The response of endothelial cells (ECs) to their hemodynamic environment strongly influences normal vascular physiology and the pathogenesis of atherosclerosis. Unique responses to the complex flow patterns in lesion-prone regions imply that the temporal and spatial features of the mechanical stimuli modulate the cellular response to flow. We report the first systematic study of the effects of temporal gradients of shear stress on ECs. Flow was applied to cultured ECs using a novel cone-and-plate device allowing precise and independent control of the shear stress magnitude and the onset rate. Intracellular free calcium concentration [Ca2+]i increased rapidly following the onset of flow, and the characteristics of the transient were modulated by both the shear stress magnitude and onset rate. ECs were most sensitive to shear stress applied at physiological onset rates. Furthermore, the relative contribution of extracellular calcium and IP3-mediated release were dependent upon the specific flow regime. [S0148-0731(00)01003-7]

1.
Fuster, V., Ross, R., Topol, E. J., et al., eds., 1996, Atherosclerosis and Coronary Artery Disease, 1, Lippincott-Raven, Philadelphia.
2.
Giddens
,
D. P.
,
Zarins
,
C. K.
, and
Glagov
,
S.
,
1993
, “
The Role of Fluid Mechanics in the Localization and Detection of Atherosclerosis
,”
ASME J. Biomech. Eng.
,
115
, pp.
588
594
.
3.
Ojha
,
M.
,
1993
, “
Spatial and Temporal Variations of Wall Shear Stress Within an End-to-Side Arterial Anastomosis Model
,”
J. Biomech.
,
26
, pp.
1377
1388
.
4.
Ojha
,
M.
,
1994
, “
Wall Shear Stress Temporal Gradient and Anastomotic Intimal Hyperplasia
,”
Circ. Res.
,
74
, pp.
1227
1231
.
5.
Ku
,
D. N.
,
Giddens
,
D. P.
,
Zarins
,
C. K.
, and
Glagov
,
S.
,
1985
, “
Pulsatile Flow and Atherosclerosis in the Human Carotid Bifurcation. Positive Correlation Between Plaque Location and Low Oscillating Shear Stress
,”
Arteriosclerosis
,
5
, pp.
293
302
.
6.
Zarins
,
C. K.
,
Giddens
,
D. P.
,
Bharadvaj
,
B. K.
,
Sottiurai
,
V. S.
,
Mabon
,
R. F.
, and
Glagov
,
S.
,
1983
, “
Carotid Bifurcation Atherosclerosis. Quantitative Correlation of Plaque Localization With Flow Velocity Profiles and Wall Shear Stress
,”
Circ. Res.
,
53
, pp.
502
514
.
7.
Davies
,
P. F.
,
Barbee
,
K. A.
,
Volin
,
M. V.
, et al.
,
1997
, “
Spatial Relationships in Early Signaling Events of Flow-Mediated Endothelial Mechanotransduction
,”
Annu. Rev. Physiol.
,
59
, pp.
527
549
.
8.
Helmlinger
,
G.
,
Geiger
,
R. V.
,
Schreck
,
S.
, and
Nerem
,
R. M.
,
1991
, “
Effects of Pulsatile Flow on Cultured Vascular Endothelial Cell Morphology
,”
ASME J. Biomech. Eng.
,
113
, pp.
123
131
.
9.
DePaola
,
N.
,
Gimbrone
, Jr.,
M. A.
,
Davies
,
P. F.
, and
Dewey
, Jr.,
C. F.
,
1992
, “
Vascular Endothelium Responds to Fluid Shear Stress Gradients
,”
Arterioscler. Thromb.
,
12
, pp.
1254
1257
.
10.
Davies
,
P. F.
,
Remuzzi
,
A.
,
Gordon
,
E. J.
,
Dewey
, Jr.,
C. F.
, and
Gimbrone
, Jr.,
M. A.
,
1986
, “
Turbulent Fluid Shear Stress Induces Vascular Endothelial Cell Turnover in Vitro
,”
Proc. Natl. Acad. Sci. USA
,
83
, pp.
2114
2117
.
11.
Davies
,
P. F.
,
1995
, “
Flow-Mediated Endothelial Mechanotransduction
,”
Physiol. Rev.
,
75
, pp.
519
560
.
12.
Resnick
,
N.
, and
Gimbrone
, Jr.,
M. A.
,
1995
, “
Hemodynamic Forces Are Complex Regulators of Endothelial Gene Expression
,”
FASEB J.
,
9
, pp.
874
882
.
13.
Ando
,
J.
,
Ohtsuka
,
A.
,
Korenaga
,
R.
, and
Kamiya
,
A.
,
1991
, “
Effect of Extracellular ATP Level on Flow-Induced Ca2+ Response in Cultured Vascular Endothelial Cells
,”
Biochem. Biophys. Res. Commun.
,
179
, pp.
1192
1199
.
14.
Helmlinger
,
G.
,
Berk
,
B. C.
, and
Nerem
,
R. M.
,
1995
, “
Calcium Responses of Endothelial Cell Monolayers Subjected to Pulsatile and Steady Laminar Flow
,”
Am. J. Physiol.
,
269
, pp.
367
375
.
15.
James
,
N. L.
,
Harrison
,
D. G.
, and
Nerem
,
R. M.
,
1995
, “
Effects of Shear on Endothelial Cell Calcium in the Presence and Absence of ATP
,”
FASEB J.
,
9
, pp.
968
973
.
16.
Shen
,
J.
,
Luscinskas
,
F. W.
,
Connolly
,
A.
,
Dewey
, Jr.,
C. F.
, and
Gimbrone
, Jr.,
M. A.
,
1992
, “
Fluid Shear Stress Modulates Cytosolic Free Calcium in Vascular Endothelial Cells
,”
Am. J. Physiol.
,
262
, pp.
384
390
.
17.
Geiger
,
R. V.
,
Berk
,
B. C.
,
Alexander
,
R. W.
, and
Nerem
,
R. M.
,
1992
, “
Flow-Induced Calcium Transients in Single Endothelial Cells: Spatial and Temporal Analysis
,”
Am. J. Physiol.
,
262
, pp.
1411
1417
.
18.
Milner
,
P.
,
Bodin
,
P.
,
Loesch
,
A.
, and
Burnstock
,
G.
,
1990
, “
Rapid Release of Endothelin and ATP From Isolated Aortic Endothelial Cells Exposed to Increased Flow
,”
Biochem. Biophys. Res. Commun.
,
170
, pp.
649
656
.
19.
Blackman
,
B.
,
Barbee
,
K.
, and
Thibault
,
L.
,
2000
, “
In Vitro Cell Shearing Device to Investigate the Dynamic Response of Cells in a Controlled Hydrodynamic Environment
,”
Ann. Biomed. Eng.
,
28
, No.
4
, pp.
1
11
.
20.
Grynkiewicz
,
G.
,
Poenie
,
M.
, and
Tsien
,
R. Y.
,
1985
, “
A New Generation of Ca2+ Indicators With Greatly Improved Fluorescence Properties
,”
J. Biol. Chem.
,
260
, pp.
3440
3450
.
21.
Schacht
,
J.
,
1976
, “
Effect of Neomycin on Phosphoinositide Labelling and Calcium Binding in Guinea-Pig Inner Ear Tissues in Vivo and in Vitro
,”
J. Neurochem.
,
27
, pp.
1119
1124
.
22.
Prentki
,
M.
,
Deeney
,
J. T.
,
Matschinsky
,
F. M.
, and
Joseph
,
S. K.
,
1986
, “
Neomycin: a Specific Drug to Study the Inositol-Phospholipid Signalling System?
FEBS Lett.
,
197
, pp.
285
288
.
23.
Prasad
,
A. R.
,
Logan
,
S. A.
,
Nerem
,
R. M.
,
Schwartz
,
C. J.
, and
Sprague
,
E. A.
,
1993
, “
Flow-Related Responses of Intracellular Inositol Phosphate Levels in Cultured Aortic Endothelial Cells
,”
Circ. Res.
,
72
, pp.
827
836
.
24.
Randriamampita
,
C.
, and
Tsien
,
R. Y.
,
1993
, “
Emptying of Intracellular Stores Releases a Novel Small Messenger That Stimulates Ca2+ Influx
,”
Nature
,
364
, pp.
809
814
.
25.
Schwarz
,
G.
,
Droogmans
,
G.
, and
Nilius
,
B.
,
1992
, “
Shear Stress Induced Membrane Currents and Calcium Transients in Human Vascular Endothelial Cells
,”
Pflugers Archives—Eur. J. Physiol.
,
421
, pp.
394
396
.
26.
Nollert
,
M. U.
,
Eskin
,
S. G.
, and
McIntire
,
L. V.
,
1990
, “
Shear Stress Increases Inositol Trisphosphate Levels in Human Endothelial Cells
,”
Biochem. Biophys. Res. Commun.
,
170
, pp.
281
287
.
27.
Berridge
,
M. J.
, and
Irvine
,
R. F.
,
1989
, “
Inositol Phosphates and Cell Signaling
,”
Nature
,
341
, pp.
197
205
.
28.
Helmlinger
,
G.
,
Berk
,
B. C.
, and
Nerem
,
R. M.
,
1996
, “
Pulsatile and Steady Flow-Induced Calcium Oscillations in Single Cultured Endothelial Cells
,”
J. Vasc. Res.
,
33
, pp.
360
369
.
29.
Clapham
,
D. E.
,
1995
, “
Calcium Signaling
,”
Cell
,
80
, pp.
259
268
.
30.
Putney
,
J. W.
,
1990
, “
Capacitive Calcium Entry Revisited
,”
Cell Calcium
,
11
, pp.
611
624
.
31.
Chakravarthy
,
S. R.
, and
Giorgio
,
T. D.
,
1992
, “
Shear Stress-Facilitated Calcium Ion Transport Across Lipid Bilayers
,”
Biochim. Biophys. Acta
,
1112
, pp.
197
204
.
32.
Berthiaume
,
F.
, and
Frangos
,
J. A.
,
1994
, “
Fluid Flow Increases Membrane Permeability to Merocyanine 540 in Human Endothelial Cells
,”
Biochim. Biophys. Acta
,
1191
, pp.
209
218
.
33.
Dolmetsch
,
R. E.
,
Lewis
,
R. S.
,
Goodnow
,
C. C.
, and
Healy
,
J. I.
,
1997
, “
Differential Activation of Transcription Factors Induced by Ca2+ Response Amplitude and Duration
,”
Nature
,
386
, pp.
855
858
.
34.
Kuchan
,
M. J.
, and
Frangos
,
J. A.
,
1994
, “
Role of Calcium and Calmodulin in Flow-Induced Nitric Oxide Production in Endothelial Cells
,”
Am. J. Physiol.
,
266
, pp.
628
636
.
35.
Bao
,
X.
,
Lu
,
C.
, and
Frangos
,
J.
,
1999
, “
Temporal Gradient in Shear but Not Steady Shear Stress Induces PDGF-A and MCP-1 Expression in Endothelial Cells; Role of NO, NFκB, and egr-1
,”
Arterioscler Thromb Vasc. Biol.
,
19
, pp.
996
1003
.
36.
LaPlaca
,
M.
, and
Thibault
,
L.
,
1997
, “
An in Vitro Traumatic Injury Model to Examine the Response of Neurons to a Hydrodynamically-Induced Deformation
,”
Ann. Biomed. Eng.
,
25
, pp.
665
677
.
37.
Fung
,
Y. C.
, and
Liu
,
S. Q.
,
1993
, “
Elementary Mechanics of the Endothelium of Blood Vessels
,”
ASME J. Biomech. Eng.
,
115
, pp.
1
12
.
38.
Liu
,
S. Q.
,
Yen
,
M.
, and
Fung
,
Y. C.
,
1994
, “
On Measuring the Third Dimension of Cultured Endothelial Cells in Shear Flow
,”
Proc. Natl. Acad. Sci. USA
,
91
, pp.
8782
8786
.
39.
Satcher
,
R. L.
, and
Dewey
,
C. F. J.
,
1996
, “
Theoretical Estimates of Mechanical Properties of the Endothelial Cell Cytoskeleton
,”
Biophys. J.
,
71
, pp.
109
118
.
40.
Gudi
,
S. P.
,
Clark
,
C. B.
, and
Frangos
,
J. A.
,
1996
, “
Fluid Flow Rapidly Activates G Proteins in Human Endothelial Cells
,”
Circ. Res.
,
79
, No
4
, pp.
834
839
.
41.
Gudi
,
S.
,
Nolan
,
J. P.
, and
Frangos
,
J. A.
,
1998
, “
Modulation of GTPase Activity of G Proteins by Fluid Shear Stress and Phospholipid Composition
,”
Proc. Natl. Acad. Sci. USA
,
95
, pp.
2515
2519
.
42.
Hubner
,
S.
,
Couvillon
,
A. D.
,
Kas
,
J. A.
,
Bankaitis
,
V. A.
,
Vegners
,
R.
,
Carpenter
,
C. L.
, and
Janmey
,
P. A.
,
1998
, “
Enhancement of Phosphoinositide 3-Kinase (PI 3-kinase) Activity by Membrane Curvature and Inositol-Phospholipid-Binding Peptides
,”
Eur. J. Biochem.
,
258
, pp.
846
853
.
43.
James
,
S. R.
,
Paterson
,
A.
,
Harden
,
T. K.
,
Demel
,
R. A.
, and
Downes
,
C. P.
,
1997
, “
Dependence of the Activity of Phospholipase C Beta on Surface Pressure and Surface Composition in Phospholipid Monolayers and Its Implications for Their Regulation
,”
Biochemistry
,
36
, pp.
848
855
.
44.
Machida
,
K.
, and
Ohnishi
,
S. I.
,
1980
, “
Effect of Bilayer Membrane Curvature on Activity of Phosphatidylcholine Exchange Protein
,”
Biochim. Biophys. Acta
,
596
, pp.
201
209
.
45.
Rebecchi
,
M.
,
Bonhomme
,
M.
, and
Scarlata
,
S.
,
1999
, “
Role of Lipid Packing in the Activity of Phospholipase C-Delta1 as Determined by Hydrostatic Pressure Measurements
,”
Biochem. J.
,
341
, pp.
571
576
.
You do not currently have access to this content.