Differential Activation of p38-MAPK by VEGFA165_b and VEGFA165_a Results in Different Migration Activity of Primary Human Retinal Endothelial Cells

Abstract

Purpose : Reports of switching from the expression of VEGFA165_b to VEGFA165_a in the vitreous of patients with Diabetic Retinopathy and Retinopathy of Prematurity (ROP) led us to ask if this isoform shift could potentially contribute to VEGF-driven pathology. We compared the activation kinetics of the p38-MAPK pathway and its contributions to proliferation and migration by both VEGFA165 isoforms in primary human retinal microvascular endothelial cells (HRMECs) using in situ cellular analysis methodologies.

Methods : For both VEGFA165_b and VEGFA165_a, dose-response curves for activation of the p38-MAPK pathway were determined using in situ infrared immunofluorescence assays. VEGFA165 isoforms were also compared for their effects on cell proliferation, measured with infrared-fluorescence Cell-Tag assays. Cell migration was compared using fluorescent transmembrane migration assays in the multiwell plate format. Relative contribution of the p38-MAPK pathway to cell proliferation and migration were assessed using pharmacological inhibition of p38-MAPK activation.

Results : VEGFA165_a was a much stronger activator of p38-MAPK at lower doses where VEGFA165_b had little effect (≤1000 pM). VEGFA165_a stimulated cell migration more than 6-fold compared to VEGFA165_b at 1000pM. The p38-MAPK inhibitor SB203580 increased cell proliferation but was also found to be a potent activator of ERK1/2 MAPK, even at lower doses. A more specific inhibitor, BIRB796, completely eliminated VEGFA165_a induced migration. VEGFA165_b was as good or slightly more potent than VEGFA165_a as a stimulator of cell proliferation but was not significantly different between both isoforms.

Conclusions : Dose-response analysis revealed that VEGFA165_a was significantly stronger than VEGFA165_b at activating the p38-MAPK pathway in primary HRMECs. Compared to the a-isoform, the b-isoform is a significantly weaker activator of this and several other intracellular signalling pathways at lower doses. Interestingly, while the b-isoform was not as strong an activator of cell migration, it was as potent as the a-isoform in stimulating cell proliferation. The reason for this is not yet known. We conclude that the reported switching VEGFA165 isoforms in Diabetic Retinopathy and ROP vitreous could have functional consequences because of their significantly different effects upon activation of primary HRMECs.

This is a 2020 ARVO Annual Meeting abstract.