Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the US, but its disease mechanisms remain poorly understood and for many, treatment is suboptimal.1,2,3 However, new scientific approaches such as metabolomics and proteomics are shedding light on the causes of AMD and other eye diseases.3,4,5 These powerful modern methods are also revealing novel biomarkers that are leading to the development of new diagnostic, monitoring and predictive tools, as well as potential treatment options.6,7,8 These methods rely on advanced technologies that are constantly being improved, such as mass spectrometry (MS).
Investigating AMD disease mechanisms
As the name suggests, AMD is a disease that affects older people. It progressively destroys the macula, the part of the retina responsible for sharp, central vision (see Figure 1). Without central visual acuity, people are unable to perceive facial expressions, read, drive, and use a computer. The underlying causes of AMD are still not well understood, and as the main risk factor – aging – is not modifiable, research continues to investigate its disease mechanisms and how they can be targeted. There is no cure for AMD, but management options include drugs injected into the eye, lasers, photodynamic therapy and sometimes, even surgery.
Figure 1: Age-related degeneration of the macula results in the loss of central vision and visual acuity. Credit: Lei Zhou, Singapore Eye Research Institute.
The standard treatment for wet AMD (wAMD), a rarer but more severe form of the disease than dry AMD, is a drug that targets vascular endothelial growth factor (VEGF). This disrupts VEGF function in the formation of new blood vessels, which in wAMD, leak blood and fluid into the macula. However, reports of some patients still exhibiting vascular bleeding or leakage despite anti-VEGF treatment suggest that other disease mechanisms are at play, ones without VEGF involvement.3,9 Indeed, it has been postulated that AMD is a manifestation of a systemic disease.10
To better elucidate the mechanisms involved in age-related eye diseases, Professor Hannu Uusitalo of Tampere University, Finland, investigated the proteomes of healthy individuals of various ages without eye disease. With Dr Lei Zhou, Head of the Ocular Proteomics Platform at the Singapore Eye Research Institute (SERI), Singapore, he examined the proteomic profiles of tear samples, using advanced liquid chromatography mass spectrometry (LC-MS) technologies. The LC-MS scans were performed using SWATH Acquisition, an untargeted data-independent way to comprehensively detect all the proteins and peptides present in each tiny sample.5,8 Proteins related to inflammation, immune response, and cell death were found at different quantities depending on the person’s age, which suggested that in healthy aging, cell growth and survival decreases while immune response and inflammation increase. These proteins could also help us better understand various age-related eye diseases.5
Identifying potential AMD biomarkers and treatments
Another researcher using LC-MS to investigate eye disease with Dr Lei Zhou is Professor Gemmy Cheung of the Duke-NUS Medical School at National University of Singapore (NUS), and Head of the Medical Retina Department of the Singapore National Eye Center (SNEC). With colleagues at NUS, SNEC, the Ocular Proteomics Platform at SERI, the Agency for Science, Technology and Research (A*STAR) Singapore, and the University of Colorado, USA, she is examining the metabolic profiles of serum samples obtained from wAMD patients. These LC-MS experiments were also performed using SWATH Acquisition, the label-free data-independent method for comprehensively detecting all the metabolites present in these complex samples.4,8
The comparison of serum metabolomes between wAMD patients and healthy individuals revealed that the patients had higher levels of certain molecules than the healthy controls, including glycerophospholipids, amino acids, and omega fatty acids.4 Another metabolomic comparison of sera from wAMD patients, this time between anti-VEGF drug responders and suboptimal responders, identified metabolites that differed between the two groups. Once again, the glycerophospholipid metabolic pathway was implicated as higher levels were observed in the suboptimal responders than in the responders. In particular, lysophosphatidylcholine may accumulate in the serum of suboptimal responders and thus interfere with vascular modeling and induce oxidative stress, resulting in a poor response to anti-VEGF medicines.3 These and other findings are forming the foundation for the development of a new clinical test, which will use LC-MS to detect and characterize metabolomic biomarkers for predicting which patients are likely to respond to anti-VEGF treatment.
LC-MS with SWATH Acquisition is also being used to measure the proteomes of individuals with wAMD. The study of vitreous proteomes led to the elucidation of a new pathway that contributes to the wAMD disease mechanism and excludes VEGF involvement. Unraveling this pathway identified further molecules and resulted in the discovery of a novel and potent anti-angiogenic factor. The active portion of this protein was isolated and synthesized into an investigational drug, which is being assessed in preclinical studies. Preliminary results indicate that this new experimental therapy may successfully treat wAMD, either as an alternative or in combination with anti-VEGF agents.
Proteomic biomarkers to predict treatment response in dry eye disease
MS-based proteomic studies have also been useful in the study of other ocular surface diseases, such as dry eye disease (DED) and glaucoma.5,7 In the healthy eye, the complex, extracellular fluid, commonly known as tears, is secreted from multiple sources, and serves to bathe the surface of the eye, to deliver and remove nutrients and metabolic products, to improve the retinal image, and contributes to the mechanical, antimicrobial and anti-inflammatory defense of the eye surface. In DED, the homeostatic functional unit of the lacrimal glands, one of the tear-fluid sources, is dysfunctional, resulting in an immune-based multifactorial disease of the ocular surface.11
Treatment for DED primarily involves artificial tears but in more severe disease, topical steroids, such as flourometholone (FML), or other anti-inflammatory drugs, such as polyvinyl alcohol (PA) are used.11 LC-MS based proteomic analysis of tears from DED patients undergoing treatment with either FML or PA revealed protein biomarkers that could be used to predict which patients would respond best to management with FML instead of PA. Further, potential biomarkers were also identified that may in conjunction with clinical signs provide a more accurate way of predicting whether a patient’s disease severity would be moderate or severe.11
Tear proteome guides glaucoma patient stratification
DED can also occur with other eye diseases, particularly glaucoma. Although generally known as a disease of older age, glaucoma is also a leading cause of blindness in children, and the second leading cause of blindness in the world.12,13 Although there is no cure, there are drugs that can delay the glaucomatous deterioration of the eye, often by lowering the intraocular pressure – a common characteristic of glaucoma.7 Eye drops are the most common administration route, but prolonged use of topical glaucoma medication may induce the signs and symptoms of DED.
When this happens, switching patients from a medication containing preservative to a preservative-free drug can diminish adverse reactions.7 A study of the tear proteomes from glaucoma patients identified biomarkers that could potentially be used to detect which patients with glaucoma and concomitant DED would not respond to a switch to preservative-free medication, would have moderate improvement in symptoms in response to a switch, or would benefit the most from the medication switch.7
These are just a few of the ways in which metabolomics and proteomics studies using LC-MS with SWATH Acquisition are illuminating the disease mechanisms of eye diseases and how best to ensure that the right treatments are provided at the right time for the right patients. This is precision medicine, and you do not get precision medicine without the sensitive and precise quantification of molecules.6
Dr Aaron Hudson, PhD, is vice president, global marketing and strategy, at SCIEX, a Danaher operating company and a global leader in the sensitive and precise quantification of molecules.
1. Age-related macular degeneration (AMD) data and statistics. US National Institutes of Health (NIH) National Eye Institute. https://www.nei.nih.gov/learn-about-eye-health/resources-for-health-educators/eye-health-data-and-statistics/age-related-macular-degeneration-amd-data-and-statisticsAccessed March 2021.
2. Aged-related macular degeneration: facts & figures. BrightFocus Foundation. https://www.brightfocus.org/macular/article/age-related-macular-facts-figures. Published January 5, 2019. Accessed March 2021.
3. Gao Y, Teo YCK, Beuerman RW, et al. A serum metabolomics study of patients with nAMD in response to anti-VEGF therapy. Sci Rep. 2020; 10: 1341. doi:10.1038/s41598-020-58346-3
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9. Dugel PU, Koh A, Ogura Y, et al. HAWK and HARRIER: phase 3, multicenter, randomized, double-masked trials of brolucizumab for neovascular age-related macular degeneration. Ophthalmology. 2020; 127: 72–84. doi:10.1016/j.ophtha.2019.04.017
10. Cheung CM, Wong TY. Is age-related macular degeneration a manifestation of systemic disease? New prospects for early intervention and treatment. J Intern Med. 2014; 276: 140–53. doi:10.1111/joim.12227
11. Nättinen J, Jylhä A, Aapola U, et al. Topical fluorometholone treatment and desiccating stress change inflammatory protein expression in tears. Ocul Surf. 2018; 16: 84–92. doi:10.1016/j.jtos.2017.09.003
12. Personal story: Hannah Eckstein. Glaucoma Research Foundation. https://www.glaucoma.org/personalstories/personal-story-hannah-eckstein.php Accessed March 2021.
13. Glaucoma: The silent thief of sight. University of Utah Health. https://healthcare.utah.edu/healthfeed/postings/2017/01/glaucoma.phpAccessed March 2021.