Rebekah Goodrich, Leenalitha Panneerseelan Bharath, Ting Ruan, Tetyana Forostyan, Ashot Sargsyan,
Yong Han, Robert Mueller, Pon Velayutham Anandh Babu, Sihem Boudina, Timothy Eugene Graham,
and John David Symons, University of Utah
Health
Aging is associated with suppressed endothelial cell (EC) autophagy and EC nitric oxide (NO) bioavailability. A link might exist among autophagy, mitophagy, and stimulated NO production in ECs. ECs treated with scrambled siRNA exposed to shear stress (3h x 20 dyn/cm2) exhibit an increase in LC3-II:LC3-1, LC3-GFP puncta formation (indices of increased autophagy), TOM20:LAMP1 colocalization, ROS generation, endothelial NO synthase (eNOS) phosphorylation, and NO production (p<0.05 for all). In tandem with a decrease in VDAC, m-aconitase, mitochondrial pH indicating lysosomal compartmentalization (indices of increased mitochondrial turnover), and p62 (p<0.05 for all). ECs transfected with autophagy-related protein 3 (Atg3) siRNA did not exhibit shear- induced autophagy, mitochondrial turnover, eNOS phosphorylation, or NO production, but ROS accumulation and cytokine production (ICAM-1, MCP-1, IL-8, E-selectin) were exaggerated (all p<0.05). Shear-induced increases in oxygen consumption rate, extracellular acidification rate, and ATP production (all p<0.05) in ECs were prevented by autophagy suppression, indicating impaired mitochondrial function. Dysfunctional mitochondria might be the source of ROS when autophagy is compromised because: (i) shear-induced increases in NO generation were restored, and ROS production and inflammation were normalized, when ECs +Atg3 siRNA were treated with a mitochondrial- targeted O2•- scavenger; and (ii) the EC phenotype +Atg3 siRNA could be recapitulated using approaches that limit mitophagy per se. A crosstalk exists among autophagy, mitophagy, and NO production in ECs.