Experimental evidence of a potentially increased thrombo-embolic disease risk by domestic electromagnetic field exposure.

We have used the EaHy926 endothelial cell line, able to secrete both pro and anti-aggregant platelet agents, as a model for thrombo-embolic diseases. We experimentally established, by comparing these two secretions with or without a Faraday cage, that the environmental electromagnetic field significantly increases the thrombo-embolic risks in this endothelial cell line.

Direct physical factors and PGI2 and TXA2 secretions by a human endothelial cell line: in vitro investigation of pressure and shear stress applied independently or in synergy.

The direct effect of two types of mechanical stress was measured through the prostacyclin (PGI2) and thromboxane A2 (TXA2) secretions by a confluent monolayer of cells from the EA.hy926 line. Eight values of constant pressure were applied in the gas phase above the culture medium, around atmospheric pressure taken as a control (0 mm Hg), from -500 to +760 mm Hg. Three amplitudes of sinewave modulated pressure (+/- 40; +/- 80; +/- 160 mm Hg) were explored at a frequency of 1 Hz. Modulated pressure (+/- 40 mm Hg) was also applied synergetically to a shear stress generated under steady state conditions by a rectilinear laminar motion of the medium. The cells remained adherent and exhibited unchanged morphology and viability. Constant pressure or depressure increased both PGI2 and TXA2 release but to an extent depending on the pressure value. Under pressure, the PGI2/TXA2 ratio was unchanged, but was higher under depressure, compared to the control. Pressure modulation strongly stimulated the secretion of PGI2 but had no effect on TXA2. Modulation strongly increased the PGI2/TXA2 ratio to a similar extent for the three amplitudes. Pressure-shear synergy enhanced secretion of PGI2 markedly more than shear stress alone, but the level reached was similar to the one induced by pressure modulation. No cumulative effect on the secretion of PGI2 was observed, whereas TXA2 synthesis undergoes a more than cumulative effect. The PGI2/TXA2 ratio remained unchanged under shear alone or under combined shear-pressure modulation but was higher with the modulated pressure alone. These results demonstrate that pressure has an outstanding effect on secretion that may be origin to local disturbances of the vascular system, thus inducing pathologies such as thrombosis or atherosclerosis.

Influence of a constant or a modulated pressure on insulin secretion by intact murine islets of Langerhans: existence of a threshold effect.

Pressure effect on cultured islets was investigated to simulate the behavior of the endocrine pancreas when subjected to an anatomical change in its environment. Insulin secretion from islets seeded on glass was determined by radioimmunoassays after 12-h exposure to constant or sine-wave pressure perturbation, at 37 degrees C and pH 7.4, under well-defined conditions applied through the culture medium. A specially devised setup allowed control of the constant component Po, the amplitude A, and the frequency f of the pressure perturbation. Under constant conditions, three Po values (760, 760 + 110, 760 + 250 mm Hg) were explored. Under sine-wave conditions, two frequencies (f = 0.2 and 4.33 Hz) were investigated for Po = 760 + 110 mm Hg and A = 10 mm Hg, as well as three amplitude values (A = +/-10, +/-50, and +/-110 mm Hg) for Po = 760 mm Hg and f = 0.2 Hz. Constant pressure inhibits secretion, by a factor of up to 60%, as well as low frequency (also by approximately 60%). Low amplitudes enhance secretion (by 41 and 31%), whereas a higher amplitude has a marked inhibiting effect (by 70%). In all cases, no linearity was observed, showing the existence of a threshold pressure effect. These results suggest that pressure may be an important parameter in certain forms of endocrine pancreatic dysfunctions.

Effect of blood storage on erythrocyte/wall interactions: implications for surface charge and rigidity.

In this report, we study, under flow conditions, the interactions of stored erythrocytes with an artificial surface: a microelectrode whose charge density ranges from -15 to +27 microC/cm2. Interactions consist of red cells slowly circulating on the microelectrode and exerting a real contact with the electrode. Interaction is detected and measured by transient fluctuations of the electrolyte resistance obtained by impedance measurement of the microelectrode. Effects of aging induced by storage of whole blood at 4 degrees C show that the surface charge of erythrocytes rapidly decreases when blood is stored for more than 6 days under our experimental conditions. In comparison with trypsin-treated erythrocytes, an eight day storage induces a 60% decrease in the surface charge of red cells. After two weeks of storage, red cells are no longer negatively charged, presumably because of removal of sialic acid. Cells rigidity is significant after 6 days of storage and influences the electrical contact. Membrane rigidity increase could arise from the surface charge decrease. Finally the surface charge decrease could be importance in the use of stored blood.

Vascular endothelial cell responses to different electrically charged poly(vinylidene fluoride) supports under static and oscillating flow conditions.

We investigated the effect of electrically charged surface copolymers on endothelialization of four types of poly(vinylidene fluoride) (PVDF) copolymer surface films with different electrical characteristics. PVDF films without a surface charge, with a remanent surface (5 and 7 microC) and with piezoelectric characteristics were studied through the secretion by an endothelial cell (EC) line culture, under static and oscillating flow conditions of prostacyclin (PGI2) and thromboxane (TXA2), two metabolites which have directly opposing actions on platelet function. The surface electrical properties of PVDF are suitable for promoting cell adhesion. Secretion of thrombomodulatory mediators varied, depending on the surface electrical charge and on the molecular structure of the PVDF substrate. Under static conditions the ECs respond to the substrates by a similar increase of PGI2. Under oscillating flow conditions, the ratio of PGI2 to TXA2 is higher with the piezoelectric PVDF film. The piezoelectricity generated from shear stress along the entire length of the fibres may be appropriate in vivo to keep the [PGI2]/[TXA2] ratio at a level which could counteract the build-up of surface deposits which could be at the origin of thrombosis.

Morphological aspects of endothelial cells cultured in the absence of fetal calf serum under controlled electrical charges applied to the support.

The effect of constant and modulated electrical charges applied through the culture support on endothelial cell (EC) morphology was investigated. Endothelial cells tolerated electrical charges for 48 hours, in a serum-free medium. The EC had normal morphology, except at high stationary charges, for which nucleus and cell size increased. The observed cell divisions and cellular secretions indicated that the cells rapidly adapted to the imposed stresses.

Interactions of erythrocytes with an artificial wall: influence of the electrical surface charge.

Electrical charge on any biological surface plays a crucial role in its interaction with other molecules or surfaces. Here, we study, under flow conditions, the interactions of erythrocytes with an artificial surface: a platinum microelectrode whose charge density ranges from -15 to +27 microC/cm2. This artificial surface could be similar in surface charge to an endothelium or a biomaterial. In this model, interactions are measured as a transient relative increase of the electrolyte resistance obtained by impedance measurement of a microelectrode. A maximal interaction of erythrocytes with the charged surface is calculated in the 0 to +10 microC/cm2 charge density range. At negative surface charge, a less efficient contact was obtained because of electrostatic repulsion forces. High positive surface charge (charge density > 10 microC/cm2) does not improve the contact but induces a progressive decrease in the contact efficiency, which could be explained by a rearrangement of macromolecules on the erythrocyte surface or an effect of positive groups on the cell membrane. This work suggests that a greater surface area of contact is obtained in the 0 to +10 microC/cm2 charge density range and that this is provided by more molecular bridges.

Electrochemical analysis of blood cell/substrate interactions under flow conditions.

Interactions of blood cells (RBCs) with a microelectrode of 50 microns diameter have been examined under flow conditions using impedance measurements at high frequencies. At such frequencies, the electrolyte resistance (Re) is assimilated to the real part of impedance, and interactions are associated with transient fluctuations of Re. Sedimentation experiments suggest that one erythrocyte contributes to a 1.1% Re increase. Effects of wall shear rate (from 25 to 140 s-1) and RBC concentration (from 8.4 x 10(5) to 2.7 x 10(6) cells/ml) have been investigated; the number of interactions rapidly decreases with wall shear rate. Event frequency is proportional to RBC concentration ranging from 3.1 x 10(6) cells/ml to 1.3 x 10(7) cells/ml. At high concentrations of RBCs, some transient events overlap. Videotaped images help to determine how many RBCs interact with the microelectrode at the same time on separate surface areas. Under flow conditions, the contribution of one RBC on the Re increase is similar to the mathematical value obtained by sedimentation and decreases slightly with wall shear rate.

Effect of constant and modulated electrical charges applied to the culture material on PGI2 and TXA2 secretion by endothelial cells.

Electrical stimulation of an endothelial cell culture applied through the culture support biomaterial induced strong secretion of prostaglandin I2 (PGI2) and, to a lesser extent, thromboxane A2 (TXA2), which varied with time, the sign of the stationary electrical charge and the amplitude and frequency of a superimposed sine wave. In stationary conditions, the response is more rapid at positive electrical charges (+ some tens of microC cm-2) than at negative ones (-some tens of microC cm-2). In sine wave conditions, the ratio [PGI2]/[TXA2] is strongly increased and is maximum for an amplitude of 50 mV and a frequency of 1 Hz. These results suggest that the extracellular matrix is not only involved in cellular anchoring but also participates actively in secretion of thrombomodulatory substances, an effect apparently related to the piezoelectric properties of its components.

Adsorption of bovine serum albumin onto glassy carbon in a Couette flow. Effect of shear rate on the adsorption kinetics and on the structure of the adsorbed proteic layer.

Adsorption of bovine serum albumin onto glassy carbon is investigated by analysing the time-variation of the double-layer capacitance recorded during the adsorption process. The effect of shear rate is investigated under laminar conditions in a Couette flow. Stationary and sinusoidally modulated values of the shear rate are imposed over the (0-200 s-1) range. The flow conditions are shown to play an essential role by markedly modifying the rate of all the steps (three at most) involved in the adsorption mechanism. Moreover, the structure of the adsorbed layers in the intermediate and final states are also strongly modified, an increase of the shear rate increasing the interaction between the protein and the electrode. Piezoelectric properties of albumin are invoked to account for the experimental results.

Fractionation of charge-modified low density lipoproteins by fast protein liquid chromatography.

We describe a methodology developed to separate different forms of charge-modified low density lipoproteins (LDL) using the fast protein liquid chromatography (FPLC) system from Pharmacia. Lipoproteins were isolated by sequential ultracentrifugation and introduced onto an anion-exchange column (Mono Q HR 5/5). The multistep NaCl gradient elution was optimized and the analytical variables were determined on copper-oxidized LDL. After oxidation by copper for various times (up to 48 h), five forms were obtained (fractions A, B, C, D, and E). Within-run and day-to-day reproducibility were better than 8.6% and 10%, respectively. Protein and cholesterol recovery after the chromatographic separation was good (greater than 82%) and the detection limit was about 1 microgram. The more negative forms of collected LDL were mainly characterized by an increase in the lipid peroxidation product content, a depletion of vitamin E, an alteration of apoB and increased degradation by macrophages. The proposed methodology was applied to the study of LDL modifications generated by human umbilical endothelial cells and the protective effect of antioxidants (vitamin E and probucol).

Influence of surface charge on adsorption of fibrinogen and/or albumin on a rotating disc electrode of platinum and carbon.

The adsorption of fibrinogen on to platinum and carbon and of albumin on to carbon was investigated for various changes of the surface by recording the variations of the double-layer capacitance of the electrochemical interface during adsorption, as a function of time. The rate of the second step of the adsorption decreased with increasing negative charge on to platinum but was charge-independent on to carbon. In contrast, on both surfaces, the rate of the first step and the area of the electrode surface in close contact with adsorbed proteins were both found to increase with increasing negative charge of the surface, although at pH 7.4 albumin and fibrinogen are negatively charged. The hypothesis of ion coadsorption inside the proteic layer has been proposed to account for this result.

Shear stress affects expression of major histocompatibility complex antigens on human endothelial cells.

Endothelialization of the inner face of a prosthesis appears to improve patency of small caliber arterial substitutes. The importance of understanding the factors that affect human endothelial cell behavior is highlighted by failure of vascular prosthetic grafts to endothelialize when implanted in man. In the present study, endothelial cells isolated from microvasculature are used for their ability to be easily selected from human adult fat, their proliferative capacity, and for their immunologic properties relevant to human pathology: allograft implantation, vessel injury or atherosclerosis. The system described provides a tool for assessing the individual roles of shear stress in modulating endothelial cell morphology and major histocompatibility complex (MHC) antigen expression. Using indirect immunofluorescent staining, initial results showed a homogenous increase of class I and appearance of class II expression after an exposure for 30 hr to physiologic arterial values. Significantly increased staining intensity was observed following exposure to supraphysiologic values. Moreover, precoating of substrate with fibronectin instead of poly-L-Lysine enhanced MHC straining intensity. Scanning electron microscopy (SEM) confirmed the activated morphology of stained cells. This provides a model to study involvement of MHC expression in endothelial cell activation under physical constraints. It may contribute to the development of biomaterial for implantation.