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1.
FASEB J ; 24(1): 158-72, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19741171

RESUMO

Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS, including a prooxidant status and a shorter mean life span compared to wild-type animals. Here, we show that Wrn mutant mice also develop premature liver sinusoidal endothelial defenestration along with inflammation and metabolic syndrome. Vitamin C supplementation rescued the shorter mean life span of Wrn mutant mice and reversed several age-related abnormalities in adipose tissues and liver endothelial defenestration, genomic integrity, and inflammatory status. At the molecular level, phosphorylation of age-related stress markers like Akt kinase-specific substrates and the transcription factor NF-kappaB, as well as protein kinase Cdelta and Hif-1alpha transcription factor levels, which are increased in the liver of Wrn mutants, were normalized by vitamin C. Vitamin C also increased the transcriptional regulator of lipid metabolism PPARalpha. Finally, microarray and gene set enrichment analyses on liver tissues revealed that vitamin C decreased genes normally up-regulated in human WS fibroblasts and cancers, and it increased genes involved in tissue injury response and adipocyte dedifferentiation in obese mice. Vitamin C did not have such effect on wild-type mice. These results indicate that vitamin C supplementation could be beneficial for patients with WS.


Assuntos
Envelhecimento/efeitos dos fármacos , Ácido Ascórbico/uso terapêutico , Síndrome de Werner/tratamento farmacológico , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Envelhecimento/genética , Envelhecimento/metabolismo , Animais , Sequência de Bases , DNA Mitocondrial/genética , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glutationa/sangue , Glutationa/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Longevidade/efeitos dos fármacos , Longevidade/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Estresse Oxidativo , PPAR alfa/genética , RecQ Helicases/genética , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Síndrome de Werner/patologia , Helicase da Síndrome de Werner
2.
J Electron Microsc (Tokyo) ; 59(1): 65-9, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19648606

RESUMO

Old age is associated with ultrastructural changes in the hepatic sinusoid called pseudocapillarization, which include defenestration and thickening of the sinusoidal endothelium. We investigated whether such changes also occur in isolated and cultured liver sinusoidal endothelial cells. Liver sinusoidal endothelial cells were isolated from young (6-10 months, n = 4) and old (24-26 months, n = 4) F344 rats and fenestrations evaluated using scanning electron microscopy. Fenestration diameter was reduced in old age from 194 +/- 1 nm to 185 +/- 1 nm (P < 0.001) and there was an age-related increase in the number fused fenestrations and large gaps. Age-related changes in the diameter of fenestrations in the liver sinusoidal endothelial cell are maintained following isolation and culture. This suggests that this age-related change may be intrinsic to the liver endothelial cell and/or irreversible.


Assuntos
Envelhecimento/patologia , Fígado/ultraestrutura , Animais , Células Cultivadas , Células Endoteliais/ultraestrutura , Técnicas In Vitro , Masculino , Microscopia Eletrônica de Varredura , Ratos , Ratos Endogâmicos F344
3.
Int J Exp Pathol ; 89(6): 410-8, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19134050

RESUMO

The liver sinusoidal endothelial cell (LSEC) is damaged by many toxins, including oxidants and bacterial toxins. Any effect on LSECs of the Pseudomonas aeruginosa virulence factor, pyocyanin, may be relevant for systemic pseudomonal infections and liver transplantation. In this study, the effects of pyocyanin on in vivo rat livers and isolated LSECs were assessed using electron microscopy, immunohistochemistry and biochemistry. In particular, the effect on fenestrations, a crucial morphological aspect of LSECs was assessed. Pyocyanin treatment induced a dose-dependent reduction in fenestrations in isolated LSECs. In the intact liver, intraportal injection of pyocyanin (11.9 microM in blood) was associated with a reduction in endothelial porosity from 3.4 +/- 0.2% (n = 5) to 1.3 +/- 0.1% (n = 7) within 30 min. There were decreases in both diameter and frequency of fenestrations in the intact endothelium. There was also a decrease in endothelial thickness from 175.8 +/- 5.8 to 156.5 +/- 4.0 nm, an endothelial pathology finding previously unreported. Hepatocyte ultrastructure, liver function tests and immunohistochemical markers of oxidative stress (3-nitrotyrosine and malondialdehyde) were not affected. Pyocyanin induces significant ultrastructural changes in the LSEC in the absence of immunohistochemical evidence of oxidative stress or hepatocyte injury pointing to a novel mechanism for pyocyanin pathogenesis.


Assuntos
Células Endoteliais/efeitos dos fármacos , Hepatopatias/patologia , Fígado/ultraestrutura , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa , Piocianina/farmacologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Esquema de Medicação , Células Endoteliais/ultraestrutura , Imuno-Histoquímica , Fígado/microbiologia , Hepatopatias/microbiologia , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Estresse Oxidativo , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley
4.
Prog Neuropsychopharmacol Biol Psychiatry ; 32(2): 492-8, 2008 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-17976880

RESUMO

Post-mortem studies of the human brain indicate that certain GABA(A) receptor subtypes may be differentially altered in schizophrenia. Increased binding to the total population of GABA(A) receptors using [3H]muscimol is observed in the post-mortem schizophrenic brain, yet a proportion of these receptors which bind benzodiazepines and are labelled with [3H]flunitrazepam, show decreased or unaltered expression. Data from animal studies suggest that antipsychotic drugs alter GABA(A) receptor expression in a subtype selective manner, but in the opposite direction to that observed in schizophrenia. To broaden our understanding of the effects of antipsychotic drugs on GABA(A) receptors, we examined the saturation binding maximum (B(max)) and binding affinity (K(D)) of [3H]muscimol and [3H]flunitrazepam in the prefrontal cortex (PFC), hippocampus and thalamus of male SD rats that received a sucrose solution containing either haloperidol (1.5 mg/kg), olanzapine (6.5 mg/kg) or no drug daily for up to 28 days using quantitative receptor autoradiography. [3H]Muscimol binding density was increased most prominently in the PFC after 7 days, with larger and more prolonged effects being induced by the atypical antipsychotic drug olanzapine in subcortical regions. While no changes were observed in [3H]muscimol binding in any region after 28 days of drug administration, [3H]flunitrazepam binding density (B(max)) was increased for both antipsychotic treatments in the PFC only. These findings confirm that the subset of GABA(A) receptors sensitive to benzodiazepines are regulated differently from other GABA(A) receptor subtypes following antipsychotic drug administration, in a time- and region-dependent manner.


Assuntos
Antipsicóticos/farmacocinética , Benzodiazepinas/farmacocinética , Flunitrazepam/metabolismo , Haloperidol/farmacocinética , Muscimol/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptores de GABA-A/metabolismo , Animais , Antipsicóticos/administração & dosagem , Antipsicóticos/uso terapêutico , Autorradiografia , Benzodiazepinas/uso terapêutico , Densitometria , Haloperidol/uso terapêutico , Hipocampo/diagnóstico por imagem , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Masculino , Olanzapina , Córtex Pré-Frontal/diagnóstico por imagem , Cintilografia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/efeitos dos fármacos , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismo , Tálamo/diagnóstico por imagem , Tálamo/efeitos dos fármacos , Tálamo/metabolismo , Distribuição Tecidual/efeitos dos fármacos , Trítio/metabolismo
5.
Exp Gerontol ; 42(10): 1012-9, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17658234

RESUMO

In old age, the liver contains less ATP and hypoxia-responsive genes are upregulated. Age-related changes in hepatic perfusion and the liver sinusoidal endothelial cell (LSEC) could contribute to this altered hepatic oxygen-dependent metabolism by causing intrahepatocytic hypoxia. Furthermore, age-related changes in the LSEC ('pseudocapillarization') have been partially induced by ATP depletion. To investigate whether there is intracellular hypoxia in the old rat liver, pimonidazole immunohistochemistry in intact livers and ATP levels in isolated LSECs were studied from young and old rats. There were no age-related changes. To determine whether defenestration of the LSEC could impair oxygen diffusion, pimonidazole immunohistochemistry was performed in rats treated with poloxamer 407. Despite defenestration, there was no change in pimonidazole staining. Immunohistochemistry was then performed to determine whether there are age-related changes in VEGF and VEGFR2. VEGF staining was not associated with age. However, there was an increase in perisinusoidal VEGFR2 expression with increasing age. In conclusion, liver hypoxia does not occur in old age and LSEC pseudocapillarization does not constitute an oxygen-diffusion barrier. There are no age-related changes in VEGF expression but an increase in perisinusoidal VEGFR2 expression, which has implications for the effects of aging on the hepatic sinusoid.


Assuntos
Envelhecimento/metabolismo , Fígado/metabolismo , Consumo de Oxigênio/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Envelhecimento/patologia , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Técnicas Imunoenzimáticas , Fígado/ultraestrutura , Circulação Hepática/fisiologia , Masculino , Microscopia Eletrônica de Varredura , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley
6.
Schizophr Res ; 90(1-3): 76-80, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17208412

RESUMO

Changes in GABA(A) receptors are observed in schizophrenia, with benzodiazepine-sensitive GABA(A) receptor subtypes being affected differently to other subtypes. However, long-term antipsychotic drug use in schizophrenia may underlie these changes. To test this, we examined the effects of administering a typical (haloperidol) and an atypical (olanzapine) antipsychotic drug on the GABA(A) receptor agonist (orthosteric) and benzodiazepine (allosteric) binding sites in rat prefrontal cortex. As antipsychotic drugs have delayed maximal therapeutic effects we also examined different drug treatment periods. Male SD rats received a sucrose solution containing either haloperidol (1.5 mg/kg), olanzapine (6.5 mg/kg) or no drug daily for either 7, 14 or 28 days. Sections of rat brain were then labelled with [(3)H]muscimol, which labels the total population of GABA(A) receptors, or the benzodiazepine site ligand [(3)H]flunitrazepam in separate saturation binding experiments using quantitative receptor autoradiography. [(3)H]Muscimol binding was enhanced in the prefrontal cortex after 7 days but no differences were observed after longer periods of drug administration. In contrast there was a delayed increase in density of benzodiazepine-sensitive GABA(A) receptors in the PFC, suggesting that antipsychotic drugs have different effects on different GABA(A) receptor subtypes. These changes in the properties of GABA(A) receptor binding following antipsychotic drug administration are not consistent with those observed in schizophrenia and suggest a 'reshuffling' in GABA(A) receptor subtypes over time.


Assuntos
Antipsicóticos/farmacologia , Flunitrazepam/farmacocinética , Agonistas GABAérgicos/farmacocinética , Moduladores GABAérgicos/farmacocinética , Haloperidol/farmacologia , Muscimol/farmacocinética , Córtex Pré-Frontal/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Animais , Benzodiazepinas/farmacologia , Ligação Competitiva/efeitos dos fármacos , Humanos , Assistência de Longa Duração , Masculino , Olanzapina , Córtex Pré-Frontal/patologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Esquizofrenia/tratamento farmacológico , Esquizofrenia/patologia
7.
J Vis Exp ; (98): e52698, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25993325

RESUMO

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures--both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.


Assuntos
Células Endoteliais/ultraestrutura , Fígado/citologia , Microscopia Eletrônica de Varredura/métodos , Animais , Células Endoteliais/citologia , Hepatócitos/citologia , Processamento de Imagem Assistida por Computador/métodos , Fígado/ultraestrutura , Camundongos , Ratos
8.
J Gerontol A Biol Sci Med Sci ; 69(9): 1076-86, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24149428

RESUMO

Werner syndrome is a progeric syndrome characterized by premature atherosclerosis, diabetes, cancer, and death in humans. The knockout mouse model created by deletion of the RecQ helicase domain of the mouse Wrn homologue gene (Wrn(∆hel/∆hel)) is of great interest because it develops atherosclerosis and hypertriglyceridemia, conditions associated with aging liver and sinusoidal changes. Here, we show that Wrn(∆hel/∆hel) mice exhibit increased extracellular matrix, defenestration, decreased fenestration diameter, and changes in markers of liver sinusoidal endothelial cell inflammation, consistent with age-related pseudocapilliarization. In addition, hepatocytes are larger, have increased lipofuscin deposition, more frequent nuclear morphological anomalies, decreased mitochondria number, and increased mitochondrial diameter compared to wild-type mice. The Wrn(∆hel/∆hel) mice also have altered mitochondrial function and altered nuclei. Microarray data revealed that the Wrn(∆hel/∆hel) genotype does not affect the expression of many genes within the isolated hepatocytes or liver sinusoidal endothelial cells. This study reveals that Wrn(∆hel/∆hel) mice have accelerated typical age-related liver changes including pseudocapillarization. This confirms that pseudocapillarization of the liver sinusoid is a consistent feature of various aging models. Moreover, it implies that DNA repair may be implicated in normal aging changes in the liver.


Assuntos
Fígado/patologia , Síndrome de Werner/patologia , Envelhecimento/patologia , Envelhecimento/fisiologia , Animais , Capilares/patologia , Reparo do DNA/fisiologia , Modelos Animais de Doenças , Imuno-Histoquímica , Fígado/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Mitocôndrias Hepáticas/patologia , Mitocôndrias Hepáticas/fisiologia , Mitocôndrias Hepáticas/ultraestrutura , RecQ Helicases/genética , Smegmamorpha , Helicase da Síndrome de Werner
9.
Toxicol In Vitro ; 24(1): 123-8, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19720132

RESUMO

Aging is associated with increased susceptibility to oxidative stress. To study this in the liver and to elucidate underlying mechanisms, hepatocytes from young (4-6 months) and old (24-26 months) rats were exposed to two oxidants, hydrogen peroxide and tert-butyl hydroperoxide. ATP content and mitochondrial activity were lower in old hepatocytes and decreased further with oxidative stress. Expression of Cu/Zn superoxide dismutase, Mn superoxide dismutase and catalase was not substantially influenced by oxidative stress in young and old hepatocytes, whereas glutathione peroxidase 1 expression was markedly increased only in young hepatocytes. Oxidative stress in young hepatocytes led to increased expression of apoE and movement of apoE to the early endosomes. In old hepatocytes, oxidative stress did not increase apoE expression and apoE was co-localized with early endosomes under control conditions. The results show that old age is associated with impaired hepatocyte responses of mitochondria, ATP, glutathione peroxidase 1 and apoE to oxidative stress.


Assuntos
Envelhecimento/fisiologia , Hepatócitos/efeitos dos fármacos , Peróxido de Hidrogênio/toxicidade , Oxidantes/toxicidade , terc-Butil Hidroperóxido/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Antioxidantes/metabolismo , Apolipoproteínas E/biossíntese , Apolipoproteínas E/metabolismo , Catalase/metabolismo , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Hepatócitos/metabolismo , L-Lactato Desidrogenase/metabolismo , Masculino , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/metabolismo
10.
Pathology ; 42(3): 255-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20350219

RESUMO

AIMS: Fenestrations are pores in the liver sinusoidal endothelial cell that facilitate the transfer of substrates between blood and hepatocytes. The aim of this study was to determine the effect of nutritional state on the morphology of fenestrations. METHODS: Scanning electron microscopy was used to investigate fenestrations in livers from fasted and fed rats. RESULTS: Fasting for 48 hours in rats was associated with an increase in the diameter of fenestrations from 90.7 +/- 11.7 nm in the fed state to 99.0 +/- 12.11 nm (p < 0.005). There was a concomitant reduction in the frequency of fenestrations from 8.45 +/- 2.43 to 7.39 +/- 2.28 fenestrations per microm(2) (p =0.05). CONCLUSIONS: Fasting was associated with increased diameter of fenestrations. The results provide evidence that fenestrations are dynamic structures that respond in vivo to physiological stimuli such as nutritional status.


Assuntos
Células Endoteliais/ultraestrutura , Comportamento Alimentar/fisiologia , Privação de Alimentos/fisiologia , Fígado/irrigação sanguínea , Fígado/ultraestrutura , Animais , Circulação Hepática , Masculino , Microscopia Eletrônica de Varredura , Ratos , Ratos Endogâmicos F344
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