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1.
Nat Immunol ; 21(7): 756-765, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32572240

RESUMO

The molecular basis for the propensity of a small number of environmental proteins to provoke allergic responses is largely unknown. Herein, we report that mite group 13 allergens of the fatty acid-binding protein (FABP) family are sensed by an evolutionarily conserved acute-phase protein, serum amyloid A1 (SAA1), that promotes pulmonary type 2 immunity. Mechanistically, SAA1 interacted directly with allergenic mite FABPs (Der p 13 and Blo t 13). The interaction between mite FABPs and SAA1 activated the SAA1-binding receptor, formyl peptide receptor 2 (FPR2), which drove the epithelial release of the type-2-promoting cytokine interleukin (IL)-33 in a SAA1-dependent manner. Importantly, the SAA1-FPR2-IL-33 axis was upregulated in nasal epithelial cells from patients with chronic rhinosinusitis. These findings identify an unrecognized role for SAA1 as a soluble pattern recognition receptor for conserved FABPs found in common mite allergens that initiate type 2 immunity at mucosal surfaces.


Assuntos
Asma/imunologia , Rinite Alérgica/imunologia , Proteína Amiloide A Sérica/metabolismo , Transdução de Sinais/imunologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alérgenos/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Asma/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais , Proteínas de Ligação a Ácido Graxo/imunologia , Feminino , Humanos , Imunidade Humoral , Imunidade Inata , Interleucina-33/metabolismo , Pulmão/citologia , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Cultura Primária de Células , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Rinite Alérgica/patologia , Proteína Amiloide A Sérica/genética , Regulação para Cima , Adulto Jovem
2.
Chem Res Toxicol ; 34(6): 1681-1692, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34085520

RESUMO

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


Assuntos
Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Óxido Nítrico/farmacologia , Peroxidase/antagonistas & inibidores , Poliaminas/farmacologia , Biocatálise , Células Endoteliais/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxirredução , Peroxidase/metabolismo , Poliaminas/química , Poliaminas/metabolismo
3.
Int J Mol Sci ; 22(21)2021 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-34769194

RESUMO

Osteosarcoma (OS) is the most common type of bone tumor, and has limited therapy options. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) has striking anti-tumor effects in various tumors. Here, we investigated molecular mechanisms that mediate anti-tumor effects of 15d-PGJ2 in different OS cell lines. Human U2-OS and Saos-2 cells were treated with 15d-PGJ2 and cell survival was measured by MTT assay. Cell proliferation and motility were investigated by scratch assay, the tumorigenic capacity by colony forming assay. Intracellular ROS was estimated by H2DCFDA. Activation of MAPKs and cytoprotective proteins was detected by immunoblotting. Apoptosis was detected by immunoblotting and Annexin V/PI staining. The ex ovo CAM model was used to study growth capability of grafted 15d-PGJ2-treated OS cells, followed by immunohistochemistry with hematoxylin/eosin and Ki-67. 15d-PGJ2 substantially decreased cell viability, colony formation and wound closure capability of OS cells. Non-malignant human osteoblast was less affected by 15d-PGJ2. 15d-PGJ2 induced rapid intracellular ROS production and time-dependent activation of MAPKs (pERK1/2, pJNK and pp38). Tempol efficiently inhibited 15d-PGJ2-induced ERK1/2 activation, while N-acetylcystein and pyrrolidine dithiocarbamate were less effective. Early but weak activation of cytoprotective proteins was overrun by induction of apoptosis. A structural analogue, 9,10-dihydro-15d-PGJ2, did not show toxic effects in OS cells. In the CAM model, we grafted OS tumors with U2-OS, Saos-2 and MG-63 cells. 15d-PGJ2 treatment resulted in significant growth inhibition, diminished tumor tissue density, and reduced tumor cell proliferation for all cell lines. Our in vitro and CAM data suggest 15d-PGJ2 as a promising natural compound to interfere with OS tumor growth.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/tratamento farmacológico , Osteossarcoma/tratamento farmacológico , Prostaglandina D2/análogos & derivados , Animais , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Galinhas , Ativação Enzimática/efeitos dos fármacos , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Osteossarcoma/metabolismo , Prostaglandina D2/farmacologia , Espécies Reativas de Oxigênio/metabolismo
4.
J Neuroinflammation ; 17(1): 127, 2020 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-32326963

RESUMO

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-1ß, 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.


Assuntos
Inflamação/metabolismo , Lisofosfolipídeos/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Microglia/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Diester Fosfórico Hidrolases/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo
5.
Pharmacol Res ; 158: 104870, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32434052

RESUMO

AIMS: Sodium-glucose co-transporter 2 (SGLT2) were originally developed as kidney-targeting anti-diabetic drugs. However, due to their beneficial cardiac off-target effects (as SGLT2 is not expressed in the heart), these antagonists currently receive intense clinical interest in the context of heart failure (HF) in patients with or without diabetes mellitus (DM). Since the mechanisms by which these beneficial effects are mediated are still unclear yet, inflammation that is present in DM and HF has been proposed as a potential pharmacological intervention strategy. Therefore, we tested the hypothesis that the SGLT2 inhibitor, empagliflozin, displays anti-inflammatory potential along with its glucose-lowering property. METHODS AND RESULTS: Lipopolysaccharide (LPS) was used to induce inflammation in vitro and in vivo. In cardiomyocytes and macrophages empagliflozin attenuated LPS-induced TNFα and iNOS expression. Analysis of intracellular signalling pathways suggested that empagliflozin activates AMP kinase (AMPK) in both cell types with or without LPS-treatment. Moreover, the SGLT2 inhibitor increased the expression of anti-inflammatory M2 marker proteins in LPS-treated macrophages. Additionally, empagliflozin-mediated AMPK activation prevented LPS-induced ATP/ADP depletion. In vivo administration of LPS in mice impaired cardiac contractility and aortic endothelial relaxation in response to acetylcholine, whereby co-administration of empagliflozin preserved cardiovascular function. These findings were accompanied by improved cardiac AMPK phosphorylation and ATP/ADP, reduced cardiac iNOS, plasma TNFα and creatine kinase MB levels. CONCLUSION: Our data identify a novel cardio protective mechanism of SGLT2 inhibitor, empagliflozin, suggesting that AMPK activation-mediated energy repletion and reduced inflammation contribute to the observed cardiovascular benefits of the drug in HF.


Assuntos
Compostos Benzidrílicos/farmacologia , Cardiotônicos/farmacologia , Glucosídeos/farmacologia , Miócitos Cardíacos/metabolismo , Proteínas Quinases/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Quinases Proteína-Quinases Ativadas por AMP , Animais , Compostos Benzidrílicos/uso terapêutico , Cardiotônicos/uso terapêutico , Relação Dose-Resposta a Droga , Metabolismo Energético , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Glucosídeos/uso terapêutico , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/prevenção & controle , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Células RAW 264.7 , Transportador 2 de Glucose-Sódio/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico
6.
Int J Mol Sci ; 21(23)2020 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-33287422

RESUMO

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 (N3-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.


Assuntos
Aldeídos/metabolismo , Endotoxemia/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Peroxidase/metabolismo , Animais , Biomarcadores , Química Click , Endotoxemia/etiologia , Ácidos Graxos/metabolismo , Ácido Hipocloroso/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Proteoma , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo
7.
Int J Mol Sci ; 21(3)2020 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-32050431

RESUMO

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.


Assuntos
Encéfalo/irrigação sanguínea , Células Endoteliais/metabolismo , Peroxidase/metabolismo , Sepse/metabolismo , Esfingolipídeos/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Capilares/metabolismo , Capilares/patologia , Linhagem Celular , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/patologia , Homeostase , Humanos , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Sepse/patologia
8.
Hum Mol Genet ; 23(15): 4015-23, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24626631

RESUMO

We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development.


Assuntos
Deficiência Intelectual/genética , Metiltransferases/genética , Mutação , Sequência de Bases , Criança , Cromossomos Humanos Par 17 , Consanguinidade , Exoma , Feminino , Genes Recessivos , Homozigoto , Humanos , Deficiência Intelectual/fisiopatologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem
9.
J Neuroinflammation ; 13(1): 205, 2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27565558

RESUMO

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-1ß, 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.


Assuntos
Polaridade Celular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Microglia/classificação , Microglia/efeitos dos fármacos , Actinas/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Células Cultivadas , Córtex Cerebral/citologia , Citocinas/genética , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fatores de Tempo
10.
Arch Biochem Biophys ; 571: 1-9, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25731855

RESUMO

Peroxynitrite, a potent pro-inflammatory and cytotoxic species, interacts with a variety of heme containing proteins. We addressed the question whether (i) the interaction of myeloperoxidase (MPO, an enzyme generating hypochlorous acid from hydrogen peroxide and chloride ions) with peroxynitrite affects the clearance of peroxynitrite, and (ii) if peroxynitrite could modulate the chlorinating activity of MPO. Our results show that this interaction promotes the decomposition of the highly reactive pro-inflammatory oxidant, whereby MPO Compound II (but not Compound I) is formed. The efficiency of MPO to remove peroxynitrite was enhanced by L-tyrosine, nitrite and (-)-epicatechin, substances known to reduce Compound II with high reaction rate. Next, peroxynitrite (added as reagent) diminished the chlorinating activity of MPO in the presence of hydrogen peroxide. Alternatively, SIN-1, a peroxynitrite donor, reduced hypochlorous acid formation by MPO, as measured by aminophenyl fluorescein oxidation (time kinetics) and taurine chloramine formation (end point measurement). At inflammatory loci, scavenging of peroxynitrite by MPO may overcome the uncontrolled peroxynitrite decomposition and formation of reactive species, which lead to cell/tissue damage.


Assuntos
Anti-Inflamatórios/química , Peroxidase/química , Ácido Peroxinitroso/química , Compostos de Anilina/química , Catequina/química , Fluoresceínas/química , Halogenação , Humanos , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Cinética , Molsidomina/análogos & derivados , Molsidomina/química , Nitritos/química , Oxidantes/química , Oxirredução , Tirosina/química
11.
Biochem J ; 459(2): 313-22, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24517414

RESUMO

ECM (extracellular matrix) materials, such as laminin, perlecan, type IV collagen and fibronectin, play a key role in determining the structure of the arterial wall and the properties of cells that interact with the ECM. The aim of the present study was to investigate the effect of peroxynitrous acid, an oxidant generated by activated macrophages, on the structure and function of the ECM laid down by HCAECs (human coronary artery endothelial cells) in vitro and in vivo. We show that exposure of HCAEC-derived native matrix components to peroxynitrous acid (but not decomposed oxidant) at concentrations >1 µM results in a loss of antibody recognition of perlecan, collagen IV, and cell-binding sites on laminin and fibronectin. Loss of recognition was accompanied by decreased HCAEC adhesion. Real-time PCR showed up-regulation of inflammation-associated genes, including MMP7 (matrix metalloproteinase 7) and MMP13, as well as down-regulation of the laminin α2 chain, in HCAECs cultured on peroxynitrous acid-treated matrix compared with native matrix. Immunohistochemical studies provided evidence of co-localization of laminin with 3-nitrotyrosine, a biomarker of peroxynitrous acid damage, in type II-III/IV human atherosclerotic lesions, consistent with matrix damage occurring during disease development in vivo. The results of the present study suggest a mechanism through which peroxynitrous acid modifies endothelial cell-derived native ECM proteins of the arterial basement membrane in atherosclerotic lesions. These changes to ECM and particularly perlecan and laminin may be important in inducing cellular dysfunction and contribute to atherogenesis.


Assuntos
Vasos Coronários/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Ácido Peroxinitroso/farmacologia , Aterosclerose/metabolismo , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Oxidantes/farmacologia , Oxirredução
12.
J Mol Cell Cardiol ; 72: 64-73, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24583250

RESUMO

Lower heart rate is associated with better survival in patients with multiple organ dysfunction syndrome (MODS), a disease mostly caused by sepsis. The benefits of heart rate reduction by ivabradine during MODS are currently being investigated in the MODIfY clinical trial. Ivabradine is a selective inhibitor of the pacemaker current If and since If is impaired by lipopolysaccharide (LPS, endotoxin), a trigger of sepsis, we aimed to explore If blocking potency of ivabradine under elevated endotoxin levels in human atrial cardiomyocytes. Treatment of myocytes with S-LPS (containing the lipid A moiety, a core oligosaccharide and an O-polysaccharide chain) but not R595 (an O-chain lacking LPS-form) caused If inhibition under acute and chronic septic conditions. The specific interaction of S-LPS but not R595 to pacemaker channels HCN2 and HCN4 proves the necessity of O-chain for S-LPS-HCN interaction. The efficacy of ivabradine to block If was reduced under septic conditions, an observation that correlated with lower intracellular ivabradine concentrations in S-LPS- but not R595-treated cardiomyocytes. Computational analysis using a sinoatrial pacemaker cell model revealed that despite a reduction of If under septic conditions, ivabradine further decelerated pacemaking activity. This novel finding, i.e. If inhibition by ivabradine under elevated endotoxin levels in vitro, may provide a molecular understanding for the efficacy of this drug on heart rate reduction under septic conditions in vivo, e.g. the MODIfY clinical trial.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Benzazepinas/farmacologia , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Proteínas Musculares/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Nó Sinoatrial/efeitos dos fármacos , Ensaios Clínicos como Assunto , Átrios do Coração/citologia , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Ivabradina , Modelos Biológicos , Proteínas Musculares/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , Cultura Primária de Células , Nó Sinoatrial/citologia , Nó Sinoatrial/metabolismo
13.
J Lipid Res ; 55(4): 747-57, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24534704

RESUMO

Oxidation of LDL by the myeloperoxidase (MPO)-H2O2-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H2O2-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H2O2-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.


Assuntos
Apolipoproteína B-100/metabolismo , Lipoproteínas LDL/metabolismo , Peroxidase/metabolismo , Sequência de Aminoácidos , Apolipoproteína B-100/química , Estudos de Casos e Controles , Humanos , Peróxido de Hidrogênio/química , Hidrólise , Nefropatias/sangue , Nefropatias/terapia , Lipoproteínas LDL/química , Dados de Sequência Molecular , Oxirredução , Fragmentos de Peptídeos , Peroxidase/química , Processamento de Proteína Pós-Traducional , Diálise Renal
14.
Biochim Biophys Acta ; 1831(12): 1665-78, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23973266

RESUMO

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.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Microdomínios da Membrana/metabolismo , Neurônios/enzimologia , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Animais , Compostos de Boro , Caveolinas/genética , Caveolinas/metabolismo , Linhagem Celular , Endocitose , Retículo Endoplasmático/efeitos dos fármacos , Corantes Fluorescentes , Regulação da Expressão Gênica , Complexo de Golgi/efeitos dos fármacos , Hidrólise , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Lipoproteínas HDL/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Camundongos , Neurônios/citologia , Neurônios/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/farmacologia , Temperatura
15.
Biochem Biophys Res Commun ; 450(4): 1643-9, 2014 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-25044109

RESUMO

The serum amyloid A (SAA) family of proteins is encoded by multiple genes, which display allelic variation and a high degree of homology in mammals. The SAA1/2 genes code for non-glycosylated acute-phase SAA1/2 proteins, that may increase up to 1000-fold during inflammation. The SAA4 gene, well characterized in humans (hSAA4) and mice (mSaa4) codes for a SAA4 protein that is glycosylated only in humans. We here report on a previously uncharacterized SAA4 gene (rSAA4) and its product in Rattus norvegicus, the only mammalian species known not to express acute-phase SAA. The exon/intron organization of rSAA4 is similar to that reported for hSAA4 and mSaa4. By performing 5'- and 3'RACE, we identified a 1830-bases containing rSAA4 mRNA (including a GA-dinucleotide tandem repeat). Highest rSAA4 mRNA expression was detected in rat liver. In McA-RH7777 rat hepatoma cells, rSAA4 transcription was significantly upregulated in response to LPS and IL-6 while IL-1α/ß and TNFα were without effect. Luciferase assays with promoter-truncation constructs identified three proximal C/EBP-elements that mediate expression of rSAA4 in McA-RH7777 cells. In line with sequence prediction a 14-kDa non-glycosylated SAA4 protein is abundantly expressed in rat liver. Fluorescence microscopy revealed predominant localization of rSAA4-GFP-tagged fusion protein in the ER.


Assuntos
Proteína Amiloide A Sérica/metabolismo , Animais , Linhagem Celular Tumoral , Fígado/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/genética
16.
Am J Pathol ; 182(3): 727-41, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23318573

RESUMO

Dyslipidemia is a frequent component of the metabolic disorder of diabetic patients contributing to organ damage. Herein, in low-density lipoprotein receptor-deficient hyperlipidemic and streptozotozin-induced diabetic mice, hyperglycemia and hyperlipidemia acted reciprocally, accentuating renal injury and altering renal function. In hyperglycemic-hyperlipidemic kidneys, the accumulation of Tip47-positive lipid droplets in glomeruli, tubular epithelia, and macrophages was accompanied by the concomitant presence of the oxidative stress markers xanthine oxidoreductase and nitrotyrosine, findings that could also be evidenced in renal biopsy samples of diabetic patients. As liver X receptors (LXRα,ß) regulate genes linked to lipid and carbohydrate homeostasis and inhibit inflammatory gene expression in macrophages, the effects of systemic and macrophage-specific LXR activation were analyzed on renal damage in hyperlipidemic-hyperglycemic mice. LXR stimulation by GW3965 up-regulated genes involved in cholesterol efflux and down-regulated proinflammatory/profibrotic cytokines, inhibiting the pathomorphology of diabetic nephropathy, renal lipid accumulation, and improving renal function. Xanthine oxidoreductase and nitrotyrosine levels were reduced. In macrophages, GW3965 or LXRα overexpression significantly suppressed glycated or acetylated low-density lipoprotein-induced cytokines and reactive oxygen species. Specifically, in mice, transgenic expression of LXRα in macrophages significantly ameliorated hyperlipidemic-hyperglycemic nephropathy. The results demonstrate the presence of lipid droplet-induced oxidative mechanisms and the pathophysiologic role of macrophages in diabetic kidneys and indicate the potent regulatory role of LXRs in preventing renal damage in diabetes.


Assuntos
Hiperglicemia/patologia , Rim/patologia , Metabolismo dos Lipídeos , Receptores Nucleares Órfãos/metabolismo , Animais , Benzoatos/farmacologia , Benzilaminas/farmacologia , Citocinas/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/fisiopatologia , Nefropatias Diabéticas/complicações , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/ultraestrutura , Fibrose , Humanos , Hiperglicemia/complicações , Hiperglicemia/genética , Hiperglicemia/fisiopatologia , Hiperlipidemias/complicações , Hiperlipidemias/genética , Hiperlipidemias/patologia , Hiperlipidemias/fisiopatologia , Inflamação/patologia , Rim/efeitos dos fármacos , Rim/fisiopatologia , Rim/ultraestrutura , Testes de Função Renal , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/patologia , Células Mesangiais/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Podócitos/ultraestrutura
17.
J Immunol ; 189(6): 3007-17, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22875802

RESUMO

Recognition of endogenous lipid Ag(s) on CD1d is required for the development of invariant NKT (iNKT) cells. Isoglobotrihexosylceramide (iGb3) has been implicated as this endogenous selecting ligand and recently suggested to control overstimulation and deletion of iNKT cells in α-galactosidase A-deficient (αGalA(-/-)) mice (human Fabry disease), which accumulate isoglobosides and globosides. However, the presence and function of iGb3 in murine thymus remained controversial. In this study, we generate a globotrihexosylceramide (Gb3)-synthase-deficient (Gb3S(-/-)) mouse and show that in thymi of αGalA(-/-)/Gb3S(-/-) double-knockout mice, which store isoglobosides but no globosides, minute amounts of iGb3 can be detected by HPLC. Furthermore, we demonstrate that iGb3 deficiency does not only fail to impact selection of iNKT cells, in terms of frequency and absolute numbers, but also does not alter the distribution of the TCR CDR 3 of iNKT cells. Analyzing multiple gene-targeted mouse strains, we demonstrate that globoside, rather than iGb3, storage is the major cause for reduced iNKT cell frequencies and defective Ag presentation in αGalA(-/-) mice. Finally, we show that correction of globoside storage in αGalA(-/-) mice by crossing them with Gb3S(-/-) normalizes iNKT cell frequencies and dendritic cell (DC) function. We conclude that, although detectable in murine thymus in αGalA(-/-)/Gb3S(-/-) mice, iGb3 does not influence either the development of iNKT cells or their interaction with peripheral DCs. Moreover, in αGalA(-/-) mice, it is the Gb3 storage that is responsible for the decreased iNKT cell numbers and impeded Ag presentation on DCs.


Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Globosídeos/fisiologia , Células T Matadoras Naturais/imunologia , Triexosilceramidas , Animais , Sequência de Carboidratos , Células Dendríticas/enzimologia , Células Dendríticas/metabolismo , Globosídeos/deficiência , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Células T Matadoras Naturais/enzimologia , Células T Matadoras Naturais/metabolismo , Baço/citologia , Baço/enzimologia , Baço/metabolismo , Timo/citologia , Timo/enzimologia , Timo/metabolismo , Triexosilceramidas/deficiência , Triexosilceramidas/fisiologia , alfa-Galactosidase/genética , alfa-Galactosidase/fisiologia
18.
Exp Cell Res ; 319(12): 1828-1838, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23541792

RESUMO

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor and is invariably fatal to affected patients. Oxysterols belong to a class of bioactive lipids that are implicated in neurological disease and are associated with various types of cancer. Here, we investigated expression and transcriptional regulation of cholesterol 25-hydroxylase (CH25H) in human U87MG and GM133 glioblastoma cell lines. We demonstrate that in both cell lines transcription and translation of CH25H are increased in response to TNFα and IL1ß. In parallel, both cell lines upregulate 25-hydroxycholesterol (25-OHC) synthesis and secretion to levels comparable to bone marrow-derived mouse macrophages under inflammatory conditions. To determine whether 25-OHC acts as chemoattractant for tumor-associated macrophages, the human THP-1 monoblastic leukemia cell line was treated with varying amounts of the oxysterol. Experiments revealed that 25-OHC and lipid extracts isolated from GM133-conditioned medium (containing 7-fold higher 25-OHC concentrations than U87MG medium) induce chemotactic migration of THP-1 cells. Of note, 25-OHC also induced the migration of primary human peripheral blood monocytes. In response to exogenously added 25-OHC, THP-1 cells reorganized intermediate filament-associated vimentin to more cortical and polarized structures. Chemotactic migration of monocytes in response to 25-OHC was pertussis toxin-sensitive, indicating the involvement of G protein-coupled receptors. Using RNA interference we demonstrated that G protein-coupled receptor 183 (EBI2) contributes to 25-OHC-mediated chemotactic migration of THP-1 cells. These in vitro data indicate that GBM-derived and secreted 25-OHC may be involved in the recruitment of immune-competent cells to a tumor via EBI2.


Assuntos
Neoplasias Encefálicas/metabolismo , Quimiotaxia/efeitos dos fármacos , Glioblastoma/metabolismo , Hidroxicolesteróis/metabolismo , Monócitos/fisiologia , Linhagem Celular Tumoral , Humanos , Biossíntese de Proteínas , RNA Interferente Pequeno , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Esteróis/farmacologia , Transcrição Gênica , Regulação para Cima , Vimentina/metabolismo
19.
Pharmacol Ther ; 262: 108710, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39179117

RESUMO

In an aging society, unveiling new anti-aging strategies to prevent and combat aging-related diseases is of utmost importance. Mitochondria are the primary ATP production sites and key regulators of programmed cell death. Consequently, these highly dynamic organelles play a central role in maintaining tissue function, and mitochondrial dysfunction is a pivotal factor in the progressive age-related decline in cellular homeostasis and organ function. The current review examines recent advances in understanding the interplay between mitochondrial dysfunction and organ-specific aging. Thereby, we dissect molecular mechanisms underlying mitochondrial impairment associated with the deterioration of organ function, exploring the role of mitochondrial DNA, reactive oxygen species homeostasis, metabolic activity, damage-associated molecular patterns, biogenesis, turnover, and dynamics. We also highlight emerging therapeutic strategies in preclinical and clinical tests that are supposed to rejuvenate mitochondrial function, such as antioxidants, mitochondrial biogenesis stimulators, and modulators of mitochondrial turnover and dynamics. Furthermore, we discuss potential benefits and challenges associated with the use of these interventions, emphasizing the need for organ-specific approaches given the unique mitochondrial characteristics of different tissues. In conclusion, this review highlights the therapeutic potential of addressing mitochondrial dysfunction to mitigate organ-specific aging, focusing on the skin, liver, lung, brain, skeletal muscle, and lung, as well as on the reproductive, immune, and cardiovascular systems. Based on a comprehensive understanding of the multifaceted roles of mitochondria, innovative therapeutic strategies may be developed and optimized to combat biological aging and promote healthy aging across diverse organ systems.


Assuntos
Envelhecimento , Mitocôndrias , Humanos , Envelhecimento/metabolismo , Envelhecimento/fisiologia , Animais , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Especificidade de Órgãos , DNA Mitocondrial/metabolismo , Antioxidantes/farmacologia
20.
Redox Biol ; 64: 102794, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37402332

RESUMO

Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO-/ONOOH. Both reagent ONOO-/ONOOH and SIN-1 (a thermal source of ONOO-/ONOOH) modified Tyr, Trp and Met residues. ONOO-/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO-/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO-/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.


Assuntos
Aterosclerose , Placa Aterosclerótica , Humanos , Oxidantes/metabolismo , Ácido Peroxinitroso/metabolismo , Versicanas/genética , Versicanas/metabolismo , Ácido Hialurônico/metabolismo , Placa Aterosclerótica/metabolismo , Matriz Extracelular/metabolismo , Aterosclerose/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Inflamação/metabolismo
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