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1.
Biomed Pharmacother ; 178: 117278, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39116784

RESUMO

BACKGROUND: Empagliflozin (EMPA), a selective sodium-glucose cotransporter type 2 (SGLT2) inhibitor, has been shown to reduce major adverse cardiovascular events in patients with heart failure of different etiologies, although the underlying mechanism still remains unclear. Ponatinib (PON) is a multi-tyrosine kinase inhibitor successfully used against myeloid leukemia and other human malignancies, but its cardiotoxicity remains worrisome. Cardiac connexins (Cxs) are both substrates and regulators of autophagy and responsible for proper heart function. Alteration in connexin expression and localization have been described in patients with heart failure. AIMS: To assess whether EMPA can mitigate PON-induced cardiac dysfunction by restoring the connexin 43-autophagy pathway. METHODS AND RESULTS: Male C57BL/6 mice, randomized into four treatment groups (CNTRL, PON, EMPA, PON+EMPA) for 28 days, showed increased autophagy, decreased Cx43 expression as well as Cx43 lateralization, and attenuated systo-diastolic cardiac dysfunction after treatment with EMPA and PON compared with PON alone. Compared with CNTRL (DMSO), cardiomyocyte-differentiated H9c2 (dH9c2) cells treated with PON showed significantly reduced cell viability to approximately 20 %, decreased autophagy, increased cell senescence and reduced DNA binding activity of serum response factor (SRF) to serum response elements (SRE), which were paralleled by reduction in cardiac actin expression. Moreover, PON induced a significant increase of Cx43 protein and its S368-phosphorylated form (pS368-Cx43), as well as their displacement from the plasma membrane to the perinuclear and nuclear cellular region. All these effects were reverted by EMPA. CONCLUSION: EMPA attenuates PON-induced cardiotoxicity by reducing senescence, enhancing the SRE-SRF binding and restoring the connexin 43-autophagy pathway. This effect may pave the way to use of SGLT2 inhibitors in attenuating tyrosine-kinase inhibitor cardiotoxicity.


Assuntos
Autofagia , Compostos Benzidrílicos , Cardiotoxicidade , Conexina 43 , Glucosídeos , Imidazóis , Miócitos Cardíacos , Piridazinas , Animais , Masculino , Camundongos , Ratos , Autofagia/efeitos dos fármacos , Compostos Benzidrílicos/farmacologia , Cardiotoxicidade/etiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Conexina 43/metabolismo , Glucosídeos/farmacologia , Imidazóis/farmacologia , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia
2.
Mov Disord ; 2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-39051733

RESUMO

BACKGROUND: Elevated levels of prokineticin-2 (PK2), regarded as a protein involved in modulating immune/inflammatory responses, have been detected in the substantia nigra, serum, and olfactory neurons of Parkinson's disease (PD) patients. Of note, emerging evidence suggests that gut alterations, including dysbiosis and enteric inflammation, play a role in PD via the gut-brain axis. OBJECTIVES: Our goal was to investigate the expression of PK2 in colonic biopsies of PD patients. METHODS: Mucosal biopsies from the descending colon were obtained in 11 PD patients and five asymptomatic subjects. Biopsy samples were processed for PK2 immunofluorescence and western blot. RESULTS: We revealed an increased PK2 expression in colonic mucosa from PD patients in the early stages compared to controls. In addition, we found that PK2 was expressed by activated enteric glial cells and macrophages. CONCLUSIONS: PK2 is highly expressed within neurogenic/inflammatory cells of colonic mucosa from early PD patients, suggesting a potential role of PK2 in gut inflammation, especially in the early stages of PD. © 2024 International Parkinson and Movement Disorder Society.

3.
Eur J Neurol ; 30(11): 3440-3450, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36263629

RESUMO

BACKGROUND AND PURPOSE: Changes in gut microbiota composition, enteric inflammation, impairments of the intestinal epithelial barrier and neuroplastic changes in the enteric nervous system have been reported in Parkinson's disease (PD) patients and could contribute to the onset of both neurological and gastrointestinal symptoms. However, their mutual interplay has rarely been investigated. This study evaluated, in an integrated manner, changes in faecal microbiota composition, morphofunctional alterations of colonic mucosal barrier and changes of inflammatory markers in blood and stools of PD patients. METHODS: Nineteen PD patients and nineteen asymptomatic subjects were enrolled. Blood lipopolysaccharide binding protein (LBP, marker of altered intestinal permeability) and interleukin-1ß (IL-1ß) levels, as well as stool IL-1ß and tumour necrosis factor (TNF) levels, were evaluated. Gut microbiota analysis was performed. Epithelial mucins, collagen fibres, claudin-1 and S100-positive glial cells as markers of an impairment of the intestinal barrier, mucosal remodelling and enteric glial activation were evaluated on colonic mucosal specimens collected during colonoscopy. RESULTS: Faecal microbiota analysis revealed a significant difference in the α-diversity in PD patients compared to controls, while no differences were found in the ß-diversity. Compared to controls, PD patients showed significant chenags in plasma LBP levels, as well as faecal TNF and IL-1ß levels. The histological analysis showed a decrease in epithelial neutral mucins and claudin-1 expression and an increased expression of acidic mucins, collagen fibres and S100-positive glial cells. CONCLUSIONS: Parkinson's disease patients are characterized by enteric inflammation and increased intestinal epithelial barrier permeability, as well as colonic mucosal barrier remodelling, associated with changes in gut microbiota composition.

4.
Open Biol ; 12(12): 220216, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36541101

RESUMO

Cell quiescence appeared early in evolution as an adaptive response to adverse conditions (i.e. nutrient depletion). In metazoans, quiescence has been involved in additional processes like tissue homeostasis, which is made possible by the presence of adult stem cells (ASCs). Cell cycle control machinery is a common hub for quiescence entrance, and evidence indicates a role for p53 in establishing the quiescent state of undamaged cells. Mechanisms responsible for waking up quiescent cells remain elusive, and nutritional stimulus, as a legacy of its original role, still appears to be a player in quiescence exit. Planarians, rich in ASCs, represent a suitable system in which we characterized a quiescent population of ASCs, the dorsal midline cord (DMC) cells, exhibiting unique transcriptional features and maintained quiescent by p53 and awakened upon feeding. The function of DMC cells is puzzling and we speculate that DMC cells, despite retaining ancient properties, might represent a functional drift in which quiescence has been recruited to provide evolutionary advantages.


Assuntos
Células-Tronco Adultas , Planárias , Animais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Células-Tronco , Divisão Celular
5.
Circ Res ; 131(6): 476-491, 2022 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-35968712

RESUMO

BACKGROUND: Experimental evidence suggests a key role of SIRT1 (silent information regulator 1) in age- and metabolic-related vascular dysfunction. Whether these effects hold true in the human microvasculature is unknown. We aimed to investigate the SIRT1 role in very early stages of age- and obesity-related microvascular dysfunction in humans. METHODS: Ninety-five subjects undergoing elective laparoscopic surgery were recruited and stratified based on their body mass index status (above or below 30 kg/m2) and age (above or below 40 years) in 4 groups: Young Nonobese, Young Obese, Old Nonobese, and Old Obese. We measured small resistance arteries' endothelial function by pressurized micromyography before and after incubation with a SIRT1 agonist (SRT1720) and a mitochondria reactive oxygen species (mtROS) scavenger (MitoTEMPO). We assessed vascular levels of mtROS and nitric oxide availability by confocal microscopy and vascular gene expression of SIRT1 and mitochondrial proteins by qPCR. Chromatin immunoprecipitation assay was employed to investigate SIRT1-dependent epigenetic regulation of mitochondrial proteins. RESULTS: Compared with Young Nonobese, obese and older patients showed lower vascular expression of SIRT1 and antioxidant proteins (FOXO3 [forkhead box protein O3] and SOD2) and higher expression of pro-oxidant and aging mitochondria proteins p66Shc and Arginase II. Old Obese, Young Obese and Old Nonobese groups endothelial dysfunction was rescued by SRT1720. The restoration was comparable to the one obtained with mitoTEMPO. These effects were explained by SIRT1-dependent chromatin changes leading to reduced p66Shc expression and upregulation of proteins involved in mitochondria respiratory chain. CONCLUSIONS: SIRT1 is a novel central modulator of the earliest microvascular damage induced by age and obesity. Through a complex epigenetic control mainly involving p66Shc and Arginase II, it influences mtROS levels, NO availability, and the expression of proteins of the mitochondria respiratory chain. Therapeutic modulation of SIRT1 restores obesity- and age-related endothelial dysfunction. Early targeting of SIRT1 might represent a crucial strategy to prevent age- and obesity-related microvascular dysfunction.


Assuntos
Arginase , Obesidade , Sirtuína 1 , Doenças Vasculares , Adulto , Arginase/metabolismo , Epigênese Genética , Humanos , Proteínas Mitocondriais/metabolismo , Óxido Nítrico/metabolismo , Obesidade/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Doenças Vasculares/etiologia
6.
Biol Open ; 9(11)2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33037013

RESUMO

Here, we present miniCoopR-I, an inducible upgrade of the constitutive miniCoopR vector. We developed miniCoopR-I-sponge-204 and miniCoopR-I-pre-miR-204 vectors and we successfully tested them for their ability to achieve time- (embryo/juvenile/adult) and space- (melanocytic lineage) restricted inhibition/overexpression of miR-204, a positive modulator of pigmentation previously discovered by us. Furthermore, melanoma-free survival curves performed on induced fish at the adult stage indicate that miR-204 overexpression accelerates the development of BRAFV600E-driven melanoma. miniCoopR-I allows study of the impact that coding and non-coding modulators of pigmentation exert on melanomagenesis in adult zebrafish, uncoupling it from the impact that they exert on melanogenesis during embryonic development.This article has an associated First Person interview with the first author of the paper.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , MicroRNAs/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Expressão Gênica , Técnicas de Inativação de Genes , Ordem dos Genes , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Melanócitos/metabolismo , Melanoma/patologia , Peixe-Zebra
7.
J Mol Biol ; 431(4): 873-884, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30664870

RESUMO

Tau displacement from microtubules is the first step in the onset of tauopathies and is followed by toxic protein aggregation. However, other non-canonical functions of Tau might have a role in these pathologies. Here, we demonstrate that a small amount of Tau localizes in the nuclear compartment and accumulates in both the soluble and chromatin-bound fractions. We show that favoring Tau nuclear translocation and accumulation, by Tau overexpression or detachment from MTs, increases the expression of VGluT1, a disease-relevant gene directly involved in glutamatergic synaptic transmission. Remarkably, the P301L mutation, related to frontotemporal dementia FTDP-17, impairs this mechanism leading to a loss of function. Altogether, our results provide the demonstration of a direct physiological role of Tau on gene expression. Alterations of this mechanism may be at the basis of the onset of neurodegeneration.


Assuntos
Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteínas tau/genética , Linhagem Celular , Linhagem Celular Tumoral , Cromatina/genética , Expressão Gênica/genética , Células HeLa , Humanos , Microtúbulos/genética , Mutação/genética , Tauopatias/genética
8.
Int J Obes (Lond) ; 43(2): 331-343, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30082748

RESUMO

BACKGROUND: The murine model of high fat diet (HFD)-induced obesity is characterized by an increment of intestinal permeability, secondary to an impairment of mucosal epithelial barrier and enteric inflammation, followed by morphofunctional rearrangement of the enteric nervous system. The present study investigated the involvement of abdominal macrophages in the mechanisms underlying the development of enteric dysmotility associated with obesity. METHODS: Wild type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD, 18% kcal from fat) for 8 weeks. Groups of mice fed with NCD or HFD were treated with clodronate encapsulated into liposomes to deplete abdominal macrophages. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. Substance P distribution was examined by confocal immunohistochemistry. The density of macrophages in the colonic wall was examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay) and IL-1ß (ELISA assay) levels were also evaluated. RESULTS: MDA and IL-1ß levels were increased in colonic tissues from HFD-treated animals. In colonic preparations, electrically evoked tachykininergic contractions were enhanced in HFD mice. Immunohistochemistry displayed an increase in substance P immunoreactivity in myenteric ganglia, as well as in the muscular layers of colonic cryosections from obese mice. Macrophage depletion in HFD mice was associated with a significant reduction of colonic inflammation. In addition, the decrease in macrophage density attenuated the morphofunctional alterations of tachykininergic pathways observed in obese mice. CONCLUSION: Obesity elicited by HFD determines a condition of colonic inflammation, followed by a marked rearrangement of motor excitatory tachykininergic enteric nerves. Macrophage depletion counteracted the morphofunctional changes of colonic neuromuscular compartment, suggesting a critical role for these immune cells in the onset of enteric dysmotility associated with obesity.


Assuntos
Colo , Dieta Hiperlipídica/efeitos adversos , Inflamação/fisiopatologia , Obesidade , Animais , Peso Corporal , Colo/citologia , Colo/patologia , Colo/fisiopatologia , Doenças do Colo/fisiopatologia , Motilidade Gastrointestinal/fisiologia , Interleucina-1beta/análise , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Masculino , Malondialdeído/análise , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/fisiopatologia
9.
Front Oncol ; 8: 555, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30574454

RESUMO

The Polycomb gene BMI1 expression exerts a negative predictive impact on several hematological malignancies, such as acute and chronic myeloid leukemia (CML), myelofibrosis, and follicular lymphoma. As already demonstrated in CML, BMI1 is responsible for the resistance to the tyrosine kinase inhibitors (TKIs) in a BCR-ABL1-independent way. Even if, it is unknown where BMI1 in CML is expressed (in progenitors or more mature cells). We decided, therefore, to evaluate if and where the BMI1 protein is located, focusing mainly on the CD34+/CD38-/CD26+ CML progenitors. To begin we measured, by flow cytometry, the proportion of CD34+/CD26+ cells in 31 bone marrow samples from 20 CML patients, at diagnosis and during treatment with imatinib. After that the bone marrow blood smears were stained with antibodies anti-CD26, BCR-ABL1, and BMI1. These smears were observed by a confocal laser microscope and a 3D reconstruction was then performed. At diagnosis, CD34+/CD26+ cells median value/µL was 0.48; this number increased from diagnosis to the third month of therapy and then reduced during treatment with imatinib. The number and behavior of the CD26+ progenitors were independent from the BCR-ABL1 expression, but they summed up what previously observed about the BMI1 expression modulation. In this work we demonstrate for the first time that in CML the BMI1 protein is co-expressed with BCR-ABL1 only in the cytoplasm of the CD26+ precursors; on the contrary, in other hematological malignancies where BMI1 is commonly expressed (follicular lymphoma, essential thrombocytemia, acute myeloid leukemia), it was not co-localized with CD26 or, obviously, with BCR-ABL1. Once translated into the clinical context, if BMI1 is a marker of stemness, our results would suggest the combination of the BMI1 inhibitors with TKIs as an interesting object of research, and, probably, as a promising way to overcome resistance in CML patients.

10.
Sci Rep ; 8(1): 10599, 2018 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006630

RESUMO

The natural alkaloid berberine has several pharmacological properties and recently received attention as a potential anticancer agent. In this work, we investigated the molecular mechanisms underlying the anti-tumor effect of berberine on glioblastoma U343 and pancreatic carcinoma MIA PaCa-2 cells. Human dermal fibroblasts (HDF) were used as non-cancer cells. We show that berberine differentially affects cell viability, displaying a higher cytotoxicity on the two cancer cell lines than on HDF. Berberine also affects cell cycle progression, senescence, caspase-3 activity, autophagy and migration in a cell-specific manner. In particular, in HDF it induces cell cycle arrest in G2 and senescence, but not autophagy; in the U343 cells, berberine leads to cell cycle arrest in G2 and induces both senescence and autophagy; in MIA PaCa-2 cells, the alkaloid induces arrest in G1, senescence, autophagy, it increases caspase-3 activity and impairs migration/invasion. As demonstrated by decreased citrate synthase activity, the three cell lines show mitochondrial dysfunction following berberine exposure. Finally, we observed that berberine modulates the expression profile of genes involved in different pathways of tumorigenesis in a cell line-specific manner. These findings have valuable implications for understanding the complex functional interactions between berberine and specific cell types.


Assuntos
Berberina/farmacologia , Carcinogênese/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Berberina/uso terapêutico , Carcinogênese/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Citrato (si)-Sintase/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia
11.
Heart Vessels ; 33(11): 1403-1410, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29789901

RESUMO

Cardiac myxomas are rare tumors with a heterogeneous cell population including properly neoplastic (lepidic), endothelial and smooth muscle cells. The assessment of neoplastic (lepidic) cell differentiation pattern is rather difficult using conventional light microscopy immunohistochemistry and/or whole tissue extracts for mRNA analyses. In a preliminary study, we investigated 20 formalin-fixed and paraffin-embedded cardiac myxomas by means of conventional immunohistochemistry; in 10/20 cases, cell differentiation was also analyzed by real-time RT-PCR after laser capture microdissection of the neoplastic cells, whereas calretinin and endothelial antigen CD31 immunoreactivity was localized in 4/10 cases by double immunofluorescence confocal microscopy. Gene expression analyses of α-smooth muscle actin, endothelial CD31 antigen, alpha-cardiac actin, matrix metalloprotease-2 (MMP2) and tissue inhibitor of matrix metalloprotease-1 (TIMP1) was performed on cDNA obtained from either microdissected neoplastic cells or whole tumor sections. We found very little or absent CD31 and α-Smooth Muscle Actin expression in the microdissected cells as compared to the whole tumors, whereas TIMP1 and MMP2 genes were highly expressed in both ones, greater levels being found in patients with embolic phenomena. α-Cardiac Actin was not detected. Confocal microscopy disclosed two different signals corresponding to calretinin-positive myxoma cells and to endothelial CD31-positive cells, respectively. In conclusion, the neoplastic (lepidic) cells showed a distinct gene expression pattern and no consistent overlapping with endothelial and smooth muscle cells or cardiac myocytes; the expression of TIMP1 and MMP2 might be related to clinical presentation; larger series studies using also systematic transcriptome analysis might be useful to confirm the present results.


Assuntos
Neoplasias Cardíacas/patologia , Microdissecção e Captura a Laser/métodos , Microscopia Confocal/métodos , Miocárdio/patologia , Mixoma/patologia , Actinas/biossíntese , Actinas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Calbindina 2/biossíntese , Calbindina 2/genética , Diferenciação Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Neoplasias Cardíacas/genética , Neoplasias Cardíacas/cirurgia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Miocárdio/metabolismo , Mixoma/genética , Mixoma/cirurgia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , RNA Neoplásico/genética , Reação em Cadeia da Polimerase em Tempo Real
12.
PLoS One ; 13(4): e0195502, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29641549

RESUMO

BACKGROUND AND PURPOSE: Apigenin can exert beneficial actions in the prevention of obesity. However, its putative action on obesity-associated bowel motor dysfunctions is unknown. This study examined the effects of apigenin on colonic inflammatory and motor abnormalities in a mouse model of diet-induced obesity. EXPERIMENTAL APPROACH: Male C57BL/6J mice were fed with standard diet (SD) or high-fat diet (HFD). SD or HFD mice were treated with apigenin (10 mg/Kg/day). After 8 weeks, body and epididymal fat weight, as well as cholesterol, triglycerides and glucose levels were evaluated. Malondialdehyde (MDA), IL-1ß and IL-6 levels, and let-7f expression were also examined. Colonic infiltration by eosinophils, as well as substance P (SP) and inducible nitric oxide synthase (iNOS) expressions were evaluated. Motor responses elicited under blockade of NOS and tachykininergic contractions were recorded in vitro from colonic longitudinal muscle preparations. KEY RESULTS: When compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1ß and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1ß and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens. CONCLUSIONS AND IMPLICATIONS: Apigenin prevents systemic metabolic alterations, counteracts enteric inflammation and normalizes colonic dysmotility associated with obesity.


Assuntos
Apigenina/farmacologia , Colo/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Flavonoides/farmacologia , Atividade Motora/efeitos dos fármacos , Obesidade/patologia , Obesidade/fisiopatologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/patologia , Animais , Peso Corporal/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/patologia , Inflamação/fisiopatologia , Inflamação/prevenção & controle , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , Óxido Nítrico Sintase Tipo II/metabolismo , Obesidade/induzido quimicamente , Obesidade/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Substância P/metabolismo
13.
Purinergic Signal ; 13(4): 497-510, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28808842

RESUMO

Adenosine A2B receptors (A2BR) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A2BR in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A2BR localization was examined by immunofluorescence. The role of A2BR in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A2BR were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A2BR antagonist MRS1754 enhanced electrically induced NK1-mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A2BR ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A2BR expression. A2BR, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Motilidade Gastrointestinal/fisiologia , Obesidade/metabolismo , Receptor A2B de Adenosina/metabolismo , Animais , Colo/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/complicações
14.
Oncotarget ; 8(15): 25395-25417, 2017 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-28445987

RESUMO

Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones. Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway.By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib. We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib's anti-motility activity by targeting AP1S2. Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway. In doing so, miR-211 serves as an effector of vemurafenib's pro-pigmentation activity. We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib.In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells. Our work challenges the widely accepted "same miRNA family = same function" rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.


Assuntos
Melanoma Amelanótico/genética , Melanoma/genética , MicroRNAs/genética , Neoplasias Cutâneas/genética , Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Subunidades sigma do Complexo de Proteínas Adaptadoras/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases , Melanoma/metabolismo , Melanoma/patologia , Melanoma Amelanótico/tratamento farmacológico , Melanoma Amelanótico/metabolismo , Melanoma Amelanótico/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Sulfonamidas/farmacologia , Transfecção , Vemurafenib
15.
Eur Neuropsychopharmacol ; 27(4): 383-398, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28283227

RESUMO

Antipsychotics (APDs) are divided into first-generation antipsychotics (FGAs) and second-generation antipsychotics (SGAs) based on the concept that SGAs have reduced motor side effects. With this premise, this study examined in HeLa and other cell lines the effects of different APDs on the activation of ERK1/2 (Extracellular signal-regulated kinases) and AKT (Protein Kinase B) kinases, which may be affected in schizophrenia and bipolar disorder. Among the SGAs, Clozapine clearly resulted as the most effective drug inducing ERK1/2 phosphorylation with potency in the low micromolar range. Quetiapine and Olanzapine showed a maximal response of about 50% compared to Clozapine, while FGAs such as Haloperidol and Sulpiride did not have any relevant effect. Among FGAs, Chlorpromazine was able to partially activate ERK1/2 at 30% compared to Clozapine. Referring to AKT activation, Clozapine, Quetiapine and Olanzapine demonstrated a similar efficacy, while FGAs, besides Chlorpromazine, were incapable to obtain any particular biological response. In relation to ERK1/2 activation, we found that 5-HT2A serotonin receptor antagonists Ketanserin and M100907, both partially reduced Clozapine effect. In addition, we also observed an increase of potency of Clozapine effect in HeLa transfected cells with recombinant 5-HT2A receptor and in rat glioma C6 cells that express a higher amount of this receptor. This indicates that ERK1/2 stimulation induced by Clozapine could, to some extent, be mediated by 5-HT2A receptor, through a novel mechanism that is called "biased agonism", even though other cellular targets are involved. This evidence may be relevant to explain the superiority of Clozapine among the APDs.


Assuntos
Antipsicóticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Agonistas do Receptor 5-HT2 de Serotonina/farmacologia , Animais , Células Cultivadas , Clozapina , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Modelos Moleculares , Fumarato de Quetiapina/farmacologia , Receptor 5-HT2A de Serotonina/metabolismo , Serotonina/farmacologia
16.
Hypertension ; 69(1): 71-78, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27802422

RESUMO

We evaluated whether vascular remodeling is present in physiological aging and whether hypertension accelerates the aging process for vascular function and structure. Small arteries from 42 essential hypertensive patients (HT) and 41 normotensive individuals (NT) were dissected after subcutaneous biopsy. Endothelium-dependent vasodilation (pressurized myograph) was assessed by acetylcholine, repeated under the nitric oxide synthase inhibitor N-nitro-l-arginine methylester or the antioxidant tempol. Structure was evaluated by media-lumen ratio (M/L). Intravascular oxidative generation and collagen deposition were assessed. Inhibition by N-nitro-l-arginine methylester on ACh was inversely related to age in both groups (P<0.0001) and blunted in HT versus NT for each age range. In NT, tempol enhanced endothelial function in the oldest subgroup; in HT, the potentiating effect started earlier. HT showed an increased M/L (P<0.001) versus control. In both groups, M/L was directly related to age (P<0.0001). M/L was greater in HT, starting from 31 to 45 years range. A significant age-hypertension interaction occurred (P=0.0009). In NT, intravascular superoxide emerged in the oldest subgroup, whereas it appeared earlier among HT. Among NT, aged group displayed an increment of collagen fibers versus young group. In HT, collagen deposition was already evident in youngest, with a further enhancement in the aged group. In small arteries, ageing shows a eutrophic vascular remodeling and a reduced nitric oxide availability. Oxidative stress and fibrosis emerge in advanced age. In HT, nitric oxide availability is early reduced, but the progression rate with age is similar. Structural alterations include wide collagen deposition and intravascular reactive oxygen species, and the progression rate with age is steeper.


Assuntos
Envelhecimento , Artérias/patologia , Endotélio Vascular/patologia , Hipertensão/fisiopatologia , Remodelação Vascular , Vasodilatação/fisiologia , Adulto , Idoso , Artérias/metabolismo , Artérias/fisiopatologia , Biópsia , Colágeno/metabolismo , Estudos Transversais , Progressão da Doença , Endotélio Vascular/fisiopatologia , Hipertensão Essencial , Feminino , Humanos , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Adulto Jovem
17.
J Crohns Colitis ; 10(10): 1194-204, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26995183

RESUMO

BACKGROUND AND AIMS: Intestinal fibrosis is a complication of inflammatory bowel disease [IBD]. Although fibrostenosis is a rare event in ulcerative colitis [UC], there is evidence that a fibrotic rearrangement of the colon occurs in the later stages. This is a retrospective study aimed at examining the histopathological features of the colonic wall in both short-lasting [SL] and long-lasting [LL] UC. METHODS: Surgical samples of left colon from non-stenotic SL [≤ 3 years, n = 9] and LL [≥ 10 years, n = 10] UC patients with active disease were compared with control colonic tissues from cancer patients without UC [n = 12] to assess: collagen and elastic fibres by histochemistry; vascular networks [CD31/CD105/nestin] by immunofluorescence; parameters of fibrosis [types I and III collagen, fibronectin, RhoA, alpha-smooth muscle actin [α-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. RESULTS: Colonic tissue from both SL-UC and LL-UC showed tunica muscularis thickening and transmural activated neovessels [displaying both proliferating CD105-positive endothelial cells and activated nestin-positive pericytes], as compared with controls. In LL-UC, the increased collagen deposition was associated with an up-regulation of tissue fibrotic markers [collagen I and III, fibronectin, vimentin, RhoA], an enhancement of proliferation [PCNA] and, along with a loss of elastic fibres, a rearrangement of the tunica muscularis towards a fibrotic phenotype. CONCLUSIONS: A significant transmural fibrotic thickening occurs in colonic tissue from LL-UC, together with a cellular fibrotic switch in the tunica muscularis. A full-thickness angiogenesis is also evident in both SL- and LL-UC with active disease, as compared with controls.


Assuntos
Colite Ulcerativa/patologia , Colo/patologia , Remodelação Vascular , Doença Aguda , Adulto , Idoso , Biomarcadores/metabolismo , Western Blotting , Estudos de Casos e Controles , Colo/irrigação sanguínea , Colo/metabolismo , Progressão da Doença , Feminino , Fibrose , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Método Simples-Cego
18.
Eur Heart J ; 36(43): 3023-30, 2015 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-26224075

RESUMO

AIMS: We assessed whether acute intra-arterial infusion of exogenous ghrelin can improve endothelial dysfunction by restoring nitric oxide (NO) availability in the forearm microcirculation of essential hypertensive patients. The effect of ghrelin on endothelial dysfunction (pressurized myograph), vascular oxidative stress generation (fluorescent dihydroethidium), and phosphorylation of p47phox (western blot), an index of NAD(P)H oxidase activation, in isolated small arteries taken from essential hypertensive patients (subcutaneous biopsy) were also investigated. METHODS AND RESULTS: In 18 normotensive control subjects and 18 essential hypertensive patients, we studied the forearm blood flow (strain-gauge plethysmography) response to intra-arterial acetylcholine, repeated under NO synthase inhibitor N(G)-monomethyl-l-arginine (l-NMMA) or the antioxidant ascorbic acid. The protocol was repeated at the end of exogenous ghrelin intra-arterial infusion. In hypertensive patients, ghrelin normalized the blunted response to acetylcholine, restored the inhibiting effect of l-NMMA and abrogated the potentiating effect of ascorbic acid on acetylcholine. In controls, ghrelin failed to modify these vascular responses. In hypertensive patients, ghrelin decreased venous levels of malondialdehyde, lipoperoxide, and interleukin-6, and concomitantly increased endogenous antioxidant capacity. Small vessels from hypertensive patients showed an enhanced intravascular oxidative stress, which was strongly and similarly decreased by incubation with ghrelin, the NAD(P)H oxidase inhibitor gp91 ds-tat, or both. Ghrelin also normalized the overexpression of p47 phosphorylation and restored the NO availability in small vessels from hypertensive patients. CONCLUSIONS: Exogenous ghrelin increases endothelial dysfunction by restoring NO availability in the forearm microcirculation of essential hypertensive patients, an effect ascribable to an antioxidant effect via inhibition of NAD(P)H oxidase activation.


Assuntos
Grelina/farmacologia , Hipertensão/fisiopatologia , NADPH Oxidases/fisiologia , Óxido Nítrico/biossíntese , Vasodilatadores/farmacologia , Análise de Variância , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Estudos de Casos e Controles , Endotélio Vascular , Inibidores Enzimáticos/farmacologia , Feminino , Antebraço/irrigação sanguínea , Grelina/administração & dosagem , Humanos , Hipertensão/enzimologia , Infusões Intra-Arteriais , Masculino , Pessoa de Meia-Idade , NADPH Oxidases/metabolismo , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Vasodilatadores/administração & dosagem , ômega-N-Metilarginina/farmacologia
19.
PLoS One ; 8(2): e57023, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23437299

RESUMO

BACKGROUND: The pathogenesis of diverticular disease (DD) is thought to result from complex interactions among dietary habits, genetic factors and coexistence of other bowel abnormalities. These conditions lead to alterations in colonic pressure and motility, facilitating the formation of diverticula. Although electrophysiological studies on smooth muscle cells (SMCs) have investigated colonic motor dysfunctions, scarce attention has been paid to their molecular abnormalities, and data on SMCs in DD are lacking. Accordingly, the main purpose of this study was to evaluate the expression patterns of molecular factors involved in the contractile functions of SMCs in the tunica muscularis of colonic specimens from patients with DD. METHODS AND FINDINGS: By means of immunohistochemistry and image analysis, we examined the expression of Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs, as well as pS368-Cx43, PKCps, RhoA and αSMA, all known to regulate the functions of gap junctions and the contractile activity of SMCs. The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors. CONCLUSION: This study demonstrates, for the first time, that an altered pattern of factors involved in SMC contractility is present at level of the tunica muscularis of DD patients. Moreover, considering that our analysis was conducted on colonic tissues not directly affected by diverticular lesions or inflammatory reactions, it is conceivable that these molecular alterations may precede and predispose to the formation of diverticula, rather than being mere consequences of the disease.


Assuntos
Colo/metabolismo , Divertículo do Colo/metabolismo , Músculo Liso/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Colo/patologia , Conexina 26 , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Divertículo do Colo/genética , Divertículo do Colo/patologia , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Músculo Liso/patologia , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
20.
J Cell Mol Med ; 16(2): 318-27, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21426484

RESUMO

Ulcerative colitis (UC) is an inflammatory bowel disease with alterations of colonic motility, which influence clinical symptoms. Although morpho-functional abnormalities in the enteric nervous system have been suggested, in UC patients scarce attention has been paid to possible changes in the cells that control colonic motility, including myenteric neurons, glial cells and interstitial cells of Cajal (ICC). This study evaluated the neural-glial components of myenteric ganglia and ICC in the colonic neuromuscular compartment of UC patients by quantitative immunohistochemical analysis. Full-thickness archival samples of the left colon were collected from 10 patients with UC (5 males, 5 females; age range 45-62 years) who underwent elective bowel resection. The colonic neuromuscular compartment was evaluated immunohistochemically in paraffin cross-sections. The distribution and number of neurons, glial cells and ICC were assessed by anti-HuC/D, -S100ß and -c-Kit antibodies, respectively. Data were compared with findings on archival samples of normal left colon from 10 sex- and age-matched control patients, who underwent surgery for uncomplicated colon cancer. Compared to controls, patients with UC showed: (i) reduced density of myenteric HuC/D(+) neurons and S100ß(+) glial cells, with a loss over 61% and 38%, respectively, and increased glial cell/neuron ratio; (ii) ICC decrease in the whole neuromuscular compartment. The quantitative variations of myenteric neuro-glial cells and ICC indicate considerable alterations of the colonic neuromuscular compartment in the setting of mucosal inflammation associated with UC, and provide a morphological basis for better understanding the motor abnormalities often observed in UC patients.


Assuntos
Colite Ulcerativa/patologia , Gânglios/patologia , Células Intersticiais de Cajal/patologia , Plexo Mientérico/patologia , Colite Ulcerativa/metabolismo , Colo/metabolismo , Colo/patologia , Proteínas ELAV/metabolismo , Proteína Semelhante a ELAV 3 , Feminino , Gânglios/metabolismo , Humanos , Células Intersticiais de Cajal/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade , Fatores de Crescimento Neural/metabolismo , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/metabolismo
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