RESUMO
First described almost 50 years ago, chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare autoimmune disorder characterized by progressive peripheral neuropathy. CIDP is difficult to diagnose, but early diagnosis can be crucial to prevent permanent nerve damage. Initial treatment options include corticosteroids, immunoglobulin given by intravenous administration, and therapeutic plasma exchange. Subcutaneous administration of immunoglobulin provides a new option for patients with CIDP that has the potential to increase independence and improve tolerability. This article reviews the epidemiology, diagnosis, treatment options for first- and second-line therapy, treatment guidelines, and monitoring parameters for CIDP.
Assuntos
Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/diagnóstico , Corticosteroides/uso terapêutico , Efeitos Psicossociais da Doença , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Aceitação pelo Paciente de Cuidados de Saúde , Troca Plasmática , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/economia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/epidemiologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/terapiaRESUMO
BACKGROUND: There is a growing body of literature documenting local herpes zoster outbreak following procedures. The mechanism underlying these outbreaks remains elusive. We present a case of zoster following onabotulinumtoxinA (BTX) for migraine and a literature review. METHODS: Chart and literature review. CASE: A 72-year-old woman with chronic migraine received BTX injections for 3 years without incident. She had a history of thoracic zoster with subsequent post-herpetic neuralgia. In August 2013, 48 hours after receiving BTX injections, she developed a painful rash in the right V1 distribution consistent with herpes zoster ophthalmicus. One week later the rash had resolved without treatment. LITERATURE REVIEW: We identified 65 (including 2 from Juel-Jenson) cases of zoster reactivation following minor procedures. These cases tend to be in young patients without specific risk factors. Outbreaks characteristically occur at the level of exposure to local trauma. DISCUSSION: Our review suggests that local trauma, regardless of the nature of stimuli, may be sufficient for zoster reactivation. We hypothesize that the stressors in these reported cases exert a local epigenetic influence on viral transcription, allowing for viral reactivation. CONCLUSION: Zoster is a potential complication of BTX administration for chronic migraine in adults. Physician awareness can reduce the significant morbidity associated with this disease.
Assuntos
Toxinas Botulínicas Tipo A/efeitos adversos , Herpes Zoster Oftálmico/etiologia , Transtornos de Enxaqueca/prevenção & controle , Fármacos Neuromusculares/efeitos adversos , Idoso , Feminino , Herpesvirus Humano 3/fisiologia , Humanos , Ativação Viral/fisiologiaRESUMO
PURPOSE: The chaperone proteins, α-crystallins, also possess antiapoptotic properties. The purpose of the present study was to investigate whether 19 to 20-mer α-crystallin-derived mini-chaperone peptides (α-crystallin mini-chaperone) are antiapoptotic, and to identify their putative transporters in human fetal RPE (hfRPE) cells. METHODS: Cell death and caspase-3 activation induced by oxidative stress were quantified in early passage hfRPE cells in the presence of 19 to 20-mer αA- or αB-crystallin-derived or scrambled peptides. Cellular uptake of fluorescein-labeled, α-crystallin-derived mini-peptides and recombinant full-length αB-crystallin was determined in confluent hfRPE. The entry mechanism in hfRPE cells for α-crystallin mini-peptides was investigated. The protective role of polycaprolactone (PCL) nanoparticle encapsulated αB-crystallin mini-chaperone peptides from H2O2-induced cell death was studied. RESULTS: Primary hfRPE cells exposed to oxidative stress and either αA- or αB-crystallin mini-chaperones remained viable and showed marked inhibition of both cell death and activation of caspase-3. Uptake of full-length αB-crystallin was minimal while a time-dependent uptake of αB-crystallin-derived peptide was observed. The mini-peptides entered the hfRPE cells via the sodium-coupled oligopeptide transporters 1 and 2 (SOPT1, SOPT2). PCL nanoparticles containing αB-crystallin mini-chaperone were also taken up and protected hfRPE from H2O2-induced cell death at significantly lower concentrations than free αB-crystallin mini-chaperone peptide. CONCLUSIONS: αA- and αB-crystallin mini-chaperones offer protection to hfRPE cells and inhibit caspase-3 activation. The oligopeptide transporters SOPT1 and SOPT2 mediate the uptake of these peptides in RPE cells. Nanodelivery of αB-crystallin-derived mini-chaperone peptide offers an alternative approach for protection of hfRPE cells from oxidant injury.
Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/fisiologia , Peptídeos/fisiologia , Epitélio Pigmentado da Retina/citologia , Cadeia A de alfa-Cristalina/fisiologia , Cadeia B de alfa-Cristalina/fisiologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/fisiologia , Caspase 3/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Peróxido de Hidrogênio/toxicidade , Oxidantes/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Proteínas Recombinantes , Epitélio Pigmentado da Retina/metabolismo , Fatores de TempoRESUMO
Endoplasmic reticulum (ER) stress is linked to several pathological conditions including age-related macular degeneration. Excessive ER stress initiates cell death cascades which are mediated, in part, through mitochondrial dysfunction. Here, we identify αB crystallin as an important regulator of ER stress-induced cell death. Retinal pigment epithelial (RPE) cells from αB crystallin (-/-) mice, and human RPE cells transfected with αB crystallin siRNA, are more vulnerable to ER stress induced by tunicamycin. ER stress-mediated cell death is associated with increased levels of reactive oxygen species, depletion of glutathione in mitochondria, decreased superoxide dismutase activity, increased release of cytochrome c, and activation of caspases 3 and 4. The ER stress signaling inhibitors, salubrinal and 4-(2-aminoethyl) benzenesulfonyl fluoride, decrease mitochondrial damage and reduce RPE apoptosis induced by ER stress. Prolonged ER stress decreases levels of αB crystallin, thus exacerbating mitochondrial dysfunction. Overexpression of αB crystallin protects RPE cells from ER stress-induced apoptosis by attenuating increases in Bax, CHOP, mitochondrial permeability transition, and cleaved caspase 3. Thus, these data collectively demonstrate that αB crystallin provides critical protection of mitochondrial function during ER stress-induced RPE apoptosis.
Assuntos
Apoptose , Retículo Endoplasmático/metabolismo , Mitocôndrias/patologia , Cadeia B de alfa-Cristalina/genética , Animais , Apoptose/efeitos dos fármacos , Cinamatos/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Sulfonas/farmacologia , Tioureia/análogos & derivados , Tioureia/farmacologia , Cadeia B de alfa-Cristalina/metabolismoRESUMO
Bone morphorgenetic protein (BMP)-4 has been shown to play a pivotal role in eye development; however, its role in mature retina or ocular angiogenic diseases is unclear. Activating downstream Smad signaling, BMP4 can be either pro-angiogenic or anti-angiogenic, depending on the context of cell types and associated microenvironment. In this study, we generated transgenic mice over-expressing BMP4 in retinal pigment epithelial (RPE) cells (Vmd2-Bmp4 Tg mice), and used the laser-induced choroidal neovascularization (CNV) model to study the angiogenic properties of BMP4 in adult eyes. Vmd2-Bmp4 Tg mice displayed normal retinal histology at 10 weeks of age when compared with age-matched wildtype mice. Over-expression of BMP4 in RPE in the transgenic mice was confirmed by real-time PCR and immunostaining. Elevated levels of Smad1,5 phosphorylation were found in BMP4 transgenic mice compared to wildype mice. Over-expression of BMP4 was associated with less severe CNV as characterized by fluorescein angiography, CNV volume measurement and histology. While control mice showed increased levels of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 after laser injury, Vmd2-Bmp4 Tg showed no increase in either VEGF or MMP-9. Further, we found that TNF-induced MMP-9 secretion in vitro was reduced by pretreatment of RPE cells with BMP4. The inhibition of MMP-9 was Smad-dependent because BMP4 failed to repress TNF-induced MMP-9 expression when Smad1,5 was silenced by siRNA. In summary, our studies identified an anti-angiogenic role for BMP4 in laser-induced CNV, mediated by direct inhibition of MMP-9 and indirect inhibition of VEGF.
Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Neovascularização de Coroide/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bestrofinas , Proteína Morfogenética Óssea 4/genética , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Lasers/efeitos adversos , Metaloproteinase 9 da Matriz/genética , Camundongos , Camundongos Transgênicos , Epitélio Pigmentado da Retina/patologia , Proteína Smad1/genética , Proteína Smad1/metabolismo , Proteína Smad5/genética , Proteína Smad5/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Absence of α-crystallins (αA and αB) in retinal pigment epithelial (RPE) cells renders them susceptible to oxidant-induced cell death. We tested the hypothesis that the protective effect of α-crystallin is mediated by changes in cellular glutathione (GSH) and elucidated the mechanism of GSH efflux. In α-crystallin overexpressing cells resistant to cell death, cellular GSH was >2 fold higher than vector control cells and this increase was seen particularly in mitochondria. The high GSH levels associated with α-crystallin overexpression were due to increased GSH biosynthesis. On the other hand, cellular GSH was decreased by 50% in murine retina lacking αA or αB crystallin. Multiple multidrug resistance protein (MRP) family isoforms were expressed in RPE, among which MRP1 was the most abundant. MRP1 was localized to the plasma membrane and inhibition of MRP1 markedly decreased GSH efflux. MRP1-suppressed cells were resistant to cell death and contained elevated intracellular GSH and GSSG. Increased GSH in MRP1-supressed cells resulted from a higher conversion of GSSG to GSH by glutathione reductase. In contrast, GSH efflux was significantly higher in MRP1 overexpressing RPE cells which also contained lower levels of cellular GSH and GSSG. Oxidative stress further increased GSH efflux with a decrease in cellular GSH and rendered cells apoptosis-prone. In conclusion, our data reveal for the first time that 1) MRP1 mediates GSH and GSSG efflux in RPE cells; 2) MRP1 inhibition renders RPE cells resistant to oxidative stress-induced cell death while MRP1 overexpression makes them susceptible and 3) the antiapoptotic function of α-crystallin in oxidatively stressed cells is mediated in part by GSH and MRP1. Our findings suggest that MRP1 and α crystallin are potential therapeutic targets in pathological retinal degenerative disorders linked to oxidative stress.
Assuntos
Apoptose , Membrana Celular/metabolismo , Glutationa/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , alfa-Cristalinas/metabolismo , Animais , Transporte Biológico Ativo/genética , Linhagem Celular , Membrana Celular/genética , Glutationa/genética , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Humanos , Camundongos , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Epitélio Pigmentado da Retina/patologia , alfa-Cristalinas/genéticaRESUMO
PURPOSE: To investigate the role of fibronectin containing extra domain A (FN-EDA) in the pathogenesis of proliferative vitreoretinopathy (PVR) and the regulation of FN-EDA by transforming growth factor (TGF)-ß and connective tissue growth factor (CTGF) in retinal pigment epithelial (RPE) cells. METHODS: Expression of FN-EDA in normal human retinas and PVR membranes was evaluated by immunohistochemistry. The effects of TGFß and CTGF on FN-EDA mRNA and protein expression in primary cultures of human RPE cells were analyzed at different time points by real-time PCR and Western blot, respectively. The interaction of CTGF with TGFß2 or with its type II receptor TGFßRII was examined by ELISA, immunoprecipitation, and solid-phase binding assays. RESULTS: FN-EDA was abundantly expressed in PVR membranes but absent from the RPE monolayer in normal human retinas. Treatment of RPE cells with TGFß2 induced FN-EDA expression in a time- and dose-dependent manner, but CTGF alone had no effect. However, CTGF, through its N-terminal half fragment, augmented TGFß2-induced expression of FN-EDA at the protein level. This effect was blocked by antibodies against TGFß2 or TGFßRII. Interaction of TGFß2 or TGFßRII with CTGF was dose dependent and specific. CTGF directly bound TGFß2 and TGFßRII at its N- and C-terminal domains, respectively. CONCLUSIONS: These findings suggest that CTGF promotes the profibrotic activities of TGFß acting as a cofactor through direct protein interactions and complex regulatory mechanisms.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/genética , Regulação da Expressão Gênica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Fator de Crescimento Transformador beta2/metabolismo , Western Blotting , Células Cultivadas , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fator de Crescimento do Tecido Conjuntivo/farmacologia , Ensaio de Imunoadsorção Enzimática , Fibronectinas , Humanos , Técnicas Imunoenzimáticas , Domínios e Motivos de Interação entre Proteínas , RNA/isolamento & purificação , RNA Mensageiro/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Retina/embriologia , Retina/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta2/farmacologia , Regulação para Cima , Vitreorretinopatia Proliferativa/metabolismoRESUMO
Intraoperative monitoring of cerebral hemodynamics during carotid endarterectomy (CEA) provides essential information for detecting cerebral hypoperfusion induced by temporary internal carotid artery (ICA) clamping and post-CEA hyperperfusion syndrome. This study tests the feasibility and sensitivity of a novel dual-wavelength near-infrared diffuse correlation spectroscopy technique in detecting cerebral blood flow (CBF) and cerebral oxygenation in patients undergoing CEA. Two fiber-optic probes were taped on both sides of the forehead for cerebral hemodynamic measurements, and the instantaneous decreases in CBF and electroencephalogram (EEG) alpha-band power during ICA clamping were compared to test the measurement sensitivities of the two techniques. The ICA clamps resulted in significant CBF decreases (-24.7 ± 7.3%) accompanied with cerebral deoxygenation at the surgical sides (n = 12). The post-CEA CBF were significantly higher (+43.2 ± 16.9%) than the pre-CEA CBF. The CBF responses to ICA clamping were significantly faster, larger and more sensitive than EEG responses. Simultaneous monitoring of CBF, cerebral oxygenation and EEG power provides a comprehensive evaluation of cerebral physiological status, thus showing potential for the adoption of acute interventions (e.g., shunting, medications) during CEA to reduce the risks of severe cerebral ischemia and cerebral hyperperfusion syndrome.
Assuntos
Encéfalo/cirurgia , Eletroencefalografia , Endarterectomia das Carótidas/métodos , Raios Infravermelhos , Fenômenos Ópticos , Análise Espectral , Adulto , Idoso , Idoso de 80 Anos ou mais , Encéfalo/irrigação sanguínea , Artéria Carótida Interna/fisiopatologia , Artéria Carótida Interna/cirurgia , Circulação Cerebrovascular , Constrição , Endarterectomia das Carótidas/efeitos adversos , Feminino , Hemodinâmica , Humanos , Hipóxia/fisiopatologia , Masculino , Pessoa de Meia-IdadeRESUMO
Bone morphogenetic protein-4 (BMP4) may be involved in the molecular switch that determines which late form of age-related macular degeneration (AMD) an individual develops. BMP4 expression is high in retinal pigment epithelium (RPE) cells in late, dry AMD patients, while BMP4 expression is low in the wet form of the disease, characterized by choroidal neovascularization (CNV). Here, we sought to determine the mechanism by which BMP4 is down-regulated in CNV. BMP4 expression was decreased within laser-induced CNV lesions in mice at a time when tumor necrosis factor (TNF) expression was high (7 d postlaser) and was reexpressed in RPE when TNF levels declined (14 d postlaser). We found that TNF, an important angiogenic stimulus, significantly down-regulates BMP4 expression in cultured human fetal RPE cells, ARPE-19 cells, and RPE cells in murine posterior eye cup explants. We identified two specificity protein 1 (Sp1) binding sites in the BMP4 promoter that are required for basal expression of BMP4 and its down-regulation by TNF. Through c-Jun NH(2)-terminal kinase (JNK) activation, TNF modulates Sp1 phosphorylation, thus decreasing its affinity to the BMP4 promoter. The down-regulation of BMP4 expression by TNF in CNV and mechanisms established might be useful for defining novel targets for AMD therapy.
Assuntos
Proteína Morfogenética Óssea 4/genética , Regulação da Expressão Gênica , Degeneração Macular/genética , Fator de Necrose Tumoral alfa/genética , Animais , Sítios de Ligação/genética , Western Blotting , Proteína Morfogenética Óssea 4/metabolismo , Linhagem Celular , Células Cultivadas , Neovascularização de Coroide/etiologia , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Regulação para Baixo , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lasers/efeitos adversos , Degeneração Macular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas/genética , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
αB crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.
Assuntos
Epitélio Pigmentado da Retina/metabolismo , alfa-Cristalinas/metabolismo , beta-Cristalinas/metabolismo , Animais , Western Blotting , Compartimento Celular , Morte Celular/efeitos dos fármacos , Polaridade Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Peróxido de Hidrogênio/farmacologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologiaRESUMO
We hypothesized that neutrophils and their secreted factors mediate breakdown of the integrity of the outer blood-retina-barrier by degrading the apical tight junctions of the retinal pigment epithelium (RPE). The effect of activated neutrophils or neutrophil cell lysate on apparent permeability of bovine RPE-Choroid explants was evaluated by measuring [3H] mannitol flux in a modified Ussing chamber. The expression of matrix metalloproteinase- (MMP-) 9 in murine peritoneal neutrophils, and the effects of neutrophils on RPE tight-junction protein expression were assessed by confocal microscopy and western blot. Our results revealed that basolateral incubation of explants with neutrophils decreased occludin and ZO-1 expression at 1 and 3 hours and increased the permeability of bovine RPE-Choroid explants by >3-fold (P < .05). Similarly, basolateral incubation of explants with neutrophil lysate decreased ZO-1 expression at 1 and 3 hours (P < .05) and increased permeability of explants by 75%. Further, we found that neutrophils prominently express MMP-9 and that incubation of explants with neutrophils in the presence of anti-MMP-9 antibody inhibited the increase in permeability. These data suggest that neutrophil-derived MMP-9 may play an important role in disrupting the integrity of the outer blood-retina barrier.
Assuntos
Corioide/fisiologia , Neutrófilos/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Bovinos , Células Cultivadas , Corioide/metabolismo , Corioide/patologia , Histocitoquímica , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Ocludina , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/patologia , Junções Íntimas/metabolismoRESUMO
alphaB-crystallin is a chaperone belonging to the small heat shock protein family. Herein we show attenuation of intraocular angiogenesis in alphaB-crystallin knockout (alphaB-crystallin(-/-)) mice in 2 models of intraocular disease: oxygen-induced retinopathy and laser-induced choroidal neovascularization. Vascular endothelial growth factor A (VEGF-A) mRNA and hypoxia inducible factor-1alpha protein expression were induced during retinal angiogenesis, but VEGF-A protein expression remained low in alphaB-crystallin(-/-) retina versus wild-type mice, whereas VEGF-R2 expression was not affected. Both alphaB-crystallin and its phosphorylated serine59 formwere expressed, and immunoprecipitation revealed alphaB-crystallin binding to VEGF-A but not transforming growth factor-beta in cultured retinal pigment epithelial (RPE) cells. alphaB-crystallin and VEGF-A are colocalized in the endoplasmic reticulum in RPE cells under chemical hypoxia. alphaB-crystallin(-/-) RPE showed low VEGF-A secretion under serum-starved conditions compared with wild-type cells. VEGF-A is polyubiquitinated in control and alphaB-crystallin siRNA treated RPE; however, mono-tetra ubiquitinated VEGF-A increases with alphaB-crystallin knockdown. Endothelial cell apoptosis in newly formed vessels was greater in alphaB-crystallin(-/-) than wild-type mice. Proteasomal inhibition in alphaB-crystallin(-/-) mice partially restores VEGF-A secretion and angiogenic phenotype in choroidal neovascularization. Our studies indicate an important role for alphaB-crystallin as a chaperone for VEGF-A in angiogenesis and its potential as a therapeutic target.
Assuntos
Neovascularização de Coroide/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Animais , Apoptose/fisiologia , Western Blotting , Neovascularização de Coroide/genética , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoprecipitação , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Epitélio Pigmentado da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator A de Crescimento do Endotélio Vascular/genética , Cadeia B de alfa-Cristalina/genéticaRESUMO
PURPOSE: EphB4 receptor (EphB4) and its ligand (EphrinB2) play an important role in the regulation of cell adhesion, growth, and migration. The purpose of this study was to determine the effects of EphB4 blockade by soluble EphB4 (sEphB4) on retinal pigment epithelial (RPE) cell migration and proliferation, induced by platelet-derived growth factor-BB (PDGF), and to establish its relevance to proliferative vitreoretinopathy (PVR). METHODS: The expression of EphB4 and EphrinB2 in early-passage human RPE cells and in human PVR membranes was evaluated by confocal microscopy. The effect of sEphB4 (0.1-3 microg/mL) on PDGF (20 ng/mL)-induced RPE migration and proliferation was evaluated using a modified Boyden chamber assay and an MTT assay, respectively. Attachment to basement membrane matrix and fibronectin was assayed by MTT. Phosphorylation of FAK and p42/44 mitogen-activated protein kinase (MAPK) in retinal pigment epithelium was determined by Western blot analysis after exposure to sEphB4. The effect of sEphB4 on the phosphorylation of EphB4/EphrinB2 was demonstrated with the use of immunoprecipitation assays. RESULTS: EphrinB2 and EphB4 were expressed on human RPE cells in vitro and in cells within human PVR membranes. sEphB4 blocked EphB4 and EphrinB2 phosphorylation in RPE cells in vitro. sEphB4 reduced RPE migration in response to PDGF stimulation (P < 0.01). Similarly, sEphB4 inhibited RPE attachment and proliferation in a dose-dependent manner (P < 0.05). PDGF-induced phosphorylation of FAK and MAPK was inhibited by sEphB4. CONCLUSIONS: EphB4 and EphrinB2 are expressed in RPE cells and PVR membranes. sEphB4 inhibits PDGF-induced RPE cell attachment, proliferation, and migration. This effect may result from the inhibition of FAK and MAPK phosphorylation.
Assuntos
Movimento Celular/efeitos dos fármacos , Efrina-B2/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptor EphB4/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Vitreorretinopatia Proliferativa/metabolismo , Western Blotting , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fibronectinas/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Humanos , Ligantes , Microscopia Confocal , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Epitélio Pigmentado da Retina/embriologiaRESUMO
We provide our detailed, standardized in vitro protocol for the culture and differentiation of human retinal pigment epithelial (RPE) cells into a highly polarized and functional monolayer. Disruption of the polarized RPE function plays an important role in the pathogenesis of common blinding disorders of the retina. The availability of this polarized RPE monolayer allows for reproducible evaluation of RPE function, modeling of RPE dysfunction in retinal disease and in vitro evaluation of new therapies. The protocol, which takes approximately 6 weeks to complete, describes the culture of RPE from human fetal donor eyes, the differentiation of these cells into a polarized monolayer with high transepithelial resistance and morphologic characteristics that mimic the RPE monolayer in vivo. By modifying the procedure for initial isolation of pure RPE cells and the culture conditions used in existing protocols, we have established a standardized protocol that provides highly reproducible RPE monolayers from the same donor eye.
Assuntos
Técnicas de Cultura de Células , Diferenciação Celular , Polaridade Celular , Epitélio Pigmentado da Retina/citologia , Células Cultivadas , Meios de Cultura , Dissecação/métodos , Humanos , Doadores de TecidosRESUMO
BACKGROUND: Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE). METHODS: Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-gamma (IFN-gamma; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1-100 ng/ml). RESULTS: CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-gamma. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-gamma stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-gamma increased CFH secretion into RPE supernatants. Dose-dependent RPE cell chemotactic migration was induced by CFH. CONCLUSION: IFN-gamma promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-gamma have the potential to play a role in the pathogenesis of AMD.
Assuntos
Movimento Celular/fisiologia , Interferon gama/farmacologia , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacos , Animais , Western Blotting , Bovinos , Diferenciação Celular , Ensaios de Migração Celular , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Fator H do Complemento/metabolismo , Fator H do Complemento/farmacologia , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Microscopia Confocal , Proteínas Recombinantes/farmacologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
The retinal pigment epithelium is a primary site of pathology in age-related macular degeneration. Oxidative stress and senescence are both thought to be important mediators of macular degeneration pathogenesis. We demonstrate here that bone morphogenetic protein-4 is highly expressed in the retinal pigment epithelium and adjacent extracellular matrix of patients with dry age-related macular degeneration. In vitro studies revealed that sublethal oxidative stress increased bone morphogenetic protein-4 expression in retinal pigment epithelial cells, and both bone morphogenetic protein-4 and persistent mild oxidative stress can induce retinal pigment epithelial cell senescence through p53-p21(Cip1/WAF1)-Rb pathway. We further demonstrate that bone morphogenetic protein-4 acts as a mediator in oxidative stress-induced senescence and that this mediator function is via Smad and the p38 signaling pathway to increase and activate p53 and p21(Cip1/WAF1) and decrease phospho-Rb. Oxidative stress-induced senescence can be blocked by Chordin-like, an antagonist of bone morphogenetic protein-4, or SB203580, a phospho-p38 inhibitor. Our results suggest that oxidative stress and bone morphogenetic protein-4 may interact to promote retinal pigment epithelial cell senescence and that bone morphogenetic protein-4 may represent a novel therapeutic target to inhibit the progressive effects of oxidative stress and senescence in dry age-related macular degeneration.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Senescência Celular/fisiologia , Degeneração Macular/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteína Morfogenética Óssea 4/genética , Ciclo Celular , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Fosforilação , Proteínas Smad/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The antiangiogenic and neurotrophic growth factor, pigment epithelial derived factor (PEDF), and the proangiogenic growth factor, vascular endothelial growth factor-A (VEGF), are released from retinal pigment epithelial (RPE) cells where they play a critical role in the pathogenesis of age-related macular degeneration (AMD). Since RPE polarity may be altered in advanced AMD, we studied the effect of polarization of differentiated, human RPE monolayer cultures on expression and secretion of PEDF and VEGF. Polarized RPE demonstrated apical microvilli, expression of tight junction proteins, apical localization of Na/K- ATPase, and high transepithelial resistance (490 +/- 17 Omega x cm(2)). PEDF secretion was about 1000 fold greater than that for VEGF in both polarized and non-polarized cultures. Polarization of the RPE monolayer increased PEDF secretion, which was predominantly apical, by 34 fold (p<0.02) and VEGF secretion, which was predominantly basolateral, by 5.7 fold (p<0.02). Treatment of non-polarized RPE cultures with bone morphogenetic protein-4 (BMP-4) had no effect on PEDF or VEGF secretion, but resulted in a dose-dependent >2-fold increase in basolateral VEGF secretion (p<0.05) in polarized cultures. Our data show that polarity is an important determinant of the level of PEDF and VEGF secretion in RPE and support the contention that loss of polarity of RPE in AMD results in marked loss of neurotrophic and vascular support for the retina potentially leading to photoreceptor loss and blindness.
Assuntos
Envelhecimento/patologia , Polaridade Celular/fisiologia , Proteínas do Olho/metabolismo , Degeneração Macular/patologia , Fatores de Crescimento Neural/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Envelhecimento/fisiologia , Ciclo Celular , Proliferação de Células , Imunofluorescência , Humanos , Degeneração Macular/metabolismo , Microscopia Eletrônica , Epitélio Pigmentado da Retina/metabolismoRESUMO
The purpose of this study was to determine the expression, regulation and signaling of a key redoxin family member thioredoxin 1 (Trx1) in normal, oxidant-stimulated and growth factor-pretreated RPE cells. Trx1 is expressed in early passage, human RPE cell cultures. RPE cells exposed to C(2)-ceramide for 24h showed no significant change in expression of Trx1 vs. controls with and without pretreatment for 24h with hepatocyte growth factor (HGF). Neither hypoxia from 1% O(2) or from CoCl(2) exposure resulted in any alteration in Trx1 expression in RPE cells. C(2)-ceramide treatment caused translocation of Trx1 from cytosol to the nucleus, which was abolished by pre-treatment of cells with a p38 MAPK-specific inhibitor. Furthermore, the gene and protein expression of thioredoxin interacting protein (Txnip) increased with ceramide treatment and was significantly (p<0.001) elevated with HGF preincubation vs. untreated controls. Prominent protection from ceramide-induced RPE cell death by exogenous rTrx1 was demonstrated. Although Trx1 directly interacts with its inhibitor, Txnip, p38 inhibition does not appear to have a role in this interaction. We found no direct interaction between apoptosis signal regulating kinase (ASK-1) and Txnip under the same experimental conditions. In summary, our data demonstrate the expression of Trx1 and Txnip in human RPE cells. Ceramide treatment results in translocation of Trx1 to the nucleus, and upregulation of Txnip expression; exogenous rTrx1 protects from ceramide-induced cell death. These results suggest that Trx1 and Txnip play an important role in the response of RPE to ceramide toxicity.