Leone Tranqui

Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo

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 μg/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. (Mol Cell Biochem 184: 81–100, 1998)

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

SummaryThis 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 αvβ3 integrin localization demonstrates that cell adhesion to fibrinogen is mediated through the αvβ3 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 αvβ3 integrin distribution.

Mechanical signalling and angiogenesis. The integration of cell-extracellular matrix couplings.

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.

The formation of tubular structures by endothelial cells is under the control of fibrinolysis and mechanical factors

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 TGFβ1 was measured in the medium (1000–1700 pg/ml). The active form of TGFβ1 was not, however, detected and the addition of anti-TGF-β1 antibody to the medium did not influence the formation of the CLS network. Yet, added activated TGF-β1 led to the formation of less organised structures, that were completely abolished by the concomitant addition of the same anti-TGF-β1 antibody. Thus, it is likely that TGF-β1 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.

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.

Quantification and macroscopic modeling of the nonlinear viscoelastic behavior of strained gels with varying fibrin concentrations

The mechanical properties of fibrin gels under uniaxial strains have been analyzed for low fibrin concentrations using a free-floating gel device. The authors 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. The authors 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.

Description of an in vitro angiogenesis model designed to test antiangiogenic molecules

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.

An in vitro model giving access to adhesion plaques.

SummaryA 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.