1 Vascular Bioengineering Laboratory, Department of Biomedical Engineering, and 2 Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Production of reactive oxygen species (ROS) by ischemic tissue after ischemia-reperfusion (I/RP) is an important factor that contributes to tissue injury. The small GTPase Rac1 mediates the oxidative burst, and ROS act on signaling pathways involved in expression of inflammatory genes. Because there is evidence implicating monocytes in the pathogenesis of I/RP injury, our objective was to determine the molecular mechanisms that regulate adhesive interactions between monocytes and hypoxia-reoxygenation (H/RO)-exposed cultured endothelial cells (ECs). When U937 cells were perfused over human umbilical vein ECs at 1 dyn/cm2, H (1 h at 1% O2)/RO (13 h) significantly increased the fluxes of rolling and stably adherent U937 cells. Either EC treatment with the antioxidant pyrrolidine dithiocarbamate (PDTC) or infection with AdRac1N17, which results in expression of the dominant-negative form of Rac1, abolished H/RO-induced ROS production, attenuated rolling, and abolished stable adhesion of U937 cells to H/RO-exposed ECs. Infection with AdRac1N17 also abolished H/RO-induced upregulation of vascular cell adhesion molecule (VCAM)-1. In turn, blocking VCAM-1 abolished U937 cell stable adhesion and slightly increased rolling. We concluded that the Rac1-dependent ROS partially regulate rolling and exclusively regulate stable adhesion of monocytic cells to ECs after H/RO and that stable adhesion, but not rolling, is mediated by ROS-induced expression of VCAM-1.
reactive oxygen species; shear stress; endothelial cell adhesion molecules
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INTRODUCTION |
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EARLY REPERFUSION is desirable after myocardial infarction, because it is an effective way to reduce infarct size and improve cardiac tissue function. However, reperfusion itself results in neutrophil adhesion in the postcapillary venules and emigration into the interstitium, increased monolayer permeability, and abnormal vasoregulation (16, 34). The pathogenesis of ischemia-reperfusion (I/RP) injury has been attributed to the production of oxygen-derived free radicals, often called reactive oxygen species (ROS), upon readmission of blood at reperfusion (19, 61). Vascular endothelial cells (ECs) are the primary target for ROS generated by themselves immediately after reperfusion and by adherent leukocytes at later times in reperfusion (43, 61).
In vitro hypoxia-reoxygenation (H/RO) studies with cultured ECs
demonstrated that ROS production is increased shortly after reoxygenation (41, 53) and is dependent on activation of
the small GTP-binding protein Rac1 (29). Rac1 regulates
the activity of the transcription factor nuclear factor (NF)-B via
production of ROS (50), believed to occur through the
plasma membrane-bound superoxide
(O
B and activator protein (AP)-1 promotes the
late phase by stimulating the expression of EC adhesion molecules
(30).
The fact that many EC adhesion molecules are upregulated after A/RO
raised the possibility that the endothelium may support adhesion of
other leukocyte subclasses, such as monocytes and lymphocytes, and that
these cells may also mediate RP injury. In accordance with this, Kokura
et al. (31) showed that T-lymphocyte adhesion to HUVECs
increases after EC exposure to A (1 h)/RO (8 h) and that this
hyperadhesivity is inhibited by antibodies against ICAM-1, vascular
cell adhesion molecule (VCAM)-1, and their counterreceptors on T cells
(2-integrins for ICAM-1 and
4-integrins
for VCAM-1). In addition, a number of in vivo studies implicated
monocytes in the pathogenesis of reperfusion injury. Birdsall et al.
(6) found that ECs released monocyte chemoattractant
protein (MCP)-1 that led to monocyte enrichment into the canine
myocardium after reperfusion. Administration of an anti-MCP-1 antibody
reduced infarct size in rat hearts (42). Blocking the
function of endothelial monocyte-activating polypeptide (EMAP)-II
prevented the onset of apoptosis and the influx of monocytes in
reperfused kidney (12). Nevertheless, there is not yet an
in vitro model that evaluates the molecular mechanisms of H/RO-induced
monocyte adhesion to ECs.
Most in vitro studies on leukocyte adhesion to A/RO-exposed ECs have been conducted under static conditions (25, 30, 31), with few exceptions (17, 47). Incorporating flow in the adhesion assay provides a more realistic environment for adhesion to occur, in which flowing cells have to form contacts within milliseconds with their counterligands and to withstand the shearing forces imposed by the flow (32). Flow studies allow examination of the three types of adhesive interactions: tethering, rolling, and stable adhesion (39). In addition, EC-released inflammatory mediators are washed away by flow, preventing the buildup of soluble mediators and leaving only the surface-bound molecules active.
In light of the advantages of in vitro flow systems, we studied the
adhesive interactions between human monocytic U937 cells and
H/RO-exposed HUVECs under a wall shear stress of 1 dyn/cm2,
a level typically encountered in postcapillary venules (28, 49). Kukreti et al. (33) studied monocyte adhesion
to interleukin (IL)-1-stimulated ECs under flow and showed that
L-selectin and
4-integrins support rolling, whereas
4- and
2-integrins support stable
adhesion. U937 cells are an appropriate model of monocytes in studying
adherent interactions with ECs, because they express
4
1- and
2-integrins as
well as L-selectin (22, 24, 26, 46). In A/RO-exposed
HUVECs, VCAM-1 expression was shown to increase at 4 h after
reoxygenation with a peak either at 12-16 h (57) or
at 8 h (31). Because it remains unclear how and to
what extent I/RP promotes monocyte adhesion, the objectives of the
present study were 1) to quantify the U937 cell
adhesive interactions to H (1 h)/RO (13 h)-exposed HUVECs,
2) to determine whether reoxygenation-induced ROS production
promotes the expression of VCAM-1, 3) to investigate whether
Rac1 activation is required for reoxygenation-induced ROS production,
and 4) to clarify the role of VCAM-1 in monocytic cell-EC
adhesive events.
Using the chemical antioxidant pyrrolidine dithiocarbamate (PDTC),
which prevents NF-B activation (37), we demonstrated that EC-derived ROS are involved both in VCAM-1 expression on H/RO-exposed HUVECs and in monocytic cell rolling and stable adhesion to these HUVECs. VCAM-1 was found to be solely responsible for stable
adhesion, but it did not mediate rolling. The effects of H/RO exposure
on VCAM-1 expression and U937 cell stable adhesion to H/RO-exposed ECs
were completely blocked in ECs infected with an adenovirus that encodes
a dominant-negative Rac1 (Rac1N17) but not with a control virus,
suggesting that reoxygenation-induced ROS production is Rac1 dependent.
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MATERIALS AND METHODS |
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U937 cell culture. U937 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI 1640 with 25 mM HEPES (GIBCO BRL, Rockville, MD) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (GIBCO). Cells were subcultured every 3 days until they reached a density of 106/ml in assay medium (RPMI 1640 with 25 mM HEPES and 1% FBS).
EC tissue culture.
Primary noncryopreserved HUVECs were purchased from GlycoTech
(Rockville, MD) and grown in T25 flasks in EGM-2 complete growth medium
containing 2% FBS and growth supplements (Clonetics, San Diego, CA).
On confluence, cells were rinsed, trypsinized (0.05% trypsin solution,
Clonetics), and seeded in different containers, depending on the
experimental protocol. All culture containers were coated with 0.2%
gelatin (Sigma, St. Louis, MO). For monocyte adhesion assays,
second-passage ECs were grown in 35-mm tissue culture dishes (Corning,
Corning, NY). For cell-based ELISA, ECs of second to fourth passage
were grown in 24-well plates (Becton Dickinson). For measurement of
O
EC infection with recombinant adenoviruses.
The replication-deficient adenovirus AdRac1N17, which encodes the
myc epitope-tagged dominant-negative allele of Rac1, was constructed with homologous recombination in HEK 293 cells as previously described (50). The control virus, AdGal,
which encodes the bacterial LacZ gene, was also described previously (51). Viruses were amplified in HEK-293 cells, purified on
double cesium gradients, and plaque-titered (20). Purified
AdRac1N17 and Ad
Gal had titers of 2 × 1010 and
5 × 1010 plaque-forming units (pfu)/ml, respectively.
ECs were infected for 48 h before H/RO exposure (~80%
confluence) at a multiplicity of infection (MOI) of 150 in EGM-2
complete growth medium. No obvious change on cell morphology was observed.
EC exposure to H/RO.
Growth medium was removed, and a minimal amount of basal medium M199
(Gibco) was added in each container to decrease the diffusion distance
for the atmospheric gases. Each container was exposed to a
"hypoxic" gas mixture (5% CO2-2% H2-93%
N2; Puritan Bennett, Linthicum Heights, MD) in a humidified
incubator (Billups-Rothenburg, Del Mar, CA) for 1 h. The incubator
was flushed with N2 at a pressure of 2 psi for 15 min
before switching to the hypoxic gas mixture. To ensure an oxygen-free
environment, the gas mixture was passed through a catalytic
deoxygenator (Gas Purification Technology, Manalapan, NJ) before entry
to the incubator. On measurement of the partial pressure of
O2 with a Clark-style probe (Hudson RCI, Temecula, CA)
placed in the fluid contained in the tissue culture container, the
starting partial pressure was 150 mmHg and dropped to 10 mmHg (1%
O2 content) within 10 min of hypoxia. Temperature inside
the incubator was maintained at 37°C by a heating pad and was
monitored throughout hypoxia. After 1 h of hypoxia, reoxygenation was initiated by returning the cells to the regular tissue culture incubator (normoxia: 21% O2-5% CO2-74%
N2). EGM-2 complete growth medium was added 30 min from the
onset of reoxygenation to maintain cell viability. Cell viability was
determined by trypan blue exclusion at 13 h from the onset of
reoxygenation. Normoxic controls were exposed to normoxia for the
duration of the experiment. PDTC (100 µM) was added to some
monolayers 60 min before hypoxia. To some monolayers, a monoclonal
antibody against VCAM-1 (1G11; Immunotech, Fullerton, CA), which was
shown to inhibit U937 cell adhesion to tumor necrosis factor
(TNF)--stimulated ECs (55), was added at a final
concentration of 40 µg/ml for 30 min before completion of reoxygenation.
Cell-based ELISA for VCAM-1. At the end of 13-h reoxygenation or normoxia, VCAM-1 expression was determined by a cell-based ELISA. Briefly, monolayers were fixed with ice-cold ethanol for 10 min and nonspecific binding was blocked by adding I-block (Tropix, Bedford, MA) in PBS for 1 h at 37°C. Subsequently, 200 µl of mouse monoclonal anti-human VCAM-1 (1:2,500 dilution; R&D Systems, Minneapolis, MN) were added to each well and incubated overnight at 4°C. Monolayers were washed in PBS, incubated with biotin-conjugated goat anti-mouse IgG (1:2,500 dilution; Zymed, San Francisco, CA) in PBS for 1 h at room temperature, washed, and incubated with horseradish peroxidase (HRP)-conjugated streptavidin (1:5,000 dilution) in PBS for 30 min at room temperature. After washing with PBS, 200 µl of tetramethyl benzidine substrate solution (TMB; Sigma) were added to each well for 10 min. The reaction was stopped with 50 µl of 8 N H2SO4, and the plates were read on a spectrophotometric plate reader at 450 nm. The mean optical density (OD) of the wells containing normoxic cells without primary antibody was considered as background OD and was subtracted from all other readings. For each condition, measurement of triplicate wells was made to obtain an average value.
Flow adhesion assay.
Each 35-mm tissue culture dish with a cell monolayer formed one side of
a parallel-plate perfusion chamber (GlycoTech), with a flow path of
0.025-cm thickness and 0.1-cm width, as determined by a silicone gasket
(GlycoTech). The tissue culture dish and the flow deck were kept
together by vacuum. The perfusion chamber and inlet and outlet tubing
were filled with assay medium and maintained at 37°C by a
thermostatic air pump (Nikon, Garden City, NY). A syringe pump (Harvard
Apparatus, South Natick, MA) was used to perfuse first assay medium for
2 min and then a suspension of U937 cells in assay medium for 5 min
through the chamber at a constant flow rate of 0.64 ml/min. The
viscosity of the assay medium was assumed to be 0.01 g · cm1 · s
1. By
considering fully developed steady laminar flow of a viscous Newtonian
fluid (5), the wall shear stress on the monolayer was
calculated at 1 dyn/cm2, as previously described (23,
49). The perfusion chamber was mounted on an inverted-stage
microscope (Diaphot 300; Nikon) equipped with a video camera (CCD-72S;
MTI, Michigan City, IN). Stage movement was controlled in the
x and y directions with two micro-stepping motors
(Multitech, West Chester, PA). For each experiment, three random
microscopic fields (×20 objective, 0.38 mm2/field) close
to the chamber inlet were chosen for recording the U937 cell-EC
adhesive interactions for 90 s per field with a JVC sVHS
videocassette recorder (Professional Products, Baltimore, MD). Nine
experimental conditions were tested by flow adhesion assay: 14-h
normoxia (N); 1 h with PDTC and 14-h normoxia (N-PDTC); infection
with Ad
Gal and 14-h normoxia (N-
Gal); infection with AdRac1N17
and 14-h normoxia (N-Rac); 1-h hypoxia followed by 13-h reoxygenation
(H/R); 1 h with PDTC and H (1 h)/RO (13 h) (H/R-PDTC); infection
with Ad
Gal and H (1 h)/RO (13 h) (H/R-
Gal); infection with
AdRac1N17 and H (1 h)/RO (13 h) (H/R-Rac); and H (1 h)/RO (13 h) and 30 min with anti-VCAM-1 (H/R-VCAM).
Quantitation of monocytic cell-EC adhesive interactions. Video recordings from flow adhesion assays were digitized, and quantitative information was obtained by image processing (IC-300 modular image processing workstation; Inovision, Durham, NC). The total of U937 interacting cells per minute was defined as the number of cells that interacted with the monolayer in the digitized image (0.38 mm2) for at least 1 s and included cells that tethered, rolled, and stably adhered. Brief contacts for <2 s were identified as tethering. Rolling cells were identified by acquiring "maximization" images for 3-5 s at 30 frames/s, as described previously (27). Rolling velocities in micrometers per second were determined by dividing the distances traveled by the acquisition time. Data from n = 4 independent experiments were pooled to give rolling velocities for 60 rolling monocytes for each condition tested. The frequency of occurrence for each velocity range was presented as a percentage relative to the total number of examined monocytes. Cells were defined as stably adherent if they remained attached to the monolayer for at least 5 s. Stably adherent cells were identified by acquiring "minimization" images for 5 s at 30 frames/s, as described previously (27), and were not included in the rolling cells. Distribution charts of the three interaction types were presented as a percentage of the total of interacting cells. Cells that interacted with other rolling or stably adherent cells before interacting with ECs were not accounted for, to study only primary adhesive events.
Measurement of EC ROS production.
Lucigenin, a compound that emits light upon interaction with
O
Statistical analysis.
The number of cells per minute that tethered, rolled, or stably adhered
are presented as means ± SE for n = 4 independent experiments in each condition. ODs from the ELISA assay were normalized to their corresponding normoxic controls and are presented as means ± SE for n = 4 independent experiments in
each condition. Statistical significance of differences among means was
determined using Statview software (Abacus Concepts, Berkeley, CA) to
perform one-way ANOVA with Bonferroni corrections for multiple
comparisons. P values 0.05 were considered significant.
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RESULTS |
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U937 cell adhesion to H/RO-exposed ECs: role of ROS.
Three types of adhesive interactions between U937 cells and ECs,
namely, tethering, rolling, and stable adhesion, were quantified (n = 4; Fig. 1,
A-C). Under normoxic
conditions (N), tethering was the predominant type of interaction,
accounting for 80% of total interacting cells (Fig. 1D). The
interaction appeared to be nonspecific, because H (1 h)/RO (13 h) did
not increase tethering (Fig. 1A). Under N, the frequency of
rolling and stable adhesion accounted for 20% and 1%, respectively,
of total interacting cells (Fig. 1D). H/RO-exposed ECs (H/R)
were capable of supporting significantly higher fluxes of rolling and
stably arrested cells: rolling increased 5.6-fold and stable adhesion
increased from 0.1 to 2.3 cells/min, relative to N (Fig. 1,
B and C). Rolling cells became the dominant interacting cell type after H/RO, accounting for >50% of total interacting cells compared with 20% under N (Fig. 1D).
Almost all monocytes that were arrested onto the monolayer had
previously rolled on it. The majority of rolling cells, however, later
detached and reentered the fluid stream. Rolling cells appeared
spherical, whereas many stably adherent cells were less regular in
shape. Rarely, a stably adherent cell (cell arrested for at least
5 s) was later released and resumed rolling. No transmigrating
cells were observed in these experiments.
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Role of Rac1 in U937 cell adhesion to H/RO-exposed ECs.
Because Rac1 is an integral part of the NAD(P)H oxidase complex and
regulates ROS production in phagocytic and nonphagocytic cells
(1, 29, 52), we asked whether monocytic cell adhesion to
H/RO-exposed ECs was dependent on Rac1-mediated ROS production (n = 4; Fig. 2). Normoxic
HUVECs infected with either AdRac1N17 (N-Rac) or AdGal (N-
Gal)
showed the same distribution of the three types of U937 cell-EC
adhesive interactions, as in noninfected N cells (Fig. 2D).
Specifically for rolling and stable adhesion, there was no significant
difference between N-Rac and N-
Gal conditions (Fig. 2, B
and C). For tethering, there was a decrease in N-Rac compared with N-
Gal conditions, but it was not significant (Fig. 2A). After H/RO exposure of Ad
Gal-infected ECs
(H/R-
Gal), tethering was not affected (Fig. 2A). In
contrast, H/R-
Gal cells showed a 3- and 10-fold increase in rolling
and stable adhesion, respectively (Fig. 2, B and
C). In H/R-
Gal cells, the distribution of interaction types was skewed toward rolling, with 50% of total interacting cells
being rolling cells, as in noninfected H/R cells (Fig. 2D). Inhibition of Rac1 in H/RO-exposed ECs (H/R-Rac) significantly reduced
the rolling flux by 62%, compared with its H/RO-exposed counterpart
H/R-
Gal (Fig. 2B). In H/R-Rac cells, the rolling flux was
1.7-fold higher than in N-Rac cells (Fig. 2B). Inhibition of
Rac1 also resulted in complete inhibition of stable adhesion after H/RO
(compare H/R-
Gal and H/R-Rac, Fig. 2C). In H/R-Rac cells,
the distribution of interaction types became as it was in N and
H/R-PDTC cells (tethering accounted for 60% of total interacting
cells; Fig. 2D). Hence, Rac1 activation may be required for
rolling and stable adhesion after H/RO but is not required for adhesive
interactions under normoxia (both N-
Gal and N-Rac showed the same
rates of tethering, rolling, and stable adhesion).
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EC OGal infection. AdRac1N17 infection led to
even lower basal O
Gal cells, suggesting that
O
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EC VCAM-1 expression after H/RO.
Because VCAM-1 is involved in monocyte-EC adhesive events under flow
(33) and is known to be upregulated by H/RO
(31, 57), we studied EC VCAM-1 expression under different
conditions by cell ELISA (n = 4; Fig.
4). In N-Gal cells, there was a
slight, but not significant, increase in VCAM-1 expression
compared with N cells. Either PDTC treatment (N-PDTC) or
infection with AdRac1N17 (N-Rac) downregulated the basal VCAM-1
expression compared with that of their respective controls (N,
N-
Gal), but the difference was not significant. Exposure to H/RO
increased VCAM-1 expression almost twofold in both infected and
noninfected cells. PDTC treatment (H/R-PDTC) inhibited by 75% and
infection with AdRac1N17 (H/R-Rac) almost completely abolished the
H/RO-induced increase in VCAM-1 expression compared with H/R and
H/R-
Gal, respectively (Fig. 4). Thus H/RO-induced VCAM-1 expression
is regulated by Rac1-dependent ROS.
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Role of VCAM-1 in U937 cell adhesion to H/RO-exposed ECs.
Because VCAM-1 supports monocyte stable adhesion to cytokine-stimulated
ECs (33) and has been implicated in tethering and rolling
(2, 3, 33), we studied the VCAM-1 contribution to U937
cell adhesive interactions with H/RO-exposed ECs by using a blocking
anti-VCAM-1 monoclonal antibody (n = 4; Fig.
5). Incubation with anti-VCAM-1 antibody
1G11 (40 µg/ml) for 30 min before completion of 13 h of
reoxygenation (H/R-VCAM) did not affect the tethering flux (Fig.
5A). Incubation with anti-VCAM-1 antibody resulted in a
measurable (40%), but not significant, increase in rolling flux
compared with H/R (Fig. 5B). Furthermore, it completely
abolished H/RO-induced stable adhesion, indicating a crucial role of
VCAM-1 in monocytic cell stable adhesion (Fig. 5C). The
shift in distribution of interaction types from tethering to rolling,
observed in H/R, was maintained in H/R-VCAM, with rolling accounting
for >60% of total interactions (Fig. 5D). In H/R-VCAM, not
only did the flux of rolling cells increase compared with H/R (Fig.
5B), but the mean rolling velocity also slightly decreased.
Figure 6 shows the distribution of
rolling velocities of U937 cells perfused at 1 dyn/cm2 over
ECs exposed to different conditions (N, H/R, or H/R-VCAM; n = 4). Under N conditions, cells rolled at a mean
rolling velocity of 133 ± 8 µm/s (±SE; Fig. 6A).
H/R caused cells to roll significantly more slowly, at 88 ± 8 µm/s on average (±SE; Fig. 6B). H/R-VCAM resulted in
further skewing of the distribution of rolling velocities to the low
side: the mean rolling velocity was 79 ± 8 µm/s (±SE; Fig.
6C), which was significantly different from N but not
significantly different from H/R.
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DISCUSSION |
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The present study provides the first evidence that 1) flowing monocytic cells will roll and firmly adhere to H/RO-exposed ECs, 2) stable adhesion is totally, and rolling partially, dependent on Rac1-mediated ROS production, and 3) VCAM-1 mediates stable adhesion, but not rolling, of monocytic cells to H/RO-exposed ECs. By incorporating flow in the adhesion assay, it is possible not only to observe and quantify each step in the cascade of adhesive events (tethering, rolling, and stable adhesion) but also to determine the molecular mechanisms that regulate individual steps.
Tethering was the most prominent adhesive interaction type under
normoxia (Fig. 1D). These brief contacts of monocytic cells with HUVEC monolayers cause a temporary halt in the movement of flowing
cells, and although most are instantly released back to the fluid
stream, some start to roll on the EC surface. Rolling decelerates the
monocytes, allowing them to sample the local environment and interact
with EC receptors that mediate firm arrest. A limited flux of stably
adherent cells was observed under N conditions (Fig. 1C). EC
exposure to H (1 h)/RO (13 h) significantly increased the fluxes of
rolling and stably adherent cells, compared with N, while maintaining
the same tethering flux (Fig. 1, A-C). Rolling and
stable adhesion fluxes increase when monocytes are perfused over
cytokine-stimulated ECs (33, 36). The flux of stably adherent U937 cells to H/RO-exposed HUVECs was low (~5
cells · mm2 · min
1 at 1 dyn/cm2) compared with that of human peripheral blood
monocytes to either 4-h IL-1
-stimulated HUVECs (~15
cells · mm
2 · min
1 at 2 dyn/cm2; Ref. 33) or 24-h IL-4-stimulated
HUVECs (~60
cells · mm
2 · min
1 at 0.8 dyn/cm2) (36). Consistent with this finding,
Kokura et al. (31) showed that A (1 h)/RO (8 h) induces an
increase in (static) T-lymphocyte adhesion of a magnitude similar to
the response induced by lower levels of cytokine exposure.
EC O200; Ref.
13), there is also a possibility that Rac1N17 expression
may have not blocked the NAD(P)H oxidase activity entirely.
EC treatment with PDTC significantly attenuated H/RO-induced rolling
and completely abolished stable adhesion (Fig. 1, B and C), suggesting that ROS regulate monocytic cell rolling and
stable adhesion to H/RO-exposed ECs. PDTC also decreased rolling under N, but the difference was not significant (Fig. 1B),
implying that ROS may, in part, regulate the expression of a
constitutively expressed adhesion molecule involved in rolling.
Normoxic ECs infected with either AdRac1N17 or AdGal showed the same
rolling and stable adhesion fluxes and similar tethering fluxes (Fig. 2). Infection with AdRac1N17 significantly reduced the H/RO-induced increase in rolling and completely abolished the H/RO-induced increase
in stable adhesion compared with H/RO-exposed ECs infected with
Ad
Gal (Fig. 2), suggesting that the Rac1-dependent ROS mainly regulate monocytic cell rolling and exclusively regulate stable adhesion. Specifically for rolling, AdRac1N17 was more potent than PDTC
in attenuating H/RO-induced rolling, compared with their respective N
counterparts (1.7-fold with H/R-Rac vs. 3.4-fold with H/R-
Gal;
5-fold with H/R-PDTC vs. 5.6-fold with H/R). This implies that
Rac1-regulated ROS-independent signaling pathways contribute to the
H/RO-induced increase in rolling. Similarly, chemical antioxidants were
shown to be effective at suppressing H/RO-induced heat shock factor
(HSF) activation but not as effective as Rac1N17, implying that
Rac1-dependent ROS-independent mechanisms partially regulate
HSF-mediated transcription (44), leading to attenuation of
proinflammatory responses (54).
From the measurements of EC viability at 13 h of reoxygenation (complete growth medium was added back to ECs at 30 min of reoxygenation), the adhesive interactions between a fraction of ECs in the monolayer and U937 cells are not due to EC death. Neither PDTC nor Rac1 inhibition, with or without H/RO exposure, caused cell death, because >90% cells were viable in each case. Furthermore, the continuation of the monolayers was always checked under the microscope right before the flow adhesion assay. In accordance with this, there is evidence that 1) EC infection with AdRac1N17 at MOIs of 50-200 does not cause apoptosis (13), 2) incubation of confluent HUVECs with PDTC (100 µM) for 24 h does not induce cell death (14), and 3) infection with AdRac1N17 protects HUVECs from H/RO-induced cell death (29). Hence, although we have not tested specifically for apoptosis, we know that our studies were performed with viable ECs.
Because VCAM-1 mediates monocyte adhesive interactions with
cytokine-stimulated ECs under flow (33) and
antioxidants inhibit NF-B activation and VCAM-1 expression on
cytokine-stimulated ECs (37, 55), we evaluated the effect
of H/RO, and the importance of H/RO-induced Rac1-dependent ROS, on
VCAM-1 expression. In agreement with published studies on the time
course of VCAM-1 on HUVECs exposed to A (1 or 4 h)/RO (31,
57), we found a twofold increase in VCAM-1 expression at 13 h of reoxygenation (Fig. 4). H/RO-induced VCAM-1 upregulation was
significantly attenuated by PDTC and almost completely abolished by
AdRac1N17 (Fig. 4), suggesting that VCAM-1 expression is mainly
regulated by Rac1-dependent ROS. NF-
B and, to a lesser extent, AP-1
are involved in A/RO-induced VCAM-1 expression (31).
Because Rac1-mediated ROS are a major part of total ROS (Fig. 3) and
both NF-
B DNA binding activity and the turnover of the inhibitor
I
B are oxidant sensitive (18), it is possible that
Rac1-mediated ROS regulate NF-
B-driven gene expression. There are
downstream effectors of Rac1, however, that may also be important in
regulating H/RO-induced expression of VCAM-1: Rac1 has been implicated
in activation of members of the family of mitogen-activated protein
(MAP) kinases, such as p38 (60) and the extracellular
signal-regulated kinases (ERK1/2) (59), and these MAP
kinases are required for NF-
B-driven gene expression (7,
8). The GTPases Rac1 and Cdc42 are also known to regulate a
protein kinase cascade initiated at p21-activated kinase (Pak)-1 and
leading to activation of c-Jun NH2-terminal kinase (JNK)
and p38 MAP kinase (10, 60). Hence, it is possible that
each of the above effectors, as well as the actin cytoskeleton
(11), differentially regulate VCAM-1 expression.
Because blocking VCAM-1 completely abolished firm adhesion (Fig.
5C), VCAM-1 is the sole mediator of monocytic cell
firm adhesion to H/RO-exposed HUVECs. This agrees with the fact that
blocking 4
1 significantly reduced
firm adhesion of flowing monocytes to IL-1
-stimulated HUVECs,
whereas blocking
2-integrins (hence, ICAM-1-mediated
pathway) did not (33). A limited number of U937 cells were
arrested on H/RO-exposed ECs without prior rolling, suggesting that
VCAM-1 is capable of firm adhesion even in the absence of
selectin-mediated adhesive interactions. The
4
1-VCAM-1 interaction can precede
activation events that are required for development of firm adhesion
(2, 15, 58). From our findings, however, VCAM-1 does not
mediate tethering or rolling (Fig. 5, A and B).
The surface density of VCAM-1 after H/RO, despite being higher than
basal, may not be high enough to increase the avidity between
4
1 and VCAM-1 to mediate tethering or rolling.
Rolling markedly increased in the presence of saturating concentrations
of anti-VCAM-1 antibody compared with H/R (Fig. 5B). Increased rolling flux may be due to decreased firm adhesion upstream of the field of view, thus more rolling cells enter the field per unit
time. This agrees with the data of Weber et al. (56), who
inhibited NF-B mobilization by overexpression of I
B in HUVECs and
found that reduced induction of VCAM-1/ICAM-1 by TNF-
resulted in a
decrease in the ability of monocytes to firmly adhere and an increase
in the rolling fraction of monocytes. However, in our case, the
increase in rolling cells after VCAM-1 blockade cannot be accounted for
solely by the reduction in stably adherent cells, as can be seen by
comparing Fig. 5, B and C. The increased flux of
rolling monocytes combined with the reduced rolling velocities after
VCAM-1 blockade (Fig. 6) suggest a greater number of receptor-ligand bonds. Antibody cross-linking of VCAM-1 on the EC surface induces a
Ca2+ flux (48) and activates NAD(P)H oxidase
to produce ROS (38). Hence, VCAM-1-mediated outside-in
signaling may cause rapid expression of the oxidant-sensitive preformed
P-selectin from Weibel-Palade bodies (45), or of a novel
receptor, leading to increased rolling interactions and reduced rolling velocities.
The U937 cell mean rolling velocity over H/RO-exposed HUVECs is
comparable to that of monocytes over (24 h) IL-4-stimulated HUVECs
(36) but is higher than that of monocytes over either (4 h) IL-1- or (6 h) TNF-
-activated HUVECs (33, 35).
Rolling is probably mediated by L-selectin binding to sialylated
determinants on the EC surface, as in the case of monocytes rolling
over IL-1
-stimulated HUVECs (33). At 13 h of
reoxygenation, P- and E-selectin are not likely to be present on the EC
surface, because their expression levels were shown to return to basal
levels at 10 h of reoxygenation (25) and neither of
the two affected static T-lymphocyte adhesion at 8 h of
reoxygenation (31).
Under our experimental conditions, besides exposure to hypoxia, ECs were exposed to serum/growth factor deprivation for 1 h during hypoxia (and the first 30 min of reoxygenation), to make hypoxia more physiologically relevant to ischemia. Because of the short time period of serum/growth factor withdrawal, it is believed that the effects of our treatment on EC signaling and EC-leukocyte adhesive events are for the most part due to H/RO exposure. In sum, we demonstrated that flowing monocytic cells tether, roll, and firmly adhere to cultured HUVECs exposed to H/RO. EC Rac1-dependent ROS partially regulate rolling and exclusively regulate stable adhesion. Stable adhesion, but not rolling, is mediated by induction of VCAM-1 expression. In vivo, physical and biological factors not present in our in vitro system may modulate leukocyte adhesion within the range of physiological shear stresses (40). In vitro systems, however, are still very useful in subjecting cultured ECs to well-defined flow conditions and in understanding the molecular basis underlying the leukocyte response to ECs after I/RP injury.
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ACKNOWLEDGEMENTS |
---|
We thank A. Hall for the Rac1N17 cDNA, R. Crystal and T. Finkel for
AdGal, and J. T. Patton for helpful discussions.
![]() |
FOOTNOTES |
---|
B. R. Alevriadou was supported by National Heart, Lung, and Blood Institute Grant HL-54089, a Whitaker Biomedical Engineering research grant, and a grant from the Johns Hopkins Medical Institutions Center for Advanced Transfusion Practices and Blood Research. K. Irani was supported by a Johns Hopkins Clinician Scientist Award, the American Heart Association, the Bernard Foundation, and an endowment from Abraham and Virginia Weiss.
Address for reprint requests and other correspondence: B. R. Alevriadou, Johns Hopkins Univ. School of Medicine, BME Dept., Traylor Bldg., Rm. 619, 720 Rutland Ave., Baltimore, MD 21205 (E-mail: ralevria{at}bme.jhu.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.
First published March 6, 2002;10.1152/ajpcell.00301.2001
Received 2 July 2001; accepted in final form 28 February 2002.
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