Targeting the degradation of angiotensin II with recombinant angiotensin-converting enzyme 2: prevention of angiotensin II-dependent hypertension

Abstract

Angiotensin (Ang)-converting enzyme 2 (ACE2) cleaves 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 3 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. After Ang II infusion (40 pmol/min), rACE2 (1 mg/kg per day) resulted in normalization of systolic blood pressure and plasma Ang II. In acute studies, rACE2 (1 mg/kg) prevented the rapid hypertensive effect of Ang II (0.2 mg/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 supraphysiologic 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 overactivity by enhancing its degradation, an approach that differs from the current focus on blocking Ang II formation and action.

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, filled bars). Panel A–C) After rACE2 administration, a dose-dependent increase in serum ACE2 activity was observed (Panel A), whereas there was no increase in either kidney cortex (Panel B) or heart ACE2 activity (Panel C); *p<0.001 vs. control, ^#p<0.001 vs. 0.1 mg/kg/d, ^&p<0.001 vs. 1 mg/kg/d; (Panel D) rACE2 did not lower systolic blood pressure significantly at any of the doses used.

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 Ang-(1-7) receptor]. (Panel A) The administration of rACE2 to mice receiving Ang II resulted in a significantly lower blood pressure as compared to mice infused with Ang II alone. The administration of A779 concomitantly with Ang II and rACE2 was associated with a blood pressure similar to that seen in animals infused with rACE2 and Ang II; *p<0.05 vs. Ang II only; (Panel B) Plasma Ang II levels in mice receiving the combined infusion of rACE2 and Ang II were markedly lower as compared to Ang II infusion alone. In the group infused concomitantly with rACE2, Ang II, and A779, plasma Ang II levels were similarly reduced, as compared to Ang II alone; *p<0.05 vs. Ang II only; (Panel C) Plasma Ang-(1-7) levels in mice receiving the combined infusion of rACE2 and Ang II were higher as compared to Ang II infusion alone, but the difference did not reach statistical significance. In the group receiving A779 and rACE2, plasma Ang-(1-7) levels were even higher but the difference was not statistically significant as compared to the other two groups.

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). (Panel A) Kidney Ang II levels in WT mice receiving rACE2 concurrently with Ang II were significantly lower than in mice infused with Ang II only, but were still higher than in controls not infused with Ang II. (Panel B) In ACE2KO mice infused with Ang II, rACE2 administration resulted in a similar partial reduction in kidney Ang II as compared to ACE2KO receiving Ang II alone; *p<0.05, **p<0.005, ***p<0.001.

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 an ACE2 inhibitor, MLN-4760 (1 mg/kg) (arrow, time point 0 min.), SBP was recorded at 30 s intervals for the following 20 min. (Panel A) There was no difference in baseline SBP between rACE2 and PBS infused animals prior to Ang II bolus. A bolus of Ang II to mice pre-treated with PBS (n=14) was associated with a rapid increase in SBP. In mice administered with rACE2 prior to Ang II injection (n=11), the SBP increase was blunted and normalized within the first 5 minutes after Ang II injection. (Panel B) In a separate group of experiments, in mice pre-treated with rACE2 (n=6) a bolus of Ang II (arrow) was associated with a blunted SBP increase as compared to mice pre-treated with PBS (n=7) or mice that were rACE2-pretreated and received a bolus of Ang II with MLN-4760 (n=6).

Figure 5

Mice received PBS or rACE2 (1 mg/kg) in a single i.p injection two hours prior to blood pressure measurement. (Panel A) A saline i.p. bolus (PBS, time point 0 min) did not alter systolic blood pressure (SBP) in anesthetized mice (n=8). An i.p. bolus of Mas receptor blocker, A779, at two doses, (0.2 mg/kg, n=8) and (1 mg/kg, n=4), to mice pre-treated with PBS was also not associated with an alteration in SBP. (Panel B) A bolus of Ang II (0.2 mg/kg, arrow) to mice pre-treated with PBS (n=11) was associated with a rapid increase in SBP. In mice pre-treated with PBS, the concomitant injection of Ang II and A779 (n=9) caused a similar SBP increase as in PBS pre-treated mice injected with Ang II only. In mice administered with rACE2 (1mg/kg i.p.) prior to a bolus of Ang II and A779 (n=9), the SBP increase was blunted and normalized within 5 minutes after Ang II injection. (Panel C). In PBS-pre-treated mice (n=7) after i.p. injection of Ang II (0.2 mg/kg, arrow), SBP increased rapidly and remained elevated for the entire 20 min period of the BP measurements. The administration of Ang-(1-7) concurrently with Ang II (both peptides at the dose of 0.2 mg/kg n=7) did not alter the pattern of blood pressure increase caused by Ang II administration.

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 5. The administration of Ang II alone (n=8) in mice pre-treated with rACE2 was associated with markedly lower plasma Ang II levels and significantly increased plasma Ang-(1-7) levels as compared to mice pre-treated with PBS (n=7). Injection of Ang II along with A779, (n=8) to mice pre-treated with rACE2 resulted in a similar reduction in plasma Ang II and an increase in Ang-(1-7) levels; ***p<0.001, *p<0.05 vs. Ang II only;