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Roles of Microtubule Dynamics and Small GTPase Rac in Endothelial Cell Migration and Lamellipodium Formation under Flow
Ying-Li Hu^a, Song Li^a, Hui Miao^a, Tsui-Chun Tsou^a, Miguel Angel del Pozo^b, Shu Chien<sup>a

a</sup>Department of Bioengineering and Whitaker Institute of Biomedical Engineering, University of California, SanDiego, and
^bDepartment of Vascular Biology, Scripps Research Institute, La Jolla, Calif., USA

Address of Corresponding Author

J Vasc Res 2002;39:465-476 (DOI: 10.1159/000067202)

 Outline

• Key Words
• Abstract
• Introduction
• Materials and Methods
• Cell Culture
• Cell Treatment
• DNA Plasmids and Transfection
• Shear Stress Experiment
• Multi-Mode Time-Lapse Microscopy
• Cell Tracking and Motility Parameters
• Rac Activity Assay
• Immunofluorescence Staining and Confocal Microscopy
• Statistics
• Results
• MT Stabilization Inhibits Lamellipodial Protrusion and Cell Motility under Static and Flow Conditions
• Quantitative Analysis of BAEC Migration under Static and Flow Conditions
• MT Stabilization Inhibits the Shear Induction of Rac Activity
• Rac Plays a Significant Role in the Shear-Induced Lamellipodial Protrusion
• Rac Mediates BAEC Migration under Static and Flow Conditions
• Discussion
• Acknowledgments
• References

• Author Contacts
• Article Information
• Publication Details
• Drug Dosage / Copyright

  Key Words

• Cell migration
• Fluid shear stress
• Lamellipodium
• Microtubule
• Rac

  Abstract

Endothelial cell (EC) migration is required for vascular development and wound healing. We investigated the roles of microtubule (MT) dynamics and the small GTPase Rac in the fluid shear stress-induced protrusion of lamellipodia and enhancement of migration of bovine aortic ECs (BAECs). Shear stress increased lamellipodial protrusion and cell migration. Treating BAECs with paclitaxel (Taxol), an MT-stabilizing agent, inhibited lamellipodial protrusion and reduced migration speed in both the static and sheared groups. After Taxol washout, both lamellipodial protrusion and cell migration increased in the flow direction. Taxol treatment also decreased the shear-induced Rac activation. Transfection of BAECs with a dominant negative mutant of Rac1 inhibited lamellipodial protrusion and cell migration under static and shear conditions. Transfection with an activated mutant of Rac1 induced lamellipodia in all directions and attenuated the shear-induced migration, suggesting that an appropriate level of Rac activity and a polarized lamellipodial protrusion are important for cell migration under static and shear conditions. Our findings suggest that MT dynamics and optimum Rac activation are required for the polarized protrusion of lamellipodia that drives the directional EC migration under flow.

Copyright © 2002 S. Karger AG, Basel

 Introduction

Endothelial cell (EC) migration is an important process in embryonic vasculogenesis, angiogenesis, and wound healing after denudation injury due to angioplasty and bypass grafting. Cell migration involves dynamic, coordinated changes in cytoskeleton organization and cell adhesion. The intracellular signaling pathways that mediate cell migration have to respond to diverse extracellular cues and translate these into finely regulated cellular activities [1, 2, 3]. ECs are constantly subjected to fluid shear stress, the tangential component of hemodynamic forces due to blood flow. Although there are several reports on the effects of shear flow on EC migration in wounded monolayers [4, 5, 6, 7], the molecular mechanism of EC migration under flow is not well understood.

There is considerable evidence that fluid shear stress can regulate EC structure and function through mechano-chemical transduction [8, 9, 10, 11, 12]. ECs subjected to shear stress undergo shape modifications and align in the flow direction [13, 14, 15, 16, 17, 18]. Accompanying these shear-induced shape alterations are the formation and distribution of actin-containing microfilament bundles, or stress fibers, and the axial realignment of microtubules (MTs), with a transient re-positioning of the MT-organizing center [19, 20, 21]. The cell alignment in the flow direction is inhibited by the disruption of actin filaments or MTs and the inhibition of Rho GTPase, tyrosine kinase and p38 mitogen-activated protein kinase [22, 23, 24]. However, the roles of cytoskeleton and signaling events in EC migration under flow remain to be determined.

The driving force for cell movement is provided by the dynamic reorganization of the actin cytoskeleton, leading to protrusion at the front of the cell and retraction at the rear [1, 3, 25]. The Rho family of small GTPases, particularly Rho, Rac, and Cdc42, play important roles in the control of actin filament assembly and disassembly [26]. Rho regulates the formation of stress fibers [27], while Rac and Cdc42 regulate the formation of lamellipodia and filopodia, respectively [28, 29, 30]. Rho is also required for the formation and maintenance of focal adhesions [31], whereas Rac and Cdc42 regulate the formation of smaller 'focal complex' structures associated with lamellipodia and filapodia [32].

The MT dynamic instability, with cyclic polymerization and depolymerization, has been linked to the regulation of specific small-GTPase-mediated signal transduction cascades that control actin organization and dynamics [33, 34, 35, 36]. Cell migration is stopped by pharmacological stabilization of MTs without their disassembly, indicating that migration requires dynamic changes of MT [37]. Depolymerization of MTs with nocodazole or stabilization of MTs with Taxol decreases lamellipodial protrusion in fibroblasts [38]. The induction of MT polymerization by the removal of nocodazole causes an activation of the small GTPase Rac1 in fibroblasts, leading to actin polymerization and lamellipodial protrusions. Thus, the growth phase of MT dynamic instability can activate a signal transduction cascade that induces lamellipodial protrusion to drive cell migration [34].

The objective of this study was to examine the effects of MT dynamic instability on lamellipodium formation and EC migration under shear stress and to assess the role of Rac in these processes. We showed that shear-induced activation of Rac is necessary for the directional lamellipodial protrusions and cell migration under flow. Taxol inhibited lamellipodium formation and the migration of subconfluent bovine aortic endothelial cells (BAECs) under static and flow conditions, and it blocked both the shear activation of Rac and the Rac modulation of the directional lamellipodial protrusions and cell migration. These results suggest that MT dynamics and Rac activation are required for the polarized protrusion of lamellipodia that drives the directional EC migration under flow.

 Materials and Methods

 Cell Culture

BAECs isolated from bovine aorta were cultured in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Gaithersburg, Md., USA) supplemented with 10% fetal calf serum, 2 mML-glutamine, and 1 mM each of penicillin-streptomycin and sodium pyruvate. The cells were grown on glass slides (3 × 1 inch) precoated with 4 µg/cm² fibronectin (Sigma, St. Louis, Mo., USA) in DMEM containing 10% fetal calf serum (Hyclone Laboratories, Logan, Utah, USA). The cells were maintained in a humidified 95% air, 5% CO₂ incubator at 37°C.

 Cell Treatment

Taxol was purchased from Sigma. The stock solution of Taxol (10^-3M) was dissolved in absolute methanol. BAECs in culture medium were treated either with Taxol (final concentration 10^-6 M) or with methanol as control. Paired studies on experimental and control groups were conducted on subconfluent (<20%) BAECs. In the Taxol experiments, BAECs were first treated with Taxol for 2 h, and then the Taxol medium was replaced by fresh Taxol-free medium. In the flow experiments, BAECs were subjected to shear stress at 12 dyn/cm², and the controls were kept under static condition.

 DNA Plasmids and Transfection

Plasmids encoding green flourescence protein (GFP)-tagged tubulin were from Clontech (Palo Alto, Calif., USA). Plasmids encoding the wild-type (WT), an activated mutant (V12), and a dominant negative mutant (N17) of the GTPase Rac1 were described previously [39]. To facilitate the detection of individual cells harboring the exogenously introduced GTPases, Rac1 was fused to the C-terminal of the enhanced GFP (EGFP) [39]. pEGFP-C1 GFP expression plasmids were from Clontech. Plasmids encoding the GFP-tagged proteins were transfected into BAECs at 80% confluence using the lipofectamine method (Life Technologies). After incubation for 6 h, the cells were washed with DMEM and incubated with fresh DMEM containing 10% serum for another 24-48 h to reach confluence. The cells in the tissue culture wells were then seeded on glass slides coated with 4 µg/cm² bovine fibronectin for use in the experiments.

 Shear Stress Experiment

The shear stress experiments were conducted in a rectangular flow chamber [40]. The chamber, a reservoir, and a circulation circuit were filled with cell culture medium. The flow of the medium was driven by a constant pressure head, which was maintained by a roller pump, such that the BAECs were subjected to a laminar shear stress at 12 dyn/cm². The medium was kept at a constant temperature of 37°C and equilibrated with a gas mixture of humidified 95% air and 5% CO₂. In the control static group, the BAECs were kept for the same duration without flow.

 Multi-Mode Time-Lapse Microscopy

Digital images of cell motility were obtained using the multi-mode microscopy system, which consists of a Nikon microscope equipped with ×10, ×20 and ×40 objectives for fluorescence and phase contrast microscopy, a CCD camera, and a computer for image acquisition. Phase contrast images were collected at 10-min intervals. Fluorescence imaging of cells expressing GFP was performed by using an excitation filter (495 nm) and a band-pass emission filter (510-530 nm). Phase contrast and fluorescence images were taken alternatively by switching the excitation light paths. The resulting image signals were displayed on the video screen and digitized with a Matrox MVP-AT image processor. The Image-1 system (Universal Imaging; West Chester, Pa., USA) was used to capture and store the images in the computer. For time-lapse recordings, the cells (static and sheared) were observed under a constant temperature of 37°C. The system automatically digitized and stored one image every 10 min on the hard disk.

 Cell Tracking and Motility Parameters

The dynamic image analysis system (DIAS; Solltech, Oakdale, La., USA) was used to analyze the cell migration. The images of the cells during the course of the experiment were tracked semiautomatically by outlining and pointing at their nuclei to create a database of their position. The x and y pixel coordinates of the centroid of the nucleus of individual cells were digitized. Such trails of the centroid of the nucleus were used to track cell migration for the calculations of the migration speed and the cumulative migration distance. The migration speed is the distance traversed per minute calculated over each 10-min interval. The cumulative distance is the running sum of the distances traversed over these 10-min intervals. To analyze the directional migrations, the distance traversed for each 10-min interval was decomposed by projecting it to axes perpendicular and parallel to the direction of flow.

 Rac Activity Assay

Rac activity was determined from the specific binding of the active Rac to the p21-binding domain (PBD) of PAK1 fused to glutathione S-transferase (GST) [41]. BAECs were washed with ice-cold phosphate-buffered saline, and lyzed in a buffer containing 25 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 100 mM NaCl, 5 mM MgCl₂, 5% glycerol, 1 µg/ml leupeptin, 1 mM orthovanadate, and 1 mM phenylmethylsulfonyl fluoride. The lysates were spun at 12,000 g for 10 min at 4°C, and the supernatants were incubated with 20 µg of glutathione-agarose beads with recombinant GST-PBD for 30 min at 4°C. The beads were washed with the lysis buffer, and the bound Rac was eluted with SDS sample buffer. Bound Rac was analyzed by Western blotting using a monoclonal anti-Rac antibody (Transduction Laboratories, Lexington, Ky., USA). Whole-cell lysates were also analyzed for the amount of Rac for normalization.

 Immunofluorescence Staining and Confocal Microscopy

For immunostaining, the BAECs were fixed with 4% paraformaldehyde for 15 min at 37°C in phosphate-buffered saline. The fixed cells were permeabilized with 0.3% Triton X-100, and nonspecific binding was blocked by 1% normal goat serum. To visualize the MTs, the cells were incubated with an anti--tubulin MAb (Sigma) in phosphate-buffered saline, followed by a tetramethylrhodamine isothiocyanate-conjugated secondary antibody. For negative controls, the samples were incubated with either the anti--tubulin MAb without the secondary antibody, or the tetramethylrhodamine isothiocyanate-labeled secondary antibody without the anti--tubulin MAb. To visualize actin microfilaments, the cells were stained with rhodamine-conjugated phalloidin (Molecular Probes, Eugene, Oreg., USA). The slides were mounted with the SlowFade^TM antifade reagent (Molecular Probes) and examined by using a Nikon diaphot microscope equipped with a Bio-Rad MRC 1024 laser scanning confocal imaging system (Bio-Rad, Richmond, Calif., USA). A ×60 oil immersion objective was used to examine the specimens. GFP was excited at a wavelength of 488 nm and detected within a band between 506 and 538 nm. Rhodamine was excited at 568 nm and detected within a band between 589 and 621 nm. By stepping the objective through the depth of the specimen, a z-series collection of optical sections was obtained and projected to an image. The images were transferred to a Power Macintosh computer for further analysis; the Adobe Photoshop (Adobe System, Mountain View, Calif., USA) was used to generate RGB images to depict the F-actin or -tubulin in red and the expressed GFP in green.

 Statistics

Mean values, together with standard deviations, and standard errors of the mean were calculated for the experimental and control groups. Analysis of variance (ANOVA) was performed by using data analysis tools of the program Microsoft Excel (Microsoft, Redmond, Wash., USA), with the aid of a Power Macintosh computer. The results are expressed as mean values ± SEM. The p values were calculated to test the level of statistical significance. A p value of less than 0.05 was taken to be statistically significant for differences between means. The difference between two groups was tested for significance by using paired t test. All plots were made with the aid of KaleidaGraph (Synergy Software, Reading, Pa., USA).

 Results

 MT Stabilization Inhibits Lamellipodial Protrusion and Cell Motility under Static and Flow Conditions

Subconfluent BAECs were used to study the effects of shear stress on EC migration. To observe directly the MT dynamics in relation to lamellipodial protrusion in living BAECs under shear stress, the cells were transfected with plasmids encoding GFP-tubulin, and the MTs labeled with green fluorescence were observed by fluorescence microscopy. As shown in figure 1, MT dynamics was observed in the lamellipodial protrusion of EC under shear stress.

Fig. 1. MT dynamics in a live BAEC subjected to shear stress (12 dyn/cm2). Cells were transfected with GFP-tubulin and subjected to fluorescence microscopy during shearing. b was taken 5 min after a. b Arrows show the dynamic changes in tubulin as compared with a. The boxed regions in a and b are shown at higher magnification in c and d, respectively. Direction of flow is from left to right. Bars: a, b = 4 µm; c, d = 2 µm.

To investigate the role of MT dynamics in lamellipodial protrusion and cell motility, we used Taxol (10^-6M) to stabilize the assembled MTs in BAECs under stationary condition. In Taxol-free medium, the cells were irregular in shape, with small areas of protrusive ruffling at random sites, and exhibited motility (fig. 2a, top row). After 2 h of Taxol treatment (fig. 2a, middle row), the cells showed little lamellipodial protrusion or migration, and became more uniform in their morphology (generally more round). After Taxol washout and incubation in a Taxol- free medium (fig. 2a, bottom row), lamellipodial protrusion and cell migration showed a slight recovery. Some lamellipodial protrusions formed along the perimeters of the cells, and some cell migration was observed.

Fig. 2. Effects of MT stabilization on EC migration under static and flow conditions. Subconfluent BAECs cultured on fibronectin were either kept under static condition (a) or subjected to shear stress at 12 dyn/cm2 (b) for 2 h. There were three groups under each condition. The cells in control groups were not treated with Taxol. In the Taxol groups, cells were treated with Taxol (10-6M) for 2 h. In Taxol washout groups, cells were first treated with Taxol (10-6M) for 2 h and then changed to a fresh Taxol-free medium. Cell migration was monitored by phase contrast microscopy. In each row, an arrow and an arrowhead point to two cells as examples to show their motility. Bars = 50 µm.

In Taxol-free medium, BAECs subjected to shear stress formed lamellipodial protrusions and migrated in the direction of flow (fig. 2b, top row). For cells kept for 2 h in the medium with Taxol (10^-6M), shear stress caused random movement of lamellipodia at cell periphery, but failed to induce lamellipodial protrusion and cell migration in the flow direction (fig. 2b, middle row), suggesting that MT dynamics is required for the directional lamellipodial protrusions and migration induced by shear stress. After Taxol washout (fig. 2b, bottom row), the application of shear stress led to cell elongation and lamellipodial protrusions in the flow direction, and cell migration also showed more recovery than in the static condition (fig. 2a).

 Quantitative Analysis of BAEC Migration under Static and Flow Conditions

Tracking of individual cells under static and flow conditions showed that shear stress had a strong effect on cell movement. The rate of migration of BAECs, as determined from the cumulative distance under shear stress (12 dyn/cm²) over a period of 2 h, was projected to directions parallel and perpendicular to the flow direction (fig. 3a, b, respectively). For cells under static condition, the cumulative distance of movement over 2 h was projected to the long axis of the flow channel and the axis perpendicular to that. The migration distance parallel to the flow direction over 2 h was 33.3 ± 5.7 µm (mean ± SEM) in sheared cells; this was significantly (p < 0.05) higher than the 18.4 ± 2.9 µm in static cells, as well as the migration distances in the perpendicular direction of 24.6 ± 3.8 µm for the sheared cells and 13.6 ± 1.7 µm for the static cells. The speed in the flow direction was significantly higher than that in the perpendicular direction in sheared cells, but there was no significant difference in speed in the two directions for static cells. When cells were treated with Taxol (10^-6M), the 2-hour migration distance parallel to the flow direction was 8.7 ± 1.5 µm (mean ± SEM) in sheared cell and 8.3 ± 1.1 µm in static cells; the migration distance in the perpendicular direction was 7.8 ± 1.1 µm for sheared cells, and 7.1 ± 1.0 µm for static cells. Thus, Taxol treatment inhibited (p < 0.05) the migration in both directions, for both static and sheared cells, to values in the range of 7-9 µm, and there was no significant difference among these groups. After Taxol washout and changing the cells into a Taxol-free medium, the 2-hour migration distance parallel to the flow direction was 19.1 ± 3.3 µm (mean ± SEM) in sheared cells; this was significantly (p < 0.05) higher than the 10.5 ± 1.4 µm in static cells, as well as the migration distances in the perpendicular direction of 13.9 ± 2.2 µm for sheared cells and 9.9 ± 1.5 µm for static cells. The migration distances in both parallel and perpendicular directions significantly increased (p < 0.05) in both the static and sheared groups over the corresponding values under Taxol, but were still below the corresponding control values (fig. 3a, b). The results indicate that the shear-induced increase of cell migration is eliminated following stabilization of the assembled MTs. Thus, cell migration requires dynamic changes in MT organization.

Fig. 3. Cumulative distances of migration projected to parallel (a) and perpendicular (b) directions for BAECs under static and flow conditions. Phase contrast images from experiments described in figure 2 were analyzed with the DIAS software. At least thirty cells from three separate experiments were used in the statistical analysis of cell migration under each treatment. Values represent means ± SEM. * p < 0.05, static vs. sheared cells.

 MT Stabilization Inhibits the Shear Induction of Rac Activity

The small GTPase Rac has been shown to regulate lamellipodial protrusion in fibroblasts in response to growth factors [28]. Since MT stabilization inhibited shear stress-induced lamellipodial protrusion, we tested whether MT stabilization would affect Rac activity under static and flow conditions. The cells were pre-incubated in the presence or absence of Taxol, and either kept under static condition or subjected to shear stress for 15 min. Rac activity in the cell lysates was assessed by using the PBD pull-down assay. Application of shear stress (12 dyn/cm²) to BAECs for 15 min caused a significant increase (about 2-fold) of Rac activity over the static cells (fig. 4). After 2 h of Taxol (10^-6M) treatment, the shear-induced increase in Rac-GTP was abolished. These results indicate that the shear induction of Rac activity is inhibited when MTs are stabilized. Thus, the shear activation of Rac requires dynamic MTs, suggesting that MTs may play a role upstream to Rac.

Fig. 4. Effects of MT stabilization on Rac activation by shear stress. Rac activity was assessed for BAECs under static and flow conditions, in the absence or presence of Taxol. Active Rac was pulled down by GST-PBD beads from cell lysates and detected by Western blotting (a). Probing of total cell lysates for Rac demonstrates equal amounts of total Rac in different groups (b). Bars represent means ± SD from densitometric results of three experiments. *p < 0.05, static vs. sheared cells.

 Rac Plays a Significant Role in the Shear-Induced Lamellipodial Protrusion

To assess the role of the small GTPase Rac1 in lamellipodium formation and cell migration, BAECs were transfected with cDNA encoding the WT of Rac1 (Rac1WT), a dominant negative mutant of Rac1 (Rac1N17), and a dominant active mutant of Rac1 (Rac1V12). These Rac1 transfectants were fused to GFP to enable the visualization of the individual BAECs transfected. Figure 5 shows the expression of GFP-tagged proteins in BAECs and their effects on actin and MT structures. Rhodamine-phalloidin staining of actin microfilaments shows the cell boundary (fig. 5a) and immunostaining of MTs shows the lamellipodium formation and lamellipodial protrusion (fig. 5b) in the untransfected cells and cells transfected with GFP-Rac1WT, GFP-Rac1N17, and GFP-Rac1V12. The cells transfected with GFP-Rac1WT showed lamellipodium formation and protrusion comparable to those of untransfected cells. In cells transfected with GFP-Rac1N17, the lamellipodium formation and protrusion were inhibited. The cells transfected with GFP-Rac1V12 were rounder and larger than the untransfected cells and cells transfected with GFP-Rac1WT and GFP-Rac1N17. GFP-Rac1V12 induced lamellipodium formation in all directions, but did not induce lamellipodial protrusion.

Fig. 5. Cytoskeletal structure of cells expressing Rac1WT, Rac1N17 and Rac1V12. a Fluorescence photomicrographs showing GFP (upper row) and actin microfilaments (lower row) in the same fields. Microfilaments were stained with rhodamine-phalloidin (red). The left, middle, and right columns show BAECs transfected with GFP-Rac1WT, GFP-Rac1N17, and GFP-Rac1V12, respectively. b Fluorescence photomicrographs showing GFP (upper row) and MTs (lower row) in the same fields. MTs were stained with antibody -tubulin followed by an rhodamine-labeled secondary antibody (red). The left, middle, and right columns show BAECs transfected with GFP-Rac1WT, GFP-Rac1N17, and GFP-Rac1V12, respectively. Bars = 25 µm.

To determine whether Rac1 is involved in the shear-induced lamellipodial protrusion, BAECs transfected with GFP-Rac1WT, Rac1N17 and Rac1V12 were subjected to shear stress. As a control of transfected cells, BAECs were transfected with pEGFP-C1. The untransfected cells and cells transfected with pEGFP-C1 or GFP-Rac1WT developed lamellipodia in the flow direction (fig. 6). Transfection with the negative mutant GFP-Rac1N17 blocked lamellipodial protrusion and cell migration. Transfection with the active mutant Rac1V12 increased lamellipodia in all directions, but inhibited the polarization of lamellipodial protrusion for migration (fig. 6). GFP-Rac1V12-transfected cells showed membrane motion at cell periphery under dynamic observation, but there was no significant net migration.

Fig. 6. Effects of EGFP-C1, Rac1WT, Rac1N17 and Rac1V12 on lamellipodial protrusion under flow condition. BAECs were transfected with EGFP-C1, GFP-Rac1WT, GFP-Rac1N17, or GFP-Rac1V12, and subjected to shear stress at 12 dyn/cm2 for 2 h. Fluorescence photomicrographs in the top row show cells expressing GFP-tagged proteins before shearing. The phase contrast photomicrographs in the second to fourth rows show all cells in the field, with arrowheads pointing to the GFP-expressing cells. Motions in the flow direction can be seen for cells expressing EGFP-C1 and GFP-Rac1WT, but not for cells expressing GFP-Rac1N17 or GFP-Rac1V12.

 Rac Mediates BAEC Migration under Static and Flow Conditions

The role of Rac in EC migration under static and flow conditions was assessed from microscopic images (fluorescence and phase contrast) of untransfected and transfected BAECs. The images were automatically stored every 10 min into the computer and analyzed with the DIAS software. Comparing untransfected cells with cells transfected with pEGFP-C1 cells, the cumulative distances of BAEC migration over 2 h were not significantly different in both static and sheared groups (not shown). The 2-hour migration distances of BAECs under static condition (mean ± SEM) were 29.3 ± 4.5, 28.0 ± 2.3, 15.9 ± 2.4, and 16.9 ± 2.6 µm in pEGFP-C1, GFP-Rac1WT, GFP-Rac1N17, and GFP-Rac1V12 groups, respectively (fig. 7). The 2-hour migration distances under flow for these respective groups were 44.5 ± 6.8, 42.0 ± 6.5, 18.2 ± 2.8, and 24.2 ± 3.7 µm, respectively (fig. 7). Compared with pEGFP-C1, cell migration was not significantly altered by GFP-Rac1WT under both static and shear conditions. When compared with pEGFP-C1 in both static and shear conditions, cell migration decreased significantly with GFP-Rac1N17 and also with GFP-Rac1V12. These results indicate that modification of Rac1 activity can modulate EC migration under static and flow conditions. Thus, cell migration decreased with either an increase or a decrease in Rac1 activity in the cells transfected with either GFP-Rac1V12 or GFP-Rac1N17, respectively.

Fig. 7. Effects of Rac1WT, Rac1N17 and Rac1V12 on EC migration under static and flow conditions. The cells had been transfected with pEGFP-C1, GFP-Rac1WT, GFP-Rac1N17, or GFP-Rac1V12 2 days before the experiments, and cell migration under static and flow conditions was monitored for 2 h. Phase contrast images from these experiments were analyzed with DIAS software. At least thirty cells from three separate experiments were used in the statistical analysis of cell migration under each treatment. Values represent means ± SEM. * p < 0.001, static vs. sheared cells within each group. Shear-induced increases in migration distance occur to similar extents for cells expressing EGFP-C1 and GFP-RacWT, but are not seen in cells expressing GFP-RacN17 or GFP-RacV12.

 Discussion

MTs play an important role in the regulation of EC morphology, including its response to shear flow. Shear stress has been shown to cause a transient preferential positioning of the MT organization center [19, 20, 21]. Taxol, which stabilizes MTs in the polymerized state, retards and attenuates the MT alignment in response to shear stress [22]. While it is well established that shear stress can modulate EC morphology and cytoskeleton organization, the relations of these changes to lamellipodial protrusion, cell migration, and intracellular signaling remain to be established. Our findings that the MT-stabilizing agent Taxol inhibited the shear-induced lamellipodial protrusion and migration enhancement indicate that EC migration under shear stress requires dynamic changes in MT organization. We also showed that the recovery of lamellipodial protrusion and cell migration after Taxol washout was greater under shear stress (fig. 2). These findings, together with our microscopy observations (fig. 1), suggest that shear stress enhanced MT dynamics to increase EC migration. MTs are required for the persistent polarized movement in many cell types during wound healing [42, 43, 44]. Studies on fibroblasts under static condition have shown that cell migration is inhibited by the disruption of MT organization or the stabilization of MTs without substantial MT polymer loss [37, 45]. Thus, MTs can regulate cell migration by their dynamic growth and shortening, in addition to serving as tracks for the directed delivery to the leading cell edge of substances required for motility [46].

What is the molecular mechanism by which the shear stress-induced MT dynamics drives the directional cell migration? We designed experiments to analyze the roles of MTs in Rac activation and their roles in regulating the morphology and migration of individual BAECs in response to shear stress. The shear-induced Rac1 activation, as assessed from GTP-bound Rac (fig. 4), was abolished by Taxol. This indicates that the shear activation of Rac requires dynamic MTs and suggests that MTs may be upstream to Rac. In fibroblasts under static condition, it has been shown that the pool of growing MT plus-ends in the cell periphery could induce local activation of Rac1 to promote actin polymerization and lamellipodial protrusion to drive cell migration [34]. On the other hand, Rac may stabilize MT extension in lamellipodia as a positive feedback. Indeed, the recent study by Daub et al. [47] has shown that Rac regulates the phosphorylation state of the MT-destabilizing protein stathmin to stabilize MT structure.

We studied ECs transfected with GFP-Rac1 and its mutants to assess the role of Rac1 in lamellipodial protrusion and EC migration under shear stress. Our data showed that the negative mutant of Rac1 (Rac1N17) inhibited both the lamellipodial protrusion and cell migration. The overactivation of Rac1 by Rac1V12 transfection induced a greater degree of lamellipodial formation, but inhibited the polarized lamellipodial protrusion and hence cell migration. The results in the four groups studied (control, Rac1WT, Rac1N17, and Rac1V12) indicate that the state of Rac1 activation plays a significant role in lamellipodial formation and cell migration. While lamellipodial formation increased directly with an increase in Rac activity, cell migration was best in the control group. These findings suggest that with a low Rac1 activity (i.e. in the Rac1N17 group), there was insufficient lamellipodial formation to form a strong enough anchor to the substrate for cell migration. On the other hand, with a high Rac1 activity (i.e. in the RacV12 group), the excessive lamellipodial formation was associated with non-polarized adhesions that are too strong for the cell to migrate. The Rac1 activity and the associated lamellipodial formation for the control group under shear appear to be optimal for cell migration. Thus, our results indicate that the state of Rac1 activation plays a significant role in modulating lamellipodium formation, and that an optimum level of Rac1 activity may be required for appropriate lamellipodial protrusion to mediate the maximum rate of cell migration. Thus, an optimum rate of cell migration requires not only a medium level of cell-matrix adhesion [48, 49], but also a medium level of signaling molecule activities. There is recent evidence implicating a role for Rho GTPases, including Rac, in chemotactic migration of ECs in response to vascular endothelial growth factor [50]. These data also suggest that Rac-mediated signaling events play significant roles in EC migration.

In summary, our experiments show that shear stress increases directional MT growth, which is important for Rac1 activation and the attendant enhancement of lamellipodial protrusion and cell migration in the flow direction, i.e. mechanotaxis [51]. Studies with various Rac1 mutants indicate that lamellipodial formation increases with Rac1 activity and that maximum cell migration occurs at the Rac1 activity found in the control group under shear, which probably provides optimal levels of cell-matrix adhesion and lamellipodial formation. Our findings on the roles of MT and Rac in EC migration have important implications in angiogenesis and vascular wound healing. Thus, MT and Rac may be potential therapeutic targets to inhibit angiogenesis during tumor growth, and the inhibitory effect of Taxol on cell migration might contribute to its anticancer effect. When EC wound healing is desired after vascular injury, e.g. after balloon angioplasty, agents that promote the functions of MT and Rac to enhance EC migration may have therapeutic values.

 Acknowledgments

We thank Mr. Casey Laris for his excellent assistance in the use of the confocal microscope. This work was supported by National Heart, Lung, and Blood Institute Research Grants HL-19454, HL-43026 (S.C.), and a Scientific Development Grant from the American Heart Association (S.L.).

  References

1.

Lauffenburger DA, Horwitz AF: Cell migration: A physically integrated molecular process. Cell 1996;84:359-369. 

2.

Ridley AJ, Allen WE, Peppelenbosch M, Jones GE: Rho family proteins and cell migration. Biochem Soc Symp 1999;65:111-123. 

3.

Sheetz MP, Felsenfeld D, Galbraith CG, Choquet D: Cell migration as a five-step cycle. Biochem Soc Symp 1999;65:233-243. 

4.

Ando J, Nomura H, Kamiya A: The effect of fluid shear stress on the migration and proliferation of cultured endothelial cells. Microvasc Res 1987;33:62-70. 

5.

Tardy Y, Resnick N, Nagel T, Gimbrone MJ, Dewey CJ: Shear stress gradients remodel endothelial monolayers in vitro via a cell proliferation-migration-loss cycle. Arterioscler Thromb Vasc Biol 1997;17:3102-3106. 

6.

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. 

7.

Hsu P-P, Li S, Li Y-S, Shunichi Usami S, Ratcliffe A, Wang X, Chien S: Effects of flow patterns on endothelial cell migration into a zone of mechanical denudation. Biochem Biophys Res Commun 2001;285:751-759. 

8.

Chien S, Li S, Shyy YJ: Effects of mechanical forces on signal transduction and gene expression in endothelial cells. Hypertension 1998;31:162-169. 

9.

Traub O, Berk BC: Laminar shear stress: Mechanisms by which endothelial cells transduce an atheroprotective force. Arterioscler Thromb Vasc Biol 1998;18:677-685. 

10.

Barakat AI, Davies PF: Mechanisms of shear stress transmission and transduction in endothelial cells. Chest 1998;114(1 suppl):58S-63S.

11.

Chappell DC, Varner SE, Nerem RM, Medford RM, Alexander RW: Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res 1998;82:532-539. 

12.

Resnick N, Yahav H, Schubert S, Wolfovitz E, Shay A: Signalling pathways in vascular endothelium activated by shear stress: Relevance to atherosclerosis. Curr Opin Lipidol 2000;11:167-177. 

13.

Dewey CF Jr, Bussolari SR, Gimbrone MA Jr, Davies PF: The dynamic response of vascular endothelial cells to fluid shear stress. J Biomech Eng 1981;103:177-185. 

14.

Remuzzi A, Dewey CJ, Davies PF, Gimbrone MJ: Orientation of endothelial cells in shear fields in vitro. Biorheology 1984;21:617-630. 

15.

Franke RP, Grafe M, Schnittler H, Seiffge D, Mittermayer C, Drenckhahn D: Induction of human vascular endothelial stress fibers by fluid shear stress. Nature 1984;307:648-649. 

16.

Eskin SG, Ives CL, McIntire LV, Navarro LT: Response of cultured endothelial cells to steady flow. Microvasc Res 1984;28:87-94. 

17.

Wechezak AR, Viggers RF, Sauvage LR: Fibronectin and F-actin redistribution in cultured endothelial cells exposed to shear stress. Lab Invest 1985;53:639-647. 

18.

Levesque MJ, Nerem RM: The elongation and orientation of cultured endothelial cells in response to shear stress. J Biomech Eng 1985;107:341-347. 

19.

Ives CL, Eskin SG, McIntire LV: Mechanical effects on endothelial cell morphology: In vitro assessment. In Vitro Cell Dev Biol 1986;22:500-507. 

20.

Coan DE, Wechezak AR, Viggers RF, Sauvage LR: Effect of shear stress upon localization of the Golgi apparatus and microtubule organizing center in isolated cultured endothelial cells. J Cell Sci 1993;104:1145-1153. 

21.

Galbraith CG, Skalak R, Chien S: Shear stress induces spatial reorganization of the endothelial cell cytoskeleton. Cell Motil Cytoskeleton 1998;40:317-330. 

22.

Malek AM, Izumo S: Mechanism of endothelial cell shape change and cytoskeletal remodeling in response to fluid shear stress. J Cell Sci 1996;109:713-726. 

23.

Li S, Chen BP, Azuma N, Hu YL, Wu SZ, Sumpio BE, Shyy JY, Chien S: Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress. J Clin Invest 1999;103:1141-1150. 

24.

Azuma N, Akasaka N, Kito H, Ikeda M, Gahtan V, Sasajima T, Sumpio BE: Role of p38 MAP kinase in endothelial cell alignment induced by fluid shear stress. Am J Physiol 2001;280:H189-H197. 

25.

Mitchison TJ, Cramer LP: Actin-based cell motility and cell locomotion. Cell 1996;84:371-379. 

26.

Hall A: Rho GTPases and the actin cytoskeleton. Science 1998;279:509-514. 

27.

Ridley AJ, Hall A: The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 1992;70:389-399. 

28.

Ridley AJ, Paterson HF, Johnston CL, Diekmann D, Hall A: The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell 1992;70:401-410. 

29.

Kozma R, Ahmed S, Best A, Lim L: The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol 1995;15:1942-1952. 

30.

Nobes CD, Hawkins P, Stephens L, Hall A: Activation of the small GTP-binding proteins rho and rac by growth factor receptors. J Cell Sci 1995:225-233.

31.

Hotchin NA, Hall A: The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J Cell Biol 1995;108:1857-1865.

32.

Nobes CD, Hall A: Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell 1995;81:53-62. 

33.

Ren XD, Kiosses WB, Schwartz MA: Regulation of the small GTP-binding protein Rho by cell adhesion and the cytoskeleton. EMBO J 1999;18:578-585. 

34.

Waterman-Storer CM, Worthylake RA, Liu BP, Burridge K, Salmon ED: Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts. Nat Cell Biol 1999;1:45-50. 

35.

Waterman-Storer CM, Salmon E: Positive feedback interactions between microtubule and actin dynamics during cell motility. Curr Opin Cell Biol 1999;11:61-67. 

36.

Elbaum M, Chausovsky A, Levy ET, Shtutman M, Bershadsky AD: Microtubule involvement in regulating cell contractility and adhesion-dependent signalling: A possible mechanism for polarization of cell motility. Biochem Soc Symp 1999;65:147-172. 

37.

Liao G, Nagasaki T, Gundersen GG: Low concentrations of nocodazole interfere with fibroblast locomotion without significantly affecting microtubule level: Implications for the role of dynamic microtubules in cell locomotion. J Cell Sci 1995;108:3473-3483. 

38.

Mikhailov A, Gundersen GG: Relationship between microtubule dynamics and lamellipodium formation revealed by direct imaging of microtubules in cells treated with nocodazole or taxol. Cell Motil Cytoskeleton 1998;41:325-340. 

39.

del Pozo MA, Vicente-Manzanares M, Tejedor R, Serrador JM, Sánchez-Madrid F: Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes. Eur J Immunol 1999;29:3609-3620. 

40.

Gallik S, Usami S, Jan KM, Chien S: Shear stress-induced detachment of human polymorphonuclear leukocytes from endothelial cell monolayers. Biorheology 1989;26:823-834. 

41.

del Pozo MA, Price LS, Alderson NB, Ren X-D, Schwartz MA: Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK. EMBO J 2000;19:2008-2014. 

42.

Gordon SR, Staley CA: Role of the cytoskeleton during injury-induced cell migration in corneal endothelium. Cell Motil Cytoskeleton 1990;16:47-57. 

43.

Ettenson DS, Gotlieb AI: Centrosomes, microtubules, and microfilaments in the reendothelialization and remodeling of double-sided in vitro wounds. Lab Invest 1992;66:722-733. 

44.

Bershadsky AD, Vaisberg EA, Vasiliev JM: Pseudopodial activity at the active edge of migrating fibroblast is decreased after drug-induced microtubule depolymerization. Cell Motil Cytoskeleton 1991;19:152-158. 

45.

Tanaka E, Ho T, Kirschner MW: The role of microtubule dynamics in growth cone motility and axonal growth. J Cell Biol 1995;128:139-155. 

46.

Goode BL, Drubin DG, Barnes G: Functional cooperation between the microtubule and actin cytoskeletons. Curr Opin Cell Biol 2000;12:63-71. 

47.

Daub H, Gevaert K, Vandekerckhove J, Sobel A, Hall A: Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16. J Biol Chem 2001;276:1677-1680. 

48.

DiMilla PA, Stone JA, Quinn JA, Albelda SM, Lauffenburger DA: Maximal migration of human smooth muscle cells on fibronectin and type IV collagen occurs at an intermediate attachment strength. J Cell Biol 1993;122:729-737. 

49.

Palecek SP, Loftus JC, Ginsberg MH, Lauffenburger DA, Horwitz AF: Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 1997;385:537-540. 

50.

Soga N, Namba N, McAllister S, Cornelius L, Teitelbaum SL, Dowdy SF, Kawamura J, Hruska KA: Rho family GTPases regulate VEGF-stimulated endothelial cell motility. Exp Cell Res 2001;269:73-87. 

51.

Li S, Butler P, Wang YX, Shiu YT, Hu YL, Han DC, Usami S, Guan JL, Chien S: The role of the dynamics of focal adhesion kinase in the mechanotaxis of endothelial cells. Proc Natl Acad Sci USA 2002;99:3546-3551. 

  Author Contacts

Dr. Shu Chien
Department of Bioengineering
University of California, San Diego
La Jolla, CA 92093-0427 (USA)
Tel. +1 858 534 5195, Fax +1 858 534 5453, E-Mail schien@bioeng.ucsd.edu

  Article Information

Received: Received: December 10, 2001
Accepted after revision: April 16, 2002
Number of Figures : 7, Number of Tables : 0, Number of References : 51

  Publication Details

Journal of Vascular Research (Incorporating International Journal of Microcirculation)
Founded 1964 as Angiologica by M. Comèl and L. Laszt (1964-1973) continued as Blood Vessels by J.A. Bevan (1974-1991)
Official Journal of the European Society for Microcirculation

Vol. 39, No. 6, Year 2002 (Cover Date: November-December 2002)

Journal Editor: U. Pohl, Munich
ISSN: 1018-1172 (print), 1423-0135 (Online)

For additional information: http://www.karger.com/jvr

  Drug Dosage / Copyright

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