© 1999 American Heart Association, Inc.
Original Contribution |
Mechanotransduction of Rat Aortic Vascular Smooth Muscle Cells Requires RhoA and Intact Actin Filaments
From the Department of Biochemistry (K.N., S.E., T.Y., T.I.), Vanderbilt University School of Medicine, and Department of Anatomy and Physiology (E.D.M.), Meharry Medical College, Nashville, Tenn.
Correspondence to Tadashi Inagami, Department of Biochemistry, Vanderbilt University School of Medicine, Rm 663 Light Hall, Nashville, TN 37232-0146. E-mail inagamit{at}ctrvax.vanderbilt.edu
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Abstract |
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Abstract—The growth-promoting effect of mechanical stress on vascular smooth muscle cells (VSMCs) has been implicated in the progress of vascular disease in hypertension. Extracellular signal–regulated kinases (ERKs) have been implicated in cellular responses, such as vascular remodeling, induced by mechanical stretch. However, it remains to be determined how mechanical stretch activates ERKs. The cytoskeleton seems the most likely candidate for force transmission into the interior of the cell. Therefore, we examined (1) whether the cytoskeleton involves mechanical stretch–induced signaling, (2) whether Rho is activated by stretch, and (3) whether Rho mediates the stretch-induced signaling in rat cultured VSMCs. Mechanical stretch activated ERKs, with a peak response observed at 20 minutes, followed by a significant increase in DNA synthesis. Treatment with the ERK kinase-1 inhibitor, PD98059, inhibited the stretch-induced increase in DNA synthesis. Cytochalasin D, which selectively disrupts the network of actin filaments, markedly inhibited stretch-induced ERK activation. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to mechanical stretch. Botulinum C3 exoenzyme, which inactivates Rho p21 (known to participate in the reorganization of the actin cytoskeleton), attenuated stretch-induced ERK activation. Inhibition of Rho kinase (p160ROCK) also suppressed stretch-induced ERK activation dose dependently. Our results suggest that mechanotransduction in VSMCs is dependent on intact actin filaments, that Rho is activated by stretch, and that Rho/p160ROCK mediates stretch-induced ERK activation and vascular hyperplasia.
Key Words: actin filament • cell growth • mitogen-activated protein kinase • RhoA • vascular smooth muscle cell
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Introduction |
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Excessive hemodynamic forces play an important role in vascular remodeling in hypertension and the genesis of atherosclerosis in vivo.1 2 In hypertension, mechanical strain on the vessel wall is increased by as much as 15%.3 Previous studies have shown that mechanical stress such as hydrostatic pressure4 and stretch5 6 induces DNA synthesis and proto-oncogene expression in vascular smooth muscle cells (VSMCs). These growth-promoting events are induced partly through an autocrine secretion of platelet-derived growth factor-AA (PDGF-AA) in neonatal VSMCs.7 Recently, it has been shown that extracellular signal–regulated kinase (ERK)-1/ERK-2, one of the important mitogenic signals, is activated by stretch in vivo and in vitro in VSMCs.8 9 10 However, the underlying mechanisms by which mechanical events are converted into biochemical signals in VSMCs remain unknown.
Physical deformation of endothelial and other cell types is associated with cytoskeletal reorganization and adaptive changes in the area of cell-matrix contact.11 12 All eukaryotic cells express actin, which often constitutes as much as 50% of the total cellular protein. Although actin filaments are dispersed throughout the cell, they are most highly concentrated just beneath the plasma membrane. Rearrangement of the actin cytoskeleton mediates, at least in part, adaptive changes of cell shape and the degree of cell attachment to the substratum in response to environmental signals. Cytoskeletal proteins are also known to modulate ion channel activity in neural tissue and fibroblasts.13 14 It has been reported that changes in the cytoskeleton are closely associated with some specific gene expression.15 16 Point mutations in adducin heterodimer, which could affect the assembly of the actin-based cytoskeleton, are involved in a form of rat primary hypertension.17 Therefore, the cytoskeleton seems the most likely candidate for force transmission into the interior of the cell.
Previous studies have shown that Rho-family small G proteins (Rho, Rac,
and Cdc42) play a central role in the organization of the actin
cytoskeleton18 and that Rho is required for G
q-induced
hypertrophic gene expression in
cardiomyocytes.19 20 21 Recently, Rho-associated
protein kinase (p160ROCK), a downstream target of Rho, has been shown
to mediate stress fiber formation in Swiss 3T3
fibroblasts.22 However, little is known about the
involvement of Rho-family small G proteins in mechanotransduction in
VSMCs.
In the present study, we investigated whether the cytoskeleton is involved in stretch-induced biochemical signals of VSMCs. We also examined the role of Rho and p160ROCK in mechanotransduction in VSMCs.
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Materials and Methods |
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Materials
DMEM, FCS, penicillin, and streptomycin were obtained from Life Technologies, Inc. Cytochalasin D, colchicine, and angiotensin II were purchased from Sigma. PDGF-AA was purchased from Upstate Biotechnology Inc. Botulinum C3 exoenzyme was purchased from Calbiochem. PD98059 was purchased from New England Biolabs. [
-33P]ATP was purchased from NEN.
Y-27632 was a gift from Yoshitomi Pharmaceutical Industries
Ltd.
Cell Culture and Stretch Protocol
VSMCs were prepared from the thoracic aorta of 12-week-old
Sprague-Dawley rats (Charles River Laboratories) by the explant method
and cultured in DMEM containing 10% FCS, penicillin, and streptomycin.
Subcultured VSMCs from passages 3 to 15, used in the experiments,
showed >99% positive immunostaining of smooth muscle
-actin antibody (Sigma) and were negative for mycoplasma infection
by the polymerase chain reaction kit (Stratagene).
The cells were seeded at 5x104 cells per culture
well on type I collagen–coated Flex I and Flex II plates (Flexercell
International Corp) and grown under nonstretch conditions. Cells were
maintained in complete medium for 4 days, achieving 
80% confluence.
The medium was then changed to serum-free DMEM for 3 days to allow the
cells to become quiescent before application of stretch. The Flex I
plates containing a flexible silicone elastomer substratum were then
mounted in the Flexcell Strain Unit (Flexercell International
Corp),23 24 which consists of a computer-controlled vacuum
unit that applies cyclic vacuum (15 to 20 kPa, 1 Hz) to the
rubber-bottomed dishes in a humidified incubator with 5%
CO2 at 37°C (10% elongation did not increase
thymidine uptake). Control Flex II plates, containing the same
collagen-coated silicone elastomer substratum plus a rigid polystyrene
bottom, were grown in parallel but not mounted in the strain unit.
ERK Activity
After stimulation, the reaction was terminated by the
replacement of medium with the ice-cold lysis buffer containing
(in mmol/L) Tris-HCl (pH 7.4) 10, NaCl 20, EGTA 2, DTT 2,
Na3VO4 1, and
phenylmethylsulfonyl fluoride (PMSF) 1, as well as leupeptin
and aprotinin (10 µg/mL each). After brief sonication (10 seconds),
the samples were centrifuged at 14 000g for 5
minutes, and the supernatant was assayed for ERK activity with an assay
kit (Amersham Corp) that measures the incorporation of
[-33P]ATP into a synthetic peptide
(KRELVEPLTPAGEAPNQALLR) as a specific ERK substrate. The reaction was
carried out with the cell lysate (1 µg of protein) in 75
mmol/L HEPES buffer (pH 7.4), containing (in mmol/L)
MgCl2 1.2, substrate peptide 2, and ATP 1.2, and
1 µCi of [-33P]ATP for 30 minutes at
30°C. The resultant solution was applied to a phosphocellulose
membrane and washed in 1% acetic acid and then
H2O. The radioactivity trapped on the membrane
was measured by liquid scintillation counting.
Subcellular Fractionation
Cell-free lysates were prepared by adding 100 µL lysis buffer
containing (in mmol/L) Tris (pH 8.0) 20, sucrose 250, and PMSF 1,
as well as aprotinin and leupeptin (10 µg/mL each). After 3 cycles of
freeze and thaw, samples were centrifuged at
100 000g at 4°C for 60 minutes. The supernatant was saved
as a "soluble" fraction. Pellets were washed twice by the same
lysis buffer and resuspended in 100 µL of lysis buffer supplemented
with 1% Triton X-100 and 0.1% SDS. Cell debris was separated by
centrifugation (14 000 rpm at 4°C for 20 minutes),
and the supernatant was saved as a "particulate" fraction. The
protein content of each fraction was determined by the Lowry
method.
Immunoblotting
In separate experiments, the reaction was terminated by the
replacement of medium with SDS-PAGE buffer, pH 6.8, containing
62.5 mmol/L Tris-HCl, 2% SDS, 10% glycerol, 50 mmol/L DTT,
and 0.1% bromphenol blue after stimulation. After brief sonication (5
seconds), samples were boiled for 5 minutes at 95°C and
centrifuged (14 000g, 5 minutes) at 4°C, and the
supernatant (25 µL) was subjected to 10% SDS-PAGE. Proteins in the
gel were transferred to a membrane (Amersham) by electroblotting. The
membrane was treated with rabbit polyclonal phosphospecific
mitogen-activated protein kinase (MAPK) antibody (New England
Biolabs Inc) that detects ERK-1/ERK-2 only when catalytically
activated by phosphorylation of Tyr204. To
detect Rho, cell lysates containing equal amounts of protein were
subjected to SDS-PAGE on 15% gels. After transferring the membrane,
it was probed with 1 µg/mL of anti-RhoA antibody (Santa Cruz
Biotechnology). After incubation with secondary anti-rabbit horseradish
peroxidase–conjugated antibody, signals were visualized with an
enhanced chemiluminescence detection system (Amersham).
[3H]Thymidine Incorporation
After 72 hours in serum-free medium, cells were stimulated
by stretch. After stretching for 24 hours,
[3H]thymidine (1 µCi/mL, Amersham
Lifescience) was added for the next 24 hours. Cells were then washed
twice with PBS, and 5% trichloroacetic acid was added at 4°C for 30
minutes to precipitate the protein. Trichloroacetic acid–precipitable
radioactivity was counted by a scintillation counter.
Statistics
Data are given as mean±SE. Statistical analyses were
performed using ANOVA. A post hoc test was performed by the method of
Bonferroni.25 Significance was accepted at the
P<0.05 level.
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Results |
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Mechanical Stretch Activated ERKs and Increased DNA Synthesis
In response to mechanical stretch, there was an apparent phosphorylation of both ERK1 and ERK2, which are p44 and p42 MAPKs, respectively, with peak activity at 20 minutes followed by return to baseline (Figure 1A
).
The time course of ERK activity induced by stretch was similar to that
of phosphorylation (Figure 1B, open bar). The
cells in the middle of the dish show little strain, but they constitute
<15% of the total cells.26 We performed control
experiments using dishes in which cells were excluded from the central,
low-stretch area by placing a 9-mm stainless-steel cloning cup in the
center of the well. Results in ERK activation using these dishes
(Figure 1B, hatched bar) showed results closely analogous with
those of stimulation of cells grown on the entire well. Therefore, we
can assume that the small-percentage presence of cells showing little
strain has little effect. It has been shown that mechanical stretch
induces growth of neonatal VSMCs via autocrine production of
PDGF.7 To investigate the possible role of prestored
growth factor, which might be released from the cells during
stretching, we applied conditioned medium harvested from stretched
cells to other wells containing quiescent VSMCs. However, the
conditioned medium had no effect on ERK activation, which suggests that
the contribution of a paracrine release of growth factors is minimal in
the present system (Figure 1C). Mechanical stretch also
significantly increased [3H]thymidine
incorporation. PD98059 (25 µmol/L), a highly selective ERK
kinase-1 inhibitor that binds to the inactive form of ERK
kinase-1 and prevents its activation,27 abolished the
increase in thymidine incorporation by stretch (Figure 2). These observations suggest a
correlation between mitogenesis and the activity of ERK.
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Stretch-Induced but Not Agonist-Induced ERK Activation Is Dependent
on Intact Actin Filaments
Cytoskeletons mainly comprise actin filaments, microtubules, and
intermediate filaments. To test whether the cytoskeleton is important
in MAPK activation by mechanical stretch,
phosphorylation of ERKs and their kinase activities at
20 minutes were determined in the absence and presence of cytochalasin
D or colchicine. Cytochalasin D is known to depolymerize actin
microfilament bundles, and colchicine is known to bind tubulin and
inhibit its assembly to form microtubules.28 Disruption of
actin filaments with cytochalasin D (0.4 µmol/L) markedly
inhibited ERK activation by mechanical stretch in rat VSMCs, whereas
inhibition of microtubule polymerization with colchicine had no
inhibitory effect (Figure 3A and 3B). To exclude the nonspecific action of cytochalasin D, we
examined its effect on ERK activation by 2 different agonists.
Angiotensin II is reportedly released from cardiac myocytes
during stretching.29 PDGF-AA is reported to be important
for strain-induced growth of neonatal rat VSMCs.7 Both
agonists induced apparent phosphorylation of
ERK-1/ERK-2. However, the dose of cytochalasin D used in the
present study had little effect on angiotensin
II–induced and PDGF-AA–induced phosphorylation of ERK
(Figure 4). Therefore, the effect of
cytochalasin D is specific for mechanical stretch–induced
signaling.
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Mechanical Stretch Induces Partial Translocation of RhoA From the
Soluble to the Particulate Fraction in VSMCs
Because Rho p21, one of the Rho-family small GTP-binding proteins,
has been reported to be critical in actin reorganization and formation
of focal adhesion,17 we investigated whether Rho is
involved in mechanical stretch–induced signaling in VSMCs. It has been
shown that Rho and Rac partially translocate from the soluble to the
particulate fraction on their activation.30 To study
whether mechanical stretch activates RhoA in VSMCs, the
subcellular localization of RhoA before and after mechanical stretch
was examined. Before stimulation, the majority of RhoA was detected in
the soluble fraction, consistent with previous
reports.31 Mechanical stretch caused a rapid and
statistically significant increase in the RhoA content of the
particulate fraction (Figure 5A and 5B).
A significant decrease in RhoA content in the soluble fraction was not
detected in our system because of the relative abundance of RhoA in the
soluble fraction. Parallel immunoblot analysis
indicates that the amount of RhoA detectable in total lysate did not
change significantly before and after mechanical stretch. These results
suggest that part of RhoA translocates from the soluble to the
particulate fraction in response to mechanical stretch.
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Botulinum C3 Exoenzyme and Inhibition of Rho-Associated Protein
Kinase Suppressed Stretch-Induced ERK Activation
Exoenzyme C3 has been a useful and well-established tool in
studying the function of Rho, because it causes ADP ribosylation at
Asn41 of Rho and, hence, specifically inactivates
Rho.32 To assess the involvement of Rho p21 in the
mechanical stretch–induced ERK activation, VSMCs were treated with 4
µg/mL of C3 for 48 hours. Pretreatment with C3 significantly
attenuated mechanical stretch–induced ERK activation (Figure 6). p160ROCK is known as one of
the main direct target molecules of Rho.18 Recently,
Uehata et al33 have developed a pyridine derivative,
Y-27632, which suppresses Rho-induced, p160ROCK-mediated formation of
stress fibers and have shown that p160ROCK is involved in various
models of hypertension. Pretreatment with Y-27632 dose-dependently
inhibited mechanical stretch–induced ERK activation in our system,
suggesting that p160ROCK mediates stretch-induced signaling in rat
VSMCs (Figure 7).
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Discussion |
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The present study demonstrates for the first time that mechanical stretch–induced ERK activation in VSMCs requires intact actin filaments. Our results also suggest that mechanical stretch activates Rho, and Rho/p160ROCK mediates mechanical stretch–induced ERK activation.
When a member of the small GTP-binding proteins is activated, the GTP-bound form is supposedly increased. However, the guanine nucleotide binding assay and determination of the guanine nucleotide exchange activity for Rho have been technically difficult, because the currently available antibodies are not suitable for immunoprecipitating the native form of Rho in the presence of Mg2+. On the other hand, Mg2+ is required for preserving its guanine nucleotide binding of Rho.34 However, results from the present study support the conclusion that mechanical stretch activates Rho in VSMCs. First, mechanical stretch partially translocated Rho from the cytosolic to the membrane fraction determined by immunoblotting. A variety of mitogens, such as angiotensin II,21 endothelin, and lysophosphatidic acid,35 have been reported to induce actin filament organization and cause translocation of Rho from the cytosolic to the membrane fraction. The level of mechanical stretch–induced RhoA translocation was comparable with that of the known stimulators mentioned above.21 35 Second, activation of ERK was suppressed by C3 treatment and inhibition of Rho kinase. Taken together, these results are consistent with RhoA activation by mechanical stretch in VSMCs.
At present, we do not know how Rho is activated by
mechanical stretch. Several growth factors, such as lysophosphatidic
acid, endothelin, PDGF,35 and angiotensin
II,21 have been reported to activate Rho. A
previous study has also suggested that mechanical stretch induces
growth of neonatal VSMCs via autocrine production of
PDGF.7 Therefore, 1 possibility is the release of growth
factors from stretched cells. However, this is less likely in our
system, because the conditioned medium had no effect on ERK activation
(Figure 1C
) and [3H]thymidine uptake
(data not shown). Another possible factor that can activate Rho
is an integrin receptor. It has been shown that antibodies to
Vß5 and
ß3 integrins block thymidine uptake induced by
mechanical stretch36 and that Rho involves
integrin-mediated activation of ERK.37 Whether antibodies
to integrins inhibit stretch-induced activation of Rho and ERK remains
to be elucidated.
The downstream signaling pathway that can connect Rho to ERK constitutes another important question. Recently, several proteins, such as p160ROCK, showing selective binding to GTP-Rho have been isolated and proposed as potential Rho targets.18 p160ROCK, which is a 160-kDa protein serine/threonine kinase, has also been reported to work as a Rho effector in the formation of focal adhesions and stress fibers.38 It has been reported that Y-27632, a recently developed inhibitor of p160ROCK, binds p160ROCK, and its affinity for p160ROCK is apparently higher than that for conventional protein kinase C or cAMP-dependent protein kinase or for myosin light-chain kinase. It has also been shown that Y-27632 can inhibit Rho-induced formation of focal adhesions and stress fibers but not Rac-induced membrane ruffling, and that Y-27632 can decrease blood pressure of various hypertensive animals but not that of normotensive ones.33 Consistent with this report, inhibition of p160ROCK suppressed stretch-induced ERK activation dose dependently in our system, which supports the effect of exoenzyme C3. However, compared with the effect of cytochalasin D, the inhibitory effect of exoenzyme C3 or the p160ROCK inhibitor on stretch-induced ERK activation was weak. These findings may be partly due to the dose of exoenzyme C3, because the cytotoxicity became apparent when we tested a dose >4 µg/mL in our preliminary studies. Another possibility is that Rho has many targets except p160ROCK.38 Also, we cannot exclude the presence of Rho/p160ROCK-independent pathways such as superoxide. Hishikawa et al39 have shown that pulsatile stretch stimulates superoxide production and antioxidants inhibit stretch-induced DNA synthesis. Rac, another GTP-binding protein of the Rho family, may affect the activity of NADPH.40 Therefore, we need to perform further studies on the Rho/p160ROCK-independent pathways in our system.
Details of the mechanotransduction pathway of mechanical stretch–induced cell growth remain unclear. The present study provides the first evidence that Rho, p160ROCK, and intact actin filaments may play an important role in mechanical stretch–induced ERK activation and cell growth.
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Acknowledgments |
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This work was supported in part by NIH Grants HL-03320, HL-85025, and (core facility) DK-20593. K.N. is the recipient of a grant from the Japan Heart Foundation and a Bayer Yakuhin Research Grant Abroad. We thank Trinita Fitzgerald and Edward Price for excellent technical assistance and Tina Stack for secretarial assistance.
Received September 24, 1998; accepted April 7, 1999.
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References |
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|---|
1. Langille BL. Remodeling of developing and mature arteries: endothelium, smooth muscle, and matrix. J Cardiovasc Pharmacol. 1993;21(suppl):S11–S17.
2.
Thubrikar MJ, Robicsek F. Pressure-induced
arterial stress and atherosclerosis.
Ann Thorac Surg. 1995;59:1594–1603.
3.
Safar ME, Peronnoeau PA, Levenson JA, Toto-Moukouno
JA, Simon AC. Pulsed doppler: diameter, blood flow velocity, and
volumic flow of the brachial artery in sustained essential
hypertension. Circulation. 1981;63:393–400.
4. Hishikawa K, Nakaki T, Marumo T, Hayashi M, Suzuki H, Kato R, Saruta T. Pressure promotes DNA synthesis in rat cultured vascular smooth muscle cells. J Clin Invest. 1994;93:1975–1980.
5. Lyall F, Deehan MR, Greer IA, Boswell F, Brown WC, McInnes GT. Mechanical stretch increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells. J Hypertens. 1994;12:1139–1145.[Medline] [Order article via Infotrieve]
6. Sudhir K, Wilson E, Chatterjee K, Ives HE. Mechanical strain and collagen potentiate mitogenic activity of angiotensin II in rat vascular smooth muscle cells. J Clin Invest. 1993;92:3003–3007.
7.
Wilson E, Mai Q, Sudhir K, Weiss RH, Ives HE.
Mechanical strain induces growth of vascular smooth muscle cells via
autocrine action of PDGF. J Cell Biol. 1993;123:741–747.
8. Reusch HP, Chan G, Ives HE, Nemenoff RA. Activation of JNK/SAPK and ERK by mechanical strain in vascular smooth muscle cells depends on extracellular matrix composition. Biochem Biophys Res Commun. 1997;237:239–244.[Medline] [Order article via Infotrieve]
9.
Birukov KG, Lehoux S, Birukova AA, Merval R, Tkachuk
VA, Tedgui A. Increased pressure induces sustained protein kinase
C-independent herbimycin A-sensitive activation of extracellular
signal-related kinase 1/2 in the rabbit aorta in organ culture.
Circ Res. 1997;81:895–903.
10.
Pyles JM, March KL, Franklin M, Mehdi K, Wilensky RL,
Adam LP. Activation of MAP kinase in vivo follows balloon overstretch
injury of porcine coronary and carotid arteries. Circ
Res. 1997;81:904–910.
11. Franke RP, Grafe M, Schnittler H, Seiffge D, Mittermayer C. Induction of human vascular endothelial stress fiber by fluid shear stress. Nature. 1984;307:648–649.[Medline] [Order article via Infotrieve]
12.
Davies PF, Tripathi SC. Mechanical stress mechanisms
and the cell: an endothelial paradigm. Circ
Res. 1993;72:239–245.
13. Johnson BD, Byerly L. A cytoskeletal mechanism for Ca2+ channel metabolic dependence on inactivation by intracellular Ca2+. Neuron. 1993;10:797–804.[Medline] [Order article via Infotrieve]
14.
Ribiero CMP, Reece J, Putney JW Jr. Role of
cytoskeleton in calcium signaling in NIH 3T3 cells. J Biol
Chem. 1997;272:26555–26561.
15. Morita T, Kurihara H, Maemura K, Yoshizumi M, Yazaki Y. Disruption of cytoskeletal structures mediates shear stress-induced endothelin-1 gene expression in cultured porcine aortic endothelial cells. J Clin Invest. 1993;92:1706–1712.
16.
Botteri FM, Ballmer-Hofer K, Rajput B, Nagamine Y.
Disruption of cytoskeletal structures results in the induction of the
urokinase-type plasminogen activator gene
expression. J Biol Chem. 1990;265:13327–13334.
17. Tripodi G, Valtorta F, Torielli L, Chieregatti E, Salardi S, Trusolino L, Menegon A, Ferrari P, Marchisio PC, Bianchi G. Hypertension-associated point mutations in the adducin alpha and beta subunits affect actin cytoskeleton and ion transport. J Clin Invest. 1996;97:2815–2822.[Medline] [Order article via Infotrieve]
18. Tapon N, Hall A. Rho, Rac, and Cdc42 GTPases regulate the organization of the actin cytoskeleton. Curr Opin Cell Biol. 1997;9:86–92.[Medline] [Order article via Infotrieve]
19.
Sah VP, Hoshijima M, Chien KR, Brown JH. Rho is
required for Gq and 1-adrenergic receptor signaling in
cardiomyocytes: dissociation of Ras and Rho pathways.
J Biol Chem. 1996;271:31185–31190.
20.
Hines WA, Thorburn A. Ras and Rho are required for
Gq-induced hypertrophic gene expression in neonatal rat cardiac
myocytes. J Mol Cell Cardiol. 1998;30:485–494.[Medline]
[Order article via Infotrieve]
21.
Aoki H, Izumo S, Sadoshima J. Angiotensin
II activates RhoA in cardiac myocytes: a critical role of RhoA
in angiotensin II-induced premyofibril formation.
Circ Res. 1998;82:666–676.
22.
Amano M, Chihara K, Kimura K, Fukata Y, Nakamura N,
Matsuura Y, Kaibuchi K. Formation of actin stress fibers and focal
adhesions enhanced by Rho-kinase. Science. 1997;275:1308–1311.
23. Banes AJ, Gilbert J, Taylor D, Monbureau O. A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro. J Cell Sci. 1985;75:35–42.[Abstract]
24. Banes AJ, Link GW, Gilbert JW, Monbureau O. Culturing cells in a mechanically active environment: the Flexercell Strain Unit can apply cyclic or static tension or compression to cells in culture. Am Biotechnol Lab. 1990;8:12–22.[Medline] [Order article via Infotrieve]
25. Glantz SA. How to use t tests to isolate differences between groups in analysis of variance. In: Primer of Biostatistics. New York, NY: McGraw-Hill; 1987:88–89.
26. Gilbert JA, Weinhold PS, Banes AJ, Link GW, Jones GL. Strain profiles for circular cell culture plates containing flexible surfaces employed to mechanically deform cells in vitro. J Biomech. 1994;27:1169–1177.[Medline] [Order article via Infotrieve]
27.
Alessi DR, Cuenda A, Cohen P, Dudley DT, Saltiel AR.
PD98059 is a specific inhibitor of the activation of
mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem. 1995;270:27489–27494.
28. Darnell J, Lodish H, Baltimore D. Molecular Cell Biology. 2nd ed. New York, NY: Scientific American Books; 1990.
29. Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell. 1993;75:977–984.[Medline] [Order article via Infotrieve]
30.
Bokoch GM, Bohl BP, Chuang TH. Guanine
nucleotide exchange regulates membrane translocation of
Rac/Rho GTP-binding proteins. J Biol Chem. 1994;269:31674–31679.
31.
Adamson P, Paterson HF, Hall A. Intracellular
localization of the p21rho proteins. J Cell Biol. 1992;119:617–627.
32.
Sekine A, Fujiwara M, Narumiya S. Asparagine residue in
the rho gene product is the modification site for botulinum
ADP-ribosyltransferase. J Biol Chem. 1989;264:8602–8605.
33. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature. 1997;389:990–994.[Medline] [Order article via Infotrieve]
34. Aktories K, Just I. In vitro ADP-ribosylation of Rho by bacterial ADP- ribosyltransferases. Methods Enzymol. 1995;256:184–195.[Medline] [Order article via Infotrieve]
35.
Fleming IN, Elliot CM, Exton JH. Differential
translocation of Rho family GTPases by lysophosphatidic acid,
endothelin-1, and platelet-derived growth factor. J Biol
Chem. 1996;271:33067–33073.
36. Wilson E, Sudhir K, Ives HE. Mechanical strain of rat vascular smooth muscle cells is sensed by specific matrix/integrin interactions. J Clin Invest. 1995;96:2364–2372.
37.
Renshaw MW, Toksoz D, Schwartz MA. Involvement of the
small GTPase Rho in integrin-mediated activation of
mitogen-activated protein kinase. J Biol Chem. 1996;271:21691–21694.
38. Narumiya S, Ishizaki T, Watanabe N. Rho effectors and reorganization of actin cytoskeleton. FEBS Lett. 1997;410:68–72.[Medline] [Order article via Infotrieve]
39.
Hishikawa K, Oemar BS, Yang Z, Luscher TF. Pulsatile
stretch stimulates superoxide production and activates
nuclear factor-
B in human coronary smooth muscle.
Circ Res. 1997;81:797–803.
40.
Diekmann D, Abo A, Johnston C, Segal AW, Hall A.
Interaction of Rac with p67phox and regulation of phagocytic NADPH
oxidase activity. Science. 1994;265:531–533.
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G. Loirand, P. Guerin, and P. Pacaud Rho Kinases in Cardiovascular Physiology and Pathophysiology Circ. Res., February 17, 2006; 98(3): 322 - 334. [Abstract] [Full Text] [PDF]
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 K. J. Salu, J. M. Bosmans, Y. Huang, M. Hendriks, M. Verhoeven, A. Levels, S. Cooper, I. K. De Scheerder, C. J. Vrints, and H. Bult Effects of cytochalasin D-eluting stents on intimal hyperplasia in a porcine coronary artery model Cardiovasc Res, February 1, 2006; 69(2): 536 - 544. [Abstract] [Full Text] [PDF]
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T. Kozai, M. Eto, Z. Yang, H. Shimokawa, and T. F. Luscher Statins prevent pulsatile stretch-induced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway Cardiovasc Res, December 1, 2005; 68(3): 475 - 482. [Abstract] [Full Text] [PDF]
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 J. C. Krepinsky, Y. Li, Y. Chang, L. Liu, F. Peng, D. Wu, D. Tang, J. Scholey, and A. J. Ingram Akt Mediates Mechanical Strain-Induced Collagen Production by Mesangial Cells J. Am. Soc. Nephrol., June 1, 2005; 16(6): 1661 - 1672. [Abstract] [Full Text] [PDF]
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 G. Burgstaller and M. Gimona Podosome-mediated matrix resorption and cell motility in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, June 1, 2005; 288(6): H3001 - H3005. [Abstract] [Full Text] [PDF]
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 N. I. Gokina, K. M. Park, K. McElroy-Yaggy, and G. Osol Effects of Rho kinase inhibition on cerebral artery myogenic tone and reactivity J Appl Physiol, May 1, 2005; 98(5): 1940 - 1948. [Abstract] [Full Text] [PDF]
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G. Loirand, M. Rolli-Derkinderen, and P. Pacaud RhoA and resistance artery remodeling Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1051 - H1056. [Abstract] [Full Text] [PDF]
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R. Moudgil, E. D. Michelakis, and S. L. Archer Hypoxic pulmonary vasoconstriction J Appl Physiol, January 1, 2005; 98(1): 390 - 403. [Abstract] [Full Text] [PDF]
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 S. Zhou, A. Schmelz, T. Seufferlein, Y. Li, J. Zhao, and M. G. Bachem Molecular Mechanisms of Low Intensity Pulsed Ultrasound in Human Skin Fibroblasts J. Biol. Chem., December 24, 2004; 279(52): 54463 - 54469. [Abstract] [Full Text] [PDF]
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K. Lockman, J. S. Hinson, M. D. Medlin, D. Morris, J. M. Taylor, and C. P. Mack Sphingosine 1-Phosphate Stimulates Smooth Muscle Cell Differentiation and Proliferation by Activating Separate Serum Response Factor Co-factors J. Biol. Chem., October 8, 2004; 279(41): 42422 - 42430. [Abstract] [Full Text] [PDF]
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C. Boer, G. P. van Nieuw Amerongen, A. B. J. Groeneveld, G. J. Scheffer, J. J. de Lange, N. Westerhof, V. W. M. van Hinsbergh, and P. Sipkema Smooth muscle F-actin disassembly and RhoA/Rho-kinase signaling during endotoxin-induced alterations in pulmonary arterial compliance Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L649 - L655. [Abstract] [Full Text] [PDF]
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S. Albinsson, I. Nordstrom, and P. Hellstrand Stretch of the Vascular Wall Induces Smooth Muscle Differentiation by Promoting Actin Polymerization J. Biol. Chem., August 13, 2004; 279(33): 34849 - 34855. [Abstract] [Full Text] [PDF]
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 P. A. Ortiz, N. J. Hong, and J. L. Garvin Luminal flow induces eNOS activation and translocation in the rat thick ascending limb Am J Physiol Renal Physiol, August 1, 2004; 287(2): F274 - F280. [Abstract] [Full Text] [PDF]
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N. Boutahar, A. Guignandon, L. Vico, and M.-H. Lafage-Proust Mechanical Strain on Osteoblasts Activates Autophosphorylation of Focal Adhesion Kinase and Proline-rich Tyrosine Kinase 2 Tyrosine Sites Involved in ERK Activation J. Biol. Chem., July 16, 2004; 279(29): 30588 - 30599. [Abstract] [Full Text] [PDF]
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 Y. Zhao, H. Xiao, W. Long, W. J. Pearce, and L. D. Longo Expression of several cytoskeletal proteins in ovine cerebral arteries: developmental and functional considerations J. Physiol., July 15, 2004; 558(2): 623 - 632. [Abstract] [Full Text] [PDF]
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J.-i. Kawabe, S. Okumura, M.-C. Lee, J. Sadoshima, and Y. Ishikawa Translocation of caveolin regulates stretch-induced ERK activity in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1845 - H1852. [Abstract] [Full Text] [PDF]
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T. R. Polte, G. S. Eichler, N. Wang, and D. E. Ingber Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress Am J Physiol Cell Physiol, March 1, 2004; 286(3): C518 - C528. [Abstract] [Full Text] [PDF]
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 E. B. Okon, M. J. Millar, C. M. Crowley, J. G. Bashir, R. C. Cook, Y. N. Hsiang, B. McManus, and C. van Breemen Effect of moderate pressure distention on the human saphenous vein vasomotor function Ann. Thorac. Surg., January 1, 2004; 77(1): 108 - 114. [Abstract] [Full Text] [PDF]
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 A. Zeidan, J. Broman, P. Hellstrand, and K. Sward Cholesterol Dependence of Vascular ERK1/2 Activation and Growth in Response to Stretch: Role of Endothelin-1 Arterioscler Thromb Vasc Biol, September 1, 2003; 23(9): 1528 - 1534. [Abstract] [Full Text] [PDF]
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S. Kawamura, S. Miyamoto, and J. H. Brown Initiation and Transduction of Stretch-induced RhoA and Rac1 Activation through Caveolae: CYTOSKELETAL REGULATION OF ERK TRANSLOCATION J. Biol. Chem., August 15, 2003; 278(33): 31111 - 31117. [Abstract] [Full Text] [PDF]
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A. Zeidan, I. Nordstrom, S. Albinsson, U. Malmqvist, K. Sward, and P. Hellstrand Stretch-induced contractile differentiation of vascular smooth muscle: sensitivity to actin polymerization inhibitors Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1387 - C1396. [Abstract] [Full Text] [PDF]
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 Y. E.G. Eskildsen-Helmond and M. J. Mulvany Pressure-Induced Activation of Extracellular Signal-Regulated Kinase 1/2 in Small Arteries Hypertension, April 1, 2003; 41(4): 891 - 897. [Abstract] [Full Text] [PDF]
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T. Seko, M. Ito, Y. Kureishi, R. Okamoto, N. Moriki, K. Onishi, N. Isaka, D. J. Hartshorne, and T. Nakano Activation of RhoA and Inhibition of Myosin Phosphatase as Important Components in Hypertension in Vascular Smooth Muscle Circ. Res., March 7, 2003; 92(4): 411 - 418. [Abstract] [Full Text] [PDF]
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A. J. Putnam, J. J. Cunningham, B. B. L. Pillemer, and D. J. Mooney External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly Am J Physiol Cell Physiol, March 1, 2003; 284(3): C627 - C639. [Abstract] [Full Text] [PDF]
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R. A. Oeckler, P. M. Kaminski, and M. S. Wolin Stretch Enhances Contraction of Bovine Coronary Arteries via an NAD(P)H Oxidase-Mediated Activation of the Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Cascade Circ. Res., January 10, 2003; 92(1): 23 - 31. [Abstract] [Full Text] [PDF]
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M. P. Massett, Z. Ungvari, A. Csiszar, G. Kaley, and A. Koller Different roles of PKC and MAP kinases in arteriolar constrictions to pressure and agonists Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2282 - H2287. [Abstract] [Full Text] [PDF]
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 I. Lavelin, N. Meiri, M. Einat, O. Genina, and M. Pines Mechanical strain regulation of the chicken glypican-4 gene expression in the avian eggshell gland Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2002; 283(4): R853 - R861. [Abstract] [Full Text] [PDF]
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N. Kobayashi, S. Horinaka, S.-i. Mita, S. Nakano, T. Honda, K. Yoshida, T. Kobayashi, and H. Matsuoka Critical role of Rho-kinase pathway for cardiac performance and remodeling in failing rat hearts Cardiovasc Res, September 1, 2002; 55(4): 757 - 767. [Abstract] [Full Text] [PDF]
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 N. Kobayashi, S. Nakano, S.-i. Mita, T. Kobayashi, T. Honda, Y. Tsubokou, and H. Matsuoka Involvement of Rho-Kinase Pathway for Angiotensin II-Induced Plasminogen Activator Inhibitor-1 Gene Expression and Cardiovascular Remodeling in Hypertensive Rats J. Pharmacol. Exp. Ther., May 1, 2002; 301(2): 459 - 466. [Abstract] [Full Text] [PDF]
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Y. Shafrir and G. Forgacs Mechanotransduction through the cytoskeleton Am J Physiol Cell Physiol, March 1, 2002; 282(3): C479 - C486. [Abstract] [Full Text] [PDF]
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I. Tamura, J. Rosenbloom, E. Macarak, and B. Chaqour Regulation of Cyr61 gene expression by mechanical stretch through multiple signaling pathways Am J Physiol Cell Physiol, November 1, 2001; 281(5): C1524 - C1532. [Abstract] [Full Text] [PDF]
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E. Songu-Mize, N. Sevieux, X. Liu, and M. Jacobs Effect of short-term cyclic stretch on sodium pump activity in aortic smooth muscle cells Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2072 - H2078. [Abstract] [Full Text] [PDF]
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I. Lavelin, N. Meiri, O. Genina, R. Alexiev, and M. Pines Na+-K+-ATPase gene expression in the avian eggshell gland: distinct regulation in different cell types Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2001; 281(4): R1169 - R1176. [Abstract] [Full Text] [PDF]
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M. A. Hill, H. Zou, S. J. Potocnik, G. A. Meininger, and M. J. Davis Signal Transduction in Smooth Muscle: Invited Review: Arteriolar smooth muscle mechanotransduction: Ca2+ signaling pathways underlying myogenic reactivity J Appl Physiol, August 1, 2001; 91(2): 973 - 983. [Abstract] [Full Text] [PDF]
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K. Matrougui, L. B. Tanko, L. Loufrani, D. Gorny, B. I. Levy, A. Tedgui, and D. Henrion Involvement of Rho-Kinase and the Actin Filament Network in Angiotensin II-Induced Contraction and Extracellular Signal-Regulated Kinase Activity in Intact Rat Mesenteric Resistance Arteries Arterioscler Thromb Vasc Biol, August 1, 2001; 21(8): 1288 - 1293. [Abstract] [Full Text] [PDF]
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Y. Funakoshi, T. Ichiki, H. Shimokawa, K. Egashira, K. Takeda, K. Kaibuchi, M. Takeya, T. Yoshimura, and A. Takeshita Rho-Kinase Mediates Angiotensin II-Induced Monocyte Chemoattractant Protein-1 Expression in Rat Vascular Smooth Muscle Cells Hypertension, July 1, 2001; 38(1): 100 - 104. [Abstract] [Full Text] [PDF]
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 E. G. Araujo, C. Bianchi, K. Sato, R. Faro, X. A. Li, and F. W. Sellke Inactivation of the MEK/ERK pathway in the myocardium during cardiopulmonary bypass J. Thorac. Cardiovasc. Surg., April 1, 2001; 121(4): 773 - 781. [Abstract] [Full Text] [PDF]
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M. J. Davis, X. Wu, T. R. Nurkiewicz, J. Kawasaki, G. E. Davis, M. A. Hill, and G. A. Meininger Integrins and mechanotransduction of the vascular myogenic response Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1427 - H1433. [Abstract] [Full Text] [PDF]
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G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh Cytoskeletal Effects of Rho-Like Small Guanine Nucleotide-Binding Proteins in the Vascular System Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 300 - 311. [Abstract] [Full Text] [PDF]
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N. ENDLICH, K. R. KRESS, J. REISER, D. UTTENWEILER, W. KRIZ, P. MUNDEL, and K. ENDLICH Podocytes Respond to Mechanical Stress In Vitro J. Am. Soc. Nephrol., March 1, 2001; 12(3): 413 - 422. [Abstract] [Full Text]
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 U. Laufs, M. Endres, F. Custodis, K. Gertz, G. Nickenig, J. K. Liao, and M. Bohm Suppression of Endothelial Nitric Oxide Production After Withdrawal of Statin Treatment Is Mediated by Negative Feedback Regulation of Rho GTPase Gene Transcription Circulation, December 19, 2000; 102(25): 3104 - 3110. [Abstract] [Full Text] [PDF]
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N. Sawada, H. Itoh, K. Ueyama, J. Yamashita, K. Doi, T.-H. Chun, M. Inoue, K. Masatsugu, T. Saito, Y. Fukunaga, et al. Inhibition of Rho-Associated Kinase Results in Suppression of Neointimal Formation of Balloon-Injured Arteries Circulation, May 2, 2000; 101(17): 2030 - 2033. [Abstract] [Full Text] [PDF]
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T. Yamakawa, S.-i. Tanaka, K. Numaguchi, Y. Yamakawa, E. D. Motley, S. Ichihara, and T. Inagami Involvement of Rho-Kinase in Angiotensin II-Induced Hypertrophy of Rat Vascular Smooth Muscle Cells Hypertension, January 1, 2000; 35(1): 313 - 318. [Abstract] [Full Text] [PDF]
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C. P. Mack, A. V. Somlyo, M. Hautmann, A. P. Somlyo, and G. K. Owens Smooth Muscle Differentiation Marker Gene Expression Is Regulated by RhoA-mediated Actin Polymerization J. Biol. Chem., January 5, 2001; 276(1): 341 - 347. [Abstract] [Full Text] [PDF]
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A. J. Ingram, L. James, L. Cai, K. Thai, H. Ly, and J. W. Scholey NO Inhibits Stretch-induced MAPK Activity by Cytoskeletal Disruption J. Biol. Chem., December 15, 2000; 275(51): 40301 - 40306. [Abstract] [Full Text] [PDF]
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N. Ashida, H. Arai, M. Yamasaki, and T. Kita Distinct Signaling Pathways for MCP-1-dependent Integrin Activation and Chemotaxis J. Biol. Chem., May 4, 2001; 276(19): 16555 - 16560. [Abstract] [Full Text] [PDF]
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M. E. Goldschmidt, K. J. McLeod, and W. R. Taylor Integrin-Mediated Mechanotransduction in Vascular Smooth Muscle Cells : Frequency and Force Response Characteristics Circ. Res., April 13, 2001; 88(7): 674 - 680. [Abstract] [Full Text] [PDF]
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S. Chrissobolis and C. G. Sobey Evidence That Rho-Kinase Activity Contributes to Cerebral Vascular Tone In Vivo and Is Enhanced During Chronic Hypertension : Comparison With Protein Kinase C Circ. Res., April 27, 2001; 88(8): 774 - 779. [Abstract] [Full Text] [PDF]
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H. Chaulet, C. Desgranges, M.-A. Renault, F. Dupuch, G. Ezan, F. Peiretti, G. Loirand, P. Pacaud, and A.-P. Gadeau Extracellular Nucleotides Induce Arterial Smooth Muscle Cell Migration Via Osteopontin Circ. Res., October 26, 2001; 89(9): 772 - 778. [Abstract] [Full Text] [PDF]
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