Abstract
Purpose :
Erythropoietin (EPO) has been implicated in pathologic and physiologic angiogenesis in retinopathy of prematurity (ROP). We found EPO receptor (EPOR)-mediated signaling protected against hyperoxia-induced vascular loss and increased intraretinal, but not intravitreal, vascular growth. We addressed the hypothesis that the scaffolding protein, MEMO1, is involved in EPO-mediated signaling in angiogenesis.
Methods :
Human retinal microvascular endothelial cells (HRMECs, passage 5) maintained in growth media with 10% FBS were transfected with MEMO1 or control siRNAs for 48 hrs. HRMECs were serum-starved overnight after 36 hrs of transfection and treated with control (PBS) or EPO (2 U/mL) for 30, 60, or 120 min. Cell lysates were processed for western blots. 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 mL subretinal lentiviruses to deliver plasmids with Cdh5 promoter-driven microRNA30 embedded short hairpin RNAs (shRNAs) to MEMO1 (L-MEMO1shRNA) or control luciferase (L-LUCshRNA). At p10, 12, and 14, pups received intraperitoneal EPO or saline. At p14, pups were moved to room air and euthanized at p20. Areas of intravitreal neovascularization (IVNV) and total retina were measured in isolectin-B4-stained retinal flat mounts and analyzed as IVNV/total retinal area (%IVNV). All statistical analyses were with multilevel linear regression models using Stata-18 software.
Results :
Compared to control, MEMO1 knockdown reduced STAT3 (p<0.05) and Src (p<0.05) activation after 60 min of EPO treatment. Compared to L-LUCshRNA, %IVNV was greater in L-MEMO1shRNA after saline (p<0.05). Compared to saline, there was no difference in %IVNV after EPO in L-LUCshRNA but a trend toward reduced %IVNV after EPO in L-MEMO1shRNA (p=0.173).
Conclusions :
MEMO1 mediated EPO-induced STAT3 and Src activation in HRMECs, but also appeared to be somewhat protective against IVNV in rat OIR. Further studies are required to understand interactions between EPO and MEMO1 in angiogenesis.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.