What does dry eye have in common with cancer, neurodegenerative disorders, normal aging and heart disease? One answer is oxidative stress. We all know what it’s like to feel stressed out over deadlines or other demands of life. Similarly, our cells experience oxidative stress in response to a variety of molecular disturbances. With dry eye, the pressure might come from exposure to environmental variables such as cigarette smoke, low humidity, sun, wind or pollutants. Certain medications or medical conditions may also contribute to dry eye. Aging itself is associated with decreased tear production and dry eye. All of these variables have the capacity to impact oxidative stress levels on the ocular surface and in so doing contribute to ophthalmic conditions beyond dry eye, including macular degeneration, cataracts, uveitis, keratitis and corneal inflammation.Wakamatsu TH, Dogru M, Tsubota K. Tearful relations: Oxidative stress, inflammation and eye diseases. Arq Bras Oftalmol 2008;71:6:S72-79.
This month, we take a look at oxidation as a root cause of ocular surface disease, examine how this occurs and consider potential steps toward an antioxidant-based approach to dry-eye treatments.
Oxidative Stress Explained
What exactly is oxidative stress? Oxidative stress occurs when the level of reactive oxygen species produced in cells and tissues exceeds normal levels. ROS are types of free radicals (an atom with one or more unpaired electrons) that play a beneficial role in cell signaling and overall cellular homeostasis. Antioxidants naturally present in tissues usually control ROS levels, but surplus ROS react with nearby proteins, lipids or other cellular components, leading to unpredictable, cumulative and often deleterious effects on normal cell function. Oxidative injury from ROS occurs in the tears and conjunctiva of Sjögren’s syndrome patients, and high levels of ROS and oxidative stress have been identified in the tear film of dry-eye patientsAugustin AJ, Spitznas M, Kaviani N, Meller D, Koch FH, Grus F, Göbbels MJ. Oxidative reactions in the tear fluid of patients suffering from dry eyes. Graefes Arch Clin Exp Ophthalmol 1995;233:11:694-698. and in animal models of dry eye.Deng R, Hua X, Li J, Chi W, Zhang Z, Lu F, Zhang L, Pflugfelder SC, Li DQ. Oxidative stress markers induced by hyperosmolarity in primary human corneal epithelial cells. PLoS One 2015; 10:5:e0126561.
A primary source of cellular ROS is mitochondria, the intracellular organelles responsible for oxidation of glucose into H20, CO2 and the chemical energy of adenosine triphosphate; ROS are often a byproduct of this process. Antioxidants such as reduced glutathione or enzymes such as fneroxide dismutase provide electrons to convert ROS into less-reactive forms, but the cellular fnply of antioxidants can be overwhelmed by too much ROS. As electrons pass along the mitochondrial electron transport chain, a fraction is lost to ROS and subsequent local oxidation events. This theft of electrons by ROS can lead to a host of cellular dysfunctions including membrane disruption. ROS can also inflict damage on DNA, RNA or cell proteins, effects that can ultimately lead to cell apoptosis.
While high ROS levels within the mitochondria lead to oxidative stress and potential organelle damage, ROS outside the mitochondria may be involved in inflammation, a primary mechanism of dry-eye disease. This inflammation can be the result of ROS exiting the mitochondria, or the generation of ROS in other cellular structures. Macrophages and other phagocytes involved in fighting infection use ROS as a weapon against foreign invaders, but control of these ROS is not always adequate. In particular, pro-inflammatory cytokines, such as IL-1β, can stimulate ROS to levels that can lead to oxidative tissue injury.
A number of preclinical models have been used to explore the relationship between oxidative stress and inflammation in dry-eye disease. In one study, increased ROS activated the NLRP3 gene, a key player in immune cell recognition of microbial pathogens and stress-related signals.Zheng Q, Ren Y, Reinach PS, She Y, Xiao B, Hua S, Qu J, Chen W. Reactive oxygen species activated NLRP3 inflammasomes prime environment-induced murine dry eye. Experimental Eye Research 2014;125:1-8. NLRP3 activation in this dry-eye study increased secretion of the pro-inflammatory cytokine IL-1β. The same inflammatory process was confirmed in another dry-eye study using stressed human corneal epithelial cells: Increased ROS activated NLRP3, which in turn stimulated IL-1β and subsequent tissue inflammation.Zheng Q, Ren Y, Reinach PS, Xiao B1, Lu H, Zhu Y, Qu J, Chen W. Reactive oxygen species activated NLRP3 inflammasomes initiate inflammation in hyperosmolarity stressed human corneal epithelial cells and environment-induced dry eye patients. Exp Eye Res 2015;134:133-40. This study also examined 20 dry-eye patients and 15 normal subjects. In the dry-eye subjects, levels of ROS, NLRP3 and IL-1β were elevated in tear samples and conjunctival epithelial cells, indicating that inflammation in dry-eye disease may occur through the ROS–NLRP3–IL-1β signaling pathway. IL-1β levels in the dry-eye patients correlated with ocular surface disease index and Schirmer’s test scores and were elevated compared to control subjects. Previous studies found that NLRP3 is involved in other ocular diseases, including macular degeneration, glaucoma and corneal ulcer, as well as non-ocular diseases. In the future, it’s possible that inhibiting the ROS–NLRP3–IL-1β pathway may turn out to be an effective approach for dry-eye relief.
Benefits and Limitations
Most of us have heard about the purportedly miraculous qualities of antioxidants in food or nutritional fnplements that allegedly can keep us healthy and help keep various diseases at bay. Antioxidant-rich foods (such as blueberries) are highly recommended by health experts, and sales of antioxidant fnplements (such as vitamin C, vitamin E and Coenzyme Q) have skyrocketed. Although the evidence for a role of oxidative damage in conditions from diabetes, cancer or heart disease is undeniable, efforts to use antioxidants as therapeutics have been hit-or-miss: Antioxidant fnplements have shown beneficial effects in some trials, while other studies have found little or no benefit. This is true both for overall health and for eye health specifically.
The intricate membrane structure of the mitochondria provides the architecture to generate proton concentration gradients; using oxygen as an electron acceptor, the gradient drives ATP synthesis and produces reactive oxygen-containing molecules as a byproduct.
In one preclinical study, nutritional polyunsaturated fatty acid fnplementation produced statistically significant changes in serum fatty acids and a dose-related inhibition of rabbit corneal infiltrates and corneal angiogenesis. This process involved modulation of eicosanoid precursors, changes in corneal neovascularization and in alkali-induced inflammation.Ormerod LD, Garsd A, Abelson MB, Kenyon KR. Effects of altering the eicosanoid precursor pool on neovascularization and inflammation in the alkali-burned rabbit cornea. Am J Pathol 1990;137:1243. A subsequent study, however, was unable to reproduce the effect of nutritional fnplementation with the same PUFAs, gamma-linolenic acid, eicosapentaenoic acid or a combination of the fatty acids used in the prior study.Ormerod LD, Garsd A, Abelson MB, Kenyon KR. Eicosanoid modulation and epithelial wound healing kinetics of the alkali-burned cornea. J of Ocular Pharm 1992;8:1:53-58. The latter study is in line with more recent publications showing little or no effect of fish oils in reducing ocular inflammation.
For macular degeneration, a combination of antioxidant vitamins C and E, plus beta-carotene and zinc, afforded a statistically significant protection in disease onset in the Age-Related Eye Disease Study.AREDS. A randomized, placebo-controlled, clinical trial of high-dose fnplementation with vitamins C and E, beta-carotene, and zinc for age-related macular degeneration and vision loss. Arch Ophthalmol 2001;119:1417-1436. A second AREDS study showed that lutein plus zeaxanthin—which are two carotenoids—can substitute for beta-carotene, which has been associated with an increased risk of some types of cancer.AREDS2. Lutein + zeaxanthin and omega-3 fatty acids for age-related macular degeneration: the Age-Related Eye Disease Study 2 (AREDS2) randomized clinical trial. JAMA 2013;15:309: 2005-2015. While the effects are modest, the AREDS studies represent the most prominent examples of the benefits of antioxidants in the eye. Other studies have suggested that selenium or lactoferrin fnplementation may similarly protect the corneal epithelium from oxidative stress.,Higuchi A, Inoue H, Kawakita T, Ogishima T, Tsubota K. Selenium compound protects corneal epithelium against oxidative stress. PLoS One 2012;7:9:e45612. Antioxidants have also been explored as potential therapy in many other conditions associated with ROS. For example, fnplements of vitamin E, vitamin C and Coenzyme Q have yielded some relief from the ocular complications associated with diabetes, although overall effects are mixed.Green K, Brand M, Murphy M. Prevention of mitochondrial oxidative damage as a therapeutic strategy in diabetes. Diabetes 2004; 53:1:S110-S118.
It’s important to note that there are also a few other studies that have suggested an increased risk of disease associated with the use of antioxidant fnplements.
Two studies showed an increase in cancer risk for people who were heavy smokers or were exposed to asbestos and were taking beta-carotene or a beta-carotene/vitamin A combination.Omenn GS, Goodman GE, Thornquist MD, Balmes J, Cullen MR, Glass A, Keogh JP, Meyskens FL, Valanis B, Williams JH, Barnhart S, Hammar S. Effects of a combination of beta-carotene and vitamin A on lung cancer and cardiovascular disease. N Engl J Med 1996;334:1150-1155. It’s possible that the mixed results of antioxidant fnplementation in reducing oxidative stress and disease may be due to the limited ability of natural antioxidants to reach a cell’s mitochondria and accumulate there, due to the relatively poor bioavailability, pharmacokinetics or stability of these antioxidant fnplements. For some degenerative diseases, for example, very large doses were necessary to show a significant treatment benefit.Bentinger M, Dallner G, Chojnacki T, Swiezewska E. Distribution and breakdown of labeled coenzyme Q(10) in rat. Free Radic Biol Med 2003;34:563-575. And although antioxidant fnplements have been beneficial for certain eye conditions, it’s also true that, except for the retina, enzymatic antioxidant activity in the eye is limited, with few protections against ROS.,Behndig A, Svensson B, Marklund SL, Karlsson K. fneroxide dismutase isoenzymes in the human eye. Invest Ophthalmol Vis Sci 1998;39:3:471-475.
Because of the limitations of natural antioxidant fnplements in reducing ROS damage, mitochondrial-targeted antioxidants have been developed that are capable of accumulating in mitochondria. These therapies have shown beneficial effects for ocular and non-ocular diseases in some animal and clinical studies, although other studies did not confirm these results.Jin H, Kanthasamy A, Ghosh A, Anantharam V, Kalyanaraman B, Kanthasamy A. Mitochondria-targeted antioxidants for treatment of Parkinson’s Disease: Preclinical and clinical outcomes. Biochim Biophys Acta 2014;1842:8:1282-1294.
Mice lacking the enzyme fneroxide dismutase displayed degenerative loss of retinal pigment epithelial cells; this defect was corrected in these fneroxide dismutase knockout mice by directed RPE expression of fneroxide dismutase, which is capable of reducing mitochondrial and extracellular ROS generation.Imamura Y, Noda S, Hashizume K, Shinoda K, Yamaguchi M, Uchiyama S, Shimizu T, Mizushima Y, Shirasawa T, Tsubota K. Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: A model of age-related macular degeneration. Proc Natl Acad Sci 2006;103:30:11282-112827. Using this same mouse model, another study showed that fneroxide dismutase knockout resulted in abnormalities in lacrimal gland tissue, tear quantity and stability and the ocular surface. This murine model may be useful for future dry-eye studies.
Although antioxidant fnplements have been beneficial for certain eye conditions, it’s also true that, except for the retina, enzymatic antioxidant activity in the eye is limited, with few protections against reactive oxygen species.
MitoQ’s mitochondrial-targeted drug MitoQ (ubiquinone, which is identical to the antioxidant moiety in Coenzyme Q10) has been tested both in animals and in humans. In rodent studies MitoQ protected cells from pathological mitochondrial oxidative changes associated with effects such as cardiac damage, hypertension, liver damage, kidney damage and processes related to Parkinson’s disease.Smith R, Murphy M. Mitochondria-targeted antioxidants as therapies. Discovery Medicine 2011;11:106-114. In human studies, MitoQ has had mixed but promising results. In subjects with hepatitis C it significantly reduced liver enzyme levels, suggesting a reduction in liver inflammation, although viral levels were not significantly reduced.Gane E, Weilert F, Orr D, Keogh GF, Gibson M, Lockhart MM, Frampton CM, Taylor KM, Smith RA, Murphy MP. The mitochondria-targeted anti-oxidant mitoquinone decreases liver damage in a phase II study of hepatitis C patients. Liver International 2010;30:7:1019-1026. MitoQ didn’t slow the progression of Parkinson’s disease in an Australian/New Zealand study, possibly because it may be too late to rescue remaining dopamine neurons once the clinical signs of Parkinson’s are present.Snow B, Rolfe F, Lockhart M, Frampton CM, O’Sullivan JD, Fung V, Smith RA, Murphy MP, Taylor KM; Protect Study Group. A double-blind, placebo-controlled study to assess the mitochondria-targeted antioxidant MitoQ as a disease-modifying therapy in Parkinson’s disease. Mov Disord 2010;25:1670-1674.
Compounds with known antioxidant activity are chemically diverse. For example, another approach is the use of Szeto-Schiller peptides, short sequences of alternating aromatic and basic amino acids that are selectively taken up by mitochondria and are capable of reducing ROS at nano-molar concentrations.Zhao K, Zhao GM, Wu D, Soong Y, Birk AV, Schiller PW, Szeto HH. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem 2004;279:33:34682-34690. These peptides have shown promise in treating several conditions associated with inflammation or oxidative stress, including cardiac ischemia/reperfusion injury, insulin resistance and Parkinson’s. This approach may also be relevant as an ocular therapeutic, although no studies have been published to date.
Other antioxidant therapeutics targeting mitochondria include plastoquinone derivatives such as SKQ1 (Mitotech, Luxembourg). This compound is an approved treatment for dry eye in Russia. At the cellular level, SKQ1 reduces cell damage caused by excessive ROS by modulating the mitochondria’s membrane electrical potential, the driving force for the electron transfer chain, ATP production and ROS formation.Skulachev VP. Cationic antioxidants as a powerful tool against mitochondrial oxidative stress. Biochem Biophys Res Comm 2013;441:2:275-9. An important feature of SKQ1 is that its oxidation chemistry is such that it is recycled in the mitochondria, allowing it to serve as a renewable antioxidant. In addition to its antioxidant properties, studies in cell cultures of human conjunctival epithelial cells showed that SKQ1 reduced the production of prostaglandin E2, a pro-inflammatory signaling molecule that has been implicated in dry eye. Other SKQ1 studies of human endothelial cells indicated that mitochondria ROS are involved in regulation of the immune response.Zinovkin RA, Romaschenko VP, Galkin II, Zakharova VV, Pletjushkina OY, Chernyak BV, Popova EN. Role of mitochondrial-reactive oxygen species in age-related inflammatory activation of endothelium. Aging 2014;6:661-674.
In mouse models of dry eye, SKQ1 reduced corneal staining and appeared to have a rapid onset and long duration of action. Other studies of the compound showed SKQ1 or related plastoquinones had beneficial therapeutic effects in animal models of retinopathy, glaucoma, macular degeneration and UV damage to the lens;Iomdina EN, Khoroshilova-Maslova IP, Robustova OV, Averina OA, Kovaleva NA, Aliev G, Reddy VP, Zamyatnin AA Jr, Skulachev MV, Senin II, Skulachev VP. Mitochondria-targeted antioxidant SkQ1 reverses glaucomatous lesions in rabbits. Frontiers in Bioscience 2015;20:892-901.,Muraleva NA, Kozhevnikova OS, Zhdankina AA, Stefanova NA, Karamysheva TV, Fursova AZ, Kolosova NG. The mitochondria-targeted antioxidant SkQ1 restores alphaB-crystallin expression and protects against AMD-like retinopathy in OXYS rats. Cell cycle 2014;13:22:3499-3505. systemic benefits in ischemia-related diseases have also been documented.Plotnikov EY, Silachev DN, Jankauskas SS, Rokitskaya TI, Chupyrkina AA, Pevzner IB, Zorova LD, Isaev NK, Antonenko YN, Skulachev VP, Zorov DB. Mild uncoupling of respiration and phosphorylation as a mechanism providing nephro- and neuroprotective effects of penetrating cations of the SkQ family. Biochemistry(Mosc) 2012;77:1029-1037.
In Russian clinical trials, SKQ1-induced reductions in dry-eye signs and symptoms were significantly greater than those seen with an artificial tear control.Brzheskiy V, Efimova E, Vorontsova T, Alekseev VN, Gusarevich OG, Shaidurova KN, Ryabtseva AA, Andryukhina OM, Kamenskikh TG, Sumarokova ES, Miljudin ES, Egorov EA, Lebedev OI, Surov AV, Korol AR. Results of a multicenter, randomized, double-masked, placebo-controlled clinical study of the efficacy and safety of visomitin eye drops in patients with dry-eye syndrome. Adv Ther 2015;32:12:1263-1279. SKQ1 improved corneal cell function, increased tear-film stability and reduced dryness, burning, grittiness and blurred vision. In a subsequent U.S. clinical trial, SKQ1 reduced corneal and conjunctival staining, improved ocular discomfort scores and was generally fnerior to placebo control treatment.Petrov A, Perekhvatova N, Skulachev M, Stein L, Ousler G. SkQ1 ophthalmic solution for dry eye treatment: Results of a phase 2 safety and efficacy clinical study in the environment and during challenge in the Controlled Adverse Environment model Adv Ther 2016;33:1:96-115. Of note, this study demonstrated SKQ1 improvements in both signs and symptoms of dry eye evoked through the use of the controlled adverse environment, a model that is designed to exacerbate dry-eye instigators, including oxidative stress effects. In both clinical trials, the compound exhibited a good safety profile and was well-tolerated by subjects.
Results with the compound SKQ1 confirm the importance of the mitochondria as a target for reducing oxidative stress in the body, and also fnport the notion that ROS are important contributors to dry-eye disease.
Novel treatments for dry eye hold promise in the not-to-distant future; treatments that hone in on the initial damaging events of intracellular oxidation could halt dry eye signs and symptoms in their tracks. Treating dry eye from the inside out may very well de-stress the cells that are under the onslaught of oxidative stress, allowing both the patient and the ophthalmologist some much needed respite from this disease.
Dr. Abelson is a clinical professor of ophthalmology at Harvard Medical School. Mr. Ousler is vice president of dry eye at Ora Inc. Ms. Stein is a medical writer at Ora. Dr. Abelson may be reached at MarkAbelsonMD@gmail.com.