Traction Forces of Endothelial Cells under Slow Shear Flow

Abstract

Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress.

Full text

Biophysical Letter
Traction Forces of Endothelial Cells under Slow Shear Flow
Cecile M. Perrault,
1,2,3,
*Agusti Brugues,
3
Elsa Bazellieres,
3
Pierre Ricco,
2
Damien Lacroix,
1,2,3
and Xavier Trepat
3,4,5,6
1
Institute for In Silico Medicine and
2
Department of Mechanical Engineering, University of Sheffield, Sheffield, United Kingdom;
3
Institute for
Bioengineering of Catalonia, Barcelona, Spain;
4
Facultat de Medicina, Universitat de Barcelona, Barcelona, Spain;
5
Centro de Investigacio
´n
Biome
´dica en Red en Bioingenierı
´a, Biomateriales y Nanomedicina, Barcelona, Spain; and
6
Institucio
´Catalana de Recerca i Estudis Avanc¸ats,
Barcelona, Spain
ABSTRACT Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeosta-
sis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial
circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as
that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we
used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell
monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces
and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps
reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow
regime in which endothelial cell mechanics is acutely responsive to shear stress.
Received for publication 4 March 2015 and in final form 11 August 2015.
*Correspondence: c.perrault@sheffield.ac.uk
The ability of endothelial cells (ECs) to sense and adapt to
shear flow is one of the best-studied phenomena in all me-
chanobiology. In response to flow, ECs are known to change
their orientation, remodel cell-cell and cell-matrix adhe-
sions, modify patterns of gene expression, and alter protein
localization at the cell membrane (1). Because these re-
sponses are downstream of a mechanical stimulus, a number
of studies have analyzed the time evolution of cell-matrix
tractions during the application of shear flow. Some of these
studies reported increases in traction forces with shear flow
(2–4), whereas others reached the opposite conclusion (5,6).
Previous studies of traction forces exerted by ECs in the
presence of constant shear flow focused on the application
of shear stresses >1 Pa (10 dyn/cm
2
). Shear stresses in this
range are characteristic of arterial flow during physiological
function. In many other physiological and pathological
conditions, however, shear stresses are much weaker. This
is the case of shear stresses during venous (7) and interstitial
flow (8), as well as during atherosclerosis (9) and intracranial
aneurysm (10). The biochemical and structural responses of
ECs to high versus low shear stress have been extensively
shown to differ in terms of cell morphology, orientation,
and expression of vasoactive agents, antioxidant enzymes,
growth regulators, inflammatory mediators, and adhesion
molecules (reviewed by Malek et al. (9)). Moreover, in the
presence of ultraslow flow such as interstitial flow (11),
ECs are capable of forming numerous capillary-like struc-
tures and have a greater rate of invasion (12). Many of the
phenomena described above are likely to involve a synergy
between flow sensing and force generation (13), but the
link between slow flow and cell contractility is unknown.
To address this question, we combined traction micro-
scopy (TM) and monolayer stress microscopy (MSM)
with microfluidic techniques and explored cellular traction
forces in reaction to slow shear flow (Fig. 1). TM maps
the magnitude, location, and direction of the forces exerted
by cells against their underlying soft substrate (14). Sub-
strate displacements caused by cell tractions are mapped
using fiduciary markers embedded in the soft substrate.
The displacement fields are then used to compute tractions
by inverting the elasticity equations in Fourier space.
TM was integrated into a microfluidic chamber, created in
PDMS (polydimethylsiloxane) by soft lithography. A mold
was machined from Plexiglas to create rectangular flow
channel of 2 mm in width and 2 cm in length. The channel
was designed to have two different heights over its length,
thus creating a channel with two different shear stress values
(15). The circulating media entered a chamber with an
initial height of 300 mm, and moved into a chamber with
a height of 600 mm. Corresponding shear stress values can
be found in Table S1 in the Supporting Material. Cell trac-
tions were monitored in both chambers, away from the tran-
sition zone between the two.
Editor: Gijsje Koenderink.
Ó2015 by the Biophysical Society
http://dx.doi.org/10.1016/j.bpj.2015.08.036
Biophysical Journal Volume 109 October 2015 1–4 1
Please cite this article in press as: Perrault et al., Traction Forces of Endothelial Cells under Slow Shear Flow, Biophysical Journal (2015), http://dx.doi.org/
10.1016/j.bpj.2015.08.036
BPJ 6761

Monolayers of human umbilical vein endothelial cells
were exposed to a time-varying protocol alternating no-
flow and applied flow in the range 0.014–0.133 Pa (see
Fig. 2 and the Supporting Material). The temporal stress
pattern consisted of two consecutive flow steps of 30-min
duration and increasing magnitude (5 and 10 mL/h), fol-
lowed by a 30-min period of no flow. After this period, we
applied a second pulse of flow (10 mL/h) lasting 30 min.
Upon exposure to flow, we observed an acute increase in
strain energy (the total energy transmitted by the cells on
the substrate) with no significant differences between the
two flow levels (Fig. 2). Subsequent doubling of the shear
flow did not trigger a second increase in traction forces.
Instead, cells tended to plateau at values that were 50–
100% higher than baseline levels. Quickly after stopping
the flow, the strain energy relaxed toward baseline levels,
thus indicating reversibility of responses to flow. Applica-
tion of an additional flow pulse triggered a second acute in-
crease in tractions, with pronounced differences between
the two flow levels, which is suggestive of a memory effect.
Finally, stopping the flow led to a relaxation toward base-
line levels.
The responses shown in Fig. 2 are spatial averages of trac-
tion maps. As previously shown in a diversity of cell types
(6,16), these maps exhibited a punctate distribution with
large spatial heterogeneities (Fig. 3,Aand B). The response
to shear flow was also heterogeneous; although the overall
traction of the monolayer increased, several cells displayed
significant traction drops (Fig. 3 C). The magnitude of local
changes in traction in response to shear was similar to the
global traction average. Upon flow application, tractions
showed a weak but significant tendency to orient perpendic-
ular to the direction of flow (p<0.001, Rayleigh test, Fig. 3,
Dand E).
Web 3C
FIGURE 2 ECs display acute responses to
slow shear flow. Strain energy was normalized
to its baseline (t¼0). (Green line) Flow values.
(Red and black lines) High shear (HS) stress
and low shear (LS) stress; both shear stresses
were at least one order-of-magnitude lower
than previously reported shear stresses in
TM experiments. n¼6 monolayers per condi-
tion. Differences between HS and LS are only
significant during the second shear pulse
(t¼105 min and t¼125 min, p%0.05). The
slopes of the strain energy between the first
and second flow periods were not significant.
To see this figure in color, go online.
Web 3C
FIGURE 1 Microfluidic traction assay. A
PDMS flow chamber is assembled over a
strip of polyacrylamide (E¼1.25 KPa) poly-
merized on a glass coverslip. The chamber
is divided into two sections of varying
heights. The flow is controlled by a syringe
pump. To see this figure in color, go online.
Biophysical Journal 109(8) 1–4
2Biophysical Letters
Please cite this article in press as: Perrault et al., Traction Forces of Endothelial Cells under Slow Shear Flow, Biophysical Journal (2015), http://dx.doi.org/
10.1016/j.bpj.2015.08.036
BPJ 6761

Finally, we used MSM to measure cell-cell stresses (14).
As reported previously (6,14), cell-cell stresses showed
supracellular spatial fluctuations (see Fig. S1). Upon flow
application, these fluctuations increased in magnitude but
cell-cell stresses did not change in orientation. Unlike
cell-substrate tractions, cell-cell stresses did not show sig-
nificant differences depending on flow magnitude.
Traction forces in the presence of constant shear stresses
of relatively high magnitude (>1 Pa) have been extensively
characterized in previous studies, with conflicting results
(2–6). Here we used shear stresses between one and two
orders-of-magnitude smaller than those applied in previous
studies based on TM. In response to these low stresses, ECs
exhibited acute but reversible increases in traction. These re-
sponses were fast and more pronounced than those reported
in previous studies using higher shear flows (2–6). The low
shear stresses applied here fall within the range of physio-
logical interstitial flow (8). Because interstitial flow induces
angiogenesis, we speculate that increased traction forces
observed here might recapitulate those required to initiate
the formation of new blood vessels in vivo (17). Low flows
are also characteristic of pathological conditions such as
intracranial aneurysm (10) and atherosclerosis (9); our find-
ings raise the question of whether increases in traction
forces might be protective or disruptive in these conditions.
A remarkable feature of our experiments was the hetero-
geneity of the responses. Heterogeneous responses of ECs to
flow have been previously reported (18) in terms of protein
and mRNA levels, calcium signaling, and organelle locali-
zation. Heterogeneity has been attributed to the topography
of the monolayer (19), the heterogeneous location, proper-
ties of cytoskeleton elements (20), and flow sensors. Our
A
B
C
D
E
Web 3C
FIGURE 3 Instantaneous maps of traction
forces of an endothelial monolayer at (A)t¼
96 min and (B)t¼126 min. (C) Difference
between (A) and (B). Distribution of the angle
between traction vectors and the direction of
flow at (D)t¼96 min and (E)t¼126 min. To
see this figure in color, go online.
Biophysical Journal 109(8) 1–4
Biophysical Letters 3
Please cite this article in press as: Perrault et al., Traction Forces of Endothelial Cells under Slow Shear Flow, Biophysical Journal (2015), http://dx.doi.org/
10.1016/j.bpj.2015.08.036
BPJ 6761

findings of heterogeneous force distributions might underlie
heterogenous responses in signaling and molecular localiza-
tion through mechanotransduction activity. Whether such
activity is dominated by cell-matrix or cell-cell stresses
could be elicited based on the differences in the character-
istic lengths of the corresponding fluctuations.
In conclusion, we used a new device, to our knowledge, to
combine microfluidics, TM, and MSM to assess the me-
chanical response of ECs to slow shear flow. The observed
acute increases in traction generation provide fresh insights
into the synergy between flow and the biomechanical reac-
tion of cells, with potential implications in morphogenesis
and disease.
SUPPORTING MATERIAL
Supporting Materials and Methods, one table, and one figure, are available at
http://www.biophysj.org/biophysj/supplemental/S0006-3495(15)00871-1.
AUTHOR CONTRIBUTIONS
C.M.P., D.L., and X.T. designed the study; C.M.P. implemented the flow
chamber and carried out experiments; C.M.P., A.B., and E.B. processed
data; C.M.P. and X.T. wrote the article; P.R. calculated shear and gel
displacement in the chamber; and all authors discussed and interpreted re-
sults and commented on the article.
ACKNOWLEDGMENTS
We thank Daniel Navajas, and members of the Integrative Cell and Tissue
Dynamics Lab and the MechanoBio Lab at the Institute for Bioengineering
of Catalonia, for fruitful discussions.
C.M.P. received funding from the Institute for Bioengineering of Catalonia.
This work was funded by the Spanish Ministry of Economy and Competi-
tiveness (under grant No. BFU2012-38146), the Generalitat de Catalunya
(under grant No. 2014-SGR-927), and the European Research Council (un-
der grant No. CoG-616480).
SUPPORTING CITATIONS
References (21–24) appear in the Supporting Material.
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Biophysical Journal 109(8) 1–4
4Biophysical Letters
Please cite this article in press as: Perrault et al., Traction Forces of Endothelial Cells under Slow Shear Flow, Biophysical Journal (2015), http://dx.doi.org/
10.1016/j.bpj.2015.08.036
BPJ 6761

TRACTION FORCES OF ENDOTHELIAL CELLS UNDER SLOW
SHEAR FLOW
C.M. Perrault1,2,3, A. Brugués3, E. Bazellieres3 , P. Ricco2 , D. Lacroix1,2,3 and X. Trepat3,4,5
1 INSIGNEO Institute for in-silico Medicine, University of Sheffield, Sheffield, UK; 2 Department of Mechanical Engineering,
University of Sheffield, Sheffield, UK; 3 Institute for Bioengineering of Catalonia, Barcelona, Spain; 4 Facultat de Medicina,
Universitat de Barcelona, and Ciber-BBN, Barcelona, Spain; 5 Institució Catalana de Recerca i Estudis Avançats, Barcelona,
Spain.
Supplementary material
1. Materials and Methods
1.1. Preparation of polyacrylamide gels and cell culture
Large rectangular coverslips were activated by using a 1:1:14 solution of acetic acid/bind-
silane/ethanol. The dishes were washed twice with ethanol and air-dried for 10 min.
Polyacrylamide gels (E= 1.25 kPa) were prepared as described in Kandow et al (1) and Yeung et al
(2). Briefly, a solution containing 5% acrylamide, 0.1% bis acrylamide, 0.5% ammonium
persulphate, 0.05% tetramethylethylenediamine, 0.4% of 200-nm-diameter red fluorescent
carboxylate-modified beads and 2 mg ml−1 NH-acrylate was prepared. A molding channel was
created on the coverslip using double sided-tape and transparency paper to create polyacrylamide gels
with dimensions of 1mm in width and 2cm in length. A drop of 10 μl was placed on one end of the
channel, which then fills by capillary action.
After polymerization, tape and paper were carefully removed and the gels were washed with PBS and
incubated with 100μl of a collagen I solution (0.1 mg/ml, Millipore) overnight at 4°C. The gels were
washed afterwards with PBS and cells were seeded and incubated with cell culture media with 10%
FBS for 6h.
HUVEC cells were cultured in EGM™ BulletKit™ (Lonza, MA).
1.2. Flow experiments
A small volume (8 μl) containing 150,000 cells was placed on the polyacrylamide gel. Once the cells
were attached to the polyacrylamide gel (20 min), the unattached cells were washed away and 2 ml of
medium were added. Twelve hours after seeding the cells, the coverslip was attached to the PDMS
flow chamber and held together by a custom-made holder. The ensemble was then placed on the
microscope and connected on one side to a syringe pump (World Precision Instruments Aladdin 1000,
WPI, FL) and to the other side to a reservoir of degassed medium.
1.3. Time-lapse microscopy
Multidimensional acquisition routines were performed on an automated inverted microscope (Nikon
Eclipse Ti) equipped with thermal, CO2 and humidity control, using MetaMorph (Universal Imaging)

software. Time-lapse recording started approximately 10 min after assembly. The interval between
image acquisition was 1 min.
1.4. Traction microscopy (TM)
Cell tractions were evaluated using monolayer Fourier-transform traction microscopy (3). Briefly, the
displacement field was calculated by comparing fluorescent microbead images obtained during the
experiment with a reference image taken at the end of the experiment after the trypsinization and the
consequent detachment of the cells from the underlying substrate. A particle imaging velocimetry
algorithm (3) was used to determine the deformation of the substrate caused by the traction forces.
1.5 Monolayer Stress Microscopy (MSM)
In a 2D approximation, monolayer stress is fully captured by a tensor possessing two independent
normal components ( and ) and two identical shear components ( and ). At every pixel
of the monolayer, these four components of the stress tensor define two particular directions of the
plane, one in which the normal stress is maximum and one in which it is minimum. These directions,
which are mutually orthogonal, are called principal stress orientations, and the stress values in each
principal orientation are called maximum () and minimum () stress components. The average
normal stress is defined as =(+)/2, while the maximum shear stress is defined as =(-
)/2. The spatial resolution and force precision of MSM are formally set by those in the original
traction maps. How the reconstructed stress field is affected by the choice of boundary conditions and
by the assumptions of continuity, incompressibility, and homogeneity was extensively studied
elsewhere (3,6).
1.6 Statistical analysis
Summary data are expressed as mean ± SEM (standard error of the mean). Unless noted otherwise,
statistical comparisons were computed by Student’s t-test. Traction angles were compared to an
isotropic distribution using a Raleigh test. A value of p ≤ 0.05 was considered statistically significant.
2. Flow analysis in the microfluidic chamber
The microfluidic chamber was created to limit contact of the cells with PDMS prior to the experiment.
As a result, the system was designed with two components: 1) a strip of PAA gels on a glass slide and
2) the PDMS flow chamber. To maintain both sides together during operation, a customized
aluminium holder was created to apply mechanical forces over the assembly. To facilitate alignment
and limit leakage, the PAA gels were created slightly less wide than the flow channel, but the gap was
small enough to avoid fluctuations in the fluid distribution, as observed by flow of fluorescent beads.
To ensure that the displacement of the beads in the polyacrylamide gels was only caused by the cells,
the displacement of the upper layer of the gels due to shear stress was calculated. As the Reynolds
number of the flow (  , where u is the velocity, d is the hydraulic diameter and v is the
kinematic viscosity) is low, the flow can be assumed to be laminar. Assuming Poiseuille flow, the
flow rate per unit width (m2/s) is related to the streamwise mean pressure gradient as follows (4):

 (1)

Where Px represents the pressure gradient along the streamwise direction and μ is the viscosity of the
fluid.
As the flow rate per unit width is related to the volumetric flow rate  , the pressure
gradient is readily found. The laminar flow is given by

  (2)
where the y coordinate is taken from the middle of the gap so that the wall-shear stress τw is found

 
 (3)
The angle γ of deformation of the surface is

 (4)
where G is the shear modulus of the flexible surface.
The streamwise displacement Δs of the flexible surface is thus
∆  tan (5)
The results for flow rates of 5 and 10 mL/hr in the small and large region of the chambers are
summarized in the table 1.
Small chamber (high shear) Large chamber (low shear)
Wall shear stress Gel displacement Wall shear stress Gel displacement
Q= 5mL/hr 0.066 Pa 2.7x10-9 m 0.014 Pa 5.5x10-10 m
Q= 10 mL/hr 0.133 Pa 5.3x10-9 m 0.028 Pa 1.1x10-9 m
Table1.Wallshearstressandgeldisplacementvaluesfortheflowratesappliedduringtheexperimentsinboth
chambersoftheflowdevice.Gelstiffnessis1.25kPa.
The displacement of the gel due to the flow is thus minimal and the shear stress values correspond
to a low flow regime (5).

3. Supplementary Figure
Supplementary Fig S1. Cell-cell stresses display acute and reversible responses to low shear flow.
A-B) Maps of the average normal stress at t= 96 min and t=126 min (same time points as in Fig. 3
in the main text). C) Time evolution of the average cell-cell stresses (norm of the average normal
stress) in response to the flow pattern depicted in green (n=6 per condition). D) Angular change of
the maximum principal stress between t=96 min and t=126min. These data show that changes in
cell-cell stress orientation in response to shear flow were minimal.
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guidance by cooperative intercellular forces. Nat. Mater. 10: 469–75.
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5. Swartz, M.A., and M.E. Fleury. 2007. Interstitial flow and its effects in soft tissues. Annu.
Rev. Biomed. Eng. 9: 229–56.
6. Tambe DT, Croutelle U, Trepat X, Park CY, Kim JH, Millet E, Butler JP, and Fredberg JJ.
Monolayer stress microscopy: limitations, artifacts, and accuracy of recovered intercellular
stresses. PloS one 8, e55172 (2013).