Aviva Gamliel-Lazarovich

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 &mgr;mol/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.

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.

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

Abstract: 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−1·d−1) 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 &agr;, 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.

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.

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.

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.

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.

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.

Letter by Keidar and Gamliel-Lazarovich Regarding Article, “Effects of A Novel Aldosterone Synthase Inhibitor for Treatment of Primary Hypertension: Results of a Randomized, Double-Blind, Placebo- and Active-Controlled Phase 2 Trial”

To the Editor: We read with great interest the recent report by Calhoun et al1 in which the authors tested the effect of a novel aldosterone synthase inhibitor, LCI699, for treatment of primary hypertension. An 8-week course of treatment with LCI699 indeed resulted in a significant decrease in blood pressure for all doses tested, although this effect was not consistent with dose. The …