*3.3. Angiotensin-II*

In addition to the ACE-dependent cleavage of Ang-I, Ang-II can also be produced by the direct cleavage of AGT by cathepsin G [54], tonin, and kallikrein, or through Ang-I cleavage by chymase and cathepsin G<sup>216</sup> (Figure 1 and Table 1). Of importance, chymase, a serine protease that is highly specific to the Phe8-His9 bond [17] of Ang-I, was shown to be more active than ACE in generating Ang-II in human heart [55] and diabetic kidneys [56].

In addition to AT1R (Figure 1 and Table 1), Ang-II also acts through AT2R, a seven transmembrane receptor that acts mainly through Gi and tyrosine phosphatases to exert inhibitory actions on cellular responses mediated by AT1R, mainly by inhibiting cell growth and proliferation while promoting cell di fferentiation, in addition to vasodilation and reducing blood pressure [57].

Ang-II was thought for a long time to be only a circulating peptide, exerting its e ffects through endocrine mechanisms. However, many studies identified Ang-II in several tissues and showed that it was produced locally independent of systemic RAS. The first demonstration of tissue Ang-II was in the arterial wall in sheep in 1980 [58]. Studies quantifying tissue Ang-II synthesis, using radiolabeled angiotensin, revealed that Ang-II in the heart, kidney, and adrenal gland [59,60] almost completely originates from local synthesis, both under normal and pathological conditions [60,61]. ACE was shown to be expressed in multiple tissues, including vascular endothelium, renal proximal tubular endothelium, heart, lung, small intestine, colon, activated macrophages, and several regions of the brain [62], where physiologic e ffects of ACE are the result of tissue rather than circulating ACE activity [63].

ACE is generally considered the main Ang-II-forming enzyme in the circulation. However, in tissues, various serine proteases were shown to play a role in Ang-II generation [53]. Not only chymase, but also trypsin [64] and kallikrein [65] serine proteases were shown to generate Ang-II in vitro and in vivo in ischemic dog hearts, ischemic human hearts, and even in normal healthy individuals during exercise [66]. In fact, chymase has been a focus of interest because of its specificity and potency in the human cardiovascular system [55,67].

Ang-II may exert local paracrine or autocrine e ffects through its locally expressed AT1 and AT2 receptors (Figure 1 and Table 1). AT1R was found to be expressed in several adult tissues, including blood vessels, heart, kidney, adrenal glands, and liver [2]. On the other hand, AT2R is mainly expressed in fetal tissue and decreases through fetal development [2] to be restricted to certain tissues, mainly the heart, vessels, brainstem, liver, and kidney [68]. At the tissue level, AT1R and AT2R exert opposite e ffects; therefore, the final local e ffects of Ang-II are defined by the combined net result obtained from the activation of both receptors. For instance, AT1R induces vasoconstriction and sodium retention in the kidney whereas AT2R promotes vasodilation and natriuresis [69]. On the other hand, in the gastrointestinal tract, AT2R opposes the actions of AT1R in sodium and water absorption, which contributes to the regulation of the finely tuned sodium transport in this tissue [70]. In general, AT2R stimulates protein dephosphorylation, which counterbalances protein phosphorylation induced by AT1R, thus, a ffecting the signaling pathways inside the cell, leading mainly to opposite cellular actions [69]. Despite this general "antagonistic" view of AT2R, certain studies on cardiac myocytes showed that its overexpression may complement, rather than antagonize, the AT1R e ffects in cellular hypertrophy [71,72]. Similarly, AT1R and AT2R synergistically act to induce adipogenesis and lipid storage in adipose tissue, wherein AT1R inhibit lipolysis, while AT2R induces the expression of lipogenic enzymes [73]. Interestingly, this adds a new level of complexity to RAS in which the effects of the system depend not only on the "inter-molecular" balance between the antagonistic arms, but also on the "intra-molecular" balance of the levels of the same molecule in certain cells under specific conditions.

Ang-II is the most studied pathway in RAS and additional research on it would open new avenues in understanding the complexity of the system and inter-pathway interactions.
