NDRG1 activates VEGF-A-induced angiogenesis through PLCγ1/ERK signaling in mouse vascular endothelial cells

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1−/− mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling.


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October 2018
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All studies must disclose on these points even when the disclosure is negative. No data were excluded from the analyses.
All attempts at replication were successful. Each individual experiment was repeated independently with similar results at least three times.
Age/sex matched litter mates were assigned to groups by genotype. Groups were not randomized.
In vivo analysis was performed by experimenters who were blinded to the identity of the groups.
All antibodies (supplier name, catalog number/clone name, dilution) are provided in the Methods section under "Reagents and antibodies" as follows: Polyclonal antibody against full-length Ndrg1 (1:1000) was a kind gift from Dr. Kokame (National Cerebral and Cardiovascular Center, Suita, Japan). We purchased anti-mouse VEGFR-2 antibody ( Primary antibodies were validated for use based on the position the antigen in SDS-PAGE gels. We also used the NDRG1 KO or siRNAs to specifically deplete endogenous protein to verify anti-NDRG1 antibodies. All other commercial antibodies were validated by manufacturers.
The cell lines used are described in the Methods section under "Cell culture" as follows: We obtained murine RENCA cells from the American Type Culture Collection (Manassas, VA). We cultured cancer cells in RPMI-1640 medium (Nissui, Tokyo, Japan) supplemented with 10% FBS (Hyclone, Logan, UT). We cultured mouse lung ECs in endothelial cell basal medium (EBM-2) supplemented with the EGM-2MV Bullet Kit (15% FBS) (CC-3202; Lonza Walkersville Inc, Walkersville, MD). We purchased HUVECs from Lonza Walkersville Inc. and cultured them in EBM-2 supplemented with