RESUMO
AIMS: Endothelial progenitor cells (EPCs) are widely accepted to be applied in ischemic diseases. However, the therapeutic potency is largely impeded because of its inviability in these ischemic conditions. Autophagy is recognized to be vital in cell activity. Therefore, we explore the role and the mechanism of autophagy in ischemic EPCs. METHODS AND RESULTS: We applied 7d-cultured bone marrow EPCs to investigate the autophagy status under the oxygen and glucose deprivation (OGD) conditions in vitro, mimicking the in-vivo harsh ischemia and anoxia microenvironment. We found increased EPC apoptosis, accompanied by an impaired autophagy activation. Intriguingly, mTOR inhibitor Rapamycin was incapable to reverse this damped autophagy and EPC damage. We further found that autophagy pathway downstream Vps34-Beclin1-Atg14 complex assembly and activity were impaired in OGD conditions, and an autophagy-inducing peptide Tat-Beclin1 largely recovered the impaired complex activity and attenuated OGD-stimulated EPC injury through restoring autophagy activation. CONCLUSIONS: The present study discovered that autophagy activation is inhibited when EPCs located in the ischemia and anoxia conditions. Restoration of Vps34 complex activity obtains sufficient autophagy, thus promoting EPC survival, which will provide a potential target and advance our understanding of autophagy manipulation in stem cell transplantation.
Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Isquemia/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Células Progenitoras Endoteliais/efeitos dos fármacos , Glucose/deficiência , Masculino , Camundongos Endogâmicos C57BL , Oxigênio , Sirolimo/farmacologia , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Modulating the biological status of endothelial progenitor cells (EPCs), such as function and survival, is essential for therapeutic angiogenesis in ischemic vascular disease environments. This study aimed to explore the role and molecular mechanisms underlying Netrin1 in the viability and angiogenic function of EPCs. EPCs were isolated from the bone barrow of adult C57/BL6 mice. The apoptosis and various functions of EPCs were analyzed in vitro by manipulating the expression of Netrin1. The TUNEL assay was performed to detect apoptotic EPCs. Cell migration and tube formation assays were performed to detect EPC function. Trypan blue staining was performed to detect cell viability. Western blot analysis was performed to detect the protein expression levels of Netrin1, CD146 and apoptotic factors. Quantitative PCR analysis was performed to detect the expression levels of Netrin1 receptors. The results demonstrated that treatment with exogenous Netrin1 promoted EPC migration and tube formation, whereas transfection with small interfering (si)RNA targeting Netrin1 exhibited the opposite effects. Exogenous Netrin1 protected EPCs from hypoxiainduced apoptosis, whereas the interruption of endogenous Netrin1 enhancement under hypoxia by Netrin1siRNA exacerbated the apoptosis of EPCs. Furthermore, CD146, one of the immunoglobulin receptors activated by Netrin1, was screened for in the present study. Results demonstrated that CD146 did not participate in Netrin1promoted EPC function, but mediated the antiapoptotic effects of Netrin1 in EPCs. In conclusion, Netrin1 enhanced the angiogenic function of EPCs and alleviated hypoxiainduced apoptosis, which was mediated by CD146. This biological function of Netrin1 may provide a potential therapeutic option to promote EPCs for the treatment of ischemic vascular diseases.