Crosstalk between exosomes signaling pathway and autophagy flux in senescent human endothelial cells

The intracellular endomembrane system contributes to maintaining cell homeostasis. We investigate the dynamic of exosomal and autophagy pathways in Human Umbilical Vein Cells (HUVECs) following incubation with H2O2 in vitro. Cellular senescence was induced to HUVECs using 100 µM H₂O₂. ELISA and AChE assay was used to calculate the number of exosomes. Exosomes were isolated and characterized by dynamic light scattering, flow cytometry, and SEM. Transcript and protein levels of genes involved in the exosomal and autophagy pathways were measured by real-time PCR (Q-PCR) and western blotting. Subcellular distribution of CD63 was monitored by immunofluorescence microscopy. We also measured the expression of miR-182 and miR-155 by qPCR assay. Results showed that secretion of exosomes was increased in treated cells (p < 0.05). Exosomes were confirmed by size and positive for CD63 marker. Molecular analysis of the exosomal secretory pathway has revealed a significant induction of CD63, CD81, TSAP6, Rab11, Rab27a, and Rab27b in response to H₂O₂ (p < 0.05). The distribution of CD63 was increased inside treated cells. The western blotting technique revealed a significant up-regulation in Becline-1 and P62 and a significant decrease in LC3 II/I ratio in treated cells (p < 0.05)s. Concomitant with an up-regulation of common molecules in exosomes biogenesis and autophagy including Atg5, P62 and P53, expression of miR-182 decreased and miR-155 increased in cells incubated with H2O2 (p < 0.05). Data suggested an induction in the exosomal secretory pathway coincided with a block in autophagy progress, accelerating senescence, which might be targeted for the treatment of age-related diseases.