Cardiovascular actions of angiotensin-( 1-7 )

Angiotensin-(1-7) (Ang-(1-7)) is now considered to be a biologically active member of the renin-angiotensin system. The functions of Ang-(1-7) are often opposite to those attributed to the main effector component of the renin-angiotensin system, Ang II. Chronic administration of angiotensin-converting enzyme inhibitors (ACEI) increases 10- to 25-fold the plasma levels of this peptide, suggesting that part of the beneficial effects of ACEI could be mediated by Ang-(1-7). Ang-(1-7) can be formed from Ang II or directly from Ang I. Other enzymatic pathways for Ang-(1-7) generation have been recently described involving the novel ACE homologue ACE2. This enzyme can form Ang-(1-7) from Ang II or less efficiently by the hydrolysis of Ang I to Ang-(1-9) with subsequent Ang-(1-7) formation. The biological relevance of Ang-(1-7) has been recently reinforced by the identification of its receptor, the G-protein-coupled receptor Mas. Heart and blood vessels are important targets for the formation and actions of Ang-(1-7). In this review we will discuss recent findings concerning the biological role of Ang-(1-7) in the heart and blood vessels, taking into account aspects related to its formation and effects on these tissues. In addition, we will discuss the potential of Ang-(1-7) and its receptor as a target for the development of new cardiovascular drugs.


Introduction
The renin-angiotensin system (RAS) is a major regulator of renal and cardiovascular function, playing a pivotal role in the homeostasis of arterial pressure and of the hydroelectrolyte balance.In the past few years the combination of classical physiopharmacological techniques with modern genomics and protein chemistry methods has led to the identification of many novel components of the RAS: the angiotensin (Ang) IV binding site insulin-regulated aminopeptidase (1), angiotensin-converting enzyme 2 (ACE2) (2,3), the renin receptor (4), and the Ang-(1-7) receptor Mas (5).Recent reports have also suggested that Des-Asp 1 -Ang I (Ang-(2-10)) has cardiovascular actions which may be direct or depend on its processing to smaller peptides (6).Ang-(1-7) is one of the most interesting peptide fragments of the RAS because it can be formed directly from Ang I independent of ACE (Figure 1) and because it has actions which are often opposite to those of Ang II (7).The recent identification of the Ang-(1-7)-forming enzyme ACE2 and of Mas as an Ang-(1-7) receptor has added further support and wider acceptance (8)(9)(10) to a new concept of the RAS, formally supported by several groups (7,(11)(12)(13).According to this novel concept, the RAS has two major types of action, each using a different RAS peptide: a vasoconstrictor/proliferative one in which the major player is Ang II and a vasodilator/anti-proliferative one in which the major effector is Ang- (1)(2)(3)(4)(5)(6)(7).A further layer of complexity in this concept is represented by the AT 2 -mediated actions which are often opposite to those elicited by activation of AT 1 receptors (9), and possibly involves interaction with the AT 1-7 receptor Mas (14).Thus, the dual effects of the RAS can be ascribed to its peptides (mainly Ang II vs Ang-(1-7)) as well as to their receptors (mainly AT 1 vs AT 2 + Mas) (Table 1).In this article, we will briefly review novel aspects related to the cardiovascular actions of Ang-(1-7), focusing on the possible physiological role of the ACE2-Ang-(1-7)-Mas axis.

Ang-(1-7) formation and actions in the heart
Recent reports have indicated heart and blood vessels as the main targets for the actions of Ang-(1-7).These actions include biochemical and functional alterations leading to vasodilation and improved cardiac function (15)(16)(17).Several studies suggest that the heptapeptide Ang-(1-7) has beneficial cardiovascular effects either directly or indirectly through bradykinin (BK) potentiation or by counter-regulation of the actions of Ang II (7).The presence and local generation of Ang-(1-7) in the myocardium was first reported by Santos et al. (18)  A novel ACE homologue (ACE2) was described a few years ago (2,3).ACE2 can form Ang-(1-7) from Ang II (22) or less efficiently by hydrolysis of Ang I to Ang-(1-9) (3,10,22) with subsequent Ang-(1-7) generation by ACE and neprilysin hydrolysis (23).ACE2 is expressed extensively in the heart but is also located in the endothelium (22).Recent studies have indicated that ACE2 is an important regulator of the RAS and apparently plays an important role in heart function (10,24).Interestingly, heart ACE2 is increased by treatment with AT 1 antagonists and in failing human heart ventricles (25,26).Averill et al. (27) reported that Ang-(1-7) immunoreactivity was limited to cardiac myocytes and absent in interstitial cells and vessels.Myocardial infarction induced by left coronary artery occlusion significantly increased Ang-(1-7) immunoreactivity in the ventricular tissue surrounding the infarcted area.These studies suggest that Ang-(1-7) is part of a functional RAS in the heart.
Concerning the effects of Ang-(1-7) in the coronary bed there are marked differences related to dose and species.In isolated canine coronary artery rings precontracted with the thromboxane A 2 analogue U46,619, Ang-(1-7) elicited a dose-dependent vasorelaxation (10 nM to 100 µM).This effect was completely blocked by the nonselective Ang II antagonist (Sar 1 , Thr 8 )-Ang II, but not by the selective AT 1 or AT 2 Ang II antagonists CV11974 and PD123319, respectively, sug- The vascular endothelial dysfunction observed in aortic rings from rats with myocardial infarction was also markedly reversed by chronic infusion of Ang-(1-7) (17).The mechanisms of the coronary vascular actions of Ang-(1-7) have not been fully characterized, but probably involve release of prostacyclin and/or NO (11).One possible pathway involved in the coronary effects of Ang-(1-7) is the amplification of the vasodilator effects of BK.The vasodilator effect produced by BK injected in bolus into isolated perfused rat hearts or into isolated canine coronary arteries was potentiated by Ang-(1-7) (15,30).
Ang-(1-7) produced a significant increase in cardiac output and stroke volume in anesthetized Wistar rats as assessed by a fluorescent microsphere methodology.These effects were partially attenuated by the Ang-(1-7) antagonist, A-779 (31).We have shown that Ang-(1-7) at 220 pM concentration decreased the incidence and duration of ischemia-reperfusion arrhythmias in isolated perfused rat hearts (16).These cardioprotective effects were blocked by A-779 and by the cyclooxygenase inhibitor indomethacin.Furthermore, at the same low concentration, Ang-(1-7) improved the post-ischemic con- tractile function in isolated perfused rat hearts by a mechanism apparently involving its receptor Mas (5) and release of BK and prostaglandins (Figure 2) (32).Of note is the fact that at higher concentration (27 nM) Ang-(1-7) exerts an opposite effect.Neves et al. (28) reported that Ang-(1-7) facilitated reperfusion arrhythmias in isolated perfused rat hearts.Accordingly, transgenic mice overexpressing ACE2 in the heart presented sudden death due to cardiac arrhythmias (33).These observations suggest that high local concentrations of Ang-(1-7) have deleterious effects on the heart.Using the engineered fusion protein strategy, we have generated a transgenic rat model expressing an Ang-(1-7)-producing fusion protein (34).These TGR(A1-7)3292 rats presented a 2.5-fold increase in plasma Ang-(1-7) concentration.Radiotelemetry measurements showed that transgenic rats presented a slight but significant increase in diurnal and nocturnal dP/dt measurements.TGR(A1-7)3292 rats were significantly more resistant than control animals to induction of cardiac hypertrophy by isoproterenol.Moreover, these transgenic rats showed a reduced duration of reperfusion arrhythmias and an improved post-ischemic function in isolated perfused hearts.Therefore, this model provided further evidence for an important role of Ang-(1-7) in cardiac function (34).

Ang-(1-7) formation and actions in blood vessels
Endothelial cells are an important site for the formation (35) and metabolism (36) of Ang-(1-7).It has been shown that Ang-(1-7) produces relaxation in several vascular beds, including canine and porcine coronary arteries, canine middle cerebral artery, piglet pial arterioles, feline systemic vasculature, rabbit renal afferent arterioles, rat aortic rings, and mesenteric microvessels of normotensive and hypertensive rats (37,38; see also Ref. 7 for a review).In human vessels, con-tradictory results have been reported.Sasaki and co-workers (39) reported vasodilation in the human forearm while Davie and Mc-Murray (40) did not observe any effect of Ang-(1-7) in the same territory in ACEItreated patients.In keeping with previous reports by Ferrario's group (41), a role for Ang-(1-7) in mediating the chronic hypotensive effect of losartan in normal rats has been proposed by Collister and Hendel (42).
Many studies have shown that the vascular actions of Ang-(1-7) appear to involve increased production of vasodilatory prostanoids, NO and endothelium-derived hyperpolarizing factor (15,43,44).Moreover, some vascular effects involve simultaneous participation of these vasodilator mediators.A crosstalk between AT 1-7 and other receptors such as kinin B 2 and AT 2 receptors may also activate these pathways in blood vessels.For example, it has been demonstrated that the vasodilator action of Ang-(1-7) can be blocked by the B 2 receptor antagonist Hoe 140 (7,15).Moreover, a recent study showed that the link between Ang II and BK in endothelium-dependent vasorelaxation could involve receptors that bind the AT 2 and Ang-(1-7) receptor antagonists, PD123319 and A-779, respectively (45).The participation of vasodilating prostanoids in the vascular actions of Ang-(1-7) is not restricted to vasorelaxation, since they are also apparently involved in the antiproliferative actions of Ang-(1-7) in vascular smooth muscle cells and cardiomyocytes (44,46).Recently, it has been demonstrated that Ang-(1-7) inhibits vascular growth by the production of prostaglandin-mediated intracellular events, which include cAMP production and reduction of Ang II-stimulated ERK1/2 activities (47).

Ang-(1-7) and its receptor in the heart and blood vessels
Many of the cardiovascular effects of Ang-(1-7) are completely blocked by the selective Ang-(1-7) antagonist A-779, suggesting that the effects of Ang-(1-7) on the heart and blood vessels are mediated by a specific receptor sensitive to A-779 (16,(30)(31)(32)37,38; see also Ref. 7 for a review).Recently, Santos et al. (5) reported that Ang-(1-7) is an endogenous ligand for the G-proteincoupled receptor Mas.The specific binding of 125 I-Ang-(1-7) in mouse kidney slices was abolished by genetic deletion of Mas while the binding of 125 I-Ang II and 125 I-Ang IV was fully preserved.Furthermore, 125 I-Ang-(1-7) bound with high affinity to Mas-transfected COS cells and Ang-(1-7) treatment elicited arachidonic acid release from CHO and COS cells transfected with the Mas re-ceptor.The binding of 125 I-Ang-(1-7) and the effect of Ang-(1-7) on arachidonic acid release were blocked by the Ang-(1-7) antagonist A-779.Mas mRNA was detected in the heart, testis, kidney, and brain (55).In keeping with the putative cardioprotective role of Ang-(1-7), isolated hearts of Masdeficient mice presented marked changes in cardiac function.The systolic tension and positive and negative dT/dt were importantly decreased.Furthermore, a significant decrease in heart rate and an increase in coronary vascular resistance were also observed (56).In addition, the left ventricular pressure, positive dP/dt, and cardiac output were significantly lower in Mas-knockout mice assessed by means of a micro-conductance catheter placed inside the left ventricle (57).A reduced heart rate variability associated with an increased sympathetic tone was also found in Mas-deficient animals (58). Thee findings indicate that the Ang-(1-7) receptor Mas plays an important role in cardiac function and that the interaction of Ang-(1-7) with its specific receptor Mas is an important step for the actions of this peptide in the heart.
Evidence for a role of the Ang-(1-7) receptor Mas in the vascular effects of Ang-(1-7) was also obtained.Experiments on aortic rings derived from Mas-deficient mice demonstrated that the Ang-(1-7)-induced endothelium-dependent relaxation was abolished (5).This observation agrees with the results observed in Mas-transfected CHO cells, where Ang-(1-7), as well as its non-peptide agonist, AVE 0991 (59), cause a concentration-dependent increase in NO release (14).This effect was also blocked by A-779 but not by AT 1 or AT 2 antagonists.The Mas receptor appears to be also involved in the antiproliferative effect of Ang-(1-7) in vascular smooth muscle cells (47).It should be pointed out that, besides a Mas-Ang-(1-7) interaction, other mechanisms may be involved in the Ang-(1-7) vasodilatory effect, depending on the vessel diameter and the vascular regional bed (31).

Perspectives
Considering our current knowledge of the actions of Ang-(1-7), we could divide its cardiovascular actions into Mas-mediated and non-Mas-mediated ones.The fact that some actions of Ang-(1-7) are also blocked by AT 1 or AT 2 antagonists (7,11,60) is in favor of this hypothesis and may represent an indirect interaction of Ang-(1-7) with AT 1 and AT 2 receptors via Mas.In addition, the possibility of Ang-(1-7) directly interacting with AT 1 or AT 2 receptors as it does with ACE (7,53), cannot be excluded, especially at high Ang-(1-7) concentrations.The availability of non-peptide Ang-(1-7) receptor agonists, such as AVE 0991 (59), is a novel and important tool that will permit us to better understand its biological role and to clarify its complex mechanism of action (14).More importantly, these drugs may represent a new possibility for the development of novel therapeutic strategies for the treatment of cardiovascular diseases by interfering with the ACE2-Ang-(1-7)-Mas axis.

Figure 2 .
Figure 2. Time course of changes in systolic tension in isolated perfused rat heart submitted to temporary coronary occlusion.The hearts were perfused with Krebs-Ringer solution (KRS, control), KRS containing 0.22 nM angiotensin (Ang)-(1-7), or KRS containing 0.20 nM Ang II.The systolic tension was preserved in hearts perfused with Ang-(1-7) in the reperfusion period (A).This effect was completely blocked by A-779 (2 nM), Hoe 140 (100 nM), and pretreatment with indomethacin (5 mg/kg, ip, 1 h before heart removal) (B).*P < 0.05 compared to the control group and + P < 0.05 compared to the Ang II group (Student ttest).
in the

Table 1 .
Cardiovascular effects mediated by angiotensin receptors.