ABSTRACT
PURPOSE: To investigate the extracellular matrix and cellular components in lens capsules extracted from patients with dead bag syndrome (DBS) through immunohistochemistry. SETTING: Department of Ophthalmology, Wakayama Medical University School of Medicine, Wakayama, Japan, and Department of Ophthalmology and Visual Sciences, John A. Moran Eye Center, University of Utah, Salt Lake City, Utah. DESIGN: Immunohistochemical experimental study. METHODS: 9 capsular bag specimens from DBS cases, as well as 2 control specimens from late-postoperative in-the-bag intraocular lens dislocation cases related to previous vitrectomy, pseudoexfoliation, and blunt trauma were included. They were processed for histopathology; unstained sections were obtained from each one and analyzed by immunohistochemistry targeting collagen type IV, laminin, vimentin, collagen type I, and fibronectin. RESULTS: Immunohistochemistry in DBS showed lens capsule stained for basement membrane components. The outer part of the anterior capsule that was split from the inner part was more markedly stained for type IV collagen as compared with the posterior part. Faint staining for fibrous posterior capsular opacification (PCO) components, for example, collagen type I and fibronectin, was detected in limited areas, but the major portion of the capsule was free from these components. Small spotty vimentin-positive materials, suggesting the presence of cell debris, were also detected in limited samples. CONCLUSIONS: Small amounts of fibrotic PCO components were detected in capsules extracted from patients with DBS, but their major parts were free from PCO components. Current findings suggest small amounts of lens epithelial cells were present after surgery and secreted fibrous components before undergoing cell death process.
Subject(s)
Collagen Type IV , Collagen Type I , Fibronectins , Lens Capsule, Crystalline , Vimentin , Humans , Collagen Type IV/metabolism , Fibronectins/metabolism , Male , Vimentin/metabolism , Aged , Female , Lens Capsule, Crystalline/pathology , Lens Capsule, Crystalline/metabolism , Middle Aged , Collagen Type I/metabolism , Laminin/metabolism , Lens Subluxation/surgery , Lens Subluxation/diagnosis , Lens Subluxation/metabolism , Aged, 80 and over , Phacoemulsification , Exfoliation Syndrome/metabolism , Capsule Opacification/metabolism , Immunoenzyme Techniques , Syndrome , Lens Implantation, Intraocular , AdultABSTRACT
To explore the role of lncRNA m6A methylation modification in aqueous humour (AH) of patients with pseudoexfoliation glaucoma (PXG). Patients with open-angle PXG under surgery from June 2021 to December 2021 were selected. Age- and gender-matched patients with age-related cataract (ARC) were chosen as control. Patients underwent detailed ophthalmic examinations. 0.05-0.1 ml AH were extracted during surgery for MeRIP-Seq and RNA-Seq. Joint analysis was used to screen lncRNAs with differential m6A methylation modification and expression. Online software tools were used to draw lncRNA-miRNA-mRNA network (ceRNA). Expression of lncRNAs and mRNAs was confirmed using quantitative real-time PCR. A total of 4151 lncRNAs and 4386 associated m6A methylation modified peaks were identified in the PXG group. Similarly, 2490 lncRNAs and 2595 associated m6A methylation modified peaks were detected in the control. Compared to the ARC group, the PXG group had 234 hypermethylated and 402 hypomethylated m6A peaks, with statistically significant differences (| Fold Change (FC) |≥2, p < 0.05). Bioinformatic analysis revealed that these differentially methylated lncRNA enriched in extracellular matrix formation, tight adhesion, TGF- ß signalling pathway, AMPK signalling pathway, and MAPK signalling pathway. Joint analysis identified 10 lncRNAs with differential m6A methylation and expression simultaneously. Among them, the expression of ENST000000485383 and ROCK1 were confirmed downregulated in the PXG group by RT-qPCR. m6A methylation modification may affect the expression of lncRNA and participate in the pathogenesis of PXG through the ceRNA network. ENST000000485383-hsa miR592-ROCK1 May be a potential target pathway for further investigation in PXG m6A methylation.
Subject(s)
Adenosine , Exfoliation Syndrome , RNA, Long Noncoding , Humans , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Female , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Male , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/genetics , Aged , Aqueous Humor/metabolism , Gene Regulatory Networks , rho-Associated Kinases/genetics , rho-Associated Kinases/metabolism , Middle Aged , RNA, Messenger/genetics , RNA, Messenger/metabolism , DNA Methylation , Glaucoma, Open-Angle/genetics , Glaucoma, Open-Angle/metabolismABSTRACT
Pseudoexfoliation syndrome (PEX) is characterized by the deposition of fibrous pseudoexfoliation material (PEXM) in the eye, and secondary glaucoma associated with this syndrome has a faster and more severe clinical course. The incidence of PEX and pseudoexfoliative glaucoma (PEXG) exhibits ethnic clustering; however, few proteomic studies related to PEX and PEXG have been conducted in Asian populations. Therefore, we aimed to conduct proteomic analysis on the aqueous humor (AH) obtained from Uyghur patients with cataracts, those with PEX and cataracts, and those with PEXG and cataracts to better understand the molecular mechanisms of the disease and identify its potential biomarkers. To this end, AH was collected from patients with cataracts (n = 10, control group), PEX with cataracts (n = 10, PEX group), and PEXG with cataracts (n = 10, PEXG group) during phacoemulsification. Label-free quantitative proteomic techniques combined with bioinformatics were used to identify and analyze differentially expressed proteins (DEPs) in the AH of PEX and PEXG groups. Then, independent AH samples (n = 12, each group) were collected to validate DEPs by enzyme-linked immunosorbent assay (ELISA). The PEX group exhibited 25 DEPs, while the PEXG group showed 44 DEPs, both compared to the control group. Subsequently, we found three newly identified proteins in both PEX and PEXG groups, wherein FRAS1-related extracellular matrix protein 2 (FREM2) and osteoclast-associated receptor (OSCAR) exhibited downregulation, whereas coagulation Factor IX (F9) displayed upregulation. Bioinformatics analysis suggested that extracellular matrix interactions, abnormal blood-derived proteins, and lysosomes were mainly involved in the process of PEX and PEXG, and the PPI network further revealed F9 may serve as a potential biomarker for both PEX and PEXG. In conclusion, this study provides new information for understanding the proteomics of AH in PEX and PEXG.
Subject(s)
Aqueous Humor , Exfoliation Syndrome , Eye Proteins , Proteomics , Humans , Exfoliation Syndrome/metabolism , Aqueous Humor/metabolism , Proteomics/methods , Male , Female , Aged , Eye Proteins/metabolism , China/epidemiology , Glaucoma, Open-Angle/metabolism , Middle Aged , Biomarkers/metabolism , Enzyme-Linked Immunosorbent Assay , Cataract/metabolism , Intraocular Pressure/physiologyABSTRACT
Pseudoexfoliation is a complex, progressive, and systemic age-related disorder. The early stage of deposition of extracellular fibrillar material on ocular and extraocular tissues is termed as pseudoexfoliation syndrome (PEXS). The severe advanced stage is known as pseudoexfoliation glaucoma (PEXG), which involves increased intraocular pressure and optic nerve damage. Through genome-wide association and candidate gene studies, PEX has been associated with numerous genetic risk variants in various gene loci. However, the genetic basis of the disease fails to explain certain features of PEX pathology, such as the progressive nature of the disease, asymmetric ocular manifestation, age-related onset, and only a subset of PEXS individuals developing PEXG. Increasing evidence shows an interplay of genetic and epigenetic factors in the pathology of complex, multifactorial diseases. In this review, we have discussed the genetic basis of the disease and the emerging contribution of epigenetic regulations in PEX pathogenesis, focusing on DNA methylation and non-coding RNAs. Aberrant methylation patterns, histone modifications, and post-transcriptional regulation by microRNAs lead to aberrant gene expression changes. We have reviewed these aberrant epigenetic changes in PEX pathology and their effect on molecular pathways associated with PEX. We have further discussed some possible genetic/epigenetic-based diagnoses and therapeutics for PEX. Although studies to understand the role of epigenetic regulations in PEX are just emerging, epigenetic modifications contribute significantly to PEX pathogenesis and may pave the way for better and targeted therapeutics.
Subject(s)
Exfoliation Syndrome , Glaucoma , Humans , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Exfoliation Syndrome/pathology , Genome-Wide Association Study , Glaucoma/genetics , Risk Factors , Epigenesis, GeneticABSTRACT
Pseudoexfoliation glaucoma (PEXG) is characterized by dysregulated extracellular matrix (ECM) homeostasis that disrupts conventional outflow function and increases intraocular pressure (IOP). Prolonged IOP elevation results in optic nerve head damage and vision loss. Uniquely, PEXG is a form of open angle glaucoma that has variable penetrance, is difficult to treat and does not respond well to common IOP-lowering pharmaceuticals. Therefore, understanding modulators of disease severity will aid in targeted therapies for PEXG. Genome-wide association studies have identified polymorphisms in the long non-coding RNA lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) as a risk factor for PEXG. Risk alleles, oxidative stress and mechanical stretch all alter LOXL1-AS1 expression. As a long non-coding RNA, LOXL1-AS1 binds hnRNPL and regulates global gene expression. In this study, we focus on the role of LOXL1-AS1 in the ocular cells (trabecular meshwork and Schlemm's canal) that regulate IOP. We show that selective knockdown of LOXL1-AS1 leads to cell-type-specific changes in gene expression, ECM homeostasis, signaling and morphology. These results implicate LOXL1-AS1 as a modulator of cellular homeostasis, altering cell contractility and ECM turnover, both of which are well-known contributors to PEXG. These findings support LOXL1-AS1 as a key target for modifying the disease.
Subject(s)
Exfoliation Syndrome , Glaucoma, Open-Angle , RNA, Long Noncoding , Humans , Glaucoma, Open-Angle/genetics , RNA, Long Noncoding/genetics , Protein-Lysine 6-Oxidase/genetics , Genome-Wide Association Study , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Amino Acid Oxidoreductases/geneticsABSTRACT
Pseudoexfoliation (PEX) is a multifactorial age-related disease characterized by the deposition of extracellular fibrillar aggregates in the anterior ocular tissues. This study aims to identify the genetic and epigenetic contribution of clusterin (CLU) in PEX pathology. CLU is a molecular chaperone upregulated in PEX and genetically associated with the disease. Sequencing of a 2.9 kb region encompassing the previously associated rs2279590 in 250 control and 313 PEX [(207 pseudoexfoliation syndrome (PEXS) and 106 pseudoexfoliation glaucoma (PEXG)] individuals identified three single nucleotide polymorphisms (SNPs), rs9331942, rs9331949 and rs9331950, in the 3'-UTR of CLU of which rs9331942 and rs9331949 were found to be significantly associated with PEXS and PEXG as risk factors. Following in silico analysis, in vitro luciferase reporter assays in human embryonic kidney cells revealed that risk alleles at rs9331942 and rs9331949 bind to miR-223 and miR-1283, respectively, suggesting differential regulation of clusterin in the presence of risk alleles at the SNPs. Further, through bisulfite sequencing, we also identified that CLU promoter is hypomethylated in DNA from blood and lens capsules of PEX patients compared to controls that correlated with decreased expression of DNA methyltransferase 1 (DNMT1). Promoter demethylation of CLU using DNMT inhibitor, 5'-aza-dC, in human lens epithelial cells increased CLU expression. Chromatin immunoprecipitation assays showed that the demethylated CLU promoter provides increased access to the transcription factor, Sp1, which might lead to enhanced expression of CLU. In conclusion, this study highlights the different molecular mechanisms of clusterin regulation in pseudoexfoliation pathology.
Subject(s)
Clusterin , DNA Methylation , Exfoliation Syndrome , Glaucoma , Humans , Clusterin/genetics , Clusterin/metabolism , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Exfoliation Syndrome/pathology , Glaucoma/genetics , MicroRNAs/genetics , 3' Untranslated Regions/genetics , Polymorphism, Genetic , Promoter Regions, GeneticABSTRACT
AIMS: The etiology of pseudoexfoliation (PEX), a stress-induced fibrillopathy and a leading cause of secondary glaucoma worldwide, remains limited. This study aims to understand the role of the Wnt antagonist Dickkopf-related protein 1 (DKK1) in PEX pathophysiology and assess its candidature as a biomarker for PEX. MAIN METHODS: Expression levels of DKK1 and Wnt signaling genes were assayed in the anterior ocular tissues of study subjects by qRT-PCR, Western blotting, and immunohistochemistry. Protein aggregation was studied through Proteostat staining. Role of DKK1 in protein aggregation and regulation of target Wnt signaling genes was elucidated through overexpression and knockdown studies in Human Lens Epithelial cells (HLEB3). Levels of DKK1 in circulating fluids were assayed through ELISA. KEY FINDINGS: DKK1 upregulation was observed in lens capsule and conjunctiva tissues of PEX individuals compared to controls correlating with an upregulation of the Wnt signaling target, ROCK2. Proteostat staining showed increased protein aggregates in lens epithelial cells of PEX patients. HLE B-3 cells overexpressed with DKK1 showed increased protein aggregates along with upregulation of ROCK2, and knockdown of DKK1 in HLE B-3 cells demonstrated downregulation of ROCK2. Further, ROCK2 inhibition by Y-27632 in DKK1 overexpressed cells showed that DKK1 regulated protein aggregation via ROCK2. Also, increased levels of DKK1 were observed in patients' plasma and aqueous humor compared to controls. SIGNIFICANCE: This study shows that DKK1 and ROCK2 might play a role in protein aggregation in PEX. Further, elevated levels of DKK1 in aqueous humor serve as a fair classifier of pseudoexfoliation glaucoma.
Subject(s)
Exfoliation Syndrome , Glaucoma , Humans , Epithelial Cells/metabolism , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Glaucoma/metabolism , Protein Aggregates , rho-Associated Kinases/metabolism , Up-RegulationABSTRACT
N6-methyladenosine (m6A) modification is one of the most common types of methylation modifications in eukaryotic mRNA. However, its role in the pathogenesis of pseudoexfoliation glaucoma (PXG) has not yet been reported. To enhance understanding in this regard, we assessed the m6A methylome in the aqueous humor of patients with PXG. MeRIP-Seq and RNA-Seq analyses were performed to compare the m6A methylomes and gene expression profiles of the aqueous humor of patients with PXG with those of patients with age-related cataract (ARC). Colorimetric m6A quantification was performed to detect global m6A levels. Quantitative reverse transcription PCR confirmed the expression of m6A-related enzymes and mRNAs in both groups. Results showed significantly higher aqueous humor m6A levels in the PXG group than in the ARC group. Five m6A-related enzymes, including METTL3, YTHDC2, HNRNPA2B1, HNRNPC, and LRPPRC, were significantly up-regulated in PXG specimens. We also observed 9728 m6A-modified peaks related to 6126 gene transcripts in the PXG group, with more than 250 genes containing one m6A peak (hypomethylated or hypermethylated). The distribution of the m6A peaks was enriched in coding sequences and 3'-untranslated regions for both groups. GGAC motif structures were also significantly enriched. Bioinformatics analysis further revealed that m6A plays a critical role in extracellular matrix formation and histone deacetylation. Additionally, MMP14, ADAMTSL1, FN1, and HDAC1 showed significant changes in m6A methylation and mRNA expression in the PXG group. Therefore, m6A methylation may regulate extracellular matrix composition in PXG and METTL3 may be a pivotal regulator of this process. In the future, it would be necessary to investigate MMP14, ADAMTSL1, FN1, and HDAC1, which are potential target genes.
Subject(s)
Cataract , Exfoliation Syndrome , Glaucoma , Humans , Transcriptome , Matrix Metalloproteinase 14/genetics , Matrix Metalloproteinase 14/metabolism , Methylation , Aqueous Humor/metabolism , Exfoliation Syndrome/metabolism , Cataract/genetics , Cataract/metabolism , Glaucoma/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Methyltransferases/geneticsABSTRACT
BACKGROUND: Exfoliation syndrome (XFS) is an age-related systemic disorder characterized by excessive production and progressive accumulation of abnormal extracellular material, with pathognomonic ocular manifestations. It is the most common cause of secondary glaucoma, resulting in widespread global blindness. The largest global meta-analysis of XFS in 123,457 multi-ethnic individuals from 24 countries identified seven loci with the strongest association signal in chr15q22-25 region near LOXL1. Expression analysis have so far correlated coding and a few non-coding variants in the region with LOXL1 expression levels, but functional effects of these variants is unclear. We hypothesize that analysis of the contribution of the genetically determined component of gene expression to XFS risk can provide a powerful method to elucidate potential roles of additional genes and clarify biology that underlie XFS. RESULTS: Transcriptomic Wide Association Studies (TWAS) using PrediXcan models trained in 48 GTEx tissues leveraging on results from the multi-ethnic and European ancestry GWAS were performed. To eliminate the possibility of false-positive results due to Linkage Disequilibrium (LD) contamination, we i) performed PrediXcan analysis in reduced models removing variants in LD with LOXL1 missense variants associated with XFS, and variants in LOXL1 models in both multiethnic and European ancestry individuals, ii) conducted conditional analysis of the significant signals in European ancestry individuals, and iii) filtered signals based on correlated gene expression, LD and shared eQTLs, iv) conducted expression validation analysis in human iris tissues. We observed twenty-eight genes in chr15q22-25 region that showed statistically significant associations, which were whittled down to ten genes after statistical validations. In experimental analysis, mRNA transcript levels for ARID3B, CD276, LOXL1, NEO1, SCAMP2, and UBL7 were significantly decreased in iris tissues from XFS patients compared to control samples. TWAS genes for XFS were significantly enriched for genes associated with inflammatory conditions. We also observed a higher incidence of XFS comorbidity with inflammatory and connective tissue diseases. CONCLUSION: Our results implicate a role for connective tissues and inflammation pathways in the etiology of XFS. Targeting the inflammatory pathway may be a potential therapeutic option to reduce progression in XFS.
Subject(s)
Exfoliation Syndrome , Humans , Exfoliation Syndrome/genetics , Exfoliation Syndrome/complications , Exfoliation Syndrome/metabolism , Amino Acid Oxidoreductases/genetics , RNA, Messenger , Mutation, Missense , Gene Expression , Polymorphism, Single Nucleotide , DNA-Binding Proteins/genetics , B7 Antigens/geneticsABSTRACT
Oxidative stress (OS) affects the anterior ocular tissues, rendering them susceptible to several eye diseases. On the other hand, protection of the eye from harmful factors is achieved by unique defense mechanisms, including enzymatic and non-enzymatic antioxidants. The imbalance between oxidants and antioxidants could be the cause of pseudoexfoliation syndrome (PEXS), a condition of defective extracellular matrix (ECM) remodeling. A systematic English-language literature review was conducted from May 2022 to June 2022. The main antioxidant enzymes protecting the eye from reactive oxygen species (ROS) are superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), which catalyze the reduction of specific types of ROS. Similarly, non-enzymatic antioxidants such as vitamins A, E and C, carotenoids and glutathione (GSH) are involved in removing ROS from the cells. PEXS is a genetic disease, however, environmental and dietary factors also influence its development. Additionally, many OS products disrupting the ECM remodeling process and modifying the antioxidative defense status could lead to PEXS. This review discusses the antioxidative defense of the eye in association with PEXS, and the intricate link between OS and PEXS. Understanding the pathways of PEXS evolution, and developing new methods to reduce OS, are crucial to control and treat this disease. However, further studies are required to elucidate the molecular pathogenesis of PEXS.
Subject(s)
Exfoliation Syndrome , Oxidative Stress , Humans , Exfoliation Syndrome/metabolism , Glutathione/metabolism , Reactive Oxygen Species/metabolism , Superoxide Dismutase/metabolismABSTRACT
Pseudoexfoliation syndrome (PEX) is an important systemic disorder of the extracellular matrix, in which granular amyloid-like protein fibers accumulate in the anterior segment of the eyeball as well as in other organs. PEX is currently considered to be a multifactorial systemic disorder with genetic and environmental risk factors. The aim of this manuscript was to analyze miR expression in PEX. In recent years, an attempt has been made to investigate and describe the level of expression of selected miRs in PEX. Four polymorphisms of genes isolated from the blood that may be related to PEX were identified and miR-122-5p was found to be upregulated in patient blood. Furthermore, 18 miRs were identified with a statistically different expression in the aqueous humor. A significantly elevated expression of miR-125b was found in the anterior lens capsule, and four miRs were described, which may have a significant impact on the development of PEX. Regulatory miR molecules are gaining more and more importance in research aimed at identifying and isolating molecular markers related to the pathogenesis and prognosis of PEX, but further studies are needed.
Subject(s)
Exfoliation Syndrome , MicroRNAs , Humans , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Exfoliation Syndrome/pathology , Aqueous Humor/metabolism , Extracellular Matrix/metabolism , MicroRNAs/genetics , MicroRNAs/metabolismABSTRACT
Exfoliation syndrome presents as an accumulation of insoluble fibrillar aggregates that commonly correlates with age and causes ocular complications, most notably open-angle glaucoma. Despite advances in understanding the pathogenesis and risk factors associated with exfoliation syndrome, there has been no significant progress in curative pharmacotherapy of this disease. It is thought that the ability to target the fibrillar aggregates associated with exfoliation may offer a new therapeutic approach, facilitating their direct removal from affected tissues. Phage display techniques yielded two peptides (LPSYNLHPHVPP, IPLLNPGSMQLS) that could differentiate between exfoliative and non-affected regions of the human lens capsule. These peptides were conjugated to magnetic particles using click chemistry to investigate their ability in targeting and removing exfoliation materials from the anterior human lens capsule. The behavior of the fibrillar materials upon binding to these magnetic particles was assessed using magnetic pins and rotating magnetic fields of various strengths. Ex vivo studies showed that the magnetic particle-peptide conjugates could generate enough mechanical force to remove large aggregates of exfoliation materials from the lens capsule when exposed to a low-frequency rotating magnetic field (5000 G, 20 Hz). Biocompatibility of targeting peptides with and without conjugated magnetic particles was confirmed using MTT cell toxicity assay, live/dead cell viability assay, and DNA fragmentation studies on primary cultured human trabecular meshwork cells. This is a novel, minimally invasive, therapeutic approach for the treatment of exfoliation glaucoma via the targeting and removal of exfoliation materials that could be applied to all tissues within the anterior segment of the eye.
Subject(s)
Exfoliation Syndrome , Glaucoma, Open-Angle , Humans , Exfoliation Syndrome/complications , Exfoliation Syndrome/metabolism , Exfoliation Syndrome/pathology , Glaucoma, Open-Angle/complications , Glaucoma, Open-Angle/pathology , Cytoskeleton/metabolism , Myocytes, Cardiac/metabolismABSTRACT
Pseudoexfoliation (PEX) is a multifactorial age-related disease involving deposition of extracellular fibrillar material on anterior ocular tissues. If left untreated, the early stage of deposition termed as pseudoexfoliation syndrome (PEXS) may lead to the advanced stage of pseudoexfoliation glaucoma (PEXG) characterised by increased intraocular pressure, damage to the optic nerve and subsequent irreversible blindness. The etiology of PEX is complex and identification of novel factors associated with the disease is needed. This study aimed to identify the involvement of vimentin in pseudoexfoliation pathology and assess the levels of vimentin, clusterin and fibulin-5 in the circulating fluids of PEX patients compared to controls. Eighty-seven participants (35 controls, 35 PEXS and 17 PEXG) were enrolled for this case-control study. The expression of vimentin in lens capsules of patients and age-sex matched controls was assayed by qRT-PCR, western blotting and immunohistochemistry. Aqueous humor (AH) vimentin levels and plasma levels of vimentin, clusterin and fibulin-5 were assayed through ELISA. Increased vimentin was observed in the lens capsule of patients compared to controls at mRNA and protein levels. Compared to control (11.5 ± 1.4 ng/ml [±SEM]), the AH vimentin concentrations were significantly higher (ANOVA p = 0.01) in PEXS (16.4 ± 1.8 ng/ml) and PEXG (20.1 ± 2.5 ng/ml). Compared to controls (372.2 ± 15.1 ng/ml), plasma vimentin levels were significantly higher (ANOVA, p < 0.001) in PEXS (449.9 ± 15.7 ng/ml) and PEXG (535.5 ± 25.0 ng/ml). The plasma and aqueous humor levels of vimentin showed a positive correlation of 0.31. The plasma levels of clusterin were 298.9 ± 19.0 µg/ml, 367.8 ± 25.6 µg/ml and 272.9 ± 16.8 µg/ml in controls, PEXS and PEXG, respectively and were significantly higher in PEXS (p = 0.03) compared to control. Plasma fibulin-5 levels were 149 ± 32.2 pg/ml, 187.6 ± 32.3 pg/ml and 203.8 ± 27.3 pg/ml in controls, PEXS and PEXG, respectively and there was no significant difference in its levels between the groups (ANOVA p = 0.49). In conclusion, vimentin is upregulated in PEX affected eyes. Increased vimentin levels in plasma and AH differentiate PEXS and PEXG from controls.
Subject(s)
Exfoliation Syndrome , Glaucoma , Humans , Exfoliation Syndrome/metabolism , Clusterin/metabolism , Case-Control Studies , Vimentin , Glaucoma/complications , RNA, MessengerABSTRACT
Small nucleolar RNAs (snoRNAs) are small non-coding regulatory RNAs that have been investigated extensively in recent years. However, the relationship between snoRNA and glaucoma is still unknown. This study aims to analyze the levels of snoRNA expression in the aqueous humor (AH) of patients with pseudoexfoliation glaucoma (PEXG) compared to a control group and identify hypothetical snoRNA-dependent mechanisms contributing to PEXG. The AH was obtained from eighteen Caucasian patients, comprising nine PEXG and nine age-matched control patients. RNA was isolated, and a microarray system was used to determine the snoRNA expression profiles. Functional and enrichment analyses were performed. We identified seven snoRNAs, SNORD73B, SNORD58A, SNORD56, SNORA77, SNORA72, SNORA64, and SNORA32, in the AH of the PEXG and control group patients. Five snoRNAs showed statistically significantly lower expression in the PEXG group, and two snoRNAs had statistically significantly higher expression in the PEXG group compared to the control group. In addition, we identified two factors-CACNB3 for SNORA64 and TMEM63C for SNORA32, similar to PEX-related genes (CACNA1A and TMEM136). The enrichment analysis for four genes targeted by snoRNAs revealed possible mechanisms associated with glaucoma and/or PEX, but the direct role of snoRNAs in these biological processes was not proven.
Subject(s)
Exfoliation Syndrome , Glaucoma, Open-Angle , Glaucoma , Aqueous Humor/metabolism , Exfoliation Syndrome/complications , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Glaucoma/genetics , Glaucoma/metabolism , Glaucoma, Open-Angle/genetics , Humans , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolismABSTRACT
Pseudoexfoliation (PEX) syndrome, a stress-induced fibrotic matrix process, is the most common recognizable cause of open-angle glaucoma worldwide. The recent identification of PEX-associated gene variants uncovered the vitamin A metabolic pathway as a factor influencing the risk of disease. In this study, we analyzed the role of the retinoic acid (RA) signaling pathway in the PEX-associated matrix metabolism and evaluated its targeting as a potential candidate for an anti-fibrotic intervention. We provided evidence that decreased expression levels of RA pathway components and diminished RA signaling activity occur in an antagonistic crosstalk with TGF-ß1/Smad signaling in ocular tissues and cells from PEX patients when compared with age-matched controls. Genetic and pharmacologic modes of RA pathway inhibition induced the expression and production of PEX-associated matrix components by disease-relevant cell culture models in vitro. Conversely, RA signaling pathway activation by natural and synthetic retinoids was able to suppress PEX-associated matrix production and formation of microfibrillar networks via antagonization of Smad-dependent TGF-ß1 signaling. The findings indicate that deficient RA signaling in conjunction with hyperactivated TGF-ß1/Smad signaling is a driver of PEX-associated fibrosis, and that restoration of RA signaling may be a promising strategy for anti-fibrotic intervention in patients with PEX syndrome and glaucoma.
Subject(s)
Exfoliation Syndrome , Glaucoma, Open-Angle , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Exfoliation Syndrome/pathology , Glaucoma, Open-Angle/metabolism , Humans , Signal Transduction , Transforming Growth Factor beta1/genetics , Tretinoin/pharmacologyABSTRACT
We aimed to obtain microRNA (miRNA) profiles of patients with pseudoexfoliation (PEX) glaucoma or normal-tension glaucoma (NTG) compared to normal controls using individual aqueous humor (AH) samples and investigate the role of miRNAs in the pathogenesis of PEX glaucoma compared to NTG in Korean. AH (80-120 µl) was collected before cataract surgery or trabeculectomy from 26 Korean subjects (eleven with PEX glaucoma, age-matched eight NTG, and seven controls). RNA sequencing was conducted for RNA samples extracted from 26 AH samples. Bioinformatics analysis was performed for targets and related pathways. A total of 334 and 291 discrete miRNAs were detected in AH samples of PEX glaucoma and NTG patients, respectively. Two significantly upregulated miRNAs (hsa-miR-30d-5p and hsa-miR-320a) and ten significantly downregulated miRNAs (hsa-miR-3156-5p, hsa-miR-4458, hsa-miR-6717-5p, hsa-miR-6728-5p, hsa-miR-6834-5p, hsa-miR-6864-5p, hsa-miR-6879-5p, hsa-miR-877-3p, hsa-miR-548e-3p, and hsa-miR-6777-5p) in PEX glaucoma patients compared to control (fold-change > 2, p < 0.05) were found. In NTG patients, ten significantly upregulated and two downregulated miRNAs compared to control were found. Only hsa-miR-6777-5p was commonly downregulated in both PEX glaucoma and NTG patients. Related pathways were proteoglycans in cancer, glioma, and TGF-beta signaling pathway in PEX glaucoma. These differentially expressed miRNAs between PEX glaucoma and NTG samples suggest the possible role of miRNA in the pathogenesis of glaucoma, further implying that pathogenic mechanisms may differ between different types of glaucoma.
Subject(s)
Exfoliation Syndrome , Glaucoma , Low Tension Glaucoma , MicroRNAs , Aqueous Humor/metabolism , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Glaucoma/metabolism , Humans , Low Tension Glaucoma/genetics , Low Tension Glaucoma/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , Republic of KoreaABSTRACT
Pseudoexfoliation syndrome (PEXS) is an age-related condition manifesting mainly in ocular tissues. PEXS is manifested through excess aggregation of fibrillary extracellular material at the anterior part of the eye that consists of a plethora of biomolecules, such as different proteoglycans (PGs) and glycosaminoglycans. PEXS is often linked to increased intraocular pressure, and can also lead to pseudoexfoliation glaucoma with very poor prognosis. Various stimuli are known to affect PEXS, including oxidation stress (OS), UV radiation and osmotic pressure. OS, is prominently involved on the progression of the syndrome as it promotes fibrogenesis, possibly via the induction of transforming growth factor-ß (TGF-ß) and other biomolecular effectors. In addition, PEXS initiation is tightly connected with the dysregulation of extracellular matrix (ECM) homeostasis since aberrant expression of ECM molecules is linked to both the accumulation and low degradation of pseudoexfoliation material. This article aims at uncovering the crucial role of various ECM effectors such as lysyl oxidase-like proteins, matrix metalloproteinases, and TGF-ß1, as well as the biochemical pathways involved in the development and the progression of the PEXS.
Subject(s)
Exfoliation Syndrome , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Exfoliation Syndrome/therapy , Extracellular Matrix/metabolism , Glycosaminoglycans , Humans , Matrix Metalloproteinases , Protein-Lysine 6-Oxidase , Proteoglycans/genetics , Transforming Growth Factor beta , Transforming Growth Factor beta1/genetics , Transforming Growth FactorsABSTRACT
Glaucoma is the second leading cause of blindness. Exfoliation syndrome (XFN) is a risk factor for exfoliation glaucoma (XFG) which is a secondary open angle glaucoma. XFG is difficult to manage with a worse prognosis. Though 40% of the XFN progress to XFG, there are no predictive markers to identify the susceptible patients. Herein, we analyze clinical data, ATP levels in aqueous humor and cytokines in plasma to identify alteration that help distinguish XFN from XFG. Our results show characteristic clinical features of XFG compared to XFN and controls. Elevated levels of ATP in aqueous humor were observed in XFG compared to XFN and cataract controls while elevated levels of plasma cytokines were observed in XFG compared to XFN, cataract controls and healthy controls. Microglia are immune cells in the retina implicated in glaucoma. TNFα plays a predominant role in microglial inflammation and is implicated in neurodegeneration. Using in vitro N9 microglial cell culture model, we demonstrate that TNFα modulated expression of cytokines and chemotaxis is dependent on P2 receptors like P2X7, P2Y12 and P2Y6. In addition, ATP also induce expression of TNFα which might act as a feed forward loop. The TNFα induced inflammation is dependent on downstream signaling modules like PI3K, JNK and ROS. Taken together, our results show that elevated ATP in aqueous humor, plasma cytokines and inflammation potentially involving microglia distinguish XFG from XFN. Purinergic receptors might be potential therapeutic targets in XFG.
Subject(s)
Exfoliation Syndrome , Glaucoma, Open-Angle , Adenosine Triphosphate , Cytokines/metabolism , Exfoliation Syndrome/metabolism , Glaucoma, Open-Angle/diagnosis , Glaucoma, Open-Angle/metabolism , Humans , Inflammation , Microglia/metabolismABSTRACT
Pseudoexfoliation syndrome (PXF) is an idiopathic disease with a high prevalence rate. The elastosis disorder is contributed by genetic and non-genetic factors. Elastin dysregulation associated with the disease mechanism is incompletely understood. This study evaluated the molecules of the elastogenesis machinery in PXF. Lens capsule and aqueous humor (aqH) samples (age/sex-matched) were collected from the eyes with PXF alone and PXF with glaucoma (PXF-G) undergoing Extra Capsular Cataract Extraction (ECCE) surgery. The Elastin turnover was assessed by estimating Desmosine levels in the lens capsules by HPLC analysis. Expression of elastogenesis genes [EMILIN1, CLU, FBN1, FN1, FBLN5, FBLN4 and LOXL1] were evaluated in the lens capsule by qPCR while the proteins were assessed in aqH by western blot analysis. The Desmosine content in the lens capsules were 3-fold and 6-fold elevated in PXF (P = 0.02) and PXF-G (P = 0.01) respectively compared to the cataract-alone, indicating increased elastin degradation. A significant increase in the transcript levels of the CLU, FBLN4, EMILIN1, FBLN5, FN1, FBN1, LOXL1 along with significant changes in protein expression of CLU, FBLN5, FBN1 and LOXL1 signified up-regulation of the elastogenesis machinery. The study provides direct evidence of augmented elastin degradation and turnover in the lens capsule of PXF marked by increased Desmosine content and the expression of proteins involved in mature elastin formation.
Subject(s)
Cataract , Exfoliation Syndrome , Glaucoma , Lens Capsule, Crystalline , Capsules/metabolism , Cataract/metabolism , Desmosine/metabolism , Elastin/genetics , Exfoliation Syndrome/genetics , Exfoliation Syndrome/metabolism , Glaucoma/metabolism , Humans , Lens Capsule, Crystalline/metabolismABSTRACT
Purpose: We compared cytokines, chemokines, and growth factors in the aqueous humor (AH) of patients with exfoliation syndrome (XFS), with exfoliation glaucoma (XFG), with primary open angle glaucoma (POAG), and healthy controls. Methods: AH samples were collected from 21 patients with XFS, 28 with XFG, 14 with POAG, and 17 healthy controls during routine cataract surgery. The protein levels of 21 cytokines and growth factors, together with TGF-ß1, 2, and 3, were quantified using the multiplex immunoassay. The levels of each protein in the four groups were compared using the Kruskal-Wallis test. Results: Among the 24 cytokines and growth factors, 16 were out of the detectable range in >50% of samples in at least one group; the remaining 8 cytokines and growth factors (IL-8, MIP-1α, fractalkine, Flt3 ligand, PDGF-AA, VEGF, TGF-ß1, and TGF-ß2) were included in the analysis. TGF-ß1 and TGF-ß2 levels were the highest in patients with XFG and those with POAG, respectively. Expression levels of the inflammatory chemokines IL-8, MIP-1α, and fractalkine, as well as levels of the immune cell growth factor Flt3 ligand, were significantly higher in the XFG group than in the other groups. The protein levels of PDGF-AA and VEGF were not significantly different among the 4 groups. Conclusions: Both TGF-ß1 and inflammatory cytokines were highly expressed in the AH of patients with XFG. Considering that the levels of these cytokines are increased by oxidative stress and that they regulate the extracellular matrix, they may also play a role in intraocular pressure elevation in XFG.