Eplerenone reduced lesion size in early but not advanced atherosclerosis in apolipoprotein E-deficient mice.

The beneficial effects of eplerenone, a specific mineralocorticoid receptor blocker, were previously demonstrated in early atherosclerosis (ATS). The aim of the present study was to evaluate the effect of eplerenone in advanced versus early ATS. Apolipoprotein E knockout mice aged 16 or 32 weeks were randomly divided into eplerenone (100 mg·kg·d) or vehicle treatment for 14 weeks. Eplerenone reduced atherosclerotic lesion size by 51% only in early ATS. In peritoneal macrophages obtained from these mice, eplerenone reduced messenger RNA expression of pro-inflammatory markers, interleukin 6, tumor necrosis factor α, monocyte chemotactic protein 1, and increased anti-inflammatory marker arginase 1 to a greater extent in early compared with advanced ATS. These changes correspond to macrophage polarization toward alternative inflammatory phenotype. Messenger RNA expression of the mineralocorticoid receptor and aldosterone synthase were also reduced by eplerenone to a greater extent in early ATS, and these might increase the sensitivity of macrophages to mineralocorticoid blockade in early ATS. The results of the present study point to the benefits of early initiation of treatment with eplerenone in reducing experimental ATS.

Aldosterone up-regulates 12- and 15-lipoxygenase expression and LDL oxidation in human vascular smooth muscle cells.

Several lines of evidence suggest that aldosterone excess may have detrimental effects in the cardiovascular system, independent of its interaction with the renal epithelial cells. Here we examined the possibility that aldosterone modulates 12- and/or 15-lipoxygenase (LO) expression/activity in human vascular smooth muscle cells (VSMC), in vitro, thereby potentially contributing to both vascular reactivity and atherogenesis. Following 24 h treatment of VSMC with aldosterone (1 nmol/L), there was a approximately 2-fold increase in the generation rate of 12 hydroxyeicosatetraenoic acid (12-HETE), 70% increase in platelet type 12-LO mRNA expression (P < 0.001) along with a approximately 3-fold increase in 12-LO protein expression, which were blocked by the mineralocorticoid receptor (MR) antagonists spironolactone (100 nmol/L) and eplerelone (100 nmol/ml). Additionally, aldosterone (1 nmol/L; 24 h) increased the production of 15-HETE (50%; P < 0.001) and the expression of 15-LO type 2 mRNA (50%; P < 0.05) (in VSMC). Aldosterone also increased the 12- and 15-LO type 2 mRNA expression in a line of human aortic smooth muscle cells (T/G HA-VSMC) (60% and 50%, respectively). Aldosterone-induced 12- and 15-LO type 2 mRNA expressions were blocked by the EGF-receptor antagonist AG 1478 and by the MAPK-kinase inhibitor UO126. Aldosterone-treated VSMC also showed increased LDL oxidation, (approximately 2-fold; P < 0.001), which was blocked by spironolactone. In conclusion, aldosterone increased 12- and 15-LO expression in human VSMC, in association with increased 12- and 15-HETE generation and enhanced LDL oxidation and may directly augment VSMC contractility, hypertrophy, and migration through 12-HETE and promote LDL oxidation via the pro-oxidative properties of these enzymes.

Vitamin E supplementation reduces cardiovascular events in a subgroup of middle-aged individuals with both type 2 diabetes mellitus and the haptoglobin 2-2 genotype: a prospective double-blinded clinical trial.

OBJECTIVE: Clinical trials of vitamin E have failed to demonstrate a decrease in cardiovascular events. However, these studies did not address possible benefit to subgroups with increased oxidative stress. Haptoglobin (Hp), a major antioxidant protein, is a determinant of cardiovascular events in patients with Type 2 diabetes mellitus (DM). The Hp gene is polymorphic with 2 common alleles, 1 and 2. The Hp 2 allelic protein product provides inferior antioxidant protection compared with the Hp 1 allelic product. We sought to test the hypothesis that vitamin E could reduce cardiovascular events in DM individuals with the Hp 2-2 genotype, a subgroup that comprises 2% to 3% of the general population. METHODS AND RESULTS: 1434 DM individuals > or = 55 years of age with the Hp 2-2 genotype were randomized to vitamin E (400 U/d) or placebo. The primary composite outcome was myocardial infarction, stroke, and cardiovascular death. At the first evaluation of events, 18 months after initiating the study, the primary outcome was significantly reduced in individuals receiving vitamin E (2.2%) compared with placebo (4.7%; P=0.01) and led to early termination of the study. CONCLUSIONS: Vitamin E supplementation appears to reduce cardiovascular events in individuals with DM and the Hp 2-2 genotype (ClinicalTrials.gov NCT00220831).

Aspirin promotes low density lipoprotein susceptibility to oxidative modification in healthy volunteers.

BACKGROUND: Low density lipoprotein oxidation is a major pathogenic pathway in atherosclerosis. Previous studies suggested that aspirin, a commonly prescribed drug in patients with atherosclerosis, when given in a dose of 300 mg/day may decrease LDL susceptibility to oxidative modification. However, the effect of the more common lower dose aspirin on LDL oxidation is not known. OBJECTIVES: To examine the effect of aspirin administration (low dosage) on the susceptibility of LDL to oxidative modification in healthy volunteers. METHODS: Aspirin 75 mg was administered daily for 2 weeks to 10 healthy volunteers selected from the medical staff and students at the faculty of medicine. The main outcome measure was ex vivo oxidation of LDL by ultraviolet C irradiation or by peroxyl free radicals generated by AAPH (2,2'-azobis 2-amidinopropane hydrochloride). The extent of LDL oxidation was determined by measuring the formed amounts of thiobarbituric acid-reactive substances, lipid peroxides and conjugated dienes. RESULTS: Following exposure to UVc irradiation there was a significant (P 0.01) increase (10.8%) in TBARS concentrations and a significant (P < 0.05) increase (5.4%) in PD concentrations in LDL withdrawn after aspirin treatment as compared to LDL withdrawn before aspirin treatment. Following incubation with AAPH there was a significant (P < 0.05) increase (15%) in PD concentrations and a significant (P < 0.05) reduction (10%) of the LDL oxidation lag time in LDL withdrawn after aspirin intake as compared to LDL withdrawn before aspirin treatment. CONCLUSIONS: Aspirin treatment given to healthy volunteers at a dose of 75 mg/day increased the susceptibility of their plasma LDL to oxidative modification ex vivo. Our study provides, for the first time, in vivo evidence of pro-oxidative properties of aspirin already suggested by previous in vitro trials.

ACE2 of the heart: From angiotensin I to angiotensin (1-7).

Angiotensin II (Ang II), a bioactive peptide of the renin-angiotensin system (RAAS), plays an important role in the development of cardiovascular diseases (CVD). Pharmacological inhibition of angiotensin-converting enzyme (ACE), the Ang II forming enzyme, or specific blockade of Ang II binding to angiotensin type 1 receptor (AT1R) through which it exerts its deleterious effects, were shown to provide some protection against progression of CVD. The ACE-Ang II-AT1R axis has been challenged over the last few years with RAAS components able to counterbalance the effects of the main axis. The ACE homologue ACE2 efficiently hydrolyses Ang II to form Ang (1-7), a peptide that exerts actions opposite to those of Ang II. In contrast to the Ang II axis, the role of the ACE2-Ang (1-7) axis in cardiac function is largely obscure. Ang (1-7) is present in the viable myocardium, and its formation depends on Ang II as a substrate. The expression of this peptide is associated with cardiac remodeling: it is lost in the infarcted area and significantly increased in the border area. Low doses of Ang (1-7) improve cardiac output and antagonize Ang II-induced vasoconstriction. The type of Ang (1-7) biological activity is tissue specific and dose dependent. These findings point to a possible protective role for Ang (1-7) in abating the Ang II-induced actions. The elevated expression of Ang (1-7) in failing heart tissue paralleled the expression of its forming enzyme, ACE2. Several observations and experimental evidence suggest a beneficial role for ACE2 in cardiovascular function. Elevated ACE2 expression at the initial stage of several pathologies which decline with progression of disease might indicate a protective role for ACE2. Genetic manipulation of ACE2 expression, either targeted disruption or overexpression, point to the possible significance of this enzyme in cardiac function. Based on the above, a therapeutic approach that will amplify the ACE2-Ang (1-7) axis could provide further protection against the development of CVD. It turns out that the merits of currently used drugs--ACE inhibitors, AT1R blockers and mineralocorticoid receptor blockers (MRB) - lay beyond their direct effects on suppression of the ACE-Ang II-AT1R axis as they also increase cardiac ACE2 and Ang (1-7) significantly.

Measurement of carotid artery intima-media thickness in dyslipidemic patients increases the power of traditional risk factors to predict cardiovascular events.

A longitudinal observational study investigated whether the measurement, in clinical practice, of carotid maximum intima-media thickness (Max-IMT) could be combined with the Framingham risk score (FRS) to improve the predictability of cardiovascular events in dyslipidemic patients who are at low or intermediate risk. Max-IMT was measured by ultrasound in 1969 patients attending a lipid clinic. The "best threshold values" (BTVs) above which we considered the Max-IMT to be abnormally high were calculated for our dyslipdemic population for each 10-year age interval in men and women. Two hundred and forty-two patients (age 54+/-10 years; 43.8% women) with an FRS <20%, i.e. at low or intermediate risk, were monitored for more than 5 years. Twenty-four of these patients suffered a cardiovascular event within 5.1+/-2.3 years. Both FRS and Max-IMT proved to be independent outcome predictors (p<0.04, both), with a hazard ratio (HR) of 6.7 (95% CI 1.43, 31.04; p=0.015) in patients in whom FRS was 10-20% and Max-IMT was above the BTV (60th percentile of Max-IMT distribution for men or 80th for women). In Kaplan-Meier analysis, the Max-IMT significantly improved the predictive value of the FRS (chi(2)=8.13, p=0.04). Patients with FRS 10-20% (currently considered intermediate-risk) and also elevated Max-IMT values came into the same high-risk category as patients with FRS 20-30%. The combination of FRS with Max-IMT measurement can be used in routine clinical practice to greatly enhance the predictability of cardiovascular events in the large number of patients who fall into the intermediate-risk category, which currently does not call for aggressive preventive measures.

Mineralocorticoid receptor blocker increases angiotensin-converting enzyme 2 activity in congestive heart failure patients.

Aldosterone plays an important role in the pathophysiology of congestive heart failure (CHF), and spironolactone improves cardiovascular function and survival rates in patients with CHF. We hypothesized that the mineralocorticoid receptor blockade (MRB) exerted its beneficial effects by reducing oxidative stress and changing the balance between the counter-acting enzymes angiotensin-converting enzyme (ACE) and ACE2. Monocyte-derived macrophages were obtained from 10 patients with CHF before and after 1 month of treatment with spironolactone (25 mg/d). Spironolactone therapy significantly (P<0.005) reduced oxidative stress, as expressed by reduced lipid peroxide content, superoxide ion release, and low-density lipoprotein oxidation by 28%, 53%, and 70%, respectively. Although spironolactone significantly (P<0.01) reduced macrophage ACE activity by 47% and mRNA expression by 53%, ACE2 activity and mRNA expression increased by 300% and 654%, respectively. In mice treated for 2 weeks with eplerenone (200 mg.kg(-1).d(-1)), cardiac ACE2 activity significantly (P<0.05) increased by 2-fold and was paralleled by increased ACE2 activity in macrophages. The mechanism of aldosterone antagonist action was studied in mouse peritoneal macrophages (MPMs) in vitro. Although ACE activity and mRNA were significantly increased by 250 nmol/L aldosterone, ACE2 was significantly reduced. Cotreatment with eplerenone (2 micromol/L) attenuated these effects. In MPM obtained from p47 knockout mice, where NADPH oxidase is inactive, as well as in control MPMs treated with NADPH oxidase inhibitor, aldosterone did not increase ACE or decrease ACE2. MRB reduced oxidative stress, decreased ACE activity, and increased ACE2 activity, suggesting a protective role for MRB by possibly increasing generation of angiotensin (1-7) and decreasing formation of angiotensin II. These effects are mediated, at least in part, by NADPH oxidase.

Atherosclerosis and the protective role played by different proteins in apolipoprotein E-deficient mice.

The aim of the present study was to analyze the early events in atherogenesis and the role of pro- or anti-atherosclerotic proteins in the development of atherosclerotic lesions. We used apolipoprotein E-deficient (E(0)) mice that spontaneously develop hypercholesterolemia and atherosclerotic lesions in the aorta in a time-dependent manner. Aortas of mice aged 6, 8, 10 and 12 weeks were examined to determine histopathological changes. In mice aged 8-12 weeks, developing atherosclerotic lesions were present in different regions of the aortas. These lesions protruded into the lumen of the vessel and showed lipid deposits, lipid-filled macrophages and extensive accumulation of collagen and elastic fibers throughout the entire arterial wall. A parallel immunohistochemical study included analysis of three proteins known to be involved in atherosclerosis, i.e. inducible nitric oxide synthase (iNOS, NOS2), vascular endothelial growth factor (VEGF) and matrix metalloproteinase-2 (MMP2). Increased immunolabelling of iNOS and VEGF accompanied atherosclerosis development in E(0) mice aged 8, 10 and 12 weeks. On the contrary, immunolabelling for MMP2 was negative in E(0) mice aged 10 and 12 weeks. Our results indicate morphological alterations in the Tunica intima and Tunica media of atherosclerotic aortas and possible protective roles for iNOS and VEGF proteins against atherosclerosis development. These data may be relevant for developing therapeutic strategies for atherosclerosis development.

[The importance of ACE2 in regulating the cardiovascular system].

A recently discovered homologue of the angiotensin-converting enzyme, ACE2, insensitive to ACE inhibitors, was found in rodents and humans. ACE2 is expressed mainly in the vasculature, heart and kidney. ACE2 removes a single amino acid of the carboxy terminal of peptides. In the renin-angiotensin-aldosterone system (RAAS), it is responsible for the conversion of angiotensin I (Ang I) and angiotensin II (Ang II) to Ang 1-9 and Ang 1-7, respectively. While ACE forms Ang II, a potent vasoconstrictor, ACE2 degrades this peptide to form Ang 1-7 which has an opposite action. Therefore, ACE2 counteracts ACE in the balance of vasopressor/vasodilator as well as heart and kidney function. The importance of ACE2 in physiological and pathophysiological conditions is unclear and is currently being studied.

A mouse model for human atherosclerosis: long-term histopathological study of lesion development in the aortic arch of apolipoprotein E-deficient (E0) mice.

Apolipoprotein E-deficient mice, since their introduction in the early 1990s, have proved to be a very popular model for studying spontaneous hypercholesterolemia and the subsequent development of atherosclerotic lesions. The pathogenesis of atherosclerotic lesions in these mice mimics that found in humans on a very short time-scale. Atherosclerotic lesion development is especially prominent in the aortic arch. We have followed the progressive histopathological development of atherosclerotic lesions in the aortic arch of apolipoprotein E-deficient mice aged from 6 weeks to 18 months in 1 microm epoxy-resin sections stained with alkaline toluidine blue, which gives greatly improved resolution over wax sections. During the early stages of lesion formation, lipid-filled macrophages appear in the subendothelium, and accumulate leading to "fatty streaks". Macrophage degeneration and the formation of lipid pools are accompanied by accumulation of cholesterol deposits. Disruptions of elastic laminae of the Tunica media are accompanied by structural changes in the myocytes. More advanced lesions involve fibrous cap development, calcification of the vessel wall and progressive occlusion of the lumen. Unstable plaque may also be found. Various approaches for quantitative determination of lesion size are considered. The study provides a histopathological baseline for spontaneous atherosclerosis associated with hypercholesterolemia, which can be used in connection with experimental interventional studies on the efficacy of drugs or foodstuffs in retardation of atherosclerosis.

Aldosterone administration to mice stimulates macrophage NADPH oxidase and increases atherosclerosis development: a possible role for angiotensin-converting enzyme and the receptors for angiotensin II and aldosterone.

BACKGROUND: The renin-angiotensin-aldosterone system is involved in the pathogenesis of atherosclerosis, partially because of its pro-oxidative properties. We questioned the effect and mechanisms of action of administration of aldosterone to apolipoprotein E-deficient (E(0)) mice on their macrophages and aorta oxidative status and the ability of pharmacological agents to block this effect. METHODS AND RESULTS: Aldosterone (0.2 to 6 microg. mouse(-1) x d(-1)) was administered to E(0) mice alone or in combination with eplerenone (200 mg x kg(-1) x d(-1)), ramipril (5 mg x kg(-1) x d(-1)), or losartan (25 mg x kg(-1) x d(-1)). Mouse aortic atherosclerotic lesion area and macrophage and aortic oxidative status were evaluated. Aldosterone administration enhanced the mouse atherosclerotic lesion area by 32%. Mouse peritoneal macrophages and aortic segments from aldosterone-treated mice exhibited increased superoxide anion formation by up to 155% and 69%, respectively, and this effect was probably mediated by NADPH oxidase activation, because increased translocation of its cytosolic component p47phox to the macrophage plasma membrane was observed. THP-1 macrophages incubated in vitro with aldosterone (10 micromol/L) exhibited a higher capacity to release superoxide ions by 110% and increased ability to oxidize LDL by 74% compared with control cells. Aldosterone administration enhanced mouse peritoneal macrophage ACE activity and mRNA expression by 2.3-fold and 2.4-fold, respectively. Only cotreatment of eplerenone with ramipril or losartan completely blocked the oxidative effects of aldosterone. CONCLUSIONS: Aldosterone administration to E(0) mice increased macrophage oxidative stress and atherosclerotic lesion development. Blocking of the mineralocorticoid receptor and inhibition of tissue ACE and/or the angiotensin receptor-1 reduced aldosterone deleterious pro-oxidative and proatherogenic effects.

Macrophage foam-cell formation in streptozotocin-induced diabetic mice: stimulatory effect of glucose.

BACKGROUND: Diabetes is associated with an increased risk for atherosclerosis. We investigated the effect of diabetes induction on atherogenesis and on macrophage-foam cell formation. METHODS AND RESULTS: Atherosclerotic apolipoprotein-E-deficient mice were converted into diabetic mice by streptozotocin injection. Aortic atherosclerotic lesion area was significantly enhanced by 67% and 106% in mice that were diabetic for 1 and 3 months, respectively, compared to the non-diabetic mice. Moreover, mouse peritoneal macrophages (MPM) from diabetic mice for 1 and 3 months exhibit higher lipid peroxides content by 55% and 63%, respectively, in association with the MPM glucose content. Oxidized LDL (Ox-LDL) uptake by MPM obtained from diabetic mice for 1 and 3 months was significantly increased by 36% and 45%, respectively, in association with the increased macrophage cholesterol content. To determine whether the accelerated foam cell formation in diabetic mice could result from a direct effect of glucose on macrophages, J-774-A.1 macrophages were incubated with increasing glucose concentrations (2.5-62 mM). Glucose-enriched macrophages exhibit dose-dependent higher peroxides content up to 7.5-fold and increased Ox-LDL cellular uptake associated with up-regulation of the scavenger receptor CD36 at the mRNA level. CONCLUSION: Induction of diabetes in atherosclerotic mice led to an accelerated atherosclerosis and macrophage-derived foam cell formation, probably by involving a glucose-dependent related mechanism.

Angiotensin II increases the expression of lectin-like oxidized low-density lipoprotein receptor-1 in human vascular smooth muscle cells via a lipoxygenase-dependent pathway.

BACKGROUND: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a membrane protein that can act as a surface endocytosis receptor for oxidized LDL (ox-LDL). As increased cellular uptake of ox-LDL by macrophages and activated smooth muscle cells may transform these cells into foam cells, potential interactions among LDL oxidation, ox-LDL uptake, and regulators of vascular smooth muscle cell function are of obvious interest. The objective of this study was to examine the effect of angiotensin II (AII) on the expression of LOX-1 and ox-LDL degradation in human vascular smooth muscle cells (VSMC) METHODS: We performed in vitro experiments in a human VSMC line (T/G HA-VSMC) derived from normal aortic VSMC, using standards methods. RESULTS: We found that AII (10(-7) mol/L) increased the expression of LOX-1 (approximately 2.5-fold, P < .0001) in association with higher degradation of ox-LDL by HA-SMC (from 4019 +/- 529 ng/mg cell protein to 6207 +/- 287 ng/mg cell protein; P = .0033). AII also increased the expression of 12-lipoxygenase (12-LO) and 15-lipoxygenase (15-LO) by approximately 2.2-fold (P = .03) and approximately 3-fold (P = .006), respectively. In addition, AII (10(-7) mol/L) increased the release of 12- and 15-hydroxyeicosatetraenoic acid from VSMC within 10 min approximately 3-fold (P = .03) and 50% (P < .05), respectively. CONCLUSIONS: Our study findings provide evidence that angiotensin II upregulates LOX-1 and 12-LO and 15-LO expression in human VSMC, thereby potentially providing mechanisms for both accelerated LDL oxidation within the cell and the internalization of exogenous ox-LDL, two processes that could increase the susceptibility of human VSMC to further transformation into foam cells.

Tissue angiotensin-converting-enzyme (ACE) deficiency leads to a reduction in oxidative stress and in atherosclerosis: studies in ACE-knockout mice type 2.

UNLABELLED: Background- Angiotensin II, produced by angiotensin-converting-enzyme (ACE), enhances oxidative stress and atherogenesis. In this study, we analyzed whether tissue ACE deficiency in ACE-knockout mice type-2 would affect their oxidative status. Moreover, by crossbreeding the ACE-knockout mice with atherosclerotic apolipoprotein E (apo E)-deficient (E0) mice, we questioned whether tissue ACE deficiency affects atherogenesis. METHODS AND RESULTS: ACE-deficient mice type-2 (ACE+/-) exhibited reduced serum lipid peroxidation compared with ACE+/+ mice. Peritoneal macrophages from ACE+/- mice demonstrated lower oxidative status, as exhibited by decreases of 47%, 33% 56%, and 51%, in their lipid peroxides, superoxide release, dichlorofluorescein fluorescence, and LDL oxidation, respectively, compared with ACE+/+ mice. ACE+/- mice crossbred with E0 mice, resulting in atherosclerotic mice heterozygous for ACE (ACE+/-/E0 mice), exhibited reduced lipid peroxidation, increased paraoxonase activity, and lower macrophage LDL oxidation compared with E0 and ACE+/+/E0 mice. ACE+/-/E0 mice also exhibited reduced NADPH-induced aortic superoxide ion production by 52% and a reduction of 43% in their atherosclerotic lesion size compared with E0 mice. Finally, 2 animals genotyped as homozygous-knockout for both ACE and APOE genes (ACE-/-/E0), exhibited a striking reduction of 86% in their atherosclerotic lesion area compared with E0 mice. CONCLUSIONS: Reduction of tissue ACE with the ACE-knockout mouse type-2 model inhibited oxidative stress and atherogenesis.

Ramipril administration to atherosclerotic mice reduces oxidized low-density lipoprotein uptake by their macrophages and blocks the progression of atherosclerosis.

Foam cell formation, the hallmark of early atherosclerosis, results from cholesterol accumulation in arterial macrophages. Angiotensin-II stimulates foam cell formation and angiotensin converting enzyme (ACE) inhibitors reduce atherosclerosis in animal models. The goal of the present study was to determine the effect of the ACE inhibitor Ramipril on the progression of atherosclerosis in apolipoprotein-E-deficient (E0) mice with already advanced atherosclerosis. Therefore, 4-month-old atherosclerotic E0 mice were treated with Ramipril for 2 and 4 months and compared to age-matched placebo-treated mice, as well as to control young (4-month-old) non-treated E0 mice, for their atherosclerosis. Histomorphometry showed that Ramipril treatment substantially inhibited atherogenesis as shown by 48 and 72% reduction in lesion size at 6 and 8 months of age, respectively, compared to the lesion size in age-matched placebo-treated mice. Moreover, the size of the atherosclerotic lesions in 6- and 8-month-old Ramipril-treated mice was almost identical to the size of atherosclerosis of the 4-month-old control mice. Moreover, Ramipril treatment of E0 mice, significantly reduced oxidized low-density lipoprotein (Ox-LDL) uptake by their peritoneal macrophages (MPM) by 32%, compared to Ox-LDL uptake by MPM from 6-month-old placebo mice, and even reduced it by 12% in comparison to Ox-LDL uptake by MPM from 4-month-old control mice. A significant decrease in the mRNA levels of the Ox-LDL receptor CD36 by 58% was observed in macrophages from 6-month-old Ramipril-treated mice compared to macrophages from the 6-month-old placebo-treated mice. There was even a significant reduction (by 32%) in CD36 mRNA levels in macrophages from the 6-month-old Ramipril-treated mice, compared to the CD36 mRNA levels in macrophages from the 4-month-old control mice. We thus conclude that administration of the ACE inhibitor Ramipril to E0 mice, which already exhibit significant atherosclerosis, blocked the progression of the atherosclerotic lesion build-up, a phenomenon that could be related to Ramipril-induced inhibition of Ox-LDL uptake by macrophages.

Atorvastatin therapy in hypercholesterolemic patients suppresses cellular uptake of oxidized-LDL by differentiating monocytes.

Atherosclerosis is characterized by macrophage foam cells formation, which originate from differentiating blood monocytes that have taken up oxidized LDL (Ox-LDL) at enhanced rate. Statin therapy exhibit pleiotropic effects on many components of atherosclerosis. We have studied the effect of atorvastatin therapy in hypercholesterolemic patients, on the cellular uptake of Ox-LDL by their monocytes during differentiation into macrophages. Eleven hypercholesterolemic men were treated with 20 mg/day of atorvastatin for a period of 1 month. Peripheral blood monocytes harvested from control subjects and from patients before and after atorvastatin therapy were allowed to differentiate in culture for up to 9 days in the presence of 20% autologous serum. In control monocytes/macrophages the cellular uptake of Ox-LDL and the scavenger receptors CD36, SRA-I and SRA-II mRNA expression were upregulated during differentiation, and this upregulation was significantly enhanced in cells from hypercholesterolemic patients. Atorvastatin therapy suppressed the upregulation in Ox-LDL degradation and scavenger receptors expression in differentiating monocytes. These effects could be related at least in part to antioxidant characteristics of atorvastatin. Reduced susceptibility of plasma to free radical-induced lipid peroxidation (by 35%), increased plasma total antioxidant status (TAS; by 30%), and increased serum paraoxonase activity (by 53%), were noted following drug therapy. We conclude that atorvastatin therapy in hypercholesterolemic patients reduces the enhanced cellular uptake of Ox-LDL during ex-vivo differentiation of monocytes into macrophages, and decreases cellular scavenger receptors gene expression. These effects may account for the attenuation of atherogenesis in hypercholesterolemic patients following atorvastatin treatment.

Oxidative stress increases the expression of the angiotensin-II receptor type 1 in mouse peritoneal macrophages.

Angiotensin II (Ang II) has been shown to accelerate atherogenesis, and the cellular Ang II type 1 (AT(1))-receptor mediates most of Ang II-induced pro-atherogenic effects. In this study we have examined the effect of macrophage oxidative stress on cellular AT(1)-receptor expression. Mouse peritoneal macrophages (MPM) from apolipoprotein-E deficient (E(0)) mice at increasing ages (1 6 months) demonstrated an age-dependent increase in cellular lipid-peroxides (PD) content. In parallel, the AT(1)-receptor mRNA and protein levels both increased by up to 3.7-fold and 1.7-fold, respectively, in MPM from 6-month old mice compared with 1-month old mice. Vitamin E supplementation to E(0) mice significantly decreased the MPM PD content and macrophage AT(1)-receptor mRNA expression compared with placebo-treated mice. The role of oxidative stress in the cellular expression of AT(1)-receptors was further demonstrated by manipulation of macrophage glutathione content. Buthionine-sulfoximine, a glutathione synthesis inhibitor, increased MPM PD content and AT(1)-receptor mRNA expression, whereas L-2-oxothiazolidine-4-carboxylic acid, that contributes to glutathione synthesis, reduced macrophage PD and AT(1)-receptor mRNA expression. Incubation of MPM with oxidised low-density lipoproteins (LDL) led to a significant, dose-dependent and time-dependent increase in macrophage AT(1)-receptor mRNA and protein expression, compared with control cells. In contrast, native LDL or acetylated LDL did not significantly affect macrophage AT(1)-receptor mRNA expression. In conclusion, our findings suggest that oxidative stress in macrophages induces AT(1)-receptor expression. This phenomenon can stimulate the interaction of Ang II with macrophages and hence accelerate macrophage foam cell formation and early atherogenesis.

Cardiovascular diseases in patients with high levels of plasma high density lipoprotein: association with increased plasma oxidative state.

BACKGROUND: Increased levels of high density lipoprotein (over 60 mg/dl) are considered to be a negative risk factor for ischemic heart disease. However, some patients with high HDL still develop cardiovascular diseases. OBJECTIVE: To explore why patients with very high HDL still suffer from cardiovascular diseases. METHODS: We analyzed several risk factors, such as increased lipid peroxidation, hyperhomocysteinemia and increased release of inflammatory molecules, that could be related to the development of vascular disease in patients with high serum HDL levels. Patients with HDL cholesterol levels above 75 mg/dl were selected for this study and were separated into two groups based on the presence of atherosclerotic vascular disease, i.e., those with vascular disease (patients) and those without (controls). RESULTS: Plasma isolated from the patient group exhibited significantly increased lipid peroxidation by 21% and decreased total antioxidant status by 17%, but there were no differences regarding their serum or their paraoxonase activity. Moreover, both groups exhibited similar levels of serum C-reactive protein, fibrinogen and homocysteine, enabling us to eliminate these risk factors in the etiology of cardiovascular disease in the patient group. CONCLUSION: Increased oxidative stress could be one of the factors leading to cardiovascular diseases in patients with high serum HDL levels.