Glucocorticoids Induce The Dysfunction Of Endothelial Progenitor Cells Through The PI3K/Akt Pathway

【Objectives】 Glucocorticoids (GCs) play an important role in non-traumaticosteonecrosis of the femoral head (ONFH), and patients with ONFH accompanied bychanges in the number and function of endothelial progenitor cells (EPCs). Taken together,the hypothesis that GCs contribute to the reduced number and dysfunction of EPCs can beproposed. Here, we aim to explore the effect of GCs on EPCs and its possible mechanisms.【Methods】(1) Mononuclear cells (MNCs) from umbilical cord blood isolated by densitygradient centrifugation were plated on human fibronectin-coated culture plates and culturedat37℃,5%CO2in EGM-2medium. Morphological changes were observed with aninverted microscope. The EPCs were identified by the combined FITC-labeled Ulexeuropaeus agglutinin-1(UEA-1) and Dil-labeled acetylated low density lipoprotein(Dil-ac-LDL) double staining tests, in vitro tube formation assay, and endothelial nitricoxide synthase (eNOS) was detected by quantum dots immunofluorescence technology.(2)EPCs treated with different concentrations of dexamethasone (DXM), cell proliferation wasexamined by EdU assay and Cell Counting Kit-8(CCK-8), cell migration was examinedusing transwell assay, tube formation ability was examined by in vitro tube formation assayand nitric oxide (NO) produced by EPCs was detected using nitric oxide assay kit.(3)pretreated the EPCs with phosphatidylinositol3-kinase (PI3K) specific inhibitorsLY294002and wortmannin, and mitogen-activated protein kinase (MAPK) specificinhibitor PD98059, then treated with5μmol/Lol/L DXM, Akt, p-Akt, eNOS were detectedby western blot and real time quantitative reverse transcription polymerase chain reaction(RT-PCR).【Results】(1) mononuclear cells isolated from umbilical cord blood cultured in EGM-2 medium, typically formed the colony forming units (CFU), combined to FITC-labeledUEA-1and uptook Dil-ac-LDL, expressed eNOS and tube formation in vitro, confirmedthat the cultured cells to EPCs.(2) Short-term treated EPCs with GCs canconcentration-dependently inhibit the proliferation of EPCs, as well as migration, in vitrotube formation and NO generation.(3) PI3K specific inhibitors LY294002and wortmanninfurther inhibited phosphorylation of Akt, reduced the expression of eNOS, but MAPK/ERKspecific inhibitor PD98059had no such effect.【Conclusion】(1) MNCs isolated from umbilical cord blood cultured in EGM-2mediumcan proliferate and differentiate to EPCs;(2) Short-term treat EPCs canconcentration-dependently inhibit the proliferation of EPCs, as well as migration, in vitrotube formation and NO generation;(3) These findings suggest that GCs induceddysfunction of EPCs via PI3K/Akt, rather than via MAPK/ERK signal transductionpathway.