Lens Epithelium Research Articles

Metformin alleviates oxidative stress-induced senescence of human lens epithelial cells via AMPK activation and autophagic flux restoration.

Cataracts are the leading cause of blindness worldwide owing to the increasing proportion of elderly individuals in the population. The purpose of this study was to investigate whether metformin could alleviate the occurrence and development of age‐related cataract (ARC) and the underlying mechanism. In the present study, we established a senescence model induced by oxidative stress, which was confirmed by measuring β‐galactosidase activity, qRT‐PCR and Western blotting. In addition, we showed that metformin alleviated the oxidative stress‐induced senescence of HLE‐B3 cells via the activation of AMPK. Next, we provided evidence that oxidative stress impaired autophagic flux and induced lysosomal dysfunction. Subsequently, we found that metformin restored autophagic flux that had been impaired by oxidative stress by activating AMPK. Additionally, we found that metformin suppressed HLE‐B3 cell senescence by improving lysosomal function and inactivating mTOR. Furthermore, the inactivation of AMPK, impairment of autophagic flux and lysosomal dysfunction were observed in the human lens epithelium of ARC. In summary, our data suggest that the activation of AMPK may be a potential strategy for preventing ARC, and metformin may be an emerging candidate to alleviate the formation and development of ARC.

Attenuation of Transforming Growth Factor-? Expression by Alteplase in Anterior Lens Capsule Fibrosis Model with Fibrin Reaction In Vitro

Background: Underlying pathophysiology of posterior capsule opacification, a frequent visual disturbance postop complication of cataract surgery, is multifactorial. Several cytokines and growth factors, such as Transforming Growth Factor-? (TGF-?), Fibroblast Growth Factor 2 (FGF2), and Hepatocyte Growth Factor (HGF), have been found to play roles in the formation of PCO. Multiple advancements have been made including researches in therapeutic agents. Alteplase, a fibrinolytic agent, has been studied to decrease fibrin formation, which is a mechanism causing PCO. This study aimed to investigate the effect of alteplase on TGF-? expression in an anterior lens capsule fibrosis model resembling PCO. Methods: An experimental study was performed using cultured anterior lens capsule tissue taken during cataract surgery. Primary cell isolation culture and lens epithelial lens characterization were conducted. Following identification of lens epithelial cell and 90% confluency rate on cell culture, lens epithelial cells were divided into four groups and then scratched to induce inflammatory reaction resembling PCO process after cataract surgery. Doses of 25 mcg/ml, 50 mcg/ml, and 100 mcg/ml were then applied to each group of cell cultures with the other group serving as negative control. Using a fluorescein microscope, measurements to asses the influence of alteplase were taken 7 days after scratching by analyzing the intensity of fluoresce using FITC antibody of TGF-?. Results: The attenuation effect of alteplase to TGF-? expression was observed in all treatment groups; 25 µg/ml (15.5716x106 ±2.56558x106 ), 50 µg/ml (12.7364x106 ±1.67067x10 6 ), and 100 µg/ml group (7.8422x106 ±2.24941x106). The Tukey HSD posthoc tests found significant decrease between control group and each treatment group (p=0.019, p=0.000, and p=0.000; respectively). Conclusion: Alteplase within measure dose range holds potential effect in inhibiting fibrosis formation in PCO through attenuation of TGF-?

New sign of anterior zonular dehiscence

A new anterior capsule sign was encountered at the time of phacoemulsification surgery in an eye that had previously sustained blunt trauma. A combination of traumatic dehiscence of the anterior zonular fibers, dehiscence between the lens and capsular epithelium, and recumbency at the time of surgery is posited to be the cause of the appearance. To the authors' knowledge, this is the first reported instance of this sign.

Aging-dependent loss of GAP junction proteins Cx46 and Cx50 in the fiber cells of human and mouse lenses accounts for the diminished coupling conductance.

The homeostasis of the ocular lens is maintained by a microcirculation system propagated through gap junction channels. It is well established that the intercellular communications of the lens become deteriorative during aging. However, the molecular basis for this change in human lenses has not been well defined. Here, we present evidence to show that over 90% of Cx46 and Cx50 are lost in the fiber cells of normal human lenses aged 50 and above. From transparent to cataractous lenses, while Cx43 was upregulated, both Cx46 and Cx50 were significantly down-regulated in the lens epithelia. During aging of mouse lenses, Cx43 remained unchanged, but both Cx46 and Cx50 were significantly downregulated. Under oxidative stress treatment, mouse lenses develop in vitro cataractogenesis. Associated with this process, Cx43 was significantly upregulated, in contrast, Cx46 and Cx50 were sharply downregulated. Together, our results for the first time reveal that downregulation in Cx46 and Cx50 levels appears to be the major reason for the diminished coupling conductance, and the aging-dependent loss of Cx46 and Cx50 promotes senile cataractogenesis.

Filaggrin Expression in the Lid Margin During Contact Lens Wear.

To investigate the expression of the keratinization-related protein, filaggrin, in the lid margin epithelium of contact lens (CL) wearers compared with nonwearers. This was a cross-sectional study of 100 individuals with different exposures to CL wear: short, moderate, and long experience; previous CL wearers; and nonwearers as controls. Impression cytology samples were collected from the lid wiper (LW) area of the central upper lid margin. After fixing, an equal, random sample was selected from each group (n=13) for immunocytochemistry analysis using antihuman primary anybody (mouse filaggrin), then stained with secondary antibody (fluorescein isothiocyanate-conjugated donkey anti-mouse immunoglobulin G horseradish peroxidase) to detect filaggrin. Imaging was performed with the 3i-Vivo 2-photon microscope equipped with a Zeiss 20×-objective and SlideBook-reader software. Sixty-five samples from 65 participants (37 women; mean age±SD: 25.1±4.1 years) were collected. Filaggrin was detected in all 65 randomly selected immunostained marginal epithelium samples. All samples were similar in showing patchy areas of filaggrin immunostaining, regardless of CL wear, symptoms or epithelium morphology. Because the filaggrin immunostaining showed similar patterns across almost all the observed samples, comparison between subject groups was impractical. The presence of filaggrin in the healthy LW was additionally confirmed by an independent laboratory. Filaggrin expression seems to be a normal part of epithelial cell differentiation in the lid margin and may not be a useful keratinization/stress biomarker in the marginal epithelium. Investigating other keratinization biomarkers that are not detected in the normal mucocutaneous junction/LW may help to understand the keratinization nature of LW epithelium changes in CL wearers.

Early Onset of Age-Related Cataracts in Cystine/Glutamate Antiporter Knockout Mice.

PurposeThe purpose of this study was to determine the importance of the xCT is a subunit. The cystine/glutamate antiporter is actually system xc-xCT subunit of the cystine/glutamate antiporter in maintaining redox balance by investigating the effects of the loss of xCT on lens transparency and cystine/cysteine balance in the aqueous humour.MethodsC57Bl/6 wild-type and xCT knockout mice at five age groups (6 weeks to 12 months) were used. Lens transparency was examined using a slit-lamp and morphological changes visualized by immunolabelling and confocal microscopy. Quantification of glutathione in lenses and cysteine and cystine levels in the aqueous was conducted by liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsSlit-lamp examinations revealed that 3-month-old wild-type mice and xCT knockout mice lenses exhibited an anterior localized cataract. The frequency of this cataract significantly increased in the knockout mice compared to the wild-type mice. Morphological studies revealed a localized swelling of the lens fiber cells at the anterior pole. Glutathione levels in whole lenses were similar between wild-type and knockout mice. However, glutathione levels were significantly decreased at 3 months in the knockout mice in the lens epithelium compared to the wild-type mice. Aqueous cysteine levels remained similar between wild-type and knockout mice at all age groups, whereas cystine levels were significantly increased in 3-, 9-, and 12-month-old knockout mice compared to wild-type mice.ConclusionsLoss of xCT resulted in the depletion of glutathione in the epithelium and an oxidative shift in the cysteine/cystine ratio of the aqueous. Together, these oxidative changes may contribute to the accelerated development of an anterior cataract in knockout mice, which appears to be a normal feature of aging in wild-type mice.

Qki activates Srebp2-mediated cholesterol biosynthesis for maintenance of eye lens transparency

Defective cholesterol biosynthesis in eye lens cells is often associated with cataracts; however, how genes involved in cholesterol biosynthesis are regulated in lens cells remains unclear. Here, we show that Quaking (Qki) is required for the transcriptional activation of genes involved in cholesterol biosynthesis in the eye lens. At the transcriptome level, lens-specific Qki-deficient mice present downregulation of genes associated with the cholesterol biosynthesis pathway, resulting in a significant reduction of total cholesterol level in the eye lens. Mice with Qki depletion in lens epithelium display progressive accumulation of protein aggregates, eventually leading to cataracts. Notably, these defects are attenuated by topical sterol administration. Mechanistically, we demonstrate that Qki enhances cholesterol biosynthesis by recruiting Srebp2 and Pol II in the promoter regions of cholesterol biosynthesis genes. Supporting its function as a transcription co-activator, we show that Qki directly interacts with single-stranded DNA. In conclusion, we propose that Qki-Srebp2–mediated cholesterol biosynthesis is essential for maintaining the cholesterol level that protects lens from cataract development.

Studies on the Involvement of Integrin and the Tubulin Cytoskeleton in the Mechanism Responsible for TRPV1‐dependent NKCC1 Activation in Mouse Lens

Previously we showed shrinkage of the mouse lens by hyperosmotic solution causes TRPV1-dependent activation of NKCC1. In various tissues the cytoskeleton has been shown to play a role in responses to osmotic stress. Here we present evidence for the involvement of integrin and tubulin in the activation of NKCC1 in mouse lens epithelium. Studies were carried out on lenses from wild-type (WT) and TRPV1 knockout (KO) mice. Rubidium (Rb) uptake, measured using an atomic absorption spectrophotometer, was used as a measure of NKCC activity. ERK1/2 phosphorylation was measured by quantitative Western blot analysis. Intracellular calcium was measured in Fura-2 loaded cultured lens epithelium by a ratiometric imaging technique. In WT mice, the integrin agonist leukadherin 1 (LA-1, 25 μM) increased Rb uptake by 33% in intact lenses and increased intracellular calcium concentration in cultured lens epithelium from 197±18 to 341±25 nM in (n=5, P<0.001). In contrast, LA-1 failed to produce any significant change of Rb uptake in TRPV1 KO lenses and did not increase calcium in cultured lens epithelium from TRPV1 KO animals. The increase of Rb uptake elicited by LA-1 in WT lenses was abolished by the putative tubulin stabilizer, paclitaxel (100 nm). The TRPV1 antagonist, A889425 (1.0 μM) also abolished the Rb uptake response to LA-1. LA-1, the TRPV1 agonist capsaicin, and hyperosmotic solution (350 mOsm) all caused ERK1/2 activation in WT cultured lens epithelium. In each case, the ERK1/2 response was transient with a peak response at ~5 min. The ERK1/2 activation responses to hyperosmotic stress and LA-1 induced were markedly suppressed by A889425 and by paclitaxel. Taken together, the results point to a link between integrin, the tubulin cytoskeleton, and TRPV1-dependent activation of NKCC1 in the mouse lens. It is noteworthy that the integrin agonist LA-1 as well as hyperosmotic stress both were found to activate ERK1/2, the upstream kinase that was shown previously associated with activation of NKCC1 in the lens.

PAX8 Expression in the Crystalline Lens and Lens-Derived Lesions.

PurposeTo evaluate PAX8 expression by immunohistochemistry in the normal pediatric and adult crystalline lens and to assess the usefulness of PAX8 immunohistochemical stain in the diagnosis of morphologically challenging lesions of lenticular origin.DesignRetrospective, observational case series.ParticipantsFourteen congenital and acquired lens-derived lesions and 10 control crystalline lenses.MethodsHematoxylin–eosin and periodic acid–Schiff stains and an immunohistochemical panel of PAX8, vimentin, S100, smooth muscle actin, AE1/AE3, cytokeratin 7, and cytokeratin 5/6 antibodies were performed on all tissues.Main Outcome MeasuresDistribution of PAX8 expression in normal crystalline lens and in lens-derived lesions.ResultsRecords search identified 10 normal pediatric and adult crystalline lenses, 1 phakomatous choristoma, 1 Peters anomaly with adherent leukoma, 1 lens capsule with congenital pyramidal cataract formation, 2 lenses with anterior and posterior subcapsular cataract formation, 3 postsurgical cataractous lenses (Soemmerring ring cataract and capsular fibrosis), and 6 retrocorneal membranes that incorporated various components of metaplastic corneal endothelium, metaplastic lens epithelium, corneal stroma, and epithelial downgrowth. Strong nuclear PAX8 expression was observed in the lens epithelium and in the equatorial lens bow of normal pediatric and adult lenses. Nuclear PAX8 expression also was observed in the lesions that retained some of the epithelial morphologic features, such as phakomatous choristoma, adherent leukoma, congenital pyramidal cataract, and components of intraocular membranes with lens epithelial differentiation. PAX8 expression was lost in lens epithelial lesions that had undergone mesenchymal transition, such as anterior subcapsular cataract and capsular fibrosis.ConclusionsPAX8 antibody may be a useful adjunct to the immunohistochemical panels in morphologically challenging lens epithelial-derived lesions that retain epithelial morphologic features. PAX8 is not useful in the diagnosis of lens-derived lesions that feature epithelial–mesenchymal transition.

RERE deficiency causes retinal and optic nerve atrophy through degeneration of retinal cells.

The arginine-glutamic acid dipeptide repeats gene (RERE) encodes a nuclear receptor coregulator that modulates gene expression through its interaction with transcriptional machinery. In humans, RERE deficiency causes neurodevelopmental disorder with or without structural defects of the brain, eye, heart, and kidney (NEDBEH). Ophthalmological defects are seen in approximately one third of individuals with NEDBEH and in RERE-deficient mice which can serve as a useful animal model. In mice, RERE is expressed in a subset of retinal ganglion cells (RGC), the lens epithelium, and the ciliary body during the embryonic period. RERE expression expands into the outer nuclear layer and the inner nuclear layer during the postnatal period. RERE-deficient mice have retinal and optic nerve atrophy. We show that RERE deficiency causes progressive loss of retinal cells and apoptosis of retinal cells in the ganglion cell layer as early as E17.5. The number of RGCs is also reduced in RERE-deficient embryos and mice. We conclude that RERE is required to control the apoptosis of retinal cells in the developing retina, and that RERE deficiency results in the retina atrophy through degeneration of the retinal cells and optic nerve atrophy through the loss of RGCs.

Aging lens epithelium is susceptible to ferroptosis

Age-related cataracts (ARC) are the primary cause of blindness worldwide, and oxidative stress is considered the central pathogenesis of age-related cataractogenesis. Interestingly, ample evidence suggests that there is no remarkable apoptosis present in aged and cataractous human lenses despite the profound disruption of redox homeostasis, raising an essential question regarding the existence of other cell death mechanisms. Here we sought to explore the lens epithelial cell's (LEC) susceptibility to ferroptosis after documentation has concluded that aged and cataractous human lenses manifest with increased reactive oxygen species (ROS) formation, elevated lipid peroxidation, and accumulative intracellular redox-active iron, constituting the three hallmarks of ferroptosis during aging and cataractogenesis. Here we show that very low concentrations of system Xc- inhibitor Erastin (0.5 μM) and glutathione peroxidase 4 (GPX4) inhibitor RSL3 (0.1 μM) can drastically induce human LEC (FHL124) ferroptosis in vitro and mouse lens epithelium ferroptosis ex vivo. Depletion of intracellular glutathione (GSH) in human LECs and mouse lens epithelium significantly sensitizes ferroptosis, particularly under RSL3 challenge. Intriguingly, both human LECs and the mouse lens epithelium demonstrate an age-related sensitization of ferroptosis. Transcriptome analysis indicates that clusters of genes are up-or down-regulated in aged LECs, impacting cellular redox and iron homeostases, such as downregulation of both cystine/glutamate antiporter subunits SLC7A11 and SLC3A2 and iron exporter ferroportin (SLC40A1). Here, for the first time, we are suggesting that LECs are highly susceptible to ferroptosis. Moreover, aged and cataractous human lenses may possess more pro-ferroptotic criteria than any other organ in the human body.

Pseudoexfoliation syndrome in diabetic patients: transmission electron microscopy study of anterior lens epithelial cells.

Purpose: to examine the lens epithelial cells in diabetic patients with pseudoexfoliation to ultramicroscope and to compare the findings with those of patients without diabetes mellitus (DM) and/or without pseudoexfoliation (PEX).Materials and Methods: Forty patients aged 65-86 years were enrolled in the study. All patients had senile cataract and were divided into four groups of ten patients in each group. Group I: patients without pseudoexfoliation, without DM, Group II: without pseudoexfoliation, with DM, Group III: with pseudoexfoliation, without DM, Group IV (Pseudoexfoliation-Diabetic Group): with pseudoexfoliation, with DM. In all cases, part of the central portion of anterior lens capsule was removed during routine cataract surgery, and was properly prepared in order to be examined under a transmission electron microscope.Results: In the control group, mainly degenerative alterations to varying extents were observed. In all groups, intracellular and extracellular oedema, multilayering, apoptosis, completely destroyed cells adjacent to normal cellswere described. In the diabetic group, alterations were more severe with respect to group I. In PEX cases, the additionalirregularity of the epithelium surface, loose intercellular connection, as well as the loose connection between cells and basement membrane were described with the presence of PEX material free and within the basement membrane. In cases with PEX and DM, degenerative alterations and PEX material were observed as well, but the epithelium was better conserved compared to the PEX group.Conclusion: the observed lesions were more extended and more frequent in the pseudoexfoliation group, followed by the diabetic group. The pseudoexfoliation-diabetic group presented less intense modifications raising questions about the interaction of these different diseases.Abbreviations: DM = Diabetes Mellitus, PEX = Pseudoexfoliation, PXM = Pseudoexfoliative Material, AD = Alzheimer disease, TGF-β1 = Transforming Growth Factor beta 1, WHO = World Health Organization, LEC = Lens Epithelium Cells, BM = Basement Membrane, CM = Cytoplasmic Membrane

Resident immune cells of the avascular lens: Mediators of the injury and fibrotic response of the lens.

Tissues typically harbor subpopulations of resident immune cells that function as rapid responders to injury and whose activation leads to induction of an adaptive immune response, playing important roles in repair and protection. Since the lens is an avascular tissue, it was presumed that it was absent of resident immune cells. Our studies now show that resident immune cells are a shared feature of the human, mouse, and chicken lens epithelium. These resident immune cells function as immediate responders to injury and rapidly populate the wound edge following mock cataract surgery to function as leader cells. Many of these resident immune cells also express MHCII providing them with antigen presenting ability to engage an adaptive immune response. We provide evidence that during development immune cells migrate on the ciliary zonules and localize among the equatorial epithelial cells of the lens adjacent to where the ciliary zonules associate with the lens capsule. These findings suggest that the vasculature‐rich ciliary body is a source of lens resident immune cells. We identified a major role for these cells as rapid responders to wounding, quickly populating each wound were they can function as leaders of lens tissue repair. Our findings also show that lens resident immune cells are progenitors of myofibroblasts, which characteristically appear in response to lens cataract surgery injury, and therefore, are likely agents of lens pathologies to impair vision like fibrosis.

Radiation-induced DNA Damage and Repair in Lens Epithelial Cells of both Ptch1(+/-) and Ercc2(+/-) Mutated Mice.

Epidemiological studies suggest an increased incidence and risk of cataract after low-dose (<2 Gy) ionizing radiation exposures. However, the biological mechanism(s) of this process are not fully understood. DNA damage and repair are thought to have a contributing role in radiation-induced cataractogenesis. Recently we have reported an inverse dose-rate effect, as well as the low-dose response, of DNA damage and repair in lens epithelial cells (LECs). Here, we present further initial findings from two mutated strains (Ercc2+/- and Ptch1+/-) of mice, both reportedly susceptible to radiation-induced cataract, and their DNA damage and repair response to low-dose and low-dose-rate gamma rays. Our results support the hypothesis that the lens epithelium responds differently to radiation than other tissues, with reported radiation susceptibility to DNA damage not necessarily translating to the LECs. Genetic predisposition and strain(s) of mice have a significant role in radiation-induced cataract susceptibility.

Investigation of MicroRNA Expression in Anterior Lens Capsules of Senile Cataract Patients and MicroRNA Differences According to the Cataract Type.

PurposeWe investigated the microRNAs (miRNAs) expression in the anterior lens capsules of patients with senile cataract and compared it to that in the anterior lens capsules of healthy controls. Moreover, we compared the differences in miRNAs expression according to the types of cataracts.MethodsIndividual lens epithelium samples were collected from 33 senile patients and 10 controls. The cataract patients were classified into cortical, nuclear, posterior and anterior subcapsular and mixed. The expression of 12 different miRNAs in lens epithelium was measured using real-time polymerase chain reaction and compared between the senile cataract patients and controls. The differences of miRNA levels according to cataract type were analyzed.ResultsThe expression levels of let-7g-5p, miR-23a-3p, miR-23b-3p, and miR-125a-5p were significantly upregulated in patients with senile cataract when compared with those in the control group (P < 0.05). The expressions of let-7a-5p, let-7d-5p, miR-16-5p and miR-22-3p were significantly downregulated in the senile cataracts (P < 0.05). Let-7a-5p, let-7d-5p, let-7g-5p and mir-23b-3p had significant difference in expression between nuclear and anterior subcapsular cataracts.ConclusionsThe eight differentially expressed miRNAs may be involved in the pathogenesis of senile cataract, in particular, related to oxidative stress and autophagy.Translational RelevanceWe infer that several miRNAs in lens epithelial cells are promising candidate biomarkers of senile cataracts.

Protective Effect and Mechanism of Bone Morphogenetic Protein-4 on Apoptosis of Human Lens Epithelium Cells under Oxidative Stress.

Bone morphogenetic proteins (BMPs), a member of the transforming growth factor β (TGF-β) superfamily, are abundant in human ocular tissues and play an important role in lens development. Targeted deletion of BMP-4 in mice results in failure of lens placode formation. Following lens maturation, the formation of senile cataracts is demonstrably associated with free radical-related oxidative stress. Previous studies reported that BMPs play an antiapoptotic role in cells under oxidative stress, and the BMP-4 signal is important in inflammation regulation and homeostasis. BMP-4 evidently suppressed the apoptosis of human lens epithelial cells (HLECS) under oxidative stress induced by H2O2. This protective antiapoptotic effect is partly due to a decrease in caspase-3 activity and reactive oxygen species (ROS) level. Furthermore, the expression of activating transcription factor- (ATF-) 6 and Krüppel-like factor- (KLF-) 6 increased under oxidative stress and decreased after BMP-4 treatment.

Unlike Its Paralog LEDGF/p75, HRP-2 Is Dispensable for MLL-R Leukemogenesis but Important for Leukemic Cell Survival.

HDGF-related protein 2 (HRP-2) is a member of the Hepatoma-Derived Growth Factor-related protein family that harbors the structured PWWP and Integrase Binding Domain, known to associate with methylated histone tails or cellular and viral proteins, respectively. Interestingly, HRP-2 is a paralog of Lens Epithelium Derived Growth Factor p75 (LEDGF/p75), which is essential for MLL-rearranged (MLL-r) leukemia but dispensable for hematopoiesis. Sequel to these findings, we investigated the role of HRP-2 in hematopoiesis and MLL-r leukemia. Protein interactions were investigated by co-immunoprecipitation and validated using recombinant proteins in NMR. A systemic knockout mouse model was used to study normal hematopoiesis and MLL-ENL transformation upon the different HRP-2 genotypes. The role of HRP-2 in MLL-r and other leukemic, human cell lines was evaluated by lentiviral-mediated miRNA targeting HRP-2. We demonstrate that MLL and HRP-2 interact through a conserved interface, although this interaction proved less dependent on menin than the MLL-LEDGF/p75 interaction. The systemic HRP-2 knockout mice only revealed an increase in neutrophils in the peripheral blood, whereas the depletion of HRP-2 in leukemic cell lines and transformed primary murine cells resulted in reduced colony formation independently of MLL-rearrangements. In contrast, primary murine HRP-2 knockout cells were efficiently transformed by the MLL-ENL fusion, indicating that HRP-2, unlike LEDGF/p75, is dispensable for the transformation of MLL-ENL leukemogenesis but important for leukemic cell survival.

Moderate oxidative stress promotes epithelial-mesenchymal transition in the lens epithelial cells via the TGF-β/Smad and Wnt/β-catenin pathways.

The epithelial-mesenchymal transition (EMT) plays a significant role in fibrosis and migration of lens epithelial cells (LECs), and eventually induces posterior capsule opacification (PCO). In the past, it was generally believed that the TGF-β/Smad pathway regulates lens EMT. A recent study found that attenuated glutathione level promotes LECs EMT via the Wnt/β-catenin pathway, which suggests a more complex pathogenesis of PCO. To test the hypothesis, we used the mouse cataract surgery PCO model and tested both canonical Wnt/β-catenin and TGF-β/Smad signaling pathways. The results showed that both TGF-β/Smad and Wnt/β-catenin pathways were activated during the lens capsule fibrosis. Compared with the freshly isolated posterior capsule, the expression level of phosphorylated Smad2 was highest at day3 and then slightly decreased, but the expression level of Wnt10a gradually increased from day0 to day7. It shows that these two pathways are involved in the lens epithelium's fibrotic process and may play different roles in different periods. Subsequently, we established oxidative stress-induced EMT model in primary porcine lens epithelial cells and found that both the TGF-β/Smad and Wnt/β-catenin pathways were activated. Further study suggests that block Wnt/β-catenin pathway using XAV939 alone or block TGF-β/Smad pathway using LY2109761 could partially block pLECs fibrosis, but blocking Wnt/β-catenin and TGF-β/Smad pathway using combined XAV939 and LY2109761 could completely block pLECs fibrosis. In conclusion, this study demonstrates that both TGF-β/Smad and canonical Wnt/β-catenin pathways play a significant role in regulating epithelial-mesenchymal transformation of lens epithelial cells but might be in a different stage.

Geometric mapping of the eye lens and its epithelium reveals changes that accompany emmetropia

PurposeTo investigate changes in both lens shape and the epithelial distribution as emmetropia is established in C57Bl6J mice between postnatal weeks 4 to 6.MethodsWe used a computational approach to produce geometrical maps of the mouse lens and its epithelium. Conveniently our approach ulitises data collected from intact lenses imaged using a conventional laser scanning confocal microscope. It delivers cartesian coordinates for every epithelial cell in the lens epithelium. We chose to investigate lenses during postnatal weeks 4 to 6, a time during lens development when emmetropia is established in this inbred strain of mice (Tkatchenko et al., 2010).ResultsGeometric mapping standardises the measurement and analysis of lens epithelial cell densities. Emmetropia is established in C57Bl6J inbred mice, but many other strains exhibit considerable refractive error (Tkatchenko et al., 2019). The eye lens plays its part in the development and establishment of emmetropia in mice (Tkatchenko et al., 2010; Pardue et al., 2013). We record a change in the shape of the eye lens from a spheroid to a lentoid shape between postnatal weeks 4 and 6. The distribution of the epithelial cells in the lens epithelium also changes from a non‐zonal to a zonal distribution, with characteristics that typify the epithelia of adult animals. We correlate these observations regarding lens geometry with genetic mutations in lens cytoskeletal proteins found in those strains that present with refractive error, but which are absent from C57Bl6J (Sandilands et al., 2004). We discuss the potential significance of these observations in the context of the mechanistic basis of the lens contribution to emmetropia.ConclusionsThe development of emmetropia in the C57Bl6J inbred strain of mice correlates with changes in epithelial cell organisation and changes in lens geometry, which when combined with strain‐specific genetic differences offer a mechanistic understanding involving the lintermediate filament cytoskeleton.Pardue, M. T., et al., (2013) Exp Eye Res, 114, 96–105.Sandilands, A., et al (2004). Eye Res. 78, 109–123.Tkatchenko, T. V. et al. (2019). BMC Medical Genomics, 12, 1–24.Tkatchenko, T. V., Shen, Y. and Tkatchenko, A. V. (2010) Invest Ophth Vis Sci, 51, 21–27. https://doi.org/10.1167/iovs.08‐2767

Whole mount staining of lenses for visualization of lens epithelial cell proteins

Whole mount imaging of the lens allows for high spatial resolution visualization of lens epithelial structures by using small molecule fluorescent probes. However, the visualization of specific proteins in lens epithelial cells within whole lenses remains a challenge as the capsule that surrounds the lens does not allow penetration of antibodies. Here we describe a whole mount imaging method that allows us to overcome this challenge by digesting the lens capsules of paraformaldehyde fixed lenses using collagenase. This method enables the penetration of antibodies for effective visualization of proteins in the epithelium of whole lenses.•A limitation to lens whole mount imaging is the ability to visualize specific proteins as the collagen capsule surrounding the lens impedes the penetration of antibodies•This protocol helps overcome this limitation by a light collagenase digestion of the capsule of fixed lenses prior to immunostaining•This method allows for the imaging of specific proteins in the epithelium of the whole lens tissue