VEGFA₁₆₅ Regulation of PLVAP and Early Permeability of Primary Human Retinal Endothelial Cells via the p38-MAPK and AKT Signaling Pathways

Abstract

Purpose : Vascular endothelial growth factor (VEGFA₁₆₅) is elevated and exhibits changes in isoform expression (from VEGFA₁₆₅b to VEGFA₁₆₅a) in the vitreous of patients with diabetic retinopathy and retinopathy of prematurity. VEGFA₁₆₅ elevates the gene expression and protein concentration of Plasmalemma Vesicle-Associated Protein (PLVAP), a protein which facilitates transcytosis across an endothelial barrier. The aim of this study was to understand how VEGFA₁₆₅ mediated activation of the AKT and p38-MAPK intracellular signaling pathways are involved in the regulation of PLVAP and permeability of primary human retinal microvascular endothelial cells (HRMECs).

Methods : Primary HRMECs (26 year old male) were cultured using EndoGRO-LS 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) increased PLVAP gene expression 15- and 7.5-fold, respectively at 24 hrs (early), but continued to increase at 48 hrs. VEGFA₁₆₅a and VEGFA₁₆₅b increased PLVAP protein content over 14- and 5-fold by 48 hrs. Inhibition of AKT and p38-MAPK together suppressed VEGFA₁₆₅a and VEGFA₁₆₅b-mediated increase of early PLVAP gene expression by 73% and 70%, respectively. Blocking either pathway did not prevent changes to junctional gene-expression. VEGFA₁₆₅a and VEGFA₁₆₅b increased early transcellular permeability by 22% and 40% respectively, and inhibition of the AKT and p38-MAPK pathways did not prevent the this permeability increase.

Conclusions : VEGFA₁₆₅a was a stronger activator of PLVAP gene and protein expression relative to VEGFA₁₆₅b, suggesting the reported isoform switching in retinal vascular diseases could contribute to disease mechanisms. While most of the VEGFA₁₆₅-mediated increase to PLVAP gene expression involves the p38-MAPK and AKT pathways, these pathways were not implicated in the VEGFA₁₆₅a-mediated early (24 hr) increase to HRMEC permeability. This suggests that junctional disruption instead of PLVAP may be involved in early VEGFA₁₆₅a mediated increase HRMEC permeability at the 70 kDa molecular weight range.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.