The role of dietary vitamin A in mechanisms of cataract development in the teleost lumpfish (Cyclopterus lumpus L).

Lumpfish (Cyclopterus lumpus L) are highly prone to cataract development in the wild and in culture. There is evidence that cataract in farmed fish is related to nutrition. However, both the nutrients and the mechanisms involved in cataract development in lumpfish are not clear. Here we investigated the mechanisms involved and the role of dietary vitamin A in cataract development in a cultured lumpfish population. Cultured lumpfish were fed three diets differing only in vitamin A supplementation level (5000, 15,000 and 120,000 IU/kg) over an 18-month period, and fish weight, cataract frequencies and severities were determined. Western blotting and immunohistochemistry were performed on lens tissue to measure the levels of oxidative stress, and apoptosis. The lowest levels of vitamin A significantly reduced cataract frequencies in adult lumpfish and resulted in less severe cataract and increased weight in males. Oxidative stress levels in the lens were positively correlated with vitamin A intake. Apoptosis was observed at high levels in lenses with severe cataract. Oxidative stress and apoptosis levels were the highest in regions of the lens with severe, advanced cataract pathology when compared to regions with no visible pathology. These results suggest that higher vitamin A intake contributes to cataract development through an oxidative stress pathway, and that both oxidative stress and apoptosis are involved in advanced stages of cataract in lumpfish.

Systematic analysis of ocular features and responses of cultured spotted wolffish (Anarhichas minor).

A better understanding of unique anatomical and functional features of the visual systems of teleost fish could provide key knowledge on how these systems influence the health and survival of these animals in both wild and culture environments. We took a systematic approach to assess some of the visual systems of spotted wolffish (Anarhichas minor), a species of increasing importance in North Atlantic aquaculture initiatives. The lumpfish (Cyclopterus lumpus) was included in these studies in a comparative manner to provide reference. Histology, light and electron microscopy were used to study the spatial distribution and occurrence of cone photoreceptor cells and the nature of the retinal tissues, while immunohistochemistry was used to explore the expression patterns of two photoreceptor markers, XAP-1 and XAP-2, in both species. A marine bacterial infection paradigm in lumpfish was used to assess how host-pathogen responses might impact the expression of these photoreceptor markers in these animals. We define a basic photoreceptor mosaic and present an ultrastructural to macroscopic geographical configuration of the retinal pigment tissues in both animals. Photoreceptor markers XAP-1 and XAP-2 have novel distribution patterns in spotted wolffish and lumpfish retinas, and exogenous pathogenic influences can affect the normal expression pattern of XAP-1 in lumpfish. Live tank-side ophthalmoscopy and spectral domain optical coherence tomography (SD-OCT) revealed that normal cultured spotted wolffish display novel variations in the shape of the retinal tissue. These two complementary imaging findings suggest that spotted wolffish harbour unique ocular features not yet described in marine teleosts and that visual function might involve specific retinal tissue shape dynamics in these animals. Finally, extensive endogenous biofluorescence is present in the retinal tissues of both animals, which raises questions about how these animals might use retinal tissue in novel ways for visual perception and/or communication. This work advances fundamental knowledge on the visual systems of two economically important but now threatened North Atlantic teleosts and provides a basic foundation for further research on the visual systems of these animals in health versus disease settings. This work could also be useful for understanding and optimizing the health and welfare of lumpfish and spotted wolffish in aquaculture towards a one health or integrative perspective.

Novel segmentation algorithm for high-throughput analysis of spectral domain-optical coherence tomography imaging of teleost retinas.

Spectral domain-optical coherence tomography (SD-OCT) has become an essential tool for assessing ocular tissues in live subjects and conducting research on ocular development, health, and disease. The processing of SD-OCT images, particularly those from non-mammalian species, is a labor-intensive manual process due to a lack of automated analytical programs. This paper describes the development and implementation of a novel computer algorithm for the quantitative analysis of SD-OCT images of live teleost eyes. Automated segmentation processing of SD-OCT images of retinal layers was developed using a novel algorithm based on thresholding. The algorithm measures retinal thickness characteristics in a large volume of imaging data of teleost ocular structures in a short time, providing increased accuracy and repeatability of SD-OCT image analysis over manual measurements. The algorithm also generates hundreds of retinal thickness measurements per image for a large number of images for a given dataset. Meanwhile, heat mapping software that plots SD-OCT image measurements as a color gradient was also created. This software directly converts the measurements of each processed image to represent changes in thickness across the whole retinal scan. It also enables 2D and 3D visualization of retinal thickness across the scan, facilitating specimen comparison and localization of areas of interest. The study findings showed that the novel algorithm is more accurate, reliable, and repeatable than manual SD-OCT analysis. The adaptability of the algorithm makes it potentially suitable for analyzing SD-OCT scans of other non-mammalian species.

CD45 in ocular tissues during larval and juvenile stages and early stages of V. anguillarum infection in young lumpfish (Cyclopterus lumpus).

Immune responses to infectious diseases impacting lumpfish (Cyclopterus lumpus) eye tissue are only starting to be studied at a molecular and histopathological level. In this study, we extend our understanding of lumpfish sensory organ anatomy, of components of the lumpfish nasal and ocular immune system and the nature of the intraocular response to Vibrio anguillarum infection. We have evaluated the expression of cluster of differentiation (CD) 45 protein, a tyrosine phosphatase, in larval and juvenile lumpfish tissues in order to spatially survey ocular and related head structures that may participate in early stages of intraocular immune responses. We provide here a histological mapping of the larval lumpfish nasal chamber system since its connectively with the eye though mucosal epithelia have not been explored. These results build upon our growing understanding of the lumpfish intraocular immune response to pathogens, exemplified herein by experimental nasally delivered V. anguillarum infection. CD45 is developmentally regulated in lumpfish eyes and periocular anatomy with early expression appearing in larvae in corneal epithelium and in nasal structures adjacent to the eye. Normal juvenile and adult lumpfish eyes express CD45 in the corneal epithelium, in leukocyte cells within blood vessel lumens of the rete mirabile, choroid body and choriocapillaris vasculatures. Experimental nasally delivered V. anguillarum infection led to qualitative and quantitative changes in CD45 expression in head kidney renal tubule tissues by 7 days post infection (dpi). The same animals showed redistribution and upregulation of corneal epithelial CD45 expression, corneal epithelial dysplasia and an increased frequency of CD45+ cells in ocular vasculature. Interestingly, while CD45 upregulation and/or CD45+ cell infiltration into inner ocular and retinal tissues was not observed under this experimental scenario, subtle neural retinal changes were observed in infected fish. This work provides new fundamental knowledge on North Atlantic teleost visual systems and vision biology in general.

Use of three-dimensional printing for adapting and optimizing smartphone ophthalmoscopy to existing SD-OCT instrumentation for rodent and teleost ocular research.

Use of animal models for human vision research is now pervasive. To address a range of technical challenges, laboratories either modify existing equipment or purchase products that are purpose designed. Three-dimensional (3D) printing technology now allows the do-it-yourself capability to invent, innovate, and manufacture for a specific purpose. Ophthalmic imaging is often used with a range of other sophisticated experimental retinal imaging techniques, such as spectral domain optical coherence tomography (SD-OCT). The handheld smartphone camera and cost-effective, readily available professional-quality apps now allow accessible high-definition video ophthalmic image recording. However, to our knowledge, there are few reports of adapting smartphone ophthalmic imaging to existing experimental SD-OCT imaging instrumentation. This would offer better accuracy, reproducibility, and most importantly, precision. The objective of the present study was to use 3D printing to enhance the functionality and precision of existing SD-OCT instrumentation and smartphone-based ophthalmic imaging through construction of a custom 3D-printed assembly. The assembly can be controlled either manually or by the highly precise rodent stage of the SD-OCT instrument. Using this technical approach, 3D printing facilitated a novel methodology for high-quality ophthalmic imaging with low cost and ease of production either manually or by enhancing existing SD-OCT instrumentation.

Ocular tissue changes associated with anterior segment opacity in lumpfish (Cyclopterus lumpus L) eye.

Lumpfish use their vision to hunt prey or, in the case of aquaculture, to see and eat pelleted diets. A common anterior ocular opacity abnormality designated in the literature as "cataract" described in both farmed and wild lumpfish has not yet been characterized in detail at the pathobiological level. We describe here lens tissue changes associated with cataract in cultured and domesticated lumpfish. Methodology included gross observations, ophthalmoscopy and histology. Young adult cultured animals approaching 400 days post-hatch presented a range of anterior segment opacities associated with lenticular abnormalities observable at a histological level. Wild aged domesticated animals also displayed cataracts characterized mainly by abnormalities of the lens observed by both ophthalmoscopy and histology. Dysplastic lesions of the lens in one aged domesticated lumpfish were accompanied with both retinal and optic nerve degeneration. These novel naturally occurring anatomical changes in lumpfish present both commonalities and unique features associated with cataract in young adult cultured animals versus aged domesticated broodstock animals.

Novel characteristics of the cultured Lumpfish Cyclopterus lumpus eye during post-hatch larval and juvenile developmental stages.

We systematically analysed the characteristics of the Cyclopterus lumpus eye and retina during cultured post-hatch developmental stages using gross observations, histology, immunohistochemistry, microscopy, fundus imaging and spectral domain optical coherence tomography retinal imaging. Post-hatch developing cultured C. lumpus eye and retinal tissues share a number of features typically conserved in other teleost fish. However, cultured C. lumpus possess some novel ocular and retinal features different from previous descriptions of other teleosts, including a prominent retractor lentis pigmented tissue closely associated with the vascular rete mirabile, peripherally located lobes of separate retinal tissue containing proliferative cells, extensive tapetum material of varying thickness, prominent fundus stripes and an elongated rod-shaped optic nerve stalk. Post-hatch developing cultured C. lumpus also developmentally regulate a protein homologous to alpha smooth-muscle actin in strikingly dense continuous bands in the plexiform layers of the retina. The novel features of the eye and retina of cultured C. lumpus described here could contribute to our understanding of fitness and survival of C. lumpus in a widely ranging habitat.

High rates of glucose utilization in the gas gland of Atlantic cod (Gadus morhua) are supported by GLUT1 and HK1b.

The gas gland of physoclistous fish utilizes glucose to generate lactic acid that leads to the off-loading of oxygen from haemoglobin. This study addresses characteristics of the first two steps in glucose utilization in the gas gland of Atlantic cod (Gadus morhua). Glucose metabolism by isolated gas gland cells was 12- and 170-fold higher, respectively, than that in heart and red blood cells (RBCs) as determined by the production of (3)H2O from [2-(3)H]glucose. In the gas gland, essentially all of the glucose consumed was converted to lactate. Glucose uptake in the gas gland shows a very high dependence upon facilitated transport as evidenced by saturation of uptake of 2-deoxyglucose at a low extracellular concentration and a requirement for high levels of cytochalasin B for uptake inhibition despite the high efficacy of this treatment in heart and RBCs. Glucose transport is via glucose transporter 1 (GLUT1), which is localized to the glandular cells. GLUT1 western blot analysis from whole-tissue lysates displayed a band with a relative molecular mass of 52 kDa, consistent with the deduced amino acid sequence. Levels of 52 kDa GLUT1 in the gas gland were 2.3- and 33-fold higher, respectively, than those in heart and RBCs, respectively. Glucose phosphorylation is catalysed by hexokinase Ib (HKIb), a paralogue that cannot bind to the outer mitochondrial membrane. Transcript levels of HKIb in the gas gland were 52- and 57-fold more abundant, respectively, than those in heart and RBCs. It appears that high levels of GLUT1 protein and an unusual isoform of HKI are both critical for the high rates of glycolysis in gas gland cells.

Genetic Variants of the Beta-Adrenergic Receptor Pathways as Both Risk and Protective Factors for Retinopathy of Prematurity.

PURPOSE: There is strong evidence that genetic factors influence retinopathy of prematurity (ROP), a neovascular eye disease. It has been previously suggested that polymorphisms in the genes involved in ß-adrenergic receptor (ADRß) pathways could protect against ROP. Antagonists for the ADRß are actively tested in clinical trials for ROP treatment, but not without controversy and safety concerns. This study was designed to assess whether genetic variations in components of the ADRß signaling pathways associate with risk of developing ROP. DESIGN: An observational case-control targeted genetic analysis. METHODS: A study was carried out in premature participants with (n = 30) or without (n = 34) ROP and full-term controls (n = 20), who were divided into a discovery cohort and a validation cohort. ROP was defined using International Classification of Retinopathy of Prematurity criteria (ICROP). Targeted sequencing of 20 genes in the ADRß pathways was performed in the discovery cohort. Polymerase chain reaction (PCR)/restriction enzyme analysis for some of the discovered ROP-associated variants was performed for validation of the results using the validation cohort. RESULTS: The discovery cohort revealed 543 bi-allelic variants within 20 genes of the ADRß pathways. Ten single-nucleotide variants (SNVs) in 5 genes including protein kinase A regulatory subunit 1α (PRKAR1A), rap guanine exchange factor 3 (RAPGEF3), adenylyl cyclase 4 (ADCY4), ADCY7, and ADCY9 were associated with ROP (P < .05). The most significant SNV was found in PRKAR1A (P = .001). Multiple variants located in the 3'-untranslated region (3'UTR) of RAPGEF3 were also associated with ROP (P < .05). PCR/restriction enzyme analysis of the 3'UTR of RAPGEF3 methodologically validated these findings. CONCLUSION: SNVs in PRKAR1A may represent protective factors whereas SNVs in RAPGEF3 may represent risk factors for ROP. PRKAR1α has previously been implicated in retinal vascular development whereas the RAPGEF3 product has a role in the maintenance of vascular barrier function, 2 processes important in ROP. Multicenter validation of these newly discovered risk factors could lead to valuable tools for predicting and preventing the development of severe ROP.

Increased water temperature contributes to a chondrogenesis response in the eyes of spotted wolffish.

Adult vertebrate cartilage is usually quiescent. Some vertebrates possess ocular scleral skeletons composed of cartilage or bone. The morphological characteristics of the spotted wolffish (Anarhichas minor) scleral skeleton have not been described. Here we assessed the scleral skeletons of cultured spotted wolffish, a globally threatened marine species. The healthy spotted wolffish we assessed had scleral skeletons with a low percentage of cells staining for the chondrogenesis marker sex-determining region Y-box (Sox) 9, but harboured a population of intraocular cells that co-express immunoglobulin M (IgM) and Sox9. Scleral skeletons of spotted wolffish with grossly observable eye abnormalities displayed a high degree of perochondrial activation as evidenced by cellular morphology and expression of proliferating cell nuclear antigen (PCNA) and phosphotyrosine. Cells staining for cluster of differentiation (CD) 45 and IgM accumulated around sites of active chondrogenesis, which contained cells that strongly expressed Sox9. The level of scleral chondrogenesis and the numbers of scleral cartilage PCNA positive cells increased with the temperature of the water in which spotted wolffish were cultured. Our results provide new knowledge of differing Sox9 spatial tissue expression patterns during chondrogenesis in normal control and ocular insult paradigms. Our work also provides evidence that spotted wolffish possess an inherent scleral chondrogenesis response that may be sensitive to temperature. This work also advances the fundamental knowledge of teleost ocular skeletal systems.

Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax).

PURPOSE: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt. METHODS: Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes. RESULTS: Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8-10 °C. Compared to the 8-10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry. CONCLUSIONS: This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol.

CD10+ Cells and IgM in Pathogen Response in Lumpfish (Cyclopterus lumpus) Eye Tissues.

Lumpfish (Cyclopterus lumpus), a North Atlantic "cleaner" fish, is utilized to biocontrol salmon louse (Lepeophtheirus salmonis) in Atlantic salmon (Salmo salar) farms. Lumpfish require excellent vision to scan for and eat louse on salmon skin. The lumpfish eye immune response to infectious diseases has not been explored. We examined the ocular response to a natural parasite infection in wild lumpfish and to an experimental bacterial infection in cultured lumpfish. Cysts associated with natural myxozoan infection in the ocular scleral cartilage of wild adult lumpfish harbored cells expressing cluster of differentiation 10 (CD10) and immunoglobulin M (IgM). Experimental Vibrio anguillarum infection, which led to exophthalmos and disorganization of the retinal tissues was associated with disruption of normal CD10 expression, CD10+ cellular infiltration and IgM expression. We further describe the lumpfish CD10 orthologue and characterize the lumpfish scleral skeleton in the context of myxozoan scleral cysts. We propose that lumpfish develop an intraocular response to pathogens, exemplified herein by myxozoan and V. anguillarum infection involving novel CD10+ cells and IgM+ cells to contain and mitigate damage to eye structures. This work is the first demonstration of CD10 and IgM expressing cells in a novel ocular immune system component in response to disease in a teleost.

Tubedown expression correlates with the differentiation status and aggressiveness of neuroblastic tumors.

PURPOSE: The discovery and validation of new prognostic factors and further refinement of risk group stratification are needed to improve clinical interpretation of neuroblastoma. Our laboratory isolated and characterized a developmentally regulated gene named TUBEDOWN against which we have raised a monoclonal antibody (OE5). Tubedown becomes down-regulated postnatally yet remains strongly expressed in some neuroblastomas. The purpose of this study is to define the utility of Tubedown expression as a new measure of the differentiation status and aggressiveness of neuroblastic tumors. EXPERIMENTAL DESIGN: Tubedown protein expression was quantitatively assessed in neuroblastic tumors (neuroblastomas, ganglioneuroblastomas, and ganglioneuromas) and normal adrenal tissues using Western blot and OE5 immunohistochemistry. Regulation of Tubedown expression during retinoic acid-induced neuronal differentiation in neuroblastoma cell lines was assessed by Western blotting. RESULTS: High levels of Tubedown expression are observed in tumors with significant neuroblastic component, unfavorable histopathology, advanced stage, high-risk group, and poor outcome. In contrast, more differentiated subsets of neuroblastic tumors, ganglioneuroblastomas with favorable histopathology and ganglioneuromas, express low levels of Tubedown. In vitro, marked retinoic acid-induced neuronal differentiation responses of neuroblastoma cells are associated with a significant decrease in Tubedown expression, whereas limited neuronal differentiation responses to retinoic acid were associated with little or no decrease in Tubedown expression. CONCLUSIONS: Our results indicate that the levels of Tubedown expression are linked to the differentiation status and aggressiveness of neuroblastic tumors and represent an independent prognostic factor for neuroblastoma. Tubedown expression may be useful to more accurately define different neuroblastic tumor subsets and ultimately provide more adequate assessment and treatment for neuroblastoma patients.

Tubedown-1 (Tbdn-1) suppression in oxygen-induced retinopathy and in retinopathy of prematurity.

PURPOSE: Identification of unique proteins involved in retinopathy of prematurity (ROP) may facilitate new and more effective diagnostic tools and molecular-based treatments for ROP. Tubedown-1 (Tbdn-1), a novel homeostatic protein which copurifies with an acetyltransferase activity, is expressed in normal retinal endothelium and is specifically suppressed in retinal endothelial cells from patients with proliferative diabetic retinopathy. Furthermore, recent in vivo knockdown studies in mice have revealed that Tbdn-1 is important for retinal blood vessel homeostasis and for preventing retinal neovascularization in adults. The purpose of the present study was to determine if the expression pattern of Tbdn-1 is altered during oxygen-induced retinal neovascularization in mice and in a specimen of stage 3 human ROP. METHODS: Specimens of oxygen-induced retinal neovascularization in mice, and a single specimen of active stage 3 ROP were studied by immunohistochemistry and digital image analysis using antibodies raised against Tbdn-1 and other blood vessel markers. RESULTS: The pattern of Tbdn-1 expression during the course of oxygen-induced retinal neovascularization in mice suggests a regulating role in neonatal retinopathy. Retinal lesions from oxygen-induced retinal neovascularization in mice display suppression of retinal endothelial Tbdn-1 protein expression in conjunction with an increase in expression of proliferating cell nuclear antigen (a marker of proliferation) and alpha smooth muscle actin (a marker of myofibroblastic cells). Abnormal blood vessels within vitreoretinal neovascular lesions in a human specimen of active stage 3 ROP did not show Tbdn-1 protein expression. CONCLUSIONS: These results suggest that the loss of retinal endothelial Tbdn-1 expression may be a contributing factor in retinal blood vessel proliferation in ROP.

Tubedown regulation of retinal endothelial permeability signaling pathways.

Tubedown (Tbdn; Naa15), a subunit of the N-terminal acetyltransferase NatA, complexes with the c-Src substrate Cortactin and supports adult retinal homeostasis through regulation of vascular permeability. Here we investigate the role of Tbdn expression on signaling components of retinal endothelial permeability to understand how Tbdn regulates the vasculature and supports retinal homeostasis. Tbdn knockdown-induced hyperpermeability to Albumin in retinal endothelial cells was associated with an increase in the levels of activation of the Src family kinases (SFK) c-Src, Fyn and Lyn and phospho-Cortactin (Tyr421). The knockdown of Cortactin expression reduced Tbdn knockdown-induced permeability to Albumin and the levels of activated SFK. Inhibition of SFK in retinal endothelial cells decreased Tbdn knockdown-induced permeability to Albumin and phospho-Cortactin (Tyr421) levels. Retinal lesions of endothelial-specific Tbdn knockdown mice, with tissue thickening, fibrovascular growth, and hyperpermeable vessels displayed an increase in the levels of activated c-Src. Moreover, the retinal lesions of patients with proliferative diabetic retinopathy (PDR) associated with a loss of Tbdn expression and hyperpermeability to Albumin displayed increased levels of activated SFK in retinal blood vessels. Taken together, these results implicate Tbdn as an important regulator of retinal endothelial permeability and homeostasis by modulating a signaling pathway involving c-Src and Cortactin.

Conditional knockdown of tubedown-1 in endothelial cells leads to neovascular retinopathy.

PURPOSE: Identification of novel proteins involved in retinal neovascularization may facilitate new and more effective molecular-based treatments for proliferative retinopathy. Tubedown-1 (Tbdn-1) is a novel protein that shows homology to the yeast acetyltransferase subunit NAT1 and copurifies with an acetyltransferase activity. Tbdn-1 is expressed in normal retinal endothelium but is specifically suppressed in retinal endothelial cells from patients with proliferative diabetic retinopathy. The purpose of this study was to investigate the importance of Tbdn-1 expression in retinal blood vessels in vivo. METHODS: A bitransgenic mouse model that enables conditional knockdown of Tbdn-1 specifically in endothelial cells was produced and studied using molecular, histologic, and immunohistochemical techniques and morphometric analysis. RESULTS: Tbdn-1-suppressed mice exhibited retinal and choroidal neovascularization with intra- and preretinal fibrovascular lesions similar to human proliferative retinopathies. Retinal lesions observed in Tbdn-1-suppressed mice increased in severity with prolonged suppression of Tbdn-1. In comparison to normal retina, the retinal lesions displayed alterations in the basement membrane of blood vessels and in the distribution of glial and myofibroblastic cells. Moreover, the pathologic consequences of Tbdn-1 knockdown in endothelium were restricted to the retina and the choroid. CONCLUSIONS: These results indicate that the maintenance of Tbdn-1 expression is important for retinal blood vessel homeostasis and for controlling retinal neovascularization in adults. Restoration of Tbdn-1 protein expression and/or activity may provide a novel approach for treating proliferative retinopathies.

Loss of tubedown expression as a contributing factor in the development of age-related retinopathy.

PURPOSE: Tubedown (Tbdn), a cortactin-binding acetyltransferase subunit, regulates retinal vascular permeability and homeostasis in adulthood. Here the authors explore whether Tbdn loss during aging might contribute to the mechanisms underlying age-related neovascular retinopathy. METHODS: A conditional endothelial-specific transgenic model of Tbdn loss was compared with aged mouse and human specimens from 5- to 93-year-old individuals. Specimens were analyzed by morphometric measurements and for functional markers using immunohistochemistry and Western blot analysis. RESULTS: An age-dependent decrease in Tbdn expression in endothelial cells of the posterior pole of the eye correlated with pathologic changes in choroidal and retinal tissues of aged mice. In humans, aged specimens without eye disease exhibited a moderate decrease in retinal and choroidal endothelial Tbdn expression compared with younger persons, whereas a greater decrease in choroid vascular Tbdn expression was observed in patients with age-related macular degeneration. In mice, Tbdn loss resulting from old age or conditional Tbdn knockdown was associated with retinal lesions showing significant extravascularly localized albumin and correlated with increased activity of senescence-associated ß-galactosidase in the retinal pigment epithelium. A range of abnormalities in RPE, Bruch's membrane, and choriocapillaris observable at the ultrastructural level in Tbdn-knockdown eyes recapitulate those present in human AMD. CONCLUSIONS: This work provides evidence that the marked decrease in the level of expression of Tbdn in the retinal and choroidal vasculature during aging contributes to the multifactorial process that leads to the development of age-related retinopathy and choroidopathy.

Tubedown associates with cortactin and controls permeability of retinal endothelial cells to albumin.

Tubedown (Narg1, Tbdn), a member of the Nat1 family of proteins, associates with the acetyltransferase Ard1 and exerts an angiostatic function in adult retinal-blood-vessel homeostasis. The purpose of the present study was to gain a better understanding of the nature of the Tbdn protein complex and how it might exert a homeostatic influence on blood vessels. Immunoprecipitation of Tbdn from endothelial cells followed by gel electrophoresis and liquid-chromatography-tandem-mass-spectrometry identified the actin-cytoskeleton-binding protein cortactin as a co-immunopurifying species. Western blotting confirmed the association between Tbdn and cortactin. Immunofluorescence confocal microscopy revealed that Tbdn colocalizes with cortactin and F-actin in cytoplasmic regions and at the cortex of cultured endothelial cells. Because cortactin is known to regulate cellular permeability through its interaction with the actin cytoskeleton, a process that is crucial for endothelial cell homeostasis, the role of Tbdn on endothelial cell permeability was examined. Knockdown of Tbdn expression in endothelial cells led to the co-suppression of Ard1 protein expression and to a significant increase in cellular permeability measured by the transit of FITC-albumin across the cellular monolayer. Furthermore, the proliferative retinal neovascularization and thickening resulting from induction of Tbdn knockdown in endothelium in transgenic mice was associated with a significant increase in extravasation or leakage of albumin from abnormal retinal blood vessels in vivo. These results provide evidence that an association occurs between Tbdn and cortactin, and that Tbdn is involved in the regulation of retinal-endothelial-cell permeability to albumin. This work implicates a functional role for Tbdn in blood-vessel permeability dynamics that are crucial for vascular homeostasis.

Role of Cys41 in the N-terminal domain of lumican in ex vivo collagen fibrillogenesis by cultured corneal stromal cells.

The keratan sulphate proteoglycan lumican regulates collagen fibrillogenesis to maintain the integrity and function of connective tissues such as cornea. We examined the role of a highly conserved cysteine-containing domain proximal to the N-terminus of lumican in collagen fibrillogenesis using site-specific mutagenesis to prepare plasmid DNA encoding wild-type murine lumican (Cys(37)-Xaa(3)-Cys(41)-Xaa-Cys-Xaa(9)-Cys) and a Cys-->Ser (C/S) mutant (Cys(37)-Xaa(3)-Ser(41)-Xaa-Cys-Xaa(9)-Cys). cDNAs were cloned into the pSecTag2A vector, and cultures of MK/T-1 cells (an immortalized cell line from mouse keratocytes) were transfected with the cDNAs. Stable transformants were selected and cloned in the presence of Zeocin. All stable transformants maintained a dendritic morphology and growth rate similar to those of parental MK/T-1 cells. Western blot analysis with anti-lumican antibody detected a 42 kDa lumican protein secreted into the culture medium of both wild-type and C/S mutant lumican cell lines. Ultrastructural analyses by transmission electron microscopy showed both cell lines to form a multi-layered stroma ex vivo, but the matrix assembled by the two cell lines differed. Compared with the mutant cell line, the wild-type cells assembled a more organized matrix with regions containing orthogonal collagen fibrils. In addition, the fibrils in the extracellular matrix formed by the mutant cell line exhibited alterations in fibril packing and structure. Immunostaining analysed by confocal microscopy showed a further difference in this matrix, with the marked occurrence of lumican and collagen I co-localization in the lumican wild-type cells, but a lack thereof in the lumican C/S mutant cells. The results indicate that the cysteine-rich domain of lumican is important in collagen fibrillogenesis and stromal matrix assembly.