Abstract
Vascular endothelial growth factor A (VEGF-A) is one of the most important factors controlling angiogenesis. Although the functions of exogenous VEGF-A have been widely studied, the roles of endogenous VEGF-A remain unclear. Here we focused on the mechanistic functions of endogenous VEGF-A in endothelial cells. We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. Endogenous VEGF-A also controls expression of key endothelial specific genes including VEGFR-2, Tie-2, and vascular endothelial cadherin. Of importance, endogenous VEGF-A differs from exogenous VEGF-A by regulating VEGFR-2 transcription through mediation of FoxC2 binding to the FOX:ETS motif, and the complex formed by endogenous VEGF-A with VEGFR-2 is localized within the EEA1 (early endosome antigen 1) endosomal compartment. Taken together, our results emphasize the importance of endogenous VEGF-A in endothelial cells by regulating key vascular proteins and maintaining the endothelial homeostasis.
Highlights
Vascular endothelial growth factor A (VEGF-A) is an important mediator of angiogenesis; the role of endogenous VEGF-A in endothelial cells is unclear
For Western Blotting—VEGF receptor 2 (VEGFR-2) and VEGFR-1 primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.); -actin was purchased from Sigma; p-VEGFR-2 Y1059 and Tie-2 were purchased from Upstate Biologicals (Millipore, Lake Placid, NY); p-VEGFR-2 Tyr-1175, Src, p-Src Tyr-416, Tyr-527, p-paxillin Tyr-118, PLC␥, p-PLC␥ Tyr-783, and VEcadherin were purchased from Cell Signaling; paxillin was purchased from BD Biosciences
Endogenous VEGF-A Controls VEGFR-2 Expression and VEGFR-2 Downstream Signaling—To investigate the mechanistic functions displayed by endogenous VEGF-A in endothelial cells, we utilized siRNA to knock down endogenous VEGF-A in Human Umbilical Vein Endothelial Cells (HUVECs)
Summary
VEGF-A is an important mediator of angiogenesis; the role of endogenous VEGF-A in endothelial cells is unclear. Results: Endogenous VEGF-A maintains expression of VEGFR-2 and other endothelial-specific proteins via transcriptional regulation. We focused on the mechanistic functions of endogenous VEGF-A in endothelial cells We found that it is complexed with VEGF receptor 2 (VEGFR-2) and maintains a basal expression level for VEGFR-2 and its downstream signaling activation. A new transcriptional enhancer regulated by the FOX:ETS motif has been recently discovered and shown to be sufficient to direct expression and exclusively to the developing vascular endothelium [21]. This transcriptional control has not yet been connected with VEGF-A. Lack of intracellular VEGF-A can influence VEGFR-2 expression regardless of outside conditions and eventually influences endothelial functions
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