Dexamethasone (DEX) regulates aqueous humor outflow by inducing a reorganization of the cytoskeleton to form cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells. Rho-associated protein kinase (ROCK) has been demonstrated to have an important role in this process, but the upstream components leading to its activation remain elusive. The purpose of the study is to demonstrate that noncanonical Wnt signaling mediates the DEX-induced CLAN formation in TM cells.
The TM cells were treated with 100 nM DEX in low serum medium for over 7 days. The medium was changed every 3 days. The cells were harvested and subjected to molecular analysis for the expression of Wnt ligands. Stress fiber structures were revealed by Phalloidin staining. Lentivirus-based shRNA against noncanonical Wnt receptor (Ror2) was used to determine the role of noncanonical Wnt signaling in DEX-induced CLAN formation.
The DEX induced stress fiber rearrangement in TM cells. A noncanonical Wnt ligand (Wnt5a) was upregulated by DEX as demonstrated by Wnt ligand degenerate PCR, real-time quantitative PCR (qRT-PCR), and Western blotting. Knocking-down Ror2, the receptor of noncanonical Wnt signaling, abolished the effects of DEX on the TM cells.
Our data suggest that DEX induces the upregulation of noncanonical Wnt ligand Wnt5a. Recombinant WNT5a protein induces CLAN formation through the noncanonical Wnt receptor ROR2/RhoA/ROCK signaling axis. Given the similarities between DEX-induced ocular hypertension and primary open-angle glaucoma, our results provide a mechanism of action for applying ROCK inhibitor to treat primary open-angle glaucoma.
Noncanonical Wnt ligand (Wnt5a) was upregulated by DEX and WNT5a protein induced CLAN formation, while knocking-down Wnt5a receptor (Ror2) abolished the CLAN formation induced by DEX. These observations demonstrate that dexamethasone induces CLAN formation through noncanonical Wnt signaling in TM cells.
trabecular meshwork; steroid; glaucoma; noncanonical Wnt
To investigate whether high glucose (HG)-induced downregulation of connexin 43 (Cx43), a gap junction protein, alters ZO-1 and occludin expression and cell monolayer permeability.
Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM) medium, high glucose (HG; 30 mM) medium, N medium transfected with Cx43 siRNA, or N medium transfected with scrambled siRNA. To determine Cx43, occludin, and ZO-1 protein expression, Western blot (WB) analysis and immunostaining were performed. Gap junction intercellular communication (GJIC) was determined using scrape load dye transfer (SLDT) assay. In parallel, cell monolayer permeability was assessed in the four groups of cells, and in cells transfected with Cx43 plasmid or dominant negative Cx43 plasmid.
Connexin 43 protein expression was significantly reduced in cells grown in HG (67 ± 15% of control), and a significant reduction in Cx43 was achieved when cells grown in N medium were transfected with Cx43 siRNA (76 ± 12% of control), with concomitant decrease in GJIC activity. Cells grown in HG showed significant reduction in occludin (77 ± 9% of control) and ZO-1 (80 ± 11% of control) protein level compared with cells grown in N media. Importantly, cells transfected with Cx43 siRNA and grown in N medium showed significant downregulation in occludin (78 ± 8% of control) and ZO-1 (81 ± 6% of control) expression, and exhibited increased cell monolayer permeability. Furthermore, Cx43 upregulation protected cells against HG-induced excess cell monolayer permeability.
Our findings indicate that HG-induced downregulation of Cx43 expression and GJIC may contribute to the breakdown of endothelial barrier tight junctions associated with diabetic retinopathy.
In this study, the effect of high glucose–induced connexin 43 (Cx43) downregulation on tight junction protein levels and barrier characteristics was investigated. The findings indicate that Cx43 levels can regulate tight junction protein expression and affect endothelial cell monolayer permeability.
connexins; gap junctions; tight junctions
The goal of this study was to functionally evaluate three previously uncharacterized heat shock factor protein 4 (HSF4) mutations (c.595_599delGGGCC, c.1213C>T, c.1327+4A>G) encoding mutant HSF4 proteins (G199EfsX15, R405X, and M419GfsX29) with missing C-terminal ends. These HSF4 mutations were previously identified in families with congenital autosomal recessive cataracts.
FLAG-tagged recombinant wild type (WT) and mutant HSF4 proteins were analyzed using the protein stability assay, cellular immunofluorescence, Western blotting, electrophoretic mobility shift assay (EMSA), and reporter activation.
HSF4 mutant proteins did not differ in the protein turnover rate when compared with WT HSF4. Immunofluorescence revealed that WT and mutant HSF4 proteins were properly trafficked to the nucleus. EMSA analysis revealed that the G199EfsX15 and M419GfsX29 proteins exhibited decreased heat shock element (HSE)-mediated DNA binding, whereas the R405X mutant exhibited increased HSE-mediated DNA binding when compared with WT HSF4. All three HSF4 mutant proteins exhibited abolished HSE-mediated luciferase reporter activation. Detailed evaluation of the C-terminal region identified three novel domains: two activation domains and one repression domain.
The three HSF4 autosomal recessive mutations evaluated here result in a loss of HSF4 function due to a loss of regulatory domains present at the C-terminal end. These findings collectively indicate that the transcriptional activation of HSF4 is mediated by interactions between activator and repressor domains within the C-terminal end.
This study focuses on functional analysis of three HSF4 mutations previously identified in families with autosomal recessive cataracts. The analysis of these mutations facilitated identification of three novel regulatory regions within the HSF4 C-terminal end.
congenital; cataracts; HSF4; mutation
Pigment epithelium-derived factor (PEDF) regulates blood–retinal barrier function. As a constituent of aqueous humor, the role of PEDF in conventional outflow function is unknown. The goals of the study were to examine the effects of PEDF on barrier function of cultured Schlemm's canal (SC) endothelia and outflow facility in mouse eyes in situ.
To model the inner wall of SC, transendothelial electrical resistance (TEER) of human SC and porcine angular aqueous plexus (AAP) cells was monitored. To examine an intact conventional outflow pathway, enucleated eyes from culled C57BL/6 mice were perfused with PEDF using a computer-controlled system. Purified PEDF (0.1 and 1 μg/mL) was perfused at four different pressure steps (4, 8, 15, 20 mm Hg), measuring flow to determine outflow facility (slope of flow/pressure relationship).
Pigment epithelium-derived factor increased TEER of porcine AAP cells in a dose-dependent fashion (0.3–3 μg/mL), and 1 μg/mL recombinant PEDF or conditioned media from pigmented retinal pigment epithelial monolayers stabilized TEER of human SC monolayers over time (0–48 hours). In perfusion experiments, we observed a 43.7% decrease in outflow facility (0.016 vs. 0.029 μL/min/mm Hg, P = 4.5 × 10−5) in eyes treated with 1 μg/mL PEDF compared to vehicle-perfused controls, and a 19.9% decrease (0.021 vs. 0.027 μL/min/mm Hg, P = 0.003) at 100 ng/mL PEDF.
Pigment epithelium-derived factor increased barrier function in both the in vitro and in situ models of the inner wall of SC. Modification of PEDF signaling in SC cells may be therapeutically exploited to increase outflow facility in people with ocular hypertension or decrease outflow facility in those with hypotony.
Results show that PEDF has the greatest acute increase in outflow resistance of any endogenous constituent of the aqueous humor. This finding points to a possible role in the pathogenesis or treatment of glaucoma.
aqueous humor; outflow facility; glaucoma; growth factors; trabecular meshwork; Schlemm's canal; VEGF; PEDF
We applied a recently reported next-generation sequencing (NGS) strategy for screening the ABCA4 gene in a British cohort with ABCA4-associated disease and report novel mutations.
We identified 79 patients with a clinical diagnosis of ABCA4-associated disease who had a single variant identified by the ABCA4 microarray. Comprehensive phenotypic data were obtained, and the NGS strategy was applied to identify the second allele by means of sequencing the entire coding region and adjacent intronic sequences of the ABCA4 gene. Identified variants were confirmed by Sanger sequencing and assessed for pathogenicity by in silico analysis.
Of the 42 variants detected by prescreening with the microarray, in silico analysis suggested that 34, found in 66 subjects, were disease-causing and 8, found in 13 subjects, were benign variants. We detected 42 variants by NGS, of which 39 were classified as disease-causing. Of these 39 variants, 31 were novel, including 16 missense, 7 splice-site–altering, 4 nonsense, 1 in-frame deletion, and 3 frameshift variants. Two or more disease-causing variants were confirmed in 37 (47%) of 79 patients, one disease-causing variant in 36 (46%) subjects, and no disease-causing variant in 6 (7%) individuals.
Application of the NGS platform for ABCA4 screening enabled detection of the second disease-associated allele in approximately half of the patients in a British cohort where one mutation had been detected with the arrayed primer extension (APEX) array. The time- and cost-efficient NGS strategy is useful in screening large cohorts, which will be increasingly valuable with the advent of ABCA4-directed therapies.
PCR-enrichment–based next-generation sequencing with an amplicon tagging protocol revealed two or more disease-causing variants in 37 of 79 patients with ABCA4-associated retinal disease, who had only one variant detected in prescreening with arrayed primer extension technology.
ABCA4; next generation sequencing; Stargardt disease
To determine whether the age-regulating protein klotho was expressed in the retina and determine whether the absence of klotho affected retinal function.
Immunohistochemistry and qPCR of klotho knockout and wild-type mice were used to detect klotho expression in retina. Immunohistochemistry was used to probe for differences in expression of proteins important in synaptic function, retinal structure, and ionic flux. Electroretinography (ERG) was conducted on animals across lifespan to determine whether decreased klotho expression affects retinal function.
Klotho mRNA and protein were detected in the wild-type mouse retina, with protein present in all nuclear layers. Over the short lifespan of the knockout mouse (∼8 weeks), no overt photoreceptor cell loss was observed, however, function was progressively impaired. At 3 weeks of age neither protein expression levels (synaptophysin and glutamic acid decarboxylase [GAD67]) nor retinal function were distinguishable from wild-type controls. However, by 7 weeks of age expression of synaptophysin, glial fibrillary acidic protein (GFAP), and transient receptor potential cation channel subfamily member 1 (TRPM1) decreased while GAD67, post synaptic density 95 (PSD95), and wheat germ agglutinin staining, representative of glycoprotein sialic acid residues, were increased relative to wild-type mice. Accompanying these changes, profound functional deficits were observed as both ERG a-wave and b-wave amplitudes compared with wild-type controls.
Klotho is expressed in the retina and is important for healthy retinal function. Although the mechanisms for the observed abnormalities are not known, they are consistent with the accelerating aging phenotype seen in other tissues.
The absence of age-regulating protein klotho does not cause overt retinal cell loss; however, retinal function and protein expression are dramatically altered.
aging; protein expression; knockout
Previous studies that measured liquefaction and oxygen content in human vitreous suggested that exposure of the lens to excess oxygen causes nuclear cataracts. Here, we developed a computational model that reproduced available experimental oxygen distributions for intact and degraded human vitreous in physiologic and environmentally perturbed conditions. After validation, the model was used to estimate how age-related changes in vitreous physiology and structure alter oxygen levels at the lens.
A finite-element model for oxygen transport and consumption in the human vitreous was created. Major inputs included ascorbate-mediated oxygen consumption in the vitreous, consumption at the posterior lens surface, and inflow from the retinal vasculature. Concentration-dependent relations were determined from experimental human data or estimated from animal studies, with the impact of all assumptions explored via parameter studies.
The model reproduced experimental data in humans, including oxygen partial pressure (Po2) gradients (≈15 mm Hg) across the anterior-posterior extent of the vitreous body, higher oxygen levels at the pars plana relative to the vitreous core, increases in Po2 near the lens after cataract surgery, and equilibration in the vitreous chamber following vitrectomy. Loss of the antioxidative capacity of ascorbate increases oxygen levels 3-fold at the lens surface. Homogeneous vitreous degeneration (liquefaction), but not partial posterior vitreous detachment, greatly increases oxygen exposure to the lens.
Ascorbate content and the structure of the vitreous gel are critical determinants of lens oxygen exposure. Minimally invasive surgery and restoration of vitreous structure warrant further attention as strategies for preventing nuclear cataracts.
A finite-element model of oxygen transport and consumption in the vitreous body predicts the effects of vitreous liquefaction, oxygen influx from the retinal vasculature, and ascorbate levels on the exposure of the posterior surface of the lens to oxygen, a major risk factor for nuclear cataract formation.
vitreous; oxygen; consumption; transport; modeling; liquefaction; aging
The objective of this study was to elucidate the signaling pathway through which cationic antimicrobial protein of 37 kDa (CAP37) mediates human corneal epithelial cell (HCEC) chemotaxis.
Immortalized HCECs were treated with pertussis toxin (10 and 1000 ng/mL), protein kinase C (PKC) inhibitors (calphostin c, 50 nM and Ro-31-8220, 100 nM), phorbol esters (phorbol 12,13-dibutyrate, 200 nM and phorbol 12-myristate 13-acetate, 1 μM) known to deplete PKC isoforms, and siRNAs (400 nM) before a modified Boyden chamber assay was used to determine the effect of these inhibitors and siRNAs on CAP37-directed HCEC migration. PKCδ protein levels, PKCδ-Thr505 phosphorylation, and PKCδ kinase activity was assessed in CAP37-treated HCECs using immunohistochemistry, Western blotting, and a kinase activity assay, respectively.
Chemotaxis studies revealed that treatment with pertussis toxin, PKC inhibitors, phorbol esters, and siRNAs significantly inhibited CAP37-mediated chemotaxis compared with untreated controls. CAP37 treatment increased PKCδ protein levels and led to PKCδ phosphorylation on residue Thr505. Direct activation of PKCδ by CAP37 was demonstrated using a kinase activity assay.
These findings lead us to conclude that CAP37 is an important regulator of corneal epithelial cell migration and mediates its effects through PKCδ.
This study strongly indicates that CAP37, upon binding to a GPCR receptor, activates the PKC signaling cascade through the PKCδ isoform leading to CAP37-directed HCEC chemotaxis.
cationic antimicrobial proteins; protein kinase C; migration; signaling; inflammation
Fuchs endothelial corneal dystrophy (FECD) is an oxidative stress disorder that leads to age-related and gradual loss of corneal endothelial cells resulting in corneal edema and loss of vision. To date, other than surgical intervention, there are no treatment options for patients with FECD. We have shown that in FECD, there is a deficiency in nuclear factor erythroid 2-related factor 2 (Nrf2)–regulated antioxidant defense due to decreased Nrf2 nuclear translocation and activation of antioxidant response element (ARE). In this study, we used sulforaphane (SFN) and D3T to investigate a strategy of targeting Nrf2-ARE in FECD.
FECD and normal ex vivo corneas and human corneal endothelial cell lines were pretreated with SFN or D3T and exposed to oxidative stress with tert-Butyl hydroperoxide (tBHP). Apoptosis was detected with TUNEL. Cellular localization of Nrf2 and p53 was assessed by immunohistochemistry. Effect of SFN was determined by using DCFDA assay, Western blot and real-time PCR.
After pretreatment with SFN, oxidative stress was induced with tBHP. In ex vivo FECD specimens, SFN decreased CEC apoptosis by 55% in unstressed group and by 43% in tBHP-treated specimens. SFN enhanced nuclear translocation of Nrf2 in FECD specimens and decreased p53 staining under oxidative stress. Pretreatment with SFN enhanced cell viability by decreasing intracellular reactive oxygen species production. Upregulation of Nrf2 levels led to increased synthesis of DJ-1, heme oxygenase 1, and nicotinamide adenine dinucleotide quinone oxidoreductase-1. SFN significantly upregulated major ARE-dependent antioxidants and ameliorated oxidative stress–induced apoptosis in FECD.
Our results suggest that targeting Nrf2-ARE pathway may arrest degenerative cell loss seen in FECD.
In this study, pretreatment with sulforaphane significantly upregulated major ARE-dependent antioxidants and ameliorated oxidative stress–induced apoptosis seen in FECD, suggesting that targeting the Nrf2-ARE pathway may arrest FECD degenerative cell loss.
Fuchs endothelial corneal dystrophy; sulforaphane; apoptosis; DJ-1; Nrf2; oxidative stress; Hmox-1; NQO1; corneal endothelial cells; reactive oxygen species
We have previously shown that TGF-β3 (T3) stimulates extracellular matrix (ECM) assembly while maintaining antifibrotic characteristics in a model using human corneal fibroblasts (HCFs). This model, however, requires non-physiological levels of serum. In the current study, we tested whether T3 could stimulate human corneal keratocytes (HCKs) in vitro to assemble a functional ECM, while maintaining their characteristics.
Human corneal keratocytes and HCFs were isolated and cultured using 1% or 10% serum, respectively ±T3. The constructs were processed for indirect immunofluorescence (IF), transmission electron microscopy (TEM), and qRT-PCR, analyzing for keratocyte marker, keratocan, and ECM components, collagen (col) types I, III, and V.
Quantitative reverse transcriptase PCR data showed that keratocan, col I, and V were all upregulated in HCKs compared with HCFs, whereas col III was expressed at low levels in HCKs. Transforming growth factor beta 3 stimulation further enhanced the level of change. Without T3, HCK constructs were very thin, approximately 5 μm; however, as with HCFs, upon stimulation with T3, HCK constructs increased in thickness by approximately 5-fold. Cell counts and ECM production revealed that HCKs assembled more ECM per unit area compared with HCFs, and IF revealed downregulation of fibrotic markers, col III, and thrombospondin-1, with T3 stimulation. Transmission electron microscopy data revealed aligned ECM with long fibrils for all conditions except HCK Controls. Human corneal keratocytes+T3 also showed denser collagen fibrils with more consistent fibril diameter.
Overall, the data suggests that it is possible to stimulate matrix secretion and assembly by HCKs in vitro by using a single growth factor, T3.
In the current study, we stimulated human corneal keratocytes (HCKs) in order to test whether HCKs can assemble a functional extracellular matrix, while maintaining their characteristics. Transforming growth factor-beta 3 stimulation enhanced matrix secretion and assembly by HCKs in vitro without losing keratocyte markers and phenotype.
human corneal keratocytes; TGF-β3; extracellular matrix
To determine the effects of serial expansion on the cellular, molecular, and functional properties of human iPS cell (hiPSC)-derived RPE cultures.
Fibroblasts obtained from four individuals were reprogrammed into hiPSCs and differentiated to RPE cells using previously described methods. Patches of deeply pigmented hiPSC-RPE were dissected, dissociated, and grown in culture until they re-formed pigmented monolayers. Subsequent passages were obtained by repeated dissociation, expansion, and maturation of RPE into pigmented monolayers. Gene and protein expression profiles and morphological and functional characteristics of hiPSC-RPE at different passages were compared with each other and to human fetal RPE (hfRPE).
RPE from all four hiPSC lines could be expanded more than 1000-fold when serially passaged as pigmented monolayer cultures. Importantly, expansion of hiPSC-RPE monolayers over the first three passages (P1–P3) resulted in decreased expression of pluripotency and neuroretinal markers and maintenance of characteristic morphological features and gene and protein expression profiles. Furthermore, P1 to P3 hiPSC-RPE monolayers reliably demonstrated functional tight junctions, G-protein–coupled receptor-mediated calcium transients, phagocytosis and degradation of photoreceptor outer segments, and polarized secretion of biomolecules. In contrast, P4 hiPSC-RPE cells failed to form monolayers and possessed altered morphological and functional characteristics and gene expression levels.
Highly differentiated, pigmented hiPSC-RPE monolayers can undergo limited serial expansion while retaining key cytological and functional attributes. However, passaging hiPSC-RPE cultures beyond senescence leads to loss of such features. Our findings support limited, controlled passaging of patient-specific hiPSC-RPE to procure cells needed for in vitro disease modeling, drug screening, and cellular transplantation.
Human hiPSC-RPE maintain their key cellular and functional characteristics with limited passaging and expansion.
induced pluripotent stem cell; retinal pigment epithelium; passaging
Levels of TGF-β2 are higher in POAG aqueous humor, causing deposition of extracellular matrix (ECM) proteins, including fibronectin (FN), in the glaucomatous human trabecular meshwork (HTM) that may be responsible for elevated IOP. The purpose of this study was to identify the expression of cellular FN (cFN) isoforms (EDA and EDB) in HTM cells and tissues, and to determine whether TGF-β2 can induce cFN expression and fibril formation in cultured HTM cells.
Expression of cFN mRNA isoforms and induction by recombinant TGF-β2 (5 ng/mL) were determined by quantitative RT-PCR. The TGF-β2 induction of EDA isoform protein expression and FN fibril formation were analyzed using Western immunoblots and immunocytochemistry (ICC), respectively. Immunohistochemistry (IHC) analysis was used to examine total FN and EDA isoform expression in normal (NTM) and glaucomatous (GTM) trabecular meshwork (TM) tissues.
Both cFN mRNA isoforms were expressed in cultured HTM cells and were induced by TGF-β2 after 2, 4, and 7 days (P < 0.05). Similarly, EDA isoform protein and fibril formation were increased after 4 and 7 days of TGF-β2 treatment. Finally, GTM tissues had significantly greater EDA isoform protein levels (1.7-fold, P < 0.05) compared to NTM tissues.
This study demonstrated that cFN isoforms are expressed and induced in HTM cells by TGF-β2. Also, increased EDA isoform protein levels were seen in GTM tissues. Our findings suggest that induction of cFN isoform expression in the TM ECM may be a novel pathologic mechanism involved in the TM changes associated with glaucoma.
We demonstrate that cellular fibronectin is expressed in human normal and glaucomatous trabecular meshwork cells and tissues, and that EDA isoform levels are elevated in glaucomatous tissue. We also show that TBF-β2 induces cellular fibronectin expression in human trabecular meshwork cells.
glaucoma; trabecular meshwork; TGF-β2; fibronectin
Epidemiologic studies have linked UV-B exposure to development of cortical cataracts, but the underlying molecular mechanism(s) is unresolved. Here, we used a mouse model to examine the nature and distribution of DNA photolesions produced by ocular UV-B irradiation.
Anesthetized mice, eye globes, or isolated lenses were exposed to UV-B. Antibodies specific for 6-4 photoproducts (6-4 PPs) or cyclobutane pyrimidine dimers (CPDs) were used to visualize DNA adducts.
Illumination of intact globes with UV-B–induced 6-4 PP and CPD formation in cells of the cornea, anterior iris, and central lens epithelium. Photolesions were not detected in retina or lens cells situated in the shadow of the iris. Photolesions in lens epithelial cells were produced with radiant exposures significantly below the minimal erythemal dose. Lens epithelial cells rapidly repaired 6-4 PPs, but CPD levels did not markedly diminish, even over extended postirradiation recovery periods in vitro or in vivo. The repair of 6-4 PPs did not depend on the proliferative activity of the epithelial cells, since the repair rate in the mitotically-active germinative zone (GZ) was indistinguishable from that of quiescent cells in the central epithelium.
Even relatively modest exposures to UV-B produced 6-4 PP and CPD photolesions in lens epithelial cells. Cyclobutane pyrimidine dimer lesions were particularly prevalent and were repaired slowly if at all. Studies on sun-exposed skin have established a causal connection between photolesions and so-called UV-signature mutations. If similar mechanisms apply in the lens, it suggests that somatic mutations in lens epithelial cells may contribute to the development of cortical cataracts.
Ultraviolet B exposure has been linked to cortical cataract formation. In this study the generation and repair of UV-B–induced DNA photolesions was examined in the mouse lens epithelium in vitro and in vivo.
UV-B; cortical cataract; DNA damage
To report distinct characteristics of fundus autofluorescence (AF) patterns inferior to the optic disc in recessive Stargardt disease (STGD1) and retinitis pigmentosa (RP).
Short-wavelength (SW) and near-infrared (NIR) AF images were acquired from patients with STGD1 and RP. In SW- and NIR-AF images of STGD1 patients, gray levels (GL) on both sides of the demarcation line were measured.
In STGD1, a demarcation line, which has been assigned to the closed optic fissure, was visible on SW-AF and NIR-AF inferior to the optic disc. In healthy subjects, this demarcation line is only visible by SW-AF. At 20° inferior to the disc center, AF levels on the nasal side were 25% (±11%) lower than on the temporal side in SW-AF images and 18% (±11%) lower in NIR-AF images. For both STGD1 and RP, the inferonasal quadrant exhibited distinct SW- and NIR-AF patterns compared with other fundus areas. Disease-related AF changes, such as flecks, appeared to respect the demarcation line as a boundary.
Disease-related AF patterns originating in RPE in STGD1 and RP appear to respect the demarcation line in the inferonasal quadrant of the fundus as a border. The visibility of the inferonasal demarcation line by NIR-AF in STGD1 but not in healthy eyes may indicate that increased levels of RPE lipofuscin modulate the melanin-related NIR-AF signal. This feature of NIR-AF images may aid in the diagnosis of STGD1 patients.
Disease-related SW-AF exhibits an abrupt discontinuity inferior to the optic disc along a line attributable to the closed optic fissure. This line is visible with NIR-AF imaging in recessive Stargardt disease but not in healthy subjects and thus may aid in identifying patients.
ABCA4; fundus autofluorescence; lipofuscin; melanin; optic fissure; retinal pigment epithelium; retinitis pigmentosa; scanning laser ophthalmoscope; recessive Stargardt disease
To describe Heidelberg Retina Tomograph (HRT) measures, their principal components, and their associations in a British population.
The European Prospective Investigation of Cancer (EPIC)-Norfolk Eye Study is nested within a multicenter cohort study. Measurements were taken with the HRT-2 and the software subsequently updated to yield HRT-3 parameters. Principal components analysis (PCA) was used to identify distinct components of the HRT variables. Generalized estimating equation models were used to examine associations of these components with age, sex, height, body mass index (BMI), blood pressure, social class, education, alcohol intake, smoking status, axial length, IOP, and lens status.
Complete data were available from 10,859 eyes of 6430 participants with a mean age of 68 years. Principal components analysis identified three components with an eigenvalue greater than 1, explaining 79.9% of the variance of all the HRT measures. These were named cup, retinal nerve fiber layer (RNFL), and rim based on the factor loadings they were most correlated with. Older age was significantly associated with a greater cup (P = 0.003), smaller RNFL (P < 0.001), and smaller rim (P < 0.001). Female sex (P = 0.001), higher education (P < 0.001), and shorter axial length (P < 0.001) were associated with a greater RNFL. Lower BMI and higher IOP were associated with a greater cup (both, P < 0.001) and a smaller rim (BMI, P = 0.001; IOP, P < 0.001).
Heidelberg Retina Tomograph measures in this cohort were largely explained by three principal components related to optic disc cup, RNFL, and rim. Associations with cup and rim were distinct to associations with RNFL, suggesting different underlying determinants.
Analysis of Heidelberg Retina Tomograph measurements from a large population-based sample identified three distinct components (optic disc “cup”, “rim” and retinal nerve fiber layer [“RNFL”]). Associations with “cup” and “rim” were distinct to associations with “RNFL”, suggesting different underlying determinants.
diagnostic techniques; epidemiology; axial length; glaucoma; body mass index; optic disk
We examined the signaling mechanisms involved in δ-opioid–receptor agonist, SNC-121–mediated attenuation of TNF-α–induced matrix metalloproteinase-2 (MMP-2) secretion from human optic nerve head (ONH) astrocytes.
Human ONH astrocytes were treated with SNC-121 (1 μmol/L) for 15 minutes followed by TNF-α (25 ng/mL) treatment for 6 or 24 hours. Cells were pretreated with inhibitors of p38 mitogen-activated protein (MAP) kinase (SB-203580) or NF-κB (Helenalin) prior to TNF-α treatment. Changes in phosphorylation and expression of p38 MAP kinase, IκBα, NF-κB, and MMP-2 were measured by Western blotting. Translocation of NF-κB was determined by immunocytochemistry.
TNF-α treatment increased MMP-2 secretion from ONH astrocytes to 236% ± 17% and 142% ± 8% at 6 and 24 hours, respectively; while SNC-121 treatment reduced MMP-2 secretion to 149% ± 11% and 108% ± 7% at 6 and 24 hours, respectively. The SNC-121-mediated inhibitory response was blocked by the δ-opioid–receptor antagonist naltrindole. TNF-α treatment resulted in a sustained phosphorylation of p38 MAP kinase up to 24 hours (226% ± 15% over control levels), which was reduced to 150% ± 20% by SNC-121 treatment. TNF-α treatment increased the expression of NF-κB to 179% ± 21% and 139% ± 6% at 6 and 24 hours, respectively, which was significantly blocked by SNC-121 treatment. Furthermore, TNF-α–induced MMP-2 secretion was blocked by 100% and 78% in the presence of SB-203580 and Helenalin, respectively.
Evidence is provided that SNC-121 attenuated TNF-α–induced MMP-2 secretion from ONH astrocytes. Data also supported the idea that p38 MAP kinase and NF-κB played central roles in TNF-α–induced MMP-2 secretion, and both were negatively regulated by SNC-121.
δ-opioid agonist can directly mitigate the destabilization/remodeling of the optic nerve by blocking the TNF-α–induced production of MMP-2.
optic nerve head; astrocytes; glaucoma; matrix metalloproteinases; opioids
We examined the treatment period necessary to restore retinal and visual stability following trauma to the optic nerve.
Cats received unilateral optic nerve crush and no treatment (NT), treatment of the injured eye with brain-derived neurotrophic factor (BDNF), or treatment of the injured eye combined with treatment of visual cortex for 2 or 4 weeks. After 1-, 2-, 4-, or 6-week survival periods, pattern electroretinograms (PERGs) were obtained and retinal ganglion cell (RGC) survival determined.
In the peripheral retina, RGC survival for NT, eye only, and eye + cortex animals was 55%, 78%, and 92%, respectively, at 1 week, and 31%, 60%, and 93%, respectively, at 2 weeks. PERGs showed a similar pattern of improvement. After 4 weeks, RGC survival was 7%, 29%, and 53% in each group, with PERGs in the dual-treated animals similar to the 1- to 2-week animals. For area centralis (AC), the NT, eye only, and eye + cortex animals showed 47%, 78%, and 82% survival, respectively, at 2 weeks, and 13%, 54%, and 81% survival, respectively, at 4 weeks. Removing the pumps at 2 weeks resulted in ganglion cell survival levels of 76% and 74% in the AC at 4 and 6 weeks postcrush, respectively. The PERGs from 2-week treated, but 4- and 6-week survival animals were comparable to those of the 2-week animals.
Treating the entire central visual pathway is important following optic nerve trauma. Long-term preservation of central vision may be achieved with as little as 2 weeks of treatment using this approach.
These studies emphasize the importance of treating the entire central visual pathway following trauma to the optic nerve. They also indicate that, by doing so, one might achieve long-term preservation of central vision with minimal intervention.
neurotrophins; retina; optic neuropathy
We compared gene expression signatures in tree shrew sclera produced by three different visual conditions that all produce ocular elongation and myopia: minus-lens wear, form deprivation, and dark treatment.
Six groups of tree shrews (n = 7 per group) were used. Starting 24 days after normal eye-opening (days of visual experience [DVE]), two minus-lens groups wore a monocular −5 diopter (D) lens for 2 days (ML-2) or 4 days (ML-4); two form-deprivation groups wore a monocular translucent diffuser for 2 days (FD-2) or 4 days (FD-4). A dark-treatment (DK) group was placed in continuous darkness for 11 days after experiencing a light/dark environment until 17 DVE. A normal colony-reared group was examined at 28 DVE. Quantitative PCR was used to measure the relative differences in mRNA levels for 55 candidate genes in the sclera that were selected, either because they showed differential expression changes in previous ML studies or because a whole-transcriptome analysis suggested they would change during myopia development.
The treated eyes in all groups responded with a significant myopic shift, indicating that the myopia was actively progressing. In the ML-2 group, 27 genes were significantly downregulated in the treated eyes, relative to control eyes. In the treated eyes of the FD-2 group, 16 of the same genes also were significantly downregulated and one was upregulated. The two gene expression patterns were significantly correlated (r2 = 0.90, P < 0.001). After 4 days of treatment, 31 genes were significantly downregulated in the treated eyes of the ML-4 group and three were upregulated. Twenty-nine of the same genes (26 down- and 3 up-regulated) and six additional genes (all downregulated) were significantly affected in the FD-4 group. The response patterns were highly correlated (r2 = 0.95, P < 0.001). When the DK group (mean of right and left eyes) was compared to the control eyes of the ML-4 group, the direction and magnitude of the gene expression patterns were similar to those of the ML-4 (r2 = 0.82, P < 0.001, excluding PENK). Similar patterns also were found when the treated eyes of the ML-4, FD-4, and DK groups were compared to the age-matched normal eyes.
The very similar gene expression signatures produced in the sclera by the three different myopiagenic visual conditions at different time points suggests that there is a “scleral remodeling signature” in this mammal, closely related to primates. The scleral genes examined did not distinguish between the specific visual stimuli that initiate the signaling cascade that results in axial elongation and myopia.
Three different visual conditions (minus-lens wear, form deprivation, darkness) that cause axial elongation and myopia produce very similar gene expression signatures in the sclera in 55 genes. In the sclera, the differing visual signals have merged into a single “GO” remodeling signature.
myopia; animal models; refractive error; emmetropization; axial elongation; gene expression; sclera
Histological evidence suggests a role for the central nervous system in controlling episcleral venous pressure (EVP). Based on prior studies that identified candidate regions in the brain stem, the present study assessed the effect of electrical stimulation at the location of the superior salivatory nucleus (SSN) on EVP in rats.
Male Sprague-Dawley rats (n = 11) were anesthetized using pentobarbital sodium (50 mg/kg intraperitoneally initially, supplemented intravenously [IV] as needed) and paralyzed with gallamine triethiodide (1 mg/kg, IV). The animals were artificially ventilated and the femoral artery and vein were cannulated for blood pressure measurement and drug administration. Carotid blood flow was measured with an ultrasound flow probe and heart rate with a cardiotachometer. IOP was measured through a cannula in the vitreous compartment and EVP was measured through a micropipette in episcleral veins using the servonull technique. After a craniotomy was performed, a unipolar stainless steel electrode was inserted into the brainstem at the coordinates of the SSN using a stereotactic instrument. Stimulations were performed at 20Hz, 9 μA, 1 ms pulse duration, and 200 pulses.
Stimulation at the SSN coordinates increased IOP from 10.6 ± 0.4 to 11.8 ± 0.6 mm Hg (P < 0.01) and EVP from 7.8 ± 1.3 to 10.7 ± 1.1 mm Hg (P < 0.01). Mean arterial pressure, carotid blood flow, and heart rate remained unaltered.
The present study indicates that the SSN may participate in regulating EVP.
Episcleral venous pressure increases in response to electrical stimulation at the coordinates of the superior salivatory nucleus.
episcleral venous pressure; superior salivatory nucleus; stimulation; IOP
To report the application of a novel imaging technique, pump-probe microscopy, to analyze patterns of pigment chemistry of conjunctival melanocytic lesion biopsies.
Histopathologic specimens of eight previously excised conjunctival melanocytic lesions were analyzed with pump-probe microscopy. The technique uses a laser scanning microscope with a two-color pulsed laser source to distinguish hemoglobin, eumelanin, and pheomelanin pigment based on differences in transient excited state and ground state photodynamics. The pump-probe signatures of conjunctival melanins were compared with cutaneous melanins. The distributions of hemoglobin, eumelanin, and pheomelanin were analyzed, and pump-probe images were correlated with adjacent hematoxylin and eosin (H&E)-stained sections.
The pump-probe signatures of conjunctival melanins are similar, but not identical to cutaneous melanins. In addition, there are qualitative and quantitative differences in the structure and pigment chemistry of conjunctival benign nevi, primary acquired melanosis of the conjunctiva (PAM), and conjunctival melanomas. The pump-probe images correlated well with histopathologic features observed in the adjacent H&E-stained sections, and provided a label-free means of discerning conjunctival anatomic features and pathologic benign or malignant tissue.
Pump-probe laser microscopy shows promise as an adjuvant diagnostic tool in evaluation of ocular melanocytic lesions based on morphologic correlation with the histopathology results and pigment chemistry. This initial study suggests systematic differences in pigmentation patterns among conjunctival benign nevi, primary acquired melanosis, and melanomas. In addition, pump-probe microscopy has the potential for use as a noninvasive “in vivo” optical biopsy technique to aid clinical and surgical management of conjunctival melanocytic lesions.
Using pump-probe microscopy, a multiphoton technique that images absorptive pigments, we analyzed the microscopic distribution of melanins in biopsy sections of conjunctival melanocytic lesions, and found differences among conjunctival benign nevi, primary acquired melanosis, and melanomas.
conjunctival melanoma; primary acquired melanosis; benign conjunctival nevus; pump-probe microscopy; multiphoton microscopy
Autoimmune inflammation of the retina causes vision loss in the majority of affected individuals. Th1 or Th17 cells initiate the disease on trafficking from the circulation into the eye across the retinal vascular endothelium. We investigated the ability of human Th1- and Th17-polarized cells to cross a simulated human retinal endothelium, and examined the role of IgG superfamily members in this process.
Th1- and Th17-polarized cell populations were generated from human peripheral blood CD4+ T cells, using two Th1- and Th17-polarizing protocols. Transendothelial migration assays were performed over 18 hours in Boyden chambers, after seeding the transwell membrane with human retinal endothelial cells. In some assays intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), or activated leukocyte cell adhesion molecule (ALCAM) blocking antibody, or isotype- and concentration-matched control antibody, was added to the upper chambers.
Th1- and Th17-polarized cells migrated equally efficiently across the human retinal endothelial monolayer. The percentage of IL-17+ IFN-γ− Th17-polarized cells was reduced following migration. Blocking ICAM-1, but not VCAM-1 or ALCAM, significantly reduced migration of Th1- and Th17-polarized cells for a majority of human donors.
Taken in the context of other literature on transendothelial migration, our results illustrate the importance of investigating the specific tissue and vascular endothelium when considering helper T cell migration in autoimmune inflammation. Our findings further indicate that while generalizations about involvement of specific adhesion molecules in uveitis and other autoimmune disease may be possible, these may not apply to individual patients universally. The observations are relevant to the use of adhesion blockade for therapeutic purposes.
This work shows that human Th1- and Th17-polarized cells migrate equally efficiently across human retinal vascular endothelium, and that ICAM-1 is involved in coordinating the movement of both cell subsets.
uveitis; retina; endothelial cell; Th1 cell; Th17 cell
The role of microRNA (miRNA) regulation in corneal wound healing and scar formation has yet to be elucidated. This study analyzed the miRNA expression pattern involved in corneal wound healing and focused on the effect of miR-133b on expression of several profibrotic genes.
Laser-ablated mouse corneas were collected at 0 and 30 minutes and 2 days. Ribonucleic acid was collected from corneas and analyzed using cell differentiation and development miRNA PCR arrays. Luciferase assay was used to determine whether miR-133b targeted the 3′ untranslated region (UTR) of transforming growth factor β1 (TGFβ1) and connective tissue growth factor (CTGF) in rabbit corneal fibroblasts (RbCF). Quantitative real-time PCR (qRT-PCR) and Western blots were used to determine the effect of miR-133b on CTGF, smooth muscle actin (SMA), and collagen (COL1A1) in RbCF. Migration assay was used to determine the effect of miR-133b on RbCF migration.
At day 2, 37 of 86 miRNAs had substantial expression fold changes. miR-133b had the greatest fold decrease at −14.33. Pre-miR-133b targeted the 3′ UTR of CTGF and caused a significant decrease of 38% (P < 0.01). Transforming growth factor β1–treated RbCF had a significant decrease of miR-133b of 49% (P < 0.01), whereas CTGF, SMA, and COL1A1 had significant increases of 20%, 54%, and 37% (P < 0.01), respectively. The RbCF treated with TGFβ1 and pre-miR133b showed significant decreases in expression of CTGF, SMA, and COL1A1 of 30%, 37%, and 28% (P < 0.01), respectively. Finally, there was significant decrease in migration of miR-133b–treated RbCF.
Significant changes occur in key miRNAs during early corneal wound healing, suggesting novel miRNA targets to reduce scar formation.
Substantial changes occurred in levels of 37 miRNAs in mouse corneas 2 days following excimer ablation, including a 14-fold decrease in miR-133b. Addition of pre-miR-133b suppressed expression of CTGF, COL1A1, and SMA in RbCF cultures.
CTGF; microRNA; corneal wound healing; gene expression
Oxygenation abnormalities are implicated in the development of retinopathy of prematurity (ROP). The purpose of this study is to report in vivo retinal vascular oxygen tension (PO2) measurements and fluorescein angiography (FA) findings in the mouse model of oxygen-induced retinopathy (OIR).
We exposed 19 neonatal mice to 77% oxygen from postnatal day 7 (P7) to P12 (OIR), while 11 neonatal mice were kept under room air (control). Using phosphorescence lifetime imaging, retinal vascular PO2 was measured followed by FA. Repeated measures ANOVA was performed to determine the effects of blood vessel type (artery and vein) and group (OIR and control) on PO2. Avascular retinal areas were measured from FA images in OIR mice.
There was a significant effect of vessel type on PO2 (P < 0.001). The effect of group on PO2 was not significant (P = 0.3), indicating similar PO2 between OIR and control mice. The interaction between group and vessel type was significant (P = 0.03), indicating a larger arteriovenous PO2 difference in OIR mice than control mice. In control mice, FA displayed normal vascularization, while FA of OIR mice showed abnormalities, including dilation and tortuosity of major retinal blood vessels, and avascular regions. In OIR mice, the mean percent avascular retinal area was 33% ± 18%.
In vivo assessment of retinal vascular oxygen tension and vascularization patterns demonstrated abnormalities in the mouse model of OIR. This approach has the potential to improve understanding of retinal vascular development and oxygenation alterations due to ROP and other ischemic retinal diseases.
In vivo assessment of retinal vascular oxygen tension and vascularization patterns demonstrated abnormalities in the neonatal mouse model of oxygen-induced retinopathy.
retina; vascular oxygen tension; oxygen-induced retinopathy; retinopathy of prematurity; fluorescein angiography
Complete congenital stationary night blindness (CSNB1) is characterized by loss of night vision due to a defect in the retinal ON-bipolar cells (BCs). Mutations in GPR179, encoding the G-protein–coupled receptor 179, have been found in CSNB1 patients. In the mouse, GPR179 is localized to the tips of ON-BC dendrites. In this study we determined the ultrastructural localization of GPR179 in human retina and determined the functional consequences of mutations in GPR179 in patients and mice.
The localization of GRP179 was analyzed in postmortem human retinas with immunohistochemistry. The functional consequences of the loss of GPR179 were analyzed with standard and 15-Hz flicker ERG protocols.
In the human retina, GPR179 is localized on the tips of ON-BC dendrites, which invaginate photoreceptors and terminate juxtaposed to the synaptic ribbon. The 15-Hz flicker ERG abnormalities found in patients with mutations in GPR179 more closely resemble those from patients with mutations in either TRPM1 or NYX than in GRM6. 15-Hz flicker ERG abnormalities of Gpr179nob5 and Grm6nob3 mice were comparable.
GRP179 is expressed on dendrites of ON-BCs, indicating that GRP179 is involved in the ON-BCs' signaling cascade. The similarities of 15-Hz flicker ERGs noted in GPR179 patients and NYX or TRPM1 patients suggest that the loss of GPR179 leads to the loss or closure of TRPM1 channels. The difference between the 15-Hz flicker ERGs of mice and humans indicates the presence of important species differences in the retinal activity that this signal represents.
Mutations in the gene encoding GRP179 lead to congenital stationary night blindness type 1. Here we show that on ultrastructural level GPR179 colocalizes with other members the ON-bipolar signaling cascade. Mutated GPR179 seems to lead to nonfunctional or mislocalized TRPM1 channels.
GPR179; CSNB1; missense mutations; G-protein–coupled receptor; trafficking defect; pathogenicity
To compare the reproducibility of spectral-domain optical coherence tomography (SD-OCT)–based ganglion cell–layer-plus-inner plexiform–layer (GCL+IPL) thickness measurements for glaucoma patients obtained using both a publicly available and a commercially available algorithm.
Macula SD-OCT volumes (200 × 200 × 1024 voxels, 6 × 6 × 2 mm3) were obtained prospectively from both eyes of patients with open-angle glaucoma or with suspected glaucoma on two separate visits within 4 months. The combined GCL+IPL thickness was computed for each SD-OCT volume within an elliptical annulus centered at the fovea, based on two algorithms: (1) a previously published graph-theoretical layer segmentation approach developed at the University of Iowa, and (2) a ganglion cell analysis module of version 6 of Cirrus software. The mean overall thickness of the elliptical annulus was computed as was the thickness within six sectors. For statistical analyses, eyes with an SD-OCT volume with low signal strength (<6), image acquisition errors, or errors in performing the commercial GCL+IPL analysis in at least one of the repeated acquisitions were excluded.
Using 104 eyes (from 56 patients) with repeated measurements, we found the intraclass correlation coefficient for the overall elliptical annular GCL+IPL thickness to be 0.98 (95% confidence interval [CI]: 0.97–0.99) with the Iowa algorithm and 0.95 (95% CI: 0.93–0.97) with the Cirrus algorithm; the intervisit SDs were 1.55 μm (Iowa) and 2.45 μm (Cirrus); and the coefficients of variation were 2.2% (Iowa) and 3.5% (Cirrus), P < 0.0001.
SD-OCT–based GCL+IPL thickness measurements in patients with early glaucoma are highly reproducible.
SD-OCT–based ganglion cell–layer plus inner plexiform–layer thickness measurements for glaucoma patients, obtained with both a publicly available and a commercially available algorithm, are highly reproducible.
ganglion cell layer; glaucoma; image segmentation; reproducibility