RESUMO
Metabolic syndrome is a disorder characterized by a constellation of clinical findings such as elevated blood glucose, hyperinsulinemia, dyslipidemia, hypertension, and obesity. A positive correlation has been found between metabolic syndrome or its components and retinopathy, mainly at microvascular level, in patients without a history of diabetes. Here, we extend the investigations beyond the vascular component analyzing functional changes as well as neuronal and glial response in retinas of Apolipoprotein E knockout (ApoE-KO) mice fed with 10% w/v fructose diet. Given that autophagy dysfunction is implicated in retinal diseases related to hyperglycemia and dyslipidemia, the activation of this pathway was also analyzed. Two months of fructose intake triggered metabolic derangements in ApoE-KO mice characterized by dyslipidemia, hyperglycemia and hyperinsulinemia. An increased number of TUNEL positive cells, in addition to the ganglion cell layer, was observed in the inner nuclear layer in retina. Vascular permeability, evidenced by albumin-Evans blue leakage and extravasation of albumin was also detected. Furthermore, a significant decrease of the glial fibrillary acidic protein expression was confirmed by Western blot analysis. Absence of both Müller cell gliosis and pro-angiogenic response was also demonstrated. Finally, retinas of ApoE-KO FD mice showed defective autophagy activation as judged by LC3B mRNA and p62 protein levels correlating with the increased cell death. These results demonstrated that FD induced in ApoE-KO mice biochemical alterations compatible with metabolic syndrome associated with neuronal impairment and mild vascular alterations in the retina.
RESUMO
An increased risk of pregnancy disorders has been reported in women and animal models exposed to organophosphate pesticides. However, less information is available on impacts to human placental function. Here, we addressed the impact of chlorpyrifos (CPF) on extravillous cytotrophoblasts (evCTB) employing HTR8/SVneo cells as an in vitro model. Cell proliferation, migration and invasion were not affected by CPF under conditions where cell viability was not compromised; however, we observed reduced expression of genes for vascular endothelial growth factor receptor 1, hypoxia-inducible factor 1-alpha, peroxisome proliferator activated receptor gamma, and the ß-subunit of human chorionic gonadotropin. These results are the first effects reported by organophosphate pesticide in evCTB cells and show altered expression of several genes important for placental development that could serve as potential biomarkers for future research.
Assuntos
Clorpirifos/toxicidade , Inseticidas/toxicidade , Trofoblastos/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Gonadotropina Coriônica Humana Subunidade beta/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , PPAR gama/genética , Trofoblastos/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Hypoxia is one of the main insults in proliferative retinopathies, leading to neovascularization and neurodegeneration. To maintain homeostasis, neurons require efficient degradation and recycling systems. Autophagy participates in retinal cell death, but it is also a cell survival mechanism. Here, we analyzed the role of autophagy at the three characteristic time periods in the oxygen-induced retinopathy (OIR) mouse model and determined if its modulation can improve vascular and non-vascular alterations. Experiments were performed with chloroquine (CQ) in order to monitor autophagosome accumulation by lysosomal blockade. Post natal day (P)17 OIR mouse retinas showed a significant increase in autophagy flux. In particular, an intense LC3B and p62 staining was observed in inner layers of the retina, mainly proliferating endothelial cells. After a single intraocular injection of Rapamycin at P12 OIR, a decreased neovascular area and vascular endothelial growth factor (VEGF) protein expression were observed at P17 OIR. In addition, whereas the increased expression of glial fibrillary acidic protein (GFAP) was reversed at P26 OIR, the functional alterations persisted. Using a similar therapeutic schedule, we analyzed the effect of anti-VEGF therapy on autophagy flux. Like Rapamycin, VEGF inhibitor treatment not only reduced the amount of neovascular tufts, but also activated autophagy flux at P17 OIR, mainly in ganglion cell layer and inner nuclear layer. Finally, the effects of the disruption of autophagy by Spautin-1, were evaluated at vascular, glial, and neuronal levels. After a single dose of Spautin-1, Western blot analysis showed a significant decrease in LC3B II and p62 protein expression at P13 OIR, returning both autophagy markers to OIR control levels at P17. In addition, neither gliosis nor functional alterations were attenuated. In line with these results, TUNEL staining showed a slight increase in the number of positive cells in the outer nuclear layer at P17 OIR. Overall, our results demonstrate that all treatments of induction or inhibition of the autophagic flux reduced neovascular area but were unable to completely reverse the neuronal damage. Besides, compared to current treatments, rapamycin provides a more promising therapeutic strategy as it reduces both neovascular tufts and persistent gliosis.
RESUMO
Müller glial cells (MGCs) are known to participate actively in retinal development and to contribute to homoeostasis through many intracellular mechanisms. As there are no homologous cells in other neuronal tissues, it is certain that retinal health depends on MGCs. These macroglial cells are located at the centre of the columnar subunit and have a great ability to interact with neurons, astrocytes, microglia and endothelial cells in order to modulate different events. Several investigations have focused their attention on the role of MGCs in diabetic retinopathy, a progressive pathology where several insults coexist. As expected, data suggest that MGCs display different responses according to the severity of the stimulus, and therefore trigger distinct events throughout the course of the disease. Here, we describe physiological functions of MGCs and their participation in inflammation, gliosis, synthesis and secretion of trophic and antioxidant factors in the diabetic retina. We invite the reader to consider the protective/deleterious role of MGCs in the early and late stages of the disease. In the light of the results, we open up the discussion around and ask the question: Is it possible that the modulation of a single cell type could improve or even re-establish retinal function after an injury?
Assuntos
Retinopatia Diabética , Células Ependimogliais/fisiologia , Gliose , Inflamação , Fatores de Crescimento Neural/fisiologia , Estresse Oxidativo/fisiologia , Animais , Retinopatia Diabética/imunologia , Retinopatia Diabética/metabolismo , Retinopatia Diabética/fisiopatologia , Células Ependimogliais/imunologia , Células Ependimogliais/metabolismo , Gliose/imunologia , Gliose/metabolismo , Gliose/fisiopatologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/fisiopatologia , Fatores de Crescimento Neural/imunologia , Fatores de Crescimento Neural/metabolismo , Estresse Oxidativo/imunologiaRESUMO
Neovascular retinopathies are leading causes of irreversible blindness. Although vascular endothelial growth factor (VEGF) inhibitors have been established as the mainstay of current treatment, clinical management of these diseases is still limited. As retinal impairment involves abnormal neovascularization and neuronal degeneration, we evaluated here the involvement of galectin-1 in vascular and non-vascular alterations associated with retinopathies, using the oxygen-induced retinopathy (OIR) model. Postnatal day 17 OIR mouse retinas showed the highest neovascular profile and exhibited neuro-glial injury as well as retinal functional loss, which persisted until P26 OIR. Concomitant to VEGF up-regulation, galectin-1 was highly expressed in P17 OIR retinas and it was mainly localized in neovascular tufts. In addition, OIR induced remodelling of cell surface glycophenotype leading to exposure of galectin-1-specific glycan epitopes. Whereas VEGF returned to baseline levels at P26, increased galectin-1 expression persisted until this time period. Remarkably, although anti-VEGF treatment in P17 OIR improved retinal vascularization, neither galectin-1 expression nor non-vascular and functional alterations were attenuated. However, this functional defect was partially prevented in galectin-1-deficient (Lgals1-/-) OIR mice, suggesting the importance of targeting both VEGF and galectin-1 as non-redundant independent pathways. Supporting the clinical relevance of these findings, we found increased levels of galectin-1 in aqueous humor from patients with proliferative diabetic retinopathy and neovascular glaucoma. Thus, using an OIR model and human samples, we identified a role for galectin-1 accompanying vascular and non-vascular retinal alterations in neovascular retinopathies.
Assuntos
Galectina 1/metabolismo , Retinose Pigmentar/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Fenótipo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Chlorpyrifos (CPF) is an organophosphorous pesticide widely used in agricultural, industrial, and household applications. We have previously shown that JEG-3 cells are able to attenuate the oxidative stress induced by CPF through the adaptive activation of the Nrf2/ARE pathway. Considering that there is a relationship between oxidative stress and endoplasmic reticulum stress (ER), herein we investigated whether CPF also induces ER stress in JEG-3 cells. Cells were exposed to 50µM or 100µM CPF during 24h in conditions where cell viability was not altered. Western blot and PCR assays were used to explore the protein and mRNA levels of ER stress biomarkers, respectively. CPF induced an increase of the typical ER stress-related proteins, such as GRP78/BiP and IRE1α, a sensor for the unfolded protein response, as well as in phospho-eIF2α and XBP1 mRNA splicing. Additionally, CPF led to a decrease in p53 protein expression. The downregulation of p53 levels induced by CPF was partially blocked when cells were exposed to CPF in the presence of the proteasome inhibitor MG132. Altogether, these findings point out that CPF induces ER stress in JEG-3 cells; however these cells are able to attenuate it downregulating the levels of the pro-apoptotic protein p53.
Assuntos
Clorpirifos/toxicidade , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inseticidas/toxicidade , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Endorribonucleases/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação a X-Box/genéticaRESUMO
StarD7 is an intracellular lipid transport protein identified as up-regulated in the choriocarcinoma JEG-3 cell line. StarD7 facilitates the delivery of phosphatidylcholine (PC) to the mitochondria, and StarD7 knockdown causes a reduction in phospholipid synthesis. Since inhibition of PC synthesis may lead to endoplasmic reticulum (ER) stress we hypothesized that StarD7 may be involved in maintaining cell homeostasis. Here, we examined the effect of StarD7 silencing on ER stress response and on the levels of reactive oxygen species (ROS) in the human hepatoma cell line HepG2. StarD7 knockdown induced alterations in mitochondria and ER morphology. These changes were accompanied with an ER stress response as determined by increased expression of inositol-requiring enzyme 1α (IRE1α), calnexin, glucose regulated protein 78/immunoglobulin heavy chain-binding protein (Grp78/BiP), protein kinase-like ER kinase (PERK) as well as the phosphorylated eukaryotic translation initiation factor 2, subunit 1α (p-eIF2α). Additionally, a downregulation of the tumor suppressor p53 by a degradation mechanism was observed in StarD7 siRNA cells. Furthermore, StarD7 silencing induced ROS generation and reduced cell viability after H2O2 exposure. Decreased expression of StarD7 was associated to increased levels of the heme oxygenase-1 (HO-1) and catalase enzymes as well as in catalase enzymatic activity. Finally, no changes in levels of autophagy and apoptosis markers were observed in StarD7 siRNA treated cells respect to control cells. Taken together, these results indicate that StarD7 contributes to modulate cellular redox homeostasis.
Assuntos
Proteínas de Transporte/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica , Espécies Reativas de Oxigênio/metabolismo , Transporte Biológico , Calnexina/genética , Calnexina/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Catalase/genética , Catalase/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Endorribonucleases/genética , Endorribonucleases/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Células Hep G2 , Homeostase/genética , Humanos , Peróxido de Hidrogênio/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Fosfatidilcolinas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/agonistas , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND: Lysine acetylation is an important post-translational modification that plays a central role in eukaryotic transcriptional activation by modifying chromatin and transcription-related factors. Human pregnancy-specific glycoproteins (PSG) are the major secreted placental proteins expressed by the syncytiotrophoblast at the end of pregnancy and represent early markers of cytotrophoblast differentiation. Low PSG levels are associated with complicated pregnancies, thus highlighting the importance of studying the mechanisms that control their expression. Despite several transcription factors having been implicated as key regulators of PSG gene family expression; the role of protein acetylation has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Here, we explored the role of acetylation on PSG gene expression in the human placental-derived JEG-3 cell line. Pharmacological inhibition of histone deacetylases (HDACs) up-regulated PSG protein and mRNA expression levels, and augmented the amount of acetylated histone H3 associated with PSG 5'regulatory regions. Moreover, PSG5 promoter activation mediated by Sp1 and KLF6, via the core promoter element motif (CPE, -147/-140), was markedly enhanced in the presence of the HDAC inhibitor trichostatin A (TSA). This effect correlated with an increase in Sp1 acetylation and KLF6 nuclear localization as revealed by immunoprecipitation and subcellular fractionation assays. The co-activators PCAF, p300, and CBP enhanced Sp1-dependent PSG5 promoter activation through their histone acetylase (HAT) function. Instead, p300 and CBP acetyltransferase domain was dispensable for sustaining co-activation of PSG5 promoter by KLF6. CONCLUSIONS/SIGNIFICANCE: Results are consistent with a regulatory role of lysine acetylation on PSG expression through a relaxed chromatin state and an increase in the transcriptional activity of Sp1 and KLF6 following an augmented Sp1 acetylation and KLF6 nuclear localization.
Assuntos
Histonas/genética , Placenta/metabolismo , Glicoproteínas beta 1 Específicas da Gravidez/genética , Regulação para Cima/fisiologia , Acetilação/efeitos dos fármacos , Linhagem Celular , Feminino , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Lisina/genética , Lisina/metabolismo , Placenta/efeitos dos fármacos , Gravidez , Glicoproteínas beta 1 Específicas da Gravidez/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Regulação para Cima/efeitos dos fármacos , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
The effects of organophosphate pesticides on human placenta remain poorly investigated although an increased risk of pregnancy alterations has been reported in women chronically exposed to these pesticides. Here, we have addressed whether chlorpyrifos (CPF) modifies the expression of genes relevant for placental function. Human placental JEG-3 cells were exposed to increasing CPF concentrations up to 100 µM for 24 and 48 h and cell viability, mRNA, protein and hormone levels were analyzed. Quantitative RT-PCR assays revealed that CPF increased the expression of ABCG2, GCM1 and, even more significantly, ßhCG mRNAs in conditions where cell viability and morphology were not compromised. In addition, ßhCG protein synthesis and secretion were time-dependently augmented. Present results may reflect a CPF nocive effect on placenta cells or a placental-defense mechanism to preserve its function. These novel CPF trophoblast target genes should be considered in future studies of pregnancy outcomes associated with in vivo exposures.
Assuntos
Clorpirifos/toxicidade , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inseticidas/toxicidade , Placenta/efeitos dos fármacos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gonadotropina Coriônica Humana Subunidade beta/genética , Proteínas de Ligação a DNA , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Placenta/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Krüppel-like factor-6 (KLF6) is a widely expressed member of the Sp1/KLF family of transcriptional regulators involved in differentiation, cell cycle control and proliferation in several cell systems. Even though the highest expression level of KLF6 has been detected in human and mice placenta, its function in trophoblast physiology is still unknown. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we explored KLF6 expression and sub-cellular distribution in human trophoblast cells differentiating into the syncytial pathway, and its role in the regulation of genes associated with placental development and pregnancy maintenance. Confocal immunofluorescence microscopy demonstrated that KLF6 is expressed throughout human cytotrophoblast differentiation showing no evident modifications in its nuclear and cytoplasmic localization pattern. KLF6 transcript and protein peaked early during the syncytialization process as determined by qRT-PCR and western blot assays. Overexpression of KLF6 in trophoblast-derived JEG-3 cells showed a preferential nuclear signal correlating with enhanced expression of human ß-chorionic gonadotropin (ßhCG) and pregnancy-specific glycoprotein (PSG) genes. Moreover, KLF6 transactivated ßhCG5, PSG5 and PSG3 gene promoters. Deletion of KLF6 Zn-finger DNA binding domain or mutation of the consensus KLF6 binding site abolished transactivation of the PSG5 promoter. CONCLUSIONS/SIGNIFICANCE: Results are consistent with KLF6 playing a role as transcriptional regulator of relevant genes for placental differentiation and physiology such as ßhCG and PSG, in agreement with an early and transient increase of KLF6 expression during trophoblast syncytialization.