RESUMO
AIMS: In calcific aortic valve disease (CAVD), progressive valvular sclerosis and calcification cause narrowing of the orifice and an impairment of the valve's function. We applied high-resolution cine-MRI to perform quantitative analysis of the dynamics of the aortic valve in a mice model of CAVD. METHODS AND RESULTS: LDLr-/- ApoB100/100 mice were fed a Western diet (WD) or a standard diet (control) for 22 weeks. The mice were imaged in a 7 T horizontal MRI scanner, and aortic valve dynamics was examined by imaging the cross-section of the aorta at valve level using cine sequences. From these images, the area of the aortic valve orifice was determined during the heart cycle. MRI results were compared with echocardiographic and histopathologic results. The data revealed evidence of clear aortic valve dysfunction in WD mice as compared with control mice (interaction P < 0.001). MRI showed narrowing (14%, P < 0.05) of the orifice area, and this was also seen in histology (34%, P < 0.05), indicating more severe aortic stenosis after WD than in controls. Additionally, MRI revealed a reduction in the ejection fraction (EF) (-11%, P < 0.01), a result confirmed with echocardiography (-27%, P < 0.001) in mice fed with WD. EF detected by MRI and echocardiography also correlated strongly with the degree of stenosis assessed by histology. CONCLUSIONS: Cine-MRI can be used for quantitative analysis of the aortic valve orifice over the cardiac cycle in mice. MRI showed the cusps clearly, and we were able to detect aortic valve dysfunction over time through the cardiac cycle.
Assuntos
Valva Aórtica/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética , Animais , Antígenos de Diferenciação/metabolismo , Valva Aórtica/patologia , Valva Aórtica/fisiopatologia , Apolipoproteínas B/metabolismo , Eletrocardiografia , Hipercolesterolemia/patologia , Imageamento por Ressonância Magnética , Camundongos , Receptores de LDL/metabolismo , Proteínas S100/metabolismo , Volume SistólicoRESUMO
BACKGROUND: Calcific aortic valve disease (CAVD) is an atheroinflammatory process; finally it leads to progressive calcification of the valve. There is no effective pharmacological treatment for CAVD and many of the underlying molecular mechanisms remain unknown. We conducted a proteomic study to reveal novel factors associated with CAVD. METHODS: We compared aortic valves from patients undergoing valvular replacement surgery due to non-calcified aortic insufficiency (control group, n = 5) to a stenotic group (n = 7) using two-dimensional difference gel electrophoresis (2D-DIGE). Protein spots were identified with mass spectrometry. Western blot and immunohistochemistry were used to validate the results in a separate patient cohort and Ingenuity Pathway Analysis (IPA) was exploited to predict the regulatory network of CAVD. RESULTS: We detected an upregulation of complement 9 (C9), serum amyloid P-component (APCS) and transgelin as well as downregulation of heat shock protein (HSP90), protein disulfide isomerase A3 (PDIA3), annexin A2 (ANXA2) and galectin-1 in patients with aortic valve stenosis. The decreased protein expression of HSP90 was confirmed with Western blot. CONCLUSIONS: We describe here a novel data set of proteomic changes associated with CAVD, including downregulation of the pro-inflammatory cytosolic protein, HSP90.
Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/química , Valva Aórtica/patologia , Calcinose/metabolismo , Proteínas de Choque Térmico HSP90/análise , Adulto , Idoso , Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Calcinose/patologia , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Proteômica , Transdução de SinaisRESUMO
BACKGROUND: The transforming growth factor (TGF)-ß is one of the key mediators in cardiac remodelling occurring after myocardial infarction (MI) and in hypertensive heart disease. The TGF-ß-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains unknown. METHODS: The aim of the present study was to characterize cardiac TSC-22 expression in vivo in cardiac remodelling and in myocytes in vitro. In addition, we used TSC-22 gene transfer in order to examine the effects of TSC-22 on cardiac gene expression and function. RESULTS: We found that TSC-22 is rapidly up-regulated by multiple hypertrophic stimuli, and in post-MI remodelling both TSC-22 mRNA and protein levels were up-regulated (4.1-fold, P <0.001 and 3.0-fold, P <0.05, respectively) already on day 1. We observed that both losartan and metoprolol treatments reduced left ventricular TSC-22 gene expression. Finally, TSC-22 overexpression by local intramyocardial adenovirus-mediated gene delivery showed that TSC-22 appears to have a role in regulating collagen type IIIα1 gene expression in the heart. CONCLUSIONS: These results demonstrate that TSC-22 expression is induced in response to cardiac overload. Moreover, our data suggests that, by regulating collagen expression in the heart in vivo, TSC-22 could be a potential target for fibrosis-preventing therapies.
Assuntos
Colágeno Tipo III/genética , Hipertensão/metabolismo , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Proteínas Repressoras/metabolismo , Regulação para Cima , Animais , Anti-Hipertensivos/uso terapêutico , Células Cultivadas , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Losartan/uso terapêutico , Masculino , Metoprolol/uso terapêutico , Células Musculares/metabolismo , RNA Mensageiro/metabolismo , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Ratos Sprague-Dawley , Transdução de Sinais , Remodelação VentricularRESUMO
For more than 100 years, examinations of pathology specimens have relied on the use of the light microscope. The technological progress of the last few years is enabling the digitizing of histologic specimen slides and application of the virtual microscope in diagnostics. Virtual microscopy will facilitate consultation possibilities, and digital image analysis serves to enhance the level of diagnostics. Organizing and monitoring clinicopathological meetings will become easier. Digital archive of histologic specimens and the virtual microscopy network are expected to benefit training and research as well, particularly what applies to the Finnish biobank network which is currently being established.
Assuntos
Microscopia/instrumentação , Microscopia/tendências , Patologia Clínica/instrumentação , Patologia Clínica/tendências , Interface Usuário-Computador , Bancos de Espécimes Biológicos , Finlândia , Humanos , Processamento de Imagem Assistida por ComputadorRESUMO
The use of molecular markers in the diagnostics of gliomas aids histopathological diagnosis and allows their further classification into clinically significant subgroups. The aim of this study was to characterize the methylation pattern of the O(6) -methylguanine-DNA methyltransferase (MGMT) promoter, gene copy number aberrations, and isocitrate dehydrogenase I (IDH1) mutation in gliomas. We studied 51 gliomas (15 oligodendrogliomas, 18 oligoastrocytomas, 3 astrocytomas, and 15 glioblastomas) by pyrosequencing, array comparative genome hybridization (CGH), and immunohistochemistry. MGMT hypermethylation was observed in 100% of oligoastrocytomas, 93% of oligodendrogliomas, and 47% of glioblastomas. The most frequently altered chromosomal regions were deletions of 1p31.1/21.1-22.2 and 19q13.3qter in oligodendroglial tumors, and losses of 9p21.3, 10q25.3qter, and 10q26.13-26.2 in glioblastomas. Deletions on 9p and 10q, and gain of 7p were associated with the unmethylated MGMT phenotype, whereas deletion of 19q and oligodendroglial morphology was associated with MGMT hypermethylation. IDH1 mutation showed positive correlation with MGMT hypermethylation and loss of 1p/19q. Our results suggest that MGMT promoter methylation, analyzed by pyrosequencing, is a frequent event in oligodendroglial tumors, and it correlates with IDH1 mutation and 19q loss in gliomas. Pyrosequencing proved a good method for assessing the degree of MGMT methylation in formalin-fixed paraffin-embedded glioma samples. However, further studies are needed to confirm a clinically relevant cut-off point for MGMT methylation in gliomas.
Assuntos
Hibridização Genômica Comparativa , Metilação de DNA , Glioma/genética , Isocitrato Desidrogenase/genética , Mutação , O(6)-Metilguanina-DNA Metiltransferase/genética , Regiões Promotoras Genéticas , Adulto , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Feminino , Glioma/classificação , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
OBJECTIVE: Both obesity and exposure to chemicals may induce non-alcoholic fatty liver disease (NAFLD). Pregnane X Receptor (PXR) is a central target of metabolism disrupting chemicals and disturbs hepatic glucose and lipid metabolism. We hypothesized that the metabolic consequences of PXR activation may be modified by existing obesity and associated metabolic dysfunction. METHODS: Wildtype and PXR knockout male mice were fed high-fat diet to induce obesity and metabolic dysfunction. PXR was activated with pregnenolone-16α-carbonitrile. Glucose metabolism, hepatosteatosis, insulin signaling, glucose uptake, liver glycogen, plasma and liver metabolomics, and liver, white adipose tissue, and muscle transcriptomics were investigated. RESULTS: PXR activation aggravated obesity-induced liver steatosis by promoting lipogenesis and inhibiting fatty acid disposal. Accordingly, hepatic insulin sensitivity was impaired and circulating alanine aminotransferase level increased. Lipid synthesis was facilitated by increased liver glucose uptake and utilization of glycogen reserves resulting in dissociation of hepatosteatosis and hepatic insulin resistance from the systemic glucose tolerance and insulin sensitivity. Furthermore, glucagon-induced hepatic glucose production was impaired. PXR deficiency did not protect from the metabolic manifestations of obesity, but the liver transcriptomics and metabolomics profiling suggest diminished activation of inflammation and less prominent changes in the overall metabolite profile. CONCLUSIONS: Obesity and PXR activation by chemical exposure have a synergistic effect on NAFLD development. To support liver fat accumulation the PXR activation reorganizes glucose metabolism that seemingly improves systemic glucose metabolism. This implies that obese individuals, already predisposed to metabolic diseases, may be more susceptible to harmful metabolic effects of PXR-activating drugs and environmental chemicals.
Assuntos
Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Masculino , Receptor de Pregnano X , Camundongos Obesos , Obesidade/metabolismo , Glucose/metabolismoRESUMO
Histological analysis with microscopy is the gold standard to diagnose and stage cancer, where slides or whole slide images are analyzed for cell morphological and spatial features by pathologists. The nuclei of cancerous cells are characterized by nonuniform chromatin distribution, irregular shapes, and varying size. As nucleus area and shape alone carry prognostic value, detection and segmentation of nuclei are among the most important steps in disease grading. However, evaluation of nuclei is a laborious, time-consuming, and subjective process with large variation among pathologists. Recent advances in digital pathology have allowed significant applications in nuclei detection, segmentation, and classification, but automated image analysis is greatly affected by staining factors, scanner variability, and imaging artifacts, requiring robust image preprocessing, normalization, and segmentation methods for clinically satisfactory results. In this paper, we aimed to evaluate and compare the digital image analysis techniques used in clinical pathology and research in the setting of gastric cancer. A literature review was conducted to evaluate potential methods of improving nuclei detection. Digitized images of 35 patients from a retrospective cohort of gastric adenocarcinoma at Oulu University Hospital in 1987-2016 were annotated for nuclei (n = 9085) by expert pathologists and 14 images of different cancer types from public TCGA dataset with annotated nuclei (n = 7000) were used as a comparison to evaluate applicability in other cancer types. The detection and segmentation accuracy with the selected color normalization and stain separation techniques were compared between the methods. The extracted information can be supplemented by patient's medical data and fed to the existing statistical clinical tools or subjected to subsequent AI-assisted classification and prediction models. The performance of each method is evaluated by several metrics against the annotations done by expert pathologists. The F1-measure of 0.854 ± 0.068 is achieved with color normalization for the gastric cancer dataset, and 0.907 ± 0.044 with color deconvolution for the public dataset, showing comparable results to the earlier state-of-the-art works. The developed techniques serve as a basis for further research on application and interpretability of AI-assisted tools for gastric cancer diagnosis.
Assuntos
Corantes , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/patologia , Artefatos , Estudos Retrospectivos , Algoritmos , Processamento de Imagem Assistida por Computador/métodos , Núcleo Celular/metabolismoRESUMO
Reactive oxygen species (ROS) have been implicated in many aspects of tissue/cellular metabolic signaling and pathology, including cardioprotection against ischemia-reperfusion damage. Recent reports of enhanced ROS production under global or simulated ischemia in intact heart or isolated cardiomyocytes, respectively, and its decrease again upon reperfusion are paradoxical. Mechanisms for increasing ROS production with decreasing reactant (oxygen) concentration remain elusive, making it important to critically evaluate the experimental methods used to measure ROS production. In the present paper superoxide production in isolated perfused rat hearts was monitored by lucigenin chemiluminescence or dihydroethidine (DHE) oxidation product fluorescence in parallel with redox state of flavin and cytochrome oxidase. Lucigenin luminescence decreased in ischemia and increased again upon reperfusion, transiently reaching values eightfold the control value coincidently with an overshoot of mitochondrial oxygen concentration. Hypoxic perfusion decreased lucigenin chemiluminescence in spite of coronary flow increase, whereas change in lucigenin concentration in the perfusate had negligible effect. In contrast to lucigenin luminescence, the fluorescence of the DHE oxidation product increased continuously during a 30-min global ischemia and decreased precipitously upon reperfusion, this change is coincident with absorption changes of the oxygen-binding protein myoglobin. The time course of DHE oxidation product fluorescence during ischemia and reperfusion was similar to that of the mitochondrial membrane potential probe safranin as shown in perfused heart previously [Ylitalo KV, Ala-Rämi A, Liimatta EV, Peuhkurinen KJ, Hassinen IE. J Mol Cell Cardiol 2000;32:1223-38]. In solution under high oxygen partial pressure DHE was mainly oxidized to a product, whose fluorescence, absorbance and mass spectra were similar to ethidium, and this product behaved like a mitochondrial membrane potential probe in isolated mitochondria. As a membrane permeable cation it accumulates into the mitochondria when the membrane potential is high (high intramitochondrial concentration quenches fluorescence) and then is released (increased fluorescence) during hypoxia/ischemia. Upon reperfusion it is re-accumulated in the mitochondria as the membrane potential recovers. The non-specific oxidation of DHE makes this dye less suitable for superoxide detection in experiments on isolated perfused hearts that necessitate high oxygen partial pressure in the perfusate. The time course of lucigenin luminescence during ischemia/reperfusion is consistent with decreased ROS production during ischemia/hypoxia, while the oxygen concentration is decreased, followed by an overshoot when the heart tissue is reperfused and the oxygen pressures return to normal or above normal.
Assuntos
Traumatismo por Reperfusão Miocárdica/metabolismo , Superóxidos/metabolismo , Acridinas , Animais , Circulação Coronária , Dicarbetoxi-Di-Hidrocolidina/análogos & derivados , Dicarbetoxi-Di-Hidrocolidina/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Flavoproteínas/metabolismo , Técnicas In Vitro , Fígado/metabolismo , Substâncias Luminescentes , Masculino , Potencial da Membrana Mitocondrial , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mioglobina/metabolismo , Oxirredução , Consumo de Oxigênio , RatosRESUMO
BACKGROUND: Cardiovascular diseases are the most important cause of death in patients with impaired kidney function. Left ventricular hypertrophy (LVH), cardiac interstitial fibrosis and cardiovascular calcifications are characteristic of chronic renal insufficiency (CRI). Periostin is a fibrogenesis- and calcification-related matricellular protein re-expressed in adult tissues undergoing remodelling in response to pathological stimuli. The role of periostin in CRI-induced LVH is unknown. METHODS: Rats were 5/6-nephrectomized (NX), and after 15 weeks of disease progression high-calcium, high-phosphate or paricalcitol treatment was given for 12 weeks. Cardiac tissue and blood samples were taken to study periostin gene expression and to determine factors contributing to its reactivation, respectively. Left ventricular (LV) periostin expression was also examined in response to angiotensin II or arginine(8)-vasopressin (AVP)-induced pressure overload and in spontaneously hypertensive rats. RESULTS: CRI resulted in a 6.5-fold increase in LV periostin messenger RNA (mRNA) levels. Positive extracellular immunostaining for periostin was detected in areas of infiltrated inflammatory cells and fibrotic lesions. There was a significant correlation between LV periostin mRNA levels and plasma biomarkers of impaired kidney function, LVH, fibrogenesis-related proteins osteopontin and osteoactivin, and anti-calcific matrix Gla protein. Moreover, LV periostin gene expression in CRI correlated positively with systolic blood pressure (BP) and was activated rapidly in response to angiotensin II or AVP infusions. CONCLUSIONS: Periostin is involved in fibrotic cardiac remodelling in CRI. The re-expression of periostin is localized to the fibrotic and inflammatory lesions and is most likely the consequence of elevated BP.
Assuntos
Biomarcadores/metabolismo , Moléculas de Adesão Celular/metabolismo , Fibrose/etiologia , Fibrose/patologia , Expressão Gênica , Ventrículos do Coração/metabolismo , Insuficiência Renal/patologia , Animais , Western Blotting , Conservadores da Densidade Óssea/farmacologia , Calcinose , Moléculas de Adesão Celular/genética , Modelos Animais de Doenças , Ergocalciferóis/farmacologia , Fibrose/metabolismo , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/patologia , Técnicas Imunoenzimáticas , Testes de Função Renal , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Nefrectomia/efeitos adversos , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos SHR , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Insuficiência Renal/complicações , Insuficiência Renal/metabolismoRESUMO
AIMS: Aortic valve calcification is an actively regulated process with endothelial dysfunction displaying hallmarks of atherosclerosis. C-type natriuretic peptide (CNP) system has been reported to have a role in the pathogenesis of vascular atherosclerosis and to be distinctly downregulated in aortic valve stenosis (AS). Here we studied gene expressions of CNP and is target receptor natriuretic peptide receptor type B (NPR-B) in human aortic valves. Furthermore, we compared gene expression of CNP system in patients with HMG-coenzyme-A reductase (statin) treatment to non-statin-treated patients in AS group. METHODS AND RESULTS: With the study population of 108 patients, we characterized expression of CNP and NPR-B in human aortic valves and compared normal control valves (n = 12) with valves obtained from patients with aortic regurgitation (AR, n = 16), AR with fibrosis (AR+fibr., n = 19) and AS (n = 61). By reverse transcription-polymerase chain reaction (RT-PCR), CNP mRNA levels were 89% lower (p = 0.022) in stenotic valves, when compared to AR group. Moreover, the mRNA levels of NPR-B, the target receptor of CNP, were 62% lower (p < 0.001) in stenotic valves when compared to control group and 54% lower (p = 0.002) in stenotic valves, when compared to AR group. There was no statistical significant difference in CNP and NPR-B levels in AS group when the statin-treated patients were compared to untreated patients. CONCLUSIONS: These results show for the first time that the gene expression of anti-atherogenic CNP system did not differ between statin-treated and non-statin-treated patients in AS. The research data supports the results of clinical trials with the same drug class.
Assuntos
Estenose da Valva Aórtica/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Peptídeo Natriurético Tipo C/metabolismo , Adulto , Idoso , Valva Aórtica/patologia , Calcinose/metabolismo , Regulação para Baixo/fisiologia , Feminino , Regulação da Expressão Gênica/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Tipo C/antagonistas & inibidores , Peptídeo Natriurético Tipo C/biossíntese , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Receptores do Fator Natriurético Atrial/metabolismoRESUMO
In calcific aortic valve disease (CAVD) progressive valvular calcification causes aortic valve dysfunction. CAVD has several risk factors such as age and dyslipidemia. Vitamin K was shown to inhibit vascular calcification in mice and valvular calcification in patients with CAVD. We studied the effect of menaquinone 4 (MK4/vitamin K2) on valvular calcification in the hypercholesterolemic mouse model of CAVD. LDLr-/-ApoB100/100 male mice were fed with a Western diet for 5 months, with (n = 10) or without (n = 10) added 0.2 mg/g MK4. Body weight gain was followed weekly. Morphology of aortic valves and liver was assessed with immunohistochemistry. Plasma cholesterol levels and cytokines from hepatic tissue were assessed in the end of the study. Hepatic gene expression of lipid metabolism regulating genes were assessed after 18 h diet. MK4 exacerbated the lipoprotein lipid profile without affecting aortic valve morphology in hypercholesterolemic LDLr-/- ApoB100/100 mice. The MK4-containing WD diet increased plasma levels of LDL and triglycerides, hepatic steatosis, and mRNA expression of genes required for triglyceride and cholesterol synthesis. MK4 diminished levels of several cytokines and chemokines in liver, including IL-6, TNFα and MCP1, as measured by hepatic cytokine array. Consequently, MK4 may exert non-beneficial effects on circulating lipid levels, especially in hypercholesterolemic individuals.
Assuntos
Estenose da Valva Aórtica/tratamento farmacológico , Valva Aórtica/patologia , Apolipoproteínas B/genética , Calcinose/tratamento farmacológico , Hipercolesterolemia/tratamento farmacológico , Receptores de LDL/genética , Vitamina K 2/farmacologia , Animais , Estenose da Valva Aórtica/sangue , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Calcinose/sangue , Calcinose/genética , Calcinose/patologia , Modelos Animais de Doenças , Humanos , Hipercolesterolemia/sangue , Hipercolesterolemia/genética , Hipercolesterolemia/patologia , Lipídeos/sangue , Lipoproteínas LDL/sangue , Camundongos , Camundongos Knockout , Fatores de Risco , Triglicerídeos/sangueRESUMO
AIMS: Aortic valve stenosis (AS) is an actively regulated process like atherosclerosis, which is accompanied by changes e.g. in endothelin-related genes. However, the role of endothelin peptides in AS is unknown. METHODS AND RESULTS: We characterized the expression of the endothelin system in aortic valves of patients with normal valves (n = 12), regurgitation, and fibrosis (n = 6) and AS (n = 18) by reverse-transcriptase-polymerase chain reaction and immunohistochemistry. The number of endothelin-1 (ET-1) positive cells was higher in AS than in control valves, while levels of ET-1 mRNA did not differ between groups. Endothelin receptor-A (ET(A)) mRNA levels were upregulated in stenotic valves (4.3-fold, P = 0.032) associated with a remarkable increase in number of ET(A)-immunopositive cells. ET(B)-receptor mRNA levels did not change during disease progression. Endothelin-converting enzyme-1 (ECE-1) mRNA levels were 42% lower (P = 0.007) in stenotic valves. Finally, because ET-1 and ECE-1 have binding site for activator protein-1 (AP-1), we measured AP-1 DNA binding by gel shift assays, which showed significantly lower (76%, P = 0.003) activity in AS. CONCLUSION: AS is characterized by distinct upregulation of ET-1 and its target receptor ET(A), promoting growth, inflammation, and fibrosis. These findings suggest therapeutic potential for ET(A)-receptor antagonists in aortic valve calcification.
Assuntos
Estenose da Valva Aórtica/metabolismo , Ácido Aspártico Endopeptidases/genética , Endotelina-1/metabolismo , Metaloendopeptidases/genética , Receptor de Endotelina A/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Estenose da Valva Aórtica/genética , Ácido Aspártico Endopeptidases/metabolismo , Regulação para Baixo , Endotelina-1/genética , Enzimas Conversoras de Endotelina , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Metaloendopeptidases/metabolismo , Pessoa de Meia-Idade , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor de Endotelina A/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND AND AIM OF THE STUDY: Aortic valve stenosis (AS) is an actively regulated pathobiological process that shows some hallmarks of atherosclerosis. Apelin and its receptor, APJ, are highly expressed in the heart, and the proposed effects of the apelin-APJ system are opposite to those of the angiotensin II-AT1-receptor pathway. The role of the apelin-APJ signaling pathway in calcified aortic valve disease is unknown. METHODS: The study involved the characterization and comparison of expression of apelin and APJ as well as angiotensin II receptors (AT1 and AT2) in the aortic valves of patients with normal valves (n = 6), aortic regurgitation (n = 9 AR), regurgitation and fibrosis/mild sclerosis (n = 14), and AS (n = 25). RESULTS: By employing the reverse-transcriptase polymerase chain reaction (RT-PCR), the gene expression of apelin (3.63-fold, p = 0.001) and the APJ receptor (2.70-fold, p = 0.01) were shown to be significantly up-regulated in stenotic valves when compared to controls. In addition, APJ receptor mRNA levels were higher (2.9-fold, p = 0.010) in the AR + sclerosis group when compared to controls. Using immunohistochemistry, apelin was shown to be localized in stenotic aortic valves to the valvular endothelial layer of the aortic valve, to vascular endothelial cells in neovessels, and to fibroblasts and macrophages adjacent to vessels in the stromal area. AT2-receptor mRNA levels were 90% (p < 0.001) lower in stenotic valves. In contrast, the gene expression of AT1-receptors did not differ significantly among the groups. CONCLUSION: Aortic valve stenosis is characterized by an up-regulation of the apelin-APJ signaling pathway, revealing a possible novel target for drug discovery in calcified aortic valve disease by suppressing chemotaxis, angiogenesis and osteoblast activity, all of which are well-documented phenomena in the disease process.
Assuntos
Estenose da Valva Aórtica/metabolismo , Valva Aórtica/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Valva Aórtica/patologia , Estenose da Valva Aórtica/patologia , Apelina , Receptores de Apelina , Calcinose/metabolismo , Calcinose/patologia , Estudos de Casos e Controles , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
BACKGROUND AND AIM OF THE STUDY: Calcific aortic valve disease (CAVD) is a progressive disease starting from mild valvular sclerosis and progressing to severe aortic stenosis (AS) with calcified valves. The origin of the calcification is proposed to be mesenchymal cells which have differentiated towards an osteoblastic phenotype. Podoplanin is a glycoprotein expressed in the endothelium of lymphatic vessels and in osteoblasts and osteocytes, mesenchymal cells, as well as in many carcinomas and aortic atherosclerotic lesions. In CAVD, its expression has been evaluated only as a marker of the lymphatic vasculature. MATERIALS AND METHODS: We determined podoplanin expression in human aortic valves in four patient groups: control (C, n=7), aortic regurgitation (AR, n=8), aortic regurgitation and fibrosis (ARâ¯+â¯f, n=15) and AS (n=49) by immunohistochemistry and quantitative real-time PCR (RT-PCR). RESULTS: Immunohistochemically, podoplanin expression was significantly increased in ARâ¯+â¯f and AS groups when compared with the control and AR groups and the level of expression positively correlated with the extent of calcification and vascularity. Podoplanin mRNA levels were 1.7-fold higher in the AS group as compared with the control group (P=.05). Podoplanin-positivity was present not only in lymphatic vessel endothelium but also in osteoblasts, osteocytes, chondrocytes, macrophages and extracellular matrix. The majority of the podoplanin-positivity was in spindle cells with a myofibroblastic phenotype, often associated with calcifications. Tricuspid valves had more calcification-associated podoplanin than bi/unicuspid valves (median 1.52 vs 1.16, P<.001). CONCLUSIONS: CAVD is characterized by an increased expression of podoplanin; this is associated with the differentiation of valvular interstitial cells into calcium-producing, myofibroblast-like cells. In addition, tricuspid valves express relatively more podoplanin than bi/unicuspid valves.
Assuntos
Insuficiência da Valva Aórtica/metabolismo , Valva Aórtica/química , Valva Aórtica/patologia , Glicoproteínas de Membrana/análise , Mesoderma/química , Adulto , Idoso , Valva Aórtica/anormalidades , Insuficiência da Valva Aórtica/genética , Insuficiência da Valva Aórtica/patologia , Estenose da Valva Aórtica/genética , Estenose da Valva Aórtica/patologia , Biomarcadores/análise , Calcinose/genética , Calcinose/patologia , Estudos de Casos e Controles , Feminino , Fibrose , Humanos , Masculino , Glicoproteínas de Membrana/genética , Mesoderma/patologia , Pessoa de Meia-Idade , Miofibroblastos/química , Miofibroblastos/patologia , RNA Mensageiro/genética , Regulação para CimaRESUMO
Aims: Oxidative stress and inflammation play an important role in the progression of atherosclerosis. Transcription factor NF-E2-related factor 2 (Nrf2) has antioxidant and anti-inflammatory effects in the vessel wall, but paradoxically, global loss of Nrf2 in apoE deficient mice alleviates atherosclerosis. In this study, we investigated the effect of global Nrf2 deficiency on early and advanced atherogenesis in alternative models of atherosclerosis, LDL receptor deficient mice (LDLR-/-), and LDLR-/- mice expressing apoB-100 only (LDLR-/- ApoB100/100) having a humanized lipoprotein profile. Methods and results: LDLR-/- mice were fed a high-fat diet (HFD) for 6 or 12 weeks and LDLR-/-ApoB100/100 mice a regular chow diet for 6 or 12 months. Nrf2 deficiency significantly reduced early and more advanced atherosclerosis assessed by lesion size and coverage in the aorta in both models. Nrf2 deficiency in LDLR-/- mice reduced total plasma cholesterol after 6 weeks of HFD and triglycerides in LDLR-/-ApoB100/100 mice on a chow diet. Nrf2 deficiency aggravated aortic plaque maturation in aged LDLR-/-ApoB100/100 mice as it increased plaque calcification. Moreover, â¼36% of Nrf2-/-LDLR-/-ApoB100/100 females developed spontaneous myocardial infarction (MI) or sudden death at 5 to 12 months of age. Interestingly, Nrf2 deficiency increased plaque instability index, enhanced plaque inflammation and calcification, and reduced fibrous cap thickness in brachiocephalic arteries of LDLR-/-ApoB100/100 female mice at age of 12 months. Conclusions: Absence of Nrf2 reduced atherosclerotic lesion size in both atherosclerosis models, likely via systemic effects on lipid metabolism. However, Nrf2 deficiency in aged LDLR-/-ApoB100/100 mice led to an enhanced atherosclerotic plaque instability likely via increased plaque inflammation and oxidative stress, which possibly predisposed to MI and sudden death.
Assuntos
Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Hipercolesterolemia/complicações , Fator 2 Relacionado a NF-E2/deficiência , Placa Aterosclerótica , Fatores Etários , Animais , Aorta/patologia , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Apolipoproteína B-100/genética , Apolipoproteína B-100/metabolismo , Aterosclerose/etiologia , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Células Cultivadas , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Feminino , Hipercolesterolemia/genética , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Receptores de LDL/deficiência , Receptores de LDL/genética , Triglicerídeos/sangueRESUMO
BACKGROUND: Whole slide images (WSIs, digitized histopathology glass slides) are large data files whose long-term storage remains a significant cost for pathology departments. Currently used WSI formats are based on lossy image compression alogrithms, either using JPEG or its more efficient successor JPEG 2000. While the advantages of the JPEG 2000 algorithm (JP2) are commonly recognized, its compression parameters have not been fully optimized for pathology WSIs. METHODS: We defined an optimized parametrization for JPEG 2000 image compression, designated JP2-WSI, to be used specifically with histopathological WSIs. Our parametrization is based on allowing a very high degree of compression on the background part of the WSI while using a conventional amount of compression on the tissue-containing part of the image, resulting in high overall compression ratios. RESULTS: When comparing the compression power of JP2-WSI to the commonly used fixed 35:1 compression ratio JPEG 2000 and the default image formats of proprietary Aperio, Hamamatsu, and 3DHISTECH scanners, JP2-WSI produced the smallest file sizes and highest overall compression ratios for all 17 slides tested. The image quality, as judged by visual inspection and peak signal-to-noise ratio (PSNR) measurements, was equal to or better than the compared image formats. The average file size by JP2-WSI amounted to 15, 9, and 16 percent, respectively, of the file sizes of the three commercial scanner vendors' proprietary file formats (3DHISTECH MRXS, Aperio SVS, and Hamamatsu NDPI). In comparison to the commonly used 35:1 compressed JPEG 2000, JP2-WSI was three times more efficient. CONCLUSIONS: JP2-WSI allows very efficient and cost-effective data compression for whole slide images without loss of image information required for histopathological diagnosis.
RESUMO
Pregnane X receptor (PXR) is a nuclear receptor that senses chemical environment and is activated by numerous clinically used drugs and environmental contaminants. Previous studies have indicated that several drugs known to activate PXR appear to induce glucose intolerance. We now aimed to reveal the role of PXR in drug-induced glucose intolerance and characterize the mechanisms involved. We used PXR knockout mice model to investigate the significance of this nuclear receptor in the regulation of glucose tolerance. PXR ligand pregnenolone-16É-carbonitrile (PCN) impaired glucose tolerance in the wildtype mice but not in the PXR knockout mice. Furthermore, DNA microarray and bioinformatics analysis of differentially expressed genes and glucose metabolism relevant pathways in PCN treated primary hepatocytes indicated that PXR regulates genes involved in glucose uptake. PCN decreased the expression of glucose transporter 2 (GLUT2) in mouse liver and in the wildtype mouse hepatocytes but not in the PXR knockout cells. Data mining of published chromatin immunoprecipitation-sequencing results indicate that Glut2 gene is a direct PXR target. Furthermore, PCN induced internalization of GLUT2 protein from the plasma membrane to the cytosol in the liver in vivo and repressed glucose uptake in the primary hepatocytes. Our results indicate that the activation of PXR impairs glucose tolerance and thus PXR represents a novel diabetogenic pathway. PXR activation dysregulates GLUT2 function by two different mechanisms. These findings may partly explain the diabetogenic effects of medications and environmental contaminants.
Assuntos
Transportador de Glucose Tipo 2/metabolismo , Fígado/metabolismo , Receptor de Pregnano X/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Intolerância à Glucose , Transportador de Glucose Tipo 2/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Pregnano X/genética , Carbonitrila de Pregnenolona/farmacologia , Transporte Proteico , TranscriptomaRESUMO
Cancer cell budding at the invasive margin has been associated with poor prognosis in rectal cancer. beta-Catenin is an adhesion protein involved in the nuclear Wnt/beta-catenin pathway, and mesenchymal transition of colorectal cancer cells. Hence, we investigated the relationship between cancer cell budding at the invasive margin, beta-catenin expression, and 5-year-survival in colorectal cancer. Four hundred and sixty six colorectal cancer specimens were analysed for budding margin, and 108 specimens from the same set for beta-catenin by immunohistochemistry. A budding margin was present in 24.0% of the cases and predicted a poor 5-year-survival (15.4%, P < 0.00001). Nuclear beta-catenin expression increased from the central area towards the invasive margin (P < 0.001), but did not predict budding. Budding margin is an independent factor associated with poor prognosis in colorectal cancer, and could be utilised in diagnostic pathology. Nuclear beta-catenin was often found at the invasive margin, but is unlikely to be the sole cause of budding.
Assuntos
Neoplasias Colorretais/metabolismo , Proteínas de Neoplasias/metabolismo , beta Catenina/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Humanos , Imuno-Histoquímica , Invasividade Neoplásica/patologia , Recidiva Local de Neoplasia/metabolismo , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Variações Dependentes do Observador , Prognóstico , Análise de Regressão , Análise de SobrevidaRESUMO
Background: The repair of a segmental peripheral nerve injury is a clinical challenge. Several studies have been performed to determine superior methods for overcoming nerve gaps. The purpose of this study was to investigate if the inside-out slided epineurium of the distal segment of an injured nerve can serve as a conduit to bridge a short nerve defect (10 mm). Methods: Nineteen sciatic nerves in Sprague-Dawley rats were transected, and a 10-mm gap was left between the ends. A section of distal epineurium was pulled inside out to bridge the gap. Walking track analysis was performed, and the sciatic function index (SFI) was calculated. Wet muscle mass and withdrawal reflex were measured. The density of axon fibers at different levels of repaired nerves was determined, and histological analysis was performed at 16 weeks. Results: The mean SFI improved from -81.0 at 4 weeks to 36.3 at 16 weeks. The axon densities showed regeneration through the epineural tube, and 5 of the rats demonstrated a withdrawal reflex. The weight of the tibialis anterior muscle of the injured limb at 16 weeks was 59% that of the uninjured side. Conclusions: The distal epineural sheath tube provided a size-matched conduit between the nerve stumps, with no histological donor-site morbidity. Histologically, regeneration occurred through the epineural tube without neuroma formation, and functional recovery was comparable to that of previous studies of nerve repair techniques. Technique may be an addition to the armamentarium of tools used to treat segmental nerve defects.
Assuntos
Regeneração Nervosa , Traumatismos dos Nervos Periféricos/cirurgia , Nervo Isquiático/lesões , Animais , Masculino , Procedimentos Neurocirúrgicos/métodos , Ratos , Ratos Sprague-Dawley , Reflexo/fisiologia , Nervo Isquiático/fisiologiaRESUMO
Regenerating islet-derived 3γ (Reg3γ) is a multifunctional protein, associated with various tissue injuries and inflammatory states. Since chronic inflammation is characteristics also for heart failure, the aim of this study was to characterize Reg3γ expression in cardiac inflammatory conditions. Reg3γ expression was studied in experimental rat models of myocardial infarction (MI) and pressure overload in vivo. For cell culture studies neonatal rat cardiac myocytes (NRCMs) were used. In addition, adenovirus-mediated gene transfer of upstream mitogen-activated protein kinase (MAPK) kinase 3b and p38α MAPK in vivo and in vitro was performed. Reg3γ mRNA (12.8-fold, P < 0.01) and protein (5.8-fold, P < 0.001) levels were upregulated during the postinfarction remodeling at day 1 after MI, and angiotensin II (Ang II) markedly increased Reg3γ mRNA levels from 6 h to 2 weeks. Immunohistochemistry revealed that the Ang II-induced expression of Reg3γ was localized into the cardiac fibroblasts and myofibroblasts of the proliferating connective tissue in the heart. Stretching and treatments with endothelin-1, lipopolysaccharide (LPS), and fibroblast growth factor-1 increased Reg3γ mRNA levels in NRCMs. SB203580, a selective p38 MAPK inhibitor, markedly attenuated LPS and mechanical stretch-induced upregulation of Reg3γ gene expression. Moreover, combined overexpression of MKK3bE and WT p38α increased Reg3γ gene expression in cultured cardiomyocytes in vitro and in the rat heart in vivo. Our study shows that cardiac stress activates Reg3γ expression and p38 MAPK is an upstream regulator of Reg3γ gene expression in heart. Altogether our data suggest Reg3γ is associated with cardiac inflammatory signaling.