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S. K. Mitter, H. Vittal Rao, J. Cai, P. Thampi, X. Qi, W. A. Dunn, M. B. Grant, M. E. Boulton; Nitric Oxide Affects the Circadian Rhythmicity of Autophagy in Retinal Microvascular Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5624.
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Autophagy plays a critical role in cellular housekeeping in non-dividing and long-lived cells. Nitric oxide (NO) has been shown to play a critical role in the regulation of both autophagy and circadian rhythm in non-vascular cells. The aim of this study was to determine if autophagy exhibited circadian rhythmicity in retinal microvascular endothelial cells and how this was affected by changing NO levels.
Retinal microvascular endothelial cells were exposed to 100µM dexamethasone for 2 hr to resets the peripheral clock. Once the clock had been reset, retinal microvascular endothelial cells were exposed to the NO donor DETA or NO inhibitor L-NAME (1 - 100 µM) and samples taken every 2 hours for up to 48 hours. QRT-PCR was used to analyze expression of the clock genes Bmal1 and Per2 and values normalized against GAPDH. To determine autophagy flux, microvascular endothelial cells were transfected with an RFP-GFP-LC3 construct prior to resetting the clock and the number of autophagosomes under the different treatments determined. The expression of autophagy proteins Atg7, LC3 and Beclin were determined by Western blot. siRNA against Bmal 1 was transfected into RMECs followed by detecting autophage flux.
Dexamethasone reset the peripheral clock for at least 48 hours. RT-PCR for Bmal1 and Per2 showed that both clock genes exhibited a circadian rhythm with Bmal1 peaking at 18 and 42 hr post reset and Per2 peaking at 4 and 28 hours. NO had a significant effect on the timing and height of the circadian peaks. The NO donor caused Bmal1 to peak later and greatly reduced the amplitude of the Per2 peak. The NO inhibitor abolished the Bmal1 peak and caused irregular expression of Per2. The number of autophagosomes showed a circadian rhythm with a peak in autophagy flux at 4 hours and 28 hours and was confirmed by an increased expression of autophagy proteins at these peak periods. siRNA knockdown of Bmal1 significantly reduced the peak amplitude in autophagy and made it broader. Similarly, the amplitude of the peak was significantly reduced in the presence of the NO inhibitor.
We conclude that autophagy flux is under circadian regulation in retinal microvascular endothelial cells and this is regulated by NO levels. Since diabetics show abnormal NO levels and impaired circadian rhythmicity we believe that autophagy may be impaired in the diabetic retinal vasculature and may represent a therapeutic target in diabetic retinopathy.
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