protein kinase CK2 known to suppress induction of ATF4 and VEGF by OxPLs. Our data show that different species of OxPLs, which are present in the human eye are capable of stimulating expression of VEGF in fetal and primary RPE cells via ATF4-dependent mechanisms., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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28. Permissive role of miR-663 in induction of VEGF and activation of the ATF4 branch of unfolded protein response in endothelial cells by oxidized phospholipids.
- Author
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Afonyushkin T, Oskolkova OV, and Bochkov VN
- Subjects
- Endothelial Cells metabolism, Human Umbilical Vein Endothelial Cells, Humans, MicroRNAs antagonists & inhibitors, Oxidation-Reduction, Phosphatidylcholines, Phospholipids metabolism, Tunicamycin pharmacology, Activating Transcription Factor 4 metabolism, MicroRNAs physiology, Unfolded Protein Response drug effects, Vascular Endothelial Growth Factor A biosynthesis
- Abstract
Objective: Atherosclerotic lesions contain high concentrations of oxidized phospholipids (OxPLs) known to induce VEGF via the ATF4 arm of unfolded protein response (UPR), and to promote angiogenic reactions thus potentially contributing to the progression and destabilization of atherosclerotic plaques. In order to get further insights into the mechanisms of cellular stress-induced angiogenesis we studied the role of a specific microRNA (miR-663) in the mechanisms of VEGF induction by OxPLs and inducers of UPR., Methods: miRNA and mRNA levels were determined using microarray profiling and qRT-PCR methods. Proteins were analyzed by Western blotting. miR-663 levels were changed by transfecting cells with synthetic oligonucleotides., Results: OxPAPC elevated miR-663 in two types of human endothelial cells (ECs). Knockdown of miR-663 inhibited upregulation of VEGF mRNA in ECs treated by OxPAPC, OxPAPS or OxPAPA. In addition, silencing of miR-663 suppressed upregulation by OxPAPC of ATF4 mRNA and protein, as well as a downstream gene TRIB. Similarly to the inhibition of OxPAPC effects, knockdown of miR-663 suppressed elevation of ATF4, VEGF and TRIB in response to another inducer of UPR, tunicamycin. Overexpression of miR-663 reversed the inhibition of VEGF induction by miR-663 inhibitor., Conclusion: miR-663 is critically important for 2 key events induced in ECs by stress agents and oxidized lipids, namely induction of transcription factor ATF4 and its downstream gene VEGF. These findings allow hypothesizing that miR-663 plays a general role in control of the ATF4 branch of UPR induced by different agents., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2012
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29. Pharmacophore-based discovery of FXR-agonists. Part II: identification of bioactive triterpenes from Ganoderma lucidum.
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Grienke U, Mihály-Bison J, Schuster D, Afonyushkin T, Binder M, Guan SH, Cheng CR, Wolber G, Stuppner H, Guo DA, Bochkov VN, and Rollinger JM
- Subjects
- Animals, HEK293 Cells, Hep G2 Cells, Humans, Mice, Stereoisomerism, Structure-Activity Relationship, Receptors, Cytoplasmic and Nuclear agonists, Reishi chemistry, Triterpenes isolation & purification, Triterpenes pharmacology
- Abstract
The farnesoid X receptor (FXR) belonging to the metabolic subfamily of nuclear receptors is a ligand-induced transcriptional activator. Its central function is the physiological maintenance of bile acid homeostasis including the regulation of glucose and lipid metabolism. Accessible structural information about its ligand-binding domain renders FXR an attractive target for in silico approaches. Integrated to natural product research these computational tools assist to find novel bioactive compounds showing beneficial effects in prevention and treatment of, for example, the metabolic syndrome, dyslipidemia, atherosclerosis, and type 2 diabetes. Virtual screening experiments of our in-house Chinese Herbal Medicine database with structure-based pharmacophore models, previously generated and validated, revealed mainly lanostane-type triterpenes of the TCM fungus Ganoderma lucidum Karst. as putative FXR ligands. To verify the prediction of the in silico approach, two Ganoderma fruit body extracts and compounds isolated thereof were pharmacologically investigated. Pronounced FXR-inducing effects were observed for the extracts at a concentration of 100 μg/mL. Intriguingly, five lanostanes out of 25 secondary metabolites from G. lucidum, that is, ergosterol peroxide (2), lucidumol A (11), ganoderic acid TR (12), ganodermanontriol (13), and ganoderiol F (14), dose-dependently induced FXR in the low micromolar range in a reporter gene assay. To rationalize the binding interactions, additional pharmacophore profiling and molecular docking studies were performed, which allowed establishing a first structure-activity relationship of the investigated triterpenes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)
- Published
- 2011
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30. Oncostatin M-enhanced vascular endothelial growth factor expression in human vascular smooth muscle cells involves PI3K-, p38 MAPK-, Erk1/2- and STAT1/STAT3-dependent pathways and is attenuated by interferon-γ.
- Author
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Demyanets S, Kaun C, Rychli K, Pfaffenberger S, Kastl SP, Hohensinner PJ, Rega G, Katsaros KM, Afonyushkin T, Bochkov VN, Paireder M, Huk I, Maurer G, Huber K, and Wojta J
- Subjects
- Adult, Aged, Atherosclerosis metabolism, Cells, Cultured, Coronary Vessels metabolism, Female, Humans, Interleukin-10 metabolism, Interleukin-4 metabolism, MAP Kinase Signaling System, Male, Mitogen-Activated Protein Kinase 1 metabolism, Phosphatidylinositol 3-Kinases metabolism, RNA, Messenger metabolism, STAT1 Transcription Factor metabolism, STAT3 Transcription Factor metabolism, Up-Regulation, p38 Mitogen-Activated Protein Kinases metabolism, Interferon-gamma metabolism, Muscle, Smooth, Vascular metabolism, Myocytes, Smooth Muscle metabolism, Oncostatin M metabolism, Vascular Endothelial Growth Factor A metabolism
- Abstract
The pleiotropic cytokine oncostatin M (OSM), a member of the glycoprotein (gp)130 ligand family, plays a key role in inflammation and cardiovascular disease. As inflammation precedes and accompanies pathological angiogenesis, we investigated the effect of OSM and other gp130 ligands on vascular endothelial growth factor (VEGF) production in human vascular smooth muscle cells (SMC). Human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) were treated with different gp130 ligands. VEGF protein was determined by ELISA. Specific mRNA was detected by RT-PCR. Western blotting was performed for signal transducers and activators of transcription1 (STAT1), STAT3, Akt and p38 mitogen-activated protein kinase (p38 MAPK). OSM mRNA and VEGF mRNA expression was analyzed in human carotid endaterectomy specimens from 15 patients. OSM increased VEGF production in both HCASMC and HASMC derived from different donors. OSM upregulated VEGF and OSM receptor-specific mRNA in these cells. STAT3 inhibitor WP1066, p38 MAPK inhibitors SB-202190 and BIRB 0796, extracellular signal-regulated kinase1/2 (Erk1/2) inhibitor U0126, and phosphatidylinositol 3-kinase (PI3K) inhibitors LY-294002 and PI-103 reduced OSM-induced VEGF synthesis. We found OSM expression in human atherosclerotic lesions where OSM mRNA correlated with VEGF mRNA expression. Interferon-γ (IFN-γ), but not IL-4 or IL-10, reduced OSM-induced VEGF production in vascular SMC. Our findings that OSM, which is present in human atherosclerotic lesions and correlates with VEGF expression, stimulates production of VEGF by human coronary artery and aortic SMC indicate that OSM could contribute to plaque angiogenesis and destabilization. IFN-γ reduced OSM-induced VEGF production by vascular SMC.
- Published
- 2011
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31. Involvement of CK2 in activation of electrophilic genes in endothelial cells by oxidized phospholipids.
- Author
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Afonyushkin T, Oskolkova OV, Binder BR, and Bochkov VN
- Subjects
- Activating Transcription Factor 4 genetics, Activating Transcription Factor 4 metabolism, Casein Kinase II genetics, Casein Kinase II metabolism, Cells, Cultured, Endothelial Cells metabolism, Gene Silencing, Humans, NF-E2-Related Factor 2 genetics, NF-E2-Related Factor 2 metabolism, Oxidation-Reduction, Oxygen metabolism, Up-Regulation, Vascular Endothelial Growth Factor A genetics, Vascular Endothelial Growth Factor A metabolism, Casein Kinase II physiology, Endothelial Cells enzymology, Phospholipids metabolism
- Abstract
Oxidized phospholipids (OxPLs) are increasingly recognized as pleiotropic lipid mediators demonstrating a variety of biological activities. In particular, OxPLs induce electrophilic stress response and stimulate expression of NF-E2-related factor 2 (NRF2)-dependent genes. The mechanisms of NRF2 upregulation in response to OxPLs, however, are incompletely understood. Here we show that upregulation of NRF2 by OxPLs depends on the activity of the CK2 protein kinase. Inactivation of CK2 by chemical inhibitors or gene silencing resulted in diminished accumulation of NRF2 and its target genes, GCLM, HMOX1, and NQO1, downstream in response to OxPLs. Furthermore, inhibition of CK2 suppressed NRF2-dependent induction of ATF4 and its downstream gene VEGF. Thus, inactivation of CK2 in OxPL-treated endothelial cells results in inhibition of the NRF2-ATF4-VEGF axis and is likely to produce antiangiogenic effects. This work characterizes novel cross-talk between CK2 and cellular stress pathways, which may provide additional insights into the mechanisms of beneficial action and side-effects of CK2 inhibitors.
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- 2011
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32. Oxidized phospholipids are more potent antagonists of lipopolysaccharide than inducers of inflammation.
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Oskolkova OV, Afonyushkin T, Preinerstorfer B, Bicker W, von Schlieffen E, Hainzl E, Demyanets S, Schabbauer G, Lindner W, Tselepis AD, Wojta J, Binder BR, and Bochkov VN
- Subjects
- Animals, Cytokines biosynthesis, Cytokines immunology, E-Selectin biosynthesis, E-Selectin immunology, Female, Inflammation chemically induced, Inflammation metabolism, Inflammation Mediators immunology, Inflammation Mediators metabolism, Mice, Phosphatidylcholines immunology, Phosphatidylcholines metabolism, RNA, Messenger biosynthesis, RNA, Messenger immunology, Toll-Like Receptor 4 agonists, Toll-Like Receptor 4 metabolism, Inflammation immunology, Lipopolysaccharides toxicity, Phosphatidylcholines pharmacology, Toll-Like Receptor 4 immunology
- Abstract
Polyunsaturated fatty acids are precursors of multiple pro- and anti-inflammatory molecules generated by enzymatic stereospecific and positionally specific insertion of oxygen, which is a prerequisite for recognition of these mediators by cellular receptors. However, nonenzymatically oxidized free and esterified polyunsaturated fatty acids also demonstrate activities relevant to inflammation. In particular, phospholipids containing oxidized fatty acid residues (oxidized phospholipids; OxPLs) were shown to induce proinflammatory changes in endothelial cells but paradoxically also to inhibit inflammation induced via TLR4. In this study, we show that half-maximal inhibition of LPS-induced elevation of E-selectin mRNA in endothelial cells developed at concentrations of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) 10-fold lower than those required to induce proinflammatory response. Similar concentration difference was observed for other classes and molecular species of OxPLs. Upon injection into mice, OxPAPC did not elevate plasma levels of IL-6 and keratinocyte chemoattractant but strongly inhibited LPS-induced upregulation of these inflammatory cytokines. Thus, both in vitro and in vivo, anti-LPS effects of OxPLs are observed at lower concentrations than those required for their proinflammatory action. Quantification of the most abundant oxidized phosphatidylcholines by HPLC/tandem mass spectrometry showed that circulating concentrations of total oxidized phosphatidylcholine species are close to the range where they demonstrate anti-LPS activity but significantly lower than that required for induction of inflammation. We hypothesize that low levels of OxPLs in circulation serve mostly anti-LPS function and protect from excessive systemic response to TLR4 ligands, whereas proinflammatory effects of OxPLs are more likely to develop locally at sites of tissue deposition of OxPLs (e.g., in atherosclerotic vessels).
- Published
- 2010
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33. T-cadherin attenuates the PERK branch of the unfolded protein response and protects vascular endothelial cells from endoplasmic reticulum stress-induced apoptosis.
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Kyriakakis E, Philippova M, Joshi MB, Pfaff D, Bochkov V, Afonyushkin T, Erne P, and Resink TJ
- Subjects
- Cell Survival, Endoplasmic Reticulum Chaperone BiP, Endothelial Cells enzymology, Endothelium, Vascular cytology, Endothelium, Vascular enzymology, Humans, JNK Mitogen-Activated Protein Kinases metabolism, Oxidative Stress, Phosphatidylinositol 3-Kinases metabolism, Signal Transduction, eIF-2 Kinase antagonists & inhibitors, Apoptosis, Cadherins metabolism, Endoplasmic Reticulum metabolism, Endothelial Cells metabolism, Endothelium, Vascular metabolism, Unfolded Protein Response
- Abstract
Endoplasmic reticulum (ER) stress activated by perturbations in ER homeostasis induces the unfolded protein response (UPR) with chaperon Grp78 as the key activator of UPR signalling. The aim of UPR is to restore normal ER function; however prolonged or severe ER stress triggers apoptosis of damaged cells to ensure protection of the whole organism. Recent findings support an association of ER stress-induced apoptosis of vascular cells with cardiovascular pathologies. T-cadherin (T-cad), an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily is upregulated in atherosclerotic lesions. Here we investigate the ability of T-cad to influence UPR signalling and endothelial cell (EC) survival during ER stress. EC were treated with a variety of ER stress-inducing compounds (thapsigargin, dithiothereitol, brefeldin A, tunicamycin, A23187 or homocysteine) and induction of ER stress validated by increases in levels of UPR signalling molecules Grp78 (glucose-regulated protein of 78kDa), phospho-eIF2alpha (phosphorylated eukaryotic initiation factor 2alpha) and CHOP (C/EBP homologous protein). All compounds also increased T-cad mRNA and protein levels. Overexpression or silencing of T-cad in EC respectively attenuated or amplified the ER stress-induced increase in phospho-eIF2alpha, Grp78, CHOP and active caspases. Effects of T-cad-overexpression or T-cad-silencing on ER stress responses in EC were not affected by inclusion of either N-acetylcysteine (reactive oxygen species scavenger), LY294002 (phosphatidylinositol-3-kinase inhibitor) or SP6000125 (Jun N-terminal kinase inhibitor). The data suggest that upregulation of T-cad on EC during ER stress attenuates the activation of the proapoptotic PERK (PKR (double-stranded RNA-activated protein kinase)-like ER kinase) branch of the UPR cascade and thereby protects EC from ER stress-induced apoptosis., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)
- Published
- 2010
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34. Oxidized phospholipids regulate expression of ATF4 and VEGF in endothelial cells via NRF2-dependent mechanism: novel point of convergence between electrophilic and unfolded protein stress pathways.
- Author
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Afonyushkin T, Oskolkova OV, Philippova M, Resink TJ, Erne P, Binder BR, and Bochkov VN
- Subjects
- Activating Transcription Factor 4 genetics, Binding Sites, Blotting, Western, Cells, Cultured, Chromatin Immunoprecipitation, Humans, Lipoproteins, LDL metabolism, NF-E2-Related Factor 2 genetics, Oxidation-Reduction, Promoter Regions, Genetic, Prostaglandins metabolism, RNA Interference, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Time Factors, Transcriptional Activation, Up-Regulation, Activating Transcription Factor 4 metabolism, Endothelial Cells metabolism, NF-E2-Related Factor 2 metabolism, Neovascularization, Physiologic, Phospholipids metabolism, Stress, Physiological, Unfolded Protein Response, Vascular Endothelial Growth Factor A metabolism
- Abstract
Objective: The ATF4 arm of the unfolded protein response is increasingly recognized for its relevance to pathology, and in particular to angiogenic reactions. Oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, were shown to upregulate vascular endothelial growth factor (VEGF) and induce angiogenesis via an ATF4-dependent mechanism. In this study, we analyzed the mechanism of ATF4 upregulation by OxPLs and more specifically the involvement of NRF2, the major transcriptional mediator of electrophilic stress response., Methods and Results: Using reverse transcription/real-time polymerase chain reaction and Western blotting, we found that OxPLs induced upregulation of ATF4 mRNA and protein in several types of endothelial cells and that these effects were suppressed by short interfering RNA (siRNA) against NRF2. Electrophilic (iso)prostaglandins and oxidized low-density lipoprotein, similarly to OxPLs, elevated ATF4 mRNA levels in an NRF2-dependent mode. Chromatin immunoprecipitation revealed OxPL-dependent binding of NRF2 to a putative antioxidant response element site in the ATF4 gene promoter. Knockdown of NRF2 inhibited OxPL-induced elevation of VEGF mRNA and endothelial cell sprout formation., Conclusion: Our data characterize NRF2 as a positive regulator of ATF4 and identify a novel cross-talk between electrophilic and unfolded protein responses, which may play a role in stress-induced angiogenesis.
- Published
- 2010
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35. ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response.
- Author
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Oskolkova OV, Afonyushkin T, Leitner A, von Schlieffen E, Gargalovic PS, Lusis AJ, Binder BR, and Bochkov VN
- Subjects
- Cells, Cultured, Endothelium, Vascular cytology, Humans, Oxidation-Reduction, Up-Regulation, Activating Transcription Factor 4 physiology, Phospholipids physiology, Protein Denaturation, Transcription, Genetic, Vascular Endothelial Growth Factor A genetics
- Abstract
We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques.
- Published
- 2008
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36. Nrf2 regulates antioxidant gene expression evoked by oxidized phospholipids in endothelial cells and murine arteries in vivo.
- Author
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Jyrkkänen HK, Kansanen E, Inkala M, Kivelä AM, Hurttila H, Heinonen SE, Goldsteins G, Jauhiainen S, Tiainen S, Makkonen H, Oskolkova O, Afonyushkin T, Koistinaho J, Yamamoto M, Bochkov VN, Ylä-Herttuala S, and Levonen AL
- Subjects
- Active Transport, Cell Nucleus drug effects, Active Transport, Cell Nucleus physiology, Animals, Carotid Arteries cytology, Cell Nucleus genetics, Endothelial Cells cytology, Gene Expression Regulation, Enzymologic physiology, Glutamate-Cysteine Ligase biosynthesis, Glutamate-Cysteine Ligase genetics, Heme Oxygenase-1 biosynthesis, Heme Oxygenase-1 genetics, Mice, Mice, Mutant Strains, NAD(P)H Dehydrogenase (Quinone), NADPH Dehydrogenase biosynthesis, NADPH Dehydrogenase genetics, NF-E2-Related Factor 2 antagonists & inhibitors, NF-E2-Related Factor 2 genetics, Oxidation-Reduction drug effects, Phosphatidylcholines metabolism, RNA, Small Interfering genetics, Antioxidants metabolism, Carotid Arteries enzymology, Cell Nucleus metabolism, Endothelial Cells enzymology, Gene Expression Regulation, Enzymologic drug effects, NF-E2-Related Factor 2 metabolism, Phosphatidylcholines pharmacokinetics
- Abstract
Besides their well-characterized proinflammatory and proatherogenic effects, oxidized phospholipids, such as oxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine) have been shown to have beneficial responses in vascular cells via induction of antioxidant enzymes such as heme oxygenase-1. We therefore hypothesized that oxPAPC could evoke a general cytoprotective response via activation of antioxidative transcription factor Nrf2. Here, we show that oxPAPC increases nuclear accumulation of Nrf2. Using the small interfering RNA approach, we demonstrate that Nrf2 is critical in mediating the induction of glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase-1 (NQO1) by oxPAPC in human endothelial cells, whereas the contribution to the induction of heme oxygenase-1 was less significant. The induction of GCLM and NQO1 was attenuated by reduction of electrophilic groups with sodium borohydrate, as well as treatment with thiol antioxidant N-acetylcysteine, suggesting that the thiol reactivity of oxPAPC is largely mediating its effect on Nrf2-responsive genes. Moreover, we show that oxidized phospholipid having a highly electrophilic isoprostane ring in its sn-2 position is a potent inducer of Nrf2 target genes. Finally, we demonstrate that the oxPAPC-inducible expression of heme oxygenase-1, GCLM, and NQO1 is lower in Nrf2-null than wild-type mouse carotid arteries in vivo. We suggest that the activation of Nrf2 by oxidized phospholipids provides a mechanism by which their deleterious effects are limited in the vasculature.
- Published
- 2008
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37. Identification of proteins associating with glycosylphosphatidylinositol- anchored T-cadherin on the surface of vascular endothelial cells: role for Grp78/BiP in T-cadherin-dependent cell survival.
- Author
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Philippova M, Ivanov D, Joshi MB, Kyriakakis E, Rupp K, Afonyushkin T, Bochkov V, Erne P, and Resink TJ
- Subjects
- Biotinylation, Cell Membrane, Cell Survival, Endoplasmic Reticulum Chaperone BiP, Glycosylphosphatidylinositols metabolism, Humans, Integrin beta3 metabolism, Mass Spectrometry, Membrane Microdomains metabolism, Microscopy, Confocal, Models, Biological, Proto-Oncogene Proteins c-myc metabolism, Signal Transduction, Cadherins metabolism, Endothelial Cells cytology, Heat-Shock Proteins metabolism, Molecular Chaperones metabolism
- Abstract
There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor alpha1 subunit, integrin beta3, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin beta3 with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone.
- Published
- 2008
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38. Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5'-leader.
- Author
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Resch A, Afonyushkin T, Lombo TB, McDowall KJ, Bläsi U, and Kaberdin VR
- Subjects
- Alternative Splicing, Base Sequence, Binding Sites genetics, DNA Primers genetics, Escherichia coli genetics, Escherichia coli metabolism, Genes, Bacterial, Models, Biological, Molecular Sequence Data, Mutation, RNA Processing, Post-Transcriptional, RNA Stability, RNA, Small Untranslated, Ribonuclease III genetics, Ribosomes metabolism, Transcriptional Activation, Bacterial Proteins genetics, Bacterial Proteins metabolism, RNA, Bacterial genetics, RNA, Bacterial metabolism, RNA, Untranslated genetics, RNA, Untranslated metabolism, Ribonuclease III metabolism, Sigma Factor genetics, Sigma Factor metabolism
- Abstract
The intricate regulation of the Escherichia coli rpoS gene, which encodes the stationary phase sigma-factor sigmaS, includes translational activation by the noncoding RNA DsrA. We observed that the stability of rpoS mRNA, and concomitantly the concentration of sigmaS, were significantly higher in an RNase III-deficient mutant. As no decay intermediates corresponding to the in vitro mapped RNase III cleavage site in the rpoS leader could be detected in vivo, the initial RNase III cleavage appears to be decisive for the observed rapid inactivation of rpoS mRNA. In contrast, we show that base-pairing of DsrA with the rpoS leader creates an alternative RNase III cleavage site within the rpoS/DsrA duplex. This study provides new insights into regulation by small regulatory RNAs in that the molecular function of DsrA not only facilitates ribosome loading on rpoS mRNA, but additionally involves an alternative processing of the target.
- Published
- 2008
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39. In human macrophages the complement component C5a induces the expression of oncostatin M via AP-1 activation.
- Author
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Kastl SP, Speidl WS, Kaun C, Katsaros KM, Rega G, Afonyushkin T, Bochkov VN, Valent P, Assadian A, Hagmueller GW, Hoeth M, de Martin R, Ma Y, Maurer G, Huber K, and Wojta J
- Subjects
- Analysis of Variance, Atherosclerosis complications, Blotting, Western, Cells, Cultured, Gene Expression Regulation, Humans, Inflammation complications, Inflammation Mediators metabolism, Macrophages cytology, Oncostatin M genetics, Probability, Protein Binding, RNA, Messenger analysis, Reverse Transcriptase Polymerase Chain Reaction, Sensitivity and Specificity, Transcription Factor AP-1 genetics, Up-Regulation, Atherosclerosis metabolism, Complement C5a metabolism, Inflammation metabolism, Macrophages metabolism, Oncostatin M metabolism, Transcription Factor AP-1 metabolism
- Abstract
Objective: Macrophages produce the cytokine oncostatin M (OSM), which beside other functions is also involved in inflammation. The complement component C5a mobilizes and activates these cells at inflammatory sites. We examined the effect of C5a on OSM production in human monocytes and in human monocyte-derived macrophages., Methods and Results: For macrophage transformation peripheral blood monocytes were cultivated for 8 to 10 days in the presence of human serum. C5a significantly increased in these cells OSM antigen as determined by specific ELISA and mRNA as quantitated by real-time polymerase chain reaction in these cells as well as in plaque macrophages. This effect was blocked by antibodies against the receptor C5aR/CD88 and by pertussis toxin. The C5a-induced phosphorylation of p38 and JNK and the C5a-induced increase in OSM production in macrophages was abolished by 2 p38 inhibitors and by a JNK inhibitor. Furthermore C5a increased the nuclear translocation of c-fos and c-jun. Using different OSM promoter deletion mutant constructs we show that the putative AP-1 element is responsible for activation of OSM promoter activity by C5a., Conclusions: Our data establish a link between the complement system and the gp130 receptor cytokine family with possible implications for the pathology of inflammatory diseases.
- Published
- 2008
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40. The inflammatory cytokine oncostatin M induces PAI-1 in human vascular smooth muscle cells in vitro via PI 3-kinase and ERK1/2-dependent pathways.
- Author
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Demyanets S, Kaun C, Rychli K, Rega G, Pfaffenberger S, Afonyushkin T, Bochkov VN, Maurer G, Huber K, and Wojta J
- Subjects
- Aorta cytology, Aorta metabolism, Cell Proliferation drug effects, Cells, Cultured, Chromones pharmacology, Coronary Vessels cytology, Coronary Vessels metabolism, Enzyme Inhibitors pharmacology, Flavonoids pharmacology, Humans, Mitogen-Activated Protein Kinase 3 antagonists & inhibitors, Morpholines pharmacology, Muscle, Smooth, Vascular cytology, Phosphoinositide-3 Kinase Inhibitors, Proto-Oncogene Proteins c-akt metabolism, Tissue Plasminogen Activator metabolism, Urokinase-Type Plasminogen Activator metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Muscle, Smooth, Vascular metabolism, Oncostatin M pharmacology, Oncostatin M physiology, Phosphatidylinositol 3-Kinases metabolism, Plasminogen Activator Inhibitor 1 metabolism, Signal Transduction physiology
- Abstract
Plasminogen activator inhibitor-1 (PAI-1) plays a pivotal role in the regulation of the fibrinolytic system and in the modulation of extracellular proteolysis. Increased PAI-1 was found in atherosclerotic lesions, and high PAI-1 plasma levels were associated with coronary heart disease. Smooth muscle cells (SMC) are a major source of PAI-1 within the vascular wall, and PAI-1 was implicated in SMC migration, proliferation, and apoptosis. We treated human coronary artery SMC (HCASMC) and human aortic SMC (HASMC) with the glycoprotein 130 (gp130) ligands cardiotrophin-1, interleukin-6 (IL-6), leukemia inhibitory factor (LIF), or oncostatin M (OSM). Only OSM increased PAI-1 antigen and activity production significantly in these cells up to 20-fold. OSM upregulated mRNA specific for PAI-1 up to 4.5-fold in these cells. HCASMC and HASMC express gp130, OSM receptor, IL-6 receptor, and LIF receptor. OSM induced extracellular signal-regulated kinase (ERK) 1/2 and Akt phosphorylations in HASMC. A phosphatidylinositol 3-kinase inhibitor and a mitogen-activated protein/extracellular signal-regulated kinase inhibitor reduced Akt and ERK1/2 phosphorylation, respectively, and abolished OSM-induced PAI-1 upregulation. A janus kinase/signal transducer and activator of transcription inhibitor, a p38 mitogen-activated protein kinase inhibitor, or c-Jun NH(2)-terminal kinase inhibitor I did not inhibit the OSM-dependent PAI-1 induction. OSM enhanced proliferation of both HCASMC and HASMC by 77 and 90%, respectively. We hypothesize that, if the effect of OSM on PAI-1 expression in smooth muscle cells is operative in vivo, it could, via modulation of fibrinolysis and extracellular proteolysis, be involved in the development of vascular pathologies such as plaque progression, destabilization and subsequent thrombus formation, and restenosis and neointima formation.
- Published
- 2007
- Full Text
- View/download PDF
41. Oxidized phospholipids stimulate angiogenesis via autocrine mechanisms, implicating a novel role for lipid oxidation in the evolution of atherosclerotic lesions.
- Author
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Bochkov VN, Philippova M, Oskolkova O, Kadl A, Furnkranz A, Karabeg E, Afonyushkin T, Gruber F, Breuss J, Minchenko A, Mechtcheriakova D, Hohensinner P, Rychli K, Wojta J, Resink T, Erne P, Binder BR, and Leitinger N
- Subjects
- ADAM Proteins biosynthesis, ADAMTS1 Protein, Angiogenesis Inducing Agents metabolism, Animals, Cell Movement, Cells, Cultured, Cyclooxygenase 2 biosynthesis, Endothelial Cells drug effects, Endothelial Cells metabolism, Female, Interleukin-8 biosynthesis, Mice, Mice, Inbred C57BL, Monocytes drug effects, Monocytes metabolism, Neoplasms metabolism, Neoplasms pathology, Oxidation-Reduction, Phospholipids pharmacology, Skin cytology, Skin drug effects, Skin metabolism, Vascular Endothelial Growth Factor A biosynthesis, Atherosclerosis etiology, Autocrine Communication, Lipid Metabolism, Neovascularization, Pathologic etiology, Phospholipids chemistry, Phospholipids metabolism
- Abstract
Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2-generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.
- Published
- 2006
- Full Text
- View/download PDF
42. Functional effects of variants of the RNA chaperone Hfq.
- Author
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Sonnleitner E, Napetschnig J, Afonyushkin T, Ecker K, Vecerek B, Moll I, Kaberdin VR, and Bläsi U
- Subjects
- Allolevivirus growth & development, Amino Acid Substitution, Bacterial Outer Membrane Proteins genetics, Escherichia coli genetics, Escherichia coli virology, Molecular Chaperones genetics, Mutagenesis, Site-Directed, RNA, Small Untranslated, RNA, Untranslated genetics, RNA, Untranslated metabolism, Recombinant Proteins metabolism, Structure-Activity Relationship, Bacterial Outer Membrane Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Host Factor 1 Protein genetics, Host Factor 1 Protein metabolism, Molecular Chaperones metabolism, RNA genetics, RNA metabolism
- Abstract
The ring-shaped RNA chaperone Hfq has recently received much attention owing to its multiple roles in RNA metabolism. In this study we have performed a mutational analysis of the Escherichia coli hfq gene, and have studied the effects of amino acid substitutions at several positions in the Hfq protein as well as of C-terminal truncations on its role in phage Qbeta replication, in repression of a target mRNA, and on the stability of the small regulatory RNA DsrA. These functional studies provided insights into the interaction of Hfq with RNA and suggested a role for the C-terminus of Hfq in DsrA stability.
- Published
- 2004
- Full Text
- View/download PDF
43. Temperature-dependent stability and translation of Escherichia coli ompA mRNA.
- Author
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Afonyushkin T, Moll I, Bläsi U, and Kaberdin VR
- Subjects
- 5' Untranslated Regions, Bacterial Outer Membrane Proteins genetics, Base Sequence, Blotting, Northern, Blotting, Western, Cell Division, Endoribonucleases chemistry, Molecular Sequence Data, Nucleic Acid Conformation, RNA, Messenger metabolism, Temperature, Time Factors, Bacterial Outer Membrane Proteins chemistry, Escherichia coli metabolism, Protein Biosynthesis
- Abstract
RNase E is known to affect the turnover of ompA mRNA in a growth rate-dependent manner. Here, we show that this enzyme also plays a role in the temperature-dependent stability of the transcript, thereby maintaining comparable levels of OmpA at 28 and 37 degrees C. An increase in the efficiency of RNase E cleavages at 37 degrees C within the 5(') UTR of the transcript in vitro was found to correlate with a decreased half-life and steady-state level at elevated temperature in vivo. However, measurements of de novo OmpA synthesis and in vitro toeprinting experiments suggest that translation of ompA mRNA is more efficient at 37 degrees C when compared to 28 degrees C. Thus, the enhanced translation apparently counteracts the decreased half-life at elevated temperature. Moreover, we propose that the temperature-dependent inverse correlation between ompA mRNA stability and translation can result from structural changes induced in the 5(') UTR of the transcript.
- Published
- 2003
- Full Text
- View/download PDF
44. Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs.
- Author
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Moll I, Afonyushkin T, Vytvytska O, Kaberdin VR, and Bläsi U
- Subjects
- 5' Untranslated Regions, Base Sequence, Binding Sites, Hydrolysis, Molecular Sequence Data, Nucleic Acid Conformation, Promoter Regions, Genetic, RNA, Messenger chemistry, Endoribonucleases metabolism, Host Factor 1 Protein metabolism, RNA, Messenger metabolism
- Abstract
The Escherichia coli RNA chaperone Hfq was discovered originally as an accessory factor of the phage Qbeta replicase. More recent work suggested a role of Hfq in cellular physiology through its interaction with ompA mRNA and small RNAs (sRNAs), some of which are involved in translational regulation. Despite their stability under certain conditions, E. coli sRNAs contain putative RNase E recognition sites, that is, A/U-rich sequences and adjacent stem-loop structures. We show herein that an RNase E cleavage site coincides with the Hfq-binding site in the 5'-untranslated region of E. coli ompA mRNA as well as with that in the sRNA, DsrA. Likewise, Hfq protects RyhB RNA from in vitro cleavage by RNase E. These in vitro data are supported by the increased abundance of DsrA and RyhB sRNAs in an RNase E mutant strain as well as by their decreased stability in a hfq(-) strain. It is commonly believed that the RNA chaperone Hfq facilitates or promotes the interaction between sRNAs and their mRNA targets. This study reveals another role for Hfq, that is, protection of sRNAs from endonucleolytic attack.
- Published
- 2003
- Full Text
- View/download PDF
45. Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli.
- Author
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Vecerek B, Moll I, Afonyushkin T, Kaberdin V, and Bläsi U
- Subjects
- 5,10-Methylenetetrahydrofolate Reductase (FADH2) genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Base Sequence, Carrier Proteins genetics, Cyclophilins genetics, Escherichia coli genetics, Escherichia coli Proteins genetics, Gene Expression Regulation, Bacterial, Host Factor 1 Protein genetics, Molecular Chaperones genetics, Molecular Chaperones metabolism, Molecular Sequence Data, Peptidylprolyl Isomerase, Protein Biosynthesis, Proteomics methods, RNA Stability, RNA, Small Nuclear genetics, RNA, Small Nuclear metabolism, Repressor Proteins genetics, Repressor Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Host Factor 1 Protein metabolism, Iron metabolism, RNA, Messenger metabolism
- Abstract
The Escherichia coli Sm-like host factor I (Hfq) is thought to play direct and indirect roles in post-transcriptional regulation by targeting small regulatory RNAs and mRNAs. In this study, we have used proteomics to identify new mRNA targets of Hfq. We have identified 11 candidate proteins, synthesis of which was differentially affected in a hfq- background. The effect of Hfq on some of the corresponding mRNAs including fur, gapA, metF, ppiB and sodB mRNA was assessed, using different in vitro and in vivo methods. This allowed us to distinguish between direct and indirect effects of Hfq in modulating the translational activities of these mRNAs. From the collection of mRNAs tested, only fur and sodB mRNA, encoding the master regulator of iron metabolism and the iron superoxide dismutase, respectively, were found to be regulated by Hfq. Fur is known to be a negative regulator of transcription of the small RNA RyhB. Mutations in the sodB leader and compensating mutations in RyhB revealed that RyhB in turn represses translation of sodB mRNA, explaining the previously reported positive control of sodB by Fur. These data assign a role to Hfq in regulation of iron uptake and in switching off of iron scavenger genes.
- Published
- 2003
- Full Text
- View/download PDF
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