| Cell-bound membrane vesicles(CBMVs)exist on surfaces of many types of cells.Due to the lack of effective methods for isolation and purification,the components,structure,production,and functions of CBMVs are poorly understood.Recently,it was found that CBMVs are resistant to multiple detergents(e.g.Triton X-100 and SDS).This study seeks to isolate and purify CBMVs by using the detergent-resistant property of CBMVs,to detect the protein components of CBMVs under nomal and oxidative stress conditions by utilizing proteomic methods,and to preliminarily test a potential function of CBMVs.Based on detergent treatments and sucrose density gradient ultracentrifugation,this study isolated and purified CBMVs from the surfaces of human umbilical vascular endothelial cells(HUVECs).The nanometer-sized spherical vesicles were observed by confocal microscopy and transmission electron microscopy,and their sizes(an average diamether of 442.9 ± 6.3 nm ranging from 220 nm to 825 nm)were measured by dynamic light scattering(DLS).By using proteomic techniques(LC-MS/MS),1335 proteins were detected in the isolated CBMVs,among which 12 proteins including 8 high-abundance proteins(PRDX 1,PRDX 3,PRDX 4,PRDX 6,COP I,PPIA,P4HB,and Talin 1)and 4 low-abundance proteins(PRDX 2,PRDX 5,Dynamin 2,and CHORDC 1)were selected for verification by western blot and immunofluorescence imaging.The western blot data showed that the levels of 7 proteins(PRDX 1,PRDX 3,PRDX 4,PRDX 6,COP I,PPIA,and P4HB)were relatively high whereas the levels of 5 proteins(PRDX 2,PRDX 5,Dynamin 2,CHORDC 1,and Talin 1)were relatively low or not expressed in isolated CBMVs;the immunofluorescence imaging showed that CBMVs were colocalized with each of the former 7 proteins in the plasma membrane of HUVECs but non-colocalized with the latter 5 proteins.These data conform the proteomic results.Recently,single-vesicle tracking study revealed that oxidized low-density lipoprotein(oxLDL)could increase the amount and motion of CBMVs on HUVECs,implying the potential correlation of oxLDL with CBMVs.Therefore,after confirming the effectiveness of oxLDL by detecting the expression of intercellular cell adhesion molecule 1(ICAM-1)at protein and mRNA levels on/in HUVECs,this study also investigated the effects of oxLDL stimulation on the protein components in CBMVs by using proteomic techniques.LC-MS/MS detected 381 differential proteins between oxLDL and control groups,including 221 upregulated proteins and 160 downregulated proteins.Further bioinformatic analyses were performed on the differential proteins.GO analysis showed that the differential proteins were involved in multiple biological processes and multiple molecular functions;KEGG Pathway analysis displayed that the differential proteins participated in many signal pathways among which some are closely related with cell apoptosis and cardiovascular diseases;PPI analysis found that some upregulated Prdx family members(e.g.PRDX 2,PRDX 3,PRDX 5,and PRDX 6)played important roles in 3 key protein-protein interaction network.These Prdx family members have an antioxidant property and can regulate the oxidative stress of cells together with other proteins(e.g.metabolic enzymes).It implies that CBMVs probably have a function related with oxidative stress(e.g.antioxidation).To test the potential antioxidant function of CBMVs,this study developed a cell model of oxidative damage by using HUVECs treated with hydrogen peroxide(H2O2).Dynamic single-vesicle tracking analyses found that H2O2 treatment could significantly change the motion(e.g.average speed and displacement)of CBMVs,implying the potential correlation of CBMVs with H2O2-induced oxidative damage of cells.Further MTT detection revealed that isolated CBMVs could reverse the H2O2-induced decrease in cell viability(or oxidative damage)in a concentration-dependent manner.Although more experimental evidence will be needed,this study preliminarily reveals a potential antioxidant function of cell-bound membrane vesicles. |
PDF Full Text Request