Inflammation is an immune response triggered by microbial invasion and/or tissue injury. While acute inflammation is directed toward invading pathogens and injured cells, thus enabling tissue regeneration, chronic inflammation can lead to severe pathologies and tissue dysfunction. These processes are linked with macrophage polarization into specific inflammatory "M1-like" or regulatory "M2-like" subsets. Nitro-fatty acids (NO2-FAs), produced endogenously as byproducts of metabolism and oxidative inflammatory conditions, may be useful for treating diseases associated with dysregulated immune homeostasis. The goal of this study was to characterize the role of nitro-oleic acid (OA-NO2) in regulating the functional specialization of macrophages induced by bacterial lipopolysaccharide or interleukin-4, and to reveal specific signaling mechanisms which can account for OA-NO2-dependent modulation of inflammation and fibrotic responses. Our results show that OA-NO2 inhibits lipopolysaccharide-stimulated production of both pro-inflammatory and immunoregulatory cytokines (including transforming growth factor-β) and inhibits nitric oxide and superoxide anion production. OA-NO2 also decreases interleukin-4-induced macrophage responses by inhibiting arginase-I expression and transforming growth factor-β production. These effects are mediated via downregulation of signal transducers and activators of transcription, mitogen-activated protein kinase and nuclear factor-кB signaling responses. Finally, OA-NO2 inhibits fibrotic processes in an in vivo model of angiotensin II-induced myocardial fibrosis by attenuating expression of α-smooth muscle actin, systemic transforming growth factor-β levels and infiltration of both "M1-" and "M2-like" macrophage subsets into afflicted tissue. Overall, the electrophilic fatty acid derivative OA-NO2 modulates a broad range of "M1-" and "M2-like" macrophage functions and represents a potential therapeutic approach to target diseases associated with dysregulated macrophage subsets.
- MeSH
- aktivace makrofágů účinky léků MeSH
- fibróza MeSH
- interleukin-4 farmakologie MeSH
- kultivované buňky MeSH
- kyseliny olejové farmakologie MeSH
- lipopolysacharidy farmakologie MeSH
- myokard patologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- oxid dusnatý biosyntéza MeSH
- PPAR gama fyziologie MeSH
- superoxidy metabolismus MeSH
- transkripční faktor STAT3 fyziologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
AIM: Atrial fibrosis, one of the most striking features in the pathology of atrial fibrillation (AF), is promoted by local and systemic inflammation. Electrophilic fatty acid nitroalkenes, endogenously generated by both metabolic and inflammatory reactions, are anti-inflammatory mediators that in synthetic form may be useful as drug candidates. Herein we investigate whether an exemplary nitro-fatty acid can limit atrial fibrosis and AF. METHODS AND RESULTS: Wild-type C57BL6/J mice were treated for 2 weeks with angiotensin II (AngII) and vehicle or nitro-oleic acid (10-nitro-octadec-9-enoic acid, OA-NO2, 6 mg/kg body weight) via subcutaneous osmotic minipumps. OA-NO2 significantly inhibited atrial fibrosis and depressed vulnerability for AF during right atrial electrophysiological stimulation to levels observed for AngII-naive animals. Left atrial epicardial mapping studies demonstrated preservation of conduction homogeneity by OA-NO2. The protection from fibrotic remodelling was mediated by suppression of Smad2-dependent myofibroblast transdifferentiation and inhibition of Nox2-dependent atrial superoxide formation. CONCLUSION: OA-NO2 potently inhibits atrial fibrosis and subsequent AF. Nitro-fatty acids and possibly other lipid electrophiles thus emerge as potential therapeutic agents for AF, either by increasing endogenous levels through dietary modulation or by administration as synthetic drugs.
- MeSH
- akční potenciály účinky léků MeSH
- angiotensin II farmakologie MeSH
- dusíkaté sloučeniny farmakologie MeSH
- fibrilace síní prevence a kontrola MeSH
- fibróza MeSH
- konexin 43 analýza MeSH
- kultivované buňky MeSH
- kyseliny linolové farmakologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- protein Smad2 antagonisté a inhibitory MeSH
- remodelace síní účinky léků MeSH
- srdeční síně patologie MeSH
- transdiferenciace buněk účinky léků MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
RATIONALE: Pulmonary hypertension (PH) represents a serious health complication accompanied with hypoxic conditions, elevated levels of asymmetric dimethylarginine (ADMA), and overall dysfunction of pulmonary vascular endothelium. Since the prevention strategies for treatment of PH remain largely unknown, our study aimed to explore the effect of nitro-oleic acid (OA-NO2), an exemplary nitro-fatty acid (NO2-FA), in human pulmonary artery endothelial cells (HPAEC) under the influence of hypoxia or ADMA. METHODS: HPAEC were treated with OA-NO2 in the absence or presence of hypoxia and ADMA. The production of nitric oxide (NO) and interleukin-6 (IL-6) was monitored using the Griess method and ELISA, respectively. The expression or activation of different proteins (signal transducer and activator of transcription 3, STAT3; hypoxia inducible factor 1α, HIF-1α; endothelial nitric oxide synthase, eNOS; intercellular adhesion molecule-1, ICAM-1) was assessed by the Western blot technique. RESULTS: We discovered that OA-NO2 prevents development of endothelial dysfunction induced by either hypoxia or ADMA. OA-NO2 preserves normal cellular functions in HPAEC by increasing NO production and eNOS expression. Additionally, OA-NO2 inhibits IL-6 production as well as ICAM-1 expression, elevated by hypoxia and ADMA. Importantly, the effect of OA-NO2 is accompanied by prevention of STAT3 activation and HIF-1α stabilization. CONCLUSION: In summary, OA-NO2 eliminates the manifestation of hypoxia- and ADMA-mediated endothelial dysfunction in HPAEC via the STAT3/HIF-1α cascade. Importantly, our study is bringing a new perspective on molecular mechanisms of NO2-FAs action in pulmonary endothelial dysfunction, which represents a causal link in progression of PH. Graphical Abstract ᅟ.
- MeSH
- arginin analogy a deriváty farmakologie MeSH
- arteria pulmonalis cytologie MeSH
- buněčná adheze účinky léků MeSH
- endoteliální buňky účinky léků metabolismus fyziologie MeSH
- faktor 1 indukovatelný hypoxií - podjednotka alfa metabolismus MeSH
- hypoxie buňky účinky léků MeSH
- interleukin-6 metabolismus MeSH
- kultivované buňky MeSH
- kyseliny olejové farmakologie MeSH
- lidé MeSH
- mezibuněčná adhezivní molekula-1 metabolismus MeSH
- oxid dusnatý metabolismus MeSH
- pohyb buněk účinky léků MeSH
- synthasa oxidu dusnatého, typ III metabolismus MeSH
- transkripční faktor STAT3 antagonisté a inhibitory metabolismus MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Caveolins are specific proteins involved in regulation of signal transduction to intracellular space. Still, their contribution to immune functions has not been completely clarified. Thus, we decided to characterize the expression of caveolins in bone marrow-derived macrophages (BMDMs) under resting and inflammatory conditions. The effect of classical activators (lipopolysaccharide, LPS; interferon-gamma, IFN-γ) was further potentiated with hypoxic (5% O2) conditions. The activation of p44/42-extracellular signal-regulated kinases 1 and 2 (ERK1/2) and expression of caveolin-1, -2, and -3, hypoxia inducible factor-1 alpha (HIF-1α), as well as inducible nitric oxide synthase (iNOS) was monitored using the Western blot technique. The production of nitric oxide (NO) and tumor necrosis factor-alpha (TNFα) was analyzed by Griess method or ELISA, respectively. BMDMs were also transfected with siRNA against caveolin-2. Importantly, our study showed for the first time that BMDMs expressed only caveolin-2, and its level decreased after activation of macrophages with LPS, IFN-γ, and/or hypoxia. The expression of caveolin-2 negatively correlates with the iNOS and HIF-1α protein levels, as well as with the LPS/IFN-γ- and hypoxia-induced activation of ERK1/2. We concluded that caveolin-2 is most probably involved in regulation of pro-inflammatory responses of BMDMs, triggered via activation of ERK1/2.
- MeSH
- aktivace makrofágů genetika imunologie MeSH
- cytokiny metabolismus MeSH
- exprese genu * MeSH
- fenotyp MeSH
- hypoxie metabolismus MeSH
- kaveolin 2 genetika metabolismus MeSH
- makrofágy imunologie metabolismus MeSH
- malá interferující RNA genetika MeSH
- mitogenem aktivovaná proteinkinasa 1 metabolismus MeSH
- mitogenem aktivovaná proteinkinasa 3 metabolismus MeSH
- myši MeSH
- oxid dusnatý metabolismus MeSH
- regulace genové exprese MeSH
- RNA interference MeSH
- synthasa oxidu dusnatého, typ II genetika metabolismus MeSH
- viabilita buněk MeSH
- zánět genetika imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Two major effector systems are frequently implicated in the immune and endothelial cell alternations associated with inflammation. They include the enhanced production of reactive oxygen species and diminished bioavailability of nitric oxide (NO). Importantly, these processes can be regulated by endogenously produced methylarginines, inhibitors for NO derived from macrophages and endothelial cells. Therefore, the aim of this study was to show the potential pharmacological intervention of methylarginines (N(G)-methyl-L-arginine, L-NMMA; N(G), N(G)'-dimethyl-L-arginine-symmetric dimethylarginine, SDMA; and N(G), N(G)-dimethyl-L-arginine-asymmetric dimethylarginine, ADMA) in activation of murine peritoneal (RAW 264.7) and alveolar (MHS) macrophages with lipopolysaccharide from Gram-negative bacteria (LPS). The data presented in this study clearly declare that L-NMMA (1-50μM) and ADMA (10-50 μM) significantly inhibited the LPS-induced NO production from macrophages in a concentration-dependent manner. It was demonstrated, for the first time, that the ADMA- and L-NMMA-induced down regulation of NO production was accompanied by reduced expression of mRNA and protein for inducible NO synthase as well as decreased activation of nuclear factor-κB. Importantly, we found a negative correlation between the ADMA-dependent reduction of NO production and ADMA-increased superoxide formation, which indicates that ADMA can negatively affect the balance in LPS-induced macrophage-derived production of reactive mediators. The only effect of SDMA was observed for LPS-triggered superoxide production, which was significantly decreased in its highest concentration (50 μM). In summary, L-NMMA and ADMA can mediate their effects on macrophage activation via regulation of intracellular signaling pathways, which can affect critical functions in activated macrophages.
- MeSH
- alveolární makrofágy účinky léků imunologie metabolismus MeSH
- arginin analogy a deriváty chemie farmakologie MeSH
- buněčné kultury MeSH
- buněčné linie MeSH
- exprese genu účinky léků MeSH
- lipopolysacharidy toxicita MeSH
- myši MeSH
- NF-kappa B antagonisté a inhibitory biosyntéza genetika MeSH
- oxid dusnatý biosyntéza MeSH
- peritoneální makrofágy účinky léků imunologie metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- superoxidy metabolismus MeSH
- synthasa oxidu dusnatého, typ II antagonisté a inhibitory biosyntéza genetika MeSH
- viabilita buněk účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
It is known that cells and organisms can indirectly "sense" changes in L-arginine availability via changes in the activity of various metabolic pathways. However, the mechanism(s) by which genes can be directly regulated by L-arginine in mammalian cells have not yet been elucidated. We investigated the effect of L-arginine in the in vivo model of peritoneal inflammation in mice and in vitro in RAW 264.7 macrophages. A detailed analysis of basic physiological functions and selected intracellular signaling cascades revealed that L-arginine is crucial for the acceleration of macrophage activation by bacterial lipopolysaccharide. L-arginine increased the production of reactive oxygen species, nitric oxide, release of Ca(2+), as well as inducible nitric oxide synthase expression. Interestingly, the effect of L-arginine on macrophage activation was dependent on the phosphorylation of mitogen-activated protein kinases and activity of phospholipase C. In RAW 264.7 cells, L-arginine was shown to modulate the response of macrophages toward lipopolysaccharide via the activation of G-protein-coupled receptors. According to our data, we concluded that L-arginine availability plays a key role in the initiation of intracellular signaling pathways that trigger the lipopolysaccharide-induced inflammatory responses in murine macrophages. Although macrophages are partially stimulated in the absence of extracellular L-arginine, the presence of this amino acid significantly accelerates the sensitivity of macrophages to bacterial endotoxin.
- MeSH
- arginin imunologie farmakologie MeSH
- buněčné linie MeSH
- dieta MeSH
- endotoxiny imunologie MeSH
- extracelulárním signálem regulované MAP kinasy metabolismus MeSH
- fosfolipasy typu C metabolismus MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- oxid dusnatý metabolismus MeSH
- peritoneální makrofágy účinky léků imunologie MeSH
- peritonitida imunologie MeSH
- signální transdukce účinky léků imunologie MeSH
- synthasa oxidu dusnatého genetika metabolismus MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O(2)(∙-) formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O(2)(∙-) and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O(2)(∙-) soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O(2) (∙-) formation in inflammatory macrophages.
- MeSH
- aktivace enzymů MeSH
- arginin imunologie MeSH
- biopteriny analogy a deriváty metabolismus MeSH
- buněčné linie MeSH
- časové faktory MeSH
- Escherichia coli imunologie MeSH
- inhibitory enzymů farmakologie MeSH
- lipopolysacharidy škodlivé účinky MeSH
- makrofágy účinky léků imunologie metabolismus MeSH
- myši MeSH
- NADPH-oxidasy metabolismus MeSH
- NG-nitroargininmethylester farmakologie MeSH
- oxid dusnatý metabolismus MeSH
- respirační vzplanutí MeSH
- superoxidy metabolismus MeSH
- synthasa oxidu dusnatého, typ II antagonisté a inhibitory metabolismus MeSH
- tyrosin analogy a deriváty metabolismus MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
