Mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apo)A-I from murine RAW 264.7 macrophages

<p>Abstract</p> <p>Background</p> <p>Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function.</p> <p>Methods</p> <p>Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [³H]cholesterol to apolipoprotein (apo) A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, <it>Gapdh</it>, and combined with studies of this molecule on cholesterol esterification, <it>de novo</it> lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett’s or Bonferroni post <it>t</it>-tests, as appropriate.</p> <p>Results</p> <p>The positive control, resveratrol (24 h), significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; <it>p</it><0.01) and oligomycin (55%; <it>p</it><0.01), under conditions (10 μM, 3 h) which did not induce cellular toxicity or deplete total cellular ATP content. Levels of ATP binding cassette transporter A1 (ABCA1) protein were repressed by oligomycin under optimal efflux conditions, despite paradoxical increases in <it>Abca1</it> mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (<it>Abca1</it>, <it>Abcg4</it>, <it>Stard1</it>) and cholesterol biosynthesis (<it>Hmgr</it>, <it>Mvk</it>, <it>Scap</it>, <it>Srebf2</it>), indicating profound dysregulation of cholesterol homeostasis.</p> <p>Conclusions</p> <p>Acute loss of mitochondrial function, and in particular Δψ_m, reduces cholesterol efflux to apoA-I and dysregulates macrophage cholesterol homeostasis mechanisms. Bioavailable antioxidants, targeted to mitochondria and capable of sustaining effective mitochondrial function, may therefore prove effective in maintenance of arterial health.</p

Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

10.1371/journal.pone.0053147PLoS ONE82

Potential Involvement of LOX-1 in Functional Consequences of Endothelial Senescence

Numerous studies have described the process of senescence associated with accumulation of oxidative damage, mutations and decline in proliferative potential. Although the changes observed in senescent cells are likely to result in significant phenotypic alterations, the studies on consequences of endothelial senescence, especially in relation to aging-associated diseases, are scarce. We have analyzed effects of senescence on the functions of endothelial cells relevant to the development of atherosclerosis including angiogenesis, adhesion, apoptosis and inflammation. In the course of progressing through the passages, human umbilical vein endothelial cells (HUVECs) displayed significant increase in size (+36% passage 12 vs. passage 4 , p<0.001) and reduction in both basal and VEGF-stimulated tube formation. The analysis of a scavenger receptor LOX-1, a key molecule implicated in atherogenesis, revealed a significant decline of its message (mRNA) and protein content in senescent endothelial cells (−33%) and in aortas of 50 wk (vs. 5 wk) old mice (all p<0.01). These effects were accompanied by a marked reduction of the basal expression of VCAM-1 and ICAM-1. Compared to early cultures, late passage HUVECs also exhibited nuclear translocation of NF-κB (p65) and reciprocal shifts in BAX and BCL2 protein content resulting in almost 2-fold increase in BAX/BCL2 ratio and 3-fold increase in apoptotic response to TNFα exposure (p<0.04). These changes in senescent endothelial cells are suggestive of aberrant responses to physiological stimuli resulting in a less permissive environment for tissue remodeling and progression of diseases requiring angiogenesis and cell adhesion in elderly, possibly, mediated by LOX-1

Smoking Dose Modifies the Association between C242T Polymorphism and Prevalence of Metabolic Syndrome in a Chinese Population

Background: The C242T polymorphism of the CYBA gene that encodes p22phox, a component of NADPH oxidase, has been found to modulate superoxide production. Oxidase is a major source of the superoxide anion that contributes to individual components of metabolic syndrome. We examined the relationship of the C242T polymorphism with the prevalence of metabolic syndrome in a Chinese population, taking account of consumed cigarette amounts. Methodology/Principal Findings: In 870 participants, we collected biomarkers related to metabolic syndrome and detailed history of smoking and genotyped the C242T polymorphisms. After adjustment for covariates, the CT/TT genotypes were associated with a lower risk of metabolic syndrome (P = 0.0008). The odds of having metabolic syndrome in the CT/TT participants were 0.439 (95%CI: 0.265, 0.726), while for CC participants the odds were 1.110 (95%CI: 0.904, 1.362). There was significant (P = 0.014) interaction between the C242T polymorphism and smoking status in relation to the prevalence of metabolic syndrome. For smokers who smoke no less than 25 pack-years, those with CT/TT genotypes had lower risk of metabolic syndrome as compared with CC polymorphism carriers (P = 0.015). In the multiple regression analysis, the CT/TT genotypes were significantly associated with lower serum concentration of triglycerides both in all subjects and smokers; furthermore, the CT/TT genotypes were also related to smaller waist circumference in smokers. Conclusions: Our study suggests that the C242T gene polymorphism is indeed related to the prevalence of metaboli

Mitochondrial DNA haplogroup T is associated with coronary artery disease and diabetic retinopathy: a case control study

<p>Abstract</p> <p>Background</p> <p>There is strong and consistent evidence that oxidative stress is crucially involved in the development of atherosclerotic vascular disease. Overproduction of reactive oxygen species (ROS) in mitochondria is an unifying mechanism that underlies micro- and macrovascular atherosclerotic disease. Given the central role of mitochondria in energy and ROS production, mitochondrial DNA (mtDNA) is an obvious candidate for genetic susceptibility studies on atherosclerotic processes. We therefore examined the association between mtDNA haplogroups and coronary artery disease (CAD) as well as diabetic retinopathy.</p> <p>Methods</p> <p>This study of Middle European Caucasians included patients with angiographically documented CAD (n = 487), subjects with type 2 diabetes mellitus with (n = 149) or without (n = 78) diabetic retinopathy and control subjects without clinical manifestations of atherosclerotic disease (n = 1527). MtDNA haplotyping was performed using multiplex PCR and subsequent multiplex primer extension analysis for determination of the major European haplogroups. Haplogroup frequencies of patients were compared to those of control subjects without clinical manifestations of atherosclerotic disease.</p> <p>Results</p> <p>Haplogroup T was significantly more prevalent among patients with CAD than among control subjects (14.8% vs 8.3%; p = 0.002). In patients with type 2 diabetes, the presence of diabetic retinopathy was also significantly associated with a higher prevalence of haplogroup T (12.1% vs 5.1%; p = 0.046).</p> <p>Conclusion</p> <p>Our data indicate that the mtDNA haplogroup T is associated with CAD and diabetic retinopathy in Middle European Caucasian populations.</p

Genome-Wide Association Analysis of Oxidative Stress Resistance in Drosophila melanogaster

Background: Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress. Methods and Findings: We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genomewide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79 % and 56–66 % of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis. Conclusions: We identified novel candidate genes associated with variation in resistance to oxidative stress that hav

Humanin, a Cytoprotective Peptide, Is Expressed in Carotid Artherosclerotic Plaques in Humans

The mechanism of atherosclerotic plaque progression leading to instability, rupture, and ischemic manifestation involves oxidative stress and apoptosis. Humanin (HN) is a newly emerging endogenously expressed cytoprotective peptide. Our goal was to determine the presence and localization of HN in carotid atherosclerotic plaques.Plaque specimens from 34 patients undergoing carotid endarterectomy were classified according to symptomatic history. Immunostaining combined with digital microscopy revealed greater expression of HN in the unstable plaques of symptomatic compared to asymptomatic patients (29.42±2.05 vs. 14.14±2.13% of plaque area, p<0.0001). These data were further confirmed by immunoblot (density of HN/β-actin standard symptomatic vs. asymptomatic 1.32±0.14 vs. 0.79±0.11, p<0.01). TUNEL staining revealed a higher proportion of apoptotic nuclei in the plaques of symptomatic patients compared to asymptomatic (68.25±3.61 vs. 33.46±4.46% of nuclei, p<0.01). Double immunofluorescence labeling revealed co-localization of HN with macrophages (both M1 and M2 polarization), smooth muscle cells, fibroblasts, and dendritic cells as well as with inflammatory markers MMP2 and MMP9.The study demonstrates a higher expression of HN in unstable carotid plaques that is localized to multiple cell types within the plaque. These data support the involvement of HN in atherosclerosis, possibly as an endogenous response to the inflammatory and apoptotic processes within the atheromatous plaque