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Guorui Dou, Xingcheng Zhao, Na Li, Yusheng Wang, Hua Han; Inhibition of ocular angiogenesis by the DSL domain of Dll1 targeted to endothelial cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5396.
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Several therapies targeting ocular angiogenesis have been developed. However, poor response in some cases and emerging resistance necessitate further investigations of new drug targets. Recent progress highlights Notch signaling in ocular vascular development and pathologic angiogenesis. As blocking Notch pathway results in the formation of vascular neoplasm, activation of Notch pathway to prevent ocular angiogenesis might be an alternative choice. However, an in vivo deliverable reagent with highly efficient Notch-activating capacity has not been developed. Here, we generated a polypeptide, D1R, which consists of the Delta-Serrate-Lag-2 fragment of the Notch ligand Delta-like 1 and an arginine-glycine-aspartate (RGD) motif targeting endothelial cells (ECs), to investigate its role on mice models of choroidal neovascularization (CNV) and oxygen induced retinopathy (OIR) by specific activation of Notch signaling in EC.
D1R was manufactured in E.Coli. The specification of this recombinant to EC was observed by adherence assay. Its Notch activating activity was detected in HUVECs and confirmed by the downstream genes. The roles of D1R and DAPT (Notch signal inhibitor) on vascular growth were observed in neonatal C57BL/6J mice injected with D1R subcutaneously. Bead assay, transwell chamber and tube formation were conducted to study biological behaviors of HUVEC treated by D1R. Mice with laser-induced CNV were injected with Dll1-RGD intravitrously and choroid flatmount were examined 7 days later. Neonatal C57BL/6J mice was taken into 75% oxygen chamber from P7-P12 and were injected with Dll1-RGD on P12. The area of retinal angiogenesis was analyzed on P17.
We showed that D1R could bind to ECs specifically through its RGD motif and effectively triggered Notch signaling in ECs. Both in vitro and in vivo D1R inhibited angiogenic sprouting and EC proliferation. In mice CNV model D1R effectively repressed CNV growth, most likely through decreasing EC proliferation and sprouting. In OIR model treated with D1R, the amount and width of neo-vessels in non-perfusion area were remarkably reconstructed. Moreover, these vessels in regrowth area of OIR mice recruited more NG2(+) perivascular cells and were better perfused.
Our data provide Dll1-RGD as a potential therapeutic strategy, by enhancing Notch signaling in EC, to inhibit ocular angiogenesis and promote vascular reconstruction.
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