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1 The Whitaker Institute of Biomedical Engineering and Department of Bioengineering, University of California, San Diego, La Jolla, California 92093-0427; 2 Center for Biomedical Engineering and Department of Mechanical Engineering, City College of New York, New York, New York 10031
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Blood flow-associated
shear stress may modulate cellular processes through its action on the
plasma membrane. We quantified the spatial and temporal aspects of the
effects of shear stress (
) on the lipid fluidity of
1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(13)]-stained plasma membranes
of bovine aortic endothelial cells in a flow chamber. A confocal
microscope was used to determine the DiI diffusion coefficient
(D) by fluorescence recovery after photobleaching on cells
under static conditions, after a step- of 10 or 20 dyn/cm2, and after the cessation of . The method
allowed the measurements of D on the upstream and downstream
sides of the cell taken midway between the respective cell borders and
the nucleus. In <10 s after a step- of 10 dyn/cm2,
D showed an upstream increase and a downstream decrease, and both changes disappeared rapidly. There was a secondary, larger increase in upstream D, which reached a peak at 7 min and decreased thereafter, despite the maintenance of .
D returned to near control values within 5 s after
cessation of . Downstream D showed little secondary
changes throughout the 10-min shearing, as well as after its cessation.
Further investigations into the early phase, with simultaneous
measurements of upstream and downstream D, confirmed that a
step- of 10 dyn/cm2 elicited a rapid (5-s) but transient
increase in upstream D and a concurrent decrease in
downstream D, yielding a significant difference between the
two sites. A step- of 20 dyn/cm2 caused D to
increase at both sites at 5 s, but by 30 s and 1 min the
upstream D became significantly higher than the downstream D. These results demonstrate shear-induced changes in
membrane fluidity that are time dependent and spatially heterogeneous. These changes in membrane fluidity may have important implications in
shear-induced membrane protein modulation.
mechanotransduction; membrane fluidity; fluorescence recovery after photobleaching; cholesterol; alcohol
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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BLOOD FLOW IMPARTS ON VASCULAR endothelium a tangential shear stress, which initiates cellular processes related to vessel wall homeostasis and pathophysiology. While many cellular structures, including the cell membrane (6, 7, 13), the cytoskeleton (33), focal adhesions (10), integrins (31), and glycocalyx (18), have been proposed to respond to shear stress and initiate the cellular signaling processes, there have been few experimental attempts to quantify the direct effects of shear stress on these structures. Recent studies have suggested that at least one site of mechanotransduction may be the cell membrane via force-induced changes in fluidity (15, 18, 22).
We developed a new technique to determine the shear-induced changes in membrane fluidity at specific locations on the cell at various time intervals by measuring fluorescence recovery after photobleaching (FRAP) with a Bio-Rad 1024 confocal microscope and Time-Course software. FRAP was chosen over other methods of fluidity measurements, e.g., fluorescence polarization, electron spin resonance, and fluorescence correlation spectroscopy, because rapid measurements (temporal resolution of 1-2 s) with high spatial resolution (~2 µm) can be made. A simplified one-dimensional (1-D) model was developed to derive diffusion coefficients from the FRAP curves. The diffusion coefficient (D) of a lipophilic fluorophore is a quantitative measurement of membrane lipid fluidity (29). This novel approach of performing FRAP analysis of cells in a flow chamber led to measurements of the temporal and spatial variations of shear-induced changes in membrane-lipid fluidity, thus providing insights into mechanotransduction of shear stress by vascular endothelium.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Cell cultures. Bovine aortic endothelial cells (BAECs) were cultured in DMEM supplemented with 2 mM L-glutamine, 50 U/ml penicillin, 50 mg/ml streptomycin, 1 mM sodium pyruvate, and 10% FCS. BAEC cultures were maintained at 37°C in a gas mixture of 95% air-5% CO2. Cells used were from passages 6-17.
Serum-free DMEM containing 100 µM of the lipophilic probe, 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC16(3); Molecular Probes, Eugene, OR] was used to stain the cell membrane. The endothelial cells were washed with warm, serum-free medium, incubated at 37°C in the staining solution for 10 min, and then washed three times with serum-free medium and twice with complete medium. This procedure resulted in a uniform staining of the membrane surface as assessed by confocal microscopy (Fig. 1B, top). Fluidity measurements were obtained within the first 20 min after staining.
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Shear stress apparatus.
Immediately after staining, the glass coverslip was assembled into a
parallel-plate flow chamber with dimensions of 2 mm × 5 cm × 100 µm. An infusion-withdrawal pump with adjustable rate (Harvard
Apparatus, Holliston, MA) was used to induce flow through the chamber
by withdrawing fluid from the downstream side of the flow chamber, with
the upstream side of the flow chamber connected to a syringe barrel
open to atmospheric pressure (Fig. 1A). A flow rate was
chosen to yield a shear stress of 10 or 20 dyn/cm2 using
the equation = 6Qµ/wh2, where Q is
flow rate, µ is medium viscosity (0.007 poise), w is
channel width, and h is channel height. Temperature was
maintained at 37°C by a control loop consisting of a thermocouple, a
temperature controller (Omega, Stamford, CT ), and a heat gun (Master
Appliance, Racine, WI ) in the ultraviolet protection hood of the
confocal microscope. The medium was preequilibrated with 95% air-5%
CO2 overnight in the incubator. The upstream reservoir of
the flow apparatus was covered with mineral oil to maintain gas tension.
Confocal-FRAP system.
BAECs were sheared at 10 or 20 dyn/cm2 at 37°C using
complete medium (DMEM with FCS). FRAP analysis was performed using a
Bio-Rad 1024 confocal microscope. A thin line (0.902 µm wide)
parallel to the flow direction was bleached in the otherwise uniformly fluorescent membrane midway between the nuclear region and the cell
border by using repeated scans with all visible wavelengths from an
argon-ion laser at 10% power (5 scans at 2 ms/scan; Fig. 1B,
bottom). Diffusion-mediated recovery of DiI fluorescence into the
bleached region was monitored using the krypton/argon laser (1% power,
excitation = 568 nm, emission = 585 nm) using line scans at 2 ms/scan for up to 2 s. Diffusion
coefficients were calculated by using the theory for 1-D diffusion with
a Gaussian initial condition for the bleach profile and a Gaussian
profile for the monitoring beam (21). The resulting
equation used to fit the recovery curves was
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(1) |
= f(t 
) is the asymptotic value of fluorescence recovery reached in infinite time, Lb is initial Gaussian
(e
2) half-width of the bleached line,
Lm is Gaussian half-width of the monitoring beam
(krypton/argon), and D is diffusion coefficient. The details
of the mathematical derivation of Eq. 1 are given in the
APPENDIX. From f0, f, and
fi we calculated the fraction of fluorophore available for
diffusion, 
, where = (f 
f0)/(fi f0).
In separate experiments we measured the profiles of the bleached line
(using immobile fluorophores) and the monitoring beam [using a
variation of the point-scan technique (30)]. The Gaussian (e2) half-width of the bleached line was 0.451 µm for a bleach depth similar to that induced on live cells, and the
Gaussian (e2) half-width of the monitoring
beam was 0.316 µm. The bleached line extended in length beyond the
region of interest which was 1.7 µm in length. The slope of the
membrane at the bleach location ( ~0.3 µm/µm) was estimated from
three-dimensional reconstruction of confocal slices of DiI-stained
cells (Fig. 2B). The error in the FRAP measurement associated with this slope is evaluated in the APPENDIX.
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Experimental protocols.
As positive controls, we tested the effects of a membrane fluidizing
agent, benzyl alcohol (BA), and a membrane rigidifying agent,
cholesterol, on endothelial cell membrane fluidity. Cells were cultured
to confluence in 2 × 2-cm wells. For BA experiments, DiI-stained
cells were incubated for 10 min in complete medium containing 30 mM BA
(Sigma Chemicals). For cholesterol experiments, cells were incubated
for 3 h in 0.1 mM cholesterol (cholesterol in
methyl-
-cyclodextrin; Sigma) in complete medium and stained with
DiI. For both experiments, up to three FRAP measurements were taken per
cell on multiple cells in multiple wells, all done within 3 min after staining.
Data and statistical analysis.
D, f0 , and f were evaluated by
fitting Eq. 1 to the raw recovery data using a
Levenberg-Marquardt nonlinear least-squares regression with the aid of
SlideWrite software (Advanced Graphics Software, Encinitas, CA) or a
custom program written in LabVIEW programming language (Fig.
3; National Instruments, Austin, TX). The
diffusion coefficients for shear experiments were normalized by using
the preshear value (Dinit). These ratios
(D/Dinit) were averaged and expressed as
means ± SE. For statistical analysis that involved multiple
pairwise comparisons, ANOVA was performed using SigmaStat software
(SPSS, Chicago, IL). For those groups showing significance among
groups, the significance of differences between each experimental group
was assessed using Tukey's post hoc test. For analysis of differences
between simultaneous upstream and downstream fluidity measurements,
upstream and downstream measurements of D/Dinit
for each cell were paired and plotted as means ± SE, and a paired
t-test was used to assess significant differences at each
time point. P 
0.05 was considered to be significant. In
addition, 95% confidence intervals were computed for
D/Dinit for each group and each time point to
assess differences from initial D/Dinit values.
All values presented in the text and figures are means ± SE.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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Positive controls.
Table 1 shows the means ± SE values
of the DiI diffusion coefficients for control cells (no treatment),
BA-treated cells, and cholesterol-treated cells (all without shear).
Incubation of cells in 30 mM BA for 10 min resulted in a significant
increase in lateral mobility of DiI in the apical membrane of BAECs
over the control values. Conversely, incubation of cells in the
rigidifying agent, cholesterol (0.1 mM for 3 h), resulted in a
significant reduction in the DiI diffusion coefficient relative to
controls. The mobile fraction was nearly 100% for control cells,
and decreased slightly to 90% for BA-treated cells and to a
greater extent (70%) for cholesterol-treated cells.
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Effects of shear stress on membrane fluidity.
For control experiments, repeated FRAP measurements resulted in a
gradual decrease in D/Dinit (38% over 15 min;
Fig. 4). Following the application of a
step-shear stress of 10 dyn/cm2,
D/Dinit measurements on the downstream side of
the cell resulted in an abrupt decrease in fluidity followed by
essentially the same time course as in the control experiments.
Measurements on the upstream side of the cell, however, showed that
D/Dinit rapidly increased after step-shear,
returned immediately to control values, and then began to rise by 3 min
and became significantly higher than control at 7 min
(D/Dinit = 2.01 ± 0.47) after the
application of step-shear. Thereafter, the
D/Dinit value decreased with time, despite the
maintenance of shear stress, but remained significantly elevated over
control for the 10-min duration of shear. On cessation of shear stress,
D/Dinit returned to near control values. While the earliest changes in upstream and downstream
D/Dinit were not significantly different
from control values, they were significantly different from each other
as assessed by ANOVA and a Tukey's post hoc pairwise comparison. The
mobile fraction of cells subjected to shear, as for the control cells,
was ~100% in all cases.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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In the present study we demonstrate that a confocal laser scanning microscope can be used to perform simultaneous, multipoint FRAP measurements with three-dimensional spatial specificity. We confirmed the utility of this system through rigorous comparisons with more specialized FRAP systems (see discussion below) and by showing that BA and cholesterol cause an increase and decrease, respectively, of endothelial cell membrane fluidity in terms of DiI diffusion coefficient D. We used this system to measure the time course and spatial distribution of shear-induced changes in membrane fluidity. The main findings of the present study are as follows: 1) a shear stress of 10 dyn/cm2 was sufficient to induce both early (5 s) and delayed (>5 min) increases in D; 2) this moderate shear of 10 dyn/cm2 elicited an increase and decrease in up- and downstream D, respectively, with the change being rapid and transient; 3) a higher shear stress of 20 dyn/cm2 caused increases in both up- and downstream D, with the upstream increase being greater and more sustained than the downstream increase; and 4) the upstream increases in D resulting from a step-shear of 20 dyn/cm2 were sustained for a longer period than those resulting from 10 dyn/cm2.
Shear stress initiates many cellular processes ranging from immediate changes in ion conductance and G protein activation (seconds) to alterations in cytoskeletal organization and gene expression (minutes to hours) (9). The most proximal events mediating this mechanotransduction have not been clearly established and may occur via multiple structures. Furthermore, long-term effects of shear stress may be transduced by signaling pathways and cellular structures different from those mediating the immediate events. One logical candidate for a mechanotransducer is the cell membrane because of its proximity to the flowing blood. It was the goal of the present study to measure the direct effects of shear stress on the cell membrane fluidity, a property that has been shown to be strongly correlated with cell functions [by modifying membrane protein diffusion (1) and function (11)]. Membrane fluidity has been proposed as a modulator of shear-related cellular processes (11, 18), and, recently, Haidekker et al. (17) published results on an early increase in membrane fluidity due to shear stress. To our knowledge, the present study represents the first experimental quantification of the temporal (short-term and long-term) and spatial (upstream vs. downstream) aspects of shear-induced increases in fluidity on the apical plasma membrane.
To assess diffusion of unbleached fluorophore into a bleached line, we
solved the 1-D diffusion equation (2, 20). The effects of
convection of fluorophores via lipid flow or of cell deformation in the
direction of flow on the FRAP measurements were neglected, because the
lines were made parallel to the flow direction. The membrane where the
FRAP measurement was made was considered flat and devoid of
corrugations and/or microvilli (see APPENDIX for estimation
of the error associated with the assumption of a flat membrane). To
measure the initial bleach geometry, we performed the bleaching
experiments on fluorophores made immobile by drying the stained cells
and then returning them to culture medium. We measured the beam shape
using a variation of the point scan method (30) by
scanning a laser past a small stationary (0.17-µm diameter)
fluorescent latex microsphere (Molecular Probes) and then fitting the
fluorescent profile with a Gaussian curve. Alignment of bleaching and
monitoring lasers was verified each day by projecting the beams through
a target-labeled prism mounted on the microscope nose piece. Finally,
we compared the ability and accuracy of our model to assess
D with that of Stolpen et al. (32), who
bleached with an extended elliptical beam to approximate a line bleach.
For the same D, f0/fi,
f/fi, Lb, and
Lm, fluorescence recovery curves generated by
our model agreed with those generated by Stolpen et al. within 4%.
While 46 terms were summed in their model for this comparison, our
model does not require any summation and hence is convenient for curve
fitting using standard curve-fitting software.
We report that both physiologically moderate (10 dyn/cm2) and high (20 dyn/cm2) shear stress elicits a significant immediate (5 s) increase in membrane fluidity as measured by FRAP. This increase subsided by 10 s for 10 dyn/cm2 and by 2 min for 20 dyn/cm2. The transient nature of the response suggests that the early increase in membrane fluidity is in response to the sudden onset of shear and that this response is dissipated. Recently, Haidekker et al. (17), using a molecular rotor, 9-(dicyanovinyl)-julolidine (DCVJ), to assess membrane fluidity, also showed an increase in fluidity by 5 s after step-shear, but there the increase in fluidity was found to be sustained. The reason for the differences in persistence of this early change is not clear at this time but may be due to differences in cell types (human umbilical vein endothelial cells vs. BAECs) or perfusion medium (Hanks' balanced salt solution vs. DMEM-FCS), or methods of measurement (DCVJ fluorescence vs. FRAP). The DCVJ measurement represents fluidity averaged over the entire cell culture, whereas our FRAP measurement was localized to specific subcellular regions on the cell membrane observed under confocal microscopy. In any event, the present data support, as did Haidekker et al., the suggestion that membrane fluidity may play a role in modulating some of the earliest events known to be related to the mechotransduction of shear stress (e.g., G protein hydrolysis, ion channel conductance).
Simultaneous upstream and downstream measurements of DiI diffusion revealed a spatial distribution of the effects of shear stress on the membrane, and the magnitude and persistence of the change were related to the level of shear stress. A shear stress of 10 dyn/cm2 caused a rapid (5 s) increase and decrease in DiI diffusion on the upstream and downstream portions of the membrane, respectively, and both returned to initial values by 10 s. A step-shear of 20 dyn/cm2, however, caused increases in DiI diffusion on both the upstream and downstream parts of the cell, with the upstream increase persisting for 1 min and the downstream increase falling to initial values by 10 s. These differences in spatial distribution and persistence of the shear-induced increases in fluidity may provide insights into how the cell can sense differences in shear magnitude.
This novel finding that the increase in membrane fluidity is predominantly found in the upstream side of the cell correlates well with the location of positive shear stress gradient distributions, which were computed by Barbee et al. (5), but not with the absolute shear stress distributions. Using measured cell topography and computational fluid dynamics, Barbee et al. showed that shear stress is symmetrically distributed on the upstream and downstream side of the cell, whereas positive temporal shear stress gradients are concentrated on the upstream side of the cell. Hence our observation that shear stress induced a differential spatial (upstream vs. downstream) change in membrane fluidity suggests that shear stress gradients, in addition to shear stress per se, may play an important role in modulating membrane fluidity.
The results here, demonstrating a delayed (>5 min) increase in membrane fluidity, also support the hypothesis that the membrane fluidity, as measured by DiI-FRAP, may play a role in modulating later responses to shear stress. The only other study to investigate the later perturbing effects of shear stress on the cell membrane was performed by Berthiaume and Frangos (6), who used the shear-induced incorporation of MC540 (a lipophilic dye) into the endothelial cells to reflect an increase in membrane permeability. They found a significant increase in the incorporation of the dye beginning at 5 min after shearing with medium 199 supplemented with 20% FCS. Our results on the time course of fluidity changes with shear stress are in excellent agreement with theirs on the shear-induced incorporation of this lipophilic dye.
The causes of shear-induced increases in membrane fluidity remain unclear. It is likely that fluid shear would cause a time-dependent cell deformation in the direction of flow, thus leading to temporally varying and spatially heterogeneous stresses in the cell membrane. These may, in turn, induce time- and position-dependent fluidity changes. In support of this hypothesis, Sato et al. (28) showed that, when endothelial cells are suctioned with a small pipette, a portion of the cell exhibits an immediate elastic deformation followed by a slower viscous deformation. The time scales of these deformations agree well with the early and late increases in membrane fluidity shown in the present study. Wang et al. (34), modeling cell deformation with shear stress, have suggested that shear causes the nuclear bulge to deform in the direction of flow. Such deformation may lead to increased tension in the upstream cell membrane. Experimental support of such cell deformation has been given by Helmke et al. (19), who showed that intermediate filaments close to the apical membrane are displaced in the direction of flow within the first 3 min after the application of a step-shear stress. Finally, the link between membrane strain and membrane fluidity is suggested by the increase in membrane fluidity of human fibroblasts in response to hypotonic swelling (3).
Physiological processes in the cell may be caused by increases in membrane fluidity. Prostacyclin production has been shown to be enhanced by an increase in membrane fluidity (3). Hence, the increases in fluidity after a step-shear observed here may partially explain the prostaglandin-mediated vasodilation seen in our recent study (8). The physiological implications of the more sustained component of increases in membrane fluidity shown here are suggested by a study in which cell apoptosis was caused by agents that induced a sustained increase in membrane fluidity (12).
The cytoskeleton may modulate membrane dynamics. There is evidence that actin filaments remodel on the time scale of the later fluidity changes seen in this study (23). Morita et al. (24) showed that a low shear stress of 5 dyn/cm2 could elicit the depolymerization of F-actin to G-actin in as early as 5 min. Although the shear-induced actin depolymerization is not expected to alter membrane fluidity directly (29), it is likely to alter cell deformation with flow (25). In an earlier study from our laboratory, Galbraith et al. (14) noted that microtubule remodeling due to shear stress occurred preferentially in the upstream side of the cell, and Hage Chahine et al. (16) showed that microtubule disassembly increases membrane fluidity of fibroblasts as measured with FRAP. Finally, Sato et al. (27) noted that the cell surface was stiffer on the upstream side of the cell after 6 h of flow and attributed this polarization to localized stress fiber development. Together, these studies suggest that the cytoskeleton has an intimate association with the membrane and may modulate its functions via membrane stabilization/destabilization cycles.
In summary, we have introduced a novel quantitative measurement of the temporal changes in membrane fluidity and its spatial distribution in response to shear stress. The time course and heterogeneous distribution of the increase in fluidity with shear stress may be related to calcium signaling, shear-induced phospholipid metabolism, and cytoskeletal remodeling. Fluidity changes are likely to have a significant effect on membrane proteins and their interactions (1, 26).
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APPENDIX |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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FRAP Model
The following model describes isotropic, 1-D diffusion into a bleached line with a Gaussian profile initial condition. We first write the 1-D diffusion equation
<IT>,</IT>](/content/vol280/issue4/fulltext/C962/img002.gif)
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(A1) |
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![c(<IT>x, t</IT>)<IT>=</IT>c<SUB><IT>∞</IT></SUB><IT>+</IT>[c<SUB><IT>0</IT></SUB>(<IT>0</IT>)<IT>−</IT>c<SUB><IT>∞</IT></SUB>] <FR><NU><IT>1</IT></NU><DE><RAD><RCD><IT>8 </IT><FR><NU><IT>Dt</IT></NU><DE><IT>L</IT><SUP><IT>2</IT></SUP><SUB>b</SUB></DE></FR><IT>+1</IT></RCD></RAD></DE></FR> exp<FENCE><FR><NU>−<IT>2 </IT><FR><NU><IT>x<SUP>2</SUP></IT></NU><DE><IT>L</IT><SUP><IT>2</IT></SUP><SUB>b</SUB></DE></FR></NU><DE><IT>8 </IT><FR><NU><IT>Dt</IT></NU><DE><IT>L</IT><SUP><IT>2</IT></SUP><SUB>b</SUB></DE></FR><IT>+1</IT></DE></FR></FENCE>](/content/vol280/issue4/fulltext/C962/img004.gif)
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(A2) |
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(A3) |
![f(<IT>t</IT>)<IT>=2I</IT><SUB>m0</SUB>c<SUB>∞</SUB><IT>+</IT>[<IT>2I</IT><SUB>m0</SUB>c<SUB>0</SUB>(<IT>0</IT>)<IT>−2I</IT><SUB>m0</SUB>c<SUB><IT>∞</IT></SUB>]](/content/vol280/issue4/fulltext/C962/img006.gif)
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(A4) |
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) = f and f(t = 0) = f0 and recognize that the error function,
erf(), approaches 1. Because a (the confocal
aperture distance) is large, the erf term goes to 1 and Eq. A4 simplifies to
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(A5) |
The error associated with the assumption of a flat membrane can be
estimated by using the slope of the membrane, the length of the region
of interest for fluorescence measuring, and the depth of the confocal
field. If the membrane has a slope of 0.3 µm/µm (see Fig. 2 and
Ref. 4), and the region of interest (ROI) is 1.7 µm
long, then the left and right edges of the ROI will be out of the focal
plane by 0.25 µm. (Note that the bleached line is longer than the ROI
and, therefore, there is no diffusion of fluorophores from the upstream
and downstream edges of the ROI). The depth of the confocal field for a
×60 1.4 numerical aperture (NA) objective is ~0.61 µm
(14), or 0.3 µm above and below the center of the ROI.
The error arises from the broadening of the laser beam with distance
from the focal point and the consequent broadening of
Lb and Lm. From the
definition of numerical aperture, NA= 1.5 sin 
(where 1.5 is the
refractive index of the immersion oil, is the half angle subtended
by the laser beam, and NA = 1.4), we can estimate that the beam
will broaden by ~0.1 µm at 0.3 µm from the focal point. We then
compute the area increase for the bleaching and monitoring beams and
compute effective Lm and
Lb that yield the effective areas. These widths
are 0.476 µm for the bleaching beam and 0.340 µm for the monitoring
beam. Replacing Lb and
Lm with these values yields an ~12%
underestimation of D for control values and an ~10%
underestimation of the peak values of D. When normalized,
these errors yield an ~4% overestimation of the twofold increase
seen on the upstream side at 7 min after a step-shear stress of
10 dyn/cm2.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeffrey H. Price, of the National Science Foundation-Whitaker Quantitative Imaging and Confocal Microscopy Resource at University of California San Diego for assistance in confocal microscopy and valuable suggestions regarding FRAP, and we thank Dr. Shunichi Usami for expert technical advice and assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-19454 and HL-43026.
P. J. Butler is a recipient of an National Institutes of Health National Research Service Award.
Address for reprint requests and other correspondence: S. Chien, The Whitaker Institute of Biomedical Engineering and Department of Bioengineering, University of California, San Diego, 9500 Gilman Drive MC 0427, La Jolla, CA 92093-0427 (E-mail: shuchien{at}ucsd.edu; pbutler{at}be-research.ucsd.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 2 December 1999; accepted in final form 20 October 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES
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