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Oxidative stress causes ERK phosphorylation and cell death in cultured retinal pigment epithelium: prevention of cell death by AG126 and 15-deoxy-delta 12, 14-PGJ2.

Garg TK, Chang JY - BMC Ophthalmol (2003)

Bottom Line: The retina, which is exposed to both sunlight and very high levels of oxygen, is exceptionally rich in polyunsaturated fatty acids, which makes it a favorable environment for the generation of reactive oxygen species.The cytotoxic effects of hydrogen peroxide (H2O2) induced oxidative stress on retinal pigment epithelium were characterized in this study.This cell death was prevented partially by the prostaglandin derivative, 15d-PGJ2 and by the protein kinase inhibitor, AG126. 15d-PGJ2 and AG126 may be useful pharmacological tools in the future capable of preventing oxidative stress induced RPE cell death in human ocular diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Anatomy & Neurobiology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. gargtarunk@uams.edu

ABSTRACT

Background: The retina, which is exposed to both sunlight and very high levels of oxygen, is exceptionally rich in polyunsaturated fatty acids, which makes it a favorable environment for the generation of reactive oxygen species. The cytotoxic effects of hydrogen peroxide (H2O2) induced oxidative stress on retinal pigment epithelium were characterized in this study.

Methods: The MTT cell viability assay, Texas-Red phalloidin staining, immunohistochemistry and Western blot analysis were used to assess the effects of oxidative stress on primary human retinal pigment epithelial cell cultures and the ARPE-19 cell line.

Results: The treatment of retinal pigment epithelial cells with H2O2 caused a dose-dependent decrease of cellular viability, which was preceded by a significant cytoskeletal rearrangement, activation of the Extracellular signal-Regulated Kinase, lipid peroxidation and nuclear condensation. This cell death was prevented partially by the prostaglandin derivative, 15d-PGJ2 and by the protein kinase inhibitor, AG126.

Conclusion: 15d-PGJ2 and AG126 may be useful pharmacological tools in the future capable of preventing oxidative stress induced RPE cell death in human ocular diseases.

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Related in: MedlinePlus

H2O2 induced lipid peroxidation in RPE. ARPE-19 cells were treated with 1.5 mM H2O2 for 4 hours then processed for immunofluorescent staining using antibodies against 4-HNE. Fig. 3A: untreated cells. Scale bar: 50 μm. Fig. 3B: H2O2 treated cells. Note the overall enhanced staining in nucleus and cytoplasm. Fig. 3C: ARPE-19 cells were treated with various concentrations (0.5, 1, 1.5, 2 mM) of H2O2 for 24 hours then processed for Western blot analysis using anti-4-HNE antibodies. These polyclonal antibodies recognize cysteine-, histidine- and lysine-4-HNE Michael adducts. This is a representative result from 6 similar experiments.
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Figure 3: H2O2 induced lipid peroxidation in RPE. ARPE-19 cells were treated with 1.5 mM H2O2 for 4 hours then processed for immunofluorescent staining using antibodies against 4-HNE. Fig. 3A: untreated cells. Scale bar: 50 μm. Fig. 3B: H2O2 treated cells. Note the overall enhanced staining in nucleus and cytoplasm. Fig. 3C: ARPE-19 cells were treated with various concentrations (0.5, 1, 1.5, 2 mM) of H2O2 for 24 hours then processed for Western blot analysis using anti-4-HNE antibodies. These polyclonal antibodies recognize cysteine-, histidine- and lysine-4-HNE Michael adducts. This is a representative result from 6 similar experiments.

Mentions: Subsequent experiments with immunofluorescent staining indicated that 1.5 mM H2O2 treatment of ARPE-19 cells for 4 hours led to the formation of 4-HNE protein adducts both in the cytoplasm and nucleus, presumably as a result of lipid-peroxidation (Fig. 3A,3B). Various protein bands on Western blots were positive for anti-HNE antibody reactivity, further intensified as a result of H2O2 treatment (Fig 3C). Densitometry measurements were performed to estimate the collective increase of band intensity in each lane between 39 Kd and 87 Kd. Results derived from a total of six independent experiments indicated that treatment of cells with 0.5, 1, 1.5 or 2 mM H2O2 caused an increase of intensity to 2.5-, 3.1-, 3.7- or 7.1-fold of control, respectively.


Oxidative stress causes ERK phosphorylation and cell death in cultured retinal pigment epithelium: prevention of cell death by AG126 and 15-deoxy-delta 12, 14-PGJ2.

Garg TK, Chang JY - BMC Ophthalmol (2003)

H2O2 induced lipid peroxidation in RPE. ARPE-19 cells were treated with 1.5 mM H2O2 for 4 hours then processed for immunofluorescent staining using antibodies against 4-HNE. Fig. 3A: untreated cells. Scale bar: 50 μm. Fig. 3B: H2O2 treated cells. Note the overall enhanced staining in nucleus and cytoplasm. Fig. 3C: ARPE-19 cells were treated with various concentrations (0.5, 1, 1.5, 2 mM) of H2O2 for 24 hours then processed for Western blot analysis using anti-4-HNE antibodies. These polyclonal antibodies recognize cysteine-, histidine- and lysine-4-HNE Michael adducts. This is a representative result from 6 similar experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153521&req=5

Figure 3: H2O2 induced lipid peroxidation in RPE. ARPE-19 cells were treated with 1.5 mM H2O2 for 4 hours then processed for immunofluorescent staining using antibodies against 4-HNE. Fig. 3A: untreated cells. Scale bar: 50 μm. Fig. 3B: H2O2 treated cells. Note the overall enhanced staining in nucleus and cytoplasm. Fig. 3C: ARPE-19 cells were treated with various concentrations (0.5, 1, 1.5, 2 mM) of H2O2 for 24 hours then processed for Western blot analysis using anti-4-HNE antibodies. These polyclonal antibodies recognize cysteine-, histidine- and lysine-4-HNE Michael adducts. This is a representative result from 6 similar experiments.
Mentions: Subsequent experiments with immunofluorescent staining indicated that 1.5 mM H2O2 treatment of ARPE-19 cells for 4 hours led to the formation of 4-HNE protein adducts both in the cytoplasm and nucleus, presumably as a result of lipid-peroxidation (Fig. 3A,3B). Various protein bands on Western blots were positive for anti-HNE antibody reactivity, further intensified as a result of H2O2 treatment (Fig 3C). Densitometry measurements were performed to estimate the collective increase of band intensity in each lane between 39 Kd and 87 Kd. Results derived from a total of six independent experiments indicated that treatment of cells with 0.5, 1, 1.5 or 2 mM H2O2 caused an increase of intensity to 2.5-, 3.1-, 3.7- or 7.1-fold of control, respectively.

Bottom Line: The retina, which is exposed to both sunlight and very high levels of oxygen, is exceptionally rich in polyunsaturated fatty acids, which makes it a favorable environment for the generation of reactive oxygen species.The cytotoxic effects of hydrogen peroxide (H2O2) induced oxidative stress on retinal pigment epithelium were characterized in this study.This cell death was prevented partially by the prostaglandin derivative, 15d-PGJ2 and by the protein kinase inhibitor, AG126. 15d-PGJ2 and AG126 may be useful pharmacological tools in the future capable of preventing oxidative stress induced RPE cell death in human ocular diseases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departments of Anatomy & Neurobiology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. gargtarunk@uams.edu

ABSTRACT

Background: The retina, which is exposed to both sunlight and very high levels of oxygen, is exceptionally rich in polyunsaturated fatty acids, which makes it a favorable environment for the generation of reactive oxygen species. The cytotoxic effects of hydrogen peroxide (H2O2) induced oxidative stress on retinal pigment epithelium were characterized in this study.

Methods: The MTT cell viability assay, Texas-Red phalloidin staining, immunohistochemistry and Western blot analysis were used to assess the effects of oxidative stress on primary human retinal pigment epithelial cell cultures and the ARPE-19 cell line.

Results: The treatment of retinal pigment epithelial cells with H2O2 caused a dose-dependent decrease of cellular viability, which was preceded by a significant cytoskeletal rearrangement, activation of the Extracellular signal-Regulated Kinase, lipid peroxidation and nuclear condensation. This cell death was prevented partially by the prostaglandin derivative, 15d-PGJ2 and by the protein kinase inhibitor, AG126.

Conclusion: 15d-PGJ2 and AG126 may be useful pharmacological tools in the future capable of preventing oxidative stress induced RPE cell death in human ocular diseases.

Show MeSH
Related in: MedlinePlus