Abstract
Objective To examine 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 vascular smooth muscle cells (VSMCs) were cultured in vitro, and calcification was induced by treatment with aldosterone at a concentration of 100 nM. The level of calcification in mouse VSMCs was evaluated using colorimetric assays assessing the presence of ALP activity, and qRT-PCR identifying the expression of calcification-related markers such as RUNX2, α-SMA, OCN, and ALP mRNA. Western blot analysis was performed to determine the protein level of expression associated with the Wnt/β-catenin pathway (LRP6, p-LRP6, GSK3β, p-GSK3β, β-catenin), as well as 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) Comparing the aldosterone treatment group with the control group, the ALP activity increased significantly. In conjunction with this increase, RUNX2, OCN, and ALP mRNA levels increased, as did LRP6, p-LRP6, GSK3, p-GSK3, -catenin, and AIF-1 protein levels. Additionally, an important decrease in the expression of -SMA mRNA was observed (P< 0.05). (2) Comparing the aldosterone + oe-AIF-1 with the aldosterone + oe-NC group showed significant increases in the ALP activity, whereas the aldosterone + sh-AIF-1 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 In the process of calcification, aldosterone affects mouse VSMCs, and AIF-1/Wnt/β-catenin signaling pathway activation is the mechanism behind its action.