Background

Calcified aortic valve disease (CAVD) is a heart valvular disease commonly occurring in the elderly over 65 years old [1]. The pathological process of CAVD involves inflammation, matrix remodeling, thickening and calcification of valve leaflets, and more serious, it may progress to calcified aortic valve stenosis (CAVD) [2]. Currently, there is no clinically effective treatments for CAVD except aortic valve replacement surgery [3]. Therefore, therapeutic targets and drugs for CAVD need to be further developed and explored.

NE (Neutrophil elastase) is a serine protease mainly existed in neutrophils azurophilic granules [4], which is involved in a variety of physiological processes like formation of neutrophil extracellular trap and degradation of extracellular matrix and proteins [5, 6]. Due to it can rapidly released from neutrophils in response to inflammatory signals, NE is often thought to be a sign of inflammation and significantly contributes to some inflammatory diseases such as bronchiectasis [7] and chronic obstructive pulmonary disease [8]. Besides, NE also actively participates in cardiovascular disease. In patients with coronary heart disease, especially in patients with unstable angina and acute myocardial infarction, the level of NE is significantly increased, which is considered to be a predictor of cardiovascular events [9]. NE increases myocardial damage by inducing excessive inflammation [10]. It has been reported that NE was highly expressed in atherosclerotic plaques and its inhibitor effectively remitted the instability of atherosclerotic plaques, suggesting that it plays an important role in atherosclerosis [16]. In terms of pathogenic factors, CAVD shares many pathophysiological features with atherosclerosis. Aortic valve tissue is divided into three layers: fibrosa, spongiosa and ventricularis [17]. During the disease progression, the valve leaflets gradually become thicker and stiff, with structural and collagen disorders [18]. In line with this, we found that compared with normal valve tissue, calcified valves were significantly thickened with increased elastin fiber fragments and calcium nodules. The valve interstitial cells (VICs) are the main cell type of valve. VICs exhibits mesenchymal phenotype characterised by high expression of α-SMA (alpha-smooth muscle actin) and vimentin instead of CD31 [19]. Therefore, we used these mesenchymal markers to phenotypic identify isolated porcine valve interstitial cells prior to the initiation of in vitro experiments (Additional file 1: Fig. S1).

Inflammation is essential for initiating valve calcification and accompanying the entire process of calcification [20]. Meanwhile, NE is closely associated with some inflammatory diseases such as arthritis [21] and acute pancreatitis [22]. NE levels are rapidly upregulated during inflammatory responses. The first finding of this study was that the expression and activity of NE of CAVD-patients was higher than that of control group. Besides, In vitro osteogenic medium also promoted the expression and activity of NE in pVICs, suggesting that it may play an important role in valve calcification. This experiment confirmed for the first time that valve interstitial cells could also secrete NE. In fact, NE was not only produced by neutrophils, but also secreted by macrophages and endothelial cells [23]. Since previous studies have reported that there are infiltration of macrophages, monocytes and other inflammatory cells in the pathological valve tissue [24], we consider that these inflammatory cells may be one of the sources of NE in calcified valve tissues. Our team will further investigate the mechanism of NE elevation in calcified valves.

Apoptosis is a crucial regulator of initiation and progression of CAVD [25]. Meanwhile, it is reported that NE could induce endothelial cells apoptosis [26]. Here, we found that NE promoted bax protein level with or without osteogenic medium induction. NE had no significant effect on the expression of bcl-2 under the conditions of OM induction. We speculate that it may be due to the negative feedback regulation mechanism of pVICs activated by OM. In general, NE exhibited the effect of promoting pVICs apoptosis, which suggests that NE may partly influence the osteogenic differentiation of pVICs through apoptosis.

In healthy valves, pVICs remain in a quiescent state [27]. With the change of valve homeostasis, pVICs are activated and further differentiated into myofibroblast-VIC characteristiced by high expression of α-SMA and osteogenic-VIC characteristiced by high expression of RUNX2 [28]. Therefore, we examined the effect of NE treatment on VIC phenotypic transformation at different time points. Our results demonstrated that NE promoted the activation of pVICs in the early stage, and then promoted the osteogenic differentiation of pVICs accompanied by an increase in inflammatory factors.

We then evaluated the role of NE on VIC during OM induction. As expected, NE aggravated the osteogenic differentiation, apoptosis and infalmmation of pVICs induced by OM, together with increased ALP and calcium deposits. However, these results were reversed by NE inhibitor Alvelestat. Alvelestat (AZD9668) is a novel, orally bioavailable NE inhibitor [29]. Early NE inhibitors, such as Sivelestat, bind irreversibly to NE, which causes serious side effects [30]. Compared to Sivelestat, the interaction between Alvelestat and NE is rapidly reversible. Moreover, clinical studies were conducted on pharmacology, tolerability and safety of Alvelestat. The results showed that Alvelestat could be quickly absorbed after oral administration, eliminating half-life between 5 and 15 h, and most of Alvelestat could be eliminated by the kidneys [31]. Therefore, it is less toxic than other early NE inhibtors. At present, the application of Alvelestat in the treatment of COPD and bronchiectasis has entered the clinical phase II study, suggesting its better clinical application prospect [32,33,34]. Alvelestat also has been reported to be protective in some inflammatory diseases, such as abdominal aortic aneurysms [35] and cystic fibrosis [36]. However, its role in cardiovascular diseases, especially CAVD, has not been studied. Hence, we further studied the role of Alvelestat in OM-induced osteogenic differentiation of pVICs. We chose a concentration of 40 nmol/L Alvelestat for this experiment, because previous studies has reported that the pIC50 values of Alvelestat for the whole-blood and cell-associated assays was about 40 nmol/L [33]. Our analysis indicated that Alvelestat could effectively inhibit the increase in NE activity and expression induced by OM. Meanwhile, Alvelestat also had a certain inhibitory effect on apoptosis and inflammation. These results suggest the potential of Alvelestat to inhibit osteogenic differentiation of pVICs. To achieve satisfactory inhibition of NE, we next used siRNA against NE to knockdown the NE mRNA expression during OM induction. Similarly, NE silencing also reduced OM-induced apoptosis, inflammation and osteogenic differentiation of pVICs. In addition, in order to further increase the clinical value of Alvelestat, we then explored the effect of Alvelestat on APOE−/− mice fed with western diet. Our results showed that Alvelestat markly remitted the thickening and fibrosis of the valves. The results of echocardiography were further revealed that Alvelestat could decreased the transaortic peak velocity to alleviate cardiac function in APOE−/− mice fed with western diet. Although there was no statistical difference in trends, mice in the WD group showed decline in cusp separation and ejection fraction. The reason for this phenomenon may be due to the small number of mice in the WD group. Therefore, the number of mice in WD group needs to be expanded to obtain more stable results.

Our group previously investigated the role of the inflammatory regulator PGRN in valve calcification and we found for the first time that 45KD GRN, a degraded fragment of PGRN, had a characteristic increase in calcified valve tissues. We demonstrated that full-length PGRN antagonized TNF-α and inhibited valve calcification while 45KD GRN played the opposite biological function of pro-inflammatory and pro-calcification [12]. So why does the degradation of PGRN to 45KD GRN increase when the valve is calcified? We hypothesized that this phenomenon may be caused by the elevation of a certain enzyme related to PGRN degradation. It is well known that PGRN can be cleaved by neutrophil elastase and degraded into short peptides called GRN [13]. Our experimental results showed that NE levels and activity were significantly increased during valve calcification. In vitro experiments showed that NE promoted the production of 45KD GRN in the presence or absence of OM. Overall, NE plays its role in promoting valve calcification at least partially by degrading PGRN to 45KD GRN.

We then explored the signaling pathways involved in NE promoting osteogenic differentiation in pVICs. The effect of NE seems to be mediated by several pathways. It has been reported that NE increased the nuclear translocation of NF-κB p-P65 and thus relieved LPS-induced lung injury of rats [37]. Another research reported that NE activated ERK and induced IL-8 production [38]. In addition, NE also induced tumor cell survival and migration via Src/PI3K-dependent activation of AKT signaling [39]. However, the effect of NE on Smad 1/5/8 signaling pathway is unclear. In our current study, we found that NE did not affect the classical osteogenesis Smad 1/5/8 signaling pathway and the non-classical osteogenesis-related ERK signaling pathway, but activated the NF-κB and AKT signaling pathway in the presence or absence of OM. Further verification was carried out by using BAY11-7082, the inhibitor of NF-KB, and LY294002, the inhibitor of AKT pathway.

Conclusion

We have discovered that NE accelerates osteogenic differentiation of pVICs through promoting the production of 45KD GRN and activating the NF-KB and AKT pathways, thus enhancing valve calcification progression. In addition, we found that inhibition of NE by Alvelestat significantly attenuates valve calcification induced by OM. These findings open the possibility that NE may be a potential therapeutic targets for CAVD.