Expression data from isolated mouse lung endothelial cells

Exaggerated signaling by vascular endothelial growth factor (VEGF) A and its receptor, VEGFR2, in pathologies results in poor vessel function. Still, pharmacological suppression of VEGFA/VEGFR2 may aggravate disease. Delineating VEGFR2 signaling in vivo provides strategies for suppression of specific VEGFR2-induced pathways. Three VEGFR2 tyrosine residues (Y949, Y1212, Y1173) induce downstream signaling. Here, we show that knock-in of phenylalanine to create VEGFR2 Y1212F in C57Bl/6 and FVB mouse strains leads to loss of growth factor receptor bound (GRB)2- and phosphoinositide 3´kinase (PI3K)p85-signaling. C57Bl/6 Vegfr2Y1212F/Y1212F show reduced embryonic endothelial cell (EC) proliferation and partial lethality. FVB Vegfr2Y1212F/Y1212F show reduced postnatal EC proliferation. Reduced EC proliferation in Vegfr2Y1212F/Y1212F explants is rescued by c-Myc overexpression. We conclude that VEGFR2 Y1212 signaling induces activation of extracellular-signal regulated kinase (ERK)1/2 and Akt pathways required for c-Myc-dependent gene regulation, endothelial proliferation and vessel stability. We use microarray to determine the differences in gene expression in endothelial cells between WT or Vegfr2Y1212F/Y1212F mice. Mouse lung ECs were isolated from WT or Vegfr2Y1212F/Y1212F mice (both C57Bl/6 and FVB strains) using MACS lung disassociation kit and CD31 microbeads. The cells from each mouse were starved for 3 hours in MV2 medium with 0.2% FCS and then either left untreated (basal) or treated with VEGFA (50 ng/mL) for 1 or 3 hours. Microarray was performed by using ClariomTM D Assays, mouse (ThermoFisher Scientific) with RNA samples from 5 WT and 5 Vegfr2Y1212F/Y1212F mice for each time point.