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Introduction
Endothelial progenitor cells (EPCs) can differentiate into endothelial cells (ECs) and are important in angiogenesis (1). It has been demonstrated that oxidative stress-induced endothelial dysfunction can cause ischemia/reperfusion injury, and the proliferation and differentiation of EPCs are essential for the treatment of this type of injury. Hypoxia has been demonstrated to mobilize EPCs from the bone marrow into the peripheral blood (2), and induce ECs to secret chemokines for the recruitment and migration of EPCs to hypoxic tissues (3). In addition, hypoxia can induce the expression of hypoxia-inducible factor-1α (HIF-1α), an important transcriptional factor that is involved in the proliferation and differentiation of EPCs (4).
Apelin is an endogenous ligand for the G protein-coupled receptor, APLNR (5). It has been suggested that Apelin/APLNR signaling can be activated by HIF-1α in hypoxia neonatal cardiomyocytes (6). In addition, downregulated serum levels of Apelin are associated with EPC mobilization induced by acute myocardial infarction (7) and Apelin deficiency impairs EPC sprouting in ischemia-reperfusion injury (8). These findings suggest that Apelin may be involved in the regulation of EPC mobilization and sprouting. However, whether the Apelin/APLNR signaling is involved in hypoxia-induced EPC proliferation has not been investigated previously.
The mitogen-activated protein kinase (MAPK) signaling pathway is important in the regulation of cellular survival, proliferation, differentiation and migration (9). It has been demonstrated that the MAPK signaling pathway is involved in the regulation of EPC proliferation, for example, the effect of granulocyte macrophage colony-stimulating factor on the modulation of EPC proliferation is mediated by the MAPK signaling pathway (10). Our previous study revealed that Apelin/APLNR signaling promotes hypoxia-induced proliferation of EPCs via phosphoinositide 3 kinase/Akt signaling (11). However, whether Apelin/APLNR signaling regulates EPC proliferation via MAPK signaling remains to be elucidated
The aim of the present study was to determine the underlying molecular mechanisms by which Apelin/APLNR signaling regulates the hypoxia-induced proliferation of EPCs.
Materials and methods
Cell isolation and culture
EPCs were isolated from the umbilical cord at the Second Xiangya Hospital of Central South University in September 2012. Briefly, umbilical cord tissue sample was diluted with Dulbecco's phosphate-buffered saline at a ratio of 1:1 and overlaid onto 1.077 g/ml Ficoll-Paque™ Premium (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) prior to centrifugation for 30 min at 400 × g....