Hydrogen sulfide inhibits calcification of heart valves; implications for calcific aortic valve disease

Background and Purpose Calcification of heart valves is a frequent pathological finding in chronic kidney disease and in elderly patients. Hydrogen sulfide (H2S) may exert anti‐calcific actions. Here we investigated H2S as an inhibitor of valvular calcification and to identify its targets in the pathogenesis. Experimental Approach Effects of H2S on osteoblastic transdifferentiation of valvular interstitial cells (VIC) isolated from samples of human aortic valves were studied using immunohistochemistry and western blots. We also assessed H2S on valvular calcification in apolipoprotein E‐deficient (ApoE−/−) mice. Key Results In human VIC, H2S from donor compounds (NaSH, Na2S, GYY4137, AP67, and AP72) inhibited mineralization/osteoblastic transdifferentiation, dose‐dependently in response to phosphate. Accumulation of calcium in the extracellular matrix and expression of osteocalcin and alkaline phosphatase was also inhibited. RUNX2 was not translocated to the nucleus and phosphate uptake was decreased. Pyrophosphate generation was increased via up‐regulating ENPP2 and ANK1. Lowering endogenous production of H2S by concomitant silencing of cystathionine γ‐lyase (CSE) and cystathionine β‐synthase (CBS) favoured VIC calcification. analysis of human specimens revealed higher Expression of CSE in aorta stenosis valves with calcification (AS) was higher than in valves of aortic insufficiency (AI). In contrast, tissue H2S generation was lower in AS valves compared to AI valves. Valvular calcification in ApoE−/− mice on a high‐fat diet was inhibited by H2S. Conclusions and Implications The endogenous CSE‐CBS/H2S system exerts anti‐calcification effects in heart valves providing a novel therapeutic approach to prevent hardening of valves. Linked Articles This article is part of a themed section on Hydrogen Sulfide in Biology & Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.4/issuetoc


| INTRODUCTION
In the developed countries, calcific aortic valve disease (CAVD), an actively regulated disease process, is the most common valvular heart disease with high morbidity and mortality (Yutzey et al., 2014).
Numerous studies show that CAVD is accompanied by calcification, lipid accumulation, and inflammation resulting in heterogeneous lesions within the heart valvular tissue. The ratio of the calcified and non-calcified regions could guide in distinguishing the state of calcification (Chester, 2011;Lusis, Mar, & Pajukanta, 2004;Mohler, 2004;Mohler et al., 2001;Speer & Giachelli, 2004). Cardiovascular calcification is a complex, chronic disease of the major and medium-sized arteries including those in aortic valves. Such calcification is involved in the increased risk of cardiovascular morbidity and mortality. Pathologically, it is well known that its progression is much more pronounced in patients with diabetes and chronic kidney disease (CKD; Davignon & Ganz, 2004;Libby, Ridker, & Maseri, 2002;Rajamannan et al., 2011;Stocker & Keaney, 2004).
Hydrogen sulfide (H 2 S) is the third endogenous gasotransmitter, along with NO and carbon monoxide (Wang, 2002). Previously, our laboratory demonstrated that NaSH (a H 2 S donor compound) significantly inhibits the mineralization of vascular smooth muscle cells (Zavaczki et al., 2011). Jiang, Wu, Li, Geng, & Tang (2005) and now many groups have shown cystathionine γ-lyase (CSE) in cardiac tissue and it is important for normal heart function (Chen, Xin, & Zhu, 2007). They demonstrated that disturbance of H 2 S production contributed to the development of heart diseases as manifested by lower levels of H 2 S in plasma, in patients with coronary heart disease (Jiang et al 2005;Shen, Shen, Luo, Guo, & Zhu, 2015). Furthermore, Abe and Kimura (1996) presented for the first time that endogenous H 2 S production by cystathionine β-synthase (CBS) contributed to normal brain function. Kang, Neill, and Xian (2017) synthesized a new generation, slow release, H 2 S donor compound (GYY4137) from Lawesson's reagent and morpholine. Protonation of GYY4137 resulted in more stable H 2 S-releasing compounds such as AP67 and AP72. AP72 has an excellent water solubility and a very slow generation of H 2 S, compared to the fast H 2 S-releasing donors such as NaSH and Na 2 S (Chitnis et al., 2013;Kang et al., 2017;Nagy et al., 2014).
In calcified valves, the valvular interstitial cells (VIC) transdifferentiate into osteoblast-like cells, identified by up-regulation of alkaline phosphatase (ALP) activity, and increased levels of osteocalcin expression at later stages (Rajamannan et al., 2003). One of the most potent, recognized inducers of vascular calcification is raised levels of plasma phosphate, in CKD patients (Becs et al., 2016). The increase in its intracellular level promotes nuclear translocation of the osteogenic transcription factor RUNX2 resulting in transition of cells towards an osteoblast phenotype (Ducy, Zhang, Geoffroy, Ridall, & Karsenty, 1997). Phosphate uptake occurs via phosphate carriers Pit1 and Pit2 (Crouthamel et al., 2013;X. Li, Yang, & Giachelli, 2006

What this study adds
• CSE-and CBS-derived H 2 S and H 2 S-releasing donors inhibit mineralization of aortic valves.
• Anti-calcification occurs via inhibiting phosphate uptake, preventing nuclear translocation of RUNX2, and increasing pyrophosphate levels.

What is the clinical significance
• H 2 S-releasing donors and CSE/CBS-derived H 2 S have therapeutic potential in calcific aortic valve disease.
crystals. Ectonucleotide pyrophosphatase/PDE-2 (ENPP2) is a cell membrane glycoprotein that generates PPi via cleaving ATP. Ankyrin G1 (ANK1) is a transmembrane protein which has a critical role in the regulation of pyrophosphate levels. The main function of ANK1 is transporting intracellular PPi into the extracellular space (Jansen et al., 2012;Mitton-Fitzgerald, Gohr, Bettendorf, & Rosenthal, 2016).
The purpose of this study was to explore the effects of H 2 S donors (NaSH, Na 2 S, GYY4137, AP67, or AP72) on the development of calcification in human VIC and ApoE −/− mice fed with atherogenic diet and to investigate its underlying mechanism with regard to the development of atherosclerosis and mineralization. Our study identifies a novel potential treatment for preventing and/or reversing valvular calcification. N = 9) on every other day as previously described (Potor et al., 2018

| Western blot
To show RUNX2 translocation into the nucleus, we separated the nuclei and cytoplasm fractions with the nuclear extraction kit. After that, nucleus and cytoplasm lysates were separated by electrophoresis with 10% SDS-PAGE. After blotting, the membrane was incubated with rabbit anti-human RUNX2 (Cbfa-1) antibody at 1:600 dilution

| Intracellular phosphate uptake measurement
The VIC were cultured on 12-well plates exposed to calcification medium, with or without of phenol red using DMEM supplemented with/without AP72 (20 μmol·L −1 ) for 5 days. Cells were lysed with 0.5% NP40 and 1% Triton-X100. Whole cell lysate centrifuged at

| Determination of sulfide level from AS and AI valve tissues with zinc precipitation assay
Sulfide levels were measured with the zinc precipitation method developed by Gilboa-Garber (1971) and improved by Ang, Konigstorfer, Giles, and Bhatia (2012). The human valves were pulverized under liquid N 2 . Next, the samples were taken up in PBS (pH 7.4), followed by sonication. After that, the sample was centrifuged at 12,000× g for 15 min, and the lipid-free, clear supernatant was collected; 200-μl sample was mixed with 350 μl 1% zinc acetate and 50 μl 1.5-mol·L −1 sodium hydroxide and incubated for 60 min on a shaker. The incubation step was followed by centrifugation at 2,000× g for 5 min to pellet the generated zinc sulfide. The supernatant was then removed, and the pellet was washed with 1 ml of distilled water by vortexing extensively, followed by centrifugation at 2,000× g for 5 min. The supernatant was then aspirated off, and the 2.14 | CSE and CBS double gene silencing CSE and CBS genes were silenced, using appropriate siRNAs (Ambion, 4392420; s3710). Briefly, the VIC were cultured on 12-well plates in antibiotic-free medium (DMEM, Sigma). At about 70% of confluence, cells were transfected with siRNA against CSE and CBS (Ambion, 4390824; s289). Transfection occurred for 4 hr in minimal serumcontent medium (Opti-MEM; Gibco). At the end of transfection, 30% FBS containing antibiotic-free DMEM was added. Next day, cells were washed and treated with AP72 every second day until 5 days. The sequences of the siRNAs were inserted in Data S1.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMA-COLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander, Fabbro et al., 2017; 3 | RESULTS

| H2S prevents calcification of VIC
We compared the potential of different H 2 S donors for inhibiting calcification of VIC isolated from human aortic valves. Cells were maintained in calcification medium containing 2.5-mmol·L −1 inorganic phosphate and 1.8-mmol·L −1 calcium-chloride. VIC were treated with H 2 S from two sources. One source of H 2 S was the simple sulfide salts NaSH and Na 2 S that instantaneously generates H 2 S via pH-dependent salt dissociation and the other was the novel slow-release sulfide donors (AP67 and AP72) and the more commonly used donor GYY4137 (synthesized in-house). As expected, the transition of VIC into osteoblasts occurred in the calcifying environment, which is reflected by the calcium accumulation in the extracellular matrix ( Figure 1) and the increased expression of osteocalcin and ALP ( Figure 2). Importantly, all H 2 S donors decreased calcium deposition in a dose-dependent fashion (Figure 1). NaSH reached the maximum inhibition at 150 μmol·L −1 (Figure 1a), Na 2 S and GYY4137 attenuated calcification at 100 μmol·L −1 (Figure 1b,c), while AP67 suppressed calcification at 50 μmol·L −1 (Figure 1d), compared to calcification medium without H 2 S supplementation. Among the H 2 S donors, AP72 fully prevented calcium deposition in the extracellular matrix of VIC at 20 μmol·L −1 concentration (Figure 1e,f). Furthermore, osteocalcin accumulation and expression of ALP in VIC along with calcium deposition were also prevented by AP72 (20 μmol·L −1 ; Figure S3A-C).
Similarly, other fast (NaSH and Na 2 S) and slow (GYY4137 and AP67) sulfide-releasing compounds significantly attenuated the secretion of osteocalcin ( Figure S1A). ALP and Alizarin Red S staining showed pronounced osteoblastic transformation of VIC in the calcific environment, and this effect was prevented by AP72 ( Figure S3C,D). As demonstrated in Figure S3E, AP72 did not exhibit any cytotoxic effects on VIC at the applied dose. We observed a "U" shape curve in the inhibition of mineralization. The use of H 2 S donors at concentrations in excess of that stated above resulted in a concentrationdependent decline in protection. From these studies, we then selected the most effective H 2 S donor (AP72) for further investigation to explore the mechanism by which H 2 S regulates the calcification processes.
As phenol is known to capture H 2 S (Huang, Zhang, Zhou, Tao, & Fan, 2017), we tested if AP72 affects calcification at lower concentrations in phenol red-free medium, compared to calcification medium with phenol red. Without phenol red, AP72 significantly inhibited calcification of VIC at concentration of 2.5 nmol·L −1 to 5 μmol·L −1 ( Figure S1B). Accordingly, osteocalcin accumulation was prevented, and phosphate uptake was also decreased by AP72 in phenol red-free condition (Figure 2a,b). ALP and Alizarin Red S staining demonstrated the inhibitory effect of AP72 at a concentration of 2 μmol·L −1 (Figure 2d,e). As shown in Figure S3, in phenol red-containing medium, AP72 exhibited inhibitory effect on calcification in VIC at one-order magnitude higher concentration. There was no cytotoxicity in the VIC cultures due to the sulfide donor compounds, at the most effective concentrations ( Figure S1C).

| AP72 inhibits the phosphate-induced nuclear translocation of RUNX2
RUNX2 is a key transcription factor associated with an early osteoblastic differentiation of vascular smooth muscle cells and VIC. We therefore assessed the effects of AP72 treatment on the localization of RUNX2 in VIC cultured in calcification medium. Immunofluorescence staining indicated that RUNX2 was located in the cytoplasm of VIC cultured in growth medium (control medium; Figure 3a; upper panels). Phosphate exposure of VIC triggered the translocation of RUNX2 from the cytoplasm to the nucleus (Figure 3a; middle panels).
As demonstrated in Figure 3a (lower panel), AP72 prevented the appearance of RUNX2 in the nucleus of VIC maintained in calcification medium. To support our immunofluorescence observation, cytoplasmic and nuclear fractions of VIC were examined for RUNX2 using western blot analysis. We found that RUNX2 appeared in the nucleus in response to calcification medium (Figure 3b; left panel), while its level was decreased in the cytoplasmic fraction (Figure 3b; right panel).
Importantly, AP72 treatment prevented the translocation of RUNX2 into the nucleus of VIC exposed to phosphate (Figure 3b).
Next, we examined the nuclear location of the RUNX2 in VIC derived from human valves with visible calcification (AS) and without calcification (AI). Confocal microscopy and western blot analysis showed that RUNX2 was mainly located in the nucleus of VIC derived from AS human valve tissue, whereas RUNX2 was detected in the cytoplasm of VIC derived from AI tissue (Figure 3c,d). Importantly, exposure of cells to 200 μmol·L −1 of AP72 did not restrain the nuclear translocation of RUNX2 from the cytoplasm to the nucleus ( Figure S4).

| Hydrogen sulfide enhances PPi production
PPi is a well-known anti-calcification molecule which is regulated by ENPP2 and ANK1. Therefore, we tested the effects of H 2 S on PPi production, through regulating the expression of ENPP2 and ANK1 in VIC. As shown in Figure 4a Figure 4c). We also tested GYY4137 and AP67 for their effects on the PPi levels in VIC. These donors enhanced the PPi level less effectively than AP72 (Figure 4c). The fast sulfide-releasing molecules (NaSH and Na 2 S) were able to enhance the PPi level only to the baseline ( Figure S5). Moreover, our measurements in human heart valve tissue samples indicated significantly lower ENPP2 protein levels and lower PPi content in AS valve specimens than those in samples from AI valves (Figure 4d,e).

| Attenuated CSE and CBS expression promotes VIC calcification
To investigate potential anti-calcification effects of endogenously produced H 2 S we first silenced CSE production in VIC using siRNA.  Figure S8). Furthermore, we monitored the progression of calcification in extracellular matrix on the first and third days. On the first day, we did not find significant alteration in the calcium content of VIC maintained in calcifying condition compared to control. In contrast, we observed a significantly increased extracellular calcium content in VIC silenced with CSE/CBS siRNA (Figure 5e).
Mineralization was more robust in the double silenced VIC by day three (Figure 5e). As shown in Figure S7B, production of H 2 S was lowered in calcifying conditions, compared to cells cultured in growth media and that was further decreased by double silencing for CSE and CBS. Finally, we examined the expression of 3-MST in VIC treated with CSE/CBS siRNA. We found that siRNA specific to CSE and CBS decreased 3-MST protein level in VIC ( Figure S7C). Figure S8.

| Hydrogen sulfide inhibits phosphate uptake through affecting the functions of phosphate channels
As cellular phosphate uptake is a key event in the mineralization process, we therefore next measured intracellular phosphate levels in VIC and observed a significant elevation in cells cultured in calcification medium, compared to cells kept in control medium (Figure 6a). Exposure of VIC to AP72 diminished this increase in phosphate content to the level observed in control cells (Figure 6a). In order to explain the inhibition of phosphate uptake, we measured the expression of phosphate channels (Pit1 and Pit2) in VIC maintained in calcification medium, with or without AP72. We found that AP72 did not affect the expression of Pit1 and Pit2 channels (Figure 6b,c). Therefore, we hypothesize that sulfide-induced post-translational modification of these channels might affect phosphate uptake. Measurements to support or disprove this hypothesis are underway.

FIGURE 2
Phenol red impairs anti-calcification effect of H 2 S. Cultured VIC in (a) phenol red-containing DMEM (Sigma) or (b) phenol red-free media were supplemented with AP72 (2 μmol·L −1 ; 20 μmol·L −1 ) for 5 days, and calcium content of the cells was measured and normalized to protein content of the cells. Alizarin Red S staining represents the microscopic image of calcium deposition of extracellular matrix. Data shown are means ± SEM of five independent experiments. *P < .05, significantly different as indicated 3.6 | Expression of CSE and generation of H2SH2S in human aortic valves CSE is one of the main endogenous hydrogen sulfide producing proteins in the human body. Using western blot analyses, we investigated the expression of CSE in tissue lysates of human AS and AI valves. We found higher expression of CSE in AS valves with massive calcification, compared with the expression in AI valves known to lack calcification ( Figure 7a). In contrast, sulfide levels that can be precipitated by Zn 2+ under alkaline conditions from tissue lysates of valves were markedly and significantly lower in calcified AS specimens compared to not calcified AI specimens (Figure 7b). Next, we performed dual immunohistochemistry analyses (CSE-SMA and CSE-ALP) on human AI and AS valves to localize CSE. As shown in Figure 7, less SMA+ and more CSE+ cells were present in calcified AS tissue than in AI tissue (upper panels). ALP-CSE double staining revealed the appearance of ALP+ cells expressing high levels of CSE protein in AS valve samples, while ALP+ cells were not detected in AI valve (Figure 7c; lower panels).
The potential of osteoblastic differentiation was dependent upon the origin of VIC. Under the same calcifying conditions, VIC derived from AS exhibited earlier mineralization than AI. The higher CSE level found in AI was accompanied by delayed calcification (Figure 7c).
Human CSE recombinant protein was used as a control for the CSE western blot ( Figure S8B).

| DISCUSSION
CAVD is the most common valvular heart disease, found mostly in patients with CKD and in the elderly (Freeman & Otto, 2005 O'Brien et al., 1995). More recently it has been proposed that therapies targeting the molecular processes of aortic valve calcification (Yutzey et al., 2014) could increase the durability of surgically implanted and transcatheter bioprosthetic valves (Leopold, 2012).
Increased phosphate levels are a significant risk factor for CAVD and have been demonstrated in many studies to act as a key regulator of vascular calcification (Adeney et al., 2009;Giachelli, 2009;Hruska, Mathew, Lund, Qiu, & Pratt, 2008). It provokes calcification of vascular cells in a process mediated by sodium-dependent phosphate cotransporters (Pit1 and Pit2) which facilitate the entry of phosphate into the cells (Zarjou et al., 2009). This induces osteoblastic transition of vascular smooth muscle cells via a process that is accompanied by translocation of RUNX2 from the cytosol into the nucleus required for osteoblast differentiation, bone matrix gene expression, and, consequently, bone mineralization (Komori, 2006;Zarjou et al., 2009).
There is also induction of ALP, an important enzyme in early osteogenesis and of osteocalcin, a major non-collagenous protein found in bone matrix that is demonstrated to regulate mineralization (Zarjou et al., 2009).
We have earlier demonstrated that CSE expression was elevated in human atheroma, derived from the carotid artery, with lipid FIGURE 4 AP72 enhances generation of PPi. VIC were cultured in growth medium or calcification medium alone or supplemented with AP72 (20 μmol·L −1 ) for 5 days. Differences in (a) ENPP2 protein and mRNA levels, (b) ANK1 protein and mRNA levels, (c) pyrophosphate level measured using a PPiLight pyrophosphate detection kit are presented. (d) Representative ENPP2 western blot from AI and AS tissue lysate of heart valves. (e) Pyrophosphate levels of heart valve tissues were measured using a pyrophosphate detection kit. Data shown are means ± SEM of five independent experiments. *P < .05, significantly different as indicated; ns, not significant accumulation without calcification (Potor et al., 2018). In the present study, we found that CSE expression was higher in human AS valves known to be associated with calcification, compared with that in AI valves lacking calcification (Figure 5a). Furthermore, ALP+ cells were shown to exhibit CSE positivity (Figure 7a) in AS valves. Importantly, the generation of H 2 S was decreased in AS heart valve tissue ( Figure 7b) Figure 5a). The interaction between CSE/CBS expression was reported by Nandi and Mishra (2017), demonstrating that CBS deficiency up-regulates CSE protein levels. Therefore, we investigated the expression of CBS in CSE silenced VIC and found that CSE silencing resulted in an increased CBS protein expression in VIC (Figure 5b).
We therefore silenced both CSE and CBS, which further increased calcium accumulation in the extracellular matrix of VIC cultured in high phosphate containing medium (Figure 5c). Furthermore, when we followed the progression of calcification, we found that CSE+CBS silenced VIC underwent calcification earlier compared to cells maintained in calcification medium without siRNA (Figure 5e). Treatment of VIC with a synthetic CSE inhibitor (PPG) together with a CBS inhibitor (AOAA) also enhanced the extracellular calcium deposition To gain insight into the contribution of endogenous sulfide production to prevent mineralization of VIC, we employed several H 2 S generating molecules. For example, we used NaSH and Na 2 S which instantly generate H 2 S in aqueous solution as well as the slow sulfide release donor molecules (GYY4137, AP67, and AP72). It is important to note that the concentrations of donors do not represent the total amount of released sulfide, and all slow donors have different sulfide-releasing potential. In addition, it is increasingly recognized that slow-releasing H 2 S donors are likely to more closely mimic the effects of the endogenous H 2 S buffer system, because of their slow generation of low sulfide levels (Nagy et al., 2014;Whiteman et al., 2015). In particular, we have shown that slow-releasing H 2 S molecules such as AP72 (Nagy et al., 2014) exhibited a greater inhibition of the calcification, compared with GYY4137, possibly because the rate of H 2 S release from AP72 is faster than that of GYY4137, a poorly efficient H 2 S donor . It is important to note that phenols are capable of absorbing a high amount of H 2 S from liquids (Huang et al., 2017). Accordingly, in our experiments, AP72 inhibited VIC mineralization at one-order magnitude lower concentration in phenol red-free media. Additionally, phosphate uptake of the cells was   (Figure 4b). Taken together, the regulation of PPI generation by H 2 S represents a novel additional mechanism to control calcification.
The ApoE −/− mouse is the most widely studied animal model for atherosclerosis (Massy et al., 2005;Rattazzi et al., 2005). We showed that calcification occurred in the aorta and aortic valves after a highfat diet with the most pronounced calcification seen in the aortic arch.
In our current study, the development of mineralization in aortic valves of ApoE −/− mice on a high-fat diet was strongly associated with the increase of extracellular matrix and the formation of hydroxyapatite nodules, and the expansion of extracellular matrix in the heart valves was prevented by AP72. We also observed that similar to human aortic stenosis, the expression of CSE in VIC was increased in mice on a high-fat diet. Several triggers were previously identified by our group for inducing CSE expression in resident cells of atherosclerotic lesions including plaque lipids, oxidized LDL, TNF-α, and IL-1β (Potor et al., 2018). Therefore, the induction of CSE might represent an adaptive cellular response to lipid deposition and inflammation, serving as a clinical biomarker of CAVD. In the process of the calcification, vascular smooth muscle cells were shown to transform into osteoblastic-like cells and lose their SMA (Johnson, Leopold, & Loscalzo, 2006). In our experiments, immunostaining of the aortic valves of ApoE −/− mice demonstrated that AP72 treatment preserved α-SMA (Figure 7, lower panels). In keeping with our hypothesis that CAVD is a condition of perturbed H 2 S bioavailability, we found a deficiency in the generation of H 2 S in human AS valves in spite of the elevated expression of CSE. This contradiction can be resolved by the observations of Bibli et al., (2019), who found a markedly decreased H 2 S production after phosphorylation of Ser 377 of CSE, resulting in enzyme inactivation. As such, therapeutic supplementation of H 2 S using H 2 S donor molecules such as AP72 or other novel compounds that are currently under preclinical or clinical investigation by us and others may offer a novel approach to prevent valvular calcification in CAVD and related conditions.
The pKa values of protein cysteines and their nucleophilicities are mainly dependent on charged states, orientations, inductive effects of neighbouring functional groups, and solvent accessibility of the thiol group (Nagy, 2013). Unfortunately, the accurate crystal structures of Pit1 and RUNX2 are still not known, which makes it virtually impossible to appropriately predict reactive cysteine residues on these proteins, which might be persulfidated with a functional effect on their activities. However, persulfidation of RUNX2 on Cys 123 and Cys 132 by FIGURE 8 Hydrogen sulfide inhibits valvular calcification in ApoE −/− knockout mice. Haematoxylin and eosin (upper panels), von Kossa (middle panels), CSE (second middle panels), and α smooth muscle actin (α-SMA; lower panels) staining was performed on aortic valves of mice fed a normal diet (first column; N = 5), on a high-fat diet (second column, N = 9); and on a high-fat diet treated with AP72 (third column; N = 5). Comparison (>) designates the calcified regions, and arrow indicates CSE positive cells CSE-produced endogenous sulfide enhanced its transactivation (Zheng et al., 2017). This effect is in contrast to our observations that endogenously produced or exogenously administered sulfide inhibited nuclear translocation of RUNX2, suggesting that the anti-calcification effects of H 2 S are more likely due to the inhibition of phosphate uptake, and the inhibition of RUNX2 translocation in our model is likely to be a secondary effect of this, as we proposed above.
In our study, we identify three separate mechanisms for the anticalcification effects of H 2 S (a) inhibiting phosphate uptake, (b) preventing nuclear translocation of RUNX2, and (c) increasing pyrophosphate level. Although all these pathways are important and related to each other, some caveats are acknowledged. AP72 exhibits slight but significant anti-calcification action at 200 μmol·L −1 , although it did not affect nuclear translocation of RUNX2 indicating the importance of pyrophosphate for preventing the formation of mineralized nodules in extracellular matrix.
In conclusion, our study has provided evidence that endogenous H 2 S limits calcification of VIC. Pharmacologically generated H 2 S, derived from novel H 2 S-releasing molecules, may have the potential to control calcification in heart valves and osteoblastic differentiation of VIC.