Abstract
Purpose :
Diabetes has been reported to disrupt circadian rhythms with circadian disruption emerging as an important factor in the disease prognosis and treatment success. Endothelial cells are central in the progression of diabetic retinopathy and have a robust circadian clock with rhythms in diverse cellular processes. Thus, we asked whether diabetes alters the expression of circadian genes in these cells and if so, which aspects of the diabetic microenvironment drive these changes.
Methods :
Induced pluripotent stem cell derived endothelial cells (iPS-ECs) from healthy and diabetic patients were sequenced and differential analysis was performed, identifying genes related to circadian rhythms. Primary human retinal endothelial cells (hRECs) were cultured in vitro in hyperglycaemia or normoglycaemia for 7 days. Cultures were then synchronised with 50% serum shock and repeated samples collected every 2 hours over 25 hours in either hypoxia or atmospheric oxygen levels. Circadian gene expression was measured with RT-PCR.
Results :
iPS-ECs from diabetic patients have 4.03-fold higher Bmal-2 mRNA expression and 5.7-fold less Dec2. Hypoxia, and not hyperglycaemia, affects circadian gene expression in synchronised hRECs. In hypoxia, Bmal1 expression is increased at all time points and phase advanced. The opposite is true of its negative regulator, Per2, which is instead reduced in hypoxia with weakened rhythmicity. Hyperglycaemia alone slightly increases the rhythmicity of Per2 oscillation. Circadian rhythmicity in gene expression persists in all treatments for both Bmal1 and Per2 but is lost in Cry1 expression when cells are treated with hypoxia (normoxia p=0.02 vs hypoxia p=0.18), as analysed using JTK_Cycle.
Conclusions :
Diabetes can alter the expression and rhythmicity of the circadian molecular clock in endothelial cells. In separately testing some elements of the diabetic microenvironment, we have shown that hypoxia alone drives major changes in key core loop genes of the molecular clock. More, hypoxia alters the positive and negative arms of this molecular clock in different ways, with potential implications for the period of the clock and transcription of clock-controlled genes, many of which are immediately relevant to diabetic retinopathy including VEGF. Bmal1, a master transcription factor, appears to have bolstered expression in hypoxia while its negative regulator, Per2, is reduced.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.