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

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.

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 1α (HIF-1α) in both types of cells, associated with drastic alternations in normal cellular functions (e.g., nitric oxide production, cell proliferation/Ca(2+) 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-1α cascade. Therefore this signaling pathway represents the potential pathway for future clinical interventions in PH.

Asymmetric dimethylarginine regulates the lipopolysaccharide-induced nitric oxide production in macrophages by suppressing the activation of NF-kappaB and iNOS expression.

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.

The unique role of dietary L-arginine in the acceleration of peritoneal macrophage sensitivity to bacterial endotoxin.

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.