422109

VMJ16510.1177/1358863X11422109Johnson BD et al.Vascular Medicine

Mechanotransduction of shear in the

endothelium: Basic studies and clinical
implications

Vascular Medicine
16(5) 365377
The Author(s) 2011
Reprints and permission: sagepub.
co.uk/journalsPermissions.nav
DOI: 10.1177/1358863X11422109
vmj.sagepub.com

Blair D Johnson1, Kieren J Mather2 and Janet P Wallace1

Abstract
The endothelium plays an integral role in the development and progression of atherosclerosis. Hemodynamic forces,
particularly shear stress, have a powerful influence on endothelial phenotype and function; however, there is no clear
consensus on how endothelial cells sense shear. Nevertheless, multiple endothelial cell signal transduction pathways
are activated when exposed to shear stress in vitro. The type of shear, laminar or oscillatory, impacts which signal
transduction pathways are initiated as well as which subsequent genes are up- or down-regulated, thereby influencing
endothelial phenotype and function. Recently, human studies have examined the impact of shear stress and different
shear patterns at rest and during exercise on endothelial function. Current evidence supports the theory that augmented
exercise-induced shear stress contributes to improved endothelial function following acute exercise and exercise training,
whereas retrograde shear initiates vascular dysfunction. The purpose of this review is to examine the current theories
on how endothelial cells sense shear stress, to provide an overview on shear stress-induced signal transduction pathways
and subsequent gene expression, and to review the current literature pertaining to shear stress and shear patterns at
rest as well as during exercise in humans and the related effects on endothelial function.
Keywords
endothelial function; exercise; flow-mediated dilation; shear rate; signal transduction

Introduction
Hemodynamic forces, including shear stress (SS), are influential factors that affect endothelial cell (EC) phenotype
and function. Increased cardiac output and blood flow during exercise modulate hemodynamic forces against the
arterial wall, which include circumferential strain and SS.
Furthermore, these hemodynamic forces differ throughout
the arterial tree owing to bends and bifurcations. The purpose of this review is to examine how EC sense SS, signal
transduction pathways in EC which are activated by SS,
and examine the effects of various types of SS that are produced in cell culture on EC phenotype and the resulting
factors that affect atherosclerotic etiology. Additionally,
research examining SS or shear rate (SR) in humans, with
specific emphasis on SR patterns during and after exercise,
is reviewed.

How do endothelial cells sense

shear stress?
Several theories have been proposed as to how endothelial
cells sense SS as a physical force and transmit this information as an intracellular chemical signal. These theories
include ion channel activation, caveolae-mediated regulation of Ca2+, G-protein-coupled receptor activation, tyrosine

kinase receptor activation, adhesive protein activation, glycocalyx elongation, and bending of primary cilia.1,2

Shear stress sensing overview and

integrative theories
Ion channels
There are several flow-responsive ion channels that participate in sensing SS. The sequence of events begins with activation of inward rectifying K+ channels, followed by
activation of outward rectifying Cl channels. The K+ flux
initiates transmembrane hyperpolarization and drives Ca2+
entry into the cell;35 whereas non-selective cation channels
1 Indiana
2 Indiana

University, Bloomington, IN, USA

University School of Medicine, Bloomington, IN, USA

Corresponding author:
Blair D Johnson
1025 E. 7th Street
HPER 112
Bloomington, IN 47405
USA
Email: bj33@indiana.edu

366
control the magnitude of Ca2+ influx.6,7 Extracellular Ca2+
passage through the cell membrane follows activation of
two SS-dependent ion channels, namely P2X purinoceptors
and transient receptor potential channels.811 P2X puri
noceptors are activated by increases in endogenous
intracellular ATP, which has been shown to be SS dosedependent.1214 The SS-induced augmentation of intracellular ATP appears to be the result of cell surface ATP
synthase bound to caveolae and lipid rafts within the cell
surface.15 The influx of Ca2+ into the EC leads to activation
of Ca2+-dependent signaling pathways. Endotheliumderived nitric oxide (NO), which modulates vascular tone,
flow-dependent dilation, and vascular remodeling, is generated due to the Ca2+-induced release of caveolae-bound
endothelial NO synthase (eNOS).16 Removal of extracellular Na+, as well as the addition of a voltage-gated Na+
antagonist, attenuated the SS-induced activation of extracellular signal-regulated kinase (ERK).17 These results suggest that this class of voltage-gated ion channels is involved
with sensing SS despite concerns raised over the low membrane potential of EC and the slow changes in membrane
polarization, both of which argue against the involvement
of SS sensing voltage-gated channels.18
There are currently three theories on how these channels
are activated in response to SS. The first theory is that there
is a direct physical interaction between blood flow and the
ion channel. The ion channel is pushed open due to the
drag force that is elicited by blood flow, presumably in a
non-specific way related to the magnitude of force generated by SS. In a review by Barakat and colleagues, the
authors explore this theory by mathematically modeling the
energy due to blood flow drag and concluded that blood
flow drag energy is far below the energy required to force
open the membrane-bound channel.2
A second theory on how ion channels sense SS is through
ion channel interaction with the cytoskeleton, which
appears crucial for SS-induced production of NO.19 As SS
changes the mechanical tension on the cytoskeleton, the ion
channel that is associated with the cytoskeleton is activated
allowing ion flux through the cell membrane.20 Indeed,
mathematical modeling data suggest that flow may deform
sensors and cytoskeletal elements, which may initiate
SS-induced intracellular signaling cascades; however, the
presumed flow patterns were not representative of flow
through the arterial system.21 EC are exposed to both SS
and circumferential stretch in vivo, which complicates the
ability to decipher the effects of cytoskeleton deformation
on EC ion channel conductance. Although the mechanisms
underlying these interactions are not well understood, the
potential viability of this theory is supported by observations that shear-induced cellular deformation and cytoskeleton reorientation can activate eNOS and increase NO
production.4,22
The third prevailing theory on how ion channels sense
SS is through changes in cell membrane fluidity. As blood
flow moves across the cell surface the viscosity of the lipid
bilayer is altered.23,24 Evidence in support of this theory
includes observations that reducing EC membrane fluidity
by depleting the cellular membrane of cholesterol leads to

Vascular Medicine 16(5)

ERK activation and abolition of the eNOS response to
SS.25,26
Although Na+ channels have not been studied at length,
these channels may also be involved in detecting SS. Na+
channels activated by flow have been found in mammalian
EC,27 and Na+ influx through voltage-gated Na+ channels
has been observed to inhibit shear induced ERK1/2 activation,17 indicating that EC shear-related signal transduction
may be altered by extracellular Na+.

Summary
The physicochemical mechanisms that underlie the transduction of shear forces into effects on endothelial cell transmembrane channel activation and intracellular signaling
responses are incompletely understood. The current best
evidence supports participation of membrane cytoskeleton
and membrane fluidity in this effect.

Shear stress sensing cell

membrane components
Caveolae
The plasma membrane of endothelial cells is internally lined
with small invaginations called caveolae, which may work
in conjunction with ion channels and Ca2+ signaling,28 as
well as house ATP synthase.15 A role for caveolae in sensing
SS is suggested by observations that flow-induced Ca2+
responses start at the caveolae and then migrate as a Ca2+
wave across the entire cell membrane.16 Also, caveolae are
well recognized as providing functional compartmentalization of eNOS, and caveolae-bound eNOS is released into the
cytoplasm in response to the Ca2+ wave allowing eNOS to
become phosphorylated and subsequently produce NO.16

G-protein-coupled receptors
G-protein-coupled receptors have been implicated in
ligand-independent mechanotransduction of SS. SS activates bradykinin B2 G-protein-coupled receptors via a conformational change, possibly due to changes in cell
membrane fluidity.29 Mice lacking B2 G-protein-coupled
receptors exhibited a blunted response to blood flow.30
However, purified G proteins have been found to be activated in response to SS without their associated receptor,31
suggesting that G proteins sense SS independently.

Tyrosine kinase receptors

SS can activate tyrosine kinase receptors (i.e. VEGFR2,
Tie-2) through phosphorylation, independent of their cognate ligands.3235 A possible mechanism for the activation
of VEGFR2 is the SS-induced movement of VEGF2 monomers that initiate dimerization of VEGFR2.33 The phosphorylation of VEGFR2 could also be caused by the SS-induced
ATP release from caveolae and lipid raft-bound ATP synthase.15 Several signal transduction pathways are initiated
through the phosphorylation of tyrosine kinase receptors

367

Johnson BD et al.
including ERK, c-Jun N-terminal kinases (JNK), PI3kinase, and Akt.1 Activation of PI3-kinase and Akt signal
transduction pathways are involved with eNOS phosphorylation and subsequent NO production.36

Summary of intracellular events

A number of signaling pathways and other intracellular
events have been shown to contribute to the responses to
shear stress. The known interaction of NOS with caveolae
is recognized as an important component of the SS response.
Signaling through G-protein-coupled receptors and tyro
sine kinase receptors contribute as well, but the relative
importance of these mechanisms is not known, and it is not
known whether these pathways are sufficient to explain the
observed responses.

Shear stress sensing extracellular

components
Cell adhesion molecules
Several cell adhesion molecules have been associated with
sensing SS. Integrins have been found to be activated by SS
and integrin activation in turn subsequently leads to the activation of the Ras-ERK signal transduction pathway.37,38 The
integrins may transmit SS signals to the cytoskeleton.39,40
The Ras-ERK signal transduction pathway is also initiated
through the SS-induced phosphorylation of the platelet EC
adhesion molecule (PECAM)-1 located in cell junctions.41
Vascular endothelial cadherins may be integral in the activation of PI3-kinase and Akt pathways through the SS-induced
phosphorylation of PECAM-1. These cadherins appear to
form a tertiary complex with VEGFR2 and PECAM-1 in
response to SS.42 Within this complex, cadherins act as an
adaptor to form signaling complexes.42

sensing SS in both mouse and human EC, and the resulting

SS mechanotransduction activates Ca2+-dependent signaling cascades.48,49 Primary cilia also appear to disassemble
in response to laminar SS.50 Additionally, when primary
cilia are found in vivo, they appear to localize in areas
which are prone to atherosclerosis,51 which indicates they
may be involved with sensing low SS.

Summary of extracellular events

It is very likely that more than one of these proposed
mechanisms explain how EC sense SS, as increases of intracellular Ca2+ can result from several mechanosensor mechanisms. Interactions among these mechanosensors are
thought to coordinate the initiation of SS-induced signal
transduction pathways which can modify EC phenotype,
gene expression, and EC anti-atherogenic functions.52
Further in vitro research is warranted to determine how
multiple sensors collectively interact to detect SS of various
magnitudes and flow patterns and how the resulting interaction influences a pro- or anti-atherogenic environment.

Shear stress intracellular responses

Shear stress-induced signal transduction in
endothelial cells
SS activates a number of signal transduction pathways in
EC, which are summarized in Figure 1. Several of these
pathways have been previously mentioned; other important
components of the response include focal adhesion kinase
(FAK),53 Rho family GTPases,54 PI3-kinase,55 mitogenactivated protein kinases (MAPKs),56,57 protein kinase C
(PKC),56 and nuclear factor-B (NF-B).58
FAK, a tyrosine kinase, is involved with the activation
of the Ras/MAPK pathway and is co-localized with

Glycocalyx
The glycocalyx is a network of glycosylated and sialated
transmembrane proteins that protrude from the EC surface
into the arterial lumen and are coiled under no-flow conditions. As blood flow increases, the glycocalyx, with a net
negative charge on the surface layer,43 becomes uncoiled in
the direction of flow causing a conformational change
which increases Na+ ion binding sites that initiate signal
transduction pathways.44,45 Another postulated role of the
glycocalyx in SS mechanotransduction is the transmission
of SS through the core of the glycocalyx protein, glypican,
to the caveolae,43 where phosphorylation of eNOS likely
occurs through the Src pathway.46

Primary cilia
Another proposed sensing mechanism involves bending of
primary cilia located on the EC surface in response to SS.
This bending is thought to allow Ca2+ flux by increasing
permeability through ion channels resulting in Ca2+-induced
signal transduction.47 Indeed, polycystin-1 and -2, which
are localized on EC cilia, are integrally involved with

Shear Stress

G-ProteinCoupled
Receptors

Ion
Channels

Caveolae

Tyrosine
Kinase
Receptors

Glycocalyx

Primary
Cilia

Cellular
Adhesion
Molecules

Signal Transduction Pathways

MAPK, Ras-ERK, C-JNK, PI3-Kinase, Akt, FAK,
Rho Family GTPases, NF-B, & PKC

Endothelial
Function

Gene
Expression

Figure 1. Shear stress appears to be sensed in concert by

multiple mechanotransducers located on the membrane of
endothelial cells, which initiate several signal transduction pathways
that alter gene expression and function. (MAPK, mitogen-activated
protein kinase; ERK, extracellular signal-regulated kinase; C-JNK,
c-Jun N-terminal kinases; PI3, phosphoinositide 3; FAK, focal
adhesion kinase; GTP, guanosine triphosphate; NF-B, nuclear
factor-kappaB; PKC, protein kinase C.)

368
integrins. SS causes an increase in FAK phosphorylation
along with FAK activity and it is associated with growth
factor receptor binding protein 2/son of sevenless complex,
which is involved with Ras/MAPK activation in integrinmediated cellular adhesion.37,38
SS causes activation of Cdc42 and RhoA, which are
members of the Rho family GTPases, which require integrin and extracellular matrix interactions.59 The major functions of this family of GTPases in response to SS on EC are
to activate JNK/activator protein (AP)-1, NF-B, and
c-fos.6062 c-Fos encodes the AP-1,63 which appears to regulate endothelin-164 and monocyte chemoattractant protein-1
gene expression,65 and NF-B controls the expression of
inflammatory cytokines,66 and adhesion molecules.67
PI3-kinase is rapidly activated by SS in EC, which
leads to the production of phosphoinositide 3,4,5trisphosphate and phosphoinositide 3,4-bisphosphate,
which are involved with several downstream signaling
pathways.68 Akt, which is downstream of PI3-kinase, is
involved with cellular proliferation,69 protection against
apoptotic stimuli,70 and eNOS phosphorylation, which
leads to NO production in response to SS.36 Therefore,
the PI3-kinase-Akt signaling pathway is vitally important for flow-dependent dilation and providing the antiatherogenic properties of NO.
MAPKs are a family of Ser/Thr kinases that include
p38, ERK and JNK. p38 is involved with MAPK signaling
and is activated in response to SS along with ERK71 and
JNK.72 ERK activation is dependent on kinases and Ca2+
signaling pathways, whereas JNK is mediated through the
PI3-kinase/Akt pathway.55 Activation of the p38 pathway
induces the production of pro-inflammatory cytokines73
and adhesion molecules,74 and is involved with cellular
apoptosis.75 JNK activation also appears to be involved
with cellular apoptosis, and ERK has been implicated in
mediating cell growth.76 MAPKs are involved with several
EC gene transcription factors, which include AP-157 and
c-myc.77 AP-1 is involved with mediating thrombin activation of endothelin-164 and monocyte chemoattractant
protein-1 gene expression.65
The PKC pathway is sensitive to SS in EC. PKC activation by SS subsequently activates ERK downstream78
along with activating the monocyte chemotactic protein 1
promotor.79 PKC activation is also involved with gene
expression of endothelin-180 and platelet-derived growth
factor.81 Endothelin-1 induces vasoconstriction and
increases the activity of the renin angiotensin system, sympathetic nerve activity, and macrophage activation,82 while
platelet-derived growth factor has trophic effects on
smooth muscle cells and endothelial cells and is implicated
in atherogenesis.83
NF-B is a transcription factor located in the cytoplasm
of EC and is tonically inhibited by binding to the inhibitor
of B (IB). SS causes the phosphorylation of IB, which
subsequently dissociates from NF-B.84 The free NF-B
translocates to the nucleus of the EC to control the expression of cytokines (i.e. IL-6 and IL-8) and adhesion molecules (i.e. ICAM-1 and VCAM-1) involved in the
atherosclerotic process.85

Vascular Medicine 16(5)

SS alters the expression of approximately 3000 endothel
ial cell genes.86 In general, SS induces EC expression of
genes that affect growth factors, adhesion molecules, vasoactive substances, endogenous antioxidants, coagulation
factors, and chemoattractants. mRNA of growth factors
which are increased in response to EC SS are plateletderived growth factor-A and -B,87 basic fibroblast growth
factor,88 heparin-binding epidermal growth factor-like
growth factor,89 and transforming growth factor-.90
Adhesion molecule gene expression is down-regulated in
response to SS. Vascular adhesion molecule-1 (VCAM1)91 is down-regulated in response to steady SS, which
results in a decrease of leukocyte adhesion to the vascular
wall.1,52,67 Vasodilator production of NO92,93 and prostacyclin94 are increased in response to steady SS and the vasoconstrictor endothelin-1 is decreased.95 Anti-thrombotic
genes are also up-regulated after EC exposure to steady
SS. Tissue plasminogen activator,96,97 thrombomodulin,98
and cylcooxygenase-299 gene expression are increased in
response to sustained SS. Two forms of superoxide dismutase (SOD) (Mn and Cu/Zn) genes are increased
following SS exposure, which enhances the capacity to
mitigate reactive oxygen species.99103103 In summary, SS
tends to induce EC gene expression, which results in an
anti-atherogenic environment. However, the expression of
some of these EC genes can be differentially regulated
when patterns of SS are manipulated or when blood flow is
disturbed at bifurcations in the arterial tree.

Methods of applying shear stress in

cell culture
Cone and plate flow systems
Manipulating SS patterns in cell culture can be performed
by using a cone and plate flow system. This system utilizes
a Teflon cone positioned in the center of a tissue culture
dish.104 The tip of the cone is placed in the medium and the
cone is rotated to produce uniform flow conditions across
the cultured endothelium. The SS is calculated as follows:
w = /
where is the rotation speed, is the viscosity of the
fluid, and is the angle of the cone.104

Parallel plate flow systems

The most common method of subjecting SS to cultured cells
is the parallel plate flow system.104 A gasket is placed on the
bottom of a tissue culture dish with a standard rectangular
cut-out channel which can be modified to induce various
areas of disturbed flow within the flow domain. Inlet and
outlet ports are located at either end of the rectangular cutout flow domain in order to create flow across the cultured
EC. The SS applied to the endothelium is calculated as
follows:
= 6Q/wh2

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Johnson BD et al.
where Q is flow, is the fluid viscosity, w is the width of
the channel, and h is the thickness of the gasket.104

Table 1. Summary of the cellular effects of oscillatory and

laminar shear on endothelial cells in vitro
Oscillatory shear

Types of shear stress

Endothelial cells are subjected to various forms and magnitudes of SS in vivo. Mimicking these patterns produces differences in the resulting EC phenotypes and in the factors
that are produced by EC due to SS, differing by the type of
SS applied. Laminar blood flow produces predominantly
antegrade SS along the EC surface, whereas bifurcations in
the arterial tree disturb the laminar blood flow to produce
areas of low SS, retrograde SS, and oscillatory SS.

Laminar blood flow

Laminar blood flow is characteristic of steady, undisturbed
blood flow that creates a constant SS along the EC surface.
A steady laminar flow activates both K+ and Cl channels
that leads to depolarization of the cell membrane.105 Flowdependent K+ channels are initially activated and reach
peak activation at different magnitudes of SS than Cl
dependent channels.105 EC may sense the magnitude of SS
based on the relative amplitudes of the K+ and Cl currents105 or through the SS-induced dose-dependent influx
of Ca2+ through cation channels.12,13,106 The influx of Ca2+
into the EC causes activation of PKC and subsequent activation of MAPKs, which modulate transcription factors
and gene expression along with other signal transduction
pathways which may be activated through additional methods of SS detection.52
EC exposed to laminar blood flow and SS typically
exhibit an anti-atherogenic phenotype that is accompanied
by expression of anti-atherogenic genes. One of the most
important anti-atherogenic molecules produced in vivo is
NO, which is catalyzed by eNOS in response to SS, insulin,
and acetylcholine. eNOS mRNA expression in cultured EC
is up-regulated in response to 1 hour of laminar SS.107
Isolated and cannulated porcine coronary arterioles were
subjected to three doses of laminar SS (no shear, low shear,
and high shear) for 2 hours and 4 hours, and only high SS
induced an increase of eNOS mRNA following both time
periods.108 Additionally, exercise training has been shown
to increase eNOS protein and activation through a
SS-dependent mechanism;100,109,110 however, the increased
SS during and after a single session of exercise has antegrade and retrograde flow patterns that create oscillatory
shear across the EC surface.111 The increased production of
NO through the up-regulation and activity of eNOS produces an anti-atherogenic environment as NO inhibits several aspects of atherogenesis, including smooth muscle cell
proliferation and migration, platelet aggregation and leukocyte adhesion to the vascular wall, improves fibrinolysis,
regulates permeability and vasomotor tone, as well as act as
an antioxidant under situations of increased superoxide
anion concentrations.112
As previously mentioned, SS increases SOD mRNA and
protein content to reduce oxidative stress, a key component
in atherogenesis. Indeed, Woodman and colleagues observed

Laminar shear

E-selectin expression
ICAM-1 expression
Endothelin-1 expression
Macromolecule uptake
NF-B

Prostacyclin
Nitric oxide production
eNOS mRNA
Superoxide dismutase mRNA
Tissue plasminogen activator gene
expression
Superoxide release
Thrombomodulin gene expression
NADH oxidase activity Cyclooxygenase-2 gene
expression

Activation of p53

Inhibitors of apoptosis proteins 1

and 2

Retinoblastoma phosphorylation

Plasminogen activator inhibitor

type-1 gene expression

an increased SOD mRNA in porcine arterioles following

4 hours of high laminar SS; whereas stagnant SS and low
SS conditions did not affect SOD mRNA.108 However, the
increased antioxidant capacity may be a secondary response
to increased superoxide radical production by SS-stimulated
nicotinamide adenine dinucleotide (NADH) oxidase,113 and
not necessarily a direct effect of SS on EC.
Atherosclerosis progression is also mediated through the
coagulation cascade, which is also affected by laminar SS.
As previously mentioned, tissue plasminogen activator,
thrombomodulin, and cylcooxygenase-2 gene expression
are up-regulated and plasminogen activator inhibitor type-1
is down-regulated in response to steady SS. SS also rapidly
induces the production of the vasodilator NO, which appears
to inhibit platelet aggregation by increasing platelet cyclic
GMP and through the S-nitrosylation of platelet proteins.114
Laminar SS has profound effects on EC proliferation
and apoptosis. Twenty-four hours of laminar SS has been
observed to be a potential inhibitor of EC proliferation by
activating p53, which increases growth arrest proteins and
reduces EC proliferation.115 Exposed to 18 hours of
SS-suppressed EC apoptosis despite being treated with proapoptotic stimuli such as TNF-, oxygen radicals, and oxidized LDL, through inhibition of interleukin 1-converting
enzyme and capase-3.116 The PI3-kinase/Akt survival pathway has also been observed to be activated following 1 h of
SS exposure.70 Furthermore, the up-regulated activity of Cu/
Zn SOD and eNOS due to SS appears to suppress cellular
apoptosis.100 Inhibitor of apoptosis protein 1 is up-regulated
in response to 24 hours of laminar SS,117 whereas inhibitor
of apoptosis protein 2 is increased following 4 hours of laminar SS.118 Retinoblastoma protein, a cellular regulator of
cell cycles, is hypophosphorylated in response to 24 hours
of laminar SS, thus reducing cellular proliferation.115 These
effects collectively produce an anti-atherogenic environment by reducing EC turnover, thus preventing lesions in
straight segments of the arterial tree where laminar flow is
prevalent. A summary of the cellular effects of laminar SS
on endothelial cells in vitro is provided in Table 1.

370

Oscillatory blood flow

Bifurcations in the arterial tree modify laminar blood flow
and SS into areas of low SS and oscillatory flow patterns
beyond the bifurcation. These areas of low and oscillatory
SS are prone to atherosclerotic lesions.119 Cheng and colleagues manipulated the SS pattern in vivo.120 Mice were
outfitted with a cast on the carotid artery to manipulate SS
to produce areas of high laminar SS, low SS and oscillatory
SS while on an atherogenic diet. The areas of low SS produced vulnerable atherosclerotic lesions whereas oscillatory SS produced stable atherosclerotic lesions after 6
weeks of the atherogenic diet.120 The low SS areas also had
larger lesions which contained more lipids, fewer smooth
muscle cells, less collagen, and protruded less into the
lumen, produced more pro-inflammatory mediators and
intraplaque hemorrhages when compared to the oscillatory
regions. On the other hand, the areas of high laminar SS did
not exhibit atherosclerotic lesions following 6 weeks of the
experiment.120 This SS manipulation in vivo provides potent
evidence for the negative effects of oscillatory and low SS.
The frequency of oscillatory SS may have important
implications in EC function and gene expression.
Oscillatory SS with frequencies of 0.2 or 1.0 Hz have been
shown to fully activate K+ channels while only minimally
affecting Cl channels and hyperpolarizing the cell membrane.105 A greater frequency of oscillatory SS, 5 Hz has
been shown to not activate either K+ or Cl channels. A
high frequency of oscillatory SS with little net directional
flow may be too fast for the EC ion channels to detect2 as
there is no net influx of Ca2+ into the cell during periods
of pure oscillation.121 The recognition of oscillatory SS by
EC may be sensed by the hyperpolarized cell membrane
which in turn induces the pro-atherogenic environment.
Oscillatory flow has been shown to induce the expression of ICAM-1, E-selectin, and endothelin-1, increase
monocyte-EC attachment through NF-B, as well as
increase EC macromolecule uptake; whereas steady laminar
flow down-regulates VCAM-1 and endothelin-1.52 The
expression of ICAM-1 and VCAM-1 may be sensitive to the
oxidative state of the cell as antioxidant treatment of
N-acetylcysteine prevented the 24-hour oscillatory
SS-induced expression of VCAM-1 and reduced ICAM-1
expression by nearly 70%.122 Indeed, oxidative stress is
induced by oscillatory SS through the increase of intracellular superoxide radicals and NADH oxidase activity.113 It
has also been suggested that NAD(P)H oxidase maintains
the function of xanthine oxidase during periods of oscillatory SS to produce superoxide radicals in EC.123 The complex interaction between oxidative stress and adhesion
molecule gene expression during periods of oscillatory SS
underscore the importance of reducing superoxide radicals
in EC by increasing antioxidant capacity or reducing superoxide generation. A summary of the cellular effects of oscillatory SS on endothelial cells in vitro is provided in Table 1.
The recognition and transmission of SS to biochemical
processes in EC is a complex and integrated series of events
which generates a wide range of pro- and anti-atherogenic
functions and phenotypes contingent upon the type of SS
exposure. Additional research that examines the regulation

Vascular Medicine 16(5)

and interaction of downstream intracellular signaling pathways which affect EC phenotype and function in response
to SS which accurately mimics in vivo patterns merits further investigation.
Although extensive research has focused on SS in vitro,
the extrapolation of these results to in vivo events has its
limitations. In vitro research commonly attempts to mimic
the vascular milieu in vivo; however, it may over simplify the
effects of SS on EC as the combined magnitude of SS and the
corresponding circumferential strain, or lack thereof, may
not be applicable in vivo. Furthermore, in vitro research does
not account for additional mechanisms which may regulate
EC phenotype and function in vivo. Estimated SS at rest in
mice conduit arteries (1.28.4 Pa)124 is far greater than shear
exhibited in human conduit arteries (0.41.3 Pa),125 further
confounding the extension of animal results to humans.

Human studies
The data presented above were derived from studies conducted on EC from various animals or human umbilical
veins in cell culture. We now turn to studies on SS and SS
patterns at rest as well as during and following aerobic exercise in humans and, where applicable, how the SS affects
endothelial function. However, SS is not commonly assessed
in humans due to the difficulty of measuring the viscosity of
blood in direct contact with the endothelium. Owing to this
restraint, shear rate (SR) is used as an estimation of SS in
humans and is generally defined as blood velocity divided
by arterial diameter. Arterial blood velocity (both antegrade
and retrograde) and diameters are typically viewed using a
duplex ultrasound which images simultaneous pulsed-wave
Doppler blood velocities and two-dimensional B-mode
diameters.111 Additional Doppler envelope detection and
arterial wall tracking software is used to analyze the magnitude of antegrade and retrograde blood velocities and measure arterial diameters, respectively.

Shear rate in conduit arteries feeding the

working limbs
Tinken and colleagues performed a study to address the significance of exercise-induced SR in the brachial artery following 8 weeks of bilateral handgrip exercise for the
improvement of flow-mediated dilation (FMD),126 which is
an assessment of endothelium derived NO127130 (Table 2).
One forearm served as the non-cuffed control, whereas the
contralateral arm had a blood pressure cuff inflated to 60
mmHg to alter the magnitude of exercise-induced SR.
Mean SR, antegrade SR, and retrograde SR were increased
above baseline in the non-cuffed arm whereas the cuffed
arm did not receive a change in mean, antegrade, or retrograde SR during acute handgrip exercise.126 An increased
FMD was seen in the non-cuffed arm at weeks 2, 4, and 6.
The cuffed arm maintained the same FMD throughout the
8-week training program.126 These findings emphasize the
importance of augmented SR during exercise as a means to
improve FMD. Studies which have focused on the effects
of shear on FMD are summarized in Table 2.

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Johnson BD et al.
Table 2. Summary of the effects of shear in humans on endothelial function
Author (year)

Research question

Thijssen et al.
(2009)144

How do different
magnitudes of acute
retrograde SR affect EF?
How do different acute
SR stimuli affect EF?

Tinken et al.
(2009)134

Padilla et al.
(2009)148
Tinken et al.
(2010)126

Intervention

30 minutes of partial
forearm cuff occlusion
vs control arm
30 minutes of forearm
heating, handgrip exercise,
and leg cycling with partial
forearm cuff occlusion on
one arm
Does acute exposure to
3 hours of arm hanging
low SR and high hydrostatic (brachial artery) vs sitting
pressure influence EF?
(popliteal artery)
Is exercise-induced
8 weeks of bilateral
SR responsible for
handgrip training with SR
improvements in EF after restriction in one arm
training?

Hemodynamic influence

Outcome

Three distinct doses of

mean and retrograde SR
in cuffed arm
Reduced mean and
antegrade SR in cuffed
arm for all conditions

Progressive doseresponse
decline of FMD in cuffed
arm
Reduced FMD in cuffed arm
following leg cycling only

Low SR with high

hydrostatic pressure

Reduction of FMD in
brachial artery only

Attenuated exerciseinduced SR in one arm

during training

Improved FMD at 2, 4, and

6 weeks in non-restricted
arm; unaltered FMD in
shear-restricted arm

SR, shear rate; EF, endothelial function; FMD, flow-mediated dilation.

Shear rate in conduit arteries feeding

non-working limbs during exercise
Blood flow and SR increase in the non-exercising limbs
during exercise, but not nearly to the same extent as in exercising limbs.111,131133 It is postulated that this increased SR
to non-exercising limbs during exercise is partially responsible for the improvement in vascular function assessed in
non-exercise trained limbs.134,135
The magnitude of SR in non-working limbs during exercise appears to be dependent on exercise intensity.
Antegrade blood flow through the brachial artery was
measured during incremental lower body cycle exercise
and significantly increased at a somewhat low power output of 60 W and continued to increase up to 160 W (end of
data collection).111 Green and colleagues had also found
that brachial artery retrograde blood flow progressively
increased, starting at 40 W during 3-minute stages of incremental cycle ergometer exercise.111 Tanaka and colleagues
found a similar increase in brachial artery blood flow and
SS starting at 60 W during 3-minute stages of incremental
lower body cycle ergometer exercise, and found a progressive increase in femoral blood flow and SS starting at 20 W
during 5-minute stages of incremental arm crank exercise.133
Five 3-minute stages were used to observe dose-dependent
increases of brachial artery mean blood flow and SR during
walking at low speeds (3, 4, and 5 km/h), repeated kicking
with three different loads (5 kg, 7.5 kg, and 10 kg), and
cycling at three various workloads (60 W, 80 W, and 120
W).131 Three 30-minute interventions (forearm heating,
handgrip exercise, and lower body cycling) with partial
lower arm occlusion randomly placed on one arm were performed by healthy volunteers134 (Table 2). Although retrograde flow was not significantly altered in the two exercise
treatments, mean SR and antegrade SR were both significantly reduced due to the lower arm occlusion. The change
in SR patterns during the exercise treatments altered local
FMD. The non-cuffed arm significantly increased FMD
following both exercise conditions, whereas the cuffed arm
during cycling exercise had decreased FMD and the cuffed

arm during handgrip exercise was not significantly

altered.134 These results suggest that mean and antegrade
SR increase in an exercise intensity dose-dependent manner during exercise of short duration (35 minutes), and
reduced mean or antegrade SR during exercise can directly
impact endothelial function. These results also underscore
the importance of increased SR during exercise for
improved FMD.
Exercise intensity is associated with the magnitude of
antegrade SR in non-working limbs and thus high-intensity
exercise may generate SR, which produces the greatest
improvement in endothelial function. However, this is not
necessarily the case. Acute high-intensity exercise has been
observed to increase oxidative stress136 and exercise training
has been shown to elicit decreased circulating antioxidants.137
A decreased endothelial function following both acute and
chronic high-intensity exercise have also been reported.136,137
Conversely, acute moderate intensity exercise appears to
reduce oxidative stress and increase FMD,136 and moderate
intensity exercise training appears to augment antioxidant
defense138 and endothelial function.139 Collectively these
results suggest that the hormesis theory for adaptation
applies to antioxidant and endothelial alterations following
acute and chronic exercise.140,141
The increased oxidative stress from high-intensity exercise bouts may be initiated by the augmented oscillatory and
retrograde SS that is associated with greater work rates.
Oscillatory SS has been observed to increase superoxide
generation in cultured EC by increasing NADH oxidase and
xanthine oxidase activity.113,123 Retrograde blood flow and
SR have been reported to be exercise intensity-dependent to
the same extent as antegrade blood flow. Green and colleagues earlier work observed the progressive increase in
retrograde blood flow and SR in the brachial artery during
incremental cycle exercise, which started slightly earlier in
the protocol (40 W) when compared to antegrade blood flow
(60 W).111,132 Walking is also a mode of exercise that progressively increases brachial artery retrograde blood flow and SR
with increasing exercise intensity, which elicits a greater

372
oscillatory SS on the EC surface.131 However, retrograde SS
in the brachial artery during leg kicking does not appear to
increase with exercise intensity.131 This mode of exercise
provides a possible model to examine the effect of exercise
training, without the impact of oscillatory SR, on endothelial
function. It could be speculated that if indeed repeated bouts
of oscillatory SR negatively affect long-term EC function,
then leg kick training may exhibit greater EC function
improvements when compared to other modes of training.

The effects of oscillatory shear at rest on

endothelial function
Oscillatory SR has been identified as a possible mechanism
for reduced endothelial function. Healthy older adults tend to
exhibit greater retrograde and oscillatory SR in the femoral
artery142 and typically have lower femoral artery endothelial
function when compared to healthy young adults.143 This suggests that increased retrograde blood flow in older adults is part
of the natural aging process and that the augmented retrograde
blood flow may have a negative impact on endothelial function. Thijssen et al. manipulated blood flow by partial forearm
occlusion to induce three distinct doses of increased retrograde
blood flow and SR in healthy men at rest144 (Table 2). There
was a progressive decrease of FMD that corresponded to the
increased retrograde SR. A significant correlation between
the change of FMD and the change of retrograde SR was also
found.144 These results indicate that increased retrograde SR
at rest, which increases oscillatory SR, causes a relative
reduction of FMD. Table 2 summarizes the effects of oscillatory SR on endothelial function in humans.

Potential modulators of shear rate profiles

What modulates retrograde and oscillatory SR? Thijssen
et al. recently demonstrated that healthy, able-bodied controls and spinal cord-injured subjects display similar SR
patterns in the femoral artery during arm crank exercise,
thus possibly ruling out increased sympathetic nervous system activation to the non-working limbs.145 However,
Padilla and colleagues subjected healthy men to several
sympathoexcitatory maneuvers which resulted in augmented muscle sympathetic nerve activity, retrograde and
oscillatory shear patterns, as well as an increase in arterial
blood pressure.146 Recent evidence also suggests that thermoregulatory vasodilation of the forearm vasculature modulates the magnitude of retrograde SR during prolonged leg
cycling.147 This leads to speculation that results obtained by
Green et al.,111 Tanaka et al.,133 and Thijssen et al.131 cannot
be extended beyond the 35-minute data collection periods.
Collectively, these findings indicate that there may be an
interaction among the degree or duration of sympathetic
activity (or lack thereof), arterial blood pressure, and thermoregulatory mechanisms which may alter shear patterns.
Alternative theories include increased blood flow reflection
due to amplified systolic arterial pressure accompanied by
augmented antegrade velocity, or an enhanced myogenic
response during diastole due to heightened systolic arterial
pressure during exercise.145

Vascular Medicine 16(5)

Shear rate and hydrostatic pressure

A reduction of shear with an accompanying increased
hydrostatic pressure also impacts endothelial function at
rest; however, the response appears to be limb-specific.
Padilla et al. investigated the impact of acute arm hanging
(a low SR combined with high arterial pressure used to
mimic the vascular conditions of the lower leg in a seated
position) in the brachial artery and acute sitting in the popliteal artery on FMD148 (Table 2). The observed response
was limb-specific; the brachial artery exhibited a reduction
of FMD, whereas FMD in the popliteal artery remained
unchanged.148 These findings suggest a possible adaptation
in the popliteal artery to these vascular conditions and a
possible increased sensitivity to the hemodynamic conditions observed in the brachial artery.148

Role of nitric oxide production during

dynamic aerobic exercise
NO appears to be vital in allowing SR to increase in the
brachial artery during lower body exercise. Green et al.
infused the eNOS inhibitor l-NMMA in the brachial artery
to reduce NO production during incremental lower body
cycle ergometer exercise.132 The reduced NO production,
due to l-NMMA, resulted in a decrease in antegrade blood
flow in the brachial artery at the highest intensity and
increased retrograde blood flow during moderate intensity
workloads. Goto and colleagues also examined the NO
response in the brachial artery during lower body cycle
exercise using l-NMMA as well.149 During the control condition, moderate intensity exercise (50% maximal oxygen
consumption) induced a decreased forearm vascular resistance and increased forearm blood flow; whereas mild
intensity exercise (25% maximal oxygen consumption) did
not alter the forearm vascular resistance or forearm blood
flow response.149 l-NMMA administration attenuated the
decrease in forearm vascular resistance during moderate
intensity exercise. The results of Green et al. and Goto and
et al. demonstrate the importance of NO in the regulation of
blood flow and blood flow patterns during exercise in the
non-working limbs.132,149 The increased NO production in
response to increased SS during exercise may be vital in the
protection against developing atherosclerosis due to the
anti-atherogenic properties of NO.

Brachial artery shear rate following aerobic

exercise
The increased SR during exercise appears to play a major role
in the prevention of atherosclerosis; furthermore, SR appears
to remain elevated following acute bouts of aerobic exercise
as well. Despite the possible beneficial role of elevated postexercise SR in endothelial function, literature is presently
scarce. Post-exercise SR was characterized by Padilla et al.
following 45 minutes of low, moderate, and high-intensity
exercise in older, overweight or obese men.150 High-intensity
exercise produced the greatest SR when compared to low and
moderate intensities. Unfortunately, baseline SR and retrograde SR were not reported and thus it is unclear if and/or

Johnson BD et al.
when SR returned to pre-exercise values within the 3-hour
window and if post-exercise retrograde SR was altered.150
Endothelial function in conduit arteries which feed
both the working and non-working limbs following acute
exercise and exercise training appears to be heavily influenced by the magnitude and pattern of the exerciseinduced SR. Additional research is warranted to investigate
the relationship between SR magnitude and patterns during various exercise modalities and the degree to which
the exercise-induced SR exerts anti- or pro-atherogenic
functions of the endothelium. Furthermore, potential in
vivo mechanisms (i.e. oxidative stress, adhesion molecule
expression, cytokines) responsible for altering endothelial
function following periods of augmented retrograde SR
deserve attention to further validate in vitro findings.

Summary
Endothelial cells appear to sense shear stress through several different mechanisms, which modulate many signal
transduction pathways to influence cell phenotype and
function. The type of shear stress to which the endothelial
cell is exposed determines the shear-induced changes in
intracellular signaling and gene expression, which in turn
elicits a spectrum of pro- or anti-atherogenic effects on the
endothelial cell. Arteries exposed to retrograde and oscillatory shear stress appear to activate the endothelium in vitro;
these types of shear patterns appear to impair endothelial
function in humans. Further studies are needed to investigate approaches to modulating shear stress with the goals
of mitigating adverse effects of unfavorable forms of shear
and improving vasodilator and anti-atherosclerotic functions of the vascular endothelium.
Funding
This research received no specific grant from any funding agency
in the public, commercial, or not-for-profit sectors.

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