YMVRE-03087; No. of pages: 10; 4C:
Microvascular Research xxx (2011) xxx–xxx
Contents lists available at ScienceDirect
Microvascular Research
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y m v r e
Temporal variations of the cell-free layer width may enhance NO bioavailability in
small arterioles: Effects of erythrocyte aggregation
Peng Kai Ong, Swati Jain, Sangho Kim ⁎
Division of Bioengineering & Department of Surgery, National University of Singapore, Singapore
a r t i c l e
i n f o
Article history:
Received 2 December 2010
Revised 11 February 2011
Accepted 12 February 2011
Available online xxxx
Keywords:
Nitric oxide
Microcirculation
Wall shear stress
Vasodilation
a b s t r a c t
Recently, we have shown that temporal variations in the cell-free layer width can potentially enhance nitric
oxide (NO) bioavailability in small arterioles. Since the layer width variations can be augmented by red blood
cell aggregation, we tested the hypothesis that an increase in the layer width variations due to red blood cell
aggregation could provide an underlying mechanism to improve NO bioavailability in the endothelium and
promote vasodilatory effects. Utilizing cell-free layer width data acquired from arterioles of the rat cremaster
muscle before and after dextran infusion in reduced flow conditions (wall shear stress = 0.13–0.24 Pa), our
computational model predicted exponential enhancements of NO bioavailability in the endothelium and
soluble guanylyl cyclase (sGC) activation in the smooth muscle layer with increasing temporal variability of
the layer width. These effects were mediated primarily by the transient responses of wall shear stress and NO
production rate to the layer width variations. The temporal variations in the layer width were significantly
enhanced (P b 0.05) by aggregation, leading to significant improvements (P b 0.05) in NO bioavailability and
sGC activation. As a result, the significant reduction (P b 0.05) of sGC activation due to the increased width of
the layer after aggregation induction was diminished by the opposing effect of the layer variations. These
findings highlighted the possible enhancement of NO bioavailability and vascular tone in the arteriole by the
augmented layer width variations due to the aggregation.
© 2011 Elsevier Inc. All rights reserved.
Introduction
The overall nitric oxide (NO) bioavailability in the arterioles is a
function of NO synthesis in the endothelium and NO degradation by
various NO sinks in the surrounding tissues that include the reaction
with superoxides, oxygen or free radicals (Vallance and Leiper, 2002),
the binding to heme-containing proteins (Buerk, 2001) and the
scavenging by red blood cells (Butler et al., 1998; Lancaster, 1994; Liao
et al., 1999). NO production in the arteriolar endothelium is
dependent on fluid shear stress acting on the luminal wall of the
vessel. In the microvessels, red blood cells tend to migrate away from
the vessel wall, leading to formation of a plasma layer (cell-free layer)
adjacent to the luminal vessel wall. The extent of this phase separation
phenomenon can be enhanced by red blood cell aggregation, resulting
in formation of a thicker cell-free layer with greater variability in its
width (Alonso et al., 1995; Cokelet and Goldsmith, 1991; Ong et al.,
2010b). The cell-free layer is capable of modulating the shear stress
exerted by blood flow on the endothelium, which in turn alters the
molecular mechanisms involved in NO production (Gori and Forconi,
2007).
⁎ Corresponding author at: Division of Bioengineering, Faculty of Engineering
National University of Singapore 9 Engineering Drive 1, Block EA #03-12 117576,
Singapore. Fax: +65 6872 3069.
E-mail address: [email protected] (S. Kim).
Previous theoretical studies (Chen et al., 2006; Lamkin-Kennard
et al., 2004) have introduced numerical models that simulate the NO
transport in the arteriolar blood flow with existence of the cell-free
layer. However, those studies did not consider temporal variations of
model parameters such as the cell-free layer, wall shear stress, and
core hematocrit. During blood flow in arterioles, the cell-free layer
formation is highly dynamic (Kim et al., 2007), resulting in a timevarying diffusion barrier that would be capable of modulating both
the rates of NO production and NO scavenging by changing the wall
shear stress and core hematocrit temporally. Therefore, it would be
important to consider temporal changes of the model parameters
resulting from the variations of the cell-free layer width. In our recent
simulation study (Ong et al., 2010a), we have shown that the
temporal variations in the cell-free layer width may enhance NO
preservation in small arterioles primarily by mediating the variations
in wall shear stress. In our earlier experimental study (Ong et al.,
2010b), we showed that temporal variations of the layer width in
small arterioles became significantly larger due to red blood cell
aggregation.
In this study, we tested the hypothesis that the increase in the
temporal variations of the cell-free layer width by the aggregation
could produce a greater enhancement of NO bioavailability in the
arterioles. This in turn could potentially affect the autoregulation of
blood flow in the arterioles by enhancing vasodilation. In vivo cell-free
layer data (Ong et al., 2010b) reported previously together with new
0026-2862/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.mvr.2011.02.002
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
Materials and methods
Some of the in vivo cell-free layer data used in this study were
acquired from our previous study (Ong et al., 2010b). Additional
data were used to increase the number of data sets for analyses. The
cell-free layer widths and flow parameters in unbranched arteriolar
vessel segments in reduced flow conditions both before and after
dextran infusion were utilized in the current study. Brief descriptions of the animal preparation and experimental protocol are
provided below.
around the abdominal aorta using an air-filled syringe. The reduction
of arterial pressure creates a reduced flow situation that would be
more favorable for formation of red blood cell aggregates in the
arteriolar flows and thus aggregation effects become more distinct
A
8
data sets obtained from arterioles of the rat cremaster muscle were
applied to our computational model of NO transport in an arteriole. To
examine the effect of red blood cell aggregation on NO bioavailability,
two scenarios were considered, one (CASE I) where no temporal
changes in physiological responses (NO production and red blood cell
scavenging) occur and the other (CASE II) where both physiological
responses occur in synchronization with the layer width variations.
Tissue
y3
y2
y1
Endothelium
CFL
y (µm)
2
Blood Lumen
Animal preparation
Data from 11 male Wistar–Furth rats (202.8 ± 12.9 g) were used in
this study. All animal handling and care procedures were in
accordance with that outlined in the Guide for the Care and Use of
Laboratory animals (Institute for Laboratory Animal Research,
National Research Council, Washington, DC: National Academy
Press, 1996) and approved by the local Animal Subjects Committee.
A detailed description of the animal preparation procedures can be
referred to our prior study (Kim et al., 2007). In brief, the rat cremaster
muscle was exposed for the visualization of arteriolar blood flow.
Vessel center
0
0.0
Experimental protocol
1.0
1.5
2.0
Time (s)
B
y2
y1
y (µm)
Physiological and rheological parameters
HC
0
Hematocrit (%)
C
y2
y1
k = 2.8
y (µm)
A blood sample (~0.1 ml) was withdrawn from the femoral artery
for hematocrit and aggregation measurements. Hematocrit was
determined with a microhematocrit centrifuge (Readacrit, Clay
Adams) while the degree of red blood cell aggregation was measured
periodically during the experiment using an aggregometer (Myrenne
aggregometer; Myrenne, Roetgen, Germany). Arterial pressure was
continuously recorded with a physiological data-acquisition system
(MP100, BIOPAC Systems, Goleta, CA).
Red blood cell aggregation was elevated to levels observed in
healthy humans by infusion of Dextran 500 (average molecular mass
460 kDa; Sigma) dissolved in saline (6%). A total of 200 mg/kg body
wt. was infused over the course of 1–2 min to achieve a plasmadextran concentration of ~0.6%. Another blood sample (~ 0.1 ml) was
withdrawn for hematocrit and aggregation level determination
~ 10 min after the dextran infusion.
The centerline velocity (Vc) was obtained from high-speed video
recordings by the dual-window method via a video sampler (Model
204A, Vista Electronics) and velocity correlator system (Model 102BC,
Vista Electronics) (Baker and Wayland, 1974; Intaglietta et al., 1975;
Wayland and Johnson, 1967). The edge velocity (Vedge) of the red
blood cell core was determined by tracking movements of outermost
cells manually across 10 digital frames using image analysis software
(SigmaScan Pro 5) (Namgung et al., 2011). Pseudoshear
rate γ̇ used in
this study was defined by the relation: γ̇ = Vc D where D is vessel
diameter.
0.5
k = 2.0
0
Velocity (mm/s)
The rat was mounted on the microscopic stage and an unbranched
region of an arteriole with an inner diameter ranging from 45 to 55 μm
was chosen for the study based on the criteria of stable flow, clear
focus and good contrast of the image. Blood flow in the arteriole was
reduced by increasing the pressure in the pneumatic cuff pre-inserted
Fig. 1. (A) Computational model of an arteriole in a Cartesian coordinate system which
consists of blood lumen, cell-free layer (CFL), endothelium and tissue. Temporal
variations in the blood lumen-CFL border (y1) for a time period of 2 s are shown.
(B) Hematocrit profile. Hc is the hematocrit in the blood lumen. (C) Velocity profile
before (k = 2.0) and after (k = 2.8) dextran infusion.
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
3
(Ong et al., 2010b). An intravital microscope (Ortholux II, Leitz) was
used in conjunction with a water immersion objective (40×,
Olympus) and a long working distance condenser (Instec, Boulder,
CO) with numerical apertures of 0.7 and 0.35, respectively. In
addition, a blue filter (transmission peak 400 nm, model no. 59820)
was used to enhance light absorption by the red blood cells to
improve the contrast between the cells and background. A high-speed
video camera (FASTCAM ultima SE, Photron USA) was utilized to
record blood flow in the arteriole at a frame rate of 2250/s. A total of
4500 frames were obtained for a vessel which corresponded to a video
recording time of 2 s. The above protocol was repeated after the
Dextran 500 infusion.
In addition, NO concentration is assumed to be symmetrical about
the vessel center at y = 0 and an asymptotic NO concentration is
assumed far away from the vessel as follows:
Cell-free layer width measurement
At y = y2 ; qCFLjEC;NO = DCFL
dCCFL;NO
dCEC;NO
−DEC
;
dy
dy
The cell-free layer width was defined by the distance from the
edge of the red blood cell core to the inner wall of the vessel. The
procedures for determination of the temporal layer width variations
were reported in previous studies (Kim et al., 2006, 2007) and will not
be repeated here. The spatial resolution of this cell-free layer
measurement was ~0.4 μm with the current system.
At y = y3 ;
dCEC;NO
dCT;NO
−DT
dy
dy
Mathematical model
The transient computational model used in this study follows the
simulation procedure reported in our previous study (Ong et al.,
2010a) and only a brief description of the model is provided here. As
shown in Fig. 1A, the model consists of four compartments (from the
vessel center outward) representing the blood lumen (LU), cell-free
layer (CFL), endothelial cell layer (EC) and tissue layer (T). A Cartesian
coordinate system is used to describe NO transport in the radial
direction (y) of the arteriole. Convective transport of nitric oxide (NO)
is neglected as described in previous studies (Kavdia and Popel, 2003;
Sriram et al., 2010). Therefore, our computational model can be
simplified to a system of transient reaction–diffusion equations. The
general form of this equation is shown in Eq. (1).
∂CC;NO
∂ CC;NO
−kC;NO CC;NO
= DC
2
∂t
∂y
2
ð1Þ
where CC,NO is the concentration of NO, DC is the diffusion
coefficient and kC,NO is the NO reaction rate constant in the
compartment (c) where applicable. The left hand term of the equation
accounts for the time dependent change in CC,NO while the first and
second terms on the right hand side of the equation depict the
diffusion and reaction of NO, respectively in the compartment. For
simplicity, the same diffusion coefficient is used in all compartments
(Lamkin-Kennard et al., 2004). A 1st order reaction rate constant is
used to describe the loss of NO by various sinks in the different
compartments (Chen et al., 2006; Vaughn et al., 1998) except in the
CFL where no biochemical reaction is assumed to occur due to the
absence of red blood cells (Lamkin-Kennard et al., 2004).
Continuity of NO concentration and conservation of NO mass
applies at the boundaries of all compartments so that
At y = y1 ; CCFL;NO = CLU;NO ; DNO
∂CCFL;NO
∂CLU;NO
= DNO
;
∂y
∂y
At y = y2 ;
CEC;NO = CCFL;NO ;
DNO
∂CEC;NO
∂CCFL;NO
= DNO
∂y
∂y
At y = y3 ;
CT;NO = CEC;NO ;
DNO
∂CT;NO
∂CEC;NO
= DNO
:
∂y
∂y
ð2Þ
At y = 0;
dCLU;NO
= 0;
dy
At y→∞;
dCT;NO
= 0:
dy
ð3Þ
NO production occurs at both CFL-EC and EC-T borders and equal
production rate is assumed at each border (Vaughn et al., 1998).
qECjT;NO = DEC
ð4Þ
where qCFL|EC,NO and qEC|T,NO are NO production rates at the luminal
and abluminal boundaries of the endothelium, respectively.
The hematocrit (Hc) in the blood core and shear stress (τw) acting
on the luminal surface of the endothelial cell layer are assumed to vary
with the temporal variation in the cell-free layer width (Ong et al.,
2010a). Therefore, both NO scavenging rate of the blood core and NO
production rate in the endothelium vary with instantaneous changes
in the layer width. To obtain Hc at a given time point, we consider the
mass conservation of red blood cells in Hc and systemic hematocrit
(HSYS) using Eq. (5) (Chen et al., 2006; Sriram et al., 2010).
Mass balance equation:
y
y
2π∫02 HðyÞVðyÞydy = HSYS 2π∫02 VðyÞydy
ð5Þ
where H(y) and V(y) refer to the hematocrit and velocity profiles,
respectively.
Unlike our previous simulation study (Ong et al., 2010a), in this
study, reduced flow conditions have been used to promote red blood
cell aggregation in the arteriolar flows. Thus, several modifications
have been made in the velocity and hematocrit profiles to better
simulate the rheological characteristics of blood under low shear
conditions before and after dextran infusion. A step hematocrit
function (Eq. (6)) (Chen et al., 2006; Lamkin-Kennard et al., 2004) is
used for both conditions, which depicts the absence of red blood cells
in the cell-free layer (see Fig. 1B).
Hematocrit function:
HðyÞ = Hc for 0≤y≤y1 ;
HðyÞ = 0 for y1 by≤y2
ð6Þ
where y1 is the interface between the cell-free layer and red blood cell
core and y2 is the location of the luminal vessel wall.
Velocity function:
"
k #
y
for 0≤y≤y2
VðyÞ = Vc 1−
y2
ð7Þ
where k is a bluntness parameter that describes the shape of the
velocity profile. k = 2 denotes a parabolic profile whereas k N 2 refers
to a blunted profile. Based on the experimental findings by Bishop and
coworkers (Bishop et al., 2001), in this study as shown in Fig. 1C, k
values of 2 and 2.8 have been used to describe the shape of the
velocity profiles of non-aggregating and aggregating blood flows in
low shear conditions, respectively. Using the experimental data of the
cell-free layer widths (y2 − y1) at incremental time points and mean
centerline flow velocity (Vc), temporal variations in Hc can be
obtained.
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
4
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
A transient profile of NO scavenging rate can then be derived using
Eq. (8) (Lamkin-Kennard et al., 2004).
kLU;NO =
Hc
× kSYS;NO
HSYS;ref
ð8Þ
where kSYS,NO refers to the reference NO scavenging rate by red
blood cells defined at HSYS,ref = 45%.
Wall shear stress (τw) at a particular time point can be obtained by
using Eq. (9). This form of τw determination has been validated and
reported in our previous study (Namgung et al., 2011).
τw = γ̇w × μ p
ð9Þ
where γ̇w and μp are the wall shear rate and plasma viscosity,
respectively. γ̇w is derived from Eq. (10) by assuming a linear velocity
gradient in the cell-free layer. The same magnitude of μp is used before
and after dextran infusion, neglecting the change in μp that should
result from dextran intervention. Therefore, the simulation results
obtained in this study would not be directly affected by plasma
viscosity changes.
γ̇w =
Vedge
:
y2 −y1
ð10Þ
By repeating the same procedure for individual cell-free layer
widths at consecutive time points, the temporal variation in τw can be
derived. Experimental data suggest that the rate of NO production
increases with the shear stress acting on the endothelium (Kanai et al.,
1995; Kuchan and Frangos, 1994; Wessells et al., 2006) and this
relation appears to be linear (Ando and Yamamoto, 2010). Thus, in
this study, the wall shear stress was assumed to vary linearly with NO
production as shown in Eq. (11). A transient profile in NO production
rate (qCFL|EC,NO) can then be obtained using Eq. (11) (Kavdia and
Popel, 2003).
qCFLjEC;NO =
τw
× qNO;control
τw;ref
ð11Þ
where τw,ref is defined as the reference wall shear stress that leads
to basal rate of NO production (qNO,control) at each boundary source. In
the case where red blood cells came into contact with the wall, τw was
assigned as the reference value.
The smooth muscle layer is assumed to be 6-μm thick and
incorporated as part of the tissue layer, adjacent to the endothelium.
Using the averaged NO concentration (CSM,NO) obtained in this layer,
the NO activation of sGC (sGC Activation) can be calculated as follows
(Kavdia and Popel, 2003):
n
CSM;NO
n
sGC Activationð%Þ ¼ × 100%
CSM;NO + ðKm Þn
ð12Þ
where Km and n are the apparent Michealis constant and Hill
coefficient, respectively. On the basis of a previous work (Condorelli
and George, 2001), Km and n are 23 nM and 1.3, respectively. Thus, sGC
Activation could be assumed to be half maximum (50%) at a CSM,NO of
23 nM. Table 1 summarizes the model parameters used in this
simulation.
Numerical solution
The set of linear partial differential equations for time-dependent
NO transport in the arteriole was solved to find mean peak NO
concentrations using the finite difference method. A grid with 100
points was used for each compartment and could be adjusted
accordingly to better define regions with steeper concentration
gradients. A finite distance of 2500 μm was used to simulate an
infinite domain (y = ∞). The validity of the numerical method and the
grid independence of the solution have been ensured in our earlier
study (Ong et al., 2010a). Initial value conditions for this transient
problem were obtained by first simulating NO concentration profile to
steady state based on a constant mean magnitude of the cell-free layer
width.
Statistical analysis and data presentation
Temporal variations in the cell-free layer width were characterized
×
by the coefficient of variation (Cv) such that CV ð%Þ = CFL sd CFL
mean
100% where CFLsd and CFLmean are the standard deviation and mean of
the layer widths, respectively. Statistical analyses including regression
fits of the experimental data were performed using a statistical
software package (Prism 4.0, Graphpad). Unpaired t-tests were used
to determine differences in responses with or without association of
the layer width variations. A one-way ANOVA test with post
Bonferroni and Tukey tests were utilized to compare two or more
groups (with or without the layer width variations, before and after
aggregation induction). All physiological and rheological data were
reported as means ± SD. For all statistical tests and regression fits,
P b 0.05 was regarded statistically significant.
Table 1
Parameters used in the computational model.
Parameters
Symbol used
Value
Units
Source
Blood lumen width
Cell-free layer width
Vessel radius
Edge velocity of blood core
Centerline velocity
Systemic hematocrit
Bluntness parameter of velocity profile
Endothelial cell width
Tissue layer width
Apparent Michealis constant
Control NO production rate
Diffusion coefficient in all compartments
Hill coefficient
NO scavenging rate at HSYS,ref 45%
NO scavenging rate in endothelium
NO scavenging rate in tissue
Plasma viscosity
Reference wall shear stress
y1
y 2 − y1
y2
Vedge
Vc
HSYS
k
y 3 − y2
y(∞) − y3
Km
qNO,control
Dc
n
kSYS,NO
kEC,NO
kT,NO
μp
τw,ref
–
μm
μm
μm
mm s− 1
mm s− 1
%
–
μm
μm
μM
μmol μm− 2 s− 1
μm2 s− 1
–
s− 1
s− 1
s− 1
cP
Pa
Previous (Ong et al., 2010b) and new
experimental data
2 and 2.8
2.5
2500
0.023
2.65 × 10− 14
3300
1.3
382.5
0.1
0.1
1.2
2.4
Bishop et al. (2001)
Kuo et al. (1990)
Vaughn et al. (1998)
Kavdia and Popel, (2003)
Vaughn et al. (1998)
Vaughn et al. (1998)
Kavdia and Popel (2003)
Chen et al. (2006)
Lamkin-Kennard et al. (2004)
Lamkin-Kennard et al. (2004)
Zhang et al. (2009)
Kavdia and Popel (2003)
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
5
Results
Effect of dextran infusion on the layer width variations
Physiological and rheological parameters
Figs. 2A and B show a typical example of temporal variation of the
cell-free layer width in an arteriole (ID = 46.6 μm) before and after
dextran infusion, respectively at pseudoshear rates of 19.1 ± 0.5 s− 1.
Both CFLmean and CFLsd increased after dextran infusion. However,
since the percentage increase in CFLsd was ~ 2 folds larger than that in
CFLmean, an augmentation in Cv was obtained after dextran treatment.
When the cell-free layer width data from all the arterioles were
combined (Fig. 2C), an overall significant augmentation (P b 0.05) in
mean Cv from 41.2 ± 8.8% to 53.1 ± 12.5% was observed after dextran
treatment. The significantly larger mean Cv values after dextran
infusion indicate that the layer width variations can be enhanced by
aggregation induction.
16
CFLmean = 2.73 µm
CFLsd = 1.21 µm
Cv = 44.3%
14
Edge of red blood cell core
CFLmean
12
10
8
6
4
2
0
0.0
0.5
1.0
1.5
2.0
Time (s)
Distance from wall (µm)
B
16
14
CFLmean = 3.22 µm
CFLsd = 1.64 µm
Cv = 50.9%
A
14
13
12
Δ+
11
Mean Peak NONSS
10
Mean Peak NOSS
9
8
7
6
0.0
12
0.5
1.0
10
8
B
6
4
2
0
0.0
0.5
C 100
1.0
1.5
2.0
*
80
60
40
2.0
2.5
2.0
2.5
14
13
12
11
Δ+
10
Mean Peak NONSS
9
8
Mean Peak NOSS
7
6
0.0
0.5
1.0
1.5
Time (s)
20
0
1.5
Time (s)
Time (s)
Mean CV (%)
Fig. 3 shows simulated changes of mean peak NO concentration in
an arteriole in response to a temporal varying layer width (A) before
and (B) after dextran treatment. In both cases, mean peak NO
concentration (Mean Peak NOss) was initially maintained at a steady
state value when subjected to a constant layer width from 0 to 0.5 s.
Mean Peak NOss was obtained by carrying out the NO simulation
based on the mean value of all temporal layer width measurements
until the NO concentration reaches a steady value. When physiological responses were introduced into the transient model (0.5 to 2.5 s),
Peak NO concentration (nM)
Distance from wall (µm)
A
Influence of the layer width variations on NO bioavailability
Peak NO concentration (nM)
After the flow reduction in the abdominal aorta, mean arterial
pressures in the femoral artery for the rats used for this study
became 43.5 ± 8.1 and 44.3 ± 6.2 mm Hg before and after dextran
infusion, respectively whereas corresponding hematocrits were
40 ± 2% and 38 ± 1%, respectively. The steady-state mean wall
shear stress values were 0.19 ± 0.04 and 0.17 ± 0.03 Pa before and
after dextran infusion, respectively. These parameters were not
significantly different before and after dextran treatment. Red blood
cell aggregation degree index (M) was elevated from 0.0 to 11.8 ±
2.8 after dextran infusion, which is similar to levels reported for
healthy humans (Lee et al., 2007; Vaya et al., 2003). The respective
mean pseudoshear rates and mean edge velocities at reduced
arterial pressures were 19.1 ± 0.3 s− 1 and 0.45 ± 0.2 mm/s before
dextran infusion and 18.9 ± 0.3 s− 1 and 0.43 ± 0.2 mm/s after
dextran infusion. No significant effect of dextran treatment on
both mean pseudoshear rates and mean edge velocities was found.
The diameter of the arterioles was 47.2 ± 0.8 (N = 11) and 46.6 ±
0.8 μm (N = 10) before and after dextran infusion, respectively with
no significant difference.
Before dextran infusion
After dextran infusion
Fig. 2. (A) and (B) A typical example of temporal variations in the cell-free layer width
in an arteriole (46.6 μm) before and after dextran infusion, respectively. (C) Effect of red
blood cell aggregation on mean Cv. *P b 0.05.
Fig. 3. (A) and (B) Simulated peak NO concentration profile in an arteriole (46.6 μm) in
response to temporal changes in the cell-free layer width before and after dextran
treatment, respectively. Mean Peak NOSS and Mean Peak NONSS denote the averaged
peak NO concentration based on a constant layer width (0 to 0.5 s) and a varying layer
width (0.5 to 2.5 s), respectively. Δ+ corresponds to an increase in mean peak NO
concentration.
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
an augmentation in mean peak NO concentration (Mean Peak NONSS)
from 11.2 to 12.2 nM and from 9.5 to 10.9 nM was obtained before
and after dextran treatment, respectively. The magnitude of this
increase was found to be greater after dextran treatment (14.2%) than
before (9.1%). It is of note that mean peak NO concentration
consistently decreased after dextran treatment under the effects of
both constant and temporally varying layer widths.
To examine the effect of the layer width variations on NO
bioavailability, Mean peak NONSS was compared with Mean peak
NOSS. Any discrepancy in mean peak NO concentration (ΔMean Peak NO) between the two could be obtained as follows:
ΔMean Peak NO ð%Þ ¼
A
Δ Mean Peak NOCASE II (%)
6
Mean Peak NONSS −Mean Peak NOSS
× 100%: ð13Þ
Mean Peak NOSS
50
Before dextran infusion
After dextran infusion
40
30
20
10
0
0
20
40
60
80
CV (%)
CV (%)
0
20
40
60
80
Δ Mean Peak NOCASE I (%)
0
-2
B
Mean Peak NO (nm)
For CASE I (Fig. 4), the negative values of ΔMean Peak NO indicated
an attenuation of mean peak NO concentration in the arterioles when
temporal variations in the layer width were considered. Based on
linear regression, this diminution effect was significantly enhanced
with increasing Cv for both before (P b 0.0001) and after (P b 0.002)
dextran infusion. Although Cv values were significantly larger after
dextran infusion, the extent of NO diminution by the layer width
variations was not significantly (P = 0.65) altered by aggregation
induction. Mean magnitude of ΔMean Peak NO was −2.7 ± 1.4% and
−3.0 ± 1.4% before and after dextran infusion, respectively.
This diminution effect was however offset when transient wall
shear stress responses to the layer width variations were considered
(CASE II), resulting in an exponential enhancement of NO bioavailability with increasing Cv (Fig. 5A). Based on the curve fits, the ΔMean
Peak NO increased from 4.6% to 33.9% in the Cv range of 29.1%–56.2%
before dextran infusion while it increased from 8.2% to 35.2% in the
range of 33.9%–75.4% after dextran treatment. Although Cv values
became significantly larger after aggregation induction, the maximum
ΔMean Peak NO in the arterioles appeared to be slightly increased by
aggregation. However, a pronounced enhancement of mean peak NO
concentration in the arterioles by the layer width variations under
aggregating conditions was apparent in Fig. 5B. Accordingly, only after
dextran infusion, mean peak NO concentration obtained with a
transiently changing layer was significantly larger (P b 0.05) than that
acquired with a steady state layer. In addition, mean peak NO
15
Steady state
Transient
*
10
5
0
Before dextran infusion
Fig. 5. (A) Relationship between ΔMean Peak NO and Cv before and after dextran
infusion in CASE II. Exponential curve fit was used: y = 0.33e0.08x; R2 = 0.88 and
y = 1.75e0.04x; R2 = 0.87 before and after dextran treatment, respectively. (B) Effect of
transient changes in the layer width on mean peak NO concentration before and after
dextran infusion. *P b 0.05.
concentration was generally lowered after dextran infusion for both
steady state (9.4 ± 1.2 → 8.7 ± 0.8 nM) and transient (10.7 ±
2.0 → 10.2 ± 1.8 nM) cases of the layer width.
Influence of the layer width variations on sGC activity
The effect of the layer width variations on vascular tone can be
predicted from the level of sGC activation in the smooth muscle cells.
An increase in sGC activation is associated with enhanced smooth
muscle relaxation which is capable of eliciting vasodilatory effects.
ΔsGC activation was defined as the percentage change in sGC
activation comparing the effects of transient (sGC ActivationNSS) and
steady state (sGC ActivationSS) layer widths on sGC activation as
follows:
ΔsGC Activationð%Þ ¼
-4
-6
Before dextran infusion
-8
After dextran infusion
Fig. 4. Relationship between ΔMean Peak NO and Cv before and after dextran infusion in
CASE I. Linear regression fit was used: y = − 0.14x + 3.03; R2 = 0.82 and y = − 0.09x +
1.95; R2 = 0.72 before and after dextran treatment, respectively.
After dextran infusion
sGC ActivationNSS −sGC ActivationSS
× 100%:
sGC ActivationSS
ð14Þ
As shown in Fig. 6A, positive magnitudes of ΔsGC activation were
generally obtained, implying that sGC activity could be enhanced by
temporal variations in the layer width. Similar to Fig. 5A, sGC
activation increased from 6.5% to 27.7% in the Cv range of 29.1%–
56.2% before dextran infusion whereas it increased from 9.0% to 28.6%
in the range of 33.9%–75.4% after dextran infusion. The direct effect of
aggregation on sGC activation was not clearly seen in Fig. 6A.
However, as shown in Fig. 6B, mean sGC activation obtained with a
transient layer width was significantly augmented (P b 0.05) from that
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
Δ sGC Activation (%)
A
40
7
flow stream. Microscopic observations of temporal and spatial
changes in the cell-free layer formation for aggregating blood flow
(Maeda et al., 1996; Ong et al., 2010b; Reinke et al., 1987; Soutani
et al., 1995; Suzuki et al., 1996) have shown more irregular contours
of the cell-free layer interface with the red blood cell core after flow
reduction. In the present study, a significantly larger mean Cv was
obtained after aggregation induction, indicating an increase in the
temporal variation of the layer width due to aggregation. In addition,
our finding suggested that the standard deviation of the layer width
could be a more important statistical parameter than its mean in
defining the temporal changes of the layer width since an overall
increase in C v due to aggregation was found even with an
augmentation in mean layer width.
Before dextran infusion
After dextran infusion
30
20
10
0
0
20
40
60
80
CV (%)
B
40
sGC Activation (%)
35
30
Steady state
Transient
*
*
25
20
15
10
5
0
Before dextran infusion
After dextran infusion
Fig. 6. (A) Relationship between ΔsGC activation and Cv before and after dextran
infusion. Exponential curve fit was used: y = 0.69e0.07x; R2 = 0.83 and y = 2.66e0.03x;
R2 = 0.79 before and after dextran treatment, respectively. (B) Effect of transient
changes in the layer width on sGC activation before and after dextran infusion. *P b 0.05.
with a steady state layer width after dextran infusion, but not before.
It is of note that sGC activation generally decreased after dextran
treatment (24.0 ± 3.1% → 20.7 ± 2.2% and 26.9 ± 4.8% → 23.9 ± 3.8%
for steady state and transient layer widths, respectively) and the
magnitude of this mitigation was found to be statistically significant
(P b 0.05) only for the steady state layer width.
Discussion
Principal findings
The induction of red blood cell aggregation in arteriolar blood flow
at reduced flow rates can augment the temporal variations in the cellfree layer width and this effect can contribute to the enhancement of
NO bioavailability in the arteriole. This improvement in NO bioavailability by the layer width variations may present a mechanism for
altering the local vascular tone since reduction of sGC activation due
to the increased width of the layer after aggregation induction was
diminished by the effect of the layer variation.
Effect of aggregation on temporal variations of cell-free layer
It is well established that in microcirculatory blood flow, red blood
cells are not homogeneously distributed over the cross-section of the
vessel, but the cells tend to migrate towards the center of the vessel.
When red blood cell aggregation is induced, this axial migration can
be enhanced by the growth of compact multicellular aggregates in the
Influence of the layer variations on NO bioavailability
The enhanced cell-free layer formation due to the red blood cell
aggregation at reduced flow rates could lead to opposing influences
on NO bioavailability in the arteriole. A thicker cell-free layer could
lower the effective blood viscosity at the luminal vessel wall and thus
reduce the endothelial production of NO by attenuating the wall shear
stress (Baskurt and Meiselman, 2007). Furthermore, the thicker layer
corresponds to a reduction in red blood core width, resulting in a
more concentrated red blood cell core (higher Hc) that potentially
enhances NO scavenging and attenuates NO bioavailability (Azarov
et al., 2005). Conversely, an increase in the layer width can improve
NO preservation in the arteriole by creating a larger diffusion barrier
that protects NO in the endothelium from scavenging by the blood
lumen (Liao et al., 1999). The present study suggests an alternative
mechanism that could enhance NO bioavailability by considering the
concomitant changes in wall shear stress and NO synthesis with the
enhanced temporal variation of the layer due to aggregation. The
concept that transient production of NO may enhance NO bioavailability has been previously modeled by Tsoukias et al. (2004). In their
model, the endothelium is assumed to have a time-varying NO
production rate which supports the possible enhancement of NO
bioavailability by the time-varying cell-free layer.
A flow dependent effect on the extent of NO preservation by the
layer width variations was apparent by comparing the results in this
study with those obtained in our previous study under high flow
conditions (Ong et al., 2010b). In contrast to the linear augmentation
of mean peak NO concentration with the layer variability obtained at
high flow rates, an exponential increase was found at low flow rates in
the present study, suggesting a larger possible enhancement of NO
bioavailability in the arterioles by the layer width variations under
reduced flow conditions.
Although NO bioavailability could be enhanced by a larger
temporal variability of the layer width after aggregation induction,
such an effect was secondary to the attenuation of NO imposed by
formation of a thicker layer. As shown in Fig. 5B, mean peak NO
concentration in the arteriole was reduced after dextran treatment
even though a greater improvement in NO concentration was
obtained with larger temporal variations in the layer width. This
finding is consistent with previous experimental studies (Baskurt
et al., 2004; Yalcin et al., 2008) which reported a downregulation in
the expression of eNOS protein or relevant markers on NO synthesis
in the vascular wall after aggregation induction without an
elevation of plasma viscosity. A predominant reduction in NO
bioavailability due to an increase in the layer width may have
important clinical implications by triggering non-hemodynamic
related mechanisms. For instance, reduced vascular NO bioavailability may stimulate platelet adhesion and aggregation that could
impair the capacity of vascular endothelium to react to vasodilator
stimuli, leading to prognosis of vascular diseases (Gori and Forconi,
2007).
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
8
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
Comparison of NO values with previous models and experimental studies
The steady-state NO values predicted from the present model are
lower than those predicted by previous NO transport models. Direct
comparison between steady-state NO values obtained by our model
and previous models would be limited by the different methods used
to compute the NO production rate. NO production rate in previous
models (Chen et al., 2006; Lamkin-Kennard et al., 2004) is dependent
on O2 availability in the arteriole based on the Michaelis–Menten
kinetics. On the other hand in our model, NO production rate is a
function of the shear stress at the arteriolar wall and thus it is directly
dependent on the wall shear rate which is determined by experimental hemodynamic parameters such as the cellular velocity at the
edge of the blood core and the thickness of the cell-free layer. Since
our study is conducted under reduced flow conditions (~17 s− 1)
where the cellular velocity is small and the cell-free layer formation is
prominent, we would expect the NO production rate to substantially
decrease from those found under normal flow conditions (Ong et al.,
2010a), resulting in the lower steady-state NO values than those
reported in the previous studies. It is of note that, in the present
model, the shear-dependent relationship for the NO production rate is
based on the study by Kavdia and Popel (2003) where a maximum NO
concentration of 100 nM in the endothelium was obtained at a
reference wall shear stress value of 2.4 Pa. The smaller steady-state
wall shear stress values (0.13–0.24 Pa) obtained in the present study
which are approximately 6%–10% of this reference value would thus
justify the lower steady state values of NO concentration.
The steady-state models by Lamkin-Kennard et al. (2004) and
Chen et al. (2006) predicted increasingly higher NO levels in the
vascular wall with greater cell-free layer width in the arterioles.
However, their studies did not consider the effects of changes in the
wall shear stress on NO production rate caused by changes in the cellfree layer width. With a thicker cell-free layer due to aggregation, an
attenuation of wall shear stress would be expected which result in a
decrease in NO production. This effect, as shown in the present study,
could reduce NO bioavailability. On the other hand, an improved NO
preservation that could arise from decreased NO scavenging by the
blood lumen due to a thicker diffusion barrier would be possible when
the NO production does not change with the cell-free layer width,
which is not the case in our simulation.
In this study, a slightly lower systemic hematocrit was found after
dextran infusion although the change was not significant. The reduced
hematocrit after dextran treatment may lower the rate of NO
consumption by the blood which seems to slightly improve NO
bioavailability. This effect, however, could similarly be offset by the
predominant attenuation of wall shear stress and NO production due
to the lower effective blood viscosity. A previous study (Vazquez et al.,
2010) reported constriction of the vasculature with a small decrease
(10%) in the systemic hematocrit from baseline level (47.9%) which
could support the diminished release of vasodilators such as NO for
promoting vasodilation.
In vivo measurements of perivascular NO in the hamster skin
chamber by Tsai et al. (2005) showed an increase in NO concentration
with hemodilution using Dextran 500 which does not seem to support
the predicted decrease in NO concentration by dextran treatment in
the present study. However, it should be noted that direct comparison
of their study with our simulation is limited by different objectives
and simulated conditions. In their study, plasma viscosity was tested
as a possible mediator of microvascular perfusion under extreme
hemodilution conditions and therefore, hemodilution by the exchange of whole blood with Dextran 500 solution was used to
simulate the plasma hyperviscosity condition. On the other hand in
the present study, Dextran 500 was infused into the animal to
simulate the desired levels of aggregation. As a result, in comparison
with the present study, their mean plasma viscosity (1.99 cP) was
greater by ~55% and their hematocrit (11%) was lower by more than 3
folds. This substantially higher plasma viscosity and lower hematocrit
could predominantly enhance NO bioavailability in the arteriole,
possibly through the augmentation of NO production by an increase in
wall shear stress and the reduction of NO scavenging by red blood
cells.
Plasma viscosity effect on NO bioavailability
The use of high molecular weight dextrans to induce red blood
cell aggregation can elevate the blood plasma viscosity which could
also contribute to an increase in NO production by augmenting the
wall shear stress (Baskurt and Meiselman, 2008). Yalcin and
coworkers (Yalcin et al., 2008) have shown that a greater activation
of eNOS protein can occur in aggregating blood flow where the
plasma viscosity was elevated, suggesting an enhancement in NO
production. A greater NO-mediated dilation of arterioles has been
reported after the perfusion of red blood cells suspended in a
dextran solution of higher molecular weight (515 kDa vs 39.5 kDa)
(de Wit et al., 1997). This discrepancy has been attributed to an
increase in wall shear stress associated with the appreciable rise
(64%) in viscosity of the suspending phase containing the higher
molecular weight dextran. However, in the present study, the effect
of plasma viscosity change on NO bioavailability and vascular
responses is expected to be less prominent since the increase
(~ 7.5%) in viscosity was substantially smaller. Furthermore, it is of
note that, in this simulation study, the effect of plasma viscosity on
NO transport was isolated by using the same viscosity value for all
the conditions, with the assumption that the cell-free layer
formation is not significantly altered by the small increase of
plasma viscosity. We are aware that a small increase (~ 7.5%) in
plasma viscosity after dextran infusion may lead to an elevation in
wall shear stress, resulting in an increase in NO production rate.
However, since the primary objective of this study is to elucidate the
sole effect of red blood cell aggregation on NO bioavailability in the
arteriole by considering the temporal variations in cell-free layer
width, we did not consider the plasma viscosity effects in the NO
simulations which would result from dextran-induced aggregation.
Thus, our predicted values of the NO concentration after dextran
treatment might be slightly lower than when the plasma viscosity
effects are considered.
Influence of temporal variations of the layer on vascular tone
One important form of NO transport from the endothelial source
that influences the vascular tone is the diffusion of NO into the smooth
muscle layer. Smooth muscle cells in this layer are affected by
exposure to NO concentrations ranging from 5 to 100 nM (Condorelli
and George, 2001). These levels of NO concentration can activate the
target hemoprotein, sGC, which is responsible for the catalytic
conversion of guanosine triphosphate (GTP) to cyclic guanosine
monophosphate (cGMP) (Arnold et al., 1977). Consequently, these
small signaling molecules are responsible for relaxation of the smooth
muscle cells, which elicit vasodilatory responses. In the present study,
the simulated sGC activation was augmented by the temporal
variations in the layer width after aggregation induction due to the
associated enhancement in NO bioavailability although this effect
seemed to be secondary to the layer thickness effect. It is of note that
the significant reduction of sGC activation by aggregation seen in the
absence of the layer width variation was eliminated by the
enhancement of sGC activation resulting from the larger layer
variations associated with the aggregating condition (see Fig. 6B).
This suggested that the suppression effect of vasodilatory responses
by the thicker layer width due to aggregation could be offset by the
effect of layer width variations.
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
Other potential physiological implications
Although a NO concentration of 23 nM is used in the present study
to give half-maximal activation of sGC (EC50), a wide range of NO
concentrations with EC50 from 1 to 250 nM were reported in previous
studies (Bellamy et al., 2002; Stone and Marletta, 1996). It was shown
previously that NO could activate sGC with an EC50 of as low as 1–
4 nM which catalyzes cGMP formation (Griffiths and Garthwaite,
2001; Griffiths et al., 2003), eliciting many physiological effects such
as the inhibition of platelet aggregation and synaptic plasticity
(Garthwaite and Boulton, 1995; Koesling and Friebe, 1999). Furthermore, NO is known to be an inhibitor of mitochondrial respiration at
sufficiently high concentrations (Hall and Attwell, 2008). Accordingly,
an estimated NO concentration of 11 nM could elicit 50% inhibition
(Koivisto et al., 1997). It seems that small changes in NO concentration of the order obtained in this study would be capable of
implicating physiological functions.
Limitations of current study
The approximation of wall shear stress was based on the mean
velocity of red blood cells at the edge of the blood core although the
velocity of the cells could vary with time depending on their radial
positions. If a linear velocity profile is assumed within the cell-free
layer, the error can be approximated by the root mean square (RMS)
deviation (Goldsmith et al., 1999) in the layer for each time interval
(~0.44 ms) in a time period of 2 s which was found to be ~ 2.4% and
~ 2.1% before and after dextran infusion, respectively. Due to the
increasing non-linearity of the velocity profile farther away from the
vessel wall (Bishop et al., 2001), further refinement of the numerical
model would be required to provide a more realistic account of wall
shear stress for larger thickness of the layer.
Our model did not consider the effects that would be imposed by
the presence of the endothelial surface layer (ESL) on NO bioavailability in the vessel. This layer (~0.5 μm width) (Smith et al., 2003)
consists of macromolecules (glycolipids, proteoglycans and glycoproteins) bound to the endothelial membrane and is responsible for
mediating the transduction of hemodynamic signals from the flowing
blood to the endothelium. Thus, it could play a critical role in
modulating the shear stress acting on the endothelium and the
subsequent activation of molecular pathways responsible for the
synthesis of NO. A decrease in the ESL width may impair the
biochemical production of NO in the endothelium, resulting in
reduced NO production (Weinbaum et al., 2003).
Acknowledgments
This work was supported by NUS FRC grant R-397-000-076-112
and URC grant R-397-000-091-112.
References
Alonso, C., et al., 1995. Transient rheological behavior of blood in low-shear tube flow:
velocity profiles and effective viscosity. Am. J. Physiol. 268, H25–H32.
Ando, J., Yamamoto, K., 2010. Shear-stress-sensing and response mechanisms in vascular
endothelial cells. Interface Oral Health Science10.1007/978-4-431-99644-6_1
Arnold, W.P., et al., 1977. Nitric oxide activates guanylate cyclase and increases
guanosine 3′:5′-cyclic monophosphate levels in various tissue preparations. Proc.
Natl Acad. Sci. USA 74, 3203–3207.
Azarov, I., et al., 2005. Nitric oxide scavenging by red blood cells as a function of
hematocrit and oxygenation. J. Biol. Chem. 280, 39024–39032.
Baker, M., Wayland, H., 1974. On-line volume flow rate and velocity profile
measurement for blood in microvessels. Microvasc. Res. 7, 131–143.
Baskurt, O.K., Meiselman, H.J., 2007. In Vivo Hemorheology. Handbook of Hemorheology and Hemodynamics, vol. 69. IOS Press, pp. 322–335.
Baskurt, O.K., Meiselman, H.J., 2008. RBC aggregation: more important than RBC
adhesion to endothelial cells as a determinant of in vivo blood flow in health and
disease. Microcirculation 15, 585–590.
Baskurt, O.K., et al., 2004. Modulation of endothelial nitric oxide synthase expression by
red blood cell aggregation. Am. J. Physiol. Heart Circ. Physiol. 286, H222–H229.
.
9
Bellamy, T.C., et al., 2002. Differential sensitivity of guanylyl cyclase and mitochondrial
respiration to nitric oxide measured using clamped concentrations. J. Biol. Chem.
277, 31801–31807.
Bishop, J.J., et al., 2001. Effect of erythrocyte aggregation on velocity profiles in venules.
Am. J. Physiol. Heart Circ. Physiol. 280, H222–H236.
Buerk, D.G., 2001. Can we model nitric oxide biotransport? A survey of mathematical
models for a simple diatomic molecule with surprisingly complex biological
activities. Annu. Rev. Biomed. Eng. 3, 109–143.
Butler, A.R., et al., 1998. Diffusion of nitric oxide and scavenging by blood in the
vasculature. Biochim. Biophys. Acta 1425, 168–176.
Chen, X., et al., 2006. The influence of radial RBC distribution, blood velocity profiles,
and glycocalyx on coupled NO/O2 transport. J. Appl. Physiol. 100, 482–492.
Cokelet, G.R., Goldsmith, H.L., 1991. Decreased hydrodynamic resistance in the twophase flow of blood through small vertical tubes at low flow rates. Circ. Res. 68,
1–17.
Condorelli, P., George, S.C., 2001. In vivo control of soluble guanylate cyclase activation
by nitric oxide: a kinetic analysis. Biophys. J. 80, 2110–2119.
de Wit, C., et al., 1997. Elevation of plasma viscosity induces sustained NO-mediated
dilation in the hamster cremaster microcirculation in vivo. Pflugers Arch. 434,
354–361.
Garthwaite, J., Boulton, C.L., 1995. Nitric oxide signaling in the central nervous system.
Annu. Rev. Physiol. 57, 683–706.
Goldsmith, H.L., et al., 1999. Effect of red blood cells and their aggregates on platelets
and white cells in flowing blood. Biorheology 36, 461–468.
Gori, T., Forconi, S., 2007. Endothelium and Hemorheology. Handbook of Hemorheology
and Hemodynamics, vol. 69. IOS Press, Amsterdam, pp. 339–350.
Griffiths, C., Garthwaite, J., 2001. The shaping of nitric oxide signals by a cellular sink. J.
Physiol. 536, 855–862.
Griffiths, C., et al., 2003. A new and simple method for delivering clamped nitric oxide
concentrations in the physiological range: application to activation of guanylyl
cyclase-coupled nitric oxide receptors. Mol. Pharmacol. 64, 1349–1356.
Hall, C.N., Attwell, D., 2008. Assessing the physiological concentration and targets of
nitric oxide in brain tissue. J. Physiol. 586, 3597–3615.
Intaglietta, M., et al., 1975. Capillary flow velocity measurements in vivo and in situ by
television methods. Microvasc. Res. 10, 165–179.
Kanai, A.J., et al., 1995. Shear stress induces ATP-independent transient nitric oxide
release from vascular endothelial cells, measured directly with a porphyrinic
microsensor. Circ. Res. 77, 284–293.
Kavdia, M., Popel, A.S., 2003. Wall shear stress differentially affects NO level in arterioles
for volume expanders and Hb-based O2 carriers. Microvasc. Res. 66, 49–58.
Kim, S., et al., 2006. A computer-based method for determination of the cell-free layer
width in microcirculation. Microcirculation 13, 199–207.
Kim, S., et al., 2007. Temporal and spatial variations of cell-free layer width in arterioles.
Am. J. Physiol. Heart Circ. Physiol. 293, H1526–H1535.
Koesling, D., Friebe, A., 1999. Soluble guanylyl cyclase: structure and regulation. Rev.
Physiol. Biochem. Pharmacol. 135, 41–65.
Koivisto, A., et al., 1997. Kinetics of the inhibition of mitochondrial respiration by NO.
FEBS Lett. 417, 75–80.
Kuchan, M.J., Frangos, J.A., 1994. Role of calcium and calmodulin in flow-induced nitric
oxide production in endothelial cells. Am. J. Physiol. 266, C628–C636.
Kuo, L., et al., 1990. Coronary arteriolar myogenic response is independent of
endothelium. Circ. Res. 66, 860–866.
Lamkin-Kennard, K.A., et al., 2004. Impact of the Fahraeus effect on NO and O2
biotransport: a computer model. Microcirculation 11, 337–349.
Lancaster Jr., J.R., 1994. Simulation of the diffusion and reaction of endogenously
produced nitric oxide. Proc. Natl Acad. Sci. USA 91, 8137–8141.
Lee, B.K., et al., 2007. Red blood cell aggregation quantitated via Myrenne aggregometer
and yield shear stress. Biorheology 44, 29–35.
Liao, J.C., et al., 1999. Intravascular flow decreases erythrocyte consumption of nitric
oxide. Proc. Natl Acad. Sci. USA 96, 8757–8761.
Maeda, N., et al., 1996. Erythrocyte flow and elasticity of microvessels evaluated by
marginal cell-free layer and flow resistance. Am. J. Physiol. 271, H2454–H2461.
Namgung, B., et al., 2011. Effect of cell-free layer variation on arteriolar wall shear
stress. Ann. Biomed. Eng. 39, 359–366.
Ong, P.K., et al., 2010a. Modulation of NO bioavailability by temporal variation of the
cell-free layer width in small arterioles. Ann. Biomed. Eng.10.1007/s10439-0100216-y
Ong, P.K., et al., 2010b. Effect of erythrocyte aggregation and flow rate on cell-free layer
formation in arterioles. Am. J. Physiol. Heart Circ. Physiol. 298, H1870–H1878.
Reinke, W., et al., 1987. Blood viscosity in small tubes: effect of shear rate, aggregation,
and sedimentation. Am. J. Physiol. 253, H540–H547.
Smith, M.L., et al., 2003. Near-wall micro-PIV reveals a hydrodynamically relevant
endothelial surface layer in venules in vivo. Biophys. J. 85, 637–645.
Soutani, M., et al., 1995. Quantitative evaluation of flow dynamics of erythrocytes in
microvessels: influence of erythrocyte aggregation. Am. J. Physiol. 268,
H1959–H1965.
Sriram, K., et al., 2010. The effect of small changes in hematocrit on nitric oxide
transport in arterioles. Antioxid. Redox Signal. 14, 175–185.
Stone, J.R., Marletta, M.A., 1996. Spectral and kinetic studies on the activation of soluble
guanylate cyclase by nitric oxide. Biochemistry 35, 1093–1099.
Suzuki, Y., et al., 1996. Flow behavior of erythrocytes in microvessels and glass
capillaries: effects of erythrocyte deformation and erythrocyte aggregation. Int. J.
Microcirc. Clin. Exp. 16, 187–194.
Tsai, A.G., et al., 2005. Elevated plasma viscosity in extreme hemodilution increases
perivascular nitric oxide concentration and microvascular perfusion. Am. J. Physiol.
Heart Circ. Physiol. 288, H1730–H1739.
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002
10
P.K. Ong et al. / Microvascular Research xxx (2011) xxx–xxx
Tsoukias, N.M., et al., 2004. A theoretical model of nitric oxide transport in arterioles:
frequency- vs. amplitude-dependent control of cGMP formation. Am. J. Physiol.
Heart Circ. Physiol. 286, H1043–H1056.
Vallance, P., Leiper, J., 2002. Blocking NO synthesis: how, where and why? Nat. Rev.
Drug Discov. 1, 939–950.
Vaughn, M.W., et al., 1998. Effective diffusion distance of nitric oxide in the
microcirculation. Am. J. Physiol. 274, H1705–H1714.
Vaya, A., et al., 2003. Erythrocyte aggregation determined with the Myrenne
aggregometer at two modes (M0, M1) and at two times (5 and 10 sec). Clin.
Hemorheol. Microcirc. 29, 119–127.
Vazquez, B.Y., et al., 2010. The variability of blood pressure due to small changes of
hematocrit. Am. J. Physiol. Heart Circ. Physiol. 299, H863–H867.
Wayland, H., Johnson, P.C., 1967. Erythrocyte velocity measurement in microvessels by
a two-slit photometric method. J. Appl. Physiol. 22, 333–337.
Weinbaum, S., et al., 2003. Mechanotransduction and flow across the endothelial
glycocalyx. Proc. Natl Acad. Sci. USA 100, 7988–7995.
Wessells, H., et al., 2006. Fluid shear stress-induced nitric oxide production in human
cavernosal endothelial cells: inhibition by hyperglycaemia. BJU Int. 97,
1047–1052.
Yalcin, O., et al., 2008. Nitric oxide generation by endothelial cells exposed to shear
stress in glass tubes perfused with red blood cell suspensions: role of aggregation.
Am. J. Physiol. Heart Circ. Physiol. 294, H2098–H2105.
Zhang, J., et al., 2009. Effects of erythrocyte deformability and aggregation on the cell free
layer and apparent viscosity of microscopic blood flows. Microvasc. Res. 77, 265–272.
Please cite this article as: Ong, P.K., et al., Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles:
Effects of erythrocyte aggregation, Microvasc. Res. (2011), doi:10.1016/j.mvr.2011.02.002