© 2000 American Heart Association, Inc.
Cellular Biology |
Redox Changes of Cultured Endothelial Cells and Actin Dynamics
From the Heart and Lung Institute and Division of Cardiology, Department of Internal Medicine (L.M., N.I.M., P.J.G.-C.), The Ohio State University, Columbus, Ohio; Johns Hopkins University School of Medicine (R.H.S., S.A.P.), Baltimore, Md.
Correspondence to Pascal J. Goldschmidt-Clermont, MD, 514 Medical Research Facility, 420 W 12th Ave, Columbus, OH 43210. E-mail Goldschmidt-1{at}medctr.osu.edu
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Abstract |
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Abstract—We studied the association between the production of reactive oxygen species, actin organization, and cellular motility. We have used an endothelial cell monolayer–wounding assay to demonstrate that the cells at the margin of the wound thus created produced significantly more free radicals than did cells in distant rows. The rate of incorporation of actin monomers into filaments was fastest at the wound margin, where heightened production of free radicals was detected. We have tested the effect of decreasing reactive oxygen species production on the migration of endothelial cells and on actin polymerization. The NADPH inhibitor diphenylene iodonium and the superoxide dismutase mimetic manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) virtually abolished cytochalasin D–inhibitable actin monomer incorporation at the fast-growing barbed ends of filaments. Moreover, endothelial cell migration within the wound was significantly retarded in the presence of both diphenylene iodonium and MnTMPyP. We conclude that migration of endothelial cells in response to loss of confluence includes the intracellular production of reactive oxygen species, which contribute to the actin cytoskeleton reorganization required for the migratory behavior of endothelial cells.
Key Words: actin • endothelium • migration • polymerization • reactive oxygen species
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Introduction |
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The endothelium plays major roles in controlling the traffic of molecules and cells to and from underlying tissues, in smooth muscle cell tone and proliferation, in the generation of extracellular matrix proteins, and in the formation of endovascular thrombi. As a consequence, any morphological or functional disruption of the endothelium resulting from a variety of injuries can lead to dramatic effects on the integrity of the cardiovascular system.1
In an effort to dissect the responses of endothelial cells (ECs) to various types of injury, we have previously shown that the combination of hypoxia and reoxygenation of ECs leads to the generation of superoxide and induces the polymerization of actin filaments. Actin polymerization in these conditions could be blocked by overexpression of Cu,Zn superoxide dismutase by using a replication-incompetent adenovirus vector.2 These data suggest that the production of superoxide, and probably of other free radicals, might contribute to important cellular responses to injury, such as actin reorganization. Next, we demonstrated that the overexpression of the constitutively activated form of the small GTP-binding protein rac1 in human or mouse ECs induces an increase in F-actin.3 This process was found to require superoxide production, presumably via an NADPH oxidase,3 whose presence in nonphagocytic cells, such as ECs, has been confirmed.4 5 6 7 8 9
For years, the regulation of the actin cytoskeleton has been reconstituted in vitro in privileged redox conditions, in which a large excess of antioxidants, such as dithiothreitol, is added to protein preparations.10 The reason for this redox bias is that in the absence of reducing agents, actin preparations are too unstable for studies of protein-protein interaction. Although these studies have provided invaluable information concerning the regulation of actin polymerization and organization, it has remained speculative to interpret them as being relevant to the mechanism of actin regulation in vivo. On the basis of our previous observations, we hypothesized that in many instances the actin response to various stimuli is taking place in oxidative conditions and that this redox change may play a previously unsuspected role in the remodeling of the cytoskeleton—remodeling that is a prerequisite for cellular motility. In the present report, we have examined this hypothesis by monitoring the concomitant production of reactive oxygen species (ROS), actin reorganization, and cellular motility in ECs that are responding to a wound applied to the confluent monolayer in vitro.
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Materials and Methods |
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Cells and Chemicals
We used a mouse aortic endothelial cell (MAEC) line.11 Unless otherwise stated, all chemicals were from Sigma Chemical Co or Fisher Scientific and were of the best grade available. Manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) was from Alexis Corp; diphenylene iodonium sulfate (DPI) was from Toronto Research Chemicals Inc; and 5- (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-DCF-DA), Alexa 488-actin (A488A), Alexa 568-actin (A568A), rhodamine phalloidin, and Calcein Blue AM (CBAM) were from Molecular Probes.
Cell-Wounding Assay
Confluent monolayers were wounded with a pipette tip. All
microscopic analyses were performed on the heated stage of a
Nikon Eclipse 800 fluorescence microscope. Time-lapse
microscopy was performed in a closed bath imaging chamber (Warner
Instrument Corp) on wounded monolayers of MAECs. In the experiments
using MnTMPyP and DPI, the inhibitors were present in
the media 1 hour before wounding and during the assay. Images were
acquired at 90-second intervals for at least 5 hours. Individual cells
were tracked with the "track object" menu of the MetaMorph image
analysis system, and the mean speed of individual cells
(µm/min) was computed by the program. We determined the module of the
resultant vector of the cell path by measuring the distance between the
start and end points.
For free radical detection, MAECs were loaded with CM-DCF-DA at a final concentration of 5 µmol/L in HBSS (GIBCO) for 20 to 30 minutes at 37°C in 5% CO2. At the end of the incubation, the monolayer was rinsed with HBSS, and the cells were examined alive at 37°C with a 490-nm excitation wavelength. Digital images were recorded on a SenSys digital camera. The integrated fluorescence intensity was measured in 10 rows (50 µm wide) parallel to the wound edge with the MetaMorph image analysis system. In some experiments, we loaded the cells concomitantly with CM-DCF-DA and CBAM. Ratios of the CM-DCF-DA and CBAM digital images were obtained and analyzed.12
Study of Actin Polymerization in MAECs
We adapted a published method13 to study the
incorporation of fluorescent actin into filaments; we used 1 to
3 µmol/L A568A rather than rhodamine actin. In some experiments,
cells were also stained with the nuclear probe Hoechst 33342.
The inhibitors DPI and MnTMPyP were added at 10
µmol/L and 25 µmol/L, respectively, for 1 hour before the
assay and were present at the same concentrations in the loading
buffer.
For the quantification of exposed barbed ends by flow cytometry, we used the same method, except that MAECs were at 60% confluence. A488A was added at a final concentration of 1 µmol/L in loading buffer for 0, 1, 2, and 3 minutes. At the end of the incubation period, fluorescence-activated cell sorting analysis was performed on trypsinized cells in a FACSCalibur flow cytometer (Beckton Dickinson Immunocytometry Systems). The inhibitors were added as indicated. In parallel samples, we added 2 µmol/L cytochalasin D (CyD)14 30 minutes before the addition of A488A. The number of exposed barbed ends was determined by using published methods.15 16
An expanded Materials and Methods section is available online at http://www.circresaha.org.
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Results |
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Cells at Wound Margin Produce More Free Radicals
Confluent monolayers of MAECs11 were scratched with a glass tip to create a wound. The formation of intracellular ROS was monitored by preloading the cells with CM-DCF-DA, a derivative of dichlorofluorescein diacetate that has improved retention within live cells and fluoresces on exposure to oxidants, in particular H2O2.17 18 Although cells before scratching or cells distant from the scratched area displayed low CM-DCF-DA fluorescence, MAECs flanking the wound were consistently and significantly more fluorescent (Figure 1a
and 1b). Quantification of CM-DCF-DA fluorescence by morphometric
analysis of digital images demonstrated increased free radical
production in the cell rows closest to the wound margin. The
fluorescence increased progressively over time after wounding
and peaked when the cells became actively involved in migrating to
restore the confluence of the monolayer (Figure 1c). The
ROS-producing cells contained an intact actin cytoskeleton, as detected
by rhodamine phalloidin staining of actin filaments within these cells
(not shown). Moreover, time-lapse fluorescence microscopy of
the monolayer flanking the wound margins, which had been preloaded with
CM-DCF-DA, showed that the cells with the highest fluorescent
signal were also the most actively motile. They had an intense and
transient membrane-ruffling activity. Free radical production
was detected not only within the cell body but also at the edge of the
membrane ruffles, the most motile part of the cell (Figure 1d
through 1j, arrowheads).
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The increased CM-DCF-DA fluorescence detected within the cells
flanking the wound margin could have been due to a volume effect, in
view of the fact that confluent ECs from inner rows are generally more
spread out (therefore more flattened) than the migrating cells. To rule
out a volume effect, we loaded the cells with the cell-permeant dye
CBAM, whose fluorescence is not influenced by the oxidative
status of the cell or by other intracellular variables, such as
pH.18 After acquiring digital images corresponding to
identical areas for both fluorophores, we performed the arithmetic
operation of dividing the pixel intensity of the CM-DCF-DA images by
the pixel intensity of the CBAM images, resulting in a new image whose
brightness was proportional to the ratio. Thus, dark regions
represent low ratios of CM-DCF-DA to CBAM fluorescence,
whereas bright regions are indicative of specific accumulation of
oxidized CM-DCF-DA. A representative image is
presented in Figure 2, where the
upper panels show, from left to right, CM-DCF-DA, CBAM, the ratio
image, and the differential interference contrast (DIC) image for a
cell from the wound margin. The lower panels show matching images for a
cell from the interior of the monolayer. The graphs present pixel
intensity levels as a function of the position along the scanned line.
Two conclusions may be drawn from these data: (1) The intensity of the
CM-DCF-DA fluorescence, relative to CBAM fluorescence,
is much higher within the cell flanking the wound margin (in Figure 2, compare the pixel intensity levels for the wound margin cell
[LS1] and for the cell away from the margin [LS2]),
consistent with increased production of ROS in these
cells. (2) There is specific accumulation of CM-DCF-DA at the edge of
migrating EC, whereas the higher fluorescence detected in the
perinuclear area represents a volume effect.
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Actin Polymerization in Motile MAECs Is Regulated by ROS
Production
A488A incorporation into filaments was measured by flow cytometry
in the presence and absence of the inhibitor CyD (2
µmol/L). We considered that an increase in A488A fluorescence
was secondary to actin polymerization at barbed ends if it could be
specifically inhibited by the addition of CyD. We used subconfluent
MAECs, which are actively motile and exhibit intense membrane
ruffling.19 The CyD-sensitive incorporation of A488A into
filaments within motile MAECs increased significantly during the first
3 minutes of incubation (preliminary experiments showed that after this
time point, the detected fluorescence begins to plateau in this
assay).
Many laboratories, including our own, have shown that a major
source of superoxide and derived radicals within ECs is the enzyme
NADPH oxidase.2 3 6 17 Therefore, we tested the effect of
the flavoprotein inhibitor DPI20 on A488A
incorporation into filaments. We also examined the effect of MnTMPyP, a
cell-permeant superoxide scavenger that mimics the effect of the enzyme
superoxide dismutase.21 22 Both inhibitors,
DPI and MnTMPyP, significantly reduced the elongation of actin
filaments at the barbed (fast growing) ends (Figure 3a). Moreover, we have measured the
number of opened barbed ends in ECs with and without antioxidants. In
control, freely moving, subconfluent ECs, we measured 3534±1109 opened
actin filament barbed ends. This number was decreased to 503±629 in
the presence of DPI and 731±343 in the presence of MnTMPyP
(P<0.05) (Figure 3b).
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To support the flow cytometric data, we used microscopy to study
monomeric actin incorporation within the cells flanking the wound
margins, and as an intrinsic control, we used the quiescent cells from
distant rows of the confluent monolayer on the same coverslip. We
examined MAECs 1 to 4 hours after wounding. The cells were loaded with
CM-DCF-DA, examined alive under the microscope to detect ROS
production, and then processed with the incorporation of A568A.
The cells were then fixed and examined again. A568A was most rapidly
incorporated within the spreading cells at the margin of the wound
(Figures 4a through 4c). The pattern of
actin distribution was typical of migrating cells23 :
arc-shaped arrays of actin filaments at the base of lamellipodia at the
advancing edge and microspikes parallel to the direction of migration
(Figure 4a). Cells that exhibited the highest actin staining
also stained stronger for free radicals, as detected by CM-DCF-DA
fluorescence (Figures 4b and 4c). In contrast, cells
away from the wound accumulated little A568A, mostly in a punctate
pattern with no subcellular compartmentalization, and produced little,
if any, free radicals (Figures 4d through 4f).
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We also assessed A568A incorporation in the presence of
inhibitors: CyD (2 µmol/L), DPI (10 µmol/L),
and MnTMPyP (25 µmol/L). A568A incorporation was nearly
completely inhibited by CyD, indicating that the fluorescent
signal was indeed secondary to the polymerization of A568A (in Figure 5, compare panels 5a and 5d). CyD had no
detectable effect on free radical production (not shown). In
the presence of DPI, A568A incorporation nearly vanished (in Figure 5, compare panels 5a and 5f). The presence of the superoxide
dismutase mimetic MnTMPyP also reduced markedly the polymerization of
actin (Figure 5h), and many cells acquired a polarized shape,
contrasting with the usual cobblestone appearance of cultured ECs. In
cells with a detectable residual A568A fluorescent signal,
stress fibers, instead of lamellipodia, were labeled (Figure 5h, inset). Thus, MnTMPyP affected not only the rate of polymerization of
labeled actin but also the specific site of actin incorporation within
ECs.
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Some actin accumulation was also detected within the cell body, in the
perinuclear region, particularly with a higher concentration of A568A
(3 µmol/L), a previously described finding in microinjected
cells.24 To rule out that such actin accumulation was
taking place within the nucleus,25 we labeled the nuclei
with Hoechst 33342 and performed both standard fluorescence
microscopy (Figure 5c
) and confocal analysis of the
cells (not shown) to define more accurately the localization of the
fluorescent actin within the cell body. Figure 5c is
representative and shows that the A568A
fluorescence is perinuclear.
Superoxide Scavenging Reduces Cell Motility
If increased production of free radicals is necessary for
the dynamic actin turnover within lamellipodia, a process required for
the migration of cells into the wound, then superoxide scavenging
should reduce the motility of ECs. To test the relation between
increased free radical production and cellular motility, we
examined the speed of cells moving into the wound, in the presence or
absence of DPI or MnTMPyP, by time-lapse microscopy. We also measured
the resultant vector for each individual cell path, as a measure of the
efficiency of migration, for 5 to 6 hours after wounding.
The parameters measured for control cells were as follows:
mean speed, 0.45±0.02 µm/min, which agrees well with the
reported speed for subconfluent ECs of 
0.5
µm/min,19 and mean resultant vector, 97.9±10.9
µm for 5 hours. In the presence of DPI, these 2
parameters were significantly reduced to 0.26±0.009
µm/min and 36.8±3.0 µm, respectively (P<0.001 for
both parameters), and in the presence of MnTMPyP, they were
reduced to 0.28±0.02 µm/min and 20.3±2.6 µm,
respectively (P<0.001 for both parameters)
(Figure 6). Interestingly, although both
inhibitors reduced the speed of the cells (by 42.3% for
DPI and by 37.8% for MnTMPyP), these agents decreased even more the
effective distance migrated by the cells (by 62.4% and 79.3%,
respectively). This could be explained by the fact that the migration
of cells exposed to superoxide scavengers is more random (Figure 7, cell tracks). We ruled out the
possibility that this effect was due to a cytotoxic effect of the
inhibitors on the cells, because after their removal, the
speed of migration was gradually restored to normal values within the
next 20 hours (data not shown).
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Discussion |
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In the present study, we report that ECs respond to a stresslike mechanical disruption of the confluent monolayer by producing increased amounts of free radicals. For years, the impact of ROS has been considered solely as harmful for cells, and in many instances, it may be.26 27 28 However, more recent evidence has accumulated showing that many physiological processes require the production of minute amounts of various ROS, such as H2O2,29 superoxide,30 or NO.31 In our experiments, the increase in ROS production is more prominent within the ECs flanking the wound margin. As opposed to the quiescent cells from a confluent monolayer, the cells in this position start migrating within the "empty" space of the wound. This process requires actin cytoskeleton reorganization,32 and our findings show that fluorescent monomeric actin is more actively incorporated into filaments within cells that are producing heightened amounts of oxidants.
In a model of hypoxia and reoxygenation of human ECs, we have previously shown that the reorganization of the actin superstructure can be controlled by the oxidative status of the cell.2 Moreover, when we analyzed the mechanism by which the small GTP-binding protein rac1 controls the reorganization of actin cytoskeleton and the formation of membrane ruffles in human ECs, we found that actin polymerization requires superoxide as well. The actin changes induced by overexpression of the constitutively activated mutant of rac1, racV12, could be reversed by either overexpression of Cu,Zn superoxide dismutase or incubation with MnTMPyP and by the antioxidant N-acetylcysteine.3
In the present study, we attest that actin reorganization
within cells exposed to a stresslike confluence disruption takes place
in the presence of upregulated intracellular ROS production, as
detected by free radical–induced CM-DCF-DA fluorescence
(Figures 1 and 2). A key question is whether the presence
of ROS is causative for, independent of, or secondary to the actin
changes. Experimental limitations do not allow for a definition of the
temporal relation between free radical production and the
transition from the "quiescent" actin cytoskeleton, mainly
characterized by the presence of stress fibers, to the migratory
phenotype, with the formation of a distinctive lamellipodium.
However, our findings suggest that removal of ROS by 2 different
mechanisms, either inhibiting the enzyme that produces superoxide
(by DPI) or scavenging already produced superoxide (by MnTMPyP), has
significant effects on actin reorganization and cell migration.
The inhibition of free radical production and of actin
incorporation into the filaments in the presence of DPI (Figures 3 and 5) suggests that (1) an endothelial
NAD(P)H oxidase-like enzyme is the leading source of the free radicals
detected by CM-DCF-DA,2 3 6 33 34 and (2) superoxide has a
role in the polymerization of specific actin structures. The detection
of CM-DCF-DA fluorescence at the tips of the membrane ruffles
(Figure 1e through 1j) is consistent with the activation
of a nonphagocytic NADPH oxidase via rac1 bound to GTP, in view of the
fact that rac1 was also detected in the membrane
ruffles.3 35 DPI, which is a flavoprotein
inhibitor, is not absolutely specific for NADPH oxidase. NO
synthase is another major flavoprotein in ECs. We have ruled out the
possibility that DPI affected the production of free radicals
by ECs through inhibition of NO synthase, because a classic
inhibitor of NO synthase
(N
-nitro-L-arginine
methyl ester) did not affect EC production of ROS in our assay
(data not shown). This finding is consistent with the fact that
NO has been, in general, associated with the inhibition of cell
migration.36
The effect of MnTMPyP showed that removal of superoxide prevented
efficient migration, reduced motility to a lesser extent, and inhibited
actin polymerization because of the limitation of available barbed ends
(Figures 3 and 5 through 7). However, the detection of
stained stress fibers (Figure 5g, inset) suggests that the
effect of MnTMPyP is more complex than the effect of DPI. We consider
this particular finding significant because (1) the detection of
fluorescent stress fibers was markedly enhanced in the presence
of MnTMPyP, and (2) this result is consistent with our previous
observation that when superoxide dismutase was overexpressed in human
aortic ECs via adenovirus transduction, a similar pattern of cell
polarization and parallel actin fibers occurred.3
Dismutation of superoxide results in the formation of
H2O2,21 and it
is known that treatment of cells with
H2O2 induces an increase in
actin polymerization in various cells, including
ECs,37 38 39 perhaps through a process that involves the
small GTP-binding protein Rho and the actin-binding protein
cofilin.40
A key result of the present study is the requirement of ROS
for the exposure of barbed ends (Figure 3). This finding
suggests a possible mechanism by which ROS may promote the actin
polymerization observed in this and our previous work.3
Specifically, ROS may either promote the severing of actin filaments,
the uncapping of barbed ends, or both, and whatever the mechanism for
this effect might be, the net result is the increase in the exposure of
barbed ends, which dramatically increases the speed of actin
polymerization.40
The physiological production of ROS
by ECs at the margins of the wound is also necessary for migration to
take place, because the removal of superoxide either by inhibition of
the enzyme that generates it (DPI) or by dismutation (MnTMPyP) reduces
the motility of the exposed cells (Figures 6 and 7). It
is interesting to compare this behavior with that of neutrophils from
patients with chronic granulomatous disease.41 Although an
NADPH oxidase from these patients is nonfunctional because of various
mutations, the migratory ability of these cells is intact. However,
there are major differences between superoxide production in
phagocytic versus nonphagocytic cells: (1) The phagocytic NADPH oxidase
evolved to maximize the production of the reactive species
required for bacteria killing, whereas much lower amounts are produced
in nonphagocytic cells.41 42 (2) The site where superoxide
is produced in phagocytic cells is either the extracellular space or
the phagosome, which is also a space excluded from the cell cytoplasm.
In contrast, in our experiments, the probe we used (ie, CM-DCF-DA)
specifically detected the intracellular production of oxidants.
This is in agreement with other recent reports showing that
ECs,43 as well as smooth muscle cells,44
produce oxidant species within the cytoplasmic compartment.
We propose that although there are possible instances when actin
reorganization occurs in the absence of any specific production
of ROS, in other instances the targeted production of ROS, in a
timely manner, modulates actin cytoskeleton reorganization. The
localized production of oxidants could selectively affect the
function of actin monomer–sequestering proteins, –capping proteins,
or –severing proteins, reversibly or irreversibly, in a process that
may be analogous to the activation of nuclear factor-
B by
oxidant-triggered degradation of IB.45 In turn,
oxidant-modified actin binding proteins may affect the
parameters of the actin
polymerization/depolymerization processes.
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Acknowledgments |
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This study was supported by National Institutes of Health grants GM-53236 and HL-52315 (to Dr Goldschmidt-Clermont), by the American Heart Association (Dr Goldschmidt-Clermont is an Established Investigator), and by the Scleroderma Research Foundation. We thank Dr R. Auerbach, University of Wisconsin, for kindly providing us with the mouse aortic EC line.
Received October 4, 1999; accepted December 20, 1999.
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