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1.
Hypoxia (Auckl) ; 4: 81-90, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27800510

RESUMO

Glycosaminoglycan hyaluronan (HA), a major constituent of the endothelial glycocalyx, helps to maintain vascular integrity. Preconditioning the heart with volatile anesthetic agents protects against ischemia/reperfusion injury. We investigated a possible protective effect of sevoflurane on the glycocalyx, especially on HA. The effect of pre-ischemic treatment with sevoflurane (15 minutes at 2% vol/vol gas) on shedding of HA was evaluated in 28 isolated, beating guinea pig hearts, subjected to warm ischemia (20 minutes at 37°C) followed by reperfusion (40 minutes), half with and half without preconditioning by sevoflurane. HA concentration was measured in the coronary effluent. Over the last 20 minutes of reperfusion hydroxyethyl starch (1 g%) was continuously infused and the epicardial transudate collected over the last 5 minutes for measuring the colloid extravasation. Additional hearts were fixed by perfusion after the end of reperfusion for immunohistology and electron microscopy. Sevoflurane did not significantly affect post-ischemic oxidative stress, but strongly inhibited shedding of HA during the whole period, surprisingly even prior to ischemia. Immunohistology demonstrated that heparan sulfates and SDC1 of the glycocalyx were also preserved by sevoflurane. Electron microscopy revealed shedding of glycocalyx caused by ischemia and a mostly intact glycocalyx in hearts exposed to sevoflurane. Coronary vascular permeability of the colloid hydroxyethyl starch was significantly decreased by sevoflurane vs the control. We conclude that application of sevoflurane preserves the coronary endothelial glycocalyx, especially HA, sustaining the vascular barrier against ischemic damage. This may explain beneficial effects associated with clinical use of volatile anesthetics against ischemia/reperfusion injury.

2.
Ann Anat ; 196(6): 456-63, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25175150

RESUMO

No systematic, histologically confirmed data are available concerning the association between magnitude of direct dynamic impact caused by vertical impact trauma and the resulting injury to cartilage and subchondral bone. The aim of this study was to investigate the association between dynamic impact and the resulting patterns of osteochondral injury in an ex-vivo model. A mechanical apparatus was employed to perform ex-vivo controlled dynamic vertical impact experiments in 110 pig knees with the femur positioned in a holding fixture. A falling body with a thrust plate and photo sensor was applied. The direct impact to the trochlear articular surface was registered and the resulting osteochondral injuries macroscopically and histologically correlated and categorized. The relationship between magnitude of direct impact and injury severity could be classified as stage I injuries (impact <7.3MPa): elastic deformation, no histological injury; stage II injuries (impact 7.3-9.6MPa): viscoelastic imprint of the cartilaginous surface, subchondral microfractures; stage III injuries (impact 9.6-12.7MPa): disrupted cartilage surface, chondral fissures and subchondral microfractures; stage IV injuries (impact >12.7MPa): osteochondral impression, histologically imprint and osteochondral macrofractures. The impact ranges and histologic injury stages determined from this vertical dynamic impact experiment allowed for a biomechanical classification of direct, acute osteochondral injury. In contrast to static load commonly applied in ex-vivo experiments, dynamic impact more realistically represents actual trauma to the knee joint.


Assuntos
Traumatismos do Joelho/patologia , Traumatismos do Joelho/fisiopatologia , Articulação do Joelho/patologia , Articulação do Joelho/fisiopatologia , Ferimentos não Penetrantes/patologia , Ferimentos não Penetrantes/fisiopatologia , Aceleração , Acidentes por Quedas , Animais , Técnicas In Vitro , Estresse Mecânico , Suínos
3.
Basic Res Cardiol ; 108(3): 347, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23563917

RESUMO

Atrial natriuretic peptide (ANP) is a peptide hormone released from the cardiac atria during hypervolemia. Though named for its well-known renal effect, ANP has been demonstrated to acutely increase vascular permeability in vivo. Experimentally, this phenomenon was associated with a marked shedding of the endothelial glycocalyx, at least for supraphysiological intravascular concentrations. This study investigates the impact and mechanism of action of physiological doses of ANP and related peptides on the vascular barrier. In isolated guinea pig hearts, prepared and perfused in a modified Langendorff mode with and without the intravascular presence of the colloid hydroxyethyl starch (HES), we measured functional changes in vascular permeability and glycocalyx shedding related to intracoronary infusion of physiological concentrations of A-, B- and C-type natriuretic peptide (ANP, BNP and CNP). Significant coronary venous washout of glycocalyx constituents (syndecan-1 and heparan sulfate) was observed. As tested for ANP, this effect was positively related to the intracoronary concentration. Intravascular shedding of the glycocalyx was morphologically confirmed by electron microscopy. Also, functional vascular barrier competence decreased, as indicated by significant increases in transudate formation and HES extravasation. Ortho-phenanthroline, a non-specific inhibitor of matrix metalloproteases, was able to reduce ANP-induced glycocalyx shedding. These findings suggest participation of natriuretic peptides in pathophysiological processes like heart failure, inflammation or sepsis. Inhibition of metalloproteases might serve as a basis for future therapeutical options.


Assuntos
Fator Natriurético Atrial/metabolismo , Permeabilidade Capilar , Células Endoteliais/metabolismo , Glicocálix/metabolismo , Peptídeo Natriurético Encefálico/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/ultraestrutura , Exsudatos e Transudatos/metabolismo , Glicocálix/efeitos dos fármacos , Glicocálix/ultraestrutura , Cobaias , Derivados de Hidroxietil Amido/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloproteinases da Matriz/metabolismo , Microscopia Eletrônica , Perfusão , Fenantrolinas/farmacologia , Substitutos do Plasma/farmacologia , Fatores de Tempo
4.
Biol Cell ; 105(7): 289-303, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23521563

RESUMO

BACKGROUND INFORMATION: Exosomes are small RNA- and protein-containing extracellular vesicles (EVs) that are thought to mediate hetero- and homotypic intercellular communication between normal and malignant cells.Tumour-derived exosomes are believed to promote re-programming of the tumour-associated stroma to favour tumour growth and metastasis. Currently, exosomes have been intensively studied in carcinomas. However, little is known about their existence and possible role in sarcomas. RESULTS: Here, we report on the identification of vesicles with exosomal features derived from Ewing's sarcoma(ES), the second most common soft-tissue or bone cancer in children and adolescents. ES cell line-derived EV shave been isolated by ultracentrifugation and analysed by flow-cytometric assessment of the exosome-associated proteins CD63 and CD81 as well as by electron microscopy. They proved to contain ES-specific transcripts including EWS-FLI1, which were suitable for the sensitive detection of ES cell line-derived exosomes by qRT-PCRin a pre-clinical model for patient plasma. Microarray analysis of ES cell line-derived exosomes revealed that they share a common transcriptional signature potentially involved in G-protein-coupled signalling, neurotransmitter signalling and stemness. CONCLUSIONS: In summary, our results imply that ES-derived exosomes could eventually serve as biomarkers for minimal residual disease diagnostics in peripheral blood and prompt further investigation of their potential biological role in modification of the ES-associated microenvironment


Assuntos
Exossomos/metabolismo , Proteínas de Fusão Oncogênica/sangue , Proteína Proto-Oncogênica c-fli-1/sangue , Proteína EWS de Ligação a RNA/sangue , Sarcoma de Ewing/sangue , Neoplasias de Tecidos Moles/sangue , Tetraspanina 28/sangue , Tetraspanina 30/sangue , Biomarcadores/sangue , Linhagem Celular Tumoral , Exossomos/genética , Humanos , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/genética , Neoplasias de Tecidos Moles/diagnóstico , Neoplasias de Tecidos Moles/genética , Tetraspanina 28/genética , Tetraspanina 30/genética
5.
EMBO J ; 30(21): 4356-70, 2011 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-22009199

RESUMO

Mitochondria are organelles with a complex architecture. They are bounded by an envelope consisting of the outer membrane and the inner boundary membrane (IBM). Narrow crista junctions (CJs) link the IBM to the cristae. OMs and IBMs are firmly connected by contact sites (CS). The molecular nature of the CS remained unknown. Using quantitative high-resolution mass spectrometry we identified a novel complex, the mitochondrial contact site (MICOS) complex, formed by a set of mitochondrial membrane proteins that is essential for the formation of CS. MICOS is preferentially located at the CJs. Upon loss of one of the MICOS subunits, CJs disappear completely or are impaired, showing that CJs require the presence of CS to form a superstructure that links the IBM to the cristae. Loss of MICOS subunits results in loss of respiratory competence and altered inheritance of mitochondrial DNA.


Assuntos
Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Sítios de Ligação/fisiologia , DNA Mitocondrial/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Mitocôndrias/química , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/fisiologia , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/fisiologia , Organismos Geneticamente Modificados , Ligação Proteica/genética , Ligação Proteica/fisiologia , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
6.
Cancer Immunol Immunother ; 60(5): 639-48, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21293856

RESUMO

In order to grow within an immunocompetent host, tumour cells have evolved various strategies to cope with the host's immune system. These strategies include the downregulation of surface molecules and the secretion of immunosuppressive factors like IL-10 and PGE2 that impair the maturation of immune effector cells, among other mechanisms. Recently, tumour exosomes (TEX) have also been implicated in tumour-induced immune suppression as it has been shown that TEX can induce apoptosis in T lymphocytes. In this study, we extend our knowledge about immunosuppressive features of these microvesicles in that we show that TEX efficiently bind and sequester tumour-reactive antibodies and dramatically reduce their binding to tumour cells. Moreover, we demonstrate that this antibody sequestration reduces the antibody-dependent cytotoxicity by immune effector cells, which is among the most important anti-tumour reactions of the immune system and a significant activity of therapeutic antibodies. Taken together, these data point to the fact that tumour-derived exosomes interfere with the tumour-specific function of immune cells and constitute an additional mechanism how tumours escape from immune surveillance.


Assuntos
Anticorpos Antineoplásicos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Neoplasias da Mama/imunologia , Exossomos/imunologia , Evasão Tumoral , Antígenos de Neoplasias/imunologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Molécula de Adesão da Célula Epitelial , Exossomos/metabolismo , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Microscopia Eletrônica , Neoplasias Ovarianas/imunologia , Receptor ErbB-2/imunologia
7.
Cardiovasc Res ; 90(1): 157-64, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21106562

RESUMO

AIMS: Oxidative processes and vascular inflammation underlying atherosclerosis lead to an accumulation of lysophosphatidic acid (LPA) molecules in the atheromatous intima. LPA, a platelet-activating component of human atherosclerotic plaques, possibly contributes to atherothrombus formation after plaque rupture. Human platelets express mRNA for the G protein-coupled receptors LPA1₋7 that derive from megakaryocytes. The aim of our study was to identify the functional LPA receptor(s) in human platelets by silencing individual LPA receptors in megakaryocytic (MK) cells. METHODS AND RESULTS: We studied shape change of two human MK cell lines (Meg-01, Dami) by turbidometry, phase-contrast and scanning electron microscopy. They showed upon LPA stimulation a rapid, Rho-kinase-mediated shape change similar to that of human platelets. By qRT-PCR analysis we found expression of LPA1₋7 in both cell lines; LPA4 and LPA5 were the most abundant receptor transcripts. In both Meg-01 and Dami cells, the rank order of activation by LPA species was similar to that found in platelets: alkyl-LPA 18:1 > alkyl-LPA 16:0 > acyl-LPA 18:1 >> alkyl-LPA 18:0. Knock-down of individual LPA receptors by siRNA showed that LPA-mediated activation of MK cells was mediated by LPA5, but not by LPA1₋4,6,7. Importantly, we found that human atherosclerotic plaque and lipid-rich core induced shape change of Dami cells, and that this effect was inhibited after LPA5 silencing. CONCLUSIONS: Our findings indicate that LPA5 mediates LPA-induced shape change of MK cells and support its involvement in atherosclerotic plaque and lipid-rich core-mediated platelet activation. This receptor could be an attractive novel target for antithrombotic therapy.


Assuntos
Aterosclerose/metabolismo , Forma Celular , Lisofosfolipídeos/metabolismo , Megacariócitos/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Aterosclerose/patologia , Linhagem Celular , Humanos , Metabolismo dos Lipídeos , Megacariócitos/patologia , Microscopia Eletrônica de Varredura , Nefelometria e Turbidimetria , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transfecção , Quinases Associadas a rho/metabolismo
8.
J Vasc Res ; 48(3): 219-26, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21088428

RESUMO

BACKGROUND: Heterogeneity of vascular permeability has been suggested for the coronary system. Whereas arteriolar and capillary segments are tight, plasma proteins pass readily into the interstitial space at venular sites. Fittingly, lymphatic fluid is able to coagulate. However, heart tissue contains high concentrations of tissue factor, presumably enabling bleeding to be stopped immediately in this vital organ. The distribution of pro- and anti-coagulatively active factors in human heart tissue has now been determined in relation to the types of microvessels. METHODS AND RESULTS: Samples of healthy explanted hearts and dilated cardiomyopathic hearts were immunohistochemically stained. Albumin was found throughout the interstitial space. Tissue factor was packed tightly around arterioles and capillaries, whereas the tissue surrounding venules and small veins was practically free of this starter of coagulation. Thrombomodulin was present at the luminal surface of all vessel segments and especially at venular endothelial cell junctions. Its product, the anticoagulant protein C, appeared only at discrete extravascular sites, mainly next to capillaries. These distribution patterns were basically identical in the healthy and diseased hearts, suggesting a general principle. CONCLUSIONS: Venular extravasation of plasma proteins probably would not bring prothrombin into intimate contact with tissue factor, avoiding interstitial coagulation in the absence of injury. Generation of activated protein C via thrombomodulin is favored in the vicinity of venular gaps, should thrombin occur inside coronary vessels. This regionalization of distribution supports the proposed physiological heterogeneity of the vascular barrier and complies with the passage of plasma proteins into the lymphatic system of the heart.


Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Permeabilidade Capilar , Cardiomiopatia Dilatada/sangue , Vasos Coronários/metabolismo , Miocárdio/metabolismo , Albumina Sérica/metabolismo , Adulto , Arteríolas/metabolismo , Capilares/metabolismo , Estudos de Casos e Controles , Humanos , Imuno-Histoquímica , Lactente , Sistema Linfático/metabolismo , Proteína C/metabolismo , Trombomodulina/metabolismo , Tromboplastina/metabolismo , Vênulas/metabolismo
9.
Histol Histopathol ; 26(2): 177-84, 2011 02.
Artigo em Inglês | MEDLINE | ID: mdl-21154231

RESUMO

The apocrine secretory mechanism is a mode of secretion by which the apical part of the cell cytoplasm is pinched off, which leads to the formation of an aposome. The distinct mechanism of formation and decapitation of the aposome is not well investigated. Only few proteins are known that are involved in this secretory mechanism. We studied the human axillary apocrine gland and looked at proteins associated with cytokinesis, a process that is comparable to the pinching-off mechanism of apocrine glandular cells. By immunohistochemistry, we detected actin, myosin II, cytokeratin 7 and 19, α- and ß-tubulin, anillin, cofilin, syntaxin 2, vamp8/endobrevin and septin 2. In highly active glandular cells, these proteins are located at the base of the apical protrusion when the aposome is in the process of being released or are concentrated in the cap of the apical protrusion. These findings demonstrate new insights on apocrine secretory mechanisms and point to similarities to the terminal step of cytokinesis, which is regulated by a SNARE-mediated membrane fusion event.


Assuntos
Glândulas Apócrinas/metabolismo , Secreções Corporais/metabolismo , Proteínas/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Glândulas Apócrinas/anatomia & histologia , Axila , Biomarcadores/metabolismo , Citocinese/fisiologia , Citoesqueleto/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
Biomaterials ; 31(26): 6692-700, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20619783

RESUMO

The increasing interest in biomedical applications of semiconductor quantum dots (QDs) is closely linked to the use of surface modifications to target specific sites of the body. The immense surface area of vascular endothelium is a possible interaction platform with systemically administered QDs. Therefore, the aim of this study was to investigate the microvascular distribution of neutral, cationic, and anionic QDs in vivo. QDs with carboxyl-, amine- and polyethylene glycol surface coatings were injected into the blood circulation of mice. In vivo microscopy of the cremaster muscle, two-photon microscopy of skeletal and heart muscle, as well as quantitative fluorescence measurements of blood, excreta, and tissue samples were performed. Transmission electron microscopy was used to detect QDs at the cellular level. The in vitro association of QDs with cultured endothelial cells was investigated by flow cytometry and confocal microscopy. Anionic QDs exhibited a very low residence time in the blood stream, preferably accumulated in organs with a prominent mononuclear phagocytic component, but were also found in other tissues with low phagocytic properties where they were predominantly associated with capillary endothelium. This deposition behavior was identified as a new, phagocyte-independent principle contributing to the rapid clearance of anionic QDs from the circulation.


Assuntos
Endotélio Vascular/metabolismo , Pontos Quânticos , Animais , Ânions , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Hemodinâmica , Injeções Intra-Arteriais , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia , Microvasos/citologia , Microvasos/metabolismo , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/ultraestrutura , Fótons , Distribuição Tecidual
11.
Shock ; 34(2): 133-9, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20634656

RESUMO

Adhesion of polymorphonuclear neutrophils (PMN) to coronary endothelium is a key event for cardiac ischemia/reperfusion injury. Adhesion molecules are normally harbored within the glycocalyx, clothing every healthy vascular endothelium, but shed by ischemia/reperfusion. Our aim was to show whether protection of the glycocalyx with either hydrocortisone or antithrombin can reduce postischemic leukocyte adhesion. Isolated guinea pig hearts, perfused with Krebs-Henseleit buffer, were subjected to 20 min of warm (37 degrees C) no-flow ischemia and consecutive 10 min of reperfusion, either in the absence or presence of hydrocortisone (10 microg/mL) or antithrombin (1 U/mL). An intracoronary bolus of 3 x 10 PMN was applied at the end of reperfusion but without prior contact to the drugs. The sequestration of PMN was calculated from the difference between coronary input and output of cells. Expression of the integrin CD11b on PMN was measured before and after coronary passage. Ischemia/reperfusion induced severe degradation of the glycocalyx (coronary venous syndecan-1 release, 171 +/- 15 ng/g heart vs. basal, 19 +/- 2 ng/g; heparan sulfate, 5.27 +/- 0.28 microg/g vs. basal, 0.26 +/- 0.06 microg/g) and increased PMN adhesion (38.1% +/- 3.5% vs. basal, 11.7% +/- 3.1%). Hydrocortisone and antithrombin both not only reduced glycocalyx shedding (syndecan-1 release, 34 +/- 6 ng/g and 26 +/- 5 ng/g; heparan sulfate, 1.96 +/- 0.24 microg/g and 1.28 +/- 0.2 microg/g, respectively), but also PMN adhesion (17.3% +/- 2.2% and 25.4% +/- 3.3%, respectively) after ischemia/reperfusion. Electron microscopy revealed a mostly intact coronary glycocalyx after pretreatment with either drug. Activation of PMN upon coronary passage was not influenced. Preservation of the glycocalyx mitigates postischemic PMN adhesion. Preconditioning with either hydrocortisone or antithrombin should, thus, alleviate vascular leakage, tissue edema, and inflammation.


Assuntos
Antitrombinas/farmacologia , Adesão Celular/efeitos dos fármacos , Glicocálix/efeitos dos fármacos , Hidrocortisona/farmacologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Neutrófilos/fisiologia , Animais , Antitrombinas/uso terapêutico , Antígeno CD11b/biossíntese , Glicocálix/metabolismo , Glicocálix/ultraestrutura , Cobaias , Coração/efeitos dos fármacos , Humanos , Hidrocortisona/uso terapêutico , Técnicas In Vitro , Masculino , Microscopia Eletrônica , Neutrófilos/efeitos dos fármacos
12.
Eur J Endocrinol ; 162(4): 813-8, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20133447

RESUMO

CONTEXT: Only occasionally, endocrine-active tumors develop directly from hepatic tissue, and may lead to paraneoplastic syndromes (PNS). PNS mostly accompany malignancy of adulthood and are exceedingly rare in children. PATIENT: A girl aged 6 years and 9 months presented with a 2-month history of rapidly progressive weight gain, abdominal distension, and polyuria/pollakiuria accompanied by short episodes of abdominal pain. She showed the typical clinical features of Cushing's syndrome and a huge hepatic mass. An abdominal computed tomography (CT) scan revealed a large liver tumor. Blood glucose and serum calcium were greatly elevated. DESIGN AND OBJECTIVE: Case report describing the causative relationship of the clinical findings. METHODS: Physical examination; ultrasound of the abdomen; CT scan of the abdomen and the chest; conventional X-rays; routine hematology; blood chemistry and multiple parameters of calcium and phosphorus metabolism; multisteroid analysis in serum and urine; adrenocortical stimulation and suppression tests; histopathological assessment of the resected tumor; immunohistochemistry for ACTH, beta-endorphin, corticotrophin-releasing hormone (CRH), and PTH-related peptide (PTHrP); electron microscopy of tumor cells; ACTH and CRH extraction from the tumor tissue; and clinical follow-up for more than 20 years. RESULTS: Giant hepatoblastoma (HB; approximately 1000 ml volume) of the right lobe of the liver with combined ectopic ACTH syndrome and PTHrP-induced tumor-associated hypercalcemia. Wide local excision and polychemotherapy led to complete reversal of the paraneoplastic phenotype. CONCLUSIONS: This is the first report of an endocrine-active HB causing both Cushing's syndrome and PTHrP-related 'humoral hypercalcemia of malignancy'. This information should be added to the well-known beta-human chorionic gonadotropin-related paraneoplastic effects of HB in children.


Assuntos
Síndrome de ACTH Ectópico/etiologia , Hepatoblastoma/complicações , Hipercalcemia/etiologia , Neoplasias Hepáticas/complicações , Proteína Relacionada ao Hormônio Paratireóideo/sangue , Síndrome de ACTH Ectópico/sangue , Síndrome de ACTH Ectópico/terapia , Criança , Feminino , Hepatoblastoma/sangue , Hepatoblastoma/terapia , Humanos , Hipercalcemia/sangue , Hipercalcemia/terapia , Imuno-Histoquímica , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/terapia
13.
Cardiovasc Pathol ; 19(6): 353-60, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19747851

RESUMO

BACKGROUND: Increased proliferation, mitigated apoptosis, and recruitment of primarily extravascular cells to injured vessels are important processes during neointima formation. Therefore, the goal of this study was to assess the spatiotemporal balance between proliferation and apoptosis and the influence of apoptosis on the survival of primarily extravascular cells in in-stent neointima. METHODS: Minipigs underwent stent implantation to abdominal aortic segments. At Days 1, 7, 14, 30, 60, and 90 after injury, arterial cross sections were analyzed by TUNEL assay to detect apoptotic cells. For immunohistochemical detection of Ki67+/proliferating cell nuclear antigen (PCNA)+ proliferative, caspase3+ apoptotic, S100+/fascin+ dendritic, GFAP+ neural crest-derived cells and CD14+ monocytes/macrophages, the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method was used. RESULTS: In the incipient cell-rich neointima, both frequency of proliferation and apoptosis was maximal (Ki67, 28.5±2.2%; PCNA, 25.4±3.8%; TUNEL, 8.6±0.4%; caspase3, 7.9±4.3%). With time, parallel to the decline in the neointima cellularity, signaling for proliferation and apoptosis decreased. Throughout the time course of neointima development, the apoptotic activity was detected in primarily extravascular cells. CONCLUSIONS: Imbalance between proliferation and apoptosis and recruitment of dendritic cells, monocytes/macrophages, and neural crest-derived cells to the injured vessels may partly explain the formation of the hypercellular in-stent neointima. Herein, apoptosis is an important factor that regulates survival of primarily extravascular neointimal cells.


Assuntos
Angioplastia com Balão/instrumentação , Aorta Abdominal/patologia , Apoptose , Proliferação de Células , Stents , Túnica Íntima/patologia , Animais , Aorta Abdominal/metabolismo , Biomarcadores/metabolismo , Sobrevivência Celular , Hiperplasia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Masculino , Suínos , Porco Miniatura , Fatores de Tempo , Túnica Íntima/metabolismo
14.
Circ Res ; 104(11): 1313-7, 2009 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-19423849

RESUMO

Potter and Damiano recently assessed the hydrodynamic dimensions of the endothelial glycocalyx in vivo (mouse cremaster muscle venules) and in vitro (human umbilical vein and bovine aorta endothelium cultured in perfused microchannels) using fluorescent microparticle image velocimetry (Circ Res. 2008;102:770-776). Great discrepancy was observed, the glycocalyx presenting a zone of interaction extending approximately 0.52 microm into the vessel lumen in vivo, but only 0.02 to 0.03 microm from cultured cells. In an accompanying editorial, Barakat cautioned that the difference in hydrodynamic interaction did not allow one to conclude that the cultured cells totally lack a physical cell surface layer capable of mechanotransduction (Circ Res. 2008;102:747-748). To stabilize the glycocalyx for electron microscopic investigation, we perfusion-fixed 6 human umbilical veins and confluent and nonconfluent cultures (5 each) of human umbilical vein endothelial cells (HUVECs) with lanthanum/glutaraldehyde solution. Ex vivo, the thickness of glycocalyx of umbilical vein endothelium averaged 878 nm. HUVECs in vitro presented a glycocalyx with a dense-zone thickness of only 29.4 nm, plus sparse filaments reaching out on average to 118 nm, there being no difference between the nonconfluent and confluent cells. Immunohistology demonstrated the presence of heparan sulfates and syndecan-1, main constituents of the glycocalyx, both ex vivo and in vitro. These results support the observed discrepancy between glycocalyx thickness in vivo and in vitro, now for one and the same type of human cell. The presence of heparan sulfates and syndecan-1 also on cultured cells may explain why mechanotransduction phenomena can be observed even with a nonmature glycocalyx.


Assuntos
Endotélio Vascular/fisiologia , Glicocálix/fisiologia , Veias Umbilicais/fisiologia , Técnicas de Cultura de Células/métodos , Células Cultivadas , Endotélio Vascular/ultraestrutura , Eritrócitos/fisiologia , Eritrócitos/ultraestrutura , Glicocálix/ultraestrutura , Heparitina Sulfato/análise , Humanos , Microscopia Eletrônica , Sindecana-1/análise , Veias Umbilicais/ultraestrutura
15.
Transplantation ; 87(7): 956-65, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19352113

RESUMO

BACKGROUND: Major causes of death after heart transplantation are right ventricular pump failure and, chronically, cardiac allograft vasculopathy. Traditional preservation techniques focus on immediate cardioplegia, without particularly considering vascular demands. Recently, the endothelial surface layer, composed of the endothelial glycocalyx and plasma proteins, was discovered to play a major role in vascular barrier function, edema formation, and leukocyte-to-endothelial interaction. The impact of augmenting a traditional preservation solution with plasma colloid albumin was therefore investigated. METHODS: Guinea pig hearts underwent cold ischemic storage for 4 hr using Bretschneider's solution (histidine-tryptophan-ketoglutarate [HTK]) without and with augmentation with 1 g% human albumin. After reperfusion, intracoronary adhesion of polymorphonuclear granulocytes, edema formation, left and right heart performance of pressure-to-volume work, and glycocalyx shedding were assessed. RESULTS: Intracoronary retention of leukocytes was doubled in the traditional group (36.4+/-6.6%), whereas it remained at basal values after albumin preservation (23.5+/-2.4%; P<0.05). Addition of albumin to HTK significantly decreased edema formation (wet to dry weight ratio 6.9+/-0.1 vs. 7.2+/-0.2; P<0.05). Although left heart performance was comparable, right heart cardiac output was doubled in hearts having received HTK containing albumin versus HTK alone (94+/-14 vs. 50+/-11 mL/min/g; P<0.05). Glycocalyx shedding was significantly reduced when the hearts were stored under albumin protection. CONCLUSIONS: Augmenting HTK with human albumin improves endothelial integrity and heart performance after 4 hr cold ischemia, because of a marked protection of the endothelial glycocalyx. For the prevention of acute and chronic graft failure, the glycocalyx might represent a new target.


Assuntos
Transplante de Coração/efeitos adversos , Coração/efeitos dos fármacos , Isquemia Miocárdica/tratamento farmacológico , Miocárdio/imunologia , Albumina Sérica/uso terapêutico , Animais , Débito Cardíaco , Adesão Celular , Vasos Coronários/fisiologia , Vasos Coronários/fisiopatologia , Edema/etiologia , Endotélio Vascular/fisiopatologia , Glucose , Cobaias , Humanos , Manitol , Modelos Animais , Neutrófilos/fisiologia , Preservação de Órgãos/métodos , Soluções para Preservação de Órgãos , Cloreto de Potássio , Procaína , Falha de Tratamento
16.
Cardiovasc Res ; 83(2): 388-96, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19307232

RESUMO

AIMS: Antithrombin is an important inhibitor of the coagulation system, additionally exerting specific anti-inflammatory effects on endothelial cells. Healthy vascular endothelium is coated by the endothelial glycocalyx, diminution of which increases capillary permeability, e.g. after ischaemia. Antithrombin is known to infiltrate the glycocalyx, binding to glycosaminoglycans, and to preserve the glycocalyx after application tumour necrosis factor-alpha. We investigated the influence of antithrombin on glycocalyx subjected to ischaemia/reperfusion. METHODS AND RESULTS: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer (KHB). Antithrombin was applied to achieve physiological levels (1 U/mL) before inducing 20 min of ischaemia (37 degrees C). Hearts were reperfused for 20 min at constant flow (baseline perfusion pressure 70 cmH(2)O) with KHB or KHB plus 2 g% hydroxyethyl starch (130 kDa). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Post-ischaemic coronary release of syndecan-1 and heparan sulfate was quantified by ELISA. Hearts were perfusion-fixed to visualize the glycocalyx by electron microscopy. Ischaemia/reperfusion caused degradation of the glycocalyx, enhanced coronary perfusion pressure, and increased vascular permeability. Antithrombin significantly reduced post-ischaemic glycocalyx shedding, coronary perfusion pressure, coronary leak, and tissue oedema formation compared to untreated hearts. Additional application of colloid augmented these actions of antithrombin. Electron microscopy revealed a mostly intact glycocalyx after antithrombin treatment. CONCLUSION: Antithrombin preserves the endothelial glycocalyx, sustaining the vascular barrier function and reducing interstitial oedema. The potentiated effect of colloid in these hearts suggests that the prevention of shedding should be of functional benefit also in vivo.


Assuntos
Antitrombinas/farmacologia , Vasos Coronários/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Glicocálix/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Animais , Permeabilidade Capilar/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/ultraestrutura , Edema Cardíaco/patologia , Edema Cardíaco/prevenção & controle , Células Endoteliais/metabolismo , Células Endoteliais/ultraestrutura , Glicocálix/metabolismo , Glicocálix/ultraestrutura , Cobaias , Derivados de Hidroxietil Amido/farmacologia , Imuno-Histoquímica , Técnicas In Vitro , Ácido Láctico/metabolismo , Masculino , Microscopia Eletrônica , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Perfusão , Substitutos do Plasma/farmacologia , Purinas/metabolismo , Ácido Úrico/metabolismo
17.
Basic Res Cardiol ; 104(1): 78-89, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18836678

RESUMO

BACKGROUND: Healthy vascular endothelium is clothed by the endothelial glycocalyx. This structure plays a key role in the regulation of inflammation and vascular permeability and is known to be degraded by ischemic and inflammatory stress. Our aim was to show whether hydrocortisone and antithrombin stabilize the glycocalyx and, therefore, the vascular barrier, against damage induced by the inflammatory stimulus TNF-alpha, thus improving the cardio-vascular situation. METHODS: Isolated guinea pig hearts were perfused with Krebs-Henseleit buffer for 20 min at constant flow (baseline perfusion pressure 70 cmH(2)O). Hydrocortisone in a stress dose (10 microg/ml) or antithrombin in a physiological dose (1 U/ml) were then applied for 15 min before infusion of TNF-alpha (4 ng/ml, 10 min). Coronary net fluid filtration was assessed directly by measuring transudate formation on the epicardial surface. Hearts were perfusion-fixed to visualize the glycocalyx. RESULTS: TNF-alpha induced severe degradation of the glycocalyx, increased coronary resistance, heightened vascular leak and permeability to hydroxyethyl starch and caused mast-cell degranulation. Hydrocortisone and antithrombin both reduced all of these effects. Electron microscopy revealed a mostly intact glycocalyx after treatment with either drug. CONCLUSIONS: Both hydrocortisone and antithrombin clearly preserve the endothelial glycocalyx in the face of inflammatory degradation initiated by TNF-alpha, however, with different mechanisms. This is an important new facet in the pathophysiology and therapy of sepsis, since preservation of the glycocalyx should help prevent vasoconstriction, tissue edema as well as leukocyte and platelet adhesion, thus mitigating inflammation and tissue hypoxia.


Assuntos
Antitrombinas/farmacologia , Endotélio Vascular/fisiologia , Glicocálix/fisiologia , Hidrocortisona/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Endotélio Vascular/efeitos dos fármacos , Exsudatos e Transudatos/fisiologia , Glicocálix/química , Glicocálix/efeitos dos fármacos , Cobaias , Coração/efeitos dos fármacos , Coração/fisiologia , Histamina/análise , Humanos , Derivados de Hidroxietil Amido/análise , Isquemia/fisiopatologia , Lactatos/análise , Purinas/análise , Valores de Referência , Ácido Úrico/análise
18.
Buenos Aires; Médica Panamericana; 2 ed; 2009. 676 p. ilus, tab, graf.
Monografia em Espanhol | MINSALCHILE | ID: biblio-1545303
19.
Crit Care ; 12(3): R73, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18518977

RESUMO

INTRODUCTION: Postischemic injury to the coronary vascular endothelium, in particular to the endothelial glycocalyx, may provoke fluid extravasation. Shedding of the glycocalyx is triggered by redox stress encountered during reperfusion and should be alleviated by the radical scavenger nitric oxide (NO). The objective of this study was to investigate the effect of exogenous administration of NO during reperfusion on both coronary endothelial glycocalyx and vascular integrity. METHODS: Isolated guinea pig hearts were subjected to 15 minutes of warm global ischemia followed by 20 minutes of reperfusion in the absence (Control group) and presence (NO group) of 4 microM NO. In further experiments, the endothelial glycocalyx was enzymatically degraded by means of heparinase followed by reperfusion without (HEP group) and with NO (HEP+NO group). RESULTS: Ischemia and reperfusion severely damaged the endothelial glycocalyx. Shedding of heparan sulfate and damage assessed by electron microscopy were less in the presence of NO. Compared with baseline, coronary fluid extravasation increased after ischemia in the Control, HEP, and HEP+NO groups but remained almost unchanged in the NO group. Tissue edema was significantly attenuated in this group. Coronary vascular resistance rose by 25% to 30% during reperfusion, but not when NO was applied, irrespective of the state of the glycocalyx. Acute postischemic myocardial release of lactate was comparable in the four groups, whereas release of adenine nucleotide catabolites was reduced 42% by NO. The coronary venous level of uric acid, a potent antioxidant and scavenger of peroxynitrite, paradoxically decreased during postischemic infusion of NO. CONCLUSION: The cardioprotective effect of NO in postischemic reperfusion includes prevention of coronary vascular leak and interstitial edema and a tendency to forestall both no-reflow and degradation of the endothelial glycocalyx.


Assuntos
Endotélio Vascular/metabolismo , Sequestradores de Radicais Livres/farmacologia , Glicocálix/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Óxido Nítrico/farmacologia , Nucleotídeos de Adenina/metabolismo , Animais , Vasos Coronários/metabolismo , Endotélio Vascular/ultraestrutura , Cobaias , Heparina Liase/metabolismo , Heparitina Sulfato/metabolismo , Ácido Láctico/metabolismo , Masculino , Microscopia Eletrônica , Reperfusão Miocárdica , Ácido Úrico/sangue
20.
J Heart Valve Dis ; 17(2): 187-93, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18512489

RESUMO

BACKGROUND AND AIM OF THE STUDY: Although degenerative calcific aortic valve stenosis is the most common valvular disease among the elderly, neither the etiology underlying the condition nor degeneration of the bioprostheses is yet fully understood. The study aim was to assess the expression profile of those OPG/RANKL/RANK-system determinants known to act as key regulators of bone metabolism and the immune system in calcific aortic valve stenosis and porcine aortic bioprostheses. METHODS: Valve probes from a total of 69 patients (41 with end-stage aortic stenosis, 11 with mild-to-moderate aortic sclerosis, 17 with degenerative porcine aortic bioprostheses) were explanted either during surgery or at autopsy. The presence and localization of OPG, RANKL, RANK and NF-kappaB were analyzed by immunostaining and morphometry. RESULTS: The majority of stenotic and sclerotic valves exhibited cell-bound signals of OPG, RANKL, RANK and NF-kappaB, while bioprostheses showed only sparse signaling. As key findings, the percentage of cells labeled by OPG, RANK and NF-kappaB was increased in sclerotic valves compared with stenotic valves (each p < 0.001), whereas the frequency of RANKL was higher in stenotic compared to sclerotic valves (p < 0.001). As a consequence, the OPG/RANKL ratio was decreased in stenotic (0.83) compared to sclerotic valves (20.2). CONCLUSION: The differential expression profile of specific members of the OPG/RANKL/RANK axis suggests an involvement of their determinants in native valve calcification, but not in the degeneration of porcine bioprostheses. Thus, these mediators of bone homeostasis may represent new targets for a more specified prevention and/or therapy of native aortic stenosis.


Assuntos
Estenose da Valva Aórtica/metabolismo , Próteses Valvulares Cardíacas , Osteoprotegerina/metabolismo , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Idoso , Estenose da Valva Aórtica/patologia , Bioprótese , Calcinose/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Estudos Retrospectivos
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