VEGFA₁₆₅ Regulates Plasmalemma Vesicle-Associated Protein in Primary Human Retinal Endothelial Cells via the p38-MAPK and AKT Signaling Pathways

Abstract

Purpose : Elevated concentrations of Vascular Endothelial Growth Factor (VEGF) decrease blood-retinal barrier properties of the retinal endothelium and increase retinal endothelium permeability. Elevation of total VEGFA165 and switching of VEGFA165 isoform expression from VEGFA165b to VEGFA165a have been reported in the aqueous and vitreous humors of eyes with diabetic retinopathy and ROP. VEGFA165 elevates the concentration of Plasmalemma Vesicle-Associated Protein (PLVAP), a protein which facilitates transcytosis across an endothelial barrier. PLVAP regulation in Human Retinal Microvascular Endothelial Cells (HRMECs) is poorly understood. We hypothesized that both VEGFA165 isoforms increase PLVAP gene expression in primary HRMECs and that one or more of the following intracellular signaling pathways are involved: ERK1/2, AKT, p38-MAPK.

Methods : Primary HRMECs (27 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. 96-well format was used for in situ assays of PLVAP protein content. Pharmacological inhibition of signaling pathways used the following inhibitors: MK2206 (AKT), BIRB796 (p38-MAPK), U0126 (ERK1/2).

Results : Our results show that VEGFA165a (5000 pM) increased PLVAP gene and protein expression by 27% and 63%, respectively, compared to VEGFA165b. AKT pathway inhibition with 0.02 µM MK2206 suppressed VEGFA165a or VEGFA165b-mediated increase of PLVAP gene expression by 51% and 54%, respectively. P38-MAPK pathway inhibition with 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 suppressed VEGFA165a or VEGFA165b-mediated increase of PLVAP gene expression by 73% and 70%, respectively.

Conclusions : VEGFA165a is a more potent 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. Our results implicate PLVAP as a potential contributor to VEGFA165-mediated disruption of the retinal endothelial barrier.

This is a 2020 ARVO Annual Meeting abstract.