Carlos H. Castro

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.

Evidence for a Functional Interaction of the Angiotensin-(1–7) Receptor Mas With AT1 and AT2 Receptors in the Mouse Heart

The aim of this study was to evaluate the angiotensin (Ang)-(1–7) effects in isolated mouse hearts. The hearts of male C57BL/6J and knockout mice for the Ang-(1–7) receptor Mas were perfused by the Langendorff method. After a basal period, the hearts were perfused for 20 minutes with Krebs-Ringer solution (KRS) alone (control) or KRS containing Ang-(1–7) (0.22 pmol/L), the Mas antagonist A-779 (115 nmol/L), the angiotensin type 1 receptor antagonist losartan (2.2 &mgr;mol/L), or the angiotensin type 2 receptor antagonist PD123319 (130 nmol/L). To evaluate the involvement of Ang receptors, prostaglandins, and nitric oxide in the Ang-(1–7) effects, the hearts were perfused for 20 to 30 minutes with KRS containing either A-779, losartan, PD123319, indomethacin, or NG-nitro-l-arginine methyl ester (l-NAME) alone or in association with subsequent Ang-(1–7) perfusion. In addition, hearts from Mas-knockout mice were perfused for 20 minutes with KRS containing Ang-(1–7) (0.22 pmol/L) and losartan. Ang-(1–7) alone did not change the perfusion pressure. Strikingly, in the presence of losartan, 0.22 pmol/L Ang-(1–7) induced a significant decrease in perfusion pressure, which was blocked by A-779, indomethacin, and l-NAME. Furthermore, this effect was not observed in Mas-knockout mice. In contrast, in the presence of PD123319, Ang-(1–7) produced a significant increase in perfusion pressure. This change was not modified by the addition of A-779. Losartan reduced but did not abolish this effect. Our results suggest that Ang-(1–7) produces complex vascular effects in isolated, perfused mouse hearts involving interaction of its receptor with angiotensin type 1- and type 2-related mechanisms, leading to the release of prostaglandins and nitric oxide.

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.

New cardiovascular and pulmonary therapeutic strategies based on the Angiotensin-converting enzyme 2/angiotensin-(1-7)/mas receptor axis.

Angiotensin (Ang)-(1–7) is now recognized as a biologically active component of the renin-angiotensin system (RAS). The discovery of the angiotensin-converting enzyme homologue ACE2 revealed important metabolic pathways involved in the Ang-(1–7) synthesis. This enzyme can form Ang-(1–7) from Ang II or less efficiently through hydrolysis of Ang I to Ang-(1–9) with subsequent Ang-(1–7) formation. Additionally, it is well established that the G protein-coupled receptor Mas is a functional ligand site for Ang-(1–7). The axis formed by ACE2/Ang-(1–7)/Mas represents an endogenous counter regulatory pathway within the RAS whose actions are opposite to the vasoconstrictor/proliferative arm of the RAS constituted by ACE/Ang II/AT1 receptor. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1–7)/Mas arm in the cardiovascular and pulmonary system. Also, we will highlight the initiatives to develop potential therapeutic strategies based on this axis.

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.

Attenuation of isoproterenol-induced cardiac fibrosis in transgenic rats harboring an angiotensin-(1-7)-producing fusion protein in the heart

Objective: It has been shown that Ang-(1-7) has cardioprotective actions. To directly investigate the effects of Ang-(1-7) specifically in the heart, we generated and characterized transgenic (TG) rats which express an Ang-(1-7)-producing fusion protein driven by the α-MHC promoter. Methods and Results: After microinjection of the transgene into fertilized rat zygotes, we obtained four different transgenic lines. Homozygous animals were analyzed with regard to the expression profile of the transgene by ribonuclease protection assay. Transgene expression was detected mainly in the heart with weak or no expression in other organs. Heterozygous TG(hA-1-7)L7301 rats presented a significant increase in cardiac Ang-(1-7) concentration compared with control rats (17.1±2.1 versus 3.9±1.4 pg/mg protein in SD rats). Radiotelemetry analysis revealed that TG rats presented no significant changes in blood pressure and heart rate compared with normal rats. Overexpression of Ang-(1-7) in the heart produced slight improvement in resting cardiac function (+ dT/dt: 81530±1305.0 versus 77470±345.5 g/s bpm in SD rats, p < 0.05), which was in keeping with the enhanced [Ca2+] handling observed in cardiomyocytes of TG rats. TG(hA-1-7)L7301 rats also showed a greater capacity to withstand stress since TG rats showed a less pronounced deposition of collagen type III and fibronectin induced by isoproterenol treatment in the subendocardial area than in corresponding controls. In addition, hearts from TG rats showed reduced incidence and duration of reperfusion arrhythmias in comparison with SD rats. Conclusion: These results indicate that Ang-(1-7) has blood pressure-independent, antifibrotic effects, acting directly in the heart.

Angiotensin-(1-7) receptor Mas is an essential modulator of extracellular matrix protein expression in the heart

In this study we investigated the effects of genetic deletion of the Angiotensin-(1-7) receptor Mas or the Angiotensin II receptor AT(2) on the expression of specific extracellular matrix (ECM) proteins in atria, right ventricles and atrioventricular (AV) valves of neonatal and adult mice. Quantification of collagen types I, III and VI and fibronectin was performed using immunofluorescence-labeling and confocal microscopy. Picrosirius red staining was used for the histological assessment of the overall collagen distribution pattern. ECM proteins, metalloproteinases (MMP), ERK1/2 and p38 levels were quantified by western blot analysis. Gelatin zymography was used to evaluate the activity of MMP-2 and MMP-9. We observed that the relative levels of collagen types I and III and fibronectin are significantly higher in both the right ventricle and AV valves of neonatal Mas(-/-) mouse hearts (e.g., collagen type I: 85.28±6.66 vs 43.50±4.41 arbitrary units in the right ventricles of Mas(+/+) mice). Conversely, the level of collagen type VI was lower in the right ventricle and AV valves of Mas(-/-) mice. Adult Mas(-/-) mouse hearts presented similar patterns as observed in neonates. No significant differences in ECM protein level were detected in atria. Likewise, no changes in ECM levels were observed in AT(2) knockout mouse hearts. Although deletion of Mas induced a significant reduction in the level of the active form of MMP-2 in neonate hearts and a reduction of both MMP-2 and MMP-9 in adult Mas(-/-) mice, no significant differences were observed in MMP enzymatic activities when compared to controls. The levels of the active, phosphorylated forms of ERK1/2 and p38 were higher in hearts of both neonatal and adult Mas(-/-) mice. These observations suggest that Mas is involved in the selective expression of specific ECM proteins within both the ventricular myocardium and AV valves. The changes in the ECM profile may alter the connective tissue framework and contribute to the decreased cardiac performance observed in Mas(-/-) mice.

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.

Angiotensin II Type 1 receptor blockade restores angiotensin-(1-7)-induced coronary vasodilation in hypertrophic rat hearts.

The aim of the present study was to investigate the coronary effects of Ang-(1-7) [angiotensin-(1-7)] in hypertrophic rat hearts. Heart hypertrophy was induced by abdominal aorta CoA (coarctation). Ang-(1-7) and AVE 0991, a non-peptide Mas-receptor agonist, at picomolar concentration, induced a significant vasodilation in hearts from sham-operated rats. These effects were blocked by the Mas receptor antagonist A-779. Pre-treatment with L-NAME (N(G)-nitro-L-arginine methyl ester) or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one) [NOS (NO synthase) and soluble guanylate cyclase inhibitors respectively] also abolished the effect of Ang-(1-7) in control hearts. The coronary vasodilation produced by Ang-(1-7) and AVE 0991 was completely blunted in hypertrophic hearts. Chronic oral administration of losartan in CoA rats restored the coronary vasodilation effect of Ang-(1-7). This effect was blocked by A-779 and AT2 receptor (angiotensin II type 2 receptor) antagonist PD123319. Acute pre-incubation with losartan also restored the Ang-(1-7)-induced, but not BK (bradykinin)-induced, coronary vasodilation in hypertrophic hearts. This effect was inhibited by A-779, PD123319 and L-NAME. Chronic treatment with losartan did not change the protein expression of Mas and AT2 receptor and ACE (angiotensin-converting enzyme) and ACE2 in coronary arteries from CoA rats, but induced a slight increase in AT2 receptor in aorta of these animals. Ang-(1-7)-induced relaxation in aortas from sham-operated rats was absent in aortas from CoA rats. In vitro pre-treatment with losartan restored the Ang-(1-7)-induced relaxation in aortic rings of CoA rats, which was blocked by the Mas antagonist A-779 and L-NAME. These data demonstrate that Mas is strongly involved in coronary vasodilation and that AT1 receptor (angiotensin II type 1 receptor) blockade potentiates the vasodilatory effects of Ang-(1-7) in the coronary beds of pressure-overloaded rat hearts through NO-related AT2- and Mas-receptor-dependent mechanisms. These data suggest the association of Ang-(1-7) and AT1 receptor antagonists as a potential therapeutic avenue for coronary artery diseases.

Manganese ferrite-based nanoparticles induce ex vivo, but not in vivo, cardiovascular effects

Magnetic nanoparticles (MNPs) have been used for various biomedical applications. Importantly, manganese ferrite-based nanoparticles have useful magnetic resonance imaging characteristics and potential for hyperthermia treatment, but their effects in the cardiovascular system are poorly reported. Thus, the objectives of this study were to determine the cardiovascular effects of three different types of manganese ferrite-based magnetic nanoparticles: citrate-coated (CiMNPs); tripolyphosphate-coated (PhMNPs); and bare magnetic nanoparticles (BaMNPs). The samples were characterized by vibrating sample magnetometer, X-ray diffraction, dynamic light scattering, and transmission electron microscopy. The direct effects of the MNPs on cardiac contractility were evaluated in isolated perfused rat hearts. The CiMNPs, but not PhMNPs and BaMNPs, induced a transient decrease in the left ventricular end-systolic pressure. The PhMNPs and BaMNPs, but not CiMNPs, induced an increase in left ventricular end-diastolic pressure, which resulted in a decrease in a left ventricular end developed pressure. Indeed, PhMNPs and BaMNPs also caused a decrease in the maximal rate of left ventricular pressure rise (+dP/dt) and maximal rate of left ventricular pressure decline (−dP/dt). The three MNPs studied induced an increase in the perfusion pressure of isolated hearts. BaMNPs, but not PhMNPs or CiMNPs, induced a slight vasorelaxant effect in the isolated aortic rings. None of the MNPs were able to change heart rate or arterial blood pressure in conscious rats. In summary, although the MNPs were able to induce effects ex vivo, no significant changes were observed in vivo. Thus, given the proper dosages, these MNPs should be considered for possible therapeutic applications.