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239
Mini Review
Mechanical Stress Mechanisms and the Cell
An Endothelial Paradigm
Peter F. Davies and Satish C. Tripathi
There are important physiological and pathological cardiovascular consequences related to endothelial
biomechanical properties. The endothelium, however, is not unique in responding to external forces;
virtually all cells accommodate or respond to the mechanical environment. (Circulation Research
1993;72:239-245)
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T he evolution of organisms has been both constrained and facilitated by many internal and
external mechanical limitations. Mechanical
sensing systems used by primitive cells to ensure survival have been retained, often in modified form, in
multicellular animals and in the tissues and organ
systems of mammals to form specialized sensors that
monitor both the internal state of the organism as well
as its interactions with the external environment. Mechanical stresses alter the structural and functional
properties of cells (mechanotransduction) at the cellular, molecular, and genetic levels, leading to both rapid
responses in the adjacent tissue and slower adaptive
changes to a sustained mechanical environment. The
latter often results in an alteration of intracellular
tension to counterbalance the external stress.
Cellular responses to direct mechanical stresses appear to involve an interplay between structural elements
and biochemical second messengers. Cell surface proteins and extracellular matrix, linked by transmembrane
proteins to the cytoskeleton, activate ion channels and
enzymes by mechanical deformation. The signal transduction mechanisms by which mechanical stresses can
be converted to electrophysiological and biochemical
responses in the sensing cells and the adaptation of such
cells to the external forces by altered gene expression is
receiving increased attention. A change in the extracellular concentration of bioactive ligands at the cell
surface as a result of fluid movement has also been
identified as an indirect mechanism of mechanotransduction. Using the paradigm of endothelial cell responses to flow forces, this review attempts to 1) define
the nature of direct biomechanical stresses, 2) identify
key examples of bioresponses, and 3) discuss signal
transduction mechanisms of mechanical stress.
From the Department of Pathology (P.F.D.), The University of
Chicago, and the Department of Life Sciences (S.C.T.), IIT
Research Institute, Chicago.
Supported by National Institutes of Health grants HL-15062
and HL-36028 and American Heart Association Grant-in-Aid
91-1557.
Address for correspondence: Peter F. Davies, PhD, Department
of Pathology, The University of Chicago, MC6079, 5841 S. Maryland Ave., Chicago, IL 60637.
Received July 29, 1992; accepted September 30, 1992.
Mechanical Stress
Stress is force per unit area, which may be manifested
as pressure (and compression) acting inward upon a
structure, frictional shear at the surface, and tensile
reactive forces acting outward, often to equalize an
externally imposed stress. Tissues and cells also exhibit
definitive stresses characteristic of solid bodies; e.g.,
torsional stresses resulting from internal shear stresses
may be imposed across an entire tissue or organ when
subjected to opposing external shearing forces on opposite faces. Hydrostatic forces, both inside and outside
the cell, are another example of pressure.
Breadth of Mechanical Responses
In organisms as diverse as microbes, plants, and
animals, the retention of cellular responses to mechanical stress factors reflects the fundamental importance
of this process. Stretch-sensitive ion channels occur in
Bacillus subtilis, Escherichia coli, rust fungi, yeast, mollusks, insects, amphibians, birds, reptiles, and mammals.
In mammals, interconnecting forces on muscles, bones,
and connective tissues are additive to the effects of
gravity; when gravitational forces are decreased in
space, tissues remodel to adapt to the altered load. In
the pressurized cardiovascular system, where mechanical demands on the heart and arteries vary enormously,
adaptive mechanisms are essential to facilitate the
optimal distribution of blood. The endothelium plays a
key role in this by regulating vascular tone.
Mechanical Stress Responses in Endothelial Cells
The arterial endothelium is located at the interface
between blood flow and the vessel wall; consequently, it
is uniquely exposed to forces that incorporate diverse
mechanical characteristics. Blood flow regulates the
internal diameter of arteries both acutely, by relaxation/
contraction of smooth muscle cells,' and chronically, by
the reorganization of vascular wall cellular and extracellular components.2 In both cases, the presence of the
endothelium is required. Thus, the endothelium functions as a mechanically sensitive signal transduction
interface between blood and artery wall. Endothelial
cells hold a unique position among biomechanically
responsive cells because, in addition to pressure and
associated stretch and solid mechanical forces, the cells
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Circulation Research Vol 72, No 2 February 1993
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are continuously exposed to relatively high fluid shear
stresses to which they readily respond. Consequently,
they represent an excellent model for studies of the
effects of fluid and solid-phase stress on cell function in
general.3
When endothelial cells are subjected to shear stress
or mechanical stretching, a diverse set of responses are
generated; some are extremely fast, whereas others
develop over many hours. These are outlined in Table 1
in the approximate order of response time. Rapid
changes in ionic conductance, adenylate cyclase activity,
inositol trisphosphate generation, and intracellular free
calcium in response to mechanical stress are similar to
second messenger responses resulting from agonistreceptor coupling, suggesting that they may share common transduction pathways. The rapid responses are
followed after several hours by altered gene expression
(growth factors, endothelin, and regulators of fibrinolysis) and by major structural reorganization (cytoskeletal
redistribution and cell-shape change) if the forces are
sustained. How are such stresses manifested in the
blood?
Blood flow in arteries is generally laminar; however,
the vessel geometry near branches, bifurcations, and
curved regions promotes flow separation and vortex
formation. At such locations, the fluid shear stresses
and pressures fluctuate greatly in magnitude and direction over short distances; elsewhere, the forces are
unidirectional and pulsatile. A schematic of forces
acting on the endothelial cell is shown in Figure 1; the
stresses are similar to those imposed on other cells,
except that blood flow imparts much higher levels of
fluid shear stress than in any other mammalian tissue. In
the fluid phase, the force vectors of pressure/stretch and
wall shear stress act on the endothelial surface. In
endothelium, as in all anchorage-dependent cells, these
external mechanical stresses are imposed on a preexisting force equilibrium generated by the cytoskeletal
tension.4 Overall, the forces acting on the cell must be
balanced by resistance to the action. The forces are
transmitted as solid mechanical stresses that are distributed throughout the cell via the cytoskeleton. Intracellular tensile stresses may then be transmitted to adjacent cells and to the underlying extracellular matrix
structures via sites of focal adhesion at the ventral cell
surface. Thus, the spatial relations of the cell to its
neighbors and the surrounding matrix will influence cell
tension and mechanotransduction. How then are these
interrelated?
Stress Transmission and Transduction
There is strong evidence that the force transduction
mechanisms in endothelial cells, like anchorage-dependent cells in general, are a combination of force transmission via cytoskeletal elements and transduction of
the physical forces to biochemical signals at mechanotransducer sites. The most compelling evidence implicates F-actin microfilaments as the principal transmission structure. F-actin also appears to be required for
transduction; e.g., in several cell types, stimulation of
adenylate cyclase and stretch-activated ion channels in
response to deformation is inhibited by disruption of
actin microfilaments.5 Endothelial focal adhesion remodeling, cell-shape change, and realignment to flow
are all inhibited by drugs that interfere with microfil-
ament turnover. In this regard, the endothelium behaves like other anchorage-dependent nontransformed
cells. How does the mechanochemical transduction take
place such that the mechanisms are consistent with
diverse response times?
The microfilament network confers tension to the
cell, which, together with microtubular rigidity, determines cell shape.4 Actin filaments are anchored in the
plasma membrane at several sites (Figure 1), most
notably in association with 1) focal adhesions on the
ventral surface, 2) intercellular adhesion proteins at
the cell periphery, 3) integral membrane proteins at
the apical cell surface, and 4) the nuclear membrane.
Although the stress transmission pathway is shared,
different responses may be determined by the nature
of the association between cytoskeleton and mechanotransducers at the different locations in the cell and
possibly also by transducers that are independent of
the cytoskeleton. Figure 1 illustrates the likely sites of
transduction, and Figure 2 summarizes the principal
pathways linking stress transmission and transduction.
The existence of mechanically activated ion channels6
provides a prototypic mechanism for rapid transduction
of stress to an electrophysiological response. Recently,
Schultz et a17 have demonstrated that adenylate cyclase
purified from Paramecium flagella can act as a K'
channel in which the hyperpolarized state of the enzyme regulates cAMP production. Thus, ion flux and
generation of an important biochemical second messenger are regulated by an enzyme that exhibits some of the
functional characteristics of an ion channel. Since in
many cells adenylate cyclase activation occurs in response to mechanical stimulation,5 a direct effect of
stress on integral membrane enzymes and ion channels
may generate second messenger activity by similar
mechanisms. Stretch-activated,6 stretch-inactivated,8
and shear stress-activated9 ion channels are candidates
for direct activation.
Are cytoskeletal elements necessary for ion channel
responses? Although the presence of cytoskeleton is not
energetically necessary to activate the ion channels
(stretch activated channels can solely use the free
energy stored in the transmembrane electrochemical
gradient and are sensitive to the tension imposed by
membrane lipids6), disruption of actin microfilaments
inhibits their responses. It is unclear whether the cytoskeleton-channel association is by direct linkage or is
an indirect effect of changing the "background" cytoskeletal tension exerted on the membrane in general.
Other membrane proteins, both integral as well as those
linked to the glycocalyx extension of the cell surface,
could act as flow sensors that, on deformation, activate
membrane enzymes and channels.
Integral membrane proteins of the integrin superfamily are the most extensively studied sites of microfilament anchorage to the plasma membrane. The
integrins are linked to F-actin on the cytoplasmic side
via a-actinin, talin, and vinculin.10 On the cell surface,
integrins bind to the extracellular matrix, thereby
facilitating a series of protein-protein interactions
extending from outside the cell to the filamentous
cytoskeleton. Endothelial cell shape and DNA synthesis are regulated by integrin-extracellular matrix associations that determine the tensional integrity (tensegrity) of the cell. 4 When the endothelial cell is
Davies and Tripathi Endothelial Stress Mechanisms
241
Shear Stress
(Endothielial cell)
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1. SAC / SIC
2. Shear stress
JK
DAG 1P3
K+
Na+K +
cytoskeleton
Ca++
3. Focal adhesions
FIGURE 1. Diagrams showing mechanical
stress sites in cells. SAC, stretch-activated ion
channel; SIC, stretch-inactivated ion channel;
IMP, integral membrane protein; PL, phospholipid; DAG, diacylglycerol; IP3, inositol 1,4,5trisphosphate; prots., proteins; EC-CAM, endothelial cell to cell adhesion molecule;
PECAM, platelet endothelial cell adhesion
molecule.
Adenyate
Cyclwse
(K1)
4. Cell-cell
5. Nucleus
F-Acdin
Nuclear L
pore
Link prots.
Integrins
=
7
matrix
exposed to directional fluid shear stress, the force
distribution throughout the cytoskeleton must ultimately be balanced by resistance at the focal adhesion
sites if the cell is to remain stationary. Endothelial
focal adhesions are directionally remodeled by shear
stress," consistent with the tensegrity hypothesis of
mechanochemical transduction proposed by Ingber4 in
1991. But do integrins function as mechanotransducers as well as mechanotransmitters?
Recent evidence suggests that the integrins may elicit
biochemical signals by tyrosine kinase phosphorylation
as well as by interaction with closely associated intracellular proteins. Endothelial integrin proteins are composed primarily of the subunits a,43. The P subunit
binds a-actinin, which in turn binds to F-actin. Tyrosine-phosphorylated proteins (including integrins,
talin, vinculin, a-actinin, and paxillin) and the cytoplasmic tyrosine kinase pp60src have been localized to focal
adhesions.12 Since tyrosine phosphorylation is a prominent chemical transducer for receptor signaling, induc-
tion of phosphorylation by mechanical stress at these
sites in endothelial cells would demonstrate convergence of mechanotransduction and hormone receptor
transduction pathways in the cell.
Stimulation of phosphatidylinositol metabolism, a
prominent response to shear stress and stretch,13 is
likely to be linked to plasma membrane protein deformation. Association of the actin-binding protein profilin with specific phosphoinositides has recently been
shown to inhibit phospholipase C,14 thus providing a
direct link between phospholipid metabolism and the
cytoskeleton. The consequences of phospholipase activation and the release of inositol trisphosphate and
diacylglycerol are immediate (Ca'+ influx and mobilization), rapid (Ca'+ as second messenger regulates
rapid gene expression in excitable cells15), and delayed
(protein kinase C activation leading to altered gene
expression'6).
Whatever the transduction system, the fundamental
unambiguous signal is the stress force acting on the
242
Circulation Research Vol 72, No 2 February 1993
TABLE 1. Mechanical Stress Responses in Endothelial Cells
Force
LSS (0.2-16.5 dyne/cm2)
LSS (10-120 dyne/cm2)
Suction (pressure, stretch)
(10-20 mm Hg)
Mechanical poking and dimpling
LSS (8 dyne/cm2)
Flow through microcarrier bed
LSS (30 and 60 dyne/cm2)
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LSS (0.2-4.0 dyne/cm2)
Effect
K' channel activation,
hyperpolarization (whole-cell
recording)
Hyperpolarization
(Vm-sensitive dyes)
Activation of nonselective
cation channels (membrane
patch)
Intracellular Ca2+ rise
(fluo-3)
50-fold increase in release of
nitric oxide
Release of ATP,
acetylcholine, and
substance P
Transient elevation of 1P3,
biphasic (BAECs)
Intracellular Ca2+ rise, Ca 2+
oscillations
Cell type and
response time
BAECs (msec)
BPAECs (steady state
at 60 seconds)
PAECs (msec)
Earliest response to flow,
related to vasorelaxation
Endothelial stretch-activated
channels
HUVECs (seconds)
BAECs (seconds)
Stretch-activated Ca2+
channels, depolarization
Flow-mediated vasorelaxation
HUVECs (seconds)
Neurotransmitter release
BAECs and HUVECs
(15-30 seconds),
major peak at 5
minutes (BAECs)
BAECs (15-40
Phosphoinositides and Ca2+
as second messengers for
shear stress transduction
seconds)
Cyclic strain (24%), deformation
(1 Hz)
Transient elevation of IP3
HSVECs
LSS (pulsatile) (mean, 10
Pulsed PGI2 release
HUVECs (<1 minute)
LSS (0.9 and 14.0 dyne/cm2)
Sustained PGI2 release at
lower rate than pulsatile flow
HUVECs (2 minutes)
Cyclic stretching, osmotic
swelling
LSS (10 dyne/cm2)
LSS (10 dyne/cm2)
Activation of adenylate
cyclase
Induction of c-myc expression
Directional remodeling of
focal adhesion sites,
realignment with flow
(>8 hours)
Tension control of cell shape,
pH, and growth via
ECM-integrin binding
10-fold enhancement of
PDGF-A mRNA, 2-3 fold
increase of PDGF-B
expression
Pinocytosis stimulated,
adaptation by 6 hours
BAECs and HUVECs
dyne/cm2)
Modulation of inherent cell
tension
LSS (0-51 dyne/cm2)
LSS (>5 dyne/cm2)
LSS (10 dyne/cm2)
LSS (5 dyne/cm2)
LSS (15 and 25 dyne/cm2)
LSS (15 and 25 dyne/cm2)
Turbulent flow (average shear
stress, 1.5-15.0 dyne/cm2)
LSS (>5 dyne/cm2)
LSS (>5 dyne/cm2 and in vivo)
Induction of c-fos and c-jun
Endothelin mRNA and
protein secretion stimulated
t-PA mRNA expression and
secretion stimulated
PAI-1 mRNA expression and
secretion elevated
Cell proliferation in quiescent
monolayer
Cell alignment in direction of
flow, function of time and
magnitude of shear stress
Cytoskeletal and fibronectin
rearrangement
Significance
Earliest response to flow,
related to vasorelaxation
Phosphoinositides and Ca 2+
as second messengers for
shear stress transduction
Phosphoinositides as second
messengers for strain
deformation
PGI2 regulation of vascular
tone, antithrombotic
properties
PGI2 regulation of vascular
tone, antithrombotic
properties
cAMP as second messenger
(minutes)
BAECs (minutes)
BAECs (minutes)
Early growth response gene
Cell attachment sites as
transmitters and/or
transducers of stress
Capillary EC
(<1 hour)
Integrins regulate cell growth
via cell tension
HUVECs (PDGF-A
peak, 1.5-2 hours)
Enhanced mitogen secretion,
regulation of SMC growth
BAECs (<2 hours)
HUVECs (5 hours)
Plasma membrane vesicle
formation rate transiently
elevated
Growth response genes
Regulation of
vasoconstriction
Enhancement of fibrinolytic
HUVECs (?)
Antagonizes t-PA effects
BAECs (>3 hours)
Loss of contact inhibition of
growth by disturbed flow
Minimizes drag on cell
BAECs (2 hours)
PAECs (peak at 2-4
hours)
activity
All types (>6 hours)
All types
(>6 hours)
Associated with cell
realignment
Davies and Tripathi Endothelial Stress Mechanisms
243
TABLE 1. (continued)
Force
Cyclic biaxial deformation
(0.78-12%, 1-Hz frequency,
20-24% strain, 0.9-1.0 Hz)
LSS (24 dyne/cm2+20 mm Hg
hydrostatic pressure)
Effect
Cell realignment
perpendicular to strain,
protein synthesis increase,
F-actin redistribution
perpendicular to strain
Downregulation of
fibronectin synthesis
Cell type and
response time
BPAECs (>7 hours)
HUVECs and
HSVECs (15 minutes)
Significance
Stretching of artery by blood
pulsation, separation of strain
and shear stress effects
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Altered cell adhesion,
platelet-endothelium
interactions
Disturbed laminar flow (flow
Regional cell cycle
BAECs (12 hours)
Steep shear gradient
separation, vortex,
stimulation in confluent
stimulation of cell turnover,
reattachment) (0-10 dyne/cm2)
monolayer
focal hemodynamic effects
LSS (10-85 dyne/cm2)
Mechanical stiffness of cell
BAECs (24 hours)
Decreased deformability of
surface proportional to extent
subplasma membrane cortical
of realignment to flow
complex
LSS (30 and 60 dyne/cm2)
LDL metabolism stimulated
BAECs (24 hours)
Endothelial cholesterol
balance
Cyclic biaxial stretch (3
Inhibition of collagen
Inverse relation related to
BAECs (5 days)
endothelial repair
cycles/min, 24% deformation)
synthesis and stimulation of
cell growth
mechanisms
LSS, laminar shear stress; BAECs, bovine aortic endothelial cells; Vm, membrane potential; BPAECs, bovine pulmonary artery
endothelial cells; PAECs, porcine aortic endothelial cells; HUVECs, human umbilical vein endothelial cells; 1P3, inositol trisphosphate;
HSVECs, human saphenous vein endothelial cells; PGI2, prostaglandin I2 (prostacyclin); t-PA, tissue plasminogen activator; PAI-1,
plasminogen activator inhibitor-i; ECM, extracellular matrix; EC, endothelial cell; PDGF-A and PDGF-B, platelet-derived growth factor
A and B chains; SMC, vascular smooth muscle cell; LDL, low density lipoprotein. (A list of references relating to the above table may be
obtained by writing to the authors.)
primary transducer. How does the physical deformation
of a membrane protein or cytoskeletal component lead
to a biochemical response? Conformational changes
within the proteins appear to be the likeliest mechanism. The intrinsic tension within the cell generated by
cytoskeletal anchoring and the molecular interactive
forces of components in the plasma membrane provide
HUVECs (12 and 48
hours)
a framework against which extrinsic forces act. An
existing thermodynamic equilibrium would be disturbed
by deformation and a new more favorable one would be
established that alters the conformation of the proteins.
Thus, conformational changes could occur 1) directly
within the mechanotransducer at the plasma membrane
(ion channel, enzyme, and integrin), 2) within proteins
Transduction
0
ce
FIGURE 2. Diagram showing
mechanisms of mechanical stress
transmission and transduction in
cells. ECM, extracellular matrix; DAG, diacylglycerol; PKC,
protein kinase C; IP3, inositol
1,4,5-trisphosphate.
Cor.t
s:3
k~
'I
L
+
IOaysequilibnarI
fee
Cyokeeo -j>
|Cytoskeq-letoi|
Intermed.
filaments
Microfilaments
Microtubules
Protein
Iconformationail
changes |
244
Circulation Research Vol 72, No 2 February 1993
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associated with integral membrane proteins but not in
the membrane, (a-actinin, talin, vinculin, and paxillin),
or 3) within the cytoskeletal proteins themselves, leading to changes in the subunit dissociation rates of actin
and tubulin. These are not mutually exclusive mechanisms, and as noted above, all active cell responses to
stress require assembled microfilaments. The transducer-actin interaction must therefore be physically very
close or be mediated by intermediate metabolites. In
some cases, GTP-binding proteins, classically associated
with the cytoplasmic domain of receptors, can influence
mechanotransduction. While deformation-induced activation of adenylate cyclase in S49 mouse lymphoma
cells occurs independent of GTP-binding proteins,5
tubulin and Gi protein complex can inhibit adenylate
cyclase.'7 Evidence for direct binding of cytoskeleton to
ion channels is lacking and needs to be addressed. At
present, the integrins are the best example of cytoskeletal-transducer coupling, particularly in focal adhesion
sites.
When a stress stimulus is sustained, gene regulatory
changes often follow the immediate responses; several
examples for endothelial cells are included in Table 1.
The mechanisms probably involve protein kinase C
activation. For example, the immediate early genes c-fos
and c-myc and the late responsive genes a-actin and
,B-myosin heavy chain are induced in cardiac myocytes
by mechanical loading.16 Both c-fos and actin responses
were associated with activated protein kinase C and
were suppressed by downregulation of the enzyme.
These responses did not appear to involve Ca`+. In
contrast, rapid "touch-sensitive" gene expression occurs
in the plant Arabidopsis by mechanisms involving Ca ` /
calmodulin.18 Ten to 30 minutes after stimulation,
mRNA levels for four touch-induced genes coding for
calmodulin-related proteins increased 100-fold. Diverse
biochemical pathways therefore appear to influence
gene expression, and these are derived from the immediate responses (inositol trisphosphate and diacylglycerol). In what ways does the cell determine if a mechanical response will lead to gene expression? Perhaps the
mechanical properties of the cell itself or the tissue in
which it is imbedded may act as a filter that is differentially responsive to selective frequencies.
Indirect Mechanisms of Stress Responses
Although this review is focused on direct mechanical
perturbation of cells, mechanical forces acting on the
fluid around the cell can also stimulate intracellular
responses; e.g., changes of flow influence the endothelial boundary layer concentrations of adenine nucleotides by altering the mass transport of exogenous or
endogenous agonists and consequently influence their
interactions with cell surface receptors.19 This indirect
mechanism may contribute to some cellular responses
that are considered to result from direct deformation.
Analyses of the relative contributions of the two types of
mechanisms to endothelial flow responses are currently
proceeding in several laboratories.
Concluding Remarks
There has been a large increase of interest in
mechanotransduction over the last few years as investigators in well-developed research areas, particularly
cytoskeletal, endothelial, cardiac, and focal adhesion
biology and signal transduction, have begun to study
mechanical stress mechanisms in cells, often in collaboration with physical scientists. The importance of
hemodynamic forces has focused much attention on
the endothelium, but only recently has attention narrowed from the observational to the mechanistic level.
The role of cytoskeleton and focal adhesion proteins is
likely to be particularly important in these shear
responsive cells. Are phosphorylation events altered
by flow? Can mechanotransduction be influenced by
integrin subunit manipulation (e.g., truncation or antibody block)? Can the precise topography of the
apical cell surface and its deformation by flow be
measured in real time to determine shear stresses
localized to specific domains? Could there be tensionrelated conformational changes in the nucleus, transmitted via cytoskeletal anchorage to the nuclear membrane? Within tissues, can the tension distribution be
more precisely evaluated throughout a cell in relation
to the matrix that surrounds it? In the near future,
cloning of the genes that code for stretch- and shearsensitive ion channels will provide valuable probes,
and parallels between hormone/growth factor transduction and mechanotransduction should continue to
be a rich source for comparative studies.
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Mechanical stress mechanisms and the cell. An endothelial paradigm.
P F Davies and S C Tripathi
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Circ Res. 1993;72:239-245
doi: 10.1161/01.RES.72.2.239
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1993 American Heart Association, Inc. All rights reserved.
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