# Prevention of Oxidative Stress-Induced Retinal Pigment Epithelial Cell Death by the PPARγ

Authors: Jason Y. Chang; Puran S. Bora; Nalini S. Bora
Journal: PPAR Research
 (2008)

---

## 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 PPARγ mRNA in these cells.  These studies suggest that activation of PPARγ 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 PPARγ ligand, 15d-PGJ2, 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 PPARγ agonists.  Nonetheless, this property of 15d-PGJ2 may be useful in future development of pharmacological tools against retinal diseases caused by oxidative stress.


---

## Body

## 1. Age-Related Macular Degeneration: Possible Involvement of RPE

Age-related macular degeneration (AMD) is the leading cause of legal blindness in individuals 50 years of age or
older in the United States and developed countries. AMD can be divided into two major forms as follows: (i) nonneovascular form, also known as
“dry” or “nonexudative” form; as clinical findings of this form include
drusen and abnormalities of the retinal pigment epithelium (RPE) and  (ii) neovascular form, also known as “wet” or “exudative”
form, which is defined by the appearance of choroidal
neovascularization with subsequent subretinal fibrosis or disciform scarring.
Patients with drusen larger than 63 μm in diameter (termed “soft drusen”) have a high risk of
developing choroidal neovascularization [1].There is evidence that pathological alterations of RPE around macula area may be
partially responsible for the development of AMD [2, 3]. Clinical abnormalities of RPE in AMD include
clumping and atrophy of these cells. RPE
is involved in the ingestion of photoreceptor outer segments and the general
health of photoreceptors. As a result, pathological changes of RPE can lead to photoreceptor cell death and visual
impairment. Study with human cadaver eyes indicates that there is an age-dependent RPE apoptosis as evidenced by
TUNEL staining [4]. A separate study further indicates that eye
specimens from patients with AMD show statistically more macular RPE apoptosis
than those without AMD [5].


## 2. Possible Roles of Oxidative Stress in AMD

Retina is exposed to a combination
of sunlight, high concentrations of polyunsaturated fatty acids, and high
oxygen environment. It is proposed that
reactive oxygen species (such as hydrogen peroxide, superoxide anion, hydroxyl
radicals, and singlet oxygen) are constantly generated in this
environment. As a result, oxidative
stress is believed to have an important role in RPE apoptosis and in the
development of AMD [2, 3].An increase of oxidative stress in
RPE is associated with an increase of cellular catalase, metallothionein [6], and glutathione
S-transferase [7], which should serve as a
protective mechanism to decrease the cytotoxicity caused by H2O2 and other reactive oxygen species. This
protective mechanism declines with age.For example, a study analyzing metallothionein levels in RPE of macular
region showed a significant (68%) decrease in aged donors (mean age = 80-year-old) as compared to those from younger donors (mean age = 58-year-old) [8]. A separate report also concluded that there
was an age-dependent decrease of catalase activity in RPE [9]. These studies suggest that RPE cells  in the elderly are more susceptible to
oxidative stress-induced damage.


## 3. Studies of Oxidative Stress on RPE: Prevention by Pharmacological Agents

Given the observations that RPE
might be the prime targets for oxidative stress, a number of studies are conducted
to study this issue. A majority of research use direct oxidative agents, such
as hydrogen peroxide (H2O2) or t-butylhydroperoxide (tBH),
to initiate cellular oxidative stress, as further discussed below. Other conditions of experimental oxidative stress
include: intense light [10–12], iron [13], and oxidative metabolites
that are toxic to cells, such as A2E [14, 15], acrolein [16], and oxysterols [17–19].By using H2O2 or tBH as the direct source of oxidative stress on RPE, a number of studies
focus on strategies to build up cellular defense mechanisms against the insult.
Several reports explore the importance of cellular antioxidative enzymes, such
as catalase [20], glutathione-S-transferase [21, 22], superoxide dismutase [23], and methionine sulfoxide
reductase [24]. Growth factors including lens
epithelium-derived growth factor [25], keratinocyte growth factor [26], and pigment
epithelium-derived factor [27] are also protective against
oxidative stress. Other proteins that can enhance RPE antioxidative mechanism
against H2O2 include bcl-2 [28], alpha B-crystallin [29], melatonin [30], and poly(ADP-ribose)
polymerase [31].In addition to those protein factors
discussed above, many investigators seek the use of small-molecule
pharmacological agents to prevent RPE damage caused by H2O2 or tBH. Examples of these
pharmacological agents include: (R)-alpha-lipoic acid [32],
17-beta-estradiol [33], flavonoids [34],
and L-carnitine [35]. The endogenous PPARγ ligand, 15-deoxy-delta-12,14-prostaglandin J2(15d-PGJ2), is also very effective in preventing RPE
oxidative stress, as further discussed below.


## 4. Prevention of Oxidative Stress-Induced RPE Death by 15d-PGJ2

15d-PGJ2, a prostaglandin
derivative, is normally present in tissues at low levels (<1 nM), but can
reach high concentrations during infection and inflammation [36]. Under in vitro conditions, it can be
induced by chemical [37] or physical [38] stress. It has a very potent anti-inflammatory effect
[39]. For example, it is a potent
inhibitor of macrophage [40–42] and microglia [43–45] activation.During RPE ingestion of rod outer
segments, there is a generation of H2O2 [6, 46] and a 10-fold upregulation of
PPARγ mRNA [47]. Based on these observations, it is likely
that PPARγ is involved in RPE cellular responses toward H2O2. One can hypothesize that PPARγ agonists
should modulate cellular defense against oxidative stress.We reported earlier that the PPARγ agonist,
15d-PGJ2, protected H2O2-induced RPE cell
death [48]. With primary human RPE cells, pretreatment of
cells overnight with 15d-PGJ2 dose-dependently prevented H2O2-induced
cytotoxicity, such that the viability raised from ∼25% (H2O2 only) to ∼80% of control. Maximal
protection was observed at ∼2 μM 15d-PGJ2. Similar protection was made in the human
ARPE-19 cell line. While H2O2 caused significant nuclear condensation, a sign of apoptosis; this was largely
prevented by 1 μM 15d-PGJ2 (see Figure 1). However, it should be mentioned that the
protective effect by 15d-PGJ2 was not shared by other PPARγ agonists, such as ciglitazone, azelaoyl PAF,
or LY171883. These results raised the
possibility that the protective effect by 15d-PGJ2 was not mediated
through PPARγ activation. This idea was supported by other investigators, as further discussed below.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.
(a)(b)(c)(d)(e)The cytoprotective effect of 15d-PGJ2 on H2O2-treated
RPE was further studied by Qin et al. [49].These investigators confirmed that 1 μM
15d-PGJ2 effectively prevented H2O2-induced
cell death. Other PPARγ agonists, such as AGN195037 or Roziglitazone,
had no protective effects. Importantly, reduction of PPARγ by siRNA did not block the protective
effect of 15d-PGJ2. This set of experiments together with those
described above strongly suggests
that 15d-PGJ2 protect RPE cells through a PPARγ-independent mechanism. Some properties of
15d-PGJ2 are independent of PPARγ activation, as reviewed by Straus and Glass [39].Subsequent studies by Qin et al. [49] indicated that 15d-PGJ2
could upregulate glutamylcyteine synthetase, the rate-limiting enzyme that regulates glutathione
(GSH) synthesis. These investigators reported that 15d-PGJ2 at 1-2 μM induced
GSH levels to ∼300% of control. With 1 μM 15d-PGJ2, the maximal induction occurred at 18–24
hours after treatment. This GSH induction appeared to depend on JNK and p38 pathways because inhibitors of
these pathways greatly reduced GSH induction by 15d-PGJ2. Induction
of GSH by 15d-PGJ2 is also observed in other cell types [37, 50, 51]. Since intracellular GSH is very important in
cellular defense against oxidative stress, the induction of GSH should have an
important role in the protective effect caused by 15d-PGJ2 treatment. Even though induction of heme
oxygenase-1 (HO-1) was associated with cytoprotective effects of 15d-PGJ2 in other studies [52], this enzyme had no roles in
the protection observed in this experimental system.If 15d-PGJ2 greatly induced intracellular GSH, one would expect that this agent should reduce
oxidant-induced intracellular reactive oxygen species generation. Indeed, we reported earlier
that 15d-PGJ2 could reduce H2O2- and tBH-induced
reactive oxygen species in human ARPE-19 cells [53]. For example, pretreatment of cells
with 1 μM 15d-PGJ2 reduced 1 mM H2O2-generated reactive
oxygen species to ∼80% of untreated cells challenged with H2O2. Similar reduction was observed in cells
challenged with tBH. This reduction
apparently was enough to keep free radical levels under a critical threshold,
thus rendering cells survive an otherwise detrimental oxidant insult.Our study further indicated that
15d-PGJ2 helped RPE cells to maintain mitochondrial integrity [53]. This is significant because mitochondria are
intimately involved in apoptosis.Oxidative stress can induce mitochondria dysfunction, which is a
critical event that leads to cytochrome c release and subsequent activation of
caspases, a group of enzymes that executes apoptosis [54, 55].
An important event associated with mitochondrial dysfunction is a drop of mitochondrial membrane potential (ΔΨm), that is, mitochondrial depolarization. This event initiated by oxidative stress was
largely prevented by 1 μM 15d-PGJ2 (see Figure 2). This is likely to prevent cytochrome c
release and subsequent activation of the apoptosis pathway.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)).
(a)(b)(c)(d)(e)(f)


## 5. Cytoprotective versus Cytotoxic Effects of 15d-PGJ2

In addition to those studies described above regarding the protective effect of
15d-PGJ2 against oxidative stress on RPE, this agent is
cytoprotective toward other retinal cells.
For example, Aoun et al. [56] reported that glutamate could
induce oxidative stress and cell death in the rat retinal ganglion cell line,
RGC-5 cells. This cell death was
prevented by 1–5 μM 15d-PGJ2. Outside of retina, 15d-PGJ2 was effective in preventing
glutamate-induced cell death of primary cortical neurons [51]. Both groups attributed the protective effect
through the antioxidative property of 15d-PGJ2. In this respect, it should be noted that this
agent can also prevent cell death caused by toxic metabolites of oxidative
stress. For example, we reported earlier
that 15d-PGJ2 prevented cytotoxicity of oxysterols, toxic
cholesterol metabolites generated under oxidative stress [57]. The cytoprotective effect of 15d-PGJ2 in other experimental systems were also described in reports by Kawamoto et al.
[58] and Itoh et al. [59].It is clear now that 15d-PGJ2 can induce intracellular oxidative stress
[60, 61]. It is likely that this agent at low
concentrations (1–5 μM) can cause low levels of oxidative stress, thus inducing
the build up of cellular defense mechanisms against oxidative stress. However, at high concentrations, this agent
can cause severe oxidative stress and cell death [60, 61]. Induction of apoptosis by this agent was
reported in several cell types [62–64]. This interesting
bifunctional property of 15d-PGJ2 has been reported [50], and is a subject of review by Na and Surh [65]. This also prompts a recent
microarray study analyzing the regulation of prosurvival and prodeath genes by
this agent [66].


## 6. Concluding Remarks

Oxidative stress is believed to play an important role in RPE cell death during aging and
the development of age-related macular degeneration. During phagocytosis of rod outer segments,
there is an upregulation of PPARγ in RPE
cells. The natural PPARγ ligand 15d-PGJ2 has a potent
protective effect for RPE under oxidative stress. This agent can upregulate GSH and prevent
oxidant-induced intracellular reactive oxygen species accumulation,
mitochondrial depolarization, and apoptosis (see Figure 3).
There is also evidence that 15d-PGJ2 can prevent glutamate-induced death of cultured retinal ganglion cells.
Current data suggests that this
cytoprotection is not mediated through the activation of PPARγ. The
antioxidative property of 15d-PGJ2 may be useful in future
development of pharmacological tools against retinal diseases caused by
oxidative stress.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.Finally, based on anti-inflammatory effects of 15d-PGJ2, we would like to
speculate that this agent might be effective in the treatment of other ocular
diseases such as idiopathic autoimmune anterior uveitis. To confirm our hypothesis, we intend to
explore the effect of 15d-PGJ2 on experimental autoimmune anterior
uveitis (EAAU) which serves as an animal model of idiopathic human autoimmune
anterior uveitis [67, 68].



---

*Source: 720163-2008-03-16.xml*