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Lili Xu, John Penn, Douglas McMahon; Circadian Clock Gene Regulation of Retinal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2013;54(15):606.
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Abnormal neovascularization of the retina is a fundamental cause of blindness in a number of eye diseases including retinopathy of prematurity (ROP). Expression and secretion of VEGF by Müller glial cells is a critical element in the development of aberrant neovascularization. We have shown previously that mouse and human retinal Müller cells exhibit self-sustained clock gene and these rhythms were suppressed by treated with siRNA knockdown of Per1. Here we demonstrated that genetic knockout of the specific circadian clock genes Period1 (Per1) and Period2 (Per2) results in increased hypoxia-induced VEGF in Müller cells and an increased neovascularization phenotype in the mouse oxygen-induced retinopathy (OIR) model.
Purified primary Müller cells from KO and WT were cultured in low-glucose DMEM medium containing 10% FBS at 37°C in a 5% CO2 incubator. The cells were serum starved for 12 hours and then were grown at 37°C for 24 hours in growth medium under both normoxic and hypoxic (O2 < 5%) conditions; P7 KO and WT pups with nursing mothers were subjected to hyperoxia (75% oxygen) or room air for 5 days and then oxygen-exposed mice were returned to room air. Gene expression, vascular development and neovascularization were measured in mouse Müller cells and retinas by quantitative RT-PCR and whole-mount retina staining on vary time points, respectively.
The baseline VEGF expression in WT and KO Müller cells did not differ, but was significantly increased in KO cells in hypoxia. There was a day/night difference in VEGF expression levels in WT cells in hypoxia. In vivo, consistent with in vitro, WT and KO mice had similar baseline levels of VEGF expression and angiogenic development, but KO mice in OIR protocol exhibited higher retinal VEGF levels and blunted VEGF rhythms. Vascular development of WT and KO retinas did not differ in OIR or control mice at P13, but OIR-induced neovascularization was significantly increased in KO mice at P18 when measured as proportion of vascular retinal area or whole retinal area.
These data suggest that the Period clock genes act to negatively regulate VEGF are protective against neovascularization, and may constitute novel targets for intervention in the neovascularization process. We will determine that precise clock genes are critical in regulating VEGF, and determine if Müller cell clocks are the key locus of clock gene regulation of neovascularization.
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