Oxidative stress has been shown to be a factor in the progression of a number of retinal diseases, including those affecting photoreceptors and RPE cells, such as RP and AMD. Due to rapid growth in the aging population, the prevalence of visual disabilities is expected to drastically increase during the next 20 years.44
For example, AMD is the leading cause of visual impairment in the elderly and accounts for over 54% of all irreversible vision loss in Caucasians.45
Progression of AMD and RP may occur at least in part because of light exposure, although other factors are clearly also critically important. The fact that excessive light evokes the generation of ROS coupled with the susceptibility of RPE and photoreceptor cells to oxidative stress suggests involvement of this process as one pathway to cell injury in the outer retina.1,2,46
The RPE cell layer plays a crucial role in the maintenance and survival of adjacent photoreceptors via phagocytosis of the photoreceptor outer segments.47
These cells also provide trophic support to the neural retina by supplying neurotrophic factors.48,49
Thus, rescue of both photoreceptor and RPE cells will be critical for preventing the onset and progression of AMD. Here, we present evidence that treatment with CA prior to exposure to oxidative stress or damaging light protects both form and function in the rat retina.
Our group previously reported that CA protects cerebrocortical neurons from oxidative stress, excitotoxicity, and ischemic injury via Nrf2-mediated induction of phase 2 antioxidant enzymes.19
Importantly, GSH, the major cell reductant, was increased in the brain following CA treatment, rather than decreased as observed with previously studied electrophilic compounds. We showed that CA was different from these other compounds by virtue of being a pro-electrophile that becomes activated to an electrophile by oxidative stress, the very injury that it then protects from by inducing various antioxidant genes. These findings and the strong association of progression of retinal damage with oxidative stress in the dry form of macular degeneration and in RP suggested that CA-induced antioxidant pathways could prove beneficial in these diseases. Based on our previous results, we hypothesized that CA would be activated by the oxidative stress associated with light-induced damage in the retina and subsequently boost the expression of endogenous phase 2 genes without depleting the endogenous reducing capacity of normal cells. This approach of boosting endogenous phase 2 gene expression, which results in synthesis of proteins with a broad spectrum of antioxidant, detoxification, and reductive activities, is attractive due to the well-known and long-lasting cytoprotective effects of these gene products. In contrast, an antioxidant-based therapy would have to be continuously supplied to the injured cells in order to remain effective, as the agents are continuously consumed in the neutralization process.
In this study, we found that the pro-electrophilic drug CA promoted survival of retina-derived cell lines, 661W photoreceptors and ARPE-19 RPEs, against H2O2-induced cell death in vitro. Accounting for this effect, CA induced nuclear translocation of Nrf2 and then, as shown in our reporter gene assays, activated transcription of phase 2 genes. We confirmed these results by microarray and qPCR analyses. Transcriptional activation resulted in upregulation of antioxidant/detoxifying proteins, including HO-1, NQO1, GCLM, xCT, and SRXN1.
Several electrophilic compounds have been found to activate the Nrf2 pathway in RPE cells, which have the potential of protecting photoreceptors against oxidative stress.20–22
Unlike these compounds, however, an advantage of CA-induced activation of the Nrf2 pathway is that it preserves the endogenous reducing capacity of healthy cells by avoiding reaction with glutathione; this is accomplished by the targeted activation of CA in the oxidatively stressed tissue.
One of the phase 2 proteins that we found to be upregulated by CA was SRXN1, which is responsible for enzymatically reversing hyperoxidation of the active site cysteine of Prxs from the sulfinic acid derivative to the free thiol form (from -SO2H to -SOH). CA should thus afford cytoprotection in part by allowing Prxs to detoxify additional ROS.50
Indeed, our data demonstrate that CA protects the retina-derived cell lines ARPE-19 and 661W from H2
-induced hyperoxidation of Prx2.
Importantly, our data also indicate that CA crosses the blood–retina barrier when administered by IP injection and further accumulates in the retina after multiple doses. After CA treatment, compared to control, we found histological evidence that a significantly greater number of photoreceptors survived in rats after exposure to damaging light. Additionally, we observed significantly increased amplitudes of the ERG a-wave and b-wave after CA treatment in the face of a light-damaging insult, indicating functional preservation of photoreceptors and more distal retinal elements, predominantly bipolar and Müller cells, respectively. Hence, our ERG findings are also consistent with the notion that CA attenuated light-induced retinal dysfunction in photoreceptors and the inner retina.
In summary, our results suggest that the pro-electrophilic drug CA, after conversion to its active electrophilic form, can attenuate oxidative damage to the retina in the face of light-induced insult. CA acts, at least in part, via activation of the Nrf2 transcriptional pathway to activate endogenous antioxidant phase 2 genes. Hence, such pro-electrophilic drug therapy may prove to be a useful strategy to ameliorate retinal oxidative stress that occurs as a result of exposure to light or other factors and, hence, to prevent progression of AMD and RP.