Abstract
Apoptotic exosome-like vesicles (ApoExos) are a novel type of extracellular vesicle that contribute to the propagation of inflammation at sites of vascular injury when released by dying cells. ApoExos are characterized by the presence of the C-terminal perlecan LG3 fragment and 20S proteasome, and they are produced downstream of caspase-3 activation. In the present study, we assessed the relative roles of autophagy and caspase-3-mediated pathways in controlling the biogenesis and secretion of immunogenic ApoExos. Using electron microscopy and confocal immunofluorescence microscopy in serum-starved endothelial cells, we identified large autolysosomes resulting from the fusion of lysosomes, multivesicular bodies, and autophagosomes as a site of ApoExo biogenesis. Inhibition of autophagy with ATG7 siRNA or biochemical inhibitors (wortmannin and bafilomycin) coupled with proteomics analysis showed that autophagy regulated the processing of perlecan into LG3 and its loading onto ApoExos but was dispensable for ApoExo biogenesis. Caspase-3 activation was identified using caspase-3-deficient endothelial cells or caspase inhibitors as a pivotal regulator of fusion events between autolysosomes and the cell membrane, therefore regulating the release of immunogenic ApoExos. Collectively, these findings identified autolysosomes as a site of ApoExo biogenesis and caspase-3 as a crucial regulator of autolysosome cell membrane interactions involved in the secretion of immunogenic ApoExos.
Highlights
Autophagy is generally viewed as an autodigestive process aimed at providing energy to cells exposed to stress or facing reduced access to nutrients
The apoptotic exosome-like vesicles (ApoExos) expressed several exosome markers, such as TSG101, syntenin-1, and TCTP (Fig. S1), but unlike classical exosomes produced by normal endothelial cells, they expressed a set of specific markers, including LAMP2, LG3, and the active 20S proteasome
Mounting evidence associates graft rejection and ischemia–reperfusion injury with the presence of autoantibodies reactive to components of endothelial cells or apoptotic cells in general [12, 24,25,26,27,28]
Summary
Autophagy is generally viewed as an autodigestive process aimed at providing energy to cells exposed to stress or facing reduced access to nutrients. In addition to its degradative function, autophagy contributes to the secretion of various types of mediators, some of which play important roles in shaping the immune response. Our group showed that the protein constituents of autophagosomes are released by endothelial cells downstream of caspase activation induced by serum starvation [3]. In this system, serum-starved endothelial cells mount an autophagic response leading to the formation of a network of large autophagic vacuoles that fuse with the cell membrane once caspases are activated [3]. The LC3-conjugation machinery at play during autophagy has recently been found to regulate the loading of RNA-binding proteins and small noncoding RNAs into membrane-bound vesicles that, in turn, are released by autophagic cells [7]
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