Abstract
Homocysteine (Hcy) is an independent risk factor for atherosclerosis (AS). Hcy induces the transformation of vascular smooth muscle cells (VSMCs) into foam cells, which play a crucial role in this process. However, the detailed mechanism is still unclear. To identify the key regulatory proteins during this process and clarify the possible mechanism of Hcy-induced foam cell formation in VSMCs, thereby providing theoretical support for the intervention of AS. VSMCs were allocated into two groups: a control cohort and a group exposed to Hcy to simulate an AS-like state. Quantitative proteomic profiling was performed using the label-free quantitative DIA (LFQ-DIA) approach to detect differentially expressed proteins between these groups. To explore functional implications, enrichment analyses involving Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were conducted. Protein-protein interaction networks were constructed using the STRING database to identify central interactors. Target proteins were subsequently validated through parallel reaction monitoring (PRM). Furthermore, histological analyses (hematoxylin and eosin (HE) staining, Oil Red O staining), biochemical assays of lipid content (total cholesterol (TC) and triglycerides (TG)), and Western blot analysis were utilized to confirm the role and mechanism of identified proteins in the context of Hcy-driven foam cell conversion. The results showed that proteomic analysis identified 4804 proteins in total, of which 4799 passed missing-value filtering and were retained for downstream quantitative analysis. A total of 54 proteins were identified as differentially expressed using thresholds of adjusted p-value < 0.05 and fold change > 1.5. Among them, 13 proteins were upregulated, while 41 were downregulated in response to Hcy treatment. For PRM validation, 20 candidate proteins were selected according to proteomic evidence, biological relevance, and technical feasibility. Among them, 16 proteins (COX7C, STX5, UBQLN2, DDX50, TBCB, GSR, PCNP, CDV3, PEBP1, PPIA, S100A6, EIF4E2, UBQLN1, ARMC1, NUDCD2, and H1-2) showed the same direction of fold-change values as in the LFQ-DIA dataset, thereby underscoring the reliability of the proteomic analysis. Data are available via ProteomeXchange with identifier PXD064315. Histological staining demonstrated enhanced lipid accumulation, and the protein expression of the contraction phenotype marker a-SMA decreased, while the protein expression of the synthesis phenotype marker OPN increased. This indicates that Hcy induces VSMCs to transform from a contraction phenotype to a synthesis phenotype, resulting in the formation of foam cells. The protein levels of COX7C and sterol regulatory element-binding proteins (SREBP1C and SREBP2) were elevated upon Hcy exposure. Overexpression of COX7C further augmented the expression of SREBP1C and SREBP2, exacerbated lipid accumulation, and promoted foam cell transformation in Hcy-treated VSMCs. On the other hand, knockdown of COX7C had the opposite effect. Overall, the results of the present study suggest that COX7C plays a crucial regulatory role in Hcy-induced transformation of VSMCs into foam cells. Its pathogenic role is likely mediated through the upregulation of SREBP1C and SREBP2, thereby promoting lipid accumulation. These findings provide new insights into AS pathogenesis and identify COX7C maybe a potential therapeutic target.
Similar content being viewed by others
Data availability
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at PXD064315.Log in to the PRIDE website (https://www.ebi.ac.uk/pride/) using the following details: Project accession: PXD064315Token: CNMIH8RVbehGAlternatively, the reviewer can access the dataset by logging in to the PRIDE website using the following account details: Username: [reviewer_pxd064315@ebi.ac.uk](mailto: reviewer_pxd064315@ebi.ac.uk)Password: 1ArBWo5E6bs7.
References
Kong, P. et al. Inflammation and atherosclerosis: signaling pathways and therapeutic intervention. Signal. Transduct. Target. Ther. 7, 131. https://doi.org/10.1038/s41392-022-00955-7 (2022).
Jebari-Benslaiman, S. et al. Pathophysiology of atherosclerosis. Int. J. Mol. Sci. 23 https://doi.org/10.3390/ijms23063346 (2022).
Owens, G. K. Regulation of differentiation of vascular smooth muscle cells. Physiol. Rev. 75, 487–517. https://doi.org/10.1152/physrev.1995.75.3.487 (1995).
Yang, X. H. et al. Sprouty1 has a protective role in atherogenesis and modifies the migratory and inflammatory phenotype of vascular smooth muscle cells. Atherosclerosis 23, 373:17–28. https://doi.org/10.1016/j.atherosclerosis.2023.04.007 (2023).
Wang, T. M. et al. microRNA let-7 g suppresses PDGF-induced conversion of vascular smooth muscle cell into the synthetic phenotype. J. Cell Mol. Med. 21(12), 3592–3601. (2017). https://doi.org/10.1111/jcmm.13269
Xin, H. et al. Calcified decellularized arterial scaffolds impact vascular smooth muscle cell transformation via downregulating alpha-SMA expression and upregulating OPN expression. Exp. Ther. Med. 29 (1), 705–710. https://doi.org/10.3892/etm.2019.7626 (2019).
Zhang, L. et al. Role of the balance of Akt and MAPK pathways in the Exercise-Regulated phenotype switching in spontaneously hypertensive rats. Int. J. Mol. Sci. 13 (22), 5690. https://doi.org/10.3390/ijms20225690 (2019).
Grootaert, M. O. J. & Bennett, M. R. Vascular smooth muscle cells in atherosclerosis: time for a re-assessment. Cardiovasc. Res. 117, 2326–2339. https://doi.org/10.1093/cvr/cvab046 (2021).
Allahverdian, S., Chehroudi, A. C., McManus, B. M., Abraham, T. & Francis, G. A. Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis. Circulation 129, 1551–1559. https://doi.org/10.1161/circulationaha.113.005015 (2014).
Wang, Y. et al. Smooth muscle cells contribute the majority of foam cells in ApoE (Apolipoprotein E)-Deficient mouse atherosclerosis. Arterioscler. Thromb. Vasc Biol. 39, 876–887. https://doi.org/10.1161/atvbaha.119.312434 (2019).
Galindo, C. L. et al. Lipid-laden foam cells in the pathology of atherosclerosis: shedding light on new therapeutic targets. Expert Opin. Ther. Targets. 27, 1231–1245. https://doi.org/10.1080/14728222.2023.2288272 (2023).
Paganelli, F. et al. Hyperhomocysteinemia and cardiovascular disease: is the adenosinergic system the missing link? Int. J. Mol. Sci. 22 https://doi.org/10.3390/ijms22041690 (2021).
Boushey, C. J., Beresford, S. A., Omenn, G. S. & Motulsky, A. G. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. Jama 274, 1049–1057. https://doi.org/10.1001/jama.1995.03530130055028 (1995).
Welch, G. N. & Loscalzo, J. Homocysteine and atherothrombosis. N Engl. J. Med. 338, 1042–1050. https://doi.org/10.1056/nejm199804093381507 (1998).
McCully, K. S. Chemical pathology of homocysteine. IV. Excitotoxicity, oxidative stress, endothelial dysfunction, and inflammation. Ann. Clin. Lab. Sci. 39, 219–232 (2009).
Shi, Y. F. et al. Effects of Rosuvastatin on the production and activation of matrix metalloproteinase-2 and migration of cultured rat vascular smooth muscle cells induced by homocysteine. J. Zhejiang Univ. Sci. B. 14, 696–704. https://doi.org/10.1631/jzus.BQICC703 (2013).
Wang, X. Y., Ma, X., Zeng, Y., Xu, L. B. & Zhang, M. Hypermethylation of the CTRP9 promoter region promotes Hcy induced VSMC lipid deposition and foam cell formation via negatively regulating ER stress. Sci. Rep. 13, 19438. https://doi.org/10.1038/s41598-023-46981-5 (2023).
Wang, X. Y. et al. Proliferation, migration and phenotypic transformation of VSMC induced via Hcy related to up-expression of WWP2 and p-STAT3. PLOS ONE. 19, e0296359. https://doi.org/10.1371/journal.pone.0296359 (2024).
Xu, L. et al. Aberrant MFN2 transcription facilitates homocysteine-induced VSMCs proliferation via the increased binding of c-Myc to DNMT1 in atherosclerosis. J. Cell. Mol. Med. 23 (7), 4611–4626 (2019).
Yang, A. N. et al. High-methionine diets accelerate atherosclerosis by HHcy-mediated FABP4 gene demethylation pathway via DNMT1 in ApoE(–/–) mice. FEBS Lett. 589, 3998–4009 (2015).
Perez-Riverol, Y. et al. The PRIDE database at 20 years: 2025 update. Nucleic Acids Res. 6;53 (D1), D543–D553. https://doi.org/10.1093/nar/gkae1011 (2025).
Luo, S. S. et al. Endothelial HDAC1-ZEB2-NuRD complex drives aortic aneurysm and dissection through regulation of protein S-Sulfhydration. Circulation 2 (18), 1382–1403. https://doi.org/10.1161/CIRCULATIONAHA.122.062743 (2023).
Kanehisa, M., Furumichi, M., Sato, Y., Kawashima, M. & Ishiguro-Watanabe, M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 6 (D1), D587–D592. https://doi.org/10.1093/nar/gkac963 (2023).
Guo, W. et al. Homocysteine accelerates atherosclerosis by inhibiting scavenger receptor class B member1 via DNMT3b/SP1 pathway. J. Mol. Cell. Cardiol. 138, 34–48 (2020).
Tam, M. et al. Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation. J. Lipid Res. 53 (1), 95–104. https://doi.org/10.1194/jlr.M018820 (2012).
Alaa Sirwi, M., Mahmood & Hussain Lipid transfer proteins in the assembly of apoB-containing lipoproteins. J. Lipid Res. 59 (7), 1094–1102. https://doi.org/10.1194/jlr.R083451 (2018).
Du, X. M. et al. A role for oxysterol-binding protein-related protein 5 in endosomal cholesterol trafficking. J. Cell. Biol. 10 (1), 121–135. https://doi.org/10.1083/jcb.201004142 (2011).
Judith Alonso, M. & Galán Ingrid Martí-Pàmies. NOR-1/NR4A3 regulates the cellular inhibitor of apoptosis 2 (cIAP2) in vascular cells: role in the survival response to hypoxic stress. Sci. Rep. 22, 634056. https://doi.org/10.1038/srep34056 (2016).
Masashi Masuda, S. et al. Saturated phosphatidic acids mediate saturated fatty acid-induced vascular calcification and lipotoxicity. J. Clin. Invest. 26 (12), 4544–4558. https://doi.org/10.1172/JCI82871 (2015).
Hiroki Ono, T., Ichiki, H. & Ohtsubo Critical role of Mst1 in vascular remodeling after injury. Arterioscler. Thromb. Vasc Biol. 25 (9), 1871–1876. https://doi.org/10.1161/01.ATV.0000174588.50971.1a (2005).
Jing, S. H. et al. Raf kinase inhibitor protein (RKIP) inhibits tumor necrosis Factor-α (TNF-α) induced adhesion molecules expression in vascular smooth muscle bells by suppressing (Nuclear transcription Factor-κB (NF-kappaB) pathway. Med. Sci. Monit. 6, 23:4789–4797. https://doi.org/10.12659/msm.903661 (2017).
Nageswara, R., Madamanchi, Marschall, S. & Runge Mitochondrial dysfunction in atherosclerosis. Circ Res. 100(4), 460 – 473. (2007). https://doi.org/10.1161/01.RES.0000258450.44413.96
Peng, W. X. et al. Mitochondrial dysfunction in atherosclerosis. DNA Cell. Biol. 38 (7), 597–606. https://doi.org/10.1089/dna.2018.4552 (2019).
Zou, T. et al. Homocysteine enhances cell proliferation in vascular smooth muscle cells: role of p38 MAPK and p47phox. Acta Biochim. Biophys. Sin (Shanghai). 42 (12), 908–915. https://doi.org/10.1093/abbs/gmq102 (2010).
Peter Kaplan, Z. et al. Homocysteine and Mitochondria in Cardiovascular and Cerebrovascular Systems. Int. J. Mol. Sci. 18; 21(20), 7698. (2020). https://doi.org/10.3390/ijms21207698
Karasawa, T. et al. Sterol regulatory element-binding protein-1 determines plasma remnant lipoproteins and accelerates atherosclerosis in low-density lipoprotein receptor-deficient mice. Arterioscler. Thromb. Vasc Biol. 31, 1788–1795. https://doi.org/10.1161/atvbaha.110.219659 (2011).
Gopoju, R., Panangipalli, S. & Kotamraju, S. Metformin treatment prevents SREBP2-mediated cholesterol uptake and improves lipid homeostasis during oxidative stress-induced atherosclerosis. Free Radic Biol. Med. 118, 85–97. https://doi.org/10.1016/j.freeradbiomed.2018.02.031 (2018).
Visseren, F. L. J. et al. ESC Guidelines on cardiovascular disease prevention in clinical practice. Eur. Heart J. 42, 3227–3337, (2021). https://doi.org/10.1093/eurheartj/ehab484 (2021).
Mach, F. et al. 2019 ESC/EAS guidelines for the management of dyslipidaemias: lipid modification to reduce cardiovascular risk. Eur. Heart J. 41, 111–188. https://doi.org/10.1093/eurheartj/ehz455 (2020).
Hetherington, I. & Totary-Jain, H. Anti-atherosclerotic therapies: Milestones, challenges, and emerging innovations. Mol. Ther. 30, 3106–3117. https://doi.org/10.1016/j.ymthe.2022.08.024 (2022).
Wu, G., Yu, G., Zheng, M., Peng, W. & Li, L. Recent advances for Dynamic-Based therapy of atherosclerosis. Int. J. Nanomed. 18, 3851–3878. https://doi.org/10.2147/ijn.S402678 (2023).
Bruun, K. & Mortensen, M. B. Rethinking atherosclerotic cardiovascular disease prevention in the era of expanding therapies: could plaque stabilization reduce the need for lifelong treatments and polypharmacy? Curr. Opin. Cardiol. 40, 50–55. https://doi.org/10.1097/hco.0000000000001188 (2025).
Zhang, S. et al. Homocysteine promotes atherosclerosis through macrophage pyroptosis via Endoplasmic reticulum stress and calcium disorder. Mol. Med. 29, 73. https://doi.org/10.1186/s10020-023-00656-z (2023).
Habib, S. S., Al-Khlaiwi, T., Almushawah, A., Alsomali, A. & Habib, S. A. Homocysteine as a predictor and prognostic marker of atherosclerotic cardiovascular disease: a systematic review and meta-analysis. Eur. Rev. Med. Pharmacol. Sci. 27, 8598–8608. https://doi.org/10.26355/eurrev_202309_33784 (2023).
Ma, S. et al. Homocysteine promotes the pathogenesis of atherosclerosis through the Circ-PIAS1-5/miR-219a-2-3p/TEAD1 axis. Adv. Sci. (Weinh). e2415563. https://doi.org/10.1002/advs.202415563 (2025).
Pham, L. et al. Regulation of mitochondrial oxidative phosphorylation through tight control of cytochrome c oxidase in health and disease - Implications for ischemia/reperfusion injury, inflammatory diseases, diabetes, and cancer. Redox Biol. 10, 78: 103426. https://doi.org/10.1016/j.redox.2024.103426 (2024).
Jia, J. et al. Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury. Front Pharmacol. 22(14), 1084564. (2023). https://doi.org/10.3389/fphar. (2023).
Li, L. et al. Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition. Development. 26;145(18):dev168609. (2018). https://doi.org/10.1242/dev.168609
Wang, Z. et al. Mitochondrial ROS Produced by Skeletal Muscle Mitochondria Promote the Decisive Signal for UPRmt Activation. Biomed Res Int. 21; : 7436577. (2022). https://doi.org/10.1155/2022/7436577 (2022).
Yuan, X. et al. Altered mitochondrial unfolded protein response and FGF21 secretion in MASLD progression and the effect of exercise intervention. Sci. Rep. 29 (1), 3686. https://doi.org/10.1038/s41598-025-87190-6( (2025).
Song, Y. F. et al. Dietary Choline Alleviates High-Fat Diet-Induced Hepatic Lipid Dysregulation via UPRmt Modulated by SIRT3-Mediated mtHSP70 Deacetylation. Int J Mol Sci. 11;23(8):4204. (2022). https://doi.org/10.3390/ijms23084204
Jamerson, L. E. & Bradshaw, P. C. The Roles of White Adipose Tissue and Liver NADPH in Dietary Restriction-Induced Longevity. Antioxidants (Basel). 8; 13(7), 820. (2024). https://doi.org/10.3390/antiox13070820
Yu, Q., Zhang, Y., Xu, C. B. & Apolipoprotein, B. The villain in the drama? Eur. J. Pharmacol. 748, 166–169. https://doi.org/10.1016/j.ejphar.2014.08.037 (2015).
Nguyen, T. M., Sawyer, J. K., Kelley, K. L., Davis, M. A. & Rudel, L. L. Cholesterol esterification by ACAT2 is essential for efficient intestinal cholesterol absorption: evidence from thoracic lymph duct cannulation. J. Lipid Res. 53, 95–104. https://doi.org/10.1194/jlr.M018820 (2012).
Iqbal, J. et al. Increased intestinal lipid absorption caused by Ire1β deficiency contributes to hyperlipidemia and atherosclerosis in Apolipoprotein E-deficient mice. Circ. Res. 110, 1575–1584. https://doi.org/10.1161/circresaha.112.264283 (2012).
Aguilar-Ballester, M., Herrero-Cervera, A., Vinué, Á., Martínez-Hervás, S. & González-Navarro, H. Impact of cholesterol metabolism in immune cell function and atherosclerosis. Nutrients 12 https://doi.org/10.3390/nu12072021 (2020).
Wu, Y. P., Sun, D. D., Wang, Y., Liu, W. & Yang, J. Herpes simplex virus type 1 and type 2 infection increases atherosclerosis risk: evidence based on a Meta-Analysis. Biomed. Res. Int. 2016 (2630865). https://doi.org/10.1155/2016/2630865 (2016).
Erl, W. et al. Nuclear factor-kappa B regulates induction of apoptosis and inhibitor of apoptosis protein-1 expression in vascular smooth muscle cells. Circ. Res. 84, 668–677. https://doi.org/10.1161/01.res.84.6.668 (1999).
Xiang, H. et al. Targeting autophagy-related protein kinases for potential therapeutic purpose. Acta Pharm. Sin B. 10, 569–581. https://doi.org/10.1016/j.apsb.2019.10.003 (2020).
Kadenbach, B. & Hüttemann, M. The subunit composition and function of mammalian cytochrome c oxidase. Mitochondrion 24, 64–76. https://doi.org/10.1016/j.mito.2015.07.002 (2015).
Wang, H. X. & Zhao, Y. X. Prediction of genetic risk factors of atherosclerosis using various bioinformatic tools. Genet. Mol. Res. 15 https://doi.org/10.4238/gmr.15027347 (2016).
Linders, P. T., Horst, C. V., Beest, M. T. & van den Bogaart, G. Stx5-Mediated ER-Golgi transport in mammals and yeast. Cells 8 https://doi.org/10.3390/cells8080780 (2019).
Lin, Y. et al. SNARE-Mediated cholesterol movement to mitochondria supports steroidogenesis in rodent cells. Mol. Endocrinol. 30, 234–247. https://doi.org/10.1210/me.2015-1281 (2016).
Couto, N., Wood, J. & Barber, J. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radic Biol. Med. 95, 27–42. https://doi.org/10.1016/j.freeradbiomed.2016.02.028 (2016).
Qiao, M. et al. Increased expression of glutathione reductase in macrophages decreases atherosclerotic lesion formation in low-density lipoprotein receptor-deficient mice. Arterioscler. Thromb. Vasc Biol. 27, 1375–1382. https://doi.org/10.1161/atvbaha.107.142109 (2007).
Chen, W. et al. NudCL2 regulates cell migration by stabilizing both myosin-9 and LIS1 with Hsp90. Cell. Death Dis. 11, 534. https://doi.org/10.1038/s41419-020-02739-9 (2020).
Jiang, C. et al. Homocysteine promotes vascular smooth muscle cell migration by induction of the adipokine resistin. Am. J. Physiol. Cell. Physiol. 297, C1466–1476. https://doi.org/10.1152/ajpcell.00304.2009 (2009).
Acknowledgements
The authors express their gratitude to PTM Bio for providing technical support for mass spectrometry.
Funding
The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This research was funded by the Ningxia Natural Science Foundation Project (2024AAC03200), Scientific Research Project of Higher Education Institutions of the Department of Education of Ningxia Hui Autonomous Region (NYG2024116), Open competition mechanism to select the best candidates for key research projects of Ningxia Medical University (XJKF240312).
Author information
Authors and Affiliations
Contributions
ZMH designed the experiments. WXY, MXP, ZX, and MX performed the experiments and analyzed the data. WXY and ZMH wrote the manuscript. All authors have read and approved the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Wang, X., Ma, X., Zhang, X. et al. Study on biomarkers of homocysteine-induced transformation of vascular smooth muscle cells into foam cells. Sci Rep (2026). https://doi.org/10.1038/s41598-026-38763-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-026-38763-6
