Histamine alters endothelial barrier function at cell-cell and cell-matrix sites

Alan B. Moy1,2, Michael Winter1, Anant Kamath1, Ken Blackwell1, Gina Reyes1, Ivar Giaever3, Charles Keese3, and D. M. Shasby1

Departments of 1 Internal Medicine and 2 Biomedical Engineering, University of Iowa, Iowa City, Iowa 52242; and 3 Department of Biology and Physics, School of Science, Rensselaer Polytechnic Institute, Troy, New York 12180


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To determine how histamine regulates endothelial barrier function through an integrative cytoskeletal network, we mathematically modeled the resistance across an endothelial cell-covered electrode as a function of cell-cell, cell-matrix, and transcellular resistances. Based on this approach, histamine initiated a rapid decrease in transendothelial resistance predominantly through decreases in cell-cell resistance in confluent cultured human umbilical vein endothelial cells (HUVECs). Restoration of resistance was characterized by initially increasing cell-matrix resistance, with later increases in cell-cell resistance. Thus histamine disrupts barrier function by specifically disrupting cell-cell adhesion and restores barrier function in part through direct effects on cell-matrix adhesion. To validate the precision of our technique, histamine increased the resistance in subconfluent HUVECs in which there was no cell-cell contact. Exposure of confluent monolayers to an antibody against cadherin-5 caused a predominant decrease in cell-cell resistance, whereas the resistance was unaffected by the antibody to cadherin-5 in subconfluent cells. Furthermore, we observed an increase predominantly in cell-cell resistance in ECV304 cells that were transfected with a plasmid containing a glucocorticoid-inducible promoter controlling expression of E-cadherin. Transmission electron microscopy confirmed tens of nanometer displacements between adjacent cells at a time point in which histamine maximally decreased cell-cell resistance.

electrical resistance; cadherin; electron microscopy; modeling; transfection


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HISTAMINE TRANSIENTLY increases microvascular permeability in vivo (12, 27). The transient changes in permeability are associated with a decrease followed by a restoration of cell adhesion in endothelial cells (3, 11, 14, 15, 17). Although these rapidly resolving changes in cell adhesion are clearly described, the mechanisms by which they occur are uncertain.

Changes in cell adhesion require changes in cell shape. These changes in cell shape imply a change in the balance of forces that round cells up and forces that keep cells in a spread configuration (10, 25). For example, when extracellular calcium is chelated, sites of cell adhesion dependent on calcium for binding are weakened. The resting centripetally directed tension in the cells is less opposed, and adjacent cells retract from each other (20, 22, 23).

Histamine causes a small and transient increase in myosin light chain phosphorylation in endothelial cells (17, 18), but the level of phosphorylation does not reach those that increase the activity of the actomyosin ATPase (19). Consistent with the level of myosin light chain phosphorylation, histamine decreased endothelial cell adhesion without causing a detectable increase in centripetal tension (18). These data imply that histamine changes barrier function by altering sites of adhesion, similar to the effects of chelating extracellular calcium rather than through contraction of the actin cytoskeleton. If histamine mediates direct effects on cell adhesion, then further identification of the sites at which histamine affects cell adhesion is necessary to better understand how inflammatory edema is regulated.

Identification of the adhesion sites and the mechanisms by which histamine disrupts and restores barrier function is complex because the cytoskeleton is an integrated three-dimensional network of filaments and adhesion proteins in which mechanical forces at cell-cell regions are mechanically coupled to cell-matrix regions (25). If the cytoskeleton is viewed as an integrative structure, histamine could disrupt cell-cell adhesion through two basic mechanisms. Activation of signal transduction events could decrease adhesion at cell-matrix sites and cause cell rounding, which in turn could result in a secondary or reactive loss in cell-cell adhesion. Alternatively, activation of signal transduction pathways may directly target cell-cell sites and cause a direct loss in cell-cell adhesion with a reactive loss in cell-matrix adhesion. Because there are distinct adhesion proteins at cell-cell and cell-matrix sites that could be affected differently by signaling pathways, it is important to identify the specific sites that histamine directly affects.

To understand how histamine directly affects endothelial cell-cell and cell-matrix adhesion, we modeled the electrical resistance across a confluent endothelial monolayer as a function of cell-cell resistance, cell-matrix resistance, and transcellular resistance. This approach was first introduced by Giaever and Keese (8) and Lo et al. (13) to model the resistance of unstimulated cultured fibroblasts and epithelial cells. We have now extended this approach to cultured endothelial cells to derive a real-time evaluation of cell-cell and cell-matrix adhesion in response to a physiological stimulus. We validated the precision of this approach by testing the model with specific biological interventions. We have correlated cell-cell resistance with ultrastructural details of cell-cell strain using electron microscopy. Using this model, we were able to demonstrate that histamine transiently disrupts barrier function by first disrupting cell-cell adhesion and later by increasing cell-matrix adhesion.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. For these studies, we used a commercial anti-cadherin-5 antibody (Transduction Laboratories; Lexington, KY) that was prepared in a carrier buffer that did not contain glycerol and sodium azide. The antibody recognizes an epitope at amino acids 26---194 of the extracellular domain of cadherin-5.

Cell cultures. Cultured human umbilical vein endothelial cells (HUVECs) were prepared by collagenase treatment of freshly obtained human umbilical veins as described (3). Harvested primary cultures designated for cell adhesion assays were plated on 60-mm tissue culture plates that were coated with 100 µg/ml of fibronectin (Collaborative Research; Bedford, MA). All cells were cultured in medium 199 and supplemented with 20% heat-inactivated fetal calf serum, basal medium Eagle vitamins and amino acids, glucose (5 mM), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 µg/ml). Cultures were identified as endothelial cells by their characteristic uniform morphology, uptake of acetylated low-density lipoprotein, and indirect immunofluorescent staining for factor Vlll.

Cell adhesion assay. Cell adhesion was measured using a previously reported technique (6-8, 18, 24). In this system, referred to as electric cell-substrate impedance sensing, cells were cultured on a small gold electrode (5 × 10-4 cm2) using culture medium as the electrolyte, and barrier function was measured dynamically by determining the electrical impedance of a cell-covered electrode. The total impedance of the monolayer is composed of the impedance between the ventral surface of the cell and the electrode, the impedance between the cells, and the impedance of the cell membranes dominated by the membrane capacitance (8). Membrane impedance is very large, and thus most of the current flows under and between the cells. Furthermore, membrane impedance is not expected to change on addition of histamine. Thus measured changes in impedance represent alterations primarily in cell-cell adhesion and/or cell-matrix adhesion.

A 1-V, 4,000-Hz alternating current signal was supplied through a 1-MOmega resistor to approximate a constant-current source. Voltage and phase data were measured with a SRS830 lock-in amplifier (Stanford Research Systems) stored and processed with a personal computer. The same computer also controlled the output of the amplifier and relay switches to different electrodes. Critical features of the setup are the current frequency of 4,000 Hz and the small area of the active electrode (a surface area of 10-4 cm2).

For experiments, electrodes were coated with adsorbed fibronectin by exposure to a 100 µg/ml solution for 30 min. HUVECs were inoculated on electrodes at a confluent density of 105 cells/cm2. The in-phase voltage (proportional to the resistance) and the out-of-phase voltage (proportional to the capacitive reactance) were measured. We chose to express barrier integrity as a function of resistance, normalized to the initial value and expressed as a fractional change, because there were greater changes in resistance than impedance or reactance. Thus a 10% decline in resistance, for example, would represent a fractional resistance of 0.9. Electrical resistance increased after cells attached and covered the electrodes, and the resistance achieved a steady-state level by 24 h.

In some studies, we directly measured changes in cell-matrix adhesion by measuring the resistance in subconfluent cells that had not formed cell-cell contact. Microelectrodes were inoculated at a density of 2.5 × 104 cells/cm2. Microelectrodes were viewed under a microscope 24 h later, and experiments were conducted in wells in which cell-cell contact was not observed. The signal was amplified and typically reflected the change in resistance in less than 10 cells on the active electrode, which approximately represented one-tenth of the electrode area.

Mathematical model to resolve experimental resistance into changes in cell-cell and cell-matrix adhesion. We used a previously derived mathematical model to calculate specific cell-cell and cell-substrate adhesion (5, 8, 13). The model assumes that current flows radially from under the ventral surface of the cell and the electrode and escapes between cells, with a minor amount going directly through the cell membrane by capacitive coupling. In this model (Fig. 1, inset) the total impedance across a cell-covered electrode is composed of the impedance between the ventral surface of the cell and the electrode (related to alpha ), the impedance between cells (indicated by Rb), the transcellular impedance (Zm), and the impedance of a naked electrode (Zn). For these calculations, the cells are regarded as circular disks and Zm is inversely related to membrane capacitance (Cm). alpha  can also be defined as
&agr; = <IT>R</IT><SUB>c</SUB><RAD><RCD><FR><NU>&rgr;</NU><DE><IT>h</IT></DE></FR></RCD></RAD>
where Rc is the cell radius, rho  is the resistivity of the medium, and h is the average separation distance between the cell and the underlying matrix (8).


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Fig. 1.   Inset: different contributions to cell-covered impedance, Zc, when electrical circuit is modeled as a resistor and a capacitor in series. Cells are regarded as circular disks. We assume that current flows radially from under the ventral surface of the cell and around the cells. A small amount of current passes capacitively through the cell. Total impedance across monolayer is composed of impedance between ventral surface of cell and electrode (related to alpha ), impedance between cells (Rb), and impedance created from transcellular conduction, Zm, which is defined by expression Zm = -i/2pi upsilon (Cm/2), where Cm represents capacitance of single cell membrane and i, in complex notation, is the square root of -1, and upsilon  is the current frequency. The figure demonstrates 5 models in which transendothelial resistance could theoretically be created across confluent endothelial monolayer based on combination of different values of alpha , Rb, and Cm. Zn, impedance of naked electrode. A: model in which transendothelial resistance is not dependent on cell-cell and cell-matrix adhesion. B: model in which transendothelial resistance is dependent on cell-matrix adhesion. C: model in which transendothelial resistance is dependent on cell-cell adhesion. D: model in which resistance is created by cell-cell adhesion and cell-matrix adhesion with Cm of 1 µF/cm2. E: same as D except Cm is greater than 3 µF/cm2.

Because Zn and the cell-covered impedance (Zc) are measured and Zm is the impedance of the two cell membranes in series, alpha , Rb and Cm are the only adjustable parameters in the model. We first identified the values of alpha , Rb and Cm before the addition of test agents. The alpha  and Rb cannot be explicitly solved because they are dependent on modified Bessel functions and thus have to be derived by curve fitting. First, Zc and Zn are measured as a function of current frequency (upsilon ) in untreated cells. By iteratively choosing values for Cm, Rb, and alpha  we can arrive at the best fit to the experimental impedance. Because resistance is also dependent on the same values for Cm, Rb, and alpha  as they are for impedance or reactance, Cm, Rb, and alpha  were measured by finding the best fit of the calculated resistance to the experimental resistance. The resistance was measured at 13 separate frequencies between 22 and 90,000 Hz and is expressed as the ratio of normalized resistance of cell-covered to cell-free electrodes.

Real-time changes in cell-cell adhesion (Rb) and cell-matrix adhesion (alpha ) in response to histamine or other interventions were determined at a single frequency of 4,000 Hz. Zn and Zm were presumed constant at 4,000 Hz, and Rb and alpha  were assumed to be the only variables that change during the course of the experiment. Even if Rb(t) and alpha (t) changed significantly over time, by using repeated linearization, the error using this procedure could be kept low. We herein refer to this method as a dynamic optimization procedure.

Custom instrumentation software to perform the impedance measurements and the modeling analyses were first developed by Applied Biophysics. Custom software written in the LabVIEW programming language (National Instruments) was later developed to perform the same operations, and it achieved equivalent results.

Scanning electron microscopy. Confluent monolayers were exposed to test agents for the prescribed duration. The medium was rapidly removed, and the cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 5 min at room temperature. Cells were subsequently rinsed for three 5-min intervals in 0.1 M sodium cacodylate. Cultured cells were then secondarily fixed with a solution containing 1% osmium tetroxide and 1% potassium ferrocyanide in 0.1 M sodium cacodylate for 30 min at room temperature. Monolayers were then subsequently washed with 0.1 M sodium cacodylate, followed by double-distilled water. Next, monolayers were subjected to serial dehydration in 25, 50, 75, 95, and finally 100% ethanol for 15 min each. Cells were then exposed to fresh 100% ethanol for 1 h. The tissue dish was cut into 2 × 2-cm sections with a hot scalpel under 100% ethanol and air-dried under hexamethyldisilazane-100% ethanol at a 1:1 volume ratio for 30 min and then repeated for 1 h. Dried specimens were mounted onto aluminum stubs that were painted with graphite. Stubs were coated with gold-palladium using an argon-based sputter. Images were taken with a Hitachi S-4000 electron microscope with an accelerating voltage from 1 to 5 kV at a magnification of ×1,000. Final prints were made on Polaroid 55 electron microscopy grade film.

Transmission electron microscopy. Studies were conducted on cultured cells grown on plastic dishes coated with 0.01% gelatin for 30 min, followed by 30 µg/ml of fibronectin for 30 min. Control monolayers and monolayers exposed to 10 µM histamine for 1 and 20 min were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate at 37°C for 10 min. Monolayers were washed with 0.1 M sodium cacodylate. Cells were then secondarily fixed for 30 min in a solution consisting of two parts 0.1 M sodium cacodylate, one part 4% osmium tetroxide in water and one part 6% potassium ferrocyanide in water. Cells were washed three times with 0.1 M sodium cacodylate buffer for 10 min each and once with distilled water for 10 min at room temperature. Cells were then exposed to 2.5% uranyl acetate at room temperature for 20 min. After the cells were rinsed with distilled water, monolayers were sequentially dehydrated in 25, 50, 75, 95, and 100% ethanol at room temperature for 30 min. Cells were embedded in resin consisting of one part eponate resin and one part 100% ethanol at room temperature for 1 h. The latter step was repeated. Finally, cells were subjected twice to 100% eponate for 2 h at room temperature. The resins were baked overnight at 70°C. Specimens were pivoted 90 degrees and cut into sections with a diamond knife followed by poststaining with Reynolds lead citrate. The images were then acquired at 75 kV in a Hitachi H7000 electron microscope. Prints were then developed on multigrade deluxe type 4 Ilford paper.

To measure the average separation distance between adjacent cells, we performed the following procedure. Electron micrographs were scanned with an Apple One Scanner using Ofoto Software, and image analysis was accomplished with National Institutes of Health Image. The entire length of the gap between two adjacent cells was subdivided into 10 equidistant regions. After pixels were calibrated into a nanometer scale, a plot of radiance intensities and distance measurements was obtained across a gap at a regional point of interest. The separation distance was determined by measuring the distance between the two radiance maxima.

Plasmid transfection. Immortalized cultured HUVECs, ECV304 cells, were transfected with a pLKneo vector containing the cDNA for E-cadherin (graciously provided by James Nelson) using Lipofectamine (as per the manufacturer's protocol) (1). The pLKneo vector contains a glucocorticoid-inducible promoter (9). Transfected cells were selected with G-418 (1 mg/ml) in medium 199 with 10% fetal bovine serum. Surviving cells were isolated, and clones were expanded in the presence of G-418. Clones were tested for expression of E-cadherin after stimulation with dexamethasone for 18 h using either Western blotting or fluorescent-activated cell sorting (FACS; University of Iowa Core FACS Facility) of cells prepared to examine surface expression of the E-cadherin.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Modeling transendothelial resistance across endothelial cell-covered electrode. Giaever and Keese (8) modeled the resistance across a monolayer of fibroblasts as a circuit with three components, the membrane capacitance (Cm), the resistance created by the apposition of the ventral surface of the cell to the matrix (alpha ), and the resistance created by the apposition of the lateral surfaces of adjacent cells to each other (Rb). We had to determine if these same components contributed to the resistance across an endothelial cell monolayer, and if so, how much each component contributed in an unperturbed state.

A model based on the assumption that there was no resistance due to cell-cell and cell-matrix adhesion (Fig. 1A; Rb and alpha  approaches 0, Cm = 1 µF/cm2, the Cm of a theoretical smooth membrane) was a poor fit to the experimental resistance. Under these circumstances, the calculated resistance, expressed as the normalized ratio of the resistance of the cell-covered electrode to a naked electrode, did not change between frequencies of 22 and 90,000 Hz. In contrast, the experimental normalized ratio was maximum at 4,000 Hz, consistent with previous reports (6-8).

The experimental results also did not fit a model based on the assumption that the resistance was due to cell-matrix adhesion and not to cell-cell adhesion (Fig. 1B; Rb approaches 0, alpha  = 5.4 Omega 1/2 · cm, and Cm = 1 µF/cm2). Similarly, the experimental results also did not precisely fit a model based on the assumption that resistance was predominantly due to cell-cell adhesion (Fig. 1C; Rb = 2.2 Omega  · cm2, alpha  approaches 0, Cm = 1 µF/cm2).

In contrast to these three models, a model in which resistance was created by cell-cell adhesion and cell-matrix adhesion and membrane capacitance (Fig. 1D; Rb = 0.9 Omega  · cm2, alpha  = 4 Omega 1/2 · cm, and Cm =1 µF/cm2) was a much better fit to the experimental data. This model was further improved by increasing the Cm to >3 µF/cm2 (Fig. 1E). This increase in cell Cm is consistent with a folded or convoluted membrane with a greater surface area than a simple smooth membrane.

Characterizing Rb, alpha , and Cm in monolayers of different ages of confluency. We next compared the values of Rb, alpha , and Cm used to fit data from young and older postconfluent cultured monolayers. Rb increased from 1.205 ± 0.17 Omega  · cm2 in cells 1 day postconfluent to 2.28 ± 0.17 Omega  · cm2 in cells 5 days postconfluent. alpha  decreased from 3.62 ± 0.15 Omega 1/2 · cm in cells 1 day postconfluent to 3.15 ± 0.10 Omega 1/2 · cm in cells 5 days postconfluent. Cm also decreased from 3.57 ± 0.14 µF/cm2 in cells 1 day postconfluent to 3.09 ± 0.15 µF/cm2 in cells 5 days postconfluent. These data suggest that mechanical forces were directed toward increased horizontal tethering with older postconfluent monolayers.

Identifying dynamic changes in cell-cell and cell-matrix resistance to predictable perturbations. To validate the ability of the model to predict real-time changes in cell-cell and cell-matrix adhesion, we measured dynamic changes in alpha  and Rb in response to a predictable perturbation in transendothelial resistance. We added an antibody to cadherin-5 to cause a predictable decrease in cell-cell adhesion. An antibody against cadherin-5, which disrupts homeotypic cadherin binding, decreased the resistance by 15-20% across the cell-covered electrode, whereas neither control serum nor the special vehicle for the antibody altered the resistance (Fig. 2A). As anticipated, the anti-cadherin-5 antibody decreased Rb, which preceded decreases in the experimental resistance or in alpha  (Fig. 2B). The anti-cadherin-5 antibody caused a small (10-15%) secondary decline in alpha , which lagged behind the change in experimental resistance and which is consistent with a paradigm in which a primary loss in cell-cell tethering induced a reactive stress that in turn strained cell-matrix adhesion sites. Thus the optimization procedure detected the expected response for alpha  and Rb.


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Fig. 2.   A: effect of 50 µg/ml of anti-cadherin-5 antibody, nonimmune antibody, and carrier vehicle on electrical resistance in postconfluent monolayers measured at 4,000 Hz. Tracings represent means of >= 3 separate experiments. B: experimental resistance comparison with changes in Rb and alpha  in response to antibody to cadherin-5 in postconfluent monolayers, based on dynamic optimization procedure. C: effect of 50 µg/ml of the antibody to cadherin-5 in subconfluent cells. As predicted, there is no detectable decline in resistance under conditions in which cell-cell tethering is eliminated. Subsequent exposure to 2 mM EGTA decreased the resistance to level of a naked electrode, indicating that EGTA completely released cell-matrix adhesion. Tracings are representative of 5 separate experiments.

Because the model indicated that the antibody to cadherin-5 mediated a primary decrease in cell-cell adhesion, we further validated the analysis by measuring the resistance of subconfluent cells that had not established cell-cell contact. Under these conditions Rb is negligible, and thus changes in transendothelial resistance would reflect primary changes in alpha . As expected, when we added the antibody to cadherin-5 to subconfluent cells, we did not detect a decline in resistance (Fig. 2C). However, we did detect a decline in resistance in these same subconfluent cells in response to 2 mM EGTA, which would affect sites of cell-matrix adhesion. The final resistance on exposure to EGTA approached the resistance of a naked electrode (1,700 Omega ), which is consistent with the notion that EGTA mediated a loss in cell-matrix adhesion. Hence, the failure of the anti-cadherin-5 antibody to decrease the resistance of subconfluent cells was not due to a sensitivity limit of the system.

To validate our assumption that Cm does not significantly change, we measured Cm before and 15 min after the addition of the anti-cadherin-5 antibody at frequencies between 22 and 90,000 Hz (as described for Fig. 1). In a separate series of experiments, the Cm was 2.85 ± 0.05 µF/cm2 in untreated cells and 2.87 ± 0.35 µF/cm2 in cells exposed to the antibody.

To further validate the precision of our model, we also measured the change in Rb and alpha  in confluent human endothelial cells that were transfected with an E-cadherin expression vector that was under the control of a glucocorticoid-inducible promoter. We observed an increase in resistance on exposure to dexamethasone in cultured ECV304 cells that were transfected with a pLKneo vector that contained the cDNA insert for E-cadherin (Fig. 3). The resistance increased from 8,396 ± 876 Omega  in control monolayers to 13,014 ± 582 Omega  after 18 h of exposure to 1 µM dexamethasone. In contrast, the resistance did not increase in response to dexamethasone when cultured monolayers were transfected with the pLKneo vector without the E-cadherin insert (26). As expected, exposure of dexamethasone to cultured ECV304 cells mediated a greater effect on Rb than on alpha  (Fig. 3).


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Fig. 3.   Change in transendothelial resistance, alpha , and Rb in confluent ECV304 cells that were transfected with a pLKneo vector with cDNA insert of E-cadherin after 18 h of exposure to 1 µM dexamethasone (Dex). Actual values of Rb and alpha  were reported (Rb is expressed in units of Omega  · cm2 and alpha  is expressed in units of Omega 1/2 · cm). Tracing is representative of more than 25 separate experiments. See text for explanations. Impedance was measured at 4,000 Hz.

Localizing the effects of histamine to cell-cell and cell-matrix sites. We next measured dynamic changes in alpha  and Rb in response to histamine by measuring the resistance and reactance at a single frequency. Figure 4 illustrates the effect of 10 µM histamine on transendothelial resistance, reactance, and impedance. Histamine caused a rapid but transitory decrease in transendothelial resistance. Histamine decreased the transendothelial resistance by 20-30% within 30-60 s. The resistance recovered to basal levels within 3-5 min and subsequently increased to above initial basal levels. In contrast, histamine mediated smaller changes in transendothelial impedance and even smaller changes in transendothelial reactance.


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Fig. 4.   Effect of 10 µM histamine on transendothelial resistance, reactance, and impedance. Histamine had greater effect on resistance than on impedance or reactance. Transendothelial resistance was most sensitive index of barrier function, which is why resistance was reported with Rb and alpha . Tracing represents means of 5 separate experiments.

When we used our dynamic optimization procedure to arrive at measured values of alpha  and Rb, the decrease in resistance occurred primarily at sites of cell-cell adhesion, especially in more mature monolayers. Histamine initiated decreases in alpha  and Rb, with much greater fractional decreases in Rb, which paralleled the decline in the experimental resistance in monolayers 1 day postconfluent (Fig. 5A). In monolayers 5 days postconfluent, histamine caused a very little decrease in alpha  (Fig. 5B), with predominant decreases in Rb.


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Fig. 5.   Experimental resistance with change in alpha  and Rb in response to 10 µM histamine based on dynamic optimization procedure. A: change in alpha  and Rb compared with change in experimental resistance in 1-day postconfluent cells. B: change in alpha  and Rb compared with change in experimental resistance in 5-day postconfluent cells. C: change in the experimental resistance compared with change in alpha  and Rb within first 60 s in 1-day postconfluent cells exposed to histamine. Means are plotted for 5 separate experiments.

When changes in Rb and alpha , which occurred within 1 min after exposure to histamine, were examined with finer time resolution, the decline in experimental resistance temporally correlated with a decline in Rb (50-60% change) (Fig. 5C). The decline in Rb preceded the decline in experimental resistance. In contrast, the decline in alpha  (20-30% change) lagged behind the experimental resistance, similar to the reactive decline in alpha  observed in cells exposed to the cadherin-5 antibody.

Although histamine decreased the resistance predominantly through effects on cell-cell adhesion, histamine initially restored the resistance at cell-matrix sites in cells 1 day postconfluent. We detected increases in alpha  before increases in the experimental resistance (Fig. 5A). In contrast, the initial increases in Rb lagged behind increases in the experimental resistance. Hence, during the initial restoration phase, histamine engaged cell-matrix sites and increased resistance, whereas early restoration of cell-cell adhesion occurred later.

Whereas histamine engaged cell-matrix sites during the initial part of the restoration phase, later increases in the resistance to above basal levels were due primarily to increases in cell-cell adhesion. After 5 min, alpha  remained constant, whereas the experimental resistance continued to increase in monolayers 1 day postconfluent (Fig. 5A). In contrast, changes in Rb paralleled the increase in the experimental resistance after this time (Fig. 5A).

There was a closer association between the restoration phase of the experimental resistance and increases in Rb in cells 5 days postconfluent (Fig. 5B). There was very little increase in alpha .

To further support the idea that the primary effects of histamine on the endothelium were to decrease cell-cell adhesion and to increase cell-matrix adhesion, we measured the resistance in subconfluent cells that had not established cell-cell contact. Because the contribution of Rb would be negligible, the change in resistance would represent the direct effect of histamine on cell-matrix adhesion. An additional advantage of this approach is that it removes the reactive effects of histamine on cell-matrix adhesion by eliminating the mechanical coupling between cell-cell and cell-matrix sites. As predicted from the model, we observed no significant decline in resistance in response to histamine, which validates the detected direct effect of histamine on cell-cell adhesion. Histamine did not decrease resistance but instead increased the resistance (Fig. 6). Taken together, these data demonstrate that histamine disrupts barrier function by directly decreasing cell-cell tethering and inducing a reactive loss in cell-matrix adhesion. In contrast, histamine restores barrier function by first increasing cell-matrix adhesion.


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Fig. 6.   Effect of 10 µM histamine on electrical resistance across subconfluent cells. Microelectrode was inoculated with sparse number of endothelial cells. Resistance was measured in subconfluent cultures (verified microscopically) in which there was no cell-cell contact. Electrical resistance was amplified by a factor of 10. Under these conditions, changes in resistance were dominated by changes in cell-matrix adhesion. Tracing is representative of 6 separate experiments. Sham, control cells.

Correlation between changes in Rb and separation distance between adjacent cells. Because a decrease in Rb is dependent on increased separation distance or strain between adjacent cells, we asked whether functional changes in Rb correlated with structural changes in cell-cell separation. At ×1,000 magnification using scanning electron microscopy (SEM), we did not observe the formation of discrete gaps between adjacent cells in monolayers exposed to histamine when viewed from the cell surface (Fig. 7). Cell-cell contact was preserved between control cells (Fig. 7A), cells exposed to histamine for 1 min (Fig. 7B), and cells exposed to histamine for 20 min (7C). In contrast, we observed large generalized gaps between adjacent endothelial cells exposed to 2 mM EGTA (Fig. 7D). As expected, we observed the formation of focal gaps between adjacent endothelial cells on neutralization of cadherin-5 (Fig. 7E).


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Fig. 7.   Cell-cell contact based on scanning electron microscopy in control cells (A), cells treated with histamine for 60 s (B), cells exposed to histamine for 20 min (C), cells exposed to 2 mM EGTA for 10 min (D), and cells exposed to 50 µg/ml of the anti-cadherin-5 antibody for 10 min (E). Magnification, ×1,000. Bars, 10 µm. Arrowheads in E, areas of disruption between adjacent cells. See text for explanation.

Histamine-induced gap formation between adjacent cells could not be detected when viewed from the surface using SEM because the cell membranes of adjacent cells overlapped. To resolve gap formations, cross sections were prepared by cutting the specimen in the y-z axis and were subsequently viewed under transmission electron microscopy (TEM; Fig. 8A). Interestingly, we did not observe a complete full-thickness separation between adjacent cells in monolayers exposed to histamine for 60 s (Fig. 8B). Instead, we observed only focal and nonspecific areas of separation between adjacent cells. Within 20 min of exposure to histamine, cell-cell contact was restored toward its basal state (Fig. 8C).


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Fig. 8.   Cell-cell separation between adjacent cells in postconfluent monolayers based on transmission electron microscopy in control cells (A), cells exposed to histamine for 60 s (B), and cells exposed to histamine for 20 min (C). Scale bars, 200 nm (A and B) and 124 nm (C). Arrowheads, adjacent cell membranes. See text for explanation.

The average separation distance increased from 18.9 ± 0.93 nm in control cells to 103 ± 14.9 nm in cells treated with histamine for 60 s (Fig. 9). In cells exposed to histamine for 20 min, the average separation distance recovered to 24.8 ± 1.18 nm. Whereas the initial loss in Rb correlated with the increase in the average separation distance, the final separation distance approached but did not fully explain the final increase in Rb (Fig. 9).


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Fig. 9.   Correlation between changes in average separation distances (A) and Rb (B) among control cells, cells treated with histamine for 1 min, and cells treated with histamine for 20 min. To quantitate average separation distance, entire length of gap between 2 adjacent cells was subdivided into 10 equidistant regions. After calibrating pixels into nanometer scale, plots of radiance intensities and distance measurements were obtained across each gap point. Separation distance was determined by measuring distance between 2 radiance maxima. Data for each reported group of separation distance represent means ± SE of >80 regional points of interest. See text for explanation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Histamine initiates transitory increases in endothelial permeability in situ and in vitro (3, 18, 27). Under in situ conditions, increased permeability is associated with the development of small gaps between adjacent endothelial cells, and restored barrier function is associated with the reapposition of adjacent cells (12, 27). In recent years, several investigators have hypothesized that these gaps might develop as the result of an increase in centripetal tension developed by endothelial cells. However, we found that histamine decreases barrier function in confluent human endothelial cells without increasing centripetally directed tension (18). Rather than tearing the cells apart by an increase in centripetal tension, histamine appears to act by reducing tethering forces and utilizes the resting tension to create the development of small gaps. This paradigm is consistent in part with the gaps that develop between adjacent endothelial and epithelial cells when calcium-dependent adhesion sites are severed by chelating extracellular calcium (22, 23).

Resolving the sites at which histamine remodels tethering in cultured endothelial cells is particularly complex for two reasons. First, cell-cell and cell-matrix sites are mechanically coupled by an intervening series of filamentous cytoskeleton that can transfer local mechanical forces to distal sites (16, 25). This makes it difficult to resolve primary stresses, which are consequent to the specific targeting of signal transduction, from reactive forces, which are consequent to expressed primary stresses. Second, changes in tethering in response to edemagenic stimuli are rapid. We previously reported rapid decreases in transendothelial resistance in response to histamine that occur over a period of only 60 s (18). Thus to dynamically and quantitatively localize primary stresses in response to edemagenic stimuli, changes in cell-cell and cell-matrix adhesion need to be separately measured but simultaneously compared. To begin to localize the effects of histamine, we modeled the resistance across a cell-covered electrode as a resistor and capacitor in series (5, 8). In this model, the transendothelial resistance is a function of the resistance created by cell-matrix adhesion (alpha ), the resistance created by cell-cell adhesion (Rb), and Cm.

The hypothesis that the total resistance is dependent on alpha , Rb and Cm in cultured endothelial cells is supported by several observations. First, the experimental resistance did not fit a model based on the assumption that there was no cell-cell and cell-matrix adhesion (alpha  and Rb approaching zero). Second, the experimental resistance did not fit models that were dependent on alpha  or Rb alone. Instead, the experimental resistance best fit a model that was dependent on Rb, alpha , and a cell Cm greater than 3 µF/cm2. The higher Cm suggests that the plasma membrane was not smooth but rather convoluted with great surface area. This is consistent with the observations of Schmid-Schonbein et al. (21), who reported numerous surface membrane folds in endothelial cells using electron microscopy.

We observed quantitative differences in alpha , Rb, and Cm in confluent monolayers of different ages. We observed higher values of Rb in cells 5 days postconfluent compared with cells 1 day postconfluent. In contrast, alpha  and Cm decreased in older cultures. Consistent with our observation, Davies et al. (4) reported a decrease in focal contacts in older confluent monolayers based on interference reflection microscopy. Our data suggest that with increased age, there is increased horizontal tethering and less vertical tethering.

Dynamic changes in cell-cell and cell-matrix adhesion were resolved by the measured resistance and reactance determined at a single frequency because resistance and reactance are dependent on the same solution of alpha  and Rb. Inasmuch as there are an equal number of measurements for the number of the unknown solutions for alpha  and Rb (resistance and reactance), solutions for alpha  and Rb can be computed at multiple time points. Based on this approach, the model indicated that histamine decreased transendothelial resistance predominantly through decreases in cell-cell adhesion. This was consistent in cells 1 and 5 days postconfluent.

We also identified a smaller decrease in alpha  in cells 1 day postconfluent, which temporally lagged behind the experimental resistance and Rb. These data alone suggest that the loss in cell-matrix adhesion was mechanically coupled to the loss in cell-cell adhesion or that the loss of cell-matrix adhesion occurred independent of the loss of cell-cell adhesion but at a slower rate. However, additional data support the former paradigm. Histamine did not decrease the resistance in subconfluent cells, a condition in which cell-matrix adhesion is mechanically uncoupled from cell-cell adhesion. Also, the cadherin antibody similarly mediated a secondary and smaller decrease in alpha  in confluent cells, whereas the same antibody had no effect in subconfluent cells.

Although the model indicated that histamine decreased the resistance primarily through a loss in cell-cell adhesion, restoration of the resistance was initiated by cell-matrix-directed forces that increased cell-matrix adhesion in monolayers 1 day postconfluent. The model detected increases in alpha  before there were detectable increases in the experimental resistance. In contrast, increases in Rb lagged behind increases in the experimental resistance.

The lag between the initial increase in Rb and alpha  is particularly interesting because it suggests two possible paradigms for the initial restoration of endothelial barrier function. In one paradigm, histamine independently restores cell-cell and cell-matrix sites, but it restores cell-matrix sites first. Alternatively, restoring cell-matrix sites may help to restore cell-cell adhesion. Remodeling of cell-matrix adhesion sites might alter the distribution of tensile stress on cell-cell adhesion sites, which would redirect forces to the periphery and facilitate restoration of cell-cell adhesion.

Although the optimization procedure indicated that the initial restoration of the experimental resistance after histamine treatment was due to increased cell-matrix adhesion, further increases in resistance that developed after 3-5 min were due to cell-cell-directed forces that increased cell-cell adhesion in cells 1 day postconfluent. At these later time points, alpha  was constant, whereas changes in Rb paralleled changes in the experimental resistance.

The relationship between alpha  and Rb to the experimental resistance during the restoration phase was slightly different in cells 5 days postconfluent. The experimental resistance increased, with less of an increase in alpha . Instead, there was a temporal correlation between the increases in experimental resistance and increases in Rb. The differences between cells 1 and 5 days postconfluent suggest that the pattern of remodeling of barrier function may be dependent on the age of the monolayer, which is consistent with the observed differences in alpha , Rb, and Cm between cells 1 and 5 days postconfluent.

We validated the ability of the model to measure dynamic changes in alpha  and Rb through a number of controlled studies. First, we applied the optimization procedure to localize changes in resistance in monolayers exposed to an antibody against cadherin-5. Because the antibody disrupts homeotypic cadherin tethering, the model should detect a predominant decrease in Rb that temporally parallels a decline in experimental resistance. This paradigm was supported by our experimental results. Second, dexamethasone-mediated expression of E-cadherin in ECV304 cells resulted in predominant changes in cell-cell resistance. Third, the primary changes in alpha  and Rb detected in confluent cells when exposed to histamine or the cadherin-5 antibody are consistent with the observed responses in subconfluent cells exposed to these same agents. Inasmuch as there was no cell-cell contact, the resistance was dominated by alpha  in subconfluent cells. According to our model, histamine should increase resistance because of its effect on alpha  and histamine should not decrease resistance because Rb is negligible. Exposure to the cadherin-5 antibody should not affect resistance because homeotypic cadherin binding was not established. As anticipated, these responses were observed, further supporting the ability of the model to identify site-specific changes in endothelial barrier function.

Although the decrease in Rb correlated with increased separation distance between adjacent cells in response to histamine, the measured separation distance did not fully explain the final increase in Rb at later time points. The separation distance only approached control levels (Delta  ~6 nm) at time points corresponding to values of Rb above basal levels. There are several potential explanations for this discrepancy. First, Rb is an approximated value of cell-cell adhesion. The precision of Rb is dependent on the initial assumptions of the model and the mathematical approach used to generate the curve fit. The model assumes that the cell is disk shaped, which departs from the typical cuboidal shape of an endothelial cell. Our eventual goal is to use various constitutive equations to evaluate whether different geometries alter the contribution of alpha  and Rb to barrier function. Also, we must assume that the Cm does not significantly change or, if it did change, such changes would have a minor influence on the interpretable effects of histamine on alpha  and Rb.

Second, adhesion assays were performed on gold, whereas TEM studies were performed on plastic. Thus minor differences in cell motility may be attributed to differential effects from different substrates.

Third, the method used to quantitate cell-cell separation distance may underestimate cell-cell impedance. These gaps were focal and not uniform. Histamine-mediated cell-cell gaps required the resolution of TEM to detect the tens of nanometers of displacement. Changes in Rb actually reflect three-dimensional displacements in cell-cell adhesion, whereas our measured cell-cell separation distance was based on a two-dimensional measurement. The electrical current may take a convoluted course between adjacent cells, which may not be fully appreciated because of the overlapping cell membranes.

Although Cm was not included in our evaluation of the measured impedance at 4,000 Hz, the solutions for alpha  and Rb in confluent cells were still predictive for the cadherin interventions, validated by the subconfluent responses, and paralleled changes in cell-cell separation distance. To include Cm in the evaluation, at least three different measurements of resistance and reactance are required. One approach would be to measure resistance and reactance in real time at multiple frequencies, but at this point, the data acquisition among the lock-in amplifier, relay stations, and the computer would need to be restructured.

Although alpha  is directly related to Rc (cell radius) and inversely related to the square root of h (the average separation distance between the substrate and the ventral surface of the cell), changes in Rc likely had little effect on measured changes in alpha . There are several reasons that support this notion. First, the scale of h is 1,000 times smaller than Rc. Thus the factor that includes h has a much greater effect than Rc on the measured alpha . Second, if histamine decreased alpha  exclusively through effects on Rc, then we should have observed greater changes in Rc. Histamine decreased cell radius by 84 nm, which compared with the initial Rc (~10-11 µm) represents only a 1% change. The alpha  should have decreased by only 1% rather than the observed decrease of 25%. Thus very large changes in Rc would have to occur, which would have been easily detected by SEM. Third, if large changes in Rc occurred, the monolayer would unlikely have been able to restore resistance to its basal level in such a short period of time.

Giaever and Keese (8) reported that Rc would have to change 400 times more than h to achieve the same unit change in alpha . Based on their report in cultured fibroblasts, they argued that alpha  should be more sensitive to changes in h unless there are very large changes in Rc.

It is important to distinguish h, which was used in our model, from other reported measurements of cell-substrate separation. Davies et al. (4) reported a quantitative method using interference reflection microscopy to define focal contact adhesion. They defined focal contact sites as radiances associated with cell-substrate separation distances of 15-30 nm. Yet, cell matrix adhesion is also dependent on nonfocal contact adhesion or close contact adhesion, which is defined by the absence of focal contact proteins and is associated with cell-substrate distances of greater than 30 nm (2). The value of h used in our model represents an integration of the entire cell-substrate interface, which consists of both focal and nonfocal contact sites.

Consistent with the results of our analysis, Winter et al. (26) reported that histamine did not decrease transcellular resistance in ECV304 cells, which do not express cadherin-5 or E-cadherin in wild-type cells. Yet, they reported that histamine decreased resistance in cells transfected with a pLKneo vector expressing E-cadherin or cadherin-5. The effect of histamine on resistance in ECV304 cells transfected with these cadherin-expression vectors was similar to its effect on resistance in cultured HUVECs. Thus this report suggests that the cadherin complex may be an important target site to decrease cell-cell apposition in response to histamine.

In summary, fitting resistance measurements with a model of cell-electrode interactions indicates that histamine rapidly and transiently altered endothelial barrier function through sequential changes at cell-cell and cell-matrix sites. These changes in response to histamine were best modeled by a sequential decrease in cell-cell adhesion, then increased cell-matrix adhesion, and finally increased cell-cell adhesion. This model now facilitates identifying the specific molecules and mechanisms that regulate inflammatory edema formation.


    ACKNOWLEDGEMENTS

We thank Randy Nessler for technical assistance on the electron microscopy studies and Jeff VanEngelenhoven and Matthew Lorson for technical assistance on the electric cell-substrate impedance sensing studies.


    FOOTNOTES

Scanning and transmission electron microscopic studies were conducted in the Central Microscopy, Central Research Facility.

This work was supported by a University of Iowa College of Medicine Grant, an AHA Grant-in-Aid (A. B. Moy), and National Heart, Lung, and Blood Institute Grant HL-33540 (D. M. Shasby).

This work was conducted during A. B. Moy's tenure as a recipient of the American Heart Association (AHA) Clinician Scientist Award and a Clinical Investigator Award from the American Lung Association.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. B. Moy, Dept. of Internal Medicine, C-33 GH, Univ. of Iowa College of Medicine, Iowa City, IA 52242 (E-mail: alan-moy{at}uiowa.edu).

Received 23 April 1999; accepted in final form 6 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Angres, B, Barth A, and Nelson WJ. Mechanism for transition from initial to stable cell-cell adhesion: kinetic analysis of E-cadherin-mediated adhesion using a quantitative adhesion assay. J Cell Biol 134: 549-557, 1996[Abstract].

2.   Burridge, K. Substrate adhesion in normal and transformed fibroblasts: organization and regulation of cytoskeletal membrane and extracellular matrix components at focal contacts. Cancer Rev 4: 18-78, 1986.

3.   Carson, M, Shasby S, and Shasby DM. Histamine and inositol phosphate accumulation in endothelium: cAMP and G protein. Am J Physiol Lung Cell Mol Physiol 257: L259-L264, 1989[Abstract/Free Full Text].

4.   Davies, P, Robotewskyj A, and Griem M. Endothelial cell adhesion in real time: measurement in vitro by tandem scanning confocal image analysis. J Clin Invest 91: 2640-2652, 1993[ISI][Medline].

5.   Giaever, I, and Keese C. Toxic? Cells can tell. Chemtech Feb: 116-125, 1992.

6.   Giaever, I, and Keese CR. Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc Natl Acad Sci USA 81: 3761-3764, 1984[Abstract].

7.   Giaever, I, and Keese CR. Use of electric fields to monitor the dynamical aspect of cell behavior in tissue culture. IEEE Trans Biomed Eng 33: 242-247, 1986[ISI][Medline].

8.   Giaever, I, and Keese CR. Micromotion of mammalian cells measured electrically. Proc Natl Acad Sci USA 88: 7896-7900, 1991[Abstract].

9.   Hirt, RP, Poulain-Godefroy O, Billotte J, Kraehenbuhl JP, and Fasel N. Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector. Gene 111: 199-206, 1992[ISI][Medline].

10.   Ingber, D. Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J Cell Sci 104: 613-627, 1993[Free Full Text].

11.   Laposata, M, Dovnarsky D, and Shin H. Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro. Blood 62: 549-556, 1983[Abstract].

12.   Leach, L, Eaton B, Wescott D, and Firth J. Effect of histamine on endothelial permeability and structure and adhesion molecules of the paracellular junctions of perfused human placental microvessels. Microvasc Res 50: 323-337, 1995[ISI][Medline].

13.   Lo, C, Keese C, and Giaever I. Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing. Biophys J 69: 2800-2807, 1995[Abstract].

14.   Majno, G, and Palade G. Studies on inflammation. 1. Effect of histamine and serotonin on vascular permeability: an electron microscopic study. J Biophys Biochem Cytol 11: 571-605, 1961[Abstract/Free Full Text].

15.   Majno, G, Shea S, and Leventhal M. Endothelial contraction induced by histamine type mediators: an electron microscopic study. J Cell Biol 42: 647-672, 1969[Abstract/Free Full Text].

16.   Maniotis, AJ, Chen CS, and Ingber DE. Demonstration of mechanical connections between integrins cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure. Proc Natl Acad Sci USA 94: 849-854, 1997[Abstract/Free Full Text].

17.   Moy, A, Scott B, Shasby S, and Shasby DM. The effect of histamine and cyclic adenosine monophosphate on myosin light chain phosphorylation in human umbilical vein endothelial cells. J Clin Invest 92: 1198-1206, 1993[ISI][Medline].

18.   Moy, AB, VanEngelenhoven J, Bodmer J, Kamath J, Keese C, Giaever I, Shasby S, and Shasby DM. Histamine and thrombin modulates endothelial focal adhesion through centripetal and centrifugal forces. J Clin Invest 97: 1020-1027, 1996[Abstract/Free Full Text].

19.   Persechini, A, and Hartshorne D. Phosphorylation of smooth muscle myosin: evidence for cooperativity between the myosin heads. Science 213: 1383-1385, 1981[ISI][Medline].

20.   Pitelka, D, Taggart B, and Hamamoto S. Effects of extracellular calcium depletion on membrane topography and occluding junctions of mammary epithelial cells in culture. J Cell Biol 96: 613-624, 1983[Abstract].

21.   Schmid-Schonbein, G, Kosawada T, Skalak R, and Chien S. Membrane model of endothelial cells and leukocytes. A proposal for the origin of a cortical stress. J Biomech Eng 117: 171-178, 1995[ISI][Medline].

22.   Shasby, DM, and Shasby S. Effects of calcium on transendothelial albumin transfer and electrical resistance. J Appl Physiol 60: 71-79, 1986[Abstract/Free Full Text].

23.   Sheldon, R, Moy A, Lindsley K, Shasby S, and Shasby DM. Role of myosin light-chain phosphorylation in endothelial cell retraction. Am J Physiol Lung Cell Mol Physiol 265: L606-L612, 1993[Abstract/Free Full Text].

24.   Tiruppathi, C, Malik AB, Vecchio PD, Keese C, and Giaever I. Electrical method for detection of endothelial cell shape change in real time: assessment of endothelial barrier function. Proc Natl Acad Sci USA 89: 7919-7923, 1992[Abstract].

25.   Wang, N, Butler J, and Ingber D. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260: 1124-1127, 1993[ISI][Medline].

26.   Winter, M, Kamath A, Ries D, Shasby S, Chen Y, and Shasby D. Histamine alters cadherin-mediated sites of endothelial adhesion. Am J Physiol Lung Cell Mol Physiol 277: L988-L995, 1999[Abstract/Free Full Text].

27.   Wu, NZ, and Baldwin AL. Transient venular permeability increase and endothelial gap formation induced by histamine. Am J Physiol Heart Circ Physiol 262: H1238-H1247, 1992[Abstract/Free Full Text].


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