bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cx43 internalization is mediated by the ubiquitin‐binding adaptor protein Eps15 in retinal endothelial cells
Authors: GIRAO H , CATARINO S , LOPES C , PEREIRA P 
Published: September 2008

Abstract Purpose Intercellular communication through gap junctions (GJIC) is important in maintaining the integrity of the blood‐retinal barrier. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. The purpose of this study is to investigate the molecular mechanisms involved in the regulation of connexin 43 (Cx43) stability at plasma membrane by eps15 in retinal endothelial cells. Methods To investigate the interaction of Cx43 with eps15, Cx43 was selectively immunoprecipitated (IP), followed by western blot (WB) and probing with antibodies against eps15. To investigate the role of eps15 in Cx43 stability and subcellular distribution cells were transfected with a dominant negative form of eps15 or siRNA targeted against eps15, cell extracts were WB and probed with antibodies against Cx43. Alternatively, cells were fixed and simultaneously stained with antibodies against Cx43 and eps15, and imaged by confocal microscopy. Results Data obtained in this study indicates that eps15 interacts with Cx43 mediating its internalization from the plasma membrane. When the normal function of eps15 is disrupted by the presence of a dominant negative form of the protein or through silencing of the eps15 gene with siRNA, Cx43 accumulates at the plasma membrane. Conclusion Data suggests a novel protein interaction between Cx43 and eps15 that mediates the plasma membrane stability of Cx43. Disruption of this interaction may impact on GJIC, contributing to endothelial cell dysfunction associated with the breakdown of the blood‐retinal barrier, as observed in diabetic retinopathy. Supported by POCI/SAU‐MMO/57216/2004
Receptor retinoid‐binding protein (IRBP) binds cone outer segments differentially in light and dark
Authors: GONZALEZ‐FERNANDEZ F , GARLIPP MA , KEATING AM 
Published: September 2008

Abstract Purpose Vision requires the continuous exchanges of visual cycle retinoids through the interphotoreceptor matrix (IPM). IRBP, which is synthesized by the rods and cones, is the major soluble protein component of the IPM. In the matrix, IRBP protects and targets retinoids trafficking between the photoreceptors, RPE and Muller cells. Methods Organ culture of isolated Xenopus retina was used to characterize the cellular binding of recombinant full‐length IRBP protein free of fusion tags. Xenopus was selected for these studies because of their large photoreceptors which are easily detached in both light and dark‐adapted states. xIRBP was covalently bound to the fluorescent dye Alexa‐647 at 1:1 molar ratio. Labeling had no effect on the retinoid ligand‐binding properties of the protein. Xenopus retinas were detached and incubated with xIRBP‐647 for 1 hour at room temperature in light and dark. The distribution of xIRBP‐647 was studied by confocal microscopy in retinal flat whole mounts and cryosections. Results In light, xIRBP‐647 bound to cone outer segments with lighter labeling of the rod outer segments. Retinas incubated with the xIRBP‐647 in the presence of a 50 fold excess of unlabeled protein showed reduced cone and rod outer segment labeling. In dark, outer segment binding was not appreciated. Positive staining near the level of the external‐limiting membrane was noted in dark but not light. Conclusion The binding of xIRBP‐647 to cells of the isolated retina was different in light versus dark. Our data suggests that the direct interaction of IRBP with the outer segments and Muller cells plays an important role in retinoid trafficking in the the visual cycle.
Selective estrogen receptor modulators regulate IL‐6 inflammatory response in ARPE‐19 cells
Authors: PAIMELA T , RYHÄNEN T , VIIRI J , HYTTINEN J , UNKILA M , UUSITALO HMT , SALMINEN A , KAARNIRANTA K 
Published: September 2008

Abstract Purpose Recent findings reveal that Toll‐like receptors (TLRs) and innate immunity participate in the pathology of age‐related macular degeneration (AMD). Many studies indicate that estrogens and selective estrogen modulators (SERMs) modulate inflammatory responses, but their effect on the development of AMD is weakly understood. In this study, we investigated the regulatory role of various SERMs (provided by Hormos Medical Ltd.) on IL‐6 expression in human retinal pigment epithelial cells (ARPE‐19). Methods ARPE‐19 cells were exposed to lipopolysaccharide (LPS; TLR 4 agonist) with simultaneous exposure to various SERMs and the secretion of IL‐6 cytokine was analyzed by ELISA. The estrogen receptor alpha and beta were qualitatively measured by RT‐PCR. To study the effect of various SERM treatments of estrogen response element (ERE) ‐mediated transcription, the ARPE‐19 cells were transiently transfected with ERE‐luciferase vector. The activity of ERE was measured by Luciferase assay. Results Simultaneous exposures to LPS and SERM‐320 reduced the IL‐6 expression levels in ARPE‐19 cells compared to LPS exposure alone. The RT‐PCR analysis showed that ARPE‐19 cells expressed estrogen receptor alpha but not beta proteins. Interestingly, SERM‐320 did not increase the activity of ERE in ARPE cells. This reveals that SERM‐320 is implicated in regulation of IL‐6 expression, but is not mediated through estrogen response element. Conclusion Our findings reveal that SERM‐320 is a novel compound to suppress innate immunity response in human retinal pigment epithelial cells.
Activation of autophagy induced by 7‐Ketocholesterol and dysfunction of RPE in AMD
Authors: OLIVEIRA FERREIRA J , GIRAO H , PEREIRA P 
Published: September 2008

Abstract Purpose Retinal pigment epithelial (RPE) cell death is a hallmark of age‐related macular degeneration (AMD). Lysosomal impairment in RPE cells is one of the traits present in the pathology of AMD. Authophagy, a process whereby proteins and subcellular organelles are degraded in the lysosome is activated by several stress stimuli. RPEs internalize large amounts of oxidized lipids including 7‐ketocholesterol, the most cytotoxic of oxysterols. The purpose of this study is to determine if exposing RPE to 7‐ketocholesterol can act as a stimulus to induce autophagy and thus account to the dysfunction of the RPE. Methods RPE cells where incubated with 7‐ketocholesterol (20 ug/ml) for 6 or 12 h, or deprived from nutrients for 12h. To assess Macroautophagy cells were transfected with EGFP‐LC3 plasmid. Formation of autophagic vesicles was assessed by confocal microscopy. LC3 processing was measured by western blot with antibodies against GFP. Chaperone mediated autophagy (CMA) was assessed by determining if chaperones interacted with Lamp2, performing Lamp2 immunoprecipitation. Results 7‐ketocholesterol induces the formation of autophagic vesicles and the processing of the LC3‐I into LC3‐II, both hallmarks of Macroautophagy, Moreover treatment with 7‐ketocholesterol increases the interaction between Lamp2 and Hsc70, Hsp90 and Hsp40, suggesting that this cholesterol oxide also stimulates CMA. Conclusion RPE cells undergo Macroautophagy and CMA activation when exposed to 7‐ketocholesterol. At early stages both may act as a cell survival mechanism whereas prolonged exposure may result in macroautophagic cell death.
New developments in OCT evaluations
Authors: BERNARDES R , CUNHA‐VAZ J 
Published: September 2008

AbstractDiabetic macular edema (DME) is one of the most common causes for visual loss (Ciulla et al., Diabetes Care, 2003). DME is characterized by an abnormal increase of the retinal thickness (RT) due to the accumulation of liquids in the retinal tissue, consequence of the first response to the inflammatory process occurring in diabetic retinopathy (DR). It is therefore considered one of the most important indicators of DR progression. Other indicators, such as microaneurysms and retinal leakage, were also observed and quantified in a prospective longitudinal 3‐year follow‐up study involving type 2 diabetic patients with nonproliferative DR. Patterns of progression were established based on these ophthalmic indicators (Lobo et al., Arch Opthalmol., 2004). OCT plays an important role in the assessment and quantification of DME. Stratus OCT version established a 9 area map for RT, being the central area of 1000µm in diameter. However, there is a lack of detailed information when studying DME. This problem was addressed by the use of an atlas of the human macula in a recent work from our group, where the merging of radial and circular scans was made possible (Bernardes et al., IOVS, 2008). Recently, Carl Zeiss introduced Cirrus HD‐OCT, a Fourier domain OCT, allowing to achieve more detailed information of the macular area, performing either 512×128 or 200×200 B‐scans, and allowing to export an eye fundus image for reference. It is now possible to assess local RT and correlate it with any imaging modalities available.
Role of inflammation in blood‐retinal barrier breakdown: animal models
Authors: AMBROSIO AF , LEAL EC , MANIVANNAN A , CUNHA‐VAZ J , FORRESTER JV 
Published: September 2008

Abstract Purpose Animal models are valuable tools to study the pathogenic mechanisms of many diseases. Diabetic retinopathy is a low‐grade chronic inflammatory disease. Nitric oxide (NO) is involved in leukostasis and blood‐retinal barrier (BRB) breakdown in the early stages of the disease. However, the role of the different NO synthase (NOS) isoforms was not elucidated. We aimed to clarify the involvement of constitutive (eNOS, nNOS) and inducible NOS (iNOS) isoforms and the mechanisms underlying NO‐mediated leukostasis and BRB breakdown, by using an experimental model of diabetes and iNOS KO mice. Methods Diabetes was induced by streptozotocin in normal and KO mice (2 weeks of diabetes). Normal mice were treated with L‐NAME (NOS inhibitor). Vessel leakage was assessed with Evans blue. Leukostasis was quantified in flat‐mounted retinas and in vivo. ICAM‐1, occludin, ZO‐1 and nitrotyrosine levels were assessed by Western blotting or immunohistochemistry. Results Diabetes increased the permeability of BRB and leukostasis, which were reduced by L‐NAME. Similar effects were observed in diabetic iNOS KO mice. In diabetic mice, the immunoreactivity of tight junction proteins, occludin and ZO‐1, decreased, whereas ICAM‐1 protein levels increased. Those effects were prevented by L‐NAME and in diabetic iNOS KO mice. Diabetes also upregulated all NOS isoforms and increased nitrotyrosine levels in normal mice, but did not significantly increase eNOS and nNOS, and nitrotyrosine levels, in iNOS KO mice. Conclusion These data demonstrate that iNOS plays a predominant role in leukostasis and BRB breakdown. The mechanism involves ICAM‐1 upregulation and tight junction proteins downregulation. Support: FCT, Portugal
The role of FGF2 in the in vitro proliferation and angiogenesis of human choroidal endothelial cells
Authors: STEWART EA , AMOAKU WMK 
Published: September 2008

Abstract Purpose Age‐related macular degeneration (AMD) is the most common cause of irreversible visual loss in elderly populations in the western world. The mechanism of this disease is not yet fully understood, although vascular endothelial growth factor (VEGF) is thought to be the most significant growth factor in AMD, other growth factors, including FGF2 also stimulates the proliferation of human choroidal endothelial cells (hCEC). These factors may explain the only partial success of current anti‐VEGF AMD treatments. Methods hCEC were isolated from cadaver eyes and cultured in EBM2‐MV medium as previously published. hCEC proliferation after exposure to VEGF and FGF2 was measured using the WST‐1 assay and angiogenesis assessed using a double layer Matrigel technique. Western blotting was used to examine the intracellular signalling stimulated by FGF2 and VEGF. Results hCEC proliferation was increased in response to both FGF2 and VEGF. This effect was cumulative, indicating that these work through different intracellular signalling pathways. Western blotting identified common and differential intracellular signalling between the two growth factors. Conclusion Currently VEGF is thought to be the most important growth factor which stimulates neovascularisation leading to AMD and treatments developed reflect this. However, these results show that FGF2 also stimulates neovascularisation and may play a role in AMD development. A treatment which targets downstream signalling common to both growth factors may provide a more targeted and therefore successful treatment.
Form deprivation amblyopia ‐ a treatable cause of blindness
Authors: SJÖSTRAND JB 
Published: September 2008

Abstract Purpose Form deprivation amblyopia is associated with the obstruction or deformation of the passage of light causing degraded images on the retina. This lack of normal visual experience delays or blocks the visually driven development. If the obstacle is removed within the critical period the developing visual system has the capacity to regain visual function. Left untreated the deprivation and blocked visual experience may cause severe visual impairment and blindness. Methods To review our current understanding of the pathophysiology and risk factors in form deprivation amblyopia and to evaluate the effectiveness of treatment. The main conditions obstructing light passage during early development of vision will be described. Form deprivation amblyopia and its functional consequences are defined. Factors of importance for a satisfactory long term visual outcome are evaluated. Results Valid experimental and clinical models of form deprivation amblyopia are reviewed with focus on the role of visual deprivation per se and unequal visual input to the visual cortex, the primary site of functional change. Recent clinical observations of the visual outcome following early surgery of congenital dense bilateral and unilateral cataracts are used to evaluate the factors involved. Conclusion Form deprivation amblyopia is a complex condition with a hallmark of impaired visual acuity. For a satisfactory visual outcome early detection and intervention is important.
Development of immunostaining of cell cycle related proteins in flat mounted corneal endothelium
Authors: HE Z , THURET G , PIPPARELLI A , PISELLI S , DUMOLLARD JM , PEOC'H M , GAIN P 
Published: September 2008

Abstract Purpose Immunostaining of cell cycle proteins is necessary for the study of the proliferative status of corneal endothelial cells (EC). Most studies use cross sections, which offer direct access to intracellular antigens but allow visualization of only few cells, without giving a global view of an intact endothelium. We developed protocols for immunostaining of ECs of flat mounted corneas Methods Studied proteins: 4 proteins with a known expression pattern (intra‐membranous ZO‐1, cytoplasmic α actin, nuclear hnRNP and H3 histone) and 8 others, already described within EC: 3 cell cycle inhibitors (P27, P21, P16); 3 proliferation markers (PCNA, MCM2, Ki67), cyclins D1, and E. Fresh and organ cultured (OC) human corneas were used. Fixation/permeabilization: paraformaldehyde (PFA), acetone, methanol, ethanol, Hg, Zn, acetic acid, triton, sodium docecyl sulfate, alone or in combination. Antigen retrieval: heating and biochemical or chemical agents. Patterns of expression were also compared between endothelium and epithelium Results There was no universal protocol. Most of the time 0.5% PFA gave the best results, but for p21, and cyclins, specific protocols were necessary. Homogeneous staining was always obtained with a clear subcellular localization (nuclear or cytoplasmic). OC did not globally modify expression within EC whereas several nuclear or cytoplasmic translocations were observed within the epithelium Conclusion The use of protocols specifically designed for the endothelium of whole intact flat mounted corneas would allow a better localization of cell cycle proteins. They will be especially useful during attempts to alter the cell cycle and trigger EC proliferation. Grant: Fondation de l’Avenir 2007 ET7‐468
Inflammation in AMD pathology
Authors: NOWAK JZ 
Published: September 2008

AbstractAge‐related macular degeneration (AMD) is a progressive retinal disease that leads to substantial irreversible vision loss in elderly patients. Two clinical categories of AMD are distinguished: the “dry” atrophic form and the exudative neovascular or “wet” form. There is neither a preventive therapy nor a cure for both forms, although recent efforts succeeded in a more effective treatment of the wet AMD with PDT and anti‐VEGF drugs. AMD is a multifactorial pathology which involves complex interaction of metabolic, genetic and environmental factors, with major biochemical‐clinical abnormalities seen in four functionally interrelated tissues: photoreceptors, retinal pigment epithelium, Bruch’s membrane and choriocapilaries. Four processes specifically contribute to the development of AMD pathology: lipofuscinogenesis (in RPE cells), drusogenesis (with drusen located between RPE and Bruch’s membrane), inflammation (local) and choroidal neovascularization (in wet form). Although the role of immune system and inflammation has been implicated in AMD pathogenesis for many years, an impetus to intensify the research in this direction gave a recent discovery of polymorphisms in genes that encode for elements of the complement system, including factor H (CFH; Y402H), factor B, and complement component 2. An increased activity of the complement alternative pathway due to the lack of or insufficient control by CFH appears to contribute to AMD progression via immunologic mechanism which drives inflammatory response. An arising question is whether blockade of overactive complement system will be a therapeutic strategy safe for patients and effective to prevent or slowing down the macula‐devastating and vision‐threatening disease. Supported by grant no. 503‐1023‐1 from Medical University of Lodz.
Complement factor H and factor B expression in RPE cells
Authors: XU H , CHEN M , FORRESTER JV 
Published: September 2008

Abstract Purpose Age‐related macular degeneration (AMD) is the leading cause of untreatable blindness in the developed world. The pathogenesis of AMD is not fully understood. Recent evidence suggests that local inflammation in particular complement activation plays an important role. We aim to understand how complement activation is regulated at retina/choroidal interface. Methods The expression and distribution of complement factor H (CFH) and factor B (CFB) in mouse ocular tissues were examined by immunohistochemistry. Regulation of CFH and CFB gene expression by various cytokines or photoreceptor outer segments (POS) was investigated in vitro in cultured RPE cells. Changes in CFH or CFB gene expression after treatment were evaluated by RT‐PCR. Results In normal mouse eyes, CFH was detected in corneal epithelial cells, ciliary body, RPE cells, Bruch’s membrane and choroidal vessels. There is no significant change in either the expression level or the distribution pattern of CFH in ocular tissues of different ages of mice. CFB was exclusively detected in RPE cells in normal mice. The expression of CFB in RPE cells increases with age. In vitro in RPE cultures, the expression of CFH was negatively regulated by cytokine TNF‐alpha and IL‐6, whereas the expression of CFB was positively regulated by TNF‐alpha and IFN‐gamma. Short‐term incubation of RPE cells with POS did not alter the expression of CFH or CFB, whereas long‐term incubation of RPE cells with POS significantly down‐regulated CFH expression but up‐regulated CFB expression. Conclusion Complement regulatory factors CFH and CFB are produced locally in the retina/choroidal interface by RPE cells. The production of CFH and CFB in RPE cells is regulated differently by various cytokines and oxidized POS.
Inflammatory gene defects in AMD
Authors: IMMONEN I 
Published: September 2008

AbstractInflammatory markers have been raported in AMD patients at the serum, gene an tissue levels. Of the inflammatory cytokines especially IL‐6, IL‐8 and IL‐10 have been associated with AMD. Inflammatory cytokines are multifunctional molecules potentially stimulating macrophages and other inflammatory cells and choroidal neovascularization (CNV) in AMD lesions. The cytokines a modulate the expression of other inflammatory markers and are associated e.g with elevated levels of CRP in AMD. Targeting these pathways with non‐steroidal anti‐inflammatory agents may be used in future pharmacologic treatments for AMD before and after CNV formation. This presentation reviews current knowledge of inflammatory cytokines in relation to the RPE and AMD in general.
Cell cycle genes expression in human corneal endothelium: study by microarray and qRT‐PCR
Authors: PIPPARELLI A , THURET G , PISELLI S , PEOC'H M , ACQUART S , GARRAUD O , GAIN P 
Published: September 2008

Abstract Purpose To use microarray and qRT‐PCR to identify changes in cell cycle genes expression in human corneal endothelial cells (CEC) from “ in vivo”, post mortem, and organ cultured (OC) corneas, and also in confluent primary culture and immortalized CEC Methods Total RNAs were extracted. “in vivo” corneas were obtained during penetrating grafts for keratoconus, immediately after trephination, post mortem corneas (body donation to Science) within 24H after death. OC corneas were stored for 15D. Descemet was peeled off to avoid contamination with other cell types. Biotin‐labeled cRNA probe was synthesized from RNAs extract and hybridized to the pretreated Oligo GEArray Human Cell Cycle Microarrays, which covered 112 key genes of cell cycle. Microarrays were performed in duplicate. Gene expression of 'in vivo' CEC served as a reference. A difference >1,5 fold of the transcriptional level was considered significant. qRT‐PCR were done on cyclin E1; cyclin D1; p16INK4a; p21CIP1 and p27Kip1 to confirm the microarrays Results Compared to ‘in vivo’, CEC from OC corneas had 1 upregulated gene (CDK7) and 26 downregulated (CCNE1; CDKN1B; MCM2; RB1…), post mortem corneas had 2 up(CDC28; CDK7) genes and 16 downregulated (ATM; BAX; CDC16; CDKN1B…), primary culture had 1 up(CKS2) and 9 downregulated (BAX; CCNE1; CDK2; MCM2…), and the immortalized cell line had 16 up (ATM; CCNH; CKS2; MCM2…) and 2 downregulated genes (BAX; CDK5R2). qRT‐PCR of the 5 aforementioned genes validated the Microarrays data Conclusion Microarray seems to be a powerful tool to better understand the proliferative status of human CEC. It will help to choose the targets we need to alter in order to trigger CEC proliferation. Grant: Fondationdel’Avenir2007 ET7‐468