Abstract
BackgroundCalcific 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.MethodsWe 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.ResultsWe 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.ConclusionsThis 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.
Highlights
Calcific aortic valve disease (CAVD) is the most prevalent valvular disease worldwide
Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis reflected that PI3K/AKT and NF-κB signaling pathways might be closely associated with the occurrence and development of CAVD (Additional file 1: Fig. S2)
western blot (WB) assays showed that the expression of PI3K, AKT, endothelial nitric oxide synthase (eNOS), AMPK, and Bcl2 were all down-regulated, but BMP2, OPN, and Bax were significantly up-regulated in calcific valves (Fig. 3)
Summary
Calcific aortic valve disease (CAVD) is the most prevalent valvular disease worldwide. No effective treatment could delay or prevent the progression of the disease due to the poor understanding of its pathological mechanism. Calcific aortic valve disease (CAVD), the most common valve disease, is characterized by aortic valve calcification and progressive stenosis, leading to left ventricular. When the disease progresses to severe aortic stenosis, it may present with symptoms including the sudden onset of chest tightness, angina pectoris, syncope, and dyspnea (Bonow et al 2016). Patients with mechanical valves need to take anticoagulants for life; the bioprosthetic valve will inevitably deteriorate and lead to the possibility of reoperation less than 15 years (Head et al 2017). No effective drugs can delay or prevent the disease progression of CAVD due to the unclear understanding of the pathological mechanism. It is extremely important to discover potential biomarkers, pathways, and drugs through basic experimental research
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