Abstract P241: Calpain-1 Is Increased in Mitochondria and Contributes to Mitochondrial ROS Generation in High Glucose--Stimulated Endothelial Cells and Endothelial Dysfunction in Mouse Models of Diabetes

Abstract

Objectives: Elevated levels of reactive oxygen species (ROS) are the initial source of endothelial dysfunction in diabetes. Calpain has been implicated in diabetic vascular complications. The present study was to investigate the role of calpain in mitochondrial ROS generation in endothelial cells and vascular dysfunction in diabetic mice. Methods: Endothelial cells cultured from human umbilical vein (HUVEC) were stimulated with high glucose. Calpain activity and protein were determined in mitochondria of HUVEC. Intracellular and mitochondrial ROS generation as well as apoptosis were measured. Type I diabetic OVE 26 mice and type II diabetic db/db mice with calpastatin over-expression (OVE26/CAST and db/db-CAST) were generated, respectively. Type I diabetes was also induced in both wild-type and Tg-CAST mice by injection of streptozocin (STZ). The endothelium-dependent relaxation of aortic ring was measured. Results: High glucose significantly increased calpain-1 protein, calpain activity and ROS generation in mitochondria of HUVEC. Pharmacological inhibition of calpain or over-expression of calpastatin abrogated high glucose-induced intracellular ROS production, mitochondrial ROS generation and apoptosis in HUVEC. Incubation of isolated mitochondria with calpain-1 protein significantly induced its ROS generation and the membrane potential. In diabetic mice, calpain activity was induced in aortic vessels, which correlated with an increase in ROS production and protein tyrosine nitration. Over-expression of calpastatin prevented calpain activity, reduced ROS production and inhibited protein tyrosine nitration in diabetic mice. Aortic ring segments from diabetic mice exhibited a significant reduction in vascular relaxation to acetylcholine, which was reversed by over-expression of calpastatin in Tg-CAST, OVE26/CAST and db/db-CAST mice. Conclusions: This study has demonstrated a novel role of calpain in mitochondrial ROS generation, which contributes to apoptosis in endothelial cells during hyperglycemia. Thus, over-expression of calpastatin inhibits reduces ROS production and ameliorates endothelium-dependent vascular dysfunction in mouse models of diabetes.