Contribution of Fluid Shear Response in Leukocytes to
Hemodynamic Resistance in the Spontaneously
Hypertensive Rat
Shunichi Fukuda, Takanori Yasu, Nobuhiko Kobayashi, Nahoko Ikeda, Geert W. Schmid-Schönbein
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Abstract—The mechanisms for elevation of peripheral vascular resistance in spontaneously hypertensive rats (SHR), a
glucocorticoid-dependent form of hypertension, are unresolved. An increase in hemodynamic resistance caused by
circulating blood may be a factor. Physiological fluid shear stress induces a variety of responses in circulating
leukocytes, including pseudopod retraction. Due to high rigidity, leukocytes with pseudopods have greater difficulty to
pass through capillaries. Because SHR have more circulating leukocytes with pseudopods, we hypothesize that
inhibition of the leukocyte shear response by glucocorticoids in SHR impairs normal leukocyte passage through
capillaries and causes enhanced resistance in capillary channels. Fluid shear leads to retraction of pseudopods in normal
leukocytes, whereas shear induces pseudopod projection in SHR and dexamethasone-treated Wistar rats. The high
incidence of circulating leukocytes with pseudopods results in slower cell passage through capillaries under normal
blood flow and during reduced flow enhanced capillary plugging both in vivo and in vitro. SHR blood requires higher
pressure (90.0⫾8.2 mm Hg) than Wistar Kyoto rat (WKY, 69.6⫾6.5 mm Hg; P⬍0.0001) or adrenalectomized SHR
(73.5⫾2.1 mm Hg; P⫽0.0009) at the same flow rate in the resting hemodynamically isolated skeletal muscle
microcirculation. Intravenous injection of blood from SHR, but not WKY, causes blood pressure increase in normal rats,
which depends on pseudopod formation. We conclude that in addition to enhanced vascular tone, pseudopod formation
with lack of normal fluid shear response may serve as mechanisms for an elevated hemodynamic resistance in SHR.
(Circ Res. 2004;95:100-108.)
Key Words: capillaries 䡲 glucocorticoids 䡲 pseudopod formation 䡲 mechanotransduction
T
here is a widely held opinion that elevation of blood
pressure and peripheral resistance in arterial hypertension is due to an increase in vasoconstriction and restructuring of arteries and arterioles.1–3 But no conclusive evidence
exists that demonstrates that the shift in resistance of arteries/
arterioles is actually the sole cause for the blood pressure
elevation.
In addition to vascular factors, fluid stresses in circulating
blood may contribute to vascular resistance elevation. Perhaps most surprisingly, the relatively small number of leukocytes in the circulation has a powerful effect on the hemodynamic resistance in capillaries with single file cells.4 –5
Because leukocytes are larger and less deformable than
erythrocytes, leukocytes move slower through capillaries.
Erythrocytes are forced to slow down,4 disturbing their
position in the capillary lumen and sharply increasing their
apparent viscosity.5 The reduced leukocyte velocity causes an
increase in microvascular resistance produced by erythrocytes. The effect depends on leukocytes biomechanical properties, it requires no adhesion to the endothelium and can also
be simulated by microspheres with similar dimensions as
leukocytes and with similar low numbers.4
An important aspect of this mechanism relates to the ability
of leukocytes to form pseudopods. Because the resting
diameter of leukocytes is larger than most capillary diameters, leukocytes are required to deform in most capillary
networks. The deformation depends on cell mechanical properties6,7 and as rigid structures in the cell cytoplasm is
strongly influenced by pseudopods.7,8
Furthermore, we have recently demonstrated that physiological levels of fluid shear promote pseudopod retraction and keep
circulating leukocytes in a spherical shape.9 –11 Continued shear
exposure contributes to formation of passive spherical cell
shapes without pseudopods. Therefore, impairment of the shear
response may cause an enhanced number of leukocytes with
pseudopods, slowing of leukocyte passage through capillaries,
and elevated microvascular resistance.6 – 8,12,13
SHR have an abnormal response to glucocorticoids.14 –18
SHR have an elevated leukocyte count, reduced number of
leukocyte rolling on and adhering to venules due to P-selectin
Original received December 5, 2003; revision received May 11, 2004; accepted May 14, 2004.
From the Department of Bioengineering (S.F., N.K., G.W.S.-S.), The Whitaker Institute of Biomedical Engineering University of California San Diego,
La Jolla, Calif; and the Department of Integrated Medicine (T.Y., N.I.), Omiya Medical Center, Jichi Medical School, Saitama, Japan.
Correspondence to Dr Geert W. Schmid-Schönbein, Department of Bioengineering, University of California San Diego, 9500 Gilman Dr, La Jolla, CA
92093-0412. E-mail [email protected]
© 2004 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000133677.77465.38
100
Fukuda et al
suppression, an impaired dilation of vascular smooth muscle,
enhanced oxidative stress, and apoptosis, all of which are
mediated by glucocorticoids. By chance, we recently observed that glucocorticoids have a profound effect on shear
response of circulating leukocytes.19
Thus, we hypothesize that glucocorticoid reverses the
leukocyte shear response in SHR, which causes an increase in
the counts of leukocytes with pseudopods. The effect leads to
impaired passage through single file capillaries and elevated
hemodynamic resistance.
We present a sequence of in vivo and in vitro studies
designed to explore key elements of the circulatory consequences of reversed shear response in leukocytes of SHR and
dexamethasone-treated rats. The results suggest that, in addition to enhanced vascular tone, a shear-mediated hemorheological mechanism in capillaries may contribute to the elevated peripheral resistance in SHR.
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Materials and Methods
Leukocyte separation by centrifugation was avoided because it
impairs the shear response.12 For an expanded Materials and Methods section, see the online data supplement available at
http://circres.ahajournals.org.
Animals
After general anesthesia (sodium-pentobarbital, 50 mg/kg), the
femoral veins and arteries of mature male Wistar rats (n⫽100), SHR
(n⫽50), Wistar Kyoto rats (WKY, n⫽29), adrenalectomized Wistar
rats (n⫽4), adrenalectomized SHR (n⫽7), and adrenalectomized
WKY (n⫽4) (290 to 390 g; Charles River Laboratories, Wilmington,
Mass) were cannulated. Mean blood pressure was measured in the
femoral artery (MABP). Adrenalectomized animals were purchased
from the breeder and received 0.9% saline in the drinking water
1-week before the experiments. The experimental protocol was
approved by the University of California San Diego Animal
Subjects Committee.
Shear Response of Suspended Rat Leukocytes in a
Cone-and-Plate Device
Arterial blood (30 U/mL ammonium heparin) was collected with or
without dexamethasone (Sigma Chemical Corp). Fifty minutes after
collection, whole blood was sheared in a cone-and-plate device at a
physiological range of 5.0 dyn/cm2 for 10 minutes,10 and immediately fixed with 2% glutaraldehyde. Unsheared samples were fixed
simultaneously. The fraction of leukocytes with pseudopods was
counted after staining with 0.02% crystal violet.
Shear Response of SHR Leukocytes After
Treatment With a ␤-Blocker
MABP and fraction of leukocytes with pseudopods in arterial blood
were examined in SHR 1 hour after treatment with 1 mg propranolol
hydrochloride (Inderal) and treatment for 7 days (1 mg/12 hours) and
in control SHR (n⫽4 rats/group).
Shear Response of Leukocytes In Vivo
After DX-Treatment
The fraction of leukocytes with pseudopods was examined in Wistar
treated with dexamethasone (0.5 mg/kg, IV) at time 0, Wistar treated
with dexamethasone for 7 days (0.5 mg/kg per day, IM), and in
control (n⫽4/group). In all 3 groups, FMLP (10⫺9 mol/kg) was
intravenously injected at time 0. Femoral arterial blood (0.1 mL) was
collected every 15 minutes up to 60 minutes and immediately fixed
with 2% glutaraldehyde.
Fluid Shear Response in SHR
101
Leukocyte Kinetics in the Rat Microcirculation
The mesenteric microcirculation was visualized through an intravital
fluorescence microscope.9 –11 Carboxyfluorescein succinimidyl ester
(1.0 mg/kg, CFSE; Molecular Probes) served as label for circulating
leukocytes.
Relative Transit Time in Mesenteric Microcirculation
Relative transit time (leukocytes/erythrocytes transit time) of CFSElabeled leukocytes from WKY, SHR, Wistar rats, and Wistar rats
treated with 0.5 mg/kg dexamethasone for 7 days (DX-Wistar) was
determined in selected true capillaries at constant MABP. CFSElabeled leukocytes in whole blood from either WKY or SHR (or
normotensive Wistar or DX-Wistar) were observed for 10 minutes
after injection. When no more labeled cells were encountered, a
second aliquot of blood was administered. In the same capillary
network, the transit time of 50 PKH-26 –labeled (Sigma) erythrocytes of the recipient rat and 30 leukocytes of each donor rat was
measured.20
In Vivo Leukocyte Kinetics in Capillaries During
Reduced Flow
The velocity in mesenteric capillaries in Wistar or DX-Wistar was
reduced to 0.2 to 1.5 mm/sec by partial occlusion of the celiac artery
using an extravascular balloon (Medtronic PS Medical). PKH-26 –
labeled erythrocytes were used to measure velocity. The number of
plugging leukocytes per capillary in each observation field was
determined. “Plugging leukocytes” were defined as cells that obstruct a capillary for at least 30 seconds.
Measurement of Pressure-Flow Relation in Rat
Gracilis Muscle
The gracilis muscle of normotensive Wistar was hemodynamically
isolated from the central circulation using microsurgery.4,5,21 The
feeder was connected to a precise syringe pump (Harvard Apparatus)
and the draining vein held at 0 mm Hg. Plasma-Lyte (Baxter Health
Care) with 5% bovine serum albumin (Sigma), 10 U/mL ammonium
heparin, and 0.015% papaverine hydrochloride was perfused to fully
dilate the microvessels.21
Blood (30 U/mL ammonium heparin) was collected from either
Wistar, DX-Wistar, SHR, WKY, or adrenalectomized SHR. The
blood (with 0.015% papaverine) was divided into 4 groups: plasma,
plasma⫹erythrocytes, whole blood, and whole blood⫹0.1 ␮mol/L
cytochalasin-D (Sigma) to prevent pseudopod formation.6 In the
plasma⫹erythrocytes group, the hematocrit was adjusted to the same
values as in donor blood. The steady-state pressure-flow relationship
for each group was measured from 0 to ⬇350 ␮L/min. At a constant
flow rate, additional pressure drops for each cell group (see expanded Materials and Methods) were determined as a measure of
their impact on the pressure-flow relationship.
In another set of experiments, gracilis muscle of SHR was
hemodynamically isolated in a resting condition, but without vasodilation, and the steady-state pressure-flow relationship for plasma,
whole blood, and whole blood⫹cytochalasin-D of Wistar or SHR
was measured without papaverine. The pressure-flow curves were
fitted with third-order polynomials, a fundamental relationship for
skeletal muscle microvessels.22
Injection of SHR Blood Into Normal Rats
Whole blood with or without 0.1 ␮mol/L cytochalasin-D from SHR
or WKY was injected into normal Wistar (4.5 mL/kg), while MABP
was measured. In selected samples, erythrocytes were infused after
removal of leukocytes.
MABP After Blood Exchange Between WKY and SHR
Between WKY and SHR (n⫽5/group), or WKY and WKY (n⫽6),
10 mL of arterial blood was exchanged every 5 minutes for 5 times,
and MABP was measured 5 minutes after each blood exchange.
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July 9, 2004
Leukocyte Passage Through an In Vitro
Microchannel Array
Due to the enhanced propensity for rat blood to coagulate, human
blood was used in this study. Human blood was diluted in physiological saline with 1 mmol/L MgSO4 (1:1), and divided into 5
groups: unsheared and sheared controls, 1 ␮mol/L dexamethasonetreated blood without and with shear, and dexamethasone-treated
blood with 5 mmol/L EDTA (Sigma) with shear exposure.
One-minute after blood collection, the sheared groups were
exposed to shear stress (5.0 dyn/cm2 for 3 or 13 minutes) in a
cone-and-plate device. Four, 14, and 19 minutes after blood collection, the passage time of 0.1 mL blood through the Microchannel
Flow Analyzer (Kowa Co) was determined.23 The parallel array of
microchannels (3100 channels, equivalent diameter 5 ␮m, equivalent
length 20 ␮m) was examined under constant suction (15, 20, and 25
cmH2O). Light microscopic images of the blood passage through the
microchannels were recorded on videotape for off-line analysis.
Fractions of occluded microchannels due to leukocyte plugging and
leukocytes with pseudopods were evaluated 14 minutes after blood
collection.
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Statistics
Values are shown as mean⫾SD. Differences were analyzed by
ANOVA and Fischer’s PLSD. A value of P⬍0.05 was
considered significant.
Results
In Vitro Shear Response of SHR Leukocytes
The fraction of leukocytes with pseudopods in Wistar was
significantly lower with shear than without shear (Figure 1A).
In contrast, the number of leukocytes with pseudopods in
SHR was significantly increased after shear. Adrenalectomy
inhibited the increase in the number of SHR leukocytes with
pseudopods after fluid shear. The shear response in WKY
was still noticeable, but slightly suppressed compared with
that in Wistar (Figure 1A).
With dexamethasone, the shear response in all animal
groups was reversed (Figure 1B).
Blood Pressure
MABP in SHR was significantly higher than in Wistar or
WKY. Treatment of Wistar with dexamethasone caused a
significant rise in MABP (Figure 2A). Adrenalectomy reduced MABP in SHR to normal levels, whereas MABP
changed little in Wistar.
In Vivo Pseudopod Formation of Circulating
Leukocytes in SHR and DX-Wistar
The average fraction of leukocytes with pseudopods in
arterial blood of DX-Wistar was higher than that of control
Wistar (Figure 2B). The fraction of cells with pseudopods in
SHR (30.4⫾9.2%) was higher than in WKY (13.8⫾5.5%) or
Wistar. The fraction in WKY was also higher than that in
Wistar. Adrenalectomy caused a significant decrease in the
fraction of cells with pseudopods in SHR, but not in Wistar
(Figure 2B).
Effect of ␤-Blocker on MABP and Pseudopods in
Arterial Blood
Both acute (1-hour) and chronic (7-day) treatment of SHR
with a ␤-blocker, propranolol hydrochloride, significantly
reduced MABP; however, there was no change in fraction of
circulating leukocytes with pseudopods (Figure 2C).
Figure 1. Fraction of leukocytes with pseudopods in rat whole
blood with and without shear exposure for 10 minutes. n⫽4 animals in each group. A, Control nontreated blood. Wistar (E),
WKY (‚), SHR (✖), adrenalectomized Wistar (ad Wistar, F),
adrenalectomized WKY (ad WKY,Œ), and adrenalectomized SHR
(ad SHR, 多). B, 10 ␮mol/L dexamethasone-treated blood. Wistar
(DX-Wistar, E), WKY (DX-WKY, ‚), SHR (DX-SHR, ✖), adrenalectomized Wistar (DX-ad Wistar, F), and adrenalectomized SHR
(DX-ad SHR, 多).
In Vivo Effect of Dexamethasone on the Fraction
of Leukocytes With Pseudopods
Although 7-day treatment of Wistar with dexamethasone
enhanced the fraction of leukocytes with pseudopods, acute
(1-hour) dexamethasone treatment caused only a modest, but
insignificant, increase in this fraction (Figure 2D).
Human Leukocyte Kinetics in Narrow
Microchannels In Vitro
After 13-minute shear exposure in the cone-and-plate device,
0.1 mL of dexamethasone-treated blood required significantly
longer channel passage times than the same amount of control
blood at all pressure-drops (Figure 3A).
The passage time of unsheared control blood gradually
increased in time after blood collection (Figure 3B). Fluid
shear modestly suppressed the passage time. In contrast, the
passage time in unsheared dexamethasone-treated blood
gradually decreased, whereas the passage time in sheared
dexamethasone-treated blood significantly increased. Five
minutes after shear, we saw a significant decrease in passage
time of dexamethasone-treated blood group. EDTA inhibited
the ability of dexamethasone to increase the passage time
with shear.
Fukuda et al
Fluid Shear Response in SHR
103
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Figure 2. A, Mean arterial blood pressure
(MABP) of rats. DX-Wistar indicates
DX-treated Wistar; ad Wistar, adrenalectomized Wistar; ad SHR, adrenalectomized
SHR. n⫽54 (Wistar), 11 (DX-Wistar), 15
(WKY), 15 (SHR), 4 (ad-Wistar), and 7 (adSHR). *P⬍0.0001 vs Wistar; #P⬍0.0001 vs
WKY; **P⬍0.0001) vs SHR. B, Fraction of
leukocytes with pseudopods in arterial
blood. n⫽17 (Wistar), 9 (DX-Wistar), 8
(WKY), 10 (SHR), 6 (ad-Wistar), and 8 (adSHR). *Pⱕ0.0010 vs Wistar; #P⬍0.0001 vs
WKY; **P⬍0.0001 vs SHR. C, MABP and
fraction of circulating leukocytes with
pseudopods after treatment with propranolol hydrochloride in Wistar. 1 hour indicates 1 hour after treatment with 1 mg
propranolol hydrochloride; 7 days, 7 days
after treatment with propranolol hydrochloride (1 mg/12 hours). n⫽4 in each group.
*Pⱕ0.0027 vs control. D, Fraction of circulating leukocytes with pseudopods after
0.5 mg/kg dexamethasone-treatment and
after FMLP (10⫺9 mol/kg) was intravenously injected into all 3 groups at time 0.
n⫽4 rats per group. *P⬍0.0001 vs Wistar.
In all groups, we observed a parallel response between the
fraction of leukocytes with pseudopods, fraction of occluded
microchannels, and passage time (Figure 3C).
In Vivo Transit Time of Leukocytes
The relative transit time in DX-Wistar was significantly
longer than that in Wistar (Figure 4A). The average leukocyte
transit time in WKY was 1.21⫾0.06, similar to previous
measurements. 24 The relative transit time in SHR
(1.55⫾0.18) was significantly longer (Figure 4A).
Leukocyte Behavior in Capillaries During Reduced
Blood Flow
In Wistar, the fraction of leukocytes plugging capillaries was
less than 1% even under low flow velocity, whereas capillary
plugging in DX-Wistar was significantly more frequent at
reduced flow rates (Figure 4B). Some capillaries were obstructed by leukocytes from DX-Wistar (Figure 4C).
In SHR, most capillaries were easily occluded during
reduced flow rates so that the fraction of leukocytes plugging
capillaries could not be counted (results not shown).
Pressure-Flow Relation in Resting
Hemodynamically Isolated Gracilis Muscle
The zero-flow pressure was subtracted from the arterial
pressures at each flow21 (Figure 5A). There was no significant
difference in zero-flow pressure between groups of blood
samples in vasodilated and hemodynamically isolated Wistar
skeletal muscle. In each group, the data revealed the same
trend (Figure 5B and 5C).
Both in WKY and SHR blood, the pressure-flow curve was
shifted equally to the right after addition of erythrocytes to
plasma at a hematocrit adjusted to the same value as in whole
blood. However, SHR whole blood caused a greater right
shift than WKY whole blood. Treatment with cytochalasin-D,
to eliminate pseudopods,6 reduced the degree of right shift for
SHR whole blood, but not WKY blood (Figure 5B and 5C).
At equal flow (150 ␮L/min per g), there was no
significant difference between WKY and SHR in the
perfusion pressure for plasma or plasma with erythrocytes.
However, at the same flow the pressure for SHR whole
blood was significantly higher than that of WKY whole
blood, an effect that was inhibited by cytochalasin-D
(Figure 6A). Whole blood from adrenalectomized SHR
caused a decrease in pressure compared with that from
SHR. Dexamethasone-treatment also induced a rise in
pressure of whole blood, but not plasma or plasma with
erythrocytes (Figure 6B). The increased pressure due to
dexamethasone-treatment was reduced by cytochalasin-D.
The pressure with plasma (PPL) or the additional pressure
drops due to erythrocytes (PRBC–PPL) were not significantly different between WKY and SHR blood (Figure
6C). In contrast, the additional pressure drop induced by
leukocytes (PWB–PRBC) were significantly different in
WKY and SHR.
When SHR blood samples were perfused into resting, but
not vasodilated, skeletal muscle of SHR, perfusion pressure
of whole blood in SHR was significantly higher than that in
Wistar at the same flow (Figure 6D). Cytochalasin-D significantly reduced the perfusion pressure of SHR whole blood.
There was no difference in perfusion pressure between Wistar
whole blood with and without cytochalasin-D.
The hematocrit of Wistar was significantly lower than that
of all other groups. Although SHR as donor animals had a
higher leukocyte count than WKY, there was no difference in
leukocyte counts between other animal groups (Figure 7).
MABP After Injection of SHR Blood
Injection of whole blood from SHR, but not WKY, significantly increased MABP of Wistar (Figure 8A). The increase
in MABP by SHR blood was not present when leukocytes
were removed from the blood. Cytochalasin-D significantly
reduced the ability of SHR blood to raise MABP (from 1 to
15 minutes after blood injection; Figure 8A). There was no
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Figure 3. A, Average passage time of 0.1 mL human blood
through capillaries of a microchannel array analyzer at 15, 20,
and 25 cmH2O pressure drops. Blood was sheared at 5 dyn/
cm2 for 13 minutes in a cone-and-plate device before passage
through the microchannel array. Control (E) and 1 ␮mol/L dexamethasone (DX, ✖). n⫽5 samples in each. *Pⱕ0.0384. B, Time
course of passage time of 0.1 mL human blood. Unsheared
control (E), sheared control (F), 1 ␮mol/L dexamethasone without shear (unsheared DX; ‚), sheared DX (Œ), dexamethasone
and 5 mmol/L EDTA with shear (sheared DX/EDTA, ✖). n⫽5
samples in each. #Pⱕ0.0001 vs unsheared control at time 14
minutes, sheared-DX at 4 minutes, or sheared DX/EDTA at 14
minutes; *P⫽0.0035 vs sheared DX at 14 minutes. C, Passage
time, fraction of leukocytes with pseudopods, and fraction of
occluded microchannels 14 minutes after blood collection. n⫽5
samples in each. *Pⱕ0.0276 vs unsheared control; #Pⱕ0.0007
vs sheared DX.
significant difference in MABP between WKY blood with
and without cytochalasin-D.
MABP After Blood Exchange Between WKY
and SHR
Blood exchange between WKY and SHR (50 mL) significantly increased MABP of WKY and modestly reduced
MABP in SHR. Blood exchange between WKY gave no
significant shift (Figure 8B and 8C).
Discussion
SHR have a higher fraction of circulating leukocytes with
pseudopods as well as a reversal of their shear response. This
Figure 4. Leukocytes kinetics in microcirculation. A, Relative
transit time of leukocytes from Wistar, DX-Wistar, WKY, and
SHR in a Wistar microcirculation. n⫽6 capillary networks in 3
rats (50 erythrocytes and 30 leukocytes per network in each
group). *P⫽0.0016 vs Wistar; #P⫽0.0033 vs WKY. B, Fraction of
leukocytes plugging mesentery capillaries under reduced flow
rates. Controls (F) and DX-Wistar (✖). n⫽5 animals per group. C
and D, Micrographs of labeled leukocytes in Wistar (C) and
DX-Wistar (D) mesentery capillaries under reduced flow rates. In
the control, no plugging leukocytes are found. In the DX-Wistar,
two CFSE-labeled leukocytes plugging in capillaries (arrows) are
shown, a regular observation under reduced flow rates.
leads to a rise of the resistance in a resting hemodynamically
isolated skeletal muscle microcirculation, which could be
eliminated by adrenalectomy or cytochalasin-D treatment.
The current evidence suggests that the enhanced flow resistance caused by the reversal of leukocyte shear response due
to glucocorticoids may be associated with an elevated capillary flow resistance, contributing to a rise in peripheral
resistance in SHR.
Although the number of leukocytes in the circulation is
much smaller than that of erythrocytes, leukocytes have a
Fukuda et al
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Figure 5. Pressure-flow relation in a resting vasodilated gracilis
muscle of Wistar. Plasma (E), plasma with erythrocytes
(plasma⫹RBC, F), whole blood (✖), and whole blood with
0.1 ␮mol/L cytochalasin-D (whole blood⫹cyD, ‚). Zero-flow
pressure is subtracted from arterial pressure. A, Example of
pressure-flow relationship in gracilis muscle during perfusion
with SHR blood. Third-order least-square polynomials are fitted
to data. PPL, PRBC, PWB, and PcyD are pressure values at 150
␮L/min per g of plasma, plasma⫹RBC, whole blood, and whole
blood⫹cyD, respectively (see Figure 6). B and C, Combined
pressure-flow relationships in gracilis muscles during perfusions
with WKY (B) or SHR (C) blood. n⫽5 animals/group.
significant impact on the hemodynamic resistance in the
microcirculation. Reduction of erythrocyte velocity in capillaries by leukocyte causes a substantial increase in microvascular resistance.4,5 The current data in the resting muscle of
normotensive Wistar with and without leukocytes also support this evidence seen exclusively in capillaries (Figure 6).
The microvascular resistance derived from blood depends on
the velocity of leukocytes.4,5 Events during pseudopod formation, such as cell shape change and reduced cell deformability, serve to further reduce the velocity of leukocytes and
Fluid Shear Response in SHR
105
even cause plugging in capillaries.6 – 8,12 SHR and DX-Wistar
leukocytes, about one-third of which have pseudopods (Figure 2), had longer relative transit times than WKY or Wistar
in identical capillary networks (Figure 4A), indicating a
reduced velocity in the presence of SHR leukocytes with
pseudopods. Although leukocytes without pseudopods rarely
obstructed capillaries, leukocytes with pseudopods during
reduced flow produced more frequent capillary plugging.7,8
The incidence of capillary plugging in DX-Wistar was higher
than that in controls under reduced blood flow velocity
(Figure 4B through 4D). Because plugging is largely dependent on the size of the leukocytes, neutrophils and monocytes
might be more involved than lymphocytes. The motion of
leukocytes in capillaries may also be influenced by the
endothelial glycocalyx.25
What mechanism may raise the count of circulating leukocytes with pseudopods in the SHR? Although there is no
significant difference in circulating levels of corticosteroids
between WKY and SHR,26 glucocorticoid receptors have
higher density27 and sensitivity in SHR than WKY,28 suggesting that the abnormal response to glucocorticoids is a
requirement for sustained elevation of blood pressure in SHR.
Fluid shear stress serves to inactivate leukocytes by downregulation of membrane CD18 molecules29 and retraction of
pseudopods.9 –11 The latter event is a requirement to keep
circulating leukocytes in a spherical shape without pseudopods. Impairment of the shear response has a profound effect
on dynamics of circulating leukocytes. Our results indicate
that the shear response of leukocytes is reversed in the
presence of glucocorticoids: fluid shear induces pseudopod
projection rather than retraction (Figure 1).
Glucocorticoids reduce leukocyte activation and
leukocyte-endothelium interaction by reduction of adhesion
molecule expression, inhibiting leukocyte emigration in postcapillary venules.30,31 In the presence of fluid shear, however,
glucocorticoids may cause dissociation between reduced
expression of adhesion molecules and enhanced pseudopod
formation. Excess levels (in DX-Wistar) of, or excess response (in SHR) to glucocorticoids make leukocytes less
adhesive but cause them to project more pseudopods, leading
to the elevated fraction of leukocytes with pseudopods in the
circulation (Figure 2). The number of leukocytes with pseudopods was significantly higher in SHR and DX-Wistar,
models in which the leukocyte shear response was impaired
(Figures 1 and 2). In SHR, both phenomena were prevented
by adrenalectomy. The elevated blood pressure may not
stimulate pseudopod formation in SHR because reduction of
MABP by beta-blocker did not cause any change in fraction
of leukocyte with pseudopods (Figure 2C). Although the
reversal of the shear response is rapid in vitro,19 longer
periods are required to reverse the response in vivo (Figure
2D). Because glucocorticoids have a number of effects in
vivo, multiple factors may be associated with the difference
between in vitro and in vivo.
The reduction of the leukocyte velocity due to pseudopod
formation in association with glucocorticoids may cause a
substantial increase in microvascular resistance. In order to
examine this hypothesis, we first evaluated the association
between pseudopod formation and microvascular resistance
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Figure 6. A and B, Additional pressuredrop at 150 ␮L/min per g flow rate of
blood from WKY, SHR, adrenalectomized SHR (ad SHR) (A), and Wistar and
DX-Wistar (B) in a vasodilated gracilis
muscle of Wistar rats. n⫽5 animals/
group except ad SHR (n⫽3). *P⬍0.0001
vs WKY PWB in A, or vs Wistar PWB in
B; # vs SHR PWB (P⬍0.0001), or vs
DX-Wistar PWB (P⫽0.0103); **P⫽0.0009
vs SHR PWB. C, Arterial pressure with
plasma (PPL), additional pressure-drop
for erythrocytes (PRBC–PPL), leukocytes
(PWB–PRBC), and leukocytes after
0.1 ␮mol/L cytochalasin-D (PcyD–PWB)
of WKY and SHR in vasodilated muscle
of Wistar. n⫽5 animals/group.
*P⬍0.0001 vs WKY PWB–PRBC;
#P⬍0.0001 vs WKY PcyD–PWB. D,
Pressure values at 150 ␮L/min per g in a
resting, but not vasodilated, SHR muscle
with Wistar and SHR blood. *P⬍0.0001
vs Wistar PWB; #P⫽0.0013 vs SHR
PcyD.
in the microchannel flow array analyzer in vitro (Figure 3).
After dexamethasone-treatment, shear application caused significant increases in blood passage time, in the fraction of
cells with pseudopods, and in the frequency with which
leukocytes are plugging microchannels (Figure 3C). In this
device the microchannel resistance is directly related to the
cell passage time. EDTA, which suppresses pseudopod formation in leukocytes,9 inhibited all these effects by dexamethasone. Therefore, pseudopod formation in leukocytes
due to glucocorticoid-mediated reversal of the shear response
elevates microvascular resistance.
We then examined in the vasodilated skeletal muscle to
what degree pseudopod formation contributes to the hemodynamic resistance in vivo independent of the arterial/arteriolar control (Figure 5). At a constant flow rate, there was no
significant difference in additional pressure drops induced by
plasma or erythrocytes between WKY and SHR. But SHR
leukocytes in whole blood caused a significantly higher
pressure-drop than WKY ones, similar to DX-Wistar leuko-
Figure 7. Hematocrit (A) and leukocyte (B) count of donor rat
blood used in resting vasodilated gracilis muscle of Wistar rats.
n⫽5 animals/group except ad SHR (n⫽3). *Pⱕ0.0203 vs Wistar.
cytes compared with Wistar leukocytes. Both adrenalectomy
and treatment with cytochalasin-D, an agent that reduces
cytoplasmic stiffness and pseudopod formation,6 eliminated
the increase in additional pressure drop induced by SHR
leukocytes.
Furthermore, when the microvascular resistance was examined in resting, but not vasodilated skeletal muscle of SHR, close
to its normal physiological conditions, cytochalasin-D treatment
caused a significant reduction of perfusion pressure of whole
blood of SHR, but not of Wistar (Figure 6D). These collective
data then support our hypothesis that, in addition to the elevated
number of leukocyte count (Figure 7), an elevated fraction of
circulating leukocytes with pseudopods enhances peripheral
hemodynamic resistance in SHR.
In addition, adrenalectomy in SHR serves to recover the shear
response of leukocytes and reduce both MABP and pseudopod
formation in circulating leukocytes (Figures 1 and 2). Moreover,
injection of blood aliquots derived from SHR into normal
animals caused a significant rise in MABP, whereas neither
blood aliquots from WKY nor SHR blood without leukocytes
would cause a blood pressure elevation (Figure 8A). The
increase in blood pressure by a SHR blood aliquot was inhibited
by cytochalasin-D, although cytochalasin-D had no effect when
WKY blood was injected.
Although the mechanisms for glucocorticoid-induced hypertension are still uncertain, peroxisome proliferator–activated receptor-␣ may be involved.32 Blood pressure depends
on several factors, including blood volume, peripheral vascular resistance, cardiac output, and blood viscosity. Among
them, excess levels of glucocorticoids induce hypertension in
humans and rats due to a rise in cardiac output and peripheral
resistance.33,34 A rise in total peripheral resistance in SHR
results mainly from the enhanced resistance in the terminal
arterioles.35 Previously this was attributed exclusively to
elevated arteriolar tone caused by increased reactivity to
vasoconstrictors, decreased response to vasodilators, and
rarefaction.1–3 Although vascular control might be one major
factor responsible for an increase in blood pressure in SHR,
Fukuda et al
Fluid Shear Response in SHR
107
WKY and SHR caused a significant increase in MABP in
WKY (Figure 8B), which is consistent with previous reports,36 suggesting that pseudopod formation in SHR is in
part responsible for the development of hypertension.
Because glucocorticoids are used as antiinflammatory
agents, they were expected to be effective in myocardial
infarction or cerebral ischemia due to inhibition of leukocyteendothelial cell interaction.30,31 However, several trials have
failed to confirm any beneficial effect.37–39 Multidose treatment with glucocorticoids even enhanced infarct volume
during myocardial ischemia.37 This may be partly associated
with enhanced capillary leukostasis by glucocorticoids in
microvascular regions with reduced blood flow, such as
penumbra around cerebral infarct areas. The same mechanism
may be involved in several organ injuries in SHR.
Acknowledgments
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
NIH Grant HL-10881 and HL-43026 supported this research. We are
very grateful to Dr Herbert H Lipowsky, Department of Bioengineering, Penn State University and Dr Shunichi Usami in our
Department for valuable suggestions.
References
Figure 8. A, MABP in Wistar rats after injection of whole blood
from WKY without (WKY, E) or with (WKY cyD, ‚) 0.1 ␮mol/L
cytochalasin-D, or SHR without (SHR, F) or with (SHR cyD, Œ)
cytochalasin-D, and blood without leukocytes from WKY (WKY
wo WBC, 䉫) or SHR (SHR wo WBC, ✖). Average MABP for 5
minutes before blood injection in each was set to 0 mm Hg. In
SHR, values between 1 and 30 minutes are significantly higher
than before injection (Pⱕ0.0330). n⫽4 animals/group.
*Pⱕ0.0237 vs SHR cyD. B and C, MABP after blood exchange
between WKY and WKY (B), or WKY (E) and SHR (F) (C). n⫽6
(3 pairs) (B), or 5 (C) in each group. *P⫽0.0074 vs MABP at time
0 in WKY.
we now demonstrate that the elevated hemodynamic resistance due to glucocorticoid-induced pseudopod formation of
circulating leukocytes, and its impact on the apparent viscosity of the erythrocyte in single file capillaries, might be
another contributor in this hypertensive animal model. Interestingly, calcium channel blockers,11 cGMP/nitric oxide,10
and cAMP19 enhance the leukocyte shear response and/or
suppress the impairment of shear response, all of which are
known to be vasodilators. This may be physiologically a
favorable coincidence, but it makes it difficult to examine
inhibition of glucocorticoid-induced reversal of shear response of leukocytes without influencing vascular tone. In
this study, we used cytochalasin-D at a dose without detectable effect on vascular tone but still prevention of pseudopod
formation. There was no significant difference between WKY
blood and WKY blood with cytochalasin-D (Figures 6D and
8A) although further study may be required using other
microfilament blockers. In addition, blood exchange between
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Contribution of Fluid Shear Response in Leukocytes to Hemodynamic Resistance in the
Spontaneously Hypertensive Rat
Shunichi Fukuda, Takanori Yasu, Nobuhiko Kobayashi, Nahoko Ikeda and Geert W.
Schmid-Schönbein
Circ Res. 2004;95:100-108; originally published online May 27, 2004;
doi: 10.1161/01.RES.0000133677.77465.38
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2004 American Heart Association, Inc. All rights reserved.
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Supplemental Methods
Since even mild levels of centrifugation interfere with the shear response (1), leukocyte
separation by centrifugation was avoided in all the experiments.
Animals: After general anesthesia (sodium-pentobarbital, 50 mg/kg), the femoral veins
and arteries of mature male Wistar rats (Wistar, n=100), SHRs (n=50), Wistar Kyoto rats
(WKYs, n=29), adrenalectomized Wistar rats (n=4), adrenalectomized SHRs (n=7), and
adrenalectomized WKYs (n=4) (290-390 g, Charles River Laboratories, Wilmington,
M.A.) were cannulated.
Mean blood pressure was measured in the femoral artery
(MABP). Adrenalectomized animals were purchased from the breeder and received
0.9% saline in the drinking water before the experiments (1 week). The experimental
protocol was approved by the University of California San Diego Animal Subjects
Committee.
Shear response of rat leukocytes in suspension in a cone-and-plate device: Arterial
blood with ammonium heparin (30 U/ml) was collected (n=4 in each animal group) and
treated with or without dexamethasone (Sigma Chemical Corporation, St. Louis, MO) 30min after blood collection. Fifty minutes after blood collection, whole blood (0.3 ml)
was sheared in a cone-and-plate device at 5.0 dyn/cm2 for 10 min (2), and immediately
fixed with 2 % glutaraldehyde. Unsheared samples were fixed at the same time. Stained
with 0.02 % crystal violet, the fraction of leukocytes with pseudopods was counted by
microscopy in a blinded manner. For microphotography, erythrocytes were removed by
FACS Lysing solution (Becton Dickinson), which after fixation does not affect the
leukocyte shapes.
Shear response of SHR leukocytes after treatment with a beta-blocker: MABP and
fraction of leukocytes with pseudopods in arterial blood were examined in SHR 1-hour
after treatment with 1 mg propranolol hydrochloride (Inderal, Sumitomo, Osaka, Japan),
SHR after treatment with propranolol hydrochloride (1 mg/12 hours) for 7 days, and
control SHR (n=4 in each group).
Shear response of leukocytes in vivo after DX-treatment: In all groups FMLP (10-9
mol/kg) was intravenously injected at time “0”.
The fraction of leukocytes with
pseudopods in arterial blood was examined in Wistar treated with dexamethasone (0.5
mg/kg, i.v.) at time “0”, Wistar treated with dexamethasone for 7 days (0.5 mg/kg/day,
i.m.), and control Wistar (n=4 in each group). Arterial blood (0.1 ml) was collected from
the femoral artery every 15 min from time “0” to “60” min, and immediately fixed with 2
% glutaraldehyde, and the fraction of leukocytes with pseudopods was counted with
microscopy in a blinded manner.
Leukocytes kinetics in the rat microcirculation: The ileocecal portion of the rat
mesentery was exteriorized and superfused with a Krebs-Henseleit bicarbonate-buffered
solution saturated with a 95% N2 and 5% CO2 mixture (36.5OC, pH 7.4). The mesenteric
microcirculation was visualized through a digital intravital fluorescence microscope with
silicone intensified target camera (SIT Model 66, Dage-MTI, Inc., Michigan City,
Indiana) (1-3). Carboxyfluorescein succinimidyl ester (1.0 mg/kg, CFSE; Molecular
Probes) served as label for circulating leukocytes.
Relative transit in mesenteric microcirculation Relative transit time (ratio of leukocytes
and erythrocytes transit time) of CFSE-labeled leukocytes from WKYs and SHRs, or
normotensive Wistar rats and Wistar rats treated with 0.5 mg/kg dexamethasone for 7
days (DX-Wistar) (n=3 in each) was determined. Dexamethasone (0.5 mg/kg in saline)
was intramuscularly injected into the animal every day for 7 days. The measurements
were carried out in the same capillary network of recipient rats (n=6) under identical
MABP (4). Since transit time is a measurement of the flow rates through the capillary
network, the inverse of the transit time serves as measure for microvascular resistance.
The cell membrane of erythrocytes from recipient rats was labeled with the fluorescent
dye PKH-26 (Sigma) and injected into the recipient rats (0.5 ml) (5). CFSE-labeled
leukocytes in whole blood from either WKY or SHR (or normotensive Wistar or DXWistar) were observed for 10 min after a first intravenous injection. When no more
labeled cells were encountered, a second aliquot of CFSE-labeled leukocytes in whole
blood was injected. The order of blood sample injection was kept at random. The transit
time of 50 PKH-26-labeled erythrocytes of the recipient rat and 30 leukocytes of each
donor rat through the same capillary network was measured after each injection.
In vivo leukocyte kinetics in capillaries during reduced blood flow The flow velocity in
mesenteric capillaries in Wistar (n=5) or DX-Wistar (n=5) was reduced to 0.2-1.5
mm/sec by partial occlusion of the celiac artery using an extravascular balloon
(Medtronic PS Medical, Mentor Corporation, Goleta, CA). PKH-26-labeled erythrocytes
were used to measure flow velocity with frame by frame analysis (5). The number of
plugging leukocytes per capillary in each observation field was determined for 5 min.
“Plugging leukocytes” were defined as cells that obstructed a capillary for at least 30 sec.
Measurement of pressure-flow relation in resting hemodynamically isolated rat
gracilis muscle:
The left gracilis muscle of normotensive Wistar (n=23) was
hemodynamically isolated from the central circulation using microsurgical techniques (6-
9). After hemodynamic isolation of the muscle by ligation of communicating vessels, the
feeder (femoral artery) was connected to a precise syringe pump (Pump 22, Multiple
syringe pump, Harvard Apparatus, Holliston, MA) and the draining vein (femoral vein)
was held at 0 mmHg throughout the experiments. The surgical isolation of the muscle
required approximately 60 to 90 min, and a set of experiments was completed within 4
hours after the recipient animal was anesthetized. A buffer containing Plasma-Lyte
(Baxter Health Care, Deerfield, IL) with 5% bovine serum albumin (Sigma), 10 U/ml
ammonium heparin, and 0.015% papaverine hydrochloride (Bedford Laboratories,
Bedford, OH), was perfused for 20 min to fully dilate the microvessels (9).
Blood with 30 U/ml ammonium heparin was collected from either normotensive
Wistar, DX-Wistar, SHR, WKY (n=5 animals in each case), or adrenalectomized SHR
(n=3).
The blood was divided into 4 groups (each with 0.015% papaverine
hydrochloride): 1. plasma, 2. plasma + erythrocytes, 3. whole blood, and 4. whole blood
+ 0.1 µM cytochalasin D (Sigma) to prevent pseudopod formation (10). Plasma was
obtained from blood after centrifugation at 900 g for 10 min. Erythrocytes were obtained
from the bottom part of mixed solution with blood and Histopaque-1077 (Sigma) after 1
g sedimentation for 30 min. In the “plasma + erythrocytes” group, the hematocrit was
adjusted to the same values as that of donor animal blood. Cytochalasin-D was applied
to the blood samples for 15 min immediately after the blood collection. The steady-state
pressure-flow relationship for each blood sample was measured by stepping the input
arterial flow rate in 8-10 steps up and down from 0 to ~350 µl/min in intervals of 15-20
s.
At a constant flow rate, “additional pressure drops” were determined from these
curves to describe the relative effects of erythrocytes and leukocytes on the pressure-flow
relationships.
The additional pressure drop for erythrocytes was determined by
subtraction of the arterial pressure (at constant flow rate of 150 µl/min/g) during
perfusion with red cells (PRBC) from the arterial pressure for plasma only (PPL). In a
similar fashion the additional pressure drop for leukocytes (PWB) was determined by
subtraction of the arterial pressure for whole blood (erythrocytes and leukocytes) (PWB)
from the arterial pressure for erythrocytes only (PRBC) (see Fig 5A).
In another set of experiments, the gracilis muscle of SHRs (n=10) was also
hemodynamically isolated from the central circulation in a resting condition, but without
vasodilation. The steady-state pressure-flow relationship for plasma, whole blood, and
whole blood + cytochalasin-D of Wistar or SHR (n=5 animals in each) was measured
without papaverine hydrochloride-treatment.
At the end of each set of experiments, 0.02 % crystal violet was infused to mark
and weigh the perfused muscle tissue.
In each animal the number of circulating
leukocytes was counted (Unopette, Becton Dickinson, Franklin Lakes, NJ).
Injection of SHR blood into normal rats
Whole blood with or without 0.1 µM
cytochalasin-D (to prevent pseudopod formation) from SHR (n=6) or WKY (n=6) was
injected into normal recipient Wistar (4.5 ml/kg, n=24), while MABP was measured. In
selected blood samples leukocytes were removed after centrifugation (at 300 g for 5
min). Cytochalasin-D was applied to the blood samples for 15 min immediately after the
blood collection.
MABP after blood exchange between WKY and SHR Between WKY and SHR (n=5 in
each group), or WKY and WKY (n=6), 10 ml of arterial blood was exchanged every 5
min for 5 times, and MABP was measured 5 min after each blood exchange.
Leukocyte passage through an in vitro microchannel flow array: Due to the strong
tendency for rat blood to coagulate, human blood was used in this study. Human blood
with 30 U/ml ammonium heparin from volunteers was diluted in physiological saline
with 1mM MgSO4 (1:1), and divided into 5 groups; unsheared and sheared control
samples, 1 µM dexamethasone-treated blood without and with shear application, and
dexamethasone-treated blood with 5 mM EDTA (Sigma) with shear exposure.
One-minute after blood collection, the sheared groups were exposed to a shear
stress of 5.0 dyn/cm2 for 3 or 13 min in a cone-and-plate device. . Four-, 14-, and 19-min
after blood collection, the passage time of 0.1 ml blood through the Microchannel Flow
Analyzer (Kowa Co. Ltd.) was determined (11). The array of identical microchannels
(equivalent diameter 5 µm, length 20 µm, 3100 parallel channels) was examined under
constant suction (negative pressure of -15, -20, and -25 cmH2O). Light microscopic
images of the blood passage through the microchannels were recorded on videotapes with
a charge-coupled–device camera (HV-D28S, Hitachi, Tokyo) for off-line analysis.
Fractions of occluded microchannels due to leukocyte plugging and leukocytes with
pseudopods were evaluated 14-min after blood collection.
Statistics:
All values are shown as mean ± standard deviation. Differences between
groups were analyzed by ANOVA and Fischer's protected least significant difference
test. p<0.05 was considered significant.
References for Supplemental Methods
1. Fukuda S, Schmid-Schönbein GW. Centrifugation attenuates the fluid shear response
of circulating leukocytes. J Leukoc Biol. 2002;72:133-139.
2. Fukuda S, Yasu T, Predescu DN, Schmid-Schönbein GW. Mechanisms for regulation
of fluid shear stress response in circulating leukocytes. Circ Res. 2000;86:E13-E18.
3. Moazzam F, Delano FA, Zweifach BW, Schmid-Schönbein GW. The leukocyte
response to fluid stress. Proc Natl Acad Sci USA. 1997;94:5338-5343.
4. Eppihimer MJ, Lipowsky HH. Leukocyte sequestration in the microvasculature in
normal and low flow state. Am J Physiol. 1994;267: H1122-H1134.
5. Yasu T, Schmid-Schönbein GW, Cotter B, DeMaria AN. Flow dynamics of QW7437,
a new dodecafluoropentane ultrasound contrast agent, in the microcirculation. J Am
Coll Cardiol. 34:578-586.
6. Helmke BP, Bremner SN, Zweifach BW, Skalak R, Schmid-Schönbein GW.
Mechanisms for increased blood flow resistance due to leukocytes. Am J Physiol.
1997;273:H2884-H2890.
7. Helmke BP, Sugihara-Seki M, Skalak R, Schmid-Schönbein GW. A mechanism for
elevation of erythrocyte apparent viscosity by leukocytes in-vivo without adhesion to
the endothelium. Biorheology. 1998;35:437-448.
8. Sutton DW, Schmid-Schönbein GW. Elevation of organ resistance due to leukocyte
perfusion. Am J Physiol. 1992;262:H1646-H1650.
9. Sutton DW, Schmid-Schönbein GW. Hemodynamics at low flow in resting
vasodilated rat skeletal muscle. Am J Physiol. 1989;257:H1419-H1427.
10. Harris AG, Skalak TC. Leukocyte cytoskeletal structure determines capillary
plugging and network resistance. Am J Physiol. 1993;265: H1670-H1675.
11. Kikuchi Y. Effect of leukocytes and platelets on blood flow through a parallel array
of microchannels: micro- and macroflow relation and rheological measures of
leukocyte and platelet activities. Microvasc Res. 1995;50:288-300.