Caffeic Acid Phenethyl Ester Ameliorates Calcification by Inhibiting Activation of the AKT/NF-κB/NLRP3 Inflammasome Pathway in Human Aortic Valve Interstitial Cells.

Ming Liu,Si Chen,Fei Li,Tingwen Zhou,Nianguo Dong,Jiawei Shi,Kang Xu,Geng Li,Yuming Huang

doi:10.3389/fphar.2020.00826

Abstract

Calcific aortic valve disease (CAVD) occurs via a pathophysiological process that includes inflammation-induced osteoblastic differentiation of aortic valvular interstitial cells (AVICs). Here, we investigated the role of the anti-inflammatory compound caffeic acid phenethyl ester (CAPE) in inhibiting CAVD. Human AVICs were isolated and cultured in osteogenic induction medium (OM) with or without 10 μM CAPE. Cell viability was assessed using CCK8 assays and calcified transformation of AVICs was evaluated by Alizarin Red staining and osteogenic gene/protein expression. RNA-sequencing was conducted to identify differentially expressed genes (DEGs) and enrichment in associated pathways, as potential molecular targets through which CAPE inhibits osteogenic induction. The regulatory effects of CAPE on activation of the AKT/NF-κB and NLRP3 inflammasome were evaluated by Western blot analysis and immunofluorescent staining. CAPE slowed the growth of AVICs cultured in OM but did not show significant cytotoxicity. In addition, CAPE markedly suppressed calcified nodule formation and decreased gene/protein expression of RUNX2 and ALP in AVICs. Gene expression profiles of OM-induced AVICs cultured with or without CAPE revealed 518 common DEGs, which were highly enriched in the NOD-like receptor, PI3K-AKT, and NF-κB signaling pathways. Furthermore, CAPE inhibited phosphorylation of AKT, ERK1/2, and NF-κB, and suppressed NLRP3 inflammasome activation in AVICs cultured in OM. Thus, CAPE is implicated as a potent natural product for the prevention of CAVD by inhibiting activation of the AKT/NF-κB pathway and NLRP3 inflammasome.

Highlights

Calcified aortic valve disease (CAVD), the most common Cardiac valvular disease worldwide, is characterized by valvular calcification, leading to aortic stenosis and subsequent heart failure (Nkomo et al, 2006)
Increasing evidence obtained over the past decade suggests that CAVD is not a passive degenerative process, but an active pathological condition similar to that associated with atherosclerosis, including processes such as lipoprotein deposition, chronic inflammation, and osteoblastic differentiation of aortic valve interstitial cells (AVICs) (Li et al, 2013; P et al, 2014; Rutkovskiy et al, 2017)
We found that caffeic acid phenethyl ester (CAPE) significantly inhibited osteogenic medium (OM)-induced calcification in human AVICs

Summary

Introduction

Calcified aortic valve disease (CAVD), the most common Cardiac valvular disease worldwide, is characterized by valvular calcification, leading to aortic stenosis and subsequent heart failure (Nkomo et al, 2006). There is no effective pharmacological therapy for CAVD other than surgical or interventional aortic valve replacement (Da et al, 2015) Both in vitro and clinical studies have suggested that a sequence of active osteogenic processes contribute to CAVD, and that osteogenic activity is initiated by inflammation (Nadra et al, 2005; Marincheva-Savcheva et al, 2011; New and Aikawa, 2011; Pawade et al, 2015). AVICs are the principle cell type found within aortic valve leaflets and participate in the process of CAVD primarily by inducing both inflammation and osteoblastic differentiation (Rutkovskiy et al, 2017). This inflammatory damage is a critical factor that causes CAVD. The search for effective treatment modalities for valvular calcification, such as the use of medication to regulate inflammatory responses, has important clinical value and significance, and may effectively delay the onset of aortic valve calcification

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