Endomucin deletion leads to disorganized choroidal capillary fenestration and reduces laser-induced choroidal neovascularization in mice

Abstract

Purpose : The type I integral membrane glycoprotein endomucin (EMCN) is selectively expressed by capillary and venous endothelium. We have previously reported that knockdown of EMCN in vitro prevents VEGF165-induced VEGFR2 internalization and downstream activities. This study aims to investigate the effect of EMCN deletion on the choriocapillaris and angiogenesis in laser-induced model of choroidal neovascularization (CNV).

Methods : Global EMCN knock-out (EMCN^-/-) mice were generated by crossing EMCN-floxed mice with ROSA26-Cre mice. Choroid, retina, kidney, lung, spleen, liver and thyroid were harvested and mRNA levels of EMCN were examined. Eyes from adult EMCN^-/- mice and EMCN^+/+ littermates were fixed for transmission electron microscopy (TEM). CNV was induced in EMCN^+/+ and EMCN^-/- mice (6-8 weeks old) using 532 nm laser photocoagulation. Mice were imaged using fluorescein angiography (FA) and optical coherence tomography (OCT) seven days after laser and then sacrificed. The lesion leakage was calculated by subtracting the integrated intensity of the earlier time point from the later time point. Expression of IB4, VEGF and Iba1 were examined on retinal pigment epithelium/choroid flat mounts using an SP8 confocal microscope.

Results : The choroid had the highest level of EMCN mRNA level of the tissues examined (n=4). The fenestrations of the choriocapillaris of EMCN^-/- mice were disorganized and the endothelial cells were thickened compared to wildtype (n=3) on TEM. EMCN^-/- mice had significantly reduced CNV leakage compared to EMCN^+/+ mice (22.2 x 10⁴ ± 2.2 x 10⁴ pixels vs 41.06 x 10⁴ ± 3.6 x 10⁴ pixels, n=24, p<0.001, Student’s t-test). EMCN^-/- mice had significantly smaller lesion area compared to EMCN^+/+ mice on OCT (2.7 x 10⁴ ± 841.2 um² vs 3.5 x 10⁴ ± 1119 um², n=24, p<0.0001, Student’s t-test). Quantification of IB4-stained lesion area corroborated the OCT findings confirming that EMCN^-/- mice had smaller lesions compared to EMCN^+/+ mice(2.2 x 10⁴ ± 1639 um² vs 3.2 x 10⁴ ± 2226 um², n=23, p<0.001, Student’s t-test).

Conclusions : Lack of EMCN in vivo leads to disorganized fenestrations in choroidal endothelium, structures known to be dependent on continuous signaling. In addition, the absence of EMCN interferes with angiogenesis and permeability, processes that are driven by VEGF signaling, pointing to EMCN as an alternative therapeutic target.

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