Objective We have previously shown that genetic deletion of the transcription factor E2F1 increases the expression of vascular endothelial growth factor (VEGF) and enhances blood flow recovery in the ischaemic limb (Qin et al, PNAS 2006). However, the physiological significance of this regulation in ischaemic heart disease and the molecular mechanisms of E2F1-mediated VEGF regulation are still unknown. The purpose of this study is to understand the role of E2F1 in cardiac neovascularization following ischaemic injury.
Methods and results Myocardial infarction (MI) was induced by surgical ligation of the Left Anterior Descending (LAD) coronary artery in wild-type (WT) and E2F1–/– mice. At day 5 after surgery, angiogenic factors at the infarct border zone were analysed by qRT-PCR and Western blotting. At day 28, the vascular density and infarct size were evaluated histologically. VEGF mRNA and protein levels were significantly higher in E2F1–/– than in WT mice (p<0.01, n=5). E2F1–/– mice displayed a greater vessel density in the infarct border area (p<0.01, n=5) and a smaller infarct size (p<0.01, n=15). In vitro, hypoxia treatment (0.5% O2 for 24 h) increased VEGF mRNA expression to a higher level in E2F1–/– cardiac fibroblasts than in WT control cells (p<0.01, n=3). Overexpression of E2F1 suppressed the hypoxia-induced VEGF promoter activity in WT cells, however, (del) but not in p53–/– cells, suggesting that p53 is required for E2F1 to suppress VEGF transcription. Hypoxia treatment (0.5% O2) for 24 h dramatically increased the level of both E2F1 and p53 proteins; overexpression of E2F1 further enhanced the hypoxia-induced accumulation of p53. To understand whether E2F1 regulates p53 protein stability, we treated WT and E2F1–/– cardiac fibroblasts with hypoxia for 6h, pulsed the cells with cyclohexamide (40 mg/ml) and chased p53 degradation. The p53 protein level declined gradually in WT cells (half-life: ∼4 h), but, significantly faster in E2F1–/– cells (half-life: ∼1h) (p<0.01 at 1, 2, and 4 h, n=4). Interestingly, addition of Lactacystin significantly delayed the rates of p53 degradation in both WT and E2F1–/– cells and eliminated the difference between the two groups of cells, suggesting that under hypoxia, E2F1 promotes p53 accumulation by attenuating its ubiquitin-proteasomal degradation. Furthermore, co-immunoprecipitation (co-IP) experiments indicated that hypoxia treatment induced physical associations between E2F1 and p53. In the E2F1–/– fibroblasts transiently transfected with HA-tagged E2F1 or E2F1 truncation mutants, p53 co-precipitated with the ectopically expressed WT E2F1 and the E2F1 mutants with deletions in the transactivaton domain and/or DP-dimerisation domain, but not with the E2F1 mutant with a deletion in the N-terminus (amino acids 1−109), suggesting that the N-terminal region is essential for E2F1 to interact with p53.
Conclusions The E2F1 stabilises p53 protein, thereby suppressing VEGF expression and new vessel formation in the ischaemic heart. Targeting E2F1:p53 interaction (eg, by E2F1 N-terminal peptide) may protect heart from ischaemic injury.
- VEGF expression
- ischaemic myocardium
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