*3.6. Angiotensin A*/*Alamandine*/*MrgD*

Ang-A is a recently discovered angiotensin peptide detected in the plasma of patients with end-stage renal disease, where Ang-A/Ang-II ratio was found to be higher compared to healthy individuals [113]. Ang-A is an octapeptide with the sequence Ala-Arg-Val-Tyr-Ile-His-Pro-Phe, which can be produced from Ang-II by conversion of the first amino acid, aspartic acid, into alanine [113]. Ang-A can bind to AT1R and AT2R with equal a ffinity as Ang-II [114]. Intravenous and intrarenal administration of Ang A induced dose-dependent increase in blood pressure and renal vasoconstrictor responses in normotensive and spontaneously hypertensive rats [114,115]. In isolated perfused rat kidney, Ang-A induced smaller vasoconstrictive e ffects compared to Ang-II, which were inhibited using AT1R inhibitor, but not AT2R inhibitor [113,114].

In fact, the importance of Ang-A is increasing due to its junctional position in the system. Despite its vasoconstrictive and pro-proliferative actions, Ang-A is also a precursor of alamandine, a recently discovered peptide identified in rats, mice, and humans [116]. Alamandine can also be produced from Ang-(1–7) by ACE2 and was shown to produce several physiological actions that resemble those produced by Ang-(1–7) including vasodilation, antifibrotic, antihypertensive, and CNS e ffects, independent of MasR and AT2R [116]. The e ffects of alamandine were shown to be mediated through the activation of a specific receptor, the member D of Mas1-related G-protein-coupled receptor (MasDR) [116], which has been recently demonstrated to also be an alternative receptor to

Ang-(1–7) [117]. Interestingly, alamandine was shown to exert opposite effects in central nervous system where microinjection of alamandine into the rostral ventrolateral medulla of rats induced a vasopressor effect, whereas its administration into the caudal ventrolateral medulla elicited a vasodilatory effect. Of importance, similar effects were obtained after Ang-(1–7) injection [116]. On the other hand, in control but not diseased blood vessels, alamandine enhanced acetylcholine-mediated vasodilation in normal thoracic aorta and the iliac artery, whereas it reduced it in the renal artery [118]. Interestingly, these effects were absent in blood vessels from atherogenic rabbits, which also showed a reduced vasoconstrictive response toward Ang-A.

The finding of MasDR receptor has added another level of complexity into RAS, especially with regards to the anti-inflammatory Ang-(1–7) axis. This warrants further studies that may explain additional interactions and would balance between the different axes of RAS.

#### *3.7. Other Angiotensin Peptides*

Ang-(1–9) was considered for a long time as an intermediate peptide with no biological significance. However, recent evidence suggests that Ang-(1–9) can exert several effects in vivo and in vitro independent of Ang-(1–7)-mediated MasR activation, possibly through AT2R [2]. Indeed, a new study showed that Ang-(1–9) exerts beneficial cardiovascular effects via the AT2R in hypertensive rats independent of blood pressure modulation, where it ameliorated structural alterations (hypertrophy and fibrosis) and oxidative stress in the heart and aorta and improved cardiac and endothelial function [2]. These effects were inhibited by an AT2R antagonist, but not a MasR one. On the contrary, in another study on rats, Ang-(1–9) enhanced thrombosis, decreased plasma concentrations of tissue plasminogen activator (tPA), and increased the levels of its inhibitor (PAI-1) through indirect activation of AT1R [119]. These effects were reversed by selective antagonists to AT1R, but not to that of Ang-(1–7).

Ang-(3–7) is an angiotensin peptide that was shown to bind to AT4R, with lower affinity compared to Ang-IV, leading to important effects in the brain and kidney [2]. Ang-(3–7) can be produced by cleavage of Ang-(1–7), Ang-II, or Ang-IV by aminopeptidases or carboxypeptidases [2]. Administration of Ang (3–7) intracerebroventricularly (i. c. v.) significantly enhanced learning and behavioral activity in rats [120]. Co-treatment withthe ARB, losartan, only affected learning ability, without altering the behavioral activity. This suggests that Ang (3–7) is an active peptide that exerts its effects through different receptors, one of which is AT1R [120]. Moreover, Ang-(1–7) induced inhibitory effects on the energy-dependent solute transport in proximal tubules of the rat kidney [121] were shown to be mediated by the metabolism of Ang-(1–7) into Ang (3–7), by binding to AT4R [121]. Such results may raise questions about the previously described "direct" effects of certain angiotensin peptides.

#### **4. Conclusion and Future Directions**

The concept of tissue RAS could be defined as a specific combination of RAS enzymes that are locally expressed in a tissue, which results in the production of a specific quantitative and qualitative combination of peptides that can bind to their corresponding locally expressed receptors, thus leading to a locally balanced paracrine/autocrine effect that plays a role in tissue physiology and homeostasis. A change in local RAS expression will consequently lead to alterations in the balance obtained, and thus, to pathophysiological consequences (Figure 2). In this regard, studies on RAS need to be shifted from the one peptide-one pathway approach, toward a more general approach that considers the tissue-specific pathways and their respective local and systemic interactions. Indeed, the knowledge obtained from the former approach may lead to misleading conclusions that rely on the used model, with a lack of information on other pathways that may balance the effect of the pathway in question. Therefore, for a better understanding of the "real" global physiological effects of RAS, it is necessary to measure the different components of RAS in a specific tissue, under specific physiological conditions.

**Figure 2.** A specific combination of locally expressed RAS enzymes in a tissue results in the production of a specific combination of peptides that can bind to their corresponding receptors, leading to a locally balanced paracrine/autocrine effect that plays a role in tissue physiology and homeostasis. A change in local balance of RAS components will consequently lead to pathophysiological consequences.

Using transcriptomics meta-analysis, we have recently established the atlas of tissue RAS, which includes the transcriptional maps of RAS in 23 normal human tissues [7,122]. The maps provide information on the favored pathways of RAS in each tissue, but also on the co-expression of RAS genes, which may provide the basis for the discovery of potential regulatory mechanisms involved in the global expression of RAS components at the tissue level. In this regard, we have recently created the transcriptional maps of RAS in normal and atherosclerotic vascular wall showing the differences in angiotensin metabolism between both tissues [123]. Also, by analyzing the promoters of co-expressed genes, we identified potential transcription factors that could play a role in the global expression of RAS components in atheroma. Therefore, the atlas needs to be extended and studied at the protein level. In addition, RAS maps should be established from studies on each tissue under pathophysiological conditions, which will help understand the way the system is altered in each tissue under specific conditions, and thus, a better understanding of the mechanisms by which the system is involved in local tissue pathophysiology.

**Funding:** This work was supported by grants to KZ from Coopération pour l'Évaluation et le Développement de la Recherche (CEDRE), the Lebanese National Council for Scientific Research (CNRS) and Lebanese University grants. A.N. was awarded a scholarship from "La Nouvelle Société Francophone d'Athérosclérose" (NSFA).

**Conflicts of Interest:** The authors declare no conflict of interest.
