Tissue origin of endothelial cells determines immune system modulation and regulation of HIF-1α-, TGF-β-, and VEGF signaling

Tight junctions of vascular endothelial cells in the central nervous system form the blood-brain and inner blood-retinal barriers, the integrity of which are further influenced by neighboring cells such as pericytes, astrocytes/Mu<euro>ller glial processes, and immune cells. In addition, the retina is shielded from the fenestrated endothelium of the choriocapillaris by the epithelial barrier of the retinal pigment epithelium. Dysfunction of the blood retinal barriers and/or proliferation of retinal and choroidal endothelial cells are caused by late stages of diabetic retinopathy (DR) and neovascular age-related macular degeneration (nAMD), the main causes of blindness in western countries. To elucidate endothelial-derived pathomechanisms in DR and nAMD, we established immortalized mouse cell lines of retinal and choroidal endothelial cells and immortalized brain endothelial cells as CNS-derived controls. We then used immunofluorescence staining, state-ofthe-art long-range RNA sequencing and monolayer permeability assays to compare the functional state of these cells depending on their tissue origin. We furthermore demonstrate that activation of the wingless- type MMTV integration site (Wnt)/b-catenin signaling pathway restored blood brain/retinal barrier properties in brain and retinal endothelial cells, but unexpectedly increased permeability of choroidal endothelial cells. Transcriptome profiling showed that depending on the tissue origin of endothelial cells, regulation of the immune system was altered and pathways such as hypoxia-inducible factor (HIF)-1/2a, transforming growth factor (TGF)-b, and vascular endothelial growth factor (VEGF) were differentially regulated, strongly indicating their contribution in the molecular pathogenesis of DR and nAMD. These findings significantly increase the understanding of the vascular biology of endothelial cells, highlighting the fact that depending on their tissue origin, their contribution to vascular pathologies varies.