Pharmacological Inhibition of Dll4 Ameliorates Pathological Retinal Neovascularization and Improves Retinal Revascularization in the OIR Model

Abstract

Purpose:: We previously reported that Delta-like 4 ligand is prominently expressed in the vascular endothelium of retinal arteries and capillaries, most strongly in the tips and stalks of angiogenic sprouts. Genetic deletion of a single Dll4 allele resulted in a significant increase in vessel sprouting, indicating that Dll4 is a potent endogenous inhibitor of angiogenesis (IOVS 2006 47: E-Abstract 3877). To evaluate the therapeutic potential of Dll4 inhibition in ocular vascular diseases, we tested the effects of a Dll4-Fc chimeric recombinant protein and an anti-Dll4 blocking antibody on normal retinal vascular development, and in the murine model of oxygen-induced retinopathy (OIR).

Methods:: OIR was produced following the method developed by Smith et al. (1994). Intravitreal microinjections were performed using a Drummond nanoinjector equipped with a glass needle. Lycopersicon esculentum (LE) lectin labeled with Texas Red was injected into the left cardiac ventricle and allowed to circulate for 5 minutes to assess blood vessel patency. After enucleation, retinas were dissected, stained with FITC-labeled Griffonia simplicifolia lectin I to visualize the vasculature and flat mounted.

Results:: Pharmacological inhibition of Dll4/Notch signaling by intravitreal administration of either soluble Dll4-Fc or a blocking antibody against Dll4 produced the same set of characteristic abnormalities in the developing retinal vasculature described previously for genetic deletion of a single Dll4 allele. Most notably Dll4 blockade enhanced angiogenic sprouting and endothelial cell proliferation, resulting in the formation of a denser and more highly interconnected superficial capillary plexus. In the OIR model, inhibition of Dll4/Notch signaling also enhanced angiogenic sprouting and the re-growth of superficial retinal vessels, while suppressing ectopic pathological neovascularization (intravitreal tufts) and the formation of retinal arterial-venous shunts. Perfusion-staining with LE lectin showed that the new blood vessels formed during Dll4 inhibition are functional.

Conclusions:: Pharmacological inhibition of Dll4/Notch interaction may afford a novel approach to treatment of retinal diseases characterized by vascular pathology and/or insufficient angiogenesis.

Keywords: retina • retinal neovascularization • growth factors/growth factor receptors