Calpain inhibition prevents high-glucose-induced autophagy blockade and mitochondrial fragmentation in endothelial cells and preserves vascular functions in diabetic mice

Abstract

Endothelial dysfunction is a major contributor to diabetic vascular complications, yet the mechanisms linking hyperglycemia to impaired endothelial homeostasis remain insufficiently defined. This study identifies calpains as key regulators of endothelial autophagy and mitophagy under diabetic conditions. In HUVECs and primary lung endothelial cells, hyperglycemia activated calpains and induced autophagic flux blockade, characterized by SQSTM1 accumulation, reduced LC3B expression, and impaired mitophagy associated with mitochondrial fragmentation. Pharmacological calpain inhibition or genetic calpastatin overexpression restored autophagic flux and preserved mitochondrial network architecture. Transcriptomic profiling revealed that calpain inhibition counteracted major hyperglycemia‑driven pathways, including oxidative phosphorylation, ROS signaling, and the unfolded protein response. Collectively, our findings demonstrate that calpain overactivation disrupts endothelial autophagy and mitophagy in diabetes, contributing to mitochondrial dysfunction and altered vascular reactivity. Restoring autophagic flux through calpain inhibition emerges as a promising strategy to preserve endothelial homeostasis and limit diabetic vascular complications.