Central subfield thickness of diabetic macular edema: Correlation with the aqueous humor proteome.

Purpose: Diabetic macular edema (DME) is a sight-threatening complication of diabetes. Consequently, studying the proteome of DME may provide novel insights into underlying molecular mechanisms. Methods: In this study, aqueous humor samples from eyes with treatment-naïve clinically significant DME (n = 13) and age-matched controls (n = 11) were compared with label-free liquid chromatography-tandem mass spectrometry. Additional aqueous humor samples from eyes with treatment-naïve DME (n = 15) and controls (n = 8) were obtained for validation by enzyme-linked immunosorbent assay (ELISA). Best-corrected visual acuity (BCVA) was evaluated, and the severity of DME was measured as central subfield thickness (CST) employing optical coherence tomography. Control samples were obtained before cataract surgery. Significantly changed proteins were identified using a permutation-based calculation, with a false discovery rate of 0.05. A human donor eye with DME and a control eye were used for immunofluorescence. Results: A total of 101 proteins were differentially expressed in the DME. Regulated proteins were involved in complement activation, glycolysis, extracellular matrix interaction, and cholesterol metabolism. The highest-fold change was observed for the fibrinogen alpha chain (fold change = 17.8). Complement components C2, C5, and C8, fibronectin, and hepatocyte growth factor-like protein were increased in DME and correlated with best-corrected visual acuity (BCVA). Ceruloplasmin and complement component C8 correlated with central subfield thickness (CST). Hemopexin, plasma kallikrein, monocyte differentiation antigen CD14 (CD14), and lipopolysaccharide-binding protein (LBP) were upregulated in the DME. LBP was correlated with vascular endothelial growth factor. The increased level of LBP in DME was confirmed using ELISA. The proteins involved in desmosomal integrity, including desmocollin-1 and desmoglein-1, were downregulated in DME and correlated negatively with CST. Immunofluorescence confirmed the extravasation of fibrinogen at the retinal level in the DME. Conclusion: Elevated levels of pro-inflammatory proteins, including the complement components LBP and CD14, were observed in DME. DME was associated with the loss of basal membrane proteins, compromised desmosomal integrity, and perturbation of glycolysis.

Large-Scale Protein Analysis of Experimental Retinal Artery Occlusion.

Retinal artery occlusion (RAO) is a devastating condition with no effective treatment. The management of RAO could potentially be improved through an in-depth understanding of the molecular alterations in the condition. This study combined advanced proteomic techniques and an experimental model to uncover the retinal large-scale protein profile of RAO. In 13 pigs, RAO was induced with an argon laser and confirmed by fluorescein angiography. Left eyes serving as controls received a sham laser without inducing occlusion. Retinal samples were collected after one, three, or six days and analyzed with liquid chromatography-tandem mass spectrometry. In RAO, 36 proteins were differentially regulated on day one, 86 on day three, and 557 on day six. Upregulated proteins included clusterin, vitronectin, and vimentin, with several proteins increasing over time with a maximum on day six, including clusterin, vimentin, osteopontin, annexin-A, signal transducer, and the activator of transcription 3. On day six, RAO resulted in the upregulation of proteins involved in cellular response to stress, hemostasis, innate immune response, and cytokine signaling. Downregulated proteins were involved in transmission across chemical synapses and visual phototransduction. This study identified the upregulation of multiple inflammatory proteins in RAO and the downregulation of proteins involved in visual pathways.

Meibomian Gland Dysfunction Is Associated with Low Levels of Immunoglobulin Chains and Cystatin-SN.

Meibomian gland dysfunction (MGD) is a highly prevalent condition and the most common cause of evaporative dry eye disease. Studying the proteome of MGD can result in important advances in the management of the condition. Here, we collected tear film samples from treatment naïve patients with MGD (n = 10) and age-matched controls (n = 11) with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography-tandem mass spectrometry. The proteins were considered differentially expressed if p < 0.05. A total of 88 proteins were significantly regulated. The largest change was observed in cystatin-SN, which was downregulated in MGD and correlated negatively with tear meniscus height. The downregulation of cystatin-SN was confirmed with targeted mass spectrometry by single reaction monitoring (SRM). Eighteen immunoglobulin components involved in B cell activation, phagocytosis, and complement activation were downregulated in MGD including Ig alpha-1 chain C region, immunoglobulin J chain, immunoglobulin heavy variable 3-15, and Ig mu chain C region. The changes in cystatin-SN and immunoglobulin chains are likely to result from the inflammatory changes related to tear film evaporation, and future studies may assess their association with the meibum quality.

Laser-Induced Porcine Model of Experimental Retinal Vein Occlusion: An Optimized Reproducible Approach.

Retinal vein occlusion (RVO) is a frequent visually disabling condition. The management of RVO continues to challenge clinicians. Macular edema secondary to RVO is often recurrent, and patients typically require intravitreal injections for several years. Understanding molecular mechanisms in RVO is a key element in improving the treatment of the condition. Studying the molecular mechanisms in RVO at the retinal level is possible using animal models of experimental RVO. Most studies of experimental RVO have been sporadic, using only a few animals per experiment. Here, we report on 10 years of experience of the use of argon laser-induced experimental RVO in 108 porcine eyes from 65 animals, including 65 eyes with experimental branch retinal vein occlusion (BRVO) and 43 eyes with experimental central retinal vein occlusion (CRVO). Reproducibility and methods for evaluating and controlling ischemia in experimental RVO are reviewed. Methods for studying protein changes in RVO are discussed in detail, including proteomic analysis, Western blotting, and immunohistochemistry. Experimental RVO has brought significant insights into molecular changes in RVO. Testing intravitreal interventions in experimental RVO may be a significant step in developing personalized therapeutic approaches for patients with RVO.

Dexamethasone Intravitreal Implant Is Active at the Molecular Level Eight Weeks after Implantation in Experimental Central Retinal Vein Occlusion.

Central retinal vein occlusion (CRVO) is a visually disabling condition resulting from a thrombus in the major outflow vessel of the eye. The inflammatory response in CRVO is effectively treated with a dexamethasone (DEX) intravitreal implant. Uncovering the proteome changes following DEX implant intervention in CRVO may identify key proteins that mediate the beneficial effects of DEX. In six Göttingen minipigs, CRVO was induced in both eyes with an argon laser using a well-established experimental model. The right eyes were treated with a DEX intravitreal implant (Ozurdex, Allergan), while the left control eyes received a sham injection. Eight weeks after DEX intervention, retinal samples were collected and analyzed with tandem mass tag-based mass spectrometry. DEX implant intervention resulted in the upregulation of peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) and ubiquilin-4. Immunohistochemistry showed expression of FKBP5 in the nuclei in all cellular layers of the retina. Cell adhesion molecule 3, tumor necrosis factor receptor superfamily member 16, and trans-1,2-dihydrobenzene-1,2-diol dehydrogenase were downregulated following DEX intervention. The upregulation of the corticosteroid-sensitive protein FKBP5 suggests that the implant remained active at the molecular level after eight weeks of treatment. Future studies may investigate if FKBP5 regulates the efficacy and duration of the DEX implant.

Proteome Analysis of Aflibercept Intervention in Experimental Central Retinal Vein Occlusion.

Aflibercept is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the treatment of macular edema following central retinal vein occlusion (CRVO). Retinal proteome changes following aflibercept intervention in CRVO remain largely unstudied. Studying proteomic changes of aflibercept intervention may generate a better understanding of mechanisms of action and uncover aspects related to the safety profile. In 10 Danish Landrace pigs, CRVO was induced in both eyes with an argon laser. Right eyes were treated with intravitreal aflibercept while left control eyes received isotonic saline water. Retinal samples were collected 15 days after induced CRVO. Proteomic analysis by tandem mass tag-based mass spectrometry identified a total of 21 proteins that were changed in content following aflibercept intervention. In retinas treated with aflibercept, high levels of aflibercept components were reached, including the VEGF receptor-1 and VEGF receptor-2 domains. Fold changes in the additional proteins ranged between 0.70 and 1.19. Aflibercept intervention resulted in a downregulation of pigment epithelium-derived factor (PEDF) (fold change = 0.84) and endoplasmin (fold change = 0.91). The changes were slight and could thereby not be confirmed with less precise immunohistochemistry and Western blotting. Our data suggest that aflibercept had a narrow mechanism of action in the CRVO model. This may be an important observation in cases when macular edema secondary to CRVO is resistant to aflibercept intervention.

Proteomics reveals a set of highly enriched proteins in epiretinal membrane compared with inner limiting membrane.

Few data exist regarding the protein composition of idiopathic epiretinal membrane (iERM). In the present study we compared the proteome of epiretinal membrane of iERM with the proteome of the inner limiting membrane (ILM) of idiopathic macular hole (iMH). Twelve epiretinal membrane samples were obtained from patients with iERM undergoing therapeutic vitrectomy. Twelve ILM samples from patients with iMH were used as controls. Proteomic analysis was conducted with discovery-based label-free quantitative nano-liquid chromatography - tandem mass spectrometry (LFQ nLC-MS/MS). Verification of results was performed with targeted MS using selected reaction monitoring on a different set of samples. Discovery data were searched against the Uniprot Homo sapiens protein database using MaxQuant Software. Identified proteins were filtered with Perseus software. Bioinformatic analysis of the differences in protein expression between epiretinal membrane from iERM and ILM from iMH was performed using STRING. A total of 2,183 different proteins were identified. 357 proteins were found to be present in all samples. The protein profile of iERM was highly different from iMH with 62 proteins found at significantly higher levels in iERM. The proteins upregulated more than 10-fold in iERM were: fibrillin-1, tenascin, prolargin, biglycan, opticin, collagen alpha-1(II) chain, protein-glutamine gamma-glutamyltransferase 2, fibronectin, filamin-A, collagen alpha-2(IX) chain, spectrin alpha chain, transforming growth factor beta induced protein ig-h3, dihydropyrimidinase - related protein 3, endoplasmin and glutamate dehydrogenase 1. Proteins with high level in iERM consisted of proteins that especially localized to the actin cytoskeleton, the extracellular matrix and the mitochondrion. Analysis of all proteins indicated that the disease process in iERM at least in part can be characterized as skin formation with perturbation of nucleotide metabolism. Our study identified proteins that have not earlier been associated with iERM. Fifteen proteins are found at very high concentration, 10-fold or more, and amongst these four proteins, fibrillin-1, tenascin, prolargin and biglycan were found at more than a 100-fold higher content compared to ILM of iMH. These proteins may be potential therapeutic targets. Data are available via ProteomeXchange with identifier PXD014286.

Intravitreal bevacizumab upregulates transthyretin in experimental branch retinal vein occlusion.

Purpose: To identify retinal protein changes that mediate beneficial effects of intravitreal bevacizumab in experimental branch retinal vein occlusion (BRVO). Methods: In six Danish Landrace pigs, BRVO was induced with argon laser in both eyes. After BRVO was induced, the right eye of each animal was given an intravitreal injection of bevacizumab while the left eye was treated with saline water. The retinas were collected 15 days after BRVO, and differentially expressed proteins were analyzed with tandem mass tags-based mass spectrometry. Validation of statistically significantly changed proteins was performed with immunohistochemistry and western blotting. Results: Fluorescein angiography showed no recanalization of the occluded vessels. A total of 4,013 proteins were successfully identified and quantified. Nine proteins were statistically significantly changed following bevacizumab intervention. In experimental BRVO, bevacizumab treatment resulted in upregulation of transthyretin (TTR) and pantothenate kinase 3. Bevacizumab downregulated protocadherin 7, protein FAM192A, and ATP synthase protein 8. Immunohistochemistry revealed that TTR was highly abundant in the choroid following bevacizumab intervention. Conclusions: Bevacizumab intervention in experimental BRVO resulted in an increased level of TTR. This is the second study in which we showed an increased retinal level of TTR following anti-vascular endothelial growth factor (VEGF) intervention in experimental BRVO. We hypothesize that there is an interaction between TTR and VEGF and that bevacizumab may exert a beneficial effect on the retina by upregulating TTR.

Dexamethasone intravitreal implant downregulates PDGFR-α and upregulates caveolin-1 in experimental branch retinal vein occlusion.

A dexamethasone (DEX) intravitreal implant (OZURDEX) provides an effective treatment of inflammation secondary to branch retinal vein occlusion (BRVO). Retinal proteome changes which mediate the beneficial effects of the implant remain poorly understood. To study retinal proteome changes in BRVO following an intervention with a DEX implant this study combined an experimental model of BRVO with proteomic techniques. In eight Danish Landrace pigs experimental BRVO was induced in both eyes using argon laser. After inducing BRVO a DEX implant was injected into the right eye of each animal while the left control eye was given an identical injection without an implant. Fifteen days after BRVO and DEX implant intervention the retinas were excised and analyzed with tandem mass tag based mass spectrometry. A total of 26 significantly changed proteins were identified. DEX intervention reduced the retinal levels of platelet-derived growth factor receptor-α (PDGFR-α) and vascular endothelial growth factor receptor 2 (VEGFR-2). DEX treatment resulted in increased levels of caveolin-1, peptidyl-prolyl cis-trans isomerase FKBP5 and transgelin. Changes in PDGFR-α and caveolin-1 were confirmed with immunohistochemistry. In BRVO treated with the DEX implant a strong reaction for caveolin-1 was observed in the innermost retinal layers. DEX implant intervention may inhibit PDGF signaling by decreasing the retinal level of PDGFR-α while an increased content of caveolin-1 may help maintain the integrity of the blood-retinal barrier.

IL-18 and S100A12 Are Upregulated in Experimental Central Retinal Vein Occlusion.

Retinal vein occlusion (RVO) is a common retinal vascular disease. RVO may be complicated by pronounced ischemia that often leads to severe loss of visual function. The present work aimed at studying the retinal proteome of RVO complicated by ischemia. In six Danish Landrace pigs RVO was induced with argon laser in the right eye of each animal. As four retinal veins were occluded, the RVO best corresponded to a central retinal vein occlusion (CRVO). Left control eyes received a similar laser treatment without inducing occlusion. RVO and retinal ischemia were verified by angiography. The retinas were collected 15 days after RVO for proteomic analysis. RVO resulted in a downregulation of proteins involved in visual perception, including rhodopsin, transducin alpha chain, and peripherin-2. RVO also caused a downregulation of proteins involved in neurotransmitter transport, including glutamate decarboxylase 1 (GAD1), glutamate decarboxylase 2 (GAD2), and complexins 2⁻4. RVO lead to increased contents of proteins involved in inflammation, including interleukin-18 (IL-18), S100A12, and annexin A1 (ANXA1). Immunohistochemistry revealed a general retinal upregulation of IL-18 and ANXA1 while S100A12 was highly abundant in retinal ganglion cells in RVO. IL-18 and S100A12 are likely to be driving forces in the inflammatory response of RVO complicated by ischemia. Our findings also suggest that RVO results in compromised neurotransmission and a downregulation of proteins involved in visual perception.

A Review: Proteomics in Retinal Artery Occlusion, Retinal Vein Occlusion, Diabetic Retinopathy and Acquired Macular Disorders.

Retinal artery occlusion (RAO), retinal vein occlusion (RVO), diabetic retinopathy (DR) and age-related macular degeneration (AMD) are frequent ocular diseases with potentially sight-threatening outcomes. In the present review we discuss major findings of proteomic studies of RAO, RVO, DR and AMD, including an overview of ocular proteome changes associated with anti-vascular endothelial growth factor (VEGF) treatments. Despite the severe outcomes of RAO, the proteome of the disease remains largely unstudied. There is also limited knowledge about the proteome of RVO, but proteomic studies suggest that RVO is associated with remodeling of the extracellular matrix and adhesion processes. Proteomic studies of DR have resulted in the identification of potential therapeutic targets such as carbonic anhydrase-I. Proliferative diabetic retinopathy is the most intensively studied stage of DR. Proteomic studies have established VEGF, pigment epithelium-derived factor (PEDF) and complement components as key factors associated with AMD. The aim of this review is to highlight the major milestones in proteomics in RAO, RVO, DR and AMD. Through large-scale protein analyses, proteomics is bringing new important insights into these complex pathological conditions.

Retinal proteome changes following experimental branch retinal vein occlusion and intervention with ranibizumab.

Animal models of experimental branch retinal vein occlusion (BRVO) provide a unique opportunity to study protein changes directly in retinal tissue. Results from these experimental models suggest that experimental BRVO is associated with an upregulation of extracellular matrix remodeling and adhesion signaling processes. To study whether these processes could be blocked by inhibition of VEGF-A, a porcine model of experimental BRVO was combined with proteomic analyses. In six Danish Landrace pigs experimental BRVO was induced with argon laser in both eyes. After 24 h an injection of 0.05 mL ranibizumab was given in the right eyes of the animals while left eyes received an injection of 0.05 mL 9 mg/mL sodium chloride water. Retinas were dissected three days after BRVO and the retinal samples were analyzed with label-free quantification as well as tandem mass tag based proteomics. In retinas treated with ranibizumab five proteins exhibited statistically significant changes in content with both proteomic techniques. These five proteins, which were all decreased in content, included integrin ß-1, peroxisomal 3-ketoacyl-CoA thiolase, OCIA domain-containing protein 1, calnexin and 40S ribosomal protein S5. As anti-integrin therapies are under development for inhibition of angiogenesis in retinal diseases it is interesting that inhibition of VEGF-A in itself resulted in a small decrease in the content of integrin ß-1. The decreased content of integrin ß-1 indicates that extracellular matrix remodeling and adhesion processes associated with BRVO are at least partly reversed through inhibition of VEGF-A.

Proteins involved in focal adhesion signaling pathways are differentially regulated in experimental branch retinal vein occlusion.

Branch retinal vein occlusion (BRVO) is a common retinal vascular disease, but global protein changes following the condition remain largely unelucidated. To bring new insights into pathological processes and identify potential therapeutic targets, large-scale retinal protein changes following BRVO were studied by combining a porcine model of experimental BRVO with proteomic analysis by label-free liquid chromatography mass spectrometry. Among a total set of 1974 proteins, 52 significantly upregulated proteins and 10 significantly downregulated proteins were identified in retinas with BRVO after 15 days. Significantly upregulated proteins were involved in signaling pathways of focal adhesion via integrin and blood coagulation. Proteins involved in focal adhesion signaling included collagen α-2 chain, laminin subunit ß-2, laminin subunit γ-1, lipocalin-7, nidogen-2, osteopontin, integrin-ß, α-actinin-1, isoform 2 of α-actinin-1, talin-2 and filamin C. The identified proteins indicate that BRVO was associated with extracellular matrix remodeling processes. The present study identified focal adhesion signaling and ECM remodeling as important biological mechanisms to evaluate in the search for signaling pathways that promote neovascularisation and macular edema following BRVO.

Impact of the Valsalva manoeuvre on the choroid: A systematic review with meta-analyses.

A variety of daily activities can intentionally or unintentionally cause the Valsalva manoeuvre, which induces a physiological response of elevated peripheral venous pressure. Studies have speculated that it may ultimately affect the choroidal anatomy. This is particularly important from a clinical point-of-view since patients occasionally hold their breath while undergoing macular optical coherence tomography (OCT). In this study, we systematically reviewed the literature to understand the impact of the Valsalva manoeuvre on the choroid and conducted meta-analyses on the changes induced in the subfoveal choroidal thickness (SFCT) and the choroidal vascularity index (CVI). We searched 12 literature databases for studies in healthy participants undergoing Valsalva manoeuvre with choroidal OCT scans before and during the manoeuvre. Seven studies with a total of 444 eyes of 279 individuals were eligible for the review. The Valsalva manoeuvre led to a statistically significant but numerically small increase in the SFCT of 6.5 µm (95% CI: 1.6-11.4 µm; p = 0.01) and a statistically significant increase in the CVI of 1.48 (95% CI: 1.23-1.73; p = 0.0002). Thus, the Valsalva manoeuvre has a measurable impact on the choroid, and we recommend careful observation of how the patient sits and behaves behind the OCT scanner while scanning in order to allow accurate measurements of the choroid for diagnosis and monitoring.

Proteome Analysis of Bevacizumab Intervention in Experimental Central Retinal Vein Occlusion.

Bevacizumab is a frequently used inhibitor of vascular endothelial growth factor (VEGF) in the management of macular edema in central retinal vein occlusion (CRVO). Studying retinal protein changes in bevacizumab intervention may provide insights into mechanisms of action. In nine Danish Landrace pigs, experimental CRVO was induced in both eyes with argon laser. The right eyes received an intravitreal injection of 0.05 mL bevacizumab (n = 9), while the left control eyes received 0.05 mL saline water (NaCl). Retinal samples were collected 15 days after induced CRVO. Label-free quantification nano-liquid chromatography-tandem mass spectrometry identified 59 proteins that were regulated following bevacizumab treatment. Following bevacizumab intervention, altered levels of bevacizumab components, including the Ig gamma-1 chain C region and the Ig kappa chain C region, were observed. Changes in other significantly regulated proteins ranged between 0.58-1.73, including for the NADH-ubiquinone oxidoreductase chain (fold change = 1.73), protein-transport protein Sec24B (fold change = 1.71), glycerol kinase (fold change = 1.61), guanine-nucleotide-binding protein G(T) subunit-gamma-T1 (fold change = 0.67), and prefoldin subunit 6 (fold change = 0.58). A high retinal concentration of bevacizumab was achieved within 15 days. Changes in the additional proteins were limited, suggesting a narrow mechanism of action.

Macular Edema in Central Retinal Vein Occlusion Correlates With Aqueous Fibrinogen Alpha Chain.

Purpose: The global protein profile of the aqueous humor has been found to correlate with the severity of retinal vascular disease. Studying the aqueous humor in central retinal vein occlusion (CRVO) with proteomic techniques may bring insights to the molecular mechanisms underlying the condition. Methods: Aqueous humor samples from treatment naïve patients with CRVO complicated by macular edema (n = 28) and age-matched controls (n = 20) were analyzed by label-free quantification liquid chromatography - tandem mass spectrometry. Best corrected visual acuity (BCVA) was measured as logMAR, and the severity of macular edema was evaluated as central retinal thickness (CRT) with optical coherence tomography. Control samples were obtained prior to cataract surgery. Significantly changed proteins were identified by a permutation-based calculation with a false discovery rate of 0.05. Results: A total of 177 proteins were differentially expressed in CRVO. Regulated proteins were involved in complement activation, innate immune response, blood coagulation, and cell adhesion. Upregulated proteins that correlated with BCVA and CRT included fibrinogen alpha, beta, and gamma chains, fibronectin, Ig lambda-6 chain C region, Ig alpha-1 chain C region, and complement C7. Downregulated proteins that correlated negatively with BCVA, and CRT, included procollagen C-endopeptidase enhancer 1, clusterin, opticin, reelin, fibrillin-1, and cadherin-2. Monocyte differentiation antigen CD14 and lipopolysaccharide-binding protein were increased in CRVO. Conclusions: Fibrinogen chains, fibronectin, and immunoglobulin components correlated with BCVA and CRT, suggesting a multifactorial response. Protective anti-angiogenic proteins, including procollagen C-endopeptidase enhancer 1, clusterin, and opticin, were downregulated in CRVO and correlated negatively with BCVA and CRT.

Tear film proteome changes following Tobradex® therapy in anterior blepharitis.

PURPOSE: The management of blepharitis continues to challenge clinicians due to the poorly understood aetiology of the condition. We recently identified the family of intracellular plakin proteins as essential driving forces underlying anterior blepharitis. A large-scale protein analysis was used to study if a topical dexamethasone/tobramycin solution could be used to reverse the expression of plakin proteins. METHODS: Tear film samples from treatment naïve patients with anterior blepharitis (n = 15) were collected with Schirmer filtration paper. A subgroup of the patients (n = 10) received treatment with a dexamethasone/tobramycin 1 + 3 mg/mL ophthalmic suspension (Tobradex® ) for 3 weeks and collection of tear film samples was repeated. The samples were analysed with label-free quantification nano liquid chromatography-tandem mass spectrometry requiring quantification in at least 70% of the samples in each group. Proteins were considered differentially expressed if p < 0.05. RESULTS: Following Tobradex® intervention, 27 proteins were upregulated while 61 proteins were downregulated. Regulated proteins after Tobradex® treatment were involved in intermediate filament cytoskeleton organization including downregulation of the plakin proteins envoplakin, epiplakin and periplakin. Plectin, a protein of the plakin family, remained unchanged after Tobradex® therapy. Tobradex® treatment resulted in the regulation of proteins involved in translation including a cluster of downregulated ribosomal proteins. Tobradex® intervention was associated with the regulation of proteins involved in fructose metabolism and glycolytic processes including fructose-1.6-bisphosphatase 1, fructose-bisphosphate aldolases A and B, pyruvate kinase PKM and transketolase. Ig lambda chain V-I region, prominin-1, and protein Niban were upregulated after Tobradex® treatment. CONCLUSIONS: Tobradex treatment reversed the expression of plakin proteins in anterior blepharitis. Topical solutions which inhibit the expression of plakin proteins may have the potential to restore the ocular surface integrity in anterior blepharitis and should be explored further.

Anterior blepharitis is associated with elevated plectin levels consistent with a pronounced intracellular response.

PURPOSE: Anterior blepharitis is a frequent ocular condition which may result in severe ocular surface disease. In this study, advanced proteome analysis was performed to elucidate biological mechanisms underlying anterior blepharitis. METHODS: All patients underwent full ophthalmological examination including Ocular Surface Disease Index score (OSDI). Measurement of non-invasive break-up time (NBUT), Oxford score, and meibography were performed. Tear film samples from treatment naïve patients with anterior blepharitis (n = 15) and age-matched controls (n = 11) were collected with Schirmer filtration paper. The samples were analyzed with label-free quantification nano liquid chromatography - tandem mass spectrometry (LFQ nLC-MS/MS). Significantly regulated proteins were identified with a permutation-based calculation with a false discovery rate at 0.05. RESULTS: Among the 927 proteins detected, a total of 162 proteins were significantly changed. Regulated proteins were involved in cytoplasmic translation, positive regulation of B cell activation, complement activation and phagocytosis. High levels of plakin proteins, a group of proteins involved in cytoskeleton organization, were observed in anterior blepharitis, including plectin, desmoplakin, envoplakin, epiplakin, periplakin, and vimentin. The upregulation of plectin was confirmed with single reaction monitoring. Patients with anterior blepharitis had lower levels of immunoglobulin chains, VEGF coregulated chemokine 1 (CXCL17), and platelet-derived growth factor C. CONCLUSIONS: Anterior blepharitis was associated with a high level of plectin indicating a pronounced intracellular response with cytoskeletal reorganization. Our data suggest a lack of immunoglobulin chains and CXCL17 in anterior blepharitis with potential alterations in the ocular surface immune response.

Proteomic analysis of vitreous humour of eyes with diabetic macular oedema: a systematic review.

The pathophysiology of diabetic macular oedema (DME) remains poorly understood. Proteomic analysis of the vitreous using mass spectrometry (MS) can potentially identify proteins of pathophysiological importance. In this systematic review, we summarize the available evidence on protein changes in DME detected by MS. We systematically searched 13 literature databases on 19 September 2021. Eligible studies were defined as those using samples from human eyes with DME analysed with MS. Two authors assessed the studies for eligibility, extracted data and evaluated risk of bias independently. Six eligible studies were identified. All were designed in a cross-sectional fashion comparing results to either a non-diabetic control group or a control group without DME. A total of 62 eyes from 60 patients contributed as study group and 48 eyes from 48 patients served as control group. Proteomic analyses revealed significant differences in the vitreous protein levels in patients with DME when compared with controls. Three studies or more identified increased contents of apolipoprotein A-I, apolipoprotein A-II, apolipoprotein A-IV, apolipoprotein C-III, gelsolin, pigment epithelium-derived factor, serum albumin, transthyretin, vitamin D-binding protein in DME. Two studies found increased levels of complement factors B and C3. Protein changes reproduced across the studies suggested that DME was associated with retinal lipid accumulation, angiogenesis, retinal protective mechanisms, inflammation and complement activation. Proteome studies support the multifactorial pathogenesis of DME as proteins with highly different biological functions are regulated in DME. An important number of proteins differ, provide pathophysiological insight and suggest the direction for future research.

Aflibercept Intervention in Experimental Branch Retinal Vein Occlusion Results in Upregulation of DnaJ Homolog Subfamily C Member 17.

Aflibercept is an inhibitor of vascular endothelial growth factor (VEGF) used to treat macular edema following branch retinal vein occlusion (BRVO). Despite well-documented efficacy, there is limited knowledge about proteome changes following aflibercept intervention in BRVO. Proteome changes may provide insights into mechanisms of action as well as aspects related to safety profile. In seven Danish Landrace pigs, BRVO was induced with a well-established experimental model of argon laser-induced BRVO. BRVO was induced in both eyes. Three days after the induced BRVO, aflibercept was injected intravitreally in the right eyes, while the left eyes received intravitreal isotonic saline water. Retinas were collected 15 days after the induced BRVO and analyzed with label-free quantification liquid chromatography tandem mass spectrometry (LFQ LC-MS/MS). Fourteen proteins were changed in expression following aflibercept intervention in the BRVO model. LFQ LC-MS/MS identified an upregulation of DnaJ homolog subfamily C member 17 (DNAJC17) (fold change = 6.19) and a modest downregulation of isoform 2 of the protein encoded by N-myc downstream regulated gene 2 (NDRG2) (fold change = 0.40). NDRG2 was unchanged by Western blotting. In the additional significantly regulated proteins, only discrete changes were observed (fold changes 0.52-1.59). Our study is the first to report an association between aflibercept intervention and the heat shock protein DNAJC17. Our results indicate that the role of heat shock proteins in the treatment of BRVO should be further explored.