KRAS depletion suppresses ferroptosis and affects Hippo pathway in cataract.

Cataract, a painless and progressive disorder is manifested as the opacification of the lens that represents the most significant cause of blindness worldwide. The objective of this study is to unveil the function of Kirsten rat sarcoma (KRAS) and potential action mechanisms against cataract. The ferroptosis-associated differentially expressed genes (DEGs) and pivot genes were extracted through the comprehensive bioinformatics methods. Erastin was applied for inducing ferroptosis in hydrogen peroxide (H2O2)-treated SRA01/04 cells, and validated by detecting content of intracellular iron, glutathione (GSH), malondialdehyde (MDA). Additionally, the effects of KRAS deficiency on ferroptosis were determined by functional assays. The proteins expression related to ferroptosis and Hippo pathway were determined by Western blotting. A total of 73 ferroptosis-related DEGs were discovered, and 6 critical core genes were confirmed upregulation in cataract cell model. The H2O2-treated SRA01/04 cells exhibited decrease of cell viability and proliferation, iron accumulation, MDA increase, GSH consumption, rise of COX2 and decline of GPX4, with further aggravated under erastin treatment, while the phenomena were improved by KRAS knockdown. Additionally, KRAS deficiency was involved in the Hippo signalling pathway activation. Downregulation of KRAS might restrain ferroptosis and affect Hippo pathway in cataract.

The Functional Significance of High Cysteine Content in Eye Lens γ-Crystallins.

Cataract disease is strongly associated with progressively accumulating oxidative damage to the extremely long-lived crystallin proteins of the lens. Cysteine oxidation affects crystallin folding, interactions, and light-scattering aggregation especially strongly due to the formation of disulfide bridges. Minimizing crystallin aggregation is crucial for lifelong lens transparency, so one might expect the ubiquitous lens crystallin superfamilies (α and ßγ) to contain little cysteine. Yet, the Cys content of γ-crystallins is well above the average for human proteins. We review literature relevant to this longstanding puzzle and take advantage of expanding genomic databases and improved machine learning tools for protein structure prediction to investigate it further. We observe remarkably low Cys conservation in the ßγ-crystallin superfamily; however, in γ-crystallin, the spatial positioning of Cys residues is clearly fine-tuned by evolution. We propose that the requirements of long-term lens transparency and high lens optical power impose competing evolutionary pressures on lens ßγ-crystallins, leading to distinct adaptations: high Cys content in γ-crystallins but low in ßB-crystallins. Aquatic species need more powerful lenses than terrestrial ones, which explains the high methionine content of many fish γ- (and even ß-) crystallins. Finally, we discuss synergies between sulfur-containing and aromatic residues in crystallins and suggest future experimental directions.

Metabonomics analysis of aqueous humor samples from cataract patients with branch retinal vein occlusion.

Cataract (CAT) has a very high incidence rate among the middle-aged and elderly, with most patients complicated by branch retinal vein occlusion (BRVO), a key cause of blindness. In this study, through metabolomic analysis of aqueous humor samples from CAT patients with BRVO, a total of 319 different metabolites were found, most of which belonged to the categories of carboxylic acids and derivatives, fatty acyls, and organooxygen compounds. The most typical metabolites were 3-methylhistidine and biliverdin, which were up-regulated, as well as the down-regulated beta-glycerophosphoric acid. Tricosanoic acid showed the most significant correlation with CAT+BRVO. According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, the most commonly related keywords for differentially expressed metabolites were biosynthesis of unsaturated fatty acids and synaptic vesicle cycle. These results can not only help to further understand the pathogenesis of CAT complicated by BRVO in clinical practice, but also provide some new therapeutic research directions.

Prevention of age-related truncation of γ-glutamylcysteine ligase catalytic subunit (GCLC) delays cataract formation.

A sharp drop in lenticular glutathione (GSH) plays a pivotal role in age-related cataract (ARC) formation. Despite recognizing GSH's importance in lens defense for decades, its decline with age remains puzzling. Our recent study revealed an age-related truncation affecting the essential GSH biosynthesis enzyme, the γ-glutamylcysteine ligase catalytic subunit (GCLC), at aspartate residue 499. Intriguingly, these truncated GCLC fragments compete with full-length GCLC in forming a heterocomplex with the modifier subunit (GCLM) but exhibit markedly reduced enzymatic activity. Crucially, using an aspartate-to-glutamate mutation knock-in (D499E-KI) mouse model that blocks GCLC truncation, we observed a notable delay in ARC formation compared to WT mice: Nearly 50% of D499E-KI mice remained cataract-free versus ~20% of the WT mice at their age of 20 months. Our findings concerning age-related GCLC truncation might be the key to understanding the profound reduction in lens GSH with age. By halting GCLC truncation, we can rejuvenate lens GSH levels and considerably postpone cataract onset.

miR-26 Deficiency Causes Alterations in Lens Transcriptome and Results in Adult-Onset Cataract.

Purpose: Despite strong evidence demonstrating that normal lens development requires regulation governed by microRNAs (miRNAs), the functional role of specific miRNAs in mammalian lens development remains largely unexplored. Methods: A comprehensive analysis of miRNA transcripts in the newborn mouse lens, exploring both differential expression between lens epithelial cells and lens fiber cells and overall miRNA abundance, was conducted by miRNA sequencing. Mouse lenses lacking each of three abundantly expressed lens miRNAs (miR-184, miR-26, and miR-1) were analyzed to explore the role of these miRNAs in lens development. Results: Mice lacking all three copies of miR-26 (miR-26TKO) developed postnatal cataracts as early as 4 to 6 weeks of age. RNA sequencing analysis of neonatal lenses from miR-26TKO mice exhibited abnormal reduced expression of a cohort of genes found to be lens enriched and linked to cataract (e.g., Foxe3, Hsf4, Mip, Tdrd7, and numerous crystallin genes) and abnormal elevated expression of genes related to neural development (Lhx3, Neurod4, Shisa7, Elavl3), inflammation (Ccr1, Tnfrsf12a, Csf2ra), the complement pathway, and epithelial to mesenchymal transition (Tnfrsf1a, Ccl7, Stat3, Cntfr). Conclusions: miR-1, miR-184, and miR-26 are each dispensable for normal embryonic lens development. However, loss of miR-26 causes lens transcriptome changes and drives cataract formation.

The importance of the fourth Greek key motif of human γD-crystallin in maintaining lens transparency-the tale told by the tail.

Purpose: Congenital cataract affects 1-15 per 10,000 newborns worldwide, and 20,000-40,000 children are born every year with developmental bilateral cataracts. Mutations in the crystallin genes are known to cause congenital cataracts. Crystallins, proteins present in the eye lens, are made up of four Greek key motifs separated into two domains. Greek key motifs play an important role in compact folding to provide the necessary refractive index and transparency. The present study was designed to understand the importance of the fourth Greek key motif in maintaining lens transparency by choosing a naturally reported Y134X mutant human γD- crystallin in a Danish infant and its relationship to lens opacification and cataract. Methods: Human γD-crystallin complementary DNA (cDNA) was cloned into the pET-21a vector, and the Y134X mutant clone was generated by site-directed mutagenesis. Wild-type and mutant proteins were overexpressed in the BL21 DE3 pLysS cells of E. coli. Wild-type protein was purified from the soluble fraction using the ion exchange and gel filtration chromatography methods. Mutant protein was predominantly found in insoluble fraction and purified from inclusion bodies. The structure, stability, aggregational, and amyloid fibril formation properties of the mutant were compared to those of the wild type using the fluorescence and circular dichroism spectroscopy methods. Results: Loss of the fourth Greek key motif in human γD-crystallin affects the backbone conformation, alters the tryptophan micro-environment, and exposes a nonpolar hydrophobic core to the surface. Mutant is less stable and opens its Greek key motifs earlier with a concentration midpoint (CM) of unfolding curve of 1.5 M compared to the wild type human γD-crystallin (CM: 2.5 M). Mutant is capable of forming self-aggregates immediately in response to heating at 48.6 °C. Conclusions: Loss of 39 amino acids in the fourth Greek key motif of human γD-crystallin affects the secondary and tertiary structures and exposes the hydrophobic residues to the solvent. These changes make the molecule less stable, resulting in the formation of light-scattering particles, which explains the importance of the fourth Greek key in the underlying mechanism of opacification and cataract.

Dysregulation of Autophagy Occurs During Congenital Cataract Development in ßA3ΔG91 Mice.

Purpose: To examine lens phenotypic characteristics in ßA3ΔG91 mice and determine if ßA3ΔG91 affects autophagy in the lens. Methods: We generated a ßA3ΔG91 mouse model using CRISPR/Cas9 methodology. Comparative phenotypic and biochemical characterizations of lenses from postnatal day 0 (P0), P15, and 1-month-old ßA3ΔG91 and wild-type (WT) mice were performed. The methodologies used included non-invasive slit-lamp examination, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot, and immunohistochemical (IHC) analyses to determine the levels of autophagy-related genes and proteins. Transmission electron microscopy (TEM) analysis of lenses was performed to assess organelle degradation and the presence of autophagic vesicles. TUNEL staining was used to determine apoptosis in the lens. Results: Relative to WT lenses, 1-month-old ßA3ΔG91 mice developed congenital nuclear cataract and microphthalmia and showed an early loss of endoplasmic reticulum (ER) in the cortex and attenuation of nuclei degradation. This observation was confirmed by TEM analysis, as was the presence of autophagic vesicles in ßA3ΔG91 lenses. Comparative IHC and RT-qPCR analyses showed relatively higher levels of autophagy markers (ubiquitinated proteins and p62, LC3, and LAMP2 proteins) in ßA3ΔG91 lenses compared to WT lenses. Additionally, ßA3ΔG91 lenses showed relatively greater numbers of apoptotic cells and higher levels of cleaved caspase-3 and caspase-9. Conclusions: The deletion of G91 in ßA3ΔG91 mice leads to higher levels of expression of autophagy-related proteins and their transcripts relative to WT lenses. Taken together, G91 deletion in ßA3/A1-crystallin is associated with autophagy disruption, attenuation of nuclei degradation, and cellular apoptosis in the lens, which might be congenital cataract causative factors.

TGFß overcomes FGF-induced transinhibition of EGFR in lens cells to enable fibrotic secondary cataract.

To cause vision-disrupting fibrotic secondary cataract (PCO), lens epithelial cells that survive cataract surgery must migrate to the posterior of the lens capsule and differentiate into myofibroblasts. During this process, the cells become exposed to the FGF that diffuses out of the vitreous body. In normal development, such relatively high levels of FGF induce lens epithelial cells to differentiate into lens fiber cells. It has been a mystery as to how lens cells could instead undergo a mutually exclusive cell fate, namely epithelial to myofibroblast transition, in the FGF-rich environment of the posterior capsule. We and others have reported that the ability of TGFß to induce lens cell fibrosis requires the activity of endogenous ErbBs. We show here that lens fiber-promoting levels of FGF induce desensitization of ErbB1 (EGFR) that involves its phosphorylation on threonine 669 mediated by both ERK and p38 activity. Transinhibition of ErbB1 by FGF is overcome by a time-dependent increase in ErbB1 levels induced by TGFß, the activation of which is increased after cataract surgery. Our studies provide a rationale for why TGFß upregulates ErbB1 in lens cells and further support the receptor as a therapeutic target for PCO.

A grape-supplemented diet prevented ultraviolet (UV) radiation-induced cataract by regulating Nrf2 and XIAP pathways.

The purpose of this study is to investigate if grape consumption, in the form of grape powder (GP), could protect against ultraviolet (UV)-induced cataract. Mice were fed with the regular diet, sugar placebo diet, or a grape diet (regular diet supplemented with 5%, 10%, and 15% GP) for 3 months. The mice were then exposed to UV radiation to induce cataract. The results showed that the GP diet dose-dependently inhibited UV-induced cataract and preserved glutathione pools. Interestingly, UV-induced Nrf2 activation was abolished in the groups on the GP diet, suggesting GP consumption may improve redox homeostasis in the lens, making Nrf2 activation unnecessary. For molecular target prediction, a total of 471 proteins regulated by GP were identified using Agilent Literature Search (ALS) software. Among these targets, the X-linked inhibitor of apoptosis (XIAP) was correlated with all of the main active ingredients of GP, including resveratrol, catechin, quercetin, and anthocyanins. Our data confirmed that GP prevented UV-induced suppression of XIAP, indicating that XIAP might be one of the critical molecular targets of GP. In conclusion, this study demonstrated that GP protected the lens from UV-induced cataract development in mice. The protective effects of GP may be attributed to its ability to improve redox homeostasis and activate the XIAP-mediated antiapoptotic pathway.

Increased molar ratio of free fatty acids to albumin in blood as cause and early biomarker for the development of cataracts and Alzheimer's disease.

Cataracts and Alzheimer's disease (AD) are closely linked and are associated with aging and with systemic diseases that increase the molar ratio of free fatty acids to albumin (mFAR) in the blood. From the results of our earlier studies on the development of senile cataracts and from results recently published in the literature on the pathogenesis of Alzheimer's disease, we suggest that there is a common lipotoxic cascade for both diseases, explaining the strong connection between aging, an elevated mFAR in the blood, cataract formation, and AD. Long-chain free fatty acids (FFA) are transported in the blood as FFA/albumin complexes. In young people, vascular albumin barriers in the eyes and brain, very similar in their structure and effect, reduce the FFA/albumin complex concentration from around 650 µmol/l in the blood to 1-3 µmol/l in the aqueous humour of the eyes as well as in the cerebrospinal fluid of the brain. At such low concentrations the fatty acid uptake of the target cells - lens epithelial and brain cells - rises with increasing FFA/albumin complex concentrations, especially when the fatty acid load of albumin molecules is mFAR>1. At higher albumin concentrations, for instance in blood plasma or the interstitial tissue spaces, the fatty acid uptake of the target cells becomes increasingly independent of the FFA/albumin complex concentration and is mainly a function of the mFAR (Richieri et al., 1993). In the blood plasma of young people, the mFAR is normally below 1.0. In people over 40 years old, aging increases the mFAR by decreasing the plasma concentration of albumin and enhancing the plasma concentrations of FFA. The increase in the mFAR in association with C6-unsaturated FFA are risk factors for the vascular albumin barriers (Hennig et al., 1984). Damage to the vascular albumin barrier in the eyes and brain increases the concentration of FFA/albumin complex in the aqueous humour as well as in the cerebrospinal fluid, leading to mitochondrial dysfunction and the death of lens epithelial and brain cells, the development of cataracts, and AD. An age-dependent increase in the concentration of FFA/albumin complex has been found in the aqueous humour of 177 cataract patients, correlating with the mitochondria-mediated apoptotic death of lens epithelial cells, lens opacification and cataracts (Iwig et al., 2004). Mitochondrial dysfunction is also an early crucial event in Alzheimer's pathology, closely connected with the generation of amyloid beta peptides (Leuner et al., 2012). Very recently, amyloid beta production has also been confirmed in the lenses of Alzheimer's patients, causing cataracts (Moncaster et al., 2022). In view of this, we propose that there is a common lipotoxic cascade for senile cataract formation and senile AD, initiated by aging and/or systemic diseases, leading to an mFAR>1 in the blood.

Proteomic characteristics of the aqueous humor in Uyghur patients with pseudoexfoliation syndrome and pseudoexfoliative glaucoma.

Pseudoexfoliation syndrome (PEX) is characterized by the deposition of fibrous pseudoexfoliation material (PEXM) in the eye, and secondary glaucoma associated with this syndrome has a faster and more severe clinical course. The incidence of PEX and pseudoexfoliative glaucoma (PEXG) exhibits ethnic clustering; however, few proteomic studies related to PEX and PEXG have been conducted in Asian populations. Therefore, we aimed to conduct proteomic analysis on the aqueous humor (AH) obtained from Uyghur patients with cataracts, those with PEX and cataracts, and those with PEXG and cataracts to better understand the molecular mechanisms of the disease and identify its potential biomarkers. To this end, AH was collected from patients with cataracts (n = 10, control group), PEX with cataracts (n = 10, PEX group), and PEXG with cataracts (n = 10, PEXG group) during phacoemulsification. Label-free quantitative proteomic techniques combined with bioinformatics were used to identify and analyze differentially expressed proteins (DEPs) in the AH of PEX and PEXG groups. Then, independent AH samples (n = 12, each group) were collected to validate DEPs by enzyme-linked immunosorbent assay (ELISA). The PEX group exhibited 25 DEPs, while the PEXG group showed 44 DEPs, both compared to the control group. Subsequently, we found three newly identified proteins in both PEX and PEXG groups, wherein FRAS1-related extracellular matrix protein 2 (FREM2) and osteoclast-associated receptor (OSCAR) exhibited downregulation, whereas coagulation Factor IX (F9) displayed upregulation. Bioinformatics analysis suggested that extracellular matrix interactions, abnormal blood-derived proteins, and lysosomes were mainly involved in the process of PEX and PEXG, and the PPI network further revealed F9 may serve as a potential biomarker for both PEX and PEXG. In conclusion, this study provides new information for understanding the proteomics of AH in PEX and PEXG.

Metabolomic profiling of the aqueous humor in patients with pediatric cataract.

Pediatric cataract, including congenital and developmental cataract, is a kind of pediatric vision-threatening disease with extensive phenotypic heterogeneity and multiple mechanisms. We aimed to investigate the metabolite profile of aqueous humor (AH) in patients with pediatric cataracts, and identify underlying mutual correlations between differential metabolites. Metabolomic profiles of AH were analyzed and compared between pediatric cataract patients (n = 33) and age-related cataract patients without metabolic diseases (n = 29), using global untargeted metabolomics with ultra-high-performance liquid chromatography tandem mass spectrometry. Principal component analysis, partial least squares discriminant analysis and heat map were applied. Enriched pathway analysis was conducted using Kyoto Encyclopedia of Genes and Genomes. Receiver-operating characteristic (ROC) analyses were employed to select potential biomarkers. A total of 318 metabolites were identified, of which 54 differential metabolites (25 upregulated and 29 downregulated) were detected in pediatric cataract group compared with controls (variable importance of projection >1.0, fold change ≥1.5 or ≤ 0.667 and P < 0.05). A significant accumulation of N-Acetyl-Dl-glutamic acid was observed in pediatric cataract group. The differential metabolites were mainly enriched in histidine metabolism (increased L-Histidine and decreased 1-Methylhistamine) and the tryptophan metabolism (increased N-Formylkynurenine and L-Kynurenine). 5-Aminosalicylic acid showed strong positive mutual inter-correlation with L-Tyrosinemethylester and N,N-Diethylethanolamine, both of which were down-regulated in pediatric cataract group. The ROC analysis implied 11 metabolites served as potential biomarkers for pediatric cataract patients (all area under the ROC curve ≥0.900). These results illustrated novel potential metabolites and metabolic pathways in pediatric cataract, which provides new insights into the pathophysiology of pediatric cataract.

MicroRNA-1225-5p Promotes the Development of Fibrotic Cataracts via Keap1/Nrf2 Signaling.

PURPOSE: Fibrotic cataracts, including anterior subcapsular cataract (ASC) as well as posterior capsule opacification (PCO), are a common vision-threatening cause worldwide. Still, little is known about the underlying mechanisms. Here, we demonstrate a miRNA-based pathway regulating the pathological fibrosis process of lens epithelium. METHODS: Gain- and loss-of-function approaches, as well as multiple fibrosis models of the lens, were applied to validate the crucial role of two miR-1225 family members in the TGF-ß2 induced PCO model of human LECs and injury-induced ASC model in mice. RESULTS: Both miR-1225-3p and miR-1225-5p prominently stimulate the migration and EMT process of lens epithelial cells (LECs) in vitro as well as lens fibrosis in vivo. Moreover, we demonstrated that the underlying mechanism for these effects of miR-1225-5p is via directly targeting Keap1 to regulate Keap1/Nrf2 signaling. In addition, evidence showed that Keap1/Nrf2 signaling is activated in the TGF-ß2 induced PCO model of human LECs and injury-induced ASC model in mice, and inhibition of the Nrf2 pathway can significantly reverse the process of LECs EMT as well as lens fibrosis. CONCLUSIONS: These results suggest that blockade of miR-1225-5p prevents lens fibrosis via targeting Keap1 thereby inhibiting Nrf2 activation. The 'miR-1225-Keap1-Nrf2' signaling axis presumably holds therapeutic promise in the treatment of fibrotic cataracts.

Dichloroacetate ameliorates apoptosis, EMT and oxidative stress in diabetic cataract via inhibiting the IDO1-dependent p38 MAPK pathway.

As an oral antidiabetic agent, dichloroacetate (DCA) has been proven to improve diabetes and related complications. However, its functional role in diabetic cataract (DC) remains to be elucidated. This study was to define the role of DCA and its underlying molecular mechanism in DC in vitro and in vivo. In this study, it was shown that DCA dose-dependently ameliorated DC formation and development in DM rats. In addition, DCA significantly increased cell viability, reduced apoptosis, and inhibited EMT and oxidative stress of high glucose (HG)-treated SRA-01/04 cells in a concentration-dependent manner. Besides, it was revealed that Indoleamine 2,3-dioxygenase 1 (IDO1) expression was upregulated in lenses of DM rats and HG-treated SRA-01/04 cells, which was reversed by DCA. In addition, DCA abrogated the activation of the p38 MAPK signaling in the lenses of DM rats and HG-treated SRA-01/04 cells. Further experiments showed that IDO1 upregulation activated the p38 MAPK signaling in HG-challenged SRA-01/04 cells. Moreover, IDO1 overexpression partially reversed DCA-mediated inactivation of p38 MAPK signaling and suppression of HG-induced damage to SRA-01/04 cells. To sum up, our findings showed that DCA prevented DC-related apoptosis, EMT, and oxidative stress via inactivating IDO1-dependent p38 MAPK signaling.

Methyltransferase METTL3-mediated maturation of miR-4654 facilitates high glucose-induced apoptosis and oxidative stress in lens epithelial cells via decreasing SOD2.

N6-methyladenosine (m6 A) modification has been reported to have roles in modulating the development of diabetic cataract (DC). Methyltransferase-like 3 (METTL3) is a critical m6 A methyltransferase involving in m6 A modification activation. Here, we aimed to explore the action and mechanism of METTL3-mediated maturation of miR-4654 in DC progression. Human lens epithelial cells (HLECs) were exposed to high glucose (HG) to imitate DC condition in vitro. Levels of genes and proteins were tested via qRT-PCR and western blotting assays. The proliferation and apoptosis of HLECs were evaluated by cell counting kit-8, 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays, respectively. Oxidative stress was analyzed by detecting the contents of reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA). The binding of miR-4654 and SOD2 was confirmed by dual-luciferase reporter assay. The m6 A-RNA immunoprecipitation (MeRIP) assay detected the m6 A modification profile. Thereafter, we found that miR-4654 expression was elevated in DC samples and HG-induced HLECs. MiR-4654 knockdown reversed HG-mediated apoptosis and oxidative stress in HLECs. Mechanistically, miR-4654 directly targeted SOD2, silencing of SOD2 abolished the protective effects of miR-4654 knockdown on HLECs under HG condition. In addition, METTL3 induced miR-4654 maturation through promoting pri-miR-4654 m6 A modification, thereby increasing miR-4654 content in HLECs. METTL3 was highly expressed in DC samples and HG-induced HLECs, METTL3 deficiency protected HLECs against HG-mediated apoptotic and oxidative injury via down-regulating miR-4654. In all, METTL3 induced miR-4654 maturation in a m6 A-dependent manner, which was then reduced SOD2 expression, thus promoting apoptosis and oxidative stress in HLECs, suggesting a novel path for DC therapy.

High glucose promotes osteogenic differentiation of human lens epithelial cells through hypoxia-inducible factor (HIF) activation.

Cataract, a leading cause of blindness, is characterised by lens opacification. Type 2 diabetes is associated with a two- to fivefold higher prevalence of cataracts. The risk of cataract formation increases with the duration of diabetes and the severity of hyperglycaemia. Hydroxyapatite deposition is present in cataractous lenses that could be the consequence of osteogenic differentiation and calcification of lens epithelial cells (LECs). We hypothesised that hyperglycaemia might promote the osteogenic differentiation of human LECs (HuLECs). Osteogenic medium (OM) containing excess phosphate and calcium with normal (1 g/L) or high (4.5 g/L) glucose was used to induce HuLEC calcification. High glucose accelerated and intensified OM-induced calcification of HuLECs, which was accompanied by hyperglycaemia-induced upregulation of the osteogenic markers Runx2, Sox9, alkaline phosphatase and osteocalcin, as well as nuclear translocation of Runx2. High glucose-induced calcification was abolished in Runx2-deficient HuLECs. Additionally, high glucose stabilised the regulatory alpha subunits of hypoxia-inducible factor 1 (HIF-1), triggered nuclear translocation of HIF-1α and increased the expression of HIF-1 target genes. Gene silencing of HIF-1α or HIF-2α attenuated hyperglycaemia-induced calcification of HuLECs, while hypoxia mimetics (desferrioxamine, CoCl2) enhanced calcification of HuLECs under normal glucose conditions. Overall, this study suggests that high glucose promotes HuLEC calcification via Runx2 and the activation of the HIF-1 signalling pathway. These findings may provide new insights into the pathogenesis of diabetic cataracts, shedding light on potential factors for intervention to treat this sight-threatening condition.

Cataract-related variant R114C increases ßA3-crystallin susceptibility to environmental stresses by disrupting the protein senior structure.

Congenital cataract is a major cause of childhood blindness worldwide, with crystallin mutations accounting for over 40 % of gene-mutation-related cases. Our research focused on a novel R114C mutation in a Chinese family, resulting in bilateral coronary cataract with blue punctate opacity. Spectroscopic experiments revealed that ßA3-R114C significantly altered the senior structure, exhibiting aggregation, and reduced solubility at physiological temperature. The mutant also displayed decreased resistance and stability under environmental stresses such as UV irradiation, oxidative stress, and heat. Further, cellular models confirmed its heightened sensitivity to environmental stresses. These data suggest that the R114C mutation impairs the hydrogen bond network and structural stability of ßA3-crystallin, particularly at the boundary of the second Greek-key motif. This study revealed the pathological mechanism of ßA3-R114C and may help in the development of potential treatment strategies for related cataracts.

Fuch's Endothelial Corneal Dystrophy in Cataract Patients Is Associated with Elevated Levels of Inflammatory Chemokines, but Not Growth Factors, in the Aqueous Humor.

The study investigated a profile of chemokines and growth factors in the aqueous humor (AH) of eyes with Fuch's endothelial corneal dystrophy (FECD) and cataracts in comparison with cataract patients as a control group. A total of 52 AH samples (26 FECD + cataract and 26 cataract/control) were collected before cataract surgery. None of the patients had any clinically apparent inflammation at the time of AH collection. The AH levels of MCP-1 (CCL2), MIP-1α (CCL3), MIP-1ß(CCL4), RANTES (CCL5), eotaxin (CCL11), IP-10 (CXCL10), FGF basic, G-CSF, GM-CSF, PDGF-bb, and VEGF were compared between the groups. The analyses were performed using the Bio-Plex 200 System from Bio-Rad. Among the studied parameters, the AH levels of RANTES, eotaxin, and IP-10 significantly increased in the FECD + cataract eyes, compared with the cataract controls (p < 0.05). Elevated levels of the RANTES, Eotaxin, and IP-10 indicate more intense inflammation in the eyes of patients in the FECD + cataract group. Moreover, these factors exhibit potential as predictive biomarkers for early detection of FECD in cataract patients. The discovery of elevated concentrations of biochemical markers in a patient, who has not yet received a clinical diagnosis, may suggest the need for heightened observation of the other eye to monitor the potential development of FECD.

Cholesterol Content Regulates the Interaction of αA-, αB-, and α-Crystallin with the Model of Human Lens-Lipid Membranes.

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The Ka of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: Ka (αBc) ≈ Ka (αABc) > Ka (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation.

CircSTRBP contributes to H2O2-induced lens epithelium cell dysfunction through increasing NOX4 mRNA stability by recruiting IGF2BP1.

Previous studies have shown that the development of age-related cataract (ARC) is involved in lens epithelium dysfunction, which is associated with abnormally expressed circular RNAs (circRNAs). The current work aims to probe the role of circSTRBP (hsa_circ_0088,427) in hydrogen peroxide (H2O2)-induced lens epitheliums. Lens epithelium tissues were harvested from ARC or normal subjects (n = 23). CircSTRBP, spermatid perinuclear RNA binding protein (STRBP), and nicotinamide adenine dinucleotide phosphate oxidase subunit 4 (NOX4) levels were measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Cell proliferation, cycle progression, and apoptosis were assessed using 5-ethynyl-2'-deoxyuridine (EdU), Cell Counting Kit-8 (CCK-8), and flow cytometry assays. Caspase 3 activity, reactive oxygen species (ROS), malondialdehyde (MDA), and Glutathione peroxidases (GSH-PX) levels were detected using corresponding kits. NOX4 protein level was determined using Western blot. The interaction between insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) and circSTRBP or NOX4 was assessed through RNA immunoprecipitation (RIP). CircSTRBP and NOX4 abundances were increased in lens epithelium samples from ARC patients and H2O2-treated SRA01/04 cells. CircSTRBP knockdown might abolish H2O2-triggered SRA01/04 cell proliferation repression and apoptosis and oxidative stress promotion. In mechanism, circSTRBP is bound with IGF2BP1 and improves the stability and expression of NOX4 mRNA in SRA01/04 cells. CircSTRBP facilitated H2O2-induced SRA01/04 cell apoptosis and oxidative stress through by enhancing NOX4 mRNA stability via recruiting IGF2BP1, providing novel insights for ARC progression and treatment.