Calcific aortic valve disease is the most common valvular disease globally, with a disease spectrum ranging from asymptomatic aortic valve sclerosis (valve thickening without impaired leaflet movement) to complete left ventricular outflow obstruction (aortic valve stenosis) (Rajamannan et al., 2011; Dweck et al., 2012; Yutzey et al., 2014). According to World Health Organization epidemiological data, cardiovascular diseases are the leading cause of death globally, accounting for 32% of total global deaths in 2019 (Benjamin et al., 2019). Calcific aortic valve stenosis (CAVS) is a progressive disorder that requires proper diagnosis and treatment; without appropriate treatment, the patient’s condition deteriorates, increasing the risk of cardiac hypertrophy and, eventually, heart failure (Rajamannan et al., 2011; Yutzey et al., 2014). The only treatment option available is valve replacement, either surgical valve replacement or transcatheter, which has no lifetime guarantee and is not appropriate for all patients (Hutcheson et al., 2014; Pawade et al., 2015). Various approaches can be used to effectively manage CAVS, such as risk factor management and the development of novel therapies targeting CAVS signaling pathways that can inhibit osteogenic differentiation of valve interstitial cells (VICs) and delay/or prevent CAVS.

CAVS is known to be an inflammatory disease with pathobiology slightly similar to atherosclerosis (Rutkovskiy et al., 2017; Bogdanova et al., 2018). The phenotype and function of VICs in calcified aortic valves differ from healthy VICs (Bogdanova et al., 2019). When activated by external factors, VICs, which are present in all three layers of aortic valve leaflets, play an important role in CAVS progression by undergoing myofibroblastic and osteoblastic differentiation (Rajamannan et al., 2011; Lerman et al., 2015). This differentiation results in active calcium deposition in aortic valve leaflets, which is controlled by the transcription factors runt-related transcription factor 2 (Runx2) and osteopontin (OPN) (Rutkovskiy et al., 2017). During the pathogenesis of CAVS, the expression of fibrotic and osteogenic genes is increased as a result of ROS activating several signaling pathways, such as p38, ERK 1/2, AKT, GSK-3β/β-catenin, and NFκB, which in turn causes VICs to differentiate into an osteoblastic phenotype (Adhikari et al., 2022). The pathology of CAVS is complex and various biological processes such as oxidative stress, chronic inflammation, lipoprotein deposition and renin-angiotensin system are involved in the development of CAVS, however, several existing therapies, such as statins, antihypertensives, and drugs targeting calcium and phosphate metabolism, have failed to show benefit in large randomized controlled trials (Houslay et al., 2006; Vossen et al., 2020). There are currently no effective drug interventions to effectively impede its progression (Hutcheson et al., 2014), so it is critical to find other clinical therapies to prevent CAVS, which can only be accomplished through a thorough study of pathogenesis.

Niclosamide (5-chloro-N-(2-chloro-4-nitrophenyl)-2-hydroxybenzamide) is an anti-helminthic drug approved by the FDA (Andrews et al., 1982). Niclosamide has recently been shown to be effective as an anti-bacterial, anti-hypertensive, rheumatoid arthritis, and systemic sclerosis treatment (Chen et al., 2018). Furthermore, niclosamide demonstrated anti-inflammatory properties by decreasing cytokine expression in synoviocytes targeting MAP kinase and NFκB pathway (Liang et al., 2015), and inhibiting osteoclast and osteoblastic differentiation (Liu et al., 2017). Furthermore, niclosamide is recently shown to inhibit vascular calcification and the expression of the calcification marker Runx2, as well as alkaline phosphatase and o-octanoyl transferase mRNA expression (Tanaka et al., 2022). However, its impact on CAVS and VICs calcification has received little attention. In this study, we investigated the effect of niclosamide on calcification in porcine VICs induced by a pro-calcifying medium (PCM), and elucidated the underlying mechanisms involved in calcification inhibition.

VICs isolation, culture, and treatment

Porcine hearts were brought to the lab immediately after sacrifice, within 30 minutes, and stored at 4°C in Krebs bicarbonate solution (composition in mmol/L: KCl 4.7, NaCl 119, KH₂PO₄ 1.18, CaCl₂ 1.25, MgSO₄ 1.18, D-glucose 11 and NaHCO₃ 1.25; pH 7.4). Tissues for this study were obtained postmortem from a local commercial slaughterhouse (Mokpo, Korea), which follows the US Department of Agriculture’s Animal Cruelty and Slaughter Act guidelines for animal cruelty and slaughter and thus was not subject to institutional animal protocol approval. Primary VICs were isolated and cultured using a previously described protocol (Gould and Butcher, 2010). In brief, aortic valve leaflets were isolated immediately after arrival, washed with phosphate-buffered saline (PBS), and incubated in type I collagenase solution (600 µ/mL; Worthington) for 15 min at 37°C, 5% CO₂. The endothelial layer was then removed by dabbing a cotton swab onto the leaflet surface and incubating it with collagenase overnight at 37°C, 5% CO₂. VICs were then isolated and cultured in T-flasks with DMEM (Gen depot, Katy, TX, USA) culture medium containing 5% fetal bovine serum, Penicillin (100 U/mL), streptomycin (100 U/mL), and fungizone (250 μg/mL) until confluent. VICs were treated with PCM containing 2 mM sodium dihydrogen phosphate (pH 7.4) and 50 µg/mL ascorbic acid in a culture medium to induce calcification. After reaching the desired confluence, cells were treated for 10 days with varying doses of niclosamide (Daewoong Therapeutics, Inc., Hwaseong, Korea) in PCM. Every two days, the culture medium was changed.

Cell viability analysis

VICs were seeded in 96 well plates (1×10⁴ cells/well) and starved overnight before being treated with various doses of niclosamide (0.01, 0.03, 0.1 µM). The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega Corporation, Madison, WI, USA) was used to determine cell viability. To determine cell viability, a 20 µL MTS tetrazolium solution was added per well and incubated for 2-3 h at 37°C 5% CO₂, with absorbance measured at 490 nm in an Enspire Multilabel Reader (PerkinElmer, Inc., Waltham, MA, USA).

Calcification analysis

The level of calcification was determined using alizarin red s staining. A solution of 40 mM alizarin red s (Sigma-Aldrich, St Louis, MO, USA) in 3X distilled water was prepared, and the pH was kept at 4.1-4.2 with a 10% ammonium hydroxide solution (Biosolution, Seoul, Korea). Briefly, VICs were seeded in 96 well plates and cultured for 10 days in PCM with or without different doses of niclosamide (0.01, 0.03, 0.1 µM). The control group received no treatment and was cultured in a standard control medium (CM). Cells were then fixed with 4% paraformaldehyde (Thermo Scientific, Franklin, MA, USA) and stained for 20 min at room temperature with an alizarin red s solution. Images were taken after washing twice to assess the degree of calcification. Cells were fixed with 70% ethanol at 4°C and incubated for 1 h with 10% cetylpyridinium chloride (Sigma-Aldrich) for quantitative analysis. The amount of alizarin red s released was measured using an Enspire Multilabel Reader (Perkin Elmer Ltd.) at 550 nm.

RT-qPCR assay

The Trizol reagent was used to isolate total RNA (Takara Bio, Shiga, Japan). The cDNA was created using the PrimeScript 1st strand cDNA synthesis kit according to the manufacturer’s instructions (Takara Bio). RT-qPCR was used to quantify genes of interest using TB green premix Ex Taq II (Takara Bio) and a Bio-Rad CFX384 Touch Real-time PCR detection system (Bio-Rad, Hercules, CA, USA). 2^-ΔΔCt was used to assess relative expression. All experiments have been conducted a minimum of three times. The primers used were as follows:

Runx2 forward, 5’-CCC TGA ACT CTG CAC CAA G-3’ and reverse, 5’- TCT GGC TCA AGT AGG AGG GA-3’; OPN forward, 5’-GGA GGA AAC GGA CGA CTT CAA ACA-3’ and reverse, 5’-GGC TTC GGA TCT GCG GAA CTT C-3’; Nox2 forward, 5’-GTG CAC CAT GAT GAG GAG AA-3’ and reverse, 5’-AGT TAG GCC GTC CGT ACA AG-3’; p22^phox Forward, 5’-AGT TCA CGC AGT GGT ACC TG-3’ and reverse, 5’-AGG CCC GGA TGT AGT AGT T-3’

Western blot analysis

VICs were lysed and harvested after being cultured for 10 days in various treatment conditions. Each lane was loaded with 20 µg of protein, and gel electrophoresis was performed on SDS-polyacrylamide gels. After that, the blots were transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% bovine serum albumin and incubated with primary antibodies overnight before being incubated with relevant secondary antibodies for 1 h. An enhanced chemiluminescence substrate was then used to visualize the membranes (Supersignal West Pico PLUS, Thermo Scientific). Each protein’s expression level was determined in at least three independent experiments. The following are the primary antibodies used in this study: Runx2 (1:2,000, LsBio, Seattle, WA, USA), OPN (1:2,000, Abcam, Cambridge, UK), Nox2 (1:2,000, Proteintech, Rosemont, IL, USA), p22^phox (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin (1:2,000, Cell Signaling Technology, Beverly, MA, USA), p-AKT (1:2,000, Cell Signaling Technology), t-AKT (1:2,000, Cell Signaling Technology), p-ERK (1:2,000, Cell Signaling Technology), t-ERK (1:2,000, Cell Signaling Technology) and secondary antibodies [horseradish peroxidase (HRP) - conjugated anti-mouse or anti-rabbit immunoglobulin G (1:20,000; Cell Signaling Technology)].

Determination of the level of oxidative stress

The level of intracellular reactive oxygen species (ROS) was measured using two different redox-sensitive cell-permeable dye, 2’,7’- dichlorodihydrofluorescein diacetate (DCF-DA), and dihydroethidium (DHE), in a microplate assay or fluorescence microscopy, as previously described (Shiwakoti et al., 2022). For fluorescence microscopy, VICs were seeded in a 12-well plate (1×10⁵ cells/well) and cultured for 10 days with various doses of niclosamide (0.01, 0.03, 0.1 µM) in PCM. After staining with DCF-DA (10μM) and DHE (10μM) for 30 min at 37°C, cells were washed with 1X PBS and counter-stained with DAPI (10 μM) for 10 min. The fluorescence intensity was measured using fluorescence microscopy and a fluorescein-specific filter (FITC). For the microplate assay, VICs were seeded in 96 well plates (1×10⁴ cells/well) and treated for 10 days with the above-mentioned niclosamide dose in PCM. The fluorescence intensity of the oxidation products was measured at excitation/emission wavelengths of 485/535 nm to quantify the level of ROS.

Determination of nicotinamide adenine dinucleotide phosphate oxidase activity

Lucigenin chemiluminiscence assay was carried out to determine the nicotinamide adenine dicnucletide phosphate (NADPH) oxidase activity as previously described (Richer and Ford, 2001) with some modifications. Chemiluminescence was monitored using an Enspire multimode plate reader (PerkinElmer, Inc.). Briefly, isolated VICs were seeded in 96 well plates and treated with different concentration of niclosamide (0.01, 0.03, 0.1 µM), and inhibitors apocynin (10 µM), allopurinol (10 µM), and L-N^G Nitroarginine methyl ester (L-NAME) (100 µM) (Kang et al., 2006; Richards et al., 2013; Sánchez-Esteban et al., 2022). 5µM lucigenin in PBS was added in each well and basal signal was recorded. Then, 100 µM NADPH was added and chemiluminiscence was recorded upto 20 min. Figures for induced LDCL were calculated from the mean signal recorded over a 5 min period of maximum light output. Chemiluminescence was expressed as percentage of control of chemiluminiscence intensity.

Statistical analysis

The mean ± standard error of mean is used to express the results. Statistical evaluation was performed by using either one-way analysis of variance (ANOVA) with Tukey’s post hoc test or two-way ANOVA with a Bonferroni post hoc test. All statistical analyses were performed using the Prism software (v8.0, GraphPad, San Diego, CA, USA). Values of p<0.05 were considered substantial.

Niclosamide attenuates PCM-induced calcification of VICs

Fig. 1A depicts the molecular structure of niclosamide. Firstly, the cytotoxicity of niclosamide in VICs was investigated. There were no signs of cytotoxicity up to 0.1 µM of niclosamide treatment as there was no significant difference in cell viability compared to the control group (Fig. 1B). Thus, additional experiments were conducted with 0.1µM as the highest concentration of niclosamide. Cells were cultured with different doses of niclosamide (0.01, 0.03, 0.1 µM) in PCM for 10 days to determine the role of niclosamide in the calcification of VICs. Inorganic phosphate-containing PCM has been shown to induce calcification and is used as an osteogenic differentiation model to determine the level of calcification (Goto et al., 2019; Goody et al., 2020). Following a 10-day culture with PCM, the cells were stained with alizarin red S. The results showed that PCM-induced cells stained positively with alizarin red S, in contrast to CM-treated cells. Pertinently, different niclosamide doses significantly reduced PCM-induced calcification (Fig. 1C, 1D). The level of calcification was also measured using the cetylpyridinium chloride assay, which revealed that niclosamide treatment prevented the PCM-induced increase in calcified nodule formation. These findings imply that niclosamide can reduce PCM-induced calcification of VICs.

Figure 1. Inhibitory effect of niclosamide on VICs calcification. (A) Molecular structure of niclosamide. (B) Cell Viability with different dose of niclosamide (0.01 µM, 0.03 µM, 0.1 µM) compared to control (CM). (C) The relative quantification of Alizarin red s staining results by cetyl pyridinium chloride assay. Results are expressed as mean ± SEM (n=4). ***p<0.001 vs. control CM; ^##p<0.01 vs. PCM. (D) Alizarin red s staining of VICs with different treatment groups: CM (control medium); PCM (pro-calcifying medium); PCM with different dose of niclosamide (Scale bar=200 µm).

Niclosamide reduces PCM-induced expression of calcification specific markers

PCM-induced cells have been shown to express the calcification marker OPN as well as the classic calcification regulator Runx2 (Liu et al., 2020). Thus, after 10 days, the calcification-specific genes Runx2 and OPN were tested in VICs cultured in PCM with or without niclosamide. The results showed that Runx2 and OPN mRNA expression were significantly increased in PCM-treated cells. However, niclosamide treatment downregulated the expression of Runx2 and OPN mRNA in PCM-induced VICs (Fig. 2A, 2B). Similarly, after 10 days treatment period, the protein expression of Runx2 and OPN was assessed. The results were consistent with the mRNA expression results and revealed a significant upregulation of Runx2 and OPN protein expression levels in PCM-treated cells, which were significantly downregulated in niclosamide-treated cells (Fig. 2C, 2D). These findings show that niclosamide can effectively prevent PCM-induced Runx2 and OPN upregulation, resulting in reduced calcification.

Figure 2. Effect of niclosamide on PCM induced calcification specific gene/protein expression in VICs. (A, B) The mRNA expression levels of calcification genes Runx2 and OPN were detected by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) in CM and PCM induced VICs with or without niclosamide treatment (0.01 µM, 0.03 µM, 0.1 µM). Results are expressed as mean ± SEM (n=3). *p<0.05; ***p<0.001 vs. CM; ^#p<0.05; ^###p<0.001 vs. PCM. (C, D) The protein expression of Runx2 and OPN were determined by western blot and quantification analysis. Results are expressed as mean ± SEM (n=4). **p<0.01; ***p<0.001 vs. CM; ^#p<0.05; ^##p<0.01; ^###p<0.001 vs. PCM.

Niclosamide reduces PCM-induced oxidative stress in VICs

CAVS pathogenesis is influenced by oxidative stress (Liu et al., 2020). One of the driving mechanisms in PCM or inorganic phosphate-induced calcification has been identified as an increase in oxidative stress caused by the increased generation of ROS (Khalid et al., 2020). Hence, experiments were conducted to ascertain the role of niclosamide in PCM-induced oxidative stress. Two redox sensitive indicators DHE and DCF-DA which primarily reflects the formation of intracellular ROS superoxide and hydrogen peroxide, respectively, were used to measure the level of ROS (Huang et al., 2020). VICs cultured with PCM produced more ROS, as evidenced by an increase in DHE and DCF-DA fluorescence intensity. This shows the increased levels of superoxide and hydrogen peroxide species which were significantly suppressed with various doses of niclosamide (0.01, 0.03, 0.1 µM), suggesting that niclosamide may attenuate PCM-induced calcification, at least in part, by reducing excessive oxidative stress (Fig. 3A, 3B). The primary sources of ROS in the cardiovascular system are proteins from the nicotinamide adenine dinucleotide phosphate oxidase (Nox) family (Liu et al., 2020). Therefore, NADPH oxidase activity and the expression levels of the Nox family subunits Nox2 and p22^phox were measured in various treatment groups. Lucigenin chemiluminescence assay was performed to determine the effect of niclosamide on NADPH oxidase. Lucigenin chemiluminescence assay is widely used to detect the levels of superoxide anion radical production by biological systems (Li et al., 1998). In consistent with other studies the level of NADPH oxidase was upregulated in calcified cells (Liberman et al., 2008; Okamoto et al., 2014; Liu et al., 2020; Adhikari et al., 2023). This increase in NADPH oxidase was downregulated with niclosamide treatment dose dependently. Our results coincided with a previous study that reported niclosamide possesses antioxidant function. This antioxidant function of niclosamide may be attributed to its ability to inhibit NADPH oxidase 2 (Park et al., 2019; Serrano et al., 2019). Similarly, we used NADPH oxidase inhibitor apocynin, xanthine oxidase inhibitor allopurinol and nitric oxide synthase inhibitor L-N^G Nitroarginine methyl ester (L-NAME) to observe the effect of inhibitors on NADPH oxidase activity. We found that apocynin could significantly reduce the NADPH oxidase levels which aligned with previous study (Sun et al., 2015) while, other inhibitors, although tend to reduce NADPH oxidase levels but not significantly (Fig. 3C). Additionally, in comparison to CM, VICs cultured with PCM showed increased mRNA expression of both Nox2 and p22^phox, which was significantly downregulated by niclosamide treatment (Fig. 3D, 3E). Furthermore, western blotting revealed an increase in Nox2 and p22^phox protein expression in the PCM-treated group, which was significantly reduced in the niclosamide-treated group (Fig. 3F, 3G).

Figure 3. Niclosamide inhibits PCM induced ROS generation in VICs. (A) Representative fluorescence microscopy images of DCFDA and DHE in live-adherent VICs with corresponding DAPI staining. (B) Relative level of ROS in VICs determined by microplate assay and were calculated as a percentage of CM. Results are expressed as mean ± SEM (n=3). ***p<0.001 vs. CM; ^#p<0.05; ^##p<0.01 vs. PCM. (C) Relative level of NADPH oxidase in VICs was determined by lucigenin chemiluminiscence assay and chemiluminiscence intensities were calculated as a percentage of CM. Results are expressed as mean ± SEM (n=3). **p<0.01 vs. CM; ^#p<0.05; ^##p<0.01; ^###p<0.001 vs. PCM. (D, E) The gene expression of NOX2 and its catalytic subunit p22^phox were determined by RT-qPCR. Results are expressed as mean ± SEM (n=4). **p<0.01; ***p<0.001 vs. CM; ^#p<0.05; ^##p<0.01; ^###p<0.001 vs. PCM. (F, G) The protein expression of Nox2 and p22^phox were determined by western blotting and quantification analysis. Results are expressed as mean ± SEM (n=4). *p<0.05; **p<0.01; vs. CM; ^#p<0.05; ^##p<0.01 vs. PCM.

Niclosamide inhibits the calcification of VICs by interfering with the GSK-3β/β-catenin signaling pathway

To induce calcification, inorganic phosphate generates ROS, which activates multiple signaling pathways including Wnt/β-catenin, AKT, and ERK (Byon et al., 2008; Khalid et al., 2020). A Wnt/β-catenin signaling pathway is involved in the induction of osteoblastic differentiation and Runx2-mediated calcification due to the inactivation of the destruction complex formed to degrade the β-catenin, resulting in β-catenin accumulation and translocation to the nucleus, which may contribute to the pathogenesis of CAVS (Caira et al., 2006; Liu et al., 2022; Tanaka et al., 2022). The protein expression of β-catenin and phosphorylated GSK-3β was examined in the PCM-treated group with or without niclosamide to assess the effects of niclosamide in the GSK-3β/β-catenin signaling pathway. The results showed that niclosamide could effectively reverse PCM-induced increases in β-catenin and phosphorylated GSK-3β protein expression (Fig. 4A, 4B). When activated by ROS, the AKT and ERK signaling pathways also induce calcification and the expression of calcification markers (Gu and Masters, 2009; Ning et al., 2022). The AKT signaling pathway and its downstream effector GSK-3β have been shown to be critical mediators of multiple cellular events such as cell proliferation, differentiation and apoptosis (Hou et al., 2021). Activation of ERK has also shown to inhibit GSK-3β, thus resulting in the β-catenin translocation into the nucleus (Ding et al., 2005). To elucidate the specific pathway through which niclosamide inhibits aortic valve calcification, we examined the potential role of AKT and ERK, which has been shown to regulate β-catenin (Caraci et al., 2008; Zheng et al., 2017). In the current study, in addition to β-catenin and phosphorylated GSK-3β, phosphorylated AKT and phosphorylated ERK protein expression was increased in PCM-treated cells and significantly decreased with niclosamide treatment (Fig. 4C, 4D). These findings suggest that niclosamide may inhibit AKT and ERK phosphorylation resulting in its inactivation. Taken together, our findings suggest that niclosamide may help to alleviate PCM-induced calcification by targeting oxidative stress mediated GSK-3β/β-catenin signaling via inhibiting activation of AKT and ERK (Fig. 5).

Figure 4. Niclosamide inhibits calcification of VICs via interfering GSK-3β/β-catenin. (A, B) The protein expression of β-catenin and phosphorylated GSK-3β in VICs induced with PCM with or without niclosamide treatment and the relative quantification of obtained blots. Results are expressed as mean ± SEM (n=3). **p<0.01; ***p<0.001 vs. CM; ^#p<0.05; ^##p<0.01; ^###p<0.001 vs. PCM. (C, D) The protein expression of phosphorylated AKT and ERK in PCM induced VICs with or without niclosamide determined by western blotting and the relative quantification of obtained blots. Phosphorylated proteins were normalized against total proteins. Results are expressed as mean ± SEM (n=3). ***p<0.001 vs. CM; ^#p<0.05; ^##p<0.01 vs. PCM.

Figure 5. Graphical scheme of niclosamide inhibiting PCM-induced aortic valve interstitial cell calcification by interfering GSK-3β/β-catenin signaling pathway.

In this study, we discovered that niclosamide acts as a negative regulator of VICs calcification. The mechanism involved is interference with the GSK-3β/β-catenin signaling pathway, at least in part. For the first time, we show that niclosamide inhibits PCM-induced VICs calcification.

CAVS is a slowly progressive disorder for which no effective medical therapy has been developed to date (Hutcheson et al., 2014; Pasquale et al., 2019). With valve replacement as the only treatment option, developing a novel therapeutic strategy is deemed critical, which necessitates a thorough understanding of the molecular mechanisms involved in CAVS pathobiology (Rutkovskiy et al., 2017). CAVS pathogenesis is multifaceted, involving multiple mechanisms like dysregulated calcium-phosphate metabolism, inflammation, endothelial dysfunction, and endothelial nitric oxide synthase alterations (Kraler et al., 2021). When VICs are activated, the disease progresses from endothelial dysfunction to slow reorganization of the valve matrix to myofibroblastic and osteoblastic differentiation of VICs, culminating in CAVS with stiff and fibrosed aortic valve leaflets and expression of bone-related proteins like osteocalcin, alkaline phosphatase, and OPN (Rutkovskiy et al., 2017). Hence, we used porcine VICs as a model to investigate the role of niclosamide in aortic valve calcification. Niclosamide has been shown to have anticancer and anti-inflammatory properties by regulating various cellular pathways such as STAT3, mTORC1, NF- κB, and Wnt/β-catenin signaling pathways (Liu et al., 2017; Tanaka et al., 2022). Because these pathways are directly involved in regulating calcification, niclosamide may alter these pathways and inhibit calcification in VICs. Furthermore, niclosamide has recently been shown to inhibit vascular calcification and reduce Runx2, a key factor in calcification (Tanaka et al., 2022). However, the effect of niclosamide on valvular calcification yet remains to be explored. In this study, we found that niclosamide could inhibit VICs calcification via the GSK-3β/β-catenin signaling pathway.

PCM induces calcification and calcium deposits through the phenotypic transition of VICs to myofibroblasts or osteoblast-like cells, as well as the expression of calcification markers such as Runx2, BMP2, and OPN (Yang et al., 2009; Goto et al., 2019; Goody et al., 2020; Lu et al., 2020). Runx2 is a transcription factor that regulates the expression of various osteoblast proteins such as osterix, OPN, bone sialoprotein, type I collagen, and osteocalcin. BMP2 controls Runx2 expression by activating multiple signaling pathways (Jang et al., 2012). In this study, calcification levels were significantly increased in PCM-treated cells, as were mRNA and protein expression of calcification markers Runx2 and OPN, which were suppressed by niclosamide treatment. Our findings were consistent with recent research on niclosamide’s ability to inhibit vascular calcification (Tanaka et al., 2022). These findings suggest that niclosamide could be a promising new therapeutic intervention for preventing aortic valve calcification.

Oxidative stress is associated with several cardiovascular diseases such as endothelial dysfunction, hypertension, vascular calcification, atherosclerosis, cardiac remodeling, stroke, and diabetes (Sugamura and Keaney, 2011; Münzel et al., 2017; Incalza et al., 2018; Shiwakoti et al., 2020; Adhikari et al., 2022). Furthermore, oxidative stress is directly involved in the initiation and progression of CAVS (Greenberg et al., 2022). By activating osteogenic and profibrotic genes such as Runx2, oxidative stress promotes osteoblast differentiation of VICs (Branchetti et al., 2013). This strongly suggests that oxidative stress occurs before VICs transdifferentiation into an osteoblastic phenotype. There are several species of ROS in vasculature like superoxide anions, hydrogen peroxide, and hydroxyl radicals, in addition to many others. Among them, superoxide ion and hydrogen peroxide are important components that regulate the differentiation of osteoclasts, and increased level of superoxide ion and hydrogen peroxide has shown to contribute to increased oxidative stress in calcific aortic valvular stenosis in humans (Miller et al., 2008; Agidigbi and Kim, 2019). Similar to those findings, intracellular ROS superoxide and hydrogen peroxide levels in VICs cultured with PCM were significantly increased, emphasizing the role of oxidative stress in VICs calcification. This increase in ROS level was significantly reduced by niclosamide treatment, implying that niclosamide may attenuate PCM-induced calcification by reducing excessive oxidative stress. Uncoupled nitric oxide synthases, nicotinamide adenine dinucleotide phosphate oxidases (Nox), and mitochondria are the major sources of ROS in the cardiovascular system (Vásquez-Vivar et al., 1998; Lassègue et al., 2012; Zhang et al., 2013). Nox2 (a Nox family protein) is the most abundant and is involved in CAVS pathogenesis through its superoxide generation mechanism (Lassègue et al., 2012; Liu et al., 2020). Several studies have also found that Nox2 expression levels in calcified aortic valves are altered (Liberman et al., 2008; Chu et al., 2013). In PCM-induced calcification, NADPH oxidase activity and the expression levels of Nox2 and p22^phox (a membrane catalytic subunit of Nox) mRNA and protein were determined. The level of NADPH oxidase was upregulated in PCM treated cells which was significantly downregulated by niclosamide treatment dose-dependently. To confirm the roles of NADPH oxidase, effects of different ROS inhibitors apocynin (NADPH oxidase inhibitor), allopurinol (xanthine oxide inhibitor) and L-NAME (nitric oxide synthase inhibitor) were studied. The level NADPH oxidase was significantly reduced with apocynin treatment although, allopurinol and L-NAME did not show significant reduction of NADPH oxidase levels. Furthermore, VICs cultured with PCM significantly increased the mRNA and protein expression of Nox2 and p22^phox, which was significantly reduced by niclosamide treatment. These findings suggest that niclosamide can effectively reduce ROS production in VICs by downregulating the Nox family. This also suggests that the NADPH oxidase may be the major source of ROS during calcification induced by PCM. In addition, NADPH oxidase inhibitors are known to inhibit VICs calcification (Liu et al., 2020). Taken together, niclosamide inhibits VICs calcification at least in part via inhibition of NADPH oxidase.

It has been demonstrated that increased Nox2 expression activates the GSK-3β/β-catenin pathway as a downstream signal of oxidative stress to induce calcification (Liu et al., 2020). When the GSK-3β/β-catenin pathway is activated, the β-catenin destruction complex is destroyed due to GSK3β inactivation, resulting in the accumulation of β-catenin in the cytoplasm, which translocates to the nucleus, activating transcriptional regulator Runx2 and calcification marker BMP2-mediated calcification (Liu et al., 2022). In this study, niclosamide prevented a significant increase in β-catenin protein expression in PCM-induced calcification of VICs. Numerous studies have found that increased β-catenin expression promotes osteogenic differentiation and thus speeds up the development of vascular calcification (Beazley et al., 2012; Rong et al., 2014). Thus, the inhibitory role of niclosamide in the calcification of VICs could be linked to a downregulation of β-catenin expression. Similarly, niclosamide inhibited GSK-3β activity by decreasing the protein expression of p-GSK-3β, the inactive form of GSK-3β, implying that niclosamide may inhibit calcification via the GSK-3β/β-catenin pathway. Aside from the GSK-3β/β-catenin pathway, oxidative stress increases the Runx2 expression via the PI3K/AKT, ERK, and NFκB signaling pathways (Greenberg et al., 2022; Qiao, 2022). H2O2-induced oxidative stress was found to activate AKT signaling in a variety of cell types (Byon et al., 2008). Furthermore, oxidative stress and inorganic phosphate both activate ERK1/2 signaling pathways in osteogenic cells, as well as extracellular matrix mineralization (Chaudhary et al., 2016; Khalid et al., 2020). AKT and ERK phosphorylation can also trigger the inactivation of GSK-3β, leading to the intracellular aggregation of GSK-3β and transfer of β-catenin into the nucleus, leading to the expressions of downstream target genes of calcification (Caraci et al., 2008; Zheng et al., 2017). In this study, along with β-catenin and phosphorylated GSK-3β, PCM-induced phosphorylation of both AKT and ERK in VICs was significantly inhibited by niclosamide treatment. ERK is a member of the mitogen-activated protein kinases family (Khalid et al., 2020), which is known to play an important role in osteoblastic differentiation and mineralization (Ge et al., 2007). When activated, phosphorylated ERK enters the nucleus and regulates transcription factor activity and gene expression, as well as calcification in osteoblast or osteoblast precursors cells via Runx2 transcription factor activation (Gu and Masters, 2009). Similarly, activation of AKT signaling regulates Runx2 expression as well as the expression of other bone markers (Byon et al., 2008). Thus, niclosamide may inhibit VICs calcification by interfering with AKT and ERK phosphorylation. Overall, we used PCM as a calcification inducer and investigated the effect of niclosamide on aortic valve calcification, discovering that niclosamide could inhibit aortic valve calcification, at least in part, by targeting oxidative stress mediated GSK-3β/β-catenin signaling pathway by inhibiting the activation of AKT and ERK.

The current study used in vitro experiments to determine the role of niclosamide in the calcification of VICs, which must be extrapolated to see the effects of niclosamide in vivo before using it as a potential candidate for calcification treatment. Most animals, to the best of our knowledge, do not naturally develop CAVS, and the genetically modified or high-fat diet model is known to have reproducibility issues (Goto et al., 2019; Goody et al., 2020). Hence, newer in vivo models with higher reliability is required to assess the effects of niclosamide on CAVS. Furthermore, niclosamide limitations as a potential drug candidate include poor solubility as well as low absorption and oral bioavailability (Chang et al., 2006). Thus, there is a great deal of interest in discovering new niclosamide derivates with improved bioavailability.

In summary, we investigated the effect of niclosamide in CAVS and discovered that niclosamide could inhibit VICs calcification induced by PCM. The inhibition mechanism may involve interfering with signaling pathways involved in the pathogenesis of CAVS, specifically the GSK-3β/β-catenin signaling pathway via inhibition of AKT and ERK. The activation of AKT and ERK has shown to modulate several downstream pathways beside GSK-3β/β-catenin signaling pathway such as NFκB and mTORC1 pathway (Winter et al., 2011; Sui et al., 2014). Additionally, niclosamide is reported to target various signaling pathways including STAT3, mTORC1, NFκB and Notch (Tanaka et al., 2022). Thus, the protective effect of niclosamide on calcification of VICs might not solely depend on GSK-3β activation, but it could be one of the downstream targets. Further studies are required to better understand the mechanistic pathways in depth, and to establish safety profile when intending to use niclosamide for cardiovascular treatment.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.