VEGFA165 Activation of PLVAP Expression Utilizes the p38-MAPK and AKT Signaling Pathways in Primary Human Retinal Endothelial Cells

Abstract

Purpose : Elevation of total VEGFA165 and switching of VEGFA165b isoform expression to the VEGFA165a isoform have been reported in the aqueous and vitreous humors of eyes with diabetic retinopathy and ROP. VEGFA165a elevates the gene expression and protein concentration of Plasmalemma Vesicle-Associated Protein (PLVAP), a protein involved in transcytosis across the endothelial barrier. PLVAP regulation in primary Human Retinal Microvascular Endothelial Cells (HRMECs) is poorly understood. We hypothesize that both VEGFA165 isoforms increase permeability and PLVAP gene and protein expression in primary HRMECs, and AKT and p38-MAPK intracellular signaling pathways are involved in PLVAP gene expression in primary HRMECs.

Methods : Primary HRMECs (26 year old male) were cultured using EndoGRO-LS Complete Culture Media, in 6-well format for total RNA extraction. T-25 flasks were used for PLVAP immunoblotting. 24-well 0.4 µM pore polyester transwell inserts with 70 kDa RITC dextran were used for transcellular permeability assays. Pharmacological inhibition of signaling pathways used MK2206 (AKT) and BIRB796 (p38-MAPK) inhibitors.

Results : VEGFA165a and VEGFA165b (5000 pM, saturating) increased PLVAP gene expression over 19-fold and 13-fold, respectively. VEGFA165a and VEGFA165b (5000 pM) increased PLVAP protein expression over 14-fold and 5-fold, respectively. AKT pathway inhibition (0.02 µM MK2206) suppressed VEGFA165a or VEGFA165b -mediated increase of PLVAP gene expression by 51% and 54%, respectively. P38-MAPK pathway inhibition (0.01 µM BIRB796) suppressed VEGFA165a or VEGFA165b -mediated increase of PLVAP gene expression by 63% and 58%, respectively. Double inhibition of the AKT and p38-MAPK pathways further suppressed VEGFA165a or VEGFA165b -mediated increase of PLVAP gene expression by 73% and 70%, respectively. VEGFA165a and VEGFA165b (5000 pM) increased transcellular permeability by 22% and 40%, respectively.

Conclusions : VEGFA165a was a stronger activator of PLVAP gene and protein expression relative to VEGFA165b. Differences between the effects of VEGFA165a and VEGFA165b on PLVAP gene and protein expression suggest that the reported isoform switching in retinal vascular diseases could contribute to disease mechanisms. Most of the VEGFA165-mediated increase to PLVAP gene expression involves the p38-MAPK and AKT pathways.

This is a 2021 ARVO Annual Meeting abstract.