Mitochondrial coupling regulates responses of vascular endothelial cells to oxidative stress.

Abstract

Failure to normalize generation of reactive oxygen species (ROS) is the most important factor in development of cardiovascular disease. Uncoupling of oxidative phosphorylation (OXPHOS) reduces ROS generation. We hypothesized that in human coronary artery endothelial cells (HCAECs) uncoupling of mitochondria reduces ability to generate ROS. Mitochondria were uncoupled using FCCP (100 nM), a chemical uncoupler. Oxidative stress was induced by doxorubicin (DOX, 5 μM, 1 hr) a redox cycling drug. HCAECs were treated with FCCP (10 min) or FCCP followed by DOX. The levels of ATP, LDH release and MTT assay were monitored 1‐ and 24 hours after treatment. FCCP alone had no effects on total ATP level, LDH release or cells viability. Treatment with DOX caused 20% (n=3, P<0.05) reduction in viability of HCAECs. Treatment with FCCP prior to the addition of DOX maintained cells viability at the control levels (n=3, NS). Treatment with DOX increased expression of Cu, Zn‐SOD and Mn‐SOD (2‐fold vs. control). Treatment with FCCP prior to addition of DOX maintained expression of Cu, Zn‐SOD and Mn‐SOD at the control levels. Functional importance of uncoupling was tested in the whole‐cell mode of the patch‐clamp. The addition of FCCP depolarized HCAECs by 40 mV (n=10, P<0.05). The dialysis of cells with a re‐coupling agent GDP (500 mM) caused a repolarization of HCAECs. Taken together, oxidative stress related changes of vascular function could be normalized by a short‐term, mild uncoupling of mitochondrial OXPHOS. The ability to control the level and time of mitochondrial coupling could be a way of delaying or preventing a vascular damage.