RESUMO
Oxygen-regulated protein 150 kD (ORP150) is a novel endoplasmic-reticulum-associated chaperone induced by hypoxia/ischemia. Although ORP150 was sparingly upregulated in neurons from human brain undergoing ischemic stress, there was robust induction in astrocytes. Cultured neurons overexpressing ORP150 were resistant to hypoxemic stress, whereas astrocytes with inhibited ORP150 expression were more vulnerable. Mice with targeted neuronal overexpression of ORP150 had smaller strokes compared with controls. Neurons with increased ORP150 demonstrated suppressed caspase-3-like activity and enhanced brain-derived neurotrophic factor (BDNF) under hypoxia signaling. These data indicate that ORP150 is an integral participant in ischemic cytoprotective pathways.
Assuntos
Morte Celular/fisiologia , Hipóxia Celular , Neurônios/patologia , Proteínas/fisiologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Choque Térmico HSP70 , Humanos , Camundongos , Neurônios/metabolismo , Proteínas/antagonistas & inibidoresRESUMO
To elucidate mechanisms underlying neuroprotective properties of astrocytes in brain ischemia, production of neurotrophic mediators was studied in astrocytes exposed to hypoxia/reoxygenation (H/R). Rat astrocytes subjected to H/R released increased amounts of interleukin (IL) 6 in a time-dependent manner, whereas levels of tumor necrosis factor and IL-1 remained undetectable. IL-6 transcripts were induced in hypoxia and the early phase of reoxygenation, whereas synthesis and release of IL-6 antigen/activity occurred during reoxygenation. Elevated levels of IL-6 mRNA were due, at least in part, to increased transcription, as shown by nuclear runoff analysis. The mechanism stimulating synthesis and release of IL-6 antigen by astrocytes was probably production of reactive oxygen intermediates (ROIs), which occurred within 15-20 minutes after placing hypoxia cultures back into normoxia, as the inhibitor diphenyl iodonium inhibited the burst of ROIs and subsequent IL-6 generation (blockade of nitric oxide formation had no effect on ROI generation or IL-6 production). Enhanced IL-6 generation was also observed in human astrocytoma cultures exposed to H/R. Survival of differentiated PC12 cells exposed to H/R was potentiated by conditioned medium from H/R astrocytes, an effect blocked by neutralizing anti-IL-6 antibody. In a gerbil model of brain ischemia, IL-6 activity was lower in the hippocampus, an area sensitive to ischemia, compared with IL-6 activity in the cortex, an area more resistant to ischemia. IL-6 antigen, demonstrated immunohistochemically, was increased in astrocytes from ischemic regions of gerbil brain. These data suggest that H/R enhances transcription of IL-6, resulting in increased translation and release of IL-6 antigen after the burst of ROI generated early during reoxygenation. Release of IL-6 from astrocytes could exert a paracrine neurotrophic effect in brain ischemia.
Assuntos
Astrócitos/fisiologia , Isquemia Encefálica/fisiopatologia , Encéfalo/fisiologia , Sobrevivência Celular/imunologia , Interleucina-6/biossíntese , Ataque Isquêmico Transitório/fisiopatologia , Neurônios/citologia , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/imunologia , Sequência de Bases , Encéfalo/imunologia , Isquemia Encefálica/imunologia , Células Cultivadas , Meios de Cultivo Condicionados , Primers do DNA , Expressão Gênica , Ataque Isquêmico Transitório/imunologia , Microglia/imunologia , Modelos Neurológicos , Dados de Sequência Molecular , Células PC12 , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Transcrição Gênica , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Neurodegenerative disorders are caused by progressive neuronal loss, and there is no complete treatment available yet. Neuroinflammation is a common feature across neurodegenerative disorders and implicated in the progression of neurodegeneration. Dysregulated activation of microglia causes neuroinflammation and has been highlighted as a treatment target in therapeutic strategies. Here, we identified novel therapeutic candidate ALGERNON2 (altered generation of neurons 2) and demonstrate that ALGERNON2 suppressed the production of proinflammatory cytokines and rescued neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease model. ALGERNON2 stabilized cyclinD1/p21 complex, leading to up-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which contributes to antioxidative and anti-inflammatory responses. Notably, ALGERNON2 enhanced neuronal survival in other neuroinflammatory conditions such as the transplantation of induced pluripotent stem cell-derived dopaminergic neurons into murine brains. In conclusion, we present that the microglial potentiation of the p21-Nrf2 pathway can contribute to neuronal survival and provide novel therapeutic potential for neuroinflammation-triggered neurodegeneration.
Assuntos
Microglia , Doenças Neurodegenerativas , Animais , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/terapia , Doenças NeuroinflamatóriasRESUMO
Application of differential display to cultured rat astrocytes subjected to hypoxia allowed cloning of a novel cDNA, termed stress-associated endoplasmic reticulum protein 1 (SERP1). Expression of SERP1 was enhanced in vitro by hypoxia and/or reoxygenation or other forms of stress, causing accumulation of unfolded proteins in endoplasmic reticulum (ER) stress, and in vivo by middle cerebral artery occlusion in rats. The SERP1 cDNA encodes a 66-amino acid polypeptide which was found to be identical to ribosome-associated membrane protein 4 (RAMP4) and bearing 29% identity to yeast suppressor of SecY 6 protein (YSY6p), suggesting participation in pathways controlling membrane protein biogenesis at ER. In cultured 293 cells subjected to ER stress, overexpression of SERP1/RAMP4 suppressed aggregation and/or degradation of newly synthesized integral membrane proteins, and subsequently, facilitated their glycosylation when the stress was removed. SERP1/RAMP4 interacted with Sec61alpha and Sec61beta, which are subunits of translocon, and a molecular chaperon calnexin. Furthermore, Sec61alpha and Sec61beta, but not SERP1/RAMP4, were found to associate with newly synthesized integral membrane proteins under stress. These results suggest that stabilization of membrane proteins in response to stress involves the concerted action of a rescue unit in the ER membrane comprised of SERP1/RAMP4, other components of translocon, and molecular chaperons in ER.
Assuntos
Hipóxia Celular/fisiologia , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Astrócitos/patologia , Isquemia Encefálica/patologia , Isquemia Encefálica/fisiopatologia , Proteínas de Ligação ao Cálcio/metabolismo , Calnexina , Linhagem Celular , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Homeostase/fisiologia , Humanos , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Desnaturação Proteica , Renaturação Proteica , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Canais de Translocação SEC , Homologia de Sequência de AminoácidosRESUMO
Expression of angiogenic factors such as VEGF under conditions of hypoxia or other kinds of cell stress contributes to neovascularization during wound healing. The inducible endoplasmic reticulum chaperone oxygen-regulated protein 150 (ORP150) is expressed in human wounds along with VEGF. Colocalization of these two molecules was observed in macrophages in the neovasculature, suggesting a role of ORP150 in the promotion of angiogenesis. Local administration of ORP150 sense adenovirus to wounds of diabetic mice, a treatment that efficiently targeted this gene product to the macrophages of wound beds, increased VEGF antigen in wounds and accelerated repair and neovascularization. In cultured human macrophages, inhibition of ORP150 expression caused retention of VEGF antigen within the endoplasmic reticulum (ER), while overexpression of ORP150 promoted the secretion of VEGF into hypoxic culture supernatants. Taken together, these data suggest an important role for ORP150 in the setting of impaired wound repair and identify a key, inducible chaperone-like molecule in the ER. This novel facet of the angiogenic response may be amenable to therapeutic manipulation.
Assuntos
Hipóxia Celular/fisiologia , Fatores de Crescimento Endotelial/fisiologia , Linfocinas/fisiologia , Chaperonas Moleculares/fisiologia , Neovascularização Fisiológica/fisiologia , Proteínas/fisiologia , Fatores de Transcrição , Adenoviridae/genética , Animais , Células Cultivadas , Meios de Cultivo Condicionados , Proteínas de Ligação a DNA/fisiologia , Complicações do Diabetes , Diabetes Mellitus/genética , Retículo Endoplasmático/metabolismo , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Fator 2 de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Terapia Genética , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Proteínas de Choque Térmico HSP70 , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Linfocinas/biossíntese , Linfocinas/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Neovascularização Patológica/fisiopatologia , Proteínas Nucleares/fisiologia , Oxigênio/farmacologia , Transporte Proteico , Proteínas/genética , RNA Antissenso/farmacologia , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/fisiologia , Método Simples-Cego , Pele/irrigação sanguínea , Pele/lesões , Fator de Crescimento Transformador beta/fisiologia , Fator de Crescimento Transformador beta1 , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Cicatrização/fisiologiaRESUMO
An important component of amyloid fibrils in dialysis-related amyloidosis is a form of beta2microglobulin modified with advanced glycation end products (AGEs) of the Maillard reaction, known as AGE-beta2M. We demonstrate here that the interaction of AGE-beta2M with mononuclear phagocytes (MPs), cells important in the pathogenesis of the inflammatory arthropathy of dialysis-related amyloidosis, is mediated by the receptor for AGEs, or RAGE. 125I-AGE-beta2M bound to immobilized RAGE or to MPs in a specific, dose-dependent manner (Kd approximately 53.5 and approximately 81.6 nM, respectively), a process inhibited in the presence of RAGE blockade. AGE-beta2M-mediated monocyte chemotaxis was prevented by excess sRAGE or anti-RAGE IgG. Induction of tumor necrosis factor-alpha (TNF) expression by MPs exposed to AGE-beta2M resulted from engagement of RAGE, as appearances of TNF transcripts and TNF antigen release into culture supernatants were prevented by addition of sRAGE, a process mediated, at least in part, by oxidant stress. AGE-beta2M reduced cytochrome c and the elaboration of TNF by MPs was inhibited by N-acetylcysteine. Consistent with these data, immunohistochemical studies of AGE-laden amyloid deposits of a long-term hemodialysis patient revealed positive staining for RAGE in the MPs infiltrating these lesions. These data indicate that RAGE is a central binding site for AGEs formed in vivo and suggest that AGE-beta2M-MP-RAGE interaction likely contributes to the initiation of an inflammatory response in amyloid deposits of long-term hemodialysis patients, a process which may ultimately lead to bone and joint destruction.
Assuntos
Amiloidose/etiologia , Produtos Finais de Glicação Avançada/metabolismo , Artropatias/etiologia , Leucócitos Mononucleares/metabolismo , Fagócitos/metabolismo , Receptores Imunológicos/metabolismo , Microglobulina beta-2/metabolismo , Antígenos CD/isolamento & purificação , Antígenos de Diferenciação Mielomonocítica/isolamento & purificação , Movimento Celular , Humanos , Oxirredução , Estresse Oxidativo , Ligação Proteica , Receptor para Produtos Finais de Glicação Avançada , Diálise Renal/efeitos adversos , Transdução de Sinais , Pele/patologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150-kDa oxygen-regulated protein (ORP150), is expressed both in the human brain after seizure attack and in mouse hippocampus after kainate administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of mu-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a possible role for ER stress in glutamate toxicity and pinpoint a key ER chaperone, ORP150, which contributes to the stress response critical for neuronal survival.
Assuntos
Retículo Endoplasmático/metabolismo , Ácido Glutâmico/toxicidade , Hipocampo/efeitos dos fármacos , Chaperonas Moleculares/metabolismo , Neurônios/efeitos dos fármacos , Proteínas/metabolismo , Animais , Proteínas de Choque Térmico HSP70 , Heterozigoto , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genética , Neurônios/metabolismo , Proteínas/genéticaRESUMO
Vascular cell adhesion molecule-1 (VCAM-1), an inducible cell-cell recognition protein on the endothelial cell surface (EC), has been associated with early stages of atherosclerosis. In view of the accelerated vascular disease observed in patients with diabetes, and the enhanced expression of VCAM-1 in diabetic rabbits, we examined whether irreversible advanced glycation endproducts (AGEs), could mediate VCAM-1 expression by interacting with their endothelial cell receptor (receptor for AGE, RAGE). Exposure of cultured human ECs to AGEs induced expression of VCAM-1, increased adhesivity of the monolayer for Molt-4 cells, and was associated with increased levels of VCAM-1 transcripts. The inhibitory effect of anti-RAGE IgG, a truncated form of the receptor (soluble RAGE) or N-acetylcysteine on VCAM-1 expression indicated that AGE-RAGE-induced oxidant stress was central to VCAM-1 induction. Electrophoretic mobility shift assays on nuclear extracts from AGE-treated ECs showed induction of specific DNA binding activity for NF-kB in the VCAM-1 promoter, which was blocked by anti-RAGE IgG or N-acetylcysteine. Soluble VCAM-1 antigen was elevated in human diabetic plasma. These data are consistent with the hypothesis that AGE-RAGE interaction induces expression of VCAM-1 which can prime diabetic vasculature for enhanced interaction with circulating monocytes.
Assuntos
Moléculas de Adesão Celular/biossíntese , Angiopatias Diabéticas/fisiopatologia , Endotélio Vascular/fisiologia , Expressão Gênica , Produtos Finais de Glicação Avançada/metabolismo , Produtos Finais de Glicação Avançada/farmacologia , Receptores Imunológicos/fisiologia , Animais , Sequência de Bases , Adesão Celular , Moléculas de Adesão Celular/análise , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Primers do DNA , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Modelos Biológicos , Dados de Sequência Molecular , Estresse Oxidativo , Reação em Cadeia da Polimerase , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Transcrição Gênica , Veias Umbilicais , Molécula 1 de Adesão de Célula VascularRESUMO
Dysfunctional endothelium is associated with and, likely, predates clinical complications of diabetes mellitus, by promoting increased vascular permeability and thrombogenicity. Irreversible advanced glycation end products (AGEs), resulting from nonenzymatic glycation and oxidation of proteins or lipids, are found in plasma, vessel wall, and tissues and have been linked to the development of diabetic complications. The principal means through which AGEs exert their cellular effects is via specific cellular receptors, one of which, receptor for AGE (RAGE), is expressed by endothelium. We report that blockade of RAGE inhibits AGE-induced impairment of endothelial barrier function, and reverse, in large part, the early vascular hyperpermeability observed in diabetic rats. Inhibition of AGE- and diabetes-mediated hyperpermeability by antioxidants, both in vitro and in vivo, suggested the central role of AGE-RAGE-induced oxidant stress in the development of hyperpermeability. Taken together, these data support the concept that ligation of AGEs by endothelial RAGE induces cellular dysfunction, at least in part by an oxidant-sensitive mechanism, contributing to vascular hyperpermeability in diabetes, and that RAGE is central to this pathologic process.
Assuntos
Permeabilidade Capilar , Diabetes Mellitus Experimental/metabolismo , Endotélio Vascular/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Receptores Imunológicos/metabolismo , Adulto , Albuminas/farmacocinética , Animais , Antioxidantes/farmacologia , Permeabilidade Capilar/efeitos dos fármacos , Bovinos , Células Cultivadas , Diabetes Mellitus/sangue , Endotélio Vascular/citologia , Transfusão de Eritrócitos , Eritrócitos/fisiologia , Humanos , Inulina/farmacocinética , Pessoa de Meia-Idade , Probucol/farmacologia , Ratos , Receptor para Produtos Finais de Glicação Avançada , Solubilidade , Vitamina E/farmacologiaAssuntos
Astrócitos/efeitos dos fármacos , Chalconas/química , Chalconas/farmacologia , Lipopolissacarídeos/farmacologia , Óxido Nítrico/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Morte Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Fatores de TempoRESUMO
Expression of angiogenic factors such as vascular endothelial growth factor (VEGF) under conditions of cell stress involves both transcriptional and translational events, as well as an important role for inducible endoplasmic reticulum (ER) chaperones. Coexpression of VEGF and 150-kDa oxygen-regulated protein (ORP), a novel ER chaperone, in human glioblastoma suggested a link between angiogenesis and ORP150. C6 glioma cells stably transfected with ORP150 antisense displayed selectively reduced ORP150 expression. Tumors raised after inoculation of immunocompromised mice with ORP150 antisense C6 glioma transfectants demonstrated an initial phase of growth comparable to wild-type C6 glioma cells which was followed by marked regression within 8 days. Decreased density of platelet/endothelial cell adhesion molecule 1-positive structures within the tumor bed was consistent with reduced angiogenesis in C6 gliomas expressing ORP150 antisense, compared with tumors derived from C6 cells overexpressing ORP150 sense or vector controls. In vitro, inhibition of ORP150 expression decreased release of VEGF into culture supernatants; in ORP150 antisense transfectants, VEGF accumulated intracellularly within the ER. These findings demonstrate a critical role for the inducible ER chaperone ORP150 in tumor-mediated angiogenesis via processing of VEGF, and, thus, highlight a new facet of angiogenic mechanisms amenable to therapeutic manipulation in tumors.
Assuntos
Retículo Endoplasmático/metabolismo , Neovascularização Patológica/metabolismo , Proteínas/fisiologia , Animais , Autoanticorpos/biossíntese , Autoanticorpos/sangue , Divisão Celular/fisiologia , Hipóxia Celular/fisiologia , DNA Antissenso/genética , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/metabolismo , Glioblastoma/irrigação sanguínea , Glioblastoma/imunologia , Glioblastoma/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Choque Térmico HSP70 , Humanos , Linfocinas/biossíntese , Linfocinas/metabolismo , Camundongos , Neovascularização Patológica/imunologia , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas/genética , Ratos , Transfecção , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Exposure of proteins to reducing sugars results in nonenzymatic glycation with the ultimate formation of advanced glycation end products (AGEs). One means through which AGEs modulate cellular functions is through binding to specific cell surface acceptor molecules. The receptor for AGEs (RAGE) is such a receptor and is a newly identified member of the immunoglobulin superfamily expressed on endothelial cells (ECs), mononuclear phagocytes (MPs), and vascular smooth muscle cells (SMCs) in both vivo and in vitro. Binding of AGEs to RAGE results in induction of cellular oxidant stress, as exemplified by the generation of thiobarbituric acid-reactive substances, expression of heme oxygenase type I, and activation of the transcription factor NF-kB, with consequences for a range of cellular functions. AGEs on the surface of diabetic red cells enhance binding to endothelial RAGE and result in enhanced oxidant stress in the vessel wall. By using reagents to selectively block access to RAGE, the role of this receptor in AGE-mediated perturbation of cellular properties can be dissected in detail.
Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Receptores Imunológicos/fisiologia , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Humanos , Macrófagos/metabolismo , Malondialdeído/metabolismo , Oxirredução , Ratos , Receptor para Produtos Finais de Glicação AvançadaRESUMO
Based on the neurotrophic properties of astrocytes in response to ischemia, the current work focuses on the mechanism for cultured astrocytes to adapt to a hypoxic environment. Intracellular glucose levels in primary cultured rat astrocytes exposed to hypoxia fell by 30% within 24 h, in parallel with a decrease in glycogen stores. Glycolytic metabolism was crucial for cell survival during hypoxia, as 2-deoxyglucose resulted in rapid ATP depletion and cell death. The mechanism for maintaining glucose levels under these conditions appeared to be mobilization of glycogen stores, rather than increased extracellular uptake of glucose, as gluconolactone (an inhibitor of beta1-4 amyloglucosidase) induced a rapid fall in cellular ATP in cultures subjected to hypoxia, whereas cytochalasin B was without affect. Addition of cycloheximide diminished the viability of astrocytes in hypoxia, suggesting an obligatory role of de-novo gene expression to respond to hypoxia. Consistently, the results of differential display suggested the induction of glycolytic enzymes, including aldolase A (EC 4.1.2.13), hexokinase II (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), and triosephosphate isomerase (EC 5.3.1.1) in the hypoxic culture. Marked induction of these glycolytic enzymes in hypoxic astrocytes was confirmed by Northern blot analysis. These data provide a theoretical basis to understand the ability of astrocytes to tolerate ischemic condition.
Assuntos
Astrócitos/metabolismo , Enzimas/metabolismo , Glucose/metabolismo , Hipóxia/fisiopatologia , Animais , Animais Recém-Nascidos , Antimetabólitos/farmacologia , Astrócitos/citologia , Astrócitos/enzimologia , Northern Blotting , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Desoxiglucose/farmacologia , Indução Enzimática , Enzimas/genética , Frutose-Bifosfato Aldolase/genética , Frutose-Bifosfato Aldolase/metabolismo , Regulação da Expressão Gênica , Glucose/farmacocinética , Glicogênio/metabolismo , Glicólise , Hexoquinase/genética , Hexoquinase/metabolismo , Oxigênio/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA/genética , RNA/metabolismo , Ratos , Ratos Sprague-Dawley , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismoRESUMO
Vertebro-basilar insufficiency (VBI) is a vague clinical entity including several symptoms such as faintness, dizziness, vertigo. Millikan and Siekert reported a 'syndrome of intermittent insufficiency of basilar arterial system'. But vascular abnormalities responsible for such symptoms are often hardly diagnosed by conventional angiography, because vertebro-basilar artery systems have many kinds of anomalous congenital origins. Conventional angiography gives us arterial informations such as stenosis, occlusion and malformations, but it often fails to reveal capillary perfusions because of its relatively poor density resolution. Digital subtraction angiography (DSA) is considered to have improved density resolution, and it may enable us to detect vascular perfusions more sensitively in the capillary phase. In this study, we evaluated the usefulness of the capillary filling analysis in digital subtraction angiography for diagnosis of VBI.
Assuntos
Angiografia Cerebral , Insuficiência Vertebrobasilar/diagnóstico por imagem , Capilares/diagnóstico por imagem , Humanos , Insuficiência Vertebrobasilar/fisiopatologiaRESUMO
We have examined changes in rectal temperature of mice after subcutaneous administrations of d-methamphetamine alone or methamphetamine plus morphine. Methamphetamine 5 mg/kg produced slight hyperthermia, while simultaneous administration of morphine (25-100 mg/kg), which alone produces hypothermia, potentiated markedly the increase in body temperature by methamphetamine. Methamphetamine showed a hyperthermic effect in a dose-dependent manner in the presence of morphine. The hyperthermia due to methamphetamine plus morphine was avoided by pretreatment with 10 mg/kg naloxone. When animals were pretreated with 2.5 mg/kg haloperidol, hyperthermia due to methamphetamine alone was completely abolished, while that due to methamphetamine plus morphine was still observed. These results showed that dopamine may be implicated in methamphetamine hyperthermia and a haloperidol-nonsensitive mechanism may be involved in the methamphetamine-morphine hyperthermia.
Assuntos
Regulação da Temperatura Corporal/efeitos dos fármacos , Febre/induzido quimicamente , Haloperidol/farmacologia , Metanfetamina/toxicidade , Morfina/toxicidade , Naloxona/farmacologia , Animais , Sinergismo Farmacológico , Masculino , Camundongos , Atividade Motora/efeitos dos fármacosAssuntos
Astrócitos/fisiologia , Isquemia Encefálica , Neurônios/fisiologia , Estresse Fisiológico/fisiopatologia , Animais , Apoptose/fisiologia , Encéfalo/citologia , Isquemia Encefálica/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiologia , HumanosRESUMO
Advanced glycation endproducts (AGEs), which accumulate in diabetic vasculature, result in enhanced expression of endothelial cell-associated vascular cell adhesion molecule-1 (VCAM-1) as well release of a soluble form of VCAM-1 (sVCAM-1) into culture supernatants. We hypothesized that sVCAM-1 in diabetic plasma might reflect early vascular perturbation in diabetic vasculopathy. Diabetic patients with microalbuminuria, a group with a high incidence of vascular complications, had increased plasma levels of sVCAM-1, approximately 1.5-fold greater than diabetic patients without microalbuminuria; P < 0.05. sVCAM-1 may be an indicator of ongoing cellular dysfunction in diabetes, as well as a dynamic surrogate marker for the effectiveness of therapeutic interventions.
Assuntos
Albuminúria/sangue , Diabetes Mellitus/sangue , Angiopatias Diabéticas/diagnóstico , Molécula 1 de Adesão de Célula Vascular/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: To investigate the relationship between the expression of the 150-kDa oxygen-regulated protein (ORP150, which functions as a molecular chaperone in the endoplasmic reticulum for the folding and trafficking of newly synthesized proteins) and the aggressiveness of bladder cancer, and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), as the former is a secreting protein through the endoplasmic reticulum and the latter are closely involved in tumour invasion. MATERIALS AND METHODS: Thirty-nine cystectomy specimens, comprising 12 superficial (pT1) and 27 invasive (pT2-pT4) tumours, were immunohistochemically analysed using antibodies against ORP150, VEGF, MMP-1, MMP-2 and MMP-9. Staining was scored from 0 to 3, according to the ratio of positively staining cells. RESULTS: Staining was positive (score 1-3) for ORP150 in 10 of 12 superficial and 25 (93%) of the invasive tumours, with a significantly higher staining score for stage T4 than stage T1 tumours. The trend was the same for the staining score of MMP-2, and there was a significant correlation between ORP150 and MMP-2 expression. CONCLUSIONS: The expression of ORP150 was common in bladder cancer, with a tendency for greater expression in higher stages. The significant correlation between ORP150 and MMP-2 expression suggests that ORP150 acts as a molecular chaperone for MMP-2 secretion and thus tumour invasion.
Assuntos
Proteínas/metabolismo , Neoplasias da Bexiga Urinária/diagnóstico , Fatores de Crescimento Endotelial/metabolismo , Proteínas de Choque Térmico HSP70 , Humanos , Imuno-Histoquímica , Linfocinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Chaperonas Moleculares/metabolismo , Invasividade Neoplásica , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Advanced glycation end products (AGEs) exert their cellular effects on cells by interacting with specific cellular receptors, the best characterized of which is the receptor for AGE (RAGE). The transductional processes by which RAGE ligation transmits signals to the nuclei of cells is unknown and was investigated. AGE-albumin, a prototypic ligand, activated p21(ras) in rat pulmonary artery smooth muscle cells that express RAGE, whereas nonglycated albumin was without effect. MAP kinase activity was enhanced at concentrations of AGE-albumin, which activated p21(ras) and NF-kappaB. Depletion of intracellular glutathione rendered cells more sensitive to AGE-mediated activation of this signaling pathway. In contrast, signaling was blocked by preventing p21(ras) from associating with the plasma membrane or mutating Cys118 on p21(ras) to Ser. Signaling was receptor-dependent, because it was prevented by blocking access to RAGE with either anti-RAGE IgG or by excess soluble RAGE. These data suggest that RAGE-mediated induction of cellular oxidant stress triggers a cascade of intracellular signals involving p21(ras) and MAP kinase, culminating in transcription factor activation. The molecular mechanism that triggers this pathway likely involves oxidant modification and activation of p21(ras).
Assuntos
Alquil e Aril Transferases , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Estresse Oxidativo , Transferases/metabolismo , Animais , Ativação Enzimática , Glutationa/metabolismo , Células PC12 , Ratos , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo , Transdução de SinaisRESUMO
Advanced glycation end products (AGEs) form by the interaction of aldoses with proteins and the subsequent molecular rearrangements of the covalently linked sugars, eventuating in a diverse group of fluorescent compounds of yellow-brown color. This heterogeneous class of nonenzymatically glycated proteins or lipids is found in the plasma and accumulates in the vessel wall and tissues even in normal aging. As a consequence of hyperglycemia, AGE formation and deposition are much enhanced in diabetes, in which their presence has been linked to secondary complications, especially microvascular disease. This review summarizes the cellular interactions of AGEs and describes the central role of a novel receptor for AGE (RAGE). RAGE, an immunoglobulin superfamily member, mediates the binding of AGEs to endothelial cells and mononuclear phagocytes, interacts with a lactoferrin-like polypeptide that also binds AGEs, and appears to activate intracellular signal transduction mechanisms consequent to its interaction with the glycated ligand. RAGE is expressed by ECs, mononuclear phagocytes, smooth muscle cells, mesangial cells, and neurons, indicating a potential role in the regulation of their properties in homeostasis and/or their dysfunction in the development of diabetic complications. Since AGEs have been shown to generate reactive oxygen intermediates, tethering of AGEs to the cell surface by their receptors focuses oxidant stress on cellular targets, resulting in changes in gene expression and the cellular phenotype. The discovery of RAGE and development of reagents to block its interaction with AGEs should provide insights into the role of this ligand-receptor interaction in the pathogenesis of diabetic complications and, potentially, atherosclerosis.