Cinzia Myriam Calabrese

Obesity and Hypercholesterolemia are Associated with NOX2 Generated Oxidative Stress and Arterial Dysfunction

OBJECTIVE To analyze the interplay among oxidative stress, NOX2, the catalytic core of nicotinamide-adenine dinucleotide phosphate oxidase, and endothelial dysfunction in children with obesity and/or hypercholesterolemia. STUDY DESIGN We performed a cross-sectional study comparing flow-mediated arterial dilation (FMD), oxidized low-density lipoprotein, and urinary excretion of isoprostanes (8-iso-PGF2α), as markers of oxidative stress, and NOX2 activity, as assessed by blood levels of soluble NOX2-dp (sNOX2-dp), in a population of 100 children, matched for age and sex, including 40 healthy subjects (HS), 20 children with hypercholesterolemia (HC), 20 obese children (OC), and 20 children with coexistence of hypercholesterolemia and obesity (HOC). RESULTS HOC had higher sNOX2-dp and oxidized low-density lipoprotein levels compared with HS, HC, and OC. HC, OC, and HOC had lower FMD values compared with HS. Urinary 8-iso-PGF2α excretion was higher in HOC compared with HS. FMD was inversely correlated with sNOX2-dp levels (r = -0.483; P < .001) and with the number of cardiovascular risk factors (r = -0.617; P < .001). Multiple linear regression analysis showed that the number of cardiovascular risk factors was the only independent predictive variable associated with FMD (β: -0.585; P < .001; R(2) = 35%) and sNOX2-dp (β: 0.587; P < .001; R(2) = 34%). CONCLUSION The study suggests that NOX2-generating oxidative stress may have a pathogenic role in the functional changes of the arterial wall occurring in HOC.

NOX2 up-regulation is associated with artery dysfunction in patients with peripheral artery disease.

OBJECTIVE Oxidative stress seems to play a role in impairing flow-mediated dilation (FMD) in patients with peripheral artery disease (PAD) but the underlying mechanism is still undefined. We evaluated whether NOX2, the catalytic core of NADPH oxidase, the most important producer of reactive oxidant species (ROS), is implicated in impairing FMD. METHODS We measured FMD, urinary isoprostanes, a marker of oxidative stress, nitric oxide generation by serum levels of nitrite/nitrate (NOx), and serum levels of soluble NOX2-derived peptide (sNOX2-dp), a marker of NOX2 activation, in 50 PAD patients and 50 controls. Also, we performed an interventional cross-over study to assess if propionyl-L-carnitine (PLC) (6g/day), vs. placebo, was able to affect FMD via an oxidative stress-mediated mechanism. RESULTS Compared to controls, patients with PAD had enhanced sNOX2-dp and isoprostanes and reduced NOx and FMD. Multiple linear regression analysis showed that FMD was independently associated with sNOX2-dp. After PLC infusion FMD increased while sNOX2-dp and isoprostanes significantly decreased; no changes were observed after placebo. In vitro study by incubating platelets or white cells with PLC demonstrated a significant inhibition of p47(phox) translocation on cellular surface and ROS generated by NOX2 activation. CONCLUSION This study suggests that in PAD patients ROS generated by NOX2 contribute to reduce FMD and that the administration of an antioxidant is able to improve arterial dilatation via NOX2 inhibition.

Adherence to Mediterranean Diet and Non-Alcoholic Fatty Liver Disease: Effect on Insulin Resistance

Objectives:The prevalence of cardiometabolic disorders, including non-alcoholic fatty liver disease (NAFLD), is increasing in western countries, because of changes in lifestyle and dietary habits. Mediterranean Diet (Med-Diet) is effective for cardiovascular prevention, but its relationship with NAFLD has been scarcely investigated.Methods:We included 584 consecutive outpatients presenting with one or more cardiovascular risk factor such as type 2 diabetes mellitus (T2DM), arterial hypertension, overweight/obesity, and dyslipidemia. Liver steatosis was assessed using ultrasonography. Med-Diet adherence was investigated by a validated semiquantitative nine-item dietary questionnaire; patients were divided into low, intermediate, and high adherence. Insulin resistance was defined by the 75th percentile of homeostasis model of insulin resistance (HOMA-IR; ≥3.8).Results:The mean age was 56.2±12.4 years and 38.2% were women. Liver steatosis was present in 82.7%, and its prevalence decreased from low to high adherence group (96.5% vs. 71.4%, P<0.001). In a multiple logistic regression analysis, hypertriglyceridemia (odds ratio (OR): 2.913; P=0.002), log (ALT) (OR: 6.186; P<0.001), Med-Diet adherence (intermediate vs. low OR: 0.115; P=0.041, high vs. low OR: 0.093; P=0.030), T2DM (OR: 3.940; P=0.003), and high waist circumference (OR: 3.012; P<0.001) were associated with NAFLD. Among single foods, low meat intake (OR: 0.178; P<0.001) was inversely significantly associated with NAFLD. In 334 non-diabetic NAFLD patients, age (OR: 1.035, P=0.025), high waist circumference (OR: 7.855, P<0.001), hypertriglyceridemia (OR: 2.152, P=0.011), and Log (ALT) (OR: 2.549, P=0.002) were directly associated with HOMA-IR, whereas Med-Diet score was inversely associated (OR: 0.801, P=0.018).Conclusions:We found an inverse relationship between Med-Diet and NAFLD prevalence. Among NAFLD patients, good adherence to Med-Diet was associated with lower insulin resistance. Our findings suggest that Med-Diet may be a beneficial nutritional approach in NAFLD patients.

Is NOX2 Upregulation Implicated in Myocardial Injury in Patients with Pneumonia

In the present study, we tested the hypothesis that oxidative stress could be implicated in myocardial damage during the acute phase of pneumonia. NOX2 activation, the catalytic subunit of NADPH oxidase, and high-sensitivity cardiac troponin T (hs-cTnT) elevation have been analyzed in two hundred forty-eight consecutive patients hospitalized for community-acquired pneumonia. Serum NOX2-derived peptide (sNOX2-dp), a marker of NOX2 activation, and 8-isoprostaglandin F2α (8-iso-PGF2α), a marker of oxidative stress, were measured upon admission; serum hs-cTnT and ECG were measured every 12 and 24 h, respectively. One hundred thirty-five patients (54%) showed elevated serum levels of hs-cTnT (>0.014 μg/L). A logistic regression analysis showed sNOX2-dp (p<0.001), Pneumonia Severity Index score (p<0.001), renal failure (p=0.024), and ejection fraction (p<0.001) as independent predictors of elevated serum levels of hs-cTnT. Serum sNOX2-dp was linearly correlated with hs-cTnT (Rs=0.538; p<0.001) and 8-iso-PGF2α (Rs=0.354; p<0.001). The study provides the first evidence of a significant association between serum cardiac Troponin T elevation and NOX2 upregulation in patients with pneumonia. This finding raises the hypothesis that NOX2-derived oxidative stress may be implicated in myocardial injury and that its inhibition could be a novel therapeutic strategy to limit it.

Role of platelets in NOX2 activation mediated by TNFα in heart failure

Tumor necrosis factor (TNF) α may contribute to the deterioration of cardiovascular function in heart failure (HF) through various mechanisms, including the generation of reactive oxygen species (ROS). NADPH oxidase is the major source of ROS in the vascular system, but the interplay between TNFα and NADPH oxidase activation is elusive. As platelets possess NADPH oxidase enzyme, they represent an important tool to investigate the interplay between NADPH oxidase and TNFα in patients with HF. Serum gp91phox (NOX2), the catalytic core of NADPH oxidase, and serum TNFα were measured in 120 HF patients and in 60 healthy subjects. Compared with healthy subjects, HF patients had higher blood levels of NOX2 and TNFα with a progressive increase from NYHA I to NYHA IV classes. NOX2 levels in blood were independently associated with TNFα in HF patients. An in vitro study, performed on platelets from a subgroup of HF patients, shows that TNFα, at concentrations commonly found in HF patients’ peripheral circulation, activates platelet NOX2. Thus, TNFα increases ROS production and the extracellular levels of NOX2. These phenomena are inhibited by the NOX2-specific blocking peptide gp91ds-tat. The study provides evidence that circulating NOX2, as well as the activation of NOX2 on platelets, is increased in HF likely as a consequence of the underlying inflammatory process.

Impaired flow-mediated dilation in hospitalized patients with community-acquired pneumonia

BACKGROUND Community-acquired pneumonia (CAP) is complicated by cardiovascular events as myocardial infarction and stroke but the underlying mechanism is still unclear. We hypothesized that endothelial dysfunction may be implicated and that endotoxemia may have a role. METHODS Fifty patients with CAP and 50 controls were enrolled. At admission and at discharge, flow-mediated dilation (FMD), serum levels of endotoxins and oxidative stress, as assessed by serum levels of nitrite/nitrate (NOx) and isoprostanes, were studied. RESULTS At admission, a significant difference between patients with CAP and controls was observed for FMD (2.1±0.3 vs 4.0±0.3%, p<0.001), serum endotoxins (157.8±7.6 vs 33.1±4.8pg/ml), serum isoprostanes (341±14 vs 286±10 pM, p=0.009) and NOx (24.3±1.1 vs 29.7±2.2μM). Simple linear correlation analysis showed that serum endotoxins significantly correlated with Pneumonia Severity Index score (Rs=0.386, p=0.006). Compared to baseline, at discharge CAP patients showed a significant increase of FMD and NOx (from 2.1±0.3 to 4.6±0.4%, p<0.001 and from 24.3±1.1 to 31.1±1.5μM, p<0.001, respectively) and a significant decrease of serum endotoxins and isoprostanes (from 157.8±7.6 to 55.5±2.3pg/ml, p<0.001, and from 341±14 to 312±14 pM, p<0.001, respectively). Conversely, no changes for FMD, NOx, serum endotoxins and isoprostanes were observed in controls between baseline and discharge. Changes of FMD significantly correlated with changes of serum endotoxins (Rs=-0.315; p=0.001). CONCLUSIONS The study provides the first evidence that CAP is characterized by impaired FMD with a mechanism potentially involving endotoxin production and oxidative stress.