Showing papers in "Molecular Vision in 2006"

MicroRNAs of the mammalian eye display distinct and overlapping tissue specificity.

Abstract: Purpose: In mammals, endogenous, noncoding RNAs, designated as microRNAs (miRNAs), inhibit the translation of a target messenger RNA, thereby silencing protein production. MiRNAs have been shown to regulate many aspects of development and differentiation in a wide range of tissues. Surprisingly, little consideration has been directed towards characterizing the expression of miRNAs in mammalian ocular tissues. Methods: Low molecular weight (LMW) RNA isolated from the adult mouse corneal epithelium, lens/ciliary body, and a retina fractions of the eye was analyzed by miRNA arrays. The validity of the miRNA expression profiles were confirmed by northern blots and the tissue distribution of selected miRNAs was determined by in situ hybridization. Results: MiRNAs exhibited distinct tissue and cell-type specificity in the ocular regions studied. MiRNA (mir)-184 had the highest hybridization signal in the corneal and lens arrays. In situ hybridization analysis revealed that mir-184 was expressed in the basal and immediately suprabasal cells of the corneal epithelium. In contrast, expression of mir-205 was detected throughout the anterior segmental epithelia as well as in the epidermis. Within the lens, expression of mir-184 was more strongly expressed in the epithelial cells of the germinative zone, whereas expression of mir-204 was uniformly expressed in all lens epithelial cells. Mir-181, -182, and -183 were detected in retinal and brain tissues, and their distribution patterns within the retina were both distinct and overlapping. Conclusions: The tissue and cell specificity of ocular miRNAs suggests that these noncoding RNAs may be regulating aspects of development and differentiation.

Intraretinal lipid transport is dependent on high density lipoprotein-like particles and class B scavenger receptors.

Abstract: PURPOSE In our companion paper we demonstrated that circulating lipoproteins enter the retina via the retinal pigment epithelium (RPE) and possibly Muller cells. In order to understand how these lipids are transported within the retina, expression and localization of the main proteins known to be involved in systemic lipid transport was determined. METHODS Expression of ABCA1, apoA1 (the major HDL protein), SR-BI, SR-BII, CD36, lecithin:cholesterol acyltransferase (LCAT), and cholesteryl ester transfer protein (CETP) was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblots. Localization was determined by immunohistochemistry using fresh monkey vibrotome sections and imaged by confocal microscopy. RESULTS ABCA1 and apoA1 were localized to the ganglion cell layer, retinal pigment epithelium (RPE), and rod photoreceptor inner segments. ApoA1 was also observed associated with rod photoreceptor outer segments, presumably localized to the interphotoreceptor matrix (IPM). The scavenger receptors SR-BI and SR-BII localized mainly to the ganglion cell layer and photoreceptor outer segments; in the latter they appear to be associated with microtubules. LCAT and CETP localized mainly to the IPM. CONCLUSIONS The presence and specific localization of these well-known lipid transport proteins suggest that the retina employs an internal lipid transport mechanism that involves processing and maturation of HDL-like particles.

Characterization of cytokine responses to retinal detachment in rats

Abstract: Purpose: Photoreceptor apoptosis is associated with retinal detachment (RD) induced photoreceptor degeneration. Previously, we demonstrated the importance of caspase activation for RD-induced photoreceptor death in a rat model of RD. However, extracellular signals that precede the activation of caspases and photoreceptor degeneration remain unclear. The aim of this study is to characterize the molecular and cellular responses that occur after RD. The expression of cytokines, chemokines, and growth factors were examined in a rat model of RD. Methods: RD was induced in adult rats by subretinal injection of sodium hyaluronate. Retinal tissues were collected at various times (1, 3, 6, 24, and 72 h) after the induction of detachment. To screen for expressional changes in response to RD, major candidates for cytokines, chemokines, and growth factors were broadly examined by quantitative real time polymerase chain reaction (QPCR). To identify the cellular sources of the expressed genes, cells from various layers of the retina were obtained using laser capture microdissection (LCM), and their mRNAs were isolated. Protein expression was quantified by immunohistochemistry and Enzyme Linked-Immuno-Sorbent Assay (ELISA). To assess the potential of early response genes after RD to induce photoreceptor degeneration, exogenous recombinant proteins were subretinally injected and the photoreceptor cell death was assessed using a TdT-dUTP terminal nick-end labeling (TUNEL) assay at 24 h after RD. Results: At 72 h after RD a significant increase in mRNA levels for tumor necrosis factor α (TNF-α), interleukin-1β (IL1β), monocyte chemotactic protein-1 (MCP-1), and basic fibroblast growth factor (bFGF) were detected in the neural retina. LCM revealed increased expression of mRNA for bFGF and MCP-1 in all retinal layers, though bFGF was especially evident in the outer nuclear layer (ONL) and MCP-1 in the inner nuclear layer (INL). TNF- α was increased in the ONL and the INL, and IL-1β was increased in the ganglion cell layer. Time course experiments showed that TNF-α, IL1β and MCP-1 increased within 1 h after RD, while bFGF was increased by 24 h. Increased protein expression for TNFα, IL-1β, and MCP-1 was demonstrated by ELISA at 6 h after RD. Immunohistochemistry showed TNF-α and bFGF expression in the whole retina, with IL-1β specifically expressed in astrocytes and MCP-1 in Muller cells. Subretinal administration of MCP-1 significantly increased TUNEL-positive cells in the ONL 24 h after RD, while injection of vehicle control, TNF-α, or IL-1β showed no effect. Conclusions: Retinal glial cells, including astrocytes and Muller cells, are a major source of cytokine induction after RD. The increased expression and release of MCP-1 may be an important cause of photoreceptor degeneration associated with RD. This study helps to understand the mechanisms of RD-induced photoreceptor degeneration. Our results may provide new therapeutic targets to prevent photoreceptor degeneration following RD.

Automated tool for the detection of cell nuclei in digital microscopic images: application to retinal images.

Abstract: Purpose: To develop an automated tool that provides reliable, consistent, and accurate results for counting cell nuclei in tissue sections. Methods: We propose a novel method based on an image processing algorithm to analyze large sets of digital micrographs. The nucleus detector design is based on a Laplacian of Gaussian filter. We use the leave-one-out cross validation method for estimating the generalization error, which is then used to choose the model and parameters of the proposed nucleus detector with both fluorescent and dye stained images. We also evaluate the performance of a nucleus detector by comparing the results with manual counts. Results: When our nucleus detector is applied to previously unanalyzed images of feline retina, it correctly counts nuclei within the outer nuclear layer (ONL) with an average error of 3.67% ranging from 0 to 6.07%, and nuclei within the inner nuclear layer (INL) with an average error of 8.55% ranging from 0 to 13.76%. Our approach accurately identifies the location of cell bodies. Even though we have a relatively large error in the INL due to the large intra-observer variation, both manual counting and nucleus detector result in the same conclusion. This is the first time that cell death in the INL in response to retinal detachment is analyzed quantitatively. We also test the proposed tool with various images and show that it is applicable to a wide range of image types with nuclei varying in size and staining intensity. Conclusions: The proposed method is simple and reliable. It also has widespread applicability to a variety of sample preparation and imaging methods. Our approach will be immediately useful in quantifying cell number in large sets of digital micrographs and from high-throughput imaging. The tool is available as a plug-in for Image J.

Uptake of cholesterol by the retina occurs primarily via a low density lipoprotein receptor-mediated process.

Abstract: Purpose In this study we examined the uptake of circulating lipoproteins into the retina, using a naturally fluorescent cholesterol analog for imaging and deuterated cholesterol for quantification by mass spectroscopy. The purpose of this study was to better understand cholesterol uptake, transport and homeostasis in the retina. Methods Human low density lipoprotein (LDL) and high density lipoprotein (HDL) were labeled with the fluorescent cholesterol analog cholesta-5,7,9(11)-trien-3beta-ol (CTL) and deuterated cholesterol (25,26,26,26,27,27,27-[2H]cholesterol, D7Ch). Rats were injected intravenously with CTL-LDL, CTL-HDL and D7Ch-LDL. Fluorescent confocal microscopy was used to image the uptake of CTL and mass spectroscopy was used to quantify D7Ch. Immunohistochemistry and fluorescent confocal microscopy were used to localize apoB (an LDL marker protein) and LDL receptor (LDLR) protein in rat and monkey retinas. Results CTL-specific fluorescence was imaged by confocal microscopy in the retinal pigment epithelium (RPE), choriocapillaris and parts of the neural retina within 2 h post-injection and was visualized in the photoreceptor outer segments by 4 h. Replacing LDL with HDL as the CTL carrier gave a less robust and more delayed labeling of retinal layers. Human apolipoprotein B (apoB) was also localized in the rat choriocapillaris and RPE by 4 h post-injection. Human apoB was detected by immunoblot analysis in the rat retina primarily as a about 70 kDa protein, suggesting proteolytic degradation. LDL-mediated uptake of cholesterol was quantified by mass spectroscopy using deuterated cholesterol in place of CTL. In addition, apoB and LDLR were localized in monkey retina by immunohistochemistry. Conclusions The retina is capable of rapid uptake of circulating LDL via an LDLR-mediated process primarily occurring in the RPE and also possibly Muller cells. Despite the dominance of HDL over LDL in rat serum, LDL appears to be the preferred carrier for cholesterol transport to and uptake by the retina. The results also suggest that blood-borne LDL represents a significant contributor to the steady-state levels of cholesterol and possibly other lipids in the retina.

VEGF165b, an endogenous C-terminal splice variant of VEGF, inhibits retinal neovascularization in mice.

Abstract: Purpose Hypoxia driven ocular angiogenesis occurs in a range of ischemic retinopathies including proliferative diabetic retinopathy and retinopathy of prematurity. These conditions are initiated and sustained by hypoxia dependent vascular endothelial growth factor (VEGF) expression in the eye. There are two families of VEGF isoforms formed by differential splicing, the pro-angiogenic VEGF family, known to contribute to ocular neovascularization, and the anti-angiogenic VEGF family, which are downregulated in diabetic retinopathy in humans. The first member of the VEGF family to be isolated was VEGF165b. To determine whether VEGF165b could inhibit hypoxia driven angiogenesis in the eye, the oxygen induced retinopathy mouse model of ocular neovascularization was used. Methods 1 ng of recombinant human VEGF165b peptide was injected intraocularly upon return to normoxia after 5 days exposure to 95% oxygen, and neovascularization assessed. Results VEGF165b significantly inhibited the percentage area of retinal neovascularization from 23+/-3% to 12+/-3.3%, and significantly increased normal vascular areas from 62+/-4% to 74+/-4%. The percentage area of residual ischemic retina was not affected. Conclusions These results show that a single injection of VEGF165b can significantly reduce preretinal neovascularization without inhibition of physiological intraretinal angiogenesis. Controlling the balance of VEGF(xxx) to VEGF(xxx) isoforms may therefore be therapeutically valuable in the treatment of proliferative eye diseases such as diabetic retinopathy and age related macular degeneration. The regulation of splicing between these two families of isoforms may provide a novel therapeutic strategy for proliferative eye disease.

Association of VEGF and eNOS gene polymorphisms in type 2 diabetic retinopathy.

Abstract: 2test. Haplotype estimation and multiple logistic regression analysis were carried out to analyze the significance of polymorphisms. Results: We investigated four reported polymorphisms in the VEGF (5' UTR, promoter) and one reported polymorphism (intron 4) in the eNOS gene in Type 2 diabetes patients with (n=120) and without (n=90) retinopathy. The genotype distribution of the C(-7)T, T(-1498)C, and C(-634)G polymorphisms of VEGF differed significantly between patients with DR and DWR (p=0.001, p=0.0001, and p=0.021, respectively). Allele C in the -1498 region (p=0.0001) and T in -7 region (p=0.002) were also found to be significantly increased in patients with retinopathy. Calculated odds ratios (OR) for three heterozygous genotypes of C(-7)T, T(-1498)C, and C(-634)G regions were 4.17 (95% CI: 1.90-9.18, p=0.0001), 4.37 (95% CI: 2.44-7.84, p=0.0001), and 2.33 (95% CI: 1.24-4.36, p=0.008), respectively, and was found to be significantly higher in the DR group when compared with the DWR group. Multiple logistic regression analysis revealed that the nongenetic parameters, age (p=0.024) and duration of diabetes (p=0.009), and the genetic parameters, like VEGF C(-7)T (p=0.002) and T(-1498)C (p=0.001) polymorphisms, were significantly associated with DR. The frequencies of haplotype consisting of the majority of alleles in VEGF were found to be significantly associated with DR. The genotype distribution of eNOS did not differ significantly between the two study groups, and therefore the eNOS intron 4 polymorphism was considered to be less significant. Conclusions: This is the first study to report VEGF and eNOS gene polymorphisms in patients with DR in the Indian population. The data suggest that the polymorphisms in the 5' UTR and promoter region of VEGF could be regarded as a major genetic risk factor for DR.

Complement factor H polymorphisms in Japanese population with age-related macular degeneration.

Abstract: Purpose: To study the frequency of five haplotypes previously reported in the complement factor H ( CFH) gene for Japanese patients with age-related macular degeneration (AMD). Methods: Genomic DNA was isolated from peripheral blood samples taken from 96 Japanese AMD patients and 89 agematched controls. All patients were diagnosed as having exudative (wet-type) AMD. The amplified polymerase chain reaction (PCR) products of CFH exons 2, 9, and 13, and intron 6 were analyzed by temperature gradient capillary electrophoresis (TGCE) and by direct sequencing. The haplotypes were identified, and their frequencies were calculated and compared with reported results. Results: Five haplotypes were identified in the Japanese population including four already reported in the American population. The frequencies of these haplotypes were significantly different between Japanese and American in both control and case groups. The haplotype containing Y402H, which was previously reported to be associated with AMD, was only 4% in the control and case population, with a p value of 0.802. However, two other haplotypes were found as risk factors, which gave an increased likelihood of AMD of 1.9 and 2.5 fold (95% CI 1.12-3.69 and 1.42-6.38). One protective haplotype that decreased the likelihood of AMD by 1.6 fold (95% CI 0.26-0.67) was identified. Conclusions: The frequencies for five haplotypes previously identified were analyzed in a Japanese population with AMD. Four previously found haplotypes were identified and one additional haplotype was found. The frequencies of each haplotype were significantly different from that in found Americans affected with AMD. Two of the haplotypes were identified as risk factors and one was considered protective.

Microglia and macrophages are increased in response to ischemia-induced retinopathy in the mouse retina.

Abstract: Purpose The ability of microglial cells (MG) and macrophages (MAC) to release cytokines, induce apoptosis, as well as perform phagocytic functions suggests a possible role in wound healing following oxygen-induced injury. This study was performed to determine the temporal and spatial expression of F4/80 (F4/80+) positive microglia/macrophages (MG/MAC) in areas of retinal damage in the mouse model of oxygen-induced retinopathy. Methods C57BL/6 postnatal day 7 (P7) mice were exposed to 75% O2 for 5 days (P12) then allowed to recover in room air. Hyperoxia-exposed (O2) mice (O2 refers to hyperoxia exposure from P7 to P12 only) were sacrificed on P12, P14, P17, and P21 and their eyes were examined. Localization of F4/80+ cells in FITC-dextran-perfused retinas allowed coordinate visualization of retinal vessels and MG/MAC via fluorescence microscopy. BrdU, a cellular proliferation marker, was injected intraperitoneally 1 h prior to sacrifice. Immunostaining was performed for a microglia and macrophage-specific antigen (F4/80) and BrdU. CCL2 (monocyte chemoattractant protein-1; MCP-1) expression was examined by quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR). Results There was a marked increase (>500%) in MG/MAC in hyperoxia-exposed retinas on P17O2 and P21O2 compared to control retinas. At P17O2, MG/MAC were localized in areas of neovascularization (NV), revealing an intimate relationship between MG/MAC and neovascular tufts. However, P21O2 retinas demonstrated MG/MAC associated with avascular regions in the outer layers of the retina. Immunostaining for F4/80 and BrdU revealed rare co-localization in hyperoxia-exposed retinas. Real time RT-PCR results demonstrated increased expression of CCL2 in P14O2- and P17O2- exposed retinas. Conclusions Our results suggest that resident retinal microglia proliferation occurs at a low frequency in response to injury in this model. The substantial increase in total F4/80+ cells in hyperoxia-exposed retinas in conjunction with the upregulation of CCL2 is consistent with recruitment of hematogenous macrophages into the retina. The temporal and spatial localization of MG/MAC adjacent to neovascular tufts suggests these cells are modulating the retinal response to ischemia-induced retinopathy.

Stimulation of apical and basolateral VEGF-A and VEGF-C secretion by oxidative stress in polarized retinal pigment epithelial cells.

Abstract: PURPOSE To investigate whether oxidative stress modulates vascular endothelial growth factor (VEGF)-A and VEGF-C expression and polarized secretion in a human retinal pigment epithelium cell line (ARPE-19). METHODS Long-term culture of ARPE-19 cells in Dulbecco's modified Eagle medium (DMEM)/F12 containing 1% fetal bovine serum (FBS) on transwell filters (12 mm or 6 mm, pore size 0.4 microm) was performed to produce polarized retinal pigment epithelium (RPE) monolayers. The integrity of polarized monolayer was established by measurement of transepithelial resistance (TER) and presence of tight junctions assessed by zonula occludens (ZO-1) and occludin expression and apical Na/K ATPase localization. Paracellular permeability was studied using radiolabeled mannitol. Confluent cells were treated with tertiary butyl hydrogen peroxide (tBH) for varying durations (0-5 h) and doses (50-200 microM). VEGF-A and -C expression was evaluated by western blot and quantitative RT-PCR, while secretion to the apical and basolateral surfaces was quantitated by ELISA. RESULTS Polarity of ARPE-19 cells was verified by the localization of tight junction proteins, ZO-1 and its binding partner occludin by confocal microscopy as well as by localization of Na,K-ATPase at the apical surface. The TER in confluent ARPE-19 cells averaged 48.7+/-2.1 Omega. cm(2) and tBH treatment (0-5 h) did not alter TER significantly (46.9+/-1.9 Omega. cm(2); p>0.05 versus controls) or ZO-1 expression. Whole cell mRNA in nonpolarized ARPE-19 increased with tBH at 5 h both for VEGF-A and VEGF-C and the increase was significant (p<0.05 vs controls). A similar, maximal increase at 5 h tBH treatment was also observed for VEGF-A and VEGF-C cellular protein levels. The secretion of VEGF-A and VEGF-C in nonpolarized ARPE showed an increase with tBH exposure. The levels of secretion of VEGF-A and -C were significantly higher in polarized monolayers and were stimulated significantly with tBH at both apical and basolateral domains. The secretion of VEGF-A increased with 150 microM of tBH treatment as a function of time (1-5 h) with maximal increases at 5 h from 410 to 2080 pg/10(6) cells on the apical and 290 to 1680 pg/10(6) cells on basolateral domains. The pattern of VEGF-C secretion was similar. VEGF-A secretion was dose-dependent for the tBH range of 50-200 microM and apical secretion tended to be higher than basolateral secretion. CONCLUSIONS Our data show that oxidative stress to RPE from tBH upregulates secretion of both VEGF-A and C. The secretion to the apical side was higher than that of basolateral side for VEGF-A and C. Given the role of VEGF in choroidal neovascularization, these data may be of value in understanding pathogenic mechanisms and designing antiangiogenic therapies.

Pre-mRNA splicing and retinitis pigmentosa.

Abstract: Retinitis pigmentosa (RP) is a group of genetically and clinically heterogeneous retinal diseases and a common cause of blindness. Among the 12 autosomal dominant RP (adRP) genes identified, four encode ubiquitously expressed proteins involved in pre-mRNA splicing, demonstrating the important role that pre-mRNA splicing plays in the pathogenesis of retinal degeneration. This review focuses on recent progress in identifying adRP mutations in genes encoding pre-mRNA splicing factors and the potential underlying molecular mechanisms.

Gene expression profile of human trabecular meshwork cells in response to long-term dexamethasone exposure

Abstract: PURPOSE Topical use of dexamethasone has long been associated with steroid induced-glaucoma, although the mechanism is unknown We applied a strict filtering of comparative microarray data to more than 18,000 genes to evaluate global gene expression of cultured human trabecular meshwork cells in response to treatment with dexamethasone METHODS Three human trabecular meshwork cell primary cultures from nonglaucomatous donors were incubated with and without dexamethasone for 21 days Relative gene expression was evaluated by analysis of U133A GeneChip and the results validated using quantitative polymerase chain reaction (PCR) RESULTS Application of strict filtering to include only genes with statistically significant differences in gene expression across all three trabecular meshwork cell cultures produced a list of 1,260 genes Significant changes in signal level were observed, including 23 upregulated and 18 downregulated genes that changed greater than three fold in each of three cell cultures Using quantitative PCR we found changes greater than a thousand fold for two genes (SLP1 and SAA2) and changes greater than a hundred fold for another five genes (ANGPTL7, MYOC, SAA1, SERPINA3, and ZBTB16) CONCLUSIONS Expression changes in trabecular meshwork cells in response to dexamethasone treatment indicate that a group of actins and actin-associated proteins are involved in the development of cross-linked actin networks that form in response to dexamethasone A trend was identified toward decreased expression of protease genes accompanied by an increased expression of protease inhibitors Such a trend in nonproteasomal proteolysis conceivably affects gene product levels above the level of transcription Only two genes, MYOC and IGFBP2, showed significantly elevated expression after dexamethasone treatment in our study and the other three previously published reports of primary culture trabecular meshwork cell gene expression

Genome-wide expression profile of human trabecular meshwork cultured cells, nonglaucomatous and primary open angle glaucoma tissue.

Abstract: Purpose To contrast genome-wide gene expression profiles of cultured human trabecular meshwork (HTM) cells to that of control and primary open angle glaucoma (POAG) HTM tissues. Methods Cultured HTM cells, HTM tissue dissected from control donors, and HTM tissue from POAG donors receiving medication for glaucoma were fixed in RNA latertrade mark. Total RNA extracted from these samples was linearly amplified with the Ovation Biotin RNA Amplification and Labeling System and individually hybridized to Affymetrix Human Genome U133 Plus 2.0 high density microarrays. Data analysis was performed using GeneSpring Software 7.0. Selected genes showing significant differential expression were validated by quantitative real-time PCR in nonamplified RNA. Results Cultured HTM cells retained the expression of some genes characteristic of HTM tissue, including chitinase 3-like 1 and matrix Gla protein, but demonstrated downregulation of physiologically important genes such as myocilin. POAG HTM tissue showed relatively small changes compared to that of control donors. These changes included the statistically significant upregulation of several genes associated with inflammation and acute-phase response, including selectin-E (ELAM-I), as well as the downregulation of the antioxidants paraoxonase 3 and ceruloplasmin. Conclusions Downregulation in cultured HTM cells of genes potentially relevant for outflow pathway function highlights the importance of developing new conditions for the culture of TM cells capable of preserving the characteristics of TM cells in vivo. Comparative analysis between control and POAG tissues suggests that the upregulation of inflammation-associated genes might be involved in the progression of glaucoma.

Association of complement factor H polymorphisms with exudative age-related macular degeneration.

Abstract: PURPOSE Variants in the complement factor H (CFH) gene have been reported to be associated with age-related macular degeneration (AMD). We conducted a case-control association study to investigate the association of 6 single nucleotide polymorphisms (SNPs) in CFH with exudative AMD in the Chinese population. METHODS We recruited 163 cases and 244 controls, all ethnic Chinese, with complete ophthalmic examination including fundus investigation. Cigarette smoking was recorded. Six SNPs (dbSNP ID: rs3753394, rs800292, rs1061147, rs1061170, rs380390, and rs1329428) in the CFH gene were genotyped by Taqman assays. RESULTS Y402H (1277 T>C) has low frequencies in our study population, 5.8% in patients and 3.9% in controls. It was not associated with exudative AMD adjusted for age, gender and smoking. Significant associations were detected for AMD with rs3753394 (p=0.003, p(corr)=0.018), rs800292 (p=0.00053, p(corr)=0.0032), and rs1329428 (p=0.00092, p(corr)=0.0028), p(corr) values obtained after adjustment for multicomparison. A haplotype containing these four SNPs (TGTC) was found to confer a significantly increased likelihood of exudative AMD with an odds ratio of 1.68 (95% CI: 1.26-2.23) p=0.0003 (p(corr)=0.0026 after correction by permutation test). Logistic regression analysis detected no interactions between the SNPs and age, gender or smoking. CONCLUSIONS We have found differences in the association between the CFH gene and exudative AMD in Chinese from Caucasians and Japanese. We detected SNP rs3753394 in the CFH promoter carrying a significantly increased risk for exudative AMD.

A new autosomal recessive syndrome consisting of posterior microphthalmos, retinitis pigmentosa, foveoschisis, and optic disc drusen is caused by a MFRP gene mutation

Abstract: PURPOSE To describe the clinical and genetic characteristics of a new ophthalmic syndrome, which consists of posterior microphthalmos, retinitis pigmentosa, foveoschisis, and optic disc drusen, that segregates as an autosomal recessive trait in a family with four affected siblings. The membrane-type frizzled-related protein (MFRP) and CEH10 homeodomain-containing homolog (CHX10) genes, previously implicated in autosomal recessive forms of nanophthalmos/microphthalmos, were analyzed as candidate genes for this novel disease. METHODS Complete ophthalmologic examinations were performed in four affected siblings and their parents. Ophthalmologic manifestations, fundus photographs, ultrasonographic (US) assessment, electroretinography (ERG), fluorescein retinal angiography (FA), Goldmann kinetic perimetry (GKP), and optical coherence tomography (OCT), as well as mutational status of MFRP and CHX10 genes in genomic DNA. RESULTS In all affected siblings, ophthalmologic examination demonstrated normal horizontal corneal diameters and high hyperopia; funduscopy, ERG, and FA evidenced a progressive retinal dystrophy compatible with retinitis pigmentosa; A- and B-mode ultrasonography revealed decreased axial eye length and optic disc drusen; OCT showed localized macular retinoschisis. MFRP molecular analysis disclosed a one base pair insertion in exon 5 (c.498_499insC) in all affected individuals, a mutation that predicts a truncated protein (P165fsX198). Both parents were heterozygous for this mutation. CONCLUSIONS A distinct autosomal recessive ophthalmic syndrome characterized by microphthalmos, retinitis pigmentosa, foveoschisis, and optic disc drusen is described. We demonstrated that this clinical association is caused by a mutation in MFRP, a gene previously implicated in isolated nanophthalmos. Our data indicate that defects in MFRP could be responsible for syndromic forms of microphthalmos/retinal degeneration and that this gene is necessary for photoreceptor maintenance.

The retinal carotenoids zeaxanthin and lutein scavenge superoxide and hydroxyl radicals: a chemiluminescence and ESR study.

Abstract: Purpose Carotenoids are present in many biological systems, often decreasing the formation of products of oxidative damage to biological molecules. In the macula their concentration is so high that it has been believed that the yellow color filters out damaging blue light. Recent reports that dietary lutein reduces the risk of cataract in the eye lens suggested that the antioxidant action of carotenoids, which has been inferred from decreased oxidative damage, warranted further direct investigation. Methods Superoxide and hydroxyl radical scavenging by lutein and zeaxanthin (retinal carotenoids), beta-carotene, lycopene, lutein esters (from marigolds), and a commercial mixture of soy carotenoids were compared to scavenging by ascorbate and ascorbyl palmitate. Radical scavenging was measured with a chemiluminescent assay (luminol) and by electron spin resonance, ESR. Inhibitory concentrations, IC(50), were determined with the luminescent assay. Results All of the carotenoids scavenged both superoxide (in ESR 30-50% at 16.7 microM) and hydroxyl radicals (in ESR 50-70% at 16.7 microM, in a luminescent assay 90-99%). Conclusions While crocin may be unable to scavenge superoxide, some of the other carotenoids do so quite effectively. The mixtures of 15,15'-cis and all-trans-carotenoids studied by ESR and luminescent assay scavenge both superoxide and hydroxyl radicals. Lycopene and beta-carotene both scavenge superoxide more effectively than the xanthophylls of the retina, zeaxanthin and lutein. All of the carotenoids examined scavenged the hydroxyl radicals more effectively than superoxide radicals. The predominant carotenoid in the fovea of the retina, zeaxanthin, scavenged hydroxyl radicals more effectively than the other retinal carotenoid, lutein. Possible mechanisms of radical scavenging by the carotenoids are discussed.

Primary role of CYP1B1 in Indian juvenile-onset POAG patients.

Abstract: Purpose CYP1B1, a member of the cytochrome P450 superfamily of enzymes, has been implicated in primary congenital glaucoma (PCG). Recent studies suggest a role of CYP1B1 in primary open-angle glaucoma (POAG) as a modifier locus. The purpose of the study was to further investigate the potential role of CYP1B1 in POAG patients. Methods Two hundred unrelated Indian POAG patients and 100 unrelated ethnically matched controls were enrolled in this study. The coding sequence of CYP1B1 was amplified by polymerase chain reaction (PCR) from genomic DNA, followed by direct DNA sequencing to identify the allelic variants. Results Six mutations were identified in nine patients and none of the controls examined. One novel mutation (R523T) was detected in the homozygous condition while three reported (W57C, E229K, and R368H) and two novel mutations (S515L and D530G) were found in the heterozygous state. The homozygous mutation of a conserved residue, detected in a familial juvenile onset POAG (JOAG) patient (lacking MYOC or OPTN mutations), cosegregated with the disease locus in an autosomal recessive mode of transmission. All the novel mutations (R523T, S515L and D530G) were detected in a region of CYP1B1 that did not harbor any of the 34 point mutations implicated in PCG. In addition, six previously reported (p.R48G, p.A119S, p.V432L, p.D449D, p.N453S, and 372-12C>T in intron 1) and four novel (p.V395V, p.P400P, p.V518A, and c.2016C>G in the 3'-UTR) single nucleotide polymorphism (SNPs) were also observed in POAG patients and controls. Conclusions Our observation suggests that on rare occasions CYP1B1 may be primarily responsible for JOAG by possible monogenic association, and this observation emphasizes the importance of screening for mutation in this gene of JOAG patients that are determined not to harbor mutations in previously characterized candidate genes and loci for POAG.

Tool from ancient pharmacopoeia prevents vision loss

Abstract: PURPOSE Bear bile has been used in Asia for over 3,000 years to treat visual disorders, yet its therapeutic potential remains unexplored in Western vision research. The purpose of this study was to test whether treatment of mice undergoing retinal degeneration with tauroursodeoxycholic acid (TUDCA), a primary constituent of bear bile, alters the course of degeneration. METHODS Two retinal degeneration models were tested: the rd10 mouse, which has a point mutation in the gene encoding the beta subunit of rod phosphodiesterase, and light induced retinal damage (LIRD). For LIRD studies, albino Balb/C adult mice were subcutaneously injected with TUDCA (500 mg/kg body weight) or vehicle (0.15 M NaHCO(3)). Sixteen h later, each mouse received repeat injections. Half of each treatment group was then placed in bright light (10,000 lux) or dim light (200 lux) for seven h. At the end of exposure, animals were transferred to their regular housing. Electroretinograms (ERGs) were assessed 24 h later, mice sacrificed, eyes embedded in paraffin and sectioned, and retina sections assayed for morphology and apoptosis by TUNEL and anti-active caspase-3 immunoreactivity via fluorescent confocal microscopy. A subset of mice were sacrificed 8 and 15 days after exposure and retina sections analyzed for morphology and apoptosis. For rd10 studies, mice were injected subcutaneously with TUDCA or vehicle at postnatal (P) days 6, 9, 12, and 15. At p18, ERGs were recorded, mice were euthanized and eyes were harvested, fixed, and processed. Retinal sections were stained (toluidine blue), and retinal cell layers morphometrically analyzed by light microscopy. Consecutive sections were analyzed for apopotosis as above. RESULTS By every measure, TUDCA greatly slowed retinal degeneration in LIRD and rd10 mice. ERG a-wave and b-wave amplitudes were greater in mice treated with TUDCA compared to those treated with vehicle. Retinas of TUDCA-treated mice had thicker outer nuclear layers, more photoreceptor cells, and more fully-developed photoreceptor outer segments. Finally, TUDCA treatments dramatically suppressed signs of apoptosis in both models. CONCLUSIONS Systemic injection of TUDCA, a primary constituent of bear bile, profoundly suppressed apoptosis and preserved function and morphology of photoreceptor cells in two disparate mouse models of retinal degeneration. It may be that bear bile has endured so long in Asian pharmacopeias due to efficacy resulting from this anti-apoptotic and neuroprotective activity of TUDCA. These results also indicate that a systematic, clinical assessment of TUDCA may be warranted.

Proximal renal tubular acidosis and ocular pathology: a novel missense mutation in the gene (SLC4A4) for sodium bicarbonate cotransporter protein (NBCe1).

Abstract: Molecular Vision 2006; 12:324-30 Received 12 January 2006 | Accepted 3 April 2006 | Published 10 April 2006 ©2006 Molecular Vision Proximal renal tubular acidosis and ocular pathology: a novel missense mutation in the gene (SLC4A4) for sodium bicarbonate cotransporter protein (NBCe1) F. Yesim K. Demirci, 1,2 Min-Hwang Chang, 3 Tammy S. Mah, 1 Michael F. Romero, 3,4 Michael B. Gorin 1,2 Department of Ophthalmology, UPMC Eye Center, Ophthalmology and Visual Science Research Center, School of Medicine and Department of Human Genetics, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA; Departments of Physiology and Biophysics and 4 Pharmacology, Case Western Reserve University, Cleveland, OH Purpose: The electrogenic Na + /HCO 3- cotransporter (NBCe1) plays a major role in renal bicarbonate absorption via proximal tubules and therefore is crucial for maintaining normal blood pH. The human gene for NBCe1 (SLC4A4) pro- duces two major transcripts by alternative promoter usage (kNBCe1, originally cloned from kidney and pNBCe1, pancre- atic/general form). Though rare, recessive SLC4A4 mutations have been reported in patients with proximal renal tubular acidosis, short stature, and ocular pathology. A 27-year-old male presented with these findings. The purpose of this study was to investigate the molecular pathology responsible for this patient’s clinical findings. Methods: A comprehensive ophthalmic examination was performed, detailed ocular and systemic medical histories were taken and past medical records were obtained. Mutation screening was performed by using direct PCR sequencing of SLC4A4 exons and flanking intronic regions. Functional characterization of the mutation was made by expressing the wild-type and mutant NBCe1 proteins in Xenopus oocytes. Results: We identified a novel, homozygous, missense SLC4A4 mutation (Leu522Pro in kNBCe1) in our patient who had pRTA, short stature, enamel hypoplasia, and bilateral ocular disease (cataract, glaucoma, and band keratopathy). The patient also had a medical history of ataxia, migraine with transient hemiparesis attacks, and slight hypothyroidism. The mutant RNA failed to induce electrogenic transport activity. The L522P-protein was not effectively transported to the oocyte membrane and thus was unable to act as a transmembrane transporter. Conclusions: This novel mutation increases our understanding of the structural/functional aspects of the NBCe1 protein and the molecular basis of the multiorgan pathologies associated with its defects. expression has also been detected in other tissues such as heart, stomach, intestine, lung, thyroid, salivary glands, and pros- tate [1,4]. We have adopted the previously used nomenclature for SLC4A4 mutations based upon their locations in kNBCe1. The kidney seems to express all NBCe1 variants and their function accounts for 80-90% of renal HCO 3- absorption [1]. Therefore, the renal findings constitute the major phenotype for SLC4A4 mutations. Renal tubular acidosis (RTA) is a clini- cal syndrome characterized by hyperchloremic, metabolic aci- dosis resulting from defective renal acidification. Proximal RTA (pRTA) is caused by impairment of bicarbonate absorp- tion in proximal tubules with decreased renal HCO 3- thresh- old, while the distal tubule acidification remains intact. A rare syndrome was described in patients with SLC4A4 mutations that was characterized by autosomal recessive (AR) perma- nent isolated pRTA and hypokalemia (blood pH <7.2, blood HCO 3- =5-11 mM/L, serum K + =2.6-3.3 mEq/l), short stature, and ocular pathology (glaucoma, band keratopathy, cataract) [5-8]. Additional features may include enamel defects of per- manent teeth and mental retardation [5-7]. Calcification of basal ganglia, hyperamylasemia, and hypothyroidism were also noted in some patients [4]. Eight homozygous SLC4A4 muta- tions (6 missense-R298S, S427L, T485S, R510H, A799V, R881C; one nonsense-Q29X; and one frameshift-2311delA) have been published to date [5-9]. One additional mutation (a Maintenance of blood pH within a narrow range (7.35- 7.45) is crucial for essential biochemical and metabolic func- tions. The kidneys have a key role in homeostasis because of their ability to reabsorb HCO 3- and excrete acid. The basolateral membrane, electrogenic Na + /HCO 3- cotransporter (NBCe1) plays a major role in renal bicarbonate absorption via the proxi- mal tubules [1]. The human gene for NBCe1 protein (SLC4A4) is located on chromosome 4q21 and produces two major tran- scripts with 5'-end variants resulting from alternative promoter usage [2]. The pNBCe1 transcript is generated from the pri- mary promoter and is expressed in most tissues, including the pancreas [1]. The kNBCe1 transcript uses an alternative pro- moter and transcription initiation site within intron 3 of SLC4A4 and was originally cloned from kidney [2]. kNBCe1 and pNBCe1 are composed of 1,035 and 1,079 amino acid residues, respectively. kNBCe1 differs from pNBCe1 with its unique NH 2 -terminal that harbors 41 amino acids (in lieu of the first 85 amino acids of pNBCe1) [2]. A third isoform, re- ported in rat but not yet in human, is found in brain and uses the pNBCe1 promoter but a novel C-terminus [3]. NBCe1 Correspondence to: Michael B. Gorin, MD, PhD, UPMC Eye Cen- ter, University of Pittsburgh, 203 Lothrop Street, EEINS Building, Rm 1025, Pittsburgh, PA, 15213; Phone: (412) 647-7726; FAX: (412) 647-5880; email: gorinmb@upmc.edu

A genome-wide scan maps a novel juvenile-onset primary open angle glaucoma locus to chromosome 5q.

Abstract: PURPOSE To map the disease-associated locus of a family with autosomal dominant juvenile-onset primary open angle glaucoma (JOAG) and to screen the novel glaucoma gene WD repeat domain 36 (WDR36). METHODS Complete ophthalmic examination and genomic DNA were obtained from 27 family members, in which nine were confirmed JOAG patients. Myocilin (MYOC), optineurin (OPTN), and WDR36 were screened for mutations by polymerase chain reaction and direct sequencing. Genome-wide scanning was carried out using the ABI PRISM Linkage Mapping Set MD-10. Two-point and multipoint linkage analyses were performed with the MLINK, ILINK, and LINKMAP programs. For fine mapping, additional markers flanking the most promising region on chromosome 5q were also analyzed. The significance of LOD scores was tested with simulation analyses using FASTLINK. Haplotypes were constructed using Simwalk2. RESULTS MYOC or OPTN mutations were excluded in all family members. A maximum LOD score value of 4.82 at theta=0.00 was obtained for the marker D5S2011. Markers D5S2065, D5S1384, D5S471, D5S503, D5S2098, and D5S638 had LOD score values over 4.0 at theta=0.00. Haplotype analysis and recombination mapping further confined this region to 5q22.1-q32 within a region of 36 Mb flanked by D5S2051 and D5S2090. Screening of the novel WDR36 glaucoma-associated gene, which lies centromeric to the disease interval, revealed no mutations within any of the 23 coding exons or splicing junctions. CONCLUSIONS Our results provided the mapping of a novel locus for JOAG at 5q and excluded coding or splicing junctions mutations within the WDR36 gene.

Luteinizing hormone-releasing hormone agonist and transferrin functionalizations enhance nanoparticle delivery in a novel bovine ex vivo eye model.

Abstract: Purpose To determine whether topical ocular delivery of Methods A novel ex vivo bovine eye model was validated for its integrity up to 60 min. Using this model, the uptake of 20 nm polystyrene nanoparticles (administered as a single 50 mul drop) before and after surface conjugation with deslorelin, a luteinizing hormone-releasing hormone (LHRH) agonist, or transferrin was determined at 5 and 60 min in individual layers of cornea and aqueous humor. Selected studies were done in the absence of corneal epithelium in the ex vivo model or using excised cornea and conjunctiva. LHRH and transferrin receptor mRNA and protein expression in corneal epithelium and conjunctiva were determined by real-time PCR and western blot, respectively. Results Corneal histology, ZO-1 immunostain pattern, and mannitol permeability were similar in controls and at the end of the ex vivo study. Corneal epithelial nanoparticle uptake in the absence of surface modification was 1.1-1.6% at 5 min and remained at about this level even at 60 min. Removal of the corneal epithelium resulted in about 22% particle uptake in the corneal stroma at 5 and 60 min compared to about 0.5% in the presence of epithelium, indicating the barrier nature of corneal epithelium. Deslorelin and transferrin conjugation enhanced corneal epithelial uptake of nanoparticles by 3- and 4.5 fold at 5 min and by 4.5- and 3.8 fold at 60 min, respectively. The total corneal uptake in 5 min is approximately 2.4, 9, and 16% with plain, deslorelin-functionalized, and transferrin-functionalized nanoparticles. In all groups, the nanoparticle uptake per unit tissue weight was in the order: corneal epithelium>stroma>endothelium with levels in the aqueous humor being undetectable. In excised cornea and conjunctiva studies, nanoparticle transport and uptake was elevated for both deslorelin and transferrin conjugated nanoparticles. Expression of LHRH and transferrin receptors was observed in corneal epithelium as well as conjunctiva. Conclusions The ex vivo bovine eye model is a useful tool in understanding disposition of nanoparticles after topical delivery. The corneal epithelium is a significant barrier for topical nanoparticle delivery to the anterior segment. Surface modification of nanoparticles by conjugating an LHRH agonist or transferrin is a useful approach to provide rapid, efficient delivery of intact nanoparticles into and/or across cornea and conjunctiva.

Novel mutations in GJA8 associated with autosomal dominant congenital cataract and microcornea.

Abstract: Purpose: The purpose of this study was to estimate the importance of mutations in the connexin50 gene ( GJA8) as a cause of congenital or developmental cataracts in the Indian population and to identify novel mutations in GJA8 that cause cataract in this population. Methods: The coding region of GJA8 was analyzed for mutation by single strand conformational polymorphism in 60 probands affected with congenital or developmental cataract of which 11 probands’ corneal diameter measured less than 11.00 mm. Direct sequencing was performed for samples that displayed an abnormal electrophoresis pattern. The segregation of the change with the diseased phenotype was analyzed in the entire pedigree by restriction fragment length polymorphism (RFLP) analysis. Results: Molecular analysis of GJA8 revealed two novel missense mutations V44E and R198Q, in the population screened. The mutations cosegregated with the diseased phenotype in an autosomal dominant manner and were absent in 400 normal control chromosomes analyzed. GJA8 mutations were seen in two of the 60 unrelated probands with cataracts. Affected individuals in both of whose families also had microcornea and variable myopia. Conclusions: This is the first report of mutations in GJA8 to be associated with autosomal dominant cataract and microcornea. Mutations in GJA8 cause 3.3% of congenital cataracts in the population of India.

Analysis of variants in the complement factor H, the elongation of very long chain fatty acids-like 4 and the hemicentin 1 genes of age-related macular degeneration in the Finnish population.

Abstract: PURPOSE: A strong association of a Tyr402His polymorphism in the complement factor H (CFH) gene and a Met299Val polymorphism in the elongation of very long chain fatty acids-like 4 (ELOVL4) gene with age-related macular degeneration (AMD) has been identified in Caucasian populations in the United States. Earlier a Gln5345Arg variant in the hemicentin 1 (HMCN1) gene was reported in a large AMD family in the United States. We wanted to investigate whether the polymorphisms of the CFH and the ELOVL4 genes or the mutation of the HMCN1 gene are associated with AMD in patients originating from the Finnish population with characteristics of a genetic isolate. METHODS: The material consisted of familial (n=181) and sporadic cases (n=154) with AMD, a control group with no AMD (non-AMD controls, n=105), and a control group of anonymous blood donors (blood donor controls, n =350). The DNA of the subjects was sequenced to analyze the variants of the three genes. RESULTS: We detected a strong association between the C/C-genotype compared to the T/T-genotype of Tyr402His polymorphism (first base of the Tyr-codon changes) of the CFH gene and AMD in the AMD cases compared to the non-AMD (p=8.86x10(-12)) or to blood donor controls (p=2.02x10(-13)). The frequency of the C/C genotype was significantly increased in both familial cases compared to non-AMD controls with non-adjusted odds ratio (OR) 10.1 (confidence intervals [CI] 95% 4.64-22.2) or compared to blood donor controls with non-adjusted OR 5.50 (CI 95% 3.17-9.55) and in sporadic cases with non-adjusted OR 9.33 (CI 95% 4.10-21.3; non-AMD-controls), OR 5.06 (CI 95% 2.75-9.28; blood donor controls). Frequency of C allele differed significantly between cases and controls (p=1.32x10(-11); non-AMD-controls and p=3.94x10(-14); blood donor controls). No association with AMD was detected with Met299Val polymorphism in the ELOVL4 gene in the familial or sporadic cases compared to non-AMD or blood donor controls. None of our subjects (258 AMD cases, 72 non-AMD controls) had the Gln5345Arg variant in the HMCN1 gene. CONCLUSIONS: The CFH gene polymorphism seems to be an important etiologic factor for AMD also in the isolated Finnish population.

Heterozygous CYP1B1 gene mutations in Spanish patients with primary open-angle glaucoma.

Abstract: PURPOSE: To investigate CYP1B1 gene mutations in Spanish patients with ocular hypertension (OHT) or primary open angle glaucoma (POAG). METHODS: The two coding exons of CYP1B1 were screened for sequence alterations by direct PCR DNA sequencing in 37 and 82 unrelated Spanish subjects diagnosed with OHT and POAG, respectively. As a control we used a group of 93 subjects from whom OHT or glaucoma were ruled out. RESULTS: We found three different predicted amino acid substitutions (Ala189Pro, Ala330Ser, and Ala443Gly) in three (8.1%) OHT subjects, and seven different mutations (Ser28Trp, Gly61Glu, Tyr81Asn, Gln144His, Arg145Trp, Glu229Lys, and Val409Phe) in nine (10.9%) glaucoma patients. These sequence variations showed higher frequencies in cases than in controls (as recently reported in French patients). They are predicted to produce a significant change in the amino acid sequence and affect conserved regions of the protein. All these missense mutations were found as heterozygots. In addition, four of them have been previously found in PCG and/or POAG patients, whereas the other six mutations (Ser28Trp, Gln144His, Arg145Trp, Ala189Pro, Ala330Ser, and Val409Phe) have not been previously described. Clinically, these mutations are associated with an age at diagnosis ranging from 12 to 58 years (mean 34.3 years) and from 48 to 77 years (mean 59.9 years) among OHT and glaucoma patients, respectively. CONCLUSIONS: Heterozygous CYP1B1 mutations could confer increased susceptibility to the development of POAG in the Spanish population.

Identification of a novel, putative cataract-causing allele in CRYAA (G98R) in an Indian family.

Abstract: PURPOSE The aim of the present study was to investigate the molecular basis underlying a nonsyndromic presenile autosomal dominant cataract in a three-generation pedigree. The phenotype was progressive from a peripheral ring-like opacity to a total cataract with advancing age from teenage to adulthood. The visual impairment started as problem in distant vision at the age of 16 years, to diminishing vision by the age of 24. METHODS Clinical interventions included complete ophthalmological examination, a collection of case history, and pedigree details. Blood samples were collected from available family members irrespective of their clinical status. A functional candidate gene approach was employed for PCR screening and sequencing of the exons and their flanking regions of CRYGC, CRYGD, and CRYAA genes. For structural consequences of the mutated alphaA-crystallin we used the bioinformatics tool of the ExPASy server. RESULTS Sequence analysis of CRYGC and CRYGD genes excluded possible causative mutations but identified known polymorphisms. Sequencing of the exons of the CRYAA gene identified a sequence variation in exon 2 (292 G->A) with a substitution of Gly to Arg at position 98. All three affected members revealed this change but it was not observed in the unaffected father or sister. The putative mutation obliterated a restriction site for the enzyme BstDSI. The same was checked in controls representing the general population of the same ethnicity (n=30) and of randomly selected DNA samples from ophthalmologically normal individuals from the population-based KORA S4 study (n=96). Moreover, the Gly at position 98 is highly conserved throughout the animal kingdom. For the mutant protein, the isoelectric point was raised from pH 5.77 to 5.96. Moreover, an extended alpha-helical structure is predicted in this region. CONCLUSIONS The G98R mutation segregates only in affected family members and is not seen in representative controls. It represents very likely the fourth dominant cataract-causing allele in CRYAA. In all reported alleles the basic amino acid Arg is involved, suggesting the major importance of the net charge of the alphaA-crystallin for functional integrity in the lens.

Macular and peripheral distribution of ICAM-1 in the human choriocapillaris and retina.

Abstract: PURPOSE In order to understand the extent of choriocapillary endothelial cell activation in different topographic regions of the eye, we sought to compare the localization of intercellular adhesion molecule-1 (ICAM-1) in macular and peripheral regions of human eyes. METHODS Sections of sucrose-embedded human donor eyes that included the macula and ora serrata were evaluated for ICAM-1 and ICAM-2 immunoreactivity with monoclonal and polyclonal antibodies. Patterns of ICAM-1 labeling in peripheral and macular regions were examined in 20 eyes. Morphometric analyses of anti-ICAM-1 labeling intensity in the choriocapillaris were performed using ImageJ software on a series of macular and extramacular punches from nine eyes. Quantitative PCR analysis for ICAM-1 mRNA was performed on the RPE-choroid from the same regions from six of the same eyes, and Western blots of samples treated or untreated with N-glycosidase were performed to compare retinal and choroidal ICAM-1. RESULTS ICAM-1 labeling of the choriocapillaris was typically more intense in the macula than in the peripheral choroid in human donor eyes (14/20). ICAM-2 was also detected in the choriocapillaris and retinal vessels. Morphometric measurements confirmed a significant macular-extramacular difference in ICAM-1 in six of nine eyes (p<0.05), with 1 of 9 eyes showing the opposite pattern. This pattern was not noted for endogenous alkaline phosphatase or ICAM-2. The opposite pattern was noted in the external limiting membrane (ELM), which exhibited more intense ICAM-1 labeling in the far periphery than in the macula. On Western blots, choroidal ICAM-1 exhibited a greater molecular weight than the retinal form, with most of the apparent weight difference due to N-linked carbohydrate chains. CONCLUSIONS The regional differences in ICAM-1 distribution in the choriocapillaris may indicate that this region is subject to increased leukocyte trafficking. In view of the role of inflammatory processes in age-related macular degeneration (AMD), we propose that the higher level of ICAM-1 protein in the macular choriocapillaris may impart greater susceptibility of the macula to immune cell-mediated damage in AMD.

Mouse opsin promoter-directed Cre recombinase expression in transgenic mice.

Abstract: Purpose Gene inactivation with homologous recombination in mice is a widely used tool to study gene function. However, many proteins play essential roles in a number of tissues and germline gene inactivation often results in embryonic lethality. To overcome this limitation and to dissect the functions of essential genes beyond embryonic development, we generated mouse rod opsin promoter-controlled cre transgenic mice with a goal of obtaining transgenic lines with a range of Cre activity in rod photoreceptors. Methods Transgenic mice expressing Cre recombinase directed by a long or short mouse opsin promoter were generated. Candidate Cre-expressing lines were identified with RT-PCR and Western blot analysis. Potentially useful Cre-expressing lines were characterized further with immunohistochemistry, PCR, and functional analysis using a Cre-activatable lacZ reporter mouse strain (R26R) to determine temporal and spatial patterns of Cre expression. Retinal function and morphology in these mouse lines were analyzed with electroretinography (ERG) and light microscopy of hematoxylin and eosin stained retinal sections. Results Transgenic mice expressing Cre in rod photoreceptors were generated. Characterization of candidate photoreceptor-specific Cre mice using immunohistochemistry and functional assays demonstrated that an efficient Cre-mediated recombination occurred in rod photoreceptor cells in one mouse line and a mosaic Cre-mediated recombination occurred in rod photoreceptors and rod bipolar cells in another mouse line. Further analysis of these mice with ERG and morphological examination suggested that the retinas of eight-month-old adults were normal. Conclusions We have generated transgenic mice expressing Cre recombinase in rod photoreceptors. One transgenic mouse line was capable of carrying out efficient Cre-mediated recombination in rod photoreceptors. Another transgenic mouse line was capable of carrying out mosaic Cre-mediated recombination in rod photoreceptors and bipolar cells across the whole retina. These mice will be useful tools for Cre/lox-based gene activation and inactivation, as well as genetic mosaics, in rod photoreceptors and rod bipolar cells.

Proceedings of the Third International Symposium on Retinopathy of Prematurity: an update on ROP from the lab to the nursery (November 2003, Anaheim, California).

Abstract: The Third International Symposium on Retinopathy of Prematurity (ROP) was convened with the aim of cross fertilizing the horizons of basic and clinical scientists with an interest in the pathogenesis and management of infants with ROP. Ten speakers in the clinical sciences and ten speakers in the basic sciences were recruited on the basis of their research to provide state of the art talks. The meeting was held November 9, 2003 immediately prior to the American Academy of Ophthalmology meeting; scholarships were provided for outreach to developing countries and young investigators. This review contain the summaries of the 20 platform presentations prepared by the authors and the abstracts of presented posters. Each author was asked to encapsulate the current state of understanding, identify areas of controversy, and make recommendations for future research. The basic science presentations included insights into the development of the human retinal vasculature, animal models for ROP, growth factors that affect normal development and ROP, and promising new therapeutic approaches to treating ROP like VEGF targeting, inhibition of proteases, stem cells, ribozymes to silence genes, and gene therapy to deliver antiangiogenic agents. The clinical presentations included new insights into oxygen management, updates on the CRYO-ROP and ETROP studies, visual function in childhood following ROP, the neural retina in ROP, screening for ROP, management of stage 3 and 4 ROP, ROP in the third world, and the complications of ROP in adult life. The meeting resulted in a penetrating exchange between clinicians and basic scientists, which provided great insights for conference attendees. The effect of preterm delivery on the normal cross-talk of neuroretinal and retinal vascular development is a fertile ground for discovering new understanding of the processes involved both in normal development and in retinal neovascular disorders. The meeting also suggested promising potential therapeutic interventions on the horizon for ROP.

Ocular gene therapy: a review of nonviral strategies.

Abstract: Along with viral vectors, non-viral strategies have been developed in order to efficiently deliver nucleic acids to ocular cells. During the last decade, we have observed that the outcome of these non-viral delivery systems depends on the genetic material used, the targeted tissue or cells, the expected effect duration, and the routes of administration. Assessment of efficiency has been evaluated in normal eyes or in animal models of ocular diseases. The chemical and physical methods that have been adapted for the delivery of nucleic acids to ocular tissues are highlighted and discussed in this review. Also, the results obtained with different non-viral strategies from their initial conception to their present development are summarized. At the present, selective targeting of ocular tissues and cells can be achieved using the most yielding route of administration to the eye in combination with an appropriate drug delivery technique.

Autologous transplantation of rabbit limbal epithelia cultured on fibrin gels for ocular surface reconstruction

Abstract: Purpose Regeneration of the corneal epithelium could be severely impaired in patients suffering from limbal stem cell deficiency. The purpose of this study was to evaluate the restoration of the corneal epithelium by grafting onto denuded corneas autologous limbal cells cultured on fibrin gels. The rabbit model was chosen to allow the microscopic evaluation over time after grafting. Methods Rabbit limbal epithelial cells (RLECs) were isolated and cultured from small limbal biopsies (3 mm2). The epithelium was separated from stroma after dispase digestion and put in culture on lethally irradiated fibroblasts used as a feeder layer. At the first passage, RLECs were cultured on a fibrin gel matrix. At confluence, the cultured epithelia were grafted in vivo on denuded autologous rabbit corneas. At different postoperative times, grafted and control (without graft or grafted with fibrin gels only) rabbit corneas were compared in vivo with a slit lamp microscope, and in situ by histological and immunohistological microscopy of harvested biopsies. Results A small limbal biopsy was sufficient to generate enough RLECs to prepare several grafts and to perform cell analysis. Only two weeks were required to produce a cultured epithelium suitable for autologous transplantation. One month after grafting, a normal corneal phenotype was observed on the ocular surface of grafted rabbits in contrast to the control rabbits (ungrafted or grafted with fibrin gel only) where histological signs of conjunctivalization were found. The absence of goblet cells and negative staining for keratin 4 confirmed that the cultured cells persisted and that the epithelium regenerated after grafting was not from conjunctival origin. Conclusions Our results demonstrate that an autologous epithelium cultured on a physiologically biodegradable matrix can be prepared from a small biopsy and grafted on denuded cornea. The autologous graft allows epithelial regeneration from cultured cells and promotes corneal healing of unilateral total stem cell deficiency.