Cultured Lens Epithelial Cells Research Articles

Tonic ErbB signaling underlies TGFβ-induced activation of ERK and is required for lens cell epithelial to myofibroblast transition.

Fibrosis is a major, but incompletely understood, component of many diseases. The most common vision-disrupting complication of cataract surgery involves differentiation of residual lens cells into myofibroblasts. In serum-free primary cultures of lens epithelial cells (DCDMLs), inhibitors of either ERK or of ErbB signaling prevent TGFβ from upregulating both early (fibronectin) and late (αSMA) markers of myofibroblast differentiation. TGFβ stimulates ERK in DCDMLs within 1.5 h. Kinase inhibitors of ErbBs, but not of several other growth factor receptors in lens cells, reduce phospho ERK to below basal levels in the absence or presence of TGFβ. This effect is attributable to constitutive ErbB activity playing a major role in regulating the basal levels pERK. Additional studies support a model in which TGFβ-generated reactive oxygen species serve to indirectly amplify ERK signaling downstream of tonically active ErbBs to mediate myofibroblast differentiation. ERK activity is in turn essential for expression of ErbB1 and ErbB2, major inducers of ERK signaling. By mechanistically linking TGFβ, ErbB, and ERK signaling to myofibroblast differentiation, our data elucidate a new role for ErbBs in fibrosis and reveal a novel mode by which TGFβ directs lens cell fate.

Synchrotron‐based Fourier transform infrared microspectroscopy examined differences between the cultured and post‐operative human lens epithelial cells important for understanding posterior capsular opacification and lens regeneration

Aims/Purpose: To compare the bio‐macromolecular composition and the differences between the human cultured and post‐operative lens epithelial cells (LECs) on lens capsules (LCs) for a better understanding of posterior capsular opacification (PCO)—the most common complication of cataract surgery, and lens regeneration.Methods: The explants of the anterior portion of the LC containing the LECs, obtained from cataract surgery and cultivated under adherent conditions, as well as the post‐operative LC with LECs, were analysed by using synchrotron radiation‐based Fourier transform infrared (SR‐FTIR) microspectroscopy, a vibrational spectroscopic technique that allows monitoring of the entire biochemical status of the biological processes. The SR‐FTIR microspectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA was used, where measurements were set to achieve single‐cell resolution, with high spectral stability and high photon flux.Results: We found that the differences exist between the composites of cultured and post‐operative LECs on LCs on the level of all chemical constituents: proteins, lipids, nucleic acids and carbohydrates as well as oxidative stress. The strongest differences are found in protein secondary structure contribution where the composite of primary LECs cultures on LC have more α‐helix (1652 cm−1) and post‐operative LC have more β‐sheet (1624 cm−1 and 1697 cm−1) secondary structures. The oxidative stress is more expressed in the composite of primary LECs cultures on LC and relatively less in the composite of post‐operative lens epithelium LECs on LC.Conclusions: Our results obtained by SR‐FTIR increase the knowledge about the total proteins, lipids, and nucleic acids in human primary cultures LECs while offering also evidence that SR‐FTIR is sensitive to the pathologic processes of LECs transdifferentiation. We showed that the composite of primary cultures LECs on LC have a distinct bio‐macromolecular composition compared to the composite of post‐operative LECs on LC, which gives additional information for a better understanding of PCO and lens regeneration.

Towards the Identification and Characterization of Putative Adult Human Lens Epithelial Stem Cells.

The anterior lens epithelium has the ability to differentiate into lens fibres throughout its life. The present study aims to identify and functionally characterize the adult stem cells in the human lens epithelium. Whole mounts of lens epithelium from donor eyes (normal/cataract) were immunostained for SOX2, gap junction protein alpha 1 (GJA1), PAX6, α, β and γ-crystallins, followed by a confocal analysis. The functional property of adult stem cells was analysed by their sphere forming ability using cultured lens epithelial cells from different zones. Based on marker expression, the lens epithelium was divided into four zones: the central zone, characterized by a small population of PAX6+, GJA1-, β-crystallin- and γ-crystallin- cells; the germinative zone, characterized by PAX6+, GJA1+, β-crystallin- and γ-crystallin-; the transitional zone, characterized by PAX6+, GJA1+, β-crystallin+ and γ-crystallin-; and the equatorial zone, characterized by PAX6+/-, GJA1+, β-crystallin+, and γ-crystallin+ cells. The putative lens epithelial stem cells identified as SOX2+ and GJA1 membrane expression negative cells were located only in the central zone (1.89 ± 0.84%). Compared to the other zones, a significant percentage of spheres were identified in the central zone (1.68 ± 1.04%), consistent with the location of the putative adult lens epithelial stem cells. In the cataractous lens, an absence of SOX2 expression and a significant reduction in sphere forming ability (0.33 ± 0.11%) were observed in the central zone. The above findings confirmed the presence of putative stem cells in the central zone of the adult human lens epithelium and indicated their probable association with cataract development.

ErbBs in Lens Cell Fibrosis and Secondary Cataract.

TGFβ-induced epithelial-to-myofibroblast transition (EMyT) of lens cells has been linked to the most common vision-disrupting complication of cataract surgery-namely, posterior capsule opacification (PCO; secondary cataract). Although inhibitors of the ErbB family of receptor tyrosine kinases have been shown to block some PCO-associated processes in model systems, our knowledge of ErbB signaling in the lens is very limited. Here, we investigate the expression of ErbBs and their ligands in primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) and how TGFβ affects ErbB function. DCDMLs were analyzed by immunofluorescence microscopy and Western blotting under basal and profibrotic conditions. Small-molecule ErbB kinase blockers, including the human therapeutic lapatinib, selectively inhibit TGFβ-induced EMyT of DCDMLs. Lens cells constitutively express ErbB1 (EGFR), ErbB2, and ErbB4 protein on the plasma membrane and release into the medium ErbB-activating ligand. Culturing DCDMLs with TGFβ increases soluble bioactive ErbB ligand and markedly alters ErbBs, reducing total and cell surface ErbB2 and ErbB4 while increasing ErbB1 expression and homodimer formation. Similar, TGFβ-dependent changes in relative ErbB expression are induced when lens cells are exposed to the profibrotic substrate fibronectin. A single, 1-hour treatment with lapatinib inhibits EMyT in DCDMLs assessed 6 days later. Short-term exposure to lower doses of lapatinib is also capable of eliciting a durable response when combined with suboptimal levels of a mechanistically distinct multikinase inhibitor. Our findings support ErbB1 as a therapeutic target for fibrotic PCO, which could be leveraged to pharmaceutically preserve the vision of millions of patients with cataracts.

The Differential Expression of Circular RNAs and the Role of circAFF1 in Lens Epithelial Cells of High-Myopic Cataract.

High-myopic cataract (HMC) is a complex cataract with earlier onset and more rapid progress than age-related cataract (ARC). Circular RNAs (circRNAs) have been implicated in many diseases. However, their involvement in HMC remain largely unexplored. To investigate the role of dysregulated circRNAs in HMC, lens epithelium samples from 24 HMC and 24 ARC patients were used for whole transcriptome sequencing. Compared with ARC, HMC had 3687 uniquely expressed circRNAs and 1163 significantly differentially expressed circRNAs (DEcRs) (|log2FC| > 1, p < 0.05). A putative circRNA-miRNA-mRNA network was constructed based on correlation analysis. We validated the differential expression of 3 DEcRs by quantitative polymerase chain reaction (qPCR) using different sets of samples. We further investigated the role of circAFF1 in cultured lens epithelial cells (LECs) and found that the overexpression of circAFF1 promoted cell proliferation, migration and inhibited apoptosis. We also showed that circAFF1 upregulated Tropomyosin 1 (TPM1) expression by sponging miR-760, which was consistent with the network prediction. Collectively, our study suggested the involvement of circRNAs in the pathogenesis of HMC and provide a resource for further study on this topic.

The role of reduced glutathione on oxidative stress, reticulum endoplasmic stress and glycation in human lens epithelial cell culture

Context: Cataracts are currently the main cause of blindness worldwide. This condition caused by a low concentration of reduced glutathione (GSH) in the lens. Aims: To evaluate the effect of glutathione (GSH) on the hyperglycemia-induced oxidative stress, reticulum endoplasmic stress, and glycation in human lens epithelial cells. Methods: Human lens epithelial cells were cultured under high-glucose conditions. Malondialdehyde (MDA), protein carbonyl content (PCC), glucose reactive protein (GRP), and advanced glycation end product (AGE) were measured by enzyme-linked immunosorbent assay after 72 h of incubation for MDA, PCC, GRP and after 2 weeks incubation for AGE. Results: The MDA and PCC levels increased in response to high-dose glucose administration compared to the control group. MDA and PCC levels were decreased at all GSH doses, whereas the lowest mean MDA and PCC levels were observed at GSH doses of 10 and 100 μM, respectively. GRP levels increased after high-glucose administration as compared to the control group. Additionally, the groups co-treated with 30 and 100 μM GSH showed reduced PCC. The AGE level was reduced at all doses of GSH compared to those in the control group. Conclusions: The results suggest that GSH inhibits oxidative stress, reticulum endoplasmic stress, and AGE formation, which may lead to the progression of diabetic cataract. Additionally, GSH may maintain lens transparency by acting as an antiglycation and controlling the AGE formation.

Protein kinase CK2 in TGFβ-induced lens epithelial-mesenchymal transition

Cataract is a leading cause of reversible blindness. Secondary cataract is the result of migration, proliferation, and epithelial-mesenchymal transformation (EMT) of lens epithelial cells (LECs), induced by transforming growth factor β (TGFβ). The extracellular signal-regulated kinases 1 and 2 (ERK 1/2) and casein kinase 2 (CK2) are important for fundamental cellular processes. This study aimed to evaluate the role of CK2 in the EMT of LECs. Primary LECs cultures were obtained from age-related and type 2 diabetes-induced cataracts. Diabetic LECs were capable of spontaneous in vitro differentiation. Treatment of LECs with TGFβ2-stimulated ERK1/2 activity, which correlated with the expressed α-smooth muscle actin (α-SMA). siRNA-mediated attenuation of the endogenous catalytic subunit (α) of CK2-inhibited ERK1/2 activity and decreased α-SMA expression. This study shows evidence that the function of ERK1/2 is regulated by the constitutively active heterotetrameric protein kinase CK2 in this cellular phenotype. This study enriches the knowledge on LECs physiology in normal and in pathological conditions.

Increased Association of Deamidated \u03b1A-N101D with Lens membrane of transgenic \u03b1AN101D vs. wild type \u03b1A mice: potential effects on intracellular ionic imbalance and membrane disorganization

We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization.MethodsLenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.ResultsCompared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice.ConclusionsThe results show that an increased lens membrane association of αAN101D-−relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

Heterozygous Loss of Yap1 in Mice Causes Progressive Cataracts.

PurposeYap1 encodes an evolutionarily conserved transcriptional coactivator and functions as a down-stream effector of the Hippo signaling pathway that controls tissue size and cell growth. Yap1 contributes to lens epithelial development. However, the effect of Yap1 haplodeficiency on the lens epithelium and its role in the development of cataracts has not been reported. The aim of the current study is to investigate Yap1 function and its regulatory mechanisms in lens epithelial cells (LECs).MethodsLens phenotypes were investigated in Yap1 heterozygous mutant mice by visual observation and histological and biochemical methods. Primary LEC cultures were used to study regulatory molecular mechanism.ResultsThe heterozygous inactivation of Yap1 in mice caused cataracts during adulthood with defective LEC phenotypes. Despite a normal early development of the eye including the lens, the majority of Yap1 heterozygotes developed cataracts in the first six months of age. Cataract was preceded by multiple morphological defects in the lens epithelium, including decreased cell density and abnormal cell junctions. The low LEC density was coincident with reduced LEC proliferation. In addition, expression of the Yap1 target gene Crim1 was reduced in the Yap1+/− LEC, and overexpression of Crim1 restored Yap1+/− LEC cell proliferation in vitro.ConclusionsHomozygosity of the Yap1 gene was critical for adequate Crim1 expression needed to maintain the constant proliferation of LEC and to maintain a normal-sized lens. Yap1 haplodeficiency leads to cataracts.

TGF-β regulation of microRNA miR-497-5p and ocular lens epithelial cell mesenchymal transition.

The purpose of this study was to investigate the role of a human lens microRNA (miR-497-5p) in regulating epithelialmesenchymal transition (EMT) under the control of transforming growth factor beta (TGF-β). A microRNA array was used to evaluate the microRNA profiles of untreated and TGF-β-treated human lens epithelial cells in culture. This showed that TGF-β treatment led to the upregulation of 96 microRNAs and downregulation of 39 microRNAs. Thirteen microRNAs were predicted to be involved in the pathogenesis of posterior capsule opacification (PCO). Meanwhile, overexpression of miR-497-5p suppressed cell proliferation and EMT 48 h post-transfection, and inhibition of miR-497-5p accelerated cell proliferation and EMT. Treatment with TGF-β inhibited the expression of miR-497-5p, but not cell proliferation. miR-497-5p was also found to regulate the level of CCNE1 and FGF7, which are reported to be actively involved in EMT. CCNE1 and FGF7 were bona fide targets of miR-497-5p. The results suggest that miR-497-5p participates in the direct regulation of lens epithelial cell EMT and is regulated by TGF-β. miR-497-5p may be a novel target for PCO therapy.

Connexin 50-R205G Mutation Perturbs Lens Epithelial Cell Proliferation and Differentiation.

PurposeTo investigate the underlying mechanisms for how the mouse Cx50-R205G point mutation, a homologue of the human Cx50-R198W mutation that is linked to cataract-microcornea syndrome, affects proper lens growth and fiber cell differentiation to lead to severe lens phenotypes.MethodsEdU labeling, immunostaining, confocal imaging analysis, and primary lens epithelial cell culture were performed to characterize the lens epithelial cell (LEC) proliferation and fiber cell differentiation in wild-type and Cx50-R205G mutant lenses in vivo and in vitro.ResultsThe Cx50-R205G mutation severely disrupts the lens size and transparency. Heterozygous and homozygous Cx50-R205G mutant and Cx50 knockout lenses all show decreased central epithelium proliferation while only the homozygous Cx50-R205G mutant lenses display obviously decreased proliferating LECs in the germinative zone of neonatal lenses. Cultured Cx50-R205G lens epithelial cells reveal predominantly reduced Cx50 gap junction staining but no change of the endoplasmic reticulum stress marker BiP. The heterozygous Cx50-R205G lens fibers show moderately disrupted Cx50 and Cx46 gap junctions while the homozygous Cx50-R205G lens fibers have drastically reduced Cx50 and Cx46 gap junctions with severely altered fiber cell shape in vivo.ConclusionsThe Cx50-R205G mutation inhibits both central and equatorial lens epithelial cell proliferation to cause small lenses. This mutation also disrupts the assembly and functions of both Cx50 and Cx46 gap junctions in lens fibers to alter fiber cell differentiation and shape to lead to severe lens phenotypes.

Calponin-3 deficiency augments contractile activity, plasticity, fibrogenic response and Yap/Taz transcriptional activation in lens epithelial cells and explants

The transparent ocular lens plays a crucial role in vision by focusing light on to the retina with loss of lens transparency leading to impairment of vision. While maintenance of epithelial phenotype is recognized to be essential for lens development and function, knowledge of the identity of different molecular mechanisms regulating lens epithelial characteristics remains incomplete. This study reports that CNN-3, the acidic isoform of calponin, an actin binding contractile protein, is expressed preferentially and abundantly relative to the basic and neutral isoforms of calponin in the ocular lens, and distributes predominantly to the epithelium in both mouse and human lenses. Expression and MEKK1-mediated threonine 288 phosphorylation of CNN-3 is induced by extracellular cues including TGF-β2 and lysophosphatidic acid. Importantly, siRNA-induced deficiency of CNN3 in lens epithelial cell cultures and explants results in actin stress fiber reorganization, stimulation of focal adhesion formation, Yap activation, increases in the levels of α-smooth muscle actin, connective tissue growth factor and fibronectin, and decreases in E-cadherin expression. These results reveal that CNN3 plays a crucial role in regulating lens epithelial contractile activity and provide supporting evidence that CNN-3 deficiency is associated with the induction of epithelial plasticity, fibrogenic activity and mechanosensitive Yap/Taz transcriptional activation.

Protective Effect of β-Glucogallin on Damaged Cataract Against Methylglyoxal Induced Oxidative Stress in Cultured Lens Epithelial Cells.

Backgroundβ-glucogallin (GG) is one of the major plant polyphenolic antioxidants that have been associated with positive effects on human health and are crucial in the developing defense mechanism against the risk of diseases. However, reports on the protective mechanism of GG in lens epithelial cells are limited.Material/MethodsARPE-19 cells (a human retinal epithelial cell line) were exposed to methylglyoxal (MG) with or without GG to illuminate the protective role of GG in counteracting the cataract signaling.ResultsCells predisposed to MG demonstrated an increase in oxidative stress with augmented (P<0.01) inflammatory cytokines such as cyclooxygenase (COX)-2, chemokine receptor CXCR4, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule 1 (ICAM-1) genes. In addition, the expression of aldose reductase (AR) was increased to 2-fold with accumulated sorbitol in MG exposed cells compared to control. On the other hand, cells exposed to MG evidenced a 3-fold increase in RAGE (receptor for advanced glycation end products) and a 2-fold increase in NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) expression compared to control cells. Intriguingly, lens epithelial cells pre-treated with GG attenuated the reactive oxygen species levels with improved antioxidant enzymes. Simultaneously, the levels of AR and other inflammatory cytokines were observed in the levels closer to control cells in GG pre-treated cells.ConclusionsThus, the results of the present investigation show that GG may be a potential drug for the prevention of cataract development and progression.

Determination of trypan blue efficacy in the mitigation of ex vivo canine PCO formation.

To determine whether trypan blue (TB) reduces canine lens epithelial cell (LEC) or corneal endothelial cell (CEC) viability in vitro; if cell death is noted, to subsequently evaluate the molecular mechanism. Cellular viability was determined using a lactate dehydrogenase (LDH) assay. In TB-treated LECs, caspase 3/7 activity was assessed to evaluate apoptosis; autophagy was evaluated using immunoblotting against LC3 and p62. To evaluate the effects of TB on ex vivo posterior capsule opacification (PCO), following mock cataract surgery, lens capsules were treated with TB and subsequently maintained in culture to determine LEC migration and proliferation. Following acute exposure, TB did not significantly reduce LEC or CEC viability at any of the concentrations tested. Increased caspase 3/7 activity was found in LEC cultures treated with TB for an extended period of time; no change in LC3 or p62 expression was noted. Ex vivo PCO formation was not significantly altered by TB treatment. Acute exposure to TB did not reduce LEC or CEC viability, and only longer exposure to TB was able to initiate apoptosis. Treatment with intraocular TB at the time of cataract surgery is likely safe to the CECs but will not prevent PCO formation.

Inhibition of histone deacetylase protects the damaged cataract via regulating the NF-κB pathway in cultured lens epithelial cells

Induction of oxidative damage by the activation of histone deacetylase (HDAC) is an integral event that causes major membrane damage of ocular tissues and leads to the pathogenesis of cataract. It is elucidated that nuclear factor-κB is a mediator in the process of cataract development. However, studies on the role played by epigenetic proteins in regulating cataract pathogenesis are limited. Hence, in the current investigation, ARPE-19 human retinal epithelial cells were used as an experimental model to elucidate the role of HDAC inhibition and its mechanism behind the cataract pathogenesis. ARPE-19 cells were exposed to H2O2, with and without Trichostatin A (TSA), a pan-HDAC inhibitor, and maintained along with control cells without any treatment. On exposure to H2O2, cells were susceptible to oxidative stress as it is evident from the reduced expression levels of superoxide dismutase (SOD), catalase, and GSH levels. Simultaneously, H2O2-exposed cells showed the nuclear translocation of NF-κB with the activation of inflammatory cytokines such as CXCL1 and IL-6. In addition, the mRNA expression analysis revealed that the GADD45α, COX-2, MCP-1, and ICAM-1 expressions were increased in H2O2 group. Moreover, the activity of HDAC was increased to 2-fold with a significant reduction in the histone acetyltransferase (HAT) activity in cells that were maintained under oxidative conditions. However, TSA was able to inhibit the critical cytokines’ expression with attenuated HDAC activity and limited NF-κB translocation. Furthermore, pre-treatment of TSA significantly suppressed the transcript levels of up-regulated inflammatory markers in cells. Together, these findings offer new insight into the role of HDACs in regulating cellular processes involved in the pathogenesis of cataract as well as the potential use of HDAC inhibitors as therapeutics for controlling the disease progression.

DNA hypermethylation-mediated downregulation of antioxidant genes contributes to the early onset of cataracts in highly myopic eyes

High myopia is recognized as a risk factor for earlier onset of nuclear cataracts. One possible explanation for this is that lenses in highly myopic eyes are exposed to higher levels of oxygen than normal eyes owing to earlier vitreous liquefaction and, hence, are subjected to oxidative insults. Here, we first compared the methylation levels of six essential antioxidant genes (GSTP1, NRF2, OGG1, TXN, TXNRD1 and TXNRD2) between highly myopic cataract (HMC) and age-related cataract (ARC) lens epithelial samples via Sequenom MassARRAY. We found that specific CpG units in the promoters of GSTP1 and TXNRD2 were hypermethylated and that the expression levels of these two genes were lower in the HMC group than in the ARC group. A luciferase reporter assay confirmed the significance of differentially methylated fragments in the activation of transcription. The importance of GSTP1 and TXNRD2 in antioxidant capacity was confirmed by overexpression or knockdown experiments on cultured lens epithelial cells (LECs). In addition, the expression of DNA methyl transferase 1 (DNMT1) was higher in the lens epithelium of HMC patients than that of ARC patients, and the expression of GSTP1 and TXNRD2 was upregulated by use of a DNMT inhibitor in cultured LECs. Finally, we mimicked the intraocular environment of highly myopic eyes by treating LECs with hydrogen peroxide (H2O2) and observed both alterations in the methylation status of the GSTP1 and TXNRD2 promoters and time-dependent altered expression levels. Therefore, we propose that in an environment with high oxygen, in which lenses in highly myopic eyes are immersed, there exists a vicious cycle composed of increased oxidative stress and decreased enzymatic antioxidants via the hypermethylation of antioxidant genes.

Effect of deacetylation degree on controlled pilocarpine release from injectable chitosan-g-poly(N-isopropylacrylamide) carriers

Development of biodegradable thermogels as intracameral injectable carriers for ocular delivery of antiglaucoma medications can provide a better treatment modality with low dosing frequency than eye drop formulations. For the first time, this study investigates the effect of deacetylation degree (DD) of the polysaccharide component in chitosan-g-poly(N-isopropylacrylamide) (CN) carriers on controlled release of pilocarpine in the management of glaucoma. Our results showed that increasing the chitosan DD from 60.7% to 98.5% leads to enhanced biodegradation resistance of carrier and prolonged release profile of the drug. Significant DNA damage and caspase-3 activation could be detected in lens epithelial cell cultures exposed to CN made from highly deacetylated polysaccharides, indicating apoptosis-related cytotoxicity due to relatively high positive charge density of the graft copolymers. Postoperative outcomes demonstrated that long-term therapeutic efficacy in glaucomatous rabbits is governed by intraocular pressure changes in response to intracamerally administered pilocarpine-loaded CN, strongly suggesting the usefulness of deacetylation in this injectable drug delivery carrier.

Fibronectin regulates growth factor signaling and cell differentiation in primary lens cells.

Lens epithelial cells are bound to the lens extracellular matrix capsule, of which laminin is a major component. After cataract surgery, surviving lens epithelial cells are exposed to increased levels of fibronectin, and so we addressed whether fibronectin influences lens cell fate, using DCDML cells as a serum-free primary lens epithelial cell culture system. We found that culturing DCDMLs with plasma-derived fibronectin upregulated canonical TGFβ signaling relative to cells plated on laminin. Fibronectin-exposed cultures also showed increased TGFβ signaling-dependent differentiation into the two cell types responsible for posterior capsule opacification after cataract surgery, namely myofibroblasts and lens fiber cells. Increased TGFβ activity could be identified in the conditioned medium recovered from cells grown on fibronectin. Other experiments showed that plating DCDMLs on fibronectin overcomes the need for BMP in fibroblast growth factor (FGF)-induced lens fiber cell differentiation, a requirement that is restored when endogenous TGFβ signaling is inhibited. These results demonstrate how the TGFβ-fibronectin axis can profoundly affect lens cell fate. This axis represents a novel target for prevention of late-onset posterior capsule opacification, a common but currently intractable complication of cataract surgery.

Calcium signaling in human lens epithelial cells after mechanical stimulation

PurposeThe purpose of this study is to explore and identify intra‐ and inter‐cellular calcium (Ca2+) signaling in human lens epithelial cells (LECs) upon local mechanical stimulation, to understand better the role of Ca2+ in intercellular communication related to cataract formation, lens regeneration and posterior capsular opacification (PCO).MethodsThe anterior lens capsule (aLC: basement membrane and associated LECs) were obtained from cataract surgery. Primary human LEC cultures were established by placing adherently the intact human aLC onto Petri dishes. LECs were stained with Fura‐2 dye, the fluorescence of which was imaged to monitor spatio‐temporal changes in cytosolic free Ca2+ concentrations in response to localized, micropipette induced mechanical stimulation.ResultsAnalysis of the intra‐ and inter‐cellular Ca2+ signaling from postoperative aLCs and cultures showed that the Ca2+ signal spreads radially and its propagation speed increases with the degree of cataract. While in aLC Ca2+ signals travels between the first few neighbor LECs in the order of seconds, in confluent culture the propagation is slower and it covers smaller distances. LECs from aLC with less developed cataracts exhibit faster and bigger changes in intracellular Ca2+ concentration. Moreover, in aLC and in LEC cultures, the duration of Ca2+ transients prolongs with increasing distance from stimulation site, whereas their amplitude decreases.ConclusionsThe modifications of Ca2+ homeostasis in LECs, which are associated with different degrees of cataract, affect Ca2+ signaling upon the local mechanical stimulation. Impairment of Ca2+ signaling might be the basis of cataract formation. LEC's cultures have less developed Ca2+ signaling capabilities, what might be reflected on lens regeneration and PCO.

Chitosan-g-poly(N-isopropylacrylamide) copolymers as delivery carriers for intracameral pilocarpine administration

This study reports, for the first time, the development of a chitosan-g-poly(N-isopropylacrylamide) (Chi-PN) biodegradable in situ gelling delivery system for ocular pilocarpine administration through intracameral injection. The number of thermo-responsive polymer segments grafted onto the chitosan via carbodiimide-mediated formation of amide linkages was greatly affected by varying the feeding amount of carboxyl-terminated poly(N-isopropylacrylamide) in the synthesis, thereby determining the phase transition temperature and enzymatic degradability of Chi-PN materials. The increase in grafting ratio facilitated temperature triggered gelation and drug encapsulation at physiological conditions. Additionally, the slow biodegradation process of delivery carriers was responsible for the delayed pilocarpine release, which allowed that the drug concentration could reach minimum therapeutic level for treating glaucoma during 42days of the study. All of the synthesized Chi-PN carriers demonstrated good ocular biocompatibility with lens epithelial cell cultures. In a rabbit model of experimental glaucoma, the intraocular pressure-lowering and miotic as well as corneal endothelial preservation responses to pilocarpine strongly depended on the drug release profiles. It is concluded that injectable biodegradable chitosan-based thermogels can be potentially utilized as intracameral biomaterials for extended release of antiglaucoma medications and improved performance of delivery carriers.