Search

Purpose: Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process., Methods: To study how gene expression is altered in focal areas of pathology, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We performed differential expression to identify genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes., Results: Within the area of neovascularization, endothelial cells demonstrated increased expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we investigated regional gene expression patterns within the macular neural retina and between the macular and peripheral choroid., Conclusions: Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

Purpose: Age-related macular degeneration (AMD), the most common cause of incurable blindness in the western world, is characterized by the dysfunction and eventual death of choroidal endothelial (CECs), RPE, and photoreceptor cells. Stem cell-based treatment strategies designed to replace photoreceptor and RPE cells currently are a major scientific focus. However, the success of these approaches likely also will require replacement of the underlying, supportive choroidal vasculature. The purpose of this study was to generate stem cell-derived CECs to develop efficient differentiation and transplantation protocols., Methods: Dermal fibroblasts from the Tie2-GFP mouse were isolated and reprogrammed into two independent induced pluripotent stem cell (iPSC) lines via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Tie2-GFP iPSCs were differentiated into CECs using a coculture method with either the RF6A CEC line or primary mouse CECs. Induced pluripotent stem cell-derived CECs were characterized via RT-PCR and immunocytochemistry for EC- and CEC-specific markers., Results: Induced pluripotent stem cells generated from mice expressing green fluorescent protein (GFP) under control of the endothelial Tie2 promoter display classic pluripotency markers and stem cell morphology. Induced pluripotent stem cell-derived CECs express carbonic anhydrase IV, eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF., Conclusions: Induced pluripotent stem cell-derived CECs will be a valuable tool for modeling of choriocapillaris-specific insults in AMD and for use in future choroidal endothelial cell replacement approaches.

Purpose: The purpose of this study was to identify the structural and histological effects of a Tano diamond-dusted membrane scraper (DDMS) on the retinal surface after internal limiting membrane (ILM) abrasion in macular hole surgery., Methods: Institutional experimental study was performed in 11 eyes. All eyes underwent ILM abrasion in the operating room with a DDMS for macular hole repair as an alternative to traditional ILM peeling. Three human donor eyes underwent an identical procedure in the laboratory. Retinal tissues were removed by ILM abrasion with a DDMS during vitrectomy for macular hole repair and retinal tissues remaining in human donor eyes. Main outcome measures were microscopic and immunohistological characteristics of instrument tip tissues and retinal structure after ILM abrasion., Results: The tips of the Tano DDMS showed evidence of cellular membranes and ILM removal. The retinas showed distinct areas of lamellar ILM removal without penetration of the retinal nerve fiber layer (RNFL)., Conclusions: Application of the Tano DDMS instrument is sufficient to remove membranes from the surface of the ILM and layers of the ILM without disruption of the underlying RNFL. Internal limiting membrane abrasion can be a useful and effective alternative to complete ILM removal for macular surgery.

Purpose: To evaluate the validity of a novel fully automated three-dimensional (3D) method capable of segmenting the choroid from two different optical coherence tomography scanners: swept-source OCT (SS-OCT) and spectral-domain OCT (SD-OCT)., Methods: One hundred eight subjects were imaged using SS-OCT and SD-OCT. A 3D method was used to segment the choroid and quantify the choroidal thickness along each A-scan. The segmented choroidal posterior boundary was evaluated by comparing to manual segmentation. Differences were assessed to test the agreement between segmentation results of the same subject. Choroidal thickness was defined as the Euclidian distance between Bruch's membrane and the choroidal posterior boundary, and reproducibility was analyzed using automatically and manually determined choroidal thicknesses., Results: For SS-OCT, the average choroidal thickness of the entire 6- by 6-mm2 macular region was 219.5 μm (95% confidence interval [CI], 204.9-234.2 μm), and for SD-OCT it was 209.5 μm (95% CI, 197.9-221.0 μm). The agreement between automated and manual segmentations was high: Average relative difference was less than 5 μm, and average absolute difference was less than 15 μm. Reproducibility of choroidal thickness between repeated SS-OCT scans was high (coefficient of variation [CV] of 3.3%, intraclass correlation coefficient [ICC] of 0.98), and differences between SS-OCT and SD-OCT results were small (CV of 11.0%, ICC of 0.73)., Conclusions: We have developed a fully automated 3D method for segmenting the choroid and quantifying choroidal thickness along each A-scan. The method yielded high validity. Our method can be used reliably to study local choroidal changes and may improve the diagnosis and management of patients with ocular diseases in which the choroid is affected.

Purpose: We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions., Methods: Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed., Results: Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78)., Conclusions: Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.

Purpose: In animal models of degenerative photoreceptor disease, there has been some success in restoring photoreception by transplanting stem cell-derived photoreceptor cells into the subretinal space. However, only a small proportion of transplanted cells develop extended outer segments, considered critical for photoreceptor cell function. The purpose of this study was to determine whether photoreceptor cells that lack a fully formed outer segment could usefully contribute to vision., Methods: Retinal and visual function was tested in wild-type and Rds mice at 90 days of age (Rds(P90)). Photoreceptor cells of mice homozygous for the Rds mutation in peripherin 2 never develop a fully formed outer segment. The electroretinogram and multielectrode recording of retinal ganglion cells were used to test retinal responses to light. Three distinct visual behaviors were used to assess visual capabilities: the optokinetic tracking response, the discrimination-based visual water task, and a measure of the effect of vision on wheel running., Results: Rds(P90) mice had reduced but measurable electroretinogram responses to light, and exhibited light-evoked responses in multiple types of retinal ganglion cells, the output neurons of the retina. In optokinetic and discrimination-based tests, acuity was measurable but reduced, most notably when contrast was decreased. The wheel running test showed that Rds(P90) mice needed 3 log units brighter luminance than wild type to support useful vision (10 cd/m(2))., Conclusions: Photoreceptors that lack fully formed outer segments can support useful vision. This challenges the idea that normal cellular structure needs to be completely reproduced for transplanted cells to contribute to useful vision.

Purpose: The choroid plays a vital role in the health of the outer retina. While measurements of choroid using optical coherence tomography show altered thickness in aging and macular disease, detailed histopathologic and proteomic analyses are lacking. In this study we sought to evaluate biochemical differences in human donor eyes between very thin and thick choroids., Methods: One hundred forty-one eyes from 104 donors (mean age ± standard deviation, 81.5 ± 12.2) were studied. Macular sections were collected, and the distance between Bruch's membrane and the inner surface of the sclera was measured in control, early/dry age-related macular degeneration (AMD), neovascular AMD, and geographic atrophy eyes. Proteins from the RPE-choroid of eyes with thick and thin choroids were analyzed using two-dimensional electrophoresis and/or mass spectrometry. Two proteins with altered abundance were confirmed using Western blot analysis., Results: Donor eyes showed a normal distribution of thicknesses. Eyes with geographic atrophy had significantly thinner choroids than age-matched controls or early AMD eyes. Proteomic analysis showed higher levels of the serine protease SERPINA3 in thick choroids and increased levels of tissue inhibitor of metalloproteinases-3 (TIMP3) in thin choroids., Conclusions: Consistent with clinical imaging observations, geographic atrophy was associated with choroidal thinning. Biochemical data suggest an alteration in the balance between proteases and protease inhibitors in eyes that lie at the extremes of choroidal thickness. An improved understanding of the basic mechanisms associated with choroidal thinning may guide the development of new therapies for AMD.

Purpose: Several lines of evidence suggest the involvement of antibodies and immune complex inflammation in AMD, a blinding disease with a strong inflammatory component. To examine this further, we developed a novel experimental mouse model of retinal inflammation and evaluated whether inflammation associated with immune complex formation was present in eyes of AMD donors., Methods: A localized immune complex-mediated reaction was induced in the retina of wild-type (WT), Fc receptor γ chain-deficient (γ(-/-)), and C1q-deficient (C1q(-/-)) mice, and donor eyes were obtained after death from donors with early or wet AMD and from healthy control subjects. The presence of immune complexes, Fcγ receptors (FcγRs), and markers of macrophage/microglia activation was investigated by immunohistochemistry., Results: In WT and C1q(-/-) mice, immune complex deposition in the retina led to a robust inflammatory response with activation of microglia, recruitment of myeloid cells, and increased expression of FcγRI through FcγRIV and major histocompatibility complex class II. This response was not observed in γ(-/-) mice lacking activating FcγRs. We found that early AMD was associated with deposition of IgG, C1q, and membrane attack complex in the choriocapillaris and with increased numbers of CD45+ cells expressing FcγRIIa and FcγRIIb. Furthermore, FcγRIIa and FcγRIIb were observed in eyes of donors with wet AMD., Conclusions: Our studies suggest that immune complexes may contribute to AMD pathogenesis through interaction of IgG with FcγRs and might inform about possible adverse effects associated with therapeutic antibodies.

Purpose: To study safety and efficacy of subretinal adeno-associated virus (AAV) vector AAV-Bbs1 injection for treatment of a mouse model of Bardet-Biedl syndrome type 1 (BBS1)., Methods: Constructs containing a wild-type (WT) Bbs1 gene with and without a FLAG tag in AAV2/5 vectors were generated. Viral genomes were delivered by subretinal injection to right eyes and sham injections to left eyes at postnatal day 30 (P30) to P60. Transgene expression and BBSome reconstitution were evaluated by immunohistochemistry and Western blotting following sucrose gradient ultracentrifugation. Retinal function was analyzed by electroretinogram (ERG) and structure by optical coherence tomography (OCT). Histology and immunohistochemistry were performed on selected eyes., Results: Expression of FLAG-tagged Bbs1 was demonstrated in photoreceptor cells using antibody directed against the FLAG tag. Coinjection of AAV-GFP demonstrated transduction of 24% to 32% of the retina. Western blotting demonstrated BBS1 protein expression and reconstitution of the BBSome. ERG dark-adapted bright flash b-wave amplitudes were higher in AAV-Bbs1-injected eyes than in sham-injected fellow eyes in more than 50% of 19 animals. Anti-rhodopsin staining demonstrated improved localization of rhodopsin in AAV-Bbs1-treated eyes. WT retinas injected with AAV-Bbs1 with or without a FLAG tag showed outer retinal degeneration on ERG, OCT, and histology., Conclusions: In a knock-in model of BBS1, subretinal delivery of AAV-Bbs1 rescues BBSome formation and rhodopsin localization, and shows a trend toward improved ERG. BBS is challenging to treat with gene therapy due to the stoichiometry of the BBSome protein complex and overexpression toxicity.

Purpose: We investigated the evidence of recent positive selection in the human phototransduction system at single nucleotide polymorphism (SNP) and gene level., Methods: SNP genotyping data from the International HapMap Project for European, Eastern Asian, and African populations was used to discover differences in haplotype length and allele frequency between these populations. Numeric selection metrics were computed for each SNP and aggregated into gene-level metrics to measure evidence of recent positive selection. The level of recent positive selection in phototransduction genes was evaluated and compared to a set of genes shown previously to be under recent selection, and a set of highly conserved genes as positive and negative controls, respectively., Results: Six of 20 phototransduction genes evaluated had gene-level selection metrics above the 90th percentile: RGS9, GNB1, RHO, PDE6G, GNAT1, and SLC24A1. The selection signal across these genes was found to be of similar magnitude to the positive control genes and much greater than the negative control genes., Conclusions: There is evidence for selective pressure in the genes involved in retinal phototransduction, and traces of this selective pressure can be demonstrated using SNP-level and gene-level metrics of allelic variation. We hypothesize that the selective pressure on these genes was related to their role in low light vision and retinal adaptation to ambient light changes. Uncovering the underlying genetics of evolutionary adaptations in phototransduction not only allows greater understanding of vision and visual diseases, but also the development of patient-specific diagnostic and intervention strategies.

Purpose: Best disease is a macular dystrophy caused by mutations in the BEST1 gene. Affected individuals exhibit a reduced electro-oculographic (EOG) response to changes in light exposure and have significantly longer outer segments (OS) than age-matched controls. The purpose of this study was to investigate the anatomical changes in the outer retina during dark and light adaptation in unaffected and Best disease subjects, and to compare these changes to the EOG., Methods: Unaffected (n = 11) and Best disease patients (n = 7) were imaged at approximately 4-minute intervals during an approximately 40-minute dark-light cycle using spectral domain optical coherence tomography (SD-OCT). EOGs of two subjects were obtained under the same conditions. Automated three-dimensional (3-D) segmentation allowed measurement of light-related changes in the distances between five retinal surfaces., Results: In normal subjects, there was a significant decrease in outer segment equivalent length (OSEL) of -2.14 μm (95% confidence interval [CI], -1.77 to -2.51 μm) 10 to 20 minutes after the start of light adaptation, while Best disease subjects exhibited a significant increase in OSEL of 2.07 μm (95% CI, 1.79-2.36 μm). The time course of the change in OS length corresponded to that of the EOG waveform., Conclusions: Our results strongly suggest that the light peak phase of the EOG is temporally related to a decreased OSEL in normal subjects, and the lack of a light peak phase in Best disease subjects is associated with an increase in OSEL. One potential role of Bestrophin-1 is to trigger an increase in the standing potential that approximates the OS to the apical surface of the RPE to facilitate phagocytosis.

Purpose: We developed and evaluated a fully automated 3-dimensional (3D) method for segmentation of the choroidal vessels, and quantification of choroidal vasculature thickness and choriocapillaris-equivalent thickness of the macula, and evaluated repeat variability in normal subjects using standard clinically available spectral domain optical coherence tomography (SD-OCT)., Methods: A total of 24 normal subjects was imaged twice, using clinically available, 3D SD-OCT. A novel, fully-automated 3D method was used to segment and visualize the choroidal vasculature in macular scans. Local choroidal vasculature and choriocapillaris-equivalent thicknesses were determined. Reproducibility on repeat imaging was analyzed using overlapping rates, Dice coefficient, and root mean square coefficient of variation (CV) of choroidal vasculature and choriocapillaris-equivalent thicknesses., Results: For the 6 × 6 mm(2) macula-centered region as depicted by the SD-OCT, average choroidal vasculature thickness in normal subjects was 172.1 μm (95% confidence interval [CI] 163.7-180.5 μm) and average choriocapillaris-equivalent thickness was 23.1 μm (95% CI 20.0-26.2 μm). Overlapping rates were 0.79 ± 0.07 and 0.75 ± 0.06, Dice coefficient was 0.78 ± 0.08, CV of choroidal vasculature thickness was 8.0% (95% CI 6.3%-9.4%), and of choriocapillaris-equivalent thickness was 27.9% (95% CI 21.0%-33.3%)., Conclusions: Fully automated 3D segmentation and quantitative analysis of the choroidal vasculature and choriocapillaris-equivalent thickness demonstrated excellent reproducibility in repeat scans (CV 8.0%) and good reproducibility of choriocapillaris-equivalent thickness (CV 27.9%). Our method has the potential to improve the diagnosis and management of patients with eye diseases in which the choroid is affected.

Purpose: Time and spectrally resolved measurements of autofluorescence have the potential to monitor metabolism at the cellular level. Fluorophores that emit with the same fluorescence intensity can be discriminated from each other by decay time of fluorescence intensity after pulsed excitation. We performed time-resolved autofluorescence measurements on fundus samples from a donor with significant extramacular drusen., Methods: Tissue sections from two human donors were prepared and imaged with a laser scanning microscope. The sample was excited with a titanium-sapphire laser, which was tuned to 860 nm, and frequency doubled by a BBO crystal to 430 nm. The repetition rate was 76 MHz and the pulse width was 170 femtoseconds (fs). The time-resolved autofluorescence was recorded simultaneously in 16 spectral channels (445-605 nm) and bi-exponentially fitted., Results: RPE can be discriminated clearly from Bruch's membrane, drusen, and choroidal connective tissue by fluorescence lifetime. In RPE, bright fluorescence of lipofuscin could be detected with a maximum at 510 nm and extending beyond 600 nm. The lifetime was 385 ps. Different types of drusen were found. Most of them did not contain lipofuscin and exhibited a weak fluorescence, with a maximum at 470 nm. The lifetime was 1785 picoseconds (ps). Also, brightly emitting lesions, presumably representing basal laminar deposits, with fluorescence lifetimes longer than those recorded in RPE could be detected., Conclusions: The demonstrated differentiation of fluorescent structures by their fluorescence decay time is important for interpretation of in vivo measurements by the new fluorescence lifetime imaging (FLIM) ophthalmoscopy on healthy subjects as well as on patients.

Purpose: Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous condition characterized by progressive loss of retinal photoreceptor cells. In order to gain new insights into the pathogenesis of ARRP, we evaluated the morphological, biochemical, and gene expression changes in eyes from a human donor with ARRP due to mutations in the ABCA4 gene., Methods: Eyes were obtained postmortem from a donor with end-stage retinitis pigmentosa. The coding sequences of the RDS, RHO, and ABCA4 genes were screened for disease-causing mutations. Morphological changes in different regions of the retina were examined histologically, and levels of lipofuscin-associated bisretinoids were measured. Gene expression was examined in retinal/choroidal tissue using microarray analysis, and all parameters were compared to those in unaffected control donors., Results: Genetic analysis of the donor's DNA identified two mutations in the ABCA4 gene, IVS14+1G > C and Phe1440del1 cT, each on a separate allele. Morphological evaluation revealed complete loss of the outer nuclear layer, remodeling of the inner retina, loss of retinal vasculature, and regional neovascularization. The retinal pigment epithelium and choriocapillaris exhibited regional preservation. Microarray analysis revealed loss of photoreceptor cell-associated transcripts, with preservation of multiple genes expressed specifically in inner retinal neurons., Conclusions: The persistence of transcripts expressed by inner retinal neurons suggests that despite significant plasticity that occurs during retinal degeneration, bipolar cells and ganglion cells remain at least partially differentiated. Findings from this study suggest that some forms of therapy currently under investigation may have benefit even in advanced retinal degeneration.

Purpose: To investigate whether the benefit of Age-Related Eye Disease Study (AREDS) formula multivitamins and zinc in the progression of age-related macular degeneration (AMD) may occur through inhibiting inflammatory events in the choroid., Methods: Mouse C166 endothelial cells (ECs) and, for some experiments, human retinal pigment epithelium (RPE)-choroid organ cultures were treated with AREDS multivitamin solution (MVS) or ZnCl(2). The cytotoxicity of MVS was evaluated using a lactate dehydrogenase colorimetric assay. Cell motility was assessed using a scratch assay. Macrophage adhesion to EC monolayers or ICAM-1 protein was determined after MVS and zinc treatment and with or without lipopolysaccharide (LPS). Quantitative reverse transcription PCR and Western blot analysis were used to determine the effects of MVS on the expression of proinflammatory molecules in treated and untreated cells., Results: AREDS MVS and zinc did not affect C166 EC viability until the 56th hour after treatment. Scratch assays showed partial inhibition of MVS and zinc on EC migration. In cell adhesion assays, MVS and zinc decreased the number of macrophages bound to EC and to ICAM-1 protein. Quantitative PCR showed that LPS increased the expression of ICAM-1 in both C166 and human RPE-choroid cultures, which was partially offset by MVS and zinc. MVS and zinc also mitigated LPS-induced ICAM-1 protein expression on Western blot analysis., Conclusions: Treatment with AREDS MVS and zinc may affect both angiogenesis and endothelial-macrophage interactions. These results suggest that AREDS vitamins and zinc ions may slow the progression of AMD, in part through the attenuation of EC activation.

Purpose: Age-related macular degeneration (AMD) is a common, potentially blinding disease characterized by the presence of extracellular deposits beneath the retinal pigment epithelium (RPE). Choroidal vascular changes have also been noted in AMD. This study examined the relationship between the choroidal vasculature and extent of drusen and other sub-RPE deposits, the key pathologic landmarks of AMD., Methods: Sections of the maculas of 45 human eyes (21 early AMD and 24 age-matched control) were evaluated morphometrically. The cross-sectional area of sub-RPE deposits, vascular density, number of CD45+ leukocytes, and number of "ghost vessels" were determined in a masked fashion and evaluated by regression analysis. In addition, the extramacular vascular density either directly beneath drusen or adjacent to drusen was evaluated in a separate set of donor eyes., Results: The vascular density of the choriocapillaris showed a trend toward decreasing in association with AMD status. By linear regression analysis, vascular density was inversely associated with sub-RPE deposit density (r(2) = 0.22, P < 0.01). The number of ghost vessels was negatively correlated with vascular density (r(2) = 0.55, P < 0.001) and positively correlated with sub-RPE deposit density (r(2) = 0.57, P < 0.001). In morphologic studies of extramacular solitary drusen, vascular density beneath drusen was found to be 45% lower than adjacent to drusen (P < 0.01)., Conclusions: These findings support the concept that microvascular changes are related to the pathogenesis of AMD and suggest that vascular endothelial cell loss occurs in association with sub-RPE deposit formation. Whether microvascular events are a cause or consequence of drusen or other deposit formation remains to be determined.

Purpose: Detection of light in the eye contributes both to spatial awareness (form vision) and to responses that acclimate an animal to gross changes in light (irradiance detection). This dual role means that eye disease that disrupts form vision can also adversely affect physiology and behavioral state. The purpose of this study was to investigate how inner retinal circuitry mediating rod-cone photoreceptor input contributes to functionally distinct irradiance responses and whether that might account for phenotypic diversity in retinal disease., Methods: The sensitivity of the pupillary light reflex and negative masking (activity suppression by light) was measured in wild-type mice with intact inner retinal circuitry, Nob4 mice that lack ON-bipolar cell function, and rd1 mice that lack rods and cones and, therefore, have no input to ON or OFF bipolar cells., Results: An expected increase in sensitivity to negative masking with loss of photoreceptor input in rd1 was duplicated in Nob4 mice. In contrast, sensitivity of the pupillary light reflex was more severely reduced in rd1 than in Nob4 mice., Conclusions: Absence of ON-bipolar cell-mediated rod-cone input can fully explain the phenotype of outer retina degeneration for negative masking but not for the pupillary light reflex. Therefore, inner retinal pathways mediating rod-cone input are different for negative masking and the pupillary light reflex.

Purpose: The complement system plays a crucial role in the progression of age-related macular degeneration (AMD). In this study, the authors sought to evaluate the pathophysiologic roles of complement components C3a and C5a in the human choroid in AMD., Methods: Human RPE/choroid was assayed for the presence of C3a and C5a receptors (C3aR and C5aR) using RT-PCR and immunohistochemistry. Choroidal endothelial cell migration and proliferation were evaluated in the presence of C5a. Organ cultures of human choroid were incubated in C5a or bovine serum albumin (BSA) followed by quantitative immunohistochemistry and quantitative PCR for ICAM-1. AMD patients and controls were genotyped at SNPs in the C5R1 and C3AR1 genes., Results: C5aR, but not C3aR, was detected in human choroid. C5a did not promote endothelial cell migration or proliferation. However, choriocapillaris endothelial cells in organ culture responded to C5a by increasing ICAM-1 mRNA and protein. No significant association of SNP genotypes was detected in AMD patients at the C3AR1 and C5R1 genes., Conclusions: The generation of C5a peptides may lead to activation of choriocapillaris endothelial cells in AMD. Activation of the choroidal endothelium may affect the progression of AMD by recruitment of monocytes, leading to additional sequelae of AMD pathogenesis.

Purpose: Endothelial cell (EC) migration is a key event in angiogenesis, and is likely to play an important role in choroidal neovascularization in age-related macular degeneration (AMD). Altered elastin metabolism has been described in AMD, and the present study sought to determine the effects of elastin-derived peptides (EDPs) on choroidal EC migration and proliferation., Methods: Migration of the chorioretinal EC line Rf/6a and a primary culture of human choroidal ECs through polycarbonate membrane inserts was quantified in the presence of elastin bioactive hexapeptides (BPs), EDPs, bovine serum albumin (BSA), or balanced salt solution. Proliferation assays and in vitro wound closure experiments were also performed in the presence of elastin fragments or balanced salt solution (control). Elastin overlay experiments were performed on sections of human eyes., Results: For both Rf/6a and human primary choroidal ECs exposed to EDPs or BPs, the number of ECs that migrated through the polycarbonate membrane was significantly higher than ECs exposed to balanced salt solution alone or to BSA (P < 0.05) in all experiments. In contrast, the rate of EC proliferation did not significantly change in comparison to controls. Elastin binding sites were identified on choroidal ECs in human eyes., Conclusions: Elastin fragments increase choroidal EC migration, whereas they do not appear to increase or decrease EC proliferation. Local or systemic abnormalities in elastin physiology may participate in pathologic neovascular membrane formation in AMD.

Purpose: Tests of vision for mice remain limited and the visual phenotype of some retinal disorders in mice remain poorly understood. A novel assay of vision was used to determine how the form and extent of retinal disease affects visual phenotype in mice., Methods: Retinal histology, the suppression of locomotion by light and visual guidance of locomotion, were assessed in mice with progressive photoreceptor degeneration (rd/rd) or visual cycle dysfunction (Rpe65 rd12)., Results: In wild-type mice, there was visual guidance of locomotor activity in dim light and suppression of activity (negative masking) in bright light. In rd/rd mice, vision was sufficient to guide locomotion at postnatal day (P)34 but was lost from P46 onward. In bright light rd/rd mice had enhanced negative masking. Although Rpe65 rd12 mice had no dim light response, with high illumination, vision was sufficient to guide locomotion at all ages tested., Conclusions: A major concern for gene and cell replacement therapies is the development of visual pathways through which restored retinal function can connect to visual centers of the brain. The residual retinal response to high illumination in Rpe65 rd12 mice translates into useful vision, and visual pathways remain functional--a prerequisite for restoring vision in disorders of the retina. Similarly, useful vision in young rd/rd mice shows that there is visual pathway function before photoreceptor degeneration and suggests the potential for early therapy. Together, these findings recommend observation of masking responses in the assessment of gene and cell replacement therapies.

Purpose: To identify and characterize gene expression changes associated with photoreceptor cell loss in a Bbs4-knockout mouse model of retinal degeneration., Methods: Differential gene expression in the eyes of 5-month-old Bbs4(-/-) mice undergoing retinal degeneration were analyzed using gene microarrays (Affymetrix, Santa Clara, CA). Elevated ocular transcripts were confirmed by Northern blotting of RNA from Bbs4(-/-) and three additional mouse models of Bardet-Biedl Syndrome (BBS). TUNEL assays and transmission electron microscopy were used to study cell death and photoreceptor morphology in these mice., Results: Three hundred fifty-four probes were differentially expressed in Bbs4(-/-) eyes compared with controls using a twofold cutoff. Numerous vision-related transcripts decreased because of photoreceptor cell loss. Increased expression of the stress response genes Edn2, Lcn2, Serpina3n, and Socs3 was noted at 5 months of age and as early as postnatal week 4 in the eyes of four BBS mouse model strains. A burst of apoptotic activity in the photoreceptor outer nuclear layer at postnatal week 2 and highly disorganized outer segments by postnatal weeks 4 to 6 was observed in all four strains., Conclusions: The specific loss of photoreceptors in Bbs4(-)(/)(-) mice allows us to identify a set of genes that are preferentially expressed in photoreceptors compared with other cell types found in the eye and is a valuable resource in the continuing search for genes involved in retinal disease. The molecular and morphologic changes observed in young BBS animal model eyes implies that BBS proteins play a critical, early role in establishing the correct structure and function of photoreceptors.

Purpose: Best disease, or vitelliform macular degeneration, is an autosomal dominant form of macular degeneration that is caused by mutations in the gene encoding bestrophin. On clinical examination, Best disease is characterized by an elevated lesion beneath the neurosensory retina, resembling an egg yolk. The lesions in Best disease are primarily restricted to the macula, a small region of the retina responsible for central vision. The nature of the vitelliform material and the reason the development of such lesions is usually restricted to the macula are two unsolved questions in the pathogenesis of this disorder., Methods: The expression of bestrophin protein and mRNA was evaluated by immunohistochemistry, Western blot, and quantitative PCR in a series of normal human eyes. The ultrastructure of the retinal pigment epithelium and the histopathology of two donors with clinically diagnosed Best disease were also examined., Results: An eye from a Best disease donor with a T6R mutation was found to have deposits containing lipid and glycoconjugates within the central retinal scar. These deposits may be remnants of the vitelliform lesion. Immunohistochemical localization of bestrophin in a series of 22 unaffected eyes revealed a pattern in which macular labeling was less robust than labeling outside the macula in most (18/22) cases. This pattern was confirmed using quantitative PCR and Western blotting., Conclusions: Topographic differences in the levels of bestrophin protein may in part explain the propensity for the macula to develop lesions in Best disease.

Purpose: Canine multifocal retinopathy (cmr) is an autosomal recessive disorder of multiple dog breeds. The disease shares a number of clinical and pathologic similarities with Best macular dystrophy (BMD), and cmr is proposed as a new large animal model for Best disease., Methods: cmr was characterized by ophthalmoscopy and histopathology and compared with BMD-affected patients. BEST1 (alias VMD2), the bestrophin gene causally associated with BMD, was evaluated in the dog. Canine ortholog cDNA sequence was cloned and verified using RPE/choroid 5'- and 3'-RACE. Expression of the canine gene transcripts and protein was analyzed by Northern and Western blotting and immunocytochemistry. All exons and the flanking splice junctions were screened by direct sequencing., Results: The clinical phenotype and pathology of cmr closely resemble lesions of BMD. Canine VMD2 spans 13.7 kb of genomic DNA on CFA18 and shows a high level of conservation among eukaryotes. The transcript is predominantly expressed in RPE/choroid and encodes bestrophin, a 580-amino acid protein of 66 kDa. Immunocytochemistry of normal canine retina demonstrated specific localization of protein to the RPE basolateral plasma membranes. Two disease-specific sequence alterations were identified in the canine VMD2 gene: a C(73)T stop mutation in cmr1 and a G(482)A missense mutation in cmr2., Conclusions: The authors propose these two spontaneous mutations in the canine VMD2 gene, which cause cmr, as the first naturally occurring animal model of BMD. Further development of the cmr models will permit elucidation of the complex molecular mechanism of these retinopathies and the development of potential therapies.

Purpose: To evaluate whether HLA genotypes are associated with age-related macular degeneration (AMD)., Methods: HLA class I-A, -B, and -Cw and class II DRB1 and DQB1 principal allele groups were genotyped in two stages: initially for principal allele groups in a cohort of 100 AMD cases and 92 control subjects, and then, in the next 100 cases and controls from the same cohort, for alleles or allele groups with P < 0.1 on initial typing. Genotype frequencies were compared by 2 x 2 contingency tables. The strongest associations for individual HLA alleles were calculated with two-locus stratification analysis and logistic regression for all possible pair-wise HLA combinations. Bonferroni corrections were applied for multiple measurements (P(c)). Each HLA allele was subjected to logistic regression for known AMD covariates. HLA immunohistochemistry for class I antigens was performed on elderly donor eyes., Results: Allele Cw*0701 (P = 0.004, P(c) = 0.036) correlated positively with AMD, whereas alleles B*4001 (P = 0.003, P(c) = 0.027) and DRB1*1301(P = 0.001, P(c) = 0.009) were negatively associated. These HLA associations were independent of any linkage disequilibrium. Immunohistochemistry demonstrated differential HLA class I expression in choriocapillary endothelial cells., Conclusions: Significant positive and negative associations exist between HLA alleles and AMD. HLA polymorphisms influence the development of AMD, possibly via modulating choroidal immune function.

Purpose: To determine whether vitronectin (Vn), a plasma protein and extracellular matrix molecule that is also a prominent constituent of drusen, is synthesized by cells in the adult human retina., Methods: The distribution of Vn in the normal adult human retina was examined using antibodies to circulating plasma Vn and to the multimeric, heparin-binding form that is most prevalent in extravascular tissues. Evidence of Vn transcription by retinal cells was analyzed by in situ hybridization and also by reverse transcription of total RNA derived from dissociated human or mouse photoreceptors followed by amplification using polymerase chain reaction (RT-PCR)., Results: Cytoplasmic immunoreactivity for plasma Vn or multimeric Vn was detected in photoreceptors, in a subpopulation of neurons situated in the inner retina, and in vitreous hyalocytes. Extracellular labeling was limited primarily to Bruch's membrane and the retinal vasculature. At the transcriptional level, Vn mRNA was localized to both photoreceptors and ganglion cells by in situ hybridization. The in situ findings were corroborated by RT-PCR using total RNA from dissociated mouse or human photoreceptor cells., Conclusions: The results constitute the first evidence for Vn gene expression by adult neurons in the mammalian central nervous system. The identification of the photoreceptors as a cellular source of Vn suggests that these cells have the potential to make a biosynthetic contribution to the Vn that is found in drusen.