Abstract
Purpose:
Period gene knockout mice have an increased hypoxia-induced retinal neovascularization phenotype. Müller cells (MMC) are an important source of VEGF in the retina. The retinal endothelial cell (MEC) is a key participant in retinal ischemic vasculopathies. We will test the hypotheses that retinal circadian clock genes including Period1 and 2, Bmal1, VEGF and RORa are critical modulators of retinal vascularizing responses and may be important participants in proliferative neovascularizing diseases. We will use both a cellular model of cultured MMC/MEC and the Oxygen Induced Retinopathy (OIR) model in intact mice, combined with genetic and molecular manipulations to test this hypothesis.
Methods:
Purified MEC were cultured in EBM medium containing 10% FBS and EGM kit. MMC 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, or transfected with siRNA. Gene expression and protein levels were measured by quantitative RT-PCR and western blot.
Results:
The baseline VEGF and RORa expression in WT and Per1 KD MEC did not differ, but were significantly increased in hypoxia and in Per1 siRNA KD, also, they are significantly increased in Per1/Per2 KO MMC in hypoxia; consistent with in vitro results, KO mice in response to the OIR protocol exhibited higher retinal VEGF levels and blunted VEGF rhythms. Per1 mRNA was significantly increased in hypoxia and significantly decreased in Per1 siRNA KD in hypoxia. It is interesting that there was no amplification of Per2 mRNA and Bmal1 mRNA in MEC suggesting low expression. HIF1a levels in hypoxia-treated MMC cultures are significantly decreased in Per1/Per2. Bmal1 mRNA was dramatically increased in Per1/Per2 KO MMC treated in hypoxia and decreased in normoxia.
Conclusions:
These data suggest that mouse retinal PERIOD proteins may interact directly with HIF1a to affect protein stability; circadian clock gene network influences retinal neovascularizing responses, and may therefore represent significant unexplored molecular targets for intervention in neovascularizing diseases. The Period clock genes act to negatively regulate VEGF and RORa are protective against neovascularization, and may constitute novel targets for intervention in the neovascularization process.