Autophagic Changes Of Endothelial Progenitor Cells Under Ischemia-hypoxia And Effects On The Survival And Proliferation Of The Cells

Objective To study the autophagic changes of EPCs under ischemia and hypoxia and explore the effects of autophagy on the survival and proliferation of EPCs. Methods Mononuclear cells were isolated from umbilical cord blood using density centrifugation with Percoll solution. VEGFR-2+CD34+and VEGFR-2+CD133+cells were sorted with flow cytometry (FCM), the coexpression of VEGFR-2, CD34and CD133on EPC was observed through laser scanning confocal microscope and then EPCs were induced and proliferated under the effection of VEGF. Rat models of myocardial infarction were established and then EPCs loaded by FG were transplanted into normal cardiac tissues, peripheral fields of infarction cardiac tissues and infarction cardiac tissues respecticely. The distribution of EPCs laoded by FG was detected by semithin sections. The autophagic changes of EPCs were viewed by transmission electron microscope (TEM). For clarifying the effects of autophagy on proliferation and activity of the cells, EPCs were pretreated with3-MA and then cells cycles and activity were examined by FCM and MTT assay respectively. In order to study the effects of hypoxia on autophagy, hypoxic microenvironment was imitated in vitro and the cells sorted and proliferated were divided into control, hypoxia1, hypoxia2, hypoxia3and3-MA groups. Hypoxia1,2,3groups were treated under hypoxia for30min,1h,2h respectively, cells in3-MA group were pretreated with3-MA solution (5mmol/L) for1h and then cultivated under hypoxia for1h. After hypoxia treatment, the expression of autophagy-related gene Beclin-1was examined through immunocytochemistry and RT-PCR. The number of autophagosomes and positive cells containing autophagosomes was detected by LC3immunocytochemistry and MDC staining. The morphological characteristics of autophagosome precursor, autophagosome and autolysosome were observed through TEM and then the sectional areas of autophagic structures in the cells were measured with VLCDS image analyzer. The number of lysosomes was detected by labelling lysosomes with lysoSensorTMND-189. For clarifying the relationship between autophagy and apoptosis, AO/EB staining and FCM were applied to examine the apoptotic changes of EPCs after blocking autophagy with3-MA. Results Comparing with EPCs transplanted into normal cardiac tissues, there was not significant difference in the number of EPCs transplanted into peripheral areas of infarction cardiac tissues, the number of EPCs in infarction cardiac tissues, however, decreased remarkably. Through TEM, no autophagic structures were detected in the cells transplanted into normal cardiac tissues, few but abundant autophagosomes were detected in the cells transplanted into peripheral areas of infarction cardiac tissues and infarction cardiac tissues respectively, some necrotic EPCs in infarction cardiac tissues were observed. The proliferation index and activity of the cells decreased remarkably after bloking autophagy. In studying autophagic changes induced by hypoxia, following detected indexes were detected increased with extension of hypoxia time but decreased after blocking autophagy, they were the expression of Beclin-1, the number of autophagosomes and positive cells containing LC3or MDC-labeled autophagosomes, the ratios of the cross-section areas of the autophagic structures to that of the cytoplasm of EPCs and the number of lysosomes. In study of the relationship between autophagy and apoptosis, we found that the number of apoptotic cells induced by hypoxia1h increased slightly but increased significantly after blocking autophagy. Conclusion Autophagy participates in sustaining the proliferation and activity of the cells. Ischemia and hypoxia can induce autophgy. Autophagy plays a positive role in retarding apoptosis induced by hypoxia.