Metformin alleviates the calcification of aortic valve interstitial cells through activating the PI3K/AKT pathway in an AMPK dependent way

Abstract

Background Calcific aortic valve disease (CAVD) is the most prevalent valvular disease worldwide. However, no effective treatment could delay or prevent the progression of the disease due to the poor understanding of its pathological mechanism. Many studies showed that metformin exerted beneficial effects on multiple cardiovascular diseases by mediating multiple proteins such as AMPK, NF-κB, and AKT. This study aims to verify whether metformin can inhibit aortic calcification through the PI3K/AKT signaling pathway. Methods We first analyzed four microarray datasets to screen differentially expressed genes (DEGs) and signaling pathways related to CAVD. Then aortic valve samples were used to verify selected genes and pathways through immunohistochemistry (IHC) and western blot (WB) assays. Aortic valve interstitial cells (AVICs) were isolated from non-calcific aortic valves and then cultured with phosphate medium (PM) with or without metformin to verify whether metformin can inhibit the osteogenic differentiation and calcification of AVICs. Finally, we used inhibitors and siRNA targeting AMPK, NF-κB, and AKT to study the mechanism of metformin. Results We screened 227 DEGs; NF-κB and PI3K/AKT signaling pathways were implicated in the pathological mechanism of CAVD. IHC and WB experiments showed decreased AMPK and AKT and increased Bax in calcific aortic valves. PM treatment significantly reduced AMPK and PI3K/AKT signaling pathways, promoted Bax/Bcl2 ratio, and induced AVICs calcification. Metformin treatment ameliorated AVICs calcification and apoptosis by activating the PI3K/AKT signaling pathway. AMPK activation and NF-κB inhibition could inhibit AVICs calcification induced by PM treatment; however, AMPK and AKT inhibition reversed the protective effect of metformin. Conclusions This study, for the first time, demonstrates that metformin can inhibit AVICs in vitro calcification by activating the PI3K/AKT signaling pathway; this suggests that metformin may provide a potential target for the treatment of CAVD. And the PI3K/AKT signaling pathway emerges as an important regulatory axis in the pathological mechanism of CAVD.

Integrated Analysis of LncRNA-Mediated ceRNA Network in Calcific Aortic Valve Disease

“…The function and pathway enrichment showed ECM receptor interaction signaling pathways were significantly enriched in our analysis. Compared to other microarray datasets, enrichment analysis found PI3K/AKT signaling pathway in an AMPK-dependent manner was inhibited in CAVD [30], our enrichment results also revealed that the downregulated DEmRNAs could enriched in AMPK signaling pathway. Among the DEmRNAs involved in the lncRNA-miRNA-mRNA ceRNA network, COL4A1, COL4A2, COL4A3, COL1A1, COL6A3 and COL9A1 were belong to constitute of type I, IV, VI and IX collagen, which are structural proteins in the ECM.…”

Nesfatin-1 mitigates calcific aortic valve disease via suppressing ferroptosis mediated by GSH/GPX4 and ZIP8/SOD2 axes

“…The PI3K-AKT signaling pathway has been implicated in CAVD. En et al [47,48] found that DEGs between normal aortic valves and calcific aortic valves were mainly enriched in the PI3K-AKT signaling pathway. Moreover, they observed a decrease in the expression of P-AKT and the ratio of P-AKT to AKT in calcific aortic valves.…”

Inhibition of miR-101-3p prevents human aortic valve interstitial cell calcification through regulation of CDH11/SOX9 expression

“…It was reported that miR-101-3p regulates the PTEN/AKT axis in trophoblast cells in the placenta (Zhang et al 2022 ). Metformin can inhibit AVICs calcification in vitro by activating the PI3K/AKT signaling pathway (En et al 2021 ). Yang et al ( 2022 ) found that LEVs might inhibit neuron apoptosis via the miR-101a-3p/c-Fos/TGF-β axis, and has-miR-101-3p is a potential marker of neurological recovery in IS patients.…”