Mineralocorticoid receptor blockade inhibits accelerated atherosclerosis induced by a low sodium diet in apolipoprotein E-deficient mice.

INTRODUCTION: A low-sodium diet (LSD) was shown to increase both angiotensin II (AngII) and aldosterone levels, and to accelerate atherosclerosis in apolipoprotein E-deficient (E0) mice. The aim of the present study was to examine whether accelerated atherosclerosis in E0 mice fed a LSD is mediated by aldosterone, using the mineralocorticoid receptor blocker, eplerenone (Epl). METHODS AND RESULTS: Mice were divided into three groups: normal diet (ND), LSD and LSD treated with Epl at 100 mg/kg per day (LSD+Epl) for 10 weeks. LSD significantly enhanced plasma renin and aldosterone levels, which were further increased in mice fed LSD+Epl. The aortic lesion area increased three-fold with LSD, while LSD+Epl significantly reduced the lesion area to values similar to ND. Serum and peritoneal macrophages obtained from LSD-fed mice exhibited pro-atherogenic properties including increased inflammation, oxidation and cholesterol accumulation, which were inhibited in mice fed LSD+Epl. In a J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, Epl was shown to have a direct anti-inflammatory effect. CONCLUSION: In E0 mice, Epl inhibited LSD-accelerated atherosclerosis, despite the elevation of renin and aldosterone levels. It is therefore suggested that the atherogenic action of LSD could be mediated, at least in part, by activation of the mineralocorticoid receptor. In addition, eplerenone may have direct anti-inflammatory actions.

Viewpoint: personalizing statin therapy.

Cardiovascular disease (CVD), associated with vascular atherosclerosis, is the major cause of death in Western societies. Current risk estimation tools, such as Framingham Risk Score (FRS), based on evaluation of multiple standard risk factors, are limited in assessment of individual risk. The majority (about 70%) of the general population is classified as low FRS where the individual risk for CVD is often underestimated but, on the other hand, cholesterol lowering with statin is often excessively administered. Adverse effects of statin therapy, such as muscle pain, affect a large proportion of the treated patients and have a significant influence on their quality of life. Coronary artery calcification (CAC), as assessed by computed tomography, carotid artery intima-media thickness (CIMT), and especially presence of plaques as assessed by B-mode ultrasound are directly correlated with increased risk for cardiovascular events and provide accurate and relevant information for individual risk assessment. Absence of vascular pathology as assessed by these imaging methods has a very high negative predictive value and therefore could be used as a method to reduce significantly the number of subjects who, in our opinion, would not benefit from statins and only suffer from their side-effects. In summary, we suggest that in very-low-risk subjects, with the exception of subjects with low FRS with a family history of coronary artery disease (CAD) at young age, if vascular imaging shows no CAC or normal CIMT without plaques, statin treatment need not be administered.

The effects of aldosterone on diet-induced fatty liver formation in male C57BL/6 mice: comparison of adrenalectomy and mineralocorticoid receptor blocker.

OBJECTIVE: Obesity, diabetes, fatty liver, and hypertension are major determinants of the metabolic syndrome. The effects of aldosterone and mineralocorticoid receptor blockers on fatty liver are largely unknown. The aim of the present study was to evaluate the relationships between aldosterone and the development of fatty liver. MATERIALS AND METHODS: In our experiments, we performed adrenalectomy (ADX) or administered 100 mg/kg/day eplerenone, a specific mineralocorticoid receptor blocker, to male C57BL/6 mice fed with a 60% fat diet for 20 weeks. RESULTS: High-fat diet led to metabolic syndrome as indicated by increased body weight, elevated systolic blood pressure, impaired glucose tolerance, elevated insulin levels, and development of fatty liver. A marked reduction of aldosterone by ADX or blockade of aldosterone interaction with its receptor by eplerenone, which increased serum aldosterone considerably, resulted in reduced blood pressure, and reduced serum insulin and levels of triglycerides. However, differential effects were found on reduction of blood glucose to normal levels, which was observed only in ADX. Neither ADX nor eplerenone affected fatty liver formation or body weight. In cultured hepatocytes, triglyceride loading induced by high glucose, oleic acid or very low density lipoprotein was not affected by aldosterone, spironolactone or eplerenone. CONCLUSION: Our results suggest that whereas aldosterone might be involved in some of the diet-induced insulin and glucose metabolic effects, it played no role in the development of fatty liver.

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.

Paraoxonase1 deficiency in mice is associated with hypotension and increased levels of 5,6-epoxyeicosatrienoic acid.

AIM: Serum paraoxonase 1 (PON1) is an HDL-associated lipolactonase and its association with hypertension is controversial. We studied the possible role of PON1 in blood pressure (BP) regulation, by using PON1 knockout (PON1KO) mice. METHODS AND RESULTS: Both, systolic and diastolic BPs were lower in PON1KO compared to WT mice. Hypotension detected in PON1KO is probably neither related to nitric oxide/guanylate cyclase-mediated vasodilation nor to angiotensin II or aldosterone-mediated vasoconstriction. Surprisingly, when challenged by high-salt diet, BP was further reduced in PON1KO mice. The later, pointed to a possible involvement of transient receptor potential vanilloid 4 (TRPV4), and indeed, administration of ruthenium red, a TRPV4 blocker, resulted in a sharp rise in BP. The protein levels of TRPV4 in kidneys of PON1KO were not higher than in WT. However, the renal level of 5,6-epoxyeicosatrienoic acid (5,6-EET), a TRPV4 specific agonist, was significantly higher in PON1KO compared with WT mice. 5,6-EET levels were further elevated under high-salt diet or administration of arachidonic acid. Injection of inhibitor of CYP450 epoxygenase resulted in increased BP in PON1KO mice. Injection of recombinant human PON1 resulted in elevation of BP and a concomitant reduction in renal content of 5,6-EET. PON1, in vitro, metabolized 5,6-EET, but not other EETs, to its corresponding diol. Vasodilation, blocked by excess of dietary K(+) but not reversed by depletion of cellular Ca(2+) stores, point to endothelial-derived hyperpolarization-like response. CONCLUSION: The present study shows causal, direct relationship between PON1 and blood pressure which is mediated, at least in part, by the regulation of 5,6-EET.

FAD286, an aldosterone synthase inhibitor, reduced atherosclerosis and inflammation in apolipoprotein E-deficient mice.

INTRODUCTION: Aldosterone is known to be involved in atherosclerosis and cardiovascular disease and blockade of its receptor was shown to improve cardiovascular function. It was, therefore, hypothesized that inhibition of aldosterone synthesis would also reduce atherosclerosis development. METHOD: To test this hypothesis, we examined the effect of FAD286 (FAD), an aldosterone synthase inhibitor, on the development of atherosclerosis in spontaneous atherosclerotic apolipoprotein E-deficient mice. Mice were divided into three treatment groups: normal diet, low-salt diet (LSD) and LSD treated with FAD at 30 mg/kg per day (LSD + FAD) for 10 weeks. RESULTS AND CONCLUSION: Histomorphometry of the aortas obtained from these mice showed that atherosclerotic lesion area increased by three-fold under LSD compared with normal diet and FAD significantly reduced lesion area to values similar to normal diet. Changes in atherosclerosis were paralleled by changes in the expression of the inflammation markers (C-reactive protein, monocyte chemotactic protein-1, interleukin-6, nuclear factor kappa B and intercellular adhesion molecule-1) in peritoneal macrophages obtained from these mice. Surprisingly, whereas LSD increased serum or urine aldosterone levels, FAD did not alter these levels when evaluated at the end of the study. In J774A.1 macrophage-like cell line stimulated with lipopolysaccharide, FAD was shown to have a direct dose-dependent anti-inflammatory effect. In apolipoprotein E-deficient mice, FAD reduces atherosclerosis and inflammation. However, these actions appeared to be dissociated from its effect on inhibition of aldosterone synthesis.

Paraoxonase 1 deficiency in mice is associated with reduced steroid biosynthesis: effects on HDL binding, cholesteryl ester accumulation and scavenger receptor type BI expression.

OBJECTIVE: Selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) is considered as the major source of cholesterol for production of steroids in the adrenal gland in rodents. As paraoxonase 1 (PON1) is an HDL-associated lipo-lactonase that has been shown to increase binding of HDL to macrophages, we used PON1 knock-out (PON1KO) mice to test the possible role of PON1 in corticosterone (CS) biosynthesis. METHODS AND RESULTS: PON1 deficiency was associated with reduced serum CS concentration. Adrenal glands obtained from PON1KO mice had significantly lower CE content compared to adrenals from C57Bl6 control mice. Binding of HDL obtained from PON1KO mice to human adrenocortical carcinoma cell line was found to be significantly lower than that of control HDL, and was associated with decreased CS biosynthesis. Addition of purified PON1 to HDL from PON1KO mice increased HDL binding and CS synthesis. Furthermore, the expression of the HDL receptor, SR-BI, protein and mRNA, was reduced in adrenals from PON1KO mice compared to control mice. When challenged with low salt diet, PON1KO mice demonstrated an increase in adrenal SR-BI gene expression and in serum corticosterone which reached levels similar to those obtained in control mice. CONCLUSION: PON1 regulates adrenal CS biosynthesis at two levels: (a) via an accessory role in HDL binding properties, and (b) a supportive role in SR-BI expression and CE supply to the cells.

Apolipoprotein E and its role in aging and survival.

The study of biological aging has seen spectacular progress in the last decade and markers are increasingly employed for understanding physiological processes that change with age. Recently, it has been demonstrated that apolipoprotein E (apoE) has a major impact on longevity, but its mechanisms are still not fully understood. ApoE-deficient (E(o)) mice have proved to be a very popular model for studying spontaneous hypercholesterolemia and the subsequent development of atherosclerotic lesions, but only limited data are available with regard to aging and aging changes. We used this murine model to better characterize the involvement of apoE in aging and to evaluate its role in the maintenance of normal organ morphology. Our results show that E(0) mice at different ages (6, 12, 20 weeks old) developed age-dependent morphological and biochemical alterations, including fibrosis (newly formed collagen), pro-inflammatory cytokine (IL-6 and iNOS), lipofuscin accumulation, and decrease of antioxidant enzymes (superoxide dismutase and catalase) in several organs (kidney, liver and heart). It is significant that the observed degenerative findings in E(0) mice at different ages (6, 12, 20 weeks old) were not identified in control mice (C57BL), at 6, 12 and 20 weeks of age. Consequently, since these mice showed enzymatic and structural alterations, normally linked to the age, such as increase of lipofuscin, pro-inflammatory cytokines and decrease of antioxidant enzymes, we can conclude that apoE is a useful player in studies of longevity and age-related diseases, such as inflammatory status and atherosclerosis that are known risk factors for functional decline and early mortality. Moreover, it is possible that apoE may also play a role in other pathological conditions including, for example, cancer, rheumatoid arthritis and macular degeneration.

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.

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.

High plasma high-density lipoprotein levels, very low cardiovascular risk profile, and subclinical carotid atherosclerosis in postmenopausal women.

BACKGROUND: Low plasma concentrations of high-density lipoprotein (HDL) are associated with increased risk of cardiovascular disease. However, recently several studies have questioned the protective role of high plasma HDL levels. OBJECTIVE: This study was designed to evaluate HDL functions in women with high plasma HDL cholesterol and very low risk profile with relation to subclinical carotid atherosclerosis (ATS). METHODS: Included were 158 middle-aged women with plasma HDL >60 mg/dL and Framingham risk score <7% who had B-mode ultrasound of the carotid arteries. Subclinical ATS was determined by the presence of plaques and/or intima-media thickness (IMT) >1.0 mm. RESULTS: ATS was observed in 51 women, with the majority (n=41) having carotid plaques, some with advanced morphology. In a multivariable model analysis, each, HDL or age, were independently associated with increased prevalence of ATS. Odds ratios for ATS were 3.1 and 2.5 greater for age>60 years and HDL >70 mg/dL, respectively. None of HDL functions determined by its antioxidative properties, reverse-cholesterol transport, or activities of HDL-associated enzyme were different between -ATS and +ATS. C-reactive protein was similar in both groups. CONCLUSION: Subclinical carotid ATS is present in one-third of middle-aged women independently of conventional risk factors. A greater ATS prevalence was associated with very high HDL values. We could not find association between ATS and HDL dysfunction.

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).

[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.

High incidence of reduced plasma HDL cholesterol in diabetic patients treated with rosiglitazone and fibrate.

BACKGROUND: A paradoxical plasma HDL-Cholesterol (HDL-C) reducing effect following combined fibrate and thiazolidinediones (TZDs) therapy was recently reported in occasional cases. As HDL-C level is inversely related to cardiovascular disease (CVD) risk, we have studied the incidence of reduced HDL-C level following mono- and combined therapy with these drugs in a large diabetic population. METHODS: This study was designed as a retrospective 5-year study. Lipid profile records of 54 000 diabetic patients were searched for transient reduction of HDL-C to levels lower than 17 mg/dL, which was correlated with fibrates and/or TZD treatment. RESULTS: Transient reduction in plasma HDL-C to values lower than 17 mg/dL was observed in 0.02% (2/11 175) of the patients treated with fibrates alone, none of the rosiglitazone-treated patients (0/3213) and in 1.39% (9/649) of patients treated with combination of fibrate and TZD. HDL-C lowering effect was reversible upon stopping either fibrate or rosiglitazone and in some patients it occurred within 2 weeks. In two of the patients, the effect was dose-dependent. CONCLUSION: Severe reduction in plasma HDL-C is not rare when TZD and fibrates are co-administrated to diabetic hyperlipidemic patients. As low plasma HDL cholesterol is a risk factor for CVD, the physician should be alert to this phenomenon.

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.

ACE2 activity is increased in monocyte-derived macrophages from prehypertensive subjects.

BACKGROUND: Hypertension is a major risk factor for cardiovascular disease and the renin-angiotensin-aldosterone system (RAAS) plays a central pathophysiological role in its formation. Angiotensin-converting enzyme (ACE) and its homologue ACE2 control the formation of counteracting effectors, angiotensin II (AngII), a potent vasopressor and Ang-(1-7) which has vasodilatory action. It is therefore hypothesized that the balance of the activities of these two enzymes, ACE and ACE2, could be important for the control of blood pressure (BP). METHODS: Monocyte-derived macrophages were isolated from blood samples of normotensives (NT), prehypertensives (preHTN) and untreated hypertensive (HTN) male patients (n = 28, 18 and 11, respectively). The activities of ACE2 were determined by measuring leucine or phenylalanine released following hydrolysis of Ang I and Ang II, respectively. The activity of ACE was measured using a synthetic substrate. RESULTS: The levels of BP were 112.6 +/- 1.4/74.8 +/- 1.2, 128.3 +/- 0.8/78.1 +/- 1.2 and 151.4 +/- 2.7/99.3 +/- 2.4 mmHg in the NT, preHTN and HTN, respectively (P < 0.001). The ACE2-mediated Ang II degrading activity (ACE2-II) was 1201 +/- 241 fmol/min/mg cell protein in NT subjects and was significantly (P < 0.01) increased by 2.4-fold in preHTN. ACE2-II activity in HTN and NT was not significantly different. ACE2-mediated Ang I hydrolysis (ACE2-I) was 85-fold lower than the ACE2-II activity. ACE activity in the human monocyte-derived macrophages (HMDM) averaged 21.6 +/- 3.0 mU/mg cell protein and did not differ among the three groups. CONCLUSIONS: PreHTN subjects have higher ACE2-II activity compared with HTN subjects, suggesting a protective role for ACE2 in the early stage of HTN development, probably by accelerated degradation of the vasopressor AngII.

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.

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.