Defining the proteome of human iris, ciliary body, retinal pigment epithelium, and choroid.

The iris is a fine structure that controls the amount of light that enters the eye. The ciliary body controls the shape of the lens and produces aqueous humor. The retinal pigment epithelium and choroid (RPE/choroid) are essential in supporting the retina and absorbing light energy that enters the eye. Proteins were extracted from iris, ciliary body, and RPE/choroid tissues of eyes from five individuals and fractionated using SDS-PAGE. After in-gel digestion, peptides were analyzed using LC-MS/MS on an Orbitrap Elite mass spectrometer. In iris, ciliary body, and RPE/choroid, we identified 2959, 2867, and 2755 nonredundant proteins with peptide and protein false-positive rates of <0.1% and <1%, respectively. Forty-three unambiguous protein isoforms were identified in iris, ciliary body, and RPE/choroid. Four "missing proteins" were identified in ciliary body based on ≥2 proteotypic peptides. The mass spectrometric proteome database of the human iris, ciliary body, and RPE/choroid may serve as a valuable resource for future investigations of the eye in health and disease. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001424 and PXD002194.

Identification of lymphatics in the ciliary body of the human eye: a novel "uveolymphatic" outflow pathway.

Impaired aqueous humor flow from the eye may lead to elevated intraocular pressure and glaucoma. Drainage of aqueous fluid from the eye occurs through established routes that include conventional outflow via the trabecular meshwork, and an unconventional or uveoscleral outflow pathway involving the ciliary body. Based on the assumption that the eye lacks a lymphatic circulation, the possible role of lymphatics in the less well defined uveoscleral pathway has been largely ignored. Advances in lymphatic research have identified specific lymphatic markers such as podoplanin, a transmembrane mucin-type glycoprotein, and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1). Lymphatic channels were identified in the human ciliary body using immunofluorescence with D2-40 antibody for podoplanin, and LYVE-1 antibody. In keeping with the criteria for lymphatic vessels in conjunctiva used as positive control, D2-40 and LYVE-1-positive lymphatic channels in the ciliary body had a distinct lumen, were negative for blood vessel endothelial cell marker CD34, and were surrounded by either discontinuous or no collagen IV-positive basement membrane. Cryo-immunogold electron microscopy confirmed the presence D2-40-immunoreactivity in lymphatic endothelium in the human ciliary body. Fluorescent nanospheres injected into the anterior chamber of the sheep eye were detected in LYVE-1-positive channels of the ciliary body 15, 30, and 45 min following injection. Four hours following intracameral injection, Iodine-125 radio-labeled human serum albumin injected into the sheep eye (n = 5) was drained preferentially into cervical, retropharyngeal, submandibular and preauricular lymph nodes in the head and neck region compared to reference popliteal lymph nodes (P < 0.05). These findings collectively indicate the presence of distinct lymphatic channels in the human ciliary body, and that fluid and solutes flow at least partially through this system. The discovery of a uveolymphatic pathway in the eye is novel and highly relevant to studies of glaucoma and other eye diseases.

The differing embryonic origins of retinal and uveal (iris/ciliary body and choroid) melanosomes are mirrored by their phospholipid composition.

The phospholipids present in uveal (iris/ciliary body and choroid) and retinal bovine ocular melanosomes were identified using mass spectrometry. Similar phospholipid content is found for the two types of uveal melanosome, with sphingomyelin being the major species. Significant differences are found between the uveal and retinal melanosome. Glycerophosphoethanolamine (GPEtn) is the major species in the retinal pigment epithelium (RPE); 93% of the GPEtn contain polyunsaturated fatty acids, notably docosahexanoic acid and arachidonic acid, in the sn-2 position. RPE melanosomes also contain detectable quantities of glycerophosphoserine and glycerophosphate; these species were not detected in the uveal samples. While the structural and functional roles of melanosomal lipids largely remain to be determined, these different lipid compositions reported herein offer new insights into the roles of melanosomes in the different ocular tissues.

Characterisation of WE-14 in porcine ocular tissue.

WE-14 is derived from the cell-specific posttranslational processing of chromogranin A (CgA) in subpopulations of neuroendocrine cells and neurons. Region- and site-specific chromogranin A, pancreastatin and WE-14 antisera were employed to study the generation of WE-14 in porcine ocular tissues. No chromogranin A or pancreastatin immunostaining was detected in ocular tissue. Immunohistochemistry detected WE-14 immunostaining in a network of nerve fibre bundles and nerve fibres throughout the limbus, cornea, iris and ciliary body with sparse nerve fibres detected throughout the choroid and sclera. Retinal analysis detected intense WE-14 immunostaining in large ovoid cells in the ganglion cell layer with weak immunostaining in a population of small cells in the inner nuclear layer; weak immunostaining was detected within the fibre layers in the inner plexiform layer. Quantitatively, the highest WE-14 tissue concentration was recorded in aqueous retinal and corneal extracts with lower concentrations in the sclera, choroid and anterior uveal tissues. Chromatographic profiling resolved a minor chromogranin A-like immunoreactant and a predominant immunoreactant co-eluting with synthetic human WE-14. This is the first study to demonstrate that WE-14 is generated in neuronal fibres primarily innervating the anterior chamber and in select cell populations in the retina.

Angiotensin converting anzyme (ACE) activity in porcine ocular tissue: effects of diet and ACE inhibitors.

The aim of the present experiments was to determine angiotensin converting enzyme (ACE) activity in different parts of the porcine eye, and to examine whether an atherogenic diet influenced ACE activity. Female mini-pigs were fed a standard diet or a diet with high cholesterol to produce atherosclerosis. The animals were killed by an overdose of pentobarbital, and the eyes were enucleated and dissected into iris, ciliary body, retina, and choroid. Crude tissue homogenates were used for determination of ACE activity, which was done with a radioenzymatic assay. In pigs fed a normal diet, basal ACE activity was 18.1 +/- 1.6, 13.6 +/- 1.9, 4.4 +/- 0.6, and 44.7 +/- 8.5 units/mg for iris, ciliary body, retina, and choroid, respectively. The ACE activities in ocular tissues from the pigs that had been fed an atherogenic diet were not significantly different. Nor was the ACE activity in the abdominal aorta and serum significantly different between the two groups. In both groups, the ACE inhibitors captopril and enalaprilat, caused a significant inhibition of the ACE activity in the choroid and ciliary body, with enalaprilat being more potent. In the retina, ACE activity was inhibited significantly only in the group fed a normal diet, whereas ACE activity in the iris was not significantly inhibited in either group. We did not find any differences in ACE activity between pigs fed a normal diet and pigs fed an atherogenic diet, which is in disagreement with previous studies that showed an increased ACE activity in aorta from atherosclerotic mini-pigs. The reason for this discrepancy is not clear, but lower cholesterol levels are one possibility.

Identification and localization of muscarinic acetylcholine receptors in the ocular tissues of the chick.

The purpose of this study was to characterize the distribution of muscarinic acetylcholine receptors (mAChRs) in the ocular tissues of hatched chicks. In the chick, different isoforms of these receptors have been detected in the brain, heart, and retina, and mAChRs in ocular tissues have been implicated in the pathogenesis of form-deprivation myopia. However, the precise anatomical distribution of mAChRs within the retina, retinal pigment epithelium, choroid, ciliary body, and ciliary ganglion remains unknown. We used affinity-purified, type-specific antibodies directed to three different chick mAChR subtypes (cm2, cm3, and cm4) to detect receptor immunoreactivity in sections and extracts of these ocular tissues. We found cm2, cm3, and cm4 in the retina, retinal pigment epithelium, choroid, and ciliary body. Within the retina, cm2 was expressed in numerous amacrine and ganglion cells; cm3 was expressed in many bipolar cells and small subsets of amacrine cells; and cm4 was found in most, if not all, amacrine and ganglion cells. Each mAChR was localized to distinct strata within the inner plexiform layer that cumulatively form three broad bands that closely match previously described localizations of subtype-nonspecific muscarinic ligand binding. Only cm3 was detected in the outer plexiform layer, and only cm4 was detected in the ciliary ganglion. We propose that each mAChR subtype has unique functions in each ocular tissue.

Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours.

The immunolocalization and gene expression of vascular endothelial growth factor (VEGF) and its cognate tyrosine kinase receptors, Flt-1 and KDR, has been studied in ocular melanomas and retinoblastomas using in situ hybridization and immunohistochemistry. Tumour-related alterations in VEGF/VEGF-receptor expression have also been examined in separate and uninvolved iris, retina and choroid of the same eyes. Although VEGF immunoreactivity in the normal retina was virtually absent, low-level VEGF expression was evident in the ganglion cell-bodies, Müller cells and in a distinct population of amacrine cells. VEGF gene expression was absent in the iris and choroid of normal eyes. In tumour-bearing eyes, high levels of VEGF protein and gene expression were observed within the vascularized regions of the tumours, while the adjacent retina and choroid showed increased VEGF levels when compared with normals. Flt-1 and KDR gene expression and immunolocalization occurred in VEGF-expressing ganglion, Müller and amacrine cells in normal eyes. Within the intra-ocular tumours, VEGF-receptor gene expression and protein was evident in the endothelial cells and also in cells close to the vessels, while in the adjacent retina, Flt-1 and KDR levels were elevated over normal, especially in the blood vessels. Flt-1 and KDR were both observed at elevated levels in the choroid and iris blood vessels. This study suggests that VEGF, Flt-1 and KDR are expressed by neural, glial and vascular elements within normal human retina. Intra-ocular tumours demonstrate a high level of VEGF and VEGF-receptor expression; within uninvolved, spatially separate retina, choroid and iris in the same eyes, expression is also elevated, especially within the vasculature. Retinal vascular endothelia may respond to high intra-ocular levels of VEGF by increasing expression of their VEGF receptors, a phenomenon which could have relevance to neoplasm-related ocular neovascularization.

Immunocytochemical localization of prostaglandin E2 receptor subtypes in porcine ocular tissues. I. Uveal tissues.

Polyclonal antibodies were raised against 15-residue sequences in the carboxyl terminal region of mouse EP1, EP2, and EP3 subtypes. The selected sequences are well conserved in different species. Using the antibodies, the localization of the receptor subtypes in porcine uveal tissues was investigated by immunoperoxidase reaction (by light microscopy) and immunogold labeling (by electron microscopy). EP1 immunoreactivity was found in ciliary nonpigmented epithelium and iris muscles (both sphincter and dilator). EP2 was localized to ciliary nonpigmented epithelium and muscle, iris sphincter muscle, and trabecular meshwork. EP3 immunoreactivity was detected in all uveal tissues examined.

Lipids of human retina, retinal pigment epithelium, and Bruch's membrane/choroid: comparison of macular and peripheral regions.

PURPOSE: To understand the difference between macular and peripheral regions, tissue samples of human retina, retinal pigment epithelium, and Bruch's membrane/choroid were dissected and analyzed for lipid composition. METHOD: To facilitate dissections and enhance the recovery of tissues, eyecups were prefixed for 1 hour in 10% formalin (pH 7). Lipids were extracted from 4-mm trephined punches of tissues and analyzed by high-performance liquid chromatography. After separation of neutral lipids and phospholipids, total fatty acids in both lipid classes were quantitated. RESULTS: The major phospholipid classes in retina, retinal pigment epithelium, and Bruch's membrane/choroid were phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidyl serine; the major fatty acids were palmitic (16:0), stearic (18:0), and oleic (18:1). Although the three tissues had similar total fatty acid and phospholipid components, their relative compositions were different. Neutral lipid/phospholipid ratios in retinal pigment epithelium and Bruch's membrane/choroid were almost three times higher than in the retina. CONCLUSIONS: This study provides information about the lipid composition of macular and peripheral regions of the human retina, retinal pigment epithelium, and Bruch's membrane/choroid. The methodology employed enabled study of lipids in small amounts of tissue, which will be of value in investigating the biochemical aspects of age-related macular degeneration.

Quantification of melanin and iron content in uveal malignant melanomas and correlation with magnetic resonance image.

Eleven patients with uveal malignant melanomas (MM) were studied by magnetic resonance (MR) imaging before enucleation. The MR appearances varied, but often were different from those previously reported to be characteristic of these tumours. Using an image analyser to assess quantitatively the melanin and iron content of each tumour, a wide range of tumour melanin concentrations was found, but universally low tumour iron concentrations. These values were compared with MR appearances that were quantified and expressed as contrast to noise ratios. The correlation between T1 and T2 shortening and increasing melanin content did not reach statistical significance. There was no correlation between MR appearances and iron content. The theories postulated to explain the diverse MR appearances of uveal MMs are discussed and variations in tumour melanin content and differences in scanner strengths are suggested as the most likely explanations.