Background
Calcific aortic valve disease (CAVD) is the most prevalent valvular disease in individuals over 65 years of age. Progressive aortic valve calcification will develop into aortic stenosis, and 82.7% of deaths in individuals with aortic valve disease in developed countries are attributed to aortic stenosis [
1‐
3]. To date, there is no pharmacological therapy for retarding CAVD progression, and valve replacement remains the sole treatment for CAVD patients in the advanced stage [
4]. Discovering potential targets and effective agents for treating aortic valve calcification is urgently needed. It has been recognized that osteogenic differentiation of aortic valve interstitial cells (AVICs) is the fundamental hallmark of CAVD [
5,
6]. Therefore, an effective therapeutic strategy for CAVD may be prevent the phenotypic switching of AVICs to an osteoblast-like phenotype.
Protein tyrosine phosphorylation is an essential mechanism for numerous biological processes, including immunity, proliferation, differentiation, and calcification [
7]. Aberrant protein tyrosine phosphatases (PTPs) may cause several human diseases, such as cardiovascular diseases, diabetes, and autoimmune disorders [
8,
9]. The complement of PTPs includes the classic, phosphotyrosine (pTyr)-specific PTP and the VH1-like or dual specificity protein phosphatase (DUSP) subfamilies [
10]. In particular, PTPN1, also known as protein tyrosine phosphatase nonreceptor type 1, has been associated with several diseases, especially those that affect the metabolic and cardiovascular systems. Dawn Thompson et al. showed that PTPN1 ablation attenuated the formation of atherosclerotic plaques in an LDLR-/- mouse model [
11]. Targeted inhibition of PTPN1 is known to protect against cardiovascular dysfunction and mortality [
12]. Additionally, Chiu-Fen Yang et al. found that PTPN12 levels were increased in the myocardium during ischaemia/reperfusion, and PTPN12 activation was harmful in ischaemia/reperfusion [
13]. After treatment with auranofin, an inhibitor of PTPN12, the infarct size was significantly reduced and cardiac function was improved in a mouse model of ischaemia/reperfusion, suggesting that PTPN12 contributes to myocardial ischaemia/reperfusion injury [
13]. A study showed that DUSP26, a member of the PTP family, was expressed at significantly higher levels in the calcific aortic valve, and ablation of DUSP26 ameliorated aortic valve calcification in human AVICs and in ApoE
-/- mice fed a high cholesterol diet [
14]. Indeed, PTPs are involved in cardiovascular diseases, including aortic valve calcification.
Protein tyrosine phosphatase nonreceptor type 22 (PTPN22), which is an intracellular nonreceptor-like PTP, dephosphorylates threonine and tyrosine residues in proteins [
15]. PTPN22 has been linked to several chronic inflammatory disorders, such as systemic lupus erythematosus, rheumatoid arthritis, Crohn’s disease, and diabetes [
16]. It has been established that PTPN22 regulates T-cell receptor signalling [
17]. Furthermore, Won Jin Ho et al. found that inhibition of PTPN22 protected against tumour growth [
18]. Recent studies have shown that PTPN22 ablation reduces tail-bleeding time and accelerates arterial thrombus formation, suggesting that PTPN22 is a novel potential target for thrombotic or cardiovascular diseases [
19]. Furthermore, several lines of evidence suggest that PTPN22 is implicated in atherosclerosis [
20,
21], a disease that shares common risk factors and pathogenesis with CAVD. Hence, the potential effect of PTPN22 on CAVD remains to be investigated using comprehensive approaches.
In the present study, we examined the role of PTPN22 in CAVD by utilizing genetic and pharmacologic approaches in vitro and in vivo. More importantly, we discovered a new PTPN22 inhibitor, 13-hydroxypiericidin A 10-O-α-D-glucose (1 → 6)-β-D-glucoside (S18), which is a marine-derived product. The results of our study reveal that PTPN22 is crucial to the pathogenesis of CAVD, and S18, a novel PTPN22 inhibitor, is identified as a potential therapeutic drug.
Discussion
The major novel findings in the present study are as follows: (1) PTPN22 expression is increased in aortic valve from CAVD patients and in human AVICs stimulated with OM; (2) PTPN22 plays a vital role in mitochondrial stress, and PTPN22 silencing alleviates mitochondrial dysfunction and osteogenic responses in vitro as well as aortic valve calcification in vivo; (3) piericidin diglycoside S18, a novel PTPN22 inhibitor, was discovered; and (4) pharmacological piericidin diglycoside S18 significantly attenuates aortic valve calcification in animal experiments and in vitro studies. On the basic of our knowledge, our study provides direct evidence for a novel target for therapeutic intervention in CAVD and demonstrates the anti-CAVD properties of piericidin diglycoside S18.
Increasing evidence indicates that PTPs exert great effects on the progression of cardiovascular diseases. Dong N et al. found that DUSP26 expression is related to the severity of CAVD and is increased in calcified aortic valves and in ApoE
−/− mice. DUSP26 ablation markedly reduces osteogenic markers in vitro and mitigates aortic valve calcification in ApoE
−/− mice [
14]. Some studies show that PTPN22 is associated with chronic inflammatory diseases, and PTPN22 knockdown reduces IL-1β secretion [
18]. PTPN22 knockdown reduced the levels of inflammatory factors, including ICAM-1, IL-8 and MCP-1, in THP1 monocytes stimulated with IFN-γ [
39,
40]. Furthermore, PTPN22 interacts with TRAF3, promotes TRAF3 polyubiquitination, and then activates the serine-threonine kinases TBK1 and IKK. Subsequently, the transcription factors IRF3 and IRF7, which are substrates for TBK1 and IKK, translocate into the nucleus and activate type I interferon transcription [
15,
41,
42]. In this regard, PTPN22 is likely involved in the pathogenesis of CAVD. Our study is the first to report that PTPN22 expression is increased in aortic valve tissue from CAVD patients, ApoE
−/− mice fed with HFD, and a wire injury-induced CAVD mouse model, which is in line with the effects of DUSP26. Moreover, PTPN22 expression was induced in human AVICs treated with OM. These results indicate that the upregulation of PTPN22 is a molecular pathogenic feature in calcific aortic valves.
In view of PTPN22 induction with OM, it is important to elucidate the potential effect of PTPN22 activation on valvular cells. We showed that PTPN22 overexpression was sufficient to induce osteogenic marker expression in human AVICs. Consistent with this notion, siRNA-mediated knockdown or S18-mediated inhibition dramatically suppresses OM-induced osteogenic responses in cells. These observations indicate a strong pro-calcific action of PTPN22. Moreover, PTPN22 ablation protected mice against the progression of CAVD after AVI, providing strong evidence for PTPN22-mediated aortic valve calcification. These results are also consistent with the effect of DUSP26 and in harmony with observations in vitro. Furthermore, similar to the genetic approach, the PTPN22 inhibitor S18 suppressed ALP, Runx2 and BMP2 levels in human AVICs and alleviated calcification in wire injury-induced aortic valves. Taken together, the results of our study on the role of PTPN22 in CAVD are quite cohesive, as shown by in vivo and in vitro data, as well as genetic and pharmacologic techniques.
The mitochondria provide the majority of energy in cells and maintain myocardial excitation–contraction coupling [
37]. Mitochondrial dysfunction and alterations in mitochondrial morphology result in abnormal ROS production, causing mitochondrial stress and inflammatory responses [
43]. An overt increase in ROS was observed in ApoE
−/− mice fed with HFD. In cardiac tissue, excessive ROS promote postischemic inflammatory infiltration, leading to cardiac hypertrophy, fibrosis, and necrosis [
23,
44]. Additionally, it has been demonstrated in animal and in vitro models that mitochondrial stress is associated with the progression of CAVD [
30,
45]. Oxidative stress, especially ROS, facilitates lipid infiltration and inflammation, which induce calcification and promote the progression of CAVD [
46]. Moreover, ROS also induce BMP2 expression, which then modulates osteogenic responses through Runx2 [
30]. Some studies have shown that the pharmacologic induction of mitochondrial stress modulates calcification in the aortic valve. Huibing Liu et al. found that Celastrol alleviates aortic valve calcification by reducing ROS generation in a rabbit model of CAVD and in AVICs [
47]. Moreover, in our previous study, 4-Octyl itaconate alleviated OM-induced calcification in vitro and alleviated aortic stenosis in mice with aortic wire injury by reducing ROS production [
26]. Furthermore, N-acetyl-L-cysteine (NAC) decreased OM-induced ROS induction and then downregulated ALP and Runx2 expression in human AVICs, suggesting that OM-induced osteogenic differentiation occurs in a ROS-dependent manner. Therefore, targeting mitochondrial stress may be an effective strategy for treating CAVD.
Previous studies have shown that PTPN1 knockout reduces the levels of ROS and augments mitochondrial mass in palmitate-treated oval cells, suggesting that PTPN1 deficiency prevents oxidative stress [
3]. However, the role of PTPN22 in mitochondrial dysfunction is unclear. In this study, we found that treatment with PTPN22 siRNA attenuated the overexpression of ROS and enhanced the MMP in human AVICs, providing independent evidence that PTPN22 inhibition protects against mitochondrial dysfunction in human AVICs.
We also discovered that PTPN22 silencing decreased the phosphorylation of NF-κB and ERK signalling, which are well-known pathologic pathways involved in the progression of CAVD [
48]. Loss of PTPN22 reduced NF-κB p65 phosphorylation in response to IFN-γ and LPS stimulation [
39], which is consistent with our findings. Furthermore, it has been reported that SOCS1 interacts with nuclear NF-κB p65 through polyubiquitination and proteasomal degradation [
49]. However, PTPN22 knockdown induces SOCS1 activation and suppresses NF-κB p65 phosphorylation [
39]. Here, we found that PTPN22 ablation prevented against mitochondrial stress, which may explain the reduction in NF-κB p65 activation in cells treated with PTPN22 siRNA. Accordingly, the mechanism by which PTPN22 regulates NF-κB and ERK signalling may be involved in mitochondrial stress in AVICs.
Another major finding of this study is the discovery of a novel PTPN22 inhibitor, piericidin diglycoside S18, which was obtained from marine-derived
Streptomyces [
34]
. The present study demonstrated that piericidin diglycoside S18 bound to the active site residues of PTPN22 through hydrogen bonds. Consistent with the binding relationship with PTPN22, we found that piericidin diglycoside S18 decreased the protein and mRNA levels of PTPN22, suggesting its potent inhibitory effect on PTPN22. Furthermore, piericidin diglycoside S18 not only alleviated OM-induced osteogenic responses of human AVICs but also ameliorated aortic valve thickening and calcium deposition in mice with CAVD. It is worth highlighting that OM-induced mitochondrial stress in human AVICs was significantly attenuated by S18 administration, and as stated previously, S18 could moderate osteogenic responses in AVICs and aortic valve calcification by mitigating mitochondrial dysfunction and excessive ROS production. Thus, S18 is a novel and rare marine-derived compound with therapeutic potential, and in which S18 ameliorates osteogenic differentiation in AVICs and aortic valve lesions in mice in this study. As anticipated, these findings shed light on the therapeutic potential of S18 for CAVD.
There are some limitations in this study. First, we did not establish cell-type-specific gene-modified mice in vivo. Data from PTPN22-knockout mice harbouring a cell-type specific deletion of PTPN22 in human AVICs will be more convincing. Second, given that CAVD is a multifactorial disease and S18 may have multiple targets in CAVD, it cannot be excluded that S18 protects the aortic valve from lesions through other pathways. Collectively, our results indicate that piericidin diglycoside S18 might be a therapeutic candidate for CAVD.
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