Diabetes is a widely spread metabolic disorder and is intimately associated with major micro- and macro-vascular complications. Several previous studies including ours highlight the complex interplay between Endoplasmic Reticulum (ER) stress and oxidative stress in the pathogenesis of cardiovascular complications associated with obesity and diabetes. Hemeoxygenase-1 (HO-1) induction has been shown to protect against oxidative stress in diabetes, however the underlying molecular mechanisms have not yet been fully elucidated. We aim in this project to test the hypothesis that HO-1 induction will protect against high glucose-mediated ER stress and oxidative stress in endothelial cells and will enhance cell survival.
Endothelial cells, EA.hy926 cell line and Human Umbilical Vein Endothelial cells (HUVECs), were cultured in physiological or high concentrations of glucose to mimic diabetic environment in the presence of cobalt protoporphyrin IX (CoPP, HO-1 inducer), 4-phenylbutyrate (PBA, chemical chaperone to inhibit ER stress), Tauroursodeoxycholic acid (TUDCA, another chemical chaperone to inhibit ER stress) or vehicle. Then, ER stress response was assessed (PCR and western blot). The productions of reactive oxygen species (ROS) and Nitric Oxide (NO) were analysed by flow cytometer and Griess assay, respectively. Furthermore, we analysed apoptosis and caspase 3/7activity in endothelial cells. Expression of inflammatory cytokines was also assessed by PCR.
High glucose treatment in endothelial cells induced an increase of mRNA expression of several ER stress response markers (BIP, CHOP, ATF4) in addition to an increase in ROS production and a reduction in NO release. Interestingly, the pre-treatment of cells with TUDCA/PBA or CoPP significantly reduced high glucose-mediated ER stress and oxidative stress in cells. In addition, endothelial cells incubated with high glucose exhibited enhanced cell death and increased caspase 3/7 activity while cells pre-treated with either PBA or CoPP were partially or totally protected. The mRNA expression of inflammatory cytokine IL-6 was significantly enhanced in cells incubated with high glucose while this was completely reversed in cells pre-treated with TUDCA or CoPP.
Overall, these results highlight the importance of oxidative stress both in initiating or maintaining ER stress response and in mediating ER stress-induced damage and cell death in endothelial cells. This work also underscores the therapeutic potential of HO-1 induction against hyperglycaemia-mediated endothelial dysfunction.
- High glucose
- Oxidative stress and endoplasmic reticulum stress