Targeting the Degradation of Angiotensin II With Recombinant Angiotensin-Converting Enzyme 2

Angiotensin (Ang)-converting enzyme 2 (ACE2) is the only known enzymatically active homologue of Ang-converting enzyme (ACE).^1–3 ACE2 is a monocarboxypeptidase that removes single amino acids from the C terminus of its substrates.^1–3 ACE, by contrast, is a peptidyl dipeptidase that removes C-terminal dipeptides. ACE promotes Ang II formation from Ang I, whereas ACE2 converts Ang I to Ang-(1-9) and Ang II to Ang-(1-7), respectively.^1–3 The catalytic efficiency of human ACE2 is 400-fold higher with Ang II than with Ang I as a substrate.³ Moreover, because 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 ACE2.^4–9 A protective effect of ACE2 against acute lung injury^10,11 and cardiovascular disease^12,13 has also been proposed. Ang-(1-7) is a blood-vessel dilator identified as an endogenous ligand for a G protein–coupled Mas receptor.^14–16 Ang 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 Ang II type 1 receptor using specific antagonists has provided a rationale for modern antihypertensive and cardiovascular therapies.¹⁷ 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 on the basis of reducing Ang II overactivity. 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 2 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 has been documented in models of rat hypertension.^12,18,19 Indeed, in one of those models, the spontaneously hypertensive stroke-prone rats, transgenic ACE2 overexpression in vascular smooth muscle led to attenuation of hypertension.²⁰ Moreover, in ACE2-deficient mice, the infusion of Ang II has been shown to increase blood pressure above the level of WT control.²¹

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 whether 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 rACE2

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 of the studies were approved by the Institutional Animal Care and Use Committee. To study the dose response of rACE2, 3 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 per day) using osmotic minipumps (model 1003, ALZET). 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 per day), Ang II (40 pmol/min), or concurrently with rACE2 (1 mg/kg per day) and Ang II (40 pmol/min). An additional group of mice received rACE2 (1 mg/kg per day) by minipumps for 14 days (please see the online Data Supplement).

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 also induced in 10-week–old C57BL/6J mice by the subcutaneous infusion of Ang II (Sigma-Aldrich; 40 pmol/min; 1000 ng/kg per minute).²¹ Two additional groups were infused simultaneously with either Ang II (40 pmol/min) and rACE2 (1 mg/kg per day) or concurrently with Ang II (40 pmol/min), A779 (100 ng/kg per minute; Bachem), and rACE2 (1.0 mg/kg per day) through separate osmotic minipumps. The compound A779 has been shown to selectively block the Ang-(1-7)/Mas receptor in several studies both in vitro¹⁴ and in vivo.^22,23 The dose that we used in this study (100 ng/kg per minute), moreover, has been reported to reverse the antifibrotic effect of Ang-(1-7) in vivo.²⁴

Systolic blood pressure (SBP) was measured noninvasively in conscious and anesthetized mice by determining the tail blood volume with a volume-pressure recording sensor and an occlusion tail-cuff using a computerized system (CODA System, Kent Scientific); mice were conditioned previously to the blood pressure monitoring procedure for ≥4 consecutive days before the day of the experiment. The volume-pressure recording system has been validated and provides a high correlation with telemetry and direct arterial blood pressure measurements.²⁵

Acute Studies

To study the acute effect of rACE2 on SBP and Ang II degradation, male C57BL/6J mice also 10 to 13 weeks old were anesthetized with an IP ketamine injection (200 mg/kg of body weight). Two hours before anesthesia, mice were pretreated with an IP injection of either sterile PBS or rACE2 (1 mg/kg). Immediately after inducing anesthesia, mice were placed on a heating platform for 10 minutes. SBP was monitored noninvasively every 30 seconds for a period of 25 minutes. After 5 minutes of baseline SBP recording, acute hypertension in anesthetized mice was induced with an IP bolus of Ang II (0.2 mg/kg), and the SBP was monitored for the remaining 20 minutes. In additional experiments, Ang II (0.2 mg/kg) was infused together with an ACE2 inhibitor (MLN-4760, Millennium Pharmaceuticals; 1 mg/kg) after rACE2 infusion 2 hours earlier, as described above. In separate experiments, mice pretreated with PBS were injected with A779 at 2 different doses (0.2 and 1.0 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 0.2 mg/kg).

In a set of mice under ketamine anesthesia (pretreated with PBS or rACE2, as above), 5 minutes 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 activities were measured by an enzymatic assay that uses a fluorogenic peptide substrate 7-Mca-YVADAPK(Dnp; R&D Systems), as described previously.²⁶

Measurements of Plasma and Whole Kidney Ang II and Ang-(1-7)

Please see the online Data Supplement for details.

Determination of Plasma Antihuman rACE2 Antibodies

Please see the online Data Supplement for details.

Statistical Analysis

Results are presented as mean±SEM. Differences in the means among multiple groups were compared using 1-way ANOVA. The blood pressure curves were compared with the use of general linear model multivariate analysis. Pairwise multiple comparisons were made with the Bonferroni post hoc analysis to detect significant differences between groups. For data exhibiting nonnormal distribution, a nonparametric Kruskal-Wallis test was used, followed by a Mann–Whitney test for pairwise 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 (Figure S1, available in the online Data Supplement). By high-performance liquid chromatography 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 with Ang-(1-9) (Figure S1B). The effect of rACE2 on Ang I cleavage is modest as compared with the marked effect on Ang II (compare Figure 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 (Figure S2A). By contrast, the disappearance of Ang I over time was very slow and not significantly affected by MLN-4760 (Figure S2B). Altogether, these findings show that rACE2 effectively digests Ang II, whereas 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 3 days to 3 groups of animals at 3 different doses, 0.1 mg/kg per day (n=11), 1 mg/kg per day (n=13), and 5 mg/kg per day (n=10), resulted in a dose-dependent increase in serum ACE2 activity (1.30±0.34, 6.56±0.84, and 22.21±1.67 relative fluorescence units [RFU]/μL per hour, respectively; P<0.001 by ANOVA; Figure 1A). In contrast to serum ACE2 activity, there was no significant increase in ACE2 activity in the kidney cortex after rACE2 administration (Figure 1B). In heart tissue, ACE2 activity was much lower than in the kidney, as reported previously,²⁶ and also ACE2 activity did not increase in animals infused with the 3 different doses of rACE2 (Figure 1C).

• Download figure
• Download PowerPoint

Recombinant ACE2 had no significant effect on SBP in conscious animals infused with 0.1, 1.0, or 5.0 mg/kg per day 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 per day of rACE2 (Figure S3).

Serum ACE activity was not significantly affected by any of the doses of rACE2 (Figure S4A). Plasma Ang II decreased modestly but significantly with the 2 highest doses, whereas Ang-(1-7) levels also increased modestly but not significantly (Figure S4B and S4C).

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, SBP was significantly higher than in sham-operated controls not infused with Ang II (150.7±5.2 mm Hg, n=12, versus 132±2.2 mm Hg, n=8, respectively; P<0.05). In mice infused with Ang II and rACE2 (1 mg/kg per day), SBP was significantly lower than in animals infused with Ang II alone (132.4±4.3 mm Hg, n=12 versus 150.7±5.2 mm Hg, n=12; P<0.05; Figure 2A). When A779 was given to block the Ang-(1-7)/Mas receptor,¹⁴ 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 mm Hg, n=9, versus 132.4±4.3 mm Hg, n=12, respectively; Figure 2A).

• Download figure
• Download PowerPoint

The infusion of Ang II alone for 3 days resulted in plasma Ang II levels ≈3-fold higher than those of controls (173.1±38.3 versus 54.8±21.6 fmol/mL, respectively; P<0.05). 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 3 groups of Ang II–infused animals. Plasma Ang-(1-7) tended to be higher in mice infused with Ang II and rACE2 as compared with 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 Ang II.^21,27 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 with noninfused sham controls (71.6±7.7 versus 9.5±1.4 fmol/mg, respectively; P<0.001). The concomitant infusion of rACE2 and Ang II resulted in a reduction of renal Ang II as compared with mice infused with Ang II only (42.5±5.0 and 71.6±7.7 fmol/mg, respectively; P<0.005), 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 versus 10.3±2.0 pg/mg of protein, respectively).

Renal ACE2 enzymatic activity in mice infused concurrently with Ang II and rACE2 (20.1±1.3 RFU/μg of protein per hour) was not significantly different from either sham-operated controls (23.2±1.8 RFU/μg of protein per hour) or mice receiving Ang II alone (25.9±1.6 RFU/μg of protein per hour). 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 ACE2 KO After Infusion of Ang II

In ACE2-deficient mice receiving rACE2 (1 mg/kg per day), serum ACE2 activity was significantly higher from ACE2 KO mice not infused with rACE2 (3.75±0.70 versus 0.05±0.23 RFU/μL per hour, respectively; P<0.005). By contrast, in kidney cortex from the ACE2 KO mice infused with rACE2, ACE2 activity was undetectable and remained not significantly different from ACE2 KO mice not infused with rACE2 (−0.70±0.22 versus 0.13±0.19 RFU/μL per hour). This is consistent with the studies in kidneys from WT 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 with ACE2 KO mice not infused with Ang II (100.8±16.4 versus 23.05±4.8 fmol/mg, respectively; P<0.005; Figure 3). The concomitant infusion of rACE2 and Ang II resulted in a significant reduction of Ang II as compared with mice infused with Ang II only (55.2±9.3 and 100.8±16.4 fmol/mg, respectively; P<0.05), but it remained higher than in KO not infused with Ang II (55.2±9.3 versus 23.05±4.8 fmol/mg; P<0.05; Figure 3).

• Download figure
• Download PowerPoint

The concordant behavior of kidney Ang II levels after rACE2 infusions in wild-type (WT) and ACE2 KO mice (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 ACE2 KO and WT mice.

Recombinant ACE2 Infusion for 2 Weeks

To examine whether a more prolonged human rACE2 infusion can produce a sustained effect on serum ACE2 activity, mice were infused with rACE2 (1 mg/kg per day) for 14 days. Because of the development of antibodies against human rACE2 after 14 days of its administration, the initial increase in serum ACE2 activity was not sustained (Figure S5). Because of 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-day experiments described above, where there was no evidence of antihuman rACE2 antibodies.

Acute Studies in Anesthetized Mice

Effect of rACE2 on Blood Pressure

The 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 pretreated with rACE2 2 hours before Ang II infusion showed a marked increase in serum ACE2 activity when compared with animals pretreated the same way with PBS (21.7±1.2 versus 0.28±0.09 RFU/μL per hour, respectively; P<0.001). Baseline SBP in rACE2-infused mice, however, was not significantly different from mice not pretreated with rACE2 (111±4.4 versus 108±3.0 mm Hg, respectively; P value not significant), which is consistent with the 3-day rACE2 infusion protocol (see Figure 1).

Administration of a bolus of Ang II (n=14; time: 0 minutes in Figure 4A), resulted in a rapid increase in SBP. The SBP peak in the first minute was markedly higher than in the group of Ang II–infused animals pretreated with rACE2 (Δ_SBP 68.3±3.9 versus Δ_SBP 29.2±3.8 mm Hg; P<0.05). The difference in blood pressure between the 2 groups persisted throughout the continuous monitoring at 30-second intervals for 20 minutes (Figure 4A). After 20 minutes, the SBP remained significantly higher than baseline in the group infused with Ang II alone (Δ_SBP=19.7±3.4 mm Hg; P<0.001), whereas it was not significantly different from baseline in the group pretreated with rACE2 (Δ_SBP=−3.3±4.7 mm Hg; P value not significant). In similar experiments in a group of animals infused with Ang II and an ACE2 inhibitor (MLN-4760, 1 mg/kg), rACE2 failed to lower blood pressure such that the peak increase was not significantly different from that in mice infused with Ang II alone (Δ_SBP 81.8±6.4 versus Δ_SBP 73.8±7.8 mm Hg, respectively; P value not significant; Figure 4B).

• Download figure
• Download PowerPoint

SBP was unchanged by the Ang-(1-7) receptor blocker, A779, at 2 different concentrations (0.2 and 1.0 mg/kg) as compared with 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 with mice that received bolus of Ang II only (Δ_SBP=62±6.5 versus Δ_SBP=63.6±4.5 mm Hg, respectively; P value not significant; Figure 5B).

• Download figure
• Download PowerPoint

In mice pretreated with rACE2 that received A779, the Ang II bolus produced a significantly lower peak increase in blood pressure than in mice pretreated with PBS (Δ_SBP=19.8±8.5 versus 62±6.5 mm Hg; 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 minutes 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 pretreated with PBS were administered a supraphysiologic dose of Ang-(1-7) (0.2 mg/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 with that in 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) (Δ_SBP=79.4±9.0 versus 71.4±9.0 mm Hg, respectively). Moreover, Ang-(1-7) did not enhance the blood pressure recovery after Ang II bolus, which was incomplete in both groups as compared with 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

Because in rACE2-infused mice SBP normalized within 5 minutes after Ang II bolus (see Figures 4 and 5), we chose this time point to examine plasma Ang peptides (Figure 6). In rACE2-pretreated mice (for 2 hours) plasma Ang II levels were markedly reduced as compared with animals infused with Ang II that had not received rACE2 (6.1±0.9 versus 76.7±39 fmol/mL, respectively; P<0.001). The concomitant administration of Ang II and A779 to mice pretreated 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).

• Download figure
• Download PowerPoint

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 previous rACE2 pretreatment (1.52±0.32 versus 0.74±0.08 pmol/mL; P<0.05). In the group also pretreated 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 the Chinese hamster ovary cell line, which has a calculated half-life in vivo of 8.5 hours (please see the supplementary Methods section). Our protocols involved acute studies in anesthetized animals and studies where rACE2 was given by osmotic minipumps for 3 days to conscious animals. Studies of long duration with human rACE2 administration were precluded because we found that mouse antihuman rACE2 antibodies developed over time, and this resulted in a decrease in serum ACE2 activity despite continued rACE2 infusion (please see the supplementary Results section).

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) (Figures S1 and S2). We were able to show that plasma Ang II levels, after Ang II infusion, can be lowered, whereas 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 because the plasma levels of Ang II were completely normalized after rACE2 administration (Figure 6).

Ang-(1-7), a peptide with vasodilatory properties,¹⁶ is the only known product of ACE2-driven Ang II cleavage.^2,3 Increasing ACE2 activity could lower blood pressure, by 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 completely abrogated the blood pressure increase caused by Ang II infusion and to the same extent as in the absence of A779 (Figures 2, 4, and 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 pretreatment (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 output.²⁸ 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 article in centrally denervated animals, where Ang-(1-7) appeared to have blood-lowering effect.²⁹ Because Ang-(1-7) significantly decreased cardiac fibrosis and prevented cardiac remodeling,²⁴ 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 show that, during Ang II infusions, administration of rACE2 lowers blood pressure by a mechanism independent of Ang-(1-7), but also show that this blood pressure–lowering effect is attributable 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 attributable 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 (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 after rACE2 infusions. The failure of rACE2 to increase tissue ACE2 activity was confirmed in ACE2 KO mice, where it should have been easier to demonstrate any detectable increases in ACE2 activity attributable to the lack of any baseline activity. The soluble form of ACE2 differs from the full-length form 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 was 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, on the basis of 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. Because 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 (Figures 2 and 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 overexpression in the vasculature of spontaneously hypertensive stroke-prone rats, blood pressure in this model of hypertension was reduced.²⁰ 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 reported recently that chronic administration of xanthenone, a compound that has properties as ACE2 activator, reduced blood pressure and reversed myocardial fibrosis in the spontaneously hypertensive rat.³³

Perspectives

Ang 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 overactive. Agents that increase ACE2 activity at the tissue level may be particularly suitable for conditions associated with local tissue ACE2 downregulation and overactivity of the renin-Ang system. 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 overactivity.

Footnotes