Vascular Effects of Low-Dose ACE2 Inhibitor MLN-4760—Benefit or Detriment in Essential Hypertension?

Figure 1. The effect of MLN-4760 treatment on the activity of soluble RAS enzymes determined from angiotensin product/substrate ratios in plasma–plasma renin activity, PRA (a); soluble angiotensin converting enzyme, ACE-S (b); and the total alternative renin angiotensin system activity, Alt-S (c).

Figure 2. The correlation between the plasma level of the total alternative renin angiotensin system activity (Alt-S) and the weight of the visceral fat (a) and body weight (b). The correlation between the plasma level of angiotensin 1–7 (Ang 1–7) and the weight of the visceral fat (c) and body weight (d) in SHRs and SHRs treated with MLN-4760 (SHR + MLN).

Figure 3. The effect of MLN-4760 treatment on the integrated pressure responses of the cardiovascular system. The changes in mean blood pressure (ΔMAP) were followed after infusion of acetylcholine (1 µg/kg, (a)), noradrenaline (1 µg/kg, (b)), NOS inhibitor (L-NAME, 30 mg/kg, (c)), ACE inhibitor-captopril (10 mg/kg, (e)) and H2S scavenger bismuth(III) subsalicylate (BSC, 0.25 µg/kg, (f)). The effect of acetylcholine (1 µg/kg) also was followed before and after pretreatment with L-NAME (30 mg/kg, (d)). The results are presented as the mean ± S.E.M., and differences between groups were analyzed by Student´s t-test. * p < 0.05, ** p < 0.01 vs. SHR; ++ p < 0.01 vs. SHR + MLN.

Figure 4. Endothelium-dependent vasorelaxant response of the thoracic aorta (a), femoral artery (d) and mesenteric artery ((g,h)-AUC (area under curve) values). Noradrenaline-induced contractile response of the thoracic aorta ((b)-absolute response, (c)-percent values of the maximum response), femoral artery ((e)-absolute response, (f)-percent values of the maximum response) and mesenteric artery ((i)-absolute response). Arteries were isolated from control SHRs and SHRs treated with MLN-4760. The results are presented as the mean ± S.E.M., and differences between groups were analyzed by repeated two-way repeated measures ANOVA or Student´s t-test, as appropriate. * p < 0.05 vs. SHR; # p < 0.05 vs. maximal response to Ach.

Figure 5. The effect of acute Mas receptor inhibition on the endothelium-dependent vasorelaxation (a) and contractile response (b) of TA isolated from control SHRs and SHRs treated with MLN-4760. The mRNA levels of the Mas1 and ACE2 genes (c,e) and the protein expression of the Mas receptor and ACE2 (d,f) were measured in aortic tissue. The results are presented as the mean ± S.E.M., and differences between groups were analyzed by three-way ANOVA or Student’s t-test, as appropriate. * p < 0.05 vs. SHRs, ** p < 0.01 vs. SHRs; + p < 0.05, +++ p < 0.001 vs. SHR + MLN.

Figure 6. The effect of acute NO synthase (NOS) inhibition on endothelium-dependent vasorelaxation (a) and the contractile response (b) of TA isolated from control SHRs and SHRs treated with MLN-4760. The total NOS activity (c) and the protein expression of endothelial eNOS (d), neuronal nNOS (e) and inducible iNOS (f) were measured in aortic tissue. The results are presented as the mean ± S.E.M., and differences between groups were analyzed by three-way ANOVA or Student´s t-test, as appropriate, * p < 0.05, ** p < 0.01 vs. SHR, *** p < 0.001 vs. SHR; + p < 0.05 vs. SHR + MLN.

Figure 7. The effect of the H2S scavenger on endothelium-dependent vasorelaxation (a) and the contractile response (b) of TA isolated from control SHRs and SHRs treated with MLN-4760. The protein expression of cystathionine γ-lyase CSE (c) and cystathionine-β-synthase CBS (d) was measured in aortic tissue. The results are presented as the mean ± S.E.M., and differences between groups were analyzed by three-way ANOVA or Student´s t-test, as appropriate. * p < 0.05 vs. SHR, ** p < 0.01 vs. SHR; + p < 0.05 vs. SHR + MLN.

Figure 8. MLN-4760 reduces vascularization in the model of chick chorionallantoic membrane, determined as the number of vascular branching points in the area of MLN-4760 application compared to the area adjacent to the area of application (a). * p < 0.02 vs. SHR, x p < 0.02 vs. AAppl in the given group. Results are analyzed by two-way ANOVA. Next pictures are examples of chick chorionallantoic membrane in native yellow color with black Teflon circle placed on chick chorionallantoic membrane (b), and examples of evaluations of vascular branching in pictures of chick chorioallantoic membrane with colors inverted to blue in the control group (c) and in MLN-4760-treated chorioallantoic membranes (d) after the removal of Teflon circles. Abbreviations: AAppl, area of drug application; AAdj, area adjacent to area of drug application.