Vitreous humor biomarkers in Age-related Macular Degeneration

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Refinement of two-dimensional electrophoresis for vitreous proteome profiling using an artificial neural network

Publication . Santos, Fátima Raquel Milhano dos; Albuquerque, Tânia Gonçalves; Gaspar, Leonor Isabel Mesquita ; Dias, Joao ML; Sousa, João Paulo Castro De; Paradela, Alberto; Tomaz, C. T.; Passarinha, LA

Despite technological advances, two-dimensional electrophoresis (2DE) of biological fluids, such as vitreous, remains a major challenge. In this study, artificial neural network was applied to optimize the recovery of vitreous proteins and its detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer), temperature, and total voltage. The highest protein recovery (94.9% ± 4.5) was achieved using 4% (w/v) CHAPS, 0.1% (v/v) Genapol, 20 mM DTT, and 2% (v/v) IPG buffer. Two iterations were required to achieve an optimized response (580 spots) using 4% (w/v) CHAPS, 0.2% (v/v) Genapol, 60 mM DTT, and 0.5% (v/v) IPG buffer at 35 kVh and 25 °C, representing a 2.4-fold improvement over the standard initial conditions of the experimental design. The analysis of depleted vitreous using the optimized protocol resulted in an additional 1.3-fold increment in protein detection over the optimal output, with an average of 761 spots detected in vitreous from different vitreoretinopathies. Our results clearly indicate the importance of combining the appropriate amount of solubilizing agents with a suitable control of the temperature and voltage to obtain high-quality gels. The high-throughput of this model provides an effective starting point for the optimization of 2DE protocols. This experimental design can be adapted to other types of matrices. Graphical abstract.

2019Journal article

Open access

Proteome analysis of vitreous humor in retinal detachment using two different flow-charts for protein fractionation

Publication . Gaspar, Leonor Isabel Mesquita ; Santos, F.M.; Albuquerque, Tânia; Sousa, João P. Castro de; Passarinha, L A; Tomaz, Cândida T.

The deeper understanding of retinal detachment (RD) pathogenesis may improve the visual outcome after surgery. Given the main role of the vitreous in retinal eye diseases, two strategies were explored to identify its proteome in RD. Fractionation techniques such as anion exchange chromatography (IEX) and SDS-PAGE combined with MALDI-TOF/TOF analysis allowed to identify 127 proteins in vitreous of RD patients. From these proteins, 19 were identified using only the IEX fractionation strategy, and 117 using a bidimensional (IEX and SDS-PAGE) fractionation. Of these proteins, 68 had not yet been found in other vitreous proteomic studies. The fractionation with IEX and SDS-PAGE largely improved the number of identified proteins proving that it is crucial to combine several methodologies to cover vitreous proteome.

2017Journal article

Open access

Human vitreous proteome in vitreoretinal diseases

Publication . Santos, Fátima Raquel Milhano dos; Passarinha, Luís António Paulino; Tomaz, Cândida Ascensão Teixeira; Elizalde, Alberto Paradela

Vitreous, also termed vitreous body or vitreous humor, is a transparent fluid that fills the posterior cavity of the eye, surrounded by the neurosensorial retina, and lens. For a long time, the vitreous was not appreciated for its role in health and disease, and its function was thought to be merely structural. Nevertheless, the analysis of vitreous proteome has gained a growing interest in recent years. These studies proved that vitreous is highly complex and biologically more active than initially thought. As a matter of fact, changes in vitreous proteome reflect the physiological and pathological state of the eye, and, therefore, it is the ideal matrix for studying vitreoretinal diseases. Although the search for sensitive and specific vitreous biomarkers in ocular disease has not been successful so far, the analysis of vitreous proteome has been seen to be promising in elucidating some of the pathological mechanisms underlying vitreoretinal diseases. In this project, several gel-based and gel-free techniques were developed and applied for the analysis of vitreous proteome in retinal detachment (RD), diabetic retinopathy (DR), and age-related macular degeneration (AMD). Since the early proteomic studies, two-dimensional gel electrophoresis (2DE) has been the preferential method for the separation and identification of vitreous proteins. If combined with more sensitive detection techniques, refined gel image processing, and proper sample preparation, 2DE is still a valuable tool for high-resolution separation and routine analysis of proteoforms. Despite technological advances, 2DE of biological fluids, such as vitreous, remains a major challenge. Therefore, in the first part of this work, an artificial neural network was applied to optimize the recovery of vitreous proteins and their detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer) and physical parameters (temperature and total voltage). Using a mathematical model created by ANN, both the protein recovery and the number of spots detected in 2DE gels were significantly improved. The optimized response (580 spots) represents a 2.4-fold improvement over the standard conditions applied for vitreous analysis by 2DE. Our results clearly indicate that it is crucial to combine appropriate amounts of solubilizing agents to improve the extraction, solubilization, and detection of vitreous proteins, and to obtain well-resolved gels. Beyond that, our results also indicate that physical parameters have a significant influence on isoelectric focusing and, thereby, should be adjusted and monitored. When working with biological fluids, it is also important to reduce their complexity before 2DE analysis to facilitate the detection of low-abundant proteins, and to increase the detected in the gel increased 1.3-fold over the optimal output refined by the ANN model, with an average of 761 spots detected in vitreous from different vitreoretinopathies, including rhegmatogenous retinal detachment (RRD) and the proliferative diabetic retinopathy (PDR). In the second task of this Ph.D. project, the performance of gel-free proteomic techniques combined with stable-isotope labeling was tested for the analysis of vitreous samples in RRD. RRD is a potentially blinding condition characterized by a physical separation between the neurosensory retina and retinal pigment epithelium. Vitreous has a central role in the onset of RRD, which may be triggered by vitreous liquefaction. It reduces the vitreoretinal adhesion, leading to the accumulation of vitreous fluid in subretinal space, and, subsequently, to the physical separation between the neuronal retina and the retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur in the eye, providing additional information about the molecular mechanisms underlying RRD pathogenesis. In this study, the proteome of vitreous collected from patients with RRD was analyzed and compared to epimacular membranes (MEM) using iTRAQ reagents (Isobaric tags for relative and absolute quantitation) combined with analysis by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS). Using this strategy, we identified 6078 peptides corresponding to 1030 proteins, with 2613 out of these corresponded to unique peptides. Overall, 150 proteins were found differentially expressed in the RRD vitreous, including 96 overexpressed and 54 underexpressed. Among overexpressed proteins, several glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), and protease inhibitors (metalloproteinase inhibitor 1, plasminogen activator inhibitor 1) are regulated by hypoxia-inducible factor-1 (HIF-1), which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the overexpression of proteins of carbon metabolism or molecular chaperones or, among others, suggests that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses, e.g. the activation of the HIF-1 signaling pathway. In the third task, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR and dry AMD. DR and AMD are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Although Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and PDR, no treatment options exist for dry AMD. Therefore, quantitative proteomics can help to recognize the biological mechanisms underlying these pathologies and to find new potential biomarkers and/or pharmaceutical targets. For this purpose, the proteome of vitreous collected from patients with PDR (n=4) were compared to dry AMD (n=4) and epiretinal membranes (ERM) (n=4) using an LFQ method that combines a fractionation by short SDS–polyacrylamide gel electrophoresis and analysis by LC-MS/MS. A total of 680 proteins were identified, of which 586 were identified using the software search engine MASCOT and 580 using MaxQuant. Subsequently, post hoc tests, hierarchical clustering, and multiple t-tests were performed for differentiating the three disease groups in terms of protein expression based on their intensity. Post hoc tests revealed that 96 proteins are capable of differentiating among the different groups, whereas 118 proteins (17 up- and 101 down-regulated) were found differentially regulated in PDR compared to ERM and 95 proteins (10 up- and 85 down-regulated) in PDR compared to dry AMD. Functional enrichment analysis indicates that these underexpressed proteins are correlated to pathways/ biological processes, such as extracellular matrix (ECM) disassembly and organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development. In turn, mediators of complement and coagulation cascades and acute-phase inflammatory responses were found enriched in PDR vitreous, reinforcing the role of these pathways in its pathogenesis of PDR. For last, some potential biomarkers were selected according to iTRAQ and LFQ experiments and validated by multiple reaction monitoring (MRM) in a larger set of vitreous samples. Therefore, we develop a scheduled MRM method for the analysis of 35 proteins in vitreous samples collected from patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and RRD (with and without proliferative vitreoretinopathy) (n=13). Of these, 26 proteins have been shown the potential to differentiate between different disease groups according to MRM results and respective receiver operating characteristic curves. Complement and coagulation components (C6, C8B, prothrombin), acute-phase proteins (alpha-1-antichymotrypsin), adhesion molecules (galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2) stand out as the more efficient biomarkers to discriminate among the different disease groups. In conclusion, several gel-based and gel-free strategies were develop ed and implemented for the preparation and analysis of the proteome of vitreous in different vitreoretinal diseases. Concerning the gel-based method, a mathematical model created by ANN provided an effective 2DE protocol for high-resolution analysis of vitreous proteome, which can be advantageous for analysis of specific proteoforms, including different isoforms and post-translational modified proteins. On the other hand, high-throughput methods, such as iTRAQ and LFQ, provided a more in-depth analysis of vitreous proteome. In these techniques, we identified 1030 proteins by iTRAQ and 680 by LFQ, some of them have not been previously identified. Even more relevant is the fact that vitreous analysis using these techniques provided new insights on the pathogenesis of RRD, PDR, and AMD. Beyond that, they provided fundamental information regarding potential biomarkers, which enabled the successful validation of 26 proteins by MRM. Nevertheless, it must be taken into consideration that vitreous biomarkers cannot be used for regular diagnosis due to invasive sampling. However, they can be candidates for new pharmaceutical targets and, when the samples are obtained as part of the clinical routine, be used for the prognosis of the patient's disease evolution and/or to predict the proper response to treatment.

2020-12-22Doctoral thesis

Open access

VEGF-B Levels in the Vitreous of Diabetic and Non-Diabetic Patients with Ocular Diseases and Its Correlation with Structural Parameters

Publication . Mesquita, Joana; Sousa, João Paulo Castro De; Pereira, Sara Vaz; Neves, Arminda; Ratado, Paulo; Santos, F.M.; Passarinha, LA; Tomaz, C. T.

Vascular endothelial growth factor B (VEGF-B) is one of the enigmatic members of the VEGF family. The knowledge gap about VEGF-B expression and how its levels are altered in diabetic eyes were the focus of this investigation that was addressed by comparing and correlating vitreous VEGF-B between diabetic and non-diabetic patients. VEGF-B levels were measured by enzyme-linked immunosorbent assay in vitreous samples (n = 33) from diabetic (n = 25) and non-diabetic (n = 8) patients. Results were compared between groups. Optical coherence tomography from diabetic patients was evaluated for central retinal thickness (CRT) and macular volume (MV). Mean vitreous VEGF-B concentration was higher in diabetic (18.82 ± 1.44 pg/mL ) vs. non-diabetic patients (17.90 ± 0.32 pg/mL) (p = 0.006), and in proliferative diabetic retinopathy (PDR) (19.03 ± 1.52 pg/mL) vs. non-PDR (NPDR) patients (18.18 ±0.96 pg/mL) (p = 0.025). In diabetic retinopathy (DR) patients, correlation between VEGF-B and CRT (μm) was positive and moderate: rs = 0.441 (p ≤ 0.05) and the correlation between VEGF-B and MV (mm³) was positive and robust: rs = 0.716 (p ≤ 0.01). VEGF-B levels are overexpressed in vitreous of diabetic patients, and the levels are higher in developed stages of DR. Correlation results show that CRT and MV increase with increased levels of VEGF-B. Targeting VEGF-B inhibition may have therapeutic beneficial implications.

2017Journal article

Open access

iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

Publication . Santos, F.M.; Gaspar, Leonor Isabel Mesquita ; Ciordia, Sergio; Rocha, Ana; Sousa, João Paulo Castro De; Paradela, Alberto; Passarinha, LA; Tomaz, C. T.

Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.

2018Journal article

Open access