Genetic Deletion of ACE2 Induces Vascular Dysfunction in C57BL/6 Mice: Role of Nitric Oxide Imbalance and Oxidative Stress

Abstract

Accumulating evidence indicates that angiotensin-converting enzyme 2 (ACE2) plays a critical role in cardiovascular homeostasis, and its altered expression is associated with major cardiac and vascular disorders. The aim of this study was to evaluate the regulation of vascular function and assess the vascular redox balance in ACE2-deficient (ACE2-/y) animals. Experiments were performed in 20-22 week-old C57BL/6 and ACE2-/y male mice. Evaluation of endothelium-dependent and -independent relaxation revealed an impairment of in vitro and in vivo vascular function in ACE2-/y mice. Drastic reduction in eNOS expression at both protein and mRNA levels, and a decrease in •NO concentrations were observed in aortas of ACE2-/y mice in comparison to controls. Consistently, these mice presented a lower plasma and urine nitrite concentration, confirming reduced •NO availability in ACE2-deficient animals. Lipid peroxidation was significantly increased and superoxide dismutase activity was decreased in aorta homogenates of ACE2-/y mice, indicating impaired antioxidant capacity. Taken together, our data indicate, that ACE2 regulates vascular function by modulating nitric oxide release and oxidative stress. In conclusion, we elucidate mechanisms by which ACE2 is involved in the maintenance of vascular homeostasis. Furthermore, these findings provide insights into the role of the renin-angiotensin system in both vascular and systemic redox balance.

Fig 1. Vascular function in ACE2^-/y mice.

Mean arterial pressure (A) and heart rate (B) were measured by radiotelemetry in ACE2^-/y and WT (n = 6) mice. Data represent means±SEMs of 3 days of recording. **P<0.01. Vascular response to increasing concentrations [50–200 μg.kg^-1] of ACh (C) and 10 μg.kg^-1 SNP (D) in ACE2^-/y (n = 5) and WT (n = 6) mice. (E) Vascular response to ACh normalized by SNP. Data represent means±SEMs. **P<0.01 (Student t test); §§ P<0.01 (2-way ANOVA). Similar data were obtained in 2 independent experiments. (F) ACh and (G) SNP dose-response curve of aortic rings from ACE2^-/y (KO, n = 8–10) and WT (n = 13) mice. Rings were precontracted with phenylephrine (1 μmol.L^-1) and then stepwise relaxed with ACh or SNP (3–3000 nmol.L^-1). *P<0.05.

Fig 2. Nitric oxide availability and eNOS expression in ACE2^-/y mice.

Plasma nitrite content (A) and 24-hour urinary excretion (B) of nitrate plus nitrite (NOx) in ACE2^-/y (n = 6) and WT (n = 6) mice. (C) eNOS activity, evaluated by ^•NO release in aorta rings upon application of ACh (DAF-FM bioassay) (ACE2 n = 5; WT n = 6). (D) arginase activity in aorta homogenates of ACE2^-/y (n = 6) and WT (n = 6) mice. Representative western blot and densitometric analysis of eNOS (E) and phosphorylated Ser1177-eNOS (F) protein levels in thoracic aortas from ACE2^-/y (n = 5) and WT (n = 5) mice. (G) Relative expression of phospho-eNOS normalized to the total eNOS levels. (H) eNOS mRNA expression levels (real time PCR analysis). Data are expressed as mean ± SEM. *P<0.05, **P<0.01, **P<0.001, Student’s t test.

Fig 3. AKT expression in ACE2^-/y mice.

Representative western blots and densitometric analysis of HSP90 (A), AKT (B) and P-AKT (C) aortic protein levels. (D) P-AKT/AKT ratio. Results are representative of four to six separate experiments. Data are expressed as mean ± SEM.

Fig 4. Oxidative stress and ROS degrading enzymes in ACE2^-/y mice.

Concentration of the TBARS malondialdehyde (MDA) in plasma (A), urine (B), and aorta (C) of ACE2^-/y (n = 5) and WT (n = 5) mice. (D) Representative fluorescent photomicrographs (left) and quantification (right) showing in situ detection of ^⚫O₂^- by confocal microscopy (7 μm sections of thoracic aorta). Aortic sections were labeled with the oxidative dye dihydroethidium (2 μM/L), which is oxidized to EtBr in the presence of ^⚫O₂^- and gives red fluorescence. High EtBr fluorescence was found in aortic walls of ACE2^-/y mice, whereas the signal was almost undetectable in aortas of WT animals. (n = 5–6). (E) H₂O₂ levels in aorta of ACE2^-/y mice. Activity (F, I) and mRNA expression by real time PCR (G, H, J) of ROS degrading enzymes, SOD (F, G, H) and catalase (I, J) in aorta of ACE2^-/y (n = 4) and WT (n = 5) mice. (K) aorta GPx activity. mRNA expression level of UCP1 (L), UCP2 (M), and UCP3 (N) in aorta of ACE2^-/y mice. *P<0.05, **P<0.01 (Student t test).