eNOS controls angiogenic sprouting and retinal neovascularization through the regulation of endothelial cell polarity

Abstract

The roles of nitric oxide (NO) and endothelial NO synthase (eNOS) in the regulation of angiogenesis are well documented. However, the involvement of eNOS in the sprouting of endothelial tip-cells at the vascular front during sprouting angiogenesis remains poorly defined. In this study, we show that downregulation of eNOS markedly inhibits VEGF-stimulated migration of endothelial cells but increases their polarization, as evidenced by the reorientation of the Golgi in migrating monolayers and by the fewer filopodia on tip cells at ends of sprouts in endothelial cell spheroids. The effect of eNOS inhibition on EC polarization was prevented in Par3-depleted cells. Importantly, downregulation of eNOS increased the expression of polarity genes, such as PARD3B, PARD6A, PARD6B, PKCΖ, TJP3, and CRB1 in endothelial cells. In retinas of eNOS knockout mice, vascular development is retarded with decreased vessel density and vascular branching. Furthermore, tip cells at the extremities of the vascular front have a marked reduction in the number of filopodia per cell and are more oriented. In a model of oxygen-induced retinopathy (OIR), eNOS deficient mice are protected during the initial vaso-obliterative phase, have reduced pathological neovascularization, and retinal endothelial tip cells have fewer filopodia. Single-cell RNA sequencing of endothelial cells from OIR retinas revealed enrichment of genes related to cell polarity in the endothelial tip-cell subtype of eNOS deficient mice. These results indicate that inhibition of eNOS alters the polarity program of endothelial cells, which increases cell polarization, regulates sprouting angiogenesis and normalizes pathological neovascularization during retinopathy.

The injured sciatic nerve atlas (iSNAT), insights into the cellular and molecular basis of neural tissue degeneration and regeneration

“…Because SNC inflicts vascular damage, we searched CellChat for injury-induced angiogenic signaling pathways; top hits include VEGF, ANGPT (angiopoietin), ANGPTL (angiopoietin-like proteins), SEMA3 (semaphorin 3), EPHA, and EPHB (ephrin receptors). Independent evidence for angiogenic sprouting is the upregulation of endothelial NO synthase ( Nos3 ) in EC of the injured nerve ( Smith et al, 2021 ). While the importance of angiogenesis and angiogenic factors in PNS repair has been appreciated, CellChat informs on specific ligand–receptor systems and shows which cell types produce these factors and how expression is regulated upon nerve injury.…”

Inducible miR-1224 silences cerebrovascular Serpine1 and restores blood flow to the stroke-affected site of the brain

“… 69 This, unlike observations discussed above in the tumor biology setting, assigns an angiostatic function to PAI-1. 69 , 70 Both eNOS and tPA are negatively regulated by PAI-1. Thus, low PAI-1 conditions would favor optimal and augment functioning of eNOS and tPA simultaneously.…”

Identification of Arhgef12 and Prkci as genetic modifiers of retinal dysplasia in the Crb1rd8 mouse model

“…Crb1 transcripts are detected in retinal progenitor cells is expressed starting at embryonic day 11.5 [ 89 ]. Crb1 is also expressed in vascular endothelial cells of the mouse retina and upregulated under conditions associated with increased endothelial cell polarization [ 90 ]. Mouse PRKCI is widely distributed among retinal progenitor cells at the apical surface of the NBL at P0, but mainly in the inner nuclear and ganglion cell layers at P9 [ 69 ].…”

“…Mouse PRKCI is widely distributed among retinal progenitor cells at the apical surface of the NBL at P0, but mainly in the inner nuclear and ganglion cell layers at P9 [ 69 ]. Like Crb1 , Prkci transcripts are detected in retinal endothelial cells and increase in abundance with cell polarization [ 90 ]. There is genetic evidence that PRKCI functions in these cells [ 91 ].…”