Aldosterone promotes calcification of vascular smooth muscle cells in mice through the AIF-1/Wnt/β-catenin signaling pathway

Objective: This study aimed to investigate the impact of aldosterone on calcification in murine vascular smooth muscle cells (VSMCs) via the allograft inflammatory factor-1 (AIF-1)/Wnt/β-catenin signaling pathway.

Methods: Mouse VSMCs were cultured in vitro, and calcification was induced by treatment with 100 nM aldosterone. The level of calcification in mouse VSMCs was evaluated using colorimetric assays to assess ALP activity and qRT-PCR to identify the expression of calcification-related markers, such as Runx2, α-SMA, OCN, and ALP mRNA. Western blot analysis was performed to determine the protein expression levels associated with the Wnt/β-catenin pathway (LRP6, p-LRP6, GSK3β, p-GSK3β, β-catenin) and AIF-1. Plasmid transfection techniques were utilized to either knock down or overexpress AIF-1, and the subsequent alterations in these markers were observed.

Results: (1) Compared to the control group, the aldosterone treatment group with exhibited a significant increase in ALP. Concurrently, Runx2, OCN, and ALP mRNA levels increased, as did LRP6, p-LRP6, GSK3β, p-GSK3β, β-catenin, and AIF-1 protein levels. Additionally, a significant decrease in the expression of α-SMA mRNA was observed (P < 0.05). (2) The aldosterone + oe-AIF-1 group showed significant increases in ALP activity compared to the aldosterone + oe-NC group, whereas the aldosterone + sh-AIF-1 group showed significant decreases (P < 0.05). (3) The aldosterone + oe-AIF-1 group exhibited significantly upregulated expression of AIF-1, p-LRP6/LRP6, p-GSK3β/GSK3β, and β-catenin proteins relative to the aldosterone + oe-NC group (P < 0.05). This was concurrent with increased mRNA expression of Runx2, OCN, and ALP, and decreased α-SMA mRNA expression (P < 0.05).

Conclusion: Aldosterone affects the calcification process in mouse VSMCs, and the activation of the AIF-1/Wnt/β-catenin signaling pathway is the mechanism behind its action.

Aldosterone; Allograft inflammatory factor-1 (AIF-1); Vascular calcification; Wnt/β-catenin signaling pathway.