Rho mediates the shear-enhancement of endothelial cell migration and traction force generation

The migration of vascular endothelial cells in vivo occurs in a fluid dynamic environment due to blood flow, but the role of hemodynamic forces in cell migration is not yet completely understood. Here we investigated the effect of shear stress, the frictional drag of blood flowing over the cell surface, on the migration speed of individual endothelial cells on fibronectin-coated surfaces, as well as the biochemical and biophysical bases underlying this shear effect. Under static conditions, cell migration speed had a bell-shaped relationship with fibronectin concentration. Shear stress significantly increased the migration speed at all fibronectin concentrations tested and shifted the bell-shaped curve upwards. Shear stress also induced the activation of Rho GTPase and increased the traction force exerted by endothelial cells on the underlying substrate, both at the leading edge and the rear, suggesting that shear stress enhances both the frontal forward-pulling force and tail retraction. The inhibition of a Rho-associated kinase, p160ROCK, decreased the traction force and migration speed under both static and shear conditions and eliminated the shear-enhancement of migration speed. Our results indicate that shear stress enhances the migration speed of endothelial cells by modulating the biophysical force of tractions through the biochemical pathway of Rho-p160ROCK.     

Mechanoresponse of stem cells for vascular repair

Stem cells have shown great potential in vascular repair. Numerous evidence indicates that mechanical forces such as shear stress and cyclic strain can regulate the adhesion, proliferation, migration, and differentiation of stem cells via serious signaling pathways. The enrichment and differentiation of stem cells play an important role in the angiogenesis and maintenance of vascular homeostasis. In normal tissues, blood flow directly affects the microenvironment of vascular endothelial cells (ECs); in pathological status, the abnormal interactions between blood flow and vessels contribute to the injury of vessels. Next, the altered mechanical forces are transduced into cells by mechanosensors to trigger the reformation of vessels. This process occurs when signaling pathways related to EC differentiation are initiated. Hence, a deep understanding of the responses of stem cells to mechanical stresses and the underlying mechanisms involved in this process is essential for clinical translation. In this the review, we provide an overview of the role of stem cells in vascular repair, outline the performance of stem cells under the mechanical stress stimulation, and describe the related signaling pathways.     

Cells in 3D matrices under interstitial flow: Effects of extracellular matrix alignment on cell shear stress and drag forces

Interstitial flow is an important regulator of various cell behaviors both in vitro and in vivo, yet the forces that fluid flow imposes on cells embedded in a 3D extracellular matrix (ECM), and the effects of matrix architecture on those forces, are not well understood. Here, we demonstrate how fiber alignment can affect the shear and pressure forces on the cell and ECM. Using computational fluid dynamics simulations, we show that while the solutions of the Brinkman equation accurately estimate the average fluid shear stress and the drag forces on a cell within a 3D fibrous medium, the distribution of shear stress on the cellular surface as well as the peak shear stresses remain intimately related to the pericellular fiber architecture and cannot be estimated using bulk-averaged properties. We demonstrate that perpendicular fiber alignment of the ECM yields lower shear stress and pressure forces on the cells and higher stresses on the ECM, leading to decreased permeability, while parallel fiber alignment leads to higher stresses on cells and increased permeability, as compared to a cubic lattice arrangement. The Spielman–Goren permeability relationships for fibrous media agreed well with CFD simulations of flow with explicitly considered fibers. These results suggest that the experimentally observed active remodeling of ECM fibers by fibroblasts under interstitial flow to a perpendicular alignment could serve to decrease the shear and drag forces on the cell.     

The effects of external compression on venous blood flow and tissue deformation in the lower leg

External pneumatic compression of the lower legs is effective as prophylaxis against deep vein thrombosis. In a typical application, inflatable cuffs are wrapped around the patient's legs and periodically inflated to prevent stasis, accelerate venous blood flow, and enhance fibrinolysis. The purpose of this study was to examine the stress distribution within the tissues, and the corresponding venous blood flow and intravascular shear stress with different external compression modalities. A two-dimensional finite element analysis (FEA) was used to determine venous collapse as a function of internal (venous) pressure and the magnitude and spatial distribution of external (surface) pressure. Using the one-dimensional equations governing flow in a collapsible tube and the relations for venous collapse from the FEA, blood flow resulting from external compression was simulated. Tests were conducted to compare circumferentially symmetric (C) and asymmetric (A) compression and to examine distributions of pressure along the limb. Results show that A compression produces greater vessel collapse and generates larger blood flow velocities and shear stresses than C compression. The differences between axially uniform and graded-sequential compression are less marked than previously found, with uniform compression providing slightly greater peak flow velocities and shear stresses. The major advantage of graded-sequential compression is found at midcalf. Strains at the lumenal border are approximately 20 percent at an external pressure of 50 mmHg (6650 Pa) with all compression modalities.     

Factors influencing the nonuniform localization of monocytes in the arterial wall

Adhesion of monocytes to arterial endothelium may contribute to the asymmetric distribution of atherosclerotic lesions. Possible mechanisms for adhesion in the relatively high shear stress environment found in arteries include greater monocyte deformation and/or more frequent penetration of microvilli through steric and charge barriers. In vivo, secondary flows generate forces acting normal to the endothelial cell surface. These forces may cause compression of the microvilli or enable cells to overcome steric or electrostatic barriers, increasing adhesion. To investigate this, we examined monocyte adhesion to activated endothelium in recirculating flow. Adhesion was characterized by short arrests in a narrow region on either side of the reattachment line. The median arrest time was longer than that observed at comparable shear stresses in a linear shear flow. The lifetimes of adhesion were analyzed using a model for multiple bond formation. For cells adhering near the reattachment line, the bond number per cell was greater than the value found for similar shear stresses under shear flow. Thus, multiple bond formation arising from greater normal forces in recirculating flow permits monocytes to adhere at higher shear stresses.     

In Vivo Wall Shear Measurements within the Developing Zebrafish Heart

Physical forces can influence the embryonic development of many tissues. Within the cardiovascular system shear forces resulting from blood flow are known to be one of the regulatory signals that shape the developing heart. A key challenge in investigating the role of shear forces in cardiac development is the ability to obtain shear force measurements in vivo. Utilising the zebrafish model system we have developed a methodology that allows the shear force within the developing embryonic heart to be determined. Accurate wall shear measurement requires two essential pieces of information; high-resolution velocity measurements near the heart wall and the location and orientation of the heart wall itself. We have applied high-speed brightfield imaging to capture time-lapse series of blood flow within the beating heart between 3 and 6 days post-fertilization. Cardiac-phase filtering is applied to these time-lapse images to remove the heart wall and other slow moving structures leaving only the red blood cell movement. Using particle image velocimetry to calculate the velocity of red blood cells in different regions within the heart, and using the signal-to-noise ratio of the cardiac-phase filtered images to determine the boundary of blood flow, and therefore the position of the heart wall, we have been able to generate the necessary information to measure wall shear in vivo. We describe the methodology required to measure shear in vivo and the application of this technique to the developing zebrafish heart. We identify a reduction in shear at the ventricular-bulbar valve between 3 and 6 days post-fertilization and demonstrate that the shear environment of the ventricle during systole is constantly developing towards a more uniform level.     

Effect of the endothelial surface layer on transmission of fluid shear stress to endothelial cells

Responses of vascular endothelial cells to mechanical shear stresses resulting from blood flow are involved in regulation of blood flow, in structural adaptation of vessels, and in vascular disease. Interior surfaces of blood vessels are lined with a layer of bound or adsorbed macromolecules, known as the endothelial surface layer (ESL). In vivo investigations have shown that this layer has a width of order 1 microm, that it substantially impedes plasma flow, and that it excludes flowing red blood cells. Here, the effect of the ESL on transmission of shear stress to endothelial cells is examined using a theoretical model. The layer is assumed to consist of a matrix of molecular chains extending from the surface, held in tension by a slight increase in colloid osmotic pressure relative to that in free-flowing plasma. It is shown that, under physiological conditions, shear stress is transmitted to the endothelial surface almost entirely by the matrix, and fluid shear stresses on endothelial cell membranes are very small. Rapid fluctuations in shear stress are strongly attenuated by the layer. The ESL may therefore play an important role in sensing of shear stress by endothelial cells.     

Effects of local heat on blood flow in infected and normal hands

Changes in response to heat in the dermal, subcutaneous, and muscle blood flow in the hands of 10 patients with hand infections were studied using 133Xe and recording of clearance data. A further 15 normal hands were studied in a similar manner. The application of topical heat to normal hands resulted in a decrease in the dermal blood flow (p less than 0.001), an increase in the subcutaneous blood flow (p less than 0.05), and perhaps an increase in the intramuscular flow (p less than 0.1). This suggests that surface heat promotes a shunting of the blood from the skin to deeper tissue layers. In infected hands, the blood flow was found to be significantly increased threefold in the intradermal circulation (p less than 0.01) and eightfold in the subcutaneous circulation (p less than 0.03) when compared to controls. In contrast to normal hands, in the infected hands, the blood flow decreased in all three compartments by 50 percent following application of heat. The blood flow of the normal hand in patients with infection did not respond to heat in the normal pattern. We conclude that the application of local heat to normal tissues results in shunting of blood flow from superficial tissues such as dermis to deeper ones such as subcutaneous fat and muscle. In infected tissues, the blood flow was found to be much higher than normal; however, the traditional belief in the improvement in blood flow by the application of heat was not confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental measurement of dynamic fluid shear stress on the aortic surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. Although exact causes and mechanisms of AV calcification are unclear, previous studies suggest that mechanical forces play a role. Since calcium deposits occur almost exclusively on the aortic surfaces of AV leaflets, it has been hypothesized that adverse patterns of fluid shear stress on the aortic surface of AV leaflets promote calcification. The current study characterizes AV leaflet aortic surface fluid shear stresses using Laser Doppler velocimetry and an in vitro pulsatile flow loop. The valve model used was a native porcine valve mounted on a suturing ring and preserved using 0.15% glutaraldehyde solution. This valve model was inserted in a mounting chamber with sinus geometries, which is made of clear acrylic to provide optical access for measurements. To understand the effects of hemodynamics on fluid shear stress, shear stress was measured across a range of conditions: varying stroke volumes at the same heart rate and varying heart rates at the same stroke volume. Systolic shear stress magnitude was found to be much higher than diastolic shear stress magnitude due to the stronger flow in the sinuses during systole, reaching up to 20 dyn/cm² at mid-systole. Upon increasing stroke volume, fluid shear stresses increased due to stronger sinus fluid motion. Upon increasing heart rate, fluid shear stresses decreased due to reduced systolic duration that restricted the formation of strong sinus flow. Significant changes in the shear stress waveform were observed at 90 beats/min, most likely due to altered leaflet dynamics at this higher heart rate. Overall, this study represents the most well-resolved shear stress measurements to date across a range of conditions on the aortic side of the AV. The data presented can be used for further investigation to understand AV biological response to shear stresses.     

Numerical investigation of the role of intercellular interactions on collective epithelial cell migration

During collective cell migration, the intercellular forces will significantly affect the collective migratory behaviors. However, the measurement of mechanical stresses exerted at cell–cell junctions is very challenging. A recent experimental observation indicated that the intercellular adhesion sites within a migrating monolayer are subjected to both normal stress exerted perpendicular to cell–cell junction surface and shear stress exerted tangent to cell–cell junction surface. In this study, an interfacial interaction model was proposed to model the intercellular interactions for the first time. The intercellular interaction model-based study of collective epithelial migration revealed that the direction of cell migration velocity has better alignment with the orientation of local principal stress at higher maximum shear stress locations in an epithelial monolayer sheet. Parametric study of the effects of adhesion strength indicated that normal adhesion strength at the cell–cell junction surface has dominated effect on local alignment between the direction of cell velocity vector and the principal stress orientation, while the shear adhesion strength has little effect, which provides compelling evidence to help explain the force transmission via cell–cell junctions between adjacent cells in collective cell motion and provides new insights into “adhesive belt” effects at cell–cell junction.     

An MRI-compatible foot-loading device for assessment of internal tissue deformation

It is well known that mechanical forces acting within the soft tissues of the foot can contribute to the formation of neuropathic ulcers in people with diabetes. Presently, only surface measurements of plantar pressure are used clinically to estimate risk status due to mechanical loading. It is currently not known how surface measurements relate to the three-dimensional (3-D) internal stress/strain state of the foot. This article describes the development of a foot-loading device that allows for the direct observation of the internal deformation of foot tissues under known forces. Ground reaction forces and plantar pressure distributions during normal walking were measured in ten healthy young adults. One instant in the gait cycle, when pressure under the metatarsal heads reached a peak, was extracted for simulation in an MR imager. T1-weighted 3-D gradient echo MRI sets were collected as the simulated walking ground reaction force was incrementally applied to the foot by the novel foot-loading device. The sub-metatarsal head soft-tissue thickness decreased rapidly at first and then reached a plateau. Peak plantar pressure measurements collected within the loading device (161+/-75kPa) were lower in magnitude and less focal than pressures measured during walking (492+/-91kPa). This finding implies that although the device successfully applied full peak walking ground reaction forces to the foot, they were not distributed in the same manner as during walking. Although not representative of gait, the data collected from this in vivo mechanical test are suitable for determination of foot tissue material properties or, when combined with finite element modeling, to examine the relationship between surface loading and internal stress.     

Experimental measurement of dynamic fluid shear stress on the ventricular surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. The exact causes and mechanisms of AV calcification are unclear, although previous studies suggest that mechanical forces play a role. It has been clinically demonstrated that calcification preferentially occurs on the aortic surface of the AV. This is hypothesized to be due to differences in the mechanical environments on the two sides of the valve. It is thus necessary to characterize fluid shear forces acting on both sides of the leaflet to test this hypothesis. The current study is one of two studies characterizing dynamic shear stress on both sides of the AV leaflets. In the current study, shear stresses on the ventricular surface of the AV leaflets were measured experimentally on two prosthetic AV models with transparent leaflets in an in vitro pulsatile flow loop using two-component Laser Doppler Velocimetry (LDV). Experimental measurements were utilized to validate a theoretical model of AV ventricular surface shear stress based on the Womersley profile in a straight tube, with corrections for the opening angle of the valve leaflets. This theoretical model was applied to in vivo data based on MRI-derived volumetric flow rates and valve dimension obtained from the literature. Experimental results showed that ventricular surface shear stress was dominated by the streamwise component. The systolic shear stress waveform resembled a half-sinusoid during systole and peaks at 64–71 dyn/cm², and reversed in direction at the end of systole for 15–25?ms, and reached a significant negative magnitude of 40–51 dyn/cm². Shear stresses from the theoretical model applied to in vivo data showed that shear stresses peaked at 77–92 dyn/cm² and reversed in direction for substantial period of time (108–110?ms) during late systole with peak negative shear stress of 35–38 dyn/cm².     

The hemodynamic and embolizing forces acting on thrombi--II. The effect of pulsatile blood flow

A previous analysis (Basmadjian, J. Biomechanics 17, 287-298, 1984) of the embolizing forces acting on thrombi in steady Poiseuille flow has been extended to pulsatile blood flow conditions in the major blood vessels. We show that for incipient and small compact thrombi up to 0.1 mm height, the maximum embolizing stresses can be calculated from the corresponding 'quasi-steady' viscous drag forces and measured maximum wall shear. Their magnitude is from 5 to 30 times (tau w)Max, the maximum wall shear stress during the cardiac cycle in the absence of thrombi. For larger thrombi, inertial and 'history' effects have to be taken into account, leading to embolizing stresses in excess of 100 Pa (1000 dyn cm-2).     

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. (c) 1993 John Wiley & Sons, Inc.     

Elevated shear stress protects Escherichia coli cells adhering to surfaces via catch bonds from detachment by soluble inhibitors

Soluble inhibitors find widespread applications as therapeutic drugs to reduce the ability of eukaryotic cells, bacteria, or viruses to adhere to surfaces and host tissues. Mechanical forces resulting from fluid flow are often present under in vivo conditions, and it is commonly presumed that fluid flow will further add to the inhibitive effect seen under static conditions. In striking contrast, we discover that when surface adhesion is mediated by catch bonds, whose bond life increases with increased applied force, shear stress may dramatically increase the ability of bacteria to withstand detachment by soluble competitive inhibitors. This shear stress-induced protection against inhibitor-mediated detachment is shown here for the fimbrial FimH-mannose-mediated surface adhesion of Escherichia coli. Shear stress-enhanced reduction of bacterial detachment has major physiological and therapeutic implications and needs to be considered when developing and screening drugs.     

Integrins may serve as mechanical transducers for low-frequency electric fields

A hypothesis is presented that a transduction mechanism for low frequency electric fields of physiological strength ( approximately 1 V/cm) is the same as that for sinusoidal fluid shear stresses, the force exerted on an integrin. Simple calculations show that the forces exerted on a model integrin by transverse electric fields and fluid shears that produce cellular effects are comparable in magnitude, about 1 fN. The electric force is provided by the interaction of the surface charges on the integrin with the tangential component of the applied field. The mechanical shear force is the transverse fluid drag force exerted on the cylindrical surface of the integrin. Either force is coupled mechanically to the actin cortex within the cell. The mechanical network which exists within a cell and connects a cell to its surroundings would then be directly coupled to an applied electric field. The fundamental transduction mechanism for some electric field effects may then be ultimately mechanical in nature.     

Cytoplasmic calcium response to fluid shear stress in cultured vascular endothelial cells

Summary Vascular endothelial cells modulate their structure and functions in response to changes in hemodynamic forces such as fluid shear stress. We have studied how endothelial cells perceive the shearing force generated by blood flow and the substance(s) that may mediate such a response. We identify cytoplasmic-free calcium ion (Ca⁺⁺), a major component of an internal signaling system, as a mediator of the cellular response to fluid shear stress. Cultured monolayers of bovine aortic endothelial cells loaded with the highly fluorescent Ca⁺⁺-sensitive dye Fura 2 were exposed to different levels of fluid shear stress in a specially designed flow chamber, and simultaneous changes in fluorescence intensity, reflecting the intracellular-free calcium concentration ([Ca⁺⁺] _i ), were monitored by photometric fluorescence microscopy. Application of shear stress to cells by fluid perfusion led to an immediate severalfold increase in fluorescence within 1 min, followed by a rapid decline for about 5 min, and finally a plateau somewhat higher than control levels during the entire period of the stress application. Repeated application of the stress induced similar peak and plateau levels of [Ca⁺⁺] _i but at reduced magnitudes of response. These responses were observed even in Ca⁺⁺-free medium. Thus, a shear stress transducer might exist in endothelial cells, which perceives the shearing force on the membrane as a stimulus and mediates the signal to increase cytosolic free Ca⁺⁺. This work was partly supported by a grant-in-aid, for Special Project Research no. 61132008, from the Japanese Ministry of Education, Science and Culture and a research fund from the Atherosclerosis Study Association.     

Radial artery wall shear stress evaluation in patients with arteriovenous fistula for hemodialysis access

It has been extensively documented that changes in blood flow induce vascular remodeling and this phenomenon seems to be correlated to the shear forces imposed on the vessel wall by motion of blood. Wall shear stress, the tractive force that acts on the endothelium, has been shown to influence endothelial cell function. To study changes in wall shear stress that develop on the vessel wall upon changes of blood flow, we set up a technique that allows estimation of shear stress in the radial artery of patients on chronic hemodialysis therapy. The technique is based on color-flow Doppler examination of the radial artery before and after surgical creation of radiocephalic fistula for hemodialysis. Calculation of time function wall shear stress and blood flow rate in the radial artery is performed on the basis of arterial diameter, center-line velocity waveform and blood viscosity, using a numerical method developed according to Womersley's theory for pulsatile flow in tubes. The results presented confirm that the model developed is suitable for calculation of the wall shear stress that develops in the radial artery of patients before and after surgical creation of an arteriovenous fistula for hemodialysis. This methodology was developed for characterization of wall shear stress in the radial artery but may be well applied to other vessels that can be examined by echo-Doppler technique.     

The potential influence of the heel counter on internal stress during static standing: a combined finite element and positional MRI investigation

Confinement of the heel due to the counter of the shoe is believed to influence heel pad biomechanics. Using a two-dimensional finite element model of the heel pad and shoe during a simulation of static standing, the aim of this study was to quantify the potential effect of confinement on internal heel pad stress. Non-weightbearing MRI and weightbearing MRI with plantar pressure and ground reaction force data were recorded for a single subject. The non-weightbearing MRI was used to create two FE models of the heel pad, using either homogeneous or composite material properties. The composite model included a distinction in material properties between fat pad and skin. Vertical and medial-lateral forces, as measured on the subject's heel, were applied to the models and vertical compressive strains for both models were comparable with those observed by weightbearing MRI. However, only for the composite model was the predicted plantar pressure distribution comparable with measured data. The composite model was therefore used in further analyses. In this composite model, the internal stresses were located mainly in the skin and were predominantly tensile in nature, whereas the stress state in the fat pad approached hydrostatic conditions. A representation of a running shoe, including an insole, midsole and heel counter was then added to the composite heel pad to form the shod model. In order to investigate the counter effect, the load was applied to the shod model with and without the heel counter. The effect of the counter on peak stress was to elevate compression (0-50%), reduce tension (22-34%) and reduce shear (22-28%) in the skin. In addition, the counter reduced both compressive (20-40%) and shear (58-80%) stress in the fat pad and tension in the fat pad remained negligible. Taken together the results indicate that a well-fitted counter works in sympathy with the internal structure of the heel pad and could be an effective reducer of heel pad stress. However, further research needs to be undertaken to assess the long-term effects on the soft-tissues, practicalities of achieving good fit and behavior under dynamic events.