(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:677-685.)
© 1998 American Heart Association, Inc.
Laminar Shear Stress
Mechanisms by Which Endothelial Cells Transduce an Atheroprotective Force
Oren Traub;
; Bradford C. Berk
From the Departments of Pathology (O.T.) and Medicine (B.C.B.), Division
of Cardiology, The University of Washington, Seattle.
Correspondence to Bradford C. Berk, Cardiology Division, Box 357710, The University of Washington, Seattle, WA 98195. E-mail bcberk{at}u.washington.edu
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Abstract
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AbstractMechanical forces are
important modulators of cellular function in many tissues and are
particularly important in the cardiovascular system.
The endothelium, by virtue of its unique location in
the vessel wall, responds rapidly and sensitively to the mechanical
conditions created by blood flow and the cardiac cycle. In this study,
we examine data which suggest that steady laminar shear stress
stimulates cellular responses that are essential for
endothelial cell function and are atheroprotective. We
explore the ability of shear stress to modulate atherogenesis via its
effects on endothelial-mediated alterations in
coagulation, leukocyte and monocyte migration, smooth muscle growth,
lipoprotein uptake and metabolism, and
endothelial cell survival. We also propose a model of
signal transduction for the endothelial cell response
to shear stress including possible mechanotransducers (integrins,
caveolae, ion channels, and G proteins), intermediate signaling
molecules (c-Src, ras, Raf, protein kinase C) and the mitogen
activated protein kinases (ERK1/2, JNK, p38, BMK-1), and
effector molecules (nitric oxide). The endothelial cell
response to shear stress may also provide a mechanism by which risk
factors such as hypertension, diabetes,
hypercholesterolemia, and sedentary lifestyle
act to promote atherosclerosis.
Key Words: endothelium shear stress atherosclerosis mechanotransduction signal transduction
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Introduction
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Numerous studies
suggest that normal functioning of the endothelium is
critical in limiting the development of
atherosclerosis, as illustrated by the correlation
between risk factors for atherosclerosis (smoking, high
cholesterol, high homocysteine, decreased estrogen,
increasing age, and hypertension) and endothelial
dysfunction.1 Endothelial cells
play a critical role in vascular homeostasis by performing many
functions. They sense and integrate hemodynamic and
hormonal stimuli and effect alterations in vascular function through
the secretion of various mediator proteins and
molecules.2 As a result of these properties,
endothelial cells modulate biological processes related
to the blood vessel wall, including regulation of the permeability of
plasma lipoproteins, adhesion of leukocytes, and release of
prothrombotic and antithrombotic factors, growth factors, and
vasoactive substances.3 Impairment of these
endothelial cellmediated processes has been
postulated to play a central role in the pathogenesis of
atherosclerosis.1
Just as other tissues have developed mechanisms to detect changes in
the physiological conditions to which they are
exposed, endothelial cells respond not only to humoral
factors in the circulation but also to the mechanical conditions
created by blood flow and the cardiac cycle.4 As
a result of their unique location, endothelial cells
experience three primary mechanical forces: pressure, created by the
hydrostatic forces of blood within the blood vessel; circumferential
stretch or tension, created as a result of defined intercellular
connections between the endothelial cells that exert
longitudinal forces on the cell during vasomotion; and shear stress,
the dragging frictional force created by blood flow. Of these forces,
shear stress appears to be a particularly important
hemodynamic force because it stimulates the release of
vasoactive substances and changes gene expression, cell
metabolism, and cell morphology.4
The nature and magnitude of shear stress plays an important role in
long-term maintenance of the structure and function of the
blood vessel. The nature of shear stress experienced by
endothelial cells is a function of blood flow patterns
throughout the vasculature generated by the cardiac cycle. In
"linear" areas of the vasculature, blood flows in ordered laminar
patterns in a pulsatile fashion dependent on the cardiac cycle, and
endothelial cells experience pulsatile shear stress
with fluctuations in magnitude that yields a mean positive shear
stress. This flow pattern should be distinguished from the flow pattern
that is often used in experimental preparations and that generates a
steady positive shear stress, being temporally and spatially uniform.
While steady shear stress generally stimulates many of the same
endothelial cell responses as pulsatile stress, there
are some qualitative and quantitative
differences.5 6 7 Cells exposed to positive shear
stress undergo reorientation, with their longitudinal axis parallel to
the direction of blood flow.8 9 This
reorientation streamlines the endothelial cell,
decreasing the effective resistance and lowering shear
stress,10 a phenomenon which may or may not be
important in terms of adaptation or filtering of shear stress
stimuli.4 At areas of abrupt curvatures in the
vasculature, as in the carotid bifurcation, the laminar flow of blood
is disrupted and separated flow patterns result. Specifically, the
medial wall of the carotid bulb experiences higher shear stress, while
the lateral wall experiences recirculation vortexes that vary with the
cardiac cycle, resulting in flow reversal.11
Thus, the lateral area of the carotid bulb experiences oscillatory
shear stress (periodic flow reversal with time-averaged shear stress
approaching zero) and low mean shear stress. As a result of the low
magnitude of the time-averaged shear stress, these cells do not
reorient12 and may be exposed to high shear
gradients (differences in shear stress magnitude on a cell
scale)4 because their lack of streamlining yields
a membrane topology in opposition to the mean shear vector. Several
investigators have demonstrated that endothelial cells
may actually be sensitive to the magnitude of the shear
gradient.12 13 14 Whether these shear gradients or
the time-averaged mean shear stress is more critical in terms of
atherogenesis remains to be defined. Nevertheless, the significance of
these flow patterns is demonstrated by studies that correlate
development of atherosclerotic lesions (fatty streaks and small
plaques) with areas of the carotid that experience these flow reversals
with low time-averaged shear stress.11 15 Regions
of the carotid bifurcation that experience pulsatile and mean positive
shear stress as the result of laminar blood flow patterns, however,
were relatively protected from atherosclerosis. Other
investigators have confirmed these observations throughout the
vasculature.16 The mechanism by which the
physical force generated by fluid shear stress is transduced into
biological signals remains unclear.
Below we will briefly review the atheroprotective effects of the
endothelium that are influenced by shear stress and
then discuss several signal-transduction mechanisms by which shear
stress exerts its beneficial effects on endothelial
function.
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Shear Stress and Endothelial Cell Biology:
Relevance to Atherosclerosis
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The hypothesis that physical injury to the
endothelium might precipitate the atherosclerotic
process was introduced over two decades ago. More recently this concept
has been modified to include biochemical and cellular alterations in
endothelial cell function. There is a strong
correlation between endothelial cell dysfunction and
areas of low mean shear stress and oscillatory flow with flow reversal
(Fig 1
). Manifestations of dysfunctional
endothelium can be readily observed in certain areas of
the arterial tree, such as branch points, which experience
low mean shear stress and flow reversal.11 These
sites demonstrate increased uptake of lipoproteins, appearance of
leukocyte adhesion molecules on the surface of the
endothelial cells, and leukocyte transmigration.
Secretion of chemotactic factors and growth factors causes
proliferation of resident monocyte/macrophages, as well as
smooth muscle cells. Smooth muscle cells synthesize a connective tissue
matrix comprised of elastic fibers, proteins, collagen, and
proteoglycans, and the accumulation of lipids and free and esterified
cholesterol follows.17 Recent data
suggest that low shear stress and, more importantly, oscillatory flow
and flow reversal are permissive or even causative in this pathogenic
process.4 19

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Figure 1. Endothelial cell biology and shear
stress. Steady laminar shear stress promotes release of factors from
endothelial cells that inhibit coagulation, migration
of leukocytes, and smooth muscle proliferation, while
simultaneously promoting endothelial cell
survival. Conversely, low shear stress and flow reversal shift the
profile of secreted factors and expressed surface molecules to one that
favors the opposite effects, thereby contributing to the development of
atherosclerosis. PGI2 indicates
prostacyclin; tPA, tissue plasminogen
activator; TGF-ß, transforming growth factor-ß; Ang II,
angiotensin II; and PDGF, platelet-derived growth
factor.
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In areas downstream of vessel bifurcations, laminar shear stress
predominates, and the endothelial cells experience
pulsatile flow, with shear stress on the order of 10 to 30
dyne/cm2.4 The
endothelium in these regions maintains circulatory and
blood vessel integrity through its ability to regulate several
different processes: coagulation, growth of underlying smooth muscle,
leukocyte adhesion to and transmigration into the blood vessel wall,
and lipoprotein uptake and metabolism.
Coagulation
Coagulation stimulates the release of powerful antithrombotic
agents from endothelial cells. Prostacyclin was the
first inhibitor of platelet aggregation shown to be
released from endothelial cells on exposure to shear
stress.7 18 Secretion of prostacyclin from
endothelial cells is enhanced when the shear stress is
pulsatile compared with steady.7 Numerous
investigators have demonstrated that shear stress is one of the most
powerful stimuli for release of the vasodilator
NO,2 20 21 which also possesses strong
antiplatelet aggregation properties.22
Shear stress can also stimulate release of factors that
inactivate the clotting cascade.5
Recent studies have shown that shear stress regulates generation of
thrombomodulin, which interacts with protein C and protein S to
inactivate certain clotting factors. Malek et
al23 reported that steady shear resulted in a
small transient increase in thrombomodulin expression but continued
exposure to shear resulted in decreased thrombomodulin expression in
bovine aortic endothelial cells. However, two other
laboratories reported that steady shear stress results in sustained
increased thrombomodulin expression in human umbilical vein
endothelial cells.24 25 The
reason for this disparity in species response is unclear. In addition
to the potential effect on thrombomodulin, fluid shear stress has also
been shown to stimulate expression of tissue plasminogen
activator23 24 26 and reduce
secretion of plasminogen activator
inhibitor type-1.24 Importantly,
endothelial cells exposed to turbulent flow failed to
show increases in thrombomodulin and tissue plasminogen
activator.
Leukocyte Adhesion and Migration
Endothelial cells regulate leukocyte adhesion and
migration of monocytes and leukocytes into the blood vessel wall by
secretion of chemotactic factors and expression of cell-surface
molecules. ICAM-1 binds ß2-integrins on various white blood cell
derivatives, while VCAM-1 mediates adhesion of monocytes to the
endothelium.27 VCAM-1 is one of
the earliest markers for fatty streaks and is upregulated in areas of
the endothelium surrounding atherosclerotic
plaques.27 Several investigators have
demonstrated an inverse relationship between VCAM-1 expression and
shear stress,28 29 30 31 32 33 which suggests that leukocyte
binding should be decreased under conditions of high shear stress.
However, other investigators have demonstrated that ICAM-1 expression
is upregulated by high shear stress34 35 36 and
that leukocyte binding after exposure to shear stress is
increased.35 36
The leukocyte binding experiments described above used static cultures
of endothelial cells that were exposed to shear stress
prior to the binding of leukocytes. However, these experimental
conditions do not accurately simulate the
physiological conditions of leukocyte binding and
the complex interplay between expression of ICAM-1, VCAM-1, and the
physical disruption of the leukocyte-endothelial cell
interaction by high levels of shear stress.37 38
A study performed by Walpola et al28 is helpful
in analyzing which of these parameters may be of
physiological importance. In this study, shear
stress in rabbit carotid arteries was altered and expression of
endothelial cell adhesion molecules, as well as
leukocyte binding, was measured. Low shear stress resulted in VCAM-1
expression 30 times greater than that of control vessels, while ICAM-1
expression fell to approximately 30% of control vessels. High levels
of shear stress also increased VCAM-1 expression (to 3.5 times that of
control vessels), while ICAM-1 expression levels increased (to 1.6
times that of control). Importantly, extensive monocyte adhesion was
noted under low shear stress, which colocalized to areas of VCAM-1
expression, while no monocyte adhesion was noted at high shear
conditions, indicating that low mean shear stress promotes leukocyte
binding in vivo compared with higher shear stress.
Another key factor in monocyte recruitment is the chemoattractant
peptide MCP-1.1 Shyy et
al39 showed that MCP-1 expression was transiently
increased in human umbilical vein endothelial cells on
exposure to shear stress. However, MCP-1 gene expression then decreased
to basal levels at 4 hours, and once gene expression was fully
suppressed, it remained so even after static incubation, leading the
authors to suggest that MCP-1 expression is likely suppressed in
endothelial cells exposed to steady pulsatile shear
stress. Thus, it seems probable that for conduit vessels, high shear
stress inhibits leukocyte binding and chemoattractant protein
expression while low shear stress and flow reversal promote leukocyte
binding and transmigration.
Proliferation
Smooth muscle proliferation is increased in atherosclerotic
lesions1 and is likely stimulated by
endothelial cell factors that are regulated by shear
stress. Kraiss et al40 showed that
endothelial platelet-derived growth factor-A mRNA
levels and smooth muscle proliferation were increased in areas that
experience low blood flow in an arteriovenous fistula model of altered
flow in baboons. Endothelin-1, a smooth muscle mitogen that acts
synergistically with platelet-derived growth factor, was shown to
be dramatically reduced by exposure to 25
dyne/cm2.41
NO42 and transforming growth
factor-ß,43 both inhibitors of
vascular smooth muscle cell growth, are secreted by
endothelial cells in response to shear stress.
Angiotensin II is an important growth factor for vascular
smooth muscle and may also be
antiapoptotic.44 Shear stress regulates
tissue levels of angiotensin II by virtue of changes in
angiotensin-converting enzyme expression. Rieder et
al45 recently demonstrated that prolonged
exposure to shear stress significantly reduced
angiotensin-converting enzyme mRNA and activity. With its
ability to regulate these disparate smooth muscle cell growth factors,
it seems likely that shear stress plays a role in the increased
proliferation of smooth muscle seen at areas of low shear stress and
flow reversal.
Lipoproteins
Unlike the effects of shear stress on growth factor secretion, the
role of shear stress in lipoprotein transport and LDL
metabolism is less well defined. Deng et
al46 reported that the concentration of LDL at
the surface of canine carotid arteries was inversely related to wall
shear stress rate and suggested that increased surface LDL
concentration results in an increased rate of lipid infiltration into
the blood vessel. This hypothesis is complemented by studies which
demonstrate that areas exposed to flow reversal are relatively
permeable to macromolecules including LDL and that LDL accumulation
within the vascular wall is preferentially localized to these areas of
disturbed flow. Berceli et al47 reported that the
LDL incorporation in the rabbit aortailiac bifurcation was elevated
in the lateral region that experiences flow reversal versus the medial
regions that experience higher steady shear, while no differences were
present in the transitional or unidirectional zone that experiences
relatively steady shear. Other investigators have confirmed these
findings in different areas of the vasculature for both
rabbits48 and pigs.49
Mechanistically, it appears that compromised
endothelial cell integrity, and hence increased
macromolecule permeability, results from high shear gradients that are
present in low shear stress/flow reversal conditions (See Weinbaum
and Chien50 for review). A study by Sprague et
al51 showed that 125I-LDL
internalization increased in bovine aortic endothelial
cells exposed to steady stress conditions for 24 hours; however, this
likely reflects an increased metabolic need for LDL under
steady shear conditions rather than increased LDL incorporation into
the arterial wall.52 Additional
studies to define the mechanistic details by which LDL accumulation is
linked to low shear stress and flow reversal conditions are
warranted.
Endothelial Cell Survival
Finally, shear stress may be critical for
endothelial cell survival. Early studies performed by
Davies et al12 demonstrated increased
endothelial cell turnover in areas that experience
turbulent shear stress conditions, suggesting compromised
endothelial cell integrity under these conditions.
Several recent studies report that the lack of shear stress triggers
apoptosis in endothelial
cells.53 54 Other investigators have demonstrated
that shear stress is required for optimal regeneration of an injured
endothelium. Vyalov et al55
reported that under low shear stress conditions,
endothelial cells on the border of a wound edge failed
to maintain contact with neighboring cells and were oriented randomly.
Further, the cells spread and migrated into wound sites more slowly.
While steady shear seems to be necessary for
endothelial cell integrity, several investigators have
demonstrated that steady shear inhibits proliferation of cultured
endothelial cells.56 Thus, it
appears that shear stress acts as an endothelial cell
"survival" factor rather than as a "growth" factor.
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NO: A Critical Factor in Shear StressMediated
Atheroprotection
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NO appears to be a key mediator of the atheroprotective effects of
shear stress on the blood vessel wall. NO has been reported to play a
role in platelet aggregation and leukocyte binding to the
endothelium, in inhibition of vascular smooth muscle
tone and growth, and in alteration of lipoprotein
metabolism.2 The ability of shear
stress to regulate these processes is abrogated by
inhibitors of NO production, suggesting that shear
stress exerts its effects through the release of NO. Further, it has
been postulated that the beneficial effects of regular aerobic
training, including its antiatherogenic properties, may be mediated
through shear-induced increases in NO
secretion.57
NO is produced by a unique enzyme present in the
endothelium, termed ecNOS.58 59 60
Shear stress is the most potent physiological
stimulus for NO production in endothelial
cells. Rapid increases in NO production are due to
posttranslational activation of ecNOS, while chronic alterations in
ecNOS expression are due to changes in gene expression. Experiments by
our laboratory and others61 62 indicate that two
distinct signaling pathways (a Ca2+-dependent and
a Ca2+-independent pathway) seem to be involved
in rapid shear-mediated increases in NO
production.63 We compared NO
production in response to the Ca2+
ionophore A23187 with shear stress. While A23187 increased NO
production by 3-fold to 6-fold, shear stress stimulated NO
production by 10-fold to 30-fold above static levels. The
initial rapid increase in NO required Ca2+, while
the sustained increase in NO production was independent of
changes in intracellular Ca2+.64 Further
experiments by our laboratory have demonstrated that ecNOS was
phosphorylated in response to
shear.64 Although the relationship between ecNOS
phosphorylation and NO production is unclear,
phosphorylation may regulate the activity of ecNOS. To
better understand how shear stress influences ecNOS activity and
expression, it will be necessary to identify upstream mediators of
ecNOS function that are activated by shear stress, such as
protein kinases.
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Mitogen-Activated Protein Kinases: Likely Signaling
Molecules in the Transduction of Shear Stress
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Several features of the endothelial cell
response to shear stress are analogous to receptor-mediated signaling:
dependence on G proteins, increase in intracellular
Ca2+, and changes in gene expression. The family
of kinases termed MAP kinases are potential candidates to mediate some
of the effects of shear stress on endothelial cells.
MAP kinases are ubiquitously expressed serine/threonine protein kinases
that are activated in response to a variety of extracellular
stimuli involved in cell growth, transformation, and differentiation
(Fig 2
). The extracellular
signalregulated kinases (ERK1/2), members of the MAP kinase family,
have many potential substrates, including other protein kinases
(p90rsk, MAPKAP, Raf-1, MEK), transcription factors (c-myc, c-jun,
c-fos, p62TCF), enzymes (cPLA2), and cell-surface
proteins (EGF receptor), and thus have many effects on cellular
physiology and gene expression.63

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Figure 2. MAP kinase activation pathways. A common theme in
the stimulation of MAP kinase family members is activation by an
immediate upstream MAP kinase kinase (MEK), which is in turn
activated by an immediate upstream MAP kinase kinase (MEKK).
Different stimuli activate different signaling pathways,
leading to individual MAP kinase activation.
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The pathway for ERK1/2 activation in response to growth factors has
been well characterized (Fig 2
). The MAP and ERK kinase (MEK-1) is a
dual-specificity kinase that phosphorylates ERK1/2 on
T-E-Y. MEK-1 is itself regulated by a MAP kinase kinase kinase, one of
which has been identified as Raf-1. Raf-1 is activated by
translocation to the membrane and association with the small
GTP-binding protein, ras. The GTPase activity of ras is regulated by a
complex involving Grb2 and mSOS which are recruited and
activated by a tyrosine kinase
receptor.65
We have recently reported that ERK1/2 is activated by shear
stress in endothelial cells in a time- and
force-dependent manner.66 Shear stress
stimulation of ERK1/2 was unaffected by treatment with the
Ca2+ chelator BAPTA-AM, indicating the response
was Ca2+ independent. These data, combined with
observations that ecNOS contains multiple consensus sites for
phosphorylation by a variety of kinases including
ERK1/2,63 make this pathway a likely candidate to
participate in the stimulation of sustained NO production in
response to shear stress. Additionally, several shear
stressresponsive genes contain elements (eg,
AP-1)39 67 that may be influenced by
ERK1/2-mediated phosphorylation of transcription
factors,63 such as c-fos, c-jun, and c-myc.
Another member of the MAP kinase family shown to be regulated by shear
stress is the stress-activated protein kinase JNK/SAPK. Two
laboratories have shown increases in JNK activity by shear stress,
although with varying kinetics.68 69 Preliminary
results in our laboratory show that shear stress inhibits tumor
necrosis factorstimulated JNK activity in endothelial
cells,69A a finding consistent with the recently
reported ability of shear stress to inhibit endothelial
cell apoptosis.53 54 Additional
experiments by our laboratory indicate that other members of the MAP
kinase family, p38 and BMK-1 (ERK5), are also activated by
shear stress in endothelial cells.69B
Experiments to determine the roles of individual MAP kinase family
members in endothelial gene expression will be
important areas for future research.
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Upstream Effectors of ERK1/2 Activity
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PKC is required for ERK1/2 activation in response to shear stress
because the ERK1/2 activation by shear stress reported by our group was
attenuated when endothelial cells were pretreated with
either phorbol ester for 24 hours or with staurosporine for
30 minutes66 (Fig 3
). PKCs are well characterized
serine/threonine kinases that are activated by a variety of
stimuli.70 A classification system for the PKC
family separates the different isoforms into distinct classes: the
"classical" PKC isoforms, (PKC-
, -ßI, -ßII, -
) are
Ca2+ independent and phorbol ester responsive;
the "novel" PKC isoforms (PKC-
, -
, -
, -
) are
Ca2+ independent and phorbol ester responsive;
and the "atypical" PKC isoforms (including
,
/
), are
Ca2+ independent and phorbol ester unresponsive.
Human umbilical vein and bovine aortic endothelial
cells express primarily three PKC isoforms: PKC-
, PKC-
, and
PKC-
.71 Through experiments using antisense
oligonucleotides to inhibit expression of the various
PKC isoforms in endothelial cells, we identified
PKC-
as the isoform necessary for the ERK1/2 stimulation by shear
stress.71

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Figure 3. Proposed model of shear stressmediated
mechanotransduction in endothelial cells. Primary
mechanosensors (eg, integrins, caveolae, G proteins, ion channels)
transduce physical stimuli into biochemical signals. Several stimuli
serve to activate Raf-1, including tyrosine
phosphorylation by c-Src or c-Srclike kinases, serine
and threonine phosphorylation by PKC, and GTP-bound
ras. Raf-1 activates MEK, which in turn activates
ERK1/2. Sustained generation of NO may result from the effects of
ERK1/2 or through direct effects of mechanosensors (eg, caveolae)
themselves.
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A recent study by Cai et al72 provides evidence
for the mechanism by which PKC activates ERK1/2. These
investigators demonstrated that activation of the MAP kinase kinase
kinase, Raf-1, a key activator of ERK1/2, is dependent on
several criteria: (1) recruitment of Raf-1 to the plasma membrane; (2)
activation of Raf-1 by the GTP-bound form of ras; (3) tyrosine
phosphorylation of Raf-1, presumably by c-Src or a
c-Src-family kinase; and (4) phosphorylation of Raf-1
on serine and threonine residues. Both PKC-
and PKC-
were
directly responsible for phosphorylation of serine and
threonine residues on Raf-1 in response to various stimuli. This
redundancy in PKC signaling to Raf-1 would allow for signaling
regardless of a rise in intracellular Ca2+, as
PKC-
is Ca2+ independent. Thus, it seems
likely that activation of PKC isoforms, particularly PKC-
, by shear
stress results in stimulation of ERK1/2 through their action on Raf-1,
regardless of intracellular Ca2+
concentration.
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Potential Shear Stress Receptors
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A question of great importance in the field of mechanotransduction
pertains to the identity of the primary mechanoreceptor(s) responsible
for initiating signal transduction. Transduction of mechanical forces
in anchorage-dependent cells is due to a combination of force
transmission via the cytoskeletal elements and transduction of the
physical forces to biochemical signals at mechanotransducer
sites.63 Based on the data presented
above, the candidate mechanotransducer molecules should be responsive
to shear stress over the physiological range and
result in the activation of a tyrosine kinase (eg, c-Src), PKC, and
ERK1/2. Due to their interaction with specific signaling molecules
already implicated in signal transduction, four candidates have been
proposed as likely mechanotransducers: integrin-matrix interactions,
specialized membrane microdomains, ion channels, and G proteins.
To sense and transduce signals in response to shear stress,
endothelial cells must be anchored to their
matrix.73 Integrins are ubiquitous
/ß
heterodimeric transmembrane glycoproteins that act as
adhesion receptors involved in the interaction between cells and
extracellular matrix. Integrins play an important role in biological
processes, including cell adhesion, cell migration, cell growth, tissue
organization, blood clotting, inflammation, target recognition by
leukocytes, and cell differentiation.74 Studies
performed by Wang et al75 and
Ingber76 using magnetic torsion have demonstrated
that integrins are capable of transducing mechanical stimuli to
biochemical signals. A recent study by Muller et
al77 showed that flow-induced vasodilation in
coronary arteries, which is mediated by NO release, could be
blocked with RGD peptides, which compete with the matrix for integrin
interactions. Similar attenuation of flow-induced vasodilation was
obtained if a blocking antibody against the ß3 integrin was employed,
supporting the hypothesis that integrins are involved in the
mechanotransduction of shear stress. Integrins are also a particularly
attractive candidate in that they have been reported to associate with
PKC71 and c-Srcfamily tyrosine
kinases.78 Through the use of antisense
oligonucleotides against the various PKC isoforms, our
laboratory has shown that adhesion-mediated activation of ERK1/2 in
human umbilical vein endothelial cells is dependent on
PKC-
and PKC-
,71 a finding that parallels
the data from Cai's group. Other studies by our laboratory have
demonstrated that activation of ß1 integrins (the predominant ß
isoform on endothelial cells) with an activating
antibody also stimulated ERK1/2, although at levels less than that
observed with shear stress.79 Further, human
endothelial cells showed adhesion-mediated ERK1/2
activation when plated on a matrix of fibronectin, which engages ß1
integrins, but showed no ERK1/2 activation when they adhered to matrix
consisting of poly-L-lysine.73 The
relatively small magnitude of ERK1/2 stimulation by integrin activation
does not preclude a key role for integrins in shear-mediated ERK1/2
activation; based on the importance of shear stress to
endothelial cell function and integrity, it is likely
that redundant pathways with different mechanotransducer molecules
mediate the full ERK/12 response to shear stress.
Another possible candidate for the transduction of shear stress into
biochemical signals are caveolae, specialized domains of the plasma
membrane that are rich in cholesterol. Because of their
high cholesterol content, caveolae are more rigid than
other portions of the plasma membrane. Caveolae are abundant in
endothelial cells and have been implicated in
transcytosis, ion movement across the membrane, and signal
transduction.80 The principal component of
caveolae is a 21- to 24-kD integral membrane protein called caveolin.
Caveolin seems to function as a scaffold for the recruitment and
sequestration of signaling molecules. Among signaling molecules known
to associate with caveolae are G proteins, c-Srcfamily tyrosine
kinases, ras, PKC, ecNOS,81 shc, Grb2, mSOS,
Raf-1, and ERK1/2 (see Reference 8282 ) Caveolae represent an
attractive site for mechanotransduction on the basis of their
biophysical characteristics and interactions with signaling molecules.
Experiments to determine the significance of caveolae and what effect
changes in caveolae number and properties may have in shear-mediated
signaling should prove an exciting area for future research.
Recent data reported by Gudi et al83 indicate
that G proteins may act as primary mechanosensors in
endothelial cells. This laboratory showed that
treatment of endothelial cells with antisense G
q
oligonucleotides inhibited shear stressinduced
ras-GTPase activity, while scrambled oligonucleotide
treatment had no effect. Another study reported that treatment of
endothelial cells with pertussis toxin prevented shear
stressmediated activation of ERK1/2,69 also
suggesting that G proteins are activated in response to shear
stress. Further, Gudi et al demonstrated that G proteins reconstituted
in liposomes, in the absence of protein receptors, showed an increase
in activity in response to shear stress.84 This
shear stressmediated increase in G protein activity could be
attenuated if the lipid bilayer was made more rigid by the addition of
cholesterol, a significant finding in the context of
caveolae as shear stress signaling domains.
A common mechanism that has evolved to sense changes in mechanical
stimuli are the mechanosensitive ion channels. These channels are
widely distributed in tissues and participate in processes such as
hearing, balance, and reflex contraction of both smooth and skeletal
muscle. Endothelial cells exhibit ion channel responses
to mechanical forces that are likely to participate in the signaling
response to shear stress. Several different mechanosensitive ion
channels are present in endothelial cells,
including a shear-responsive K+ channel and a
stretch-activated Ca2+
channel.4 Studies have shown that blockade of
mechanosensitive K+ channels with barium chloride
or tetraethylammonium blocked
shear-mediated increases in NO
production85 and transforming growth
factor-ß release,43 suggesting that
transmembrane ion flux and intracellular ion homeostasis are important
mediators of the endothelial cell response to shear
stress. However, efforts to clone the mechanosensitive
K+ channel from the endothelial
cell have not yet been successful.
Based on the demonstrated importance of shear stress to
endothelial cell function and integrity, it is likely
that each of these putative mechanoreceptors activates
intracellular signaling pathways to effect the complete
endothelial response to shear stress. Differential
coupling of signaling mechanisms and subsequent
endothelial cell response to the individual shear
stress receptor "subtypes" may provide a flexibility to the
endothelial cells in terms of responding to varying
types and degrees of shear stress that they may encounter.
 |
Conclusions
|
|---|
We have reviewed data showing that shear stress has direct
influences on the pathogenesis of atherosclerosis via
regulation of endothelial cell function and integrity.
Shear stress influences many of the processes relevant to development
of the atherosclerotic lesion, including secretion of growth factors,
regulation of coagulation, and transmigration of leukocytes. Regulation
of these processes (Fig 3
) is proposed to occur via
shear-activated endothelial cell
signal-transduction pathways that involve primary mechanotransducers,
resulting in the activation of ERK1/2, and possibly ecNOS, through
signaling molecules such as nonreceptor tyrosine kinases, ras, and
PKC.
While hemodynamic considerations are important in
atherogenesis, it is unlikely that fluid mechanical forces are the sole
positive or negative atherogenic stimuli. The potential influence of
local and systemic biochemical factors and their interplay with
mechanical factors must also be considered.86 87
Apart from the direct effects of shear stress on
endothelial cell function, flow reversal results in
alterations in mass transport, increasing the probability of leukocyte
localization and altering delivery of biochemical factors such as
inflammatory mediators that may contribute to the local atherogenic
state. The conclusion that the hemodynamic force of
fluid shear stress plays an important role in the pathogenesis of
atherosclerosis, however, does provide a framework by
which independent risk factors for atherosclerosis may
be understood. Studies suggest that
hypertension,88 diabetes,89
and
hypercholesterolemia90
promote atherosclerosis by disrupting the ability of
the endothelium to respond to shear stress, while
regular aerobic exercise exerts atheroprotective effects through
shear-mediated increases in NO.57 Further
elucidation of the mechanisms of shear stressmediated signal
transduction and its alteration with these risk factors will greatly
advance our understanding of atherosclerosis.
 |
Selected Abbreviations and Acronyms
|
|---|
| ecNOS |
= |
endothelial constitutive NO synthase |
| ICAM-1 |
= |
intracellular adhesion molecule-1 |
| MAP |
= |
mitogen-activated protein |
| MCP-1 |
= |
monocyte chemoattractant protein-1 |
| NO |
= |
nitric oxide |
| PKC |
= |
protein kinase C |
| VCAM-1 |
= |
vascular cell adhesion molecule-1 |
|
 |
Acknowledgments
|
|---|
This work was supported by a Medical Scientist Training Program
grant (NIGM-GM07266) and Poncin Fellowship to O.T., an AHA National
Grant-in-Aid (94014290) to B.C.B., and NIH PO1 Hl18645. B.C.B. is an
Established Investigator of the American Heart Association
Received July 14, 1997;
accepted December 3, 1997.
 |
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