VEGF stimulated the angiogenesis by promoting the mitochondrial functions

Abstract

The vascular endothelial growth factor (VEGF) signaling pathway involved in angiogenesis which plays a pivotal role in normal development and also represents a major therapeutic target for tumors and intraocular neovascular disorders. The aims of the present study were to evaluate the effects of VEGF on endothelial cells and clarify the mechanism. Here, we showed that VEGF significantly stimulated the proliferation, migration and cell cycle of endothelial cells, and it also induced tube formation in vitro significantly. What's more, the mitochondrial functions were enhanced in response to VEGF, including mitochondrial oxidative respiration and intracellular ATP levels. The reactive oxygen species (ROS) production decreased, while the enzymes of ROS defence system, including catalase and glutathione peroxidase (GPX1), whose expression both increased in the VEGF stimulation. VEGF activated mammalian target of rapamycinm (mTOR) signaling pathway to promote the function of mitochondria. Rapamycin, the inhibitor of mTOR pathway could inhibit the proliferation and cell cycle induced by VEGF. In summary, our study identified that VEGF promoted the angiogenesis and provided evidence for mitochondria as new therapeutic target of VEGF signaling in the angiogenic vascular disorders.

Keywords: VEGF; angiogenesis; mitochondria.

Figure 1. VEGF significantly promoted the proliferation, migration, angiogenesis and cell cycle of endothelial cells

(A) The proliferation of endothelial cells treated with different concentration of VEGF (n=12). (B) Migration of HUVEC in the absence and presence of VEGF (20 ng/ml) was assessed by wound healing assay. a) Representative micrographs of wound healing assays at 0 hrs and 8 hrs after creating a wound field. Scale bar, 200um. b) Quantitative assessment of percentage of cells migrating into the wound field (n=3). (C) PI staining followed by flow cytometry was used to assess cell cycle. Cell cycle phase length was calculated from the percentage of cells present in each cell cycle phase (n=3). (D) ECs were seeded on extracellular matrigel and exposed to VEGF treatments. a) Representative bright field micrographs of EC matrigel angiogenesis after 5h of VEGF treatment. b) Quantitative analysis of the number of branching points and total tube length (n=3). Scale bar, 100um. Results represented mean ± SEM of n independent experiments. **P<0.01, *P<0.05, 1-way ANOVA.

Figure 2. VEGF enhanced the mitochondrial function

(A) Oxygen consumption rate (OCR) in control and VEGF-treated ECs using by seahorse. Statistics of FCCP-coupled OCR was shown in the graph (n=10). (B) Intracellular ATP levels in control and VEGF-treated ECs (n=16). (C) Catalase and GPX1 mRNA were analysed by means of quantitative RT-PCR. Relative expression values of the VEGF-untreated cells were taken as 1.0 (n=4). (D) ROS staining was performed using DCFH-DA in ECs in the absence and presence of VEGF. Left: Representative images of ROS staining; right: Statistics of ROS fluorescence intensity in per cell (n=8). Scale bar, 100um. Results represented mean ± SEM of n independent experiments. **P<0.01, *P<0.05, 1-way ANOVA.

Figure 3. VEGF activated mTOR signaling pathway

(A) HUVECs cultured in M199 medium with 5%FBS were induced with VEGF for 24 hours. P-S6 and S6 were determined by western blots. (B) HUVECs cultured in M199 medium with 5%FBS were induced with VEGF and rapamycin for 24 hours. P-S6 and S6 were determined by western blots. (C) Cell proliferation in cultured HUVECs treated with VEGF and the inhibitor of mTOR signaling, rapamycin (50 nM) for 24 hours (n=12). (D) Cell cycle in cultured HUVECs treated with VEGF and an inhibitor of mTOR signaling, rapamycin (50 nM) for 24h determining by PI staining (n=3).