Abstract 12185: Restoration of Autophagy in Endothelial Cells From Patients With Diabetes Improves Nitric Oxide Signaling

Abstract

Diabetes is associated with oxidative stress and decreased nitric oxide bioactivity in the vasculature. Autophagy is a critical multistep pathway that eliminates damaged proteins and organelles from the cell. Emerging evidence suggests impaired autophagy in non-vascular tissues contributes to the pathogenesis of diabetes. We hypothesized that impaired autophagy contributes to endothelial dysfunction associated with diabetes in humans. We measured vascular function and autophagy markers in freshly isolated endothelial cells (ECs) from patients with diabetes and non-diabetic controls. Diabetes was associated with endothelial dysfunction characterized by lower brachial artery flow-mediated dilation and impaired activation of eNOS by insulin in ECs. ECs from diabetic patients had higher levels of p62 (Figure), a protein that accumulates with reduced autophagic flux. Measures of autophagy initiation including rapamycin stimulation, beclin 1 levels, and LC3 puncta were not different. Global activation of autophagy with spermidine reversed endothelial dysfunction in freshly isolated ECs from diabetic patients (Figure). In ECs from controls, inhibiting autophagy with bafilomycin impaired eNOS activation confirming that intact autophagy promotes NO signaling. Evidence from cultured endothelial cells in high glucose conditions suggested that lysosomal function is intact as measured by lysosomal number and acidification. Blocking the terminal step of autophagy with bafilomycin in ECs from diabetics led to a further accumulation of p62, suggesting intact but insufficient levels of autophagy. Lamp2a, which facilitates the merger of autophagosomes and lysosomes, was higher in diabetic cells, possibly reflecting a compensatory response to reduced flux. These findings provide evidence for inadequate autophagic flux in ECs from diabetic patients that contributes to impaired NO signaling and may be a target for therapy of diabetic vascular disease.