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Angiotensin-converting enzyme 2 (ACE2) is thought to act in an opposing manner to its homologue, angiotensin-converting enzyme (ACE), by inactivating the vasoconstrictor peptide angiotensin II and generating the vasodilatory fragment, angiotensin(1–7). Both ACE and ACE2 are membrane-bound ectoenzymes and may circulate in plasma as a consequence of a proteolytic shedding event. In this study, we show that ACE2 circulates in human plasma, but its activity is suppressed by the presence of an endogenous inhibitor. Partial purification of this inhibitor indicated that the inhibitor is small, hydrophilic and cationic, but not a divalent metal cation. These observations led us to develop a method for removal of the inhibitor, thus allowing detection of plasma ACE2 levels using a sensitive quenched fluorescent substrate-based assay. Using this technique, ACE2 activity measured in plasma from healthy volunteers (n= 18) ranged from 1.31 to 8.69 pmol substrate cleaved min−1 ml−1 (mean ±s.e.m., 4.44 ± 0.56 pmol min−1 ml−1). Future studies of patients with cardiovascular, renal and liver disease will determine whether plasma ACE2 is elevated in parallel with increased tissue levels observed in these conditions.
The generation of angiotensin II (Ang II) within the renin–angiotensin system (RAS) is recognized as a critical point in the regulation of cardiovascular function. The final step in the production of Ang II is catalysed by the membrane-bound ectoenzyme angiotensin converting enzyme (ACE), and inhibitors of ACE are the most widely prescribed therapy in the treatment of hypertension, heart failure and myocardial infarction. Several years ago, the homologous enzyme ACE2 was discovered (Donoghue et al. 2000; Tipnis et al. 2000). Evidence suggests that ACE2 may work in a counter-regulatory role to ACE, via the inactivation of Ang II, and the formation of the putative vasodilator, Ang(1–7) (see Ferrario et al. 2005 for review). Within the cardiovascular system, ACE2 expression is normally relatively low and restricted to endothelial cells of the coronary and renal circulation, and epithelial cells of the renal distal tubules (Donoghue et al. 2000; Hamming et al. 2004; Burrell et al. 2005). However, we and others have shown that ACE2 levels in these and other tissues increase markedly in a number of pathologies, including myocardial infarction (Ishiyama et al. 2004; Burrell et al. 2005), athlerosclerosis (Zulli et al. 2006), diabetes (Ye et al. 2004), renal disease (Lely et al. 2004) and liver cirrhosis (Paizis et al. 2005), suggesting a role for the enzyme in limiting the damage associated with RAS activation in these conditions.
It has long been recognized that in addition to its localization on endothelial cell membranes, ACE is also present in plasma (Alhenc-Gelas et al. 1983; Hooper 1991). This soluble ACE arises from proteolytic ‘shedding’ of the membrane-bound enzyme (Parkin et al. 2004). Angiotensin-converting enzyme 2 is also shed from cells in culture (Lambert et al. 2005) and has been reported to circulate in plasma in rats (Ocaranza et al. 2006; Herath et al. 2007), sheep (Shaltout et al. 2007) and humans (Rice et al. 2006), as well as in transgenic, but not wild-type, mice (Donoghue et al. 2003). Levels of circulating ACE2 are very low compared with ACE levels and, in humans, ACE2 activity could only be detected in 7.5% of a large cohort of subjects (Rice et al. 2006). Our own initial attempts to measure ACE2 activity directly in plasma from healthy individuals were unsuccessful; however, during the course of these studies, we made the observation that plasma itself potently inhibits the activity of purified recombinant ACE2, suggesting the presence of an endogenous inhibitor of ACE2. In this report, we describe both the preliminary characterization of this inhibitory substance and the quantification of plasma ACE2 activity following inhibitor depletion by a simple anion exchange step.
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Since its first description in 2000, the research effort into the biochemistry, physiology and pathophysiology of ACE2 has been steadily growing, reflecting its role in cardiovascular regulation as well as its identification as a functional severe-acute respiratory syndrome coronavirus receptor (Li et al. 2003). Evidence for an important role for the enzyme in the renin–angiotensin system is accumulating, particularly in the inactivation of Ang II and the formation of Ang(1–7). Several studies indicate that tissue levels of ACE2 are significantly elevated in a range of diseases, suggesting the enzyme may serve to ameliorate some of the detrimental effects of excessive Ang II in these conditions. In the present study, we describe the presence of an endogenous inhibitor of ACE2, which obscures detection of the enzyme by catalytic activity assays. Removal of this inhibitory substance by anion exchange chromatography allowed ACE2 activity to be readily detected in human plasma samples. There have been several previous reports of circulating ACE2 in a number of species; in all cases, levels in normal animals are low, especially in comparison with ACE (Ocaranza et al. 2006; Shaltout et al. 2007). This agrees with studies of our own in rats showing that plasma levels of ACE2 are normally very low, but are elevated in certain disease states (Herath et al. 2007; E. Velkoska, R. Dean & L. M. Burrell, unpublished observations). Interestingly, in these studies, rat plasma did not inhibit recombinant ACE2, and endogenous ACE2 activity could be readily detected (data not shown), suggesting that the inhibitory component may be restricted to humans.
Initial characterization of the inhibitory substance suggests that it is small, hydrophilic and cationic. These findings suggest that it might be a divalent cation, which can inhibit metallopeptidases, including ACE (Conroy et al. 1978), at high concentrations (Barrett et al. 1998). However, other related peptidases, such as neprilysin, thimet oligopeptidase and endothelin-converting enzyme, were at most only modestly affected. Indeed, the closest relative, ACE, is fully active in plasma, suggesting the inhibitor shows a degree of specificity for ACE2. Furthermore, most divalent metal ions circulate in plasma at levels far below those that inhibit metallopeptidases, including ACE2 (R. A. Lew, I. Hanchapola & A. I. Smith, unpublished observations), and often bound to plasma proteins. Indeed, chelation of divalent cations from the 40% acetonitrile fraction of plasma using Chelex-100 resin did not diminish inhibition of ACE2. Other possibilities for the inhibitor currently being considered include basic amino acids or small peptides, possibly even competing substrates as yet unidentified. It is also possible that the levels of this inhibitor may vary, either in a regulated fashion or in disease; modulating the level of circulating inhibitor may allow for the fine control of the activity of both plasma and endothelial ACE2.
The shedding of ACE2 from cells in culture has recently been demonstrated by Lambert et al. (2005), who suggest that the phorbol ester-stimulated cleavage of the enzyme from the cell surface is mediated by tumour necrosis factor-α (TNF-α)-converting enzyme (TACE, also called ADAM17), a widely expressed metalloprotease implicated in the cleavage/secretion of a number of membrane-bound proteins, including TNF-α and EGFR (Epidermal growth factor receptor) ligands (Blobel, 2005). Other metalloproteases which are responsible for the basal or unstimulated release of ACE2 have not yet been identified, although they probably also belong to the ADAM (adisintegrin and metalloprotease) family. The present study, together with that of Rice et al. (2006) in plasma and our previous work in urine (Warner et al. 2005; Lew et al. 2006), suggest that shedding also occurs in vivo in humans. Western blot analysis not only confirmed the very low levels of ACE2 present in plasma, undetectable without prior extraction by anion exchange and concentration, but also indicated that most of the immunoreactive ACE2 is smaller than the full-length enzyme, and thus is likely to result from proteolytic cleavage (Fig. 5). Whether this is also true in disease states such as myocardial infarction, where tissue necrosis may lead to the release of membrane-bound ACE2, remains to be investigated.
Angiotensin-converting enzyme activity was originally discovered in plasma over 50 years ago (Skeggs et al. 1954), but it is generally believed that the circulating enzyme plays little, if any, significant role in angiotensin metabolism relative to tissue ACE, which represents more than 90% of the total ACE content of the body (Ng & Vane, 1967; Xiao et al. 2004). Overall, ACE2 expression is much lower and with a more restricted distribution than its homologue, and this is reflected in normal plasma levels at least 10-fold lower than ACE (Alhenc-Gelas et al. 1983). From our initial observations regarding the presence of an ACE2 inhibitor in plasma, it appears unlikely that circulating ACE2 contributes significantly to peptide metabolism in the normal situation. However, given the marked increase in tissue levels of ACE2 as a consequence of disease, circulating levels may rise sufficiently to overcome any inhibition, with a subsequent increase in the degradation of angiotensin within the circulation. Indeed, one potential function of the inhibitor may be to counteract the effect of large increases in both plasma and membrane-bound ACE2 in certain diseases, which would otherwise lead to vasodilatation and hypotension. Although tissue ACE2 may not be affected by the inhibitor in plasma, the same may not be true of the enzyme residing on vascular endothelial cells. Further work is necessary to determine the catalytic activity of both circulating and membrane-bound ACE2 in the intact vasculature and the effect of the plasma inhibitor on the increased levels of enzyme that occur with disease.
Analysis of plasma from healthy volunteers indicates a normally low level of circulating ACE2, with measured levels falling within a fairly tight range (1.31–8.69 pmol substrate cleaved min−1 ml−1; Fig. 6). A recent family study of circulating ACE in 534 subjects reported mean ACE levels of 6.7 nmol l−1 (Rice et al. 2006). The same study also reported that plasma ACE2 activity was detectable in only 7.5% of subjects (mean, 33 pmol l−1; range, 3 to 460 pmol l−1; n= 40). These subjects tended to be older and have higher waist-to-hip ratios, blood pressure, fasting glucose levels and other cardiovascular risk factors. Furthermore, half of these subjects had at least one other family member with detectable levels of ACE2. These observations suggest that plasma ACE2 activity levels are typically low, as we have found in the present study, but may be increased in individuals with underlying cardiovascular or other diseases, or with a genetic predisposition to shedding of the enzyme.
Given our previous findings that ACE2 expression is markedly upregulated in the heart following myocardial infarction (Burrell et al. 2005) and in the cirrhotic liver (Paizis et al. 2005), we have begun to examine plasma ACE2 levels in these patients. Preliminary results suggest that plasma ACE2 is indeed elevated in these conditions, as well as in rat models of chronic liver disease (Herath et al. 2007; R. A. Lew, J. S. Lubel, C. Herath, L. M. Burrell, P. W. Angus & A. I. Smith, unpublished observations) and acute renal failure (E. Velkoska, R. Dean & L. M. Burrell, unpublished observations). Thus the appearance of ACE2 in plasma may serve as a biomarker of pathologies where increased tissue ACE2 expression is observed. Future studies aimed at determining the relationship between plasma ACE2 levels and myocardial or liver disease are currently underway.