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Introduction The renin angiotensin system plays a crucial role in erectile function. It has been shown that elevated angiotensin‐II levels contribute to the development of erectile dysfunction (ED). Oppositely, angiotensin‐(1‐7) (Ang‐[1‐7]) mediates penile erection by activation of receptor Mas. Recently, we have developed a formulation based on Ang‐(1‐7) inclusion in cyclodextrin (CyD) [Ang‐(1‐7)‐CyD], which allows for the oral administration of Ang‐(1‐7). Aim In the present study, we evaluated the effects of chronic treatment with Ang‐(1‐7)‐CyD on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemic mice. Methods Apolipoprotein(Apo)E −/− mice fed a Western‐type diet for 11 weeks received Ang‐(1‐7)‐CyD or vehicle during the final 3 weeks. Collagen content and reactive oxygen species (ROS) production within the corpus cavernosum were evaluated by Sirius red and dihydroethidium staining, respectively. Protein expression of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate (NADPH) subunits (p67‐phox and p22‐phox), and AT1 and Mas receptors in the penis was assessed by Western blotting. Nitric oxide (NO) production was measured by Griess assay in the mice serum. Cavernosal strips were mounted in an isometric organ bath to evaluate the endothelial function. Main Outcome Measures The effect of Ang‐(1‐7)‐CyD treatment on penile fibrosis, oxidative stress, and endothelial function in hypercholesterolemia‐induced ED. Results Ang‐(1‐7)‐CyD treatment reduced collagen content in the corpus cavernosum of ApoE −/− mice. This effect was associated with an attenuation of ROS production and a diminished expression of NADPH. Furthermore, Ang‐(1‐7)‐CyD treatment augmented the expression of nNOS and eNOS in the penis and elevated vascular NO production. Importantly, these effects were accompanied by an improvement in cavernosal endothelial function. Conclusion Long‐term treatment with Ang‐(1‐7)‐CyD reduces penile fibrosis associated with attenuation of oxidative stress. Additionally, cavernosal endothelial function in hypercholesterolemic mice was markedly improved. These results suggest that Ang‐(1‐7)‐CyD might have significant therapeutic benefits for the treatment of erectile dysfunction. Fraga‐Silva RA, Costa‐Fraga FP, Savergnini SQ, De Sousa FB, Montecucco F, da Silva D, Sinisterra RD, Mach F, Stergiopulos N, da Silva RF, and Santos RAS. An oral formulation of angiotensin‐(1–7) reverses corpus cavernosum damages induced by hypercholesterolemia. J Sex Med 2013;10:2430–2442.

Little is known about age-related differences in short-term effects of estradiol on ischemia-reperfusion (I/R) insults. The present study was designed to evaluate the effects of short-term treatment with estradiol on reperfusion arrhythmias in isolated hearts of 6-7-week-old and 12-14-month-old female rats. Wistar rats were sham-operated, ovariectomized and treated with vehicle or ovariectomized and treated with 17β-estradiol (E2; 5 µg·100 g-1·day-1) for 4 days. Hearts were perfused by the Langendorff technique. Reperfusion arrhythmias, i.e., ventricular tachycardia and/or ventricular fibrillation, were induced by 15 min of left coronary artery ligation and 30 min of reperfusion. The duration and incidence of I/R arrhythmias were significantly higher in young rats compared to middle-aged rats (arrhythmia severity index: 9.4 ± 1.0 vs 3.0 ± 0.3 arbitrary units, respectively, P < 0.05). In addition, middle-aged rats showed lower heart rate, systolic tension and coronary flow. Four-day E2 treatment caused an increase in uterine weight. Although E2 administration had no significant effect on the duration of I/R arrhythmias in middle-aged rats, it induced a marked reduction in the rhythm disturbances of young rats accompanied by a decrease in heart rate of isolated hearts. Also, this reduction was associated with an increase in QT interval. No significant changes were observed in the QT interval of middle-aged E2-treated rats. These data demonstrate that short-term estradiol treatment protects against I/R arrhythmias in hearts of young female rats. The anti-arrhythmogenic effect of estradiol might be related to a lengthening of the QT interval.

It is well known that the renin-angiotensin system (RAS) plays a pivotal role in the control of the cardiovascular and renal systems and in the pathogenesis of cardiovascular and renal diseases. The classical concept of the RAS has been changed in the recent years by the inclusion of novels components. Among the novel components of the RAS, Angiotensin(Ang)-(1-7), its major forming enzyme angiotensin-converting enzyme (ACE) 2 and its receptor, the G protein-coupled receptor Mas, have been the subject of many recent studies aimed to clarify the role of this novel axis of the RAS. This axis appears to act as a counter-regulatory mechanism for the classical axis represented by ACE/Ang II/AT1 receptor in several conditions, including hypertension and others cardiovascular diseases. In this review we will focus on the potential use of Mas agonists as anti-hypertensive drugs.

Aims: We hypothesized that a high-carbohydrate diet affects the cardiac performance by interfering in the metabolic steps involved in energy transfer in this organ. To verify this, we investigated the myocardial utilization of different substrates and contractile function in rats fed a high-carbohydrate diet, under normal flow and ischemia. Methods and Results: Male Wistar rats were fed over 9 days with standard (39.5% carbohydrate, 8% fiber) or high-carbohydrate diet (58% carbohydrate) and, afterwards, their cardiac function was examined using isolated heart preparations. The high-carbohydrate diet decreased the activity of the lipoprotein lipase, utilization of fatty acids, expression of the gene of peroxisome proliferator-activated receptor α and its target enzymes. In addition, decreased GLUT4 mass, glucose uptake, glycogen content and glycolytic intermediates were also observed. High-carbohydrate hearts displayed weaker activation of the glycolytic pathway during ischemia, according to minor production of lactate, in relation to control hearts. The functional impairment caused by high-carbohydrate diet shown by the decrease in the ventricular systolic strength, +d T/d t and −d T/d t was, at least in part, due to the low availability of adenosine triphosphate (ATP). Conclusion: Our data suggest that a high-carbohydrate diet can damage myocardial contractile function by decreasing the cardiac utilization of glucose and fatty acids and, consequently, the ATP pool.

Mas stimulation with angiotensin (Ang)-(1-7) produces cardioprotective effects and vasorelaxation. Using a computational discovery platform for predicting novel naturally occurring peptides that may activate G protein–coupled receptors, we discovered a novel Mas agonist peptide, CGEN-856S. An endothelium- and NO-dependent vasodilating effect was observed for CGEN-856S in thoracic aorta rings of rats (maximal value for the relaxant effect: 39.99±5.034%), which was similar to that produced by Ang-(1-7) (10 −10 to 10 −6 mol/L). In addition, the vasodilator activity of this peptide depended on a functional Mas receptor, because it was abolished in aorta rings of Mas-knockout mice. CGEN-856S appears to bind the Mas receptor at the same binding domain as Ang-(1-7), as suggested by the blocking of its vasorelaxant effect with the Ang-(1-7) analogue d -Ala 7 -Ang-(1-7), and by its competitive inhibition of Ang-(1-7) binding to Mas -transfected cells. The effect of CGEN-856S on reperfusion arrhythmias and cardiac function was studied on ischemia reperfusion of isolated rat hearts. We found that picomolar concentration of CGEN-856S (0.04 nmol/L) had an antiarrhythmogenic effect, as demonstrated by a reduction in the incidence and duration of reperfusion arrhythmias. Furthermore, acute infusion of CGEN-856S produced a shallow dose-dependent decrease in mean arterial pressure of conscious spontaneously hypertensive rats. The maximum change during infusion was observed at the highest dose. Strikingly, blood pressure continued to drop in the postinfusion period. The results presented here indicate that the novel Mas agonist, CGEN-856S, might have a therapeutic value, because it induces vasorelaxing, antihypertensive, and cardioprotective effects.

In this study was evaluated the chronic cardiac effects of a formulation developed by including angiotensin(Ang)-(1–7) in hydroxypropylβ-cyclodextrin (HPβCD), in infarcted rats. Myocardial infarction (MI) was induced by left coronary artery occlusion. HPβCD/Ang-(1–7) was administered for 60 days (76 μg/Kg/once a day/gavage) starting immediately before infarction. Echocardiography was utilized to evaluate usual cardiac parameters, and radial strain method was used to analyze the velocity and displacement of myocardial fibers at initial time and 15, 30, and 50 days after surgery. Real-time PCR was utilized to evaluate the fibrotic signaling involved in the remodeling process. Once-a-day oral HPβCD/Ang-(1–7) administration improved the cardiac function and reduced the deleterious effects induced by MI on TGF-βand collagen type I expression, as well as on the velocity and displacement of myocardial fibers. These findings confirm cardioprotective effects of Ang-(1–7) and indicate HPβCD/Ang-(1–7) as a feasible formulation for long-term oral administration of this heptapeptide.

CGEN-856S is a novel Mas agonist. Herein, we examined the effects of this peptide on isoproterenol (ISO)-induced cardiac remodeling and myocardial infarction (MI) injury. We also sought to determine whether CGEN-856S activates the underlying mechanisms related to Mas receptor activation. Heart hypertrophy and fibrosis were induced by ISO (2 mg·kg(-1)·day(-1)) in Wistar rats. After a 7-day treatment period with CGEN-856S (90 µg·kg(-1)·day(-1)) or vehicle, the cardiomyocyte diameter was evaluated in left ventricular sections stained with hematoxylin and eosin, and immunofluorescence labeling and quantitative confocal microscopy were used to quantify the deposition of type I and III collagen and fibronectin in the left ventricles. MI was induced by coronary artery ligation, and CGEN-856S (90 µg·kg(-1)·day(-1)) or saline was administered for 14 days. The Langendorff technique was used to evaluate cardiac function, and left ventricular sections were stained with Masson's trichrome dye to quantify the infarct area. Using Chinese hamster ovary cells stably transfected with Mas cDNA, we evaluated whether CGEN-856S alters AKT and endothelial nitric oxide synthase (eNOS) phosphorylation. CGEN-856S reduced the degree of ISO-induced hypertrophy (13.91±0.17 µm vs. 12.41±0.16 µm in the ISO+CGEN-856S group). In addition, the Mas agonist attenuated the ISO-induced increase in collagen I, collagen III, and fibronectin deposition. CGEN-856S markedly attenuated the MI-induced decrease in systolic tension, as well as in +dT/dt and -dT/dt. Furthermore, CGEN-856S administration significantly decreased the infarct area (23.68±2.78% vs. 13.95±4.37% in the MI+CGEN-856S group). These effects likely involved the participation of AKT and NO, as CGEN-856S administration increased the levels of p-AKT and p-eNOS. Thus, our results indicate that CGEN-856S exerts cardioprotective effects on ISO-induced cardiac remodeling and MI-mediated heart failure in rats through a mechanism likely involving the eNOS/AKT pathway.

Cannabinoids are considered as key mediators in the pathophysiology of inflammatory diseases, including atherosclerosis. In particular, they have been shown to reduce the ischemic injury after acute cardiovascular events, such as acute myocardial infarction and ischemic stroke. These protective and anti-inflammatory properties on peripheral tissues and circulating inflammatory have been demonstrated to involve their binding with both selective cannabinoid type 1 (CB1) and type 2 (CB2) transmembrane receptors. On the other hands, the recent discoveries of novel different classes of cannabinoids and receptors have increased the complexity of this system in atherosclerosis. Although only preliminary data have been reported on the activities of novel cannabinoid receptors, several studies have already investigated the role ofCB1andCB2receptors in ischemic stroke. WhileCB1receptor activation has been shown to directly reduce atherosclerotic plaque inflammation, controversial data have been shown on neurotransmission and neuroprotection after stroke. Given its potent anti-inflammatory activities on circulating leukocytes, theCB2activation has been proven to produce protective effects against acute poststroke inflammation. In this paper, we will update evidence on different cannabinoid-triggered avenues to reduce inflammation and neuronal injury in acute ischemic stroke.

Purpose Cardiac dysfunction is one of the possible causes of sudden unexpected death in epilepsy (SUDEP). Therefore, the objective of this study was to evaluate cardiac and electrocardiographic parameters in rats with audiogenic epileptic seizures (WAR – Wistar audiogenic rats). Methods In vivo arterial pressure, heart rate (HR), autonomic tone and electrocardiography (ECG) were measured in awake animals in order to examine cardiac function and rhythm. Ex vivo , the Langendorff technique was used to analyze the cardiac function and the severity of reperfusion arrhythmias. In vitro , confocal microscopy was used to evaluate calcium transient parameters of isolated ventricular cardiomyocytes. Results In vivo autonomic tone evaluation revealed enhanced sympathetic activity, changes in cardiac function with increased systolic arterial pressure and higher basal HR in WAR. In addition, ECG analysis demonstrated electrical alterations with prolongation of the QT interval and QRS complex in these animals. Ex vivo , we observed a decrease in systolic tone and HR and an increase in the duration of ischemia/reperfusion arrhythmias in WAR. Moreover, intracellular Ca 2+ handling analysis revealed an increase in the peak of calcium and calcium transient decay in audiogenic rats. Treatment with atenolol (β1-adrenergic antagonist) normalized the systolic tone, reduced cardiac hypertrophy and the associated increase in the susceptibility to reperfusion arrhythmias observed in WAR. Conclusion We present evidence that chronic disturbances in sympathetic tone in WAR cause increases the risk to life-threatening arrhythmias. Our results support a relationship between seizures, cardiac dysfunction and cardiac arrhythmias, which may contribute to the occurrence of SUDEP.

SummaryAngiotensin (Ang)-(1–7), acting through the receptor Mas, has atheroprotective effects; however, its role on plaque vulnerability has been poorly studied. Here, we investigated the expression of the renin-angiotensin system (RAS) components in stable and unstable human carotid plaques. In addition, we evaluated the effects of the chronic treatment with an oral formulation of Ang-(1–7) in a mouse model of shear stress-determined carotid atherosclerotic plaque. Upstream and downstream regions of internal carotid plaques were obtained from a recently published cohort of patients asymptomatic or symptomatic for ischaemic stroke. Angiotensinogen and renin genes were strongly expressed in the entire cohort, indicating an intense intraplaque modulation of the RAS. Intraplaque expression of the Mas receptor mRNA was increased in the downstream portion of asymptomatic patients as compared to corresponding region in symptomatic patients. Conversely, AT1 receptor gene expression was not modified between asymptomatic and symptomatic patients. Treatment with Ang-(1–7) in ApoE-/- mice was associated with increased intraplaque collagen content in the aortic root and low shear stress-induced carotid plaques, and a decreased MMP-9 content and neutrophil and macrophage infiltration. These beneficial effects were not observed in the oscillatory shear stress-induced plaque. In vitro incubation with Ang-(1–7) did not affect ICAM-1 expression and apoptosis on cultured endothelial cells. In conclusion, Mas receptor is up regulated in the downstream portions of human stable carotid plaques as compared to unstable lesions. Treatment with the oral formulation of Ang-(1–7) enhances a more stable phenotype in atherosclerotic plaques, depending on the local pattern of shear stress forces.