Prevention of Oxidative Stress-Induced Retinal Pigment Epithelial Cell Death by the PPARgamma Agonists, 15-Deoxy-Delta 12, 14-Prostaglandin J(2)

Abstract

Cellular oxidative stress plays an important role in retinal pigment epithelial (RPE) cell death during aging and the development of age-related macular degeneration. Early reports indicate that during phagocytosis of rod outer segments, there is an increase of RPE oxidative stress and an upregulation of PPARgamma mRNA in these cells. These studies suggest that activation of PPARgamma may modulate cellular oxidative stress. This paper presents a brief review of recent studies that investigate RPE oxidative stress under various experimental conditions. This is followed by a detailed review on those reports that examine the protective effect of the natural PPARgamma ligand, 15d-PGJ(2), against RPE oxidative stress. This agent can upregulate glutathione and prevent oxidant-induced intracellular reactive oxygen species accumulation, mitochondrial depolarization, and apoptosis. The cytoprotective effect of this agent, however, is not shared by other PPARgamma agonists. Nonetheless, this property of 15d-PGJ(2) may be useful in future development of pharmacological tools against retinal diseases caused by oxidative stress.

Figure 1

Prevention of H₂O₂-induced nuclear condensation by 15d-PGJ₂. The human RPE cell line ARPE-19 cells were treated with 1.5 mM H₂O₂ for various periods of time, and then processed for nuclear staining by bisbenzimide (Hoechst 33258) to identify apoptotic cells [48]; (a): untreated cells; (b): 4 hours; (c): 12 hours; (d): 16 hours after treatment. Arrows in (c) point to representative cells with condensed nuclei, an indication of apoptosis. (e): Cells were pretreated with 1 μM 15d-PGJ₂ overnight, followed by 1.5 mM H₂O₂ for 16 hours (without 15d-PGJ₂). The number of apoptotic cells was greatly reduced by 15d-PGJ₂. Scale bar: 100 μm.

Figure 2

Prevention of H₂O₂-induced mitochondrial membrane depolarization by 15d-PGJ₂. Binding of the JC-1 dyes to mitochondria leads to the appearance of two peaks. The green peak (at ∼545 nm) represents JC-1 monomers of this dye. The red peak (at ∼595 nm) represents JC-1 aggregates, which is caused by the negative charge of mitochondrial membrane. Depolarization of mitochondrial membrane causes a shift in the emission spectrum from red to green color, which can be quantified by a fluorescence plate reader. The relative intensity of these two peaks is a measurement of relative mitochondrial potential such that a higher ratio represents more mitochondrial membrane depolarization. (a)–(d): The JC-1 emission spectra between 520 nm to 620 nm were determined for cells under various conditions [53]; (a): untreated cells; (B): cells treated with 1 μM 15d-PGJ₂ overnight; (c): cells treated with 1.5 mM H₂O₂ for 2 hours; (d): Cells treated with 1 μM 15d-PGJ₂ overnight, then with 1.5 mM H₂O₂ (without 15d-PGJ₂) for 2 hours. Note H₂O₂ caused a shift of the relative intensity of the peaks, and 15d-PGJ₂ pretreatment restored membrane potential to a condition closer to untreated cells. (e)-(f): Cells were pretreated with 1 μM 15d-PGJ₂ overnight, then with 1.5 mM H₂O₂ (without 15d-PGJ₂) for 2 hours (e) or 4 hours (f); then the 545/595 emission intensity ratios were determined. Note in either 2-hour or 4-hour treatment, H₂O₂ caused an increase of the 545/595 emission intensity ratio, indicating mitochondrial depolarization. 15d-PGJ₂ pretreatment restored the ratio to that similar to control value (P < .001 between H₂O₂-treated and 15d-PGJ₂+H₂O₂-treated cells in (e) and (f)).

Figure 3

Protective effects of 15d-PGJ₂ against oxidative stress. Oxidative stress on RPE cells can lead to intracellular accumulation of reactive oxygen species. This can result in mitochondrial dysfunction, which in turn causes activation of the apoptosis pathway. Current data suggests that 15d-PGJ₂ can block each of these events. One mechanism that causes this protection is through upregulation of GSH synthesis by activation of the glutamylcystein synthetase. There is a possibility that other cytoprotective mechanisms are also activated that lead to prevention of apoptosis. This remains to be studied.