Endocrinol Metab.  2022 Oct;37(5):800-809. 10.3803/EnM.2022.1462.

DN200434 Inhibits Vascular Smooth Muscle Cell Proliferation and Prevents Neointima Formation in Mice after Carotid Artery Ligation

Affiliations
  • 1Research Institute of Aging and Metabolism, Kyungpook National University, Daegu, Korea
  • 2Department of Internal Medicine, Kyungpook National University Hospital, School of Medicine, Kyungpook National University, Daegu, Korea
  • 3Department of Biomedical Science, Graduate School, Kyungpook National University, Daegu, Korea
  • 4New Drug Development Center, Daegu Gyeongbuk Medical Innovation Foundation, Daegu, Korea
  • 5Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Chilgok Hospital, Daegu, Korea

Abstract

Background
Excessive proliferation and migration of vascular smooth muscle cells (VSMCs), which contributes to the development of occlusive vascular diseases, requires elevated mitochondrial oxidative phosphorylation to meet the increased requirements for energy and anabolic precursors. Therefore, therapeutic strategies based on blockade of mitochondrial oxidative phosphorylation are considered promising for treatment of occlusive vascular diseases. Here, we investigated whether DN200434, an orally available estrogen receptor-related gamma inverse agonist, inhibits proliferation and migration of VSMCs and neointima formation by suppressing mitochondrial oxidative phosphorylation.
Methods
VSMCs were isolated from the thoracic aortas of 4-week-old Sprague-Dawley rats. Oxidative phosphorylation and the cell cycle were analyzed in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated VSMCs using a Seahorse XF-24 analyzer and flow cytometry, respectively. A model of neointimal hyperplasia was generated by ligating the left common carotid artery in male C57BL/6J mice.
Results
DN200434 inhibited mitochondrial respiration and mammalian target of rapamycin complex 1 activity and consequently suppressed FBS- or PDGF-stimulated proliferation and migration of VSMCs and cell cycle progression. Furthermore, DN200434 reduced carotid artery ligation-induced neointima formation in mice.
Conclusion
Our data suggest that DN200434 is a therapeutic option to prevent the progression of atherosclerosis.

Keyword

Muscle, smooth, vascular; Neointima; Estrogen-related receptor gamma protein; Oxidative phosphorylation; Mechanistic target of rapamycin complex 1

Figure

  • Fig. 1. DN200434 inhibits fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cell (VSMC) proliferation and cell cycle progression. (A) Chemical structure of DN200434. (B, C) Inhibitory effects of DN200434 on FBS-stimulated (B) and PDGF-stimulated (C) VSMC proliferation. (D, E) Representative flow cytometric data and cell cycle distribution analysis of FBS-stimulated (D) and PDGF-stimulated (E) VSMCs treated with or without DN200434. Data are expressed as the mean±standard error of the mean (n=3 technical replicates). PI, propidium iodide. aP<0.001; bP<0.0001.

  • Fig. 2. DN200434 reduces mitochondrial oxidative phosphorylation in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs). (A, C) The oxygen consumption rates (OCRs) of FBS-stimulated (A) and PDGF-stimulated (C) VSMCs in the presence or absence of DN200434 were determined following sequential injection of oligomycin, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and rotenone. (B, D) Basal respiration, maximal respiration, and adenosine triphosphate (ATP)-linked respiration were plotted in bar charts using the data in Fig. 2A and C. Data are expressed as the mean±standard error of the mean (n=3 technical replicates). aP<0.05; bP<0.01; cP<0.001.

  • Fig. 3. DN200434 decreases mammalian target of rapamycin complex 1 (mTORC1) activity and attenuates migration in fetal bovine serum (FBS)- or platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs). (A, B) Representative western blots showing the effects of DN200434 on expression of phospho-70 kDa ribosomal protein S6 kinase (p70S6K), phospho-4E-binding protein (4E-BP), and proliferating cell nuclear antigen (PCNA) in FBS-stimulated (A) and PDGF-stimulated (B) VSMCs. (C, D) Effects of DN200434 on expression of cyclin D1 in FBS-stimulated (C) and PDGF-stimulated (D) VSMCs. (E, F) Transwell migration (upper) and wound healing (lower) assays showing the effect of DN200434 on migration of FBS-stimulated (E) and PDGF-stimulated (F) VSMCs. Data in the bar graphs are mean±standard error of the mean of three independent experiments. aP<0.01; bP<0.001; cP<0.0001.

  • Fig. 4. DN200434 attenuates carotid artery ligation-induced neointimal hyperplasia. (A) Representative cross-sections of mouse carotid arteries stained with H&E (upper) and Elastic van Gieson (EVG; lower) in the sham, ligation only, and ligation with DN200434 treatment groups. Scale bars represent 50 µm. (B) The intimal area, medial area, and ratio of the intimal area to the medial area were evaluated by morphometric analysis. (C) Immunohistochemical (IHC) staining of phospho-4E-binding protein 1 (4E-BP1; T37/46) and proliferating cell nuclear antigen (PCNA) in mouse carotid arteries. Scale bars represent 20 µm. (D) Cells immunohistochemically positive for phospho-4E-BP (T37/46) and PCNA in arteries were quantified. Data are expressed as the mean±standard error of the mean (n=4 per group). NS, not significant. aP<0.05; bP<0.01; cP<0.001; dP<0.0001.


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