Abstract
Neointimal hyperplasia, driven by abnormal proliferation and survival of vascular smooth muscle cells (VSMCs), underlies atherosclerotic stenosis and restenosis after angioplasty or stenting. Edelfosine (ET-18-OCH₃) is an alkylphospholipid with pro-apoptotic activity. We tested whether edelfosine limits pathological VSMC growth and neointimal lesion formation by enforcing cell-cycle arrest and apoptosis. Primary VSMCs from mouse and rat aortas were exposed to edelfosine (0–15 µM). Viability (MTT) and DNA synthesis (BrdU) were quantified. DNA content and binucleation were assessed by laser scanning cytometry and immunofluorescence. Apoptosis was measured by TUNEL and by cleavage of caspase-9, -7, and -3, with the pan-caspase inhibitor Z-VAD-FMK to test caspase dependence. Apoptosis in live cells was also analyzed using Annexin V and propidium iodide staining. Intracellular Ca²⁺ was imaged and measured in fluorescent Ca²⁺ indicator-loaded cells. In vivo, carotid artery ligation in mice induced neointimal hyperplasia; edelfosine or vehicle was delivered locally, and lesion size was measured morphometrically. Vascular apoptosis was further evaluated by TUNEL. Edelfosine reduced VSMC viability and proliferation in a dose- and time-dependent manner; at 5–10 µM it suppressed BrdU incorporation by > 90% and triggered extensive cell death. Cells accumulated with 4 N DNA content and showed increased binucleation, consistent with G₂/M arrest and failed cytokinesis. Approximately 40% of edelfosine-treated VSMCs were TUNEL-positive versus ~ 5% with vehicle (p < 0.001), coincident with activation of caspase-9, -7, and -3; Z-VAD-FMK prevented caspase-3 cleavage and reduced TUNEL positivity. Mechanistically, edelfosine induced endoplasmic reticulum (ER) stress (increased phospho-eIF2α), upregulated Bax, and evoked a rapid rise in intracellular Ca²⁺ in the presence of extracellular Ca²⁺. Edelfosine-induced Ca²⁺ elevation was reduced by extracellular Ca²⁺ chelation with EGTA, blockade of VGCCs with nifedipine, and perturbation of IP₃ receptor-linked Ca²⁺ pathways with 2-APB. In vivo, edelfosine significantly reduced neointimal lesion size after carotid ligation, lowering the intima-to-lumen ratio (p < 0.05) and increasing TUNEL positivity within the vessel wall. Edelfosine enforces G₂/M cell-cycle arrest and caspase-dependent apoptosis in VSMCs, linked to ER stress and Ca²⁺ influx, thereby limiting neointimal hyperplasia after blood flow cessation. Local edelfosine delivery may offer a dual anti-proliferative and pro-apoptotic strategy to prevent restenosis.
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files. Additional raw data and materials are available from the corresponding author on reasonable request.
Abbreviations
- 2-APB:
-
2-aminoethoxydiphenyl borate
- BrdU:
-
5-bromo-2′-deoxyuridine
- DES:
-
drug-eluting stent(s)
- DMSO:
-
dimethyl sulfoxide
- EGTA:
-
ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid
- ER:
-
endoplasmic reticulum
- FBS:
-
fetal bovine serum
- IP₃:
-
inositol trisphosphate
- MTT:
-
thiazolyl blue tetrazolium bromide
- PI:
-
propidium iodide
- SOCE:
-
store-operated calcium entry
- TUNEL:
-
terminal deoxynucleotidyl transferase dUTP nick end labeling
- VGCCs:
-
voltage-gated calcium channels
- VSMC:
-
vascular smooth muscle cell
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Funding
This work was supported by the Canadian Institutes of Health Research (CIHR; Project Grants PJT-178010 and PJT-165941 to X-L.Z.), the Heart & Stroke Foundation of Canada (G-22-0032035 to X-L.Z.), and the Natural Sciences and Engineering Research Council of Canada (RGPIN-2020-04592 to X-L.Z.).
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Xi-Long Zheng, Jiaxing Sun and Shenghua Zhou conceived and designed the study. Jiaxing Sun, Yu Gui and Yuxin Liu carried out the in vitro experiments and data analysis. Yanan Guo and Jiaxing Sun performed the animal surgery and in vivo data collection. Rosana González Granado, Liam Guetg, and Warren Peng assisted with data interpretation and provided critical reagents. Jiaxing Sun, Xi-Long Zheng and Yu Gui drafted the manuscript with input from all authors. All authors read and approved the final manuscript.
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All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Calgary and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. This article does not contain any studies with human participants; therefore, human consent was not required.
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Healthy, age-matched adult mice as detailed in Materials and Methods.
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Mice with dysplastic teeth.
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Sun, J., Gui, Y., Liu, Y. et al. Edelfosine induces cell cycle arrest and apoptosis in vascular smooth muscle cells to suppress neointimal hyperplasia. Sci Rep (2026). https://doi.org/10.1038/s41598-026-44632-z
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DOI: https://doi.org/10.1038/s41598-026-44632-z
