*Purpose: Ischemia-reperfusion and rejection favor endothelial apoptosis, which in turn contributes to maladaptive vascular repair leading to transplant vasculopathy. Apoptotic endothelial cells (EC) release exosome-like vesicles (ApoExo) that, when uptaken by neighboring EC, shape gene expression and functions in directions conducive to endothelial dysfunction and mesenchymal transition. However, the mechanisms by which EC internalize these vesicles remain ill-defined.

*Methods: ApoExo were isolated by ultracentrifugation from serum-free media conditioned by primary human EC labeled with CellTracker Orange CMRA dye. Serum-starved primary human EC were then exposed to purified ApoExo in vitro. The cellular uptake of ApoExo was observed by confocal microscopy and flow cytometry using selective pharmacological inhibitors and gene knockdown. EC morphology was assessed by electronic microscopy.

*Results: ApoExo displayed time and concentration-dependent uptake kinetics which were completely abolished when cells were incubated at 4°C (11.6%, P<0.001) suggesting an energy-dependent uptake process. Concomitant exposure with unlabeled ApoExo also reduced the uptake of fluorescent ApoExo (44.3%, P<0.05). Disruption of the actin cytoskeleton by exposing EC to cytochalasin D significantly attenuated ApoExo uptake (77.8%, P<0.05). siRNA-mediated knockdown of clathrin and caveolin 1 did not affect ApoExo internalization suggesting a non-classical endocytosis pathway. The disruption of ApoExo membrane integrity using methyl-β-cyclodextrin efficiently attenuated vesicles internalization (25.8%, P<0.001). EIPA, a macropinocytosis inhibitor, and Annexin V, a protein with a high affinity for phosphatidylserine also significantly decreased ApoExo uptake by EC (58.5%, P<0.05 and 67.6%, P<0.05) suggesting a phosphatidylserine-dependent fluid‐phase uptake via macropinocytosis. Electronic microscopy analysis detected membrane ruffling in EC hallmark of macropinocytosis.

*Conclusions: Collectively, these results suggest that ApoExo are internalized by EC through macropinocytosis. This characterization highlights potential strategies to target this internalization pathway and prevent endothelial dysfunction that contributes to transplant vasculopathy.

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