Angiotensin (1-7) Inhibits Ang II-mediated ERK1/2 Activation by Stimulating MKP-1 Activation in Vascular Smooth Muscle Cells

The renin–angiotensin system (RAS) exerts profound physiological effects on blood pressure regulation and fluid homeostasis, mainly by modulating renal, cardiovascular, and central nervous systems. Angiotensin (Ang)-(1-7), an end-product of RAS, is recognized by its cardiovascular protective properties through stimulation of the Mas receptor, including vasodilation, anti-inflammatory, and antihypertensive actions, and consequently, counter-regulating the well-known Ang II-elicited actions. The overall hypothesis of this study is that Ang-(1-7) inhibits Ang II-induced ERK1/2 activation in vascular smooth muscle cells (VSMCs), via regulation of mitogen-activated protein phosphatase-1 (MKP-1) activity. Aortas from male Wistar rats were incubated with Ang-(1-7) or vehicle. Concentration-response curves to Ang II were performed in endothelium-denuded aortas, in the presence or absence of ERK1/2 (PD98059) inhibitor or Mas receptor (A-779) antagonist. Expression of proteins was assessed by western blot, and immunohistochemistry was conducted in VSMCs. Ang-(1-7) incubation decreased Ang II-induced contractile response in aortas, and this effect was not observed in the presence of PD98059 or A-779. Stimulation of VSMCs with Ang-(1-7) prevented Ang II-induced ERK1/2 phosphorylation, but not C-Raf-activation. Furthermore, Ang II decreased MKP-1 phosphorylation in VSMCs. Interestingly, simultaneous incubation of Ang-(1-7) with Ang II favored MKP-1 phosphorylation, negatively modulating ERK1/2 activation in VSMCs. The results suggest that Ang-(1-7) counter-regulates actions evoked by Ang II overproduction, as observed in cardiovascular diseases, mainly by modulating MKP-1 activity. This evidence suggests that the role of Ang-(1-7) in MKP-1-regulation represents a target for new therapeutic development.

he renin-angiotensin system (RAS) is well recognized for its physiological effects in the regulation of blood pressure and fluid homeostasis.
In the vasculature, angiotensin (Ang)-II, one of the most abundant component of this system, promotes several pro-hypertensive actions primarily via activation of vascular-resistance mechanisms (1,2).
However, the RAS complexity has been demonstrated, and other members from the RAS also display the capacity to modulate vascular effects, while some others display Ang-II counter actions. For example, Ang-(1-7) is accepted as an important biologically active peptide in the RAS.
At the molecular level, a cascade of phosphorylation and dephosphorylation modulates the activation status of ERK1/2 (18). Activated ERK1/2 phosphorylates substrates in the cytoplasm or nucleus, and thereby is involved in various physiological and pathological processes, such as cell growth, development, proliferation, and differentiation (19,20). When activated, the ERK1/2 also modulates the activity of caldesmon and calponin, allowing the actin-myosin interaction, eliciting vascular contraction (20,21).

Many phosphatases act as major regulators of
MAPKs pathway, and are likely to be one of the most energy-efficient controllers in their deactivation process (18,22). In mammalian cells, the dual-specificity protein phosphatases, also known as the MAP kinase phosphatases (MKPs), are the primary phosphatases responsible for deactivation of ERK1/2 (18). In this regard, MKP-1 was the first MKP identified which belongs to a family of inducible nuclear dual-specificity phosphatases exerting catalytic activity to dephosphorylation and inactivation of MAP kinase isoforms in mammalian cells and tissues (23,24).
Since MKP-1 deactivates MAPKs, this phosphatase regulates a number of physiological and pathophysiological processes including immunity, metabolic homeostasis, cellular responses to anticancer drugs, muscle regeneration, and neuronal function (18,24).
A body of evidence suggests that Ang-II decreases MKP-1 activity (25), favoring ERK1/2 activation. Besides that, the interplay between Ang-T II and Ang-(1-7), and their action on MKP-1 activity has not been investigated in VSMCs.
Therefore, in this study, we test the hypothesis that Ang-(1-7) counterbalances Ang II-mediated ERK1/2 activation, which attenuates the vascular contractile response evoked by Ang II, via modulation of MKP-1 activity.

Animals
Male Wistar rats, 10-12 weeks-old, obtained from the colony of the Federal University of Mato Grosso, Brazil, were used in this study. All

Vascular functional studies
After euthanasia, the thoracic aorta was

VSMCs isolation and culture
VSMCs were isolated from rat thoracic aortas by the explant technique, as previously described

Western blot analysis
Proteins (40 μg Sigma-USA) for 15 min to detect nuclei, as previously described (34,35). Cover slips were mounted, and labeled cells were examined using a Zeiss microscope and software.

Data Analysis
The results are shown as mean ± SEM (n), Values of P<0.05 were considered statistically significant.
The effect of Ang-(1-7) was also determined  The results are presented as mean ± SEM for each experimental group. The statistical significance of the data was determined using the Student's t test. * P < 0.05 vs. vehicle.

Discussion
Ang-(1-7) is considered as the main antagonist of Ang-II actions (7,8). However, our current understanding of the interplay between Ang-II and  to the opposite cellular actions (7). Besides that, it has been shown that Ang II evokes ERK1/2 activation via both angiotensin type 1 and type 2 receptors (53). Therefore, the use of reninangiotensin-aldosterone system inhibitors can also play a relevant role in these signaling pathways, in a synergic manner as Ang-(1-7).
In this sense, studies have been showing that the combination of Ang-(1-7) along with an angiotensin II type 1 receptor blocker (losartan) was more effective in vascular protective effect, regression of glomerulosclerosis, and antiatherosclerosis effects relative to either separate treatment (54,55). Therefore, the different reninangiotensin-aldosterone system inhibitors may promote cardiovascular protective properties through different mechanisms, consequently that combined treatment with two renin-angiotensinaldosterone system inhibitors might have a favorable effect than monotherapy.
Considering that activation of ERK1/2 in vascular tissues is observed in several pathological conditions including hypertension, diabetes, and atherosclerosis, our study provides further evidence that the role of Ang-(1-7) in MKP-1-regulation may represent a target for new therapeutic development.
Together, these findings pave the way for the development of new combination strategies that use Ang-(1-7), along with other currently used reninangiotensin-aldosterone system inhibitors, for the treatment of cardiovascular diseases (56).
In conclusion, these findings show that Ang-