Molecular Biomarkers for Glaucoma

Abstract

Purpose of review

This article summarizes the current studies on molecular biomarkers with potential implications in diagnosis, prognosis, and response to treatment in patients with glaucoma.

Recent findings

Important advances have occurred in the understanding of the pathogenesis of glaucomatous neurodegeneration. Protein biomarkers associated with inflammatory, neurodegenerative, and other molecular pathways have been described in glaucoma patients in tear film, aqueous fluid, vitreous fluid, and serum, however, we are still far from having a clear picture of the whole molecular network that relates to the disease and its implications in clinical use.

Summary

Although more studies are needed, current and emerging molecular biomarkers candidates in glaucoma may eventually transition into clinical use and contribute to outline the concept of precision medicine and precision health in glaucoma.

Introduction

Glaucoma is one of the main causes of irreversible visual field loss and blindness worldwide (1). Vision loss in this multifactorial neurodegenerative disease results from progressive degeneration of retinal ganglion cells (RGCs). Currently, there is no single gold standard test for glaucoma screening and diagnosis. For example, basing screening on high intraocular pressure (IOP) of > 21 mmHg will lead to missing the diagnosis for more than 50% of patients due to poor sensitivity (2). Glaucoma severity classification, monitoring for disease progression and response to pharmacological or surgical treatment are based on experts’ assessment of IOP measurements, clinical exam, optic nerve structure and visual field results. In early glaucoma stages, when irreversible RGC damage is already occurring but symptoms and signs may be absent, diagnosis may be delayed or missed. In addition, there is limitation of progression detection by visual fields due to high variability and retrospective nature. Therefore, there is a significant unmet need to establish clinically useful glaucoma-related biomarkers to drive advances in risk classification, earlier glaucoma diagnosis and monitoring and predicting treatment responses and prognosis.

Biomarkers are biological markers defined as an objective and quantifiable characteristics of any biological processes. The National Institutes of Health Biomarkers Definitions Working Group defines a biomarker as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” (3). There are many examples of biomarkers, including everything from body temperature and heart rate to laboratory tests on samples of blood, urine, or other tissues. To identify biomarkers for clinical utility a potential biomarker must be confirmed as valid and should be reproducible, specific, and also sensitive (4, 5).

Could such new biomarkers target the molecular changes that accompany glaucoma? Although progress over the past several decades in understanding the pathophysiology of glaucomatous neurodegeneration has occurred, molecular mechanisms are still in some cases unclear or in others unproven. Glaucoma is a multifactorial disease which may involve multiple factors, including but not limited to immune reaction, ischemia and oxidative stress (6–11). In spite of the fact that the exact molecules to target are still unknown, the search for potential biomarkers may actually lead to a better understanding of glaucoma pathophysiology itself.

Where should we search for molecular biomarker candidates? The process of glaucomatous neurodegeneration occurs in the optic nerve and RGC in the inner retina, but sampling these tissues in living humans would not be feasible. Surveying the vitreous fluid or blood serum would be less invasive, and even more accessible would be sampling the aqueous humor or the tear film, although slightly more distant sites from glaucomatous neurodegeneration process. Here we review the progress towards discovering molecular biomarkers and discuss the potential clinical implications with a focus on sources for liquid biopsy such as the tear film, aqueous humor, vitreous body and whole blood or serum.

Tear Film Biomarkers

The tear film is a thin fluid layer that covers the outer mucosal surfaces of the eye. It contains a large variety of molecules including proteins, some of which have been reported to be glaucoma- or therapy-associated. For instance, endothelin-1, which has a functional role in glaucoma pathophysiology promoting vasoconstriction and decreasing aqueous humor outflow agent (12), was found to be increased in tear film of patients with primary open-angle glaucoma (POAG) compared to controls (13). Similarly, ciliary neurotrophic factor (CNTF), a factor that promotes proliferation, differentiation, survival, and functioning of neurons (14) and that was shown to have neuroprotective effect on RGCs (15), was reported to be reduced in the tear film and aqueous humor, and with a strong and inverse correlation with glaucoma severity estimated by visual field loss (16). It is thought-provoking to consider CNTF as a biomarker for glaucoma, as a randomized clinical trial assessing CNTF as a an experimental drug that appears to slow neurodegeneration in glaucoma patients is currently undergoing (Clinicaltrials.gov #NCT02862938), emphasizing the possible convergence of biomarker studies and clinical trials.

The role of apoptotic factors in glaucoma has also been widely studied. For instance, apoptosis stimulating fragment (FAS) ligand/receptor-related interactions were found to have an important role in RGC death, and in onset and progression of POAG (17–19). Another study reported of differential protein expression in tears of patients with primary open angle and pseudoexfoliative glaucoma (PEXG), highlighting inflammation pathways in both diseases but a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies (20).

Several studies have reported of tear biomarkers that may be related to IOP or anti-hypertensive therapy, for example finding a higher level of kalikrein and angiotensin converting enzyme (ACE) activity in aqueous and tears of glaucoma patients (21, 22). In addition, it was shown in animal studies that drugs affecting renin-angiotensin system by blocking its action as ACE inhibitors may lower intraocular pressure when locally administered (23). Therefore, renin-angiotensin system related biomarkers may relate to therapeutic pathways. Comparing topically treated versus untreated glaucoma patients has also revealed specific protein patterns between these groups (24), including metalloproteinases (MMP) as well as tissue metalloproteinases inhibitor (25), S100-A8 (26); pro-inflammatory (interleukin (IL)-1β, IL-6, IL-12, tumor necrosis factor (TNF)-α), T helper (Th)1 (Interferon (INF)-γ, IL-2) and Th2 (IL-5, IL-10, IL-4) type cytokines (27), and other cytokines (28). Such data raise a key question of whether such biomarkers are disease- or treatment-related, particularly when studying ocular surface inflammation known to be provoked by topical glaucoma medications.

Although relatively fewer groups have compered different topical formulations directly, a few studies have begun to investigate differences attributable to the drug or its preservatives’ effects. For instance, comparing two PGAs directly, it was found that latanoprost-treated eyes had higher MMP-9 expression and marginally elevated tear cytokines involved in tissue remodelling while bimatoprost eyes showed higher MMP-2 expression and elevated cytokines regulating allergic pathways (29). Another thought-provoking finding in patients with POAG is that IL-1β expression was found to be lower in glaucoma patients receiving IOP-lowering eye drops with no preservatives compared with those patients receiving preserved eye drops (30). All together these data may suggest that topical anti-glaucoma medications may promote ocular surface inflammation, although untwisting effects of IOP-lowering versus medications’ side effect remains a challenge in understanding such experiments. Taken together, tear film molecular profiling in glaucoma patients may disclose promising pathophysiology-, therapy-, or pharmacogenetic-related biomarkers.

Aqueous humor biomarkers:

Moving inside the eye, the aqueous humor is a clear fluid formed by the ciliary epithelium that fills the anterior and posterior chambers of the human eye; its circulation serves many functions. The aqueous fluid contains various proteins secreted by anterior segment tissues and the total protein concentrations in aqueous humor are affected by many systemic and ocular diseases, including POAG (31–33). Aqueous humor may contain relevant biomarkers related to or even originating from molecular mechanisms involved in glaucomatous processes such as immune and oxidative stress reactions and, eventually, RGC neurodegeneration.

Differences in the aqueous humor proteome of glaucoma patients and controls have been widely studied. For example, superoxide dismutase (SOD) and glutathione transferase levels in aqueous humor were significantly lower in glaucoma patients than in controls, and both nitric oxide synthase and glutamine synthase were found to be significantly higher in the former. (34). In contrast, another study found a significant increase in SOD and glutathione peroxidase activities in aqueous humor of glaucoma patients (35). In parallel, low expression of SOD-1 was detected in the serum of glaucoma patients (36). Serum studies are discussed further below, but clearly more validation will be needed.

As the extracellular matrix (ECM) in the trabecular meshwork (TM) is important in IOP regulation, aqueous humor proteins related to TM’s ECM may reveal biomarkers important in glaucomatous pathophysiology. For example, transforming growth factor β2 (TGF-β2), which participates in regulation of ECM composition and the TM actin cytoskeleton, was found in higher levels in aqueous humor of POAG patients (37). Moreover, TGF-β2 concentrations are correlated to the increase aqueous outflow resistance in the TM, leading to IOP elevation (38).

A number of studies have assessed cytokines and chemokines differential expression in the aqueous humor as well as in anterior chamber tissues of eyes with POAG. For example, some studies have reported of increased levels of IL-8 and other cytokines in the aqueous humor of POAG and PEXG patients (39, 40). Similar finding were found in angle closure patients (41). As with tear film studies discussed above, disease staging may affect levels of cytokines or other biomarkers, shown for example for monocyte chemotactic protein-1 (42). All together these data support the important role of immune activation in the pathophysiology of glaucoma.

Other molecular pathways have also been studied in aqueous humor of glaucoma patients. In a recent proteomic study of the aqueous humor, 87 proteins were found to be in significantly different concentrations between glaucomatous and control eyes, part of various molecular pathways such as cholesterol- and proteolysis-related cascades, in addition to inflammatory, metabolic, and antioxidant pathways (43). Importantly, the relationship between different cytokines, various disease mechanisms, IOP and its control by medications or surgical intervention requires further research. However, because aqueous humor sampling is invasive and generally limited to patients undergoing an independent intraocular surgery, its utility as a clinical diagnostic tool may require alternative non-invasive detection approaches, such as through molecular imaging modalities under development.

Vitreous Biomarkers

In spite of the fact that vitreous body sampling is more invasive and considerably less frequently surgically approached in glaucoma patients, its juxtaposition to RGCs makes it a more attractive sampling target to learn about glaucoma molecular pathophysiology and biomarkers. For example, vitreous levels of IL-2, IL-5, monocyte chemoattractant protein-1, TNF-α and interferon-γ-inducible protein (IP)-10 were higher in acute angle closure glaucoma (AACG), emphasizing the role of inflammation and immune reaction in AACG pathophysiology (44). In another proteome study, about 5000 different proteins were quantified from retina and vitreous of glaucoma and control eyes. Interestingly, out of the differentially regulated proteins between glaucoma patients and age-matched controls, 122 were found to be linked with the pathophysiology of Alzheimer’s disease. Identification of pathways in glaucoma that overlap with other CNS neurodegenerative diseases may provide deeper understanding of the molecular mechanisms and pathogenesis of neurodegenerative diseases (45).

Although inadequate access to vitreous samples in human glaucoma and control patients limits such studies in humans, molecular discoveries in animal models could help in guiding subsequent human subjects research to specific target molecules or pathways, e.g. searching for specific molecules in the vitreous body, aqueous humor, tear film or serum. For example, elevated IOP resulted in a 25-fold increase of catalase in the vitreous compared to the control contralateral eye in an experimental mouse model of hypertensive glaucoma (46). Studies integrating data across animal models and human patients are likely to bear fruits and guide direction for future research.

Serum Biomarkers

There are multiple routes by which blood serum might molecularly reflect underlying RGC degeneration process and therefore serve as a potential biomarker reservoir or even reflect underlying pathophysiology of glaucoma. For instance, RGC degeneration may directly release neuronal-specific proteins potentially detectable in the bloodstream, or consequentially influence immune cell activation, which could also contribute the pathophysiology of degeneration in reverse (47). Studies are beginning to explore candidate for serum biomarkers, and investigate their potential links to glaucoma pathophysiology. Findings of altered serum profiles in glaucoma patients have ranged from serum oxidation degradation products (48), vascular autoregulation signaling molecules (49–51), inflammatory pathway molecules (52), and neurotrophin family proteins (53). Although minimally invasive, collection of blood samples is a safe procedure, therefore serum glaucoma established biomarkers may one day enter into practical clinical use.

Crossover with Other Optic Neuropathies

Glaucoma, a neurodegenerative subtype of optic neuropathy, is likely to share cellular and molecular alterations with other optic neuropathies such as ischemic optic neuropathy or even different types of optic neuritis. Although the pathophysiology may differ in each type of optic neuropathy, in all of these, RGCs degeneration, acute or progressive, followed by death of RGCs occurs and may lead to overlapping molecular allterations detectable in the eye or peripheral serum. For example, one study reported of oligoclonal immunoglobulin G (IgG) bands serving as a potential biomarker to predicted progression to clinically definite multiple sclerosis after an episode of optic neuritis (54). Thus although shared molecular pathophysiological processes may aid in discovery of biomarkers, the specificity across optic neuropathies subtypes may be limited using such biomarkers, and the use of combinations or multiplexed molecular probes may be required for -practical clinical value.

Conclusions

Currently, no definitive molecular biomarker is established to diagnose glaucoma or predict progression, however clearly tremendous progress has been made. In glaucoma management, biomarkers could potentially aid in earlier diagnosis, severity grading and/or progression risk stratification, in identifying adequate responders to medications from those patients who are refractory, and in research on potential new medical therapies, for better patient selection or outcome measures. It will be important in future studies to understand whether a specific biomarker is sensitive not only to disease stage in cohort studies, but also to response to therapy, and/or helpful in progression prediction in longitudinal studies (5). We anticipate that validated molecular biomarkers will offer a new clinical tool useful in glaucoma diagnosis, in prediction of disease prognosis, and in monitor clinical responses to standard treatments or investigational therapies.