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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3445-3454
Enhanced Adherence of Sickle Erythrocytes to Thrombin-Treated
Endothelial Cells Involves Interendothelial Cell Gap Formation
By
Annamaria B. Manodori,
Neil M. Matsui,
James Y. Chen, and
Stephen
H. Embury
From the Hematology Division, the Department of Medicine, The
University of California-San Francisco and The San Francisco General
Hospital, San Francisco; and the Northern California Comprehensive
Sickle Cell Center, San Francisco, CA.
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ABSTRACT |
The adherence of sickle erythrocytes to vascular endothelium has the
capacity to initiate vasoocclusion. The known effects of thrombin on
endothelial cell function and the increased activity of thrombin in
sickle cell disease led us to examine the effect of thrombin on the
adhesivity of cultured endothelial cells for sickle erythrocytes. In
particular, we studied whether the effect of thrombin on
interendothelial cell gap formation (ICGF) was involved in endothelial
cell adhesivity for sickle erythrocytes. Those endothelial cell
monolayers stimulated by thrombin to maximal levels of static sickle
erythrocyte adherence also underwent striking cell contraction and
enlargement of interendothelial cell gaps. Adhesivity also increased
when gaps were induced with antilaminin antibodies or EDTA. Maximally
adhesogenic thrombin conditions failed to increase adhesivity when gap
formation was prevented by pretreatment of the monolayers with
8-bromo-cyclic adenosine monophosphate (bromo-cAMP) or
glutaraldehyde, agents that respectively inhibit
actin-myosin-dependent cell contraction or cross-link adjacent cells
in the monolayer. The influence of these two agents on EDTA-enhanced
adhesivity was linked to their ability to prevent gap formation.
Glutaraldehyde prevented both increased adherence and gap formation;
bromo-cAMP prevented neither. Interendothelial cell gap formation may
contribute to vasoocclusion by facilitating sickle erythrocyte
adherence.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
VASOOCCLUSION IN SICKLE cell anemia is
unpredictable, often painful, and potentially fatal.1,2
Thoughtful analyses of the polymerization of deoxyhemoglobin S and
sickling of erythrocytes (red blood cells [RBC]) have
delimited the basis of sickle cell disease.3-5 Despite
their sophistication and detail, these interpretations have not
identified determinants of sickle cell vasoocclusion that can be relied
on to predict vasoocclusive events. In addition to the doctrine of
sickling-induced vasoocclusion, there exists experimental evidence that
vasoocclusion can be initiated by the adherence of sickle RBC (SS RBC)
to vascular endothelium.6 Several RBC and endothelial cell
(EC) receptors, subendothelial matrix adhesive proteins, and plasma
ligands that contribute to the adherence have been
elaborated.7,8 Further complexity is added to our
understanding of this process by the action of a number of modifying
influences on EC adhesivity8,9 and by uncertainty regarding
whether sickle cell vasoocclusion occurs in the smallest capillaries or
in larger vessels.10 Because of the direct and indirect
evidence that sickle cell disease is associated with increased
production and action of thrombin11-14 and of the numerous
recognized effects of thrombin on EC function,15 we
investigated the possible role of thrombin in potentiating the
adherence of SS RBC to cultured human umbilical vein EC
(HUVEC).
Thrombin affects the endothelium in ways that are predicted to
influence adherence of SS RBC to vascular walls. Thrombin induces neutrophil (polymorphonuclear leukocyte [PMN]) adhesive
molecules.16-18 In sickle cell disease, this occurs in the
setting of commonly elevated leukocyte counts19 and PMN
activated by SS RBC.20 The consequent adherence of PMN to
EC is associated with a burst of reactive oxygen species and release of
proteases,18,21 which loosen EC from basement membranes,
causing interendothelial cell gap formation (ICGF) and endothelial
barrier dysfunction.22 The altered EC adhesion
properties22,23 and exposure of adhesive matrix
proteins,24,25 inherent in this process, led us to the hypothesis that these inflammatory response elements26 also may be involved in thrombin-enhanced SS RBC adherence to vascular walls.
We studied the effects of thrombin on adherence of SS and nonsickle
(AA) RBC to cultured EC, on the induction of cellular contraction and
ICGF, and the possible interdependence of these effects. The integrity
of confluent EC monolayers is the result of a dynamic equilibrium
between constitutive low-level actin-myosin contraction and tethering
forces provided by cell-cell adherin anchors and cell-matrix integrin
moorings.27 Thrombin actively perturbs this equilibrium by
triggering actin-myosin shortening22 by a rapid,
reversible, receptor-mediated process.28 This active transduction of EC contraction and ICGF22 can be blocked by specific inhibition of signal transduction or contractile elements and
by nonspecific prevention of EC contraction.29 ICGF can be
engendered permissively by severing cell-cell and cell-matrix tethers,
leaving basal actin-myosin tension unopposed.27 For instance, tethers can be disrupted by chelation of divalent cations or
by interruption of EC-matrix associations using RGD
peptides, antibodies directed against integrin-binding domains of
matrix proteins,22 or antibodies against EC receptors for
fibronectin (Fn) or vitronectin.30
To investigate the proposed linkage between EC adhesivity and gap
formation, we analyzed the effects of thrombin, of agents that promote
ICGF permissively, and of inhibitors of ICGF on both ICGF and EC
adherence of SS RBC. Our findings suggest a contribution of ICGF to SS
RBC adherence and vasoocclusion.
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MATERIALS AND METHODS |
Reagents.
Gelatin, heparin, thrombin, M199 medium, Fn from human plasma, bovine
serum albumin (BSA), HEPES buffer, bromo-cAMP (8-bromo-cyclic adenosine
monophosphate), IBMX (isobutyl-methylxanthine), and Evans Blue dye were
from Sigma Chemicals Co (St Louis, MO). Hanks' buffered saline
solution, L-glutamine, 100 × penicillin/streptomycin, calcium/magnesium-free phosphate-buffered saline (PBS) were from the
University of California San Francisco (UCSF) cell culture facility.
Trypsin 0.025%/EDTA 0.01% and endothelial cell growth medium (EGM)
were from Clonetics Corp (San Diego, CA). Dispase II and EC growth
factor were from Boehringer Mannheim Corp (Indianapolis, IN). Fetal
bovine serum was from HyClone Laboratories, Inc (Logan, UT). Serva
electron microscopic grade glutaraldehyde was a 25% solution in water
from Boehringer Ingelheim (Heidelberg, Germany). Polyclonal anti-von
Willebrand factor (vWF) and polyclonal antilaminin were purchased from
DAKO A/S (Glostrup, Denmark). Rhodamine-conjugated phalloidin was from
Molecular Probes, Inc (Eugene, OR). Four-chambered culture slides were
from NUNC (Naperville, IL). Biomeda Gel Mount was from Biomeda Corp
(Foster City, CA), Vectashield mounting medium was from Vector
Laboratories, Inc (Burlingame, CA), and Transwell multiple well culture
plates with 3.0 µmol/L pore polycarbonate membranes were from Corning
Costar Corp (Cambridge, MA).
Cell culture.
HUVEC cultures were obtained from anonymous umbilical cord donors at
the San Francisco General Hospital Labor and Delivery Ward with
approval from the UCSF Human Research Committee. HUVEC were isolated
within 3 days of cord collection according to the method of Jaffe et
al31 with the following modifications. Enzymatic digestion
to free the HUVEC was for 10 minutes at 37°C with 0.15% dispase in
M199 medium. Cells were grown at 37°C in 5% CO2/95% air in gelatin-coated flasks in M199 medium supplemented with 10%
human serum pooled from normal male donors and 10% fetal bovine serum,
20 µg/mL EC growth factor, 100 µg/mL heparin, 2 mmol/L L-glutamine,
100 mg/mL penicillin/100 mg/mL streptomycin solution (G-10 medium).
Cobblestone morphology and positive staining by an antibody to vWF
identified the resulting culture as EC. At 85% confluence, the cells
were subcultured to Fn-coated 4-chambered slides. The second to the
fourth passages were used for adherence assays no later
than 24 hours after HUVEC had reached confluence. For the
photographic/morphometric analyses and some of the monolayer permeability studies, HUVEC were obtained from a commercial source (Clonetics) and grown in EGM.
Sickle cell and control blood.
Blood samples were drawn from homozygous sickle cell patients of the
San Francisco General Hospital Hematology Clinic or from volunteer
control subjects after obtaining informed consent, as approved by the
UCSF Committee on Human Research. All subjects were in steady state
condition, none had received blood transfusions within 4 months, and
none were being treated with hydroxyurea.
Gravity adherence assays.
Our method of assaying static adherence of RBC to HUVEC monolayers was
adapted from a published method.32 Heparinized blood from
normal donors and sickle cell subjects was washed three times in PBS
and once in Hanks' buffered saline solution, 1% BSA, 50 mmol/L HEPES
pH 7.4 (HAH), taking care to deplete the samples of leukocytes and
platelets by removing the buffy coat with each wash, and RBC were
suspended to 3.5% hematocrit in HAH containing 17% autologous
platelet-free plasma from the heparinized blood sample. Confluent HUVEC
monolayers were washed with HAH to remove traces of serum and treated
with the following agents: thrombin at concentrations spanning 0.01 to
0.5 U/mL in HAH or thrombin-free HAH for 5 minutes; 0.5 mmol/L EDTA in
calcium, magnesium-free PBS or EDTA-free calcium, magnesium-free PBS
for 10 seconds; or 140 mg/L polyclonal antilaminin antibody in EGM or
EGM alone for 1 hour. For studies of the influence of cell contraction
or gap induction, after washing away traces of serum and medium, the monolayers were incubated at 37°C with 200 µmol/L bromo-cAMP and 200 µmol/L IBMX in EGM for 20 minutes or with 3% glutaraldehyde in
PBS for 10 minutes. These reagents were washed away twice with HAH and
gentle shaking for 5 minutes and the EC were then treated as above with
thrombin or EDTA. Immediately after washing with HAH to remove traces
of the above agents, the monolayers were covered with AA RBC or SS RBC
suspensions and incubated at 37°C for 25 minutes. The wells were
then filled completely with HAH, sealed with packing tape, and inverted
at 37°C for 20 minutes. While still inverted, the well walls and
gaskets of the slide chambers were removed. The slides were rinsed
twice with HAH to remove nonadherent RBC, fixed in 3% glutaraldehyde,
stained, and mounted. RBC adherence was monitored visually by
microscopy at 100× magnification and quantified by counting RBC
adherent to HUVEC monolayers in 16 fields marked by a grid (same random
fields for each sample). The mean adherence and SEM for each sample (n = 16) was calculated and results were used to determine the reported means from at least four experiments, only when the standard error of
mean (SEM) of each sample was less than 20% of the means.
Results were analyzed by the Student's t-test, with which
P < .05 was considered significant.
Distribution of RBC adherence to EC monolayers.
The distribution of SS RBC adherent to EC monolayers that had or had
not been exposed to 0.1 U/mL thrombin was assessed in three
representative static assays. An arbitrary number of adherent SS RBC
were scored as "at the edges of EC" when they were on the subendothelium, but touching or overlapping at least one EC, RBC "in
the gaps" had no physical contact with an EC, and RBC "on an
EC" was completely on top of an EC and had no part of it touching the edge of the cell. Percent is of the total RBC counted on the EC
monolayers.
Photomicrographs of HUVEC monolayers and morphometric analysis of
ICGF.
HUVEC monolayers were grown to 95% confluence on 35 mm tissue culture
plates then observed using a Nikon Diaphot Inverted Microscope (Nikon Corporation, Tokyo, Japan). Cells were
washed with PBS three times then treated for 5 minutes with
concentrations of thrombin spanning 0.01 to 0.5 U/mL in HAH or with HAH
alone. After 5 minutes, the monolayers again were washed three times with PBS, and EGM was added. At selected intervals before and after
treatment, photomicrographs were taken using a Polaroid MicroCam with
Polaroid 337 film (Polaroid Corporation, Cambridge, MA).
To retain the same field for successive pictures, the microscope stage
was immobilized. Photomicrographs were scanned using a UMAX 1200S
scanner (UMAX Technologies, Inc, Fremont, CA) and cell areas measured
using the Melanie gel analysis program (BioRad, Hercules, CA), whereby
cells were detected as spots. Gap percentages were determined as:
Monolayer permeability assay.
The permeability of HUVEC monolayers to Evans Blue Albumin (EBA; 0.67 mg/mL Evans Blue dye, 4% bovine serum albumin, in G-10 medium), as a
measure of ICGF, was determined using an adaptation of the method of
the Garcia laboratory.33 HUVEC monolayers
were grown on tissue culture-treated Transwells (Corning Costar) in 24-well microtiter plates. The upper Transwell and lower culture plate
wells were filled with G-10 medium, 50 µL and 500 µL, respectively. Initially the medium was removed entirely from the Transwell and replaced by 50 µL EBA. At 10-minute intervals for 1 hour before treatment of the monolayer, the entire bottom well medium was removed
for analysis by spectrophotometry and replaced with 500 µL fresh
G-10. EBA in the Transwells also was replaced with 50 µL fresh EBA
every 10 minutes. For treatment of the monolayers, the Transwells were
washed three times with PBS then treated for 5 minutes with 0.1 U/mL
thrombin in HAH or with HAH alone, after which the Transwells were
again washed three times with PBS. The 0 time point for treatment
samples was when the monolayers first were exposed to thrombin or HAH.
After treatment, medium from the bottom well was collected for analysis
and replaced at times 0, 5, 10, 20, and 40 minutes, and EBA was
replaced in the Transwell at the same time intervals. All samples were
diluted 1:1 in G-10 medium, and mean optical density (OD)
(630 nm) values were derived from multiple identical experimental
samples.
EBA standards were used to create a calibration curve of OD versus
mg/mL of albumin. The diffusion of EBA from the luminal buffer in the
Transwell to the abluminal buffer in the lower well was expressed as mg
albumin/minute/cm2 of monolayer. Replicate samples, six for
control and five for thrombin-treated, were taken at several 10-minute
time points before and after treatment. The start of the 5-minute
treatment was used as the 0 time point.
Immunofluorescence microscopy (IF).
HUVEC grown on Fn-coated 4-chambered slides were washed and treated
with thrombin, EDTA, or HAH, with or without inhibitors of ICGF, as
described above, and fixed in acetone for 5 seconds at  20°C.
After washing in PBS/1% BSA, the cells were blocked in PBS/3% BSA for
1 hour at room temperature, and labeled in blocking solution with
rhodamine-conjugated phalloidin (1:100 dilution) for 1 hour at room
temperature. After the chamber walls and gaskets were removed, the
slides were washed twice, mounted with coverslips, and photographed at
500× magnification with automatically adjusted shutter speeds
using a Nikon epifluorescence microscope equipped with a Nikon UFX-35A
camera.
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RESULTS |
We assessed the effects of different thrombin concentrations by
treating HUVEC with increasing concentrations from 0.01 to 0.5 NIH U/mL
for 5 minutes and quantifying SS RBC adherence using a static assay. In
four experiments, adherence increased in a dose-dependent manner,
peaking at 0.1 U/mL, and decreased at higher concentrations to the
levels of unstimulated control HUVEC (Fig 1). This dose-response curve for SS RBC adherence is similar to a
published curve for thrombin-induced ICGF, including curve shape, maximum concentration, and minimum exposure time.28
Consistent with other studies of the effects of thrombin on primary
HUVEC cultures, individual cultures varied considerably according to the monolayer age and cell density. Some cultures did not respond, and
others were destroyed by usually effective concentrations and durations
of exposure to thrombin. We alleviated this problem in part by using
cultures within 24 hours of reaching "cobblestone" morphology. We
chose as our working conditions of thrombin exposure those with maximal
adhesogenic effect, 0.1 U/mL for 5 minutes. Our findings and
conclusions pertain to those conditions.
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| Fig 1.
Dose response curve for thrombin-induced adherence. Each
point represents the mean number from four static assays of SS RBC
adherent to 1 mm2 of HUVEC treated with thrombin
concentrations ranging from 0 to 0.5 NIH U/mL.
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Adherence of SS RBC was compared with that of AA RBC
(Fig 2). The number of AA RBC adherent to
unstimulated HUVEC varied from 0.1 to 7.9 with a mean of 2.2 RBC/mm2 (n = 9). Adherence of SS RBC ranged from
1.2 to 17.9 with a mean of 6.3 RBC/mm2 (n = 9). The greater
extent and variability of SS RBC adherence, compared with AA RBC, is
comparable to the findings of others.9 A 5-minute treatment
of HUVEC with 0.1 U/mL thrombin caused a significant increase in RBC
adherence with both RBC populations. Mean values of adherence increased
from 2.2 to 5.8 AA RBC/mm2 (n = 9) and from 6.3 to 12.4 SS
RBC/mm2 (n = 9). Thrombin was found to influence EC
adhesivity for both AA and SS RBC, but was quantitatively greater for
the latter.
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| Fig 2.
AA and SS RBC adherence to HUVEC that had been treated
with thrombin or with control buffer. The mean ± SEM is shown
(n = 9).
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In our studies of adherence, it appeared that RBC adhered mainly to the
edges of EC, which were separated from neighboring EC and almost never
to areas of the endothelium that remained tightly adherent, a
relationship we observed in both unperturbed monolayers and those in
which ICGF had been induced by thrombin. To corroborate this finding,
in three representative experiments, we determined the site of
adherence of an arbitrary number of adherent SS RBC, 219 RBC on
unperturbed monolayers and 308 RBC on thrombin-treated monolayers. As
shown in Table 1, the distribution of
adherent SS RBC to unperturbed or thrombin-treated monolayers was,
respectively, 65% and 66% of SS to EC edges, 22% and 21% on top of
EC, and 12% and 13% in the gaps with no contact to an EC. The
persistence of the identical distribution pattern after thrombin
treatment, in the context of a twofold overall increase in adherence,
indicates commensurate increases at all three sites, EC edges, EC
luminal surfaces, and exposed matrix.
To confirm the effects of thrombin on EC contraction and ICGF that was
apparent during the adherence assays, we used computerized morphometry
and an assay of monolayer permeability to EBA. Serial photographs of
the same microscopic field taken at 0, 2, 5, 10, 20, and 40 minutes
after a 5-minute exposure to 0.1 U/mL thrombin showed a prompt and
striking contraction of EC and ICGF (Fig 3A through F). In contrast, a 5-minute exposure to HAH alone resulted in
neither EC contraction nor ICGF, as seen by comparing photographs taken
before and 40 minutes after a 5-minute exposure to HAH alone (Fig 3G
and H). Quantification of these observations by computerized morphometry using the Melanie gel analysis system (BioRad, Hercules, CA) to define the relative areas of gaps and cells in
scans of the above photomicrographs demonstrated a prompt 14-fold
increase in ICGF (Table 2).
We also used this method to assess the influence of different thrombin
concentrations on ICGF. We found that a degree of ICGF matching that
observed with 0.1 U/mL thrombin was induced by 0.2 and 0.5 U/mL
thrombin (data not shown), concentrations at which adherence was
substantially diminished (Fig 1). Thrombin also induced a prompt
increase in permeability of monolayers to EBA.
Figure 4 shows that the same thrombin
conditions resulted in a threefold increase in EBA permeability within
5 minutes, which persisted at approximately that level through the
40-minute period of measurement, and that there was no change induced
by HAH alone. The permeability data presented are those of a single representative experiment, which was corroborated in two other studies.
These visual, morphometric, and permeability data confirm that, in
addition to its effect on EC adhesivity for AA and SS RBC, thrombin
induced ICGF.
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| Fig 3.
Photomicrographs of HUVEC monolayers before and at
intervals after treatment with HAH buffer with or without 0.1 U/mL
thrombin. (A) Nearly confluent monolayer before treatment with
thrombin. (B through F) The same microscope field 2 minutes (B), 5 minutes (C), 10 minutes (D), 20 minutes (E), and 40 minutes (F) after a
5-minute exposure to thrombin. (G) Nearly confluent monolayer before
treatment with HAH buffer. (H) The same microscope field as in (G) 40 minutes after a 5-minute exposure to HAH alone. The monolayer treated
with thrombin (B through F) demonstrates a progressive and
pronounced ICGF compared with the pretreatment monolayer (A). The
monolayer exposed to HAH alone demonstrated no detectable ICGF after 40 minutes (G and H). Morphometric quantification of gap formation is
tabulated in Table 2.
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| Fig 4.
EBA permeability of HUVEC monolayers. The diffusion of
EBA from the luminal buffer in the Transwell to the abluminal buffer in
the lower well is shown as mg albumin/minute/cm2 monolayer
from 60 minutes before until 40 minutes after exposure to 0.1 U/mL
thrombin in HAH buffer (black bars) or to HAH alone (shaded bars).
Samples were taken at several time points before and after treatment;
the 0 time point is equivalent to the start of the 5-minute treatment.
Thrombin exposure resulted in a rapid increase in monolayer
permeability to EBA, which was sustained for the duration of these
measurements.
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Based on the apparent association of increased adherence and ICGF, we
tested the influence of nonspecific mechanisms of ICGF on RBC
adherence. We treated EC monolayers briefly with a low concentration of
EDTA to remove the divalent cations necessary for cell-matrix and
cell-cell interactions or with an antilaminin polyclonal antibody to
disrupt EC-matrix tethers. A 10-second exposure to 0.5 mmol/L EDTA
caused the cells to separate slightly. After washing away traces of
EDTA and monitoring visually by microscopy for initial cell rounding,
the adherence assay was performed as usual in the presence of divalent
cations. This treatment resulted in a significant (P < .05)
increase in the mean adherence of SS RBC from 5.1 to 15.5 RBC/mm2 (n = 3). Exposure of EC to antilaminin antibody
before the adherence assay also caused a significant (P < .05) increase in mean adherence (n = 3). The control adherence data to
which treated data were compared were those from all of the experiments
(n = 6). These results suggested that ICGF is associated with increased
RBC adherence.
To test this association further, before treating with thrombin or
EDTA, we preincubated EC with one of two different agents that prevent
ICGF, bromo-cAMP or glutaraldehyde. Bromo-cAMP is a cAMP analog that
inhibits cell contraction and protects EC barrier function,34 and glutaraldehyde is a cross-linking agent
that fortifies cell-cell and cell-matrix tethers, in addition to other likely effects. Buffer treatment was used as a negative control, and
EDTA was used as a positive control for ICGF. The cellular effects of
these agonists and antagonists are shown in the composite IF photograph
of rhodamine-labeled actin filaments (Fig
5), which shows the effects of the various treatments on the
endothelial cytoskeleton and monolayer integrity. Untreated cells (Fig
5A) were closely adjacent with no intercellular gaps and had
well-defined peripheral bands of actin stress fibers. Thrombin-treated
HUVEC (Fig 5B) had large intercellular gaps, disrupted and reorganized stress fibers, and occasional actin fibers distributed across the gaps.
EDTA-treated cells also had retracted to create intercellular gaps, had
fewer defined actin fibers than the thrombin-treated cells, and
remained attached by fine processes (Fig 5C). Pretreatment with
bromo-cAMP plus the phosphodiesterase inhibitor IBMX caused no
discernible changes in intercellular gaps or actin fibers (Fig 5D),
prevented thrombin-elicited changes in actin stress fibers and ICGF
(compare Fig 5E with 5B), but did not prevent the effect of EDTA on
ICGF (compare Fig 5F with 5C). Pretreatment with the concentration of
glutaraldehyde we used allowed minimal changes in actin stress fibers,
but no increased separation of EC (Fig 5G). Despite slight further
stress fiber condensation, glutaraldehyde prevented the induction of
ICGF by both thrombin (compare Fig 5H with 5B) and EDTA (compare Fig 5I
with 5C).
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| Fig 5.
Immunofluorescence microscopy (IF) shows the effect of
thrombin and EDTA, and inhibitors of EC contraction on actin and ICGF.
Actin was stained with rhodamine-labeled phalloidin. The camera shutter
speed was set automatically so the intensities are not relative. These
representative photos show that thrombin- but not EDTA-induced ICGF was
blocked by the inhibitor, bromo-cAMP, and that both were blocked by the
cross-linker, glutaraldehyde. HUVEC in the photos were (A) untreated;
(B) thrombin-stimulated; (C) EDTA-treated; (D) incubated with
bromo-cAMP before treating with control buffer; (E) incubated with
bromo-cAMP before treating with thrombin; (F) incubated with bromo-cAMP
before treating with EDTA; (G) incubated with glutaraldehyde before
treating with control buffer; (H) incubated with glutaraldehyde before
treating with thrombin; and (I) incubated with glutaraldehyde before
treating with EDTA.
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Due to the variability among SS RBC samples, the effect of bromo-cAMP
pretreatment on thrombin- and EDTA-induced SS RBC adherence shown in
Fig 6 is presented as a percent of control
adherence. In these studies adherence results were considered only when
thrombin-enhanced RBC adherence was significantly different from the
buffer-treated negative control. Again EDTA was used as a positive
control for ICGF. Bromo-cAMP reduced mean thrombin-enhanced SS RBC
adherence from 243% of baseline to 122% of baseline, but had no
significant effect on EDTA-induced adherence. There were no significant
differences in adherence to control EC treated with buffer alone
(100%), to bromo-cAMP pretreated EC treated with buffer (92%), or to
bromo-cAMP pretreated EC treated with thrombin (122%). These adherence
effects paralleled the effects of bromo-cAMP on ICGF observed by IF
(Fig 5D through F); bromo-cAMP blocked both adherence and gap formation induced by thrombin, but neither effect of EDTA. Pretreatment with
glutaraldehyde reduced mean thrombin-enhanced SS RBC adherence from
238% to 120% of baseline, a level that was not significantly different from adherence control EC treated with buffer alone (100%)
or to EC treated with glutaraldehyde (85%). Unlike bromo-cAMP, however, glutaraldehyde also significantly reduced EDTA-induced adherence from 323% to 120% of baseline. Like bromo-cAMP,
glutaraldehyde did not alter baseline adherence. Again, the adherence
effects paralleled the effects of glutaraldehyde on ICGF observed by IF (Figs 5G, H, and I); glutaraldehyde blocked both adherence and gap
formation induced by either thrombin or EDTA.
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| Fig 6.
Effect of inhibition of cell contraction by bromo-cAMP
and glutaraldehyde on SS RBC adherence. Treatment of EC with bromo-cAMP
prevented thrombin- but not EDTA-induced adherence. Treatment of EC
with glutaraldehyde blocked both thrombin- and EDTA-induced adherence.
Results are presented as a percent of control adherence.
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These data suggest a correlation between ICGF and thrombin-induced
static SS RBC adherence to cultured HUVEC. The link between ICGF and
adherence is demonstrated by the cognate effects of bromo-cAMP and
glutaraldehyde on both processes, whether stimulated by thrombin or by
EDTA. Under conditions of thrombin exposure of 0.1 U/mL for 5 minutes,
the doubling of static sickle cell adherence in selected endothelial
cell monolayers was dependent on striking EC contraction and ICGF.
| |
DISCUSSION |
Our finding of a thrombin dose-response curve for SS RBC adherence to
HUVEC (Fig 1) whose close resemblance to that for gap formation28 hints of an association between adherence and
ICGF. Consequently, we confirm that our thrombin conditions also cause ICGF (Figs 3-5, Table 2). In the context of an overall doubling of SS RBC adherence in response to thrombin (Fig 2), the equivalent patterns of distribution of adherent SS RBC on untreated and
thrombin-treated HUVEC monolayers (Table 1) indicates that there are
comparable increases in adherence at all three locations, EC edges, EC
apical surfaces, and intercellular matrix. The nonlinear nature of
these changes is illustrated by comparing the 14-fold increase in
exposed matrix with the twofold increase in adherence to this location and the corresponding, but reciprocal, changes of the area covered by
EC. The lessening of adherence seen with higher thrombin concentrations (Fig 1) may relate to a proteolytic effect of thrombin on EC adhesive proteins, to the binding by EC of antithrombin III or
 2-macroglobulin and consequent neutralization of thrombin
effect,35-37 or to the loss of some cells in posttreatment
washing, which we observed (data not shown).
The above relationships suggest that different adhesogenic mechanisms
are likely to pertain for different locations of the vascular
endothelium. Specifically, the doubling of adherence to EC edges and
luminal surfaces may be due to the liberation of adhesive molecules
normally occupied in EC tethering, such as  1 and
3 integrins,38 for binding RBC directly or
via RBC-bound thrombospondin or Fn.7 Such increased
adhesivity may be the result of increased availability of adhesive
molecules, such as integrins, which have specificity for
RBC,39 but which ordinarily reside on the abluminal EC
surface,40 or of increased expression, adhesivity, or
ligand affinity of those molecules.41-43 Increased binding
within the induced gaps is understood reasonably as due to greater
physical availability of adhesive matrix proteins that bind RBC, such
as Fn,7 or perhaps as a result of residual EC adhesive
proteins retained in the gaps after forceful cellular contraction.
While thrombin is reported to modify matrix proteins by changing their
synthetic rate,44 alternately spliced
isoforms,45 and physical accessibility,46 some
of these effects may require longer durations of thrombin exposure than
those used in this study. The specific adhesive molecules involved in
thrombin-stimulated adherence and their modulation by different
thrombin concentrations are important issues that we are now
investigating.
The significant increase in RBC adherence associated with the
permissive induction of ICGF by EDTA or antilaminin polyclonal antibodies demonstrates that ICGF participates in enhanced adherence. The concordance between the effects of bromo-cAMP and glutaraldehyde on
the ICGF and adherence induced by thrombin or EDTA (Figs 5 and 6)
further supports the notion that ICGF participates in thrombin-enhanced adherence. Taken together, these findings demonstrate that under the
conditions we used, thrombin increases SS RBC adherence and ICGF, that
ICGF induced by other means also enhances SS RBC adherence, and that
blocking the ICGF induced by thrombin also blocks its induction of
adherence. These associations provide the basis for our conclusion that
increased adherence and ICGF are interrelated.
The correlation between ICGF and thrombin-enhanced adherence breaks
down with higher concentrations of thrombin, at which there was less
adherence, but a similar degree of ICGF. This reduction of adherence
(Fig 1), whether the result of a proteolytic effect of higher doses of
thrombin on EC adhesive molecules or of neutralization of thrombin by
factors having antithrombin activity,35-37 clearly demonstrates an imperfect association between thrombin-enhanced adherence and ICGF. However, this divergence of effects occurs at
thrombin concentrations greatly exceeding levels predicted in vivo. The
concentration we use, while similar to that used in other published
studies on EC effects,15,28 actually exceeds those reported
during clotting in vitro.47 Another potential dissociation
of thrombin-induced effects stems from our having examined for gaps
only those monolayers that demonstrated an adhesogenic response to
thrombin. Documentation of ICGF in monolayers without adhesive
responses also would have provided evidence against an absolute
interdependence of adhesivity and ICGF responses. Our conclusions
regarding thrombin action pertain to those monolayers that underwent an
adhesogenic response.
Additional considerations support the pertinence in vivo of the ICGF we
observed in vitro. First, the generation of thrombin on cell
membranes48 inevitably increases local concentrations of
thrombin. Second, EC effects of thrombin likely are potentiated by the
abnormal levels of inflammatory cytokines and oxygen radicals present
in sickle cell disease.49-52 Consistent with our
proposition are the ICGF induced by thrombin in intact vessels ex
vivo,53 the interendothelial cell gaps observed in
pathologic sections from autopsied sickle cell patients,54
and the finding in sickle cell blood of substantial numbers of
circulating EC23 whose discharge into the circulating blood
is predicted to have exposed vascular matrix between the retained
cells. Further support for the potential pathophysiologic relevance of
our observations derives from the occurrence of ICGF in vivo as part of
the altered vascular permeability during acute inflammatory
responses.26 In this regard, the long held association of
vasoocclusive crises with infection and inflammation may relate to the
abnormal levels of inflammatory cytokines in sickle cell disease and to
the participation of PMN in SS RBC adherence and vasoocclusion. The
participation of inflammatory mediators and PMN in both processes, ICGF
and SS RBC adherence, support the notion that ICGF may be a part of the
link by which inflammation and vasoocclusion may be related. In our in
vitro adherence studies, the possible influence of PMN is minimized by
their removal during the preparation of SS RBC for those experiments.
However, these interacting influences raise important questions and
deserve independent study.
Other EC effects of thrombin that are predicted to influence adherence
include the secretion of vWF,55 which mediates SS RBC
adherence,56 the induction of nitric oxide
synthesis,57 which modulates the expression of certain
cytoadhesive molecules,58 and the production of
prostacyclin, which protects against PMN adherence and vascular
damage.59 The protective effect of prostacyclin on vascular
endothelium does not seem to pertain in our experiments, possibly
because the amount of prostacyclin produced relative to the
concentration of thrombin used is insufficient to prevent ICGF. Our use
of 17% heparinized plasma in the adherence assay may have inhibited
thrombospondin-mediated adherence60,61 and, thereby,
restricted the group of adhesive molecules tested. This limitation
would not be expected to affect our fundamental observations and
conclusions, partly because these proteins, eg, P-selectin, are less
important to firm than to rolling adhesion.
In view of the uncertain distinctions between EC stimulation,
activation, and injury,62,63 our findings are consistent with the report of enhanced SS RBC adherence to EC "injured" by interleukin-1.64 Our findings contrast, however, with
evidence that thrombin has no effect on the flow adherence of SS RBC
suspended in plasma-free buffer to microvascular EC exposed
continuously to thrombin during the 10 minutes of the
assay.39 Experimental differences between the studies
include the assay methods, properties of HUVEC and microvascular EC,
enhancement of adherence by autologous plasma (our data not shown), and
likely abrogation of thrombin effect by prolonged exposure of EC to the
protease. The differences in results could be accounted for by any of
these factors independently,9 which would include shear
forces minimizing thrombin-enhanced adherence and exposed matrix
proteins having a greater effect on static adherence. These factors
deserve separate investigation.
The much greater variability of SS RBC adherence compared with that of
AA RBC we describe has been observed by others.9 This may
result in part from the reported variance in SS RBC adherence, which
correlates with clinical severity65 or from the greater adhesiveness found during crises.66 The determinants of
this variability remain to be defined fully. It is probable that the degree of adherence may relate to changes in the quantity or adhesivity of receptors born on SS RBC and EC, variation in the representation of
SS RBC subpopulations having different adhesive properties, and to
fluctuations in the concentrations of adhesive ligands in the plasma.
Regarding the relevance of our observations to sickle cell disease, it
is clear that the adhesivity of HUVEC for AA RBC, as well as for SS
RBC, increases in response to thrombin (albeit to a lesser degree). The
direct effects of thrombin on EC do not require the presence of SS RBC.
However, it is not necessary that pathophysiologic relevant processes
be specifically unique to sickle cell disease. In this regard,
nonspecific RBC adherence impacts more severely on microcirculatory
flow of poorly deformable SS RBC, which are prone to being trapped
behind a nidus of occlusion, than on normally deformable AA
RBC.6 Our observations may be relevant to an adherent event
capable of initiating the "vicious cycle of
erythrostasis."67
| |
FOOTNOTES |
Submitted February 27, 1998;
accepted June 30, 1998.
Supported in part by Grant No. HL 20985 from the National Institutes of
Health, Bethesda, MD.
Address reprint requests to Stephen H. Embury, MD, Building 100, Rm
263, San Francisco General Hospital, San Francisco, CA 94110; e-mail
sembury{at}itsa.ucsf.edu.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We wish to thank Drs Robert Pytela, Irena Klimanskaya, Gilda Barabino,
and Tim Wick for valuable scientific discussions and advice, Glen Ozoa
for technical assistance, the staff of San Francisco General Hospital
Labor and Delivery for the supply of umbilical cords, and Dr William C. Mentzer for the use of his fluorescent microscope.
| |
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Abstract
Introduction
Abstract
