Role of Rapamycin and 3-MA in oxidative damage of HLECs caused by two doses of UVB radiation.

Background: This study compared the role of autophagy regulators Rapamycin and 3-MA in oxidative damage and apoptosis of human lens epithelial cells (HLECs) caused by two doses of Ultraviolet Radiation B (UVB). Methods: HLECs were irradiated with UVB, and two doses of UVB damage models were constructed. After treatment with autophagy regulators, cell damage tests such as CCK-8, LDH activity, and Ros detection were performed. Western blotting was used to detect the levels of autophagy-related proteins and apoptosis-related proteins. Quantitative real-time PCR (RT-qPCR) was used to detect the mRNA leve of secondary antioxidant enzymes.Flow cytometry was used to examine cell viability and apoptosis. Finally, the proportion of autophagy and apoptosis was observed by electron microscope. Results: Autophagy inhibitor 3-MA promoted oxidative damage and apoptosis of HLECs at low doses of UVB (5 mJ/cm2), which corresponds to 1.3 â€‹h of exposure to sunlight in human eyes. Under the high dose of UVB (50mJ/cm2), which is equivalent to 13 â€‹h of exposure to sunlight in human eyes, the autophagy inducer Rapamycin caused more extensive oxidative damage and apoptosis of HLECs. 3-MA was able to reduce this damage, indicating that moderate autophagy is necessary for HLECs to cope with mild oxidative stress. For high dose UVB-induced oxidative stress, the use of 3-MA inhibiting autophagy is more beneficial to reduce cell damage and apoptosis. The mechanisms include degradation of damaged organelles, regulation of the expression of antioxidant enzymes HO-1, NQO1, GCS and regulation of apoptosis-related proteins. Conclusions: Autophagy played different roles in HLECs oxidative stress induced by two doses of UVB. It provides new ideas for reducing oxidative damage and apoptosis of HLECs to prevent or delay the progression of age-related cataract (ARC).

Glycolysis Aids in Human Lens Epithelial Cells' Adaptation to Hypoxia.

Hypoxic environments are known to trigger pathological damage in multiple cellular subtypes. Interestingly, the lens is a naturally hypoxic tissue, with glycolysis serving as its main source of energy. Hypoxia is essential for maintaining the long-term transparency of the lens in addition to avoiding nuclear cataracts. Herein, we explore the complex mechanisms by which lens epithelial cells adapt to hypoxic conditions while maintaining their normal growth and metabolic activity. Our data show that the glycolysis pathway is significantly upregulated during human lens epithelial (HLE) cells exposure to hypoxia. The inhibition of glycolysis under hypoxic conditions incited endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production in HLE cells, leading to cellular apoptosis. After ATP was replenished, the damage to the cells was not completely recovered, and ER stress, ROS production, and cell apoptosis still occurred. These results suggest that glycolysis not only performs energy metabolism in the process of HLE cells adapting to hypoxia, but also helps them continuously resist cell apoptosis caused by ER stress and ROS production. Furthermore, our proteomic atlas provides possible rescue mechanisms for cellular damage caused by hypoxia.

Znf469 Plays a Critical Role in Regulating Synthesis of ECM: A Zebrafish Model of Brittle Cornea Syndrome.

Purpose: Brittle cornea syndrome (BCS) is a rare autosomal recessive disorder characterized by extreme thinning and fragility of the cornea, and mutations in ZNF469 cause BCS-1. We aimed to establish a znf469 mutant zebrafish line to explore its roles and possible pathogenic mechanism in cornea development and disorder. Methods: znf4694del/4del mutant zebrafish was generated using the CRISPR/Cas9 technology. Transmission electron microscopy (TEM) was performed to examine the phenotype of the cornea in different developmental stages. RNA sequencing and quantitative real-time polymerase chain reaction were used to reveal the molecular mechanism. Results: Macroscopically, homozygous znf469 mutant zebrafish larvae exhibited a curved body from 72 hours postfertilization, similar to kyphoscoliosis, and a noninflated swimbladder at 7 days postfertilization (dpf). TEM revealed an extreme reduction of corneal stroma thickness in homozygous znf469 mutant zebrafish in both the central and peripheral cornea from the early development stage. RNA-sequencing analysis demonstrated that the znf469 mutation leads to the decreased synthesis of various extracellular matrix (ECM) components, such as collagens and proteoglycans, but increased synthesis of 26S proteasome family members. Conclusions: The results of our work indicate that znf469 is a critical gene that, as a widely considered transcription factor, may regulate the synthesis and degradation of a large number of ECM components that play an important role in corneal development.

TP53INP2 Contributes to TGF-ß2-Induced Autophagy during the Epithelial-Mesenchymal Transition in Posterior Capsular Opacification Development.

BACKGROUND: Posterior capsule opacification (PCO) is the most common complication after cataract surgery, in which increased levels of transforming growth factor-beta 2 (TGF-ß2) accelerate PCO formation; however, the pathological mechanisms are not fully understood. This study aims to explore the regulation mechanism of TGF-ß2 in PCO formation via its autophagic functions. METHODS: The autophagic effect of TGF-ß2 was detected by transmission electron microscopy (TEM), Western blotting, and immunofluorescence analysis. The association between autophagy and the epithelial-mesenchymal transition (EMT) was evaluated by qPCR and Western blotting. The transcriptome analysis was used to uncover the molecular mechanism of TGF-ß2-induced PCO formation. RESULTS: TGF-ß2 specifically promotes autophagy flux in human lens epithelial cells. The activation of autophagy by rapamycin can promote EMT marker synthesis and improve cell migration. However, the inhibition of autophagy by 3-MA attenuates EMT. To uncover the molecular mechanisms, we performed RNA sequencing and found that TGF-ß2 elevated tumor protein p53-inducible nuclear protein2 (TP53INP2) expression, which was accompanied by a nuclear-to-cytoplasm translocation. Moreover, the knockdown of TP53INP2 blocked the TGF-ß2-induced autophagy and EMT processes, revealing that TP53INP2 plays an important role in TGF-ß2-induced autophagy during EMT. CONCLUSIONS: Taken together, the results of this study suggested that TP53INP2 was a novel regulator of PCO development by TGF-ß2, and notably, TP53INP2, may be a potential target for the pharmacological treatment of PCO.

KPNA4 is involved in cataract formation via the nuclear import of p53.

KPNA4 (also called importin-α3) belongs to the importin α adaptor proteins family, which orchestrates classical nuclear transport processes, importin-α/importin-ß1 pathway, and involves in cellular homeostasis. Disruption of balanced transport pathways may result in ectopic nuclear proteins and eventually cause diseases, mainly under the situation of cellular stress, such as oxidative stress. Little evidence is available on its cellular functions for high specific expression in lens. We firstly studied the role of KPNA4 in cataract formation. Lens defects were observed at an early age in kpna4 gene knockout zebrafish, generated by the CRISPR/Cas9 system. Those phenotype, including cloudy center part of the lens, via bright field microscopy, and the thinning of the LE layer, wider space between the adjacent LE and LF cells, irregular cells morphology and the increased number of holes inside the LE cells, which were detected by transmission electron microscopy, recapitulate the clinical features of cataract patients. As the p53-specific adaptor of the nuclear import, KPNA4 upregulated with the same pattern of p53 in hydrogen peroxide-induced apoptosis in human lens epithelia cells. Furthermore, the loss of Kpna4 resulted in the accumulation of p53 in the center of lens. Taken together, we showed that KPNA4 was involved in the formation of cataract, likely by mediating p53 nuclear transport.

Rapamycin relieves the cataract caused by ablation of Gja8b through stimulating autophagy in zebrafish.

Macroautophagy/autophagy is known to be important for intracellular quality control in the lens. GJA8 is a major gap junction protein in vertebrate lenses. Mutations in GJA8 cause cataracts in humans. The well-known cataractogenesis mechanism is that mutated GJA8 leads to abnormal assembly of gap junctions, resulting in defects in intercellular communication among lens cells. In this study, we observed that ablation of Gja8b (a homolog of mammalian GJA8) in zebrafish led to severe defects in organelle degradation, an important cause of cataractogenesis in developing lens. The role of autophagy in organelle degradation in lens remains disputable. Intriguingly, we also observed that ablation of Gja8b induced deficient autophagy in the lens. More importantly, in vivo treatment of zebrafish with rapamycin, an autophagy activator that inhibits MAPK/JNK and MTORC1 signaling, stimulated autophagy in the lens and relieved the defects in organelle degradation, resulting in the mitigation of cataracts in gja8b mutant zebrafish. Conversely, inhibition of autophagy by treatment with the chemical reagent 3-MA blocked these recovery effects, suggesting the important roles of autophagy in organelle degradation in the lens in gja8b mutant zebrafish. Further studies in HLE cells revealed that GJA8 interacted with ATG proteins. Overexpression of GJA8 stimulated autophagy in HLE cells. These data suggest an unrecognized cataractogenesis mechanism caused by ablation of Gja8b and a potential treatment for cataracts by stimulating autophagy in the lens.Abbreviations: 3-MA: 3-methyladenine; ATG: autophagy related; AV: autophagic vacuoles; Dpf: days post fertilization; GJA1: gap junction protein alpha 1; GJA3: gap junction protein alpha 3; GJA8: gap junction protein alpha 8; Hpf: hours post fertilization; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PtdIns3K: class III phosphatidylinositol 3-kinase; WT: wild type.

Dystrophia canthorum in Waardenburg syndrome with a novel MITF mutation.

AIM: To reveal a novel MITF gene mutation in Waardenburg syndrome (WS), which is an autosomal dominant inherited neurogenic disorder that consists of various degrees of sensorineural deafness and pigmentary abnormalities in the eyes, hair and skin. METHODS: The genetic analysis of the Chinese family was conducted by whole-exome sequencing, then the results were confirmed by Sanger sequencing. RESULTS: WS is classified into type I to IV, which are identified by the W index, clinical characteristics and additional features. The MITF gene mostly accounts for WS type II. In this study, a de novo heterozygous mutation in the MITF gene, c.638A>G in exon 7, was identified in the patient diagnosed with WS type I features, as the W index was 2.17 (over 2.10), with dystrophia canthorum, congenital bilateral profound hearing loss, bilateral heterochromia irides, premature greying of the hair, and excessive freckling on the face at birth. She also underwent refractive errors and esotropia, reduced pigmentation of the choroid and visible choroid vessels. The mutation was not found in previous studies or mutation databases. CONCLUSION: The novel mutation in the MITF gene, which altered the protein in amino acids 213 from the glutamic acid to glycine, is the genetic pathological cause for WS features in the patient. Those characteristics of this family revealed a novel genetic heterogeneity of MITF in WS, which expanded the database of MITF mutations and offered a possible in correcting the W index value of WS in distinct ethnicities. Moreover, ocular symptoms should be emphasized in all types of WS patients.

Three novel variants identified within ECM-related genes in Chinese Han keratoconus patients.

As the primary indication for corneal transplantation, the pathogenesis of keratoconus remains elusive. Aiming to identify whether any mutation from extracellular-matrix (ECM)-related genes contributes to the patients with sporadic cases of keratoconus (KC) from Chinese Han population, one hundred and fifty-three participants in total were enrolled in our study, including fifty-three KC patients and one hundred healthy controls. Mutational analysis of three ECM-related genes (LOX, COL5A1 and TIMP3) with next-generation sequencing and Sanger sequencing was performed. To further confirm the function of three ECM-related genes in the pathogenesis of keratoconus, we performed Real-time Quantitative PCR in vitro. Results showed that three new sequence variants (c.95 G > A in LOX, c.1372 C > T in COL5A1 and c.476 C > T in TIMP3) were identified in aforementioned ECM-related genes in KC patients without being detected among the healthy controls. According to the results of QPCR, we found that the expression levels of LOX and TIMP3 were decreased in the KC patients, while COL5A1 showed no significant difference of expression. This is the first time to screen so many ECM-related genes in Chinese keratoconus patients using next-generation sequencing. We find numerous underlying causal variants, enlarging lots of mutation spectrums and thus providing new sites for other investigators to replicate and for further research.

Association of IGF1R polymorphisms (rs1546713) with susceptibility to age-related cataract in a Han Chinese population.

AIM: To explore the susceptible association between the insulin-like growth factor-1 receptor (IGF1R) single nucleotide polymorphism (SNP) and age-related cataract (ARC), and investigate the underlying mechanisms in human lens epithelium (HLE) cells. METHODS: Totally 1190 unrelated participants, comprising 690 ARC patients and 500 healthy individuals in Han Chinese population were recruited and genotyped for target SNP. The χ 2-test was used to detect genotypic distribution between the patient and control groups and the logistic regression was performed to adjust the age and gender. Meanwhile, different biological experimental methods, such as cell counting kit 8 (CCK-8) assay, flow cytometry, quantitative real time polymerase chain reaction (Q-PCR) and Western blot, were used to detect cell viability, cell cycle progression and apoptosis in HLE cells or IGF1R knockdown HLE cells. RESULTS: The rs1546713 in IGF1R gene was identified (P=0.046, OR: 1.606, 95%CI: 1.245-2.071), which shown a significant relevance with ARC risk under the dominant model. The results demonstrated that IGF1R knockdown inhibited cell proliferation by inducing cell cycle arrested at S phase and promoting apoptosis. Mechanistically, the cell cycle blocked at S phase was linked with the alterations of cyclin A, cyclin B, cyclin E and P21. The pro-apoptosis function of IGF1R may related with stimulating the activation of Caspase-3 and altering the expression levels of apoptotic proteins, including Bcl-2, Bax and Caspase-3. CONCLUSION: This study first report that IGF1R polymorphisms may affect susceptibility to ARCs in Han Chinese population and provide new clues to understand the pathogenic mechanism of ARCs. Notably, IGF1R is likely a potential target for ARC prevention and treatment.

Scanning and transmission electron microscopy study of anterior lens epithelium in presenile cataract.

PURPOSE: To study the structure of lens epithelial cells (LECs) in the anterior lens epithelium of presenile cataract and to further explore the possible reasons for presenile cataract development. METHODS: The anterior lens capsules (aLCs) of patients with presenile cataracts and patients with ordinary age-related cataracts were obtained from routine cataract surgery, and the 5-5.5 mm circles of the central aLC were cut in half and prepared for transmission electron microscopy (TEM) and scanning electron microscopy (SEM). RESULTS: The most obvious structural changes in the LECs observed in both cataract groups by TEM were uneven thickness of the anterior lens epithelium, vacuolated cytoplasm and elongated nuclei. SEM showed abnormal structural changes in the LECs, with swollen cells and spheres on the anterior lens epithelium observed in both groups and holes formed by the LECs stretching observed only in the presenile cataract patients. The degeneration of the anterior lens epithelium and the structural changes in the LECs were observed more prominently in presenile cataract patients. CONCLUSIONS: Abnormal and prominently affected structural features of LECs were observed in the presenile compared to age-related cataract patients by TEM and SEM. We suppose that ultrastructural pathological changes in the anterior lens epithelial cells are one of the important reasons for the development of presenile and age-related cataract.

Socioeconomic disparities in the global burden of glaucoma: an analysis of trends from 1990 to 2016.

PURPOSE: To assess the global burden of glaucoma by year, age, sex, regions, socioeconomic development, and mean years of schooling (MYS) by using disability-adjusted life year (DALY), then to explore the health inequality with socioeconomic status in glaucoma. METHODS: Global, national, and regional DALY data of glaucoma by year, age, and sex were extracted from the Global Health Data Exchange. Human development index (HDI) and national MYS in 2015 were obtained from the Human Development Report (HDR) 2016. Mann-Whitney U test was performed to explore the sexual difference in global DALYs. Kruskal-Wallis tests were performed to explore the difference of age-standardized DALY rates across WHO regions and HDI-related country groups. Linear regression analyses were performed to explore the association between age-standardized DALY rates with HDI and MYS. Health-related Gini coefficients and concentration indexes were calculated to evaluate the trends in health inequality of glaucoma since 1990. RESULTS: DALY numbers, crude DALY rates, and age-standardized DALY rates increased by 118.0%, 55.22%, and 12.12%, respectively, since 1990. Global DALY numbers and crude DALY rates increased with age, and Mann-Whitney U test revealed no significant sex difference in global DALY numbers (P = 0.807) and global crude DALYs rates (P = 0.976) for each age group in 2016. Africa and Eastern Mediterranean had higher age-standardized DALY rates than the global one in 2016. Kruskal-Wallis test indicated significant difference in age-standardized DALY rates across WHO regions (χ2 = 94.227, P < 0.001). Linear regression analysis indicated that HDI (adjusted R2 = 0.079; F = 16.722, P < 0.001) and MYS (adjusted R2 = 0.108; F = 23.048, P < 0.001) had a significant effect on age-standardized DALY rates. Gini coefficients rose from 0.290 in 1990 to 0.292 in 2015 with a peak value 0.299 in 2005, concentration index declined from 1990 (- 0.099) to 2000 (- 0.077) with reaching a low peak value, then rapidly increased to - 0.097 in 2015. CONCLUSIONS: With population growth and aging, global burden of glaucoma is increasing and older age, lower socioeconomic status, and lower MYS are associated with higher glaucoma burden. Our results help to gain a better understanding of glaucoma and guide future health policies tailored for public.

The impact of GJA3 SNPs on susceptibility to age-related cataract.

AIM: To determine the association of gap junction protein alpha 3 (GJA3) gene tag single-nucleotide polymorphisms (SNPs) with susceptibility to age-related cataract (ARC). METHODS: In total, 486 ARC patients were matched with 500 healthy controls. All the participants underwent complete ophthalmic examinations. Haplotype-tagging SNPs of GJA3 gene were selected from the HapMap Beijing Han Chinese population. Genomic DNA was extracted from the peripheral blood leukocytes of all the subjects. Under three different genetic models: dominant, recessive, and additive, the association between SNPs and ARC was examined. After adjusting for age and sex, the genetic effects of the GJA3 SNPs were evaluated with logistic regression analysis. RESULTS: Four tag GJA3 SNPs (rs6490519, rs9506430, rs9509053, and rs9552089) were included in the present study. None of the SNPs showed a significant relationship with an altered risk of total ARC under the dominant, recessive, or additive models. In the subgroup analysis, rs9506430 had a significant effect on the formation of a posterior subcapsular cataract (P=0.002, OR: 0.227, 95%CI: 0.088-0.590) under the recessive model. CONCLUSION: Our study indicates that GJA3 variants may influence the development of posterior subcapsular cataracts. Further studies need to be designed to confirm this possibility.

The impact of GJA8 SNPs on susceptibility to age-related cataract.

The gap junction protein alpha 8 (GJA8) gene has been widely studied in human congenital cataracts. However, little is known about its relationship with age-related cataract (ARC). In this study, three GJA8-tagged single nucleotide polymorphisms related to an increased ARC risk were identified: rs2132397 for general ARC under both dominant and additive models; rs7541950 for general ARC under both recessive and additive models; and rs6657114 for cortical cataract under the recessive model. To uncover the underlying mechanisms, this study also sought to explore whether GJA8 is involved in the autophagy process in human lens epithelial cells. The results showed that GJA8 may participate in autophagy to maintain the intracellular environment, which may be a novel mechanism for cataract formation induced by GJA8. In conclusion, this study identified the genetic susceptibility of GJA8 polymorphisms on ARC and provides new clues for fully understanding the pathological mechanism of GJA8 variants in affecting lens opacity.

Hydrogen peroxide-induced apoptosis of human lens epithelial cells is inhibited by parthenolide.

AIM: To explore the effect of parthenolide on hydrogen peroxide (H2O2)-induced apoptosis in human lens epithelial (HLE) cells. METHODS: The morphology and number of apoptotic HLE cells were assessed using light microscopy and flow cytometry. Cell viability was tested by MTS assay. In addition, the expression of related proteins was measured by Western blot assay. RESULTS: Apoptosis of HLE cells was induced by 200 µmol/L H2O2, and the viability of these cells was similar to the half maximal inhibitory concentration (IC50), as examined by MTS assay. In addition, cells were treated with either different concentrations (6.25, 12.5, 25 and 50 µmol/L) of parthenolide along with 200 µmol/L H2O2 or only 50 µmol/L parthenolide or 200 µmol/L H2O2 for 24h. Following treatment with higher concentrations of parthenolide (50 µmol/L), fewer HLE cells underwent H2O2-induced apoptosis, and cell viability was increased. Further, Western blot assay showed that the parthenolide treatment reduced the expression of caspase-3 and caspase-9, which are considered core apoptotic proteins, and decreased the levels of phosphorylated nuclear factor-κB (NF-κB), ERK1/2 [a member of the mitogen-activated protein kinase (MAPK) family], and Akt proteins in HLE cells. CONCLUSION: Parthenolide may suppress H2O2-induced apoptosis in HLE cells by interfering with NF-κB, MAPKs, and Akt signaling.

Experimental assessment of novel PAX6 splicing mutations in two Chinese families with aniridia.

Aniridia is a rare, congenital ocular disorder caused by the mutations of the paired box gene-6 (PAX6) (OMIM 607108), which encodes a highly conserved transcriptional regulator. In order to investigate the clinical characterizations and genetic defects of two Chinese families affected with aniridia, we recruited the family members and 200 ethnically matched controls. The entire exons and flanking intronic sequences of the PAX6 gene (NG_008679.1) were analyzed and effects of variants on splicing were assessed in silico and in vitro using exon trapping assay with pET01. The donor site (c.1183+1G>A) mutation identified in family 1 would result in a complete skipping of exon 12 and cause a frameshift and run-on translation past the normal termination codon, creating an enlarged PAX6 protein with extended COOH-terminal domain. Novel c.1033-1_1033delinsCT mutation was detected in family 2. This mutation provoked both complete exon 12 skipping and partial skipping of exon 12 deleting 7bp. This would lead to a frameshift translation and the introduction of pre-mature termination code, which resulted in severely truncated PAX6 protein likely to be degraded. Our study further expands the spectrum of genetic pathology underlying PAX6.