Journal of the American College of Cardiology Vol. 37, No. 4, 2001 © 2001 by the American College of Cardiology ISSN 0735-1097/01/$20.00 Published by Elsevier Science Inc. PII S0735-1097(01)01111-1 Converting Enzyme (ACE) and Non-ACE Dependent Angiotensin II Generation in Resistance Arteries From Patients With Heart Failure and Coronary Heart Disease Mark C. Petrie, BSC, MB, CHB, MRCP,* Neal Padmanabhan, MA, BM, BCH, MRCP,† John E. McDonald, BSC, MB, CHB, MRCP,*† Chris Hillier, BSC,PHD,* John M. C. Connell, MD, FRCP,† John J. V. McMurray, BSC, MD, FRCP, FESC, FACC* Glasgow, United Kingdom

OBJECTIVES We sought to demonstrate non-angiotensin converting enzyme (ACE) dependent angioten- sin II (AII) generating pathways in resistance arteries from patients with chronic heart failure (CHF). BACKGROUND Non-ACE dependent AII generation occurs in resistance arteries from normal volunteers. Inhibition of non-ACE dependent AII generation may have therapeutic potential in CHF. METHODS Resistance arteries were dissected from gluteal biopsies from patients with coronary heart disease (CHD) and preserved left ventricular function and from patients with CHF. Using wire myography, concentration response curves to angiotensin I (AI) and AII were constructed in the presence of 1) vehicle, 2) chymostatin [an inhibitor of chymase], 3) enalaprilat, and 4) the combination of chymostatin and enalaprilat. RESULTS In resistance arteries from patients with CHD, the vasoconstrictor response to AI was not inhibited by either inhibitor alone (chymostatin [p Ն 0.05] or enalaprilat [p Ն 0.05]) but was significantly inhibited by the combination (p Ͻ 0.001). In arteries from patients with CHF, AI responses were inhibited by enalaprilat (p Ͻ 0.05) but not by chymostatin alone (p Ͼ 0.05). The combination of chymostatin and enalaprilat markedly inhibited the response to AI (p Ͻ 0.001) to a greater degree than enalaprilat alone (p Յ 0.01). CONCLUSIONS Non-ACE dependent AII generating pathways exist in resistance arteries from patients with both CHF and CHD. In resistance arteries from patients with CHD, inhibition of either the ACE or chymase pathway alone has no effect on AII generation, and both pathways must be blocked before the vasoconstrictor action of AI is inhibited. In CHF, blockade of ACE results in marked inhibition of responses to AI, but this is enhanced by coinhibition of chymase. These studies suggest that full suppression of the -angiotensin system cannot be achieved by ACE inhibition alone and provide a rationale for developing future therapeutic strategies. (J Am Coll Cardiol 2001;37:1056–61) © 2001 by the American College of Cardiology

The traditional view that angiotensin II (AII) formation is has recently been demonstrated by Voors et al. (5) in large solely dependent on angiotensin converting enzyme (ACE) arteries and by our group in small resistance arteries from has recently been challenged. Non-ACE dependent conver- normal human volunteers (6). sion of angiotensin I (AI) to AII has been demonstrated in Demonstration of this dual pathway for AII generation homogenates of human myocardial tissue (1,2). In vitro, has important potential implications for the treatment of a non-ACE dependent conversion of AI to AII is brought number of cardiovascular diseases, especially chronic heart about by one or more serine proteases. The most important failure (CHF). Blockade of the renin-angiotensin- of these is thought to be chymase (3,4) as non-ACE aldosterone system (RAAS) improves symptoms and sur- mediated AII formation is substantially blocked by chymase vival in heart failure (7,8), and greater inhibition brings inhibitors such as chymostatin (2). That these non-ACE about greater benefit (9). The current approach to RAAS pathways are functionally important in human blood vessels interruption is ACE inhibition. However, the existence of a dual pathway means that that AII generation might persist in CHF, despite ACE inhibition, and raises the possibility From the *Clinical Research Initiative in Heart Failure, University of Glasgow, Glasgow, Scotland; and the †Medical Research Council Blood Pressure Group, that either the syndrome itself, or ACE inhibition, might Department of Medicine and Therapeutics, Western Infirmary, Glasgow, Scotland. also up-regulate the alternative pathway. It is known that Dr. Petrie was the recipient of a British Heart Foundation Junior Research Fellowship treatment of CHF with an ACE inhibitor does not result in (FS/97031:1997), and Dr. Padmanabhan was the recipient of a Wellcome Trust Junior Research Fellowship. Also supported by the Medical Research Council long-term suppression of AII plasma levels (10). Further- (Programme Grants held by J.J.V.M. and J.M.C.C.) and the Chief Scientist Office of more, in one study of patients with CHF, deterioration in the Scottish Executive (project grant held jointly by J.J.V.M., J.M.C.C, N.P. and left ventricular (LV) function occurred despite ACE inhi- M.C.P.). Manuscript received August 4, 1999; revised manuscript received November 8, bition, and this was associated with plasma concentrations 2000, accepted December 13, 2000. of AII that not only failed to show suppression but were JACC Vol. 37, No. 4, 2001 Petrie et al. 1057 March 15, 2001:1056–61 ACE and Non-ACE AII Generation in CHF

four-channel wire myograph (J.P. Trading, Aarhus, Den- Abbreviations and Acronyms mark). ACE ϭ angiotensin converting enzyme Experimental protocol. BLOOD SAMPLING, BIOPSY PROCE- ϭ ACh acetylcholine DURE AND ARTERY PREPARATION. After 15 min supine ϭ AI angiotensin I rest, blood was drawn from a cannula in an antecubital vein AII ϭ angiotensin II BK ϭ bradykinin for estimation of blood chemistry and serum cholesterol. CHD ϭ coronary heart disease Subcutaneous gluteal biopsies were then obtained from each CHF ϭ chronic heart failure patient under local anesthesia (1% lidocaine) by the method KPSS ϭ Kreb’s solution with KCl substituted for NaCl previously described (12). Dissected tissue was placed im- on an equimolar basis mediately into cold 0.9% NaCl and then transferred to cold LV ϭ left ventricle, left ventricular LVEF ϭ left ventricular ejection fraction Kreb’s solution (composition in mM: NaCl 118.4, KCl 4.7, NE ϭ norepinephrine MgSO4.H2O 1.2, KH2PO4 1.2. Na HCO3 24.9, CaCl2 RAAS ϭ renin-angiotensin-aldosterone system 2.5, glucose 11.1, EDTA 0.023, which gives a pH of 7.4

when gassed with a 5% CO2/95% O2 mixture). Where possible, four resistance arteries approximately 2 mm in elevated compared with those found in age and gender- length were dissected free of fat. Dissected arteries were matched controls without CHF (11). stored in Kreb’s solution overnight at 4°C. Approximately ␮ The objective of this study was to determine whether or 24 h after the biopsy they were mounted on two 40 m not non-ACE dependent AI to AII conversion occurs in the diameter stainless steel wires in a four-channel myograph in resistance arteries of patients with CHF who receive long- which the wires are attached to a force transducer and term treatment with ACE inhibitors. We also studied micrometer, respectively. The temperature was raised to whether or not non-ACE dependent AII generation occurs 37°C, and a gas mixture (composition above) was bubbled in in resistance arteries from patients with coronary heart for the duration of the experiment. Dissection of resistance disease (CHD) and preserved ventricular function who have arteries from gluteal biopsies and myography protocols were not been treated with ACE inhibitors. performed by an operator who was blind to the patient type (i.e., CHF or CHD). METHODS Myography protocol: ACE and chymase inhibition. Af- ter a rest period of 30 min, each artery was stretched at Patients. All patients with renal failure (creatinine Ͼ200 1-min intervals to determine the passive exponential wall ␮mol/l) and diabetes mellitus were excluded. Written in- tension-internal circumference (L) relationship. From the formed consent was obtained from each patient, and all Laplace equation, where P ϭ T/r (P is the effective pressure, protocols were approved by the local committee on medical T is the wall tension and r is the internal radius), the

ethics. equivalent circumference (L100) for a transmural pressure of Patients with CHF. Ambulatory patients with New York 100 mm Hg was calculated for each vessel by an iterative Heart Association class II/III CHF were studied. All were computer method. Each vessel was then set to the normal- Ͼ ϭ ϫ ␲ on long-term ( 3 months) ACE inhibitor and diuretic ized internal diameter, L1 0.9 L100/ , at which treatment. The etiology of CHF was CHD in all cases, and contraction is thought to be optimal (13). each patient had an echocardiographic LV ejection fraction After the above normalization procedure, the arteries (LVEF) Ͻ40% (Simpson’s biplane method). All patients were exposed twice to KPSS (Kreb’s solution with KCl had suffered a previous myocardial infarction. The patient’s substituted for NaCl on an equimolar basis) and once to NE usual medication (including ACE inhibitor therapy) was 10 ␮M. After a plateau contraction had been attained with taken on the study morning. These patients underwent NE, ACh 3 ␮M was added to the bath in order to stimulate gluteal biopsy and study of subcutaneous resistance arteries. endothelium-dependent vasodilation. Vessels that were un- Patients with CHD. Patients with chronic stable angina able to contract to either KPSS or NE, or that showed no attending outpatient clinics were studied. All patients had relaxation to ACh (and were, therefore, considered to have preserved LV systolic function, determined as an echocar- no functionally intact endothelium), were discarded. diographic LVEF Ն40% (Simpson’s biplane method), and Vessels were then incubated for a further 30 min in either none was treated with an ACE inhibitor. Patients under- Kreb’s solution alone (vehicle, vessel 1) or with enalaprilat Ϫ Ϫ went gluteal biopsy and study of subcutaneous resistance 10 6M (vessel 2), chymostatin 10 5M (vessel 3) or both Ϫ Ϫ arteries. enalaprilat 10 6M and chymostatin 10 5M (vessel 4), Materials. Human AI, AII, bradykinin (BK), norepineph- respectively. Cumulative concentration response (contrac- Ϫ rine (NE) and acetylcholine (ACh) were purchased from tion) curves were then obtained with AI (10 11Mto3ϫ Ϫ Sigma (Poole, United Kingdom). Chymostatin was pur- 10 6M). After exposure to AI, vessels were washed with chased from Bachem (Safron-Walden, United Kingdom), Kreb’s solution to reestablish baseline, and inhibitors were and enalaprilat was a gift from Merck, Sharp and Dohme again added maintaining the same relationship between Ltd. The studies were performed on a Mulvany-Halpern vessel and inhibitor. Responses to AI are expressed as the 1058 Petrie et al. JACC Vol. 37, No. 4, 2001 ACE and Non-ACE AII Generation in CHF March 15, 2001:1056–61

Table 1. Patient Characteristics Angiotensin I Angiotensin II Concentration Response Curves CHF CHD CHF CHD Number 10 10 8 8 Gender (M/F) 8/2 9/1 6/2 7/1 Age (mean [SD]) 70.1 (Ϯ6.1) 60.3 (Ϯ7.4) 70.5 (Ϯ11.1) 66.2 (Ϯ6.3) Previous MI 9282 Previous CABG 6121 NYHA functional class 5 III; 5 II NA 4 II; 4III NA Hypertension 1123 NIDDM 0000 Ejection fraction (mean [SD]) 24.5 (Ϯ6.1) 59.3 (Ϯ8.4) 21.4 (Ϯ8.6) 57.9 (Ϯ9.3) Drug therapy ACE inhibitor 10080 Diuretic 10080 Digoxin 3020 Calcium channel blocker 0213 Nitrate 4434 Beta-blocker 1927 HMGCoA reductase inhibitor 5845 Aspirin 8 10 4 8 Systolic BP (mean [SD]) 125 (Ϯ18) 149.2 (Ϯ9.6) 131 (Ϯ19) 151 (Ϯ17) Diastolic BP (mean [SD]) 70 (Ϯ9) 82.6 (Ϯ0.9) 80 (Ϯ8) 79.5 (Ϯ2.1) Glucose (mean [SD]) 5.5 (Ϯ1.8) 5.9 (Ϯ1.7) 5.5 (Ϯ1.0) 5.3 (Ϯ0.9) Cholesterol (mean [SD]) 5.0 (Ϯ0.9) 4.7 (Ϯ0.8) 4.7 (Ϯ1.1) 5.0 (Ϯ1.1) Creatinine (mean [SD]) 116 (Ϯ17) 89 (Ϯ13) 100 (Ϯ28) 94 (Ϯ12)

ACE ϭ angiotensin converting enzyme; BP ϭ blood pressure; CABG ϭ coronary artery bypass grafting; CHD ϭ coronary heart disease; CHF ϭ chronic heart failure; HMGCoA ϭ 3-hydroxy-3-methylglutaryl coenzyme A; MI ϭ myocardial infarction; NIDDM ϭ noninsulin dependent diabetes mellitus; NYHA ϭ New York Heart Association.

percent contraction to that elicited by 123 mM KCl (second tified for each curve (5). These values were expressed as the exposure) in that vessel. negative logarithm (to ease computation) and entered into a Responses to BK and ACh. After 30 min, a cumulative one-way analysis of variance (ANOVA) with a Bonferroni concentration response (contraction) curve was constructed correction for multiple comparisons (GraphPad Prism, to NE, then to ACh and finally to BK, with further washes GraphPad Software, Inc., San Diego, California). Re- and incubation periods between curves. Responses to BK sponses to BK and ACh were expressed by the pD2 and ACh are expressed as percent change to precontraction (negative logarithm of the EC50) and maximum response with NE. (% vasodilation). Responses to BK and ACh in vessels 2, 3 Responses to AII. The above protocol was repeated in and 4 were then compared with vehicle by one-way arteries from different patients studying the effect of these ANOVA with a Bonferroni correction for multiple com- inhibitors on cumulative concentration response curves to parisons. A similar analysis was performed for responses to AII in place of AI. Responses to AI are expressed as the AII. All data in the text are expressed as mean (Ϯ SD), and percent contraction to that elicited by 123 mM KCl (second all data in graphical form are expressed as mean Ϯ SE, exposure) in that vessel. unless stated otherwise. Although pD2 values were used Effect of AII type-1 receptor blockade (with ). when comparing curves, the equivalent EC50 values (or Ϫ6 The effect of losartan (10 M) on the contractile response threshold concentrations) are stated in the text to ease to AI was studied in resistance arteries taken from patients comprehension. with CHD. The vessels were prepared as described above but were pre-incubated with losartan rather than enalaprilat or chymostatin before construction of an AI concentration RESULTS response curve. Statistical analysis. A maximal response was not observed Patients. The clinical characteristics of patients taking part to AI in arteries in the presence of the combination of in the study are given in Table 1. Baseline characteristics of enalaprilat and chymostatin. This prevented calculation of patients with CHD and CHF differed in only two aspects EC50 (concentration for half-maximum response) or com- (except their previously defined differences in LV function parisons of maximum responses. Instead, the threshold and ACE inhibitor and diuretic prescriptions). First, more concentration for the response to AI (defined as the first patients with CHF were treated with digoxin, and more concentration at which a response was detected) was iden- patients with CHD were treated with beta-adrenergic JACC Vol. 37, No. 4, 2001 Petrie et al. 1059 March 15, 2001:1056–61 ACE and Non-ACE AII Generation in CHF

Ϫ Ϫ Figure 1. Response to AI (10 11Mto3ϫ 10 6M) in arteries from patients with CHF in the presence of vehicle, enalaprilat, chymostatin or both chymostatin and enalaprilat. AI ϭ angiotensin I; CHF ϭ chronic heart failure; KPSS ϭ Kreb’s solution with KCl substituted for NaCl on an equimolar basis. blocking agents. Second, the patients with CHF were older However, in the presence of the combination of enalaprilat than the patients with CHD (p ϭ 0.004). and chymostatin (n ϭ 8), the threshold was 0.77 (1.07) ϫ Ϫ Characteristics of small resistance arteries from patients 10 6M, representing a 1,500-fold increase compared with with CHF and CHD. There were no significant differ- control. Thus, enalaprilat alone induced a significant shift to ences between the normalized diameters of the arteries from the right of the dose-response curve to AI (p Ͻ 0.05), but the different patient groups. Similarly, there were no differ- no inhibition was observed with chymostatin alone (p Ͼ ences in contractile responsiveness or endothelium- 0.05) compared with vehicle. The combination of enalapri- dependent vasodilator responsiveness to KPSS, NE and lat and chymostatin significantly inhibited the response to ACh, respectively, between experimental groups (unpaired t AI (p Ͻ 0.001) compared with vehicle. The shift to the test). right of the dose-response curve observed in the presence of AI concentration response curves: effect of enalaprilat the combination of enalaprilat and chymostatin was signif- and chymostatin. CHF. Responses to AI in arteries from icantly greater than that obtained with enalaprilat alone patients with CHF are shown in Figure 1. Angiotensin I (p Ͻ 0.01). induced a dose-dependent contraction in subcutaneous resistance arteries from CHF patients (n ϭ 6) with a CHD. Responses to AI in arteries from patients with CHD Ϫ threshold concentration of 5.03 (3.82) ϫ 10 10M and a are shown in Figure 2. Angiotensin I induced a dose- maximum at 0.1 ␮M in the absence of inhibitors. In the dependent contraction in subcutaneous resistance arteries presence of enalaprilat (n ϭ 6), the threshold concentration from patients with CHD (group 1, n ϭ 6) with a threshold Ϫ Ϫ for AI was 2.44 (3.86) ϫ 10 8M, representing a fifty-fold concentration of 2.76 (3.36) ϫ 10 9M and a maximum increase compared with control. In the presence of chymo- response at 0.1 ␮M in the absence of any inhibitors. The Ϫ statin (n ϭ 6), the threshold was 0.56 (1.20) ϫ 10 8M. thresholds in the presence of enalaprilat (n ϭ 6), chymo-

Ϫ Ϫ Figure 2. Response to AI (10 11Mto3ϫ 10 6M) in arteries from patients with CHD in the presence of vehicle, enalaprilat, chymostatin or both chymostatin and enalaprilat. AI ϭ angiotensin I; CHD ϭ coronary heart disease. KPSS ϭ Kreb’s solution with KCl substituted for NaCl on an equimolar basis. 1060 Petrie et al. JACC Vol. 37, No. 4, 2001 ACE and Non-ACE AII Generation in CHF March 15, 2001:1056–61

Table 2. EC50 and Maximum Responses to Bradykinin in Arteries from CHF and CHD CHD CHF Max Max EC50 (% resp NE) EC50 (% resp NE) Ϫ Ϫ Vehicle 1.99 (0.33) ϫ 10 7 96.9 (7.0) 1.78 (0.23) ϫ 10 7 92.6 (7.0) Ϫ Ϫ Enalaprilat 3.08 (0.12) ϫ 10 8 84.8 (14.0) 4.0 (0.12) ϫ 10 8 95.3 (5.0) Ϫ Ϫ Chymostatin 3.09 (0.13) ϫ 10 7 89.2 (5.0) 1.20 (0.32) ϫ 10 7 94.4 (7.0) Ϫ Ϫ Combination 5.25 (0.254) ϫ 10 8 88.6 (15.0) 5.57 (0.13) ϫ 10 8 94.4 (6.0)

No significant difference was found in the maximum responses or EC50s between control and each experimental group in either CHF or CHD patients (p Ͼ 0.05 by ANOVA). CHD ϭ coronary heart disease; CHF ϭ chronic heart failure; NE ϭ norepinephrine. statin (n ϭ 6) and combination (n ϭ 8) were, respectively, small resistance arteries from healthy volunteers to patients Ϫ Ϫ 2.20 (3.85) ϫ 10 8M, 1.32 (1.33) ϫ 10 9M and 2.90 with CHD (6). More importantly, however, we now report Ϫ (2.70) ϫ 10 7M. Thus, no inhibition was observed in the on patients with CHF receiving long-term treatment with presence of chymostatin alone (p Ͼ 0.05) compared with ACE inhibitors—a group in whom intense and persistent vehicle. In contrast with the response in vessels from CHF, blockade of the renin-angiotensin system is thought to be no inhibition of the response to AI was observed in the therapeutically desirable. Ͼ presence of enalaprilat alone (p 0.05) compared with Our findings in small resistance arteries from patients vehicle. However, there was a very significant shift in the with CHD are qualitatively very similar to our earlier dose-response curve to the right when enalaprilat and observations in younger healthy subjects. Collectively, the Ͻ chymostatin were combined (p 0.001). available data in healthy volunteers and patients with CHD BK concentration response curves. In the arteries of show that neither an ACE inhibitor alone, nor a chymase patients with CHF, neither enalaprilat, chymostatin or the inhibitor alone, substantially inhibits the conversion of AI combination changed the maximum response to BK (p Ͼ to AII in small arteries, medium-sized arteries or veins (5,6). 0.05). However, there was a trend toward potentiation of Only the combination of an ACE inhibitor and a chymase the response to BK in the presence of enalaprilat alone and inhibitor effectively blocks AI to AII conversion. These enalaprilat in combination with chymostatin (Table 2). This findings imply that both the limbs of the dual pathway for did not reach significance (p Ͼ 0.05). A similar situation was observed in the arteries of patients conversion of AI to AII have a high capacity and that with CHD. Thus, there was a trend toward potentiation in substrate can normally be rapidly and completely shunted the presence of both enalaprilat and the combination of through one limb, should the other be blocked. enalaprilat and chymostatin (Table 2). Again, this did not Responses to AII, BK and ACh. It is important to note reach significance. There was no change in the maximum that the response of small resistance vessels to AII was not response to BK in the presence of any inhibitor. affected by enalaprilat, chymostatin or the combination of ACh concentration response curves. There was no effect the two. Thus, there is no evidence that potentiation of the of either inhibitor, alone or in combination, on responses to action of vasodilator substances by any of these inhibitors ACh in arteries of patients with CHF and CHD. No indirectly influences the response to AI. We have previously difference was observed between the response to ACh in the demonstrated that enalaprilat potentiates the response to absence of inhibitors in arteries from patients with CHF BK in resistance arteries from normal subjects (6). In these compared with those from patients with CHD. studies we were able to confirm that, in both CHF and AII concentration response curves. No significant inhi- CHD subjects, there was a trend toward potentiation of the bition of the AII response was observed with either inhib- dilator response to BK in the presence of enalaprilat— itor alone, or in combination, in arteries from patients with confirming that ACE is involved in the degradation of this CHF and CHD (data not shown). peptide and that enalaprilat can inhibit this. There was no Study of the effect of AII receptor blockade on responses evidence that chymostatin had any effect on responses to to AI. In the presence of losartan, the response of subcu- BK. taneous resistance arteries to AI was completely abolished We also showed that vessels from patients with CHF and ϭ (n 7, data not shown). CHD exhibited a vasodilator response to ACh. There was no apparent difference between the patient groups, suggest- DISCUSSION ing that endothelial function in CHF does not differ from In this study we have been able to confirm that a non-ACE CHD. However, in the absence of a normal control group, pathway that generates AI from AII exists in human small it is not possible to make any inference about vascular subcutaneous resistance arteries. Our findings support those endothelial function in these patients with cardiovascular of Voors et al. (5) in internal mammary arteries taken from disease compared with healthy subjects. Our results do raise patients with CHD and also extend our previous findings in the possibility that previous reports of endothelial dysfunc- JACC Vol. 37, No. 4, 2001 Petrie et al. 1061 March 15, 2001:1056–61 ACE and Non-ACE AII Generation in CHF tion in CHF may, in fact, reflect endothelial dysfunction in from ACE inhibition can occur in a relevant clinical CHD rather than in CHF per se. situation. Responses to AI in arteries from patients with CHF and Conclusions. Even though the chymase pathway may be CHD. In contrast with the responses seen in arteries from less active in CHF, we did find an incremental and CHD patients, the response to AI in arteries from patients statistically significant inhibition of AI to AII conversion with CHF proved to be quite different. Contrary to our with the combination of chymostatin and enalaprilat com- original expectation, we did not find “up-regulation” of the pared with enalaprilat alone in these patients. This finding chymostatin-sensitive pathway in these subjects and instead provides a rationale for using drugs that inhibit the action of found that the small resistance arteries from these patients AII directly in the treatment of patients with CHD and were more sensitive to ACE inhibition than those from CHF; an alternative, and potentially worthwhile approach, healthy volunteers or patients with CHD. It is unlikely that would be the development of agents that inhibit non-ACE this finding is due to the minor age and therapy differences AII generating pathways. between CHF and CHD patients. There are, however, at least two potential explanations. Reprint requests and correspondence: Dr. Neal Padmanabhan, The first possibility is that the chymostatin-sensitive Medical Research Council Blood Pressure Group, Department of non-ACE pathway is actually down-regulated in patients Medicine and Therapeutics, Western Infirmary, University of with CHF treated with an ACE inhibitor. Why this should Glasgow, Glasgow, United Kingdom, G11 6NT. E-mail: occur is not clear, though it could occur if the ACE pathway [email protected]. is up-regulated. The second possibility, that ACE activity is induced, could also explain our findings in CHF, and it is known that both CHF and ACE inhibitor treatment, REFERENCES experimentally, can induce ACE. 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