Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases

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

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

Proteomic Interactions in the Mouse Vitreous-Retina Complex

<div><p>Purpose</p><p>Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina. </p> <p>Methods</p><p>Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software. </p> <p>Results</p><p>We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor. </p> <p>Conclusions</p><p>Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics. </p> </div

Mouse retina and vitreous protein networks.

<p><b>A</b>-<b>B</b>. Circular layout of the mouse retina and vitreous networks. Nodes (individual proteins; orange filled circles) with no edges (interactions) were not visualized. Nodes in the vitreous and retina are sized to the same scale to emphasize the difference in complexities of the two tissues. Enlarged blue box regions showing examples of hubs from the retina (<b>C</b>) and vitreous (<b>D</b>). Edges denote interactions (binding, substrate, transcriptional target) and are color coded (green = activation; red = inhibition; black = unknown).</p

Proteomic analysis pipeline.

<p><b>A</b>. The vitreous and retina were dissected from 16 mouse eyes. Each tissue type was pooled. <b>B</b>. Protein fractions were isolated and digested with trypsin. <b>C</b>. Peptide fragments were analyzed by multi-dimensional LC-MS/MS. <b>D</b>. MASCOT and SEAQUEST were used for peptide fragmentation finger-printing. <b>E</b>. Proteins with at least 2 peptide hits were analyzed for differential expression, ontology, pathway representation, and protein interactions.</p

Structure of mouse vitreous.

<p><b>A</b>. Illustration of the mouse eyeball. The lens composes a large portion of the eye, leaving a small portion of the eye to be filled with vitreous. <b>B</b>. The posterior mouse vitreous has a cortex and body similar to the human vitreous. The cortex defines the vitreoretinal boundary in both human and mouse. <b>C</b>. The mouse anterior hyaloid lies between the ciliary body and lens, posterior to the zonules, anterior to the vitreous base.</p