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Sample records for mouse retinal endothelial

  1. BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

    Science.gov (United States)

    Mondragon, Albert A; Betts-Obregon, Brandi S; Moritz, Robert J; Parvathaneni, Kalpana; Navarro, Mary M; Kim, Hong Seok; Lee, Chi Fung; LeBaron, Richard G; Asmis, Reto; Tsin, Andrew T

    2015-01-01

    Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFβ to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFβ, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFβ-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFβ1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFβ1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFβ showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFβ, as well as TGFβ receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFβ or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFβ released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

  2. Critical Endothelial Regulation by LRP5 during Retinal Vascular Development

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    Huang, Wei; Li, Qing; Amiry-Moghaddam, Mahmood; Hokama, Madoka; Sardi, Sylvia H.; Nagao, Masashi; Warman, Matthew L.; Olsen, Bjorn R.

    2016-01-01

    Vascular abnormalities in the eye are the leading cause of many forms of inherited and acquired human blindness. Loss-of-function mutations in the Wnt-binding co-receptor LRP5 leads to aberrant ocular vascularization and loss of vision in genetic disorders such as osteoporosis-pseudoglioma syndrome. The canonical Wnt-β-catenin pathway is known to regulate retinal vascular development. However, it is unclear what precise role LPR5 plays in this process. Here, we show that loss of LRP5 function in mice causes retinal hypovascularization during development as well as retinal neovascularization in adulthood with disorganized and leaky vessels. Using a highly specific Flk1-CreBreier line for vascular endothelial cells, together with several genetic models, we demonstrate that loss of endothelium-derived LRP5 recapitulates the retinal vascular defects in Lrp5-/- mice. In addition, restoring LRP5 function only in endothelial cells in Lrp5-/- mice rescues their retinal vascular abnormalities. Furthermore, we show that retinal vascularization is regulated by LRP5 in a dosage dependent manner and does not depend on LRP6. Our study provides the first direct evidence that endothelium-derived LRP5 is both necessary and sufficient to mediate its critical role in the development and maintenance of retinal vasculature. PMID:27031698

  3. Investigation of retinal morphology alterations using spectral domain optical coherence tomography in a mouse model of retinal branch and central retinal vein occlusion.

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    Andreas Ebneter

    Full Text Available Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001 compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001. Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.

  4. Apelin is a novel angiogenic factor in retinal endothelial cells

    International Nuclear Information System (INIS)

    Kasai, Atsushi; Shintani, Norihito; Oda, Maki; Kakuda, Michiya; Hashimoto, Hitoshi; Matsuda, Toshio; Hinuma, Shuji; Baba, Akemichi

    2004-01-01

    There has been much focus recently on the possible functions of apelin, an endogenous ligand for the orphan G-protein-coupled receptor APJ, in cardiovascular and central nervous systems. We report a new function of apelin as a novel angiogenic factor in retinal endothelial cells. The retinal endothelial cell line RF/6A highly expressed both apelin and APJ transcripts, while human umbilical venous endothelial cells (HUVECs) only expressed apelin mRNA. In accordance with these observations, apelin at concentrations of 1 pM-1 μM significantly enhanced migration, proliferation, and capillary-like tube formation of RF/6A cells, but not those of HUVECs, whereas VEGF stimulates those parameters of both cell types. In vivo Matrigel plug assay for angiogenesis, the inclusion of 1 nM apelin in the Matrigel resulted in clear capillary-like formations with an increase of hemoglobin content in the plug. This is the first report showing that apelin is an angiogenic factor in retinal endothelial cells

  5. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

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    Do, Ji Yeon; Choi, Young Keun [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Kook, Hyun [Department of Pharmacology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Suk, Kyoungho [Department of Pharmacology, Brain Science & Engineering Institute, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Lee, In-Kyu [Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease, Kyungpook National University School of Medicine, Kyungpook National University, Daegu (Korea, Republic of); Division of Endocrinology and Metabolism, Department of Internal Medicine, Research Institute of Aging and Metabolism, Kyungpook National University School of Medicine, Daegu (Korea, Republic of); Park, Dong Ho, E-mail: sarasate2222@gmail.com [Department of Ophthalmology, Kyungpook National University School of Medicine, Daegu (Korea, Republic of)

    2015-05-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O{sub 2}). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice.

  6. Retinal hypoxia induces vascular endothelial growth factor through induction of estrogen-related receptor γ

    International Nuclear Information System (INIS)

    Do, Ji Yeon; Choi, Young Keun; Kook, Hyun; Suk, Kyoungho; Lee, In-Kyu; Park, Dong Ho

    2015-01-01

    Ischemic retinopathies causing overexpression of pro-angiogenic factors, including vascular endothelial growth factor (VEGF), are the most common cause of blindness. Thus, understanding the pathophysiology of targetable pathways that regulate retinal VEGF is of great interest. A conserved binding site for estrogen-related receptor γ (ERRγ) has been identified in the promoter of the Vegfa gene. ERRγ is a constitutively active orphan nuclear receptor and its expression is increased by hypoxic stimuli in metabolically active tissues. This study evaluated the role of ERRγ in the ischemic retina and the anti-VEGF potential of GSK5182, a selective inverse agonist of ERRγ. In an oxygen-induced retinopathy (OIR) mouse model, immunohistochemistry showed significantly increased ERRγ expression in the ganglion cell layer at postnatal day (P) 17. In a ganglion cell line (RGC-5), mRNA and protein levels of ERRγ were increased by desferrioxamine treatment and hypoxic conditions (1% O 2 ). Transient transfection of RGC-5 cells revealed that ERRγ regulated Vegfa expression and this was inhibited by GSK5182. Intravitreal injection of GSK5182 into the OIR model at P14 inhibited retinal Vegfa mRNA expression at P17. GSK5182 suppresses hypoxia-induced VEGF expression via ERRγ; therefore, ERRγ could be a treatment target for ischemic retinopathies. - Highlights: • OIR mice exhibited increased ERRγ expression in the ganglion cell layer. • Hypoxia-induced ERRγ expression was observed in retinal ganglion cells. • ERRγ overexpression increased VEGFA expression in retinal ganglion cells. • An ERRγ inverse agonist suppressed VEGFA expression in retinal ganglion cells. • Intravitreal injection of an ERRγ inverse agonist suppressed VEGFA in OIR mice

  7. Endothelial Protein C–Targeting Liposomes Show Enhanced Uptake and Improved Therapeutic Efficacy in Human Retinal Endothelial Cells

    DEFF Research Database (Denmark)

    Arta, Anthoula; Eriksen, Anne Z.; Melander, Fredrik

    2018-01-01

    PURPOSE. To determine whether human retinal endothelial cells (HRECs) express the endothelial cell protein C receptor (EPCR) and to realize its potential as a targeting moiety by developing novel single and dual corticosteroid–loaded functionalized liposomes that exhibit both enhanced uptake by H...... of cell tube formations in contrast to nontargeting liposomes. CONCLUSIONS. We show that HRECs express EPCR and this receptor could be a promising nanomedicine target in ocular diseases where the endothelial barrier of the retina is compromised....

  8. Mitochondrial Protection by Exogenous Otx2 in Mouse Retinal Neurons

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    Hyoung-Tai Kim

    2015-11-01

    Full Text Available OTX2 (orthodenticle homeobox 2 haplodeficiency causes diverse defects in mammalian visual systems ranging from retinal dysfunction to anophthalmia. We find that the retinal dystrophy of Otx2+/GFP heterozygous knockin mice is mainly due to the loss of bipolar cells and consequent deficits in retinal activity. Among bipolar cell types, OFF-cone bipolar subsets, which lack autonomous Otx2 gene expression but receive Otx2 proteins from photoreceptors, degenerate most rapidly in Otx2+/GFP mouse retinas, suggesting a neuroprotective effect of the imported Otx2 protein. In support of this hypothesis, retinal dystrophy in Otx2+/GFP mice is prevented by intraocular injection of Otx2 protein, which localizes to the mitochondria of bipolar cells and facilitates ATP synthesis as a part of mitochondrial ATP synthase complex. Taken together, our findings demonstrate a mitochondrial function for Otx2 and suggest a potential therapeutic application of OTX2 protein delivery in human retinal dystrophy.

  9. Downregulation of Lysyl Oxidase Protects Retinal Endothelial Cells From High Glucose-Induced Apoptosis.

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    Kim, Dongjoon; Mecham, Robert P; Trackman, Philip C; Roy, Sayon

    2017-05-01

    To investigate the effect of reducing high glucose (HG)-induced lysyl oxidase (LOX) overexpression and increased activity on retinal endothelial cell apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N) or HG (30 mM glucose) medium for 7 days. In parallel, RRECs were grown in HG medium and transfected with LOX small interfering RNA (siRNA), scrambled siRNA as control, or exposed to β-aminopropionitrile (BAPN), a LOX inhibitor. LOX expression, AKT activation, and caspase-3 activity were determined by Western blot (WB) analysis and apoptosis by differential dye staining assay. Moreover, to determine whether diabetes-induced LOX overexpression alters AKT activation and promotes apoptosis, changes in LOX expression, AKT phosphorylation, caspase-3 activation, and Bax expression were assessed in retinas of streptozotocin (STZ)-induced diabetic mice and LOX heterozygous knockout (LOX+/-) mice. WB analysis indicated significant LOX overexpression and reduced AKT activation under HG condition in RRECs. Interestingly, when cells grown in HG were transfected with LOX siRNA or exposed to BAPN, the number of apoptotic cells was significantly decreased concomitant with increased AKT phosphorylation. Diabetic mouse retinas exhibited LOX overexpression, decreased AKT phosphorylation, and increased Bax and caspase-3 activation compared to values in nondiabetic mice. In LOX+/- mice, reduced LOX levels were observed with increased AKT activity, and reduced Bax and caspase-3 activity. Furthermore, decreased levels of LOX in the LOX+/- mice was protective against diabetes-induced apoptosis. Findings from this study indicate that preventing LOX overexpression may be protective against HG-induced apoptosis in retinal vascular cells associated with diabetic retinopathy.

  10. Genomic analysis of mouse retinal development.

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    Seth Blackshaw

    2004-09-01

    Full Text Available The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE. The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Müller glia and mitotic progenitor cells were found to be highly similar, suggesting that Müller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length ("noncoding RNAs" were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.

  11. Trypsin digest protocol to analyze the retinal vasculature of a mouse model.

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    Chou, Jonathan C; Rollins, Stuart D; Fawzi, Amani A

    2013-06-13

    Trypsin digest is the gold standard method to analyze the retinal vasculature (1-5). It allows visualization of the entire network of complex three-dimensional retinal blood vessels and capillaries by creating a two-dimensional flat-mount of the interconnected vascular channels after digestion of the non-vascular components of the retina. This allows one to study various pathologic vascular changes, such as microaneurysms, capillary degeneration, and abnormal endothelial to pericyte ratios. However, the method is technically challenging, especially in mice, which have become the most widely available animal model to study the retina because of the ease of genetic manipulations (6,7). In the mouse eye, it is particularly difficult to completely remove the non-vascular components while maintaining the overall architecture of the retinal blood vessels. To date, there is a dearth of literature that describes the trypsin digest technique in detail in the mouse. This manuscript provides a detailed step-by-step methodology of the trypsin digest in mouse retina, while also providing tips on troubleshooting difficult steps.

  12. Radioprotection of mouse CNS endothelial cells in vivo

    International Nuclear Information System (INIS)

    Lyubimova, N.; Coultas, P.; Martin, R.

    1996-01-01

    Full text: Radioprotection using the minor groove binding DNA ligand Hoechst 33342 has been demonstrated in vitro, and more recently in vivo, in mouse lung. Intravenous administration was used for the lung studies, and both endothelial and alveolar epithelial cells-showed good up-take. Radiation damage to the endothelial cell population has also been postulated as important in late developing radionecrosis of spinal cord and brain. Endothelial cell density in brain can be readily determined by a fluorescent-histochemical technique. Treatment with a monoamine oxidase inhibitor and subsequent injection with L-DOPA results in an accumulation of dopamine (DA) in CNS endothelial cells. DA is converted to a fluorophore by exposure to paraformaldehyde, and cell numbers assayed by fluorescence microscopy. Earlier studies used this technique to monitor post-irradiation changes in endothelial cell density in rodent brain and showed the loss, within 24 hours, of a sensitive subpopulation comprising about 15% of the endothelial cells. Ten minutes after intravenous injection of Hoechst 33342 (80mg/kg) the ligand is confined by its limited penetration to the endothelial cells in mouse brain. When we irradiated at this time, there was protection against early endothelial cell loss. Ablation of the sensitive subpopulation in unprotected mice takes place over a dose range of 1 to 3 Gy γ-rays, but doses between 12 to 20 Gy are required in the presence of ligand. This protection equates to a very high dose modification factor of about 7 and possibly reflects a suppression of apoptosis in the sensitive endothelial subpopulation. The extent to which there is enhanced survival in the endothelial population as a whole and how the observed protection affects late CNS necrosis development has yet to be determined. However present results clearly show potential for the use of DNA-binding radioprotectors with limited penetration for investigations into the relative significance of

  13. Bevacizumab treatment reduces retinal neovascularization in a mouse model of retinopathy of prematurity

    Institute of Scientific and Technical Information of China (English)

    Fei; Feng; Yan; Cheng; Qing-Huai; Liu

    2014-01-01

    ·AIM: To evaluate the effect of different bevacizumab concentrations on retinal neovascularization in a retinopathy of prematurity(ROP) mouse model.·METHODS: A total of 60 of C57BL/6 J mice were exposed to 75% ±2% oxygen from postnatal d7 to postnatal d12. Fifteen nonexposed mice served as negative controls(group A). On d12, 30 mice(group C)were injected with 2.5 μg intravitreal bevacizumab(IVB),30 mice(group D) were injected with 1.25 μg IVB in one eye. The contralateral eyes were injected with balanced salt solution(BSS)(control group =group B). The adenosine diphosphatase(ADPase) histochemical technique was used for retinal flat mount to assess the oxygen-induced changes of retinal vessels.Neovascularization was quantified by counting the endothelial cell proliferation on the vitreal side of the inner limiting membrane of the retina. Histological changes were examined by light microscopy. The mRNA levels of vascular endothelial growth factor(VEGF) were quantified by Real-time PCR. Western-blotting analysis was performed to examine the expression of P-VEGFR.· RESULTS: Comparing with the control group B,regular distributions and reduced tortuosity of vessels were observed in our retinal flat mounts in groups C and D. The endothelial cell count per histological section was lower in groups C(P <0.0001) and D(P <0.0001) compared with the control group B. Histological evaluation showed no retinal toxicity in any group. In all oxygen treated groups VEGF mRNA expression was significantly increased as compared to age-matched controls. No significant change in VEGF mRNA expression could be achieved in either of the treatments or the oxygen controls. The results of the Western blot were consistent with that of the Real-time PCR analysis.·CONCLUSION: An intravitreal injection of bevacizumab is able to reduce angioproliferative retinopathy in a mouse model for oxygen-induced retinopathy.

  14. INTRAOCULAR AND SERUM LEVELS OF VASCULAR ENDOTHELIAL GROWTH FACTOR IN ACUTE RETINAL NECROSIS AND OCULAR TOXOPLASMOSIS

    NARCIS (Netherlands)

    Wiertz, Karin; De Visser, Lenneke; Rijkers, Ger; De Groot-Mijnes, Jolanda; Los, Leonie; Rothova, Aniki

    2010-01-01

    Purpose: To determine the intraocular and serum vascular endothelial growth factor (VEGF) levels in patients with acute retinal necrosis (ARN) and compare those with VEGF levels found in patients with ocular toxoplasmosis (OT). Methods: Paired intraocular fluid and serum samples of 17 patients with

  15. Adaptive optics retinal imaging in the living mouse eye

    Science.gov (United States)

    Geng, Ying; Dubra, Alfredo; Yin, Lu; Merigan, William H.; Sharma, Robin; Libby, Richard T.; Williams, David R.

    2012-01-01

    Correction of the eye’s monochromatic aberrations using adaptive optics (AO) can improve the resolution of in vivo mouse retinal images [Biss et al., Opt. Lett. 32(6), 659 (2007) and Alt et al., Proc. SPIE 7550, 755019 (2010)], but previous attempts have been limited by poor spot quality in the Shack-Hartmann wavefront sensor (SHWS). Recent advances in mouse eye wavefront sensing using an adjustable focus beacon with an annular beam profile have improved the wavefront sensor spot quality [Geng et al., Biomed. Opt. Express 2(4), 717 (2011)], and we have incorporated them into a fluorescence adaptive optics scanning laser ophthalmoscope (AOSLO). The performance of the instrument was tested on the living mouse eye, and images of multiple retinal structures, including the photoreceptor mosaic, nerve fiber bundles, fine capillaries and fluorescently labeled ganglion cells were obtained. The in vivo transverse and axial resolutions of the fluorescence channel of the AOSLO were estimated from the full width half maximum (FWHM) of the line and point spread functions (LSF and PSF), and were found to be better than 0.79 μm ± 0.03 μm (STD)(45% wider than the diffraction limit) and 10.8 μm ± 0.7 μm (STD)(two times the diffraction limit), respectively. The axial positional accuracy was estimated to be 0.36 μm. This resolution and positional accuracy has allowed us to classify many ganglion cell types, such as bistratified ganglion cells, in vivo. PMID:22574260

  16. Suppression of Retinal Neovascularization in vivo by Inhibition of Vascular Endothelial Growth Factor (VEGF) Using Soluble VEGF-Receptor Chimeric Proteins

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    Aiello, Lloyd Paul; Pierce, Eric A.; Foley, Eliot D.; Takagi, Hitoshi; Chen, Helen; Riddle, Lavon; Ferrara, Napoleone; King, George L.; Smith, Lois E. H.

    1995-11-01

    The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-um section averaged 47% ± 4% (P < 0.001) and 37% ± 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66

  17. Fundus autofluorescence findings in a mouse model of retinal detachment.

    Science.gov (United States)

    Secondi, Roberta; Kong, Jian; Blonska, Anna M; Staurenghi, Giovanni; Sparrow, Janet R

    2012-08-07

    Fundus autofluorescence (fundus AF) changes were monitored in a mouse model of retinal detachment (RD). RD was induced by transscleral injection of hyaluronic acid (Healon) or sterile balanced salt solution (BSS) into the subretinal space of 4-5-day-old albino Abca4 null mutant and Abca4 wild-type mice. Images acquired by confocal scanning laser ophthalmoscopy (Spectralis HRA) were correlated with spectral domain optical coherence tomography (SD-OCT), infrared reflectance (IR), fluorescence spectroscopy, and histologic analysis. Results. In the area of detached retina, multiple hyperreflective spots in IR images corresponded to punctate areas of intense autofluorescence visible in fundus AF mode. The puncta exhibited changes in fluorescence intensity with time. SD-OCT disclosed undulations of the neural retina and hyperreflectivity of the photoreceptor layer that likely corresponded to histologically visible photoreceptor cell rosettes. Fluorescence emission spectra generated using flat-mounted retina, and 488 and 561 nm excitation, were similar to that of RPE lipofuscin. With increased excitation wavelength, the emission maximum shifted towards longer wavelengths, a characteristic typical of fundus autofluorescence. In detached retinas, hyper-autofluorescent spots appeared to originate from photoreceptor outer segments that were arranged within retinal folds and rosettes. Consistent with this interpretation is the finding that the autofluorescence was spectroscopically similar to the bisretinoids that constitute RPE lipofuscin. Under the conditions of a RD, abnormal autofluorescence may arise from excessive production of bisretinoid by impaired photoreceptor cells.

  18. Suppressing thyroid hormone signaling preserves cone photoreceptors in mouse models of retinal degeneration

    OpenAIRE

    Ma, Hongwei; Thapa, Arjun; Morris, Lynsie; Redmond, T. Michael; Baehr, Wolfgang; Ding, Xi-Qin

    2014-01-01

    Photoreceptors degenerate in a wide array of hereditary retinal diseases and age-related macular degeneration. There is currently no treatment available for retinal degenerations. While outnumbered roughly 20:1 by rods in the human retina, it is the cones that mediate color vision and visual acuity, and their survival is critical for vision. In this communication, we investigate whether thyroid hormone (TH) signaling affects cone viability in retinal degeneration mouse models. TH signaling is...

  19. Primary amines protect against retinal degeneration in mouse models of retinopathies.

    Science.gov (United States)

    Maeda, Akiko; Golczak, Marcin; Chen, Yu; Okano, Kiichiro; Kohno, Hideo; Shiose, Satomi; Ishikawa, Kaede; Harte, William; Palczewska, Grazyna; Maeda, Tadao; Palczewski, Krzysztof

    2011-12-25

    Vertebrate vision is initiated by photoisomerization of the visual pigment chromophore 11-cis-retinal and is maintained by continuous regeneration of this retinoid through a series of reactions termed the retinoid cycle. However, toxic side reaction products, especially those involving reactive aldehyde groups of the photoisomerized product, all-trans-retinal, can cause severe retinal pathology. Here we lowered peak concentrations of free all-trans-retinal with primary amine-containing Food and Drug Administration (FDA)-approved drugs that did not inhibit chromophore regeneration in mouse models of retinal degeneration. Schiff base adducts between all-trans-retinal and these amines were identified by MS. Adducts were observed in mouse eyes only when an experimental drug protected the retina from degeneration in both short-term and long-term treatment experiments. This study demonstrates a molecular basis of all-trans-retinal-induced retinal pathology and identifies an assemblage of FDA-approved compounds with protective effects against this pathology in a mouse model that shows features of Stargardt's disease and age-related retinal degeneration.

  20. Anti vascular endothelial growth factor (bevacizumab) in central retinal vein occlusion: an interventional case series

    International Nuclear Information System (INIS)

    Jan, S.; Khan, M.N.; Karim, S.; Khan, M.T.; Hussain, Z.; Khan, S.; Nazim, M.

    2010-01-01

    Vascular endothelial growth factor plays major role in ocular angio genesis and retinal edema production and is a step forward in the management of ocular neovascularization and retinal edematous pathologies. To determine the efficacy and safety of intra-vitreal Avastin (Bevacizumab) in cases having central retinal vein occlusion. All patients with central retinal occlusion occurring in the past 3 months and seen between the study period were included in the study. Diagnosis of central retinal vein occlusion was made clinically by slit lamp biomicroscopy with 78D examination Patients who had received any treatment for and eyes which already had developed Anterior Segment Neovascularization, Neovascularization elsewhere or Neovascularization on disc at presentation were excluded. Dose of 0.05 ml (1.25mg) of Avastin (Bevacizumab) was used as intra vitreal injection every month for 3 months in cases that presented within a month of occlusion and less injections were given in dose presenting later. Follow-up was done at 30th, 60th, 90th and 120th day after the onset of disease. Visual outcome was defined as Snellen's or LogMar Best Corrected Visual Acuity at final follow up, of 120th day, compared to the visual acuity at presentation. Data were analyzed by SPSS version 17. Total of 17 eyes of 17 patients were included in this study. Eleven (64.7%) patients were males while 6(35.3%) were females. Total of 40 intra-vitreal injections of Avastin were given to patients with a mean of 2.35 injections per eye. Good visual outcome was achieved in 10(58.8%) eyes, while 7(41.2%) had stable visual outcome. Mean initial Best Corrected Visual Acuity (LogMar) in all 17 eyes was 1.79(SD+0.87) which significantly improved to a mean of 1.18 (SD+0.77) at final follow up. Mean improvement in Best Corrected Visual Acuity (LogMar) after paired sample test in all patients at final follow up on day 120 was 0.61(SD+0.84). Retinal hemorrhages and macular edema decreased clinically on

  1. Vision deficits precede structural losses in a mouse model of mitochondrial dysfunction and progressive retinal degeneration.

    Science.gov (United States)

    Laliberté, Alex M; MacPherson, Thomas C; Micks, Taft; Yan, Alex; Hill, Kathleen A

    2011-12-01

    Current animal models of retinal disease often involve the rapid development of a retinal disease phenotype; however, this is at odds with age-related diseases that take many years to manifest clinical symptoms. The present study was performed to examine an apoptosis-inducing factor (Aif)-deficient model, the harlequin carrier mouse (X(hq)X), and determine how mitochondrial dysfunction and subsequent accelerated aging affect the function and structure of the mouse retina. Vision and eye structure for cohorts of 6 X(hq)X and 6 wild type mice at 3, 11, and 15 months of age were studied using in vivo electroretinography (ERG), and optical coherence tomography (OCT). Retinal superoxide levels were determined in situ using dihydroethidium (DHE) histochemistry. Retinal cell counts were quantified post mortem using hematoxylin and eosin (H&E) staining. ERG analysis of X(hq)X retinal function indicated a reduction in b-wave amplitude significant at 3 months of age (p retina (p retina may account for the early and significant reduction in retinal function. This remodeling of retinal neurochemistry in response to stress may be a relevant mechanism in the progression of normal retinal aging and early stages of some retinal degenerative diseases. Copyright © 2011 Elsevier Ltd. All rights reserved.

  2. Biology and therapy of inherited retinal degenerative disease: insights from mouse models

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    Veleri, Shobi; Lazar, Csilla H.; Chang, Bo; Sieving, Paul A.; Banin, Eyal; Swaroop, Anand

    2015-01-01

    Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases. PMID:25650393

  3. Biology and therapy of inherited retinal degenerative disease: insights from mouse models

    Directory of Open Access Journals (Sweden)

    Shobi Veleri

    2015-02-01

    Full Text Available Retinal neurodegeneration associated with the dysfunction or death of photoreceptors is a major cause of incurable vision loss. Tremendous progress has been made over the last two decades in discovering genes and genetic defects that lead to retinal diseases. The primary focus has now shifted to uncovering disease mechanisms and designing treatment strategies, especially inspired by the successful application of gene therapy in some forms of congenital blindness in humans. Both spontaneous and laboratory-generated mouse mutants have been valuable for providing fundamental insights into normal retinal development and for deciphering disease pathology. Here, we provide a review of mouse models of human retinal degeneration, with a primary focus on diseases affecting photoreceptor function. We also describe models associated with retinal pigment epithelium dysfunction or synaptic abnormalities. Furthermore, we highlight the crucial role of mouse models in elucidating retinal and photoreceptor biology in health and disease, and in the assessment of novel therapeutic modalities, including gene- and stem-cell-based therapies, for retinal degenerative diseases.

  4. Effect of essential fatty acids on glucose-induced cytotoxicity to retinal vascular endothelial cells

    Directory of Open Access Journals (Sweden)

    Shen Junhui

    2012-07-01

    Full Text Available Abstract Background Diabetic retinopathy is a major complication of dysregulated hyperglycemia. Retinal vascular endothelial cell dysfunction is an early event in the pathogenesis of diabetic retinopathy. Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by docosahexaenoic acid (DHA, 22:6 ω-3 and eicosapentaenoic acid (EPA, 20:5 ω-3. The influence of dietary omega-3 PUFA on brain zinc metabolism has been previously implied. Zn2+ is essential for the activity of Δ6 desaturase as a co-factor that, in turn, converts essential fatty acids to their respective long chain metabolites. Whether essential fatty acids (EFAs α-linolenic acid and linoleic acid have similar beneficial effect remains poorly understood. Methods RF/6A cells were treated with different concentrations of high glucose, α-linolenic acid and linoleic acid and Zn2+. The alterations in mitochondrial succinate dehydrogenase enzyme activity, cell membrane fluidity, reactive oxygen species generation, SOD enzyme and vascular endothelial growth factor (VEGF secretion were evaluated. Results Studies showed that hyperglycemia-induced excess proliferation of retinal vascular endothelial cells can be abrogated by both linoleic acid (LA and α-linolenic acid (ALA, while the saturated fatty acid, palmitic acid was ineffective. A dose–response study with ALA showed that the activity of the mitochondrial succinate dehydrogenase enzyme was suppressed at all concentrations of glucose tested to a significant degree. High glucose enhanced fluorescence polarization and microviscocity reverted to normal by treatment with Zn2+ and ALA. ALA was more potent that Zn2+. Increased level of high glucose caused slightly increased ROS generation that correlated with corresponding decrease in SOD activity. ALA suppressed ROS generation to a significant degree in a dose dependent fashion and raised SOD activity significantly. ALA suppressed

  5. Neonatal disease environment limits the efficacy of retinal transplantation in the LCA8 mouse model

    OpenAIRE

    Cho, Seo-Hee; Song, Ji Yun; Shin, Jinyeon; Kim, Seonhee

    2016-01-01

    Background Mutations of Crb1 gene cause irreversible and incurable visual impairment in humans. This study aims to use an LCA8-like mouse model to identify host-mediated responses that might interfere with survival, retinal integration and differentiation of grafted cells during neonatal cell therapy. Methods Mixed retinal donor cells (1?~?2???104) isolated from neural retinas of neonatal eGFP transgenic mice were injected into the subretinal space of LCA8-like model neonatal mice. Markers of...

  6. Noninvasive, in vivo assessment of mouse retinal structure using optical coherence tomography.

    Directory of Open Access Journals (Sweden)

    M Dominik Fischer

    Full Text Available BACKGROUND: Optical coherence tomography (OCT is a novel method of retinal in vivo imaging. In this study, we assessed the potential of OCT to yield histology-analogue sections in mouse models of retinal degeneration. METHODOLOGY/PRINCIPAL FINDINGS: We achieved to adapt a commercial 3(rd generation OCT system to obtain and quantify high-resolution morphological sections of the mouse retina which so far required in vitro histology. OCT and histology were compared in models with developmental defects, light damage, and inherited retinal degenerations. In conditional knockout mice deficient in retinal retinoblastoma protein Rb, the gradient of Cre expression from center to periphery, leading to a gradual reduction of retinal thickness, was clearly visible and well topographically quantifiable. In Nrl knockout mice, the layer involvement in the formation of rosette-like structures was similarly clear as in histology. OCT examination of focal light damage, well demarcated by the autofluorescence pattern, revealed a practically complete loss of photoreceptors with preservation of inner retinal layers, but also more subtle changes like edema formation. In Crb1 knockout mice (a model for Leber's congenital amaurosis, retinal vessels slipping through the outer nuclear layer towards the retinal pigment epithelium (RPE due to the lack of adhesion in the subapical region of the photoreceptor inner segments could be well identified. CONCLUSIONS/SIGNIFICANCE: We found that with the OCT we were able to detect and analyze a wide range of mouse retinal pathology, and the results compared well to histological sections. In addition, the technique allows to follow individual animals over time, thereby reducing the numbers of study animals needed, and to assess dynamic processes like edema formation. The results clearly indicate that OCT has the potential to revolutionize the future design of respective short- and long-term studies, as well as the preclinical

  7. Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

    Science.gov (United States)

    Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

    2017-10-01

    Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Vascular endothelial growth factor levels in tears of patients with retinal vein occlusion.

    Science.gov (United States)

    Kasza, M; Balogh, Z; Biro, L; Ujhelyi, B; Damjanovich, J; Csutak, A; Várdai, J; Berta, A; Nagy, V

    2015-09-01

    We measured vascular endothelial growth factor (VEGF) levels in tear fluid and serum in patients with retinal vein occlusion (RVO). Eight patients with RVO due to secondary macular oedema were examined. VEGF levels were measured by enzyme-linked immunosorbent assay. All patients had a full ophthalmic examination (visual acuity, slit lamp biomicroscopy, perimetry, and fluorescein angiography). Central retinal thickness (CRT) was examined using optical coherence tomography (OCT). Tear and serum samples were collected and examinations were performed at diagnosis and 1 and 4 weeks later. VEGF levels in the tears of RVO eyes were significantly higher than in fellow eyes at diagnosis and after both 1 and 4 weeks (paired t test, p1 = 0.01, p2 = 0.02, p3 = 0.006). We found a weak but significant positive correlation between VEGF levels in tear fluid and serum of patients with RVO (r = 0.21), while this correlation tended to be stronger between the fellow eyes and serum levels (r = 0.33). To the best of our knowledge, we are the first to report an increased level of VEGF in the tear fluid of patients with RVO. Alterations of VEGF levels in tears may be useful for determining stages of RVO. This non-invasive and objective method may also be helpful for estimating the severity of macular oedema and efficacy of treatment.

  9. Association of parental blood pressure with retinal microcirculatory abnormalities indicative of endothelial dysfunction in children.

    Science.gov (United States)

    Islam, Muhammad; Jafar, Tazeen H; Bux, Rasool; Hashmi, Shiraz; Chaturvedi, Nish; Hughes, Alun D

    2014-03-01

    Microcirculatory abnormalities precede the onset of hypertension and may explain its familial nature. We examined the relationship between parental blood pressure (BP) and offspring retinal microvasculature in Pakistani trios [father, mother, and child (aged 9-14 years)]. This is a substudy of a population-based trial of BP reduction. Data were available on 358 normotensive, and 410 offspring of at least one hypertensive parent. Retinal vessel characteristics were measured from digital images. Multivariable linear regression models were built to assess the associations between maternal and paternal BP and offspring retinal microvasculature. Optimality deviation was greatest in offspring of two hypertensive parents, compared with those with one or no hypertensive parent (P=0.030 for trend). Paternal SBP and DBP were each significantly associated with optimality deviation in offspring (P=0.023 and P=0.006, respectively). This relationship persisted after accounting for offspring cardiovascular risk factors [increase in optimality deviation (95% confidence interval, CI) 0.0053 (0.0001-0.0106, P=0.047) and 0.0109 (0.0025-0.0193, P=0.011), for each 10 mmHg increase in paternal SBP and DBP, respectively]. Maternal DBP was inversely associated with offspring arteriovenous ratio -0.0102 (-0.0198 to -0.0007, P=0.035). Microvascular endothelial dysfunction in children is associated with increasing levels of parental hypertension. The association with paternal BP is independent of other cardiovascular risk factors, including the child's BP. Higher maternal DBP is associated with evidence of arteriolar narrowing in offspring. These early microcirculatory changes may help explain familial predisposition to hypertension in people of Pakistani origin at an early age. :

  10. Mouse embryonic stem cell culture for generation of three-dimensional retinal and cortical tissues.

    Science.gov (United States)

    Eiraku, Mototsugu; Sasai, Yoshiki

    2011-12-15

    Generation of compound tissues with complex structures is a major challenge in cell biology. In this article, we describe a protocol for mouse embryonic stem cell (ESC) culture for in vitro generation of three-dimensional retinal tissue, comparing it with the culture protocol for cortical tissue generation. Dissociated ESCs are reaggregated in a 96-well plate with reduced cell-plate adhesion and cultured as floating aggregates. Retinal epithelium is efficiently generated when ESC aggregates are cultured in serum-free medium containing extracellular matrix proteins, spontaneously forming hemispherical vesicles and then progressively transforming into a shape reminiscent of the embryonic optic cup in 9-10 d. In long-term culture, the ESC-derived optic cup generates a fully stratified retinal tissue consisting of all major neural retinal components. In contrast, the cortical differentiation culture can be started without exogenous extracellular matrix proteins, and it generates stratified cortical epithelia consisting of four distinct layers in 13 d.

  11. Lenalidomide, an anti-tumor drug, regulates retinal endothelial cell function: Implication for treating ocular neovascular disorder

    International Nuclear Information System (INIS)

    Dong, Ling-Feng; Yao, Jin; Wang, Xiao-Qun; Shan, Kun; Yang, Hong; Yan, Biao; Jiang, Qin

    2015-01-01

    Ocular angiogenesis is an important pathologic character of several ocular diseases, such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (AMD). Inhibition of ocular angiogenesis has great therapeutic value for treating these dieses. Here we show that lenalidomide, an anti-tumor drug, has great anti-angiogenic potential in ocular diseases. Lenalidomide inhibits retinal endothelial cell viability in normal and pathological condition, and inhibits VEGF-induced endothelial cell migration and tube formation in vitro. Moreover, lenalidomide inhibits ocular angiogenesis in vivo through the reduction of angiogenesis- and inflammation-related protein expression. Collectively, lenalidomide is a promising drug for treating ocular angiogenesis through its anti-proliferative and anti-inflammatory property. - Highlights: • Lenalidomide inhibits retinal endothelial cell viability in vitro. • Lenalidomide inhibits retinal endothelial cell migration and tube formation. • Lenalidomide inhibits pathological ocular angiogenesis in vivo. • Lenalidomide inhibits angiogenesis- and inflammation-related protein expression.

  12. Lenalidomide, an anti-tumor drug, regulates retinal endothelial cell function: Implication for treating ocular neovascular disorder

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Ling-Feng; Yao, Jin; Wang, Xiao-Qun; Shan, Kun; Yang, Hong [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China); Yan, Biao, E-mail: yanbiao1982@hotmail.com [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China); Jiang, Qin, E-mail: jiangqin710@126.com [Eye Hospital, Nanjing Medical University, Nanjing (China); The Fourth School of Clinical Medicine, Nanjing Medical University, Nanjing (China)

    2015-10-02

    Ocular angiogenesis is an important pathologic character of several ocular diseases, such as retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration (AMD). Inhibition of ocular angiogenesis has great therapeutic value for treating these dieses. Here we show that lenalidomide, an anti-tumor drug, has great anti-angiogenic potential in ocular diseases. Lenalidomide inhibits retinal endothelial cell viability in normal and pathological condition, and inhibits VEGF-induced endothelial cell migration and tube formation in vitro. Moreover, lenalidomide inhibits ocular angiogenesis in vivo through the reduction of angiogenesis- and inflammation-related protein expression. Collectively, lenalidomide is a promising drug for treating ocular angiogenesis through its anti-proliferative and anti-inflammatory property. - Highlights: • Lenalidomide inhibits retinal endothelial cell viability in vitro. • Lenalidomide inhibits retinal endothelial cell migration and tube formation. • Lenalidomide inhibits pathological ocular angiogenesis in vivo. • Lenalidomide inhibits angiogenesis- and inflammation-related protein expression.

  13. Vascular endothelial growth factor up-regulates the expression of intracellular adhesion molecule-1 in retinal endothelial cells via reactive oxygen species, but not nitric oxide

    Institute of Scientific and Technical Information of China (English)

    ZHANG Xiao-ling; WEN Liang; CHEN Yan-jiong; ZHU Yi

    2009-01-01

    Background The vascular endothelial growth factor (VEGF) is involved in the initiation of retinal vascular leakage and nonperfusion in diabetes. The intracellular adhesion molecule-1 (ICAM-1) is the key mediator of the effect of VEGFs on retinal leukostasis. Although the VEGF is expressed in an early-stage diabetic retina, whether it directly up-regulates ICAM-1 in retinal endothelial cells (ECs) is unknown. In this study, we provided a new mechanism to explain that VEGF does up-regulate the expression of ICAM-1 in retinal ECs.Methods Bovine retinal ECs (BRECs) were isolated and cultured. Immunohistochemical staining was performed to identify BRECs. The cultured cells were divided into corresponding groups. Then, VEGF (100 ng/ml) and other inhibitors were used to treat the cells. Cell lysate and the cultured supernatant were collected, and then, the protein level of ICAM-1 and phosphorylation of the endothelial nitric oxide synthase (eNOS) were detected using Western blotting. Griess reaction was used to detect nitric oxide (NO).Results Western blotting showed that the VEGF up-regulated the expression of ICAM-1 protein and increased phosphorylation of the eNOS in retinal ECs. Neither the block of NO nor protein kinase C (PKC) altered the expression of ICAM-1 or the phosphorylation of eNOS. The result of the Western blotting also showed that inhibition of phosphatidylinositol 3-kinase (PI3K) or reactive oxygen species (ROS) significantly reduced the expression of ICAM-1. Inhibition of PI3K also reduced phosphorylation of eNOS. Griess reaction showed that VEGF significantly increased during NO production. When eNOS was blocked by L-NAME or PI3K was blocked by LY294002, the basal level of NO production and the increment of NO caused by VEGF could be significantly decreased.Conclusion ROS-NO coupling in the retinal endothelium may be a new mechanism that could help to explain why VEGF induces ICAM-1 expression and the resulting leukostasis in diabetic retinopathy.

  14. E2f1 mediates high glucose-induced neuronal death in cultured mouse retinal explants.

    Science.gov (United States)

    Wang, Yujiao; Zhou, Yi; Xiao, Lirong; Zheng, Shijie; Yan, Naihong; Chen, Danian

    2017-10-02

    Diabetic retinopathy (DR) is the most common complication of diabetes and remains one of the major causes of blindness in the world; infants born to diabetic mothers have higher risk of developing retinopathy of prematurity (ROP). While hyperglycemia is a major risk factor, the molecular and cellular mechanisms underlying DR and diabetic ROP are poorly understood. To explore the consequences of retinal cells under high glucose, we cultured wild type or E2f1 -/- mouse retinal explants from postnatal day 8 with normal glucose, high osmotic or high glucose media. Explants were also incubated with cobalt chloride (CoCl 2 ) to mimic the hypoxic condition. We showed that, at 7 days post exposure to high glucose, retinal explants displayed elevated cell death, ectopic cell division and intact retinal vascular plexus. Cell death mainly occurred in excitatory neurons, such as ganglion and bipolar cells, which were also ectopically dividing. Many Müller glial cells reentered the cell cycle; some had irregular morphology or migrated to other layers. High glucose inhibited the hyperoxia-induced blood vessel regression of retinal explants. Moreover, inactivation of E2f1 rescued high glucose-induced ectopic division and cell death of retinal neurons, but not ectopic cell division of Müller glial cells and vascular phenotypes. This suggests that high glucose has direct but distinct effects on retinal neurons, glial cells and blood vessels, and that E2f1 mediates its effects on retinal neurons. These findings shed new light onto mechanisms of DR and the fetal retinal abnormalities associated with maternal diabetes, and suggest possible new therapeutic strategies.

  15. Transplantation of adult mouse iPS cell-derived photoreceptor precursors restores retinal structure and function in degenerative mice.

    Directory of Open Access Journals (Sweden)

    Budd A Tucker

    2011-04-01

    Full Text Available This study was designed to determine whether adult mouse induced pluripotent stem cells (iPSCs, could be used to produce retinal precursors and subsequently photoreceptor cells for retinal transplantation to restore retinal function in degenerative hosts. iPSCs were generated using adult dsRed mouse dermal fibroblasts via retroviral induction of the transcription factors Oct4, Sox2, KLF4 and c-Myc. As with normal mouse ES cells, adult dsRed iPSCs expressed the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following transplantation into the eye of immune-compromised retinal degenerative mice these cells proceeded to form teratomas containing tissue comprising all three germ layers. At 33 days post-differentiation a large proportion of the cells expressed the retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers, CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were shown to exhibit characteristics of normal retinal physiology, responding to delivery of neurotransmitters. Following subretinal transplantation into degenerative hosts differentiated iPSCs took up residence in the retinal outer nuclear layer and gave rise to increased electro retinal function as determined by ERG and functional anatomy. As such, adult fibroblast-derived iPSCs provide a viable source for the production of retinal precursors to be used for transplantation and treatment of retinal degenerative disease.

  16. Honokiol inhibits pathological retinal neovascularization in oxygen-induced retinopathy mouse model

    Energy Technology Data Exchange (ETDEWEB)

    Vavilala, Divya Teja [Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, MO (United States); O’Bryhim, Bliss E. [Department of Ophthalmology, University of Kansas Medical Center, Kansas City, KS (United States); Ponnaluri, V.K. Chaithanya [Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, MO (United States); White, R. Sid; Radel, Jeff [Department of Ophthalmology, University of Kansas Medical Center, Kansas City, KS (United States); Symons, R.C. Andrew [Department of Ophthalmology, University of Kansas Medical Center, Kansas City, KS (United States); Ophthalmology Department, Royal Melbourne Hospital, University of Melbourne, Victoria (Australia); Department of Surgery, Royal Melbourne Hospital, University of Melbourne, Victoria (Australia); Mukherji, Mridul, E-mail: mukherjim@umkc.edu [Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, MO (United States)

    2013-09-06

    Highlights: •Aberrant activation of HIF pathway is the underlying cause of ischemic neovascularization. •Honokiol has better therapeutic index as a HIF inhibitor than digoxin and doxorubicin. •Daily IP injection of honokiol in OIR mouse model reduced retinal neovascularization. •Honokiol also prevents vaso-obliteration, the characteristic feature of the OIR model. •Honokiol enhanced physiological revascularization of the retinal vascular plexuses. -- Abstract: Aberrant activation of the hypoxia inducible factor (HIF) pathway is the underlying cause of retinal neovascularization, one of the most common causes of blindness worldwide. The HIF pathway also plays critical roles during tumor angiogenesis and cancer stem cell transformation. We have recently shown that honokiol is a potent inhibitor of the HIF pathway in a number of cancer and retinal pigment epithelial cell lines. Here we evaluate the safety and efficacy of honokiol, digoxin, and doxorubicin, three recently identified HIF inhibitors from natural sources. Our studies show that honokiol has a better safety to efficacy profile as a HIF inhibitor than digoxin and doxorubicin. Further, we show for the first time that daily intraperitoneal injection of honokiol starting at postnatal day (P) 12 in an oxygen-induced retinopathy (OIR) mouse model significantly reduced retinal neovascularization at P17. Administration of honokiol also prevents the oxygen-induced central retinal vaso-obliteration, characteristic feature of the OIR model. Additionally, honokiol enhanced physiological revascularization of the retinal vascular plexuses. Since honokiol suppresses multiple pathways activated by HIF, in addition to the VEGF signaling, it may provide advantages over current treatments utilizing specific VEGF antagonists for ocular neovascular diseases and cancers.

  17. An optimized method for mouse liver sinusoidal endothelial cell isolation

    Energy Technology Data Exchange (ETDEWEB)

    Meyer, Jeremy, E-mail: jeremy.meyer@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Lacotte, Stéphanie, E-mail: stephanie.lacotte@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Morel, Philippe, E-mail: philippe.morel@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Gonelle-Gispert, Carmen, E-mail: carmen.gonelle@unige.ch [Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland); Bühler, Léo, E-mail: leo.buhler@hcuge.ch [Division of Digestive and Transplantation Surgery, University Hospitals of Geneva, Rue Gabrielle-Perret-Gentil 4, 1211 Genève 14 (Switzerland); Unit of Surgical Research, University of Geneva, Rue Michel-Servet 1, 1206 Genève (Switzerland)

    2016-12-10

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  18. An optimized method for mouse liver sinusoidal endothelial cell isolation

    International Nuclear Information System (INIS)

    Meyer, Jeremy; Lacotte, Stéphanie; Morel, Philippe; Gonelle-Gispert, Carmen; Bühler, Léo

    2016-01-01

    The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions. - Highlights: • This protocol provides an efficient method to prepare primary mouse LSEC for studying their biological functions. • The liver cell dispersion step was improved by performing a retrograde cannulation of the liver. • The cell yield and the purity obtained were higher than comparative techniques in mice. • Contaminating macrophages were removed by introducing a CD11b- magnetic

  19. Epigalloccatechin-3-gallate Inhibits Ocular Neovascularization and Vascular Permeability in Human Retinal Pigment Epithelial and Human Retinal Microvascular Endothelial Cells via Suppression of MMP-9 and VEGF Activation

    Directory of Open Access Journals (Sweden)

    Hak Sung Lee

    2014-08-01

    Full Text Available Epigalloccatechin-3-gallate (EGCG is the main polyphenol component of green tea (leaves of Camellia sinensis. EGCG is known for its antioxidant, anti-inflammatory, antiviral, and anti-carcinogenic properties. Here, we identify EGCG as a new inhibitor of ocular angiogenesis and its vascular permeability. Matrix metalloproteinases (MMPs and vascular endothelial growth factor (VEGF play a key role in the processes of extracellular matrix (ECM remodeling and microvascular permeability during angiogenesis. We investigated the inhibitory effects of EGCG on ocular neovascularization and vascular permeability using the retina oriented cells and animal models induced by VEGF and alkaline burn. EGCG treatment significantly decreased mRNA and protein expression levels of MMP-9 in the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA and tumor necrosis factor alpha (TNF-α in human retinal pigment epithelial cells (HRPECs. EGCG also effectively protected ARPE-19 cells from cell death and attenuated mRNA expressions of key angiogenic factors (MMP-9, VEGF, VEGF Receptor-2 by inhibiting generation of reactive oxygen species (ROS. EGCG significantly inhibited proliferation, vascular permeability, and tube formation in VEGF-induced human retinal microvascular endothelial cells (HRMECs. Furthermore, EGCG significantly reduced vascular leakage and permeability by blood-retinal barrier breakdown in VEGF-induced animal models. In addition, EGCG effectively limited upregulation of MMP-9 and platelet endothelial cell adhesion molecule (PECAM/CD31 on corneal neovascularization (CNV induced by alkaline burn. Our data suggest that MMP-9 and VEGF are key therapeutic targets of EGCG for treatment and prevention of ocular angiogenic diseases such as age-related macular degeneration, diabetic retinopathy, and corneal neovascularization.

  20. NOX2-Induced Activation of Arginase and Diabetes-Induced Retinal Endothelial Cell Senescence

    Directory of Open Access Journals (Sweden)

    Modesto Rojas

    2017-06-01

    Full Text Available Increases in reactive oxygen species (ROS and decreases in nitric oxide (NO have been linked to vascular dysfunction during diabetic retinopathy (DR. Diabetes can reduce NO by increasing ROS and by increasing activity of arginase, which competes with nitric oxide synthase (NOS for their commons substrate l-arginine. Increased ROS and decreased NO can cause premature endothelial cell (EC senescence leading to defective vascular repair. We have previously demonstrated the involvement of NADPH oxidase 2 (NOX2-derived ROS, decreased NO and overactive arginase in DR. Here, we investigated their impact on diabetes-induced EC senescence. Studies using diabetic mice and retinal ECs treated with high glucose or H2O2 showed that increases in ROS formation, elevated arginase expression and activity, and decreased NO formation led to premature EC senescence. NOX2 blockade or arginase inhibition prevented these effects. EC senescence was also increased by inhibition of NOS activity and this was prevented by treatment with a NO donor. These results indicate that diabetes/high glucose-induced activation of arginase and decreases in NO bioavailability accelerate EC senescence. NOX2-generated ROS contribute importantly to this process. Blockade of NOX2 or arginase represents a strategy to prevent diabetes-induced premature EC senescence by preserving NO bioavailability.

  1. Effects of Subretinal Gene Transfer at Different Time Points in a Mouse Model of Retinal Degeneration.

    Science.gov (United States)

    Dai, Xufeng; Zhang, Hua; Han, Juanjuan; He, Ying; Zhang, Yangyang; Qi, Yan; Pang, Ji-Jing

    2016-01-01

    Lysophosphatidylcholine acyltransferase 1 (LPCAT1) is necessary for photoreceptors to generate an important lipid component of their membranes. The absence of LPCAT1 results in early and rapid rod and cone degeneration. Retinal degeneration 11 (rd11) mice carry a mutation in the Lpcat1 gene, and are an excellent model of early-onset rapid retinal degeneration (RD). To date, no reports have documented gene therapy administration in the rd11 mouse model at different ages. In this study, the AAV8 (Y733F)-smCBA-Lpcat1 vector was subretinally injected at postnatal day (P) 10, 14, 18, or 22. Four months after injection, immunohistochemistry and analysis of retinal morphology showed that treatment at P10 rescued about 82% of the wild-type retinal thickness. However, the diffusion of the vector and the resulting rescue were limited to an area around the injection site that was only 31% of the total retinal area. Injection at P14 resulted in vector diffusion that covered approximately 84% of the retina, and we found that gene therapy was more effective against RD when exposure to light was limited before and after treatment. We observed long-term preservation of electroretinogram (ERG) responses, and preservation of retinal structure, indicating that early treatment followed by limited light exposure can improve gene therapy effectiveness for the eyes of rd11 mice. Importantly, delayed treatment still partially preserved M-cones, but not S-cones, and M-cones in the rd11 retina appeared to have a longer window of opportunity for effective preservation with gene therapy. These results provide important information regarding the effects of subretinal gene therapy in the mouse model of LPCAT1-deficiency.

  2. Modulation of radiation injury response in retinal endothelial cells by quinic acid derivative KZ-41 involves p38 MAPK.

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    Jordan J Toutounchian

    Full Text Available Radiation-induced damage to the retina triggers leukostasis, retinal endothelial cell (REC death, and subsequent hypoxia. Resultant ischemia leads to visual loss and compensatory retinal neovascularization (RNV. Using human RECs, we demonstrated that radiation induced leukocyte adhesion through mechanisms involving p38MAPK, p53, and ICAM-1 activation. Additional phenotypic changes included p38MAPK-dependent tyrosine phosphorylation of the focal adhesion scaffolding protein, paxillin (Tyr118. The quinic acid derivative KZ-41 lessened leukocyte adhesion and paxillin-dependent proliferation via inhibition of p38MAPK-p53-ICAM-1 signaling. Using the murine oxygen-induced retinopathy (OIR model, we examined the effect of KZ-41 on pathologic RNV. Daily ocular application of a KZ-41-loaded nanoemulsion significantly reduced both the avascular and neovascular areas in harvested retinal flat mounts when compared to the contralateral eye receiving vehicle alone. Our data highlight the potential benefit of KZ-41 in reducing both the retinal ischemia and neovascularization provoked by genotoxic insults. Further research into how quinic acid derivatives target and mitigate inflammation is needed to fully appreciate their therapeutic potential for the treatment of inflammatory retinal vasculopathies.

  3. The Ins2Akita mouse as a model of early retinal complications in diabetes.

    Science.gov (United States)

    Barber, Alistair J; Antonetti, David A; Kern, Timothy S; Reiter, Chad E N; Soans, Rohit S; Krady, J Kyle; Levison, Steven W; Gardner, Thomas W; Bronson, Sarah K

    2005-06-01

    This study tested the Ins2(Akita) mouse as an animal model of retinal complications in diabetes. The Ins2(Akita) mutation results in a single amino acid substitution in the insulin 2 gene that causes misfolding of the insulin protein. The mutation arose and is maintained on the C57BL/6J background. Male mice heterozygous for this mutation have progressive loss of beta-cell function, decreased pancreatic beta-cell density, and significant hyperglycemia, as early as 4 weeks of age. Heterozygous Ins2(Akita) mice were bred to C57BL/6J mice, and male offspring were monitored for hyperglycemia, beginning at 4.5 weeks of age. After 4 to 36 weeks of hyperglycemia, the retinas were analyzed for vascular permeability, vascular lesions, leukostasis, morphologic changes of micro- and macroglia, apoptosis, retinal degeneration, and insulin receptor kinase activity. The mean blood glucose of Ins2(Akita) mice was significantly elevated, whereas the body weight at death was reduced compared with that of control animals. Compared with sibling control mice, the Ins2(Akita) mice had increased retinal vascular permeability after 12 weeks of hyperglycemia (P microglia, but no changes in expression of Muller cell glial fibrillary acidic protein. Increased apoptosis was identified by immunoreactivity for active caspase-3 after 4 weeks of hyperglycemia (P cell bodies in the retinal ganglion cell layer (P retinal complications of diabetes.

  4. Endothelial and lipoprotein lipases in human and mouse placenta

    DEFF Research Database (Denmark)

    Lindegaard, Marie L S; Olivecrona, Gunilla; Christoffersen, Christina

    2005-01-01

    Placenta expresses various lipase activities. However, a detailed characterization of the involved genes and proteins is lacking. In this study, we compared the expression of endothelial lipase (EL) and LPL in human term placenta. When placental protein extracts were separated by heparin-Sepharos...

  5. Mouse lung contains endothelial progenitors with high capacity to form blood and lymphatic vessels

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    Barleon Bernhard

    2010-07-01

    Full Text Available Abstract Background Postnatal endothelial progenitor cells (EPCs have been successfully isolated from whole bone marrow, blood and the walls of conduit vessels. They can, therefore, be classified into circulating and resident progenitor cells. The differentiation capacity of resident lung endothelial progenitor cells from mouse has not been evaluated. Results In an attempt to isolate differentiated mature endothelial cells from mouse lung we found that the lung contains EPCs with a high vasculogenic capacity and capability of de novo vasculogenesis for blood and lymph vessels. Mouse lung microvascular endothelial cells (MLMVECs were isolated by selection of CD31+ cells. Whereas the majority of the CD31+ cells did not divide, some scattered cells started to proliferate giving rise to large colonies (> 3000 cells/colony. These highly dividing cells possess the capacity to integrate into various types of vessels including blood and lymph vessels unveiling the existence of local microvascular endothelial progenitor cells (LMEPCs in adult mouse lung. EPCs could be amplified > passage 30 and still expressed panendothelial markers as well as the progenitor cell antigens, but not antigens for immune cells and hematopoietic stem cells. A high percentage of these cells are also positive for Lyve1, Prox1, podoplanin and VEGFR-3 indicating that a considerabe fraction of the cells are committed to develop lymphatic endothelium. Clonogenic highly proliferating cells from limiting dilution assays were also bipotent. Combined in vitro and in vivo spheroid and matrigel assays revealed that these EPCs exhibit vasculogenic capacity by forming functional blood and lymph vessels. Conclusion The lung contains large numbers of EPCs that display commitment for both types of vessels, suggesting that lung blood and lymphatic endothelial cells are derived from a single progenitor cell.

  6. The Time Course of Deafness and Retinal Degeneration in a Kunming Mouse Model for Usher Syndrome.

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    Yao, Lu; Zhang, Lei; Qi, Lin-Song; Liu, Wei; An, Jing; Wang, Bin; Xue, Jun-Hui; Zhang, Zuo-Ming

    2016-01-01

    Usher syndrome is a group of autosomal recessive diseases characterized by congenital deafness and retinitis pigmentosa. In a mouse model for Usher syndrome, KMush/ush, discovered in our laboratory, we measured the phenotypes, characterized the architecture and morphology of the retina, and quantified the level of expression of pde6b and ush2a between postnatal (P) days 7, and 56. Electroretinograms and auditory brainstem response were used to measure visual and auditory phenotypes. Fundus photography and light microscopy were used to measure the architecture and morphology of the retina. Quantitative real-time PCR was used to measure the expression levels of mRNA. KMush/ush mice had low amplitudes and no obvious waveforms of Electroretinograms after P14 compared with controls. Thresholds of auditory brainstem response in our model were higher than those of controls after P14. By P21, the retinal vessels of KMush/ush mice were attenuated and their optic discs had a waxy pallor. The retinas of KMush/ush mice atrophied and the choroidal vessels were clearly visible. Notably, the architecture of each retinal layer was not different as compared with control mice at P7, while the outer nuclear layer (ONL) and other retinal layers of KMush/ush mice were attenuated significantly between P14 and P21. ONL cells were barely seen in KMush/ush mice at P56. As compared with control mice, the expression of pde6b and ush2a in KMush/ush mice declined significantly after P7. This study is a first step toward characterizing the progression of disease in our mouse model. Future studies using this model may provide insights about the etiology of the disease and the relationships between genotypes and phenotypes providing a valuable resource that could contribute to the foundation of knowledge necessary to develop therapies to prevent the retinal degeneration in patients with Usher Syndrome.

  7. The Time Course of Deafness and Retinal Degeneration in a Kunming Mouse Model for Usher Syndrome.

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    Lu Yao

    Full Text Available Usher syndrome is a group of autosomal recessive diseases characterized by congenital deafness and retinitis pigmentosa. In a mouse model for Usher syndrome, KMush/ush, discovered in our laboratory, we measured the phenotypes, characterized the architecture and morphology of the retina, and quantified the level of expression of pde6b and ush2a between postnatal (P days 7, and 56. Electroretinograms and auditory brainstem response were used to measure visual and auditory phenotypes. Fundus photography and light microscopy were used to measure the architecture and morphology of the retina. Quantitative real-time PCR was used to measure the expression levels of mRNA. KMush/ush mice had low amplitudes and no obvious waveforms of Electroretinograms after P14 compared with controls. Thresholds of auditory brainstem response in our model were higher than those of controls after P14. By P21, the retinal vessels of KMush/ush mice were attenuated and their optic discs had a waxy pallor. The retinas of KMush/ush mice atrophied and the choroidal vessels were clearly visible. Notably, the architecture of each retinal layer was not different as compared with control mice at P7, while the outer nuclear layer (ONL and other retinal layers of KMush/ush mice were attenuated significantly between P14 and P21. ONL cells were barely seen in KMush/ush mice at P56. As compared with control mice, the expression of pde6b and ush2a in KMush/ush mice declined significantly after P7. This study is a first step toward characterizing the progression of disease in our mouse model. Future studies using this model may provide insights about the etiology of the disease and the relationships between genotypes and phenotypes providing a valuable resource that could contribute to the foundation of knowledge necessary to develop therapies to prevent the retinal degeneration in patients with Usher Syndrome.

  8. Sildenafil restores endothelial function in the apolipoprotein E knockout mouse

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    Balarini Camille M

    2013-01-01

    Full Text Available Abstract Background Atherosclerosis is an inflammatory process of the arterial walls and is initiated by endothelial dysfunction accompanied by an imbalance in the production of reactive oxygen species (ROS and nitric oxide (NO. Sildenafil, a selective phosphodiesterase-5 (PDE5 inhibitor used for erectile dysfunction, exerts its cardiovascular effects by enhancing the effects of NO. The aim of this study was to investigate the influence of sildenafil on endothelial function and atherosclerosis progression in apolipoprotein E knockout (apoE−/− mice. Methods ApoE−/− mice treated with sildenafil (Viagra®, 40 mg/kg/day, for 3 weeks, by oral gavage were compared to the untreated apoE−/− and the wild-type (WT mice. Aortic rings were used to evaluate the relaxation responses to acetylcholine (ACh in all of the groups. In a separate set of experiments, the roles of NO and ROS in the relaxation response to ACh were evaluated by incubating the aortic rings with L-NAME (NO synthase inhibitor or apocynin (NADPH oxidase inhibitor. In addition, the atherosclerotic lesions were quantified and superoxide production was assessed. Results Sildenafil restored the vasodilator response to acetylcholine (ACh in the aortic rings of the apoE−/− mice. Treatment with L-NAME abolished the vasodilator responses to ACh in all three groups of mice and revealed an augmented participation of NO in the endothelium-dependent vasodilation in the sildenafil-treated animals. The normalized endothelial function in sildenafil-treated apoE−/− mice was unaffected by apocynin highlighting the low levels of ROS production in these animals. Moreover, morphological analysis showed that sildenafil treatment caused approximately a 40% decrease in plaque deposition in the aorta. Conclusion This is the first study demonstrating the beneficial effects of chronic treatment with sildenafil on endothelial dysfunction and atherosclerosis in a model of spontaneous

  9. Novel VCP modulators mitigate major pathologies of rd10, a mouse model of retinitis pigmentosa

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    Ikeda, Hanako Ohashi; Sasaoka, Norio; Koike, Masaaki; Nakano, Noriko; Muraoka, Yuki; Toda, Yoshinobu; Fuchigami, Tomohiro; Shudo, Toshiyuki; Iwata, Ayana; Hori, Seiji; Yoshimura, Nagahisa; Kakizuka, Akira

    2014-01-01

    Neuroprotection may prevent or forestall the progression of incurable eye diseases, such as retinitis pigmentosa, one of the major causes of adult blindness. Decreased cellular ATP levels may contribute to the pathology of this eye disease and other neurodegenerative diseases. Here we describe small compounds (Kyoto University Substances, KUSs) that were developed to inhibit the ATPase activity of VCP (valosin-containing protein), the most abundant soluble ATPase in the cell. Surprisingly, KUSs did not significantly impair reported cellular functions of VCP but nonetheless suppressed the VCP-dependent decrease of cellular ATP levels. Moreover, KUSs, as well as exogenous ATP or ATP-producing compounds, e.g. methylpyruvate, suppressed endoplasmic reticulum stress, and demonstrably protected various types of cultured cells from death, including several types of retinal neuronal cells. We then examined their in vivo efficacies in rd10, a mouse model of retinitis pigmentosa. KUSs prevented photoreceptor cell death and preserved visual function. These results reveal an unexpected, crucial role of ATP consumption by VCP in determining cell fate in this pathological context, and point to a promising new neuroprotective strategy for currently incurable retinitis pigmentosa. PMID:25096051

  10. Development and degeneration of cone bipolar cells are independent of cone photoreceptors in a mouse model of retinitis pigmentosa.

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    Miao Chen

    Full Text Available Retinal photoreceptors die during retinal synaptogenesis in a portion of retinal degeneration. Whether cone bipolar cells establish regular retinal mosaics and mature morphologies, and resist degeneration are not completely understood. To explore these issues, we backcrossed a transgenic mouse expressing enhanced green fluorescent protein (EGFP in one subset of cone bipolar cells (type 7 into rd1 mice, a classic mouse model of retinal degeneration, to examine the development and survival of cone bipolar cells in a background of retinal degeneration. Our data revealed that both the development and degeneration of cone bipolar cells are independent of the normal activity of cone photoreceptors. We found that type 7 cone bipolar cells achieved a uniform tiling of the retinal surface and developed normal dendritic and axonal arbors without the influence of cone photoreceptor innervation. On the other hand, degeneration of type 7 cone bipolar cells, contrary to our belief of central-to-peripheral progression, was spatially uniform across the retina independent of the spatiotemporal pattern of cone degeneration. The results have important implications for the design of more effective therapies to restore vision in retinal degeneration.

  11. Polycomb repressive complex 2 regulates MiR-200b in retinal endothelial cells: potential relevance in diabetic retinopathy.

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    Michael Anthony Ruiz

    Full Text Available Glucose-induced augmented vascular endothelial growth factor (VEGF production is a key event in diabetic retinopathy. We have previously demonstrated that downregulation of miR-200b increases VEGF, mediating structural and functional changes in the retina in diabetes. However, mechanisms regulating miR-200b in diabetes are not known. Histone methyltransferase complex, Polycomb Repressive Complex 2 (PRC2, has been shown to repress miRNAs in neoplastic process. We hypothesized that, in diabetes, PRC2 represses miR-200b through its histone H3 lysine-27 trimethylation mark. We show that human retinal microvascular endothelial cells exposed to high levels of glucose regulate miR-200b repression through histone methylation and that inhibition of PRC2 increases miR-200b while reducing VEGF. Furthermore, retinal tissue from animal models of diabetes showed increased expression of major PRC2 components, demonstrating in vivo relevance. This research established a repressive relationship between PRC2 and miR-200b, providing evidence of a novel mechanism of miRNA regulation through histone methylation.

  12. A PEDF-Derived Peptide Inhibits Retinal Neovascularization and Blocks Mobilization of Bone Marrow-Derived Endothelial Progenitor Cells

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    Richard Longeras

    2012-01-01

    Full Text Available Proliferative diabetic retinopathy is characterized by pathological retinal neovascularization, mediated by both angiogenesis (involving mature endothelial cells and vasculogenesis (involving bone marrow-derived circulating endothelial progenitor cells (EPCs. Pigment epithelium-derived factor (PEDF contains an N-terminal 34-amino acid peptide (PEDF-34 that has antiangiogenic properties. Herein, we present a novel finding that PEDF-34 also possesses antivasculogenic activity. In the oxygen-induced retinopathy (OIR model using transgenic mice that have Tie2 promoter-driven GFP expression, we quantified Tie2GFP+ cells in bone marrow and peripheral blood by fluorescence-activated cell sorting (FACS. OIR significantly increased the number of circulating Tie2-GFP+ at P16, correlating with the peak progression of neovascularization. Daily intraperitoneal injections of PEDF-34 into OIR mice decreased the number of Tie2-GFP+ cells in the circulation at P16 by 65% but did not affect the number of Tie2-GFP+ cells in the bone marrow. These studies suggest that PEDF-34 attenuates EPC mobilization from the bone marrow into the blood circulation during retinal neovascularization.

  13. Relationship Between Visual Acuity and Retinal Thickness During Anti-Vascular Endothelial Growth Factor Therapy for Retinal Diseases.

    Science.gov (United States)

    Ou, William C; Brown, David M; Payne, John F; Wykoff, Charles C

    2017-08-01

    To investigate the relationship between best-corrected visual acuity (BCVA) and central retinal thickness (CRT) in eyes receiving ranibizumab for 3 common retinal diseases. Retrospective analysis of clinical trial data. Early Treatment Diabetic Retinopathy Study BCVA and spectral-domain optical coherence tomography-measured CRT of 387 eyes of 345 patients enrolled in 6 prospective clinical trials for management of neovascular age-related macular degeneration (AMD), diabetic macular edema (DME), and retinal vein occlusion (RVO) were evaluated by Pearson correlation and linear regression. At baseline, there was a small correlation between BCVA and CRT in pooled AMD trial data (r = -0.24). A medium correlation was identified in pooled DME trial data (r = -0.42). No correlation was found in pooled RVO trial data. At month 12, no correlation was found between changes from baseline in BCVA and CRT in pooled AMD trial data. Medium correlations were identified in both pooled DME (r = -0.45) and pooled RVO (r = -0.35) trial data at month 12. Changes in BCVA and CRT associated with edema recurrence upon transition from monthly to pro re nata (PRN) dosing were correlated in AMD (r = -0.27) and RVO (r = -0.72) trials, but not in DME trial data. DME demonstrated a convincing relationship between BCVA and CRT. Correlations appear to be more complex in AMD and RVO. At the inflection point between monthly and PRN dosing, when recurrence of edema is anticipated in many patients, CRT appears strongly correlated with loss of BCVA in RVO. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Long-term results of repeated anti-vascular endothelial growth factor therapy in eyes with retinal pigment epithelial tears.

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    Moreira, Carlos A; Arana, Luis A; Zago, Rommel J

    2013-02-01

    To evaluate the long-term results of retinal pigment epithelium tears in eyes treated with repeated anti-vascular endothelial growth factor (VEGF) therapy. Five patients with retinal pigment epithelial tears (without foveal center involvement) after anti-VEGF injection were studied retrospectively. Mean follow-up time was 52 months, with measurements of visual acuity and evaluation of macular findings by angiography and optical coherence tomography during this period. All eyes had a persistent submacular neovascular membrane 30 days after the tear. An anti-VEGF drug was reinjected until the membranes stopped leaking. The mean initial visual acuity immediately after the tear was 20/160, and the mean final visual acuity was 20/60. The number of anti-VEGF reinjections varied from two to eight during the follow-up period. Long-term optical coherence tomography analysis showed reduced fluid and remodeling of the torn retinal pigment epithelium. Long-term visual results with repeated anti-VEGF therapy are not as devastating as suggested previously. Visual acuity and metamorphopsia improve with time as long as the neovascular membrane is inactive. Optical coherence tomography changes in the macular area reflect the visual acuity improvement.

  15. Retinal cone photoreceptors of the deer mouse Peromyscus maniculatus: development, topography, opsin expression and spectral tuning.

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    Patrick Arbogast

    Full Text Available A quantitative analysis of photoreceptor properties was performed in the retina of the nocturnal deer mouse, Peromyscus maniculatus, using pigmented (wildtype and albino animals. The aim was to establish whether the deer mouse is a more suitable model species than the house mouse for photoreceptor studies, and whether oculocutaneous albinism affects its photoreceptor properties. In retinal flatmounts, cone photoreceptors were identified by opsin immunostaining, and their numbers, spectral types, and distributions across the retina were determined. Rod photoreceptors were counted using differential interference contrast microscopy. Pigmented P. maniculatus have a rod-dominated retina with rod densities of about 450.000/mm(2 and cone densities of 3000-6500/mm(2. Two cone opsins, shortwave sensitive (S and middle-to-longwave sensitive (M, are present and expressed in distinct cone types. Partial sequencing of the S opsin gene strongly supports UV sensitivity of the S cone visual pigment. The S cones constitute a 5-15% minority of the cones. Different from house mouse, S and M cone distributions do not have dorsoventral gradients, and coexpression of both opsins in single cones is exceptional (<2% of the cones. In albino P. maniculatus, rod densities are reduced by approximately 40% (270.000/mm(2. Overall, cone density and the density of cones exclusively expressing S opsin are not significantly different from pigmented P. maniculatus. However, in albino retinas S opsin is coexpressed with M opsin in 60-90% of the cones and therefore the population of cones expressing only M opsin is significantly reduced to 5-25%. In conclusion, deer mouse cone properties largely conform to the general mammalian pattern, hence the deer mouse may be better suited than the house mouse for the study of certain basic cone properties, including the effects of albinism on cone opsin expression.

  16. Role of endoplasmic reticulum stress in 12/15-lipoxygenase-induced retinal microvascular dysfunction in a mouse model of diabetic retinopathy.

    Science.gov (United States)

    Elmasry, Khaled; Ibrahim, Ahmed S; Saleh, Heba; Elsherbiny, Nehal; Elshafey, Sally; Hussein, Khaled A; Al-Shabrawey, Mohamed

    2018-05-01

    Our earlier studies have established the role of 12/15-lipoxygenase (LO) in mediating the inflammatory reaction in diabetic retinopathy. However, the exact mechanism is still unclear. The goal of the current study was to identify the potential role of endoplasmic reticulum (ER) stress as a major cellular stress response in the 12/15-LO-induced retinal changes in diabetic retinopathy. We used in vivo and in vitro approaches. For in vivo studies, experimental diabetes was induced in wild-type (WT) mice and 12/15-Lo (also known as Alox15) knockout mice (12/15-Lo -/- ); ER stress was then evaluated after 12-14 weeks of diabetes. We also tested the effect of intravitreal injection of 12-hydroxyeicosatetraenoic acid (HETE) on retinal ER stress in WT mice and in mice lacking the catalytic subunit of NADPH oxidase, encoded by Nox2 (also known as Cybb) (Nox2 -/- mice). In vitro studies were performed using human retinal endothelial cells (HRECs) treated with 15-HETE (0.1 μmol/l) or vehicle, with or without ER stress or NADPH oxidase inhibitors. This was followed by evaluation of ER stress response, NADPH oxidase expression/activity and the levels of phosphorylated vascular endothelial growth factor receptor-2 (p-VEGFR2) by western blotting and immunoprecipitation assays. Moreover, real-time imaging of intracellular calcium (Ca 2+ ) release in HRECs treated with or without 15-HETE was performed using confocal microscopy. Deletion of 12/15-Lo significantly attenuated diabetes-induced ER stress in mouse retina. In vitro, 15-HETE upregulated ER stress markers such as phosphorylated RNA-dependent protein kinase-like ER-regulated kinase (p-PERK), activating transcription factor 6 (ATF6) and protein disulfide isomerase (PDI) in HRECs. Inhibition of ER stress reduced 15-HETE-induced-leucocyte adhesion, VEGFR2 phosphorylation and NADPH oxidase expression/activity. However, inhibition of NADPH oxidase or deletion of Nox2 had no effect on ER stress induced by the 12/15-LO

  17. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

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    Tagami, Mizuki [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Kusuhara, Sentaro, E-mail: kusu@med.kobe-u.ac.jp [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Imai, Hisanori [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Uemura, Akiyoshi [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Department of Vascular Biology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan); Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira [Department of Surgery Related, Division of Ophthalmology, Kobe University Graduate School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017 (Japan)

    2010-10-01

    Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  18. Loss of Ikbkap Causes Slow, Progressive Retinal Degeneration in a Mouse Model of Familial Dysautonomia.

    Science.gov (United States)

    Ueki, Yumi; Ramirez, Grisela; Salcedo, Ernesto; Stabio, Maureen E; Lefcort, Frances

    2016-01-01

    Familial dysautonomia (FD) is an autosomal recessive congenital neuropathy that is caused by a mutation in the gene for inhibitor of kappa B kinase complex-associated protein ( IKBKAP ). Although FD patients suffer from multiple neuropathies, a major debilitation that affects their quality of life is progressive blindness. To determine the requirement for Ikbkap in the developing and adult retina, we generated Ikbkap conditional knockout (CKO) mice using a TUBA1a promoter-Cre ( Tα1-Cre ). In the retina, Tα1-Cre expression is detected predominantly in retinal ganglion cells (RGCs). At 6 months, significant loss of RGCs had occurred in the CKO retinas, with the greatest loss in the temporal retina, which is the same spatial phenotype observed in FD, Leber hereditary optic neuropathy, and dominant optic atrophy. Interestingly, the melanopsin-positive RGCs were resistant to degeneration. By 9 months, signs of photoreceptor degeneration were observed, which later progressed to panretinal degeneration, including RGC and photoreceptor loss, optic nerve thinning, Müller glial activation, and disruption of layers. Taking these results together, we conclude that although Ikbkap is not required for normal development of RGCs, its loss causes a slow, progressive RGC degeneration most severely in the temporal retina, which is later followed by indirect photoreceptor loss and complete retinal disorganization. This mouse model of FD is not only useful for identifying the mechanisms mediating retinal degeneration, but also provides a model system in which to attempt to test therapeutics that may mitigate the loss of vision in FD patients.

  19. Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.

    Science.gov (United States)

    Rops, Angelique L; van der Vlag, Johan; Jacobs, Cor W; Dijkman, Henry B; Lensen, Joost F; Wijnhoven, Tessa J; van den Heuvel, Lambert P; van Kuppevelt, Toin H; Berden, Jo H

    2004-12-01

    The culture and establishment of glomerular cell lines has proven to be an important tool for the understanding of glomerular cell functions in glomerular physiology and pathology. Especially, the recent establishment of a conditionally immortalized visceral epithelial cell line has greatly boosted the research on podocyte biology. Glomeruli were isolated from H-2Kb-tsA58 transgenic mice that contain a gene encoding a temperature-sensitive variant of the SV40 large tumor antigen, facilitating proliferative growth at 33 degrees C and differentiation at 37 degrees C. Glomerular endothelial cells were isolated from glomerular outgrowth by magnetic beads loaded with CD31, CD105, GSL I-B4, and ULEX. Clonal cell lines were characterized by immunofluorescence staining with antibodies/lectins specific for markers of endothelial cells, podocytes, and mesangial cells. Putative glomerular endothelial cell lines were analyzed for (1) cytokine-induced expression of adhesion molecules; (2) tube formation on Matrigel coating; and (3) the presence of fenestrae. As judged by immunostaining for Wilms tumor-1, smooth muscle actin (SMA), podocalyxin, and von Willebrand factor (vWF), we obtained putative endothelial, podocyte and mesangial cell lines. The mouse glomerular endothelial cell clone #1 (mGEnC-1) was positive for vWF, podocalyxin, CD31, CD105, VE-cadherin, GSL I-B4, and ULEX, internalized acetylated-low-density lipoprotein (LDL), and showed increased expression of adhesion molecules after activation with proinflammatory cytokines. Furthermore, mGEnC-1 formed tubes and contained nondiaphragmed fenestrae. The mGEnC-1 represents a conditionally immortalized cell line with various characteristics of differentiated glomerular endothelial cells when cultured at 37 degrees C. Most important, mGEnC-1 contains nondiaphragmed fenestrae, which is a unique feature of glomerular endothelial cells.

  20. BMP7 and SHH regulate Pax2 in mouse retinal astrocytes by relieving TLX repression.

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    Sehgal, Rachna; Sheibani, Nader; Rhodes, Simon J; Belecky Adams, Teri L

    2009-08-15

    Pax2 is essential for development of the neural tube, urogenital system, optic vesicle, optic cup and optic tract. In the eye, Pax2 deficiency is associated with coloboma, a loss of astrocytes in the optic nerve and retina, and abnormal axonal pathfinding of the ganglion cell axons at the optic chiasm. Thus, appropriate expression of Pax2 is essential for astrocyte determination and differentiation. Although BMP7 and SHH have been shown to regulate Pax2 expression, the molecular mechanism by which this regulation occurs is not well understood. In this study, we determined that BMP7 and SHH activate Pax2 expression in mouse retinal astrocyte precursors in vitro. SHH appeared to play a dual role in Pax2 regulation; 1) SHH may regulate BMP7 expression, and 2) the SHH pathway cooperates with the BMP pathway to regulate Pax2 expression. BMP and SHH pathway members can interact separately or together with TLX, a repressor protein in the tailless transcription factor family. Here we show that the interaction of both pathways with TLX relieves the repression of Pax2 expression in mouse retinal astrocytes. Together these data reveal a new mechanism for the cooperative actions of signaling pathways in astrocyte determination and differentiation and suggest interactions of regulatory pathways that are applicable to other developmental programs.

  1. The long noncoding RNA RNCR2 directs mouse retinal cell specification

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    Blackshaw Seth

    2010-05-01

    Full Text Available Abstract Background Recent work has identified that many long mRNA-like noncoding RNAs (lncRNAs are expressed in the developing nervous system. Despite their abundance, the function of these ncRNAs has remained largely unexplored. We have investigated the highly abundant lncRNA RNCR2 in regulation of mouse retinal cell differentiation. Results We find that the RNCR2 is selectively expressed in a subset of both mitotic progenitors and postmitotic retinal precursor cells. ShRNA-mediated knockdown of RNCR2 results in an increase of both amacrine cells and Müller glia, indicating a role for this lncRNA in regulating retinal cell fate specification. We further report that RNCR2 RNA, which is normally nuclear-retained, can be exported from the nucleus when fused to an IRES-GFP sequence. Overexpression of RNCR2-IRES-GFP phenocopies the effects of shRNA-mediated knockdown of RNCR2, implying that forced mislocalization of RNCR2 induces a dominant-negative phenotype. Finally, we use the IRES-GFP fusion approach to identify specific domains of RNCR2 that are required for repressing both amacrine and Müller glial differentiation. Conclusion These data demonstrate that the lncRNA RNCR2 plays a critical role in regulating mammalian retinal cell fate specification. Furthermore, we present a novel approach for generating dominant-negative constructs of lncRNAs, which may be generally useful in the functional analysis of this class of molecules.

  2. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

    NARCIS (Netherlands)

    Bennis, A.; Gorgels, T.G.M.F.; ten Brink, J.B.; van der Spek, P.J.; Bossers, K.; Heine, V.M.; Bergen, A.A.

    2015-01-01

    Background The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to

  3. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles : Potential Implications for Age-Related Macular Degeneration

    NARCIS (Netherlands)

    Bennis, Anna; Gorgels, Theo G M F; Ten Brink, Jacoline B; van der Spek, Peter J; Bossers, Koen; Heine, Vivi M; Bergen, Arthur A

    2015-01-01

    BACKGROUND: The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to

  4. Comparison of Mouse and Human Retinal Pigment Epithelium Gene Expression Profiles: Potential Implications for Age-Related Macular Degeneration

    NARCIS (Netherlands)

    Bennis, Anna; Gorgels, Theo G. M. F.; ten Brink, Jacoline B.; van der Spek, Peter J.; Bossers, Koen; Heine, Vivi M.; Bergen, Arthur A.

    2015-01-01

    The human retinal pigment epithelium (RPE) plays an important role in the pathogenesis of age related macular degeneration (AMD). AMD is the leading cause of blindness worldwide. There is currently no effective treatment available. Preclinical studies in AMD mouse models are essential to develop new

  5. Cardiac endothelial cells isolated from mouse heart - a novel model for radiobiology

    International Nuclear Information System (INIS)

    Jelonek, K.; Walaszczyk, A.; Gabrys, D.; Pietrowska, M.; Widlak, P.; Kanthou, Ch.

    2011-01-01

    Cardiovascular disease is recognized as an important clinical problem in radiotherapy and radiation protection. However, only few radiobiological models relevant for assessment of cardiotoxic effects of ionizing radiation are available. Here we describe the isolation of mouse primary cardiac endothelial cells, a possible target for cardiotoxic effects of radiation. Cells isolated from hearts of juvenile mice were cultured and irradiated in vitro. In addition, cells isolated from hearts of locally irradiated adult animals (up to 6 days after irradiation) were tested. A dose-dependent formation of histone γH 2 A.X foci was observed after in vitro irradiation of cultured cells. However, such cells were resistant to radiation-induced apoptosis. Increased levels of actin stress fibres were observed in the cytoplasm of cardiac endothelial cells irradiated in vitro or isolated from irradiated animals. A high dose of 16 Gy did not increase permeability to Dextran in monolayers formed by endothelial cells. Up-regulated expression of Vcam1, Sele and Hsp70i genes was detected after irradiation in vitro and in cells isolated few days after irradiation in vivo. The increased level of actin stress fibres and enhanced expression of stress-response genes in irradiated endothelial cells are potentially involved in cardiotoxic effects of ionizing radiation. (authors)

  6. Impact of Lycium Barbarum Polysaccharide and Danshensu on vascular endothelial growth factor in the process of retinal neovascularization of rabbit

    Directory of Open Access Journals (Sweden)

    Xue-Min Tian

    2013-02-01

    Full Text Available AIM:To discuss the impact of Lycium Barbarum Polysaccharide (LBP and Danshensu purified from Traditional Chinese Medicine (TCM on vascular endothelial growth factor (VEGF of rabbits with retinal neovascularization.METHODS:Forty rabbits were divided into normal control group, model control group, LBP group and Danshensu group. Animals in the normal control group were fed in the normal oxygen environment. Animals in the other three groups were put into the environment with 70% oxygen for 5 days in order to build the model of oxygen-induced vascular proliferation retinopathy. And then different TCM extract was injected into the abdominal cavities of these annimals. After 7 days, the VEGF content of in the serum of rabbit was measured by double antibody sandwich method.RESULTS:Data analysis indicated that VEGF content was as follows:Danshensu group was lower than model control group (12.92±3.84ng/L vs 19.32±4.15ng/L, Pvs 19.32±4.15ng/L, P<0.01; total blood viscosity, plasma viscosity, cholesterol content, fibrinogen content and triacylglycerol content after peritoneal injection of LBP and Danshensu were obviously lower than before injection.CONCLUSION:TCM extract-LBP and Danshensu can prominently reduce the content of VEGF in the process of vascular proliferative retinopathy of rabbit; can prevent the occurrence of retinal microvascular disease by improving partial oxygen-deficient environment or affecting all kinds of new growth factor.

  7. The db/db mouse: a useful model for the study of diabetic retinal neurodegeneration.

    Directory of Open Access Journals (Sweden)

    Patricia Bogdanov

    Full Text Available BACKGROUND: To characterize the sequential events that are taking place in retinal neurodegeneration in a murine model of spontaneous type 2 diabetes (db/db mouse. METHODS: C57BLKsJ-db/db mice were used as spontaneous type 2 diabetic animal model, and C57BLKsJ-db/+ mice served as the control group. To assess the chronological sequence of the abnormalities the analysis was performed at different ages (8, 16 and 24 weeks. The retinas were evaluated in terms of morphological and functional abnormalities [electroretinography (ERG]. Histological markers of neurodegeneration (glial activation and apoptosis were evaluated by immunohistochemistry. In addition glutamate levels and glutamate/aspartate transporter (GLAST expression were assessed. Furthermore, to define gene expression changes associated with early diabetic retinopathy a transcriptome analyses was performed at 8 week. Furthermore, an additional interventional study to lower blood glucose levels was performed. RESULTS: Glial activation was higher in diabetic than in non diabetic mice in all the stages (p<0.01. In addition, a progressive loss of ganglion cells and a significant reduction of neuroretinal thickness were also observed in diabetic mice. All these histological hallmarks of neurodegeneration were less pronounced at week 8 than at week 16 and 24. Significant ERG abnormalities were present in diabetic mice at weeks 16 and 24 but not at week 8. Moreover, we observed a progressive accumulation of glutamate in diabetic mice associated with an early downregulation of GLAST. Morphological and ERG abnormalities were abrogated by lowering blood glucose levels. Finally, a dysregulation of several genes related to neurotransmission and oxidative stress such as UCP2 were found at week 8. CONCLUSIONS: Our results suggest that db/db mouse reproduce the features of the neurodegenerative process that occurs in the human diabetic eye. Therefore, it seems an appropriate model for investigating the

  8. Oxygen-induced retinopathy in mice with retinal photoreceptor cell degeneration.

    Science.gov (United States)

    Zhang, Qian; Zhang, Zuo-Ming

    2014-04-25

    It is reported that retinal neovascularization seems to rarely co-exist with retinitis pigmentosa in patients and in some mouse models; however, it is not widely acknowledged as a universal phenomenon in all strains of all animal species. We aimed to further explore this phenomenon with an oxygen-induced retinopathy model in mice with retinal photoreceptor cell degeneration. Oxygen-induced retinopathy of colored and albino mice with rapid retinal degeneration were compared to homologous wild-type mice. The retinas were analyzed using high-molecular-weight FITC-dextran stained flat-mount preparation, hematoxylin and eosin (H&E) stained cross-sections, an immunohistochemical test for vascular endothelial growth factor (VEGF) distribution and Western blotting for VEGF expression after exposure to hyperoxia between postnatal days 17 (P17) and 21. Leakage and areas of non-perfusion of the retinal blood vessels were alleviated in the retinal degeneration mice. The number of preretinal vascular endothelial cell nuclei in the retinal degeneration mice was smaller than that in the homologous wild-type mice after exposure to hyperoxia (Poxygen-induced retinopathy was positively correlated with the VEGF expression level. However, the VEGF expression level was lower in the retinal degeneration mice. Proliferative retinopathy occurred in mice with rapid retinal degeneration, but retinal photoreceptor cell degeneration could partially restrain the retinal neovascularization in this rapid retinal degeneration mouse model. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. A CTRP5 gene S163R mutation knock-in mouse model for late-onset retinal degeneration.

    Science.gov (United States)

    Chavali, Venkata R M; Khan, Naheed W; Cukras, Catherine A; Bartsch, Dirk-Uwe; Jablonski, Monica M; Ayyagari, Radha

    2011-05-15

    Late-onset retinal macular degeneration (L-ORD) is an autosomal dominant inherited disorder caused by a single missense mutation (S163R) in the CTRP5/C1QTNF5 protein. Early phenotypic features of L-ORD include: dark adaptation abnormalities, nyctalopia, and drusen deposits in the peripheral macular region. Apart from posterior segment abnormalities, these patients also develop abnormally long anterior lens zonules. In the sixth decade of life the rod and cone function declines, accompanied by electroretinogram (ERG) abnormalities. Some patients also develop choroidal neovascularization and glaucoma. In order to understand the disease pathology and mechanisms involved in retinal dystrophy, we generated a knock-in (Ctrp5(+/-)) mouse model carrying the disease-associated mutation in the mouse Ctrp5/C1QTNF5 gene. These mice develop slower rod-b wave recovery consistent with early dark adaptation abnormalities, accumulation of hyperautofluorescence spots, retinal pigment epithelium abnormalities, drusen, Bruch's membrane abnormalities, loss of photoreceptors, and retinal vascular leakage. The Ctrp5(+/-) mice, which have most of the pathological features of age-related macular degeneration, are unique and may serve as a valuable model both to understand the molecular pathology of late-onset retinal degeneration and to evaluate therapies.

  10. Sirtuin1 Over-Expression Does Not Impact Retinal Vascular and Neuronal Degeneration in a Mouse Model of Oxygen-Induced Retinopathy

    Science.gov (United States)

    Michan, Shaday; Juan, Aimee M.; Hurst, Christian G.; Cui, Zhenghao; Evans, Lucy P.; Hatton, Colman J.; Pei, Dorothy T.; Ju, Meihua; Sinclair, David A.; Smith, Lois E. H.; Chen, Jing

    2014-01-01

    Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy. PMID:24416337

  11. Sirtuin1 over-expression does not impact retinal vascular and neuronal degeneration in a mouse model of oxygen-induced retinopathy.

    Science.gov (United States)

    Michan, Shaday; Juan, Aimee M; Hurst, Christian G; Cui, Zhenghao; Evans, Lucy P; Hatton, Colman J; Pei, Dorothy T; Ju, Meihua; Sinclair, David A; Smith, Lois E H; Chen, Jing

    2014-01-01

    Proliferative retinopathy is a leading cause of blindness, including retinopathy of prematurity (ROP) in children and diabetic retinopathy in adults. Retinopathy is characterized by an initial phase of vessel loss, leading to tissue ischemia and hypoxia, followed by sight threatening pathologic neovascularization in the second phase. Previously we found that Sirtuin1 (Sirt1), a metabolically dependent protein deacetylase, regulates vascular regeneration in a mouse model of oxygen-induced proliferative retinopathy (OIR), as neuronal depletion of Sirt1 in retina worsens retinopathy. In this study we assessed whether over-expression of Sirtuin1 in retinal neurons and vessels achieved by crossing Sirt1 over-expressing flox mice with Nestin-Cre mice or Tie2-Cre mice, respectively, may protect against retinopathy. We found that over-expression of Sirt1 in Nestin expressing retinal neurons does not impact vaso-obliteration or pathologic neovascularization in OIR, nor does it influence neuronal degeneration in OIR. Similarly, increased expression of Sirt1 in Tie2 expressing vascular endothelial cells and monocytes/macrophages does not protect retinal vessels in OIR. In addition to the genetic approaches, dietary supplement with Sirt1 activators, resveratrol or SRT1720, were fed to wild type mice with OIR. Neither treatment showed significant vaso-protective effects in retinopathy. Together these results indicate that although endogenous Sirt1 is important as a stress-induced protector in retinopathy, over-expression of Sirt1 or treatment with small molecule activators at the examined doses do not provide additional protection against retinopathy in mice. Further studies are needed to examine in depth whether increasing levels of Sirt1 may serve as a potential therapeutic approach to treat or prevent retinopathy.

  12. Bone marrow endothelial progenitors augment atherosclerotic plaque regression in a mouse model of plasma lipid lowering

    Science.gov (United States)

    Yao, Longbiao; Heuser-Baker, Janet; Herlea-Pana, Oana; Iida, Ryuji; Wang, Qilong; Zou, Ming-Hui; Barlic-Dicen, Jana

    2012-01-01

    The major event initiating atherosclerosis is hypercholesterolemia-induced disruption of vascular endothelium integrity. In settings of endothelial damage, endothelial progenitor cells (EPCs) are mobilized from bone marrow into circulation and home to sites of vascular injury where they aid endothelial regeneration. Given the beneficial effects of EPCs in vascular repair, we hypothesized that these cells play a pivotal role in atherosclerosis regression. We tested our hypothesis in the atherosclerosis-prone mouse model in which hypercholesterolemia, one of the main factors affecting EPC homeostasis, is reversible (Reversa mice). In these mice normalization of plasma lipids decreased atherosclerotic burden; however, plaque regression was incomplete. To explore whether endothelial progenitors contribute to atherosclerosis regression, bone marrow EPCs from a transgenic strain expressing green fluorescent protein under the control of endothelial cell-specific Tie2 promoter (Tie2-GFP+) were isolated. These cells were then adoptively transferred into atheroregressing Reversa recipients where they augmented plaque regression induced by reversal of hypercholesterolemia. Advanced plaque regression correlated with engraftment of Tie2-GFP+ EPCs into endothelium and resulted in an increase in atheroprotective nitric oxide and improved vascular relaxation. Similarly augmented plaque regression was also detected in regressing Reversa mice treated with the stem cell mobilizer AMD3100 which also mobilizes EPCs to peripheral blood. We conclude that correction of hypercholesterolemia in Reversa mice leads to partial plaque regression that can be augmented by AMD3100 treatment or by adoptive transfer of EPCs. This suggests that direct cell therapy or indirect progenitor cell mobilization therapy may be used in combination with statins to treat atherosclerosis. PMID:23081735

  13. RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

    Directory of Open Access Journals (Sweden)

    Li eJiang

    2014-04-01

    Full Text Available RNA interference (RNAi knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementary (sc AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C establishing a rapid RP-like phenotype, whereas the nonallele-specific model expresses mouse GCAP1(L151F producing a slowly progressing cone/rod dystrophy (CORD. The late onset GCAP1(L151F-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse GCAP1 showed strong expression at one week post-injection. In both allele-specific (GCAP1(Y99C-RP and nonallele-specific (GCAP1(L151F-CORD models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a proof of concept toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAP1 mutation. Further, nonallele-specific RNAi knockdown of GCAP1 may prove generally applicable toward the rescue of any human GCAP1-based dominant cone-rod dystrophy.

  14. Thioredoxin plays a key role in retinal neuropathy prior to endothelial damage in diabetic mice

    OpenAIRE

    Ren, Xiang; Li, Chen; Liu, Junli; Zhang, Chenghong; Fu, Yuzhen; Wang, Nina; Ma, Haiying; Lu, Heyuan; Kong, Hui; Kong, Li

    2017-01-01

    Diabetes is a chronic metabolic syndrome that results in changes in carbohydrate, lipid and protein metabolism. With diabetes for a long time, it increases the risk of diabetic retinopathy (DR) and long-term morbidity and mortality. Moreover, emerging evidence suggests that neuron damage occurs earlier than microvascular complications in DR patients, but the underlying mechanism is unclear. We investigated diabetes-induced retinal neuropathy and elucidated key molecular events to identify new...

  15. A novel podoplanin-GFPCre mouse strain for gene deletion in lymphatic endothelial cells.

    Science.gov (United States)

    Gil, Hyea Jin; Ma, Wanshu; Oliver, Guillermo

    2018-04-01

    The lymphatic vascular system is a one-direction network of thin-walled capillaries and larger vessels covered by a continuous layer of endothelial cells responsible for maintaining fluid homeostasis. Some of the main functions of the lymphatic vasculature are to drain fluid from the extracellular spaces and return it back to the blood circulation, lipid absorption from the intestinal tract, and transport of immune cells to lymphoid organs. A number of genes controlling the development of the mammalian lymphatic vasculature have been identified in the last few years, and their functional roles started to be characterized using gene inactivation approaches in mice. Unfortunately, only few mouse Cre strains relatively specific for lymphatic endothelial cells (LECs) are currently available. In this article, we report the generation of a novel Podoplanin (Pdpn) GFPCre transgenic mouse strain using its 5' regulatory region. Pdpn encodes a transmembrane mucin-type O-glycoprotein that is expressed on the surface of embryonic and postnatal LECs, in addition to few other cell types. Our detailed characterization of this novel strain indicates that it will be a valuable additional genetic tool for the analysis of gene function in LECs. © 2018 Wiley Periodicals, Inc.

  16. Intraocular Injection of ES Cell-Derived Neural Progenitors Improve Visual Function in Retinal Ganglion Cell-Depleted Mouse Models

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    Mundackal S. Divya

    2017-09-01

    Full Text Available Retinal ganglion cell (RGC transplantation is a promising strategy to restore visual function resulting from irreversible RGC degeneration occurring in glaucoma or inherited optic neuropathies. We previously demonstrated FGF2 induced differentiation of mouse embryonic stem cells (ESC to RGC lineage, capable of retinal ganglion cell layer (GCL integration upon transplantation. Here, we evaluated possible improvement of visual function by transplantation of ES cell derived neural progenitors in RGC depleted glaucoma mice models. ESC derived neural progenitors (ES-NP were transplanted into N-Methyl-D-Aspartate (NMDA injected, RGC-ablated mouse models and a pre-clinical glaucoma mouse model (DBA/2J having sustained higher intra ocular pressure (IOP. Visual acuity and functional integration was evaluated by behavioral experiments and immunohistochemistry, respectively. GFP-expressing ES-NPs transplanted in NMDA-injected RGC-depleted mice differentiated into RGC lineage and possibly integrating into GCL. An improvement in visual acuity was observed after 2 months of transplantation, when compared to the pre-transplantation values. Expression of c-Fos in the transplanted cells, upon light induction, further suggests functional integration into the host retinal circuitry. However, the transplanted cells did not send axonal projections into optic nerve. Transplantation experiments in DBA/2J mouse showed no significant improvement in visual functions, possibly due to both host and transplanted retinal cell death which could be due to an inherent high IOP. We showed that, ES NPs transplanted into the retina of RGC-ablated mouse models could survive, differentiate to RGC lineage, and possibly integrate into GCL to improve visual function. However, for the survival of transplanted cells in glaucoma, strategies to control the IOP are warranted.

  17. Chronic hydroxychloroquine improves endothelial dysfunction and protects kidney in a mouse model of systemic lupus erythematosus.

    Science.gov (United States)

    Gómez-Guzmán, Manuel; Jiménez, Rosario; Romero, Miguel; Sánchez, Manuel; Zarzuelo, María José; Gómez-Morales, Mercedes; O'Valle, Francisco; López-Farré, Antonio José; Algieri, Francesca; Gálvez, Julio; Pérez-Vizcaino, Francisco; Sabio, José Mario; Duarte, Juan

    2014-08-01

    Hydroxychloroquine has been shown to be efficacious in the treatment of autoimmune diseases, including systemic lupus erythematosus. Hydroxychloroquine-treated lupus patients showed a lower incidence of thromboembolic disease. Endothelial dysfunction, the earliest indicator of the development of cardiovascular disease, is present in lupus. Whether hydroxychloroquine improves endothelial function in lupus is not clear. The aim of this study was to analyze the effects of hydroxychloroquine on hypertension, endothelial dysfunction, and renal injury in a female mouse model of lupus. NZBWF1 (lupus) and NZW/LacJ (control) mice were treated with hydroxychloroquine 10 mg/kg per day by oral gavage, or with tempol and apocynin in the drinking water, for 5 weeks. Hydroxychloroquine treatment did not alter lupus disease activity (assessed by plasma double-stranded DNA autoantibodies) but prevented hypertension, cardiac and renal hypertrophy, proteinuria, and renal injury in lupus mice. Aortae from lupus mice showed reduced endothelium-dependent vasodilator responses to acetylcholine and enhanced contraction to phenylephrine, which were normalized by hydroxychloroquine or antioxidant treatments. No differences among all experimental groups were found in both the relaxant responses to acetylcholine and the contractile responses to phenylephrine in rings incubated with the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. Vascular reactive oxygen species content and mRNA levels of nicotinamide adenine dinucleotide phosphate oxidase subunits NOX-1 and p47(phox) were increased in lupus mice and reduced by hydroxychloroquine or antioxidants. Chronic hydroxychloroquine treatment reduced hypertension, endothelial dysfunction, and organ damage in severe lupus mice, despite the persistent elevation of anti-double-stranded DNA, suggesting the involvement of new additional mechanisms to improve cardiovascular complications. © 2014 American Heart Association, Inc.

  18. Progranulin, a major secreted protein of mouse adipose-derived stem cells, inhibits light-induced retinal degeneration.

    Science.gov (United States)

    Tsuruma, Kazuhiro; Yamauchi, Mika; Sugitani, Sou; Otsuka, Tomohiro; Ohno, Yuta; Nagahara, Yuki; Ikegame, Yuka; Shimazawa, Masamitsu; Yoshimura, Shinichi; Iwama, Toru; Hara, Hideaki

    2014-01-01

    Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose-derived stem cells (ASCs) and ASC-conditioned medium (ASC-CM) against retinal damage and identified the neuroprotective factors in ASC-CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light-induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC-CM and mature adipocyte-conditioned medium were collected after 72 hours of culture. In vitro, H2O2- and light-induced cell death was reduced in a photoreceptor cell line with ASC-CM but not with mature adipocyte-conditioned medium. In vivo, light-induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC-CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal-regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC-CM and progranulin have neuroprotective effects in the light-induced retinal-damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.

  19. Human pericyte-endothelial cell interactions in co-culture models mimicking the diabetic retinal microvascular environment.

    Science.gov (United States)

    Tarallo, Sonia; Beltramo, Elena; Berrone, Elena; Porta, Massimo

    2012-12-01

    Pericytes regulate vascular tone, perfusion pressure and endothelial cell (EC) proliferation in capillaries. Thiamine and benfotiamine counteract high glucose-induced damage in vascular cells. We standardized two human retinal pericyte (HRP)/EC co-culture models to mimic the diabetic retinal microvascular environment. We aimed at evaluating the interactions between co-cultured HRP and EC in terms of proliferation/apoptosis and the possible protective role of thiamine and benfotiamine against high glucose-induced damage. EC and HRP were co-cultured in physiological glucose and stable or intermittent high glucose, with or without thiamine/benfotiamine. No-contact model: EC were plated on a porous membrane suspended into the medium and HRP on the bottom of the same well. Cell-to-cell contact model: EC and HRP were plated on the opposite sides of the same membrane. Proliferation (cell counts and DNA synthesis), apoptosis and tubule formation in Matrigel were assessed. In the no-contact model, stable high glucose reduced proliferation of co-cultured EC/HRP and EC alone and increased co-cultured EC/HRP apoptosis. In the contact model, both stable and intermittent high glucose reduced co-cultured EC/HRP proliferation and increased apoptosis. Stable high glucose had no effects on HRP in separate cultures. Both EC and HRP proliferated better when co-cultured. Thiamine and benfotiamine reversed high glucose-induced damage in all cases. HRP are sensitive to soluble factors released by EC when cultured in high glucose conditions, as suggested by conditioned media assays. In the Matrigel models, addition of thiamine and benfotiamine re-established the high glucose-damaged interactions between EC/HRP and stabilized microtubules.

  20. Eliminating Glutamatergic Input onto Horizontal Cells Changes the Dynamic Range and Receptive Field Organization of Mouse Retinal Ganglion Cells.

    Science.gov (United States)

    Ströh, Sebastian; Puller, Christian; Swirski, Sebastian; Hölzel, Maj-Britt; van der Linde, Lea I S; Segelken, Jasmin; Schultz, Konrad; Block, Christoph; Monyer, Hannah; Willecke, Klaus; Weiler, Reto; Greschner, Martin; Janssen-Bienhold, Ulrike; Dedek, Karin

    2018-02-21

    In the mammalian retina, horizontal cells receive glutamatergic inputs from many rod and cone photoreceptors and return feedback signals to them, thereby changing photoreceptor glutamate release in a light-dependent manner. Horizontal cells also provide feedforward signals to bipolar cells. It is unclear, however, how horizontal cell signals also affect the temporal, spatial, and contrast tuning in retinal output neurons, the ganglion cells. To study this, we generated a genetically modified mouse line in which we eliminated the light dependency of feedback by deleting glutamate receptors from mouse horizontal cells. This genetic modification allowed us to investigate the impact of horizontal cells on ganglion cell signaling independent of the actual mode of feedback in the outer retina and without pharmacological manipulation of signal transmission. In control and genetically modified mice (both sexes), we recorded the light responses of transient OFF-α retinal ganglion cells in the intact retina. Excitatory postsynaptic currents (EPSCs) were reduced and the cells were tuned to lower temporal frequencies and higher contrasts, presumably because photoreceptor output was attenuated. Moreover, receptive fields of recorded cells showed a significantly altered surround structure. Our data thus suggest that horizontal cells are responsible for adjusting the dynamic range of retinal ganglion cells and, together with amacrine cells, contribute to the center/surround organization of ganglion cell receptive fields in the mouse. SIGNIFICANCE STATEMENT Horizontal cells represent a major neuronal class in the mammalian retina and provide lateral feedback and feedforward signals to photoreceptors and bipolar cells, respectively. The mode of signal transmission remains controversial and, moreover, the contribution of horizontal cells to visual processing is still elusive. To address the question of how horizontal cells affect retinal output signals, we recorded the light

  1. Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

    Science.gov (United States)

    Wavre-Shapton, Silène T; Tolmachova, Tanya; Lopes da Silva, Mafalda; da Silva, Mafalda Lopes; Futter, Clare E; Seabra, Miguel C

    2013-01-01

    The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

  2. Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Silène T Wavre-Shapton

    Full Text Available The retinal pigment epithelium (RPE is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1. REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox, Tyr-Cre+. Transmission electron microscopy (TEM was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

  3. Rescue of retinal function by BDNF in a mouse model of glaucoma.

    Directory of Open Access Journals (Sweden)

    Luciano Domenici

    Full Text Available Vision loss in glaucoma is caused by progressive dysfunction of retinal ganglion cells (RGCs and optic nerve atrophy. Here, we investigated the effectiveness of BDNF treatment to preserve vision in a glaucoma experimental model. As an established experimental model, we used the DBA/2J mouse, which develops chronic intraocular pressure (IOP elevation that mimics primary open-angle glaucoma (POAG. IOP was measured at different ages in DBA/2J mice. Visual function was monitored using the steady-state Pattern Electroretinogram (P-ERG and visual cortical evoked potentials (VEP. RGC alterations were assessed using Brn3 immunolabeling, and confocal microscope analysis. Human recombinant BDNF was dissolved in physiological solution (0.9% NaCl; the effects of repeated intravitreal injections and topical eye BDNF applications were independently evaluated in DBA/2J mice with ocular hypertension. BDNF level was measured in retinal homogenate by ELISA and western blot. We found a progressive decline of P-ERG and VEP responses in DBA/2J mice between 4 and 7 months of age, in relationship with the development of ocular hypertension and the reduction of Brn3 immunopositive RGCs. Conversely, repeated intravitreal injections (BDNF concentration = 2 µg/µl, volume = 1 µl, for each injection; 1 injection every four days, three injections over two weeks and topical eye application of BDNF eye-drops (12 µg/µl, 5 µl eye-drop every 48 h for two weeks were able to rescue visual responses in 7 month DBA/2J mice. In particular, BDNF topical eye treatment recovered P-ERG and VEP impairment increasing the number of Brn3 immunopositive RGCs. We showed that BDNF effects were independent of IOP reduction. Thus, topical eye treatment with BDNF represents a promisingly safe and feasible strategy to preserve visual function and diminish RGC vulnerability to ocular hypertension.

  4. Chemically-induced photoreceptor degeneration and protection in mouse iPSC-derived three-dimensional retinal organoids

    Directory of Open Access Journals (Sweden)

    Shin-ichiro Ito

    2017-10-01

    Full Text Available Induced pluripotent stem cells (iPSCs, which can be differentiated into various tissues and cell types, have been used for clinical research and disease modeling. Self-organizing three-dimensional (3D tissue engineering has been established within the past decade and enables researchers to obtain tissues and cells that almost mimic in vivo development. However, there are no reports of practical experimental procedures that reproduce photoreceptor degeneration. In this study, we induced photoreceptor cell death in mouse iPSC-derived 3D retinal organoids (3D-retinas by 4-hydroxytamoxifen (4-OHT, which induces photoreceptor degeneration in mouse retinal explants, and then established a live-cell imaging system to measure degeneration-related properties. Furthermore, we quantified the protective effects of representative ophthalmic supplements for treating the photoreceptor degeneration. This drug evaluation system enables us to monitor drug effects in photoreceptor cells and could be useful for drug screening.

  5. Antioxidant and Anti-Inflammatory Effects of Blueberry Anthocyanins on High Glucose-Induced Human Retinal Capillary Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Wuyang Huang

    2018-01-01

    Full Text Available Blueberries possess abundant anthocyanins, which benefit eye health. The purpose of this study was to explore the protective functional role of blueberry anthocyanin extract (BAE and its predominant constituents, malvidin (Mv, malvidin-3-glucoside (Mv-3-glc, and malvidin-3-galactoside (Mv-3-gal, on high glucose- (HG- induced injury in human retinal capillary endothelial cells (HRCECs. The results showed that BAE, Mv, Mv-3-glc, and Mv-3-gal enhanced cell viability (P<0.05 versus the HG group at 24 h; decreased the reactive oxygen species (ROS, P<0.01 versus the HG group both at 24 and 48 h; and increased the enzyme activity of catalase (CAT and superoxide dismutase (SOD (P<0.05 versus the HG group both at 24 and 48 h. Mv could greatly inhibit HG-induced Nox4 expression both at 24 and 48 h (P<0.05, while BAE and Mv-3-gal downregulated Nox4 only at 48 h (P<0.05. Mv, Mv-3-glc, and Mv-3-gal also changed nitric oxide (NO levels (P<0.05. BAE and Mv-3-glc also influenced angiogenesis by decreasing the vascular endothelial cell growth factor (VEGF level and inhibiting Akt pathway (P<0.05. Moreover, Mv and Mv-3-glc inhibited HG-induced intercellular adhesion molecule-1 (ICAM-1, P<0.001 and nuclear factor-kappa B (NF-κB (P<0.05. It indicated that blueberry anthocyanins protected HRCECs via antioxidant and anti-inflammatory mechanisms, which could be promising molecules for the development of nutraceuticals to prevent diabetic retinopathy.

  6. Differentiation/Purification Protocol for Retinal Pigment Epithelium from Mouse Induced Pluripotent Stem Cells as a Research Tool.

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    Yuko Iwasaki

    Full Text Available To establish a novel protocol for differentiation of retinal pigment epithelium (RPE with high purity from mouse induced pluripotent stem cells (iPSC.Retinal progenitor cells were differentiated from mouse iPSC, and RPE differentiation was then enhanced by activation of the Wnt signaling pathway, inhibition of the fibroblast growth factor signaling pathway, and inhibition of the Rho-associated, coiled-coil containing protein kinase signaling pathway. Expanded pigmented cells were purified by plate adhesion after Accutase® treatment. Enriched cells were cultured until they developed a cobblestone appearance with cuboidal shape. The characteristics of iPS-RPE were confirmed by gene expression, immunocytochemistry, and electron microscopy. Functions and immunologic features of the iPS-RPE were also evaluated.We obtained iPS-RPE at high purity (approximately 98%. The iPS-RPE showed apical-basal polarity and cellular structure characteristic of RPE. Expression levels of several RPE markers were lower than those of freshly isolated mouse RPE but comparable to those of primary cultured RPE. The iPS-RPE could form tight junctions, phagocytose photoreceptor outer segments, express immune antigens, and suppress lymphocyte proliferation.We successfully developed a differentiation/purification protocol to obtain mouse iPS-RPE. The mouse iPS-RPE can serve as an attractive tool for functional and morphological studies of RPE.

  7. Endothelial ATP-binding cassette G1 in mouse endothelium protects against hemodynamic-induced atherosclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Xue, Shanshan [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Department of Pediatrics, Baodi District People’s Hospital of Tianjin City, Tianjin, 301800 (China); Wang, Jiaxing [Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); Zhang, Xu; Shi, Ying; Li, Bochuan; Bao, Qiankun [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Pang, Wei [Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); Ai, Ding [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Zhu, Yi [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China); Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing, 100191 (China); He, Jinlong, E-mail: hejinlong@tmu.edu.cn [Department of Physiology and Pathophysiology, Tianjin Medical University, Tianjin, 300070 (China)

    2016-08-19

    Activated vascular endothelium inflammation under persistent hyperlipidemia is the initial step of atherogenesis. ATP-binding cassette G1 (ABCG1) is a crucial factor maintaining sterol and lipid homeostasis by transporting cholesterol efflux to high-density lipoprotein. In this study, we investigated the protective effects of ABCG1 in endothelial inflammation activation during early-stage atherogenesis in mice and the underlying mechanisms. Endothelial cell (EC)-specific ABCG1 transgenic (EC-ABCG1-Tg) mice were generated and cross-bred with low-density lipoprotein receptor–deficient (Ldlr{sup −/−}) mice. After a 4-week Western-type diet, the mice were sacrificed for assessing atherosclerosis. Human umbilical vein ECs were treated with different flows, and ABCG1 was adenovirally overexpressed to investigate the mechanism in vitro. Compared with Ldlr{sup −/−} mouse aortas, EC-ABCG1-Tg/Ldlr{sup −/−} aortas showed decreased early-stage lesions. Furthermore, the lesion area in the EC-ABCG1-Tg/Ldlr{sup −/−} mouse aortic arch but not thoracic aorta was significantly reduced, which suggests a protective role of ABCG1 under atheroprone flow. In vitro, overexpression of ABCG1 attenuated EC activation caused by oscillatory shear stress. Overexpression of ABCG1 blunted cholesterol-activated ECs in vitro. In exploring the mechanisms of ABCG1 attenuating endothelial inflammation, we found that ABCG1 inhibited oscillatory flow-activated nuclear factor kappa B and NLRP3 inflammasome in ECs. ABCG1 may play a protective role in early-stage atherosclerosis by reducing endothelial activation induced by oscillatory shear stress via suppressing the inflammatory response. - Highlights: • EC-ABCG1-Tg mice in a Ldlr{sup −/−} background showed decreased atherosclerosis. • Overexpression of ABCG1 in ECs decreased OSS-induced EC activation. • NLRP3 and NF-κB might be an underlying mechanism of ABCG1 protective role.

  8. Protective effect of sulforaphane against retinal degeneration in the Pde6rd10 mouse model of retinitis pigmentosa.

    Science.gov (United States)

    Kang, Kai; Yu, Minzhong

    2017-12-01

    Retinitis pigmentosa (RP) is a group of inherited diseases characterized by the death of rod photoreceptors, followed by the death of cone photoreceptors, progressively leading to partial or complete blindness. Currently no specific treatment is available for RP patients. Sulforaphane (SFN) has been confirmed to be an effective antioxidant in the treatment of many diseases. In this study, we tested the therapeutic effects of SFN against photoreceptor degeneration in Pde6b rd10 mice. rd10 mice and C57/BL6 wild-type (WT) mice were treated with SFN and saline, respectively, from P6 to P20. Electroretinography (ERG), terminal deoxynucleotidyl transferase dUTP nick end labeling and western blot were tested, respectively, at P21 for the analysis of retinal function, retinal cell apoptosis or death and the protein express of GRP78/BiP (TUNEL) as a marker of endoplasmic reticulum (ER) stress. Compared with the saline group, the SFN-treated group showed significantly higher ERG a-wave and b-wave amplitudes, less photoreceptor death, and the downregulation of GRP78/BiP. Our data showed that SFN ameliorated the retinal degeneration of rd10 mice, which is possibly related to the downregulation of GRP78 expression.

  9. An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina.

    Science.gov (United States)

    Rana, T; Shinde, V M; Starr, C R; Kruglov, A A; Boitet, E R; Kotla, P; Zolotukhin, S; Gross, A K; Gorbatyuk, M S

    2014-12-18

    Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1β, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with

  10. Effects of elevated glucose concentration on cultured bovine retinal endothelial (BRE) cells

    International Nuclear Information System (INIS)

    Capetandes, A.; Gerritsen, M.E.

    1986-01-01

    Salient clinical features of diabetic retinopathy include capillary microaneurysm and neovascularization, which progress with the severity of the disease. It has been suggested that exposure of the retinal vascular cells to high glucose concentrations may play a causative role in the retinopathy. In the present study, the effects of variant media glucose concentrations on BRE cell growth were determined. Normal growth curves were obtained with glucose concentrations of 100, 450 and 600 mg%, but the replication rate was decreased with 600 mg%. To determine if elevated glucose concentrations also altered DNA synthesis, BRE cells cultivated with 100 and 600 mg% glucose demonstrated increased thymidine uptake and total DNA content compared to the 100 mg% group. Furthermore, vacuolation and increased cell diameter occurred in BRE cells cultivated 600 mg% compared to 100 mg% glucose. In conclusion, increases in media glucose concentrations result in a decreased cellular replication rate, increased DNA synthesis and increased cell diameter during the log phase of growth

  11. Anti-angiogenic mechanism of cordycepin on rhesus macaque choroid-retinal endothelial cell line cultured in high glucose condition

    Directory of Open Access Journals (Sweden)

    Xiao-Li Zhu*

    2016-07-01

    Full Text Available AIM: To investigate the angiogenesis effect and protective mechanism of cordycepin on rhesus macaque choroid-retinal endothelial(RF/6Acell line cultured in high glucose condition. METHODS: Cultured RF/6A cells were divided into normal control group, high glucose group and high glucose(HG+ different concentration cordycepin groups(HG+10μg/mL group, HG+50μg/mL group, HG+100μg/mL group. The cell proliferation was assessed using cholecystokinin octapeptide dye after treated for 48h. The cell migration was investigated by a Transwell assay. The tube formation was measured on Matrigel. Furthermore, the impact of cordycepin on high glucose-induced activation of VEGF and VEGF receptor 2(VEGFR-2was tested by Western blot analysis. RESULTS: Compared with normal control group, cell viability markedly increased in high glucose group(PPPPPPvs normal control group, oppositely gradually decreased with the increase of cordycepin concentrations, and had a statistically significant difference vs high glucose group(PCONCLUSION: Cordycepin can suppress the proliferation, migration and tubu formation of RF/6A in high glucose condition, might via inhibiting expression of VEGF and VEGFR-2.

  12. The MTT assays of bovine retinal pericytes and human microvascular endothelial cells on DLC and Si-DLC-coated TCPS.

    Science.gov (United States)

    Okpalugo, T I T; McKenna, E; Magee, A C; McLaughlin, J; Brown, N M D

    2004-11-01

    MTT (Tetrazolium)-assay suggests that diamond-like carbon (DLC) and silicon-doped DLC (Si-DLC) films obtained under appropriate deposition parameters are not toxic to bovine retinal pericytes, and human microvascular endothelial cells (HMEC). The observed frequency distributions of the optical density (OD) values indicative of cell viability are near Gaussian-normal distribution. One-way ANOVA indicates that at 0.05 levels the population means are not significantly different for the coated and control samples. The observed OD values depend on the cell line (cell growth/metabolic rate), possibly cell cycle stage, the deposition parameters-bias voltage, ion energy, pressure, argon precleaning, and the dopant. For colored thin films like DLC with room temperature photoconductivity and photoelectric effects, it is important to account for the OD contribution from the coating itself. MTT assay, not surprisingly, seems not to be highly sensitive to interfacial cellular interaction resulting from the change in the film's nanostructure, because the tetrazolium metabolism is mainly intracellular and not interfacial. The thin films were synthesized by 13.56 MHz RF-PECVD using argon and acetylene as source gases, with tetramethylsilane (TMS) vapor introduced for silicon doping. This study could be relevant to biomedical application of the films in the eye, peri-vascular, vascular compartments, and for cell-tissue engineering. (c) 2004 Wiley Periodicals, Inc.

  13. Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

    DEFF Research Database (Denmark)

    Castillo, M B; Berchtold, M W; Rülicke, T

    1997-01-01

    To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical...... methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent...... vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased...

  14. Receptor-mediated endocytosis and intracellular trafficking of insulin and low-density lipoprotein by retinal vascular endothelial cells.

    Science.gov (United States)

    Stitt, A W; Anderson, H R; Gardiner, T A; Bailie, J R; Archer, D B

    1994-08-01

    The authors investigated the receptor-mediated endocytosis (RME) and intracellular trafficking of insulin and low-density lipoprotein (LDL) in cultured retinal vascular endothelial cells (RVECs). Low-density lipoprotein and insulin were conjugated to 10 nm colloidal gold, and these ligands were added to cultured bovine RVECs for 20 minutes at 4 degrees C. The cultures were then warmed to 37 degrees C and fixed after incubation times between 30 seconds and 1 hour. Control cells were incubated with unconjugated gold colloid at times and concentrations similar to those of the ligands. Additional control cells were exposed to several concentrations of anti-insulin receptor antibody or a saturating solution of unconjugated insulin before incubation with gold insulin. Using transmission electron microscopy, insulin gold and LDL gold were both observed at various stages of RME. Insulin-gold particles were first seen to bind to the apical plasma membrane (PM) before clustering in clathrin-coated pits and internalization in coated vesicles. Gold was later visualized in uncoated cytoplasmic vesicles, corresponding to early endosomes and multivesicular bodies (MVBs) or late endosomes. In several instances, localized regions of the limiting membrane of the MVBs appeared coated, a feature of endosomal membranes not previously described. After RME at the apical PM and passage through the endosomal system, the greater part of both insulin- and LDL-gold conjugates was seen to accumulate in large lysosome-like compartments. However, a small but significant proportion of the internalized ligands was transcytosed and released as discrete membrane-associated quanta at the basal cell surface. The uptake of LDL gold was greatly increased in highly vacuolated, late-passage RVECs. In controls, anti-insulin receptor antibody and excess unconjugated insulin caused up to 89% inhibition in gold-insulin binding and internalization. These results illustrate the internalization and intracellular

  15. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas.

    Science.gov (United States)

    Chintalapudi, Sumana R; Djenderedjian, Levon; Stiemke, Andrew B; Steinle, Jena J; Jablonski, Monica M; Morales-Tirado, Vanessa M

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2(hi)CD48(neg)CD15(neg)CD57(neg) surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

  16. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    Science.gov (United States)

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases. PMID:27242509

  17. Kaempferol targets estrogen-related receptor α and suppresses the angiogenesis of human retinal endothelial cells under high glucose conditions.

    Science.gov (United States)

    Wu, Yan; Zhang, Qinmei; Zhang, Rui

    2017-12-01

    Diabetic retinopathy (DR) is the most common complication of diabetes and a major cause of new-onset blindness in the developed world. The present study aimed to examine the effect of kaempferol on high glucose-induced human retinal endothelial cells (HRECs) in vitro . The expression levels of various mRNAs and proteins were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, respectively. The target of kaempferol was determined using a luciferase reporter assay. In addition, HREC proliferation, migration and cell sprouting were determined using Cell Counting kit-8, wound scratch and tube formation assays, respectively. RT-qPCR and western blotting results showed that treatment with 30 mM glucose for 12, 24 and 48 h increased the expression level of estrogen-related receptor α (ERRα) mRNA and protein. The luciferase reporter assay demonstrated that kaempferol inhibited ERRα activity in HRECs. Compared with 5 mM normal glucose treatment, high (30 mM) glucose significantly promoted the proliferation, migration and tube formation of HRECs, which was antagonized by 10 and 30 µM kaempferol in a dose-dependent manner. Treatment with 30 mM glucose also increased the expression of vascular endothelial growth factor (VEGF) mRNA and protein, and the expression levels of VEGF mRNA and protein were suppressed by kaempferol (10 and 30 µM). Kaempferol (30 µM) treatment also increased the expression levels of thrombospondin 1 (TSP-1) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS-1) mRNA; however, TSP-1 and ADAMTS-1 levels did not differ between high glucose and normal (5 mM) glucose conditions. The results of this study suggest that kaempferol targets ERRα and suppresses the angiogenesis of HRECs under high glucose conditions. Kaempferol may be a potential drug for use in controlling the progression of DR; however, in vivo studies are required to evaluate its efficacy and safety.

  18. Therapeutic Effect of Novel Single-Stranded RNAi Agent Targeting Periostin in Eyes with Retinal Neovascularization

    Directory of Open Access Journals (Sweden)

    Takahito Nakama

    2017-03-01

    Full Text Available Retinal neovascularization (NV due to retinal ischemia remains one of the principal causes of vision impairment in patients with ischemic retinal diseases. We recently reported that periostin (POSTN may play a role in the development of preretinal fibrovascular membranes, but its role in retinal NV has not been determined. The purpose of this study was to examine the expression of POSTN in the ischemic retinas of a mouse model of oxygen-induced retinal NV. We also studied the function of POSTN on retinal NV using Postn KO mice and human retinal endothelial cells (HRECs in culture. In addition, we used a novel RNAi agent, NK0144, which targets POSTN to determine its effect on the development of retinal NV. Our results showed that the expression of POSTN was increased in the vascular endothelial cells, pericytes, and M2 macrophages in ischemic retinas. POSTN promoted the ischemia-induced retinal NV by Akt phosphorylation through integrin αvβ3. NK0144 had a greater inhibitory effect than canonical double-stranded siRNA on preretinal pathological NV in vivo and in vitro. These findings suggest a causal relationship between POSTN and retinal NV, and indicate a potential therapeutic role of intravitreal injection of NK0144 for retinal neovascular diseases.

  19. Sulodexide prevents activation of the PLA2/COX-2/VEGF inflammatory pathway in human retinal endothelial cells by blocking the effect of AGE/RAGE.

    Science.gov (United States)

    Giurdanella, Giovanni; Lazzara, Francesca; Caporarello, Nunzia; Lupo, Gabriella; Anfuso, Carmelina Daniela; Eandi, Chiara M; Leggio, Gian Marco; Drago, Filippo; Bucolo, Claudio; Salomone, Salvatore

    2017-10-15

    Diabetic retinopathy is characterized by the breakdown of endothelial blood-retinal barrier. We tested the hypothesis that sulodexide (SDX), a highly purified glycosaminoglycan composed of 80% iduronylglycosaminoglycan sulfate and 20% dermatan sulfate, protects human retinal endothelial cells (HREC) from high glucose (HG)-induced damage, through the suppression of inflammatory ERK/cPLA2/COX-2/PGE 2 pathway, by blocking the effect of advanced glycation end-products (AGEs). HREC were treated with HG (25mM) or AGEs (glycated-BSA, 2mg/ml) for 48h, with or without SDX (60μg/ml) or aflibercept (AFL, 40μg/ml), a VEGF-trap. SDX protected HREC from HG-induced damage (MTT and LDH release) and preserved their blood-retinal barrier-like properties (Trans Endothelial Electrical Resistance and junction proteins, claudin-5, VE-cadherin and occludin, immunofluorescence and immunoblot) as well as their angiogenic potential (Tube Formation Assay). Both HG and AGEs increased phosphoERK and phospho-cPLA 2 , an effect counteracted by SDX and, less efficiently, by AFL. Both HG and exogenous VEGF (80ng/ml) increased PGE 2 release, an effect partially reverted by SDX for HG and by AFL for VEGF. Analysis of NFκB activity revealed that HG increased the abundance of p65 in the nuclear fraction (nuclear translocation), an effect entirely reverted by SDX, but only partially by AFL. SDX, AFL and SDX+AFL protected HREC even when added 24h after HG. These data show that SDX protects HREC from HG damage and suggest that it counteracts the activation of ERK/cPLA2/COX-2/PGE 2 pathway by reducing AGE-related signaling and downstream NFκB activity. This mechanism, partially distinct from VEGF blockade, may contribute to the therapeutic effect of SDX. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Differential expression and role of hyperglycemia induced oxidative stress in epigenetic regulation of β1, β2 and β3-adrenergic receptors in retinal endothelial cells

    Science.gov (United States)

    2014-01-01

    Background Aberrant epigenetic profiles are concomitant with a spectrum of developmental defects and diseases. Role of methylation is an increasingly accepted factor in the pathophysiology of diabetes and its associated complications. This study aims to examine the correlation between oxidative stress and methylation of β1, β2 and β3-adrenergic receptors and to analyze the differential variability in the expression of these genes under hyperglycemic conditions. Methods Human retinal endothelial cells were cultured in CSC complete medium in normal (5 mM) or high (25 mM) glucose to mimic a diabetic condition. Reverse transcription PCR and Western Blotting were performed to examine the expression of β1, β2 and β3-adrenergic receptors. For detections, immunocytochemistry was used. Bisulfite sequencing method was used for promoter methylation analysis. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to measure reactive oxygen species (ROS) production in the cells. Results β1 and β3-adrenergic receptors were expressed in retinal endothelial cells while β2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on β1 and β2-adrenergic receptors methylation and expression however β3-adrenergic receptors showed a significantly higher expression (p adrenergic receptors methylation with no significant effect on β1 and β2-adrenergic receptors. β2-adrenergic receptor was hypermethylated with halted expression. Conclusion Our study demonstrates that β1 and β3-adrenergic receptors expressed in human retinal endothelial cells. Oxidative stress and apoptosis are inversely proportional to the extent of promoter methylation, suggesting that methylation loss might be due to oxidative stress-induced DNA damage. PMID:24885710

  1. Advanced multiphoton methods for in vitro and in vivo functional imaging of mouse retinal neurons (Conference Presentation)

    Science.gov (United States)

    Cohen, Noam; Schejter, Adi; Farah, Nairouz; Shoham, Shy

    2016-03-01

    Studying the responses of retinal ganglion cell (RGC) populations has major significance in vision research. Multiphoton imaging of optogenetic probes has recently become the leading approach for visualizing neural populations and has specific advantages for imaging retinal activity during visual stimulation, because it leads to reduced direct photoreceptor excitation. However, multiphoton retinal activity imaging is not straightforward: point-by-point scanning leads to repeated neural excitation while optical access through the rodent eye in vivo has proven highly challenging. Here, we present two enabling optical designs for multiphoton imaging of responses to visual stimuli in mouse retinas expressing calcium indicators. First, we present an imaging solution based on Scanning Line Temporal Focusing (SLITE) for rapidly imaging neuronal activity in vitro. In this design, we scan a temporally focused line rather than a point, increasing the scan speed and reducing the impact of repeated excitation, while maintaining high optical sectioning. Second, we present the first in vivo demonstration of two-photon imaging of RGC activity in the mouse retina. To obtain these cellular resolution recordings we integrated an illumination path into a correction-free imaging system designed using an optical model of the mouse eye. This system can image at multiple depths using an electronically tunable lens integrated into its optical path. The new optical designs presented here overcome a number of outstanding obstacles, allowing the study of rapid calcium- and potentially even voltage-indicator signals both in vitro and in vivo, thereby bringing us a step closer toward distributed monitoring of action potentials.

  2. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Pourgholami, Mohammad H., E-mail: mh.pourgholami@unsw.edu.au [University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney (Australia); Khachigian, Levon M.; Fahmy, Roger G. [Centre for Vascular Research, The University of New South Wales, Department of Haematology, The Prince of Wales Hospital, Sydney (Australia); Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson [University of New South Wales, Department of Surgery, St George Hospital (SESIAHS), Sydney (Australia)

    2010-07-09

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  3. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis

    International Nuclear Information System (INIS)

    Pourgholami, Mohammad H.; Khachigian, Levon M.; Fahmy, Roger G.; Badar, Samina; Wang, Lisa; Chu, Stephanie Wai Ling; Morris, David Lawson

    2010-01-01

    The angiogenic process begins with the cell proliferation and migration into the primary vascular network, and leads to vascularization of previously avascular tissues and organs as well to growth and remodeling of the initially homogeneous capillary plexus to form a new microcirculation. Additionally, an increase in microvascular permeability is a crucial step in angiogenesis. Vascular endothelial growth factor (VEGF) plays a central role in angiogenesis. We have previously reported that albendazole suppresses VEGF levels and inhibits malignant ascites formation, suggesting a possible effect on angiogenesis. This study was therefore designed to investigate the antiangiogenic effect of albendazole in non-cancerous models of angiogenesis. In vitro, treatment of human umbilical vein endothelial cells (HUVECs) with albendazole led to inhibition of tube formation, migration, permeability and down-regulation of the VEGF type 2 receptor (VEGFR-2). In vivo albendazole profoundly inhibited hyperoxia-induced retinal angiogenesis in mice. These results provide new insights into the antiangiogenic effects of albendazole.

  4. Effect of surface charge of immortalized mouse cerebral endothelial cell monolayer on transport of charged solutes.

    Science.gov (United States)

    Yuan, Wei; Li, Guanglei; Gil, Eun Seok; Lowe, Tao Lu; Fu, Bingmei M

    2010-04-01

    Charge carried by the surface glycocalyx layer (SGL) of the cerebral endothelium has been shown to significantly modulate the permeability of the blood-brain barrier (BBB) to charged solutes in vivo. The cultured monolayer of bEnd3, an immortalized mouse cerebral endothelial cell line, is becoming a popular in vitro BBB model due to its easy growth and maintenance of many BBB characteristics over repeated passages. To test whether the SGL of bEnd3 monolayer carries similar charge as that in the intact BBB and quantify this charge, which can be characterized by the SGL thickness (L(f)) and charge density (C(mf)), we measured the solute permeability of bEnd3 monolayer to neutral solutes and to solutes with similar size but opposite charges: negatively charged alpha-lactalbumin (-11) and positively charged ribonuclease (+3). Combining the measured permeability data with a transport model across the cell monolayer, we predicted the L(f) and the C(mf) of bEnd3 monolayer, which is approximately 160 nm and approximately 25 mEq/L, respectively. We also investigated whether orosomucoid, a plasma glycoprotein modulating the charge of the intact BBB, alters the charge of bEnd3 monolayer. We found that 1 mg/mL orosomucoid would increase SGL charge density of bEnd3 monolayer to approximately 2-fold of its control value.

  5. Vascular endothelial growth factors enhance the permeability of the mouse blood-brain barrier.

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    Shize Jiang

    Full Text Available The blood-brain barrier (BBB impedes entry of many drugs into the brain, limiting clinical efficacy. A safe and efficient method for reversibly increasing BBB permeability would greatly facilitate central nervous system (CNS drug delivery and expand the range of possible therapeutics to include water soluble compounds, proteins, nucleotides, and other large molecules. We examined the effect of vascular endothelial growth factor (VEGF on BBB permeability in Kunming (KM mice. Human VEGF165 was administered to treatment groups at two concentrations (1.6 or 3.0 µg/mouse, while controls received equal-volume saline. Changes in BBB permeability were measured by parenchymal accumulation of the contrast agent Gd-DTPA as assessed by 7 T magnetic resonance imaging (MRI. Mice were then injected with Evans blue, sacrificed 0.5 h later, and perfused transcardially. Brains were removed, fixed, and sectioned for histological study. Both VEGF groups exhibited a significantly greater signal intensity from the cerebral cortex and basal ganglia than controls (P<0.001. Evans blue fluorescence intensity was higher in the parenchyma and lower in the cerebrovasculature of VEGF-treated animals compared to controls. No significant brain edema was observed by diffusion weighted MRI (DWI or histological staining. Exogenous application of VEGF can increase the permeability of the BBB without causing brain edema. Pretreatment with VEGF may be a feasible method to facilitate drug delivery into the CNS.

  6. Coenzyme Q10 instilled as eye drops on the cornea reaches the retina and protects retinal layers from apoptosis in a mouse model of kainate-induced retinal damage.

    Science.gov (United States)

    Lulli, Matteo; Witort, Ewa; Papucci, Laura; Torre, Eugenio; Schipani, Christian; Bergamini, Christian; Dal Monte, Massimo; Capaccioli, Sergio

    2012-12-17

    To evaluate if coenzyme Q10 (CoQ10) can protect retinal ganglion cells (RGCs) from apoptosis and, when instilled as eye drops on the cornea, if it can reach the retina and exert its antiapoptotic activity in this area in a mouse model of kainate (KA)-induced retinal damage. Rat primary or cultured RGCs were subjected to glutamate (50 μM) or chemical hypoxia (Antimycin A, 200 μM) or serum withdrawal (FBS, 0.5%) in the presence or absence of CoQ10 (10 μM). Cell viability was evaluated by light microscopy and fluorescence-activated cell sorting analyses. Apoptosis was evaluated by caspase 3/7 activity and mitochondrion depolarization tetramethylrhodamine ethyl ester analysis. CoQ10 transfer to the retina following its instillation as eye drops on the cornea was quantified by HPLC. Retinal protection by CoQ10 (10 μM) eye drops instilled on the cornea was then evaluated in a mouse model of KA-induced excitotoxic retinal cell apoptosis by cleaved caspase 3 immunohistofluorescence, caspase 3/7 activity assays, and quantification of inhibition of RGC loss. CoQ10 significantly increased viable cells by preventing RGC apoptosis. Furthermore, when topically applied as eye drops to the cornea, it reached the retina, thus substantially increasing local CoQ10 concentration and protecting retinal layers from apoptosis. The ability of CoQ10 eye drops to protect retinal cells from apoptosis in the mouse model of KA-induced retinal damage suggests that topical CoQ10 may be evaluated in designing therapies for treating apoptosis-driven retinopathies.

  7. AFM-based detection of glycocalyx degradation and endothelial stiffening in the db/db mouse model of diabetes.

    Science.gov (United States)

    Targosz-Korecka, Marta; Jaglarz, Magdalena; Malek-Zietek, Katarzyna E; Gregorius, Aleksandra; Zakrzewska, Agnieszka; Sitek, Barbara; Rajfur, Zenon; Chlopicki, Stefan; Szymonski, Marek

    2017-11-21

    Degradation of the glycocalyx and stiffening of endothelium are important pathophysiological components of endothelial dysfunction. However, to our knowledge, these events have not been investigated in tandem in experimental diabetes. Here, the mechanical properties of the glycocalyx and endothelium in ex vivo mouse aorta were determined simultaneously in indentation experiments with an atomic force microscope (AFM) for diabetic db/db and control db/+ mice at ages of 11-19 weeks. To analyze highly heterogeneous aorta samples, we developed a tailored classification procedure of indentation data based on a bi-layer brush model supplemented with Hertz model for quantification of nanomechanics of endothelial regions with and without the glycocalyx surface. In db/db mice, marked endothelial stiffening and reduced glycocalyx coverage were present already in 11-week-old mice and persisted in older animals. In contrast, reduction of the effective glycocalyx length was progressive and was most pronounced in 19-week-old db/db mice. The reduction of the glycocalyx length correlated with an increasing level of glycated haemoglobin and decreased endothelial NO production. In conclusion, AFM nanoindentation analysis revealed that stiffening of endothelial cells and diminished glycocalyx coverage occurred in early diabetes and were followed by the reduction of the glycocalyx length that correlated with diabetes progression.

  8. Posttranslational inactivation of endothelial nitric oxide synthase in the transgenic sickle cell mouse penis

    Science.gov (United States)

    Musicki, Biljana; Champion, Hunter C.; Hsu, Lewis L.; Bivalacqua, Trinity J.; Burnett, Arthur L.

    2017-01-01

    INTRODUCTION Sickle cell disease (SCD)-associated priapism is characterized by endothelial nitric oxide synthase (eNOS) dysfunction in the penis. However, the mechanism of decreased eNOS function/activation in the penis in association with SCD is not known. AIMS Our hypothesis in the present study was that eNOS is functionally inactivated in the SCD penis in association with impairments in eNOS posttranslational phosphorylation and the enzyme’s interactions with its regulatory proteins. METHODS Sickle cell transgenic (sickle) mice were used as an animal model of SCD. Wild type (WT) mice served as controls. Penes were excised at baseline for molecular studies. eNOS phosphorylation on Ser-1177 (positive regulatory site) and Thr-495 (negative regulatory site), total eNOS, and phosphorylated AKT (upstream mediator of eNOS phosphorylation on Ser-1177) expressions, and eNOS interactions with heat shock protein 90 (HSP90) and caveolin-1 were measured by Western blot. Constitutive NOS catalytic activity was measured by conversion of L-[14C]arginine-to-L-[14C]citrulline in the presence of calcium. MAIN OUTCOME MEASURES Molecular mechanisms of eNOS dysfunction in the sickle mouse penis. RESULTS eNOS phosphorylated on Ser-1177, an active portion of eNOS, was decreased in the sickle mouse penis compared to WT penis. eNOS interaction with its positive protein regulator HSP90, but not with its negative protein regulator caveolin-1, and phosphorylated AKT expression, as well as constitutive NOS activity, were also decreased in the sickle mouse penis compared to WT penis. eNOS phosphorylated on Thr-495, total eNOS, HSP90, and caveolin-1 protein expressions in the penis were not affected by SCD. CONCLUSION These findings provide a molecular basis for chronically reduced eNOS function in the penis by SCD, which involves decreased eNOS phosphorylation on Ser-1177 and decreased eNOS-HSP90 interaction. PMID:21143412

  9. Pericytes derived from adipose-derived stem cells protect against retinal vasculopathy.

    Directory of Open Access Journals (Sweden)

    Thomas A Mendel

    Full Text Available Retinal vasculopathies, including diabetic retinopathy (DR, threaten the vision of over 100 million people. Retinal pericytes are critical for microvascular control, supporting retinal endothelial cells via direct contact and paracrine mechanisms. With pericyte death or loss, endothelial dysfunction ensues, resulting in hypoxic insult, pathologic angiogenesis, and ultimately blindness. Adipose-derived stem cells (ASCs differentiate into pericytes, suggesting they may be useful as a protective and regenerative cellular therapy for retinal vascular disease. In this study, we examine the ability of ASCs to differentiate into pericytes that can stabilize retinal vessels in multiple pre-clinical models of retinal vasculopathy.We found that ASCs express pericyte-specific markers in vitro. When injected intravitreally into the murine eye subjected to oxygen-induced retinopathy (OIR, ASCs were capable of migrating to and integrating with the retinal vasculature. Integrated ASCs maintained marker expression and pericyte-like morphology in vivo for at least 2 months. ASCs injected after OIR vessel destabilization and ablation enhanced vessel regrowth (16% reduction in avascular area. ASCs injected intravitreally before OIR vessel destabilization prevented retinal capillary dropout (53% reduction. Treatment of ASCs with transforming growth factor beta (TGF-β1 enhanced hASC pericyte function, in a manner similar to native retinal pericytes, with increased marker expression of smooth muscle actin, cellular contractility, endothelial stabilization, and microvascular protection in OIR. Finally, injected ASCs prevented capillary loss in the diabetic retinopathic Akimba mouse (79% reduction 2 months after injection.ASC-derived pericytes can integrate with retinal vasculature, adopting both pericyte morphology and marker expression, and provide functional vascular protection in multiple murine models of retinal vasculopathy. The pericyte phenotype demonstrated

  10. Spontaneous oscillatory rhythms in the degenerating mouse retina modulate retinal ganglion cell responses to electrical stimulation

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    Yong Sook eGoo

    2016-01-01

    Full Text Available Characterization of the electrical activity of the retina in the animal models of retinal degeneration has been carried out in part to understand the progression of retinal degenerative diseases like age-related macular degeneration (AMD and retinitis pigmentosa (RP, but also to determine optimum stimulus paradigms for use with retinal prosthetic devices. The models most studied in this regard have been the two lines of mice deficient in the β-subunit of phosphodiesterase (rd1 and rd10 mice, where the degenerating retinas exhibit characteristic spontaneous hyperactivity and oscillatory local field potentials (LFPs. Additionally, there is a robust ~10 Hz rhythmic burst of retinal ganglion cell (RGC spikes on the trough of the oscillatory LFP. In rd1 mice, the rhythmic burst of RGC spikes is always phase-locked with the oscillatory LFP and this phase-locking property is preserved regardless of postnatal ages. However, in rd10 mice, the frequency of the oscillatory rhythm changes according to postnatal age, suggesting that this rhythm might be a marker of the stage of degeneration. Furthermore when a biphasic current stimulus is applied to rd10 mice degenerate retina, distinct RGC response patterns that correlate with the stage of degeneration emerge. This review also considers the significance of these response properties.

  11. Multipronged approach to identify and validate a novel upstream regulator of Sncg in mouse retinal ganglion cells.

    Science.gov (United States)

    Chintalapudi, Sumana R; Morales-Tirado, Vanessa M; Williams, Robert W; Jablonski, Monica M

    2016-02-01

    Loss of retinal ganglion cells (RGCs) is one of the hallmarks of retinal neurodegenerative diseases, glaucoma being one of the most common. Mechanistic studies on RGCs are hindered by the lack of sufficient primary cells and consensus regarding their signature markers. Recently, γ-synuclein (SNCG) has been shown to be highly expressed in the somas and axons of RGCs. In various mouse models of glaucoma, downregulation of Sncg gene expression correlates with RGC loss. To investigate the role of Sncg in RGCs, we used a novel systems genetics approach to identify a gene that modulates Sncg expression, followed by confirmatory studies in both healthy and diseased retinae. We found that chromosome 1 harbors an expression quantitative trait locus that modulates Sncg expression in the mouse retina, and identified the prefoldin-2 (PFDN2) gene as the candidate upstream modulator of Sncg expression. Our immunohistochemical analyses revealed similar expression patterns in both mouse and human healthy retinae, with PFDN2 colocalizing with SNCG in RGCs and their axons. In contrast, in retinae from glaucoma subjects, SNCG levels were significantly reduced, although PFDN2 levels were maintained. Using a novel flow cytometry-based RGC isolation method, we obtained viable populations of murine RGCs. Knocking down Pfdn2 expression in primary murine RGCs significantly reduced Sncg expression, confirming that Pfdn2 regulates Sncg expression in murine RGCs. Gene Ontology analysis indicated shared mitochondrial function associated with Sncg and Pfdn2. These data solidify the relationship between Sncg and Pfdn2 in RGCs, and provide a novel mechanism for maintaining RGC health. © 2015 FEBS.

  12. The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases.

    Science.gov (United States)

    Rueda, Elda M; Johnson, Jerry E; Giddabasappa, Anand; Swaroop, Anand; Brooks, Matthew J; Sigel, Irena; Chaney, Shawnta Y; Fox, Donald A

    2016-01-01

    The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The

  13. Effects of the PPARγ agonist troglitazone on endothelial cells in vivo and in vitro: Differences between human and mouse

    International Nuclear Information System (INIS)

    Kakiuchi-Kiyota, Satoko; Vetro, Joseph A.; Suzuki, Shugo; Varney, Michelle L.; Han, Huai-Yun; Nascimento, Merielen; Pennington, Karen L.; Arnold, Lora L.; Singh, Rakesh K.; Cohen, Samuel M.

    2009-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) agonists and PPARγ/α dual agonists have been or are being developed for clinical use in the treatment of type 2 diabetes mellitus and hyperlipidemias. A common tumor finding in rodent carcinogenicity studies for these agonists is hemangioma/hemangiosarcoma in mice but not in rats. We hypothesized that increased endothelial cell proliferation may be involved in the mechanism of PPAR agonist-induced vascular tumors in mice, and we investigated the effects on endothelial cells utilizing troglitazone, the first clinically used PPARγ agonist, in vivo and in vitro. Troglitazone (400 and 800 mg/kg/day) induced hemangiosarcomas in mice in a 2-year bioassay. We showed that troglitazone increased endothelial cell proliferation in brown and white adipose tissue and liver in mice at sarcomagenic doses after 4 weeks of treatment. Troglitazone was cytotoxic both to human dermal microvascular endothelial cells (HMEC1) and mouse mammary fat pad microvascular endothelial cells (MFP MVEC) at high concentrations. However, MFP MVEC were more resistant to the cytotoxic effects of troglitazone based on the much lower LC 50 in HMEC1 (17.4 μM) compared to MFP MVEC (92.2 μM). Troglitazone increased the proliferation and survival of MFP MVEC but not HMEC1 in growth factor reduced conditions. Our data demonstrate that troglitazone may induce hemangiosarcomas in mice, at least in part, through enhancement of survival and proliferation of microvascular endothelial cells. Such an effect does not occur with human cells, suggesting that human may react differently to exposure to PPAR agonists compared with mice.

  14. ROCK-1 mediates diabetes-induced retinal pigment epithelial and endothelial cell blebbing: Contribution to diabetic retinopathy.

    Science.gov (United States)

    Rothschild, Pierre-Raphaël; Salah, Sawsen; Berdugo, Marianne; Gélizé, Emmanuelle; Delaunay, Kimberley; Naud, Marie-Christine; Klein, Christophe; Moulin, Alexandre; Savoldelli, Michèle; Bergin, Ciara; Jeanny, Jean-Claude; Jonet, Laurent; Arsenijevic, Yvan; Behar-Cohen, Francine; Crisanti, Patricia

    2017-08-18

    In diabetic retinopathy, the exact mechanisms leading to retinal capillary closure and to retinal barriers breakdown remain imperfectly understood. Rho-associated kinase (ROCK), an effector of the small GTPase Rho, involved in cytoskeleton dynamic regulation and cell polarity is activated by hyperglycemia. In one year-old Goto Kakizaki (GK) type 2 diabetic rats retina, ROCK-1 activation was assessed by its cellular distribution and by phosphorylation of its substrates, MYPT1 and MLC. In both GK rat and in human type 2 diabetic retinas, ROCK-1 is activated and associated with non-apoptotic membrane blebbing in retinal vessels and in retinal pigment epithelium (RPE) that respectively form the inner and the outer barriers. Activation of ROCK-1 induces focal vascular constrictions, endoluminal blebbing and subsequent retinal hypoxia. In RPE cells, actin cytoskeleton remodeling and membrane blebs in RPE cells contributes to outer barrier breakdown. Intraocular injection of fasudil, significantly reduces both retinal hypoxia and RPE barrier breakdown. Diabetes-induced cell blebbing may contribute to ischemic maculopathy and represent an intervention target.

  15. Pars plana vitrectomy combined with internal limiting membrane peeling for recurrent macular edema due to branch retinal vein occlusion after antivascular endothelial growth factor treatments

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    Shirakata Y

    2016-02-01

    Full Text Available Yukari Shirakata,1 Kouki Fukuda,1 Tomoyoshi Fujita,1 Yuki Nakano,1 Hiroyuki Nomoto,2 Hidetaka Yamaji,3 Fumio Shiraga,4 Akitaka Tsujikawa1 1Department of Ophthalmology, Faculty of Medicine, Kagawa University, Miki-cho, 2Nomoto Eye Clinic, Himeji, 3Department of Ophthalmology, Shirai Eye Hospital, Mitoyo, 4Department of Ophthalmology, Okayama University, Okayama, Japan Purpose: To evaluate the anatomic and functional outcomes of pars plana vitrectomy combined with internal limiting membrane peeling for recurrent macular edema (ME due to branch retinal vein occlusion (BRVO after intravitreal injections of antivascular endothelial growth factor (anti-VEGF agents. Methods: Twenty-four eyes of 24 patients with treatment-naive ME from BRVO were treated with intravitreal injections of anti-VEGF agents. Recurred ME was treated with pars plana vitrectomy combined with internal limiting membrane peeling. Results: After the surgery, ME was significantly reduced at 1 month (P=0.031 and the reduction increased with time (P=0.007 at the final visit. With the reduction in ME, treated eyes showed a slow improvement in visual acuity (VA. At the final visit, improvement in VA was statistically significant compared with baseline (P=0.048. The initial presence of cystoid spaces, serous retinal detachment, or subretinal hemorrhage under the fovea, as well as retinal perfusion status, showed no association with VA improvement. However, the presence of epiretinal membrane showed a significant association with the visual recovery. Although eyes without epiretinal membrane showed visual improvement (-0.10±0.32 in logarithm of the minimum angle of resolution [logMAR], eyes with epiretinal membrane showed greater visual improvement (-0.38±0.12 in logMAR, P=0.012. Conclusion: For recurrent ME due to BRVO after anti-VEGF treatment, particularly when accompanied by epiretinal membrane, pars plana vitrectomy combined with internal limiting membrane peeling might be a

  16. Diabetes Accelerates Retinal neuronal cell Death in A Mouse Model of endogenous Hyperhomocysteinemia

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    Preethi S. Ganapathy

    2009-01-01

    Full Text Available Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase ( cbs gene, demonstrate loss of neurons in the retinal ganglion cell (RGC layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs +/– mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB was induced in 3 wk old cbs +/– and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs +/– non-DB cbs +/– DB cbs +/+ ; non-DB cbs +/+ . One group of diabetic cbs +/– mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs +/– had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs +/– and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 μm retina length, respectively. Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.

  17. Diabetes Accelerates Retinal Neuronal Cell Death In A Mouse Model of Endogenous Hyperhomocysteinemia

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    Preethi S. Ganapathy

    2009-07-01

    Full Text Available Hyperhomocysteinemia has been implicated in visual dysfunction. We reported recently that mice with endogenous hyperhomocysteinemia, due to mutation of the cystathionine-β-synthase (cbs gene, demonstrate loss of neurons in the retinal ganglion cell (RGC layer and other retinal layers as homocysteine levels increase. Some clinical studies implicate hyperhomocysteinemia in the pathogenesis of diabetic retinopathy, which is also characterized by RGC loss. The present study used cbs+/- mice to determine whether modest elevation of plasma homocysteine, in the presence of diabetes, accelerates neuronal cell loss. Diabetes (DB was induced in 3 wk old cbs+/- and wildtype mice using streptozotocin; four groups of mice were studied: DB cbs+/-; non-DB cbs+/-; DB cbs+/+; non-DB cbs+/+. One group of diabetic cbs+/- mice was maintained on a high methionine diet (HMD, 0.5% methionine drinking water to increase plasma homocysteine slightly. Eyes were harvested at 5, 10 and 15 weeks post-onset of diabetes; retinal cryosections were examined by light microscopy and subjected to systematic morphometric analysis. Diabetic cbs+/- had significantly fewer RGCs at 5 weeks compared to age-matched, non-diabetic cbs+/- and wildtype controls (10.0 ± 0.5 versus 14.9 ± 0.5 and 15.8 ± 0.6 cells/100 µm retina length, respectively. Significant differences in retinas of DB/high homocysteine versus controls were obtained 15 wks post-onset of diabetes including fewer RGCS and decreased thickness of inner nuclear and plexiform layers. Moderate increases in plasma homocysteine coupled with diabetes cause a more dramatic alteration of retinal phenotype than elevated homocysteine or diabetes alone and suggest that diabetes accelerates the retinal neuronal death in hyperhomocysteinemic mice.

  18. Mechanism of Retinal Pigment Epithelium Tear Formation Following Intravitreal Anti–Vascular Endothelial Growth Factor Therapy Revealed by Spectral-Domain Optical Coherence Tomography

    DEFF Research Database (Denmark)

    Nagiel, Aaron; Freund, K Bailey; Spaide, Richard F

    2013-01-01

    to the retracted RPE. In all eyes, the RPE ruptured along a segment of bare RPE not in contact with the CNV or Bruch membrane. CONCLUSIONS: Eyes with vascularized PEDs secondary to AMD may show specific OCT findings that increase the risk for RPE tear following intravitreal anti-VEGF injection. Rapid involution......PURPOSE: To demonstrate the mechanism by which retinal pigment epithelium (RPE) tears occur in eyes with neovascular age-related macular degeneration (AMD) treated with intravitreal anti-vascular endothelial growth factor (VEGF) agents using spectral-domain optical coherence tomography (OCT......). DESIGN: Retrospective observational case series. METHODS: OCT images of 8 eyes that developed RPE tears following the administration of intravitreal anti-VEGF agents for neovascular AMD were evaluated. Pretear and posttear images were compared in order to elucidate the mechanism by which RPE tears occur...

  19. Interferon-gamma (IFN-γ-mediated retinal ganglion cell death in human tyrosinase T cell receptor transgenic mouse.

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    Shahid Husain

    Full Text Available We have recently demonstrated the characterization of human tyrosinase TCR bearing h3T-A2 transgenic mouse model, which exhibits spontaneous autoimmune vitiligo and retinal dysfunction. The purpose of current study was to determine the role of T cells and IFN-γ in retina dysfunction and retinal ganglion cell (RGC death using this model. RGC function was measured by pattern electroretinograms (ERGs in response to contrast reversal of patterned visual stimuli. RGCs were visualized by fluorogold retrograde-labeling. Expression of CD3, IFN-γ, GFAP, and caspases was measured by immunohistochemistry and Western blotting. All functional and structural changes were measured in 12-month-old h3T-A2 mice and compared with age-matched HLA-A2 wild-type mice. Both pattern-ERGs (42%, p = 0.03 and RGC numbers (37%, p = 0.0001 were reduced in h3T-A2 mice when compared with wild-type mice. The level of CD3 expression was increased in h3T-A2 mice (h3T-A2: 174 ± 27% vs. HLA-A2: 100%; p = 0.04. The levels of effector cytokine IFN-γ were also increased significantly in h3T-A2 mice (h3T-A2: 189 ± 11% vs. HLA-A2: 100%; p = 0.023. Both CD3 and IFN-γ immunostaining were increased in nerve fiber (NF and RGC layers of h3T-A2 mice. In addition, we have seen a robust increase in GFAP staining in h3T-A2 mice (mainly localized to NF layer, which was substantially reduced in IFN-γ ((-/- knockout h3T-A2 mice. We also have seen an up-regulation of caspase-3 and -9 in h3T-A2 mice. Based on our data we conclude that h3T-A2 transgenic mice exhibit visual defects that are mostly associated with the inner retinal layers and RGC function. This novel h3T-A2 transgenic mouse model provides opportunity to understand RGC pathology and test neuroprotective strategies to rescue RGCs.

  20. Pattern of retinal morphological and functional decay in a light-inducible, rhodopsin mutant mouse.

    Science.gov (United States)

    Gargini, Claudia; Novelli, Elena; Piano, Ilaria; Biagioni, Martina; Strettoi, Enrica

    2017-07-18

    Hallmarks of Retinitis Pigmentosa (RP), a family of genetic diseases, are a typical rod-cone-degeneration with initial night blindness and loss of peripheral vision, followed by decreased daylight sight and progressive visual acuity loss up to legal blindness. Great heterogeneity in nature and function of mutated genes, variety of mutations for each of them, variability in phenotypic appearance and transmission modality contribute to make RP a still incurable disease. Translational research relies on appropriate animal models mimicking the genetic and phenotypic diversity of the human pathology. Here, we provide a systematic, morphological and functional analysis of Rho Tvrm4 /Rho + rhodopsin mutant mice, originally described in 2010 and portraying several features of common forms of autosomal dominant RP caused by gain-of-function mutations. These mice undergo photoreceptor degeneration only when exposed briefly to strong, white light and allow controlled timing of induction of rod and cone death, which therefore can be elicited in adult animals, as observed in human RP. The option to control severity and retinal extent of the phenotype by regulating intensity and duration of the inducing light opens possibilities to exploit this model for multiple experimental purposes. Altogether, the unique features of this mutant make it an excellent resource for retinal degeneration research.

  1. Limitations of the colloidal silica method in mapping the endothelial plasma membrane proteome of the mouse heart.

    Science.gov (United States)

    Arjunan, Selvam; Reinartz, Michael; Emde, Barbara; Zanger, Klaus; Schrader, Jürgen

    2009-01-01

    The endothelial cell (EC) membrane is an important interface, which plays a crucial role in signal transduction. Our aim was to selectively purify luminal EC membrane proteins from the coronary vasculature of the isolated perfused mouse heart and analyze its composition with mass spectrometry (MS). To specifically label coronary ECs in the intact heart, the colloidal silica method was applied, which is based on the binding of positively charged colloidal silica to the surface of EC membranes. Transmission electron microscopy revealed the specific labeling of ECs of macro and microvessels. Two different methods of tissue homogenization (Teflon pestle and ultra blade) together with density centrifugation were used for membrane protein enrichment. Enrichment and purity was controlled by Western blot analysis using the EC-specific protein caveolin 1 and various intracellular marker proteins. The ultra blade method resulted in a tenfold enrichment of caveolin 1, while there was negligible contamination as judged by Western blot. However, protein yield was low and required pooling of ten hearts for MS. When enriched endothelial membrane proteins were digested with trypsin and analyzed by LC-MS, a total of 56 proteins could be identified, of which only 12 were membrane proteins. We conclude that coronary endothelial membranes can be conveniently labeled with colloidal silica. However, due to the ionic nature of interaction of colloidal silica with the EC membrane the shear rate required for cardiac homogenization resulted in a substantial loss of specificity.

  2. Intravitreal administration of HA-1077, a ROCK inhibitor, improves retinal function in a mouse model of huntington disease.

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    Mei Li

    Full Text Available Huntington disease (HD is an inherited neurodegenerative disease that affects multiple brain regions. It is caused by an expanded polyglutamine tract in huntingtin (Htt. The development of therapies for HD and other neurodegenerative diseases has been hampered by multiple factors, including the lack of clear therapeutic targets, and the cost and complexity of testing lead compounds in vivo. The R6/2 HD mouse model is widely used for pre-clinical trials because of its progressive and robust neural dysfunction, which includes retinal degeneration. Profilin-1 is a Htt binding protein that inhibits Htt aggregation. Its binding to Htt is regulated by the rho-associated kinase (ROCK, which phosphorylates profilin at Ser-137. ROCK is thus a therapeutic target in HD. The ROCK inhibitor Y-27632 reduces Htt toxicity in fly and mouse models. Here we characterized the progressive retinopathy of R6/2 mice between 6-19 weeks of age to determine an optimal treatment window. We then tested a clinically approved ROCK inhibitor, HA-1077, administered intravitreally via liposome-mediated drug delivery. HA-1077 increased photopic and flicker ERG response amplitudes in R6/2 mice, but not in wild-type littermate controls. By targeting ROCK with a new inhibitor, and testing its effects in a novel in vivo model, these results validate the in vivo efficacy of a therapeutic candidate, and establish the feasibility of using the retina as a readout for CNS function in models of neurodegenerative disease.

  3. Correspondence between visual and electrical input filters of ON and OFF mouse retinal ganglion cells

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    Sekhar, S.; Jalligampala, A.; Zrenner, E.; Rathbun, D. L.

    2017-08-01

    Objective. Over the past two decades retinal prostheses have made major strides in restoring functional vision to patients blinded by diseases such as retinitis pigmentosa. Presently, implants use single pulses to activate the retina. Though this stimulation paradigm has proved beneficial to patients, an unresolved problem is the inability to selectively stimulate the on and off visual pathways. To this end our goal was to test, using white noise, voltage-controlled, cathodic, monophasic pulse stimulation, whether different retinal ganglion cell (RGC) types in the wild type retina have different electrical input filters. This is an important precursor to addressing pathway-selective stimulation. Approach. Using full-field visual flash and electrical and visual Gaussian noise stimulation, combined with the technique of spike-triggered averaging (STA), we calculate the electrical and visual input filters for different types of RGCs (classified as on, off or on-off based on their response to the flash stimuli). Main results. Examining the STAs, we found that the spiking activity of on cells during electrical stimulation correlates with a decrease in the voltage magnitude preceding a spike, while the spiking activity of off cells correlates with an increase in the voltage preceding a spike. No electrical preference was found for on-off cells. Comparing STAs of wild type and rd10 mice revealed narrower electrical STA deflections with shorter latencies in rd10. Significance. This study is the first comparison of visual cell types and their corresponding temporal electrical input filters in the retina. The altered input filters in degenerated rd10 retinas are consistent with photoreceptor stimulation underlying visual type-specific electrical STA shapes in wild type retina. It is therefore conceivable that existing implants could target partially degenerated photoreceptors that have only lost their outer segments, but not somas, to selectively activate the on and off

  4. Photodegradation of retinal bisretinoids in mouse models and implications for macular degeneration.

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    Ueda, Keiko; Zhao, Jin; Kim, Hye Jin; Sparrow, Janet R

    2016-06-21

    Adducts of retinaldehyde (bisretinoids) form nonenzymatically in photoreceptor cells and accumulate in retinal pigment epithelial (RPE) cells as lipofuscin; these fluorophores are implicated in the pathogenesis of inherited and age-related macular degeneration (AMD). Here we demonstrate that bisretinoid photodegradation is ongoing in the eye. High-performance liquid chromatography (HPLC) analysis of eyes of dark-reared and cyclic light-reared wild-type mice, together with comparisons of pigmented versus albino mice, revealed a relationship between intraocular light and reduced levels of the bisretinoids A2E and A2-glycero-phosphoethanolamine (A2-GPE). Analysis of the bisretinoids A2E, A2-GPE, A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE), and all-trans-retinal dimer-phosphatidylethanolamine (all-trans-retinal dimer-PE) also decreases in albino Abca4(-/-) mice reared in cyclic light compared with darkness. In albino Abca4(-/-) mice receiving a diet supplemented with the antioxidant vitamin E, higher levels of RPE bisretinoid were evidenced by HPLC analysis and quantitation of fundus autofluorescence; this effect is consistent with photooxidative processes known to precede bisretinoid degradation. Amelioration of outer nuclear layer thinning indicated that vitamin E treatment protected photoreceptor cells. Conversely, in-cage exposure to short-wavelength light resulted in reduced fundus autofluorescence, decreased HPLC-quantified A2E, outer nuclear layer thinning, and increased methylglyoxal (MG)-adducted protein. MG was also released upon bisretinoid photodegradation in cells. We suggest that the lower levels of these diretinal adducts in cyclic light-reared and albino mice reflect photodegradative loss of bisretinoid. These mechanisms may underlie associations among AMD risk, oxidative mechanisms, and lifetime light exposure.

  5. Edaravone suppresses retinal ganglion cell death in a mouse model of normal tension glaucoma

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    Akaiwa, Kei; Namekata, Kazuhiko; Azuchi, Yuriko; Guo, Xiaoli; Kimura, Atsuko; Harada, Chikako; Mitamura, Yoshinori; Harada, Takayuki

    2017-01-01

    Glaucoma, one of the leading causes of irreversible blindness, is characterized by progressive degeneration of optic nerves and retinal ganglion cells (RGCs). In the mammalian retina, excitatory amino-acid carrier 1 (EAAC1) is expressed in neural cells, including RGCs. Loss of EAAC1 leads to RGC degeneration without elevated intraocular pressure (IOP) and exhibits glaucomatous pathology including glutamate neurotoxicity and oxidative stress. In the present study, we found that edaravone, a free radical scavenger that is used for treatment of acute brain infarction and amyotrophic lateral sclerosis (ALS), reduces oxidative stress and prevents RGC death and thinning of the inner retinal layer in EAAC1-deficient (KO) mice. In addition, in vivo electrophysiological analyses demonstrated that visual impairment in EAAC1 KO mice was ameliorated with edaravone treatment, clearly establishing that edaravone beneficially affects both histological and functional aspects of the glaucomatous retina. Our findings raise intriguing possibilities for the management of glaucoma by utilizing a widely prescribed drug for the treatment of acute brain infarction and ALS, edaravone, in combination with conventional treatments to lower IOP. PMID:28703795

  6. Substituting mouse transcription factor Pou4f2 with a sea urchin orthologue restores retinal ganglion cell development.

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    Mao, Chai-An; Agca, Cavit; Mocko-Strand, Julie A; Wang, Jing; Ullrich-Lüter, Esther; Pan, Ping; Wang, Steven W; Arnone, Maria Ina; Frishman, Laura J; Klein, William H

    2016-03-16

    Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures. © 2016 The Authors.

  7. Characterisation of a C1qtnf5 Ser163Arg knock-in mouse model of late-onset retinal macular degeneration.

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    Xinhua Shu

    Full Text Available A single founder mutation resulting in a Ser163Arg substitution in the C1QTNF5 gene product causes autosomal dominant late-onset retinal macular degeneration (L-ORMD in humans, which has clinical and pathological features resembling age-related macular degeneration. We generated and characterised a mouse "knock-in" model carrying the Ser163Arg mutation in the orthologous murine C1qtnf5 gene by site-directed mutagenesis and homologous recombination into mouse embryonic stem cells. Biochemical, immunological, electron microscopic, fundus autofluorescence, electroretinography and laser photocoagulation analyses were used to characterise the mouse model. Heterozygous and homozygous knock-in mice showed no significant abnormality in any of the above measures at time points up to 2 years. This result contrasts with another C1qtnf5 Ser163Arg knock-in mouse which showed most of the features of L-ORMD but differed in genetic background and targeting construct.

  8. Microglia in the mouse retina alter the structure and function of retinal pigmented epithelial cells: a potential cellular interaction relevant to AMD.

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    Wenxin Ma

    2009-11-01

    Full Text Available Age-related macular degeneration (AMD is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.In this study, we examined the effects of retinal microglia on RPE cells using 1 an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2 an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1 changes in RPE structure and distribution, 2 increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3 increased extent of in vivo choroidal neovascularization in the subretinal space.These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.

  9. Detection of DNA Double Strand Breaks by γH2AX Does Not Result in 53bp1 Recruitment in Mouse Retinal Tissues

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    Brigitte Müller

    2018-05-01

    Full Text Available Gene editing is an attractive potential treatment of inherited retinopathies. However, it often relies on endogenous DNA repair. Retinal DNA repair is incompletely characterized in humans and animal models. We investigated recruitment of the double stranded break (DSB repair complex of γH2AX and 53bp1 in both developing and mature mouse neuroretinas. We evaluated the immunofluorescent retinal expression of these proteins during development (P07-P30 in normal and retinal degeneration models, as well as in potassium bromate induced DSB repair in normal adult (3 months retinal explants. The two murine retinopathy models used had different mutations in Pde6b: the severe rd1 and the milder rd10 models. Compared to normal adult retina, we found increased numbers of γH2AX positive foci in all retinal neurons of the developing retina in both model and control retinas, as well as in wild type untreated retinal explant cultures. In contrast, the 53bp1 staining of the retina differed both in amount and character between cell types at all ages and in all model systems. There was strong pan nuclear staining in ganglion, amacrine, and horizontal cells, and cone photoreceptors, which was attenuated. Rod photoreceptors did not stain unequivocally. In all samples, 53bp1 stained foci only rarely occurred. Co-localization of 53bp1 and γH2AX staining was a very rare event (< 1% of γH2AX foci in the ONL and < 3% in the INL, suggesting the potential for alternate DSB sensing and repair proteins in the murine retina. At a minimum, murine retinal DSB repair does not appear to follow canonical pathways, and our findings suggests further investigation is warranted.

  10. Late gestational hypoxia and a postnatal high salt diet programs endothelial dysfunction and arterial stiffness in adult mouse offspring.

    Science.gov (United States)

    Walton, Sarah L; Singh, Reetu R; Tan, Tiffany; Paravicini, Tamara M; Moritz, Karen M

    2016-03-01

    Gestational hypoxia and high dietary salt intake have both been associated with impaired vascular function in adulthood. Using a mouse model of prenatal hypoxia, we examined whether a chronic high salt diet had an additive effect in promoting vascular dysfunction in offspring. Pregnant CD1 dams were placed in a hypoxic chamber (12% O2) or housed under normal conditions (21% O2) from embryonic day 14.5 until birth. Gestational hypoxia resulted in a reduced body weight for both male and female offspring at birth. This restriction in body weight persisted until weaning, after which the animals underwent catch-up growth. At 10 weeks of age, a subset of offspring was placed on a high salt diet (5% NaCl). Pressurized myography of mesenteric resistance arteries at 12 months of age showed that both male and female offspring exposed to maternal hypoxia had significantly impaired endothelial function, as demonstrated by impaired vasodilatation to ACh but not sodium nitroprusside. Endothelial dysfunction caused by prenatal hypoxia was not exacerbated by postnatal consumption of a high salt diet. Prenatal hypoxia increased microvascular stiffness in male offspring. The combination of prenatal hypoxia and a postnatal high salt diet caused a leftward shift in the stress-strain relationship in both sexes. Histopathological analysis of aortic sections revealed a loss of elastin integrity and increased collagen, consistent with increased vascular stiffness. These results demonstrate that prenatal hypoxia programs endothelial dysfunction in both sexes. A chronic high salt diet in postnatal life had an additive deleterious effect on vascular mechanics and structural characteristics in both sexes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  11. Curcumin induces therapeutic angiogenesis in a diabetic mouse hindlimb ischemia model via modulating the function of endothelial progenitor cells.

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    You, Jinzhi; Sun, Jiacheng; Ma, Teng; Yang, Ziying; Wang, Xu; Zhang, Zhiwei; Li, Jingjing; Wang, Longgang; Ii, Masaaki; Yang, Junjie; Shen, Zhenya

    2017-08-03

    Neovascularization is impaired in diabetes mellitus, which leads to the development of peripheral arterial disease and is mainly attributed to the dysfunction of endothelial progenitor cells (EPCs). Previous studies proved the promotional effect of curcumin on neovascularization in wound healing of diabetes. Thus, we hypothesize that curcumin could promote neovascularization at sites of hindlimb ischemia in diabetes and might take effect via modulating the function of EPCs. Streptozotocin-induced type 1 diabetic mice and nondiabetic mice both received unilateral hindlimb ischemic surgery. Curcumin was then administrated to the mice by lavage for 14 days consecutively. Laser Doppler perfusion imaging was conducted to demonstrate the blood flow reperfusion. Capillary density was measured in the ischemic gastrocnemius muscle. In addition, angiogenesis, migration, proliferation abilities, and senescence were determined in EPCs isolated from diabetic and nondiabetic mice. Quantitative PCR was then used to determine the mRNA expression of vascular endothelial growth factor (VEGF) and angiopoetin-1 (Ang-1) in EPCs. Curcumin application to type 1 diabetic mice significantly improved blood reperfusion and increased the capillary density in ischemic hindlimbs. The in-vitro study also revealed that the angiogenesis, migration, and proliferation abilities of EPCs and the number of senescent EPCs were reversed by curcumin application. Quantitative PCR confirmed the overexpression of VEGF-A and Ang-1 in EPCs after curcumin treatment. Curcumin could enhance neovascularization via promoting the function of EPCs in a diabetic mouse hindlimb ischemia model.

  12. Proliferation studies of the endothelial and smooth muscle cells of the mouse mesentery after irradiation

    International Nuclear Information System (INIS)

    Hirst, D.G.; Denekamp, J.; Hobson, B.

    1980-01-01

    A continuous tritium labelling technique was employed to study the effects of external β-radiation on the proliferation of endothelial cells and smooth muscle cells in the mesenteric arterioles of mice. Calculations showed very long turnover times for the two cell populations in control animals (> 2 years for endothelium and > 3 years for smooth muscle). After single doses of 20 and 45 Gy, no significant increase in endothelial proliferation was seen except at 3 weeks. No significant increase in labelling was observed in smooth muscle up to 48 weeks after irradiation. These labelling data have been compared with the pattern of cell depletion of the irradiated endothelium. It was concluded that the depletion was much earlier than expected for a slowly proliferating tissue, if all the cells were cycling very slowly. Such an early depletion is, however, consistent with cell death resulting from a small proportion of the cells having a short cell cycle. The recovery of the endothelial cell numbers between 9 and 12 months was not accompanied by a rise in the fraction of labelled cells. It is suggested that repopulation may occur from outside the treated area. (author)

  13. Human Usher 1B/mouse shaker-1: the retinal phenotype discrepancy explained by the presence/absence of myosin VIIA in the photoreceptor cells.

    Science.gov (United States)

    el-Amraoui, A; Sahly, I; Picaud, S; Sahel, J; Abitbol, M; Petit, C

    1996-08-01

    Usher syndrome type 1 (USH1) associates severe congenital deafness, vestibular dysfunction and progressive retinitis pigmentosa leading to blindness. The gene encoding myosin VIIA is responsible for USH1B. Mutations in the murine orthologous gene lead to the shaker-1 phenotype, which manifests cochlear and vestibular dysfunction, without any retinal defect. To address this phenotypic discrepancy, the expression of myosin VIIA in retinal cells was analyzed in human and mouse during embryonic development and adult life. In the human embryo, myosin VIIA was present first in the pigment epithelium cells, and later in these cells as well as in the photoreceptor cells. In the adult human retina, myosin VIIA was present in both cell types. In contrast, in mouse, only pigment epithelium cells expressed the protein throughout development and adult life. Myosin VIIA was also found to be absent in the photoreceptor cells of other rodents (rat and guinea-pig), whereas these cells expressed the protein in amphibians, avians and primates. These observations suggest that retinitis pigmentosa of USH1B results from a primary rod and cone defect. The USH1B/shaker-1 paradigm illustrates a species-specific cell pattern of gene expression as a possible cause for the discrepancy between phenotypes involving defective orthologous genes in man and mouse. Interestingly, in the photoreceptor cells, myosin VIIA is mainly localized in the inner and base of outer segments as well as in the synaptic ending region where it is co-localized with the synaptic vesicles. Therefore, we suggest that myosin VIIA might play a role in the trafficking of ribbon-synaptic vesicle complexes and the renewal processes of the outer photoreceptor disks.

  14. The role of whiskers in compensation of visual deficit in a mouse model of retinal degeneration.

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    Voller, Jaroslav; Potužáková, Barbora; Šimeček, Vojtěch; Vožeh, František

    2014-01-13

    Sensory deprivation in one modality can enhance the development of the remaining modalities via mechanisms of synaptic plasticity. Mice of the C3H strain suffer from RD1 retinal degeneration that leads to visual impairment at weaning age. We examined a role of whiskers in compensation of the visual deficit. In order to differentiate the contribution of the whiskers from other mechanisms that can take part in the compensation, we investigated the effect of both chronic and acute tactile deprivation. Three-month-old mice were used. We examined motor skills (rotarod, beam walking test), gait control (CatWalk system), spontaneous motor activity (open field) and CNS excitability to an acoustic stimulus for assessment of compensatory changes in auditory system (audiogenic epilepsy). In the sighted mice, the only effect was a decline in their rotarod test performance after acute whisker removal. In the blind animals, chronic tactile deprivation caused changes in their gait and impaired the performance in motor tests. Some other compensatory mechanisms were involved but the whiskers are essential for the compensation as it emerged from more marked change of gait and the worsening of the motor performance after the acute whisker removal. Both chronic and acute tactile deprivation induced anxiety-like behaviour. Only a combination of blindness and chronic tactile deprivation led to an increased sense of hearing. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. Morphology and Topography of Retinal Pericytes in the Living Mouse Retina Using In Vivo Adaptive Optics Imaging and Ex Vivo Characterization

    Science.gov (United States)

    Schallek, Jesse; Geng, Ying; Nguyen, HoanVu; Williams, David R.

    2013-01-01

    Purpose. To noninvasively image retinal pericytes in the living eye and characterize NG2-positive cell topography and morphology in the adult mouse retina. Methods. Transgenic mice expressing fluorescent pericytes (NG2, DsRed) were imaged using a two-channel, adaptive optics scanning laser ophthalmoscope (AOSLO). One channel imaged vascular perfusion with near infrared light. A second channel simultaneously imaged fluorescent retinal pericytes. Mice were also imaged using wide-field ophthalmoscopy. To confirm in vivo imaging, five eyes were enucleated and imaged in flat mount with conventional fluorescent microscopy. Cell topography was quantified relative to the optic disc. Results. We observed strong DsRed fluorescence from NG2-positive cells. AOSLO revealed fluorescent vascular mural cells enveloping all vessels in the living retina. Cells were stellate on larger venules, and showed banded morphology on arterioles. NG2-positive cells indicative of pericytes were found on the smallest capillaries of the retinal circulation. Wide-field SLO enabled quick assessment of NG2-positive distribution, but provided insufficient resolution for cell counts. Ex vivo microscopy showed relatively even topography of NG2-positive capillary pericytes at eccentricities more than 0.3 mm from the optic disc (515 ± 94 cells/mm2 of retinal area). Conclusions. We provide the first high-resolution images of retinal pericytes in the living animal. Subcellular resolution enabled morphological identification of NG2-positive cells on capillaries showing classic features and topography of retinal pericytes. This report provides foundational basis for future studies that will track and quantify pericyte topography, morphology, and function in the living retina over time, especially in the progression of microvascular disease. PMID:24150762

  16. Progressive retinal degeneration and glial activation in the CLN6 (nclf mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation.

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    Myriam Mirza

    Full Text Available Neuronal ceroid lipofuscinosis (NCL is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6 (nclf mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6 (nclf retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6 (nclf retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA, could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6 (nclf mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6 (nclf retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6 (nclf retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.

  17. Expression of the growth factor progranulin in endothelial cells influences growth and development of blood vessels: a novel mouse model.

    Science.gov (United States)

    Toh, Huishi; Cao, Mingju; Daniels, Eugene; Bateman, Andrew

    2013-01-01

    Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2-promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5-14.5) and later (E15.5-17.5) developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with subsequent effects in

  18. Expression of the growth factor progranulin in endothelial cells influences growth and development of blood vessels: a novel mouse model.

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    Huishi Toh

    Full Text Available Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2-promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5-14.5 and later (E15.5-17.5 developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with

  19. Oxalomalate reduces expression and secretion of vascular endothelial growth factor in the retinal pigment epithelium and inhibits angiogenesis: Implications for age-related macular degeneration

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    Sung Hwan Kim

    2016-12-01

    Full Text Available Clinical and experimental observations indicate a critical role for vascular endothelial growth factor (VEGF, secreted by the retinal pigment epithelium (RPE, in pathological angiogenesis and the development of choroidal neovascularization (CNV in age-related macular degeneration (AMD. RPE-mediated VEGF expression, leading to angiogenesis, is a major signaling mechanism underlying ocular neovascular disease. Inhibiting this signaling pathway with a therapeutic molecule is a promising anti-angiogenic strategy to treat this disease with potentially fewer side effects. Oxalomalate (OMA is a competitive inhibitor of NADP+-dependent isocitrate dehydrogenase (IDH, which plays an important role in cellular signaling pathways regulated by reactive oxygen species (ROS. Here, we have investigated the inhibitory effect of OMA on the expression of VEGF, and the associated underlying mechanism of action, using in vitro and in vivo RPE cell models of AMD. We found that OMA reduced the expression and secretion of VEGF in RPE cells, and consequently inhibited CNV formation. This function of OMA was linked to its capacity to activate the pVHL-mediated HIF-1α degradation in these cells, partly via a ROS-dependent ATM signaling axis, through inhibition of IDH enzymes. These findings reveal a novel role for OMA in inhibiting RPE-derived VEGF expression and angiogenesis, and suggest unique therapeutic strategies for treating pathological angiogenesis and AMD development.

  20. Light adaptation alters the source of inhibition to the mouse retinal OFF pathway

    Science.gov (United States)

    Mazade, Reece E.

    2013-01-01

    Sensory systems must avoid saturation to encode a wide range of stimulus intensities. One way the retina accomplishes this is by using both dim-light-sensing rod and bright-light-sensing cone photoreceptor circuits. OFF cone bipolar cells are a key point in this process, as they receive both excitatory input from cones and inhibitory input from AII amacrine cells via the rod pathway. However, in addition to AII amacrine cell input, other inhibitory inputs from cone pathways also modulate OFF cone bipolar cell light signals. It is unknown how these inhibitory inputs to OFF cone bipolar cells change when switching between rod and cone pathways or whether all OFF cone bipolar cells receive rod pathway input. We found that one group of OFF cone bipolar cells (types 1, 2, and 4) receive rod-mediated inhibitory inputs that likely come from the rod-AII amacrine cell pathway, while another group of OFF cone bipolar cells (type 3) do not. In both cases, dark-adapted rod-dominant light responses showed a significant contribution of glycinergic inhibition, which decreased with light adaptation and was, surprisingly, compensated by an increase in GABAergic inhibition. As GABAergic input has distinct timing and spatial spread from glycinergic input, a shift from glycinergic to GABAergic inhibition could significantly alter OFF cone bipolar cell signaling to downstream OFF ganglion cells. Larger GABAergic input could reflect an adjustment of OFF bipolar cell spatial inhibition, which may be one mechanism that contributes to retinal spatial sensitivity in the light. PMID:23926034

  1. Dendritic thickness: a morphometric parameter to classify mouse retinal ganglion cells

    Directory of Open Access Journals (Sweden)

    L.D. Loopuijt

    2007-10-01

    Full Text Available To study the dendritic morphology of retinal ganglion cells in wild-type mice we intracellularly injected these cells with Lucifer yellow in an in vitro preparation of the retina. Subsequently, quantified values of dendritic thickness, number of branching points and level of stratification of 73 Lucifer yellow-filled ganglion cells were analyzed by statistical methods, resulting in a classification into 9 groups. The variables dendritic thickness, number of branching points per cell and level of stratification were independent of each other. Number of branching points and level of stratification were independent of eccentricity, whereas dendritic thickness was positively dependent (r = 0.37 on it. The frequency distribution of dendritic thickness tended to be multimodal, indicating the presence of at least two cell populations composed of neurons with dendritic diameters either smaller or larger than 1.8 µm ("thin" or "thick" dendrites, respectively. Three cells (4.5% were bistratified, having thick dendrites, and the others (95.5% were monostratified. Using k-means cluster analysis, monostratified cells with either thin or thick dendrites were further subdivided according to level of stratification and number of branching points: cells with thin dendrites were divided into 2 groups with outer stratification (0-40% and 2 groups with inner (50-100% stratification, whereas cells with thick dendrites were divided into one group with outer and 3 groups with inner stratification. We postulate, that one group of cells with thin dendrites resembles cat ß-cells, whereas one group of cells with thick dendrites includes cells that resemble cat a-cells.

  2. Role of the mouse retinal photoreceptor ribbon synapse in visual motion processing for optokinetic responses.

    Science.gov (United States)

    Sugita, Yuko; Araki, Fumiyuki; Chaya, Taro; Kawano, Kenji; Furukawa, Takahisa; Miura, Kenichiro

    2015-01-01

    The ribbon synapse is a specialized synaptic structure in the retinal outer plexiform layer where visual signals are transmitted from photoreceptors to the bipolar and horizontal cells. This structure is considered important in high-efficiency signal transmission; however, its role in visual signal processing is unclear. In order to understand its role in visual processing, the present study utilized Pikachurin-null mutant mice that show improper formation of the photoreceptor ribbon synapse. We examined the initial and late phases of the optokinetic responses (OKRs). The initial phase was examined by measuring the open-loop eye velocity of the OKRs to sinusoidal grating patterns of various spatial frequencies moving at various temporal frequencies for 0.5 s. The mutant mice showed significant initial OKRs with a spatiotemporal frequency tuning (spatial frequency, 0.09 ± 0.01 cycles/°; temporal frequency, 1.87 ± 0.12 Hz) that was slightly different from the wild-type mice (spatial frequency, 0.11 ± 0.01 cycles/°; temporal frequency, 1.66 ± 0.12 Hz). The late phase of the OKRs was examined by measuring the slow phase eye velocity of the optokinetic nystagmus induced by the sinusoidal gratings of various spatiotemporal frequencies moving for 30 s. We found that the optimal spatial and temporal frequencies of the mutant mice (spatial frequency, 0.11 ± 0.02 cycles/°; temporal frequency, 0.81 ± 0.24 Hz) were both lower than those in the wild-type mice (spatial frequency, 0.15 ± 0.02 cycles/°; temporal frequency, 1.93 ± 0.62 Hz). These results suggest that the ribbon synapse modulates the spatiotemporal frequency tuning of visual processing along the ON pathway by which the late phase of OKRs is mediated.

  3. Role of the mouse retinal photoreceptor ribbon synapse in visual motion processing for optokinetic responses.

    Directory of Open Access Journals (Sweden)

    Yuko Sugita

    Full Text Available The ribbon synapse is a specialized synaptic structure in the retinal outer plexiform layer where visual signals are transmitted from photoreceptors to the bipolar and horizontal cells. This structure is considered important in high-efficiency signal transmission; however, its role in visual signal processing is unclear. In order to understand its role in visual processing, the present study utilized Pikachurin-null mutant mice that show improper formation of the photoreceptor ribbon synapse. We examined the initial and late phases of the optokinetic responses (OKRs. The initial phase was examined by measuring the open-loop eye velocity of the OKRs to sinusoidal grating patterns of various spatial frequencies moving at various temporal frequencies for 0.5 s. The mutant mice showed significant initial OKRs with a spatiotemporal frequency tuning (spatial frequency, 0.09 ± 0.01 cycles/°; temporal frequency, 1.87 ± 0.12 Hz that was slightly different from the wild-type mice (spatial frequency, 0.11 ± 0.01 cycles/°; temporal frequency, 1.66 ± 0.12 Hz. The late phase of the OKRs was examined by measuring the slow phase eye velocity of the optokinetic nystagmus induced by the sinusoidal gratings of various spatiotemporal frequencies moving for 30 s. We found that the optimal spatial and temporal frequencies of the mutant mice (spatial frequency, 0.11 ± 0.02 cycles/°; temporal frequency, 0.81 ± 0.24 Hz were both lower than those in the wild-type mice (spatial frequency, 0.15 ± 0.02 cycles/°; temporal frequency, 1.93 ± 0.62 Hz. These results suggest that the ribbon synapse modulates the spatiotemporal frequency tuning of visual processing along the ON pathway by which the late phase of OKRs is mediated.

  4. Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model.

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    Kevin P Kennelly

    Full Text Available PURPOSE: Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure "transplant conditions" (TC. We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE cell line, DH01. METHODS: Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL and cleaved caspase 3 immunolabeling of subretinal allografts. RESULTS: Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01. Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1% that did not increase following TC (4.8%±0.5%. However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%. Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001. Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001. CONCLUSION: Pre-confluent cells should be used to maximize graft cell viability.

  5. Long-term consequences of developmental vascular defects on retinal vessel homeostasis and function in a mouse model of Norrie disease.

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    Susanne C Beck

    Full Text Available Loss of Norrin signalling due to mutations in the Norrie disease pseudoglioma gene causes severe vascular defects in the retina, leading to visual impairment and ultimately blindness. While the emphasis of experimental work so far was on the developmental period, we focus here on disease mechanisms that induce progression into severe adult disease. The goal of this study was the comprehensive analysis of the long-term effects of the absence of Norrin on vascular homeostasis and retinal function. In a mouse model of Norrie disease retinal vascular morphology and integrity were studied by means of in vivo angiography; the vascular constituents were assessed in detailed histological analyses using quantitative retinal morphometry. Finally, electroretinographic analyses were performed to assess the retinal function in adult Norrin deficient animals. We could show that the primary developmental defects not only persisted but developed into further vascular abnormalities and microangiopathies. In particular, the overall vessel homeostasis, the vascular integrity, and also the cellular constituents of the vascular wall were affected in the adult Norrin deficient retina. Moreover, functional analyses indicated to persistent hypoxia in the neural retina which was suggested as one of the major driving forces of disease progression. In summary, our data provide evidence that the key to adult Norrie disease are ongoing vascular modifications, driven by the persistent hypoxic conditions, which are ineffective to compensate for the primary Norrin-dependent defects.

  6. Long-term consequences of developmental vascular defects on retinal vessel homeostasis and function in a mouse model of Norrie disease.

    Science.gov (United States)

    Beck, Susanne C; Feng, Yuxi; Sothilingam, Vithiyanjali; Garcia Garrido, Marina; Tanimoto, Naoyuki; Acar, Niyazi; Shan, Shenliang; Seebauer, Britta; Berger, Wolfgang; Hammes, Hans-Peter; Seeliger, Mathias W

    2017-01-01

    Loss of Norrin signalling due to mutations in the Norrie disease pseudoglioma gene causes severe vascular defects in the retina, leading to visual impairment and ultimately blindness. While the emphasis of experimental work so far was on the developmental period, we focus here on disease mechanisms that induce progression into severe adult disease. The goal of this study was the comprehensive analysis of the long-term effects of the absence of Norrin on vascular homeostasis and retinal function. In a mouse model of Norrie disease retinal vascular morphology and integrity were studied by means of in vivo angiography; the vascular constituents were assessed in detailed histological analyses using quantitative retinal morphometry. Finally, electroretinographic analyses were performed to assess the retinal function in adult Norrin deficient animals. We could show that the primary developmental defects not only persisted but developed into further vascular abnormalities and microangiopathies. In particular, the overall vessel homeostasis, the vascular integrity, and also the cellular constituents of the vascular wall were affected in the adult Norrin deficient retina. Moreover, functional analyses indicated to persistent hypoxia in the neural retina which was suggested as one of the major driving forces of disease progression. In summary, our data provide evidence that the key to adult Norrie disease are ongoing vascular modifications, driven by the persistent hypoxic conditions, which are ineffective to compensate for the primary Norrin-dependent defects.

  7. Retinal vascular oximetry during ranibizumab treatment of central retinal vein occlusion

    DEFF Research Database (Denmark)

    Traustason, Sindri; la Cour, Morten; Larsen, Michael

    2014-01-01

    PURPOSE: To investigate the effect of intravitreal injections of the vascular endothelial growth factor inhibitor ranibizumab on retinal oxygenation in patients with central retinal vein occlusion (CRVO). METHODS: Retinal oxygen saturation in patients with CRVO was analysed using the Oxymap Retin...

  8. RETINAL PIGMENT EPITHELIAL TEAR AND ANTI-VASCULAR ENDOTHELIAL GROWTH FACTOR THERAPY IN EXUDATIVE AGE-RELATED MACULAR DEGENERATION: Clinical Course and Long-Term Prognosis.

    Science.gov (United States)

    Heimes, Britta; Farecki, Marie-Louise; Bartels, Sina; Barrelmann, Anna; Gutfleisch, Matthias; Spital, Georg; Lommatzsch, Albrecht; Pauleikhoff, Daniel

    2016-05-01

    To document the long-term outcome in cases of retinal pigment epithelial (RPE) tears after treatment of vascularized pigment epithelial detachments with anti-vascular endothelial growth factor therapy. A retrospective analysis of the long-term outcome of a consecutive series of eyes with RPE tear developed during anti-vascular endothelial growth factor therapy for pigment epithelial detachment associated with choroidal neovascularization or retinal angiomatous proliferation (vascularized pigment epithelial detachment) was performed. Best-corrected visual acuity (BCVA), spectral domain optical coherence tomography, and autofluorescence images and also fluorescein angiograms were analyzed to determine the functional and morphologic development over time. The long-term outcome of 22 eyes (21 patients, 13 women and 8 men; 65-85 years; mean: 76 years) with RPE tear was performed with minimal follow-up of 3 years (range: 3-5 years, mean: 44 months) and re-treatment with different therapeutic strategies. The eyes were differentiated in 2 groups according to the course of BCVA after the first 2 years of follow-up: Group 1 (11 eyes) demonstrated a stabilized or improved BCVA after 2 years and Group 2 (11 eyes) demonstrated a decrease in BCVA after 2 years. The initial BCVA between both groups was comparable. Also the mean initial size of the RPE tear was the same between the 2 groups, the area of the RPE tear decreased continuously during follow-up in Group 1, whereas this was the case in Group 2 only at the beginning of treatment with a further increase of the size of the RPE tear with longer follow-up. This corresponded with a different morphologic development between the two groups. In Group 1, increasing recovery of autofluorescence at the RPE-free area was visible beginning from the outer border, whereas in Group 2, further growth of the neovascular complex in the area of the RPE tear was observed resulting in larger fibrovascular scars. In addition, in both groups

  9. Pharmacologic Activation of Wnt Signaling by Lithium Normalizes Retinal Vasculature in a Murine Model of Familial Exudative Vitreoretinopathy.

    Science.gov (United States)

    Wang, Zhongxiao; Liu, Chi-Hsiu; Sun, Ye; Gong, Yan; Favazza, Tara L; Morss, Peyton C; Saba, Nicholas J; Fredrick, Thomas W; He, Xi; Akula, James D; Chen, Jing

    2016-10-01

    Familial exudative vitreoretinopathy (FEVR) is characterized by delayed retinal vascular development, which promotes hypoxia-induced pathologic vessels. In severe cases FEVR may lead to retinal detachment and visual impairment. Genetic studies linked FEVR with mutations in Wnt signaling ligand or receptors, including low-density lipoprotein receptor-related protein 5 (LRP5) gene. Here, we investigated ocular pathologies in a Lrp5 knockout (Lrp5(-/-)) mouse model of FEVR and explored whether treatment with a pharmacologic Wnt activator lithium could bypass the genetic defects, thereby protecting against eye pathologies. Lrp5(-/-) mice displayed significantly delayed retinal vascular development, absence of deep layer retinal vessels, leading to increased levels of vascular endothelial growth factor and subsequent pathologic glomeruloid vessels, as well as decreased inner retinal visual function. Lithium treatment in Lrp5(-/-) mice significantly restored the delayed development of retinal vasculature and the intralaminar capillary networks, suppressed formation of pathologic glomeruloid structures, and promoted hyaloid vessel regression. Moreover, lithium treatment partially rescued inner-retinal visual function and increased retinal thickness. These protective effects of lithium were largely mediated through restoration of canonical Wnt signaling in Lrp5(-/-) retina. Lithium treatment also substantially increased vascular tubular formation in LRP5-deficient endothelial cells. These findings suggest that pharmacologic activation of Wnt signaling may help treat ocular pathologies in FEVR and potentially other defective Wnt signaling-related diseases. Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  10. LPS, but not Angiotensin ll, lnduces Direct Pro-lnflammatory Effects in Cultured Mouse Arteries and Human Endothelial and Vascular Smooth Muscle Cells

    DEFF Research Database (Denmark)

    Outzen, Emilie M; Zaki, Marina; Mehryar, Rahila

    2017-01-01

    resistance-sized arteries (MRA) supported by experiments in cultured human primary endothelial and vascular smooth muscle cells. Results showed that 24-hr organ culture of mouse MRA with 10 nM Ang II had, unlike 100 ng/mL LPS, no effects on IL-6 or MCP-1 secretion, VCAM1 mRNA expression or endothelial......]-Ang II had no concentration- or time-dependent effects on IL-6 and MCP-1 secretion in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC). AGTR1 or AGTR2 mRNA expression were undetectable in HUVEC, whereas HASMC expressed only AGTR1 mRNA. In summary, contrary...... rights reserved....

  11. Tlx acts as a proangiogenic switch by regulating extracellular assembly of fibronectin matrices in retinal astrocytes.

    Science.gov (United States)

    Uemura, Akiyoshi; Kusuhara, Sentaro; Wiegand, Stanley J; Yu, Ruth T; Nishikawa, Shin-ichi

    2006-02-01

    In response to hypoxia, hypoxia-inducible factors act as the primary proangiogenic triggers by regulating transcription levels of target genes, including VEGF. However, little is known about the specific factors that control other components of the angiogenic process, particularly formation of matrix scaffolds that promote adhesion and migration of endothelial cells. We show that in the postnatal mouse retina, the orphan nuclear receptor tailless (Tlx) is strongly expressed in the proangiogenic astrocytes, which secrete VEGF and fibronectin. Tlx expression by retinal astrocytes is controlled by oxygen concentration and rapidly downregulated upon contact with blood vessels. In mice null for Tlx, retinal astrocytes maintain VEGF expression; however, the extracellular assembly of fibronectin matrices by astrocytes is severely impaired, leading to defective scaffold formation and a complete failure of normal retinal vascular development. This work identifies Tlx as an essential component of the molecular network involved in the hypoxia-inducible proangiogenic switch in retinal astrocytes.

  12. Hyperglycemia Induces Toll-Like Receptor-2 and -4 Expression and Activity in Human Microvascular Retinal Endothelial Cells: Implications for Diabetic Retinopathy

    Directory of Open Access Journals (Sweden)

    Uthra Rajamani

    2014-01-01

    Full Text Available Diabetic retinopathy (DR causes visual impairment in working age adults and hyperglycemia-mediated inflammation is central in DR. Toll-like receptors (TLRs play a key role in innate immune responses and inflammation. However, scanty data is available on their role in DR. Hence, in this study, we examined TLR2 and TLR4 mRNA and protein expression and activity in hyperglycemic human retinal endothelial cells (HMVRECs. HMVRECs were treated with hyperglycemia (HG or euglycemia and mRNA and protein levels of TLR-2, TLR-4, MyD88, IRF3, and TRIF as well as NF-κB p65 activation were measured. IL-8, IL-1β, TNF-α and MCP-1, ICAM-1, and VCAM-1 as well as monocyte adhesion to HMVRECs were also assayed. HG (25 mM significantly induced TLR2 and TLR4 mRNA and protein in HMVRECs. It also increased both MyD88 and non-MyD88 pathways, nuclear factor-κB (NF-κB, biomediators, and monocyte adhesion. This inflammation was attenuated by TLR-4 or TLR-2 inhibition, and dual inhibition by a TLR inhibitory peptide as well as TLR2 and 4 siRNA. Additionally, antioxidant treatment reduced TLR-2 and TLR4 expression and downstream inflammatory markers. Collectively, our novel data suggest that hyperglycemia induces TLR-2 and TLR-4 activation and downstream signaling mediating increased inflammation possibly via reactive oxygen species (ROS and could contribute to DR.

  13. Identification and functional analysis of endothelial tip cell-enriched genes.

    Science.gov (United States)

    del Toro, Raquel; Prahst, Claudia; Mathivet, Thomas; Siegfried, Geraldine; Kaminker, Joshua S; Larrivee, Bruno; Breant, Christiane; Duarte, Antonio; Takakura, Nobuyuki; Fukamizu, Akiyoshi; Penninger, Josef; Eichmann, Anne

    2010-11-11

    Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4(+/-) mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4(+/-) and wild-type mice, we identified 3 clusters of tip cell-enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.

  14. Microultrasound Molecular Imaging of Vascular Endothelial Growth Factor Receptor 2 in a Mouse Model of Tumor Angiogenesis

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    Joshua J. Rychak

    2007-09-01

    Full Text Available High-frequency microultrasound imaging of tumor progression in mice enables noninvasive anatomic and functional imaging at excellent spatial and temporal resolution, although microultrasonography alone does not offer molecular scale data. In the current study, we investigated the use of microbubble ultrasound contrast agents bearing targeting ligands specific for molecular markers of tumor angiogenesis using high-frequency microultrasound imaging. A xenograft tumor model in the mouse was used to image vascular endothelial growth factor receptor 2 (VEGFR-2 expression with microbubbles conjugated to an anti-VEGFR-2 monoclonal antibody or an isotype control. Microultrasound imaging was accomplished at a center frequency of 40 MHz, which provided lateral and axial resolutions of 40 and 90 μm, respectively. The B-mode (two-dimensional mode acoustic signal from microbubbles bound to the molecular target was determined by an ultrasound-based destruction-subtraction scheme. Quantification of the adherent microbubble fraction in nine tumor-bearing mice revealed significant retention of VEGFR-2-targeted microbubbles relative to control-targeted microbubbles. These data demonstrate that contrast-enhanced microultrasound imaging is a useful method for assessing molecular expression of tumor angiogenesis in mice at high resolution.

  15. Immunohistochemical Examination on the Distribution of Cells Expressed Lymphatic Endothelial Marker Podoplanin and LYVE-1 in the Mouse Tongue Tissue

    Science.gov (United States)

    Noda, Yuya; Amano, Ikuko; Hata, Minoru; Kojima, Hiroshi; Sawa, Yoshihiko

    2010-01-01

    The clinical study for lingual disease requires the detailed investigation of the lingual lymphatic network and lymphatic marker-positive cells. Recently, it has been reported that several tissue cells and leukocytes express lymphatic markers, LYVE-1 and podoplanin. This study was aimed to clarify the lingual distribution of cells expressing LYVE-1 and podoplanin. In the mouse tongue, podoplanin is expressed in nerve sheaths, lingual gland myoepithelial cells, and lymphatic vessels. LYVE-1 is expressed in the macrophage marker Mac-1-positive cells as well as lymphatic vessels, while factor-VIII was detected in only blood endothelial cells. α-SMA was detected in vascular smooth muscle and myoepithelial cells. Therefore, identification of lymphatic vessels in lingual glands, the combination of LYVE-1 and factor-VIII, or LYVE-1 and Mac-1 is useful because myoepithelial cells express podoplanin and α-SMA. The immunostaining of factor-VIII on lymphatic vessels was masked by the immunostaining to LYVE-1 or podoplanin because lymphatic vessels express factor-VIII to a far lesser extent than blood vessels. Therefore, except for the salivary glands, the combination of podoplanin and α-SMA, or factor-VIII is useful to identify lymphatic vessels and blood vessels with smooth muscle, or blood capillaries. PMID:20514293

  16. Indian hedgehog signaling from endothelial cells is required for sclera and retinal pigment epithelium development in the mouse eye.

    Science.gov (United States)

    Dakubo, Gabriel D; Mazerolle, Chantal; Furimsky, Marosh; Yu, Chuan; St-Jacques, Benoit; McMahon, Andrew P; Wallace, Valerie A

    2008-08-01

    The development of extraocular orbital structures, in particular the choroid and sclera, is regulated by a complex series of interactions between neuroectoderm, neural crest and mesoderm derivatives, although in many instances the signals that mediate these interactions are not known. In this study we have investigated the function of Indian hedgehog (Ihh) in the developing mammalian eye. We show that Ihh is expressed in a population of non-pigmented cells located in the developing choroid adjacent to the RPE. The analysis of Hh mutant mice demonstrates that the RPE and developing scleral mesenchyme are direct targets of Ihh signaling and that Ihh is required for the normal pigmentation pattern of the RPE and the condensation of mesenchymal cells to form the sclera. Our findings also indicate that Ihh signals indirectly to promote proliferation and photoreceptor specification in the neural retina. This study identifies Ihh as a novel choroid-derived signal that regulates RPE, sclera and neural retina development.

  17. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

    2017-01-01

    Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

  18. Clinical utilization of anti-vascular endothelial growth-factor agents and patient monitoring in retinal vein occlusion and diabetic macular edema

    Directory of Open Access Journals (Sweden)

    Kiss S

    2014-08-01

    .03; IVTA, 3.3 versus 3.0, P<0.05.Conclusion: During the study period (2008–2011, bevacizumab was the main anti-VEGF therapy used in clinical practice for BRVO, CRVO, and DME. Patients treated with bevacizumab were monitored less frequently and received fewer injections than patients in major clinical trials of ranibizumab. Keywords: anti-vascular endothelial growth factor, bevacizumab, ranibizumab, diabetic macular edema, retinal vein occlusion, intravitreal

  19. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Kawaguchi, Manami [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Kitajima, Kenji [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kanokoda, Mai [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Suzuki, Hidenori [Division of Morphological and Biomolecular Research, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo, 113-8602 (Japan); Miyashita, Kazuya; Nakajima, Marino [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan); Nuriya, Hideko [Core Technology and Research Center, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Kasahara, Kohji [Laboratory of Biomembrane, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Hara, Takahiko, E-mail: hara-tk@igakuken.or.jp [Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, 2-1-6 Kamikitazawa, Setagaya-ku, Tokyo, 156-8506 (Japan); Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8510 (Japan)

    2016-06-03

    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit{sup −}Tie2{sup −}CD41{sup +} Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41{sup +}CD42b{sup +}CD61{sup +} platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit{sup −}Tie2{sup −}CD41{sup +}. •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  20. Efficient production of platelets from mouse embryonic stem cells by enforced expression of Gata2 in late hemogenic endothelial cells

    International Nuclear Information System (INIS)

    Kawaguchi, Manami; Kitajima, Kenji; Kanokoda, Mai; Suzuki, Hidenori; Miyashita, Kazuya; Nakajima, Marino; Nuriya, Hideko; Kasahara, Kohji; Hara, Takahiko

    2016-01-01

    Platelets are essential for blood circulation and coagulation. Previous study indicated that overexpression of Gata2 in differentiated mouse embryonic stem cells (ESCs) resulted in robust induction of megakaryocytes (Mks). To evaluate platelet production capacity of the Gata2-induced ESC-derived Mks, we generated iGata2-ESC line carrying the doxycycline-inducible Gata2 expression cassette. When doxycycline was added to day 5 hemogenic endothelial cells in the in vitro differentiation culture of iGata2-ESCs, c-Kit − Tie2 − CD41 + Mks were predominantly generated. These iGata2-ESC-derived Mks efficiently produced CD41 + CD42b + CD61 + platelets and adhered to fibrinogen-coated glass coverslips in response to thrombin stimulation. Transmission electron microscopy analysis demonstrated that the iGata2-ESC-derived platelets were discoid-shaped with α-granules and an open canalicular system, but were larger than peripheral blood platelets in size. These results demonstrated that an enforced expression of Gata2 in late HECs of differentiated ESCs efficiently promotes megakaryopoiesis followed by platelet production. This study provides valuable information for ex vivo platelet production from human pluripotent stem cells in future. -- Highlights: •Megakaryocytes are efficiently induced by Gata2 from ESC-derived day 5 HECs. •Gata2-induced ESC-derived megakaryocytes are c-Kit − Tie2 − CD41 + . •Gata2-induced ESC-derived megakaryocytes produce larger discoid-shaped platelets. •Gata2-induced ESC-derived platelets bind fibrinogen upon thrombin stimulation.

  1. Cadherins in the retinal pigment epithelium (RPE revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo.

    Directory of Open Access Journals (Sweden)

    Xue Yang

    Full Text Available The retinal pigment epithelium (RPE supports the health and function of retinal photoreceptors and is essential for normal vision. RPE cells are post-mitotic, terminally differentiated, and polarized epithelial cells. In pathological conditions, however, they lose their epithelial integrity, become dysfunctional, even dedifferentiate, and ultimately die. The integrity of epithelial cells is maintained, in part, by adherens junctions, which are composed of cadherin homodimers and p120-, β-, and α-catenins linking to actin filaments. While E-cadherin is the major cadherin for forming the epithelial phenotype in most epithelial cell types, it has been reported that cadherin expression in RPE cells is different from other epithelial cells based on results with cultured RPE cells. In this study, we revisited the expression of cadherins in the RPE to clarify their relative contribution by measuring the absolute quantity of cDNAs produced from mRNAs of three classical cadherins (E-, N-, and P-cadherins in the RPE in vivo. We found that P-cadherin (CDH3 is highly dominant in both mouse and human RPE in situ. The degree of dominance of P-cadherin is surprisingly large, with mouse Cdh3 and human CDH3 accounting for 82-85% and 92-93% of the total of the three cadherin mRNAs, respectively. We confirmed the expression of P-cadherin protein at the cell-cell border of mouse RPE in situ by immunofluorescence. Furthermore, we found that oxidative stress induces dissociation of P-cadherin and β-catenin from the cell membrane and subsequent translocation of β-catenin into the nucleus, resulting in activation of the canonical Wnt/β-catenin pathway. This is the first report of absolute comparison of the expression of three cadherins in the RPE, and the results suggest that the physiological role of P-cadherin in the RPE needs to be reevaluated.

  2. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    OpenAIRE

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes ...

  3. Biocompatibility of Poly-ε-caprolactone-hydroxyapatite composite on mouse bone marrow-derived osteoblasts and endothelial cells

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    Wooley Paul H

    2009-02-01

    Full Text Available Abstract Background Tissue-engineered bone may be developed by seeding the cells capable of both osteogenesis and vascularization on biocompatible composite scaffolds. The current study investigated the performance of mice bone marrow-derived osteogenic cells and endothelial cells as seeded on hydroxyapatite (HA and poly-ε-caprolactone (PCL composite scaffolds. Methods Mononuclear cells were induced to osteoblasts and endothelial cells respectively, which were defined by the expression of osteocalcin, alkaline phosphatase (ALP, and deposits of calcium-containing crystal for osteoblasts, or by the expression of vascular endothelial growth factor receptor-2 (VEGFR-2 and von Willebrand factor (vWF, and the formation of a capillary network in Matrigel™ for endothelial cells. Both types of cell were seeded respectively on PCL-HA scaffolds at HA to PCL weight ratio of 1:1, 1:4, or 0:1 and were evaluated using scanning electron microscopy, ALP activity (of osteoblasts and nitric oxide production (of endothelial cells plus the assessment of cell viability. Results The results indicated that HA led to a positive stimulation of osteoblasts viability and ALP activity, while HA showed less influence on endothelial cells viability. An elevated nitric oxide production of endothelial cells was observed in HA-containing group. Conclusion Supplement of HA into PCL improved biocompatible for bone marrow-derived osteoblasts and endothelial cells. The PCL-HA composite integrating with two types of cells may provide a useful system for tissue-engineered bone grafts with vascularization.

  4. Accumulation of phosphorylated alpha-synuclein (p129S) and retinal pathology in a mouse model of Parkinson's disease

    Science.gov (United States)

    Aims: Parkinson's disease (PD) is a neurodegenerative disorder characterized by accumulation of misfolded alpha-synuclein within the CNS. Although non-motor clinical phenotypes of PD such as visual dysfunction have become increasingly apparent, retinal pathology associated with PD is not well under...

  5. Detection and analysis of apoptosis- and autophagy-related miRNAs of mouse vascular endothelial cells in chronic intermittent hypoxia model.

    Science.gov (United States)

    Liu, Kai-Xiong; Chen, Gong-Ping; Lin, Ping-Li; Huang, Jian-Chai; Lin, Xin; Qi, Jia-Chao; Lin, Qi-Chang

    2018-01-15

    Endothelial dysfunction is the main pathogenic mechanism of cardiovascular complications induced by obstructive sleep apnea/hyponea syndrome (OSAHS). Chronic intermittent hypoxia (CIH) is the primary factor of OSAHS-associated endothelial dysfunction. The hypoxia inducible factor (HIF) pathway regulates the expression of downstream target genes and mediates cell apoptosis caused by CIH-induced endothelial injury. miRNAs play extensive and important negative regulatory roles in this process at the post-transcriptional level. However, the regulatory mechanism of miRNAs in CIH tissue models remains unclear. The present study established a mouse aortic endothelial cell model of CIH in an attempt to screen out specific miRNAs by using miRNA chip analysis. It was found that 14 miRNAs were differentially expressed. Of them, 6 were significantly different and verified by quantitative real-time PCR (Q-PCR), of which four were up-regulated and two were down-regulated markedly. To gain an unbiased global perspective on subsequent regulation by altered miRNAs, we established signaling networks by GO to predict the target genes of the 6 miRNAs. It was found that the 6 identified miRNAs were apoptosis- or autophagy-related target genes. Down-regulation of miR-193 inhibits CIH induced endothelial injury and apoptosis- or autophagy-related protein expression. In conclusion, our results showed that CIH could induce differential expression of miRNAs, and alteration in the miRNA expression pattern was associated with the expression of apoptosis- or autophagy-related genes. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Differentiation of retinal ganglion cells and photoreceptor precursors from mouse induced pluripotent stem cells carrying an Atoh7/Math5 lineage reporter.

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    Bin-Bin Xie

    Full Text Available The neural retina is a critical component of the visual system, which provides the majority of sensory input in humans. Various retinal degenerative diseases can result in the permanent loss of retinal neurons, especially the light-sensing photoreceptors and the centrally projecting retinal ganglion cells (RGCs. The replenishment of lost RGCs and the repair of optic nerve damage are particularly challenging, as both RGC specification and their subsequent axonal growth and projection involve complex and precise regulation. To explore the developmental potential of pluripotent stem cell-derived neural progenitors, we have established mouse iPS cells that allow cell lineage tracing of progenitors that have expressed Atoh7/Math5, a bHLH transcription factor required for RGC production. These Atoh7 lineage reporter iPS cells encode Cre to replace one copy of the endogenous Atoh7 gene and a Cre-dependent YFP reporter in the ROSA locus. In addition, they express pluripotent markers and are capable of generating teratomas in vivo. Under anterior neural induction and neurogenic conditions in vitro, the Atoh7-Cre/ROSA-YFP iPS cells differentiate into neurons that co-express various RGC markers and YFP, indicating that these neurons are derived from Atoh7-expressing progenitors. Consistent with previous in vivo cell lineage studies, the Atoh7-Cre/ROSA-YFP iPS cells also give rise to a subset of Crx-positive photoreceptor precursors. Furthermore, inhibition of Notch signaling in the iPSC cultures results in a significant increase of YFP-positive RGCs and photoreceptor precursors. Together, these results show that Atoh7-Cre/ROSA-YFP iPS cells can be used to monitor the development and survival of RGCs and photoreceptors from pluripotent stem cells.

  7. Cre recombinase expression or topical tamoxifen treatment do not affect retinal structure and function, neuronal vulnerability or glial reactivity in the mouse eye.

    Science.gov (United States)

    Boneva, S K; Groß, T R; Schlecht, A; Schmitt, S I; Sippl, C; Jägle, H; Volz, C; Neueder, A; Tamm, E R; Braunger, B M

    2016-06-14

    Mice with a constitutive or tamoxifen-induced Cre recombinase (Cre) expression are frequently used research tools to allow the conditional deletion of target genes via the Cre-loxP system. Here we analyzed for the first time in a comprehensive and comparative way, whether retinal Cre expression or topical tamoxifen treatment itself would cause structural or functional changes, including changes in the expression profiles of molecular markers, glial reactivity and photoreceptor vulnerability. To this end, we characterized the transgenic α-Cre, Lmop-Cre and the tamoxifen-inducible CAGG-CreER™ mouse lines, all having robust Cre expression in the neuronal retina. In addition, we characterized the effects of topical tamoxifen treatment itself in wildtype mice. We performed morphometric analyses, immunohistochemical staining, in vivo ERG and angiography analyses and realtime RT-PCR analyses. Furthermore, the influence of Cre recombinase or topical tamoxifen exposure on neuronal vulnerability was studied by using light damage as a model for photoreceptor degeneration. Taken together, neither the expression of Cre, nor topical tamoxifen treatment caused detectable changes in retinal structure and function, the expression profiles of investigated molecular markers, glial reactivity and photoreceptor vulnerability. We conclude that the Cre-loxP system and its induction through tamoxifen is a safe and reliable method to delete desired target genes in the neural retina. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  8. Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

    Science.gov (United States)

    Herz, Katia; Becker, Alexandra; Shi, Chenyue; Ema, Masatsugo; Takahashi, Satoru; Potente, Michael; Hesse, Michael; Fleischmann, Bernd K; Wenzel, Daniela

    2018-05-01

    Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP + signals specifically in Ki67 + /PECAM + endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

  9. Involvement of insulin-degrading enzyme in insulin- and atrial natriuretic peptide-sensitive internalization of amyloid-β peptide in mouse brain capillary endothelial cells.

    Science.gov (United States)

    Ito, Shingo; Ohtsuki, Sumio; Murata, Sho; Katsukura, Yuki; Suzuki, Hiroya; Funaki, Miho; Tachikawa, Masanori; Terasaki, Tetsuya

    2014-01-01

    Cerebral clearance of amyloid-β peptide (Aβ), which is implicated in Alzheimer's disease, involves elimination across the blood-brain barrier (BBB), and we previously showed that an insulin-sensitive process is involved in the case of Aβ1-40. The purpose of this study was to clarify the molecular mechanism of the insulin-sensitive Aβ1-40 elimination across mouse BBB. An in vivo cerebral microinjection study demonstrated that [125I]hAβ1-40 elimination from mouse brain was inhibited by human natriuretic peptide (hANP), and [125I]hANP elimination was inhibited by hAβ1-40, suggesting that hAβ1-40 and hANP share a common elimination process. Internalization of [125I]hAβ1-40 into cultured mouse brain capillary endothelial cells (TM-BBB4) was significantly inhibited by either insulin, hANP, other natriuretic peptides or insulin-degrading enzyme (IDE) inhibitors, but was not inhibited by phosphoramidon or thiorphan. Although we have reported the involvement of natriuretic peptide receptor C (Npr-C) in hANP internalization, cells stably expressing Npr-C internalized [125I]hANP but not [125I]hAβ1-40, suggesting that there is no direct interaction between Npr-C and hAβ1-40. IDE was detected in plasma membrane of TM-BBB4 cells, and internalization of [125I]hAβ1-40 by TM-BBB4 cells was reduced by IDE-targeted siRNAs. We conclude that elimination of hAβ1-40 from mouse brain across the BBB involves an insulin- and ANP-sensitive process, mediated by IDE expressed in brain capillary endothelial cells.

  10. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

    2017-04-01

    Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

  11. The P2Y12 Receptor Antagonist Ticagrelor Reduces Lysosomal pH and Autofluorescence in Retinal Pigmented Epithelial Cells From the ABCA4-/- Mouse Model of Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    Wennan Lu

    2018-04-01

    Full Text Available The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt’s disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.

  12. Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

    Science.gov (United States)

    Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

    2009-01-01

    Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

  13. Differentiation and Transplantation of Embryonic Stem Cell-Derived Cone Photoreceptors into a Mouse Model of End-Stage Retinal Degeneration

    Directory of Open Access Journals (Sweden)

    Kamil Kruczek

    2017-06-01

    Full Text Available The loss of cone photoreceptors that mediate daylight vision represents a leading cause of blindness, for which cell replacement by transplantation offers a promising treatment strategy. Here, we characterize cone differentiation in retinas derived from mouse embryonic stem cells (mESCs. Similar to in vivo development, a temporal pattern of progenitor marker expression is followed by the differentiation of early thyroid hormone receptor β2-positive precursors and, subsequently, photoreceptors exhibiting cone-specific phototransduction-related proteins. We establish that stage-specific inhibition of the Notch pathway increases cone cell differentiation, while retinoic acid signaling regulates cone maturation, comparable with their actions in vivo. MESC-derived cones can be isolated in large numbers and transplanted into adult mouse eyes, showing capacity to survive and mature in the subretinal space of Aipl1−/− mice, a model of end-stage retinal degeneration. Together, this work identifies a robust, renewable cell source for cone replacement by purified cell suspension transplantation.

  14. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    International Nuclear Information System (INIS)

    Lee, Hae-June; Yoon, Changhwan; Park, Do Joong; Kim, Yeo-Jung; Schmidt, Benjamin; Lee, Yoon-Jin; Tap, William D.; Eisinger-Mathason, T.S. Karin; Choy, Edwin; Kirsch, David G.; Simon, M. Celeste

    2015-01-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm 3 within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm 3 for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature

  15. Inhibition of Vascular Endothelial Growth Factor A and Hypoxia-Inducible Factor 1α Maximizes the Effects of Radiation in Sarcoma Mouse Models Through Destruction of Tumor Vasculature

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hae-June [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Yoon, Changhwan [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Park, Do Joong [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Department of Surgery, Seoul National University Bundang Hospital, Sungnam (Korea, Republic of); Kim, Yeo-Jung [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Schmidt, Benjamin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Lee, Yoon-Jin [Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Division of Radiation Effects, Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of); Tap, William D. [Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York (United States); Eisinger-Mathason, T.S. Karin [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Choy, Edwin [Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts (United States); Kirsch, David G. [Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (United States); Department of Radiation Oncology, Duke University Medical Center, Durham, North Carolina (United States); Simon, M. Celeste [Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (United States); Howard Hughes Medical Institute (United States); and others

    2015-03-01

    Purpose: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. Methods and Materials: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. Results: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm{sup 3} within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm{sup 3} for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. Conclusions: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

  16. EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat.

    Directory of Open Access Journals (Sweden)

    Marisa Zallocchi

    Full Text Available Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific, EIAV-CMV-MYO7A (UshStat or EIAV-CMV-Null (control vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.

  17. EIAV-based retinal gene therapy in the shaker1 mouse model for usher syndrome type 1B: development of UshStat.

    Science.gov (United States)

    Zallocchi, Marisa; Binley, Katie; Lad, Yatish; Ellis, Scott; Widdowson, Peter; Iqball, Sharifah; Scripps, Vicky; Kelleher, Michelle; Loader, Julie; Miskin, James; Peng, You-Wei; Wang, Wei-Min; Cheung, Linda; Delimont, Duane; Mitrophanous, Kyriacos A; Cosgrove, Dominic

    2014-01-01

    Usher syndrome type 1B is a combined deaf-blindness condition caused by mutations in the MYO7A gene. Loss of functional myosin VIIa in the retinal pigment epithelia (RPE) and/or photoreceptors leads to blindness. We evaluated the impact of subretinally delivered UshStat, a recombinant EIAV-based lentiviral vector expressing human MYO7A, on photoreceptor function in the shaker1 mouse model for Usher type 1B that lacks a functional Myo7A gene. Subretinal injections of EIAV-CMV-GFP, EIAV-RK-GFP (photoreceptor specific), EIAV-CMV-MYO7A (UshStat) or EIAV-CMV-Null (control) vectors were performed in shaker1 mice. GFP and myosin VIIa expression was evaluated histologically. Photoreceptor function in EIAV-CMV-MYO7A treated eyes was determined by evaluating α-transducin translocation in photoreceptors in response to low light intensity levels, and protection from light induced photoreceptor degeneration was measured. The safety and tolerability of subretinally delivered UshStat was evaluated in macaques. Expression of GFP and myosin VIIa was confirmed in the RPE and photoreceptors in shaker1 mice following subretinal delivery of the EIAV-CMV-GFP/MYO7A vectors. The EIAV-CMV-MYO7A vector protected the shaker1 mouse photoreceptors from acute and chronic intensity light damage, indicated by a significant reduction in photoreceptor cell loss, and restoration of the α-transducin translocation threshold in the photoreceptors. Safety studies in the macaques demonstrated that subretinal delivery of UshStat is safe and well-tolerated. Subretinal delivery of EIAV-CMV-MYO7A (UshStat) rescues photoreceptor phenotypes in the shaker1 mouse. In addition, subretinally delivered UshStat is safe and well-tolerated in macaque safety studies These data support the clinical development of UshStat to treat Usher type 1B syndrome.

  18. Expression of SPIG1 reveals development of a retinal ganglion cell subtype projecting to the medial terminal nucleus in the mouse.

    Directory of Open Access Journals (Sweden)

    Keisuke Yonehara

    Full Text Available Visual information is transmitted to the brain by roughly a dozen distinct types of retinal ganglion cells (RGCs defined by a characteristic morphology, physiology, and central projections. However, our understanding about how these parallel pathways develop is still in its infancy, because few molecular markers corresponding to individual RGC types are available. Previously, we reported a secretory protein, SPIG1 (clone name; D/Bsp120I #1, preferentially expressed in the dorsal region in the developing chick retina. Here, we generated knock-in mice to visualize SPIG1-expressing cells with green fluorescent protein. We found that the mouse retina is subdivided into two distinct domains for SPIG1 expression and SPIG1 effectively marks a unique subtype of the retinal ganglion cells during the neonatal period. SPIG1-positive RGCs in the dorsotemporal domain project to the dorsal lateral geniculate nucleus (dLGN, superior colliculus, and accessory optic system (AOS. In contrast, in the remaining region, here named the pan-ventronasal domain, SPIG1-positive cells form a regular mosaic and project exclusively to the medial terminal nucleus (MTN of the AOS that mediates the optokinetic nystagmus as early as P1. Their dendrites costratify with ON cholinergic amacrine strata in the inner plexiform layer as early as P3. These findings suggest that these SPIG1-positive cells are the ON direction selective ganglion cells (DSGCs. Moreover, the MTN-projecting cells in the pan-ventronasal domain are apparently composed of two distinct but interdependent regular mosaics depending on the presence or absence of SPIG1, indicating that they comprise two functionally distinct subtypes of the ON DSGCs. The formation of the regular mosaic appears to be commenced at the end of the prenatal stage and completed through the peak period of the cell death at P6. SPIG1 will thus serve as a useful molecular marker for future studies on the development and function of ON DSGCs.

  19. 1,4-Anhydro-4-seleno-d-talitol (SeTal) protects endothelial function in the mouse aorta by scavenging superoxide radicals under conditions of acute oxidative stress

    DEFF Research Database (Denmark)

    Ng, Hooi Hooi; Leo, Chen Huei; O'Sullivan, Kelly

    2017-01-01

    and decreased basal nitric oxide (NO) availability. SeTal (1mM) co-treatment prevented high glucose-induced endothelial dysfunction and oxidative stress in the mouse aorta. The presence of a cyclooxygenase inhibitor, indomethacin significantly improved the sensitivity to ACh in high glucose-treated aortae......, but had no effect in SeTal-treated aortae. Our data show that SeTal has potent antioxidant activity in isolated mouse aortae and prevents high glucose-induced endothelial dysfunction by decreasing superoxide levels, increasing basal NO availability and normalising the contribution of vasoconstrictor......Hyperglycaemia increases the generation of reactive oxidants in blood vessels and is a major cause of endothelial dysfunction. A water-soluble selenium-containing sugar (1,4-Anhydro-4-seleno-d-talitol, SeTal) has potent antioxidant activity in vitro and is a promising treatment to accelerate wound...

  20. Differential effects of vascular endothelial growth factor A isoforms in a mouse brain metastasis model of human melanoma.

    NARCIS (Netherlands)

    Kusters, B.; Waal, R.M.W. de; Wesseling, P.; Verrijp, K.; Maass, C.N.; Heerschap, A.; Barentsz, J.O.; Sweep, C.G.J.; Ruiter, D.J.; Leenders, W.P.J.

    2003-01-01

    We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood

  1. CCR7 signaling pathway and retinal neovascularization

    Directory of Open Access Journals (Sweden)

    Lin-Hui Yuan

    2015-11-01

    Full Text Available Retinal neovascularization diseases are the major causes of blindness. C-C chemokine receptor type 7(CCR7can promote the expression of vascular endothelial growth factor(VEGFthrough the extracellular signal regulated kinase(ERKpathway, leading to vascular leakage, proliferation of vascular endothelial cell, neovascularization and etc. The detection of CCR7 can guide the diagnosis and treatments of retinal neovascularization diseases.

  2. Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co-regulate axonal outgrowth of mouse retinal ganglion cells

    DEFF Research Database (Denmark)

    Gaublomme, Djoere; Buyens, Tom; De Groef, Lies

    2014-01-01

    regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model......, but not MMP-9, are involved in this process. Furthermore, administration of a novel antibody to MT1-MMP that selectively blocks pro-MMP-2 activation revealed a functional co-involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP-2 and MT1...... nervous system is lacking in adult mammals, thereby impeding recovery from injury to the nervous system. Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteases that were sporadically reported to influence axon outgrowth. Inhibition of specific MMPs reduced neurite outgrowth from...

  3. One amino acid in mouse activated factor VII defines its endothelial protein C receptor (EPCR) binding and modulates its EPCR-dependent hemostatic activity in vivo.

    Science.gov (United States)

    Pavani, G; Zintner, S M; Ivanciu, L; Small, J C; Stafford, K A; Szeto, J H; Margaritis, P

    2017-03-01

    Essentials The lack of factor (F) VIIa-endothelial protein C receptor (EPCR) binding in mice is unresolved. A single substitution of Leu4 to Phe in mouse FVIIa (mFVIIa) enables its interaction with EPCR. mFVIIa with a Phe4 shows EPCR binding-dependent enhanced hemostatic function in vivo vs. mFVIIa. Defining the FVIIa-EPCR interaction in mice allows for further investigating its biology in vivo. Background Human activated factor VII (hFVIIa), which is used in hemophilia treatment, binds to the endothelial protein C (PC) receptor (EPCR) with unclear hemostatic consequences. Interestingly, mice lack the activated FVII (FVIIa)-EPCR interaction. Therefore, to investigate the hemostatic consequences of this interaction in hemophilia, we previously engineered a mouse FVIIa (mFVIIa) molecule that bound mouse EPCR (mEPCR) by using three substitutions from mouse PC (mPC), i.e. Leu4→Phe, Leu8→Met, and Trp9→Arg. The resulting molecule, mFVIIa-FMR, modeled the EPCR-binding properties of hFVIIa and showed enhanced hemostatic capacity in hemophilic mice versus mFVIIa. These data implied a role of EPCR in the action of hFVIIa in hemophilia treatment. However, the substitutions in mFVIIa-FMR only broadly defined the sequence determinants for its mEPCR interaction and enhanced function in vivo. Objectives To determine the individual contributions of mPC Phe4, Met8 and Arg9 to the in vitro/in vivo properties of mFVIIa-FMR. Methods The mEPCR-binding properties of single amino acid variants of mFVIIa or mPC at position 4, 8 or 9 were investigated. Results and conclusions Phe4 in mFVIIa or mPC was solely critical for interaction with mEPCR. In hemophilic mice, administration of mFVIIa harboring a Phe4 resulted in a 1.9-2.5-fold increased hemostatic capacity versus mFVIIa that was EPCR binding-dependent. This recapitulated previous observations made with triple-mutant mFVIIa-FMR. As Leu8 is crucial for hFVIIa-EPCR binding, we describe the sequence divergence of this interaction in

  4. Arsenic augments the uptake of oxidized LDL by upregulating the expression of lectin-like oxidized LDL receptor in mouse aortic endothelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Ekhtear [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Ota, Akinobu, E-mail: aota@aichi-med-u.ac.jp [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Karnan, Sivasundaram; Damdindorj, Lkhagvasuren [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Takahashi, Miyuki [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Division of Hematology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan); Konishi, Yuko; Konishi, Hiroyuki; Hosokawa, Yoshitaka [Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi (Japan)

    2013-12-15

    Although chronic arsenic exposure is a well-known risk factor for cardiovascular diseases, including atherosclerosis, the molecular mechanism underlying arsenic-induced atherosclerosis remains obscure. Therefore, this study aimed to elucidate this molecular mechanism. We examined changes in the mRNA level of the lectin-like oxidized LDL (oxLDL) receptor (LOX-1) in a mouse aortic endothelial cell line, END-D, after sodium arsenite (SA) treatment. SA treatment significantly upregulated LOX-1 mRNA expression; this finding was also verified at the protein expression level. Flow cytometry and fluorescence microscopy analyses showed that the cellular uptake of fluorescence (Dil)-labeled oxLDL was significantly augmented with SA treatment. In addition, an anti-LOX-1 antibody completely abrogated the augmented uptake of Dil-oxLDL. We observed that SA increased the levels of the phosphorylated forms of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB)/p65. SA-induced upregulation of LOX-1 protein expression was clearly prevented by treatment with an antioxidant, N-acetylcysteine (NAC), or an NF-κB inhibitor, caffeic acid phenethylester (CAPE). Furthermore, SA-augmented uptake of Dil-oxLDL was also prevented by treatment with NAC or CAPE. Taken together, our results indicate that arsenic upregulates LOX-1 expression through the reactive oxygen species-mediated NF-κB signaling pathway, followed by augmented cellular oxLDL uptake, thus highlighting a critical role of the aberrant LOX-1 signaling pathway in the pathogenesis of arsenic-induced atherosclerosis. - Highlights: • Sodium arsenite (SA) increases LOX-1 expression in mouse aortic endothelial cells. • SA enhances cellular uptake of oxidized LDL in dose-dependent manner. • SA-induced ROS generation enhances phosphorylation of NF-κB. • SA upregulates LOX-1 expression through ROS-activated NF-κB signaling pathway.

  5. Protective effects of a composition of Chinese herbs-Gurigumu-13 on retinal ganglion cell apoptosis in DBA/2J glaucoma mouse model

    Directory of Open Access Journals (Sweden)

    Qiu-Li Zhang

    2018-03-01

    Full Text Available AIM: To explore the concrete mechanism of a Mongolian compound medicine-Gurigumu-13 (GRGM for glaucoma treatment. METHODS: DBA/2J mice, as glaucoma models, were intragastric administrated with GRGM to study the effect of GRGM on retinal ganglion cells (RGCs. The loss of RGCs was evaluated with the number of RGCs and axons. The expression of the target protein of RGCs or mouse retinas was determined by Western blot. The relative content of malondialdehyde (MDA was examined by ELISA assay. RESULTS: GRGM distinctly improved retina damage via increasing the number of neurons, RGCs and axons in a concentration dependent manner. Meanwhile, GRGM obviously decreased the high level of MDA and the expression of oxidative stress-related proteins in retinas of DBA/2J mice, but promoted the expression of antioxidant proteins. Additionally, GRGM also significantly inhibited the protein expression of Bip and Chop, which were markers of endoplasmic reticulum stress-induced apoptosis. CONCLUSION: GRGM have obvious protective effects on RGCs in DBA/2J mice, and increase the number of RGCs and axons via inhibiting oxidative stress and endoplasmic reticulum stress.

  6. Circulating Reactive Oxidant Causes Apoptosis of Retinal Pigment Epithelium and Cone Photoreceptors in the Mouse Central Retina

    Directory of Open Access Journals (Sweden)

    Wei Wang

    2011-01-01

    Full Text Available Reactive oxidants damage the retinal pigment epithelium (RPE, which is required for viability of overlying photoreceptors. Smoking which leads to chronic accumulation of reactive oxidants in the circulation is linked to age-related macular degeneration (AMD where RPE death is seen along with photoreceptor loss in the central macular region of the retina. It is unclear why this damage is concentrated in the central retina. We asked whether circulating oxidant might specifically target the central retina. Mice were administered the classic reactive oxidant iodate through tail vein injection, and visual acuity was followed by optokinetic response. Histology and apoptosis was examined by H&E and immunostaining. Iodate indeed selectively damaged the central retina, and this damage was highlighted by early apoptosis of RPE in the central retina followed by apoptosis of photoreceptors adjacent to the region of RPE loss–-cones were lost preferentially. The pattern and extent of this damage was independent of exposure to light. We then conclude that circulating oxidant is sufficient to selectively damage the central retina highlighted by sequential apoptosis of RPE and photoreceptors, with cones being the most sensitivity to this RPE loss.

  7. Cell Therapy Applications for Retinal Vascular Diseases: Diabetic Retinopathy and Retinal Vein Occlusion.

    Science.gov (United States)

    Park, Susanna S

    2016-04-01

    Retinal vascular conditions, such as diabetic retinopathy and retinal vein occlusion, remain leading causes of vision loss. No therapy exists to restore vision loss resulting from retinal ischemia and associated retinal degeneration. Tissue regeneration is possible with cell therapy. The goal would be to restore or replace the damaged retinal vasculature and the retinal neurons that are damaged and/or degenerating from the hypoxic insult. Currently, various adult cell therapies have been explored as potential treatment. They include mesenchymal stem cells, vascular precursor cells (i.e., CD34+ cells, hematopoietic cells or endothelial progenitor cells), and adipose stromal cells. Preclinical studies show that all these cells have a paracrine trophic effect on damaged ischemic tissue, leading to tissue preservation. Endothelial progenitor cells and adipose stromal cells integrate into the damaged retinal vascular wall in preclinical models of diabetic retinopathy and ischemia-reperfusion injury. Mesenchymal stem cells do not integrate as readily but appear to have a primary paracrine trophic effect. Early phase clinical trials have been initiated and ongoing using mesenchymal stem cells or autologous bone marrow CD34+ cells injected intravitreally as potential therapy for diabetic retinopathy or retinal vein occlusion. Adipose stromal cells or pluripotent stem cells differentiated into endothelial colony-forming cells have been explored in preclinical studies and show promise as possible therapies for retinal vascular disorders. The relative safety or efficacy of these various cell therapies for treating retinal vascular disorders have yet to be determined.

  8. Progesterone amplifies oxidative stress signal and promotes NO production via H2O2 in mouse kidney arterial endothelial cells.

    Science.gov (United States)

    Yuan, Xiao-Hua; Fan, Yang-Yang; Yang, Chun-Rong; Gao, Xiao-Rui; Zhang, Li-Li; Hu, Ying; Wang, Ya-Qin; Jun, Hu

    2016-01-01

    The role of progesterone on the cardiovascular system is controversial. Our present research is to specify the effect of progesterone on arterial endothelial cells in response to oxidative stress. Our result showed that H2O2 (150 μM and 300 μM) induced cellular antioxidant response. Glutathione (GSH) production and the activity of Glutathione peroxidase (GPx) were increased in H2O2-treated group. The expression of glutamate cysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) was induced in response to H2O2. However, progesterone absolutely abolished the antioxidant response through increasing ROS level, inhibiting the activity of Glutathione peroxidase (GPx), decreasing GSH level and reducing expression of GClC and GCLM. In our study, H2O2 induced nitrogen monoxide (NO) production and endothelial nitric oxide synthase (eNOS) expression, and progesterone promoted H2O2-induced NO production. Progesterone increased H2O2-induced expression of hypoxia inducible factor-α (HIFα) which in turn regulated eNOS expression and NO synthesis. Further study demonstrated that progesterone increased H2O2 concentration of culture medium which may contribute to NO synthesis. Exogenous GSH decreased the content of H2O2 of culture medium pretreated by progesterone combined with H2O2 or progesterone alone. GSH also inhibited expression of HIFα and eNOS, and abolished NO synthesis. Collectively, our study demonstrated for the first time that progesterone inhibited cellular antioxidant effect and increased oxidative stress, promoted NO production of arterial endothelial cells, which may be due to the increasing H2O2 concentration and amplified oxidative stress signal. Copyright © 2015. Published by Elsevier Ltd.

  9. The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1.

    Science.gov (United States)

    Meijles, Daniel N; Sahoo, Sanghamitra; Al Ghouleh, Imad; Amaral, Jefferson H; Bienes-Martinez, Raquel; Knupp, Heather E; Attaran, Shireen; Sembrat, John C; Nouraie, Seyed M; Rojas, Mauricio M; Novelli, Enrico M; Gladwin, Mark T; Isenberg, Jeffrey S; Cifuentes-Pagano, Eugenia; Pagano, Patrick J

    2017-10-17

    Senescent cells withdraw from the cell cycle and do not proliferate. The prevalence of senescent compared to normally functioning parenchymal cells increases with age, impairing tissue and organ homeostasis. A contentious principle governing this process has been the redox theory of aging. We linked matricellular protein thrombospondin 1 (TSP1) and its receptor CD47 to the activation of NADPH oxidase 1 (Nox1), but not of the other closely related Nox isoforms, and associated oxidative stress, and to senescence in human cells and aged tissue. In human endothelial cells, TSP1 promoted senescence and attenuated cell cycle progression and proliferation. At the molecular level, TSP1 increased Nox1-dependent generation of reactive oxygen species (ROS), leading to the increased abundance of the transcription factor p53. p53 mediated a DNA damage response that led to senescence through Rb and p21 cip , both of which inhibit cell cycle progression. Nox1 inhibition blocked the ability of TSP1 to increase p53 nuclear localization and p21 cip abundance and its ability to promote senescence. Mice lacking TSP1 showed decreases in ROS production, p21 cip expression, p53 activity, and aging-induced senescence. Conversely, lung tissue from aging humans displayed increases in the abundance of vascular TSP1, Nox1, p53, and p21 cip Finally, genetic ablation or pharmacological blockade of Nox1 in human endothelial cells attenuated TSP1-mediated ROS generation, restored cell cycle progression, and protected against senescence. Together, our results provide insights into the functional interplay between TSP1 and Nox1 in the regulation of endothelial senescence and suggest potential targets for controlling the aging process at the molecular level. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  10. Studies of Scleral Biomechanical Behavior Related to Susceptibility for Retinal Ganglion Cell Loss in Experimental Mouse Glaucoma

    Science.gov (United States)

    Nguyen, Cathy; Cone, Frances E.; Nguyen, Thao D.; Coudrillier, Baptiste; Pease, Mary E.; Steinhart, Matthew R.; Oglesby, Ericka N.; Jefferys, Joan L.; Quigley, Harry A.

    2013-01-01

    Purpose. To study anatomical changes and mechanical behavior of the sclera in mice with experimental glaucoma by comparing CD1 to B6 mice. Methods. Chronic experimental glaucoma for 6 weeks was produced in 2- to 4-month-old CD1 (43 eyes) and B6 mice (42 eyes) using polystyrene bead injection into the anterior chamber with 126 control CD1 and 128 control B6 eyes. Intraocular pressure (IOP) measurements were made with the TonoLab at baseline and after bead injection. Axial length and scleral thickness were measured after sacrifice in the CD1 and B6 animals and compared to length data from 78 eyes of DBA/2J mice. Inflation testing of posterior sclera was conducted, and circumferential and meridional strain components were determined from the displacement response. Results. Experimental glaucoma led to increases in axial length and width by comparison to fellow eyes (6% in CD1 and 10% in B6; all P glaucoma, the remainder of the sclera uniformly thinned in CD1, but thickened in B6. Peripapillary sclera in CD1 controls had significantly greater temporal meridional strain than B6 and had differences in the ratios of meridional to effective circumferential strain from B6 mice. In both CD1 and B6 mice, exposure to chronic IOP elevation resulted in stiffer pressure–strain responses for both the effective circumferential and meridional strains (multivariable regression model, P = 0.01–0.03). Conclusions. Longer eyes, greater scleral strain in some directions at baseline, and generalized scleral thinning after glaucoma were characteristic of CD1 mice that have greater tendency to retinal ganglion cell damage than B6 mice. Increased scleral stiffness after glaucoma exposure in mice mimics findings in monkey and human glaucoma eyes. PMID:23404116

  11. Intimal cushions and endothelial nuclear elongation around mouse aortic branches and their spatial correspondence with patterns of lipid deposition

    Science.gov (United States)

    Bond, Andrew R.; Ni, Chih-Wen; Jo, Hanjoong

    2010-01-01

    Spatial variation in hemodynamic stresses acting on the arterial wall may explain the nonuniform distribution of atherosclerosis. In thoracic aortas of LDL receptor/apolipoprotein E double knockout mice, lesions develop preferentially around the entire circumference of intercostal branch ostia, regardless of age, with the highest prevalence occurring upstream. Additional chevron-shaped lesions occur further upstream of the ostia. This pattern differs from the age-related ones occurring in people and rabbits. In the present study, patterns of near-wall blood flow around intercostal ostia in wild-type mice were estimated from the morphology of endothelial nuclei, which were shown in vitro to elongate in response to elevated shear stress and to align with the flow, and wall structure was assessed from confocal and scanning electron microscopy. A triangular intimal cushion surrounded the upstream part of most ostia. Nuclear length-to-width ratios were lowest over this cushion and highest at the sides of branches, regardless of age. Nuclear orientations were consistent with flow diverging around the branch. The pattern of nuclear morphology differed from the age-related ones observed in rabbits. The intimal cushion and the distribution of shear stress inferred from these observations can partly account for the pattern of lesions observed in knockout mice. Nuclear elongation in nonbranch regions was approximately constant across animals of different size, demonstrating the existence of a mechanism by which endothelial cells compensate for the dependence of mean aortic wall shear stress on body mass. PMID:19933414

  12. Retinitis Pigmentosa.

    Science.gov (United States)

    Carr, Ronald E.

    1979-01-01

    The author describes the etiology of retinitis pigmentosa, a visual dysfunction which results from progressive loss of the retinal photoreceptors. Sections address signs and symptoms, ancillary findings, heredity, clinical diagnosis, therapy, and research. (SBH)

  13. Retinitis Pigmentosa

    Science.gov (United States)

    ... Linked Retinoschisis (XLRS) X-Linked Retinitis Pigmentosa (XLRP) Usher Syndrome Other Retinal Diseases Glossary News & Research News & Research ... degenerate. Forms of RP and related diseases include Usher syndrome, Leber congenital amaurosis, and Bardet-Biedl syndrome, among ...

  14. Retinal Diseases

    Science.gov (United States)

    ... Linked Retinoschisis (XLRS) X-Linked Retinitis Pigmentosa (XLRP) Usher Syndrome Other Retinal Diseases Glossary News & Research News & Research ... central portion of the retina called the macula. Usher Syndrome Usher syndrome is an inherited condition characterized by ...

  15. miR-214-Dependent Increase of PHLPP2 Levels Mediates the Impairment of Insulin-Stimulated Akt Activation in Mouse Aortic Endothelial Cells Exposed to Methylglyoxal

    Directory of Open Access Journals (Sweden)

    Cecilia Nigro

    2018-02-01

    Full Text Available Evidence has been provided linking microRNAs (miRNAs and diabetic complications, by the regulation of molecular pathways, including insulin-signaling, involved in the pathophysiology of vascular dysfunction. Methylglyoxal (MGO accumulates in diabetes and is associated with cardiovascular complications. This study aims to analyze the contribution of miRNAs in the MGO-induced damaging effect on insulin responsiveness in mouse aortic endothelial cells (MAECs. miRNA modulation was performed by transfection of specific miRNA mimics and inhibitors in MAECs, treated or not with MGO. miRNA-target protein levels were evaluated by Western blot. PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2 regulation by miR-214 was tested by luciferase assays and by the use of a target protector specific for miR-214 on PHLPP2-3′UTR. This study reveals a 4-fold increase of PHLPP2 in MGO-treated MAECs. PHLPP2 levels inversely correlate with miR-214 modulation. Moreover, miR-214 overexpression is able to reduce PHLPP2 levels in MGO-treated MAECs. Interestingly, a direct regulation of PHLPP2 is proved to be dependent by miR-214. Finally, the inhibition of miR-214 impairs the insulin-dependent Akt activation, while its overexpression rescues the insulin effect on Akt activation in MGO-treated MAECs. In conclusion, this study shows that PHLPP2 is a target of miR-214 in MAECs, and identifies miR-214 downregulation as a contributing factor to MGO-induced endothelial insulin-resistance.

  16. Overexpression of catalase delays G0/G1- to S-phase transition during cell cycle progression in mouse aortic endothelial cells.

    Science.gov (United States)

    Onumah, Ogbeyalu E; Jules, George E; Zhao, Yanfeng; Zhou, LiChun; Yang, Hong; Guo, ZhongMao

    2009-06-15

    Although it is understood that hydrogen peroxide (H(2)O(2)) promotes cellular proliferation, little is known about its role in endothelial cell cycle progression. To assess the regulatory role of endogenously produced H(2)O(2) in cell cycle progression, we studied the cell cycle progression in mouse aortic endothelial cells (MAECs) obtained from mice overexpressing a human catalase transgene (hCatTg), which destroys H(2)O(2). The hCatTg MAECs displayed a prolonged doubling time compared to wild-type controls (44.0 +/- 4.7 h versus 28.6 +/- 0.8 h, pcatalase inhibitor, prevented the observed diminished growth rate in hCatTg MAECs. Inhibition of catalase activity with aminotriazole abrogated catalase overexpression-induced antiproliferative action. Flow cytometry analysis indicated that the prolonged doubling time was principally due to an extended G(0)/G(1) phase in hCatTg MAECs compared to the wild-type cells (25.0 +/- 0.9 h versus 15.9 +/- 1.4 h, pinhibitors, p21 and p27, which inhibit the Cdk activity required for the G(0)/G(1)- to S-phase transition. Knockdown of p21 and/or p27 attenuated the antiproliferative effect of catalase overexpression in MAECs. These results, together with the fact that catalase is an H(2)O(2) scavenger, suggest that endogenously produced H(2)O(2) mediates MAEC proliferation by fostering the transition from G(0)/G(1) to S phase.

  17. Increased Retinal Expression of the Pro-Angiogenic Receptor GPR91 via BMP6 in a Mouse Model of Juvenile Hemochromatosis.

    Science.gov (United States)

    Arjunan, Pachiappan; Gnanaprakasam, Jaya P; Ananth, Sudha; Romej, Michelle A; Rajalakshmi, Veeranan-Karmegam; Prasad, Puttur D; Martin, Pamela M; Gurusamy, Mariappan; Thangaraju, Muthusamy; Bhutia, Yangzom D; Ganapathy, Vadivel

    2016-04-01

    Hemochromatosis, an iron-overload disease, occurs as adult and juvenile types. Mutations in hemojuvelin (HJV), an iron-regulatory protein and a bone morphogenetic protein (BMP) coreceptor, underlie most of the juvenile type. Hjv(-/-) mice accumulate excess iron in retina and exhibit aberrant vascularization and angiomas. A succinate receptor, GPR91, is pro-angiogenic in retina. We hypothesized that Hjv(-/-) retinas have increased BMP signaling and increased GPR91 expression as the basis of angiomas. Expression of GPR91 was examined by qPCR, immunofluorescence, and Western blot in wild-type and Hjv(-/-) mouse retinas and pRPE cells. Influence of excess iron and BMP6 on GPR91 expression was investigated in ARPE-19 cells, and wild-type and Hjv(-/-) pRPE cells. Succinate was used to activate GPR91 and determine the effects of GPR91 signaling on VEGF expression. Signaling of BMP6 was studied by the expression of Smad1/5/8 and pSmad4, and the BMP-target gene Id1. The interaction of pSmad4 with GPR91 promoter was studied by ChIP. Expression of GPR91 was higher in Hjv(-/-) retinas and RPE than in wild-type counterparts. Unexpectedly, BMP signaling was increased, not decreased, in Hjv(-/-) retinas and RPE. Bone morphogenetic protein 6 induced GPR91 in RPE, suggesting that increased BMP signaling in Hjv(-/-) retinas was likely responsible for GPR91 upregulation. Exposure of RPE to excess iron and succinate as well as BMP6 and succinate increased VEGF expression. Bone morphogenetic protein 6 promoted the interaction of pSmad4 with GPR91 promoter in RPE. G-protein-coupled receptor 91 is a BMP6 target and Hjv deletion enhances BMP signaling in retina, thus underscoring a role for excess iron and hemochromatosis in abnormal retinal vascularization.

  18. Vascular endothelial growth factor-D over-expressing tumor cells induce differential effects on uterine vasculature in a mouse model of endometrial cancer

    Directory of Open Access Journals (Sweden)

    Stacker Steven A

    2010-07-01

    Full Text Available Abstract Background It has been hypothesised that increased VEGF-D expression may be an independent prognostic factor for endometrial cancer progression and lymph node metastasis; however, the mechanism by which VEGF-D may promote disease progression in women with endometrial cancer has not been investigated. Our aim was to describe the distribution of lymphatic vessels in mouse uterus and to examine the effect of VEGF-D over-expression on these vessels in a model of endometrial cancer. We hypothesised that VEGF-D over-expression would stimulate growth of new lymphatic vessels into the endometrium, thereby contributing to cancer progression. Methods We initially described the distribution of lymphatic vessels (Lyve-1, podoplanin, VEGFR-3 and VEGF-D expression in the mouse uterus during the estrous cycle, early pregnancy and in response to estradiol-17beta and progesterone using immunohistochemistry. We also examined the effects of VEGF-D over-expression on uterine vasculature by inoculating uterine horns in NOD SCID mice with control or VEGF-D-expressing 293EBNA tumor cells. Results Lymphatic vessels positive for the lymphatic endothelial cell markers Lyve-1, podoplanin and VEGFR-3 profiles were largely restricted to the connective tissue between the myometrial circular and longitudinal muscle layers; very few lymphatic vessel profiles were observed in the endometrium. VEGF-D immunostaining was present in all uterine compartments (epithelium, stroma, myometrium, although expression was generally low. VEGF-D immunoexpression was slightly but significantly higher in estrus relative to diestrus; and in estradiol-17beta treated mice relative to vehicle or progesterone treated mice. The presence of VEGF-D over-expressing tumor cells did not induce endometrial lymphangiogenesis, although changes were observed in existing vessel profiles. For myometrial lymphatic and endometrial blood vessels, the percentage of profiles containing proliferating

  19. Magnetic Resonance Tracking of Endothelial Progenitor Cells Labeled with Alkyl-Polyethylenimine 2 kDa/Superparamagnetic Iron Oxide in a Mouse Lung Carcinoma Xenograft Model

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    Cong Chen

    2014-11-01

    Full Text Available The potential of using endothelial progenitor cells (EPCs in novel anticancer therapy and the repair of vascular injury has been increasingly recognized. In the present study, EPCs were labeled with N-alkyl-polyethylenimine 2 kDa (PEI2k-stabilized superparamagnetic iron oxide (SPIO to facilitate magnetic resonance imaging (MRI of EPCs in a mouse lung carcinoma xenograft model. EPCs derived from human peripheral blood were labeled with alkyl-PEI2k/SPIO. The viability and activity of labeled cells were evaluated using proliferation, migration, and tubulogenesis assays. Alkyl-PEI2k/SPIO-labeled EPCs were injected intravenously (group 1 or mixed and injected together with A549 cells subcutaneously (group 2 into groups of six mice with severe combined immunodeficiency. The labeling efficiency with alkyl-PEI2k/SPIO at 7 mg Fe/mL concentration was approximately 100%. Quantitative analysis of cellular iron was 6.062 ± 0.050 pg/cell. No significant effects on EPC proliferation, migration, or tubulogenesis were seen after labeling. Seventesla micro-MRI showed the presence of schistic or linear hypointense regions at the tumor margins starting from days 7 to 8 after EPC administration. This gradually extended into the inner tumor layers in group 1. In group 2, tumor growth was accompanied by dispersion of low-signal intensity regions inside the tumor. Iron-positive cells identified by Prussian blue dye were seen at the sites identified using MRI. Human CD31-positive cells and mouse CD31-positive cells were present in both groups. Labeling EPCs with alkyl-PEI2k/SPIO allows noninvasive magnetic resonance investigation of EPC involvement in tumor neovasculature and is associated with excellent biocompatibility and MRI sensitivity.

  20. Retinal Vasculitis

    Science.gov (United States)

    Rosenbaum, James T.; Sibley, Cailin H.; Lin, Phoebe

    2016-01-01

    Purpose of review Ophthalmologists and rheumatologists frequently miscommunicate in consulting on patients with retinal vasculitis. This report seeks to establish a common understanding of the term, retinal vasculitis, and to review recent papers on this diagnosis. Recent findings 1) The genetic basis of some rare forms of retinal vascular disease have recently been described. Identified genes include CAPN5, TREX1, and TNFAIP3; 2) Behçet’s disease is a systemic illness that is very commonly associated with occlusive retinal vasculitis; 3) retinal imaging including fluorescein angiography and other newer imaging modalities has proven crucial to the identification and characterization of retinal vasculitis and its complications; 4) although monoclonal antibodies to IL-17A or IL-1 beta failed in trials for Behçet’s disease, antibodies to TNF alpha, either infliximab or adalimumab, have demonstrated consistent benefit in managing this disease. Interferon treatment and B cell depletion therapy via rituximab may be beneficial in certain types of retinal vasculitis. Summary Retinal vasculitis is an important entity for rheumatologists to understand. Retinal vasculitis associated with Behçet’s disease responds to monoclonal antibodies that neutralize TNF, but the many other forms of non-infectious retinal vasculitis may require alternate therapeutic management. PMID:26945335

  1. Vasoinhibins regulate the inner and outer blood-retinal barrier and limit retinal oxidative stress.

    Science.gov (United States)

    Arredondo Zamarripa, David; Díaz-Lezama, Nundehui; Meléndez García, Rodrigo; Chávez Balderas, Jesús; Adán, Norma; Ledesma-Colunga, Maria G; Arnold, Edith; Clapp, Carmen; Thebault, Stéphanie

    2014-01-01

    Vasoinhibins are prolactin fragments present in the retina, where they have been shown to prevent the hypervasopermeability associated with diabetes. Enhanced bradykinin (BK) production contributes to the increased transport through the blood-retina barrier (BRB) in diabetes. Here, we studied if vasoinhibins regulate BRB permeability by targeting the vascular endothelium and retinal pigment epithelium (RPE) components of this barrier. Intravitreal injection of BK in male rats increased BRB permeability. Vasoinhibins prevented this effect, as did the B2 receptor antagonist Hoe-140. BK induced a transient decrease in mouse retinal and brain capillary endothelial monolayer resistance that was blocked by vasoinhibins. Both vasoinhibins and the nitric oxide (NO) synthase inhibitor L-NAME, but not the antioxidant N-acetyl cysteine (NAC), blocked the transient decrease in bovine umbilical vein endothelial cell (BUVEC) monolayer resistance induced by BK; this block was reversed by the NO donor DETANONOate. Vasoinhibins also prevented the BK-induced actin cytoskeleton redistribution, as did L-NAME. BK transiently decreased human RPE (ARPE-19) cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC. DETANONOate reverted the blocking effect of vasoinhibins. Similar to BK, the radical initiator Luperox induced a reduction in ARPE-19 cell monolayer resistance, which was prevented by vasoinhibins. These effects on RPE resistance coincided with actin cytoskeleton redistribution. Intravitreal injection of vasoinhibins reduced the levels of reactive oxygen species (ROS) in retinas of streptozotocin-induced diabetic rats, particularly in the RPE and capillary-containing layers. Thus, vasoinhibins reduce BRB permeability by targeting both its main inner and outer components through NO- and ROS-dependent pathways, offering potential treatment strategies against diabetic retinopathies.

  2. Vasoinhibins regulate the inner and outer blood-retinal barrier and limit retinal oxidative stress

    Directory of Open Access Journals (Sweden)

    David eArredondo Zamarripa

    2014-10-01

    Full Text Available Vasoinhibins are prolactin fragments present in the retina, where they have been shown to prevent the hypervasopermeability associated with diabetes. Enhanced bradykinin (BK production contributes to the increased transport through the blood-retina barrier (BRB in diabetes. Here, we studied if vasoinhibins regulate BRB permeability by targeting the vascular endothelium and retinal pigment epithelium (RPE components of this barrier. Intravitreal injection of BK in male rats increased BRB permeability. Vasoinhibins prevented this effect, as did the B2 receptor antagonist Hoe-140. BK induced a transient decrease in mouse retinal and brain capillary endothelial monolayer resistance that was blocked by vasoinhibins. Both vasoinhibins and the nitric oxide (NO synthase inhibitor L-NAME, but not the antioxidant N-acetyl cysteine (NAC, blocked the transient decrease in bovine umbilical vein endothelial cell (BUVEC monolayer resistance induced by BK; this block was reversed by the NO donor DETANONOate. Vasoinhibins also prevented the BK-induced actin cytoskeleton redistribution, as did L-NAME. BK transiently decreased human RPE (ARPE-19 cell monolayer resistance, and this effect was blocked by vasoinhibins, L-NAME, and NAC. DETANONOate reverted the blocking effect of vasoinhibins. Similar to BK, the radical initiator Luperox induced a reduction in ARPE-19 cell monolayer resistance, which was prevented by vasoinhibins. These effects on RPE resistance coincided with actin cytoskeleton redistribution. Intravitreal injection of vasoinhibins reduced the levels of reactive oxygen species (ROS in retinas of streptozotocin-induced diabetic rats, particularly in the RPE and capillary-containing layers. Thus, vasoinhibins reduce BRB permeability by targeting both its main inner and outer components through NO- and ROS-dependent pathways, offering potential treatment strategies against diabetic retinopathies.

  3. An evidence-based meta-analysis of vascular endothelial growth factor inhibition in pediatric retinal diseases: part 1. Retinopathy of prematurity.

    Science.gov (United States)

    Mititelu, Mihai; Chaudhary, Khurram M; Lieberman, Ronni M

    2012-01-01

    Recently there has been interest in the novel, off-label use of anti-vascular endothelial growth factor (anti-VEGF) agents for various stages of retinopathy of prematurity (ROP). The authors report on the quality and depth of new evidence published from 2009 to 2011 concerning the treatment of retinopathy of prematurity (ROP) with bevacizumab (Avastin; Genentech Inc., South San Francisco, CA) as either primary or adjunctive treatment for ROP. There is significant variability in the evidence, quality, and design of the studies available in the literature. There has been a trend in the scientific literature of the past 2 years toward larger, multi-center, randomized studies investigating the role of bevacizumab in the treatment of ROP. More recent evidence suggests that monotherapy with intravitreal bevacizumab may be a viable first-line treatment for select cases of zone I ROP and possibly for posterior zone II disease. Adjunctive treatment with bevacizumab may enhance outcomes in patients treated with laser photocoagulation or pars plana vitrectomy. However, there are significant concerns regarding its long-term safety profile. Further prospective studies are warranted to more fully determine the role of anti-VEGF therapy in this disease. Copyright 2012, SLACK Incorporated.

  4. Cholesterol enhances amyloid {beta} deposition in mouse retina by modulating the activities of A{beta}-regulating enzymes in retinal pigment epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jiying [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Ohno-Matsui, Kyoko, E-mail: k.ohno.oph@tmd.ac.jp [Department of Ophthalmology and Visual Science, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan); Morita, Ikuo [Section of Cellular Physiological Chemistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519 (Japan)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer Cholesterol-treated RPE produces more A{beta} than non-treated RPE. Black-Right-Pointing-Pointer Neprilysin expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer {alpha}-Secretase expression and activity decreased in cholesterol-treated RPE. Black-Right-Pointing-Pointer Cholesterol-enriched diet induced subRPE deposits in aged mice. Black-Right-Pointing-Pointer A{beta} were present in cholesterol-enriched-diet-induced subRPE deposits in aged mice. -- Abstract: Subretinally-deposited amyloid {beta} (A{beta}) is a main contributor of developing age-related macular degeneration (AMD). However, the mechanism causing A{beta} deposition in AMD eyes is unknown. Hypercholesterolemia is a significant risk for developing AMD. Thus, we investigated the effects of cholesterol on A{beta} production in retinal pigment epithelial (RPE) cells in vitro and in the mouse retina in vivo. RPE cells isolated from senescent (12-month-old) C57BL/6 mice were treated with 10 {mu}g/ml cholesterol for 48 h. A{beta} amounts in culture supernatants were measured by ELISA. Activity and expression of enzymes and proteins that regulate A{beta} production were examined by activity assay and real time PCR. The retina of mice fed cholesterol-enriched diet was examined by transmission electron microscopy. Cholesterol significantly increased A{beta} production in cultured RPE cells. Activities of A{beta} degradation enzyme; neprilysin (NEP) and anti-amyloidogenic secretase; {alpha}-secretase were significantly decreased in cell lysates of cholesterol-treated RPE cells compared to non-treated cells, but there was no change in the activities of {beta}- or {gamma}-secretase. mRNA levels of NEP and {alpha}-secretase (ADAM10 and ADAM17) were significantly lower in cholesterol-treated RPE cells than non-treated cells. Senescent (12-month-old) mice fed cholesterol-enriched chow developed subRPE deposits containing A{beta}, whereas

  5. Retinal vasculitis.

    Science.gov (United States)

    Abu El-Asrar, Ahmed M; Herbort, Carl P; Tabbara, Khalid F

    2005-12-01

    Retinal vasculitis is a sight-threatening intraocular inflammation affecting the retinal vessels. It may occur as an isolated ocular condition, as a manifestation of infectious or neoplastic disorders, or in association with a systemic inflammatory disease. The search for an underlying etiology should be approached in a multidisciplinary fashion based on a thorough history, review of systems, physical examination, and laboratory evaluation. Discrimination between infectious and noninfectious etiologies of retinal vasculitis is important because their treatment is different. This review is based on recently published articles on retinal vasculitis and deals with its clinical diagnosis, its link with systemic diseases, and its laboratory investigation.

  6. Retinal progenitor cell xenografts to the pig retina

    DEFF Research Database (Denmark)

    Warfvinge, Karin; Kiilgaard, Jens Folke; Lavik, Erin B

    2005-01-01

    To investigate the survival, integration, and differentiation of mouse retinal progenitor cells after transplantation to the subretinal space of adult pigs.......To investigate the survival, integration, and differentiation of mouse retinal progenitor cells after transplantation to the subretinal space of adult pigs....

  7. Simultaneous Fluorescein Angiography and Spectral Domain Optical Coherence Tomography Correlate Retinal Thickness Changes to Vascular Abnormalities in an In Vivo Mouse Model of Retinopathy of Prematurity

    Directory of Open Access Journals (Sweden)

    Olachi J. Mezu-Ndubuisi

    2017-01-01

    Full Text Available Background. Retinopathy of prematurity (ROP is a condition of abnormal retinal vascular development (RVD in premature infants. Fluorescein angiography (FA has depicted phases (early, mid, late, and mature of RVD in oxygen-induced retinopathy (OIR mice. We sought to establish the relationship between retinal structural and vascular changes using simultaneous FA and spectral domain optical coherence tomography (SD-OCT. Method. 63 mice were exposed to 77% oxygen at postnatal day 7 (P7 for 5 days, while 63 mice remained in room air (RA. Total retinal thickness (TRT, inner retinal thickness (IRT, and outer retinal thickness (ORT were calculated at early (P19, mid (P24, late (P32, and mature (P47 phases of RVD. Results. TRT was reduced in OIR (162.66 ± 17.75 μm, n=13 compared to RA mice at P19 (197.57 ± 3.49 μm, n=14, P24, P32, and P49 (P0.05. IRT was reduced in OIR (71.60 ± 17.14 μm compared to RA (103.07 ± 3.47 μm mice at P19 and all ages (P<0.0001. Conclusion. We have shown the spatial and temporal relationship between retinal structure and vascular development in OIR. Significant inner retinal thinning in OIR mice persisted despite revascularization of the capillary network; further studies will elucidate its functional implications in ROP.

  8. Ketamine alleviates bradykinin-induced disruption of the mouse cerebrovascular endothelial cell-constructed tight junction barrier via a calcium-mediated redistribution of occludin polymerization

    International Nuclear Information System (INIS)

    Chen, Jui-Tai; Lin, Yi-Ling; Chen, Ta-Liang; Tai, Yu-Ting; Chen, Cheng-Yu; Chen, Ruei-Ming

    2016-01-01

    Highlights: • Ketamine could suppress bradykinin-induced intracellular calcium mobilization. • Ketamine induced B1R protein and mRNA expressions but did not change B2R protein levels. • Ketamine attenuated bradykinin-induced redistribution of occludin tight junctions. • Ketamine prevented bradykinin-induced breakage of the MCEC-constructed tight junction barrier. - Abstract: Following brain injury, a sequence of mechanisms leads to disruption of the blood-brain barrier (BBB) and subsequent cerebral edema, which is thought to begin with activation of bradykinin. Our previous studies showed that ketamine, a widely used intravenous anesthetic agent, can suppress bradykinin-induced cell dysfunction. This study further aimed to evaluate the protective effects of ketamine against bradykinin-induced disruption of the mouse cerebrovascular endothelial cell (MCEC)-constructed tight junction barrier and the possible mechanisms. Exposure of MCECs to bradykinin increased intracellular calcium (Ca 2+ ) concentrations in a time-dependent manner. However, pretreatment of MCECs with ketamine time- and concentration-dependently lowered the bradykinin-induced calcium influx. As to the mechanisms, although exposure of MCECs to ketamine induced bradykinin R1 receptor protein and mRNA expression, this anesthetic did not change levels of the bradykinin R2 receptor, a major receptor that responds to bradykinin stimulation. Bradykinin increased amounts of soluble occludin in MCECs, but pretreatment with ketamine alleviated this disturbance in occludin polymerization. Consequently, exposure to bradykinin decreased the transendothelial electronic resistance in the MCEC-constructed tight junction barrier. However, pretreatment with ketamine attenuated the bradykinin-induced disruption of the tight junction barrier. Taken together, this study shows that ketamine at a therapeutic concentration can protect against bradykinin-induced breakage of the BBB via suppressing calcium

  9. Fibroblast growth factor-2-induced host stroma reaction during initial tumor growth promotes progression of mouse melanoma via vascular endothelial growth factor A-dependent neovascularization.

    Science.gov (United States)

    Tsunoda, Satoshi; Nakamura, Toshiyuki; Sakurai, Hiroaki; Saiki, Ikuo

    2007-04-01

    Fibroblast growth factor (FGF)-2 has been considered to play a critical role in neovascularization in several tumors; however, its precise role in tumor progression is not fully understood. In the present study, we have characterized the role of FGF-2 in B16-BL6 mouse melanoma cells, focusing on effects during the initial phase of tumor growth. FGF-2 was injected at the tumor inoculation site of dorsal skin during the initial phase. FGF-2 induced marked tumor growth and lymph node metastasis. This was well correlated with an increase in neovascularization in the host stroma. FGF-2 also recruited inflammatory and mesenchymal cells in host stroma. Marked tumor growth, pulmonary metastasis and intensive neovascularization in tumor parenchyma were also observed after a single injection of FGF-2 into the footpad inoculation site. In contrast, repeated injections of FGF-2 at a site remote from the footpad tumor were ineffective in promoting tumor growth and metastasis. These promoting activities of FGF-2 were blocked by local injections of a glucocorticoid hormone, suggesting that host inflammatory responses induced by FGF-2 are associated with FGF-2-induced tumor progression. In addition, although FGF-2 did not promote cellular proliferation and vascular endothelial growth factor A (VEGFA) mRNA expression in B16-BL6 cells in vitro, FGF-2 induced VEGFA expression in host stroma rather than tumor tissue, and local injections of a neutralizing antibody against VEGFA inhibited these activities of FGF-2 in vivo. These results indicate that abundant FGF-2 during the initial phase of tumor growth induces VEGFA-dependent intensive neovascularization in host stroma, and supports marked tumor growth and metastasis.

  10. Culture media-based selection of endothelial cells, pericytes, and perivascular-resident macrophage-like melanocytes from the young mouse vestibular system.

    Science.gov (United States)

    Zhang, Jinhui; Chen, Songlin; Cai, Jing; Hou, Zhiqiang; Wang, Xiaohan; Kachelmeier, Allan; Shi, Xiaorui

    2017-03-01

    The vestibular blood-labyrinth barrier (BLB) is comprised of perivascular-resident macrophage-like melanocytes (PVM/Ms) and pericytes (PCs), in addition to endothelial cells (ECs) and basement membrane (BM), and bears strong resemblance to the cochlear BLB in the stria vascularis. Over the past few decades, in vitro cell-based models have been widely used in blood-brain barrier (BBB) and blood-retina barrier (BRB) research, and have proved to be powerful tools for studying cell-cell interactions in their respective organs. Study of both the vestibular and strial BLB has been limited by the unavailability of primary culture cells from these barriers. To better understand how barrier component cells interact in the vestibular system to control BLB function, we developed a novel culture medium-based method for obtaining EC, PC, and PVM/M primary cells from tiny explants of the semicircular canal, sacculus, utriculus, and ampullae tissue of young mouse ears at post-natal age 8-12 d. Each phenotype is grown in a specific culture medium which selectively supports the phenotype in a mixed population of vestibular cell types. The unwanted phenotypes do not survive passaging. The protocol does not require additional equipment or special enzyme treatment. The harvesting process takes less than 2 h. Primary cell types are generated within 7-10 d. The primary culture ECs, PCs, and PVM/M shave consistent phenotypes more than 90% pure after two passages (∼ 3 weeks). The highly purified primary cell lines can be used for studying cell-cell interactions, barrier permeability, and angiogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Diabetes and Retinal Vascular Dysfunction

    Directory of Open Access Journals (Sweden)

    Eui Seok Shin

    2014-01-01

    Full Text Available Diabetes predominantly affects the microvascular circulation of the retina resulting in a range of structural changes unique to this tissue. These changes ultimately lead to altered permeability, hyperproliferation of endothelial cells and edema, and abnormal vascularization of the retina with resulting loss of vision. Enhanced production of inflammatory mediators and oxidative stress are primary insults with significant contribution to the pathogenesis of diabetic retinopathy (DR. We have determined the identity of the retinal vascular cells affected by hyperglycemia, and have delineated the cell autonomous impact of high glucose on function of these cells. We discuss some of the high glucose specific changes in retinal vascular cells and their contribution to retinal vascular dysfunction. This knowledge provides novel insight into the molecular and cellular defects contributing to the development and progression of diabetic retinopathy, and will aid in the development of innovative, as well as target specific therapeutic approaches for prevention and treatment of DR.

  12. Advances in Retinal Stem Cell Biology

    Directory of Open Access Journals (Sweden)

    Andrea S Viczian

    2013-01-01

    Full Text Available Tremendous progress has been made in recent years to generate retinal cells from pluripotent cell sources. These advances provide hope for those suffering from blindness due to lost retinal cells. Understanding the intrinsic genetic network in model organisms, like fly and frog, has led to a better understanding of the extrinsic signaling pathways necessary for retinal progenitor cell formation in mouse and human cell cultures. This review focuses on the culture methods used by different groups, which has culminated in the generation of laminated retinal tissue from both embryonic and induced pluripotent cells. The review also briefly describes advances made in transplantation studies using donor retinal progenitor and cultured retinal cells.

  13. [Indications for Retinal Laser Therapy Revisited].

    Science.gov (United States)

    Enders, P; Schaub, F; Fauser, S

    2017-02-10

    Background Laser therapy is an important treatment option in retinal diseases, especially in cases of vascular involvement. Most approaches are based on coagulation of retinal structures. As there is increasing use of agents targetting vascular endothelial growth factor in the treatment of macular diseases, indications for the use of laser treatment need to be reviewed carefully, especially with respect to their significance in first line therapy. This article explains recent strategies and treatment protocols. Materials and Methods Review of current literature in PubMed as well as synopsis of relevant guidelines. Results and Conclusion Retinal laser therapy is still widely used within retinal opthalmology and covers a large spectrum of indications. Despite the success of medical approaches, retinal laser therapy remains an indispensable treatment option for proliferative diabetic retinopathy, central or peripheral vein occlusion and less frequent pathologies, such as retinopathy of prematurity or Coats's disease. Georg Thieme Verlag KG Stuttgart · New York.

  14. Histological Study on the Protective Effect of Simvastatin on the Retinal Changes Induced by High-Fat Diet in Mice

    Directory of Open Access Journals (Sweden)

    Fayza Ezz Ahmad

    2017-09-01

    Full Text Available Background: High-fat diet (HFD feeding is an important model to study the changes induced by insulin resistance, Type 2 diabetes mellitus and obesity including retinopathy. Vascular endothelial growth factor (VEGF and p53 have been implicated in the development of retinopathy. Objectives: The aim of his study was to analyze histological retinal changes in a high-fat atherogenic mouse model and to evaluate the possible protective effect of simvastatin on these changes including its effects on the expression of VEGF and p53. Materials and Methods: A total of 27 mice (6 weeks old were divided into 3 study groups according to their diet and treatment given; Group I - normal balanced diet-fed mice, Group II - HFD-fed mice, and Group III - HFD-fed mice treated with simvastatin daily for 30 weeks. All mice were followed up for 30 weeks. At the end of the study at 36 weeks of age, eye tissues were collected and retinal sections were examined using light microscopy. Comparison of the thickness of retinal layers in the three groups was carried out. The localization of VEGF in the retina was determined by immunohistochemical analysis, and apoptotic cell death was assessed using the p53. Results: In the HFD-fed mice, there was an increase in the retinal thickness associated with presence of wide intercellular spaces in the outer nuclear layer. Many cells in the inner nuclear layer showed cytoplasmic vacuolations. Expression of VEGF was significantly increased in the retinal ganglion cell layers and nuclear cell layers. Elevated p53 reaction was demonstrated within the inner retina. The histological changes were significantly improved in the simvastatin treated group. Conclusions: HFD-induced structural changes in the retinal layers and simultaneous upregulation of VEGF and p53. Administration of simvastatin improved these retinal alterations. [J Interdiscip Histopathol 2017; 5(3.000: 83-91

  15. Neuroprotective and Antiapoptotic Activity of Lineage-Negative Bone Marrow Cells after Intravitreal Injection in a Mouse Model of Acute Retinal Injury

    Directory of Open Access Journals (Sweden)

    Anna Machalińska

    2015-01-01

    Full Text Available We investigated effects of bone marrow-derived, lineage-negative cell (Lin−BMC transplantation in acute retinal injury. Lin−BMCs were intravitreally injected into murine eyes at 24 h after NaIO3-induced injury. Morphology, function, and expression of apoptosis-related genes, including brain-derived neurotrophic factor (BDNF and its receptor, were assessed in retinas at 7 days, 28 days, and 3 months after transplantation. Moreover, global gene expression at day 7 was analyzed by RNA arrays. We observed that Lin−BMCs integrated into outer retinal layers improving morphological retinal structure and induced molecular changes such as downregulation of proapoptotic caspase-3 gene, a decrease in BAX/BCL-2 gene ratio, and significant elevation of BDNF expression. Furthermore, transplanted Lin−BMCs differentiated locally into cells with a macrophage-like phenotype. Finally, Lin−BMCs treatment was associated with generation of two distinct transcriptomic patterns. The first relates to downregulated genes associated with regulation of neuron cell death and apoptosis, response to oxidative stress/hypoxia and external stimuli, and negative regulation of cell proliferation. The second relates to upregulated genes associated with neurological system processes and sensory perception. Collectively, our data demonstrate that transplanted Lin−BMCs exert neuroprotective function against acute retinal injury and this effect may be associated with their antiapoptotic properties and ability to express neurotrophic factors.

  16. Chaetomium retinitis.

    Science.gov (United States)

    Tabbara, Khalid F; Wedin, Keith; Al Haddab, Saad

    2010-01-01

    To report a case of Chaetomium atrobrunneum retinitis in a patient with Hodgkin lymphoma. We studied the ocular manifestations of an 11-year-old boy with retinitis. Biomicroscopy, ophthalmoscopy, and fundus photography were done. Magnetic resonance imaging of the brain was performed. A vitreous biopsy was subjected to viral, bacterial, and fungal cultures. Vitreous culture grew C. atrobrunneum. Magnetic resonance imaging showed multiple cerebral lesions consistent with an infectious process. The patient was given intravenous voriconazole and showed improvement of the ocular and central nervous system lesions. We report a case of central nervous system and ocular lesions by C. atrobrunneum. The retinitis was initially misdiagnosed as cytomegaloviral retinitis. Vitreous biopsy helped in the early diagnosis and prompt treatment of a life- and vision-threatening infection.

  17. Retinitis pigmentosa

    Science.gov (United States)

    ... treatments for retinitis pigmentosa, including the use of DHA, which is an omega-3 fatty acid. Other ... Geme JW, Schor NF, eds. Nelson Textbook of Pediatrics . 20th ed. Philadelphia, PA: Elsevier; 2016:chap 630. ...

  18. Cytomegalovirus retinitis

    Science.gov (United States)

    ... have weakened immune systems as a result of: HIV/AIDS Bone marrow transplant Chemotherapy Drugs that suppress the immune system Organ transplant Symptoms Some people with CMV retinitis have no symptoms. ...

  19. Retinal Detachment

    Science.gov (United States)

    ... to your brain. It provides the sharp, central vision needed for reading, driving, and seeing fine detail. A retinal detachment lifts or pulls the retina from its normal position. It can occur at ...

  20. A Proinflammatory Function of Toll-Like Receptor 2 in the Retinal Pigment Epithelium as a Novel Target for Reducing Choroidal Neovascularization in Age-Related Macular Degeneration.

    Science.gov (United States)

    Feng, Lili; Ju, Meihua; Lee, Kei Ying V; Mackey, Ashley; Evangelista, Mariasilvia; Iwata, Daiju; Adamson, Peter; Lashkari, Kameran; Foxton, Richard; Shima, David; Ng, Yin Shan

    2017-10-01

    Current treatments for choroidal neovascularization, a major cause of blindness for patients with age-related macular degeneration, treat symptoms but not the underlying causes of the disease. Inflammation has been strongly implicated in the pathogenesis of choroidal neovascularization. We examined the inflammatory role of Toll-like receptor 2 (TLR2) in age-related macular degeneration. TLR2 was robustly expressed by the retinal pigment epithelium in mouse and human eyes, both normal and with macular degeneration/choroidal neovascularization. Nuclear localization of NF-κB, a major downstream target of TLR2 signaling, was detected in the retinal pigment epithelium of human eyes, particularly in eyes with advanced stages of age-related macular degeneration. TLR2 antagonism effectively suppressed initiation and growth of spontaneous choroidal neovascularization in a mouse model, and the combination of anti-TLR2 and antivascular endothelial growth factor receptor 2 yielded an additive therapeutic effect on both area and number of spontaneous choroidal neovascularization lesions. Finally, in primary human fetal retinal pigment epithelium cells, ligand binding to TLR2 induced robust expression of proinflammatory cytokines, and end products of lipid oxidation had a synergistic effect on TLR2 activation. Our data illustrate a functional role for TLR2 in the pathogenesis of choroidal neovascularization, likely by promoting inflammation of the retinal pigment epithelium, and validate TLR2 as a novel therapeutic target for reducing choroidal neovascularization. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  1. Mitochondrial transcription factor A protects human retinal ...

    African Journals Online (AJOL)

    Purpose: To investigate the impact of mitochondrial transcription factor A (TFAM), as a modulator of NF-κB, on proliferation of hypoxia-induced human retinal endothelial cell (HREC), and the probable mechanism. Methods: After exposure to hypoxia (1 % O2) for 5 days, cell proliferation and cell cycle of HREC were ...

  2. Radiation Retinopathy Associated with Central Retinal Vein Occlusion

    Institute of Scientific and Technical Information of China (English)

    Yan; Liu; FengWen

    2007-01-01

    Purpose: To report a case of radiation retinopathy associated with central retinal vein occlusion.Methods: The clinical features and fundus fluorescein angiography of this case were analyzed.Results: The patient had been treated with radiotherapy for her nasopharyngeal carcinoma, and presented with sudden visual loss in the left eye. The funduscopic examination and fluorescein angiography showed the features of radiation retinopathy in both eyes, and central retinal vein occlusion in the left eye.Conclusions: Radiation retinopathy can be associated with central retinal vein occlusion in the same eye, and it seems that the endothelial cell loss caused by radiation retinopathy may lead to retinal vein occlusion.

  3. Positive Feedback Regulation of Agonist-Stimulated Endothelial Ca2+ Dynamics by KCa3.1 Channels in Mouse Mesenteric Arteries

    DEFF Research Database (Denmark)

    Qian, Xun; Francis, Michael; Köhler, Ralf

    2014-01-01

    Intermediate and small conductance KCa channels IK1 (KCa3.1) and SK3 (KCa2.3) are primary targets of endothelial Ca(2+) signals in the arterial vasculature, and their ablation results in increased arterial tone and hypertension. Activation of IK1 channels by local Ca(2+) transients from internal ...... stores or plasma membrane channels promotes arterial hyperpolarization and vasodilation. Here, we assess arteries from genetically altered IK1 knockout mice (IK1(-/-)) to determine whether IK1 channels exert a positive feedback influence on endothelial Ca(2+) dynamics....

  4. Intracavernous Delivery of a Designed Angiopoietin-1 Variant Rescues Erectile Function by Enhancing Endothelial Regeneration in the Streptozotocin-Induced Diabetic Mouse

    Science.gov (United States)

    Jin, Hai-Rong; Kim, Woo Jean; Song, Jae Sook; Piao, Shuguang; Choi, Min Ji; Tumurbaatar, Munkhbayar; Shin, Sun Hwa; Yin, Guo Nan; Koh, Gou Young; Ryu, Ji-Kan; Suh, Jun-Kyu

    2011-01-01

    OBJECTIVE Patients with diabetic erectile dysfunction often have severe endothelial dysfunction and respond poorly to oral phosphodiesterase-5 inhibitors. We examined the effectiveness of the potent angiopoietin-1 (Ang1) variant, cartilage oligomeric matrix protein (COMP)-Ang1, in promoting cavernous endothelial regeneration and restoring erectile function in diabetic animals. RESEARCH DESIGN AND METHODS Four groups of mice were used: controls; streptozotocin (STZ)-induced diabetic mice; STZ-induced diabetic mice treated with repeated intracavernous injections of PBS; and STZ-induced diabetic mice treated with COMP-Ang1 protein (days −3 and 0). Two and 4 weeks after treatment, we measured erectile function by electrical stimulation of the cavernous nerve. The penis was harvested for histologic examinations, Western blot analysis, and cGMP quantification. We also performed a vascular permeability test. RESULTS Local delivery of the COMP-Ang1 protein significantly increased cavernous endothelial proliferation, endothelial nitric oxide (NO) synthase (NOS) phosphorylation, and cGMP expression compared with that in the untreated or PBS-treated STZ-induced diabetic group. The changes in the group that received COMP-Ang1 restored erectile function up to 4 weeks after treatment. Endothelial protective effects, such as marked decreases in the expression of p47phox and inducible NOS, in the generation of superoxide anion and nitrotyrosine, and in the number of apoptotic cells in the corpus cavernosum tissue, were noted in COMP-Ang1–treated STZ-induced diabetic mice. An intracavernous injection of COMP-Ang1 completely restored endothelial cell-cell junction proteins and decreased cavernous endothelial permeability. COMP-Ang1–induced promotion of cavernous angiogenesis and erectile function was abolished by the NOS inhibitor, N-nitro-L-arginine methyl ester, but not by the NADPH oxidase inhibitor, apocynin. CONCLUSIONS These findings support the concept of cavernous

  5. Signaling hierarchy regulating human endothelial cell development

    Science.gov (United States)

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these stud...

  6. Silver nano - a trove for retinal therapies.

    Science.gov (United States)

    Kalishwaralal, Kalimuthu; Barathmanikanth, Selvaraj; Pandian, Sureshbabu Ram Kumar; Deepak, Venkatraman; Gurunathan, Sangiliyandi

    2010-07-14

    Pathological retinal angiogenesis (neovascularization) is one of the most feared complications among retinal diseases, leading to visual impairment and irreversible blindness. Recent findings made by us on therapeutic applications of biologically synthesized silver nanoparticles (AgNPs) against VEGF induced retinal endothelial cells, elucidates the effectual inhibitory activities of AgNPs over the downstream signaling pathways (Src and AKT/PI3K) leading to retinal angiogenesis. The current review focuses on the imperative role of VEGF induced angiogenesis in the development of retinal neovascularization and despite the fact that several VEGF targeting ocular drugs are available; the review examines the need for a cost economic alternative, thereby suggesting the role of AgNPs as an emerging economic ocular drug for retinal therapies. The current technologies available for the development of targeted and controlled release of drugs is being discussed and a model has been proposed for the amenable targeting mechanism, by which Poly gamma glutamic acid (PGA) capsulated AgNPs conjugated to cyclic RGD peptides carry out a sustained controlled release specifically targeting the neovascularization cells and induce apoptosis unaffecting the normal retinal cells. These constructs consequently affirm the futuristic application of silver nanoparticles as a boon to ocular therapies. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  7. Retinal Detachment

    Directory of Open Access Journals (Sweden)

    Adnan Riaz, MD

    2018-04-01

    Full Text Available History of present illness: A 58-year-old female presented to the emergency department reporting six days of progressive, atraumatic left eye vision loss. Her symptoms started with the appearance of dark spots and “spider webs,” and then progressed to darkening of vision in her left eye. She reports mild pain since yesterday. Her review of symptoms was otherwise negative. Ocular physical examination revealed normal external appearance, intact extraocular movements, and visual acuities of 20/25 OD and light/dark sensitivity OS. Fluorescein uptake was negative and slit lamp exam was unremarkable. Significant findings: Bedside ocular ultrasound revealed a serpentine, hyperechoic membrane that appeared tethered to the optic disc posteriorly with hyperechoic material underneath. These findings are consistent with retinal detachment (RD and associated retinal hemorrhage. Discussion: The retina is a layer of organized neurons that line the posterior portion of the posterior chamber of the eye. RD occurs when this layer separates from the underlying epithelium, resulting in ischemia and progressive photoreceptor degeneration, with potentially rapid and permanent vision loss if left untreated.1 Risk factors include advanced age, male sex (60%, race (Asians and Jews, and myopia and lattice degeneration.2 Bedside ultrasound (US performed by emergency physicians provides a valuable tool that has been used by ophthalmologists for decades to evaluate intraocular disease.1,3 Findings on bedside ultrasound consistent with RD include a hyperechoic membrane floating in the posterior chamber. RD usuallyremain tethered to the optic disc posteriorly and do not cross midline, a feature distinguishing them from posterior vitreous detachments. Associated retinal hemorrhage, seen as hyperechoic material under the retinal flap, can often be seen.1,2 US can also distinguish between “mac-on” and “mac-off” detachments. If the retina is still attached to the

  8. Retinal detachment and retinal holes in retinitis pigmentosa sine pigmento.

    Science.gov (United States)

    Csaky, K; Olk, R J; Mahl, C F; Bloom, S M

    1991-01-01

    Retinal detachment and retinal holes in two family members with retinitis pigmentosa sine pigmento are reported. We believe these are the first such cases reported in the literature. We describe the presenting symptoms and management, including cryotherapy, scleral buckling procedure, and sulfur hexafluoride injection (SF6), resulting in stable visual acuity in one case and retinal reattachment and improved visual acuity in the other case.

  9. Retinitis pigmentosa

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2006-10-01

    Full Text Available Abstract Retinitis pigmentosa (RP is an inherited retinal dystrophy caused by the loss of photoreceptors and characterized by retinal pigment deposits visible on fundus examination. Prevalence of non syndromic RP is approximately 1/4,000. The most common form of RP is a rod-cone dystrophy, in which the first symptom is night blindness, followed by the progressive loss in the peripheral visual field in daylight, and eventually leading to blindness after several decades. Some extreme cases may have a rapid evolution over two decades or a slow progression that never leads to blindness. In some cases, the clinical presentation is a cone-rod dystrophy, in which the decrease in visual acuity predominates over the visual field loss. RP is usually non syndromic but there are also many syndromic forms, the most frequent being Usher syndrome. To date, 45 causative genes/loci have been identified in non syndromic RP (for the autosomal dominant, autosomal recessive, X-linked, and digenic forms. Clinical diagnosis is based on the presence of night blindness and peripheral visual field defects, lesions in the fundus, hypovolted electroretinogram traces, and progressive worsening of these signs. Molecular diagnosis can be made for some genes, but is not usually performed due to the tremendous genetic heterogeneity of the disease. Genetic counseling is always advised. Currently, there is no therapy that stops the evolution of the disease or restores the vision, so the visual prognosis is poor. The therapeutic approach is restricted to slowing down the degenerative process by sunlight protection and vitaminotherapy, treating the complications (cataract and macular edema, and helping patients to cope with the social and psychological impact of blindness. However, new therapeutic strategies are emerging from intensive research (gene therapy, neuroprotection, retinal prosthesis.

  10. Protein kinase C-α signals P115RhoGEF phosphorylation and RhoA activation in TNF-α-induced mouse brain microvascular endothelial cell barrier dysfunction

    Directory of Open Access Journals (Sweden)

    Deng Xiaolu

    2011-04-01

    Full Text Available Abstract Background Tumor necrosis factor-α (TNF-α, a proinflammatory cytokine, is capable of activating the small GTPase RhoA, which in turn contributes to endothelial barrier dysfunction. However, the underlying signaling mechanisms remained undefined. Therefore, we aimed to determine the role of protein kinase C (PKC isozymes in the mechanism of RhoA activation and in signaling TNF-α-induced mouse brain microvascular endothelial cell (BMEC barrier dysfunction. Methods Bend.3 cells, an immortalized mouse brain endothelial cell line, were exposed to TNF-α (10 ng/mL. RhoA activity was assessed by pull down assay. PKC-α activity was measured using enzyme assasy. BMEC barrier function was measured by transendothelial electrical resistance (TER. p115RhoGEF phosphorylation was detected by autoradiography followed by western blotting. F-actin organization was observed by rhodamine-phalloidin staining. Both pharmacological inhibitors and knockdown approaches were employed to investigate the role of PKC and p115RhoGEF in TNF-α-induced RhoA activation and BMEC permeability. Results We observed that TNF-α induces a rapid phosphorylation of p115RhoGEF, activation of PKC and RhoA in BMECs. Inhibition of conventional PKC by Gö6976 mitigated the TNF-α-induced p115RhoGEF phosphorylation and RhoA activation. Subsequently, we found that these events are regulated by PKC-α rather than PKC-β by using shRNA. In addition, P115-shRNA and n19RhoA (dominant negative mutant of RhoA transfections had no effect on mediating TNF-α-induced PKC-α activation. These data suggest that PKC-α but not PKC-β acts as an upstream regulator of p115RhoGEF phosphorylation and RhoA activation in response to TNF-α. Moreover, depletion of PKC-α, of p115RhoGEF, and inhibition of RhoA activation also prevented TNF-α-induced stress fiber formation and a decrease in TER. Conclusions Taken together, our results show that PKC-α phosphorylation of p115RhoGEF mediates TNF

  11. Lutein facilitates physiological revascularization in a mouse model of retinopathy of prematurity.

    Science.gov (United States)

    Fu, Zhongjie; Meng, Steven S; Burnim, Samuel B; Smith, Lois Eh; Lo, Amy Cy

    2017-07-01

    Retinopathy of prematurity is one of the leading causes of childhood blindness worldwide, with vessel growth cessation and vessel loss in phase I followed by neovascularization in phase II. Ischaemia contributes to its pathogenesis, and lutein protects against ischaemia-induced retinal damages. We aimed to investigate the effects of lutein on a murine model of oxygen-induced retinopathy. Mouse pups were exposed to 75% oxygen for 5 days and returned to room air for another 5 days. Vascular obliteration, neovascularization and blood vessel leakage were examined. Immunohistochemistry for glial cells and microglia were performed. Compared with vehicle controls, mouse pups receiving lutein treatment displayed smaller central vaso-obliterated area and reduced blood vessel leakage. No significant difference in neovascular area was found between lutein and vehicle controls. Lutein promoted endothelial tip cell formation and maintained the astrocytic template in the avascular area in oxygen-induced retinopathy. No significant changes in Müller cell gliosis and microglial activation in the central avascular area were found in lutein-treated pups. Our observations indicated that lutein significantly promoted normal retinal vascular regrowth in the central avascular area, possibly through promoting endothelial tip cell formation and preserving astrocytic template. Our results indicated that lutein might be considered as a supplement for the treatment of proliferative retinopathy of prematurity because of its role in facilitating the revascularization of normal vasculature. © 2016 Royal Australian and New Zealand College of Ophthalmologists.

  12. Signaling hierarchy regulating human endothelial cell development.

    Science.gov (United States)

    Kelly, Melissa A; Hirschi, Karen K

    2009-05-01

    Our present knowledge of the regulation of mammalian endothelial cell differentiation has been largely derived from studies of mouse embryonic development. However, unique mechanisms and hierarchy of signals that govern human endothelial cell development are unknown and, thus, explored in these studies. Using human embryonic stem cells as a model system, we were able to reproducibly and robustly generate differentiated endothelial cells via coculture on OP9 marrow stromal cells. We found that, in contrast to studies in the mouse, bFGF and VEGF had no specific effects on the initiation of human vasculogenesis. However, exogenous Ihh promoted endothelial cell differentiation, as evidenced by increased production of cells with cobblestone morphology that coexpress multiple endothelial-specific genes and proteins, form lumens, and exhibit DiI-AcLDL uptake. Inhibition of BMP signaling using Noggin or BMP4, specifically, using neutralizing antibodies suppressed endothelial cell formation; whereas, addition of rhBMP4 to cells treated with the hedgehog inhibitor cyclopamine rescued endothelial cell development. Our studies revealed that Ihh promoted human endothelial cell differentiation from pluripotent hES cells via BMP signaling, providing novel insights applicable to modulating human endothelial cell formation and vascular regeneration for human clinical therapies.

  13. Krypton laser photocoagulation induces retinal vascular remodeling rather than choroidal neovascularization.

    Science.gov (United States)

    Behar-Cohen, F; Benezra, D; Soubrane, G; Jonet, L; Jeanny, J C

    2006-08-01

    The purpose of this study is to analyze the retina and choroid response following krypton laser photocoagulation. Ninety-two C57BL6/Sev129 and 32 C57BL/6J, 5-6-week-old mice received one single krypton (630 nm) laser lesion: 50 microm, 0.05 s, 400 mW. On the following day, every day thereafter for 1 week and every 2-3 days for the following 3 weeks, serial sections throughout the lesion were systematically collected and studied. Immunohistology using specific markers or antibodies for glial fibrillary acidic protein (GFAP) (astrocytes, glia and Muller's cells), von Willebrand (vW) (vascular endothelial cells), TUNEL (cells undergoing caspase dependent apoptosis), PCNA (proliferating cell nuclear antigen) p36, CD4 and F4/80 (infiltrating inflammatory and T cells), DAPI (cell nuclei) and routine histology were carried out. Laser confocal microscopy was also performed on flat mounts. Temporal and spatial observations of the created photocoagulation lesions demonstrate that, after a few hours, activated glial cells within the retinal path of the laser beam express GFAP. After 48 h, GFAP-positive staining was also detected within the choroid lesion center. "Movement" of this GFAP-positive expression towards the lasered choroid was preceded by a well-demarcated and localized apoptosis of the retina outer nuclear layer cells within the laser beam path. Later, death of retinal outer nuclear cells and layer thinning at this site was followed by evagination of the inner nuclear retinal layer. Funneling of the entire inner nuclear and the thinned outer nuclear layers into the choroid lesion center was accompanied by "dragging" of the retinal capillaries. Thus, from days 10 to 14 after krypton laser photocoagulation onward, well-formed blood capillaries (of retinal origin) were observed within the lesion. Only a few of the vW-positive capillary endothelial cells stained also for PCNA p36. In the choroid, dilatation of the vascular bed occurred at the vicinity of the

  14. Extracellular S100A4(mts1) stimulates invasive growth of mouse endothelial cells and modulates MMP-13 matrix metalloproteinase activity

    DEFF Research Database (Denmark)

    Schmidt-Hansen, Birgitte; Ornås, Dorte; Grigorian, Mariam

    2004-01-01

    with the transcriptional modulation of genes involved in the proteolytic degradation of extracellular matrix (ECM). Treatment of SVEC 4-10 with the S100A4 protein leads to the transcriptional activation of collagenase 3 (MMP-13) mRNA followed by subsequent release of the protein from the cells. Beta-casein zymography...... demonstrates enhancement of proteolytic activity associated with MMP-13. This observation indicates that extracellular S100A4 stimulates the production of ECM degrading enzymes from endothelial cells, thereby stimulating the remodeling of ECM. This could explain the angiogenic and metastasis...

  15. In Vivo Knockout of the Vegfa Gene by Lentiviral Delivery of CRISPR/Cas9 in Mouse Retinal Pigment Epithelium Cells

    Directory of Open Access Journals (Sweden)

    Andreas Holmgaard

    2017-12-01

    Full Text Available Virus-based gene therapy by CRISPR/Cas9-mediated genome editing and knockout may provide a new option for treatment of inherited and acquired ocular diseases of the retina. In support of this notion, we show that Streptococcus pyogenes (Sp Cas9, delivered by lentiviral vectors (LVs, can be used in vivo to selectively ablate the vascular endothelial growth factor A (Vegfa gene in mice. By generating LVs encoding SpCas9 targeted to Vegfa, and in parallel the fluorescent eGFP marker protein, we demonstrate robust knockout of Vegfa that leads to a significant reduction of VEGFA protein in transduced cells. Three of the designed single-guide RNAs (sgRNAs induce in vitro indel formation at high frequencies (44%–93%. A single unilateral subretinal injection facilitates RPE-specific localization of the vector and disruption of Vegfa in isolated eGFP+ RPE cells obtained from mice five weeks after LV administration. Notably, sgRNA delivery results in the disruption of Vegfa with an in vivo indel formation efficacy of up to 84%. Sequencing of Vegfa-specific amplicons reveals formation of indels, including 4-bp deletions and 2-bp insertions. Taken together, our data demonstrate the capacity of lentivirus-delivered SpCas9 and sgRNAs as a developing therapeutic path in the treatment of ocular diseases, including age-related macular degeneration.

  16. Plasmalemma Vesicle-Associated Protein Has a Key Role in Blood-Retinal Barrier Loss

    NARCIS (Netherlands)

    Wisniewska-Kruk, Joanna; van der Wijk, Anne-Eva; van Veen, Henk A.; Gorgels, Theo G. M. F.; Vogels, Ilse M. C.; Versteeg, Danielle; van Noorden, Cornelis J. F.; Schlingemann, Reinier O.; Klaassen, Ingeborg

    2016-01-01

    Loss of blood-retinal barrier (BRB) properties induced by vascular endothelial growth factor (VEGF) and other factors is an important cause of diabetic macular edema. Previously, we found that the presence of plasmalemma vesicle-associated protein (PLVAP) in retinal capillaries associates with loss

  17. Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor

    OpenAIRE

    Hata, Yasuaki; Clermont, Allen Charles; Yamauchi, Teruaki; Pierce, Eric Adam; Suzuma, Izumi; Kagokawa, Hiroyuki; Yoshikawa, Hiroshi; Robinson, Gregory S.; Ishibashi, Tatsuro; Hashimoto, Toshihiko; Umeda, Fumio; Bursell, Sven E.; Aiello, Lloyd Paul

    2000-01-01

    Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericy...

  18. Comparison of three fluorescence labeling and tracking methods of endothelial progenitor cells in laser-injured retina

    Directory of Open Access Journals (Sweden)

    Hui Shi

    2018-04-01

    Full Text Available AIM: To compare three kinds of fluorescent probes for in vitro labeling and in vivo tracking of endothelial progenitor cells (EPCs in a mouse model of laser-induced retinal injury. METHODS: EPCs were isolated from human umbilical cord blood mononuclear cells and labeled with three different fluorescent probes: 5-(and-6-carboxyfluorescein diacetate succinimidyl ester (CFSE, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindo-carbocyanine perchlorate linked acetylated low-density lipoprotein (DiI-AcLDL, and green fluorescent protein (GFP. The fluorescent intensity of EPCs was examined by confocal microscopy. Survival rate of labeled EPCs was calculated with trypan blue staining, and their adhesive capability was assessed. A mouse model of retinal injury was induced by laser, and EPCs were injected into the vitreous cavity. Frozen section and fluorescein angiography on flat-mounted retinal samples was employed to track the labeled EPCs in vivo. RESULTS: EPCs labeled with CFSE and DiI-AcLDL exhibited an intense green and red fluorescence at the beginning; the fluorescence intensity decreased gradually to 20.23% and 49.99% respectively, after 28d. On the contrary, the florescent intensity of GFP-labeled EPCs increased in a time-dependent manner. All labeled EPCs showed normal morphology and no significant change in survival and adhesive capability. In the mouse model, transplantation of EPCs showed a protective effect against retinal injury. EPCs labeled with CFSE and DiI-AcLDL were successfully tracked in mice during the development of retinal injury and repair; however, GFP-labeled EPCs were not detected in the laser-injured mouse retina. CONCLUSION: The three fluorescent markers used in this study have their own set of advantages and disadvantages. CFSE and DiI-AcLDL are suitable for short-term EPC-labeling, while GFP should be used for long-term labeling. The choice of fluorescent markers should be guided by the purpose of the study.

  19. Bilateral retinal vein occlusion and rubeosis irides: lessons to learn.

    Science.gov (United States)

    Md Noh, Umi Kalthum; Ahem, Amin; Mustapha, Mushawiahti

    2013-01-01

    Uncontrolled hypertension is well- known to give rise to systemic complications involving multiple central organs. Artherosclerosis leads to damage of the retinal vessels wall, contributing to venous stasis, thrombosis and finally, occlusion. Retinal vein occlusions compromise vision through development of ischaemic maculopathy, macular oedema, and rubeotic glaucoma. Laser photocoagulation remains the definitive treatment for ischaemic vein occlusion with secondary neovascularization. Timely treatment with anti- vascular endothelial growth factor prevents development of rubeotic glaucoma. We hereby report an unusual case of bilateral retinal vein occlusion complicated by rubeosis irides, which was successfully managed to improve vision and prevent rubeotic glaucoma.

  20. Neuroblast survival depends on mature vascular network formation after mouse stroke: role of endothelial and smooth muscle progenitor cell co-administration.

    Science.gov (United States)

    Nih, Lina R; Deroide, Nicolas; Leré-Déan, Carole; Lerouet, Dominique; Soustrat, Mathieu; Levy, Bernard I; Silvestre, Jean-Sébastien; Merkulova-Rainon, Tatiana; Pocard, Marc; Margaill, Isabelle; Kubis, Nathalie

    2012-04-01

    Pro-angiogenic cell-based therapies constitute an interesting and attractive approach to enhancing post-stroke neurogenesis and decreasing neurological deficit. However, most new stroke-induced neurons die during the first few weeks after ischemia, thus impairing total recovery. Although the neovascularization process involves different cell types and various growth factors, most cell therapy protocols are based on the biological effects of single-cell-type populations or on the administration of heterogeneous populations of progenitors, namely human cord blood-derived CD34(+) cells, with scarce vascular progenitor cells. Tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. We hypothesized that neuroblast survival in stroke brain depends on mature vascular network formation. In this study, we injected a combination of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs), isolated from human umbilical cord blood, into a murine model of permanent focal ischemia induced by middle cerebral artery occlusion. The co-administration of SMPCs and EPCs induced enhanced angiogenesis and vascular remodeling in the peri-infarct and infarct areas, where vessels exhibited a more mature phenotype. This activation of vessel growth resulted in the maintenance of neurogenesis and neuroblast migration to the peri-ischemic cortex. Our data suggest that a mature vascular network is essential for neuroblast survival after cerebral ischemia, and that co-administration of EPCs and SMPCs may constitute a novel therapeutic strategy for improving the treatment of stroke. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  1. The pleiotropic effects of simvastatin on retinal microvascular endothelium has important implications for ischaemic retinopathies.

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    Reinhold J Medina

    Full Text Available BACKGROUND: Current guidelines encourage the use of statins to reduce the risk of cardiovascular disease in diabetic patients; however the impact of these drugs on diabetic retinopathy is not well defined. Moreover, pleiotropic effects of statins on the highly specialised retinal microvascular endothelium remain largely unknown. The objective of this study was to investigate the effects of clinically relevant concentrations of simvastatin on retinal endothelium in vitro and in vivo. METHODS AND FINDINGS: Retinal microvascular endothelial cells (RMECs were treated with 0.01-10 microM simvastatin and a biphasic dose-related response was observed. Low concentrations enhanced microvascular repair with 0.1 microM simvastatin significantly increasing proliferation (p<0.05, and 0.01 microM simvastatin significantly promoting migration (p<0.05, sprouting (p<0.001, and tubulogenesis (p<0.001. High concentration of simvastatin (10 microM had the opposite effect, significantly inhibiting proliferation (p<0.01, migration (p<0.01, sprouting (p<0.001, and tubulogenesis (p<0.05. Furthermore, simvastatin concentrations higher than 1 microM induced cell death. The mouse model of oxygen-induced retinopathy was used to investigate the possible effects of simvastatin treatment on ischaemic retinopathy. Low dose simvastatin (0.2 mg/Kg promoted retinal microvascular repair in response to ischaemia by promoting intra-retinal re-vascularisation (p<0.01. By contrast, high dose simvastatin(20 mg/Kg significantly prevented re-vascularisation (p<0.01 and concomitantly increased pathological neovascularisation (p<0.01. We also demonstrated that the pro-vascular repair mechanism of simvastatin involves VEGF stimulation, Akt phosphorylation, and nitric oxide production; and the anti-vascular repair mechanism is driven by marked intracellular cholesterol depletion and related disorganisation of key intracellular structures. CONCLUSIONS: A beneficial effect of low

  2. Missed retinal breaks in rhegmatogenous retinal detachment

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    Brijesh Takkar

    2016-12-01

    Full Text Available AIM: To evaluate the causes and associations of missed retinal breaks (MRBs and posterior vitreous detachment (PVD in patients with rhegmatogenous retinal detachment (RRD. METHODS: Case sheets of patients undergoing vitreo retinal surgery for RRD at a tertiary eye care centre were evaluated retrospectively. Out of the 378 records screened, 253 were included for analysis of MRBs and 191 patients were included for analysis of PVD, depending on the inclusion criteria. Features of RRD and retinal breaks noted on examination were compared to the status of MRBs and PVD detected during surgery for possible associations. RESULTS: Overall, 27% patients had MRBs. Retinal holes were commonly missed in patients with lattice degeneration while missed retinal tears were associated with presence of complete PVD. Patients operated for cataract surgery were significantly associated with MRBs (P=0.033 with the odds of missing a retinal break being 1.91 as compared to patients with natural lens. Advanced proliferative vitreo retinopathy (PVR and retinal bullae were the most common reasons for missing a retinal break during examination. PVD was present in 52% of the cases and was wrongly assessed in 16%. Retinal bullae, pseudophakia/aphakia, myopia, and horse shoe retinal tears were strongly associated with presence of PVD. Traumatic RRDs were rarely associated with PVD. CONCLUSION: Pseudophakic patients, and patients with retinal bullae or advanced PVR should be carefully screened for MRBs. Though Weiss ring is a good indicator of PVD, it may still be over diagnosed in some cases. PVD is associated with retinal bullae and pseudophakia, and inversely with traumatic RRD.

  3. Sphingosine-1-Phosphate/Sphingosine-1-Phosphate Receptor 2 Axis Can Promote Mouse and Human Primary Mast Cell Angiogenic Potential through Upregulation of Vascular Endothelial Growth Factor-A and Matrix Metalloproteinase-2

    Directory of Open Access Journals (Sweden)

    Alena Chumanevich

    2016-01-01

    Full Text Available Mast cells (MC are present in most vascularized tissues around the vasculature likely exerting immunomodulatory functions. Endowed with diverse mediators, resident MC represent first-line fine-tuners of local microenvironment. Sphingosine-1-phosphate (S1P functions as a pluripotent signaling sphingolipid metabolite in health and disease. S1P formation occurs at low levels in resting MC and is upregulated upon activation. Its export can result in type 2 S1P receptor- (S1PR2- mediated stimulation of MC, further fueling inflammation. However, the role of S1PR2 ligation in proangiogenic vascular endothelial growth factor- (VEGF- A and matrix metalloproteinase- (MMP- 2 release from MC is unknown. Using a preclinical MC-dependent model of acute allergic responses and in vitro stimulated primary mouse bone marrow-derived MC (BMMC or human primary skin MC, we report that S1P signaling resulted in substantial amount of VEGF-A release. Similar experiments using S1pr2-deficient mice or BMMC or selective S1P receptor agonists or antagonists demonstrated that S1P/S1PR2 ligation on MC is important for VEGF-A secretion. Further, we show that S1P stimulation triggered transcriptional upregulation of VEGF-A and MMP-2 mRNA in human but not in mouse MC. S1P exposure also triggered MMP-2 secretion from human MC. These studies identify a novel proangiogenic axis encompassing MC/S1P/S1PR2 likely relevant to inflammation.

  4. Revisiting the mouse model of oxygen-induced retinopathy

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    Kim CB

    2016-05-01

    Full Text Available Clifford B Kim,1,2 Patricia A D’Amore,2–4 Kip M Connor1,2 1Angiogenesis Laboratory, Massachusetts Eye and Ear, 2Department of Ophthalmology, Harvard Medical School, 3Schepens Eye Research Institute, Massachusetts Eye and Ear, 4Department of Pathology, Harvard Medical School, Boston, MA, USA Abstract: Abnormal blood vessel growth in the retina is a hallmark of many retinal diseases, such as retinopathy of prematurity (ROP, proliferative diabetic retinopathy, and the wet form of age-related macular degeneration. In particular, ROP has been an important health concern for physicians since the advent of routine supplemental oxygen therapy for premature neonates more than 70 years ago. Since then, researchers have explored several animal models to better understand ROP and retinal vascular development. Of these models, the mouse model of oxygen-induced retinopathy (OIR has become the most widely used, and has played a pivotal role in our understanding of retinal angiogenesis and ocular immunology, as well as in the development of groundbreaking therapeutics such as anti-vascular endothelial growth factor injections for wet age-related macular degeneration. Numerous refinements to the model have been made since its inception in the 1950s, and technological advancements have expanded the use of the model across multiple scientific fields. In this review, we explore the historical developments that have led to the mouse OIR model utilized today, essential concepts of OIR, limitations of the model, and a representative selection of key findings from OIR, with particular emphasis on current research progress. Keywords: ROP, OIR, angiogenesis

  5. Loss of CD34 expression in aging human choriocapillaris endothelial cells.

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    Elliott H Sohn

    Full Text Available Structural and gene expression changes in the microvasculature of the human choroid occur during normal aging and age-related macular degeneration (AMD. In this study, we sought to determine the impact of aging and AMD on expression of the endothelial cell glycoprotein CD34. Sections from 58 human donor eyes were categorized as either young (under age 40, age-matched controls (> age 60 without AMD, or AMD affected (>age 60 with early AMD, geographic atrophy, or choroidal neovascularization. Dual labeling of sections with Ulex europaeus agglutinin-I lectin (UEA-I and CD34 antibodies was performed, and the percentage of capillaries labeled with UEA-I but negative for anti-CD34 was determined. In addition, published databases of mouse and human retinal pigment epithelium-choroid were evaluated and CD34 expression compared between young and old eyes. Immunohistochemical studies revealed that while CD34 and UEA-I were colocalized in young eyes, there was variable loss of CD34 immunoreactivity in older donor eyes. While differences between normal aging and AMD were not significant, the percentage of CD34 negative capillaries in old eyes, compared to young eyes, was highly significant (p = 3.8×10(-6. Endothelial cells in neovascular membranes were invariably CD34 positive. Published databases show either a significant decrease in Cd34 (mouse or a trend toward decreased CD34 (human in aging. These findings suggest that UEA-I and endogenous alkaline phosphatase activity are more consistent markers of aging endothelial cells in the choroid, and suggest a possible mechanism for the increased inflammatory milieu in the aging choroid.

  6. Retinal vein occlusion in Benin City, Nigeria | Uhumwangho ...

    African Journals Online (AJOL)

    Eyes which had definitive treatment with intravitreal antivascular endothelial growth factors and laser photocoagulation for macula edema and retinal neovascularization, respectively, had better visual acuity compared to eyes which did not receive these treatment, P = 0.002. Conclusion: The incidence and visual loss that ...

  7. Protection of blood retinal barrier and systemic vasculature by insulin-like growth factor binding protein-3.

    Directory of Open Access Journals (Sweden)

    Yagna P R Jarajapu

    Full Text Available Previously, we showed that insulin growth factor (IGF-1 binding protein-3 (IGFBP-3, independent of IGF-1, reduces pathological angiogenesis in a mouse model of the oxygen-induced retinopathy (OIR. The current study evaluates novel endothelium-dependent functions of IGFBP-3 including blood retinal barrier (BRB integrity and vasorelaxation. To evaluate vascular barrier function, either plasmid expressing IGFBP-3 under the regulation of an endothelial-specific promoter or a control plasmid was injected into the vitreous humor of mouse pups (P1 and compared to the non-injected eyes of the same pups undergoing standard OIR protocol. Prior to sacrifice, the mice were given an injection of horseradish peroxidase (HRP. IGFBP-3 plasmid-injected eyes displayed near-normal vessel morphology and enhanced vascular barrier function. Further, in vitro IGFBP-3 protects retinal endothelial cells from VEGF-induced loss of junctional integrity by antagonizing the dissociation of the junctional complexes. To assess the vasodilatory effects of IGFBP-3, rat posterior cerebral arteries were examined in vitro. Intraluminal IGFBP-3 decreased both pressure- and serotonin-induced constrictions by stimulating nitric oxide (NO release that were blocked by L-NAME or scavenger receptor-B1 neutralizing antibody (SRB1-Ab. Both wild-type and IGF-1-nonbinding mutant IGFBP-3 (IGFBP-3NB stimulated eNOS activity/NO release to a similar extent in human microvascular endothelial cells (HMVECs. NO release was neither associated with an increase in intracellular calcium nor decreased by Ca(2+/calmodulin-dependent protein kinase II (CamKII blockade; however, dephosphorylation of eNOS-Thr(495 was observed. Phosphatidylinositol 3-kinase (PI3K activity and Akt-Ser(473 phosphorylation were both increased by IGFBP-3 and selectively blocked by the SRB1-Ab or PI3K blocker LY294002. In conclusion, IGFBP-3 mediates protective effects on BRB integrity and mediates robust NO release to stimulate

  8. Hypoxia inducible factor-2α regulates the development of retinal astrocytic network by maintaining adequate supply of astrocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Li-Juan Duan

    Full Text Available Here we investigate the role of hypoxia inducible factor (HIF-2α in coordinating the development of retinal astrocytic and vascular networks. Three Cre mouse lines were used to disrupt floxed Hif-2α, including Rosa26(CreERT2, Tie2(Cre, and GFAP(Cre. Global Hif-2α disruption by Rosa26(CreERT2 led to reduced astrocytic and vascular development in neonatal retinas, whereas endothelial disruption by Tie2(Cre had no apparent effects. Hif-2α deletion in astrocyte progenitors by GFAP(Cre significantly interfered with the development of astrocytic networks, which failed to reach the retinal periphery and were incapable of supporting vascular development. Perplexingly, the abundance of strongly GFAP(+ mature astrocytes transiently increased at P0 before they began to lag behind the normal controls by P3. Pax2(+ and PDGFRα(+ astrocytic progenitors and immature astrocytes were dramatically diminished at all stages examined. Despite decreased number of astrocyte progenitors, their proliferation index or apoptosis was not altered. The above data can be reconciled by proposing that HIF-2α is required for maintaining the supply of astrocyte progenitors by slowing down their differentiation into non-proliferative mature astrocytes. HIF-2α deficiency in astrocyte progenitors may accelerate their differentiation into astrocytes, a change which greatly interferes with the replenishment of astrocyte progenitors due to insufficient time for proliferation. Rapidly declining progenitor supply may lead to premature cessation of astrocyte development. Given that HIF-2α protein undergoes oxygen dependent degradation, an interesting possibility is that retinal blood vessels may regulate astrocyte differentiation through their oxygen delivery function. While our findings support the consensus that retinal astrocytic template guides vascular development, they also raise the possibility that astrocytic and vascular networks may mutually regulate each other

  9. Zika virus infection of cellular components of the blood-retinal barriers: implications for viral associated congenital ocular disease.

    Science.gov (United States)

    Roach, Tracoyia; Alcendor, Donald J

    2017-03-03

    Ocular abnormalities present in microcephalic infants with presumed Zika virus (ZIKV) congenital disease includes focal pigment mottling of the retina, chorioretinal atrophy, optic nerve abnormalities, and lens dislocation. Target cells in the ocular compartment for ZIKV infectivity are unknown. The cellular response of ocular cells to ZIKV infection has not been described. Mechanisms for viral dissemination in the ocular compartment of ZIKV-infected infants and adults have not been reported. Here, we identify target cells for ZIKV infectivity in both the inner and outer blood-retinal barriers (IBRB and OBRB), describe the cytokine expression profile in the IBRB after ZIKV exposure, and propose a mechanism for viral dissemination in the retina. We expose primary cellular components of the IBRB including human retinal microvascular endothelial cells, retinal pericytes, and Müller cells as well as retinal pigmented epithelial cells of the OBRB to the PRVABC56 strain of ZIKV. Viral infectivity was analyzed by microscopy, immunofluorescence, and reverse transcription polymerase chain reaction (RT-PCR and qRT-PCR). Angiogenic and proinflammatory cytokines were measured by Luminex assays. We find by immunofluorescent staining using the Flavivirus 4G2 monoclonal antibody that retinal endothelial cells and pericytes of the IBRB and retinal pigmented epithelial cells of the OBRB are fully permissive for ZIKV infection but not Müller cells when compared to mock-infected controls. We confirmed ZIKV infectivity in retinal endothelial cells, retinal pericytes, and retinal pigmented epithelial cells by RT-PCR and qRT-PCR using ZIKV-specific oligonucleotide primers. Expression profiles by Luminex assays in retinal endothelial cells infected with ZIKV revealed a marginal increase in levels of beta-2 microglobulin (β2-m), granulocyte macrophage colony-stimulating factor (GMCSF), intercellular adhesion molecule 1 (ICAM-1), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP

  10. Retinal Protection and Distribution of Curcumin in Vitro and in Vivo

    Directory of Open Access Journals (Sweden)

    Chiara B. M. Platania

    2018-06-01

    Full Text Available Diabetic retinopathy (DR, a secondary complication of diabetes, is a leading cause of irreversible blindness accounting for 5% of world blindness cases in working age. Oxidative stress and inflammation are considered causes of DR. Curcumin, a product with anti-oxidant and anti-inflammatory properties, is currently proposed as oral supplementation therapy for retinal degenerative diseases, including DR. In this study we predicted the pharmacodynamic profile of curcumin through an in silico approach. Furthermore, we tested the anti-oxidant and anti-inflammatory activity of curcumin on human retinal pigmented epithelial cells exposed to oxidative stress, human retinal endothelial and human retinal pericytes (HRPCs cultured with high glucose. Because currently marketed curcumin nutraceutical products have not been so far evaluated for their ocular bioavailability; we assessed retinal distribution of curcumin, following oral administration, in rabbit eye. Curcumin (10 μM decreased significantly (p < 0.01 ROS concentration and TNF-α release in retinal pigmented epithelial cells and retinal endothelial cells, respectively. The same curcumin concentration significantly (p < 0.01 protected retinal pericytes from high glucose damage as assessed by cell viability and LDH release. Among the tested formulations, only that containing a hydrophilic carrier provided therapeutic levels of curcumin in rabbit retina. In conclusion, our data suggest that curcumin, when properly formulated, may be of value in clinical practice to manage retinal diseases.

  11. Molecular Responses of Human Retinal Cells to Infection with Dengue Virus.

    Science.gov (United States)

    Carr, Jillian M; Ashander, Liam M; Calvert, Julie K; Ma, Yuefang; Aloia, Amanda; Bracho, Gustavo G; Chee, Soon-Phaik; Appukuttan, Binoy; Smith, Justine R

    2017-01-01

    Recent clinical reports indicate that infection with dengue virus (DENV) commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin- β 1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

  12. Molecular Responses of Human Retinal Cells to Infection with Dengue Virus

    Directory of Open Access Journals (Sweden)

    Jillian M. Carr

    2017-01-01

    Full Text Available Recent clinical reports indicate that infection with dengue virus (DENV commonly has ocular manifestations. The most serious threat to vision is dengue retinopathy, including retinal vasculopathy and macular edema. Mechanisms of retinopathy are unstudied, but observations in patients implicate retinal pigment epithelial cells and retinal endothelial cells. Human retinal cells were inoculated with DENV-2 and monitored for up to 72 hours. Epithelial and endothelial cells supported DENV replication and release, but epithelial cells alone demonstrated clear cytopathic effect, and infection was more productive in those cells. Infection induced type I interferon responses from both cells, but this was stronger in epithelial cells. Endothelial cells increased expression of adhesion molecules, with sustained overexpression of vascular adhesion molecule-1. Transcellular impedance decreased for epithelial monolayers, but not endothelial monolayers, coinciding with cytopathic effect. This reduction was accompanied by disorganization of intracellular filamentous-actin and decreased expression of junctional molecules, zonula occludens 1, and catenin-β1. Changes in endothelial expression of adhesion molecules are consistent with the retinal vasculopathy seen in patients infected with DENV; decreases in epithelial junctional protein expression, paralleling loss of integrity of the epithelium, provide a molecular basis for DENV-associated macular edema. These molecular processes present potential therapeutic targets for vision-threatening dengue retinopathy.

  13. Laser photocoagulation for retinal vein occlusion

    Directory of Open Access Journals (Sweden)

    K. A. Mirzabekova

    2015-03-01

    Full Text Available Retinal vein occlusion (RVO is one of the leading causes of permanent vision loss. In adults, central retinal vein occlusion (CRVO occurs in 1.8% while branch retinal vein occlusion (BRVO occurs in 0.2%. Treatment strategy and disease prognosis are determined by RVO type (ischemic/non-ischemic. Despite numerous studies and many current CRVO and BRVO treatment approaches, the management of these patients is still being debated. Intravitreal injections of steroids (triamcinolone acetate, dexamethasone and vascular endothelial growth factor (VEGF inhibitors (bevacizumab, ranibizumab were shown to be fairly effective. However, it is unclear whether anti-VEGF agents are reasonable in ischemic RVOs. Laser photocoagulation remains the only effective treatment of optic nerve head and/or retinal neovascularization. Laser photocoagulation is also indicated for the treatment of macular edema. Both threshold and sub-threshold photocoagulation may be performed. Photocoagulation performed with argon (514 nm, krypton (647 nm, or diode (810 nm laser for macular edema provides similar results (no significant differences. The treatment may be complex and include medication therapy and/or surgery. Medication therapy includes anti-aggregant agents and antioxidants, i.e., emoxypine which may be used in acute RVO as well as in post-thrombotic retinopathy. 

  14. Laser photocoagulation for retinal vein occlusion

    Directory of Open Access Journals (Sweden)

    K. A. Mirzabekova

    2015-01-01

    Full Text Available Retinal vein occlusion (RVO is one of the leading causes of permanent vision loss. In adults, central retinal vein occlusion (CRVO occurs in 1.8% while branch retinal vein occlusion (BRVO occurs in 0.2%. Treatment strategy and disease prognosis are determined by RVO type (ischemic/non-ischemic. Despite numerous studies and many current CRVO and BRVO treatment approaches, the management of these patients is still being debated. Intravitreal injections of steroids (triamcinolone acetate, dexamethasone and vascular endothelial growth factor (VEGF inhibitors (bevacizumab, ranibizumab were shown to be fairly effective. However, it is unclear whether anti-VEGF agents are reasonable in ischemic RVOs. Laser photocoagulation remains the only effective treatment of optic nerve head and/or retinal neovascularization. Laser photocoagulation is also indicated for the treatment of macular edema. Both threshold and sub-threshold photocoagulation may be performed. Photocoagulation performed with argon (514 nm, krypton (647 nm, or diode (810 nm laser for macular edema provides similar results (no significant differences. The treatment may be complex and include medication therapy and/or surgery. Medication therapy includes anti-aggregant agents and antioxidants, i.e., emoxypine which may be used in acute RVO as well as in post-thrombotic retinopathy. 

  15. Focal retinal phlebitis.

    Science.gov (United States)

    Hoang, Quan V; Freund, K Bailey; Klancnik, James M; Sorenson, John A; Cunningham, Emmett T; Yannuzzi, Lawrence A

    2012-01-01

    To report three cases of solitary, focal retinal phlebitis. An observational case series. Three eyes in three patients were noted to have unilateral decreased vision, macular edema, and a focal retinal phlebitis, which was not at an arteriovenous crossing. All three patients developed a branch retinal vein occlusion at the site of inflammation. These patients had no other evidence of intraocular inflammation, including vitritis, retinitis, retinal vasculitis, or choroiditis, nor was there any systemic disorder associated with inflammation, infection, or coagulation identified. Focal retinal phlebitis appears to be an uncommon and unique entity that produces macular edema and ultimately branch retinal vein occlusion. In our patients, the focal phlebitis and venous occlusion did not occur at an arteriovenous crossing, which is the typical site for branch retinal venous occlusive disease. This suggests that our cases represent a distinct clinical entity, which starts with a focal abnormality in the wall of a retinal venule, resulting in surrounding exudation and, ultimately, ends with branch retinal vein occlusion.

  16. Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa

    NARCIS (Netherlands)

    Alves, Celso Henrique; Pellissier, Lucie P; Vos, Rogier M; Garcia Garrido, Marina; Sothilingam, Vithiyanjali; Seide, Christina; Beck, Susanne C; Klooster, J.; Furukawa, Takahisa; Flannery, John G; Verhaagen, J.; Seeliger, Mathias W; Wijnholds, J.

    2014-01-01

    In humans, the Crumbs homolog-1 (CRB1) gene is mutated in autosomal recessive Leber congenital amaurosis and early-onset retinitis pigmentosa. In mammals, the Crumbs family is composed of: CRB1, CRB2, CRB3A and CRB3B. Recently, we showed that removal of mouse Crb2 from retinal progenitor cells, and

  17. Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk [Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul 151744 (Korea, Republic of); Kim, Kyu-Won [NeuroVascular Coordination Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151742 (Korea, Republic of); Kim, Myung Hun, E-mail: hunin315@paran.com, E-mail: ysyu@snu.ac.kr [Department of Chemistry, Yonsei University, 134 Shinchon-dong, Seodaemun-ku, Seoul 120749 (Korea, Republic of)

    2009-12-16

    The retina maintains homeostasis through the blood-retinal barrier (BRB). Although it is ideal to deliver the drug to the retina via systemic administration, it is still challenging due to the BRB strictly regulating permeation from blood to the retina. Herein, we demonstrated that intravenously administered gold nanoparticles could pass through the BRB and are distributed in all retinal layers without cytotoxicity. After intravenous injection of gold nanoparticles into C57BL/6 mice, 100 nm nanoparticles were not detected in the retina whereas 20 nm nanoparticles passed through the BRB and were distributed in all retinal layers. 20 nm nanoparticles in the retina were observed in neurons (75 {+-} 5%), endothelial cells (17 {+-} 6%) and peri-endothelial glial cells (8 {+-} 3%), where nanoparticles were bound on the membrane. In the retina, cells containing nanoparticles did not show any structural abnormality and increase of cell death compared to cells without nanoparticles. Gold nanoparticles never affected the viability of retinal endothelial cells, astrocytes and retinoblastoma cells. Furthermore, gold nanoparticles never led to any change in expression of representative biological molecules including zonula occludens-1 and glut-1 in retinal endothelial cells, neurofilaments in differentiated retinoblastoma cells and glial fibrillary acidic protein in astrocytes. Therefore, our data suggests that small gold nanoparticles (20 nm) could be an alternative for drug delivery across the BRB, which could be safely applied in vivo.

  18. Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity

    International Nuclear Information System (INIS)

    Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk; Kim, Kyu-Won; Kim, Myung Hun

    2009-01-01

    The retina maintains homeostasis through the blood-retinal barrier (BRB). Although it is ideal to deliver the drug to the retina via systemic administration, it is still challenging due to the BRB strictly regulating permeation from blood to the retina. Herein, we demonstrated that intravenously administered gold nanoparticles could pass through the BRB and are distributed in all retinal layers without cytotoxicity. After intravenous injection of gold nanoparticles into C57BL/6 mice, 100 nm nanoparticles were not detected in the retina whereas 20 nm nanoparticles passed through the BRB and were distributed in all retinal layers. 20 nm nanoparticles in the retina were observed in neurons (75 ± 5%), endothelial cells (17 ± 6%) and peri-endothelial glial cells (8 ± 3%), where nanoparticles were bound on the membrane. In the retina, cells containing nanoparticles did not show any structural abnormality and increase of cell death compared to cells without nanoparticles. Gold nanoparticles never affected the viability of retinal endothelial cells, astrocytes and retinoblastoma cells. Furthermore, gold nanoparticles never led to any change in expression of representative biological molecules including zonula occludens-1 and glut-1 in retinal endothelial cells, neurofilaments in differentiated retinoblastoma cells and glial fibrillary acidic protein in astrocytes. Therefore, our data suggests that small gold nanoparticles (20 nm) could be an alternative for drug delivery across the BRB, which could be safely applied in vivo.

  19. Plastic roles of pericytes in the blood-retinal barrier.

    Science.gov (United States)

    Park, Do Young; Lee, Junyeop; Kim, Jaeryung; Kim, Kangsan; Hong, Seonpyo; Han, Sangyeul; Kubota, Yoshiaki; Augustin, Hellmut G; Ding, Lei; Kim, Jin Woo; Kim, Hail; He, Yulong; Adams, Ralf H; Koh, Gou Young

    2017-05-16

    The blood-retinal barrier (BRB) consists of tightly interconnected capillary endothelial cells covered with pericytes and glia, but the role of the pericytes in BRB regulation is not fully understood. Here, we show that platelet-derived growth factor (PDGF)-B/PDGF receptor beta (PDGFRβ) signalling is critical in formation and maturation of BRB through active recruitment of pericytes onto growing retinal vessels. Impaired pericyte recruitment to the vessels shows multiple vascular hallmarks of diabetic retinopathy (DR) due to BRB disruption. However, PDGF-B/PDGFRβ signalling is expendable for maintaining BRB integrity in adult mice. Although selective pericyte loss in stable adult retinal vessels surprisingly does not cause BRB disintegration, it sensitizes retinal vascular endothelial cells (ECs) to VEGF-A, leading to upregulation of angiopoietin-2 (Ang2) in ECs through FOXO1 activation and triggering a positive feedback that resembles the pathogenesis of DR. Accordingly, either blocking Ang2 or activating Tie2 greatly attenuates BRB breakdown, suggesting potential therapeutic approaches to reduce retinal damages upon DR progression.

  20. Technical Brief: A comparison of two methods of euthanasia on retinal dopamine levels

    OpenAIRE

    Hwang, Christopher K.; Iuvone, P. Michael

    2013-01-01

    Purpose Mice are commonly used in biomedical research, and euthanasia is an important part of mouse husbandry. Approved, humane methods of euthanasia are designed to minimize the potential for pain or discomfort, but may also influence the measurement of experimental variables. Methods We compared the effects of two approved methods of mouse euthanasia on the levels of retinal dopamine. We examined the level of retinal dopamine, a commonly studied neuromodulator, following euthanasia by carbo...

  1. In Vivo Imaging of Retinal Hypoxia in a Model of Oxygen-Induced Retinopathy.

    Science.gov (United States)

    Uddin, Md Imam; Evans, Stephanie M; Craft, Jason R; Capozzi, Megan E; McCollum, Gary W; Yang, Rong; Marnett, Lawrence J; Uddin, Md Jashim; Jayagopal, Ashwath; Penn, John S

    2016-08-05

    Ischemia-induced hypoxia elicits retinal neovascularization and is a major component of several blinding retinopathies such as retinopathy of prematurity (ROP), diabetic retinopathy (DR) and retinal vein occlusion (RVO). Currently, noninvasive imaging techniques capable of detecting and monitoring retinal hypoxia in living systems do not exist. Such techniques would greatly clarify the role of hypoxia in experimental and human retinal neovascular pathogenesis. In this study, we developed and characterized HYPOX-4, a fluorescence-imaging probe capable of detecting retinal-hypoxia in living animals. HYPOX-4 dependent in vivo and ex vivo imaging of hypoxia was tested in a mouse model of oxygen-induced retinopathy (OIR). Predicted patterns of retinal hypoxia were imaged by HYPOX-4 dependent fluorescence activity in this animal model. In retinal cells and mouse retinal tissue, pimonidazole-adduct immunostaining confirmed the hypoxia selectivity of HYPOX-4. HYPOX-4 had no effect on retinal cell proliferation as indicated by BrdU assay and exhibited no acute toxicity in retinal tissue as indicated by TUNEL assay and electroretinography (ERG) analysis. Therefore, HYPOX-4 could potentially serve as the basis for in vivo fluorescence-based hypoxia-imaging techniques, providing a tool for investigators to understand the pathogenesis of ischemic retinopathies and for physicians to address unmet clinical needs.

  2. Retinal detachment following endophthalmitis.

    Science.gov (United States)

    Nelsen, P T; Marcus, D A; Bovino, J A

    1985-08-01

    Fifty-five consecutive patients with a clinical diagnosis of bacterial endophthalmitis were reviewed. All patients were treated with systemic, periocular, topical, and intravitreal antibiotics. In addition, 33 of the patients underwent a pars plana vitrectomy. Nine retinal detachments occurred within six months of initial diagnosis. The higher frequency of retinal detachment in the vitrectomy group (21%) as compared to those patients managed without vitrectomy (9%) may be explained by a combination of surgical complications and the increased severity of endophthalmitis in the vitrectomy group. The two patients who developed retinal detachment during vitrectomy surgery rapidly progressed to no light perception. Conversely, the repair of retinal detachments diagnosed postoperatively had a good prognosis.

  3. Retinal oximetry in patients with ischaemic retinal diseases

    DEFF Research Database (Denmark)

    Rilvén, Sandra; Torp, Thomas Lee; Grauslund, Jakob

    2017-01-01

    The retinal oximeter is a new tool for non-invasive measurement of retinal oxygen saturation in humans. Several studies have investigated the associations between retinal oxygen saturation and retinal diseases. In the present systematic review, we examine whether there are associations between...... retinal oxygen saturation and retinal ischaemic diseases. We used PubMed and Embase to search for retinal oxygen saturation and retinal ischaemic diseases. Three separate searches identified a total of 79 publications. After two levels of manual screening, 10 studies were included: six about diabetic...... retinopathy (DR) and four about retinal vein occlusion. No studies about retinal artery occlusion were included. In diabetes, all studies found that increases in retinal venous oxygen saturation (rvSatO2 ) were associated with present as well as increasing levels of DR. Four of six studies also found...

  4. In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression.

    Science.gov (United States)

    Lulli, Matteo; Cammalleri, Maurizio; Granucci, Irene; Witort, Ewa; Bono, Silvia; Di Gesualdo, Federico; Lupia, Antonella; Loffredo, Rosa; Casini, Giovanni; Dal Monte, Massimo; Capaccioli, Sergio

    2017-08-26

    Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Dorzolamide increases retinal oxygen tension after branch retinal vein occlusion

    DEFF Research Database (Denmark)

    Noergaard, Michael Hove; Bach-Holm, Daniella; Scherfig, Erik

    2008-01-01

    To study the effect of dorzolamide on the preretinal oxygen tension (RPO(2)) in retinal areas affected by experimental branch retinal vein occlusion (BRVO) in pigs.......To study the effect of dorzolamide on the preretinal oxygen tension (RPO(2)) in retinal areas affected by experimental branch retinal vein occlusion (BRVO) in pigs....

  6. Occlusion of retinal capillaries caused by glial cell proliferation in chronic ocular inflammation.

    Science.gov (United States)

    Bianchi, E; Ripandelli, G; Feher, J; Plateroti, A M; Plateroti, R; Kovacs, I; Plateroti, P; Taurone, S; Artico, M

    2015-01-01

    The inner blood-retinal barrier is a gliovascular unit in which glial cells surround capillary endothelial cells and regulate retinal capillaries by paracrine interactions. During chronic ocular inflammation, microvascular complications can give rise to vascular proliferative lesions, which compromise visual acuity. This pathologic remodelling caused by proliferating Müller cells determines occlusion of retinal capillaries. The aim of the present study was to identify qualitative and quantitative alterations in the retinal capillaries in patients with post-traumatic chronic ocular inflammation or post-thrombotic vascular glaucoma. Moreover, we investigated the potential role of vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines in retinal inflammation. Our electron microscopy findings demonstrated that during chronic ocular inflammation, thickening of the basement membrane, loss of pericytes and endothelial cells and proliferation of Müller cells occur with irreversible occlusion of retinal capillaries. Angiogenesis takes place as part of a regenerative reaction that results in fibrosis. We believe that VEGF and pro-inflammatory cytokines may be potential therapeutic targets in the treatment of this disease although further studies are required to confirm these findings.

  7. Mechanisms of Retinal Damage after Ocular Alkali Burns.

    Science.gov (United States)

    Paschalis, Eleftherios I; Zhou, Chengxin; Lei, Fengyang; Scott, Nathan; Kapoulea, Vassiliki; Robert, Marie-Claude; Vavvas, Demetrios; Dana, Reza; Chodosh, James; Dohlman, Claes H

    2017-06-01

    Alkali burns to the eye constitute a leading cause of worldwide blindness. In recent case series, corneal transplantation revealed unexpected damage to the retina and optic nerve in chemically burned eyes. We investigated the physical, biochemical, and immunological components of retinal injury after alkali burn and explored a novel neuroprotective regimen suitable for prompt administration in emergency departments. Thus, in vivo pH, oxygen, and oxidation reduction measurements were performed in the anterior and posterior segment of mouse and rabbit eyes using implantable microsensors. Tissue inflammation was assessed by immunohistochemistry and flow cytometry. The experiments confirmed that the retinal damage is not mediated by direct effect of the alkali, which is effectively buffered by the anterior segment. Rather, pH, oxygen, and oxidation reduction changes were restricted to the cornea and the anterior chamber, where they caused profound uveal inflammation and release of proinflammatory cytokines. The latter rapidly diffuse to the posterior segment, triggering retinal damage. Tumor necrosis factor-α was identified as a key proinflammatory mediator of retinal ganglion cell death. Blockade, by either monoclonal antibody or tumor necrosis factor receptor gene knockout, reduced inflammation and retinal ganglion cell loss. Intraocular pressure elevation was not observed in experimental alkali burns. These findings illuminate the mechanism by which alkali burns cause retinal damage and may have importance in designing therapies for retinal protection. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  8. Mouse adenovirus type 1 infection of macrophages

    NARCIS (Netherlands)

    Ashley, S.L.; Welton, A.R.; Harwood, K.M.; Rooijen, van N.; Spindler, K.R.

    2009-01-01

    Mouse adenovirus type 1 (MAV-1) causes acute and persistent infections in mice, with high levels of virus found in the brain, spinal cord and spleen in acute infections. MAV-1 infects endothelial cells throughout the mouse, and monocytes/macrophages have also been implicated as targets of the virus.

  9. Differential diagnosis of retinal vasculitis.

    Science.gov (United States)

    Abu El-Asrar, Ahmed M; Herbort, Carl P; Tabbara, Khalid F

    2009-10-01

    Retinal vaculitis is a sight-threatening inflammatory eye condition that involves the retinal vessels. Detection of retinal vasculitis is made clinically, and confirmed with the help of fundus fluorescein angiography. Active vascular disease is characterized by exudates around retinal vessels resulting in white sheathing or cuffing of the affected vessels. In this review, a practical approach to the diagnosis of retinal vasculitis is discussed based on ophthalmoscopic and fundus fluorescein angiographic findings.

  10. Retinal Diseases Associated with Oxidative Stress and the Effects of a Free Radical Scavenger (Edaravone

    Directory of Open Access Journals (Sweden)

    Tomomi Masuda

    2017-01-01

    Full Text Available Oxidative stress plays a pivotal role in developing and accelerating retinal diseases including age-related macular degeneration (AMD, glaucoma, diabetic retinopathy (DR, and retinal vein occlusion (RVO. An excess amount of reactive oxygen species (ROS can lead to functional and morphological impairments in retinal pigment epithelium (RPE, endothelial cells, and retinal ganglion cells (RGCs. Here we demonstrate that edaravone, a free radical scavenger, decreased apoptotic cell death, oxidative damage to DNA and lipids, and angiogenesis through inhibiting JNK and p38 MAPK pathways in AMD, glaucoma, DR, and RVO animal models. These data suggest that the therapeutic strategy for targeting oxidative stress may be important for the treatment of these ocular diseases, and edaravone may be useful for treating retinal diseases associated with oxidative stress.

  11. Retinal Diseases Associated with Oxidative Stress and the Effects of a Free Radical Scavenger (Edaravone)

    Science.gov (United States)

    Hara, Hideaki

    2017-01-01

    Oxidative stress plays a pivotal role in developing and accelerating retinal diseases including age-related macular degeneration (AMD), glaucoma, diabetic retinopathy (DR), and retinal vein occlusion (RVO). An excess amount of reactive oxygen species (ROS) can lead to functional and morphological impairments in retinal pigment epithelium (RPE), endothelial cells, and retinal ganglion cells (RGCs). Here we demonstrate that edaravone, a free radical scavenger, decreased apoptotic cell death, oxidative damage to DNA and lipids, and angiogenesis through inhibiting JNK and p38 MAPK pathways in AMD, glaucoma, DR, and RVO animal models. These data suggest that the therapeutic strategy for targeting oxidative stress may be important for the treatment of these ocular diseases, and edaravone may be useful for treating retinal diseases associated with oxidative stress. PMID:28194256

  12. Hypoxia-ischemia and retinal ganglion cell damage

    Directory of Open Access Journals (Sweden)

    Charanjit Kaur

    2008-08-01

    Full Text Available Charanjit Kaur1, Wallace S Foulds2, Eng-Ang Ling11Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore; 2Singapore Eye Research Institute, SingaporeAbstract: Retinal hypoxia is the potentially blinding mechanism underlying a number of sight-threatening disorders including central retinal artery occlusion, ischemic central retinal vein thrombosis, complications of diabetic eye disease and some types of glaucoma. Hypoxia is implicated in loss of retinal ganglion cells (RGCs occurring in such conditions. RGC death occurs by apoptosis or necrosis. Hypoxia-ischemia induces the expression of hypoxia inducible factor-1α and its target genes such as vascular endothelial growth factor (VEGF and nitric oxide synthase (NOS. Increased production of VEGF results in disruption of the blood retinal barrier leading to retinal edema. Enhanced expression of NOS results in increased production of nitric oxide which may be toxic to the cells resulting in their death. Excess glutamate release in hypoxic-ischemic conditions causes excitotoxic damage to the RGCs through activation of ionotropic and metabotropic glutamate receptors. Activation of glutamate receptors is thought to initiate damage in the retina by a cascade of biochemical effects such as neuronal NOS activation and increase in intracellular Ca2+ which has been described as a major contributing factor to RGC loss. Excess production of proinflammatory cytokines also mediates cell damage. Besides the above, free-radicals generated in hypoxic-ischemic conditions result in RGC loss because of an imbalance between antioxidant- and oxidant-generating systems. Although many advances have been made in understanding the mediators and mechanisms of injury, strategies to improve the damage are lacking. Measures to prevent neuronal injury have to be developed.Keywords: retinal hypoxia, retinal ganglion cells, glutamate receptors, neuronal injury, retina

  13. High glucose alters retinal astrocytes phenotype through increased production of inflammatory cytokines and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Eui Seok Shin

    Full Text Available Astrocytes are macroglial cells that have a crucial role in development of the retinal vasculature and maintenance of the blood-retina-barrier (BRB. Diabetes affects the physiology and function of retinal vascular cells including astrocytes (AC leading to breakdown of BRB. However, the detailed cellular mechanisms leading to retinal AC dysfunction under high glucose conditions remain unclear. Here we show that high glucose conditions did not induce the apoptosis of retinal AC, but instead increased their rate of DNA synthesis and adhesion to extracellular matrix proteins. These alterations were associated with changes in intracellular signaling pathways involved in cell survival, migration and proliferation. High glucose conditions also affected the expression of inflammatory cytokines in retinal AC, activated NF-κB, and prevented their network formation on Matrigel. In addition, we showed that the attenuation of retinal AC migration under high glucose conditions, and capillary morphogenesis of retinal endothelial cells on Matrigel, was mediated through increased oxidative stress. Antioxidant proteins including heme oxygenase-1 and peroxiredoxin-2 levels were also increased in retinal AC under high glucose conditions through nuclear localization of transcription factor nuclear factor-erythroid 2-related factor-2. Together our results demonstrated that high glucose conditions alter the function of retinal AC by increased production of inflammatory cytokines and oxidative stress with significant impact on their proliferation, adhesion, and migration.

  14. Retinal oximetry during treatment of retinal vein occlusion by ranibizumab in patients with high blood pressure and dyslipidemia.

    Science.gov (United States)

    Keilani, C; Halalchi, A; Wakpi Djeugue, D; Regis, A; Abada, S

    2016-12-01

    In the present study, we examined retinal vascular oxygen saturation in patients with retinal vein occlusion (RVO), high blood pressure (HBP) and dyslipidemia, before and during intravitreal vascular endothelial growth factor (VEGF) injection (ranibizumab). We retrospectively reviewed the medical records of six patients with visual acuity (VA) reduced by macular edema (ME) secondary to RVO with HBP and dyslipidemia, who underwent intravitreal anti-VEGF injection between October 2014 and February 2015 in the department of ophthalmology of François-Quesnay Hospital at Mantes-la-Jolie (France). The main inclusion criterion was the presence of RVO with ME and decreased VA. The primary endpoint was improvement of retinal venous oxygen saturation in patients with RVO before and 3 months after intravitreal ranibizumab injection. Secondary outcomes were improvement of retinal arterial oxygen saturation, improvement of best-corrected visual acuity (BCVA) on the Early Treatment Diabetic Retinopathy Study (ETDRS) scale, regression of ME measured by the central macular thickness (CMT) in nm and studying the correlation between blood pressure (BP) and retinal venous oxygen saturation before and after ranibizumab. Six eyes of six patients were included. Before treatment, the mean (standard deviation [SD]) of the retinal venous saturation (%) was 38.1±14.2. Three months after the injections, the mean (SD) of the retinal venous saturation (%) increased statistically significantly 49.2±11 (P=0.03). In this study, retinal venous oxygen saturation in patients with RVO, HBP and dyslipidemia was partially normalized during intravitreal ranibizumab treatment. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Retinal Detachment Vision Simulator

    Science.gov (United States)

    ... Feb 20, 2018 Gene Therapy May Be a Game-Changer for People With Inherited Retinal Disease Dec 19, 2017 ... the Academy Jobs at the Academy Financial Relationships with Industry Medical Disclaimer Privacy Policy Terms of Service For ...

  16. Learning about Retinitis Pigmentosa

    Science.gov (United States)

    Skip to main content Learning about Retinitis Pigmentosa Enter Search Term(s): Español Research Funding An Overview Bioinformatics Current Grants Education and Training Funding Extramural Research ...

  17. Progressive outer retinal necrosis-like retinitis in immunocompetent hosts.

    Science.gov (United States)

    Chawla, Rohan; Tripathy, Koushik; Gogia, Varun; Venkatesh, Pradeep

    2016-08-10

    We describe two young immunocompetent women presenting with bilateral retinitis with outer retinal necrosis involving posterior pole with centrifugal spread and multifocal lesions simulating progressive outer retinal necrosis (PORN) like retinitis. Serology was negative for HIV and CD4 counts were normal; however, both women were on oral steroids at presentation for suspected autoimmune chorioretinitis. The retinitis in both eyes responded well to oral valaciclovir therapy. However, the eye with the more fulminant involvement developed retinal detachment with a loss of vision. Retinal atrophy was seen in the less involved eye with preservation of vision. Through these cases, we aim to describe a unique evolution of PORN-like retinitis in immunocompetent women, which was probably aggravated by a short-term immunosuppression secondary to oral steroids. 2016 BMJ Publishing Group Ltd.

  18. Hypoxia-induced retinal neovascularization in zebrafish embryos: a potential model of retinopathy of prematurity.

    Science.gov (United States)

    Wu, Yu-Ching; Chang, Chao-Yuan; Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

    2015-01-01

    Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2-4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.

  19. Impaired Retinal Vasodilator Responses in Prediabetes and Type 2 Diabetes

    Science.gov (United States)

    Lott, Mary E.J.; Slocomb, Julia E.; Shivkumar, Vikram; Smith, Bruce; Quillen, David; Gabbay, Robert A.; Gardner, Thomas W.; Bettermann, Kerstin

    2013-01-01

    Purpose In diabetes, endothelial dysfunction and subsequent structural damage to blood vessels can lead to heart attacks, retinopathy and strokes. However, it is unclear whether prediabetic subjects exhibit microvascular dysfunction indicating early stages of arteriosclerosis and vascular risk. The purpose of this study was to examine whether retinal reactivity may be impaired early in the hyperglycemic continuum and may be associated with markers of inflammation. Methods Individuals with prediabetes (n = 22), type 2 diabetes (n = 25) and healthy age and body composition matched controls (n = 19) were studied. We used the Dynamic Vessel Analyzer to assess retinal vasoreactivity (percent change in vessel diameter) during a flickering light stimulation. Fasting highly sensitive c-reactive protein (hs-CRP), a marker of inflammation, was measured in blood plasma. Results Prediabetic and diabetic individuals had attenuated peak vasodilator and relative amplitude changes in retinal vein diameters to the flickering light stimulus compared to healthy controls (peak dilation: prediabetic subjects 3.3 ± 1.8 %, diabetic subjects 3.3 ± 2.1% controls 5.6 ± 2.6%, p = .001; relative amplitude: prediabetic subjects 4.3 ± 2.2%, diabetic subjects 5.0 ± 2.6% and control subjects 7.2 ± 3.2%, p = .003). Similar findings were observed in retinal arteries. Levels of hs-CRP were not associated with either retinal vessel response parameters. Conclusion Retinal reactivity was impaired in prediabetic and type 2 diabetic individuals in parallel with reduced insulin sensitivity but not associated with levels of hs-CRP. Retinal vasoreactivity measurements may be a sensitive tool to assess early vascular risk. PMID:23742315

  20. Molecular imaging of retinal endothelial injury in diabetic animals

    Directory of Open Access Journals (Sweden)

    Sonja Frimmel

    2017-01-01

    Conclusion: Results indicate that molecular imaging can be used to detect subtle changes in the diabetic retina prior to the occurrence of irreversible pathology. Thus, ICAM-1 could serve as a diagnostic target in patients with diabetes. This study provides a proof of principle for non-invasive subclinical diagnosis in experimental diabetic retinopathy. Further development of this technology could improve management of diabetic complications.

  1. Economic evaluation of an e-mental health intervention for patients with retinal exudative diseases who receive intra-ocular anti-VEGF injections (E-PsEYE): protocol for a randomised controlled trial.

    NARCIS (Netherlands)

    van der Aa, HPA; van Rens, G.H.M.B.; Verbraak, F.D.; Bosscha, M; Koopmanschap, M.A.; Comijs, H.C.; Cuijpers, P.; van Nispen, R.M.A.

    2017-01-01

    Introduction Because of the great potential of vascular endothelial growth factor inhibitors (anti-VEGF) for retinal exudative diseases, an increased number of patients receives this treatment. However, during this treatment, patients are subjected to frequent invasive intravitreal injections, and

  2. Retinal shows its true colours

    DEFF Research Database (Denmark)

    Coughlan, N. J.A.; Adamson, B. D.; Gamon, L.

    2015-01-01

    Retinal is one of Nature's most important and widespread chromophores, exhibiting remarkable versatility in its function and spectral response, depending on its protein environment. Reliable spectroscopic and photochemical data for the isolated retinal molecule are essential for calibrating theor...

  3. Retinal findings in membranoproliferative glomerulonephritis

    Directory of Open Access Journals (Sweden)

    Ahmad M. Mansour

    2017-09-01

    Conclusions and importance: Drusen remain the ocular stigmata for MPGN occuring at an early age. The retinal disease is progressive with gradual thickening of Bruch's membrane and occurrence of retinal pigment epithelium detachment.

  4. Effect of pharmacologically induced retinal degeneration on retinal autofluorescence lifetimes in mice.

    Science.gov (United States)

    Dysli, Chantal; Dysli, Muriel; Zinkernagel, Martin S; Enzmann, Volker

    2016-12-01

    Fluorescence lifetime imaging ophthalmoscopy (FLIO) was used to investigate retinal autofluorescence lifetimes in mouse models of pharmacologically induced retinal degeneration over time. Sodium iodate (NaIO 3 , 35 mg/kg intravenously) was used to induce retinal pigment epithelium (RPE) degeneration with subsequent loss of photoreceptors (PR) whereas N-methyl-N-nitrosourea (MNU, 45 mg/kg intraperitoneally) was employed for degeneration of the photoreceptor cell layer alone. All mice were measured at day 3, 7, 14, and 28 after the respective injection of NaIO 3 , MNU or NaCl (control). Fluorescence lifetime imaging was performed using a fluorescence lifetime imaging ophthalmoscope (Heidelberg Engineering, Heidelberg, Germany). Fluorescence was excited at 473 nm and fluorescence lifetimes were measured in a short and a long spectral channel (498-560 nm and 560-720 nm). Corresponding optical coherence tomography (OCT) images were consecutively acquired and histology was performed at the end of the experiments. Segmentation of OCT images and histology verified the cell type-specific degeneration process over time. Retinal autofluorescence lifetimes increased from day 3 to day 28 in mice after NaIO 3 treatment. Finally, at day 28, fluorescence lifetimes were prolonged by 8% in the short and 61% in the long spectral channel compared to control animals (p = 0.21 and p = 0.004, respectively). In mice after MNU treatment, the mean retinal autofluorescence lifetimes were already decreased at day 3 and retinal lifetimes were finally shortened by 27% in the short and 51% in the long spectral channel at day 28 (p = 0.0028). In conclusion, degeneration of the RPE with subsequent photoreceptor degeneration by NaIO 3 lead to longer mean fluorescence lifetimes of the retina compared to control mice, whereas during specific degeneration of the photoreceptor layer induced by MNU shorter lifetimes were measured. Therefore, short retinal fluorescence lifetimes may originate

  5. Consumption of Polyphenol-Rich Zingiber Zerumbet Rhizome Extracts Protects against the Breakdown of the Blood-Retinal Barrier and Retinal Inflammation Induced by Diabetes

    Directory of Open Access Journals (Sweden)

    Thing-Fong Tzeng

    2015-09-01

    Full Text Available The present study investigates the amelioration of diabetic retinopathy (DR by Zingiber zerumbet rhizome ethanol extracts (ZZRext in streptozotocin-induced diabetic rats (STZ-diabetic rats. ZZRext contains high phenolic and flavonoid contents. STZ-diabetic rats were treated orally with ZZRext (200, 300 mg/kg per day for three months. Blood-retinal barrier (BRB breakdown and increased vascular permeability were found in diabetic rats, with downregulation of occludin, and claudin-5. ZZRext treatment effectively preserved the expression of occludin, and claudin-5, leading to less BRB breakdown and less vascular permeability. Retinal histopathological observation showed that the disarrangement and reduction in thickness of retinal layers were reversed in ZZRext-treated diabetic rats. Retinal gene expression of tumor necrosis factor-α, interleukin (IL-1β, IL-6, vascular endothelial growth factor, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 were all decreased in ZZRext-treated diabetic rats. Moreover, ZZRext treatment not only inhibited the nuclear factor κB (NF-κB activation, but also downregulated the protein expression of p38 mitogen-activated protein kinase (MAPK in diabetic retina. In conclusion, the results suggest that the retinal protective effects of ZZRext occur through improved retinal structural change and inhibiting retinal inflammation. The antiretinopathy property of ZZRext might be related to the downregulation of p38 MAPK and NF-κB signal transduction induced by diabetes.

  6. Bioelectronic retinal prosthesis

    Science.gov (United States)

    Weiland, James D.

    2016-05-01

    Retinal prosthesis have been translated to clinical use over the past two decades. Currently, two devices have regulatory approval for the treatment of retinitis pigmentosa and one device is in clinical trials for treatment of age-related macular degeneration. These devices provide partial sight restoration and patients use this improved vision in their everyday lives to navigate and to detect large objects. However, significant vision restoration will require both better technology and improved understanding of the interaction between electrical stimulation and the retina. In particular, current retinal prostheses do not provide peripheral visions due to technical and surgical limitations, thus limiting the effectiveness of the treatment. This paper reviews recent results from human implant patients and presents technical approaches for peripheral vision.

  7. Probabilistic retinal vessel segmentation

    Science.gov (United States)

    Wu, Chang-Hua; Agam, Gady

    2007-03-01

    Optic fundus assessment is widely used for diagnosing vascular and non-vascular pathology. Inspection of the retinal vasculature may reveal hypertension, diabetes, arteriosclerosis, cardiovascular disease and stroke. Due to various imaging conditions retinal images may be degraded. Consequently, the enhancement of such images and vessels in them is an important task with direct clinical applications. We propose a novel technique for vessel enhancement in retinal images that is capable of enhancing vessel junctions in addition to linear vessel segments. This is an extension of vessel filters we have previously developed for vessel enhancement in thoracic CT scans. The proposed approach is based on probabilistic models which can discern vessels and junctions. Evaluation shows the proposed filter is better than several known techniques and is comparable to the state of the art when evaluated on a standard dataset. A ridge-based vessel tracking process is applied on the enhanced image to demonstrate the effectiveness of the enhancement filter.

  8. Dose response of rat retinal microvessels to proton dose schedules used clinically: a pilot study

    International Nuclear Information System (INIS)

    Archambeau, John O.; Mao, Xiao W.; McMillan, Paul J.; Gouloumet, Vanessa L.; Oeinck, Steven C.; Grove, Roger; Yonemoto, Leslie T.; Slater, Jerry D.; Slater, James M.

    2000-01-01

    Purpose: This preclinical rat pilot study quantifies retinal microvessel, endothelial, and pericyte population changes produced by proton irradiation Methods and Materials: The left eyes of rats were irradiated with single doses of 8, 14, 20, and 28 Gy protons; right eyes, with two fractions. Animals were euthanized, and eyes were removed; elastase digests were prepared, and cell populations were counted in sample fields. Results were compared with unirradiated controls. Results: Progressive time- and dose-dependent endothelial cell loss occurred following all schedules. Cell loss was significantly different from control values (p 0 phase of the mitotic cycle. 28 Gy produced photoreceptor cell loss. Conclusion: The retinal digest is an elegant bioassay to quantify the microvessel population response. Single- and split-dose schedules appear to yield similar outcomes, in terms of endothelial cell density

  9. Sector retinitis pigmentosa.

    Science.gov (United States)

    Van Woerkom, Craig; Ferrucci, Steven

    2005-05-01

    Retinitis pigmentosa (RP) is one of the most common hereditary retinal dystrophies and causes of visual impairment affecting all age groups. The reported incidence varies, but is considered to be between 1 in 3,000 to 1 in 7,000. Sector retinitis pigmentosa is an atypical form of RP that is characterized by regionalized areas of bone spicule pigmentation, usually in the inferior quadrants of the retina. A 57-year-old Hispanic man with a history of previously diagnosed retinitis pigmentosa came to the clinic with a longstanding symptom of decreased vision at night. Bone spicule pigmentation was found in the nasal and inferior quadrants in each eye. He demonstrated superior and temporal visual-field loss corresponding to the areas of the affected retina. Clinical measurements of visual-field loss, best-corrected visual acuity, and ophthalmoscopic appearance have remained stable during the five years the patient has been followed. Sector retinitis pigmentosa is an atypical form of RP that is characterized by bilateral pigmentary retinopathy, usually isolated to the inferior quadrants. The remainder of the retina appears clinically normal, although studies have found functional abnormalities in these areas as well. Sector RP is generally considered a stationary to slowly progressive disease, with subnormal electro-retinogram findings and visual-field defects corresponding to the involved retinal sectors. Management of RP is very difficult because there are no proven methods of treatment. Studies have shown 15,000 IU of vitamin A palmitate per day may slow the progression, though this result is controversial. Low vision rehabilitation, long wavelength pass filters, and pedigree counseling remain the mainstay of management.

  10. Age and diabetes related changes of the retinal capillaries: An ultrastructural and immunohistochemical study.

    Science.gov (United States)

    Bianchi, Enrica; Ripandelli, Guido; Taurone, Samanta; Feher, Janos; Plateroti, Rocco; Kovacs, Illes; Magliulo, Giuseppe; Orlando, Maria Patrizia; Micera, Alessandra; Battaglione, Ezio; Artico, Marco

    2016-03-01

    Normal human aging and diabetes are associated with a gradual decrease of cerebral flow in the brain with changes in vascular architecture. Thickening of the capillary basement membrane and microvascular fibrosis are evident in the central nervous system of elderly and diabetic patients. Current findings assign a primary role to endothelial dysfunction as a cause of basement membrane (BM) thickening, while retinal alterations are considered to be a secondary cause of either ischemia or exudation. The aim of this study was to reveal any initial retinal alterations and variations in the BM of retinal capillaries during diabetes and aging as compared to healthy controls. Moreover, we investigated the potential role of vascular endothelial growth factor (VEGF) and pro-inflammatory cytokines in diabetic retina.Transmission electron microscopy (TEM) was performed on 46 enucleated human eyes with particular attention to alterations of the retinal capillary wall and Müller glial cells. Inflammatory cytokines expression in the retina was investigated by immunohistochemistry.Our electron microscopy findings demonstrated that thickening of the BM begins primarily at the level of the glial side of the retina during aging and diabetes. The Müller cells showed numerous cytoplasmic endosomes and highly electron-dense lysosomes which surrounded the retinal capillaries. Our study is the first to present morphological evidence that Müller cells start to deposit excessive BM material in retinal capillaries during aging and diabetes. Our results confirm the induction of pro-inflammatory cytokines TNF-α and IL-1β within the retina as a result of diabetes.These observations strongly suggest that inflammatory cytokines and changes in the metabolism of Müller glial cells rather than changes in of endothelial cells may play a primary role in the alteration of retinal capillaries BM during aging and diabetes. © The Author(s) 2015.

  11. Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1

    Directory of Open Access Journals (Sweden)

    Esraa Shosha

    2018-04-01

    Full Text Available We have recently found that diabetes-induced premature senescence of retinal endothelial cells is accompanied by NOX2-NADPH oxidase-induced increases in the ureohydrolase enzyme arginase 1 (A1. Here, we used genetic strategies to determine the specific involvement of A1 in diabetes-induced endothelial cell senescence. We used A1 knockout mice and wild type mice that were rendered diabetic with streptozotocin and retinal endothelial cells (ECs exposed to high glucose or transduced with adenovirus to overexpress A1 for these experiments. ABH [2(S-Amino-6-boronohexanoic acid] was used to inhibit arginase activity. We used Western blotting, immunolabeling, quantitative PCR, and senescence associated β-galactosidase (SA β-Gal activity to evaluate senescence. Analyses of retinal tissue extracts from diabetic mice showed significant increases in mRNA expression of the senescence-related proteins p16INK4a, p21, and p53 when compared with non-diabetic mice. SA β-Gal activity and p16INK4a immunoreactivity were also increased in retinal vessels from diabetic mice. A1 gene deletion or pharmacological inhibition protected against the induction of premature senescence. A1 overexpression or high glucose treatment increased SA β-Gal activity in cultured ECs. These results demonstrate that A1 is critically involved in diabetes-induced senescence of retinal ECs. Inhibition of arginase activity may therefore be an effective therapeutic strategy to alleviate diabetic retinopathy by preventing premature senescence.

  12. Concurrent OCT imaging of stimulus evoked retinal neural activation and hemodynamic responses

    Science.gov (United States)

    Son, Taeyoon; Wang, Benquan; Lu, Yiming; Chen, Yanjun; Cao, Dingcai; Yao, Xincheng

    2017-02-01

    It is well established that major retinal diseases involve distortions of the retinal neural physiology and blood vascular structures. However, the details of distortions in retinal neurovascular coupling associated with major eye diseases are not well understood. In this study, a multi-modal optical coherence tomography (OCT) imaging system was developed to enable concurrent imaging of retinal neural activity and vascular hemodynamics. Flicker light stimulation was applied to mouse retinas to evoke retinal neural responses and hemodynamic changes. The OCT images were acquired continuously during the pre-stimulation, light-stimulation, and post-stimulation phases. Stimulus-evoked intrinsic optical signals (IOSs) and hemodynamic changes were observed over time in blood-free and blood regions, respectively. Rapid IOSs change occurred almost immediately after stimulation. Both positive and negative signals were observed in adjacent retinal areas. The hemodynamic changes showed time delays after stimulation. The signal magnitudes induced by light stimulation were observed in blood regions and did not show significant changes in blood-free regions. These differences may arise from different mechanisms in blood vessels and neural tissues in response to light stimulation. These characteristics agreed well with our previous observations in mouse retinas. Further development of the multimodal OCT may provide a new imaging method for studying how retinal structures and metabolic and neural functions are affected by age-related macular degeneration (AMD), glaucoma, diabetic retinopathy (DR), and other diseases, which promises novel noninvasive biomarkers for early disease detection and reliable treatment evaluations of eye diseases.

  13. Pharmacotherapy of retinal disease with visual cycle modulators.

    Science.gov (United States)

    Hussain, Rehan M; Gregori, Ninel Z; Ciulla, Thomas A; Lam, Byron L

    2018-04-01

    Pharmacotherapy with visual cycle modulators (VCMs) is under investigation for retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt macular dystrophy (SMD) and nonexudative age-related macular degeneration (AMD), all blinding diseases that lack effective treatment options. Areas covered: The authors review investigational VCMs, including oral retinoids, 9-cis-retinyl-acetate (zuretinol) and 9-cis-β-carotene, which restore 11-cis-retinal levels in RP and LCA caused by LRAT and RPE65 gene mutations, and may improve visual acuity and visual fields. Therapies for SMD aiming to decrease accumulation of toxic Vitamin A dimers and lipofuscin in the retina and retinal pigment epithelium (RPE) include C20-D3-vitamin A (ALK-001), isotretinoin, VM200, emixustat, and A1120. Mouse models of SMD show promising data for these treatments, though proof of efficacy in humans is currently lacking. Fenretinide and emixustat are investigational VCMs for dry AMD, though neither has been shown to reduce geographic atrophy or improve vision in human trials. A1120 prevents retinol transport into the RPE and may spare the side effects typically seen in VCMs (nyctalopia and chromatopsia) per mouse studies. Expert opinion: Oral VCMs may be feasible treatment options for degenerative retinal diseases based on pre-clinical and some early clinical studies. Further trials are warranted to assess their efficacy and safety in humans.

  14. Retinal dopamine mediates multiple dimensions of light-adapted vision.

    Science.gov (United States)

    Jackson, Chad R; Ruan, Guo-Xiang; Aseem, Fazila; Abey, Jane; Gamble, Karen; Stanwood, Greg; Palmiter, Richard D; Iuvone, P Michael; McMahon, Douglas G

    2012-07-04

    Dopamine is a key neuromodulator in the retina and brain that supports motor, cognitive, and visual function. Here, we developed a mouse model on a C57 background in which expression of the rate-limiting enzyme for dopamine synthesis, tyrosine hydroxylase, is specifically disrupted in the retina. This model enabled assessment of the overall role of retinal dopamine in vision using electrophysiological (electroretinogram), psychophysical (optokinetic tracking), and pharmacological techniques. Significant disruptions were observed in high-resolution, light-adapted vision caused by specific deficits in light responses, contrast sensitivity, acuity, and circadian rhythms in this retinal dopamine-depleted mouse model. These global effects of retinal dopamine on vision are driven by the differential actions of dopamine D1 and D4 receptors on specific retinal functions and appear to be due to the ongoing bioavailability of dopamine rather than developmental effects. Together, our data indicate that dopamine is necessary for the circadian nature of light-adapted vision as well as optimal contrast detection and acuity.

  15. Nanomaterials and Retinal Toxicity

    Science.gov (United States)

    The neuroretina should be considered as a potential site of nanomaterial toxicity. Engineered nanomaterials may reach the retina through three potential routes of exposure including; intra­ vitreal injection of therapeutics; blood-borne delivery in the retinal vasculature an...

  16. Optical modulation of transgene expression in retinal pigment epithelium

    Science.gov (United States)

    Palanker, D.; Lavinsky, D.; Chalberg, T.; Mandel, Y.; Huie, P.; Dalal, R.; Marmor, M.

    2013-03-01

    Over a million people in US alone are visually impaired due to the neovascular form of age-related macular degeneration (AMD). The current treatment is monthly intravitreal injections of a protein which inhibits Vascular Endothelial Growth Factor, thereby slowing progression of the disease. The immense financial and logistical burden of millions of intravitreal injections signifies an urgent need to develop more long-lasting and cost-effective treatments for this and other retinal diseases. Viral transfection of ocular cells allows creation of a "biofactory" that secretes therapeutic proteins. This technique has been proven successful in non-human primates, and is now being evaluated in clinical trials for wet AMD. However, there is a critical need to down-regulate gene expression in the case of total resolution of retinal condition, or if patient has adverse reaction to the trans-gene products. The site for genetic therapy of AMD and many other retinal diseases is the retinal pigment epithelium (RPE). We developed and tested in pigmented rabbits, an optical method to down-regulate transgene expression in RPE following vector delivery, without retinal damage. Microsecond exposures produced by a rapidly scanning laser vaporize melanosomes and destroy a predetermined fraction of the RPE cells selectively. RPE continuity is restored within days by migration and proliferation of adjacent RPE, but since the transgene is not integrated into the nucleus it is not replicated. Thus, the decrease in transgene expression can be precisely determined by the laser pattern density and further reduced by repeated treatment without affecting retinal structure and function.

  17. Peripapillary retinal thermal coagulation following electrical injury

    Directory of Open Access Journals (Sweden)

    Manjari Tandon

    2013-01-01

    Full Text Available In this study, we have presented the case report of a 20 year old boy who suffered an electric injury shock, following which he showed peripapillary retinal opacification and increased retinal thickening that subsequently progressed to retinal atrophy. The fluorescein angiogram revealed normal retinal circulation, thus indicating thermal damage to retina without any compromise to retinal circulation.

  18. Hmga2 regulates self-renewal of retinal progenitors.

    Science.gov (United States)

    Parameswaran, Sowmya; Xia, Xiaohuan; Hegde, Ganapati; Ahmad, Iqbal

    2014-11-01

    In vertebrate retina, histogenesis occurs over an extended period. To sustain the temporal generation of diverse cell types, retinal progenitor cells (RPCs) must self-renew. However, self-renewal and regulation of RPCs remain poorly understood. Here, we demonstrate that cell-extrinsic factors coordinate with the epigenetic regulator high-mobility group AT-hook 2 (Hmga2) to regulate self-renewal of late retinal progenitor cells (RPCs). We observed that a small subset of RPCs was capable of clonal propagation and retained multipotentiality of parents in the presence of endothelial cells (ECs), known self-renewal regulators in various stem cell niches. The self-renewing effects, also observed in vivo, involve multiple intercellular signaling pathways, engaging Hmga2. As progenitors exhaust during retinal development, expression of Hmga2 progressively decreases. Analyses of Hmga2-expression perturbation, in vitro and in vivo, revealed that Hmga2 functionally helps to mediate cell-extrinsic influences on late-retinal progenitor self-renewal. Our results provide a framework for integrating the diverse intercellular influences elicited by epigenetic regulators for self-renewal in a dynamic stem cell niche: the developing vertebrate retina. © 2014. Published by The Company of Biologists Ltd.

  19. Peripheral retinal degenerations and the risk of retinal detachment.

    Science.gov (United States)

    Lewis, Hilel

    2003-07-01

    To review the degenerative diseases of the peripheral retina in relationship with the risk to develop a rhegmatogenous retinal detachment and to present recommendations for use in eyes at increased risk of developing a retinal detachment. Focused literature review and author's clinical experience. Retinal degenerations are common lesions involving the peripheral retina, and most of them are clinically insignificant. Lattice degeneration, degenerative retinoschisis, cystic retinal tufts, and, rarely, zonular traction tufts, can result in a rhegmatogenous retinal detachment. Therefore, these lesions have been considered for prophylactic therapy; however, adequate studies have not been performed to date. Well-designed, prospective, randomized clinical studies are necessary to determine the benefit-risk ratio of prophylactic treatment. In the meantime, the evidence available suggests that most of the peripheral retinal degenerations should not be treated except in rare, high-risk situations.

  20. Msx1 is expressed in retina endothelial cells at artery branching sites

    Directory of Open Access Journals (Sweden)

    Miguel Lopes

    2012-02-01

    Msx1 and Msx2 encode homeodomain transcription factors that play a role in several embryonic developmental processes. Previously, we have shown that in the adult mouse, Msx1lacZ is expressed in vascular smooth muscle cells (VSMCs and pericytes, and that Msx2lacZ is also expressed in VSMCs as well as in a few endothelial cells (ECs. The mouse retina and choroid are two highly vascularized tissues. Vessel alterations in the retina are associated with several human diseases and the retina has been intensely used for angiogenesis studies, whereas the choroid has been much less investigated. Using the Msx1lacZ and Msx2lacZ reporter alleles, we observed that Msx2 is not expressed in the eye vascular tree in contrast to Msx1, for which we establish the spatial and temporal expression pattern in these tissues. In the retina, expression of Msx1 takes place from P3, and by P10, it becomes confined to a subpopulation of ECs at branching points of superficial arterioles. These branching sites are characterized by a subpopulation of mural cells that also show specific expression programs. Specific Msx gene inactivation in the endothelium, using Msx1 and Msx2 conditional mutant alleles together with a Tie2-Cre transgene, did not lead to conspicuous structural defects in the retinal vascular network. Expression of Msx1 at branching sites might therefore be linked to vessel physiology. The retinal blood flow is autonomously regulated and perfusion of capillaries has been proposed to depend on arteriolar precapillary structures that might be the sites for Msx1 expression. On the other hand, branching sites are subject to shear stress that might induce Msx1 expression. In the choroid vascular layer Msx1lacZ is expressed more broadly and dynamically. At birth Msx1lacZ expression takes place in the endothelium but at P21 its expression has shifted towards the mural layer. We discuss the possible functions of Msx1 in the eye vasculature.

  1. Msx1 is expressed in retina endothelial cells at artery branching sites.

    Science.gov (United States)

    Lopes, Miguel; Goupille, Olivier; Saint Cloment, Cécile; Robert, Benoît

    2012-04-15

    Msx1 and Msx2 encode homeodomain transcription factors that play a role in several embryonic developmental processes. Previously, we have shown that in the adult mouse, Msx1(lacZ) is expressed in vascular smooth muscle cells (VSMCs) and pericytes, and that Msx2(lacZ) is also expressed in VSMCs as well as in a few endothelial cells (ECs). The mouse retina and choroid are two highly vascularized tissues. Vessel alterations in the retina are associated with several human diseases and the retina has been intensely used for angiogenesis studies, whereas the choroid has been much less investigated. Using the Msx1(lacZ) and Msx2(lacZ) reporter alleles, we observed that Msx2 is not expressed in the eye vascular tree in contrast to Msx1, for which we establish the spatial and temporal expression pattern in these tissues. In the retina, expression of Msx1 takes place from P3, and by P10, it becomes confined to a subpopulation of ECs at branching points of superficial arterioles. These branching sites are characterized by a subpopulation of mural cells that also show specific expression programs. Specific Msx gene inactivation in the endothelium, using Msx1 and Msx2 conditional mutant alleles together with a Tie2-Cre transgene, did not lead to conspicuous structural defects in the retinal vascular network. Expression of Msx1 at branching sites might therefore be linked to vessel physiology. The retinal blood flow is autonomously regulated and perfusion of capillaries has been proposed to depend on arteriolar precapillary structures that might be the sites for Msx1 expression. On the other hand, branching sites are subject to shear stress that might induce Msx1 expression. In the choroid vascular layer Msx1(lacZ) is expressed more broadly and dynamically. At birth Msx1(lacZ) expression takes place in the endothelium but at P21 its expression has shifted towards the mural layer. We discuss the possible functions of Msx1 in the eye vasculature.

  2. Role of glutathione biosynthesis in endothelial dysfunction and fibrosis

    Directory of Open Access Journals (Sweden)

    Cristina Espinosa-Díez

    2018-04-01

    Full Text Available Glutathione (GSH biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL, which is composed of the catalytic (GCLc and the modulatory (GCLm subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice. In murine lung endothelial cells (MLEC derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177 and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+ male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH4. To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+ mice. We observed that obstructed kidneys from Gclc(e/+ mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Keywords: Glutamate-cysteine ligase, ROS, Glutathione, Endothelial dysfunction, Kidney Fibrosis

  3. Retinitis pigmentosa and deafness.

    OpenAIRE

    Mills, R P; Calver, D M

    1987-01-01

    Seventeen patients with retinitis pigmentosa (RP) have been investigated audiologically. Of 9 found to have a significant hearing loss, 6 were examples of Usher's syndrome; these patients had a cochlear pattern of hearing loss. The other 3 were examples of Senior's syndrome, Kearne-Sayre syndrome and Lawrence-Moon-Biedle syndrome respectively. Two of these patients had absent stapedius reflexes. It is suggested that patients with different RP-deafness syndromes may have lesions in different p...

  4. Inherited Retinal Degenerative Disease Registry

    Science.gov (United States)

    2017-09-13

    Eye Diseases Hereditary; Retinal Disease; Achromatopsia; Bardet-Biedl Syndrome; Bassen-Kornzweig Syndrome; Batten Disease; Best Disease; Choroidal Dystrophy; Choroideremia; Cone Dystrophy; Cone-Rod Dystrophy; Congenital Stationary Night Blindness; Enhanced S-Cone Syndrome; Fundus Albipunctatus; Goldmann-Favre Syndrome; Gyrate Atrophy; Juvenile Macular Degeneration; Kearns-Sayre Syndrome; Leber Congenital Amaurosis; Refsum Syndrome; Retinitis Pigmentosa; Retinitis Punctata Albescens; Retinoschisis; Rod-Cone Dystrophy; Rod Dystrophy; Rod Monochromacy; Stargardt Disease; Usher Syndrome

  5. Outcomes in bullous retinal detachment

    Directory of Open Access Journals (Sweden)

    Sarah P. Read

    2017-06-01

    Conclusions and importance: GRTs are an uncommon cause of retinal detachment. While pars plana vitrectomy with tamponade is standard in GRT management, there is variability in the use of scleral buckling and PFO in these cases. This is in contrast to retinal dialysis where scleral buckle alone can yield favorable results. Though a baseball ocular trauma is common, retinal involvement is rare compared to other sports injuries such as those occurring with tennis, soccer and golf. Sports trauma remains an important cause of retinal injury and patients should be counseled on the need for eye protection.

  6. Retinal Thickening and Photoreceptor Loss in HIV Eyes without Retinitis.

    Directory of Open Access Journals (Sweden)

    Cheryl A Arcinue

    Full Text Available To determine the presence of structural changes in HIV retinae (i.e., photoreceptor density and retinal thickness in the macula compared with age-matched HIV-negative controls.Cohort of patients with known HIV under CART (combination Antiretroviral Therapy treatment were examined with a flood-illuminated retinal AO camera to assess the cone photoreceptor mosaic and spectral-domain optical coherence tomography (SD-OCT to assess retinal layers and retinal thickness.Twenty-four eyes of 12 patients (n = 6 HIV-positive and 6 HIV-negative were imaged with the adaptive optics camera. In each of the regions of interest studied (nasal, temporal, superior, inferior, the HIV group had significantly less mean cone photoreceptor density compared with age-matched controls (difference range, 4,308-6,872 cones/mm2. A different subset of forty eyes of 20 patients (n = 10 HIV-positive and 10 HIV-negative was included in the retinal thickness measurements and retinal layer segmentation with the SD-OCT. We observed significant thickening in HIV positive eyes in the total retinal thickness at the foveal center, and in each of the three horizontal B-scans (through the macular center, superior, and inferior to the fovea. We also noted that the inner retina (combined thickness from ILM through RNFL to GCL layer was also significantly thickened in all the different locations scanned compared with HIV-negative controls.Our present study shows that the cone photoreceptor density is significantly reduced in HIV retinae compared with age-matched controls. HIV retinae also have increased macular retinal thickness that may be caused by inner retinal edema secondary to retinovascular disease in HIV. The interaction of photoreceptors with the aging RPE, as well as possible low-grade ocular inflammation causing diffuse inner retinal edema, may be the key to the progressive vision changes in HIV-positive patients without overt retinitis.

  7. Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

    Science.gov (United States)

    Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

    2018-04-17

    Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

  8. Role of calcium-activated potassium channels with small conductance in bradykinin-induced vasodilation of porcine retinal arterioles

    DEFF Research Database (Denmark)

    Dalsgaard, Thomas; Kroigaard, Christel; Bek, Toke

    2009-01-01

    PURPOSE: Endothelial dysfunction and impaired vasodilation may be involved in the pathogenesis of retinal vascular diseases. In the present study, the mechanisms underlying bradykinin vasodilation were examined and whether calcium-activated potassium channels of small (SK(Ca)) and intermediate (IK...

  9. Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis

    OpenAIRE

    Sun, Dawei; Nakao, Shintaro; Xie, Fang; Zandi, Souska; Bagheri, Abouzar; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Soheili, Zahra-Soheila; Frimmel, Sonja; Zhang, Zhongyu; Ablonczy, Zsolt; Ahmadieh, Hamid; Hafezi-Moghadam, Ali

    2014-01-01

    Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of vision loss. Biomarkers and methods for early diagnosis of DR are urgently needed. Using a new molecular imaging approach, we show up to 94% higher accumulation of custom designed imaging probes against vascular endothelial growth factor receptor 2 (VEGFR-2) in retinal and choroidal vessels of diabetic animals (P

  10. The use of bevacizumab in a multilevel retinal hemorrhage secondary to retinal macroaneurysm: a 39-month follow-up case report

    Directory of Open Access Journals (Sweden)

    Tsakpinis D

    2011-10-01

    Full Text Available Dimitrios Tsakpinis1, Mayssa B Nasr1,2, Paris Tranos3, Nikos Krassas1, Theodoros Giannopoulos2, Chrysanthos Symeonidis1, Stavros A Dimitrakos1, Anastasios GP Konstas212nd University Department of Ophthalmology, Papageorgiou Hospital; 2Glaucoma Unit, 1st University, Department of Ophthalmology, AHEPA Hospital, Thessaloniki, Greece; 3Retina Eye Center, Thessaloniki, GreecePurpose: The evaluation of long-term visual outcome after the use of bevacizumab for the management of multilevel hemorrhage due to retinal arterial macroaneurysm (MA.Case report: A 71-year-old hypertensive female presented with sudden reduction of visual acuity in her left eye (OS. Fundoscopy revealed an arterial macroaneurysm with preretinal and subretinal hemorrhage in the eye. Due to significant macular involvement, the patient received two intravitreal injections of bevacizumab within 2 months.Results: Significant visual and anatomical recovery was observed 2 months later, which was confirmed by fluorescein angiography. At the end of a follow-up period (39 months visual acuity and visual field were at normal levels.Conclusion: Retinal MA is a relatively rare condition. Anti-vascular endothelial growth factor therapy appears a safe and effective treatment option for selected symptomatic individuals that may offer faster visual rehabilitation. Herein we report, for the first time, a 39-month follow-up of a retinal MA treated with anti-vascular endothelial growth factor therapy.Keywords: arterial retinal macroaneurysm, anti-VEGF, bevacizumab, multilevel hemorrhage

  11. Edaravone Protect against Retinal Damage in Streptozotocin-Induced Diabetic Mice

    Science.gov (United States)

    Liu, Xiaoyi; Chen, Xi; Xie, Ping; Yuan, Songtao; Zhang, Weiwei; Lin, Xiaojun; Liu, Qinghuai

    2014-01-01

    Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a free radical scavenger, is used for the clinical treatment of retinal injury. In this study, we investigated the protective effects of edaravone against diabetic retinal damage in the mouse. Diabetic retinopathy in the mouse was induced by injection of streptozotocin. Edaravone was given once-daily and was intraperitoneally (i.p.) treated at a dose of 3 mg/kg from streptozotocin injection to 4 weeks after onset of diabetes. Retinal ganglion cells (RGCs) damage was evaluated by recording the pattern electroretinogram (ERG). RGCs damage was also detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and the levels of reactive oxygen species (ROS) were determined fluorometrically. The expressions of phosporylated-ERK1/2, BDNF, and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expression of BDNF was significantly decreased in the retinas of diabetic mice, compared to nondiabetic mice. Administration of edaravone significantly attenuated diabetes induced RGCs death, upregulation of ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF. These findings suggest that oxidative stress plays a pivotal role in diabetic retinal damage and that systemic administration of edaravone may slow the progression of retinal neuropathy induced by diabetes. PMID:24897298

  12. Edaravone protect against retinal damage in streptozotocin-induced diabetic mice.

    Directory of Open Access Journals (Sweden)

    Dongqing Yuan

    Full Text Available Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one, a free radical scavenger, is used for the clinical treatment of retinal injury. In this study, we investigated the protective effects of edaravone against diabetic retinal damage in the mouse. Diabetic retinopathy in the mouse was induced by injection of streptozotocin. Edaravone was given once-daily and was intraperitoneally (i.p. treated at a dose of 3 mg/kg from streptozotocin injection to 4 weeks after onset of diabetes. Retinal ganglion cells (RGCs damage was evaluated by recording the pattern electroretinogram (ERG. RGCs damage was also detected by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL staining, and the levels of reactive oxygen species (ROS were determined fluorometrically. The expressions of phosporylated-ERK1/2, BDNF, and caspase-3 were determined by Western blot analysis. Retinal levels of ROS, phosphorylated ERK1/2, and cleaved caspase-3 were significantly increased, whereas the expression of BDNF was significantly decreased in the retinas of diabetic mice, compared to nondiabetic mice. Administration of edaravone significantly attenuated diabetes induced RGCs death, upregulation of ROS, ERK1/2 phosphorylation, and cleaved caspase-3 and downregulation of BDNF. These findings suggest that oxidative stress plays a pivotal role in diabetic retinal damage and that systemic administration of edaravone may slow the progression of retinal neuropathy induced by diabetes.

  13. Gliovascular and cytokine interactions modulate brain endothelial barrier in vitro.

    Science.gov (United States)

    Chaitanya, Ganta V; Cromer, Walter E; Wells, Shannon R; Jennings, Merilyn H; Couraud, P Olivier; Romero, Ignacio A; Weksler, Babette; Erdreich-Epstein, Anat; Mathis, J Michael; Minagar, Alireza; Alexander, J Steven

    2011-11-23

    The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs). © 2011 Chaitanya et al; licensee BioMed Central Ltd.

  14. A Review: Proteomics in Retinal Artery Occlusion, Retinal Vein Occlusion, Diabetic Retinopathy and Acquired Macular Disorders.

    Science.gov (United States)

    Cehofski, Lasse Jørgensen; Honoré, Bent; Vorum, Henrik

    2017-04-28

    Retinal artery occlusion (RAO), retinal vein occlusion (RVO), diabetic retinopathy (DR) and age-related macular degeneration (AMD) are frequent ocular diseases with potentially sight-threatening outcomes. In the present review we discuss major findings of proteomic studies of RAO, RVO, DR and AMD, including an overview of ocular proteome changes associated with anti-vascular endothelial growth factor (VEGF) treatments. Despite the severe outcomes of RAO, the proteome of the disease remains largely unstudied. There is also limited knowledge about the proteome of RVO, but proteomic studies suggest that RVO is associated with remodeling of the extracellular matrix and adhesion processes. Proteomic studies of DR have resulted in the identification of potential therapeutic targets such as carbonic anhydrase-I. Proliferative diabetic retinopathy is the most intensively studied stage of DR. Proteomic studies have established VEGF, pigment epithelium-derived factor (PEDF) and complement components as key factors associated with AMD. The aim of this review is to highlight the major milestones in proteomics in RAO, RVO, DR and AMD. Through large-scale protein analyses, proteomics is bringing new important insights into these complex pathological conditions.

  15. Retinal astrocytoma in a dog.

    Science.gov (United States)

    Kuroki, Keiichi; Kice, Nathan; Ota-Kuroki, Juri

    2017-09-01

    A miniature schnauzer dog presenting with hyphema and glaucoma of the right eye had a retinal neoplasm. Neoplastic cells stained positively for glial fibrillary acidic protein, vimentin, and S-100 and largely negatively for oligodendrocyte transcription factor 2 by immunohistochemistry. The clinical and histopathological features of canine retinal astrocytomas are discussed.

  16. Non-syndromic retinitis pigmentosa

    NARCIS (Netherlands)

    Verbakel, S.K. (Sanne K.); R.A.C. van Huet (Ramon A. C.); C.J.F. Boon (Camiel); A.I. Hollander (Anneke); R.W.J. Collin (Rob); C.C.W. Klaver (Caroline); C. Hoyng (Carel); R. Roepman (Ronald); B.J. Klevering (Jeroen)

    2018-01-01

    textabstractRetinitis pigmentosa (RP) encompasses a group of inherited retinal dystrophies characterized by the primary degeneration of rod and cone photoreceptors. RP is a leading cause of visual disability, with a worldwide prevalence of 1:4000. Although the majority of RP cases are non-syndromic,

  17. Retinal Imaging and Image Analysis

    Science.gov (United States)

    Abràmoff, Michael D.; Garvin, Mona K.; Sonka, Milan

    2011-01-01

    Many important eye diseases as well as systemic diseases manifest themselves in the retina. While a number of other anatomical structures contribute to the process of vision, this review focuses on retinal imaging and image analysis. Following a brief overview of the most prevalent causes of blindness in the industrialized world that includes age-related macular degeneration, diabetic retinopathy, and glaucoma, the review is devoted to retinal imaging and image analysis methods and their clinical implications. Methods for 2-D fundus imaging and techniques for 3-D optical coherence tomography (OCT) imaging are reviewed. Special attention is given to quantitative techniques for analysis of fundus photographs with a focus on clinically relevant assessment of retinal vasculature, identification of retinal lesions, assessment of optic nerve head (ONH) shape, building retinal atlases, and to automated methods for population screening for retinal diseases. A separate section is devoted to 3-D analysis of OCT images, describing methods for segmentation and analysis of retinal layers, retinal vasculature, and 2-D/3-D detection of symptomatic exudate-associated derangements, as well as to OCT-based analysis of ONH morphology and shape. Throughout the paper, aspects of image acquisition, image analysis, and clinical relevance are treated together considering their mutually interlinked relationships. PMID:22275207

  18. Activin/Nodal Signaling Supports Retinal Progenitor Specification in a Narrow Time Window during Pluripotent Stem Cell Neuralization

    Directory of Open Access Journals (Sweden)

    Michele Bertacchi

    2015-10-01

    Full Text Available Retinal progenitors are initially found in the anterior neural plate region known as the eye field, whereas neighboring areas undertake telencephalic or hypothalamic development. Eye field cells become specified by switching on a network of eye field transcription factors, but the extracellular cues activating this network remain unclear. In this study, we used chemically defined media to induce in vitro differentiation of mouse embryonic stem cells (ESCs toward eye field fates. Inhibition of Wnt/β-catenin signaling was sufficient to drive ESCs to telencephalic, but not retinal, fates. Instead, retinal progenitors could be generated from competent differentiating mouse ESCs by activation of Activin/Nodal signaling within a narrow temporal window corresponding to the emergence of primitive anterior neural progenitors. Activin also promoted eye field gene expression in differentiating human ESCs. Our results reveal insights into the mechanisms of eye field specification and open new avenues toward the generation of retinal progenitors for translational medicine.

  19. Role of the Norrie disease pseudoglioma gene in sprouting angiogenesis during development of the retinal vasculature.

    Science.gov (United States)

    Luhmann, Ulrich F O; Lin, Jihong; Acar, Niyazi; Lammel, Stefanie; Feil, Silke; Grimm, Christian; Seeliger, Mathias W; Hammes, Hans-Peter; Berger, Wolfgang

    2005-09-01

    To characterize developmental defects and the time course of Norrie disease in retinal and hyaloid vasculature during retinal development and to identify underlying molecular angiogenic pathways that may be affected in Norrie disease, exudative vitreoretinopathy, retinopathy of prematurity, and Coats' disease. Norrie disease pseudoglioma homologue (Ndph)-knockout mice were studied during retinal development at early postnatal (p) stages (p5, p10, p15, and p21). Histologic techniques, quantitative RT-PCR, ELISA, and Western blot analyses provided molecular data, and scanning laser ophthalmoscopy (SLO) angiography and electroretinography (ERG) were used to obtain in vivo data. The data showed that regression of the hyaloid vasculature of Ndph-knockout mice occurred but was drastically delayed. The development of the superficial retinal vasculature was strongly delayed, whereas the deep retinal vasculature did not form because of the blockage of vessel outgrowth into the deep retinal layers. Subsequently, microaneurysm-like lesions formed. Several angiogenic factors were differentially transcribed during retinal development. Increased levels of hypoxia inducible factor-1alpha (HIF1alpha) and VEGFA, as well as a characteristic ERG pattern, confirmed hypoxic conditions in the inner retina of the Ndph-knockout mouse. These data provide evidence for a crucial role of Norrin in hyaloid vessel regression and in sprouting angiogenesis during retinal vascular development, especially in the development of the deep retinal capillary networks. They also suggest an early and a late phase of Norrie disease and may provide an explanation for similar phenotypic features of allelic retinal diseases in mice and patients as secondary consequences of pathologic hypoxia.

  20. Technical brief: a comparison of two methods of euthanasia on retinal dopamine levels.

    Science.gov (United States)

    Hwang, Christopher K; Iuvone, P Michael

    2013-01-01

    Mice are commonly used in biomedical research, and euthanasia is an important part of mouse husbandry. Approved, humane methods of euthanasia are designed to minimize the potential for pain or discomfort, but may also influence the measurement of experimental variables. We compared the effects of two approved methods of mouse euthanasia on the levels of retinal dopamine. We examined the level of retinal dopamine, a commonly studied neuromodulator, following euthanasia by carbon dioxide (CO₂)-induced asphyxiation or by cervical dislocation. We found that the level of retinal dopamine in mice euthanized through CO₂ overdose substantially differed from that in mice euthanized through cervical dislocation. The use of CO₂ as a method of euthanasia could result in an experimental artifact that could compromise results when studying labile biologic processes.

  1. Hypoxia-induced mitogenic factor enhances angiogenesis by promoting proliferation and migration of endothelial cells

    International Nuclear Information System (INIS)

    Tong Qiangsong; Zheng Liduan; Li Bo; Wang Danming; Huang Chuanshu; Matuschak, George M.; Li Dechun

    2006-01-01

    Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4-10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4-10 cells. Treatment of mouse aortic rings and SVEC 4-10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Δp85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways

  2. Spectrophotometric retinal oximetry in pigs

    DEFF Research Database (Denmark)

    Traustason, Sindri; Kiilgaard, Jens Folke; Karlsson, Robert

    2013-01-01

    PURPOSE: To assess the validity of spectrophotometric retinal oximetry, by comparison to blood gas analysis and intra-vitreal measurements of partial pressure of oxygen (pO2). METHODS: Female domestic pigs were used for all experiments (n=8). Oxygen fraction in inspired air was changed using...... a mixture of room air, pure oxygen and pure nitrogen, ranging from 5% to 100% oxygen. Femoral arterial blood gas analysis and retinal oximetry was performed at each level of inspiratory oxygen fraction. Retinal oximetry was performed using a commercial instrument, the Oxymap Retinal Oximeter T1 (Oxymap ehf...... arterial oxygen saturation and the optical density ratio over retinal arteries revealed an approximately linear relationship (R(2) = 0.74, p = 3.4 x 10(-9)). In order to test the validity of applying the arterial calibration to veins, we compared non-invasive oximetry measurements to invasive pO2...

  3. Oral contraceptive pills: A risk factor for retinal vascular occlusion in in-vitro fertilization patients

    Directory of Open Access Journals (Sweden)

    Rohina S Aggarwal

    2013-01-01

    Full Text Available Retinal vascular occlusion is the most common cause of retinopathy leading to severe visual loss in all age groups. Central retinal vein occlusion (CRVO is usually seen in older age group and is often associated with systemic vascular diseases. Although the exact cause and effect relationship has not been proven, central retinal vein occlusion has been associated with various systemic pathological conditions, hence a direct review of systems toward the various systemic and local factors predisposing the central retinal vein occlusion is advocated. We describe the development of central retinal venous occlusion with associated cystoid macular edema (CME in two healthy infertile women who were recruited for in vitro fertilization cycle for infertility. Predisposing risk factors associated with central retinal vein occlusion are obesity, sedentary life style, smoking, and some systemic diseases such as hyperlipidemia, hypertension, associated autoimmune disorders e.g., antiphospholipid antibody syndrome, lupus, diabetes mellitus, cardiovascular disorders, bleeding or clotting disorders, vasculitis, closed-head trauma, alcohol consumption, primary open-angle glaucoma or angle-closure glaucoma.In our patients, they were ruled out afterdoing allpertaining investigations. The cases were managed with further avoidance of oral contraceptives and intra-vitreal injections of Bevacizumab (Avastin, an anti-vascular endothelial growth factor (anti-VEGF drug and Triamcinolone acetonide (a long acting synthetic steroid. Hence, even if no systemic diseases are detected. Physical examinations are recommended periodically for young women on oral contraceptive pills.

  4. Hypothalamic neurosecretory and circadian vasopressinergic neuronal systems in the blind cone-rod homeobox knock out mouse (Crx(-/-) ) and the 129sv wild type mouse

    DEFF Research Database (Denmark)

    Rovsing, Louise; Rath, Martin Fredensborg; Møller, Morten

    2013-01-01

    circadian AVP-rhythm. We have in this study of the brown 129sv mouse and the visual blind cone-rod homeobox gene knock out mouse (Crx(-/-) ) with degeneration of the retinal rods and cones, but a preserved non-image forming optic system, studied the temporal Avp-expression in both the neurosecretory...

  5. Evolution of endothelial keratoplasty.

    Science.gov (United States)

    Price, Francis W; Price, Marianne O

    2013-11-01

    Endothelial keratoplasty has evolved into a popular alternative to penetrating keratoplasty (PK) for the treatment of endothelial dysfunction. Although the earliest iterations were challenging and were not widely adopted, the iteration known as Descemet stripping endothelial keratoplasty (DSEK) has gained widespread acceptance. DSEK combines a simplified technique for stripping dysfunctional endothelium from the host cornea and microkeratome dissection of the donor tissue, a step now commonly completed in advance by eye bank technicians. Studies show that a newer endothelial keratoplasty iteration, known as Descemet membrane endothelial keratoplasty (DMEK), provides an even faster and better visual recovery than DSEK does. In addition, DMEK significantly reduces the risk of immunologic graft rejection episodes compared with that in DSEK or in PK. Although the DMEK donor tissue, consisting of the bare endothelium and Descemet membrane without any stroma, is more challenging to prepare and position in the recipient eye, recent improvements in instrumentation and surgical techniques are increasing the ease and the reliability of the procedure. DSEK successfully mitigates 2 of the main liabilities of PK: ocular surface complications and structural problems (including induced astigmatism and perpetually weak wounds), whereas DMEK further mitigates the 2 principal remaining liabilities of PK: immunologic graft reactions and secondary glaucoma from prolonged topical corticosteroid use.

  6. Trifluoperazine: corneal endothelial phototoxicity

    International Nuclear Information System (INIS)

    Hull, D.S.; Csukas, S.; Green, K.

    1983-01-01

    Trifluoperazine is used for the treatment of psychiatric disorders. Perfusion of corneal endothelial cells with trifluoperazine-HC1 concurrent with exposure to long wavelength ultraviolet light resulted in a corneal swelling rate greater than that found in perfused corneas not exposed to ultraviolet light. Exposure of endothelial cells to 25 W incandescent light during perfusion with trifluoperazine-HC1 did not result in a higher corneal swelling rate compared to those perfused in the dark. The increased corneal swelling rate could be produced by pre-exposure of the trifluoperazine-HC1 perfusing solution to ultraviolet light suggesting the production of toxic photoproducts during exposure of trifluoperazine-HC1 to ultraviolet light. Perfusion of corneal endothelial cells with non-ultraviolet illuminated trifluoperazine-HC1 had no effect on endothelial cell membranes or ultrastructure. This is in contrast to cells perfused with trifluoperazine-HC1 that had been exposed to ultraviolet light in which there was an alteration of mitochondria and a loss of cytoplasmic homogeneity. The data imply that the trifluoperazine-HC1 photoproduct had an adverse effect on cellular transport mechanisms. The study also further demonstrates the value of the corneal endothelial cell model for identifying the physiological and anatomical changes occuring in photo-induced toxic reactions. (author)

  7. Mitochondria and Endothelial Function

    Science.gov (United States)

    Kluge, Matthew A.; Fetterman, Jessica L.; Vita, Joseph A.

    2013-01-01

    In contrast to their role in other cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, ROS production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that mechanism to endothelial control of vasomotor tone, angiogenesis, and inflammatory activation. We particularly highlight the small number of clinical and translational studies that have investigated each mechanism in human subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention and treatment of cardiovascular disease. PMID:23580773

  8. Versatile functional roles of horizontal cells in the retinal circuit.

    Science.gov (United States)

    Chaya, Taro; Matsumoto, Akihiro; Sugita, Yuko; Watanabe, Satoshi; Kuwahara, Ryusuke; Tachibana, Masao; Furukawa, Takahisa

    2017-07-17

    In the retinal circuit, environmental light signals are converted into electrical signals that can be decoded properly by the brain. At the first synapse of the visual system, information flow from photoreceptors to bipolar cells is modulated by horizontal cells (HCs), however, their functional contribution to retinal output and individual visual function is not fully understood. In the current study, we investigated functional roles for HCs in retinal ganglion cell (RGC) response properties and optokinetic responses by establishing a HC-depleted mouse line. We observed that HC depletion impairs the antagonistic center-surround receptive field formation of RGCs, supporting a previously reported HC function revealed by pharmacological approaches. In addition, we found that HC loss reduces both the ON and OFF response diversities of RGCs, impairs adjustment of the sensitivity to ambient light at the retinal output level, and alters spatial frequency tuning at an individual level. Taken together, our current study suggests multiple functional aspects of HCs crucial for visual processing.

  9. Intraretinal proliferation induced by retinal detachment

    International Nuclear Information System (INIS)

    Fisher, S.K.; Erickson, P.A.; Lewis, G.P.; Anderson, D.H.

    1991-01-01

    Cellular proliferation after retinal detachment was studied by 3 H-thymidine light microscopic autoradiography in cats that had experimental detachments of 0.5-180 days duration. The animals underwent labeling 2 hr before death with an intraocular injection of 200 microCi of 3 H-thymidine. The number of labeled nuclei were counted in 1-micron thick tissue sections in regions of detachment, in regions of the experimental eyes that remained attached, and in control eyes that had no detachments. In the normal eye, in one that had only the lens and vitreous removed, and in the eyes with 0.5- and 1-day detachments, the number of labeled nuclei ranged from 0/mm (0.5-day detachment) to 0.38/mm (lens and vitreous removed only). By 2 days postdetachment, the number of labeled nuclei increased to 2.09/mm. The highest levels of labeling occurred in two animals with detachments of 3 (7.86/mm) and 4 (7.09/mm) days. Thereafter, the numbers declined steadily until near-baseline counts were obtained at 14 days. The number of labeled nuclei was slightly elevated in the attached regions of two animals with 3-day detachments. Labeled cell types included: Mueller cells, astrocytes, pericytes, and endothelial cells of the retinal vasculature, and both resident (microglial cells) and invading macrophages. In an earlier study RPE cells were also shown to proliferate in response to detachment. Thus, these data show that proliferation is a rapid response to detachment, reaching a maximum within 4 days, and that virtually every nonneuronal cell type in the retina can participate in this response. The data suggest that events leading to such clinical manifestations as proliferative vitreoretinopathy and subretinal fibrosis may have their beginnings in this very early proliferative response

  10. Role of glutathione biosynthesis in endothelial dysfunction and fibrosis.

    Science.gov (United States)

    Espinosa-Díez, Cristina; Miguel, Verónica; Vallejo, Susana; Sánchez, Francisco J; Sandoval, Elena; Blanco, Eva; Cannata, Pablo; Peiró, Concepción; Sánchez-Ferrer, Carlos F; Lamas, Santiago

    2018-04-01

    Glutathione (GSH) biosynthesis is essential for cellular redox homeostasis and antioxidant defense. The rate-limiting step requires glutamate-cysteine ligase (GCL), which is composed of the catalytic (GCLc) and the modulatory (GCLm) subunits. To evaluate the contribution of GCLc to endothelial function we generated an endothelial-specific Gclc haplo-insufficient mouse model (Gclc e/+ mice). In murine lung endothelial cells (MLEC) derived from these mice we observed a 50% reduction in GCLc levels compared to lung fibroblasts from the same mice. MLEC obtained from haplo-insufficient mice showed significant reduction in GSH levels as well as increased basal and stimulated ROS levels, reduced phosphorylation of eNOS (Ser 1177) and increased eNOS S-glutathionylation, compared to MLEC from wild type (WT) mice. Studies in mesenteric arteries demonstrated impaired endothelium-dependent vasodilation in Gclc(e/+) male mice, which was corrected by pre-incubation with GSH-ethyl-ester and BH 4 . To study the contribution of endothelial GSH synthesis to renal fibrosis we employed the unilateral ureteral obstruction model in WT and Gclc(e/+) mice. We observed that obstructed kidneys from Gclc(e/+) mice exhibited increased deposition of fibrotic markers and reduced Nrf2 levels. We conclude that the preservation of endothelial GSH biosynthesis is not only critical for endothelial function but also in anti-fibrotic responses. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

    Science.gov (United States)

    Monaghan, Kevin; McNaughten, Jennifer; McGahon, Mary K.; Kelly, Catriona; Kyle, Daniel; Yong, Phaik Har

    2015-01-01

    Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the

  12. Retinal photography: A window into the cardiovascular-brain link in adolescent bipolar disorder.

    Science.gov (United States)

    Naiberg, Melanie R; Hatch, Jessica K; Selkirk, Beth; Fiksenbaum, Lisa; Yang, Victor; Black, Sandra; Kertes, Peter J; Goldstein, Benjamin I

    2017-08-15

    The burden of cardiovascular disease in bipolar disorder (BD) exceeds what can be explained by traditional cardiovascular risk factors (CVRFs), lifestyle, and/or medications. Moreover, neurocognitive deficits are a core feature of BD, and are also related to CVRFs. We examined retinal vascular photography, a proxy for cerebral microvasculature, in relation to CVRFs, peripheral microvascular function, and neurocognition among BD adolescents. Subjects were 30 adolescents with BD and 32 healthy controls (HC). Retinal photography was conducted using a Topcon TRC 50 DX, Type IA camera, following pupil dilation. Retinal arteriolar and venular caliber was measured, from which the arterio-venular ratio (AVR) was computed. All measures were conducted masked to participant diagnosis. Peripheral arterial tonometry measured endothelial function. Neurocognition was assessed using the Cambridge Neuropsychological Tests Automated Battery. AVR was not significantly different between groups (Cohen's d=0.18, p=0.103). Higher diastolic blood pressure (BP) was associated with lower (worse) AVR in BD (r=-0.441, p=0.015) but not HC (r=-0.192, p=0.293). Similarly, in the BD group only, higher (better) endothelial function was associated with higher AVR (r=0.375, p=0.041). Hierarchical regression models confirmed that, independent of covariates, retinal vascular caliber was significantly associated with diastolic BP and endothelial function in BD. Within the BD group, mood scores were significantly negatively correlated with AVR (β=-0.451, p=0.044). This study's limitations include a small sample size, a cross-sectional study design, and a heterogeneous sample. Retinal photography may offer unique insights regarding the cardiovascular and neurocognitive burden of BD. Larger longitudinal studies are warranted. Copyright © 2017. Published by Elsevier B.V.

  13. Overexpression of Pax6 results in microphthalmia, retinal dysplasia and defective retinal ganglion cell axon guidance

    Directory of Open Access Journals (Sweden)

    Jeffery Glen

    2008-05-01

    Full Text Available Abstract Background The transcription factor Pax6 is expressed by many cell types in the developing eye. Eyes do not form in homozygous loss-of-function mouse mutants (Pax6Sey/Sey and are abnormally small in Pax6Sey/+ mutants. Eyes are also abnormally small in PAX77 mice expressing multiple copies of human PAX6 in addition to endogenous Pax6; protein sequences are identical in the two species. The developmental events that lead to microphthalmia in PAX77 mice are not well-characterised, so it is not clear whether over- and under-expression of Pax6/PAX6 cause microphthalmia through similar mechanisms. Here, we examined the consequences of over-expression for the eye and its axonal connections. Results Eyes form in PAX77+/+ embryos but subsequently degenerate. At E12.5, we found no abnormalities in ocular morphology, retinal cell cycle parameters and the incidence of retinal cell death. From E14.5 on, we observed malformations of the optic disc. From E16.5 into postnatal life there is progressively more severe retinal dysplasia and microphthalmia. Analyses of patterns of gene expression indicated that PAX77+/+ retinae produce a normal range of cell types, including retinal ganglion cells (RGCs. At E14.5 and E16.5, quantitative RT-PCR with probes for a range of molecules associated with retinal development showed only one significant change: a slight reduction in levels of mRNA encoding the secreted morphogen Shh at E16.5. At E16.5, tract-tracing with carbocyanine dyes in PAX77+/+ embryos revealed errors in intraretinal navigation by RGC axons, a decrease in the number of RGC axons reaching the thalamus and an increase in the proportion of ipsilateral projections among those RGC axons that do reach the thalamus. A survey of embryos with different Pax6/PAX6 gene dosage (Pax6Sey/+, Pax6+/+, PAX77+ and PAX77+/+ showed that (1 the total number of RGC axons projected by the retina and (2 the proportions that are sorted into the ipsilateral and

  14. White centered retinal hemorrhages in vitamin b(12) deficiency anemia.

    Science.gov (United States)

    Zehetner, Claus; Bechrakis, Nikolaos E

    2011-05-01

    To report a case of severe vitamin B(12) deficiency anemia presenting with white centered retinal hemorrhages. Interventional case report. A 40-year-old man, general practitioner himself, presented with a 1-day history of diminished left visual acuity and a drop-shaped central scotoma. The corrected visual acuities were 20/20, OD and 20/100, OS. Ophthalmic examination revealed bilaterally pale tarsal conjunctiva, discretely icteric bulbar conjunctiva and disseminated white centered intraretinal hemorrhages with foveal involvement. OCT imaging through these lesions revealed a retinal thickening caused by a sub-ILM accumulation of hyperreflective and inhomogeneous deposits within the nerve fiber layer. Immediate laboratory work-up showed severe megaloblastic anemia caused by vitamin B(12) deficiency requiring erythrocyte transfusions. Most reports of white centered retinal hemorrhages have been described in patients with leukemic retinopathy and bacterial endocarditis. It is interesting that this case of vitamin B(12) deficiency anemia retinopathy has a clinically indistinguishable fundus appearance. This is probably due to the common pathology of capillary disruption and subsequent hemostatic fibrin plug formation. In megaloblastic anemia, direct anoxia results in endothelial dysfunction. The loss of impermeability allows extrusion of whole blood and subsequent diffusion from the disrupted site throughout and above the nerve fiber layer. Therefore the biomicroscopic pattern of white centered hemorrhages observed in anemic retinopathy is most likely due to the clot formation as the reparative sequence after capillary rupture.

  15. White Centered Retinal Hemorrhages in Vitamin B12 Deficiency Anemia

    Directory of Open Access Journals (Sweden)

    Claus Zehetner

    2011-05-01

    Full Text Available Background: To report a case of severe vitamin B12 deficiency anemia presenting with white centered retinal hemorrhages. Methods: Interventional case report. Results: A 40-year-old man, general practitioner himself, presented with a 1-day history of diminished left visual acuity and a drop-shaped central scotoma. The corrected visual acuities were 20/20, OD and 20/100, OS. Ophthalmic examination revealed bilaterally pale tarsal conjunctiva, discretely icteric bulbar conjunctiva and disseminated white centered intraretinal hemorrhages with foveal involvement. OCT imaging through these lesions revealed a retinal thickening caused by a sub-ILM accumulation of hyperreflective and inhomogeneous deposits within the nerve fiber layer. Immediate laboratory work-up showed severe megaloblastic anemia caused by vitamin B12 deficiency requiring erythrocyte transfusions. Discussion: Most reports of white centered retinal hemorrhages have been described in patients with leukemic retinopathy and bacterial endocarditis. It is interesting that this case of vitamin B12 deficiency anemia retinopathy has a clinically indistinguishable fundus appearance. This is probably due to the common pathology of capillary disruption and subsequent hemostatic fibrin plug formation. In megaloblastic anemia, direct anoxia results in endothelial dysfunction. The loss of impermeability allows extrusion of whole blood and subsequent diffusion from the disrupted site throughout and above the nerve fiber layer. Therefore the biomicroscopic pattern of white centered hemorrhages observed in anemic retinopathy is most likely due to the clot formation as the reparative sequence after capillary rupture.

  16. Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study

    Directory of Open Access Journals (Sweden)

    Liam M. Ashander

    2016-01-01

    Full Text Available Targeting the endothelial adhesion molecules that control leukocyte trafficking into a tissue has been explored as a biological therapy for inflammatory diseases. However, these molecules also participate in leukocyte migration for immune surveillance, and inhibiting the physiological level of an adhesion molecule might promote infection or malignancy. We explored the concept of targeting endothelial adhesion molecule transcription during inflammation in a human system. Intercellular adhesion molecule 1 (ICAM-1 mediates leukocyte migration across the retinal endothelium in noninfectious posterior uveitis. We observed an increase in the transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NF-κB1, in parallel with ICAM-1, in human retinal endothelial cells treated with tumor necrosis factor-alpha (TNF-α, and identified putative binding sites for NF-κB1 within the ICAM-1 regulatory region. We targeted induced NF-κB1 expression in endothelial cells with small interfering (siRNA. Knockdown of NF-κB1 significantly decreased cell surface expression of ICAM-1 protein induced by TNF-α but did not reduce constitutive ICAM-1 expression. Consistently, NF-κB1 knockdown significantly reduced leukocyte binding to cell monolayers in the presence of TNF-α but did not impact baseline binding. Findings of this proof-of-concept study indicate that induced transcription of endothelial adhesion molecules might be targeted therapeutically for inflammatory disease in humans.

  17. Nanosecond laser therapy reverses pathologic and molecular changes in age-related macular degeneration without retinal damage.

    Science.gov (United States)

    Jobling, A I; Guymer, R H; Vessey, K A; Greferath, U; Mills, S A; Brassington, K H; Luu, C D; Aung, K Z; Trogrlic, L; Plunkett, M; Fletcher, E L

    2015-02-01

    Age-related macular degeneration (AMD) is a leading cause of vision loss, characterized by drusen deposits and thickened Bruch's membrane (BM). This study details the capacity of nanosecond laser treatment to reduce drusen and thin BM while maintaining retinal structure. Fifty patients with AMD had a single nanosecond laser treatment session and after 2 yr, change in drusen area was compared with an untreated cohort of patients. The retinal effect of the laser was determined in human and mouse eyes using immunohistochemistry and compared with untreated eyes. In a mouse with thickened BM (ApoEnull), the effect of laser treatment was quantified using electron microscopy and quantitative PCR. In patients with AMD, nanosecond laser treatment reduced drusen load at 2 yr. Retinal structure was not compromised in human and mouse retina after laser treatment, with only a discrete retinal pigment epithelium (RPE) injury, and limited mononuclear cell response observed. BM was thinned in the ApoEnull mouse 3 mo after treatment (ApoEnull treated 683 ± 38 nm, ApoEnull untreated 890 ± 60 nm, C57Bl6J 606 ± 43 nm), with the expression of matrix metalloproteinase-2 and -3 increased (>260%). Nanosecond laser resolved drusen independent of retinal damage and improved BM structure, suggesting this treatment has the potential to reduce AMD progression. © FASEB.

  18. NADPH Oxidase-Mediated ROS Production Determines Insulin's Action on the Retinal Microvasculature.

    Science.gov (United States)

    Kida, Teruyo; Oku, Hidehiro; Horie, Taeko; Matsuo, Junko; Kobayashi, Takatoshi; Fukumoto, Masanori; Ikeda, Tsunehiko

    2015-10-01

    To determine whether insulin induces nitric oxide (NO) formation in retinal microvessels and to examine the effects of high glucose on the formation of NO. Freshly isolated rat retinal microvessels were incubated in normal (5.5 mM) or high (20 mM) glucose with or without insulin (100 nM). The levels of insulin-induced NO and reactive oxygen species (ROS) in the retinal microvessels were determined semiquantitatively using fluorescent probes, 4,5-diaminofluorescein diacetate, and hydroethidine, respectively, and a laser scanning confocal microscope. The insulin-induced changes of NO in rat retinal endothelial cells and pericytes cultured at different glucose concentrations (5.5 and 25 mM) were determined using flow cytometry. Nitric oxide synthase (NOS) protein levels were determined by Western blot analysis; intracellular levels of ROS were determined using fluorescence-activated cell sorting (FACS) analysis of ethidium fluorescence; and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RNA expression was quantified using real-time PCR. Exposure of microvessels to insulin under normal glucose conditions led to a significant increase in NO levels; however, this increase was significantly suppressed when the microvessels were incubated under high glucose conditions. Intracellular levels of ROS were significantly increased in both retinal microvessels and cultured microvascular cells under high glucose conditions. The expression of NOS and NADPH oxidase were significantly increased in endothelial cells and pericytes under high glucose conditions. The increased formation of NO by insulin and its suppression by high glucose conditions suggests that ROS production mediated by NADPH oxidase is important by insulin's effect on the retinal microvasculature.

  19. Wine and endothelial function.

    Science.gov (United States)

    Caimi, G; Carollo, C; Lo Presti, R

    2003-01-01

    In recent years many studies have focused on the well-known relationship between wine consumption and cardiovascular risk. Wine exerts its protective effects through various changes in lipoprotein profile, coagulation and fibrinolytic cascades, platelet aggregation, oxidative mechanisms and endothelial function. The last has earned more attention for its implications in atherogenesis. Endothelium regulates vascular tone by a delicate balancing among vasorelaxing (nitric oxide [NO]) and vasoconstrincting (endothelins) factors produced by endothelium in response to various stimuli. In rat models, wine and other grape derivatives exerted an endothelium-dependent vasorelaxing capacity especially associated with the NO-stimulating activity of their polyphenol components. In experimental conditions, reservatrol (a stilbene polyphenol) protected hearts and kidneys from ischemia-reperfusion injury through antioxidant activity and upregulation of NO production. Wine polyphenols are also able to induce the expression of genes involved in the NO pathway within the arterial wall. The effects of wine on endothelial function in humans are not yet clearly understood. A favorable action of red wine or dealcoholized wine extract or purple grape juice on endothelial function has been observed by several authors, but discrimination between ethanol and polyphenol effects is controversial. It is, however likely that regular and prolonged moderate wine drinking positively affects endothelial function. The beneficial effects of wine on cardiovascular health are greater if wine is associated with a healthy diet. The most recent nutritional and epidemiologic studies show that the ideal diet closely resembles the Mediterranean diet.

  20. Infections and endothelial cells

    NARCIS (Netherlands)

    Keller, Tymen T.; Mairuhu, Albert T. A.; de Kruif, Martijn D.; Klein, Saskia K.; Gerdes, Victor E. A.; ten Cate, Hugo; Brandjes, Dees P. M.; Levi, Marcel; van Gorp, Eric C. M.

    2003-01-01

    Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells may lead to rapid activation of

  1. Bilateral patching in retinal detachment: fluid mechanics and retinal "settling".

    Science.gov (United States)

    Foster, William J

    2011-07-20

    When a patient suffers a retinal detachment and surgery is delayed, it is known clinically that bilaterally patching the patient may allow the retina to partially reattach or "settle." Although this procedure has been performed since the 1860s, there is still debate as to how such a maneuver facilitates the reattachment of the retina. Finite element calculations using commercially available analysis software are used to elucidate the influence of reduction in eye movement caused by bilateral patching on the flow of subretinal fluid in a physical model of retinal detachment. It was found that by coupling fluid mechanics with structural mechanics, a physically consistent explanation of increased retinal detachment with eye movements can be found in the case of traction on the retinal hole. Large eye movements increase vitreous traction and detachment forces on the edge of the retinal hole, creating a subretinal vacuum and facilitating increased subretinal fluid. Alternative models, in which intraocular fluid flow is redirected into the subretinal space, are not consistent with these simulations. The results of these simulations explain the physical principles behind bilateral patching and provide insight that can be used clinically. In particular, as is known clinically, bilateral patching may facilitate a decrease in the height of a retinal detachment. The results described here provide a description of a physical mechanism underlying this technique. The findings of this study may aid in deciding whether to bilaterally patch patients and in counseling patients on pre- and postoperative care.

  2. Genetics Home Reference: retinitis pigmentosa

    Science.gov (United States)

    ... A characteristic of X-linked inheritance is that fathers cannot pass X-linked traits to their sons. ... in known genes account for 58% of autosomal dominant retinitis pigmentosa (adRP). Adv Exp Med Biol. 2008; ...

  3. Automated detection of retinal disease.

    Science.gov (United States)

    Helmchen, Lorens A; Lehmann, Harold P; Abràmoff, Michael D

    2014-11-01

    Nearly 4 in 10 Americans with diabetes currently fail to undergo recommended annual retinal exams, resulting in tens of thousands of cases of blindness that could have been prevented. Advances in automated retinal disease detection could greatly reduce the burden of labor-intensive dilated retinal examinations by ophthalmologists and optometrists and deliver diagnostic services at lower cost. As the current availability of ophthalmologists and optometrists is inadequate to screen all patients at risk every year, automated screening systems deployed in primary care settings and even in patients' homes could fill the current gap in supply. Expanding screens to all patients at risk by switching to automated detection systems would in turn yield significantly higher rates of detecting and treating diabetic retinopathy per dilated retinal examination. Fewer diabetic patients would develop complications such as blindness, while ophthalmologists could focus on more complex cases.

  4. Advances in Retinal Optical Imaging

    Directory of Open Access Journals (Sweden)

    Yanxiu Li

    2018-04-01

    Full Text Available Retinal imaging has undergone a revolution in the past 50 years to allow for better understanding of the eye in health and disease. Significant improvements have occurred both in hardware such as lasers and optics in addition to software image analysis. Optical imaging modalities include optical coherence tomography (OCT, OCT angiography (OCTA, photoacoustic microscopy (PAM, scanning laser ophthalmoscopy (SLO, adaptive optics (AO, fundus autofluorescence (FAF, and molecular imaging (MI. These imaging modalities have enabled improved visualization of retinal pathophysiology and have had a substantial impact on basic and translational medical research. These improvements in technology have translated into early disease detection, more accurate diagnosis, and improved management of numerous chorioretinal diseases. This article summarizes recent advances and applications of retinal optical imaging techniques, discusses current clinical challenges, and predicts future directions in retinal optical imaging.

  5. Prophylactic treatment of retinal breaks

    DEFF Research Database (Denmark)

    Blindbæk, Søren Leer; Grauslund, Jakob

    2015-01-01

    Prophylactic treatment of retinal breaks has been examined in several studies and reviews, but so far, no studies have successfully applied a systematic approach. In the present systematic review, we examined the need of follow-up after posterior vitreous detachment (PVD) - diagnosed by slit...... published before 2012. Four levels of screening identified 13 studies suitable for inclusion in this systematic review. No meta-analysis was conducted as no data suitable for statistical analysis were identified. In total, the initial examination after symptomatic PVD identified 85-95% of subsequent retinal......-47% of cases, respectively. The cumulated incidence of RRD despite prophylactic treatment was 2.1-8.8%. The findings in this review suggest that follow-up after symptomatic PVD is only necessary in cases of incomplete retinal examination at presentation. Prophylactic treatment of symptomatic retinal breaks...

  6. An autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA.

    Directory of Open Access Journals (Sweden)

    Guo-Xiang Ruan

    2008-10-01

    Full Text Available The influence of the mammalian retinal circadian clock on retinal physiology and function is widely recognized, yet the cellular elements and neural regulation of retinal circadian pacemaking remain unclear due to the challenge of long-term culture of adult mammalian retina and the lack of an ideal experimental measure of the retinal circadian clock. In the current study, we developed a protocol for long-term culture of intact mouse retinas, which allows retinal circadian rhythms to be monitored in real time as luminescence rhythms from a PERIOD2::LUCIFERASE (PER2::LUC clock gene reporter. With this in vitro assay, we studied the characteristics and location within the retina of circadian PER2::LUC rhythms, the influence of major retinal neurotransmitters, and the resetting of the retinal circadian clock by light. Retinal PER2::LUC rhythms were routinely measured from whole-mount retinal explants for 10 d and for up to 30 d. Imaging of vertical retinal slices demonstrated that the rhythmic luminescence signals were concentrated in the inner nuclear layer. Interruption of cell communication via the major neurotransmitter systems of photoreceptors and ganglion cells (melatonin and glutamate and the inner nuclear layer (dopamine, acetylcholine, GABA, glycine, and glutamate did not disrupt generation of retinal circadian PER2::LUC rhythms, nor did interruption of intercellular communication through sodium-dependent action potentials or connexin 36 (cx36-containing gap junctions, indicating that PER2::LUC rhythms generation in the inner nuclear layer is likely cell autonomous. However, dopamine, acting through D1 receptors, and GABA, acting through membrane hyperpolarization and casein kinase, set the phase and amplitude of retinal PER2::LUC rhythms, respectively. Light pulses reset the phase of the in vitro retinal oscillator and dopamine D1 receptor antagonists attenuated these phase shifts. Thus, dopamine and GABA act at the molecular level of PER

  7. Concentric retinitis pigmentosa: clinicopathologic correlations.

    Science.gov (United States)

    Milam, A H; De Castro, E B; Smith, J E; Tang, W X; John, S K; Gorin, M B; Stone, E M; Aguirre, G D; Jacobson, S G

    2001-10-01

    Progressive concentric (centripetal) loss of vision is one pattern of visual field loss in retinitis pigmentosa. This study provides the first clinicopathologic correlations for this form of retinitis pigmentosa. A family with autosomal dominant concentric retinitis pigmentosa was examined clinically and with visual function tests. A post-mortem eye of an affected 94 year old family member was processed for histopathology and immunocytochemistry with retinal cell specific antibodies. Unrelated simplex/multiplex patients with concentric retinitis pigmentosa were also examined. Affected family members of the eye donor and patients from the other families had prominent peripheral pigmentary retinopathy with more normal appearing central retina, good visual acuity, concentric field loss, normal or near normal rod and cone sensitivity within the preserved visual field, and reduced rod and cone electroretinograms. The eye donor, at age 90, had good acuity and function in a central island. Grossly, the central region of the donor retina appeared thinned but otherwise normal, while the far periphery contained heavy bone spicule pigment. Microscopically the central retina showed photoreceptor outer segment shortening and some photoreceptor cell loss. The mid periphery had a sharp line of demarcation where more central photoreceptors were near normal except for very short outer segments and peripheral photoreceptors were absent. Rods and cones showed abrupt loss of outer segments and cell death at this interface. It is concluded that concentric retinitis pigmentosa is a rare but recognizable phenotype with slowly progressive photoreceptor death from the far periphery toward the central retina. The disease is retina-wide but shows regional variation in severity of degeneration; photoreceptor death is severe in the peripheral retina with an abrupt edge between viable and degenerate photoreceptors. Peripheral to central gradients of unknown retinal molecule(s) may be defective

  8. Unilateral retinitis pigmentosa sine pigmento.

    Science.gov (United States)

    Pearlman, J T; Saxton, J; Hoffman, G

    1976-05-01

    A patient presented with unilateral findings of night blindness shown by impaired rod function and dark adaptation, constricted visual fields with good central acuity, a barely recordable electro-retinographic b-wave, and a unilaterally impaired electro-oculogram. There were none of the pigmentary changes usually associated with retinitis pigmentosa. The unaffected right eye was normal in all respects. Therefore the case is most probably one of unilateral retinitis pigmentosa sine pigmento.

  9. Light and inherited retinal degeneration

    OpenAIRE

    Paskowitz, D M; LaVail, M M; Duncan, J L

    2006-01-01

    Light deprivation has long been considered a potential treatment for patients with inherited retinal degenerative diseases, but no therapeutic benefit has been demonstrated to date. In the few clinical studies that have addressed this issue, the underlying mutations were unknown. Our rapidly expanding knowledge of the genes and mechanisms involved in retinal degeneration have made it possible to reconsider the potential value of light restriction in specific genetic contexts. This review summ...

  10. Iron Overload Accelerates the Progression of Diabetic Retinopathy in Association with Increased Retinal Renin Expression.

    Science.gov (United States)

    Chaudhary, Kapil; Promsote, Wanwisa; Ananth, Sudha; Veeranan-Karmegam, Rajalakshmi; Tawfik, Amany; Arjunan, Pachiappan; Martin, Pamela; Smith, Sylvia B; Thangaraju, Muthusamy; Kisselev, Oleg; Ganapathy, Vadivel; Gnana-Prakasam, Jaya P

    2018-02-14

    Diabetic retinopathy (DR) is a leading cause of blindness among working-age adults. Increased iron accumulation is associated with several degenerative diseases. However, there are no reports on the status of retinal iron or its implications in the pathogenesis of DR. In the present study, we found that retinas of type-1 and type-2 mouse models of diabetes have increased iron accumulation compared to non-diabetic retinas. We found similar iron accumulation in postmortem retinal samples from human diabetic patients. Further, we induced diabetes in HFE knockout (KO) mice model of genetic iron overload to understand the role of iron in the pathogenesis of DR. We found increased neuronal cell death, vascular alterations and loss of retinal barrier integrity in diabetic HFE KO mice compared to diabetic wildtype mice. Diabetic HFE KO mouse retinas also exhibited increased expression of inflammation and oxidative stress markers. Severity in the pathogenesis of DR in HFE KO mice was accompanied by increase in retinal renin expression mediated by G-protein-coupled succinate receptor GPR91. In light of previous reports implicating retinal renin-angiotensin system in DR pathogenesis, our results reveal a novel relationship between diabetes, iron and renin-angiotensin system, thereby unraveling new therapeutic targets for the treatment of DR.

  11. On the retinal toxicity of intraocular glucocorticoids.

    Science.gov (United States)

    Torriglia, Alicia; Valamanesh, Fatemeh; Behar-Cohen, Francine

    2010-12-15

    Corticosteroids are hormones involved in many physiological responses such as stress, immune modulation, protein catabolism and water homeostasis. The subfamily of glucocorticoids is used systemically in the treatment of inflammatory diseases or allergic reactions. In the eye, glucocorticoides are used to treat macular edema, inflammation and neovascularization. The most commonly used glucocorticoid is triamcinolone acetonide (TA). The pharmaceutical formulation of TA is not adapted for intravitreal administration but has been selected by ophthalmologists because its very low intraocular solubility provides sustained effect. Visual benefits of intraocular TA do not clearly correlate with morpho-anatomical improvements, suggesting potential toxicity. We therefore studied, non-common, but deleterious effects of glucocorticoids on the retina. We found that the intravitreal administration of TA is beneficial in the treatment of neovascularization because it triggers cell death of endothelial cells of neovessels by a caspase-independent mechanism. However, this treatment is toxic for the retina because it induces a non-apoptotic, caspase-independent cell death related to paraptosis, mostly in the retinal pigmented epithelium cells and the Müller cells. Copyright © 2010 Elsevier Inc. All rights reserved.

  12. Transgenic Mice Over-Expressing RBP4 Have RBP4-Dependent and Light-Independent Retinal Degeneration.

    Science.gov (United States)

    Du, Mei; Phelps, Eric; Balangue, Michael J; Dockins, Aaron; Moiseyev, Gennadiy; Shin, Younghwa; Kane, Shelley; Otalora, Laura; Ma, Jian-Xing; Farjo, Rafal; Farjo, Krysten M

    2017-08-01

    Transgenic mice overexpressing serum retinol-binding protein (RBP4-Tg) develop progressive retinal degeneration, characterized by microglia activation, yet the precise mechanisms underlying retinal degeneration are unclear. Previous studies showed RBP4-Tg mice have normal ocular retinoid levels, suggesting that degeneration is independent of the retinoid visual cycle or light exposure. The present study addresses whether retinal degeneration is light-dependent and RBP4-dependent by testing the effects of dark-rearing and pharmacological lowering of serum RBP4 levels, respectively. RBP4-Tg mice reared on normal mouse chow in normal cyclic light conditions were directly compared to RBP4-Tg mice exposed to chow supplemented with the RBP4-lowering compound A1120 or dark-rearing conditions. Quantitative retinal histological analysis was conducted to assess retinal degeneration, and electroretinography (ERG) and optokinetic tracking (OKT) tests were performed to assess retinal and visual function. Ocular retinoids and bis-retinoid A2E were quantified. Dark-rearing RBP4-Tg mice effectively reduced ocular bis-retinoid A2E levels, but had no significant effect on retinal degeneration or dysfunction in RBP4-Tg mice, demonstrating that retinal degeneration is light-independent. A1120 treatment lowered serum RBP4 levels similar to wild-type mice, and prevented structural retinal degeneration. However, A1120 treatment did not prevent retinal dysfunction in RBP4-Tg mice. Moreover, RBP4-Tg mice on A1120 diet had significant worsening of OKT response and loss of cone photoreceptors compared to RBP4-Tg mice on normal chow. This may be related to the very significant reduction in retinyl ester levels in the retina of mice on A1120-supplemented diet. Retinal degeneration in RBP4-Tg mice is RBP4-dependent and light-independent.

  13. Elucidating the role of AII amacrine cells in glutamatergic retinal waves.

    Science.gov (United States)

    Firl, Alana; Ke, Jiang-Bin; Zhang, Lei; Fuerst, Peter G; Singer, Joshua H; Feller, Marla B

    2015-01-28

    Spontaneous retinal activity mediated by glutamatergic neurotransmission-so-called "Stage 3" retinal waves-drives anti-correlated spiking in ON and OFF RGCs during the second week of postnatal development of the mouse. In the mature retina, the activity of a retinal interneuron called the AII amacrine cell is responsible for anti-correlated spiking in ON and OFF α-RGCs. In mature AIIs, membrane hyperpolarization elicits bursting behavior. Here, we postulated that bursting in AIIs underlies the initiation of glutamatergic retinal waves. We tested this hypothesis by using two-photon calcium imaging of spontaneous activity in populations of retinal neurons and by making whole-cell recordings from individual AIIs and α-RGCs in in vitro preparations of mouse retina. We found that AIIs participated in retinal waves, and that their activity was correlated with that of ON α-RGCs and anti-correlated with that of OFF α-RGCs. Though immature AIIs lacked the complement of membrane conductances necessary to generate bursting, pharmacological activation of the M-current, a conductance that modulates bursting in mature AIIs, blocked retinal wave generation. Interestingly, blockade of the pacemaker conductance Ih, a conductance absent in AIIs but present in both ON and OFF cone bipolar cells, caused a dramatic loss of spatial coherence of spontaneous activity. We conclude that during glutamatergic waves, AIIs act to coordinate and propagate activity generated by BCs rather than to initiate spontaneous activity. Copyright © 2015 the authors 0270-6474/15/351675-12$15.00/0.

  14. The use of bevacizumab in a multilevel retinal hemorrhage secondary to retinal macroaneurysm: a 39-month follow-up case report

    Science.gov (United States)

    Tsakpinis, Dimitrios; Nasr, Mayssa B; Tranos, Paris; Krassas, Nikos; Giannopoulos, Theodoros; Symeonidis, Chrysanthos; Dimitrakos, Stavros A; Konstas, Anastasios GP

    2011-01-01

    Purpose The evaluation of long-term visual outcome after the use of bevacizumab for the management of multilevel hemorrhage due to retinal arterial macroaneurysm (MA). Case report A 71-year-old hypertensive female presented with sudden reduction of visual acuity in her left eye (OS). Fundoscopy revealed an arterial macroaneurysm with preretinal and subretinal hemorrhage in the eye. Due to significant macular involvement, the patient received two intravitreal injections of bevacizumab within 2 months. Results Significant visual and anatomical recovery was observed 2 months later, which was confirmed by fluorescein angiography. At the end of a follow-up period (39 months) visual acuity and visual field were at normal levels. Conclusion Retinal MA is a relatively rare condition. Anti-vascular endothelial growth factor therapy appears a safe and effective treatment option for selected symptomatic individuals that may offer faster visual rehabilitation. Herein we report, for the first time, a 39-month follow-up of a retinal MA treated with anti-vascular endothelial growth factor therapy. PMID:22069349

  15. Determination of retinal surface area.

    Science.gov (United States)

    Nagra, Manbir; Gilmartin, Bernard; Thai, Ngoc Jade; Logan, Nicola S

    2017-09-01

    Previous attempts at determining retinal surface area and surface area of the whole eye have been based upon mathematical calculations derived from retinal photographs, schematic eyes and retinal biopsies of donor eyes. 3-dimensional (3-D) ocular magnetic resonance imaging (MRI) allows a more direct measurement, it can be used to image the eye in vivo, and there is no risk of tissue shrinkage. The primary purpose of this study is to compare, using T2-weighted 3D MRI, retinal surface areas for superior-temporal (ST), inferior-temporal (IT), superior-nasal (SN) and inferior-nasal (IN) retinal quadrants. An ancillary aim is to examine whether inter-quadrant variations in area are concordant with reported inter-quadrant patterns of susceptibility to retinal breaks associated with posterior vitreous detachment (PVD). Seventy-three adult participants presenting without retinal pathology (mean age 26.25 ± 6.06 years) were scanned using a Siemens 3-Tesla MRI scanner to provide T2-weighted MR images that demarcate fluid-filled internal structures for the whole eye and provide high-contrast delineation of the vitreous-retina interface. Integrated MRI software generated total internal ocular surface area (TSA). The second nodal point was used to demarcate the origin of the peripheral retina in order to calculate total retinal surface area (RSA) and quadrant retinal surface areas (QRSA) for ST, IT, SN, and IN quadrants. Mean spherical error (MSE) was -2.50 ± 4.03D and mean axial length (AL) 24.51 ± 1.57 mm. Mean TSA and RSA for the RE were 2058 ± 189 and 1363 ± 160 mm 2 , respectively. Repeated measures anova for QRSA data indicated a significant difference within-quadrants (P area/mm increase in AL. Although the differences between QRSAs are relatively small, there was evidence of concordance with reported inter-quadrant patterns of susceptibility to retinal breaks associated with PVD. The data allow AL to be converted to QRSAs, which will assist further

  16. Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis

    Science.gov (United States)

    Sun, Dawei; Nakao, Shintaro; Xie, Fang; Zandi, Souska; Bagheri, Abouzar; Kanavi, Mozhgan Rezaei; Samiei, Shahram; Soheili, Zahra-Soheila; Frimmel, Sonja; Zhang, Zhongyu; Ablonczy, Zsolt; Ahmadieh, Hamid; Hafezi-Moghadam, Ali

    2014-01-01

    Diabetic retinopathy (DR) is a microvascular complication of diabetes and a leading cause of vision loss. Biomarkers and methods for early diagnosis of DR are urgently needed. Using a new molecular imaging approach, we show up to 94% higher accumulation of custom designed imaging probes against vascular endothelial growth factor receptor 2 (VEGFR-2) in retinal and choroidal vessels of diabetic animals (PM. R., Samiei, S., Soheili, Z.-S., Frimmel, S., Zhang, Z., Ablonczy, Z., Ahmadieh, H., Hafezi-Moghadam, A. Molecular imaging reveals elevated VEGFR-2 expression in retinal capillaries in diabetes: a novel biomarker for early diagnosis. PMID:24903276

  17. [Circulating endothelial cells: biomarkers for monitoring activity of antiangiogenic therapy].

    Science.gov (United States)

    Farace, Françoise; Bidart, Jean-Michel

    2007-07-01

    Tumor vessel formation is largely dependent on the recruitment of endothelial cells. Rare in healthy individuals, circulating endothelial cells (CEC) are shed from vessel walls and enter the circulation reflecting endothelial damage or dysfunction. Increased numbers of CEC have been documented in different types of cancer. Recent studies have suggested the role for CEC in tumor angiogenesis, but whose presence could also reflect normal endothelium perturbation in cancer. Originating from the bone marrow rather than from vessel walls, endothelial progenitor cells (EPC) are mobilized following tissue ischemia and may be recruited to complement local angiogenesis supplied by existing endothelium. Recently, studies in mouse models suggest that the circulating fraction of endothelial progenitors (CEP) is involved in tumor angiogenesis but their contribution is less clear in humans. The detection of CEC and CEP is difficult and impeded by the rarity of these cells. They may have important clinical implication as novel biomarkers susceptible to predict more efficiently and rapidly the therapeutic response to anti-angiogenic treatments. However, a methodological consensus would be necessary in order to correctly evaluate the clinical interest of CEC and CEP in patients.

  18. Effect of sunitinib combined with ionizing radiation on endothelial cells

    International Nuclear Information System (INIS)

    Zhang Haiping; Jiao Xiaodong; Li Rui; Wang Jiejun; Takayama, Koichi; Su Bo

    2011-01-01

    The aims of present study were to evaluate the efficacy of combining sunitinib with ionizing radiation (IR) on endothelial cells in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were exposed to IR with or without sunitinib pretreatment. Apoptosis assay and cell cycle distribution were analyzed by flow cytometry. Clonogenic survival assay at 3 Gy dose with or without sunitinib was performed. The activity of phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway was detected by Western immunoblot. Lewis lung carcinoma mouse model was built to examine the effect of combination therapy on endothelial cells in vivo. Microvasculature changes were detected by immunohistochemistry using anti-CD31 antibody. Our results showed combination therapy of sunitinib and IR significantly increased apoptosis of endothelial cells and inhibited colony formation compared to sunitinib or radiotherapy alone. It also resulted in cell cycle redistribution (decreasing cells in S phase and increasing cells in G2/M phase). The activity of PI3K/Akt signal pathway was inhibited, which could be the potential mechanisms that account for the enhanced radiation response induced by sunitinib. In vivo analysis showed that combination therapy significantly decreased microvasculature formation. The results demonstrated that combination therapy of sunitinib and IR has the potential to increase the cytotoxic effects on endothelial cells. (author)

  19. Quantification of rat retinal growth and vascular population changes after single and split doses of proton irradiation: translational study using stereology methods

    Science.gov (United States)

    Mao, Xiao W.; Archambeau, John O.; Kubinova, Lucie; Boyle, Soames; Petersen, Georgia; Grove, Roger; Nelson, G. A. (Principal Investigator)

    2003-01-01

    This study quantified architectural and population changes in the rat retinal vasculature after proton irradiation using stereology. A 100 MeV conformal proton beam delivered 8, 14, 20 and 28 Gy as single and split doses to the whole eye. The vascular networks were prepared from retinal digests. Stereological methods were used to obtain the area of the retina and unbiased estimates of microvessel/artery/vein endothelial, pericyte and smooth muscle population, and vessel length. The retinal area increased progressively in the unirradiated, age-matched controls and in the retinas irradiated with 8 and 14 Gy, indicating uniform progressive retinal growth. No growth occurred after 20 and 28 Gy. Regression analysis of total endothelial cell number in all vessels (arteries, veins and capillaries) after irradiation documented a progressive time- and dose-dependent cell loss occurring over 15 to 24 months. The difference from controls was significant (Ppopulations after split doses. At 10 Gy, the rate of endothelial cell loss, a dose parameter used to characterize the time- and dose-dependent loss of the endothelial population, was doubled.

  20. Retinal pigmentary changes in chronic uveitis mimicking retinitis pigmentosa.

    Science.gov (United States)

    Sevgi, D Damla; Davoudi, Samaneh; Comander, Jason; Sobrin, Lucia

    2017-09-01

    To present retinal pigmentary changes mimicking retinitis pigmentosa (RP) as a finding of advanced uveitis. We retrospectively reviewed charts of patients without a family history of inherited retinal degenerations who presented with retinal pigment changes and signs of past or present intraocular inflammation. Comprehensive eye examination including best-corrected visual acuity, slit-lamp examination and dilated fundus examination was performed on all patients in addition to color fundus photography, optical coherence tomography, fluorescein angiography (FA), and full-field electroretinogram testing. We identified five patients with ages ranging from 33 to 66 years, who presented with RP-like retinal pigmentary changes which were eventually attributed to longstanding uveitis. The changes were bilateral in three cases and unilateral in two cases. Four of five cases presented with active inflammation, and the remaining case showed evidence of active intraocular inflammation during follow-up. This study highlights the overlapping features of advanced uveitis and RP including the extensive pigmentary changes. Careful review of possible past uveitis history, detailed examination of signs of past or present inflammation and ancillary testing, with FA often being most helpful, are required for the correct diagnosis. This is important, because intervention can prevent further damage if the cause of the pigmentary changes is destructive inflammation.

  1. Retinal detachment in paediatric patients

    International Nuclear Information System (INIS)

    Zafar, S. N.; Qureshi, N.; Azad, N.; Khan, A.

    2013-01-01

    Objective: To assess the causes of retinal detachment in children and the various operative procedures requiring vitreoretinal surgical intervention for the same. Study Design: Case series. Place and Duration of Study: Department of Ophthalmology, Al-Shifa Trust Eye Hospital, Rawalpindi, from January 2006 to May 2009. Methodology: A total of 281 eyes of 258 patients, (aged 0 - 18 years) who underwent vitreo-retinal surgical intervention for retinal detachment were included. Surgical log was searched for the type of retinal detachment and its causes. Frequencies of various interventions done in these patients viz. vitrectomy, scleral buckle, use of tamponading agents, laser photocoagulation and cryotherapy were noted. Results were described as descriptive statistics. Results: Myopia was the cause in 62 (22.1%) and trauma in 51 (18.1%) of the eyes. Total retinal detachment (RD) was treated in 94 (33.5%) eyes, sub total RD in 36 (12.8%), recurrent RD in 32 (11.4%), giant retinal tear in 28 (10%), tractional RD in 15 (5.3%) and exudative RD in 2 (0.7%). Prophylactic laser or cryotherapy was applied in 74 (26.3%) of the eyes. Pars plana vitrectomy (PPV) was carried out in 159 (56.6%) eyes while scleral buckle procedure was done in 129 (45.9%) eyes. Silicon oil was used in 149 (53%), perfluorocarbon liquid in 32 (11.4%) and gas tamponade in 20 (7.1%) eyes. Conclusion: The most common cause of retinal detachment in paediatric patients was myopia, followed by trauma. Total RD was more common as compared to the other types. The most common procedure adopted was pars plana vitrectomy followed by scleral buckle procedure. (author)

  2. Hydrogen sulfide metabolism regulates endothelial solute barrier function

    Directory of Open Access Journals (Sweden)

    Shuai Yuan

    2016-10-01

    Full Text Available Hydrogen sulfide (H2S is an important gaseous signaling molecule in the cardiovascular system. In addition to free H2S, H2S can be oxidized to polysulfide which can be biologically active. Since the impact of H2S on endothelial solute barrier function is not known, we sought to determine whether H2S and its various metabolites affect endothelial permeability. In vitro permeability was evaluated using albumin flux and transendothelial electrical resistance. Different H2S donors were used to examine the effects of exogenous H2S. To evaluate the role of endogenous H2S, mouse aortic endothelial cells (MAECs were isolated from wild type mice and mice lacking cystathionine γ-lyase (CSE, a predominant source of H2S in endothelial cells. In vivo permeability was evaluated using the Miles assay. We observed that polysulfide donors induced rapid albumin flux across endothelium. Comparatively, free sulfide donors increased permeability only with higher concentrations and at later time points. Increased solute permeability was associated with disruption of endothelial junction proteins claudin 5 and VE-cadherin, along with enhanced actin stress fiber formation. Importantly, sulfide donors that increase permeability elicited a preferential increase in polysulfide levels within endothelium. Similarly, CSE deficient MAECs showed enhanced solute barrier function along with reduced endogenous bound sulfane sulfur. CSE siRNA knockdown also enhanced endothelial junction structures with increased claudin 5 protein expression. In vivo, CSE genetic deficiency significantly blunted VEGF induced hyperpermeability revealing an important role of the enzyme for barrier function. In summary, endothelial solute permeability is critically regulated via exogenous and endogenous sulfide bioavailability with a prominent role of polysulfides.

  3. Zingiber officinale attenuates retinal microvascular changes in diabetic rats via anti-inflammatory and antiangiogenic mechanisms

    Science.gov (United States)

    Dongare, Shirish; Mathur, Rajani; Saxena, Rohit; Mathur, Sandeep; Agarwal, Renu; Nag, Tapas C.; Srivastava, Sushma; Kumar, Pankaj

    2016-01-01

    Purpose Diabetic retinopathy is a common microvascular complication of long-standing diabetes. Several complex interconnecting biochemical pathways are activated in response to hyperglycemia. These pathways culminate into proinflammatory and angiogenic effects that bring about structural and functional damage to the retinal vasculature. Since Zingiber officinale (ginger) is known for its anti-inflammatory and antiangiogenic properties, we investigated the effects of its extract standardized to 5% 6-gingerol, the major active constituent of ginger, in attenuating retinal microvascular changes in rats with streptozotocin-induced diabetes. Methods Diabetic rats were treated orally with the vehicle or the ginger extract (75 mg/kg/day) over a period of 24 weeks along with regular monitoring of bodyweight and blood glucose and weekly fundus photography. At the end of the 24-week treatment, the retinas were isolated for histopathological examination under a light microscope, transmission electron microscopy, and determination of the retinal tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and vascular endothelial growth factor (VEGF) levels. Results Oral administration of the ginger extract resulted in significant reduction of hyperglycemia, the diameter of the retinal vessels, and vascular basement membrane thickness. Improvement in the architecture of the retinal vasculature was associated with significantly reduced expression of NF-κB and reduced activity of TNF-α and VEGF in the retinal tissue in the ginger extract–treated group compared to the vehicle-treated group. Conclusions The current study showed that ginger extract containing 5% of 6-gingerol attenuates the retinal microvascular changes in rats with streptozotocin-induced diabetes through anti-inflammatory and antiangiogenic actions. Although precise molecular targets remain to be determined, 6-gingerol seems to be a potential candidate for further investigation. PMID:27293376

  4. Noninvasive Retinal Markers in Diabetic Retinopathy

    DEFF Research Database (Denmark)

    Blindbæk, Søren Leer; Torp, Thomas Lee; Lundberg, Kristian

    2017-01-01

    The retinal vascular system is the only part of the human body available for direct, in vivo inspection. Noninvasive retinal markers are important to identity patients in risk of sight-threatening diabetic retinopathy. Studies have correlated structural features like retinal vascular caliber...... and fractals with micro- and macrovascular dysfunction in diabetes. Likewise, the retinal metabolism can be evaluated by retinal oximetry, and higher retinal venular oxygen saturation has been demonstrated in patients with diabetic retinopathy. So far, most studies have been cross-sectional, but these can only...... retinopathy and diabetic macular edema. The Department of Ophthalmology at Odense University Hospital, Denmark, has a strong tradition of studying the retinal microvasculature in diabetic retinopathy. In the present paper, we demonstrate the importance of the retinal vasculature not only as predictors of long...

  5. Mitochondrial dysfunction underlying outer retinal diseases

    DEFF Research Database (Denmark)

    Lefevere, Evy; Toft-Kehler, Anne Katrine; Vohra, Rupali

    2017-01-01

    Dysfunction of photoreceptors, retinal pigment epithelium (RPE) or both contribute to the initiation and progression of several outer retinal disorders. Disrupted Müller glia function might additionally subsidize to these diseases. Mitochondrial malfunctioning is importantly associated with outer...

  6. Mouse adhalin

    DEFF Research Database (Denmark)

    Liu, L; Vachon, P H; Kuang, W

    1997-01-01

    . To analyze the biological roles of adhalin, we cloned the mouse adhalin cDNA, raised peptide-specific antibodies to its cytoplasmic domain, and examined its expression and localization in vivo and in vitro. The mouse adhalin sequence was 80% identical to that of human, rabbit, and hamster. Adhalin...... was specifically expressed in striated muscle cells and their immediate precursors, and absent in many other cell types. Adhalin expression in embryonic mouse muscle was coincident with primary myogenesis. Its expression was found to be up-regulated at mRNA and protein levels during myogenic differentiation...

  7. Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting

    Directory of Open Access Journals (Sweden)

    Livia S. Carvalho

    2017-09-01

    Full Text Available Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for inherited retinal degeneration due to mutations in RPE65 have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies in vitro and in vivo using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both in vitro and in vivo compared to trans-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.

  8. Endothelial RIG-I activation impairs endothelial function

    Energy Technology Data Exchange (ETDEWEB)

    Asdonk, Tobias, E-mail: tobias.asdonk@ukb.uni-bonn.de [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Motz, Inga; Werner, Nikos [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Coch, Christoph; Barchet, Winfried; Hartmann, Gunther [Institute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany); Nickenig, Georg; Zimmer, Sebastian [Department of Medicine/Cardiology, University of Bonn, Sigmund-Freud-Str. 25, 53105 Bonn (Germany)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer RIG-I activation impairs endothelial function in vivo. Black-Right-Pointing-Pointer RIG-I activation alters HCAEC biology in vitro. Black-Right-Pointing-Pointer EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 {mu}g of the RIG-ligand 3pRNA (RNA with triphosphate at the 5 Prime end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  9. Endothelial RIG-I activation impairs endothelial function

    International Nuclear Information System (INIS)

    Asdonk, Tobias; Motz, Inga; Werner, Nikos; Coch, Christoph; Barchet, Winfried; Hartmann, Gunther; Nickenig, Georg; Zimmer, Sebastian

    2012-01-01

    Highlights: ► RIG-I activation impairs endothelial function in vivo. ► RIG-I activation alters HCAEC biology in vitro. ► EPC function is affected by RIG-I stimulation in vitro. -- Abstract: Background: Endothelial dysfunction is a crucial part of the chronic inflammatory atherosclerotic process and is mediated by innate and acquired immune mechanisms. Recent studies suggest that pattern recognition receptors (PRR) specialized in immunorecognition of nucleic acids may play an important role in endothelial biology in a proatherogenic manner. Here, we analyzed the impact of endothelial retinoic acid inducible gene I (RIG-I) activation upon vascular endothelial biology. Methods and results: Wild type mice were injected intravenously with 32.5 μg of the RIG-ligand 3pRNA (RNA with triphosphate at the 5′end) or polyA control every other day for 7 days. In 3pRNA-treated mice, endothelium-depended vasodilation was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticle (EMP) numbers significantly elevated compared to controls. To gain further insight in RIG-I dependent endothelial biology, cultured human coronary endothelial cells (HCAEC) and endothelial progenitor cells (EPC) were stimulated in vitro with 3pRNA. Both cells types express RIG-I and react with receptor upregulation upon stimulation. Reactive oxygen species (ROS) formation is enhanced in both cell types, whereas apoptosis and proliferation is not significantly affected in HCAEC. Importantly, HCAEC release significant amounts of proinflammatory cytokines in response to RIG-I stimulation. Conclusion: This study shows that activation of the cytoplasmatic nucleic acid receptor RIG-I leads to endothelial dysfunction. RIG-I induced endothelial damage could therefore be an important pathway in atherogenesis.

  10. Retinal detachment in black South Africans

    African Journals Online (AJOL)

    low incidence of retinal detachment in black patients is not known. ... a retinal break. Predisposing factors include peripheral retinal degenerations, myopia, aphakia and trauma. Delay in presentation increases the difficulty in achieving adequate surgical ... On examination, note was taken of the visual acuity in both eyes, the ...

  11. Transcorneal Electrical Stimulation Therapy for Retinal Disease

    Science.gov (United States)

    2012-05-03

    Retinitis Pigmentosa; Macula Off; Primary Open Angle Glaucoma; Hereditary Macular Degeneration; Treated Retina Detachment; Retinal Artery Occlusion; Retinal Vein Occlusion; Non-Arthritic-Anterior-Ischemic Optic-Neuropathy; Hereditary Autosomal Dominant Optic Atrophy; Dry Age Related Macular Degeneration; Ischemic Macula Edema

  12. Activated ovarian endothelial cells promote early follicular development and survival.

    Science.gov (United States)

    Kedem, Alon; Aelion-Brauer, Anate; Guo, Peipei; Wen, Duancheng; Ding, Bi-Sen; Lis, Raphael; Cheng, Du; Sandler, Vladislav M; Rafii, Shahin; Rosenwaks, Zev

    2017-09-19

    New data suggests that endothelial cells (ECs) elaborate essential "angiocrine factors". The aim of this study is to investigate the role of activated ovarian endothelial cells in early in-vitro follicular development. Mouse ovarian ECs were isolated using magnetic cell sorting or by FACS and cultured in serum free media. After a constitutive activation of the Akt pathway was initiated, early follicles (50-150 um) were mechanically isolated from 8-day-old mice and co-cultured with these activated ovarian endothelial cells (AOEC) (n = 32), gel (n = 24) or within matrigel (n = 27) in serum free media for 14 days. Follicular growth, survival and function were assessed. After 6 passages, flow cytometry showed 93% of cells grown in serum-free culture were VE-cadherin positive, CD-31 positive and CD 45 negative, matching the known EC profile. Beginning on day 4 of culture, we observed significantly higher follicular and oocyte growth rates in follicles co-cultured with AOECs compared with follicles on gel or matrigel. After 14 days of culture, 73% of primary follicles and 83% of secondary follicles co-cultured with AOEC survived, whereas the majority of follicles cultured on gel or matrigel underwent atresia. This is the first report of successful isolation and culture of ovarian ECs. We suggest that co-culture with activated ovarian ECs promotes early follicular development and survival. This model is a novel platform for the in vitro maturation of early follicles and for the future exploration of endothelial-follicular communication. In vitro development of early follicles necessitates a complex interplay of growth factors and signals required for development. Endothelial cells (ECs) may elaborate essential "angiocrine factors" involved in organ regeneration. We demonstrate that co-culture with ovarian ECs enables culture of primary and early secondary mouse ovarian follicles.

  13. Retinal image quality during accommodation.

    Science.gov (United States)

    López-Gil, Norberto; Martin, Jesson; Liu, Tao; Bradley, Arthur; Díaz-Muñoz, David; Thibos, Larry N

    2013-07-01

    We asked if retinal image quality is maximum during accommodation, or sub-optimal due to accommodative error, when subjects perform an acuity task. Subjects viewed a monochromatic (552 nm), high-contrast letter target placed at various viewing distances. Wavefront aberrations of the accommodating eye were measured near the endpoint of an acuity staircase paradigm. Refractive state, defined as the optimum target vergence for maximising retinal image quality, was computed by through-focus wavefront analysis to find the power of the virtual correcting lens that maximizes visual Strehl ratio. Despite changes in ocular aberrations and pupil size during binocular viewing, retinal image quality and visual acuity typically remain high for all target vergences. When accommodative errors lead to sub-optimal retinal image quality, acuity and measured image quality both decline. However, the effect of accommodation errors of on visual acuity are mitigated by pupillary constriction associated with accommodation and binocular convergence and also to binocular summation of dissimilar retinal image blur. Under monocular viewing conditions some subjects displayed significant accommodative lag that reduced visual performance, an effect that was exacerbated by pharmacological dilation of the pupil. Spurious measurement of accommodative error can be avoided when the image quality metric used to determine refractive state is compatible with the focusing criteria used by the visual system to control accommodation. Real focusing errors of the accommodating eye do not necessarily produce a reliably measurable loss of image quality or clinically significant loss of visual performance, probably because of increased depth-of-focus due to pupil constriction. When retinal image quality is close to maximum achievable (given the eye's higher-order aberrations), acuity is also near maximum. A combination of accommodative lag, reduced image quality, and reduced visual function may be a useful

  14. The Expression Profiles of Lysophospholipid Receptors (LPLRs in Different Endothelial Cells

    Directory of Open Access Journals (Sweden)

    Yu-Wei Lee

    2006-03-01

    Full Text Available Sphingosine-1-phosphate (S1P and lysophosphatidic acid (LPA are two bioactive lysophospholipids (LPLs, stored primarily in platelets and released during platelet activation. Both LPLs are capable of regulating endothelial cell functions. The physiological functions of S1P and LPA are mediated by interacting with eight different G-protein coupled receptors: S1P1 through 5 and LPA1 through 3, which activate three different heterotrimeric GTP proteins-including Gi、Gq and G(12/13. The expression of LPL receptors in endothelial cells would affect the responses of S1P and LPA to these cells. There is no previous report discussing the expression profiles of LPL receptors in different endothelial cells from various species. In this study, we aim to investigate the expression profiles of S1P and LPA receptors in different endothelial cells isolated from human, rat, mouse and bovine origin. We used RT-PCR to determine LPLs receptors expression profiles in different endothelial cells. Our results indicated that endothelial cells from various species express different LPL receptors. Endothelial cells isolated from the same source of different species also had different LPLs receptors expression profiles. Therefore, different endothelial cells should respond to LPLs in different manners.

  15. The Volatile Anesthetic Isoflurane Increases Endothelial Adenosine Generation via Microparticle Ecto-5′-Nucleotidase (CD73) Release

    Science.gov (United States)

    Kim, Mihwa; Ham, Ahrom; Kim, Katelyn Yu-Mi; Brown, Kevin M.; Lee, H. Thomas

    2014-01-01

    Endothelial dysfunction is common in acute and chronic organ injury. Isoflurane is a widely used halogenated volatile anesthetic during the perioperative period and protects against endothelial cell death and inflammation. In this study, we tested whether isoflurane induces endothelial ecto-5′-nucleotidase (CD73) and cytoprotective adenosine generation to protect against endothelial cell injury. Clinically relevant concentrations of isoflurane induced CD73 activity and increased adenosine generation in cultured human umbilical vein or mouse glomerular endothelial cells. Surprisingly, isoflurane-mediated induction of endothelial CD73 activity occurred within 1 hr and without synthesizing new CD73. We determined that isoflurane rapidly increased CD73 containing endothelial microparticles into the cell culture media. Indeed, microparticles isolated from isoflurane-treated endothelial cells had significantly higher CD73 activity as well as increased CD73 protein. In vivo, plasma from mice anesthetized with isoflurane had significantly higher endothelial cell-derived CD144+ CD73+ microparticles and had increased microparticle CD73 activity compared to plasma from pentobarbital-anesthetized mice. Supporting a critical role of CD73 in isoflurane-mediated endothelial protection, a selective CD73 inhibitor (APCP) prevented isoflurane-induced protection against human endothelial cell inflammation and apoptosis. In addition, isoflurane activated endothelial cells Rho kinase evidenced by myosin phosphatase target subunit-1 and myosin light chain phosphorylation. Furthermore, isoflurane-induced release of CD73 containing microparticles was significantly attenuated by a selective Rho kinase inhibitor (Y27632). Taken together, we conclude that the volatile anesthetic isoflurane causes Rho kinase-mediated release of endothelial microparticles containing preformed CD73 and increase adenosine generation to protect against endothelial apoptosis and inflammation. PMID:24945528

  16. Renal-Retinal Ciliopathy Gene Sdccag8 Regulates DNA Damage Response Signaling

    DEFF Research Database (Denmark)

    Airik, Rannar; Slaats, Gisela G; Guo, Zhi

    2014-01-01

    Nephronophthisis-related ciliopathies (NPHP-RCs) are developmental and degenerative kidney diseases that are frequently associated with extrarenal pathologies such as retinal degeneration, obesity, and intellectual disability. We recently identified mutations in a gene encoding the centrosomal...... protein SDCCAG8 as causing NPHP type 10 in humans. To study the role of Sdccag8 in disease pathogenesis, we generated a Sdccag8 gene-trap mouse line. Homozygous Sdccag8(gt/gt) mice lacked the wild-type Sdccag8 transcript and protein, and recapitulated the human phenotypes of NPHP and retinal degeneration....... These mice exhibited early onset retinal degeneration that was associated with rhodopsin mislocalization in the photoreceptors and reduced cone cell numbers, and led to progressive loss of vision. By contrast, renal histologic changes occurred later, and no global ciliary defects were observed in the kidneys...

  17. CXCL10 can inhibit endothelial cell proliferation independently of CXCR3.

    Directory of Open Access Journals (Sweden)

    Gabriele S V Campanella

    2010-09-01

    Full Text Available CXCL10 (or Interferon-inducible protein of 10 kDa, IP-10 is an interferon-inducible chemokine with potent chemotactic activity on activated effector T cells and other leukocytes expressing its high affinity G protein-coupled receptor CXCR3. CXCL10 is also active on other cell types, including endothelial cells and fibroblasts. The mechanisms through which CXCL10 mediates its effects on non-leukocytes is not fully understood. In this study, we focus on the anti-proliferative effect of CXCL10 on endothelial cells, and demonstrate that CXCL10 can inhibit endothelial cell proliferation in vitro independently of CXCR3. Four main findings support this conclusion. First, primary mouse endothelial cells isolated from CXCR3-deficient mice were inhibited by CXCL10 as efficiently as wildtype endothelial cells. We also note that the proposed alternative splice form CXCR3-B, which is thought to mediate CXCL10's angiostatic activity, does not exist in mice based on published mouse CXCR3 genomic sequences as an in-frame stop codon would terminate the proposed CXCR3-B splice variant in mice. Second, we demonstrate that human umbilical vein endothelial cells and human lung microvascular endothelial cells that were inhibited by CXL10 did not express CXCR3 by FACS analysis. Third, two different neutralizing CXCR3 antibodies did not inhibit the anti-proliferative effect of CXCL10. Finally, fourth, utilizing a panel of CXCL10 mutants, we show that the ability to inhibit endothelial cell proliferation correlates with CXCL10's glycosaminoglycan binding affinity and not with its CXCR3 binding and signaling. Thus, using a very defined system, we show that CXCL10 can inhibit endothelial cell proliferation through a CXCR3-independent mechanism.

  18. Retinal vein occlusion and macular edema – critical evaluation of the clinical value of ranibizumab

    Directory of Open Access Journals (Sweden)

    Keane PA

    2011-06-01

    Full Text Available Pearse A Keane1, Srinivas R Sadda21NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of Ophthalmology, London, UK; 2Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USAAbstract: Retinal vein occlusions (RVOs constitute the second most common cause of retinal vascular disease after diabetic retinopathy, with a prevalence of between 1% and 2% in persons older than 40 years of age. Despite the existence of numerous potential therapeutic options, none is entirely satisfactory, and many patients with RVO suffer irreversible visual loss. Fortunately however, the recent introduction of antivascular endothelial growth factor (VEGF agents, such as ranibizumab (Lucentis®, Genentech, South San Francisco, CA and bevacizumab (Avastin®, Genentech, offers a potentially new treatment approach for clinicians managing this disorder. The results of the BRAVO and CRUISE trials have provided the first definitive evidence for the efficacy and safety of ranibizumab in the treatment of RVO. As a result, ranibizumab has recently been approved by the US Food and Drug Administration for the treatment of RVO-associated macular edema. In this review, we provide a critical evaluation of clinical trial data for the safety and efficacy of ranibizumab, and address unresolved issues in the management of this disorder. Keywords: ranibizumab, retinal vein occlusion, vascular endothelial growth factor, macular edema

  19. Rank Order Coding: a Retinal Information Decoding Strategy Revealed by Large-Scale Multielectrode Array Retinal Recordings.

    Science.gov (United States)

    Portelli, Geoffrey; Barrett, John M; Hilgen, Gerrit; Masquelier, Timothée; Maccione, Alessandro; Di Marco, Stefano; Berdondini, Luca; Kornprobst, Pierre; Sernagor, Evelyne

    2016-01-01

    How a population of retinal ganglion cells (RGCs) encodes the visual scene remains an open question. Going beyond individual RGC coding strategies, results in salamander suggest that the relative latencies of a RGC pair encode spatial information. Thus, a population code based on this concerted spiking could be a powerful mechanism to transmit visual information rapidly and efficiently. Here, we tested this hypothesis in mouse by recording simultaneous light-evoked responses from hundreds of RGCs, at pan-retinal level, using a new generation of large-scale, high-density multielectrode array consisting of 4096 electrodes. Interestingly, we did not find any RGCs exhibiting a clear latency tuning to the stimuli, suggesting that in mouse, individual RGC pairs may not provide sufficient information. We show that a significant amount of information is encoded synergistically in the concerted spiking of large RGC populations. Thus, the RGC population response described with relative activities, or ranks, provides more relevant information than classical independent spike count- or latency- based codes. In particular, we report for the first time that when considering the relative activities across the whole population, the wave of first stimulus-evoked spikes is an accurate indicator of stimulus content. We show that this coding strategy coexists with classical neural codes, and that it is more efficient and faster. Overall, these novel observations suggest that already at the level of the retina, concerted spiking provides a reliable and fast strategy to rapidly transmit new visual scenes.

  20. Endothelial Dll4 overexpression reduces vascular response and inhibits tumor growth and metastasization in vivo.

    Science.gov (United States)

    Trindade, Alexandre; Djokovic, Dusan; Gigante, Joana; Mendonça, Liliana; Duarte, António

    2017-03-14

    The inhibition of Delta-like 4 (Dll4)/Notch signaling has been shown to result in excessive, nonfunctional vessel proliferation and significant tumor growth suppression. However, safety concerns emerged with the identification of side effects resulting from chronic Dll4/Notch blockade. Alternatively, we explored the endothelial Dll4 overexpression using different mouse tumor models. We used a transgenic mouse model of endothelial-specific Dll4 overexpression, previously produced. Growth kinetics and vascular histopathology of several types of solid tumors was evaluated, namely Lewis Lung Carcinoma xenografts, chemically-induced skin papillomas and RIP1-Tag2 insulinomas. We found that increased Dll4/Notch signaling reduces tumor growth by reducing vascular endothelial growth factor (VEGF)-induced endothelial proliferation, tumor vessel density and overall tumor blood supply. In addition, Dll4 overexpression consistently improved tumor vascular maturation and functionality, as indicated by increased vessel calibers, enhanced mural cell recruitment and increased network perfusion. Importantly, the tumor vessel normalization is not more effective than restricted vessel proliferation, but was found to prevent metastasis formation and allow for increased delivery to the tumor of concomitant chemotherapy, improving its efficacy. By reducing endothelial sensitivity to VEGF, these results imply that Dll4/Notch stimulation in tumor microenvironment could be beneficial to solid cancer patient treatment by reducing primary tumor size, improving tumor drug delivery and reducing metastization. Endothelial specific Dll4 overexpression thus appears as a promising anti-angiogenic modality that might improve cancer control.

  1. Nrl-Cre transgenic mouse mediates loxP recombination in developing rod photoreceptors.

    Science.gov (United States)

    Brightman, Diana S; Razafsky, David; Potter, Chloe; Hodzic, Didier; Chen, Shiming

    2016-03-01

    The developing mouse retina is a tractable model for studying neurogenesis and differentiation. Although transgenic Cre mouse lines exist to mediate conditional genetic manipulations in developing mouse retinas, none of them act specifically in early developing rods. For conditional genetic manipulations of developing retinas, a Nrl-Cre mouse line in which the Nrl promoter drives expression of Cre in rod precursors was created. The results showed that Nrl-Cre expression was specific to the retina where it drives rod-specific recombination with a temporal pattern similar to endogenous Nrl expression during retinal development. This Nrl-Cre transgene does not negatively impact retinal structure and function. Taken together, the data suggested that the Nrl-Cre mouse line was a valuable tool to drive Cre-mediated recombination specifically in developing rods. © 2016 Wiley Periodicals, Inc.

  2. Assessment of ischemia in acute central retinal vein occlusion from inner retinal reflectivity on spectral domain optical coherence tomography

    Directory of Open Access Journals (Sweden)

    Browning DJ

    2016-12-01

    Full Text Available David J Browning, Omar S Punjabi, Chong Lee Department of Ophthalmology, Charlotte Eye, Ear, Nose and Throat Associates, P.A., Charlotte, NC, USA Purpose: To determine the relationship between different spectral domain optical coherence tomography (SD-OCT signs of retinal ischemia in acute central retinal vein occlusion (CRVO and whether they predict anterior segment neovascularization (ASNV.Design: Retrospective, observational study.Subjects: Thirty-nine consecutive patients with acute CRVO and 12 months of follow-up.Methods: We graded baseline SD-OCTs for increased reflectivity of the inner retina, loss of definition of inner retinal layers, presence of a prominent middle-limiting membrane (p-MLM sign, and presence of paracentral acute middle maculopathy (PAMM. Graders were masked with respect to all clinical information.Results: The intraclass correlation coefficients (ICCs of grading–regrading by graders 1 and 2 were 0.8104, 95% confidence interval (CI (0.6686, 0.8956, and 0.7986, 95% CI (0.6475, 0.8892, respectively. The intragrader coefficients of repeatability (COR for graders 1 and 2 were 0.94 and 0.92, respectively. The ICC of graders 1 compared with 2 was 0.8039, 95% CI (0.6544, 0.8916. The intergrader COR was 0.80. SD-OCT grades of baseline ischemia were not associated with baseline visual acuity (VA, central subfield mean thickness (CSMT, or relative afferent pupillary defect; 12-month VA, CSMT, change in VA, change in CSMT, number of antivascular endothelial growth factor injections or corticosteroid injections, or proportion of eyes developing ASNV. SD-OCT grades of ischemia did not correlate with the proportion of eyes having the p-MLM sign or PAMM. PAMM and p-MLM are milder signs of ischemia than increased reflectivity of the inner retinal layers. Eyes with PAMM can evolve, losing PAMM and gaining the p-MLM sign.Conclusion: Grading of ischemia from SD-OCT in acute CRVO was repeatable within graders and reproducible across

  3. Risk factor profile in retinal detachment

    Directory of Open Access Journals (Sweden)

    Azad Raj

    1988-01-01

    Full Text Available 150 cases of retinal detachment comprising 50 patients each of bilateral retinal detachment, unilateral retinal detachment without any retinal lesions in the fellow eve and unilateral retinal detachment with retinal lesions in the fellow eye were studied and the various associated risk factors were statistically analysed. The findings are discussed in relation to their aetiological and prognostic significance in the different types of retinal detachment. Based on these observations certain guidelines are offered which may be of value in decision making, in prophylactic detachment surgery. Tractional breaks in the superior temporal quadrant especially when symptomatic. mandate prophylactic treatment. Urgency is enhanced it′ the patient is aphakic. Associated myopia adds to the urgency. The higher incidence of initial right e′ e involvement in all groups suggests a vascular original possibly ischaemic.

  4. Characterization of vascular endothelial progenitor cells from chicken bone marrow

    Directory of Open Access Journals (Sweden)

    Bai Chunyu

    2012-05-01

    Full Text Available Abstract Background Endothelial progenitor cells (EPC are a type of stem cell used in the treatment of atherosclerosis, vascular injury and regeneration. At present, most of the EPCs studied are from human and mouse, whereas the study of poultry-derived EPCs has rarely been reported. In the present study, chicken bone marrow-derived EPCs were isolated and studied at the cellular level using immunofluorescence and RT-PCR. Results We found that the majority of chicken EPCs were spindle shaped. The growth-curves of chicken EPCs at passages (P 1, -5 and -9 were typically “S”-shaped. The viability of chicken EPCs, before and after cryopreservation was 92.2% and 81.1%, respectively. Thus, cryopreservation had no obvious effects on the viability of chicken EPCs. Dil-ac-LDL and FITC-UAE-1 uptake assays and immunofluorescent detection of the cell surface markers CD34, CD133, VEGFR-2 confirmed that the cells obtained in vitro were EPCs. Observation of endothelial-specific Weibel-Palade bodies using transmission electron microscopy further confirmed that the cells were of endothelial lineage. In addition, chicken EPCs differentiated into endothelial cells and smooth muscle cells upon induction with VEGF and PDGF-BB, respectively, suggesting that the chicken EPCs retained multipotency in vitro. Conclusions These results suggest that chicken EPCs not only have strong self-renewal capacity, but also the potential to differentiate into endothelial and smooth muscle cells. This research provides theoretical basis and experimental evidence for potential therapeutic application of endothelial progenitor cells in the treatment of atherosclerosis, vascular injury and diabetic complications.

  5. Retinitis Pigmentosa and Education Issues

    Science.gov (United States)

    Brown, Thomas J.

    2005-01-01

    Retinitis Pigmentosa includes a number of inherited diseases which usually result in blindness. The disease is progressive in nature and begins with the deterioration of cells in the eye responsible for peripheral vision. As the condition worsens there is a gradual loss of peripheral vision and night blindness. Proper educational planning requires…

  6. [Surgical managment of retinal detachment].

    Science.gov (United States)

    Haritoglou, C; Wolf, A

    2015-05-01

    The detachment of the neurosensory retina from the underlying retinal pigment epithelium can be related to breaks of the retina allowing vitreous fluid to gain access to the subretinal space, to exudative changes of the choroid such as tumours or inflammatory diseases or to excessive tractional forces exerted by interactions of the collagenous vitreous and the retina. Tractional retinal detachment is usually treated by vitrectomy and exudative detachment can be addressed by treatment of the underlying condition in many cases. In rhegmatogenous retinal detachment two different surgical procedures, vitrectomy and scleral buckling, can be applied for functional and anatomic rehabilitation of our patients. The choice of the surgical procedure is not really standardised and often depends on the experience of the surgeon and other more ocular factors including lens status, the number of retinal breaks, the extent of the detachment and the amount of preexisting PVR. Using both techniques, anatomic success rates of over 90 % can be achieved. Especially in young phakic patients scleral buckling offers the true advantage to prevent the progression of cataract formation requiring cataract extraction and intraocular lens implantation. Therefore, scleral buckling should be considered in selected cases as an alternative surgical option in spite of the very important technical refinements in modern vitrectomy techniques. Georg Thieme Verlag KG Stuttgart · New York.

  7. Retinal imaging and image analysis

    NARCIS (Netherlands)

    Abramoff, M.D.; Garvin, Mona K.; Sonka, Milan

    2010-01-01

    Many important eye diseases as well as systemic diseases manifest themselves in the retina. While a number of other anatomical structures contribute to the process of vision, this review focuses on retinal imaging and image analysis. Following a brief overview of the most prevalent causes of

  8. Intrinsic bursting of AII amacrine cells underlies oscillations in the rd1 mouse retina.

    Science.gov (United States)

    Choi, Hannah; Zhang, Lei; Cembrowski, Mark S; Sabottke, Carl F; Markowitz, Alexander L; Butts, Daniel A; Kath, William L; Singer, Joshua H; Riecke, Hermann

    2014-09-15

    In many forms of retinal degeneration, photoreceptors die but inner retinal circuits remain intact. In the rd1 mouse, an established model for blinding retinal diseases, spontaneous activity in the coupled network of AII amacrine and ON cone bipolar cells leads to rhythmic bursting of ganglion cells. Since such activity could impair retinal and/or cortical responses to restored photoreceptor function, understanding its nature is important for developing treatments of retinal pathologies. Here we analyzed a compartmental model of the wild-type mouse AII amacrine cell to predict that the cell's intrinsic membrane properties, specifically, interacting fast Na and slow, M-type K conductances, would allow its membrane potential to oscillate when light-evoked excitatory synaptic inputs were withdrawn following photoreceptor degeneration. We tested and confirmed this hypothesis experimentally by recording from AIIs in a slice preparation of rd1 retina. Additionally, recordings from ganglion cells in a whole mount preparation of rd1 retina demonstrated that activity in AIIs was propagated unchanged to elicit bursts of action potentials in ganglion cells. We conclude that oscillations are not an emergent property of a degenerated retinal network. Rather, they arise largely from the intrinsic properties of a single retinal interneuron, the AII amacrine cell. Copyright © 2014 the American Physiological Society.

  9. A route for direct retinal input to the preoptic hypothalamus: dendritic projections into the optic chiasm.

    Science.gov (United States)

    Silver, J; Brand, S

    1979-07-01

    With the use of Golgi, horseradish peroxidase, and electron microscopic techniques, neurons within a broad region of the preoptic hypothalamus of the mouse were shown to have dendrites that projected well into the depths of the optic chiasm. Further experimental and ultrastructural investigation demonstrated synapses between these dendrites and retinal axonal boutons within the chiasm. All synapses located in the chiasm were classified as Gray's type I. The possible function of these dendritic projections is discussed.

  10. The Impact of Macronutrients on Retinal Microvasculature among Singapore Pregnant Women during the Mid-Late Gestation.

    Science.gov (United States)

    Li, Ling-Jun; Ong, Peng Guan; Colega, Marjorelee T; Han, Chad Yixian; Chen, Ling Wei; Man Eyn Kidd, Ryan; Lamoureux, Ecosse; Gluckman, Peter; Kwek, Kenneth; Chong, Yap Seng; Saw, Seang Mei; Godfrey, Keith M; Wong, Tien Yin; Chong Foong-Fong, Mary

    2016-01-01

    Imbalanced macronutrient intakes can induce impairment of endothelial and vascular function, and further lead to metabolic and cardiovascular disease. However, little is known about the influence of such diets on endothelial and vascular dysfunction in pregnant women, even though high-fat diet is a known risk for pregnancy complications such as gestational diabetes and pre-eclampsia. We aimed to assess the association between maternal macronutrient intakes (protein, fat and carbohydrates), dietary quality and retinal microvascular changes in a multi-ethnic Asian mother-offspring cohort. Pregnant women (n = 614) with singleton pregnancies were recruited during their first trimester from June 2009 to Sep 2010. Maternal diet quality and macronutrient intakes, expressed as a percentage of total energy during pregnancy, were ascertained using 24 hr recalls and 3 d food diaries at 26-28 weeks gestation. Retinal examination was completed at the same clinic visit. Dietary quality was assessed and scored using the Health Eating Index in Asian Pregnant women (HEI-AP), while macronutrients intakes ware expressed as percentages of total energy and further log transformed for analysis. Associations were examined cross-sectionally by substitution models with the use of multiple linear regression. In adjusted model, each 20 points decrease in HEI-AP score was associated with a significant increase of 1.70 μm (p<0.05) in retinal venular calibre. Each 0.1 log increase in percentage of total fat intake was associated with a significant increment of 1.84 μm (p<0.05) in retinal venular caliber. Additionally, each 0.1 log increase in percentage of mono-unsaturated fat intake was associated with an increment of 1.84 μm (p<0.01) in retinal venular caliber. In this cross-sectional study, we found that women with higher fat and lower protein intakes, and lower diet quality tended to have wider retinal venular caliber, which is suggestive of suboptimal microvasculature.

  11. User-guided segmentation for volumetric retinal optical coherence tomography images

    Science.gov (United States)

    Yin, Xin; Chao, Jennifer R.; Wang, Ruikang K.

    2014-01-01

    Abstract. Despite the existence of automatic segmentation techniques, trained graders still rely on manual segmentation to provide retinal layers and features from clinical optical coherence tomography (OCT) images for accurate measurements. To bridge the gap between this time-consuming need of manual segmentation and currently available automatic segmentation techniques, this paper proposes a user-guided segmentation method to perform the segmentation of retinal layers and features in OCT images. With this method, by interactively navigating three-dimensional (3-D) OCT images, the user first manually defines user-defined (or sketched) lines at regions where the retinal layers appear very irregular for which the automatic segmentation method often fails to provide satisfactory results. The algorithm is then guided by these sketched lines to trace the entire 3-D retinal layer and anatomical features by the use of novel layer and edge detectors that are based on robust likelihood estimation. The layer and edge boundaries are finally obtained to achieve segmentation. Segmentation of retinal layers in mouse and human OCT images demonstrates the reliability and efficiency of the proposed user-guided segmentation method. PMID:25147962

  12. A small population of liver endothelial cells undergoes endothelial-to-mesenchymal transition in response to chronic liver injury.

    Science.gov (United States)

    Ribera, Jordi; Pauta, Montse; Melgar-Lesmes, Pedro; Córdoba, Bernat; Bosch, Anna; Calvo, Maria; Rodrigo-Torres, Daniel; Sancho-Bru, Pau; Mira, Aurea; Jiménez, Wladimiro; Morales-Ruiz, Manuel

    2017-11-01

    Rising evidence points to endothelial-to-mesenchymal transition (EndMT) as a significant source of the mesenchymal cell population in fibrotic diseases. In this context, we hypothesized that liver endothelial cells undergo EndMT during fibrosis progression. Cirrhosis in mice was induced by CCl 4 A transgenic mouse expressing a red fluorescent protein reporter under the control of Tie2 promoter (Tie2-tdTomato) was used to trace the acquisition of EndMT. Sinusoidal vascular connectivity was evaluated by intravital microscopy and high-resolution three-dimensional confocal microscopy. A modest but significant fraction of liver endothelial cells from both cirrhotic patients and CCl 4 -treated Tie2-tdTomato mice acquired an EndMT phenotype characterized by the coexpression of CD31 and α-smooth muscle actin, compared with noncirrhotic livers. Bone morphogenetic protein-7 (BMP-7) inhibited the acquisition of EndMT induced by transforming growth factor-β1 (TGF-β1) treatment in cultured primary mouse liver endothelial cells from control mice. EndMT was also reduced significantly in vivo in cirrhotic Tie2-tdTomato mice treated intraperitoneally with BMP-7 compared with untreated mice (1.9 ± 0.2 vs. 3.8 ± 0.3%, respectively; P livers correlated with a significant decrease in liver fibrosis ( P livers in both animal models and patients. BMP-7 treatment decreases the occurrence of the EndMT phenotype and has a positive impact on the severity of disease by reducing fibrosis and sinusoidal vascular disorganization. NEW & NOTEWORTHY A subpopulation of liver endothelial cells from cirrhotic patients and mice with liver fibrosis undergoes endothelial-to-mesenchymal transition. Liver endothelial cells from healthy mice could transition into a mesenchymal phenotype in culture in response to TGF-β1 treatment. Fibrotic livers treated chronically with BMP-7 showed lower EndMT acquisition, reduced fibrosis, and improved vascular organization. Copyright © 2017 the American

  13. Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

    Science.gov (United States)

    Wang, Jin-Da; An, Ying; Zhang, Jing-Shang; Wan, Xiu-Hua; Zhang, Wei; Lanza, Robert; Lu, Shi-Jiang; Jonas, Jost B; Xu, Liang

    2017-09-01

    To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. In the OIR model, the study group versus control groups showed a significantly (p < 0.001) smaller retinal avascular area [5.1 ± 2.7%;n = 18 animals versus 12.2 ± 2.8% (PBS group; n = 10 animals) and versus 11.8 ± 3.7% (EC group; n = 8 animals)] and less retinal neovascularization [6.3 ± 2.5%;n = 18 versus 15.2 ± 6.3% (n = 10; PBS group) and versus 15.8 ± 3.3% (n = 8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p = 0.001) better blood-retina barrier function as measured at 2 days after the intravitreal injections [study group: 20.2 ± 12.8 μl/(g × hr); n = 6; versus EC control group: 52.9 ± 9.9 μl/(g × hr; n = 6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7 days after baseline. By integrating into

  14. Screening for retinitis in children with probable systemic ...

    African Journals Online (AJOL)

    CMV retinitis may be prevented by timely diagnosis and treatment. This study aimed to .... retinitis are: 'a fulminant picture of retinal vasculitis and vascular sheathing with areas of yellow-white, full thickness, retinal necrosis producing retinal oedema associated ... and intravenous foscarnet as alternatives.[4] Although CMV- ...

  15. Retinitis pigmentosa-associated cystoid macular oedema: pathogenesis and avenues of intervention

    Science.gov (United States)

    Strong, S; Liew, G; Michaelides, M

    2017-01-01

    Hereditary retinal diseases are now the leading cause of blindness certification in the working age population (age 16–64 years) in England and Wales, of which retinitis pigmentosa (RP) is the most common disorder. RP may be complicated by cystoid macular oedema (CMO), causing a reduction of central vision. The underlying pathogenesis of RP-associated CMO (RP-CMO) remains uncertain, however, several mechanisms have been proposed, including: (1) breakdown of the blood-retinal barrier, (2) failure (or dysfunction) of the pumping mechanism in the retinal pigment epithelial, (3) Müller cell oedema and dysfunction, (4) antiretinal antibodies and (5) vitreous traction. There are limited data on efficacy of treatments for RP-CMO. Treatments attempted to date include oral and topical carbonic anhydrase inhibitors, oral, topical, intravitreal and periocular steroids, topical non-steroidal anti-inflammatory medications, photocoagulation, vitrectomy with internal limiting membrane peel, oral lutein and intravitreal antivascular endothelial growth factor injections. This review summarises the evidence supporting these treatment modalities. Successful management of RP-CMO should aim to improve both quality and quantity of vision in the short term and may also slow central vision loss over time. PMID:27913439

  16. Progress of stem/progenitor cell-based therapy for retinal degeneration.

    Science.gov (United States)

    Tang, Zhimin; Zhang, Yi; Wang, Yuyao; Zhang, Dandan; Shen, Bingqiao; Luo, Min; Gu, Ping

    2017-05-10

    Retinal degeneration (RD), such as age-related macular degeneration (AMD) and retinitis pigmentosa, is one of the leading causes of blindness. Presently, no satisfactory therapeutic options are available for these diseases principally because the retina and retinal pigmented epithelium (RPE) do not regenerate, although wet AMD can be prevented from further progression by anti-vascular endothelial growth factor therapy. Nevertheless, stem/progenitor cell approaches exhibit enormous potential for RD treatment using strategies mainly aimed at the rescue and replacement of photoreceptors and RPE. The sources of stem/progenitor cells are classified into two broad categories in this review, which are (1) ocular-derived progenitor cells, such as retinal progenitor cells, and (2) non-ocular-derived stem cells, including embryonic stem cells, induced pluripotent stem cells, and mesenchymal stromal cells. Here, we discuss in detail the progress in the study of four predominant stem/progenitor cell types used in animal models of RD. A short overview of clinical trials involving the stem/progenitor cells is also presented. Currently, stem/progenitor cell therapies for RD still have some drawbacks such as inhibited proliferation and/or differentiation in vitro (with the exception of the RPE) and limited long-term survival and function of grafts in vivo. Despite these challenges, stem/progenitor cells represent the most promising strategy for RD treatment in the near future.

  17. Ischemic preconditioning enhances integrity of coronary endothelial tight junctions

    International Nuclear Information System (INIS)

    Li, Zhao; Jin, Zhu-Qiu

    2012-01-01

    Highlights: ► Cardiac tight junctions are present between coronary endothelial cells. ► Ischemic preconditioning preserves the structural and functional integrity of tight junctions. ► Myocardial edema is prevented in hearts subjected to ischemic preconditioning. ► Ischemic preconditioning enhances translocation of ZO-2 from cytosol to cytoskeleton. -- Abstract: Ischemic preconditioning (IPC) is one of the most effective procedures known to protect hearts against ischemia/reperfusion (IR) injury. Tight junction (TJ) barriers occur between coronary endothelial cells. TJs provide barrier function to maintain the homeostasis of the inner environment of tissues. However, the effect of IPC on the structure and function of cardiac TJs remains unknown. We tested the hypothesis that myocardial IR injury ruptures the structure of TJs and impairs endothelial permeability whereas IPC preserves the structural and functional integrity of TJs in the blood–heart barrier. Langendorff hearts from C57BL/6J mice were prepared and perfused with Krebs–Henseleit buffer. Cardiac function, creatine kinase release, and myocardial edema were measured. Cardiac TJ function was evaluated by measuring Evans blue-conjugated albumin (EBA) content in the extravascular compartment of hearts. Expression and translocation of zonula occludens (ZO)-2 in IR and IPC hearts were detected with Western blot. A subset of hearts was processed for the observation of ultra-structure of cardiac TJs with transmission electron microscopy. There were clear TJs between coronary endothelial cells of mouse hearts. IR caused the collapse of TJs whereas IPC sustained the structure of TJs. IR increased extravascular EBA content in the heart and myocardial edema but decreased the expression of ZO-2 in the cytoskeleton. IPC maintained the structure of TJs. Cardiac EBA content and edema were reduced in IPC hearts. IPC enhanced the translocation of ZO-2 from cytosol to cytoskeleton. In conclusion, TJs occur in

  18. Development of mPMab-1, a Mouse-Rat Chimeric Antibody Against Mouse Podoplanin.

    Science.gov (United States)

    Yamada, Shinji; Kaneko, Mika K; Nakamura, Takuro; Ichii, Osamu; Konnai, Satoru; Kato, Yukinari

    2017-04-01

    Podoplanin (PDPN), the ligand of C-type lectin-like receptor-2, is used as a lymphatic endothelial marker. We previously established clone PMab-1 of rat IgG 2a as a specific monoclonal antibody (mAb) against mouse PDPN. PMab-1 is also very sensitive in immunohistochemical analysis; however, rat mAbs seem to be unfavorable for pathologists because anti-mouse IgG and anti-rabbit IgG are usually used as secondary antibodies in commercially available kits for immunohistochemical analysis. In this study, we develop a mouse-rat chimeric antibody, mPMab-1 of mouse IgG 2a , which was derived from rat PMab-1 mAb. Immunohistochemical analysis shows that mPMab-1 detects podocytes of the kidney, lymphatic endothelial cells of the colon, and type I alveolar cells of the lung. Importantly, mPMab-1 is more sensitive than PMab-1. This conversion strategy from rat mAb to mouse mAb could be applicable to other mAbs.

  19. Effects of the neuroprotective drugs somatostatin and brimonidine on retinal cell models of diabetic retinopathy.

    Science.gov (United States)

    Beltramo, Elena; Lopatina, Tatiana; Mazzeo, Aurora; Arroba, Ana I; Valverde, Angela M; Hernández, Cristina; Simó, Rafael; Porta, Massimo

    2016-12-01

    Diabetic retinopathy is considered a microvascular disease, but recent evidence has underlined early involvement of the neuroretina with interactions between microvascular and neural alterations. Topical administration of somatostatin (SST), a neuroprotective molecule with antiangiogenic properties, prevents diabetes-induced retinal neurodegeneration in animals. The α 2 -adrenergic receptor agonist brimonidine (BRM) decreases vitreoretinal vascular endothelial growth factor and inhibits blood-retinal barrier breakdown in diabetic rats. However, SST and BRM effects on microvascular cells have not yet been studied. We investigated the behaviour of these drugs on the crosstalk between microvasculature and neuroretina. Expression of SST receptors 1-5 in human retinal pericytes (HRP) was checked. We subsequently evaluated the effects of diabetic-like conditions (high glucose and/or hypoxia) with/without SST/BRM on HRP survival. Endothelial cells (EC) and photoreceptors were maintained in the above conditions and their conditioned media (CM) used to culture HRP. Vice versa, HRP-CM was used on EC and photoreceptors. Survival parameters were assessed. HRP express the SST receptor 1 (SSTR1). Glucose fluctuations mimicking those occurring in diabetic subjects are more damaging for pericytes and photoreceptors than stable high glucose and hypoxic conditions. SST/BRM added to HRP in diabetic-like conditions decrease EC apoptosis. However, neither SST nor BRM changed the response of pericytes and neuroretina-vascular crosstalk under diabetic-like conditions. Retinal pericytes express SSTR1, indicating that they can be a target for SST. Exposure to SST/BRM had no adverse effects, direct or mediated by the neuroretina, suggesting that these molecules could be safely evaluated for the treatment of ocular diseases.

  20. Levels of serum vascular endothelial growth factor in type 2 diabetics with retinopathy

    International Nuclear Information System (INIS)

    Parveen, N.; Rahman, S.; Khan, Q.

    2012-01-01

    Background: Ischemic retina in diabetic patients releases a number of chemical substances including vascular endothelial growth factor which leads to retinal vascular proliferation and blindness following rupture and bleeding of vessels. Strategies to control this action can considerably halt this process. Objectives: To determine the relationship of various stages of diabetic retinopathy with the levels vascular endothelial growth factor in the serum of type 2 diabetic patients. Study type, settings and duration: This cross sectional analytical study was done over one year (2010-2011) in three major public sector hospitals of Peshawar. Patients and Methods: Adult patients of either gender having type 2 diabetes mellitus with proliferative or non proliferative retinopathy and those without retinopathy were selected for the study. Retinopathy was diagnosed on fundoscopy. Non-diabetic patients without retinopathy were selected as controls. Serum levels of vascular endothelial growth factor were done in patients and controls using ELISA. Results: Serum vascular endothelial growth factor levels were significantly higher in all cases having retinopathy as compared to controls. These levels progressively increased with the grades of retinopathy. Levels were higher in females. Conclusions: Levels of vascular endothelial growth factor are raised in diabetic retinopathy and rising levels can alert the clinician in worsening of retinopathy so that preventive and therapeutic measures can be taken promptly. Policy message: Further larger scale studies are recommended on national level to pave way for the establishment of appropriate management paradigms for diabetic retinopathy through anti-VEGF treatment. (author)

  1. Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

    Science.gov (United States)

    De Silva, Samantha R; Charbel Issa, Peter; Singh, Mandeep S; Lipinski, Daniel M; Barnea-Cramer, Alona O; Walker, Nathan J; Barnard, Alun R; Hankins, Mark W; MacLaren, Robert E

    2016-11-01

    Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4 -/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4 -/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

  2. Retinal Astrocytes and GABAergic Wide-Field Amacrine Cells Express PDGFRα: Connection to Retinal Ganglion Cell Neuroprotection by PDGF-AA.

    Science.gov (United States)

    Takahama, Shokichi; Adetunji, Modupe O; Zhao, Tantai; Chen, Shan; Li, Wei; Tomarev, Stanislav I

    2017-09-01

    Our previous experiments demonstrated that intravitreal injection of platelet-derived growth factor-AA (PDGF-AA) provides retinal ganglion cell (RGC) neuroprotection in a rodent model of glaucoma. Here we used PDGFRα-enhanced green fluorescent protein (EGFP) mice to identify retinal cells that may be essential for RGC protection by PDGF-AA. PDGFRα-EGFP mice expressing nuclear-targeted EGFP under the control of the PDGFRα promoter were used. Localization of PDGFRα in the neural retina was investigated by confocal imaging of EGFP fluorescence and immunofluorescent labeling with a panel of antibodies recognizing different retinal cell types. Primary cultures of mouse RGCs were produced by immunopanning. Neurobiotin injection of amacrine cells in a flat-mounted retina was used for the identification of EGFP-positive amacrine cells in the inner nuclear layer. In the mouse neural retina, PDGFRα was preferentially localized in the ganglion cell and inner nuclear layers. Immunostaining of the retina demonstrated that astrocytes in the ganglion cell layer and a subpopulation of amacrine cells in the inner nuclear layer express PDGFRα, whereas RGCs (in vivo or in vitro) did not. PDGFRα-positive amacrine cells are likely to be Type 45 gamma-aminobutyric acidergic (GABAergic) wide-field amacrine cells. These data indicate that the neuroprotective effect of PDGF-AA in a rodent model of glaucoma could be mediated by astrocytes and/or a subpopulation of amacrine cells. We suggest that after intravitreal injection of PDGF-AA, these cells secrete factors protecting RGCs.

  3. Prevalence of generalized retinal dystrophy in Denmark

    DEFF Research Database (Denmark)

    Bertelsen, Mette; Jensen, Hanne; Bregnhøj, Jesper F

    2014-01-01

    of this study was to examine the prevalence and diagnostic spectrum of generalized retinal dystrophy in the Danish population. METHODS: A population-based cross-sectional study with data from the Danish Retinitis Pigmentosa Registry that comprises all patients in Denmark with generalized retinal......PURPOSE: Generalized retinal dystrophy is a frequent cause of visual impairment and blindness in younger individuals and a subject of new clinical intervention trials. Nonetheless, there are few nation-wide population-based epidemiological data of generalized retinal dystrophy. The purpose...... and chorioretinal dystrophies from the 19th century to the present. Among 3076 registered cases, the primary diagnosis of generalized retinal dystrophy was assessed by chart review, including fundus photographs and electroretinograms. Demographic data on the Danish population were retrieved from Statistics Denmark...

  4. Automatic Vessel Segmentation on Retinal Images

    Institute of Scientific and Technical Information of China (English)

    Chun-Yuan Yu; Chia-Jen Chang; Yen-Ju Yao; Shyr-Shen Yu

    2014-01-01

    Several features of retinal vessels can be used to monitor the progression of diseases. Changes in vascular structures, for example, vessel caliber, branching angle, and tortuosity, are portents of many diseases such as diabetic retinopathy and arterial hyper-tension. This paper proposes an automatic retinal vessel segmentation method based on morphological closing and multi-scale line detection. First, an illumination correction is performed on the green band retinal image. Next, the morphological closing and subtraction processing are applied to obtain the crude retinal vessel image. Then, the multi-scale line detection is used to fine the vessel image. Finally, the binary vasculature is extracted by the Otsu algorithm. In this paper, for improving the drawbacks of multi-scale line detection, only the line detectors at 4 scales are used. The experimental results show that the accuracy is 0.939 for DRIVE (digital retinal images for vessel extraction) retinal database, which is much better than other methods.

  5. Retinal Image Preprocessing: Background and Noise Segmentation

    Directory of Open Access Journals (Sweden)

    Usman Akram

    2012-09-01

    Full Text Available Retinal images are used for the automated screening and diagnosis of diabetic retinopathy. The retinal image quality must be improved for the detection of features and abnormalities and for this purpose preprocessing of retinal images is vital. In this paper, we present a novel automated approach for preprocessing of colored retinal images. The proposed technique improves the quality of input retinal image by separating the background and noisy area from the overall image. It contains coarse segmentation and fine segmentation. Standard retinal images databases Diaretdb0, Diaretdb1, DRIVE and STARE are used to test the validation of our preprocessing technique. The experimental results show the validity of proposed preprocessing technique.

  6. [Peripheral retinal degenerations--treatment recommendations].

    Science.gov (United States)

    Joussen, A M; Kirchhof, B

    2004-10-01

    This report reviews the clinical appearance of degenerative diseases of the peripheral retina in relationship to the risk of developing a rhegmatogenous retinal detachment. We present recommendations for preventive treatment in eyes at increased risk of developing retinal detachment. Retinal degenerations are common lesions involving the peripheral retina but most of them are clinically insignificant. Lattice degeneration, degenerative retinoschisis, cystic retinal tufts, and very rarely zonular traction tufts can result in rhegmatogenous retinal detachment. Therefore, these lesions have been considered for prophylactic treatment; however, adequate studies have not been performed to date. Most of the peripheral retinal degenerations may not require treatment except in rare, high-risk situations. According to current knowledge there is no higher incidence of secondary pucker or other side effects after laser coagulation. Therefore, generous laser indication is recommended if risk factors apply.

  7. [Prophylactic treatment of retinal detachment].

    Science.gov (United States)

    Binder, S; Riss, B

    1981-08-01

    The indications for and results of prophylactic treatment of retinal detachment during a period of five years are reported and compared with the results in the literature. Half of the cases (3 out of 6 eyes) which developed a retinal detachment had been horse-shoe tears combined with a vitreous hemorrhage. For this reason a small buckle operation is recommended in these cases, to prevent further traction. Lattice degeneration should rather be observed than treated, except in special cases: This includes eyes where the fellow eye had a detachment from a lattice degeneration, cases in which one eye is blind from an uncured detachment or has no useful visual acuity, and eyes whose fellow eye has giant tears. In aphakic eyes treatment of lattice degeneration is recommended, because the incidence of detachment from these areas is high, especially in young aphakic cases. In one aphakic eye which had been photocoagulated several times the formation of a preretinal membrane was observed.

  8. Apolipoprotein A-1 mimetic peptide 4F promotes endothelial repairing and compromises reendothelialization impaired by oxidized HDL through SR-B1

    Directory of Open Access Journals (Sweden)

    Dan He

    2018-05-01

    Full Text Available Disruption of endothelial monolayer integrity is the primary instigating factor for many cardiovascular diseases. High density lipoprotein (HDL oxidized by heme enzyme myeloperoxidase (MPO is dysfunctional in promoting endothelial repair. Apolipoprotein A-1 mimetic 4F with its pleiotropic benefits has been proven effective in many in vivo models. In this study we investigated whether 4F promotes endothelial repair and restores the impaired function of oxidized HDL (Cl/NO2-HDL in promoting re-endothelialization. We demonstrate that 4F and Cl/NO2-HDL act on scavenger receptor type I (SR-B1 using human aorta endothelial cells (HAEC and SR-B1 (-/- mouse aortic endothelial cells. Wound healing, transwell migration, lamellipodia formation and single cell migration assay experiments show that 4F treatment is associated with a recovery of endothelial cell migration and associated with significantly increased endothelial nitric oxide synthase (eNOS activity, Akt phosphorylation and SR-B1 expression. 4F increases NO generation and diminishes oxidative stress. In vivo, 4F can stimulate cell proliferation and re-endothelialization in the carotid artery after treatment with Cl/NO2-HDL in a carotid artery electric injury model but fails to do so in SR-B1(-/- mice. These findings demonstrate that 4F promotes endothelial cell migration and has a potential therapeutic benefit against early endothelial injury in cardiovascular diseases.

  9. Inherited Retinal Degenerative Clinical Trial Network. Addendum

    Science.gov (United States)

    2013-10-01

    inherited orphan retinal degenerative diseases and dry age-related macular degeneration (AMD) through the conduct of clinical trials and other...design and conduct of effective and efficient clinical trials for inherited orphan retinal degenerative diseases and dry AMD; • Limited number and...linica l trial in the NEER network for autosomal dominant retinitis pigmentosa, and the ProgSTAR studies for Stargardt disease ) . As new interventions b

  10. Frequency of lattice degeneration and retinal breaks in the fellow eye in retinal detachment.

    Science.gov (United States)

    Lorentzen, S E

    1988-04-01

    The fellow eye of 100 consecutively admitted cases of retinal detachment was studied with three-mirror examination for the presence of lattice degeneration and retinal breaks. Lattice degeneration was found in 18% and retinal breaks in 20% of fellow eyes.

  11. Papillary endothelial hyperplasia in angiokeratoma.

    Science.gov (United States)

    Mehta, Anurag; Sayal, Satish Kumar; Raman, Deep Kumar; Sood, Aradhana

    2003-01-01

    Papillary endothelial hyperplasia (Masson's tumour) is a reactive proliferation of endothelium producing papillary structures with fibrovascular cores. Dilatation, stasis and accompanying inflammation have been incriminated as the inciting events, evident by the presence of this lesion in haemorrhoids, urethral caruncles and laryngeal polyps. We present here a case of papillary endothelial hyperplasia in angiokeratoma hitherto undescribed despite sharing common etiopathogenetic features of dilatation and stasis with other aforementioned lesions.

  12. Retinal Cell Degeneration in Animal Models

    Directory of Open Access Journals (Sweden)

    Masayuki Niwa

    2016-01-01

    Full Text Available The aim of this review is to provide an overview of various retinal cell degeneration models in animal induced by chemicals (N-methyl-d-aspartate- and CoCl2-induced, autoimmune (experimental autoimmune encephalomyelitis, mechanical stress (optic nerve crush-induced, light-induced and ischemia (transient retinal ischemia-induced. The target regions, pathology and proposed mechanism of each model are described in a comparative fashion. Animal models of retinal cell degeneration provide insight into the underlying mechanisms of the disease, and will facilitate the development of novel effective therapeutic drugs to treat retinal cell damage.

  13. Retinal Macroglial Responses in Health and Disease

    Directory of Open Access Journals (Sweden)

    Rosa de Hoz

    2016-01-01

    Full Text Available Due to their permanent and close proximity to neurons, glial cells perform essential tasks for the normal physiology of the retina. Astrocytes and Müller cells (retinal macroglia provide physical support to neurons and supplement them with several metabolites and growth factors. Macroglia are involved in maintaining the homeostasis of extracellular ions and neurotransmitters, are essential for information processing in neural circuits, participate in retinal glucose metabolism and in removing metabolic waste products, regulate local blood flow, induce the blood-retinal barrier (BRB, play fundamental roles in local immune response, and protect neurons from oxidative damage. In response to polyetiological insults, glia cells react with a process called reactive gliosis, seeking to maintain retinal homeostasis. When malfunctioning, macroglial cells can become primary pathogenic elements. A reactive gliosis has been described in different retinal pathologies, including age-related macular degeneration (AMD, diabetes, glaucoma, retinal detachment, or retinitis pigmentosa. A better understanding of the dual, neuroprotective, or cytotoxic effect of macroglial involvement in retinal pathologies would help in treating the physiopathology of these diseases. The extensive participation of the macroglia in retinal diseases points to these cells as innovative targets for new drug therapies.

  14. Coincidence of retinitis pigmentosa and pseudoexfoliative glaucoma

    Directory of Open Access Journals (Sweden)

    Božić Marija

    2017-01-01

    Full Text Available Introduction. This is an observational case report presenting retinitis pigmentosa associated with pseudoexfoliative glaucoma. Case outline. A 69-year-old man presented with retinitis pigmentosa. On examination, pseudoexfoliative material was detected on anterior segment structures, and intraocular pressure was 26 mmHg in the right and 24 mmHg in the left eye. The patient was commenced on topical antiglaucomatous therapy (timolol + dorzolamide twice daily, latanoprost once in the evening to both eyes. Conclusion. To the best of our knowledge, this is the first reported case of retinitis pigmentosa associated with pseudoexfoliative glaucoma. Although rare, retinitis pigmentosa and glaucoma can occur in the same eye.

  15. Retinal phlebitis associated with autoimmune hemolytic anemia.

    Science.gov (United States)

    Chew, Fiona L M; Tajunisah, Iqbal

    2009-01-01

    To describe a case of retinal phlebitis associated with autoimmune hemolytic anemia. Observational case report. A 44-year-old Indian man diagnosed with autoimmune hemolytic anemia presented with a 1-week history of blurred vision in both eyes. Fundus biomicroscopy revealed bilateral peripheral retinal venous sheathing and cellophane maculopathy. Fundus fluorescent angiogram showed bilateral late leakage from the peripheral venous arcades and submacular fluid accumulation. The retinal phlebitis resolved following a blood transfusion and administration of systemic steroids. Retinopathy associated with autoimmune hemolytic anemia is not well known. This is thought to be the first documentation of retinal phlebitis occurring in this condition.

  16. Tractional retinal detachment in Usher syndrome type II.

    Science.gov (United States)

    Rani, Alka; Pal, Nikhil; Azad, Raj Vardhan; Sharma, Yog Raj; Chandra, Parijat; Vikram Singh, Deependra

    2005-08-01

    Retinal detachment is a rare complication in patients with retinitis pigmentosa. A case is reported of tractional retinal detachment in a patient with retinitis pigmentosa and sensorineural hearing loss, which was diagnosed as Usher syndrome type II. Because of the poor visual prognosis, the patient refused surgery in that eye. Tractional retinal detachment should be added to the differential diagnoses of visual loss in patients with retinitis pigmentosa.

  17. Stem Cell-Based Therapeutic Applications in Retinal Degenerative Diseases.

    OpenAIRE

    Huang Yiming; Enzmann Volker; Ildstad Suzanne T

    2011-01-01

    Retinal degenerative diseases that target photoreceptors or the adjacent retinal pigment epithelium (RPE) affect millions of people worldwide. Retinal degeneration (RD) is found in many different forms of retinal diseases including retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy, cataracts, and glaucoma. Effective treatment for retinal degeneration has been widely investigated. Gene-replacement therapy has been shown to improve visual function in inheri...

  18. Omega-3 polyunsaturated fatty acids preserve retinal function in type 2 diabetic mice.

    Science.gov (United States)

    Sapieha, P; Chen, J; Stahl, A; Seaward, M R; Favazza, T L; Juan, A M; Hatton, C J; Joyal, J-S; Krah, N M; Dennison, R J; Tang, J; Kern, T S; Akula, J D; Smith, L E H

    2012-07-23

    Diabetic retinopathy (DR) is associated with hyperglycemia-driven microvascular pathology and neuronal compromise in the retina. However, DR is also linked to dyslipidemia. As omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) are protective in proliferative retinopathy, we investigated the capacity of ω-3PUFAs to preserve retinal function in a mouse model of type 2 diabetes mellitus (T2DM). Male leptin-receptor-deficient (db/db) mice were maintained for 22 weeks (4 weeks-26 weeks of life) on calorically and compositionally matched diets, except for 2% enrichment in either ω-3 or ω-6PUFAs. Visual function was assessed at 9, 14 and 26 weeks by electroretinography. Retinal capillary and neuronal integrity, as well as glucose challenge responses, were assessed on each diet. The ω-3PUFA diet significantly preserved retinal function in the mouse model of T2DM to levels similar to those observed in nondiabetic control mice on normal chow. Conversely, retinal function gradually deteriorated in db/db mice on a ω-6PUFA-rich diet. There was also an enhanced ability of ω-3PUFA-fed mice to respond to glucose challenge. The protection of visual function appeared to be independent of cytoprotective or anti-inflammatory effects of ω-3PUFAs. This study identifies beneficial effects of dietary ω-3PUFAs on visual function in T2DM. The data are consistent with dyslipidemia negatively impacting retinal function. As ω-3PUFA lipid dietary interventions are readily available, safe and inexpensive, increasing ω-3PUFA intake in diabetic patients may slow the progression of vision loss in T2DM.

  19. Endothelial Cells Control Pancreatic Cell Fate at Defined Stages through EGFL7 Signaling

    Directory of Open Access Journals (Sweden)

    Der-I Kao

    2015-02-01

    Full Text Available Although endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.

  20. HIF-1alpha and HIF-2alpha are differentially activated in distinct cell populations in retinal ischaemia.

    Directory of Open Access Journals (Sweden)

    Freya M Mowat

    2010-06-01

    Full Text Available Hypoxia plays a key role in ischaemic and neovascular disorders of the retina. Cellular responses to oxygen are mediated by hypoxia-inducible transcription factors (HIFs that are stabilised in hypoxia and induce the expression of a diverse range of genes. The purpose of this study was to define the cellular specificities of HIF-1alpha and HIF-2alpha in retinal ischaemia, and to determine their correlation with the pattern of retinal hypoxia and the expression profiles of induced molecular mediators.We investigated the tissue distribution of retinal hypoxia during oxygen-induced retinopathy (OIR in mice using the bio-reductive drug pimonidazole. We measured the levels of HIF-1alpha and HIF-2alpha proteins by Western blotting and determined their cellular distribution by immunohistochemistry during the development of OIR. We measured the temporal expression profiles of two downstream mediators, vascular endothelial growth factor (VEGF and erythropoietin (Epo by ELISA. Pimonidazole labelling was evident specifically in the inner retina. Labelling peaked at 2 hours after the onset of hypoxia and gradually declined thereafter. Marked binding to Müller glia was evident during the early hypoxic stages of OIR. Both HIF-1alpha and HIF-2alpha protein levels were significantly increased during retinal hypoxia but were evident in distinct cellular distributions; HIF-1alpha stabilisation was evident in neuronal cells throughout the inner retinal layers whereas HIF-2alpha was restricted to Müller glia and astrocytes. Hypoxia and HIF-alpha stabilisation in the retina were closely followed by upregulated expression of the downstream mediators VEGF and EPO.Both HIF-1alpha and HIF-2alpha are activated in close correlation with retinal hypoxia but have contrasting cell specificities, consistent with differential roles in retinal ischaemia. Our findings suggest that HIF-2alpha activation plays a key role in regulating the response of Müller glia to hypoxia.

  1. Experimental Branch Retinal Vein Occlusion Induces Upstream Pericyte Loss and Vascular Destabilization.

    Directory of Open Access Journals (Sweden)

    Elisa Dominguez

    Full Text Available Branch retinal vein occlusion (BRVO leads to extensive vascular remodeling and is important cause of visual impairment. Although the vascular morphological changes following experimental vein occlusion have been described in a variety of models using angiography, the underlying cellular events are ill defined.We here show that laser-induced experimental BRVO in mice leads to a wave of TUNEL-positive endothelial cell (EC apoptosis in the upstream vascular network associated with a transient edema and hemorrhages. Subsequently, we observe an induction of EC proliferation within the dilated vein and capillaries, detected by EdU incorporation, and the edema resolves. However, the pericytes of the upstream capillaries are severely reduced, which was associated with continuing EC apoptosis and proliferation. The vascular remodeling was associated with increased expression of TGFβ, TSP-1, but also FGF2 expression. Exposure of the experimental animals to hypoxia, when pericyte (PC dropout had occurred, led to a dramatic increase in endothelial cell proliferation, confirming the vascular instability induced by the experimental BRVO.Experimental BRVO leads to acute endothelial cells apoptosis and increased permeability. Subsequently the upstream vascular network remains destabilized, characterized by pericyte dropout, un-physiologically high endothelial cells turnover and sensitivity to hypoxia. These early changes might pave the way for capillary loss and subsequent chronic ischemia and edema that characterize the late stage disease.

  2. Impairment of endothelial-myocardial interaction increases the susceptibility of cardiomyocytes to ischemia/reperfusion injury.

    Directory of Open Access Journals (Sweden)

    Thorsten M Leucker

    Full Text Available Endothelial-myocardial interactions may be critically important for ischemia/reperfusion injury. Tetrahydrobiopterin (BH4 is a required cofactor for nitric oxide (NO production by endothelial NO synthase (eNOS. Hyperglycemia (HG leads to significant increases in oxidative stress, oxidizing BH4 to enzymatically incompetent dihydrobiopterin. How alterations in endothelial BH4 content impact myocardial ischemia/reperfusion injury remains elusive. The aim of this study was to examine the effect of endothelial-myocardial interaction on ischemia/reperfusion injury, with an emphasis on the role of endothelial BH4 content. Langendorff-perfused mouse hearts were treated by triton X-100 to produce endothelial dysfunction and subsequently subjected to 30 min of ischemia followed by 2 h of reperfusion. The recovery of left ventricular systolic and diastolic function during reperfusion was impaired in triton X-100 treated hearts compared with vehicle-treated hearts. Cardiomyocytes (CMs were co-cultured with endothelial cells (ECs and subsequently subjected to 2 h of hypoxia followed by 2 h of reoxygenation. Addition of ECs to CMs at a ratio of 1∶3 significantly increased NO production and decreased lactate dehydrogenase activity compared with CMs alone. This EC-derived protection was abolished by HG. The addition of 100 µM sepiapterin (a BH4 precursor or overexpression of GTP cyclohydrolase 1 (the rate-limiting enzyme for BH4 biosynthesis in ECs by gene trasfer enhanced endothelial BH4 levels, the ratio of eNOS dimer/monomer, eNOS phosphorylation, and NO production and decreased lactate dehydrogenase activity in the presence of HG. These results demonstrate that increased BH4 content in ECs by either pharmacological or genetic approaches reduces myocardial damage during hypoxia/reoxygenation in the presence of HG. Maintaining sufficient endothelial BH4 is crucial for cardioprotection against hypoxia/reoxygenation injury.

  3. Gene expression analysis of embryonic stem cells expressing VE-cadherin (CD144 during endothelial differentiation

    Directory of Open Access Journals (Sweden)

    Libermann Towia

    2008-05-01

    Full Text Available Abstract Background Endothelial differentiation occurs during normal vascular development in the developing embryo. This process is recapitulated in the adult when endothelial progenitor cells are generated in the bone marrow and can contribute to vascular repair or angiogenesis at sites of vascular injury or ischemia. The molecular mechanisms of endothelial differentiation remain incompletely understood. Novel approaches are needed to identify the factors that regulate endothelial differentiation. Methods Mouse embryonic stem (ES cells were used to further define the molecular mechanisms of endothelial differentiation. By flow cytometry a population of VEGF-R2 positive cells was identified as early as 2.5 days after differentiation of ES cells, and a subset of VEGF-R2+ cells, that were CD41 positive at 3.5 days. A separate population of VEGF-R2+ stem cells expressing the endothelial-specific marker CD144 (VE-cadherin was also identified at this same time point. Channels lined by VE-cadherin positive cells developed within the embryoid bodies (EBs formed by differentiating ES cells. VE-cadherin and CD41 expressing cells differentiate in close proximity to each other within the EBs, supporting the concept of a common origin for cells of hematopoietic and endothelial lineages. Results Microarray analysis of >45,000 transcripts was performed on RNA obtained from cells expressing VEGF-R2+, CD41+, and CD144+ and VEGF-R2-, CD41-, and CD144-. All microarray experiments were performed in duplicate using RNA obtained from independent experiments, for each subset of cells. Expression profiling confirmed the role of several genes involved in hematopoiesis, and identified several putative genes involved in endothelial differentiation. Conclusion The isolation of CD144+ cells during ES cell differentiation from embryoid bodies provides an excellent model system and method for identifying genes that are expressed during endothelial differentiation and that

  4. Endothelial-regenerating cells: an expanding universe.

    Science.gov (United States)

    Steinmetz, Martin; Nickenig, Georg; Werner, Nikos

    2010-03-01

    Atherosclerosis is the most common cause for cardiovascular diseases and is based on endothelial dysfunction. A growing body of evidence suggests the contribution of bone marrow-derived endothelial progenitor cells, monocytic cells, and mature endothelial cells to vessel formation and endothelial rejuvenation. To this day, various subsets of these endothelial-regenerating cells have been identified according to cellular origin, phenotype, and properties in vivo and in vitro. However, the definition and biology, especially of endothelial progenitor cells, is complex and under heavy debate. In this review, we focus on current definitions of endothelial progenitor cells, highlight the clinical relevance of endothelial-regenerating cells, and provide new insights into cell-cell interactions involved in endothelial cell rejuvenation.

  5. Normal central retinal function and structure preserved in retinitis pigmentosa.

    Science.gov (United States)

    Jacobson, Samuel G; Roman, Alejandro J; Aleman, Tomas S; Sumaroka, Alexander; Herrera, Waldo; Windsor, Elizabeth A M; Atkinson, Lori A; Schwartz, Sharon B; Steinberg, Janet D; Cideciyan, Artur V

    2010-02-01

    To determine whether normal function and structure, as recently found in forms of Usher syndrome, also occur in a population of patients with nonsyndromic retinitis pigmentosa (RP). Patients with simplex, multiplex, or autosomal recessive RP (n = 238; ages 9-82 years) were studied with static chromatic perimetry. A subset was evaluated with optical coherence tomography (OCT). Co-localized visual sensitivity and photoreceptor nuclear layer thickness were measured across the central retina to establish the relationship of function and structure. Comparisons were made to patients with Usher syndrome (n = 83, ages 10-69 years). Cross-sectional psychophysical data identified patients with RP who had normal rod- and cone-mediated function in the central retina. There were two other patterns with greater dysfunction, and longitudinal data confirmed that progression can occur from normal rod and cone function to cone-only central islands. The retinal extent of normal laminar architecture by OCT corresponded to the extent of normal visual function in patients with RP. Central retinal preservation of normal function and structure did not show a relationship with age or retained peripheral function. Usher syndrome results were like those in nonsyndromic RP. Regional disease variation is a well-known finding in RP. Unexpected was the observation that patients with presumed recessive RP can have regions with functionally and structurally normal retina. Such patients will require special consideration in future clinical trials of either focal or systemic treatment. Whether there is a common molecular mechanism shared by forms of RP with normal regions of retina warrants further study.

  6. Sustained apnea induces endothelial activation.

    Science.gov (United States)

    Eichhorn, Lars; Dolscheid-Pommerich, Ramona; Erdfelder, Felix; Ayub, Muhammad Ajmal; Schmitz, Theresa; Werner, Nikos; Jansen, Felix

    2017-09-01

    Apnea diving has gained worldwide popularity, even though the pathophysiological consequences of this challenging sport on the human body are poorly investigated and understood. This study aims to assess the influence of sustained apnea in healthy volunteers on circulating microparticles (MPs) and microRNAs (miRs), which are established biomarkers reflecting vascular function. Short intermittent hypoxia due to voluntary breath-holding affects circulating levels of endothelial cell-derived MPs (EMPs) and endothelial cell-derived miRs. Under dry laboratory conditions, 10 trained apneic divers performed maximal breath-hold. Venous blood samples were taken, once before and at 4 defined points in time after apnea. Samples were analyzed for circulating EMPs and endothelial miRs. Average apnea time was 329 seconds (±103), and SpO 2 at the end of apnea was 79% (±12). Apnea was associated with a time-dependent increase of circulating endothelial cell-derived EMPs and endothelial miRs. Levels of circulating EMPs in the bloodstream reached a peak 4 hours after the apnea period and returned to baseline levels after 24 hours. Circulating miR-126 levels were elevated at all time points after a single voluntary maximal apnea, whereas miR-26 levels were elevated significantly only after 30 minutes and 4 hours. Also miR-21 and miR-92 levels increased, but did not reach the level of significance. Even a single maximal breath-hold induces acute endothelial activation and should be performed with great caution by subjects with preexisting vascular diseases. Voluntary apnea might be used as a model to simulate changes in endothelial function caused by hypoxia in humans. © 2017 Wiley Periodicals, Inc.

  7. Evaluation of the effectiveness and safety of glucocorticoids intravitreal implant therapy in macular edema due to retinal vein occlusion

    Directory of Open Access Journals (Sweden)

    Michalska-Małecka K

    2016-05-01

    Full Text Available Katarzyna Michalska-Małecka,1,2 Aneta Gaborek,2 Mariusz Nowak,3 Tomasz Halat,4 Mariola Pawłowska,2 Dorota Śpiewak2 1Department of Ophthalmology, School of Medicine in Katowice, Medical University of Silesia, Katowice, 2University Center of Ophthalmology and Oncology, Independent Public Clinical Hospital, Medical University of Silesia, Katowice, 3Pathophysiology Division, Department of Pathophysiology and Endocrinology, Medical University of Silesia, School of Medicine with Division of Density, Zabrze, 4Education and Medical Simulation Center, Medical University of Silesia, Katowice, Poland Abstract: The purpose of this study was to evaluate the impact of intravitreal dexamethasone implant (Ozurdex on macular morphology and functions in eyes with macular edema (ME secondary to retinal vein occlusion. Efficacy outcomes of the treatment were best-corrected visual acuity (BCVA and central retinal thickness (CRT. Safety outcomes were intraocular pressure and cornea endothelial cell density. The study was conducted by the prospective analysis on 36 patients (17 women and 19 men aged 28–77 years (the average age was 58±15 years treated with the injection of dexamethasone implant because of the persistent ME at the Department of Ophthalmology and Ophthalmology Outpatient Clinic of the University Centre of Ophthalmology and Oncology in Katowice. The studied group included 16 patients with central retinal vein occlusion (16 eyes, and 20 patients with branch retinal vein occlusion (20 eyes. We found a significant increase of BCVA after first, second, and third month of treatment. Six months after the treatment, BCVA decreased, although not significantly compared with the value obtained in the third month. Two months after the intravitreal implantation of dexamethasone delivery system, CRT was 338±163 µm and was significantly lower compared with pretreatment value. Between third and sixth month after the treatment, we found insignificant increase of

  8. Mouse model for Usher syndrome: linkage mapping suggests homology to Usher type I reported at human chromosome 11p15.

    OpenAIRE

    Heckenlively, J R; Chang, B; Erway, L C; Peng, C; Hawes, N L; Hageman, G S; Roderick, T H

    1995-01-01

    Usher syndrome is a group of diseases with autosomal recessive inheritance, congenital hearing loss, and the development of retinitis pigmentosa, a progressive retinal degeneration characterized by night blindness and visual field loss over several decades. The causes of Usher syndrome are unknown and no animal models have been available for study. Four human gene sites have been reported, suggesting at least four separate forms of Usher syndrome. We report a mouse model of type I Usher syndr...

  9. A CNGB1 frameshift mutation in Papillon and Phalene dogs with progressive retinal atrophy.

    Directory of Open Access Journals (Sweden)

    Saija J Ahonen

    Full Text Available Progressive retinal degenerations are the most common causes of complete blindness both in human and in dogs. Canine progressive retinal atrophy (PRA or degeneration resembles human retinitis pigmentosa (RP and is characterized by a progressive loss of rod photoreceptor cells followed by a loss of cone function. The primary clinical signs are detected as vision impairment in a dim light. Although several genes have been associated with PRAs, there are still PRAs of unknown genetic cause in many breeds, including Papillons and Phalènes. We have performed a genome wide association and linkage studies in cohort of 6 affected Papillons and Phalènes and 14 healthy control dogs to map a novel PRA locus on canine chromosome 2, with a 1.9 Mb shared homozygous region in the affected dogs. Parallel exome sequencing of a trio identified an indel mutation, including a 1-bp deletion, followed by a 6-bp insertion in the CNGB1 gene. This mutation causes a frameshift and premature stop codon leading to probable nonsense mediated decay (NMD of the CNGB1 mRNA. The mutation segregated with the disease and was confirmed in a larger cohort of 145 Papillons and Phalènes (PFisher = 1.4×10(-8 with a carrier frequency of 17.2 %. This breed specific mutation was not present in 334 healthy dogs from 10 other breeds or 121 PRA affected dogs from 44 other breeds. CNGB1 is important for the photoreceptor cell function its defects have been previously associated with retinal degeneration in both human and mouse. Our study indicates that a frameshift mutation in CNGB1 is a cause of PRA in Papillons and Phalènes and establishes the breed as a large functional animal model for further characterization of retinal CNGB1 biology and possible retinal gene therapy trials. This study enables also the development of a genetic test for breeding purposes.

  10. Inner Retinal Oxygen Delivery, Metabolism, and Extraction Fraction in Ins2Akita Diabetic Mice.

    Science.gov (United States)

    Blair, Norman P; Wanek, Justin; Felder, Anthony E; Brewer, Katherine C; Joslin, Charlotte E; Shahidi, Mahnaz

    2016-11-01

    Retinal nonperfusion and hypoxia are important factors in human diabetic retinopathy, and these presumably inhibit energy production and lead to cell death. The purpose of this study was to elucidate the effect of diabetes on inner retinal oxygen delivery and metabolism in a mouse model of diabetes. Phosphorescence lifetime and blood flow imaging were performed in spontaneously diabetic Ins2Akita (n = 22) and nondiabetic (n = 22) mice at 12 and 24 weeks of age to measure retinal arterial (O2A) and venous (O2V) oxygen contents and total retinal blood flow (F). Inner retinal oxygen delivery (DO2) and metabolism (MO2) were calculated as F ∗ O2A and F ∗ (O2A - O2V), respectively. Oxygen extraction fraction (OEF), which equals MO2/DO2, was calculated. DO2 at 12 weeks were 112 ± 40 and 97 ± 29 nL O2/min in nondiabetic and diabetic mice, respectively (NS), and 148 ± 31 and 85 ± 37 nL O2/min at 24 weeks, respectively (P < 0.001). MO2 were 65 ± 31 and 66 ± 27 nL O2/min in nondiabetic and diabetic mice at 12 weeks, respectively, and 79 ± 14 and 54 ± 28 nL O2/min at 24 weeks, respectively (main effects = NS). At 12 weeks OEF were 0.57 ± 0.17 and 0.67 ± 0.09 in nondiabetic and diabetic mice, respectively, and 0.54 ± 0.07 and 0.63 ± 0.08 at 24 weeks, respectively (main effect of diabetes: P < 0.01). Inner retinal MO2 was maintained in diabetic Akita mice indicating that elevation of the OEF adequately compensated for reduced DO2 and prevented oxidative metabolism from being limited by hypoxia.

  11. In vitro induction of protein complexes between bevacizumab, VEGF-A¹⁶⁵ and heparin: explanation for deposits observed on endothelial veins in monkey eyes.

    Science.gov (United States)

    Julien, Sylvie; Biesemeier, Antje; Schraermeyer, Ulrich

    2013-04-01

    By investigating the effects of intravitreal bevacizumab on retinal vessels of monkeys, we found that bevacizumab accumulated locally at high concentration within individual blood vessels. It formed electron-dense fibrous deposits between endothelial cells and erythrocytes or granulocytes inducing retinal vein thrombosis. To better characterise the observed deposits, we investigated in vitro whether these deposits result from a complex between bevacizumab, vascular endothelial growth factor (VEGF)-A(165) and heparin. Cynomolgus monkeys were intravitreally injected with 1.25 mg bevacizumab. The eyes were enucleated between 1 and 14 days after injection and investigated by electron microscopy and immunohistochemistry. Human umbilical vein endothelial cells (HUVEC) were incubated with bevacizumab, VEGF-A(165) and heparin at different concentrations. Treatments with ranibizumab served as control. Bevacizumab and ranibizumab were detected immunohistochemically using Cy-3 or immunogold labelled antibodies. Treated animals showed bevacizumab locally at high concentration within retinal blood vessels. Electron-dense deposits inside retinal vessels and between erythrocytes were detected in three out of four treated monkeys. In vitro, many globular aggregates heavily stained with anti-human IgG were only observed with equimolar amounts (240 nM) of bevacizumab and VEGF-A(165) and 0.2 U/ml heparin and not after ranibizumab treatment. The immunogold labelling specifically localised ultrastructurally the complexes formed between bevacizumab, VEGF-A(165) and heparin at the surfaces of HUVEC cells. Heparin promotes bevacizumab immune complex deposition on to endothelial cells. Our in vitro results could explain the presence of deposits observed on endothelial veins in monkey eyes intravitreally injected with bevacizumab.

  12. TAM receptor knockout mice are susceptible to retinal autoimmune induction.

    Science.gov (United States)

    Ye, Fei; Li, Qiutang; Ke, Yan; Lu, Qingjun; Han, Lixia; Kaplan, Henry J; Shao, Hui; Lu, Qingxian

    2011-06-16

    TAM receptors are expressed mainly by dendritic cells and macrophages in the immune system, and mice lacking TAM receptors develop systemic autoimmune diseases because of inefficient negative control of the cytokine signaling in those cells. This study aims to test the susceptibility of the TAM triple knockout (tko) mice to the retina-specific autoantigen to develop experimental autoimmune uveoretinitis (EAU). TAM tko mice that were or were not immunized with interphotoreceptor retinoid-binding protein (IRBP) peptides were evaluated for retinal infiltration of the macrophages and CD3(+) T cells by immunohistochemistry, spontaneous activation of CD4(+) T cells, and memory T cells by flow cytometry and proliferation of IRBP-specific CD4(+) T cells by [(3)H]thymidine incorporation assay. Ocular inflammation induced by IRBP peptide immunization and specific T cell transfer were observed clinically by funduscopy and confirmed by histology. Tko mice were found to have less naive, but more activated, memory T cells, among which were exhibited high sensitivity to ocular IRBP autoantigens. Immunization with a low dose of IRBP and adoptive transfer of small numbers of IRBP-specific T cells from immunized tko mice caused the infiltration of lymphocytes, including CD3(+) T cells, into the tko retina. Mice without TAM receptor spontaneously develop IRBP-specific CD4(+) T cells and are more susceptible to retinal autoantigen immunization. This TAM knockout mouse line provides an animal model with which to study the role of antigen-presenting cells in the development of T cell-mediated uveitis.

  13. STUDIES ON ENDOTHELIAL REACTIONS

    Science.gov (United States)

    Foot, Nathan Chandler

    1923-01-01

    operative. On the other hand, there may be an increase in the phagocytic activity of the endothelium of the sinusoids which might take up more bacteria under these changed conditions. Several investigators have claimed, recently, that there is an increased activity of the liver endothelium following splenectomy, their experiments being directed chiefly toward determining the fate of the erythrocytes. Pearce (1918) in reporting the effects of experimental splenectomy in dogs, states that there are definite compensatory changes in the lymph nodes, in the form of an increased proliferation of endothelial phagocytes, and that the stellate cells of the liver sinusoids often show a similar compensatory increase in number. In both cases the cells are, apparently, formed in situ rather than transported to the organs. He says: ‘Such findings suggest the development of a compensatory function on the part of the lymph-nodes and possibly the liver,’ and suggests that, in times of stress ‘the stellate cells of the liver thus assume, in part at least, the function of destroying red blood-corpuscles by phagocytosis.’ Incidentally, he presents an excellent discussion of the history and subject of splenectomy. Motohashi (1922) reports a great increase in the hemophagic power of the hepatic endothelium and an increase in the number of endothelial elements, after some 45 days following splenectomy in rabbits. Nishikawa and Takagi (1922) have observed similar phenomena with white rats, the Kupffer cells taking up erythrocytes in large numbers in splenectomized animals, whereas controls never show similar propensities on the part of these cells. It may be that different substances cause different reactions on the part of the hepatic endothelium. Contributory Experiment.—A side experiment was performed with five rabbits, two splenectomized and three controls, into which uniform doses of pneumococci were injected intravenously. They all died of septicemia after a few days. The results

  14. Retinal vein occlusion: pathophysiology and treatment options

    OpenAIRE

    Karia, Niral

    2010-01-01

    Niral KariaDepartment of Ophthalmology, Southend Hospital, Prittlewell Chase, Westcliff on Sea, Essex, United KingdomAbstract: This paper reviews the current thinking about retinal vein occlusion. It gives an overview of its pathophysiology and discusses the evidence behind the various established and emerging treatment paradigms.Keywords: central, hemispheric, branch, retinal vein occlusion, visual loss

  15. Retinitis pigmentosa, Coats disease and uveitis.

    Science.gov (United States)

    Solomon, A; Banin, E; Anteby, I; Benezra, D

    1999-01-01

    To study the anamnestic immune response to retinal specific antigens of two patients suffering from a rare triad of retinitis pigmentosa, Coats disease and uveitis. 17-year-old girl presented with an acute episode of panuveitis, and her 19-year-old brother suffered from chronic uveitis. On examination, both patients showed retinal vascular changes and subretinal exudations typical of Coats disease, with bone-spicule pigmentary changes as observed in retinitis pigmentosa. All routine examinations were unrevealing. However, the peripheral lymphocytes from these two siblings gave a specific anamnestic response to retinal antigens in vitro. A stimulation index of 4.6 was obtained when the sister's lymphocytes were stimulated with interphotoreceptor binding protein, IRBP--during the acute stage of the uveitis. The brother's lymphocytes showed a stimulation index of 2.7 towards S-Ag during the chronic phase of his uveitic condition. These results indicate that autoimmunity towards retinal antigens may play some role in specific types of retinitis pigmentosa. Whether these autoimmune reactions are a primary pathological mechanism or are secondary to the extensive destruction of the photoreceptor layer resulting from the retinitis pigmentosa remains debatable.

  16. Fundus autofluorescence applications in retinal imaging

    Science.gov (United States)

    Gabai, Andrea; Veritti, Daniele; Lanzetta, Paolo

    2015-01-01

    Fundus autofluorescence (FAF) is a relatively new imaging technique that can be used to study retinal diseases. It provides information on retinal metabolism and health. Several different pathologies can be detected. Peculiar AF alterations can help the clinician to monitor disease progression and to better understand its pathogenesis. In the present article, we review FAF principles and clinical applications. PMID:26139802

  17. Fundus autofluorescence applications in retinal imaging

    Directory of Open Access Journals (Sweden)

    Andrea Gabai

    2015-01-01

    Full Text Available Fundus autofluorescence (FAF is a relatively new imaging technique that can be used to study retinal diseases. It provides information on retinal metabolism and health. Several different pathologies can be detected. Peculiar AF alterations can help the clinician to monitor disease progression and to better understand its pathogenesis. In the present article, we review FAF principles and clinical applications.

  18. Rhegmatogenous retinal detachment and uveitis.

    Science.gov (United States)

    Kerkhoff, Frank T; Lamberts, Querin J; van den Biesen, Pieter R; Rothova, Aniki

    2003-02-01

    To evaluate the frequency, high-risk factors, and visual prognosis of rhegmatogenous retinal detachment (RRD) in patients with uveitis. Retrospective case-control study. We included 1387 consecutive patients with uveitis who consulted our uveitis clinic from January 1990 through December 1997 of whom 43 patients (46 eyes) with RRD were identified. The retinal detachment (RD) controls were 212 consecutive patients with RRD (221 eyes, first occurrence of RD, not associated with uveitis) who were admitted for surgery in the period from April 1999 to April 2000. The uveitis control group consisted of 150 age-matched patients (210 eyes) selected from the entire uveitis series. Retrospective analysis of clinical data. The presence of RRD and eventual risk factors for RRD, such as myopia, retinal lattice degeneration, prior intraocular surgery, anatomic location of uveitis, its specific diagnosis, and clinical manifestations. Furthermore, the surgical and nonsurgical outcomes of RRD, as well as the results of various treatment regimens, were analyzed. RRD was identified in 3.1% of the patients with uveitis. RRD was most frequently associated with panuveitis (6.6%). RRD was associated more frequently with infectious (7.6%) than noninfectious uveitis (2.1%). At the onset of RRD, uveitis was active in most (46%) affected eyes. Proliferative vitreoretinopathy was present in 30% of the uveitic RRD eyes at presentation in contrast to 12% of the RRD control eyes. In uveitic RRD, the retina was reattached in 59% of eyes with a single operation; the final anatomic reattachment rate was 88%. Finally, a visual acuity of less than 20/200 was present in 71% of the uveitic RRD eyes, 10% of which had no light perception. We discovered a high prevalence of RRD in patients with active panuveitis and infectious uveitis and document that uveitis in itself is a risk factor for the development of RRD. The visual prognosis of RRD in uveitis was poor because of the uveitis itself and the

  19. [To cognize retinitis pigmentosa with scientific view].

    Science.gov (United States)

    Li, Gen-lin

    2009-03-01

    Retinitis pigmentosa (RP) is the most common inherited eye disease that usually leads into blind, and is high simplex and clinical heterogeneity. Recent years, some new hereditary forms have been found, such as digenic RP, mitochondrial RP, incomplete dominant inheritance RP. The phenotype of RP is multiplicity. Incompatible phenomenon between genotype and phenotypes was shown in some genes such as peripherin/RDS, RHO, RP2 and RP3. The complicated phenotype was shown in the rare RP forms, such as centricity RP, stemma RP, retinitis pigmentosa sine pigmento, and retinal degeneration slow. Retinal transplantation, retinal implantation, drug and neurotrophic factor therapy, and gene therapy have been well studied worldwide and presented some hopeful efficacy. Ophthalmologists and practitioners should cognize the new advance and new knowledge on RP therapy with a scientific view for better serving the RP patients.

  20. Variable retinal presentations in nanophthalmos

    International Nuclear Information System (INIS)

    Khan, A.; Zafar, S.N.

    2009-01-01

    Nanophthalmos is an uncommon developmental ocular disorder characterized by a small eye with short axial length, high hyperopia and high lens/eye volume ratio due to arrested development of the globe in all directions. Different types of fundus changes can rarely occur with nanophthalmos. We describe five cases of nanophthalmos, each of them presenting with a different fundus appearance. Our case series highlights variability of pigmentary changes from retinal flecks to bone spicules and bull's eye maculopathy, which are rare in the combinations described here. (author)

  1. Regenerative Therapy for Retinal Disorders

    Directory of Open Access Journals (Sweden)

    Narsis Daftarian

    2010-01-01

    Full Text Available Major advances in various disciplines of basic sciences including embryology, molecular and cell biology, genetics, and nanotechnology, as well as stem cell biology have opened new horizons for regenerative therapy. The unique characteristics of stem cells prompt a sound understanding for their use in modern regenerative therapies. This review article discusses stem cells, developmental stages of the eye field, eye field transcriptional factors, and endogenous and exogenous sources of stem cells. Recent studies and challenges in the application of stem cells for retinal pigment epithelial degeneration models will be summarized followed by obstacles facing regenerative therapy.

  2. Heritability of Retinal Vascular Fractals

    DEFF Research Database (Denmark)

    Vergmann, Anna Stage; Broe, Rebecca; Kessel, Line

    2017-01-01

    , the retinal vascular fractal dimension was measured using the box-counting method and compared within monozygotic and dizygotic twin pairs using Pearson correlation coefficients. Falconer's formula and quantitative genetic models were used to determine the genetic component of variation. Results: The mean...... fractal dimension did not differ statistically significantly between monozygotic and dizygotic twin pairs (1.505 vs. 1.495, P = 0.06), supporting that the study population was suitable for quantitative analysis of heritability. The intrapair correlation was markedly higher (0.505, P = 0...

  3. Retinal peripheral changes after LASIK

    OpenAIRE

    Nassaralla Junior,João Jorge; Santos,Regina Cândido Ribeiro dos; Nassaralla,Belquiz Amaral

    2008-01-01

    PURPOSE: To better define the effect of laser in situ keratomileusis (LASIK) on myopic eyes and the risk and incidence of retinal complications after surgery. METHODS: In a prospective study, 200 eyes of 100 patients, 49 male and 51 female, with a mean age of 29.7 years, had a complete posterior pole examination before and at 1 week, 1, 3 and 12 months after bilateral simultaneous LASIK for the correction of myopia. Mean spherical equivalent was 7.75D (range 1.00 to -17.25D). Before LASIK, pr...

  4. Collaborative enhancement of antibody binding to distinct PECAM-1 epitopes modulates endothelial targeting.

    Directory of Open Access Journals (Sweden)

    Ann-Marie Chacko

    Full Text Available Antibodies to platelet endothelial cell adhesion molecule-1 (PECAM-1 facilitate targeted drug delivery to endothelial cells by "vascular immunotargeting." To define the targeting quantitatively, we investigated the endothelial binding of monoclonal antibodies (mAbs to extracellular epitopes of PECAM-1. Surprisingly, we have found in human and mouse cell culture models that the endothelial binding of PECAM-directed mAbs and scFv therapeutic fusion protein is increased by co-administration of a paired mAb directed to an adjacent, yet distinct PECAM-1 epitope. This results in significant enhancement of functional activity of a PECAM-1-targeted scFv-thrombomodulin fusion protein generating therapeutic activated Protein C. The "collaborative enhancement" of mAb binding is affirmed in vivo, as manifested by enhanced pulmonary accumulation of intravenously administered radiolabeled PECAM-1 mAb when co-injected with an unlabeled paired mAb in mice. This is the first demonstration of a positive modulatory effect of endothelial binding and vascular immunotargeting provided by the simultaneous binding a paired mAb to adjacent distinct epitopes. The "collaborative enhancement" phenomenon provides a novel paradigm for optimizing the endothelial-targeted delivery of therapeutic agents.

  5. BAC-Dkk3-EGFP Transgenic Mouse: An In Vivo Analytical Tool for Dkk3 Expression

    Directory of Open Access Journals (Sweden)

    Yuki Muranishi

    2012-01-01

    Full Text Available Dickkopf (DKK family proteins are secreted modulators of the Wnt signaling pathway and are capable of regulating the development of many organs and tissues. We previously identified Dkk3 to be a molecule predominantly expressed in the mouse embryonic retina. However, which cell expresses Dkk3 in the developing and mature mouse retina remains to be elucidated. To examine the precise expression of the Dkk3 protein, we generated BAC-Dkk3-EGFP transgenic mice that express EGFP integrated into the Dkk3 gene in a BAC plasmid. Expression analysis using the BAC-Dkk3-EGFP transgenic mice revealed that Dkk3 is expressed in retinal progenitor cells (RPCs at embryonic stages and in Müller glial cells in the adult retina. Since Müller glial cells may play a potential role in retinal regeneration, BAC-Dkk3-EGFP mice could be useful for retinal regeneration studies.

  6. The endothelial border to health

    DEFF Research Database (Denmark)

    Hansen, Nina Wærling; Hansen, Anker Jon; Sams, Anette

    2017-01-01

    player for maintenance of health and for development of a number of diseases. Endothelial dysfunction is known to be an important component of type 2 diabetes, but is also assumed to be involved in many other diseases, for example, rheumatoid arthritis, inflammatory bowel disease, asthma...... extracellular proteins form epitopes for potential specific antibody formation upon interactions with reducing sugars. This paper reviews the endothelial metabolism, biology, inflammatory processes, physical barrier functions, and summarizes evidence that although stochastic in nature, endothelial responses...... to hyperglycemia are major contributors to disease pathophysiology. We present molecular and mechanistic evidence that both biological and physical barriers, protein function, specific immunity, and inflammatory processes are compromised by hyperglycemic events and thus, hyperglycemic events alone should...

  7. Endothelial Targeting of Cowpea Mosaic Virus (CPMV) via Surface Vimentin

    Science.gov (United States)

    Koudelka, Kristopher J.; Destito, Giuseppe; Plummer, Emily M.; Trauger, Sunia A.; Siuzdak, Gary; Manchester, Marianne

    2009-01-01

    Cowpea mosaic virus (CPMV) is a plant comovirus in the picornavirus superfamily, and is used for a wide variety of biomedical and material science applications. Although its replication is restricted to plants, CPMV binds to and enters mammalian cells, including endothelial cells and particularly tumor neovascular endothelium in vivo. This natural capacity has lead to the use of CPMV as a sensor for intravital imaging of vascular development. Binding of CPMV to endothelial cells occurs via interaction with a 54 kD cell-surface protein, but this protein has not previously been identified. Here we identify the CPMV binding protein as a cell-surface form of the intermediate filament vimentin. The CPMV-vimentin interaction was established using proteomic screens and confirmed by direct interaction of CPMV with purified vimentin, as well as inhibition in a vimentin-knockout cell line. Vimentin and CPMV were also co-localized in vascular endothelium of mouse and rat in vivo. Together these studies indicate that surface vimentin mediates binding and may lead to internalization of CPMV in vivo, establishing surface vimentin as an important vascular endothelial ligand for nanoparticle targeting to tumors. These results also establish vimentin as a ligand for picornaviruses in both the plant and animal kingdoms of life. Since bacterial pathogens and several other classes of viruses also bind to surface vimentin, these studies suggest a common role for surface vimentin in pathogen transmission. PMID:19412526

  8. Endothelial targeting of cowpea mosaic virus (CPMV via surface vimentin.

    Directory of Open Access Journals (Sweden)

    Kristopher J Koudelka

    2009-05-01

    Full Text Available Cowpea mosaic virus (CPMV is a plant comovirus in the picornavirus superfamily, and is used for a wide variety of biomedical and material science applications. Although its replication is restricted to plants, CPMV binds to and enters mammalian cells, including endothelial cells and particularly tumor neovascular endothelium in vivo. This natural capacity has lead to the use of CPMV as a sensor for intravital imaging of vascular development. Binding of CPMV to endothelial cells occurs via interaction with a 54 kD cell-surface protein, but this protein has not previously been identified. Here we identify the CPMV binding protein as a cell-surface form of the intermediate filament vimentin. The CPMV-vimentin interaction was established using proteomic screens and confirmed by direct interaction of CPMV with purified vimentin, as well as inhibition in a vimentin-knockout cell line. Vimentin and CPMV were also co-localized in vascular endothelium of mouse and rat in vivo. Together these studies indicate that surface vimentin mediates binding and may lead to internalization of CPMV in vivo, establishing surface vimentin as an important vascular endothelial ligand for nanoparticle targeting to tumors. These results also establish vimentin as a ligand for picornaviruses in both the plant and animal kingdoms of life. Since bacterial pathogens and several other classes of viruses also bind to surface vimentin, these studies suggest a common role for surface vimentin in pathogen transmission.

  9. A novel homozygous truncating GNAT1 mutation implicated in retinal degeneration.

    Science.gov (United States)

    Carrigan, Matthew; Duignan, Emma; Humphries, Pete; Palfi, Arpad; Kenna, Paul F; Farrar, G Jane

    2016-04-01

    The GNAT1 gene encodes the α subunit of the rod transducin protein, a key element in the rod phototransduction cascade. Variants in GNAT1 have been implicated in stationary night-blindness in the past, but unlike other proteins in the same pathway, it has not previously been implicated in retinitis pigmentosa. A panel of 182 retinopathy-associated genes was sequenced to locate disease-causing mutations in patients with inherited retinopathies. Sequencing revealed a novel homozygous truncating mutation in the GNAT1 gene in a patient with significant pigmentary disturbance and constriction of visual fields, a presentation consistent with retinitis pigmentosa. This is the first report of a patient homozygous for a complete loss-of-function GNAT1 mutation. The clinical data from this patient provide definitive evidence of retinitis pigmentosa with late onset in addition to the lifelong night-blindness that would be expected from a lack of transducin function. These data suggest that some truncating GNAT1 variants can indeed cause a recessive, mild, late-onset retinal degeneration in human beings rather than just stationary night-blindness as reported previously, with notable similarities to the phenotype of the Gnat1 knockout mouse. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  10. Temporal Progression of Retinal Progenitor Cell Identity: Implications in Cell Replacement Therapies

    Directory of Open Access Journals (Sweden)

    Awais Javed

    2017-12-01

    Full Text Available Retinal degenerative diseases, which lead to the death of rod and cone photoreceptor cells, are the leading cause of inherited vision loss worldwide. Induced pluripotent or embryonic stem cells (iPSCs/ESCs have been proposed as a possible source of new photoreceptors to restore vision in these conditions. The proof of concept studies carried out in mouse models of retinal degeneration over the past decade have highlighted several limitations for cell replacement in the retina, such as the low efficiency of cone photoreceptor production from stem cell cultures and the poor integration of grafted cells in the host retina. Current protocols to generate photoreceptors from stem cells are largely based on the use of extracellular factors. Although these factors are essential to induce the retinal progenitor cell (RPC fate from iPSCs/ESCs, developmental studies have shown that RPCs alter fate output as a function of time (i.e., their temporal identity to generate the seven major classes of retinal cell types, rather than spatial position. Surprisingly, current stem cell differentiation protocols largely ignore the intrinsic temporal identity of dividing RPCs, which we argue likely explains the low efficiency of cone production in such cultures. In this article, we briefly review the mechanisms regulating temporal identity in RPCs and discuss how they could be exploited to improve cone photoreceptor production for cell replacement therapies.

  11. Retinal neurodegeneration may precede microvascular changes characteristic of diabetic retinopathy in diabetes mellitus.

    Science.gov (United States)