Transcriptome analyses of immortalized retinal, brain and choroidal endothelial cells indicate alterations in immune system modulation and regulation of HIF 1α-, TGFβ- and VEGF- signaling.

Abstract

Purpose : Diabetic retinopathy (DR) and neovascular age-related macular degeneration (nAMD) are leading causes of blindness in developed countries. To elucidate the pathomechanisms in DR and nAMD, knockout and transgenic mice are being used. Cultures of retinal and choroidal endothelial cells from these mice would be valuable tools to study endothelial-driven aspects of disease progression in vitro. Here, we describe a protocol to selectively immortalize and expand mouse retinal and choroidal endothelial cells. Moreover, we provide their in-depth transcriptional characterization and functional permeability data.

Methods : Using the polyoma virus middle T antigen we immortalized retinal, choroidal and brain endothelial cells from 129SV mice. Immunofluorescence staining, RNA sequencing analyses and monolayer permeability assays were performed to study their endothelial characteristics and their blood brain/retinal barrier properties.

Results : Immunofluorescence staining and RNA sequencing analyses confirmed essential characteristics of endothelial cells. Functional data from monolayer permeability assays initially demonstrated comparable permeabilities, but activation of the Wnt/β-catenin signaling pathway restored blood brain/retinal barrier properties in brain and retinal endothelial cells, but increased permeability of choroidal endothelial cells. RNA sequencing showed striking transcriptional similarities of retinal and brain endothelial cells but identified 493 differentially expressed genes (DEG) between choroidal and retinal/brain endothelial cells. These DEGs contribute to processes such as modulation of the immune system and dysregulation of pathways such as HIF 1/2α-, TGFβ- and VEGF- signaling.

Conclusions : Our protocol can be used to establish retinal and choroidal endothelial cell cultures from virtually any mouse line. Excitingly, the transcriptional profile of retinal/brain endothelial cells points towards certain differentially regulated pathways that may drive the molecular pathogenesis of DR and nAMD. Moreover, the cells might represent new experimental tools to investigate endothelial-derived pathomechanisms of DR and nAMD and can furthermore be used to screen for new therapeutic options.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.