doi: 10.1161/hq0102.101518
© 2002 American Heart Association, Inc.
Vascular Biology |
Shear Stress–Induced Endothelial Cell Migration Involves Integrin Signaling Via the Fibronectin Receptor Subunits 
5 and ß1
From Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Frankfurt, Germany.
Correspondence to Stefanie Dimmeler, PhD, Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor Stern-Kai 7, 60590 Frankfurt, Germany. E-mail dimmeler{at}em.uni-frankfurt.de
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Abstract |
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Endothelial cell (EC) migration is required for angiogenesis, neovascularization, and reendothelialization. Integrins, known as
ß-heterodimeric cell-surface receptors, regulate cell migration and are essential for mechanotransduction of hemodynamic forces. Therefore, we investigated the effect of shear stress on EC migration and the contribution of the integrins and integrin-dependent signaling pathways in a scratched-wound assay. Laminar shear stress–induced EC migration was significantly reduced by integrin-receptor blocking with RGD peptides or with neutralizing antibodies against integrin subunits 5 and ß1, whereas antibodies against vß3 or 2ß1 had no effect. Cell-surface levels of the integrin 5 and ß1 were specifically upregulated in migrating ECs at the wound edges. Consistent with the important role of integrins for shear stress–increased cell migration, blockade of the integrin-associated adapter protein Shc by overexpression of dominant negative construct inhibited shear stress–stimulated EC migration. Moreover, pharmacological inhibition of the integrin downstream effector signaling molecules ERK1/2 or phosphatidyl-inositol-3-kinase prevented shear stress–induced EC migration. In contrast, inhibition of the NO synthase had no effect. Taken together, our results indicate that laminar shear stress enhances EC migration via the fibronectin receptor subunits 5 and ß1, which serve as central mechanotransducers in ECs. Shear stress–induced enhancement of EC migration might contribute importantly to accelerated reendothelialization of denuded arteries.
Key Words: integrins • migration • shear stress • endothelial cells • fibronectin receptor
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Introduction |
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Integrins are
ß-heterodimeric cell-surface receptors that recognize a large number of extracellular ligands.1–3 Currently, 18 - and 8 ß-subunits have been identified, which could form more than 24 heterodimeric receptors.3,4 Ligand binding by integrins regulates such processes as cell adhesion, growth, differentiation, and migration.1,5 Cell adhesion and migration are required for angiogenesis, which was thought to depend primarily on v-integrins, for example, vß3.6 Studies in v-knockout mice, however, indicate that other receptors and their ligands are also involved in vasculogenesis and angiogenesis.7 Consistent with these findings, recent knockout studies indicate a more important role of other integrins, such as ß1 or 5.4 5-Knockout mice have severe defects in vascular development,8 and 1ß1- and 2ß1-integrins seems to contribute to angiogenesis.9,10 Integrins play a further important role in mediating mechanical stress–induced signals. Specifically, integrins like 5ß1 and vß3 are essential for the mechanotransduction of hemodynamic forces into biochemical signals.11–15
Hemodynamic forces are important regulators of blood vessel function and structure. Of these forces, the laminar blood flow (shear stress) is a potent endogenous mediator of atheroprotective signals in endothelial cells (ECs).14 Several studies have shown that the beneficial effects of shear stress, such as activation of the prosurvival kinases Akt and ERK1/2, require an intact integrin signaling.12,13,16 We have previously demonstrated that laminar shear stress upregulates the fibronectin receptor subunits 5 and ß1 in ECs and enhances EC adhesion and survival.17 In addition, integrins are important for cell migration, a process essential for angiogenesis and reendothelialization on denuding injury.18–20 A recent study provides evidence that shear stress regulates EC migration.21 The underlying mechanism, however, remains unclear. Therefore, we investigated the effect of laminar shear stress on EC migration, the involvement of the integrins 5 and ß1, and the intracellular signaling pathways. The results of the present study demonstrate that laminar shear stress enhances migration of human umbilical vein ECs (HUVECs) and human cardiac microvascular ECs (HMVEC-Cs). Shear stress–induced EC migration is mediated via the fibronectin receptor subunits 5 and ß1 and activation of phosphatidylinositol 3-kinase (PI3K) and ERK1/2 but is independent of NO. Moreover, the cell-surface levels of the integrins 5 and ß1 are upregulated in migrating ECs at the edge of the wound and additionally enhanced in migrating cells exposed to laminar shear stress. Finally, we demonstrate that the intracellular integrin signaling, as measured by the phosphorylation of focal adhesion kinase (FAK) and the mitogen-activated protein (MAP) kinase (MAPK) ERK1/2, is activated in migrating ECs that were exposed to shear stress.
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Methods |
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Cell Culture
Pooled HUVECs were purchased from Cell Systems/Clonetics and cultured in endothelial basal medium (Clonetics) supplemented with hydrocortisone (1 µg/mL), bovine brain extract (3 µg/mL), gentamicin (50 µg/mL), amphotericin B (50 µg/mL), epidermal growth factor (10 µg/mL), and 10% FCS until the third passage. After detachment with trypsin, cells (4.0x105 cells) were grown in 6-cm cell culture dishes for
18 hours. HMVEC-Cs were purchased from Cell Systems/Clonetics and cultured according to the instructions of the manufacturer in endothelial basal medium-2 (Clonetics) supplemented with epidermal growth factor, hydrocortisone, fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, ascorbic acid, gentamicin, amphotericin-B, and 5% FCS until the fifth passage. HUVECs or HMVEC-Cs were exposed to laminar fluid flow in a cone-and-plate apparatus as previously described.12,22 A constant shear stress of 5, 15, or 45 dynes/cm2 was used to simulate physiological levels of shear stress. NG-monomethyl-L-arginine (L-NMMA; Alexis), PD98059 (Biomol), Ly294002 (Biomol), the synthetic peptide Gly-Arg-Gly-Asp-Asn-Pro (GRGDNP, Gibco-BRL), or antibodies against the integrin subunits 5 and ß1 (Dianova and Gibco-BRL) were preincubated 30 minutes before shear stress exposure.
Cell Migration
Migration of HUVECs and HMVEC-Cs was detected by use of a "scratched-wound assay." Therefore, in vitro scratched wounds (14 mm wide) were created by scraping cell monolayers with a sterile disposable rubber policeman.23,24 Cells were grown on 6-cm wells previously labeled with a traced line. After injury, the cells were gently washed with medium and exposed to laminar shear stress. EC migration from the edge of the injured monolayer was quantified by measuring the distance between the wound edges before and after injury with a computer-assisted microscope (Zeiss) at 5 distinct positions (every 5 mm).
Immunostaining
HUVECs were fixed with 4% paraformaldehyde and stained with anti–integrin 5 or ß1 antibodies (1:20 in PBS/5% FCS, Transduction Laboratories) and secondary anti-mouse FITC-linked antibodies (1:20 in PBS/5% FCS, Dako). Integrin staining was quantified with a computer-assisted microscope.
Detection of Cell Proliferation
HUVECs were washed, fixed with 2% paraformaldehyde, permeabilized with 0.25% Triton X-100, and stained with an anti–Ki-67 antibody (1:50 in PBS/5% FCS, Dianova). Cells were incubated with a secondary anti-mouse antibody linked to FITC (1:20 in PBS/5% FCS, Dako) followed by counterstaining with 4',6-diamidinophenylindole (DAPI; 1 µg/mL).
Flow Cytometry
HUVECs were washed with PBS, incubated with 1 mmol/L EDTA (pH 7.4) in PBS for 20 minutes at 37°C, and harvested by gentle pipetting. After centrifugation (8 minutes, 700g), cells were washed with PBS, and centrifugation was repeated. The cell pellet was resuspended in 100 µL PBS/10% FCS and incubated for 30 minutes at 4°C with 10 µL FITC-conjugated integrin 5 or ß1 antibodies (Dianova). Subsequently, immunofluorescence-labeled cells were fixed with 4% paraformaldehyde in PBS and analyzed by flow cytometry with FACSCalibur, Perkin Elmer (CellQuest software).
Western Blot Analysis
For determination of the phosphorylated form of ERK1/2 and FAK, HUVECs were scraped off the plates on migration, first in front of the wound edge and then behind the wound edge, and were centrifuged (700g, 5 minutes), followed by incubation of the cells with 60 µL lysis buffer (mmol/L: Tris 20 [pH 7.4], NaCl 150, EDTA 1, EGTA 1, sodium pyrophosphate 2.5, ß-glycerophosphate 1, Na3VO4 1, and PMSF 1, and 1% Triton and 1 µg/mL leupeptin) for 20 minutes on ice. After centrifugation for 15 minutes at 20 000g (4°C), the protein content of the samples was determined according to Bradford. Proteins (40 µg/lane) were loaded onto SDS-polyacrylamide gels and blotted onto PVDF membranes. After blocking with 3% BSA at room temperature for 2 hours, the phospho-ERK1/2 antibody (1:2000; Biolabs) or the phospho-FAK (Tyr 397) antibody (1:500; Upstate Biotechnology) were incubated in TBS (mmol/L: Tris/HCl 50 [pH 8], NaCl 150, KCl 2.5)/0.1% Tween-20/3% BSA for 2 hours. After incubation with the secondary antibody (1:4000 anti-rabbit; Amersham) for 1 hour at room temperature, enhanced chemiluminescence was performed according to the manufacturer’s instructions. Then, the blots were reprobed with total ERK1/2 (1:1000 in TBS/0.1% Tween-20/3% BSA for 2 hours; Biolabs) or FAK (1:1000 in TBS/0.1% Tween-20/5% milk for 2 hours; Transduction Laboratories). The autoradiographs were scanned and semiquantitatively analyzed.
Plasmids and Transfection
Plasmids encoding the human full-length Shc and ERK2 were cloned by polymerase chain reaction pcDNA3.1-myc-His (InVitrogen). The mutated constructs Shc Y317F and ERK D319N were generated by site-directed mutagenesis. Clones with verified sequences were transfected in HUVECs (3.5x105 cells/6-cm well; 3 µg plasmid DNA; 25 µL Superfect) with a transfection efficiency of 
50% as previously described.25
Statistical Analysis
Data are expressed as mean±SEM from 3 independent experiments. Statistical analysis was performed by t test. ANOVA was performed for serial analyses.
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Results |
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Shear Stress Enhances EC Migration Via Integrin
5ß1To investigate the effect of laminar shear stress on EC migration, HUVECs were exposed to laminar shear stress (5, 15, or 45 dynes/cm2) for 24 hours, and cell migration was detected by use of a scratched-wound assay. As shown in Figure 1A, shear stress dose-dependently enhances migration of HUVECs, with a maximum at 45 dynes/cm2. Thereby, shear stress was at least as effective as vascular endothelial growth factor (VEGF, 10 ng/mL: 1.6±0.1 mm25). The proliferation rate of ECs in the reendothelialized areas of the wound remained unchanged as detected by Ki-67 staining of migrating cells (59.1% in the scratched wound versus 57.8% behind the wound under static conditions and 51.2% versus 45.6% with shear stress exposure). Similar results were obtained with HMVEC-Cs (Figure 1B).
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Because integrins are necessary for cell migration,10,26 we investigated the involvement of integrin 5ß1 in EC migration. Incubation of HUVECs with the peptides GRGDNP, which block the binding of integrins to fibronectin and vitronectin, or GRGDSP, which more specifically interferes with fibronectin-integrin interaction, significantly reduced the shear stress–induced EC migration (Figure 1C). The control peptides GRGESP or GRADSP had no effect (Figure 1C).
In addition, the specific inhibition of integrin 5ß1 with neutralizing antibodies against integrin 5 and ß1 prevented the shear stress–induced migration of HUVECs and HMVEC-Cs (Figure 1B/D), whereas anti-vß3 or -2ß1 antibodies did not affect shear stress–induced EC migration (Figure 1D). These results suggest a crucial involvement of the fibronectin receptor 5ß1 in shear stress–induced EC migration. To test the role of integrins in VEGF-induced cell migration, HUVECs were coincubated with RGD peptides (GRGDNP; 0.5 mmol/L) and VEGF (10 ng/mL) for 24 hours. RGD peptides completely prevented VEGF-induced cell migration (data not shown), indicating that VEGF-stimulated reendothelialization also depends on integrins.
Shear Stress Upregulates the Cell-Surface Levels of Integrin 5ß1 in Migrating ECs
Having demonstrated that the fibronectin receptor 5ß1 is necessary for EC migration, we analyzed the cell-surface levels of integrin 5 and ß1 in migrating cells by immunostaining. Therefore, HUVECs were cultured under static conditions or exposed to laminar shear stress (15 dynes/cm2) for 24 hours. Integrin 5 and ß1 cell-surface levels were measured in resting cells behind the scratched wound and in cells in the migrating front of the wound edge, as illustrated in Figure 2A. In HUVECs cultured under static conditions, integrin 5 and ß1 cell-surface levels were significantly increased in migrating cells compared with resting cells (Figure 2B and 2C). In addition, shear stress upregulated integrin 5 and ß1 cell-surface levels in migrating cells (Figure 2B and 2C). To confirm these results, the cell-surface levels of integrin 5 and ß1 were determined by FACS analysis (Figure 2D and 2E). Integrin 5 and ß1 was increased in migrating cells under static conditions (5, 165±35% of control; ß1, 163±35% of control). Additional exposure to shear stress further increased 5 and ß1 cell-surface levels to 215±47% and 189±31%, respectively (Figure 2D and 2E).
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Phosphorylation of ERK1/2 and FAK Is Enhanced in Migrating Cells
Integrins are known to activate the MAPK ERK1/2 via the FAK and Shc pathways.1,27–29 Because the total cell-surface levels of ß1-integrins and integrin 5ß1 are upregulated in migrating ECs, we analyzed the activation of the MAPK ERK1/2 in these cells. Therefore, the phosphorylation of ERK1/2 was investigated in ECs in the migrating front of the scratched wound (migrating) and behind the wound edge (resting) under static conditions or shear stress exposure, respectively. As shown in Figure 3A and 3B, ERK1/2 phosphorylation was increased in migrating cells in front of the scratched wound. Moreover, shear stress exposure for 24 hours slightly enhanced the phosphorylation of ERK1/2 in migrating cells compared with migrating cells under static conditions (Figure 3A). In addition, we analyzed the phosphorylation of FAK at tyrosine 397. As shown in Figure 3C and 3D, phosphorylation of FAK was stimulated in cells in the migrating front of the scratched wound. In addition, shear stress exposure for 24 hours increased the phosphorylation of FAK in migrating cells compared with migrating cells under static conditions (Figure 3C and 3D).
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Contribution of Shc to Shear Stress–Induced Cell Migration
The adaptor protein Shc associates with integrins and plays a crucial role for EC adhesion.15,27 To assess the contribution of Shc, we overexpressed a dominant negative p52Shc construct (Y317F).27 Dominant negative Shc completely inhibited shear stress–induced cell migration and slightly reduced basal cell migration (Figure 4A), suggesting an important role of Shc for shear stress–induced cell migration.
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Role of ERK1/2 in EC Migration
To test the role of ERK1/2 in EC migration, HUVECs were pretreated with the MEK-1 inhibitor PD98059, which prevents the activation of ERK1/2 through the inhibition of the upstream MAPK kinase MEK-1. In accordance with the activation of ERK1/2 in migrating cells (Figure 3A and 3B), incubation of ECs with PD98059 significantly reduced shear stress–induced cell migration (Figure 4B). Moreover, overexpression of the active ERK2 sevenmaker construct (D319N), which cannot be dephosphorylated and therefore not inactivated,30 was sufficient to induce EC migration (Figure 4C). In the presence of ERK, shear stress had no additional effect (Figure 4C).
Role of PI3K and NO in EC Migration
Because activation of PI3K/Akt and endothelial NO synthase (eNOS) and the release of NO are necessary for VEGF-induced EC migration,25 we investigated the role of Akt and NO in shear stress–induced EC migration. Therefore, HUVECs were pretreated with the pharmacological PI3K inhibitor Ly294002 or the NO synthase inhibitor L-NMMA 30 minutes before shear stress exposure. Ly294002 completely suppressed shear stress–induced EC migration (Figure 4B), demonstrating the involvement of PI3K. Inhibition of eNOS by L-NMMA, however, did not influence the shear stress–induced EC migration (Figure 4D), excluding the involvement of NO.
Taken together, these results indicate that shear stress–induced EC migration depends on the activation of ERK1/2 and PI3K but not on the activation of eNOS.
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Discussion |
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The data of the present study demonstrate that shear stress stimulates migration of HUVECs and microvascular ECs to the same extent as the potent angiogenic growth factor VEGF. In contrast to VEGF, which also stimulates a proliferative response,31 shear stress did not increase proliferation but specifically stimulated migration of the ECs. Shear stress–induced cell migration was shown to depend on the fibronectin receptor
5ß1. Integrins are well established to play a crucial role for cell migration18–20 and vascular and cardiac development8,32,33 and are known to sense mechanical signals.15,34 The total cell-surface levels of ß1-integrins and integrin 5ß1 were specifically increased at the edges of the wound and further augmented within these areas by shear stress exposure. The effect of shear stress on EC migration was dose-dependent, with a maximum at 45 dynes/cm2, and correlated with the expression of the integrin subunits 5 and ß1.17 Genetic studies or antibody data implied that several integrin subunits, including ß1 and the -chains v, 1, 2, and 5, are important for vascular development and/or angiogenesis.4,8–10 Shear stress–induced cell migration, however, seems to depend primarily on 5ß1, because blocking antibodies against vß3 or 2ß1 were not effective. Furthermore, previous studies revealed only a minor, nonsignificant increase in 3 or 6 subunits by flow.17
The mechanism of shear stress–induced integrin 5ß1 expression remains unclear. In previous studies, however, we excluded the contribution of shear stress–induced release of NO and growth factors to shear stress–induced upregulation of integrin expression.17 Because the homeobox gene Hox D3 is known to induce integrin vß3 expression and an angiogenic phenotype in ECs35 and shear stress was shown to upregulate Hox D3 mRNA expression (unpublished data), one may speculate that the homeobox transcription factor Hox D3 may contribute to shear stress–mediated increases in integrin expression.
The integrins 5ß1 and vß3 associate with the adaptor protein Shc, which couples integrin signaling to the MAPK cascade.27 Shc is essential to EC adhesion and cell survival.27 In line with these findings, overexpression of a dominant negative construct, which was shown to block fibronectin signaling,27 prevents shear stress–stimulated EC migration. Interestingly, previous studies revealed that short-term exposure to shear stress stimulates the interaction of vß3 and 5ß1 with Shc.15,36 Consistent with the finding that Shc is required for shear stress–induced cell migration and with the upregulation of 5ß1 cell-surface levels in the migrating cells, the downstream MAPK cascades and FAK are activated. Several studies suggest that FAK is essential for integrin-stimulated cell migration. For example, expression of the phosphatase PTEN leads to an inactivation of FAK and inhibition of cell motility.23,37 These results are in line with the established role of FAK in cell migration using FAK-knockout cells, FAK overexpression, and dominant negative FAK.38–40 FAK is thought to sustain the ERK activation compared with the initial high activation of ERK1/2 mediated by Shc1 and can be activated on short-term exposure of ECs to shear.41 In the present study, FAK phosphorylation was augmented in the migrating cells at the edge of the scratched wounds, indicating that the increased cell-surface level of the fibronectin receptor 5ß1 allows for enhanced activation of FAK, which in turn may enhance to activation of ERK1/2.
Pharmacological inhibitor studies demonstrated that the MAPK ERK1/2 and, in addition, PI3K are causally essential downstream molecules to mediate shear stress–induced EC migration and that overexpression of ERK is sufficient to induce cell migration. The activation of ERK1/2 and the PI3K pathway is well established to depend on an intact integrin signaling.12,13 The PI3K pathway has previously been shown to be involved in regulation of cell migration and angiogenesis in response to different stimuli, including VEGF.25,42–45 The downstream signaling pathways, however, are less clear. PI3K is necessary for integrin-stimulated activation of the MAPK cascade and the serine/threonine kinase Akt.46 In addition, PI3K phosphorylates and activates Akt.47–49 Akt has previously been shown to mediate VEGF-induced EC migration.25,42 The question remains of what the downstream effectors of Akt might be. Akt has been shown to stimulate eNOS activity and therefore contribute to VEGF-induced EC migration.25 Shear stress–induced increase in EC migration was independent of NO, however, thus indicating that other signaling pathways might be responsible for mediating the effect of shear stress. The stress fiber formation and reorganization of F-actin, which is induced by constitutively active Akt independent of NO,42 may explain the PI3K/Akt-dependent stimulation of EC migration by shear stress. Furthermore, via phosphorylation and thus inactivation of glycogen synthase kinase-3, Akt may also affect ß-catenin translocation to the nucleus and thereby may contribute to cell migration.50–52 Taken together, our results indicate that shear stress activates several kinase pathways that are involved in regulation of EC migration. To better understand the mechanism of EC migration, it will be necessary to identify downstream mediators of the kinases. The activation of transcription factors by Akt or ERK1/2 might contribute to alterations in gene expression, which in turn influence cell migration.
Angiogenic growth factors are known to induce EC migration as a key step in the formation of new blood vessels. In addition to various stimuli, such as growth factors, the endothelial monolayer is continuously influenced by the laminar blood flow, which exerts many beneficial effects. The upregulation of integrin cell-surface levels by shear stress not only supports cell adhesion and survival but also facilitates cell migration. As a physiological consequence, shear stress might stimulate reendothelialization of denuded arteries, as evidenced by the in vitro scratched-wound assay, and accelerate the formation of new blood vessels or modulate vessel remodeling.
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft (Di600/2-3). We would like to thank Christiane Mildner-Rihm and Melanie Näher for expert technical assistance.
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*These authors contributed equally to the present study.
Received September 5, 2001; accepted October 16, 2001.
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References |
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1. Giancotti FG, Ruoslahti E. Integrin signaling. Science. 1999; 285: 1028–1032.
2. Boudreau NJ, Jones PL. Extracellular matrix and integrin signalling: the shape of things to come. Biochem J. 1999; 339: 481–488.[CrossRef][Medline] [Order article via Infotrieve]
3.
Plow EF, Haas TA, Zhang L, Loftus J, Smith JW. Ligand binding to integrins. J Biol Chem. 2000; 275: 21785–21788.
4.
Hynes RO, Zhao Q. The evolution of cell adhesion. J Cell Biol. 2000; 150: F89–F96.
5. Calderwood DA, Shattil SJ, Ginsberg MH. Integrins and actin filaments: reciprocal regulation of cell adhesion and signaling. J Biol Chem. 2000.
6.
Friedlander M, Brooks PC, Shaffer RW, Kincaid CM, Varner JA, Cheresh DA. Definition of two angiogenic pathways by distinct alpha v integrins. Science. 1995; 270: 1500–1502.
7. Bader BL, Rayburn H, Crowley D, Hynes RO. Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins. Cell. 1998; 95: 507–519.[CrossRef][Medline] [Order article via Infotrieve]
8. Yang JT, Rayburn H, Hynes RO. Embryonic mesodermal defects in alpha 5 integrin-deficient mice. Development. 1993; 119: 1093–1105.[Abstract]
9.
Pozzi A, Moberg PE, Miles LA, Wagner S, Soloway P, Gardner HA. Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization. Proc Natl Acad Sci U S A. 2000; 97: 2202–2207.
10.
Senger DR, Claffey KP, Benes JE, Perruzzi CA, Sergiou AP, Detmar M. Angiogenesis promoted by vascular endothelial growth factor: regulation through 1ß1 and 2ß1 integrins. Proc Natl Acad Sci U S A. 1997; 94: 13612–13617.
11.
Muller JM, Chilian WM, Davies MJ. Integrin signaling transduces shear stress–dependent vasodilation of coronary arterioles. Circ Res. 1997; 80: 320–326.
12.
Dimmeler S, Assmus B, Hermann C, Haendeler J, Zeiher AM. Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res. 1998; 83: 334–342.
13. Takahashi M, Berk BC. Mitogen-activated protein kinase (ERK1/2) activation by shear stress and adhesion in endothelial cells: essential role for a herbimycin- sensitive kinase. J Clin Invest. 1996; 98: 2623–2631.[Medline] [Order article via Infotrieve]
14.
Traub O, Berk BC. Laminar shear stress: mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol. 1998; 18: 677–685.
15.
Jalali S, del Pozo MA, Chen K, Miao H, Li Y, Schwartz MA, Shyy JY, Chien S. Integrin-mediated mechanotransduction requires its dynamic interaction with specific extracellular matrix (ECM) ligands. Proc Natl Acad Sci U S A. 2001; 98: 1042–1046.
16.
Fujio Y, Walsh K. Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage-dependent manner. J Biol Chem. 1999; 274: 16349–16354.
17.
Urbich C, Walter DH, Zeiher AM, Dimmeler S. Laminar shear stress upregulates integrin expression: role in endothelial cell adhesion and apoptosis. Circ Res. 2000; 87: 683–689.
18. Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell. 1996; 84: 359–369.[CrossRef][Medline] [Order article via Infotrieve]
19.
Pepper MS. Manipulating angiogenesis: from basic science to the bedside. Arterioscler Thromb Vasc Biol. 1997; 17: 605–619.
20. Hynes RO, Bader BL, Hodivala-Dilke K. Integrins in vascular development. Braz J Med Biol Res. 1999; 32: 501–510.[Medline] [Order article via Infotrieve]
21. Albuquerque ML, Waters CM, Savla U, Schnaper HW, Flozak AS. Shear stress enhances human endothelial cell wound closure in vitro. Am J Physiol. 2000; 279: H293–H302.
22.
Ayajiki K, Kindermann M, Hecker M, Fleming I, Busse R. Intracellular pH and tyrosine phosphorylation but not calcium determine shear stressd–induced nitric oxide production in native endothelial cells [see comments]. Circ Res. 1996; 78: 750–758.
23.
Tamura M, Gu J, Matsumoto K, Aota S, Parson R, Yamada KM. Inhibition of cell migration, spreading and focal adhesions by tumor suppressor PTEN. Science. 1998; 280: 1614–1617.
24.
Chavakis E, Dernbach E, Hermann C, Mondorf UF, Zeiher AM, Dimmeler S. Oxidized LDL inhibits vascular endothelial growth factor-induced endothelial cell migration by an inhibitory effect on the akt/endothelial nitric oxide synthase pathway. Circulation. 2001; 103: 2102–2107.
25. Dimmeler S, Dernbach E, Zeiher AM. Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration. FEBS Lett. 2000; 477: 258–262.[CrossRef][Medline] [Order article via Infotrieve]
26.
Eliceiri BP, Klemke R, Stromblad S, Cheresh DA. Integrin vß3 requirement for sustained mitogen-activated protein kinase activity during angiogenesis. J Cell Biol. 1998; 140: 1255–1263.
27. Wary KK, Mainiero F, Isakoff SJ, Marcantonio EE, Giancotti FG. The adapter protein Shc couples a class of integrins to the control of cell cycle progression. Cell. 1996; 87: 733–743.[CrossRef][Medline] [Order article via Infotrieve]
28. Wary KK, Mariotti A, Zurzolo C, Giancotti FG. A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell. 1998; 94: 625–634.[CrossRef][Medline] [Order article via Infotrieve]
29.
Barberis L, Wary KK, Fiucci G, Liu F, Hirsch E, Brancaccio M, Altruda F, Tarone G, Giancotti FG. Distinct roles of the adaptor protein shc and focal adhesion kinase in integrin signaling to ERK. J Biol Chem. 2000; 275: 36532–36540.
30.
Camps M, Nichols A, Gillieron C, Antonsson B, Muda M, Chabert C, Boschert U, Arkinstall S. Catalytic activation of the phosphatase MKP-3 by ERK-2 mitogen-activated protein kinase. Science. 1998; 280: 1262–1265.
31.
Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. FASEB J. 1999; 13: 9–22.
32. George EL, Georges-Labouesse EN, Patel-King RS, Rayburn H, Hynes RO. Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin. Development. 1993; 119: 1079–1091.[Abstract]
33.
Goh KL, Yang JT, Hynes RO. Mesodermal defects and cranial neural crest apoptosis in 5 integrin-null embryos. Development. 1997; 124: 4309–4319.[Abstract]
34. Meyer CJ, Alenghat FJ, Rim P, Fong JH, Fabry B, Ingber DE. Mechanical control of cyclic AMP signalling and gene transcription through integrins. Nat Cell Biol. 2000; 2: 666–668.[CrossRef][Medline] [Order article via Infotrieve]
35.
Boudreau N, Andrews C, Srebrow A, Ravanpay A, Cheresh DA. Induction of the angiogenic phenotype by Hox D3. J Cell Biol. 1997; 139: 257–264.
36.
Chen KD, Li YS, Kim M, Li S, Yuan S, Chien S, Shyy JY. Mechanotransduction in response to shear stress: roles of receptor tyrosine kinases, integrins, and Shc. J Biol Chem. 1999; 274: 18393–18400.
37.
Gu J, Tamura M, Pankov R, Danen EH, Takino T, Matsumoto K, Yamada KM. Shc and FAK differentially regulate cell motility and directionality modulated by PTEN. J Cell Biol. 1999; 146: 389–403.
38. Sieg DJ, Hauck CR, Schlaepfer DD. Required role of focal adhesion kinase (FAK) for integrin-stimulated cell migration. J Cell Sci. 1999; 112: 2677–2691.[Abstract]
39.
Owen JD, Ruest PJ, Fry DW, Hanks SK. Induced focal adhesion kinase (FAK) expression in FAK-null cells enhances cell spreading and migration requiring both auto- and activation loop phosphorylation sites and inhibits adhesion-dependent tyrosine phosphorylation of Pyk2. Mol Cell Biol. 1999; 19: 4806–4818.
40. Gilmore AP, Romer LH. Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation. Mol Biol Cell. 1996; 7: 1209–1224.[Abstract]
41.
Li S, Kim M, Hu Y-L, Jalali S, Schlaepfer DD, Hunter T, Chien S, Shyy JY-J. Fluid shear stress activation of focal adhesion kinase. J Biol Chem. 1997; 272: 30455–30462.
42.
Morales-Ruiz M, Fulton D, Sowa G, Languino LR, Fujio Y, Walsh K, Sessa WC. Vascular endothelial growth factor-stimulated actin reorganization and migration of endothelial cells is regulated via the serine/threonine kinase Akt. Circ Res. 2000; 86: 892–896.
43. Souttou B, Raulais D, Vigny M. Pleiotrophin induces angiogenesis: involvement of the phosphoinositide-3 kinase but not the nitric oxide synthase pathways. J Cell Physiol. 2001; 187: 59–64.[CrossRef][Medline] [Order article via Infotrieve]
44. Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y, Sakaue H, Ando A, Chavanieu A, Calas B, Grigorescu F, et al. Involvement of phosphoinositide 3-kinase in insulin- or IGF-1-induced membrane ruffling. EMBO J. 1994; 13: 2313–2321.[Medline] [Order article via Infotrieve]
45. Wennstrom S, Siegbahn A, Yokote K, Arvidsson AK, Heldin CH, Mori S, Claesson-Welsh L. Membrane ruffling and chemotaxis transduced by the PDGF ß-receptor require the binding site for phosphatidylinositol 3' kinase. Oncogene. 1994; 9: 651–660.[Medline] [Order article via Infotrieve]
46. King WG, Mattaliano MD, Chan TO, Tsichlis PN, Brugge JS. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation. Mol Cell Biol. 1997; 17: 4406–4418.[Abstract]
47. Franke TF, Yang S-I, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell. 1995; 81: 727–736.[CrossRef][Medline] [Order article via Infotrieve]
48.
Franke TF, Kaplan DR, Cantley LC, Toker A. Direct regulation of the Akt proto-oncogene product by phosphatidylinositol-3,4-bisphosphate. Science. 1997; 275: 665–668.
49. Burgering BMT, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995; 376: 599–602.[CrossRef][Medline] [Order article via Infotrieve]
50.
Novak A, Hsu SC, Leung-Hagesteijn C, Radeva G, Papkoff J, Montesano R, Roskelley C, Grosschedl R, Dedhar S. Cell adhesion and the integrin-linked kinase regulate the LEF-1 and beta-catenin signaling pathways. Proc Natl Acad Sci U S A. 1998; 95: 4374–4379.
51.
Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A. 1998; 95: 11211–11216.
52. Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995; 378: 785–789.[CrossRef][Medline] [Order article via Infotrieve]
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