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1.
Proc Natl Acad Sci U S A ; 120(40): e2215421120, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37756334

RESUMO

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática , Interleucina-27 , Humanos , Camundongos , Animais , Camundongos Endogâmicos C57BL , Histonas , Plaquetas , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/genética
2.
Basic Res Cardiol ; 119(4): 1-18, 2024 08.
Artigo em Inglês | MEDLINE | ID: mdl-38554187

RESUMO

CD40L-CD40-TRAF signaling plays a role in atherosclerosis progression and affects the pathogenesis of coronary heart disease (CHD). We tested the hypothesis that CD40L-CD40-TRAF signaling is a potential therapeutic target in hyperlipidemia, diabetes, and hypertension. In mouse models of hyperlipidemia plus diabetes (db/db mice) or hypertension (1 mg/kg/d angiotensin-II for 7 days), TRAF6 inhibitor treatment (2.5 mg/kg/d for 7 or 14 days) normalized markers of oxidative stress and inflammation. As diabetes and hypertension are important comorbidities aggravating CHD, we explored whether the CD40L-CD40-TRAF signaling cascade and their associated inflammatory pathways are expressed in CHD patients suffering from comorbidities. Therefore, we analyzed vascular bypass material (aorta or internal mammary artery) and plasma from patients with CHD with diabetes and/or hypertension. Our Olink targeted plasma proteomic analysis using the IMMUNO-ONCOLOGY panel revealed a pattern of step-wise increase for 13/92 markers of low-grade inflammation with significant changes. CD40L or CD40 significantly correlated with 38 or 56 other inflammatory targets. In addition, specific gene clusters that correlate with the comorbidities were identified in isolated aortic mRNA of CHD patients through RNA-sequencing. These signaling clusters comprised CD40L-CD40-TRAF, immune system, hemostasis, muscle contraction, metabolism of lipids, developmental biology, and apoptosis. Finally, immunological analysis revealed key markers correlated with comorbidities in CHD patients, such as CD40L, NOX2, CD68, and 3-nitrotyrosine. These data indicate that comorbidities increase inflammatory pathways in CHD, and targeting these pathways will be beneficial in reducing cardiovascular events in CHD patients with comorbidities.


Assuntos
Antígenos CD40 , Ligante de CD40 , Hipertensão , Transdução de Sinais , Humanos , Animais , Ligante de CD40/metabolismo , Hipertensão/imunologia , Hipertensão/metabolismo , Antígenos CD40/metabolismo , Masculino , Inflamação/metabolismo , Inflamação/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Feminino , Pessoa de Meia-Idade , Fator 6 Associado a Receptor de TNF/metabolismo , Idoso , Doença das Coronárias/imunologia , Doença das Coronárias/metabolismo
3.
Pflugers Arch ; 475(7): 807-821, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37285062

RESUMO

Electronic cigarettes (E-cigarettes) have recently become a popular alternative to traditional tobacco cigarettes. Despite being marketed as a healthier alternative, increasing evidence shows that E-cigarette vapour could cause adverse health effects. It has been postulated that degradation products of E-cigarette liquid, mainly reactive aldehydes, are responsible for those effects. Previously, we have demonstrated that E-cigarette vapour exposure causes oxidative stress, inflammation, apoptosis, endothelial dysfunction and hypertension by activating NADPH oxidase in a mouse model. To better understand oxidative stress mechanisms, we have exposed cultured endothelial cells and macrophages to condensed E-cigarette vapour (E-cigarette condensate) and acrolein. In both endothelial cells (EA.hy 926) and macrophages (RAW 264.7), we have observed that E-cigarette condensate incubation causes cell death. Since recent studies have shown that among toxic aldehydes found in E-cigarette vapour, acrolein plays a prominent role, we have incubated the same cell lines with increasing concentrations of acrolein. Upon incubation with acrolein, a translocation of Rac1 to the plasma membrane has been observed, accompanied by an increase in oxidative stress. Whereas reactive oxygen species (ROS) formation by acrolein in cultured endothelial cells was mainly intracellular, the release of ROS in cultured macrophages was both intra- and extracellular. Our data also demonstrate that acrolein activates the nuclear factor erythroid 2-related factor 2 (Nrf2) antioxidant pathway and, in general, could mediate E-cigarette vapour-induced oxidative stress and cell death. More mechanistic insight is needed to clarify the toxicity associated with E-cigarette consumption and the possible adverse effects on human health.


Assuntos
Vapor do Cigarro Eletrônico , Sistemas Eletrônicos de Liberação de Nicotina , Animais , Camundongos , Humanos , Células Endoteliais/metabolismo , Acroleína/toxicidade , Acroleína/metabolismo , Vapor do Cigarro Eletrônico/metabolismo , Vapor do Cigarro Eletrônico/farmacologia , Espécies Reativas de Oxigênio/metabolismo , NADPH Oxidases/metabolismo , Macrófagos/metabolismo , Estresse Oxidativo , Aldeídos/metabolismo , Aldeídos/farmacologia
4.
Cell Commun Signal ; 20(1): 47, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35392923

RESUMO

BACKGROUND: NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet. METHODS: Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5'-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells. RESULTS: In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; "diff-iNOS") differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; "immuno-iNOS"). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5'-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform. CONCLUSIONS: Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. Video Abstract.


Assuntos
Células-Tronco Pluripotentes , Isoformas de RNA , Tetraciclina , Diferenciação Celular , Humanos , Isoenzimas/genética , Nitratos/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
5.
Biochem J ; 476(2): 333-352, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30578289

RESUMO

Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by different proteins binding to these AREs leading to either stabilization or destabilization of the respective target mRNA. The KH-type splicing regulatory protein KSRP (also named KHSRP) is an important negative regulator of ARE-containing mRNAs. Here, we identify the interferon lambda 3 (IFNL3) mRNA as a new KSRP target by pull-down and immunoprecipitation experiments, as well as luciferase reporter gene assays. We characterize the KSRP-binding site in the IFNL3 3'-UTR and demonstrate that KSRP regulates the mRNA half-life of the IFNL3 transcript. In addition, we detect enhanced expression of IFNL3 mRNA in KSRP-/- mice, establishing a negative regulatory function of KSRP in type III IFN expression also in vivo Besides KSRP the RNA-binding protein AUF1 (AU-rich element RNA-binding protein 1) also seems to be involved in the regulation of type III IFN mRNA expression.


Assuntos
Regiões 3' não Traduzidas , Interferons/biossíntese , Splicing de RNA , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Interferons/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a RNA/genética , Transativadores/genética
6.
Nitric Oxide ; 88: 50-60, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004763

RESUMO

The human inducible nitric oxide synthase (iNOS) gene contains an upstream open reading frame (uORF) in its 5'-untranslated region (5'-UTR) implying a translational regulation of iNOS expression. Transfection experiments in human DLD-1 cells revealed that the uORF although translatable seems not to inhibit the translation start at the bona fide ATG. Our data clearly show that human iNOS translation is cap-dependent and that the 5'-UTR of the iNOS mRNA contains no internal ribosome entry site. Translation of the bona fide coding sequence is most likely mediated by a leaky scanning mechanism. The 5'-UTR is encoded by exon 1 and exon 2 of the iNOS gene with the uORF stop codon located in front of the first intron indicating an involvement of the nonsense mediated RNA decay (NMD) in iNOS regulation. SiRNA-mediated down-regulation of Upf1 resulted in enhanced endogenous cytokine iNOS expression in human DLD-1 cells. Transfection of constructs containing iNOS exon 1, intron 1 and exon 2 in front of a luciferase gene showed a clear effect of the mutation of the uORF-ATG on luciferase reportergene expression. Our data indicate that the uORF in the 5'-UTR sequence of human iNOS gene reduces its expression via the NMD mechanism.


Assuntos
Regulação da Expressão Gênica/fisiologia , Óxido Nítrico Sintase Tipo II/metabolismo , Fases de Leitura Aberta/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Regulação para Baixo , Éxons , Humanos , Íntrons , Mutação , Óxido Nítrico Sintase Tipo II/genética , Degradação do RNAm Mediada por Códon sem Sentido/fisiologia , RNA Helicases/genética , RNA Helicases/metabolismo , Transativadores/genética , Transativadores/metabolismo
7.
Nucleic Acids Res ; 42(20): 12555-69, 2014 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-25352548

RESUMO

Resveratrol shows beneficial effects in inflammation-based diseases like cancer, cardiovascular and chronic inflammatory diseases. Therefore, the molecular mechanisms of the anti-inflammatory resveratrol effects deserve more attention. In human epithelial DLD-1 and monocytic Mono Mac 6 cells resveratrol decreased the expression of iNOS, IL-8 and TNF-α by reducing mRNA stability without inhibition of the promoter activity. Shown by pharmacological and siRNA-mediated inhibition, the observed effects are SIRT1-independent. Target-fishing and drug responsive target stability experiments showed selective binding of resveratrol to the RNA-binding protein KSRP, a central post-transcriptional regulator of pro-inflammatory gene expression. Knockdown of KSRP expression prevented resveratrol-induced mRNA destabilization in human and murine cells. Resveratrol did not change KSRP expression, but immunoprecipitation experiments indicated that resveratrol reduces the p38 MAPK-related inhibitory KSRP threonine phosphorylation, without blocking p38 MAPK activation or activity. Mutation of the p38 MAPK target site in KSRP blocked the resveratrol effect on pro-inflammatory gene expression. In addition, resveratrol incubation enhanced KSRP-exosome interaction, which is important for mRNA degradation. Finally, resveratrol incubation enhanced its intra-cellular binding to the IL-8, iNOS and TNF-α mRNA. Therefore, modulation of KSRP mRNA binding activity and, thereby, enhancement of mRNA degradation seems to be the common denominator of many anti-inflammatory effects of resveratrol.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Mediadores da Inflamação/metabolismo , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estilbenos/farmacologia , Transativadores/metabolismo , Animais , Linhagem Celular Tumoral , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Knockout , Mutação , Proteínas de Ligação a RNA/genética , Resveratrol , Transativadores/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
8.
J Biol Chem ; 289(22): 15653-65, 2014 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-24727475

RESUMO

Cardiovascular events are important co-morbidities in patients with chronic inflammatory diseases like rheumatoid arthritis. Tristetraprolin (TTP) regulates pro-inflammatory processes through mRNA destabilization and therefore TTP-deficient mice (TTP(-/-) mice) develop a chronic inflammation resembling human rheumatoid arthritis. We used this mouse model to evaluate molecular signaling pathways contributing to the enhanced atherosclerotic risk in chronic inflammatory diseases. In the aorta of TTP(-/-) mice we observed elevated mRNA expression of known TTP targets like tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1α, as well as of other pro-atherosclerotic mediators, like Calgranulin A, Cathepsin S, and Osteopontin. Independent of cholesterol levels TTP(-/-) mice showed a significant reduction of acetylcholine-induced, nitric oxide-mediated vasorelaxation. The endothelial dysfunction in TTP(-/-) mice was associated with increased levels of reactive oxygen and nitrogen species (RONS), indicating an enhanced nitric oxide inactivation by RONS in the TTP(-/-) animals. The altered RONS generation correlates with increased expression of NADPH oxidase 2 (Nox2) resulting from enhanced Nox2 mRNA stability. Although TNF-α is believed to be a central mediator of inflammation-driven atherosclerosis, genetic inactivation of TNF-α neither improved endothelial function nor normalized Nox2 expression or RONS production in TTP(-/-) animals. Systemic inflammation caused by TTP deficiency leads to endothelial dysfunction. This process is independent of cholesterol and not mediated by TNF-α solely. Thus, other mediators, which need to be identified, contribute to enhanced cardiovascular risk in chronic inflammatory diseases.


Assuntos
Aterosclerose/metabolismo , Células Endoteliais/patologia , Estresse Oxidativo/fisiologia , Tristetraprolina/genética , Fator de Necrose Tumoral alfa/genética , Vasculite/metabolismo , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/genética , Aterosclerose/imunologia , Colesterol/metabolismo , Doença Crônica , Células Endoteliais/metabolismo , Feminino , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , NADPH Oxidase 2 , NADPH Oxidases/metabolismo , Técnicas de Cultura de Órgãos , Estabilidade de RNA/fisiologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vasculite/genética , Vasculite/imunologia
9.
Kidney Int ; 86(4): 780-9, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24717299

RESUMO

Recently oxacyclododecindione (Oxa), a macrocyclic lactone isolated from the imperfect fungus Exserohilum rostratum, has been described as a potent transcription inhibitor of inducible proinflammatory and profibrotic genes in cell culture models. As kidney disease in systemic lupus erythematosus is characterized by aberrant expression of inflammatory mediators and infiltration of immune cells, we investigated the effect of Oxa in MRL-Fas(lpr) mice, a model of systemic lupus erythematosus. These mice develop a spontaneous T-cell and macrophage-dependent autoimmune disease including severe glomerulonephritis that shares features with human lupus. Comparable to the results of in vitro models, we found a reduced expression of the cytokines IFN-γ, IL-6, and TNF-α and the chemokines CCL2, RANTES, and CSF-1 on mRNA and protein level in the kidney of Oxa-treated MRL-Fas(lpr) mice. Accordingly, Oxa treatment reduced the infiltration of immune cells and the frequency of activated proinflammatory T cells in the kidney. Moreover, kidney disease, measured by histopathology, IgG and collagen deposition, and proteinuria, was ameliorated in Oxa-treated MRL-Fas(lpr) mice compared with the control group. Thus, Oxa is a new effective anti-inflammatory compound, which may serve as base structure for the development of new therapeutics for the treatment of chronic inflammatory and/or fibrotic diseases.


Assuntos
Anti-Inflamatórios/uso terapêutico , Citocinas/genética , Citocinas/metabolismo , Nefrite Lúpica/tratamento farmacológico , Compostos Macrocíclicos/uso terapêutico , RNA Mensageiro/metabolismo , Animais , Calgranulina A/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL4/metabolismo , Quimiocina CCL5/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocina CXCL9/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica/efeitos dos fármacos , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Nefrite Lúpica/genética , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , Osteopontina/genética , Análise Serial de Proteínas , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
10.
Eur Heart J ; 34(41): 3206-16, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22555214

RESUMO

AIMS: Isosorbide-5-mononitrate (ISMN) is one of the most frequently used compounds in the treatment of coronary artery disease predominantly in the USA. However, ISMN was reported to induce endothelial dysfunction, which was corrected by vitamin C pointing to a crucial role of reactive oxygen species (ROS) in causing this phenomenon. We sought to elucidate the mechanism how ISMN causes endothelial dysfunction and oxidative stress in vascular tissue. METHODS AND RESULTS: Male Wistar rats (n= 69 in total) were treated with ISMN (75 mg/kg/day) or placebo for 7 days. Endothelin (ET) expression was determined by immunohistochemistry in aortic sections. Isosorbide-5-mononitrate infusion caused significant endothelial dysfunction but no tolerance to ISMN itself, whereas ROS formation and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase activity in the aorta, heart, and whole blood were increased. Isosorbide-5-mononitrate up-regulated the expression of NADPH subunits and caused uncoupling of the endothelial nitric oxide synthase (eNOS) likely due to a down-regulation of the tetrahydrobiopterin-synthesizing enzyme GTP-cyclohydrolase-1 and to S-glutathionylation of eNOS. The adverse effects of ISMN were improved in gp91phox knockout mice and normalized by bosentan in vivo/ex vivo treatment and suppressed by apocynin. In addition, a strong increase in the expression of ET within the endothelial cell layer and the adventitia was observed. CONCLUSION: Chronic treatment with ISMN causes endothelial dysfunction and oxidative stress, predominantly by an ET-dependent activation of the vascular and phagocytic NADPH oxidase activity and NOS uncoupling. These findings may explain at least in part results from a retrospective analysis indicating increased mortality in post-infarct patients in response to long-term treatment with mononitrates.


Assuntos
Endotelina-1/metabolismo , Endotélio Vascular/efeitos dos fármacos , Dinitrato de Isossorbida/análogos & derivados , Doadores de Óxido Nítrico/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Animais , Aorta , GMP Cíclico/metabolismo , Endotelina-1/fisiologia , Inibidores Enzimáticos/farmacologia , Dinitrato de Isossorbida/toxicidade , Masculino , Camundongos , Camundongos Knockout , NADPH Oxidases/metabolismo , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Superóxidos/metabolismo
11.
Eur J Prev Cardiol ; 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39351780

RESUMO

BACKGROUND: Epidemiology links noise to increased risk of metabolic diseases like diabetes and obesity. Translational studies in humans and experimental animals showed that noise causes reactive oxygen species (ROS)-mediated cardiovascular damage. The interaction between noise and diabetes, specifically potential additive adverse effects, remains to be determined. METHODS AND RESULTS: C57BL/6 mice were treated with streptozotocin (i.p. injections, 50 mg/kg/d for 5d) to induce type-1 diabetes, with S961 (subcutaneous osmotic minipumps, 0.57 mg/kg/d for 7d) or fed a high-fat diet (HFD, 20 weeks) to induce type-2 diabetes. Control and diabetic mice were exposed to aircraft noise to an average sound pressure level of 72 dB(A) for 4d. While body weight was unaffected, noise reduced insulin production in all diabetes models. The oral glucose tolerance test showed only an additive aggravation by noise in the HFD model. Noise increased blood pressure and aggravated diabetes-induced aortic, mesenteric, and cerebral arterioles endothelial dysfunction. ROS formation in cerebral arterioles, the aorta, the heart, and isolated mitochondria was consistently increased by noise in all models of diabetes. Mitochondrial respiration was impaired by diabetes and noise, however without additive effects. Noise increased ROS and caused inflammation in adipose tissue in the HFD model. RNA sequencing data and alteration of gene pathway clusters also supported additive damage by noise in the setting of diabetes. CONCLUSION: In all three models of diabetes, aircraft noise exacerbates oxidative stress, inflammation, and endothelial dysfunction in mice with pre-existing diabetes. Thus, noise may potentiate the already increased cardiovascular risk in diabetic patients.


Traffic noise significantly contributes to an increased risk of cardiometabolic diseases (including diabetes and obesity) in the general population via stress hormones, inflammation and oxidative stress, all of which contribute to impaired vascular function and high blood pressure. However, the extent to which noise affects pre-existing diabetes is not sufficiently explained, which prompted us to investigate the molecular mechanisms responsible for noise-mediated exacerbation of cardiometabolic complications in three different animal models with diabetes mellitus: Noise exposure in diabetic mice caused further impairment of insulin signalling, increased blood pressure, and damage of small and large blood vessels as well as oxidative stress in the aorta, brain, and heart.Our functional observations were supported by gene analyses indicating combined effects of noise and diabetes on gene groups related to inflammation and metabolism, suggesting a need for further studies in humans to investigate how noise impacts cardiovascular risk in vulnerable groups such as patients with diabetes.

12.
Am J Pathol ; 180(1): 73-81, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22051774

RESUMO

We recently described a model of inflammatory cardiomyopathy in interferon (IFN)-γ overexpressing transgenic mice stably circulating IFN-γ in the serum referred to as SAP--IFN-γ mice. SAP-IFN-γ transgenic mice show cardiac infiltration by mononuclear leukocytes, culminating in dilated cardiomyopathy characterized by an increase of left ventricular end diastolic diameter and reduction of fractional shortening. We hypothesized that the pathological mechanism underlying SAP-IFN-γ cardiomyopathy might be mediated by (auto)immune processes or tumor necrosis factor (TNF)-α synthesis from IFN-γ-activated macrophages. To verify these hypotheses, we crossed SAP-IFN-γ transgenic mice with immunodeficient Rag1(-/-) or TNF-α(-/-) knockout mice and analyzed the cardiac phenotype of the resulting double-mutant offspring. Immunodeficient Rag1(-/-) SAP-IFN-γ mice had a decreased impaired life span and intensive cardiac inflammatory reactions, showing that the cardiotoxic IFN-γ effect operative in SAP-IFN-γ mice was not mediated by an adaptive immune mechanism. SAP-IFN-γ TNF-α(-/-) hearts showed virtually no histopathological alterations, a significant reduction of cardiac infiltration by CD11c(+) dendritic cells and F4/80(+) macrophages, almost complete normalization of cardiac troponin T levels in serum and of left ventricular end diastolic diameter and fractional shortening, and a dramatic increase of life span, compared with SAP-IFN-γ transgenic controls. Thus, myocarditis and cardiomyopathy developing in IFN-γ-overexpressing transgenic mice is, to a significant degree, mediated by TNF-α. TNF-α-mediated cardiotoxicity in SAP-IFN-γ transgenic mice is independent of changes of apoptosis.


Assuntos
Imunidade Adaptativa/fisiologia , Interferon gama/metabolismo , Macrófagos/imunologia , Miocardite/imunologia , Fator de Necrose Tumoral alfa/fisiologia , Alanina Transaminase/metabolismo , Animais , Apoptose/imunologia , Autoimunidade/fisiologia , Doença Crônica , Citocinas/metabolismo , Ecocardiografia , Feminino , Inativação Gênica/fisiologia , Hepatite/imunologia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Fator de Necrose Tumoral alfa/genética
13.
Nitric Oxide ; 30: 49-59, 2013 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-23471078

RESUMO

Human inducible nitric oxide synthase (iNOS) is regulated on the expressional level mostly by post-transcriptional mechanisms modulating the mRNA stability. Another important step in the control of eukaryotic gene expression is the nucleocytoplasmic mRNA transport. Most cellular mRNAs are exported via the TAP/Nxt complex of proteins. However, some mRNAs are transported by a different mechanism involving the nuclear export receptor CRM1. Treatment of DLD-1 cells with the CRM1 inhibitor leptomycin B (LMB) or anti-CRM1 siRNAs reduced cytokine-induced iNOS expression. We could demonstrate that the iNOS mRNA is exported from the nucleus in a CRM1-dependent manner. Since CRM1 itself does not possess any RNA binding affinity, an adapter protein is needed to mediate CRM1-dependent mRNA export. Western blot experiments showed that the eukaryotic translation initiation factor eIF4E is retained in the nucleus after LMB treatment. Blockade of eIF4E by ribavirin or overexpression of the promyelocytic leukemia protein (PML) decreased iNOS expression due to reduced iNOS mRNA export from the nucleus. Transfection experiments provide evidence that the 3'-untranslated region of the iNOS mRNA is involved in eIF4E-mediated iNOS mRNA transport. In summary, CRM1 and eIF4E seem to play an important role in the nucleocytoplasmic export of human iNOS mRNA.


Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Carioferinas/metabolismo , Óxido Nítrico Sintase Tipo II/biossíntese , Transporte de RNA , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transporte Ativo do Núcleo Celular , Análise de Variância , Linhagem Celular Tumoral , Fator de Iniciação 4E em Eucariotos/antagonistas & inibidores , Humanos , Luciferases/genética , Luciferases/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ribavirina/farmacologia , Proteína Exportina 1
14.
Nitric Oxide ; 32: 29-35, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23583951

RESUMO

Many of the cardiovascular protective effects of resveratrol are attributable to an enhanced production of nitric oxide (NO) by the endothelial NO synthase (eNOS). Resveratrol has been shown to enhance eNOS gene expression as well as eNOS enzymatic activity. The aim of the present study was to analyze the molecular mechanisms of eNOS transcriptional activation by resveratrol. Treatment of human EA.hy 926 endothelial cells with resveratrol led to a concentration-dependent upregulation of eNOS expression. In luciferase reporter gene assay, resveratrol enhanced the activity of human eNOS promoter fragments (3500, 1600, 633 and 263bp in length, respectively), indicating that the proximal promoter region is required for resveratrol-induced eNOS transcriptional activation. Knockdown of the NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1) by siRNA prevented the upregulation of eNOS mRNA and protein by resveratrol. Forkhead box O (FOXO) transcription factors are established downstream targets of SIRT1. siRNA-mediated knockdown of FOXO1 and FOXO3a abolished the effect of resveratrol on eNOS expression, indicating the involvement of these factors. Resveratrol treatment enhanced the expression of FOXO1 and FOXO3a in EA.hy 926 cells. Reporter gene assay using promoter containing forkhead response elements showed increased FOXO factor activity by resveratrol. In electrophoretic mobility shift assay, the enhanced binding of nuclear proteins to the eNOS promoter regions by resveratrol could be blocked by antibodies against FOXO1 and FOXO3a. In conclusion, resveratrol enhances the expression and activity of FOXO transcription factors. The SIRT1/FOXO factor axis is involved in resveratrol-induced eNOS transcriptional activation.


Assuntos
Fatores de Transcrição Forkhead/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Ativação Transcricional/efeitos dos fármacos , Análise de Variância , Linhagem Celular , Fatores de Transcrição Forkhead/genética , Técnicas de Silenciamento de Genes , Humanos , Óxido Nítrico Sintase Tipo III/genética , Regiões Promotoras Genéticas , RNA Interferente Pequeno/genética , Resveratrol , Sirtuína 1/genética , Regulação para Cima/efeitos dos fármacos
15.
Nitric Oxide ; 33: 6-17, 2013 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-23711718

RESUMO

Affinity purification using the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide synthase (iNOS) mRNA identified the cytosolic poly(A)-binding protein (PABP) as a protein interacting with the human iNOS 3'-UTR. Downregulation of PABP expression by RNA interference resulted in a marked reduction of cytokine-induced iNOS mRNA expression without changes in the expression of mRNAs coding for the major subunit of the RNA polymerase II (Pol 2A) or ß2-microglobuline (ß2M). Along with the mRNA also iNOS protein expression was reduced by siPABP-treatment, whereas in the same cells protein expression of STAT-1α, NF-κB p65, or GAPDH was not altered. Reporter gene analyses showed no change of the inducibility of the human 16kb iNOS promoter in siPABP cells. In contrast, the siPABP-mediated decline of iNOS expression correlated with a reduction in the stability of the iNOS mRNA. As the stability of the Pol 2A and ß2M mRNA was not changed, siPABP-treatment seems to have a specific effect on iNOS mRNA decay. UV-crosslinking experiments revealed that PABP interacts with one binding site in the 5'-UTR and two different binding sites in the 3'-UTR of the human iNOS mRNA. Mutation or deletion of the binding site in the 5'-UTR but not in the 3'-UTR reduced luciferase expression in DLD-1 cells transfected with iNOS-5'-UTR or iNOS-3'-UTR luciferase reporter constructs. In summary, our data demonstrate that PABP by binding to specific sequence elements in the 5'-UTR post-transcriptionally enhances human iNOS mRNA stability and thereby iNOS expression.


Assuntos
Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas , Sítios de Ligação , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/genética , Citocinas/metabolismo , Regulação para Baixo , Humanos , Mutação , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Proteínas de Ligação a Poli(A)/genética , RNA Mensageiro/química , RNA Mensageiro/genética
16.
Redox Biol ; 59: 102580, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36566737

RESUMO

Worldwide, up to 8.8 million excess deaths/year have been attributed to air pollution, mainly due to the exposure to fine particulate matter (PM). Traffic-related noise is an additional contributor to global mortality and morbidity. Both health risk factors substantially contribute to cardiovascular, metabolic and neuropsychiatric sequelae. Studies on the combined exposure are rare and urgently needed because of frequent co-occurrence of both risk factors in urban and industrial settings. To study the synergistic effects of PM and noise, we used an exposure system equipped with aerosol generator and loud-speakers, where C57BL/6 mice were acutely exposed for 3d to either ambient PM (NIST particles) and/or noise (aircraft landing and take-off events). The combination of both stressors caused endothelial dysfunction, increased blood pressure, oxidative stress and inflammation. An additive impairment of endothelial function was observed in isolated aortic rings and even more pronounced in cerebral and retinal arterioles. The increase in oxidative stress and inflammation markers together with RNA sequencing data indicate that noise particularly affects the brain and PM the lungs. The combination of both stressors has additive adverse effects on the cardiovascular system that are based on PM-induced systemic inflammation and noise-triggered stress hormone signaling. We demonstrate an additive upregulation of ACE-2 in the lung, suggesting that there may be an increased vulnerability to COVID-19 infection. The data warrant further mechanistic studies to characterize the propagation of primary target tissue damage (lung, brain) to remote organs such as aorta and heart by combined noise and PM exposure.


Assuntos
COVID-19 , Sistema Cardiovascular , Camundongos , Animais , Material Particulado/efeitos adversos , Camundongos Endogâmicos C57BL , Inflamação/induzido quimicamente , Estresse Oxidativo , Aeronaves
17.
Cardiovasc Res ; 119(6): 1416-1426, 2023 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-36702626

RESUMO

AIMS: Traffic noise may play an important role in the development and deterioration of ischaemic heart disease. Thus, we sought to determine the mechanisms of cardiovascular dysfunction and inflammation induced by aircraft noise in a mouse model of myocardial infarction (MI) and in humans with incident MI. METHODS AND RESULTS: C57BL/6J mice were exposed to noise alone (average sound pressure level 72 dB; peak level 85 dB) for up to 4 days, resulting in pro-inflammatory aortic gene expression in the myeloid cell adhesion/diapedesis pathways. The noise alone promoted adhesion and infiltration of inflammatory myeloid cells in vascular/cardiac tissue, paralleled by an increased percentage of leucocytes with a pro-inflammatory, reactive oxygen species (ROS)-producing phenotype and augmented expression of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase type 2 (Nox2)/phosphorylation of nuclear factor 'kappa light chain enhancer' of activated B-cells (phospho-NFκB) in peripheral blood. Ligation of the left anterior descending artery resulted in worsening of cardiac function, pronounced cardiac infiltration of CD11b+ myeloid cells and Ly6Chigh monocytes, and induction of interleukin (IL) 6, IL-1ß, CCL-2, and Nox2, being aggravated by noise exposure prior to MI. MI induced stronger endothelial dysfunction and more pronounced increases in vascular ROS in animals preconditioned with noise. Participants of the population-based Gutenberg Health Cohort Study (median follow-up:11.4 years) with incident MI revealed elevated C-reactive protein at baseline and worse left ventricular ejection fraction (LVEF) after MI in case of a history of noise exposure and subsequent annoyance development. CONCLUSION: Aircraft noise exposure before MI substantially amplifies subsequent cardiovascular inflammation and aggravates ischaemic heart failure, facilitated by a pro-inflammatory vascular conditioning. Our translational results suggest that measures to reduce environmental noise exposure will be helpful in improving the clinical outcome of subjects with MI.Key questionKey finding Take-home-MessageAircraft noise exposure before MI substantially amplifies cardiovascular inflammation and aggravates cardiac impairment after MI.


Assuntos
Infarto do Miocárdio , Função Ventricular Esquerda , Animais , Camundongos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Estudos de Coortes , Volume Sistólico , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Inflamação , Aeronaves
18.
Eur J Prev Cardiol ; 30(15): 1554-1568, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37185661

RESUMO

AIMS: Environmental stressors such as traffic noise represent a global threat, accounting for 1.6 million healthy life years lost annually in Western Europe. Therefore, the noise-associated health side effects must be effectively prevented or mitigated. Non-pharmacological interventions such as physical activity or a balanced healthy diet are effective due to the activation of the adenosine monophosphate-activated protein kinase (α1AMPK). Here, we investigated for the first time in a murine model of aircraft noise-induced vascular dysfunction the potential protective role of α1AMPK activated via exercise, intermittent fasting, and pharmacological treatment. METHODS AND RESULTS: Wild-type (B6.Cg-Tg(Cdh5-cre)7Mlia/J) mice were exposed to aircraft noise [maximum sound pressure level of 85 dB(A), average sound pressure level of 72 dB(A)] for the last 4 days. The α1AMPK was stimulated by different protocols, including 5-aminoimidazole-4-carboxamide riboside application, voluntary exercise, and intermittent fasting. Four days of aircraft noise exposure produced significant endothelial dysfunction in wild-type mice aorta, mesenteric arteries, and retinal arterioles. This was associated with increased vascular oxidative stress and asymmetric dimethylarginine formation. The α1AMPK activation with all three approaches prevented endothelial dysfunction and vascular oxidative stress development, which was supported by RNA sequencing data. Endothelium-specific α1AMPK knockout markedly aggravated noise-induced vascular damage and caused a loss of mitigation effects by exercise or intermittent fasting. CONCLUSION: Our results demonstrate that endothelial-specific α1AMPK activation by pharmacological stimulation, exercise, and intermittent fasting effectively mitigates noise-induced cardiovascular damage. Future population-based studies need to clinically prove the concept of exercise/fasting-mediated mitigation of transportation noise-associated disease.


Traffic noise, e.g. from aircraft, significantly contributes to an increased risk of cardiovascular or metabolic diseases in the general population by brain-dependent stress reactions leading to higher levels of circulating stress hormones and vasoconstrictors, all of which cause hypertension, oxidative stress, and inflammation. With the present experimental studies, we provide for the first time molecular mechanisms responsible for successful noise mitigation: Physical exercise, intermittent fasting, and pharmacological activation of the adenosine monophosphate-activated protein kinase (AMPK), a metabolic master regulator protein, prevent cardiovascular damage caused by noise exposure, such as hypertension, endothelial dysfunction, and reactive oxygen species formation (e.g. free radicals) and inflammation.These beneficial mitigation manoeuvers are secondary to an activation of the endothelial AMPK, thereby mimicking the antidiabetic drug metformin.


Assuntos
Endotélio Vascular , Ruído dos Transportes , Humanos , Camundongos , Animais , Endotélio Vascular/metabolismo , Estresse Oxidativo , Ruído dos Transportes/efeitos adversos , Jejum , Aeronaves , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Quinases Ativadas por AMP/farmacologia
19.
J Biol Chem ; 286(11): 8893-900, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21252222

RESUMO

Recently, mitochondrial aldehyde dehydrogenase (ALDH-2) was reported to reduce ischemic damage in an experimental myocardial infarction model. ALDH-2 activity is redox-sensitive. Therefore, we here compared effects of various electrophiles (organic nitrates, reactive fatty acid metabolites, or oxidants) on the activity of ALDH-2 with special emphasis on organic nitrate-induced inactivation of the enzyme, the biochemical correlate of nitrate tolerance. Recombinant human ALDH-2 was overexpressed in Escherichia coli; activity was determined with an HPLC-based assay, and reactive oxygen and nitrogen species formation was determined by chemiluminescence, fluorescence, protein tyrosine nitration, and diaminonaphthalene nitrosation. The organic nitrate glyceryl trinitrate caused a severe concentration-dependent decrease in enzyme activity, whereas incubation with pentaerythritol tetranitrate had only minor effects. 4-Hydroxynonenal, an oxidized prostaglandin J(2), and 9- or 10-nitrooleate caused a significant inhibition of ALDH-2 activity, which was improved in the presence of Mg(2+) and Ca(2+). Hydrogen peroxide and NO generation caused only minor inhibition of ALDH-2 activity, whereas peroxynitrite generation or bolus additions lead to severe impairment of the enzymatic activity, which was prevented by the thioredoxin/thioredoxin reductase (Trx/TrxR) system. In the presence of glyceryl trinitrate and to a lesser extent pentaerythritol tetranitrate, ALDH-2 may be switched to a peroxynitrite synthase. Electrophiles of different nature potently regulate the enzymatic activity of ALDH-2 and thereby may influence the resistance to ischemic damage in response to myocardial infarction. The Trx/TrxR system may play an important role in this process because it not only prevents inhibition of ALDH-2 but is also inhibited by the ALDH-2 substrate 4-hydroxynonenal.


Assuntos
Aldeído Desidrogenase/química , Peróxido de Hidrogênio/química , Proteínas Mitocondriais/química , Óxido Nítrico/química , Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Aldeído-Desidrogenase Mitocondrial , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Óxido Nítrico/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
J Pharmacol Exp Ther ; 343(1): 106-14, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22767531

RESUMO

In previous studies, we identified the fungal macrocyclic lactone (S)-curvularin (SC) as an anti-inflammatory agent using a screening system detecting inhibitors of the Janus kinase/signal transducer and activator of transcription pathway. The objective of the present study was to investigate whether SC is able to decrease proinflammatory gene expression in an in vivo model of a chronic inflammatory disease. Therefore, the effects of SC and dexamethasone were compared in the model of collagen-induced arthritis (CIA) in mice. Total genomic microarray analyses were performed to identify SC target genes. In addition, in human C28/I2 chondrocytes and MonoMac6 monocytes, the effect of SC on proinflammatory gene expression was tested at the mRNA and protein level. In the CIA model, SC markedly reduced the expression of a number of proinflammatory cytokines and chemokines involved in the pathogenesis of CIA as well as human rheumatoid arthritis (RA). In almost all cases, the effects of SC were comparable with those of dexamethasone. In microarray analyses, we identified additional new therapeutic targets of SC. Some of them, such as S100A8, myeloperoxidase, or cathelicidin, an antimicrobial peptide, are known to be implicated in pathophysiological processes in RA. Similar anti-inflammatory effects of SC were also observed in human C28/I2 chondrocyte cells, which are resistant to glucocorticoid treatment. These data indicate that SC and glucocorticoid effects are mediated via independent signal transduction pathways. In summary, we demonstrate that SC is a new effective anti-inflammatory compound that may serve as a lead compound for the development of new drugs for the therapy of chronic inflammatory diseases.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/fisiologia , Zearalenona/análogos & derivados , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Reumatoide/genética , Linhagem Celular Transformada , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Zearalenona/farmacologia , Zearalenona/uso terapêutico
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