MicroRNA Let-7c-5p-Mediated Regulation of ERCC6 Disrupts Autophagic Flux in Age-Related Cataract via the Binding to VCP.

Purpose: DNA damage contributes to the pathogenesis of age-related cataract (ARC) and is repaired through the nucleotide excision repair (NER) pathway, which includes ERCC6. Evidence has demonstrated that defective autophagy leads to lens organelle degradation and cataract. This study aimed to investigate the effects of ERCC6 on autophagy and determine its mechanisms in ARC.Methods: The clinical case-control study comprised 30 patients with ARC and 30 age-matched controls who received transparent lens extraction. Transmission electron microscopy was used to assess the ultrastructure of autophagic vesicles in lens anterior capsule tissues and lens epithelial cell line (SRA01/04). Real-time polymerase chain reaction and western blot analyses were performed to measure relative gene expression levels. Gene expression levels and localization were assessed by immunofluorescence. A coimmunoprecipitation assay was used to investigate the relationship between CSB which encoded by ERCC6 and VCP. ERCC6-siRNA and let-7 c-5p mimic were used to alter the expression of ERCC6 and let-7 c-5p.Results: Autophagy induction occurred in lens anterior capsule tissues of patients with ARC and in UVB-induced SRA01/04 cells, where the number of LC3B puncta was increased. Consistent with this result, the expression of beclin1 (BECN1) and LC3B, in addition to that of p62, was increased. Additionally, ERCC6 expression decreased, and silencing ERCC6 induced increases in the expression of BECN1, LC3B and p62. Moreover, CSB interacted with VCP, and let-7 c-5p induced dysregulation of autophagy by targeting ERCC6.Conclusion: In ARC, Let-7 c-5p-mediated downregulation of ERCC6 might prevent the degradation of autophagic vacuoles. CSB binds to VCP, inducing autophagosomes to combine with lysosomes and be degraded.

Structural polymorphisms in fibrillar aggregates associated with exfoliation syndrome.

Exfoliation syndrome is largely considered an age-related disease that presents with fibrillar aggregates in the front part of the eye. A growing body of literature has investigated structural diversity of amyloids and fibrillar aggregates associated with neurodegenerative disease. However, in case of exfoliation syndrome, there is a dearth of information on the biophysical characteristics of these fibrils and structural polymorphisms. Herein, structural diversity of fibrils isolated from the anterior lens capsule of patients was evaluated using transmission electron microscopy techniques. It was apparent that, despite having a low sample number of different patients, there exists a wide range of fibril morphologies. As it is not precisely understood how these fibrils form, or what they are composed of, it is difficult to postulate a mechanism responsible for these differences in fibril structure. However, it is apparent that there is a wider range of fibril structure than initially appreciated. Moreover, these data may suggest the variance in fibril structure arises from patient-specific fibril composition and/or formation mechanisms.

Long noncoding RNA glutathione peroxidase 3-antisense inhibits lens epithelial cell apoptosis by upregulating glutathione peroxidase 3 expression in age-related cataract.

Purpose: Age-related cataract (ARC) is the leading cause of visual impairment and blindness worldwide. The apoptosis of lens epithelial cells (LECs) induced by oxidative damage is a major contributing factor to ARC. Long noncoding RNAs (lncRNAs) play important roles in various biologic processes. We aimed to explore the role of glutathione peroxidase 3 (GPX3)-antisense (AS) in ARCs. Methods: We extracted total RNAs from transparent and age-matched cataractous human lenses and detected lncRNA expression profiles using high-throughput RNA sequencing. The expression of GPX3-AS and GPX3 was detected by quantitative real-time PCR (qRT-PCR). Apoptotic proteins were detected by western blot and immunofluorescence. We treated SRA01/04 cells with H2O2 to mimic oxidative stress and induce cell apoptosis, which was analyzed by flow cytometry and TdT-mediated dUTP Nick-End Labeling (TUNEL) assay. The cell counting kit-8 (CCK-8) assay was used to detect the viability of SRA01/04 cells. The location of GPX3-AS was determined by fluorescence in situ hybridization (FISH) and cell nuclear and cytoplasmic RNA separation. Results: The lncRNA GPX3-AS, which is located in the nuclei of LECs, was downregulated in cataractous human lenses compared with control lenses, and proapoptotic proteins were expressed at high levels in the anterior lens capsules of ARC tissues. An in vitro study suggested that GPX3-AS inhibited H2O2-induced SRA01/04 cell apoptosis. As GPX3-AS is transcribed from the AS strand of the GPX3 gene locus, we further revealed its regulatory role in GPX3 expression. GPX3-AS was positively correlated with GPX3 expression. In addition, GPX3-AS inhibited H2O2-induced SRA01/04 cell apoptosis by upregulating GPX3 expression. Conclusions: In summary, our study revealed that GPX3-AS downregulated the apoptosis of LECs via promoting GPX3 expression, implying a novel therapeutic target for ARCs.

Relationship between SIRT1 gene expression level and disease in age-related cataract cases

Background/aim: Age-related cataract is the most important visual impairment all over the world. Epigenetic modifications, especially overexpression of histone deacetylases, have become the focus of interest for cataract development in recent years. Sirtuin 1 (SIRT1), a class II histone deacetylase and a member of the sirtuin family, is one of the best-characterized histone deacetylases and has a pivotal role in age-related diseases. However, the association of SIRT1 with age-related cataracts has not yet been fully elucidated. Therefore, we aimed to determine the expression of SIRT1 in age-related cataract patients. Materials and methods: Expressions of SIRT1 were evaluated by quantitative polymerase chain reaction (qPCR) in patients and healthy controls. RNA samples were collected from the anterior capsule and peripheral blood samples of age-related cataract patients. Human lens epithelial cell line B3 and peripheral blood samples of healthy subjects were used as controls. Results: We determined that the expression of SIRT1 in blood and anterior capsule samples increased significantly compared to the control group (P < 0.05). Conclusion: The expression level of SIRT1 plays a vital role in the development of age-related cataract and it can be used as a biomarker. Thus, SIRT1 inhibitors can be used in the treatment of age-related cataract disease.

Expression levels of aldose reductase enzyme, vascular endothelial growth factor, and intercellular adhesion molecule-1 in the anterior lens capsule of diabetic cataract patients.

PURPOSE: To compare the levels of aldose reductase (ALR) enzyme, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF) in the anterior lens capsule of diabetic versus nondiabetic patients. SETTING: Alexandria Main University Hospital, Alexandria, Egypt. DESIGN: Prospective case-control study. METHODS: The study enrolled patients undergoing cataract extraction and divided them into 3 groups: eyes that had proliferative diabetic retinopathy (PDR), eyes that had nonproliferative diabetic retinopathy (NPDR), and nondiabetic eyes. The anterior lens capsules were obtained by performing femtosecond laser-assisted capsulorhexis. Concentrations of ALR, ICAM-1, and VEGF in the lens capsule specimens were measured using human enzyme-linked immunosorbent assay. RESULTS: This study comprised 200 patients (200 eyes); 51 eyes had PDR, 49 eyes had NPDR, and 100 eyes were nondiabetic. The mean ALR, ICAM-1, and VEGF levels in the anterior capsule of diabetic group were 2.84 nanogram (ng)/mL ± 0.51 (SD), 87.73 ± 22.84 picogram (pg)/mL, and 75.53 ± 14.95 pg/mL, respectively; whereas, in the nondiabetic group, they were 1.44 ± 0.17 ng/mL, 35.45 ± 2.8 pg/mL, and 33.55 ± 5.47 pg/mL, respectively. In comparing the concentrations of these mediators, both the PDR and NPDR groups had significantly higher levels compared with the nondiabetic eyes (P < .001). In addition, eyes with PDR had significantly higher levels of these mediators than eyes with NPDR (P < .001). CONCLUSION: The concentrations of ALR, ICAM-1, and VEGF in the anterior lens capsule of diabetic patients are significantly higher than those of nondiabetics. A significantly higher level of 3 mediators in eyes with PDR compared with those with NPDR might allow the use of them as a biomarker for severity of diabetic retinopathy.

Pharmacokinetics of Caffeine in the Lens Capsule/Epithelium After Peroral Intake: A Pilot Randomized Controlled Study.

Purpose: To determine the pharmacokinetics of perorally administered caffeine, a widely consumed and potent dietary antioxidant, in the anterior lens capsule and lens epithelial cells, a crucial cell monolayer for cataract development. Methods: Bilateral cataract patients were scheduled for cataract surgery with a caffeine abstinence of 1 week before surgery of each eye. At the day of surgery of the second eye patients were administered no drink (0-mg group) or coffee with 60-, 120-, or 180-mg caffeine. After capsulorhexis the lens capsule including lens epithelial cells was transferred to a test tube for analysis of caffeine concentration by gas chromatography-mass spectrometry (GC-MS/MS). Results: Coffee consumption significantly (P < 0.05) increased caffeine levels of the lens capsule/epithelium in the 60-, 120-, and 180-mg group. Caffeine concentrations (caffeine ng/lens capsule/epithelium) measured as difference between 1st and 2nd eye were -0.52 ± 1.16 (0-mg group, n = 7), 1.88 ± 2.02 (60-mg group, n = 8), 2.09 ± 0.67 (120-mg group, n = 9), and 3.68 ± 1.86 (180-mg group, n = 9). The increase constant of caffeine in a linear regression model was estimated as a 95% CI 0.02 ± 0.0046 (degrees of freedom; 25; r = 0.85). Conclusions: Peroral intake of coffee significantly increased caffeine concentrations in the lens capsule and lens epithelial cells in a dose-dependent manner. This information is important for further investigations on preventing cataract.

MicroRNA-26a and -26b inhibit lens fibrosis and cataract by negatively regulating Jagged-1/Notch signaling pathway.

Fibrosis is a chronic process involving development and progression of multiple diseases in various organs and is responsible for almost half of all known deaths. Epithelial-mesenchymal transition (EMT) is the vital process in organ fibrosis. Lens is an elegant biological tool to investigate the fibrosis process because of its unique biological properties. Using gain- and loss-of-function assays, and different lens fibrosis models, here we demonstrated that microRNA (miR)-26a and miR-26b, members of the miR-26 family have key roles in EMT and fibrosis. They can significantly inhibit proliferation, migration, EMT of lens epithelial cells and lens fibrosis in vitro and in vivo. Interestingly, we revealed that the mechanisms of anti-EMT effects of miR-26a and -26b are via directly targeting Jagged-1 and suppressing Jagged-1/Notch signaling. Furthermore, we provided in vitro and in vivo evidence that Jagged-1/Notch signaling is activated in TGFß2-stimulated EMT, and blockade of Notch signaling can reverse lens epithelial cells (LECs) EMT and lens fibrosis. Given the general involvement of EMT in most fibrotic diseases, cancer metastasis and recurrence, miR-26 family and Notch pathway may have therapeutic uses in treating fibrotic diseases and cancers.

The Expressions of Klotho Family Genes in Human Ocular Tissues and in Anterior Lens Capsules of Age-Related Cataract.

PURPOSE: Klotho genes are expressed in limited tissues and have been found to be a pathogenic factor of age-related diseases in mammals, but their expressions and functions in human eyes are yet to be defined. This study was performed to investigate the expression of Klotho family genes in human ocular tissues and anterior lens capsules of age-related cataract. METHODS: Individual tissues were isolated from human eyeballs collected from Henan Eye Bank. Human anterior lens capsules were collected from cortical cataract patients during phacoemulsification with the informed consent. Real-time quantitative reverse transcription was performed to detect the mRNA expressions of α-Klotho, ß-Klotho, γ-Klotho, IGFR1, AKT, and ERK; Western blot analysis for detecting the expression of ERK1/2 and phosphor-ERK1/2 protein; and Pearson correlation analysis was performed to detect the relationship between Klotho genes and IGFR1, AKT or ERK genes. RESULTS: The expressions of α-, ß-, and γ-Klotho mRNA were detected in human retina and optic nerve, but were not detected in iris, ciliary body, sclera, and choroid. Only α-and γ-Klotho mRNA expressions were detected in the lens, and the expression levels were higher than that in the retina and optic nerve. The mRNA expressions of IGFR1 and AKT were not changed among the transparent and the opaque lens capsules. The mRNA expression of ERK was significantly decreased in the opaque lens capsule. Correlation analysis showed that the ERK mRNA expression was positively correlated with the expression of the γ-Klotho gene. Western blot results showed a significant decrease of ERK1/2 and phosphor-ERK1/2 protein in opaque lens capsules. CONCLUSIONS: α- and γ-Klotho genes express in the human retina, optic nerve, and lens. The ß-Klotho gene expresses only in retina and optic nerve. The γ-Klotho gene may regulate human lens epithelial cells' function mainly by the MAPK/ERK1/2 signaling pathway.

Elevated TGF-ß2 level in aqueous humor of cataract patients with high myopia: Potential risk factor for capsule contraction syndrome.

PURPOSE: To evaluate the level of transforming growth factor-ß2 (TGF-ß2) in the aqueous humor of highly myopic cataract patients and its correlation with capsule contraction syndrome. SETTING: Eye and ENT Hospital of Fudan University, Shanghai, China. DESIGN: Prospective comparative case series. METHODS: The highly myopic cataract patients were divided into the following 2 groups according to the Lens Opacity Classification System III: nuclear color (NC) 2 to 3 and NC 5 to 6. Aqueous humor TGF-ß2 concentrations were assayed in the highly myopic cataract and age-related cataract groups. The TGF-ß2, TGF-ßRII (the type II receptor for TGF-ß2), and α-smooth muscle actin (α-SMA) expressions in lens epithelial cells (LECs) were detected by real-time polymerase chain reaction and immunofluorescent staining. RESULTS: The study comprised 40 highly myopic cataract patients (40 eyes) and 20 patients (20 eyes) with age-related cataract as the control group. Compared with the control group, the highly myopic cataract group had significantly higher TGF-ß2 concentration in the aqueous humor and increased TGF-ßRII expression in LECs, especially in NC 5 to 6 cases. Expression of α-SMA was barely detectable in both groups. CONCLUSION: In highly myopic cataract patients, especially those with dark nuclei, elevated aqueous humor TGF-ß2 levels and the upregulated TGF-ßRII expression in LECs might contribute to the pathogenesis of capsule contraction syndrome through transdifferentiation of LECs into myofibroblasts. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Anterior lens epithelial cells attachment to the basal lamina.

PURPOSE: To study the structure of the anterior lens epithelial cells (aLECs) and the contacts of the aLECs with the basal lamina (BL) in order to understand their role in the lens epithelium's function. METHODS: The aLCs (BL and associated aLECs) were obtained from routine uneventful cataract surgery, prepared for and studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and confocal microscopy. RESULTS: SEM shows that the basal surface of the aLECs (~10-15 µm) is with aLECs foldings (~1-3 µm) and extensions (~0.5-3 µm) attached to the BL. Confocal microscopy images of the basal sections of the aLECs after membrane staining also suggest that the basal part of aLECs has foldings (~1-3 µm). TEM shows in the aLECs basal parts, towards BL, the structures that look like entanglement (~1-4 µm). In cases where there is a swelling of the cytoplasm and offset of the aLECs from the BL, individual extensions (~0.5-2 µm) that extend to the BL are visible by TEM. CONCLUSIONS: We provide detail evidence about the structural organization of the aLECs, in particular about their basal side which is in contact with the BL. This is supported by the complementary use of three techniques, SEM, TEM and confocal microscopy, each of them showing the same morphological features, the extensions and the entanglements of the aLECs cytoplasmic membrane at the border with the BL. The basal surface of the aLECs is increased. It suggests the functional importance of the contact between aLECs and BL.

The cellular and molecular biology of the iris, an overlooked tissue: the iris and pseudoexfoliation glaucoma.

Located between the cornea and the lens, the Iris is fully immersed in aqueous humor. During exfoliation syndrome, a disorder of the elastic fibers, an abnormal fibrillar material (XFM) is deposited on the anterior lens capsule underneath the pigment epithelium of the Iris. Release of this material to the aqueous humor reaches the trabecular meshwork where its presence is associated with elevated intraocular pressure. Ultrastructural studies suggest that the XFM material is produced by the lens capsule, lens epithelial and iris pigment epithelial cells (IPE). The involvement of the IPE in pseudoexfoliation glaucoma has not been extensively addressed. Immunohistochemistry studies have shown higher levels of LOXL1 and clusterin in the IPE extracellular space of specimens from exfoliation patients. But studies using IPE cells to understand the formation of the XFM in vitro and/or in vivo are scarce. A focus on the Iris and its IPE cells would be key for the elucidation of XFM and the understanding of the development of pseudoexfoliation glaucoma.

Elemental analysis of sunflower cataract in Wilson's disease: a study using scanning transmission electron microscopy and energy dispersive spectroscopy.

Signature ophthalmic characteristics of Wilson's disease (WD) are regarded as diagnostically important manifestations of the disease. Previous studies have proved the common occurrence of copper accumulation in the liver of patients with WD. However, in the case of sunflower cataracts, one of the rare diagnostic signs of WD, no study has demonstrated copper accumulation in the lens capsules of sunflower cataracts in WD patients. To investigate the nanostructure and elemental composition of sunflower cataracts in WD, transmission electron microscopy (TEM) was done on the capsulorhexised anterior lens capsule of sunflower cataracts in WD in order to evaluate anatomical variation and elemental changes. We utilized energy dispersive X-ray spectroscopy (EDS) to investigate the elemental composition of the lens capsule using both point and mapping spectroscopy. Quantitative analysis was performed for relative comparison of the elements. TEM showed the presence of granular deposits of varying size (20-350 nm), appearing mainly in the posterior one third of the anterior capsule. The deposits appeared in linear patterns with scattered dots. There were no electron-dense particles in the epithelial cell layer of the lens. Copper and sulfur peaks were consistently revealed in electron-dense granular deposits. In contrast, copper and sulfur peaks were absent in other tissues, including granule-free lens capsules and epithelial tissue. Most copper was exclusively located in clusters of electron-dense particles, and the copper distribution overlapped with sulfur on mapping spectroscopy. Quantitative analysis presented inconsistent ratios of copper to sulfur in each electron-dense granule. The mean ratio of copper to sulfur was about 3.25 (with a range of 2.39-3.78). This is the first elemental analysis of single electron particles in sunflower cataracts using EDS in the ophthalmic area. Sunflower cataracts with WD are assumed to be the result of accumulation of heterogeneous compounds composed of several materials, including copper, sulfur, and/or copper-binding proteins. Linear patterns of copper and sulfur deposition were detected in various sizes and composition ratios with these elements in cases of WD.

Ocular manifestations of monoclonal copper-binding immunoglobulin.

The dense accumulation of copper in Descemet membrane and lens capsule is the characteristic manifestation of a circulating monoclonal antibody with strong affinity for copper. The overproduction of this monoclonal immunoglobulin may be associated with either multiple myeloma or a benign monoclonal gammopathy. Despite prolonged exposure to elevated serum copper, no other tissues in the body are adversely affected by this redox metal. We describe the clinical and pathological findings in a 46-year-old woman with this disorder.

Effects of antioxidant supplementation on mRNA expression of glucose-6-phosphate dehydrogenase, ß-actin and 18S rRNA in the anterior capsule of the lens in cataract patients.

This was a preliminary study of the effects of antioxidant supplementation on the peroxidation status of the lens by investigating mRNA expression of anti-oxidative enzymes in the lens. The mRNA expression levels of glucose-6-phosphate dehydrogenase (G6PDH), ß-actin (ß-ACT) and 18S rRNA (18S) were measured in this study because they are common reference genes for measuring mRNA levels by means of a real-time reverse transcription polymerase chain reaction (RT-PCR) in various tissues. Thirteen patients with binocular cataracts of the same grade were included in the study after giving informed consent. A piece of the anterior capsule, along with a sample of lenticular epithelial cells (LECs), was collected as a pre-intake sample during cataract surgery. Ocuvite + Lutein(®), an antioxidant supplement, was administered orally beginning the day after surgery. Six weeks later, a piece of the anterior capsule along with a sample of LECs, was collected as a post-intake sample during cataract surgery of the opposite eye. RNA was purified from the homogenized samples, and cDNA was reverse transcribed to measure mRNA levels. The expression levels of G6PDH, 18S and ß-ACT were measured using RT-PCR. The expression levels of G6PDH and 18S were significantly higher in the post-intake samples than they were in the pre-intake samples. Significant positive correlations between the expression levels of G6PDH and 18S were observed in both the pre- and post-intake samples. Following gender-specific analyses, the expression levels of G6PDH and 18S in the post-intake samples were found to be significantly higher among the female patients. A significant positive correlation between the expression levels of G6PDH and 18S was observed in the post-intake samples from the male patients. There were no significant changes in the gene expression levels of ß-ACT after supplementation among male or female patients. ß-ACT has been verified for use as a reference gene for measuring the effects of antioxidant supplementation in the lens by RT-PCR. Antioxidant supplementation was noted to increase G6PDH in the pentose phosphate cycle and 18S rRNA in the ribosome.

Diverse roles of Eph/ephrin signaling in the mouse lens.

Recent genetic studies show that the Eph/ephrin bidirectional signaling pathway is associated with both congenital and age-related cataracts in mice and humans. We have investigated the molecular mechanisms of cataractogenesis and the roles of ephrin-A5 and EphA2 in the lens. Ephrin-A5 knockout ⁻/⁻ mice often display anterior polar cataracts while EphA2⁻/⁻ lenses show very mild cortical or nuclear cataracts at weaning age. The anterior polar cataract of ephrin-A5⁻/⁻ lenses is correlated with multilayers of aberrant cells that express alpha smooth muscle actin, a marker for mesenchymal cells. Only select fiber cells are altered in ephrin-A5⁻/⁻ lenses. Moreover, the disruption of membrane-associated ß-catenin and E-cadherin junctions is observed in ephrin-A5⁻/⁻ lens central epithelial cells. In contrast, EphA2⁻/⁻ lenses display normal monolayer epithelium while disorganization is apparent in all lens fiber cells. Immunostaining of ephrin-A5 proteins, highly expressed in lens epithelial cells, were not colocalized with EphA2 proteins, mainly expressed in lens fiber cells. Besides the previously reported function of ephrin-A5 in lens fiber cells, this work suggests that ephrin-A5 regulates ß-catenin signaling and E-cadherin to prevent lens anterior epithelial cells from undergoing the epithelial-to-mesenchymal transition while EphA2 is essential for controlling the organization of lens fiber cells through an unknown mechanism. Ephrin-A5 and EphA2 likely interacting with other members of Eph/ephrin family to play diverse functions in lens epithelial cells and/or fiber cells.

Growth factor deposition in anterior subcapsular cataract.

PURPOSE: To characterize immunohistochemically the distribution of growth factors and extracellular matrix (ECM) components in an anterior subcapsular cataract (ASC) and to determine the role of growth factors in the development of ASC. SETTING: Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan. METHODS: During cataract surgery in 22 patients, anterior capsules with an ASC were obtained. Sections of each specimen were immunostained with a panel of antibodies against ECM components, growth factors, cytoskeletal components, and signal transduction-related molecules. RESULTS: Collagen types I, V, and VI; fibronectin; fibrillin-1; and latent transforming growth factor beta binding protein-1 (LTBP-1) were localized to the ECM in ASC tissues. Collagen IV was localized to the ECM and the capsule. Lens epithelial cells (LECs) were positive for alpha-smooth muscle actin (alphaSMA). Lens epithelial cells and ECM stained for transforming growth factor beta2 (TGFbeta2) and TGFbeta3 in all samples, but TGFbeta1 latency-associated peptide (TGFbeta1-LAP) were detected in some samples. Fibroblast growth factor-2 (FGF-2) and hepatocyte growth factor-alpha (HGF-alpha) were localized to the ECM. Lens epithelial cells with nuclear staining for Erk-1, the mitogen-activated protein kinase (MAP kinase) cascade-related molecule, and Smad3, 1 of the Smad family members involving TGFbeta signaling, were detected. CONCLUSIONS: Matrix components (ie, collagen types, fibronectin, fibrillin-1), as well as growth factors such as TGFbeta1-LAP, TGFbeta2, TGFbeta3, FGF-2, and HGF-alpha, were detected in ASC. Fibrillin-1 might serve as a repository for TGFbetas. These growth factors may modulate the phenotypic alteration and behavior of LECs. The MAP kinase cascade and TGFbeta signaling are both activated in LECs in ASC.