Antonio Bastos Peluso

Discovery and Characterization of Alamandine, a Novel Component of the Renin-Angiotensin System

Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand β-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders. # Novelty and Significance {#article-title-32}Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand &bgr;-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/&bgr;-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

Time-resolved quantitative phosphoproteomics: new insights into Angiotensin-(1-7) signaling networks in human endothelial cells.

Angiotensin-(1-7) [Ang-(1-7)] is an endogenous ligand of the Mas receptor and induces vasodilation, positive regulation of insulin, and antiproliferative and antitumorigenic activities. However, little is known about the molecular mechanisms behind these biological properties. Aiming to identify proteins involved in the Ang-(1-7) signaling, we performed a mass spectrometry-based time-resolved quantitative phosphoproteome study of human aortic endothelial cells (HAEC) treated with Ang-(1-7). We identified 1288 unique phosphosites on 699 different proteins with 99% certainty of correct peptide identification and phosphorylation site localization. Of these, 121 sites on 79 proteins had their phosphorylation levels significantly changed by Ang-(1-7). Our data suggest that the antiproliferative activity of Ang-(1-7) is due to the activation or inactivation of several target phosphoproteins, such as forkhead box protein O1 (FOXO1), mitogen-activated protein kinase 1 (MAPK), proline-rich AKT1 substrate 1 (AKT1S1), among others. In addition, the antitumorigenic activity of Ang-(1-7) is at least partially due to FOXO1 activation, since we show that this transcriptional factor is activated and accumulated in the nucleus of A549 lung adenocarcinoma cells treated with Ang-(1-7). Moreover, Ang-(1-7) triggered changes in the phosphorylation status of several known downstream effectors of the insulin signaling, indicating an important role of Ang-(1-7) in glucose homeostasis. In summary, this study provides new concepts and new understanding of the Ang-(1-7) signal transduction, shedding light on the mechanisms underlying Mas activation.

New components of the renin-angiotensin system: alamandine and the MAS-related G protein-coupled receptor D.

The renin-angiotensin system is an important component of the central and humoral mechanisms of blood pressure and hydro-electrolytic balance control. Angiotensin II is a key player of this system. Twenty-five years ago the first manuscripts describing the formation and actions of another peptide of the RAS, angiotensin-(1-7), were published. Since then several publications have shown that angiotensin-(1-7) is as pleiotropic as angiotensin II, influencing the functions of many organs and systems. The identification of the ACE homologue ACE2 and, a few years later, Mas, as a receptor for angiotensin-(1-7) contributed a great deal to establish this peptide as a key player of the RAS. Most of the actions of angiotensin-(1-7) are opposite to those described for angiotensin II. This has led to the concept of two arms of the RAS: one comprising ACE/AngII/AT1R and the other ACE2/Ang-(1-7)/Mas. More recently, we have described the identification of a novel component of the RAS, alamandine, which binds to the Mas-related G protein coupled receptor D. This peptide is formed by decarboxylation of the Asp residue of angiotensin-(1-7), leading to the formation of Ala as the N-terminal amino acid. Alternatively, it can be formed by hydrolysis of Ang A, by ACE2. Its effects include vasorelaxation, central effects similar to those produced by angiotensin-(1-7), blunting of isoproterenol-induced heart fibrosis, and anti-hypertensive action in SHR. The putative enzyme responsible for alamandine formation from angiotensin-(1-7) is under investigation. The identification of this novel component of the RAS opens new venues for understanding its physiological role and opens new putative therapeutic possibilities for treating cardiovascular diseases.

The novel Mas agonist, CGEN-856S, attenuates isoproterenol-induced cardiac remodeling and myocardial infarction injury in rats.

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.

Exercise modulates the aortic renin-angiotensin system independently of estrogen therapy in ovariectomized hypertensive rats.

HIGHLIGHTSSome components of the renin‐angiotensin system (RAS) are altered under conditions of estrogen deficiency and hypertension.Exercise training reverses the detrimental effects of ovariectomy on the aortic reactivity to ANG II and ANG‐(1–7) in hypertensive rats.Exercise training modulates RAS independently of estrogen therapy, protecting the hypertensive postmenopausal women against CVD. ABSTRACT The renin‐angiotensin‐system is an important component of cardiovascular control and is up‐regulated under various conditions, including hypertension and menopause. The aim of this study was to evaluate the effects of swimming training and estrogen therapy (ET) on angiotensin‐II (ANG II)‐induced vasoconstriction and angiotensin‐(1–7) [ANG‐(1–7)]‐induced vasorelaxation in aortic rings from ovariectomized spontaneously hypertensive rats. Animals were divided into Sham (SH), Ovariectomized (OVX), Ovariectomized treated with E2 (OE2), Ovariectomized plus swimming (OSW) and Ovariectomized treated with E2 plus swimming (OE2 + SW) groups. ET entailed the administration of 5 &mgr;g of 17&bgr;‐Estradiol three times per week. Swimming was undertaken for sixty minutes each day, five times per week. Both, training and ET were initiated seven days following ovariectomy. Forty‐eight hours after the last treatment or training session, the animals’ systolic blood pressures were measured, and blood samples were collected to measure plasma ANG II and ANG‐(1–7) levels via radioimmunoassay. In aortic rings, the vascular reactivity to ANG II and ANG‐(1–7) was assessed. Expression of ANG‐(1–7) in aortic wall was analyzed by immunohistochemistry. The results showed that both exercise and ET increased plasma ANG II levels despite attenuating systolic blood pressure. Ovariectomy increased constrictor responses to ANG II and decreased dilatory responses to ANG‐(1–7), which were reversed by swimming independently of ET. Moreover, it was observed an apparent increase in ANG‐(1–7) content in the aorta of the groups subjected to training and ET. Exercise training may play a cardioprotective role independently of ET and may be an alternative to ET in hypertensive postmenopausal women.

The angiotensin type 2 receptor and the kidney

Purpose of review Angiotensin II is a main regulator of kidney function. Renal actions mediated by the angiotensin AT1 receptor have been well known for many years. In contrast, several details of angiotensin AT2 receptor actions in kidney physiology and pathophysiology were only described very recently. These findings are reviewed in this article. Recent findings Regarding the role of the angiotensin AT2 receptor in kidney physiology, a major recent finding was that the AT2 receptor-mediated inhibition of Na+-H+ exchanger-3 and Na+/K+-ATPase in the renal proximal tubules is caused by internalisation of these transporters, thus reducing reabsorption and increasing natriuresis/diuresis. Regarding renal pathology, several studies demonstrated an attenuation of renal injury caused by diabetes or by obesity with or without high-salt diet through anti-inflammatory, antifibrotic, and antioxidative mechanisms. Generally, AT2 receptor expression seems increased and AT2 receptor-mediated effects stronger in female and obese animals. Summary The recent findings about the role of the angiotensin AT2 receptor in renal health and disease strongly suggest that pharmacological targeting of this receptor with selective agonists is a promising therapeutic strategy for inducing diuresis/natriuresis (also additive to established diuretics) and for the treatment of diabetic nephropathy or kidney disease of other pathogenesis.

Angiotensin-(1-7) Influences Tryptophan Absorption in the Rat and Mouse Intestine

1 Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. 2 Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil. 3 Departamento de Morfologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Discovery and Characterization of Alamandine

Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand β-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders. # Novelty and Significance {#article-title-32}Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand &bgr;-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/&bgr;-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.

[PS 01-28] AT2-RECEPTOR STIMULATION PROMOTES NO RELEASE THROUGH eNOS SERINE1177 PHOSPHORYLATION AND eNOS TYROSINE657 DEPHOSPHORYLATION

Objective: Angiotensin AT2 receptor (AT2R) stimulation promotes vasodilation by increased nitric oxide (NO) release from endothelial cells. However, the mechanisms underlying AT2R-induced NO synthesis are still not completely characterized. We investigated whether AT2R-stimulation activates endothelial NO synthase (eNOS) by phosphorylation at Ser1177 or dephosphorylation at Tyr657 thus increasing the production of NO, and the participation of phosphatases in mediate these effects. Design and Method: Human Aortic Endothelial Cells (HAEC) were stimulated with the AT2R-agonist C21 (1 &mgr;M) in the presence or absence of PD123319 (10 &mgr;M) (an AT2R antagonist), L-NAME (10 &mgr;M) (an eNOS inhibitor), okadaic acid (10 nM; serine/threonine phosphatase inhibitor) or sodium orthovanadate (10 nM; tyrosine phosphatase inhibitor). NO release was estimated by quantifying DAF-FM fluorescence over a period of 10 minutes. HAEC cells were also stimulated with C21 and intracellular calcium transient analysis was performed using FURA-2 probe. Furthermore, HAEC cells were stimulated with C21 1 &mgr;M for 5, 10, 15 minutes, 24 and 48 hours, and expression of phospho-Ser1177-eNOS (activation site), phospho-Tyr657-eNOS (inhibition site) and total eNOS determined by Western blotting. Best time response of eNOS activation using C21 was performed again in presence of PD123319. Results: Stimulation of HAEC by C21 resulted in a significant increase in NO release, which was blocked by PD123319 or L-NAME preincubation. Both phosphatase inhibitors were also able to block the increase in NO release promoted by AT2R stimulation. No intracellular calcium transient was observed after C21 stimulation. Moreover, a significant increase in Ser1177-eNOS phosphorylation which was blocked by PD123319, as well as a significant decrease in Tyr657-eNOS dephosphorylation was observed. AT2R stimulation did not alter expression of total eNOS. Conclusions: From these data, it was concluded that AT2R-stimulation increases NO synthesis by endothelial cells through modulation of eNOS phosphorylation resulting in increased eNOS activity, but not by modulation of total eNOS expression. This pathway is not mediated via intracellular calcium transient.

Discovery and Characterization of AlamandineNovelty and Significance

Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand β-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/β-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders. # Novelty and Significance {#article-title-32}Rationale: The renin–angiotensin system (RAS) is a key regulator of the cardiovascular system, electrolyte, and water balance. Here, we report identification and characterization of alamandine, a new heptapeptide generated by catalytic action of angiotensin-converting enzyme-2 angiotensin A or directly from angiotensin-(1–7). Objective: To characterize a novel component of the RAS, alamandine. Methods and Results: Using mass spectrometry we observed that alamandine circulates in human blood and can be formed from angiotensin-(1–7) in the heart. Alamandine produces several physiological actions that resemble those produced by angiotensin-(1–7), including vasodilation, antifibrosis, antihypertensive, and central effects. Interestingly, our data reveal that its actions are independent of the known vasodilator receptors of the RAS, Mas, and angiotensin II type 2 receptor. Rather, we demonstrate that alamandine acts through the Mas-related G-protein–coupled receptor, member D. Binding of alamandine to Mas-related G-protein–coupled receptor, member D is blocked by D-Pro7-angiotensin-(1–7), the Mas-related G-protein–coupled receptor, member D ligand &bgr;-alanine and PD123319, but not by the Mas antagonist A-779. In addition, oral administration of an inclusion compound of alamandine/&bgr;-hydroxypropyl cyclodextrin produced a long-term antihypertensive effect in spontaneously hypertensive rats and antifibrotic effects in isoproterenol-treated rats. Alamandine had no noticeable proliferative or antiproliferative effect in human tumoral cell lines. Conclusions: The identification of these 2 novel components of the RAS, alamandine and its receptor, provides new insights for the understanding of the physiological and pathophysiological role of the RAS and may help to develop new therapeutic strategies for treating human cardiovascular diseases and other related disorders.