AICAR upregulates ABCA1/ABCG1 expression in the retinal pigment epithelium and reduces Bruch's membrane lipid deposit in ApoE deficient mice.

The etiology of age-related macular degeneration (AMD) is diverse; however, recent evidence suggests that the lipid metabolism-cholesterol pathway might be associated with the pathophysiology of AMD. The ATP-binding cassette (ABC) transporters, ABCA1 and ABCG1, are essential for the formation of high-density lipoprotein (HDL) and the regulation of macrophage cholesterol efflux. The failure of retinal or retinal pigment epithelium (RPE) cholesterol efflux to remove excess intracellular lipids causes morphological and functional damage to the retina. In this study, we investigated whether treatment with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMP-activated protein kinase (AMPK) activator, improves RPE cholesterol efflux and Bruch's membrane (BM) lipid deposits. The protein and mRNA levels of ABCA1 and ABCG1 in ARPE-19 cells and retinal and RPE/choroid tissue from apolipoprotein E-deficient (ApoE-/-) mice were evaluated after 24 weeks of AICAR treatment. The cholesterol efflux capacity of ARPE-19 cells and the cholesterol-accepting capacity of apoB-depleted serum from mice were measured. The thickness of the BM and the degree of lipid deposition were evaluated using electron microscopy. AICAR treatment increased the phosphorylation of AMPK and the protein and mRNA expression of ABCA1 and ABCG1 in vitro. It promoted cholesterol efflux from ARPE-19 cells and upregulated the protein and mRNA levels of ABCA1 and ABCG1 in the retina and RPE in vivo. ApoB-depleted serum from the AICAR-treated group showed enhanced cholesterol-accepting capacity. Long-term treatment with AICAR reduced BM thickening and lipid deposition in ApoE-/- mice. In conclusion, AICAR treatment increased the expression of lipid transporters in the retina and RPE in vivo, facilitated intracellular cholesterol efflux from the RPE in vitro, and improved the functionality of HDL to accept cholesterol effluxed from the cell, possibly via AMPK activation. Collectively, these effects might contribute to the improvement of early age-related pathologic changes in the BM. Pharmacological improvement of RPE cholesterol efflux via AMPK activation may be a potential treatment strategy for AMD.

DIFFERENTIAL RESPONSE TO GLUCOCORTICOID IMMUNOSUPPRESSION OF TWO DISTINCT INFLAMMATORY SIGNS ASSOCIATED WITH PUNCTATE INNER CHOROIDOPATHY.

PURPOSE: To describe the differential response of two distinct inflammatory signs occurring in eyes with punctate inner choroidopathy. METHODS: Retrospective, observational case series using multimodal imaging. RESULTS: Four eyes of 4 myopic female patients (mean age of 35 years, range 31-42 years) presenting with retinal manifestations of punctate inner choroidopathy. All study eyes had 2 distinct signs of active disease: 1) acute focal hyperreflective lesions splitting the retinal pigment epithelium/Bruch membrane complex on optical coherence tomography which appeared hypoautofluorescent on fundus autofluorescence and 2) more diffuse areas of outer retinal disruption limited to the ellipsoid zone and interdigitation zone on optical coherence tomography and corresponding to hyperautofluorescence on fundus autofluorescence. All patients were treated with oral prednisone and demonstrated prompt regression of the retinal pigment epithelium/Bruch membrane complex lesions with a concurrent, paradoxical centrifugal expansion of outer retinal disruption. The outer retinal disruption eventually resolved in all eyes (mean time of 6 weeks, range 4-10 weeks). CONCLUSION: In patients with punctate inner choroidopathy, two distinct inflammatory signs observed with multimodal imaging display a differential response to systemic corticosteroids. Although focal inflammatory lesions splitting the retinal pigment epithelium/Bruch membrane complex seem to respond rapidly, the more diffuse, transient outer retinal disruption shows little response. This difference in treatment response may reflect different immunological phenomena with independent natural history.

The sustained delivery of resveratrol or a defined grape powder inhibits new blood vessel formation in a mouse model of choroidal neovascularization.

The objective of this study was to determine whether resveratrol or a defined, reconstituted grape powder can attenuate the formation of new blood vessels in a mouse model of choroidal neovascularization (CNV). To accomplish this objective, C57BL/6J mice were randomized into control or treatment groups which received either resveratrol or grape powder by daily oral gavage, resveratrol or grape powder delivered ad libitum through the drinking water, or resveratrol by slow release via implanted osmotic pumps. A laser was used to rupture Bruch's membrane to induce CNV which was then detected in sclerochoroidal eyecups stained with antibodies against intercellular adhesion molecule-2. CNV area was measured using fluorescence microscopy and Image J software. Ad libitum delivery of both resveratrol and grape powder was shown to significantly reduce the extent of CNV by 68% and 57%, respectively. Parallel experiments conducted in vitro demonstrated that resveratrol activates p53 and inactivates Akt/protein kinase B in choroidal endothelial cells, contributing to its anti-proliferative and anti-migratory properties. In addition resveratrol was shown to inhibit the formation of endothelial cell networks, augmenting its overall anti-angiogenic effects. The non-toxic nature of resveratrol makes it an especially attractive candidate for the prevention and/or treatment of CNV.

A rat model for studying the biological effects of circulating LDL in the choriocapillaris-BrM-RPE complex.

Retention of apolipoprotein B-containing lipoproteins in Bruch's membrane (BrM) is believed to be important in early age-related macular degeneration (AMD). The origin of the lipoproteins in BrM is a hot topic in AMD research. Some studies hypothesize an intraocular origin. BrM is in direct contact to the choriocapillaris; a plasma origin has also been suggested for the low-density lipoprotein (LDL) particles. We developed an animal model to study the biological effects of circulating LDL on the retina. After injection of LDL for 7 days, our results showed evidence of circulating apolipoprotein B100 retention in BrM and showed induction of early AMD-like alterations in the rat retina, such as thickening of BrM, photoreceptor TUNEL-positive cells, and inflammatory cell infiltration. In vitro assays showed that oxidized LDL (ox-LDL) treatment decreased ARPE-19 cell viability in a dose-dependent manner and that 10 mg/L ox-LDL induced marked apoptosis. The ratio of matrix metalloproteinase-2 to tissue inhibitors of metalloproteinase-3 was dysregulated after LDL and ox-LDL treatment in ARPE-19 cells, which can produce profound changes in the extracellular matrix, including thickening of and deposit formation in BrM. The observation that circulating LDL may be a significant, but not complete, origin of the lipoprotein in BrM suggests that these findings can be readily exploited for the development of new model systems and the future benefit of patients with AMD.

A retrospective evaluation of the effect of hydroxyquinine on RPE thickness.

BACKGROUND: To investigate a possible structural difference in the retina of hydroxychloroquine (Plaquenil)-treated patients as an explanation for the protective effect of this medication against age-related macular degeneration (AMD). METHODS: In this retrospective study, we compared the mean thickness of the retinal outer band (consisting of the Bruch's membrane and retinal pigment epithelium layer), as measured by optical coherence tomography (OCT), of 54 eyes of 27 hydroxychloroquine-treated rheumatoid arthritis patients (study group), 40 eyes of 20 healthy similar aged individuals (control group I), and 22 eyes of 11 non-hydroxychloroquine-treated rheumatoid arthritis patients (control group II). RESULTS: The mean thicknesses of the outer band of the retinal pigment epithelium layer was 60.4 ± 7.4, 43.3 ± 2.7, and 39.7 ± 3.6 µm for the study group, control group I, and control group II, respectively. P values for differences in mean thicknesses were < 0.0001 between the study group and each of the control groups, and 0.086 between the two control groups. CONCLUSION: Treatment with hydroxychloroquine was associated with increased thickness of the outer band of the retinal pigment epithelium layer. This finding may explain the protective effect of hydroxychloroquine against age-related macular degeneration (AMD).

Night blindness in Sorsby's fundus dystrophy reversed by vitamin A.

Sorsby's fundus dystrophy (SFD) is an autosomal dominant retinal degeneration caused by mutations in the tissue inhibitor of metalloproteinases-3 (TIMP3) gene. Mechanisms of the visual loss in SFD, however, remain unknown. In a SFD family with a novel TIMP3 point mutation, we tested a hypothesis that their night blindness is due to a chronic deprivation of vitamin A at the level of the photoreceptors caused by a thickened membrane barrier between the photoreceptor layer and its blood supply. Vitamin A at 50,000 IU/d was administered orally. Within a week, the night blindness disappeared in patients at early stages of disease. Nutritional night blindness is thus part of the pathophysiology of this genetic disease and vitamin A supplementation can lead to dramatic restoration of photoreceptor function.

Proteomic profiling of human retinal pigment epithelium exposed to an advanced glycation-modified substrate.

PURPOSE: The retinal pigment epithelium (RPE) and underlying Bruch's membrane undergo significant modulation during ageing. Progressive, age-related modifications of lipids and proteins by advanced glycation end products (AGEs) at this cell-substrate interface have been implicated in RPE dysfunction and the progression to age-related macular degeneration (AMD). The pathogenic nature of these adducts in Bruch's membrane and their influence on the overlying RPE remains unclear. This study aimed to identify alterations in RPE protein expression in cells exposed to AGE-modified basement membrane (AGE-BM), to determine how this "aged" substrate impacts RPE function and to map the localisation of identified proteins in ageing retina. METHODS: Confluent ARPE-19 monolayers were cultured on AGE-BM and native, non-modified BM (BM). Following 28-day incubation, the proteome was profiled using 2-dimensional gel electrophoresis (2D), densitometry and image analysis was employed to map proteins of interest that were identified by electrospray ionisation mass spectrometry (ESI MS/MS). Immunocytochemistry was employed to localise identified proteins in ARPE-19 monolayers cultured on unmodified and AGE-BM and to analyze aged human retina. RESULTS: Image analysis detected altered protein spot densities between treatment groups, and proteins of interest were identified by LC ESI MS/MS which included heat-shock proteins, cytoskeletal and metabolic regulators. Immunocytochemistry revealed deubiquitinating enzyme ubiquitin carboxyterminal hydrolase-1 (UCH-L1), which was upregulated in AGE-exposed RPE and was also localised to RPE in human retinal sections. CONCLUSIONS: This study has demonstrated that AGE-modification of basement membrane alters the RPE proteome. Many proteins are changed in this ageing model, including UCHL-1, which could impact upon RPE degradative capacity. Accumulation of AGEs at Bruch"s membrane could play a significant role in age-related dysfunction of the RPE.

Effects of chronic methamphetamine abuse on the retinal nerve fiber layer, ganglion cell layer and Bruch's membrane opening minimum rim width.

BACKGROUND: Methamphetamine (Meth) is a highly addictive and hallucinogenic agent which is used as the second most common illicit drug globally. Meth could affect the retina and optic nerve by inducing the release of vasoconstrictive agents such as endothelin 1 and induction of severe oxidative stress with accumulation of reactive oxygen species. AIM: To evaluate the effects of chronic Meth abuse on the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL) and the Bruch's membrane opening minimum rim width (MRW). METHOD: In this case-control study, we recruited 55 Meth abusers and 49 healthy individuals with mean age of 44.63 ± 0.97 and 43.08 ± 0.91 years, respectively. RNFL thickness, GCL thickness and MRW were evaluated using optical coherence tomography. RESULTS: We found statistically significant decrease in RNFL, MRW thickness in Meth abusers (P: 0.002 and P: 0.006, respectively). We did not detect statistically significant difference regarding GCL thickness between the groups (P = 0.320). Our results showed a weak but statistically significant correlation of Meth dose increment and decrement of RNFL thickness ((P: 0.005, r = -0.193) and MRW (P: 0.013, r = -0.174). We found no correlation between duration of Meth consumption with RNFL and MRW thickness (P: 0.205, r= -0.124; P: 0.771, r= -0.029, respectively). CONCLUSION: We found a statistically significant adverse association in meth abusers with RNFL thickness and MRW. These two parameters were also statistically associated with the meth dose as measured by daily dose of Meth. Although we found a decrease in the GCL thickness, it did not reach statistical significance.

Apolipoprotein A-I Mimetic Peptide L-4F Removes Bruch's Membrane Lipids in Aged Nonhuman Primates.

Purpose: Multiple evidence lines support Bruch's membrane lipid deposition as a major precursor of soft drusen and age-related macular degeneration as including a potentially treatable atherosclerosis-like progression in the subretinal pigment epithelium (RPE)-basal lamina space. We evaluated the effect of anti-inflammatory, antiatherogenic peptide L-4F on Bruch's membrane of aged nonhuman primates in a dose-escalating study. Methods: Macaca fascicularis ≥20 years of age evaluated by color fundus photography and optical coherence tomography received monocular intravitreal injections of L-4F (n = 7) or a placebo-scrambled peptide (n = 2) in 6 doses of 25 to 175 µg over 6 months. Eyes were processed for detection and masked semiquantitative assessment of macular Bruch's membrane neutral lipid (oil red O staining), esterified cholesterol (filipin histochemistry), membrane attack complex (immunofluorescence), and paramacular thickness (transmission electron microscopy). Results: Bruch's membrane neutral lipid, esterified cholesterol, and membrane attack complex were cleared and ultrastructure was improved in L-4F-injected eyes, compared to placebo-injected eyes. Fellow eyes were also affected to the same degree as the injected eyes. Punctate yellow fundus lesions without corresponding RPE elevations on optical coherence tomography correlated to RPE lipoidal degeneration (engorgement with lipid droplets), which was unchanged by this treatment. Conclusions: Clinical-stage apolipoprotein A-I mimetic peptide L-4F, delivered intravitreally in repeated doses, produced a substantial pharmacologic reduction of Bruch's membrane lipid and restoration of ultrastructure in a nonhuman primate model that exhibits an important precursor of soft drusen, if not soft drusen themselves.

[Angioid streaks in systemic disease]. / Szemfenéki angioid csíkok szisztémás betegségekben.

Angioid streaks are defined as the special morphological alteration of the fundus; the most common clinical manifestations are irregular, reddish brownish stripes around the optic nerve head or on the posterior pole. On the basis of histological examination, the cause of this phenomenon is the breaks and continuity deficiencies in the thin layer of Bruch membrane caused by the degeneration of elastic fibers. The aim of this study is to present the ocular complication of this rare entity through the description of three cases, and to draw attention to systemic diseases in the background. In our first and third cases, pseudoxanthoma elasticum (Grönblad-Strandberg syndrome) was in the background, while in our second case, hematological disease was confirmed. In our first and second cases, the ocular complication was the choroidal neovascularization, which we treated with intravitreal anti-VEGF injection. In our third case, the choroidal rupture was the ocular complication, caused by trauma. Angioid streaks on the fundus may be sub-phenomena of systemic diseases, the detection, differential diagnosis and treatment require interdisciplinary collaboration between associate physicians. Orv Hetil. 2019; 160(25): 994-1000.

Optimization of an in vitro bilayer model for studying the functional interplay between human primary retinal pigment epithelial and choroidal endothelial cells isolated from donor eyes.

OBJECTIVE: The microenvironment of outer retina is largely regulated by retinal pigment epithelium (RPE) and choroid. Damage to either of these layers lead to the development of age related macular degeneration (AMD). A simplified cell culture model that mimics the RPE/Bruch's membrane (BM) and choroidal layers of the eye is a prerequisite for elucidating the molecular mechanism of disease progression. RESULTS: We have isolated primary retinal pigment epithelial cells (hRPE) and human primary choroidal endothelial cells (hCEC) from donor eyes to construct a bilayer of hCEC/hRPE on transwell inserts. Secretion of VEGF in the insert grown bilayer was significantly higher (22 pg/ml) than hCEC monolayer (3 pg/ml). To mimic the disease condition the model was treated with 100 ng/ml of VEGF, which increased the permeability of bilayer for 20 kDa FITC dextran while addition of bevacizumab, a humanized anti-VEGF drug, reversed the effect. To conclude the transwell insert based human primary hCEC/hRPE bilayer model would be an ideal system for studying the disease mechanisms and the crosstalk between RPE and choroid. This model will also be useful in screening small molecules and performing drug permeability kinetics.

ApoA-I Mimetic Peptide 4F Reduces Age-Related Lipid Deposition in Murine Bruch's Membrane and Causes Its Structural Remodeling.

PURPOSE: Accumulation of lipoprotein-derived lipids including esterified and unesterified cholesterol in Bruch's membrane of human eyes is a major age-related change involved in initiating and sustaining soft drusen in age-related macular degeneration (AMD). The apolipoprotein (apo) A-I mimetic peptide 4F is a small anti-inflammatory and anti-atherogenic agent, and potent modifier of plasma membranes. We evaluated the effect of intravitreally-injected 4F on murine Bruch's membrane. METHODS: We tested single intravitreal injections of 4F doses (0.6 µg, 1.2 µg, 2.4 µg, and placebo scrambled peptide) in ApoEnull mice ≥10 months of age. After 30 days, mice were euthanized. Eyes were processed for either direct immunofluorescence detection of esterified cholesterol (EC) in Bruch's membrane whole mounts via a perfringolysin O-based marker linked to green fluorescent protein or by transmission electron microscopic visualization of Bruch's membrane integrity. Fluorescein isothiocyanate-conjugated 4F was traced after injection. RESULTS: All injected eyes showed a dose-dependent reduction of Bruch's membrane EC with a concomitant ultrastructural improvement compared to placebo treated eyes. At a 2.4 µg dose of 4F, EC was reduced on average by ~60% and Bruch's membrane returned to a regular pentalaminar structure and thickness. Tracer studies confirmed that injected 4F reached intraocular targets. CONCLUSION: We demonstrated a highly effective pharmacological reduction of EC and restoration of Bruch's membrane ultrastructure. The apoA-I mimetic peptide 4F is a novel way to treat a critical AMD disease process and thus represents a new candidate for treating the underlying cause of AMD.

Bone marrow-derived cells home to and regenerate retinal pigment epithelium after injury.

PURPOSE: To determine whether hematopoietic stem and progenitor cells (HSCs/HPCs) can home to and regenerate the retinal pigment epithelium (RPE) after induced injury. METHODS: Enriched HSCs/HPCs from green fluorescent protein (gfp) transgenic mice were transplanted into irradiated recipient mice to track bone marrow-derived cells. Physical damage was induced by breaching Bruch's membrane and inducing vascular endothelial growth factor A (VEGFa) expression to promote neovascularization. RPE damage was also induced by sodium iodate injection (40 mg/kg) into wild-type or albino C57Bl/6 mice. Cell morphology, gfp expression, the presence of the Y chromosome, and the presence of melanosomes were used to determine whether the injured RPE was being repaired by the donor bone marrow. RESULTS: Injury to the RPE recruits HSC/HPC-derived cells to incorporate into the RPE layer and differentiate into an RPE phenotype. A portion of the HSCs/HPCs adopt RPE morphology, express melanosomes, and integrate into the RPE without cell fusion. CONCLUSIONS: HSCs/HPCs can migrate to the RPE layer after physical or chemical injury and regenerate a portion of the damaged cell layer.

Cigarette smoke-related oxidants and the development of sub-RPE deposits in an experimental animal model of dry AMD.

PURPOSE: Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to be an important injury stimulus relevant to the accumulation of subretinal deposits in age-related macular degeneration (AMD). Cigarette smoking is a major risk factor for AMD, and cigarette smoke-related tar contains high concentrations of a potent oxidant, hydroquinone (HQ). This study was an investigation of the effects of cigarette smoke (CS) and HQ in the development of sub-RPE deposits in an experimental mouse model. METHODS: Sixteen-month-old C57BL/6 female mice were fed a high-fat diet (HFD) for 4.5 months. Mice were divided into two major experimental groups, one to examine the effects of cigarette smoke and one to study the effects of a defined cigarette smoke component such as HQ. In the first group, mice eyes were exposed to blue-green light (positive controls) or to whole cigarette smoke. A third group with no intervention served as the negative control. In the second experimental group, animals received a purified diet with HQ (0.8%) with low or high fat content for 4.5 months. Mice in both groups were euthanatized at 4.5 months and eyes processed for transmission electron microscopy. RESULTS: As previously demonstrated by our laboratory and others, most mice fed an HFD without other oxidant exposure demonstrated normal morphology or, in a few cases, small nodular basal laminar deposits. Eyes of mice exposed to whole cigarette smoke or to HQ in the food demonstrated a variable degree of basal laminar deposits and diffusely thickened Bruch's membrane. The choriocapillaris endothelium was variably hypertrophic. CONCLUSIONS: Exposure to cigarette smoke or the smoke-related redox molecule, HQ, results in the formation of sub-RPE deposits, thickening of Bruch's membrane, and accumulation of deposits within Bruch's membrane. Smoke-related oxidants may be another oxidative injury stimulus to the choriocapillaris and RPE, and may explain the association between cigarette smoking and early AMD.

Modulating the Transport Characteristics of Bruch's Membrane With Steroidal Glycosides and its Relevance to Age-Related Macular Degeneration (AMD).

PURPOSE: Beneficial expectations of supplement therapies to increase the transport of nutrients, vitamins, and antioxidants across Bruch's membrane in AMD, by mass action alone, remain inconclusive. Therefore, the potential for targeting the transport pathways themselves to improve bidirectional exchange using amphipathic steroidal glycosides (ginsenosides) has been investigated. METHODS: Bruch's choroid preparations were mounted in modified Ussing chambers and basal levels of hydraulic conductivity (23 donors, age range, 12-89 years) and diffusional transport of FITC-albumin (21 donors, age range, 12-92 years) quantified. Then, following a 24-hour incubation with ginsenoside preparations, the transport parameters were re-evaluated and the resulting data analyzed with respect to aging and modulation by ginsenosides. RESULTS: Basal hydraulic conductivity of Bruch's showed an age-related exponential decline with a half-life of 19 years. Incubation with ginsenosides improved hydraulic conductivity with levels equivalent to donors 19 years younger. Across the age range examined, hydraulic conductivities were increased to 2.05-fold ± 0.38 (P < 0.001) of basal values. Diffusional transport of albumin across Bruch's also showed an age-related exponential decline with a half-life of 18 years. The decay curves were elevated on incubation with ginsenosides and diffusional rates were equivalent to donors 15 years younger. Diffusional rates were elevated 2.01-fold ± 0.49 over basal values (P < 0.001). CONCLUSIONS: Transport characteristics of human Bruch's can be improved by ginsenosides, facilitating the bidirectional exchange of nutrients and waste products across the membrane. With improved transport pathways, the need for supplement therapies becomes redundant. Slowed aging of Bruch's is expected to delay the onset and/or progression of AMD.

Repopulation of different layers of host human Bruch's membrane by retinal pigment epithelial cell grafts.

PURPOSE: To determine the morphology of human retinal pigment epithelium (RPE) after reattachment to different ultrastructural layers of human Bruch's membrane (BM). METHODS: Bruch's membrane explants were prepared from eyes of 23 human donors (age range, 11-89 years). The basal lamina of the RPE, inner collagenous layer, and elastin layer were removed sequentially by mechanical and enzymatic techniques. First-passage cells of human RPE (15,000 cells/6 mm explant) from three donors (ages, 52, 64, and 80 years) were plated onto different layers of human BM, and the explants were examined by scanning and transmission electron microscopy up to 21 days later. RESULTS: RPE flattened and extended footplates 6 hours after plating onto basal lamina. Cells remained round 6 and 24 hours after plating onto the inner collagenous, elastin, or outer collagenous layer. The RPE cells became confluent 14 days after plating onto basal lamina but did not become confluent up to 21 days after plating onto the inner collagenous or elastin layer. Sparse round cells were observed 21 days after plating onto deeper layers, suggesting extensive loss of RPE. CONCLUSIONS: The morphology and subsequent behavior of the RPE reattached to BM depends on the anatomic layer of BM available for cell reattachment. The results suggest that the ability of transplanted RPE to repopulate BM in age-related macular degeneration and other disorders may depend on the layer of BM available to serve as a substrate for cell reattachment.

Ultrastructural aging of the RPE-Bruch's membrane-choriocapillaris complex in the D-galactose-treated mouse.

PURPOSE: Low-dose D-galactose treatment in mice induces accelerated aging due to advanced glycation endproduct (AGEs) formation. The purpose of this study was to identify ultrastructural aging in the retinal pigment epithelium (RPE)-Bruch's membrane-choriocapillaris. METHODS: Five-month-old C57Bl6 mice were injected daily with D-galactose or control buffer for 8 weeks. Eighteen-month-old mice were also treated with control buffer for 8 weeks. Eyes were prepared for electron microscopy and AGE-specific fluorescence at ex = 370 nm/em = 440 nm and ex = 330 nm/ex = 390 nm. RESULTS: D-Galactose treatment induced AGE-specific fluorescence in lens and RPE/choroid compared to buffer-treated controls. In D-galactose-treated animals, the RPE had dilated and fewer basolateral infoldings. Bruch's membrane had alterations that included significant thickening, sub-RPE and prominent outer collagenous layer deposits, and choriocapillaris basement membrane duplication/splitting and thickening. The choriocapillaris endothelium displayed fenestration loss. CONCLUSIONS: Ultrastructural aging to the RPE-Bruch's membrane-choriocapillaris developed in mice treated with low-dose D-galactose. These changes could contribute to age-related changes that promote early age-related disease.

Maculas affected by age-related macular degeneration contain increased chelatable iron in the retinal pigment epithelium and Bruch's membrane.

OBJECTIVE: To investigate whether iron is involved in the pathogenesis of age-related macular degeneration (AMD). METHODS: Postmortem AMD-affected (nonexudative or exudative) and healthy maculas were studied using the 3,3'-diaminobenzidine-enhanced Perls Prussian blue stain. The Perls Prussian blue stain was quantified by computer-assisted analysis of digital images. To determine whether the iron was chelatable, sections treated with the iron chelator deferoxamine were compared with adjacent, nonchelated sections. RESULTS: Compared with healthy maculas, AMD-affected maculas had statistically significant increases in the total iron level. Some of this iron was chelatable. The iron was present in retinal pigment epithelium and Bruch's membrane in maculas from patients who had drusen only, geographic atrophy, and exudative AMD in pathologic areas and, occasionally, in relatively healthy areas. CONCLUSIONS: Oxidative stress has been implicated in the pathogenesis of AMD by the Age-Related Eye Disease Study. Increased concentrations of iron, which generate highly reactive hydroxyl radicals via the Fenton reaction, may induce oxidative stress in the macula and lead to AMD. As the increased iron concentrations in AMD-affected eyes consist in part of a chelatable iron pool, treatment of patients who have AMD with iron chelators might be considered a potential therapy. While there are, as yet, no clinical data indicating that the treatment of patients who have AMD with iron chelators is beneficial, data presented herein indicate that further investigation of iron concentrations in postmortem tissues and the mechanisms of iron transport in the retina is warranted.

Extracellular matrix ligands promote RPE attachment to inner Bruch's membrane.

PURPOSE: To determine if resurfacing layers of human Bruch's membrane with extracellular matrix ligands increases adult human retinal pigment epithelium (RPE) attachment. METHODS: We removed successive layers of Bruch's membrane sequentially by mechanical or enzymatic means to expose the RPE basal lamina, inner collagen layer, and elastin layer. Each layer was coated with extracellular matrix proteins (4 microg/cm(2) laminin, 4 microg/cm(2) vitronectin, 20 microg/cm(2) fibronectin, 20 microg/cm(2) collagen IV) for 2 hrs at 37 degrees C. First passage adult human RPE (15000 cells/6 mm explant) were plated onto coated or uncoated Bruch's membrane layers and RPE attachment was measured 18 hrs later. RESULTS: The reattachment rate to basal lamina derived from younger (age < 50) donors was higher than older (age >/= 70) donors (56.2 +/- 4.8% versus 46.7 +/- 0.7% respectively; p < 0.05). Coating with laminin or collagen IV increased RPE attachment to older but not younger basal lamina. The reattachment rate to inner collagen derived from younger donors was higher than older donors (51.3 +/- 2.7% versus 37.4 +/- 1.7% respectively, p < 0.05). Coating with laminin, fibronectin or collagen IV increased RPE attachment to older but not younger inner collagen layer. Adding extracellular matrix ligands did not increase RPE attachment to elastin. CONCLUSIONS: RPE attachment is increased by adding ligands to the basal lamina or inner collagen layer but not to the elastin layer. This may be important if the elastin layer is exposed in AMD patients after submacular surgery.

Effects of simvastatin on retinal structure and function of a high-fat atherogenic mouse model of thickened Bruch's membrane.

PURPOSE: To determine the effect of a statin (simvastatin) on the ultrastructure and function of the RPE, Bruch's membrane (BM), and photoreceptor interface in a high-fat atherogenic mouse model of thickened BM. METHODS: Wild-type C57BL/6 mice (6-weeks old) were divided into three study groups according to their diet and treatment given; Group 1, normal chow diet-fed mice; Group 2, high fat diet (HFD) fed mice; and Group 3, HFD-fed mice treated with simvastatin daily for 30 weeks. All mice were followed-up for 30 weeks. The retinal morphology and function was examined in vivo using fundus imaging and electroretinography at 15- and 30-weeks follow-up. At the end of the study, at 36 weeks of age, eye tissues were collected and retinal sections were examined using light microscopy and transmission electron microscopy. RESULTS: Fundus images of the HFD-fed mice showed the presence of discrete, multiple white spots, which was significantly reduced by approximately 73% in the simvastatin-treated animals. In the HFD-fed mice, there was an increase in the empty cytoplasmic vacuoles of the RPE, presence of lipid droplets in the BM, thickening and fragmentation of the elastic lamina of the BM, and a reduction in retinal function; these ultrastructural and functional changes were significantly improved in the simvastatin-treated group. CONCLUSIONS: Chronic administration of simvastatin significantly improves the ultrastructure and function of the RPE, BM, and photoreceptor in a high-fat atherogenic mouse model of thickened BM.