Advanced glycation end product (AGE) accumulation on Bruch's membrane: links to age-related RPE dysfunction.

PURPOSE: Advanced glycation end products (AGEs) accumulate during aging and have been observed in postmortem eyes within the retinal pigment epithelium (RPE), Bruch's membrane, and subcellular deposits (drusen). AGEs have been associated with age-related dysfunction of the RPE-in particular with development and progression to age-related macular degeneration (AMD). In the present study the impact of AGEs at the RPE-Bruch's membrane interface was evaluated, to establish how these modifications may contribute to age-related disease. METHODS: AGEs on Bruch's membrane were evaluated using immunohistochemistry. A clinically relevant in vitro model of substrate AGE accumulation was established to mimic Bruch's membrane ageing. Responses of ARPE-19 growing on AGE-modified basement membrane (AGE-BM) for 1 month were investigated by using a microarray approach and validated by quantitative (q)RT-PCR. In addition to identified AGE-related mRNA alterations, lysosomal enzyme activity and lipofuscin accumulation were also studied in ARPE-19 grown on AGE-BM. RESULTS: Autofluorescent and glycolaldehyde-derived AGEs were observed in clinical specimens on Bruch's membrane and choroidal extracellular matrix. In vitro analysis identified a range of dysregulated mRNAs in ARPE-19 exposed to AGE-BM. Altered ARPE-19 degradative enzyme mRNA expression was observed on exposure to AGE-BM. AGE-BM caused a significant reduction in cathepsin-D activity in ARPE-19 (P < 0.05) and an increase in lipofuscin accumulation (P < 0.01). CONCLUSIONS: AGEs influence ARPE-19 mRNA expression profiles and may contribute to reduced lysosomal enzyme degradative capacity and enhanced accumulation of lipofuscin. Formation of AGEs on Bruch's membrane may have important consequences for age-related dysfunction of the RPE, perhaps leading to age-related outer retinal disease.

Prediction and verification of miRNA expression in human and rat retinas.

PURPOSE: MicroRNAs (miRNAs) play a global role in regulating gene expression and have important tissue-specific functions. Little is known about their role in the retina. The purpose of this study was to establish the retinal expression of those miRNAs predicted to target genes involved in vision. METHODS: miRNAs potentially targeting important "retinal" genes, as defined by expression pattern and implication in disease, were predicted using a published algorithm (TargetScan; Envisioneering Medical Technologies, St. Louis, MO). The presence of candidate miRNAs in human and rat retinal RNA was assessed by RT-PCR. cDNA levels for each miRNA were determined by quantitative PCR. The ability to discriminate between miRNAs varying by a single nucleotide was assessed. The activity of miR-124 and miR-29 against predicted target sites in Rdh10 and Impdh1 was tested by cotransfection of miRNA mimics and luciferase reporter plasmids. RESULTS: Sixty-seven miRNAs were predicted to target one or more of the 320 retinal genes listed herein. All 11 candidate miRNAs tested were expressed in the retina, including miR-7, miR-124, miR135a, and miR135b. Relative levels of individual miRNAs were similar between rats and humans. The Rdh10 3'UTR, which contains a predicted miR-124 target site, mediated the inhibition of luciferase activity by miR-124 mimics in cell culture. CONCLUSIONS: Many miRNAs likely to regulate genes important for retinal function are present in the retina. Conservation of miRNA retinal expression patterns from rats to humans supports evidence from other tissues that disruption of miRNAs is a likely cause of a range of visual abnormalities.

Retinopathy is reduced during experimental diabetes in a mouse model of outer retinal degeneration.

PURPOSE: Diabetic patients who also have retinitis pigmentosa (RP) appear to have fewer and less severe retinal microvascular lesions. Diabetic retinopathy may be linked to increased inner retinal hypoxia, with the possibility that this is exacerbated by oxygen usage during the dark-adaptation response. Therefore, patients with RP with depleted rod photoreceptors may encounter proportionately less retinal hypoxia, and, when diabetes is also present, there may be fewer retinopathic lesions. This hypothesis was tested in rhodopsin knockout mice (Rho-/-) as an RP model in which the diabetic milieu is superimposed. The study was designed to investigate whether degeneration of the outer retina has any impact on hypoxia, to examine diabetes-related retinal gene expression responses, and to assess lesions of diabetic retinopathy. METHODS: Streptozotocin-induced diabetes was created in male C57Bl6 (wild-type; WT) and Rho-/- mice, and hyperglycemia was maintained for 5 months. The extent of diabetes was confirmed by measurement of glycated hemoglobin (%GHb) and accumulation of advanced glycation end products (AGEs). Retinal hypoxia was assessed using the bioreductive drug pimonidazole. The retinal microvasculature was studied in retinal flatmounts stained by the ADPase reaction, and the outer retina was evaluated histologically in paraffin-embedded sections. Retinal gene expression of VEGF-A, TNF-alpha, and mRNAs encoding basement membrane component proteins were quantified by real-time RT-PCR. RESULTS: The percentage GHb increased significantly in the presence of diabetes (P < 0.001) and was not different between WT or Rho-/- mice. Hypoxia increased in the retina of WT diabetic animals when compared with controls (P < 0.001) but this diabetes-induced change was absent in Rho-/- mice. Retinal gene expression of VEGF-A was significantly increased in WT mice with diabetes (P < 0.05), but was unchanged in Rho-/- mice. TNF-alpha gene expression significantly increased (4.9-fold) in WT mice with diabetes (P < 0.05) and also increased appreciably in Rho-/- mice but to a reduced extent (1.5 fold; P < 0.05). The outer nuclear layer in nondiabetic Rho-/- mice was reduced to a single layer after 6 months, but when diabetes was superimposed on this model, there was less degeneration of photoreceptors (P < 0.05). Vascular density was attenuated in diabetic WT mice compared with the nondiabetic control (P < 0.001); however, this diabetes-related disease was not observed in Rho-/- mice. CONCLUSIONS: Loss of the outer retina reduces the severity of diabetic retinopathy in a murine model. Oxygen usage by the photoreceptors during dark adaptation may contribute to retinal hypoxia and exacerbate the progression of diabetic retinopathy.

Rod photoreceptor loss in Rho-/- mice reduces retinal hypoxia and hypoxia-regulated gene expression.

PURPOSE: This study was conducted to evaluate whether regions of the retinal neuropile become hypoxic during periods of high oxygen consumption and whether depletion of the outer retina reduces hypoxia and related changes in gene expression. METHODS: Retinas from rhodopsin knockout (Rho-/-) mice were evaluated along with those of wild-type (WT) control animals. Retinas were also examined at the end of 12-hour dark or light periods, and a separate group was treated with l-cis-diltiazem at the beginning of a 12-hour dark period. Hypoxia was assessed by deposition of hypoxyprobe (HP) and HP-protein adducts were localized by immunohistochemistry and quantified using ELISA. Also, hypoxia-regulated gene expression and transcriptional activity were assessed alongside vascular density. RESULTS: Hypoxia was observed in the inner nuclear and ganglion cell layers in WT retina and was significantly reduced in Rho-/- mice (P < 0.05). Retinal hypoxia was significantly increased during dark adaptation in WT mice (P < 0.05), whereas no change was observed in Rho-/- or with l-cis-diltiazem-treated WT mice. Hypoxia-inducible factor (HIF)-1alpha DNA-binding and VEGF mRNA expression in Rho-/- retina was significantly reduced in unison with outer retinal depletion (P < 0.05). Retina from the Rho-/- mice displayed an extensive intraretinal vascular network after 6 months, although there was evidence that capillary density was depleted in comparison with that in WT retinas. CONCLUSIONS: Relative hypoxia occurs in the inner retina especially during dark adaptation. Photoreceptor loss reduces retinal oxygen usage and hypoxia which corresponds with attenuation of the retinal microvasculature. These studies suggest that in normal physiological conditions and diurnal cycles the adult retina exists in a state of borderline hypoxia, making this tissue particularly susceptible to even subtle reductions in perfusion.

Pigmented paravenous chorioretinal atrophy is associated with a mutation within the crumbs homolog 1 (CRB1) gene.

PURPOSE: Pigmented paravenous chorioretinal atrophy (PPCRA) is an unusual retinal degeneration characterized by accumulation of pigmentation along retinal veins. The purpose of this study was to describe the phenotype of a family with PPCRA, determine the mode of inheritance, and identify the causal mutation. METHODS: Ophthalmic examination was performed on seven family members and serially detailed in the proband over a 3-year period. Blood samples were collected and DNA extracted. All 12 coding exons and the 5' promoter region of the crumbs homologue 1 (CRB1) gene were PCR amplified and DNA sequenced. In silico homology modeling was performed on the mutated protein domain. RESULTS: Subtle symmetrical chorioretinal atrophy in the inferior quadrant was the earliest clinical sign detectable within this family. Paravenous pigmentation occurred initially in the far periphery, progressing centrally, with atrophy later becoming more widespread, involving the nasal, then the temporal, and finally the upper quadrant. A novel, dominant Val162Met mutation within the fourth EGF-like domain of CRB1 cosegregates with the PPCRA phenotype. It is thought to affect domain structure, because codon 162 is involved in hydrogen bonding between the antiparallel beta-strands of the major beta-sheet, causing sufficient perturbation of the backbone that the domain-stabilizing hydrogen bond does not form or is weakened. CONCLUSIONS: PPCRA was dominantly inherited in this family, but exhibited variable expressivity. Males are more likely to exhibit a severe phenotype, whereas females may remain virtually asymptomatic even in later years. The PPCRA phenotype is associated with a Val162Met mutation in CRB1 which is likely to affect the structure of the CRB1 protein.

A novel diagnostic test detects a low frequency of the hemicentin Gln5345Arg variant among Northern Irish age related macular degeneration patients.

PURPOSE: Age related macular degeneration (AMD) is a common cause of severe vision loss. Identification of genes involved in AMD will facilitate early detection and ultimately help to identify pathways for treatment for this disorder. The A16,263G mutation in the HEMICENTIN-1 gene produces a non-conservative substitution of arginine for glutamine at codon 5345 which has been implicated in familial AMD. The aim of this study is to develop a rapid diagnostic assay for the detection of this mutation and to evaluate its frequency in a sample of AMD patients. METHODS: A primer probe set was designed from exon 104 of the HEMICENTIN-1 gene to differentiate between mutant and wild type alleles. A region spanning the mutation was amplified by PCR using a LightCycler (Roche Diagnostic). The mutation was then detected by melt curve analysis of the hybrid formed between the PCR product and a specific fluorescent probe. The frequency of the mutation within the Northern Ireland population was evaluated by assaying 508 affected AMD patients, 25 possibly affected and 163 controls. RESULTS: This assay clearly discriminates between the A16,263G mutant and wild type HEMICENTIN-1 alleles. The wild type sequence has a single base mismatch with the probe which decreases the stability of the hybrid, resulting in a lower TM (TM=51.27 degrees C) than that observed for the perfectly matched mutant allele (TM=59.9 degrees C). The mutant allele was detected in only one of the 696 subjects, an affected AMD patient. CONCLUSIONS: We describe a rapid assay for the genotyping of the Gln5345Arg mutation using real-time fluorescence PCR to facilitate rapid processing of samples through combined amplification and detection steps. These characteristics are suitable for a clinical setting where high throughput diagnostic procedures are required. The frequency of this mutation within the Northern Ireland population has been estimated at 0.2%, concurring with previous findings that this mutation is a rare variant associated with AMD. A rapid diagnostic assay will facilitate a reliable and convenient evaluation of the frequency of the Gln5345Arg mutation and its association with AMD within other populations.

Preparation of planar retinal specimens: verification by histology, mRNA profiling, and proteome analysis.

PURPOSE: Elucidation of the transcriptome and proteome of the normal retina will be difficult since it is comprised of at least 55 different cell types. However the characteristic layered cellular anatomy of the retina makes it amenable to planar sectioning, enabling the generation of enriched retinal cell populations. The aim of this study was to validate a reproducible method for preparing enriched retinal layers from porcine retina. METHODS: The thicknesses of the retinal photoreceptor, inner nuclear and ganglion cell, and fiber layers were determined by routine histology of cross sections of fresh whole retina mounted on polyvinylidene difluoride (PVDF) membrane. Dissected retina (5 mm2) was placed on PVDF membrane and a series of planar cryosections corresponding to the photoreceptor and inner nuclear layer were removed leaving the ganglion cell and fiber layer which was subsequently detached from the membrane. The retinal specimens were stored at -80 degrees C. Representative planar tissue sections were sonicated in ice-chilled 40 mM ammonium bicarbonate pH 7.9 and aliquots removed for RNA extraction. Quantitative RT-PCR was used to analyze the mRNA expression of genes indicative of specific retinal layers. Ammonium bicarbonate protein extracts were centrifuged, lyophilized and prepared for direct liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis using a Waters Q-Tof Ultima. RESULTS: Histological analysis established the parameters for planar cryosectioning: photoreceptor layer (69+/-1.8 microm), outer plexiform (11+/-0.6 microm), inner nuclear layer (28+/-0.5 microm), inner plexiform, ganglion cell and fiber layer (100+/-5.3 microm). Gene expression profiling provided an independent method for validating the respective retinal preparations. For example, glial fibrillary acidic protein (GFAP) was expressed up to 21 fold higher in the inner retinal "ganglion cell enriched" fraction than in the outer retinal "photoreceptor enriched" fraction. The pattern was reversed for blue cone opsin, which was expressed up to 24 fold higher in the "photoreceptor enriched" fraction. Endogenous protein fragments indicative of each layer were identified by mass spectrometry and de novo sequence data obtained. CONCLUSIONS: Combined histological and mRNA expression profiling has confirmed the development of a reproducible method for generating validated porcine retinal layers enriched for specific cell types. Direct proteome analysis detected endogenous peptide fragments of characteristic retinal proteins. Further analysis of these enriched retinal cell preparations will facilitate a more selective investigation of the retinal transcriptome and proteome than studies of the intact retina.

Sources of PDGF expression in murine retina and the effect of short-term diabetes.

PURPOSE: Progressive dysfunction and death of vascular smooth muscle cells and pericytes is a pathophysiological hallmark of diabetic retinopathy, although the underlying mechanisms behind this process remain ill-defined. The multifunctional peptide platelet-derived growth factor (PDGF) is known to act as an important survival factor for both of these vascular cell-types at times of physiological stress. The retinal cell source(s) of PDGF remain unknown. It is important to understand how diabetes alters expression of this important growth factor. METHODS: Streptozotocin-diabetes was established in C57 mice. Following 8 weeks of sustained diabetes, the eyes were enucleated and in situ hybridization was used to localize expression of PDGF-A and PDGF-B chains in retina from both diabetic and non-diabetic controls. mRNA levels for both forms of PDGF, and their cognate PDGF-alpha and PDGF-beta receptors, were also quantified using real-time PCR. RESULTS: In situ hybridization demonstrated that PDGF-A and PDGF-B were predominantly expressed by the retinal ganglion cells/nerve fibre layer in both normal and diabetic mice, and this localization pattern did not alter in diabetes. PDGF-A receptor was expressed exclusively in the ganglion cell layer of the retina while PDGF-B receptor was mostly localized to the Muller cell end-feet at the internal limiting membrane with lesser immunoreactivity in the ganglion cells, inner plexiform layer, and inner nuclear layer. PDGF-A and PDGF-alpha receptor mRNA expression levels remained unaltered between treatment groups, although retinal immunolocalization patterns between both receptors was distinct. However, there was a significant decrease of PDGF-B mRNA levels in diabetic retina when compared to non-diabetic controls (p<0.001), although there was no significant difference in PDGF-alpha receptor(insert space) expression. CONCLUSIONS: Previous studies have shown PDGF expression in a range of cell-types during retinal development, but these results confirm ganglion cells as the principal PDGF source in mature retina. It may be significant that diabetes can reduce PDGF-B mRNA expression since this may have serious implications for vascular survival during diabetic retinopathy progression.

Role of vascular endothelial growth factor and placental growth factors during retinal vascular development and hyaloid regression.

PURPOSE: Vascular endothelial growth factor (VEGF)-A and placental growth factor (PlGF) are members of a large group of homologous peptides identified as the VEGF family. Although VEGF-A is known to act as a potent angiogenic peptide in the retina, the vasoactive function of PlGF in this tissue is less well defined. This study has sought to elucidate the expression patterns and modulatory role of these growth factors during retinal vascular development and hyaloid regression in the neonatal mouse. METHODS: C57BL6J mice were killed at postnatal days (P)1, P3, P5, P7, P9, and P11. The eyes were enucleated and processed for in situ hybridization and immunocytochemistry and the retinas extracted for total protein or RNA. Separate groups of neonatal mice were also injected intraperitoneally daily from P2 through P9 with either VEGF-neutralizing antibody, PlGF-neutralizing antibody, isotype immunoglobulin (Ig)-G, or phosphate-buffered saline (PBS). The mice were then perfused with fluorescein isothiocyanate (FITC)-dextran, and the eyes were subsequently embedded in paraffin wax or flat mounted. RESULTS: Quantitative (real-time) reverse transcription-polymerase chain reaction (RT-PCR) demonstrated similar expression patterns of VEGF-A and PlGF mRNA during neonatal retinal development, although the fluctuation between time periods was greater overall for VEGF-A. The localization of VEGF-A and PlGF in the retina, as revealed by in situ hybridization and immunohistochemistry, was also similar. Neutralization of VEGF-A caused a significant reduction in the hyaloid and retinal vasculature, whereas PlGF antibody treatment caused a marked persistence of the hyaloid without significantly affecting retinal vascular development. CONCLUSIONS: Although having similar expression patterns in the retina, these growth factors appear to have distinct modulatory influences during normal retinal vascular development and hyaloid regression.

Application of in silico positional cloning and bioinformatic mutation analysis to the study of eye diseases.

A vast amount of DNA and protein sequence is now available and a plethora of programs have been developed to analyse the data. The bewildering variety of analyses that can be performed via the World-Wide Web can deter researchers from applying bioinformatics to augment their traditional genetic research. Focusing on the inherited eye diseases, this paper provides a guide to the appropriate software required for identification of candidate genes through to the detection and analysis of mutations.

Identification of an IMPDH1 mutation in autosomal dominant retinitis pigmentosa (RP10) revealed following comparative microarray analysis of transcripts derived from retinas of wild-type and Rho(-/-) mice.

Comparative analysis of the transcriptional profiles of approximately 6000 genes in the retinas of wild-type mice with those carrying a targeted disruption of the rhodopsin gene was undertaken by microarray analysis. This revealed a series of transcripts, of which some were derived from genes known to map at retinopathy loci, levels of which were reduced or elevated in the retinas of Rho(-/-) mice lacking functional photoreceptors. The human homologue of one of these genes, encoding inosine monophosphate dehydrogenase type 1 (IMPDH1), maps to the region of 7q to which an adRP gene (RP10) had previously been localized. Mutational screening of DNA from the Spanish adRP family, originally used to localize the RP10 gene, revealed an Arg224Pro substitution co-segregating with the disease phenotype. The amino acid at position 224 of the IMPDH1 protein is conserved among species and the substitution is not present in healthy, unrelated individuals of European origin. These data provide strong evidence that mutations within the IMPDH1 gene cause adRP, and validate approaches to mutation detection involving comparative analysis of global transcription profiles in normal and degenerating retinal tissues. Other genes showing significant alterations in expression include some with anti-apoptotic functions and many encoding components of the extracellular matrix or cytoskeleton, a possible reflection of a response by Muller cells to preserve the remaining outer nuclear layer of the retina. We suggest that those genes identified are prime candidates for etiological involvement in degenerative retinal disease.