Antioxidants in the Fight Against Atherosclerosis: Is This a Dead End?

PURPOSE OF REVIEW: The purpose of this review is to focus on the outcome of recent antioxidant interventions using synthetic and naturally occurring molecules established as adjuvant strategies to lipid-lowering or anti-inflammatory therapies designed to reduce the risk of cardiovascular disease. RECENT FINDINGS: To date, accumulated evidence regarding oxidation as a pro-atherogenic factor indicates that redox biochemical events involved in atherogenesis are indeed a very attractive target for the management of cardiovascular disease in the clinic. Nevertheless, although evidence indicates that redox reactions are important in the initiation and progression of atherosclerosis, oxidation with a pro-atherogenic context does not eliminate the fact that oxidation participates in many cases as an essential messenger of important cellular signaling pathways. Therefore, disease management and therapeutic goals require not only high-precision and high-sensitivity methods to detect in plasma very low amounts of reducing and oxidizing molecules but also a much better understanding of the normal processes and metabolic pathways influenced and/or controlled by oxidative stress. As several methodologies have been specifically described for the quantification of the total antioxidant capacity and the oxidation state of diverse biological systems, a successful way to carefully study how redox reactions influence atherosclerosis can be achieved. Since there is still a lack of standardization with many of these methods, clinical trials studying antioxidant capacity have been difficult to compare and therefore difficult to use in order to reach a conclusion. We believe a comprehensive analysis of new knowledge and its relationship with the presence of plasma antioxidants and their reducing capacity will undoubtedly open new ways to understand and develop new therapeutic pathways in the fight not only against atherosclerosis but also against other degenerative diseases.

Hepatic Accumulation of Hypoxanthine: A Link Between Hyperuricemia and Nonalcoholic Fatty Liver Disease.

BACKGROUND: An elevated level of plasma uric acid has been widely recognized as a risk factor for non-alcoholic fatty liver disease (NAFLD), where oxidative stress and inflammation play an important role in the pathophysiology of the disease. Although the complete molecular mechanisms involved remain unknown, while under physiological conditions uric acid presents antioxidant properties, hyperuricemia has been linked to oxidative stress, chronic low-grade inflammation, and insulin resistance, basic signs of NAFLD. AIM OF STUDY: Employing in vivo experimentation, we aim to investigate whether a high-fat diet rich in cholesterol (HFD), modifies the metabolism of purines in close relationship to molecular events associated with the development of NAFLD. In vitro experiments employing HepG2 cells are also carried out to study the phenomenon of oxidative stress. METHODS: Adult male rabbits were fed for 8 weeks an HFD to induce NAFLD. At the beginning of the experiment and every 15 d until the completion of the study, plasma levels of lipids, lipoproteins, and uric acid were measured. Liver tissue was isolated, and histology performed followed by the biochemical determination of hypoxanthine, protein expression of xanthine oxidoreductase (XOR) by western blot analysis, and xanthine oxidase (XO) activity using an enzymatic kinetic assay. Furthermore, we employed in vitro experimentation studying HepG2 cells to measure the effect of hypoxanthine and H2O2 upon the production of radical oxygen species (ROS), XO activity, and cell viability. RESULTS AND CONCLUSION: Hepatic tissue from rabbits fed the HFD diet showed signs of NAFLD associated with an increased ROS concentration and an altered purine metabolism characterized by the increase in hypoxanthine, together with an apparent equilibrium displacement of XOR towards the xanthine dehydrogenase (XDH) isoform of the enzyme. This protein shift visualized by a western blot analysis, associated with an increase in plasma uric acid and hepatocyte hypoxanthine could be understood as a compensatory series of events secondary to the establishment of oxidative stress associated with the chronic establishment of fatty liver disease.

Enzymatic assessment of cholesterol on electrophoresis gels for estimating HDL size distribution and plasma concentrations of HDL subclasses.

The aim of this study was to develop an enzymatic cholesterol staining method to determine HDL subclasses in a polyacrylamide gradient gel electrophoresis, which further allows staining by protein in the same electrophoresis lane. HDLs from 120 healthy individuals were separated through nondenaturing PAGE. HDLs were stained for cholesterol using an enzymatic semisolid mixture. Once the gels were unstained, they were stained again for proteins with Coomassie blue. The proportions of HDL subclasses were determined by densitometry. HDL subclasses were transformed to concentrations using as reference HDL-cholesterol plasma levels. This method is comparable in linearity and reproducibility to Coomassie blue staining, although it provides quantitative data. As expected, HDL size distribution shifted toward larger particles when determined by cholesterol as compared with protein. With this method, we observed different proportions of HDL subclasses between men and women as compared with Coomassie blue staining. We described a method to determine HDL size distribution by enzymatic cholesterol staining on polyacrylamide gels. The method allows the quantification of the cholesterol plasma concentration of each HDL subclass with the possibility to further stain the protein in the same sample. The combination of HDL staining by cholesterol and protein on electrophoresis gels provides information that may have clinical relevance.

Early Transcriptomic Response to LDL and oxLDL in Human Vascular Smooth Muscle Cells.

BACKGROUND: Although nowadays it is well known that the human transcriptome can importantly vary according to external or environmental condition, the reflection of this concept when studying oxidative stress and its direct relationship with gene expression profiling during the process of atherogenesis has not been thoroughly achieved. OBJECTIVE: The ability to analyze genome-wide gene expression through transcriptomics has shown that the genome responds dynamically to diverse stimuli. Here, we describe the transcriptome of human vascular smooth muscle cells (hVSMC) stimulated by native and oxidized low-density lipoprotein (nLDL and oxLDL respectively), with the aim of assessing the early molecular changes that induce a response in this cell type resulting in a transcriptomic transformation. This expression has been demonstrated in atherosclerotic plaques in vivo and in vitro, particularly in the light of the oxidative modification hypothesis of atherosclerosis. APPROACH AND RESULTS: Total RNA was isolated with TRIzol reagent (Life Technologies) and quality estimated using an Agilent 2100 bioanalyzer. The transcriptome of hVSMC under different experimental conditions (1,5 and 24 hours for nLDL and oxLDL) was obtained using the GeneChip Human Gene 1.0 ST (Affymetrix) designed to measure gene expression of 28,869 well-annotated genes. A fixed fold-change cut-off corresponding to ± 2 was used to identify genes exhibiting the most significant variation and statistical significance (P< 0.05), and 8 genes validated by qPCR using Taqman probes. CONCLUSIONS: 10 molecular processes were significantly affected in hVSMC: Apoptosis and cell cycle, extracellular matrix remodeling, DNA repair, cholesterol efflux, cGMP biosynthesis, endocytic mechanisms, calcium homeostasis, redox balance, membrane trafficking and finally, the immune response to inflammation. The evidence we present supporting the hypothesis for the involvement of oxidative modification of several processes and metabolic pathways in atherosclerosis is strengthen by the fact that gene expression patterns obtained when hVSMC are incubated for a long period of time in the presence of nLDL, correspond very much the same as when cells are incubated for a short period of time in the presence of chemically modified oxLDL. Our results indicate that under physiological conditions and directly related to specific environmental conditions, LDL particles most probably suffer chemical modifications that initially serve as an alert signal to overcome a harmful stimulus that with time might get transformed to a pathological pattern and therefore consolidate a pathological condition.

Normal HDL-apo AI turnover and cholesterol enrichment of HDL subclasses in New Zealand rabbits with partial nephrectomy.

OBJECTIVE: The kidney has been proposed to play a central role in apo AI catabolism, suggesting that HDL structure is determined, at least in part, by this organ. Here, we aimed at determining the effects of a renal mass reduction on HDL size distribution, lipid content, and apo AI turnover. METHODS: We characterized HDL subclasses in rabbits with a 75% reduction of functional renal mass (Nptx group), using enzymatic staining of samples separated on polyacrylamide electrophoresis gels, and also performed kinetic studies using radiolabeled HDL-apo AI in this animal model. RESULTS: Creatinine clearance was reduced to 35% after nephrectomy as compared to the basal values, but without increased proteinuria. A slight, but significant modification of the relative HDL size distribution was observed after nephrectomy, whereas cholesterol plasma concentrations gradually augmented from large HDL2b (+54%) to small HDL3b particles (+150%, P<0.05). Cholesteryl esters were the increased fraction; in contrast, free cholesterol phospholipids and triglycerides of HDL subclasses were not affected by nephrectomy. HDL-apo AI fractional catabolic rates were similar to controls. CONCLUSION: Reduction of functional renal mass is associated to enrichment of HDL subclasses with cholesteryl esters. Structural abnormalities were not related to a low apo AI turnover, suggesting renal contribution to HDL remodeling beyond being just a catabolic site for these lipoproteins.

Lipid plasma concentrations of HDL subclasses determined by enzymatic staining on polyacrylamide electrophoresis gels in children with metabolic syndrome.

BACKGROUND: The antiatherogenic role of different HDL subclasses is still controversial. HDL particles of the same size can have different lipid contents in some physiopathological situations. However, little is known about the plasma lipid levels of HDL subclasses when they are separated by their hydrodynamic diameter. METHODS: Triglycerides (Tg), phosphatidylcholine (Ph), and cholesterol (C) plasma concentrations of HDL subclasses, were determined by enzymatic staining on polyacrylamide gradient gel (PAGE) in 50 pediatric patients with metabolic syndrome (MS), and 50 control children paired by age and gender. Proteins of HDL subclasses were also stained for the assessment of the relative size distribution of HDL. RESULTS: Relative HDL size distribution was shifted to small particles in MS pediatric patients when determined per protein. In contrast, cholesterol plasma concentrations corresponding to the HDL2b, 2a, 3a, and 3b subclasses were decreased; triglycerides of HDL3b and 3c, as well as plasma phospholipids from HDL3c, were elevated in MS patients as compared to controls. The C-to-Ph ratio, considered as indicative of HDL composition, was similar among the 5 HDL subclasses in control subjects, whereas this ratio gradually decreased from large HDL2b to small HDL3c in the MS group. Cholesterol plasma concentrations of HDL subclasses correlated with the components of the MS. CONCLUSIONS: Lipids of HDL subclasses provide more and accurate information than the relative HDL size distribution determined by protein staining, and may contribute to understand better HDL metabolism and the coronary risk associated to these lipoproteins.

Rosiglitazone modifies HDL structure and increases HDL-apo AI synthesis and catabolic rates.

BACKGROUND: Rosiglitazone is an agonist of the peroxisome proliferator-activated receptor (PPAR) gamma that may modify HDL metabolism in humans, but this effect has not been completely elucidated. Therefore, we determined the effect of rosiglitazone on apo AI turnover, HDL structure, and PON1 plasma activity. METHODS: Kinetic studies of HDL-apo AI radiolabeled with (125)I were performed in 7 chow-fed, male, New Zealand white rabbits after 6 weeks of 0.32 mg/kg/d rosiglitazone-treatment vs. vehicle-treated rabbits (n=11). HDL size distribution was determined by polyacrylamide gradient electrophoresis and paraoxonase-1 (PON1); plasma activity was assessed spectrophotometrically using phenylacetate as substrate. RESULTS: Fractional catabolic rate (FCR) of HDL apo AI was higher in the rosiglitazone-treated group than in the control group (0.031+/-0.004 vs. 0.025+/-0.006 pools/h, respectively, p<0.05). The mean apo AI production rate (PR) was 62% higher in the rosiglitazone group as compared to controls (0.918+/-0.238 vs. 0.564+/-0.160 mg/kg/h, p<0.01). Accordingly, apo AI plasma levels in rosiglitazone-treated animals were about 37% higher than in the control group. Rosiglitazone-induced changes in apo AI turnover appeared concomitantly with a significant increase of phospholipids and a decrease in colesteryl esters content of the HDL. Compositional changes resulted in a relative increase of the HDL3b and HDL3c subfractions and a significant enhancement of the plasma PON1 activity (488.5+/-138.2 vs. 595.2+/-179.4 micromol/min/ml, p<0.05). CONCLUSIONS: Rosiglitazone increased apo AI plasma concentrations, resulting from an enhancement of apo AI synthesis, and induced the synthesis of smaller HDL particles with a concomitant increase of plasma PON1 activity. These modifications may contribute to the anti-atherogenic potential of rosiglitazone.