title: Ghrelin induces cell migration through GHSR1a-mediated PI3K/Akt/eNOS/NO signaling pathway in endothelial progenitor cells.
abstract: 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.
  
  
  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)~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~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.
authors.affiliation: Department of Geriatrics, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China.
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Chen
authors.forename: Qingwei
authors.initials: Q
authors.lastname: Chen
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Guiqiong
authors.initials: G
authors.lastname: Li
mesh_terms: D000818:Animals; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D054439:Ghrelin / Q000494:pharmacology* / Q000502:physiology; D008297:Male; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D051058:Oncogene Protein v-akt / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D054440:Receptors, Ghrelin / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects; D013234:Stem Cells / Q000187:drug effects* / Q000378:metabolism
chemical_list: D054439:Ghrelin; C571124:Ghsr1a protein, rat; D054440:Receptors, Ghrelin; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D019869:Phosphatidylinositol 3-Kinases; D051058:Oncogene Protein v-akt