Enhanced Apoptosis in 3D Endothelial Cell Cultures Exposed to Hyperglycaemia and Microgravity

Abstract

Objectives The space environment has specific stressors that are not found on Earth, including cosmic radiation and microgravity (µg). Astronauts, after long-term spaceflight, return with various health-related problems, including increased plasma glucose and induced diabetogenic state. It is unknown how these health-related alterations occur, and the interactions between µg and hyperglycaemia are not fully understood. In this study, we aimed to gain some insights by investigating endothelial cell changes in different gravity conditions and glucose concentrations.Materials and methods We cultured EA.hy926 endothelial cells in simulated µg (s-µg) using a 3D clinostat and static normogravity (1g) conditions exposed to physiological and hyperglycaemic glucose levels. After two weeks, the samples were collected, and the expression of various ECM, inflammation and apoptosis-related genes were analyzed by qPCR and protein expression by the Western blot method. In addition, immunofluorescence and confocal microscopy techniques were used to investigate samples' morphological differences and protein distribution. Apoptosis was assessed by TUNEL staining. Results Our results indicate that hyperglycaemia did not affect the gene and protein expression in 1g conditions. In µg-conditions, which resulted in detached multicellular structures, hyperglycaemia increased the size and the number of spheroids, decreased fibronectin, transglutaminase-2, and increased NOX4, NF-κB, and caspase-3. Conclusions These results suggest hyperglycaemia activates programmed cell death. The findings bring new knowledge into the possible molecular pathways involved in diabetogenic vascular effects in µg.