*3.1. Angiotensin-I*

Apart from Renin, several enzymes were found to cleave angiotensinogen into Ang-I, such as cathepsin D (CTSD), cathepsin G (CTSG), and tonins [25] (Figure 1). In addition, studies have shown that three main receptors can be bound and activated by renin, which are: renin-binding protein (RnBP), mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptor and the renin/prorenin receptor (R/PR) [26] (Figure 1 and Table 1). Although RnBP is known to be a renin inhibitor, the latter two are known to increase renin catalytic activity and activate intracellular signaling [20]. R/PR binding is supposed to induce full non-proteolytic activation of prorenin by a conformational change, through which prorenin active site is exposed [26]. The interaction between R/PR and prorenin is species-specific, which may explain the lack of rat prorenin activation by human R/PR [27].

The concept of local RAS was initially challenged by the fact that renin was considered the rate-limiting specific enzyme to cleave AGT into Ang-I [28]. However, renin mRNA and/or activity was detected in several extra-renal tissues, including the vascular wall [29], cardiac [30], adipose [31], and eye tissues [32]. In addition, M6P/IGFII receptor, encoded by the IGF2R gene, contribute to the uptake and activation of M6P-containing prorenin from the circulation at di fferent tissues and in a variety of cells including cardiomyocytes [33], fibroblasts, VSMCs, and ECs [26,33]. On the other hand, the P/PR was shown to be expressed and active in diverse tissues, including the kidney, vascular wall, brain, and the eye. Alterations in R/PR are associated with pathologies such as glomerulosclerosis, diabetes, hypertension, neovascularization and inflammation [26].

In fact, renin is considered the rate limiting enzyme for the generation of Ang-I in plasma, whereas in tissues, enzymes other than renin are thought to regulate Ang-I generation [34]. Of importance is CTSD, which is a ubiquitous lysosomal aspartyl protease [35] that was shown to provide an alternative angiotensin production pathway after myocardial infarction, and hence falsely increase clinical plasma renin activity determinations [36]. Cathepsin D knockdown by siRNA led to more than a two-fold reduction in intracellular and extracellular Ang-I and Ang-II production by VSMCs under both normal and high glucose concentrations [37]. Interestingly, however, silencing of tissue plasminogen activator (tPA) gene, previously associated with Ang-II production [38], moderately increased intracellular Ang-I and Ang-II levels in VSMCs only under high glucose concentrations, while strongly decreased Ang-I and Ang-II levels in the media [37]. However, a consensus on the renin-compensatory activity of CTSD has not been reached ye<sup>t</sup> due to the low specificity and e fficiency of CTSD, which has been found to only metabolize AGT under acidic conditions [39,40].

Despite the importance of this rate limiting step in RAS, little e ffort has been made to further support local Ang-I generation from renin or renin-like enzymes. Therefore, further studies should be performed to identify local mechanisms involved in angiotensinogen cleavage, either through renin recruitment from the circulation, or through local renin-independent enzymatic reactions.

#### *3.2. Angiotensin (1–12)*

One of the recent important discoveries in RAS is the identification of the Angiotensin (1–12) peptide (Ang-(1–12)), which may serve as an alternative precursor for the production of bioactive angiotensin peptides, independent from Ang-I production [41]. Ang-(1–12) was first isolated by Nagata et al. from rat small intestine, and was shown to induce constriction of aortic strips ex vivo and to raise blood pressure in rats when infused intravenously [41]. Both the vasoconstrictor and pressor responses to Ang-(1–12) were abolished by ACE inhibitors (ACEI) and AT1R blockers (ARBs), which sugges<sup>t</sup> a renin-independent pathway for angiotensin peptides production [41,42]. Despite the di fferences in amino acid sequence of rat and human Ang-(1–12), Ferrario et al. showed cardiac production of Ang-(1–12) in a rat model expressing human AGT [43].

In addition, compelling evidence suggests that Ang-(1–12) is a major source for local Ang-II production in the central nervous system. Endogenous neutralization of Ang-(1–12) using antibodies directed against the C-terminal end of Ang-(1–12) into a lateral cerebral ventricle of (mRen2)27 transgenic hypertensive rats prompted blood pressure reduction that was associated with a transient anti-dipsogenic behavior [44]. Central e ffects of Ang-(1–12) were later shown to be mediated via the Ang-II/AT1R axis in the solitary tract nucleus and hypothalamic arcuate nucleus [45–47]. Ang-(1–12) was also shown to be present abundantly in a wide range of rat organs and tissues, including heart ventricular myocytes, small intestine, spleen, kidneys, and liver [41]. Lower levels were also found in the medial layer of intracoronary arteries and vascular endothelium [48]. Despite the increase in plasma renin activity following low-salt feeding, the levels of Ang-I, Ang-II, and Ang-(1–12) in plasma and various tissues remained unchanged [49], which suggests that Ang-(1–12) metabolism is regulated in a manner that is independent of circulating renin activity.

Studies on Ang-(1–12) clearly demonstrate species-specific, tissue-specific, and condition-specific metabolism that favors one pathway or enzyme, over the others [17]. For instance, Jessup et al.

showed that Ang-(1–12) immunoreactivity was detected in both the heart and kidney of spontaneously hypertensive rats (SHR) and Wistar–Kyoto (WKY) rats. However, tissue measurements by radioimmunoassay showed higher cardiac and lower renal levels of Ang-(1–12) in SHR, compared with WKY rats [48]. It was shown that Ang-(1–12) was cleaved by ACE to generate both Ang-I and Ang-II in rat serum, independent of renin participation [50]. On the other hand, the same team showed that Ang-(1–7) and Ang-(1–4) were the main products of Ang-(1–12) metabolism in renal cortical membranes of rats, which were abolished by neprilysin inhibition [50]. In addition, myocytes of WKY rats were shown to sequester Ang-(1–12) in culture, which was mainly metabolized by ACE and membrane metallo-endopeptidase (MME) [51]. Interestingly, the uptake and metabolism were higher in cardiomyocytes obtained from SHR rats with a predominant e ffect of chymase in these cells [52]. On the other hand, chymase was shown to be the major enzyme contributing to Ang-(1–12) cleavage in an isolated heart model of cardiac ischaemia-reperfusion injury in Sprague–Dawley rats [53]. ACEI, however, but not chymostatin, inhibited circulatory Ang-(1–12) production in both SHR and WKY rats42.

Ang-(1–12) production and metabolism have raised concerns about the possible role of this peptide in mitigating the e ffects of renin and ACE inhibitors in the treatment of heart failure, which warrant further studies to identify tissue-specific RAS metabolic targets for disease treatment.
