Differences in activation of intracellular signaling in primary human retinal endothelial cells between isoforms of VEGFA 165

Purpose: There are reports that a b-isoform of vascular endothelial growth factor-A 165 (VEGFA(165)b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA(165)a) in the vitreous of some diseased eyes. Although these isoforms appear to have a different ability to activate the VEGF receptor 2 (VEGFR2) in various endothelial cells, the nature of their ability to activate intracellular signaling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signaling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA(165) target genes in primary human retinal microvascular endothelial cells (HRMECs). Methods: To determine full dose-response curves for the activation of MAPK (ERK1/2), AKT, and VEGFR2, direct in-cell western assays were developed using primary HRMECs. Potential differences in dose-response effects on gene expression markers related to endothelial cell and leukocyte adhesion (ICAM1, VCAM1, and SELE) and tight junctions (CLDN5 and OCLN) were tested with quantitative PCR. Results: Activation dose-response analysis revealed much stronger activation of MAPK, AKT, and VEGFR2 by the a-isoform at lower doses. MAPK activation in primary HRMECs displayed a sigmoidal dose-response to a range of VEGFA(165)a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA(165b). Similar maximum activation of MAPK was achieved by both isoforms at high concentrations. Maximum activation of AKT by VEGFA(165)b was only half of the maximum activation from VEGFA(165)a. At a lower intermediate dose, where VEGFA(165)a activated intracellular signaling stronger than VEGFA(165)b, the changes in VEGFA target gene expression were generally greater with VEGFA(165)a. Conclusions: In primary HRMECs, VEGFA(165)a could maximally activate MAPK and AKT at lower concentrations where VEGFA(165)b had relatively little effect. The timing for maximum activation of MAPK was similar for the isoforms, which is different from that reported for non-retinal endothelial cells. Although differences in VEGFA(165)a and VEGFA(165)b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ability to activate at least two key intracellular signaling pathways and VEGF-target gene expression in primary human retinal endothelial cells.