Vasopeptidase inhibition: a double-edged sword?

The enormous benefits of inhibition of ACE demonstrate that manipulation of the metabolism of peptide hormones is a valuable therapeutic strategy for cardiovascular disease. Recent attempts to expand these benefits have combined ACE inhibition with inhibition of other peptidases such as neutral endopeptidase (NEP) in a single molecule, a strategy known as vasopeptidase inhibition. NEP metabolizes natriuretic peptides, and NEP inhibition offers the prospect of combining the benefits of increased natriuretic peptide levels with those of ACE inhibition. However, peptidases such as ACE and NEP have many different substrates, and there are complex interactions between ACE inhibition and NEP inhibition. Both ACE and NEP metabolize the kinin peptides bradykinin and kallidin, and NEP also converts angiotensin (Ang) I to Ang-(1-7) and metabolizes Ang II and endothelin. Addition of NEP inhibition to ACE inhibition potentiates the ACE inhibitor-induced increase in kinin levels, increases Ang II levels, reduces Ang-(1-7) levels, and may increase endothelin levels. These additional consequences of combined ACE/NEP inhibition increase the risk of angioedema and may counteract any benefit of ACE inhibition that depends on reduced Ang II levels and increased Ang-(1-7) levels. Further considerations are that the ratio of ACE and NEP inhibition is fixed for vasopeptidase inhibitors, and there is uncertainty how these drugs should be compared with ACE inhibitors. Vasopeptidase inhibitors will therefore require careful evaluation before they are introduced to patient care.     

Neutral endopeptidase and angiotensin I converting enzyme insertion/deletion gene polymorphism in humans

Neutral endopeptidase (NEP) hydrolyses angiotensins (Ang) I and II and generates angiotensin-(1-7) [Ang-(1-7)]. In humans, the insertion/deletion (I/D) angiotensin-I converting enzyme (ACE) gene polymorphism determined plasma ACE levels by 40%. In rats, a similar polymorphism determines ACE levels which are inversely associated to NEP activity. The objective of this study is to evaluate the relationship between ACE expression and plasma NEP activity in normotensive subjects and in hypertensive patients. In total, 58 consecutive patients with hypertension, evaluated in our Hypertension Clinic, were compared according to their ACE I/D genotypes with 54 control subjects in terms of both plasma ACE activity and NEP activities. Plasma ACE activity was elevated 51 and 70% in both DD ACE groups (normotensives and hypertensives) compared with their respective ID and II ACE groups (P<0.001). A significant effect of the ACE polymorphism and of the hypertensive status on ACE activity was observed (P<0.001). In normotensive DD ACE subjects, NEP activity was 0.30+/-0.02 U/ml, whereas in the normotensive II ACE and in the normotensive ID ACE subjects NEP activity was increased 65 and 48%, respectively (P<0.001). In the hypertensive DD ACE patients, NEP activity was 0.47+/-0.03 U/mg. An effect of the I/D ACE genotypes on NEP activity (P<0.04) and an interaction effect between the I/D ACE genotype and the hypertensive status were also observed (P<0.001). These results are consistent with a normal and inverse relationship between the ACE polymorphism and NEP activity in normotensive humans (as is also observed in rats). This normal relationship is not observed in hypertensive patients.     

Enalapril attenuates downregulation of Angiotensin-converting enzyme 2 in the late phase of ventricular dysfunction in myocardial infarcted rat

The early and long-term effects of coronary artery ligation on the plasma and left ventricular angiotensin-converting enzyme (ACE and ACE2) activities, ACE and ACE2 mRNA levels, circulating angiotensin (Ang) levels [Ang I, Ang-(1-7), Ang-(1-9), and Ang II], and cardiac function were evaluated 1 and 8 weeks after experimental myocardial infarction in adult Sprague Dawley rats. Sham-operated rats were used as controls. Coronary artery ligation caused myocardial infarction, hypertrophy, and dysfunction 8 weeks after surgery. At week 1, circulating Ang II and Ang-(1-9) levels as well as left ventricular and plasma ACE and ACE2 activities increased in myocardial-infarcted rats as compared with controls. At 8 weeks post-myocardial infarction, circulating ACE activity, ACE mRNA levels, and Ang II levels remained higher, but plasma and left ventricular ACE2 activities and mRNA levels and circulating levels of Ang-(1-9) were lower than in controls. No changes in plasma Ang-(1-7) levels were observed at any time. Enalapril prevented cardiac hypertrophy and dysfunction as well as the changes in left ventricular ACE, left ventricular and plasmatic ACE2, and circulating levels of Ang II and Ang-(1-9) after 8 weeks postinfarction. Thus, the decrease in ACE2 expression and activity and circulating Ang-(1-9) levels in late ventricular dysfunction post-myocardial infarction were prevented with enalapril. These findings suggest that in this second arm of the renin-angiotensin system, ACE2 may act through Ang-(1-9), rather than Ang-(1-7), as a counterregulator of the first arm, where ACE catalyzes the formation of Ang II.     

An alternative strategy for the radioimmunoassay of angiotensin peptides using amino-terminal-directed antisera: measurement of eight angiotensin peptides in human plasma

We describe here a method of measuring angiotensin peptides and their carboxy-truncated metabolites in human plasma using N-terminal-directed antisera. Antisera raised against N-acetylated angiotensin (Ang) II and N-acetylated Ang III analogues were used to develop two radioimmunoassays. Extracted plasma samples were acetylated prior to separation of cross-reacting angiotensin peptides by high-performance liquid chromatography (HPLC). Fractions were assayed with both antisera to obtain measurements for eight angiotensin peptides. Angiotensin levels measured in normal males were (fmol/ml plasma, mean +/- s.e.m., n = 14): Ang-(1-7) 1.0 +/- 0.2, Ang II 13.9 +/- 2.0, Ang-(1-9) less than 0.4, Ang I 19.5 +/- 2.4, Ang-(2-7) less than 1.1, Ang III 2.9 +/- 1.0, Ang-(2-9) less than 2.1, Ang-(2-10) 2.4 +/- 0.8. Hypertensive patients receiving angiotensin converting enzyme (ACE) inhibitor therapy (n = 8) had an increase in Ang I to 187.3 +/- 107.2 fmol/ml (P = 0.002), and a reduction in Ang II to 4.8 +/- 1.2 fmol/ml (P less than 0.001). Furthermore, these patients showed a ninefold increase in Ang-(1-7) to 9.7 +/- 4.3 fmol/ml (P less than 0.001), indicating a role for prolylendopeptidase in the metabolism of Ang I in vivo. These N-terminal assays have demonstrated that carboxy-truncated metabolites of Ang I and Ang II make little contribution to plasma angiotensin peptides, except during ACE inhibitor therapy. Furthermore, these antisera allow the measurement of Ang I and Ang II in the same radioimmunoassay of fractions from HPLC, providing a highly reliable estimate of the Ang II:Ang I ratio.     

Effects of angiotensin-(1-7) and other bioactive components of the renin-angiotensin system on vascular resistance and noradrenaline release in rat kidney

OBJECTIVE: Angiotensin (Ang) is broken down enzymatically to several different metabolites which, in addition to Ang II, may have important biological effects in the kidney. This study investigates the role of Ang metabolites on vascular resistance and noradrenaline release in the rat kidney. METHODS AND RESULTS: In rat isolated kidney Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I induced pressor responses and enhanced noradrenaline release to renal nerve stimulation (RNS) in an concentration-dependent manner, with the following rank order of potency (EC(50)): Ang II >or= Ang III > Ang I = des-Asp-Ang I > Ang IV. All effects were blocked by the AT(1)-receptor antagonist EXP 3174 (0.1 micromol/l) but not by the AT(2)-receptor antagonist PD 123319 (1 micromol/l). Angiotensin-converting enzyme (ACE) inhibition by captopril (10 micromol/l) abolished the effect of Ang I and des-Asp-Ang I but had no influence on the effect of the other metabolites. Ang-(1-7) blocked the effects of Ang I and Ang II, being 10 times more potent against Ang I than Ang II. The selective Ang-(1-7) receptor blocker d-Ala7-Ang-(1-7) (10 micromol/l) did not influence the inhibitory effects of Ang-(1-7). Ang-(1-7) (10 micromol/l) by itself had no influence on vascular resistance and RNS-induced noradrenaline release. CONCLUSION: Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I regulate renal vascular resistance and noradrenaline release by activation of AT(1) receptors. In the case of Ang I and des-Asp-Ang I this depends on conversion by ACE. Ang-(1-7) may act as a potent endogenous inhibitor/antagonist of ACE and the AT(1)-receptors, respectively.     

Angiotensin-(1-7) modulates vascular resistance and sympathetic neurotransmission in kidneys of spontaneously hypertensive rats

OBJECTIVE: Angiotensin (Ang)-(1-7) generated from Ang I and II is reported to act as an endogenous angiotensin-converting enzyme (ACE) inhibitor and angiotensin type 1 (AT1)-receptor antagonist in vitro and in vivo. Ang-(1-7) has been suggested to play an important role in hypertension. METHODS AND RESULTS: Therefore, we tested whether Ang-(1-7) differentially modulates vascular resistance and neurotransmission in isolated kidneys of spontaneously hypertensive rats stroke prone (SHR-SP) and Wistar-Kyoto rats (WKY). Ang-(1-7) was administered in three concentrations (0.1, 1 and 10 micromol/l) to prevent Ang I- and Ang II-induced pressor responses and facilitation of noradrenaline release. There were indeed concentration-dependent strain differences. Ang-(1-7) prevented Ang I- and Ang II-mediated changes in vascular resistance more potently in SHR-SP than in WKY by inhibiting ACE and by blocking AT1-receptors. Ang-(1-7) by itself had no influence on renal vascular tone in both strains. Ang-(1-7) inhibited Ang I-mediated facilitation of noradrenaline release more potently than Ang II-mediated facilitation of noradrenaline release. Ang-(1-7) by itself enhanced noradrenaline release from SHR-SP, but not from WKY kidneys. CONCLUSION: Ang-(1-7) had a greater impact on Ang I and Ang II modulation of renal vascular resistance in SHR-SP than in normotensive rats. Furthermore, Ang-(1-7) by itself has facilitatory presynaptic effects on noradrenaline release but no postsynaptic effects on renal vascular resistance in SHR-SP. Since plasma levels of Ang-(1-7) accumulate during ACE-inhibitor or AT1-receptor antagonist therapy, Ang-(1-7) could contribute to antihypertensive effects of these agents.     

Olmesartan is an angiotensin II receptor blocker with an inhibitory effect on angiotensin-converting enzyme.

Angiotensin II receptor blockers (ARBs) are widely used for the treatment of hypertension. It is believed that treatment with an ARB increases the level of plasma angiotensin II (Ang II) because of a lack of negative feedback on renin activity. However, Ichikawa (Hypertens Res 2001; 24: 641-646) reported that long-term treatment of hypertensive patients with olmesartan resulted in a reduction in plasma Ang II level, though the mechanism was not determined. It has been reported that angiotensin 1-7 (Ang-(1-7)) potentiates the effect of bradykinin and acts as an angiotensin-converting enzyme (ACE) inhibitor. It is known that ACE2, which was discovered as a novel ACE-related carboxypeptidase in 2000, hydrolyzes Ang I to Ang-(1-9) and also Ang II to Ang-(1-7). It has recently been reported that olmesartan increases plasma Ang-(1-7) through an increase in ACE2 expression in rats with myocardial infarction. We hypothesized that over-expression of ACE2 may be related to a reduction in Ang II level and the cardioprotective effect of olmesartan. Administration of 0.5 mg/kg/day of olmesartan for 4 weeks to 12-week-old stroke-prone spontaneously hypertensive rats (SHRSP) significantly reduced blood pressure and left ventricular weight compared to those in SHRSP given a vehicle. Co-administration of olmesartan and (D-Ala7)-Ang-(1-7), a selective Ang-(1-7) antagonist, partially inhibited the effect of olmesartan on blood pressure and left ventricular weight. Interestingly, co-administration of (D-Ala7)-Ang-(1-7) with olmesartan significantly increased the plasma Ang II level (453.2+/-113.8 pg/ml) compared to olmesartan alone (144.9+/-27.0 pg/ml, p<0.05). Moreover, olmesartan significantly increased the cardiac ACE2 expression level compared to that in Wistar Kyoto rats and SHRSP treated with a vehicle. Olmesartan significantly improved cardiovascular remodeling and cardiac nitrite/ nitrate content, but co-administration of olmesartan and (D-Ala7)-Ang-(1-7) partially reversed this anti-remodeling effect and the increase in nitrite/nitrate. These findings suggest that olmesartan may exhibit an ACE inhibitory action in addition to an Ang II receptor blocking action, prevent an increase in Ang II level, and protect cardiovascular remodeling through an increase in cardiac nitric oxide production and endogenous Ang-(1-7) via over-expression of ACE2.     

Murine Recombinant Angiotensin-Converting Enzyme 2: Effect on Angiotensin II-Dependent Hypertension and Distinctive Angiotensin-Converting Enzyme 2 Inhibitor Characteristics on Rodent and Human Angiotensin-Converting Enzyme 2

A newly produced murine recombinant angiotensin (Ang)-converting enzyme 2 (ACE2) was characterized in vivo and in vitro. The effects of available ACE2 inhibitors (MLN-4760 and 2 conformational variants of DX600, linear and cyclic) were also examined. When murine ACE2 was given to mice for 4 weeks, a marked increase in serum ACE2 activity was sustainable. In acute studies, mouse ACE2 (1 mg/kg) obliterated hypertension induced by Ang II infusion by rapidly decreasing plasma Ang II. These effects were blocked by MLN-4760 but not by either form of DX600. In vitro, conversion from Ang II to Ang-(1-7) by mouse ACE2 was blocked by MLN-4760 (10(-6) m) but not by either form of DX600 (10(-5) m). Quantitative analysis of multiple Ang peptides in plasma ex vivo revealed formation of Ang-(1-9) from Ang I by human but not by mouse ACE2. Both human and mouse ACE2 led to the dissipation of Ang II with formation of Ang (1-7). By contrast, mouse ACE2-driven Ang-(1-7) formation from Ang II was blocked by MLN-4760 but not by either linear or cyclic DX600. In conclusion, sustained elevations in serum ACE2 activity can be accomplished with murine ACE2 administration, thereby providing a strategy for ACE2 amplification in chronic studies using rodent models of hypertension and cardiovascular disease. Human but not mouse ACE2 degrades Ang I to form Ang-(1-9). There are also species differences regarding rodent and human ACE2 inhibition by known inhibitors such that MLN-4760 inhibits both human and mouse ACE2, whereas DX600 only blocks human ACE2 activity.     

Angiotensin-(1-7) does not affect vasodilator or TPA responses to bradykinin in human forearm

Studies in isolated vessels and rat models of hypertension suggest that angiotensin (Ang)-(1-7) potentiates the vasodilator effect of bradykinin, possibly through ACE inhibition. We therefore tested the hypothesis that Ang-(1-7) potentiates the vasodilator or tissue plasminogen activator (TPA) response to bradykinin in the human forearm vasculature. Graded doses of Ang-(1-7) (10, 100, and 300 pmol/min), bradykinin (47, 94, and 189 pmol/min), and Ang I (1, 10, and 30 pmol/min) were administered through the brachial artery to 8 normotensive subjects in random order. Thirty minutes after initiation of a constant infusion of Ang-(1-7) (100 pmol/min), bradykinin and Ang I infusions were repeated. There were no systemic hemodynamic effects of the agonists. Bradykinin significantly increased forearm blood flow (P<0.001, from 3.8+/-0.5 to 13.9+/-3.1 mL/min per 100 mL at 189 pmol/min) and net TPA release (P=0.007, from 1.1+/-1.0 to 23.6+/-6.2 ng/min per 100 mL at 189 pmol/min), whereas Ang I caused vasoconstriction (P=0.003, from 3.3+/-0.4 to 2.5+/-0.3 mL/min per 100 mL at 30-pmol/min dose). There was no effect of Ang-(1-7) on either forearm blood flow (P=0.62, 3.3+/-0.4 to 3.5+/-0.4 mL/min per 100 mL at 300 pmol/min) or TPA release (P=0.52, from 0.7+/-0.8 to 1.0+/-0.7 ng/min/100 mL at 300 pmol/min). Moreover, there was no effect of 100 pmol/min Ang-(1-7) on the vasodilator [P=0.46 for Ang-(1-7) effect] or TPA [P=0.82 for Ang-(1-7) effect] response to bradykinin or the vasoconstrictor response to Ang I [P=0.62 for Ang-(1-7) effect]. These data do not support a role of Ang-(1-7), given at supraphysiological doses, in the regulation of human peripheral vascular resistance or fibrinolysis.     

Angiotensin-converting enzyme 2 deficiency is associated with impaired gestational weight gain and fetal growth restriction

Angiotensin-converting enzyme 2 (ACE2) is a key enzyme of the renin-angiotensin system that influences the relative expression of angiotensin II (Ang II) and Ang-(1-7). Although ACE2 expression increases in normal pregnancy, the impact of ACE2 deficiency in pregnancy has not been elucidated. We determined the influence of ACE2 deficiency on circulating and tissue renin-angiotensin system components, fetal and maternal growth characteristics, and maternal hemodynamics (mean blood pressure and cardiac output) at day 18 of gestation. Gestational body weight gain was lower in the ACE2 knockout (KO) versus C57BL/6 (wild-type) mice (30.3±4.7 versus 38.2±1.0 g; P<0.001). Fetal weight (0.94±0.1 versus 1.24±0.01 g; P<0.01) and length (19.6±0.2 versus 22.2±0.2 mm; P<0.001) were less in KO. Mean blood pressure was significantly reduced in C57BL/6 with pregnancy; it was elevated (P<0.05) in the KO virgin and pregnant mice, and this was associated with an increased cardiac output in both C57BL/6 and KO pregnant mice (P<0.05). Plasma Ang-(1-7) was reduced in pregnant KO mice (P<0.05). Placenta Ang II levels were higher in KO mice (52.9±6.0 versus 22.0±3.3 fmol/mg of protein; P<0.001). Renal Ang II levels were greater in KO virgin mice (30.0±1.7 versus 23.7±1.1 fmol/mg of protein; P<0.001). There was no change in the Ang-(1-7) levels in the KO placenta and virgin kidney. These results suggest that ACE2 deficiency and associated elevated placenta Ang II levels impact pregnancy by impairing gestational weight gain and restricting fetal growth.     

Opportunities for Targeting the Angiotensin-Converting Enzyme 2/Angiotensin-(1-7)/Mas Receptor Pathway in Hypertension

It is well known that the renin-angiotensin system (RAS) plays a pivotal role in the pathophysiology of cardiovascular diseases. This is well illustrated by the great success of ACE inhibitors and angiotensin (Ang) II AT₁ blockers in the treatment of hypertension and its complications. In the past decade, the classical concept of RAS orchestrated by a series of enzymatic reactions culminating in the linear generation and action of Ang II has expanded and become more complex. From the discoveries of new components such as the angiotensin converting enzyme 2 and the receptor Mas emerged a novel concept of dual opposite branches of the RAS: one vasoconstrictor and pro-hypertensive composed of ACE/Ang II/AT1; and other vasodilator and anti-hypertensive composed of ACE2/Ang-(1-7)/Mas. In this review we will discuss recent findings concerning the biological role of the ACE2/Ang-(1-7)/Mas arm in the cardiovascular system and highlight the initiatives to develop potential therapeutic strategies based on this axis for treating hypertension.     

Urinary vasodilator and vasoconstrictor angiotensins during menstrual cycle, pregnancy, and lactation

Since normal human pregnancy is characterized by normotension in the face of an increased renin-angiotensin-aldosterone system (RAAS), we evaluated the temporal pattern of urinary excretion of a novel vasodilator within this system, angiotensin-(1–7) (Ang-[1–7]), during the menstrual cycle, pregnancy, and lactation. The urinary profiles of Ang I, Ang II, human chorionic gonadotropin, 17β-estradiol, and progesterone were also determined. During the menstrual cycle, urinary Ang-(1–7) and Ang II remained stable (mean cycle value: 94.6±11.3 and 11.4±1.1 pmol/g of creatinine, respectively) in nine females. In 10 normal pregnant women, urinary Ang-(1–7) and Ang II increased throughout gestation, averaging 1499.8±310 and 224.4±58 pmol/g of creatinine, respectively (p<0.05) at wk 35 and falling during lactation to 394.0±95 and 65.7±20 pmol/g of creatinine (p<0.05), respectively. The Ang-(1–7)/Ang II ratio was unchanged in the different reproductive periods. During the menstrual cycle, Ang II and Ang-(1–7) correlated with 17β-estradiol and progesterone using multivariate analysis (r=0.31, p<0.001) and r=0.28, p<0.02, respectively). During gestation, 17β-estradiol and progesterone correlated with urinary Ang-(1–7) (r=0.48, p<0.001 and r=0.47, p<0.001, respectively) and Ang II (r=0.24, p<0.03 and r=0.25, p<0.03, respectively); by multiple regression, only Ang-(1–7) correlated with both steroids (r=0.49, p<0.001). The progressive rise of Ang-(1–7) throughout gestation, probably modulated by estrogen and progesterone, suggests a physiologic counterregulation within the RAAS.     

Vasodilator action of angiotensin-(1-7) on isolated rabbit afferent arterioles

Recent studies have shown that angiotensin-(1-7) (Ang-[1-7]), which is generated endogenously from both Ang I and II, is a bioactive component of the renin-angiotensin system and may play an important role in the regulation of blood pressure. However, little is known about its role in regulating the reactivity of the afferent arteriole or the mechanism(s) involved. We hypothesized that Ang-(1-7), acting on specific receptors, participates in the control of afferent arteriole tone. We first examined the direct effect of Ang-(1-7) on rabbit afferent arterioles microperfused in vitro, and we tested whether endothelium-derived relaxing factor/NO and cyclooxygenase products are involved in its actions. To assess the vasodilator effect of Ang-(1-7), afferent arterioles were preconstricted with norepinephrine, and increasing concentrations of Ang-(1-7) were added to the lumen. We found that 10(-10) to 10(-6) mol/L Ang-(1-7) produced dose-dependent vasodilatation, increasing luminal diameter from 8.9+/-1.0 to 16.3+/-1.1 microm (P<0.006). Indomethacin had no effect on Ang-(1-7)-induced dilatation. N(G)-nitro-L-arginine methyl ester, a NO synthesis inhibitor, abolished the dilatation induced by Ang-(1-7). We attempted to determine which angiotensin receptor subtype is involved in this process. We found that 10(-6) mol/L [d-Ala7]-Ang-(1-7), a potent and selective Ang-(1-7) antagonist, abolished the dilatation induced by Ang-(1-7). An angiotensin II type 1 receptor antagonist (L158809) and an angiotensin II type 2 receptor antagonist (PD 123319) at 10(-6) mol/L had no effect on Ang-(1-7)-induced dilatation. Our results show that Ang-(1-7) causes afferent arteriole dilatation. This effect may be due to production of NO, but not the action of cyclooxygenase products. Ang-(1-7) has a receptor-mediated vasodilator effect on the rabbit afferent arteriole. This effect may be mediated by Ang-(1-7) receptors, because angiotensin type 1 and type 2 receptor antagonists could not block Ang-(1-7)-induced dilatation. Thus, our data suggest that Ang-(1-7)opposes the action of Ang II and plays an important role in the regulation of renal hemodynamics.     

Liver disease and the renin-angiotensin system: recent discoveries and clinical implications

The renin–angiotensin system (RAS) is a key regulator of vascular resistance, sodium and water homeostasis and the response to tissue injury. Historically, angiotensin II (Ang II) was thought to be the primary effector peptide of this system. Ang II is produced predominantly by the effect of angiotensin converting enzyme (ACE) on angiotensin I (Ang I). Ang II acts mainly through the angiotensin II type-1 receptor (AT₁) and, together with ACE, these components represent the 'classical' axis of the RAS. Drug therapies targeting the RAS by inhibiting Ang II formation (ACE inhibitors) or binding to its receptor (angiotensin receptor blockers) are now in widespread clinical use and have been shown to reduce tissue injury and fibrosis in cardiac and renal disease independently of their effects on blood pressure. In 2000, two groups using different methodologies identified a homolog of ACE, called ACE2, which cleaves Ang II to form the biologically active heptapeptide, Ang-(1–7). Conceptually, ACE2, Ang-(1–7), and its putative receptor, the mas receptor represent an 'alternative' axis of the RAS capable of opposing the often deleterious actions of Ang II. Interestingly, ACE inhibitors and angiotensin receptor blockers increase Ang-(1–7) production and it has been proposed that some of the beneficial effects of these drugs are mediated through upregulation of Ang-(1–7) rather than inhibition of Ang II production or receptor binding. The present review focuses on the novel components and pathways of the RAS with particular reference to their potential contribution towards the pathophysiology of liver disease.     

Low sodium diet inhibits the local counter-regulator effect of angiotensin-(1-7) on angiotensin II

OBJECTIVE: The heptapeptide angiotensin-(1-7) [Ang-(1-7)] has been identified as a versatile, endogenous inhibitor of the renin-angiotensin system (RAS). As the therapeutic response to exogenous RAS inhibitors, such as AT1 receptor antagonists, is altered by changes in salt intake, we investigated the effect of a low, normal and high sodium diet on the antagonism of Ang II by Ang-(1-7). The role of angiotensin receptor subtypes and the endothelium was assessed. METHODS: Male Wistar rats received a normal sodium (0.3% NaCl), high sodium (2.0% NaCl) or low sodium (0.05% NaCl) diet for 10 days. Vascular responses were assessed ex vivo in thoracic aortic rings in the presence of the nitric oxide (NO) inhibitor N-monomethyl-l-arginine (l-NMMA) to avoid aspecific vasodilator effects of Ang-(1-7). RESULTS: After a normal or high salt diet, Ang-(1-7) significantly decreased maximal Ang II-induced vascular constrictions by 40-50%. After a low salt diet this non-competitive antagonism disappeared. The AT2 receptor antagonist PD 123319 and the Ang-(1-7) receptor antagonist A779 attenuated the effect of Ang-(1-7) found in rats fed with a normal or high sodium diet. Further, removal of endothelium and pretreatment with the prostaglandin synthesis inhibitor indomethacin (10 mol/l) abolished the non-competitive antagonism by Ang-(1-7). CONCLUSION: Ang-(1-7) elicits a specific, endothelium-dependent and non-competitive antagonism of Ang II, which involves AT2 and Ang-(1-7) receptors but is independent of NO production. This non-competitive antagonism of Ang-(1-7) is abolished by a low sodium intake in normotensive rats, suggesting that it serves as a negative feedback towards Ang II in response to an altered sodium intake.     

Angiotensin-converting enzyme inhibition, but not AT(1) receptor blockade, in the solitary tract nucleus improves baroreflex sensitivity in anesthetized transgenic hypertensive (mRen2)27 rats

Transgenic hypertensive (mRen2)27 rats overexpress the murine Ren2 gene and have impaired baroreflex sensitivity (BRS) for control of the heart rate. Removal of endogenous angiotensin (Ang)-(1-7) tone using a receptor blocker does not further lower BRS. Therefore, we assessed whether blockade of Ang II with a receptor antagonist or combined reduction in Ang II and restoration of endogenous Ang-(1-7) levels with Ang-converting enzyme (ACE) inhibition will improve BRS in these animals. Bilateral solitary tract nucleus (nTS) microinjections of the AT(1) receptor blocker, candesartan (CAN, 24?pmol in 120?nl, n=9), or a peptidic ACE inhibitor, bradykinin (BK) potentiating nonapeptide (Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro; BPP9α, 9?nmol in 60?nl, n=12), in anesthetized male (mRen2)27 rats (15-25 weeks of age) show that AT(1) receptor blockade had no significant effect on BRS, whereas microinjection of BPP9α improved BRS over 60-120?min. To determine whether Ang-(1-7) or BK contribute to the increase in BRS, separate experiments using the Ang-(1-7) receptor antagonist D-Ala(7)-Ang-(1-7) or the BK antagonist HOE-140 showed that only the Ang-(1-7) receptor blocker completely reversed the BRS improvement. Thus, acute AT(1) blockade is unable to reverse the effects of long-term Ang II overexpression on BRS, whereas ACE inhibition restores BRS over this same time frame. As the BPP9α potentiation of BK actions is a rapid phenomenon, the likely mechanism for the observed delayed increase in BRS is through ACE inhibition and elevation of endogenous Ang-(1-7).     

Chymase mediates angiotensin-(1-12) metabolism in normal human hearts

Identification of angiotensin-(1-12) [Ang-(1-12)] in forming angiotensin II (Ang II) by a non-renin dependent mechanism has increased knowledge on the paracrine/autocrine mechanisms regulating cardiac expression of Ang peptides. This study now describes in humans the identity of the enzyme accounting for Ang-(1-12) metabolism in the left ventricular (LV) tissue of normal subjects. Reverse phase HPLC characterized the products of ¹²⁵I-Ang-(1-12) metabolism in plasma membranes (PMs) from human LV in the absence and presence of inhibitors for chymase (chymostatin), angiotensin-converting enzyme (ACE) 1 (lisinopril) and 2 (MLN-4760), and neprilysin (SHC39370). In the presence of the inhibitor cocktail, ≥98% ± 2% of cardiac ¹²⁵I-Ang-(1-12) remained intact, whereas exclusion of chymostatin from the inhibitor cocktail led to significant conversion of Ang-(1-12) into Ang II. In addition, chymase-mediated hydrolysis of ¹²⁵I-Ang I was higher compared with Ang-(1-12). Negligible Ang-(1-12) hydrolysis occurred by ACE, ACE2, and neprilysin. A high chymase activity was detected for both ¹²⁵I-Ang-(1-12) and ¹²⁵I-Ang I substrates. Chymase accounts for the conversion of Ang-(1-12) and Ang I to Ang II in normal human LV. These novel findings expand knowledge of the alternate mechanism by which Ang-(1-12) contributes to the production of cardiac angiotensin peptides.     

Targeting the ACE2-Ang-(1-7) pathway in cardiac fibroblasts to treat cardiac remodeling and heart failure

Fibroblasts play a pivotal role in cardiac remodeling and the development of heart failure through the deposition of extra-cellular matrix (ECM) proteins and also by affecting cardiomyocyte growth and function. The renin-angiotensin system (RAS) is a key regulator of the cardiovascular system in health and disease and many of its effects involve cardiac fibroblasts. Levels of angiotensin II (Ang II), the main effector molecule of the RAS, are elevated in the failing heart and there is a substantial body of evidence indicating that this peptide contributes to changes in cardiac structure and function which ultimately lead to progressive worsening in heart failure. A pathway involving angiotensin converting enzyme 2 (ACE2) has the capacity to break down Ang II while generating angiotensin-(1-7) (Ang-(1-7)), a heptapeptide, which in contrast to Ang II, has cardioprotective and anti-remodeling effects. Many Ang-(1-7) actions involve cardiac fibroblasts and there is information indicating that it reduces collagen production and also may protect against cardiac hypertrophy. This report describes the effects of ACE2 and Ang-(1-7) that appear to be relevant in cardiac remodeling and heart failure and explores potential therapeutic strategies designed to increase ACE2 activity and Ang-(1-7) levels to treat these conditions. This article is part of a special issue entitled ‘‘Key Signaling Molecules in Hypertrophy and Heart Failure.’’     

Angiotensin-(1-7) downregulates the angiotensin II type 1 receptor in vascular smooth muscle cells

Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT(1)) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT(1) receptor. Ang-(1-7) competed poorly for [(125)I]Ang II binding to the AT(1) receptor on vascular smooth muscle cells, with an IC(50) of 2.0 micromol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [(125)I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 micromol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [B(max) 437.7+/-261.5 fmol/mg protein in Ang-(1-7)-pretreated cells compared with 607.5+/-301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT(1) receptor. Pretreatment with the AT(1) receptor antagonist L-158,809 blocked the reduction in [(125)I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II-stimulated phospholipase C activity; however, the decrease was significant (81.2+/-6.4%, P<0.01, n=5) only at 1 micromol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT(1) receptor on vascular cells and a reduction in Ang II-stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT(1) receptor or Ang-(1-7)-mediated downregulation of the vascular AT(1) receptor.     

Angiotensin 1-9 and 1-7 release in human heart: role of cathepsin A

Human heart tissue enzymes cleave angiotensin (Ang) I to release Ang 1-9, Ang II, or Ang 1-7. In atrial homogenate preparations, cathepsin A (deamidase) is responsible for 65% of the liberated Ang 1-9. Ang 1-7 was released (88% to 100%) by a metallopeptidase, as established with peptidase inhibitors. Ang II was liberated to about equal degrees by ACE and chymase-type enzymes. Cathepsin A's presence in heart tissue was also proven because it deamidated enkephalinamide substrate by immunoprecipitation of cathepsin A with antiserum to human recombinant enzyme and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. The products of Ang I cleavage, Ang 1-9 and Ang 1-7, potentiated the effect of an ACE-resistant bradykinin analog and enhanced kinin effect on the B(2) receptor in Chinese hamster ovary cells transfected to express human ACE and B(2) (CHO/AB), and in human pulmonary arterial endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and nitric oxide (NO) release by kinin. Direct assay of NO liberation by bradykinin from endothelial cells was potentiated at 10 nmol/L concentration, 2.4-fold (Ang 1-9) and 2.1-fold (Ang 1-7); in higher concentrations, Ang 1-9 was significantly more active than Ang 1-7. Both peptides had traces of activity in the absence of bradykinin. Ang 1-9 and Ang 1-7 potentiated bradykinin action on the B(2) receptor by raising arachidonic acid and NO release at much lower concentrations than their 50% inhibition concentrations (IC(50)s) with ACE. They probably induce conformational changes in the ACE/B(2) receptor complex via interaction with ACE.