*Purpose: We have previously demonstrated activation and increasing mitophagy of human endothelial cells (ECs) following exogenous mitochondrion-stimulation. The objectives of this study were to evaluate stimulator of interferon genes (STING) and mTOR signaling in regulating mitophagy in ECs following their interaction with exogenous mitochondria.

*Methods: Human mitochondria were purified from HeLa cells. Phosflow was used to detect mTOR, S6, and NF-kB phosphorylation in ECs during their interaction with exogenous mitochondria. ECs with or without inhibitors specific for STING and mTOR were incubated with mitochondria followed by FACS analysis to detect EC activation, mitochondria internalization, and mitophagy. RNA-sequencing analysis was performed to assess mitophagy/autophagy pathways in naïve and rapamycin-preconditioned ECs.

*Results: ECs internalized mitochondria and showed activation as determined by upregulation of ICAM and VCAM expression, respectively. ECs rapidly phosphorylated STING at Ser366 and NF-kB after coculture with exogenous mitochondria. The mitochondrion-stimulated ECs significantly phosphorylated mTOR at serine residue 2488 when compared to resting ECs (p<0.0001). Similarly, significant phosphorylation of ribosomal protein S6 at C-terminal serine residue 235/236 was revealed in mitochondrion-treated ECs (p<0.0001). The mitochondrion-induced phosphorylation of mTOR and S6 was inhibited by rapamycin pretreatment (p<0.001). However, rapamycin but not STING inhibitor H151 failed to prevent upregulation of CD54 and CD106 expression in ECs following incubation with exogenous mitochondria. Internalization of exogenous mitochondria by ECs was scavenger receptor-dependent, and mTOR blockade of ECs did not alter internalization of mitochondria. The activity of mitophagy/autophagy in rapamycin-preconditioned ECs was unchanged when compared to resting or vehicle-treated ECs. An increase of mitophagy/autophagy was revealed in exogenous mitochondrion-treated ECs when compared to resting cells. The activity of mitophagy/autophagy was significantly enhanced in rapamycin-preconditioned ECs (p=0.022) when compared to vehicle-conditioned ECs following mitochondrion stimulation. In contrast, STING inhibitor prevented mitophagy/autophagy in mitochondrion-stimulated ECs. RNA sequencing analysis of mitophagy signaling pathway in rapamycin-preconditioned ECs revealed upregulation of genes associated with hypoxia/mitophagy signaling.

*Conclusions: Our study demonstrates that exogenous mitochondrion-stimulated ECs rapidly phosphorylate STING, mTOR, and S6, and upregulate adhesion molecules. Internalization of exogenous mitochondria by ECs is mediated by scavenger receptor. STING signaling plays a critical role in regulating mitophagy and activation of mitochondrion-stimulated ECs. mTOR blockade in ECs fails to prevent mitochondrion-induced EC activation, but enhance mitophagy activity in maintaining cell health.

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