Unveiling Endothelial Cell Expression Profiles in FEVR: Identification of Key Genes Associated With Pathological Neovascularisation in a FZD4Mouse Model.

Abstract

BACKGROUND: Familial exudative vitreoretinopathy (FEVR) is a disorder of retinal blood vessel development characterised by impaired retinal angiogenesis. While FZD4 mutations are established genetic causes of FEVR, the molecular mechanisms underlying pathological neovascularisation remain poorly understood. METHODS: CRISPR/Cas9 was used to generate the Fzd4mouse model. Comprehensive vascular phenotyping entailed retinal wholemount imaging, analysis of hyaloid vessel regression and immunohistochemical evaluation of the deep retinal vasculature. Flow cytometry sorting and single-cell RNA sequencing (scRNA-seq) were used to characterise the expression profiles of retinal endothelial cells (ECs) in mice. RESULTS: The FZD4model recapitulated key FEVR features including retinal aneurysmal vessels in adult mice, developmental abnormalities in juveniles, impaired deep vascular formation, delayed hyaloid regression, vascular leakage and reduced endothelial proliferation. ScRNA-seq revealed distinct transcriptional profiles in pathological EC clusters and tip cells. KEGG analysis revealed that the EC clusters were enriched in focal adhesion, Rap1, PI3K-Akt and apelin signalling pathways, whereas tip cells were enriched in the VEGF, chemokine, NF-κB and TNF signalling pathways. We further identified four novel candidate markers of pathological neovascularisation: Ctss, Ccl4, Ccl3 and Apoe. Subsequent validation confirmed the upregulation of CTSS and CCL4 within neovascular lesions, supporting their functional role in pathological angiogenesis. CONCLUSIONS: We established a novel mouse model of pathological neovascularisation with a missense mutation and elucidated the expression profiles of retinal ECs. The identified pathways and novel biomarkers-particularly CTSS and CCL4-provide new insights for further pathogenesis investigation of FEVR.