Nitro-fatty acids (NO2FAs) are endogenously produced electrophiles and NRF2 activators with therapeutic potential. We developed a synthetic protocol combining a Henry reaction and base-promoted β-elimination, yielding ultrapure regio/stereoisomers of nitro-stearic (NO2SA), nitro-oleic (NO2OA), and conjugated/bis-allylic nitro-linoleic (NO2LA) acids. These were tested for NRF2 pathway activation in bone marrow cells under different oxygen conditions. We observed that 9- and 10-NO2OA, and 10-NO2LA increased NRF2 stabilization under hypoxia, while 9- and 10-NO2OA significantly upregulated Hmox1 and Gclm at all oxygen levels. 9- and 10-NO2OA enhanced HO-1 and GCLM proteins independently of oxygen, while 10-NO2LA was oxygen-dependent, boosting HO-1 under hypoxia and GCLM under ambient conditions. Moreover, 10-NO2OA and 10-NO2LA induced NRF2 nuclear translocation. In contrast, the saturated 10-NO2SA, which has lower electron-acceptor ability, was inactive. In summary, these findings suggest the biological activity of NO2FAs is dependent on oxygen level, which could be used in future research of other oxidative stress-dependent pathways.

• MeSH
• Nitro Compounds * pharmacology   chemical synthesis   chemistry   MeSH
• NF-E2-Related Factor 2 * metabolism   MeSH
• Heme Oxygenase-1 metabolism   MeSH
• Cell Hypoxia MeSH
• Linoleic Acids chemical synthesis   chemistry   pharmacology   MeSH
• Oxygen metabolism   MeSH
• Fatty Acids * pharmacology   chemical synthesis   chemistry   MeSH
• Mice MeSH
• Signal Transduction drug effects   MeSH
• Stereoisomerism MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Nitro Compounds * MeSH
• NF-E2-Related Factor 2 * MeSH
• Heme Oxygenase-1 MeSH
• Linoleic Acids MeSH
• Oxygen MeSH
• Fatty Acids * MeSH
• Nfe2l2 protein, mouse MeSH Browser

Nitro-oleic acid (NO2-OA), pluripotent cell-signaling mediator, was recently described as a modulator of the signal transducer and activator of transcription 3 (STAT3) activity. In our study, we discovered new aspects of NO2-OA involvement in the regulation of stem cell pluripotency and differentiation. Murine embryonic stem cells (mESC) or mESC-derived embryoid bodies (EBs) were exposed to NO2-OA or oleic acid (OA) for selected time periods. Our results showed that NO2-OA but not OA caused the loss of pluripotency of mESC cultivated in leukemia inhibitory factor (LIF) rich medium via the decrease of pluripotency markers (NANOG, sex-determining region Y-box 1 transcription factor (SOX2), and octamer-binding transcription factor 4 (OCT4)). The effects of NO2-OA on mESC correlated with reduced phosphorylation of STAT3. Subsequent differentiation led to an increase of the ectodermal marker orthodenticle homolog 2 (Otx2). Similarly, treatment of mESC-derived EBs by NO2-OA resulted in the up-regulation of both neural markers Nestin and β-Tubulin class III (Tubb3). Interestingly, the expression of cardiac-specific genes and beating of EBs were significantly decreased. In conclusion, NO2-OA is able to modulate pluripotency of mESC via the regulation of STAT3 phosphorylation. Further, it attenuates cardiac differentiation on the one hand, and on the other hand, it directs mESC into neural fate.

• Keywords
• STAT3, cardiomyogenesis, mouse embryonic stem cells, neurogenesis, nitro-oleic acid, pluripotency,
• MeSH
• Biomarkers metabolism   MeSH
• Cell Differentiation * drug effects   MeSH
• Nitro Compounds pharmacology   MeSH
• Embryoid Bodies drug effects   metabolism   MeSH
• Myocytes, Cardiac drug effects   metabolism   MeSH
• Oleic Acids pharmacology   MeSH
• Mouse Embryonic Stem Cells cytology   drug effects   metabolism   MeSH
• Mice MeSH
• Neurons cytology   drug effects   metabolism   MeSH
• Organogenesis drug effects   MeSH
• Pluripotent Stem Cells drug effects   metabolism   MeSH
• Signal Transduction drug effects   MeSH
• STAT3 Transcription Factor metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Biomarkers MeSH
• CXA-10 MeSH Browser
• Nitro Compounds MeSH
• Oleic Acids MeSH
• STAT3 Transcription Factor MeSH

Many diseases accompanied by chronic inflammation are connected with dysregulated activation of macrophage subpopulations. Recently, we reported that nitro-fatty acids (NO2-FAs), products of metabolic and inflammatory reactions of nitric oxide and nitrite, modulate macrophage and other immune cell functions. Bone marrow cell suspensions were isolated from mice and supplemented with macrophage colony-stimulating factor (M-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination with NO2-OA for different times. RAW 264.7 macrophages were used for short-term (1-5min) experiments. We discovered that NO2-OA reduces cell numbers, cell colony formation, and proliferation of macrophages differentiated with colony-stimulating factors (CSFs), all in the absence of toxicity. In a case of GM-CSF-induced bone marrow-derived macrophages (BMMs), NO2-OA acts via downregulation of signal transducer and activator of transcription 5 and extracellular signal-regulated kinase (ERK) activation. In the case of M-CSF-induced BMMs, NO2-OA decreases activation of M-CSFR and activation of related PI3K and ERK. Additionally, NO2-OA also attenuates activation of BMMs. In aggregate, we demonstrate that NO2-OA regulates the process of macrophage differentiation and that NO2-FAs represent a promising therapeutic tool in the treatment of inflammatory pathologies linked with increased accumulation of macrophages in inflamed tissues.

• Keywords
• Differentiation, Growth factors, Inflammation, Macrophages, Nitro-fatty acids, Nitro-oleic acid, Signaling pathways,
• MeSH
• Cell Differentiation drug effects   MeSH
• Bone Marrow Cells drug effects   MeSH
• Extracellular Signal-Regulated MAP Kinases genetics   MeSH
• Colony-Stimulating Factors genetics   MeSH
• Phosphatidylinositol 3-Kinases genetics   MeSH
• Oleic Acid administration & dosage   chemistry   MeSH
• Macrophages drug effects   MeSH
• MAP Kinase Signaling System drug effects   MeSH
• Mice MeSH
• Nitric Oxide administration & dosage   chemistry   MeSH
• RAW 264.7 Cells MeSH
• Gene Expression Regulation, Neoplastic drug effects   MeSH
• STAT5 Transcription Factor genetics   MeSH
• Inflammation drug therapy   genetics   pathology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Extracellular Signal-Regulated MAP Kinases MeSH
• Colony-Stimulating Factors MeSH
• Phosphatidylinositol 3-Kinases MeSH
• Oleic Acid MeSH
• Nitric Oxide MeSH
• STAT5 Transcription Factor 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.

• Keywords
• Asymmetric dimethylarginine, Human pulmonary artery endothelial cell, Hypoxia, Nitro-oleic acid, Pulmonary hypertension,
• MeSH
• Arginine analogs & derivatives   pharmacology   MeSH
• Pulmonary Artery cytology   MeSH
• Cell Adhesion drug effects   MeSH
• Endothelial Cells drug effects   metabolism   physiology   MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit metabolism   MeSH
• Cell Hypoxia drug effects   MeSH
• Interleukin-6 metabolism   MeSH
• Cells, Cultured MeSH
• Oleic Acids pharmacology   MeSH
• Humans MeSH
• Intercellular Adhesion Molecule-1 metabolism   MeSH
• Nitric Oxide metabolism   MeSH
• Cell Movement drug effects   MeSH
• Nitric Oxide Synthase Type III metabolism   MeSH
• STAT3 Transcription Factor antagonists & inhibitors   metabolism   MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Arginine MeSH
• Hypoxia-Inducible Factor 1, alpha Subunit MeSH
• HIF1A protein, human MeSH Browser
• IL6 protein, human MeSH Browser
• Interleukin-6 MeSH
• Oleic Acids MeSH
• Intercellular Adhesion Molecule-1 MeSH
• N,N-dimethylarginine MeSH Browser
• NOS3 protein, human MeSH Browser
• Nitric Oxide MeSH
• STAT3 protein, human MeSH Browser
• Nitric Oxide Synthase Type III MeSH
• STAT3 Transcription Factor MeSH