Study On The Role Of Endothelial Progenitor Cells In The Ischemic Stroke Therapy

Background: Peripheral blood endothelial progenitor cells (EPCs) has beenlinked to the risk and prognosis of cerebrovascular or cardiovasculardiseases. Transplantation of umbilical-cord blood derived endothelialprogenitor cells led to beneficial outcomes in a mouse model of transientmiddle cerebral artery occlusion (tMCAO). If patient-specific peripheralblood derived endothelial progenitor cells are efficious for treatingischemic stroke is unclear. The therapeuticmechanism of endothelialprogenitor cell transplantation for ischemic strokeis not fully investigated.It was demonstrated that high mobility group box1(HMGB1)-mediatedcrosstalk between astrocytes and endogenous endothelial progenitor cellspromoted post-stroke neurovascular repairing and remodeling in ischemicmouse brain. If HMGB1involves in exogenous endothelial progenitorcell-mediated stroke recovery remains unknown.Objects: The specific aims of the current study are:1) to investigatewhether microglial MHBG1enhances the pro-angiogenic properties ofendothelial progenitor cells by affecting the paracrine function ofendothelial progenitor cells; and2) to explore whether transplantatingpatient-specific endothelial progenitor cells improves the outcomes ofischemic stroke model and whether post-ischemic HMGB1up-regulationinvolves in exogenous endothelial progenitor cells-mediated strokerecovery. Materials and Methods: endothelial progenitor cells were isolated fromperipheral-blood of five hemorrhagic stroke patients and cultured in EGM-2medium. BV2cells were activated by stimulation oflipopolysaccharide(LPS). Co-culture of BV2cells and endothelialprogenitor cells was achieved using a transwell system. The cytokinesexpression in endothelial progenitor cells was examined by quantitativereal-time PCR. Glycyrrhizin was used to inhibit the HMGB1release fromBV2cells. Human umbilical vein endothelial cells (HUVECs) were treatedby conditioned medium of endothelial progenitorand tested for in vitroproliferation and tube formation. Mice underwent transient middle cerebralartery occlusion were treated with phosphate buffer solution (PBS), humanumbilical vein endothelial cells, endothelial progenitor cells alone, orendothelial progenitor cells plus glycyrrhizin.Endothelial progenitor cellswere transfected with lentivirus-GFP and tracked at day-14followingmiddle cerebral artery occlusion. Microessels density, infarct volume,atrophy volume and neurobehavioral outcomeswere examined.Results: Co-culture with lipopolysaccharide-activated BV2cellsup-regulated interleukin-8andhepatic growth factor (HGF) expression inendothelial progenitor cells (p<0.05). Suppression of HMGB1usingglycyrrhizin inhibited interleukin-8but not hepatic growth factorup-regulation in endothelial progenitor cells. Conditionedmedium fromendothelial progenitor cell co-cultured with activated BV2cells promotedproliferation and tube formation for human umbilical vein endothelial cells(p<0.05). Suppression of HMGB1in BV2cells or interleukin-8knockdown inendothelial progenitor cells by siRNA reversed the effect of endothelialprogenitor cell conditionedmedium on human umbilical vein endothelial cells (p<0.05). In vivo study showed endothelial progenitor celltransplantation led to increased vessel density in peri-infarctareas,reduced atrophy volume and improvement in neurobehavioral functionsfollowing14daysof middle cerebral artery occlusion in mice (p<0.05).Administration of glycyrrhizin (10mg/kg/d) blocked the beneficial effect ofendothelial progenitor cell transplantation (p<0.05). Further investigationshowed GFP+exogenous endothelial progenitor cells accumulated inperi-infarctareas following14days of middle cerebral artery occlusion inmice, but integration of exogenous endothelial progenitor cells with CD31+microvessels was not observed.Conclusion: Patient-specific peripheral blood-derived endothelialprogenitor cells are an ideal source of stem cell for the ischemic stroketreatment. The therapeutic mechanism of exogenous endothelialprogenitor cells is associated with paracrine function of endothelialprogenitor cells. Ischemia-induced microglial HMGB1up-regulation is animportant factor for the trigger the reparing and remodeling duringpost-ischemia.