The Angiotensin-(1-7)/Mas Axis Counteracts Angiotensin II-Dependent and -Independent Pro-inflammatory Signaling in Human Vascular Smooth Muscle Cells

FIGURE 1. Ang-(1-7) inhibits inducible nitric oxide synthase (iNOS) induction elicited by Ang II or IL-1β in human aortic smooth muscle cells (HASMC). (A) HASMC were stimulated with Ang II (100 nM) alone or in the presence of the AT1 receptor antagonist losartan (1 μM) for 18 h, after which iNOS levels were determined by Western blot and normalized to α-tubulin. (B) In another set of experiments, the impact of increasing concentrations of Ang-(1-7) (1 nM–1 μM) on iNOS induction by Ang II was tested. (C) The pro-inflammatory cytokine IL-1β (2.5 ng/mL) promoted iNOS induction, which was prevented by the IL1 receptor blocker anakinra (1 μg/mL). (D) As for Ang II, Ang-(1-7) prevented the induction of iNOS by IL-1β in a concentration-dependent manner. Results are expressed as mean ± SEM; n = 5. Representative blots are shown in the corresponding panels. ∗p < 0.05 vs. untreated cultures, #p < 0.05 vs. Ang II- or IL-1β-treated cultures, respectively.

FIGURE 2. The inhibition of iNOS induction mediated by Ang-(1-7) is dependent on Mas receptors. After exposing HASMC for 18 h to (A) Ang II (100 nM) or (B) IL-1β (2.5 ng/mL) with or without 100 nM Ang-(1-7), iNOS levels were determined by Western blot. In some experiments, cells were pre-incubated with the Mas receptor antagonist A779 (1 μM). (C) iNOS levels (red) were also determined by indirect immunofluorescence in response to the different stimuli. Nuclei were counterstained with DAPI (blue) (400×). In another set of experiments, nitric oxide (NO) production was determined in the supernatants of HASMC exposed for 18 h to Ang II (D) or IL-1β (E), with or without Ang-(1-7) and A779. The Mas receptor antagonist D-Pro7-Ang-(1-7) (1 μM) was also capable to block the inhibitory effect of Ang-(1-7) on iNOS induction by Ang II (F) or IL-1β (G). Results are expressed as mean ± SEM; n = 5. Representative blots are shown in the corresponding panels. ∗p < 0.05 vs untreated cultures, #p < 0.05 vs Ang II- or IL-1β-treated cultures, respectively.

FIGURE 3. Ang-(1-7) reduces NF-κB activation via Mas receptors in HASMC. The activation of NF-κB induced after 18 h incubation with (A) Ang II (100 nM) or (B) IL-1β (2.5 ng/mL) was blunted by the antioxidant NADPH oxidase inhibitor apocynin (30 μM) and significantly reduced by Ang-(1-7) (100 nM), as determined by EMSA. The effect of Ang-(1-7) was prevented by the Mas receptor antagonist A779 (1 μM). The induction of iNOS by Ang II (C) or IL-1β (D) was abolished by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) (100 μM). Representative blot or EMSA images are shown in the corresponding panels. Results are expressed as mean ± SEM; n = 4. ∗p < 0.05 vs untreated cultures, #p < 0.05 vs Ang II- or IL-1β-treated cultures, respectively.

FIGURE 4. Ang-(1-7) reduces NADPH oxidase activity via Mas receptors. In HASMC, Ang-(1-7) (100 nM) reduced the stimulation of NADPH oxidase observed after 18 h incubation with (A) Ang II (100 nM) or (B) IL-1β (2.5 ng/mL), and this effect was blocked by the Mas receptor antagonist A779 (1 μM). The inhibition of NADPH oxidase by the antioxidant inhibitor apocynin (30 μM) prevented the induction of iNOS by Ang II (C) or IL-1β (D). Representative blots are shown in the corresponding panels. The NADPH oxidase activity is given as relative light units (RLUs)/μg protein/min. Results are expressed as mean ± SEM; n = 4. ∗p < 0.05 vs untreated cultures, #p < 0.05 vs Ang II- or IL-1β-treated cultures, respectively.

FIGURE 5. Schematic diagram of the proposed action of the Ang-(1-7)/Mas axis on HAMSC pro-inflammatory signaling. The binding of Ang II or IL-1β to their respective membrane receptors results in NADPH oxidase activation, which consecutively leads to NF-κB activation, iNOS induction and NO release. Through an effect mediated by Mas receptors, Ang-(1-7) promotes NADPH oxidase inhibition and thus attenuates the pro-inflammatory signaling triggered by Ang II, but also by other non-RAS inflammatory stimuli, such as IL-1β.