Mouse Choroid Proteome Revisited: Focus on Aging.

Age is a major risk factor for age-related macular degeneration (AMD), and age has a role in the disease phenotypes of heritable macular dystrophies. The proteomes of C57Bl6/J mouse choroids at 2 ages were analyzed to identify biochemical processes affected by aging. Proteins of interest were identified as those contributing most to the variance in principal component analysis and those showing the largest significant differences between ages. These proteins implicated altered ECM composition, immune system function, and lipid metabolism.

Nanocarriers, Progenitor Cells, Combinational Approaches, and New Insights on the Retinal Therapy.

Progenitor cells derived from the retinal pigment epithelium (RPECs) have shown promise as therapeutic approaches to degenerative retinal disorders including diabetic retinopathy, age-related macular degeneration and Stargardt disease. However, the degeneration of Bruch's membrane (BM), the natural substrate for the RPE, has been identified as one of the major limitations for utilizing RPECs. This degeneration leads to decreased support, survival and integration of the transplanted RPECs. It has been proposed that the generation of organized structures of nanofibers, in an attempt to mimic the natural retinal extracellular matrix (ECM) and its unique characteristics, could be utilized to overcome these limitations. Furthermore, nanoparticles could be incorporated to provide a platform for improved drug delivery and sustained release of molecules over several months to years. In addition, the incorporation of tissue-specific genes and stem cells into the nanostructures increased the stability and enhanced transfection efficiency of gene/drug to the posterior segment of the eye. This review discusses available drug delivery systems and combination therapies together with challenges associated with each approach. As the last step, we discuss the application of nanofibrous scaffolds for the implantation of RPE progenitor cells with the aim to enhance cell adhesion and support a functionally polarized RPE monolayer.

Role of retinal pigment epithelium in age-related macular disease: a systematic review.

Age-related macular disease (AMD) is a major cause of blindness and there is little treatment currently available by which the progress of the basic disorder can be modulated. Histological and clinical studies show that the major tissues involved are the outer retina, retinal pigment epithelium, Bruch's membrane and choroid. Because of a wide variation of phenotype from one case to another, it has been suggested that accurate phenotyping would be necessary for assessment of the effectiveness of treatment that is tissue-directed. However, based on findings from the study of human donor material and animal models of disease and of cell culture, it is concluded that retinal pigment epithelial dysfunction plays a central role in the disease process in most, if not all, cases of early AMD. The metabolism of phagosomal material, particularly lipids, and energy generation are interdependent, and dysfunction of both appears to be important in the genesis of disease. Evidence exists to suggest that both can be modulated therapeutically. These metabolic functions are amenable to further investigation in both the normal state and in disease. Once fully characterised, it is likely that treatment could be directed towards a limited number of functions in single tissue, thus simplifying treatment strategies.

Visual Field Cluster Map Corresponding to Bruch Membrane Opening-minimum Rim Area Sectors in Open-angle Glaucoma.

PRéCIS:: We generated a new visual field (VF) cluster map corresponding to Bruch membrane opening-minimum rim area (BMO-MRA) sectors, which described in detail the structure-function relationships between the optic nerve head and VF in patients with open-angle glaucoma. PURPOSE: The purpose of this study was to investigate the structure-function relationship between BMO-MRA and VF in patients with open-angle glaucoma. MATERIALS AND METHODS: We retrospectively reviewed 67 eyes of 50 patients with open-angle glaucoma who underwent spectral-domain optical coherence tomography for BMO-MRA and the Humphrey VF test. BMO-MRA of the glaucomatous optic nerve head was divided into 12 sectors. The correlation between BMO-MRA sectors and the VF points was analyzed to generate a new VF cluster map. RESULTS: Forty-three of the 52 VF points showed a significant correlation with at least 1 BMO-MRA sector. The VF cluster map was generated using the BMO-MRA sectors and each VF point that showed the most correlation. The superior hemifield correlated with 5, 6, 7, and 8 o'clock positions (ρ=0.312 to 0.710), whereas the inferior hemifield correlated with 10, 11, 12, and 2 o'clock positions (ρ=0.241 to 0.483). The VF cluster maps of superior and inferior hemifields showed different configurations of VF clusters and topographical relationships with the glaucomatous optic nerve head. CONCLUSION: The newly generated VF cluster map corresponding to BMO-MRA sectors showed a significant structure-function relationship and could be useful in the diagnosis and evaluation of glaucoma.

Role of Fibulins 2 and 5 in Retinal Development and Maintenance.

Fibulins 2 and 5 are part of a seven-member family of proteins integral to the retinal extracellular matrix. Our study aimed to further explore the roles of both fibulins in retinal function. We obtained knockout mouse models of both fibulins and performed immunohistochemistry, electroretinography, and histology to investigate the outcome of eliminating these proteins. Immunohistochemical analysis showed that both fibulins are localized to the RPE, choroid, and Bruch's membrane. Functional testing showed a significantly reduced scotopic A response at 1 month of age, when compared to their wild-type counterpart. This functional reduction remained constant throughout the age of the animal and only declined as a result of normal aging. The functional decline was associated with reduced number of photoreceptor cells. The results presented clearly demonstrate that fibulins 2 and 5, as extracellular proteins, are necessary for normal retinal development.

The glycation of fibronectin by glycolaldehyde and methylglyoxal as a model for aging in Bruch's membrane.

The purpose of the study is to identify the sites of modification when fibronectin reacts with glycolaldehyde or methylglyoxal as a model system for aging of Bruch's membrane. A synthetic peptide consisting of the α5ß1 integrin binding region of fibronectin was incubated with glycolaldehyde for 12 h or with methylglyoxal for 1 h at 37 °C. After tryptic digestion, the samples were analyzed with liquid chromatography-mass spectrometry (LC/MS). Tandem MS was used to determine the sites of modification. The adducts, aldoamine and N (ε)-carboxymethyl-lysine, attached preferably at lysine residues when the fibronectin peptide reacted with glycolaldehyde. When the fibronectin peptide reacted with methylglyoxal, modifications occurred at lysine and arginine residues. At lysine residues, N (ε)-carboxyethyl-lysine adducts were present. At arginine residues, hydroimidazolone and tetrapyrimidine adducts were present. Several advanced glycation endproducts were generated when fibronectin was glycated via glycolaldehyde and methylglyoxal. These results can help explain the structural changes Bruch's membrane undergoes during aging.

The Rate of Vitamin A Dimerization in Lipofuscinogenesis, Fundus Autofluorescence, Retinal Senescence and Degeneration.

One of the earliest events preceding several forms of retinal degeneration is the formation and accumulation of vitamin A dimers in the retinal pigment epithelium (RPE) and underlying Bruch's membrane (BM). Such degenerations include Stargardt disease, Best disease, forms of retinitis pigmentosa, and age-related macular degeneration (AMD). Since their discovery in the 1990's, dimers of vitamin A, have been postulated as chemical triggers driving retinal senescence and degeneration. There is evidence to suggest that the rate at which vitamin A dimerizes and the eye's response to the dimerization products may dictate the retina's lifespan. Here, we present outstanding questions, finding the answers to which may help to elucidate the role of vitamin A dimerization in retinal degeneration.

Age-related macular degeneration and the role of the complement system.

Age-related macular degeneration (AMD) is a leading cause of visual impairment. It is characterised by damage to a tissue complex composed of the retinal pigment epithelium, Bruch's membrane and choriocapillaris. In early AMD extracellular debris including drusen accumulates in Bruch's membrane and then in late AMD geographic atrophy and/or neovascularisation develop. Variants in genes encoding components of the alternative pathway of the complement cascade have a major influence on AMD risk, especially at the RCA locus on chromosome 1, which contains CFH and the CFHR genes. Immunohistochemical studies have demonstrated complement components in unaffected and AMD macular tissue. Whilst other factors, including oxidative stress, play important roles in AMD pathogenesis, evidence for the central role played by complement dysregulation is discussed in this review.

A novel Bruch's membrane-mimetic electrospun substrate scaffold for human retinal pigment epithelium cells.

Various artificial membranes have been used as scaffolds for retinal pigment epithelium cells (RPE) for monolayer reconstruction, however, long-term cell viability and functionality are still largely unknown. This study aimed to construct an ultrathin porous nanofibrous film to mimic Bruch's membrane, and in particular to investigate human RPE cell responses to the resultant substrates. An ultrathin porous nanofibrous membrane was fabricated by using regenerated wild Antheraea pernyi silk fibroin (RWSF), polycaprolactone (PCL) and gelatin (Gt) and displayed a thickness of 3-5 µm, with a high porosity and an average fiber diameter of 166 ± 85 nm. Human RPE cells seeded on the RWSF/PCL/Gt membranes showed a higher cell growth rate (p < 0.05), and a typical expression pattern of RPE signature genes, with reduced expression of inflammatory mediators. With long-term cultivation on the substrates, RPE cells exhibited characteristic polygonal morphology and development of apical microvilli. Immunocytochemisty demonstrated RPE-specific expression profiles in cells after 12-weeks of co-culture on RWSF/PCL/Gt membranes. Interestingly, the cells on the RWSF/PCL/Gt membranes functionally secreted polarized PEDF and phagocytosed labeled porcine POS. Furthermore, RWSF/PCL/Gt membranes transplanted subsclerally exhibited excellent biocompatibility without any evidence of inflammation or rejection. In conclusion, we established a novel RWSF-based substrate for growth of RPE cells with excellent cytocompatibility in vitro and biocompatibility in vivo for potential use as a prosthetic Bruch's membrane for RPE transplantation.

The proteomics of drusen.

The formation of extracellular deposits known as drusen below the macular region of the retina correlates with increased risk of severe visual loss from age-related macular degeneration (AMD). Inflammation and complement dysregulation contribute to AMD progression; however, disease mechanisms remain incompletely defined. Multiple genetic and environmental factors influence AMD pathology, and although immune system processes play a central role, multiple molecular mechanisms appear to be involved. Drusen proteomics, including the analyses of constituent proteins, oxidative protein modifications, and pattern recognition receptors, provide a foundation for deciphering mechanisms of drusen biogenesis and AMD pathology.

Developing methacrylate-based copolymers as an artificial Bruch's membrane substitute.

Age-related macular degeneration (AMD) is the most common cause of blindness in the developed world. There is currently no treatment for the cellular loss, which is characteristic of AMD. Transplantation of retinal pigment epithelium (RPE) cells represents a potential therapy. Because of AMD-related pathology in the native support, Bruch's membrane, transplanted RPE cells require a scaffold to reside on. We present here the development of an electrospun fibrous scaffold derived from methyl methacrylate and poly(ethylene glycol) (PEG) methacrylate for novel application as an RPE scaffold. Scaffolds were chemically modified to improve cell adhesion by functionalization not previously reported for this type of copolymer system. A human RPE cell line was used to investigate cell-scaffold interactions for up to two weeks in vitro. Scanning electron microscopy was used to characterize the fibrous scaffolds and confirm cell attachment. By day 15, cell area was significantly (p < 0.001) enhanced on scaffolds with chemical modification of the PEG chain terminus. In addition, significantly, less-apoptotic cell death was demonstrable on these modified surfaces.

Heparan sulfate, including that in Bruch's membrane, inhibits the complement alternative pathway: implications for age-related macular degeneration.

An imbalance between activation and inhibition of the complement system has been implicated in the etiologies of numerous common diseases. Allotypic variants of a key complement fluid-phase regulatory protein, complement factor H (CFH), are strongly associated with age-related macular degeneration (AMD), a leading cause of worldwide visual dysfunction, although its specific role in AMD pathogenesis is still not clear. CFH was isolated from individuals carrying combinations of two of the nonsynonymous coding variants most strongly associated with AMD risk, V62/H402 (risk haplotype variants), I62/Y402 (nonrisk haplotype variants), and V62/Y402. These proteins were used in two functional assays (cell surface- and fluid-phase-based) measuring cofactor activity of CFH in the factor I-mediated cleavage of C3b. Although no variant-specific differences in the cofactor activity were detected, when heparan sulfate (HS) was added to these assays, it accelerated the rate of C3b cleavage, and this effect could be modulated by degree of HS sulfation. Bruch's membrane/choroid, a site of tissue damage in AMD, contains high concentrations of glycosaminoglycans, including HS. Addition of human Bruch's membrane/choroid to the fluid-phase assay accelerated the C3b cleavage, and this effect was lost posttreatment of the tissue with heparinase III. Binding of CFH variants to Bruch's membrane/choroid isolated from elderly, non-AMD donor eyes, was similar, as was the functional activity of bound CFH. These findings refine our understanding of interactions of HS and complement and support the hypothesis that these interactions play a role in the transition between normal aging and AMD in Bruch's membrane/choroid.

Histochemistry and lipid profiling combine for insights into aging and age-related maculopathy.

Aging is the major risk factor for age-related maculopathy (ARM), the biggest cause of vision loss among the elderly in industrialized societies, and a major change in the affected tissues is the age-related accumulation of neutral lipid in Bruch's membrane (BrM) of the eye throughout adulthood. Here we show that esterified cholesterol (EC) is the major neutral lipid species in this tissue, which has implications for potential sources of this material. The combination of filipin histochemistry and comprehensive lipid profiling made possible this insight on a complex tissue.

Modifications to the basement membrane protein laminin using glycolaldehyde and A2E: a model for aging in Bruch's membrane.

In a variety of retinal diseases, including age-related macular degeneration (AMD); basement membranes are susceptible to alterations in structure and function. Chemical modifications to basement membrane proteins may deleteriously affect Bruch's membrane leading to the development of AMD. The purpose of this study was to investigate modifications from glycolaldehyde and A2E, which are present in the retinal pigment epithelium (RPE), on the membrane like protein fragment, laminin, as a model for aging of Bruch's membrane in age related eye diseases. Laminin was allowed to react with either glycolaldehyde or A2E during irradiation of A2E and then tryptically digested before analysis with electrospray ionization mass spectrometry (ESI-MS). Modifications to laminin occurred preferentially on lysine or arginine residues. The A2E modified laminin fragments are consistent with additions of A2E derived aldehydes resulting from cleavages closest to the pyridinium ring in A2E and oxidized A2E. These results provide evidence that A2E and advanced glycation endproducts (AGE) may be involved in modifications to essential basement membrane proteins leading to deleterious changes in the retinal pigment epithelium extracellular matrix (RPE-ECM) environment. These preliminary experiments are essential for the identification of these modifications in vivo.

Collagen-binding peptide interaction with retinal tissue surfaces.

One of the current challenges in treating age-related macular degeneration (AMD) is the surface modification of the retinal Bruch membrane. In this study, the collagen fibers of the inner collagenous zone of the Bruch membrane were identified as type I and type III. Subsequently, the adsorption of a collagen-binding peptide onto the inner collagenous zone surface was investigated. The collagen-binding peptide was able to bind specifically to the collagen fibers while maintaining the biological activity of the N-terminus biotin tag. These results indicate that the collagen-binding peptide may be used as an anchor to immobilize bioactive molecules on the inner collagenous zone surface of the Bruch membrane.

Synthesis of complement factor H by retinal pigment epithelial cells is down-regulated by oxidized photoreceptor outer segments.

Complement activation is thought to be involved in the pathogenesis of age-related macular degeneration (AMD), in part because certain gene polymorphisms in complement factor H (CFH), an important regulator of the alternative complement activation pathway, are high risk factors for AMD. How CFH is regulated locally at the retina/choroid interface and how this contributes to AMD development remain unknown. In the present study, we have confirmed that CFH was detectable by immunohistochemistry in the choroid, and at low levels in the RPE cell and interphotoreceptor matrix, but appeared to be concentrated in dense patches in Bruch's membrane. In vitro, cultured human and mouse RPE cells expressed high levels of CFH as evidenced by immunohistochemistry and western blot. Using a stabilized mouse RPE cell line, we confirmed that RPE cells constitutively synthesise CFH. Synthesis of CFH was not affected by a short-term (2 h) photoreceptor outer segment (POS) treatment. However, long-term (24-48 h) treatment of RPE cells with oxidised POS (ox-POS) but not normal POS (n-POS) markedly down-regulated CFH mRNA expression. Phagocytosis of both ox-POS and n-POS appeared to reduce intracellular CFH protein expression in RPE cultures. Synthesis of CFH by cultured RPE cells was also reduced at the mRNA level by the proinflammatory cytokines TNF-alpha and IL-6. Other cytokines tested including IFN-gamma, IL-1alpha and IL-4 showed no effect on either CFH protein or mRNA levels. Our results support the view that RPE cells synthesise and express CFH and are probably a major local source of this protein at the retina/choroid interface, secreting CFH into the interphotoreceptor matrix as well as Bruch's membrane. Prolonged phagocytosis of POS, particularly if modified by oxidative processes as occurs in inflammation, appears to markedly impair synthesis and secretion of CFH, with potential loss of important regulatory functions in counteracting the pro-inflammatory effects of activated complement.

Carboxyethylpyrrole oxidative protein modifications stimulate neovascularization: Implications for age-related macular degeneration.

Choroidal neovascularization (CNV), the advanced stage of age-related macular degeneration (AMD), accounts for >80% of vision loss in AMD. Carboxyethylpyrrole (CEP) protein modifications, uniquely generated from oxidation of docosahexaenoate-containing lipids, are more abundant in Bruch's membrane from AMD eyes. We tested the hypothesis that CEP protein adducts stimulate angiogenesis and possibly contribute to CNV in AMD. Human serum albumin (HSA) or acetyl-Gly-Lys-O-methyl ester (dipeptide) were chemically modified to yield CEP-modified HSA (CEP-HSA) or CEP-dipeptide. The in vivo angiogenic properties of CEP-HSA and CEP-dipeptide were demonstrated by using the chick chorioallantoic membrane and rat corneal micropocket assays. Low picomole amounts of CEP-HSA and CEP-dipeptide stimulated neovascularization. Monoclonal anti-CEP antibody neutralized limbal vessel growth stimulated by CEP-HSA, whereas anti-VEGF antibody was found to only partially neutralize vessel growth. Subretinal injections of CEP-modified mouse serum albumin exacerbated laser-induced CNV in mice. In vitro treatments of human retinal pigment epithelial cells with CEP-dipeptide or CEP-HSA did not induce increased VEGF secretion. Overall, these results suggest that CEP-induced angiogenesis utilizes VEGF-independent pathways and that anti-CEP therapeutic modalities might be of value in limiting CNV in AMD.

Characterization of collagen fibers in Bruch's membrane using chemical force microscopy.

Bruch's membrane is a layer composed of collagen fibers located just beneath the retina. This study validates a strategy used to map the morphological and adhesion characteristics of collagen fibers in Bruch's membrane. Atomic force microscopy tips were functionalized with different chemical groups and used to map the hydrophilic and hydrophobic regions on the surface of the eye tissue. The largest adhesion forces were observed when tips functionalized with NH(2) groups were used. The trend in the adhesion forces was rationalized based on the distribution of different functional groups in the triple-helical structure of the collagen fibers. The results of this study can be used to design more effective strategies to treat eye diseases such as age-related macular degeneration.

Expression of endostatin in human choroidal neovascular membranes secondary to age-related macular degeneration.

Endostatin is an endogenous angiogenesis inhibitor which requires E-selectin for its antiangiogenic activity. The aim of this study was to investigate the expression of endostatin in human choroidal neovascular membranes (CNV) secondary to age-related macular degeneration (AMD) with regard to vascularization and proliferative activity. An interventional case series of 36 patients who underwent removal of CNV were retrospectively investigated. Thirty-six CNV were analyzed by light microscopic immunohistochemistry for the expression of CD34 (endothelial cells, EC), CD105 (activated EC), Ki-67 (cell proliferation), Cytokeratin 18 (epithelial cells), VEGF (vascular endothelial growth factor), E-selectin and endostatin. Donor eyes (n=7) including one with AMD were used as controls. Endostatin immunoreactivity was present in choroidal vessels of five as well as in the retinal pigment epithelium (RPE)-Bruch's membrane complex of two donor eyes without AMD. In one eye with AMD, endostatin was detected in RPE, Bruch's membrane and choroidal vessels. Ninety-two percent (33/36) of CNV disclosed endostatin staining. RPE-Bruch's membrane complex, choroidal vessels and stroma were positive in 50% (18/36), 72% (26/36), and 78% (28/36) of the membranes, respectively. Both control eyes and CNV expressed all the investigated markers except E-selectin being positive only in membranes. Endostatin, an endogenous angiogenesis inhibitor, is expressed in CNV and its therapeutic up-regulation may be a new strategy in the treatment of neovascular AMD.

Pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) in aged human choroid and eyes with age-related macular degeneration.

The purpose of this study was to examine the localization and relative levels of vascular endothelial growth factor (VEGF; an angiogenic factor) and pigment epithelium-derived factor (PEDF; an antiangiogenic factor) in aged human choroid and to determine if the localization or their relative levels changed in age-related macular degeneration (AMD). Ocular tissues were obtained from eight aged control donors (age range, 75-86 years; mean age, 79.8 years) with no evidence or history of chorioretinal disease and from 12 donors diagnosed with AMD (age range, 61-105 years; mean age, 83.9 years). Tissues were cryopreserved and streptavidin alkaline phosphatase immunohistochemistry was performed with rabbit polyclonal anti-human VEGF and rabbit polyclonal anti-human PEDF antibodies. Binding of the antibodies was blocked by preincubation of the antibody with an excess of recombinant human PEDF or VEGF peptide. Choroidal blood vessels were identified with mouse anti-human CD-34 antibody in adjacent tissue sections. Three independent observers graded the immunohistochemical reaction product. The most prominent sites of VEGF and PEDF localization in aged control choroid were RPE-Bruch's membrane-choriocapillaris complex including RPE basal lamina, intercapillary septa, and choroidal stroma. There was no significant difference in immunostaining intensity and localization of VEGF and PEDF in aged control choroids. The most intense VEGF immunoreactivity was observed in leukocytes within blood vessels. AMD choroid had a similar pattern and intensity of VEGF immunostaining to that observed in aged controls. However, PEDF immunoreactivity was significantly lower in RPE cells (p=0.0073), RPE basal lamina (p=0.0141), Bruch's membrane (p<0.0001), and choroidal stroma (p=0.0161) of AMD choroids. The most intense PEDF immunoreactivity was observed in disciform scars. Drusen and basal laminar deposits (BLDs) were positive for VEGF and PEDF. In aged control subjects, VEGF and PEDF immunostaining was the most intense in RPE-Bruch's membrane-choriocapillaris complex. In AMD, PEDF was significantly lower in RPE cells, RPE basal lamina, Bruch's membrane and choroidal stroma. These data suggest that a critical balance exists between PEDF and VEGF, and PEDF may counteract the angiogenic potential of VEGF. The decrease in PEDF may disrupt the balance and be permissive for the formation of choroidal neovascularization (CNV) in AMD.