Cross‐talk Between Autophagy and Mitophagy Regulates Shear‐induced Nitric Oxide Production in Endothelial Cells

Abstract

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 (3 h x 20 dyn/cm2) exhibit: ↑LC3‐II:LC3‐1, ↓p62, ↑LC3‐GFP puncta formation (indices of increased autophagy); ↓VDAC, ↓m‐aconitase, ↑TOM20:LAMP1 colocalization, ↓ mitochondrial pH indicating lysosomal compartmentalization (indices of increased mitochondrial turnover), ↑ROS generation, ↑endothelial NO synthase (eNOS) phosphorylation, and ↑NO production (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.