Carlos M. Ferrario

Angiotensin-converting enzyme 2 is an essential regulator of heart function

Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.

Counterregulatory Actions of Angiotensin-(1-7)

Angiotensin (Ang)-(1-7) is a bioactive component of the renin-angiotensin system that is formed endogenously from either Ang I or Ang II. The first actions described for Ang-(1-7) indicated that the peptide mimicked some of the effects of Ang II, including the release of prostanoids and vasopressin. However, Ang-(1-7) is devoid of vasoconstrictor, central pressor, or thirst-stimulating actions. In fact, new findings reveal depressor, vasodilator, and antihypertensive actions that may be more apparent in hypertensive animals or humans. Thus, the accumulating evidence suggests that Ang-(1-7) may oppose the actions of Ang II either directly or by stimulation of prostaglandins and nitric oxide. These observations are significant because they may explain the effective antihypertensive action of converting enzyme inhibitors in a variety of non-renin-dependent models of experimental and genetic hypertension as well as most forms of human hypertension. In this context, studies in humans and animals showed that the antihypertensive action of converting enzyme inhibitors correlated with increases in plasma levels of Ang-(1-7). In this review, we summarize our knowledge of the mechanisms accounting for the counterregulatory actions of Ang-(1-7) and elaborate on the emerging concept that Ang-(1-7) functions as an antihypertensive peptide within the cascade of the renin-angiotensin system.

Angiotensin-(1-7) Dilates Canine Coronary Arteries Through Kinins and Nitric Oxide

Angiotensin-(1-7) [Ang-(1-7)] was recently recognized to have novel biological functions that are distinct from those of Ang II. In these studies, we determined the vasoactive effects of Ang-(1-7) together with the endothelium-dependent mediator(s) of these responses in canine coronary arteries. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers perfused with 95% O2/5% CO2 at 37 degrees C. Ang-(1-7) caused significant concentration-dependent vascular relaxation (2.73 +/- 0.58 micromol/L, EC50) of rings precontracted with the thromboxane A2 analogue U46,619. Pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (1 mol/L) abolished the vasodilator response to Ang-(1-7), whereas treatment with the cyclooxygenase inhibitor indomethacin (10 micromol/L) was without effect. The vasodilator response produced by Ang-(1-7) was blocked by 75% with the bradykinin B2 receptor antagonist Hoe 140 (1 micromol/L) or by 80% with the nonselective Ang II antagonist [Sar1,Thr8]-Ang II (1 micromol/L). In contrast, the selective AT1 or AT2 Ang II antagonists CV 11974 (1 micromol/L), and PD 123319 (1 micromol/L), respectively, were ineffective in inhibiting the Ang-(1-7)-elicited vasodilation. Furthermore, pretreatment of the coronary rings with 2 micromol/L Ang-(1-7) markedly potentiated the bradykinin response. These results suggest that Ang-(1-7) elicits coronary vasodilation that is specifically mediated by the endothelium-dependent release of nitric oxide. These responses involve a B2 bradykinin receptor and a non-AT1, non-AT2, angiotensin receptor. These data suggest that increases in circulating levels of Ang-(1-7) accompanying long-term administration of converting enzyme inhibitors or Ang II receptor blockers may contribute to the cardioprotective actions of these drugs.

Angiotensin-(1-7) Inhibits Vascular Smooth Muscle Cell Growth

Although angiotensin II (Ang II) and the heptapeptide Ang-(1-7) differ by only one amino acid, the two peptides produce different responses in vascular smooth muscle cells. We previously showed that Ang II stimulated phosphoinositide hydrolysis, whereas Ang II and Ang-(1-7) released prostaglandins. We now report that Ang II and Ang-(1-7) differentially modulate rat aortic vascular smooth muscle cell growth. Ang-(1-7) inhibited [3H]thymidine incorporation in response to stimulation by fetal bovine serum, platelet-derived growth factor, or Ang II. The reduction in serum-stimulated thymidine incorporation by Ang-(1-7) depended on the concentration of the heptapeptide over the range of 1 nmol/L to 1 mumol/L, with a maximal inhibition of 60% by 1 mumol/L Ang-(1-7). Ang-(1-7) also inhibited the serum-stimulated increase in cell number to a maximum of 77% by 1 mumol/L Ang-(1-7). The attenuation of serum-stimulated thymidine incorporation by Ang-(1-7) was unaffected by antagonists selective for angiotensin type 1 (AT1) or type 2 (AT2) receptors; however, [Sar1,Ile1]Ang II and [Sar1,Thr2]Ang II were effective antagonists, indicating that growth inhibition by Ang-(1-7) was a result of angiotensin receptor activation. In contrast, Ang II stimulated [3H]thymidine incorporation in cultured vascular smooth muscle cells over the same concentration range, with a maximal stimulation of 314% at 1 mumol/L Ang II. Ang II also increased the total number of cells (to 145% of control), suggesting that enhanced thymidine incorporation was associated with vascular smooth muscle cell proliferation. The AT1 antagonist losartan or L-158,809 but not AT2 antagonists blocked [3H]thymidine incorporation by Ang II. These results suggest that Ang-(1-7) and Ang II exhibit opposite effects on the regulation of vascular smooth muscle cell growth. The inhibition of proliferation by Ang-(1-7) appears to be mediated by a novel angiotensin receptor that is not inhibited by AT1 or AT2 receptor antagonists.

Inhibition of Early Atherogenesis by Losartan in Monkeys With Diet-Induced Hypercholesterolemia

BACKGROUND Angiotensin II may contribute to atherogenesis by facilitating the proliferative and inflammatory response to hypercholesterolemia. This study determined, in a primate model of diet-induced atherosclerosis, the effect of AT(1) blockade on fatty-streak formation, plasma lipids, and surrogate markers of vascular injury. METHODS AND RESULTS Male cynomolgus monkeys fed a diet containing 0.067 mg cholesterol/kJ for 20 weeks were given losartan (180 mg/d, n=6) or vehicle (n=8) for 6 weeks starting at week 12 of the dietary regimen. Arterial pressure, heart rate, plasma total and lipoprotein cholesterol concentrations, and lipoprotein particle sizes and subclass distributions were unaffected by treatment. Losartan caused significant (P<0.05) increases in plasma angiotensin II and angiotensin-(1-7). Compared with vehicle-treated controls, losartan reduced the extent of fatty streak in the aorta, the coronary arteries, and the carotid arteries by approximately 50% (P<0.05). A significant (P<0.05) reduction in the susceptibility of LDL to in vitro oxidation, serum levels of monocyte chemoattractant protein-1, and circulating monocyte CD11b expression were also associated with losartan treatment. In addition, serum levels of vascular cell adhesion molecule-1 and E-selectin did not change during treatment but increased after discontinuation of losartan. Serum C-reactive protein, platelet aggregability, and white cell counts were not modified by losartan. CONCLUSIONS This study demonstrates for the first time an antiatherogenic effect of AT(1) receptor blockade in nonhuman primates. Losartan inhibited fatty-streak formation through mechanisms that may include protection of LDL from oxidation and suppression of vascular monocyte activation and recruitment factors.

Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function

Angiotensin-converting enzyme 2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein that functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the COOH-terminus of its substrates. Because ACE2 efficiently hydrolyzes the potent vasoconstrictor angiotensin II to angiotensin (1–7), this has changed our overall perspective about the classical view of the renin angiotensin system in the regulation of hypertension and heart and renal function, because it represents the first example of a feedforward mechanism directed toward mitigation of the actions of angiotensin II. This paper reviews the new data regarding the biochemistry of angiotensin-(1–7)-forming enzymes and discusses key findings such as the elucidation of the regulatory mechanisms participating in the expression of ACE2 and angiotensin-(1–7) in the control of the circulation.

Effect of Angiotensin-Converting Enzyme Inhibition and Angiotensin II Receptor Blockers on Cardiac Angiotensin-Converting Enzyme 2

Background—Angiotensin-converting enzyme 2 (ACE2) has emerged as a novel regulator of cardiac function and arterial pressure by converting angiotensin II (Ang II) into the vasodilator and antitrophic heptapeptide, angiotensin-(1–7) [Ang-(1–7)]. As the only known human homolog of ACE, the demonstration that ACE2 is insensitive to blockade by ACE inhibitors prompted us to define the effect of ACE inhibition on the ACE2 gene. Methods and Results—Blood pressure, cardiac rate, and plasma and cardiac tissue levels of Ang II and Ang-(1–7), together with cardiac ACE2, neprilysin, Ang II type 1 receptor (AT1), and mas receptor mRNAs, were measured in Lewis rats 12 days after continuous administration of vehicle, lisinopril, losartan, or both drugs combined in their drinking water. Equivalent decreases in blood pressure were obtained in rats given lisinopril or losartan alone or in combination. ACE inhibitor therapy caused a 1.8-fold increase in plasma Ang-(1–7), decreased plasma Ang II, and increased cardiac ACE2 mRNA but not cardiac ACE2 activity. Losartan increased plasma levels of both Ang II and Ang-(1–7), as well as cardiac ACE2 mRNA and cardiac ACE2 activity. Combination therapy duplicated the effects found in rats medicated with lisinopril, except that cardiac ACE2 mRNA fell to values found in vehicle-treated rats. Losartan treatment but not lisinopril increased cardiac tissue levels of Ang II and Ang-(1–7), whereas none of the treatments had an effect on cardiac neprilysin mRNA. Conclusions—Selective blockade of either Ang II synthesis or activity induced increases in cardiac ACE2 gene expression and cardiac ACE2 activity, whereas the combination of losartan and lisinopril was associated with elevated cardiac ACE2 activity but not cardiac ACE2 mRNA. Although the predominant effect of ACE inhibition may result from the combined effect of reduced Ang II formation and Ang-(1–7) metabolism, the antihypertensive action of AT1 antagonists may in part be due to increased Ang II metabolism by ACE2.

A comparison of the properties and enzymatic activities of three angiotensin processing enzymes : angiotensin converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11

The discovery of angiotensin-(1-7) [Ang-(1-7)] as a bioactive Ang II fragment of the renin-angiotensin system (RAS) alters the current understanding of the enzymatic components that comprise the RAS cascade. Two neutral endopeptidases, prolyl endopeptidase (E.C. 3.4.21.26) and neutral endopeptidase 24.11 (E.C. 3.4.24.11), are capable of forming Ang-(1-7) from Ang I and have been implicated in the in vivo processing of Ang I. This makes them putative Ang processing enzymes and part of the RAS cascade. This review summarizes the physical characteristics and distribution of angiotensin converting enzyme (E.C. 3.4.15.1), a known Ang I processing enzyme, and compares its features to what is known of prolyl endopeptidase and neutral endopeptidase 24.11.

Metabolism of Angiotensin-(1–7) by Angiotensin-Converting Enzyme

Angiotensin converting enzyme (ACE) inhibitors augment circulating levels of the vasodilator peptide angiotensin-(1-7) [Ang-(1-7)] in man and animals. Increased concentrations of the peptide may contribute to the antihypertensive effects associated with ACE inhibitors. The rise in Ang-(1-7) following ACE inhibition may result from increased production of the peptide or inhibition of the metabolism of Ang-(1-7)-similar to that observed for bradykinin. To address the latter possibility, we determined whether Ang-(1-7) is a substrate for ACE in vitro. In a pulmonary membrane preparation, the ACE inhibitor lisinopril attenuated the metabolism of low concentrations of 125I-Ang-(1-7). The primary product of 125I-Ang-(1-7) metabolism was identified as Ang-(1-5). Using affinity-purified ACE from canine lung, HPLC separation and amino acid analysis revealed that ACE functioned as a dipeptidyl carboxypeptidase cleaving Ang-(1-7) to the pentapeptide Ang-(1-5). The ACE inhibitors lisinopril and enalaprilat (1 micromol/L), as well as the chelating agents EDTA, o-phenanthroline, and DTT (0.1-1 mmol/L) abolished the generation of Ang-(1-5) and did not yield other metabolic products. Ang-(1-5) was not further hydrolyzed by ACE. Kinetic analysis of the hydrolysis of Ang-(1-7) by ACE revealed a substrate affinity of 0.81 micromol/L and maximal velocity of 0.65 micromols min(-1) mg(-1). The calculated turnover constant for the peptide was 1.8 sec(-1) with a catalytic efficiency (Kcat/Km) of 2200 sec(-1) mmol/L(-1). These findings suggest that increased levels of Ang-(1-7) following ACE inhibition may be due, in part, to decreased metabolism of the peptide.

Angiotensin-(1–7) Augments Bradykinin-Induced Vasodilation by Competing With ACE and Releasing Nitric Oxide

Recent studies have shown that angiotensin-(1-7) [Ang-(1-7)] interacts with kinins and augments bradykinin (BK)-induced vasodilator responses by an unknown mechanism. In this study, we evaluated whether the potentiation of the BK-induced vasodilation by Ang-(1-7) may be attributable to inhibition of BK metabolism, release of nitric oxide, or both. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers. 125I-[Tyr0]-BK metabolism was determined in vascular rings by assessing the degradation of the peptide by high-performance liquid chromatography. Ang-(1-7) augmented the vasodilation induced by BK in a concentration-dependent manner in rings preconstricted with the thromboxane analog U46619. The EC50 of BK (2.45 +/- 0.51 nmol/L versus 0.37 +/- 0.08 nmol/L) was shifted leftward by 6.6-fold in the presence of 2 mumol/L concentration of Ang-(1-7). The response was specific for BK. since Ang-(1-7) did not augment the vasodilation induced by either acetylcholine (0.05 mumol/L) or sodium nitroprusside (0.1 mumol/L). Moreover, neither angiotensin I nor angiotensin II (Ang II) duplicated the augmented BK response of Ang-(1-7). Pretreatment of vascular rings with the nitric oxide synthase inhibitor, N omega-nitro-L-arginine (L-NA; 100 mumol/L) completely abolished the effects of Ang-(1-7) on BK-induced vasodilation whereas pretreatment with indomethacin (10 mumol/L) was without effect. The potent specific BK B2 receptor antagonist, Hoe 140. nearly abolished the BK and the Ang-(1-7) potentiated responses at 2 mumol/L, whereas at a lower concentration (20 nmol/L) Hoe 140 shifted the response curve to the right for both Ang-(1-7) and vehicle; however, the augmented response to Ang-(1-7) persisted. Preincubation of vascular rings with 20 mumol/L of the AT1 (CV11974), AT2 (PD123319), or nonselective (Sar1 Thr8-Ang II) receptor antagonists had no significant effect on the Ang-(1-7)-enhanced vasodilator response to BK. Lisinopril (2 mumol/L) significantly enhanced the BK-induced vasodilator response while at the same time it abolished the synergistic action of Ang-(1-7) on BK. In addition, pretreatment with 2 mumol/L Ang-(1-7) significantly inhibited the degradation of 125I-[Tyr0]-BK and the appearance of the BK-(1-7) and BK-(1-5) metabolites in coronary vascular rings. Ang-(1-7) inhibited purified canine angiotensin converting enzyme activity with an IC50 of 0.65 mumol/L. In conclusion. Ang-(1-7) acts as a local synergistic modulator of kinin-induced vasodilation by inhibiting angiotensin converting enzyme and releasing nitric oxide.