Circadian Rhythmic Release of Endothelial Precursor Cells Is Lost in Diabetes: Implications for Pathogenesis of Diabetic Retinopathy

Abstract

Purpose: : Endothelial precursor cell (EPC) dysfunction has been implicated in the generation of acellular capillaries in diabetic retinopathy. In diabetes, EPC are not effective in vascular repair due to reduced ability to migrate and differentiate into endothelial cells. Recently, EPC have been shown to be released from the bone marrow in a circadian pattern with peak release approximately 5 hours following initiation of the light cycle in rodents or dark cycle in humans. An altered circadian release of EPC could result in less than optimal repair of tissues including acellular capillaries in the retina. In this study we asked whether a loss of circadian release of EPC may be occurring in diabetes.

Methods: : Type two (BBDZR) diabetic rats with 4 months duration of diabetes and age matched controls were used. All animals were maintained in standard light cycle conditions. Measurements were based on Zeiter Time (ZT), a 24-hr period divided into a 12-hr activity phase and a 12-hr rest phase. In nocturnal animals, such as rats, ZT12 is at the start of the activity phase (dark) and ZT0 is at the start of the rest phase (light). Rats underwent placement of the indwelling arterial catheter. 100µL of blood was collected using a tethered system (Dilab systems) every two hrs for a 24 hr period. Rat EPC (Thy-1 positive/CD3, CD4, CD8 negative cells) were enumerated by FACS analysis in each sample.

Results: : Control rats demonstrated a biphasic EPC release pattern with a 7 fold increase of Thy1 positive cells in the blood 3 hours after initiation of the light cycle (ZT3) relative to baseline. In diabetic rats the peak release of Thy1 positive cells was dramatically reduced (p<0.05). This was due to entrapment of EPC in the bone marrow as evidenced by increased number of Thy-1 positive cells within the bone marrow of diabetic compared to control rats (p < 0.05). Significant decrease in expression of clock genes in Thy-1 positive cells isolated from blood and bone marrow of diabetic rats compared to controls also confirms our findings.

Conclusions: : Our data support the hypothesis that diabetes is associated with markedly impaired circadian rhythmicity of EPC release. This loss of rhythmicity is likely to impact the reparative capability of EPC and can contribute to the inadequate repair of accelular capillaries in the vasodegenerative phase of diabetic retinopathy. Correction of the circadian defect may represent a novel target for the treatment of diabetic retinopathy.

Keywords: diabetic retinopathy • retinal neovascularization • flow cytometry