| Objective:To explore the effects and the underlying mechanisms of exosomes secreted by cord blood-derived endothelial progenitor cells(EPCs)treated with oscillating shear stress(OSS)on the endothelial-mesenchymal transition(EndoMT)of EPCs.Methods:(1)Human peripheral blood density gradient centrifugation was used to extract mononuclear cells from the umbilical cord blood of healthy pregnant women.Then,mononuclear cells were plated on culture flask pre-coated with fibronectin(Fn).EGM-2 complete medium(supplemented with EGM-2 bullet kit,including 10%fetal calf serum,recombinant VEGF,recombinant b FGF)was used for culture,and the cell morphology of 0 d,7 d and 14 d was observed under an inverted phase contrast microscope.EPCs were characterized by the uptake of Di L-Ac-LDL and FITC-UEA-1 staining.(2)EPCs were divided into static group(Static)and oscillating shear stress treatment group(OSS),and exosomes-free serum was used for culture before the experiment for 12h.The Flexcell flow STR-4000 parallel plate flow chamber system was employed to apply OSS(±3.5 dyne/cm~2,1 Hz)on EPCs for 12h.Then,a combination of freeze-drying and exo Easy Maxi Kit were used to extract exosomes from the cellular supernatant or perfusate.The morphology of exosomes was observed by electron microscopy.The size of exosomes was detected by nanometer particle size analyzer.The activity of exosomes was detected by PKH26 staining.The expression of exosomal specific proteins CD9,CD81 and CD63 was detected by Western Blotting(WB).(3)EPCs were treated with Static-Exos or OSS-Exos,then cell biological function,such as the ability of tube formation and proliferation were measured by Matrigel and Ed U incorporation assay,respectively.The q RT-PCR and WB were used to detect the gene and protein expression of endothelial cell marker VE-cadherin,CD31 and mesenchymal cell markerα-SMA,SM22α.(4)CM-Di L labeled EPCs pre-treated with Static-Exos or OSS-Exos were locally transplanted under the dorsal skin of nude mice.The expression of endothelial cell marker v WF,CD31 and the mesenchymal cell markerα-SMA,SM22αwere detected by immunofluorescence staining at 7 days after transplantation.And the EPCs angiogenesis in vivo was detected by HE staining.(5)The expression of Circ-1199 was detected by q RT-PCR in Static-Exos and OSS-Exos.Then the Static-Exos and OSS-Exos were added into the medium,and the CY3labeled Circ-1199 fluorescent probe and RNA.FISH kit were used to measure the location of Circ-1199 in EPCs.(6)The dual luciferase reporter system was employed to detect the interaction of Circ-1199 and let-7g m RNA,as well as the interaction of let-7g and its downstream target gene HMGA2 m RNA.The gene expression of Circ-1199,let-7g and HMGA2 were measured by q RT-PCR,and the protein expression of HMGA2,p-Smad3/Smad3,Snail were detected by WB.Results:(1)The isolated mononuclear cells that initially seeded were round in suspension.The distinct colonies formed after 96 h,reaching a peak on the 7 d.After passage,cells showed characteristic homogeneity and cobblestone-like morphology similar to mature endothelial cells.The cells were identified as double positive staining for Di L-Ac-LDL(red fluorescence)and FITC-UEA-1(green fluorescence)under an inverted fluorescence microscope,indicating that the cultured cells were EPCs.(2)Exosomes were uniform in size,showing a typical and complete vesicle-like structure.The particle size was concentrated at 30～150 nm,and the average peak value was at 109.7±15.18 nm,indicating that the size was appropriate and the purity was high.Moreover,the particle contained exosome-associated proteins,e.g,CD9,CD81 and CD63.PKH26 could bind to the membranous structure of the particle and was efficiently taken up by cells.(3)Compared with the Static-Exos,Exos devived from EPCs pre-treated with OSS decreased EPC angiogenesis(P<0.01)and increased EPCs proliferation(P<0.01),indicating that OSS-Exos inhibited the angiogenesis and promoted the proliferation of EPCs in vitro.q RT-PCR and WB showed that the expression of endothelial cell marker VE-cadherin and CD31 were decreased(P<0.05),while the expression of mesenchymal cell markerα-SMA and SM22αwere increased(P<0.05)in OSS-Exos treated EPCs,suggesting that OSS-Exos induced EndoMT in EPCs.(4)HE staining of Matrigel graft in nude mice showed the angiogenesis ability was decreased in OSS-Exos treated EPCs compared with those kept in static(P<0.05),which indicated that OSS-Exos inhibited angiogenesis of EPCs.Immunofluorescence staining of frozen sections showed that pre-treated with OSS-Exos,the expression of endothelial cell marker VE-cadherin(P<0.01)and CD31(P<0.05)was decreased,while the mesenchymal cell markerα-SMA and SM22αwas increased(P<0.01),suggesting OSS-Exos contributed EPC EndoMT.(5)q RT-PCR showed that the expression of Circ-1199 in OSS-Exos was higher than that in Static-Exos(P<0.05).Then Static-Exos and OSS-Exos were added into EPCs medium,Circ-1199RNA.FISH showed that Circ-1199 was mainly located in the cytoplasm of EPCs,and the expression level of Circ-1199 was higher in EPCs pre-treated with OSS-Exos compared with those pre-treated with Static-Exos(P<0.05).(6)The dual-luciferase reporter gene assay demonstrated that let-7g was a direct target of Circ-1199.Furthermore,HMGA2 was the target of let-7g.In EPCs pre-treated with OSS-Exos,the expression of Circ-1199 and HMGA2 was increased.However,the level of let-7g was decreased(P<0.001).In the mean time,WB showed that the protein levels of HMGA2(P<0.01),p-Smad3/Smad3(P<0.05)and Snail(P<0.05)were significantly up-regulated in EPCs pre-treated with OSS-Exos.Conclusions:(1)The combination of freeze-drying and exo Easy Maxi Kit was an efficient method for exosomes isolation.(2)OSS-Exos induced EPC EndoMT,namely inhibiting EPC angiogenic ability and promoting the proliferation.(3)Circ-1199 enriched in OSS-Exos functioned as molecular"sponge"to absorb let-7g,thereby up-regulated the expression of let-7g target gene HMGA2,then activated the expression of EndoMT-related transcription factor p-Smad3/Smad3 and Snail. |
