Herpud1 deficiency alleviates homocysteine-induced aortic valve calcification

doi:10.21203/rs.3.rs-1887767/v1

Download PDF

Research Article

Herpud1 deficiency alleviates homocysteine-induced aortic valve calcification

https://doi.org/10.21203/rs.3.rs-1887767/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 06 Feb, 2023

Read the published version in Cell Biology and Toxicology →

You are reading this latest preprint version

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 assessment of aortic valve calcification lesions, ERS activation, autophagy, and osteogenic differentiation of aortic valve interstitial cells (AVICs). We used siRNA to knock down Herpud1 in cultured Primary AVICs were isolated from mice to further validate our findings.

Results: Herpud1 was highly expressed in calcified human and mouse aortic valves as well as primary aortic valve interstitial cells (AVICs). 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. Hcy increased Herpud1 expression through the ERS pathway and promoted CAVD progression. ERS inhibitor 4-phenyl butyric acid (4-PBA) significantly attenuated aortic valve calcification in high methionine diet–fed low-density lipoprotein receptor^-/- (LDLR^-/-) mice by suppressing ERS and subsequent Herpud1 biosynthesis.

Conclusions: These findings identify a previously unknown mechanism of Herpud1 upregulation in CAVD progression, suggesting that Herpud1 silencing or inhibition is a viable therapeutic strategy for arresting CAVD progression.

Calcific aortic valve disease

Homocysteine

Herpud1

Autophagy

Endoplasmic reticulum stress

4-phenyl butyric acid

Homocysteine (hcy) promotes aortic valvular calcification in vivo and in vitro.
Herpud1 is upregulated in the leaflets of mice and humans with CAVD. Herpud1 deficiency inhibited hcy-induced CAVD in vitro and in vivo.
Herpud1 silencing activates cell autophagy, inhibiting osteogenic differentiation of primary aortic valve interstitial cells (AVICs) induced by hcy.
4-Phenyl butyric acid, a compound that suppresses ERS and subsequent Herpud1 biosynthesis, significantly decreased AVIC calcification in vitro after hcy stimulation and mitigated the severity of aortic valve fibrosis and calcification in a murine model of CAVD.

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). This 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). The amino terminus of the Herpud1 peptide chain acts as a molecular chaperone to inhibit protein aggregation, while the carboxyl terminus of Herpud1 functions as a ubiquitin ligase to degrade proteins via the proteasome pathway(Kokame et al. 2000). Herpud1 plays an important role in ERS homeostasis regulation. 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 hcy-induced 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(Martinet et al. 2007; Neutel et al. 2020; Schiattarella and Hill 2016). CAVD inhibition can be achieved by enhancing autophagy(Deng et al. 2017). 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 study explored the relationship between Herpud1 and autophagy in hcy-induced calcification. Herpud1 was highly expressed in CAVD in patients with hyperhomocysteinemia and mice fed hcy. Hcy activates Herpud1 through ERS, inhibiting autophagy and contributing to CAVD progression. Herpud1 knockout significantly alleviated CAVD by activating autophagy of primary aortic valve interstitial cells (AVICs), suggesting that it may represent a novel therapeutic strategy for CAVD.

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 2019. 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. Patients were divided into CAVD and control groups 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 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.

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 (6–8-week-old) male mice. Mice were housed under a 12-h light/dark cycle and fed a normal chow diet (ND) or an HMD (where LDLR-/- mice were fed with regular diet plus 2% methionine (wt/wt)) (Aladdin , Shanghai, China) for 28 weeks to promote CAVD.Mice were anesthetized through the intraperitoneal application of sodium pentobarbital (50 mg/kg). The adequacy of anesthesia was confirmed by the absence of reflex response to foot squeeze. 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).

Histology and immunohistochemistry

The aortic valve of animals and patients was embedded in paraffin and then cut in serial 5-μm-thick sections. Sequential sections were stained with a hematoxylin and eosin kit (G1120; Solarbio, Beijing, China), Alizarin Red S Staining Kit (G8550; Solarbio), Von Kossa Stain kit (ab150687; Abcam, Cambridge, UK), and Masson’s trichrome staining kit (BP-DL023; Sbjbio, Nanjing, China) to measure aortic valve leaflet thickness, calcium nodules, calcium deposition, and collagen content. Sections were immunostained with anti-rabbit protein kinase R-like ER kinase (PERK; ab65142, 1:400), CHOP (CST,2895, 1:400), Herpud1 (ab150424, 1:400), alkaline phosphatase (ALP; ab95462, 1:400), osteopontin (OPN; ab8448, 1:400), and collagen 1 (Col1; ab6308, 1:400) antibodies followed by detection using an horseradish peroxidase–conjugated secondary antibody and 3,3′-diaminobenzidine (Gene Tech, Shanghai, China) to measure the protein expression in the aortic root at the level of the aortic valve. The sections were imaged using a biological microscope (DM3000; Leica, Wetzlar, Germany). For dual immunofluorescence, sections were stained with anti-alpha-smooth muscle actin (α-SMA; ab5694, 1:200) and Herpud1, Runt-related transcription factor 2 (Runx2,ab76956, 1:300) antibodies followed by detection with anti-rabbit DyLight 594 and anti-rat DyLight 488 immunoglobulin G (H+L) (Abbkine Scientific, Wuhan, China). The sections were counterstained with 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI; Sigma, St. Louis, MO, USA) and images were acquired under a Leica confocal re-imagined microscope (Stellaris 5).

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% CO₂. 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; MedChemExpress). The cells were then treated with 200 μM hcy (H4628; Sigma) for 7 days. To inhibit hcy-stimulated ERS, the cells were pretreated with ER stress inhibitor 4-phenyl butric acid (4-PBA; 5 mM; MedChemExpress), and then each group was stimulated with hcy 200 μM for 7 days. To inhibit hcy-activated 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. To overexpress Herpud1, AVICs were transduced with lentivirus encoding for Herpud1.

Reverse transcription quantitative polymerase chain reaction 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 PrimeScript 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 S1. 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.

Western blotting analysis

Total protein was extracted from the AVICs using RIPA lysis buffer (Beyotime, Shanghai, China), and protein concentrations were determined by the bicinchoninic acid method according to the manufacturer’s protocol. The lysates were subjected to the sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene fluoride membranes (Millipore, MA, USA). After blocking with 5% skim milk in Tris-buffered saline with Tween-20 at 20-25℃ for 1 h, the membranes were incubated with primary antibodies against Herpud1 (ab150424; 1:1000), ATF6 (ab37149; 1:1000), Runx2 (ab76956; 1:1000), OPN (ab8448; 1:10400), PERK (ab65142; 1:1000), collagen1 (ab6308; 1:1000), Becline1 (ab210498; 1:1000)，LC3B (ab192890; 1:1000), GAPDH (ab181602; 1:1000) (Abcam) at 4°C overnight and further incubated in horseradish peroxidase–conjugated secondary antibodies (Abbkine, Inc., Redlands, CA, USA). Immune complexes were visualized with an electrochemiluminescence detection system (Beyotime). The grey value of the target strip was analyzed using Quantity One 5.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Each test was performed in triplicate.

Cell immunofluorescence staining

After treatment, AVICs grown on coverslips were collected, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS) with Tween-20. After blocking with 4% bovine serum albumin, the cells were incubated with the primary antibody against α-SMA (ab5694; 1:200), and vimentin (CST; 5741; 1;200), AffiniPure goat immunoglobulin G (H+L). The nuclei were counterstained with 0.1 μg/mL DAPI. Images were captured by an Eclipse Ti-U fluorescence microscope (×400; Nikon, Tokyo ,Japan).

Alizarin Red staining

The AVICs were sub-cultured on a 24-well plate; when confluency reached 30%, they were treated in different groups (see 4. Cell culture and treatment 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.

ALP activity measurement

After 14 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 15000 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 minutes. The maximum linear rate (∆A405nm/min) for the test, blank, and control was obtained. The units/mL solution were 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 using 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), aortic valve calcification was defined as enhanced local valve echo, valve leaf thickness >2mm, and cross-valve flow velocity ≥2.5 m/s.Patients were divided into CAVD and control groups based on the presence or absence of aortic valve calcification. 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 serum triglyceride levels (p=0.019). No statistically significant intergroup differences were noted in age, sex, smoking, body mass index, smoking, hypertension, and left ventricular ejection fraction (p>0.05 for all). Therefore, we hypothesized that hcy plays a role in CAVD pathogenesis.

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 moulding(Figure S2C). We found that, 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 (Figure 1A). The immunohistochemistry results showed that hcy increased the expression of osteogenic markers (Col1, OPN, and ALP; Figure 1B). The expressions of osteogenic markers including ALP, osteocalcin, Runx2, OPN, and Col1 were explored. The reverse transcription quantitative polymerase chain reaction and western blotting analyses demonstrated upregulation of the above markers in the hcy versus control group (Figures 1C, 1D). In vitro, we isolated AVICs from mice, which expressed high SMA and low vimentin (Figure S2A). We exposed AVICs to PCM to stimulate osteogenic differentiation(Yu et al. 2018). As shown in Figure 2A, after 7 days of PCM stimulation, the mRNA levels of osteogenic markers were significantly upregulated; interestingly, the addition of hcy further elevated the osteogenic markers at the mRNA and protein levels (Figures 2E, 2B). After 5 or 10 days, PCM induced some calcification, as detected by alizarin red staining (Figure S2B), the calcific nodules increased significantly after 14 days, and hcy notably increased calcific nodules in PCM-treated AVICs in a time-dependent manner (Figures 2C, 2D). After 21 days, the ALP activity assay showed that hcy further upregulated ALP activity based on PCM (Figure 2A). 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 is stimulated by hcy in smooth muscle cells(Lin et al. 2018). Here we also investigated the profile of Heprud1 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 2. We classified the degree of aortic valve calcification into four groups. Alizarin Red staining showed a significant increase in calcified nodules in the aortic valve in patients with CAVD versus the non-calcified group (Figure 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 (Figure S3C). As shown in Figure 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, with immunofluorescence co-staining (Figure 3B), immunohistochemistry (Figure 3C), and western blotting (Figure 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 (Figure 3E). Therefore, we concluded that Herpud1 is upregulated in hcy-mediated CAVD.

We previously found that increased Herpud1 expression in smooth muscle cells was associated with ERS(Lin et al. 2018). We explored the effect of whether hcy increases Herpud1 expression by activating ERS. First, we determined that hcy activates ERS in CAVD. The immunohistochemical analysis showed that the expression of ERS transmembrane proteins (PERK, CHOP) was upregulated in the hcy group compared to the control group (Figure 4A). Western blotting analysis also yielded the same results, with increased expression of related ERS transmembrane proteins (PERK, ATF6) both in vivo (Figure 4B) and in vitro (Figure 4C).

To further clarify the relationship between ERS and Herpud1, we fed LDLR^-/- mice an ND or an HMD with or without 4-PBA treatment (100 mg/kg/d) for 28 weeks. Daily chow consumption did not differ among the four groups. As expected, the addition of the ERS inhibitor 4-PBA inhibited the expression of ERS-related pathway signaling proteins (Figures 4D, S5B). Interestingly, we found that 4-PBA inhibited the synthesis of Herpud1, mainly manifested by the downregulation of the Herpud1 protein expression levels in the hcy+4-PBA versus hcy group (Figure 4D). This was consistent with the immunohistochemical analysis (Figure S4B) and immunofluorescence co-staining (Figure S4A) results. Consistent with the in vivo data, the ERS pathway protein and Herpud1 secretion in vitro were higher in the PCM+hcy versus PCM group and suppressed by 4-PBA (Figure 4E). Therefore, we concluded that hcy upregulated Herpud1 expression in CAVD by activating ERS.

Herpud1 deficiency prevents hcy-induced aortic valve calcification

To clarify the role of Herpud1 in hcy-induced aortic valve calcification we first used immunofluorescence co-staining. We found an almost complete overlap in Herpud1 and Runx2 localization in human and animal aortic valves, and high expression of Herpud1 and Runx2 in human calcified aortic valves versus non-calcified valves, which is consistent with the hcy group in mice (Figure 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 fedan ND and HMD for 28 weeks, respectively. We established that Herpud1 was successfully knocked out in the LDLR^-/- Herpud1^-/- group (Figure 5C). We then collected aortic valves from LDLR^-/- mice from the different groups and evaluated the morphology of the valve leaflets and the degree of calcification. The mRNA and protein levels of osteogenic markers in the aortic valve were remarkably reduced compared to the LDLR^-/- Herpud1^-/- group under concomitant HMD conditions (Figure 5E,F). This was consistent with a reduction in aortic leaflet thickness, collagen, and calcium deposition (Figure 5D). 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 siRNA against Herpud1 (primary HERP^-/-/LDLR^-/- mice from AVICs with low survival in culture) to inhibit Herpud1 expression in AVICs. The knockdown was confirmed by western blotting (Figure 6A). The data suggest that the osteogenic differentiation of AVICs induced by hcy was reversed after Herpud1 knockdown, mainly manifested by reduced osteogenic marker protein (Figure 6C) and mRNA (Figure 6D) expression levels in the aortic valve, reduced calcified nodule formation (Figures 6B, 6F), and decreased ALP activity (Figure 6E). In summary, Herpud1 was negatively correlated with aortic valve calcification under hcy induction.

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 (Figure 7A). We found that overexpression of Herpud1 could aggravate hcy-induced osteogenic differentiation of AVICs, mainly manifested by increased osteogenic marker protein (Figure 7C) and mRNA (Figure 7D) expression levels and increased calcified nodule formation in the aortic valve in the PCM+hcy+4-PBA+Herpud1-overexpression(OV)group compared with the PCM+hcy+4-PBA group (Figure 7B). We concluded that: 1. these results confirmed that Herpud1 knockdown alleviates hcy-induced CAVD; 2. hcy induces osteogenic differentiation of AVICs 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(Ma et al. 2020; Phadwal et al. 2020). Consistent with these results, the western blotting analysis revealed that hcy inhibited autophagy-related protein expression (LC3B, Becline1) in AVICs (Figure 8A), while knockdown of Herpud1 effectively abolished the effect of hcy on the autophagy proteins level in AVICs (Figure 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 and, as anticipated, the addition of 3-MA resulted in downregulation of autophagy protein expression (Figure 8C). Further, 3-MA prevented Herpud1 silencing-induced inhibition of the protein levels of the osteogenic differentiation markers (Figure 8D), calcified nodule formation (Figure 8E), and ALP activity (Figure 8F). Collectively, the above experimental results suggest that Herpud1 knockdown can alleviate osteogenic differentiation of AVICs by activating autophagy.

ERS inhibitor 4-PBA alleviates valve calcification by downregulating Heprud1

4-PBA is an ERS inhibitor that has been shown to alleviate cardiovascular calcification(Chen et al. 2021). We conducted in vivo experiments to clarify the role of 4-PBA in aortic valve calcification induced by hcy. We found that aortic valve calcification improved in HMD-fed LDLR^-/- mice after the addition of 4-PBA, and 4-PBA treatment significantly reduced protein levels of three osteogenic differentiation markers (Figure 9B), consistent with reductions in aortic leaflet thickness, collagen, and calcium deposition (Figure 9A), and osteogenic marker expression (Figure S5A). Immunofluorescence co-staining results revealed that 4-PBA inhibited the expression of Herpud1 and reduced the expression of Runx2, consistent with our expectation (Figure S5B). These findings are similar to the results we observed after Herpud1 knockdown. Combined with the results of previous experiments, we confirmed that 4-PBA could alleviate aortic valve calcification in mice by downregulating hcy-induced Herpud1 expression.

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 AVICs differentiation, mainly in the PCM+hcy+4-PBA group compared with the PCM+hcy group, which downregulated osteogenic marker expression levels (Figure 9E) and reduced calcified nodule formation (Figure 9C). Taken together, these results demonstrate that the ERS inhibitor 4-PBA alleviates valve calcification by downregulating Heprud1.

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. Here, we demonstrated for the first time that hcy levels are elevated in human CAVD. Although 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). Early activation of the PERK-eIF2α-ATF4-CHOP pathway reduces the protein translation rate to enable the ER to recover from stress; the activation of IRE1-XBP1 and the ATF6-chaperone pathway primarily regulates GRP78, thereby increasing the capacity of protein folding(Cominacini et al. 2015; Walter and Ron 2011). Therefore, the UPR is essential for the endoplasmic reticulum to maintain homeostasis from various cellular perturbations. Our data suggest that hcy stimulation induces strong endoplasmic reticulum stress 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 9.9–13.0 g/m²/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, 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 Herpud1 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 Herpud1, 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 Herpud1 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 hcy-induced 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 initiates the transition of cells towards 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). It has been explored that deletion of AMPKα1 in VSMC inhibits Runx2 SUMOyalation 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 signalling(Hou et al. 2019; Lu et al. 2015). So we speculate that Hcy may 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.

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 Figure 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).

Some limitations of this study should be noted. 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.

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.

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.

Hcy=Homocysteine

ERS=Endoplasmic reticulum stress

Herpud1=Ubiquitin like domain 1

CAVD=Calcific aortic valve disease

4-AVICs=Valve interstitial cells

5-PBA=4-phenyl butyric acid

6-ND=Normal chow diet

HMD=High methionine diet

LDLR-/- =low-density lipoprotein receptor^-/-

LDLR^-/-/Herpud1^-/-=low-density lipoprotein receptor (LDLR) and Herpud1 double knockout

UPR=Unfolded protein response

ERAD=ER-associated protein degradation

ND=Normal chow diet

3-MA=3-Methyladenine

ALP=Alkaline phosphatase

Col1=Collagen1

OPN=Osteopontin

OC= Osteocalcin

FDA=Food and Drug Administration

β-GP=β-glycerophosphate

HASMCs=Human aortic smooth muscle cells

CAVM=Congenital aortic valve malformation

AVC=Aortic valve calcification

OV= Overexpression

Ethical Approval and Consent to participate

Human Ethics: Compliance with ethical standards.

Consent for publication

Availability of supporting data: The data underlying this article are available in the article and in its online supplementary material.

Competing interests: None.

Funding: This work was supported by Natural Science Foundation of Zhejiang Province ,China(No. LGF21H020001) ; Shaoxing Science and Technology Plan Project ,China ( No.2020A13015 and No. 2020A13018).

Authors' contributions: Wenqing Xie and Yue Shan Completed most of the experiments and thesis writing.Zhuonan Wu,Nan Liu, Jinjin Yang, Hanlin Zhang, Shiming Sun, Jufang Chi and Weizhong Feng participated in the analysis of experimental data. Hui Lin and Hangyuan Guo designed the experimental idea.

Acknowledgements: The authors thank Hangyuan Guo and Hui Lin for the support and guidance of this experiment, and Yue Shan, Zhuonan Wu and others assisted in this experiment.

Authors' information: Wenqing Xie ,Zhuonan Wu,Nan Liu, Jinjin Yang, Hanlin Zhang, Shiming Sun , Hui Lin Master's in Cardiology ; Yue Shan, Master of Anesthesiology; Weizhong Feng , Master of Thoracic Surgery ;Jufang Chi , Hangyuan Guo, Doctor of Cardiology.

Cai, Z., Ding, Y., Zhang, M., Lu, Q., Wu, S., and Zhu, H., et al. 2016. Ablation of adenosine monophosphate-activated protein kinase alpha1 in vascular smooth muscle cells promotes diet-induced atherosclerotic calcification in vivo. Circ. Res. 119(3): 422–433. doi:10.1161/CIRCRESAHA.116.308301.
Cai, Z., Li, F., Gong, W., Liu, W., Duan, Q., and Chen, C., et al. 2013. Endoplasmic reticulum stress participates in aortic valve calcification in hypercholesterolemic animals. Arterioscler Thromb Vasc Biol 33(10): 2345–2354. doi:10.1161/ATVBAHA.112.300226.
Chen, Y., Zhang, H., Liu, H., Li, K., and Su, X. 2018. Homocysteine up-regulates etb receptors via suppression of autophagy in vascular smooth muscle cells. Microvasc. Res. 119: 13–21. doi:10.1016/j.mvr.2018.03.010.
Chen, Z., Li, R., Pei, L.G., Wei, Z.H., Xie, J., and Wu, H., et al. 2021. High-mobility group box-1 promotes vascular calcification in diabetic mice via endoplasmic reticulum stress. J. Cell. Mol. Med. 25(8): 3724–3734. doi:10.1111/jcmm.16075.
Cominacini, L., Mozzini, C., Garbin, U., Pasini, A., Stranieri, C., and Solani, E., et al. 2015. Endoplasmic reticulum stress and nrf2 signaling in cardiovascular diseases. Free Radic Biol Med 88(Pt B): 233–242. doi:10.1016/j.freeradbiomed.2015.05.027.
Deng, X.S., Meng, X., Fullerton, D., Stone, M., and Jaggers, J. 2021. Complement upregulates runx-2 to induce profibrogenic change in aortic valve interstitial cells. Ann. Thorac. Surg. 112(6): 1962–1972. doi:10.1016/j.athoracsur.2020.12.058.
Deng, X.S., Meng, X., Venardos, N., Song, R., Yamanaka, K., and Fullerton, D., et al. 2017. Autophagy negatively regulates pro-osteogenic activity in human aortic valve interstitial cells. J. Surg. Res. 218: 285–291. doi:10.1016/j.jss.2017.05.088.
Duan, X., Zhou, Y., Teng, X., Tang, C., and Qi, Y. 2009. Endoplasmic reticulum stress-mediated apoptosis is activated in vascular calcification. Biochem Biophys Res Commun 387(4): 694–699. doi:10.1016/j.bbrc.2009.07.085.
Eva, S.K., Combi, Z., Potor, L., Szerafin, T., Hendrik, Z., and Mehes, G., et al. 2021. Hydrogen sulfide inhibits aortic valve calcification in heart via regulating runx2 by nf-kappab, a link between inflammation and mineralization. J Adv Res 27: 165–176. doi:10.1016/j.jare.2020.07.005.
Fu, Z., Li, F., Jia, L., Su, S., Wang, Y., and Cai, Z., et al. 2019. Histone deacetylase 6 reduction promotes aortic valve calcification via an endoplasmic reticulum stress-mediated osteogenic pathway. J Thorac Cardiovasc Surg 158(2): 408–417. doi:10.1016/j.jtcvs.2018.10.136.
Fumagalli, F., Noack, J., Bergmann, T.J., Cebollero, E., Pisoni, G.B., and Fasana, E., et al. 2016. Translocon component sec62 acts in endoplasmic reticulum turnover during stress recovery. Nat. Cell Biol. 18(11): 1173–1184. doi:10.1038/ncb3423.
Go, A.S., Mozaffarian, D., Roger, V.L., Benjamin, E.J., Berry, J.D., and Borden, W.B., et al. 2013. Executive summary: heart disease and stroke statistics–2013 update: a report from the american heart association. Circulation 127(1): 143–152. doi:10.1161/CIR.0b013e318282ab8f.
Gould, R.A., and Butcher, J.T. 2010. Isolation of valvular endothelial cells. J Vis Exp(46). doi:10.3791/2158.
Guo, Q., Xu, L., Li, H., Sun, H., Wu, S., and Zhou, B. 2017. 4-pba reverses autophagic dysfunction and improves insulin sensitivity in adipose tissue of obese mice via akt/mtor signaling. Biochem Biophys Res Commun 484(3): 529–535. doi:10.1016/j.bbrc.2017.01.106.
Handy, D.E., and Loscalzo, J. 2003. Homocysteine and atherothrombosis: diagnosis and treatment. Curr. Atheroscler. Rep. 5(4): 276–283. doi:10.1007/s11883-003-0050-x.
He, H.Q., Law, B., Zhang, N., Qiu, C.L., Qu, Y.Q., and Wu, A.G., et al. 2019a. Bavachin protects human aortic smooth muscle cells against beta-glycerophosphate-mediated vascular calcification and apoptosis via activation of mtor-dependent autophagy and suppression of beta-catenin signaling. Front Pharmacol 10: 1427. doi:10.3389/fphar.2019.01427.
He, H.Q., Law, B., Zhang, N., Qiu, C.L., Qu, Y.Q., and Wu, A.G., et al. 2019b. Bavachin protects human aortic smooth muscle cells against beta-glycerophosphate-mediated vascular calcification and apoptosis via activation of mtor-dependent autophagy and suppression of beta-catenin signaling. Front Pharmacol 10: 1427. doi:10.3389/fphar.2019.01427.
Hetz, C. 2012. The unfolded protein response: controlling cell fate decisions under er stress and beyond. Nat Rev Mol Cell Biol 13(2): 89–102. doi:10.1038/nrm3270.
Hori, O., Ichinoda, F., Yamaguchi, A., Tamatani, T., Taniguchi, M., and Koyama, Y., et al. 2004. Role of herp in the endoplasmic reticulum stress response. Genes Cells 9(5): 457–469. doi:10.1111/j.1356-9597.2004.00735.x.
Hou, X., Fu, M., Cheng, B., Kang, Y., and Xie, D. 2019. Galanthamine improves myocardial ischemia-reperfusion-induced cardiac dysfunction, endoplasmic reticulum stress-related apoptosis, and myocardial fibrosis by suppressing ampk/nrf2 pathway in rats. Ann Transl Med 7(22): 634. doi:10.21037/atm.2019.10.108.
Iannitti, T., and Palmieri, B. 2011. Clinical and experimental applications of sodium phenylbutyrate. Drugs R D 11(3): 227–249. doi:10.2165/11591280-000000000-00000.
Kokame, K., Agarwala, K.L., Kato, H., and Miyata, T. 2000. Herp, a new ubiquitin-like membrane protein induced by endoplasmic reticulum stress. J. Biol. Chem. 275(42): 32846–32853. doi:10.1074/jbc.M002063200.
Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K., and Deguchi, K., et al. 1997. Targeted disruption of cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89(5): 755–764. doi:10.1016/s0092-8674(00)80258-5.
Lee, B., Rhead, W., Diaz, G.A., Scharschmidt, B.F., Mian, A., and Shchelochkov, O., et al. 2010. Phase 2 comparison of a novel ammonia scavenging agent with sodium phenylbutyrate in patients with urea cycle disorders: safety, pharmacokinetics and ammonia control. Mol. Genet. Metab. 100(3): 221–228. doi:10.1016/j.ymgme.2010.03.014.
Leitman, J., Shenkman, M., Gofman, Y., Shtern, N.O., Ben-Tal, N., and Hendershot, L.M., et al. 2014. Herp coordinates compartmentalization and recruitment of hrd1 and misfolded proteins for erad. Mol. Biol. Cell 25(7): 1050–1060. doi:10.1091/mbc.E13-06-0350.
Li, H., Liu, L., Cao, Z., Li, W., Liu, R., and Chen, Y., et al. 2021. Naringenin ameliorates homocysteine induced endothelial damage via the ampkalpha/sirt1 pathway. J Adv Res 34: 137–147. doi:10.1016/j.jare.2021.01.009.
Lin, H., Ni, T., Zhang, J., Meng, L., Gao, F., and Pan, S., et al. 2018. Knockdown of herp alleviates hyperhomocysteinemia mediated atherosclerosis through the inhibition of vascular smooth muscle cell phenotype switching. Int. J. Cardiol. 269: 242–249. doi:10.1016/j.ijcard.2018.07.043.
Lindman, B.R., Clavel, M.A., Mathieu, P., Iung, B., Lancellotti, P., and Otto, C.M., et al. 2016. Calcific aortic stenosis. Nat Rev Dis Primers 2: 16006. doi:10.1038/nrdp.2016.6.
Lu, W.W., Zhao, L., Zhang, J.S., Hou, Y.L., Yu, Y.R., and Jia, M.Z., et al. 2015. Intermedin1-53 protects against cardiac hypertrophy by inhibiting endoplasmic reticulum stress via activating amp-activated protein kinase. J. Hypertens. 33(8): 1676–1687. doi:10.1097/HJH.0000000000000597.
Ma, W.Q., Sun, X.J., Zhu, Y., and Liu, N.F. 2020. Pdk4 promotes vascular calcification by interfering with autophagic activity and metabolic reprogramming. Cell Death Dis. 11(11): 991. doi:10.1038/s41419-020-03162-w.
Martinet, W., Knaapen, M.W., Kockx, M.M., and De Meyer, G.R. 2007. Autophagy in cardiovascular disease. Trends Mol. Med. 13(11): 482–491. doi:10.1016/j.molmed.2007.08.004.
Neutel, C., Hendrickx, J.O., Martinet, W., De Meyer, G., and Guns, P.J. 2020. The protective effects of the autophagic and lysosomal machinery in vascular and valvular calcification: a systematic review. Int. J. Mol. Sci. 21(23). doi:10.3390/ijms21238933.
Phadwal, K., Feng, D., Zhu, D., and Macrae, V.E. 2020. Autophagy as a novel therapeutic target in vascular calcification. Pharmacol Ther 206: 107430. doi:10.1016/j.pharmthera.2019.107430.
Qi, X., Hosoi, T., Okuma, Y., Kaneko, M., and Nomura, Y. 2004. Sodium 4-phenylbutyrate protects against cerebral ischemic injury. Mol. Pharmacol. 66(4): 899–908. doi:10.1124/mol.104.001339.
Quiroga, C., Gatica, D., Paredes, F., Bravo, R., Troncoso, R., and Pedrozo, Z., et al. 2013a. Herp depletion protects from protein aggregation by up-regulating autophagy. Biochim Biophys Acta 1833(12): 3295–3305. doi:10.1016/j.bbamcr.2013.09.006.
Quiroga, C., Gatica, D., Paredes, F., Bravo, R., Troncoso, R., and Pedrozo, Z., et al. 2013b. Herp depletion protects from protein aggregation by up-regulating autophagy. Biochim Biophys Acta 1833(12): 3295–3305. doi:10.1016/j.bbamcr.2013.09.006.
Ron, D., and Walter, P. 2007. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8(7): 519–529. doi:10.1038/nrm2199.
Schiattarella, G.G., and Hill, J.A. 2016. Therapeutic targeting of autophagy in cardiovascular disease. J. Mol. Cell. Cardiol. 95: 86–93. doi:10.1016/j.yjmcc.2015.11.019.
Schulze, A., Standera, S., Buerger, E., Kikkert, M., van Voorden, S., and Wiertz, E., et al. 2005. The ubiquitin-domain protein herp forms a complex with components of the endoplasmic reticulum associated degradation pathway. J. Mol. Biol. 354(5): 1021–1027. doi:10.1016/j.jmb.2005.10.020.
Shanahan, C.M. 2013. Autophagy and matrix vesicles: new partners in vascular calcification. Kidney Int. 83(6): 984–986. doi:10.1038/ki.2013.75.
Shi, Y., Wang, S., Peng, H., Lv, Y., Li, W., and Cheng, S., et al. 2019. Fibroblast growth factor 21 attenuates vascular calcification by alleviating endoplasmic reticulum stress mediated apoptosis in rats. Int. J. Biol. Sci. 15(1): 138–147. doi:10.7150/ijbs.28873.
Wald, D.S., Law, M., and Morris, J.K. 2002. Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. BMJ 325(7374): 1202. doi:10.1136/bmj.325.7374.1202.
Walter, P., and Ron, D. 2011. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334(6059): 1081–1086. doi:10.1126/science.1209038.
Welch, W.J., and Brown, C.R. 1996. Influence of molecular and chemical chaperones on protein folding. Cell Stress Chaperones 1(2): 109–115. doi:10.1379/1466-1268(1996)001<0109:iomacc>2.3.co;2.
Xu, C., Bailly-Maitre, B., and Reed, J.C. 2005. Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 115(10): 2656–2664. doi:10.1172/JCI26373.
Yadgir, S., Johnson, C.O., Aboyans, V., Adebayo, O.M., Adedoyin, R.A., and Afarideh, M., et al. 2020. Global, regional, and national burden of calcific aortic valve and degenerative mitral valve diseases, 1990–2017. Circulation 141(21): 1670–1680. doi:10.1161/CIRCULATIONAHA.119.043391.
Yang, B., Fan, S., Zhi, X., Wang, Y., Wang, Y., and Zheng, Q., et al. 2014. Prevalence of hyperhomocysteinemia in china: a systematic review and meta-analysis. Nutrients 7(1): 74–90. doi:10.3390/nu7010074.
Yin, Z., Pascual, C., and Klionsky, D.J. 2016. Autophagy: machinery and regulation. Microb Cell 3(12): 588–596. doi:10.15698/mic2016.12.546.
Yu, C., Li, L., Xie, F., Guo, S., Liu, F., and Dong, N., et al. 2018. Lncrna tug1 sponges mir-204-5p to promote osteoblast differentiation through upregulating runx2 in aortic valve calcification. Cardiovasc. Res. 114(1): 168–179. doi:10.1093/cvr/cvx180.
Yutzey, K.E., Demer, L.L., Body, S.C., Huggins, G.S., Towler, D.A., and Giachelli, C.M., et al. 2014. Calcific aortic valve disease: a consensus summary from the alliance of investigators on calcific aortic valve disease. Arterioscler Thromb Vasc Biol 34(11): 2387–2393. doi:10.1161/ATVBAHA.114.302523.

Table1. Clinical characteristics of patients for analysis.

Characteristics	Control (n=87)	CAVD (n=44)	P value
Median Age (years)	70.16±6.97	75.11±8.23	0.087
Male	54 (62.1%)	25 (58.1%)	0.810
BMI (kg/m2)	24.46±1.69	24.55±0.76	0.874
Smoking (%)	22 (25.3%)	14 (32.6%)	0.507
Hypertension (%)	38 (43.7%)	24 (55.8%)	0.264
Diabetes (%)	26 (29.9%)	18 (41.9%)	0.246
Triglycerides (mmol/L)	1.79±1.28	2.07±1.85	0.019
LDL (mmol/L)	2.16±0.75	2.41±0.93	0.133
HDL (mmol/L)	1.26±1.05	1.25±0.61	0.970
LVEF (%)	64.64±8.42	62.54±10.67	0.140
Hcy (μmol/L)	11.51±4.44	18.55±11.45	0.000

Values are mean ± SD or %; P values: Student t test or Fisher exact test; BMI: body mass index; LDL: low-density lipoprotein cholesterol; HDL: high-density lipoprotein cholesterol; LVEF: left ventricular ejection fraction; CRP: C-reactive protein; Hcy: homocysteine.

Table2. The baseline characteristics of the 4 patients.

Characteristics	Patient1	Patient2	Patient3	Patient4
Median Age (years)	66	72	59	73
Sex	Male	Female	Male	Male
BMI (kg/m2)	29.4	27.4	17.82	26.4
Smoking	Without	Without	With	With
Hypertension	With	Without	Without	With
Diabetes	Without	Without	Without	Without
Triglycerides (mmol/L)	1.35	1.51	1.24	1.77
LDL (mmol/L)	3.01	3.29	2.52	3.05
HDL (mmol/L)	1.07	1.2	1.25	0.73
LVEF (%)	58	65	48	70
Hcy (μmol/L)	8.78	15.11	20.61	17.64
Ultrasound	CAVM	AVC	AVC	AVC

Patient1 with non-calcified aortic valve；Patient2-4 with CAVD. CAVM(congenital aortic valve malformation);AVC(aortic valve calcification)

No competing interests reported.

TableS1.docx
VISUALABSTRACT.jpg
FigureS1.jpg
FigureS1 Ultrasound images of aortic valve calcification at different levels.By comparing the results of transthoracic echocardiography, aortic valve calcification was defined as enhanced local valve echo, valve leaf thickness >2mm, and cross-valve flow velocity ≥2.5 m/s.
FigureS2.tif
FigureS2 (A)Merged immunofluorescence photomicrographs of AVICs with antibodies against α-SMA (green) and Vimentin(red) . Nuclei were stained by DAPI (blue). (B)Alizarin red staining of mineralization nodules in AVICs harvested at different time points (days 5, 10) under on the basis of PCM with or without Hcy(200μM). (C)Homocysteine levels in the plasma of LDLR−/− mice fed a normal chow diet (ND) or an HMD diet (HMD) for 28 weeks. Statistical significance was determined by Two-way ANOVA using the Bonferroni post hoc test.n=6 per group. *P < 0.05, **P<0.01,***P<0.001. Values are the mean ± SD.
FigureS3.tif
FigureS3 (A)Haematoxylin and eosin staining of human aortic valve leaflets(Scale bars = 100μm).(B)Alizarin red staining of mineralization nodules in human aortic valve leaflets(Scale bars = 100μm).(C)Relative expression of two osteogenic markers and Herpud1 in human aortic valve leaflets,as detected by IHC staining (Scale bars = 100μm).
FigureS4.tif
FigureS4 (A)Merged immunofluorescence photomicrographs of aortic valve leaflets from LDLR^-/- mice with antibodies against α-SMA (green) and Herpud1 (red) to visualize the level of Herpud1 of AVICs. Nuclei were stained by DAPI (blue)(Scale bars = 50μm).(B)Relative expression of two osteogenic markers and Herpud1 in aortic valve leaflets from LDLR^-/- mice,as detected by IHC staining (Scale bars = 400μm).
FigureS5.tif
FigureS5 (A)Relative expression of three osteogenic markers in aortic valve leaflets from LDLR^-/- mice,as detected by IHC staining (Scale bars= 100μm). (B)Merged immunofluorescence photomicrographs of aortic valve leaflets from LDLR^-/- mice with antibodies against Runx2(green) and Herpud1 (red) to visualize the level of Herpud1 of AVICs. Nuclei were stained by DAPI (blue) (Scale bars= 50μm).

Download PDF

Journal Publication

published 06 Feb, 2023

Read the published version in Cell Biology and Toxicology →

Editorial decision: Major revision
06 Oct, 2022
Reviews received at journal
14 Aug, 2022
Reviewers agreed at journal
07 Aug, 2022
Reviewers agreed at journal
05 Aug, 2022
Reviewers invited by journal
03 Aug, 2022
Editor assigned by journal
27 Jul, 2022
Submission checks completed at journal
27 Jul, 2022
First submitted to journal
23 Jul, 2022

You are reading this latest preprint version

Herpud1 deficiency alleviates homocysteine-induced aortic valve calcification

Status:

Journal Publication

Version 1

Abstract

Figures

Highlights

Introduction

Methods

Results

Discussion

Conclusion

Perspectives

Abbreviations

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1