Redox signaling is implicated in different physiological and pathological events in the vasculature. Among the different reactive oxygen species, hydrogen peroxide (H2O2) is a very good candidate to perform functions as an intracellular messenger in the regulation of several biological events.In this review, we summarize the main physiological sources of H2O2 in the endothelium and the molecular mechanisms by which it is able to act as a signaling mediator in the vasculature.

Objectives Endothelial dysfunction is linked to insulin resistance, inflammatory activation and increased cardiovascular risk in diabetes mellitus; however the mechanisms remain incompletely understood. Recent studies have identified pro-inflammatory signaling of Wnt5a through JNK as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. Approach We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in from 85 subjects with Type 2 diabetes mellitus (n=42) and age- and sex-matched non-diabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Results Endothelial cells from patients with diabetes displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes. In endothelial cells from non-diabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In HAECs, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Conclusions Our findings demonstrate that non-canonical Wnt5a signaling and JNK activity contributes to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes.

Background Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus. Methods and Results We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n=45) and non-diabetic controls (n=41). p62 levels were higher in cells from diabetics (34.2±3.6 vs. 20.0±1.6, P=0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (−21±5% vs. 64±22%, P=0.003) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P=0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P=0.01) in cells from diabetics to a lesser extent than in cells from controls (P=0.04), suggesting ongoing, but inadequate autophagic clearance. Conclusion Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease.

ObjectivePrior studies demonstrate mitochondrial dysfunction with increased reactive oxygen species generation in peripheral blood mononuclear cells in diabetes mellitus. Oxidative stress-mediated damage to mitochondrial DNA promotes atherosclerosis in animal models. Thus, we evaluated the relation of mitochondrial DNA damage in peripheral blood mononuclear cells s with vascular function in patients with diabetes mellitus and with atherosclerotic cardiovascular disease.Approach and resultsWe assessed non-invasive vascular function and mitochondrial DNA damage in 275 patients (age 57 ± 9 years, 60 % women) with atherosclerotic cardiovascular disease alone (N = 55), diabetes mellitus alone (N = 74), combined atherosclerotic cardiovascular disease and diabetes mellitus (N = 48), and controls age >45 without diabetes mellitus or atherosclerotic cardiovascular disease (N = 98). Mitochondrial DNA damage measured by quantitative PCR in peripheral blood mononuclear cells was higher with clinical atherosclerosis alone (0.55 ± 0.65), diabetes mellitus alone (0.65 ± 1.0), and combined clinical atherosclerosis and diabetes mellitus (0.89 ± 1.32) as compared to control subjects (0.23 ± 0.64, P < 0.0001). In multivariable models adjusting for age, sex, and relevant cardiovascular risk factors, clinical atherosclerosis and diabetes mellitus remained associated with higher mitochondrial DNA damage levels (β = 0.14 ± 0.13, P = 0.04 and β = 0.21 ± 0.13, P = 0.002, respectively). Higher mitochondrial DNA damage was associated with higher baseline pulse amplitude, a measure of arterial pulsatility, but not with flow-mediated dilation or hyperemic response, measures of vasodilator function.ConclusionsWe found greater mitochondrial DNA damage in patients with diabetes mellitus and clinical atherosclerosis. The association of mitochondrial DNA damage and baseline pulse amplitude may suggest a link between mitochondrial dysfunction and excessive small artery pulsatility with potentially adverse microvascular impact.

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Protein S-nitrosylation is a reversible post-translational modification of protein cysteines that is increasingly being considered as a signal transduction mechanism. The "biotin switch" technique marked the beginning of the study of the S-nitrosoproteome, based on the specific replacement of the labile S-nitrosylation by a more stable biotinylation that allowed further detection and purification. However, its application for proteomic studies is limited by its relatively low sensitivity. Thus, typical proteomic experiments require high quantities of protein extracts, which precludes the use of this method in a number of biological settings. We have developed a "fluorescence switch" technique that, when coupled to 2-DE proteomic methodologies, allows the detection and identification of S-nitrosylated proteins by using limited amounts of starting material, thus significantly improving the sensitivity. We have applied this methodology to detect proteins that become S-nitrosylated in endothelial cells when exposed to S-nitroso-L-cysteine, a physiological S-nitrosothiol, identifying already known S-nitrosylation targets, as well as proteins that are novel targets. This "fluorescence switch" approach also allowed us to identify several proteins that are denitrosylated by thioredoxin in cytokine-activated RAW264.7 (murine macrophage) cells. We believe that this method represents an improvement in order to approach the identification of S-nitrosylated proteins in physiological conditions.

Background Posttranslational protein modification with O‐linked N ‐acetylglucosamine (O‐Glc NA c) is linked to high glucose levels in type 2 diabetes mellitus (T2 DM ) and may alter cellular function. We sought to elucidate the involvement of O‐Glc NA c modification in endothelial dysfunction in patients with T2 DM . Methods and Results Freshly isolated endothelial cells obtained by J‐wire biopsy from a forearm vein of patients with T2 DM (n=18) was compared with controls (n=10). Endothelial O‐Glc NA c levels were 1.8‐ford higher in T2 DM patients than in nondiabetic controls ( P =0.003). Higher endothelial O‐Glc NA c levels correlated with serum fasting blood glucose level ( r =0.433, P =0.024) and hemoglobin A 1c ( r =0.418, P =0.042). In endothelial cells from patients with T2 DM , normal glucose conditions (24 hours at 5 mmol/L) lowered O‐Glc NA c levels and restored insulin‐mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O‐Glc NA c levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O‐Glc NA case inhibitor, increased O‐Glc NA c levels and blunted the improvement of insulin‐mediated endothelial nitric oxide synthase phosphorylation by glucose normalization. Conclusions Taken together, our findings indicate a role for O‐Glc NA c modification in the dynamic, glucose‐induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2 DM . O‐Glc NA c protein modification may be a treatment target for vascular dysfunction in T2 DM .

The accumulation of visceral adiposity is strongly associated with systemic inflammation and increased cardiometabolic risk. WNT5A, a non-canonical WNT ligand, has been shown to promote adipose tissue inflammation and insulin resistance in animal studies. Among other non-canonical pathways, WNT5A activates planar cell polarity (PCP) signaling. The current study investigated the potential contribution of non-canonical WNT5A/PCP signaling to visceral adipose tissue (VAT) inflammation and associated metabolic dysfunction in individuals with obesity. VAT and subcutaneous adipose tissue (SAT) samples obtained from subjects undergoing bariatric surgery were analyzed by qRT-PCR for expression of WNT/PCP genes. In vitro experiments were conducted with preadipocytes isolated from VAT and SAT biopsies. The expression of 23 out of 33 PCP genes was enriched in VAT compared to SAT. Strong positive expression correlations of individual PCP genes were observed in VAT. WNT5A expression in VAT, but not in SAT, correlated with indexes of JNK signaling activity, IL6, waist-to-hip ratio and hsCRP. In vitro, WNT5A promoted the expression of IL6 in human preadipocytes. In conclusion, elevated non-canonical WNT5A signaling in VAT contributes to the exacerbated IL-6 production in this depot and the low-grade systemic inflammation typically associated with visceral adiposity.