Physiology 20: 43-53, 2005;
doi:10.1152/physiol.00040.2004
1548-9213/05 $8.00
Physiology, Vol. 20, No. 1, 43-53,
February 2005
© 2005 Int. Union Physiol. Sci./Am. Physiol. Soc.
REVIEW
Molecular Control of Cytoskeletal Mechanics by Hemodynamic Forces
Brian P. Helmke
Department of Biomedical Engineering and Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia
helmke{at}virginia.edu
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Abstract
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The endothelium at the interface between blood and tissue acts as a primary transducer of local hemodynamic forces into signals that maintain physiological function or initiate pathological processes in vessel walls. Rapid intracellular spatial gradients of structural dynamics and signaling molecule activity suggest that mechanical cues at the molecular level guide cellular mechanotransduction and adaptation to shear stress profiles.
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Introduction
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Mechanical forces are implicated in the regulation of an increasing number of physiological and pathological processes in the cardiovascular, musculoskeletal, pulmonary, and neural systems. Hemodynamic forces acting on the blood vessel wall include frictional shear stress from flowing blood, hydrostatic pressure generated by the heart pump, and wall stretch in the circumferential and longitudinal directions. The endothelium serves as the primary interface between flowing blood and the underlying tissue, and its function in maintaining vascular homeostasis varies regionally along the vascular tree according to the local hemodynamic force profile (7, 20). This suggests that the spatiotemporal distribution of fluid shear stress acting on the endothelium promotes regional heterogeneity that controls perfusion distribution by release of vasoactive substances such as nitric oxide and restricts inflammation by downregulation of chemoattractant production and leukocyte adhesion receptor expression.
Both in vivo and in vitro measurements demonstrate that endothelial cells adapt their structure, signaling activity, and gene and protein expression profiles to changes in hemodynamic shear stress. Such adaptation seems critical to prevent dysfunction associated with proinflammatory events in vivo, but several key questions remain to be addressed. How do cells sense spatial and temporal cues in their mechanochemical environment? What molecular mechanisms guide polarization and balance the responses to mechanical and chemical cues? How do specific cellular behaviors result from cross-talk among common downstream signaling networks?
Changes in the shear stress profile acting on the apical surface of endothelial cells result in initiation of signaling at multiple locations within the cell (25). Ion channels, G proteins, and transmembrane adhesion receptors are activated within seconds after onset of shear stress (6). Because these early immediate events occur almost simultaneously at the apical, junctional, and basal surfaces of the cell, it is likely that cell structure plays an important role in transmitting mechanical forces throughout the cell to specific locations to promote assembly and activation of signaling molecule complexes. Thus spatial gradients in mechanical properties at the subcellular length scale may guide the development of cell responses to extracellular mechanical stimuli. At the molecular level, intracellular mechanical gradients may alter the distribution of structural dynamics in cytoskeleton-associated components in ways that both trigger sensing-related signals at short time scales and provide polarization cues for adaptation at longer time scales. This review explores this hypothesis by suggesting potential molecular control mechanisms in structural components of endothelial cells that integrate mechanochemical cues from the extracellular environment.
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Structural and Mechanical Adaptation to Hemodynamic Forces
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Endothelial cells in vivo exhibit an elongated shape that is oriented parallel to the direction of blood flow in relatively straight regions of conducting arteries, and cytoskeletal filaments in these cells are oriented parallel to the major axis of cell elongation (11, 19, 41). In contrast, cells in regions near arterial bifurcations or high curvature have a more polygonal shape, and cytoskeletal filament organization is more random. These regions of the artery wall correspond to locations of complex flow dynamics and upregulation of inflammatory processes that lead to increased risk of atherogenesis (20, 58). Because endothelial cells in culture reproduce the morphological adaptations to flow that are hypothesized to occur in vivo, in vitro models have enabled examination of structural and mechanical behaviors that occur as cells adapt to the regional shear stress profile.
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Flow-Induced Adaptation of Cell Structure
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Early studies of endothelial cell adaptation imposed steady, unidirectional flow on a cell monolayer in a glass tube (54), a parallel plate flow chamber (15), or a cone-and-plate viscometer (11). Each device is capable of imposing a fully developed laminar Poiseuille flow in which the average wall shear stress (
w) acting on the cell layer is computed analytically (Table 1
). Several approaches have also attempted to mimic the more complex flow profiles present near arterial bifurcations and regions of high curvature (FIGURE 1). Temporal gradients have been imposed by varying the rate of flow onset ("ramp") (81) or by superimposing sinusoidal flow on mean flow rates ranging from zero ("reversing oscillatory flow") to arterial levels ("pulsatile flow") (29). Varying either the height or width within a flow chamber produces a spatial gradient of wall shear stress ("disturbed flow") (10). More recently, adaptation of the cone-and-plate device enabled reproduction of arterial flow waveforms (2). It is important to note that all of these flow profiles are laminar in nature. In other words, the Reynolds number (Table 1), which is defined as the ratio of fluid inertial forces to viscous forces, is much smaller than unity. Although endothelial gene expression profiles also adapt to turbulent flow (20), laminar profiles exist nearly everywhere in the vascular tree in vivo.
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Table 1. Wall shear stress, Reynolds number, and characteristic length scales for devices commonly used to apply shear stress on cells in vitro
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FIGURE 1. Schematic graphs of common in vitro shear stress profiles
Some data are from Refs. 2 (E) and 10 (F). A schematic of streamlines indicates the existence of a standing vortex, representing disturbed laminar flow.
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All three major cytoskeletal networks adapt their structure as cell shape elongates and reorients parallel to the direction of flow (19). During 24-h exposure to steady unidirectional shear stress, dense peripheral bands of F-actin bundles disassemble and aligned stress fiber bundles assemble (FIGURE 2). Perinuclear bundles of vimentin intermediate filaments become less prominent and are repositioned on average to the downstream side of the nucleus, and the intermediate filament network elongates with the cell shape (FIGURE 2). Microtubules also reorient with the major axis of cell shape elongation. Transient repositioning of the microtubule organizing center (MTOC) in the upstream region of the cytoplasm has been observed (19, 47), but the MTOC returns to random positions (19) or even moves downstream of the nucleus (75) at longer time scales after adaptation (FIGURE 3). This directional reorganization depends on the state of confluence of the endothelial monolayer (48) and the structure of the substrate (63). Relocation of the MTOC toward the leading edge with respect to the nucleus also occurs in migrating endothelial cells during scratch wound healing (23), consistent with reports of MTOC position in other migrating cell types (63). Indeed, both single endothelial cells (42) and cells in confluent monolayers (2) migrate in the direction of steady laminar flow at an average speed of 0.2–0.4 µm/min, and cells in confluent monolayers exposed to more complex flow profiles tend to migrate toward regions of low spatial gradients of shear stress (69). Thus the directional positioning of the MTOC is related to the establishment of cell polarity within the monolayer during adaptation to shear stress.
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FIGURE 2. Cytoskeletal adaptation to steady unidirectional shear stress
Actin stress fibers (red) and vimentin intermediate filaments (green) in bovine aortic endothelial cells exposed to unidirectional shear stress (13 dyn/cm2, left to right) for 24 h in a glass capillary tube. A few cells in this field expressed green fluorescent protein (GFP)-vimentin (green) after transient transfection. After 24 h, cells were fixed, F-actin was labeled with rhodamine-conjugated phalloidin (red), and DNA was labeled with Hoechst 33258 (blue).
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FIGURE 3. Schematic representation of most likely microtubule organizing center positions with respect to the nucleus in endothelial cells in vitro
A: in individual endothelial cells migrating alone or in a subconfluent cell layer, the microtubule organizing center (MTOC; dots) is positioned anterior to the nucleus. Arrow indicates migration direction. B: in confluent endothelium under static fluid conditions, the location of the MTOC is random. C: after exposure to unidirectional steady laminar shear stress for 1–6 h, the average position of the MTOC is upstream from the center of the nucleus. Arrow indicates direction of shear stress. D: after >12 h exposure to shear stress, the MTOC is repositioned with a bias toward the downstream side of the nucleus, consistent with the average direction of cell migration under flow. Arrow indicates net direction of cell migration; direction of shear stress is the same as in C.
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In addition to adaptation of cytoskeletal filament networks within endothelial cells, cytoskeleton-associated structures linked to the basement membrane and intercellular adhesion sites remodel during chronic exposure to unidirectional laminar shear stress. The projected shapes of focal adhesion sites, measured by tandem-scanning confocal microscopy (80), elongate and align parallel to the flow direction on a time scale similar to that of actin cytoskeleton remodeling. For endothelial cell monolayers plated on vitronectin, both the rate of shape elongation and the rate of reorientation correlate with the shear stress magnitude. However, the rate of remodeling is significantly slowed in cells plated on gelatin (8, 80), indicating that focal adhesion remodeling depends on the extracellular matrix composition. The number of focal adhesion sites per cell decreases during remodeling, but the average area per focal adhesion site increases (8). Thus the total area associated with cell adhesion to the basement membrane remains approximately constant. These data suggest the existence of feedback mechanisms that maintain a force balance between the cell and the extracellular matrix, although experimental measurements of traction force to confirm this hypothesis have not been performed during adaptation to shear stress.
Intercellular junctional complexes include tight junctions, adherens junctions, gap junctions, and homotypic interactions between platelet-endothelial cell adhesion molecule type 1 (PECAM-1). Each of these junction types is remodeled following the onset of steady unidirectional laminar shear stress. Adaptation of gap junction assembly alters cell-cell communication (10) and may be regulated by both submembrane cytoskeleton (3) and cytoplasmic cytoskeletal filament networks (37). Tight junctions modulate endothelial barrier permeability, and shear stress induces an increase in hydraulic conductivity across both cultured endothelial cell monolayers (66) and microvascular networks in vivo (82). Decreased expression of the tight junction protein occludin has been implicated (9), but the exact mechanisms remain unknown. More evidence is available to describe flow-mediated remodeling of adherens junctions, which include the structural proteins 
- and ß-catenin, plakoglobin, and vascular endothelial (VE-) cadherin. As F-actin-dense peripheral bands dissociate after onset of steady unidirectional shear stress, VE-cadherin and associated catenins are redistributed from a continuous border to punctate spots along cell edges. After 48 h, these proteins form short dashes colocalized with the ends of newly formed F-actin stress fibers that form parallel to the flow direction (51). After 96 h, a continuous border is again formed by -catenin that closely resembles the distribution of ß-catenin from straight sections of rabbit arteries (52). The intercellular adhesion molecule PECAM-1 is phosphorylated after onset of shear stress, but its spatial distribution near cell edges appears unchanged. This suggests that homophilic PECAM-1 interactions between cells experience increased tension as adherens junctions undergo remodeling. Tension applied directly to PECAM-1 using magnetic beads induces recruitment of the signaling molecule SHP-2 to cell junctions, leading to downstream activation of the extracellular signal-regulated kinases 1 and 2 (55). Thus it is likely that PECAM-1 serves as a primary mechanotransduction molecule, but its role in structural stability of the intercellular junctions is not clear.
Endothelial cell structure adapts differently in arterial regions that are more susceptible to atherosclerosis (20). The less-elongated cell shape is similar to that measured in endothelial cell monolayers grown in static fluid culture (11, 19, 41). The actin cytoskeleton contains relatively few stress fiber bundles, and existing microfilament bundles remain randomly oriented or loosely organized into peripheral bands. The intermediate filament and microtubule networks also do not exhibit a preferred filament orientation.
Endothelial cell adaptation has also been measured in response to the in vitro models attempting to mimic more closely the complexity of flow profiles in vivo (FIGURE 1
). Endothelial cells exposed to sinusoidal oscillating flow take on a shape and cytoskeletal structure that resembles cells in static culture (29). In contrast, if the same sinusoidal shear stress waveform is superimposed on a time-averaged wall shear stress equal to that in large arteries (40 dyn/cm2) to create pulsatile flow, then cells elongate and align with the flow direction in the same manner as if flow were steady (29). Similar results are obtained after imposing a shear stress waveform that more closely mimics the average shear stress waveform in vivo (2). In a parallel-plate flow chamber containing a step expansion in height, cells in the region just downstream of the step with high shear stress spatial gradient remain polygonal in shape and cytoskeletal structure, whereas cells in a region further downstream elongate and reorient in a manner consistent with the unidirectional steady parabolic velocity profile that exists there (10). Together, the results of these models indicate that structural adaptation is sensitive to both the time-averaged magnitude and spatial gradients of wall shear stress.
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Quantitative Measurements of Mechanical Properties
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The mechanical properties of the cell cytoskeleton are most commonly represented by the behavior of a linear viscoelastic polymer network. The protein concentration, degree and nature of chemical cross-linking among polymer chains, and physical entanglements among filaments determine the elasticity and the viscous flow properties of the polymer gel (45). Shearing gels of pure cytoskeletal protein polymers in a cone-and-plate rheometer reveals that an actin gel (2 mg/ml protein) without cross-linking proteins has a shear modulus of 2,830 dyn/cm2, at least an order of magnitude stiffer than tubulin gels (34). The gel ruptures and begins to flow like a viscous fluid when strain exceeds 20%. It is important to note that shear moduli and creep rate are sensitive to filament length, degree of cross-linking, polymerization conditions, and monomer storage conditions (36, 84, 85). Vimentin gels have a shear modulus of 320 dyn/cm2 and exhibit strain-hardening behavior, i.e., stiffness increases with increasing deformation. In addition, a vimentin gel can undergo stretch to nearly twice its original dimensions without rupturing. Interestingly, a vimentin-actin copolymer gel retains the strain-hardening properties of pure vimentin, but the stiffness at each strain magnitude is higher than for vimentin alone (35). This suggests that a composite material of three cytoskeleton networks exhibits properties that are not dominated by one component, and it is likely that structural interactions with cross-linking, bundling, and scaffold proteins also contribute to the overall mechanical properties of the cell. Indeed, in vitro measurements on gels that include filamin A demonstrate that orthogonal cross-linking of actin filaments is required to establish gel-like properties (50).
How do these measurements relate to mechanical properties of intact cells? Adherent endothelial cells exist in a state of prestress (79) in which forces within the cytoplasmic cytoskeleton networks are balanced both by a cortical tension resulting from local membrane curvature (62) and by traction forces exerted on the substrate (77). To a first approximation, endothelial cells have been represented as linear viscoelastic materials whose material properties are primarily determined by the cytoskeletal polymer gels. The effective Young’s modulus of endothelial cells in static culture is 750–1,500 dyn/cm2, as measured by micropipette aspiration, and cytoplasmic viscosity lies in the range of 3–7 x 104 poise (60, 70). After adaptation to steady unidirectional shear stress for 24 h, many cells retained their elongated shape even after gentle detachment from their substrate (60). Both the elastic modulus and the viscosity of these cells were increased by approximately twofold relative to cells maintained under static fluid conditions. It is likely that this is primarily due to reorganization of the F-actin network into stress fibers, since cytochalasin B treatment significantly decreased the elasticity and viscosity (61). A shortcoming of these measurements is that the displacement of cells into the micropipette tip is quite large with respect to physiological cellular deformation; however, these remain the only direct measurements of changes in mechanical properties after adaptation to physiological shear stress.
Another method for measuring intracellular mechanical properties is laser tracking microrheometry (LTM), which is based on tracking endogenous cytoplasmic granules in living cells (86). Since these lipid-rich particles are embedded in the cytoskeletal gel, movement depends primarily on the local mechanical properties. Mathematical analysis of particle trajectories yields an elastic modulus of 721 dyn/cm2 and a viscous modulus of 382 dyn•s/cm2. Note that measurements using intracellular markers yield cytoplasmic viscosity about two orders of magnitude smaller than that measured near the cell surface (1), perhaps due to the cortical cytoskeleton and linkages to adhesion receptors. Although LTM measurements have not been completed during or after adaptation to hemodynamic shear stress, these measurements have the advantage that they are noninvasive; integrin clustering or large imposed deformations do not affect the results.
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Constitutive Models of Cytoskeleton and Cell Mechanics
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Experimental measurements of material properties of cytoskeleton polymers and intact cells suggest several models to predict the intracellular mechanical environment. Early models proposed that the cell could be viewed at the macroscale as several homogenous compartments, which included a combination of cytoplasm, sub-membrane cortex, plasma membrane, and nucleus. Based on the whole cell micropipette aspiration measurements, endothelial cells were first modeled as a homogeneous viscoelastic solid, which enabled the characterization of increased stiffness and viscosity discussed above (60, 61, 70). The standard solid model consists of a combination of elastic springs, in which displacement at equilibrium is proportional to applied force, and viscous dashpots, in which shear rate is proportional to applied force. This model has been applied to a variety of biological tissues to predict creep behavior after application of mechanical stress (17), but intracellular molecular responses cannot be represented easily. To explain transmission to the basal lamina of fluid shear force acting at the luminal surface of endothelial cells, Fung and Liu (18) proposed that the endothelial cell behaves as a thin shell under tension encasing a fluid-like interior. Under this "tension field theory," the plasma membrane serves to maintain the cell shape under the influence of hemodynamic shear forces. Although these models represent the first steps toward explaining the passive mechanical behavior of endothelial cells under applied loads, neither model considers active responses of living cells or evaluates the validity of assuming that the cell is a homogeneous continuum.
Models that incorporate structural details at a subcellular length scale may more accurately explain transmission of hemodynamic forces throughout the cell. This idea is important for a "decentralization model," which proposes that mechanotransduction results from the spatial integration of biochemical signals generated at multiple sites within the cell almost simultaneously (6). The decentralization hypothesis suggests that the cytoskeleton provides mechanical connections responsible for force transmission to sites remote from the apical surface. One possible structural model proposes that the cytoskeleton behaves as a network of cables under tension, representing actin stress fibers, and compression-resistant struts, representing microtubules. This "tensional integrity," or tensegrity, model predicts cytoskeletal reorientation and stiffening under an applied stress in a manner consistent with experimental measurements (76). In this model, the cell exists at rest in an equilibrium state of prestress, and perturbation by an extracellular applied force results in redistribution of structural element positions. Prediction of mechanical defects observed experimentally in vimentin-deficient cells (13, 78) is possible by extending the model to include intermediate filament cables that are not initially under tension but contribute to cell stiffness during large deformations (78). This strain-hardening behavior in the intermediate filament cytoskeleton is likely to prevent excessive deformation of the cell at finite strains that would otherwise rupture the actin cytoskeleton (34, 78). Satcher and Dewey (59) propose a unit cell open-lattice model for the cytoplasmic actin network that resembles a foam-like material. This model also predicts force transmission through a cytoplasm that has a bulk elastic modulus on the order of 105 dyn/cm2, which is somewhat higher than many experimental measurements. Although both the tensegrity model and the foam model are consistent with redistribution of intracellular forces, neither model considers the impact of molecular control mechanisms or spatial heterogeneity that may be important to directional sensing and polarized responses to hemodynamic stimuli. It is also worth noting that some experimental measurements do not show displacement at sites far away from large imposed deformations (24), indicating that either mechanical effects are not always transmitted throughout the cytoplasm or that other active mechanisms compensate to stabilize the cytoskeletal network elsewhere in the cell.
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Rapid Intracellular Structural Dynamics Induced by Shear Stress
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Living cells exhibit dynamic mechanical behaviors such as migration, polarization, and division on a time scale of minutes to hours. Since the above measurements and models consider only steady-state mechanical properties, they are not able to give insight into the molecular mechanisms modulating active adaptation to hemodynamic forces. Characterization of morphology in fixed cell monolayers only gives spatial and temporal averages of intracellular signaling and remodeling; sequences of events involved in mechano-transduction are not captured. High-resolution live-cell microscopy enables measurement of structural dynamics at time and length scales relevant to molecular mechanisms that control cellular adaptation. Increasing evidence suggests that force acting at the cell surface results in intracellular redistribution of mechanical responses that depend on local structure (25, 31). Although it remains unclear how intracellular mechanical gradients result in functional consequences at the cellular-to-multicellular length scale, spatial focusing of strain or stress at the subcellular length scale is likely to provide cues that directly regulate cell behavior. Mechanical focusing of this nature can only be measured by observing structural dynamics in living cells while controlling changes in applied extracellular force.
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Strain Focusing in the Intermediate Filament Cytoskeleton
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Early time-lapse microscopy observations of endothelial cell monolayers indicated that onset of unidirectional steady laminar flow enhanced the dynamic movement of cytoplasmic organelles within seconds (unpublished results). Interestingly, similar responses to shear stress have been measured in neutrophils (49). If cytoskeleton filament networks are the major contributors to intracellular mechanical properties, then such behavior suggests that forces acting at the cell surface cause rapid changes in intracellular force transmission. Indeed, application of force to the cell surface by twisting magnetic beads bound to integrin receptors results in spatial redistribution of intracellular stress computed from displacement of fluorescence-labeled actin filaments or mitochondria (31). These observations lead to the hypothesis that changes in hemodynamic forces are also transmitted to cytoplasmic locations away from the cell surface via the cytoskeleton.
Green fluorescent protein (GFP) fused to vimentin (GFP-vimentin) serves as a convenient marker of cytoskeletal morphology (FIGURE 2) in living endothelial cells, since it distributes to the endogenous intermediate filament network (87). High-resolution four-dimensional fluorescence microscopy revealed both constitutive "wiggling" of the intermediate filament network and a heterogeneous displacement field within minutes after onset of unidirectional steady laminar shear stress (26). Flow-induced displacement magnitude increased with height in the cell and was largest on average in downstream regions of the cytoplasm (28). Individual filament segments moved by as much as 1 µm, and most displacement occurred within the first few minutes after flow onset. Spatial mapping of intracellular mechanical strain revealed local strain concentrations near the basal cell surface (27). Comparison with the intermediate filament skeleton suggested that strain focusing occurred near locations consistent with cytoskeletal interaction with focal adhesion sites or intercellular junctions.
Intermediate filaments may physically interact indirectly with focal adhesion sites either by entanglement of the two polymer networks near focal adhesion sites or via cross-links to actin microfilaments, which insert into adhesion protein scaffolds (65). Several actin-bundling proteins have been implicated in tethering intermediate filaments to actin filaments. One of these, fimbrin, also appears to play a role in assembling the vimentin cytoskeleton in an adhesion-dependent manner (4), suggesting that intermediate filaments may interact more directly with focal adhesion sites. In fact, vimentin intermediate filaments are anchored to 6ß4-integrins in microvascular endothelial cells by plectin (30), an intermediate filament-associated binding protein more commonly described as a key component of epithelial hemidesmosomes. Because 6ß4 binds laminins, a primary extracellular matrix component in the normal basement membrane underlying vascular endothelium, these interactions are consistent with the hypothesis that intermediate filaments are recruited to provide mechanical stability during physiological adaptation of the microfilament network to hemodynamic shear stress. Vimentin intermediate filaments also interact with Vß3 vitronectin receptors (72), which redistribute to the upstream edges of endothelial cells after onset of steady unidirectional shear stress and may guide reorientation of actin stress fibers (21). Although these behaviors have yet to be measured in living cells, it is likely that the rapid strain focusing observed in the intermediate filament cytoskeleton following changes in shear stress represents spatial cues involved in cellular mechanical adaptation.
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Flow-Induced Dynamics in the Actin Cytoskeleton
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Intermediate filaments provide a convenient indicator of cytoplasmic movements during changes in shear stress, but the actin cytoskeleton may more closely reflect mechanical changes that are directly involved in flow-mediated mechanotransduction. Visualization of GFP-actin in living endothelial cells demonstrates that shear stress enhances migration of single endothelial cells parallel to the flow direction, a behavior termed "mechanotaxis" (42). Within 10 min after onset of unidirectional steady laminar shear stress, actin ruffles were formed preferentially in the downstream direction, and recruitment and phosphorylation of focal adhesion kinase (FAK) in the ruffles indicated that new focal adhesion sites were formed, stabilized during cell migration, and dissembled at the trailing edge of cells.
Although isolated endothelial cells may be important for understanding mechanically induced migration and endothelial wound healing, new observations of GFP-actin in cells within a confluent monolayer may represent a more physiological behavior. In transiently transfected monolayers, small GFP-actin-containing ruffles are often observed 30–60 s after onset of unidirectional steady laminar shear stress (FIGURE 4A). These ruffles disappear within a few minutes and precede lamellipodial extension consistent with downstream migration during exposure to flow. Interestingly, these rapid transient ruffles are usually directional, but their orientations appear to be randomly directed and can even occur in the upstream direction (FIGURE 4A, arrows). Preliminary observations suggest that ruffling occurs in directions that stabilize physical contact with adjacent cells in the monolayer, but this hypothesis remains to be explored.
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FIGURE 4. Flow-induced dynamics indicated by GFP-actin expressed in a confluent monolayer of bovine aortic endothelial cells
Shear stress at 13 dyn/cm2 is shown from left to right. A: transient edge ruffles along the upstream edge of the cell (arrowheads) are most prominent 3 min after onset of shear stress. B: displacement of microfilaments near the basal cell surface. False-colored images from just before (red) and 2 min after (green) onset of shear stress are superimposed to highlight displacement; yellow indicates zero displacement. Positions are normalized to a stationary fiducial marker on the coverslip (26).
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In addition to initiating actin-mediated edge ruffling, onset of shear stress induces displacement of microfilament connections, or nodes, near the basal cell surface (FIGURE 4B). As in the case of flow-induced intermediate filament displacement, microfilaments were often displaced by as much as 1 µm within minutes after onset of shear stress. Since the morphology of these actin filament connections is consistent with attachment to focal contacts, flow-induced displacement serves as additional evidence for force transmission to adhesion sites through the cytoskeleton. Although it is not yet clear whether mechanisms of microfilament displacement are similar to those involved in intermediate filament dynamics, the shape deformation is more consistent with filaments under preexisting tension than with a network of slack filaments as is expected for the intermediate filament network.
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Molecular Mechanisms Modulating Flow-Induced Structural Dynamics
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Although microtubules have been implicated in determining the state of prestress in other anchorage-dependent cells (67), the role of microtubules in flow-mediated endothelial adaptation is not clear, since colcemid-treated endothelial cells responded normally to onset of steady flow (47). However, stabilization of microtubule dynamics in endothelial cells by treatment with paclitaxel prevented lamellipodial extension and migration in the flow direction (32). Thus increased rate of lamellipodial and migratory activity after onset of shear stress may require integration of polymerization dynamics in both actin and tubulin networks. This hypothesis is supported by several recent experiments in migrating fibroblasts. The cytoplasmic linker protein CLIP-170 localizes to the plus ends of microtubules (12, 57) where it mediates rescue (38) and serves to target these growing microtubule ends to cell-substrate adhesion sites (40). Microtubule plus end capture occurs by CLIP-170 binding to the Rac1/Cdc42 effector IQ motif-containing GTPase-activating protein 1 (IQGAP1), which is localized at the leading edge in migrating cells (16). Application of tensile forces using collagen-coated magnetic beads enhanced recruitment of tubulin and CLIP-170 to associated focal adhesion sites, and CLIP-170 interacted with tubulin, actin, and the actin cross-linking protein filamin A in a force-dependent manner (5). Interestingly, filamin A binds Rac1 constitutively (46, 53) and has been implicated in Rho GTPase-dependent signaling to control actin filament assembly and bundling (68), and Rac activation occurs preferentially along downstream edges of endothelial cells after onset of shear stress (73). These events may therefore control local mechanical properties in the neighborhood of focal adhesion sites. In fibroblasts exposed to shear stress, ß1-integrin-mediated filamin A recruitment enhanced actin filament bundling and caused cell stiffening (22). These studies suggest that hemodynamic shear stress induces activation of Rac and concentration of cytoskeleton-associated chaperone proteins in downstream regions of the cytoplasm to establish spatial cues that target microtubule stabilization, enhance actin assembly, and polarize cell adaptation to directional shear stress.
In addition to the polarization of cytoskeletal assembly and edge dynamics, the formation and stabilization of new focal adhesion sites linked to the actin cytoskeleton must occur in a manner that enables downstream-directed cell migration during exposure to shear stress. Onset of shear stress induces conformation-dependent activation of integrins (22, 33, 74) that regulates both binding to extracellular matrix ligands and intracellular signaling (33, 74). The cytoplasmic termini of newly ligated, activated ß-integrins then serve to recruit and activate focal adhesion-associated signaling molecules such as Shc (33) and FAK (42, 44). Integrin-mediated activation of FAK at the leading edge of migrating fibroblasts contributes to microtubule stabilization in this region in a manner that depends on Rho signaling organized in lipid rafts (56), and indeed enrichment of RhoA and Cdc42 in the plasma membrane occurs in subconfluent endothelial cells after onset of shear stress (43). If RhoA is activated preferentially near the downstream edge of endothelial cells, then it may both enhance FAK phosphorylation (14) and promote new actin polymerization where stress fibers insert into focal adhesions (52). Interestingly, onset of shear stress induces activation of RhoA in subconfluent endothelial cell layers (83), but the spatial distribution of RhoA activity is not yet known. Live cell imaging to detect activated RhoA (64) would help to demonstrate whether the time course and spatial distribution of RhoA activity correspond to cytoskeletal reinforcement and new focal adhesion site assembly involved in endothelial ruffling and migration after onset of shear stress.
In addition to spatially regulating RhoA and Rac1 activity, shear stress induces enrichment of Cdc42 in the plasma membrane (43) as well as Cdc42 activation (83) preferentially near downstream cell edges (75). The spatially polarized recruitment and activation of Cdc42 also plays a role in directing positioning of the MTOC toward the downstream side of the nucleus (75). Broader implications of MTOC repositioning for mechanotransduction and endothelial function remain unclear.
The involvement of the Rho family of GTPases represents a unifying theme in these proposed mechanisms for endothelial adaptation to shear stress. In confluent endothelial monolayers, RhoA activity decreases within 5 min after onset of steady unidirectional shear stress (74), which is in contrast to the Rho activation that occurs in sparse populations of cells (83). Decreased Rho activity may enable disassembly of dense peripheral bands and suppress cytoskeletal stabilization of intercellular junctional complexes. On the same time scale, increased Rac activity (73) promotes rapid assembly of densely cross-linked actin mesh near cell edges, which is visible in live-cell measurements as active edge dynamics. The mobilization of Rac, Cdc42, FAK, and actin-binding proteins provides a spatial profile of molecular cues that direct cytoskeletal remodeling to establish cell polarity. Strain focusing without disassembly suggests that the intermediate filament network may provide mechanical stability and may also play a role in guiding focal adhesion assembly in response to flow-mediated integrin activation. This would provide a mechanism for polarized traction force generation that drives cell elongation and migration in the flow direction. Thus a picture of cellular mechanotransduction is beginning to emerge in which endothelial cells integrate a spatiotemporal profile of mechanical cues from the hemodynamic environment. However, specific molecular scale changes that trigger biochemical signaling cascades and the integrated control and decision-making mechanisms that guide adaptation remain to be elucidated.
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Looking to the Future: Implications for Vascular Therapeutics
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The complex feedback between biochemical signaling and mechanical forces plays a critical role in the maintenance of physiological function as well as the development of vascular wall pathologies. As more mechanoresponsive signaling networks are elucidated, evidence is building to support the idea that design of a proper spatial and temporal profile of cues at the molecular level will improve restoration of endothelial function. For example, accelerating reendothelialization after angioplasty correlates with improved patient outcomes (39). In addition, the success of tissue-engineered small artery grafts has been limited by an increase in thrombogenicity and dysregulation of permeability by the endothelium in weeks to months after implant. Thus a major challenge for the emerging field of reparative medicine is to combine genetic, pharmacological, and mechanical interventions to create novel therapeutic approaches based on molecular scale tools. The molecular mechanisms outlined here are guiding the development of promising approaches that integrate quantitative vascular cell biology and biomedical engineering.
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Acknowledgments
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I thank Anil Seetharam for assistance with in vitro flow experiments.
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-71958 and Whitaker Foundation Grant RG-02-0545.
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