Genome-wide epigenetic and proteomic analysis reveals altered Notch signaling in EPC dysfunction

Endothelial progenitor cells (EPCs) are bone-marrow-derived mononuclear cells that participate in tube formation in vitro and vessel formation in vivo. EPC transplantation, as a therapeutic approach in cardiovascular diseases, has produced mixed results likely due to underlying disease states and environmental factors affecting EPC function. In this study, we investigated the mechanisms by which a high-salt diet impairs EPC function. The number of endothelial progenitor cells (CD34(+), VEGFR2(+), CD133(+), and c-Kit(+)) was decreased in the bone marrow of Sprague-Dawley (SD) rats fed a high-salt diet (HSD; 4% NaCl) as compared to SD rats on a normal-salt diet (NSD; 0.4% NaCl). NSD EPCs augmented endothelial cell tube formation in vitro, whereas HSD EPCs did not. NSD EPCs were a potent therapeutic restoring electrical stimulation-induced angiogenesis in vivo. HSD EPCs were not able to restore angiogenesis in vivo. EPC DNA methylation was analyzed by reduced representative bisulfite sequencing and membrane proteins were analyzed using high accuracy liquid chromatography mass spectrometry. Differentially methylated genes and differentially abundant membrane proteins measured between the NSD and HSD EPCs, revealed a total of 886 gene-protein sets where reciprocal methylation and expression occurred. Based on stringent criteria, Notch4 was found to be hypermethylated in HSD EPCs and had corresponding decrease in protein expression. Suppression of Notch4 protein expression in EPCs using siRNA confirmed a role for Notch4 in EPC-mediated angiogenesis, suggesting Notch4 suppression as a mechanism by which high-salt diet inhibits EPC-mediated angiogenesis.