Nitro-oleic acid inhibits vascular endothelial inflammatory responses and the endothelial-mesenchymal transition

Background: Inflammatory-mediated pathological processes in the endothelium arise as a consequence of the dysregulation of vascular homeostasis. Of particular importance are mediators produced by stimulated monocytes/macrophages inducing activation of endothelial cells (ECs). This is manifested by excessive soluble pro-inflammatory mediator production and cell surface adhesion molecule expression. Nitro-fatty acids are endogenous products of metabolic and inflammatory reactions that display immuno-regulatory potential and may represent a novel therapeutic strategy to treat inflammatory diseases. The purpose of our study was to characterize the effects of nitro-oleic acid (OA-NO2) on inflammatory responses and the endothelial-mesenchymal transition (EndMT) in ECs that is a consequence of the altered healing phase of the immune response. Methods: The effect of OA-NO2 on inflammatory responses and EndMT was determined in murine macrophages and murine and human ECs using Western blotting, ELISA, immunostaining, and functional assays. Results: OA-NO2 limited the activation of macrophages and ECs by reducing pro-inflammatory cytokine production and adhesion molecule expression through its modulation of STAT. MAPK and NF-kappa B-regulated signaling. OA-NO2 also decreased transforming growth factor-beta-stimulated EndMT and pro-fibrotic phenotype of ECs. These effects are related to the downregulation of Smad2/3. Conclusions: The study shows the pleiotropic effect of OA-NO2 on regulating EC-macrophage interactions during the immune response and suggests a role for OA-NO2 in the regulation of vascular endothelial immune and fibrotic responses arising during chronic inflammation. General significance: These findings propose the OA-NO2 may be useful as a novel therapeutic agent for treatment of cardiovascular disorders associated with dysregulation of the endothelial immune response. (C) 2016 Elsevier B.V. All rights reserved

Nitro-oleic acid regulates growth factor-induced differentiation of bone marrow-derived macrophages

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 (1-5 min) 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 ERR. 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

Asymmetric dimethyl arginine induces pulmonary vascular dysfunction via activation of signal transducer and activator of transcription 3 and stabilization of hypoxia-inducible factor 1-alpha

Pulmonary hypertension (PH), associated with imbalance in vasoactive mediators and massive remodeling of pulmonary vasculature, represents a serious health complication. Despite the progress in treatment, PH patients typically have poor prognoses with severely affected quality of life. Asymmetric dimethyl arginine (ADMA), endogenous inhibitor of endothelial nitric oxide synthase (eNOS), also represents one of the critical regulators of pulmonary vascular functions. The present study describes a novel mechanism of ADMA-induced dysfunction in human pulmonary endothelial and smooth muscle cells. The effect of ADMA was compared with well-established model of hypoxia-induced pulmonary vascular dysfunction. It was discovered for the first time that ADMA induced the activation of signal transducer and activator of transcription 3 (STAT3) and stabilization of hypoxia inducible factor la (HIF-1 alpha) in both types of cells, associated with drastic alternations in normal cellular functions (e.g., nitric oxide production, cell proliferation/Ca2+ concentration, production of pro-inflammatory mediators, and expression of eNOS, DDAH1, and ICAM-1). Additionally, ADMA significantly enhanced the hypoxia-mediated increase in the signaling cascades. In summary, increased ADMA may lead to manifestation of PH phenotype in human endothelial and smooth muscle cells via the STAT3/HIF-1 alpha cascade. Therefore this signaling pathway represents the potential pathway for future clinical interventions in PH. (C) 2015 Elsevier Inc. All rights reserved

Nitro-Oleic Acid Prevents Hypoxia- and Asymmetric Dimethylarginine-Induced Pulmonary Endothelial Dysfunction

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. 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 1 alpha, HIF-1 alpha; endothelial nitric oxide synthase, eNOS; intercellular adhesion molecule-1, ICAM-1) was assessed by the Western blot technique. 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-1 alpha stabilization. In summary, OA-NO2 eliminates the manifestation of hypoxia- and ADMA-mediated endothelial dysfunction in HPAEC via the STAT3/HIF-1 alpha 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

Nitro-oleic acid modulates classical and regulatory activation of macrophages and their involvement in pro-fibrotic responses

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-beta) 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-beta production. These effects are mediated via downregulation of signal transducers and activators of transcription, mitogen-activated protein kinase and nuclear factor-kappa B signaling responses. Finally, OA-NO2 inhibits fibrotic processes in an in vivo model of angiotensin II-induced myocardial fibrosis by attenuating expression of alpha-smooth muscle actin, systemic transforming growth factor-beta 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. (C) 2015 Elsevier Inc. All rights reserved