Lipidomic analysis revealed n-3 polyunsaturated fatty acids suppressed choroidal thinning and myopia progression in mice.

Myopia is increasing worldwide and its preventable measure should urgently be pursued. N-3 polyunsaturated fatty acids (PUFAs) have been reported to have various effects such as vasodilative and anti-inflammatory, which myopia may be involved in. This study is to investigate the inhibitory effect of PUFAs on myopia progression. A lens-induced myopia (LIM) model was prepared using C57B L6/J 3-week-old mice, which were equipped with a -30 diopter lens to the right eye. Chows containing two different ratios of n-3/n-6 PUFA were administered to the mice, and myopic shifts were confirmed in choroidal thickness, refraction, and axial length in the n-3 PUFA-enriched chow group after 5 weeks. To exclude the possibility that the other ingredients in the chow may have taken the suppressive effect, fat-1 transgenic mice, which can produce n-3 PUFAs endogenously, demonstrated significant suppression of myopia. To identify what elements in n-3 PUFAs took effects on myopia suppression, enucleated eyes were used for targeted lipidomic analysis, and eicosapentaenoic acid (EPA) were characteristically distributed. Administration of EPA to the LIM model confirmed the inhibitory effect on choroidal thinning and myopia progression. Subsequently, to identify the elements and the metabolites of fatty acids effective on myopia suppression, targeted lipidomic analysis was performed and it demonstrated that metabolites of EPA were involved in myopia suppression, whereas prostaglandin E2 and 14,15-dihydrotestosterone were associated with progression of myopia. In conclusion, EPA and its metabolites are related to myopia suppression and inhibition of choroidal thinning.

A Potential New Role for Zinc in Age-Related Macular Degeneration through Regulation of Endothelial Fenestration.

Age-related macular degeneration (AMD) is a common blinding disease in the western world that is linked to the loss of fenestration in the choriocapillaris that sustains the retinal pigment epithelium and photoreceptors in the back of the eye. Changes in ocular and systemic zinc concentrations have been associated with AMD; therefore, we hypothesized that these changes might be directly involved in fenestrae formation. To test this hypothesis, an endothelial cell (bEND.5) model for fenestrae formation was treated with different concentrations of zinc sulfate (ZnSO4) solution for up to 20 h. Fenestrae were visualized by staining for Plasmalemmal Vesicle Associated Protein-1 (PV-1), the protein that forms the diaphragms of the fenestrated endothelium. Size and distribution were monitored by transmission electron microscopy (TEM). We found that zinc induced the redistribution of PV-1 into areas called sieve plates containing ~70-nm uniform size and typical morphology fenestrae. As AMD is associated with reduced zinc concentrations in the serum and in ocular tissues, and dietary zinc supplementation is recommended to slow disease progression, we propose here that the elevation of zinc concentration may restore choriocapillaris fenestration resulting in improved nutrient flow and clearance of waste material in the retina.

Interactions of the choroid, Bruch's membrane, retinal pigment epithelium, and neurosensory retina collaborate to form the outer blood-retinal-barrier.

The three interacting components of the outer blood-retinal barrier are the retinal pigment epithelium (RPE), choriocapillaris, and Bruch's membrane, the extracellular matrix that lies between them. Although previously reviewed independently, this review integrates these components into a more wholistic view of the barrier and discusses reconstitution models to explore the interactions among them. After updating our understanding of each component's contribution to barrier function, we discuss recent efforts to examine how the components interact. Recent studies demonstrate that claudin-19 regulates multiple aspects of RPE's barrier function and identifies a barrier function whereby mutations of claudin-19 affect retinal development. Co-culture approaches to reconstitute components of the outer blood-retinal barrier are beginning to reveal two-way interactions between the RPE and choriocapillaris. These interactions affect barrier function and the composition of the intervening Bruch's membrane. Normal or disease models of Bruch's membrane, reconstituted with healthy or diseased RPE, demonstrate adverse effects of diseased matrix on RPE metabolism. A stumbling block for reconstitution studies is the substrates typically used to culture cells are inadequate substitutes for Bruch's membrane. Together with human stem cells, the alternative substrates that have been designed offer an opportunity to engineer second-generation culture models of the outer blood-retinal barrier.

Effect of acute and chronic aldosterone exposure on the retinal pigment epithelium-choroid complex in rodents.

Preclinical and clinical evidences show that aldosterone and/or mineralocorticoid receptor (MR) over-activation by glucocorticoids can be deleterious to the retina and to the retinal pigment epithelium (RPE)-choroid complex. However, the exact molecular mechanisms driving these effects remain poorly understood and pathological consequences of chronic exposure of the retina and RPE/choroid to aldosterone have not been completely explored. We aimed to decipher the transcriptomic regulation in the RPE-choroid complex in rats in response to acute intraocular aldosterone injection and to explore the consequences of systemic chronic aldosterone exposure on the morphology and the gene regulation in RPE/choroid in mice. High dose of aldosterone (100 nM) was intravitreously injected in Lewis rat eyes in order to yield an aldosterone dose able to induce a molecular response at the apical side of the RPE-choroid complex. The posterior segment morphology was evaluated in vivo using optical coherence tomography (OCT) before and 24 h after aldosterone injection. Rat RPE-choroid complexes were used for RNA sequencing and analysis. Uninephrectomy/aldosterone/salt (NAS) model was created in wild-type C57BL/6 mice. After 6 weeks, histology of mouse posterior segments were observed ex vivo. Gene expression in the RPE-choroid complex was analyzed using quantitative PCR. Acute intravitreous injection of aldosterone induced posterior segment inflammation observed on OCT. RNA sequencing of rat RPE-choroid complexes revealed up-regulation of pathways involved in inflammation, oxidative stress and RNA procession, and down-regulation of genes involved in synaptic activity, muscle contraction, cytoskeleton, cell junction and transporters. Chronic aldosterone/salt exposure in NAS model induces retinal edema, choroidal vasodilation and RPE cell dysfunction and migration. Quantitative PCR showed deregulation of genes involved in inflammatory response, oxidative stress, particularly the NOX pathway, angiogenesis and cell contractility. Both rodent models share some common phenotypes and molecular regulations in the RPE-choroid complex that could contribute to pachychoroid epitheliopathy in humans. The difference in inflammatory status relies on different intraocular or systemic route of aldosterone administration and on the different doses of aldosterone exposed to the RPE-choroid complex.

Bone Marrow-Derived Cell Recruitment to the Neurosensory Retina and Retinal Pigment Epithelial Cell Layer Following Subthreshold Retinal Phototherapy.

Purpose: We investigated whether subthreshold retinal phototherapy (SRPT) was associated with recruitment of bone marrow (BM)-derived cells to the neurosensory retina (NSR) and RPE layer. Methods: GFP chimeric mice and wild-type (WT) mice were subjected to SRPT using a slit-lamp infrared laser. Duty cycles of 5%, 10%, 15%, and 20% (0.1 seconds, 250 mW, spot size 50 µm) with 30 applications were placed 50 to 100 µm from the optic disc. In adoptive transfer studies, GFP+ cells were given intravenously immediately after WT mice received SRPT. Immunohistochemistry was done for ionized calcium-binding adapter molecule-1 (IBA-1+), CD45, Griffonia simplicifolia lectin isolectin B4, GFP or cytokeratin). Expression of Ccl2, Il1b, Il6, Hspa1a, Hsp90aa1, Cryab, Hif1a, Cxcl12, and Cxcr4 mRNA and flow cytometry of the NSR and RPE-choroid were performed. Results: Within 12 to 24 hours of SRPT, monocytes were detected in the NSR and RPE-choroid. Detection of reparative progenitors in the RPE occurred at 2 weeks using flow cytometry. Recruitment of GFP+ cells to the RPE layer occurred in a duty cycle-dependent manner in chimeric mice and in mice undergoing adoptive transfer. Hspa1a, Hsp90aa1, and Cryab mRNAs increased in the NSR at 2 hours post laser; Hif1a, Cxcl12, Hspa1a increased at 4 hours in the RPE-choroid; and Ccl2, Il1b, Ifng, and Il6 increased at 12 to 24 hours in the RPE-choroid. Conclusions: SRPT induces monocyte recruitment to the RPE followed by hematopoietic progenitor cell homing at 2 weeks. Recruitment occurs in a duty cycle-dependent manner and potentially could contribute to the therapeutic efficacy of SRPT.

Efficacy of a Fatty Acids Dietary Supplement in a Polyethylene Glycol-Induced Mouse Model of Retinal Degeneration.

Current knowledge of the benefits of nutrition supplements for eye pathologies is based largely on the use of appropriate animal models, together with defined dietary supplementation. Here, C57BL6 mice were subretinally injected with polyethylene glycol (PEG)-400, an established model of retinal degeneration with a dry age-related macular degeneration (AMD)-like phenotype, an eye pathology that lacks treatment. In response to PEG-400, markers of the complement system, angiogenesis, inflammation, gliosis, and macrophage infiltration were upregulated in both retinas and retinal pigment epithelium (RPE)/choroids, whereas dietary supplementation with a mixture based on fatty acids counteracted their upregulation. Major effects include a reduction of inflammation, in both retinas and RPE/choroids, and an inhibition of macrophage infiltration in the choroid, yet not in the retina, suggesting a targeted action through the choroidal vasculature. Histological analysis revealed a thinning of the outer nuclear layer (ONL), together with dysregulation of the epithelium layer in response to PEG-400. In addition, immunohistofluorescence demonstrated Müller cell gliosis and macrophage infiltration into subretinal tissues supporting the molecular findings. Reduced ONL thickness, gliosis, and macrophage infiltration were counteracted by the diet supplement. The present data suggest that fatty acids may represent a useful form of diet supplementation to prevent or limit the progression of dry AMD.

Targeted Intraceptor Nanoparticle for Neovascular Macular Degeneration: Preclinical Dose Optimization and Toxicology Assessment.

Neovascular age-related macular degeneration (AMD) is treated with anti-VEGF intravitreal injections, which can cause geographic atrophy, infection, and retinal fibrosis. To minimize these toxicities, we developed a nanoparticle delivery system for recombinant Flt23k intraceptor plasmid (RGD.Flt23k.NP) to suppress VEGF intracellularly within choroidal neovascular (CNV) lesions in a laser-induced CNV mouse model through intravenous administration. In the current study, we examined the efficacy and safety of RGD.Flt23k.NP in mice. The effect of various doses was determined using fluorescein angiography and optical coherence tomography to evaluate CNV leakage and volume. Efficacy was determined by the rate of inhibition of CNV volume at 2 weeks post-treatment. RGD.Flt23k.NP had peak efficacy at a dose range of 30-60 µg pFlt23k/mouse. Using the lower dose (30 µg pFlt23k/mouse), RGD.Flt23k.NP safety was determined both in single-dose groups and in repeat-dose (three times) groups by measuring body weight, organ weight, hemoglobin levels, complement C3 levels, and histological changes in vital organs. Neither toxicity nor inflammation from RGD.Flt23k.NP was detected. No side effect was detected on visual function. Thus, systemic RGD.Flt23k.NP may be an alternative to standard intravitreal anti-VEGF therapy for the treatment of neovascular AMD.

Association Between Choroidal Thickness and Metabolic Activity on Positron Emission Tomography in Eyes With Choroidal Melanoma.

PURPOSE: To investigate the relationship between subfoveal choroidal thickness and metabolic activity in eyes with choroidal melanoma. DESIGN: Retrospective, interventional case series. METHODS: The medical records of 16 patients with unilateral choroidal melanoma who underwent ruthenium (Ru) 106 brachytherapy with adjuvant transpupillary thermotherapy and who had available pretreatment positron emission tomography-computed tomography (PET-CT) images were retrospectively reviewed. Subfoveal choroidal thickness was measured in tumor eyes and in unaffected fellow eyes using enhanced-depth imaging spectral-domain optical coherence tomography (EDI OCT). Tumor eyes were divided into 2 groups (metabolically active and inactive) based on PET-CT findings and subfoveal choroidal thickness was compared between groups. Additionally, choroidal thickness measurements were compared before and after treatment. RESULTS: Before treatment, mean choroidal thickness was 293.31 ± 46.80 µm in tumor eyes and 242.44 ± 65.37 µm in fellow eyes, a difference that was statistically significant (P = .003). Eyes with metabolically active tumors had a significantly thicker choroid (348.00 ± 17.32 µm) than eyes with metabolically inactive tumors (280.69 ± 42.04 µm, P = .019). In tumor eyes, mean choroidal thickness significantly decreased from pretreatment values to 253.56 ± 61.27 µm 6 months after treatment (P = .018). CONCLUSION: Eyes with choroidal melanoma had thicker choroids than unaffected fellow eyes. Increased choroidal thickness was more prominent in metabolically active tumors. Choroidal thickness significantly decreased in tumor eyes 6 months after treatment.

Modifying Choroidal Neovascularization Development with a Nutritional Supplement in Mice.

We examined the effect of nutritional supplements (modified Age Related Eye Disease Study (AREDS)-II formulation containing vitamins, minerals, lutein, resveratrol, and omega-3 fatty acids) on choroidal neovascularization (CNV). Supplements were administered alone and combined with intravitreal anti-VEGF in an early-CNV (diode laser-induced) murine model. Sixty mice were evenly divided into group V (oral vehicle, intravitreal saline), group S (oral supplement, intravitreal saline), group V + aVEGF (oral vehicle, intravitreal anti-VEGF), and group S + aVEGF (oral supplement, intravitreal anti-VEGF). Vehicle and nutritional supplements were administered daily for 38 days beginning 10 days before laser. Intravitreal injections were administered 48 h after laser. Fluorescein angiography (FA) and flat-mount CD31 staining evaluated leakage and CNV lesion area. Expression of VEGF, MMP-2 and MMP-9 activity, and NLRP3 were evaluated with RT-PCR, zymography, and western-blot. Leakage, CNV size, VEGF gene and protein expression were lower in groups V + aVEGF, S + aVEGF, and S than in V (all p < 0.05). Additionally, MMP-9 gene expression differed between groups S + aVEGF and V (p < 0.05) and MMP-9 activity was lower in S + aVEGF than in V and S (both p < 0.01). Levels of MMP-2 and NLRP3 were not significantly different between groups. Nutritional supplements either alone or combined with anti-VEGF may mitigate CNV development and inhibit retinal disease involving VEGF overexpression and CNV.

Dynamic increase in extracellular ATP accelerates photoreceptor cell apoptosis via ligation of P2RX7 in subretinal hemorrhage.

Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.

Characterization of the R162W Kir7.1 mutation associated with snowflake vitreoretinopathy.

KCNJ13 encodes Kir7.1, an inwardly rectifying K(+) channel that is expressed in multiple ion-transporting epithelia. A mutation in KCNJ13 resulting in an arginine-to-tryptophan change at residue 162 (R162W) of Kir7.1 was associated with snowflake vitreoretinal degeneration, an inherited autosomal-dominant disease characterized by vitreous degeneration and mild retinal degeneration. We used the Xenopus laevis oocyte expression system to assess the functional properties of the R162W (mutant) Kir7.1 channel and determine how wild-type (WT) Kir7.1 is affected by the presence of the mutant subunit. Recordings obtained via the two-electrode voltage-clamp technique revealed that injection of oocytes with mutant Kir7.1 cRNA resulted in currents and cation selectivity that were indistinguishable from those in water-injected oocytes, suggesting that the mutant protein does not form functional channels in the plasma membrane. Coinjection of oocytes with equal amounts of mutant and WT Kir7.1 cRNAs resulted in inward K(+) and Rb(+) currents with amplitudes that were ∼17% of those in oocytes injected with WT Kir7.1 cRNA alone, demonstrating a dominant-negative effect of the mutant subunit. Similar to oocytes injected with WT Kir7.1 cRNA alone, coinjected oocytes exhibited inwardly rectifying Rb(+) currents that were more than seven times larger than K(+) currents, indicating that mutant subunits did not alter Kir7.1 channel selectivity. Immunostaining of Xenopus oocytes or Madin-Darby canine kidney cells expressing mutant or WT Kir7.1 demonstrated distribution of both proteins primarily in the plasma membrane. Our data suggest that the R162W mutation suppresses Kir7.1 channel activity, possibly by negatively impacting gating by membrane phosphadidylinositol 4,5-bisphosphate.

Hyperglycaemia exacerbates choroidal neovascularisation in mice via the oxidative stress-induced activation of STAT3 signalling in RPE cells.

Choroidal neovascularisation (CNV) that occurs as a result of age-related macular degeneration (AMD) causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG) antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and vascular endothelial growth factor (VEGF), in mice with CNV. The production of reactive oxygen species (ROS) in retinal pigment epithelial (RPE) cells that had been cultured under high glucose was quantitated using the 2',7'-dichlorofluorescein diacetate (DCFH-DA) method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC) rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV. Thus, our results reveal a potential strategy for the treatment and prevention of diseases involving CNV.

[Comparative experimental study of morphological and histochemical changes of chorioretinal complex after subthreshold laser exposure].

The influence of several subthreshold laser procedures on chorioretinal complex (CRC) tissues was experimentally studied in rabbits using histological and histochemical methods. Subthreshold micropulse laser radiation and transpupillar thermotherapy were found to have the most attenuated and advantageous effect on CRC structure. The feature of subthreshold laser exposure is partly reversible selective changes in CRC, that have temporary stimulating effect on cellular metabolism and local regenerative processes providing an opportunity for still viable tissues to function adequately.

A new strategy to identify and annotate human RPE-specific gene expression.

BACKGROUND: To identify and functionally annotate cell type-specific gene expression in the human retinal pigment epithelium (RPE), a key tissue involved in age-related macular degeneration and retinitis pigmentosa. METHODOLOGY: RPE, photoreceptor and choroidal cells were isolated from selected freshly frozen healthy human donor eyes using laser microdissection. RNA isolation, amplification and hybridization to 44 k microarrays was carried out according to Agilent specifications. Bioinformatics was carried out using Rosetta Resolver, David and Ingenuity software. PRINCIPAL FINDINGS: Our previous 22 k analysis of the RPE transcriptome showed that the RPE has high levels of protein synthesis, strong energy demands, is exposed to high levels of oxidative stress and a variable degree of inflammation. We currently use a complementary new strategy aimed at the identification and functional annotation of RPE-specific expressed transcripts. This strategy takes advantage of the multilayered cellular structure of the retina and overcomes a number of limitations of previous studies. In triplicate, we compared the transcriptomes of RPE, photoreceptor and choroidal cells and we deduced RPE specific expression. We identified at least 114 entries with RPE-specific gene expression. Thirty-nine of these 114 genes also show high expression in the RPE, comparison with the literature showed that 85% of these 39 were previously identified to be expressed in the RPE. In the group of 114 RPE specific genes there was an overrepresentation of genes involved in (membrane) transport, vision and ophthalmic disease. More fundamentally, we found RPE-specific involvement in the RAR-activation, retinol metabolism and GABA receptor signaling pathways. CONCLUSIONS: In this study we provide a further specification and understanding of the RPE transcriptome by identifying and analyzing genes that are specifically expressed in the RPE.

Effect of zeaxanthin and antioxidant supplementation on vascular endothelial growth factor (VEGF) expression in apolipoprotein-E deficient mice.

PURPOSE: Apolipoprotein E(-/-) deficient (apoE(-/-)) mice develop hypercholesterolemia, atherosclerosis, and retinal alterations. We studied the oxidative status and vascular endothelial growth factor (VEGF) expression in murine retinal pigment epithelium-choroid (RPE) and Bruch's membrane (BM) ultrastructure and the effect of zeaxanthin. METHODS: Ten 6-month-old C57BL/6 and 40 apoE(-/-) mice were divided into four groups (n = 10 each) and fed different diets for 12 weeks based on body weight: wild type (WT) and apoE(-/-) (AE-Con) mice standard rodent chow; apoE(-/-) mice (AES) standard rodent chow with ascorbate (800 mg/kg), tocopherol (1053 mg/kg), and zinc (135 mg/kg); and apoE(-/-) mice the last diet plus zeaxanthin with either 0.4 g/kg (AES-Z04) or 4 g/kg feed (AES-Z4). RESULTS: Plasma total cholesterol (TC) and triglycerides (TG) and urine lipid peroxidation (isoprostanes) were measured. VEGF expression was determined in RPE-choroid homogenates. Zeaxanthin uptake was assessed in liver and retina by high-performance liquid chromatography; the retinal ultrastructure was analyzed by electron microscopy. AE-Con mice had higher plasma TC (p < 0.001) and TG (p < 0.001) values than WT mice. AE-Con mice had higher RPE-choroid-VEGF levels than WT mice (p < 0.05), BM thickness (p < 0.001) and presence of basal laminar deposits (BLamD). AES-Z4 resulted in lower urinary isoprostanes (p = 0.054) and lower VEGF expression in the RPE-choroid (p < 0.01). BM in the AES-Z4 animals had less confluent BLamD than AE-Con, AES, or AES-Z04 animals. CONCLUSIONS: We have reported that supplementation with zeaxanthin and antioxidants may delay or reverse alterations in the RPE and deposits in BM, and reduced VEGF expression observed in apoE(-/-) mice.

Reduced zinc and copper in the retinal pigment epithelium and choroid in age-related macular degeneration.

PURPOSE: To measure zinc and copper levels in the retinal pigment epithelium (RPE) and choroid complex and in the neural retina in subjects with and without age-related macular degeneration (AMD). DESIGN: Laboratory investigation. METHODS: Eighty-eight donor eyes (44 subjects) were analyzed. After retinal dissection, the RPE and choroid complex was photographed. Using the Minnesota Grading System (MGS), the RPE and choroid complex was classified into 1 of 4 stages as defined by the Age-Related Eye Disease Study. Subjects without AMD were defined as both eyes having MGS stage 1; subjects with AMD were defined as both eyes having MGS stages 2 through 4. Zinc and copper levels were determined by using an inductively coupled plasma-mass spectrometer. Metal levels from two eyes of the same subject were averaged and treated as one observation. Differences in metal levels were examined by using Wilcoxon rank-sum tests. RESULTS: The mean RPE and choroid complex zinc level in subjects with AMD (+/- standard deviation, 223.7 +/- 94.0 microg/g; n = 15) was reduced 24% when compared with that of subjects without AMD (292.1 +/- 98.5 microg/g; n = 29; P = .01). The mean RPE and choroid complex copper level in subjects with AMD (5.1 +/- 1.1 microg/g) was reduced 23% when compared with that of subjects without AMD (6.6 +/- 1.4 microg/g; P = .002). No difference was detected in retinal zinc and copper levels in subjects with and without AMD (P > .09). CONCLUSIONS: Reduced RPE and choroid complex zinc and copper levels in AMD eyes combined with previous information that oral supplementation of zinc plus copper reduces the risk of progression of AMD suggests that metal homeostasis plays a role in AMD and in retinal health.

Lipoprotein particles of intraocular origin in human Bruch membrane: an unusual lipid profile.

PURPOSE: Throughout adulthood, Bruch membrane (BrM) accumulates esterified cholesterol (EC) associated with abundant 60- to 80-nm-diameter lipoprotein-like particles (LLP), putative apolipoprotein B (apoB) lipoproteins secreted by the retinal pigment epithelium (RPE). In the present study, neutral lipid, phospholipids, and retinoid components of human BrM-LLP were assayed. METHODS: Particles isolated from paired choroids of human donors were subjected to comprehensive lipid profiling (preparative liquid chromatography [LC] gas chromatography [GC]), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), Western blot analysis, and negative stain electron microscopy. Results were compared to plasma lipoproteins isolated from normolipemic volunteers and to conditioned medium from RPE-J cells supplemented with palmitate to induce particle synthesis and secretion. RESULTS: EC was the largest component (32.4+/-7.9 mol%) of BrM-LLP lipids. EC was 11.3-fold more abundant than triglyceride (TG), unlike large apoB lipoproteins in plasma. Of the fatty acids (FA) esterified to cholesterol, linoleate (18:2n6) was the most abundant (41.7+/-4.7 mol%). Retinyl ester (RE) was detectable at picomolar levels in BrM-LLP. Notably scarce in any BrM-LLP lipid class was the photoreceptor-abundant FA docosahexaenoate (DHA, 22:6n3). RPE-J cells synthesized apoB and numerous EC-rich spherical particles. CONCLUSIONS: BrM-LLP composition resembles plasma LDL more than it does photoreceptors. An EC-rich core is possible for newly synthesized lipoproteins as well as those processed in plasma. Abundant EC could contribute to a transport barrier in aging and lesion formation in age-related maculopathy (ARM). Analysis of BrM-LLP composition has revealed new aspects of retinal cholesterol and retinoid homeostasis.

Inhibition of choroidal neovascularization with an anti-inflammatory carotenoid astaxanthin.

PURPOSE: Astaxanthin (AST) is a carotenoid found in marine animals and vegetables. The purpose of the present study was to investigate the effect of AST on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms. METHODS: Laser photocoagulation was used to induce CNV in C57BL/6J mice. Mice were pretreated with intraperitoneal injections of AST daily for 3 days before photocoagulation, and treatments were continued daily until the end of the study. CNV response was analyzed by volumetric measurements 1 week after laser injury. Retinal pigment epithelium-choroid levels of IkappaB-alpha, intercellular adhesion molecule (ICAM)-1, monocyte chemotactic protein (MCP)-1, interleukin (IL)-6, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)-1, and VEGFR-2 were examined by Western blotting or ELISA. AST was applied to capillary endothelial (b-End3) cells, macrophages, and RPE cells to analyze the activation of NF-kappaB and the expression of inflammatory molecules. RESULTS: The index of CNV volume was significantly suppressed by treatment with AST compared with that in vehicle-treated animals. AST treatment led to significant inhibition of macrophage infiltration into CNV and of the in vivo and in vitro expression of inflammation-related molecules, including VEGF, IL-6, ICAM-1, MCP-1, VEGFR-1, and VEGFR-2. Importantly, AST suppressed the activation of the NF-kappaB pathway, including IkappaB-alpha degradation and p65 nuclear translocation. CONCLUSIONS: AST treatment, together with inflammatory processes including NF-kappaB activation, subsequent upregulation of inflammatory molecules, and macrophage infiltration, led to significant suppression of CNV development. The present study suggests the possibility of AST supplementation as a therapeutic strategy to suppress CNV associated with AMD.

Intrinsic tissue fluorescence in an organotypic perfusion culture of the porcine ocular fundus exposed to blue light and free radicals.

BACKGROUND: A wide variety of pathological pathways may result in age-related macular degeneration. Because of its complexity, there is no comprehensive model of the disease yet. One key feature is the accumulation of the autofluorescent pigment lipofuscin in the retinal pigment epithelium (RPE). Thus, we developed an organotypic perfusion culture model of the porcine ocular fundus, generating lipofuscin under exposure to blue light and hydrogen peroxide. METHODS: Porcine fundi (choroid, Bruch's membrane, RPE, and retina) were explanted in toto, transferred into a perfusion culture chamber, perfused with cell culture medium and kept at 37 degrees C. Free radical stress was induced by supplementation of H(2)O(2), and/or the specimens were exposed to blue light, or kept untreated as controls. After a culture period of 7 days, the specimens were subject to microscopic inspection, histology, fluorescence microscopy, and measurement of fluorescence spectra as well as fluorescence decay times. RESULTS: Histology showed atrophic ganglion cells and rod outer segments. All other tissue structures were morphologically intact. Compared to the controls, RPE and retina exposed to light showed increased fluorescence, which was shifted towards shorter wavelengths. The fluorescence spectra and decays resembled that of lipofuscin granules isolated from human donor eyes. HPLC analysis revealed the abundance of the lipofuscin component N-retinylidene-N-retinylethanolamine (A2E), its precursor products, as well as two new, green-emitting fluorophores. CONCLUSIONS: Porcine ocular fundi were successfully preserved in an organotypic perfusion culture for 7 days, and exhibited remarkable autofluorescence after light and free radical exposure, making the model suitable for investigations of lipofuscinogenesis.