〔Abstract〕 Objective To investigate ATG5 regulates VSMC proliferation by mediating endothelial exosomes. Methods HUVECs were separated by type II collagenase digestion at 37℃ and then cultivated in DMEM/F-12 culture containing 10% FBS. Tissue explants adherent method was applied to obtain VSMCs. VSMCs was cultivated in DMEM/F-12 culture containing 20% FBS. The uptake of endothelial exosomes by VSMCs was tracked by high-content cell imaging system. SiRNA was used to knock down the expression of ATG5. Results HUVECs with high purity and good viability were isolated successfully by 0. 1% type Ⅱ collagenase digestion. The cell displayed typical cobblestone-like morphology under the inverted phase-contrast microscope. Immunofluorescence technique validated that the isolated cells with CD31/Pan-Cadherin/Factor Ⅷ-positive phenotype account for 96%. VSMCs were observed on the 3 rd day of the culture. The cells showed long shuttle and polygonal morphology. Immunofluorescence technique identified that the expression frequency of SM22-α and α-actin was about 95%. Endothelial exosomes obtained by ultracentrifugation displayed Hsp70/TSG101/CD9/CD63 positive phenotype. VSMCs started to uptake endothelial exosomes after 30 minutes co-culture showed by high-content cell imaging system. After 4 hours,the numbers of endothelial exosomes uptake by VSMCs increased significantly. Cell growth and proliferation curve showed that,at the concentration of 200 μg/ml,the exosomes secreted by HUVEC in which ATG5 decreased could inhibit the proliferation of VSMC. Conclusion ATG5 regulates VSMC proliferation by mediating endothelial exosomes.