Departments of 1 Internal Medicine and 2 Biomedical Engineering, University of Iowa College of Medicine, Iowa City, Iowa 52242
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ABSTRACT |
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Exposure of cultured human umbilical vein endothelial cells to the cAMP agonists theophylline and forskolin decreased constitutive isometric tension of a confluent monolayer inoculated on a collagen membrane, but it did not prevent increased tension in cells exposed to thrombin. The inability of cAMP agonists to prevent tension development correlated with an inability of cAMP stimulation to prevent increased 20-kDa myosin light chain (MLC20) phosphorylation in response to thrombin. Although cAMP did not prevent tension development or increased MLC20 phosphorylation, cAMP attenuated the effect of thrombin on transendothelial electrical resistance across a confluent monolayer inoculated on a gold microelectrode. Activation of cAMP-dependent signal transduction did not prevent a decline in resistance in thrombin-treated cells, but it more promptly restored transendothelial resistance to initial basal levels (10 min) compared with thrombin only (60 min). ML-7, an MLC kinase antagonist, at doses that attenuate increased MLC20 phosphorylation and tension development, did not prevent a decline in resistance in thrombin-treated cells. Yet, ML-7 also restored transendothelial resistance more rapidly than thrombin alone (20 min) but at a slower rate than cAMP. These data demonstrate that activation of cAMP-dependent signal transduction protects barrier function independent of inhibition of MLC20-dependent tension development.
adenosine 3',5'-cyclic monophosphate; 20-kDa myosin light chain; histamine; thrombin; adhesion; isometric tension; resistance
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INTRODUCTION |
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INCREASED INTRACELLULAR cAMP protects endothelial barrier function and attenuates inflammatory edema formation (2, 3, 9, 18). The mechanisms by which cAMP protects barrier function are not well understood. Carson et al. (2) and later others (13) demonstrated that cAMP agonists attenuate inflammatory edema formation through effects downstream from the activation of signal transduction pathways. Because edemagenic agents increase endothelial permeability by altering endothelial cell-cell and cell-matrix adhesion (11, 19), it is reasonable to hypothesize that cAMP enhances barrier function through effects on endothelial cell adhesion.
There are several mechanisms by which cAMP could attenuate inflammatory edema (10). Breakdown of tethered cell-cell and cell-matrix sites results in decreased cell apposition to adjacent cells and/or substrate through expression of constitutive centripetal tension (12, 14, 15, 17). cAMP could attenuate the loss of cell apposition to adjacent cell and matrix sites by reducing the level of constitutive centripetal tension.
cAMP might also attenuate inflammatory edema by preventing an increase in a centripetally directed actomyosin tension. Although expression of actomyosin contraction did not initiate a decrease in endothelial cell adhesion, it did slow the restoration of cell adhesion after thrombin exposure (11). Because edema represents the diffusion and convection of water and solutes over time, a more prompt restoration of cell adhesion would result in less edema.
Last, cAMP could attenuate inflammatory edema by acting directly on adhesion sites. cAMP could increase tethering, thereby decreasing the extent of the loss in cell adhesion or accelerate the restoration of cell adhesion.
Moy et al. (11) previously reported that thrombin and histamine initiate a disruption in transendothelial electrical resistance across cultured human umbilical vein endothelial cells (HUVECs) independent of tension development (11). Thrombin, which mediated 20-kDa myosin light chain (MLC20)-dependent increases in centripetal tension, caused a more prolonged decline in transendothelial resistance than did histamine, which did not increase tension. Inhibition of MLC kinase (MLCK)-dependent tension development and MLC20 phosphorylation more promptly restored the resistance to initial basal levels, but it did not prevent the decline in resistance in thrombin-treated cells (11). Thus cAMP could protect barrier function by accelerating restoration of barrier function through an MLC20-sensitive pathway. Alternatively, cAMP might act by preventing an MLC20-insensitive disruption of barrier function or by accelerating restoration of barrier function through an MLC20-insensitive pathway.
To examine these possibilities, we examined the effects of cAMP on thrombin-stimulated MLC20 phosphorylation, thrombin-stimulated tension development, and thrombin-initiated changes in endothelial cell adhesion in real time. We found that activation of cAMP-dependent signal transduction accelerates the restoration of endothelial barrier function independent of inhibition of MLC20 phosphorylation or tension development.
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METHODS AND MATERIALS |
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Materials. Tissue culture supplies were obtained from the Cancer Center, University of Iowa (Iowa City). Fetal bovine serum was obtained from Hyclone Laboratories (Logan, UT). Cytochalasin D, histamine, theophylline, and forskolin were obtained from Sigma (St. Louis, MO). Human alpha 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 cultures. Cultured HUVECs were prepared by collagenase treatment of freshly obtained human umbilical veins as previously described (2, 11). Cultures were identified as endothelial cells by their characteristic uniform morphology, uptake of acetylated low-density lipoproteins, and indirect immunofluorescent staining for factor VIII.
Measurement of isometric tension. Isometric tension of cultured endothelial cells was measured with a newly designed prototype previously described by Bodmer et al. (1). Three milliliters of a previously defined, cold unpolymerized collagen mixture (11) were poured between two 35-mm polyethylene bars and allowed to polymerize at 37°C. The collagen matrix was inoculated with 2.0 × 106 cells after the surface of the membranes was coated with 100 µg/ml of fibronectin for 15 min. The cells were grown in M199 supplemented with 10% fetal calf serum and antibiotics. Tension experiments were conducted in at least 2-day postconfluent monolayers.
Tension experiments were conducted in a new generation system previously described by Bodmer et al. (1). In this system, isometric tension was simultaneously monitored in two separate isometric vectors. Isometric tension was measured in more than one vector to confirm that a decline in isometric tension represented a true relaxation and not an anisotropic contraction because endothelial cells can express tension multiaxially (1).
We measured isometric tension under nonprestressed conditions with a previously described protocol (1). Before addition of the test agents, a sham response consisting of a comparable volume of carrier vehicle was introduced to rule out nonspecific mechanical effects. Force is expressed as the absolute change in tension (in mg) after addition of the test agents.
Measurement of MLC phosphorylation. Identification and quantitation of the phosphorylation of MLC20 were accomplished by laser densitometry of two-dimensional gels of MLC20 isoforms immunoprecipitated from 35S-labeled cells according to a previously described procedure (10). Stoichiometry was calculated by determining the relative fraction of phosphorylated isoforms to all isoforms according to the expression stoichiometry = fraction of monophosphorylated isoform + (2 × fraction of diphosphorylated isoforms).
A dynamic and quantitative measurement of
endothelial barrier function. Cell adhesion was
measured with the technique previously reported by Giaever and Keese
(4-6), Moy et al. (11), and Tiruppathi et al. (19).
In this system, referred to as electric cell-substrate impedance
sensing, cells were cultured on a small gold electrode (5 × 104
cm2) with 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 that is
dominated by the membrane capacitance (6). 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 experimental agents. Thus measured changes in impedance represent
alterations primarily in cell-cell adhesion and/or cell-matrix
adhesion.
A 1-V, 4,000-Hz AC signal was supplied through a 1-M resistor to
approximate a constant-current source. Voltage and phase data were
measured with an SRS830 lock-in amplifier (Stanford Research Systems)
and then later stored and processed with an IBM-compatible computer.
The same computer also controlled the output of the amplifier and relay
switches to different electrodes. At a surface area of
10
4
cm2 and at 4,000 Hz, this
microelectrode can resolve changes in cell adhesion.
For the experiments, the 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 in impedance or reactance. Electrical resistance increased after the cells attached and covered the electrodes, and the resistance achieved a steady-state level by 24 h.
Statistical analysis. Data are means ± SE. Comparisons between groups were made with Student's
t-test. Comparisons between more than
two groups were made with analysis of variance. Individual group
comparisons were done with Tukey's honestly significant difference
test for post hoc comparison of means. Differences were considered
significant at the P 0.05 level.
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RESULTS |
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Effect of cAMP stimulation on thrombin-mediated tension development. To determine whether increased intracellular cAMP protects endothelial barrier function through inhibition of actomyosin contraction, we first asked whether increased intracellular cAMP prevents thrombin-dependent tension development. To measure endothelial tension, we measured isometric tension in two separate vectors in a confluent monolayer of cultured HUVECs inoculated on the surface of a polymerized collagen membrane. Isometric tension was measured in two separate vectors because endothelial cells express force multiaxially, and a decrease in isometric tension may represent an anisotropic contraction (1). Figure 1 shows a representative experiment illustrating the effect of thrombin on isometric tension in which the monolayer was pretreated with theophylline and forskolin. Theophylline (1 mM) and forskolin (20 µM) caused a simultaneous decrease in isometric tension in both vectors. The decrease in isometric tension in both vectors validates that the decrease in isometric tension is attributed to a true relaxation (1). Surprisingly, pretreatment with these cAMP agonists did not prevent tension development on subsequent exposure to 7 U/ml of thrombin. Subsequently, exposure to 3 µM cytochalasin D abolished tension development, indicating that the actin cytoskeleton mediated thrombin-generated tension.
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Pretreatment with cAMP agonists did not reduce the amount of tension generated in response to thrombin (Fig. 2). The amount of tension expressed in thrombin-treated cells was not statistically different between cells treated with cAMP agonists and those that were not exposed to cAMP agonists. In contrast, the basal tension was significantly reduced in monolayers treated with cAMP agonists alone.
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The reduction in isometric tension in cAMP-treated cells was not due to nonspecific effects induced by the carrier vehicle because the carrier vehicle alone did not reduce tension. Similarly, the increase in tension in thrombin-treated cells was not due to the carrier vehicle for thrombin.
Effect of cAMP on thrombin-mediated MLC phosphorylation. We next asked whether the inability of cAMP agonists to inhibit thrombin-dependent tension development was due to an inability to inhibit increased MLC20 phosphorylation. Thrombin increased MLC20 phosphorylation from 0.43 ± 0.04 mol of phosphate/mol of MLC (mP/mMLC) in control cells to 0.76 ± 0.06 mP/mMLC in cells treated with 7 U/ml of thrombin for 10 min (Fig. 3). MLC20 phosphorylation decreased to 0.28 ± 0.04 mP/mMLC in the cells treated with cAMP agonists only for 20 min. However, MLC20 phosphorylation increased to 0.57 ± 0.08 mP/mMLC in the cells pretreated with cAMP agonists for 20 min followed by exposure to thrombin for 10 min. Although the stoichiometry in the thrombin-treated cells pretreated with cAMP agonists was lower than in the cells treated with thrombin alone, the differences were not statistically significant. In fact, thrombin increased phosphorylation in the control cells by almost the same amount (0.33 mP/mMLC) that it increased phosphorylation in the cells pretreated with cAMP agonists (0.29 mP/mMLC).
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Effect of cAMP stimulation on transendothelial resistance in thrombin-treated cells. Because the amount of edema formation is dependent on the magnitude of the decline in cell adhesion and the time it takes to restore cell adhesion (11), we asked whether activation of cAMP-dependent signal transduction attenuated the decline in transendothelial resistance and/or accelerated the restoration of the resistance to initial basal levels in cells treated with thrombin. On exposure to theophylline and forskolin, the transendothelial resistance increased, indicating that cAMP stimulation had a direct effect on cell-cell and/or cell-matrix adhesion (Fig. 4). The increase in resistance was greater than the effect of the carrier vehicle on resistance, indicating that the effect of cAMP stimulation on resistance was specific. To support the specificity of the effects of theophylline and forskolin on cAMP activation, we observed similar effects on the transendothelial resistance in cells exposed to 1 mM dibutyryl cAMP (Fig. 4). At a dose that attenuates increased isometric tension and increased MLC20 phosphorylation (11), 100 µM ML-7, an inhibitor of MLCK, did not increase the electrical resistance (Fig. 4).
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In cells that were pretreated with forskolin and theophylline or dibutyryl cAMP, thrombin still caused a decline in transendothelial resistance (Fig. 4). Similarly, thrombin also decreased the resistance in cells pretreated with 100 µM ML-7 (Fig. 4). The magnitude of the decline in resistance was similar in thrombin-treated cells that were exposed to thrombin alone, ML-7, or theophylline and forskolin (Fig. 5). These data demonstrate that neither cAMP stimulation nor primary inhibition of MLCK prevents a loss in cell adhesion on exposure to thrombin.
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However, cAMP stimulation had a dramatic effect on the rate of restoration of the resistance in thrombin-treated cells (Fig. 5). The transendothelial resistance returned to basal levels within 5-10 min in thrombin-treated cells that were pretreated with cAMP agonists, whereas the electrical resistance required 60 min to recover to basal levels in cells treated with thrombin alone. The transendothelial resistance also returned to basal levels much more promptly (20 min) in thrombin-treated cells that were pretreated with 100 µM ML-7 but at a much slower rate than that observed in cAMP-treated cells. The rate of restoration of the resistance in thrombin-treated cells pretreated with cAMP agonists was similar to the rate of restoration in cells treated with 10 µM histamine, an agent that Moy et al. (11) previously demonstrated did not increase isometric tension.
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DISCUSSION |
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The mechanism by which increased intracellular cAMP protects endothelial barrier function has not been precisely determined. Edemagenic agents increase endothelial permeability through a loss of endothelial cell-cell and cell-matrix adhesion. Adhesion plaques localized at cell-cell and cell-matrix sites are coupled to the actin cytoskeleton. Thus cAMP could attenuate inflammatory edema through direct effects on adhesion plaques or through the coupled actin cytoskeleton.
In support of the hypothesis that cAMP attenuates inflammatory edema through effects on the actin cytoskeleton, Moy et al. (10) and Sheldon et al. (17) previously reported that the phosphorylation state of MLC20 correlated with the changes in albumin flux across cultured HUVECs exposed to cAMP agonists and to histamine. These effects suggest that activation of cAMP-dependent signal transduction events might modulate barrier function through the inhibition of MLC20-dependent tension development. However, this hypothesis had not been directly validated, and Moy et al. (11) subsequently found that histamine altered barrier function without increasing MLC20-dependent tension in these cells.
To address this important question, it was necessary to choose a cell model in which we could resolve the effect of increased MLC20-dependent tension on barrier function. Moy et al. (11) previously reported that thrombin and histamine initiate a transient disruption in transendothelial resistance across cultured HUVECs independent of MLC20-dependent tension development. Although the disruption of electrical resistance was independent of tension development, expression of MLC20-dependent tension development was associated with a more prolonged disruption of barrier function. Because measured changes in permeability are dependent on the formation and resolution of decreases in adhesion between adjacent cells and between cells and the underlying matrix, cAMP could protect barrier function by inhibiting the decline in resistance by a MLC20-insensitive mechanism; by accelerating the restoration of resistance to initial basal levels by an MLC20-sensitive mechanism; or by accelerating the restoration independent of MLC20 phosphorylation. In these studies, we found that activation of cAMP-dependent signal transduction accelerates the restoration of barrier function independent of inhibition of MLC20-dependent tension development.
Activation of cAMP-dependent signal transduction events did not prevent thrombin-mediated tension development, a mediator that increases endothelial isometric tension through increased MLC20 phosphorylation (7, 11, 16). The inability of cAMP stimulation to prevent an increase in thrombin-mediated tension development is unlikely a methodological problem because cAMP alone decreased constitutive tension.
The inability of activation of cAMP-dependent signal transduction to inhibit thrombin-mediated tension development may be attributed, in part, to an inability of cAMP to fully prevent an increase in MLC20 phosphorylation. The inability to detect a decrease in MLC20 phosphorylation is unlikely a methodological problem for two reasons. First, cAMP agonists alone decreased basal phosphorylation. Second, MLC20 phosphorylation in response to thrombin is regulated by mechanisms other than the activation of MLCK in this model (16).
Shasby et al. (16) recently reported that thrombin increased
MLC20 phosphorylation in cultured
HUVECs, in part, through inhibition of a calyculin
A-sensitive phosphatase. Thrombin increased MLC20 phosphorylation in
-toxin-permeabilized cells in which MLCK stimulation was held
constant by clamping intracellular calcium levels at 600 nM.
Thrombin-mediated increases in
MLC20 phosphorylation are
dependent on the calcium-calmodulin-regulated MLCK and a
myosin-associated phosphatase. Thus activation of cAMP-dependent signal
transduction with consequent inhibition of MLCK will not completely
prevent increased MLC20
phosphorylation after thrombin because thrombin also inhibits
MLC20 dephosphorylation.
Increased cell cAMP also had little effect on the initial decrease in transendothelial resistance. Yet, increased cell cAMP did accelerate the restoration of transendothelial resistance to initial basal levels in response to thrombin. ML-7, at doses that attenuated increases in tension development and increases in MLC20 phosphorylation (11), also restored the resistance to initial basal levels more rapidly than thrombin alone but at a slower rate than that observed in cells pretreated with cAMP agonists. The rate of restoration in cAMP-treated cells was similar to the restoration in cells exposed to histamine, an agent that did not increase isometric tension (11).
The dissociation between isometric tension and transendothelial resistance in cultured HUVECs in the presence of cAMP agonists demonstrates that cAMP protects barrier function through mechanisms other than through inhibition of actomyosin cross-bridge formation. If cAMP did not have an effect on barrier function other than modulating the amplitude of the actomyosin force, then the time that it takes for the transendothelial resistance to return to basal levels should be the same as for cells treated with thrombin alone because cAMP did not prevent increased centripetally directed tension. Instead, cAMP stimulation more promptly restored barrier function despite expression of increased tension. This indicates that activation of cAMP signal transduction pathways must uncouple the load of the increased actomyosin force from slowing the rate of restoration of barrier function.
cAMP attenuates edema formation neither by preventing actomyosin contraction nor by preventing a loss in cell adhesion. cAMP attenuates edema formation by accelerating the restoration of cell adhesion. Our data raise speculation of an alternative model by which cAMP could attenuate inflammatory edema. This model needs to account for the observations that expression of actomyosin contraction in thrombin-treated cells contributes to the prolonged disruption of barrier function, and activation of cAMP-dependent signal transduction more promptly restores barrier function despite expression of actomyosin tension development. Ingber (8) and Moy et al. (11) previously utilized the concept of cellular tensegrity to explain how cellular mechanical forces remodel endothelial-cell shape. In tensegrity, cell shape is dependent on the balance between a continuous series of prestressed centripetal forces (actomyosin filaments) that cause cell rounding and a discontinuous series of compressive-resistive elements (microtubules, intermediate filaments, and adhesion plaques) that maintain cell spreading. Based on the concept of tensegrity, activation of thrombin-dependent signal transduction would increase actomyosin tension that would stress cell adhesion sites and slow restoration of barrier function (11). Activation of cAMP-dependent transduction events could directly affect adhesion plaques and prevent any strain from increased actomyosin tension at these sites. Alternatively, activation of cAMP-dependent transduction events could directly increase compressive-resistive forces at microtubules and intermediate filaments, which would also oppose increased actomyosin tension and prevent strain at adhesion sites. Further studies are needed to resolve these mechanisms.
In summary, increased intracellular cAMP attenuates endothelial barrier function independent of inhibition of MLC20-dependent tension development. Despite expression of active contraction, cAMP uncoupled the load of the actomyosin contraction from disrupting barrier function. The potential target sites and the mechanism by which cAMP restores barrier function independent of expressed actomyosin tension development requires further investigation.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-33540 (to D. M. Shasby) and an American Heart Association Grant-in-Aid (to A. B. Moy).
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FOOTNOTES |
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This work was conducted during the tenure of A. B. Moy as a Recipient of the American Heart Association Clinician Scientist Award.
Address for reprint requests: A. B. Moy, Dept. of Internal Medicine and Dept. of Biomedical Engineering, C33 GH, Univ. of Iowa College of Medicine, Iowa City, IA 52242.
Received 31 July 1997; accepted in final form 6 March 1998.
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