Loss of ceramide synthase 5 inhibits the development of experimentally induced aortic valve stenosis

Inflammation and calcification are hallmarks in the development of aortic valve stenosis (AVS). Ceramides mediate inflammation and calcification in the vascular tissue. The highly abundant d18:1,16:0 ceramide (C16) has been linked to increased cardiovascular mortality and obesity. In this study, we investigate the role of ceramide synthase 5 (CerS5), a critical enzyme for C16 ceramide synthesis, in the development of AVS, particularly in conjunction with a high‐fat/high‐cholesterol diet (Western diet, WD).


| INTRODUCTION
Aortic valve stenosis (AVS) is the most common and most deadly valvular heart disease in humans, with a prevalence of 2.8% in patients above 75 years of age.AVS is the cause of considerable mortality, with a two-year mortality rate of severe AVS of around 50% for symptomatic patients. 1,2Until now, no pharmacological treatment of AVS has been available, leaving the replacement of the aortic valve (AV) the only treatment option.AVS development is an inflammatory process that involves immune cell infiltration and calcification of valvular interstitial cells. 3,4ell established risk factors for the development of AVS are, among others, obesity and insulin resistance. 5,6eramides are biologically active sphingolipids that are produced in a complex network of enzymes.The main regulators of ceramide synthesis are sphingomyelinases and ceramide synthases (CerS).][9][10] CerS5 and CerS6 predominantly produce C16 ceramide, which has been associated with adverse cardiovascular outcomes in observational studies. 113][14][15] Loss of CerS5 function has been shown to reduce diet-induced obesity in mice. 147][18][19] In addition, it has been suggested that ceramides play a critical role in calcification, a hallmark of AVS development. 20owever, to date, it remains unclear if the ceramide metabolism influences the development of AVS and how dietary factors interfere with AVS development in ceramide-deficient organisms.This is particularly relevant as ceramides mediate inflammation and diet-related effects, which are thought to promote AVS development independently. 5,21,22In this article, we describe the effect of a loss of CerS5 function in combination with high-fat/ high-cholesterol diet (Western diet, WD) on AVS development.We show that loss of CerS5 function reduces the inflammation of the AV and the development of AVS in mice with WD, but not with normal chow.We also find CerS5 to drive calcification of valvular interstitial cells (VIC).

| Animals and wire-injury
CerS5 constitutional knockout (CerS5 −/− ) mice were generated by Gosejacob et al. on a C57BL/6-J background. 147BL/6-J wild type (WT) mice were purchased from Janvier Labs.For the experiments, 14-16 -week-old mice were kept at 22° in a room with 12-h-light/dark cycle and fed with a high-fat/high-cholesterol diet (Western diet, WD) containing 21% fat and 0.25% cholesterol (Sniff Cat# E15721-347) or with normal chow ad libitum.The WD diet was started 3 days before the wire injury.For the induction of AVS development, we used the wire injury model, originally published by Honda et al. 23 A wire injury of the AV was performed according to the protocol of Niepmann et al. 24 Under echocardiographic control, a coronary angiography wire with a 15° angled tip was inserted into the left ventricle, pushed back and forth 20 times over the AV, and then rotated 200 times on the AV level.All animal experiments were performed according to the German animal protection law and were approved by the National Office for Nature, Environment and Consumer Protection in Recklinghausen, North Rhine-Westphalia.

| Echocardiography
Echocardiography was performed as previously reported. 24Mice were anesthetized with 1.5% isoflurane.For echocardiographic analysis, a Fujifilm Visualsonics Vevo 2100 Ultra High Frequency Imaging Platform was used.The left ventricular ejection fraction was measured in parasternal long-axis views with the Vevo LV-Trace function.AV peak velocity was measured in suprasternal view using a pulse-wave doppler.The development of AV regurgitation was monitored and only mild regurgitation was detected.

| Histology and Immunofluorescence
Forty-two days after the wire injury, the mice were sacrificed for histological analysis.The mice were anesthetized with ketamine and xylazine, followed by the collection of blood samples.The heart was flushed with a 0.9% sodium chloride solution, embedded in tissue freezing medium, and sectioned at 8 μm.H&E staining was performed to measure the AV area, as previously reported. 24For von Kossa staining to detect calcification, the slides were incubated for 30 min in 2% silver nitrate (Sigma, Cat# S6506-25G), followed by washing with tap water, 5 min incubation with sodium thiosulfate (Sigma, Cat# 217263-250G) and counterstaining with nuclear fast red-aluminum solution 0.1% (Millipore Sigma, Cat# 1001210500) for 3 min.Anti-CD68 staining to detect macrophage infiltration was performed by fixation of the slides for 20 min in −20° acetone, washing in PBS, blocking the slides with 2% BSA in PBS for 30 min, followed incubation with the primary antibody (rat anti-mouse, antibodies online, Cat# ABIN 181836) 1:100 diluted in 2% BSA in PBS at 4° overnight.After washing, the slides were incubated with the secondary antibody (Cy3, AffiniPure donkey anti-rat, Jackson ImmunoResearch, Cat#712-165-153) 1:500 diluted in 2% BSA in PBS for 1 h at room temperature.Images were taken with an Axio observer microscope (Zeiss, Germany) at 10X magnification.Von Kossa positive area and Anti-CD68 positive area were automatically quantified by Zen3.2 pro software.Using von Kossa staining, AV pigmentation can be mistaken, as calcium.To assure that this does not distort our results, we measured the AV pigmentation and found a low pigmentation that is similar between the groups.

| In vitro calcification
Human valvular interstitial cells (VICs) were isolated from Lonza upon individual request.At least three independent donors were used per experiment.In vitro calcification experiments with VICs were incubated with pro-calcifying medium (PCM) for 7 or 21 days, refreshed three times per week.PCM contains DMEM with 5% FBS, 2 mmol/L NaH 2 PO 4 (pH 7.4), and 50 μg/mL Lascorbic acid. 30For calcium staining in vitro, alizarin red (Sigma Cat# A5533-25G) was dissolved in distilled water to generate a 2% solution with an adjusted PH of 4.2.The cells were fixed with 4% formaldehyde solution (EMD Millipore Cat# 1.00496.5000)for 30 min at 4°, followed by washing the cells with 200 μL distilled water, and incubation with 100 μL alizarin red solution for 30 min at room temperature.After this, the cells were washed again two times with distilled water and alizarin red was detected in an Axio observer microscope (Zeiss, Germany).Quantification was carried out by dissolving Alizarin staining in a 100 mmol/L Hexadecylpyridinium chloride monohydrate (Sigma Cat# C9002) aqueous solution.The solution was transferred to a 48-well plate, and absorption was measured in a TECAN, Infinite M Plex at a wavelength of 540 nm.The absorption was normalized using the control wells with PCM treatment only.

| Statistical analysis
For all statistical analyses, Graph Pad Prism10 was used.All data are represented as mean ± standard error of mean (SEM).For statistical analysis of two groups, an unpaired t-test was performed; for more than two groups, a one-way ANOVA followed by Tukey's multiple comparisons test was performed.For time-course experiments, a two-way ANOVA followed by Tukey's multiple comparisons test was performed.All p values are two-sided.

| CerS5 −/− reduces echocardiographic development of AVS in mice on WD
To evaluate the effect of loss of CerS5 function on the development of AVS in mice on WD, an echocardiographic analysis was performed before, 14, 28, and 42 days after wire injury (Figure 1).Two weeks after wire injury, peak velocity was significantly increased in WT and CerS5 −/− mice compared to baseline.CerS5 −/− mice showed a significantly lower peak velocity at 28 and 42 days after injury in comparison to WT mice.In CerS5 −/− mice, the effect of the wire injury was completely abolished after 42 days, as the peak velocity returned to baseline.(Figure 2A).Left ventricular function, evaluated by ejection fraction, did not change in both groups after wire injury and showed no difference between CerS5 −/− and WT (Figure 2B).The corrected left ventricular mass increased only in WT mice after wire injury, but without difference between WT and CerS5 −/− mice 42 days after wire injury (Figure 2B).The WD resulted in a body weight increase of around 20% without a significant difference between CerS5 −/− and WT mice (Figure S1).

| CerS5 −/− counteracts inflammation and calcification of the AV in mice with WD
Histological staining against CD68 showed that CerS5 −/− exhibits significantly lower levels of macrophage infiltration in the AV after wire injury (Figure 3A).Von Kossa staining revealed a lower amount of calcification of the AV of CerS5 −/− mice (Figure 3B).AV thickness and fibrosis, stained with Sirius red, did not show any difference between WT and CerS5 −/− (Figure 3C + D).

| CerS5 −/− has no effect on AVS development in mice with normal chow
In order to evaluate whether the effect of reduced AVS development in CerS5 −/− mice is dependent on WD, the same experiments were also performed with normal chow.In this experiment, the peak velocity exhibited no significant difference between CerS5 −/− and WT (Figure 4A).Again, neither wire injury nor CerS5 −/− significantly affected the left ventricular function (Figure 4B).At baseline, the corrected left ventricular mass was higher in CerS5 −/− mice compared to WT. Forty-two days after wire injury, the corrected left  ventricular mass increased in WT but not in CerS5 −/− mice, without differences between WT and CerS5 −/− (Figure 4B).Histological assessment of the AV using anti-CD68 staining revealed no difference in macrophage infiltration between both groups (Figure 5A).Moreover, von Kossa staining showed comparable amounts of AV calcification in WT and CerS5 −/− mice (Figure 5B).AV area and fibrosis were not significantly different between CerS5 −/− and WT animals (Figure 5C + D).

| Knockdown of CerS5 reduces calcification in vitro
To analyze calcification in VICs, the cells were treated with pro-calcifying medium for 7 or 21 days.After stimulation of VICS with pro-calcifying medium for 7 days, CerS5 was slightly upregulated and CerS6 was strongly upregulated (Figure 6A).Stimulation for 7 days with procalcifying medium led to an upregulation of RUNX-2, a promotor of AV calcification, while this effect was not present after 21 days of stimulation (Figure 6B).In order to analyze the effect of CerS5 and CerS6 on the calcification of VICs, the cells were treated with RNAi for 24 h and consecutively with PCM for 7 or 21 days.The downregulation was shown to be stable for at least 1 week (Figure S3).Alizarin red staining of the VICs revealed that CerS6 KD and CerS5 KD alone, as well as the combination of both, reduced the calcification of VICs after treatment with pro-calcifying medium after 7 and 21 days of stimulation (Figure 6C).

| Lipids and knockdown of CerS5 reduce TNF-α and IL-1β secretion in THP-1
In order to further investigate potential mechanisms how CerS5 is involved in immune cell activation as observed in murine aortic valves, we performed in vitro experiments with THP-1 cells.TNF-α and IL-1β are important cytokines that regulate inflammation.To stimulate IL-1β and TNF-α secretion, THP-1 cells were stimulated with 50 ng/mL LPS. 28,29A high-fat/high-cholesterol diet was mimicked in vitro by the use of a lipid mixture (LM), as previously reported. 26,27THP-1 cells were pre-incubated with LM for 12 h before LPS treatment for 24 h (LPS + LM) (Figure 7A + B).In all groups, CerS5 KD strongly reduces IL-1β and TNF-α secretion when cells are stimulated with LPS.Interestingly, the lipid mixture attenuated IL-1β and TNF-α secretion.The MTT assay for viability shows no reduced viability of cells treated with LM (Figure S4B).

CerS2 or CerS6 upregulation
Additionally, we investigated if loss of CerS5 would lead to a compensatory upregulation of either CerS6 or CerS2.Neither in the aortic tissue of CerS5 −/− mice on WD or normal chow nor in vitro, a significant upregulation of CerS2 or CerS6 was detected (Figure S2).

| DISCUSSION
In the present manuscript, we show that the loss of CerS5 reduces the development of AVS in animals on WD.Ceramides have been shown to be involved in cardiovascular disease and obesity, but their impact on AVS has not been investigated. 13,15he impact of ceramides on obesity and lipid metabolism is well studied. 14,31,32It has been shown that CerS5 is essential to maintain systemic C16 ceramide levels in mice. 14CerS5 −/− mice are partially protected from an increase in ceramide serum levels due to high-fat diet. 14nterestingly, in our experiments, we did not detect a difference in the body weight between CerS5 −/− and WT animals on WD.This could be explained by the shorter observation time of only 6 weeks, where the mice showed only moderate weight gain.Our study revealed antiinflammatory effects of CerS5 −/− in mice on WD, which were not present in mice with normal chow.This effect appears to be independent of body weight.
Inflammation is described as a hallmark of early AVS development. 3,4In other non-cardiovascular contexts, C16 functions as a modulator of inflammation. 8Previous work indicates that in CerS5-deficient animals high-fat diet might induce an anti-inflammatory phenotype similar to our observations in the aortic valve. 14Likewise, we found a strongly reduced valvular immune cell infiltration in CerS5 −/− mice on WD compared to WT mice on WD.In murine macrophages, the impact of lipid excess on ceramide-regulated inflammation has already been studied: Schilling et al. described how lipopolysaccharide, only together with the saturated fatty acid palmitate, leads to a C16 ceramide production of the macrophages, which then leads to an increased inflammatory response of these macrophages. 18This mechanism could explain why only CerS5 −/− mice on WD, in comparison to mice on normal chow, showed less valvular inflammation than WT.Here, for the first time, we show that loss of a ceramide synthase in combination with WD can reduce the inflammatory response to an injury to the AV in vivo.As described for macrophages, we also found this effect to be dependent on the high presence of fat.Our experiments with THP-1 cells were performed with a lipid mixture containing cholesterol, saturated, and unsaturated fatty acids in order to simulate the situation in our animals on WD as closely as possible.We detect reduced secretion of TNF-α and IL-1β from monocytes after treatment with fatty acids and LPS.There are previous reports that lipids activate pro-inflammatory ceramide producing pathways. 18,33,34In line with our in vivo experiments, CerS5 KD strongly reduced the inflammatory response of monocytes, independent of lipid excess.While it has been reported that inhibition of neutral sphingomyelinase 2, another ceramide-producing enzyme, reduces the inflammatory response, these effects have not yet been described for CerS5. 16The anti-inflammatory effect of loss of CerS5 function was not abrogated but at least partially enhanced by additional lipid supply.The stronger antiinflammatory effect of a loss of CerS5 function on monocytes in the presence of excess lipids may explain why only the combination of WD and CerS5 knockout leads to a significant reduction in aortic valve inflammation and stenosis formation in our in vivo model.
Calcification of the AV is a fundamental mechanism in the development of AVS. 4,35In general, ceramides influence calcification, but the specific role of CerS5 has not been investigated yet. 20,31,36Our work shows that inhibition of CerS5 leads to reduced calcification of VICs in vitro.Moreover, we showed a reduced calcification of the AV in CerS5 −/− mice with WD.
As previous studies have shown a certain amount of cross-compensation of different CerS isoenzymes, we hypothesized that loss of CerS5 function could lead to an increased expression of CerS6, which also produces C16 ceramide, or CerS2, which would produce ceramides with longer acyl chains that exert anti-inflammatory properties. 10,37This could have explained the anti-inflammatory effect of the loss of CerS5 function under WD. 37In our models, compensation for the loss of CerS5 by upregulation of CerS6 or CerS2 was not observed and does not seem to be a relevant mechanism to explain this effect.
In our experiments, we found a reduction in AVS development only in mice on WD.It appears that loss of CerS5 protects from an enhanced inflammation caused by WD.However, our experiments show that the AV inflammation in CerS5 −/− mice on WD is even lower than in WT mice with normal chow.Therefore, it can be speculated that loss of CerS5 function in combination with WD has anti-inflammatory effects with yet-unknown mechanisms.Our THP-1 experiments suggest anti-inflammatory effects of downregulation of CerS5 in combination with lipid excess, which could explain why inflammation in CerS5 −/− mice on WD is even lower than in WT mice with normal chow.
THP-1 cells are a well-established model cell line for human monocytes, and as their overall inflammatory responses are similar to those of circulating monocytic cells, they are representative of human inflammatory cells.However, there are some limitations, as the LPS responses of THP-1 cells are substantially lower compared to other peripherally circulating monocytic cells. 16,25,38he human AVS pathophysiology and AV anatomy differ from those of mice.Mice do not have the trilayer morphology of human, porcine, or rabbit aortic valves. 4,39n addition, C57BL/6-J wild-type mice do not develop aortic valve calcification with age. 39Yet, the murine wire injury model is well established and reflects the hallmarks of human AVS development, like inflammation, calcification, and accelerated aortic valve flow. 24In contrast to other AVS models, the wire injury model does not require dietary intervention or extreme hypercholesterolemia. 40 To date, it is unknown how the findings of the murine wire injury model translate to humans.
In our model, we showed both a lower infiltration of macrophages in the AV of mice on WD as well as a direct reduction of calcification related to the loss of CerS5.This was mirrored by an abrogation of AVS development in CerS5 −/− mice on WD.In summary, our work highlights the importance of ceramides in the development of AVS in mice on a highfat/high-cholesterol diet by influencing inflammatory processes.Although the underlying mechanism of these effects is only incompletely understood, CerS5 could serve as an interesting target to counteract AVS development.

F I G U R E 2
Echocardiographic analysis of mice fed with Western diet (WD).(A) Aortic valve (AV) peak velocity of mice with wire injury and WD, Wild-type (WT) n = 8 versus CerS5 −/− n = 13, with representative images of the AV Doppler signal at 6 weeks.(B) Left ventricular (LV) function and hypertrophy of mice with wire injury and WD measured as ejection fraction in % and the corrected left ventricular mass at baseline and 6 weeks after the wire injury, WT n = 8 versus CerS5 −/− n = 13, with representative echocardiographic images.All data are presented as individual experiments with the mean ± SEM; ns not significant, *p < 0.05, † p < 0.01.A two-way ANOVA + Tukey's multiple comparison was used for B + C. F I G U R E 3 Histological analysis of the Aortic valve (AV) of the mice fed with Western diet (WD).(A) Histological analysis of immune cell infiltration using CD68 staining.(B) Calcification, detected with von Kossa staining; (C + D) AV thickness and fibrosis, detected by Sirius red staining (A-D) Wild-type (WT) n = 9 versus CerS5 −/− n = 12.Representative images in the right panel.All data are presented as individual experiments with the mean ± SEM; ns not significant, *p < 0.05, ‡ p < 0.001, and an unpaired t-test was used for (A-D).

F
I G U R E 4 (A) Echocardiographic analysis of peak velocity at baseline, 14, 28, and 42 days after wire injury in mice with normal chow.Wild-type (WT) n = 10 versus CerS5 −/− n = 13, with representative images of the AV Doppler signal at 6 weeks.(B) Left ventricular function and hypertrophy of mice with wire injury and normal chow measured as ejection fraction in % and the corrected left ventricular mass, baseline and 42 days after the wire injury, WT n = 10 versus CerS5 −/− n = 13, with representative echocardiographic images.All data are presented as individual experiments with the mean ± SEM; ns not significant, *p < 0.05, ‡ p < 0.001, ANOVA + unpaired t-test was used for (A), and ANOVA + Tukey's multiple comparison test was used for (B).

F I G U R E 5
Histological analysis of the AV of the mice fed with normal chow.(A) Histological analysis of immune cell infiltration using CD68 staining.(B) Calcification, detected with von Kossa staining (C + D) AV thickness and fibrosis, detected by Sirius red staining (A-D) Wild-type n = 10 versus CerS5 −/− n = 13.Representative images in the right panel.All data are presented as individual experiments with the mean ± SEM; ns not significant; unpaired t-test was used for (A-D).

F
I G U R E 6 (A + B) RT PCR analysis of CerS5 and CerS6 after treatment with control medium (CM) or pro-calcifying medium (PCM) in A or RUNX-2 in B in VICs was shown as relative gene expression presented as 2 −ddCT versus GAPDH, N = 3 for A and N = 4 for B. (C) Alizarin red staining for calcium of VICs with CerS5 knockdown (KD), CerS6 KD, or CerS5 KD + CerS6 KD after treatment with procalcifying medium or control medium.All data are presented as individual experiments with the mean ± SEM; ns, not significant, *p < 0.05, † p < 0.01.An unpaired t-test was used for (A and B).ANOVA + Tukey's multiple comparison test was used for (C).

F
I G U R E 7 (A + B) IL-1β and TNF-α secretion by THP-1 cells with CerS5 knockdown (KD) or control (sicont) after treatment with LPS for 12 h (LPS), lipid mixture for 12 h followed by LPS for 24 h (LPS LM).IL-1β and TNF-α concentrations in THP-1 cell supernatants were analyzed using ELISA.All data are presented as individual experiments with the mean ± SEM; ns, not significant, † p < 0.01, ‡ p < 0.001, ****p < 0.0001.ANOVA + Tukey's multiple comparison test was used for (A and B).