Calcific aortic valve disease (CAVD) is the most common valvular heart disease in the aging population of the developed world and is becoming more prevalent as the population demographics show an increasing population over 65 years of age [1,2]. Because the pathogenesis of CAVD has not been fully elucidated, the only treatment for symptomatic severe CAVD is surgical or transcatheter aortic valve replacement (AVR) [3]. Therefore, to find specific targets and drugs for CAVD, it is imperative to explore its pathogenesis. CAVD is considered to be a chronic inflammatory response and an osteogenic process [4]. CAVD can be divided into two distinct phases, the initiation and propagation phases. Each phase is dominated by different mechanisms [3]. The initiation phase is ignited by endothelial activation/damage and an inflammatory response [5]. The propagation phase is characterized by fibrosis and accelerated calcification [3]. Aortic valve interstitial cells (AVICs) are the major cellular components of aortic valve, which contribute to an essential role in valvular inflammation and calcification [6]. The endothelial activation/damage causes inflammatory cell infiltration in valvular matrix, which mediates inflammatory responses of AVICs. Previous studies confirmed that pro-inflammatory molecules induced the expression of osteogenic transcription factors, such as bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX2) in AVICs [7]. Moreover, it has been reported that osteogenic response of AVICs contributed a critical role to aortic valve calcification [8]. Thus, elucidating the proinflammatory signaling pathway and osteogenic mechanisms could contribute to therapeutic options for CAVD.

Enhancer of zeste homolog 2 (EZH2), an important histone methyltransferase, forms the polycomb repressive complex-2 (PRC2) complex with EED and Suz12, which is linked to silencing of gene expression [9]. Histone H3 lysine 27 trimethylation (H3K27me3) is an important chromatin modification induced by EZH2, which is associated with silenced gene expression [10]. In the beginning, overexpressed EZH2 was discovered in renal cell carcinoma, bladder, and breast cancer among others [11]. Then, it was found that EZH2 associated with proliferation, migration, and invasion of various human cells [12,13]. Moreover, previous studies confirmed that EZH2 could regulate inflammation [14], fibrosis [15] and osteogenesis [16]. In the cardiovascular field, EZH2 plays a critical role in orchestrating heart development and cardiac gene expression [17]. However, the understanding of EZH2 in CAVD is limited. Although our RNA-sequencing data showed non-statistically significant elevation in CAVD valvular tissues, we discovered a differential mRNA expression and protein levels of EZH2 between calcific aortic valves and non-calcific aortic leaflets by larger sample size. Thus, our study aim to examine the effect and mechanism of EZH2 on the osteogenic responses induced by osteogenic medium (OM) in AVICs.

Suppressor of cytokine signaling 3(SOCS3) is a member of the STAT-induced STAT inhibitor (SSI), also known as suppressor of cytokine signaling (SOCS), family [18]. SSI family members are cytokine-inducible negative regulators of cytokine signaling [19]. SOCS3 is a well-known anti-inflammatory mediator, which has been shown to negatively regulate TLR-induced macrophage activation and autoimmune inflammation [20]. Previous studies showed that TLR-dependent signaling pathways contributed to AVICs inflammatory responses, which was associated with calcific aortic valve disease [21,22]. Our results of high throughout mRNA sequencing found a divergence of SOCS3 in non-CAVD and CAVD valvular tissues. The present study assessed the function of SOCS3 in the regulation of inflammation and ossification in AVICs.

In this project, we discovered that EZH2-mediated H3K27me3 enhanced osteogenic differentiation in aortic valve interstitial cells (AVICs) by inhibiting SOCS3 expression, which was considered as anti-inflammatory cytokines in previous studies [23].