RESUMO
Suspensions of peripheral blood mononuclear cells (PBMC), monocytes, T or B lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a property not shared by erythrocytes. PBMC also inhibited HF activation by glass or sulfatides. Contaminating platelets may have contributed to inhibition by PBMC. Elaboration of agents inhibiting HF activation required metabolically active cells. The inhibitor(s) in PBMC supernates were not identified with known agents, but had properties of a nonenzymatic protein. PBMC supernates did not contain fibrinogen, nor alter the thrombin, prothrombin, or partial thromboplastin times of normal plasma, amidolysis by activated plasma thromboplastin antecedent (Factor XIa) or activated Stuart factor (Factor Xa) or esterolysis by C1 (C1 esterase); they inhibited plasmin minimally. These experiments suggest that peripheral blood cells may impede intravascular coagulation. Whether this property helps maintain the fluidity of blood is unclear.
Assuntos
Células Sanguíneas/metabolismo , Coagulação Sanguínea , Fator XII/metabolismo , Serina Endopeptidases/metabolismo , Adulto , Azidas/farmacologia , Linfócitos B/metabolismo , Plaquetas/metabolismo , Cicloeximida/farmacologia , Ácido Elágico/farmacologia , Fator XIIa , Feminino , Granulócitos/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Monócitos/metabolismo , Azida Sódica , Linfócitos T/metabolismoRESUMO
We examined the effect of bovine aortic endothelial cell culture supernatants upon the generation of procoagulant activity by human blood monocytes. Confluent endothelial monolayers were cultured for up to 96 h. At timed intervals, culture supernatants were collected and incubated for 5 h with lipopolysaccharide-stimulated human peripheral blood mononuclear cells. The procoagulant activity of mononuclear cell lysates was determined in a one-stage clotting assay. In five experiments, procoagulant activity with culture supernatant (time 0) was 2,294 +/- 761 U/ml (mean +/- SEM). Culture supernatants from endothelial cells incubated for 24-96 h strongly inhibited mononuclear cell generation of procoagulant activity. Indomethacin (10 microM) added to endothelial cells delayed the appearance of procoagulant inhibitor for 72 h. Bovine aortic smooth muscle cell culture supernatants did not inhibit procoagulant activity. The inhibitor was heat stable, effective at 1:50 dilution, soluble, and acid sensitive, with a molecular weight of less than 1,500. Further studies on subpopulations of mononuclear cells demonstrated that endothelial inhibitor selectively decreased the generation of monocyte procoagulant activity and interfered with T lymphocyte amplification of monocyte production of procoagulant activity. Thus, we have demonstrated that endothelial cells elaborate a potent inhibitor of monocyte procoagulant activity.
Assuntos
Aorta/fisiologia , Coagulação Sanguínea , Lipopolissacarídeos/farmacologia , Monócitos/fisiologia , Animais , Bovinos , Células Cultivadas , Endotélio/fisiologia , Humanos , Monócitos/efeitos dos fármacos , Músculo Liso Vascular/fisiologiaRESUMO
Incubated human peripheral blood monocytes release a factor(s) which specifically stimulates homologous vascular smooth muscle (SMC) but not endothelial cells (EC) to proliferate in vitro. Conditioned medium from activated monocytes significantly (P less than 0.001) stimulated proliferation of SMC within 6 days of culture while media from non-activated monocytes was less effective (P less than 0.01). Non-vascular smooth muscle cells (human myometrium) were less responsive to such media (P less than 0.01 and P less than 0.05, respectively). These findings suggest that blood monocytes may play a key and until now unsuspected role in arterial smooth muscle proliferation, a recognized event in the initial stages of atherogenesis.
Assuntos
Células Sanguíneas/citologia , Monócitos , Divisão Celular , Endotélio/citologia , Feminino , Humanos , Látex/farmacologia , Músculo Liso Vascular/citologia , Miométrio/citologia , Veias Umbilicais/citologiaRESUMO
The biocompatibility of materials at an implant site involves a complex interaction of cells and tissues with the biomaterial. This cell-cell and cell-polymer interaction evokes the release of mediators such as chemotactic and growth factors that elicit and sustain inflammatory responses at the implant site. In this review, we summarize the interaction of cells with biomaterials in vitro and in vivo.
Assuntos
Materiais Biocompatíveis , Fenômenos Fisiológicos Celulares , Animais , Osso e Ossos/citologia , Comunicação Celular , Endotélio/citologia , Fibroblastos/fisiologia , Reação a Corpo Estranho , Humanos , Macrófagos/fisiologiaRESUMO
Human vascular endothelial cells (ECs) are exposed to various levels of hemodynamic forces, cyclic strain, and shear stress in vivo. Here, we examined the in vitro effects of the various levels (0-6%, 7-16%, and 17-25%) of strain at 60, 30, and 15 cycles per minute (cpm) on human monocyte adherence to endothelial cells and extracellular matrix protein preabsorbed surfaces. Monocyte adhesion to endothelial cells under cyclic strain significantly increased. At both 30 and 60 cpm, ECs under strains of 7-16% and 17-25% showed >52% and >117% higher monocyte adhesion than endothelial cells under static condition when monocytes were added for 0.5 h. This increase in monocyte adhesion to ECs under cyclic strain remained significantly higher even after 24 h of incubation. Human monocyte adhesion to extracellular matrix protein preabsorbed surfaces differed depending on the specific extracellular matrix protein. Monocytes adhered to collagen type I and fibronectin preabsorbed surfaces >50% under 0-6% strain, >23% under 7-16% strain, and >52% under 17-25% strain at 15 and 30 cpm compared to the collagen type V preabsorbed surface. However, when extracellular protein preabsorbed surfaces under cyclic strain were compared to the control static condition, monocyte adhesion did not significantly change on most of other surfaces. These results suggest that cyclic strain may play a role in the regulation of monocyte-endothelial cells/extracellular matrix interactions in vivo.
Assuntos
Endotélio Vascular/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Monócitos/fisiologia , Adesão Celular , Células Cultivadas , Endotélio Vascular/citologia , Humanos , Monócitos/citologia , Neutrófilos/citologia , Neutrófilos/fisiologia , Estresse Mecânico , Veias UmbilicaisRESUMO
Endothelial cell seeding of synthetic small diameter vascular grafts (SSDVG) has been shown to diminish thrombosis and intimal hyperplasia, resulting in improved graft patency. However, endothelial cell retention on seeded grafts when exposed to physiological shearing conditions remains poor. We report that the genetic engineering of endothelial cells to overexpress endothelial nitric oxide synthase (eNOS), may create improved anti-thrombotic and anti-hyperplastic endothelial cell phenotypes for SSDVG seeding. eNOS-overexpressing endothelial cells may potentially overcome the biochemical loss due to shear induced reduction in endothelial cell coverage on SSDVG. Bovine aortic endothelial cells (BAEC) were transfected with the human eNOS gene, and co-incubated with either human whole blood or bovine aortic smooth muscle cells (BASMC) in vitro. eNOS-transfected BAEC significantly overexpressed eNOS compared to control beta-Gal-transfected and untransfected BAEC up to 120 h post transfection. In co-incubation and co-culture assays, human platelet aggregation decreased by 46% and BASMC proliferation decreased by 67.2% when compared to incubation with untransfected BAEC.
Assuntos
Plaquetas/fisiologia , Comunicação Celular , Endotélio Vascular/fisiologia , Engenharia Genética , Músculo Liso Vascular/fisiologia , Óxido Nítrico Sintase/fisiologia , Agregação Plaquetária , Animais , Bovinos , Divisão Celular/fisiologia , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/citologia , Humanos , Músculo Liso Vascular/citologia , Óxido Nítrico Sintase Tipo IIIRESUMO
Tissue culture techniques commonly applied to the study of human vascular smooth muscle were used to evaluate in vitro survival and proliferation of normal and neoplastic human myometrial cells. Despite their growth advantage in vivo, leiomyoma cells displayed a growth disadvantage in vitro compared with normal myometrium from the same patient. Hormonal supplementation with alpha-estradiol, progesterone, and insulin-stimulated myometrial proliferation, whereas beta-estradiol appeared ineffective at the doses tested. Hormonal supplementation also stimulated leiomyoma proliferation in vitro, but there appeared to be heterogeneity in hormonal responsiveness. Heterogeneity in the host hormonal milieu and in the ability of uterine leiomyomas to respond to various hormones may be important factors contributing to the wide variation in growth potential observed in leiomyomas.
Assuntos
Divisão Celular , Leiomioma/patologia , Miométrio/citologia , Neoplasias Uterinas/patologia , Adulto , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Estradiol/farmacologia , Feminino , Humanos , Insulina/farmacologia , Leiomioma/metabolismo , Pessoa de Meia-Idade , Miométrio/metabolismo , Progesterona/farmacologia , Receptores de Estrogênio/análise , Receptores de Progesterona/análise , Neoplasias Uterinas/metabolismoRESUMO
Lipid microtubules that self-assemble from a diacetylenic lipid are suitable structures for the sustained release of bioactive agents. Microtubules were loaded with agents under aqueous conditions and embedded in an agarose hydrogel for localization at areas of interest. Protein release from our microtubule-hydrogel delivery system was characterized in vitro, and in vivo biocompatibility was examined. The influences of protein molecular weight and initial loading concentration on release profile were evaluated by releasing test proteins myoglobin, albumin, and thyroglobulin. Protein molecular weight inversely affected the release rate, and loading with a higher protein concentration increased the mass but not the percent of initially loaded protein released daily. Preservation of protein activity was demonstrated by the ability of a neurotrophic factor released from the delivery system to induce neurite extension in PC12 cells. Bovine aortic smooth muscle cells co-cultured with the microtubule-hydrogel system showed no evidence of cytotoxicity and proliferated in the presence of the microtubules. Subcutaneous implantation of microtubules in rodents revealed no significant inflammatory response after 10 days. Our microtubule-hydrogel system is useful for applications where sustained release without contact between agent and organic solvents is desired.
Assuntos
Microtúbulos/metabolismo , Animais , Técnicas de Cocultura , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Hidrogéis , Lipídeos , Teste de Materiais , Peso Molecular , Células PC12 , Veículos Farmacêuticos , Ratos , Ratos Sprague-DawleyRESUMO
A new method for isolation and culture of endothelial cells from bovine coronary artery (BCoAEC) is presented. This method involves in situ perfusion and digestion of main coronary arteries with a collagenase solution. The isolated cells were cultured and maintained through many cell passages in Dulbecco's Modified Eagle's Medium supplemented with fetal bovine serum derived from either whole blood or plasma. Confirmation of these cells' endothelial origin was obtained by demonstration of typical morphologic and growth characteristics of endothelium, immunofluorescent staining with antibodies to von Willebrand factor (Factor VIII: vWF), and measurement of plasminogen activator (PA). In addition, production of PA was inhibited by enzymatically active thrombin as has been previously described with bovine aortic endothelial cells in culture.
Assuntos
Vasos Coronários/metabolismo , Técnicas Citológicas , Ativadores de Plasminogênio/biossíntese , Animais , Artérias , Bovinos , Divisão Celular , Separação Celular , Vasos Coronários/citologia , Endotélio/citologia , Endotélio/metabolismo , Ativadores de Plasminogênio/antagonistas & inibidores , Inativadores de Plasminogênio , Trombina/farmacologiaRESUMO
OBJECTIVE: To study the effects of estrogen on transendothelial paracellular permeability in women. METHODS: Human umbilical vein endothelial cells (HUVEC) obtained from women were grown on filters. The paracellular permeability characteristics were determined in terms of changes in the permeability to the polar acid pyranine (Ppyr) and as changes in the transendothelial electrical resistance (RTE). Tight junctional resistance characteristics were assayed by lowering luminal NaCl and measuring the dilution potential, and were expressed as the ratio of monoion mobility uCl/uNa (cation selectivity). RESULTS: Low extracellular calcium and hyperosmolarity increased Ppyr and decreased RTE. The former but not the latter condition abolished the endothelium-specific cation selectivity. Treatment with 10 nM of estradiol-17 beta had no effect on RTE, but it increased the cation selectivity. The effect of estradiol required 1-6 hours' incubation with the hormone; it was dose dependent and saturable, with a median effective concentration of estradiol of 1 nM. Diethylstilbestrol, but not estriol, could mimic the effect of estradiol, and the estrogen receptor antagonist ICI-182, 780 blocked it. CONCLUSION: Cultured HUVEC cells form patent tight junctions. Estrogens increase the cation selectivity across HUVEC cultures. The effect of estrogen may be mediated by an estrogen receptor. These effects may be important for vasculoprotection in cases of sudden changes in ions levels across the capillary wall, such as ischemia or reperfusion.
Assuntos
Endotélio Vascular/ultraestrutura , Estradiol/farmacologia , Junções Íntimas/efeitos dos fármacos , Veias Umbilicais , Cálcio/administração & dosagem , Cátions , Permeabilidade da Membrana Celular , Células Cultivadas , Cloretos/metabolismo , Dietilestilbestrol/farmacologia , Impedância Elétrica , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Antagonistas de Estrogênios/farmacologia , Feminino , Fulvestranto , Humanos , Cinética , Concentração Osmolar , Gravidez , Sódio/metabolismo , Junções Íntimas/fisiologiaRESUMO
The design and development of new biomedical polymers for clinical application in devices, prostheses, and artificial organs requires a basic and fundamental understanding of biological interactions with biomedical polymers. Efforts in our laboratory have been directed towards appreciating the humoral and cellular interactions which govern protein adsorption and cellular adhesion and activation on biomedical polymers. Information and data are presented on protein adsorption from whole human blood, complement activation and receptors, and monocyte/macrophage adhesion and activation with growth factor release. Supported by experimental evidence, concepts regarding protein/polymer, cell/polymer, cell/protein/polymer, and cell/cell interactions as they are related to in vivo events are presented.
Assuntos
Materiais Biocompatíveis , Proteínas Sanguíneas/farmacocinética , Polímeros , Adsorção , Adesão Celular , Ativação do Complemento , Dimetilpolisiloxanos , Substâncias de Crescimento , Humanos , Ativação de Macrófagos , Polietilenotereftalatos , Polietilenos , Politetrafluoretileno , Receptores de Complemento , Elastômeros de Silicone , SiliconesRESUMO
PURPOSE: Human vascular prostheses develop a pseudointima that after time is devoid of a complete endothelial lining. The composition of this pseudointima consists of both cellular and noncellular components such as coagulation proteins and extracellular matrix proteins. Of these extracellular molecules, type V collagen has been reported to be localized to surfaces of vascular prostheses. We have hypothesized that type V collagen may be involved in the mechanisms of inhibition of endothelialization on vascular prostheses. In this study, interactions of human protein and cells with clinically used material and reference surfaces were analyzed in vitro. METHODS AND RESULTS: Human collagen types IV and V or human fibronectin was coated on disks punched from expanded polytetrafluoroethylene, bacteriologic polystyrene, and tissue culture-treated polystyrene. Fibronectin adsorbed equally to these surfaces, but differential adsorption of type V collagen occurred. The attachment and growth of human saphenous vein and umbilical vein endothelial cells and of human skin fibroblasts were also evaluated on protein-coated or uncoated surfaces. Type IV collagen and fibronectin promoted the attachment of these cells, but type V collagen reduced cellular adhesion. Growth of endothelial cells was significantly inhibited on surfaces coated with type V collagen even when additional growth substances such as serum, retinal-derived growth factor, and heparin were present in the medium. Human adult dermal fibroblast adhesion and cell growth were not affected by coating the surfaces with type V collagen. CONCLUSIONS: The components of the extracellular matrix of the pseudointima may directly influence endothelial cells by inhibiting cell proliferation, migration from within the graft or from anastomoses, or both. Type V collagen, a matrix protein found at luminal surfaces of vascular prostheses, may be one protein responsible for control of endothelial responses.
Assuntos
Colágeno/farmacologia , Endotélio Vascular/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Depressão Química , Endotélio Vascular/citologia , Fibronectinas/farmacologia , Humanos , Poliestirenos/farmacologia , Politetrafluoretileno/farmacologia , Propriedades de Superfície , Fatores de TempoRESUMO
Synthetic vascular grafts implanted into humans fail to develop a complete endothelial lining. In previous studies, we have shown that high-molecular-weight kininogens (HMWK) adsorb to the surfaces of biomaterials. In addition, it has been demonstrated that these proteins modulate cellular function. In the present study, we report on the adhesion and proliferation of human umbilical-vein endothelial cells (HUVEC) on tissue culture polystyrene, glass, polyurethane, and Mylar(trade mark) surfaces coated with human HMWK, either single-chain HMWK (SC-HMWK) or double-chain HMWK (DC-HMWK). Surfaces coated with fibronectin served as a positive control for these experiments. Parallel experiments were performed in which HUVEC were allowed to migrate from crosslinked dextran microcarrier beads (Cytodex 2) onto HMWK-coated surfaces. Our results indicate that HMWK-coated surfaces inhibit endothelial cell adhesion, proliferation, and migration at 24 and 72 h, and this inhibition is concentration dependent. To determine a potential mechanism for this inhibitory phenomenon, cells were stained for cytoskeletal actin filaments using rhodamine-phalloidin. Endothelial cells on HMWK-coated surfaces displayed F-actin filament reorganization/disassembly, characterized by the absence of peripheral actin bands in focal adhesion contacts. We conclude that HMWK inhibit endothelial cell adhesion, proliferation, and migration on a variety of biomaterial surfaces. This inhibitory effect may play a role in promoting the lack of endothelialization in synthetic vascular grafts, which is thought to play a significant role in the failure of these devices.
Assuntos
Materiais Biocompatíveis , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Cininogênios/farmacologia , Adsorção , Vasos Sanguíneos/transplante , Adesão Celular/efeitos dos fármacos , Contagem de Células , Divisão Celular/efeitos dos fármacos , Movimento Celular , Citoesqueleto/fisiologia , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Humanos , Cininogênios/química , Proteínas/química , Veias Umbilicais/citologiaRESUMO
The failure of synthetic vascular grafts in humans is complex and related not only to the type of material but also to the accumulation of cellular and noncellular components onto or within the material. Proteins that adsorb on biomaterials such as vascular grafts may be involved in modulating cell function. Of these components, type V collagen and high molecular weight kininogens (HMWKs) are known to localize on the surface of vascular grafts. We have investigated the effect of type V collagen and HMWKs in the prevention of endothelialization of vascular prostheses by inhibiting cell migration. Inhibition of endothelial cell migration may lead to incomplete endothelialization and thus, failure of these devices. Various concentrations of type V collagen and HMWK solutions were placed at the bottom of 24 well tissue culture plates with porous membrane inserts (8 microns pore size) placed on top. Suspended human umbilical vein endothelial cells (HUVECs) were placed on top of the insert and incubated at 37 degrees C for 2 hours. Following incubation, the inserts were fixed and the cells that migrated through the pores were counted by microscopy. Our results showed that migration of endothelial cells was significantly inhibited in a dose dependent manner by type V collagen and HMWKs.
Assuntos
Movimento Celular/efeitos dos fármacos , Colágeno/farmacologia , Endotélio Vascular/citologia , Cininogênio de Alto Peso Molecular/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Monócitos/citologia , Neutrófilos/citologiaRESUMO
Vascular endothelial cells (ECs) are constantly subjected to hemodynamic forces that may regulate monocyte-endothelial interaction in vivo. To examine the effects of cyclic strain on endothelial expression of monocyte adhesion molecules, E-selectin, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) ECs were exposed to physiologically relevant levels of cyclic strain. When ECs were under 25% maximal strain at 30 cycles/min for 24 h, the expression of E-selectin significantly (p<0.05) increased, by 83%, compared to control ECs under static conditions. Similarly, monocyte adhesion to ECs under strain (maximum of 15 or 25% at 30 and 60 cycles/min for 24 h) also significantly (p<0.05) increased, by >82%. This cyclic-strain-induced monocyte adhesion was substantially inhibited (83.5%) by anti-E-selectin antibody. ICAM-1 expression also significantly increased, by 62%, when ECs were under 25% maximal strain at 30 cycles/min for 3 h whereas VCAM-1 expression by ECs under strain (for 0.5, 3, and 24 h) did not change compared to static ECs. When ECs were treated with anti-ICAM-1 antibody and monocytes with anti-VLA-4 antibody, an increase in monocyte adhesion to ECs under cyclic strain was reduced significantly. These results demonstrate that cyclic strain can induce EC expression of monocyte adhesion molecules E-selectin, ICAM-1, and VCAM-1 in a time-dependent manner and thus can mediate monocyte adhesion.
Assuntos
Adesão Celular/fisiologia , Selectina E/genética , Endotélio Vascular/fisiologia , Molécula 1 de Adesão Intercelular/genética , Monócitos/fisiologia , Molécula 1 de Adesão de Célula Vascular/genética , Adesão Celular/efeitos dos fármacos , Células Cultivadas , Cicloeximida/farmacologia , Selectina E/análise , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Técnicas In Vitro , Molécula 1 de Adesão Intercelular/análise , Interleucina-1/farmacologia , Monócitos/citologia , Estresse Mecânico , Veias Umbilicais , Molécula 1 de Adesão de Célula Vascular/análiseRESUMO
Protein adsorption from human plasma was investigated on phospholipid polymers, poly (2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA) or glass by radioimmunoassay and immunogold labeling techniques. In the present studies the focus was to determine the composition and distribution of proteins at the surface of these materials after contact with human blood plasma. On all materials, protein adsorption was detected and included identification of albumin, IgG, fibrinogen, fibronectin, Hageman factor (factor XII), factor VIII/von Willebrand factor, high-molecular-weight kininogen (HMWK) and the complement protein C5. The amount of protein adsorbed decreased with an increase in the MPC composition and appeared to adsorb to the surfaces in a uniform and evenly distributed manner. Therefore, we suggest that MPC moieties play an important role in suppression of protein adsorption. From these findings, it is concluded that the reduction of protein adsorption at the blood contacting surface of phospholipid polymers may result in the inhibition of thrombus formation.
Assuntos
Proteínas Sanguíneas/química , Fosfolipídeos/química , Adsorção , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Radioisótopos do Iodo , Masculino , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Polímeros , Radioimunoensaio , Trombose/prevenção & controleRESUMO
Commercial samples of atactic polypropylene (aPP) and polyvinyl methyl ether (PVME) were purified and spin-cast onto glass coverslips with a view to using these as model surfaces in thrombogenicity studies. These materials differ from polyvinyl alcohol (PVA) in a single functional group and are similarly amorphous: with the same backbone they have a hydroxyl, a methoxy, or a methyl group. The objective was to understand the role of the hydroxyl group in the platelet reactivity of PVA. Surface characterization showed that they were chemically pure (as determined by X-ray photoelectron spectroscopy) but not smooth (as determined by scanning electron microscopy or interferometry), presumably due to the difficulties of spin-casting optically clear films from hot solutions (aPP or polyethylene [PE]) or because of imperfect adhesion to the saline-treated substrate (PVME). PVME was also gamma-irradiated to insolubilize it. Fewer platelets adhered to PVA than to PVME or to aPP and PE, but roughness effects and limited data preclude definitive conclusions regarding the effect of functional groups. Less protein was found on PVA than on the hydrophobic surfaces, but the significance of this observation is unclear. Further studies with more sensitive protocols are called for to examine the extent of platelet activation and its relationship to surface chemistry.
Assuntos
Anticorpos , Materiais Biocompatíveis , Proteínas Sanguíneas , Polipropilenos , Polivinil , Adsorção , Animais , Fator XII , Fibrinogênio , Raios gama , Humanos , Interferometria , Microscopia Eletrônica de Varredura , Análise de Ativação de Nêutrons , Ovalbumina , Álcool de Polivinil , Coelhos , Albumina Sérica , Espectrometria por Raios X , Propriedades de Superfície , alfa-Fetoproteínas , Fator de von WillebrandRESUMO
Estradiol had a biphasic effect on permeability across cultures of human umbilical vein endothelial cells (HUVEC): at nanomolar concentrations it decreased the HUVEC culture permeability, but at micromolar concentrations it increased the permeability. The objective of the present study was to test the hypothesis that the changes in permeability were mediated by nitric oxide (NO)-related mechanisms. The results revealed dual modulation of endothelial paracellular permeability by estrogen. 1) An endothelial NO synthase (eNOS)-, NO-, and cGMP-related, Ca2+-dependent decrease in permeability was activated by nanomolar concentrations of estradiol, resulting in enhanced Cl- influx, increased cell size, and increases in the resistance of the lateral intercellular space (RLIS) and in the resistance of the tight junctions (RTJ); these effects appeared to be limited by the ability of cells to generate cGMP in response to NO. 2) An inducible NO synthase (iNOS)- and NO-related, Ca2+-independent increase in permeability was activated by micromolar concentrations of estradiol, resulting in enhanced Cl- efflux, decreased cell size, and decreased RLIS and RTJ. We conclude that the net effect on transendothelial permeability across HUVEC depends on the relative contributions of each of these two systems to the total paracellular resistance.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Endotélio Vascular/metabolismo , Estradiol/farmacologia , Óxido Nítrico Sintase/fisiologia , Sulfonatos de Arila/farmacocinética , Células Cultivadas , Cloretos/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Condutividade Elétrica , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Inibidores Enzimáticos/farmacologia , Espaço Extracelular/metabolismo , Feminino , Humanos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo II , Óxido Nítrico Sintase Tipo III , Nitroprussiato/farmacologia , RNA Mensageiro/metabolismo , Sódio/metabolismoRESUMO
Protein adsorption to surfaces occurs whenever blood comes into contact with biomaterials, prosthetic devices, and artificial organs. The plasma protein Hageman factor (factor XII) present at the interface between blood and foreign surfaces can be qualitatively and quantitatively detected after in vitro perfusion of anticoagulated human blood through important biomedical polymers. We have determined protein adsorption by a modified radioimmunoassay and by scanning electron microscopy using immunogold labeling techniques. The materials used included vascular graft materials (Dacron and expanded polytetrafluoroethylene) and the National Heart, Lung, and Blood Institute-Devices and Technology Branch reference materials polydimethylsiloxane, polyethylene, and silicone rubber. Factor VIII-von Willebrand factor, another trace plasma protein, and other plasma proteins such as fibrinogen, immunoglobulin G, albumin, fibronectin, and hemoglobin were also detected at the blood-contacting surface. At physiologic flow rates, the adsorption of these proteins from the circulating blood to the surface of these materials appears to be a function of time, with certain materials, as well as of the physical and chemical characteristics of the material surface. Hageman factor adsorption to surfaces, quantified under static conditions, occurs at nanogram concentrations. These data suggest that trace proteins, such as those important in the activation of the coagulation cascade, can significantly affect the blood compatibility or thrombogenicity of an implanted device.
Assuntos
Materiais Biocompatíveis , Proteínas Sanguíneas/química , Fator XII/química , Polímeros , Adsorção , Dimetilpolisiloxanos , Fibrinogênio/química , Fibronectinas/química , Hemoglobinas/química , Humanos , Imunoglobulina G/química , Imuno-Histoquímica , Cinética , Microscopia Eletrônica de Varredura , Polietilenotereftalatos , Polietilenos , Politetrafluoretileno , Radioimunoensaio , Albumina Sérica/química , Elastômeros de Silicone , SiliconesRESUMO
Oligosaccharides of heparin with high affinity for antithrombin III (ATIII) have been immobilized onto surface-modified NHLBI Primary Reference low density polyethylene (PE). PE was modified by radiofrequency plasma polymerized (< 150 nm thick) films derived from N-vinyl-2-pyrrolidone (PPNVP) or allyl alcohol (PPAA), and coupled by chemical derivatization to either 3-aminopropyltriethoxysilane or amino-terminated poly(ethylene oxide). High affinity heparin oligosaccharides (HA-heparin, anti-factor Xa activity of 592 +/- 120 IU/mg) prepared by partial deaminative cleavage of commercial crude heparin and fractionated by agarose-ATIII affinity chromatography, were immobilized to surface-modified PE by reductive amination. The anticoagulant activity, as determined by a chromogenic assay for the inhibition of factor Xa, was estimated to be 30-70 mIU/cm2, with binding estimated to be 56-119 ng/cm2. The highest activity was obtained for the HA-heparin immobilized to PE modified by PPNVP with a PEO spacer. Visual confirmation of ATIII binding to immobilized HA-heparin was demonstrated by a gold-labeled double antibody method with imaging by SEM.