Unravelling gender-specific factors that link obesity to albuminuria.

BACKGROUND: Obesity is a major public health problem, which continues to be diagnosed and classified by BMI, excluding the most elemental concepts of the precision medicine approach. Obesity does not equally affect males and females, even with the same BMI. Microalbuminuria is a risk marker of cardiovascular disease closely related to obesity. The aim of this study was to evaluate the gender-dependent differences in the development of early obesity-related disease, focusing on pathologic microalbuminuria (PMA). MATERIAL AND METHODS: We developed a single-centre cross-sectional study including 1068 consecutive adults from May 2016 to January 2018, divided into two groups: one including the first 787 patients attended, evaluated as a description population; the second group included 281 subjects analysed as an external validation population. Collected data included medical history, anthropometric measures, abdominal bioimpedance and routine laboratory tests. RESULTS: First, we confirmed the lack of accuracy of classic obesity measures in predicting microalbuminuria. Second, we tested the utility of a tailored evaluation to predict PMA, with an area under the ROC curve of 0.78 for females and 0.82 for males. We also confirmed the different physiology of visceral adiposity for males when compared to females, in which small variations of fat mass entail major changes in the clinical repercussion. Third, we performed an external validation of our results, achieving a 77% accuracy rate. CONCLUSIONS: Our findings support that there is an individual threshold of fat amount necessary to develop obesity-dependent PMA and that gender plays a major role in the interplay between PMA and adiposity.

NADPH Oxidase Overactivity Underlies Telomere Shortening in Human Atherosclerosis.

Telomere shortening and oxidative stress are involved in the pathogenesis of atherosclerosis. Different studies have shown that phagocytic NADPH oxidase is associated with this disease. This study aimed to investigate the association between phagocytic NADPH oxidase and telomere shortening in human atherosclerosis. To assess this potential association, telomere length and phagocytic NADPH oxidase activity were determined by PCR and chemiluminescence, respectively, in a population of asymptomatic subjects free of overt clinical atherosclerosis. We also measured serum 8-hydroxy-2-deoxyguanosine (8-OHdG) levels (an index of oxidative stress) and carotid intima-media thickness (IMT), a surrogate marker of atherosclerosis. After adjusting them for age and sex, telomere length inversely correlated (p < 0.05) with NADPH oxidase-mediated superoxide production, with 8-OHdG values, and with carotid IMT. Interestingly, the asymptomatic subjects with plaques have a lower telomere length (p < 0.05), and higher values of plasma 8-OHdG and superoxide production (p < 0.05). These data were confirmed in a second population in which patients with coronary artery disease showed lower telomere length and higher 8-OHdG and superoxide production than the asymptomatic subjects. In both studies, NADPH oxidase-dependent superoxide production in phagocytic cells was only due to the specific expression of the Nox2 isoform. In conclusion, these findings suggest that phagocytic NADPH oxidase may be involved in oxidative stress-mediated telomere shortening, and that this axis may be critically involved in human atherosclerosis.

Whole-body computed tomography: a new point of view in a hospital check-up unit? Our experience in 6516 patients.

BACKGROUND: There is a growing awareness that prevention and early diagnosis may reduce the high mortality associated with cancer, cardiovascular and other diseases. The role of whole-body computed tomography (WB-CT) in self-referred and asymptomatic patients has been debated. AIM: To determine frequency and spectrum of WB-CT findings in average-risk subjects derived from a Medical-Check-Up-Unit, to evaluate recommendations reported and distribution according to sex and age-groups. MATERIALS AND METHODS: We retrospectively reviewed 6516 subjects who underwent WB-CT (June 2004/February 2015). All were > 40 years and referred by Medical-Check-Up-Unit of our hospital. The main findings were categorized and classified as normal or not. Its distribution according to sex and age-groups was evaluated using Chi-square test and linear-by-linear association test, respectively. Number of recommendations, type and interval of follow-up were recorded. Descriptive statistics were used. RESULTS: WB-CT performed in 6516 patients (69% men, 31% women, mean age = 58.4 years) revealed chest (81.4%), abdominal (93.06%) and spine (65.39%) abnormalities. Only 1.60% had completely normal exploration. Abnormal WB-CT in men was significantly higher than women (98.64% vs. 97.87%; p = 0.021), with significant increase as age was higher (40-49 years: 95.65%; 50-59 years: 98.33%; 60-69 years: 99.47%; > 69 years: 99.89%) (p < 0.001). Although most findings were benign, we detected 1.47% primary tumors (96, mainly 35 kidneys and 15 lungs). 17.39% of patients received at least one recommendation predominantly in chest (78.19%) and follow-up imaging (69.89%). CONCLUSION: The most common WB-CT findings in asymptomatic subjects are benign. However, this examination allows identifying an important number of relevant and precocious findings that significantly increase with age, involving changes in lifestyle and precocious treatment.

Evaluation of measured and calculated small dense low-density lipoprotein in capillary blood and association with the metabolic syndrome.

BACKGROUND AND AIMS: Small-dense-low-density-lipoprotein cholesterol (sdLDL-C) is proatherogenic and not commonly measured. The aims were to evaluate capillary blood and its stability for sdLDL-C measurement and measure sdLDL-C in patients with metabolic syndrome (MS). METHODS: 182 patients were studied (49 with MS). sdLDL-C was measured by electrophoresis (LipoPrint®), direct measurement (Roche Diagnostics) and Sampson equation. Intima-media thickness (IMT) and presence of atheroma was evaluated. sdLDL-C was compared in paired venous and capillary blood according to CLSI-EP09c (n = 40). sdLDL-C stability was studied after 24 h at room temperature (RT). RESULTS: sdLDL-C in capillary blood and venous blood showed agreement with the direct measurement (bias: 4.17 mg/dL, LOA 95 %:-5.66; 13.99) and estimation (bias:8.12 mg/dL, LOA 95 %:-8.59; 24.82). sdLDL-C is stable in capillary blood for 24 h at RT. The electrophoretic method yielded lower (p < 0.05) sdLDL-C than the equation or direct measurement. Patients with MS had (p < 0.05) higher sdLDL-C (%) than patients without MS. Patients with atheroma plaques had higher sdLDL-C (p < 0.05). Estimated sdLDL-C correlated with IMT (r = 0.259, p < 0.001). CONCLUSIONS: Capillary blood is an alternative to venous blood for sdLDL-C measurement and is stable for 24 h after collection. Estimated and directly measured sdLDL-C associate with the MS being accessible tools for cardiovascular risk assessment.

Association of Intima-Media Thickness Measured at the Common Carotid Artery With Incident Carotid Plaque: Individual Participant Data Meta-Analysis of 20 Prospective Studies.

Background The association between common carotid artery intima-media thickness (CCA-IMT) and incident carotid plaque has not been characterized fully. We therefore aimed to precisely quantify the relationship between CCA-IMT and carotid plaque development. Methods and Results We undertook an individual participant data meta-analysis of 20 prospective studies from the Proof-ATHERO (Prospective Studies of Atherosclerosis) consortium that recorded baseline CCA-IMT and incident carotid plaque involving 21 494 individuals without a history of cardiovascular disease and without preexisting carotid plaque at baseline. Mean baseline age was 56 years (SD, 9 years), 55% were women, and mean baseline CCA-IMT was 0.71 mm (SD, 0.17 mm). Over a median follow-up of 5.9 years (5th-95th percentile, 1.9-19.0 years), 8278 individuals developed first-ever carotid plaque. We combined study-specific odds ratios (ORs) for incident carotid plaque using random-effects meta-analysis. Baseline CCA-IMT was approximately log-linearly associated with the odds of developing carotid plaque. The age-, sex-, and trial arm-adjusted OR for carotid plaque per SD higher baseline CCA-IMT was 1.40 (95% CI, 1.31-1.50; I2=63.9%). The corresponding OR that was further adjusted for ethnicity, smoking, diabetes, body mass index, systolic blood pressure, low- and high-density lipoprotein cholesterol, and lipid-lowering and antihypertensive medication was 1.34 (95% CI, 1.24-1.45; I2=59.4%; 14 studies; 16 297 participants; 6381 incident plaques). We observed no significant effect modification across clinically relevant subgroups. Sensitivity analysis restricted to studies defining plaque as focal thickening yielded a comparable OR (1.38 [95% CI, 1.29-1.47]; I2=57.1%; 14 studies; 17 352 participants; 6991 incident plaques). Conclusions Our large-scale individual participant data meta-analysis demonstrated that CCA-IMT is associated with the long-term risk of developing first-ever carotid plaque, independent of traditional cardiovascular risk factors.

CCL20 is increased in hypercholesterolemic subjects and is upregulated by LDL in vascular smooth muscle cells: role of NF-κB.

OBJECTIVE: Our aim was to analyze the regulation of CC Chemokine ligand 20 (CCL20) by LDL in human vascular smooth muscle cells (VSMC). METHODS AND RESULTS: In asymptomatic subjects, circulating CCL20 levels were higher in patients with hypercholesterolemia (18.5±3.2 versus 9.1±1.3 pg/mL; P<0.01). LDL induced the expression of CCL20 in VSMC in a dose- and time-dependent manner. Increased levels of CCL20 secreted by LDL-treated VSMC significantly induced human lymphocyte migration, an effect reduced by CCL20 silencing. The upregulation of CCL20 by LDL was dependent on the activation of kinase signaling pathways and NF-κB. By site-directed mutagenesis, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified a NF-κB site (-80/-71) in CCL20 promoter critical for LDL responsiveness. Lysophosphatidic acid mimicked the upregulation of CCL20 induced by LDL, and minimal oxidation of LDL increased the ability of LDL to induce CCL20 through a mechanism that involves lysophosphatidic acid receptors. CCL20 was overexpressed in atherosclerotic lesions from coronary artery patients, colocalizing with VSMC. CCL20 was detected in conditioned media from healthy human aorta and its levels were significantly higher in secretomes from carotid endarterectomy specimens. CONCLUSION: This study identifies CCL20 in atherosclerotic lesions and recognizes this chemokine as a mediator highly sensitive to the inflammatory response elicited by LDL.

Serum levels of matrix metalloproteinase-10 are associated with the severity of atherosclerosis in patients with chronic kidney disease.

Cardiovascular disease is the leading cause of mortality in chronic kidney disease (CKD). As matrix metalloproteinases have a major role in atherosclerosis, we hypothesized that alterations in metalloproteinases-8, -10 and their tissue inhibitor-1 can be associated with the severity of atherosclerosis in patients with kidney disease. This was evaluated in a cross-sectional, observational study of 111 patients with stages I-V kidney disease, 217 patients on dialysis and 50 healthy controls. The severity of atherosclerosis was estimated with the atherosclerosis score (AS), combining the results of ankle-brachial index and carotid ultrasound. Serum levels of the two metalloproteinases and tissue inhibitor-1 were measured by enzyme-linked immunosorbent assay and were significantly increased in patients with kidney disease compared with the healthy controls, and higher in patients on dialysis than in earlier stages of CKD. The severity of the AS was also more prevalent in the dialysis group, in which serum levels of both metalloproteinases and tissue inhibitor-1 were significantly higher. After multivariate analysis, metalloproteinase-10, dialysis, C-reactive protein, age, and male gender were associated with increased risk of atherosclerosis. Thus, patients with CKD exhibit elevated levels of circulating metalloproteinase-10, and this was independently associated with the severity of atherosclerosis and may represent a new biomarker of atherosclerotic diseases.

Does Chronic Kidney Disease Facilitate Malignant Myocardial Fibrosis in Heart Failure with Preserved Ejection Fraction of Hypertensive Origin?

In hypertensive patients with heart failure (HF) a serum biomarker combination of high carboxy-terminal propeptide of procollagen type-I (PICP) and low carboxy-terminal telopeptide of collagen type-I to matrix metalloproteinase-1 (CITP:MMP-1) ratio identifies a histomolecular phenotype of malignant myocardial fibrosis (mMF) associated with severe diastolic dysfunction (DD) and poor outcomes. As chronic kidney disease (CKD) facilitates MF and DD, we investigated the influence of CKD on the mMF biomarker combination in HF patients with preserved ejection fraction (HFpEF). Hypertensives (n = 365), 232 non-HF and 133 HFpEF, were studied, and 35% non-HF and 46% HFpEF patients had CKD (estimated glomerular filtration rate < 60 mL/min/1.73 m2 or urine albumin-to-creatinine ratio ≥ 30 mg/g). Specific immunoassays were performed to determine biomarkers. Medians were used to establish the high PICP and low CITP:MMP-1 combination. A comparison with non-HF showed that the biomarker combination presence was increased in HFpEF patients, being associated with CKD in all patients. CKD influenced the association of the biomarker combination and HFpEF (p for interaction ≤ 0.019). The E:e' ratio was associated with the biomarker combination in CKD patients. Among CKD patients with HFpEF, those with the biomarker combination exhibited higher (p = 0.016) E:e' ratio than those without the pattern. These findings suggest that CKD facilitates the development of biomarker-assessed mMF and DD in hypertensive HFpEF patients.

The angiotensin-converting enzyme insertion/deletion polymorphism is associated with phagocytic NADPH oxidase-dependent superoxide generation: potential implication in hypertension.

The objective of the present study was to analyse the influence of the ACE (angiotensin-converting enzyme) gene I/D (insertion/deletion) polymorphism on NADPH oxidase-dependent O(2)(*-) (superoxide radical) production, and to investigate the clinical implication of this association in hypertensive subjects. A case-control study was performed in a random sample of the general population composed of 189 normotensive subjects and 223 hypertensive subjects. The ACE polymorphism was determined by PCR. NADPH oxidase-dependent O(2)(*-) production was quantified in phagocytic cells by chemiluminescence. MMP-9 (matrix metalloproteinase-9), a marker of atherosclerosis previously reported to be associated with NADPH oxidase overactivity, was quantified by ELISA in plasma samples. The distribution of genotypes was in Hardy-Weinberg equilibrium. The I/D polymorphism was not associated with hypertension. NADPH oxidase-dependent O(2)(*-) production was significantly higher in D/D (deletion/deletion) than in I/I (insertion/insertion) and I/D, both in normotensive and hypertensive subjects. Interestingly, plasma levels of angiotensin II were significantly higher in D/D than in I/I and I/D, both in normotensive and hypertensive subjects. Plasma levels of MMP-9 and systolic blood pressure values were significantly higher in D/D than in I/I and I/D hypertensive subjects, whereas no differences were found among genotypes in normotensive subjects. Interestingly, NADPH oxidase-dependent O(2)(*-) production positively associated with plasma MMP-9 levels in hypertensive subjects, which remained significant after adjustment for age and gender. In conclusion, in the present study we have reported for the first time an association of the D/D genotype of the ACE I/D polymorphism with phagocytic NADPH oxidase-mediated O(2)(*-) overproduction. Within the group of hypertensive patients, D/D cases also associated with increased blood pressure values and with enhanced plasma levels of MMP-9.

Molecular mechanisms of atherosclerosis in metabolic syndrome: role of reduced IRS2-dependent signaling.

OBJECTIVE: The mechanisms underlying accelerated atherosclerosis in metabolic syndrome (MetS) patients remain poorly defined. In the mouse, complete disruption of insulin receptor substrate-2 (Irs2) causes insulin resistance, MetS-like manifestations, and accelerates atherosclerosis. Here, we performed human, mouse, and cell culture studies to gain insight into the contribution of defective Irs2 signaling to MetS-associated alterations. METHODS AND RESULTS: In circulating leukocytes from insulin-resistant MetS patients, Irs2 and Akt2 mRNA levels inversely correlate with plasma insulin levels and HOMA index and are reduced compared to insulin-sensitive MetS patients. Notably, a moderate reduction in Irs2 expression in fat-fed apolipoprotein E-null mice lacking one allele of Irs2 (apoE(-/-)Irs2(+/-)) accelerates atherosclerosis compared to apoE-null controls, without affecting plaque composition. Partial Irs2 inactivation also increases CD36 and SRA scavenger receptor expression and modified LDL uptake in macrophages, diminishes Akt2 and Ras expression in aorta, and enhances expression of the proatherogenic cytokine MCP1 in aorta and primary vascular smooth muscle cells (VSMCs) and macrophages. Inhibition of AKT or ERK1/2, a downstream target of RAS, upregulates Mcp1 in VSMCs. CONCLUSIONS: Enhanced levels of MCP1 resulting from reduced IRS2 expression and accompanying defects in AKT2 and Ras/ERK1/2 signaling pathways may contribute to accelerated atherosclerosis in MetS states.

NADPH oxidase CYBA polymorphisms, oxidative stress and cardiovascular diseases.

Oxidative stress plays a key role in the pathophysiology of several major cardiovascular diseases, including atherosclerosis, hypertension, heart failure, stroke and diabetes. ROS (reactive oxygen species) affect multiple tissues either directly or through NO depletion. ROS induce cardiovascular dysfunction by modulating cell contraction/dilation, migration, growth/apoptosis and extracellular matrix protein turnover, which contribute to vascular and cardiac remodelling. Of the several sources of ROS within the cardiovascular system, a family of multisubunit NADPH oxidases appears to be a predominant contributor of superoxide anion. Recent findings suggest a significant role of the genetic background in NADPH oxidase regulation. Common genetic polymorphisms within the promoter and exonic sequences of CYBA, the gene that encodes the p22(phox) subunit of NADPH oxidase, have been characterized in the context of cardiovascular diseases. This review aims to present the current state of research into these polymorphisms in their relationship to cardiovascular diseases.

Phagocytic NADPH oxidase-dependent superoxide production stimulates matrix metalloproteinase-9: implications for human atherosclerosis.

OBJECTIVE: Data suggest that matrix metalloproteinase-9 (MMP-9) has a role in atherosclerosis. The phagocytic NADPH oxidase has been also associated with atherosclerosis. This study aimed to investigate the association between phagocytic NADPH oxidase and MMP-9 in human atherosclerosis. METHODS AND RESULTS: In vitro experiments performed in human monocytes showed that NADPH oxidase activation enhanced MMP-9 secretion and activity, determined by enzyme-linked immunosorbent assay and zymography, respectively. Immunohistochemical study showed that phagocytic NADPH oxidase localized with MMP-9 in endarterectomies from patients with carotid stenosis. In addition, a positive relationship (P<0.001) was found between phagocytic NADPH oxidase-dependent superoxide production determined with lucigenin and plasma MMP-9 levels in 188 asymptomatic subjects free of overt clinical atherosclerosis. In multivariate analysis, this association remained significant after adjustment for cardiovascular risk factors. Interestingly, subjects in the upper quartile of superoxide production exhibited the highest values of MMP-9, oxidized low-density lipoprotein, nitrotyrosine, carotid intima media thickness, and an increased presence of carotid plaques. CONCLUSIONS: Enhanced NADPH oxidase-dependent *O2(-) production stimulates MMP-9 in monocytes and this relationship may be relevant in the atherosclerotic process. Moreover, MMP-9 emerges as an important mediator of the phagocytic NADPH oxidase-dependent oxidative stress in atherosclerosis.

[Association of age, inflammatory markers and subclinical atherosclerosis in subjects free from cardiovascular disease]. / Asociación de marcadores inflamatorios y aterosclerosis subclínica en relación con la edad en sujetos sin enfermedad cardiovascular.

BACKGROUND AND OBJECTIVE: We assessed whether an independent association between inflammatory markers and age-related subclinical atherosclerosis could be found in subjects free from cardiovascular disease. PATIENTS AND METHOD: Metabolic parameters, inflammatory and endothelial markers, such as high-sensitivity C-reactive protein, interleukin-6, fibrinogen and von Willebrand factor, as well as the carotid intima-media thickness were assessed in 890 asymptomatic subjects (mean age: 55 years; range: 20-80 years; 80% men) with cardiovascular risk factors. RESULTS: Subjects in the upper quartile (age 61-80 years) showed a significant increase of traditional risk factors, particularly arterial pressure and glucose levels (p < 0.01) as compared with lower quartiles. We also found a significant increase in the levels on inflammatory and endothelial markers (p < 0.001) and intima-media thickness (p < 0.001) in older adults. In the multivarate analysis, after adjustment for cardiovascular risk factors, intima-media thickness was independently associated with inflammation and endothelial dysfunction in older adults (p < 0.01). CONCLUSIONS: Besides age, systemic inflammation and vascular damage are associated with subclinical atherosclerosis in asymptomatic subjects. The age-related inflammatory profile may predispose to cardiovascular complications.

Predictive value for cardiovascular events of common carotid intima media thickness and its rate of change in individuals at high cardiovascular risk - Results from the PROG-IMT collaboration.

AIMS: Carotid intima media thickness (CIMT) predicts cardiovascular (CVD) events, but the predictive value of CIMT change is debated. We assessed the relation between CIMT change and events in individuals at high cardiovascular risk. METHODS AND RESULTS: From 31 cohorts with two CIMT scans (total n = 89070) on average 3.6 years apart and clinical follow-up, subcohorts were drawn: (A) individuals with at least 3 cardiovascular risk factors without previous CVD events, (B) individuals with carotid plaques without previous CVD events, and (C) individuals with previous CVD events. Cox regression models were fit to estimate the hazard ratio (HR) of the combined endpoint (myocardial infarction, stroke or vascular death) per standard deviation (SD) of CIMT change, adjusted for CVD risk factors. These HRs were pooled across studies. In groups A, B and C we observed 3483, 2845 and 1165 endpoint events, respectively. Average common CIMT was 0.79mm (SD 0.16mm), and annual common CIMT change was 0.01mm (SD 0.07mm), both in group A. The pooled HR per SD of annual common CIMT change (0.02 to 0.43mm) was 0.99 (95% confidence interval: 0.95-1.02) in group A, 0.98 (0.93-1.04) in group B, and 0.95 (0.89-1.04) in group C. The HR per SD of common CIMT (average of the first and the second CIMT scan, 0.09 to 0.75mm) was 1.15 (1.07-1.23) in group A, 1.13 (1.05-1.22) in group B, and 1.12 (1.05-1.20) in group C. CONCLUSIONS: We confirm that common CIMT is associated with future CVD events in individuals at high risk. CIMT change does not relate to future event risk in high-risk individuals.

Correction: Predictive value for cardiovascular events of common carotid intima media thickness and its rate of change in individuals at high cardiovascular risk - Results from the PROG-IMT collaboration.

[This corrects the article DOI: 10.1371/journal.pone.0191172.].

Phagocytic NADPH oxidase overactivity underlies oxidative stress in metabolic syndrome.

Oxidative stress plays a critical role in the pathogenesis of atherosclerosis in patients with metabolic syndrome. This study aimed to investigate whether a relationship exists between phagocytic NADPH oxidase activity and oxidative stress and atherosclerosis in metabolic syndrome patients. The study was performed in 56 metabolic syndrome patients (metabolic syndrome group), 99 patients with one or two cardiovascular risk factors (cardiovascular risk factor group), and 28 healthy subjects (control group). NADPH oxidase expression and activity was augmented (P < 0.05) in metabolic syndrome compared with cardiovascular risk factor and control groups. Insulin was enhanced (P < 0.05) in metabolic syndrome patients compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. Insulin stimulated NADPH oxidase activity; this effect was abolished by a specific protein kinase C inhibitor. Oxidized LDL and nitrotyrosine levels and carotid intima-media thickness were increased (P < 0.05) in the metabolic syndrome group compared with cardiovascular risk factor and control groups and correlated with NADPH oxidase activity in the overall population. These findings suggest that phagocytic NADPH oxidase overactivity is involved in oxidative stress and atherosclerosis in metabolic syndrome patients. Our findings also suggest that hyperinsulinemia may contribute to oxidative stress in metabolic syndrome patients through activation of NADPH oxidase.

A novel CYBA variant, the -675A/T polymorphism, is associated with essential hypertension.

OBJECTIVE: Oxidative stress is implicated in hypertension and the NADPH oxidase systems constitute the main source of superoxide in vascular wall. We searched for new polymorphisms within the CYBA promoter, the human gene that encodes the p22phox protein, and studied their potential association with essential hypertension. DESIGN: A case-control study in a random sample of the general population. METHODS: CYBA polymorphisms were determined by restriction fragment length polymorphism and allelic discrimination. NADPH oxidase activity was quantified in phagocytic cells by chemiluminescence. RESULTS: We identified three novel polymorphisms, at positions -852, -675 and -536 from the ATG codon. Only the -675(A/T) polymorphism associated with essential hypertension. The prevalence of the TT genotype and the T allele frequency were significantly higher (P < 0.05) in hypertensives than in normotensives. Furthermore, TT hypertensives exhibited higher (P < 0.05) systolic blood pressure values than TA/AA hypertensives. Increased phagocytic NADPH oxidase activity was observed in TT subjects compared to TA and AA individuals (P < 0.05). Enhanced carotid intima-media thickness, a surrogate marker of atherosclerosis, was found in TT subjects compared to TA and AA individuals (P < 0.05). Finally, mutagenesis experiments demonstrated a functional role of this polymorphism on the CYBA promoter activity. CONCLUSION: The -675 (A/T) CYBA polymorphism may be a novel genetic marker associated with essential hypertension. Furthermore, TT subjects exhibit features of NADPH oxidase-mediated oxidative stress and asymptomatic atherosclerosis.

Increased phagocytic NADPH oxidase activity associates with coronary artery calcification in asymptomatic men.

Vascular calcification is a common feature in atherosclerosis and associates with cardiovascular events. Oxidative stress may be involved in the pathogenesis of vascular calcification. Previous studies have shown that the phagocytic NADPH oxidase is associated with atherosclerosis. The objective of the present study was to investigate the association between phagocytic NADPH oxidase-mediated superoxide production and coronary artery calcium (CAC). NADPH oxidase-mediated superoxide production was determined by chemiluminescence and CAC by computed tomography in 159 asymptomatic men free of overt clinical atherosclerosis. Multivariate linear regression analyses were used to assess the relationship between CAC and NADPH oxidase-mediated superoxide production. Compared with individuals in the lowest score of CAC (= 0 Agatston units), those in the upper score (>400 Agatston units) showed higher superoxide production (p < 0.05). In correlation analysis, superoxide production positively (p < 0.01) correlated with CAC, which in multivariate analysis remained significant after adjusting for age, HDL-cholesterol, triglycerides, body mass index, smoking, arterial hypertension and diabetes mellitus. In conclusion, in a population of men without clinically overt atherosclerotic disease, increased NADPH oxidase-mediated superoxide production associated with enhanced CAC. Albeit descriptive, these findings suggest a potential involvement of phagocytic NADPH oxidase-mediated oxidative stress in CAC.

The C242T CYBA polymorphism of NADPH oxidase is associated with essential hypertension.

OBJECTIVE: Oxidative stress is implicated in hypertension. The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are the main source of superoxide in phagocytic and vascular cells. The C242T polymorphism of CYBA, the human gene that encodes p22phox, has been found to be functionally associated with vascular NADPH oxidase activity in atherosclerotic patients. We investigated the association of the C242T polymorphism with hypertension and its potential impact on NADPH oxidase activity. We also analysed the interaction of C242T polymorphism with the -930A/G CYBA variant. DESIGN: Case-control study in a random sample of 623 subjects (326 hypertensive patients and 297 normotensive controls) from the general population. METHODS: CYBA polymorphisms were determined by restriction fragment length polymorphism (RFLP) or allelic discrimination. NADPH oxidase activity and p22phox expression were quantified in phagocytic cells by chemiluminescence and by northern and western blots, respectively. RESULTS: The prevalence of the CC genotype and the C allele frequency were significantly higher (P < 0.05) in hypertensives than in normotensives. CC genotype remained associated with hypertension after adjusting for potential confounders in a logistic regression analysis. Increased phagocytic NADPH oxidase activity was observed in CC hypertensives compared with CT and TT hypertensives (P < 0.05). Enhanced plasma levels of von Willebrand factor were found in CC hypertensives compared with TT hypertensives (P < 0.05). The C242T polymorphism was not in linkage disequilibrium with the -930A/G CYBA promoter variation, which also associates with hypertension. CONCLUSION: The C242T CYBA polymorphism is associated with essential hypertension. Furthermore, hypertensives carrying the CC genotype of this polymorphism exhibit features of NADPH oxidase-mediated oxidative stress and endothelial damage.

NADPH oxidase-dependent superoxide production is associated with carotid intima-media thickness in subjects free of clinical atherosclerotic disease.

OBJECTIVE: Oxidative stress plays a critical role in the pathogenesis of atherosclerosis. The NADPH oxidase constitutes the main source of superoxide in phagocytic and vascular cells. This study aimed to investigate the levels of NADPH oxidase-mediated superoxide production in phagocytic cells and the association between phagocytic superoxide production and carotid intima-media thickness (IMT), a surrogate marker of asymptomatic atherosclerosis. METHODS AND RESULTS: NADPH oxidase-mediated superoxide production was determined by a chemiluminescence assay using lucigenin and associated with IMT for 184 asymptomatic subjects free of overt clinical atherosclerotic disease. Compared with individuals in the lowest tertile of superoxide production, those in the upper tertile (>20 counts/sec) showed significantly higher IMT (P<0.05). In correlation analysis, a positive relationship was found between superoxide production and carotid IMT. Superoxide production also correlated positively (P<0.05) with body mass index (BMI). In multivariate analysis, the association of superoxide production with carotid IMT remained significant after adjustment for age, sex, systolic blood pressure, BMI, triglycerides, glucose, and smoking. CONCLUSIONS: In a population sample of adults without clinically overt atherosclerotic disease, increased NADPH oxidase activity was associated with enhanced carotid IMT, suggesting a relationship between phagocytic NADPH oxidase-mediated oxidative stress and the development of atherosclerosis.