In vitro assessment of the severity of deoxyribonucleic acid damage in different types of cataracts directly in lens epithelial cells.

Purpose: This study aimed to assess the severity of deoxyribonucleic acid (DNA) damage in lens epithelial cells (LECs) of senile cortical, nuclear, and posterior subcapsular cataracts. Methods: LECs were obtained from senile cortical, nuclear, and subcapsular types of cataracts after surgery. DNA damage in the cells was immediately assessed quantitatively using the CometScore™ software. Results: Comets were found in cataractous LECs. The formation of "comets" in the DNA of LECs can be visualized using single-cell gel electrophoresis and indicates DNA strand breaks because the damaged DNA migrates at a different rate than the nondamaged DNA. Maximal damage was observed in Grade 3 cortical, nuclear, and subcapsular forms of cataracts. Statistically significant DNA damage was seen between grades 1 and 3 of cortical type of cataract, grades 1 and 3 of nuclear type of cataract, and grades 2 and 3 and grades 1 and 3 of posterior subcapsular type of cataract. Conclusion: In patients with senile cataract, DNA of LECs was randomly damaged, and this type of damage was possibly caused by reactive oxygen species (ROS). Maximum DNA damage was found in patients with Grade 3 senile cortical, nuclear, and subcapsular type cataracts. The pathogenesis of senile cataracts is multifactorial and includes continuous molecular stress resulting from photooxidative stress, UV irradiation, and oxidative reactions.

Pre-hyperglycemia immune cell trafficking underlies subclinical diabetic cataractogenesis.

BACKGROUND: This work elucidates the first cellular and molecular causes of cataractogenesis. Current paradigm presupposes elevated blood glucose as a prerequisite in diabetic cataractogenesis. Novel evidence in our model of diabetic cataract challenges this notion and introduces immune cell migration to the lens and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs) as underlying causes. METHODS: Paucity of suitable animal models has hampered mechanistic studies of diabetic cataract, as most studies were traditionally carried out in acutely induced hyperglycemic animals. We introduced diabetic cataract in the Nile grass rat (NGR) that spontaneously develops type 2 diabetes (T2D) and showed its closeness to the human condition. Specialized stereo microscopy with dual bright-field illumination revealed novel hyperreflective dot-like microlesions in the inner cortical regions of the lens. To study immune cell migration to the lens, we developed a unique in situ microscopy technique of the inner eye globe in combination with immunohistochemistry. RESULTS: Contrary to the existing paradigm, in about half of the animals, the newly introduced hyper reflective dot-like microlesions preceded hyperglycemia. Even though the animals were normoglycemic, we found significant changes in their oral glucose tolerance test (OGTT), indicative of the prediabetic stage. The microlesions were accompanied with significant immune cell migration from the ciliary bodies to the lens, as revealed in our novel in situ microscopy technique. Immune cells adhered to the lens surface, some traversed the lens capsule, and colocalized with apoptotic nuclei of the lens epithelial cells (LECs). Extracellular degradations, amorphous material accumulations, and changes in E-cadherin expressions showed epithelial-mesenchymal transformation (EMT) in LECs. Subsequently, lens fiber disintegration and cataract progression extended into cortical, posterior, and anterior subcapsular cataracts. CONCLUSIONS: Our results establish a novel role for immune cells in LEC transformation and death. The fact that cataract formation precedes hyperglycemia challenges the prevailing paradigm that glucose initiates or is necessary for initiation of the pathogenesis. Novel evidence shows that molecular and cellular complications of diabetes start during the prediabetic state. These results have foreseeable ramifications for early diagnosis, prevention and development of new treatment strategies in patients with diabetes.

Surface modification of commercial intraocular lens by zwitterionic and antibiotic-loaded coating for preventing postoperative endophthalmitis.

After cataract surgery, to prevent possible postoperative endophthalmitis (POE) caused by attached pathogenic bacteria onto the surface of implanted intraocular lens (IOL), various antibiotic-loaded IOLs have been proposed and widely studied to inhibit bacterial infection. However, most of these developed antibiotic-loaded IOLs still suffer from shortcomings such as insufficient drug loading, short release time, poor biocompatibility, and risk of secondary infection. Herein, we propose a zwitterionic and high-drug loading coating for surface modification of commercial hydrophobic IOL with both antifouling and antibacterial properties to effectively prevent POE. In this strategy, zwitterionic poly(carboxylbetaine-co-dopamine methacrylamide) copolymers (pCBDA) and dopamine (DA) were first robustly co-deposited onto IOL surface via facile mussel-inspired chemistry, resulting in a hydrophilic coating (defined as PCB) without sacrificing the high light transmittance of the native IOL. Subsequently, amikacin (AMK), an amine-rich antibiotic was reversibly conjugated onto the coating through the acid-sensitive Schiff base bonds formed by the reaction between amino and catechol groups, with high-drug payload over ∼35.5 µg per IOL and 30 days of sustained drug release under weak acid environment. Benefiting from the antifouling property of zwitterionic pCBDA copolymers, the intraocularly implanted PCB/AMK-coated IOL could effectively resist the adhesion and proliferation of residual LECs to inhibit the development of posterior capsule opacification (PCO) without affecting the normal ocular tissues, demonstrating excellent in vivo biocompatibility. Moreover, the synergy of zwitterionic pCBDA and conjugated AMK with acidic-dependent release behavior endowed this PCB/AMK-coated IOL strong antibacterial activity against both in vitro biofilm formation and in vivo postoperative Staphylococcus aureus infection, suggesting its promising application in preventing POE.

Expression of oxysterols in human lenses: Implications of the sterol pathway in age-related cataracts.

Lanosterol, an oxysterol molecule, has been proposed to help maintain lens transparency by inhibiting the formation of protein aggregates. This sterol is produced by the enzyme lanosterol synthase and is part of a metabolic pathway that forms cholesterol as a final step. Abnormalities in lanosterol synthase are responsible for congenital cataracts. The αA-crystallin protein, which acts as a molecular chaperone to lanosterol synthase, has been reported to have anti-protein aggregation, anti-inflammatory and anti-apoptotic properties. In this work, we evaluated the correlation of lanosterol synthase and αA-crystallin in human cataractous lenses with the grade of opacity, as well as the expression of lanosterol synthase, farnesyl DPP, geranyl synthase and squalene epoxidase genes. Lanosterol synthase and αA-crystallin were overexpressed in cataractous lenses as well as farnesyl-DP synthase, squalene epoxidase, lanosterol synthase and geranyl synthase genes in cataratous lenses in comparison with normal lenses. Our data confirm that lanosterol synthase and the sterol pathway are upregulated in cataractous lenses. This argues for a functional role of the oxysterol pathway and its products as an important mediator in the pathogenesis of human cataracts.

Intravitreal chemotherapy-induced cataract in retinoblastoma: challenges and outcome.

A toddler with bilateral retinoblastoma (Rb) received intravenous chemotherapy for advanced tumours in both eyes. The right eye required enucleation due to a poor response, but the left eye was salvaged by additional treatment with intravitreal chemotherapy for tumour vitreous seeds. Though the vitreous seeds regressed, a gradually progressive cataract developed likely due to drug toxicity. On ensuring continued regression of tumour within the eye, cataract extraction was performed. Surgery was uneventful and the child recovered good functional vision in the left eye. Salvaging the eyeball followed by vision restoration through cataract surgery in the only remaining eye of the child necessitated a multidisciplinary approach involving the ocular oncologist, the paediatric medical oncologist and the paediatric ophthalmologist. Ensuring tumour quiescence within the eye clinically before cataract surgery was the most crucial element of management. Additionally, close monitoring of the progress of the cataract to decide when to intervene was also necessary to keep the risk of amblyopia at bay. The patient recovered good functional vision in the left eye after the surgery and remained free of any tumour recurrence or systemic metastasis at 10 months of follow-up.

Broadening the genotypic and phenotypic spectrum of MAF in three Chinese Han congenital cataracts families.

Pathogenic variants in the v-maf avian musculoaponeurotic fibrosarcoma oncogene homologue (MAF) encoding a transcription factor (from a unique subclass of basic leucine zipper transcription factors) are associated with isolated congenital cataracts (CCs) and Aymé-Gripp syndrome (AYGRPS). We collected detailed disease histories from, and performed comprehensive ophthalmic and systemic examinations in 269 patients with CCs; we then performed whole-exome sequencing. Pathogenicity assessments were evaluated using multiple predictive tools. The clinical validities of the reported gene-disease relationships for MAF genes (MAF-CCs and MAF-AYGRPS) were assessed using the ClinGen gene curation framework. We identified two novel (c.173C>A, p.Thr58Asn and c.947T>C, p. Leu316Pro) variants and one known (c.173C>T, p.Thr58Ile) MAF missense variant in three patients. We described novel phenotypes including cleft palate, macular hypoplasia, and retinal neovascularization in the peripheral avascular area and analyzed the genotype-phenotype correlations. We demonstrated associations of variants in the MAF C-terminal DNA-binding domain with CCs and associations of variants in the N-terminal transactivation domain of MAF with AYGRPS. We thus expand the genotypic and phenotypic spectrum of the MAF gene. The ClinGen gene curation framework results suggested that variants in different domains of MAF are associated with different diseases.

FOXO4 mediates resistance to oxidative stress in lens epithelial cells by modulating the TRIM25/Nrf2 signaling.

Oxidative stress damage to the lens is a key factor in most cataracts. Forkhead box O 4 (FOXO4), a member of the forkhead box O family, plays a pivotal role in oxidative stress. FOXO4 is upregulated in lens of age-related cataract patients, but its role in cataract has not been elucidated. Herein, we investigated the role and mechanism of FOXO4 during oxidative stress damage in lens epithelial cells. H2O2 treatment enhanced FOXO4 expression in HLEpiC cells. Short hairpin RNAs mediated FOXO4 silence aggravated H2O2-induced cell apoptosis. In addition, upon H2O2 exposure, silencing of FOXO4 reduced SOD and CAT activities, as well as increased intracellular MDA and ROS levels. FOXO4 silencing also inhibited Nrf2 nuclear translocation, followed by reducing the expressions of Nrf2-governed antioxidant genes HO-1 and NOQ-1. Exogenous overexpression of FOXO4 was also involved in this study and exhibited opposite effects of FOXO4-silencing. Mechanistically, FOXO4 directly bound the promoter of TRIM25 and regulated its transcription, thereby activating the Nrf2 signaling. Taken together, in the condition of oxidative stress, the expression of FOXO4 showed a compensatory upregulation and it exhibited an anti-oxidative effect by modulating the transcription of TRIM25, thus activating the Nrf2 signaling. The FOXO4/TRIM25/Nrf2 axis may be associated with the pathological mechanisms of cataract.

Biliverdin Reductase A Protects Lens Epithelial Cells against Oxidative Damage and Cellular Senescence in Age-Related Cataract.

Age-related cataract (ARC) is the common cause of blindness globally. Reactive oxygen species (ROS), one of the greatest contributors to aging process, leads to oxidative damage and senescence of lens epithelial cells (LECs), which are involved in the pathogenesis of ARC. Biliverdin reductase A (BVRA) has ROS-scavenging ability by converting biliverdin (BV) into bilirubin (BR). However, little is known about the protective effect of BVRA against ARC. In the present study, we measured the expression level of BVRA and BR generation in human samples. Then, the antioxidative property of BVRA was compared between the young and senescent LECs upon stress condition. In addition, we evaluated the effect of BVRA on attenuating H2O2-induced premature senescence in LECs. The results showed that the mRNA expression level of BVRA and BR concentration were decreased in both LECs and lens cortex of age-related nuclear cataract. Using the RNA interference technique, we found that BVRA defends LECs against oxidative stress via (i) restoring mitochondrial dysfunction in a BR-dependent manner, (ii) inducing heme oxygenase-1 (HO-1) expression directly, and (iii) promoting phosphorylation of ERK1/2 and nuclear delivery of nuclear factor erythroid 2-related factor 2 (Nrf2). Intriguingly, the antioxidative effect of BVRA was diminished along with the reduced BR concentration and repressed nuclear translocation of BVRA and Nrf2 in senescent LECs, which would be resulted from the decreased BVRA activity and impaired nucleocytoplasmic trafficking. Eventually, we confirmed that BVRA accelerates the G1 phase transition and prevents against H2O2-induced premature senescence in LECs. In summary, BVRA protects LECs against oxidative stress and cellular senescence in ARC by converting BV into BR, inducing HO-1 expression, and activating the ERK/Nrf2 pathway. This trial is registered with ChiCTR2000036059.

Identification and functional analysis of two GJA8 variants in Chinese families with eye anomalies.

In this study, we report on two different GJA8 variants related to congenital eye anomalies in two unrelated families, respectively. GJA8 (or Cx50) encoding a transmembrane protein to form lens connexons has been known as a common causative gene in congenital cataracts and its variants have recently been reported related to a wide phenotypic spectrum of eye defects. We identified two GJA8 variants, c.134G>T (p.Try45Leu, W45L) detected in a cataract family by Sanger sequencing and c.281G>A (p.Gly94Glu, G94E) found in a family with severe eye malformations including microphthalmia by whole-exome sequencing. These two variants were absent in healthy population and predicted deleterious by bioinformatic analysis. Furthermore, we compared the expression in cell lines between these mutants and the wildtype to explore their potential mechanism. Cell counting kit-8 assay showed that overexpression of either W45L or G94E decreased cell viability compared with wild-type Cx50 and the control. A lower protein level in W45L found by western blotting and fewer punctate fluorescent signals showed by fluorescence microscopy suggested that W45L may have less protein expression. A higher G94E protein level and abundant dotted distribution indicated that G94E may cause aberrant protein degradation and accumulation. Such results from in vitro assays confirmed the impact of these two variants and gave us a hint about their different pathogenic roles in different phenotypes. In conclusion, our study is the first to have the functional analysis of two GJA8 variants c.134G>T and c.281G>A in Chinese pedigrees and explore the impact of these variants, which can help in prenatal diagnosis and genetic counseling as well in basic studies on GJA8.

Further delineation of GEMIN4 related neurodevelopmental disorder with microcephaly, cataract, and renal abnormalities syndrome.

Pathogenic variants in GEMIN4 have recently been linked to an inherited autosomal recessive neurodevelopmental disorder characterized with microcephaly, cataracts, and renal abnormalities (NEDMCR syndrome). This report provides a retrospective review of 16 patients from 11 unrelated Saudi consanguineous families with GEMIN4 mutations. The cohort comprises 11 new and unpublished clinical details from five previously described patients. Only two missense, homozygous, pathogenic variants were found in all affected patients, suggesting a founder effect. All patients shared global developmental delay with variable ophthalmological, renal, and skeletal manifestations. In addition, we knocked down endogenous Drosophila GEMIN4 in neurons to further investigate the mechanism of the functional defects in affected patients. Our fly model findings demonstrated developmental defects and motor dysfunction suggesting that loss of GEMIN4 function is detrimental in vivo; likely similar to human patients. To date, this study presents the largest cohort of patients affected with GEMIN4 mutations. Considering that identifying GEMIN4 defects in patients presenting with neurodevelopmental delay and congenital cataract will help in early diagnosis, appropriate management and prevention plans that can be made for affected families.

Elevated endothelin-1 levels as risk factor for an impaired ocular blood flow measured by OCT-A in glaucoma.

The purpose of this study was to ascertain whether a correlation exists between glaucoma-associated alteration of ocular vascular haemodynamics and endothelin-1 (ET-1) levels exist. Eyes of patients with cataract (n = 30) or glaucoma (n = 68) were examined with optical coherence tomography (OCT) and OCT-angiography (OCT-A; AngioVue™-RTVue-XR; Optovue, Fremont, California, USA). The peripapillary and the macular vessel density (VD) values were measured. Inferior and superior retinal nerve fibre layer (RNFL) thickness loss was used for further OCT staging. Aqueous humour of the examined eye and plasma were sampled during cataract or glaucoma surgery and analysed by means of ELISA to determine their ET-1 level. Glaucoma eyes are characterised by reductions in RNFL thickness and VD that correlate significantly with the OCT GSS score. Peripheral and ocular ET-1 level were significantly elevated in patients with glaucoma and correlate positively with the OCT-GSS score of the entire study population. Peripapillary and macula VD of glaucoma patients correlates negatively with plasma ET-1 levels. Multivariable analysis showed a subordinate role of intraocular pressure predictive factor for impaired retinal blood flow compared with plasma ET-1 level in glaucoma. Peripheral ET-1 level serves as risk factor for detection of ocular blood flow changes in the optic nerve head region of glaucomatous eyes.

The human lens: An antioxidant-dependent tissue revealed by the role of caffeine.

Cataract is the leading cause of blindness worldwide and surgery is the only option to treat the disease. Although the surgery is considered to be relatively safe, complications may occur in a subset of patients and access to ophthalmic care may be limited. Due to a growing and ageing population, an increase in cataract prevalence is expected and its management will become a socioeconomic challenge. Hence, there is a need for an alternative to cataract surgery. It is well known that oxidative stress is one of the main pathological processes leading to the generation of the disease. Antioxidant supplementation may, therefore, be a strategy to delay or to prevent the progression of cataract. Caffeine is a widely consumed high-potency antioxidant and may be of interest for the prevention of the disease. This review aims to give an overview of the anatomy and function of the lens, its antioxidant and reactive oxygen species (ROS) composition, and the role of oxidative stress in cataractogenesis. Also, the pharmacokinetics and -dynamics of caffeine will be described and the literature will be reviewed to give an overview of its anti-cataract potential and its possible role in the prevention of the disease.

Crystallins as Important Pathogenic Targets for Accumulation of Structural Damages Resulting in Protein Aggregation and Cataract Development: Introduction to This Special Issue of Biochemistry (Moscow).

This issue of Biochemistry (Moscow) is dedicated to the role of protein misfolding and aggregation in cataract development. In fact, many genetic mutations or chemical and physical deleterious factors can initiate alterations in the macrostructural order and proper folding of eye lens proteins, which in some cases result in the formation of large light-scattering aggregates, affecting the quality of vision and making lens more prone to cataract development. Diabetes mellitus, which is associated with oxidative stress and mass production of highly reactive compounds, can accelerate unfolding and aggregation of eye lens proteins. This journal issue contains reviews and research articles that describe the destructive effects of mutations and highly reactive metabolites on the structure and function of lens crystallin proteins, as well important molecules in the lens's natural defense system involved in protection against deleterious effects of the physical and chemical factors.

Targeted gene sequencing of FYCO1 identified a novel mutation in a Pakistani family for autosomal recessive congenital cataract.

BACKGROUND: Congenital cataract is causing one-third of blindness worldwide. Congenital cataract is heterogeneous in its inheritance patterns. The current study is aimed to explore the unknown genetic causes underlying congenital cataracts. METHODS: Blood samples from affected and normal individuals of n = 25 Pakistani families identified with congenital cataracts were collected. Genomic DNA was extracted and Sanger sequencing was performed to identify novel pathogenic variants in the FYCO1 (MIM#607182) gene. Later structural bioinformatics tools and molecular dynamics simulations were performed to analyze the impact of these variants on protein structure and function. RESULTS: Sanger sequencing resulted in the identification of a novel splice site mutation (NM_024513.3: c.3151-29_3151-7del) segregating in an autosomal recessive manner. This novel variant was confirmed to be absent in the n = 300 population controls. Further, bioinformatics tools revealed the formation of a mutant protein with a loss of the Znf domain. In addition, we also found a previously known (c.4127 T > C; p.Leu1376Pro) mutation in four families. We also report a novel heterozygous variant (c.3419G > A; p.Arg1140Gln) in another family. CONCLUSIONS: In conclusion, we report a novel deletion (NM_024513.3: c.3151-29_3151-7del) in one family and a frequent homozygous missense mutation (c.4127 T > C; p.Leu1376Pro) in four Pakistani families. The current research highlights the importance of autophagy in lens development and maintaining its transparency.

[Research progress on the relationship between circular RNAs and cataract].

Cataract is the leading cause of visual impairment and blindness on a global scale. The pathogenesis of cataract is not completely understood. Circular RNAs (circRNAs) are a special kind of non-coding RNAs with high stability and conservation. They are widely involved in a variety of biological processes and diseases. Abnormal expression of circRNAs can participate in the development of cataract, affecting the function of lens epithelial cells through interacting with proteins and sponging microRNAs. They are possible targets for cataract prevention and treatment. This article reviews the research progress on the role of circRNAs in the occurrence and development of cataract.

MDM2-mediated ubiquitination of LKB1 contributes to the development of diabetic cataract.

Diabetic cataract (DC) is a common complication of diabetes mellitus. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a crucial event in the development of DC. Murine double minute 2 (MDM2) is an E3 ubiquitin ligase that promotes EMT by regulating diverse targets. However, little is known about how MDM2 is involved in the pathogenesis of DC. We found the mRNA and protein levels of MDM2 were up-regulated in the lens of DC patients and rats. Thus, high glucose (HG)-induced human lens epithelial cells (HLECs) were constructed for further investigation. The results showed that the level of MDM2 was increased in HG-cultured HLECs, and the MDM2 knockdown alleviated HG-induced abnormal migration, EMT, and oxidative stress damage. Moreover, co-immunoprecipitation and ubiquitination assays demonstrated that MDM2 down-regulated LKB1 expression by ubiquitination degradation. LKB1 was found to be lower expressed in human and rat DC lenses, and HG-stimulated HLECs. Also, LKB1 overexpression mitigated HG-induced dysfunction of HLECs. Finally, our data showed that the changes related to EMT and oxidative stress induced by MDM2 knockdown were restored by down-regulation of LKB1. Together, MDM2 may involve in the pathogenesis of DC through down-regulating LKB1. MDM2 might be an effective therapeutical target of DC.

Senescence marker protein30 protects lens epithelial cells against oxidative damage by restoring mitochondrial function.

Etiology and pathogenesis of age-related cataract is not entirely clear till now. Senescence marker protein 30 (SMP30) is a newly discovered anti-aging factor, which plays an important role in preventing apoptosis and reducing oxidative stress damage. Mitochondria are located at the intersection of key cellular pathways, such as energy substrate metabolism, reactive oxygen species (ROS) production and apoptosis. Oxidative stress induced by 4-hydroxynonenal (4-HNE) is closely related to neurodegenerative diseases and aging. Our study focused on the effect of SMP30 on mitochondrial homeostasis of human lens epithelial cells (HLECs) induced by 4-HNE. Western blots and qPCR were used to compare the expression of SMP30 protein in the residual lens epithelial cells in the lens capsule of age-related cataract (ARC) patients and the donated transparent lens capsule. On this basis, SMP30 overexpression plasmid and SMP30 shRNA interference plasmid were introduced to explore the effect of SMP30 on the biological behavior in HLECs under the condition of oxidative stress induced by 4-HNE through immunohistochemistry, ROS evaluation, metabolic spectrum analysis and JC-1 fluorescence measurement. Given that Nuclear Factor erythroid 2-Related Factor 2 (Nrf2)/Kelch Like ECH Associated Protein 1 (KEAP1) signaling pathway is the most important antioxidant stress pathway, we further analyzed the regulatory effect of SMP30 by WB to explore its molecular mechanism. Our study indicated that SMP30 may inhibit ROS accumulation, restore mitochondrial function, activate Nrf2/Keap1 signaling pathway, therefore protecting lens epithelial cells from oxidative stress-induced cell damage.