Abstract only
Electrophilic nitro‐fatty acids, the nitration products of unsaturated fatty acids are generated in cardiomyocytes after ischemia/reperfusion injury and protects against myocardial ...infarction. Herein, we determine whether nitro‐oleic acid (OA‐NO
2
) is equally cardioprotective in hypertrophic remodeling. Cardiac hypertrophy were established in mice by transverse aortic constriction (TAC). Mice received either OA‐NO
2
, or OA as non‐nitrated fatty acid control. OA‐NO
2
delivery reduces hypertrophic remodeling and significantly reduces cardiac fibrosis compared to OA. Overall cardiac function, e.g. ejection fraction, is significantly improved by OA‐NO
2
. Electrophilic signiling through Nrf2 is a plausible mechanism of cardioprotection by nitroalkenes. However, in Nrf2 knockout mice, which develop exacerbated cardiac hypertrophy after TAC, OA‐NO
2
delivery persistently confer cardioprotection indicating an alternative pathway for the improved cardiac function mediated by OA‐NO
2
. Rather, OA‐NO
2
primarily targets cardiac fibrosis and extracellular matrix gene expression profiles. Thus, protection against cardiac hypertrophic by nitro‐fatty acids
via
improving cardiac fibrosis supports further promise on nitro‐fatty acids derivatives as therapeutic agents for cardiovascular disease. Supported by the NIH (R01‐HL68878, R01‐HL058115, R01‐AT006822A), and the American Heart Association (10SDG4150085).
Abstract only
Inflammatory responses result in the expression and activation of pro‐oxidant enzymes such as the inducible isoform of nitric oxide synthase (iNOS) and NAPDH oxidases, leading to the ...generation of the nitrating species nitrogen dioxide (
•
NO
2
). Conjugated linoleic acid is a primary substrate for biological lipid nitration in the gastric compartment giving rise to the formation of conjugated nitro‐linoleic acid (NO
2
‐CLA). NO
2
‐CLA exhibit reversible electrophilic reactivity and interact covalently with nucleophilic cysteines in proteins and low molecular weight compounds. This reactivity is pivotal for their biological actions including Nrf2 induction, partial PPARγ agonism, heat shock response activation and TLR4/NF‐κB/STAT1 inhibition. Herein we show that macrophage activation leads to NO
2
‐CLA formation both
in vitro
and
in vivo
, as characterized by high resolution mass spectrometry and quantified using isotopic dilution HPLC‐MSMS. CLA nitration by activated RAW264.7 cells results not only in the dose‐ and time‐dependent formation of the parent NO
2
‐CLA compound but also in the generation of secondary electrophilic and non‐electrophilic b‐oxidation metabolites. The formation of two positional isomers 9‐NO
2
‐CLA and 12‐NO
2
‐CLA with different metabolization rates but similar signaling potency was also observed. Addition of either mixed or pure NO
2
‐CLA positional isomers to cells in culture led to antioxidant response element (ARE) activation and to dose‐dependent expression of Nrf2‐regulated genes. Similarly, NO
2
‐CLA attenuated TNF‐a mediated NF‐kB response element activation and decreased iNOS expression and pro‐inflammatory cytokine release in LPS/IFN‐g activated RAW264.7 cells. To assess the physiological relevance of these observations, a mouse zymosan A peritonitis model that promotes CLA nitration was utilized. Animals were administered vehicle, or synthetic NO
2
‐CLA and then inflammatory cell recruitment and clearance from the peritoneal cavity was monitored. NO
2
‐CLA administration led to decreased recruitment as assessed by flow cytometry. Our work demonstrates that NO
2
‐CLA generation might constitute an endogenous adaptive mechanism capable of modulating the severity of the inflammatory response by favoring resolution and inducing the expression of both cytoprotective and antioxidant genes.
Support or Funding Information
This study was supported by NIH grants R01‐HL123333 (LV), R01‐AT006822 (FJS) and K01‐HL133331 (DAV), and AHA #14GRNT20170024 (FJS).
RESUMEN La utilización subproductos agroindustriales y cereales andinos en elaboración de alimentos funcionales se evidencian en actuales investigaciones. Las barras de cereales se destacan como ...comidas rápidas de elevado valor nutricional. El objetivo de la investigación fue formular barras de cereales con ingredientes de estudio salvado de avena, polvo de cáscara de piña y copos de quinua; con valores adecuados de proteína, firmeza, aceptación sensorial, fibra dietética y compuestos fenólicos. Se aplicó diseño de mezclas simplex con centroide ampliado con rango de trabajo de 0 - 31,34%. Encontrándose efecto significativo del salvado de avena, polvo de cáscara de piña y copos de quinua, sobre las variables respuesta. El modelo apropiado para representar el comportamiento de las variables respuestas fue seleccionado considerando valores p < 0,05; R2 > 0,80 y falta de ajuste > 0,05; siendo el modelo especial cúbico el que mejor ajustó datos para proteína y aceptabilidad general; mientras que para fibra dietética fue el modelo cuadrático. Se determinó que el tratamiento óptimo fue constituido por 4,12% salvado de avena; 10,04% polvo cáscara de piña y 17,18% copos de quinua, presentando contenido de proteína (11,37%), fibra dietética (13,28%) y aceptabilidad general (7,47 puntos) que equivale a percepción muy bueno.
Inflammatory responses result in the activation of pro-oxidant enzymes such as the inducible isoform of nitric oxide synthase (iNOS) and NAPDH oxidases, leading to the generation of the nitrating ...species nitrogen dioxide (●NO2). Conjugated linoleic acid is a primary substrate for biological nitration in the gastric compartment giving rise to the formation of conjugated nitro-linoleic acid (NO2-CLA). Herein we show that macrophage activation leads to NO2-CLA formation both in vitro and in vivo, as determined by selective reaction monitoring analysis using isotopically labeled internal standards and confirmed by high resolution mass spectrometry. CLA nitration by activated RAW264.7 cells results not only in the dose- and time-dependent formation of the parent NO2-CLA compound but also in the generation of secondary electrophilic and non-electrophilic β-oxidation metabolites. The formation of two positional NO2-CLA isomers with different metabolization rates but similar signaling potency was also observed. Addition of either mixed or pure NO2-CLA positional isomers to cells in culture led to antioxidant response element (ARE) activation and to dose-dependent expression of Nrf2-regulated genes. Similarly, NO2-CLA also attenuated TNF-α mediated NF-κB response element activation and decreased iNOS expression and pro-inflammatory cytokine release in LPS/IFN-γ activated RAW264.7 cells. To test the physiological relevance of these observations, a mouse zymosan A peritonitis model that promotes CLA nitration was utilized. Animals were administered vehicle, CLA or synthetic NO2-CLA and then polymorphonuclear cell (PMN) recruitment and clearance from the peritoneal cavity was monitored. Both synthetic and in situ CLA-derived NO2-CLA led to decreased recruitment and enhanced PMN clearance, which coincided with higher expression of Nrf2-dependent genes in peritoneal cells. Our work demonstrates that NO2-CLA generation might constitute an endogenous adaptive mechanism capable of modulating the severity of the inflammatory response by favoring resolution and inducing the expression of both cytoprotective and antioxidant genes.
12/15-Lipoxygenases (LOXs) in monocytes and macrophages generate novel phospholipid-esterified eicosanoids. Here, we report the generation of two additional families of related lipids comprising ...15-ketoeicosatetraenoic acid (KETE) attached to four phosphatidylethanolamines (PEs). The lipids are generated basally by 15-LOX in IL-4-stimulated monocytes, are elevated on calcium mobilization, and are detected at increased levels in bronchoalveolar lavage fluid from cystic fibrosis patients (3.6 ng/ml of lavage). Murine peritoneal macrophages generate 12-KETE-PEs, which are absent in 12/15-LOX-deficient mice. Inhibition of 15-prostaglandin dehydrogenase prevents their formation from exogenous 15-hydroxyeicosatetraenoic acid-PE in human monocytes. Both human and murine cells also generated analogous hydroperoxyeicosatetraenoic acid-PEs. The electrophilic reactivity of KETE-PEs is shown by their Michael addition to glutathione and cysteine. Lastly, both 15-hydroxyeicosatetraenoic acid-PE and 15-KETE-PE activated peroxisome proliferator-activated receptor-γ reporter activity in macrophages in a dose-dependent manner. In summary, we demonstrate novel peroxisome proliferator-activated receptor-γ-activating oxidized phospholipids generated enzymatically by LOX and 15-prostaglandin dehydrogenase in primary monocytic cells and in a human Th2-related lung disease. The lipids are a new family of bioactive mediators from the 12/15-LOX pathway that may contribute to its known anti-inflammatory actions in vivo.
Background: Lipoxygenases (LOXs) generate eicosanoids in inflammation.
Results: Monocyte/macrophage LOXs generate novel phospholipid-esterified eicosanoids containing ketoeicosatetraenoic acid or hydroperoxyeicosatetraenoic acid. They activate peroxisome proliferator-activated receptor-γ transcriptional activity and are found in cystic fibrosis bronchoalveolar fluid.
Significance: LOXs generate esterified eicosanoids in vitro and in vivo.
Conclusion: These new lipids represent new families of bioactive mediators.
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