Figure 1.
Senescent cells accumulated in heart after infarction.
(A) Myocardial infarction (MI) was induced by ligation of left coronary artery (LAC) in mice. After 1 week of MI, the heart sections were evaluated by hematoxylin/eosin (H&E) and Masson staining. (B) Senescent cells were detected by SA-β-Gal staining in the heart (left). Bar graph shows the percentage of SA-β-Gal positive cells in the heart sections (right). (C) A panel of senescence markers, including p16, p19, p21 and p53, was immunostained with specific antibodies (left). Scale bars: 50 µm. The percentage of positive cells was measured by selecting 6 random fields. Bar graphs show the percentage of p16-, p19-, p21- and p53-positive cells in heart sections (right). Data expressed as mean±SEM (n=5 per group). *P<0.05, **P<0.01, ***P<0.001 vs. sham.
Figure 2.
Senescent cells derive from cardiac myofibroblasts.
(A, B) Heart tissues at day 7 after infarction were double-stained using anti-p53 or p16 (markers for senescence, green) and anti-a-SMA and DDR2 (markers for myofibroblasts) antibodies, and counterstained with DAPI (blue), and then examined by a fluorescence microscopy. (C) The heart sections were immunostained using the combination of anti-p53 (green) and anti-Troponin (a marker for cardiomyocytes, red) antibodies and counterstained with DAPI (blue), and examined by a fluorescence microscopy. Scale bars: 50 µm. (D) Bar graph shows the percentage of staining positive cells.
Figure 3.
Hypoxia/reoxygenation induces senescence in cardiac fibroblasts.
(A) Neonatal cardiac fibroblasts from wild-type (WT) mice were treated with hypoxia/reoxygenation (H/R) for 0-10 days. The growth of viable cells was measured by using Trypan blue staining. Bar graphs show the number of viable cells at day 0-10 of H/R treatment. (B) Cells were culture as in A for 3 days. Cell proliferation was measured by using BrdU incorporation assay. Bar graphs show the percentage of BrdU positive cells. (C) Morphology and SA-b-Gal staining of fibroblasts treated with hypoxia were viewed and performed (left). Bar graph shows the percentage of SA-β-Gal positive cells (right). (D) Cells treated with hypoxia were subjected to immunostaining using anti-p16, p19, p21 and p53 (markers of senescence). DAPI was used for counterstaining (left). Bar graphs show the percentage of senescence marker positive cells (right). (E, F) qPCR analysis was used to quantify the mRNA expression of MMP2, MMP9, collagen I and collagen III in fibroblasts treated with hypoxia. Bar graphs show the relative mRNA levels in hypoxia-treated cells compared with normoxia group. (G) p53 protein levels were detected by Western Blot analysis in fibroblasts treated with hypoxia for 3 h, 6 h and 3 d. Bar graphs show the quantitative analysis of p53 protein (right). Scale bars: 50 µm. Data expressed as mean±SEM (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. normoxia.
Figure 4.
Effects of p53 on cell senescence and p21 expression in cardiac fibroblasts induced by hypoxia/reoxygenation.
(A) Cardiac fibroblasts were infected with scrambled siRNA (Scr-siRNA, 100 nmol/L), p53-siRNA (100 nmol/L), or adenovirus GFP control (Ad-GFP, MOI=50) or p53 (Ad-p53, MOI=50) for 24 h and then exposed to hypoxia/reoxygenation (H/R) for 3 days. The infection efficiency was detected by Western blot analysis using anti-p53 antibody. (B) Senescent cells were detected by SA-β-Gal staining (left). Bar graph shows the percentage of SA-β-Gal-positive cells (middle and right). (C) Senescent cells were subjected to immunostaining using anti-p21 antibody (top). DAPI was used for counterstaining (middle). Bar graph shows the percentage of p21-positive cells (bottom). Scale bars: 50 µm. Data expressed as mean±SEM (n=3). ***P<0.001 vs. normoxia; *P<0.05, **P<0.01 vs. Scr-siRNA+hypoxia or Ad-GFP+hypoxia.
Figure 5.
Effect of p53 on the expression of inflammatory factors.
(A) Myocardial infarction (MI) was induced by ligation of left coronary artery (LAC) in mice for 1 week. The mRNA expression of IL-6, IL-8 and CXCL1 was examined by qPCR analysis in the heart. Bar graphs show the relative mRNA levels in p53 KO mice compared with WT mice. Data expressed as mean±SEM (n=5 per group). (B) Cardiac fibroblasts were treated as in Figure 4A. The protein levels of IL-6, IL-11, CXCL1, MCP-1, GCP-2, M-CSF and CXCL2 were measured by Bio-Plex assay kit. (D) Cardiac fibroblasts were treated as in Figure 5A. The protein levels of IL-6, IL-11, CXCL1, MCP-1, GCP-2, M-CSF and CXCL2 were measured as in B. Data expressed as mean±SEM (n=3). ***P<0.001 vs. normoxia; *P<0.05, **P<0.01, ***P<0.001 vs. Scr-siRNA+hypoxia or Ad-GFP+hypoxia.
Figure 6.
Deficiency of p53 inhibits fibroblast senescence but enhances cardiac fibrosis after infarction.
(A) Wild-type (WT) and p53 knockout (p53 KO) mice underwent left coronary artery ligation for 7 days. Heart sections were stained by SA-β-Gal kit (left). Bar graph shows the percentage of SA-β-Gal-positive cells in the heart (right). Heart sections were examined using Masson’s trichrome (B) or Sirius Red staining (C) (left). Bar graphs show the areas of collagen deposition in the heart (right). (D) Heart sections were stained by immunohistochemistry with anti-Mac-2 antibody (left). Bar graph shows the percentage of Mac-2 positive cells in the heart. Scale bars: 50 µm. (E, F) qPCR analysis was used to quantify the mRNA expression of MMP2, MMP9, collagen I and collagen III in the heart tissue. Bar graphs show the relative mRNA levels in p53 KO mice compared with WT mice. Data expressed as mean ± SEM (n=5 per group). *P<0.05, **P<0.01 vs. WT+MI.