ABSTRACT
BACKGROUND AND OBJECTIVES: The redox system is an important anti-oxidative system composed of thioredoxin, thioredoxin reductase, and peroxiredoxin (PRx). The fine details of PRx expression and its protective effects in various cells in cardiovascular tissue under oxidative stress created by hydrogen peroxide have not been fully elucidated. SUBJECTS AND METHODS: Oxidative stress was induced by adding hydrogen peroxide at 0.25 mM for 2 hours to rat neonatal cardiomyocytes (rCMCs), rat vascular smooth muscle cells (rVSMCs), and human umbilical vein endothelial cells (HUVECs). Apoptosis was quantified by flow cytometry and the expression patterns of the six PRx isoforms were evaluated by western blotting in the three cell lines after hydrogen peroxide stimulation. Apoptosis and the cell survival signal pathway were evaluated by PRx1 gene delivery using lentiviral vector in hydrogen peroxide stimulated rCMCs versus green fluorescence protein gene delivery. RESULTS: Hydrogen peroxide induced 25% apoptosis in rCMCs. Furthermore, the PRx1 and 5 isoforms were found to be overexpressed in hydrogen peroxide treated rCMCs, and PRx1 overexpression by gene delivery was found to reduce hydrogen peroxide induced rCMCs apoptosis significantly. In addition, this effect was found to originate from cell survival pathway modification. CONCLUSION: Hydrogen peroxide induced significant oxidative stress in rCMCs, rVSMCs, and HUVECs, and PRx1 overexpression using a lentiviral vector system significantly reduced hydrogen peroxide induced rCMCs apoptosis by upregulation of cell survival signals and downregulation of apoptotic signals. These findings suggest that PRx1 could be used as a treatment strategy for myocardial salvage in conditions of oxidative stress.
Subject(s)
Animals , Rats , Apoptosis , Blotting, Western , Cell Line , Cell Survival , Down-Regulation , Flow Cytometry , Fluorescence , Human Umbilical Vein Endothelial Cells , Hydrogen , Hydrogen Peroxide , Muscle, Smooth, Vascular , Myocytes, Cardiac , Oxidation-Reduction , Oxidative Stress , Peroxiredoxins , Protein Isoforms , Signal Transduction , Thioredoxin-Disulfide Reductase , Thioredoxins , Up-RegulationABSTRACT
BACKGROUND AND OBJECTIVES: The thioredoxin (TRx) system is a ubiquitous thiol oxidoreductase pathway that regulates cellular reduction/oxidation status. Although endothelial cell (EC) hypoxic damage is one of the important pathophysiologic mechanisms of ischemic heart disease, its relationship to the temporal expression pattern of the TRx system has not yet been elucidated well. The work presented here was performed to define the expression pattern of the TRx system and its correlation with cellular apoptosis in EC lines in hypoxic stress. These results should provide basic clues for applying aspects of the TRx system as a therapeutic molecule in cardiovascular diseases. SUBJECTS AND METHODS: Hypoxia was induced with 1% O2, generated in a BBL GasPak Pouch (Becton Dickinson, Franklin Lakes, NJ, USA) in human endothelial progenitor cells (hEPC) and human umbilical vein endothelial cells (HUVEC). Apoptosis of these cells was confirmed by Annexin-V: Phycoerythrin flow cytometry. Expression patterns of TRx; TRx reductase; TRx interacting protein; and survival signals, such as Bcl-2 and Bax, in ECs under hypoxia were checked. RESULTS: Apoptosis was evident after hypoxia in the two cell types. Higher TRx expression was observed at 12 hours after hypoxia in hEPCs and 12, 36, 72 hours of hypoxia in HUVECs. The expression patterns of the TRx system components showed correlation with EC apoptosis and cell survival markers. CONCLUSION: Hypoxia induced significant apoptosis and its related active changes of the TRx system were evident in human EC lines. If the cellular impact of TRx expression pattern in various cardiovascular tissues under hypoxia or oxidative stress was studied meticulously, the TRx system could be applied as a new therapeutic target in cardiovascular diseases, such as ischemic heart disease or atherosclerosis.