Cyclic AMP-dependent cell shape changes induced by mechanical forces.

Biomaterials used in some biomedical devices are exposed to flow of physiological fluids. The flow-induced forces may influence the morphological and the biochemical responses of adhering cells. The objective of this work is to examine the capacity of a mechanical stress to cause changes in cell/substratum and cell/cell interactions via the second messenger cAMP pathway (cyclic Adenosine Monophosphate). Cyclic AMP is known to modulate cell shape, cell adhesion and intercellular communication in static conditions. A specially designed flow chamber was used to analyze the responses of mouse 3T3 fibroblasts spread on biocompatible substrata and submitted to controlled shear stresses. A 1.1-Pa shear stress induced: cell rounding, disruption of vitronectin receptors clusters and clustering of connexins 43 at cell-cell apposition points. These cell responses were cAMP-dependent. These investigations should help provide a better understanding of the early biochemical events triggered by mechanical forces.

Interactions between red blood cells and a lethal, partly quaternized tertiary polyamine.

Partially quaternized poly[thio-1-(N,N-diethyl-aminomethyl) ethylene]s, Q-P(TDAE)(x) with x indicating the percentage of quaternized subunits, have been proposed as potential carriers for drugs insoluble in water. However these cationic polyelectrolytes form emboli upon intravenous administration. In order to study the mechanism, Q-P(TDAE)(11) was incubated in vitro with red blood cells (RBCs) suspended in various aqueous media such as autologous plasma, autologous serum, albumin dissolved in phosphate buffer, plasma-serum mixtures and Tris buffer. The deformability of the RBC membrane studied by viscometry was not affected by the polycation. Q-P(TDAE)(11)-induced hemagglutination was studied by optical microscopy. It depended on the polymer concentration and on the presence of plasma proteins. As ghosts were formed in some cases, hemolysis was investigated by measuring potassium and hemoglobin released from RBCs. Fibrinogen and serum proteins, except albumin, protected RBCs from hemolysis. Moreover the order of addition of the suspension components modulated dramatically the Q-P(TDAE)(11)-induced hemolysis. Addition of Q-P(TDAE)(11) to whole blood caused hemolysis whereas addition of the polymer to plasma prior to contact with RBCs did not affect the cell integrity. In contrast, addition of the polymer to RBCs suspended in albumin solution caused greater hemolysis than the addition to whole blood, and the contact between Q-P(TDAE)(11) and albumin prior to RBC addition still enhanced cell lysis. Two conclusions can be drawn from these observations: (i) Q-P(TDAE)(11) induces both hemagglutination, probably through electrostatic interaction, and hemolysis, because Q-P(TDAE)(11) disrupted the RBC lipid bilayer; (ii) proteins can decrease or increase the deleterious effects of Q-P(TDAE)(11) on RBCs.

Viscous filtration of red blood cell suspensions.

Filtration experiments on red blood cell suspensions are usually conducted in a saline buffer solution. As a result, the flow of a particle in a pore is largely dominated by viscous effects, and it is not possible to distinguish between normal and membrane altered cells. A new approach to red cell filtration is proposed here, whereby the cells are suspended in a Dextran solution that has roughly the same viscosity as the internal hemoglobin solution. It is thus aimed to detect alterations of the membrane properties. In order to prove this point, filtration measurements are conducted with a hemorheometer on dilute (hematocrit 8%) suspensions of normal and membrane hardened (diamide treatment) cells, suspended either in a 8 mPa.s Dextran solution or in a 1 mPa.s buffer solution. As expected when the cells are suspended in buffer, there is no detectable difference in the filtration index for normal and treated cells. However, when they are suspended in the 8 mPa.s solution, the filtration index is significantly larger for treated than for normal cells. This shows that filtration in a viscous liquid can be used to measure changes in cell deformability due to membrane modifications.

Determination of the red blood cell apparent membrane elastic modulus from viscometric measurements.

Rhelogical measurements on a dilute suspension of red blood cells (RBCs) are interpreted by means of a microheological model that relates the shear evolution of the apparent viscosity to the intrinsic properties of the suspended particles. It is then possible to quantify the average deformability of a RBC population in terms of a mean value of the membrane shear elastic modulus, Es. Dilute suspensions of erthrocytes exhibit shear-thinning behavior with a constant high shear viscosity. This behavior is identical to the one predicted for a suspension of spherical capsules where the same phenomena of deformation and orientation prevail. A comparison between theoretical and experimental curves yields a mean value of Es, assuming all other cell properties--internal viscosity, geometry--to be otherwise equal. In Dextran, the values of Es for normal RBCs are found to be of order 3.10(-6) N/m. For erythrocytes hardened by heat exposure for 15 minutes at 48 degrees C, the increase in Es reaches 45 percent. This procedure of shear elastic modulus determination is easy to perform and seems to give a good discrimination between normal and altered erythrocytes.