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Updated: Aug 16, 2026
Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells
Published on: December 11, 2020
SOD2 protects against oxidation-induced apoptosis in mouse retinal pigment epithelium: implications for age-related
Emiko Kasahara1, Li-Ren Lin, Ye-Shih Ho
1Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105, USA.
Purpose:
Oxidative stress from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration (AMD), in which the retinal pigment epithelium (RPE) is considered a primary target. Because manganese superoxide dismutase (SOD2), localized in mitochondria, is known to be a key enzyme that protects the cells against oxidative stress, this study was undertaken to examine oxidation-induced apoptosis in cultured RPE cells with various levels of SOD2.
Methods:
Primary cultures of RPE cells were established from wild-type (WT), heterozygous Sod2-knockout mouse (HET) and hemizygous Sod2 mice with overexpression of the enzyme (HEMI). Purity of the RPE cell cultures was verified by immunostaining with antibody to RPE65 and quantified by flow cytometry. Oxidative stress was induced in RPE cells by exposing them to H(2)O(2) (0-500 muM) for 1 hour and reculturing them in normal medium for various times (0-24 hours). Apoptosis in the RPE was examined by TUNEL staining and quantified by cell-death-detection ELISA. Mitochondrial transmembrane potential (MTP) was measured by a cationic dye, and cytochrome c leakage from mitochondria was analyzed by Western blot analysis.
Results:
More than 95% of the cells in each culture were RPE65 positive, and the relative SOD2 levels in HET, WT, and HEMI cells were 0.6, 1.0, and 3.4, respectively. H(2)O(2)-induced apoptotic cell death was both dose and time dependent, and apoptosis in these cells was related to the cellular SOD2 level. Disruption of MTP and release of cytochrome c were observed to occur before apoptotic cell death, and they correlated with cellular SOD2.
Conclusions:
The results demonstrate a critical role of SOD2 in protection against oxidative challenge. Cells from HET mice showed greater apoptotic cell death, whereas in those from HEMI mice, cell death induced by oxidative injury was suppressed.
Insights
Manganese superoxide dismutase (SOD2) protects retinal cells from oxidative stress. Lower SOD2 levels increase apoptosis, while higher levels suppress cell death, highlighting SOD2
Area of Science:
- Cell Biology
- Oxidative Stress Research
- Ophthalmology
Background:
- Oxidative stress from reactive oxygen species (ROS) is implicated in diseases like age-related macular degeneration (AMD).
- The retinal pigment epithelium (RPE) is a primary target of oxidative damage in AMD.
- Manganese superoxide dismutase (SOD2), located in mitochondria, is crucial for cellular protection against oxidative stress.
Purpose of the Study:
- To investigate the role of varying manganese superoxide dismutase (SOD2) levels in oxidative stress-induced apoptosis in cultured retinal pigment epithelium (RPE) cells.
- To understand how SOD2 expression impacts RPE cell susceptibility to hydrogen peroxide (H2O2)-induced damage.
Main Methods:
- Primary RPE cell cultures were established from wild-type, SOD2-heterozygous knockout, and SOD2-overexpressing mice.
- Oxidative stress was induced using hydrogen peroxide (H2O2), followed by assessment of apoptosis via TUNEL staining and cell death ELISA.
- Mitochondrial transmembrane potential (MTP) and cytochrome c release were analyzed to understand the cell death pathway.
Main Results:
- Apoptotic cell death in RPE cells was dose- and time-dependent on H2O2 exposure and correlated with cellular SOD2 levels.
- Disruption of mitochondrial transmembrane potential and cytochrome c release preceded apoptosis and were linked to SOD2 levels.
- RPE cells with reduced SOD2 exhibited increased apoptosis, while those with overexpression showed suppressed cell death.
Conclusions:
- Manganese superoxide dismutase (SOD2) plays a critical role in protecting RPE cells against oxidative injury.
- Modulating SOD2 levels can significantly influence the outcome of oxidative stress-induced apoptosis in retinal cells.
- These findings underscore the therapeutic potential of targeting SOD2 in diseases involving RPE oxidative damage, such as AMD.