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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Hypertension. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2827767
NIHMSID: NIHMS166894

Targeting the Degradation of Angiotensin II with Recombinant ACE2: Prevention of Angiotensin II-dependent Hypertension

Abstract

Angiotensin converting enzyme 2 (ACE2) cleaves angiotensin II (Ang II) to form Ang-(1-7). Here we examined whether soluble human recombinant ACE2 (rACE2) can efficiently lower Ang II and increase Ang-(1-7), and whether rACE2 can prevent hypertension caused by Ang II infusion as a result of systemic versus local mechanisms of ACE2 activity amplification.

rACE2 was infused via osmotic minipumps for three days in conscious mice or acutely in anesthetized mice. rACE2 caused a dose-dependent increase in serum ACE2 activity but had no effect on kidney or cardiac ACE2 activity. Following Ang II infusion (40pmol/min), rACE2 (1mg/kg/d) resulted in normalization of systolic blood pressure and plasma Ang II. In acute studies, rACE2 (1mg/kg) prevented the rapid hypertensive effect of Ang II (0.2mg/kg), and this was associated with both a decrease in Ang II and an increase in Ang-(1-7) in plasma. Moreover, during infusion of Ang II, the effect of rACE2 on blood pressure was unaffected by a specific Ang-(1-7) receptor blocker, A779 (0.2 mg/kg), and infusing supra-physiologic levels of Ang-(1-7) (0.2 mg/kg) had no effect on blood pressure.

We conclude that during Ang II infusion rACE2 effectively degrades Ang II and in the process normalizes blood pressure. The mechanism of rACE2 action results from an increase in systemic, not tissue, ACE2 activity and the lowering of plasma Ang II rather than the attendant increase in Ang-(1-7). Increasing ACE2 activity may provide a new therapeutic target in states of Ang II over-activity by enhancing its degradation, an approach that differs from the current focus on blocking Ang II formation and action.

Keywords: ACE2, Soluble, Recombinant, Angiotensin II, Angiotensin-(1-7)

INTRODUCTION

Angiotensin-converting enzyme 2 (ACE2) is the only known enzymatically active homologue of angiotensin-converting enzyme (ACE)13. ACE2 is a mono-carboxypeptidase that removes single amino acids from the C-terminus of its substrates13. ACE, by contrast, is a peptidyl dipeptidase that removes C-terminal dipeptides. While ACE promotes angiotensin (Ang) II formation from Ang I, ACE2 converts Ang I to Ang-(1-9) and Ang II to Ang-(1-7), respectively13. The catalytic efficiency of human ACE2 is 400-fold higher with Ang II than with Ang I as a substrate3. Moreover, since the product of Ang I cleavage by ACE2, Ang-(1-9), has no known biological action, it seems logical to postulate that cleavage of Ang II to Ang-(1-7) is a major action of ACE2.

There is increasing interest in the possible renoprotective effects of ACE249. A protective effect of ACE2 against acute lung injury10, 11 and cardiovascular disease12, 13 has also been proposed. Ang-(1-7) is a blood-vessel dilator identified as an endogenous ligand for a G protein-coupled Mas receptor1416. Angiotensin II, among its many other known biological effects, is a potent vasoconstrictor and promotes renal sodium retention both of which lead to hypertension.

The blockade of steps leading to Ang II formation using ACE inhibitors and renin inhibitors or blocking the action of Ang II on the AT1 receptor using specific antagonists has provided a rationale for modern anti-hypertensive and cardiovascular therapies17. We reasoned that enhancing the degradation of Ang II likewise may provide an effective approach to lower Ang II levels and thus provide the basis for novel therapies based on reducing Ang II over-activity. Currently there are no in vivo studies, to our knowledge, showing that amplification of ACE2 activity results in lowering of Ang II and increasing Ang-(1-7) levels. Therefore, it has been difficult to examine the relative biological importance of the two anticipated effects of ACE2 on Ang II: lowering Ang II versus increasing Ang-(1-7).

Increasing ACE2 activity could provide an effective approach to reduce high blood pressure, particularly in situations when levels of ACE2 may be reduced, as it has been documented in models of rat hypertension12, 18, 19. Indeed, in one of those models, the spontaneously hypertensive stroke-prone rats, transgenic ACE2 over-expression in vascular smooth muscle led to attenuation of hypertension20. Moreover, in ACE2-deficient mice the infusion of Ang II has been shown to increase blood pressure above the level of wild-type control21.

The purpose of this study was therefore to examine whether the administration of soluble human recombinant ACE2 (rACE2) can effectively degrade Ang II in vivo, and if rACE2 can prevent Ang II-induced hypertension. We sought also to unravel the relative contributions of lowering Ang II versus increasing Ang-(1-7) by rACE2 on blood pressure regulation during Ang II administration both acutely and chronically.

METHODS

Soluble human recombinant ACE2

Details on the production and characterization of rACE2 are provided in the expanded Materials and Methods section in the online data supplement (please see http://hyper.ahajournals.org).

Chronic Studies

All studies were approved by the Institutional Animal Care and Use Committee. To study dose-response of rACE2, three groups of male mice (C57BL/6J) of 10 weeks of age were given rACE2 for 3 days (at doses 0.1, 1.0 and 5.0 mg/kg/d) using osmotic minipumps (Model #1003, Alzet, Cupertino, CA). Male ACE2 knockout (KO) mice also on C57BL/6J background (donated by Drs. S. Gurley and T. Coffman, Duke University, Durham, NC21) were either sham operated or infused for 3 days with rACE2 (1 mg/kg/d), Ang II (40 pmol/min), or concurrently with rACE2 (1 mg/kg/d) and Ang II (40 pmol/min).

An additional group of mice received rACE2 (1mg/kg/d) by mini-pumps for 14 days (please see http://hyper.ahajournals.org).

Pumps were implanted subcutaneously on the back between the shoulder blades and hips while animals were anesthetized by inhalation of isoflurane anesthetic. Control mice were sham operated the same way as mice that were implanted with osmotic minipumps containing rACE2. In separate experiments, hypertension was induced also in 10 weeks old C57BL/6J mice by the subcutaneous infusion of Ang II (Sigma-Aldrich, St Louis, MO) [40 pmol/min (1000 ng/kg/min)]21. Two additional groups were infused simultaneously with either Ang II (40 pmol/min) and rACE2 (1 mg/kg/d), or concurrently with Ang II (40 pmol/min), A779 (100 ng/kg/min) (Bachem) and rACE2 (1.0 mg/kg/d) through separate osmotic mini pumps. The compound A779 has been shown to selectively block Ang-(1-7)/Mas receptor in several studies both in vitro14 and in vivo22, 23. The dose we used in this study (100 ng/kg/min), moreover, has been reported to reverse the anti-fibrotic effect of Ang-(1-7) in vivo24.

Systolic blood pressure was measured non-invasively in conscious and anesthetized mice by determining the tail blood volume with a volume-pressure recording (VPR) sensor and an occlusion tail-cuff using a computerized system (CODA System, Kent Scientific, Torrington, CT); mice were previously conditioned to the blood pressure monitoring procedure for at least 4 consecutive days prior to the day of experiment. The VPR recording system has been validated and provides a high correlation with telemetry and direct arterial blood pressure measurements25.

Acute Studies

To study the acute effect of rACE2 on systolic blood pressure (SBP) and Ang II degradation, male C57BL/6J mice also 10–13 weeks old were anesthetized with an i.p. ketamine injection (200 mg/kg BW). Two hours before anesthesia, mice were pre-treated with an i.p. injection of either sterile PBS or rACE2 (1mg/kg). Immediately after inducing anesthesia, mice were placed on a heating platform for 10 minutes. SBP was monitored non-invasively every 30 sec for a period of 25 minutes. After 5 minutes of baseline SBP recording, acute hypertension in anesthetized mice was induced with an i.p. bolus of Ang II (0.2 mg/kg) and the SBP was monitored for the remaining 20 minutes. In additional experiments, Ang II (0.2mg/kg) was infused together with an ACE2 inhibitor (MLN-4760, Millennium Pharmaceuticals, Cambridge, MA, USA) (1mg/kg) following rACE2 infusion two hours earlier, as described above. In separate experiments, mice pre-treated with PBS were injected with A779 at two different doses (0.2 mg/kg and 1 mg/kg) or concomitantly with Ang II and Ang-(1-7) (both at a dose of 0.2 mg/kg). A group of mice receiving rACE2 were also injected with Ang II and A779 (both at the dose of 0.2mg/kg).

In a set of mice under ketamine anesthesia (pre-treated with PBS or rACE2 as above), 5 min after Ang II injection euthanasia was performed by cervical dislocation and blood was rapidly drawn by cardiac puncture for measurements of ACE2 activity and Ang II and Ang-(1-7) levels.

ACE2 activity

ACE and ACE2 activity was measured by an enzymatic assay that uses a fluorogenic peptide substrate 7-Mca-YVADAPK(Dnp) (R&D Systems), as previously described26.

Measurements of plasma and whole kidney Angiotensin II and Angiotensin-(1-7)

Please see http://hyper.ahajournals.org

Determination of plasma anti-human rACE2 antibodies

Please see http://hyper.ahajournals.org

Statistical Analysis

Results are presented as mean ± SEM. Differences in the means among multiple groups were compared using 1-way ANOVA. The BP curves were compared with the use of general linear model multivariate analysis. Pair-wise multiple comparisons were made with the Bonferroni post hoc analysis to detect significant differences between groups. For data exhibiting non-normal distribution, non-parametric Kruskal-Wallis test was employed and followed by Mann-Whitney test for pair-wise comparisons. P<0.05 was considered statistically significant. SPSS version 17.0 for Windows was used for statistical analyses.

RESULTS

Enzymatic properties of rACE2

The ability of rACE2 to cleave Ang II and Ang I was evaluated in vitro (Supplemental Figure S1). By HPLC analysis at 0, 30 and 60 minutes, Ang-(1-7) and phenylalanine were identified as the only products of Ang II cleavage by rACE2. rACE2 was also capable of cleaving Ang I in vitro with the emergence of a very small peak that corresponds to Ang-(1-9) (Supplemental Figure S1B). The effect of rACE2 on Ang I cleavage is modest as compared to the marked effect on Ang II (compare Figures S1A and S1B).

The cleavage of Ang II during incubation with rACE2, measured by Ang II disappearance over time, was blocked by a specific ACE2 inhibitor, MLN-4760 (Supplemental Figure S2A). By contrast, the disappearance of Ang I over time was very slow and not significantly affected by MLN-4760 (Supplemental Figure S2B). Altogether these findings show that rACE2 effectively digests Ang II while it has only a modest effect on Ang I digestion.

Chronic Studies

Effect of rACE2 administration on serum and tissue ACE2 activity and blood pressure in normotensive mice

Infusion of rACE2 for three days to three groups of animals at three different doses, 0.1 mg/kg/day (n=11), 1 mg/kg/day (n=13), and 5 mg/kg/day (n=10) resulted in a dose-dependent increase in serum ACE2 activity(1.30±0.34 RFU/uL/hr; 6.56±0.84 RFU/uL/hr; and 22.21±1.67 RFU/uL/hr, respectively) (p<0.001 by ANOVA) (Figure 1A). In contrast to serum ACE2 activity, there was no significant increase in ACE2 activity in kidney cortex after rACE2 administration (Figure 1B). In heart tissue, ACE2 activity was much lower than in the kidney, as previously reported 26, and also ACE2 activity did not increase in animals infused with the three different doses of rACE2 (Figure 1C).

Figure 1
Serum (Panel A), kidney cortex (Panel B) and heart tissue ACE2 activity (Panel C), and systolic blood pressure (Panel D) in sham-operated mice (empty bar) and mice infused for 3 days subcutaneously with recombinant ACE2 at doses 0.1, 1 or 5 mg/kg/d (rACE2, ...

Recombinant ACE2 had no significant effect on SBP in conscious animals infused with either 0.1, 1, or 5 mg/kg/d for 3 days (Figure 1D). In a subgroup of these animals, SBP was also measured under short duration anesthesia. Under these conditions, SBP was lower than in conscious animals but it was also not significantly affected by the infusion of either 1 or 5 mg/kg/d of rACE2 (supplementary Figure S3).

Serum ACE activity was not significantly affected by any of the doses of rACE2 (Supplemental Figure S4A). Plasma Ang II decreased modestly but significantly with the two highest doses while Ang-(1-7) levels increased also modestly but not significantly (Figure S4 B and C).

Effect of rACE2 on blood pressure and plasma Ang II and Ang-(1-7) levels during sustained infusion of Ang II

In conscious mice that had been infused with Ang II for 3 days, systolic blood pressure was significantly higher than in sham operated controls not infused with Ang II (150.7±5.2, n=12, vs. 132±2.2 mmHg, n=8, respectively, p<0.05). In mice infused with Ang II and rACE2 (1mg/kg/d), systolic blood pressure was significantly lower than in animals infused with Ang II alone (132.4±4.3, n=12 vs. 150.7±5.2 mmHg, n=12, p<0.05) (Figure 2A). When A779 was given to block the Ang-(1-7)/Mas receptor14, together with Ang II and rACE2, blood pressure was reduced to the same extent as in animals infused with rACE2 and Ang II (130.5±4.4, n=9 vs. 132.4±4.3mm Hg, n=12, respectively) (Figure 2A).

Figure 2
Systolic blood pressure (Panel A), plasma Ang II (Panel B) and Ang-(1-7) levels (Panel C) after 3 days infusion of either Ang II alone or simultaneously with Ang II and rACE2, or concurrently with Ang II, rACE2 and A779 [a selective antagonist of the ...

The infusion of Ang II alone for 3 days resulted in plasma Ang II levels about 3 fold higher than those of controls (173.1±38.3 vs. 54.8±21.6 fmol/mL, p<0.05, respectively). The combination of rACE2 and Ang II resulted in a reduction of plasma Ang II to the level observed in controls (52.7±27.8 and 54.8±21.6 fmol/mL, respectively). In the group infused concomitantly with rACE2, Ang II, and A779, plasma Ang II was reduced to 47.9±19.4 fmol/mL, a value also similar to that of controls (54.8±21.6 fmol/mL). Figure 2B and 2C summarizes plasma Ang II and Ang-(1-7) levels, respectively, in the three groups of Ang II infused animals. Plasma Ang-(1-7) tended to be higher in mice infused with Ang II and rACE2 as compared to the mice infused with Ang II only, but this difference was not statistically significant. In the Ang II infused group that received rACE2 and A779, plasma Ang-(1-7) levels were higher than in the other groups, but the difference also did not reach statistical significance (Figure 2C).

Effect of rACE2 on kidney Ang II and Ang-(1-7) levels and ACE2 activity after infusion of Ang II

In previous studies it has been shown that the systemic infusion of Ang II results in intrarenal accumulation of angiotensin II21, 27. We sought to determine whether rACE2 infusion decreases intrarenal Ang II accumulation in mice infused with Ang II.

The infusion of Ang II alone was associated with a marked increase in kidney Ang II levels as compared to non-infused sham controls (71.6±7.7 vs. 9.5±1.4 fmol/mg, p<0.001, respectively). The concomitant infusion of rACE2 and Ang II resulted in a reduction of renal Ang II as compared to mice infused with Ang II only (42.5±5.0 and 71.6±7.7 fmol/mg, p<0.005, respectively), but it remained higher than in controls not infused with Ang II (9.5±1.4 fmol/mg, p<0.001). Kidney Ang-(1-7) levels were not significantly different between the groups infused with Ang II alone and Ang II and rACE2 (15.8±3.5 vs. 10.3±2.0 pg/mg protein, respectively).

Renal ACE2 enzymatic activity in mice infused concurrently with Ang II and rACE2 (20.1±1.3 RFU/ug prot/hr) was not significantly different from either sham operated controls (23.2±1.8 RFU/ug prot/hr) or mice receiving Ang II alone (25.9±1.6 RFU/ug prot/hr). Accordingly, increases in kidney ACE2 activity could not account for the observed decrease in intrarenal Ang II levels after rACE2 administration in animals infused with Ang II.

Effect of rACE2 on kidney Ang II levels and rACE2 activity in an ACE2KO after infusion of Ang II

In ACE2 deficient mice receiving rACE2 (1 mg/kg/d), serum ACE2 activity was significantly higher from ACE2 KO mice not infused with rACE2 (3.75±0.70 RFU/ul/hr vs. 0.05±0.23 RFU/ul/hr, p<0.005, respectively). By contrast, in kidney cortex from the ACE2 KO infused with rACE2, ACE2 activity was undetectable and remained not significantly different from ACE2 KO not infused with rACE2 (−0.70±0.22 vs. 0.13±0.19 RFU/ul/hr). This is consistent with the studies in kidneys from wild-type C57BL/6 mice when ACE2 activity was high but also unaffected by rACE2 (see Figure 1).

The infusion of Ang II alone to ACE2 deficient mice was associated with a marked increase in kidney Ang II levels as compared to ACE2 KO mice not infused with Ang II (100.8±16.4 vs. 23.05±4.8 fmol/mg, p<0.005, respectively) (Figure 3). The concomitant infusion of rACE2 and Ang II resulted in a significant reduction of Ang II as compared to mice infused with Ang II only (55.2±9.3 and 100.8±16.4 fmol/mg, p<0.05, respectively) but it remained higher than in KO not infused with Ang II (55.2±9.3 vs. 23.05±4.8 fmol/mg, p<0.05) (Figure 3).

Figure 3
Kidney Ang II levels in WT (Panel A) and ACE2KO (Panel B). Mice were either sham-operated (control) or infused for 3 days with either Ang II (40pmol/min) alone or simultaneously with Ang II and rACE2(1mg/kg/d).

The concordant behavior of kidney Ang II levels after rACE2 infusions in WT and ACE2KO (Figure 3) further suggests that the observed effect of rACE2 on intrarenal Ang II levels is the result of degrading circulating Ang II as a result of increased serum ACE2 activity because tissue ACE2 activity is completely unaffected by rACE2 in both ACE2KO and WT.

Recombinant ACE2 infusion for two weeks

To examine whether a more prolonged human rACE2 infusion can produce a sustained effect on serum ACE2 activity, mice were infused with rACE2 (1mg/kg/day) for 14 days. Because of the development of antibodies against hrACE2 after 14 days of its administration, the initial increase in serum ACE2 activity was not sustained (supplementary Figure S5). Owing to the observed immunogenicity effect of human rACE2 on mouse antibody titers and the attendant loss of serum ACE2 activity at 14 days, the maximal duration of infusion in this study was limited to the 3 days experiments described above, where there was no evidence of anti-human rACE2 antibodies.

Acute Studies in anesthetized mice

Effect of rACE2 on blood pressure

Effect of rACE2 on blood pressure was further examined in studies in anesthetized mice in response to a bolus of Ang II.

Mice that were pre-treated with rACE2 two hours prior to Ang II infusion showed a marked increase in serum ACE2 activity when compared to animals pre-treated the same way with PBS (21.7±1.2 vs. 0.28±0.09 RFU/ul/hr, p<0.001, respectively). Baseline systolic blood pressure in rACE2-infused mice, however, was not significantly different from mice not pre-treated with rACE2 (111±4.4 vs. 108±3.0 mmHg, p=NS, respectively) which is consistent with the 3 days rACE2 infusion protocol (see Figure 1).

Administration of a bolus of Ang II (n=14) (time 0 min in Figure 4A), resulted in a rapid increase in systolic BP. The SBP peak in the first minute was markedly higher than in the group of Ang II infused animals pre-treated with rACE2 (ΔSBP 68.3±3.9 vs. ΔSBP 29.2±3.8 mmHg, p<0.05). The difference in blood pressure between the two groups persisted throughout the continuous monitoring at 30 seconds intervals for 20 minutes (Figure 4A). After 20 minutes, the SBP remained significantly higher than baseline in the group infused with Ang II alone (ΔSBP=19.7±3.4 mmHg, p<0.001) whereas it was not significantly different from baseline in the group pre-treated with rACE2 (ΔSBP= − 3.3±4.7 mmHg, p=NS). In similar experiments in a group of animals infused with Ang II and an ACE2 inhibitor (MLN-4760, 1mg/kg), rACE2 failed to lower blood pressure such that the peak increase was not significantly different from mice infused with Ang II alone (ΔSBP 81.8±6.4 vs. ΔSBP 73.8±7.8 mmHg, p=NS, respectively) (Figure 4B).

Figure 4
Continuous systolic blood pressure measurement in anesthetized mice. Mice received PBS or rACE2 (1 mg/kg) in a single i.p injection two hours prior to blood pressure measurement. After an i.p. bolus of Ang II (0.2 mg/kg) or Ang II (0.2 mg/kg) along with ...

Systolic blood pressure was unchanged by the Ang-(1-7) receptor blocker, A779, at two different concentrations (0.2 and 1 mg/kg) as compared to control (saline infused) animals (Figure 5A). The administration of A779 also did not alter the peak increase in SBP caused by Ang II bolus as compared to mice that received bolus of Ang II only (ΔSBP= 62±6.5 vs. ΔSBP= 63.6±4.5 mmHg, NS, respectively) (Figure 5B).

Figure 5
Mice received PBS or rACE2 (1 mg/kg) in a single i.p injection two hours prior to blood pressure measurement.

In mice pre-treated with rACE2 that received A779, the Ang II bolus produced a significantly lower peak increase in blood pressure than in mice pre-treated with PBS (ΔSBP=19.8±8.5 mmHg vs. ΔSBP=62±6.5 mmHg, p<0.001) (Figure 5B). SBP returned to the baseline values within 5 minutes after Ang II administration and was kept at this level throughout the 20 min of observation (Figure 5B). Thus, the Mas receptor blocker, A779, did not further potentiate the increase in blood pressure induced by Ang II or interfere with the recovery from Ang II-induced hypertension.

To further examine any possible effect related to Ang-(1-7) on blood pressure, mice pre-treated with PBS were administered with a supra-physiologic dose of Ang-(1-7) (0.2mg/kg) in addition to Ang II (Figure 5C). When blood pressure was measured in mice administered concomitantly with Ang-(1-7) and Ang II, there was an identical pattern of blood pressure as compared to animals given only the Ang II bolus (Figure 5C). The peak blood pressure increase induced by Ang II was similar in the presence and absence of Ang-(1-7) (ΔSBP=79.4±9.0 vs. 71.4±9.0 mmHg, respectively). Moreover, Ang-(1-7) did not enhance the blood pressure recovery after Ang II bolus which was incomplete in both groups as compared to baseline (Figure 5C). This suggests that under these experimental conditions, any increases in Ang-(1-7) related to rACE2 infusion did not have a detectable effect on blood pressure.

Effect of rACE2 on plasma Ang II and Ang-(1-7) levels in Ang II-infused mice

Since in rACE2 infused mice systolic BP normalized within 5 minutes after Ang II bolus (see Figure 4 and Figure 5), we chose this time point to examine plasma Ang peptides (Figure 6). In rACE2-pretreated mice (for two hours) plasma Ang II levels were markedly reduced as compared to animals infused with Ang II that had not received rACE2 (6.1±0.9 vs. 76.7±39 fmol/mL, respectively, p<0.001). The concomitant administration of Ang II and A779 to mice pre-treated with rACE2 did not affect plasma Ang II levels which were similar to mice infused with Ang II and rACE2 (6.8±1.0 and 6.1±09 fmol/mL, respectively) (Figure 6).

Figure 6
Plasma Ang II (Panel A) and plasma Ang-(1-7) levels (Panel B) measured 5 minutes after administration of Ang II bolus to anesthetized mice pre-treated with either PBS or rACE2 in a manner similar to the groups described in Figure 4 and and5.5 ...

In the group pretreated with rACE2 and given a bolus of Ang II, plasma Ang-(1-7) levels were significantly higher than in the group infused with Ang II without prior rACE2 pretreatment (1.52±0.32 vs. 0.74±0.08 pmol/mL, p<0.05). In the group also pre-treated with rACE2 that received Ang II and the Mas receptor blocker, A779, plasma Ang-(1-7) increased further (1.93±0.44 and 1.52±0.32 pmol/mL, respectively) although this difference did not reach statistical significance (see Figure 6).

DISCUSSION

This study shows that the increase in blood pressure triggered by Ang II infusion can be completely prevented by the administration of soluble human rACE2. We used a highly purified soluble human rACE2 produced in Chinese hamster ovary cell line which has a calculated half-life in vivo of 8.5 hours (supplementary Methods). Our protocols involved acute studies in anesthetized animals and studies where rACE2 was given by osmotic mini-pumps for 3 days to conscious animals. Studies of long duration with human rACE2 administration were precluded because we found that mouse anti-human rACE2 antibodies developed over time and this resulted in a decrease in serum ACE2 activity despite continued rACE2 infusion (supplementary results).

As expected from the known effect of ACE2 on Ang II 2, 3, human rACE2 was shown in vitro to cleave a single amino acid phenylalanine from Ang II which led to the formation of Ang-(1-7). Recombinant ACE2 also acted on Ang I to form Ang-(1-9), albeit with a much lower affinity than for Ang II to form Ang-(1-7) (Supplemental Figures S1 and S2). We were able to show that plasma Ang II levels, following Ang II infusion, can be lowered while Ang-(1-7) levels can be increased in mice infused with rACE2. This directly demonstrates the importance of ACE2 as a pathway in the degradation of Ang II in vivo since the plasma levels of Ang II were completely normalized following rACE2 administration (Figure 6).

Ang-(1-7), a peptide with vasodilatory properties16, is the only known product of ACE2-driven Ang II cleavage 2, 3. Increasing ACE2 activity could lower blood pressure, by either lowering Ang II levels, by the generation of Ang-(1-7), or both. In the face of blockade of the Ang-(1-7) receptor with A779, the administration of rACE2 still abrogated completely the blood pressure increase caused by Ang II infusion and to the same extent as in the absence of A779 (Figure 2, Figure 4 and Figure 5). A significant increase in plasma Ang-(1-7) was seen in the acute studies, such that 5 minutes after a bolus of Ang II, plasma levels of Ang-(1-7) increased after rACE2 pre-treatment (Figure 6). Neither the administration of A779 nor the administration of Ang-(1-7) at pharmacological doses had an effect on blood pressure (Figure 5).

This lack of effect of Ang-(1-7) on blood pressure should not be interpreted against the role of this peptide in circulatory control. This peptide has been shown to decrease resistance of peripheral blood vessels but also increases cardiac output28. The latter effect possibly counterbalances any blood pressure lowering effect of Ang-(1-7) derived from its vasodilatory action. Our findings showing a lack of effect of Ang-(1-7) on reducing blood pressure are consistent with the results of long-term Ang-(1-7) infusion studies in a rat model of Ang II-dependent hypertension24 but contrary to an earlier paper in centrally denervated animals where Ang-(1-7) appeared to have blood-lowering effect29. Since Ang-(1-7) significantly decreased cardiac fibrosis and prevented cardiac remodeling24 one may expect a potential benefit of increasing Ang-(1-7) by rACE2 administration from this action rather than from any blood pressure lowering effect of this peptide.

Our data not only shows that during Ang II infusions administration of rACE2 lowers blood pressure by a mechanism independent of Ang-(1-7), but also shows that this blood pressure-lowering effect is due to an increase in circulating ACE2 activity. The specific ACE2 inhibitor, MLN-4760, completely abrogated the effect of rACE2 on Ang II-mediated hypertension thereby demonstrating the ACE2 dependency of its effect on blood pressure during Ang II infusion. rACE2 had no effect on tissue ACE2 activity but effectively normalized plasma Ang II levels. This shows an effect on Ang II degradation owing to an increase in circulating ACE2 activity. Moreover, Plasma Ang II levels could not have been influenced by ACE-driven formation because rACE2 did not result in changes in serum ACE activity (Supplementary Figure S4).

It is noteworthy that rACE2 has only a partial effect on kidney Ang II levels (Figure 3) despite complete normalization of plasma Ang II levels (Figure 2). This shows that only the amount of Ang II that was circulating could be degraded by rACE2 present in serum because at the kidney level there was no change in ACE2 activity following rACE2 infusions. The failure of rACE2 to increase tissue ACE2 activity was confirmed in ACE2 knockout where it should have been easier to demonstrate any detectable increases in ACE2 activity because of the lack of any baseline activity. The soluble form of ACE2 differs from the full-length in that it lacks the anchoring site which normally would retain ACE2 on the cell plasma membrane 30. This may explain, in part, why neither kidney nor heart ACE2 activity were increased with different doses of rACE2 whereas serum ACE2 activity increased markedly (Figure 1).

Regardless of the explanation for the lack of an effect of rACE2 on tissue ACE2 activity, the finding in itself sheds light on the mechanism of action of rACE2. We surmise, based on this finding, that the therapeutic potential of rACE2 stems from its ability to increase serum ACE2 activity, which normally is very low, and thus enhanced degradation of circulating Ang II. Since the levels of this hormone are usually not elevated in normotensive mice, the administration of rACE2 would not be expected to have any major effect on blood pressure. In keeping with this prediction, blood pressure was unchanged by rACE2 even at very high doses (Figure 1). This is in contrast to its effect in Ang II infused animals where lowering of blood pressure was easily demonstrable when plasma Ang II levels were increased (Figure 2 and Figure 6).

Amplification of ACE2 activity at the tissue level should have important effects that cannot be achieved by rACE2 administration which has an action restricted to increasing serum ACE2 activity. In this regard, in a transgenic model of ACE2 over-expression in the vasculature of SHRSP rats, blood pressure in this model of hypertension was reduced20. Other studies have also shown the potential of ACE2 amplification within the central nervous system in lowering blood pressure.19, 31 In addition, lentiviral overexpression of ACE2 reversed cardiac hypertrophy and fibrosis induced by Ang II in Sprague Dawley rats 32. The same group recently reported that chronic administration of xanthenone, a compound which has properties as ACE2 activator, reduced blood pressure and reversed myocardial fibrosis in the spontaneously hypertensive rat33.

PERSPECTIVES

Angiotensin II-induced hypertension can be prevented by rACE2 as a result of ACE2-driven Ang II degradation within the circulation. It is possible that relative or absolute deficiency of ACE2 is involved in the pathogenesis of certain forms of hypertension by limiting the degradation of Ang II at certain tissues or systemically. Therapies aimed at amplifying ACE2 activity will hopefully be developed and will find its place in the management of forms of hypertension where Ang II may be over-active. Agents that increase ACE2 activity at the tissue level may be particularly suitable for conditions associated with local tissue ACE2 down-regulation and over-activity of the RAS. Moreover, ACE2 amplification may provide a complement to existing therapies aimed at inhibiting Ang II formation and action which are only partially effective in suppressing Ang II over-activity.

Supplementary Material

Supp1

Acknowledgments

Sources of support

Part of this work was supported by grants to D.B. from the American Diabetes Association (7-05-RA-06), the Juvenile Diabetes Research Foundation (1-2007-636), and the National Institutes of Health (1 R01 DK080089). J.W. was supported by postdoctoral research award F32DK079546 from the NIDDKD. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIDDKD or the NIH.

Footnotes

Disclosures

Manfred Schuster — ownership and income in Apeiron Biologics. Dr. Schuster has Apeiron stocks and receives his salary from Apeiron as CSO of the company.

Hans Loibner — ownership and income in Apeiron Biologics. Dr. Loibner has Apeiron stocks and receives compensation from Apeiron for CEO activities.

Josef Penninger — founder of Apeiron Biologics. Dr. Penninger has Apeiron shares.

Daniel Batlle — submitted an application for a patent entitled “Methods for Achieving a Protective ACE2 Expression Level to Treat Kidney Disease and Hypertension.”

Reference List

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