Showing papers in "Molecular Vision in 2010"

Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease.

Abstract: Purpose Inflammatory molecules have been demonstrated in the tear film of patients with severe dry eye disease (DED). However, little attention has been paid to the most frequent moderate forms of DED. This study analyzes tear cytokine levels and their clinical correlations in patients with moderate evaporative-type DED due to meibomian gland disease (MGD).

Cat-Map: putting cataract on the map.

Abstract: Lens opacities, or cataract(s), may be inherited as a classic Mendelian disorder usually with early-onset or, more commonly, acquired with age as a multi-factorial or complex trait. Many genetic forms of cataract have been described in mice and other animal models. Considerable progress has been made in mapping and identifying the genes and mutations responsible for inherited forms of cataract, and genetic determinants of age-related cataract are beginning to be discovered. To provide a convenient and accurate summary of current information focused on the increasing genetic complexity of Mendelian and age-related cataract we have created an online chromosome map and reference database for cataract in humans and mice (Cat-Map).

Reconstruction of the corneal epithelium with induced marrow mesenchymal stem cells in rats.

Abstract: Purpose To explore the feasibility of bone marrow mesenchymal stem cells (MSCs) transdifferentiating into corneal epithelial cells in a limbal stem cell deficiency (LSCD) model in rats. Methods Rat MSCs were isolated and purified using a gradient isolation procedure. The cells were induced by rat corneal stromal cells (CSCs) in a transwell co-culture system. The induced MSCs were identified by immunofluorescence staining, flow cytometry, and scanning electron microscopy (SEM). A corneal LSCD model was produced in the right eyes of 48 rats by alkali injury. The eyes of 12 rats without any transplant served as controls (Group 1). Amniotic membranes (AM; Group 2), uninduced MSCs (Group 3), or MSCs induced by CSCs (Group 4), were transplanted onto the cornea of the model (n=12 each). The therapeutic effects of the four groups were evaluated by slit lamp observation, hematoxylin and eosin staining, immunohistochemistry staining, and confocal laser corneal microscopy. Results Cultivated MSCs were positive for CD29, CD44, and CD90, but negative for CD34, CD45, CD133, and CK12, with typical MSCs characteristics revealed by SEM. After co-culture with CSCs, the induced MSCs expressed positive staining for CK12 with corneal epithelial cell characteristics confirmed by SEM; the induced MSCs were unchanged on the amnion. Compared with the other three groups, the corneal opacity, fluorescence staining, and neovascularization grades were significantly decreased in the induced MSCs group, both on postoperative week four and ten. Conclusion MSCs induced by CSCs can transdifferentiate into corneal epithelial cells in vitro. The induced MSCs on an amniotic membrane have remarkable effects on the treatment of corneal alkali burn and the reconstruction of the corneal surface of rats.

Resveratrol protects human lens epithelial cells against H2O2-induced oxidative stress by increasing catalase, SOD-1, and HO-1 expression

Abstract: Purpose Oxidative damage induced by H2O2 treatment can irreversibly damage the lens epithelium, resulting in cell death and cataract. Whether the effects of oxidative stress could be attenuated in cultured human lens epithelial cells by incubation with resveratrol (RES) is still unknown. In the present study, we examined the function of resveratrol in protecting human lens epithelial B-3 (HLEB-3) cells against H2O2 induced cell death and cell apoptosis, its role in reducing H2O2 induced intracellular reactive oxygen species (ROS) accumulation, and investigated the mechanism by which resveratrol underlies the effect.

Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types

Abstract: Purpose The purpose of this study was to produce and characterize human tissue-engineered corneas reconstructed using all three corneal cell types (epithelial, stromal, and endothelial cells) by the self-assembly approach. Methods Fibroblasts cultured in medium containing serum and ascorbic acid secreted their own extracellular matrix and formed sheets that were superposed to reconstruct a stromal tissue. Endothelial and epithelial cells were seeded on each side of the reconstructed stroma. After culturing at the air-liquid interface, the engineered corneas were fixed for histology and transmission electron microscopy (TEM). Immunofluorescence labeling of epithelial keratins, basement membrane components, Na+/K+-ATPase α1, and collagen type I was also performed. Results Epithelial and endothelial cells adhered to the reconstructed stroma. After 10 days at the air-liquid interface, the corneal epithelial cells stratified (4 to 5 cell layers) and differentiated into well defined basal and wing cells that also expressed Na+/K+-ATPase α1 protein, keratin 3/12, and basic keratins. Basal epithelial cells from the reconstructed epithelium formed many hemidesmosomes and secreted a well defined basement membrane rich in laminin V and collagen VII. Endothelial cells formed a monolayer of tightly-packed cells and also expressed the function related protein Na+/K+-ATPase α1. Conclusions This study demonstrates the feasibility of producing a complete tissue-engineered human cornea, similar to native corneas, using untransformed fibroblasts, epithelial and endothelial cells, without the need for exogenous biomaterial.

Gene and noncoding RNA regulation underlying photoreceptor protection: microarray study of dietary antioxidant saffron and photobiomodulation in rat retina

Abstract: Purpose To identify the genes and noncoding RNAs (ncRNAs) involved in the neuroprotective actions of a dietary antioxidant (saffron) and of photobiomodulation (PBM). Methods We used a previously published assay of photoreceptor damage, in which albino Sprague Dawley rats raised in dim cyclic illumination (12 h 5 lux, 12 h darkness) were challenged by 24 h exposure to bright (1,000 lux) light. Experimental groups were protected against light damage by pretreatment with dietary saffron (1 mg/kg/day for 21 days) or PBM (9 J/cm(2) at the eye, daily for 5 days). RNA from one eye of four animals in each of the six experimental groups (control, light damage [LD], saffron, PBM, saffronLD, and PBMLD) was hybridized to Affymetrix rat genome ST arrays. Quantitative real-time PCR analysis of 14 selected genes was used to validate the microarray results. Results LD caused the regulation of 175 entities (genes and ncRNAs) beyond criterion levels (p 2). PBM pretreatment reduced the expression of 126 of these 175 LD-regulated entities below criterion; saffron pretreatment reduced the expression of 53 entities (50 in common with PBM). In addition, PBM pretreatment regulated the expression of 67 entities not regulated by LD, while saffron pretreatment regulated 122 entities not regulated by LD (48 in common with PBM). PBM and saffron, given without LD, regulated genes and ncRNAs beyond criterion levels, but in lesser numbers than during their protective action. A high proportion of the entities regulated by LD (>90%) were known genes. By contrast, ncRNAs were prominent among the entities regulated by PBM and saffron in their neuroprotective roles (73% and 62%, respectively). Conclusions Given alone, saffron and (more prominently) PBM both regulated significant numbers of genes and ncRNAs. Given before retinal exposure to damaging light, thus while exerting their neuroprotective action, they regulated much larger numbers of entities, among which ncRNAs were prominent. Further, the downregulation of known genes and of ncRNAs was prominent in the protective actions of both neuroprotectants. These comparisons provide an overview of gene expression induced by two neuroprotectants and provide a basis for the more focused study of their mechanisms.

Adipose-derived stem cells differentiate to keratocytes in vitro.

Abstract: Purpose: Adipose-derived stem cells (ADSC) are an abundant population of adult stem cells with the potential to differentiate into several specialized tissue types, including neural and neural crest-derived cells. This study sought to determine if ADSC express keratocyte-specific phenotypic markers when cultured under conditions inducing differentiation of corneal stromal stem cells to keratocytes. Methods: Human subcutaneous adipose tissue was obtained by lipoaspiration. ADSC were isolated by collagenase digestion and differential centrifugation. Side population cells in ADSC were demonstrated using fluorescence-activated cell sorting after staining with Hoechst 33342. Differentiation to keratocyte phenotype was induced in fibrin gels or as pellet cultures with serum-free or reduced-serum media containing ascorbate. Keratocyte-specific gene expression was characterized using western blotting, quantitative RT–PCR, and immunostaining. Results: ADSC contained a side population and exhibited differentiation to adipocytes and chondrocytes indicating adult stem-cell potential. Culture of ADSC in fibrin gels or as pellets in reduced-serum medium with ascorbate and insulin induced expression of keratocan, keratan sulfate, and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1), products highly expressed by differentiated keratocytes. Expression of differentiation markers was quantitatively similar to corneal stromal stem cells and occurred in both serum-free and serum containing media. Conclusions: ADSC cultured under keratocyte-differentiation conditions express corneal-specific matrix components. Expression of these unique keratocyte products suggests that ADSC can adopt a keratocyte phenotype and therefore have potential for use in corneal cell therapy and tissue engineering. The cornea is the outermost tissue of the eye, providing a protective barrier and a clear path for transmission and refraction of light. This organ comprises three distinct cellular layers: epithelium, stroma, and endothelium. The stroma is primarily responsible for the strength and the refractive properties of the cornea [1]. The optical properties of the stroma result from layers of parallel aligned heterotypic collagen fibrils composed of Types I and V collagen. This collagen is associated with several proteoglycans from the small leucine-rich (SLRP) family, including decorin, lumican, keratocan, and mimecan (osteoglycin) [2-4]. The latter three of these proteins are modified with long, highly sulfated keratan sulfate glycosaminoglycan comprising the corneal keratan sulfate proteoglycans (KSPG), a family of molecules unique to corneal stromal extracellular matrix (ECM). After embryonic development, corneal collagen synthesis decreases, but synthesis of the KSPGs is maintained at a high level, suggesting an essential role for these ECM components in homeostasis of stromal transparency. This role has been confirmed by the loss of transparency associated with reduced sulfated keratan sulfate [5,6] or the loss of keratan sulfate core protein, lumican [7,8]. During stromal wound healing,

TRPM1 mutations are associated with the complete form of congenital stationary night blindness.

Abstract: Purpose To identify human transient receptor potential cation channel, subfamily M, member 1 (TRPM1) gene mutations in patients with congenital stationary night blindness (CSNB).

Mutation analysis of 272 Spanish families affected by autosomal recessive retinitis pigmentosa using a genotyping microarray.

Abstract: PURPOSE: Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive loss of vision. The aim of this study was to identify the causative mutations in 272 Spanish families using a genotyping microarray. METHODS: 272 unrelated Spanish families, 107 with autosomal recessive RP (arRP) and 165 with sporadic RP (sRP), were studied using the APEX genotyping microarray. The families were also classified by clinical criteria: 86 juveniles and 186 typical RP families. Haplotype and sequence analysis were performed to identify the second mutated allele. RESULTS: At least one-gene variant was found in 14% and 16% of the juvenile and typical RP groups respectively. Further study identified four new mutations, providing both causative changes in 11% of the families. Retinol Dehydrogenase 12 (RDH12) was the most frequently mutated gene in the juvenile RP group, and Usher Syndrome 2A (USH2A) and Ceramide Kinase-Like (CERKL) were the most frequently mutated genes in the typical RP group. The only variant found in CERKL was p.Arg257Stop, the most frequent mutation. CONCLUSIONS: The genotyping microarray combined with segregation and sequence analysis allowed us to identify the causative mutations in 11% of the families. Due to the low number of characterized families, this approach should be used in tandem with other techniques.

Expression profiles of nestin and synemin in reactive astrocytes and Müller cells following retinal injury: a comparison with glial fibrillar acidic protein and vimentin.

Abstract: Purpose: To examine the expression patterns of the intermediate filament (IF) proteins nestin and synemin following retinal injury. Methods: Wide-scale retinal injuries were created by experimental retinal detachment of 1, 3, 7, or 30 days’ duration. Injuries were induced in the right eyes of Long Evans rats, while the left eyes served as internal controls. Vibratome sections of control and injured retinas were labeled with fluorescent probes using a combination of anti–glial fibrillary acidic protein, -vimentin, -nestin, -synemin, -bromodeoxyuridine, and the lectin probe, isolectin B4. Additionally, antibody specificity, as well as protein and mRNA levels of nestin and synemin were determined and quantified using standard western blotting and real time polymerase chain reaction (RT–PCR) techniques. Results: Immunocytochemistry showed increased Muller cell labeling at 1, 3, and 7 days post injury for all four IFs, although the relative levels of nestin expression varied dramatically between individual Muller cells. Nestin was consistently observed in the foremost processes of those Muller cells that grew into the subretinal space, forming glial scars. Elevated levels of nestin expression were also observed in bromodeoxyuridine-labeled Muller cells following retinal insult. Quantitative polymerase chain reaction (qPCR) showed a twofold increase in nestin mRNA 1 day after injury, a level maintained at 3 and 7 days. Western blotting using anti-nestin showed a single band at 220 kDa and the intensity of this band increased following injury. Anti-synemin labeling of control retinas revealed faint labeling of astrocytes; this increased after injury, demonstrating an association with blood vessels. Additionally, there was an upregulation of synemin in Muller cells. qPCR and western blotting with anti-synemin showed a continuous increase in both gene and protein expression over time. Conclusions: Retinal injury induces an upregulation of a complement of four intermediate filament proteins, including synemin and nestin, in Muller cells. The latter provides suggestive support for the concept that these cells may revert to a more developmentally immature state, since these two IF proteins are developmentally regulated and expressed, and thus may serve as cell cycle reentry markers. Nestin and its differential expression patterns with glial fibrillary acidic protein and vimentin networks, as well as its association with proliferating Muller cells and those extending into the subretinal space, suggest a significant role of this protein in glial scar formation and perhaps gliogenesis. Synemin immunopositive astrocytes demonstrate a close relationship to the retinal vasculature, and illustrate a remarkable ability to reorganize their morphology in response to injury. Further examination of the changes in the cytoskeletal signatures of both of these glial cell types may lead to a more comprehensive understanding of mechanisms underway following retinal and other central nervous system injuries.

Genistein attenuates retinal inflammation associated with diabetes by targeting of microglial activation

Abstract: Purpose: Diabetic retinopathy (DR) is associated with microglial activation and increased levels of inflammatory cytokines. Genistein, a tyrosine kinase inhibitor, has been shown to possess anti-inflammatory potential that so far untested in animal models of diabetes. The aims of this study are to evaluate the efficacy of genistein for alleviation of diabetesinduced retinal inflammation and also to gain insight into the molecular mechanisms involved therein by analyzing the effect of genistein on concomitant microglia activation in the diabetic retina and in isolated cells. Methods: Streptozotocin (STZ)-induced diabetic Sprague Dawley rats were used. After diabetes was established for two weeks a single intravitreal injection of genistein or vehicle was performed. Forty-eight hours later, rats were killed, their retinal and vitreal samples were processed for Quantitative Real Time-PCR (qRT–PCR) and Enzyme-linked immunosorbent assay (ELISA) analyses, respectively. For the in vitro study, isolated microglial cells from retinas of newborn rats were used. Results: mRNA as well as protein levels for tumor necrosis factor α (TNF-α), a robust marker of inflammation, were increased in the retina early in the course of diabetes. Moreover, diabetes resulted in elevation of ionized calcium binding adaptor molecule-1 (Iba1) mRNA, known to be upregulated in activated microglia. These effects of diabetes in retina were all reduced by intervention treatment with genistein. Using an in vitro bioassay, we demonstrated the release of TNFα from microglia activated by glycated albumin, a risk factor for diabetic disorders. This inflammatory signal involves the activation of tyrosine kinase and its subsequent events, ERK and P38 MAPKs. Genistein represses the release of TNFα and significantly inhibits ERK and P38 phosphorylation in activated microglial cells by acting as a tyrosine kinase inhibitor. Conclusions: These findings show genistein to be effective in dampening diabetes-induced retinal inflammation by interfering with inflammatory signaling (ERK and P38 MAPKs) that occurs in activated microglia. This beneficial effect of genistein may represent a new intervention therapy to modulate early pathological pathways long before the occurrence of vision loss among diabetics.

Differential gene expression profile of retinoblastoma compared to normal retina.

Abstract: Purpose The retinoblastoma gene (RB1) is a tumor suppressor gene that was first discovered in a rare ocular pediatric tumor called retinoblastoma (RB). The RB1 gene is essential for normal progression through the cell cycle and exerts part of its function through the family of transcription factors (E2F) and many other intermediaries. In the absence of normal RB1, genomic instability and chromosomal aberrations accumulate, leading to tumor initiation, progression, and ultimately metastasis. The purpose of this report was to identify the molecular pathways that are deregulated in retinoblastoma.

Proteomic analysis of the tear film in patients with keratoconus.

Abstract: Purpose: To identify proteins differentially expressed between the tear film of keratoconus (KC) patients and control subjects using two dimensional electrophoresis (2-DE) and mass spectrometry-based techniques. Methods: Twenty two patients (44 eyes) diagnosed with bilateral KC and 22 control subjects (44 eyes) were studied in a prospective case-control study. Keratoconus screening programs and Orbscan II topographies were performed on all participants. Tear samples were collected by the Schirmer I method using filter paper. Proteins were extracted from the Schirmer strips and separated by 2-DE. Comparison of protein patterns was performed using PDQuest Software and protein differences were identified by mass spectrometry. Finally, results were validated by western-blot. Results: Four spots were identified to be differentially expressed between KC patients and control subjects. Three of them were more expressed in healthy subjects and they were identified as zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain). The other spot was more expressed in KC patients and it was identified as ZAG. Differences in ZAG seem controversial in two different spots because different posttranslational modifications, however, analysis of both spots revealed that globally, ZAG is overexpressed in healthy subjects. Founded differences in ZAG, lactoferrin, and IGKC expression were subsequently validated by western blot. Conclusions: IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects. These differences could contribute to the knowledge of the pathophysiology of this disease. Keratoconus (KC) is a primary corneal ectasia, generally bilateral and progressive, with a conical shape as a result of the thinning of the stroma. This thinning induces irregular astigmatism, myopia, and protusion, leading to mild to marked impairment in the quality of vision [1]. KC has its onset at puberty, and it progresses until the third or fourth decade of life. The calculated incidence of KC is between 1/500 and 1/2,000 in the general population [1,2]. KC affects young persons in a very productive period of their lives. Yet the cause of this disorder, considered to be the most common indication for penetrating keratoplasty in developed countries [3], remains unknown. KC may be isolated or may be associated with various clinical conditions, or even form part of a syndrome. Its etiology remains poorly understood, but it has been reported to arise as a consequence of biochemical alterations to corneal collagenase, which breaks down collagen leading to stromal thinning [4]. Other authors have suggested that it may arise as a consequence of keratocyte loss [5], which is on average about 19% lower in contact lens users [6]. In some atopic diseases, KC may be due to apoptosis of corneal cells, possibly

Novel mutations in MERTK associated with childhood onset rod-cone dystrophy

Abstract: Purpose: To report the clinical phenotype in patients with a retinal dystrophy associated with novel mutations in the MER tyrosine kinase (MERTK) gene.Methods: A consanguineous family of Middle Eastern origin was identified, and affected members underwent a full clinical evaluation. Linkage analysis was performed using the Affymetrix 50K chip. Regions of homozygosity were identified. The positional candidate genes protocadherin 21 (PCDH21), retinal G protein-coupled receptor (RGR), and MERTK were polymerase chain reaction (PCR) amplified and sequenced. Long-range PCR was performed to characterize the deletion. Two hundred and ninety-two probands with autosomal recessive, childhood onset, retinal dystrophies were analyzed using the Asper Ophthalmics Leber congenital amaurosis chip to screen for known MERTK mutations.Results: Analysis of a 50K-Affymetrix whole genome scan identified three regions of homozygosity on chromosomes 2 and 10. Screening of the candidate gene MERTK showed a possible deletion of exon 8. Long-range PCR identified a similar to 9 kb deletion within MERTK that removes exon 8. Screening of DNA from a panel of Saudi Arabian patients with autosomal recessive retinitis pigmentosa identified a second consanguineous family with the same mutation. One patient with a known MERTK mutation (p.R651X) was identified using the Asper Ophthalmics Leber congenital amaurosis chip. Further screening of the gene identified a second novel splice site mutation in intron 1. The phenotype associated with these identified MERTK mutations is of a childhood onset rod-cone dystrophy with early macular atrophy. The optical coherence tomography (OCT) appearance is distinctive with evidence of debris beneath the sensory retina.Conclusions: Mutations in MERTK are a rare cause of retinal dystrophy. Non homologous recombination between Alu Y repeats near or within disease genes may be an important cause of retinal dystrophies.

Antiglycating potential of Zingiber officinalis and delay of diabetic cataract in rats.

Abstract: Purpose: Advanced glycation end products (AGE) are associated in the development of several pathophysiologies including diabetic cataract. Earlier we have reported that some common dietary agents have antiglycating activity and ginger (Zingiber officinalis) was one of the few prominent agents that effectively prevented AGE formation in vitro. In this study we investigated the potential of ginger to prevent diabetic cataract in rats. Methods: Diabetes was induced in Wistar-NIN rats by intraperitoneal injection of streptozotocin (35 mg/kg bodyweight) and the control rats received vehicle alone. While a set of diabetic animals received AIN-93 diet, another set received either 0.5 or 3% ginger in their diet for a period of two months. Cataract progression was monitored by slit-lamp biomicroscope. At the end of two months, the animals were sacrificed to evaluate non-enzymatic glycation and osmotic stress in the eye lens. Results: Slit-lamp examination revealed that feeding of ginger not only delayed the onset but also the progression of cataract in rats. Molecular analyses indicated that feeding of ginger significantly inhibited the formation of various AGE products including carboxymethyl lysine in the eye lens. In addition, ginger also countered hyperglycemia-induced osmotic stress in the lens. Conclusions: The results indicated that ginger was effective against the development of diabetic cataract in rats mainly through its antiglycating potential and to a lesser extent by inhibition of the polyol pathway. Thus, ingredients of dietary sources, such as ginger, may be explored for the prevention or delay of diabetic complications.

Major LOXL1 risk allele is reversed in exfoliation glaucoma in a black South African population.

Abstract: PURPOSE To investigate whether variants in the lysyl oxidase-like 1 (LOXL1) gene are associated with exfoliation glaucoma (XFG) and primary open-angle glaucoma (POAG) in an ancestral population from South Africa. METHODS Black South African subjects with XFG, POAG, and age matched unaffected controls were recruited from the St. John Eye Hospital in Soweto, Johannesburg, South Africa, using standard clinical examination techniques. Fifty individuals were collected for each of the three groups: XFG, POAG, and normal controls. The complete coding region of LOXL1 was sequenced using the PCR-based Sanger method. The allele frequencies of the identified sequence variants were compared between XFG or POAG and controls using Fisher's exact test. RESULTS A large number of coding variants were identified, including rs1048661 (R141L), rs3825942 (G153D), S159A, S161L, rs41435250 (A320A), rs13329473 (F489F), and T567A. The allele frequencies of both rs3825942 and rs1048661 differed significantly between the XFG and control subjects from South Africa (p=5.2 x 10(-13) and 1.7 x 10(-5), respectively). The G allele for rs1048661 (encoding arginine) was the risk allele which is similar to other populations. The A allele of rs3825942 (encoding aspartic acid) was the risk allele, in sharp contrast to the G allele (encoding glycine) reported in multiple other populations. There was no significant difference in the allele frequencies of coding variants in LOXL1 between POAG and control subjects. CONCLUSIONS This represents the first genetic association study of LOXL1 in an ancestral African population with XFG. We have confirmed the association between variants of LOXL1 and XFG. To date, the G allele of the major susceptibility variant rs3825942 has consistently been shown in multiple populations to increase the risk of XFG. Surprisingly, we have found a strong association with the opposite allele in the South African population. This suggests that other as yet unknown causal variants of LOXL1 contribute to the genetic risk of XFG.

The fate of Müller's glia following experimental retinal detachment: nuclear migration, cell division, and subretinal glial scar formation.

Abstract: Purpose: To study the fate of Muller’s glia following experimental retinal detachment, using a “pulse/chase” paradigm of bromodeoxyuridine (BrdU) labeling for the purpose of understanding the role of Muller cell division in subretinal scar formation. Methods: Experimental retinal detachments were created in pigmented rabbit eyes, and 3 days later 10 µg of BrdU was injected intravitreally. The retinas were harvested 4 h after the BrdU was administered (i.e., day 3) or on days 4, 7, and 21 post detachment. The tissue was fixed, embedded in agarose, and sectioned at 100 µm. The sections were labeled with various combinations of probes, including anti-vimentin and anti-S100 (as markers for Muller cells), anti-BrdU, antiphosphohistone H3 (to identify mitotic cells), and the isolectin B4 (to identify macrophages and microglia). Images were captured using an Olympus Fluoview 500 confocal microscope. To aid in our understanding of how Muller cell nuclei undergo cell division, two additional procedures were used: 1) electron microscopy of normal cat and rabbit retinas and 2) a new method using 5-fluorouracil and subsequent anti-BrdU labeling to detect all Muller cell nuclei, using confocal imaging. Results: Three days after detachment, anti-vimentin labeled all Muller cells, some of which were also labeled with antiBrdU. On day 4, many of the anti-BrdU-labeled Muller cell nuclei appeared in columns with one labeled nucleus in the inner nuclear layer and another directly sclerad to it in the outer nuclear layer. By day 7, most anti-BrdU-labeled nuclei were observed in subretinal scars. At 3 weeks, some anti-BrdU-labeled nuclei that remained within the retina did not express vimentin or S100. Anti-phosphohistone H3-labeled (i.e., mitotic) cells, some of which were also labeled with antiBrdU, were only observed in the outer nuclear layer on day 4, and these nuclei were surrounded by an accumulation of vimentin filaments. Isolectin B4-labeled microglia and macrophages also incorporated BrdU and were observed throughout the retina and in subretinal scars during all times of detachment. Electron microscopy and immunofluorescence labeling of the 5-fluorouracil-injected eyes revealed the presence of a unique structural relationship between Muller cell nuclei and intermediate filament proteins. Conclusions: Following retinal detachment, many Muller cell nuclei initially migrate to the outer retina, undergo mitosis, and eventually reside in subretinal glial scars, suggesting a possible link between the early division of Muller cells and the process of subretinal gliosis. In addition, a subpopulation of anti-BrdU-labeled cells, presumably once Muller cells, appears to stop expressing well accepted Muller cell marker proteins, suggesting a potential dedifferentiation of some of these cells over time. Additionally, Muller cell nuclei may use intermediate filaments as a “track” for migration into the outer retina and later as an important component of cell division by the accumulation of vimentin filaments around the mitotic nuclei.

Autosomal recessive congenital cataract linked to EPHA2 in a consanguineous Pakistani family.

Abstract: Ophthalmic Genetics and Visual Function Branch, National Eye Institute,National Institutes of Health, Bethesda, MDPurpose: To investigate the genetic basis of autosomal recessive congenital cataracts in a consanguineous Pakistanifamily.Methods: All affected individuals underwent a detailed ophthalmological and clinical examination. Blood samples werecollected and genomic DNAs were extracted. A genome-wide scan was performed with polymorphic microsatellitemarkers. Logarithm of odds (LOD) scores were calculated, and Eph-receptor type-A2 (EPHA2), residing in the criticalinterval, was sequenced bidirectionally.Results: The clinical and ophthalmological examinations suggested that all affected individuals have nuclear cataracts.Genome-wide linkage analyses localized the critical interval to a 20.78 cM (15.08 Mb) interval on chromosome 1p, witha maximum two-point LOD score of 5.21 at θ=0. Sequencing of EPHA2 residing in the critical interval identified a missensemutation: c.2353G>A, which results in an alanine to threonine substitution (p.A785T).Conclusions: Here, we report for the first time a missense mutation in EPHA2 associated with autosomal recessivecongenital cataracts.

LOC387715/HTRA1 gene polymorphisms and susceptibility to age-related macular degeneration: A HuGE review and meta-analysis.

Abstract: Purpose: To examine the association of age-related macular degeneration (AMD) with HtrA serine peptidase 1 (HTRA1) gene rs11200638 G→A polymorphism and LOC387715/ ARMS2 gene rs10490924 G→T polymorphisms, and to evaluate the magnitude of the gene effect and the possible genetic mode of action. Methods: We searched the US National Library of Medicine’s PubMed, Embase, OMIM, ISI Web of Science, and CNKI databases in a systematic manner to retrieve all genetic association studies on the HTRA1 (rs11200638) and LOC387715/ ARMS2 (rs10490924) gene polymorphisms and AMD. We performed a meta-analysis conducted with Stata software, version 9.0. Results: Individuals who carried the AA and AG genotypes of HTRA1 gene rs11200638 G→A polymorphism had 2.243 and 8.669 times the risk of developing AMD, respectively, when compared with those who carry the GG genotype. Individuals carrying the TT and TG genotypes of LOC387715/ ARMS2 gene rs10490924 G→T polymorphism had 7.512 and 2.353 times the risk of developing AMD, respectively, compared with those who carry GG genotype. These results suggested a “moderate” codominant, multiplicative genetic mode; that is, both HTRA1 rs11200638 G→A polymorphism and LOC387715/ARMS2 rs10490924 G→T polymorphism play important roles in the pathogenesis of AMD. We found no evidence of publication bias. Between-study heterogeneity was found in both allele-based analysis and genotype-based analysis. Conclusions: HTRA1 rs11200638 G→A polymorphism and LOC387715/ARMS2 rs10490924 G→T polymorphism play important roles in AMD. Gene-gene and gene-environmental interactions, as well as precise mechanisms underlying common variants in the HTRA1 gene and LOC387715/ ARMS2 gene, potentially increase the risk of AMD and need further exploration.

Tear proteomics in keratoconus

Abstract: Purpose: The purpose of this work was to identify potential tear-film based proteins expressed in keratoconus. Methods: Recruited subjects were normal gas permeable (GP) contact lens wearers, keratoconus subjects wearing GP contact lenses, and keratoconus subjects without contact lenses. Subjects wearing soft lenses or having previous ocular surgeries were excluded from participating. Approximately 5 µl of tears were sampled from both eye of each subject using glass microcapillaries. Additional testing included a brief history, visual acuity, slit lamp examination, and topography. Proteomic analyses used to compare samples included Bradford assays, cytokine arrays, SDS–PAGE, and mass spectrometry. Results: Forty-four subjects were enrolled in the study including 20 normals (GP wearers), 18 with keratoconus and wearing GPs, and six with keratoconus (non-lens wearers). Across all proteomic approaches, several proteins were identified as possibly being unique to keratoconus. Increased expression of matrix metalloproteinase-1 (MMP-1) was found in keratoconus subjects with and without gas permeable contact lenses (p=0.02). Unique proteins more associated with keratoconus included several keratins, immunoglobulins alpha and kappa, precursors to prolactin, lysozyme C, and lipocalin. Conclusions: Initial analyses indicate that keratoconus may be associated with the differential expression of several proteins. Further testing is needed to determine any causal relationship or correlation with the etiology of this condition. Keratoconus is an asymmetric condition of corneal ectasia and thinning with onset usually in early teens to early twenties, with an incidence of about 1/2,000 [1]. The condition can lead to significant visual impairment with high amounts of irregular astigmatism and myopia. Classic objective signs seen by biomicroscopy include corneal stromal thinning, central corneal scarring, vertical lines in the posterior cornea (Vogt’s striae), and prominent corneal nerves; quite often a brownish or olive green colored ring of iron deposition (Fleischer’s ring) is seen at the base of the “cone” or apex of the protrusion [2]. Although improved with pinhole, the best corrected visual acuity in keratoconus subjects is often reduced with spectacle correction; therefore, most subjects are managed with rigid gas permeable (GP) contact lenses in a wide range of specifications. Some subjects may require penetrating keratoplasty if contact lenses are no longer a management option [2]. Keratoconus is historically defined as a non

Involvement of genetic factors in the response to a variable-dosing ranibizumab treatment regimen for age-related macular degeneration.

Abstract: Purpose: To determine whether gene polymorphisms of the major genetic risk factor for age-related macular susceptibility 2 (ARMS2 A69S) and the complement factor H Y402H influence the response to a variable-dosing treatment regimen with ranibizumab for age-related macular degeneration Methods: This prospective cohort study included 90 patients (90 eyes) with exudative age related macular degeneration (AMD) treated with ranibizumab Patients underwent a 1-year treatment as in the Study of Ranibizumab in Patients with Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration (Mitchell et al) Injections were administered monthly when a patient lost five letters on the Early Treatment Diabetic Retinopathy Study chart or gained 100 μm in central subfield retinal thickness (CSRT) Genotypes (rs10490924 and rs1061170) were analyzed using gene sequence analysis Best-corrected visual acuity (BCVA) and CSRT values were compared between ARMS2 and complement factor H genotypes Multiple regression analysis was used to assess the statistical significance Results: Mean increase in visual acuity was 444±812 letters with a 10363±947 µm decrease in CSRT BCVA improvement was statistically significant in all genotype groups except in homozygous 69S in the AMRS2 gene CSRT and BCVA changes were correlated (r=02521; 95% CI: 004746–04364, p=00165) Multiple regression analysis revealed a significant impact of 69S (p=0015) on the change in BCVA Conclusions: Visual acuity did not improve during the study in patients homozygous for ARMS2 69S, despite a decrease in CSRT Further investigation is needed to confirm our findings and understand the mechanisms involved

Variability of serum oxidative stress biomarkers relative to biochemical data and clinical parameters of glaucoma patients.

Abstract: Purpose: The importance of oxidative stress in both the formation and the course of glaucoma has been known. Among the antioxidants, vitamin E possesses the specific effects and regulatory mechanisms of a neurohormone. The serum oxidant/antioxidant profile is reportedly altered in ocular pathologies. In this study, we analyzed the effect of the clinical parameters of glaucoma and biochemical data on antioxidants and serum oxidative stress markers as oxidation degradation products. Methods: In this multicenter case control study, control and patient groups consisted of 31 healthy individuals and 160 glaucoma patients with no known additional abnormalities, respectively. We analyzed the oxidation degradation products malonyl dialdehyde (MDA), advanced oxidation protein products (AOPP), antioxidants, vitamins E and A, Serine (Ser), superoxide dismutase (SOD), glutathione peroxidase (Gpx), transferrine (TF), and total antioxidant capacity (TADA). All of these parameters and their relationships with serum cholesterol, glucose, protein, albumin, triglyceride levels, age, gender, visual acuities, intraocular pressure (IOP), c/d ratio, gonioscopic findings, medications, presence of pseudoexfoliation (px), central visual field and Optical Coherence Tomography (OCT) data, pachymetry, and Laplace values, were evaluated individually. Statistical comparisons were performed among them, and with the control group as well. Results: TADA, AOPP, SOD, and Gpx were found to be decreased, and MDA, Ser, TF, vitamins A and E increased in

Changes to tear cytokines of type 2 diabetic patients with or without retinopathy.

Abstract: Purpose: To investigate changes in cytokine levels in tears of type 2 diabetics with or without retinopathy. Methods: Tears were collected from 15 type 2 diabetics without retinopathy (DNR), 15 patients with retinopathy (DR), and 15 age and gender matched non-diabetic controls. Tear concentrations of 27 cytokines were measured by multiplex bead immunoassay. Cytokine differences between groups, ratios of type-1 T helper (Th1)/type-2 T helper (Th2) cytokines and anti-angiogenic/pro-angiogenic cytokines were analyzed statistically. Results: The most abundant cytokine detected in tears was interferon-induced protein-10 (IP-10). In comparison with controls, IP-10 and monocyte chemoattracant protein-1 (MCP-1) levels were significantly elevated in DR (p=0.016 and 0.036, respectively) and DNR groups (p=0.021 and 0.026, respectively). Interleukin-1 (IL-1) receptor antagonist (IL-1ra) levels were significantly increased in DNR (p=0.016). Th1/Th2 cytokines interferon-gamma (IFN-γ)/IL-5 and IL-2/IL-5 ratios were significantly increased in DR compared to controls (p=0.037 and 0.031, respectively). Anti-angiogenic/ angiogenic cytokines IFN-γ/MCP-1 and IL-4/MCP-1 ratios in DR and DNR were significantly decreased compared to controls (p<0.05). IL-4/IL-8 and IL-12p70/IL-8 ratios were also significantly decreased in DR compared to controls (p=0.02 and 0.045, respectively). No significant correlation was demonstrated between tear cytokine concentrations and glycosylated hemoglobin (HbA1c) or fasting plasma glucose (FPG). Conclusions: Diabetic tears exhibited elevated levels of IP-10 and MCP-1. The Th1/Th2 cytokine balance may shift to a predominantly Th1 state in DR patients. Pro-angiogenic cytokines are more highly represented than anti-angiogenic cytokines in the tears of diabetic patients.

Toll-like receptor 4 contributes to retinal ischemia/reperfusion injury.

Abstract: PURPOSE We investigated whether retinal ischemia and inflammation produced by raising the intraocular pressure above normal systolic levels differs in mice that lack a functional toll-like receptor 4 (Tlr4) signaling pathway METHODS In this work we used the murine strain B6B10ScN-Tlr4(lps-del)/JthJ, which does not express functional Tlr4 C57BL/6J was considered as the control We induced retinal ischemia by unilateral elevation of intraocular pressure for 1 h by direct corneal cannulation The changes in expression of proinflammatory genes 24 h postreperfusion were assessed by quantitative PCR Corresponding changes in protein abundances were analyzed by western blot and immunohistochemistry Cell death was evaluated by direct counting of neurons in the ganglion cell layer of flat-mounted retinas seven days postreperfusion RESULTS We showed that Tlr4-deficient mice display significantly reduced expression of proinflammatory genes, including RelA, tumor necrosis factor (Thf), interleukin 6 (Il6), chemokine (C-C motif) ligand 2 (Ccl2), chemokine (C-C motif) ligand 5 (Ccl5), chemokine (C-X-C motif) ligand 10 (Cxcl10), Cybb, nitric oxide synthase 2 (Nos2), and intercellular adhesion molecule 1 (Icam1) 24 h after reperfusion The mice that lacked Tlr4 showed significantly increased survival of neurons in the ganglion cell layer following ischemic injury, as compared to wild-type controls CONCLUSIONS Our results indicate that Tlr4 signaling is involved in retinal damage and inflammation triggered by ischemic injury

Associations of smoking, body mass index, dietary lutein, and the LIPC gene variant rs10468017 with advanced age-related macular degeneration.

Abstract: OBJECTIVE A novel locus in the hepatic lipase (LIPC) gene was found to be significantly related to advanced age-related macular degeneration (AMD) in our genome-wide association study. We evaluated its association and interaction with previously identified genetic variants and modifiable factors. METHODS Participants in the Age-Related Eye Disease Study with advanced AMD (n=545 cases) or no AMD (n=275 controls) were evaluated. AMD status was determined using fundus photography. Covariates included cigarette smoking, body mass index (BMI), and dietary lutein. Individuals were genotyped for the rs10468017 polymorphism in LIPC as well as seven previously identified AMD genetic loci. Unconditional logistic regression analyses were then performed. RESULTS The TT genotype of the LIPC variant was associated with a reduced risk of AMD, with odds ratios (OR) of 0.50 (95% confidence interval (CI) 0.20-0.90) and p=0.014 for the TT genotype versus the CC genotype, controlling for age, gender, smoking, body mass index (BMI), and nutritional factors. Controlling for seven other AMD genetic variants, the OR was 0.50, 95% (CI 0.20-1.1, p=0.077). The magnitude of the effect was similar for both atrophic and neovascular forms of AMD. Cigarette smoking and higher BMI increased the risk, while higher dietary lutein reduced the risk of advanced AMD, adjusting for genetic variants. There were no significant interactions between LIPC and smoking, BMI, or lutein. There was a possible association between LIPC and complement factor H (CFH) rs1410996, and a possible interaction effect between LIPC and both CFH rs10033900 and the complement factor I (CFI) variants in terms of risk of AMD. CONCLUSIONS LIPC is associated with reduced risk of advanced AMD, independent of demographic and environmental variables. Both genetic susceptibility and behavioral and lifestyle factors modify the risk of developing AMD.

Bevacizumab neutralizes the protective effect of vascular endothelial growth factor on retinal ganglion cells

Abstract: Purpose: Vascular endothelial growth factor (VEGF) is well known for its role in pathologic neovascularization, including wet age-related macular degeneration. However, a growing body of evidence indicates that VEGF is also neuroprotective of non-vascular cells in various animal models through reduction of oxidative stress. In light of the widespread use of intraocular anti-VEGF therapies for age-related macular degeneration (AMD), we evaluated the impact of anti-VEGF agents on the neuroprotective effect of VEGF on retinal ganglion cells. Methods: Staurosporine differentiated retinal ganglion cells were treated with increasing doses of VEGF in the presence of hydrogen peroxide. After optimization, an increasing concentration of bevacizumab was added to neutralize VEGFmediated protection. The degree of oxidative damage was measured at various time points using buthionine sulfoxime (BSO), a glutathione reductase inhibitor. Cell viability was assessed using WST-1 and Crystal violet assays. Results: VEGF (200 ng/ml) protected differentiated retinal ganglion cells (RGC)-5 against H202-mediated oxidative stress. This effect was eliminated by co-treatment with bevacizumab (2.0 mg/ml), which by itself was not cytotoxic. Conclusions: These results indicate an important role for VEGF in the maintenance of retinal ganglion cells.

Validation of structural and functional lesions of diabetic retinopathy in mice

Abstract: Diabetic retinopathy is a serious long-term complication of diabetes mellitus. There is considerable interest in using mouse models, which can be genetically modified, to understand how retinopathy develops and can be inhibited. Not all retinal lesions that develop in diabetic patients have been reproduced in diabetic mice; conversely, not all abnormalities found in diabetic mice have been studied or identified in diabetic patients. Thus, it is important to recognize which structural and functional abnormalities that develop in diabetic mice have been validated against the lesions that characteristically develop in diabetic patients. Those lesions that have been observed to develop in the mouse models to date are predominantly characteristic of the early stages of retinopathy. Identification of new therapeutic ways to inhibit these early lesions is expected to help inhibit progression to more advanced and clinically important stages of retinopathy.

p62/sequestosome 1 as a regulator of proteasome inhibitor-induced autophagy in human retinal pigment epithelial cells

Abstract: Purpose The pathogenesis of age-related macular degeneration involves impaired protein degradation in retinal pigment epithelial (RPE) cells. The ubiquitin-proteasome pathway and the lysosomal pathway including autophagy are the major proteolytic systems in eukaryotic cells. Prior to proteolysis, heat shock proteins (HSPs) attempt to refold stress-induced misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. Recently, p62/sequestosome 1 (p62) has been shown to be a key player linking the proteasomal and lysosomal clearance systems. In the present study, the functional roles of p62 and HSP70 were evaluated in conjunction with proteasome inhibitor-induced autophagy in human RPE cells (ARPE-19). Methods The p62, HSP70, and ubiquitin protein levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. The p62 and HSP70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. Results Proteasome inhibition evoked the accumulation of perinuclear aggregates that strongly colocalized with p62 and HSP70. The p62 perinuclear accumulation was time- and concentration-dependent after MG-132 proteasome inhibitor loading. The silencing of p62, rather than Hsp70, evoked suppression of autophagy, when related to decreased LC3-II levels after bafilomycin treatment. In addition, the p62 silencing decreased the ubiquitination level of the perinuclear aggregates. Recently, we showed that hsp70 mRNA depletion increased cell death in ARPE-19 cells. Here, we demonstrate that p62 mRNA silencing has similar effects on cellular viability. Conclusions Our findings open new avenues for understanding the mechanisms of proteolytic processes in retinal cells, and could be useful in the development of novel therapies targeting p62 and HSP70.

Ethnicity-based subgroup meta-analysis of the association of LOXL1 polymorphisms with glaucoma

Abstract: PURPOSE: To investigate the association and ethnic heterogeneity of lysyl oxidase-like 1 (LOXL1) single nucleotide polymorphisms (SNPs) with exfoliation syndrome (XFS)/exfoliation glaucoma (XFG) and other types of glaucoma. METHODS: We performed meta-analysis and ethnicity-based subgroup analyses according to published studies. Allele and genotype frequencies of SNPs rs1048661, rs2165241, and rs3825942 were extracted for analysis in Reviewer Manager: (1) comparison of the allelic distributions between XFS and XFG, (2) allelic association of LOXL1 SNPs with XFS/XFG, (3) associations in homozygote, heterozygote, and dominant and recessive models, and (4) allelic association with primary open angle glaucoma (POAG). RESULTS: In total 24 reported articles were retrieved, including Caucasian, African, Japanese, Indian, and Chinese populations. There was no significant difference in the distributions of rs1048661, rs2165241, and rs3825942 between XFS and XFG. The G allele of rs3825942 was the common at-risk allele for XFS/XFG in all populations with a total odds ratio (OR) of 10.89. The total homozygote OR of rs3825942 was 9.06 for XFS/XFG combined, but the total heterozygote OR was not significant. We also found that in the recessive model, the total OR was 14.70. There was no association of the three SNPs with POAG. CONCLUSIONS: The association of rs3825942, but not rs2165241 or rs1048661, with XFS/XFG is consistent in different ethnic populations in the recessive model. LOXL1 is not associated with POAG in all study populations.

FOXO1 plays an essential role in apoptosis of retinal pericytes.

Abstract: Purpose: An early and significant event in diabetic retinopathy is the loss of retinal microvascular pericytes. Studies were performed to investigate pathways through which an advanced glycation endproduct and tumor necrosis factor (TNF)-α stimulate apoptosis in retinal pericytes through the activation of the pro-apoptotic transcription factor Forkhead box O1 (FOXO1). Methods: Human retinal pericytes were stimulated by carboxymethyllysine (CML)-collagen, an advanced glycation endproduct, or TNF-α in vitro. Apoptosis was assessed by measuring cytoplasmic histone-associated DNA. The role of FOXO1 was examined by RNA interference (RNAi), and specific inhibitors were used to investigate the role of p38 and Jun N-terminal kinase mitogen-activated protein kinase (JNK MAP) kinases, Akt, and nuclear factor kappa B (NF-κB). Caspase-3 activity was measured with a luminescent substrate, and FOXO1 DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). Results: TNF-α and CML-collagen but not control collagen stimulated apoptosis, caspase-3 activity, and FOXO1 DNAbinding activity in pericytes. Silencing FOXO1 by small interfering RNA prevented apoptosis of pericytes in response to both TNF-α and CML-collagen. By use of specific inhibitors, we demonstrated that both FOXO1 activation and subsequent apoptosis was mediated, in part, by p38 and JNK MAP kinases. In contrast Akt and NF-κB inhibitors had the opposite effect on pericyte apoptosis. Conclusions: The results demonstrate pathways through which two different mediators, TNF-α and an advanced glycation endproduct, can induce pericyte apoptosis through activation of the transcription factor FOXO1.