Influences of vesicular transport inhibition on proliferation and store-operated calcium entry in rat endothelial progenitor cells / 中国病理生理杂志

ABSTRACT

AIM:To investigate the effects of vesicular transport inhibition on the proliferation and regulation of store-operated calcium entry ( SOCE) in rat endothelial progenitor cells ( EPCs) .METHODS:EPCs were isolated from the rats with density-gradient centrifugation and confirmed via double fluorescence staining with acLDL-DiI and FITC-UEA-I.After inhibition of vesicular transport with brefeldin A ( BFA) , the proliferation of EPCs was measured by CCK-8 assay and real-time cell analyzer instrument, apoptosis was analyzed by flow cytometry, and the expression of ADP-ribosylation factor GTPase-activating protein 1 (ARFGAP1), a key protein to vesicular transport, was also detected.SOCE was ob-served under laser scanning confocal microscope after the vesicular transport was inhibited, and the protein expression of SOCC complex was determined by Western blot.Furthermore, the influences of vesicular transport inhibition on the expres-sion of transient receptor potential channel 1 ( TRPC1 ) and SOCE were examined with a RNA interference method.RE-SULTS:The acLDL-DiI and FITC-UEA-I double positive rate of the cells was 82.53%±6.12%.BFA insult significantly inhibited the proliferation of EPCs and down-regulated the expression of ARFGAP1, and no influence on the apoptosis of the EPCs was observed, suggesting that vesicular transport of EPCs was inhibited.Vesicular transport inhibition remarkably down-regulated the expression of TRPC1 and decreased SOCE level.No evident difference in the level of SOCE between siTRPC1 group and siTRPC1+BFA group, in which the cells were pretreated with siTRPC1 before BFA addition, was ob-served.CONCLUSION:Vesicular transport inhibition in EPCs reduces the proliferation of EPCs and decreases SOCE lev-el through down-regulation of TRPC1.