Poly(2-hydroxyethyl methacrylate) surface chemistry and modulus differentially modulate neutrophils and lens epithelial cells-possible implications in cellular responses to intraocular lenses.

During cataract surgery, diseased lenses in the eye are surgically removed and replaced with polymeric artificial intraocular lenses (IOLs). Patients can experience a complication called posterior capsular opacification (PCO) that is corrected through the removal of part of the posterior capsule using a neodymium: yttrium-aluminum-garnet (Nd-YAG) laser to restore the optical path. These interventions have increased costs and can damage the retina and the IOL. PCO develops when lens epithelial cells (LECs) proliferate, migrate, and undergo epithelial-to-mesenchymal transition. Neutrophils involved in the immune response triggered during implantation impact LEC behavior and produce damaging neutrophil extracellular traps (NETs). In this research, poly(2-hydroxyethyl methacrylate) (PHEMA) -based disks were synthesized with varying amounts of comonomer (HEMA with 0, 2, and 12 mol% MMA) and functionalized with carboxyl and amine groups, yielding nine different hydrogels. Material and chemical properties of the disks were characterized, and neutrophil-like HL60 cells and B3 LECs were incubated with the disks. HL60 cell behavior was more strongly influenced by chemical functionalization than by mechanical properties with increases in adherence and NET accumulation. Conversely, the behavior and viability of B3 LECs were more strongly influenced by mechanical properties with increases in cell adhesion and α-SMA expression with increasing compressive moduli. Interestingly, B3 LECs had decreased viability and increased α-SMA expression when cultured on PHEMA2 disks pretreated with isolated NETs. Critical to the understanding of PCO and its prevention are both surface chemistry and mechanics as well as the inflammatory response.

The Immediate Early Response of Lens Epithelial Cells to Lens Injury.

Cataracts are treated by lens fiber cell removal followed by intraocular lens (IOL) implantation into the lens capsule. While effective, this procedure leaves behind numerous lens epithelial cells (LECs) which undergo a wound healing response that frequently leads to posterior capsular opacification (PCO). In order to elucidate the acute response of LECs to lens fiber cell removal which models cataract surgery (post cataract surgery, PCS), RNA-seq was conducted on LECs derived from wild type mice at 0 and 6 h PCS. This analysis found that LECs upregulate the expression of numerous proinflammatory cytokines and profibrotic regulators by 6 h PCS suggesting rapid priming of pathways leading to inflammation and fibrosis PCS. LECs also highly upregulate the expression of numerous immediate early transcription factors (IETFs) by 6 h PCS and immunolocalization found elevated levels of these proteins by 3 h PCS, and this was preceded by the phosphorylation of ERK1/2 in injured LECs. Egr1 and FosB were among the highest expressed of these factors and qRT-PCR revealed that they also upregulate in explanted mouse lens epithelia suggesting potential roles in the LEC injury response. Analysis of lenses lacking either Egr1 or FosB revealed that both genes may regulate a portion of the acute LEC injury response, although neither gene was essential for expression of either proinflammatory or fibrotic markers at later times PCS suggesting that IETFs may work in concert to mediate the LEC injury response following cataract surgery.

Spin-Coating-Based Facile Annular Photodynamic Intraocular Lens Fabrication for Efficient and Safer Posterior Capsular Opacification Prevention.

Posterior capsular opacification (PCO) is the most common complication after cataract surgery, which is primarily caused by the proliferation of the residual lens epithelial cells (LECs) in the lens capsule. Previous studies have demonstrated that a drug-eluting intraocular lens (IOL), aimed to in situ eliminate LECs, are an effective and promising way to prevent PCO. However, because of the potential toxicities of the antiproliferative drugs to the adjacent tissues, the safety of such drug-eluting IOLs is still a highly important issue to be solved. In this investigation, a facile photodynamic coating-modified IOL was developed for effective and safer PCO prevention. An annular poly(lactide-co-glycolic acid) (PLGA) coating loaded with photosensitizer chlorin e6 (Ce6) was prepared by a spin-coating technique. The optical property investigations showed that the Ce6@PLGA coating was particularly suitable for the IOL surface modification. The in vitro cell culture investigation showed that Ce6@PLGA coating-modified IOLs effectively eliminated LECs when treated with light illumination, whereas it appeared to have good cytocompatibility without irradiation. The investigation of the cell elimination mechanism showed that the apoptosis of HLECs may be associated with the cytomembrane disruption induced by ROS, which is generated by the photodynamic coating during light illumination. The in vivo implantation experiments confirmed the desired PCO prevention effect, as well as the safety to and biocompatibility with the surrounding tissues. Thus, the facile Ce6@PLGA coating will provide an effective yet safe alternative of IOL surface modification for PCO prevention.

Increased Desmosine in the lens capsules is associated with augmented elastin turnover in Pseudoexfoliation syndrome.

Pseudoexfoliation syndrome (PXF) is an idiopathic disease with a high prevalence rate. The elastosis disorder is contributed by genetic and non-genetic factors. Elastin dysregulation associated with the disease mechanism is incompletely understood. This study evaluated the molecules of the elastogenesis machinery in PXF. Lens capsule and aqueous humor (aqH) samples (age/sex-matched) were collected from the eyes with PXF alone and PXF with glaucoma (PXF-G) undergoing Extra Capsular Cataract Extraction (ECCE) surgery. The Elastin turnover was assessed by estimating Desmosine levels in the lens capsules by HPLC analysis. Expression of elastogenesis genes [EMILIN1, CLU, FBN1, FN1, FBLN5, FBLN4 and LOXL1] were evaluated in the lens capsule by qPCR while the proteins were assessed in aqH by western blot analysis. The Desmosine content in the lens capsules were 3-fold and 6-fold elevated in PXF (P = 0.02) and PXF-G (P = 0.01) respectively compared to the cataract-alone, indicating increased elastin degradation. A significant increase in the transcript levels of the CLU, FBLN4, EMILIN1, FBLN5, FN1, FBN1, LOXL1 along with significant changes in protein expression of CLU, FBLN5, FBN1 and LOXL1 signified up-regulation of the elastogenesis machinery. The study provides direct evidence of augmented elastin degradation and turnover in the lens capsule of PXF marked by increased Desmosine content and the expression of proteins involved in mature elastin formation.

Compositional Analysis of Extracellular Aggregates in the Eyes of Patients With Exfoliation Syndrome and Exfoliation Glaucoma.

Purpose: Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods: Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography-mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results: A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-ß-binding protein-2 and latent transforming growth factor-ß-binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-ß superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions: This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease.

Spatially Resolved Proteomic Analysis of the Lens Extracellular Diffusion Barrier.

Purpose: The presence of a physical barrier to molecular diffusion through lenticular extracellular space has been repeatedly detected. This extracellular diffusion barrier has been proposed to restrict the movement of solutes into the lens and to direct nutrients into the lens core via the sutures at both poles. The purpose of this study is to characterize the molecular components that could contribute to the formation of this barrier. Methods: Three distinct regions in the bovine lens cortex were captured by laser capture microdissection guided by dye penetration. Proteins were digested by Lys C and trypsin. Mass spectrometry-based proteomic analysis followed by gene ontology and protein interaction network analysis was performed. Results: Dye penetration showed that fiber cells first shrink the extracellular spaces of the broad sides followed by closure of the extracellular space between narrow sides at a normalized lens distance (r/a) of 0.9. Accompanying the closure of extracellular space of the broad sides, dramatic proteomic changes were detected, including upregulation of several cell junctional proteins. AQP0 and its interacting partners, Ezrin and Radixin, were among a few proteins that were upregulated, accompanying the closure of extracellular space of the narrow sides, suggesting a particularly important role for AQP0 in controlling the narrowing of the extracellular spaces between fiber cells. The results also provided important information related to biological processes that occur during fiber cell differentiation such as organelle degradation, cytoskeletal remodeling, and glutathione synthesis. Conclusions: The formation of a lens extracellular diffusion barrier is accompanied by significant membrane and cytoskeletal protein remodeling.

Advanced glycation end products in human diabetic lens capsules.

Advanced glycation end products (AGEs) accumulate with age in human lens capsules. AGEs in lens capsules potentiate the transforming growth factor beta-2-mediated mesenchymal transition of lens epithelial cells, which suggests that they play a role in posterior capsule opacification after cataract surgery. We measured AGEs by liquid chromatography-mass spectrometry in capsulorhexis specimens obtained during cataract surgery from nondiabetic and diabetic patients with and without established retinopathy. Our data showed that the levels of most AGEs (12 out of 13 measured) were unaltered in diabetic patients and diabetic patients with retinopathy compared to nondiabetic patients. There was one exception: glucosepane, which was significantly higher in diabetic patients, both with (6.85 pmol/µmol OH-proline) and without retinopathy (8.32 pmol/µmol OH-proline), than in nondiabetic patients (4.01 pmol/µmol OH-proline). Our study provides an explanation for the similar incidence of posterior capsule opacification between nondiabetic and diabetic cataract patients observed in several studies.

The effect of sex on the mouse lens transcriptome.

The transcriptome of mammalian tissues differs between males and females, and these differences can change across the lifespan, likely regulating known sexual dimorphisms in disease prevalence and severity. Cataract, the most prevalent disease of the ocular lens, occurs at similar rates in young individuals, but its incidence is elevated in older women compared to men of the same age. However, the influence of sex on the lens transcriptome was unknown. RNAseq based transcriptomic profiling of young adult C57BL/6J mouse lens epithelial and fiber cells revealed that few genes are differentially expressed between the sexes. In contrast, lens cells from aged (24 month old) male and female C57BL/6J mice differentially expressed many genes, including several whose expression is lens preferred. Like cataracts, posterior capsular opacification (PCO), a major sequela of cataract surgery, may also be more prevalent in women. Lens epithelial cells isolated from mouse eyes 24 h after lens fiber cell removal exhibited numerous transcriptomic differences between the sexes, including genes implicated in complement cascades and extracellular matrix regulation, and these differences are much more pronounced in aged mice than in young mice. These results provide an unbiased basis for future studies on how sex affects the lens response to aging, cataract development, and cataract surgery.

Profiling and Integrated Analysis of the ERCC6-regulated circRNA-miRNA-mRNA Network in Lens Epithelial Cells.

Purpose: To explore the regulatory role of ERCC6 in the circRNA-miRNA-mRNA network using a cellular ERCC6 overexpression model (OE-ERCC6) in lens epithelial cells.Methods: The expression profiles of circRNAs, miRNAs and mRNAs were determined by RNA-seq, and a regulatory circRNA-miRNA-mRNA network was constructed via bioinformatics. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were used for the functional annotation of circRNA host genes, differentially expressed (DE) genes, and miRNA targets.Results: The DE molecules between the OE-ERCC6 and control groups included 269 circRNAs, 241 miRNAs and 3500 mRNAs. We validated 5 selected DE reads of circRNAs (hsa_circ_0001009, hsa_circ_0002024, hsa_circ_0004592, hsa_circ_0001900 and hsa_circ_0001017). Subsequent bioinformatics analysis revealed that the DE circRNAs are mainly involved in oxidative stress- and cell death-related signaling pathways. Finally, a circRNA-miRNA-mRNA network focusing on DNA damage and cell death, which involved 5 circRNAs, 13 miRNAs and 107 mRNAs, was constructed.Conclusion: We constructed a circRNA-miRNA-mRNA network that is regulated by ERCC6. DE circRNAs have the potential to become therapeutic targets related to the lens lesions observed in ARC. The establishment of related in vivo and in vitro models could be a future direction to confirm these hypotheses.

Proteomic analysis of aqueous humor from cataract patients with retinitis pigmentosa.

A postcataract surgery complication in patients with retinitis pigmentosa (RP) is lens capsular contraction. To identify potential proteins contributing to this phenomenon, high-performance liquid chromatography/mass spectrometry-based proteomic analysis was conducted with aqueous humor samples collected from 11 patients who underwent cataract surgeries, with four patients diagnosed as RP and cataract (RP group) and the other seven with only senile cataract group. The upregulated proteins in the RP group were enriched in wound response, while downregulated proteins were enriched in cell adhesion and lens crystallins. Receptors of two dramatically upregulated proteins tenascin-C (TNC) and serotransferrin were found expressed in human lens epithelial cells (HLEs). TNC can promote primary HLEs proliferation and cell line HLE-B3 migration. This study indicates aqueous humor proteomic analysis serves as an effective way to unveil the pathogenesis of RP complications. TNC is a potential target of stimulating HLEs proliferation in RP concomitant cataract patients that worth further research.

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

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

The human Descemet's membrane and lens capsule: Protein composition and biomechanical properties.

The Descemet's membrane (DM) and the lens capsule (LC) are two ocular basement membranes (BMs) that are essential in maintaining stability and structure of the cornea and lens. In this study, we investigated the proteomes and biomechanical properties of these two materials to uncover common and unique properties. We also screened for possible protein changes during diabetes. LC-MS/MS was used to determine the proteomes of both BMs. Biomechanical measurements were conducted by atomic force microscopy (AFM) in force spectroscopy mode, and complemented with immunofluorescence microscopy. Proteome analysis showed that all six existing collagen IV chains represent 70% of all LC-protein, and are thus the dominant components of the LC. The DM on the other hand is predominantly composed of a single protein, TGF-induced protein, which accounted for around 50% of all DM-protein. Four collagen IV-family members in DM accounted for only 10% of the DM protein. Unlike the retinal vascular BMs, the LC and DM do not undergo significant changes in their protein compositions during diabetes. Nanomechanical measurements showed that the endothelial/epithelial sides of both BMs are stiffer than their respective stromal/anterior-chamber sides, and both endothelial and stromal sides of the DM were stiffer than the epithelial and anterior-chamber sides of the LC. Long-term diabetes did not change the stiffness of the DM and LC. In summary, our analyses show that the protein composition and biomechanical properties of the DM and LC are different, i.e., the LC is softer than DM despite a significantly higher concentration of collagen IV family members. This finding is unexpected, as collagen IV members are presumed to be responsible for BM stiffness. Diabetes had no significant effect on the protein composition and the biomechanical properties of both the DM and LC.

Requirement for Crk and CrkL during postnatal lens development.

The Crk and CrkL adaptor proteins have SH2 and SH3 domains and play essential overlapping, as well as distinct, roles in many biological processes, ranging from cell structure and motility to proliferation. Conditional ablation of both Crk and CrkL in neuronal progenitor cells, using a Nestin-Cre transgene, resulted in severe defects in postnatal eye development, including progressive eye closure, lens rupture, and retinal malformation. These phenotypes were not observed in the presence of a single wild-type allele of either Crk or CrkL. We found that the lens in knockout mice started to rupture and disintegrate between postnatal days 7 and 12, although the structure of the retina was relatively well maintained. As the lens deteriorated further, the outer nuclear layer in the posterior of the retina enlarged and developed ruffles. Cre recombination occurred in the lens and retina of the knockout mice. Furthermore, the posterior lens capsule of the knockout mouse was thinner at postnatal days 0.5 and 3, suggesting that the defective lens capsule caused rupturing of the lens near the posterior pole. These results indicate that Crk and CrkL play essential overlapping roles in postnatal lens development.

Down-regulation of CRTAC1 attenuates UVB-induced pyroptosis in HLECs through inhibiting ROS production.

Pyroptosis has been found to be related to diverse ocular diseases, including cataract. Abnormal CRTAC1 expression has been reported to involve in cataract formation by affecting cell apoptosis. Whether CRTAC1 regulates pyroptosis in the formation progress of cataract is completely unknown. Here, we aimed to investigate the regulatory effects of CRTAC1 on pyroptosis and the potential mechanism in the UVB-induced cell damage model. The results showed that the levels of the established pyroptosis markers (NLRP3, active Caspase-1, pro Caspase-1, GSDMD-N, IL-1ß and IL-18) were significantly increased in cataract patients. The above pyroptosis markers could be obviously induced by UVB-irradiation in human lens epithelial cells (HLECs), while down-regulation of CRTAC1 significantly reversed the UVB-induced pyroptosis. Up-regulation of CRTAC1 promoted HLECs pyroptosis, while the ROS inhibitor N-acetyl-l-cysteine blocked the effects of CRTAC1 overexpression. In conclusion, our findings further suggested that the prominent role of CRTAC1 in cataract formation.

The Evaluation of TUNEL, PCNA and SOX2 Expressions in Lens Epithelial Cells of Cataract Patients with Pseudoexfoliation Syndrome.

Purpose: This study aims to determine the expression patterns of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), proliferating cell nuclear antigen (PCNA) and SOX2 in lens epithelial cells (LEC) of cataract patients with pseudoexfoliation syndrome (PEX), and to determine the effect of apoptosis, proliferative activity and stem/progenitor cells on cataract formation in patients with PEX. This is a prospective, randomized clinical trial.Materials and Methods: Setting: institutional. 50 eyes of 50 patients were included. Anterior capsule samples were obtained during phacoemulsification surgery. The specimens of LEC were also examined using the TUNEL, PCNA and SOX2 immunohistochemical staining method. To detect the number of immunopositive cells, the total number of cells in a 3 mm2 area was counted using a microscope under x20 magnification and the percentage of cells stained positive was determined.Results: In Group 1, increased expression was observed with TUNEL, while decreased expression was detected with PCNA (p = .008, p = .015). The average percentage of TUNEL immunopositive cells was significantly higher in Group 1 than in Group 2, but there was no statistically meaningful SOX2 expression in Group 1 and Group 2 (P = .44). Apoptosis rates were 61.75 ± 14.5% and 36.91 ± 14.6% in Groups 1 and 2, respectively. Proliferation rates were 40.96 ± 16.8% and 65.45 ± 16.9% in Groups 1 and 2, respectively.Conclusion: We found increased apoptosis and decreased proliferation of LECs in cataract patients with PEX. We suspected that this could be related to oxidative stress.

Lysyl hydroxylase 3 is required for normal lens capsule formation and maintenance of lens epithelium integrity and fate.

Lens abnormalities are a major cause of reduced vision and blindness. One mechanism that can lead to reduced lens transparency, i.e. cataract, is abnormal behavior of lens epithelial cells (LECs), the precursors of the transparent lens fiber cells. Here we describe a zebrafish mutation causing the embryonic lens epithelium to generate cellular masses comprising partially differentiated lens fiber cells. We identify the mutant gene as plod3, which encodes for Lysyl hydroxylase 3 (Lh3), an enzyme essential for modification of collagens, including Collagen IV, a main component of the lens capsule. We show that plod3-deficient lenses have abnormal lens epithelium from an early developmental stage, as well as abnormal lens capsules. Subsequently, upregulation of TGFß signaling takes place, which drives the formation of lens epithelial cellular masses. We identify a similar phenotype in Collagen IVα5-deficient embryos, suggesting a key role for the defective lens capsule in the pathogenesis. We propose that plod3 and col4a5 mutant zebrafish can serve as useful models for better understanding the biology of LECs during embryonic development and in formation of lens epithelium-derived cataract.

Pigment epithelial-derived factor in the lens anterior capsule of patients with senile cataract with pseudoexfoliation.

BACKGROUND: This study sought to evaluate pigment epithelial-derived factor (PEDF) levels in lens anterior capsule material taken during cataract surgery from patients with senile cataract with pseudoexfoliation. METHODS: The study included 90 eyes of 86 patients who were diagnosed with, and underwent surgery for, cataracts. Sixty of the eyes included in the study had senile cataract. Thirty eyes of 30 young patients with other forms of cataract were included as a control group. Pseudoexfoliation was present in 21 patients with senile cataract. PEDF levels in the lens anterior capsule material - extracted with capsulorhexis in the classical phacoemulsification procedure - were measured by the enzyme-linked immunosorbent assay method and compared between the groups. RESULTS: The PEDF level in the lens anterior capsule in the senile cataract patient group was 149.36 ± 17.46 pg/ml. A statistically significant lower level of PEDF was found in the lens anterior capsule of patients with senile cataract compared with the other groups. In the patient group with pseudoexfoliation, the PEDF level in the lens anterior capsule was found to be statistically significantly lower than the patient group without pseudoexfoliation. CONCLUSION: PEDF levels decrease with senile cataract and pseudoexfoliation. These findings may clarify the pathogenesis of these conditions and point toward alternative treatment modalities.

Intraocular Deposition of Copper and ERG Findings in a Patient With Progressive Vision Loss From Hypercupremia.

A 46-year-old woman who presented for progressive glare was found to have dense deposition of copper within Descemet's membrane and lens capsule in both eyes (OU). Systemic workup revealed elevated serum copper secondary to multiple myeloma. Following bilateral Descemet stripping automated endothelial keratoplasty and cataract extraction, a green discoloration of the vitreous was noted. The patient was followed for 3 years with serial exams and electroretinograms. Electroretinograms showed declining photopic response amplitude OU, indicative of progressive retinal toxicity from copper. Although retinal toxicity and vitreous copper deposition are common in chalcosis, this appears to be the first case of hypercupremia associated with these findings. [Ophthalmic Surg Lasers Imaging Retina. 2019;50:e324-e326.].

Age-dependency of molecular diffusion in the human anterior lens capsule assessed using fluorescence recovery after photobleaching.

Purpose: To quantify the partition coefficient and the diffusion coefficient of metal-carrier proteins in the human lens capsule as a function of age. Methods: Whole lenses from human donors were incubated overnight in a solution of fluorescently labeled transferrin, albumin, or ceruloplasmin. In the central plane of the capsule thickness, fluorescence recovery after photobleaching (FRAP) experiments were conducted to measure the diffusion of the protein within the lens capsule. The anterior portion of the lens was recorded before the FRAP experiments to locate the boundaries of the anterior lens capsule and to measure the partition coefficient of the labeled proteins. The partition coefficient (P), the time to half maximum recovery of the fluorescent intensity (τ1/2), and the diffusion coefficient (D) for each protein were analyzed as a function of donor age. Results: There was no statistically significant relationship between the half maximum recovery time or the diffusion coefficient and age for transferrin (molecular weight [MW]=79.5 kDa, τ1/2=17.26±4.840 s, D=0.17±0.05 µm2/s), serum albumin (MW=66.5 kDa, τ1/2=18.45±6.110 s, D=0.17±0.06 µm2/s), or ceruloplasmin (MW=120 kDa, τ1/2=36.57±5.660 s, D=0.08±0.01 µm2/s). As expected, the larger protein (ceruloplasmin) took longer to recover fluorescent intensity due to its slower movement within the lens capsule. The partition coefficient statistically significantly increased with age for each protein (Palbumin: 0.09-0.71, Pceruloplasmin: 0.42-0.95, Ptransferrin: 0.19-1.17). Conclusions: The diffusion of heavy-metal protein carriers within the anterior lens capsule is not dependent on age, but it is dependent on the size of the protein. The permeability of the lens capsule to these heavy-metal protein carriers increases with age, suggesting that there will be a higher concentration of heavy metals in the older lens. This behavior may favor the formation of cataract, because heavy metals enhance protein oxidation through the Fenton reaction.

Resveratrol Inhibits Wound Healing and Lens Fibrosis: A Putative Candidate for Posterior Capsule Opacification Prevention.

Purpose: Posterior capsule opacification (PCO) is a common complication of cataract surgery. In addition to improved surgical methods and IOL designs, it is likely additional agents will be needed to improve patient outcomes. Presently no pharmacological agent is in clinical use to prevent PCO. Here we investigate the putative ability of resveratrol (RESV), a naturally occurring polyphenol, as a therapeutic agent. Methods: The human lens epithelial cell line FHL124, a human lens capsular bag model, and central anterior epithelium were used as experimental systems. Standard culture was in 5% fetal calf serum Eagle's minimum essential medium; 10 ng/mL transforming growth factor-ß2 (TGFß2) was used to induce fibrotic changes. A scratch wound assay was used to measure cell migration and the patch assay was used to assess matrix contraction by FHL124 cells. Protein expression was assessed by immunocytochemistry and Western blot and gene expression by quantitative RT-PCR. In capsular bags, cell growth across the posterior lens capsule, capsular wrinkling, and epithelial-to-mesenchymal transition were determined by image analysis. Results: In FHL124 cells, addition of 30 µM RESV significantly impeded cell migration in a wound-healing assay. RESV significantly inhibited TGFß2-induced expression of the myofibroblast marker alpha-smooth muscle actin (α-SMA) at both the message and protein levels, as well as significantly inhibiting matrix contraction induced by TGFß2. In human capsular bags, 30 µM RESV significantly inhibited cell growth. TGFß2-induced α-SMA expression and capsular wrinkling were also significantly inhibited by RESV treatment. RESV significantly suppressed expression of TGFß2-induced genes associated with fibrotic disease, including matrix metalloproteinase-2 in FHL124 cells, capsular bags, and central anterior epithelium. Conclusions: RESV can counter PCO-related physiological events in two human lens model systems. RESV therefore has the potential to be used as a candidate agent for the prevention of PCO, which in turn could benefit millions of cataract patients.