Prestress and adhesion site dynamics control cell sensitivity to extracellular stiffness.

This study aims at improving the understanding of mechanisms responsible for cell sensitivity to extracellular environment. We explain how substrate mechanical properties can modulate the force regulation of cell sensitive elements primarily adhesion sites. We present a theoretical and experimental comparison between two radically different approaches of the force regulation of adhesion sites that depends on their either stationary or dynamic behavior. The most classical stationary model fails to predict cell sensitivity to substrate stiffness whereas the dynamic model predicts extracellular stiffness dependence. This is due to a time dependent reaction force in response to actomyosin traction force exerted on cell sensitive elements. We purposely used two cellular models, i.e., alveolar epithelial cells and alveolar macrophages exhibiting respectively stationary and dynamic adhesion sites, and compared their sensitivity to theoretical predictions. Mechanical and structural results show that alveolar epithelial cells exhibit significant prestress supported by evident stress fibers and lacks sensitivity to substrate stiffness. On the other hand, alveolar macrophages exhibit low prestress and exhibit sensitivity to substrate stiffness. Altogether, theory and experiments consistently show that adhesion site dynamics and cytoskeleton prestress control cell sensitivity to extracellular environment with an optimal sensitivity expected in the intermediate range.

Mechanical and structural assessment of cortical and deep cytoskeleton reveals substrate-dependent alveolar macrophage remodeling.

The sensitivity of alveolar macrophages to substrate properties has been described in a recent paper (Féréol et al., Cell Motil. Cytoskel. 63 (2006), 321-340). It is presently re-analyzed in terms of F-actin structure (assessed from 3D-reconstructions in fixed cells) and mechanical properties (assessed by Magnetic Twisting Cytometry experiments in living cells) of cortical and deep cytoskeleton structures for rigid plastic (Young Modulus: 3 MPa) or glass (70 MPa) substrates and a soft (approximately 0.1 kPa) confluent monolayer of alveolar epithelial cells. The cortical cytoskeleton component (lowest F-actin density) is represented by the rapid and softer viscoelastic compartment while the deep cytoskeleton component (intermediate F-actin density) is represented by the slow and stiffer compartment. Stiffness of both cortical and deep cytoskeleton is significantly decreased when soft confluent monolayer of alveolar epithelial cells replace the rigid plastic substrate while F-actin reconstructions reveal a consistent actin cytoskeleton remodeling observable on both cytoskeleton components.

Global analysis of endothelial cell line proliferation patterns based on nutrient-depletion models: implications for a standardization of cell proliferation assays.

It is known that cell populations growing in different environmental conditions may exhibit different proliferation patterns. However, it is not clear if, despite the diversity of the so-observed patterns, inherent cellular growth characteristics of the population can nevertheless be determined. This study quantifies the proliferative behaviour of the permanent endothelial human cell line, Eahy926, and establishes to which extent the estimation of the cell proliferation rate depends on variations of the experimental protocols. Cell proliferation curves were obtained for cells cultured over 16 days and the influences of cell seeding densities, foetal bovine serum content and frequency of culture medium changes were investigated. Quantitative dynamic modelling was conducted to evaluate the kinetic characteristics of this cell population. We proposed successive models and retained a nutrient-depletion toxicity dependant model, which takes into account the progressive depletion of nutrients, as well as the increase of toxicity in the cell culture medium. This model is shown to provide a very good and robust prediction of the experimental proliferation curves, whatever are the considered frequency of culture medium changes and serum concentrations. Thus, the model enables an intrinsic quantification of the parameters driving in vitro EAhy926 proliferation, including proliferation, nutrient consumption and toxicity increase rates, rather independently of the experiments design. We therefore propose that such models could provide a basis for a standardized quantification of intrinsic cell proliferation kinetics.

Integrin-mediated adhesion as self-sustained waves of enzymatic activation.

Integrin receptors mediate interaction between the cellular actin-cytoskeleton and extracellular matrix. Based on their activation properties, we propose a reaction-diffusion model where the kinetics of the two-state receptors is modulated by their lipidic environment. This environment serves as an activator variable, while a second variable plays the role of a scaffold protein and controls the self-sustained activation of the receptors. Due to receptor diffusion which couples dynamically the activator and the inhibitor, our model connects major classes of reaction diffusion systems for excitable media. Spot and rosette solutions, characterized by receptor clustering into localized static or dynamic structures, are organized into a phase diagram. It is shown that diffusion and kinetics of receptors determines the dynamics and the stability of these structures. We discuss this model as a precursor model for cell signaling in the context of podosomes forming actoadhesive metastructures, and we study how generic signaling defects influence their organization.

Urokinase receptor mediates mechanical force transfer across the cell surface.

The tripartite complex formed by the urokinase receptor, urokinase, and its inhibitor is an enzymatic system that controls plasmin formation involved in degradation of extracellular matrix proteins. With the use of magnetic twisting cytometry with urokinase-coated ferromagnetic beads, we applied mechanical stress directly to the urokinase receptor on the surface of human myogenic cells in culture. The stiffness and the stiffening response measured through the urokinase receptor resembled those of integrins, which are linked mechanically to the cytoskeleton. Furthermore, stiffness decreased with disruption of actin microfilaments. These results demonstrate that the urokinase receptor is coupled mechanically to the cytoskeleton. Inhibition of the tripartite complex formation with antibodies led to a twofold increase in cytoskeletal stiffness. A stiffened cytoskeleton might impede cytoskeletal remodeling and reorganization and thus impede cell motility. Our results demonstrate that the urokinase receptor mediates mechanical force transfer across the cell surface. As such, it is a novel pathway to regulate cytoskeletal stiffness and, thereby, possibly to modulate motility of normal and abnormal adherent cells.

Binding of urokinase to plasminogen activator inhibitor type-1 mediates cell adhesion and spreading.

Urokinase plasminogen activator and its receptor are both found at the surface of the cell membrane in many cell types. The plasminogen activator inhibitor type-1 (PAI-1) is often associated with the extracellular matrix. The spatial localization of these three molecules could account for their involvement in cell adhesion and/or migration. We have shown previously that the urokinase receptor mediates mechanical force transmission across the cell surface to the cytoskeleton. Here we investigated whether immobilized plasminogen activator inhibitor type 1 (PAI-1) could regulate cell spreading and cytoskeleton reorganization. Serum deprived human myogenic cells were plated in serum free medium onto bacteriologic dishes precoated with different extracellular matrix ligands (fibronectin, vitronectin, or type 1 collagen) or PAI-1 at increasing concentrations. The number of adherent cells and their projected area were quantitated after 3 hours of plating. PAI-1 promoted cell adhesion and spreading in a dose dependent manner. Addition of antibodies to PAI-1 inhibited the adhesion on PAI-1 coated dishes in a dose dependent way. The PAI-1 mediated cell adhesion required the presence of urokinase at the cell surface. Removal of the glycosylphosphatidylinositol (GPI)-linked proteins abolished cell adhesion on PAI-1 dish, suggesting its dependence on the presence of the urokinase receptor, a GPI-linked receptor. Furthermore, addition of antibodies against alpha v beta3 integrin completely inhibited cell adhesion on PAI-1, suggesting that alpha v beta3 might be the transmembrane molecule that physically connects the complex of PAI-1, urokinase, and urokinase receptor to the cytoskeleton. Visualization of spread cells stained for filamentous actin with confocal microscopy showed a dose-dependent increase of filopodia on PAI-1 coated dishes and cytoskeletal reorganization, suggesting a migratory profile. These data indicate that PAI-1 plays a direct role in dynamic cell adhesion particularly at the leading edge, where increased levels of urokinase plasminogen activator (uPA) and its receptor (uPAR) are localized in migrating cells. Immobilized PAI-1 could therefore serve to bridge the cell surface with the extracellular matrix via the formation of a multimolecular complex that includes alpha v beta3 integrins in myogenic cells.

Increased expression of gelatinases and alteration of basement membrane in rat soleus muscle following femoral artery ligation.

Acute severe muscle ischaemia is characterized by significant remodelling of basement membranes of myofibres. It was hypothesized that peripheral artery insufficiency is accompanied by similar muscle extracellular matrix (ECM) changes involving matrix metalloproteinase gelatinases. Using a model of femoral artery ligation, both gelatinase activity and basement membrane component degradation were studied in hindlimb skeletal muscles. SDS-PAGE zymography of muscle homogenates showed that acute moderate ischaemia was followed by a significant transient increase in expression of 72- and 92-kDa gelatinases during 48 h; the latter probably originated from inflammatory cells. In situ zymography showed that this increase occurred chiefly at the periphery of myofibres. Immunolocalization demonstrated 72-kDa gelatinase in interspaces and at the periphery of myofibres, and suggested that this enzyme may explain the gelatinolytic activities found by in situ zymography. Type IV collagen and laminin staining showed that the gelatinase expression increase correlated with dramatic basement membrane component alterations. Our data show that even moderate ischaemia results in significant muscle basement membrane remodelling due to matrix degrading enzymes matrix metalloproteinases (MMP) gelatinases.

Pharmacological activation changes stiffness of cultured human airway smooth muscle cells.

Using magnetic twisting cytometry (MTC), we measured the cytoskeletal stiffness of adherent human airway smooth muscle (HASM) cells. We hypothesized that modulation of actin-myosin interactions by application of contractile agonists would induce changes in cytoskeletal stiffness. In cells plated on high-density collagen, bradykinin (10(-6) M) and histamine (10(-4) M) increased stiffness by 85 +/- 15 and 68 +/- 16%, respectively. Increases in cell stiffness were also consistently observed after acetylcholine, substance P, and KCl. The bronchodilator agonists isoproterenol, prostaglandin E2, forskolin, dibutryl adenosine 3', 5'-cyclic monophosphate, and 8-bromoguanosine 3', 5'-cyclic monophosphate each caused a dose-dependent decrease in cell stiffness in unstimulated as well as bradykinin-treated cells. HASM cells plated on high-density collagen were stiffer than cells plated on low-density collagen (126 +/- 16 vs. 43 +/- 3 dyn/cm2) and developed more pronounced increases in stiffness in response to bradykinin as well as more pronounced decreases in stiffness in response to isoproterenol. These results are consistent with the hypothesis that modulation of actin-myosin interactions by application of contractile agonists causes changes in cytoskeletal stiffness of HASM cells. MTC may be a valuable tool for evaluating the mechanisms of pharmacomechanical coupling in airway smooth muscle cells in culture.

Evidence of a non-conventional role for the urokinase tripartite complex (uPAR/uPA/PAI-1) in myogenic cell fusion.

Urokinase can form a tripartite complex binding urokinase receptor (uPAR) and plasminogen activator inhibitor type-1 (PAI-1), a component of the extracellular matrix (ECM). The components of the tripartite complex are modulated throughout the in vitro myogenic differentiation process. A series of experiments aimed at elucidating the role of the urokinase tripartite complex in the fusion of human myogenic cells were performed in vitro. Myogenic cell fusion was associated with increased cell-associated urokinase-type plasminogen activator (uPA) activity, cell-associated uPAR, and uPAR occupancy. Incubation of cultures with either uPA anticatalytic antibodies, or the amino-terminal fragment of uPA (ATF), which inhibits competitively uPA binding to its receptor, or anti-PAI-1 antibodies, which inhibit uPA binding to PAI-1, resulted in a 30 to 47% decrease in fusion. Incubation of cultures with the plasmin inhibitor aprotinin did not affect fusion. Decreased fusion rates induced by interfering with uPAR/uPA/PAI-1 interactions were not associated with significant changes in mRNA levels of both the myogenic regulatory factor myogenin and its inhibitor of DNA binding, Id. Incubation of cultures with purified uPA resulted in a decrease in fusion, likely due to a competitive inhibition of PAI-1 binding of endogenous uPA. We conclude that muscle cell fusion largely depends on interactions between the members of the urokinase complex (uPAR/uPA/PAI-1), but does not require proteolytic activation of plasmin. Since the intrinsic muscle cell differentiation program appears poorly affected by the state of integrity of the urokinase complex, and since cell migration is a prerequisite for muscle cell fusion in vitro, it is likely that the urokinase system is instrumental in fusion through its connection with the cell migration process. Our results suggest that the urokinase tripartite complex may be involved in cell migration in a non conventional way, playing the role of an adhesion system bridging cell membrane to ECM.

Evaluation of basement membrane degradation during TNF-alpha-induced increase in epithelial permeability.

We evaluated whether tumor necrosis factor (TNF)-alpha induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-alpha-stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-alpha induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 +/- 0.04 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 1.47 +/- 0.05 x 10(-6) cm/s, P < 0.05). The permeability increase in the TNF-alpha-stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 +/- 0.07 x 10(-6) cm/s; 1 microg/ml TNF-alpha: 2.82 +/- 0.02.10(-6) x cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 microg/ml TIMP-1 or 10(-6) M BB-3103 was found. TNF-alpha failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-alpha of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-alpha-induced increase in alveolar epithelial monolayer permeability.

Role of collagenase in mediating in vitro alveolar epithelial wound repair.

Type II pneumocytes are essential for repair of the injured alveolar epithelium. The effect of two MMP collagenases, MMP-1 and MMP-13 on alveolar epithelial repair was studied in vitro. The A549 alveolar epithelial cell line and primary rat alveolar epithelial cell cultures were used. Cell adhesion and cell migration were measured with and without exogenous MMP-1. Wound healing of a cell monolayer of rat alveolar epithelial cell after a mechanical injury was evaluated by time lapse video analysis. Cell adhesion on type I collagen, as well as cytoskeleton stiffness, was decreased in the presence of exogenous collagenases. A similar decrease was observed when cell adhesion was tested on collagen that was first incubated with MMP-1 (versus control on intact collagen). Cell migration on type I collagen was promoted by collagenases. Wound healing of an alveolar epithelial cell monolayer was enhanced in the presence of exogenous collagenases. Our results suggest that collagenases could modulate the repair process by decreasing cell adhesion and cell stiffness, and by increasing cell migration on type I collagen. Collagen degradation could modify cell adhesion sites and collagen degradation peptides could induce alveolar type II pneumocyte migration. New insights regarding alveolar epithelial cell migration are particularly relevant to investigate early events during alveolar epithelial repair following lung injury.

Matrix metalloproteinase gelatinases in sulfur mustard-induced acute airway injury in guinea pigs.

Respiratory tract lesions induced by sulfur mustard (SM), a chemical warfare agent, are characterized by epithelial damage associated with inflammatory cell infiltration. To test the potential role of matrix metalloproteinase gelatinases in these lesions, we evaluated gelatinase activity, albumin content, and total cell count in bronchoalveolar lavage fluid of guinea pigs 24 h after an intratracheal injection of 0.2 mg/kg of SM. The bronchial lavage and alveolar lavage fluids were analyzed separately. The increase in inflammatory cell content of the bronchial lavage fluid, mainly macrophages, observed in SM-intoxicated guinea pigs was accompanied by an increase in albumin and in 92-kDa gelatinase activity. There was a significant correlation between albumin content and 92-kDa gelatinase activity (r = 0.67) and between 92-kDa gelatinase and the number of macrophages. Immunohistochemistry performed on tracheal sections showed the presence of 92-kDa gelatinase at the site of intraepithelial cleavages. Zymography analysis of culture medium conditioned by guinea pig tracheal epithelial cells demonstrated that these cells produced in vitro 92-kDa gelatinase on stimulation. Culture of human bronchial epithelial cells obtained by the explant technique showed a marked increase in 92-kDa gelatinase after exposure to 5 x 10(-5) M SM that reinforced the relevance of our animal results to human exposure to SM. These results suggest that in SM respiratory intoxication, 92-kDa gelatinase of both inflammatory and epithelial cell origins could be involved in epithelial cell detachment.