MAS-mediated antioxidant effects restore the functionality of angiotensin converting enzyme 2-angiotensin-(1-7)-MAS axis in diabetic rat carotid

Abstract

We hypothesized that endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species during type I-diabetes impairs carotid ACE2-angiotensin-(1-7)-Mas axis functionality, which accounts for the impaired carotid flow in diabetic rats. We also hypothesized that angiotensin-(1-7) chronic treatment of diabetic rats restores carotid ACE2-angiotensin-(1-7)-Mas axis functionality and carotid flow. Relaxant curves for angiotensin II or angiotensin-(1-7) were obtained in carotid from streptozotocin-induced diabetic rats. Superoxide or hydrogen peroxide levels were measured by flow cytometry in carotid endothelial cells. Carotid flow was also determined. We found that endothelial AT1-activated NAD(P)H oxidase-driven generation of superoxide and hydrogen peroxide in diabetic rat carotid impairs ACE2-angiotensin-(1-7)-Mas axis functionality, which reduces carotid flow. In this mechanism, hydrogen peroxide derived from superoxide dismutation inhibits ACE2 activity in generating angiotensin-(1-7) seemingly by activating I(Cl,SWELL0, while superoxide inhibits the nitrergic Mas-mediated vasorelaxation evoked by angiotensin-(1-7). Angiotensin-(1-7) treatment of diabetic rats restored carotid ACE2-angiotensin-(1-7)-Mas axis functionality by triggering a positive feedback played by endothelial Mas receptors, that blunts endothelial AT1-activated NAD(P)H oxidase-driven generation of reactive oxygen species. Mas-mediated antioxidant effects also restored diabetic rat carotid flow, pointing to the contribution of ACE2-angiotensin-(1-7)-Mas axis in maintaining carotid flow.

Figure 1

Concentration-response curves for angiotensin II in endothelium-intact (E+) or -denuded (E−) carotid rings from control or diabetic rats, over the precontraction induced by phenylephrine (PE). Representative traces from angiotensin II-evoked relaxation in E+ control (a) or diabetic (b) rat carotid. Effect of the in vitro pretreatment with DX600, A779, or PD123,319 in carotid rings from control (c) or diabetic (d) rats. Effect of the in vitro pretreatment with apocynin, tiron, PEG-catalase, or the chronic (in vivo) treatment with angiotensin-(1–7) in carotid rings from control (e) or diabetic (f) rats. Angiotensin II E _max⁡ in carotid arteries from control or diabetic rats before or after the in vitro pretreatment with DX600, A779, PD123,319 (g), apocynin, tiron, or PEG-catalase or the chronic treatment (in vivo) with angiotensin-(1–7) (h). The values are significantly different (P < 0.01; n = 9) from nonpretreated E+ (∗) or E− (∗∗) carotid rings from nontreated control rats, from nonpretreated E+ (#) or E− (##) carotid rings from nontreated diabetic rats, or from PEG-catalase pretreated E+ (†) carotid rings from nontreated control rats.

Figure 2

Concentration-response curves for angiotensin-(1–7) in endothelium-intact (E+) or -denuded (E−) carotid rings from control or diabetic rats, over the precontraction induced by phenylephrine (PE). Representative traces from angiotensin-(1–7)-evoked relaxation in E+ control (a) or diabetic (b) rat carotid. Effect of the in vitro pretreatment with A779, PD123,319 or hydroxocobalamin in carotid rings from control (c) or diabetic (d) rats. Effect of the in vitro pretreatment with apocynin, tiron, PEG-catalase, or the chronic (in vivo) treatment with angiotensin-(1–7) in carotid rings from control (e) or diabetic (f) rats. Angiotensin-(1–7) Emax in carotid from control or diabetic rats before or after the in vitro pretreatment with A779, PD123,319, hydroxocobalamin (g), apocynin, tiron, or PEG-catalase or the chronic (in vivo) treatment with angiotensin-(1–7) (h). The values are significantly different (P < 0.01; n = 9) from nonpretreated E+ (∗) or E− (∗∗) carotid rings from nontreated control rats, from nonpretreated E+ (#) or E− (##) carotid rings from nontreated diabetic rats, or from PEG-catalase pretreated E+ (†) carotid rings from nontreated control rats.

Figure 3

Concentration-response curves for angiotensin II or angiotensin-(1–7) in endothelium-intact (E+) carotid from control or diabetic rats after the in vitro pretreatment with losartan or DCPIB. Effect of the in vitro pretreatment with losartan or DCPIB on the relaxation induced by angiotensin II (a) or angiotensin-(1–7) (b). Angiotensin II E _max⁡ (c) or angiotensin-(1–7) E _max⁡ (d) before or after the in vitro pretreatment with losartan or DCPIB. The values are significantly different (P < 0.01; n = 9) from nonpretreated carotid rings (E+) from nontreated control (∗) or diabetic (#) rats.

Figure 4

O₂ ⁻ levels in carotid endothelial cells from control or diabetic rats. Dot plots show the gates of carotid endothelial cells from nontreated control rats (a) or from nontreated (b) or angiotensin-(1–7)-treated (c) diabetic rats. Histograms show the fluorescence emitted by DHE-loaded carotid endothelial cells from nontreated control rats (d) or from nontreated (e) or angiotensin-(1–7)-treated diabetic rats (f). Effects of the in vitro pretreatment with tiron, apocynin, or losartan or the chronic (in vivo) treatment with angiotensin-(1–7) combined or not with A779 on the fluorescence emitted by DHE-loaded endothelial cells samples (g). The values are significantly different (P < 0.01; n = 5) from the respective blank samples from nontreated rats (∗), from nonpretreated DHE-loaded endothelial cells samples from nontreated control (∗∗) or diabetic (∗∗∗) rats, or from the respective DHE-loaded endothelial cells samples from angiotensin-(1–7)-treated rats (#).

Figure 5

H₂O₂ levels in carotid endothelial cells from control or diabetic rats. Dot plots show the gates of carotid endothelial cells from nontreated control rats (a) or from nontreated (b) or angiotensin-(1–7)-treated diabetic rats (c). Histograms show the fluorescence emitted by CDCF-DA-loaded carotid endothelial cells from nontreated control rats (d) or from nontreated (e) or angiotensin-(1–7)-treated diabetic rats (f). Effects of the in vitro pretreatment with PEG-catalase, tiron, apocynin, or losartan or the chronic (in vivo) treatment with angiotensin-(1–7) combined or not with A779 on the fluorescence emitted by CDCF-DA-loaded endothelial cells samples (g). The values are significantly different (P < 0.01; n = 5) from the respective blank samples from nontreated rats (∗), from nonpretreated CDCF-DA-loaded endothelial cells samples from nontreated control (∗∗) or diabetic (∗∗∗) rats, or from CDCF-DA-loaded endothelial cells samples from angiotensin-(1–7)-treated diabetic rats (#).

Figure 6

ACE2, angiotensin-(1–7) and Mas receptors staining in carotid arteries from nontreated control or diabetic rats. ACE2 expression in nontreated control (a) or diabetic (b) rat carotid, angiotensin-(1–7) levels in nontreated control (c) or diabetic (d) rat carotid, and Mas receptors expression in nontreated control (e) or diabetic (f) rat carotid. E: endothelium; M: media; Adv: adventitia. The immunostaining is denoted in red (magnification: 100x).

Figure 7

Conclusive graphical abstract. Endothelial AT₁-activated NAD(P)H oxidase-driven generation of O₂ ⁻ and H₂O₂ in carotid arteries from type I-diabetic rats impairs the functionality of the local vasoprotective ACE2-angiotensin-(1–7)-Mas axis, which in turn impairs carotid blood flow. In this mechanism, H₂O₂ derived from O₂ ⁻ dismutation inhibits ACE2 activity in generating angiotensin-(1–7) by activating I _Cl,SWELL, while O₂ ⁻ inhibits the nitrergic vasorelaxant effect evoked by angiotensin-(1–7) upon Mas receptors activation. The chronic treatment of diabetic rats with angiotensin-(1–7) restores the functionality of carotid ACE2-angiotensin-(1–7)-Mas axis by triggering a positive feedback on this axis, played by a residual population of endothelial Mas-receptors that blunts the endothelial AT₁-activated NAD(P)H oxidase-driven generation of reactive oxygen species in rat carotid. Mas-mediated antioxidant effects evoked by the chronic treatment with angiotensin-(1–7) also restores carotid resistance and blood flow in diabetic rats, pointing the important contribution of the ACE2-angiotensin-(1–7)-Mas axis in maintaining carotid function.