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1.
Traffic ; 25(4): e12934, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38613404

RESUMO

Alzheimer's disease (AD) pathology is characterized by amyloid beta (Aß) plaques and dysfunctional autophagy. Aß is generated by sequential proteolytic cleavage of amyloid precursor protein (APP), and the site of intracellular APP processing is highly debated, which may include autophagosomes. Here, we investigated the involvement of autophagy, including the role of ATG9 in APP intracellular trafficking and processing by applying the RUSH system, which allows studying the transport of fluorescently labeled mCherry-APP-EGFP in a systematic way, starting from the endoplasmic reticulum. HeLa cells, expressing the RUSH mCherry-APP-EGFP system, were investigated by live cell imaging, immunofluorescence, and Western blot. We found that mCherry-APP-EGFP passed through the Golgi faster in ATG9 knockout cells. Furthermore, ATG9 deletion shifted mCherry-APP-EGFP from early endosomes and lysosomes toward the plasma membrane concomitant with reduced endocytosis. Importantly, this alteration in mCherry-APP-EGFP transport resulted in increased secreted mCherry-soluble APP and C-terminal fragment-EGFP. These effects were also phenocopied by pharmacological inhibition of ULK1, indicating that autophagy is regulating the intracellular trafficking and processing of APP. These findings contribute to the understanding of the role of autophagy in APP metabolism and could potentially have implications for new therapeutic approaches for AD.


Assuntos
Doença de Alzheimer , Precursor de Proteína beta-Amiloide , Humanos , Peptídeos beta-Amiloides , Células HeLa , Transporte Biológico , Autofagia
2.
Mol Psychiatry ; 28(9): 3966-3981, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-37907591

RESUMO

Accumulation of amyloid ß-peptide (Aß) is a driver of Alzheimer's disease (AD). Amyloid precursor protein (App) knock-in mouse models recapitulate AD-associated Aß pathology, allowing elucidation of downstream effects of Aß accumulation and their temporal appearance upon disease progression. Here we have investigated the sequential onset of AD-like pathologies in AppNL-F and AppNL-G-F knock-in mice by time-course transcriptome analysis of hippocampus, a region severely affected in AD. Strikingly, energy metabolism emerged as one of the most significantly altered pathways already at an early stage of pathology. Functional experiments in isolated mitochondria from hippocampus of both AppNL-F and AppNL-G-F mice confirmed an upregulation of oxidative phosphorylation driven by the activity of mitochondrial complexes I, IV and V, associated with higher susceptibility to oxidative damage and Ca2+-overload. Upon increasing pathologies, the brain shifts to a state of hypometabolism with reduced abundancy of mitochondria in presynaptic terminals. These late-stage mice also displayed enlarged presynaptic areas associated with abnormal accumulation of synaptic vesicles and autophagosomes, the latter ultimately leading to local autophagy impairment in the synapses. In summary, we report that Aß-induced pathways in App knock-in mouse models recapitulate key pathologies observed in AD brain, and our data herein adds a comprehensive understanding of the pathologies including dysregulated metabolism and synapses and their timewise appearance to find new therapeutic approaches for AD.


Assuntos
Doença de Alzheimer , Aplicativos Móveis , Animais , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Autofagia/genética , Modelos Animais de Doenças , Camundongos Transgênicos
3.
Mol Cell Neurosci ; 125: 103843, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36935047

RESUMO

Proteins must fold into three-dimensional structures to execute their biological functions. Therefore, maintenance of protein homeostasis, proteostasis, including prevention of protein misfolding is essential for cellular activity and health. Molecular chaperones are key actors in proteostasis. BRICHOS domain is an intramolecular chaperone that also interferes with several aggregation-prone proteins including amyloid ß (Aß), involved in Alzheimer's disease (AD). To extend the knowledge about Bri2 BRICHOS interactome we here used recombinant human (rh) Bri2 BRICHOS-mCherry fusion protein to probe for potential binding partners. Firstly, exogenously added Bri2 BRICHOS-mCherry was used to stain brain sections of wildtype and amyloid precursor protein (App) knock-in AD mice exhibiting robust Aß pathology. Unexpectedly, we found that rh Bri2 BRICHOS-mCherry stained the cytoplasm of neurons which are devoid of Aß deposits. To identify these intraneuronal proteins that bind to the rh Bri2 BRICHOS domain, we performed co-immunoprecipitation (co-IP) of mouse brain hippocampi homogenates using the Bri2 BRICHOS-mCherry probe and analyzed co-IP proteins by LC-MS/MS. This identified several cytoskeletal proteins including spectrin alpha and beta chain, drebrin, tubulin ß3, and ß-actin as binding partners. The interactions were confirmed by a second round of pulldown experiments using rh Bri2 BRICHOS linked to magnetic beads. The interaction of rh Bri2 BRICHOS and tubulin ß3 was further investigated by staining both mouse brain sections and SH-SY5Y neuroblastoma cells with rh Bri2 BRICHOS-mCherry and tubulin ß3 immunostaining, which revealed partial co-localization. These data suggest a possible interplay of extracellular chaperone Bri2 BRICHOS domain in the intracellular space including the cytoskeleton.


Assuntos
Doença de Alzheimer , Neuroblastoma , Animais , Humanos , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cromatografia Líquida , Proteínas do Citoesqueleto , Glicoproteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Espectrometria de Massas em Tandem , Tubulina (Proteína)
4.
BMC Biol ; 19(1): 57, 2021 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-33761951

RESUMO

BACKGROUND: Mitochondrial dysfunction is a common feature of aging, neurodegeneration, and metabolic diseases. Hence, mitotherapeutics may be valuable disease modifiers for a large number of conditions. In this study, we have set up a large-scale screening platform for mitochondrial-based modulators with promising therapeutic potential. RESULTS: Using differentiated human neuroblastoma cells, we screened 1200 FDA-approved compounds and identified 61 molecules that significantly increased cellular ATP without any cytotoxic effect. Following dose response curve-dependent selection, we identified the flavonoid luteolin as a primary hit. Further validation in neuronal models indicated that luteolin increased mitochondrial respiration in primary neurons, despite not affecting mitochondrial mass, structure, or mitochondria-derived reactive oxygen species. However, we found that luteolin increased contacts between mitochondria and endoplasmic reticulum (ER), contributing to increased mitochondrial calcium (Ca2+) and Ca2+-dependent pyruvate dehydrogenase activity. This signaling pathway likely contributed to the observed effect of luteolin on enhanced mitochondrial complexes I and II activities. Importantly, we observed that increased mitochondrial functions were dependent on the activity of ER Ca2+-releasing channels inositol 1,4,5-trisphosphate receptors (IP3Rs) both in neurons and in isolated synaptosomes. Additionally, luteolin treatment improved mitochondrial and locomotory activities in primary neurons and Caenorhabditis elegans expressing an expanded polyglutamine tract of the huntingtin protein. CONCLUSION: We provide a new screening platform for drug discovery validated in vitro and ex vivo. In addition, we describe a novel mechanism through which luteolin modulates mitochondrial activity in neuronal models with potential therapeutic validity for treatment of a variety of human diseases.


Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Luteolina/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/metabolismo , Animais , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Retículo Endoplasmático/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Transdução de Sinais
5.
J Neurochem ; 156(6): 1003-1019, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32750740

RESUMO

Protein aggregation into amyloid fibrils is a key feature of a multitude of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Prion disease. To detect amyloid fibrils, fluorophores with high sensitivity and better efficiency coupled with the low toxicity are in high demand even to date. In this pursuit, we have unveiled two benzimidazole-based fluorescence sensors ([C15 H15 N3 ] (C1) and [C16 H16 N3 O2 ] (C2), which possess exceptional affinity toward different amyloid fibrils in its submicromolar concentration (8 × 10-9  M), whereas under a similar concentration, the gold standard Thioflavin-T (ThT) fails to bind with amyloid fibrils. These fluorescent markers bind to α-Syn amyloid fibrils as well as amyloid fibrils forming other proteins/peptides including Aß42 amyloid fibrils. The 1 H-15 N heteronuclear quantum correlation spectroscopy nuclear magnetic resonance data collected on wild-type α-Syn monomer with and without the fluorophores (C1 and C2) reveal that there is weak or no interactions between C1 or C2 with residues in α-Syn monomer, which indirectly reflects the specific binding ability of C1 and C2 to the α-Syn amyloid fibrils. Detailed studies further suggest that C1 and C2 can detect/bind with the α-Syn amyloid fibril as low as 100 × 10-9  M. Extremely low or no cytotoxicity is observed for C1 and C2 and they do not interfere with α-Syn fibrillation kinetics, unlike ThT. Both C1/C2 not only shows selective binding with amyloid fibrils forming various proteins/peptides but also displays excellent affinity and selectivity toward α-Syn amyloid aggregates in SH-SY5Y cells and Aß42 amyloid plaques in animal brain tissues. Overall, our data show that the developed dyes could be used for the detection of amyloid fibrils including α-Syn and Aß42 amyloids with higher sensitivity as compared to currently used ThT.


Assuntos
Amiloidose/patologia , Benzimidazóis/química , Benzotiazóis/química , Corantes Fluorescentes/química , Peptídeos beta-Amiloides/química , Animais , Benzimidazóis/síntese química , Benzimidazóis/toxicidade , Benzotiazóis/toxicidade , Linhagem Celular , Dicroísmo Circular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/toxicidade , Técnicas de Introdução de Genes , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Microscopia Eletrônica de Transmissão , Fragmentos de Peptídeos/química , Teoria Quântica , Padrões de Referência , alfa-Sinucleína/química
6.
Biochem Biophys Res Commun ; 527(4): 1050-1056, 2020 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-32439160

RESUMO

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.


Assuntos
Adesão Celular , Membrana Celular/metabolismo , Movimento Celular , Fosfatidilinositol 4,5-Difosfato/metabolismo , Actinas/metabolismo , Linhagem Celular , Colesterol/metabolismo , Humanos , Fosfatidilinositol 4,5-Difosfato/análise , Domínios de Homologia à Plecstrina , Esfingomielinas/metabolismo , Fosfolipases Tipo C/análise , Fosfolipases Tipo C/metabolismo
7.
Cancer Sci ; 108(12): 2405-2412, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29024195

RESUMO

Identification of specific drug targets is very important for cancer therapy. We recently identified zinc finger protein of the cerebellum 5 (ZIC5) as a factor that promotes melanoma aggressiveness by platelet-derived growth factor D (PDGFD) expression. However, its roles in other cancer types remain largely unknown. Here we determined the roles of ZIC5 in prostate cancer (PCa) and colorectal cancer (CRC) cells. Results showed that ZIC5 was highly expressed in CRC and dedifferentiated PCa tissues, whereas little expression was observed in relevant normal tissues. Knockdown of ZIC5 decreased proliferation of several PCa and CRC cell lines with induction of cell death. ZIC5 knockdown significantly suppressed PDGFD expression transcriptionally, and PDGFD suppression also decreased proliferation of PCa and CRC cell lines. In addition, suppression of ZIC5 or PDGFD expression decreased levels of phosphorylated focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) which are associated with PCa and CRC aggressiveness. Furthermore, knockdown of ZIC5 or PDGFD enhanced death of PCa and CRC cells induced by the anti-cancer drugs docetaxel or oxaliplatin, respectively. These results suggest that ZIC5 and PDGFD promote survival of PCa and CRC cells by enhancing FAK and STAT3 activity, and that the roles of ZIC5 are consistent across several cancer types.


Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Fatores de Transcrição/metabolismo , Adulto , Idoso , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
8.
Biochem Biophys Res Commun ; 488(4): 578-583, 2017 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-28528980

RESUMO

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Kirsten rat sarcoma viral oncogene homolog (KRAS) is frequently mutated in CRC, and KRAS mutations promote cell motility, growth, and survival. We previously revealed that the expression of phospholipase C (PLC) δ1, one of the most basal PLCs, is down-regulated in colon adenocarcinoma, and that the KRAS signaling pathway suppresses PLCδ1 expression. Although recent studies revealed that KRAS mutations activate autophagy in cancer cells, a relation between PLCδ1 and autophagy remains unclear. Here, we found that PLCδ1 overexpression suppresses the formation of autophagosomes, which are key structures of autophagy, whereas endogenous PLCδ1 knockdown increases autophagosome formation in CRC cells. We also showed that PLCδ1 overexpression promotes cell death under nutrient deprivation. Furthermore, PLCδ1 overexpression suppresses the autophagy induced by the anti-cancer drug oxaliplatin and promotes cell death under oxaliplatin treatment. These data suggest that PLCδ1 negatively regulates autophagy, and PLCδ1 suppression contributes to the tolerance of CRC cells harboring KRAS mutations to nutrient deprivation and anti-cancer drug treatment.


Assuntos
Autofagia , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Fosfolipase C delta/metabolismo , Neoplasias Colorretais/metabolismo , Células HCT116 , Humanos , Células Tumorais Cultivadas
9.
Biology (Basel) ; 12(6)2023 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-37372090

RESUMO

BACKGROUND: The endocannabinoid system (ECS) and associated lipid transmitter-based signaling systems play an important role in modulating brain neuroinflammation. ECS is affected in neurodegenerative disorders, such as Alzheimer's disease (AD). Here we have evaluated the non-psychotropic endocannabinoid receptor type 2 (CB2) and lysophosphatidylinositol G-protein-coupled receptor 55 (GPR55) localization and expression during Aß-pathology progression. METHODS: Hippocampal gene expression of CB2 and GPR55 was explored by qPCR analysis, and brain distribution was evaluated by immunofluorescence in the wild type (WT) and APP knock-in AppNL-G-F AD mouse model. Furthermore, the effects of Aß42 on CB2 and GPR55 expression were assessed in primary cell cultures. RESULTS: CB2 and GPR55 mRNA levels were significantly upregulated in AppNL-G-F mice at 6 and 12 months of age, compared to WT. CB2 was highly expressed in the microglia and astrocytes surrounding the Aß plaques. Differently, GPR55 staining was mainly detected in neurons and microglia but not in astrocytes. In vitro, Aß42 treatment enhanced CB2 receptor expression mainly in astrocytes and microglia cells, whereas GPR55 expression was enhanced primarily in neurons. CONCLUSIONS: These data show that Aß pathology progression, particularly Aß42, plays a crucial role in increasing the expression of CB2 and GPR55 receptors, supporting CB2 and GPR55 implications in AD.

10.
J Pharm Biomed Anal ; 232: 115465, 2023 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-37220701

RESUMO

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, that can attenuate Aß pathology in the brain. As a result, the identification of novel Bri2 BRICHOS client proteins has been sought to help elucidate signaling pathways and the potential identification of novel therapeutic targets. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, in combination with proteomic analysis on cytosolic and membrane fractions of cortical homogenates from C57BL/6 J WT mouse. We identified 4 proteins from the cytosolic fractions and 44 proteins from the membrane fractions that had significant interactions (p < 0.05) with Bri2 BRICHOS domain, of which 11 proteins were previously identified as proteins that interacted with Bri2 BRICHOS domain. Enrichment analysis of the retained proteins identified glycolysis/gluconeogenesis as the most enriched pathway, with several proteins identified playing roles in carbon metabolism, amino acid synthesis. The data suggested that Bri2 BRICHOS may have a role in cellular energy demands in the brain via glycolysis and mitochondrial oxidative phosphorylation and may play a role in mitochondrial homeostasis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Ligação Proteica , Animais , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteômica
11.
Front Aging Neurosci ; 14: 878303, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35663567

RESUMO

Alzheimer's disease (AD) is characterized by impaired protein homeostasis leading to amyloid-ß peptide (Aß) amyloidosis. Amyloid precursor protein (APP) knock-in mice exhibit robust Aß pathology, providing possibilities to determine its effect on protein homeostasis including autophagy. Here we compared human AD postmortem brain tissue with brains from two different types of App knock-in mice, App NL-F and App NL-G-F mice, exhibiting AD-like pathology. In AD postmortem brains, p62 levels are increased and p62-positive staining is detected in neurons, including potential axonal beadings, as well as in the vasculature and in corpora amylacea. Interestingly, p62 is also increased in the neurons in 12-month-old App NL-G-F mice. In brain homogenates from 12-month-old App NL-G-F mice, both p62 and light chain 3 (LC3)-II levels are increased as compared to wildtype (WT) mice, indicating inhibited autophagy. Double immunostaining for LC3 and Aß revealed LC3-positive puncta in hippocampus of 24-month-old App NL-F mice around the Aß plaques which was subsequently identified by electron microscopy imaging as an accumulation of autophagic vacuoles in dystrophic neurites around the Aß plaques. Taken together, autophagy is impaired in App knock-in mice upon increased Aß pathology, indicating that App knock-in mouse models provide a platform for understanding the correlation between Aß and autophagy.

12.
Nat Commun ; 13(1): 2347, 2022 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-35534464

RESUMO

Epithelial cells provide cell-cell adhesion that is essential to maintain the integrity of multicellular organisms. Epithelial cell-characterizing proteins, such as epithelial junctional proteins and transcription factors are well defined. However, the role of lipids in epithelial characterization remains poorly understood. Here we show that the phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is enriched in the plasma membrane (PM) of epithelial cells. Epithelial cells lose their characteristics upon depletion of PM PI(4,5)P2, and synthesis of PI(4,5)P2 in the PM results in the development of epithelial-like morphology in osteosarcoma cells. PM localization of PARD3 is impaired by depletion of PM PI(4,5)P2 in epithelial cells, whereas expression of the PM-targeting exocyst-docking region of PARD3 induces osteosarcoma cells to show epithelial-like morphological changes, suggesting that PI(4,5)P2 regulates epithelial characteristics by recruiting PARD3 to the PM. These results indicate that a high level of PM PI(4,5)P2 plays a crucial role in the maintenance of epithelial characteristics.


Assuntos
Osteossarcoma , Fosfatidilinositóis , Adesão Celular , Membrana Celular/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Osteossarcoma/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo
13.
Cell Res ; 32(2): 157-175, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34789895

RESUMO

A major obstacle in Alzheimer's disease (AD) research is the lack of predictive and translatable animal models that reflect disease progression and drug efficacy. Transgenic mice overexpressing amyloid precursor protein (App) gene manifest non-physiological and ectopic expression of APP and its fragments in the brain, which is not observed in AD patients. The App knock-in mice circumvented some of these problems, but they do not exhibit tau pathology and neuronal death. We have generated a rat model, with three familiar App mutations and humanized Aß sequence knocked into the rat App gene. Without altering the levels of full-length APP and other APP fragments, this model exhibits pathologies and disease progression resembling those in human patients: deposit of Aß plaques in relevant brain regions, microglia activation and gliosis, progressive synaptic degeneration and AD-relevant cognitive deficits. Interestingly, we have observed tau pathology, neuronal apoptosis and necroptosis and brain atrophy, phenotypes rarely seen in other APP models. This App knock-in rat model may serve as a useful tool for AD research, identifying new drug targets and biomarkers, and testing therapeutics.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Disfunção Cognitiva/genética , Modelos Animais de Doenças , Progressão da Doença , Humanos , Camundongos , Camundongos Transgênicos , Ratos
14.
Eur J Pharmacol ; 535(1-3): 283-90, 2006 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-16545368

RESUMO

BACKGROUND: Angiotensin II has been implicated in the pathogenesis of vascular inflammation in various organs. The aim of the present study was to examine the effect of angiotensin II type I receptor antagonist, CV-11974, on reperfusion-induced small intestinal injury in rats. METHODS: Intestinal damage was induced by clamping both the superior mesenteric artery and the celiac trunk for 30 min followed by reperfusion for 60 min in male Wistar rats. CV-11974 was given to the rats by intravenous injection 1 h before the vascular clamping. The intestinal mucosal injury and inflammation were evaluated by biochemical markers and histological findings. Thiobarbituric acid reactive substances and tissue-associated myeloperoxidase (MPO) activity were measured in the gastric mucosa as indices of lipid peroxidation and neutrophil infiltration. The expressions of pro-inflammatory cytokines (CINC-1) in intestinal mucosa were measured by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-PCR (RT-PCR). In additional experiments with an in vitro flow system, human neutrophils were perfused on human umbilical vein endothelial cells (HUVEC) pretreated with anoxia-reoxygenation with or without CV-11974 and then the adhesive neutrophils were counted. RESULTS: Reperfusion after ischemia resulted in an increase in luminal protein concentrations, hemoglobin concentrations, thiobarbituric acid reactive substances, and MPO activity. Pretreatment with CV-11974 significantly inhibited the increases in these parameters. CV-11974 also inhibited increases in intestinal CINC-1 protein and mRNA expression induced by ischemia-reperfusion. Moreover, in an in vitro study, CV-11974 significantly inhibited the adherence of neutrophils to HUVEC exposed to reoxygenation after anoxia. CONCLUSIONS: These results suggest that the blockade of angiotensin II type I receptor by treatment with CV-11974 remarkably reduced the reperfusion-induced intestinal injury.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Benzimidazóis/farmacologia , Enteropatias/prevenção & controle , Intestino Delgado/efeitos dos fármacos , Traumatismo por Reperfusão/complicações , Tetrazóis/farmacologia , Animais , Compostos de Bifenilo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Quimiocina CXCL1 , Quimiocinas CXC/genética , Quimiocinas CXC/metabolismo , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Hemoglobinas/metabolismo , Humanos , Enteropatias/etiologia , Enteropatias/metabolismo , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/lesões , Mucosa Intestinal/metabolismo , Intestino Delgado/lesões , Intestino Delgado/metabolismo , Masculino , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peroxidase/metabolismo , Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
15.
Eur J Pharmacol ; 546(1-3): 11-8, 2006 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-16919261

RESUMO

Oxidized low-density lipoprotein (ox-LDL) plays an important in the development of atherosclerosis by stimulating the production of reactive oxygen species in endothelial cells, and thereby up-regulating vascular cell adhesion molecule-1 (VCAM-1). The objectives of the present study were to determine the effects of azelnidipine, a new calcium channel blocker, on the expression of VCAM-1 induced by 7-ketocholesterol, components of ox-LDL, and tumor necrosis factor-alpha (TNF-alpha). The scavenging activities of azelnidipine against superoxide, hydroxyl, and carbon-centered radicals were determined by electron spin resonance assay. The levels of intracellular reactive oxygen species were determined fluorometrically with the use of dichlorodihydrofluorescein diacetate (H(2)DCF-DA). Human aortic endothelial cells and U937 were used as endothelial cells and monocytic cells, respectively. The surface expression and mRNA levels of VCAM-1 were determined by enzyme immunoassay and RT-PCR performed on endothelial cell monolayers stimulated with 7-ketocholesterol or TNF-alpha. The numbers of monocytic cells adhering on the stimulated endothelial cells were counted in the microscopic fields. Translocation of p65 protein to the nucleus was estimated by fluorescence microscopy. Azelnidipine, but not nifedipine, reduced the signal intensity of 1,1-diphenyl-2-picrylhydrazyl radicals. Azelnidipine scavenged hydroxyl radicals, but not superoxide radicals. Intracellular levels of reactive oxygen species and RelA (p65) nuclear translocation in stimulated endothelial cells were reduced by azelnidipine. Azelnidipine significantly inhibited the expression of protein and mRNA of VCAM-1, and prevented the U937 cell adhesion to endothelial cells treated with 7-ketocholesterol or TNF-alpha. These results suggest that azelnidipine works as an anti-atherogenic agent by inhibiting the reactive oxygen species-dependent expression of VCAM-1 induced by 7-ketocholesterol and TNF-alpha.


Assuntos
Aorta/efeitos dos fármacos , Ácido Azetidinocarboxílico/análogos & derivados , Bloqueadores dos Canais de Cálcio/farmacologia , Di-Hidropiridinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Inflamação/prevenção & controle , Espécies Reativas de Oxigênio/metabolismo , Aorta/citologia , Aorta/metabolismo , Ácido Azetidinocarboxílico/farmacologia , Ácido Azetidinocarboxílico/uso terapêutico , Compostos de Bifenilo , Bloqueadores dos Canais de Cálcio/uso terapêutico , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Óxidos N-Cíclicos , Di-Hidropiridinas/uso terapêutico , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Fluoresceínas , Sequestradores de Radicais Livres/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hidrazinas , Indicadores e Reagentes , Inflamação/metabolismo , Cetocolesteróis/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Subunidade p50 de NF-kappa B/metabolismo , Nifedipino/farmacologia , Picratos , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/farmacologia , Células U937 , Molécula 1 de Adesão de Célula Vascular/metabolismo
16.
Atherosclerosis ; 180(1): 19-25, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15823271

RESUMO

The migration of circulating monocytes into the subendothelial space occurs through the expressing of some adhesion molecules on endothelial cells. In the present study, using human aortic endothelial cells (HAECs), we investigated whether a model compound for oxysterols, 25-hydroxycholesterol, can enhance the monocyte adherence to HAECs exposed to 25-hydroxycholesterol via increasing expression of vascular cell adhesion molecule-1 (VCAM-1). We also aimed to determine the in vitro effects of tocotrienols on the enhanced interaction between monocytes and endothelial cells. We found that 25-hydroxycholesterol enhances surface expression determined by ELISA, induces VCAM-1 mRNA expression by real time-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells in a dose-dependent fashion. The combination treatment with anti-VCAM-1 and anti-CD11b monoclonal antibodies significantly reduced the monocyte adherence to 25-hydroxycholesterol-stimulated HAECs. Compared to alpha-tocopherol, tocotrienols displayed a more profound inhibitory effect on adhesion molecule expression and monocytic cell adherence. We observed that delta-tocotrienol exerted a most profound inhibitory action on monocytic cell adherence when compared to alpha-tocopherol and alpha-, beta-, and gamma-tocotrienols. Tocotrienols accumulated in HAECs to levels approximately 25-95-fold greater than that of alpha-tocopherol. In conclusion, these results indicate that a model compound 25-hydroxycholesterol can enhance the interaction between monocytes and HAECs, and that tocotrienols had a profound inhibitory effect on monocytic cell adherence to HAECs relative to alpha-tocopherol via inhibiting the VCAM-1 expression. These superior inhibitory effects of tocotrienols may be dependent on their intracellular accumulation.


Assuntos
Antioxidantes/farmacologia , Endotélio Vascular/citologia , Hidroxicolesteróis/farmacologia , Monócitos/citologia , Tocotrienóis/farmacologia , Molécula 1 de Adesão de Célula Vascular/genética , Anticorpos Monoclonais/farmacologia , Aorta/citologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/imunologia , Expressão Gênica/efeitos dos fármacos , Humanos , Monócitos/imunologia , RNA Mensageiro/análise , Células U937 , alfa-Tocoferol/farmacologia
17.
Redox Rep ; 9(6): 370-5, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15720835

RESUMO

The aim of the present study was to assess the expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), monocytic adhesion of human aortic endothelial cells (HAECs), and the production of intracellular reactive oxygen species (ROS), when HAECs were stimulated by 7-ketocholesterol. 7-ketocholesterol enhances surface expression of ICAM-1 and VCAM-1 as determined by EIA, induces their mRNA expression by RT-PCR, and stimulates adhesiveness of HAECs to U937 monocytic cells. We confirmed up-regulation of ROS production of HAECs treated with 7-ketocholesterol. Although the surface expression of ICAM-1 and VCAM-1 on HAECs treated with 7-ketocholesterol increased in a time-dependent manner, alpha-tocopherol inhibited this increase of the surface expression of ICAM-1 and VCAM-1. In the monocytic adhesion assay, adhesion of U937 to HAECs treated with 7-ketocholesterol was enhanced, but monoclonal anti-ICAM-1 and VCAM-1 antibodies reduced the endothelial adhesiveness. In conclusion, this study suggests that the endothelial adhesiveness to monocytic cells that was increased by 7-ketocholesterol was associated with enhanced expression of ICAM-1 and VCAM-1 mediated by ROS production.


Assuntos
Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Cetocolesteróis/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese , Aorta , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Fluoresceínas , Humanos , Lipoproteínas LDL/farmacologia , Monócitos/citologia , Monócitos/efeitos dos fármacos
18.
Redox Rep ; 9(6): 337-42, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15720829

RESUMO

Recent investigations have demonstrated that the signaling of hypoxia-re-oxygenation is a major contributing pathway leading to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis on the gastric mucosal cellular response to hypoxia-reoxygenation using a high-density oligonucleotide array. Cells were subjected to hypoxia with 95% N(2) and 5% CO(2) at 37 degrees C for 2 h. Reoxygenation was initiated by placing the cells in an environment of normoxia for 2 h. Total RNA was extracted, and differences in gene expression profiles between the normoxia and hypoxia-reoxygenation groups were investigated using a GeneChip of Rat Toxicology U34 array (Affymetrix). Hypoxia-reoxygenation up-regulated the stress-related genes (heat shock protein-70 [HSP-70], catalase). The enhanced expression of HSP-70 was confirmed by Western blot analysis. In conclusion, these results suggest that up-regulation of the HSP-70 gene after reoxygenation may play a role in maintaining cell survival and supporting cell function as a molecular chaperone.


Assuntos
Hipóxia Celular , Células Epiteliais/metabolismo , Mucosa Gástrica/metabolismo , Regulação da Expressão Gênica , Proteínas de Choque Térmico/genética , Animais , Catalase/genética , Hipóxia Celular/genética , Hipóxia Celular/fisiologia , Linhagem Celular , Regulação para Baixo , Mucosa Gástrica/citologia , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSP70/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Regulação para Cima
19.
Redox Rep ; 9(6): 354-9, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15720832

RESUMO

The objectives were to determine the effects of alacepril, an angiotensin-converting enzyme inhibitor, on the expression of adhesion molecules and monocyte adherence to endothelial cells induced by 7-ketocholesterol (7-KC) and tumor necrosis factor (TNF)-alpha. We used human aortic endothelial cells (HAECs) and U937 monocytic cells. Surface expression and mRNA levels of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) were determined by EIA and RT-PCR. Adherence of U937 to HAECs was assessed by adhesion assay. Incubation of HAEC with 7-KC increased the surface expression of protein and mRNA levels of ICAM-1 and VCAM-1 on HAECs and the production of reactive oxygen species (ROS) in HAECs. Pretreatment with alacepril reduced the enhanced expression of these molecules in a dose-dependent manner. The inhibitory effect of alacepril against 7-KC or TNF-alpha-induced CAMs expression was stronger than that of captopril or enalapril. Alacepril inhibited the production of ROS in HAECs stimulated by 7-KC or TNF-alpha. These results suggest that alacepril works as anti-atherogenic agent through inhibiting endothelial-dependent adhesive interactions with monocytes induced by 7-KC and TNF-alpha.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Captopril/análogos & derivados , Captopril/farmacologia , Adesão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Inflamação/induzido quimicamente , Cetocolesteróis/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Relação Dose-Resposta a Droga , Enalapril/farmacologia , Endotélio Vascular/citologia , Inflamação/tratamento farmacológico , Molécula 1 de Adesão Intercelular/biossíntese , Monócitos/citologia , Espécies Reativas de Oxigênio/metabolismo , Molécula 1 de Adesão de Célula Vascular/biossíntese
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