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1 Introduction

Age-related macular degeneration (AMD) is a leading cause of blindness in the industrial world. Lifelong oxidative stress of human retinal pigment epithelium (hRPE) has been implicated in the pathogenesis of AMD (Kothary et al. 2014) by production of reactive oxygen species (ROS), which can result in damage to hRPE.

The hRPE form a single layer of mitotically inactive cells that lie between the choroid and the neural retina. Pigment epithelial cells transport and store toxic nutrients for the photoreceptors and remove waste products such as shed photoreceptor segments. Damage to the RPE can affect the functioning of neurosensory retina.

Valproic acid (VPA) , an epigenetic factor, is a drug that is widely used to treat patients with epilepsy (Monti et al. 2009 and it also inhibits growth of some cancer cells. In addition, VPA has been shown to reduce cell death in ARPE19 cells that were subjected to oxidative injury. It is postulated that a cascade of signaling molecules may be involved in beneficial effect of VPA in the treatment of epilepsy and reduced cell death in ARPE 19 cells during oxidative stress.

MAP kinases are involved in cell proliferation and apoptosis (Wang et al. 1998; Kothary et al. 2008) . Previous studies have shown that extracellular signal-regulated kinase (ERK) is involved in proliferation where as P38 and STAT 3 (Kothary et al. 2004) are involved in cell death and cell survival (Gutierrez-Uzquiza et al. 2012) . In the present study, we have used hydrogen peroxide to induce oxidative stress in hRPE cells and investigated the effect of VPA on hRPE cell viability and P38 production, to determine if these factors may be involved in the molecular mechanisms related to cell survival.

2 Materials and Methods

2.1 Establishment and Maintenance of hRPE Cell Cultures

hRPE cells were collected from donor human eyes obtained from the Michigan Eye Bank, and differentiated primary cultures were established as described previously (Weng et al. 2009). In brief, cells were grown in an incubator at 37 °C in Ham’s F12 nutrient media until confluent, and then trypsinized and plated. The media in the cultured plates was changed every 3 days until experimental reagents were added.

2.2 Trypan Blue Exclusion Method

The procedure described in previous publication (Kothary et al. 2006) . Briefly, cell media was aspirated and cells were washed twice with F12. 3.0 mL Experimental reagents were added to each well. Plates were incubated at 37 °C for 48 h, and then media was aspirated. Cells were washed with 1 mL PBS and 1 mL Hank’s Buffer, and then 750 μL trypsin was added and mixed. After incubating 37 °C for 10 min, cell detachment was verified under a microscope and 10 uL trypan blue dye was added and mixed. Samples of cell mixture from each well were placed on a slide and transferred to a hemocytometer, where unstained and stained cells were counted in four different fields.

2.3 14 C-Methionine Assay

The procedure described in previous publication (Kothary et al. 2010) . Briefly, cell media was aspirated and cells were washed twice with F12. Experimental reagents were added, 0.5 mL to each well. After incubating at 37 °C for 1 h, 50 μL 14 C-methionine was added. Plates were incubated at 37°C for 24 h, then media was aspirated and cells were washed with 0.5 mL PBS and 200 μL Zwitteragent in 0.2 % BSA. Upon mixing, cells in Zwitteragent were transferred to microfuge tubes and 10 μL anti-P38 was added. Plates were refrigerated for 24 h, then 10 μL Protein A was added. After 1 h, tubes were centrifuged at 14,000 rpm for 5 min at 4 °C. The supernatant fluid was discarded, and 0.5 mL NaOH was added. Cells in NaOH were transferred to scintillation vials, and 10 mL Ecolite was added. After 1 h, 14 C-methionine incorporation was counted by a scintillation counter.

2.4 Nuclear Staining

Nuclear staining of hRPE cells after H2O2 and VPA treatment was performed by method described previously described by Weng et al 2009. Nuclear staining showed that H2O2 and VPA decreased the hRPE cell number (data not shown).

3 Results

3.1 Effect of FBS on hRPE Cell Viability

Figure 102.1 shows hRPE cell proliferation is stimulated by increasing concentrations of FBS in a dose dependent manner.

Fig. 102.1
figure 1

Effect of FBS on hRPE cell viability

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3.2 Effect of H2O2 and VPA on hRPE Cell Viability

Figure 102.2a shows increasing concentrations of H2O2 decrease hRPE cell viability and proliferation to a limited extent.

Fig. 102.2
figure 2

a Effect of H2O2 on hRPE cell viability, b Effect of VPA on hRPE cell viability

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Figure 102.2b shows increasing concentrations of VPA decrease hRPE cell proliferation.

3.3 Effect of VPA in Presence of H2O2 on hRPE Cell Viability

Figure 102.3 shows VPA (1 mM) eliminates the H2O2 (1 mM) reduction in hRPE cell proliferation.

Fig. 102.3
figure 3

Effect of VPA on H2O2 treated hRPE cell viability

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3.4 Effect of VPA in 14 C-P38 Production

Figure 102.4 shows increasing concentrations of VPA increase 14 C-P38 synthesis in hRPE cells.

Fig. 102.4
figure 4

Effect of VPA on 14 C-P38 synthesis in hRPE cells

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3.5 Effect of VPA in Presence of H2O2 on P38 Production

Figure 102.5 shows VPA (1 mM) eliminates the H2O2 (1 mM) induced increased 14 C-P38 synthesis in hRPE cells back to baseline.

Fig. 102.5
figure 5

Effect of VPA on 14 C-P38 synthesis in H2O2 treated hRPE cells

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4 Discussion

AMD affects millions of older people in the industrial world resulting in loss of central reading vision often to legal blindness. AMD is associated with progressive deterioration of the retinal pigment epithelium and lifelong oxidative stress seems to play a role. Therapeutically, invasive surgery e.g. laser photocoagulation of neovascular membranes, macular translocation surgery and recently discovered anti-VEGF medications have been used to treat these patients for stabilization of vision loss, but no successful preventive or fully restorative treatment has been discovered. Additional investigation of the molecular mechanism of this disease is required to develop better treatments. Therefore, our study aimed at understanding the role of the signaling molecule P38 MAPK in the survival of hRPE may aid in the development of pharmacological treatments for macular degeneration.

We have examined the nature of hydrogen peroxide induced oxidative stress in hRPE cells. Our goal was to determine the molecular expression of P38 in hRPE cells in presence and absence of hydrogen peroxide induced acute oxidative stress and the effect of adding VPA, a known inhibitor of oxidative damage, on P38 expression. We have shown that hRPE cells treated with H2O2 and VPA separately decreases hRPE cell proliferation and viability and increases P38 production. Xie et al. (2010) has shown that VPA increases P38 synthesis in microglia and that VPA induced microgia cell death is mediate by P38. Previously, we have shown that VPA treatment also increases caspase-3, a marker for apoptosis in hRPE cells.

We found that VPA reduces P38 synthesis and decreases cell death caused by H2O2 oxidative stress in cultured differentiated hRPE cells. Our data is in agreement with Gutierrez-Uzquiza et al. (2012) who showed P38 alpha mediates cell survival in response to oxidative stress. Others have shown that P38 activation may be linked mTOR inhibition (Chen et al. 2010; Pocrnich et al. 2009) . Further investigation of effect of VPA and H2O2 on mTOR expression may clarify the role of mTOR in P38 signaling. P38 may also be up regulating antioxidant gene expression, Gutierrez-Uzquiza et al. 2012) .

We conclude that VPA has a pro-survival function in H2O2 induced hRPE cell death because of its ability to down regulate P38. VPA is commonly used in the treatment of epilepsy, bipolar disease and cancers. These studies suggest that VPA may also have therapeutic value in the prevention or treatment of AMD as well.