Abstract
Purpose :
Endomucin (EMCN) is a type I integral membrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that EMCN knockdown in vitro significantly inhibits VEGF165-induced VEGFR2 internalization and downstream activities including proliferation, migration and tube formation. The goal of this study is to characterize the role of EMCN in normal retinal vascular development and in pathologic neovascularization using the EMCN knock-out mice.
Methods :
Homozygous EMCN knock-out (EMCN-/-) mice were obtained by crossing EMCN-floxed mice with the ROSA26-Cre strain. Eyes from adult (8-16 weeks) EMCN-/- mice and EMCN+/+ control littermates were collected and retinas and RPE/choroids complex were dissected for RNA extraction, and qPCR to determine gene expression. Eyes from P5 mice and adult mice (12-16 weeks) were collected for retinal flat mounts; retinal vasculature was stained with Isolectin-B4 (IB4). For the oxygen-induced retinopathy (OIR) model, P7 mice were housed in 75% oxygen for five consecutive days and returned to room air at P12, eyes at P12 and P17 were collected and the retinal vasculature was stained with IB4 and the avascular and neovascular areas were quantified using photoshop.
Results :
EMCN mRNA in both retinas and RPE/choroids from the EMCN-/- mice was undetectable (n>4, p<0.0001), compared to EMCN+/+ mice by qPCR. The area of retinal vascularization at P5 was significantly lower in the EMCN-/- pups compared to EMCN+/+ controls (0.14±0.01 vs 0.2±0.013, p<0.0001, n>10 for both groups). Adult retinal vascular density remained lower in the EMCN-/- mice compared to EMCN+/+ mice (0.135 0.015± vs 0.154±0.017, p<0.05, n= 13). In the OIR model, the avascular area generated by high oxygen exposure at P12 was similar in the EMCN-/- and EMCN+/+ mice (24.57±1.4% vs 23.18±1.0%, p=0.9, n>6). However, pathological neovascularization at P17 was significantly reduced in the EMCN-/- mice compared to EMCN+/+ (8.98 ± 2.9% vs 11.98±1.4%, p<0.05, n>10) while the avascular area at P17 was comparable between the EMCN-/- and controls (9.9± 1.5% vs 8.85±1.0%, p>0.5, n>10).
Conclusions :
Genetic ablation of the EMCN gene reduces retinal vascularization under normal and pathological conditions. As a critical regulator of retinal angiogenesis, EMCN represents a novel therapeutic target for ocular diseases characterized by pathological blood vessel growth.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.