RESUMEN
BACKGROUND: Synthetic graft materials are commonly used for shunts and cardiovascular reconstruction in neonates, but are prone to thrombosis and scarring. The umbilical vein is a potential source of autologous, endothelialized tissue for neonatal shunts and tissue reconstruction, but requires preservation before implantation. METHODS: Umbilical cords were collected in UW solution with antibiotics at 4°C until dissection. Umbilical vein segments were tested for burst pressure before and after 2 weeks of preservation. Umbilical veins segments were preserved under static or flow conditions at 4°C in UW solution with 5% human plasma lysate for 7 days. Veins were evaluated with histopathology, scanning electron microscopy, and platelet adhesion testing. RESULTS: Umbilical veins have no difference in burst pressure at harvest (n = 16) compared with 2 weeks of preservation (n = 11; 431 ± 229 versus 438 ± 244 mm Hg). After 1 week, static and flow-preserved veins showed viability of the vessel segments with endothelium staining positive for CD31, von Willebrand factor, and endothelial nitric oxide synthase. Scanning electron microscopy demonstrated preservation of normal endothelial morphology and flow alignment in the flow-preserved samples compared with cobblestone endothelial appearance and some endothelial cell loss in the static samples. Static samples had significantly more platelet adhesion than flow-preserved samples did. CONCLUSIONS: Umbilical veins have adequate burst strength to function at neonatal systemic pressures. Preservation under flow conditions demonstrated normal endothelial and overall vascular morphology with less platelet adhesion compared with static samples. Preserved autologous umbilical veins are potential source for endothelialized shunts or cardiovascular repair tissue for neonates.
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Endotelio Vascular/diagnóstico por imagen , Endotelio Vascular/fisiología , Soluciones Preservantes de Órganos/química , Procedimientos de Cirugía Plástica/métodos , Conservación de Tejido/métodos , Venas Umbilicales/trasplante , Biopsia con Aguja , Procedimientos Quirúrgicos Cardíacos/métodos , Femenino , Humanos , Inmunohistoquímica , Recién Nacido , Masculino , Microscopía Electrónica de Rastreo/métodos , Sensibilidad y Especificidad , Recolección de Tejidos y Órganos/métodos , Trasplante Autólogo/métodos , Venas Umbilicales/cirugía , Venas Umbilicales/ultraestructuraRESUMEN
Sphingosine-1-phosphate (S1P) has been known to promote endothelial cell (EC) proliferation and protect Syndecan-1 (SDC1) from shedding, thereby maintaining this antithrombotic signal. In the present study, we investigated the effect of S1P in the construction of a functional tissue-engineered blood vessel by using human endothelial cells and decellularized human umbilical vein (DHUV) scaffolds. Both human umbilical vein endothelial cells (HUVEC) and human cord blood derived endothelial progenitor cells (EPC) were seeded onto the scaffold with or without the S1P treatment. The efficacy of re-cellularization was determined by using the fluorescent marker CellTracker CMFDA and anti-CD31 immunostaining. The antithrombotic effect of S1P was examined by the anti-aggregation tests measuring platelet adherence and clotting time. Finally, we altered the expression of SDC1, a major glycocalyx protein on the endothelial cell surface, using MMP-7 digestion to explore its role using platelet adhesion tests in vitro. The result showed that S1P enhanced the attachment of HUVEC and EPC. Based on the anti-aggregation tests, S1P-treated HUVEC recellularized vessels when grafted showed reduced thrombus formation compared to controls. Our results also identified reduced SDC1 shedding from HUVEC responsible for inhibition of platelet adherence. However, no significant antithrombogenic effect of S1P was observed on EPC. In conclusion, S1P is an effective agent capable of decreasing thrombotic risk in engineered blood vessel grafts. STATEMENT OF SIGNIFICANCE: Sphingosine-1phosphate (S1P) is a low molecular-weight phospholipid mediator that regulates diverse biological activities of endothelial cell, including survival, proliferation, cell barrier integrity, and also influences the development of the vascular system. Based on these characters, we the first time to use it as an additive during the process of a small caliber blood vessel construction by decellularized human umbilical vein and endothelial cell/endothelial progenitor. We further explored the function and mechanism of S1P in promoting revascularization and protection against thrombosis in this tissue engineered vascular grafts. The results showed that S1P could not only accelerate the generation but also reduce thrombus formation of small caliber blood vessel.
Asunto(s)
Prótesis Vascular , Endotelio Vascular/fisiología , Lisofosfolípidos/farmacología , Esfingosina/análogos & derivados , Sindecano-1/metabolismo , Trombosis/patología , Venas Umbilicales/citología , Coagulación Sanguínea/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Células Progenitoras Endoteliales/citología , Endotelio Vascular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Glicocálix/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Cinética , Metaloproteinasa 7 de la Matriz/metabolismo , Modelos Biológicos , Adhesividad Plaquetaria/efectos de los fármacos , Esfingosina/farmacología , Andamios del Tejido/química , Venas Umbilicales/ultraestructuraRESUMEN
A growing body of epidemiological evidence has supported the association between maternal exposure to airborne fine particulate matter (PM2.5) during pregnancy and adverse pregnancy outcomes. However, the specific biological mechanisms implicated in the causes of adverse pregnancy outcomes are not well defined. In this study, a pregnant rat model of exposure to different doses of cooking oil fumes (COFs)-derived PM2.5 by tail intravenous injection in different pregnant stages was established. The results indicated that exposure to COFs-derived PM2.5 was associated with adverse pregnancy outcomes, changed the structure of umbilical cord blood vessels, decreased the diameter and lumen area, and increased wall thickness. What's more, a significant increase of maximum contraction tension was observed in the early pregnancy high-dose exposure group and pregnant low-dose exposure group compared to the control group. Based on the maximum contraction tension, acetylcholine (ACh) did not induce vasodilation but caused a dose-dependent constriction, and there were significant differences in the two groups compared to the control group. Exposure to COFs-derived PM2.5 impaired the vasomotor function of umbilical veins by affecting the expression of NO and ET-1. This is the first study that evaluated the association of risk of adverse pregnancy outcomes and pregnant rats exposed to COFs-derived PM2.5 and primarily explored the potential mechanisms of umbilical cord blood vessels injury on a rat model. More detailed vitro and vivo studies are needed to further explore the mechanism in the future.
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Contaminantes Atmosféricos/toxicidad , Culinaria , Sangre Fetal/fisiología , Material Particulado/toxicidad , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/fisiología , Animales , Femenino , Gases , Humanos , Masculino , Exposición Materna/efectos adversos , Aceites , Material Particulado/efectos adversos , Material Particulado/análisis , Embarazo , Resultado del Embarazo , Ratas , Riesgo , Pruebas de Toxicidad , Venas Umbilicales/ultraestructuraRESUMEN
In vitro perfusion systems have exposed vascular constructs to mechanical conditions that emulate physiological pulse pressure and found significant improvements in graft development. However, current models maintain constant, or set pulse/shear mechanics that do not account for the natural temporal variation in frequency. With an aim to develop clinically relevant small diameter vascular grafts, these investigations detail a perfusion culture model that incorporates temporal pulse pressure variation. Our objective was to test the hypothesis that short-term variation in heart rate, such as changes in respiratory activity, plays a significant role in vascular remodeling and graft development. The pulse rate of a healthy volunteer was logged to model the effect of daily activities on heart rate. Vascular bioreactors were used to deliver perfusion conditions based on modeled frequencies of temporal pulse variability, termed Physiologically Modeled Pulse Dynamics (PMPD). Acellular scaffolds derived from the human umbilical vein were seeded with human vascular smooth muscle cells and perfused under defined pulsatile conditions. vSMC exposed to constant pulse frequencies expressed a contractile phenotype, while exposure to PMPD drove cells to a synthetic state with continued cell proliferation, increased tensile strength and stiffness as well as diminished vasoactivity. Results show the temporal variation associated with normal heart physiology to have a profound effect on vascular remodeling and vasoactive function. While these models are representative of vascular regeneration further investigation is required to understanding these and other key regulators in vSMC phenotype switching in non-pathological or wound healing states. This understanding has important clinical implications that may lead to improved treatments that enhance vessel regeneration.
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Técnicas de Cultivo de Célula/instrumentación , Técnicas de Cultivo de Célula/métodos , Frecuencia Cardíaca/fisiología , Músculo Liso Vascular/fisiología , Músculo Liso Vascular/ultraestructura , Reactores Biológicos , Células Cultivadas , Expresión Génica , Humanos , Músculo Liso Vascular/irrigación sanguínea , Músculo Liso Vascular/metabolismo , Perfusión/instrumentación , Fenotipo , Andamios del Tejido , Venas Umbilicales/ultraestructura , Remodelación VascularRESUMEN
Processing ex vivo derived tissues to reduce immunogenicity is an effective approach to create biologically complex materials for vascular reconstruction. Due to the sensitivity of small diameter vascular grafts to occlusive events, the effect of graft processing on critical parameters for graft patency, such as peripheral cell adhesion and wall mechanics, requires detailed analysis. Isolated human umbilical vein sections were used as model allogenic vascular scaffolds that were processed with either: 1. sodium dodecyl sulfate (SDS), 2. ethanol/acetone (EtAc), or 3. glutaraldehyde (Glu). Changes in material mechanics were assessed via uniaxial tensile testing. Peripheral cell adhesion to the opaque grafting material was evaluated using an innovative flow chamber that allows direct observation of the blood-graft interface under physiological shear conditions. All treatments modified the grafts tensile strain and stiffness properties, with physiological modulus values decreasing from Glu 240±12 kPa to SDS 210±6 kPa and EtAc 140±3 kPa, P<.001. Relative to glutaraldehyde treatments, neutrophil adhesion to the decellularized grafts increased, with no statistical difference observed between SDS or EtAc treatments. Early platelet adhesion (% surface coverage) showed no statistical difference between the three treatments; however, quantification of platelet aggregates was significantly higher on SDS scaffolds compared to EtAc or Glu. Tissue processing strategies applied to the umbilical vein scaffold were shown to modify structural mechanics and cell adhesion properties, with the EtAc treatment reducing thrombotic events relative to SDS treated samples. This approach allows time and cost effective prescreening of clinically relevant grafting materials to assess initial cell reactivity.
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Fenómenos Fisiológicos Sanguíneos/efectos de los fármacos , Prótesis Vascular , Andamios del Tejido , Trasplantes/efectos de los fármacos , Trasplantes/fisiología , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/fisiología , Acetona/farmacología , Adhesión Celular/efectos de los fármacos , Etanol/farmacología , Glutaral/farmacología , Humanos , Ensayo de Materiales , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Adhesividad Plaquetaria/efectos de los fármacos , Dodecil Sulfato de Sodio/farmacología , Resistencia a la Tracción/efectos de los fármacos , Trasplantes/ultraestructura , Venas Umbilicales/ultraestructuraRESUMEN
Endothelial cells are tightly fitted to each other and lining the interior surface of all vessels of living organism to provide vascular permeability regulation and interchange between the blood circulating in vessels and tissue fluids of those organs in which these vessels are located. In vitro endothelial monolayer conserve it's basic barrier function which is native for vessels endothelium. Based on this fact we used endothelial cells growing in vitro as a model system in experimental studies of cytoskeletal and adhesion cell components interaction. In current paper, cultured human vein endothelial cells monolayer was used to quantify cytoskeleton alterations in the of endothelial cells from spreading and formation of the first cell-cell contacts to confluent monolayer formation. The system of actin filaments formed two different cytoskeletal structures in the cells of venous endothelium: 1) cortical actin network; 2) actin stress fibers (bundles) arranged parallel to the substrate. Two actin isoforms, ß- and γ-cytoplasmic (non-muscle) actins, are expressed in endothelial cells. The bundles of actin stress fibers were detected by immunofluorescent staining with antibody against ß-actin, whereas antibodies against γ-actin identified cortical and lamellar networks. For assessment of the actin cytoskeleton organization it's fluorescence intensity on the area of 10 µM2 located (1) near the free edge, and (2) in the zone of cell-cell contacts were analyzed. Fluorescence intensity of ß-actin structures was higher in the areas of cell-cell contact. The fluorescence of γ-actin structures was more intensive at the leading edges of the lamellae, and was the lowest on the stable edges of the cells with formed cell-cell contacts. The endothelial monolayer formation was accompanied by microtubule system alteration: the number of microtubules increased at the cell edge, and besides the microtubules quantity in the area of already formed cell-cell contact was always higher than in free lamella region.
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Citoesqueleto de Actina/ultraestructura , Células Endoteliales/ultraestructura , Microtúbulos/ultraestructura , Venas Umbilicales/ultraestructura , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Comunicación Celular , Línea Celular , Células Endoteliales/metabolismo , Humanos , Microscopía Fluorescente , Microtúbulos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Venas Umbilicales/metabolismoRESUMEN
Click chemistry plays a dual role in the design of new citrate-based biodegradable elastomers (CABEs) with greatly improved mechanical strength and easily clickable surfaces for biofunctionalization. This novel chemistry modification strategy is applicable to a number of different types of polymers for improved mechanical properties and biofunctionality.
Asunto(s)
Materiales Biocompatibles/química , Química Clic , Elastómeros/química , Andamios del Tejido/química , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citratos/química , Módulo de Elasticidad , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Humanos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Polímeros/química , Resistencia a la Tracción , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/fisiología , Venas Umbilicales/ultraestructuraRESUMEN
Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium. In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear. Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body. We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.
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Cilios/fisiología , Cilios/ultraestructura , Células Endoteliales de la Vena Umbilical Humana/ultraestructura , Morfogénesis , Venas Umbilicales/ultraestructura , Técnicas de Cultivo de Célula , Células Cultivadas , Tomografía con Microscopio Electrónico , Humanos , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Modelos BiológicosRESUMEN
A concise synthesis of APDOEGCg (3) was accomplished. Due to the reactivity of its amine group, the compound could be easily converted to the fluorescein probe 21 and immunogen probe 22 efficiently. We then demonstrated the usefulness of the probes for imaging studies and the generation of antibodies.
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Catequina/análogos & derivados , Catequina/síntesis química , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Té/química , Aminas/química , Antioxidantes/química , Antioxidantes/farmacología , Catequina/análisis , Catequina/química , Catequina/farmacología , Células Endoteliales/citología , Células Endoteliales/ultraestructura , Humanos , Venas Umbilicales/citología , Venas Umbilicales/ultraestructuraRESUMEN
Our objective was to investigate the endothelial nitric oxide synthase (eNOS) immuno-reactivity and the ultrastructure of endothelial cells of a human umbilical artery in both normal and preeclamptic pregnancies. The umbilical cords from normal and preeclamptic pregnancies were collected immediately after vaginal and abdominal deliveries. Umbilical arteries were isolated and fixed in 10% neutral formaline solution, embedded in paraffin, and then stained with hematoxylin and eosin (H&E) for the histologic investigation, and eNOS activation were examined in samples by streptavidine-biotine immunohistochemical methods. The arterial sections were also fixed in phosphate-buffered 2.5% glutaraldehyde solution (pH 7.2) for 3 h and post-fixed with 1% osmium tetroxide at 4°C for 2 h for the investigation of the ultrastructural examination. In the umbilical artery of preeclamptic pregnancies, endothelial cells were oval, triangular, or polygonal, and were disorganized. Some endothelial cells were separated by enlarged intercellular spaces. A dilated endoplasmic reticulum, swollen mitochondria, and vanished mitochondrial cristae were observed. The nuclei of some endothelial cells displayed deep invaginations and irregular outlines. Most endothelial cells had a high number of cytoplasmic vacuoles. In preeclampsia, eNOS immunoreactivity increased considerably in endothelial cells when compared to normal pregnancies. We believe that preeclampsia plays an important role in the pathogenesis of endothelial cell dysfunction and activation in the umbilical artery. However, the disturbance mechanism of endothelial cells is not known, and further studies are necessary to clarify the exact mechanism.
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Óxido Nítrico Sintasa de Tipo III/análisis , Preeclampsia/enzimología , Preeclampsia/patología , Embarazo , Células Endoteliales/enzimología , Células Endoteliales/ultraestructura , Endotelio Vascular/enzimología , Endotelio Vascular/ultraestructura , Femenino , Humanos , Inmunohistoquímica , Óxido Nítrico/metabolismo , Orgánulos/ultraestructura , Preeclampsia/fisiopatología , Transducción de Señal , Arterias Umbilicales/enzimología , Arterias Umbilicales/ultraestructura , Venas Umbilicales/enzimología , Venas Umbilicales/ultraestructuraRESUMEN
These investigations describe the development of a novel ex vivo three-dimensional scaffold derived from the human umbilical vein (HUV), and its potential as a regenerative matrix for tissue regeneration. Unique properties associated with the vascular wall have shown potential to function as a surgical barrier for guided tissue regeneration, particularly with the regeneration of periodontal tissues. HUV was isolated from umbilical cords using a semiautomated machining technology, decellularized using 1% sodium dodecyl sulfate, and then opened longitudinally to form tissue sheets. Uniaxial tensile testing, stress relaxation, and suture retention tests were performed on the acellular matrix to evaluate the HUV's biomechanical properties, followed by an evaluation of cellular interactions by seeding human gingival fibroblasts to assess adhesion, metabolic function, and proliferation on the scaffold. The scaffold's biomechanical properties were shown to display anisotropic behavior, which is attributed to the ex vivo material's composite structure. Detailed results indicated that the ultimate tensile strength of the longitudinal strips was significantly higher than that of the circumferential strips (p < 0.001). The HUV also exhibited significantly higher stress relaxation response in the longitudinal direction than in the circumferential orientation (p < 0.05). The ablumenal and lumenal surfaces of the material were also shown to differentially influence cell proliferation and metabolic activity, with both cellular functions significantly increased on the ablumenal surface (p < 0.05). Human gingival fibroblast migration into the scaffold was also influenced by the organization of extracellular matrix components, where the lumenal surface inhibits cell migration, acting as a barrier, while the ablumenal surface, which is proposed to interface with the wound site, promotes cellular invasion. These results show the HUV bioscaffold to be a promising naturally derived surgical barrier that may function well as a resorbable guided tissue regeneration membrane as well as in other clinical applications.
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Fibroblastos/citología , Fibroblastos/metabolismo , Regeneración Tisular Guiada Periodontal/métodos , Andamios del Tejido/química , Venas Umbilicales/citología , Venas Umbilicales/metabolismo , Fenómenos Biomecánicos , Proliferación Celular , Encía/citología , Humanos , Suturas , Resistencia a la Tracción , Factores de Tiempo , Venas Umbilicales/ultraestructuraRESUMEN
With the exception of its most proximal segment, the human umbilical cord lacks innervation. It might be expected, therefore, that a paracrine effect through the direct contact between the smooth muscle cells and the endothelium may be particularly important in the control of the fetoplacental circulation. In this study, electron microscopy and immunohistochemistry were applied to examine umbilical veins immediately after full-term and pre-term delivery. The smooth muscle cells in the upper layer of the tunica media exhibited long, foot-like processes with c-kit immunoreactivity. In the umbilical vein of full-term neonates more than 50% of these cell processes display a normal ultrastructure and they were closely associated with the lamina elastica interna. Whereas in pre-term infants more than 60% of these cell processes exhibit signs of severe shrinkage and detachedness from the lamina elastica interna. At the same time, the high level of immunoreactivity of the endothelial cells as regards the proapoptotic gene product Bax in pre-term infants is indicative of an enhanced apoptotic process in these cells.
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Nacimiento Prematuro , Nacimiento a Término , Venas Umbilicales/ultraestructura , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Embarazo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
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.
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Endotelio Vascular/fisiología , Glicocálix/fisiología , Venas Umbilicales/fisiología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Endotelio Vascular/ultraestructura , Eritrocitos/fisiología , Eritrocitos/ultraestructura , Glicocálix/ultraestructura , Heparitina Sulfato/análisis , Humanos , Microscopía Electrónica , Sindecano-1/análisis , Venas Umbilicales/ultraestructuraRESUMEN
With advantageous biomechanical properties, materials derived from ex vivo tissues are being actively investigated as scaffolds for tissue engineering applications. However, decellularization treatments are required before implantation to reduce the materials immune impact. The aim of these investigations was to assess a convective flow model as an enhanced methodology to decellularize ex vivo tissue. Isolated human umbilical veins were decellularized using two methods: rotary agitation at 100rpm on orbital shaker plates, and convective flow run at 5, 50, and 150mmHg within perfusion bioreactors. Extracted phospholipids and total soluble protein were assessed over time. Histology, SEM, and uniaxial tensile testing analysis were carried out to evaluate variation in the tissues. After 72h, samples exposed to traditional rotary agitation showed retention of whole cells and cellular components, whereas pressure-based systems showed no visual sign of cells. The convective flow method was significantly more effective at removing phospholipid and total protein than the agitation model. High transmembrane pressure (150mmHg) resulted in higher phospholipids extraction. However, a more efficient protein extraction occurred at 50mmHg. Variation in extraction rates was dependent on tissue permeability, which varied as pressure increased. Collectively, these findings show significant improvements in decellularization efficiency that may lead to more immune compliant ex vivo-derived biomaterials.
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Materiales Biocompatibles/síntesis química , Separación Celular/métodos , Convección , Ingeniería de Tejidos/métodos , Venas Umbilicales/citología , Módulo de Elasticidad , Humanos , Hidroxiprolina/metabolismo , Permeabilidad , Fosfolípidos/aislamiento & purificación , Proteínas/aislamiento & purificación , Solubilidad , Resistencia a la Tracción , Andamios del Tejido , Venas Umbilicales/ultraestructuraRESUMEN
VCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruited to cell-cell contact sites on the apical membrane of activated endothelial cells. In this study, we show that this recruitment is independent of ligand engagement, actin cytoskeleton anchorage, and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruited by inclusion within specialized preformed tetraspanin-enriched microdomains, which act as endothelial adhesive platforms (EAPs). Using advanced analytical fluorescence techniques, we have characterized the diffusion properties at the single-molecule level, nanoscale organization, and specific intradomain molecular interactions of EAPs in living primary endothelial cells. This study provides compelling evidence for the existence of EAPs as physical entities at the plasma membrane, distinct from lipid rafts. Scanning electron microscopy of immunogold-labeled samples treated with a specific tetraspanin-blocking peptide identify nanoclustering of VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecular organization that regulates the leukocyte integrin-binding capacity of both endothelial receptors during extravasation.
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Adhesión Celular/fisiología , Endotelio Vascular/fisiología , Leucocitos/fisiología , Proteínas de la Membrana/fisiología , Antígenos CD/fisiología , Adhesión Celular/efectos de los fármacos , Membrana Celular/fisiología , Membrana Celular/ultraestructura , Endotelio Vascular/ultraestructura , Humanos , Integrinas/genética , Integrinas/fisiología , Molécula 1 de Adhesión Intercelular/fisiología , Leucocitos/efectos de los fármacos , Microscopía Confocal , Microscopía Electrónica de Rastreo , Receptores de Superficie Celular/fisiología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Venas Umbilicales/fisiología , Venas Umbilicales/ultraestructura , Molécula 1 de Adhesión Celular Vascular/fisiologíaRESUMEN
Angiogenin, a potent angiogenic factor, was cloned and expressed by Escherichia coli and then purified with gel filtration chromatography. Approximately 90% pure angiogenin was obtained to generate a monoclonal antibody. Using western immunoblotting and ELISA, we confirmed that monoclonal antibody C46 secreted from hybridoma cells stably and specifically binds to angiogenin. The fused protein angiogenin-EGF was then expressed in HUVECs, and the subcellular localization of the recombinant protein was determined by confocal microscopy and TEM assay. Recombinant angiogenin was found to mainly concentrate in the pars granulosa of the nucleus, where the protein accumulates to form ribonucleoprotein particles.
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Nucléolo Celular/ultraestructura , Células Endoteliales/ultraestructura , Endotelio Vascular/ultraestructura , Ribonucleasa Pancreática/ultraestructura , Venas Umbilicales/ultraestructura , Anticuerpos Monoclonales/biosíntesis , Nucléolo Celular/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Escherichia coli/metabolismo , Vectores Genéticos , Humanos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Plásmidos , Proteínas Recombinantes de Fusión/biosíntesis , Ribonucleasa Pancreática/biosíntesis , Ribonucleasa Pancreática/aislamiento & purificación , Venas Umbilicales/metabolismoRESUMEN
The current study investigated whether the mode of delivery and the mode of sample collection affect the functional properties of umbilical veins as scaffolds for vascular tissue engineering purposes. Human umbilical vein (HUV) from planned cesarean-sections (PCS) showed a 1.7-fold higher maximum contraction with potassium chloride compared to spontaneous vaginal deliveries (VDs, p=0.029). The maximum contractions with histamine were 2.0- and 2.9-fold higher in the PCS and emergency c-section (ECS) groups, respectively, compared to the VD group (p=0.003). The dose-response curves of serotonin were shifted to the right approx. 6- and 5-fold in the VD group compared to PCS and ECS, respectively (p=0.009). There were no differences between the birth groups in terms of tetrazolium dye reduction, platelet adhesion, and the structural integrity. The release of the antithrombotic compound prostacyclin from vessels of the PCS and ECS groups was 6.6- and 3.5-fold higher, respectively, than in the VD group (p<0.001). There was no correlation between the duration of ischemia and any of the functional parameters. This study provides evidence that vessels obtained from PCS are to be preferred for tissue engineering purposes, as they can be harvested in a sterile fashion and show superior vasoconstrictor responses and antithrombotic properties. The data also support a once-per-day pickup schedule for umbilical cords without a deterioration of the functional properties.
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Cesárea , Parto Normal , Andamios del Tejido , Venas Umbilicales/fisiología , Adulto , Epoprostenol/metabolismo , Femenino , Histamina/farmacología , Humanos , Isquemia/fisiopatología , Masculino , Microscopía Electrónica de Rastreo , Adhesividad Plaquetaria , Cloruro de Potasio/farmacología , Embarazo , Serotonina/farmacología , Sales de Tetrazolio/metabolismo , Ingeniería de Tejidos/métodos , Venas Umbilicales/fisiopatología , Venas Umbilicales/ultraestructura , Vasoconstricción/efectos de los fármacos , Vasoconstricción/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
BACKGROUND: Pregnancy Induced Hypertension (PIH) is associated with placental morphological changes, alterations in the blood flow patterns in the umbilical vessels and adverse fetal and maternal outcome. Studies have demonstrated changes in the structure of the umbilical vessels but these have not been described across the length of the cord or correlated with the severity of disease. STUDY DESIGN: A case control study. SETTING: Kenyatta National Hospital. MATERIALS AND METHODS: Thirty six umbilical cords from newborns of women with and without PIH (18 cases, 18 controls) were obtained and studied with light microscopy. Of the cases 9 women had severe PIH and 9 had mild PIH. Means and standard deviations for the various parameters of the various groups were obtained. Student's t-test and ANOVA were used to compare means, a p value of <0.05 being significant. RESULTS: The structure of the umbilical vessels changes from the placental end to the fetal end. The umbilical vein in PIH had a greater wall thickness and a smaller luminal area than in the controls. The vein's wall-luminal ratio increased from the placental to the fetal end. Duplication of the elastic subintimal lamina (ESL) was higher in the cases. The ESL was more commonly duplicated in the fetal end. There were no structural differences between the umbilical arteries in PIH and in the controls. CONCLUSION: PIH is associated with structural changes in the umbilical vessels. These changes are more predominant in the vein than in the artery and in the vein, they are more obvious in the fetal end. The observed increase in wall-luminal ratio from the placental to the fetal end suggests that the fetal end of the umbilical vein has a more refined role in the regulation of blood flow to the fetus.
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Hipertensión Inducida en el Embarazo/patología , Venas Umbilicales/patología , Adulto , Estudios de Casos y Controles , Femenino , Edad Gestacional , Humanos , Recién Nacido , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/patología , Embarazo , Resultado del Embarazo , Venas Umbilicales/ultraestructuraRESUMEN
Recent evidence suggests that apoptosis of endothelial cells contributes to lumen formation during angiogenesis, but the biological mechanism remains obscure. In this study, we investigated the effect of nerve growth factor (NGF), a member of the neurotrophin family and a potential angiogenic factor, on human umbilical vein endothelial cells (HUVEC) apoptosis and the formation of lumen-like structures (LLS) by cultured HUVEC on Matrigel. We demonstrate that NGF induces cell apoptosis. NGF treatment has no significant effect on the expression level of its two receptors, TrkA and p75NTR. Blockade of both TrkA and p75NTR, but not that of either receptor alone significantly decreases NGF-induced cell apoptosis. NGF significantly increases formation of LLS which consist substantially of apoptotic cells. Application of NGF-neutralizing antibody or simultaneous blockade of TrkA and p75NTR significantly blocks spontaneous and NGF-induced LLS formation. These data support a role for NGF-induced cell apoptosis in LLS formation in vitro.
Asunto(s)
Células Endoteliales/metabolismo , Células Endoteliales/ultraestructura , Factor de Crecimiento Nervioso/administración & dosificación , Receptor de Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Humanos , Venas Umbilicales/efectos de los fármacos , Venas Umbilicales/metabolismo , Venas Umbilicales/ultraestructuraRESUMEN
Tissue-on-tissue friction testing was used to determine how instillation of hydrophilic polymer-containing formulations between the "blinking" tissues would compare with lubrication by saline, alone, or an oil-emulsion preparation. Best results were obtained for a formulation that contained active demulcents polyethylene glycol (PEG 400) and propylene glycol (PG), as well as a gellable polymer hydroxypropyl guar (HP-Guar) in a borate-buffered solution, in comparison with hydroxypropylcellulose-containing and carboxymethylcellulose-containing formulations. Superior performance of all the formulations was found for lubricating tissue-on-tissue couples, compared with metal-oxide-to-metal oxide interfaces, or metal oxide-to-tissue interfaces. A reciprocating pin-on-disc type friction/wear test device articulated the intimal faces of preserved human umbilical cord vein segments under increasing loads during simulated continuous "eye-blinking" with addition of increasing weights up to 60 g/cm2, simulating maximal eyelid force on the orbital globe. The tissue-on-tissue couples moved from liquid phase lubrication to boundary lubrication. After residual formulations were rinsed away with saline, persistence of low friction at the highest loads was indicative of formulation substantivity. Human umbilical cord vein segments were utilized in saline-wetted tissue-on-tissue couples that showed variable starting coefficients of friction in the range 0.2-0.4, producing moderate tearing and disruption of the interfacial layers above the medial collagen zone. The best-performing formulations instilled to the tissues pre-wetted with saline apparently reacted separately with each tissue face to produce a lower final and persistent coefficient of friction of about 0.05. Scanning electron microscopy and light microscopy of these guar-modified tissue specimens showed only a few superficial tissue disruptions, and some interphase swelling consistent with polymer uptake. The frictional values for lubricated couples having non-tissue members were considerably higher than the coefficients of friction measured for the similarly lubricated tissue-on-tissue couples, emphasizing the requirement that appropriate simulations are critical to obtaining clinically predictive data.