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Lili Xu, Douglas McMahon, John S Penn; Retinal Clock Genes Influence Retinal Neovascularization. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5384.
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Multiple circadian clock genes can interact with the hypoxic signaling pathway in non-ocular tissues. Here we tested whether retinal circadian clock genes including Period1 and 2, Bmal1, and RORa are critical modulators of retinal vascularizing responses and may be important participants in proliferative neovascularizing diseases. Müller cells are an important source of VEGF and vascularizing signals in the retina. We used both a cellular model of cultured retinal Müller cells and the Oxygen Induced Retinopathy (OIR) model in intact mice to test this hypothesis.
Primary Müller cells were purified from KO and WT mice. 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 conditions; P7 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, protein level, bioluminescent and neovascularization were measured in mouse Müller cells and retina by RT-PCR, Western blot, LumiCycle and whole-mount retina staining.
VEGF expression is significantly increased in Per1/Per2 KO Müller cells in hypoxia. In vivo, consistent with in vitro, Per1/Per2 KO mice in response to the OIR protocol exhibited higher retinal VEGF levels and blunted VEGF rhythms. OIR-induced neovascularization was significantly increased in Per1/Per2 KO mice at P18 when measured as proportion of vascular retinal area or whole retinal area. HIF1a levels in hypoxia-treated Müller cell cultures were significantly decreased in Per1/Per2 KO compared to WT. Compared to WT; Bmal1 mRNA was dramatically increased in Per1/Per2 KO Müller cells in hypoxia and decreased in normoxia. RORa was increased in both of WT and KO Muller cells in hypoxia and increased in KO Muller cells in normoxia and hypoxia.
These data indicate that the Period clock genes influence the retinal neovascularizing response and suggest that retinal PERIOD proteins may interact with HIF1a to affect protein stability. As the circadian clock gene network influences retinal neovascularizing responses, it may represent unexplored molecular targets for intervention in neovascularizing diseases.
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