Author + information
- Sang-Bing Ong1,2,
- Won Hee Lee3,
- Nur Izzah Ismail4,1,
- Khairunnisa Katwadi1,2,
- Xiu Yi Kwek1,2,
- Kroekkiat Chinda5 and
- Sang Ging Ong3
- 1Cardiovascular & Metabolic Disorders Program, Duke-NUS Medical School, Singapore
- 2National Heart Research Institute of Singapore, National Heart Centre of Singapore, Singapore
- 3Stanford Cardiovascular Institute, Stanford University School of Medicine, United States
- 4Department of Biomedical Engineering, Faculty of Engineering, University of Malaya, Malaysia
- 5Department of Physiology, Faculty of Medical Science, Naresuan University, Phitsanulok, Thailand
Diabetes mellitus remains one of the worldwide leading causes of morbidity and mortality, resulting in huge social and economic burden. Hyperglycemia and hyperlipidemia are associated with endothelial dysfunction and contributes to the progression of diabetes and its associated complications. The pathophysiology of diabetic endothelial dysfunction remains incompletely characterized, thus limiting effective therapeutic interventions. Calpain – a calcium-dependent protease, has been shown to increase in endothelial cells under diabetic conditions. Nevertheless, the role of calpain in diabetic endothelial dysfunction remains elusive. This present study was to investigate the effects of calpain inhibition in endothelial cells derived from human induced pluripotent stem cells (iPSCs) subjected to a diabetogenic microenvironment in vitro.
Human iPSCs were differentiated into endothelial cells (iPSC-ECs) and subjected to a hyperglycemic microenvironment for 72 hours to simulate a diabetic condition. Tube formation was quantified by measuring the number, length, and area of the capillary-like structures. Reactive oxygen species (ROS) formation and ATP levels were also quantified. The levels of autophagic proteins were detected using Western blot. Mitochondrial morphology in the cells were monitored using MitoTracker Red staining. To simulate ischemia-reperfusion, the cells were cultured in a buffer containing no glucose, no serum in the presence of sodium-lactate in an airtight chamber (for ischemia) followed by a normoxic buffer containing the required components (for reoxygenation).
iPSC-ECs exposed to hyperglycemia had impaired tube formation (35 ± 3% vs 92 ± 5%), increased ROS (1.4 ± 0.3 vs 1.0 ± 0.1 RLU) and reduced ATP levels (0.8 + 0.1 vs 1.0 ± 0.1 RLU), compared to cells in control conditions. In line with an increase in calpain activity, iPSC-ECs exposed to hyperglycemia had reduced autophagy – findings which were validated in primary HAECs. Treatment with the calpain inhibitor MDL28170 (5 μM) reversed the detrimental phenotypes observed in the cells. The mitochondrial morphology in iPSC-ECs exposed to hyperglycemia also switched from a fragmented phenotype (20% of cells with elongated mitochondria) to an elongated phenotype (80% of cells with elongated mitochondria) following calpain inhibition, leading to protection against simulated ischemia-reperfusion injury.
Calpain inhibition in hyperglycemic iPSC-ECs restored autophagy and inhibited hyperglycemia-induced mitochondrial fragmentation. This led to reduced susceptibility to ischemia-reperfusion injury in the hyperglycemic cells, thus revealing a potential therapeutic strategy for diabetic patients prone to cardiovascular complications.