Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130-3932
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ABSTRACT |
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The influence of reductions in venular shear
rate on platelet-endothelial (P/E) cell adhesion has not been
previously addressed. The objectives of this study were to define the
effects of reductions in venular shear rate on P/E cell adhesion and to
determine the interdependence of P/E cell adhesion and
leukocyte-endothelial (L/E) cell adhesion at low shear rates.
Intravital videomicroscopy was used to quantify P/E and L/E cell
adhesion in rat mesenteric venules exposed to shear rates ranging
between 118 ± 9 and 835 ± 44 s1. Shear rate
was altered in postcapillary venules by rapid, graded blood withdrawal,
without retransfusion of shed blood. Reducing shear rate from >600
s
1 to <200 s
1 resulted in an eightfold
increase in L/E cell adhesion, whereas P/E cell adhesion increased
18-fold. A blocking antibody directed against P-selectin blunted both
the P/E and L/E cell adhesion elicited by low shear rates.
Immunoneutralization of CD11/CD18 on leukocytes or rendering animals
neutropenic also blocked the shear rate-dependent recruitment of both
platelets and leukocytes. These findings indicate that 1)
low shear rates promote P/E and L/E cell adhesion in mesenteric
venules, and 2) adherent neutrophils (mediated by CD11/CD18)
create a platform onto which platelets can bind to the venular wall at
low shear rates.
P-selectin; neutrophils; CD18; ischemia
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INTRODUCTION |
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BLOOD FLOW AND THE ACCOMPANYING shear forces generated by blood moving within venules can exert a significant influence on the behavior of both platelets and neutrophils (5, 10, 30). The laminar flow profile of blood flowing in microvessels results in generation of lateral displacement forces that allow red blood cells to push leukocytes and platelets toward the vessel wall where interactions with endothelial cells can occur. Blood flow-induced shear forces also play an important role in disrupting the adhesive bonds between blood cells (leukocytes and platelets) and vascular endothelium (19). Hence, high shear rates might be expected to oppose blood cell-to-endothelial cell adhesion, whereas low shear rates should promote this cell-to-cell adhesion. This phenomenon has potentially important implications for the recruitment of circulating blood cells onto the wall of blood vessels under inflammatory conditions associated with elevated (e.g., chronic inflammatory diseases) or reduced (e.g., in postischemic tissues) blood flow (and shear rate). For example, reperfusion of ischemic tissues is generally associated with an enhanced adhesion of both leukocytes (9) and platelets (20, 21) within postcapillary venules. These adhesive interactions are generally accompanied by a reduction in venular shear rate. It appears likely, therefore, that the lower shear rates experienced by postischemic venules will contribute to the recruitment of the leukocytes and platelets during the reperfusion period.
Although the influence of low venular shear rates on
leukocyte-endothelial (L/E) cell adhesion has been extensively studied (3, 4, 26), it remains unclear if and how low shear rates modulate platelet-endothelial (P/E) cell adhesion in postcapillary venules. Although some attention has been devoted to the influence of
shear rate on platelet adhesion, most of this effort has been directed
toward defining the role of high shear rates in promoting thrombus
formation in large arteries (5, 30). Because platelets are
considered to be exposed to lower shear forces compared with leukocytes
due to their smaller diameter (20), one might expect that
even weak receptor-ligand interactions will allow for P/E cell adhesion
when venular shear rate is reduced below normal levels. In vitro
studies suggest that the relative contributions of different platelet
adhesion molecules [GPIIb/IIIa, von Willebrand factor (vWF), and
P-selectin]-to-P/E cell adhesion are significantly influenced by the
prevailing shear rate. For example, GPIIb/IIIa has been proposed to be
a more important determinant of P/E cell adhesion at shear rates <600
s1 (a normal resting value for venules), whereas vWF
plays no role at normal venular shear rates (27, 29).
However, a recent in vivo study (1) of murine mesenteric
venules activated with histamine has revealed a dominant role for vWF
in mediating P/E cell adhesion at shear rates <100 s
1.
In addition to the adhesive interactions that platelets and leukocytes
can establish with vascular endothelium, they can also bind to one
another. These heterotypic interactions are possible, because
leukocytes express receptors [e.g., P-selectin glycoprotein ligand-1
(PSGL-1)] for adhesive ligands on platelets (e.g., P-selectin) and
vice versa (15). Indeed, flow chamber experiments have
revealed that circulating leukocytes can roll on and adhere to
immobilized platelets (17), and there is evidence that
circulating platelets will bind to adherent leukocytes
(11). Furthermore, these studies indicate that the ability
of one population of immobilized blood cells (e.g., platelets) to bind
other moving blood cells (e.g., leukocytes) is more pronounced at low
(<100 s1) shear rates (7). These findings
raise the possibility that the recruitment of adherent platelets and/or
leukocytes into postcapillary venules exposed to low shear rates may
involve both a direct adhesive interaction between circulating cells
and endothelial cells as well as an indirect interaction mediated by
already adherent blood cells.
Major objectives of the present study were to: 1) define the dependence of P/E and L/E cell adhesion on venular shear rate, 2) determine whether shear rate-induced P/E cell adhesion affects (or is affected by) the corresponding recruitment of adherent leukocytes (L/E cell adhesion), and 3) estimate the contribution of reduced shear rate to the P/E and L/E cell adhesion seen in venules exposed to ischemia-reperfusion (I/R). These objectives were addressed by applying the technique of intravital videomicroscopy to rat mesenteric venules exposed to graded reductions in shear rate or to an I/R insult.
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MATERIALS AND METHODS |
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Surgical procedure. Male Sprague-Dawley rats (160-260 g) were fasted 18-24 h before the experiment. The animals were anesthetized by intraperitoneal injection of 120 mg/kg thiobutabarbitol (Inactin). A tracheotomy was performed to facilitate breathing during the experiment. The right carotid artery was cannulated for blood withdrawal and blood pressure measurement. The jugular vein was cannulated for administration of platelets and monoclonal antibodies. A midline abdominal incision was made to allow for exteriorization of the small bowel and associated mesentery (18).
Blood sampling and platelet preparation.
Approximately 0.9 ml of blood was collected via the carotid artery from
the experimental animal and anticoagulated with 0.1 ml acid citrate
dextrose buffer (blood volume was replaced with 2.0 ml of
bicarbonate-buffered saline). Platelet-rich plasma was obtained and
centrifuged at 600 g. The platelet pellet was resuspended in
PBS and fluorescently labeled by incubation with 90 µM
carboxyfluorescein diacetate succinimidyl ester (CFSE) for 10 min. The
fluorescently labeled platelet solution was then centrifuged,
resuspended in 500 µl of PBS (pH 7.4), and protected from light. With
the aid of a hemocytometer, blood cell counts yielded 6 × 105% leukocytes in the platelet suspension
(6).
Flow cytometry. To determine whether the platelet isolation procedure influenced platelet activation status, flow cytometry was used to compare P-selectin expression on both isolated washed platelets and platelets in whole blood. Blood was collected via the carotid artery into acid citrate dextrose buffer at a ratio of 1:10. Platelets were isolated as described above for analysis of washed platelets. Before staining with antibodies, isolated washed platelets were suspended at a concentration of 1 × 108 cells/ml in FACS buffer (2% FCS in PBS), and whole blood was diluted 1:8 in FACS buffer. Both preparations were divided into nonstimulated and thrombin-stimulated samples. Before thrombin stimulation, Gly-Pro-Arg-Pro (GPRP; 0.8 mM final concentration; Sigma, St. Louis, MO) was added to whole blood to prevent fibrin polymerization and platelet aggregation. Human thrombin was then added to both isolated washed platelets and GPRP-treated blood at a final concentration of 1 U/ml. An anti-P-selectin monoclonal antibody RMP-1 provided by Dr. D. C. Anderson (Pharmacia and Upjohn, Kalamazoo, MI) and the F(ab')2 fragment of the anti-GPII/IIIa monoclonal antibody 7E3' (Centocor, Malvern, PA) were conjugated with FITC as previously described (12). Nonstimulated and thrombin-stimulated samples were incubated with either the FITC-conjugated anti-GPIIb/IIIa antibody for identification of platelet populations, or FITC-conjugated anti-P-selectin antibody to assess platelet activation, for 20 min at room temperature. Cells were then washed twice with FACS buffer and analyzed on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences, San Jose, CA).
Intravital microscopy. Rats were placed in a supine position on an adjustable acrylic microscope stage, and the mesentery was prepared for microscopic observation as described previously (18). Briefly, the mesentery was draped over a nonfluorescent coverslip that allowed for observation of a 2-cm2 segment of tissue. The exposed bowel wall was covered with Saran Wrap (Dow Chemical), and then the mesentery was superfused with bicarbonate-buffered saline (37°C, pH 7.4) that was bubbled with a mixture of 5% CO2-95% N2.
An upright multipurpose microscope system (Zeiss, Thornwood, NY) with a 40× water immersion objective lens (Achroplan 40×/0.75 W) was used to observe the mesenteric microcirculation. The mesentery was either transilluminated (with a 12-V, 100-W direct current-stabilized light source) for visualization of adherent leukocytes or epi-illuminated (HBO 50W mercury lamp) for visualization of the fluorescently labeled platelets. CFSE (excitation: 490 nm, emission: 518 nm) was viewed with a reflector slider equipped with an excitation filter of 450-490 nm, a dichroic mirror of 510 nm, and a barrier filter of 515-565 nm. The fluorescent microscopic images were received by a charge-coupled device (CCD) video camera and optimized by a CCD camera control (Dage MTI, Michigan City, IN) attached to an intensifier with a controller (Dage MTI). The transilluminated images were received by a chromachip camera (model JE3362; Javelin) and viewed on a monitor. The images were then recorded on a video recorder (JVC, Elmwood, NJ) for off-line evaluation. A video time/date generator (Panasonic, Secaucus, NJ) projected the time, date, and stopwatch function on the monitor. Single, unbranched venules with diameters ranging between 25 and 35 µm and a length of >150 µm were selected for study. Venular diameter (Dv) was measured either on- or off-line using a video caliper (Microcirculation Research Institute, Texas A & M University, College Station, TX). Red blood cell centerline velocity (VRBC) was measured in venules using an optical Doppler velocimeter (Microcirculation Research Institute). Venular blood flow was calculated from the product of mean red blood cell velocity [Vmean = VRBC/1.6] and microvascular cross-sectional area, assuming cylindrical geometry. Wall shear rate (WSR) was calculated on the basis of the Newtonian definition: WSR = 8(Vmean/Dv). The numbers of adherent leukocytes and platelets were determined off-line during playback of recorded images. A leukocyte was considered to be adherent to venular endothelium if it remained stationary for a period ofExperimental protocols.
After a 30-min stabilization period, fluorescently labeled platelets
(4-5 × 108) were infused over a 5-min period and
allowed to circulate for 5 min before baseline measurement. Criteria
for acceptance of a vessel preparation were shear rates >500
s1 and L/E cell adhesion of
5/100-µm length. To study
the relationship between L/E or P/E cell adhesion and shear rate,
venular blood flow was altered from baseline by graded reductions in
blood volume without retransfusion of shed blood (no reperfusion),
thereby exposing mesenteric venules to one normal and three reduced
shear rates, each for a period of 5-10 min. This protocol was
selected to achieve the following desired shear rates expressed
relative to baseline: 100 (baseline), 50, 37.5, and 20%. In some
experiments, rats were pretreated with either a blocking MAb directed
against P-selectin (2 mg/kg RMP-1) (31) or CD18 (2 mg/kg
WT3) (2) or antineutrophil serum (ANS; 1.0 ml/kg; Accurate
Chemicals and Scientific, Westbury, NY) (13). The MAbs
RMP-1 and WT3 were administered 5 min before the baseline measurements
of shear rate and P/E and L/E cell adhesion. Rats receiving ANS were
given 1 ml/kg ip 3 h before the induction of anesthesia. The
mesenteric area was scanned for 3-5 venules/shear rate and each
was recorded for a period of 1.5 min.
Statistics. Data were analyzed using standard statistical analysis, i.e., one-way ANOVA and Fisher's post hoc test. All values are reported as means ± SE from 5-9 rats, and statistical significance was set at P < 0.05.
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RESULTS |
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Flow cytometric analysis of platelets isolated for intravital microscopy revealed no significant differences in either percentage of P-selectin-positive platelets (3.8 ± 1.1 vs. 3.4 ± 0.9%) or the mean fluorescence intensity of P-selectin expression (31.4 ± 0.7 vs. 32.9 ± 1.4) on unstimulated washed platelets vs. platelets in whole blood. On stimulation with thrombin, the percentage of P-selectin-positive platelets increased significantly in both washed platelets and whole blood (35.2 ± 6.5 and 73.3 ± 6.3%, respectively).
Figure 1 summarizes results of the
studies designed to assess the dependence of L/E and P/E cell adhesion
in rat mesenteric venules on shear rate. The blood cell adhesion data
were analyzed for four ranges of venular shear rate, 835 ± 44, 467 ± 19, 319 ± 9, and 118 ± 9 s1
induced by brief, graded reductions in blood volume. Data reveal that
graded reductions in venular shear rate are associated with recruitment
of increasing numbers of adherent leukocytes and platelets. Figure
1A presents the actual recorded data and illustrates that reducing shear rate appears to exert a more profound influence on the
recruitment of leukocytes compared with platelets. Figure 1B
platelet data are corrected for the fact that only 5% of the total
circulating platelets in our experiments were fluorescently labeled.
Assuming that the fluorescent and nonfluorescent platelets behaved
identically in these experiments, then a much larger number of adherent
platelets are predicted at lower shear rates, greatly exceeding the
density of adherent leukocytes.
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Effects of pretreatment with a P-selectin blocking MAb on the
recruitment of adherent platelets (Fig. 1A) and leukocytes
(Fig. 1B) at low shear rates are summarized in Fig.
2. P-selectin blockade significantly
attenuated the recruitment of both platelets and leukocytes at low
shear rates. The observation that P-selectin immunoneutralization
reduced the adhesion of both populations of circulating cells raised
the possibility that the shear rate-dependent recruitment of platelets
may be dependent on P-selectin-mediated L/E cell adhesion rather than
P-selectin on the platelets themselves. Therefore, additional
experiments were performed to determine whether leukocyte-directed
interventions (CD18 immunoneutralization or ANS) altered the
recruitment of platelets induced by low shear rates. Figure
3B illustrates that either
blocking L/E cell adhesion with a CD18-specific MAb or rendering the
rats neutropenic with ANS profoundly reduced the L/E cell adhesion
normally seen at low shear rates. In Fig. 3A, effects of
these interventions on shear rate-dependent recruitment of platelets
are shown. Both CD18 immunoneutralization and neutropenia resulted in
highly significant (P < 0.01) reductions in platelet
recruitment at low shear rates.
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Figure 4 summarizes the changes in L/E
cell adhesion, P/E cell adhesion, and shear rate in mesenteric venules
exposed to I/R. In sham rats experiencing the same protocol as the I/R
group but without superior mesenteric artery ligation, 140 ± 40 adherent leukocytes/mm2 and 7 ± 7 adherent
platelets/mm2 were detected with shear rates of 728 ± 71 s1. In venules exposed to I/R, the number of adherent
leukocytes and platelets increased to 687 ± 104 and 272 ± 82 cells/mm2, respectively. The venular shear rate at 60 min after reperfusion was reduced to 324 ± 45 s
1.
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On the basis of the effects of brief reductions in venular shear rate
on L/E and P/E cell adhesion predicted in Fig. 1A and the
shear rate values detected in venules exposed to I/R (Fig. 4B), we estimated the proportion of recruited leukocytes and
platelets induced by this I/R insult that can be attributed to shear
rate dependent and independent mechanisms (Fig.
5). This analysis revealed that ~95%
of the I/R-induced leukocyte recruitment may be due to the
correspondingly lower shear rate, whereas the reduced shear rate may
account for ~65% of the I/R-induced platelet recruitment.
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DISCUSSION |
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I/R results in the recruitment of adherent leukocytes and platelets in the microcirculation (9, 20). These blood cells have been implicated in the microvascular dysfunction (9, 20), tissue necrosis (8, 9), and apoptosis (28) that commonly occur in postischemic tissues. Recognition that blood cells contribute to the end organ damage elicited by I/R and other pathological conditions has generated much interest in defining the factors that regulate blood cell-endothelial cell adhesion in microvessels. Some of the major factors known to modulate blood cell-endothelial cell interactions include adhesion molecules (e.g., P-selectin) expressed on endothelial cells and/or blood cells, products of endothelial cell (e.g., nitric oxide) and blood cell (e.g., superoxide) activation, and hydrodynamic dispersal forces (e.g., shear rate) generated by the movement of blood in the microcirculation (10). Although all of these factors have been implicated in the recruitment of adherent platelets and leukocytes in postischemic tissues, relatively little is known about how the reduced venular shear rates that accompany reperfusion affect P/E cell adhesion and whether leukocytes influence the recruitment of platelets at low venular shear rates. These issues were addressed in the present study.
Findings of our study indicate that low shear rates (without
reperfusion) promote the recruitment of adherent platelets
and leukocytes in postcapillary venules of the rat mesentery. The shear
rate-dependent recruitment of adherent leukocytes noted in this study
is both qualitatively and quantitatively similar to that previously
reported for rat (18) and cat (3, 4, 26)
mesentery. A novel finding of the present report is that low shear
rates also elicit the adhesion of platelets in postcapillary venules,
with the largest increment in platelet recruitment seen when shear rate
is reduced <300 s1. The number of fluorescently labeled
platelets detected at the lowest shear rates (~412
cells/mm2 venule) is more than twice the density of
adherent platelets measured in mouse intestinal venules after
intraperitoneal administration of 0.5 mg/kg of Escherichia
coli lipopolysaccharide (6), a potent inducer of P/E
cell adhesion (14).
Although it appears that low shear rates result in the recruitment of
more adherent leukocytes than platelets (Fig. 1A), because only ~5% of the platelets were fluorescent, one would predict that a
far larger number of platelets are actually adherent to the venular
wall at low shear rates (Fig. 1B). This suggests that approximately eight times more platelets than leukocytes bind the
venular wall at the lowest shear rates studied. Difference in magnitude
of platelet vs. leukocyte recruitment cannot be explained by cell size,
because in vitro evaluation of the attachment rates of different-sized
PSGL-1-coated particles to immobilized P-selectin under flow conditions
have revealed a more profound influence of lowering shear rate on the
attachment of larger particles (10 µm) compared with 5-µm
particles (24). An alternative explanation is that
expression of a receptor for platelet attachment on the vessel wall is
increased when shear rate is reduced.
Because P-selectin has been previously implicated in the recruitment of
both leukocytes and platelets in postischemic mesenteric venules (16, 20), we evaluated the contribution of this
adhesion molecule to the P/E and L/E cell adhesion observed at low
venular shear rates. Immunoneutralization of P-selectin with a
rat-specific MAb RMP-1 (31) resulted in a 50-60%
reduction in L/E and P/E cell adhesion at the lowest shear rate
studied, suggesting that this adhesion molecule contributes to the
shear rate-dependent recruitment of both cell populations. Inasmuch as
venular endothelial cells can capture leukocytes using endothelial
P-selectin-leukocyte-PSGL-1 interactions and platelets can bind to
leukocytes via P-selectin (platelet)-PSGL-1 (leukocyte) interactions,
the effectiveness of the P-selectin MAb in reducing the recruitment of
both platelets and leukocytes at low shear rates may reflect a linkage
between the two recruitment processes. To address this possibility, we examined whether depletion of circulating neutrophils with ANS or
immunoneutralization of the 2-integrin CD11/CD18, which
has previously been shown to mediate shear rate-dependent L/E cell adhesion (3, 26), affect the recruitment of platelets at low shear rates. Rendering rats neutropenic with ANS virtually abolished L/E cell adhesion at low shear rates, indicating that neutrophils are the dominant leukocyte participants in this process. Because ANS treatment (which did not alter the number of circulating platelets) also abolished the recruitment of platelets at low shear
rates, it appears that platelets are recruited into venules at low
shear rates through a leukocyte-dependent mechanism.
Immunoneutralization of CD11/CD18, an adhesion molecule not known to
directly mediate P/E cell adhesion, was also effective in reducing (but
not abolishing) the recruitment of both leukocytes and platelets, which
is consistent with leukocyte-dependent platelet recruitment.
A model that can be proposed to explain the dependence of platelet
recruitment on L/E cell adhesion is shown in Fig.
6. At low shear rates, leukocytes utilize
endothelial P-selectin as well as CD11/CD18 to bind to venular
endothelium. Adherent leukocytes, which constitutively express PSGL-1,
then create a platform onto which platelets can bind using P-selectin.
Such a model would explain why P-selectin immunoneutralization
attenuates the adhesion of both platelets and leukocytes when shear
rate is low. It would also explain the ability of CD11/CD18
immunoneutralization to blunt the P/E cell adhesion response and for
neutrophil depletion to completely abolish it.
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Several experimental models, including mesentery, cremaster muscle, and
retina, have revealed that venular shear rate is significantly reduced
after reperfusion of ischemic tissue (22, 23, 25). Because reperfusion is also associated with a pronounced P/E and L/E
cell adhesion, the question arises as to what proportion of the
reperfusion-induced L/E and P/E cell adhesion can be attributed to the
reduced shear rate. Results of the present study suggest that in the
mesentery ~95 and 65% of the L/E and P/E cell adhesion, respectively, may be due to the low shear rate (~300
s1) that venules are exposed to during reperfusion. Our
previous work on cat mesentery indicates that, whereas reduced shear
rates induced by low flow ischemia are indeed associated with
the significant recruitment of adherent leukocytes (10),
the magnitude of the L/E cell adhesion is not as intense as that seen
after reperfusion. This difference likely results from the production
and release of inflammatory mediators during the early reperfusion
period and suggests that one cannot attribute the exaggerated adhesion response simply to a reduced venular shear rate. Nonetheless, our
findings suggest that increasing shear rate during the reperfusion period may be an effective strategy for preventing or attenuating the
proinflammatory and prothrombogenic phenotypes induced by I/R.
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ACKNOWLEDGEMENTS |
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Supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant POI-DK-43785.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. N. Granger, Dept. of Molecular and Cellular Physiology, Louisiana State Univ. Health Sciences Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, Louisiana 71130-3932 (E-mail: dgrang{at}lsuhsc.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.
September 11, 2002;10.1152/ajpgi.00303.2002
Received 25 July 2002; accepted in final form 9 September 2002.
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