RESUMO
Iron has a fundamental role for cell physiology and especially in retina as a cofactor of many pathways of the visual transduction. A tightly regulated homeostasis avoids the accumulation of prooxidant and proinflammatory free iron. A dysfunction of iron retinal homeostasis is associated with many genetic or age-related degenerative diseases such as age-related macular degeneration (AMD). Here, we describe various mechanisms reported during AMD, enhanced by iron accumulation and its homeostasis dysregulation. We have investigated a local treatment with transferrin, the natural iron carrier, to control these pathological pathways and iron dysfunction, without side effects. Iron has a central role in pathogenesis of AMD and is a target for futures therapies.
TITLE: La dégénérescence maculaire liée à l'âge: La piste du fer. ABSTRACT: En raison de l'intense activité physiologique de la fonction visuelle, l'homéostasie du fer dans la rétine y est contrôlée localement. Sous l'effet de sa dérégulation (qui a des origines génétiques, environnementales, ou due au vieillissement), le fer libre s'accumule et devient, par ses propriétés oxydantes et inflammatoires, toxique, comme cela est observé au cours de la dégénérescence maculaire liée à l'âge (DMLA). Le rétablissement d'un métabolisme du fer équilibré est donc une possibilité thérapeutique. Néanmoins, la toxicité oculaire des chélateurs chimiques oriente les recherches vers des chélateurs biologiques naturels. Nos travaux montrent que la transferrine, le transporteur du fer, préserve la rétine des mécanismes associés à la DMLA.
Assuntos
Ferro/fisiologia , Degeneração Macular/etiologia , Homeostase/genética , Humanos , Ferro/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/terapia , Redes e Vias Metabólicas/genética , Retina/metabolismo , Retina/patologia , Terapias em Estudo/métodos , Terapias em Estudo/tendências , Transferrina/genética , Transferrina/fisiologiaRESUMO
Iron is essential for cell survival and function. It is a transition metal, that could change its oxidation state from Fe2+ to Fe3+ involving an electron transfer, the key of vital functions but also organ dysfunctions. The goal of this review is to illustrate the primordial role of iron and local iron homeostasis in retinal physiology and vision, as well as the pathological consequences of iron excess in animal models of retinal degeneration and in human retinal diseases. We summarize evidence of the potential therapeutic effect of iron chelation in retinal diseases and especially the interest of transferrin, a ubiquitous endogenous iron-binding protein, having the ability to treat or delay degenerative retinal diseases.
Assuntos
Ferro/fisiologia , Retina/fisiopatologia , Animais , Transporte Biológico , Homeostase , Humanos , Ferro/metabolismo , Ferro/toxicidade , Quelantes de Ferro/uso terapêutico , Oxigênio/metabolismo , Retina/anatomia & histologia , Retina/metabolismo , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Degeneração Retiniana/prevenção & controle , Transferrina/genética , Transferrina/metabolismo , Transferrina/uso terapêuticoRESUMO
Iron is essential for retinal function but contributes to oxidative stress-mediated degeneration. Iron retinal homeostasis is highly regulated and transferrin (Tf), a potent iron chelator, is endogenously secreted by retinal cells. In this study, therapeutic potential of a local Tf delivery was evaluated in animal models of retinal degeneration. After intravitreal injection, Tf spread rapidly within the retina and accumulated in photoreceptors and retinal pigment epithelium, before reaching the blood circulation. Tf injected in the vitreous prior and, to a lesser extent, after light-induced retinal degeneration, efficiently protected the retina histology and function. We found an association between Tf treatment and the modulation of iron homeostasis resulting in a decrease of iron content and oxidative stress marker. The immunomodulation function of Tf could be seen through a reduction in macrophage/microglial activation as well as modulated inflammation responses. In a mouse model of hemochromatosis, Tf had the capacity to clear abnormal iron accumulation from retinas. And in the slow P23H rat model of retinal degeneration, a sustained release of Tf in the vitreous via non-viral gene therapy efficently slowed-down the photoreceptors death and preserved their function. These results clearly demonstrate the synergistic neuroprotective roles of Tf against retinal degeneration and allow identify Tf as an innovative and not toxic therapy for retinal diseases associated with oxidative stress.
Assuntos
Modelos Animais de Doenças , Inflamação/prevenção & controle , Ferro/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Degeneração Retiniana/prevenção & controle , Transferrina/farmacologia , Animais , Células Cultivadas , Homeostase/efeitos dos fármacos , Técnicas Imunoenzimáticas , Inflamação/induzido quimicamente , Masculino , Camundongos , RNA Mensageiro/genética , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Degeneração Retiniana/induzido quimicamente , Degeneração Retiniana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Iron is necessary for cell metabolism, but excess iron can be toxic Iron can generate oxygen free radicals through the Fenton reaction. Iron accumulation has been observed in the retina of patients with age-related macular degeneration (AMD). We have shown its accumulation in photoreceptor segments in two animal models of genetic retinal degeneration (RCS rats and Rd10 mice). In these rodents, hTf, injected intraperitoneally or expressed by genetic modification, delayed photoreceptor degeneration. Our studies highlight the therapeutic potential of Tf in degenerative processes such as retinitis pigmentosa and AMD.
Assuntos
Proteínas Reguladoras de Ferro/metabolismo , Ferro/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Ratos , Degeneração Retiniana/genética , Degeneração Retiniana/patologiaRESUMO
PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.
Assuntos
Envelhecimento/fisiologia , Apoferritinas/genética , Regulação da Expressão Gênica/fisiologia , Proteínas de Ligação ao Ferro/genética , Lesões Experimentais por Radiação/genética , Retina/efeitos da radiação , Degeneração Retiniana/genética , Animais , Apoferritinas/metabolismo , Proteínas de Transporte de Cátions/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Ferro/metabolismo , Luz , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , RNA Mensageiro/genética , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Retinal degeneration has been associated with iron accumulation in age-related macular degeneration (AMD), and in several rodent models that had one or several iron regulating protein impairments. We investigated the iron concentration and the protective role of human transferrin (hTf) in rd10 mice, a model of retinal degeneration. METHODS: The proton-induced X-ray emission (PIXE) method was used to quantify iron in rd10 mice 2, 3, and 4 weeks after birth. We generated mice with the ß-phosphodiesterase mutation and hTf expression by crossbreeding rd10 mice with TghTf mice (rd10/hTf mice). The photoreceptor loss and apoptosis were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling in 3-week-old rd10/hTf mice and compared with 3-week-old rd10 mice. The neuroprotective effect of hTf was analyzed in 5-day-old rd10 mice treated by intraperitoneal administration with hTf for up to 25 days. The retinal hTf concentrations and the thickness of the outer nuclear layer were quantified in all treated mice at 25 days postnatally. RESULTS: PIXE analysis demonstrated an age-dependent iron accumulation in the photoreceptors of rd10 mice. The rd10/hTf mice had the rd10 mutation, expressed high levels of hTf, and showed a significant decrease in photoreceptor death. In addition, rd10 mice intraperitoneally treated with hTf resulted in the retinal presence of hTf and a dose-dependent reduction in photoreceptor degeneration. CONCLUSIONS: Our results suggest that iron accumulation in the retinas of rd10 mutant mice is associated with photoreceptor degeneration. For the first time, the enhanced survival of cones and rods in the retina of this model has been demonstrated through overexpression or systemic administration of hTf. This study highlights the therapeutic potential of Tf to inhibit iron-induced photoreceptor cell death observed in degenerative diseases such as retinitis pigmentosa and age-related macular degeneration.
Assuntos
Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/prevenção & controle , Transferrina/administração & dosagem , Transferrina/uso terapêutico , Animais , Transporte Biológico/efeitos dos fármacos , Modelos Animais de Doenças , Microanálise por Sonda Eletrônica , Humanos , Injeções Intraperitoneais , Ferro/metabolismo , Camundongos , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Células Fotorreceptoras Retinianas Cones/efeitos dos fármacos , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Degeneração Retiniana/patologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia , Espectrometria por Raios X , Transferrina/farmacologiaAssuntos
Antígenos CD36/metabolismo , Lipoproteínas LDL/metabolismo , Degeneração Macular/metabolismo , Fagocitose , Epitélio Pigmentado da Retina/metabolismo , Fatores Etários , Envelhecimento/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/patologia , Antígenos CD36/deficiência , Antígenos CD36/genética , Gorduras na Dieta/metabolismo , Humanos , Degeneração Macular/genética , Degeneração Macular/patologia , Camundongos , Epitélio Pigmentado da Retina/patologiaRESUMO
Fibroblast growth factor 1 (FGF1) is a differentiation and survival factor for neuronal cells both in vitro and in vivo. FGF1 activities can be mediated not only by paracrine and autocrine pathways involving FGF receptors but also by an intracrine pathway, which is an underestimated mode of action. Indeed, FGF1 lacks a secretion signal peptide and contains a nuclear localization sequence (NLS), which is consistent with its usual intracellular and nuclear localization. To progress in the comprehension of the FGF1 intracrine pathway in neuronal cells, we examined the role of the nuclear translocation of FGF1 for its neurotrophic activity as well as for its protective activity against p53-dependent apoptosis. Thus, we have transfected PC12 cells with different FGF1 expression vectors encoding wild type or mutant (Delta NLS) FGF1. This deletion inhibited both FGF1 nuclear translocation and FGF1 neurotrophic activity (including differentiation and serum-free cell survival). We also show that endogenous FGF1 protection of PC12 cells against p53-dependent cell death requires FGF1 nuclear translocation. Strikingly, wild type FGF1 is found interacting with p53, in contrast to the mutant FGF1 deleted of its NLS, suggesting the presence of direct and/or indirect interactions between FGF1 and p53 pathways. Thus, we present evidences that FGF1 may act by a nuclear pathway to induce neuronal differentiation and to protect the cells from apoptosis whether cell death is induced by serum depletion or p53 activation.
Assuntos
Apoptose/fisiologia , Núcleo Celular/metabolismo , Fator 1 de Crescimento de Fibroblastos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Comunicação Autócrina/fisiologia , Diferenciação Celular/fisiologia , Núcleo Celular/genética , Sobrevivência Celular/fisiologia , Fator 1 de Crescimento de Fibroblastos/genética , Mutação , Células PC12 , Comunicação Parácrina/fisiologia , Ratos , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
PURPOSE: Transferrin (Tf) expression is enhanced by aging and inflammation in humans. We investigated the role of transferrin in glial protection. METHODS: We generated transgenic mice (Tg) carrying the complete human transferrin gene on a C57Bl/6J genetic background. We studied human (hTf) and mouse (mTf) transferrin localization in Tg and wild-type (WT) C57Bl/6J mice using immunochemistry with specific antibodies. Müller glial (MG) cells were cultured from explants and characterized using cellular retinaldehyde binding protein (CRALBP) and vimentin antibodies. They were further subcultured for study. We incubated cells with FeCl(3)-nitrilotriacetate to test for the iron-induced stress response; viability was determined by direct counting and measurement of lactate dehydrogenase (LDH) activity. Tf expression was determined by reverse transcriptase-quantitative PCR with human- or mouse-specific probes. hTf and mTf in the medium were assayed by ELISA or radioimmunoassay (RIA), respectively. RESULTS: mTf was mainly localized in retinal pigment epithelium and ganglion cell layers in retina sections of both mouse lines. hTf was abundant in MG cells. The distribution of mTf and hTf mRNA was consistent with these findings. mTf and hTf were secreted into the medium of MG cell primary cultures. Cells from Tg mice secreted hTf at a particularly high level. However, both WT and Tg cell cultures lose their ability to secrete Tf after a few passages. Tg MG cells secreting hTf were more resistant to iron-induced stress toxicity than those no longer secreted hTf. Similarly, exogenous human apo-Tf, but not human holo-Tf, conferred resistance to iron-induced stress on MG cells from WT mice. CONCLUSIONS: hTf localization in MG cells from Tg mice was reminiscent of that reported for aged human retina and age-related macular degeneration, both conditions associated with iron deposition. The role of hTf in protection against toxicity in Tg MG cells probably involves an adaptive mechanism developed in neural retina to control iron-induced stress.
Assuntos
Ferro/toxicidade , Neuroglia/metabolismo , Transferrina/metabolismo , Animais , Apoproteínas/metabolismo , Sobrevivência Celular , Células Cultivadas , Meios de Cultura , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Transferrina/genética , Transferrina/farmacologiaRESUMO
PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.
Assuntos
Diester Fosfórico Hidrolases/genética , Mutação Puntual , Degeneração Retiniana/genética , Degeneração Retiniana/terapia , Reparo Gênico Alvo-Dirigido , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Olho/enzimologia , Imuno-Histoquímica/métodos , Iontoforese , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Oligonucleotídeos/administração & dosagem , Oligonucleotídeos/uso terapêutico , Diester Fosfórico Hidrolases/metabolismo , Retina/enzimologia , Retina/patologia , Degeneração Retiniana/enzimologia , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Coloração e RotulagemRESUMO
In 1990, following an idea arising from an Inserm study section on aging, the Diplôme d'études approfondies (DEA) de Biologie du vieillissement was created. Since then, more than 300 students have followed these courses which cover the cellular mechanisms of aging and associated diseases, from basic causes of aging to CNS and sensory organs aging, as well as nutritional aspects, sarcopenia and osteoporosis, vascular and neuroendocrine aging. More than 150 thesis have been defended and more than a quarter of students has been recruited on permanent positions in French universities and research institutions (10 %) and hospitals (16 %). Since its creation, one of the particularities of the DEA was the formal links between academia and industry since teaching takes place on private laboratory settings.
Assuntos
Envelhecimento , Educação de Pós-Graduação/tendências , Pesquisa/tendências , Ensino/métodos , Universidades/tendências , Animais , França , HumanosRESUMO
PURPOSE: The antiangiogenic effect of an antisense oligodeoxynucleotide (ODN) targeting insulin receptor substrate (IRS)-1 was evaluated on rat corneal neovascularization. METHODS: Eyes with neovessels were treated with subconjunctival injections of IRS-1 antisense oligonucleotide (ASODN), IRS-1 sense ODN (SODN), or PBS. At 8 and 24 hours after the first subconjunctival injection, the expression of IRS-1, VEGF, and IL-1beta mRNA was evaluated. IRS-1 protein levels were also measured at 8 hours by Western blot analysis (n = 4/group). On day 10, corneal neovascularization was quantified in flatmount corneas of rats treated daily from days 4 to 9. RESULTS: On day 10, new vessels covered 95.5% +/- 4% of the corneal area in PBS-treated eyes, 92% +/- 7% in SODN-treated eyes and 59% +/- 20% in ASODN-treated eyes (P < 0.001). In the ASODN-treated group, the expression and synthesis of IRS-1 were significantly downregulated when compared with the control groups. ASODN did not significantly affect the expression of VEGF but significantly decreased the expression of IL-1beta at 24 hours (P = 0.04). CONCLUSIONS: Subconjunctival injections of IRS-1 antisense ODN significantly inhibit rat corneal neovascularization. This effect may be mediated by a downregulation of IL-1beta. IRS-1 proteins may be interesting targets for the regulation of angiogenesis mediated by insulin, hypoxia, or inflammation.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Neovascularização da Córnea/prevenção & controle , Oligodesoxirribonucleotídeos Antissenso/uso terapêutico , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Animais , Western Blotting , Túnica Conjuntiva/efeitos dos fármacos , Neovascularização da Córnea/metabolismo , Neovascularização da Córnea/patologia , Modelos Animais de Doenças , Regulação para Baixo , Imuno-Histoquímica , Injeções , Proteínas Substratos do Receptor de Insulina , Interleucina-1/genética , Interleucina-1/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Organismos Livres de Patógenos Específicos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PURPOSE: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo. METHODS: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination. RESULTS: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells. CONCLUSIONS: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.
Assuntos
Neoplasias da Coroide/metabolismo , Técnicas de Transferência de Genes , Luz , Melanoma/metabolismo , Oligonucleotídeos Antissenso/genética , Epitélio Pigmentado Ocular/metabolismo , Proteínas Estruturais Virais/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Expressão Gênica , Vetores Genéticos , Humanos , Masculino , Microscopia Confocal , Oligonucleotídeos Antissenso/metabolismo , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas c-raf/genética , Ratos , Ratos Endogâmicos LewRESUMO
Retinal death induced by light seems to be a caspase-independent process. In this work we investigate the LEI/L-DNase II pathway, a caspase-independent pathway, in light-induced retinal degeneration in Fischer rats. Measurement of DNase activity in total retinal extracts of light exposed Fischer rats was performed by analysing a plasmid degradation on an agarose gel. The same method was used to measure the in vitro activity of recombinant LEI (reticulocyte lysate) after incubation with calpains. L-DNase II activity is observed in retinal extracts of light exposed Fischer rats and increases with time illumination. In this apoptotic death, the activation of calpains has been shown. Here we show that L-DNase II activation is not catalized by calpains. The present study indicates that the LEI/L-DNase II may be a possible pathway activated during photoreceptor apoptosis in light-induced retinal degeneration but that this pathway is not directly activated by calpains.
Assuntos
Endodesoxirribonucleases/metabolismo , Luz/efeitos adversos , Proteínas/metabolismo , Degeneração Retiniana/enzimologia , Animais , Anexina A1/farmacologia , Western Blotting/métodos , Proteínas de Ligação ao Cálcio/farmacologia , Interações Medicamentosas , Técnicas In Vitro , Masculino , Proteínas dos Microfilamentos , Elastase Pancreática/farmacologia , Proteínas Secretadas Inibidoras de Proteinases , Proteínas/farmacologia , Ratos , Ratos Endogâmicos F344 , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia , Retina/efeitos da radiação , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Fatores de Tempo , CalponinasRESUMO
PURPOSE: To analyse the mechanism of ethanol-induced cell death, and particularly, the activation of the leucocyte elastase inhibitor (LEI) pathway. METHODS: Cultured ARPE-19 cells were exposed to 0-13% ethanol for 24 h. Cytotoxicity was estimated by morphologic changes within the nucleus and breakdown of DNA, assessed by agarose gel electrophoresis or flow cytometry cell sorter. Poly(ADP-ribose)polymerase cleavage (PARP) was determined by western blot analysis. Changes in transcription and translation of LEI were assessed by analysis of mRNA levels and expression of protein product (immunohistochemistry), respectively. RESULTS: We established the ability of ethanol to induce cell death in ARPE-19 cells. After a 24 h incubation with 4% ethanol, 50% of the cells died; all the cells died in the presence of 10% ethanol. After ethanol incubation, we observed nuclear condensation and DNA fragmentation; the amount of fragmentation was proportional to the ethanol level. By flow cytometry analysis and agarose gel electrophoresis, the pattern of DNA cleavage exhibited a sub-G1 peak, suggesting necrotic cell death. However, other observations, i.e. nuclei shrinkage, PARP cleavage and inhibition of cell death by cycloheximide, and activation of a caspase independent LEI/DNase II pathway were observed and are features associated with apoptotic cell death. During ethanol stress, an LEI/L-DNase II intermediate was lost, leading to complete activation L-DNase II (24 kDa). RT-PCR analysis showed an early and specific increase of the LEI mRNA. Cycloheximide inhibited LEI synthesis and protected cells against apoptosis. CONCLUSIONS: Our data indicate that ethanol stress on ARPE-19 cells can induce a pathway which is a form of programmed cell death with characteristics of both apoptosis and necrosis, possibly by triggering conversion of LEI to L-DNase II.
Assuntos
Endodesoxirribonucleases/metabolismo , Etanol/farmacologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Serpinas/metabolismo , Western Blotting , Morte Celular/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Epitélio Pigmentado Ocular/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/genéticaRESUMO
BACKGROUND: Since the introduction of cold corneoscleral segment storage prior to keratoplasty there have been continuous efforts to ameliorate the preservation media in order to better maintain the quality of the corneal epi- and endothelium. Recent studies have shown that basic fibroblast growth factor (FGF-2) preserves the viability of, for example, retinal ganglion cells and pigment epithelium cells. Therefore, we investigated the effect of different concentrations of FGF-2 added to a modified Optisol storage medium on endothelial damage after corneal storage at 4 degrees C. METHODS: . Bovine corneas were stored at 4 degrees C for 14 days and for another 24 h at 34 degrees C. Various FGF-2 concentrations (4, 20 and 40 ng/ml) were added to the medium either at day (D) 1, D14, or both D1 and D14. Quantitative evaluation of corneal damage after 14+1 days of storage was conducted by means of the Janus green photometry assay. Histological and ultrastructural investigations of the preserved endothelium were also performed. Bovine cell culture experiments using the TUNEL assay aimed to elucidate the role of FGF-2 on prevention of endothelial apoptosis. RESULTS: The mean endothelial damage in control corneas increased from 4.9 +/- 1.8% (fresh corneas) to 13.4 +/- 2.4% after 14+1 days of storage. FGF-2 at 20 ng/ml or 40 ng/ml added at any of the indicated time points significantly reduced the overall endothelial damage by 5.1-7.3%, corresponding to 38-54% less endothelial damage than in control corneas (P<0.001). Light- and electron microscopic investigations confirmed this protective effect of FGF-2 on corneal endothelial cells. The TUNEL assay revealed a true anti-apoptotic effect of FGF-2 on endothelial cells in culture. CONCLUSION: Our study clearly demonstrates the effectiveness of FGF-2 to enhance cell survival of the corneal endothelium after storage at 4 degrees C. A clinical interest could be seen in the potential future application of FGF-2 as an adjuvant to corneal preservation media in order to better maintain endothelial viability during corneal storage.
Assuntos
Criopreservação , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/patologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Bovinos , Citoproteção , Relação Dose-Resposta a Droga , Endotélio Corneano/fisiopatologia , Endotélio Corneano/ultraestrutura , Fator 2 de Crescimento de Fibroblastos/administração & dosagem , Humanos , Técnicas In Vitro , Microscopia Eletrônica de Varredura , Concentração Osmolar , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologiaAssuntos
Apoptose , Endodesoxirribonucleases/metabolismo , Serina Endopeptidases/fisiologia , Fator de Necrose Tumoral alfa/toxicidade , Compostos de Boro/farmacologia , Ativação Enzimática , Humanos , Oligopeptídeos/farmacologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/farmacologia , Células U937RESUMO
Ischemic proliferative retinopathy (e.g., diabetes mellitus, retinopathy of prematurity, or retinal vein occlusion) is a major cause of blindness worldwide. Apart from neovascularization, ischemic proliferative retinopathy leads to retinal degeneration. Apoptosis has been ascribed to be the leading mechanism in ischemic retinal degeneration. We showed recently that inducible nitric oxide synthase (iNOS) is expressed in the avascular retina in proliferative retinopathy in vivo and that iNOS expression in retinal glial cells is responsible for retinal neuronal cell death in vitro. Here we show that retinal apoptosis and subsequent degeneration occur in the murine model of ischemic proliferative retinopathy. Furthermore, because NO can have beneficial or detrimental effects in the retina, we analyzed the role of iNOS on retinal apoptosis in ischemic proliferative retinopathy. Using iNOS knock-out mice and iNOS inhibitor 1400W, we demonstrate in vivo that iNOS expression induces apoptosis locally in the inner nuclear layer of the avascular retina and that protein nitration may be involved in this process. These findings are the first evidence for retinal apoptosis in an animal model of ischemic proliferative retinopathy, demonstrating that iNOS plays a crucial role not only in retinal neovascular disease but also in retinal degeneration. We show that it is an ideal target to protect the hypoxic retina from degeneration and to improve its vascularization.
Assuntos
Apoptose , Isquemia/enzimologia , Óxido Nítrico Sintase/metabolismo , Doenças Retinianas/metabolismo , Tirosina/análogos & derivados , Envelhecimento/metabolismo , Amidinas/administração & dosagem , Animais , Animais Recém-Nascidos , Benzilaminas/administração & dosagem , Contagem de Células , Cruzamentos Genéticos , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Modelos Animais de Doenças , Vias de Administração de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Isquemia/complicações , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Oxigênio/toxicidade , Proteínas/metabolismo , Retina/efeitos dos fármacos , Retina/enzimologia , Retina/patologia , Doenças Retinianas/complicações , Doenças Retinianas/tratamento farmacológico , Doenças Retinianas/patologia , Oclusão da Veia Retiniana/metabolismo , Oclusão da Veia Retiniana/patologia , Tirosina/metabolismoRESUMO
PURPOSE: To determine whether iron homeostasis disorder accompanies retinal degeneration in Royal College of Surgeons (RCS) rats. METHODS: The presence of iron was revealed directly by proton-induced X-ray emission (PIXE) and indirectly by electron microscopy (EM). Ferritin, transferrin (Tf), and transferrin receptor (Tf-R) were localized by immunohistochemistry. Ferritin and Tf proteins were analyzed by Western blot analysis. Comparative study of Tf-R content was performed by slot-blot analysis and ferritin content was evaluated by enzyme-linked immunosorbent assay (ELISA). Ferritin and Tf-R expression was studied by reverse transcription-polymerase chain reaction (RT-PCR) and Tf expression by in situ hybridization (ISH). All studies were performed in RCS and control retinas from postnatal days (PN)20 to PN55. RESULTS: PIXE analysis showed iron accumulation in outer retina of RCS rats in a time-dependent manner. EM studies revealed irregular iron inclusions on partially degenerated outer segments (OS) of photoreceptors and lamellar whorls at PN35 and very large iron deposits on membranes from a debris layer at PN55. No such deposits were found in the inner retina. Ferritin and Tf-R expression and protein levels seemed to be unaffected in the inner part of the retina. Iron accumulation was preceded by Tf degradation, as revealed by immunohistochemistry and Western blot analysis. Tf mRNA was detected in RCS rat retinal pigment epithelium (RPE) at all stages studied. CONCLUSIONS: This study presents the first evidence for a correlation of iron homeostasis imbalance with the neurodegenerative state of the retina in RCS rats. The iron imbalance is not the underlying genetic defect but is the result of impaired RPE-photoreceptor interaction, which leads to debris accumulation and subsequent blockage of the outer retina's iron delivery pathway. The increase of iron in the photoreceptor area may enhance the vulnerability of cells to oxidative stress.