Ghrelin induces cell migration through GHSR1a-mediated PI3K/Akt/eNOS/NO signaling pathway in endothelial progenitor cells

Objective. The purpose of this research was to investigate the effects of ghrelin on circulating endothelial progenitor cells (EPC) directional migration and its underlying molecular mechanisms involved in this process. Materials/Methods. EPC were isolated from bone marrow of SD rats by using Percoll density gradient centrifugation, and characterized by double positive for acLDL-Dil uptake and FITC-UEA-1 binding and immunocytochemistry for CD34, CD133, vWF and Flk-1. EPC were treated with different concentrations of ghrelin (10(-9)similar to 10(-6) M) with or without GHSR1a inhibitor [D-Lys3]-GHRP-6, PI3K inhibitor LY294002 and endothelial nitric oxide synthase (eNOS) inhibitor L-NAME, migration of EPC was detected by transwell assay, levels of phosphorylated and total Akt and eNOS were determined by Western-blot analysis and Nitric Oxide (NO) production was measured by Griess assay, respectively. Results. EPC were successfully obtained by Percoll density gradient centrifugation and ghrelin at 10(-8)M similar to 10(-7) M promoted EPC migration. Ghrelin-induced EPC migration was accompanied by phosphorylation of Akt and eNOS, as well as an increase in NO production. These biochemical events and EPC directional migration induced by ghrelin were completely inhibited by GHSR-1a blocker [D-Lys3]-GHRP-6. PI3K inhibitor LY294002 attenuated ghrelin-induced EPC migration, phosphorylation of Akt and eNOS, and NO production. eNOS inhibitor L-NAME blocked ghrelin-induced EPC migration, phosphorylation of eNOS, and NO production, but had no effect on Akt phosphorylation. Conclusions. These findings suggest that ghrelin stimulates EPC directional migration via GHSR1a-mediated PI3K/Akt/eNOS/NO signal pathway. It indicates that ghrelin may be used as a therapeutic strategy to treat ischemic diseases by promoting EPC directional migration. Crown Copyright (C) 2013 Published by Elsevier Inc. All rights reserved.