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1.
J Physiol Sci ; 62(1): 63-9, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22109690

RESUMO

In response to atherogenic stimuli, blood monocytes transmigrate across the vascular endothelium not only through endothelial cell-cell junctions (para-cellular) but also through endothelial cells themselves (trans-cellular). The molecular mechanism of the latter is mostly unknown, because it rarely happens, especially in vitro. Although many reports have recognized trans-cellular migration from snapshot images of leukocytes halfway across the endothelium at non-junctional locations, it often produces a false-positive result, because some leukocytes that initiate trans-cellular migration withdraw and return to the apical endothelial surface. Thus, analyzing the entire process is essential. In this study, complete monocyte trans-cellular migration was successfully captured for live cells, with simultaneous visualization of endothelial PECAM-1. We suggest the possible existence of both PECAM-1-related migration at peri-junctional sites and PECAM-1-unrelated migration at sites remote from junctions. This is the first report to describe the entire process of monocyte trans-cellular migration for live cells and its relationship with endothelial PECAM-1.


Assuntos
Movimento Celular/fisiologia , Endotélio Vascular/citologia , Monócitos/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Linhagem Celular , Células Endoteliais/citologia , Humanos , Junções Intercelulares
2.
Int J Cardiol ; 149(2): 232-239, 2011 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-21190742

RESUMO

BACKGROUND: Although the importance of monocyte trans-endothelial migration in early atherogenesis is well recognized, it is unclear whether and how one transmigration event affects endothelium to facilitate subsequent ones. In this study, we tested the hypothesis that monocyte transmigration alters endothelial junctional organization to facilitate subsequent transmigration. METHODS AND RESULTS: When human monocytes were added twice at intervals of ≈30 min to IL-1beta-prestimulated human umbilical vein endothelial cells in vitro, significant augmentation of transmigration was observed at the second addition (≈1.5-fold, analyzed from a total of 231 monocytes in 3 experiments). Endothelial surface expressions of two major junctional molecules, PECAM-1 and VE-cadherin, increased and decreased respectively, in response to monocyte addition, which could facilitate subsequent transmigration. To further investigate spatiotemporal dynamics of the increasing molecule, PECAM-1, we constructed a PECAM-1-GFP expression system and found that monocyte transmigration induced local accumulation of endothelial PECAM-1 around the transmigration spot, which was followed by transmigration of subsequent monocyte around the same location. Detailed analysis revealed that within the defined region around one transmigration event, 50% of later transmigrating monocytes used the same or similar location as the previous one (10 out of 20 transmigrating monocytes in 11 experiments). CONCLUSIONS: These findings show that monocyte trans-endothelial migration alters endothelial junctional organization to a more monocyte-permeable state (increased PECAM-1 and decreased VE-cadherin), resulting in the augmented transmigratory activity at a later stage. This positive feedback mechanism is partially associated with monocyte transmigration-induced local accumulation of endothelial PECAM-1, which promotes transmigration of following monocytes at the same location.


Assuntos
Antígenos CD/biossíntese , Caderinas/biossíntese , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Junções Intercelulares/metabolismo , Monócitos/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Migração Transendotelial e Transepitelial/fisiologia , Caderinas/antagonistas & inibidores , Células Cultivadas , Regulação para Baixo/fisiologia , Células Endoteliais/citologia , Endotélio Vascular/citologia , Humanos , Junções Intercelulares/fisiologia , Monócitos/citologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Regulação para Cima/fisiologia
3.
J Mech Behav Biomed Mater ; 2(2): 173-85, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19627821

RESUMO

Previous studies on the intracellular force balance that forms the adherent cell structure have paid much attention to the mechanical behavior of cells seen in the horizontal project plane. By contrast, there are only few quantitative considerations on that in the vertical plane. Particularly, the contribution of the nucleus to the bearing of the vertical cell structure remains unclear. Here, we investigated the determinant of the vertical cell morphology from experimental and numerical approaches. The effect of cytoskeleton-affecting agents on the vascular endothelial cell height, as a measure of the vertical force balance, was examined by atomic force microscope indentation, demonstrating that actin depolymerization caused an increase in cell height. In contrast, disruption of microtubules lowered the cell height, whereas their stabilization elevated the cell plasma membrane. Time-lapse microscopy showed that intracellular vesicles moved radially outward after the microtubule disruption, together with an enlargement of the nuclear area in the project plane, that is probably associated with the decrease in cell height. Finite element analyses employing a 3D model were carried out to interpret the experimental results and examine potent parameters (such as prestress, elastic modulus, and Poisson's ratio) that affect vertical cell morphology. How the prestress in subcellular components influences cells subjected to extracellular tensile forces was also examined. These results indicate that the nuclear/cytoplasmic mechanical properties and degrees of prestress determine the vertical section structure of adhering cells.


Assuntos
Células Endoteliais/citologia , Análise de Elementos Finitos , Modelos Biológicos , Animais , Fenômenos Biomecânicos , Bovinos , Adesão Celular , Forma do Núcleo Celular , Citoesqueleto/metabolismo , Células Endoteliais/metabolismo , Microscopia , Microscopia de Força Atômica , Movimento , Estresse Mecânico , Fatores de Tempo
4.
Bone ; 43(1): 19-24, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18424248

RESUMO

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.


Assuntos
Linhagem da Célula , Osteoblastos/citologia , Osteócitos/citologia , Animais , Embrião de Galinha , Microscopia Confocal , Oligopeptídeos/farmacologia
5.
Atherosclerosis ; 194(2): e9-17, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17194459

RESUMO

It is poorly understood how oxidized LDL (oxLDL) promotes monocyte dynamics in transendothelial migration (TEM) in atherogenesis. We developed an in vitro 3D-live-single cell TEM assay system with subendothelial oxLDL embedded in ultra-thin collagen gels, mimicking subendothelial oxLDL accumulation in vivo. With dividing monocyte dynamics into three stages (1: adhesion on endothelium, 2: invasion and 3: complete transmigration below endothelium), we analyzed the stage transition dynamics of individual living human monocytes. OxLDL did not enhance initial monocyte adhesion to endothelium (stage 1), but it specifically primed adherent monocytes to start invasion (stage 1-->2). Once invasion started, it had no effect thereafter on monocyte stage transition (stage 2-->3). OxLDL upregulated PECAM-1 and downregulated VE-cadherin on endothelial junctions without monocyte addition, both of which could promote monocyte entry by enhanced homophilic binding to monocyte PECAM-1, and by disrupted junctional barrier, respectively. Meanwhile, monocyte speed at neither locomotion on endothelium (stage 1) nor subendothelial migration (stage 3) was altered by oxLDL. These data indicate that before monocyte adhesion, endothelial junctions changed their conformation to more monocyte-acceptable state in response to oxLDL, resulting the stage-specific promotion of monocyte TEM (stage 1-->2; initiation of invasion) with no enhancement of its initial adhesion or migration speed.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Quimiotaxia de Leucócito/fisiologia , Lipoproteínas LDL/fisiologia , Monócitos/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Aterosclerose , Adesão Celular/fisiologia , Técnicas de Cultura de Células/métodos , Movimento Celular/fisiologia , Células Cultivadas , Regulação para Baixo , Células Endoteliais , Humanos , Imageamento Tridimensional , Junções Intercelulares/fisiologia , Veias Umbilicais/citologia , Veias Umbilicais/fisiologia , Regulação para Cima
6.
Am J Physiol Renal Physiol ; 288(6): F1144-52, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15687247

RESUMO

Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or L-arginine and was partially reduced by the addition of diphenylene iodonium or N(G)-nitro-L-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2',7'-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker) with L-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (BH4), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of BH4 availability. These mechanisms are potential key targets for therapeutic interventions.


Assuntos
Biopterinas/análogos & derivados , Nefropatias Diabéticas/metabolismo , Glomérulos Renais/enzimologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase/metabolismo , Tirosina/análogos & derivados , Animais , Antioxidantes/farmacologia , Biopterinas/farmacologia , Nefropatias Diabéticas/tratamento farmacológico , Regulação Enzimológica da Expressão Gênica , Masculino , NADPH Oxidases/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo III , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-Dawley , Superóxidos/metabolismo , Tirosina/metabolismo
7.
Hypertension ; 45(4): 710-6, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15699469

RESUMO

We demonstrated recently that chronic administration of aldosterone to rats induces glomerular mesangial injury and activates mitogen-activated protein kinases including extracellular signal-regulated kinases 1/2 (ERK1/2). We also observed that the aldosterone-induced mesangial injury and ERK1/2 activation were prevented by treatment with a selective mineralocorticoid receptor (MR) antagonist, eplerenone, suggesting that the glomerular mesangium is a potential target for injuries induced by aldosterone via activation of MR. In the present study, we investigated whether MR is expressed in cultured rat mesangial cells (RMCs) and involved in aldosterone-induced RMC injury. MR expression and localization were evaluated by Western blotting analysis and fluorolabeling methods. Cell proliferation and micromechanical properties were determined by [3H]-thymidine uptake measurements and a nanoindentation technique using an atomic force microscope cantilever, respectively. ERK1/2 activity was measured by Western blotting analysis with an anti-phospho-ERK1/2 antibody. Protein expression and immunostaining revealed that MR was abundant in the cytoplasm of RMCs. Aldosterone (1 to 100 nmol/L) dose-dependently activated ERK1/2 in RMCs with a peak at 10 minutes. Pretreatment with eplerenone (10 micromol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation and decreased the elastic modulus, indicating cellular proliferative and deforming effects of aldosterone, respectively. These aldosterone-induced changes in cellular characteristics were prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor, PD988059 (100 micromol/L). The results indicate that aldosterone directly induces RMC proliferation and deformability through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.


Assuntos
Aldosterona/farmacologia , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Mesângio Glomerular/citologia , Mesângio Glomerular/metabolismo , Receptores de Mineralocorticoides/fisiologia , Animais , Forma Celular/fisiologia , Células Cultivadas , Ativação Enzimática/fisiologia , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Mineralocorticoides/metabolismo
8.
Auton Neurosci ; 116(1-2): 46-53, 2004 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-15556837

RESUMO

Prodromal signs such as cardiac rhythm disturbance and changes in gastric motility are generally induced before and during nausea in humans. These autonomic reactions were compared in subjects who were or were not experiencing nausea. Nausea was induced by having the subjects view a movie of oscillating pictures. Seventeen healthy volunteers were asked to relax their muscles and watch the movie. Electrogastrogram (EGG), electrocardiogram (ECG), palmar and metopic perspiration, digital blood flow and thoracic movement related to respiration were simultaneously measured while the subjects viewed the movie. A total of 11 of 17 subjects complained of nausea after watching the movie. The characteristic changes in their autonomic responses during exposure to the movie were as follows. The power of the EGG, heart rate and metopic perspiration significantly increased compared to those before watching the movie. The respiratory cycle gradually increased during and even after watching the movie. In contrast, no significant changes in the power of the EGG, heart rate and metopic perspiration were observed in the remaining six subjects who did not experience nausea. The role of the autonomic nervous system in nausea is discussed. These results suggest that these symptoms regarding the sympathetic nervous system could actually be defensive reactions against the sensation of nausea.


Assuntos
Sistema Nervoso Autônomo/fisiologia , Enjoo devido ao Movimento/fisiopatologia , Náusea/fisiopatologia , Gravação em Vídeo , Adulto , Análise de Variância , Eletrocardiografia/métodos , Feminino , Motilidade Gastrointestinal/fisiologia , Frequência Cardíaca/fisiologia , Humanos , Masculino , Fatores de Tempo
9.
Atherosclerosis ; 177(1): 19-27, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15488861

RESUMO

OBJECTIVE: To visualize and quantitatively analyze spatiotemporal dynamics of individual living monocytes during transendothelial migration (TEM). METHODS AND RESULTS: We developed an in vitro new experimental system using confocal laser scanning microscope with following two improvements: (1) ultra thin collagen gel layer (30-50 microm thick) constructed under human umbilical vein endothelial cell layer for three-dimensional observation with high magnification; (2) appropriate fluorescent labeling of living monocytes and endothelial cells to keep highest cell activity. Individual monocytes behaved quite diversely. Approximately 70% of adhered monocytes directionally crawled to intercellular junction, and started invasion. Time from adhesion to start of invasion was 8.6 +/- 5.4 min (mean +/- S.D., n=61 monocytes). Approximately 80% of such invading monocytes completed TEM, but remaining 20% of once invading monocytes hesitated transmigration, and returned onto the endothelial surface. Time from start to finish of invasion was 6.3 +/- 3.2 min (mean +/- S.D., n=53 monocytes). CONCLUSIONS: Using our collagen gel-based newly-developed system, we visualized and quantitatively analyzed detailed spatiotemporal, three-dimensional dynamics of individual living monocytes during TEM. We revealed that monocytes encountered at least two hurdles, at starting invasion, and leaving endothelium, to achieve complete TEM. Approximately 56% (80% of 70% of adhered monocytes) passed both hurdles.


Assuntos
Movimento Celular , Monócitos/citologia , Células Cultivadas , Endotélio Vascular/citologia , Humanos
10.
Biophys J ; 86(4): 2286-94, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15041667

RESUMO

We studied x-ray diffraction from the left ventricular wall of an excised, perfused whole heart of a rat using x rays from the third-generation synchrotron radiation facility, SPring-8. With the beam at right angles to the long axis of the left ventricle, well-oriented, strong equatorial reflections were observed from the epicardium surface. The reflections became vertically split arcs when the beam passed through myocardium deeper in the wall, and rings were observed when the beam passed into the inner myocardium of the wall. These diffraction patterns were explained by employing a layered-spiral model of the arrangement of muscle fibers in the heart. In a quiescent heart with an expanded left ventricle, the muscle fibers at the epicardium surface were found to have a (1,0) lattice spacing smaller than in the rest of the wall. The intensity ratio of the (1,0) and (1,1) equatorial reflections decreased on contraction with a similar time course in all parts of the wall. The results show that it is possible to assign the origin of reflections in a diffraction diagram from a whole heart. This study offers a basis for interpretation of x-ray diffraction from a beating heart under physiologically and pathologically different conditions.


Assuntos
Contração Muscular/fisiologia , Fibras Musculares Esqueléticas/citologia , Músculos/citologia , Miocárdio/citologia , Difração de Raios X/métodos , Animais , Ventrículos do Coração , Modelos Teóricos , Fibras Musculares Esqueléticas/química , Músculos/química , Miocárdio/química , Ratos , Ratos Wistar
11.
Pflugers Arch ; 448(2): 153-60, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-14767773

RESUMO

We examined the sarcomere length-dependence of the spacing of the hexagonal lattice of the myofilaments and the mass transfer of myosin cross-bridges during contraction of right ventricular papillary muscle of the rat. The lattice spacing and mass transfer were measured by using X-ray diffraction, and the sarcomere length was monitored by laser diffraction at the same time. Although the lattice spacing and the sarcomere length were inversely related, their relationship was not exactly isovolumic. The cell volume decreased by about 15% when the sarcomere length was shortened from 2.3 micro m to 1.8 micro m. Twitch tension increased with sarcomere length (the Frank-Starling law). At the peak tension, the ratio of the intensity of the (1,0) equatorial reflection to that of the (1,1) reflection was smaller when the tension was greater, showing that the larger tension at a longer sarcomere length accompanies a larger amount of mass transfer of cross-bridges from the thick to the thin filament. The result suggests that the Frank-Starling law is due to an increase in the number of myosin heads attached to actin, not in the average force produced by each head.


Assuntos
Coração/fisiologia , Miocárdio/ultraestrutura , Miosinas/fisiologia , Sarcômeros/fisiologia , Sarcômeros/ultraestrutura , Animais , Fenômenos Biofísicos , Biofísica , Técnicas In Vitro , Masculino , Microscopia Confocal , Contração Miocárdica/efeitos dos fármacos , Músculos Papilares/fisiologia , Músculos Papilares/ultraestrutura , Ratos , Ratos Wistar , Difração de Raios X
12.
J Appl Physiol (1985) ; 96(4): 1561-8, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-14660511

RESUMO

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.


Assuntos
Velocidade do Fluxo Sanguíneo/efeitos da radiação , Circulação Cerebrovascular/efeitos da radiação , Eritrócitos/fisiologia , Luz , Oxigênio/sangue , Animais , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Meia-Vida , Medições Luminescentes , Masculino , Mesoporfirinas/sangue , Mesoporfirinas/química , Metaloporfirinas/sangue , Metaloporfirinas/química , Microcirculação/efeitos da radiação , Ratos , Ratos Wistar , Ressuscitação , Choque Hemorrágico/fisiopatologia , Choque Hemorrágico/terapia
13.
Biochem Biophys Res Commun ; 306(2): 505-8, 2003 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-12804593

RESUMO

Intra-aortic measurement of nitric oxide (NO) would provide valuable insights into NO bioavailability in systemic circulation and vascular endothelial function. In the present study, we thus developed a catheter-type NO sensor to measure intra-aortic NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter to measure intra-aortic plasma NO concentration in vivo in anesthetized dogs. Infusion of acetylcholine (10 microg/kg) increased base-to-peak plasma NO level in the aorta by 2.4+/-0.4 nM (n=7). After 20-min infusion of N(G)-methyl-L-arginine (NO synthase inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8+/-0.4 nM, P<0.003, n=7). In conclusion, the newly developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo and demonstrated applicability to direct evaluation of intravascular NO bioavailability.


Assuntos
Acetilcolina/farmacologia , Aorta/metabolismo , Óxido Nítrico/análise , Acetilcolina/metabolismo , Animais , Cateterismo/instrumentação , Cães , Eletrodos , Endotélio/citologia , Endotélio/metabolismo , Feminino , Masculino , Óxido Nítrico/sangue , Fatores de Tempo
15.
Proc Natl Acad Sci U S A ; 99(24): 15638-43, 2002 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-12434019

RESUMO

The interaction between monocytes and endothelial cells is considered to play a major role in the early stage of atherosclerosis, and the involved endothelial cell micromechanics may provide us with important aspects of atherogenesis. In the present study, we evaluated (i) the endothelial cell-to-cell and cell-to-substrate gaps with the electric cell-substrate impedance sensing system, which can detect the nanometer order changes of cell-to-cell and cell-to-substrate distances separately, and (ii) the endothelial cell micromechanical properties with an atomic force microscope after application of monocytes to endothelial cells. Application of monocytic THP-1 cells to IL-1beta-stimulated human umbilical vein endothelial cells immediately decreased the electrical resistance of the endothelial cell-to-substrate (increase of the cell-to-substrate gap), whereas the endothelial cell-to-cell resistance (cell-to-cell gap) did not change. The elastic modulus of the endothelial cells decreased after 2-h monocyte application, indicating an increase of endothelial cell deformability. In conclusion, the interaction of the monocytes to the endothelial cells reduced the adhesiveness to the substrate and increased the deformability of endothelial cells. These changes in the adhesiveness and the deformability may facilitate migration of monocytes, a key process of atherogenesis in the later stage.


Assuntos
Adesão Celular/fisiologia , Endotélio Vascular/citologia , Monócitos/citologia , Actinas/análise , Arteriosclerose/patologia , Fenômenos Biomecânicos , Movimento Celular , Células Cultivadas , Sistemas Computacionais , Elasticidade , Impedância Elétrica , Endotélio Vascular/química , Endotélio Vascular/efeitos dos fármacos , Corantes Fluorescentes/análise , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Adesões Focais , Proteínas de Fluorescência Verde , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Interleucina-1/farmacologia , Proteínas Luminescentes/análise , Microscopia de Força Atômica , Nanotecnologia , Proteínas Tirosina Quinases/análise
16.
Physiol Meas ; 23(2): 261-8, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-12051298

RESUMO

Direct measurement of nitric oxide (NO) is of great importance and value for both in vitro and in vivo studies on dynamic NO bioactivity. Here, we evaluated the basic performance of a newly developed NO sensor (Innovative Instruments, Inc.). Unlike other NO sensors, the new NO sensor has a highly durable, gas-permeable coating and is affected much less by electrical interference due to its integrated structure where working and reference electrodes are combined in a single element. Calibration with NO gas showed high sensitivity of about 580 pA per nmol-NO l(-1) (the detection limit 0.08 nmol-NO l(-1), S/N = 3). This sensor also showed high selectivity (25,000 times and more) to NO, compared with NO-related reagents such as L-arginine, N(G)-monomethyl-L-arginine, acetylcholine, nitroglycerin (NTG) and tetrahydrobiopterin as well as dissolved oxygen. As an in vivo application, the sensor was located in the anaesthetized rat abdominal aorta to measure NTG-derived plasma NO. lntra-aortic infusion of 0.5 mg NTG caused a measurable increase in plasma NO level (2.0 +/- 2.2 nmol l(-1), mean +/- SD, n = 3). In conclusion, the new NO sensor demonstrated a satisfying performance for both in vitro and in vivo applications.


Assuntos
Biopterinas/análogos & derivados , Eletrodos , Desenho de Equipamento , Óxido Nítrico/análise , Óxido Nítrico/sangue , Acetilcolina/farmacologia , Animais , Antioxidantes/farmacologia , Arginina/farmacologia , Biopterinas/farmacologia , Calibragem , Inibidores Enzimáticos/farmacologia , Óxido Nítrico Sintase/metabolismo , Nitroglicerina/farmacologia , Oxigênio/análise , Permeabilidade , Ratos , Ratos Endogâmicos WKY , Vasodilatadores/farmacologia , ômega-N-Metilarginina/farmacologia
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