The classic pathway and the novel components of the renin–angiotensin–aldosterone system (RAAS)

In recent decades, cardiovascular disease has been considered the main cause of morbidity and mortality worldwide. Hypertension is a critical risk factor for these diseases, which include coronary and peripheral arterial disease, stroke and heart failure.1 One of the major regulatory mechanisms of cardiovascular homeostasis is the RAAS.2, 3 The classic pathway involves a two-step enzymatic pathway (Figure 1). First, the aspartyl protease renin, which is primarily released by the kidneys, cleaves a hepatic protein, angiotensinogen, to angiotensin I (Ang I).2, 3 The second step involves hydrolysis of Ang I by angiotensin-converting enzyme (ACE), resulting in the production of the bioactive octapeptide angiotensin II (Ang II), which is a potent vasoconstrictor and stimulates the release of aldosterone from the adrenal cortex.2, 3, 4, 5 Moreover, ACE inactivates the vasodilator bradykinin by degradation of the peptide.2

Figure 1
figure 1

Classic pathway and new components of the RAAS. Three main enzymes are involved in the generation of active angiotensin peptides: First, renin cleaves angiotensinogen into angiotensin I (Ang I). The second step involves hydrolysis of Ang I by angiotensin-converting enzyme (ACE), resulting in the production of the bioactive octapeptide angiotensin II (Ang II), which interacts with angiotensin type I (AT1-R) and angiotensin type II (AT2-R) receptors. Third, ACE2 catalyzes the conversion of Ang II to Ang-(1–7), which mediates its effects by interaction with the G-protein-coupled receptor Mas.

The discovery of angiotensin-(1–7) (Ang-(1–7)) by Santos et al.6 and the subsequent cloning of angiotensin-converting enzyme 2 (ACE2)7, 8 shed new light on angiotensin metabolism and the regulation of the RAAS. ACE2, a zinc metalloprotease with carboxypeptidase activity, catalyzes the conversion of Ang I to the non-apeptide Ang-(1–9) or the conversion of Ang II to Ang-(1–7) by the removal of a single carboxy-terminal amino acid (phenylalanine; Figure 1).8 This heptapeptide can also be formed from Ang I by the action of neprilysin (also known as neutral endopeptidase 24.11).9 It has been suggested that Ang-(1–7) mediates its effects by interacting with the G-protein-coupled receptor Mas,10 a prototypic seven-transmembrane domain receptor (Figure 1), which is predominantly expressed in the brain and testis11 but is also found in the kidney, heart and vessels.11, 12, 13 Moreover, several studies have shown that the interaction of Ang-(1–7) with Mas evokes numerous protective cardiovascular actions, such as nitric oxide (NO)14, 15 release, Akt phosphorylation16 and vasodilation (Figure 2).17 Nevertheless, other studies indicate that Ang-(1–7) may function through angiotensin type 2 receptor18 and that Mas can antagonize the actions of the angiotensin type 1 receptor.19, 20

Figure 2
figure 2figure 2

Role of ACE2–Ang-(1–7)–Mas axis in vascular function and redox signaling in vessels. Under normal physiological conditions (a), NO exerts pleiotropic effects in the regulation of vascular function. The balance between levels of O2 and released NO has a critical role in the maintenance of normal endothelial function. However, in pathological conditions (b), excessive O2, mainly produced by NAD(P)H oxidase, stimulates vasoconstriction and inflammation, resulting in endothelial dysfunction, mainly through the reduction of NO bioavailability and the imbalance between ROS and antioxidant capacity. Thus, the classic RAAS is a potent prooxidant system in vessels, causing endothelial dysfunction. The ACE2–Ang-(1–7)–Mas axis counteracts these effects. For more details, see text. AT1-R, angiotensin type I receptor; Cat, catalase; COX, cyclooxygenase; GPX, glutathione peroxidase; Ec-SOD; extracellular-superoxide dismutase; LPO, lipid peroxidation; MPO, myeloperoxidase; XO, xanthine oxidase.

The local activity of ACE2 determines the relative levels of the vasoconstrictor and pro-oxidative peptide Ang II and its vasodilatory and antioxidative metabolite Ang-(1–7) at the corresponding receptors (Figure 2).21, 22 There is now a very large body of evidence showing that the newly discovered angiotensin system, ACE2–Ang-(1–7)–Mas, is pivotal for physiological homeostasis.23

Endothelial dysfunction and oxidative stress in the etiology of cardiovascular disease

Strategically located between the circulating blood and the other vascular layers, the endothelium is a sensor of hemodynamic changes and is known to have a central role in vascular homeostasis. However, for a long time, this layer was seen ‘only’ as a cluster of cells that separates the circulating blood from the other layers. Exactly 30 years ago, a seminal paper revised this foundational idea. The seminal experiments of Furchgott and Zawadzki24 first demonstrated the existence of endothelium-derived relaxing factor, which was subsequently identified as nitric oxide (NO; nitrogen monoxide).25 In the endothelium, this free radical is produced from L-arginine by endothelial nitric oxide synthase (eNOS) in the presence of cofactors, mainly tetrahydrobiopterin (BH4) (Figure 2).26, 27, 28, 29 Interestingly, Landmesser et al.,30 in an elegant study, showed that oxidation of this biopterin induces eNOS uncoupling. In this structural state, the enzyme is an important source of reactive oxygen species (ROS).31, 32 Thus, in the absence of sufficient levels of cofactors, such as BH4 for enzymatic catalysis, eNOS may reduce molecular oxygen rather than transfer electrons to the substrate L-arginine, which results in the generation of superoxide anion (O2).30, 31, 32 Interestingly, BH4 is believed to be deficient in conditions associated with altered endothelial function,30 such as hypercholesterolemia,33 diabetes,34 high blood pressure35 and cigarette smoking.36, 37

Both NO and O2 are radicals. These molecules react rapidly with each other to generate peroxynitrite, ONOO38 (Figure 2), which is an important lipid peroxidation mediator. Peroxynitrite oxidizes low-density lipoprotein, a pivotal event for atherogenesis.39 However, it is well established that NO exerts pleiotropic effects in the regulation of vascular function. Under normal physiological conditions, this molecule serves as a vasodilator and platelet inhibitor, exhibiting antiproliferative,40 antithrombotic,41 antiatherogenic42 and antioxidant capacities.43 Thus, the balance between levels of O2 and released NO has a critical role in the maintenance of normal endothelial function.39, 43, 44

In physiological conditions, a certain amount of intracellular O2 is required for normal redox homeostasis in the vessel wall.45, 46, 47 Therefore, this reactive species is an important scavenger of the free radical signaling performed by NO.48 However, in pathological conditions, the extracellular increase in O2 decreases the bioavailability of NO, reducing its diffusion into the vascular smooth muscle.49 Indeed, in the endothelium, excessive O2 stimulates vasoconstriction and inflammation,46 which mutually reinforce each other, resulting in endothelial dysfunction, mainly through the reduction of NO bioavailability and the imbalance between ROS and antioxidant capacity.48 Therefore, a vicious cycle pivotal in numerous disease processes is established.

According to Dröge,45 the term redox signaling is used to describe a regulatory process in which the signal is delivered through reduction–oxidation (redox) chemistry. The cellular concentration of ROS is determined by the balance between producing sources and the rate of clearance by antioxidant compounds and enzymes. Direct ROS scavenging antioxidant enzymes include superoxide dismutase, glutathione peroxidase and catalase.44, 45, 46, 47 Superoxide dismutase represents the first defense against O2 and converts superoxide radicals to hydrogen peroxide (H2O2); catalase and glutathione peroxidase, are two different but kinetically complementary enzymes that can eliminate H2O2 by breaking it down directly to H2O and O2 (Figure 2).45, 46 On the other hand, oxidative stress emerges when the production of ROS, most notably O2, exceeds the quenching antioxidant capacity of the protective systems of the cell.44, 45, 46, 47

Recent evidence suggests that oxidative stress, which is elevated in cardiovascular disease, contributes to endothelial dysfunction. This disorder is a common feature of hypertension and results from the imbalance in the release of endothelium-derived relaxing factors, mainly NO32, 44, 45, 46 and endothelium-derived contracting factors, such as cyclooxygenase-derived constrictors, endothelins and O248, 49, 50 (Figure 2). Indeed, clinical studies show that ROS has a significant role in several cardiovascular and metabolic diseases.27, 51, 52, 53 On the other hand, impaired endothelium-mediated vasodilation in hypertension has been linked to decreased NO bioavailability. This may be secondary to decreased synthesis or to increased NO degradation because of its interaction of NO with O2 to form ONOO.38 Thus, the status of the redox system and NO bioavailability are key factors controlling the influence of oxidative stress on cardiovascular function.

Accumulating evidence suggests that NAD(P)H oxidase is a major source of ROS, most notably O2, in both endothelial and vascular smooth muscle cells.54, 55, 56, 57, 58, 59, 60 Ang II promotes ROS production by the activation of membrane-bound NAD(P)H oxidase.54, 57, 59 This activation was first demonstrated by Griendling et al.59 in vascular smooth muscle cells. The H2O2 generated from the O2 produced by NAD(P)H oxidase is involved in vasoconstriction and vascular hypertrophy57 (Figure 2). Thus, the classic RAAS is a potent prooxidant system in vessels causing endothelial dysfunction and consequently cardiovascular disease.

Redox-sensitive signaling by the ACE2–Ang-(1–7)–Mas axis

In 1992, Santos et al.61 demonstrated for the first time that Ang-(1–7) is present in vessels. After this first report, vasodilatory actions of the heptapeptide have been demonstrated in animals in several vascular beds.21, 22, 62 In addition, numerous studies showed that Ang-(1–7) functions mostly as an antithrombotic,63, 64 antiproliferative65, 66 and antioxidative agent.67 The mechanisms of action in the vasculature has not yet been well established. However, several studies have focused on control of the NO and O2 balance, which regulates cardiovascular function.

We have recently demonstrated a reduction in superoxide dismutase and catalase activity in Mas-deficient mice from two different genetic backgrounds, demonstrating impaired antioxidant properties in these animals.67 In addition, TBARS levels, which are most widely used as lipid peroxidation markers, were increased in aorta of Mas-null mice on the FVB/N genetic background. Accordingly, isoprostanes, other sensitive and stable oxidative stress biomarkers, are significantly upregulated in the urine of this strain. Moreover, ROS levels are increased in both FVB/N- and C57Bl/6 Mas-deficient animals,67, 68 but this difference is more pronounced in mice on the FVB/N background. Consequently, these Mas-knockout animals exhibit impaired in vivo endothelial function and increased blood pressure. One potential mechanism accounting for these observations is a decrease in NO bioavailability. To find out if more O2 is produced by NAD(P)H oxidases, the expression level of the main catalytic subunit of this enzyme, gp91phox (NOX2), was quantified and found to be increased in Mas-null mice. Furthermore, tempol, a stable superoxide dismutase mimetic, reduced blood pressure in Mas−/− mice to levels comparable with those observed in wild-type animals.67 Taken together, these data suggested a causative link between ROS/NO imbalance and endothelial dysfunction in Mas-deficient mice.

To confirm these findings, our group developed a transgenic model overexpressing human ACE2 in vascular smooth muscle cells, using spontaneously hypertensive stroke-prone rats as a background strain.69 These transgenic rats showed improved endothelial function and decreased vasoconstriction response to Ang II. One potential mechanism underlying these actions involves a reduction in oxidative stress by a decrease in Ang II and/or an increase in Ang-(1–7). Nevertheless, additional studies are needed to clarify this issue.

Benter et al.70 showed that streptozotocin-treated spontaneously hypertensive rats (diabetic spontaneously hypertensive rats) chronically treated with Ang-(1–7) showed improved endothelial dysfunction of the renal artery, mediated primarily by the reduction of NAD(P)H-mediated oxidative stress, which resulted in improved renal function. In contrast, another study suggests that Ang-(1–7) stimulates oxidative stress in rat kidney.71 Thus, the role of the ACE2–Ang-(1–7)–Mas axis and oxidative stress in kidney pathophysiology is still controversial.

It has been demonstrated that the vascular actions of the ACE2–Ang-(1–7)–Mas axis could be mediated by different pathways, such as antagonism of angiotensin type 1 receptor,19, 20 by vasoactive peptides and increases in the release and/or bioavailability of NO. Indeed, a bradykinin-potentiating effect of Ang-(1–7) was reported in certain rodent models.72, 73 This peptide evokes endothelium-dependent relaxation in several vascular beds.74, 75, 76 In addition, Ang-(1–7) improves endothelial function in vivo,77 induces coronary vasodilation by NO release72, 78 and activates eNOS in Chinese hamster ovary cells.16 Carvalho et al.79 showed that short-term infusions of AVE 0991, a Mas agonist, increase the hypotensive effect of bradykinin in normotensive rats; one plausible mechanism for this effect involves an increase in NO levels.

In atherosclerosis, ACE2 protects endothelial cells from Ang II-mediated macrophage infiltration and oxidative stress in an Ang-(1–7)-dependent manner.80, 81 Corroborating these findings, Tesanovic et al.82 recently reported that long-term Ang-(1–7) treatment induces atheroprotective effects in ApoE−/− mice, the most widely used animal model for atherosclerosis. According to the authors, the improvement in endothelial function resulted from an increase in NO bioavailability, as the heptapeptide increased NOS expression and decreased O2 production. Concurrently, it was recently shown that genetic deletion of ACE2 significantly increases83 plaque formation in atherosclerotic animals, which is decreased by targeted vascular ACE2 overexpression.80, 84 Consequently, these actions of the ACE2–Ang-(1–7)–Mas axis may be exploited in the treatment of atherosclerosis and other vascular diseases.

As mentioned above, the activation of NAD(P)H oxidase has a key role in the intracellular signaling that leads to hypertension-induced oxidative stress and endothelial dysfunction.85, 86, 87, 88 Sampaio et al.89 showed that the activation of this enzymatic complex by Ang II is attenuated by Ang-(1–7) in human endothelial cells. The authors suggest that the principal mechanism involves the interaction of this peptide with Mas, leading to the activation of AKT. This kinase mediates the dual phosphorylation of the Ser1177/Thr495 sites in eNOS and thereby stimulates eNOS activity and increases NO release.

Current findings also suggest that the ACE2–Ang-(1–7)–Mas axis may participate in pathomechanisms of cerebrovascular disorders. Increased levels of ACE2 and Ang-(1–7) were found in the cerebrospinal fluid of multiple sclerosis patients.90 Moreover, Lu et al.91 showed that chronic central administration of Ang-(1–7) reduces the infarct area and provides cerebroprotection following focal cerebral ischemia/reperfusion in rats. The authors suggest that the heptapeptide modulates in vivo NO release and eNOS expression in this stroke model. Corroborating these findings, another group showed in transgenic mice that the overexpression of ACE2 was associated with significant upregulation of neuronal NOS and eNOS in the brainstem.92 In addition, ACE2 overexpression in the brain prevented the Ang II-mediated decrease in NOS expression in regions involved in blood pressure regulation.93 Zhang et al.94 reported that the central administration of Ang-(1–7) stimulates NO release and upregulates eNOS in a rat model of cerebral ischemia/reperfusion, further corroborating the antioxidant actions of ACE2 in the brain.

On the basis of above findings, there are at least three potential mechanisms by which ACE2 may exert protective cardiovascular effects via the reduction and/or prevention of oxidative stress: first, Ang II is cleaved to Ang-(1–7) by ACE2, thereby attenuating Ang II-induced actions; second, Ang-(1–7) may reduce the effects of Ang II by downregulating the signaling of angiotensin type 1 receptor; and third, the production of NO is potently induced by Ang-(1–7), which might protect against endothelial dysfunction and oxidative stress (Figure 2).3, 84, 85, 86, 87, 88, 89

Conclusions

In summary, Ang-(1–7) is generated in the vascular wall and other tissues from Ang II by ACE2, resulting in a decrease in Ang II levels and an increase in Ang-(1–7) concentrations in the vessels. Thus, the ACE2–Ang-(1–7)–Mas axis counterregulates the classic RAAS in tissues responsible for blood pressure regulation and cardiovascular homeostasis. It decreases the production of ROS and increases endothelial NO synthesis, improving the antioxidant capacity of cardiovascular tissues. This leads to an improvement of endothelial function and, consequently, to a decrease in arterial stiffness and, therefore, beneficially alters overall arterial function.

The net cardiovascular effect of angiotensin peptides in the vascular wall depends on the relative expression of classic and novel components of the RAAS in vascular layers, as well as their effects on the redox balance. As the ACE2–Ang-(1–7)–Mas axis exerts primarily beneficial effects in the vasculature, it may become an important therapeutic target for cardiovascular protection.