In the circulation, flow-responsive endothelial cells (ECs) lining the lumen of blood vessels are continuously exposed to complex hemodynamic forces. To increase our understanding of EC response to these dynamic shearing forces, a novel in vitro flow model was developed to simulate pulsatile shear stress waveforms encountered by the endothelium in the arterial circulation. A modified waveform modeled after flow patterns in the human abdominal aorta was used to evaluate the biological responsiveness of human umbilical vein ECs to this new type of stimulus. Arterial pulsatile flow for 24 hours was compared to an equivalent time-average steady laminar shear stress, using no flow (static) culture conditions as a baseline. While both flow stimuli induced comparable changes in cell shape and alignment, distinct patterns of responses were observed in the distribution of actin stress fibers and vinculin-associated adhesion complexes, intrinsic migratory characteristics, and the expression of eNOS mRNA and protein. These results thus reveal a unique responsiveness of ECs to an arterial waveform and begin to elucidate the complex sensing capabilities of the endothelium to the dynamic characteristics of flows throughout the human vascular tree.

1.
Nichols, W. W., O’Rourke, M. F., Hartley, C., and McDonald, D. A., 1998, McDonald’s Blood Flow In Arteries: Theoretical, Experimental, and Clinical Principles, Oxford Press, New York.
2.
Davies
,
P. F.
,
1995
, “
Flow-mediated Endothelial Mechanotransduction
,”
Physiol. Rev.
,
75
(
3
), pp.
519
560
.
3.
Traub
,
O.
, and
Berk
,
B. C.
,
1998
, “
Laminar Shear Stress: Mechanisms by Which Endothelial Cells Transduce an Atheroprotective Force
,”
Arterioscler., Thromb., Vasc. Biol.
,
18
(
5
), pp.
677
685
.
4.
Haidekker
,
M. A.
,
L’Heureux
,
N.
, and
Frangos
,
J. A.
,
2000
, “
Fluid Shear Stress Increases Membrane Fluidity in Endothelial Cells: A Study with DCVJ Fluorescence
,”
Am J Physiol Heart Circ Physiol
,
278
(
4
), pp.
H1401–H1406
H1401–H1406
.
5.
Jalali
,
S.
,
del Pozo
,
M. A.
,
Chen
,
K.
,
Miao
,
H.
,
Li
,
Y.
,
Schwartz
,
M. A.
,
Shyy
,
J. Y.
, and
Chien
,
S.
,
2001
, “
Integrin-mediated Mechanotransduction Requires its Dynamic Interaction With Specific Extracellular Matrix Ligands
,”
Proc. Natl. Acad. Sci. U.S.A.
,
98
(
3
), pp.
1042
1046
.
6.
Dewey
,
C. F.
, Jr.
,
Bussolari
,
S. R.
,
Gimbrone
,
M. A.
, Jr.
, and
Davies
,
P. F.
,
1981
, “
The Dynamic Response of Vascular Endothelial Cells to Fluid Shear Stress
,”
J. Biomech. Eng.
,
103
(
3
), pp.
177
185
.
7.
Helmlinger
,
G.
,
Geiger
,
R. V.
,
Schreck
,
S.
, and
Nerem
,
R. M.
,
1991
, “
Effects of Pulsatile Flow on Cultured Vascular Endothelial Cell Morphology
,”
J. Biomech. Eng.
,
113
(
2
), pp.
123
131
.
8.
Girard
,
P. R.
, and
Nerem
,
R. M.
,
1995
, “
Shear Stress Modulates Endothelial Cell Morphology and f-actin Organization Through the Regulation of Focal Adhesion-associated Proteins
,”
J. Cell Physiol.
,
163
, pp.
179
193
.
9.
Thoumine
,
O.
,
Nerem
,
R. M.
, and
Girard
,
P. R.
,
1995
, “
Oscillatory Shear Stress and Hydrostatic Pressure Modulate Cell-matrix Attachment Proteins in Cultured Endothelial Cells
,”
In Vitro Cellular & Developmental Biology, Animal
,
31
(
1
), pp.
45
54
.
10.
DePaola
,
N.
,
Gimbrone
,
M. A.
Jr.
,
Davies
,
P. F.
, and
Dewey
,
C. F.
, Jr.
,
1992
, “
Vascular Endothelium Responds to Fluid Shear Stress Gradients [published erratum appears in Arterioscler Thromb 1993 Mar; 13(3) p. 465]
,”
Arterioscler. Thromb.
,
12
(
11
), pp.
1254
1257
.
11.
Davies
,
P. F.
,
Remuzzi
,
A.
,
Gordon
,
E. J.
,
Dewey
,
C. F.
, Jr.
, and
Gimbrone
,
M. A.
, Jr.
,
1986
, “
Turbulent Fluid Shear Stress Induces Vascular Endothelial Cell Turnover In Vitro
,”
Proc. Natl. Acad. Sci. U.S.A.
,
83
(
7
), pp.
2114
2117
.
12.
Noris
,
M.
,
Morigi
,
M.
,
Donadelli
,
R.
,
Aiello
,
S.
,
Foppolo
,
M.
,
Todeschini
,
M.
,
Orisio
,
S.
,
Remuzzi
,
G.
, and
Remuzzi
,
A.
,
1995
, “
Nitric Oxide Synthesis by Cultured Endothelial Cells is Modulated by Flow Conditions
,”
Circ. Res.
,
76
(
4
), pp.
536
543
.
13.
Topper
,
J.
,
Cai
,
J.
,
Falb
,
D.
, and
Gimbrone
,
M. A.
, Jr.
,
1996
, “
Identification of Vascular Endothelial Genes Differentially Responsive to Fluid Mechanical Stimuli: Cyclooxygenase-2, Manganese Superoxide Dismutase, and Endothelial Cell Nitric Oxide Synthase are Selectively Up-regulated by Steady Laminar Shear Stress
,”
Proc. Natl. Acad. Sci. U.S.A.
,
98
, pp.
10417
10422
.
14.
Garcia-Cardena
,
G.
,
Comander
,
J.
,
Anderson
,
K. R.
,
Blackman
,
B. R.
, and
Gimbrone
,
M. A.
, Jr.
,
2001
, “
Biomechanical Activation of Vascular Endothelium as a Determinant of its Functional Phenotype
,”
Proc. Natl. Acad. Sci. U.S.A.
,
98
(
8
), pp.
4478
4485
.
15.
Gimbrone
,
M. A. J.
,
Topper
,
J. N.
,
Nagel
,
T.
,
Anderson
,
K. R.
, and
Garcia-Cardena
,
G.
,
2000
, “
Endothelial Dysfunction, Hemodynamic Forces, and Atherogenesis
,”
Ann NY Acad Sci
,
902
, pp.
230
239
.
16.
Moore
,
J. E.
, Jr.
,
Burki
,
E.
,
Suciu
,
A.
,
Zhao
,
S.
,
Burnier
,
M.
,
Brunner
,
H. R.
, and
Meister
,
J. J.
,
1994
, “
A Device for Subjecting Vascular Endothelial Cells to Both Fluid Shear Stress and Circumferential Cyclic Stretch
,”
Ann. Biomed. Eng.
,
22
(
4
), pp.
416
422
.
17.
Peng
,
X.
,
Recchia
,
F. A.
,
Byrne
,
B. J.
,
Wittstein
,
I. S.
,
Ziegelstein
,
R. C.
, and
Kass
,
D. A.
,
2000
, “
In Vitro System to Study Realistic Pulsatile Flow and Stretch Signaling in Cultured Vascular Cells
,”
Am J Physiol Cell Physiol
,
279
, pp.
C797–C805
C797–C805
.
18.
Qiu
,
Y.
, and
Tarbell
,
J. M.
,
2000
, “
Interaction Between Wall Shear Stress and Circumferential Strain Affects Endothelial Cell Biochemical Production
,”
J. Vasc. Res.
,
37
(
3
), pp.
147
157
.
19.
Langille
,
B. L.
,
1984
, “
Integrity of Arterial Endothelium Following Acute Exposure to High Shear Stress
,”
Biorheology
,
21
, pp.
333
346
.
20.
Schnittler
,
H. J.
,
Franke
,
R. P.
,
Akbay
,
U.
,
Mrowietz
,
C.
, and
Drenckhahn
,
D.
,
1993
, “
Improved In Vitro Rheological System for Studying the Effect of Fluid Shear Stress on Cultured Cells
,”
Am. J. Physiol.
,
265
(1 Pt 1), pp.
289
298
.
21.
Blackman
,
B. R.
,
Barbee
,
K. A.
, and
Thibault
,
L. E.
,
2000
, “
In Vitro Cell Shearing Device to Investigate the Dynamic Response of Cells in a Controlled Hydrodynamic Environment
,”
Ann. Biomed. Eng.
,
28
(
4
), pp.
363
372
.
22.
Maier
,
S. E.
,
Meier
,
D.
,
Boesiger
,
P.
,
Moser
,
U. T.
, and
Vieli
,
A.
,
1989
, “
Human Abdominal Aorta: Comparative Measurements of Blood Flow with MR Imaging and Multigated Doppler US.
,”
Radiology
,
171
(
2
), pp.
487
492
.
23.
Oshinski
,
J. N.
,
Ku
,
D. N.
,
Mukundan
,
S. J.
,
Loth
,
F.
, and
Pettigrew
,
R. I.
,
1995
, “
Determination of Wall Shear Stress in the Aorta With the Use of MR Phase Velocity Mapping
,”
J. Magn. Reson Imaging
,
5
(
6
), pp.
640
647
.
24.
Oyre
,
S.
,
Pedersen
,
E. M.
,
Ringgaard
,
S.
,
Boesiger
,
P.
, and
Paaske
,
W. P.
,
1997
, “
In Vivo Wall Shear Stress Measured by Magnetic Resonance Velocity Mapping in the Normal Human Abdominal Aorta
,”
Eur. J. Vasc. Endovasc Surg.
,
13
(
3
), pp.
263
271
.
25.
Ling
,
S. C.
,
Atabek
,
H. B.
,
Letzing
,
W. G.
, and
Patel
,
D. J.
,
1973
, “
Nonlinear Analysis of Aortic Flow in Living Dogs
,”
Circ. Res.
,
33
(
2
), pp.
198
212
.
26.
Ling
,
S. C.
, and
Atabek
,
H. B.
,
1972
, “
A Nonlinear Analysis of Pulsatile Flow in Arteries
,”
J. Fluid Mech.
,
55
, pp.
493
511
.
27.
Dunn, G. A., 1983, Characterizing a Kinesis Response: Time Averaged Measures of Cell Speed and Directional Persistence. Leukocyte Locomotion and Chemotaxis, H. O. Keller and G. O. Till. Basel, Birkhauser: pp. 14–33.
28.
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 (Dallas)
,
5
(
3
), pp.
293
302
.
29.
Moore
,
J. E. J.
,
Xu
,
C.
,
Glagov
,
S.
,
Zarins
,
C. K.
, and
Ku
,
D. N.
,
1994
, “
Fluid Wall Shear Stress Measurements in a Model of the Human Abdominal Aorta: Oscillatory Behavior and Relationship to Atherosclerosis
,”
Arteriosclerosis (Dallas)
,
110
(
2
), pp.
225
240
.
30.
Fewell
,
M. E.
, and
Hellums
,
J. D.
,
1977
, “
The Secondary Flow of Newtonian Fluids in a Cone-and-Plate Viscometers
,”
Trans. Soc. Rheol.
,
21
, pp.
535
565
.
31.
Sdougos
,
H. P.
,
Bussolari
,
S. R.
, and
Dewey
,
C. F.
,
1984
, “
Secondary Flow and Turbulence in a Cone-and-Plate Device
,”
J. Fluid Mech.
,
138
, pp.
379
404
.
32.
Silkworth
,
J. B.
, and
Stephbens
,
W. E.
,
1975
, “
The Shape of Endothelial Cells in en face Preparations of Rabbit Blood Vessels
,”
Angiology
,
26
, pp.
474
487
.
33.
Langille
,
B. L.
, and
Adamson
,
S. L.
,
1981
, “
Relationship Between Blood Flow Direction and Endothelial Orientation at Arterial Branch Sites in Rabbits and Mice
,”
Circ. Res.
,
48
, pp.
481
488
.
34.
Burridge
,
K.
,
Molony
,
L.
, and
Kelly
,
T.
,
1987
, “
Adhesion Plaques: Sites of Transmembrane Interaction Between the Extracellular Matrix and the Actin Cytoskeleton
,”
J. Cell Sci. Suppl.
,
8
, pp.
211
229
.
35.
Girard
,
P. R.
, and
Nerem
,
R. M.
,
1993
, “
Endothelial Cell Signaling and Cytoskeletal Changes in Response to Shear Stress
,”
Front Med. Biol. Eng.
,
5
(
1
), pp.
31
36
.
36.
Galbraith
,
C. G.
,
Skalak
,
R.
, and
Chien
,
S.
,
1998
, “
Shear Stress Induces Spatial Reorganization of the Endothelial Cell Cytoskeleton
,”
Cell Motil. Cytoskeleton
,
40
(
4
), pp.
317
330
.
37.
Rubanyi
,
G. M.
,
Romero
,
J. C.
, and
Vanhoutte
,
P. M.
,
1986
, “
Flow-induced Release of Endothelium-Derived Relaxing Factor
,”
Am. J. Physiol.
,
250
, pp.
H1145–H1149
H1145–H1149
.
38.
Nishida
,
K.
,
Harrison
,
P. G.
,
Navas
,
J. P.
,
Fisher
,
A. A.
,
Dockery
,
S. P.
,
Uematsu
,
M.
,
Nerem
,
R. M.
,
Alexander
,
R. W.
, and
Murphy
,
T. J.
,
1992
, “
Molecular Cloning and Characterization of the Constitutive Bovine Aortic Endothelial Cell Nitric Oxide Synthase
,”
J. Clin. Invest.
,
90
(
5
), pp.
2092
2096
.
39.
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
(3 Pt 1), pp.
628
636
.
40.
Sessa
,
W. C.
,
Pritchard
,
K.
,
Seyedi
,
N.
,
Wang
,
J.
, and
Hintze
,
T.
,
1994
, “
Chronic Exercise in Dogs Increases Coronary Vascular Nitric Oxide Production and Endothelial Cell Nitric Oxide Synthase Gene Expression
,”
Circ. Res.
,
74
(
2
), pp.
349
353
.
41.
Davis
,
M. E.
,
Cai
,
H.
,
Drummond
,
G. R.
, and
Harrison
,
D. G.
,
2001
, “
Shear Stress Regulates Endothelial Nitric Oxide Synthase Expression Through C-src by Divergent Signaling Pathways
,”
Circ. Res.
,
89
, pp.
1073
1080
.
42.
Helmlinger
,
G.
,
Berk
,
B. C.
, and
Nerem
,
R. M.
,
1995
, “
Calcium Responses of Endothelial Cell Monolayers Subjected to Pulsatile and Steady Laminar Flow Differ
,”
Am. J. Physiol.
,
269
(2 Pt 1), pp.
367
375
.
43.
Dieterich
,
P.
,
Odenthal-Schnittler
,
M.
,
Mrowietz
,
C.
,
Kramer
,
M.
,
Sasse
,
L.
,
Oberleithner
,
H.
, and
Schnittler
,
H. J.
,
2000
, “
Quantitative Morphodynamics of Endothelial Cells Within Confluent Cultures in Response to Fluid Shear Stress
,”
Biophys. J.
,
79
, pp.
1285
1297
.
44.
Davies
,
P. F.
,
Robotewskyj
,
A.
, and
Griem
,
M. L.
,
1994
, “
Quantitative Studies of Endothelial Cell Adhesion. Directional Remodeling of Focal Adhesion Sites in Response to Flow Forces
,”
J. Clin. Invest.
,
93
(
5
), pp.
2031
2038
.
45.
Rodriquez Fernandez
,
J. L.
,
Geiger
,
B.
,
Salomon
,
D.
, and
Ben-Ze’ev
,
A.
,
1992
, “
Overexpression of Vinculin Suppresses Cell Moltility in BALB/c 3T3 Cells
,”
Cell Motil. Cytoskeleton
,
22
, pp.
127
134
.
46.
Rodriquez Fernandez
,
J. L.
,
Geiger
,
B.
,
Salomon
,
D.
,
Sabanay
,
I.
,
Zoller
,
M.
, and
Ben-Ze’ev
,
A.
,
1992
, “
Suppression of Tumorigenicity in Transformed Cells After Transfection With Vinculin cDNA
,”
J. Cell Biol.
,
119
(
2
), pp.
427
438
.
47.
Ezzell
,
R. M.
,
Goldmann
,
W. H.
,
Wang
,
N.
,
Parasharama
,
N.
, and
Ingber
,
D. E.
,
1997
, “
Vinculin Promotes Cell Spreading by Mechanically Coupling Integrins to the Cytoskeleton
,”
Exp. Cell Res.
,
231
, pp.
14
26
.
48.
McGrath
,
J. L.
,
Osborn
,
E. A.
,
Tardy
,
Y. S.
,
Dewey
,
C. F.
, Jr.
, and
Hartwig
,
J. H.
,
2000
, “
Regulation of the Actin Cycle In Vivo by Actin Filament Severing
,”
Proc. Natl. Acad. Sci. USA
,
97
(
12
), pp.
6532
6537
.
49.
Tardy
,
Y.
,
Resnick
,
N.
,
Nagel
,
T.
,
Gimbrone
, Jr.,
M. A.
, and
Dewey
, Jr.,
C. F.
,
1997
, “
Shear Stress Gradients Remodel Endothelial Monolayers In Vitro Via a Cell Proliferation-Migration-Loss Cycle
,”
Arterioscler., Thromb., Vasc. Biol.
,
17
(
11
), pp.
3102
3106
.
50.
White
,
G. E.
, and
Gimbrone
,
M. A.
, Jr.
,
1983
, “
Factors Influencing the Expression of Stress Fibers In Vascular Endothelial Cells In Situ
,”
J. Cell Biol.
,
97
, pp.
416
424
.
51.
Wong
,
A. J.
, and
Herman
,
I. M.
,
1983
, “
Actin Filament Stress Fibers In Vascular Endothelial Cells In Vivo
,”
Science
,
219
, pp.
867
869
.
52.
Kim
,
D. W.
,
Gotlieb
,
A. I.
, and
Langille
,
B. L.
,
1989
, “
In Vivo Modulation of Endothelial F-actin Microfilaments by Experimental Alterations in Shear Stress
,”
Arteriosclerosis (Dallas)
,
9
(
4
), pp.
439
445
.
53.
Franke
,
R. P.
,
Grafe
,
M.
,
Schnittler
,
H.
,
Seiffge
,
D.
, and
Mittermayer
,
C.
,
1984
, “
Induction of Human Vascular Endothelial Stress Fibers by Fluid Shear Stress
,”
Nature
,
307
, pp.
648
649
.
54.
Wechezak
,
A. R.
,
Viggers
,
R. F.
, and
Sauvage
,
L. R.
,
1985
, “
Fibronectin and f-actin Redistribution in Cultured Endothelial Cells Exposed to Shear Stress
,”
Lab Invest
,
53
(
6
), pp.
639
647
.
55.
White
,
G. E.
, and
Fujiwara
,
K.
,
1986
, “
Expression and Intracellular Distribution of Stress Fibers in Aortic Endothelium
,”
J. Cell Biol.
,
103
(
1
), pp.
63
70
.
56.
Rudic
,
R. D.
,
Shesely
,
E. G.
,
Maeda
,
N.
,
Smithies
,
O.
,
Segal
,
S. S.
, and
Sessa
,
W. C.
,
1998
, “
Direct Evidence for the Importance of Endothelium-derived Nitric Oxide In Vascular Remodeling
,”
J. Clin. Invest.
,
101
(
4
), pp.
731
736
.
57.
Papapetropoulos
,
A.
,
Rudic
,
R. D.
, and
Sessa
,
W. C.
,
1999
, “
Molecular Control of Nitric Oxide Synthases in the Cardiovascular System
,”
Cardiovasc. Res.
,
43
(
3
), pp.
509
520
.
58.
Malek
,
A. M.
,
Izumo
,
S.
, and
Alper
,
S. L.
,
1999
, “
Modulation by Pathophysiological Stimuli of the Shear Stress-induced Up-regulation of Endothelial Nitric Oxide Synthase Expression in Endothelial Cells
,”
Neurosurgery
,
45
(
2
), pp.
334
344
.
59.
Wedgewood
,
S.
,
Bekker
,
J. M.
, and
Black
,
S. M.
,
2001
, “
Shear Stress Regulation of Endothelial NOS in Fetal Pulmonary Arterial Endothelial Cells Involves PKC
,”
Am J Physiol Lung Cell Mol Physiol
,
281
, pp.
L490–L498
L490–L498
.
60.
Uematsu
,
M.
,
Ohara
,
Y.
,
Navas
,
J. P.
,
Nishida
,
K.
,
Murphy
,
T. J.
,
Alexander
,
R. W.
,
Nerem
,
R. M.
, and
Harrison
,
D. G.
,
1995
, “
Regulation of Endothelial Cell Nitric Oxide Synthase mRNA Expression by Shear Stress
,”
Am. J. Physiol.
,
269
, pp.
C1371–C1378
C1371–C1378
.
61.
Ziegler
,
T.
,
Bouzourene
,
K.
,
Harrison
,
V. J.
,
Brunner
,
H. R.
, and
Hayoz
,
D.
,
1998
, “
Influence of Oscillatory and Unidirectional Flow Environments on the Expression of Endothelin and Nitric Oxide Synthase in Cultured Endothelial Cells
,”
Arterioscler., Thromb., Vasc. Biol.
,
18
(
5
), pp.
686
692
.
62.
Blackman
,
B. R.
,
Thibault
,
L. E.
, and
Barbee
,
K. A.
,
2000
, “
Selective Modulation of Endothelial Cell [Ca2+]i Response to Flow by the Onset Rate of Shear Stress
,”
J. Biomech. Eng.
,
122
(
3
), pp.
274
282
.
63.
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
.
64.
Bao
,
X.
,
Clark
,
C. B.
, and
Frangos
,
J. A.
,
2000
, “
Temporal Gradient in Shear-induced Signaling Pathway: Involvement of MAP Kinase, c-fos, and Connexin-43
,”
Am J Physiol Heart Circ Physiol
,
278
, pp.
H1598–H1605
H1598–H1605
.
65.
Bao
,
X.
,
Lu
,
C.
, and
Frangos
,
J. A.
,
2001
, “
Mechanism of Temporal Gradients in Shear-induced ERK1/2 Activation and Proliferation in Endothelial Cells
,”
Am J Physiol Heart Circ Physiol
,
281
(
1
), pp.
22
29
.
You do not currently have access to this content.