Core Circadian Regulatory Genes Regulate Endothelial Differentiation of CD34+ Cells

Abstract

Purpose: : Endothelial progenitor cell (EPC) differentiation is critical for vascular repair function. We reported that in diabetes there is a loss of circadian rhythmicity of EPC release due to bone marrow neuropathy which is accompanied by a loss of circadian regulatory proteins (CCRP). Clock genes encoding CCRP were shown to control differentiation in neuronal stem cells, we asked whether clock genes similarly control the differentiation of EPCs into endothelial cells (EC).

Methods: : CD34⁺ cells isolated from healthy volunteers (n=6) were maintained in either EPC differentiating or control medium. At 12 hr intervals for 4 days, CD34⁺ cells were collected and stained for CD34, CD45, CD133, CD144, CD146 to study their hematopoietic and endothelial characteristics using multicolor flow cytometry and parallel studies were performed to examine clock gene expression (Clock, Bmal, Per1, Per2, Per3, Cry1, Cry2) every 4 hrs using real time RT-PCR.

Results: : Endothelial differentiation of CD34⁺ cells resulted in a decrease in the CD133⁺CD34⁺CD45⁺ population, while control medium maintained stem cell characteristics with 94.1% of cells expressing the stem cell marker CD133. Differentiation of CD34⁺ cells into ECs was associated with robust alteration in clock gene expression. The positive arm of the clock genes involving clock and Bmal1 showed suppression (p<0.001) in their expression with endothelial differentiation. In contrast, control media maintained the oscillatory pattern of the clock genes over the 96 hrs examined. The clock genes in negative loop (Per1, Cry1, Cry2) of clock gene assembly started oscillating in rhythmic pattern (p<0.001), in particular Cry1 (15 fold; p<0.001) and Cry2 (5 fold; p<0.001)exhibited a robust response when the cells were placed in EC differentiating medium.

Conclusions: : These novel findings support that CCRP are involved in endothelial differentiation of CD34⁺ cells, a step that is reduced in diabetic CD34⁺ and may be responsible for their dysfunction. Modifying the period and amplitude of circadian oscillations in diabetic CD34⁺ cells may directly impact there reparative potential.

Keywords: diabetic retinopathy • circadian rhythms • differentiation