RESUMO
Since tissue oxygenation has a profound effect on capillary growth, the effect of pO2 on endothelial cell functions was studied. Under normoxic conditions, EA.hy926 endothelial cells and HUVEC plated onto fibrin gels in low-serum culture medium underwent rapid and profound morphological changes within 12 to 48 hours depending on the cell line used. Their characteristic cobblestone organisation was transformed into a network of cord-like or tube-like structures. We showed that when exposed to low oxygen concentrations for 3 days, HUVEC and EA.hy926 have their ability to rearrange reduced to around 50 %. With EA.hy926 this effect was amplified by 79% after 9 days of hypoxia. The altered behaviour of hypoxia-adapted cells was not caused by a loss in their fibrinolytic activity. In fact, the fibrin degradation rate and the generated fibrin fragments appeared identical in normoxia and hypoxia. Confocal microscopy and gel densitometry showed that in normoxia the remaining undegraded fibrin gel underwent a dynamic remodeling whereas in hypoxia it remained undisturbed. It is likely that hypoxia induces modification in the factors that integrate matrix information and cytoskeletal organisation in order to contract fibrin.
Assuntos
Endotélio Vascular/citologia , Fibrina/metabolismo , Oxigênio/fisiologia , Divisão Celular , Hipóxia Celular/fisiologia , Linhagem Celular , Géis , Humanos , Fatores de TempoRESUMO
The mechanical properties of fibrin gels under uniaxial strains have been analyzed for low fibrin concentrations using a free-floating gel device. We were able to quantify the viscous and elastic moduli of gels with fibrin concentration ranging from 0.5 to 3 mg/ml, reporting significant differences of biogels moduli and dynamical response according to fibrin concentration. Furthermore, considering sequences of successively imposed step strains has revealed the strain-hardening properties of fibrin gels for strain amplitude below 5%. This nonlinear viscoelastic behavior of the gels has been precisely analyzed through numerical simulations of the overall gel response to the strain steps sequences. Phenomenological power laws relating the instantaneous and relaxed elasticity moduli to fibrin concentration have been validated, with concentration exponent in the order of 1.2 and 1.0, respectively. This continuous description of strain-dependent mechanical moduli was then used to simulate the biogel behavior when continuously time-varying strains are applied. We discuss how this experimental setup and associated macroscopic modeling of fibrin gels enable a further quantification of cell traction forces and mechanotransduction processes induced by biogel compaction or stretching.
Assuntos
Fibrina/química , Engenharia Biomédica , Biopolímeros/química , Elasticidade , Géis , Humanos , Técnicas In Vitro , Modelos Químicos , ViscosidadeRESUMO
In vitro angiogenesis assays have shown that the couplings between fibrin gel and cell traction forces trigger biogel pre-patterning, consisting, in the formation of lacunae which evolve toward capillary-like structures (CLS) networks. Depending on the experimental conditions (number of seeded cells, gel elasticity,...), this pre-patterning can be enhanced or inhibited. A theoretical model based on a description of the cell-biogel biochemical and mechanical interactions is proposed as a basis for understanding how integrating these interactions can lead to the pre-patterning of the biogel. We showed that the critical parameter values corresponding to the bifurcation of the model solutions correspond to threshold values of the experimental variables. Furthermore, simulations of the mechanocellular model give rise to dynamic remodelling patterns of the biogel which are in good agreement both with the lacunae morphologies and with the time and space scales derived from the in vitro angiogenesis assays. Special attention has been paid in the simulations to cell proteolytic activity and to the amplitude of cell traction forces. We finally discussed how modelling guided experiments can be inferred from these results.
Assuntos
Fenômenos Fisiológicos Celulares , Matriz Extracelular/fisiologia , Morfogênese , Neovascularização Fisiológica , Transdução de Sinais , Fenômenos Biomecânicos , Capilares , Endotélio Vascular , Humanos , Técnicas In Vitro , Matemática , Modelos Biológicos , Veias UmbilicaisRESUMO
Low-concentration biogels, which provide an extracellular matrix for cells in vitro, are involved in a number of important cell biological phenomena, such as cell motility and cell differentiation. In order to characterize soft tissues, which collapse under their own weight, we developed and standardized a new experimental device that enabled us to analyze the mechanical properties of floating biogels with low concentrations, i.e., with values ranging from 2 g/L to 5 g/L. In order to validate this approach, the mechanical responses of free floating agarose gel samples submitted to compression as well as stretching tests were quantified. The values of the Young's moduli, measured in the range of 1000 to 10,000 Pa, are compared to the values obtained from other experimental techniques. Our results showed indeed that the values we obtained with our device closely match those obtained independently by performing compression tests on an Instron device. Thus, the floating gel technique is a useful tool first to characterize and then to model soft tissues that are used in biological science to study the interaction between cell and extracellular matrix.
Assuntos
Géis/química , Teste de Materiais/métodos , Teste de Materiais/normas , Sefarose/química , Força Compressiva , Elasticidade , Reprodutibilidade dos Testes , Resistência à TraçãoRESUMO
In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50-100 microg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon--tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given.
Assuntos
Permeabilidade da Membrana Celular , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/ultraestrutura , Difosfato de Adenosina/metabolismo , Animais , Respiração Celular , Células Cultivadas , Creatina Quinase/metabolismo , Grupo dos Citocromos c/metabolismo , Humanos , Cinética , Microscopia Eletrônica , Microscopia de Fluorescência , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , NADP/metabolismo , Rotenona/farmacologia , Saponinas/farmacologia , Tripsina/metabolismoRESUMO
This study highlights the importance of several factors involved in the formation of capillary-like structure formation (CLS) using Human Umbilical Vein Endothelial Cells (HUVEC) and Bovine Retinal Endothelial Cells (BREC) cultured on fibrin gels. The fibrin concentration inducing (CLS) was 0.5 mg/ml for HUVEC and 8 mg/ml for BREC. The high fibrin concentration required for the latter cells appeared necessary to counterbalance the extensive fibrinolysis of the gel by the BREC. Fibrin degradation products measured in the culture media showed that fibrin degradation was mandatory but not sufficient for CLS formation. Fibrin degradation acted in concert with the mechanical, concentration dependent properties of the gels to induce CLS. For example, HUVEC did not form CLS on a rigid fibrin of 8 mg/ml in spite of fibrinolysis. As cell reorganisation occurred, the fibrin was disrupted (HUVEC) or pleated (BREC) giving indirect proof of the development of mechanical forces. During CLS formation, an increasing amount of latent TGFbeta1 was measured in the medium (1000-1700 pg/ml). The active form of TGFbeta1 was not, however, detected and the addition of anti-TGF-beta1 antibody to the medium did not influence the formation of the CLS network. Yet, added activated TGF-beta1 led to the formation of less organised structures, that were completely abolished by the concomitant addition of the same anti-TGF-beta1 antibody. Thus, it is likely that TGF-beta1 secreted by the endothelial cells remained in its latent form. In conclusion, a balance between the mechanical properties of fibrin and the fibrinolytic activity of each cell type may regulate CLS formation in our models. We think that the high fibrinolitic activity of the BREC may represent a defense mechanism to protect the retina against thrombosis-induced damage in vivo.
RESUMO
The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).
Assuntos
Difosfato de Adenosina/metabolismo , Respiração Celular/fisiologia , Citoesqueleto/metabolismo , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Musculares/metabolismo , Porinas , Animais , Anticorpos/imunologia , Células Cultivadas , Creatina/farmacologia , Citoesqueleto/ultraestrutura , Desmina/genética , Desmina/fisiologia , Difusão , Cinética , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Músculo Esquelético/metabolismo , Músculo Esquelético/ultraestrutura , Oxigênio/metabolismo , Permeabilidade , Ratos , Ratos Wistar , Azida Sódica/farmacologia , Tripsina/metabolismo , Tripsina/farmacologia , Canais de Ânion Dependentes de VoltagemRESUMO
This study deals with the role of the mechanical properties of matrices in in vitro angiogenesis. The ability of rigid fibrinogen matrices with fibrin gels to promote capillarylike structures was compared. The role of the mechanical properties of the fibrin gels was assessed by varying concentration of the fibrin gels. When the concentration of fibrin gels was decreased from 2 mg/ml to 0.5 mg/ml, the capillarylike network increased. On rigid fibrinogen matrices, capillarylike structures were not formed. The extent of the capillarylike network formed on fibrin gels having the lowest concentration depended on the number of cells seeded. The dynamic analysis of capillarylike network formation permitted a direct visualization of a progressive stretching of the 0.5 mg/ml fibrin gels. This stretching was not observed when fibrin concentration increases. This analysis shows that 10 h after seeding, a prearrangement of cells into ringlike structures was observed. These ringlike structures grew in size. Between 16 and 24 h after seeding, the capillarylike structures were formed at the junction of two ringlike structures. Analysis of the alpha(v)beta3 integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the alpha(v)beta3 integrin localized into adhesion plaques. Conversely, cell adhesion to fibrin shows a diffuse and dot-contact distribution. We suggest that the balance of the stresses between the tractions exerted by the cells and the resistance of the fibrin gels triggers an angiogenic signal into the intracellular compartment. This signal could be associated with modification in the alpha(v)beta3 integrin distribution.
Assuntos
Endotélio Vascular/fisiologia , Fibrina/fisiologia , Neovascularização Fisiológica , Receptores de Vitronectina/análise , Fenômenos Biomecânicos , Capilares/anatomia & histologia , Capilares/fisiologia , Adesão Celular , Divisão Celular , Células Cultivadas , Endotélio Vascular/química , Endotélio Vascular/citologia , Fibrina/administração & dosagem , Fibrinogênio/fisiologia , Géis , Humanos , Fenótipo , Veias UmbilicaisRESUMO
Traction forces developed by most cell types play a significant role in the spatial organisation of biological tissues. However, due to the complexity of cell-extracellular matrix interactions, these forces are quantitatively difficult to estimate without explicitly considering cell properties and extracellular mechanical matrix responses. Recent experimental devices elaborated for measuring cell traction on extracellular matrix use cell deposits on a piece of gel placed between one fixed and one moving holder. We formulate here a mathematical model describing the dynamic behaviour of the cell-gel medium in such devices. This model is based on a mechanical force balance quantification of the gel visco-elastic response to the traction forces exerted by the diffusing cells. Thus, we theoretically analyzed and simulated the displacement of the free moving boundary of the system under various conditions for cells and gel concentrations. This model is then used as the theoretical basis of an experimental device where endothelial cells are seeded on a rectangular biogel of fibrin cast between two floating holders, one fixed and the other linked to a force sensor. From a comparison of displacement of the gel moving boundary simulated by the model and the experimental data recorded from the moving holder displacement, the magnitude of the traction forces exerted by the endothelial cell on the fibrin gel was estimated for different experimental situations. Different analytical expressions for the cell traction term are proposed and the corresponding force quantifications are compared to the traction force measurements reported for various kind of cells with the use of similar or different experimental devices.
Assuntos
Endotélio Vascular/fisiologia , Matriz Extracelular/fisiologia , Modelos Biológicos , Modelos Teóricos , Animais , Contagem de Células , Linhagem Celular , Elasticidade , Fibrina/fisiologia , Géis , Humanos , Tração , ViscosidadeRESUMO
Angiogenesis is involved in numerous pathologies. Studies with in vitro models allow the description and analysis of the different steps involved in this process under defined culture conditions. We describe a controllable and reproducible in vitro model. We assessed the usefulness of this model with two different cell lines: human umbilical vein endothelial cells and bovine retinal endothelial cells. These cells reorganize themselves and change their phenotypes within 24 h after seeding under our culture conditions (low human serum percentage, defined cell density, fibrin matrix) to form 'capillary-like structures' (CLS) in vitro. We showed that, depending on the cell line used, the fibrinolytic activity of the cells was a determining factor which could induce or prevent the formation of the CLS. Inhibitors of angiogenesis can be tested using such a model.
Assuntos
Indutores da Angiogênese/antagonistas & inibidores , Indutores da Angiogênese/farmacologia , Endotélio Vascular/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Bovinos , Células Cultivadas , Fibrinólise/fisiologia , Humanos , Modelos Biológicos , Retina/citologia , Retina/efeitos dos fármacos , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacosRESUMO
We have studied the role of 2 exogenous cytokines, the TGF beta 1 and the VEGF, on the in vitro angiogenesis process. Endothelial cells were plated on fibrin matrices either with 2% or with 10% human serum in 199 medium. Forty-eight hours later, with 10% human serum, the capillary-like network index (the percentage of the culture area covered by the capillary network) was about 50%. Under these culture conditions, when TGF-beta 1 or VEGF were added, the capillary-like network index augmented and was nearing 75%. When the index is high, using confocal microscopy, we show that hollow capillaries are formed. Moreover, the addition of VEGF increased the kinetics of the capillary-like network formation. With 2% human serum, 48 h after seeding the capillary-like network index was about 75%. In this case, the addition of TGF-beta 1 decreased the network index, whereas the addition of VEGF increased the kinetics of its formation. These in vitro angiogenesis experiments show that the serological factors underline the 2 antagonist effects of TGF-beta 1, but have no detectable effects on the activator effects on the VEGF.
Assuntos
Fatores de Crescimento Endotelial/química , Linfocinas/química , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Transformador beta/química , Divisão Celular/efeitos dos fármacos , Endotélio Vascular/citologia , Humanos , Técnicas In Vitro , Microscopia Confocal , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
CHO cells spread out on fibronectin-coated plastic were perforated with the bacterial toxin alveolysin. This treatment preserves the integrity of the cells but opens large and stable hydrophilic pores on the plasma membrane. With these semi-intact cells, it has been possible to have a direct access to adhesion plaques. Furthermore, with this procedure one can determine the distribution of a single intracellular protein between membrane-associated and cytosolic pools. The introduction within the perforated cells of polyclonal antibodies raised against talin induced the detachment of the cells. This provides direct evidence that talin is required to stabilize the adhesion plaques. Furthermore, immunoprecipitation of talin in the membrane-associated and cytosolic fractions indicated that the membrane-associated talin was cleaved and reduced to a stable 200-kDa fragment. Conversely, soluble talin remained intact. This 200-kDa fragment was similar to the proteolytic fragment produced from talin by calpain II. Analysis of whole cell extracts and pulse chase experiments indicated that this proteolysis also occurred in vivo, although to a smaller extent. The kinetics of the limited proteolytic cleavage of talin suggested that the 200-kDa fragment was first produced within focal adhesion, and secondarily released into the cytosol.
Assuntos
Adesão Celular/fisiologia , Processamento de Proteína Pós-Traducional/fisiologia , Talina/metabolismo , Animais , Anticorpos , Anticorpos Monoclonais , Células CHO , Permeabilidade da Membrana Celular/fisiologia , Cricetinae , Citosol/metabolismo , Magnésio/fisiologia , Talina/fisiologiaRESUMO
We have investigated the dynamics between free fibronectin receptors and clusters of them organized into adhesion plaques on CHO cells using the ability of these free integrins to be endocytosed and recycled to the plasma membrane. Indirect inhibition of the endocytic cycle by monensin resulted in the subsequent internalization of free receptors, which we followed by indirect immunostaining and confocal microscopy. Consequently, all the adhesive structures that were in equilibrium with free integrins became progressively disorganized. The cellular morphological changes were analyzed and correlated with the distribution of cell-substratum contacts viewed by confocal images obtained after immunostaining with antibodies raised against the fibronectin receptor, talin, vinculin and actin. After cell adhesion to fibronectin, blockage of the endocytic cycle induced disruption of the adhesion plaques that were mainly localized at the cell periphery, and disappearance of the stress fibers. However, the cells remained firmly attached to the substratum through focal contacts localized in the central part of the cell. These central focal contacts, but not the peripheral adhesion plaques, could form when the vesicular traffic was blocked prior to adhesion and they allowed the cells to attach and flatten onto the substratum. Whereas both adhesive structures contained the same receptors linked to talin and vinculin, the central adhesive structures were attached to a short stretch of actin but never permitted the organization of stress fibers.
Assuntos
Células CHO/citologia , Fibronectinas/metabolismo , Receptores de Fibronectina/metabolismo , Animais , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Células CHO/ultraestrutura , Adesão Celular/efeitos dos fármacos , Cloroquina/farmacologia , Cricetinae , Cricetulus , Endocitose/efeitos dos fármacos , Humanos , Integrinas/metabolismo , Microscopia de Fluorescência , Monensin/farmacologia , Agregação de Receptores , Transdução de Sinais , Talina/metabolismoRESUMO
A new approach was investigated to study the interaction between integrins and actin via intracytoplasmic proteins. Because intracellular processes are hampered by the limiting plasma membrane, we developed an in vitro model with cells perforated by a bacterial toxin, streptolysin O. The specific conditions for the use of permeabilized cells to study the intramolecular associations occurring at adhesion plaques are described. The two cell types used, HUVEC and CHO, showed that the choice of the perforation method is of great importance. After perforation of cells in a monolayer, 75 +/- 10% of the cells remained adherent to a fibronectin substrate; after perforation of cells in suspension, only 25 +/- 10% of the cells readhered. Specific conditions were required however to maintain these adhesive properties up to 4 h: the presence of 1 mM Mg++ in the medium was crucial, and it was necessary to layer the cells on a specific coat rather than a substitute such as gelatin. Immunofluorescence investigations of actin, talin and vinculin, and Normarsky differential interference contrast microscopy showed retention of focal adhesion plaques in perforated cells. Moreover, in perforated cells antibodies directed against actin led to actin disorganization, showing that our model of perforated cells in a monolayer can give new insight to adhesion study.
Assuntos
Adesão Celular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Estreptolisinas/farmacologia , Actinas/análise , Animais , Proteínas de Bactérias , Células CHO , Células Cultivadas , Cricetinae , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Fibronectinas , Imunofluorescência , Humanos , Magnésio/farmacologia , Talina/análise , Vinculina/análiseRESUMO
An in vitro assay has been developed using semi-intact cells, made with the bacterial toxin streptolysin O, in order to measure integrin activity in relation to the cytosol environment. In this assay, the cytosolic content can easily be modified while the receptor binding activity is measured by monitoring the interaction of specific radiolabeled substrates with the cell surface. Using two different cell types, i.e., wild-type Chinese hamster ovary cells and human endothelial cells in culture, it has been shown that the binding activities of the fibronectin and fibrinogen receptors become cytosol-dependent on perforated cells. Furthermore, this control depends on micromolar concentrations of intracellular calcium, suggesting that calcium or calcium binding protein(s) may play a key role in controlling integrin activity.
Assuntos
Integrinas/fisiologia , Glicoproteínas da Membrana de Plaquetas/fisiologia , Receptores Imunológicos/fisiologia , Animais , Proteínas de Bactérias , Cálcio/fisiologia , Linhagem Celular , Permeabilidade da Membrana Celular , Técnicas Citológicas , Citosol/fisiologia , Endotélio Vascular/citologia , Fibrinogênio/metabolismo , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores de Fibronectina , Receptores Imunológicos/metabolismo , Estreptolisinas/farmacologiaRESUMO
The cytoadhesins represent a group of RGD receptors that belongs to the integrin superfamily of adhesion molecules. Members of this cytoadhesin family include the platelet GPIIb-IIIa and the vitronectin receptors. These glycoproteins share the same beta-subunit, which is associated with different alpha subunits to form an alpha/beta heterodimer. In the present study, we have analyzed the fine recognition specificy of the cytoadhesins from platelets and endothelial cells for the adhesive protein, fibrinogen. Two sets of synthetic peptides, RGDX peptides and peptides corresponding to the COOH terminus of the fibrinogen gamma chain, were compared for their structure-function relationships in the two cellular systems. The results indicate that: (a) both RGDX and gamma-chain peptides inhibit the binding of fibrinogen to platelets and endothelial cells; (b) a marked influence of the residue at the COOH- and NH2-terminal positions of each peptide set can be demonstrated on the two types; and (c) RGDX and gamma peptides have differential effects on platelets and endothelial cells with respect to fine structural requirements. These results clearly indicate that while the platelet and endothelial cytoadhesins may interact with similar peptidic sequences, they express a different fine structural recognition.
Assuntos
Plaquetas/metabolismo , Adesão Celular , Endotélio Vascular/metabolismo , Fibrinogênio/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Citoesqueleto de Actina/ultraestrutura , Sequência de Aminoácidos , Ligação Competitiva , Humanos , Técnicas In Vitro , Oligopeptídeos/metabolismo , Agregação Plaquetária , Solubilidade , Relação Estrutura-AtividadeRESUMO
The effect of mitomycin C (MMC) on the biosynthesis of prostacyclin was tested in culture of human umbilical cord vein endothelial cells. A 30% inhibition of the thrombin-stimulated prostacyclin synthesis by MMC was observed at concentrations of the same order as those found in MMC-treated patients (3 micrograms/ml as compared with the peak plasma concentration varying between 0.4 and 3.2 micrograms/ml (J. Den Hartigh et al., Cancer Res 40:5017-5021, 1983)). This inhibition was found for incubation times ranging from 15 to 30 min during which the cell viability was unaltered. Under these conditions it was found that the release of von Willebrand factor by the endothelial cell was unaffected. Since MMC toxicity in man is expressed by a chronic haemolytic and uraemic syndrome, the inhibitory capacity of MMC on prostacyclin synthesis favours the hypothesis that a deficiency in prostacyclin synthesis leads to the development of this syndrome in man.
Assuntos
Endotélio Vascular/metabolismo , Epoprostenol/biossíntese , Mitomicinas/farmacologia , 6-Cetoprostaglandina F1 alfa/farmacologia , Aspirina/farmacologia , Calcimicina/farmacologia , Humanos , Técnicas In Vitro , Mitomicina , Trombina/farmacologia , Fator de von Willebrand/metabolismoRESUMO
The structure of proteolytically modified fibrin and a closely related modified fibrinogen aggregate have been studied by analysis of electron microscope images. For both structures, we propose a model that consists of double-stranded, 2-fold helical protofibrils, which are associated laterally to form ordered fibrils, with a C222 space group: a = 44.0 nm, b = c = 9.4 nm. Each fibril is 80 nm or less in diameter, and twists along its length in a right-handed sense, with a pitch from 7 to 12 times the molecular length. The fibrils associate laterally to form bundles, which tend to twist in a left-handed sense, with a pitch of the order of 40 times the molecular length. The specific volume of modified fibrin calculated from this model is 3.9 A3 per dalton, which is comparable to the specific volume of 3.6 A3 per dalton for modified fibrinogen crystals but is lower than the 6 A3 per dalton determined for fibrin from light-scattering experiments. Comparison of our electron microscope results with X-ray and neutron diffraction data suggest a similar, but less well-ordered, structure for native fibrin, with a smaller fibril, approximately 18.4 nm wide, consisting of eight protofibrils.