Unveiling Endothelial Cell Expression Profiles in FEVR : Identification of Key Genes Associated With Pathological Neovascularisation in a FZD4 M105V Mouse Model

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: mouse 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: model 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.