ALYREF inhibits ferroptosis in vascular endothelial cells and improves atherosclerosis by epigenetic modification of CISD1

Abstract

Background

Atherosclerosis (AS) is a disease marked by lipid metabolism dysfunction. Methylation of 5-methylcytosine (m⁵C) mRNA can regulate AS progression. We investigated the role and mechanism of m⁵C reader ALYREF in AS.

Methods

ApoE^−/− mice AS models were constructed. Oil Red O staining evaluated the degree of aortic plaque. Serum LDL and TG levels were assessed. ELISA detected vascular inflammation and ATP production. Expressions of ALYREF, CISD1, and ferroptosis-related proteins were detected by RT-qPCR and Western blot. CCK-8, EdU, and flow cytometry were used to detect cell viability and apoptosis. RIP assay validated the direct binding of ALYREF to CISD1. Mitochondrial morphology was observed by transmission electron microscopy (TEM). Mitochondrial membrane potential was determined by JC-1. Mitochondrial ROS and cytoplasmic ROS were tested by immunofluorescence staining. Oxidative stress damage (MDA), antioxidant enzymes (SOD/GSH), and Fe²⁺ levels were detected by kits. Methylated RNA was immunoprecipitated with m⁵C-specific antibody (MeRIP).

Results

ALYREF expression declined in AS mice and human primary coronary artery endothelial cells (HCAEC) induced by oxidized low-density lipoprotein (ox-LDL). Elevated ALYREF improved ox-LDL-induced HCAEC apoptosis, inflammation, and lipid metabolism. ALYREF elevation attenuated mitochondrial damage and ferroptosis in ox-LDL-exposed HCAEC. ALYREF facilitated the stability and expression of CISD1 mRNA through m⁵C methylation. Reversing CISD1 expression negated the protective effects of ALYREF overexpression against ox-LDL-induced HCAEC damage. ALYREF-mediated epigenetic modification of CISD1 alleviated AS progression by reducing lipid levels and inhibiting ferroptosis in vivo.

Conclusion

By enhancing m⁵C modification, ALYREF enhances the stability and expression of CISD1 mRNA, which impedes lipid metabolism and endothelial cell ferroptosis in AS and alleviates AS-associated pathological changes.

Graphical abstract

ALYREF promotes CISD1 mRNA stability by enhancing its m⁵C modification, which increases its expression, thus inhibiting lipid metabolism dysfunction and endothelial cell ferroptosis in AS and improving AS-related pathological alterations.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13148-025-01955-4.

Introduction

Atherosclerosis (AS), a chronic inflammatory and metabolic disorder affecting blood vessels, leads to heart attacks, ischemic strokes, and peripheral artery diseases [1]. AS and its associated clinical outcomes, such as aneurysms and heart attacks, persist as the primary global cause of death [2]. AS is marked by the accumulation of excessive lipids within the arterial intima, closely linked to chronic inflammatory reaction [3]. In AS, an overabundance of reactive oxygen species (ROS) contributes to vascular endothelial cell (VEC) dysfunction and the production of inflammatory molecules [4]. Mitochondrial abnormalities also play a role in the progression of AS. The transformation of ADP into ATP disrupts membrane potential, leading to decreased ATP synthesis and increased generation of ROS, thus promoting the advancement of AS [5]. Recent studies have uncovered novel cellular and molecular mechanisms associated with the progression of AS, including ferroptosis, pyroptosis, epigenetic alterations, and regulation of ubiquitination [6]. Nevertheless, to date, the precise molecular and cellular mechanisms influencing the onset and progression of AS remain elusive.

Methylation of mRNAs plays a crucial regulatory role in numerous cellular processes by controlling RNA stability, subcellular localization, and protein translation efficiency [7]. RNA methylation levels are governed by writers, readers, and erasers in the regulatory process [8]. 5-methylcytosine (m⁵C) stands out as a prevalent RNA alteration within eukaryotic cells, extensively present in RNAs participating in diverse biological functions [9]. Studies have shown that m⁵C alterations play a significant role in cardiac conditions, encompassing cardiomyopathy, heart failure, and AS [10]. An evident dysregulation of m⁵C methylation is observed in acute myocardial infarction, with various m⁵C marker gene clusters serving as predictors for the disease status in animal models and patients [11]. In addition, m⁵C methyltransferase NSUN2 can diminish the mRNA stability of the antioxidant factor Nrf2 by facilitating m⁵C methylation, enhancing its structure, increasing its expression levels, thereby providing antioxidant effects and minimizing tissue damage in myocardial injury [12]. As one of the m⁵C readers, Aly/REF export factor (ALYREF) has been found to stabilize target mRNA and up-regulate its expression in various cancers [13, 14]. Additionally, ALYREF can affect adipogenesis by regulating m⁵C levels in adipocytes [15]. Nevertheless, the role of ALYREF and its m⁵C methylation in AS has not been studied.

Ferroptosis, a form of cell death regulated by iron dependency, arises from the accumulation of lipid-associated ROS [16]. Within AS, the initiation of ferroptosis pathway in endothelial cells markedly accelerates diverse cellular alterations, including lipid accumulation, inflammatory reactions, and oxidative stress in AS. Conversely, suppressing endothelial ferroptosis can ameliorate these aforementioned transformations [17]. CDGSH iron sulfur domain-containing protein 1 (CISD1), a protein localized on the outer membrane of mitochondria, plays a crucial role in controlling cell respiration and ferroptosis [18]. In the context of hypoxic–ischemic encephalopathy and liver damage, CISD1 can contribute to anti-inflammatory effects, the mitigation of oxidative stress, and the prevention of cell death [19, 20]. In our previous study, we found that in AS endothelial cells, CISD1 can mitigate lipid accumulation, inflammatory responses, and oxidative reactions while enhancing mitochondrial function [21]. Thus, CISD1 may ameliorate disease progression by suppressing ferroptosis in AS endothelial cells. In addition, we found that the m⁵C methylation process of CISD1 can be influenced by ALYREF through the RM2Target bioinformatics analysis database. This implies that the m⁵C modification of CISD1 may impact endothelial cell ferroptosis through ALYREF.

Based on the previous research of our group, we propose that ALYREF enhances the mRNA stability of CISD1 and up-regulates its expression through m⁵C modification, thereby inhibiting endothelial cell ferroptosis and alleviating AS pathological changes. Therefore, we try to provide potential therapeutic targets in AS treatment.

Materials and methods

1. Cell culture

Human primary coronary artery endothelial cells (HCAEC) were purchased from ScienCell (Carlsbad, CA, USA). Cells were incubated in endothelial cell medium (ECM) containing 5% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and 1% endothelial cell growth supplement (ECGS, ScienCell) at 37 °C in an atmosphere of 5% CO₂ and 95% humidity. HCAEC were induced by 50 μg/mL ox-LDL (Sigma, St. Louis, MO, USA) for 24 h to establish an in vitro model of AS.

• 2.Animals and treatments

As described previously [21, 22], 8-week-old male ApoE^−/− mice on a C57BL/6 background (weighing 18–22 g) and C57BL/6 mice (WT group) were acquired from Jiangsu ALF Biotechnology Co., Ltd. and housed in a specific pathogen-free environment. ApoE^−/− mice were randomly divided into five groups: WT, ApoE^−/−, ApoE^−/−  + NC, ApoE^−/−  + oe-ALYREF, ApoE^−/−  + oe-ALYREF + sh-CISD1 (n = 8 per group). ApoE^−/− mice were fed with high fat diet (0.15% cholesterol + 25% fat; Fuzhou Junke Biological Technology Co., Ltd.) for 12 weeks and simultaneously injected with lentivirus (LV)-NC, LV-oe-ALYREF or LV-sh-CISD1 (2 × 10⁹ TU/ mL) through tail vein once every 3 weeks. C57BL/6 mice in the control group were given standard diet and administered with 0.5% CMC-Na via gavage. At the end of the experiment, euthanasia was performed on the mice through cervical dislocation. Subsequently, the whole aorta, aortic root, and blood samples were obtained for additional investigations. The animal trials received approval from Institutional Animal Care and Use Committee of The First Affiliated Hospital, Jiangxi Medical College, Nanchang University (Approval number: 202510032).

• 3.Immunofluorescence staining

Cells and frozen tissue sections were initially fixed in 4% paraformaldehyde and then subjected to permeabilization with 0.2% Triton X-100 in PBS. After blocking with 5% BSA (Invitrogen) at room temperature for 30 min, the samples underwent incubation with anti-ALYREF antibodies (PA5-96489, 1:1000, Invitrogen) overnight at 4 °C and then exposed to fluorescent secondary antibody under dark conditions for 1 h. Subsequent to washing, nuclear staining with DAPI was conducted, and images were captured utilizing a fluorescence microscope (Olympus, Tokyo, Japan). For immunofluorescence co-localization analysis, the blocked slice samples were incubated with primary antibodies against ALYREF (PA5-96489, 1:1000, Invitrogen) and PECAM-1 (14-0311-82, 1:1000, Invitrogen) at 4 °C overnight and then incubated with appropriate fluorescent secondary antibodies for 1 h. The nuclei were stained with DAPI. Images were captured with a fluorescence microscope (Olympus).

• 4.RNA m⁵C dot blotting

Following treatment with RNase-free DNase set (Qiagen, Hilden, Germany), the isolated mRNA was employed for m⁵C dot blot analysis. The mRNA concentration was determined by NanoDrop Nd-1000 spectrophotometer (Agilent, Santa Clara, CA, USA). The mRNA underwent denaturation through heating at 65 °C for 5 min. Subsequently, a Bio-Dot apparatus (Bio-Rad, Hercules, CA, USA) was employed to transfer mRNA onto a nitrocellulose membrane (Amersham, GE Healthcare, Pittsburgh, PA, USA). Following ultraviolet cross-linking and milk blocking, the membrane was subjected to overnight incubation at 4 °C with anti-m⁵C antibody (1:1000, ab10805, Abcam, Cambridge, MA, USA). Subsequently, the membrane was treated with anti-mouse antibody (1:3000, GB23301, Servicebio, Wuhan, China). The visualization of membrane was achieved using chemiluminescence system from Bio-Rad. To ensure consistency, a membrane stained with 0.02% methylene blue in 0.3 mol/L sodium acetate (pH 5.2) served as a loading control.

Methylated RNA immunoprecipitation (MeRIP) assay

MeRIP assay was conducted using the m5C MeRIP kit (BersinBio, Guangzhou, China) in adherence to the provided guidelines. In summary, RNA was extracted from 2 × 10⁷ cells and fragmented into 200 nt units randomly. An “Input” fraction representing 1/10 of fragmented RNA sample was retained. Following a 4-h incubation at 4 °C with either 5 μg of m5C antibody or 5ug of IgG antibody, the immunoprecipitation complex was subsequently exposed to protein A/G magnetic beads at 4 °C for 1 h. Subsequently, the magnetic beads underwent a triple wash before being exposed to Proteinase K buffer at 55 °C for 30 min. The RNA was then purified utilizing RNA extraction reagent from ACMEC (Shanghai, China) and subjected to RT-qPCR analysis, normalized against the Input sample.

• 5.Cell transfection

CISD1 shRNA and negative control shRNA were purchased from Shhebio (Shanghai, China). Overexpression of ALYREF (oe-ALYREF) and the control (oe-NC) were synthesized by RIBOBIO (Guangzhou, China). Transfection of plasmid was carried out for 48 h using Lipofectamine™ 2000 (Invitrogen) in accordance with the manufacturer’s guidelines.

• 6.Cell counting kit-8 (CCK-8) and 5-Ethynyl-2′-deoxyuridine (EdU) assays

For CCK-8 assay, in each well of a 96-well plate, two thousand cells were plated with five replicates. Culture medium was substituted with fresh complete medium supplemented with CCK-8 (1:9, Beyotime, Shanghai, China). Following a 2-h incubation at 37 °C, the absorbance at 450 nm was measured utilizing a microplate reader (Bio-Rad). For EdU assay, cells were exposed to 50 mM EdU (Beyotime) for 2 h at 37 °C, followed by PBS rinsing and fixation in 4% paraformaldehyde for 30 min. Subsequently washed with PBS, the cells underwent incubation with Hoechst staining solution at room temperature for another 30 min. The proportion of EdU-positive cells was determined using a fluorescence microscope (Olympus).

• 7.Flow cytometry for cell apoptosis

Upon adhering to specific conditions of each group, cell trypsinization was conducted, followed by collection and double washing with pre-chilled PBS at 4 °C. Subsequent to centrifugation and removal of the PBS supernatant, the cells were reconstituted in buffer. Next, 200 μL Annexin V-FITC mixture (Vazyme Biotech Co., Ltd., Nanjing, China) was added to 1 × 10⁵ cells suspension. Subsequently, 10 μL of propidium iodide (PI) staining solution (Vazyme Biotech Co., Ltd.) was added and the mixture was incubated for 20 min. Cell apoptosis was assessed using flow cytometry (BD Biosciences, San Diego, CA, USA) and further analyzed with Flowjo software (Tree Star, San Carlos, CA, USA).

• 8.Enzyme-linked immunosorbent assay (ELISA)

Blood from mice or cell culture of HCAEC was centrifuged at 2000 rpm for 5 min in a 4 °C centrifuge. The supernatant was collected. The levels of inflammatory cytokines TNF-α, IL-6, IL-8, and IL-1β were evaluated by ELISA kits (R&D Systems, Abingdon, UK) following respective protocols. Measurement of absorbance at 450 nm was conducted using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

• 9.Transmission electron microscopy (TEM) for mitochondrial ultrastructure

After centrifugation of cells at 3000 rpm/min for 20 min, the supernatant was removed. Subsequently, the cells underwent post-fixation in cacodylate-buffered 1% osmium tetroxide, dehydration in an ethanol series, and embedding in Poly/Bed 812. Mitochondrial morphology was observed using TEM (H-600, Hitachi, Japan).

• 10.Adenosine triphosphate (ATP) detection

We assessed intracellular ATP levels using an ATP assay kit (Jiancheng Corporation, Nanjing, Jiangsu, China) based on the manufacturer’s instruction. Briefly, 1 × 10⁶ cells were homogenized in 100 mL of designated assay buffer from the kit, followed by centrifugation and analysis of soluble fraction.

• 11.Mitochondrial membrane potential (MMP)

In 24-well plates, sterilized coverslips were positioned. Subsequently, the transfected HCAEC were seeded onto the coverslips. After a 2-h exposure to ox-LDL, MMP assessment was conducted utilizing the JC-1 assay. Initially, the cells underwent a cold PBS wash before being treated with JC-1 solution (Beyotime) in the dark at 37 °C for 20 min. Following this, the cells were rinsed with JC-1 staining buffer and provided with fresh culture medium. An Olympus fluorescence microscope was employed to examine the monomer (green) and aggregate (red) fluorescence. Cell pictures were captured with a confocal fluorescence microscope from Olympus. The fluorescence intensity was evaluated through ImageJ software (National Institutes of Health, NIH, Bethesda, MD, USA).

• 12.Detection of mitochondrial and cytoplasmic reactive oxygen species (ROS)

In HCAEC cultured on six-well plates, the levels of ROS were quantified. To assess the ROS levels, cells were first rinsed with PBS and subsequently exposed to mitochondrial ROS marker MitoSOX™ Red (2 μmol/L, Invitrogen) and cytoplasmic ROS indicator CM-H2DCFDA (Invitrogen) at 37 °C for 15 min. Subsequently, PBS was used to wash the cells before mounting them with DAPI. Images were captured using an Olympus fluorescence microscope and then processed with ImageJ software (NIH). Following sacrifice, mouse aortic root was swiftly extracted for embedding and quick freezing to prepare frozen sections. Subsequently, the aortic root tissue was sliced into 5-μm sections, followed by dewaxing and rehydration. These rehydrated sections were then treated with dihydroethidium (DHE, 3 μmol/L, Invitrogen) or MitoSOX™ Red (2 μmol/L, Invitrogen) at 37 °C for 15 min. Finally, the sections were dehydrated, sealed, and subjected to fluorescence imaging using an Olympus fluorescence microscope. Signal intensity was quantified with ImageJ software (NIH).

• 13.Malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), and Fe²⁺ measurement

A kit for antioxidant enzyme activity and malondialdehyde was provided by Nanjing Jianjian Bioengineering Institute (Nanjing, China) following instructions from the manufacturer. After sonication of cell lysate, centrifugation at 3000×g for 10 min was performed on cells, and the resultant supernatant was used to determine the concentrations of MDA, GSH, and SOD. In a 24-well plate, the cells were plated at a density of 2 × 10³ cells per well for iron content analysis. Levels of iron in blank, iron standard solution, and cell lysates were assessed using Intracellular Iron Colorimetric Assay Kit (Applygen Technologies Inc., Beijing, China) following the manufacturer’s guidelines. Following a 30-min incubation at room temperature, reaction mixture was assessed for absorbance at 550 nm using a microplate reader (Thermo Fisher Scientific). Furthermore, mouse aortic root tissue was lysed with RIPA lysis buffer (Beyotime) under cold conditions. After centrifugation at 12,000×g and 4 °C for 15 min, the ensuing supernatant was utilized to measure the concentrations of SOD, MDA, GSH, and Fe²⁺ as described above.

• 14.Real-time quantitative PCR (RT-qPCR)

Tissues and cells underwent total RNA extraction utilizing TRIzol® reagent (Invitrogen), followed by reverse transcription into 1 μl of cDNA (60 ng/μl) employing a One_Step RT-PCR kit (TransGen Biotech Co., Ltd., Beijing, China). Real-time PCR was performed by FastStart Universal SYBR Green Master (Takara, Kyoto, Japan) utilizing an Applied Biosystems 7500 machine. The following primers are used: human ALYREF F: 5′-GCAGGCCAAAACAACTTCCC-3′, R: 5′-AGTTCCTGAATATCGGCGTCT-3′; LOX-1 F: 5′-TTGCCTGGGATTAGTAGTGACC-3′, R: 5′-GCTTGCTCTTGTGTTAGGAGGT-3′; LOX-1 F: 5′-TGGGTGGCCAGTTACTACAA-3′, R: 5′-CAAGGCCAACATGCTTTACA-3′; human ABCA1 F: 5′-ACCCACCCTAT GAACAACATGA-3′, R: 5′-GAGTCGGGTAACGGAAACAGG-3′; mouse ABCA1 F: 5′-GGGTGGTGTTCTTCCTCATTAC-3′, R: 5′-CACATCCTCATCCTCGTCATTC-3′; human ABCG1 F: 5′-ATTCAGGG ACCTTTCCTATTCGG-3′, R: 5′-CTCACCACTATTGAACTTCCCG-3′; mouse ABCG1 F: 5′-CTTTCCTACTCTGTACCCGAGG-3′, R: 5′-CGGGGCATTCCATTGATAAGG-3′; human CISD1 F: 5′-CTGACTTCCAGTTCCAGCGT-3′, R: 5′-TGATCAGAGGGCCCACATTG-3′; human ACSL4 F: 5′-AGGTGCTCCAACTCTGCCAGTA-3′, R: 5′-GCTATCTCCTCAGACACACCGA-3′; mouse ACSL4 F: 5′-CCGACCTAAGGGAGTGATGA-3′, R: 5′-CCTGCAGCCATAGGTAAAGC-3′; human GPX4 F: 5′-AGTGGATGAAGATCCAACCCAAGG-3′, R: 5′-GGGCCACACACTTGTGGAGCTAGA-3′; mouse GPX4 F: 5′-GTAACCAGTTCGGGAAGCAG-3′, R: 5′-TGTCGATGAGGAACTGTGGA-3′; human SLC7A11 F: 5′-GGTCCATTACCAGCTTTTGTACG-3′, R: 5′-AATGTAGCGTCCAAATGCCAG-3′; mouse SLC7A11 F: 5′-TTGTTTTGCACCCTTTGACA-3′, R: 5′-AAAGCTGGGATGAACAGTGG-3′. Relative mRNA expression levels were normalized to that of GAPDH using 2^−∆∆Ct method.

• 15.Western blot

Proteins were isolated from tissues and cells with RIPA buffer (Beyotime). The protein concentration was determined with BCA kit (Beyotime). The protein samples were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Next, the protein was transferred onto PVDF membranes (Millipore, Bedford, MA, USA). Following blocking by 5% skim milk for 1 h, the protein was incubated overnight at 4 °C with antibodies: ALYREF (PA5-96489, 1:1000, Invitrogen), LOX-1 (PA5-102452, 1:1000, Invitrogen), ABCA1 (PA1-32129, 1:1000, Invitrogen), ABCG1 (PA5-56757, 1:1000, Invitrogen), CISD1 (PA5-106281, 1:1000, Invitrogen), ACSL4 (PA5-27137, 1:1000, Invitrogen), GPX4 (MA5-32827, 1:1000, Invitrogen), SLC7A11 (711589, 1:1000, Invitrogen). Subsequently, the membranes underwent incubation with HRP-conjugated secondary antibodies (#7074, 1:1000, Cell Signaling Technology, Danvers, MA, USA) for 1 h. Detection of signals was carried out with an ECL kit (Bio-Rad). Band intensity was quantified using ImageJ (NIH), and protein expression levels were standardized against GAPDH.

• 16.RNA immunoprecipitation (RIP)

RIP assay was conducted using the Magna RIP Kit (Millipore) in accordance with the provided guidelines. HCAEC were lysed in the complete RIP lysis buffer, and cell extract was subjected to incubation with magnetic beads linked to anti-ALYREF (PA5-96489, Invitrogen) or the control anti-IgG antibody (MA5-42729, Invitrogen) overnight at 4 °C. Following protein removal by incubating beads with proteinase K, RNA was purified utilizing RNA extraction reagent (ACMEC, Shanghai, China). Subsequently, the purified RNA underwent RT-qPCR analysis.

• 17.RNA stability assay

To test RNA stability, the transfected cells were exposed to Actinomycin D (ActD, Sigma) at a concentration of 5 μg/mL. They were collected at 0, 2, 4, and 6 h. Total RNA was extracted, and the expression level of CISD1 mRNA was analyzed using RT-qPCR.

• 18.Oil red O staining

The mouse aortic tissues were fixed in 4% formalin and immersed in oil red O (Sigma) solution for 30 min, and subsequently rinsed with water. Images were then recorded with a camera. Frozen sections air-dried at room temperature were immersed in oil red O for 15 min, followed by a 2-min rinse with tap water. These sections were then stained with hematoxylin solution to highlight the nuclei for 2 min and sealed using glycerin gelatin. Subsequently, the images were captured using a microscope (Olympus).

• 19.Double immunofluorescence staining

Aortic root sections were incubated overnight at 4 °C with primary antibodies against CD31 (MA1-40074, Invitrogen), CISD1 (PA5-87804, Invitrogen). The next day, slices were washed three times with PBS and incubated with corresponding secondary antibodies (Alexa Fluor 488 and Alexa Fluor 594, Invitrogen) for 1 h in the dark. The nuclei were stained with DAPI. Sections were examined under an Olympus inverted microscope.

• 20.FerroOrange staining

Fe²⁺ levels were evaluated using FerroOrange fluorescent probe (Dojindo, Kumamoto, Japan). Briefly, tissue sections and cells underwent fixation and permeabilization utilizing 4% paraformaldehyde with 0.1% Triton X-100 for 10 min at 4 °C. A working solution of 1 μmol/l FerroOrange fluorescent probe, prepared in serum-free medium, was then added to the samples. Next, the samples were incubated at 37 °C in a dark environment for 30 min. The nuclei were stained with DAPI. Finally, images were acquired using a fluorescence microscope (Olympus).

• 21.Biochemical detection

Serum levels of low-density lipoprotein cholesterol (LDL) and triglycerides (TG) were determined utilizing commercial kits purchased from Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions. Lipid levels were assessed using a plate reader (Bio-Rad) to detect the absorbance at 500 nm.

• 22.Statistical analysis

Statistical analysis was conducted with SPSS 26.0 (IBM, SPSS, Chicago, IL, USA). Data are expressed as mean ± standard deviation (SD). Experiments were independently repeated at least three times. Student t-test was used to compare differences between two groups. Differences between multiple groups were compared utilizing one-way ANOVA. P value less than 0.05 was considered statistically significant.

Results

1. Expression of ALYREF was down-regulated in AS in vivo and in vitro

ApoE^−/− mouse model is a widely accepted AS animal model [23]. We first investigated the expression of ALYREF in ApoE^−/− mouse model. Compared with WT mice, m⁵C level was significantly decreased in aortic tissue of ApoE^−/− mice (Fig. 1A). In addition, Western blot analysis demonstrated that ALYREF protein expression was decreased in aortic tissue of ApoE^−/− mice compared to WT mice (Fig. 1B). Immunofluorescence staining of aortic tissue showed that compared with WT mice, ApoE^−/− mice displayed decreased expression of ALYREF (Fig. 1C). Furthermore, the co-localization of endothelial marker (PECAM-1) and ALYREF in aortic tissue was verified by immunofluorescence staining. Notably, compared with WT group, the fluorescence intensity of PECAM-1 and ALYREF in ApoE^−/− group was both significantly weakened (Fig. 1D). Next, we explored the levels of m⁵C-related genes, including NSUN1-7, DNMT1-2, DNMT3a, DNMT3b, ALYREF, and TET2, in aortic tissue of WT and ApoE^−/− mice. The results of RT-qPCR showed that compared with WT mice, ALYREF level was significantly decreased in aortic tissue of ApoE^−/− mice (Fig. 1E). Subsequently, HCAEC were induced by ox-LDL to establish an in vitro AS model. Similarly, there was a great decrease in ALYREF expression in HCAEC after ox-LDL treatment (Fig. 1F). In addition, compared with control cells, m⁵C level and ALYREF expression were significantly decreased in ox-LDL-treated HCAEC (Additional file 1: Supplementary Fig. 1A–D). After transfection with oe-ALYREF plasmid, expression of ALYREF in HCAEC was significantly increased (Additional file 1: Supplementary Fig. 1E and F). Taken together, these data indicated that ALYREF expression was reduced in AS mouse model and ox-LDL-induced vascular endothelial cells.

• 2.Effects of ALYREF overexpression on cell survival, inflammatory response, and lipid metabolism in ox-LDL-induced HCAEC

Fig. 1.

Expression of ALYREF was down-regulated in AS in vivo and in vitro. ApoE^−/− mice were fed a high fat diet (0.15% cholesterol + 25% fat) for 12 weeks. C57BL/6 mice in WT group were given standard feed. A The m5C level in aortic tissue of WT and ApoE^−/− mice was detected. n = 8 mice/group. B Western blot analysis of ALYREF in WT and ApoE^−/− mice. n = 4 mice/group. C Representative immunofluorescence images and quantification of ALYREF expression in aortic tissue of WT and ApoE^−/− mice. n = 8 mice/group. D Immunofluorescence staining on co-localization of endothelial marker (PECAM-1) and ALYREF in aortic tissue of WT and ApoE^−/− mice. Next, RT-qPCR was used to assess levels of m5C-related genes, including NSUN1-7, DNMT1-2, DNMT3a, DNMT3b, ALYREF, and TET2, in aortic tissue of WT and ApoE^−/− mice (E), or in control and ox-LDL-treated HCAEC (F). n = 8 mice/group. Data are the means ± SD for three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Subsequently, we investigated the effects of ALYREF on ox-LDL-regulated HCAEC survival, inflammatory response, and lipid metabolism. HCAEC were transfected with oe-NC or oe-ALYREF, followed by ox-LDL treatment. CCK-8 and EdU assays demonstrated that ox-LDL treatment inhibited cell proliferation, while ALYREF up-regulation significantly increased cell proliferation (Fig. 2A and B). Furthermore, apoptosis was enhanced in ox-LDL-induced cells, and ALYREF overexpression antagonized this effect (Fig. 2C). Next, the levels of proinflammatory cytokines (IL-1β, IL-6, TNF-α, and IL-8) were significantly up-regulated in ox-LDL-stimulated cells, which were inhibited by overexpressed ALYREF (Fig. 2D). Additionally, ox-LDL treatment dramatically decreased the expression of ALYREF while increased the expression of lipid metabolism markers (LOX-1 [24], ABCA1 [25], and ABCG1 [26]); however, transfection of oe-ALYREF abolished the dysregulation of these molecules (Fig. 2E–G). The data above indicated that overexpressed ALYREF improved ox-LDL-triggered HCAEC apoptosis, inflammatory response, and lipid metabolism.

• 3.Effects of ALYREF overexpression on mitochondrial dysfunction and ferroptosis in ox-LDL-induced HCAEC

Fig. 2.

Effects of ALYREF overexpression on cell survival, inflammatory response, and lipid metabolism in ox-LDL-induced HCAEC. HCAEC were transfected with oe-NC or oe-ALYREF, followed by 50 μg/mL ox-LDL treatment for 24 h. A CCK-8 assay was used to evaluate cell viability. B Cell proliferation was determined by EdU assay. C Apoptosis was detected by flow cytometry. D ELISA assay for proinflammatory cytokines: IL-1β, IL-6, TNF-α, and IL-8. E Levels of lipid metabolism markers (LOX-1, ABCA1, and ABCG1) were detected by RT-qPCR. F ALYREF expression was detected by RT-qPCR. G Western blot was adopted to detect protein levels of ALYREF and lipid metabolism markers. Error bars represent mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Next, the effects of ALYREF on ox-LDL-induced HCAEC mitochondrial dysfunction and ferroptosis were explored. HCAEC were transfected with oe-NC or oe-ALYREF, followed by ox-LDL treatment. TEM revealed that in ox-LDL-induced HCAEC, mitochondria were discontinuous and shrunk, while overexpression of ALYREF alleviated this damage (Fig. 3A). ATP production was inhibited in HCAEC by ox-LDL stimulation, and reinforced ALYREF relieved this inhibitory effect (Fig. 3B). JC-1 staining showed that compared to the control group, the mitochondrial membrane potential (MMP) was lower in ox-LDL-treated cells, and ALYREF up-regulation increased MMP level (Fig. 3C). Additionally, MitoSOX red was used to detect mitochondrial ROS levels in HCAEC. The MitoSOX fluorescence intensity induced by ox-LDL stimulation was down-regulated by ALYREF overexpression (Fig. 3D). Ox-LDL treatment significantly inhibited the expression of antioxidant enzymes (SOD/GSH) while increasing the levels of oxidative stress damage (MDA). However, overexpression of ALYREF offset these effects (Fig. 3E). Additionally, FerroOrange staining showed that ox-LDL increased Fe²⁺ levels in HCAEC, whereas reinforced ALYREF overturned this effect (Fig. 3F). Moreover, ox-LDL reduced ferroptosis markers GPX4 and SLC7A11 but promoted Fe²⁺ and ACSL4, while overexpressing ALYREF had the opposite impact (Fig. 3G–I). Taken together, these findings illustrated that ALYREF up-regulation alleviated mitochondrial damage and ferroptosis in ox-LDL-triggered HCAEC.

• 4.ALYREF regulated the stability and expression of CISD1 mRNA through m5C methylation

Fig. 3.

Effects of ALYREF overexpression on mitochondrial dysfunction and ferroptosis in ox-LDL-induced HCAEC. HCAEC were transfected with oe-NC or oe-ALYREF, followed by 50 μg/mL ox-LDL treatment for 24 h. A Mitochondrial morphology was observed by TEM. B ATP production was evaluated using ELISA. C JC-1 was used to detect mitochondrial membrane potential. D MitoSOX red was used to detect mitochondrial ROS levels. E Levels of markers of oxidative stress damage (MDA) and antioxidant enzymes (SOD/GSH). F The FerroOrange fluorescent probe was used to detect Fe²⁺ levels in HCAEC. G The content of Fe²⁺ in different groups. H, I Detection of ferroptosis marker genes by RT-qPCR and Western blot. Values were expressed as mean ± SD of three separate determinations. *P < 0.05, **P < 0.01, ***P < 0.001

We next investigated the molecular mechanism by which ALYREF regulates AS progression. HCAEC were transfected with oe-NC or oe-ALYREF, followed by ox-LDL treatment. The m⁵C level was decreased by ox-LDL treatment, and ALYREF overexpression rescued this downward trend (Fig. 4A). Bioinformatics analysis using the online tool RM2Target predicted that ALYREF may mediate the m⁵C methylation of CISD1 (Fig. 4B). We further found that ox-LDL induction markedly reduced CISD1 expression in HCAEC, and the reduced trends were partially improved by ALYREF up-regulation (Fig. 4C and D). Subsequently, RIP assay verified the enrichment of CISD1 mRNA in ALYREF immunoprecipitates relative to IgG in HCAEC (Fig. 4E). Subsequently, MeRIP uncovered that ALYREF overexpression led to a dramatic increase in the enrichment of m⁵C-modified CISD1 mRNA (Fig. 4F). Compared with the control group, the stability of CISD1 mRNA was enhanced and the half-life was prolonged after ALYREF overexpression (Fig. 4G). Taken together, these results showed that ALYREF positively regulated CISD1 mRNA stability and expression and affected its mRNA stability by m⁵C-dependent manner.

• 5.Effects of CISD1 knockdown on ALYREF overexpression-mediated protective effects in ox-LDL-induced HCAEC (cell survival, inflammatory response, and lipid metabolism)

Fig. 4.

ALYREF regulated the stability and expression of CISD1 mRNA through m⁵C methylation. HCAEC were transfected with oe-NC or oe-ALYREF, followed by 50 μg/mL ox-LDL treatment for 24 h. A The m⁵C level in HCAEC was detected. B Bioinformatics analysis using the online tool RM2Target predicted that ALYREF may mediate the m⁵C methylation of CISD1. C, D The effects of oe-ALYREF on CISD1 expression detected by RT-qPCR and Western blot. E RIP assay was used to validate the direct binding between ALYREF and CISD1. F Methylated RNA was immunoprecipitated by a m⁵C-specific antibody (MeRIP), and then, qPCR detected the level of m⁵C-modified CISD1 mRNA. G HCAEC with oe-NC or oe-ALYREF were treated with 5 μg/mL actinomycin D for 0, 2, 4, 6 h, respectively. RT-qPCR was used to detect the stability of CISD1. Each bar represents the mean ± SD calculated from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

We further assessed whether ALYREF could impact ox-LDL-regulated HCAEC survival, inflammatory response, and lipid metabolism through CISD1. Except for the normal control group, HCAEC were co-transfected with oe-ALYREF and sh-CISD1/sh-NC and stimulated by ox-LDL treatment. We found that cell proliferation was inhibited by CISD1 knockdown in ox-LDL-induced HCAEC with ALYREF overexpression (Fig. 5A and B). The apoptosis rate was increased by CISD1 depletion in ox-LDL-triggered HCAEC with ALYREF up-regulation (Fig. 5C). In ox-LDL-treated HCAEC with oe-ALYREF, the levels of proinflammatory cytokines were higher with sh-CISD1 than that in sh-NC group (Fig. 5D). Furthermore, CISD1 silencing decreased CISD1 expression, but increased levels of lipid metabolism markers in ox-LDL-stimulated HCAEC with overexpressed ALYREF (Fig. 5E–G). Taken together, these findings revealed that ALYREF suppressed ox-LDL-induced HCAEC apoptosis, inflammatory response, and lipid metabolism via up-regulating CISD1.

• 6.Effects of CISD1 knockdown on ALYREF overexpression-mediated protective effects in ox-LDL-induced HCAEC (mitochondrial dysfunction and ferroptosis)

Fig. 5.

Effects of CISD1 knockdown on ALYREF overexpression-mediated protective effects in ox-LDL-induced HCAEC (cell survival, inflammatory response, and lipid metabolism). Except for the normal control group, HCAEC were co-transfected with oe-ALYREF and sh-CISD1/sh-NC and stimulated by 50 μg/mL ox-LDL treatment for 24 h. A CCK-8 assay was used to evaluate cell viability. B Cell proliferation was determined by EdU assay. C Apoptosis was detected by flow cytometry. D ELISA assay for proinflammatory cytokines: IL-1β, IL-6, TNF-α, and IL-8. E Levels of lipid metabolism markers (LOX-1, ABCA1, and ABCG1) were detected by RT-qPCR. F CISD1 expression was detected by RT-qPCR. G Western blot was used to detect protein levels of CISD1 and lipid metabolism markers. Error bars represent mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

To evaluate whether ALYREF could impact ox-LDL-triggered HCAEC mitochondrial dysfunction and ferroptosis via CISD1, HCAEC were transfected with oe-ALYREF and sh-CISD1, followed by ox-LDL treatment. TEM discovered CISD1 knockdown reversed ALYREF-mediated mitochondrial protection, characterized by mitochondrial discontinuity and shrinkage (Fig. 6A). CISD1 silencing reduced ATP levels in ox-LDL-induced HCAEC with ALYREF overexpression (Fig. 6B). Depletion of CISD1 dramatically decreased MMP in ALYREF overexpressed cells under ox-LDL exposure (Fig. 6C). Furthermore, mitochondrial ROS levels were elevated by CISD1 down-regulation in ox-LDL-triggered HCAEC with ALYREF up-regulation (Fig. 6D). In ox-LDL-treated HCAEC with oe-ALYREF, CISD1 repression significantly decreased SOD and GSH levels while increasing MDA levels (Fig. 6E). FerroOrange staining showed that CISD1 knockdown increased Fe²⁺ levels in ox-LDL-exposed HCAEC with ALYREF up-regulation (Fig. 6F). Additionally, loss of CISD1 expression reduced GPX4 and SLC7A11 levels, and induced Fe²⁺ and ACSL4 expression in ox-LDL-stimulated HCAEC with overexpressed ALYREF (Fig. 6G–I). These findings manifested that ALYREF inhibited ox-LDL-stimulated HCAEC mitochondrial dysfunction and ferroptosis through CISD1.

• 7.In vivo verification of ALYREF-mediated CISD1 epigenetic modification affected AS progression by regulating ferroptosis

Fig. 6.

Effects of CISD1 knockdown on ALYREF overexpression-mediated protective effects in ox-LDL-induced HCAEC (mitochondrial dysfunction and ferroptosis). HCAEC were transfected with oe-ALYREF and sh-CISD1, followed by 50 μg/mL ox-LDL treatment for 24 h. A Mitochondrial morphology was observed by TEM. B ATP production was evaluated using ELISA. C JC-1 was used to detect mitochondrial membrane potential. D MitoSOX red was used to detect mitochondrial ROS levels. E Levels of MDA, SOD, and GSH. F The FerroOrange fluorescent probe was used to detect Fe²⁺ levels in HCAEC. G The content of Fe²⁺ in different groups. H, I Detection of ferroptosis marker genes by RT-qPCR and Western blot. Values were expressed as mean ± SD of three separate determinations. *P < 0.05, **P < 0.01, ***P < 0.001

We next wanted to determine whether ALYREF regulates ferroptosis and AS progression through modulating CISD1. The mice were subjected to five groups: WT, ApoE^−/−, ApoE^−/− + NC, ApoE^−/− + oe-ALYREF, ApoE^−/− + oe-ALYREF + sh-CISD1. Oil Red O staining demonstrated that compared to WT mice, the atherosclerotic plaque areas in thoracic and abdominal aorta were increased in ApoE^−/− mice, and ALYREF up-regulation decreased the atherosclerotic plaque areas, and CISD1 knockdown further partially abolished the rescue effect of oe-ALYREF (Fig. 7A). Additionally, the size of atherosclerotic plaque in the aortic sinus was also markedly decreased by oe-ALYREF in ApoE^−/− mice, while CISD1 silencing overturned this effect (Fig. 7B). Serum LDL and TG contents were increased in ApoE^−/− mice, and the introduction of oe-ALYREF reduced the level of LDL and TG, which was then countered by sh-CISD1 (Fig. 7C and D). Further, serum proinflammatory cytokines were suppressed by ALYREF overexpression in ApoE^−/− mice, and it was recovered by CISD1 knockdown (Fig. 7E). Additionally, overexpressed ALYREF dramatically decreased the expression of lipid metabolism markers in ApoE^−/− mice, and transfection of sh-CISD1 abolished the dysregulation of these molecules (Fig. 7F). In ApoE^−/− mice, the oe-ALYREF treatment-induced reduction of mito-SOX fluorescence intensity was recovered by the co-transfection of oe-ALYREF + sh-CISD1 (Fig. 7G). Reinforced ALYREF increased SOD and GSH levels while decreasing MDA levels. However, CISD1 depletion reversed these effects (Fig. 7H). Additionally, FerroOrange staining showed that reinforced ALYREF decreased Fe²⁺ levels in ApoE^−/− mice, while CISD1 silencing overturned this effect (Fig. 7I). Finally, overexpression of ALYREF elevated GPX4, SLC7A11, and CISD1 levels but reduced Fe²⁺, ACSL4, and LOX-1 levels in ApoE^−/− mice, whereas CISD1 down-regulation had the opposite impact (Fig. 7J–L). Double immunofluorescence staining revealed that CD31 and CISD1 co-localized in aortic root. Fluorescence of CD31 and CISD1 was lower in ApoE^−/− mice than in WT mice. The promotive effect of reinforced ALYREF on CD31 and CISD1 fluorescence was reversed by CISD1 depletion (Fig. 7M). The above experiments confirmed that ALYREF-mediated CISD1 epigenetic modification alleviated AS progression by reducing lipid levels and inhibiting ferroptosis in AS mice.

Fig. 7.

In vivo verification of ALYREF-mediated CISD1 epigenetic modification affected AS progression by regulating ferroptosis. Mice were subjected to five groups: WT, ApoE^−/−, ApoE^−/− + NC, ApoE^−/− + oe-ALYREF, ApoE^−/− + oe-ALYREF + sh-CISD1. ApoE^−/− mice were fed a high fat diet (0.15% cholesterol + 25% fat) for 12 weeks and simultaneously injected with lentivirus (LV)-NC, LV-oe-ALYREF or LV-sh-CISD1 (2 × 10⁹ TU/ mL) through tail vein once every 3 weeks. C57BL/6 mice in WT group were given standard feed and administered with 0.5% CMC-Na via gavage. A En face Oil Red O staining of aortas and quantification in five groups of mice. B Sections of the aortic root were stained with Oil Red O, and lesion areas were quantified. C, D Serum LDL and TG contents were assessed. E Serum proinflammatory cytokines were evaluated by ELISA. F LOX-1, ABCA1, ABCG1 levels were detected by RT-qPCR in aortic root. G DHE and MitoSOX staining in aortic root sections. Cellular ROS and mitochondrial ROS generation were quantified. H Expression levels of markers of oxidative stress damage (MDA) and antioxidant enzymes (SOD/GSH). I The FerroOrange fluorescent probe was used to detect Fe²⁺ levels in aortic root sections. J The content of Fe²⁺ in different groups. K ACSL4, GPX4, SLC7A11 levels in aortic root were measured by RT-qPCR. L Expressions of CISD1, LOX-1, ACSL4, and GPX4 in aortic root were evaluated by Western blot. M Double immunofluorescence staining (green for CD31 and red for CISD1) demonstrated the expression of CD31 and CISD1 in aortic root sections. Error bars depict mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. n = 8 mice/group

Discussion

AS stands as a primary contributor to morbidity and mortality, with high-risk plaques capable of triggering myocardial infarction, stroke, and sudden death [27]. Disorders in lipid metabolism, endothelial injury, inflammatory responses, and mitochondrial dysfunction collectively contribute to the promotion of AS [28, 29]. Recently, multiple reports have detailed the involvement of ferroptosis in the development and progression of AS [30]. In this study, we found that by enhancing the stability of CISD1 mRNA and increasing its expression via m⁵C modification, ALYREF impedes lipid metabolism and endothelial cell ferroptosis in AS, thus ameliorating AS-related pathological alterations.

RNA methylation modification is a research hotspot in recent years and has been proved to be a key factor in post-transcriptional regulation. Studies have shown that m⁵C methylation is one of the major post-transcriptional modifications of RNA [31]. RNA m⁵C methylation and its regulators are closely associated with various cardiovascular diseases, including AS [9]. It has been shown that m⁵C is specifically recognized by mRNA export adaptor ALYREF [32]. ALYREF, the initial m⁵C reader discovered, primarily resides in the nucleus. It predominantly interacts with the 3′ and 5′ sections of mRNA, playing a role in mRNA export, stabilization, and splicing [33]. The dysregulation of ALYREF has been noted in several cancer types. Elevated ALYREF levels enhance bladder cancer cell growth through PKM2-mediated glycolysis [34]. Moreover, ALYREF boosts the proliferation and invasion of urothelial bladder cancer cells in a m⁵C-related manner [14]. Nevertheless, the functional implications of ALYREF in AS remain largely unexplored. Here, we found that the expression of ALYREF was down-regulated in ApoE^−/− mouse model, and overexpression of ALYREF could up-regulate the total m⁵C level in ox-LDL-induced endothelial cell model. We further showed that elevated ALYREF expression ameliorated ox-LDL-induced HCAEC apoptosis, inflammatory reactions, lipid metabolism, and mitochondrial impairment, indicating that ALYREF plays a protective role in AS progression.

Ferroptosis, a recently identified form of programmed cell death, significantly contributes to AS progression [35]. For example, mitigating ferroptosis effectively alleviates AS by reducing lipid peroxidation and endothelial dysfunction in mouse aortic endothelial cells [36]. Ferroptosis is also a key molecular mechanism underlying the effects of m⁵C RNA methylation. NSUN2 up-regulation through epigenetic mechanisms provides resistance to ferroptosis in endometrial cancer by m⁵C modifying SLC7A11 mRNA [37]. The Niujiao Dihuang Jiedu decoction enhances m⁵C methylation modification of SLC7A11, providing protection against ferroptosis in acute-on-chronic liver failure [38]. Based on the effects of m⁵C methylation on ferroptosis and the critical role of ferroptosis in AS, the current study showed that ALYREF up-regulation attenuated AS by inhibiting ferroptosis in ox-LDL-triggered HCAEC and ApoE^−/− mouse model. These findings suggest that ALYREF-mediated ferroptosis may play a critical role in the advancement of AS.

CISD1, housing the CDGSH iron sulfur domain, resides in the outer mitochondrial membrane. It exerts a negative regulatory influence on ferroptosis, crucially modulating mitochondrial iron ion transport and oxidative stress [39]. Blocking CISD1 mitigates both mitochondrial dysfunction and ferroptosis in mice experiencing acute lung injury [40]. Metallothionein mitigates oxidative cardiomyopathy induced by glutathione depletion through CISD1-mediated regulation of ferroptosis in murine hearts [41]. We previously found that CISD1 can inhibit fat accumulation and inflammatory response in macrophages by regulating Drp1 in AS animal models and cell models, improve mitochondrial function, and inhibit ROS production during AS [21]. Here, for the first time we discovered that ALYREF improved the mRNA stability of CISD1 and up-regulated its expression through m5C modification, thereby inhibiting ferroptosis of endothelial cells during AS and reducing the pathological changes of AS. CISD1 knockdown reversed the suppressive effects of ALYREF up-regulation on ox-LDL-induced HCAEC inflammation, lipid metabolism, mitochondrial dysfunction, and ferroptosis. Hence, these results indicate that CISD1 functions as a downstream mediator of ALYREF, offering protective effects against AS.

In summary, our study found that ALYREF boosts CISD1 mRNA stability by enhancing its m⁵C modification, which increases its expression, thus hindering lipid metabolism dysfunction and endothelial cell ferroptosis in AS and mitigating AS-related pathological alterations. These discoveries offer significant insights into therapeutic approaches for this condition. This study had several limitations. The primary limitation of the present study is the absence of clinical data. Due to the relative difficulty in controlling the time and quantity in the process of clinical sample collection, we cannot obtain a suitable number of samples that meet the inclusion criteria in a short period of time. We will focus on the verification of the clinical part as the focus of follow-up research. In addition, although our studies have shown that oe-ALYREF decreased lipid levels and inhibited ferroptosis, the causal relationship between the two remains unresolved. If ALYREF directly regulates lipid metabolism, future research is needed to explore the mechanism. If lipid reduction is secondary to ferroptosis inhibition, further evidence must be provided to demonstrate that CISD1-mediated ferroptosis inhibition precedes lipid changes in time, and/or to demonstrate that ferroptosis can lead to lower lipid levels. Moreover, we cannot disregard the potential involvement of unidentified target sites for ALYREF in its anti-atherosclerotic actions. Finally, the study on the specific targeting of endothelial cells by oe-ALYREF/sh-CISD1 delivered by lentiviral vector in mice has not been fully verified. We will try to use transgenic animals to solve this specific targeting problem in the future.

Abbreviations

EdU

5-Ethynyl-2′-deoxyuridine

m⁵C

5-Methylcytosine

ActD

Actinomycin D

ATP

Adenosine triphosphate

ALYREF

Aly/REF export factor

AS

Atherosclerosis

CISD1

CDGSH iron sulfur domain-containing protein 1

CCK-8

Cell counting kit-8

DHE

Dihydroethidium

ECGS

Endothelial cell growth supplement

ECM

Endothelial cell medium

ELISA

Enzyme-linked immunosorbent assay

FBS

Fetal bovine serum

GSH

Glutathione

HCAEC

Human primary coronary artery endothelial cells

LV

Lentivirus

LDL

Low-density lipoprotein cholesterol

MDA

Malondialdehyde

MeRIP

Methylated RNA immunoprecipitation

MMP

Mitochondrial membrane potential

ox-LDL

Oxidized low-density lipoprotein

PI

Propidium iodide

ROS

Reactive oxygen species

RIP

RNA immunoprecipitation

SDS-PAGE

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

SD

Standard deviation

SOD

Superoxide dismutase

TEM

Transmission electron microscopy

TG

Triglycerides

VEC

Vascular endothelial cell

Author contributions

J.H. contributed to the conception and design of the study. J.H., L.Y. and W.X. contributed to the acquisition of data, J.H., R.Y., J.Z., X.P. and M.Q. contributed to the analysis and interpretation of data. J.H. drafted the manuscript. All authors critically revised the manuscript and approved the final version of the manuscript. X.P. and M.Q. supervised the study.

Funding

This work was supported by the Science and Technology Research Project of Jiangxi Provincial Department of Education (Grant number: GJJ2200135), the Science and Technology Plan Project of Health Commission of Jiangxi Province (Grant number: 202510032) and National Natural Science Foundation of China (Grant number: 82160132).

Availability of data and materials

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The animal trials received approval from Institutional Animal Care and Use Committee of The First Affiliated Hospital, Jiangxi Medical College, Nanchang University (Approval number: 202510032). All animal experiments were complied with the ARRIVE guidelines.

Consent for publication

All authors confirm their consent for publication.

Competing interests

The authors declare no competing interests.