Extracellular vesicles (EVs) released by endothelial cells support vascular homeostasis. To better understand endothelial cell EV biogenesis, researchers at the National Institute of Child Health and Human Development examined cultured human umbilical vein endothelial cells (HUVECs) prepared by rapid freezing, freeze-substitution, and serial thin section electron microscopy (EM). Thin sections of HUVECs revealed clusters of membrane protrusions on the otherwise smooth cell surface. The protrusions contained membrane-bound organelles, including multivesicular bodies (MVBs), and appeared to be on the verge of pinching off to form microvesicles. Beyond cell peripheries, membrane-bound vesicles with internal MVBs were observed, and serial sections confirmed that they were not connected to cells. These observations are consistent with the notion that these multi-compartmented microvesicles (MCMVs) pinch-off from protrusions. Remarkably, omega figures formed by fusion of vesicles with the MCMV limiting membrane were directly observed, apparently releasing exosomes from the MCMV.
HUVECs display a localized site of membrane protrusions that contain
MVBs and other membrane-bound organelles
(a) A low-mag transmission EM view of a portion of a HUVEC in a 70 nm-thick section cut in the en face orientation, showing its elongated cell morphology and predominantly smooth plasma membrane (arrows). The number in the upper right corner indicates the number of the section in the series of serial sections shown in (c), N indicates the nucleus. Additional serial sections through this area are shown in Supplemental Movie 1. Asterisks are placed to the right of two wrinkles in the plastic section; open arrowheads point to fuzzy material that is typically observed in freeze-substituted culture medium. (b) Boxed area in (a) from serial section 4 shows the cluster of membrane protrusions. A magenta dashed line outlines a branched protrusion; turquoise dashed line outlines an unbranched protrusion. Yellow lines indicate width in nm across protrusion necks. (c) Eight consecutive serial sections through the protrusion site with the serial section number indicted in the upper right (the greater the number, the higher above the coverslip in the z-axis). (d-h) Enlarged views of cytoplasmic organelles from orange boxed areas in (b) that can be identified based on their ultrastructure include: MVBs containing ILVs, mitochondria (M), endoplasmic reticulum (ER), round endosome (e), clathrin coated vesicle (cc), and tubular endosome (t). (i-l) Enlarged views of individual protrusions from green boxed areas in (c) are shown. Arrows indicate organelles that are structurally indistinguishable from organelles identified in the cytoplasm, shown in (d-h).
In summary, MCMVs are a novel form of EV that bud from membrane protrusions on the HUVEC surface, contain MVBs and release exosomes. These observations suggest that exosomes can be harboured within and released from transiting microvesicles after departure from the parent cell, constituting a new site of exosome biogenesis occurring from endothelial and potentially additional cell types.