Bazedoxifene Attenuates Abdominal Aortic Aneurysm Formation via Downregulation of Interleukin-6/Glycoprotein 130/Signal Transducer and Activator of Transcription 3 Signaling Pathway in Apolipoprotein E-Knockout Mice

Abstract

Abdominal aortic aneurysm (AAA) is a chronic inflammatory disease characterized by aortic dilatation and predominantly affects an elderly population. Accumulating evidence suggests that Interleukin-6 (IL-6) and the signal transducer and activator of transcription 3 (STAT3) play an important role in formation of AAAs. However, it remains unclear whether Bazedoxifene (BAZ) could suppress the activation of IL-6/GP130/STAT3 in vascular cells and the formation of AAA. Here we explored the effect of BAZ on AngII-stimulated AAA formation. ApoE^-/- mice infused with AngII for 28 days using osmotic minipumps were treated with placebo or 5mg/kg BAZ. In our results most of the AngII-induced mice developed AAA with exacerbated inflammation, degradation of elastin fibers, STAT3 phosphorylation, and increased expression of matrix metalloproteinases (MMPs). These effects were markedly attenuated by BAZ. Furthermore, BAZ suppressed the stimuli-induced (IL-6 or AngII) expression of P-STAT3, MMP2 and MMP9 in vascular smooth muscle cells (VSMCs). BAZ inhibited wound healing, colony formation and suppressed STAT3 nuclear translocation in vitro. In conclusion, these results indicated that BAZ downregulated IL-6/GP130/STAT3 signaling and interfered with AAA formation induced by AngII in ApoE^-/- mice, which indicates a novel potential strategy for the prevention and therapy of AAA.

Keywords: IL-6; STAT3; abdominal aortic aneurysm; bazedoxifene; inflammation.

Figure 1

The structure of BAZ and the effect of BAZ on the development of Ang-II-induced abdominal aortic aneurysm (A) Morphometrical change of AAA in ApoE^–/– mice. Representative images of macroscopic features in aneurysms from sacrificed mice after 28 days. Scale bar denotes 2 mm. (B) Histological change of aneurysms in mice shown by hematoxylin eosin (H&E) (Ba) and elastin van Gieson (EVG) (Bb) staining. Scale bar denotes 400 mm. (C) The incidence of AAA was calculated from all of the animals in each group. (D) BAZ treatment significantly deceased the external diameter of the aorta (n=9). (E) Relative aortic wall thickness of each group (n=9). (F) The structure of Bazedoxifene (BAZ). (G) Western blots for the expression of P-STAT3, T-STAT3, MMP2 and MMP9 in mice aneurysms. (H) Relative gray intensity to STAT3 and (I–K) relative gray intensity to GAPDH was calculated (n = 3). Data were expressed as mean ± standard error of the mean. ^#P < 0.05 compared with the control group, *p < 0.05, **p < 0.01 compared with the AngII group.

Figure 2

Effect of BAZ on the abdominal aorta via immunohistochemical staining in mice. (A) Expression and distribution of P-STAT3 and IL-6 (both brown-stained) in cross-sections of aorta from ApoE^–/–mice. (B) The expression of MMP2 and MMP9 was detected via immunohistochemical staining in the abdominal aorta of ApoE^–/– mice. (C) Representative immunohistochemical staining of CD68 in aortic cross-sections. (D) Representative images showing a-SMA expression in aortic cross-sections. The scale depicts 100μm for each interval in A(b) and 200μm in A(a) B, C, D.

Figure 3

BAZ suppresses the phosphorylation of STAT3 induced by IL-6 and AngII in VSMCs. Cells were pretreated with BAZ for 2 hours and then stimulated with IL-6 (A) and AngII (E), western blotting was used to show the expression of P-STAT3 and STAT3. (I) ELISA was used to exam the effect of AngII on the secretion of IL-6 in vitro. (J) The effect of sIL-6R at different concentrations on IL-6 signaling transduction. (N) The effect of AngII or sIL-6R on the expression of P-STAT3. (B, C, F, G, K, L, O, P) Relative gray intensity to GAPDH was calculated. (D, H, M, Q) Relative gray intensity to T–STAT3 was calculated. Data were expressed as mean ± standard error of the mean (n = 3). ^#P < 0.05 compared with the control; *P< 0.05, **P< 0.01compared with IL-6 or AngII.

Figure 4

BAZ suppresses the expression of MMP2 and MMP9 induced by IL-6 and AngII in VSMCs. Pretreatment of BAZ for 2 hours suppressed the increase in both MMP2 and MMP9 expression induced by IL-6 (A) and AngII (D). Relative gray intensity to GAPDH was calculated (n = 3) (B, C, E, F). The mRNA expression of MMP2 and MMP9 was assessed by qRT-PCR and normalized to GAPDH and is expressed in arbitrary units. BAZ suppressed the mRNA expression of MMP2 and MMP9 induced by IL-6 (I, J) and AngII (G, H). Data were presented as the mean ± SEM. ^#P < 0.05 compared with the control; *P< 0.05, **P< 0.01 compared with IL-6 or AngII.

Figure 5

BAZ suppresses STAT3 activation in nucleus and the nuclear translocation induced by IL-6 in VSMCs. After pretreatment with BAZ for 2 hours, cells were stimulated with IL-6 for another 30 minutes, and then STAT3 activation (A) and STAT3 nuclear translocation (B) were detected by immunofluorescence staining as described. Scale bar represents 200μm.

Figure 6

BAZ inhibits colony formation and wound healing in VSMCs. (A) Cells were pretreated with BAZ (15 or 20 mM) or DMSO for 4 h, then colony formation was evaluated after two weeks. Scale bar denotes 1 cm. (B) After BAZ treatment for 2h, the migration ability of VSMCs was detected using wound-healing assays. Scale bar represents 400 μm. (C) Cell migration was assessed. (D) BAZ reduced cell viability of VSMCs. *P< 0.05, **P< 0.01 compared with the control.