Proteomic Analysis Of Aqueous Humor Research Articles

Comprehensive Proteomic Profiling of Aqueous Humor in Idiopathic Uveitis and Vogt-Koyanagi-Harada Syndrome.

Idiopathic uveitis (IU) and Vogt-Koyanagi-Harada (VKH) syndrome are common types of uveitis. However, the exact pathological mechanisms of IU and VKH remain unclear. Proteomic analysis of aqueous humor (AH), the most easily accessible intraocular fluid and a key site of uveitis development, may reveal potential biomarkers and elucidate uveitis pathogenesis. In this study, 44 AH samples, including 12 IU cases, 16 VKH cases, and 16 controls, were subjected to label-free quantitative proteomic analysis. We identified 557 proteins from a comprehensive spectral library of 634 proteins across all samples. The AH proteomic profiles of the IU and VKH groups were different from those of the control group. Differential analysis revealed a shared pattern of extracellular matrix disruption and downregulation of retinal cellular proteins in the IU and VKH groups. Enrichment analysis revealed a protein composition indicative of inflammation in the AH of the IU and VKH groups but not in that of the control group. In the IU and VKH groups, innate immunity played an important role, as indicated by complement cascade activation and overexpression of innate immune cell markers. Extreme gradient boosting (XGBoost), an efficient and robust machine learning algorithm, was subsequently used to screen potential biomarkers for classifying the IU, VKH, and control groups. Transferrin and complement factor B were deemed the most important and represent a promising biomarker panel. These proteins were validated by high-resolution multiple reaction monitoring (HR-MRM) in an independent validation cohort. A classification decision tree was subsequently built for the diagnosis. Our findings further the understanding of the underlying molecular mechanisms in IU and VKH and facilitate the development of potential therapeutic and diagnostic strategies.

Comparative Proteomic Analysis of Aqueous Humor Reveals Biochemical Disparities in the Eyes of High Myopic Patients.

Myopia accounts for a significant proportion of visual lesions worldwide and has the potential to progress toward pathological myopia. This study aims to reveal the difference in protein content in aqueous humor between high myopic and nonhigh myopic patients, as well as better understand the dysregulation of proteins in myopic eyes. Aqueous humor was collected for liquid chromatograph mass spectrometer (LC/MS) analysis from 30 individual eyes that underwent phacoemulsification and intraocular lens (IOL) implantation. Results showed that a total of 190 differentially expressed proteins were identified, which revealed their involvement in cell metabolism, immune and inflammatory response, and system and anatomical structure. Further analysis focused on 15 intensively interacted hub proteins, encompassing functions related to complement cascades, lipoprotein metabolism, and fibrin biological function. Subsequent validations demonstrated elevated levels of APOE (apolipoprotein E), C3 (complement 3), and AHSG (α-2-HS-glycoprotein) in the high myopia group (31 eyes of cataracts and 45 eyes of high myopia with cataracts). AHSG had a significant positive correlation with axial length in high myopic patients, with good efficacy in distinguishing between myopic and nonmyopic groups. AHSG may be a potential indicator of the pathological severity and participator in the pathological progress of high myopia. This study depicted differential expression characteristics of aqueous humor in patients with high myopia and provided optional information for further experimental research on exploring the molecular mechanisms and potential therapeutic targets for high myopia. Data are available via ProteomeXchange with the identifier PXD047584.

Proteomic analysis of aqueous humor reveals novel regulators of diabetic macular edema

Diabetic macular edema (DME) is the most common cause of blindness in patients with diabetic retinopathy. To investigate the proteomic profiles of the aqueous humor (AH) of individuals with diabetic macular edema (DME), AH samples were collected from patients with non-diabetes mellitus (NDM), DM, nonproliferative diabetic retinopathy (NPDR), and DME. We performed comparative proteomic analyses using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatics analyses. We identified 425 proteins in these AH samples, of which 113 showed changes in expression in DME compared with NDM, 95 showed changes in expression in DME vs. DM, and 84 showed changes in expression in DME compared with NPDR. The bioinformatics analysis suggested that DME is closely associated with platelet degranulation, oxidative stress-related pathway, and vascular-related pathways. Upregulation of haptoglobin (HP) and downregulation of fibrillin 1 (FBN1) were validated by ELISA. Receiver operating characteristic (ROC) analysis showed that HP and FBN1 could distinguish DME from NPDR with areas under the curve of 0.987 (p = 0.00608) and 0.791 (p = 0.00629), respectively. The findings provide potential clues for further analysis of the molecular mechanisms and the development of new treatments for DME. HP and FBN1 may be potential key proteins and therapeutic targets in human DME. The proteomics dataset generated has been deposited to the ProteomeXchange/iProX Consortium with Identifier: PXD033404/IPX0004353001.

Proteomic analysis of aqueous humor in canine primary angle-closure glaucoma in American Cocker Spaniel dogs.

To analyze proteomic profiles of the aqueous humor (AH) of canines with primary angle-closure glaucoma (PACG) and identify associated protein alterations. Six American Cocker Spaniels with PACG and six American Cocker Spaniels without ocular diseases. Aqueous humor samples were collected from six American Cocker Spaniels with PACG at Seoul National University, VMTH, and six healthy Cocker Spaniels without ocular disease at Irion Animal Hospital. For the PACG group, AH samples were obtained by anterior chamber paracentesis prior to glaucoma treatment. For the AH control group, AH samples were collected from patients anesthetized for other reasons. Total AH protein concentration was determined by the bicinchoninic acid (BCA) assay. AH protein samples were quantified by liquid chromatography-mass spectrometry (LC-MS/MS). Raw MS spectra were processed using MaxQuant software 30, and the Gene Ontology (GO) enrichment analysis was performed using ClueGO. The AH protein concentration in the PACG group (10.49±17.98µg/µl) was significantly higher than that of the control group (0.45±0.11µg/µl; p<.05). A total of 758 proteins were identified in the AH. Several proteins both significantly increased (n=69) and decreased (n=252) in the PACG group compared to those in the control group. GO enrichment analysis showed that the "response to wounding," "negative regulation of endopeptidase activity," and "cell growth" pathways were the most enriched terms in the PACG group compared to the control group. The top 5 proteins that were significantly increased in the AH of the PACG group were secreted phosphoprotein 1 (SPP1), peptidoglycan recognition proteins 2 (PGLYRP2), tyrosine 3-monooxygenase (YWHAE), maltase-glucoamylase (MGAM), and vimentin (VIM). Gene Ontology enrichment analysis using the proteomic data showed that proteins and pathways related to inflammation were significantly upregulated in the various stage of PACG. Proteomic analysis of the AH from the PACG may provide valuable insights into PACG pathogenesis.

Proteomic analysis of aqueous humor in patients with pathologic myopia

Complications from pathologic myopia (PM) are a major cause of visual impairment and blindness. However, an efficient clinical therapeutic strategy for PM is still lacking. The aim of this study was to quantitatively compare the proteomic profiles of aqueous humor between PM and non-PM cataract patients. Twenty aqueous humor samples from each group were analyzed with label-free quantitative proteomic analysis to identify the differentially expressed proteins for function enrichment analyses and protein-protein interaction network construction. Hub protein was validated with ELISA using an independent cohort consisting of 20 samples from each group and its receiver operating characteristic (ROC) curve analysis was conducted. A total of 583 proteins were identified and 101 proteins were found to be differentially expressed, including 63 up-regulated proteins and 38 down-regulated proteins. The bioinformatics analysis suggested that PM is closely associated with immunity and inflammation interactions, and remodeling of extracellular matrix. Apolipoprotein A-I (ApoA1) was enriched as the hub protein of the network with the highest score, degree and centrality. ROC analysis showed that ApoA1 could distinguish PM from controls with an area under the curve of 0.963 (p < 0.001). The findings could provide potential clues for further study on the molecular mechanisms and developing new treatments for PM, especially related to immunity and inflammation interactions. ApoA1 may be a potential key protein and therapeutic target in human PM. SignificanceIt is important and urgent to discover the mechanisms of pathologic myopia (PM) to inhibit its progression. This study applied the quantitative proteomic analysis to study aqueous humor from patients with or without PM, aiming to discover dysregulated proteins related to PM. Our results suggested that those dysregulated proteins are closely associated with immunity and inflammation interactions, and remodeling of extracellular matrix. The findings from this study could provide potential clues for further research on the molecular mechanisms and developing new treatments for PM, especially related to immunity and inflammation. ApoA1 may be a potential key protein and therapeutic target in human PM.

In vivo intraocular biomarkers

Inflammatory, angiogenic, and immune processes have been associated with uveal melanoma (UM). The aim of the present study was to evaluate the presence of some specific aqueous humor (AH) soluble biomarkers in eyes affected by UM. Thirty-five eyes affected by primary UM and 35 control eyes, scheduled for cataract surgery, underwent full ophthalmic examination and AH sampling at time of surgery (brachytherapy or cataract surgery, respectively). AH samples were analyzed by means of ELISA, to detect the concentration of selected cytokines, chemokines, and growth factors. Compared with the control group, higher levels of IL-6 (P = .049), IL-8 (P = .006), RANTES (P = .008), EGF (P = .032), bFGF (P = .016), MIF (P = .007), and MCP (P = .020) were detected in eyes with UM. VEGF concentration between the two groups was statistically borderline (P = .058). Comparison between clinical characteristics and cytokine concentrations showed a positive correlation between tumor thickness and IL-8 (P = .032), and degree of serous retinal detachment and IL-6 (P = .021). UM is characterized by the presence of retinal neuroinflammatory, angiogenic, and immune biomarkers in AH. The proteomic analysis of AH could characterize UM microenvironment, allowing to better understand its pathophysiology.

Proteomic analysis of aqueous humor from cataract patients with retinitis pigmentosa.

A postcataract surgery complication in patients with retinitis pigmentosa (RP) is lens capsular contraction. To identify potential proteins contributing to this phenomenon, high-performance liquid chromatography/mass spectrometry-based proteomic analysis was conducted with aqueous humor samples collected from 11 patients who underwent cataract surgeries, with four patients diagnosed as RP and cataract (RP group) and the other seven with only senile cataract group. The upregulated proteins in the RP group were enriched in wound response, while downregulated proteins were enriched in cell adhesion and lens crystallins. Receptors of two dramatically upregulated proteins tenascin-C (TNC) and serotransferrin were found expressed in human lens epithelial cells (HLEs). TNC can promote primary HLEs proliferation and cell line HLE-B3 migration. This study indicates aqueous humor proteomic analysis serves as an effective way to unveil the pathogenesis of RP complications. TNC is a potential target of stimulating HLEs proliferation in RP concomitant cataract patients that worth further research.

Quantitative proteomic analysis of aqueous humor after rabbit lensectomy reveals differences in coagulation and immunomodulatory proteins.

Compared to adults, children experience increased postoperative scarring and inflammation following intraocular surgery. While the underlying causes of the exaggerated immune response in children are not understood, proteins play key roles in postoperative scarring and wound healing processes. To identify and quantify proteins associated with the robust postoperative immune response, this study applied quantitative proteomics approaches to a juvenile rabbit model of lensectomy with intraocular lens (IOL) insertion. Twenty-six 6-7 week-old New Zealand white rabbits underwent unilateral portions of lensectomy with IOL insertion including: anterior chamber paracentesis, corneal incision with wound suture, lensectomy only, and lensectomy with IOL insertion. Aqueous humor was collected immediately prior and three days after each procedure. Semi-quantitative protein discovery was achieved by label-free quantitation using data dependent and data independent acquisition modes. Based on the discovery results, targeted quantitation by parallel reaction monitoring of 3 proteins of interest, fibrinogen-beta chain, transforming growth factor beta-2, and retinol binding protein 3, was used to confirm the observed quantitative trends. Total protein concentration levels increased with each progressive surgical step of lensectomy with IOL insertion. Proteins related to the complement and coagulation cascades were found to increase in relative abundance, while proteins related to ocular immunosuppression decreased in abundance following surgery. These data provide insights into the postoperative response by providing the first surgical step-wise views of the AH proteome before and after surgery. Overall, this work provides the foundation for future investigations targeting specific proteins for therapeutic interventions aimed at minimizing postoperative complications after pediatric intraocular surgery.

ITRAQ-based proteomics analysis of aqueous humor in patients with dry age-related macular degeneration.

To preliminarily test proteomics in aqueous humor in patients with dry age-related macular degeneration (AMD) by using the proteomic technology. Aqueous humor samples were collected from patients with or without dry AMD, who underwent cataract surgery. The aqueous samples were analyzed with isobaric tags for relative and absolute quantification (iTRAQ) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS) technology. The differential expressed proteins were analyzed with gene ontology (GO) enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interaction (PPI) network analysis. The data were partly validated by ELISA and Western blot. False discovery rate (FDR) was used for statistical analysis. A total of 244 proteins were detected, in which 38 proteins were up-regulated and 51 were down-regulated significantly in patients with dry AMD compared with that in control groups (FDR value <1.0%). Several proteins, e.g., protein S100-A8 (S10A8), dystroglycan (DAG1), Ig alpha-1 chain C region (IGHA1), carbonic anhydrase 3 (CAH3) and alpha-1-acid glycoprotein (A1AG1) were increased more than 5 times of that in control group. The bioinformatics analysis showed that dry AMD is closely associated with inflammation or immune reaction, oxidative stress, blood coagulation and remodeling of extracellular matrix. iTRAQ-based proteomic analysis of aqueous humor demonstrate the differential expressions of proteins between dry AMD and control groups, providing the clues to understand the mechanisms and possible treatments of dry AMD.

Proteomic analysis of aqueous humor in acute primary angle-closure glaucoma

Objective: To analyze the difference among expression of aqueous humor proteins in acute primary angle-closure glaucoma (APACG). Methods: Case-control study. The patients with APACG combined cataract (APACG with cataract group) and patients with cataract (cataract group), who had undertaken surgical treatment at the Tianjin Medical University Eye Hospital from October 2016 to June2017 were collected. Upon receipt of patient's consent, 50 μl of aqueous humor were collected with 1 ml syringe and No.1 needle through the surgical access during the surgery, and then injected into a sterile collection tube to be stored at -80 ℃. Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry. The differential significance test was performed by Maxquant significances A approach. The differential proteins of the two groups were screened and determined with the conditions of P<0.05 and difference multiple>2. The functions and signal pathway of differential proteins in aqueous humor were annotated in biological big data, on the basis gene ontology (GO) and the Kyoto gene and genomic encyclopedia (KEGG) analyses. Results: There were 3 males and 7 females with an average age of (68±6) years in the APACG group. The cataract group included 2 males and 8 females with an average age of (71±8) years. There were no statistical differences in gender ratio and age between the two groups (both P>0.05). A total of 91 differential proteins were detected in this experiment, including 50 up-regulated proteins (annexinA1, vimentin, S100 calcium binding protein A8, interleukin 6, C reactive protein, laminin β2, etc.) and 41 down-regulated (keratin 85, γ-crystallin D, syntaxin-binding protein 5, semaphoring 4B, matrilin 2, cathepsin O, cadherin 4, semaphoring 3B, platelet-derived growth factor D, transforming growth factor β, etc.). On one hand, the functions of differential proteins involved in many aspects. AnnexinA1, CD163, S100 calcium-binding protein A8, C reactive protein, interleukin 6 are involved in the inflammatory reaction, cadherin 4 and laminin β2 regulate cell adhesion, matrilin 2, vimentin and laminin β2 participate in tissue fibrosis; on the other hand, KEGG analysis showed that the differential proteins participate diverse signaling pathways such as phosphatidylinositol-3-kinase-protein kinase B signaling pathway, transformation growth factor β signaling pathway, mitogen activated protein kinase signaling pathway, Toll-like receptor signaling pathway, the nuclear factor κ-light chain enhancer of the activated B cells signaling pathway, focal adhension and extracellular matrix receptor interaction pathway and so on. Conclusions: The expression of annexin A1 is significantly up-regulated in the aqueous humor in APACG, while some other factors such as transformation growth factor β, cadherin-4, and matrilin 2 are down-regulated. The change of proteins in aqueous humor is related with the outbreak of APACG. (Chin J Ophthalmol, 2019, 55: 687-694).

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration

BackgroundTo identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls.MethodsAfter uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups).ResultsIncreased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen.ConclusionsDifferential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Proteomic Analysis of Aqueous Humor from Primary Open Angle Glaucoma Patients on Drug Treatment Revealed Altered Complement Activation Cascade.

Primary open angle glaucoma (POAG) is a complex disease and a leading cause of irreversible blindness, and its underlying pathophysiology remains poorly understood. Proteomic characterization of the protein composition of aqueous humor (AH) may identify prognostic candidate proteins involved in pathogenesis and progression of the disease. To delineate the possible mechanisms that lead to POAG, this study adopted state-of-art mass spectrometric technique and analyzed AH of POAG and their respective controls. In total, more than 1000 proteins were identified with false discovery rate of less than 1%. Numerous proteins of complement cascade, immunoglobulin, neuronal and amyloidogenic proteins, which were part of processes like acute-phase and inflammatory response, humoral immune and acute inflammatory response, regulation of complement activation and protein processing were identified. Proteins of complement system underwent significant changes, which correlate to pathogenic events characterizing POAG, including altered complement cascade, astrocyte activation, neural degeneration, and apoptosis. Further, protein modification such as deamidation of complement subcomponent was noted, particularly in POAG. Proteomic analysis of AH allows a better understanding of the mechanism involved in the pathogenesis of POAG.

Aqueous humor protein dysregulation in primary angle-closure glaucoma.

Primary angle-closure glaucoma (PACG) is associated with increased intraocular pressure, optic nerve damage, and progressive vision loss, but the molecular mechanism that underpins retinal ganglion neuropathy in PACG remains poorly understood. To better understand the pathogenesis of human PACG, we performed the first comprehensive proteomic analysis of aqueous humor (AH) samples from PACG patients and matched control donors to study pathogenic alteration in AH composition in disease. High-resolution, label-free, liquid chromatography-tandem mass spectrometry-based quantitative proteomic analyses were performed in AH samples collected from PACG patients and a matched control cohort of patients with cataracts. The AH proteome comprised of 1363 distinct proteins, of which more than 50% were differentially expressed in PACG (773 total; 501 up-regulated, 272 down-regulated). AH from PACG patients was enriched in atypical collagens and fibronectins, suggesting that the composition of the trabecular matrix is significantly altered in disease. Pathway and cluster analyses revealed that AH protein modulation in PACG is closely associated with biological processes including platelet degranulation, cellular import/export mechanisms, and control of protease activity. In addition, critical mediators of oxygen homeostasis and neuronal function in AH were significantly dysregulated in disease, strongly implicating oxidative stress responses in PACG-associated nerve damage. Altered AH proteome in human PACG indicated oxidative stress in the neuronal damage that preceded vision loss. Identifying key mediators of PACG pathology will yield new prognostic biomarkers and novel targets for future therapeutic interventions.

Moxifloxacin Concentration and Proteomic Analysis of Aqueous Humor in Human Uveitis Associated with Oral Moxifloxacin Therapy.

Purpose:The aim was to report the aqueous humor moxifloxacin concentration and proteome profile of an individual with bilateral uveitis-like syndrome with pigment dispersion.Methods:Multiple reactions monitoring mass spectrometry quantified the aqueous concentration of moxifloxacin in the affected individual. Shotgun proteomic analysis performed via liquid chromatography tandem mass spectrometry (LC-MS/MS) defined the protein profile in the affected individual and unaffected control samples.Results:Moxifloxacin was present at higher than expected levels in aqueous humor 18 days following oral administration. One-third of the proteins were identified by significantly lower spectral counts in the aqueous of the individual with moxifloxacin associated uveitis compared to the unaffected control.Conclusion:Moxifloxacin was detected in aqueous humor 18 days following the completion of oral administration. These results suggest that moxifloxacin toxicity may be responsible for the uveitis-like syndrome with pigment dispersion syndrome induced by moxifloxacin therapy.

Proteomic analysis of aqueous humor in retinoblastoma patients: a focus on intra‐arterial chemotherapy effects

AbstractPurpose Proteomics can be a useful tool to understand how therapies act and affect on the molecular composition of ocular fluids. In this retrospective study we focus on how the aqueous humor (AH) proteic pattern from retinoblastoma (RTB) patients is modified by intra‐arterial(IA)treatment.Methods We enrolled 22 RTB patients(Reese stage VB or ABC group E)undergoing enucleation and 20 normal subjects undergoing cataract surgery(CTR).Five of 22 patients presented with associated secondary glaucoma (SG) whereas 17 had no secondary glaucoma (NSG);8 of 17 NSG received IA Melphalan (CHM). Pools were firstly investigated with Bradford method and SDS‐PAGE and then,analyzed using 2D‐DIGE coupled to mass spectrometer.DIA and BVA were fully automated with 2D DeCyder® and statistical analysis was performed with EDA® module.Significant spots were In‐Gel digested and identified with Maldi‐TOF/TOF.Results According to Bradford method,CTR protein content was not significantly different from CHM.After melphalan treatment, total protein contents decreased about 70% while in respect to all non‐treated patients were significantly different(Tamhane p&lt;0.01).PCA analysis underlines high distance either between CTR pool and SG/NSG pool or CHM and RTB.In particular the analysis of single protein shows that CHM pool is characterized by a high level of Crystallin and pro‐inflammatory proteins and CTR pool is characterized by a high level of Epithelium Growth Factor.Conclusion IA chemotherapy with Melphalan modifies the AH proteic pattern revealing the presence of Crystallin family and pro‐inflammatory proteins. This opens new horizons to further explore local pharmacological effects of new drugs.

Proteomic analysis of aqueous humor from patients with branch retinal vein occlusion-induced macular edema

The mechanisms responsible for macular edema with branch retinal vein occlusion (BRVO) remain to be elucidated. It is known that the expression profile of certain proteins in the aqueous humor (AH) changes in some diseases. Accordingly, determining the expression of these AH proteins may aid in the understanding of their potential role in this pathogenesis. The aim of this study was to identify the possible mechanisms involved in the development of BRVO-induced macular edema. A proteomic analysis of the AH composition in the eyes of patients with BRVO was performed and compared with that in the eyes of patients with cataract (non-BRVO; controls). AH from 6patients with macular edema due to BRVO and 6patients with cataract (non-BRVO) was collected. A proteomic approach which included 2‑dimensional electrophoresis (2‑DE) coupled with massspectrometry(MS) and bioinformatics analysis were used to identify AH proteins with altered expression in patients with macular edema due to BRVO compared with the controls. An enzyme‑linked immunosorbent assay was used to validate the proteomic results. The total protein concentration in the AH of patients with BRVO-induced macular edema was significantly greater than that of the controls. In the patients with BRVO, a total of 56protein spots were significantly altered on the 2D gels. A total of 49protein spots were identified by MS; many of these proteins have been implicated in angiogenesis, oxidative stress and collagen synthesis. In conclusion, the protein composition in AH differed significantly between the patients with BRVO and the controls. The identified proteins may be potential biomarkers for the development of macular edema due to BRVO and may play a role in the mechanisms responsible for it.

Proteomic analysis of aqueous humor in retinoblastoma: Final results

AbstractPurpose Proteomics in ocular fluids is gaining rapidly relevance.Since 2009 we work on the analysis of proteic composition of the aqueous humor (AH) from retinoblastoma (RTB) patients and,in this work,we use the 2D‐DIGE coupled to mass spectrometer to find new RTB markers.Methods We enrolled 22 RTB patients(Reese‐Ellsworth stage V or ABC classification group E)undergoing enucleation and 20 normal subjects undergoing cataract surgery(CTR).Five of 22 patients presented with associated secondary glaucoma whereas 17 had no secondary glaucoma;8 of 17 patients with no secondary glaucoma received chemotherapeutical treatment with melphalan.Pools were analyzed using 2D‐DIGE coupled to mass spectrometer.DIA and BVA were fully automated with 2D DeCyder® and statistical analysis was performed with EDA® module.Significant spots were In‐Gel digested and identified with Maldi‐TOF/TOF.Results PCA analysis of the data underlines high distance between CTR pool and RTB pools.In particular the analysis of single protein shows that all the RTB pools are characterized by a high level of Crystallin and pro‐inflammatory proteins and CTR pool are characterized by a high level of Epithelium Growth Factor.Conclusion This study is the last step for the clarification of AH protein pattern in patients with RTB.Although we learn from literature that the presence of inflammatory processes is widely documented in late stages of pathology,however the presence of crystalline proteins could be either a new marker or an active part of the late stage tumor‐induced panorama in RTB AH samples.Due to this,we think that several molecular biology studies are necessary to better understand involvement of this protein family in RTB pathological pathway.

Proteomic analysis of aqueous humor in retinoblastoma: a preliminary approach

AbstractPurpose Compare the basic protein composition of the aqueous humor from retinoblastoma (RTB) patients with aqueous humor from patients with cataract who served as controls.Methods The study was carried out on 18 hospitalized RTB patients, who had undergone ocular enucleation (Reese‐Ellsworth stage V or ABC classification group E RB), and 10 cataract patients. Five out of 18 RTB patients presented with associated secondary glaucoma and 5 out of 13 with no secondary glaucoma received chemotherapeutical treatment with Melphalan. The total protein concentration and electrophoresis pattern (SDS PAGE) of the aqueous humor was analysed.Results The aqueous humor from RTB patients had significantly higher total protein concentrations than controls (p&lt;0.01); patients with secondary glaucoma presented with the highest total protein concentrations, and they were significantly different from controls (p&lt;0.05), while those treated with Melphalan presented a 70% decreasing in protein concentration and, in respect to all non‐treated patients, were significantly different (p&lt;0.01). Besides, the protein content of controls was not significantly different from treated patients. The SDS PAGE pattern of RTB patients was very different from controls; yet, after chemotherapeutical treatment, patterns were similar.Conclusion This study represents a preliminary step towards a more accurate 2DE pattern, which will be combined with mass spectrometry analysis to clarify the potential role of specific proteins in tumor development and progression; although this results suggest that aqueous humor protein pattern in RTB is characteristic, several aspects of the study are still under investigation.

Proteome Alterations in Primary Open Angle Glaucoma Aqueous Humor

As the only nourishment and scavenging source for most of the anterior and posterior chamber tissues in the eye, the aqueous humor represents one of the target for glaucoma. The aim of this study is to investigate the yet unexplored relationship between aqueous humor protein content and open-angle glaucoma (POAG) pathogenesis. Aqueous humor was collected from 10 POAG patients (cases) and 14 senile cataract patients (controls), matched for age and gender, undergoing surgery for trabeculectomy and cataract, respectively. Protein samples were cyanine-labeled and hybridized with antibody microarrays. Microarray signals were revealed by laser scanner, quantified, and compared by statistical analyses. Total protein amounts were not significantly different in patients versus controls. Conversely, a proteome cluster significantly modified in patients as compared to controls was identified as highly predictive for disease status. Selected proteins underwent dramatic variation, which was correlated to pathogenetic events characterizing POAG, including oxidative damage, mitochondrial damage, neural degeneration, and apoptosis. The results obtained indicate that proteomic analysis of aqueous humor is a new tool for POAG diagnosis in the case of otherwise uncertain disease recognition. Furthermore, this study allows a better understanding of mechanisms involved in the pathogenesis of POAG, the main cause of irreversible blindness worldwide.