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.

On the origin of proteins in human drusen: The meet, greet and stick hypothesis.

Retinal drusen formation is not only a clinical hallmark for the development of age-related macular degeneration (AMD) but also for other disorders, such as Alzheimer's disease and renal diseases. The initiation and growth of drusen is poorly understood. Attention has focused on lipids and minerals, but relatively little is known about the origin of drusen-associated proteins and how they are retained in the space between the basal lamina of the retinal pigment epithelium and the inner collagenous layer space (sub-RPE-BL space). While some authors suggested that drusen proteins are mainly derived from cellular debris from processed photoreceptor outer segments and the RPE, others suggest a choroidal cell or blood origin. Here, we reviewed and supplemented the existing literature on the molecular composition of the retina/choroid complex, to gain a more complete understanding of the sources of proteins in drusen. These "drusenomics" studies showed that a considerable proportion of currently identified drusen proteins is uniquely originating from the blood. A smaller, but still large fraction of drusen proteins comes from both blood and/or RPE. Only a small proportion of drusen proteins is uniquely derived from the photoreceptors or choroid. We next evaluated how drusen components may "meet, greet and stick" to each other and/or to structures like hydroxyapatite spherules to form macroscopic deposits in the sub-RPE-BL space. Finally, we discuss implications of our findings with respect to the previously proposed homology between drusenogenesis in AMD and plaque formation in atherosclerosis.

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.

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.

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.

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.