Victor Debbas

Effects of high adherence to mediterranean or low-fat diets in medicated secondary prevention patients.

Although the Mediterranean diet (MD) and the low-fat Therapeutic Lifestyle Changes Diet (TLCD) promote equivalent increases in event-free survival in secondary coronary prevention, possible mechanisms of such complete dietary patterns in these patients, usually medicated, are unclear. The aim of this study was to investigate the effects of the MD versus the TLCD in markers of endothelial function, oxidative stress, and inflammation after acute coronary syndromes. Comparison was made between 3 months of the MD (n = 21; rich in whole grains, vegetables, fruits, nuts, and olive oil, plus red wine) and the TLCD (n = 19; plus phytosterols 2 g/day) in a highly homogenous population of stable patients who experienced coronary events in the previous 2 years (aged 45 to 65 years, all men) allocated to each diet under a strategy designed to optimize adherence, documented as >90%. Baseline demographics, body mass index and clinical data, and use of statins and other drugs were similar between groups. The MD and TLCD promoted similar decreases in body mass index and blood pressure (p ≤0.001) and particularly in plasma asymmetric dimethylarginine levels (p = 0.02) and l-arginine/asymmetric dimethylarginine ratios (p = 0.01). The 2 diets did not further enhance flow-mediated brachial artery dilation compared to baseline (4.4 ± 4.0%). Compared to the TLCD, the MD promoted decreases in blood leukocyte count (p = 0.025) and increases in high-density lipoprotein levels (p = 0.053) and baseline brachial artery diameter. Compared to the MD, the TLCD decreased low-density lipoprotein and oxidized low-density lipoprotein plasma levels, although the ratio of oxidized to total low-density lipoprotein remained unaltered. Glucose, high-sensitivity C-reactive protein, triglycerides, myeloperoxidase, intercellular adhesion molecular, vascular cell adhesion molecule, and glutathione serum and plasma levels remained unchanged with either diet. In conclusion, medicated secondary prevention patients show evident although small responses to the MD and the TLCD, with improved markers of redox homeostasis and metabolic effects potentially related to atheroprotection.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

CD100 and plexins B2 and B1 mediate monocyte-endothelial cell adhesion and might take part in atherogenesis.

Leukocyte migration is essential for the function of the immune system. Their recruitment from the vessels to the tissues involves sequential molecular interactions between leukocytes and endothelial cells (ECs). Many adhesion molecules involved in this process have already been described. However, additional molecules may be important in this interaction, and here we explore the potential role for CD100 and plexins in monocyte-EC binding. CD100 was shown to be involved in platelet-endothelial cell interaction, an important step in atherogenesis and thrombus formation. In a recent work we have described CD100 expression in monocytes and in macrophages and foam cells of human atherosclerotic plaques. In the present work, we have identified plexin B2 as a putative CD100 receptor in these cells. We have detected CD100 expression in the endothelium as well as in in vitro cultured endothelial cells. Blocking of CD100, plexin B1 and/or B2 in adhesion experiments have shown that both CD100 and plexins act as adhesion molecules involved in monocyte-endothelial cell binding. This effect may be mediated by CD100 expressed in both cell types, probably coupled to the receptors endothelial plexin B1 and monocytic plexin B2. These results can bring new insights about a possible biological activity of CD100 in monocyte adhesion and atherosclerosis, as well as a future candidate for targeting therapeutics.

Mechanisms underlying hypertriglyceridemia in rats with monosodium l-glutamate-induced obesity: Evidence of XBP-1/PDI/MTP axis activation

Non-alcoholic fatty liver disease (NAFLD) is intimately associated with insulin resistance and hypertriglyceridemia, whereas many of the mechanisms underlying this association are still poorly understood. In the present study, we investigated the relationship between microsomal triglyceride transfer protein (MTP) and markers of endoplasmic reticulum (ER) stress in the liver of rats subjected to neonatal monosodium L-glutamate (MSG)-induced obesity. At age 120 days old, the MSG-obese animals exhibited hyperglycemia, hypertriglyceridemia, insulin resistance, and liver steatosis, while the control (CTR) group did not. Analysis using fast protein liquid chromatography of the serum lipoproteins revealed that the triacylglycerol content of the very low-density lipoprotein (VLDL) particles was twice as high in the MSG animals compared with the CTR animals. The expression of ER stress markers, GRP76 and GRP94, was increased in the MSG rats, promoting a higher expression of X-box binding protein 1 (XBP-1), protein disulfide isomerase (PDI), and MTP. As the XBP-1/PDI/MTP axis has been suggested to represent a significant lipogenic mechanism in the liver response to ER stress, our data indicate that hypertriglyceridemia and liver steatosis occurring in the MSG rats are associated with increased MTP expression.

Testosterone induces leucocyte migration by NADPH oxidase-driven ROS- and COX2-dependent mechanisms.

The mechanisms whereby testosterone increases cardiovascular risk are not clarified. However, oxidative stress and inflammation seem to be determinants. Herein, we sought to determine whether exogenous testosterone, at physiological levels, induces leucocyte migration, a central feature in immune and inflammatory responses and the mediating mechanisms. We hypothesized that testosterone induces leucocyte migration via NADPH oxidase (NADPHox)-driven reactive oxygen species (ROS) and cyclooxygenase (COX)-dependent mechanisms. Sixteen-week-old Wistar rats received an intraperitoneal injection (5 ml) of either testosterone (10(-7) mol/l) or saline. Rats were pre-treated with 5 ml of sodium salicylate (SS, non-selective COX inhibitor, 1.25 × 10(-3) mol/l, 1 h prior to testosterone or saline), flutamide (androgen receptor antagonist, 10(-5) mol/l), apocynin (NADPHox inhibitor, 3 × 10(-4) mol/l), N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS398, COX2 inhibitor, 10(-4) mol/l) or saline, 4 h before testosterone or saline administration. Leucocyte migration was assessed 24 h after testosterone administration by intravital microscopy of the mesenteric bed. Serum levels of testosterone were measured by radioimmunoassay. NADPHox activity was assessed in membrane fractions of the mesenteric bed by dihydroethidium (DHE) fluorescence and in isolated vascular smooth muscle cells (VSMC) by HPLC. NADPHox subunits and VCAM (vascular cell adhesion molecule) expression were determined by immunoblotting. Testosterone administration did not change serum levels of endogenous testosterone, but increased venular leucocyte migration to the adventia, NADPHox activity and expression (P < 0.05). These effects were blocked by flutamide. SS inhibited testosterone-induced leucocyte migration (P<0.05). Apocynin and NS398 abolished testosterone-induced leucocyte migration and NADPHox activity (P<0.05). Testosterone induces leucocyte migration via NADPHox- and COX2-dependent mechanisms and may contribute to inflammatory processes and oxidative stress in the vasculature potentially increasing cardiovascular risk.

Cellular prion protein (PrPC) and superoxide dismutase (SOD) in vascular cells under oxidative stress

UNLABELLED The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall. OBJECTIVES to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity. METHODS To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter. RESULTS In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9±1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C).

Evaluation of mild hyperhomocysteinemia during the development of atherosclerosis in apolipoprotein E-deficient and normal mice.

We focused on the effect of mild hyperhomocysteinemia (HHcy) on the development of atherosclerosis, using apolipoprotein E-deficient (apoE(-/-)) and normal mice. Mice received diets enriched in methionine with low or high levels of folate, B(12) and B(6) (diets B and C, respectively), and diet only with low levels of folate, B(12) and B(6) (diets D), to induce mild HHcy. Normal mice fed on diets B, C and D presented mild HHcy, but they did not develop atherosclerotic lesions after 24 weeks of diet. In addition, increased endoplasmic reticulum stress was present in normal mice fed on diet B, compared to others groups. ApoE(-/-) mice fed on diet B for 20 weeks presented the greatest atherosclerotic lesion area at the aortic sinus than other groups. These results suggest that the methionine may have a toxic effect on endothelium, and the B-vitamins addition on diet may have a protective effect in the long term, despite the increase on homocysteine levels. Mild HHcy accelerated the development of atherosclerosis in apoE(-/-) mice, and supplementation with B-vitamins is important for prevention of vascular disease, principally in the long term.

Phage Display Identification of CD100 in Human Atherosclerotic Plaque Macrophages and Foam Cells

Atherosclerosis is a complex disease in which vessels develop plaques comprising dysfunctional endothelium, monocyte derived lipid laden foam cells and activated lymphocytes. Considering that humans and animal models of the disease develop quite distinct plaques, we used human plaques to search for proteins that could be used as markers of human atheromas. Phage display peptide libraries were probed to fresh human carotid plaques, and a bound phage homologous to plexin B1, a high affinity receptor for CD100, was identified. CD100 is a member of the semaphorin family expressed by most hematopoietic cells and particularly by activated T cells. CD100 expression was analyzed in human plaques and normal samples. CD100 mRNA and protein were analyzed in cultured monocytes, macrophages and foam cells. The effects of CD100 in oxLDL-induced foam cell formation and in CD36 mRNA abundance were evaluated. Human atherosclerotic plaques showed strong labeling of CD100/SEMA4D. CD100 expression was further demonstrated in peripheral blood monocytes and in in vitro differentiated macrophages and foam cells, with diminished CD100 transcript along the differentiation of these cells. Incubation of macrophages with CD100 led to a reduction in oxLDL-induced foam cell formation probably through a decrease of CD36 expression, suggesting for the first time an atheroprotective role for CD100 in the human disease. Given its differential expression in the numerous foam cells and macrophages of the plaques and its capacity to decrease oxLDL engulfment by macrophages we propose that CD100 may have a role in atherosclerotic plaque development, and may possibly be employed in targeted treatments of these atheromas.

Quiescin sulfhydryl oxidase (QSOX) is expressed in the human atheroma core: possible role in apoptosis

Quiescin sulfhydryl oxidases (QSOXs) catalyze the formation of disulfide bonds in peptides and proteins, and in vertebrates comprise two proteins: QSOX1 and QSOX2. QSOX1, the most extensively studied type, has been implicated in protein folding, production of extracellular matrix, redox regulation, protection from apoptosis, angiogenesis, and cell differentiation. Atherosclerosis is an immunopathological condition in which redox processes, apoptosis, cell differentiation, and matrix secretion/maturation have critical roles. Considering these data, we hypothesized that QSOX1 could be involved in this disease, possibly reducing apoptosis and angiogenesis inside the plaque. QSOX1 labeling in normal human carotid vessels showed predominant expression by endothelium, subendothelium, and adventitia. In atherosclerotic plaques, however, QSOX1 was highly expressed in macrophages at the lipid core. QSOX1 expression was also studied in terms of mRNA and protein in cell types present in plaques under apoptotic or activating stimuli, emulating conditions found in the atherosclerotic process. QSOX1 mRNA increased in endothelial cells and macrophages after the induction of apoptosis. At the protein level, the correlation between apoptosis and QSOX1 expression was not evident in all cell types, possibly because of a rapid secretion of QSOX1. Our results propose for the first time possible roles for QSOX1 in atherosclerosis, being upregulated in endothelial cells and macrophages by apoptosis and cell activation, and possibly controlling these processes, as well as angiogenesis. The quantitative differences in QSOX1 induction may depend on the cell type and also on local factors.

Fibrillin-1 mgΔlpn Marfan syndrome mutation associates with preserved proteostasis and bypass of a protein disulfide isomerase-dependent quality checkpoint

Fibrillin-1 mutations promote Marfan syndrome (MFS) via complex yet unclear pathways. The roles of endoplasmic reticulum (ER) and the major ER redox chaperone protein disulfide isomerase-A1 in the processing of normal and mutated fibrillin-1 and ensuing protein secretion and/or intracellular retention are unclear. Our results in mouse embryonic fibroblasts bearing the exon-skipping mgΔ(lox-P-neo) (mgΔ(lpn)) mutation, which associates in vivo with MFS and in vitro with disrupted microfibrils, indicate a preserved ER-dependent proteostasis or redox homeostasis. Rather, mutated fibrillin-1 is secreted normally through Golgi-dependent pathways and is not intracellularly retained. Similar results occurred for the C1039G point mutation. In parallel, we provide evidence that PDIA1 physically interacts with fibrillin-1 in the ER. Moreover, siRNA against PDIA1 augmented fibrillin-1 secretion rates in wild-type cells. However, fibrillin-1 with the mgΔ(lpn) mutation bypassed PDI checkpoint delay, while the C1039G mutation did not. This heretofore undisclosed PDIA1-mediated mechanism may be important to control the extracellular availability of function-competent fibrillin-1, an important determinant of disease phenotype. Moreover, our results may reveal a novel, holdase-like, PDI function associated with ER protein quality control.