Proteomic Insight into the Molecular Function of the Vitreous

The human vitreous contains primarily water, but also contains proteins which have yet to be fully characterized. To gain insight into the four vitreous substructures and their potential functions, we isolated and analyzed the vitreous protein profiles of three non-diseased human eyes. The four analyzed substructures were the anterior hyaloid, the vitreous cortex, the vitreous core, and the vitreous base. Proteins were separated by multidimensional liquid chromatography and identified by tandem mass spectrometry. Bioinformatics tools then extracted the expression profiles, signaling pathways, and interactomes unique to each tissue. From each substructure, a mean of 2,062 unique proteins were identified, with many being differentially expressed in a specific substructure: 278 proteins were unique to the anterior hyaloid, 322 to the vitreous cortex, 128 to the vitreous base, and 136 to the vitreous core. When the identified proteins were organized according to relevant functional pathways and networks, key patterns appeared. The blood coagulation pathway and extracellular matrix turnover networks were highly represented. Oxidative stress regulation and energy metabolism proteins were distributed throughout the vitreous. Immune functions were represented by high levels of immunoglobulin, the complement pathway, damage-associated molecular patterns (DAMPs), and evolutionarily conserved antimicrobial proteins. The majority of vitreous proteins detected were intracellular proteins, some of which originate from the retina, including rhodopsin (RHO), phosphodiesterase 6 (PDE6), and glial fibrillary acidic protein (GFAP). This comprehensive analysis uncovers a picture of the vitreous as a biologically active tissue, where proteins localize to distinct substructures to protect the intraocular tissues from infection, oxidative stress, and energy disequilibrium. It also reveals the retina as a potential source of inflammatory mediators. The vitreous proteome catalogues the dynamic interactions between the vitreous and surrounding tissues. It therefore could be an indirect and effective method for surveying vitreoretinal disease for specific biomarkers.     

Pig vitreous gel: macromolecular composition with particular reference to hyaluronan-binding proteoglycans

The aim of this study was to examine the macromolecular composition of pig vitreous body with particular emphasis on hyaluronan-binding proteoglycans. The whole pig vitreous gel was found to contain 76 microg of hyaluronan-derived uronic acid, 700 microg of total protein and 150 microg of collagen per ml of gel. The contents of neutral hexoses and sialic acids were 80 and 22 microg/ml of vitreous gel, but only a minor proportion of them were found to be associated with the proteoglycan fraction. As estimated by gel chromatography on Sepharose CL-2B, hyaluronan presents a polydisperse hydrodynamic behavior with a lower molecular mass (M(r)) value of 220 kDa. The existence of low amounts of a hyaluronan-binding proteoglycan population with structural and immunological characteristics similar to a member of the hyalectan family, versican, has also been demonstrated. The concentration of this versican-like proteoglycan in whole vitreous accounts for 50 microg proteoglycan protein per ml of vitreous gel and represents a minor proportion (about 7%) of the total protein content. The proteoglycan has an average M(r) of 360 kDa and is substituted by chondroitin sulphate (CS) side chains. Study of the CS sulphation pattern showed that the chains were composed of both type 4- and 6-sulphated disaccharide units.     

Composition, synthesis, and assembly of the embryonic chick retinal basal lamina

To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly.     

Differential Expression of Vitreous Proteins in Young and Mature New Zealand White Rabbits

Different anatomical regions have been defined in the vitreous humor including central vitreous, basal vitreous, vitreous cortex, vitreoretinal interface and zonule. In this study we sought to characterize changes in the proteome of vitreous humor (VH) related to compartments or age in New Zealand white rabbits (NZW). Vitreous humor was cryo-collected from young and mature New Zealand white rabbit eyes, and dissected into anterior and posterior compartments. All samples were divided into 4 groups: Young Anterior (YA), Young Posterior (YP), Mature Anterior (MA) and Mature Posterior (MP) vitreous. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry. Spectral count was used to determine the relative protein abundances and identify proteins with statistical differences between compartment and age groups. Western blotting was performed to validate some of the differentially expressed proteins. Our results showed that 231, 375, 273 and 353 proteins were identified in the YA, YP, MA and MP respectively. Fifteen proteins were significantly differentially expressed between YA and YP, and 11 between MA and MP. Carbonic anhydrase III, lambda crystallin, alpha crystallin A and B, beta crystallin B1 and B2 were more abundant in the anterior region, whereas vimentin was less abundant in the anterior region. For comparisons between age groups, 4 proteins were differentially expressed in both YA relative to MA and YP relative to MP. Western blotting confirmed the differential expression of carbonic anhydrase III, alpha crystallin B and beta crystallin B2. The protein profiles of the vitreous humor showed age- and compartment-related differences. This differential protein profile provides a baseline for understanding the vitreous compartmentalization in the rabbit and suggests that further studies profiling proteins in different compartments of the vitreous in other species may be warranted.     

Iron-binding proteins with vitreous humour

The soluble protein composition of Macaque monkey vitreous humour was studied in order to understand its iron-binding properties. The protein content of vitreous humour was 217 μg/ml ± 4.6%, 40% of which was serum albumin and 30% an iron-binding protein of hydrodynamic properties identical to that of trasferrin or lactoferrin. Relative to serum, the vitreous humour contained a 13-fold excess of this protein(s). Isoelectric focusing, iron-binding and immunoelectrophoretic studies indicated that both vitreous humour and aqueous humour contained lactoferrin as well as serum transferrin. The iron-binding capacity of these proteins in vitreous humour was equivalent to the mass of haemoglobin iron contained in at least 570 000 monkey erythrocytes. It was concluded that the intraocular lactoferrin originated from within the eye. These iron-binding proteins may play a protective role in ocular disturbances such as viterous haemorrahge, iron foreign body toxicity and infection.     

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.     

Profiling of vitreous proteomes from proliferative diabetic retinopathy and nondiabetic patients

Diabetes can lead to serious microvascular complications like proliferative diabetic retinopathy (PDR), which is the leading cause of blindness in adults. The proteomic changes that occur during PDR cannot be measured in the human retina for ethical reasons, but could be reflected by proteomic changes in vitreous humor. Thus, we considered that comparisons between the proteome profiles of the vitreous humors of PDR and nondiabetic controls could lead to the discovery of novel pathogenic proteins and clinical biomarkers. In this study, the authors used several proteomic methods to comprehensively examine vitreous humor proteomes of PDR patients and nondiabetic controls. These methods included immunoaffinity subtraction (IS)/2-DE/MALDI-MS, nano-LC-MALDI-MS/MS, and nano-LC-ESI-MS/MS. The identified proteins were subjected to the Trans-Proteomic Pipeline validation process. Resultantly, 531 proteins were identified, i.e., 415 and 346 proteins were identified in PDR and nondiabetic control vitreous humor samples, respectively, and of these 531 proteins, 240 were identified for the first time in this study. The PDR vitreous proteome was also found to contain many proteins possibly involved in the pathogenesis of PDR. The proteins described provide the most comprehensive proteome listing in the vitreous humor samples of PDR and nondiabetic control patients.     

Attempts to demonstrate specific antibody responses in the vitreous body of the eye

Postmortem serum and vitreous humor specimens obtained from 31 autopsied human bodies were assayed for specific antibody responses to adenoviruses, RS virus and Mycoplasma pneumoniae using the complement-fixation (CF) test and the ELISA procedure (in 23 of the bodies examined). The antibody responses as measured by the CF test were negative in all vitreous body samples tested, with the ELISA five specimens gave a positive reaction at a titre 1 : 40 and one at 1 : 80. These positive antibody titres turned out to invariably coincide with the high-titre antibody levels in the serum. Implicitly, at high-titre levels in the serum these antibodies tend to penetrate in the vitreous body of the eye.     

Post-translational modification of crystallins in vitreous body from experimental autoimmune uveitis of rats

Experimental autoimmune uveitis (EAU) is a well-known animal model of posterior uveitis that is one of the major causes of blindness. EAU could be induced in susceptible animals (i.e., Lewis rat) by immune reactions using evolutionarily conserved retinal proteins, such as interphoto-receptor retinoid binding protein (IRBP), or epitaphs of the protein. First, we prepared the following four test groups that subsequently increased or decreased inflammation. (1) Normal control group, (2) IRBP-induced uveitis group, (3) Hemin-treated uveitis group, and (4) Sn(IV) protoporphyrin IX dichloride (SnPP)-treated uveitis group. Second, in the vitreous bodies of Lewis rats, the infiltrated proteins were analyzed using two-dimensional electrophoresis (2-DE), MALDI-TOF/MS, and Micro LC/LC-MS/MS analysis. Finally, Western blotting was applied to confirm the relative amount of crystallins and phosphorylation sites of alphaB-crystallin. Thirty spots were identified in vitreous bodies, and 27 of these spots were members of the crystallin family. Unlike betaA4- and B2-crystallins (that were significantly increased without truncation), alphaA- and B-crystallins were only truncated in EAU vitreous body. Taken as a whole, in the rat EAU model, we suggest that post-translational truncations of alphaA- and alphaB-crystallins, phosphorylation of alphaB-crystallin, and new production of betaA4- and betaB2-crystallins are intercorrelated with uveitis progression and inflammatory responses.     

Characterization of the vitreous proteome in diabetes without diabetic retinopathy and diabetes with proliferative diabetic retinopathy

An understanding of the diabetes-induced alterations in vitreous protein composition in the absence and in the presence of proliferative diabetic retinopathy (PDR) may provide insights into factors and mechanisms responsible for this disease. We have performed a comprehensive proteomic analysis and comparison of vitreous samples from individuals with diabetes but without diabetic retinopathy (noDR) or with PDR and nondiabetic individuals (NDM). Using preparative one-dimensional SDS-PAGE and nano-LC/MS/MS of 17 independent vitreous samples, we identified 252 proteins from human vitreous. Fifty-six proteins were differentially abundant in noDR and PDR vitreous compared with NDM vitreous, including 32 proteins increased and 10 proteins decreased in PDR vitreous compared with NDM vitreous. Comparison of noDR and PDR groups revealed increased levels of angiotensinogen and decreased levels of calsyntenin-1, interphotoreceptor retinoid-binding protein, and neuroserpin in PDR vitreous. Biological pathway analysis revealed that vitreous contains 30 proteins associated with the kallikrein-kinin, coagulation, and complement systems. Five of them (complement C3, complement factor I, prothrombin, alpha-1-antitrypsin, and antithrombin III) were increased in PDR vitreous compared with NDM vitreous. Factor XII was detected in PDR vitreous but not observed in either NDM or noDR vitreous. PDR vitreous also had increased levels of peroxiredoxin-1 and decreased levels of extracellular superoxide dismutase, compared with noDR or NDM vitreous. These data provide an in depth analysis of the human vitreous proteome and reveal protein alterations that are associated with PDR.     

Mammalian vitreous humor contains networks of hyaluronan molecules: electron microscopic analysis using the hyaluronan-binding region (G1) of aggrecan and link protein.

Vitreous humor from human, bovine, and chicken eyes was analyzed by rotary shadowing to characterize further the supramolecular organization of the gel-like matrix which forms this tissue. Extensive filamentous networks, distinct from collagen fibrils, were found in both human and bovine vitreous but not in chicken vitreous. The networks consisted of branching structures of various diameters, due to variable numbers of hyaluronan molecules being laterally associated with each other and apparently giving rise to a three-dimensional lattice. These networks could be decorated in a specific and regular manner by the hyaluronan-binding region called G1 purified from bovine nasal septum cartilage. The extent of decoration of hyaluronan was dependent on the relative concentration of G1. In the presence of an excess of G1 the networks were destabilized giving rise to individual unbranched hyaluronan chains of varying length that were saturated with G1. One or more globular proteins, as yet uncharacterized, were seen interacting with the hyaluronan networks, often at branch points. These proteins may serve to stabilize the three-dimensional structure of the matrix although highly ordered networks were also observed without globular proteins. Link protein, which also binds to hyaluronan, bound to the networks in a fashion clearly distinct from G1. Neither G1 nor link protein bound directly to human or bovine vitreous collagen fibrils. However, link protein did bind extensively to the glycosaminoglycan coat of chicken vitreous collagen fibrils described previously (D. W. Wright, and R. Mayne J. Ultrastruct. Mol. Struct. Res. 100, 224-234, 1988), while G1 did not. Digestion of the chicken vitreous collagen fibrils with Streptomyces hyaluronidase did not result in the removal of the glycosaminoglycan coat of the collagen fibrils nor did it affect the binding of G1 or link protein to the fibrils, indicating that hyaluronan is not a component of this structure. These studies demonstrate that proteins with specific binding properties can be used as probes to investigate the structure of the native vitreous humor gel from several species and suggest that this method potentially can be used for structural studies of other connective tissue matrices.     

Investigation of the content of alpha-fetoprotein and serum albumin in the vitreous body of the eye of human embryos

The content of serum albumin and alpha-fetoprotein in the vitreous body of the eyes of human fetuses from the 16th through the 24th week was investigated. It was detected that albumin and alpha-fetoprotein in the vitreous body of human eyes are presented in equal molar concentrations in the 16th week. There is 1.5-fold increased concentration of alpha-fetoprotein in comparison to albumin during the 17th week. After the 17th week, there was a reduction in the concentration of both proteins. It was reported that cyanine dye, used for detection of albumin, does not interact with alpha-fetoprotein.     

Beyond Polarity: Functional Membrane Domains in Astrocytes and Müller Cells

Various ependymoglial cells display varying degrees of process specialization, in particular processes contacting mesenchymal borders (pia, blood vessels, vitreous body), or those lining the ventricular surface. Within the neuropil, glial morphology, cellular contacts, and interaction partners are complex. It appears that glial processes contacting neurons, specific parts of neurons, or mesenchymal or ventricular borders are characterized by specialized membranes. We propose a concept of membrane domains in addition to the existing concept of ependymoglial polarity. Such membrane domains are equipped with certain membrane-bound proteins, enabling them to function in their specific environment. This review focuses on Müller cells and astrocytes and discusses exemplary the localization of established glial markers in membrane domains. We distinguish three functional glial membrane domains based on their typical molecular arrangement. The domain of the endfoot specifically displays the complex of dystrophin-associated proteins, aquaporin 4 and the potassium channel Kir4.1. We show that the domain of microvilli and the peripheral glial process in the Müller cell share the presence of ezrin, as do peripheral astrocyte processes. As a third domain, the Müller cell has peripheral glial processes related to a specific subtype of synapse. Although many details remain to be studied, the idea of glial membrane domains may permit new insights into glial function and pathology.     

Catalogue of soluble proteins in the human vitreous humor: comparison between diabetic retinopathy and macular hole

Two-dimensional gel electrophoresis and mass spectrometry were used to make a catalogue of soluble proteins in the human vitreous humor (VH). Fifty-one different proteins were identified on silver-stained two-dimensional (2D) gel patterns with VH proteins obtained from diabetic retinopathy and macular hole. Thirty of these have not been listed in the reported 2D profiles of plasma. Immunoglobulin (Ig), alpha1-antitrypsin, alpha2-HS glycoprotein,and complement C(4) fragment showed stronger spots in VH with diabetic retinopathy patient samples than those with macular hole. Pigment epithelium-derived factor, a potent inhibitor of angiogenesis in the cornea and vitreous, was clearly detected in VH with diabetes. It is impressive that the inhibitor increases in the vitreous with proliferative angiogenesis.     

Purification of the Specific Plasma Membrane Proteins from the Surface of Sperm Cells of Zea mays

Pollen samples of 6 varieties of Zea mays L. were used to isolate the viable sperm cells. After being probed with N-hydroxysuccinimido-biotin (NHS-biotin), the sperm cell plasma membrane proteins were compared with each other using the method of Western blotting. Results showed that there was no significant difference among varieties. The molecular weights of probed plasma membrane proteins were concentrated on 91,60,43,30 and 17 kD. Immunochemical method was adopted for further purification of sperm plasma membrane protein preparation which was some- what contaminated with cell organelles. After the cell organelles were isolated from etiolated seedlings of Zea mays by sucrose density gradient super centrifugation, the crude membrane proteins of organelles, endoplasm reticulum, mitochondria, Golgi body and plasmolemma were respectively used as antigen to immunize Guinea pig. The antibody was obtained from respective antiserum, then further used to produce immuno-affinity absorbent. After the solution of membrane proteins of sperm cells passed through the column, some proteins probed whth NHS-biotin were identified. Two major proteins probed with NHS-biotin were considered to be sperm cell specific. The size of these proteins in SDS-PAGE was about 65 kD, 22 kD, respectively.     

Improved Ocular Delivery of Nepafenac by Cyclodextrin Complexation

Nepafenac is a nonsteroidal anti-inflammatory drug (NSAID), currently only available as 0.1% ophthalmic suspension (Nevanac®). This study utilized hydroxypropyl-β-cyclodextrin (HPBCD) to increase the water solubility and trans-corneal permeation of nepafenac. The nepafenac-HPBCD complexation in the liquid and solid states were confirmed by phase solubility, differential scanning calorimetry (DSC), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and nuclear magnetic resonance spectroscopy (NMR) analyses. Nepafenac 0.1% ophthalmic solution was formulated using HPBCD (same pH and osmolality as that of Nevanac®) and pig eye trans-corneal permeation was studied versus Nevanac®. Furthermore, nepafenac content in cornea, sclera, iris, lens, aqueous humor, choroid, ciliary body, retina, and vitreous humor was studied in a continuous isolated pig eye perfusion model in comparison to the suspension and Nevanac®. Permeation studies using porcine corneas revealed that the solution formulation had a permeation rate 18 times higher than Nevanac®. Furthermore, the solution had 11 times higher corneal retention than Nevanac®. Drug distribution studies using porcine eyes revealed that the solution formulation enables detectable levels in various ocular tissues while the drug was undetectable by Nevanac®. The ocular solution formulation had a significantly higher drug concentration in the cornea compared to the suspension or Nevanac®.     

Method of determining the colloidal status of the vitreous body

A new method for the quantitative estimation of the vitreous body colloidal state has been suggested. For realization of the method a fragment of the vitreous body and an equal volume of water are dried out, and the drying time is registered. The higher is the ratio between the drying time of the vitreous body fragment and water, the higher is the density of the vitreous body gel. The drying out of the vitreous body fragment and water on the analytical balance cups permits the registration of the drying out process in the time course. Thus some additional parameters are obtained that characterize the object studied more completely. The method can be used in experimental and clinical investigations of the vitreous body and other biological liquids.     

SYNAPTIC PROTEINS AND AXONAL FLOW IN THE PIGEON VISUAL PATHWAY 1

Abstract— The protein supply of the synaptic ending by its nerve cell body has been studied in the retino-tectal system of the pigeon. [3H]Leucine was injected into the vitreous humor of one eye and the protein specific activity was determined for subcellular fractions of the retina and optic lobes and for the optic tracts. The results show that the perikaryon supplies newly synthesized proteins to its terminals. Two main waves of labelled macromolecules could be distinguished: one, migrating at a speed of 40 mm/day or more, is directly destined for the synaptic ending while the other, progressing at a rate of 1 mm/day, distributes along the entire axon.     

Proteomics of Vitreous Humor of Patients with Exudative Age-Related Macular Degeneration

Background

There is absence of specific biomarkers and an incomplete understanding of the pathophysiology of exudative age-related macular degeneration (AMD).

Methods and Findings

Eighty-eight vitreous samples (73 from patients with treatment naïve AMD and 15 control samples from patients with idiopathic floaters) were analyzed with capillary electrophoresis coupled to mass spectrometry in this retrospective case series to define potential candidate protein markers of AMD. Nineteen proteins were found to be upregulated in vitreous of AMD patients. Most of the proteins were plasma derived and involved in biological (ion) transport, acute phase inflammatory reaction, and blood coagulation. A number of proteins have not been previously associated to AMD including alpha-1-antitrypsin, fibrinogen alpha chain and prostaglandin H2-D isomerase. Alpha-1-antitrypsin was validated in vitreous of an independent set of AMD patients using Western blot analysis. Further systems biology analysis of the data indicated that the observed proteomic changes may reflect upregulation of immune response and complement activity.

Conclusions

Proteome analysis of vitreous samples from patients with AMD, which underwent an intravitreal combination therapy including a core vitrectomy, steroids and bevacizumab, revealed apparent AMD-specific proteomic changes. The identified AMD-associated proteins provide some insight into the pathophysiological changes associated with AMD.