Lens autophagy protein ATG16L1: a potential target for cataract treatment.

Rationale: Cataract is the leading cause of blindness and low vision worldwide, yet its pathological mechanism is not fully understood. Although macroautophagy/autophagy is recognized as essential for lens homeostasis and has shown potential in alleviating cataracts, its precise mechanism remains unclear. Uncovering the molecular details of autophagy in the lens could provide targeted therapeutic interventions alongside surgery. Methods: We monitored autophagic activities in the lens and identified the key autophagy protein ATG16L1 by immunofluorescence staining, Western blotting, and transmission electron microscopy. The regulatory mechanism of ATG16L1 ubiquitination was analyzed by co-immunoprecipitation and Western blotting. We used the crystal structure of E3 ligase gigaxonin and conducted the docking screening of a chemical library. The effect of the identified compound riboflavin was tested in vitro in cells and in vivo animal models. Results: We used HLE cells and connexin 50 (cx50)-deficient cataract zebrafish model and confirmed that ATG16L1 was crucial for lens autophagy. Stabilizing ATG16L1 by attenuating its ubiquitination-dependent degradation could promote autophagy activity and relieve cataract phenotype in cx50-deficient zebrafish. Mechanistically, the interaction between E3 ligase gigaxonin and ATG16L1 was weakened during this process. Leveraging these mechanisms, we identified riboflavin, an E3 ubiquitin ligase-targeting drug, which suppressed ATG16L1 ubiquitination, promoted autophagy, and ultimately alleviated the cataract phenotype in autophagy-related models. Conclusions: Our study identified an unrecognized mechanism of cataractogenesis involving ATG16L1 ubiquitination in autophagy regulation, offering new insights for treating cataracts.

Targeted dexamethasone nano-prodrug for corneal neovascularization management.

BACKGROUND: To overcome the drawbacks of traditional therapy for corneal neovascularization (CNV), we evaluated the efficacy of polyethylene glycol (PEG)-conjugated Ala-Pro-Arg-Pro-Gly (APRPG) peptide modified dexamethasone (Dex), a novel nano-prodrug (Dex-PEG-APRPG, DPA). METHODS: Characterization of DPA nano-prodrug were measured with transmission electron microscopy (TEM) and dynamic light scattering (DLS) analyses. Cytotoxicity and effects on cell migration and tube formation of DPA were evaluated in vitro. A murine CNV model was established by cornea alkali burn. The injured corneas were given eye drops of DPA (0.2 mM), Dex solution (0.2 mM), Dexp (2 mM), or normal saline three times a day. After two weeks, eyes were obtained for the analysis of histopathology, immunostaining, and mRNA expression. RESULTS: DPA with an average diameter of 30 nm, presented little cytotoxicity and had good ocular biocompatibility. More importantly, DPA showed specific targeting to vascular endothelial cells with efficient inhibition on cell migration and tube formation. In a mouse CNV model, clinical, histological, and immunohistochemical examination results revealed DPA had a much stronger angiogenesis suppression than Dex, resembling a clinical drug with an order of magnitude higher concentration. This was ascribed to the significant downregulations in the expression of pro-angiogenic and pro-inflammatory factors in the corneas. In vivo imaging results also demonstrated that APRPG could prolong ocular retention time. CONCLUSIONS: This study suggests that DPA nano-prodrug occupies advantages of specific targeting ability and improved bioavailability over conventional therapy, and holds great potential for safe and efficient CNV therapy.

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.

Ferrostatin-1-loaded liposome for treatment of corneal alkali burn via targeting ferroptosis.

Alkali burn is a potentially blinding corneal injury. During the progression of alkali burn-induced injury, overwhelmed oxidative stress in the cornea triggers cell damage, including oxidative changes in cellular macromolecules and lipid peroxidation in membranes, leading to impaired corneal transparency, decreased vision, or even blindness. In this study, we identified that ferroptosis, a type of lipid peroxidation-dependent cell death, mediated alkali burn-induced corneal injury. Ferroptosis-targeting therapy protected the cornea from cell damage and neovascularization. However, the specific ferroptosis inhibitor ferrostatin-1 (Fer-1) is hydrophobic and cannot be directly applied in the clinic. Therefore, we developed Fer-1-loaded liposomes (Fer-1-NPs) to improve the bioavailability of Fer-1. Our study demonstrated that Fer-1-NPs exerted remarkable curative effects regarding corneal opacity and neovascularization in vivo. The efficacy was comparable to that of dexamethasone, but without appreciable side effects. The significant suppression of ferroptosis (induced by lipid peroxidation and mitochondria disruption), inflammation, and neovascularization might be the mechanisms underlying the therapeutic effect of Fer-1-NPs. Moreover, the Fer-1-NPs treatment showed no signs of cytotoxicity, hematologic toxicity, or visceral organ damage, which further confirmed the biocompatibility. Overall, Fer-1-NPs provide a new prospect for safe and effective therapy for corneal alkali burn.

Necroptosis contributes to airborne particulate matter-induced ocular surface injury.

In this study, we explored the role of necroptosis in the pathogenesis of ocular surface injury caused by airborne particulate matter (PM). Human corneal epithelial (HCE) cells and mouse ocular surface were treated with PM exposure and compared with non-exposed groups. The expression of necroptosis-related proteins was measured by immunoblotting in HCE cell groups. Cell damages were detected using CCK-8, flow cytometry, and immunofluorescence staining. In the mouse model, hematoxylin and eosin (H&E) staining and corneal fluorescein sodium staining were assessed. In addition, the expression of inflammatory cytokines and mucin were examined via Enzyme-linked immunosorbent assay (ELISA), immunofluorescence staining and/or quantitative RT -PCR (qRT-PCR), both in vitro and in vivo. Our research showed that PM exposure may trigger HCE cell damage via necroptosis. Necrostatin-1(Nec-1), one of the specific inhibitors of necroptosis, can markedly reduce PM-induced HCE cell damage. HCE cell damage markers included decreased cell viability, increased intracellular reactive oxygen species (ROS) levels, and loss of mitochondrial membrane potential. At the same time, Nec-1 inhibited the increased inflammatory cytokines and the decreased mucin expression caused by PM exposure in HCE cells. Nec-1 also reduced corneal inflammation and mucin underproduction in mouse ocular surface after PM exposure. Our study demonstrated that necroptosis is involved in the pathogenesis of PM exposure-related ocular surface injury, including inflammation and insufficient mucin production in the cornea, which can be rescued by inhibitor Nec-1. This suggests Nec-1 could be a novel therapeutic target for ocular surface disorders, especially dry eye disease, which is caused by the exacerbation of airborne PM pollution.

Exosomal microRNA⁃93⁃3p secreted by bone marrow mesenchymal stem cells downregulates apoptotic peptidase activating factor 1 to promote wound healing.

Wounds are soft tissue injuries, which are difficult to heal and can easily lead to other skin diseases. Bone marrow mesenchymal stem cells (BMSCs) and the secreted exosomes play a key role in skin wound healing. This study aims to clarify the effects and mechanisms of exosomes derived from BMSCs in wound healing. Exosomes were extracted from the supernatant of the BMSCs. The expression of the micro-RNA miR-93-3p was determined by qRT-PCR analysis. HaCaT cells were exposed to hydrogen peroxide (H2O2) to establish a skin lesion model. MTT, flow cytometry, and transwell assays were conducted to determine cellular functions. The binding relationship between miR-93-3p and apoptotic peptidase activating factor 1 (APAF1) was measured using a dual luciferase reporter gene assay. The results showed that BMSC-derived exosomes or BMSC-exos promoted proliferation and migration and suppressed apoptosis in HaCaT cells damaged by H2O2. However, the depletion of miR-93-3p in BMSC-exos antagonized the effects of BMSC-exos on HaCaT cells. In addition, APAF1 was identified as a target of miR-93-3p. Overexpression of APAF1 induced the dysfunction of HaCaT cells. Collectively, the results indicate that BMSC-derived exosomes promote skin wound healing via the miR-93-3p/APAF1 axis. This finding may help establish a new therapeutic strategy for skin wound healing.

Ocular Wnt/ß-Catenin Pathway Inhibitor XAV939-Loaded Liposomes for Treating Alkali-Burned Corneal Wound and Neovascularization.

Corneal wound involves a series of complex and coordinated physiological processes, leading to persistent epithelial defects and opacification. An obstacle in the treatment of ocular diseases is poor drug delivery and maintenance. In this study, we constructed a Wnt/ß-catenin pathway inhibitor, XAV939-loaded liposome (XAV939 NPs), and revealed its anti-inflammatory and antiangiogenic effects. The XAV939 NPs possessed excellent biocompatibility in corneal epithelial cells and mouse corneas. In vitro corneal wound healing assays demonstrated their antiangiogenic effect, and LPS-induced expressions of pro-inflammatory genes of IL-1ß, IL-6, and IL-17α were significantly suppressed by XAV939 NPs. In addition, the XAV939 NPs significantly ameliorated alkali-burned corneas with slight corneal opacity, reduced neovascularization, and faster recovery, which were attributed to the decreased gene expressions of angiogenic and inflammatory cytokines. The findings supported the potential of XAV939 NPs in ameliorating corneal wound and suppressing neovascularization, providing evidence for their clinical application in ocular vascular diseases.

Ultrastructural and immunofluorescence analysis of anterior lens capsules in autosomal recessive Alport syndrome.

Background: To first report and study the ultrastructural and immunofluorescence abnormalities of the lens anterior capsules in a patient with autosomal recessive Alport syndrome.Methods: Two anterior lens capsules were collected in femtosecond laser-assisted cataract surgeries from a 29-year-old male patient with bilateral lenticonus caused by autosomal recessive Alport syndrome. The left capsule was examined by transmission electron microscopy and the right capsule was serial sectioned and stained with antibodies against the α2, α3, and α4 chains of type Ⅳ collagen. Anterior lens capsules of another two uncomplicated age-related cataract patients were collected and treated in the same way as the control.Results: The novel findings are that the mitochondria in lens epithelial cells in autosomal recessive Alport syndrome patients increased, twisted, and exhibited high electron density. Characteristic ultrastructure changes of capsule thinning, vertical dehiscence, and irregular-shaped lens epithelial cells were also observed in the left anterior lens capsule. Normal reactivity against the α2 chain and decreased reactivity against the α3 and α4 chains were observed in the right anterior lens capsule.Conclusions: The homozygous c.4599 T > G mutation of COL4A4 not only affects the formation of type Ⅳ collagen networks in the extracellular matrix, but also affects the morphology and survival of the lens epithelial cells in the patient with autosomal recessive Alport syndrome. This study is the first report of the ultrastructural and immunofluorescence changes of anterior lens capsules in autosomal recessive Alport syndrome.

Cytoprotective role of humanin in lens epithelial cell oxidative stress­induced injury.

Oxidative stress-induced injury and apoptosis of human lens epithelial cells (HLECs) are early events in the development of age­related cataracts (ARCs). Humanin (HN) is a mitochondrial­related peptide that serves a cytoprotective role in various cell types and animal models. Following HN knockdown or overexpression, the level of reactive oxygen species (ROS), mitochondrial membrane potential and mitochondrial DNA copy number, cell viability, LDH activity and apoptosis of HLECs under oxidative stress were detected, and apoptosis and autophagy were detected via transmission electron microscopy. The results suggested that HN may be involved in the response of HLECs to oxidative stress, and that HN expression was significantly upregulated under oxidative stress conditions. Furthermore, exogenous HN reduced intracellular ROS content and mitochondrial damage, and enhanced mitochondrial biosynthesis; however, this protection was lost in an endogenous HN knockdown cell model. In addition, to the best of our knowledge, the present study was the first to identify that HN increased mitochondrial autophagy, which was involved in reducing ROS production under oxidative stress. The present study indicated a potential mechanism underlying the anti­oxidative damage and apoptotic effects of HN under oxidative stress. In conclusion, HN may be a potential therapeutic target for ARCs as it has a significant cellular protective effect on HLECs under oxidative stress; therefore, further study is required to investigate its role in the occurrence and development of ARCs.

Development of mucoadhesive cationic polypeptide micelles for sustained cabozantinib release and inhibition of corneal neovascularization.

Corneal neovascularization (CNV) is one of the leading risk factors for vision loss. Anti-angiogenic drugs can theoretically be extended to the treatment of CNV. However, the application of these drugs is often hindered by traditional administration methods, e.g., eye drops, which is ascribed to the unique structure of the cornea and tear film. In this study, cationic polypeptide nanoparticles with mucoadhesive ability that carry lipophilic cabozantinib (a tyrosine kinase inhibitor), called Cabo-NPs, were developed for sustained cabozantinib release and inhibition of CNV. The polypeptides were synthesized via N-carboxyanhydride ring-opening polymerization and could self-assemble into micelles with cabozantinib in aqueous solution. The Cabo-NPs possessed good biocompatibility both in corneal epithelial cells and mouse corneas. More importantly, in vitro angiogenesis assays demonstrated the strong inhibitory effect of Cabo-NPs on cell migration and tube formation. Furthermore, the Cabo-NPs exerted superior anti-angiogenic effects with remarkable reductions in the neovascular area, which were as effective as the clinical dexamethasone but without apparent side effects. The therapeutic mechanism of the Cabo-NPs is closely related to the significant decrease in proangiogenic and proinflammatory factors, suppressing neovascularization and inflammation. Overall, cationic Cabo-NPs offer a new prospect for safe and effective CNV treatment via enhancing the bioavailability of lipophilic cabozantinib.

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.