The Soluble Notch Ligand Dll4 Restricts Choroidal Neovascularisation by Inhibiting Endothelial Cell Proliferation

Abstract

Purpose: : The Notch signaling pathway regulates pathological angiogenesis and inflammation. DLL4 interaction with Notch-1 and 4 inhibits vascular endothelial cell proliferation and DLL4 activates macrophages via Notch-1. Vascular endothelial cells and macrophages are the main players in laser induced choroidal neovascularization (CNV) in age related macular degeneration (AMD). We studied Notch expression in murine and human eyes and analyzed the effect of DLL4 on vascular endothelial cells, macrophages and CNV.

Methods: : Expression of Notch-1, 4, DLL4 and target genes were studied in human umbilical aortic endothelial cell (HUAEC) and macrophages in vitro and normal and pathological human and mouse eyes (qRT-PCR, western blot analysis and immunohistochemistry). The effects of DLL4 in vitro on HUAEC and on peritoneal macrophages and in vivo on CNV in mice was analysed by immunohistochemistry and qRT-PCR.

Results: : Notch-1 is expressed on microglia and choroidal macrophages. Neurons, blood vessels, microglia and choroidal macrophages express Notch-4. In patients with wet form of AMD Notch-4 was detected in vessels in the choroid and in subretinal neovascularization. In laser induced CNV; VEGF and HES1 a target gene of the Notch signaling pathway, were overexpressed in the mouse choroid. Intravitreal injection of DLL4 reduced the area of neovascularisation 14 days following CNV. In vitro DLL4 reduced proliferation of HUAEC. In contrast, DLL4 induced pro-angiogenic genes (VEGF, and IL-1β) in macrophages in vitro and in retina and choroid in vivo (1h after laser injury).

Conclusions: : Expression of Notch-1 and 4 in blood vessels and macrophages during CNV suggests a possible role of Notch signalling in the regulation of CNV in AMD. In mice, intravitreal injection of DLL4 reduces laser induced CNV. The anti-angiogenic effect of DLL4 is likely mediated by a direct inhibitory effect on endothelial cell proliferation and not via the regulation of macrophage secretion of growth factors. Notch modulation by DLL4 might represent a promising alternative strategy to reduce ocular neovascularisation during AMD and other neovascular diseases.

Keywords: age-related macular degeneration • choroid: neovascularization • inflammation