Tomonaga Ichikawa

Nitrated Fatty Acids: Endogenous Anti-inflammatory Signaling Mediators

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-γ activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-κB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-κB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-α-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.

Diabetic Downregulation of Nrf2 Activity via ERK Contributes to Oxidative Stress–Induced Insulin Resistance in Cardiac Cells In Vitro and In Vivo

OBJECTIVE Oxidative stress is implicated in cardiac insulin resistance, a critical risk factor for cardiac failure, but the direct evidence remains missing. This study explored a causal link between oxidative stress and insulin resistance with a focus on a regulatory role of redox sensitive transcription factor NF-E2–related factor 2 (Nrf2) in the cardiac cells in vitro and in vivo. RESEARCH DESIGN AND METHODS Chronic treatment of HL-1 adult cardiomyocyte with hydrogen peroxide led to insulin resistance, reflected by a significant suppression of the insulin-induced glucose uptake. This was associated with an exaggerated phosphorylation of extracellular signal–related kinase (ERK). Although U0126, an ERK inhibitor, enhanced insulin sensitivity and attenuated oxidative stress–induced insulin resistance, LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), worsened the insulin resistance. Moreover, insulin increased Nrf2 transcriptional activity, which was blocked by LY294002 but enhanced by U0126. Forced activation of Nrf2 by adenoviral over-expression of Nrf2 inhibited the increased ERK activity and recovered the blunted insulin sensitivity on glucose uptake in cardiomyocytes that were chronically treated with H2O2. In the hearts of streptozotocin-induced diabetic mice and diabetic patients Nrf2 expression significantly decreased along with significant increases in 3-nitrotyrosine accumulation and ERK phosphorylation, whereas these pathogenic changes were not observed in the heart of diabetic mice with cardiac-specific overexpression of a potent antioxidant metallothionein. Upregulation of Nrf2 by its activator, Dh404, in cardiomyocytes in vitro and in vivo prevented hydrogen peroxide– and diabetes-induced ERK activation and insulin-signaling downregulation. CONCLUSIONS ERK-mediated suppression of Nrf2 activity leads to the oxidative stress–induced insulin resistance in adult cardiomyocytes and downregulated glucose utilization in the diabetic heart.

Nrf2 Protects Against Maladaptive Cardiac Responses to Hemodynamic Stress

Background—Reactive oxygen species (ROS) play an important role in the maintenance of cardiovascular homeostasis. The present study sought to determine whether nuclear factor erythroid-2 related factor 2 (Nrf2), a master gene of the endogenous antioxidant defense system, is a critical regulator of the cardiac hypertrophic response to pathological stress. Methods and Results—Cardiac hypertrophy and dysfunction were established in mice by transverse aortic constriction (TAC). Nrf2 expression was transiently increased and then declined to the basal level while impairment of cardiac function proceeded. The knockout of Nrf2 (Nrf2−/−) did not cause any apparent structural and functional abnormalities in the unstressed heart. However, Nrf2−/− mice after TAC developed pathological cardiac hypertrophy, significant myocardial fibrosis and apoptosis, overt heart failure, and increased mortality, which were associated with elevated myocardial levels of 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine and a complete blockade of the myocardial expression of several antioxidant genes. Overexpression of Nrf2 dramatically inhibited hypertrophic factor–induced ROS production and growth in both cardiomyocytes and cardiac fibroblasts, whereas knockdown of Nrf2 exerted opposite effects in both cells. Conclusions—These findings demonstrate that activation of Nrf2 provides a novel mechanism to protect the murine heart against pathological cardiac hypertrophy and heart failure via suppressing oxidative stress.

Targeting the Nrf2 pathway against cardiovascular disease

Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor that controls the basal and inducible expression of a battery of antioxidant genes and other cytoprotective Phase II detoxifying enzymes. Nrf2 is ubiquitously expressed in the cardiovascular system. While several Nrf2 downstream genes have been implicated in protection against the pathogenesis of cardiovascular diseases, the precise role of Nrf2 in the cardiovascular system remains to be elucidated. Nevertheless, mounting evidence has revealed that Nrf2 is a critical regulator of cardiovascular homeostasis via the suppression of oxidative stress, a major causative factor for the development and progression of cardiovascular diseases. Therefore, Nrf2 promises to be an attractive therapeutic target for the treatment of cardiovascular disease. Herein, we review the current literature that suggests that Nrf2 is a valuable therapeutic target for cardiovascular disease, as well as experiments that illustrate the mechanisms of Nrf2 cardioprotection.

Dihydro-CDDO-trifluoroethyl amide (dh404), a novel Nrf2 activator, suppresses oxidative stress in cardiomyocytes.

Targeting Nrf2 signaling appears to be an attractive approach for the treatment of maladaptive cardiac remodeling and dysfunction; however, pharmacological modulation of the Nrf2 pathway in the cardiovascular system remains to be established. Herein, we report that a novel synthetic triterpenoid derivative, dihydro-CDDO-trifluoroethyl amide (dh404), activates Nrf2 and suppresses oxidative stress in cardiomyocytes. Dh404 interrupted the Keap1-Cul3-Rbx1 E3 ligase complex-mediated Nrf2 ubiquitination and subsequent degradation saturating the binding capacity of Keap1 to Nrf2, thereby rendering more Nrf2 to be translocated into the nuclei to activate Nrf2-driven gene transcription. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 was resistant to dh404-induced stabilization of Nrf2 protein. In addition, dh404 did not dissociate the interaction of Nrf2 with the Keap1-Cul3-Rbx1 E3 ligase complex. Thus, it is likely that dh404 inhibits the ability of Keap1-Cul3-Rbx1 E3 ligase complex to target Nrf2 for ubiquitination and degradation via modifying Cys-151 of Keap1 to change the conformation of the complex. Moreover, dh404 was able to stabilize Nrf2 protein, to enhance Nrf2 nuclear translocation, to activate Nrf2-driven transcription, and to suppress angiotensin II (Ang II)-induced oxidative stress in cardiomyocytes. Knockdown of Nrf2 almost blocked the anti-oxidative effect of dh404. Dh404 activated Nrf2 signaling in the heart. Taken together, dh404 appears to be a novel Nrf2 activator with a therapeutic potential for cardiac diseases via suppressing oxidative stress.

Nitroalkenes Suppress Lipopolysaccharide-Induced Signal Transducer and Activator of Transcription Signaling in Macrophages: A Critical Role of Mitogen-Activated Protein Kinase Phosphatase 1

Nitration products of unsaturated fatty acids are formed via NO-dependent oxidative reactions and appear to be a new class of endogenous antiinflammatory mediators. Nitroalkene derivatives of nitrated linoleic acid (LNO(2)) and nitrated oleic acid (OA-NO(2)) alleviate inflammatory responses in macrophages, but the underlying mechanisms remain to be fully defined. Herein we report that LNO(2) and OA-NO(2) suppress proinflammatory signal transducer and activator of transcription (STAT) signaling in macrophages. In RAW264.7 cells, a murine macrophage cell line, LNO(2) and OA-NO(2) inhibited the lipopolysaccharide (LPS)-induced STAT1 phosphorylation and the STAT1-dependent transcriptional activity, thereby suppressing expression of its target gene such as iNOS and MCP-1. The nitroalkene-mediated inhibition of STAT1 activity was not affected by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (a NO scavenger), GW9662 (a peroxisome proliferator-activated receptor-gamma-specific antagonist) or glutathione (an antioxidant), suggesting an underlying mechanism independent of NO, peroxisome proliferator-activated receptor-gamma, or thio-nitralkylation. In contrast, LNO(2) or OA-NO(2) alone up-regulated both mRNA and protein levels of MAPK phosphatase 1 (MKP-1) and strongly augmented the LPS-induced MKP-1 protein expression. Knockdown of MKP-1 by MKP-1 small interfering RNA enhanced the LPS-induced STAT1 phosphorylation, suggesting that MKP-1 acts as a negative regulator for LPS-induced STAT signaling. In addition, the nitroalkene-mediated inhibitory effects on STAT1 phosphorylation, iNOS expression, and MCP-1 secretion were also largely attenuated by the MKP-1 small interfering RNA approach. Taken together, our data demonstrate that nitroalkenes inhibit proinflammatory STAT signaling through inducting MKP-1 in macrophages.

Electrophilic nitro-fatty acids inhibit vascular inflammation by disrupting LPS-dependent TLR4 signalling in lipid rafts

AIMS Electrophilic fatty acid nitroalkene derivatives, products of unsaturated fatty acid nitration, exert long-term cardiovascular protection in experimental models of metabolic and cardiovascular diseases. The goal of this study is to examine the effects of nitro-fatty acids in the regulation of upstream signalling events in nuclear factor-κB (NF-κB) activation and determine whether low-dose acute administration of nitro-fatty acids reduces vascular inflammation in vivo. METHODS AND RESULTS Using NF-κB-luciferase transgenic mice, it was determined that pre-emptive treatment with nitro-oleic acid (OA-NO2), but not oleic acid (OA) inhibits lipopolysaccharide (LPS)-induced NF-κB activation both in vivo and in isolated macrophages. Acute intravenous administration of OA-NO2 was equally effective to inhibit leukocyte recruitment to the vascular endothelium assessed by intravital microscopy and significantly reduces aortic expression of adhesion molecules. An acute treatment with OA-NO2 in vivo yielding nanomolar concentrations in plasma, is sufficient to inhibit LPS-induced Toll-like receptor 4 (TLR4)-induced cell surface expression in leukocytes and NF-κB activation. In vitro experiments reveal that OA-NO2 suppresses LPS-induced TLR4 signalling, inhibitor of κB (IκBα) phosphorylation and ubiquitination, phosphorylation of the IκB kinase (IKK), impairing the recruitment of the TLR4 and TNF receptor associated factor 6 (TRAF6) to the lipid rafts compartments. CONCLUSION These studies demonstrate that acute administration of nitro-fatty acids is effective to reduce vascular inflammation in vivo. These findings reveal a direct role of nitro-fatty acids in the disruption of the TLR4 signalling complex in lipid rafts, upstream events of the NF-κB pathway, leading to resolution of pro-inflammatory activation of NF-κB in the vasculature.

Ubiquitin Carboxyl-Terminal Hydrolase L1, a Novel Deubiquitinating Enzyme in the Vasculature, Attenuates NF-κB Activation

Objective— We identified a ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) gene, which encodes a deubiquitinating enzyme and is expressed in the vasculature, by functional screening of a human endothelial cell (EC) cDNA library. UCHL1 is expressed in neurons, and abnormalities in UCHL1 are responsible for inherited Parkinson’s disease via its effects on the ubiquitin-proteasome system. Therefore, the goal of present study was to clarify the role of the UCHL1 gene in vascular remodeling by evaluating nuclear factor-&kgr;B (NF-&kgr;B) inactivation in ECs and vascular smooth muscle cells (VSMCs). Methods and Results— From Northern blot and immunohistochemical analysis, the UCHL1 gene was endogenously expressed in vascular ECs, VSMCs, and brain tissue. Expression of UCHL1 was markedly increased in the neointima of the balloon-injured carotid artery and was also present in atherosclerotic lesions from human carotid arteries. Overexpression of the UCHL1 gene significantly attenuated tumor necrosis factor (TNF)-α–induced NF-&kgr;B activity in vascular cells and increased inhibitor of kappa B-α (I&kgr;B-α), possibly through the attenuation of I&kgr;B-α ubiquitination, leading to decreased neointima in the balloon-injured artery. In contrast, knockdown of UCHL1 by small interfering RNA resulted in increased NF-&kgr;B activity in VSMCs. Conclusions— These data suggest that UCHL1 may partially attenuate vascular remodeling through inhibition of NF-&kgr;B activity.

An essential role of Nrf2 in American ginseng-mediated anti-oxidative actions in cardiomyocytes.

AIM OF THE STUDY Ginseng has been used as a folk medicine for thousands of years in Asia, and has become a popular herbal medicine world-wide. Recent studies have revealed that ginseng, including American ginseng, exerts antioxidant effects in the cardiovascular system; however, the underlying mechanisms are not fully understood. Thus, we investigated role of Nrf2, a master transcription factor of endogenous anti-oxidative defense systems, in the regulation of American ginseng-mediated anti-oxidative actions in cardiomyocytes. MATERIALS AND METHODS A standardized crude extract of American ginseng was supplied by the National Research Council of Canada, Institute for National Measurement Standards. H9C2 cells, a rat cardiomyocyte cell line, were exposed to angiotensin II (Ang II) or tumor necrosis factor alpha (TNFalpha) to induce oxidative stress that was examined by measuring formation of reactive oxygen and nitrogen species. Oxidative stress-induced cell death was induced by exogenous addition of hydrogen peroxide (H(2)O(2)). Proteins were measured by Western blot and mRNA expression was determined by quantitative real time PCR. Nrf2-driven transcriptional activity was assessed by antioxidant response element (ARE)-luciferase reporter assay. Direct Nrf2 binding to its target gene promoters was determined by chromatin immunoprecipitation assay. Adenoviral over-expression of Nrf2 shRNA was utilized to knock down Nrf2 in H9C2 cells. Immunochemical staining was applied for Nrf2 expression in the heart. RESULTS American ginseng induced dramatic increases in Nrf2 protein expression, Nrf2 nuclear translocation, Nrf2 transcriptional activity, direct Nrf2 binding to its target gene promoters, and expression of a group of anti-oxidative genes driven by Nrf2 in H9C2 cells. In addition, American ginseng inhibited Ang II- or TNFalpha-induced free radical formation and H(2)O(2)-induced cell death in H9C2 cells over-expressed with control shRNA but not in the cells over-expressed with Nrf2 shRNA. Finally, oral administration of American ginseng markedly increased Nrf2 activity in murine hearts. CONCLUSION These results demonstrate that American ginseng suppresses oxidative stress and oxidative stress-induced cell death in cardiomyocytes through activating the Nrf2 pathway, thereby providing cardioprotection against pathological cardiac remodeling.

American ginseng preferentially suppresses STAT/iNOS signaling in activated macrophages

AIM OF THE STUDY Ginseng has been used as general tonic for thousands of years in Asia and becomes a popular herbal medicine all over the world. However, the cellular and molecular mechanisms underlying its benefit effects are less explored. Thus, we investigated the effect of a crude extract from Panax quinquefolius (American ginseng) on suppression of pro-inflammatory responses in macrophages with a focus on signal transducer and activator of transcription (STAT) signaling. MATERIALS AND METHODS The crude extract of American ginseng that was supplied by the National Research Council of Canada, Institute for National Measurement Standards (NRCC-INMS) was freshly solvated in Dulbeccos Modified Eagle Medium (DMEM) prior to each experiment. RAW264.7 cells, a murine macrophage cell line, were exposed to lipopolysaccharide (LPS) to induce inflammatory responses such as expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Proteins were measured by Western blot and mRNA expression was determined by quantitative real-time PCR (Q-PCR). Activator protein 1 (AP-1)-, nuclear factor-kappaB (NF-kappaB)- and STAT-mediated transcriptional activities were investigated using luciferase reporter constructs. RESULTS American ginseng inhibited LPS-induced iNOS expression; however, it did not affect LPS-induced COX2 expression. While American ginseng had no impact on LPS-induced activation of AP-1 or NF-kappaB pathways, it dramatically inhibited LPS-induced activation of STAT signaling. Moreover, American ginseng and AG490, an inhibitor of STAT cascade, synergistically suppressed the LPS-induced iNOS expression. CONCLUSION American ginseng selectively inhibits the expression of iNOS via suppression of STAT cascade but not NF-kappaB and AP-1 pathways in inflamed macrophages. Such a preferential suppression of STAT/iNOS cascade by American ginseng might have therapeutic potential for inflammatory diseases with over-activation of iNOS.