Fig 1.
EPO-B and EPO-D inhibits VSMCs proliferation.
(A-B) VSMCs were cultured in 96-well culture plate for 24 h, then serum starved for 12h. Cells were treated with 0.1 to 100 nM of EPO-B (A) and EPO-D (B) or 0.1% DMSO (vehicle) for 24h, cells were stimulated with PDGF-BB (50 ng/mL), and cell proliferation was then determined by BrdU incorporation assay (n = 8 each) at 24, 48, and 72 h intervals. Data shown as mean ± SEM. #P<0.05 indicate significantly different from serum-free/DMSO-treated control and *P<0.05 indicate significantly different from PDGF-BB/DMSO-treated control. (C) VSMCs were treated with EPO-B and EPO-D (1 to 100 nM) or 0.1% DMSO in serum-free media for 24h, then stimulated with PDGF-BB (50 ng/mL). After 24 h, cells were lysed and PCNA and p21 expression was analyzed by Western blot.
Fig 2.
EPO-B and EPO-D induces apoptotic cell death and mitotic catastrophe in VSMCs.
(A-B) VSMCs were cultured in an eight chambered glass culture slide, and then serum starved for 12h, cells were then treated with 1 to 100 nM of (A) EPO-B and (B) EPO-D or 0.1% DMSO (vehicle) for 24h, cells were stimulated with PDGF-BB (50 ng/mL). After 24h, cell apoptosis was determined by TUNEL-assay. The apoptotic index was determined as the TUNEL-positive cell number divided by the total cell number (n = 3 each). Data are shown as mean ± SEM. *P<0.05 indicate significantly different from DMSO-treated control cells. (C) Cell mitotic catastrophe were determined by nucleus DAPI-staining (blue) at 24 h post-PDGF-BB-stimulation. Mitotic catastrophe was characterized by cytoplasmic blebbing, condensation and irregularities in shape under fluorescence microscopy (magnification, 40×). The arrows indicated the mitotic catastrophe changed cells. Representative images of each experimental group are shown.
Fig 3.
EPO-B and EPO-D induces apoptosis through p53 activation in VSMCs.
(A) VSMCs were cultured in an eight chambered glass culture slide, and then serum starved for 12h. After treatment of EPO-B and EPO-D (1 to 100 nM), or 0.1% DMSO (vehicle) for 24h, cells were stimulated with PDGF-BB (50 ng/mL) for 24 h. Activation of p53 and microtubule polymerization were determined by immunofluorescence staining with p53 (red) and β-tubulin (green) antibodies. Nuclei were stained with DAPI (blue). Images shown are representative confocal-laser-scanning microscope (magnification, 63×). (B) VSMCs were treated with EPO-B and EPO-D (1 to 100 nM), or 0.1% DMSO in serum-free media for 24h, then stimulated with PDGF-BB (50 ng/mL). After 24 h, cells were lysed and phosphorylated p53 (p-p53, Ser15) and total p53 (t-p53) expression was analyzed by Western blot. To clarify the involvement of p53 on EPOs-induced apoptosis, cells were treated with EPO-B and EPO-D (10 nM) with/without 1–10 μM pifithrin-α, a p53 inhibitor, (C) or p53 siRNA (D) for 24 h, and were then stimulated with 50 ng/ml PDGF-BB for 24 h. The percent of VSMCs apoptosis was measured by TUNEL-assay. Data are shown as mean ± SEM (n = 3). *P<0.05 indicate significantly different from EPOs-alone treated control cells.
Fig 4.
EPO-B and EPO-D induces p-53 dependent apoptotic signaling pathway.
(A) VSMCs were treated with EPO-B and EPO-D (1 to 100 nM), or 0.1% DMSO in serum-free media for 24h, then stimulated with PDGF-BB (50 ng/mL). After 24 h, cells were lysed and Bax, mitochondrial cytochrome c release and cleaved caspase-3 expressions were analyzed by Western blot. MF: mitochondrial fraction, CF: cytosolic fraction. (B-C) VSMCs were cultured in an eight chambered glass culture slide, and then serum starved for 12h. After treatment of EPO-B and EPO-D (10 nM), or 0.1% DMSO (vehicle) for 24h, cells were stimulated with PDGF-BB (50 ng/mL) for 24 h. For immunofluorescence staining, cells were processed for double immunofluorescence labeling with antibodies against (B) Bax (red) or (C) cleaved-caspase-3 (red), and β-tubulin (green). Images were acquired using a confocal-laser-scanning microscope and the fluorescent micrographs (magnification, 63×). (D) Cells were starved for 12 h prior to the treatment with EPO-B or EPO-B (10 nM) with/without 10 μM pifithrin-α (p53 inhibitor). After 24h, the cells were stimulated with 50 ng/mL of PDGF-BB for 24h, and processed for single immunofluorescence labeling (active-caspase-3, red).
Fig 5.
Treatment of EPO-B and EPO-D inhibits neointimal hyperplasia and induces apoptosis in the regions of neointimal hyperplasia.
(A) Carotid sections of each indicated groups were stained with PCNA antibody. Images shown are representative microscopy images (n = 5 each; magnification, 4×). The lower panels show magnified views of neointimal hyperplasia regions. Nuclei (blue) and protein expression (brown) are shown. L = lumen of the artery. (B) Neointima/Medial area ratio was quantified. Data shown as mean ± SEM. *P<0.05 indicate significantly different from injured-control group. (C) Apoptotic cell death in carotid tissues (n = 5) was determined by in situ TUNEL-assay (green). Images shown are representative fluorescence microscopy images (magnification, 20×). Blue: DAPI and lined-green: autofluorescent elastic lamina. (D) The apoptotic index was determined as the TUNEL-positive cell number (lower panels) divided by the total cell number (DAPI-stained cells, upper panels). Data are shown as mean ± SEM (n = 5). *P<0.05 indicate significantly different from balloon-injured control group.
Fig 6.
Treatment of EPO-B and EPO-D increase the expressions of p53 and active-caspase-3 in the regions of neointimal hyperplasia.
Carotid sections of each indicated groups were stained with p53 (A) or active-caspase-3 (B) antibodies. Images shown are representative microscopy images (n = 5; magnification, 20×). The lower panels show magnified views of neointimal-hyperplasia regions (magnification, 40×). Nuclei (blue) and protein expression (brown) are shown. L = lumen of the artery.