Abstract P244: Characterization of Exosomes From Vascular Endothelial and Smooth Muscle Cells

Exosomes are small nanovesicles derived from endosomal compartments termed multivesicular bodies. These multivesicular bodies fuse with the plasma membrane, release exosomes into extracellular space, allowing them to transport their cargo of functional nucleic acids and proteins. Despite their well-documented existence, not much is known about exosomes in vascular biology. The luminal surface of the intima consists of a confluent monolayer of endothelial cells (ECs), while the medial layer is characterized by presence of vascular smooth muscle cells (VSMCs). We characterized the exosomes released by these cell types in culture to better understand the packaging of these vesicles and their capacity to participate in cell-to-cell communication. Rat aortic ECs exosomes were generated in serum-free media for 48 hours while rat aortic VSMC exosomes were generated in serum-free media for 72 hours. Media harvested from both was ultracentrifuged at 10,000xg to remove microparticles followed by centrifugation of the supernatant at 100,000xg to pellet exosomes. Exosome pellet was re-suspended in PBS, 0.22 μm filtered and spun at 100,000xg to remove contaminating particles. Transmission electron microscopic analysis of VSMC exosomes confirmed particle morphology with diameters ranging from 60-150 nm. Particle size and concentration was determined using NTA NanoSight. The modal size of EC exosomes and VSMC exosomes were 107.2±5.4 nm and 116.6±7.5 nm, respectively. Western blotting analysis was performed to characterize common and specific markers for vascular exosomes. We confirmed common exosome markers such as CD81, CD63, caveolin-1, and flotillin-1 in both populations. ECs specifically expressed VE-cadherin and TSG-101 whereas VSMCs expressed syntenin-1 and smooth muscle actin. Cell-to-cell signaling in vitro assays revealed that EC-derived exosomes can stimulate hypoxia-inducible factor 1-alpha expression in VSMCs in a dose-dependent and time-dependent manner. Maximal induction was seen at 6 hours of stimulation and using 5x10 8 particles/mL. From these data, we conclude that exosomes are released from these cell types and play a potential role in cell-to-cell communication to maintain homeostasis and/or promote pathology.