Autophagy regulation in glyoxal-treated endothelial cells

Diabetic complications are a major cause of death around the world. This is partly due to an increase in advanced glycation end products (AGEs) that damage endothelial cells (ECs). Glyoxal, an α-dicarbonyl intermediate, is a precursor of AGEs, including carboxymethyllysine (CML). Previous research from our group showed that glyoxal changes the endothelial proteome. It increases stress response proteins, reduces growth-related processes, and causes CML modification of nuclear, cytoskeletal, and mitochondrial proteins. Normally, glyoxal is detoxified, but its higher levels in diabetes lead to cellular damage.This thesis explored glycative stress-induced endothelial dysfunction, focusing on autophagy, a vital process disrupted in diabetes. We treated human umbilical vein endothelial cells (HUVECs) with glyoxal and examined the stages of autophagy using immunofluorescence microscopy, fluorescent constructs, western blotting, RNA interference (RNAi), pharmacological tools, and organelle assays. Glyoxal led to the formation of CML-modified proteins and signs of autophagy initiation. We observed increased AMPK and LC3B, decreased mTORC1, and higher autophagosome formation. RNAi experiments showed some degradation of CML-modified proteins, but they suggested a late-stage block, confirmed by P62 accumulation and reduced autophagic flux because of impaired autophagosome-lysosome fusion. The results included issues with lipophagy, shown by Plin2-positive lipid droplet accumulation, increased ER stress indicated by CHOP, and mitochondrial dysfunction without an increase in mitophagy. Overall, glycative stress interferes with autophagy at the fusion step, leading to increased AGE accumulation and endothelial dysfunction while identifying possible therapeutic targets.