Natalia de las Heras

Endothelial Dysfunction, Oxidative Stress and Inflammation in Atherosclerosis: Beneficial Effects of Statins

Atherosclerosis and its complications represent the major cause of death in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A [HMGCoA] reductase and consequently inhibitors of cholesterol biosynthesis. Statins have been described as the most potent class of drugs to reduce serum cholesterol levels. In clinical trials, statins are beneficial in primary and secondary prevention of coronary heart disease. Statins, were initially designed as cholesterol-lowering drugs. However, these drugs, besides their lipid-lowering properties, exert a number of protective effects on the cardiovascular system that emerged over the past years. The benefits observed with statin treatment appear to be greater than that might be expected from reduction in lipid levels alone, suggesting effects beyond cholesterol lowering. These cholesterol-independent effects have been called pleiotropic. The cholesterol-independent or pleiotropic effects of statins involve improvement of endothelial function, stability of atherosclerotic plaques, decrease of oxidative stress and inflammation, and inhibition of thrombogenic response. These pleiotropic effects of statins have been proposed as key properties of these drugs to reduce cardiovascular morbidity and mortality. The present review will emphasize the molecular mechanisms underlying the effects of statins on endothelial function and oxidative stress. In particular, inhibition of small GTP-binding proteins, Rho, Ras and Rac, which are regulated by isoprenoids [farnesyl pyrophosphate and geranylgeranyl pyrophosphate], seems to play an important role in mediating the pleiotropic effects of statins.

AT1 Receptor Antagonism Reduces Endothelial Dysfunction and Intimal Thickening in Atherosclerotic Rabbits

The effects of angiotensin (AT)(1) receptor antagonists on functional and morphological alterations associated with atherosclerosis are not well known. The current study was performed to examine the long-term effects of valsartan (3 or 10 mg/kg per day for 10 weeks) on endothelial function and structural changes in aorta from rabbits fed with either a control diet or a cholesterol-enriched diet. Rabbits fed with the cholesterol-rich diet showed higher (P<0.05) plasma levels of cholesterol than did controls. Treatment with valsartan (3 or 10 mg/kg per day) did not alter plasma cholesterol levels or systolic arterial pressure in any group. Contractions induced by angiotensin II were comparable in both control and hypercholesterolemic rabbits and were markedly reduced by treatment with valsartan. Relaxations induced by acetylcholine were lower in hypercholesterolemic rabbits than in controls. Treatment with valsartan (3 or 10 mg/kg per day) enhanced (P<0.05) this response in hypercholesterolemic rabbits but not in controls. Lumen and media cross-sectional areas were comparable in control and hypercholesterolemic rabbits. Vessel area was higher (P<0.05) in hypercholesterolemic rabbits than in controls. Intimal lesion was 29.5+/-6% in cholesterol-fed rabbits and nonexistent in control rabbits. Treatments with 3 and 10 mg/kg per day valsartan reduced (P<0.05) intimal lesion to 2.4+/-0.7% and 2.7+/-0.9%, respectively, and increased lumen area in hypercholesterolemic rabbits. No changes in either vessel or media cross-sectional areas were observed in these animals. In summary, angiotensin II, through AT(1) receptors, appears to play a key role in the development of the vascular functional and structural changes associated with hypercholesterolemia. AT(1) receptor antagonists, besides their antihypertensive effects, could be an important therapeutic tool to reduce the development of atherosclerosis.

Participation of aldosterone in the vascular inflammatory response of spontaneously hypertensive rats: role of the NFkappaB/IkappaB system.

Objective To investigate the participation of aldosterone in the vascular inflammatory process associated with hypertension, as well as the possible involvement of the NFκB/IκB system. Methods Male spontaneously hypertensive rats (SHR; 20–22 weeks old) untreated or treated with either the aldosterone receptor antagonist, eplerenone (100 mg/kg per day) or triple antihypertensive therapy (HHR: hydralazine + hydrochlorothiazide + reserpine; 20 + 7 + 0.15 mg/kg per day) were used in the study. Wistar–Kyoto rats (WKY) were used as a normotensive reference group. Aortic mRNA expression and plasma levels of interleukin (IL)-1β, IL-6 and tumour necrosis factor α (TNFα) were measured. Likewise, the aortic expression of the nuclear factor κB (NFκB) p50 subunit precursor, p105, and its inhibitor (IκB) were measured. Results SHR showed higher aortic expression of IL-1β, IL-6 and TNFα than WKY (P < 0.05) and higher plasma levels of IL-1β and IL-6 than WKY (P < 0.05). Moreover, SHR also presented increased aortic expression of nuclear transcription factor NFκB p50 subunit precursor (p105), and a reduction of its inhibitor IκB. Both eplerenone and HHR decreased blood pressure to a comparable extent (P < 0.05). This effect was accompanied by a reduction in plasma levels of IL-1β and IL-6 and aortic mRNA expression of IL-1β, IL-6 and TNFα. However, the effect of eplerenone was more marked, since eplerenone-treated rats showed significantly lower inflammatory parameters than SHR receiving HHR. In addition, both antihypertensive treatments increased IκB mRNA expression in a similar manner, but only eplerenone reduced NFκB mRNA expression. Conclusions Aldosterone, as well as an increase in haemodynamic forces produced by hypertension, participate in the vascular inflammatory process associated with hypertension in SHR. This effect seems to be mediated by enhanced vascular expression of cytokines through a modification of the NFκB/IκB system.

Interactions between aldosterone and connective tissue growth factor in vascular and renal damage in spontaneously hypertensive rats

Objective The aim of the present study was to investigate possible inter-relationships between connective tissue growth factor (CTGF) and aldosterone in vascular and renal damage associated with hypertension. Method Spontaneously hypertensive rats (SHR) were treated with two doses (100 and 30 mg/kg per day) of the mineralocorticoid receptor antagonist eplerenone, or with antihypertensive therapy (HHR) (20 mg/kg per day hydralazine + 7 mg/kg per day hydrochlorothiazide + 0.15 mg/kg per day reserpine). Results CTGF mRNA expression and protein levels in the aorta of SHR were upregulated (P < 0.05) compared with Wistar–Kyoto rats. Both doses of eplerenone similarly and significantly diminished CTGF upregulation, correlated with amelioration of aortic remodelling and endothelium-dependent relaxations. Only high-dose eplerenone and HHR significantly reduced arterial blood pressure. HHR treatment also diminished CTGF overexpression, suggesting a blood-pressure-mediated effect in CTGF regulation. This reduction, however, was lower (P < 0.05) than that produced by eplerenone (100 mg/kg per day). The direct effect of aldosterone on vascular smooth muscle cells was also studied. Incubation of cultured vascular smooth muscle cells with aldosterone increased CTGF production in a dose-related manner, but was reduced (P < 0.05) by the mineralocorticoid receptor antagonist spironolactone. Renal CTGF mRNA and protein levels were higher in SHR than in Wistar–Kyoto rats (P < 0.05), and were similarly diminished by all treatments (P < 0.05). Conclusions These data show that aldosterone and haemodynamic stress from elevated blood pressure levels regulate vascular and renal CTGF in SHR. The results suggest that aldosterone, through CTGF stimulation, could participate in vascular and renal structural alterations associated with hypertension, describing a novel mechanism of aldosterone in hypertensive target organ damage.

Relevance of endothelium-derived hyperpolarizing factor in the effects of hypertension on rat coronary relaxations.

Objectives To evaluate the relative participation of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF) and prostanoids in the relaxing responses induced by acetylcholine and isoproterenol in isolated coronary arteries from adult Wistar–Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Design and methods Male adult WKY rats and SHR were used in the study. Segments from left coronary arteries (approximately 350–380 μm internal diameter and 2 mm long) were mounted in an isometric myograph and pre-contracted with serotonin. Dose–response curves to acetylcholine and isoproterenol were carried out in absence and presence of the NO synthesis inhibitor NG-nitro-l-arginine methyl ester (lNAME), the inhibitor of the cyclo-oxygenase, indomethacin and KCl. Areas under the respective dose–response curves were used to calculate the approximate relative participation of NO, EDHF and prostanoids. Results Relaxations to either acetylcholine or isoproterenol were lower in SHR than in WKY rats. In WKY rats, presence of lNAME diminished (P < 0.05) relaxation to acetylcholine from 10−9 to 10−6 mol/l, and induced a contracting response at 10−5 and 10−4 mol/l of acetylcholine. Addition of indomethacin did not significantly affect dose-related relaxation to acetylcholine 10−9 to 10−6 mol/l in WKY rats, and reduced (P < 0.05) the contracting response observed at 10−5 mol/l of acetylcholine. In SHR, addition of lNAME markedly reduced (P < 0.05) acetylcholine relaxations, but did not produce any contracting effect. Addition of indomethacin on top of lNAME slightly (P < 0.05) enhanced relaxing response to acetylcholine in SHR. Presence of lNAME in the media diminished (P < 0.05) relaxation to isoproterenol in both WKY rats and SHR. Addition of indomethacin on top of lNAME increased (P < 0.05) isoproterenol-relaxing response to levels similar to and higher than control conditions in WKY rats and SHR, respectively. Addition of KCl blunted both acetylcholine- and isoproterenol-relaxations in both groups. Conclusions NO and EDHF are the main endothelium-derived relaxing factors underlying acetylcholine and isoproterenol relaxations in rat coronary arteries, respectively. EDHF reduction, and not only NO reduction play a key role in the diminished coronary relaxations induced by acetylcholine and isoproterenol in SHR. An arachidonic acid derivative with contracting activity released by acetylcholine and isoproterenol in a differential manner, could oppose the relaxing actions of NO and EDHF.

The protective effect of irbesartan in rats fed a high fat diet is associated with modification of leptin-adiponectin imbalance.

Objective It has been shown that the renin–angiotensin system participates in the development of the metabolic syndrome. This study aimed to show whether the angiotensin II type 1 receptor blocker, irbesartan, exerts a protective effect against metabolic and cardiovascular abnormalities in rats fed a high fat diet (HFD). Methods Wistar rats (n = 30) were divided into three groups: (1) rats fed a standard diet for 7 weeks were used as a control group; (2) rats fed a HFD (33.5% fat) for 7 weeks; and (3) rats fed a HFD (33.5% fat) treated with irbesartan (0.1 mg/kg per day) for 7 weeks. Body weight, white and brown adipose tissue weight, plasma concentrations and protein expression of leptin and adiponectin in white adipose tissue, and glucose metabolism were investigated. Vascular reactivity in aortic rings and heart function were also evaluated. Results HFD rats showed increased (P < 0.05) body, epididymal and lumbar adipose tissue weights, but did not experience a change in brown adipose tissue weight. Irbesartan attenuated (P < 0.05) all of these parameters, but increased brown adipose tissue weight. The leptin/adiponectin ratio of plasma concentrations and protein expression in lumbar adipose tissue increased (P < 0.05) in HFD rats, and were normalized by irbesartan. Along with these changes, irbesartan improved (P < 0.05) insulin sensitivity and exaggerated responses to angiotensin I and II in the aorta. Conclusion Irbesartan reduced body and white adipose tissue weights, improved glucose metabolism and vascular function in the aorta. The correction of leptin–adiponectin imbalance may be an important mechanism participating in the protective effect of irbesartan in HFD rats.

Inflammation: A Link Between Hypertension and Atherosclerosis

High blood pressure levels are associated with increases in circulating levels of inflammation markers which can reflect vascular inflammatory processes, suggesting that hypertension is a low-grade inflammatory process. The vascular inflammation associated with hypertension could be the link between high blood pressure levels and the atherosclerotic process, which is the principal origin of cardiovascular disease, the leading cause of worldwide mortality. High blood pressure levels are accompanied by increases in oxidative stress due to both higher reactive oxygen specie (ROS) production and reduced ROS scavenging by antioxidant defence. This situation favours endothelial function alterations which allow the expression of adhesion molecules and initiation of fatty streak, the earliest structural change in the atherosclerotic process. At the same time, this inflammation, allows endothelial dysfunction since some inflammatory mediators can negatively affect endothelial cell function. Inflammation, therefore, plays a critical role in development and in complications of the atherothrombotic process. Changes in mechanical stress and activation of humoral factors such as the reninangiotensin- aldosterone system can be underlying not only increases in oxidative stress (and consequently endothelial dysfunction) but also the development of the inflammatory process associated with hypertension.

Aged Garlic Extract Improves Adiponectin Levels in Subjects with Metabolic Syndrome: A Double-Blind, Placebo-Controlled, Randomized, Crossover Study

Background. Garlic (Allium sativum) has been shown to have important benefits in individuals at high cardiovascular risk. The aim of the present study was to evaluate the effects of the administration of aged garlic extract (AGE) on the risk factors that constitute the cluster of metabolic syndrome (MS). Methods and Design. Double-blind, crossover, randomized, placebo-controlled clinical trial to assess the effect of 1.2u2009g/day of AGE (Kyolic), for 24 weeks of treatment (12 weeks of AGE and 12 weeks of placebo), on subjects with MS. Results. The administration of AGE increased the plasma levels of adiponectin (P = 0.027). No serious side effects associated with the intervention were reported. Conclusion. The present results have shown for the first time that the administration of AGE for 12 weeks increased plasma adiponectin levels in patients with MS. This suggests that AGE might be a useful, novel, nonpharmacological therapeutic intervention to increase adiponectin and to prevent cardiovascular (CV) complications in individuals with MS.

Cardiac L-type calcium current is increased in a model of hyperaldosteronism in the rat

Accumulating evidence supports the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. It has been postulated that aldosterone could contribute to ventricular arrhythmogeneity by modulation of cardiac ionic channels. The aim of this study was to analyse ex vivo the electrophysiological characteristics of the L‐type cardiac calcium current (ICaL) in a model of hyperaldosteronism in the rat. Aldosterone was administered for 3 weeks, and cardiac collagen deposition and haemodynamic parameters were analysed. In addition, RT‐PCR and patch‐clamp techniques were applied to study cardiac L‐type Ca2+ channels in isolated cardiomyocytes. Administration of aldosterone induced maladaptive cardiac remodelling that was related to increased collagen deposition, diastolic dysfunction and cardiac hypertrophy. In addition, ventricular myocytes isolated from the aldosterone‐treated group showed increased ICaL density and conductance and prolongation of the action potential duration. No changes in kinetics or in voltage dependence of activation and inactivation of ICaL were observed, but relative expression of CaV1.2 mRNA levels was higher in cardiomyocytes isolated from the aldosterone‐treated group. The present study demonstrates that aldosterone treatment induces myocardial fibrosis, cardiac hypertrophy, increase of ICaL density, upregulation of L‐type Ca2+ channels and prolongation of action potential duration. It could be proposed that aldosterone, through these mechanisms, might exert pro‐arrhythmic effects in the pathological heart.

Structural, functional, and molecular alterations produced by aldosterone plus salt in rat heart: association with enhanced serum and glucocorticoid-regulated kinase-1 expression.

We aimed to evaluate the structural, functional, inflammatory, and oxidative alterations, as well as serum and glucocorticoid-regulated kinase-1 (SGK-1) expression, produced in rat heart by aldosterone + salt administration. Fibrosis mediators such as connective tissue growth factor, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2 were also evaluated. Treatment with spironolactone was evaluated to prove mineralocorticoid mediation. Male Wistar rats received aldosterone (1 mg·kg−1·d−1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg·kg−1·d−1). Systolic and diastolic blood pressures, left ventricle (LV) systolic pressure, and LV end-diastolic pressure were elevated (P < 0.05) in aldosterone + salt-treated rats. In aldosterone + salt-treated rats, -dP/dt decreased (P < 0.05), but +dP/dt was similar in all groups. Spironolactone normalized (P < 0.05) systolic blood pressure, diastolic blood pressure, LV systolic pressure, LV end-diastolic pressure, and -dP/dt. Relative heart weight, collagen content, messenger RNA expression of transforming growth factor beta, connective tissue growth factor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases 2, tumor necrosis factor alpha, interleukin-1β, p22phox, endothelial nitric oxide synhtase, and SGK-1 were increased (P < 0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (P < 0.05). SGK-1 might be a key mediator in the structural, functional, and molecular cardiac alterations induced by aldosterone + salt in rats. All the observed changes and mediators are related with the activation of mineralocorticoid receptors.