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J Appl Physiol 98: 2355-2362, 2005. First published February 10, 2005; doi:10.1152/japplphysiol.01136.2004
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HIGHLIGHTED TOPICS
Biomechanics and Mechanotransduction in Cells and Tissues

Vascular endothelial wound closure under shear stress: role of membrane fluidity and flow-sensitive ion channels

Andrea Gojova and Abdul I. Barakat

Department of Mechanical and Aeronautical Engineering, University of California, Davis, California

Submitted 11 October 2004 ; accepted in final form 2 February 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Sufficiently rapid healing of vascular endothelium following injury is essential for preventing further pathological complications. Recent work suggests that fluid dynamic shear stress regulates endothelial cell (EC) wound closure. Changes in membrane fluidity and activation of flow-sensitive ion channels are among the most rapid endothelial responses to flow and are thought to play an important role in EC responsiveness to shear stress. The goal of the present study was to probe the role of these responses in bovine aortic EC (BAEC) wound closure under shear stress. BAEC monolayers were mechanically wounded and subsequently subjected to either "high" (19 dyn/cm2) or "low" (3 dyn/cm2) levels of steady shear stress. Image analysis was used to quantify cell migration and spreading under both flow and static control conditions. Our results demonstrate that, under static conditions, BAECs along both wound edges migrate at similar velocities to cover the wounded area. Low shear stress leads to significantly lower BAEC migration velocities, whereas high shear stress results in cells along the upstream edge of the wound migrating significantly more rapidly than those downstream. The data also show that reducing BAEC membrane fluidity by enriching the cell membrane with exogenous cholesterol significantly slows down both cell spreading and migration under flow and hence retards wound closure. Blocking flow-sensitive K and Cl channels reduces cell spreading under flow but has no impact on cell migration. These findings provide evidence that membrane fluidity and flow-sensitive ion channels play distinct roles in regulating EC wound closure under flow.

cell migration; flow; wound healing; mechanotransduction; cell spreading

INJURY OF VASCULAR ENDOTHELIUM may occur under different pathological conditions. For instance, deployment of an endovascular stent within a stenosed arterial segment leads to extensive local injury and denudation of arterial endothelial cells (EC) at the stenosis site. Endothelial wound closure following injury is a complex and incompletely understood process that involves cellular spreading, migration, and proliferation (10). These different processes are sensitive not only to the local biochemical environment, including the concentration of various cytokines, chemokines, and growth factors, but also to the mechanical forces, most notably the fluid mechanical shear stress, to which the cells are exposed. Albuquerque et al. (4) studied the impact of different levels of steady shear stress on wound closure in human umbilical vein ECs (HUVECs) and human coronary artery ECs (HCAECs) and demonstrated that shear stress generally accelerates wound closure relative to static (no flow) conditions. Furthermore, the extent of wound closure depended to some degree on cell type and to a larger extent on the level of applied shear stress as well as on whether or not the cells were presheared. Because of the preferential localization of atherosclerotic lesions within regions of arterial flow disturbance (8, 18, 21), Hsu et al. (16) investigated the impact of flow separation and recirculation downstream of a step on the migration rates of bovine aortic ECs (BAECs) and demonstrated that disturbed flow patterns impact BAEC migration differently from nondisturbed flow. The sensitivity of EC wound closure to the magnitude and type of applied shear stress highlights the need for better characterization of the impact of flow on the wound closure process.

The mechanisms governing the regulation of endothelial wound closure by shear stress remain poorly understood. Exposure of ECs to shear stress elicits a sequence of coordinated humoral, metabolic, and structural responses that regulate cell structure and function (6, 9, 12). Changes in cell membrane fluidity (7, 15) and activation of flow-sensitive K and Cl channels (5, 19, 23) are among the most rapid endothelial responses to flow and have, therefore, been proposed to be involved in shear stress sensing and transduction (6, 9). While virtually nothing is known about the potential involvement of flow-sensitive ion channels in modulating EC migration, membrane fluidity has recently been shown to play a role in regulating EC motility under static conditions (13). We hypothesized that reducing membrane fluidity and blocking flow-sensitive ion channels in ECs under shear stress would reduce cell migration and would, therefore, retard wound closure. Moreover, we wished to develop a more complete and quantitative description of the impact of shear stress level on BAEC migration. Our results demonstrate that relatively high levels of shear stress lead to greatly faster BAEC wound closure than low levels of shear stress and that, while membrane fluidity affects both cell migration and spreading, flow-sensitive ion channels only impact cell spreading.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cell culture.   BAECs (Cell Systems, Kirkland, WA) in passages 3–10 were cultured by standard procedures in CSC complete medium (Cell Systems) containing 10% fetal bovine serum. For static (no flow) experiments, the cells were cultured on 25 x 75-mm Permanox plastic cell culture chamber slides (Nalge Nunc International, Naperville, IL). For flow experiments, the cells were plated on 25 x 75-mm Permanox plastic slides (VWR, Batavia, IL). In all experiments, the slides were precoated with attachment factor (Cell Systems) to optimize cell adhesion. In most experiments, the cells were used 1–2 days after attaining confluence, although a few experiments were performed 3–5 days postconfluence. The time postconfluence had no impact on the results. At the beginning of the experiment, the confluent monolayer was mechanically scratched with a plastic pipette tip to form four to five wounds perpendicular to the long axis of the slide. The wound width was 473 ± 93 µm (average ± SD).

Flow experiments.   BAECs were exposed to known levels of steady laminar shear stress using a standard parallel plate flow chamber, as described elsewhere (20, 28). Briefly, the flow chamber was connected via Masterflex PharMed tubing (Cole-Parmer Instrument, Vernon Hills, IL) to a recirculating flow loop. Flow in the loop was provided by a peristaltic pump drawing fluid (CSC medium) from a main reservoir, which subsequently passed into two buffer reservoirs to dampen pulsatility before entering the flow chamber. Flow exiting the chamber was recirculated back into the main reservoir. Fluid in the flow loop was maintained at 37°C by placing the main and buffer reservoirs in a temperature-controlled water bath. CO2 was bubbled directly into the main reservoir to maintain pH. Two levels of shear stress were studied: a relatively "high" shear stress of 19 dyn/cm2 typical of undisturbed flow zones within large arteries, and a relatively "low" shear stress of 3 dyn/cm2, typifying shear stress levels within disturbed flow regions. In all experiments, the flow was applied in a direction orthogonal to the wound.

Membrane fluidity and ion channel studies.   To study the impact of membrane fluidity on BAEC wound closure under both static and flow conditions, cell membrane fluidity was either decreased or increased. Decreasing BAEC membrane fluidity was accomplished by incubating the cells for 4 h at 37°C in 100 µM cholesterol, delivered as water-soluble cholesterol in methyl-{beta}-cyclodextrin (Sigma, St. Louis, MO) in CSC medium. Increasing membrane fluidity was accomplished by incubating the cells at 37°C in 30 mM benzyl alcohol (BA) (Sigma) in CSC medium for 10 min. Fluorescence recovery after photobleaching experiments have previously demonstrated that incubation in cholesterol, as described here, decreases the diffusion coefficient of the EC membrane by a factor of ~2.6 relative to control (untreated) cells, whereas BA treatment increases the diffusion coefficient by a factor of ~1.3 (7). Immediately following incubation in either cholesterol or BA, the culture medium was changed, the cells were wounded, and wound closure monitoring under either static or flow conditions was initiated. To probe the role that flow-sensitive K and Cl channels play in EC wound closure, pharmacological blockers of these ion channels were used. Flow-sensitive K and Cl channels were blocked by adding either 100 µM Ba2+ [in the form of barium chloride (BaCl2)] (Sigma) or 100 µM 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) (Sigma), respectively, to CSC medium. These pharmacological agents, which were present throughout the course of the experiment, have previously been demonstrated to completely block flow-sensitive ion channels in BAECs (19). The membrane fluidity and ion channel treatments had no visible cytotoxic effect on the cells.

Data acquisition and assessment of wound closure rates.   The wounded BAEC monolayer was placed on the stage of an inverted microscope (Nikon Eclipse TE300, Melville, NY) equipped with a charge-coupled device camera (Retiga, Q-Imaging, Burnaby, BC). Images of the wound under either static or flow conditions were acquired every 15 min for 24 h using Simple PCI C Imaging software (Compix, Cranberry Township, PA). In a few cases, complete wound closure was attained in less than 24 h, and the recording was halted at that point.

BAEC migration during the wound closure process was analyzed using image analysis software (Scion Image, version Beta 4.0.2; http://www.scioncorp.com). For each experiment, five cells were randomly selected along each edge of the wound. Cells undergoing division, death, or migration outside the field of view were excluded from the analysis. For each of the 10 selected cells, the cell contour at the end of every hour during the 24-h recording period was manually traced to yield the x- and y-coordinates of the cell center of gravity (x-, y-cog), as well as the cell area and perimeter. Cell migration velocity was computed as the total distance traversed divided by the time elapsed. The x- to y-velocity ratio (Vx/Vy) was computed as the absolute value of the ratio of x-direction migration velocity to that in the y-direction. Net x-direction migration was calculated as the net displacement in the x-direction (flow direction) over the time period of interest.

Statistical analysis.   Data are presented as means ± SE. Statistical analyses were performed by one-way ANOVA followed by Tukey's post hoc test. Differences in means were considered significant if P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effect of shear stress on BAEC wound closure.   Wounding of BAEC confluent monolayers provoked cell migration in the direction of the wound under both static and flow conditions. However, the migration patterns and cell migration velocities depended on whether or not flow was applied and on the level of shear stress to which the cells were exposed. Under static conditions, cells from both edges of the wound migrated equally to cover the wounded area (Fig. 1). Wound closure was complete within the 24-h recording period in all cases. Under a relatively high shear stress of 19 dyn/cm2, EC migration was more prominent along the upstream edge of the wound than along the downstream edge; therefore, cells along the upstream edge contributed more to wound closure than cells downstream (Fig. 1). Interestingly, exposure of BAECs to a low shear stress of 3 dyn/cm2 greatly retarded migration along both wound edges, leading to vastly incomplete wound closure (Fig. 1). These results suggest that EC wound closure rates under flow are highly sensitive to the magnitude of applied shear stress.



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  		Fig. 1. Representative photographs of endothelial cell (EC) wound closure progression under static conditions, high shear stress (19 dyn/cm2), and low shear stress (3 dyn/cm2).

 
To better understand the nature of the wound closure behavior shown in Fig. 1, we have examined the migration trajectories of individual BAECs for the different conditions studied (Fig. 2). Under static conditions, cells along both edges of the wound migrated into the wound with most of the movement in the x-direction (orthogonal to the wound) (Fig. 2). Under high shear stress, all cells along the upstream edge moved in the direction of flow into the wounded area and hence contributed to wound closure (Fig. 2). On the other hand, while some cells along the downstream edge moved against flow toward the wound, others moved in the direction of flow and thus retarded wound repair. Overall, fewer cells migrated away from the wound than into the wound during the 24-h monitoring period. Despite a clear impact of flow on the migration pattern, cell migration consisted of movement both in the flow coordinate direction (x-direction) and in the coordinate direction orthogonal to flow (y-direction). Under low shear stress, BAEC migration velocities were significantly lower (Fig. 2). In some cases, cells along both edges moved away from the wound initially before reversing direction and moving toward the wound. Under all conditions studied, individual cells maintained a relatively constant velocity throughout the 24-h recording period.



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  		Fig. 2. Bovine aortic EC (BAEC) x-y trajectories during the 24-h recording period under static conditions, high shear stress (19 dyn/cm2), and low shear stress (3 dyn/cm2). The movement of 5 cells along the downstream wound edge and 5 cells along the upstream edge is shown. All cells are assumed to originate at (0,0).

 
To more quantitatively assess BAEC wound closure under the different conditions, we determined the average cell migration velocity (as defined in MATERIALS AND METHODS) at various time points postwounding (Fig. 3). The migration velocity results provided quantitative confirmation of the migration patterns discussed above (cf. Fig. 1). More specifically, under static conditions, cells along both edges of the wound migrated at the same velocity to cover the wounded area. For instance, at the 12-h time point, the migration velocity for cells along the right edge (equivalent to cells along the upstream edge under flow conditions) and the left edge were virtually identical (13.6 ± 1.4 vs. 14.0 ± 1.3 µm/h, P > 0.05) (Fig. 3B). When the monolayers were subjected to high shear stress, cells along the upstream edge migrated significantly faster than cells along the downstream edge (13.2 ± 1.3 vs. 8.5 ± 0.8 µm/h at 12 h, P < 0.05) (Fig. 3B). Surprisingly, the migration velocities of cells along the upstream edge in response to high shear stress were not larger than those under static conditions (P > 0.05). However, the cells along the downstream edge migrated significantly more slowly than those under static conditions (P < 0.01). Finally, under low shear stress, the migration velocities of cells along both wound edges were the same (4.1 ± 0.3 µm/h upstream vs. 4.3 ± 0.4 µm/h downstream at 12 h, P > 0.05) but were significantly smaller than those under static conditions (P < 0.001) (Fig. 3B). Largely similar results were obtained at 6 h postwounding (Fig. 3A), as well as at the other time points studied (3 and 24 h) (data not shown), suggesting that the effect of flow on cell migration patterns is uniform in time.



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  		Fig. 3. Cell migration velocity along both wound edges at 2 different time points postwounding [6 h (A) and 12 h (B)] under static conditions, high shear stress (19 dyn/cm2), and low shear stress (3 dyn/cm2). The results are derived from measurements on 25 cells from 5 separate experiments. Values are means ± SE. {dagger}Statistically significant difference relative to corresponding static control, P < 0.05. *Statistically significant difference relative to corresponding "downstream" condition, P < 0.05.

 
As already described, BAEC migration during wound closure involves movement both parallel (y-direction) and orthogonal (x-direction) to the wound (cf. Fig. 2). Cell migration orthogonal to the wound is expected to be the primary contributor to wound closure; therefore, we examined the ratio of migration velocity in the x-direction to that in the y-direction (Vx/Vy) as a measure of whether or not flow impacts the directionality of cell migration. The x-direction migration velocity exceeded the y-direction velocity (i.e., Vx/Vy > 1) at every time point during the wound closure process (Fig. 4). Under high shear stress conditions, Vx/Vy was significantly higher for cells along the upstream than along the downstream edge (P < 0.05), suggesting that this level of shear stress enhances migration in the direction of flow for cells upstream more than cells downstream. Under low shear stress conditions, the same general trend was observed; however, the difference between upstream and downstream cells was smaller and did not attain statistical significance (P > 0.05).



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  		Fig. 4. Ratio of x-direction to y-direction velocity (Vx/Vy) along both wound edges at 2 different time points postwounding [6 h (A) and 12 h (B)] under static conditions, high shear stress (19 dyn/cm2), and low shear stress (3 dyn/cm2). The results are based on 25 cells from 5 separate runs. Values are means ± SE. *Statistically significant difference relative to corresponding "downstream" condition, P < 0.05.

 
We specifically examined net EC migration orthogonal to the wound (x-direction) as the most appropriate measure of wound closure progress. Under static conditions, the net x-direction migration 6 h after wounding (normalized by original wound width) was approximately the same for cells along both edges (0.15 ± 0.02 for right edge vs. 0.17 ± 0.02 for left edge, P > 0.05) (Fig. 5A). High shear stress drove cell migration, so the net x-direction migration upstream became significantly larger than that downstream (0.13 ± 0.01 vs. 0.03 ± 0.01, P < 0.001) (Fig. 5A). Under low shear stress conditions, there was no difference between the two wound edges (0.04 ± 0.01 upstream vs. 0.04 ± 0.01 downstream, P > 0.05), and the net x-direction migration was significantly lower than that observed in the absence of flow (P < 0.001) (Fig. 5A). The same behavior persisted and was even more pronounced at the 12-h time point (Fig. 5B).



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  		Fig. 5. Net x-direction BAEC migration along both wound edges at 2 different time points postwounding [6 h (A) and 12 h (B)] under static conditions, high shear stress (19 dyn/cm2), and low shear stress (3 dyn/cm2). Migration was normalized relative to original wound width. The results are derived from measurements on 25 cells from 5 separate runs. Values are means ± SE. {dagger}Statistically significant difference relative to corresponding static control, P < 0.05. *Statistically significant difference relative to corresponding "downstream" condition, P < 0.05.

 
Role of membrane fluidity and ion channels in regulating wound closure rates.   Changes in membrane fluidity and activation of flow-sensitive ion channels are among the fastest known EC responses to flow and have, therefore, been implicated in flow sensing (6, 9). We hypothesized that these responses impact EC wound closure. To test this hypothesis, we studied BAEC wound closure under both static and high shear stress (19 dyn/cm2) conditions in the presence of agents that either alter membrane fluidity or block flow-sensitive ion channels. As described in MATERIALS AND METHODS, BAEC membrane fluidity was either decreased by enriching the cell membrane with cholesterol or increased by using BA as a membrane fluidizing agent. As also detailed in MATERIALS AND METHODS, flow-sensitive K and Cl channels were blocked with Ba2+ and NPPB, respectively.

Under static conditions, neither the membrane fluidity agents nor the ion channel antagonists had a significant impact on BAEC migration velocity during the 24-h monitoring period postwounding (data not shown). Under high shear stress, however, the situation was considerably different. Preincubation of BAECs in cholesterol before application of shear stress significantly decreased cell migration velocity compared with nontreated cells; however, this effect was limited to cells along the upstream edge of the wound (Fig. 6). For instance, 12 h after wounding, upstream cells preincubated in cholesterol and exposed to flow migrated at a velocity of 5.6 ± 0.5 µm/h, whereas upstream control cells (no treatment) under the same shear stress migrated at 13.2 ± 1.3 µm/h (P < 0.001) (Fig. 6B). On the other hand, the equivalent numbers for downstream cells were 5.9 ± 0.5 and 8.5 ± 0.8 µm/h, respectively (P > 0.05) (Fig. 6B). The same trend generally persisted at the 3-, 6-, and 24-h time points. However, between 12 and 24 h, the effect of cholesterol on migration velocity under high shear stress began to diminish somewhat, although it remained statistically significant. More specifically, at the 24-h time point, the migration velocity for upstream cells was 10.1 ± 1.2 µm/h for cholesterol-treated cells and 14.9 ± 1.2 µm/h for control (no treatment) cells (P < 0.05) (data not shown). These results suggest that, while decreasing membrane fluidity has no impact on wound closure rates in the absence of flow, it retards wound closure in the presence of flow through a specific effect on the cells along the upstream edge of the wound, which are the cells that are most responsive to flow. Interestingly, BA did not significantly impact cell migration velocity at any time point (Fig. 6), suggesting that increasing membrane fluidity of cells does not augment wound closure in response to flow. Blocking flow-sensitive K channels with Ba2+ or flow-sensitive Cl channels with NPPB generally had no significant impact on BAEC migration velocity under flow. However, at the 24-h time point, ion channel blocking appeared to reduce the migration velocity of BAECs along the upstream edge of the wound compared with control cells (data not shown). It remains unclear whether this effect is a specific ion channel effect or whether it is a nonspecific consequence of long-term cell exposure to the pharmacological agents used to block the ion channels.



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  		Fig. 6. Effect of cell membrane fluidity and flow-sensitive ion channels on BAEC migration velocity along both wound edges under a shear stress of 19 dyn/cm2. Two different time points postwounding [6 h (A) and 12 h (B)] are presented. Each treatment bar represents measurements on 15–20 cells from 3–4 runs. Control (untreated) results are based on 25 cells from 5 separate runs. Values are means ± SE. {dagger}Statistically significant difference relative to control condition along the same wound edge, P < 0.05. *Statistically significant difference relative to corresponding "downstream" condition, P < 0.05. BA, benzyl alcohol; NPPB, 5-nitro-2-(3-phenylpropylamine)benzoic acid; BaCl2, barium chloride.

 
In addition to cell migration, cell spreading is an important component of the wound closure process. To quantify the extent of cell spreading, cell area was determined during the 24-h experimental period. Under both static and high shear stress (19 dyn/cm2) conditions, the general tendency was a progressive increase of cell area with time, indicating gradual and continual cell spreading (Fig. 7). Under static conditions, the cells along both wound edges exhibited a similar pattern of cell spreading with limited impact of any of the membrane fluidity or ion channel treatments (Fig. 7). Under high shear stress, however, treatment with cholesterol, NPPB, and Ba2+ decreased cell area along both wound edges compared with control (untreated) cells (Fig. 7). To further illustrate these findings, Table 1 depicts the 24-h average ratio of cell area for each of the treatments to the area for control (untreated) cells under both static and high shear conditions. Under static conditions, the ratios were near unity (range 0.74–1.30) for all of the treatments, suggesting minimal effect of the treatments on cell spreading. However, under high shear, the ratios for the cholesterol, NPPB, and BaCl2 treatments were considerably smaller (range 0.46–0.64), demonstrating inhibition of cell spreading. In contrast, BA did not appear to have a significant impact on spreading (range 0.82–0.93). Taken together, the migration and area change results (Figs. 6 and 7 and Table 1) suggest that, while changes in membrane fluidity under flow impact both BAEC migration velocity and spreading during wound closure, interfering with ion channel activation affects cell spreading to a much greater extent than migration velocity.



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  		Fig. 7. Effect of cell membrane fluidity and flow-sensitive ion channels on BAEC spreading. The evolution of cell area along both wound edges under static conditions and a shear stress of 19 dyn/cm2 are shown. The symbols are as follows: {bullet}, control (no-treatment) cells; {blacksquare}, cells treated with cholesterol; {blacktriangleup}, cells treated with BA; {blacklozenge}, cells treated with NPPB; and x, cells treated with Ba2+. Each data point for the treatment groups represents measurements on 15–20 cells from 3–4 separate runs, whereas data for control cells represent measurements on 25 cells from 5 separate runs. Values are means ± SE.

 

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  		Table 1. Ratio of average cell area for each of the treatments to the area for control (untreated) cells under both static and "high" shear conditions

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Shear stress is known to intricately regulate EC structure and function (6, 9, 12). Within this context, high shear stress leads to a cellular phenotype that is largely anti-inflammatory and antiatherogenic, whereas low shear stress appears to elicit EC dysfunction and to induce a proinflammatory and proatherosclerotic profile (25). One of the primary findings of the present work is that BAEC wound closure under flow is critically sensitive to the shear stress level to which the cells are exposed. More specifically, low levels of shear stress (3 dyn/cm2) lead to dramatically retarded wound closure rates relative to high shear stress levels (19 dyn/cm2). These results suggest that the EC dysfunction induced by low levels of shear stress includes the induction of signaling pathways that are associated with limited cell migration and hence with reduced wound closure capacity. Interestingly, BAECs not exposed to flow exhibit wound closure rates that are comparable to (and may be slightly faster than) those associated with high shear stress.

A scenario in which EC wound closure is particularly relevant is following endovascular stent deployment. We have recently demonstrated that the presence of a stent within a vascular segment significantly disturbs the flow field and results in regions of high shear stress in close proximity to regions of low shear stress (27). Significantly, the extent and nature of stent-induced flow disturbance depend, in a complex fashion, on stent design (27). Therefore, if the present findings demonstrating that low levels of shear stress lead to prominent retardation of EC wound closure also occur in vivo, then the results would suggest that stent designs that give rise to local regions of low shear stress would lead to incomplete wound closure and would, therefore, be more likely to result in restenosis. In support of this construct, recent in vivo data suggest that in-stent restenosis preferentially develops in arterial regions exposed to low shear stress (30).

Our present results demonstrating that high shear stress levels lead to higher EC migration rates than low shear stresses are consistent with the results of Albuquerque et al. (4) and those of Urbich et al. (29) on unsheared HUVECs. Interestingly, Albuquerque et al. (4) reported that preshearing the cells at 12 dyn/cm2 for a period of 18 h alters the wound closure dependence on shear stress level. However, direct comparison of our results with those of Albuquerque et al. is difficult for several reasons. First, the wound in the study of Albuquerque et al. was oriented parallel to the applied flow, whereas the wound in the present study was orthogonal to the direction of flow. Second, the behavior of HUVECs may be somewhat different from that of BAECs used in the present study. Indeed, Albuquerque et al. observed differences in wound closure behavior between HUVECs and HCAECs. Finally, the preshearing may have led to a situation where the cell starting point in the two studies may not have been identical. Indeed, there is extensive evidence that exposing ECs to flow for an extended period of time alters cell phenotype (9, 25).

In the present study, we have opted to perform the experiments on cells that had not previously been subjected to flow. In principle, flow preconditioning of the cells would appear to lead to a more physiological initial condition. However, this may not necessarily be the case, especially for studies focusing on wound closure. Endothelial wounding is most likely to occur subsequent to advanced atherosclerosis or to the deployment of endovascular devices such as stents. These pathological conditions occur preferentially in regions exposed to multidirectional and highly "disturbed" flow. On the other hand, arterial regions exposed to unidirectional and nondisturbed flow are generally spared and will, therefore, not be expected to correspond to regions of endothelial wounding. Therefore, to reproduce the physiological situation for wound healing studies, it would be essential to precondition ECs with the types of disturbed flow to which these cells are exposed in vivo. This would be impossible to implement in standard parallel-plate flow chambers or cone-and-plate viscometers and would necessitate the development of specialized flow systems. One might think that preconditioning the cells with undisturbed flow in a parallel plate flow chamber might be "better than nothing"; however, it is not at all clear that this would be the case in light of accumulating evidence that EC phenotype is intricately sensitive to the type of flow to which the cells are exposed (6). An additional and important point in this regard is that we believe that flow preconditioning would not have affected the present results. The rationale for this statement is that cell migration velocities and patterns during the last few hours of the 24-h flow period (at which point the cells were presheared) were not different from those during the first few hours of flow.

Hsu et al. (16) investigated the impact of flow disturbance downstream of a step on BAEC wound closure. Their results demonstrated considerably retarded wound closure in the vicinity of flow reattachment (at the end of the flow separation zone downstream of the step) relative to regions where the flow was undisturbed. In light of the fact that the flow reattachment region is associated with low levels of shear stress, these results would be largely consistent with the present study. On the other hand, Hsu et al. determined that wound closure under static conditions was slow. This is in contrast to the present results demonstrating that wound repair in the absence of flow is rapid. The reason for the difference remains to be determined; however, we have observed that wound closure under static conditions depends on the temperature at which the cells are maintained. Therefore, we have carried out all our static and flow experiments at 37°C.

The mechanisms by which shear stress regulates EC wound closure remain to be elucidated; however, recent studies have provided insight into some of the pathways involved. Several studies have implicated {alpha}5- and {beta}1-integrins (2, 29), as well as VE-cadherin (3). Hsu et al. (16) have demonstrated that Rho signaling plays an important role, especially under flow, and that tyrosine phosphorylation is important for cell migration under disturbed flow conditions, as well as for migration of cells along the upstream edge of the wound in undisturbed flow. These various pathways may be connected, however, as both Rho activation and tyrosine phosphorylation have been suggested to regulate the function of cell-cell junctions and focal adhesions (16). More generally, Rho and other small GTP-binding proteins have been shown to play an important role in regulating cell migration (1, 26).

In the present study, we have demonstrated that decreasing cell membrane fluidity by augmenting membrane cholesterol content significantly decreases both cell migration velocity and cell spreading under flow and hence retards wound closure. Interestingly, the cell migration effect only applied to cells along the upstream edge of the wound, whereas the cell spreading effect occurred along both wound edges. Alterations in membrane fluidity are among the fastest known EC responses to flow and have been implicated in initial flow sensing and in the regulation of flow-induced signaling in ECs (6, 9). Indeed, changes in membrane fluidity have been shown to regulate the activation of G proteins by flow (14). More generally, cell membrane cholesterol content has been shown to impact the organization and function of lipid caveolae, which are intricately involved in downstream signaling, including cell migration (11). Interestingly, increasing BAEC membrane fluidity using BA had no impact on cell migration. This, however, may not be very surprising, in light of recent findings that cell migration is enhanced only within an optimal range of membrane microviscosity (13). Viscosity levels that are below or above this range result in reduced cell motility. If the reduction in EC migration with reduced cell membrane fluidity occurs in vivo, then the present findings may also have some clinical implications. There is evidence that hypercholesterolemia is associated with increased EC membrane cholesterol content (17, 24); thus the present results suggest that wound closure in hypercholesterolemic individuals would occur significantly more slowly than in normocholesterolemic individuals.

Activation of flow-sensitive K and Cl channels in ECs also occurs very rapidly, and these ion channels have been proposed as candidate mechanosensors (6, 9). Furthermore, blocking flow-sensitive ion channels has been shown to impact specific gene and protein flow responses (22, 28). The present study has established that flow-sensitive ion channels do not have a significant effect on BAEC migration during wound closure, but that they inhibit cell spreading under flow and hence may have an effect on overall wound closure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This work was supported in part by a Biomedical Pilot Initiative grant from the Charles E. Culpeper Foundation.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
The authors acknowledge the technical help of Dr. Deborah K. Lieu.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Gojova, Dept. of Mechanical and Aeronautical Engineering, Univ. of California, Davis, One Shields Ave., Davis, CA 95616 (E-mail: agojova{at}ucdavis.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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