Effects of prolonged exposure to hypoxia on morphological changes of endothelial cells plated on fibrin gel

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.     

Semi-intact CHO and endothelial cells: a tool to probe the control of integrin activity?

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.     

In vitro angiogenesis is modulated by the mechanical properties of fibrin gels and is related to αvβ3 integrin localization

Summary 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 α_vβ₃ integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the α_vβ₃ 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 α_vβ₃ integrin distribution.     

An in vitro model giving access to adhesion plaques

Summary 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.     

Structural study of modified fibrinogen microcrystals by electron microscopy

The same molecular shape of proteolytically modified fibrinogen has been identified in two different crystalline forms: orthorhombic P2₁2₁2 with a = 44.7(± 1.7) nm, b = 11.8(± 1.0) nm, c = 3.8 nm and monoclinic P2₁ with a = 17.7(± 0.6) nm, b = 16.2(± 1.0) nm, c = 4.8 nm and β = 95 °. The shape of the molecule is more detailed than has been reported. It has the commonly accepted elongated form 44.5(± 1.5) nm long with a 2-fold axis perpendicular to the major axis of the molecule. Each end domain shows a distinctive substructure and has an asymmetry similar to that reported by Williams (1981) and Mosesson et al. (1981) from single molecule observations. The ends are flattened, having overall dimensions of approximately 12 nm × 9 nm × 4 nm. The major difference between this model and previous models is the absence of a central nodule. There are, however, two small additional protein-dense regions at 5.0 nm from the centre of the rod connecting the two ends. A rough estimate of the relative heights of the different parts of the molecule was obtained using the two different projections of the molecule obtained from the two different crystalline forms. The molecule appears to be slightly bent at the centre, as predicted by Doolittle (1977).     

The proteolytic action of bacterial proteases from Clostridium histolyticum on bovine fibrinogen.

Bacterial proteases from Clostridium histolyticum bring about the polymerization of fibrinogen into structured fibres, the period being 9 nm when the ionic strength (I) of the solution is between 0.1 and 0.2. At I=0.3 no polymerization occurs, but the successive actions of bacterial proteases and thrombin induce polymerization, the fibres obtained showing a periodic structure: the major period is 23 nm with two minor striations. The striation pattern is different from that obtained with fibrin which shows a major period of 23 nm with three minor striations. The degree of degradation of fibrinogen monitored by disc gel electrophoresis shows that the Aalpha polypeptide chain (mol. wt.=68 000) is degraded into Aalpha1 (mol. wt.=32 000) and Aalpha2 (mol. wt.=29 000), whilst the mobility of Bbeta increases and gamma remains unchanged; the molecular weight is estimated between 272 000 and 269 000. These results emphasize that the charge distribution differences which are compatible with the loss of different portions of the molecule, lead to variations in fibre structures.     

Differential structural requirements for fibrinogen binding to platelets and to endothelial cells

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.     

Electron microscope structural study of modified fibrin and a related modified fibrinogen aggregate

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.     

Modelling Biological Gel Contraction by Cells: Mechanocellular Formulation and Cell Traction Force Quantification

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 modelis 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. This revised version was published online in June 2006 with corrections to the Cover Date.