Fabiana P. Costa-Fraga

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.

Vascular Relaxation, Antihypertensive Effect, and Cardioprotection of a Novel Peptide Agonist of the Mas Receptor

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-Ala7-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.

An orally active formulation of angiotensin-(1-7) produces an antithrombotic effect

INTRODUCTION AND OBJECTIVE: The heptapeptide angiotensin-(1-7) is a component of the renin-angiotensin system, which promotes many beneficial cardiovascular effects, including antithrombotic activity. We have recently shown that the antithrombotic effect of angiotensin-(1-7) involves receptor Mas-mediated NO-release from platelets. Here, we describe an orally active formulation based on angiotensin-(1-7) inclusion in cyclodextrin [Ang-(1-7)- CyD] as an antithrombotic agent. Cyclodextrins are pharmaceutical tools that are used to enhance drug stability, absorption across biological barriers and gastric protection. METHOD: To test the antithrombotic effect of Ang-(1-7)-CyD, thrombus formation was induced in the abdominal vena cava of spontaneously hypertensive rats that were pretreated either acutely or chronically with Ang-(1-7)-CyD. Male Mas-knockout and wild-type mice were used to verify the role of the Mas receptor on the effect of Ang-(1-7)-CyD. RESULTS: Acute or chronic oral treatment with Ang-(1-7)-CyD promoted an antithrombotic effect (measured by thrombus weight; all values are, respectively, untreated vs. treated animals) in spontaneously hypertensive rats (acute: 2.86 ± 0.43 mg vs. 1.14 ± 0.40 mg; chronic: 4.27 ± 1.03 mg vs. 1.39 ± 0.68 mg). This effect was abolished in Mas-knockout mice (thrombus weight in Mas wild-type: 0.76 ± 0.10 mg vs. 0.37 ± 0.02 mg; thrombus weight in Mas-knockout: 0.96 ± 0.11 mg vs. 0.87 ± 0.14 mg). Furthermore, the antithrombotic effect of Ang-(1-7)-CyD was associated with an increase in the plasma level of Angiotensin-(1-7). CONCLUSION: These results show for the first time that the oral formulation Ang-(1-7)-CyD has biological activity and produces a Mas-dependent antithrombotic effect.

Angiotensin-Converting Enzyme 2 Activation Improves Endothelial Function

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin–angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1–7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1–7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II–induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Treatment with Angiotensin-(1-7) reduces inflammation in carotid atherosclerotic plaques

Angiotensin (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.

An oral formulation of angiotensin-(1-7) reverses corpus cavernosum damages induced by hypercholesterolemia.

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.

An Increased Arginase Activity Is Associated with Corpus Cavernosum Impairment Induced by Hypercholesterolemia

INTRODUCTION Hypercholesterolemia is a prevalent risk factor for the development of erectile dysfunction (ED), mostly due to an increase in oxidative stress and impaired nitric oxide (NO) bioavailability within the penis. Arginase is an enzyme that shares the common substrate L-arginine with NO synthase. Augmented arginase activity reduces NO production and is associated with ED development. However, the contribution of arginase hyperactivity in hypercholesterolemia-induced ED is unknown. AIM In the present study, we investigated the activity and role of arginase in the corpus cavernosum of hypercholesterolemic mice. METHODS Apolipoprotein E (ApoE) gene-deleted mice fed with a Western-type diet for 11 weeks were treated with the selective arginase inhibitor, N-ω-Hydroxy-L-norarginine (NOHA), or vehicle (saline 0.9%) during the last 9 weeks. Arginase activity and expression were measured in penis protein extraction. Reactive oxygen species (ROS) content within the corpus cavernosum was measured by dihydroethidium staining. Functional in vitro studies were performed using cavernosal strips mounted in an isometric organ bath to evaluate NO production. MAIN OUTCOME MEASURE Arginase activity and its role in modulating NO and ROS production within the corpus cavernosum of hypercholesterolemic mice is the main outcome measure. RESULTS Total arginase activity and arginase type II protein expression were increased in hypercholesterolemic mice compared with wild-type mice. The long-term treatment with NOHA normalized this alteration. Moreover, pharmacological arginase inhibition by NOHA attenuated the augmented ROS production within the corpus cavernosum of ApoE(-/-) mice, which increased the NO-dependent response in cavernosal strips. CONCLUSION These evidences indicate that arginase hyperactivity is associated with ED induced by hypercholesterolemia, suggesting that this enzyme is a potential target for treating ED.

Diminazene protects corpus cavernosum against hypercholesterolemia-induced injury.

INTRODUCTION Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin angiotensin system, which breaks down angiotensin II and forms angiotensin-(1-7). In erectile tissues, it has been documented that angiotensin II contributes to the development of erectile dysfunction (ED), while treatment with angiotensin-(1-7) improves penile erection. However, the expression and function of ACE2 in erectile tissues have never been investigated. AIM Here, we examined the expression of ACE2 in erectile tissues and its actions against hypercholesterolemia-induced corpus cavernosum (CC) injury. METHODS Hypercholesterolemic apolipoprotein E knockout (ApoE(-/-) ) mice, a well-known model of ED, were treated with diminazene aceturate (DIZE), an ACE2 activator compound, or vehicle for 3 weeks. Reactive oxygen species (ROS), collagen content, and protein expression of ACE2, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) subunits were evaluated in the penis of DIZE-treated and untreated ApoE(-/-) mice. Functional studies were performed in CC strips. MAIN OUTCOME MEASURES ACE2 expression and its role in modulating nitric oxide (NO)/ROS production and fibrosis within the CC of hypercholesterolemic mice were the main outcome measures. RESULTS ACE2 was expressed in smooth muscle and endothelial cells of mouse CC. Interestingly, ACE2 was downregulated in penis of hypercholesterolemic mice with ED, suggesting a protective role of ACE2 on the CC homeostasis. In accordance with that, pharmacological ACE2 activation by DIZE treatment reduced ROS production and NADPH oxidase expression, and elevated nNOS and eNOS expression and NO bioavailability in the penis of ApoE(-/-) mice. Additionally, DIZE decreased collagen content within the CC. These beneficial actions of DIZE on the CC were not accompanied by improvements in atherosclerotic plaque size or serum lipid profile. CONCLUSION ACE2 is expressed in erectile tissue and its reduction is associated with hypercholesterolemia-induced ED. Additionally, treatment with DIZE improved hypercholesterolemia-induced CC injury, suggesting ACE2 as a potential target for treating ED. .

Diminazene enhances stability of atherosclerotic plaques in ApoE-deficient mice

Angiotensin (Ang) II contributes to the development of atherosclerosis, while Ang-(1-7) has atheroprotective actions. Accordingly, angiotensin-converting enzyme 2 (ACE2), which breaks-down Ang II and forms Ang-(1-7), has been suggested as a target against atherosclerosis. Here we investigated the actions of diminazene, a recently developed ACE2 activator compound, in a model of vulnerable atherosclerotic plaque. Atherosclerotic plaque formation was induced in the carotid artery of ApoE-deficient mice by a shear stress (SS) modifier device. The animals were treated with diminazene (15mg/kg/day) or vehicle. ACE2 was strongly expressed in the aortic root and low SS-induced carotid plaques, but poorly expressed in the oscillatory SS-induced carotid plaques. Diminazene treatment did not change the lesion size, but ameliorated the composition of aortic root and low SS-induced carotid plaques by increasing collagen content and decreasing both MMP-9 expression and macrophage infiltration. Interestingly, these beneficial effects were not observed in the oscillatory SS-induced plaque. Additionally, diminazene treatment decreased intraplaque ICAM-1 and VCAM-1 expression, circulating cytokine and chemokine levels and serum triglycerides. In summary, ACE2 was distinctively expressed in atherosclerotic plaques, which depends on the local pattern of shear stress. Moreover, diminazene treatment enhances the stability of atherosclerotic plaques.

Treatment with CB2 Agonist JWH-133 Reduces Histological Features Associated with Erectile Dysfunction in Hypercholesterolemic Mice

Hypercholesterolemia is one of the most important risk factors for erectile dysfunction, mostly due to the impairment of oxidative stress and endothelial function in the penis. The cannabinoid system might regulate peripheral mechanisms of sexual function; however, its role is still poorly understood. We investigated the effects of CB2 activation on oxidative stress and fibrosis within the corpus cavernosum of hypercholesterolemic mice. Apolipoprotein-E-knockout mice were fed with a western-type diet for 11 weeks and treated with JWH-133 (selective CB2 agonist) or vehicle during the last 3 weeks. CB2 receptor expression, total collagen content, and reactive oxygen species (ROS) production within the penis were assessed. In vitro corpus cavernosum strips preparation was performed to evaluate the nitric oxide (NO) bioavailability. CB2 protein expression was shown in cavernosal endothelial and smooth muscle cells of wild type and hypercholesterolemic mice. Treatment with JWH-133 reduced ROS production and NADPH-oxidase expression in hypercholesterolemic mice penis. Furthermore, JWH-133 increased endothelial NO synthase expression in the corpus cavernosum and augmented NO bioavailability. The decrease in oxidative stress levels was accompanied with a reduction in corpus cavernosum collagen content. In summary, CB2 activation decreased histological features, which were associated with erectile dysfunction in hypercholesterolemic mice.