The a-isoform of VEGFA165 is a Significantly Stronger Activator of Human Retinal Endothelial Cells compared to the b-isoform

Studies show that the 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 eyes with active diabetic retinopathy or ROP. The potential of this isoform-switching to impact the retinal vasculature is not clear, particularly in endothelial cells which are important targets of VEGFA-mediated activation. We wished to determine if significant differences exist in the ability of these two isoforms to activate intracellular signalling and gene expression changes in retinal endothelial cells. Effects of VEGFA165 isoforms (a/b) on the retinal vasculature were tested by intravitreal injection in rats using fluorescein-angiography and Optical Coherence Tomography to monitor primary vein dilation and retinal edema. Dose response curves for activation of MAPK (ERK1/2), AKT and VEGFR2 were determined using primary Human Retinal Microvascular Endothelial Cells (HRMECs). The ability of both isoforms to alter gene expression markers related to endothelial cell / leukocyte adhesion and tight-junctions was tested by quantitative-PCR. In rats, dilation of primary retinal veins and edema could be induced within 24 hours by intravitreal injection of sufficient amounts of either isoform. Both isoforms activated MAPK and AKT in primary HMRECs, however dose-response analysis revealed much stronger activation of MAPK, AKT and VEGFR2 by the a-isoform. The a-isoform was also more effective at increasing ICAM1, VCAM1 and SELE gene expression and decreasing CLDN5 and OCLN gene expression in primary HRMECs. In conclusion, VEGFA165a could maximally activate the MAPK and AKT intracellular kinases in HRMECs at lower concentrations where VEGFA165b had little effect. Shifts of VEGFA165 expression from mostly b-isoform to mostly a-isoform in some human retinal vascular diseases could potentially contribute to VEGFA-driven pathology through differential effects on the activation of retinal endothelial cells.