CircUsp9x/miR-599/stim1 axis regulates proliferation and migration in vascular smooth muscle cells induced by oxidized-low density lipoprotein

ABSTRACT

Circular RNAs (circRNAs) regulate the function of vascular smooth muscle cells (VSMCs) in atherosclerosis (AS) progression. We aimed to explore the role of circUSP9X in oxidized low-density lipoprotein (ox-LDL)-induced VSMCs. Cell proliferation was assessed using cell counting kit-8 and EDU assays. Cell migration was evaluated using Transwell and wound healing assays. The interaction between circUSP9X or STIM1 and miR-599 was analyzed using dual-luciferase reporter and RNA pull-down assays. Their levels were examined using quantitative real-time PCR. CircUSP9X and STIM1 expression was increased, whereas miR-599 expression was reduced in the serum of patients with AS and ox-LDL-stimulated VSMCs. Overexpression of circUSP9X facilitated the proliferation and migration of VSMCs induced by ox-LDL. CircUSP9X sponged miR-599, which targeted STIM1. MiR-599 reversed the effects induced by circUSP9X, and STIM1 reversed the effects induced by miR-599. Taken together, CircUSP9X promoted proliferation and migration in ox-LDL-treated VSMCs via the miR-599/STIM1 axis, providing a theoretical basis for the role of circUSP9X/miR-599/STIM1 axis in AS.

KEYWORDS:

• Atherosclerosis
• circUSP9X
• oxidized low density lipoprotein
• proliferation
• migration

Introduction

Atherosclerosis (AS) is a common vascular disease with high incidence and mortality, represented by lipid accumulation in the intima of the arteries.¹ Increased arterial wall thickness and reduced lumen diameter lead to improper deposition of extracellular matrix, further promoting the progression of AS.² Bad lifestyle, environment, and other diseases are the risk factors of AS.³ The disease progresses slowly and is difficult to diagnose. When symptoms occur, it is commonly associated with a reduction of the blood flow induced by lumen stenosis or clot obstruction.⁴ As the pathogenesis of AS is quite complex, there is currently no effective therapeutic strategy.

Vascular smooth muscle cells (VSMCs) in the coronary artery are produced by the epicardium. The phenotypic transformation, proliferation, senescence, and apoptosis of VSMCs affect the development of AS.⁵ VSMCs are present in all stages of AS plaques. The controlled proliferation of VSMCs produces extracellular matrix, which forms fibrous caps and stabilizes plaques.⁶ Additionally, the migration of VSMCs is also a large event of AS. VSMC proliferation and migration are increased after changing their differentiation state in response to pro-AS stimulation.⁷ Therefore, studying the role of VSMCs can help us understand the pathogenesis of AS.

Circular RNAs (circRNAs) are noncoding RNAs with stable cyclized structures. They regulate multiple physiopathological processes at epigenetic, transcriptional, and post-transcriptional levels.⁸ CircRNAs are potential disease biomarkers due to their stable expression. Increasing evidence indicates that circRNAs are linked to the pathophysiological process of AS by regulating the function of endothelial cells (ECs), VSMCs, and macrophages.⁹ CircUSP9X (hsa_circ_0090231) is located in chrX with 19 576 bp in genomic length. Knockdown of circUSP9X facilitates cell cycle and angiogenesis, inhibits apoptosis, oxidative stress, as well as inflammation of ECs.^10,11 However, whether circUSP9X could affect the function of VSMCs remains unclear.

Herein, we attempted to clarify the effect of circUSP9X on the biological functions of VSMCs and explore the corresponding mechanism. The study will provide a theoretical basis for understanding AS pathogenesis.

Materials and methods

Clinical serum samples

Patients with AS and normal controls were enlisted from Jiaxing No.1 hospital. Patients with AS were diagnosed based on coronary angiography and at least one coronary artery was confirmed to be 50% narrow. The normal participants were diagnosed without AS through coronary angiography, and free of major diseases including autoimmune diseases, inflammation, and malignancy. The blood samples were obtained from all participants. After centrifugation at 3,000 g for 10 min, serum was obtained and stored at −80°C. Written informed consent was signed by each participant. The study was approved by the Ethics Committee of Jiaxing No.1 Hospital.

Cell culture and oxidized low-density lipoprotein (ox-LDL) stimulation

VSMCs were acquired from BeNa culture collection (Beijing, China). The cells were maintained in DMEM equipped with 10% FBS and 1% penicillin/streptomycin (Gibco, USA). Cells were cultured in a cell incubator at 37°C with 5% CO₂.

ox-LDL (purity >97%; Yeasen, China) was dissolved in complete DMEM to specific concentrations (25, 50, 75, and 100 μg/mL). VSMCs were exposed to 0 (DMEM without ox-LDL), 25, 50, 75, and 100 μg/mL ox-LDL for 24 h. Additionally, VSMCs were stimulated with 75 μg/mL ox-LDL for 0, 12, 24, and 48 h.

Transient transfection

CircUSP9X overexpression vector, STIM1 overexpression vector, empty vector, si-circUSP9X 1#, si-circUSP9X 2#, and si-nc were acquired from Genepharma (Shanghai, China). Particularly, to overexpression circUSP9X, pCD5-ciR, pCD25-ciR, and pLO5-ciR vectors were selected. These vectors carry optimized flanked ring frame and contain Alu elements to efficiently cyclize the inserted circRNA. As circUSP9X was cyclized from exon 3–12, the whole sequence of exon 3–12 and the flank region were cloned into these vectors mentioned above. According to the results of transfection efficiency (Supplementary Figure S1), pCD25-ciR could successfully overexpress circUSP9X (hsa_circ_0090231). Thus, circUSP9X-pCD25-ciR was selected for the further use. MiR-599 mimic, inhibitor, and their nc were obtained from Ribobio (Guangzhou, China). VSMCs were transfected with plasmids using Lipofectamine 3000 (Invitrogen, USA). The transfected cells were harvested 48 h later.

Determination of cell proliferation

Cell counting kit-8 (CCK-8, Yeasen) was applied to measure cell viability. VSMCs were cultured at 37°C for 24 h. CCK-8 solution (10 μL) was added to incubate with cells for 4 h. The absorbance was examined at 460 nm by a microplate reader (Bio-Rad, USA).

An EDU kit (Solarbio, China) was used for cell proliferation detection. After washing the cells with PBS, VSMCs were incubated with 4% paraformaldehyde for 0.5 h and infiltrated with Triton X-100. Glycine (50 μL) was added to neutralize paraformaldehyde. Then, the cells were incubated with EDU buffer for 2 h and DAPI for 0.5 h. The stained cells were imaged under a fluorescence microscope (Olympus, Japan).

Detection of cell migration

Serum-free DMEM containing transfected cells was added in the upper transwell chambers (Solarbio). Complete DMEM was added in the lower chambers. After 24 h, migrated cells were treated with 4% paraformaldehyde and crystal violet, and subsequently visualized under a light microscope (Olympus).

VSMCs were cultured in six-well plates until almost full of the well. Scratch was made using 10 μL pipette tips. After washing cell debris, the plates were incubated for 24 h. The migrated cells were visualized under a phase-contrast microscope (Olympus).

Bioinformatic analysis

The complementary sites of circUSP9X or STIM1 and miR-599 were predicted using http://starbase.sysu.edu.cn/ or http://www.targetscan.org/, respectively.

Dual-luciferase reporter assay

The 3’-UTR sequences of circUSP9X or STIM1 were cloned into pGL3 vectors (circUSP9X-wt, STIM1-wt; Promega, USA). Their mutant sequences were also cloned into pGL3 vectors (circUSP9X-mut, STIM1-mut). VSMCs were co-transfected with circUSP9X-wt/STIM1-wt or circUSP9X-mut/STIM1-wt and mimic or mimic nc using Lipofectamine 3000 for 48 h. A Dual Luciferase Reporter Gene Assay kit (Sangon, China) was utilized to test luciferase activity.

RNA pull-down assay

The biotin-labeled miR-599 and nc probes (Ribobio) were transfected into VSMCs for 72 h. After lysis and centrifugation, and supernatant (50 μL) was obtained as Input. The remaining supernatant was incubated with streptavidin-coupled Dynabeads (Invitrogen) at 4°C overnight. Lysis buffer, low-salt buffer, and high-salt buffer were used to wash the beads in turn. The enrichment of circUSP9X and STIM1 was detected using quantitative real-time PCR (qRT-PCR).

Fluorescence in situ hybridization (FISH)

VSMCs were fixed with 4% paraformaldehyde and infiltrated with Triton X-100. The Cy3-labeled circUSP9X or STIM1 probes and FITC-labeled miR-599 probes were incubated at 37°C overnight. After hybridization, cells were stained using DAPI. The signals were imaged under a fluorescence microscope.

qRT-PCR

Total RNA was isolated using TRIzol reagent (Invitrogen). Afterward, reverse transcription and qPCR were performed using a One-Step RT-qPCR Kit (Sangon) according to the manufacturer’s protocol. Cycle threshold (Ct) value was detected using the LightCycler 2.0 system (Roche, USA) The data were calculated using the 2^−ΔΔCt method. GAPDH (for circUSP9X and STIM1) and U6 (for miR-599) were utilized as the normalization.

Western blot

VSMCs were lysed using RIPA lysis buffer (Sangon) and the protein concentration was examined using a BCA kit (Sangon). Then, proteins were run using SDS-PAGE, electro-transferred to PVDF membranes (Millipore, USA), and blocked with 5% skim milk. The membranes were incubated with the corresponding primary antibodies at 4°C overnight and incubated with the secondary antibody at room temperature for 1 h. Finally, an ECL kit (Sangon) was used to capture the protein blots.

Statistical analysis

Each experiment was repeated triple. Data were analyzed using the GraphPad Prism v7.0 software. The expression of circUSP9X, miR-599, and STIM1 in patients with AS and healthy controls was analyzed using unpaired Student’s t-test. In addition, comparisons between two or multiple groups of cell experiments were analyzed using Mann-Whitney or Kruskal-Wallis test. The correlation was assessed using the Pearson correlation coefficient. Data were displayed as mean ± SD. P < .05 indicates a significant difference.

Results

CircUSP9X is upregulated in patients with as and ox-LDL-treated VSMCs

First, we detected the levels of circUSP9X in the serum samples from patients with AS and healthy individuals. The data showed that circUSP9X expression was markedly increased in the serum of patients with AS, compared with that in healthy control (Figure 1a). Additionally, VSMCs were treated with 0, 25, 50, 75, and 100 μg/mL of ox-LDL for 24 h, and cell viability was evaluated. The results showed that cell viability was promoted in a dose-dependent manner (Figure 1b). The most significant dose of ox-LDL (75 μg/ml) was chosen to treat VSMCs for 0, 12, 24, and 48 h. cell viability was promoted in a time-dependent manner (Figure 1c). Moreover, circUSP9X expression was detected in ox-LDL-stimulated VSMCs. The levels of circUSP9X were increased by ox-LDL in dose-dependent and time-dependent ways (Figure 1d,e).

Figure 1. CircUSP9X is upregulated in as and ox-LDL-stimulated VSMCs. (a) CircUSP9X expression in the serum acquired from patients with as and healthy individuals. (b) cell viability was analyzed after VSMC treatment with 0, 25, 50, 75, and 100 μg/ml ox-LDL. (c) cell viability was analyzed after VSMC treatment with ox-LDL for 0, 12, 24, and 36 h. (d) CircUSP9X was examined in VSMCs exposed to 0, 25, 50, 75, and 100 μg/ml ox-LDL. (e) CircUSP9X was examined in ox-LDL-induced VSMCs for 0, 12, 24, and 36 h. ***P < .001. **P < .01. *P < .05.

CircUSP9X promotes proliferation and migration in ox-LDL-induced VSMCs

To explore the role of circUSP9X, its overexpressing vector and si-circUSP9X were transfected into VSMCs. The results of transfection efficiency showed that circUSP9X overexpression vector was successfully transfected into VSMCs (Supplementary Figure S1). As indicated in (Figure 2a), circUSP9X expression was markedly elevated following overexpression of circUSP9X. In addition, circUSP9X expression was markedly reduced after knockdown, especially in the si-circUSP9X 2# transfection group. On one hand, overexpression of circUSP9X dramatically facilitated cell proliferation, while knockdown of circUSP9X dramatically suppressed cell proliferation induced by ox-LDL (Figure 2b–d). On the other hand, enforced circUSP9X notably promoted the migration, while knockdown of circUSP9X notably impeded the migration of ox-LDL-induced cells (Figure 2e–h). CDK2/cyclinE1 complex is essential for the cell cycle, and its activation promotes the transition of cells from G1 phase to S phase, accelerating cell proliferation. In AS, upregulated CDK2 and cyclineE1 are associated with the proliferation of VSMCs.^12,13 Besides, MMPs play a crucial role in AS. VSMCs release MMP2 and MMP9, which degrade protein in the extracellular matrix and modulate VCMC migration, proliferation, Ca²⁺ signaling, and contraction.^14,15 Therefore, based on changes in the proliferation and migration of VSMCs, we detected the protein levels of CDK2, cyclineE1, MMP2, and MMP9. The results showed that overexpression of circUSP9X upregulated the levels of CDK2, cyclinE1, MMP2, and MMP9 induced by ox-LDL, whereas interference with circUSP9X downregulated their levels (Figure 2i–m).

Figure 2. CircUSP9X promotes proliferation and migration in ox-LDL-induced VSMCs. (a) transfection efficiency was tested after VSMCs transfecting with vector, circUSP9X overexpressing vector, si-nc, si-circUSP9X 1#, and si-circUSP9X 2#. (b) cell viability of transfected ox-LDL-stimulated VSMCs. (c-d) EDU assay revealed cell proliferation. (E-F) Transwell assay measured cell migration, and migrated cells were quantified. (g-h) wound healing assay was used to determine cell migration. (i-m) protein levels of CDK2, cyclinE1, MMP2, and MMP9. ***P < .01 vs. The vector group, and **P < .05 vs. si-nc group in (A). *P < .01 and **P < .05 vs. The control group, #P < .05 vs. The ox-LDL + vector group, and &P < .05 vs. The ox-LDL +si-NC group in (B-M).

CircUSP9X sponges miR-599

The directly complementary regions between circUSP9X and miR-599 were searched using bioinformatic analysis (Figure 3a). MiR-599 is a vital regulator in AS development. Importantly, miR-599 regulates endothelial injury and affects proliferation and migration in VSMCs.^11,16 Therefore, we chose miR-599 for further study. Luciferase activity of VSMCs co-transfected with circUSP9X-wt and mimic was markedly decreased as compared to mimic nc (Figure 3b). Biotin-labeled miR-599 significantly pulled down circUSP9X (Figure 3c). Both circUSP9X and miR-599 were mainly located in the cytoplasm (Figure 3d). Compared with healthy controls, miR-599 was low expressed in the serum of patients with AS (Figure 3e). In patients with AS, miR-599 expression was negatively related to circUSP9X expression (Figure 3f). In VSMCs, miR-599 expression was negatively regulated by circUSP9X (Figure 3g).

Figure 3. CircUSP9X sponges miR-599. (a) the potential complementary sites of miR-599 on circUSP9X-wt sequences. CircUSP9X-mut sequences were designed. (b) luciferase activity was tested after co-transfection. (c) CircUSP9X expression after RNA pull-down. (d) FISH analyzed the location of circUSP9X and miR-599. (e) MiR-599 expression in the serum of patients with as and healthy controls. (f) the relationship between circUSP9X and miR-599 in patients with AS. (g) MiR-599 was examined after knockdown or overexpression of circUSP9X. ***P < .001. **P < .01. *P < .05.

CircUSP9X promotes biological behaviors by sponging miR-599

After transfection, miR-599 expression was markedly upregulated or reduced in mimic or inhibitor transfected cells, respectively (Figure 4a). Cell proliferation was significantly facilitated by circUSP9X and was partly counteracted by overexpression of miR-599 (Figure 4b–d). CircUSP9X notably promoted ox-LDL-stimulated VSMC migration, whereas miR-599 partly abrogated this promotion (Figure 4e–h). Furthermore, circUSP9X significantly elevated CDK2, cyclinE1, MMP2, and MMP9 protein levels, whereas miR-599 reversed their levels induced by circUSP9X (Figure 4i–m).

Figure 4. CircUSP9X promotes cellular processes by miR-599. (a) MiR-599 levels following transfection of mimic or inhibitor. (a) CCK-8 assessed cell viability. (C-D) EDU assay analyzed cell proliferation. Cell migration was analyzed using (e-f) transwell assay and (g-h) wound healing assay. (i-m) Western blot examined protein levels of CDK2, cyclinE1, MMP2, and MMP9, and their levels were quantified. ***P < .001 vs. mimic nc in (A) and vs. ox-LDL + vector in others. **P < .01 vs. The mimic nc group, and *P < .01 vs. The inhibitor nc group in (A). **P < .01 and #P<.05 vs. The ox-LDL + vector group, and **P < .05 vs. The ox-LDL + circUSP9X + mimic nc group in (B-M).

MiR-599 targets STIM1

The results of bioinformatics showed that miR-599 was complementary to STIM1 (Figure 5a). Luciferase activity of VSMCs co-transfected with STIM1-wt and mimic was markedly decreased as compared to mimic nc (Figure 5b). Biotin-labeled miR-599 significantly pulled down STIM1 (Figure 5c). The data of FISH indicated that STIM1 and miR-599 were both located in the cytoplasm (Figure 5d). The expression of STIM1 was increased in the serum from patients with AS and negatively correlated with miR-599 (Figure 5e,f). In addition, STIM1 expression was negatively regulated by miR-599 (Figure 5g).

Figure 5. MiR-599 targets STIM1. (a) the potential complementary sites of miR-599 on STIM1-wt. STIM1-mut was designed. (b) luciferase activity was examined after co-transfection. (c) STIM1 expression after RNA pull-down. (d) FISH analyzed the location of STIM1 and miR-599. (e) STIM1 expression in the serum of patients with as and healthy controls. (f) the interaction of STIM1 and miR-599 in patients with AS. (g) STIM1 was examined after overexpressing or inhibiting miR-599 in VSMCs. ***P < .001. **P < .01. *P < .01.

MiR-599 suppresses biological behaviors by targeting STIM1

The expression of STIM1 was significantly upregulated following overexpressing STIM1 (Figure 6a). MiR-599 mimic markedly impeded cell proliferation, and overexpressing STIM1 reversed miR-599’s effect (Figure 6b–d). Cell migration was markedly inhibited by miR-599, which was partly counteracted by enforced STIM1 (Figure 6e–h). The protein levels of CDK2, cyclinE1, MMP2, and MMP9 were notably decreased by miR-599, whereas STIM1 abrogated the effects caused by miR-599 (Figure 6i–m).

Figure 6. MiR-599 suppresses ox-LDL-induced biological function by STIM1. (a) STIM1 levels after transfecting of STIM1 overexpression vector. Cell proliferation was analyzed using (b) CCK-8 and (c-d) EDU assays. (e-f) Transwell assay and (g-h) wound healing assay assessed cell migration. (i-m) Western blot examined protein levels of CDK2, cyclinE1, MMP2, and MMP9, and their levels were quantified. ***P < .01 vs. The vector group in (a). **P < .01 and *P < .05 vs. The ox-LDL + mimic nc group. **P < .05 vs. The ox-LDL + mimic +vector group.

Discussion

VSMCs are responsible for regulating blood pressure and maintaining vascular tone. VSMCs proliferation and migration play a crucial role in early or late-stage AS. Migration of VSMCs from media to intima is an essential event in AS progression. VSMCs promote proliferation after migration, facilitating formation of fiber caps, or VSMCs proliferate in the medium and promotes migration of its offspring in intima

However, VSMCs exhibit low proliferative capacity in late stage of AS.¹⁷ CircRNAs are identified to be associated with the progression of AS. The accumulation of ox-LDL in intima triggers AS,¹⁸ so it is often used to establish AS-damaged cell models in vitro. Studies revealed that circRNAs regulated the biological functions of VSMCs, such as growth, metastasis, and phenotype transformation, accelerating or decelerating the progression of AS.¹⁹ For example, circ_0002984 loss suppresses the viability, migration, and cell cycle of VSMCs treated with ox-LDL by regulating miR-326-3p.²⁰ Circ_RUSC2 regulates VSMC proliferation, apoptosis, and migration by the miR-661/SYK axis.²¹ Besides, silencing of circDHCR24 inhibits VSMC proliferation, migration, as well as the phenotypic switch.²² In the present study, we clarified the role of circUSP9X in ox-LDL-induced VSMCs. CircUSP9X expression was elevated in patients with AS and ox-LDL-stimulated VSMCs. Overexpression of circUSP9X promoted VSMC proliferation and migration induced by ox-LDL, whereas knockdown of circUSP9X got the opposite results. Moreover, circUSP9X upregulated CDK2 and cyclinE1, two cell cycle markers related to proliferation,²³ whereas interference with circUSP9X downregulated their levels, further suggesting that circUSP9X facilitates cell proliferation. Additionally, circUSP9X increased MMP2 and MMP9 levels, while si-circUSP9X reduced their levels, which further suggested that circUSP9X promotes cell migration. Taken together, circUSP9X facilitates ox-LDL-treated VSMC proliferation and migration, which is inconsistent with its effects on EC cellular functions.¹¹ This may be due to the different roles of VSMCs and ECs in AS.

The competing endogenous RNA (ceRNA) network contributes to investigating the pathogenesis of AS. CircRNAs can competitively combine with miRNAs.^24,25 MiRNAs are also associated with the functions of VSMCs. For instance, miR-192-5p is upregulated in AS patients and accelerates VSMC proliferation and migration.²⁶ MiR-377-3p targets NRP2 to inhibit migration and proliferation in ox-LDL-treated VSMCs.¹⁵ MiR-141-3p participates in AS progression by modulating cell migration and proliferation.²⁷ MiR-599 exerts an important role in AS via regulating VSMC functions. MiR-599 regulated by H19 suppresses cell viability, invasion, and migration of VSMCs.²⁸ Moreover, lncRNA XIST acts as a miR-599 to affect ox-LDL-induced VSMC proliferation and apoptosis.²⁹ A previous study showed that circUSP9X inhibits EC injury by sponging miR-599.¹¹ In this study, we proved that circUSP9X sponges miR-599 in VSMCs. MiR-599 expression was downregulated in patients with AS and VSMCs stimulated to ox-LDL. Overexpression of miR-599 partly counteracted the effect on cell processes induced by circUSP9X. The data suggested that circUSP9X facilitates proliferation and migration in VSMCs via modulating miR-599.

STIM1 is considered a molecule related to the binding of B lymphocytes to stromal cells outside of the Ca²⁺ signaling pathway.³⁰ In AS, STIM1 initiates and develops plaque formation.³¹ STIM1 is regulated by miRNAs such as miR-541-3p, miR-185, and miR-641, and participates in the proliferation of VSMCs.^32–34 Herein, STIM1 was affirmed as a miR-599 target. STIM1 levels were increased in AS and ox-LDL-treated VSMCs. Enforced STIM1 partly reversed the effect on cell phenotype induced by miR-599. Taken together, CircUSP9X promotes cell proliferation and migration via modulating the miR-599/STIM1 axis.

Based on the different proliferative capacities of VSMCs in different stages of AS, the circUSP9X/miR-599/STIM1 axis may promote disease progression in early AS and may decelerate advanced AS progression. However, according to lineage-tracing study, it was found that the role of VSMCs in AS is complex and cannot simply be labeled as promoting or inhibiting AS progression.¹⁷ Moreover, the proliferation and migration of VSMCs in the AS may not affect the stability of plaques and may induce lesions in other ways. Hypertension is a risk factor of AS. Similar to AS, the abnormal proliferation and migration of VSMCs are also the pathological mechanism of hypertension.³⁵ However, whether the circUSP9X/miR-599/STIM1 axis affects hypertension and then facilitates AS progress remains unclear. We will further investigate its regulation of hypertension in our future work.

In conclusion, circUSP9X and STIM1 levels were upregulated, and miR-599 expression was downregulated in AS and ox-LDL-induced VSMCs. Functionally, circUSP9X facilitates proliferation and migration in ox-LDL-induced VSMCs by the miR-599/STIM1 axis. The findings suggested that the circUSP9X/miR-599/STIM1 axis has potential for AS therapy.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10641963.2023.2280758

Additional information

Funding

This research was supported by Zhejiang Provincial Natural Science Foundation of China under Grant (No.LGF21H020006); 2023 Zhe-jiang Province Traditional Chinese Medicine Scientific Research Fund (2023ZL700); Jiaxing Key Laboratory of Arteriosclerotic Diseases (2020-dmzdsys); Pioneer innovation team of Jiaxing Institute of Arteriosclerotic Disease (XFCX-DMYH); Jiaxing First Hospital independent research project (ZZKT2022-001, ZZKT2022-003, ZZKT2022-004, ZZKT2022-005, ZZKT2022-007, ZZKT2022-008); Jiaxing Key Clinical Specialty - Cardiovascular Medicine (2023-JXZK-001).