Related Experiment Video
Updated: Sep 13, 2026
Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing
Published on: October 11, 2021
Single-Nucleus RNA Sequencing Highlights Endothelial and Müller Cell ER Stress During Retinal Neovascularization in
Angela Galdamez1,2,3, Soyoung Park1,2,3, William Temme1,2,3
1Department of Ophthalmology, Stanford University, Stanford, California, United States.
Purpose:
Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) signaling are implicated in pathologic neovascularization, but their role in the spontaneous intraretinal/subretinal neovascularization in Vldlr-/- mice is unknown. We performed single-nucleus RNA sequencing (snRNA-seq) on adult Vldlr-/- retinas undergoing neovascularization and age-matched Vldlr+/+ control retinas. We examined differentially expressed genes in Vldlr-/- retinal cell types to identify stress signaling pathways selectively engaged in retinal cell types during pathologic neovascularization.
Methods:
The snRNA-seq was performed on P46 Vldlr-/- and Vldlr+/+ retinas, and differentially expressed genes and pathways associated with ER stress and UPR signaling were evaluated in retinal cell populations. KDEL and C/EBP homologous transcription factor protein (CHOP) immunostaining on retinal sections was performed to further identify retinal cell types with increased ER stress. Retinal neovascularization was evaluated by isolectin B4 (IB4) labeling of neovascular tufts and subretinal neovascularization. Retinal function was assessed by electroretinography (ERG), including oscillatory potentials.
Results:
IB4 staining revealed robust neovascular tufts and abnormal vessel extension into the outer retina/subretinal space. ERG and oscillatory potentials showed significantly reduced retinal function. The snRNA-seq revealed significant upregulation of ER stress/UPR signaling-associated genes selectively in endothelial cells and Müller cells. Strong KDEL and CHOP immunoreactivity was seen in IB4-positive abnormal vessels and activated Müller processes, supporting increased ER stress in these retinal cell populations.
Conclusions:
Endothelial cells and Müller cells show increased ER stress and UPR activation in Vldlr-/- retinas undergoing pathological neovascularization.
