**7. Angiotensin-converting enzyme 2**

ACE2 is a membrane-bound monocarboxypeptidase [EC 3.4.17.23; 120 kDa] that converts Ang II directly to Ang-(1-7) (**Figure 1**). ACE2 was initially characterized as a homolog to ACE and exhibits approximately ~40% homology with ACE [60–62]. ACE2 was initially reported to cleave Ang I not to Ang II, but to the nonapeptide Ang-(1-9) [63]. Subsequent studies found that Ang II exhibits far better kinetic values for ACE2 that would argue that the endogenous substrate is Ang II, not Ang I [64]. Among a number of peptide substrates (>100) that were screened for ACE2, Vickers et al. reported that only apelin 13 exhibited comparable kinetic values to that of Ang II [65]. In the murine heart, Ang II was primarily converted to Ang-(1-7) by ACE2 and that in the presence of the ACE2 inhibitor MLN-4760 or in ACE2 null mice there was essentially no metabolism of Ang II in the heart [66]. By contrast, under identical kinetic conditions, Ang I was primarily converted to Ang-(1-9) by carboxypeptidase A and not ACE2 in both the wild-type and ACE2 knockout mice.

In comparison to ACE, the circulating levels of ACE2 are typically quite low. Whether this reflects a reduced degree of shedding, lower vascular expression or both is not presently clear. Rice et al. reported that the molar concentration of ACE in human serum averaged 7 nM in over 500 subjects while ACE2 content was *200-fold lower* (33 pM) and was detectable in <10% of the patient population [67]. Circulating NEP content (290 pM) was also lower than ACE and evident in <30% of these patients [67]. Serum and urinary ACE2 activities are elevated in diabetes, heart failure, and hypertension [68–70]. Circulating ACE2 activity increased approximately threefold in the diabetic hypertensive mRen2.Lewis rat; however, serum ACE activity also increased in the diabetic rats. As measured under identical kinetic conditions, serum ACE was far higher than ACE2 suggesting that the capacity to generate Ang II (or metabolize Ang- (1-7) and bradykinin) remains greater than the capability to form Ang-(1-7) from Ang II [68].

Similar to ACE, ACE2 has a potentially significant role in the RAS pathway as a single catalytic step metabolizes Ang II to attenuate the Ang II-AT1 receptor pathway, and generate Ang-(1-7) that would stimulate the Ang-(1-7)-AT7/Mas receptor axis (**Figure 1**) [71, 72]. Indeed, ACE2 deficient animals exhibit exaggerated responses to Ang II or under chronic conditions of an activated RAS [73–76]. In turn, overexpression of ACE2 or administration of the soluble form of the peptidase, which retains full enzymatic activity, attenuates the Ang II-dependent increase in blood pressure and indices of target organ injury [77–85]. In regard to the benefits of an activated ACE2 pathway, several compounds have been identified that may act as allosteric activators of ACE2 including xanthenone (XNT) and diminazene aceturate (DIZE) to promote a higher ratio of Ang-(1-7) to Ang II [86]. Chronic treatment with DIZE ameliorated the extent of pulmonary hypertensin and fibrosis, renal tissue injury, and myocardial infarction consistent with enhanced levels of Ang-(1-7) and a reduction in Ang II [84, 87–90]. Interestingly, DIZE treatment was also associated with increased mRNA levels of ACE2 suggesting that DIZE may exhibit actions apart from the direct activation of the peptidase [87, 91]. However, it should be noted that the effects of DIZE on ACE2 activity or expression have not been confirmed by others. Haber et al. [92] found no effect of DIZE on soluble ACE2 activity or an influence on Ang II-dependent hypertension using similar doses of DIZE as previously reported. Velkosa et al. [93] also found no direct effect of various concentrations of DIZE on renal ACE2 activity and slightly increased renal ACE2 activity in the 5/6 nephrectomized rat following a 2-week administration. Of particular interest, the Velkosa study reported that DIZE normalized the marked increase in renal ACE activity suggesting that ACE may be a more relevant target than ACE2 [93]. DIZE also failed to increase ACE2 activity or enhance the local vascular actions of Ang-(1-7) in the isolated pig coronary arteries [94].

Conceptually, the use of ACE2 as a therapeutic agent to chronically alter the balance of Ang II and Ang-(1-7) is challenging. ACE activity in the circulation and the vasculature surface is significant with a very high capacity to generate Ang II. This reflects not simply the abundance of ACE but the significant capability of angiotensinogen and renin to generate the ACE substrate Ang I. Moreover, reduced Ang II levels by exogenous ACE2 should stimulate the generation of Ang II that reflects the activation of negative feedback mechanisms on renin. Therefore, it is difficult to conceive sufficiently high levels of ACE2 that can be achieved to chronically reduce Ang II *and* increase Ang-(1-7) except with the possible addition of an ACE inhibitor. In this case, supplementation of ACE2 may degrade residual levels of Ang II and the circulating levels of Ang-(1-7) may be augmented, particularly as the Ang-(1-7)-degrading pathway in the circulation is attenuated by the ACE inhibitor.
