RESUMO
During development, progenitor cells of the retina give rise to six principal classes of neurons and the Müller glial cells found within the adult retina. The pancreas transcription factor 1 subunit a (Ptf1a) encodes a basic-helix-loop-helix transcription factor necessary for the specification of horizontal cells and the majority of amacrine cell subtypes in the mouse retina. The Ptf1a-regulated genes and the regulation of Ptf1a activity by transcription cofactors during retinogenesis have been poorly investigated. Using a retrovirus-mediated gene transfer approach, we reported that Ptf1a was sufficient to promote the fates of amacrine and horizontal cells from retinal progenitors and inhibit retinal ganglion cell and photoreceptor differentiation in the chick retina. Both GABAergic H1 and non-GABAergic H3 horizontal cells were induced following the forced expression of Ptf1a. We describe Ptf1a as a strong, negative regulator of Atoh7 expression. Furthermore, the Rbpj-interacting domains of Ptf1a protein were required for its effects on cell fate specification. Together, these data provide a novel insight into the molecular basis of Ptf1a activity on early cell specification in the chick retina.
Assuntos
Proteínas Aviárias/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Retina/embriologia , Retina/metabolismo , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/metabolismo , Fatores de Transcrição/metabolismo , Animais , Proteínas Aviárias/química , Proteínas Aviárias/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular , Embrião de Galinha , Regulação da Expressão Gênica no Desenvolvimento , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/química , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/genética , Camundongos , Complexos Multiproteicos , Domínios e Motivos de Interação entre Proteínas , Retina/citologia , Células Ganglionares da Retina/classificação , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
PURPOSE: To compare the expression profiles of various cytokines and chemokines in vitreous samples from patients with retinal detachment (RD) to those from controls and to analyze their association with various clinical features. METHODS: In this prospective study, undiluted vitreous fluid was obtained from 41 patients with primary RD and 33 controls with macular hole or vitreomacular traction. A multiplex bead immunoassay was performed to determine the expression of 27 inflammatory mediators. RESULTS: Eleven mediators were significantly upregulated in the vitreous of RD patients compared with controls, including the following: cytokines IL-1ra, IL-6, IL-7, IL-8, IFN-γ; chemokines CCL2, CCL3, CCL4, CXCL10 and CCL11 and growth factor G-CSF. Correlation analyses showed that levels of IL-1ra, CXCL10, CCL11 and G-CSF were positively correlated to the extent of detachment, while those of IL-1ra and CXCL10 were associated with the duration of detachment. There was also a positive association between the concentrations of CXCL10 and CCL11 and preoperative flare values. Additional analysis revealed that flare values and both CXCL10 and CCL11 levels were significantly higher in eyes with grade B or C proliferative vitreoretinopathy (PVR). CONCLUSION: Our results confirm that RD induces a marked inflammatory response with a complex cytokine network. We identified proteins specifically linked to several clinical features that might contribute to photoreceptor degeneration and PVR-related redetachment. These proteins may represent potential therapeutic targets for improving the anatomical and functional outcomes of RD surgery.
Assuntos
Descolamento Retiniano , Vitreorretinopatia Proliferativa , Citocinas , Humanos , Estudos Prospectivos , Vitreorretinopatia Proliferativa/diagnóstico , Corpo VítreoRESUMO
BACKGROUND: An association of the Tyr402His variant of the complement factor H (CFH) gene with age-related macular degeneration (AMD) has been shown in several Caucasian populations, while studies for an association with other single nucleotide polymorphisms (SNP) of complement system genes have produced inconsistent results. We examined the distribution of several SNPs of complement system genes (CFH, C 2, C 3, factor B) in patients with exsudative AMD and healthy controls. PATIENTS/MATERIALS AND METHODS: 226 patients with exsudative AMD and 179 controls without AMD were included. Genomic DNA was extracted from saliva samples. RESULTS: A significant association with exsudative AMD was found only for SNP rs1061170 (Y402 H) in the CFH gene. For rs1047286 (P292L) and rs2230199 (R102G) in the C 3 gene, rs547154 (IVS10) and rs9332739 (E318D) in the C 2 gene and rs4151667 (L9 H) in CFB gene, no associations with exsudative AMD were found. CONCLUSIONS: We have replicated an association of the Y 402 H variant with exsudative AMD in our population. Although variants R 102G, IVS10, E 318D and L 9 H have been shown to be associated with AMD in earlier studies, we could not confirm these findings. The results show that AMD has variable association patterns with rare variants in different populations.
Assuntos
Proteínas do Sistema Complemento/genética , Predisposição Genética para Doença/epidemiologia , Predisposição Genética para Doença/genética , Degeneração Macular/epidemiologia , Degeneração Macular/genética , Polimorfismo de Nucleotídeo Único/genética , Idoso , Idoso de 80 Anos ou mais , Feminino , Marcadores Genéticos/genética , Alemanha/epidemiologia , Humanos , Masculino , Prevalência , Medição de Risco/métodos , Fatores de RiscoRESUMO
Drusen, the white yellowish deposits that can be seen in funduscopy, are a hallmark of age-related macular degeneration. Histologically, drusen are believed to be dome-shaped or more confluent lipid accumulations between the retinal pigment epithelium and the choriocapillaries. Recent advances in mouse funduscopy have revealed the presence of drusen-like structures in chemokine knockout animals in the absence of sizeable dome-shaped material below the retinal pigment epithelium. We show that aged CX3CR1-/- mice present with drusen-like appearance in funduscopy that is associated with a progressive age-related microglial cell accumulation in the subretinal space. We demonstrate that the anatomical equivalent of the drusen-like appearance in these mice are lipid-bloated subretinal microglial cells rather than subretinal pigment epithelium deposits [Combadière C, et al: J Clin Invest 2007;117:2920-2928].
Assuntos
Microglia/ultraestrutura , Epitélio Pigmentado Ocular/ultraestrutura , Receptores de Quimiocinas/fisiologia , Drusas Retinianas/patologia , Animais , Receptor 1 de Quimiocina CX3C , Modelos Animais de Doenças , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Epitélio Pigmentado Ocular/metabolismo , Drusas Retinianas/metabolismo , Índice de Gravidade de DoençaRESUMO
Intravitreal neovascular diseases are a major cause of blindness worldwide. It remains unclear why neovessels in many retinal diseases spread into the physiologically nonvascularized vitreous rather than into the ischemic retinal areas, where the angiogenic factors are released. Here we show that inducible nitric oxide synthase (iNOS) is expressed in the ischemic retina. Using iNOS knockout mice and the iNOS inhibitor 1400W, we demonstrate that iNOS expression inhibits angiogenesis locally in the avascular retina, mediated at least in part by a downregulation of VEGF receptor 2 (VEGFR2) in cells adjacent to iNOS-expressing cells. At the same time, pathological intravitreal neovascularization is considerably stronger in iNOS-expressing animals. These findings demonstrate that iNOS plays a crucial role in retinal neovascular disease and show that it offers an ideal target for the control of vitreal neovascularization through improvement of the vascularization of the hypoxic retina.
Assuntos
Isquemia/enzimologia , Neovascularização Patológica , Óxido Nítrico Sintase/fisiologia , Retina/enzimologia , Vasos Retinianos/enzimologia , Corpo Vítreo/irrigação sanguínea , Corpo Vítreo/enzimologia , Animais , Sequência de Bases , Primers do DNA/genética , Modelos Animais de Doenças , Humanos , Isquemia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/deficiência , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/patologia , Vasos Retinianos/patologiaRESUMO
BACKGROUND: Intravitreal neovascular diseases, as in ischemic retinopathies, are a major cause of blindness. Because inflammatory mechanisms influence vitreal neovascularization and cyclooxygenase (COX)-2 promotes tumor angiogenesis, we investigated the role of COX-2 in ischemic proliferative retinopathy. METHODS AND RESULTS: We describe here that COX-2 is induced in retinal astrocytes in human diabetic retinopathy, in the murine and rat model of ischemic proliferative retinopathy in vivo, and in hypoxic astrocytes in vitro. Specific COX-2 but not COX-1 inhibitors prevented intravitreal neovascularization, whereas prostaglandin E2, mainly via its prostaglandin E receptor 3 (EP3), exacerbated neovascularization. COX-2 inhibition induced an upregulation of thrombospondin-1 and its CD36 receptor, consistent with the observed antiangiogenic effects of COX-2 inhibition; EP3 stimulation reversed effects of COX-2 inhibitors on thrombospondin-1 and CD36. CONCLUSIONS: These findings point to an important role for COX-2 in ischemic proliferative retinopathy, as in diabetes.
Assuntos
Retinopatia Diabética/enzimologia , Isquemia/enzimologia , Isoenzimas/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores Imunológicos , Vitreorretinopatia Proliferativa/enzimologia , Adulto , Idoso , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Astrócitos/patologia , Antígenos CD36/metabolismo , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Ciclo-Oxigenase 2 , Retinopatia Diabética/complicações , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/patologia , Dinoprostona/metabolismo , Modelos Animais de Doenças , Fatores de Crescimento Endotelial/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Isquemia/complicações , Isquemia/patologia , Isoenzimas/antagonistas & inibidores , Linfocinas/metabolismo , Masculino , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Lipoproteínas/metabolismo , Receptores de Prostaglandina E/efeitos dos fármacos , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP3 , Receptores de Prostaglandina E Subtipo EP4 , Receptores Depuradores , Retina/efeitos dos fármacos , Retina/enzimologia , Retina/patologia , Vasos Retinianos/efeitos dos fármacos , Vasos Retinianos/patologia , Trombospondina 1/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio Vascular , Vitreorretinopatia Proliferativa/complicações , Vitreorretinopatia Proliferativa/tratamento farmacológicoRESUMO
In addition to its hemostatic functions, factor (F)VIIa exhibits cell proliferative properties as seen in angiogenesis and tumor growth. A role for tissue factor (TF) and protease-activated receptors (PAR)-1 and -2 in cell proliferation remain to be clarified. We tested the hypothesis that FVIIa induces cell proliferation by a mechanism involving TF and PAR-2. Human recombinant FVIIa induced cell proliferation of human BOSC23 cells transfected with plasmid containing human TF DNA sequence. Because DNA primase 1 (PRIM1) plays an essential role in cell proliferation, we used the cloned PRIM1 promoter upstream of the reporter gene chloramphenicol acetyl transferase (CAT) to elucidate the mode of action of FVIIa. FVIIa evoked a dose-dependent increase in cell proliferation and PRIM1 induction, which were markedly potentiated (4-5-fold) by the presence of TF and abrogated by TF antisense oligonucleotide. PRIM1 induction by FVIIa was also abolished by PAR-2 but not by PAR-1 antisense. In contrast, thrombin induced a small increase in CAT activity which was unaffected by TF, but was prevented only by PAR-1 antisense as well as the thrombin inhibitor hirudin. Proliferative properties of FVIIa were associated with a TF-dependent increase in intracellular calcium and were mediated by a concordant phosphorylation of p44/42 MAP kinase. In conclusion, data reveal that FVIIa induces PRIM1 and ensuing cellular proliferation via a TF- and of the PARs entirely PAR-2-dependent pathway, in distinction to that of thrombin which is PAR-1-dependent and TF-independent. We speculate that FVIIa-TF-PAR-2 inhibitors may be effective in suppressing cell proliferation.
Assuntos
Fator VIIa/metabolismo , Receptor PAR-2/metabolismo , Tromboplastina/fisiologia , Northern Blotting , Western Blotting , Linhagem Celular , Proliferação de Células , Cloranfenicol O-Acetiltransferase/metabolismo , Clonagem Molecular , DNA/química , DNA Primase/metabolismo , Relação Dose-Resposta a Droga , Genes Reporter , Hirudinas/metabolismo , Humanos , Oligonucleotídeos Antissenso/química , Fosforilação , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Receptor PAR-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Trombina/metabolismo , Tromboplastina/metabolismo , Timidina/química , Fatores de Tempo , TransfecçãoRESUMO
PURPOSE: Inducible nitric oxide synthase (NOS-II) is expressed in many inflammatory conditions. The implication of nitric oxide (NO) in angiogenesis remains controversial. The role of NOS-II and its influence on angiogenesis in corneal neovascularization is unknown and was investigated in this study. METHODS: A mouse model of corneal neovascularization induced by chemical cauterization was used. NOS-II mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction, and NOS-II protein was studied in situ by immunohistochemical analysis of the cornea. The influence of NOS-II on neovascularization was determined by comparison of vessel development in "normal" wild-type mice and mice with a targeted disruption of the NOS-II gene. RESULTS: NOS-II mRNA was induced to very high levels after corneal cauterization and remained upregulated throughout the disease. Migratory cells in the center of the cauterization area expressed NOS-II protein. The neovascular response in mice lacking the NOS-II gene was significantly stronger than in wild-type mice, and the difference increased over time. CONCLUSIONS: These data are the first evidence that NOS-II is expressed in this model of sterile corneal inflammation. NOS-II expression inhibited angiogenesis in severe corneal alkali burns.
Assuntos
Queimaduras Químicas/enzimologia , Córnea/enzimologia , Neovascularização da Córnea/prevenção & controle , Queimaduras Oculares/induzido quimicamente , Óxido Nítrico Sintase/biossíntese , Animais , Queimaduras Químicas/patologia , Córnea/efeitos dos fármacos , Córnea/patologia , Neovascularização da Córnea/induzido quimicamente , Neovascularização da Córnea/enzimologia , Neovascularização da Córnea/patologia , Primers do DNA/química , Queimaduras Oculares/enzimologia , Queimaduras Oculares/patologia , Feminino , Expressão Gênica , Técnicas Imunoenzimáticas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Nitrato de PrataRESUMO
Age related macular degeneration (AMD) is a pathological aging of the macula, brought about by the interaction of genetic and environmental factors. It induces geographic atrophy of the retina and/or choroidal neovascularization. In the latter, abnormal vessels develop from the choriocapillaris, with the involvement of VEGF (vascular endothelial growth factor). The VEGF family includes several factors, including VEGF-A, B, C, D, F and PlGF (placental growth factor). Their biological properties and their affinities to the VEGFR1, VEGFR2 and VEGFR3 receptors found on endothelial cells differ. Exudative AMD involves mainly VEGF-A and VEGF-R2. Anti-VEGF agents used in ophthalmology (ranibizumab, bevacizumab and aflibercept) are designed to primarily target this pathway. In vitro, all have sufficient affinity to their ligands. Their therapeutic efficacy must therefore be judged based on clinical criteria. In clinical practice, the minimum number of injections required for a satisfactory result appears to be comparable with all the three. The few available studies on therapeutic substitutions of anti-VEGF compounds suggest that some patients may benefit from substituting the anti-VEGF in cases of an unsatisfactory response to an initial molecule. Although local side effects, including increased risk of geographic atrophy, and systemic effects, including vascular accidents, have been suggested, these risks remain low, specially compared to the benefits of the treatment. Differences in safety between anti-VEGF are theoretically possible but unproven.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Degeneração Macular/tratamento farmacológico , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Envelhecimento/fisiologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Bevacizumab , Neovascularização de Coroide/tratamento farmacológico , Tolerância a Medicamentos , Humanos , Degeneração Macular/fisiopatologia , Ensaios Clínicos Controlados Aleatórios como Assunto , Ranibizumab , Receptores de Fatores de Crescimento do Endotélio Vascular/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Retina/fisiologia , Fatores de RiscoAssuntos
Retinopatia da Prematuridade/fisiopatologia , Ácidos Eicosanoicos/sangue , Endotélio Vascular/fisiopatologia , Humanos , Recém-Nascido , Estresse Oxidativo/fisiologia , Oxigênio/sangue , Espécies Reativas de Nitrogênio/sangue , Oclusão da Artéria Retiniana/fisiopatologia , Vasos Retinianos/fisiopatologia , Retinopatia da Prematuridade/diagnóstico , Estereoisomerismo , Fator A de Crescimento do Endotélio Vascular/fisiologiaRESUMO
Retinopathy of prematurity (ROP) is a major cause of visual impairment in premature infants. It is characterized by an arrest in normal retinal vascular development associated with microvascular degeneration, followed by an abnormal hypoxiainduced neovascularization. Recent studies point out that ROP is a multifactorial disease, implicating both oxygen-dependent and oxygen-independent mechanisms. Oxygen-dependent factors leading to microvascular degeneration include generation of reactive oxygen species and suppression of specific oxygen-regulated vascular survival factors, such as vascular endothelial growth factor (VEGF) and erythropoietin. The other major mechanism for the initial capillary loss is oxygen-independent and implicates a deficit in growth factor IGF-1/IGFBP3. The proliferative, second phase of ROP is triggered by increases in vascular growth factors concentrations, in an attempt to compensate for the hypoxic retina. Novel signaling pathways for vascular repair, implicating both metabolite signaling and inflammatory lipids signaling, represent new therapeutic avenues for ROP.
Assuntos
Retinopatia da Prematuridade/epidemiologia , Retinopatia da Prematuridade/fisiopatologia , Humanos , Recém-Nascido , Fator de Crescimento Insulin-Like I/metabolismo , Estresse Oxidativo/fisiologia , Oxigenoterapia/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Neovascularização Retiniana , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
In hypertension, increased peripheral vascular resistance results from vascular dysfunction with or without structural changes (vessel wall remodeling and/or microvascular rarefaction). Humans with lower birth weight exhibit evidence of vascular dysfunction. The current studies were undertaken to investigate whether in utero programming of hypertension is associated with in vivo altered response and/or abnormal vascular structure. Offspring of Wistar dams fed a normal (CTRL) or low (LP)-protein diet during gestation were studied. Mean arterial blood pressure response to ANG II was significantly increased, and depressor response to sodium nitroprusside (SNP) infusions significantly decreased in male LP adult offspring relative to CTRL. No arterial remodeling was observed in male LP compared with CTRL offspring. Capillary and arteriolar density was significantly decreased in striated muscles from LP offspring at 7 and 28 days of life but was not different in late fetal life [day 21 of gestation (E21)]. Angiogenic potential of aortic rings from LP newborn (day of birth, P0) was significantly decreased. Striated muscle expressions (Western blots) of ANG II AT(1) receptor subtype, endothelial nitric oxide synthase, angiopoietin 1 and 2, Tie 2 receptor, vascular endothelial growth factor and receptor, and platelet-derived growth factor C at E21 and P7 were unaltered by antenatal diet exposure. In conclusion, blood pressure responses to ANG II and SNP are altered, and microvascular structural changes prevail in this model of fetal programming of hypertension. The capillary rarefaction is absent in the fetus and appears in the neonatal period, in association with decreased angiogenic potential. The study suggests that intrauterine protein restriction increases susceptibility to postnatal factors resulting in microvascular rarefaction, which could represent a primary event in the genesis of hypertension.
Assuntos
Dieta com Restrição de Proteínas/efeitos adversos , Hipertensão/patologia , Hipertensão/fisiopatologia , Microcirculação/patologia , Microcirculação/fisiopatologia , Neovascularização Patológica/patologia , Neovascularização Patológica/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal/fisiopatologia , Animais , Suscetibilidade a Doenças/embriologia , Suscetibilidade a Doenças/patologia , Suscetibilidade a Doenças/fisiopatologia , Feminino , Hipertensão/embriologia , Hipertensão/etiologia , Masculino , Neovascularização Patológica/embriologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/patologia , Ratos , Ratos Wistar , Doenças Vasculares/embriologia , Doenças Vasculares/etiologia , Doenças Vasculares/patologia , Doenças Vasculares/fisiopatologiaRESUMO
Fibroblast growth factors (FGF) are multipotent cytokines with demonstrated mitogenic, neurotrophic and angiogenic properties. There is evidence that they have multiple functions during and after development of the vertebrate eye. Amongst these, the role of FGF receptor mediated signaling in the retinal pigmented epithelium (RPE) is not yet well understood. FGF-2 is produced in RPE cells and may play a role in photoreceptor development and/or survival in vivo. It may also stimulate growth of melanocytes and angiogenesis in the choroid. To address these questions, we have specifically disrupted FGF signaling by generating lines of transgenic mice expressing dominant-negative FGF receptor 1 (FGFR-1) in the pigmented cells. Histological analysis of the eyes were conducted on hemizygous and homozygous mice at different ages. In homozygotes, eye growth is strongly impaired during embryogenesis leading to massive eye degeneration seen in the early post-natal stages. In hemizygotes, the choroid is thinned and the finger-like junctions between RPE cells and photoreceptors are disrupted. Scanning electron microscopy of the choroid vasculature showed that choriocapillary density, diameter and branching are strongly affected. As mice age, they develop progressive retinal degeneration as evidenced by photoreceptor cell loss. Our results are in agreement with the hypothesis that FGF signaling in the RPE participates in photoreceptor survival in vivo. Our model provides evidence that FGF signaling is also involved in choroidal angiogenesis by a process that could relate to induction of terminal branching.