Angiotensin-(1–7) abrogates angiotensin II-induced proliferation, migration and inflammation in VSMCs through inactivation of ROS-mediated PI3K/Akt and MAPK/ERK signaling pathways

The proliferation, migration and inflammation of vascular smooth muscle cells (VSMCs) contribute to the pathogenesis and progression of several cardiovascular diseases such as atherosclerosis and hypertension. Angiotensin (Ang)-(1–7) and Ang II are identified to be involved in regulating cardiovascular activity. The present study is designed to determine the interaction between Ang-(1–7) and Ang II on VSMCs proliferation, migration and inflammation as well as their underlying mechanisms. We found that Ang-(1–7) significantly suppressed the positive effects of Ang II on VSMCs proliferation, migration and inflammation, as well as on induction of the phosphorylation of Akt and ERK1/2 and increase of superoxide anion level and NAD(P)H oxidase activity in VSMCs, whereas Ang-(1–7) alone had no significant effects. This inhibitory effects of Ang-(1–7) were abolished by Mas receptor antagonist A-779. In addition, Ang II type 1 (AT1) receptor antagonist losartan, but not A-779, abolished Ang II induced VSMCs proliferation, migration and inflammation responses. Furthermore, superoxide anion scavenger N-acetyl-L-cysteine (NAC) or NAD(P)H oxidase inhibitor apocynin inhibited Ang II-induced activation of Akt and ERK1/2 signaling. These results indicate that Ang-(1–7) antagonizes the Ang II-induced VSMC proliferation, migration and inflammation through activation of Mas receptor and then suppression of ROS-dependent PI3K/Akt and MAPK/ERK signaling pathways.


Ang-(1-7) suppressed Ang II-induced migration in VSMCs.
The transwell Boyden chamber experiment exhibited that treatment VSMCs with Ang II for 24 h markedly increased the number of VSMCs that migrated through transwell chamber. More important, after the VSMCs were pre-incubated with Ang-(1-7) for 5 min, Ang II-induced VSMC migration was significantly depressed. This inhibitory effect of Ang-(1-7) on Ang II was abolished by A-779 ( Fig. 2A). AT 1 receptor antagonist losartan effectively inhibited Ang II-induced VSMCs migration, but A-779 pretreatment was unable to prevent the VSMC migration induced by Ang II. Neither losartan nor A-779 alone has effect to induce migration of VSMCs (Fig. 2B).

Ang-(1-7) prevented Ang II-induced phosphorylation of Akt and ERK1/2. The phosphorylation of
Akt and ERK1/2 were markedly increased after treatment VSMC with Ang II for 30 min, lasting at least 60 min. However, total ERK1/2 (t-ERK1/2) and total Akt (t-Akt) protein levels were not influenced by Ang II (Fig. 4A). Pretreatment VSMCs with Ang-(1-7) for 5 min significantly inhibited Akt and ERK1/2 phosphorylation induced by Ang II, and this effect was also blocked by A-779 (Fig. 4B). The increased phosphorylation of Akt and ERK1/2 induced by Ang II were effectively blocked by pre-incubation of losartan, rather than A-779, while either losartan or A-779 alone has no effect to induce phosphorylation of Akt and ERK1/2 in VSMCs (Fig. 4C).
Ang-(1-7) diminished Ang II-induced ROS production. The ROS production was detected after the confluent VSMCs were stimulated by Ang II (100 nmol/l) for different time (0, 5, 10, 30 min). Both dihydroethidium (DHE) fluorescence intensity and lucigenin-derived chemiluminescence method results showed that the superoxide anions production in VSMCs was remarkably enhanced upon Ang II stimulation for 30 min (Fig. 5A,C). NAD(P)H oxidase activity in VSMCs was also augmented in Ang II-treated VSMCs (Fig. 5C). The dramatic increases in superoxide anion level and NAD(P)H oxidase activity of VSMCs induced by Ang II were diminished by pre-treated with Ang- (1)(2)(3)(4)(5)(6)(7), and this effect of Ang-(1-7) was blocked by A-779 (Fig. 5B,C). The increased superoxide anion level and NAD(P)H oxidase activity in VSMCs induced by Ang II were blocked by pre-incubation of losartan, but not A-779. Both losartan and A-779 alone have no effect to influence superoxide anion level and NAD(P)H oxidase activity of VSMCs (Fig. 5D).

Discussion
Growing evidences suggest that dysfunction of VSMCs including proliferation, migration and inflammation is involved in the development of several diseases such as atherosclerosis and hypertension 13 . The Ang II/AT 1 receptor activity plays an important role in the proliferation, migration and inflammation of VSMCs in the development of the above diseases 29 . The present study demonstrates new findings that Ang-(1-7)/Mas receptor activity significantly inhibited Ang II-induced VSMC proliferation, migration and inflammation through inactivation of superoxide anions-mediated PI3K/Akt and MAPK/ERK signaling pathways in VSMCs.
VSMCs are quiescent in healthy mature vascular tissue, however, they are activated and initiated abnormal proliferation and migration in response to vascular injury 30 . Vascular injury also causes vascular inflammation which combining with proliferation and migration are recently accepted to be as the key contributors in the pathophysiology of hypertension and atherosclerosis 10 . Inhibition of VSMC proliferation, migration and inflammation is an important strategy for therapy of atherosclerosis-related diseases 31 . Among various circulatory factors, Ang II is a well-characterized pathophysiological culprit peptide which has various actions for not only maintaining the blood volume, modulating blood pressure, but also promoting the VSMC proliferation, migration, inflammation and apoptosis via AT 1 receptors in hypertension and atherosclerosis 32,33 . Incubation of rat tubular epithelial cells with Ang II significantly increased key pro-inflammatory cytokines expressions, which was blocked by losartan 34 . It is known that employment of Ang II receptor blockers may be helpful to abate inflammation processes and disease progression 35 . Inhibition of Ang II/AT 1 activity has protective effects on VSMCs 36,37 . Ang-(1-7) is another major active component in the RAS, which can be converted from Ang II by ACE2 21 . Studies reported that Ang-(1-7) and Ang II have complicated interactions in different parts of the body in different animal models. Some studies have shown that Ang-(1-7) plays an opposite role to Ang II and inhibits the effects of Ang II in some peripheral tissues 38,39 . Ang-(1-7) prevents Ang II-induced fibrosis in cremaster microvessels 28 . The Ang-(1-7)/Mas receptor axis is counteractive to Ang II-induced proliferation, migration and inflammation in human brain VSMC and cerebral microvessels 40,41 . ACE2 deficiency accelerated Ang II-induced mRNA expressions of inflammatory cytokines, including MCP-1 and IL-1β 42 . In the present study, we found that Ang II drastically stimulated the VSMC proliferation and migration, and markedly up-regulated the expressions of inflammatory mediators including MCP-1, VCAM-1 and IL-1β , while Ang-(1-7) had no significant effects to induce VSMC proliferation, migration and inflammatory responses. However pretreatment with Ang-(1-7) on VSMCs significantly inhibited Ang II-induced proliferation, migration and inflammatory responses. These protective effects of Ang-(1-7) on VSMCs were blocked by Mas receptors inhibitor A-779. These results indicate that Ang-(1-7) is a potential antagonist in the disruption of Ang II induced excessive proliferation, migration and inflammation of VSMCs and these effects of Ang-(1-7) are done through activation of Mas receptors. We also found that blockade of AT 1 receptors, but not Mas receptors, obviously diminished the proliferation, migration and inflammatory responses in VSMCs induced by Ang II, which indicates that the positive effects of Ang II on the VSMCs to induce proliferation, migration and inflammation is AT 1 receptor dependent.
Oxidative stress is a critical modulator in the progression of hypertension and atherosclerotic lesions 35 . NAD(P)H oxidase is emerged as a multi-component enzyme complex and one of major origins of the superoxide anions in vascular system 43 . NAD(P)H oxidase-derived ROS play a critical role in Ang II-induced proliferation and migration of VSMCs 44,45 . Increased ROS is involved in the pathogenesis of Ang II-dependent hypertension 46 . Suppression of ROS by knockdown of NAD(P)H oxidase largely inhibited the Ang II-induced proliferation, inflammation in human mesangial cells 47 . We also previously revealed that the NAD(P)H oxidase-derived superoxide anions in the paraventricular nucleus (PVN) are responsible for Ang II-induced sympathetic outflow in hypertensive rats 48 . Ang-(1-7) is recently reported to counteract the Ang II-induced ROS over-production in cerebral endothelial cells 8 . The nonpeptide AVE0991, an agonist of the Mas receptor, significantly attenuated ROS production in Ang II-treated VSMCs 49 . Our results in the present study also showed that both NAD(P)H oxidase activity and superoxide anion level increased significantly in Ang II-treated VSMCs. Pretreatment with Ang-(1-7) inhibited Ang II induced increases in NAD(P)H oxidase activity and superoxide anion level in VSMCs. This effect of Ang-(1-7) was also abolished by Mas receptors inhibitor A-779. These results indicated that NAD(P) H oxidase derived ROS may be responsible for mediating the effects of Ang II to induce VSMCs proliferation, Both MAPK pathway and PI3K/Akt pathway are essential intracellular signaling pathways involved in the VSMCs migration and proliferation during the processes of atherogenesis 50 . The stimulation of ROS-mediated MAPKs and PI3K/Akt signaling pathways play an important role in the human gastric cancer BGC-823 cells apoptosis 51 . The ROS-dependent phosphorylation of MAPK and PI3K/Akt act as key mediators in 6-hydroxydopamine-induced neuronal cell death 52 . In present study, we found that Ang II markedly increased the phosphorylation of Akt and ERK1/2 in VSMCs, which was blocked by losartan, but not A-779. Furthermore, pretreatment with either ROS scavenger or NAD(P)H oxidase inhibitor abolished the activation of Akt and ERK1/2 induced by Ang II. These results indicated that Ang II may increase NAD(P)H oxidase activity via AT 1 receptors to induce superoxide anion over-production, then subsequent activation of PI3K/Akt and MAPK/ERK pathways, which is involved in the Ang II-elicited VSMCs proliferation, migration and inflammatory responses. More important, we found that the increased phosphorylation of Akt and ERK1/2 stimulated by Ang II were effectively inhibited by pre-incubation of Ang-(1-7) on VSMCs, which was blocked by Mas receptor inhibitor A-779. These results indicated that the activity of Ang-(1-7)/Mas receptor may have a potential role for vascular protection against Ang II effects via suppression of ROS-dependent PI3K/Akt and MAPK/ERK pathways.
In summary, the present study demonstrates that the ROS-dependent activation of PI3K/Akt and MAPK/ERK pathways may be critical contributors in mediating the Ang II-induced proliferation, migration and inflammation of VSMCs. Ang-(1-7) abrogates Ang II-induced proliferation, migration and inflammation by activation of Mas receptor in the VSMC membrane and then inactivation of ROS-mediated PI3K/Akt and MAPK/ERK signaling in cytoplasm. Application of antagonist of AT 1 receptors or agonist of Mas receptors may provide

Methods
Experiments were carried out by using male Sprague-Dawley rats. Experiments and procedures were approved by the Experimental Animal Care and Use Committee of Nanjing Medical University and conformed to the Guide for the Care and Use of Laboratory Animal published by the US National Institutes of Health (NIH publication, 8th edition, 2011). The rats were housed in a temperature-controlled room with a 12 h-12 h light/dark cycle and with standard chow and tap water ad libitum.
Culture of primary VSMCs. The primary culture of VSMCs from rats' aortas was prepared as described previously 53 . Briefly, excised aortas were cut longitudinally and placed in digestion flasks with collagenase (type 1, 2 mg/ml; Sigma-Aldrich, St Louis, Missouri, USA) for 20 min at 37 °C in a shaker bath. Aortas were then cut into 1-2 mm segments, incubated with collagenase and elastase (0.5-1 mg/aorta; Sigma-Aldrich) in Hanks balanced salt solution for 1-2 h at 37 °C until single-cell suspension was achieved. VSMCs were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS and 1% antibiotics (Gibco, MD, USA) at 37 °C in a 5% CO 2 humidified incubator. The VSMCs were passaged at a ratio of 1:3 until confluence was reached. Cells in the second to sixth passages were used and cells at 80% to 90% confluence were arrested by incubating in serum-free DMEM for 24 hours before stimulation.
Cell proliferation assays. The proliferation of VSMCs was evaluated with Cell Counting Kit-8 kits (CCK-8, Beyotime Institute of Biotechnology, Shanghai, China) as we previously reported 54 .
Cell migration assays. The VSMC migration was assessed by a Boyden chamber assay. In brief, the quiescent VSMCs were seeded onto the upper surface of an Millicell transwell chamber of 8-μ m (Merck Millipore, Billerica, Massachusetts, USA) in serum-free medium and then pretreated with chemicals for 5 minutes, then treated with the addition of Ang II only in the lower chamber. After that cells were incubated at 37 °C in air containing 5% CO 2 . After 24 h of incubation, the cells that migrated to the lower surface of the filter were fixed by methanol and stained by 1% crystal violet. The number of stained cells from at least 4 fields for each well was counted using a microscope 55 . Western Blot. The VSMCs were lysed in lysis buffer on ice for 15 min, and the soluble lysates were centrifuged at 4 °C for 10 min at 12,000 rpm. The protein concentration of supernatant in each sample was determined with BCA method. The equal amounts of proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by electrophoresis and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk in Tris buffered saline with Tween-20 (TBST) for 60 min at room temperature and then hybridized overnight with indicated antibodies against PCNA (1:200 After washing 3 times with TBST, the membranes were incubated with indicated secondary antibodies coupled to horseradish peroxidase for 1 h at room temperature. The signals of target proteins were visualized using the enhanced chemiluminescent reagent (Merck Millipore, Billerica, Massachusetts, USA). Densitometric analysis of bands was quantified with Image-J software (NIH, USA).

Detection of intracellular superoxide anion level and NAD(P)H oxidase activity.
After incubation, the VSMCs were washed with Hanks' balanced salt solution (HBSS) and incubated with DHE (10 μ M) for 30 min in a light-protected humidified chamber. The DHE fluorescence was measured at an excitation wavelength of 488 nm and an emission wavelength of 585 nm and the mean fluorescence intensity of DHE was assessed by Image-Pro Plus 6.0 by using the same parameters. The intracellular superoxide anion level and NAD(P)H oxidase activity were also measured with lucigenin-derived chemiluminescence method as we previously described 56 . Chemicals. Ang-(1-7) and A-779 were purchased from Bachem (Bubendorf, Switzerland). Losartan, NAD(P)H, apocynin, dimethyl sulfoxide (DMSO), lucigenin and Ang II were obtained from Sigma Chemical (St. Louis, MO, USA). NAC and DHE were obtained from Beyotime Biotechnology (Shanghai, China). Ang-(1-7) and Ang II were made fresh before each experiment.

Statistical analysis.
Comparisons between two groups were made by Student's t test. One-way or two-way ANOVA followed by post hoc Bonferroni test was used when multiple comparisons were made. All data were expressed as mean ± SE. A value of P < 0.05 was considered statistically significant.