Research article
Ablation of angiotensin (1-7) receptor Mas in C57Bl/6 mice causes endothelial dysfunction

https://doi.org/10.1016/j.jash.2008.05.003Get rights and content

Abstract

The Mas gene codes for an angiotensin (1-7) receptor. There is accumulating evidence that Mas is involved in vascular homeostasis. We have recently backcrossed Mas-knockout mice to two different genetic backgrounds, C57Bl/6 and FVB/N. FVB/NMas-deficient mice exhibited elevation in blood pressure (BP) and impaired endothelial function. In the present study, we aimed to address the question whether this phenotype is strain-specific. Therefore, we evaluated endothelial function in C57Bl/6Mas-deficient mice. Similar to FVB/NMas-knockout animals, Mas-deficiency in C57Bl/6 mice leads to endothelial dysfunction evaluated by the acute BP effect of acetylcholine administration. Measurements of nitric oxide (NO) and reactive oxygen species (ROS) and the systems involved in their metabolism revealed an imbalance between these vasoactive factors in C57Bl/6Mas-knockout mice, which may explain the impairment of endothelial function in these animals. However, endothelial dysfunction was less prominent in Mas-deficient mice on a C57Bl/6 background compared to FVB/N. Moreover, C57Bl/6Mas-deficient mice remained normotensive while FVB/N-based animals exhibited elevated BP. The impairment of endothelium-dependent vasodilatory response to acetylcholine in two different mouse strains with Mas deficiency indicates a key role of Mas in endothelial function by its effects on the generation and metabolism of NO and ROS.

Introduction

The Mas gene codes for a G protein-coupled cell surface receptor (GPCR). Although the first attempts to clarify the function of Mas protein characterized it as an angiotensin II (Ang II) receptor,1 our recent studies have shown that Mas binds angiotensin-(1-7) [Ang-(1-7)]2 and is involved in cardiovascular and neuronal regulation. Ang-(1-7) and Mas mainly exert protective effects in the cardiovascular system by reducing blood pressure (BP) and inhibiting cell growth,3, 4, 5, 6, 7 in part by hetero-oligomerization of Mas with the Ang II receptor AT1 and interfering with its signaling.8

The endothelium of blood vessels is an important target for the biological actions of Ang-(1-7).9 Accordingly, Mas expression was found in the endothelial layer of different vessel types such as coronaries and radial and interneural microvessels of the brain10 as well as in the vessels of the corpus cavernosum.11 Association of Mas expression with the endothelium was also shown for cultured endothelial cells derived from human aorta12 and rat cerebral resistance vessels,13 supporting an important role of this GPCR in the function of the endothelium. Sampaio et al12 recently showed that Ang-(1-7) via Mas counteracts Ang II and AT1-dependent c-Src activation and thereby extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and the generation of reactive oxygen species (ROS) by nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase in human endothelial cells. In order to test for the in-vivo relevance of these findings, we recently evaluated endothelial function in Mas gene-deleted mice on the FVB/N background.14 These mice exhibited elevation in BP and impaired endothelial function due to the imbalance between ROS and nitric oxide (NO) metabolism.

It is well known that different mouse strains show completely different responses to cardiovascular interventions such as renal ablation and deoxycorticosterone acetate salt treatment.15, 16 Therefore, in this study we tested whether the endothelial dysfunction observed in Mas-deficient animals is strain specific, by characterizing basal cardiovascular parameters, endothelial function, and NO/ROS metabolism in Mas-deficient mice, backcrossed to C57Bl/6 genetic background.17

Section snippets

Animals, BP Measurement, and Endothelial Function

Local German authorities approved the studies with standards corresponding to those prescribed by the American Physiological Society. Mice were maintained in individually ventilated cages (Tecniplast, Hohenpeissenberg, Germany) under standardized conditions with an artificial 12-hour dark-light cycle, with free access to standard chow (0.25% sodium; SSNIFF Spezialitäten, Soest, Germany) and drinking water ad libitum. All experiments were performed in adult (12 to 16 weeks old) male C57Bl/6Mas

Basal Cardiovascular Parameters and RAS Components in C57Bl/6/Mas-Deficient Mice

To determine the effects of Mas deficiency on regulation of BP, we first characterized basal cardiovascular parameters in Mas-deficient mice on the pure genetic C57Bl/6 background (C57Bl/6Mas−/−)17 by measuring BP in conscious, freely moving animals. Neither MAP nor heart rate was changed in the C57Bl/6Mas−/− mice in comparison to controls (Figures 1A and 1B). To examine the effect of the Mas mutation on main RAS components, we measured PRA and Ang I and II concentrations. None of these

Discussion

The only comprehensive way to study the importance of a gene in a physiological process is to evaluate the effect of its deletion on different genetic backgrounds. Therefore, we have backcrossed Mas-knockout mice22 to two different genetic backgrounds, FVB/N14 and C57Bl/6,17 and evaluated basal cardiovascular parameters and in-vivo endothelial function in these mouse lines.

Whereas in our recent study we have shown that Mas-knockout mice on FVB/N genetic background exhibit elevated BP,14Mas

Conclusion

Taken together, this study and our previous observation in FVB/NMas-deficient mice14 show that ablation of the Ang-(1-7) receptor Mas leads to an imbalance between ROS and NO and to endothelial dysfunction independently of genetic background, providing further evidence for a role of the ACE2/Ang-(1-7)/Mas axis in the regulation of endothelial function.12, 31 However, Mas deficiency has differential effects on the severity of endothelial dysfunction and the BP in two distinct mouse strains.

Acknowledgment

Drs. Luiza A. Rabelo and Ping Xu contributed equally to this work.

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    This study was supported by Fapemig/CNPq-Pronex (RASS) and DAAD/CAPES (PROBRAL). Part of the Proceedings of the Vasoactive Peptide Symposium, VII International Vasoactive Peptide Symposium in Ouro Preto, February 14–16, 2008.

    Conflict of interest: none.

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