Abstract
Purpose :
VEGF signaling is involved in pathologic intravitreal neovascularization (IVNV) and normal intraretinal vascularization in retinopathy of prematurity (ROP). We found VEGF-induced VEGFR2 activation reduced intraretinal vascularization but increased IVNV. We addressed the hypothesis that a scaffolding protein, MEMO1, is necessary for VEGFR2-triggered signaling in IVNV.
Methods :
Human retinal microvascular endothelial cells (HRMECs, passages 3-5) maintained in growth media with 10% FBS, were transfected with MEMO1 or control siRNAs for 48 hrs. RNA from HRMEC lysates were evaluated for transfection efficiency with RT-PCR. In parallel experiments, HRMECs were serum-starved overnight after 36 hrs of transfection and treated with control (PBS) or VEGF (25 ng/mL) for 30, 60, or 120 min. Cell lysates were processed for western blots. Scratch assays were performed 36 hrs after transfection and overnight serum starve, followed by treatment with VEGF (25 ng/mL) or PBS. Images were captured every hour for 24 hrs with phase-contrast microscopy. Newborn Sprague Dawley rat pups were placed into the 50/10 oxygen-induced retinopathy (OIR) model at birth (Biospherix). At postnatal day (p) 8, pups received bilateral 1 μL subretinal lentiviruses to deliver plasmids with VE-cadherin promoter driven microRNA30 embedded short hairpin RNAs (shRNAs) to MEMO1 (L-MEMO1shRNA) or a control luciferase (L-LUCshRNA). At p14, pups were moved to room air and euthanized at p20. IVNV to total retina ratio was measured in isolectin-B4-stained retinal flat mounts. All statistical analyses were with multilevel linear regression models using Stata-17 software.
Results :
Compared to control, MEMO1 knockdown reduced STAT3 (p<0.05) and VEGFR2 activation (p<0.05) after 30 min of VEGF; increased Src activation (p<0.05) after 1 hour of VEGF; and increased VEGFR2 activation (p<0.05) and Src activation (p<0.05) after 2 hrs of VEGF treatment. MEMO1 siRNA transfected, VEGF-treated HRMECs demonstrated greater wound closure (p=0.14) than control siRNA transfected, VEGF-treated HRMECs 24 hrs after scratch-induced injury. IVNV was greater in L-MEMO1shRNA than L-LUCshRNA at p20 (p=0.12).
Conclusions :
Our findings suggest MEMO1 limits sustained VEGF-triggered signaling in HRMECs and IVNV. Further investigations involving downstream effectors are required to determine if MEMO1 is a potential therapeutic target in ROP.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.