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1 Department of Internal Medicine and 2 Department of Biomedical Engineering, University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We compared temporal changes in isometric tension in cultured human umbilical vein endothelial cells inoculated on a polymerized collagen membrane with changes in cell-cell and cell-matrix adhesion derived by a mathematical model of transendothelial cell resistance. Thrombin and histamine disrupt barrier function by targeting a greater loss in cell-cell adhesion, which preceded losses in overall transendothelial resistance. There were minor losses in cell-matrix adhesion, which was temporally slower than the decline in the overall transendothelial resistance. In contrast, thrombin and histamine restored barrier function by initiating a restoration of cell-matrix adhesion, which occurred before an increase in overall transendothelial resistance. Thrombin mediated a second and slower decline in cell-cell adhesion, which was not observed in histamine-treated cells. This decline in cell-cell adhesion temporally correlated with expressed maximal levels of tension development, suggesting that actin-myosin contraction directly strains cell-cell adhesion sites. Pretreatment of cells with ML-7 mediated more rapid recovery of cell-cell adhesion and had no effect on cell-matrix adhesion. Taken together, expression of actin-myosin contraction affects the restoration of barrier function by straining cell-cell adhesion sites.
electrical resistance; analytic modeling
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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EDEMAGENIC AGENTS alter endothelial barrier function by remodeling centripetal-based cytoskeletal forces, which increase paracellular permeability. Additionally, these same edemagenic agents also activate the expression of centrifugal-based cytoskeletal forces, which promote the restoration of barrier function and limit the extent of inflammatory edema (12). A dynamic balance between centripetal and centrifugal forces at any moment in time defines the state of barrier function. The precise cytoskeletal elements and the mechanisms that regulate centripetal- and centrifugal-based forces are not well understood.
Majno et al. (7, 8) originally hypothesized that inflammatory mediators increased paracellular permeability by contraction of the actin-myosin cytoskeleton. Earlier reports (16, 22, 23) documented cell retraction by inducing actin-myosin contraction in permeabilized cultured endothelial cells exposed to low micromolar levels of calcium and ATP and myosin light chain kinase (MLCK). These reports demonstrated that, under these in vitro conditions, contraction of actin-myosin generates sufficient force to pull adjacent cells apart from each other. However, edemagenic stimuli typically increase cell calcium to nanomolar levels (3, 15), an issue that has challenged the notion that edemagenic agents disrupt barrier function through contraction of actin-myosin cross-bridges.
To precisely evaluate the contribution of actin-myosin contraction on barrier function in response to physiological stimuli, myosin ATPase activity needs to be quantitated in real time and correlated with an equally quantitative and dynamic measurement of cell adhesion. Measured levels of myosin light chain phosphorylation have been used to assess myosin ATPase activity. However, myosin ATPase activity is governed by the cooperative interaction between myosin heads, which increases exponentially once one myosin head is phosphorylated (13). Thus the relationship between MLC phosphorylation and myosin ATPase activity is nonlinear and represents an indirect and inaccurate measurement of myosin ATPase activity. Others have measured endothelial force by measuring the amount of wrinkling exerted by endothelial cells on a deformable silicone substratum (2, 9). Freely movable cell-collagen lattices in solution have also been used to measure endothelial contraction by measuring the reduction in the size of the lattice over periods of hours to days (5, 18). However, these assays suffer from significant limitation by being qualitative or incapable of dynamically and quantitatively measuring constitutive changes in myosin ATPase activity under steady-state conditions or in response to physiological or pharmacological stimuli.
To precisely understand how contractile forces disrupt barrier function, dynamic and quantitative measurements of cell adhesion are required. Traditional techniques of measuring voltage clamp, as previously reported in cultured epithelial cells (17, 19), lack the resolution to measure the small transendothelial resistance in cultured endothelial monolayers. Permeability assays lack the spatial and temporal resolution to measure endothelial-cell adhesion dynamically and quantitatively because of its dependence on diffusion or transport of macromolecules. To resolve these problems, we (10, 12) previously reported techniques that dynamically quantitate contractile forces by measuring isometric tension on collagen hydrogels and techniques that dynamically quantitate cell adhesion by measuring transendothelial resistance in cultured endothelial cells grown on a microelectrode.
We (12) previously reported that histamine and thrombin disrupt barrier function independent of contraction of the actin-myosin cytoskeleton. Thrombin is a particularly novel edemagenic molecule because it disrupts barrier function in association with contraction of the actin-myosin cytoskeleton (12). In contrast, histamine disrupts barrier function independent of active contraction of the actin cytoskeleton (12). On the basis of their differences on contractility, these mediators provide a useful strategy to understand the contribution of actin-myosin contraction on barrier function in the setting of edemagenic stimuli. We (10, 12) previously reported that thrombin mediates a more sustained decline in transendothelial resistance than histamine. Tension development is not necessary for the initial loss in cell adhesion. Tension development was temporally out of phase with overall changes in transendothelial resistance in thrombin-treated endothelial cells. At time points of maximal force expression, barrier function was well on its way toward being restored to its basal state, and at time points at which barrier function was restored to baseline conditions, centripetal force was still greatly expressed. Yet, inhibition of thrombin-mediated tension development accelerated the restoration of barrier function. Taken together, inflammatory edema formation is clearly more complex than simple contraction of the actin-myosin cytoskeleton, and such contraction potentially affects the restoration, not the initial disruption, in barrier function. The next important question is how actin-myosin contraction affects the restoration of barrier function even though force generation does not temporally correlate with changes in transendothelial resistance.
Transendothelial resistance across a cell-covered electrode is a complex measurement dependent, in a large part, on cell-cell and cell-matrix adhesion (11). Thrombin-mediated force generation could affect the restoration of barrier function by straining cell-cell or cell-matrix adhesion. We recently validated an analytical approach that simultaneously compares the change in cell-cell and cell-matrix adhesion as it temporally changes with overall transendothelial resistance. This technique allows a more precise understanding of how changes in cell-cell and cell-matrix adhesion contribute to overall changes in barrier function (11). With the use of this approach, we were able to demonstrate that histamine transiently disrupts barrier function by first disrupting cell-cell adhesion but restores barrier function, in part, through direct effects on cell-matrix adhesion. We now use this analytical approach to evaluate how actin-myosin contraction delays the restoration of barrier function in response to thrombin. In this study, we demonstrate that actin-myosin contraction delays the restoration in barrier function by straining cell-cell adhesion sites.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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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, which
were coated with 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 µ/ml),
and streptomycin (100 µg/ml). Cultures were identified as endothelial
cells by their characteristic uniform morphology and uptake of
acetylated low-density lipoprotein and by indirect immunofluorescent
staining for factor VIII. Cytochalasin D and histamine were obtained
from Sigma (St. Louis, Mo). Human 
-thrombin was obtained from Enzyme
Research Laboratories (South Bend, IN). Vitrogen collagen (type 1, bovine dermal collagen) was obtained from Celtrix Pharmaceuticals
(Santa Clara, CA). ML-7 was obtained from Calbiochem (La Jolla, CA).
Cell adhesion assay.
Quantitative measurements in cell adhesion were measured in real time
by quantitating transendothelial resistance using a previously reported
technique in a 1-day-old postconfluent monolayer of cultured
endothelial cells grown on the surface of microelectrodes coated with
100 µg/ml fibronectin (10-12). In this
system, cells were inoculated 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 the 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, which is
dominated by the membrane capacitance (Cm)
(4). A 1-V, 4,000-Hz alternating current signal was
supplied through a 1-M
resistor to approximate a constant current
source. Voltage and phase data were measured with a SRS830 lock-in amplifier (Stanford Instruments) and then later stored and
processed with a personal computer. The in-phase voltage (proportional to the resistance) and the out-of-phase voltage (proportional to the
capacitive reactance) were measured. We expressed cell adhesion as a
function of resistance, which was expressed as a fractional change to
the initial value at the moment test agents were given. Both histamine
and thrombin mediate a transient decline in the fractional resistance.
As a convention, we refer in the text to the disruption of barrier
function as the negative slope of the overall transendothelial
resistance, whereas restoration of barrier function refers to the
positive slope of the overall transendothelial resistance.
Mathematical model to resolve the 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 (4, 11). The model
is based on the notion that current flows radially from under the
ventral surface of the cell and the electrode, escapes between cells,
and goes directly through the cell membrane by capacitive coupling. In
this model, the total impedance across a cell-covered electrode is
composed of the impedance between the ventral surface of the cell and
the electrode [cell-matrix resistance ()], the impedance between
cells [cell-cell resistance (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 related to Cm. can also be defined as follows:
Rc
is the resistivity of
the medium, and h is the average separation distance between
the cell and the underlying matrix (4).
, Rb, and
Cm are the only adjustable parameters in the
model. We first identified the values of , Rb, and Cm before the
addition of test agents. 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 were measured
as a function of current frequency in untreated cells. By iteratively
choosing values for Cm,
Rb, and , we can arrive at the best fit to
the experimental impedance. Because resistance is also dependent on the
same values for Cm, Rb,
and as they are for impedance or reactance,
Cm, Rb, and were
measured by finding the best fit of the calculated resistance to the
experimental resistance.
We expressed barrier function from the experimental resistance rather
than impedance for two reasons. First, physiological stimuli mediate
greater effects on resistance than on impedance. Second, by measuring
resistance, the membrane resistance of Zm is
trivial because the model considers the membrane resistance and
capacitance as a parallel circuit. The resistance was measured at 13 separate frequencies between 22 and 90,000 Hz and expressed as the
ratio of normalized resistance of a cell-covered to cell-free electrode. Real-time changes in cell-cell adhesion
(Rb) and cell-matrix adhesion () in response
to thrombin or other interventions were determined at a single
frequency of 4,000 Hz.
Measurement of isometric tension. Isometric tension of cultured endothelial cells was measured as described by Bodmer et al. (1). Isometric tension was simultaneously monitored in two separate isometric vectors because endothelial cells express tension multiaxially (1). Force was averaged for both vectors and expressed as the fractional change of the maximal force generation in the absence of ML-7 or expressed as the fractional change of the constitutive force in the presence of ML-7.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Relationship between thrombin-mediated tension development and
transendothelial resistance.
To elucidate the complex interaction between force generation and
barrier function in cultured endothelial cells, we first compared the
temporal relationship between centripetal tension and overall
transendothelial resistance. To measure centripetal tension, we
measured isometric tension in postconfluent cultured endothelial cells
inoculated on a polymerized type-1 collagen membrane. To dynamically
measure endothelial barrier function, we monitored transendothelial
resistance in 1-day-old postconfluent cultured HUVECs grown on a
microelectrode. As shown in Fig. 1, 7 U/ml (6 × 108 M) thrombin immediately decreased
transendothelial resistance before any significant increase in
isometric tension. Thrombin maximally decreased barrier function within
60-90 s, whereas tension development was maximally expressed
within 5-10 min. Furthermore, at time points at which thrombin
maximally expressed tension, the restoration of barrier function was
already initiated. Additionally, at time points at which barrier
function had recovered to initial basal levels, force was still
expressed at 50% of its maximal level. Thus temporal changes in force
generation were out of phase with changes in overall barrier function,
demonstrating that actin-myosin contraction was not sufficient to
account for the disruption in transendothelial resistance or the
overall change in barrier function at any moment in time.
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Effect of thrombin and histamine on endothelial cell-cell and
cell-matrix adhesion.
Because force did not temporally correlate with overall
transendothelial resistance, we examined whether force generation better correlated with local changes in cell adhesion. To elucidate the
regions at which thrombin altered cell adhesion, we broke down
transendothelial resistance into separate measurements of cell-cell and
cell-matrix adhesion. We accomplished this approach by modeling
transendothelial resistance as a circuit composed of a resistor and
capacitor in series using a method we previously reported
(11). This model is a function of
Rb, which is dependent on cell-cell adhesion,
and , which is dependent on cell-matrix adhesion. The utility of the
model is its ability to predict the sites at which biological molecules
alter barrier function.
and Rb during the disruption and restoration
phase of barrier function (Fig. 2).
During the initial disruption phase of barrier function, thrombin
mediated rapid declines in both Rb and , but with a greater fractional decline in Rb (Fig.
2A). When the same data are more closely examined over the
first 60 s, the decline in Rb preceded the
decline in overall resistance (Fig. 2B). In contrast,
thrombin mediated a decline in that was slower and quantitatively
less than the decline in the overall resistance. Taken together, the
model predicts that thrombin initiates the disruption in barrier
function by first and specifically disrupting cell-cell adhesion sites.
However, thrombin decreased either through a reactant loss of
cell-cell adhesion, or the loss in cell-matrix adhesion is the target
of signal transduction pathways, which proceed slower than the loss of
cell-cell adhesion. Alternatively, thrombin decreased
cell-matrix adhesion through both mechanisms.
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preceded increases in overall transendothelial resistance. In contrast, after the initial rapid decline in
Rb, thrombin mediated a second decline in
Rb, at time points at which overall resistance
was recovering. Interestingly, the rate of the second decline in
Rb was much slower than that of the initial decline in Rb. After a period of 10-15 min,
thrombin mediated a restoration in Rb that took
several minutes to return to initial basal levels. These data suggest
that mechanical effects directed at cell-matrix adhesion sites, not
those directed at cell-cell adhesion sites, initiated the recovery
phase of barrier function in response to thrombin.
There are some important similarities between the effects of histamine
and thrombin on and Rb worth emphasizing. As
reported previously (11) and again reported here in
separate experiments (Fig. 3), histamine
disrupted barrier function by disrupting Rb. Also, histamine initiated recoveries in before recoveries in resistance, whereas the recovery in Rb was
temporally slower than the recovery of overall resistance. Taken
together, both histamine and thrombin disrupt barrier function by
rapidly disrupting cell-cell adhesion but initiate the recovery in
barrier function through effects on cell-matrix interaction.
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in thrombin-treated cells than that observed in
histamine-treated cells (Fig. 4B). Fourth, the restoration
in occurred more promptly in histamine-treated cells compared with
thrombin-stimulated cells. Taken together, these data suggest that
there may be common signal transduction mechanisms between histamine
and thrombin that govern the disruption and restoration of barrier
function. Yet, there may be different mechanisms between histamine and
thrombin that may explain their quantitative and temporal differences
on barrier function.
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Correlation between tension development and cell-cell and
cell-matrix adhesion.
To elucidate how tension development affects the restoration of
thrombin-mediated barrier dysfunction, we compared the temporal relationship between tension development and changes in and Rb (Fig. 5A).
Tension in Fig. 5A was expressed as the fractional change of
the maximal force (approximately equivalent to 100-150% of the
constitutive force). Tension development temporally correlated with the
slow and second decline in Rb but not with the
initial rapid decline in Rb. The initial decline
in Rb preceded increases in tension development.
Changes in tension development did not correlate with changes in .
recovered at times at which tension development was expressed. In
contrast, 100 µM ML-7 attenuated thrombin-mediated tension
development, which was expressed the fractional change of the
constitutive force (Fig. 5B). Tension increased by 15%
above the constitutive force compared with 100-150% in the
absence of ML-7. More importantly, ML-7 did not prevent thrombin-mediated losses in Rb and (Fig.
5B). We (10) previously reported that ML-7, at
doses used in this study, mediates a very modest and transient decline
in transendothelial resistance compared with thrombin alone.
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(Fig. 6A). The disruption and
the recovery phases were similar in the presence or absence of ML-7 in
thrombin-stimulated cells. However, thrombin did affect the recovery
phase of Rb in cells treated with ML-7 (Fig.
6B). There was an accelerated recovery in thrombin-treated
cells exposed to ML-7 compared with cells not exposed to ML-7.
Additionally, the second and slow decline in Rb
that was observed in cells stimulated with thrombin alone was absent in
thrombin-stimulated cells that were pretreated with ML-7. Taken
together, these data suggest that expression of actin-myosin tension
development under thrombin-stimulated condition affects the recovery
phase of barrier function by straining cell-cell adhesion sites.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The precise contribution of actin-myosin contraction on barrier function under edemagenic stimuli is not well understood. Exogenous administration of MLCK and calmodulin induces endothelial-cell retraction in permeabilized endothelial cells (22). Exposure of permeabilized endothelial cells to low micromolar calcium levels induces cell retraction through a myosin-dependent pathway (16). These reports have been the basis of extrapolating the hypothesis that edemagenic stimuli induce cell retraction through activation of signal transduction pathways that increase cell calcium and activate MLCK-dependent centripetal tension development. However, there are two major criticisms of these models in extrapolating the role of actin-myosin contraction under physiological conditions. First, the plasma membrane is stripped away by detergent treatment, which removes the ability to measure signal transduction in intact cells in response to receptor-ligand interactions. Second, the dose of calcium and ATP used in permeabilized models is typically higher than that observed in intact cells in response to physiological or pharmacological stimuli.
In contrast, the contribution of actin-myosin contraction in governing barrier function in intact cells in response to edemagenic stimuli is comparably much different. We (12) previously reported that edemagenic agents such as histamine and thrombin disrupt barrier function independent of expressed actin-myosin contraction. Second, barrier function is fully restored to initial basal conditions at time points at which significant levels of force are still expressed in thrombin-treated cells. Clearly, actin-myosin contraction is not sufficient to account for all changes in endothelial barrier function. However, thrombin-mediated actin-myosin contraction contributes to a more sustained decline in barrier function compared with histamine-treated cells. Thus actin-myosin contraction expressed under physiological conditions affects the restoration phase of barrier function, not the disruption phase of barrier dysfunction. In these series of experiments, we demonstrated that expression of actin-myosin contraction modulates the restoration phase of thrombin-mediated barrier by disrupting cell-cell adhesion.
To elucidate the temporal and spatial effects by which actin-myosin contraction regulates barrier function, we temporally compared the effect of force generation on cell-cell and cell-matrix adhesion in thrombin-stimulated HUVEC. With the use of a previously described analytical model (11), we reported that histamine decreased barrier function by first disrupting cell-cell adhesion but restored barrier function by first engaging cell-matrix interaction. Histamine induced a reactive loss in cell-matrix adhesion through the mechanical coupling between cell-cell and cell-matrix sites by an intervening filamentous cytoskeleton. In the present study, thrombin disrupts barrier function in a manner similar to histamine except that it additionally disrupts cell-cell adhesion by expressed actin-myosin contractile forces.
The analytical approach is based on the mathematical modeling of a
cell-covered electrode as a circuit composed of a resistor and
capacitor in series that breaks down transendothelial resistance into
separate resistance measurements that are dependent on cell-cell adhesion (Rb) and cell-matrix adhesion ().
This approach provides several advantages over other methodologies.
First, simultaneous and separate measurements of cell-cell and
cell-matrix adhesion can be compared within the context of overall
transendothelial resistance or barrier function. This is particularly
necessary to evaluate the response to physiological or pharmacological
stimuli that have a very rapid onset of action. Second, the approach
can resolve spatial, temporal, and quantitative effects on cell-cell and cell-matrix adhesion.
Thrombin mediated distinct spatial and temporal effects in cell adhesion during the disruption and the restoration phase of barrier dysfunction. During the disruption phase, thrombin mediated rapid declines in cell-cell and cell-matrix adhesion, with much greater quantitative effects on cell-cell adhesion than on cell-matrix adhesion. Additionally, thrombin mediated different temporal effects in cell-cell and cell-matrix adhesion during the disruption of barrier function. Decreases in cell-cell adhesion preceded the loss of overall resistance. In contrast, loss of cell-matrix adhesion was temporally slower than the loss in overall transendothelial resistance. Taken together, these data suggest that thrombin disrupts barrier function by first and predominately targeting cell-cell adhesion sites. The loss of cell-matrix adhesion may be explained by three potential mechanisms. First, the decline in cell-matrix adhesion is a reactive effect in response to a primary decline in cell-cell adhesion. This is conceivable if cell-cell adhesion is mechanically coupled to cell-matrix adhesion sites by an intervening filamentous cytoskeleton. Alternatively, thrombin could specifically target the loss of cell-matrix adhesion but at a rate slower than that of cell-cell adhesion. Finally, the loss of cell-matrix adhesion could reflect the combination of both mechanisms.
Although the initial loss in and Rb in
thrombin-stimulated cells showed temporal similarities with responses
in histamine-treated cells, there were quantitative differences in
these changes. Thrombin caused greater decreases in both
Rb and in cultured monolayers than did
histamine. The mechanisms for these differences between histamine and
thrombin cannot be resolved by these studies. Others (20)
have reported that histamine transiently decreased transcellular resistance in ECV304 cells transfected with cadherin-5 or E-cadherin. Additionally, we (11) previously reported similar
quantitative decreases in overall resistance and
Rb between cells exposed to histamine and cells
treated with an antibody that disrupts homeotypic cadherin binding.
Although the above reports support the notion that histamine disrupts
barrier function by targeting the cadherin complex, there are some
important differences between these two interventions. Histamine
transiently creates intracellular gaps on the order of 80-100 nm
between adjacent cells, which are below the resolution of light
microscopy and scanning electron microscopy (11).
Intercellular gaps induced by anti-cadherin antibodies produce
irreversible and large gaps that are of several orders of magnitude
greater than those seen with histamine (11). Taken together, these data suggest that histamine must activate other elements, other than cadherin, to mediate reversible reapposition at
sites of cell-cell contact.
The mechanism of how thrombin disrupts barrier function through effects
on cell-cell adhesion sites is not clear. Others (6) have
reported altered distribution of cadherin staining in
thrombin-stimulated endothelial cells. Ratcliffe et al.
(14) reported reduced serine and threonine
dephosphorylation of catenin (14). Ukropec et al.
(18a) reported Src homology 2 phosphatase (SHP2) tyrosine phosphorylation and dissociation from vascular endothelial-cadherin complexes. The loss of SHP2 from the cadherin complexes
correlated with increased tyrosine phosphorylation of 
-catenin,
-catenin, and p120-catenin (18a). The functional
relevance of these morphological and biochemical changes on the
cadherin-catenin system remains unclear. Also, the reason why thrombin
mediates greater declines in Rb and in the
overall measured transendothelial resistance than histamine cannot be
resolved from our present work.
Our model suggests several unique centrifugal and centripetal forces
were expressed during the restoration phase in thrombin-treated cells
in a temporal-dependent fashion. Some of these centripetal and
centrifugal effects on cell adhesion overlap with responses observed in
histamine-treated cells. In particular, the restoration in overall
transendothelial resistance was preceded by increases in , which is
consistent with the notion that direct effects at cell-matrix sites
facilitate the restoration of barrier function. This notion is even
more supported by the responses in thrombin-treated cells. Only
increases paralleled and preceded increases in the overall
transendothelial resistance at time points at which
Rb was decreasing. Although the model predicts
that direct effects on cell-matrix adhesion initiate the restoration
phase of barrier function in thrombin and histamine-treated cells, the
rate of recovery of cell-matrix adhesion was slower with thrombin than with histamine. Nonetheless, taken together, these data suggest that
histamine and thrombin share common signaling mechanisms by which they
transiently disrupt barrier function through a sequential loss of
cell-cell adhesion and restore barrier function through effects on
cell-matrix adhesion, followed by reapposition of cell-cell adhesion.
The most striking difference between histamine and thrombin during the
restoration phase was the observed second and slower, or more
prolonged, decline in Rb. Compared with changes
in isometric tension, this second and maximal decline in
Rb temporally correlated with maximal increases
in force generation in thrombin-stimulated cells. Consistent with the
notion that this second decline in cell-cell adhesion was coupled to
expressed actin-myosin tension, this phase was abolished by ML-7.
Additionally, ML-7 accelerated the recovery phase of
Rb. ML-7 did not alter in any quantitative or temporal manner. This data suggests that expression of actin-myosin contraction under thrombin-stimulated conditions delayed the
restoration of barrier function by specifically straining cell-cell
adhesion sites while it had little effect in altering cell-matrix adhesion.
The impact of force generation on cell-cell adhesion is particularly remarkable because it suggests that the mechanical behavior of the cytoskeleton needs to be considered as a three-dimensional integrated mechanical system in which cell-cell adhesion sites are mechanically coupled to cell-matrix adhesion sites by an intervening filamentous cytoskeleton. This implies that deformations at local sites have to be interpreted within the context of three-dimensional deformations rather than on local deformations of membrane-cytoskeletal systems. This conclusion is based on several pieces of data. Our tension system was designed to detect the transmission of endothelial force to the underlying matrix. To disrupt cell-cell adhesion, these forces have to be transmitted along actin cables to strain cell-cell adhesion sites. In further support of an integrated mechanical system, we (11) reported that histamine and antibodies to cadherin induce a reactant decline in cell-matrix adhesion in postconfluent cells. When cell-cell adhesion sites are mechanically uncoupled from cell-matrix adhesion sites under subconfluent cultured conditions, histamine and antibodies to cadherin do not mediate loss in cell-matrix adhesion (11).
However, our data also suggest the possibility that mechanical forces directed at cell-matrix and at cell-cell adhesion sites may be compartmentalized and act independent of expressed actin-myosin contraction force to restore barrier function. The precise identity and the mechanisms by which cytoskeletal-based forces restore barrier function remains to be elucidated. However, several potential candidate proteins such as microtubules, intermediate filaments, actin, and several adhesion receptors like cadherin and integrins may be critical in regulating these processes.
It is reasonable to suspect that the material properties of the endothelial cell, particularly near the cell periphery, have to be different before and after exposure to thrombin. At time points at which barrier function is restored to initial basal levels, there are significant levels of expressed actin-myosin contraction. The restoration of barrier function is not simply attributed to a reduction in tension development. We (10) previously reported that cAMP-stimulation restored thrombin-mediated barrier function more promptly despite the fact that cAMP did not inhibit thrombin-mediated tension development. ML-7 reduced the temporal effects of thrombin on cell-cell adhesion. Centrifugal forces must abrogate the impact of the contractile force of actin-myosin from straining cell-cell adhesion sites. Taken together, these data suggest that mechanical forces have to be recruited to counterbalance the actin-myosin contractile forces as cell-cell reapposition occurs.
In summary, expression of actin-myosin contraction in thrombin-treated endothelial cells affects the restoration of barrier function. Although thrombin shares some temporal changes in cell-cell and cell-matrix adhesion with histamine, thrombin also mediates quantitative and temporal differences in cell-cell and cell-matrix adhesion compared with cells exposed to histamine. In particular, thrombin induced a more prolonged decline in cell-cell adhesion, which temporally correlated with tension development. Pharmacological inhibition of tension development had predominate effects on cell-cell adhesion and had no effect on cell-matrix adhesion. Taken together, these data demonstrate that expression of actin-myosin contraction affects the restoration of barrier function by straining cell-cell adhesion sites. Mechanical deformations of cytoskeletal-membrane interactions need to be considered as a model in which cell-cell and cell-matrix adhesion sites are mechanically coupled by an intervening filamentous cytoskeletal network.
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ACKNOWLEDGEMENTS |
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National Institute of General Medical Sciences Grant GM-61732 and a grant from the American Lung Association supported this work. This work was conducted during A. B. Moy's tenure as a recipient of a Clinical Investigator Award from the American Lung Association.
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FOOTNOTES |
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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).
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. Section 1734 solely to indicate this fact.
Received 4 June 2001; accepted in final form 21 August 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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5 separate experiments. Tension
and resistance are expressed as fractional change. See text for
interpretation.
