Herpud1 deficiency alleviates homocysteine-induced aortic valve calcification

To evaluate the role and therapeutic value of homocysteine (hcy)-inducible endoplasmic reticulum stress (ERS) protein with ubiquitin like domain 1 (Herpud1) in hcy-induced calcific aortic valve disease (CAVD). The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. In vivo, we use the low-density lipoprotein receptor (LDLR) and Herpud1 double knockout (LDLR−/−/Herpud1−/−) mice and used high methionine diet (HMD) to assess of aortic valve calcification lesions, ERS activation, autophagy, and osteogenic differentiation of aortic valve interstitial cells (AVICs). In vitro, the role of Herpud1 in the Hcy-related osteogenic differentiation of AVICs was investigated by manipulating of Herpud1 expression. Herpud1 was highly expressed in calcified human and mouse aortic valves as well as primary aortic valve interstitial cells (AVICs). Hcy increased Herpud1 expression through the ERS pathway and promoted CAVD progression. Herpud1 deficiency inhibited hcy-induced CAVD in vitro and in vivo. Herpud1 silencing activated cell autophagy, which subsequently inhibited hcy-induced osteogenic differentiation of AVICs. ERS inhibitor 4-phenyl butyric acid (4-PBA) significantly attenuated aortic valve calcification in HMD-fed low-density lipoprotein receptor−/− (LDLR−/−) mice by suppressing ERS and subsequent Herpud1 biosynthesis. These findings identify a previously unknown mechanism of Herpud1 upregulation in Hcy-related CAVD, suggesting that Herpud1 silencing or inhibition is a viable therapeutic strategy for arresting CAVD progression. • Herpud1 is upregulated in the leaflets of Hcy-treated mice and patients with CAVD. • In mice, global knockout of Herpud1 alleviates aortic valve calcification and Herpud1 silencing activates cell autophagy, inhibiting osteogenic differentiation of AVICs induced by Hcy. • 4-PBA suppressed Herpud1 expression to alleviate AVIC calcification in Hcy treated AVICs and to mitigate aortic valve calcification in mice.


Abstract
Objectives To evaluate the role and therapeutic value of homocysteine (hcy)-inducible endoplasmic reticulum stress (ERS) protein with ubiquitin like domain 1 (Herpud1) in hcy-induced calcific aortic valve disease (CAVD).Background The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited.
Methods In vivo, we use the low-density lipoprotein receptor (LDLR) and Herpud1 double knockout (LDLR−/−/Herpud1−/−) mice and used high methionine diet (HMD) to assess of aortic valve calcification lesions, ERS activation, autophagy, and osteogenic differentiation of aortic valve interstitial cells (AVICs).In vitro, the role of Herpud1 in the Hcy-related osteogenic differentiation of AVICs was investigated by manipulating of Herpud1 expression.Results Herpud1 was highly expressed in calcified human and mouse aortic valves as well as primary aortic valve interstitial cells (AVICs).Hcy increased Herpud1 expression through the ERS pathway and promoted CAVD progression.Herpud1 deficiency inhibited hcy-induced CAVD in vitro and in vivo.Herpud1 silencing activated cell autophagy, which subsequently inhibited hcy-induced osteogenic differentiation of AVICs.ERS inhibitor 4-phenyl butyric

Introduction
The incidence of calcific aortic valve disease (CAVD) is increasing annually with the aging population and has become the third most common cardiovascular disease after hypertension and coronary heart disease (Yadgir et al. 2020), affecting up to 25% of people over the age of 65 years (Go et al. 2013).There are no known pharmacological treatments to slow or reverse the progression of CAVD; rather, surgical aortic valve replacement or transcatheter aortic valve implantation is the only option (Lindman et al. 2016).Therefore, understanding the molecular mechanisms of CAVD is crucial for developing new therapeutic approaches.
Current studies have shown that plasma homocysteine (hcy) levels correlate with atherosclerosis, cerebral artery calcification, and coronary artery calcification in Asian populations (Wald et al. 2002).Therefore, we hypothesized that hcy may also be associated with CAVD.Hcy can cause endoplasmic reticulum stress (ERS), which has been demonstrated in a variety of cells (Cai et al. 2013;Duan et al. 2009).ERS activates the unfolded protein response (UPR), and sustained UPR causes endoplasmic reticulum metabolic disorders, mitochondrial dysfunction, and oxidative stress, producing excess reactive oxygen species and inflammatory factors involved in the development of aortic valve calcification (Hetz 2012).The study found that fibroblast growth factor 21 could improve aortic valve calcification by inhibiting the C/EBP homologous protein and caspase-12 pathways in ERS [8].Histone deacetylase 6 has been shown to promote aortic valve calcification by activating the ATF4 pathway in ERS (Fu et al. 2019).However, it is unclear how ERS is involved in aortic valve calcification.
Hcy-inducible endoplasmic reticulum protein with ubiquitin like domain 1 (Herpud1), a transmembrane protein anchored to the endoplasmic reticulum, is a downstream target protein of UPR and a component and regulator of endoplasmic reticulum-associated protein degradation (ERAD) (Schulze et al. 2005).Herpud1 plays an important role in ERS homeostasis regulation, and a recent study found Herpud1 is crucial for activating the osteoblast maturation program (Americo- Da-Silva et al. 2018).Our previous results showed that plaques at the aortic sinus were significantly reduced in LDLR −/− /Herpud1 −/− mice compared to LDLR −/− mice fed a high methionine diet (HMD) and that Herpud1 knockdown inhibits the differentiation of vascular smooth muscle cells from contractile to synthetic type and attenuates hcyinduced atherosclerosis (Lin et al. 2018).Nonetheless, the role of Herpud1 in CAVD remains unknown.Studies have shown that hcy can inhibit autophagy in vascular smooth muscle cells (Chen et al. 2018).Autophagy is a highly conserved catabolic process responsible for degrading long-lived cytosolic proteins and organelles (Yin et al. 2016).It modulates many pathologies, playing an important role in cardiovascular diseases such as atherosclerosis, heart failure, and vascular calcification (Neutel et al. 2020;Schiattarella and Hill 2016).Further, Herpud1 can act as an autophagy repressor in addition to its role in the ERAD pathway as a stress integrator on an intermediate point in the crosstalk between the ERAD and the autophagic degradation systems (Quiroga et al. 2013a).This led us to further explore the relationship between Herpud1 and autophagy in Hcy-induced CAVD.
In the present study, we tested the hypothesis that Hcy activates ERS and stimulates Herpud1 expression to promotes aortic valve calcification and determined first, whether Herpud1 expression is altered in AVICs from aortic valves affected by CAVD and in Hcy treated AVICs; second, whether Herpud1 deficiency alleviates Hcy-induced AVIC calcification through regulating autophagy; and third, whether 4-PBA, a potential ERS inhibitor, suppresses the proosteogenic role of Hcy in AVICs, both in vitro and in vivo.

CAVD patient information and human aortic valve samples
We retrospectively analyzed data from patients with coronary atherosclerotic heart disease who were hospitalized at our institution between January 2017 and January 2020.Patients with valvular disease were excluded.Aortic valve calcification was defined as localized echogenic enhancement of the valve, leaflet thickness >2 mm, and transvalvular blood flow velocity ≥2.5 m/s on transthoracic cardiac ultrasonography.Finally, 212 patients were divided into CAVD group (n=85) and control group (n=127) according to the presence or absence of aortic valve calcification.Ultrasound images of different degrees of aortic valve calcification are shown in Supplementary Figure 1.Human aortic valves were obtained from the Cardiothoracic Surgery Department of the Shaoxing People's Hospital when the patients underwent valve replacement surgery.All patients provided informed consent, and the study protocol was approved by the ethics committee of Shaoxing People's Hospital (2020-56).We selected 3 representative CAVD patients (lesion specimens of patients with rheumatic valvular heart Vol:.( 1234567890) disease, endocarditis, and congenital aortic valvular disease were excluded) and one patient of congenital aortic valve malformation to show calcification of the aortic valve as well as ERS and Herpud1 expression levels.Macroscopic valve thickening with palpable calcium nodules was observed.The clinical characteristics of the above 4 patients were shown in Table S1.

Animals and treatments
The animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health and approved by the Animal Care and Use Committee of Shaoxing People's Hospital.Herpud1 −/− mice were purchased from Biomodel (Shanghai, China), while LDLR −/− mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China).The generation of LDLR −/− /Herpud1 −/− mice was described previously (Lin et al. 2018).LDLR −/− mice were used as controls and generated from the same crossings as LDLR −/− /Herpud1 −/− mice.All experiments were performed on adult (8-week-old) male mice.Mice were housed under a 12-h light/dark cycle and fed a normal chow diet (ND) or a high methionine diet (HMD: regular diet plus 2% methionine (wt/wt)) (Aladdin , Shanghai, China) for 28 weeks to promote CAVD.As we know, this is the firstly study only using methionine to induce CAVD.To clarify the role of ERS in CAVD, we treated mice with a potent ERS inhibitor 4-PBA (1g/kg/day, MedChem-Express) for continuous 28 weeks in drinking water, a dose been approved to suppress ERS (Lynn et al. 2019).At the end of the dietary treatment, we used echocardiography to determine the cardiac function of all mice.Then mice were anesthetized through the intraperitoneal application of sodium pentobarbital (50 mg/kg) after a 12-h fast.The adequacy of anesthesia was confirmed by the absence of reflex response to foot squeeze.Whole blood samples collected via the retro-orbital sinus were centrifuged at 2000× g for 10 min at 4 °C to obtain plasma.At the end of the experiments, mice aortic valves were isolated by killing mice with the intraperitoneal injection of an overdose of sodium pentobarbital (200 mg/kg).

Cell culture and treatment
Primary aortic valve interstitial cells (AVICs) were isolated from mice heart valves by type II collagenase (17101-015-1g; Gibco, Grand Island, NY, USA) as previously described (Gould and Butcher 2010) and cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere of air containing 5% CO 2 .Firstly, we established a model for the in vitro induction of AVIC differentiation to osteoblasts by pro-calcific medium (PCM) containing sodium β-glycerophosphate (200 μM; MedChemExpress, Shanghai, China), dexamethasone (100 μM; MedChemExpress), and ascorbic acid (100 μM; Med-ChemExpress).The cells were then treated with 200-μM hcy (H4628; Sigma) for 7 days.To inhibit hcystimulated ERS, the cells were pretreated with ER stress inhibitor 4-phenyl butyric acid (4-PBA; 5 mM; MedChemExpress), and then each group was stimulated with hcy 200 μM for 7 days.To inhibit hcyactivated autophagy, the cells were pretreated with the autophagy inhibitor 3-methyladenine (3-MA; 5 mM; MedChemExpress).Each group was then stimulated with hcy (200 μM) for 7 days.To knockdown Herpud1, the AVICs were transfected with siRNA against Herpud1 or scrambled siRNA (Shanghai GenePharma, Shanghai, China) using Lipofectamine 2000 Transfection Reagent (Invitrogen, Waltham, MA, USA) following the manufacturer's instructions.Lentivirus constructing of Herpud1 overexpression was obtained from OBiO (OBiO Technology Corp, China).In 100 pmol Herpud1 agomiR (OBiO Technology, Shanghai, China), AVICs were infected with Herpud1 overexpression lentivirus (Herpud1-OV), a negative control (vector).Pools of stable transductions were generated by selection using puromycin (4 μg/ml) for 2 weeks.RT-qPCR analysis TRIzol reagent (Invitrogen) was used to extract RNA from calcified aortic valve or cultured AVICs.Next, cDNA was synthesized from 2 μg of total RNA, and the target genes were quantified using a Prime-Script RT reagent kit (Takara, Otsu, Japan).The reactions were performed on an ABI 7300 RT-PCR Detection System (Applied Biosystems, Foster City, CA, USA) using a SYBR Premix Ex Taq kit (Takara).The primers used in this study are listed in Supplementary Table S2.The quantitative polymerase chain reaction conditions included one cycle of initial denaturation at 95°C for 5 min followed by 40 cycles at 95°C for 10 s; 60°C for 30 s; and 72°C for 15 s.GAPDH was used to normalize the target genes via the 2 −ΔΔCT method.All reactions were performed in triplicate.

Alizarin Red staining
The AVICs were sub-cultured on a 24-well plate; when confluency reached 30%, they were treated in different groups (see the "Cell culture and treatment" section for groupings) and cultured for 14 days.The medium was discarded, the cells were washed with PBS 3 times, and 0.4% alizarin (HAT, Xian, China) was added.Red S dye solution was used to visualize the calcium deposits.When deposition of the red calcium salt substance was observed on microscopy, the reaction was immediately stopped with deionized water and the cells washed.Analysis of the Alizarin Red staining was performed using Image J software (National Institutes of Health, Bethesda, USA).All experiments were performed in triplicate.
Vol:. ( 1234567890) ALP activity measurement After 7 days of treatment according to the above intervention factors, the cells cultured in 6-well plates were washed 3 times with PBS; 500 μL of 0.05% Tralatone X•100 (Beyotime) was added to each well; and freeze-thaw cycles were performed 3 times.The liquid in each well was collected and processed by centrifugation at 15,000 rpm for 15 min at 4°C.The supernatant was transferred to a new Eppendorf tube and used as the sample.Using the ALP kit (AP0100; Sigma) to make a standard curve, 980 μL of reaction buffer was pipetted into one cuvette (blank).Next, 960 μL of reaction buffer was pipetted into additional cuvettes (one for each test or enzyme control), 20 μL of 0.67 M pNPP solution was added to each cuvette (blank, test, and control), and 20 μL of the test sample was added to each test cuvette.Next, 20 μL of diluted ALP solution was added to the enzyme control cuvette, immediately followed by mixing by inversion.The increase in A405nm was recorded for ~5 min.The maximum linear rate (∆A405nm/min) for the test, blank, and control was obtained.The units/ mL solution was calculated as follows: (∆A405nm/ min test-∆A405nm/min blank) (df) (VF)/(18.5)(VE).

Statistical analysis
The data are presented as mean ± standard deviation and were analyzed utilizing SPSS version 21.0 (SPSS Inc., Chicago, IL, USA).Intergroup differences were analyzed using a t-test, and data among multiple groups were compared using one-way analysis of variance, followed with Tukey's post hoc analysis.Values of p<0.05 were considered statistically significant (**p<0.01,***p<0.001).

Hcy promotes valvular calcification in vivo and in vitro
First, by comparing the results of transthoracic echocardiography (Figure S1), we compared the patients' basic clinical information in the control and CAVD groups.Table 1 shows a significant increase in plasma hcy levels in patients with coronary artery disease plus CAVD compared with those in patients without CAVD (p=0.000).In addition, patients in the CAVD group had high age (p=0.000), had low-density lipoprotein cholesterol (LDL-C) levels (p=0.019), were predominantly male (p=0.029), and had reduced left ventricular ejection fraction (LVEF, p=0.000).No statistically significant intergroup differences were noted in smoking, body mass index, smoking, hypertension, triglycerides, and high-density lipoprotein cholesterol (p>0.05 for all).Consistent with our results, although plenty of studies have found the association between Hcy and CAVD (Wu et al. 2018), whether Hcy participates the progression of valvular calcification still need to be clarified.Subsequently, we analyzed the effects of hyperhomocysteinemia on valvular calcification in mice and the effect of hcy on mineralization in AVICs.We constructed a hcy-related animal model of CAVD by feeding LDLR −/− mice an HMD.The detection of hcy concentration in the plasma of mice indicates successful molding (Fig. S4f).After 28 weeks, the mice in the HMD group showed significant increases in transvalvular peak jet velocity (Fig. 1a, b) and mean transvalvular pressure gradient (Fig. S4b) and a significant decrease in the aortic valve area (AVA; Fig. S4c), whereas there were no significant differences in heart rate, left ventricular internal diameter during diastole (LVIDd), left ventricular internal diameter during systole (LVIDs), and functional shortening (FS) between the HMD and control groups (Tab.S3).Compared to those receiving an ND, mice receiving an HMD showed significant calcified valves, as evidenced by increased aortic leaflet thickness, calcific nodules, and collagen deposition (Fig. 1c-e).The immunohistochemistry results showed that hcy increased the expression of osteogenic markers (Col1, OPN, and ALP; Fig. 1f, g).The expressions of osteogenic markers including ALP, osteocalcin, Runx2, OPN, and Col1 were explored.
The RT-qPCR and Western blotting analyses demonstrated upregulation of the above markers in the hcy versus control group (Fig. 1h-j).It is also interesting to note that HMD increased the plasma levels of triglycerides (TG) in mice, while no significant differences were observed for total cholesterol (TC) and low-density lipoprotein (LDL) (Tab.S3).In vitro, we isolated AVICs from mice, which expressed high SMA and low vimentin (Fig. S2a).We exposed AVICs to PCM to stimulate osteogenic differentiation (Yu et al. 2018).After 7 days of PCM stimulation, the mRNA levels of osteogenic markers in AVICs were significantly upregulated (Fig. 2e); interestingly, the addition of hcy further elevated the osteogenic markers both at the mRNA and protein levels (Fig. 2e,  b).The ALP activity assay showed that Hcy further upregulated ALP activity based on PCM (Fig. 2a).
After 5 or 10 days, PCM induced some calcification, as detected by Alizarin Red staining (Fig. S2b), the calcific nodules increased significantly after 14 days, and hcy notably increased calcific nodules in PCMtreated AVICs in a time-dependent manner (Fig. 2c,  d).Taken together, the above data strongly indicated that Hcy participates in CAVD progression.
Herpud1 is upregulated in hcy-mediated CAVD due to ERS We previously reported that Herpud1 was stimulated by hcy in smooth muscle cells (Lin et al. 2018).Here we also investigated the profile of Herpud1 in AVICs.
We analyzed the expression of Herpud1 in three calcified human aortic valves and one non-calcified human aortic valve.The baseline characteristics of the four patients are shown in Table S1.Alizarin Red staining showed a significant increase in calcified nodules in the aortic valve in patients with CAVD versus the non-calcified group (Fig. S3b).An immunohistochemical analysis suggested an increased expression of osteogenic markers and Herpud1 in the aortic valves of patients with CAVD versus the non-calcified group (Fig. S3c).As shown in Fig. 3a, the immunofluorescence co-staining results further showed that Herpud1 was highly expressed in the calcified aortic valves versus the non-calcified group, while Herpud1 was mostly co-localized with α-SMA but still partially present in the extracellular space.To confirm these results, we also performed experimental validation in mice, which yielded the same results that Herpud1 was co-expressed with α-SMA in the valve of HMD induced mice (Fig. 3b, c) and western blotting (Fig. 3d) showing upregulation of Herpud1 expression in the hcy versus control group.Consistent with the expected result, in vitro, the Western blotting results showed that Herpud1 expression increased in the PCM group and hcy further increased Herpud1 protein expression based on PCM induction (Fig. 3e).Therefore, we concluded that Herpud1 is upregulated in hcy-mediated CAVD.
To determine whether ERS contributes to the Hcy-mediated CAVD, we used ERS inhibitor 4-PBA to treat LDLR−/− mice and AVICs.The immunohistochemical analysis and Western blotting showed that the expression of ERS transmembrane proteins (PERK, CHOP, ATF6) was upregulated in the HMD group compared to the control group (Fig. S4a,d).In contrast, PERK, ATF6, and Herpud1 protein expression were all decreased in the HMD+4-PBA group (Fig. 4a) which was consistent with the immunohistochemical analysis (Fig. 4b) and immunofluorescence co-staining (Fig. S5c) results.In vitro, Hcy treatment induced increased PERK and ATF6 protein (Fig. S4e), whereas addition of 4-PBA effectively inhibited ERS and subsequent Herpud1 expression (Fig. 4c).Therefore, we concluded that Hcy upregulated Herpud1 in CAVD by activating ERS.

Herpud1 deficiency prevents hcy-induced aortic valve calcification
To reveal the regulatory role of Herpud1 in CAVD, we firstly analyzed the relationship between Her-pud1 and Runx2, a critical marker of osteogenic differentiation.Accumulation of Herpud1 in Runx2rich regions of the human and animal aortic valves was observed and we also found high expression of Herpud1 and Runx2 in human calcified aortic valves versus non-calcified valves, which is consistent with the hcy group in mice (Fig. 5a, b).We successfully bred LDLR −/− Herpud1 −/− mice using the results of previous experiments, and we divided the mice into LDLR −/− and LDLR −/− Herpud1 −/− groups, which were fed an ND and HMD for 28 weeks, respectively.Herpud1 knockout partially restores the valve changes caused by HMD that the mice in the Herpud1 −/− +HMD group showed significant decrease in transvalvular peak jet velocity (Fig. 5g, h) and mean transvalvular pressure gradient (Fig. S5h) and a significant increase in aortic valve area (Fig. S5g) compared to the HMD group, and there was no significant difference between the two groups in terms of heart rate and LVIDd, LVIDs, and FS (Tab.S3).We then collected aortic valves from mice from the different groups and evaluated the morphology of the valve leaflets and the degree of calcification.Herpud1 silencing showed a reduction in aortic leaflet thickness, collagen, and calcium deposition (Fig. 5g, i, j).The protein and mRNA levels of osteogenic markers in the aortic valve were remarkably reduced compared to the LDLR −/− Herpud1 −/− group under concomitant HMD conditions (Fig. 5d-f).Besides, we found that Herpud1 deficiency did not cause significant changes in metabolic parameters (Tab.S3).The above data suggest that Herpud1 silencing abolished the repressive effect of hcy on aortic valve calcification.
In vitro, we conducted further validation.We used a specific Herpud1 siRNA (primary AVICs from Herpud1/LDLR−/− mice cannot passage, data not shown) to inhibit Herpud1 expression in AVICs.The knockdown was confirmed by Western blotting and RT-PCR (Fig. 6a, S5i).Knockdown of Herpud1 significantly decreased the calcific nodules and ALP activity induced by Hcy and PCM (Fig. 6b, c).The osteogenic differentiation of AVICs induced by hcy was reversed after Herpud1 knockdown, mainly manifested by reduced osteogenic marker protein (Fig. 6d).In addition, we targeted Herpud1-specific siRNA to overexpress Herpud1 within primary mouse AVICs and again confirmed the success of this overexpression by Western blotting (Fig. 7a).Herpud1-OV increased calcified nodule formation in AVICs upon indicated treatment (Fig. 7b).We found that overexpression of Herpud1 could aggravate hcy-induced osteogenic differentiation of AVICs, mainly manifested by increased osteogenic marker protein (Fig. 7c) and mRNA (Fig. 7d) expression levels.Therefore, Hcy induces osteogenic differentiation of AVICs to participate CAVD progression by activating ERS and thus upregulating Herpud1.

Herpud1 silencing inhibits hcy-induced calcification through autophagy activation in AVICs
Previous studies have shown that autophagy is inhibited in cardiovascular calcification (Phadwal et al. ).We thus investigated the role of autophagy in Hcy-induced CAVD.Consistent with these results, the Western blotting analysis revealed that hcy inhibited autophagy-related protein expression (LC3B, Becline1) in AVICs (Fig. 8a), while knockdown of Herpud1 effectively abolished the effect of hcy on the autophagy proteins level in AVICs (Fig. 8b).It is suggested that knockdown of Herpud1 may retard osteogenic differentiation of AVICs by activating autophagy.To further confirm this hypothesis, we added the autophagy inhibitor 3-MA to the AVICs that knockdown Herpud1, as anticipated, the addition of 3-MA resulted in downregulation of autophagy protein expression (Fig. 8c).On cell phenotype, 3-MA prevented Herpud1 knockdown-induced calcific nodules and ALP activity (Fig. 8d, e).Further, 3-MA treatment caused inhibition of the osteogenic differentiation (Fig. 8f).In sum, the above results indicated that Herpud1 control the osteogenic differentiation of AVICs by activating cell autophagy.

ERS inhibitor 4-PBA alleviates valve calcification by downregulating Herpud1
4-PBA is an ERS inhibitor that has been shown to alleviate cardiovascular calcification (Chen et al. 2021), but its role in CAVD is unclear.We conducted in vivo experiments to clarify the role of 4-PBA in aortic valve calcification induced by hcy.Similar to the results of knockout Herpud1, the addition of 4-PBA resulted in improved echocardiographic findings in mice (Fig. 9a, S5c-5f).Furthermore, we found that aortic valve calcification improved in HMDfed LDLR −/− mice after the addition of 4-PBA, and 4-PBA treatment significantly reduced protein levels of three osteogenic differentiation markers (Fig. 9c), consistent with reductions in aortic leaflet thickness, collagen, and calcium deposition (Fig. 9b), and osteogenic marker expression (Fig. S5a,b).Immunofluorescence co-staining results revealed that 4-PBA inhibited the expression of Herpud1 and reduced the expression of Runx2, consistent with our expectation (Fig. S5c,d).These findings are similar to the results we observed after Herpud1 knockdown.
To further verify this idea, we conducted corresponding in vitro experiments.Consistent with the in vivo results, 4-PBA inhibited Herpud1 synthesis and thus inhibited hcy-induced osteogenic AVIC differentiation, mainly in the PCM+hcy+4-PBA group compared with the PCM+hcy group, which downregulated osteogenic marker expression levels (Fig. 9d,  f) and reduced calcified nodule formation (Fig. 9e).Combined with the results of previous experiments, we confirmed that 4-PBA could alleviate aortic valve

Discussion
CAVD is a common aging-related cardiovascular disease with no pharmacological therapy presently.Here, we demonstrate that Herpud1 is highly expressed in CAVD patients with hyperhomocysteinemia and in mice fed with methionine.Hcy induced Herpud1 synthesis through activating sustained ERS and contributing to CAVD progression.Herpud1 deficiency alleviates high methionine diet induced CAVD.Mechanically, we identify that Her-pud1 knockdown significantly alleviated CAVD by activating autophagy of AVICs.Moreover, the ERS inhibitor 4-phenyl butyric acid (4-PBA) decreases Herpud1 expression through suppressing ERS, which is shown to inhibit osteogenic differentiation of AVICs and aortic valve calcification.These novel findings suggest that Herpud1 may represent an effective therapeutic target for treating CAVD.
Hcy is a sulfur-containing amino acid that forms during the metabolic demethylation of methionine.Hyperhomocysteinemia, defined as a plasma concentration of hcy exceeding 15 mol/L, is considered severe at levels >100 mol/L (Handy and Loscalzo 2003;Yang et al. 2014).There is a growing body of evidence linking cardiovascular calcification to hcy.However, its role in CAVD remains unclear.Firstly, we confirmed that Hcy levels are elevated in patients with CAVD.We then used an HMD diet to construct hyperhomocysteinemia-related CAVD in mice.Previous studies commonly fed mice with high fat diet to induce CAVD (The et al. 2022), and this is the first one validated the successful usage of HMD in CAVD modeling.However, in vitro, we found that the ability of Hcy along to induce osteogenic differentiation of AVICs is limited.Interestingly, Hcy could significantly promote PCM induced cell mineralization.The discrepancy between in vivo and in vitro may be due to several reasons, such as during feeding time (age), concentration, and some other unknown factors.
Valve calcification was previously known as a passive process characterized by calcium deposition; it is now recognized that valve calcification is a tightly regulated dynamic process driven by osteogenic chondrogenic differentiation of valve cells (Yutzey et al. 2014).Through analysis of osteogenic marker expression, we found that hcy promotes osteogenic differentiation of AVICs cultured in vitro and in vivo.
ERS activates the UPR, which involves dissociation of the chaperone BiP/GRP78 from the three ER transmembrane-associated sensor proteins, namely PERK, IRE1α, ATF6, and their subsequent activation (Ron and Walter 2007).Our data suggest that hcy stimulation induces strong ERS in AVICs, causing upregulation of downstream PERK and ATF6 expression.Although the above data suggest that ERS is associated with the induction of hcy in AVICs, how ERS is initiated remains unclear.Studies have reported that increased cytoplasmic Ca2+ leads to Ca2+ inward flow to the endoplasmic reticulum, which impedes the ability of endoplasmic reticulum proteins to fold, leading to the accumulation of unfolded or misfolded proteins, which may eventually lead to ERS (Ron and Walter 2007;Xu et al. 2005).We therefore hypothesized that hcy-induced ERS in AVICs may be mediated by increased intracytoplasmic Ca2+.
A well-tolerated small-molecule fatty acid, 4-PBA, is approved by the US Food and Drug Administration for use as an ammonia scavenger in patients with urea cycle disorders and hyperammonemia (450 mg/kg/day in patients weighing less than 20 kg and 9.9-13.0g/m 2 /day in larger patients) (Iannitti and Palmieri 2011;Lee et al. 2010).It is considered a new chemical chaperone for reducing ERS.With the recognition of human diseases caused by ERS, the role of 4-PBA in obesity, diabetes, and ischemic injury has been explored (Guo et al. 2017;Qi et al. 2004;Welch and Brown 1996), providing new drug targets and promising strategies for therapeutic interventions.The present study found that 4-PBA can modulate ERS to inhibit Herpud1, thereby alleviating the aortic valve calcification process.Thus, our findings provide new mechanistic insights into how 4-PBA regulates CAVD progression in an endogenous regulatory manner.
The present study is the first to show that Her-pud1 knockdown alleviates hcy-induced aortic valve calcification.Hcy-induced Herpud1 is a component and regulator of ERAD (Leitman et al. 2014), an intact endoplasmic reticulum membrane protein that is strongly upregulated during ERS (Hori et al. 2004) and then rapidly degraded after endoplasmic reticulum homeostasis is restored (Fumagalli et al. 2016).This is consistent with our data suggesting that hcy in AVICs upregulates Herpud1 expression by activating the ERS.By knocking down and overexpressing Her-pud1, we further confirmed that Herpud1, as a downstream pathway protein of ERS, is involved in Hcy activation of ERS to promote aortic valve calcification.Studies have found that when cells were stressed following Herpud1 knockdown, reduced proteasomal and ERAD degradation activities were observed as well as increased degradation of polyubiquitinated aggregates through increased autophagic flux (Quiroga et al. 2013b).We found that the expression of autophagy-related proteins was upregulated after Her-pud1 knockdown.To explore the role of autophagy in this process, we added the autophagy inhibitor 3-MA to the knockdown of Herpud1 and found that aortic valve calcification was inhibited.Therefore, we concluded that Herpud1 knockdown alleviates hcyinduced aortic valve calcification through upregulation of autophagy.
Runx2 is a key transcription factor that regulates osteoblastic differentiation and leads to expression of bone matrix protein genes, which initiate the transition of cells toward an osteoblast phenotype (Komori et al. 1997).Its expression has been identified in aortic valve calcification human valve tissue specimens and in calcifying AVICs in mice (Deng et al. 2021;Eva et al. 2021).This is also consistent with our findings.It has been explored that deletion of AMPKα1 in VSMC inhibits Runx2 SUMOylation, thereby increasing Runx2 expression and promoting atherosclerotic calcification in vivo (Cai et al. 2016).Hcy can induce endothelial damage via the AMPKa/Sirt1 pathway (Li et al. 2021).Furthermore, it was found that ERS could be inhibited by activating AMPK signaling (Hou et al. 2019;Lu et al. 2015).So we speculate that Hcy may also activate ERS by inhibiting AMPK expression, thereby upregulating Herpud1 protein expression, promoting autophagy, and leading to increased Runx2 expression, ultimately leading to osteogenic differentiation of AVICs.
Some limitations of this study should be noted.Firstly, we used Hcy plus PCM to induce the osteogenic differentiation of AVICs due to the minor procalcific ability of Hcy on cells.But, in this study we also found that the PCM group could also promote osteogenic differentiation of AVICs by activating ERS.β-Glycerophosphate, the primary ingredient of PCM, reportedly induces vascular smooth muscle calcification via ERS (He et al. 2019a).Therefore, we hypothesized that β-GP can also promote calcification via ERS in AVICs.This hypothesis needs to be corroborated by further studies.As shown in Fig. 8a, autophagy was upregulated in the PCM group, and our review of the relevant literature revealed that β-GP in the PCM may play a role in this process.Β-GP not only stimulates calcification but also induces autophagy in human aortic smooth muscle cells (He et al. 2019b).Furthermore, induction of autophagy by β-GP has been shown to be an adaptive response in the calcification process (Shanahan 2013).The existence of β-GP may, to some extent, influence our result in vitro.Secondly, the effects of Herpud1 overexpression and 3-MA on aortic valve calcification were only assessed in vitro, and these need to be further confirmed in vivo.Thirdly, studies have confirmed that LDL levels are associated with CAVD (Rajamannan 2015), and this has been confirmed in our human plasma specimens.However, in mouse plasma we found that HMD promoted TG levels, while LDL was not statistically significantly different between groups, and since some studies have shown that TG is associated with aortic valve calcification (Donis et al. 2021), so we speculate that Hcy can promote CAVD by elevating TG, but not LDL.In addition, there were no significant changes in T plasma metabolite levels after knockdown of Her-pud1.This suggests that Hcy may also induce the progression of CAVD by increasing plasma TG through pathways other than Herpud1.This remains to be explored further.Fourthly, to avoid disturbance of the female estrus cycle, we chose male mice.However, this resulted in us being unable to assess sex-based differences.Fifthly, our review of the relevant literature showed that ApoE−/− mice have a transvalvular peak jet velocity of 1500~2000 mm/s (Wang et al. 2021).As we used LDLR−/− mice for our experiments, we believe it may be that the different strains of mice and the variability of the test apparatus cause them to differ from the typical stenosis velocity of calcified mice.In addition, although the transvalvular peak jet velocity was shown to be ~1200 mm/s in Fig. 1a, the actual mean value of the measurements was ~1300 mm/s (Fig. 1b) and was statistically significant between the groups.The results according to Figs. 5g, h and S5c, d also suggest that we also have differences between the different groups, which we have not explored in depth due to the limited number of mice we have.In addition, we did not confirm the baseline of the aortic valve in all mice before starting the experiment.This approach might have produced bias since we cannot guarantee the baseline consistency of aortic valve, and we will further improve this in later animal experiments.Last but not least, we used Herpud1 global knockout mice to investigate the role of Herpud1 in Hcy-related CAVD.The contribution of Herpud1 in a conditional knockout mouse model regarding CAVD should be required to further approve the above results.

Conclusions
In summary, we found that Herpud1 knockdown attenuated hcy-induced aortic valve calcification and that hcy promotes CAVD via the ERS/Herpud1/ autophagy pathway.Our study provides new insights into the molecular mechanisms regulating Herpud1 in the pathogenesis of CAVD and highlights the potential clinical relevance of 4-PBA-targeted therapy for CAVD.

Perspectives
CAVD is one of the most common valvular heart diseases characterized by the osteogenesis of AVICs, and approximately 9% of aortic valve sclerosis progresses to aortic stenosis within 5 years.Our study is the first to identify that Herpud1 deficiency inhibits hcy-induced CAVD and provides new insights into the molecular regulation of Herpud1 in CAVD pathogenesis, highlighting the potential clinical significance of 4-PBA-targeted therapy for CAVD.

Fig. 2
Fig. 2 Hcy promote valvular calcification both in vitro.a ALP activity assay in AVICs.b The protein levels of three osteogenic markers in AVICs.c, d Alizarin Red staining of mineralization nodules in AVICs.e The mRNA levels of four osteogenic markers in AVICs under normal medium or on

Fig. 3
Fig. 3 Herpud1 is upregulated in Hcy-mediated CAVD. a Immunofluorescence of four human aortic valve with antibodies against α-SMA (green) and Herpud1 (red) to visualize the level of Herpud1 in congenital aortic valve malformation (patient 1) or calcific aortic valve (patients 2-4, scale bars=100μm).Nuclei were stained by DAPI (blue).b, c Her-pud1 and α-SMA were visualized in the AVICs of aortic valve leaflets from LDLR −/− mice fed a normal chow diet (control) or an HMD diet (Hcy) for 28 weeks (scale bars = 50μm) with

Fig. 4
Fig. 4 ERS inhibitor 4-phenyl butyric acid (4-PBA) suppresses Herpud1 expression in HMD-induced mice and Hcytreated AVICs.a The protein levels of PERK, ATF6, and Her-pud1 in aortic valve leaflets from LDLR −/− mice fed a normal chow diet (control) or an HMD diet (Hcy) for 28 weeks.b Relative expression of PERK and CHOP in aortic valve leaflets from LDLR−/− mice fed a normal chow diet (control) or an

Fig. 5
Fig. 5 Deficiency of Herpud1 prevents Hcy-induced aortic valve calcification in vivo.Merged immunofluorescence photomicrographs of human aortic valve (a, scale bars = 100μm) and aortic valve leaflets from LDLR −/− mice fed a normal chow diet (control) or an HMD diet (Hcy) for 28 weeks (b, scale bars = 50μm) with antibodies against Runx2 (green) and Herpud1 (red) to visualize the level of Herpud1 of AVICs.Nuclei were stained by DAPI (blue).c The protein level of Herpud1 in aortic valve leaflets from LDLR−/− mice and LDLR−/− Herpud1−/− mice.d, e The protein levels of three osteogenic markers in aortic valve leaflets from LDLR −/− mice

Fig. 6
Fig. 6 Knockdown of Herpud1 prevents Hcy-induced aortic valve calcification in AVICs.a Western blot showed the protein levels of Herpud1 in AVICs on the basis of PCM with or without Hcy (200μM).b Alizarin Red staining of mineralization nodules in AVICs.c ALP activity assay in AVICs.d The

Fig. 7
Fig. 7 Herpud1 overexpression promotes osteogenic differentiation of AVICs.a The protein levels of Herpud1 in AVICs following Herpud1 overexpression under Hcy (200μM) and ER stress inhibitor 4-PBA (5 mM).b Alizarin Red staining of mineralization nodules in AVICs.c The protein levels of three osteogenic markers in AVICs.d The mRNA levels of four

Fig. 8
Fig. 8 Herpud1 silencing inhibits Hcy-induced calcification through autophagy activation in AVICs.a The protein levels of three autophagy-related proteins (LC3B, P62, Becline) in AVICs under normal medium or on the basis of PCM with or without Hcy (200μM).b The protein levels of three autophagy-related proteins in AVICs following Herpud1 silencing under Hcy (200μM) on the basis of PCM.c The protein levels of three autophagy-related proteins in AVICs fol-

Table 1
Clinical characteristics of patients for analysis Values are mean ± SD or %; p values, chi-square test or oneway ANOVA or Welch's test; BMI, body mass index; LDL-C, low-density lipoprotein cholesterol; HDL, high-density lipoprotein cholesterol; LVEF, left ventricular ejection fraction;