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(Circulation Research. 2000;87:683.)
© 2000 American Heart Association, Inc.

Cellular Biology

Laminar Shear Stress Upregulates Integrin Expression

Role in Endothelial Cell Adhesion and Apoptosis

Carmen Urbich, Dirk H. Walter, Andreas M. Zeiher, Stefanie Dimmeler

From Molecular Cardiology, Department of Internal Medicine IV, University of 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

	Abstract

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Abstract—Laminar shear stress exerts important effects on endothelial cell (EC) function and inhibits apoptosis of ECs induced by various stimuli. The mechanism by which hemodynamic forces, such as shear stress, are transduced into cellular signaling is still not known. Located at the cell surface, integrins, which are required for cell adhesion and cell survival, are potential mechanotransducers. Therefore, we investigated the effect of shear stress on integrin expression in ECs. Shear stress time-dependently increased the mRNA expression of the fibronectin receptor subunits ₅ and ß₁ with a maximum at 6 hours (283±41% and 215±27% of control, respectively). In addition, the protein levels of the fibronectin receptor subunits ₅ and ß₁ were enhanced with a maximum at 12 hours of shear stress exposure (343±53% and 212±38% of control, respectively). The shear stress–induced upregulation of integrins is independent of nitric oxide. Furthermore, we confirmed the enhanced functional activity of ₅ß₁ integrin expression by FACS analysis. As a functional consequence, human umbilical vein ECs, which were preexposed to shear stress, revealed a significantly increased attachment (178±10% of static controls) and a more pronounced extracellular signal–regulated kinase 1 and 2 activation in response to cell attachment. Finally, we demonstrated that shear stress requires RGD-sensitive integrins to mediate its antiapoptotic effect. Taken together, these results define a novel mechanism by which shear stress may exert its atheroprotective effects via upregulation of integrins to support EC adhesion and survival.

Key Words: integrins • shear stress • endothelial cells • gene expression

	Introduction

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The interaction of cells with the extracellular matrix is mediated by integrins. Integrins, a large family of heterodimeric transmembrane receptors, are composed of and ß subunits. Currently, at least 16 and 8 ß subunits have been identified.¹ Each ß combination has specific binding and signaling properties. For example, the ₅ß₁ integrin binds to fibronectin, and laminin is the ligand for ₆ß₁, whereas other integrins are capable of binding multiple ligands.² Thus, the _vß₃ integrin is known to interact with vitronectin, fibronectin, and von Willebrand factor. Adherent cells anchor via integrins to the matrix, which is essential to maintain the survival of the cells.^{3 4} Thus, various integrins, including _vß₃ or ₅ß₁, prevent apoptosis of endothelial cells (ECs).^{5 6 7} Although the exact mechanism of the antiapoptotic effect is not clear, it has been suggested that antiapoptotic integrin signaling involves the activation of the prosurvival kinase Akt or the mitogen-activated protein (MAP) kinase extracellular signal–regulated kinase (ERK) 1/2.^{8 9 10} Furthermore, activation of the integrin ₅ß₁ was shown to transcriptionally upregulate antiapoptotic genes, such as Bcl-2.⁵ Besides their role in cell survival, integrins are necessary for cell migration, a process essential for angiogenesis or reendothelialization.^{7 8 11}

The laminar blood flow (shear stress) is a hemodynamic force, which acts on the luminal site of the vessel wall. Because of their unique location, ECs are continuously exposed to shear stress, which modulates gene expression and cellular structure and function.¹² Briefly, shear stress regulates gene expression of various proteins, including vasoactive substances (eg, nitric oxide [NO] synthase and endothelin-1), growth factors (eg, transforming growth factor-ß₁ and platelet-derived growth factor), adhesion and chemoattractant molecules (eg, intercellular adhesion molecule-1, vascular cellular adhesion molecule-1, and monocyte chemoattractant protein-1), coagulation factors (eg, tissue factor), proto-oncogenes (eg, c-fos, c-jun), and antioxidant enzymes (eg, superoxide dismutase).¹³ Furthermore, laminar shear stress completely inhibits apoptosis of ECs in response to various stimuli,¹⁴ demonstrating the potent atheroprotective effects of shear stress to preserve the integrity of the endothelium. The mechanisms by which hemodynamic forces such as shear stress are transduced into cellular signaling are still not known. Located at the cell surface, integrins are possible candidates for the transduction of hemodynamic forces into biochemical signals.^{15 16 17} For example, Muller et al¹⁸ showed that shear stress–induced vasodilation in coronary arteries could be blocked with RGD peptides that inhibit the binding of integrins to their ligands.² Moreover, shear stress–mediated ERK1/2 activation and Akt phosphorylation, which are important survival signals in ECs, depend on integrin binding to the extracellular matrix.^{10 17} Although compelling evidence suggests that integrins play an important role as mechanotransducers, there are no data concerning the regulation of integrin subunits by laminar shear stress in ECs.

Therefore, we examined whether shear stress regulates integrin expression. For the first time, we demonstrate that shear stress modulates the expression of integrin subunits on mRNA and protein levels. Furthermore, the enhanced functional activity of integrins is confirmed by FACS analysis. Specifically, shear stress induces an upregulation of the fibronectin receptor ₅ and ß₁ subunits. As a functional consequence, human umbilical vein ECs (HUVECs), which were preexposed to shear stress, revealed a significantly increased attachment and a more pronounced ERK1/2 activation in response to cell attachment. Finally, we demonstrate that integrins are required to mediate the apoptosis-inhibitory effect of shear stress. Taken together, these results define a novel mechanism by which shear stress mediates and intensifies its atheroprotective effects via upregulation of integrins as potential mechanotransducers.

	Materials and Methods

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Cell Culture
Pooled HUVECs were purchased from Cell Systems/Clonetics and were cultured and exposed to laminar fluid flow in a cone-and-plate apparatus as previously described.¹⁴ A constant shear stress of 15 dyne/cm² was used in all experiments to simulate physiological levels of shear stress. N^G-Monomethyl-L-arginine (LNMA; Alexis) and the synthetic peptide GRGDNP (GIBCO-BRL) or the neutralizing integrin ₅ and ß₁ antibodies (Dianova and GIBCO-BRL) were preincubated for 30 minutes before shear stress exposure.

Atlas cDNA Expression Array
Total RNA was isolated as described¹⁹ and radioactively labeled with a mixture of gene-specific primers. The differential gene expression was analyzed by hybridization with 588 cDNAs immobilized on a membrane according to the instructions of the manufacturer (Clontech). After a high-stringency wash, the expression profile was quantified by phosphor imaging.

Western Blot Analysis
For the detection of integrin expression, HUVECs (4.0x10⁵ cells) were lysed in buffer as previously described.¹⁹ For determination of the phosphorylated form of ERK1/2, the lysis buffer was supplemented with phosphatase inhibitors as described.¹⁰ The protein content of the samples was determined according to the Bradford method. Western blots were performed using antibodies directed against integrins ₅ and ß₁ (1:2500; Transduction Laboratories), phospho-ERK1/2, or ERK1/2 (1:2000; Biolabs) or actin (1:1000; Boehringer Mannheim).

Flow Cytometry
HUVECs were washed with PBS and detached with 1 mmol/L EDTA (pH 7.4) for 20 minutes at 37°C. After centrifugation the cell pellet was suspended in 100 µL PBS/10% FCS and incubated for 30 minutes at 4°C with 10 µL FITC-conjugated ₅ or ß₁ integrin antibodies (Dianova). Subsequently, cells were fixed with 4% paraformaldehyde in PBS and analyzed by flow cytometry using FACS Calibur (Perkin Elmer; CellQuest software). All experiments were performed with standardized instrumental settings (FL1-H [fluorescence], voltage 498, and amp gain 1.16).

Cell Adhesion Assay
After exposure to shear stress for 24 hours, HUVECs were washed with PBS and detached with 1 mmol/L EDTA (pH 7.4) in PBS for 20 minutes at 37°C. After centrifugation the cell pellet was suspended in endothelial cell basal medium complete medium in the presence or absence of PD98059 (10 µmol/L; Biomol), RGD peptides (GRGDNP; 0.5 mmol/L), or neutralizing antibodies. Identical numbers of cells were placed onto uncoated or fibronectin-coated culture dishes (10 µg/mL, 1 hour at 37°C) and incubated for 20 minutes at 37°C. Adherent cells were counted by 2 independent blinded investigators, or cells were lysed for Western blot analysis.

Detection of Apoptosis
Cell culture dishes were centrifuged (10 minutes at 700g), fixed in 4% formaldehyde, and stained with DAPI. Five hundred cells were counted by 2 independent blinded investigators, and the percentage of apoptotic cells per total number of cells was determined.

Statistical Analysis
Data are expressed as mean±SEM from at least 3 independent experiments. Statistical analysis was performed by t test. For serial analyses (time-dependency), ANOVA was performed.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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Shear Stress Modulates mRNA and Protein Expression of Integrins
To investigate the effect of shear stress on mRNA expression of integrins, HUVECs were exposed to laminar shear stress (15 dyne/cm²) for 6 and 12 hours. Total RNA was isolated and used in an Atlas cDNA Expression Array, which allowed the analysis of differential gene expression of various integrins. Shear stress time-dependently increased the expression of the integrin subunits ₅ and ß₁, which are the preferential receptor for fibronectin in HUVECs,^{20 21} with a maximum at 6 hours of shear stress exposure (Figures 1A and 1B).

View larger version (24K): [in this window] [in a new window]   Figure 1. Shear stress increases the mRNA expression of integrins. Total RNA was isolated from HUVECs exposed to laminar shear stress (15 dyne/cm2) for 6 and 12 hours. The differential gene expression of various integrin subunits was analyzed using an Atlas cDNA Expression Array. Shown are effects of shear stress on mRNA expression of fibronectin receptor 5 (A), fibronectin receptor ß1 (B), integrin 3 (C), and integrin 6 (D). Data were quantified by phosphor imaging and are expressed as integrin mRNA/GAPDH mRNA compared with static control (mean±SEM; n=3).

In addition, the integrin subunit ₃ (the receptor for fibronectin, collagen, and laminin² ) and the laminin receptor subunit ₆ are slightly upregulated in response to shear stress (Figures 1C and 1D). In contrast, mRNA expression of integrin _v, the vitronectin receptor, was not affected by shear stress (12 hours; 95±27% of static control).

To confirm the data obtained with the cDNA expression array, protein expression of fibronectin receptor ₅ (integrin ₅) and fibronectin receptor ß₁ (integrin ß₁) was analyzed by Western blotting. As shown in Figure 2A, integrin ₅ was time-dependently upregulated with a maximum of protein expression within 12 to 18 hours of shear stress exposure. Similar to the effects of shear stress on mRNA expression, prolonged application to shear stress led to an enhanced integrin ß₁ protein expression with a maximum at 12 to 18 hours (Figure 2B). The expression of the integrin subunits ₅ and ß₁ remained elevated 2-fold up to 48 hours of shear stress exposure (data not shown). Moreover, the effects of shear stress were dose-dependent, as illustrated in Figure 2C.

View larger version (37K): [in this window] [in a new window]   Figure 2. Shear stress enhances protein expression of integrins 5 and ß1. A and B, Time-dependency. HUVECs were exposed to laminar shear stress (15 dyne/cm2) for 6, 12, and 18 hours, and protein levels of integrin 5 (A) and integrin ß1 (B) were detected by Western blot analysis. C, Dose-dependent effect of shear stress on integrin expression (18 hours of incubation). Actin reprobes served as loading control. Integrin expression was densitometrically analyzed and normalized to actin. Representative blots of 4 independent experiments are shown.

Shear Stress Induces the Cell Surface Expression of Integrins
Because the presence of integrin mRNA and protein does not necessarily establish its biological function, we further determined the regulation of integrin ₅ and ß₁ by shear stress in HUVECs using FACS analysis (Figure 3). In accordance with the findings of the Atlas cDNA Expression Array and Western blot analysis, exposure of ECs to laminar shear stress for 24 hours enhanced cell surface expression of integrin ₅ (Figure 3A) and integrin ß₁ (Figure 3B). Thus, the shear stress–induced upregulation of integrin mRNA and protein results in an increased cell surface expression of integrins, which can mediate cell-matrix interactions.

View larger version (29K): [in this window] [in a new window]   Figure 3. Shear stress induces the cell surface expression of integrins 5 and ß1. After exposure to laminar shear stress (15 dyne/cm2) for 24 hours in endothelial cell basal medium+1% BSA, cell surface expression of integrin 5 (A) and integrin ß1 (B) was assessed by FACS analysis. Representative histograms of 5 independent experiments are shown (left panels). Arrows indicate unlabeled cells, control cells, and cells exposed to shear stress. Right panels show the fluorescence intensity (FL1-H) of static controls compared with cells exposed to shear stress for 24 hours. Data are mean±SEM (n=5); *P<0.01 vs static control.

Mechanisms of Shear Stress–Induced Upregulation of Integrin ₅ and Integrin ß₁: Role of NO and Paracrine Growth Factors
Murohara et al²² demonstrated that inhibition of endothelial NO synthase (eNOS) by the L-arginine analog N-nitro-L-arginine methyl ester in HUVECs inhibited surface expression of integrin _vß₃, which plays an important role in EC survival and angiogenesis. Previous studies have demonstrated that physiological levels of shear stress induced upregulation of eNOS followed by an increase of NO release.^{23 24} Therefore, we examined the role of NO in shear stress–induced upregulation of integrins ₅ and ß₁ by Western blot analysis. HUVECs were incubated for 12 hours with sodium nitroprusside (SNP) or 1-propanamine,3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) (50 µmol/L), each of which is an NO donor. Incubation with SNP or PAPA NONOate for 12 hours led to a significantly enhanced expression of integrins ₅ and ß₁, similar to the effects obtained with 12 hours of shear stress exposure (Figure 4; data not shown). However, inhibition of eNOS with LNMA (1 mmol/L) did not reduce the shear stress–mediated upregulation of integrins ₅ and ß₁ (Figures 4A and 4B). These data indicate that NO is sufficient to induce integrin expression. However, shear stress in addition activates a second NO-independent pathway leading to upregulation of integrin expression.

View larger version (24K): [in this window] [in a new window]   Figure 4. Shear stress–induced upregulation of integrins 5 and ß1 is independent of NO. HUVECs were incubated for 12 hours with the NO donor SNP (50 µmol/L) or exposed to laminar shear stress (15 dyne/cm2) for 12 hours. LNMA (1 mmol/L) was preincubated for 30 minutes before shear stress exposure. A, Integrin 5 and integrin ß1 protein expression was determined by Western blot analysis. The blot was then reprobed with actin to confirm equal loading. A representative blot of 4 independent experiments is shown. B, Integrin 5 and integrin ß1 protein expression was quantified by densitometric analysis of the Western blots from 4 independent experiments (data are normalized against actin and expressed as mean±SEM; n=4).

Because shear stress is known to stimulate the release of growth factors,¹³ one may speculate that it can induce integrin expression in a paracrine manner. However, conditioned medium from shear stress–exposed HUVECs (24 hours) did not increase integrin expression (Figure 5).

View larger version (26K): [in this window] [in a new window]   Figure 5. Effect of conditioned medium. HUVECs were exposed to laminar shear stress or kept under static conditions for 24 hours. Then, conditioned medium was removed, added to static cultures, and again incubated for 24 hours. Integrin 5 (A) and ß1 (B) expression was detected by Western blot analysis. A representative blot of 3 independent experiments is shown.

Shear Stress Induces HUVEC Adhesion via Upregulation of Integrins
Having demonstrated that shear stress stimulates the expression of integrins ₅ and ß₁, we examined the effect of integrin upregulation on EC adhesion. Therefore, HUVECs were exposed to laminar shear stress for 24 hours, detached, and replated on cell culture dishes. As shown in Figure 6A, ECs preexposed to shear stress exhibited a significant increase in the number of adherent cells after replating (178±10% of static controls). Similar results were obtained when ECs were replated on plastic dishes coated with fibronectin (data not shown). In contrast, incubation of preexposed ECs with the MAP/ERK kinase (MEK) inhibitor PD98059 (10 µmol/L) abrogated the shear stress–stimulated increase in EC adhesion (Figure 6A). Moreover, RGD peptides (GRGDNP; 0.5 mmol/L) or neutralizing antibodies against integrins ₅ and ß₁ prevented shear stress–induced increase in cell adhesion after replating (Figure 6B), whereas control peptides (GRGESP; 0.5 mmol/L) had no effect (data not shown), demonstrating a specific role of integrins in shear stress–stimulated cell adhesion.

View larger version (11K): [in this window] [in a new window]   Figure 6. Shear stress induces HUVEC adhesion via upregulation of integrins. A, HUVECs were cultured in static conditions (control) or preexposed to laminar shear stress (15 dyne/cm2) for 24 hours and replated on culture dishes for 20 minutes in the presence or absence of PD98059 (10 µmol/L) (shear stress preexposure±PD98059). Replated, adherent cells were counted by 2 independent blinded investigators. Data are mean percentage of control±SEM (n=4). Replated cells were lysed in buffer for determination of ERK1/2 phosphorylation (ERK1/2-P) using a phosphospecific ERK1/2 antibody in a Western blot analysis (right panel). The blot was reprobed with ERK1/2 to confirm equal loading. Shown is a representative blot of 3 independent experiments. B, HUVECs were preexposed to shear stress for 24 hours, detached from the culture dishes, and replated in the presence or absence of RGD peptides (0.5 mmol/L) or neutralizing antibodies directed against integrin subunits 5 and ß1 (dilution 1:100). Data are mean±SEM, n=3. *P<0.05 vs shear stress preexposure.

We further determined the effect of shear stress preexposure on ERK1/2 activation in replated ECs. As assessed by Western blot analysis using a phosphospecific ERK1/2 antibody, ERK1/2 phosphorylation was significantly enhanced in replated ECs, which were preexposed to shear stress for 24 hours, compared with static controls (206±77%; Figure 6A). In contrast, preexposure of ECs to shear stress for 20 minutes did not increase cell adhesion after replating (data not shown). These data indicate that long-term exposure to shear stress stimulates the ability of ECs to adhere to the matrix. Furthermore, HUVECs, which were preexposed to shear stress, revealed a more pronounced ERK1/2 activation in response to cell attachment.

Shear Stress Requires RGD-Sensitive Integrins to Mediate Its Antiapoptotic Effect
Physiological levels of laminar shear stress completely prevent apoptosis of human ECs in response to a variety of stimuli, including tumor necrosis factor , oxidized LDL, and angiotensin II.^{14 19} Several studies suggest that integrins are important for cell survival.^{3 5 6 7 25} Therefore, we examined the role of integrins in the antiapoptotic effect of shear stress using RGD peptides, which compete with the matrix, namely fibronectin and vitronectin, for integrin interactions.^{2 18} As shown in Figure 7, incubation of ECs with RGD peptides (GRGDNP; 0.5 mmol/L) for 18 hours potently induced apoptotic cell death (316±87% of control) as assessed by morphological analysis of fluorescence-stained nuclei, whereas control peptides (GRGESP; 0.5 mmol/L) had no effect (data not shown). Exposure of ECs to shear stress could not inhibit RGD peptide-induced apoptosis (326±61% of control), indicating that shear stress requires RGD-sensitive integrins to mediate its antiapoptotic effect. Coincubation of ECs with RGD peptides and a specific caspase-3 inhibitor (Ac-Asp-Glu-Val-Asp-aldehyde [AcDEVD-CHO]; 100 µmol/L) led to a decrease of apoptosis (183±55%), demonstrating the involvement of the caspase cascade in apoptosis induction by RGD peptides.

View larger version (13K): [in this window] [in a new window]   Figure 7. Shear stress requires RGD-sensitive integrins to mediate its antiapoptotic effect. HUVECs were incubated with RGD peptides (GRGDNP; 0.5 mmol/L) or coincubated with AcDEVD-CHO (100 µmol/L) or laminar shear stress (15 dyne/cm2) for 18 hours. Apoptotic cell death was assessed by morphological analysis of fluorescence-stained nuclei. Five hundred cells were counted by 2 independent blinded investigators, and apoptotic cells per total number of cells was determined. Data are mean percentage of control±SEM (n=3), *P<0.05 vs RGD.

	Discussion

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The data of the present study demonstrate that shear stress upregulates the expression of integrins in ECs. Moreover, preexposure of ECs to laminar shear stress profoundly enhances the attachment of the cells to the matrix. The integrins ₅ß₁ and _vß₃ play a central role in EC migration and angiogenesis and support EC survival.^{21 25 26} The biological effects induced by shear stress require an intact integrin signaling. Thus, shear stress–induced stimulation of the protein kinase Akt, which mediates the activation of the eNOS, depends on the integrin pathway.^{10 27} Consistent with this finding, shear stress–induced stimulation of blood vessel relaxation is prevented by RGD peptides, which block ₅ß₁ and _vß₃ integrins.^{2 18} Furthermore, an emerging body of evidence suggests that integrins are involved in shear stress–induced activation of focal adhesion kinase and MAP kinases.^{10 28 29 30} The present study extends these observations by demonstrating that shear stress additionally upregulates integrin expression, which may importantly contribute to the long-term effects of shear stress on EC biology.

Shear stress mainly affects the expression of the fibronectin receptor subunits ₅ and ß₁. Adhesion of HUVECs to fibronectin was shown to promote EC survival via activation of the p52Shc adapter protein, which in turn recruits the Grb2-mSOS complex to the membrane and thereby activates the MAP kinase pathway.²¹ In line with these findings, the inhibition of the integrin pathway by blockade with RGD peptides prevented the apoptosis-suppressive effect of shear stress. Preexposure of ECs to shear stress results in an enhanced adhesion of ECs after replating. Moreover, activation of the ERK1/2 MAP kinase was more pronounced in replated cells, which were preexposed to shear stress. As it has also been demonstrated that short-term exposure of ECs to shear stress for 20 minutes enhances adhesion-induced ERK1/2 phosphorylation probably by activation of additional intracellular signaling pathways,¹⁷ we also investigated the effects of short-term shear stress exposure on EC adhesion. However, after 20 minutes of preexposure, no increase in EC adhesion was detectable after replating, which indicated that the long-term shear stress–induced integrin expression may be required for the enhanced cell adhesion. Palecek et al³¹ demonstrated that an increase in integrin ₅ expression results in a dose-dependent enhancement of cell adhesion and migration. Thus, the integrin upregulation induced by shear stress might well explain the augmentation of cell attachment.

Taken together, given that EC adhesion was mediated by a RGD-dependent pathway and the activation of ERK1/2 after adhesion was suggested to be mediated by ß₁ integrin,^{17 28} the enhanced integrin expression induced by shear stress most likely facilitates binding of the ECs to the matrix, which promotes ERK1/2 activation. This conclusion is also supported by our finding that inhibition of ERK1/2 by PD98059 eliminated the enhanced attachment capacity of replated ECs.

The mechanism by which the integrin subunits are upregulated remains unclear. The data of the present study, demonstrating that exogenous NO can upregulate integrins, are in line with the findings by Murohara et al,²² who demonstrated that inhibition of NO synthase decreases the expression of the integrins _vß₃. The study by Murohara et al,²² however, only revealed an effect of NO on the cell surface expression of the vitronectin receptor, whereas the absolute protein levels were unchanged. These findings are in accordance with our results showing that the _v subunit is not regulated by shear stress on the mRNA level. However, although exogenous NO appears to be sufficient to upregulate the integrin subunits ₅ and ß₁, shear stress–induced endogenous NO synthesis does not seem to be required for the increase of integrin expression. Thus, blockade of NO synthesis did not prevent shear stress–stimulated integrin expression, suggesting that shear stress can use an additional pathway beyond NO to enhance integrin expression. Moreover, conditioned medium of shear stress–exposed HUVECs did not influence the expression of integrins ₅ and ß₁, thus excluding the possibility that shear stress induces integrin expression in a paracrine manner via the release of growth factors, which are known to modulate integrin expression.³² However, shear stress can also directly stimulate tyrosine phosphorylation of growth factor receptors and, for example, activate the VEGF receptor Flk-1 even in the presence of neutralizing VEGF antibodies.³³ Therefore, one may speculate that other growth factor–induced signaling events are triggered by shear stress in a similar manner. Thus, shear stress may directly activate downstream signaling pathways and thereby mimic growth factor stimulation.

The survival of ECs is critical for the maintenance of blood vessel integrity and angiogenesis.³⁴ Moreover, EC apoptosis may contribute to the initiation of atherogenesis.³⁵ The attachment of ECs to the matrix, which is mediated by integrins, thereby plays an essential role in maintaining EC survival.^{5 9} Thus, shear stress–induced upregulation of integrins may enhance the stimulation of integrin signaling and thereby promote EC survival. Moreover, given that integrins are also required for EC migration, one may speculate that shear stress may accelerate reendothelialization, eg, after balloon denudation, by enhancing cell migration via integrin upregulation.

	Acknowledgments

 
This work was supported by Deutsche Forschungsgemeinschaft (Di600/2-3) and Sonderforschungsbereich 553(C2). We thank Iris Henkel and Christiane Mildner-Rihm for expert technical assistance.

Received May 24, 2000; revision received August 16, 2000; accepted August 16, 2000.

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