Elastin-Mediated Choroidal Endothelial Cell Migration: Possible Role in Age-Related Macular Degeneration

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 Ͻ 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. (Invest Ophthalmol Vis Sci. 2008;49: 5574 -5580) DOI:10.1167/iovs. A ge-related macular degeneration (AMD) is a major cause of blindness. The neovascular (wet) form of AMD is characterized by the abnormal growth of choroidal blood vessels into the sub-retinal space of the macula. This multistep process is likely to be initiated by the breakdown of Bruch's membrane which, when intact, prevents pathologic angiogenesis. In this process, choroidal ECs may migrate from the choroid into the sub-RPE and/or sub-retinal space. These ECs proliferate and form tubes (tubulogenesis), ultimately reorganizing their junctions to increase permeability across the newly formed vascular wall. The neovascular process in AMD can result in serous detachment of the retinal pigmented epithelium (RPE) and/or neurosensory retinal detachment, as well as fibrous disciform scarring beneath the retina, causing a catastrophic decrease in visual acuity. 1-5 Current treatments for neovascular AMD are focused primarily on vascular endothelial growth factor (VEGF)-mediated processes. 6 While VEGF is a potent inducer of angiogenesis, understanding the role of additional angiogenic stimuli would be invaluable for the development of improved treatments. 7 Elastin is a glycoprotein consisting of cross-linked 72 kDa tropoelastin subunits and is an abundant component of the extracellular matrix (ECM) of arteries, lung, and skin. 8 Breakdown of elastin results in the formation of elastin-derived peptides (EDPs), cross-linked fragments of tropoelastin of varying sizes. 10 Currently, it is unknown how these peptides are able to activate ECs and whether they are capable of activating ECs from other tissues, such as the choroid. It is plausible that EDPs bind to elastin binding proteins on the cell surface, inducing angiogenic behaviors such as cell migration and/or proliferation. Several lines of evidence suggest abnormal elastin metabolism occurs in Bruch's membrane in AMD. First, early onset choroidal neovascularization has been shown in patients with pseudoxanthoma elasticum. These patients may be at risk for developing choroidal neovascular membranes because of abnormalities in the elastic layer of Bruch's membrane, including breaks, clinically defined as angioid streaks. 11,12 Second, fibulin-5 missense mutations have been identified in association with AMD. 13 This mutation may contribute to AMD development by affecting the elastic layer of Bruch's membrane, since fibulin-5 participates in elastogenesis There is also evidence for abnormal systemic elastin metabolism in AMD. Blumenkranz et al. 11 found a correlation between choroidal neovascularization (CNV) and elastotic degeneration. Patients with exudative AMD demonstrated a greater than twofold increase in their susceptibility to elastotic degeneration of relatively sun-protected areas of the skin in dermal biopsies, suggesting that AMD is associated with systemic elastin abnormalities. 11 Serum levels of EDPs in patients with exudative AMD have also been found to be significantly higher than levels in non-exudative AMD patients and control patients

HIV/AIDS Mobile Applications: An Assessment for At-Risk Populations in Thailand

There is not adequate case management and reporting of HIV/AIDS among the most at-risk populations in Thailand due in part to a lack of appropriate technology. The goal of this project was to determine the most effective mobile application or application features for HIV/AIDS management from the perspectives of the target populations, case managers, and Population Services International. We produced a framework for evaluating existing and future applications, developed a prototype application, and provided recommendations for related projects

An FPGA-Based System for Tracking Digital Information Transmitted Via Peer-to-Peer Protocols

This paper presents a Field Programmable Gate Array (FPGA)-based tool designed to process file transfers using the BitTorrent Peer-to-Peer (P2P) protocol and VoIP phone calls made using the Session Initiation Protocol (SIP). The tool searches selected control messages in real time and compares the unique identifier of the shared file or phone number against a list of known contraband files or phone numbers. Results show the FPGA tool processes P2P packets of interest 92% faster than a software-only configuration and is 97.6% accurate at capturing and processing messages at a traffic load of 89.6 Mbps

Bestrophin Gene Mutations Cause Canine Multifocal Retinopathy: A Novel Animal Model for 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 C73T stop mutation in cmr1 and a G482A 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

Evaluation of variants in the selectin genes in age-related macular degeneration

<p>Abstract</p> <p>Background</p> <p>Age-related macular degeneration (AMD) is a common disease of the elderly that leads to loss of the central visual field due to atrophic or neovascular events. Evidence from human eyes and animal models suggests an important role for macrophages and endothelial cell activation in the pathogenesis of AMD. We sought to determine whether common ancestral variants in genes encoding the selectin family of proteins are associated with AMD.</p> <p>Methods</p> <p>Expression of E-selectin, L-selectin and P-selectin was examined in choroid and retina by quantitative PCR and immunofluorescence. Samples from patients with AMD (n = 341) and controls (n = 400) were genotyped at a total of 34 SNPs in the <it>SELE</it>, <it>SELL </it>and <it>SELP </it>genes. Allele and genotype frequencies at these SNPs were compared between AMD patients and controls as well as between subtypes of AMD (dry, geographic atrophy, and wet) and controls.</p> <p>Results</p> <p>High expression of all three selectin genes was observed in the choroid as compared to the retina. Some selectin labeling of retinal microglia, drusen cores and the choroidal vasculature was observed. In the genetic screen of AMD versus controls, no positive associations were observed for <it>SELE </it>or <it>SELL</it>. One SNP in <it>SELP </it>(rs3917751) produced p-values < 0.05 (uncorrected for multiple measures). In the subtype analyses, 6 SNPs (one in <it>SELE</it>, two in <it>SELL</it>, and three in <it>SELP</it>) produced p-values < 0.05. However, when adjusted for multiple measures with a Bonferroni correction, only one SNP in <it>SELP </it>(rs3917751) produced a statistically significant p-value (p = 0.0029).</p> <p>Conclusions</p> <p>This genetic screen did not detect any SNPs that were highly associated with AMD affection status overall. However, subtype analysis showed that a single SNP located within an intron of <it>SELP </it>(rs3917751) is statistically associated with dry AMD in our cohort. Future studies with additional cohorts and functional assays will clarify the biological significance of this discovery. Based on our findings, it is unlikely that common ancestral variants in the other selectin genes (<it>SELE </it>and <it>SELL</it>) are risk factors for AMD. Finally, it remains possible that sporadic or rare mutations in <it>SELE</it>, <it>SELL</it>, or <it>SELP </it>have a role in the pathogenesis of AMD.</p

Bestrophinopathy: An RPE-Photoreceptor Interface Disease

Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies. © 2017 Elsevier Lt

Characterization of Choroidal Layers in Normal Aging Eyes Using Enface Swept-Source Optical Coherence Tomography

Purpose To characterize qualitative and quantitative features of the choroid in normal eyes using enface swept-source optical coherence tomography (SS-OCT). Methods Fifty-two eyes of 26 consecutive normal subjects were prospectively recruited to obtain multiple three-dimensional 12x12mm volumetric scans using a long-wavelength high-speed SS-OCT prototype. A motion-correction algorithm merged multiple SS-OCT volumes to improve signal. Retinal pigment epithelium (RPE) was segmented as the reference and enface images were extracted at varying depths every 4.13 mu m intervals. Systematic analysis of the choroid at different depths was performed to qualitatively assess the morphology of the choroid and quantify the absolute thicknesses as well as the relative thicknesses of the choroidal vascular layers including the choroidal microvasculature (choriocapillaris, terminal arterioles and venules;CC) and choroidal vessels (CV) with respect to the subfoveal total choroidal thickness (TC). Subjects were divided into two age groups: younger (= 40 years). Results Mean age of subjects was 41.92 (24-66) years. Enface images at the level of the RPE, CC, CV, and choroidal-scleral interface were used to assess specific qualitative features. In the younger age group, the mean absolute thicknesses were: TC 379.4 mu m (SD +/- 75.7 mu m),CC 81.3 mu m (SD +/- 21.2 mu m) and CV 298.1 mu m (SD +/- 63.7 mu m). In the older group, the mean absolute thicknesses were: TC 305.0 mu m (SD +/- 50.9 mu m),CC 56.4 mu m (SD +/- 12.1 mu m) and CV 248.6 mu m (SD +/- 49.7 mu m). In the younger group, the relative thicknesses of the individual choroidal layers were: CC 21.5% (SD +/- 4.0%) and CV 78.4% (SD +/- 4.0%). In the older group, the relative thicknesses were: CC 18.9% (SD +/- 4.5%) and CV 81.1% (SD +/- 4.5%). The absolute thicknesses were smaller in the older age group for all choroidal layers (TC p=0.006, CC p=0.0003, CV p=0.03) while the relative thickness was smaller only for the CC (p=0.04). Conclusions Enface SS-OCT at 1050nm enables a precise qualitative and quantitative characterization of the individual choroidal layers in normal eyes. Only the CC is relatively thinner in the older eyes. In-vivo evaluation of the choroid at variable depths may be potentially valuable in understanding the natural history of age-related posterior segment disease