Sérgio V.B. Pinheiro

Impairment of In Vitro and In Vivo Heart Function in Angiotensin-(1-7) Receptor Mas Knockout Mice

In this study we investigated the effects of the genetic deletion of the angiotensin (Ang)-(1-7) receptor Mas on heart function. Localization of Mas in the mouse heart was evaluated by binding of rhodamine-labeled Ang-(1-7). Cardiac function was examined using isolated heart preparations. Echocardiography was used to confirm the results obtained with isolated heart studies. To elucidate the possible mechanisms involved in the cardiac phenotype observed in Mas−/− mice, whole-cell calcium currents in cardiomyocytes and the expression of collagen types I, III, and VI and fibronectin were analyzed. Ang-(1-7) binding showed that Mas is localized in cardiomyocytes of the mouse heart. Isolated heart techniques revealed that Mas-deficient mice present a lower systolic tension (average: 1.4±0.09 versus 2.1±0.03 g in Mas+/+ mice), ±dT/dt, and heart rate. A significantly higher coronary vessel resistance was also observed in Mas-deficient mice. Echocardiography revealed that hearts of Mas-deficient mice showed a significantly decreased fractional shortening, posterior wall thickness in systole and left ventricle end-diastolic dimension, and a higher left ventricle end-systolic dimension. A markedly lower global ventricular function, as defined by a higher myocardial performance index, was observed. A higher delayed time to the peak of calcium current was also observed. The changes in cardiac function could be partially explained by a marked change in collagen expression to a profibrotic profile in Mas-deficient mice. These results indicate that Ang-(1-7)-Mas axis plays a key role in the maintenance of the structure and function of the heart.

Angiotensin-(1-7) and its receptor as a potential targets for new cardiovascular drugs.

The identification of novel biochemical components of the renin–angiotensin system (RAS) has added a further layer of complexity to the classical concept of this cardiovascular regulatory system. It is now clear that there is a counter-regulatory arm within the RAS that is mainly formed by the angiotensin-converting enzyme 2–angiotensin (1-7)–receptor Mas axis. The functions of this axis are often opposite to those attributed to the major component of the RAS, angiotensin II. This review will highlight the current knowledge concerning the cardiovascular effects of angiotensin-(1-7) through a direct interaction with its receptor Mas or through an indirect interplay with the kallikrein–kinin system. In addition, there will be a discussion of its role in the beneficial effects of angiotensin-converting enzyme inhibitors and angio-tensin receptor type 1 (AT1) antagonists, and the potential of this peptide and its receptor as a novel targets for new cardiovascular drugs.

Genetic deletion of the angiotensin-(1–7) receptor Mas leads to glomerular hyperfiltration and microalbuminuria

Angiotensin-(1-7), an active fragment of both angiotensins I and II, generally opposes the vascular and proliferative actions of angiotensin II. Here we evaluated effects of the angiotensin-(1-7) receptor Mas on renal physiology and morphology using Mas-knockout mice. Compared to the wild-type animals, Mas knockout mice had significant reductions in urine volume and fractional sodium excretion without any significant change in free-water clearance. A significantly higher inulin clearance and microalbuminuria concomitant with a reduced renal blood flow suggest that glomerular hyperfiltration occurs in the knockout mice. Histological analysis found reduced glomerular tuft diameter and increased expression of collagen IV and fibronectin in the both the mesangium and interstitium, along with increased collagen III in the interstitium. These fibrogenic changes and the renal dysfunction of the knockout mice were associated with an upregulation of angiotensin II AT1 receptor and transforming growth factor-beta mRNA. Our study suggests that Mas acts as a critical regulator of renal fibrogenesis by controlling effects transduced through angiotensin II AT1 receptors in the kidney.

Characterization of a New Selective Antagonist for Angiotensin-(1–7), d-Pro7-Angiotensin-(1–7)

Abstract—Angiotensin-(1–7) [Ang-(1–7)] has biological actions that can often be distinguished from those of angiotensin II (Ang II). Recent studies indicate that the effects of Ang-(1–7) are mediated by specific receptor(s). We now report the partial characterization of a new antagonist selective for Ang-(1–7), d-Pro7-Ang-(1–7). d-Pro7-Ang-(1–7) (50 pmol) inhibited the hypertensive effect induced by microinjection of Ang-(1–7) [4±1 vs 21±2 mm Hg, 25 pmol Ang-(1–7) alone] into the rostral ventrolateral medulla without changing the effect of Ang II (16±2.5 vs 19±2.5 mm Hg after 25 pmol Ang II alone). At 10−7 mol/L concentration, it completely blocked the endothelium-dependent vasorelaxation produced by Ang-(1–7) (10−10 to 10−6 mol/L) in the mouse aorta. The antidiuresis produced by Ang-(1–7) (40 pmol/100 g body weight) in water-loaded rats was also blocked by its analog [1 &mgr;g/100 g body weight; 3.08±0.8 vs 1.27±0.33 mL in Ang-(1–7)–treated rats]. d-Pro7-Ang-(1–7) at a molar ratio of 40:1 did not change the hypotensive effect of bradykinin. Moreover, d-Pro7-Ang-(1–7) did not affect the dipsogenic effect produced by intracerebroventricular administration of Ang II (11.4±1.15 vs 8.8±1.2 mL/h after Ang II) and did not show any demonstrable angiotensin-converting enzyme inhibitory activity in assays with the synthetic substrate Hip-His-Leu and rat plasma as a source of enzyme. Autoradiography studies with 125I–Ang-(1–7) in mouse kidney slices showed that d-Pro7-Ang-(1–7) competed for the binding of Ang-(1–7) to the cortical supramedullary region. In Chinese hamster ovary cells stably transfected with the AT1 receptor subtype, d-Pro7-Ang-(1–7) did not compete for the specific binding of 125I–Ang-II in concentrations up to 10−6 mol/L. There was also no significant displacement of Ang II binding to angiotensin type 2 receptors in membrane preparations of adrenal medulla. These data indicate that d-Pro7-Ang-(1–7) is a selective antagonist for Ang-(1–7), which can be useful to clarify the functional role of this heptapeptide.

Selective increase of angiotensin(1–7) and its receptor in hearts of spontaneously hypertensive rats subjected to physical training

In the present study we investigated the effects of physical training on plasma and cardiac angiotensin(1–7) [Ang(1–7)] levels. In addition, possible changes in expression of the Ang(1–7) Mas receptor in the heart were also evaluated. Normotensive Wistar rats and spontaneously hypertensive rats (SHR) were subjected to an 8 week period of 5% overload swimming training. Blood pressure was determined by a tail‐cuff system. Heart and left ventricle weights and cardiomyocyte diameter were analysed to evaluate cardiac hypertrophy. Radioimmunoassay was used to measure angiotensin levels. Expression of Mas was determined by semi‐quantitative polymerase chain reaction, immunofluorescence and Western blotting. Physical training induced cardiac hypertrophy in Wistar rats and SHR. A significant decrease of plasma angiotensin II (Ang II) levels in both strains was also observed. Strikingly, trained SHR, but not trained Wistar rats, showed a twofold increase in left ventricular Ang(1–7) levels. No significant changes were observed in plasma Ang(1–7) and left ventricular Ang II concentrations in either strain. Furthermore, Mas mRNA and protein expression in left ventricle were substantially increased in trained SHR. The physical training protocol used did not change blood pressure in either strain. These results suggest that the beneficial effects induced by swimming training in hypertensive rats might include an augmentation of Ang(1–7) and its receptor in the heart.

Chronic infusion of angiotensin-(1-7) reduces heart angiotensin II levels in rats

We tested the hypothesis that the actions of Angiotensin (Ang)-(1-7) in the heart could involve changes in tissue levels of Ang II. This possibility was addressed by determining the effect of chronic infusion of Ang-(1-7) on plasma and tissue angiotensins. Ang-(1-7) was infused subcutaneously (osmotic minipumps) in Wistar rats. Angiotensins were determined by radioimmunoassay (RIA) in plasma, heart, and kidney. Tissue and plasma angiotensin-converting enzyme (ACE) activity and plasma renin activity (PRA) were also measured. Cardiac and renal ACE2 mRNA levels and cardiac angiotensinogen mRNA levels were assessed by semi-quantitative polymerase chain reaction (PCR). AT1 receptor number was evaluated by autoradiograph. Chronic infusion of Ang-(1-7) (2 microg/h, 6 days) produced a marked decrease of Ang II levels in the heart. A less pronounced but significant decrease of Ang-(1-7) was also observed. No significant changes were observed for Ang I. Ang II was not altered in the kidney. In this tissue, a significant increase of Ang-(1-7) and Ang I concentration was observed. A significant increase of plasma Ang-(1-7) and Ang II was also observed. Ang-(1-7) infusion did not change ACE activity or PRA. A selective slight significant increase in ACE2 expression in the heart was observed. Heart angiotensinogen mRNA as well as the number of Ang II binding sites did not change. These results suggest that AT1 receptors-independent changes in heart Ang II concentration might contribute for the beneficial effects of Ang-(1-7) in the heart. Moreover, these results reinforce the hypothesis that this angiotensin plays an important site-specific role within the renin-angiotensin system.

Circulating renin Angiotensin system in childhood chronic renal failure: marked increase of Angiotensin-(1-7) in end-stage renal disease.

The aim of the present study was to evaluate plasma renin activity (PRA) and Angiotensin (Ang) levels [Ang I, Ang II and Ang-(1–7)] to examine the circulating Renin-Angiotensin System (RAS) in renal disease among children with different forms and stages of chronic renal failure (CRF). Subjects were divided as follows: 32 normotensive healthy subjects, 23 normotensive CRF subjects, 34 hypertensive CRF subjects and 21 subjects with end-stage renal disease (ESRD). Radioimmunoassays for PRA (ngAngI/mL/h) and angiotensin (pg/mL) measurements were performed on all subjects. PRA, Ang I, Ang II and Ang-(1–7) levels were significantly higher in hypertensive CRF subjects when compared with normotensive CRF and healthy subjects (p < 0.05 for all comparisons). No differences were observed between normotensive CRF and healthy subjects. ESRD subjects exhibited a dramatic increase in Ang-(1–7) (25-fold higher than control values). In hypertensive CRF subjects, treatment with angiotensin-converting enzyme inhibitors (ACEi) increased (1.4-fold) plasma Ang-(1–7) and decreased (2.4-fold) Ang II. In ESRD, the use of ACEi produced a similar (1.5-fold) elevation of Ang-(1–7), but no changes in plasma Ang II. Our data showed different circulating RAS profiles between hypertensive and in normotensive CRF subjects. Marked changes in plasma Ang-(1–7) were associated with the presence of hypertension and progression of kidney dysfunction.

The Therapeutic Potential of Angiotensin-(1-7) as a Novel Renin- Angiotensin System Mediator

In this review, we show Angiotensin-(1-7) as a novel Renin Angiotensin System mediator that antagonizes cardiovascular and proliferative effects of Angiotensin II and exerts complex renal actions. We also speculate the possibility of new drugs for the treatment of cardiovascular, genitourinary and hepatic diseases by interfering with ACE2-Angiotensin-(1-7)-Mas axis.

Renal function in transgenic rats expressing an angiotensin-(1-7)-producing fusion protein.

Transgenic rats [TGR(A1-7)3292] present a chronic 2.5-fold increase in plasma Angiotensin-(1-7) [Ang-(1-7)] concentration. In the present study, we investigated the effects of this chronic elevation on renal function, vasopressin levels, kidney morphology, expression of Ang-(1-7) and vasopressin receptors in TGR(A1-7)3292. Urine volume and water intake were measured for 24 h. At the end of this period, plasma and urine samples were collected to evaluate renal function parameters and circulating vasopressin levels. Expression of renal V2 receptors and Mas was assessed by ribonuclease protection assay. Renal slices were processed for histological analysis. The urine flow of TGR(A1-7)3292 was significantly lower in comparison with Sprague-Dawley rats. The reduced urine volume of TGR(A1-7)3292 was accompanied by a significant increase in urinary osmolality and decrease free water clearance. Glomerular filtration rate, urinary sodium and potassium excretion were similar in both strains. No significant changes were observed in vasopressin levels as well as in V2 receptor and Mas mRNA expression in renal tissue. No changes in kidney structure of TGR(A1-7)3292 were detected. These data suggest that changes in circulating renin-angiotensin system produced by chronic increase of Ang-(1-7) levels can lead to adjustments in the water balance that are independent of vasopressin release and V2 receptor expression.

The role of angiotensin-(1–7) receptor Mas in spermatogenesis in mice and rats

Evidence regarding the components of the renin–angiotensin (Ang) system suggests that this system plays an important role in male reproduction. However, there are few data available in the literature on the effects of Ang‐(1–7) on the male reproductive system. The present study investigated the effects of the genetic deletion and chronic blockage of Ang‐(1–7) receptor Mas on spermatogenesis and male fertility. The localization of Mas in mouse and rat testes was determined by binding assays and immunofluorescence, whereas the testis structure and spermatogenic process were morphologically and stereologically analysed by light microscopy. Ang‐(1–7) binding and immunofluorescence revealed the presence of Mas in the testes of mice and rats. Although the total numbers of Sertoli and Leydig cells per testis and Leydig cell size were similar in both wild‐type and Mas‐deficient mice, Mas−/– animals exhibited a significant reduction in testis weight and a greater volume of apoptotic cells, giant cells and vacuoles in the seminiferous epithelium. In both mice and rats, an increased number of apoptotic cells were found during meiosis. Due to disturbed spermatogenesis, daily sperm production was markedly reduced in Mas−/– mice. Moreover, chronic infusion of A‐779 [an Ang‐(1–7) antagonist] in rats significantly increased the total number of apoptotic cells and primary spermatocytes in particular stages of spermatogenesis. Taken together, these findings strongly suggest that Ang‐(1–7) receptor Mas plays an important role in the regulation of spermatogenesis.