Multi-Omics Approach for Studying Tears in Treatment-Naïve Glaucoma Patients

Abstract

Primary open-angle glaucoma (POAG) represents the leading cause of irreversible blindness worldwide and is a multifactorial, chronic neurodegenerative disease characterized by retinal ganglion cell and visual field loss. There are many factors that are associated with the risk of developing POAG, with increased intraocular pressure being one of the most prevalent. Due to the asymptomatic nature of the disease, the diagnosis of POAG often occurs too late, which necessitates development of new effective screening strategies for early diagnosis of the disease. However, this task still remains unfulfilled. In order to provide further insights into the pathophysiology of POAG, we applied a targeted metabolomics strategy based on a high-throughput screening method for the determination of tear amino acids, free carnitine, acylcarnitines, succinylacetone, nucleosides, and lysophospholipids in naïve to therapy glaucomatous patients and normal controls. Also, we conducted proteomic analyses of the whole lacrimal fluid and purified extracellular vesicles obtained from POAG patients and healthy subjects. This multi-omics approach allowed us to conclude that POAG patients had lower levels of certain tear amino acids and lysophospholipids compared with controls. These targeted analyses also highlighted the low amount of acetylcarnitine (C2) in POAG patient which correlated well with proteomics data. Moreover, POAG tear proteins seemed to derive from extracellular vesicles, which carried a specific pro-inflammatory protein cargo.

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…Last but not least, flow cytometry (FC), commonly used for the analysis of cells, is being actively developed for EV analysis [146,147] and is adopted by an increasing number of research groups, mainly to study the larger EVs [148,149]. Direct FC analysis using tracers that stain the whole vital EV compartment, such as lipophilic carbocyanine dyes, combined with phalloidin, that selectively binds to F-actin, accurately discriminates EVs from artifacts [42][43][44]150]. Standardized FC has a high potential for the detection of EVs in body fluids and, when combined with specific antibodies, concurrently allows EV immunophenotypic characterization [42,44,84,131,151,152] ( Figure 2).…”

“…Due to their substantial stability, EVs circulate systemically and have been detected in basically all body fluids, including blood, urine, cerebrospinal fluid, saliva, milk, and tears [16,[42][43][44][45][46][47][48]. Moreover, there is clear indication that EVs can cross multiple biological barriers, as demonstrated by the finding of glial/neuronal EVs in the cerebrospinal fluid, blood, tears, and urine [44].…”

“…Extracellular Vesicles in tears may carry relevant information related to the status of eyes and the other bordering structures. It has been recently demonstrated that EVs in tears of primary open-angle glaucoma patients (POAG) carry a specific pro-inflammatory protein cargo, possibly participating to POAG pathophysiology [43]. Interestingly, a number of evidences suggested that tear composition may reflect the health of the CNS [229].…”

Extracellular Vesicles in Feto–Maternal Crosstalk and Pregnancy Disorders

“…Combining such a detection methodology with the use of a tracer for the staining of the whole EV circulating population may allow the placement of the trigger threshold on a fluorescent channel, and therefore the identification of the smallest EVs become possible [45]. It also has been purposed to stain EV samples with phalloidin, in order to exclude damaged EVs from the flow cytometry analysis, allowing a more accurate discrimination of intact vesicles [6,19,20,46]. Finally, the use of the Rosetta Calibration system, which permits the identification of the EV compartment on the basis of the particle size values, combined to a flow cytometry standardized procedure, significantly enhance flow cytometry sensitivity [47,48].…”

“…In any case, EVs are released into the extracellular milieu and finally they could reach the blood circulation. EVs were identified in many different body fluids, such as cerebrospinal fluid, tears, saliva, urine, milk, and peripheral blood [1,6,8,[19][20][21][22][23]. For all of these reasons, EVs were pointed out as a reliable source of biomarkers.…”