VEGFA Isoform Switching in Diabetic Retinopathy and ROP is a Significant Factor in the Activation of Human Retinal Endothelial Cells.

Abstract

Purpose : Reports of switching from the expression of VEGFA165b to VEGFA165a in the vitreous of patients with Diabetic Retinopathy and Retinopathy of Prematurity led us to ask if this isoform shift contributes significantly to VEGF-driven pathology. Our initials studies reveal a large difference in dose response for the activation of VEGFR-2 in human retinal microvascular endothelial cells (HRMECs). We asked if these dose-response differences are transmitted to the level of gene expression.

Methods : For both VEGFA165a and VEGFA165b, dose-response curves (DRC) for the activation of VEGFR2 and three downstream signalling kinases were determined using intracellular infra-red immunofluorescence assays. Based on DRC data, several doses were selected, spanning concentrations with significant differences in dose-response between the two isoforms. These doses were used to stimulate HRMECs. RNA was prepared 6 hours after VEGFA treatment. First strand cDNA was synthesized and used for Taqman gene expression assays of markers for blood-retinal-barrier (BRB) status and the activation of leukocyte-endothelial cell-adhesion.

Results : VEGFA165a was a much stronger activator of VEGFR2 at lower doses compared to VEGFA165b. This difference was also seen in the activation of MAPK, AKT and p38MAPK. Both isoforms of VEGFA165 reduced Claudin-5 and Occludin gene expression, with VEGFA165a having a stronger effect than VEGFA165b at the same dose. Both isoforms could increase ICAM1 and Selectin-E gene expression but VEGFA165a was far more potent at lower doses where VEGFA165b had little effect on the expression of these genes.

Conclusions : We found very significant differences in the dose-response for activation of HRMECs at three regulatory levels. 1) Receptor activation. 2) Downstream activation of intracellular signalling. 3) The expression of VEGF target-genes that regulate the BRB and leukocyte-endothelial cell-adhesion. We conclude that the reported isoform switching from the b-isoform to the a-isoform of VEGFA165 in Diabetic Retinopathy and ROP vitreous is a significant factor that drives loss of the Blood-Retinal Barrier. This switch changes what would be otherwise mild effects from activation by VEGFA165b, to stronger activation of retinal endothelial cells. Isoform switching from VEGFA165b to VEGFA165a would be an excellent target for therapeutic development.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.