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Souska Zandi, Shintaro Nakao, Dawei Sun, Ruth Schmidt-Ullrich, Alexander Schering, Farhad Hafezi, Ali Hafezi-Moghadam; Impact of Endothelium-specific NF-B Signaling on Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5826.
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Choroidal neovascularization (CNV) is the main cause of severe vision loss in patients with age-related macular degeneration (AMD). Whether CNV development is inflammatory and/or immunomodulatory in nature remains unclear. Therefore we examined the CNV progression in floxed IΚBαΔN (loxP-ΔN) mice that have functionally suppressed endothelial NFΚB.
To induce CNV, C57BL/6, Tie-1Cre and Tie-1-ΔN mice were anesthetized and pupils were dilated. Using a 532-nm laser, four spots (100mW, 50µm, 100ms) were placed in each eye. Development of a bubble under laser confirmed the rupture of the Bruch’s membrane. Seven days after laser injury, the size of the CNV lesions was measured in choroidal flat mounts. The volume of the lesions was quantified, using confocal microscopy. The grade of leakage was determined by Fluorescein angiography (FA) 7 days after laser injury.To perform Western Blots, tissues were obtained after perfusion of mice with PBS. Eyes were enucleated immediately after perfusion. Choroids were microsurgically isolated and placed in 100µl of lysis buffer supplemented with protease and phosphatase inhibitors.
In C57BL/6 mice IΚBα phosphorylation peaked 3 days after laser injury, while NF-ΚB p65 phosphorylation levels were detectable after 3 days and its levels were maintained through 7 and 14 days.To investigate the role of NF-ΚB signaling in angiogenic CNV endothelium, we used the Tie-1-ΔN mouse, generated from breeding of the floxed IΚBαΔN (loxP-ΔN) mouse with the Tie-1-Cre knock-in mice and compared the CNV volume in these mice with controls. Neither CNV volume, nor leakage was reduced in the Tie-1-ΔN compared to WT or in the Tie-1Cre control mice (n=5, 40 lesions per group).
This work elucidates the impact of NFΚB in CNV development. We show that in Tie-1-ΔN mice that have a functionally suppressed endothelial NFΚB, CNV is the same as in control. Our results challenge the conventional wisdom of AMD being primarily an inflammatory disease.
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