AVE0991, a Nonpeptide Compound, Attenuates Angiotensin II-Induced Vascular Smooth Muscle Cell Proliferation via Induction of Heme Oxygenase-1 and Downregulation of p-38 MAPK Phosphorylation

Abstract

The nonpeptide AVE0991 is an agonist of the angiotensin-(1-7) (Ang-(1-7)) Mas receptor and is expected to be a putative new drug for treatment of cardiovascular disease. However, the mechanisms involved in the antiproliferative effects of AVE0991 are not fully understood. We saw that the compound attenuated proliferation in an angiotensin II-induced rat vascular smooth muscle cells (VSMC) proliferation model. Moreover, treatment with AVE0991 (10(-5) mol/L or 10(-7) mol/L) significantly attenuated reactive oxygen species (ROS) production, phosphorylation of p38 MAPK, and dose-dependently (10(-8) to 10(-5) mol/L) inhibited Ang II-induced VSMC proliferation. Meanwhile, heme oxygenase-1 (HO-1) expression increased in the AVE0991 + Ang II group (10(-5) mol/L or 10(-6) mol/L). However, the beneficial effects of AVE0991 were completely abolished when the VSMC were pretreated with A-779 (10(-6) mol/L). Furthermore, treatment with the HO-1 inhibitor ZnPPIX attenuated the inhibitory effect of AVE0991 on Ang II-induced p38MAPK phosphorylation. These results suggest that AVE0991 attenuates Ang II-induced VSMC proliferation in a dose-dependent fashion and that this effect is associated with the Mas/HO-1/p38 MAPK signaling pathway.

Figure 1

The analysis of cultured VSMCs in morphology and identification. (a) The morphology of cultured VSMCs detected by phase-contrast microscope. Magnification of light microscopy images is ×100. (b) SM-α actin immunocytochemical staining of cultured VSMCs. Magnification of light microscopy images is ×400.

Figure 2

The effect of various concentrations of AVE0991 on the VSMCs [³H]thymidine incorporation efficiency when stimulated with Ang II. *P < 0.05; **P < 0.01 versus control group; ^▵ P < 0.05  versus Ang II + AVE0991 (10⁻⁶mol/L) group; n = 8.

Figure 3

The effect of AVE0991 on the [³H]thymidine incorporation efficiency of VSMCs that were stimulated by Ang II. *P < 0.05; **P < 0.01 versus control group; ^▵▵ P < 0.01 versus Ang II group; ^□ P < 0.05 versus Ang II + AVE0991-H group; n = 8.

Figure 4

The effect of AVE0991 on Ang II-stimulated ROS production in VSMCs. *P < 0.05; **P < 0.01 versus control group; ^▵ P < 0.01 versus Ang II group; ^□ P < 0.05 versus Ang II + AVE0991-H group; n = 8.

Figure 5

The effect of AVE0991 on the Ang II-induced VSMCs [³H]thymidine incorporation efficiency with pretreatment with the HO-1 inhibitor ZnPPIX. **P < 0.01 versus control group; ^▵ P < 0.05; ^▵▵ P < 0.05 versus Ang II group; ^□ P < 0.01 versus Ang II + AVE0991 group; n = 8.

Figure 6

The effect of AVE0991 on Ang II-stimulated ROS production in VSMCs that were pretreated with the HO-1 inhibitor ZnPPIX. *P < 0.05; **P < 0.01 versus control group; ^▵ P < 0.01 versus Ang II group; ^□ P < 0.01 versus Ang II + AVE0991 group; n = 8.

Figure 7

The effect of AVE0991 on the p38 phosphorylation level in VSMCs that were induced by Ang II. Lanes 1–7 represent the control, AVE0991, A-779, Ang II, Ang II + AVE0991-H, Ang II + AVE0991-H + A-779, and the Ang II + AVE0991-L group. **P < 0.01 versus control group; ^▵ P < 0.01 versus Ang II group; ^□ P < 0.01 versus Ang II + AVE0991-H group; n = 8.

Figure 8

The effect of AVE0991 on HO-1 protein expression in VSMCs that were induced by Ang II. *P < 0.01 versus control group; ^▵ P < 0.01 versus Ang II + AVE0991-H group; n = 8.

Figure 9

The effect of AVE0991 on the p38 phosphorylation level in VSMCs when combined with the HO-1 inhibitor ZnPPIX. **P < 0.01 versus control group; ^▵ P < 0.01 versus Ang II group; ^□ P < 0.01 versus Ang II + AVE0991 group; n = 8.