Influences Of Vesicular Transport Inhibition On Proliferation And Store Operated Calcium Entry In Endothelial Progenitor Cells

Background and objectives:Endothelial progenitor cells(EPCs)is a subunit of monocytes which are derived from bone marrow.They could be recruited to vascular injured areas and keep the integrity of vascular endothelium through replacing damaged or senile endothelial cells.Moreover,EPCapplication in cell-based therapy has become a very important tool in cardiovascular diseases.Therefore,it is important to explore relative mechanisms for proliferation of EPCs.Previous studies from our laboratory found that store operated calcium channel(SOCC)affects various biological functions of EPCs,such as proliferation via store operated calcium entry(SOCE).Stromal interaction molecule-1(STIM1)could sense alteration in calcium in endoplasmic reticulum,and activate SOCE via interacting with Orai1 and /or TRPC1.Therefore,Orai1,TRPC1 and STIM1 play a vital role in regulation of SOCE of EPCs.Vesicle transport,a basic physiological process,is important for localization and distribution of proteinsin order.If it becomes abnormal,organisms will occur many diseases.Brefeldin A,a specific inhibitor for vesicle transport was used to inhibit vesicle transport in our study.It has been reported that vesicle transport plays a vital role in the regulation of TRPC1 expression on cell membrane.However,its role in other components of SOCC and SOCE has not been explored.Based on above reasons,we investigated the influences of vesicle transport inhibition on SOCC complex in EPCs and explored the relative mechanisms.Our findings would provide a noveltarget for the repair of vascularinjuries.Methods1.Culture and characterization of EPCs from ratspleenMonocytes were isolated from ratspleen and cultured for 5-7 days under aseptic condition.Ac LDL-Di I and FITC-UEA-I double fluorescent staining wereused to identify EPCs.2.Influences of BFA on proliferation of EPCs were detected by Real-Time Analyzer Instrument(RTCA).3.Influences of BFA on proliferation of EPCs were detected by Cell Counting Kit-8(CCK-8).4.Apoptosis was detected by flow cytometry.5.Detection of concentration of intracellular calcium Fluorescent intensity was evaluated under laser confocal microscope(excitation wavelength of 488 nm,emission wavelength of 530 nm)after fluo3-AM staining.6.Western blotting analysis Protein expression of SOCC complex and ARFGAP1 in various groups were detected by western blot.7.EPCs were transfected by small interference RNA targeting TRPC1.Results1.Characterization of EPCs from ratspleen Positive rate of EPCswas 82.53%±6.12%.2.Influences of BFA on proliferation of EPCs BFA significantly inhibited proliferation of EPCs.3.Influences of BFA on apoptosis of EPCs Apoptosis rates of control group and BFA group were 8.73%±0.63% and 9.67%±0.29% respectively.There was no statistical significance between two groups.4.Influences of BFA on expression of ARFGAP1 Compared with control group,ARFGAP1 expression in BFA group was significantly down-regulated.5.Influences of vesicle transport inhibition on SOCE and concentration of intracellular calcium SOCE in BFA was significantly lower than control group.Furthermore,intracellular calcium concentration with BFA was significantly lower than control.6.Influences of vesicle transport inhibition on main constitutive proteins of SOCC Inhibition of vesicle transport significantlydown-regulated TRPC1 expression with no change on Orai1 and STIM1.7.Influence of TRPC1-si RNA transfection on calcium flow of EPCs SOCE was inhibited by silencing of TRPC1 in EPCs.However,inhibition of vesicle transport by BFA after si TRPC1 did not further reduced SOCE.Intracellular calcium level was detected.We found that either TRPC1 deficiency or vesicle transport inhibitiondepressed [Ca2+]i compared with control.Yet no further [Ca2+]idecrease was detected with inhibitionsof TRPC1 plus vesicle transport.Conclusions1.BFA decreases EPC proliferation.2.Apoptosis due to BFA is not involved in proliferation inhibition of EPCs.3.Vesicle transport inhibition of EPCs down-regulates Icrac mediated by SOCC and lowers intracellular calcium concentration.4.Vesicle transport inhibition down-regulates TRPC1 expression with no change of Orai1 and STIM1.5.TRPC1 inhibition lowers level of SOCE.However,there is no significant difference between TRPC1/BFA double inhibition and TRPC1 alone inhibition.From what has been discussed above,vesicle transport inhibition of EPCs inhibits EPC proliferation,and reduced SOCE level through down-regulation of TRPC1.