Diabetes Results in a Reduction of Clock Gene Expression in Retina and in Endothelial Precursors: Implications for Diabetic Retinopathy

Abstract

Purpose: : Circadian rhythms are driven by a central clock (suprachiasmatic nucleus, SCN) and peripheral clocks (tissues) and determined by expression of "clock genes". Diabetic retinopathy (DR) is associated with endothelial precursor cell (EPC) dysfunction. We asked whether DR is associated with changes in clock genes expression within the retina, bone marrow (BM) and peripheral blood EPCs and whether this could influence EPC release from the BM and thus the ability of EPC to repair the vasodegenerative phase of DR.

Methods: : Type 2 diabetic (n=3) rats with 4 months duration of disease and age matched controls (n=5) were placed in standard LD conditions for two weeks prior to sacrifice. With a 24-hr period divided into a 12-hr activity phase and a 12-hr rest phase. In nocturnal animals, such as rats, Zeitgeber time (ZT) 12 is at the beginning of the activity phase (dark) and ZT 0 (light) is at the start of the rest phase. At peak release of EPC at (ZT) 4, animals were sacrificed and their retinas, BM and peripheral blood harvested for mRNA. Clock genes, Clock, Bmal-1, Per-1, Per-2, CRY-1, CRY-2, ERB, RORA, were quantitated by real time PCR in retina and in Thy-1 positive cells (rat EPC) from blood and BM. EPC number in the BM and blood was enumerated and functional changes, such as colony formation and migratory ability assessed.

Results: : Diabetic rats showed 75% (p<0.05) decrease in the numbers of Thy-1 cell released in peripheral blood, from BM at (ZT)-4 when compared to healthy animals. In addition diabetic Thy-1 positive cells formed 5 times (p<0.05) fewer colonies and showed markedly reduced migration towards to VEGF and SDF-1. Rats with diabetes demonstrated significant decrease (p<0.05) in expression of Clock, Bmal-1, Per-1, Per-2, CRY-1, CRY-2, ERB, and RORA in retina and Thy-1 positive cells isolated from both blood and BM. Finally, diabetic rats exhibited overall suppression of normal circadian rhythm for EPC release as compared to healthy controls.

Keywords: diabetic retinopathy • transcription • gene/expression