MSCs attenuate high glucose-induced oxidative stress and reduce mitochondrial dysfunction in human umbilical vein endothelial cells
Yujia Yuan1, MM Shi1, YN Chen1, JP Liu1, B Chen1, L Li1, J Zhang1, RX Luo1, YM Li1, LZ Li1, JQ Cheng1, YR Lu1.
1Laboratory of Transplant Engineering and Immunology, West China Hospital, Sichaun University, Cheng Du, People's Republic of China
Background: Hyperglycemia in diabetes mellitus is a major risk factor for endothelial dysfunction. Accumulated evidence has shown that mitochondrial impairment contributes to the endothelial dysfunction. Under high glucose condition, oxidative stress caused by excessive generation of reactive oxygen species (ROS) could result in mitochondrial dysfunction. Previous studies have demonstrated that mesenchymal stem cells (MSCs) had immuno-privileged and immunomodulatory properties, and were able to stimulate vessel formation by paracrine mechanisms. In our study, we examined whether specific cytokines and growth factors secreted by MSCs were able to prevent endothelial cells from the ROS-mediated mitochondrial dysfunction.
Methods: Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell Growth Medium BulletKit-2 (EGM-2) medium with either normal glucose (NG; 5mM) or high glucose (HG; 30mM) concentrations. After 96h induction, cell proliferation rate and the total intracellular ROS production were measured. To further identify the source of the excessive ROS generation, 0.5 μM diphenylenei-odonium (DPI), a NADPH oxidase (NOX) inhibitor and 50 μM Mito-TEMPO, the specific scavenger of mitochondrial ROS were administered to cells in HG group respectively. Conditioned media (CM) was prepared by culturing MSCs for 24h and then was added to the HG group (HG+CM) at the rate of 25%. Mitochondrial mass was estimated fluorimetrically with Mitotracker Green, mitochondrial ROS was measured using MitoSox Red, and the mitochondrial membrane potential was monitored by JC-1 probe. Results: Comparing with the NG group, treatment with high glucose induced a concentration-dependent decrease in cell proliferation rate and an increase in total intracellular ROS production in HUVECs. After inhibition of NOX, the intracellular ROS and mitochondrial ROS were slightly reduced to 70.5±8.6%. While, after the treatment of mito-TEMPO, not only the mitochondrial ROS, but also the total intracellular ROS was dramatically reduced to 45.6±2.0%. Comparing with HG group, when the cells were continously exposed to MSCs conditioned media (HG+CM group), the proliferation rate was improved from 68.8±3.1% to 92.4±3.7%, and the total intracellular ROS production was diminished from 1.9±0.1 fold to 1.3±0.1 fold of that in NG group. In the meantime, after treatment of CM, mitochondrial mass of these cells was as 1.5±0.1 fold higher as that in HG group. Additionally, the mitochondrial depolarization quantified by JC-1 probe was repressed from 38.2±6.3% to 21.3±3.6% and the ratios of ATP levels to that of NG group were restored from 64.2±5.4% to 88.7±5.0%. Conclusion: The study has shown that glucose-induced ROS was primarily originated from mitochondria and the excessive generation of ROS could furtherly deteriorate the mitochondrial biogenesis and energy metabolism in endothelial cells. Conditioned media obtained from the MSCs has potential to relieve mitochondrial dysfunction via its anti-oxidant effects. Our study may provide a clue for considering conditioned medium of heterogenetic MSCs as a potential cell-free strategy to ameliorate the oxidative injury in endothelial cell.
11:00 - 12:30
|Mesenchymal Stromal Cells||MSCs attenuate high glucose-induced oxidative stress and reduce mitochondrial dysfunction in human umbilical vein endothelial cells||Room 111-112|