Differences in Activation of Intracellular Signalling in Primary Human Retinal Endothelial Cells Between Isoforms of VEGFA-165.

Abstract

PurposeThere are reports that a b-isoform of Vascular Endothelial Growth Factor-A-165 (VEGFA165b) is predominant in normal human vitreous, switching to the a-isoform (VEGFA165a) in the vitreous of some diseased eyes. While 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 signalling pathways is not fully characterized, especially in retinal endothelial cells. We determined their activation potential for two key intracellular signalling pathways (MAPK, AKT) over complete dose-response curves and compared potential effects on the expression of several VEGFA165 target genes in primary human retinal microvascular endothelial cells (HRMECs). MethodsTo determine full dose-response curves for the activation of MAPK (ERK1/2), AKT and VEGFR2, direct in-cell western assays were developed using primary Human Retinal Microvascular Endothelial Cells (HRMECs). Potential differences in dose-response effects on gene expression markers related to endothelial cell / leukocyte adhesion (ICAM1, VCAM1 and SELE) and tight-junctions (CLDN5 and OCLN) were tested by quantitative-PCR. ResultsActivation 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 VEGFA165a concentrations spanning 10-250 pM, which shifted higher into the 100-5,000 pM range with VEGFA165b. Similar maximum activation of MAPK was achieved by both isoforms at high concentration. Maximum activation of AKT by VEGFA165b was only half of the maximum activation from VEGFA165a. At a lower intermediate dose, where VEGFA165a activated intracellular signalling stronger than VEGFA165b, the changes to VEGFA target gene expression was generally greater with VEGFA165a. ConclusionsIn primary HRMECs, VEGFA165a could maximally activate MAPK and AKT at lower concentrations where VEGFA165b had relatively little effect. The timing for maximal activation of MAPK was similar for both isoforms, which is different than reprorted for non-retinal endothelial cells. While VEGFA165a and VEGFA165b are limited to the sequence of their six C-terminal six amino acids, this results in a large difference in their ablility to activate at least two key intracellular signalling pathways and potentially VEGF target gene expression in primary human retinal endothelial cells.