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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1781-1786.)
© 1995 American Heart Association, Inc.

Articles

Synergistic Effects of Fluid Shear Stress and Cyclic Circumferential Stretch on Vascular Endothelial Cell Morphology and Cytoskeleton

Shumin Zhao; Andreas Suciu; Thierry Ziegler; James E. Moore, Jr; Ernst Bürki; Jean-Jacques Meister; Hans R. Brunner

From the Division of Hypertension, University Hospital of Lausanne (S.Z., T.Z., E.B., H.R.B.) and the Biomedical Engineering Laboratory, Swiss Federal Institute of Technology Lausanne (A.S., J.E.M., Jr, J.-J.M.), Switzerland

Correspondence to E. Bürki, PhD, Division of Hypertension, CHUV, CH-1011 Lausanne, Switzerland. E-mail eburki@ulys.unil.ch.

	Abstract

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Abstract The development of atherosclerosis is thought to be initiated by a dysfunctional state of the vascular endothelium. The proposal that mechanical forces play a role in the localization of this disease has led researchers to develop in vitro models to assess their effects on cultured endothelial cells. The arterial endothelium is exposed simultaneously to circumferential hoop stretch and wall shear stress, yet previous investigations have focused on the isolated effects of either cyclic stretch or shear stress. The influence of physiological levels of combined shear stress and hoop stretch on the morphology and F-actin organization of bovine aortic endothelial cells was investigated. Cells subjected for 24 hours to shear stresses higher than 2 dyne/cm² or to hoop stretch greater than 2% elongated significantly compared with unstressed controls and oriented along the direction of flow and perpendicular to the direction of stretch. Exposure to more than 4% stretch significantly enhanced the responses to shear stress. Both shear stress and hoop stretch induced formation of stress fibers that were aligned with the cells' long axes. Simultaneous exposure to both stimuli appeared to enhance stress fiber size and alignment. These results indicate that shear stress and hoop stretch synergistically induce morphological changes in endothelial cells, which suggests that circumferential strain might modulate sensitivity of endothelial cells towards shear stress.

Key Words: endothelium • shear stress • cell morphology • circumferential stretch • F-actin

	Introduction

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Atherosclerosis is a focal disease that principally affects the aorta and the carotid, coronary, iliac, and femoral arteries. Physiological factors such as hypertension, hypercholesterolemia, diabetes, and cigarette smoking contribute to the pathological mechanisms that render the vascular endothelium dysfunctional and thereby induce the formation of atherosclerotic lesions. However, these factors are felt throughout the vascular system and are, therefore, unlikely to account for the focal nature of this disease. In contrast, the hemodynamic forces imposed on the vascular wall by pulsatile blood flow vary locally, depending on the arterial geometry and architecture, and therefore are likely candidates for localizing stimuli. Indeed, there is evidence that the localization of atherosclerotic plaques correlates with areas of low and/or oscillating fluid wall shear stress.^{1 2 3}

The arterial endothelium is subjected to a complex mechanical environment. In addition to wall shear stress, pulsatile pressure generates a cyclic circumferential strain (hoop stretch) on the entire arterial wall. The in vitro cellular responses to these mechanical forces include a large spectrum of functional and morphological adaptations. Numerous investigators have addressed the effects of various shear stress levels on the morphological phenomena.^{4 5 6} Laminar steady shear stress induces endothelial cells to flatten, elongate, and align parallel to the direction of flow.^{7 8 9 10} This adaptive process is directly dependent on the magnitude of shear stress and the time of exposure. When the shear stress is pulsatile rather than steady, the morphological adaptation seems to progress at a slightly slower initial rate but the final response is enhanced, as long as the flow is nonreversing. Pulsatile flow with a reversing component diminishes, and purely oscillating flow totally inhibits the morphological response.¹¹ Rounded endothelial cells have indeed been found in vivo in lesion-prone areas where these latter flow patterns might occur.¹²

Circumferential cyclic strain induces endothelial cells to undergo a similar morphological adaptation in vitro.^{13 14 15} Cultured endothelial cells adapt to continuous cyclic stretch by elongating and aligning transversely to the direction of stretch. In separate studies, shear stress and cyclic stretch have each been shown to induce a rearrangement of actin microfilaments into stress fibers that are preferentially aligned with the long axis of the cells.^{4 5} In a cylindrical geometry, where stretch is mainly circumferential, this means that the cells and stress fibers should align parallel to the axis of the tube: that is, in the same direction as under unidirectional shear stress.

All these studies have focused on the effects of either shear stress or cyclic stretch. Recently, two different in vitro models have been developed that allow the creation of a physiologically more relevant mechanical environment by simultaneous exposure of endothelial cells to both forces.^{16 17} In the present study, the device developed in our laboratories¹⁶ was used to determine the combined effects of various physiological levels of shear stress and cyclic circumferential stretch on morphometric parameters and F-actin organization in bovine aortic endothelial cell (BAEC) cultures.

	Methods

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Experimental Flow System
The apparatus used in this study has been described.¹⁶ In brief, it consists of four custom manufactured compliant tubes (Sylgard 184, Dow Corning Europe) mounted onto specially designed fittings. These fittings are connected to a flow system consisting of a temperature- and gas-controlled reservoir and a gear pump (MCP-Z, Ismatec) driven by a pulse generator (HP-8116A, Hewlett-Packard). The pump system is capable of generating a constant flow rate onto which any desired waveform may be imposed. For the present study, an offset sinusoidal waveform with a frequency of 1 Hz was chosen. The flow rates were monitored downstream of all four Sylgard tubes with transit time ultrasonic flow probes (Transonic Systems, Inc). Shear stress values were calculated by use of the method outlined by He et al.¹⁸ The diameters of the tubes receiving hoop strain were monitored with a NIUS 02 ultrasonic echo tracking device (Asulab).¹⁹ The NIUS is capable of recording diameter changes with an accuracy of 3 µm (0.05% of the present tube diameters). The tubes in which radial extension was to be prevented were surrounded by rigid plastic shells. Hydrostatic pressure measured inside the tubes was approximately 100 mm Hg.

One goal of the present study was to generate duplicate sets of experiments (designated a and b; Table) in which shear stress values of approximately 2, 3, and 6 dyne/cm² were combined with a hoop strain of 2%, 4%, and 7% diameter change, respectively (experiments I through V). Mean shear stress values (1.9 to 2.2, 3.2 to 3.4, and 5.2 to 6.4 dyne/cm²) and relative hoop strain (2.0%, 3.9% to 4.3%, and 6.8% to 7.3% diameter change) could be reproduced accurately, as documented in the Table. The flow rate and hoop stretch were verified 24 hours after the start of exposure, and were found to vary by less than 5% of the initial value.

View this table: [in this window] [in a new window]   Table 1. Experimental Shear Stress and Hoop Stretch Values

Cell Culture
BAECs were prepared according to the method described by Booyse et al²⁰ from freshly excised bovine aortas by a single collagenase digestion for 15 minutes at 37°C (3 mg/mL collagenase type II [Worthington] in PBS). Primary cultures were established in minimal essential medium with Earl's salts (EMEM, Gibco, Life Technologies) containing 20% fetal calf serum (Seromed, Biochrom KG) and supplemented with glutamate, vitamins, nonessential amino acids, antibiotics, and 10 mmol/L HEPES. Medium was subsequently changed every 2 days. At passage 2, cells were characterized as endothelial by immunostaining for the factor VIII–like antigen. Morphologically, confluent cultures presented the cobblestone pattern typical for endothelial cells in static culture. Cells between passages 3 and 9 were used in the stress experiments.

Cells from one confluent culture flask (75 cm²) were seeded inside four Sylgard tubes. The tubes (70 mm in length, 6-mm inner diameter, and 0.19- to 0.24-mm wall thickness) were first hydrophylized for 1 minute in 70% sulfuric acid and then rinsed extensively with deionized water. Upon mounting of the tubes onto the fittings, an axial stretch of 10% was imposed to ensure that the tubes would remain straight under pressurization. After sterilization in an autoclave, the insides of the tubes were coated with human fibronectin (40 µg/mL in PBS, Boehringer Mannheim). BAECs were seeded at a final density of 5x to 8x10⁴ cells/cm² in a modified growth medium (10% instead of 20% fetal calf serum) in four successive additions of 30 minutes each, the fittings being rotated by 90° after each addition to allow for uniform cell attachment over the entire inside of the tubes. After seeding, normal growth medium was added and cells were grown at 37°C under 5% CO₂. The fittings were oriented vertically to allow for uniform cell growth. Cell monolayers reached confluence within 2 to 3 days.

The fittings carrying the confluent cell cultures were connected to the previously assembled flow device and submerged in a temperature-controlled water bath at 37°C. At the beginning of each stress experiment, the cell growth medium was replaced by flow medium, which was seeding medium complemented with 2% Rheomacrodex (10% Dextran 40000 in saline, Pharmacia). The viscosity of this medium at 37°C was 1.073x10^-4 m · g · s.

Silver Staining for Cell Junctions
At the end of each flow experiment, the cells were fixed with 3% paraformaldehyde in PBS for 10 minutes at room temperature. The fixed tubes were disconnected from the fittings and sliced longitudinally. Once open, the central part of each sheet was subdivided into 10x15-mm rectangular pieces. In one piece of tubing, silver staining of cell junctions was performed according to the method described by Zand et al.²¹ Cells were then analyzed under a confocal scanning microscope (MRC 500, Biorad, and Diaphot, Nikon) in the direct-light mode. For each experiment, 40 endothelial cells in randomly chosen areas of the tubes were analyzed quantitatively with a dedicated software (Biorad SOM) to determine the morphological parameters (area, perimeter, and the long axis of cells). The cell shape index (SI) and alignment angle (_A) were determined as described by Nerem et al.²² The SI value is defined as 4xxarea/perimeter² and is equal to 1 for a circle and 0 for a straight line. The angle of alignment, defined as the angle between the long axis of a cell and the flow direction, was calculated trigonometrically. The average angle in a randomly oriented cell population would be 45°, and a perfectly aligned population would have an angle of alignment of 0°.

Fluorescent Staining of Cytoskeletal Filaments
F-actin microfilaments were stained with FITC-labeled phalloidin and analyzed by confocal scanning microscopy.

Statistical Analysis
SIs and _As are average values (mean±SEM) from 40 different cells in each experiment. The SI and _A between individual experiments were compared by ANOVA with the INSTAT 2 software (Version 2.01, GraphPad Software). Multiple comparisons were performed with a Bonferroni test. Cells that were exposed to the same mechanical environment in different experiments revealed SIs and _As that were not significantly different (P>.05).

	Results

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Cells subjected to any of the experimental conditions remained attached as confluent monolayers over the entire 24-hour period of exposure. Each experiment had its own internal static control culture, in which the flow rate was kept below 30 mL/min, just enough to allow for continuous medium exchange. This flow rate generated a shear stress of less than 0.5 dyne/cm² that, by itself, did not induce morphometric changes.

The SI and _A of cells exposed to mechanical stimuli were determined from silver-stained BAEC monolayers as shown in Fig 1. The continuous silver lines demonstrate the integrity of the monolayers, because silver only deposits in areas of cell-cell contact.²¹ The four micrographs show cell boundaries in tube C (control, Fig 1a), S (shear stress, Fig 1b), H (hoop stretch, Fig 1c), and SH (shear plus hoop, Fig 1d) from experiment IVa. Control cells were irregularly shaped and the few clearly elongated cells were randomly oriented. Shear stress induced a marked cell elongation and alignment with the direction of flow. Exposure to hoop stretch resulted in a similar change in cell shape, and the cells oriented transversely to the direction of stretch. When cells were exposed to both shear stress and hoop stretch, elongation as well as alignment were significantly enhanced (P<.01). In the absence of hoop stretch, both cell elongation and orientation increased markedly (ie, values for SI and _A decreased) as the shear stress increased from 2 to 3 dyne/cm² (Fig 2). Applying a hoop stretch of 2% to low shear stresses (<0.5 to 2 dyne/cm²) did not cause significant changes in SI and _A relative to the static controls. An increase in hoop stretch to 4%, however, resulted in a significantly stronger alignment than under any of the shear stress levels alone (P<.05). Cell elongation was similarly enhanced, even though the trend was not statistically significant in all combinations. When a hoop stretch of 7% was superimposed on any of the shear stresses, both cell elongation and orientation were significantly enhanced (P<.01). However, a comparison of the effects of 4% and 7% hoop stretch demonstrated that with higher values of shear stress the hoop stretch effect became less important.

View larger version (152K): [in this window] [in a new window]   Figure 1. Micrographs show silver-stained BAECs after exposure for 24 hours to the following mechanical stimuli: Shear stresses of less than 0.5 dyne/cm2 (a) and 6.4±1.8 dyne/cm2 (b) and hoop stretch of 4%, either alone (c) or together with a shear stress of 5.8±1.7 dyne/cm2 (d). The flow direction was from right to left; the size bar represents 100 µm.

View larger version (13K): [in this window] [in a new window]   Figure 2. Plots show SIs (a) and As (b) for BAECs exposed for 24 hours to a shear stress of less than 0.5, 2, 3, and 6 dyne/cm2, together with 0% (), 2% (), 4% (), and 7% () hoop stretch. Values are mean±SEM. Asterisks are placed between the different values compared; in the case of ambiguities, vertical dashed lines indicate the points compared. *P<.05, **P<.01, and ***P<.001.

Mechanical stimuli imposed marked changes in the cytoskeleton, as evidenced by the important remodeling of intracellular F-actin structures. Under static conditions (Fig 3a), actin was assembled preferentially in dense peripheral bands lining the contour of the cells. In addition, small short filaments cross the cytoplasm as a network that exhibited no particular orientation. After exposure to hoop stretch (Fig 3c), the cytoplasmic filaments had thickened and aligned with the long axes of the cells. However, most cells appeared to maintain their peripheral actin structures. In contrast, in cells exposed to shear stress the dense peripheral bands were reduced and long dense stress fibers had formed (Fig 3b). Moreover, in numerous cells these fibers appeared to connect to the plasma membrane in particular intercellular contact sites. This anchorage evokes the impression of stress fibers extending over the length of more than one cell. A comparison of cells exposed to different levels of shear stress or hoop stretch was not successful in detecting a clear dose-dependence of the structural F-actin reorganization. At best, there was a tendency for stress fibers to increasingly align and orient with the long axes of the cells, as well as for dense peripheral bands to diminish with an increasing magnitude of either hoop stretch or shear stress.

View larger version (140K): [in this window] [in a new window]   Figure 3. Scanning confocal micrographs show fluorescein phalloidin–labeled BAECs after exposure for 24 hours to the following mechanical stimuli: Shear stresses of less than 0.5 dyne/cm2 (a) and 6.4±1.8 dyne/cm2 (b) and hoop stretch of 4%, either alone (c) or together with a shear stress of 5.8±1.7 dyne/cm2 (d). The flow direction was from right to left; the size bar represents 50 µm.

When cells were exposed to both hoop stretch and shear stress (Fig 3d), the stress fibers appeared generally thicker than in cells exposed to either force alone. In addition, these fibers seemed to connect from cell to cell over longer distances than when under shear stress alone.

	Discussion

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The results of the present study demonstrate that physiological combinations of shear stress and cyclic circumferential strain have a synergistic effect on vascular endothelial cell remodeling processes. The presence of shear stress and cyclic strain altered the structure and cytoskeleton of the cells in a dose-dependent manner with clear-cut threshold levels.

Endothelial dysfunction, as potentially mediated by these mechanical factors, is the hallmark of the early stages of vascular disease. During the past two decades, evidence has accumulated for endothelial structure and function being regulated in large part by the arterial mechanical environment. For instance, areas of particular hemodynamic conditions correlate with the location of atherosclerotic plaques^{1 2 3} and coincide with a rounded endothelial cell phenotype¹² and increased cell turnover.²³ In an effort to understand the mechanisms of cellular mechanoreception and mechanotransmission, several in vitro models have been developed and used to determine the effects of fluid shear stress, cyclic strain, or hydrostatic pressure on the vascular endothelium.^{7 8 14 24} All three forces induce structural changes and activate intracellular signaling and metabolic pathways that might ultimately affect functional aspects such as cell growth, cell adhesion, endothelial permeability, and regulation of vascular tone.^{4 5 6 25 26 27} However, looking at the effects of each component separately does not aid in understanding how these stimuli, which in vivo depend on flow, blood pressure, and vessel wall structure, interact to generate complex cellular responses.

The present study was designed as a first step in addressing this problem in comparing structural cell responses to different combinations of physiological cyclic strain and shear stress. Endothelial cell cultures exposed to pulsatile shear stress responded with a significant dose-dependent remodeling, as demonstrated by cell elongation, alignment with the direction of flow, and, to a certain extent, formation of actin stress fibers aligned with the cell long axis. The threshold level of shear stress (between 2 and 3 dyne/cm²) was surprisingly low when compared with values reported previously.⁵ Tentatively, this low value could be attributed to the particular extracellular matrix chosen. On the other hand, one cannot exclude a priori the possibility that hydrostatic pressure (100 mm Hg) added to shear stress, cyclic strain, or both might contribute to the cellular responses, even though it did not induce morphological changes by itself. Acevedo et al²⁵ and Sumpio et al²⁶ reported that exposure to pressures of up to 120 mm Hg for several days resulted in endothelial cell elongation.

Threshold levels of cyclic strain for endothelial cell remodeling have, to our knowledge, not been reported in the literature. Even if such data existed, it would be difficult to compare the results with those of the present study. The stretch model used here is fundamentally different from all predecessors. It relies on pulsatile pressure–induced uniaxial cyclic strain rather than (often) biaxial strain directly imposed on the substratum to which the cell cultures are attached.^{14 24}

The novel approach consists of combining physiological levels of circumferential strain and shear stress, which induced an increase in cellular responses compared with similar levels of either force alone. This synergy of action may have major implications in the present understanding of mechanoreception by endothelial cells and the translation of the mechanical signals into structural and functional responses. The data from this study suggest that pressure-induced circumferential cyclic strain increases endothelial cell sensitivity to shear stress, which results in a lowered threshold level of shear stress to provoke structural responses. Cellular structural integrity initiated by cyclic strain may be the mediating factor in this process. According to a current hypothesis, anchorage-dependent cells can be considered a mechanical entity whose architecture is established through tensional integrity.²⁸ Applying cyclic circumferential strain to the cell will, therefore, modify this mechanical equilibrium and result in remodeling of the cytoskeleton with a concomitant change in cell shape. Cytoskeletal components coupled to intracellular effector systems could then serve as mechanotransducers to produce complex intracellular responses.⁵ Intracellular force transmission, which has been shown to result in dynamic changes in focal adhesion sites,²⁹ and force transduction, which induces responses such as altered regulation of gene expression,⁶ might be affected either directly or as a consequence of changes in the sensitivity of stress receptors. Circumstantial evidence for a transducer function of the cytoskeleton has recently been presented in studies that revealed that an intact endothelial cytoskeleton is required for molecular responses to shear stress.⁶ Given the synergistic response of endothelial cells to cyclic strain and shear stress, it is possible that a certain combination of these two mechanical stimuli provokes cellular responses that lead to atherosclerosis.

The present study certainly emphasizes that a meaningful investigation of the vascular endothelial structure has to take into consideration the complexity of the mechanical environment to which these cells are exposed in vivo. The availability of in vitro systems that more accurately model the arterial mechanical environment will help to more clearly define the functional properties of the vascular endothelium under physiological and pathophysiological conditions.

	Acknowledgments

 
This study was supported by the Swiss National Scientific Research Fund, grant 32-3253591, and the CHUV-EPFL-UNIL Biomedical Engineering Common Collaboration Program.

Received March 22, 1995; accepted June 28, 1995.

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