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Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis in multiple cancer types; however, sEVs in the aqueous humor (AH) of uveal melanoma (UM) patients have never previously been profiled. In this study, we used single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically the tetraspanin co-expression patterns of CD9, CD63, and CD81. sEVs were analyzed from paired pre- and post-treatment (brachytherapy, a form of radiation) AH samples collected from 19 UM patients. In post-brachytherapy samples, two subpopulations, CD63/81+ and CD9/63/81+ sEVs, were significantly increased. These trends existed even when stratified by tumor location and GEP class 1 and class 2 (albeit not significant for GEP class 2). In this initial report of single vesicle profiling of sEVs in the AH of UM patients, we demonstrated that sEVs can be detected in the AH. We further identified two subpopulations that were increased post-brachytherapy, which may suggest radiation-induced release of these particles, potentially from tumor cells. Further study of the cargo carried by these sEV subpopulations may uncover important biomarkers and insights into tumorigenesis for UM.

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Altered protein levels in the aqueous humor (AH) may be a valuable source of novel biomarkers in neurodegenerative retinal disease. The proximity of this body fluid to the disease focus, and its corresponding enrichment for tissue specific proteins, renders it an excellent matrix to study underlying molecular mechanisms. Novel proteomic methods accordingly hold large potential for insight into pathologies based on the composition of the AH proteome, including primary open angle glaucoma (POAG). Recent mass spectrometry-based studies use novel approaches to tackle the challenges arising from the combination of low available sample volume and protein concentration, thereby increasing proteome coverage. But despite significant improvements in mass spectrometry (MS), a different class of proteomic technologies is poised to majorly impact the analysis of ocular biofluids. Affinity proteomic workflows, having become available commercially recently, have started to complement data obtained by MS and likely will grow into a crucial tool for ophthalmological biomarker research. This review highlights corresponding approaches in proteome analysis of aqueous humor and discusses recent findings on alterations of the AH proteome in POAG.

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Aims: Interocular comparison of the metabolomic signature of aqueous humor (AH) was performed. The aim of the study was to quantitatively evaluate the symmetry in concentrations of various metabolites belonging to different categories. Methods: The study included AH samples from 23 patients, 74.17 ± 11.52 years old, undergoing simultaneous bilateral cataract surgery at the Ophthalmology Department of the Medical University of Bialystok, Poland. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based targeted metabolomics and lipidomics analyses of AH samples were performed using the AbsoluteIDQ® p180 kit. Out of 188 metabolites available in the kit, 67 were measured in the majority (>70%) of the samples: 21/21 amino acids, 10/22 biogenic amines, 9/40 acylcarnitines, 0/14 lysophosphatidylcholines, 21/76 phosphatidylcholines, 5/15 sphingolipids, and 1/1sum of hexoses. Results: The comparison of both eyes revealed that the concentrations of metabolites did not differ significantly (p < 0.05) except for taurine (p = 0.037). There was moderate-to-strong positive interocular correlation (r > 0.5) between most metabolites regarding concentration. This was confirmed by the high intraclass correlation coefficient (ICC) values of different levels, which varied for the different metabolites. However, there were exceptions. Correlations were not significant for 2 acylcarnitines (tiglylcarnitine and decadienylcarnitine) and 3 glycerophospholipids (PC aa C32:3, PC aa C40:2, and PC aa C40:5). Conclusion: With a few exceptions, a single eye was found to be representative of the fellow eye in terms of the concentration of most of the analyzed metabolites. The degree of intraindividual variability in the AH of fellow eyes differs for particular metabolites/metabolite categories.

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BACKGROUND: Early diagnosis of vitreoretinal lymphoma (VRL) is critical for the successful treatment of this rare intraocular malignancy. However, fast and reliable diagnosis of VRL in patients presenting with intermediate or posterior non-infectious uveitis remains a challenge. A proliferation-inducing ligand (APRIL) and B-cell activating factor (BAFF) are vital factors in the pathophysiology, diagnosis, and prognosis of primary central nervous system lymphoma (PCNSL) and systemic autoimmune diseases. However, their utility as biomarkers for the diagnosis of VRL and uveitis remains unclear. METHODS: In this retrospective study, we analyzed APRIL and BAFF levels in the aqueous humor (AH) of 43 eyes of 40 patients, including 23 eyes of 20 patients with VRL, eight eyes of eight patients with uveitis, and 12 eyes of 12 patients with other ocular diseases (OODs). Additionally, we measured their levels after induction of chemotherapy in five eyes of five patients with VRL. RESULTS: AH levels of APRIL reliably distinguished VRL from uveitis, with a specificity of 78.3% and sensitivity of 75%. BAFF also showed similar potential. Serial AH analysis of patients with VRL during chemotherapy demonstrated a corresponding decline in AH levels of APRIL and BAFF. CONCLUSION: This study extends the spectrum of valuable diagnostic biomarkers for VRL and uveitis. In patients with uveitis, the assessment of AH APRIL may help accelerate the diagnosis of VRL. Moreover, our results underline the important role of APRIL and BAFF in therapeutic monitoring of VRL.

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BACKGROUND: Acute angle-closure glaucoma (AACG) is an ophthalmic emergency that occurs over the course of hours or days and may cause irreversible blindness if not treated immediately. In most cases, optic nerve damage is the cause of visual field (VF) loss in AACG. There has been no reliable biomarker found to evaluate optic nerve damage to date. Aqueous humor (AH) proteome analysis might reveal the proteomic alterations in AACG and provide helpful clues in the search for an AH biomarker of optic nerve damage and VF loss. METHODS: In this study, we used the AH proteome to explore the functions of differentially expressed proteins (DEPs) during disease progression. The AH proteins from the early-stage group and late-stage group were extracted and analyzed by the data-independent acquisition (DIA) method. The DEPs functions were annotated, and parallel reaction monitoring (PRM) was used to validate the key DEPs. RESULTS: A total of 87 DEPs were found. Gene Ontology analysis showed that most DEPs were enriched in immunology, hemodynamics, and apoptosis. Ingenuity pathway analysis found that vascular endothelial growth factor (VEGF) signaling, the production of reactive oxygen species (ROS) in macrophages, and the nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress response were active pathways in the late stage of AACG. The mechanism of retinal ganglion cell (RGC) death was hypothesized on the basis of DEP functional analysis. A total of 20 DEPs were validated by using PRM, and prostaglandin-H2 D-isomerase was found to have the potential to evaluate optic nerve damage. CONCLUSIONS: This study showed that AH proteomic analysis could reveal the proteomic alterations in the pathogenesis of VF loss in AACG and help to provide objective protein biomarkers to evaluate VF loss. These findings will benefit the application of the AH proteome to clinical research.

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The aim of the study was the multi-elemental analysis of aqueous humor (AH) collected from patients undergoing cataract surgery. The study included: 16 patients with age-related macular degeneration AMD (99 controls), 10 patients with retinopathy (105 controls), 61 patients with hypertension (54 controls), and 33 patients with coexisting diabetes (82 controls). The control groups were recruited from patients with a lack of co-existing disease characterizing the specified studied group. The measurements were performed by the use of inductively coupled plasma optical emission spectrometry (ICP-OES). The statistical analysis was carried out using non-parametric testing (Mann-Whitney U). The level of significance was set at p = 0.05. The data obtained revealed substantial variations in elemental composition between the test groups in comparison to the controls. However, the significant variations concerned only a few elements. The phosphorous (P) level and the ratio of P/Ca were significant in retinopathy and diabetes, whereas cobalt (0.091 ± 0.107 mg/L vs. 0.031 ± 0.075 mg/L; p = 0.004) was significant in AMD. In co-existing hypertension, the levels of tin (0.293 ± 0.409 mg/L vs. 0.152 ± 0.3 mg/L; p = 0.031), titanium (0.096 ± 0.059 mg/L vs. 0.152 ± 0.192 mg/L; p = 0.045), and ruthenium (0.035 ± 0.109 mg/L vs. 0.002 ± 0.007 mg/L; p = 0.006) varied in comparison to the controls. The study revealed inter-elemental interactions. The correlation matrices demonstrated the domination of the positive correlations, whereas negative correlations mainly concerned sodium.

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BACKGROUND: The purpose of this study is to investigate the aqueous humor (AH) T lymphocyte subsets and cytokines of acute retinal necrosis (ARN) to elucidate the immunologic inflammatory features of this disorder. METHODS: Three patients with ARN infected with varicella zoster virus (VZV) who underwent multiple intravitreal injections of ganciclovir were enrolled in this study. The control group consisted of four non-infectious patients with acute anterior uveitis (AAU). Flow cytometric analysis was performed on the lymphocyte subsets from the AH and peripheral blood (PB) samples during the active phase of intraocular inflammation. Five inflammatory cytokines were measured in each AH sample and various clinical characteristics were also assessed. RESULTS: VZV deoxyribonucleic acid (DNA) was detected by real-time polymerase chain reaction (PCR) in AH from all the ARN patients, who showed higher CD8+ T lymphocytes population in AH than the AAU patients (p = 0.006). CD4/CD8 ratios of T lymphocytes and the percentage of CD8 + CD25+ T lymphocytes in AH were significantly lower in ARN than in AAU (p = 0.006; p = 0.012). In the ARN patients, the percentages of CD4+ and CD8+ T lymphocytes in AH were higher than those found in PB. The percentage of CD4 + CD25+ T lymphocytes in AH was significantly higher than the proportion in PB in the AAU patients (p = 0.001). Immunoregulatory cytokine Interleukin-10 in AH was significantly elevated in the ARN patients in comparison with the case of the AAU patients (p = 0.036). In ARN, the copy number of VZV DNA in AH positively correlated with the percentage of CD8+ T lymphocytes in AH and negatively correlated with the CD4/CD8 ratio in AH during the course of disease treatment (p = 0.009, r = 0.92; p = 0.039, r = - 0.834). CONCLUSION: The ARN patients caused by VZV had different intraocular T lymphocyte subsets and cytokines profile than those of the non-infectious patients. High percentages of CD8+ T lymphocytes and low CD4/CD8 T cell ratios may be a potential biomarker for diagnosis of viral-infectious uveitis. T lymphocytes examination at the inflammatory sites has the potential to become a useful research tool for differentiating viral and non-viral uveitis.

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Assessing metabolomic alterations in age-related macular degeneration (AMD) can provide insights into its pathogenesis. We compared the metabolomic profiles of the aqueous humor between wet AMD patients (n = 26) and age- and sex-matched patients undergoing cataract surgery without AMD as controls (n = 20). A global untargeted metabolomics study was performed using ultra-high-performance liquid chromatography tandem mass spectrometry. Univariate analysis after the false discovery correction showed 18 significantly altered metabolites among the 291 metabolites measured. These differential metabolomic profiles pointed to three interconnected metabolic pathways: a compromised carnitine-associated mitochondrial oxidation pathway (carnitine, deoxycarnitine, N6-trimethyl-l-lysine), an altered carbohydrate metabolism pathway (cis-aconitic acid, itaconatic acid, and mesaconic acid), which plays a role in senescence and immunity, and an activated osmoprotection pathway (glycine betaine, creatine), which potentially contributes to the pathogenesis of the disease. These results suggested that metabolic dysfunction in AMD is mitochondrial-centered and may provide new insights into the pathophysiology of wet AMD and novel therapeutic strategies.

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Quantitative analysis of aqueous humor (AH) was performed to investigate glucose metabolism in patients with central retinal vein occlusion (CRVO), and to explore metabolic changes after anti-vascular endothelial growth factor (VEGF) treatment. AH samples were collected from 35 patients. Participants diagnosed with CRVO (n = 15) were compared to participants who underwent cataract surgery (n = 20). Thirteen of the participants with CRVO received second-round anti-VEGF treatments. Ultra-high performance liquid chromatography tandem-mass spectrometry (UHPLC-MS/MS) was used to quantify metabolites of the AH. Central macular thickness (CMT) and retinal ganglion cell layer (RGC) thickness were measured using spectral-domain optical coherence tomography. Thirteen metabolites involved in glucose metabolism were identified. Among these metabolites, succinate, glutamate, and glutamine were significantly decreased for the CRVO group (p = 0.028, 0.009, and 0.017, respectively). The α-ketoglutarate/citrate (K/C) ratio had a significant positive correlation with glutamine levels for both control (r = 0.922, p < 0.001) and CRVO groups (r = 0.674, p = 0.006). A significant increase in lactate was observed after intravitreal anti-VEGF administration (t = 2.273, p = 0.045); the change in CMT was negatively correlated with this increase (r = -0.745, p = 0.003). The alteration of RGC thickness was negatively correlated with increases in both glutamine (r = -0.619, p = 0.024) and glucose (r = -0.754, p = 0.003). These results indicate that, compared to glucose metabolism, glutamine was significantly decreased in the AH of patients with CRVO, and may therefore serve as a potential target for CRVO therapy. The glycolytic pathway might be enhanced after intravitreal anti-VEGF injection, which is an important insight into CRVO pathophysiology.

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BACKGROUND: While pars plana vitrectomy (PPV) has become the third most commonly performed surgery in the world, it can also induce multiple post complications easily. Among them, cataract progression is the most frequent one that can lead to blindness eventually. METHODS: To understand the underlying mechanisms of post PPV cataract progression, we performed comprehensive metabolic characterization of aqueous humor (AH) samples from 20 cataract patients (10 post PPV complication and 10 none PPV cataract) by a non-targeted metabolomic analysis using gas chromatography combined with time-of-flight mass spectrometer (GC/TOF MS). RESULTS: A total of 263 metabolites were identified and eight of them are determined to be significantly different (VIP ≥ 1 and p ≤ 0.05) between post PPV group and none PPV control group. The significantly changed metabolites included glutaric acid and pelargonic acid that play key roles in the regulation of oxidative stress and inflammatory responses. Furthermore, we constructed a metabolic regulatory network in each group based on metabolite-metabolite correlations, which reveals key metabolic pathways and regulatory elements including amino acids and lipids metabolisms that are related to cataract progression. CONCLUSIONS: Altogether, this work discovered some potential metabolite biomarkers for post PPV cataract diagnostics, as well as casted some novel insights into the underlying mechanisms of cataract progression after PPV.

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Diabetic mellitus (DM), commonly referred to diabetes, is a worldwide metabolic disorder, which usually causes high morbidity and mortality rates. Especially, DM may result in serious macrovascular problems including cataract. To investigate the underlying molecular mechanism, here we for the first time employed gas chromatography-time-of-flight mass spectrometry (GC-TOF MS) for an untargeted metabolomics study. Totally 263 metabolites were determined in aqueous humor (AH) samples from 30 patients: 15 for the controls and 15 with DM. Both the heat map and principal component analysis (PCA) plot showed a significantly distinct metabolomics profiles between patients with DM and the controls. Moreover, 20 metabolites were determined to be significantly altered (P ≤ 0.05) in DM patients, some of which were associated with oxidative stress. Metabolic pathway analysis of these significantly different metabolites identified ten most relevant pathways in the group of DM patients when compared with the control group. Among them, three pathways including fatty acid biosynthesis, fatty acid metabolism, and linoleic acid metabolism were the three most significantly influenced pathways (P ≤ 0.05), which probably play key roles in the formation of DM and its complication, cataracts. Altogether, this work not only indicated a distinct AH metabolomic profile in association with DM, but presented novel insights into the molecular mechanisms of DM formation, as well as formation of cataracts.

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High myopia is the common eye disorder worldwide, which may contribute to increase the risk of serious disorders including glaucoma and cataract. Although various studies including genomics, transcriptomics, and proteomics have been implicated to identify potential biomarkers (genes or proteins) for predicting high myopia and to reveal the underlying mechanism, the comprehensive metabolomics in relation to high myopia is very limited. In this study, we identified 242 metabolites in aqueous humor (AH) from a set of 40 cataract patients (including 20 with high myopia and 20 for controls), using a non-targeted metabolomic technology, gas chromatography coupled to time-of-flight mass spectrometer (GC/TOF MS). Further statistical analysis showed that 29 metabolites were significantly changed (Variable important for the projection, VIP ≥ 1 and p ≤ 0.05), between those two groups, while only 2 decreased metabolites were included. Moreover, for the first time, metabolite-metabolite correlations for AH were analyzed, which may dissect key regulatory elements or pathways involved in metabolism of amino acids, carbohydrates, and lipids. Accordingly, metabolic network was constructed based on those 29 changed metabolites in patients with high myopia. More than half of the changed metabolites were highly and positively associated, suggesting important roles of pathways involved in the metabolism of these metabolites in relation to high myopia. Altogether, this work not only provided potential biomarkers for the diagnosis and monitoring of high myopia formation, but also provided new insights into the underlying mechanisms.

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BACKGROUND: Cataract is one of the most common eye diseases that can further cause blindness. Discovering susceptibility factors contributing to cataract development is helpful in identifying predisposed patients and improving treatment efficacy. Although proteomics technology has been used in study of protein markers related to eye diseases, few were on studies of cataract development. METHODS: To explore cataract-associated susceptibility factors in aqueous humor (AH), a quantitative proteomics study using the iTRAQ methodology was employed to compare AH protein profiles between high myopia patients & controls, glaucoma surgery patients & controls, and vitrectomy surgery patients & controls, respectively. RESULTS: A total of 445 AH proteins were identified, and 210 proteins were differentially expressed between myopia patients and controls, 262 proteins were differentially expressed between glaucoma surgery patients and controls, and 161 proteins were differentially expressed between vitrectomy surgery patients and controls. Among the 445 identified proteins, 77 were considered to be cataract-associated, and 5 of the 77 proteins were randomly selected and confirmed by ELISA assay. Biological functions of these 77 proteins were annotated by GO/pathways analysis. Additionally, 17 proteins were found to be involved in protein-protein interaction networks, 5 of which were associated with nervous system disease and eye diseases including cataract. CONCLUSIONS: The identified candidate protein biomarkers associated with cataract development may lead to more insights into the underlying mechanisms of cataract disease.

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