Objectives Hypoxia inducible factor-1α (HIF-1α) is the key transcription regulator for multiple angiogenic factors, including vascular endothelial growth factor (VEGF). Cellular repressor of E1A-stimulated genes (CREG) has also been identified a potent promoter of angiogenesis. However, the mechanisms by which CREG promotes angiogenesis are not fully understood. Here, we show that CREG is an effective stimulator of HIF-1α under hypoxia in bone marrow-derived mesenchymal stem cells (BMSCs).
Methods All experiments were performed on rat BMSCs. The level of VEGF was measured by ELISA. The HIF-1α mRNA was analysed by RT-PCR. The level of HIF-1α protein and the mechanisms mediating these proangiogenic effects were determined by Western blotting.
Results We found that VEGF release from BMSCs was significantly increased in parallel with high level of HIF-1α in BMSCs following anoxia or hypoxia in time-dependent manner. Furthermore, the level of VEGF released from BMSCs overexpressing CREG and the expression of HIF-1α in BMSCs overexpressing CREG were higher than the normal BMSCs under hypoxia. Rather, HIF-1α steady-state mRNA was also affected by CREG. This effect was associated with constitutive activation of phosphatidylinositol 3-kinase (PI3K)/Akt and its effector p70 S6 kinase (p70S6K), but not extracellular-signal regulated kinase 1/2. The use of small molecule inhibitors LY294002 or rapamycin to inhibit PI3K/Akt and p70S6K activities, respectively, resulted in diminished HIF-1α activation and subsequent VEGF expression. RNA interference-mediated knockdown of HIF-1α suppressed CREG-induced VEGF synthesis and angiogenic tube formation, confirming that the effect was HIF-1α specific.
Conclusions Overexpressing CREG promote hypoxia-induced VEGF paracrine via HIF-1α in mesenchymal stem cells. therefore, CREG could play a major role in angiogenesis and vascular remodelling.