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Objective: The catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear., Methods: An in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition., Results: Autophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition., Conclusions: Our findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023. Published by Elsevier B.V.)

Retinal pigment epithelial (RPE) cells contribute to several clinical conditions resulting in retinal fibrotic scars. Myofibroblast trans-differentiation of RPE cells is a critical step in the process of retinal fibrosis. In this study, we investigated the effects of N-oleoyl dopamine (OLDA), a newer endocannabinoid with a structure distinct from classic endocannabinoids, on TGF-β2-induced myofibroblast trans-differentiation of porcine RPE cells. Using an in vitro collagen matrix contraction assay, OLDA was found to inhibit TGF-β2 induced contraction of collagen matrices by porcine RPE cells. This effect was concentration-dependent, with significant inhibition of contraction observed at 3 μM and 10 μM. OLDA did not affect the proliferation of porcine RPE cells. Immunocytochemistry showed that at 3 μM, OLDA decreased incorporation of α-SMA in the stress fibers of TGF-β2-treated RPE cells. In addition, western blot analysis showed that 3 μM OLDA significantly downregulated TGF-β2-induced α-SMA protein expression. Taken together these results demonstrate that OLDA inhibits TGF-β induced myofibroblast trans-differentiation of RPE cells. It has been established that classic endocannabinoid such as anandamide, by activating the CB1 cannabinoid receptor, promote fibrosis in multiple organ systems. In contrast, this study demonstrates that OLDA, an endocannabinoid with a chemical structure distinct from classic endocannabinoids, inhibits myofibroblast trans-differentiation, an important step in fibrosis. Unlike classic endocannabinoids, OLDA has weak affinity for the CB1 receptor. Instead, OLDA acts on non-classic cannabinoid receptors such as GPR119, GPR6, and TRPV1. Therefore, our study indicates that the newer endocannabinoid OLDA and its non-classic cannabinoid receptors could potentially be novel therapeutic targets for treating ocular diseases involving retinal fibrosis and fibrotic pathologies in other organ systems., Competing Interests: Declaration of competing interest All authors: Nothing to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

miR-183/96/182 cluster plays a critical role in the developing retina by regulating many target genes involved in signaling pathways. This study aimed to survey the miR-183/96/182 cluster-target interactions that, potentially contribute to human retinal pigmented epithelial (hRPE) cell differentiation into photoreceptors. Target genes of the miR-183/96/182 cluster were obtained from miRNA-target databases and applied to construct miRNA-target networks. Gene ontology and KEGG pathway analysis was performed. miR-183/96/182 cluster sequence was cloned into an eGFP-intron splicing cassette in an AAV2 vector and overexpressed in hRPE cells. The expression level of target genes including HES1, PAX6, SOX2, CCNJ, and RORΒ was evaluated using qPCR. Our results showed that miR-183, miR-96, and miR-182 share 136 target genes that are involved in cell proliferation pathways such as PI3K/AKT and MAPK pathway. qPCR data indicated a 22-, 7-, and 4-fold overexpression of miR-183, miR-96, and miR-182, respectively, in infected hRPE cells. Consequently, the downregulation of several key targets such as PAX6, CCND2, CDK5R1, and CCNJ and upregulation of a few retina-specific neural markers such as Rhodopsin, red opsin, and CRX was detected. Our findings suggest that the miR-183/96/182 cluster may induce hRPE transdifferentiation by targeting key genes that involve in the cell cycle and proliferation pathways., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper. The manuscript has been read and approved for submission by all authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Hairy and enhancer of split homolog-1 (Hes1) is a member of an extensive family of basic helix-loop-helix (bHLH) proteins and plays a crucial role in neurogenesis, myogenesis, hematopoiesis, and sex determination. It has been reported that Hes1 is essential for precursors maintenance, optic cup-stalk boundary maintenance, and morphogenesis of the retina. However, it still reminds questions about the role and mechanism of Hes1 in the development of retinal pigment epithelial cells. In our study, We generated Hes1 -/- human embrsyonic stem cells, and attempted to induce them into retinal pigment epithelial cells by our previous protocol, found that the cells induced by Hes1 -/- hESCs hardly expressed RPE-related genes, and rarely appeared RPE cell morphology. Additionally, Hes1 may affect the development of RPE cells via Wnt4 pathway by analyzing the RNA-seq data of differently expressed genes between normal RPE cells development and Hes1 -/- RPE cells development. Overall, depletion of Hes1 may result in the failure of Wnt4 signal activation, and contributed to the developmental disorder in retinal pigment epithelium morphogenesis and specification., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Di Zhou reports financial support, administrative support, equipment, drugs, or supplies, statistical analysis, and writing assistance were provided by National Engineering Research Center of Human Stem Cells., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Exposure to particulate matter 2.5 (PM2.5) has been linked to ocular surface diseases, yet knowledge of the molecular mechanism impacted on retina pathogenesis is limited. Therefore, the purpose of this study was to explore the effects and involved factors of PM2.5 exposure in human retinal pigment epithelial APRE-19 cells. Our data revealed a decreased cell viability and an increased migratory ability in APRE-19 cells after PM2.5 stimulation. The MMP-2 and MMP-9 protein levels were markedly increased while the MMPs regulators TIMP-1 and TIMP-2 were significantly reduced in PM2.5-exposed APRE-19 cells. PM2.5 also increased pro-MMP-2 expression in the cell culture supernatants. Additionally, PM2.5 promoted the EMT markers through the activation of PI3K/AKT/mTOR pathway. Moreover, the ICAM-1 production was also remarkably increased by PM2.5 but reduced by PI3K/AKT inhibitor LY294002 in APRE-19 cells. Taken together, these results suggest that PM2.5 promotes EMT in a PI3K/AKT/mTOR-dependent manner in the retinal pigment epithelium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Thioredoxin (Trx) family proteins are key players in redox signaling. Here, we have analyzed glutaredoxin (Grx) 1 and Grx2 in age-related macular degeneration (AMD) and in retinal pigment epithelial (ARPE-19) cells. We hypothesized that these redoxins regulate cellular functions and signaling circuits such as cell proliferation, Wnt signaling and VEGF release that have been correlated to the pathophysiology of AMD. ARPE-19 cells were transfected with specific siRNAs to silence the expression of Grx1 and Grx2 and were analyzed for proliferation/viability, migration capacity, β-catenin activation, and VEGF release. An active site-mutated C-X-X-S Grx1 was utilized to trap interacting proteins present in ARPE-19 cell extracts. In both, AMD retinas and in ARPE-19 cells incubated under hypoxia/reoxygenation conditions, Grx1 showed an increased nuclear localization. Grx1-silenced ARPE-19 cells showed a significantly reduced proliferation and migration rate. Our trapping approach showed that Grx1 interacts with β-catenin in a dithiol-disulfide exchange reaction. Knock-down of Grx1 led to a reduction in both total and active β-catenin levels. These findings add redox control to the regulatory mechanisms of β-catenin signaling in the retinal pigment epithelium and open the door to novel therapeutic approaches in AMD that is currently treated with VEGF-inhibitors., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding this paper., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Vertebrate rod and cone photoreceptors detect light via a specialized organelle called the outer segment. This structure is packed with light-sensitive molecules known as visual pigments that consist of a G-protein-coupled, seven-transmembrane protein known as opsin, and a chromophore prosthetic group, either 11-cis retinal ('A 1 ') or 11-cis 3,4-didehydroretinal ('A 2 '). The enzyme cyp27c1 converts A 1 into A 2 in the retinal pigment epithelium. Replacing A 1 with A 2 in a visual pigment red-shifts its spectral sensitivity and broadens its bandwidth of absorption at the expense of decreased photosensitivity and increased thermal noise. The use of vitamin A 2 -based visual pigments is strongly associated with the occupation of aquatic habitats in which the ambient light is red-shifted. By modulating the A 1 /A 2 ratio in the retina, an organism can dynamically tune the spectral sensitivity of the visual system to better match the predominant wavelengths of light in its environment. As many as a quarter of all vertebrate species utilize A 2 , at least during a part of their life cycle or under certain environmental conditions. A 2 utilization therefore represents an important and widespread mechanism of sensory plasticity. This review provides an up-to-date account of the A 1 /A 2 chromophore exchange system., (Copyright © 2021. Published by Elsevier Inc.)

Age related macular degeneration (AMD) is a multifactorial disease with genetic, biochemical and environmental risk factors. We observed a significant increase in copper levels in choroid-RPE from donor eyeballs with AMD. Adult retinal pigment epithelial cells (ARPE19 cells) exposed to copper in-vitro showed a 2-fold increase in copper influx transporter CTR1 and copper uptake at 50 μM concentration. Further there was 2-fold increase in cytochrome C oxidase activity and a 2-fold increase in the mRNA expression of NRF 2 with copper treatment. There was a significant increase in mitochondrial biogenesis markers PGC1β and TFAM which was confirmed by mitochondrial mass and copy number. On the contrary, in AMD choroid-RPE, the CTR1 mRNA was found to be significantly down-regulated compared to its respective controls. SCO1 and PGC1β mRNA showed an increase in choroid-RPE. Our study proposes copper to play an important role in mitochondrial biogenesis in RPE cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: Extracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells., Methods: RPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H 2 O 2 , others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry., Results: Clathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided., Conclusions: In summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in the elderly population. Variants in the HTRA1-ARMS2 locus have been linked to increased AMD risk. In the present study we investigated the impact of elevated HtrA1 levels on the retina pigment epithelial (RPE) secretome using a polarized culture system. Upregulation of HtrA1 alters the abundance of key proteins involved in angiogenesis and extracellular matrix remodeling. Thrombospondin-1, an angiogenesis modulator, was identified as a substrate for HtrA1 using terminal amine isotope labeling of substrates in conjunction with HtrA1 specificity profiling. HtrA1 cleavage of thrombospondin-1 was further corroborated by in vitro cleavage assays and targeted proteomics together with small molecule inhibition of HtrA1. While thrombospondin-1 is anti-angiogenic, the proteolytically released N-terminal fragment promotes the formation of tube-like structure by endothelial cells. Taken together, our findings suggest a mechanism by which increased levels of HtrA1 may contribute to AMD pathogenesis. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier. For quantitative secretome analysis, project accession: PXD007691, username: reviewer45093@ebi.ac.uk, password: 1FUpS6Yq. For TAILS analysis, project accession: PXD007139, username: reviewer76731@ebi.ac.uk, password: sNbMp7xK., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

With a quantum yield of 0.66±0.03 the photoisomerization efficiency of the visual pigment rhodopsin (11-cis⇒all-trans chromophore) is exceptionally high. This is currently explained by coherent coupling of the excited state electronic wavepacket with local vibrational nuclear modes, facilitating efficient cross-over at a conical intersection onto the photoproduct energy surface. The 9-cis counterpart of rhodopsin, dubbed isorhodopsin, has a much lower quantum yield (0.26±0.03), which, however, can be markedly enhanced by modification of the retinal chromophore (7,8-dihydro and 9-cyclopropyl derivatives). The coherent coupling in the excited state is promoted by torsional skeletal and coupled HOOP vibrational modes, in combination with a twisted conformation around the isomerization region. Since such torsion will strongly enhance the infrared intensity of coupled HOOP modes, we investigated FTIR difference spectra of rhodopsin, isorhodopsin and several analog pigments in the spectral range of isolated and coupled HCCH wags. As a result we propose that the coupled HOOP signature in these retinal pigments correlates with the distribution of torsion over counteracting segments in the retinylidene polyene chain. As such the HOOP signature can act as an indicator for the photoisomerization efficiency, and can explain the higher quantum yield of the 7,8-dihydro and 9-cyclopropyl-isorhodopsin analogs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chronic hyperglycemia is an important risk factor involved in the onset and progression of diabetic retinopathy (DR). Among other effectors, aldose reductase (AR) has been linked to the pathogenesis of this degenerative disease. The purpose of this study was to investigate whether the novel AR inhibitor, beta-glucogallin (BGG), can offer protection against various hyperglycemia-induced abnormalities in human adult retinal pigment epithelial (ARPE-19) cells. AR is an enzyme that contributes to cellular stress by production of reactive oxygen species (ROS) under high glucose conditions. A marked decrease in cell viability (from 100% to 78%) following long-term exposure (4 days) of RPE cells to high glucose (HG) was largely prevented by siRNA-mediated knockdown of AR gene expression (from 79% to 97%) or inhibition using sorbinil (from 66% to 86%). In HG, BGG decreased sorbitol accumulation (44%), ROS production (27%) as well as ER stress (22%). Additionally, we demonstrated that BGG prevented loss of mitochondrial membrane potential (MMP) under HG exposure. We also showed that AR inhibitor pretreatment reduced retinal microglia-induced apoptosis in APRE-19 cells. These results suggest that BGG may be useful as a therapeutic agent against retinal degeneration in the diabetic eye by preventing RPE cell death., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Low carotenoid status (especially of the xanthophylls, lutein [L], and zeaxanthin [Z]) is common in older adults and has been associated with a number of degenerative diseases of the central nervous system ranging from retina (e.g., macular degeneration) to brain (e.g., Alzheimer's disease). In this study, we tested whether retinal measures of L + Z (macular pigment optical density [MPOD]), used as a surrogate for brain L + Z levels, were related to cognitive function when comparing healthy older adults with mildly cognitively impaired older adults. Twenty-four subjects with mild cognitive impairment were compared with 24 matched controls. Subjects were matched with respect to age, body mass index, ethnicity, sex, and smoking status. Degree of cognitive impairment and cognitive ability was determined via structured clinical interview. MPOD was measured psychophysically. In healthy older adults, MPOD was only related to visual-spatial and constructional abilities (p = 0.04). For subjects with mild cognitive impairment (MCI), however, MPOD was broadly related to cognition including the composite score on the mini-mental state examination (p = 0.02), visual-spatial and constructional abilities (p = 0.04), language ability (p = 0.05), attention (p = 0.03), and the total scale on the Repeatable Battery for the Assessment of Neuropsychological Status (p = 0.03). It is possible that L/Z status may be more strongly related to cognition when individuals are considered with established onset of cognitive decline., (Copyright © 2014 Elsevier Inc. All rights reserved.)

11-cis-retinal acts as an inverse agonist stabilizing the inactive conformation of visual pigments, and upon photoactivation, it isomerizes to all-trans-retinal, initiating signal transduction. We have analyzed opsin regeneration with retinal analogs for rhodopsin and red cone opsin. We find differential binding of the analogs to the receptors after photobleaching and a dependence of the binding kinetics on the oligomerization state of the protein. The results outline the sensitivity of retinal entry to the binding pocket of visual receptors to the specific conformation adopted by the receptor and by the molecular architecture defined by specific amino acids in the binding pocket and the retinal entry site, as well as the topology of the retinal analog. Overall, our findings highlight the specificity of the ligand-opsin interactions, a feature that can be shared by other G-protein-coupled receptors., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this issue of Chemistry & Biology, Srinivasan and colleagues describe their study of ligand-protein interactions in visual pigments. Comparing the more stable isomeric ligand 9-cis retinal to the physiologically occurring 11-cis retinal, they report differences in ligand specificity and opsin conformational stability not previously described for bleached rhodopsin., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: To determine whether supplementation with lutein and zeaxanthin improves macular pigment and visual function in patients with early age-related macular degeneration (AMD)., Design: Randomized, double-masked, placebo-controlled trial., Participants: Participants with probable AMD who were 50 to 79 years of age were screened for study eligibility from the local communities. One hundred eight subjects with early AMD were recruited., Intervention: Early AMD patients were assigned randomly to receive 10 mg/day lutein (n = 27), 20 mg/day lutein (n = 27), 10 mg/day lutein plus 10 mg/day zeaxanthin (n = 27); or placebo (n = 27) for 48 weeks. Macular pigment optical density (MPOD) and visual function variables were assessed at baseline, 24 weeks, and 48 weeks., Main Outcome Measures: The primary outcome was MPOD. Secondary outcomes were visual function variables including best-corrected visual acuity (BCVA), contrast sensitivity (CS), photorecovery time, and Amsler grid testing results., Results: Macular pigment optical density increased significantly by a mean ± standard error of 0.076 ± 0.022 density unit in the 20-mg lutein group and 0.058 ± 0.027 density unit in the lutein and zeaxanthin group during 48 weeks. There was a significant dose-response effect for lutein supplementation, and the changes in MPOD from baseline to 48 weeks were correlated negatively with baseline MPOD in all active treatment groups (r = -0.56; P<0.001). At 48 weeks, a trend toward improvement was seen in BCVA, and there was a significant between-group difference in CS at 3 and 6 cycles/degree between the 20-mg lutein group and the placebo group. The increase in MPOD related positively to the reduction in the logarithm of the minimum angle of resolution BCVA (r = -0.31; P<0.01) and the increases in CS at 4 spatial frequencies (r ranging from 0.26 to 0.38; all P<0.05)., Conclusions: Among patients with early AMD, supplementation with lutein and zeaxanthin improved macular pigment, which played a causative role in boosting visual function and might prevent the progression of AMD. Future studies are required to evaluate the effect of these carotenoids on the incidence of late AMD., (Copyright © 2012 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Macrophages or activated microglia in the subretinal space are considered a hallmark of some retinal pathologies. We investigated the effects of age, pigmentation and CX(3)CR1 deficiency on the accumulation of macrophages/activated microglia in the outer retina of young and old Cx(3)cr1(gfp/gfp) (CX(3)CR1-deficient) or Cx(3)cr1(gfp/+) mice on either a pigmented (C57BL/6) or albino (BALB/c) background. Quantitative analysis of immunostained retinal-choroidal whole mounts revealed an increase in subretinal macrophage (SRMΦ) numbers in young Cx(3)cr1(gfp/gfp) mice compared with Cx(3)cr1(gfp/+) mice, however the increase was more marked in albino Cx(3)cr1(gfp/gfp) mice. In aged mice, large numbers of SRMΦ/activated microglia replete with autofluorescent debris were noted in both old pigmented Cx(3)cr1(gfp/gfp) and Cx(3)cr1(gfp/+) mice proving this accumulation was not CX(3)CR1-dependent. While CX(3)CR1 deficiency leads to an early onset of SRMΦ accumulation, our data reveal that this change occurs in both aged Cx(3)cr1(gfp/+) and Cx(3)cr1(gfp/gfp) pigmented mice in the absence of marked retinal degeneration and is likely a normal response to aging., (Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.)

Purpose: To characterize the phenotype and investigate the associations of intraretinal crystalline deposits in a large cohort with type 2 idiopathic macular telangiectasia (MacTel)., Design: Case-control study., Participants: Patients with and without retinal crystals from the Macular Telangiectasia Project, an international multicenter prospective study of type 2 MacTel., Methods: Grading of stereoscopic 30-degree color fundus (CF), confocal blue light reflectance (CBR), red-free (RF), and infrared (IR) images was performed according to the MacTel Natural History Study protocol and staged using the classification system devised by Gass and Blodi. Spectral domain-optical coherence tomography (SD-OCT) and adaptive optics imaging were used for a finer analysis of the phenotype. Associations between crystals and other characteristics of the disease, as well as potential risk factors, were investigated., Main Outcome Measures: Presence of crystals, fundus signs of MacTel, clinical characteristics, and presence of potential risk factors of MacTel., Results: Of 443 probands enrolled in the MacTel study, 203 (46%) had crystalline deposits present; 60% of the cases were bilateral at baseline. Eyes with crystals had a mean letter score of 70.7 (standard deviation [SD] = 15.9), whereas those without crystals had a mean letter score of 66.5 (SD = 15.5, P < 0.001). Crystals were present at all stages of the disease and showed high reflectivity within a wide wavelength range. They were located at the anterior surface of the nerve fiber layer, arranged along the nerve fibers, within an annular area centered on the fovea. Significant associations of crystalline deposits were found with a loss of retinal transparency, macular pigment optical density (MPOD) loss, fluorescein leakage, retinal thickness, and a break in the inner segment/outer segment junction line. Associations with environmental risk factors were not found., Conclusions: Intraretinal crystals are a frequent phenomenon associated with type 2 MacTel. They may appear at all stages and aid in the early diagnosis of the disease. Their morphology further implicates Müller cells in the pathogenesis of the disease. Insight into their physical and chemical properties may provide clues to the metabolic pathways involved in the pathogenesis of the disease., Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references., (Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: We examined changes in macular pigment optical density (MPOD) levels after cataract surgery and compared the MPOD between eyes with clear intraocular lenses (IOLs) and yellow-tinted IOLs., Design: Prospective, comparative case series., Participants: The MPOD levels were measured in 480 eyes of 337 patients after cataract surgery. Among them, the data from 259 eyes (clear IOL group, 121 eyes; yellow-tinted IOL group, 138 eyes) of 259 Japanese patients were selected for statistical analyses on the basis of the inclusion criteria: a postoperative visual acuity (VA) of ≥0.8 and no fundus diseases. Only 1 eye of each patient was enrolled. Patients provided informed consent to participate in this study on the basis of the approval of the institutional review board before surgery., Methods: The patients selected the type of IOL to be implanted. The MPOD levels were measured using resonance Raman spectroscopy on day 1 (baseline value); months 1, 3, and 6; and years 1 and 2 postoperatively., Main Outcome Measures: The difference in MPOD levels between the IOL groups was analyzed by unpaired t tests. The following parameters were analyzed by multiple regression analysis: age, gender, body mass index (BMI), smoking history, glaucoma, diabetes, preoperative VA, preoperative refractive error, and IOL power and type., Results: We found no significant differences in the baseline characteristics between the 2 groups. Until 6 months postoperatively, the MPOD levels did not differ significantly between the groups. However, from 1 year onward, the levels were significantly higher in the yellow-tinted IOL group compared with the clear IOL group. By multiple regression analysis, 1 day postoperatively, older age and diabetes were correlated with lower MPOD levels; 1 year postoperatively and thereafter, however, lower MPOD levels were correlated with clear IOLs., Conclusions: Cataract surgery with clear IOLs induced a greater decrease in macular pigment levels compared with yellow-tinted IOLs during a longer follow-up period. These findings agreed with observations that excessive light exposure is associated inversely with MPOD, because clear IOLs transmit higher intensities of blue light than yellow-tinted IOLs., (Copyright © 2011 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: To assess the histopathologic changes in a postmortem sample derived from an eye donor with macular telangiectasia (MacTel) type 2 to gain further insight into the cause of the disease., Design: Clinicopathological case report., Participants: Postmortem tissue was collected from 5 different donors: 1 MacTel type 2 patient; 1 healthy control; 2 type 2 diabetic patients, 1 with retinopathy and 1 without retinopathy; and 1 patient with unilateral Coat's disease., Methods: Macular pigment distribution in the posterior part of freshly dissected eyes was documented by macrophotography. Paraffin sections from both the macular and peripheral regions were assessed using antigen retrieval and immunohistochemistry to study the distribution of cell-specific markers. Blood vessels were visualized with antibodies directed against collagen IV and claudin 5; glial cells with antibodies against glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase (GS), and retinaldehyde binding protein (RLBP1, also known as CRALBP); microglia with an antibody against allograft inflammatory factor 1 (also known as Iba1); and photoreceptors with antibodies against rhodopsin and opsin. Using anatomic landmarks, the sections then were matched with the macular pigment distribution and a fluorescein angiogram of the patient that was obtained before the patient's death., Main Outcome Measures: Presence and distribution of macular pigment and cell-specific markers., Results: Macular pigment was absent in the macula. Furthermore, abnormally dilated capillaries were identified in a macular region that correlated spatially with regions of fluorescein leakage in an angiogram that was obtained 12 years before death. These telangiectatic vessels displayed a marked reduction of the basement membrane component collagen IV, indicating vascular pathologic features. The presence of GFAP was limited to retinal astrocytes, and no reactive Müller cells were identified. Importantly, reduced immunoreactivity with Müller cell markers (vimentin, GS, and RLBP1) in the macula was observed. The area that lacked Müller cells corresponded with the region of depleted macular pigment., Conclusions: These findings suggest that macular Müller cell loss or dysfunction is a critical component of MacTel type 2, which may have implications for future treatment strategies., (Copyright © 2010 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Background: Lutein and zeaxanthin may reduce the risk of dry, age-related macular degeneration because of their photo-oxidative role as macular pigment., Objective: The present study evaluated serum lutein, zeaxanthin, and macular pigment optical density (MPOD) responses at 0.25 degrees , 0.5 degrees , and 1 degree retinal eccentricities to the consumption of 2 and 4 egg yolks/d by older adults taking cholesterol-lowering medications., Design: Subjects consumed foods containing 2 followed by 4 egg yolks/d for 5 wk each with a 4-wk egg-free period at baseline and between the 2 interventions., Results: Changes in MPOD (n = 37) with egg yolk consumption were inversely associated (P < 0.05) with baseline MPOD. Subjects with low-baseline MPOD (defined as MPOD < or =0.5 at 0.25 degrees , < or =0.4 at 0.5 degrees , and < or =0.35 at 1 degrees ) showed increases of < or =50% (P < 0.05) with 4 egg yolks at the 3 retinal eccentricities. MPOD increased by 31% (P = 0.059) at 0.5 degrees with 2 egg yolks. Serum lutein increased by only 16% and 24% (P < 0.05) compared with increases of 36% and 82% (P < 0.001) in serum zeaxanthin (n = 52) after consumption of 2 and 4 egg yolks, respectively. Serum HDL cholesterol increased by 5% (P < 0.05) after consumption of 2 and 4 egg yolks. Serum LDL cholesterol did not change with either egg yolk treatment., Conclusions: Consumption of 4 egg yolks/d, and possibly of 2 egg yolks/d, for 5 wk benefited macular health in older adults with low MPOD. Serum HDL cholesterol increased without an increase in LDL cholesterol in this study population, most of whom were taking cholesterol-lowering statins.

In this study, we examined the impact of long-term (>8 y), low-fat, high-fruit and -vegetable diets on levels of lutein and zeaxanthin in the macula of the retina, as indicated by the OD of macular pigment. Macular pigment OD, measured by heterochromatic flicker photometry, was compared in women aged 60-87 y, who, 7-18 mo earlier (median 12 mo), had been in the dietary modification intervention (n = 158) or comparison (n = 236) groups of the Women's Health Initiative (WHI) at the Madison, WI site for a mean of 8.5 y. Women in the intervention group ate more fruits and vegetables (mean +/- SEM) (6.1 +/- 0.2 vs. 4.6 +/- 0.2 servings/d; P < 0.0001) and had higher intakes of lutein and zeaxanthin from foods and supplements (2.7 +/- 0.2 vs. 2.1 +/- 0.1 mg/d; P = 0.0003) than the comparison group. However, macular pigment density did not differ between the intervention (0.36 +/- 0.02 OD units) and comparison (0.35 +/- 0.01 OD units) groups. It tended to be higher (11%; P = 0.11) in women consuming lutein and zeaxanthin in the highest compared with the lowest quintile (median 6.4 vs. 1.1 mg/d). The increase in fruit and vegetable intake among dietary modification participants of this WHI subsample was not of sufficient magnitude to alter the mean density of retinal carotenoids, given other existing dietary conditions in this sample.

Purpose: To examine the association between intermediate age-related macular degeneration (AMD) and the optical density of macular pigment (MPOD), which is composed of lutein and zeaxanthin from the diet., Design: Cross-sectional cohort study., Participants: We included 1698 of 2005 women ages 54 to 86 years and participating in the Carotenoids in Age-Related Eye Disease Study, an ancillary study of the Women's Health Initiative., Methods: The MPOD was measured noninvasively by heterochromatic flicker photometry. Fundus photographs were taken to document prevalent AMD., Main Outcome Measures: Intermediate AMD (n = 305) and two subtypes-large drusen (n = 233) and pigmentary abnormalities (n = 157)., Results: After adjusting for covariates, the odds ratio (OR) and 95% confidence interval (CI) for AMD among women in quintile (Q) 5 (n = 339) versus 1 (n = 340) for MPOD was 1.4 (0.9, 2.1). However, after excluding women with possible unstable diets and recent supplement use due to chronic disease history, associations reversed (OR Q2-5 vs. 1, 0.8; 95% CI, 0.5-1.2), but remained nonsignificant. Associations also differed between middle-aged (54-69 years) and older (> or =70 years) women (P-interaction = 0.09), but less so, after excluding women who were likely to have unstable diets: adjusted ORs (95% CI) were 0.5 (0.3-1.0; P = 0.08) for intermediate AMD among middle-aged women (n = 516) with MPOD in Q2 to Q5 versus 1 and 1.0 (0.5-2.0; P = 0.90) for older women (n = 422)., Conclusions: The MPOD is not cross-sectionally associated with AMD. The inconsistency of relationships across age groups and in subgroups of women who are likely to have more stable diets suggests that cross-sectional associations may be biased and highlights the need to study these relationships prospectively.

Rhodopsin is currently the only available atomic-resolution template for understanding biological functions of the G protein-coupled receptor (GPCR) family. The structural basis for the phenomenal dark state stability of 11-cis-retinal bound to rhodopsin and its ultrafast photoreaction are active topics of research. In particular, the beta-ionone ring of the retinylidene inverse agonist is crucial for the activation mechanism. We analyzed a total of 23 independent, 100 ns all-atom molecular dynamics simulations of rhodopsin embedded in a lipid bilayer in the microcanonical (N,V,E) ensemble. Analysis of intramolecular fluctuations predicts hydrogen-out-of-plane (HOOP) wagging modes of retinal consistent with those found in Raman vibrational spectroscopy. We show that sampling and ergodicity of the ensemble of simulations are crucial for determining the distribution of conformers of retinal bound to rhodopsin. The polyene chain is rigidly locked into a single, twisted conformation, consistent with the function of retinal as an inverse agonist in the dark state. Most surprisingly, the beta-ionone ring is mobile within its binding pocket; interactions are non-specific and the cavity is sufficiently large to enable structural heterogeneity. We find that retinal occupies two distinct conformations in the dark state, contrary to most previous assumptions. The beta-ionone ring can rotate relative to the polyene chain, thereby populating both positively and negatively twisted 6-s-cis enantiomers. This result, while unexpected, strongly agrees with experimental solid-state (2)H NMR spectra. Correlation analysis identifies the residues most critical to controlling mobility of retinal; we find that Trp265 moves away from the ionone ring prior to any conformational transition. Our findings reinforce how molecular dynamics simulations can challenge conventional assumptions for interpreting experimental data, especially where existing models neglect conformational fluctuations.

Photoreceptor differentiation requires the coordinated expression of numerous genes. It is unknown whether those genes share common regulatory mechanisms or are independently regulated by distinct mechanisms. To distinguish between these scenarios, we have used in situ hybridization, RT-PCR, and real-time PCR to analyze the expression of visual pigments and other photoreceptor-specific genes during chick embryo retinal development in ovo, as well as in retinal cell cultures treated with molecules that regulate the expression of particular visual pigments. In ovo, onset of gene expression was asynchronous, becoming detectable at the time of photoreceptor generation (ED 5-8) for some photoreceptor genes, but only around the time of outer segment formation (ED 14-16) for others. Treatment of retinal cell cultures with activin, staurosporine, or CNTF selectively induced or down-regulated specific visual pigment genes, but many cognate rod- or cone-specific genes were not affected by the treatments. These results indicate that many photoreceptor genes are independently regulated during development, are consistent with the existence of at least two distinct stages of gene expression during photoreceptor differentiation, suggest that intrinsic, coordinated regulation of a cascade of gene expression triggered by a commitment to the photoreceptor fate is not a general mechanism of photoreceptor differentiation, and imply that using a single photoreceptor-specific "marker" as a proxy to identify photoreceptor cell fate is problematic.

Non-invasive measurement of the optical density of the human macular pigment by the autofluorescence method takes advantage of the fluorescence of lipofuscin in the human retinal pigment epithelium. Measuring the intensity of fluorescence above 550 nm, where macular pigment has essentially zero absorption, and stimulating the fluorescence with two wavelengths, one well absorbed by macular pigment and the other minimally absorbed by macular pigment, provides a single-pass measurement of the macular pigment optical density. The method is implemented either by fluorometry of lipofuscin to yield the optical density of the macular pigment in a 2 degrees diameter central area, or by autofluorescence imaging to yield a high-resolution map of the macular pigment distribution.

Fundus reflectance spectroscopy has proven to be a reliable method for objective, in vivo determination of the macular pigment optical density. This paper discusses the technique and reviews measurements, including the observed absence of an age effect.

Observational evidence is accumulating that the onset of age-related maculopathy, the leading cause of legal blindness in the Western World, could be delayed, or even averted, with antioxidant supplements. Lutein (L) and zeaxanthin (Z) are two hydroxy-carotenoids with antioxidant activity which accumulate at the macula, where they are collectively known as macular pigment (MP). It has been shown that MP is entirely of dietary origin, and that L and Z levels in serum, diet, and retina correlate. However, the nature of the relationships between L and Z in foodstuffs, blood, and macula is confounded by many variables including processes which influence digestion, absorption, and transport of the compounds in question, and accumulation and stabilization of the carotenoids in the tissues. If macular pigment is protective for age-related maculopathy, a clear understanding of the mechanisms whereby L and Z arrive at the target tissue (retina) from their source (foodstuff) is essential. In this paper, we review the literature germane to this growing area of interest.

In early age-related macular degeneration (AMD), lipid-containing deposits (drusen) accumulate in Bruch's membrane underlying the retinal pigment epithelium (RPE). Recent studies indicate that apolipoprotein E (apoE) may play a role in lipid trafficking in AMD. Compared with the apoE3 allele, the apoE4 and apoE2 alleles are associated with decreased and increased risk for AMD, respectively; drusen contain high levels of apoE, and apoE null mice develop lipid deposits in Bruch's membrane similar to those observed in AMD. Primary cultures of human RPE cells expressing the apoE3 allele were grown on Transwell culture plates. Western blotting, ELISA assay, and mass spectrometry confirmed that apoE3 was secreted into the apical and basal chambers and that secretion was upregulated by thyroid hormone, 9-cis-retinoic acid, and 22(R)-hydroxycholesterol. In addition, basally secreted apoE associated with exogenously added HDL. These results indicate that apoE secretion can be regulated by specific hormones and that apoE associates with HDL. The findings are consistent with a role for apoE in lipid trafficking through Bruch's membrane and may be relevant to AMD.

To gain insight into the genetic mechanisms of photoreceptor development, we analyzed a collection of zebrafish mutations characterized by early photoreceptor cell loss. The mutant defects impair outer segment formation and are accompanied by an abnormal distribution of visual pigments. Rods and different cone types display defects of similar severity suggesting that genetic pathways common to all photoreceptors are affected. To investigate whether these phenotypes involve cell-cell interaction defects, we analyzed genetically mosaic animals. Interaction of niezerka photoreceptors with wild-type tissues improves the survival of mutant cells and restores their elongated morphology. In contrast, cells carrying mutations in the loci brudas, elipsa, fleer, and oval retain their defective phenotypes in a wild-type environment indicating cell-autonomy. These experiments identify distinct phenotypic categories of photoreceptor mutants and indicate that zebrafish photoreceptor defects involve both cell-autonomous and cell-nonautonomous mechanisms.

(MP) that is composed of the hydroxy-carotenoids lutein and zeaxanthin. Although it appears that all humans have some quantity of these pigments within their retina, foveal concentrations tend to vary quite dramatically. This wide individual variability has prompted questions regarding possible functional consequences. At least two major nonexclusive hypotheses regarding the function of MP have been proposed. The "protection hypothesis" has received the most attention and is based on the possibility that MP could reduce the cumulative effects of damage due to light and oxygen and retard the development of age-related eye disease. The "acuity hypothesis" states that MP could improve visual resolution by absorbing short-wave light, which is easily scattered and poorly focused. In this article, we review evidence that lutein and zeaxanthin could improve human visual performance through both acute optical effects at the site of the retina and by maintaining health and functional integrity of the retina and crystalline lens.

The regulation of the reduced-folate transporter (RFT) by nitric oxide (NO) was analyzed in human retinal pigment epithelial (HRPE) cells. NO inhibited specifically and reversibly the uptake of N5-methyltetrahydrofolate by a cGMP-independent mechanism. The inhibition was associated with a decrease in substrate affinity. The NO-induced inhibition was prevented by antioxidants and NO scavengers. Agents capable of modifying thiol groups in proteins inhibited RFT, indicating that the likely mechanism of NO-induced inhibition is via modification of essential thiol groups in this protein. These studies suggest that NO produced during retinal disease may affect the function of RFT in adjacent RPE cells., (Copyright 1999 Academic Press.)

Background: Fundus flavimaculatus (Stargardt disease) is a group of inherited macular dystrophies in which central vision usually decreases in the first two decades of life. Previous histopathologic studies used light, scanning, and transmission electron microscopy to characterize the retinal pigment epithelium (RPE) in fundus flavimaculatus. The authors describe in detail the pathologic changes in the neurosensory retina, including use of specific immunocytochemical markers., Methods: The eyes of a patient with fundus flavimaculatus were processed using Medcast and JB-4 plastic for light and electron microscopy, and cryomicrotomy and LR-white resin for immunocytochemistry., Results: Changes in the RPE occurred in a peripheral/central gradient and included increased lipofuscin content and cell loss toward the macula. The changes in the retina paralleled those in the RPE, including accumulation of lipofuscin in photoreceptor inner segments, loss of photoreceptors, and reactive Müller cell hypertrophy. Immunocytochemistry using rod- and cone-specific markers showed abnormal photoreceptor morphology but qualitatively normal immunoreactivity, and there was strong reactivity for glial fibrillary acid protein in reactive Müller cells. Labeling for cellular retinaldehyde-binding protein was qualitatively normal in Müller cells, but was reduced in RPE cells that were engorged with lipofuscin., Conclusions: The histopathologic changes in the retina correlate with clinical progression of the disease process. Although abnormal lipofuscin metabolism has been implicated in the loss of vision in fundus flavimaculatus and other macular diseases, the mechanism is not understood. Based on the authors' observations and a review of recent literature on lipofuscin, the authors propose that all-trans-retinol dehydrogenase, a photoreceptor outer segment enzyme, may be defective in fundus flavimaculatus.

The opsin-like immunoreactivity in the retinas of C57BL/6J rdle mutant mice has been studied by light-microscopic immunocytochemistry. Positively labeled cells were found in the normal heterozygous and homozygous mutant mouse outer nuclear layer (ONL) as early as postnatal day 3 (PN3). Beginning at PN10, in the retinas of the homozygous mutant mice, labeled photoreceptor cells rapidly decreased in number and disappeared after PN42. In the decreased ONL, remaining opsin-positive cells were labeled at higher density than those of controls. The retinal pigment epithelium was also moderately labeled during the loss of opsin-positive photoreceptor cells. In addition, sparse opsin-immunoreactive cells were demonstrated in the inner nuclear layer (INL) in the retinas of both the mutant and non-dystrophic mice as early as PN10 and are presumed to be ectopic photoreceptor cells. However, these displaced photoreceptor cells disappeared by PN28 in mutants along the same time course as those in the ONL but were still present in the PN28 retina of controls and seemed to be more abundant at later adult ages. There was no difference in the developmental regulation of opsin in the heterozygous and normal controls.

Various mechanistic schemes for the recombination reaction of rhodopsin were designed and tested using computer modeling and simulation with data from kinetics experiments. The reaction schemes were mathematically modeled by systems of nonlinear first-order ordinary differential equations (ODEs) with unknown rate constants. Each model was fitted to the experimental data by using a modified simplex algorithm for parameter (rate constant) estimation and Gear's method for solving stiff systems of ODEs. The recombination reaction of rhodopsin was best modeled by branched, multistep reaction schemes which included formation of noncovalent complexes, acid-base equilibria, and acid and base-catalyzed dehydration of a Schiff base intermediate. The biochemical bases for these models are discussed.

Rhodopsin in rod outer segment disk membranes was enzymatically modified by erythrocyte transglutaminase, which linked small primary amines to glutamine residues. In order to avoid formation of protein crosslinks, rhodopsin was first reductively methylated to modify its lysines. From 1.9 to 2.5 mol of putrescine, ethanolamine, or dinitrophenylcadaverine were incorporated into rhodopsin by transglutaminase during 16 h reaction time. A maximum of 3.5 mol of [14C]putrescine was incorporated per mole of rhodopsin during 48 h. Essentially all of the rhodopsin sequence containing the putrescine could be removed by limited proteolysis of the membranes by thermolysin. Glutamine residues in positions 236, 237, 238, and 344 were modified to approximately equal extents, as determined by isolation of the cyanogen bromide peptides of modified rhodopsin followed by further subdigestion of the peptides. The modified glutamine residues are located in the helix V-VI (or F1-F2) connecting loop and in the carboxyl-terminal region of rhodopsin.

It has been generally accepted that transmitter molecules, which participated in the visual transduction process in retinal rod outer segments, are activated by bleached rhodopsin molecules serially one by one (serial activation model). This serial activation model accounts for some experimental observations, but not the quick and amplified activation of transmitter molecules. In this study, two models are proposed and solutions from the differential equations of the models are compared quantitatively with experimental observations. The two models proposed in this study are the serial-parallel activation model and the parallel-parallel activation model. In the serial-parallel activation model, rhodopsin molecules are activated by bleached rhodopsin molecules. Transmitter molecules are activated both by bleached and activated rhodopsin molecules. In the parallel-parallel activation model, both rhodopsin and transmitter molecules are activated by bleached and activated rhodopsin molecules. The time taken to activate 90% of transmitter molecules after flash stimuli, which bleaches 10(-4)% of rhodopsin, is 5.94 s, 3.91 ms and 23.7 microseconds for the serial, serial-parallel and parallel-parallel activation models, respectively. As contrasted to the serial activation model in which transmitter activation proceeds too slow, the present serial-parallel and parallel-parallel activation models can reproduce transmitter activation quick enough to account for the experimental observations.

Retinas obtained from 7-day-old rd mice show less reaction with antirhodopsin antisera than retinas from normal mice of the same age. Likewise, antisera prepared against synthetic peptides, which corresponds to the carboxyl terminus of rhodopsin, also react less with rd retinas from 7-day-old mice. In contrast, Western blots of denatured rhodopsin from rd vs. normal retinas of the same age indicate no change in the total quantity of this protein. These results demonstrate that in the 7-day-old rd mouse retina, rhodopsin is not altered in quantity; rather, it is less accessible to reaction with anti-rhodopsin antisera. Furthermore, these results suggest that the site of altered accessibility is on the carboxyl terminus of rhodopsin.

Multifocal lesions of the retinal pigment epithelium were observed in rabbits fed a diet contaning 0.5% lead subacetate for periods of up to 2 years. Groups of pigment epithelial cells became congested with a lipofuscin pigment which was apparently derived from phagosomes of rod outer segments. Lipofuscin granules displaced melanin granules from the apical surface of the retinal pigment epithelial cells and resulted in conspicuous brown pigmentation of these cells in albino animals. Migration of macrophages and pigment epithelial cells into the subretinal space was common in affected areas. This pathology was not observed in the pigment epithelium of the ora serrata, or ciliary body. At the latest time periods, the abnormal lipofuscin pigmentation subsided, and degeneration of photoreceptors occurred. The pathogenesis of the lesions is discussed.

Light-induced conformational changes occurring at the cytosolic surface of rhodopsin were investigated by performing limited digestions of native and illuminated visual pigment with thermolysin, Arg-C endoproteinase, papain and proteinase K. A higher susceptibility of the extradiscal regions of the bleached pigment to the proteases were observed together with altered capacities of the digested bleached rhodopsins to activate the cGMP phosphodiesterase. The overall results strongly suggest that light induces conformational changes not only in the C-terminal end but also in the second and the third extradiscal loop of rhodopsin.