Background: Autoantibodies to apoptotic antigens such as LG3 – a C-terminal fragment of perlecan– are present pre-transplantation and contribute to organ rejection in renal transplant patients. We previously showed that, in apoptotic endothelial cells (EC), caspase-3 prompts the release of immunogenic apoptotic exosome-like vesicles (ApoExo) that carry LG3 fragments and control autoimmune responses to LG3. Here, we aim at characterizing the relative importance of autophagy, apoptosis and necrosis in the export of LG3 within ApoExo.

Methods: Human umbilical vein endothelial cells (EC) were exposed to serum starvation and H₂O₂, respectively inducers of autophagy/apoptosis and necrosis. Cell death levels were evaluated by fluorescence microscopy and flow cytometry. LG3 export and LC3 II/I ratios were monitored by western blotting. Electron microscopy (EM) and confocal microscopy were used to monitor intracellular LG3 trafficking. Extracellular vesicles (EV) were evaluated by small particle flow cytometry (sFACS).

Results: The release of ApoExo carrying LG3 (p<0,0001) and Lamp-2 (p=0,01), a lysosomal marker, was increased in association with apoptosis but not with necrosis. Bafilomycin, an inhibitor of lysosome acidification which prevents autophagosome maturation, inhibited the release of LG3 within ApoExo (p=0.03). Inhibiting autophagosome formation with Wortmaninn and LY294002 also decreased LG3 release, suggesting that autophagy and lysosomal activity contribute to LG3 processing and export within ApoExo. In serum starved EC, electron and confocal microscopy identified LG3 within large vesicular networks reminiscent of autophagosomes at different stages of formation. Within these networks, LG3 was found within small membrane-bound structures reminiscent of intracellular ApoExo.

Conclusion: These results identify, in addition to caspase-3, lysosomal activity and autophagy as important pathways regulating the release of LG3 within ApoExo. These results point to inhibition of lysosomal activity as a novel target of intervention for preventing the production of immunogenic LG3 at sites of endothelial injury.

CITATION INFORMATION: Beillevaire D, Francis M, Diane G, Éric B, Mélanie D, Marie-Josée H. Autophagy Regulates LG3 Maturation and Export in Apoptotic Exosome-Like Vesicles. Am J Transplant. 2017;17 (suppl 3).

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