Acute vascular rejection of renal allografts is associated with increased endothelial apoptosis which contributes to the development of transplant vasculopathy. We previously showed that apoptotic endothelial cells (EC) release exosome–like vesicles (ApoExo) that enhance vascular rejection in murine models of transplantation. However, the impact of ApoExo on vascular and endothelial functions remains to be characterized.

Primary human EC were exposed to a pro-apoptotic stimulus in presence or not of ApoExo purified by sequential centrifugation. ApoExo uptake was measured by flow cytometry and confocal microscopy. A gene expression analysis was defined by RNA sequencing. Wound closure and angiogenic activity were monitored by scratch and tube formation assays respectively. The expression of endothelial markers was measured by flow cytometry and by immunohistochemistry in a murine model of aortic transplantation between MHC-incompatible strains.

EC displayed a rapid uptake of ApoExo (97% positive cells after 1 hr) which was efficiently attenuated by inhibitors of a non-classical endocytosis (Dynasore: 35%, p<0.01 and MβCD: 26%, p<0.01). RNA sequencing identified 139 genes differentially regulated in ApoExo-treated EC. These genes are involved in cell death (16), cell growth (15), inflammation (7) and cell movement (4). To follow up on the functional importance of these gene patterns we evaluated apoptosis, wound closure and angiogenesis in ApoExo-treated EC. ApoExo inhibited apoptosis (17% vs 24%, p<0.01), improved endothelial wound closure (20.4% vs 7.9%, p<0.0001), but inhibited angiogenic activity (Segments: 26 vs 75, p<0.01). Expression of CD31 was reduced in EC exposed to ApoExo (71%, p<0.01) and in aortic allograft sections from mice injected with ApoExo (MFI: 6.7 vs 8.6, p<0.001). ApoExo also increased phosphorylation (Arbitrary units [AU]: 0.52 vs 0.35, p<0.05) and translocation of NFkB (AU: 2.37 vs 1.01, p<0.01). Finally, inhibition of NFkB signaling impaired ApoExo-induced wound closure (siCtrl: 16% vs siNFkB: 5%, p<0.05) and restore angiogenic properties (Seg: siCtrl: 25 vs siNFkB: 52, p<0.05).

Collectively, these data suggest that ApoExo change gene expression patterns and function of EC. Although ApoExo can inhibit EC death and favor migration, this occurs at the price of endothelial dedifferentiation and dysfunction.

CITATION INFORMATION: Migneault F., Dieudé M., Turgeon J., Beillevaire D., Hardy M-.P., Perreault C., Hébert M-.J. Apoptotic Exosome-Like Vesicles Induce Endothelial Cell Plasticity Am J Transplant. 2017;17 (suppl 3).

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