Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis

Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (V-max = 0.057 +/- 0.004 ng exosome protein X 40,000 cells/h; K-m = 17.97 +/- 3.84 mu g exosomal protein/200 mu l media) and decreased by 80% when the incubation temperature was lowered from 37 degrees C to 4 degrees C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor D-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues.