BACKGROUND AND PURPOSE ApolipoproteinE‐deficient [apoE (−/−)] mice, a model of human atherosclerosis, develop endothelial dysfunction caused by decreased levels of nitric oxide (NO). The endogenous peptide, angiotensin‐(1‐7) [Ang‐(1‐7)], acting through its specific GPCR, the Mas receptor, has endothelium‐dependent vasodilator properties. Here we have investigated if chronic treatment with Ang‐(1‐7) improved endothelial dysfunction in apoE (−/−) mice. EXPERIMENTAL APPROACH ApoE (−/−) mice fed on a lipid‐rich Western diet were divided into three groups and treated via osmotic minipumps with either saline, Ang‐(1‐7) (82 µg·kg−1·h−1) or the same dose of Ang‐(1‐7) together with D‐Ala‐Ang‐(1‐7) (125 µg·kg−1·h−1) for 6 weeks. Renal vascular function was assessed in isolated perfused kidneys. KEY RESULTS Ang‐(1‐7)‐treated apoE (−/−) mice showed improved renal endothelium‐dependent vasorelaxation induced by carbachol and increased renal basal cGMP production, compared with untreated apoE (−/−) mice. Tempol, a reactive oxygen species (ROS) scavenger, improved endothelium‐dependent vasorelaxation in kidneys of saline‐treated apoE (−/−) mice whereas no effect was observed in Ang‐(1‐7)‐treated mice. Chronic treatment with D‐Ala‐Ang‐(1‐7), a specific Mas receptor antagonist, abolished the beneficial effects of Ang‐(1‐7) on endothelium‐dependent vasorelaxation. Renal endothelium‐independent vasorelaxation showed no differences between treated and untreated mice. ROS production and expression levels of the NAD(P)H oxidase subunits gp91phox and p47phox were reduced in isolated preglomerular arterioles of Ang‐(1‐7)‐treated mice, compared with untreated mice, whereas eNOS expression was increased. CONCLUSION AND IMPLICATIONS Chronic infusion of Ang‐(1‐7) improved renal endothelial function via Mas receptors, in an experimental model of human cardiovascular disease, by increasing levels of endogenous NO.

Abstract-Apolipoprotein E-deficient (apoE(−/−)) mice fed on Western diet are characterized by increased vascular resistance and atherosclerosis. Previously, we have shown that chronic angiotensin (Ang)(1-7) treatment ameliorates endothelial dysfunction in apoE(−/−) mice. However, the mechanism of Ang(1-7) on vasoconstrictor response to Ang II is unknown. To examine Ang(1-7) function, we used apoE(−/−) and wildtype mice fed on Western diet that were treated via osmotic minipumps either with Ang(1-7) (82 μg/kg per hour) or saline for 6 weeks. We show that Ang II-induced renal pressor response was significantly increased in apoE(−/−) compared with wildtype mice. This apoE(−/−)specific response is attributed to reactive oxygen species-mediated p38 mitogen-activated protein kinase activation and subsequent phosphorylation of myosin light chain (MLC 20 ), causing renal vasoconstriction. Here, we provide evidence that chronic Ang(1-7) treatment attenuated the renal pressor response to Ang II in apoE(−/−) mice to wildtype levels. Ang(1-7) treatment significantly decreased renal inducible nicotinamide adenine dinucleotide phosphate subunit p47phox levels and, thus, reactive oxygen species production that in turn causes decreased p38 mitogenactivated protein kinase activity. The latter has been confirmed by administration of a specific p38 mitogenactivated protein kinase inhibitor SB203580 (5 μmol/L), causing a reduced renal pressor response to Ang II in apoE(−/−) but not in apoE(−/−) mice treated with Ang(1-7). Moreover, Ang(1-7) treatment had no effect in Mas(−/−)/apoE(−/−) doubleknockout mice confirming the specificity of Ang (1-

ApoE-KO mice are characterized by endothelial dysfunction and increased vasoconstrictor response. Previously, we have shown that chronic Ang-(1-7) treatment ameliorates endothelial dysfunction in apoE-KO. However, the impact of Ang-(1-7) on vasoconstrictor response to AngII is unknown. To examine the role of Ang-(1-7) in vascular resistance in atherosclerosis, apoE-KO and wild type (WT) mice fed on western diet were treated via osmotic minipumps either with Ang-(1-7) (82μg/kg/hr) or saline for 6 weeks. Vascular resistance was tested in isolated perfused kidneys. AngII induced renal pressor response was significantly increased in apoE-KO compared to WT mice. Remarkably, chronic Ang-(1-7) treatment attenuated AngII induced pressor response in apoE-KO mice. Accordingly, in apoE-KO, phospho-MLC20, a marker of enhanced contractility was increased in preglomerular vessels compared to WT and reduced after Ang-(1-7) treatment. To examine the underlying cause, we measured reactive oxygen species (ROS) production and changes in NADP(H) expression levels. In apoE-KO mice, p47phox was increased (1.5-fold; P <0.05) compared to WT and reduced after chronic Ang-(1-7) treatment. Concordantly, urinary isoprostane-8 excretion and H2O2 generation were significantly decreased in Ang-(1-7) treated apoE-KO mice. ROS is known to activate the MAPK p38. Moreover, p38 has shown to modulate pressor response. To test the role of p38 in our model, we measured p38 activity and renal pressor response to AngII in the presence of SB203580, a specific p38 inhibitor. Thus, in preglomerular vessels of apoE-KO, the phospho-p38 / p38 ratio was increased compared to WT. Chronic Ang-(1-7) treatment restored this ratio almost to WT levels (2.8±0.5 vs. 1.0±0.1 vs. 1.3±0.2 P <0.05). Administration of SB203580 (5μmol/L) reduced renal pressor response to AngII in apoE-KO but not in Ang-(1-7) treated mice. Hence, chronic inhibition of p38 with BIRB796 (30mg/kg) attenuated the increased pressor response to AngII in apoE-KO mice. In summary, p38 plays a crucial role in regulating renal vascular resistance in apoE-KO mice. Moreover, Ang-(1-7) attenuates pressor response to AngII by reducing p38 activity. Thus, this study revealed a new pathway how Ang-(1-7) modulates vascular function.

No abstract