UV Effect on Human Anterior Lens Capsule Macro-Molecular Composition Studied by Synchrotron-Based FTIR Micro-Spectroscopy.

Ultraviolet (UV) irradiation is an important risk factor in cataractogenesis. Lens epithelial cells (LECs), which are a highly metabolically active part of the lens, play an important role in UV-induced cataractogenesis. The purpose of this study was to characterize cell compounds such as nucleic acids, proteins, and lipids in human UV C-irradiated anterior lens capsules (LCs) with LECs, as well as to compare them with the control, non-irradiated LCs of patients without cataract, by using synchrotron radiation-based Fourier transform infrared (SR-FTIR) micro-spectroscopy. In order to understand the effect of the UV C on the LC bio-macromolecules in a context of cataractogenesis, we used the SR-FTIR micro-spectroscopy setup installed on the beamline MIRAS at the Spanish synchrotron light source ALBA, where measurements were set to achieve a single-cell resolution with high spectral stability and high photon flux. UV C irradiation of LCs resulted in a significant effect on protein conformation with protein formation of intramolecular parallel ß-sheet structure, lower phosphate and carboxyl bands in fatty acids and amino acids, and oxidative stress markers with significant increase of lipid peroxidation and diminishment of the asymmetric CH3 band.

Bioinorganic Markers of a Loss of the Crystalline Lens Capsule Barrier Properties and Consequent Age-Related Cataract Development.

The age-related cataract development consequent upon a loss of the lens capsule barrier properties proved to be associated with accumulation of sodium, calcium, phosphorus and potassium. For the first time the use of spatial cluster and correlation analyses showed that the physical light scattering in the crystalline lens volume depends on changes in the lens matter elemental composition. The fields of elevated concentrations of sodium, calcium, phosphorus, potassium and chlorine conformed to the lens capsule geometry and their clustering was similar to that of opacity fields in the lens body. The accumulation geometry of the elements in the lens body that are commonly seen in the aqueous humor of the anterior chamber, can be considered evidence for excessive transitioning of their compounds through the lens capsule shell, while its spatial connection with transparency changes-proof of participation in cataractogenesis.

Amyloid found in human cataracts with two-dimensional infrared spectroscopy.

UV light and other factors damage crystallin proteins in the eye lens, resulting in cataracts that scatter light and affect vision. Little information exists about protein structures within these disease-causing aggregates. We examined postmortem lens tissue from individuals with and without cataracts using 2D infrared (2DIR) spectroscopy. Amyloid ß-sheet secondary structure was detected in cataract lenses along with denatured structures. No amyloid structures were found in lenses from juveniles, but mature lenses with no cataract diagnosis also contained amyloid, indicating that amyloid structures begin forming before diagnosis. Light scatters more strongly in regions with amyloid structure, and UV light induces amyloid ß-sheet structures, linking the presence of amyloid structures to disease pathology. Establishing that age-related cataracts involve amyloid structures gives molecular insight into a common human affliction and provides a possible structural target for pharmaceuticals as an alternative to surgery.

Characterizing human decellularized crystalline lens capsules as a scaffold for corneal endothelial tissue engineering.

The idea of transplanting a sheet of laboratory-grown corneal endothelium dates back to 1978; however, the ideal scaffold is still lacking. We hypothesized that human crystalline lens capsules (LCs) could qualify as a scaffold and aimed to characterize the properties of this material for endothelial tissue engineering. LCs were isolated from donor eyes, stored at -80 °C, and decellularized with water and trypsin-EDTA. The decellularization was investigated by nuclear staining and counting and the capsule thickness was determined by optical coherence tomography and compared with Descemet's membrane (DM). Transparency was examined by spectrometry, and collagenase degradation was performed to evaluate its resistance to degradation. Cell-scaffold interaction was assessed by measuring focal adhesions surface area on LC and plastic. Finally, primary corneal endothelial cells were grown on LCs to validate the phenotype. Trypsin-EDTA decellularized most effectively, removing 99% of cells. The mean LC thickness was 35.76 ± 0.43 µm, whereas DM measured 25.93 ± 0.26 µm (p < .0001). Light transmission was 90% for both LC and DM. On a collagenase challenge, LC and amniotic membrane were digested after 13 hr, whereas DM was digested after 17 hr. The surface area of focal adhesions for cells grown on coated LCs was at least double that compared with other conditions, whereas tight junctions, ion pumps, and hexagonal morphology were well maintained when endothelial cells were cultured on LCs. In conclusion, LCs demonstrate excellent scaffolding properties for tissue engineering and sustain the cell phenotype and can be considered a suitable substrate for ocular tissue engineering or as a template for future scaffolds.

Novel protein constituents of pathological ocular pseudoexfoliation syndrome deposits identified with mass spectrometry.

Purpose: Pseudoexfoliation (PEX) syndrome is an age-related progressive disease of the extracellular matrix with ocular manifestations. PEX is clinically diagnosed by the presence of extracellular exfoliative deposits on the anterior surface of the ocular lens. PEX syndrome is a major risk factor for developing glaucoma, the leading cause of irreversible blindness in the world, and is often associated with the development of cataract. PEX reportedly coexists with Alzheimer disease and increases the risk of heart disease and stroke. PEX material deposited on the anterior surface of the ocular lens is highly proteinaceous, complex, and insoluble, making deciphering the protein composition of the material challenging. Thus, to date, only a small proportion of the protein composition of PEX material is known. The aim of this study was to decipher the protein composition of pathological PEX material deposited on the ocular lens in patients and advance the understanding of pathophysiology of PEX syndrome. Methods: Liquid-chromatography and tandem mass spectrometry (LC-MS/MS) was employed to discover novel proteins in extracts of neat PEX material surgically isolated from patients (n = 4) with PEX syndrome undergoing cataract surgery. A sub-set of the identified proteins was validated with immunohistochemistry using lens capsule specimens from independent patients (n=3); lens capsules from patients with cataract but without PEX syndrome were used as controls (n=4). Expression of transcripts of the validated proteins in the human lens epithelium was analyzed with reverse transcription PCR (RT-PCR). Functional relationships among the proteins identified in this study and genes and proteins previously implicated in the disease were bioinformatically determined using InnateDB. Results: Peptides corresponding to 66 proteins, including ten proteins previously known to be present in PEX material, were identified. Thirteen newly identified proteins were chosen for validation. Of those proteins, 12 were found to be genuine components of the material. The novel protein constituents include apolipoproteins (APOA1 and APOA4), stress response proteins (CRYAA and PRDX2), and blood-related proteins (fibrinogen and hemoglobin subunits), including iron-free hemoglobin. The gene expression data suggest that the identified stress-response proteins and hemoglobin are contributed by the lens epithelium and apolipoproteins and fibrinogen by the aqueous humor to the PEX material. Pathway analysis of the identified novel protein constituents and genes or proteins previously implicated in the disease reiterated the involvement of extracellular matrix organization and degradation, elastic fiber formation, and complement cascade in PEX syndrome. Network analysis suggested a central role of fibronectin in the pathophysiology of the disease. The identified novel protein constituents of PEX material also shed light on the molecular basis of the association of PEX syndrome with heart disease, stroke, and Alzheimer disease. Conclusions: This study expands the understanding of the protein composition of pathological PEX material deposited on the ocular lens in patients with PEX syndrome and provides useful insights into the pathophysiology of this disease. This study together with the previous study by our group (Sharma et al. Experimental Eye Research 2009;89(4):479-85) demonstrate that using neat PEX material, devoid of the underlying lens capsule, for proteomics analysis is an effective approach for deciphering the protein composition of complex and highly insoluble extracellular pathological ocular deposits present in patients with PEX syndrome.

Pathology and immunohistochemistry of capsular bag in spontaneously late dislocated capsular bag-intraocular lens complex.

PURPOSE: Our study aims to evaluate the morphology, histopathology, and immunohistochemistry of the spontaneously late dislocated capsular bag-intraocular lens (CB-IOL) complex. Various etiologies and possible pathogenesis of the event are also discussed. METHODS: This was a tertiary-care setting and retrospective observational case series. The surgically explanted intact specimens of spontaneously late dislocated CB-IOL complex were studied. The demographics, duration of pseudophakia, IOL design/material, and specimen measurements were noted. Fresh specimens were photographed, and computer software was used for measurements. After processing, a detailed microscopic examination was carried out for three different sections of each specimen with hematoxylin and eosin (H and E), Masson's-trichrome, and immunohistochemistry stain for vimentin. The Mann-Whitney U-test was used for the statistical analysis. RESULTS: Of 12 specimens, the mean CB and capsulorhexis opening size were 8.32 ± 0.8 mm and 3.62 ± 0.61 mm, respectively. The average CB-IOL complex size of our study was significantly lower than the studies reported in the literature (P ≤ 0.001). All (n = 12, 100%) were acrylic IOLs with 11 (91.67%) having single-piece design. All specimens on H and E stain showed extensive subepithelial fibrosis while Masson's trichrome staining showed that none had any pseudoexfoliation material. The circumferential sphincter-like fibrous tissue arrangement was seen in all specimens. Immunohistochemical expression of vimentin suggested the mesenchymal metaplasia of epithelial A-cells. CONCLUSION: Significant fibrotic contraction of the CB and phimosis of capsulorhexis may cause a progressive zonular tear. This is probably the most important etiology of spontaneous late dislocation of the CB-IOL complex.

Immunohistochemical characteristics of the vitreolenticular interface in congenital unilateral posterior cataract.

PURPOSE: To gain insight into the histology of the vitreolenticular interface in congenital unilateral posterior cataract. SETTING: Antwerp University Hospital, Department of Ophthalmology, Edegem, and the University of Antwerp, Faculty of Medicine and Health Sciences, Antwerp, Belgium. DESIGN: Prospective case study. METHODS: Samples of the posterior lens capsule of patients with congenital posterior cataract (including opaque plaque on the anterior and adhesion to the vitreous on the posterior surface) were collected during the posterior capsulorhexis procedure. Staining for collagen types II and IV was performed using indirect immunohistochemistry. Results were compared with those of control posterior lens capsules of 3 children and 3 adults. RESULTS: Samples were collected from 3 patients. All posterior lens capsules contained collagen type IV. Samples from congenital posterior cataract patients all showed a narrow band of collagen type II on the outer surface, indicating strong adherence of the anterior hyaloid membrane to the center of the posterior lens capsule. Surprisingly, collagen type II was also found in the posterior capsule plaques. Collagen type II was not found in any control posterior lens capsule. CONCLUSION: The adherence of collagen type II to the center of the posterior lens capsule histologically supports the hypothesis that this subgroup of congenital cataract hints at an abnormality at the vitreolenticular interface. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Carbohydrates in rod and cone light absorbing segments in the firemouth cichlid Thorichthys meeki retina.

Carbohydrates in photoreceptor segments in the retina of the firemouth cichlid Thorichthys meeki are described and compared. The present periodic acid-Schiff (PAS) and lectin results revealed the occurrence of neutral carbohydrates, composed mainly of glycosamine and mannose and glucose, in the light absorbing segments in rods and cones in this species. Unlike in mammals, the retina in this teleost seems to be poor in galactose and galactose-galactosamine units in the light absorbing segments.

Impact of heparan sulfate chains and sulfur-mediated bonds on the mechanical properties of bovine lens capsule.

We assessed the importance of glycosaminoglycans and sulfur-mediated bonds for the mechanical properties of lens capsules by comparing the stress-strain responses from control and treated pairs of bovine source. No significant change in mechanical properties was observed upon reduction of disulfide bonds. However, removal of glycosaminoglycan chains resulted in a significantly stiffer lens capsule, whereas high concentrations of reducing agent, which is expected to reduce the recently reported sulfilimine bond of collagen IV, resulted in a significantly less stiff lens capsule. A comparison of the diffraction patterns of the control and strongly reduced lens capsules indicated structural rearrangements on a nanometer scale.

Identification of unknown intraocular material after cataract surgery: evaluation of a potential cause of toxic anterior segment syndrome.

PURPOSE: To describe and identify unknown opaque material between the optic of an AR40 intraocular lens (IOL) injected with the Emerald Series implantation system (both AMO, Inc.) and the posterior capsule at the conclusion of routine phacoemulsification to prevent an outbreak of toxic anterior segment syndrome (TASS). SETTING: Ambulatory care center operating room, University of North Carolina Hospitals and Department of Ophthalmology, University of North Carolina School of Medicine at Chapel Hill, Chapel Hill, North Carolina, USA. METHODS: After coaxial phacoemulsification in multiple patients, opaque material was present between the optic of a posterior chamber IOL and the posterior capsule. Although there was no TASS, the material was removed from 2 eyes and analyzed with scanning electron microscopy (SEM) and x-ray microanalysis (XRM). Similarly, crystalline lens, Klenzyme (Steris Corp.), Viscoat (sodium hyaluronate 3.0%-chondroitin sulfate 4.0%), and Provisc (sodium hyaluronate 1.0%) were analyzed. RESULTS: On SEM, the material had an irregular undulating surface similar to that of Provisc. Viscoat and the crystalline lens had smoother surfaces. On XRM, the material contained sodium, chlorine, and calcium, like Viscoat and Provisc, and phosphorous and sulfur, like Viscoat. The material also contained silicone, magnesium, aluminum, titanium, iron, and zinc. Klenzyme had smaller peaks of sodium, chlorine, and calcium and a higher carbon background than the unknown material. CONCLUSIONS: The material was likely ophthalmic viscosurgical device that was chemically and structurally altered by the cleaning and sterilization process. The silicone and metallic elements were probably from the Emerald Series implantation system as the disposable cartridge is coated with silicone and the reusable injector is metal.

Status of caveolin-1 in various membrane domains of the bovine lens.

Recent studies of the distribution and relative concentration of caveolin-1 in fractions of bovine lens epithelial and fiber cells have led to the novel concept that caveolin-1 may largely exist as a peripheral membrane protein in some cells. Caveolin-1 is typically viewed as a scaffolding protein for caveolae in plasma membrane. In this study, membrane from cultured bovine lens epithelial cells and bovine lens fiber cells were divided into urea soluble and insoluble fractions. Cytosolic lipid vesicles were also recovered from the lens epithelial cells. Lipid-raft domains were recovered from fiber cells following treatment with detergents and examined for caveolin and lipid content. Aliquots of all fractions were Western blotted for caveolin-1. Fluorescence microscopy and double immunofluorescence labeling were used to examine the distribution of caveolin-1 in cultured epithelial cells. Electron micrographs revealed an abundance of caveolae in plasma membrane of cultured lens epithelial cells. About 60% of the caveolin-1 in the epithelial-crude membrane was soluble in urea, a characteristic of peripheral membrane proteins. About 30% of the total was urea-insoluble membrane protein that likely supports the structure of caveolae. The remaining caveolin was part of cytosolic lipid vesicles. By contrast, most caveolin in the bovine lens fiber cell membrane was identified as intrinsic protein, being present at relatively low concentrations in caveolae-free lipid raft domains enriched in cholesterol and sphingomyelin. We estimate that these domains occupied 25-30% of the fiber cell membrane surface. Thus, the status of caveolin-1 in lens epithelial cells appears markedly different from that in fiber cells.

Detection of integrins in human cataract lens epithelial cells and two mammalian lens epithelial cell lines.

AIM: To compare the incidence of various integrin subunits in human cataract anterior lens epithelial cells (A-LEC) and in two mammalian LEC lines. METHODS: Circular sections of anterior capsules with attached LEC were obtained during cataract surgery. Integrin subunits were immunolocalised in these anterior LEC and in a human and rabbit LEC line, using four monoclonal antibodies specific for subunits alpha2, alpha3, and alpha5, and beta subunit 2. RESULTS: All of these subunits were found in at least a proportion A-LEC samples as follows: alpha2 71%, alpha3 92%, alpha5 62%, and beta2 24%. The human LEC line was immunoreactive for alpha2 and alpha3 only. The rabbit lens epithelial cell line was immunoreactive for alpha5 but there was no staining for alpha2, alpha3, or beta2. CONCLUSION: The A-LEC and mammalian LEC lines showed a similarity in their pattern of integrin expression. As these integrins are receptors for extracellular matrix (ECM) components, they are likely to be associated with the attachment and migration of LECs that precedes capsular opacification. Therefore these cell lines may be useful in the elucidation of mechanisms involved the pathogenesis of capsule opacification.

Immunological identification of types I and III collagen in bovine lens epithelium and its anterior lens capsule.

To define the molecular structure of bovine lens epithelium and its anterior lens capsule, we investigated the composition of lens capsule basement membrane proteins. Immunofluorescence and immunogold techniques were used to demonstrate the presence of type I and type III collagen in the lens capsule and in primary explant epithelial cultures grown on protein-binding membranes. Immunofluorescence staining with specific antibodies indicated that type I and type III collagen were constituents of lens basement membrane. We observed that deposition of type III collagen was more than type I collagen. The synthesis of fibrillar collagen by lens epithelium and its deposition in the lens capsule was established by localization of fibrillar collagen by transmission immunoelectron microscopy. These results demonstrate for the first time that normal lens epithelium synthesize fibrillar collagen which is an intrinsic component of the anterior lens capsule basement membrane.

Immunolocalisation of opticin in the human eye.

AIM: To localise the recently discovered glycoprotein opticin in the adult human eye. METHODS: Polyclonal rabbit antisera were raised against two different opticin peptides. Isolated human vitreous collagen fibrils were extracted with 8 M urea and the extract analysed by SDS-PAGE and western blotting. Paraffin embedded sections from two normal eyes were subjected to immunohistochemical analysis. RESULTS: Western blot analysis of the vitreous collagen fibril extract specifically identified opticin as a 45-50 kDa component that migrated as a doublet. Opticin was especially immunolocalised to the vitreous humour where labelling was most intense in the basal and cortical vitreous gel and less intense in the central vitreous. In addition, specific staining was observed along the surfaces of adjacent basement membranes including the internal limiting membrane (ILM) and posterior capsule of the lens. In one eye, labelling was also observed on the anterior lens capsule, but no other ocular tissues were specifically labelled. A type XVIII collagen/endostatin antibody labelled several ocular tissues including the ILM and basal vitreous gel. CONCLUSION: The immunolocalisation of opticin was confined to the vitreous humour, ILM, and lens capsule. In situ hybridisation studies have previously demonstrated opticin expression by the posterior non-pigmented ciliary epithelium. Thus, the immunolocalisation data support the proposition that the non-pigmented ciliary epithelium secretes opticin into the vitreous cavity where it associates with vitreous collagen and adjacent basement membranes. The staining along the ILM suggests a role for opticin in vitreoretinal adhesion and the co-localisation of opticin with type XVIII collagen/endostatin at the ILM raises the possibility that interactions between these two molecules might contribute to vitreoretinal adhesion.

Lens epithelial cell regeneration of a capsule-like structure during postoperative healing in rabbits.

PURPOSE: To determine whether lens epithelial cells (LECs) can regenerate the lens capsule during healing after lens extraction and intraocular lens (IOL) implantation. SETTING: Department of Ophthalmology, Wakayama Medical College, Japan. METHODS: Extracapsular lens extraction and IOL implantation were performed in 5 adult albino rabbits. Lens capsules were examined histologically and immunohistochemically 3 and 5 months later. RESULTS: Lens epithelial cells proliferated and regenerated lens fibers within the capsular bag. A multilayered homogenous capsule-like structure was present in the equatorial region. The structures contained type IV collagen but not type I collagen. CONCLUSION: Lens epithelial cells can regenerate lens capsule-like structures during healing after lens extraction. Postoperative LECs without phenotypic conversion to a fibroblastic type may produce this structure.

Comparison of Scheimpflug images of posterior capsule opacification and histological findings in rabbits and humans.

PURPOSE: To compare the posterior capsule opacification in Scheimpflug photographic images produced by an electronic anterior eye segment analysis system with the histopathological findings in rabbits and humans. SETTING: Department of Ophthalmology, Wakayama Medical College, Japan. METHODS: Opacified posterior capsules were photographed using the EAS-1000 system (Nidek) and were then extracted during vitreous surgery for proliferative diabetic retinopathy or proliferative vitreoretinopathy in 2 patients. In rabbits, phacoemulsification and aspiration (PEA) with intraocular lens (IOL) implantation was performed. The IOL was implanted in the bag or in the sulcus. After intervals of healing, the posterior capsule was photographed with the EAS-1000 and the animals were then killed. In both clinical and experimental specimens, the posterior capsule was processed for light microscopic histology and immunohistochemistry. RESULTS: Opacified human capsules were well imaged by the EAS-1000. Histology showed that lens epithelial cells proliferated with and without an accumulation of extracellular matrix. Details such as rolling of the capsulotomy edge were seen well. Regenerated lens fibers of Soemmering's ring were seen as a mass within the capsule. In the rabbit model, Scheimpflug images accurately represented the capsules as they appeared histologically. CONCLUSION: The EAS-1000 system provided faithful, relatively high-resolution images that corresponded to the histologic findings in the posterior capsules after PEA-IOL surgery in humans and rabbits.

Quantitative distribution of glycosaminoglycans in young and senile (cataractous) anterior lens capsules.

The ocular lens is surrounded by the lens capsule, which is an elastic and unusually thick basal membrane. Anionic sites are thought to be responsible for charge-selective permeability barriers in basal membranes. We have used cationic colloidal gold as a tracer for anionic binding sites to investigate the distribution of glycosaminoglycans in young and senile (cataractous) lens capsules. Using electron microscopy, combined with the cationic colloidal gold post-embedding technique, glycosaminoglycans were localized distinctively in a continuous layer immediately apposed to the lens epithelium, which has been referred to as the lamina lucida. The amount of gold particles decreased from the internal (lenticular) side of the capsule, toward the center, followed by an increase of label intensity toward the external (humoral) side. The humoral surface is characterized by a highly anionic layer measuring 1.5--4 micro m. Immunofluorescence microscopy localized three main types of glycosaminoglycans (heparan-, chondroitin- and dermatan sulfate) within this distinctive layer. Quantitative electron microscopy demonstrated reduced amounts of glycosaminoglycans at the lenticular and humoral side of senile (cataractous) lens capsules. The distinctive spatial distribution of glycosaminoglycans in human lens capsules is discussed in terms of age-related structural and functional changes.

Human lens beta-crystallin solubility.

The human lens is composed primarily of water and proteins called crystallins. Insolubility of these crystallins is correlated with aging and cataractogenesis. The alpha-crystallins have chaperone-like activity in maintaining the solubility of denatured beta- and gamma-crystallins. One established test of this chaperone activity is the ability of alpha-crystallin to prevent thermal destabilization of beta-crystallins. Several studies have addressed the effects of structural modifications of alpha-crystallin on chaperone activity, but little is known about the solubilities of the various beta-crystallins or the effects of post-translational modifications. Understanding the solubilities of different forms of beta-crystallins is important to elucidating the mechanism of chaperone activity. In this study, the solubilities of beta-crystallins were examined. The beta-crystallins included the gene products of betaB2, betaA1/A3, betaA4, and betaB1 as well as forms modified in vivo. Analysis of the beta-crystallins by high performance liquid chromatography and mass spectrometry before and after heating revealed large differences in the relative solubilities of the beta-crystallins. These results demonstrate a decreased solubility of specific beta-crystallins and post-translational modifications that may play a role in the crystallin insolubility associated with aging and cataract.

Uneven distribution and size of rabbit lens capsule proteoglycans.

To document the ultrastructural distribution of lens capsule proteoglycans, rabbit lens capsules were fixed and stained overnight in 50 mM sodium acetate, pH 5.6, containing 2.5% glutaraldehyde, 0.2% Cuprolinic Blue and 0.2 M MgCl2. They were rinsed, stained with 1% aqueous sodium tungstate, embedded in Epon, sectioned (60 nm), and examined with an electron microscope at 60 kV. Proteoglycan-Cuprolinic Blue complexes mainly appeared as networks of small electron-dense filaments throughout the posterior and anterior capsules. The posterior capsule was a single layer with a network of small proteoglycan filaments gradually decreasing in size from the humoral side (90 x 10 nm) to the lenticular side (30 x 8 nm). The humoral side of the anterior capsule had a thin lamina (400 nm) containing large (180 x 40 nm), very electron-dense proteoglycan-Cuprolinic Blue complexes plus small proteoglycans. Below this lamina, the complexes were only seen as filaments slightly smaller than those in the corresponding area of the posterior capsule. Cuprolinic Blue binding of the anterior and posterior lens capsules revealed differences in the size and distribution of their sulphated proteoglycans which do not correspond to the patterns of their immunoreactivity with anti-heparan sulphate proteoglycan. The humoral lamina in the anterior capsules, with large proteoglycan structures, might be a distinct structural and functional compartment.