Angiotensin-converting enzyme 2 (ACE2) shares some homology with angiotensin-converting enzyme (ACE) but is not inhibited by ACE inhibitors. The main role of ACE2 is the degradation of Ang II resulting in the formation of angiotensin 1–7 (Ang 1–7) which opposes the actions of Ang II. Increased Ang II levels are thought to upregulate ACE2 activity, and in ACE2 deficient mice Ang II levels are approximately double that of wild-type mice, whilst Ang 1–7 levels are almost undetectable. Thus, ACE2 plays a crucial role in the RAS because it opposes the actions of Ang II. Consequently, it has a beneficial role in many diseases such as hypertension, diabetes, and cardiovascular disease where its expression is decreased. Not surprisingly, current therapeutic strategies for ACE2 involve augmenting its expression using ACE2 adenoviruses, recombinant ACE2 or compounds in these diseases thereby affording some organ protection.

Abstract-ACE2, initially cloned from a human heart, is a recently described homologue of angiotensin-converting enzyme (ACE) but contains only a single enzymatic site that catalyzes the cleavage of angiotensin I to angiotensin 1-9 [Ang(1-9)] and is not inhibited by classic ACE inhibitors. It also converts angiotensin II to Ang(1-7). Although the role of ACE2 in the regulation of the renin-angiotensin system is not known, the renin-angiotensin system has been implicated in the pathogenesis of diabetic complications and in particular in diabetic nephropathy. Therefore, the aim of this study was to assess the possible involvement of this new enzyme in the kidney from diabetic Sprague-Dawley rats to compare and contrast it to ACE. ACE2 and ACE gene and protein expression were measured in the kidney after 24 weeks of streptozocin diabetes. ACE2 and ACE mRNA levels were decreased in diabetic renal tubules by Ϸ50% and were not influenced by ACE inhibitor treatment with ramipril. By immunostaining, both ACE2 and ACE protein were localized predominantly to renal tubules. In the diabetic kidney, there was reduced ACE2 protein expression that was prevented by ACE inhibitor therapy. The identification of ACE2 in the kidney, its modulation in diabetes, and the recent description that this enzyme plays a biological role in the generation and degradation of various angiotensin peptides provides a rationale to further explore the role of this enzyme in various pathophysiological states including diabetic complications. Key Words: angiotensin-converting enzyme Ⅲ diabetic nephropathy Ⅲ angiotensin Ⅲ diabetes mellitus A ngiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system (RAS). 1 It contains 2 active domains and converts angiotensin I to angiotensin II, which is a potent vasoconstrictor, growth modulator, and proinflammatory peptide. In addition, this enzyme degrades bradykinin, a vasodilator. 1 A chemically related enzyme, ACE-related carboxypeptidase, also known as ACE2, has recently been cloned and identified by 2 different groups. 2,3 ACE2 has 42% homology with ACE at the metalloprotease catalytic domain 2,3 but differs from ACE in having only one enzymatic site. In humans, ACE2 transcripts have been identified in the heart, kidney, and testis. 2,3 It has been shown that recombinant ACE2 hydrolyses the carboxy terminal leucine from angiotensin I to generate angiotensin(1-9). 2,3 ACE2 also has a high affinity for angiotensin II, 4 resulting in its degradation to the vasodilator, angiotensin(1-7). 2 Furthermore, ACE2 is not inhibited by classic ACE inhibitors such as captopril and lisinopril. 2 A rat homologue of ACE2 has been cloned (GenBank No. AF291820) that allows exploration of this metalloprotease in rodents in normal and disease states such as diabetes, in which the RAS is considered to play a pivotal role in the development of complications. 5

Angiotensin-converting enzyme (ACE) is a zinc metalloproteinase and a key regulator of the renin-angiotensin system (RAS). ACE2 is a newly described enzyme identified in rodents and humans with a more restricted distribution than ACE, and is found mainly in heart and kidney. ACE2 cleaves a single residue from angiotensin I (Ang I) to generate Ang 1-9, and degrades Ang II, the main effector of the RAS, to the vasodilator Ang 1-7. The importance of ACE2 in normal physiology and pathophysiological states is largely unknown. ACE2 might act in a counter-regulatory manner to ACE, modulating the balance between vasoconstrictors and vasodilators within the heart and kidney, and playing a significant role in regulating cardiovascular and renal function.

The renin-angiotensin system (RAS) has an important role in the endocrine pancreas. Although angiotensin II has significant effects on cell proliferation and apoptosis, the contribution of the RAS to changes in islet structure and function associated with type 2 diabetes is yet to be defined. This study examined the specific effects of RAS blockade on islet structure and function in diabetes. Thirty-six male Zucker diabetic fatty (ZDF) rats, 10 weeks of age, were randomized to receive the angiotensin-converting enzyme inhibitor perindopril (8 mg/l in drinking water; n ‫؍‬ 12), irbesartan (15 mg/kg via gavage; n ‫؍‬ 12), or no treatment (n ‫؍‬ 12) for 10 weeks. Results were compared with lean littermates (ZL) (n ‫؍‬ 12) studied concurrently. ZDF rats had increased intraislet expression of components of the RAS correlating with increased intraislet fibrosis, apoptosis, and oxidative stress. Disordered islet architecture, seen in ZDF rats, was attenuated after treatment with perindopril or irbesartan. Islet fibrogenesis was also diminished, as measured by picrosirius staining and expression of collagens I and IV. Gene expression of transforming growth factor-␤1 was increased in the ZDF pancreas (ZL, 1.0 ؎ 0.1; ZDF, 2.0 ؎ 0.3; P < 0.05) and reduced after blockade of the RAS (ZDF ؉ P, 1.3 ؎ 0.2; ZDF ؉ I, 1.5 ؎ 0.1; vs. ZDF, both P < 0.05). Improvements in structural parameters were also associated with functional improvements in first-phase insulin secretion. These findings provide a possible mechanism for the reduced incidence of new-onset diabetes that has been observed in clinical trials of RAS blockade. Diabetes 53: 989 -997, 2004

Objective: Given the important role of Ang II/Ang 1-7 in atherogenesis, we investigated the impact of ACE2 deficiency on the development of atherosclerosis. Methods and Results:C57Bl6, Ace2 knockout (KO), apolipoprotein E (ApoE) KO and ApoE/Ace2 double KO mice were followed until 30 weeks of age. Plaque accumulation was increased in ApoE/Ace2 double KO mice when compared to ApoE KO mice. This was associated with increased expression of adhesion molecules and inflammatory cytokines, including interleukin-6, monocyte chemoattractant protein-1, and vascular cell adhesion molecule-1, and an early increase in white cell adhesion across the whole aortae on dynamic flow assay. In the absence of a proatherosclerotic (ApoE KO) genotype, ACE2 deficiency was also associated with increased expression of these markers, suggesting that these differences were not an epiphenomenon. ACE inhibition prevented increases of these markers and atherogenesis in ApoE/ACE2 double KO mice. Bone marrow macrophages isolated from Ace2 KO mice showed increased proinflammatory responsiveness to lipopolysaccharide and Ang II when compared to macrophages isolated from C57Bl6 mice. Endothelial cells isolated from Ace2 KO mice also showed increased basal activation and elevated inflammatory responsiveness to TNF-␣. Similarly, selective inhibition of ACE2 with MLN-4760 also resulted in a proinflammatory phenotype with a physiological response similar to that observed with exogenous Ang II (10 ؊7 mol/L). Conclusions: Genetic

OBJECTIVE-The degradation of angiotensin (Ang) II by ACE2, leading to the formation of Ang 1-7, is an important step in the renin-angiotensin system (RAS) and one that is significantly altered in the diabetic kidney. This study examines the role of ACE2 in early renal changes associated with diabetes and the influence of ACE2 deficiency on ACE inhibitor-mediated renoprotection.RESEARCH DESIGN AND METHODS-Diabetes was induced by streptozotocin in male c57bl6 mice and ACE2 knockout (KO) mice. After 5 weeks of study, animals were randomized to receive the ACE inhibitor perindopril (2 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ). Wild-type mice were further randomized to receive the selective ACE2 inhibitor MLN-4760 (10 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) and followed for an additional 5 weeks. Markers of renal function and injury were then assessed.RESULTS-Induction of diabetes in wild-type mice was associated with a reduction in renal ACE2 expression and decreased Ang 1-7. In diabetic mice receiving MLN-4760 and in ACE2 KO mice, diabetes-associated albuminuria was enhanced, associated with an increase in blood pressure. However, renal hypertrophy and fibrogenesis were reduced in diabetic mice with ACE2 deficiency, and hyperfiltration was attenuated. Diabetic wild-type mice treated with an ACE inhibitor experienced a reduction in albuminuria and blood pressure. These responses were attenuated in both diabetic ACE2 KO mice and diabetic mice receiving MLN-4760. However, other renoprotective and antifibrotic actions of ACE inhibition in diabetes were preserved in ACE2-deficient mice.CONCLUSIONS-The expression of ACE2 is significantly modified by diabetes, which impacts both pathogenesis of kidney disease and responsiveness to RAS blockade. These data indicate that ACE2 is a complex and site-specific modulator of diabetic kidney disease.

ObjectiveWe evaluated the influence of the renin–angiotensin system (RAS) on intestinal inflammation and fibrosis.DesignCultured human colonic myofibroblast proliferation and collagen secretion were assessed following treatment with angiotensin (Ang) II and Ang (1–7), their receptor antagonists candesartan and A779, and the ACE inhibitor captopril. Circulating and intestinal RAS components were evaluated in patients with and without IBD. Disease outcomes in patients with IBD treated with ACE inhibitors and angiotensin receptor blockers (ARBs) were assessed in retrospective studies.ResultsHuman colonic myofibroblast proliferation was reduced by Ang (1–7) in a dose-dependent manner (p<0.05). Ang II marginally but not significantly increased proliferation, an effect reversed by candesartan (p<0.001). Colonic myofibroblast collagen secretion was reduced by Ang (1–7) (p<0.05) and captopril (p<0.001), and was increased by Ang II (p<0.001). Patients with IBD had higher circulating renin (mean 25.4 vs 18.6 mIU/L, p=0.026) and ACE2:ACE ratio (mean 0.92 vs 0.69, p=0.015) than controls without IBD. RAS gene transcripts and peptides were identified in healthy and diseased bowels. Colonic mucosal Masson’s trichrome staining correlated with Ang II (r=0.346, p=0.010) and inversely with ACE2 activity (r=−0.373, p=0.006). Patients with IBD who required surgery (1/37 vs 12/75, p=0.034) and hospitalisation (0/34 vs 8/68, p=0.049) over 2 years were less often treated with ACE inhibitors and ARBs than patients not requiring surgery or hospitalisation.ConclusionsThe RAS mediates fibrosis in human cell cultures, is expressed in the intestine and perturbed in intestinal inflammation, and agents targeting this system are associated with improved disease outcomes.

Repletion of ACE2's activities offers a new strategy to complement current clinical interventions in treating hypertension, renal and cardiovascular disease. In particular conditions where ACE inhibition and angiotensin receptor blockade are partially effective, the adjunctive actions of ACE2 may not only reduce clinical escape but also augment the efficacy of interventions.