Mechanisms of oxidative stress in human aortic aneurysms — association with clinical risk factors for atherosclerosis and disease severity

Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2radical dot−) production in human AAA. Methods and results: AAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n = 16) than in risk factor matched controls (n = 16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2radical dot− in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size. Conclusions: Increased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients

GTP cyclohydrolase I gene polymorphisms are associated with endothelial dysfunction and oxidative stress in patients with type 2 diabetes mellitus

Background: The genetic background of atherosclerosis in type 2 diabetes mellitus (T2DM) is complex and poorly understood. Studying genetic components of intermediate phenotypes, such as endothelial dysfunction and oxidative stress, may aid in identifying novel genetic components for atherosclerosis in diabetic patients.<p></p> Methods: Five polymorphisms forming two haplotype blocks within the GTP cyclohydrolase 1 gene, encoding a rate limiting enzyme in tetrahydrobiopterin synthesis, were studied in the context of flow and nitroglycerin mediated dilation (FMD and NMD), intima-media thickness (IMT), and plasma concentrations of von Willebrand factor (vWF) and malondialdehyde (MDA).<p></p> Results: Rs841 was associated with FMD (p = 0.01), while polymorphisms Rs10483639, Rs841, Rs3783641 (which form a single haplotype) were associated with both MDA (p = 0.012, p = 0.0015 and p = 0.003, respectively) and vWF concentrations (p = 0.016, p = 0.03 and p = 0.045, respectively). In addition, polymorphism Rs8007267 was also associated with MDA (p = 0.006). Haplotype analysis confirmed the association of both haplotypes with studied variables.<p></p> Conclusions: Genetic variation of the GCH1 gene is associated with endothelial dysfunction and oxidative stress in T2DM patients

Chymase-Dependent Generation of Angiotensin II from Angiotensin-(1-12) in Human Atrial Tissue

Since angiotensin-(1-12) [Ang-(1-12)] is a non-renin dependent alternate precursor for the generation of cardiac Ang peptides in rat tissue, we investigated the metabolism of Ang-(1-12) by plasma membranes (PM) isolated from human atrial appendage tissue from nine patients undergoing cardiac surgery for primary control of atrial fibrillation (MAZE surgical procedure). PM was incubated with highly purified 125I-Ang-(1-12) at 37°C for 1 h with or without renin-angiotensin system (RAS) inhibitors [lisinopril for angiotensin converting enzyme (ACE), SCH39370 for neprilysin (NEP), MLN-4760 for ACE2 and chymostatin for chymase; 50 µM each]. 125I-Ang peptide fractions were identified by HPLC coupled to an inline γ-detector. In the absence of all RAS inhibitor, 125I-Ang-(1-12) was converted into Ang I (2±2%), Ang II (69±21%), Ang-(1-7) (5±2%), and Ang-(1-4) (2±1%). In the absence of all RAS inhibitor, only 22±10% of 125I-Ang-(1-12) was unmetabolized, whereas, in the presence of the all RAS inhibitors, 98±7% of 125I-Ang-(1-12) remained intact. The relative contribution of selective inhibition of ACE and chymase enzyme showed that 125I-Ang-(1-12) was primarily converted into Ang II (65±18%) by chymase while its hydrolysis into Ang II by ACE was significantly lower or undetectable. The activity of individual enzyme was calculated based on the amount of Ang II formation. These results showed very high chymase-mediated Ang II formation (28±3.1 fmol×min−1×mg−1, n = 9) from 125I-Ang-(1-12) and very low or undetectable Ang II formation by ACE (1.1±0.2 fmol×min−1×mg−1). Paralleling these findings, these tissues showed significant content of chymase protein that by immunocytochemistry were primarily localized in atrial cardiac myocytes. In conclusion, we demonstrate for the first time in human cardiac tissue a dominant role of cardiac chymase in the formation of Ang II from Ang-(1-12)

Relationship between rs841 polymorphism in the 3'-UTR of GCH1 gene and vascular function measured as endothelium dependent FMD (panel A) and endothelium independent NMD) (panel B).

<p>AA indicates frequent homozygote, Aa - heterozygote, aa - rare homozygote.</p

Association of individual GCH1 gene polymorphisms with outcome variables.

<p>Association of individual GCH1 gene polymorphisms with outcome variables.</p

Clinical characteristics of the studied T2DM patients.

<p>Clinical characteristics of the studied T2DM patients.</p

Relationship between studied polymorphisms and plasma MDA concentration measured by mass-spec analysis.

<p>Relationship to polymorphisms forming haplotype block 1 (panel A) and haplotype block 2 (panel B) is demonstrated. Statistical significance of linear regression: * p<0.05; ** p<0.01.</p

Relationship between studied polymorphisms and plasma vWF concentration.

<p>Relationship to polymorphisms forming haplotype block 1 (panel A) and haplotype block 2 (panel B) is demonstrated. Statistical significance of linear regression: * p<0.05.</p