Traction Forces of Endothelial Cells under Slow Shear Flow

Endothelial cells are constantly exposed to fluid shear stresses that regulate vascular morphogenesis, homeostasis, and disease. The mechanical responses of endothelial cells to relatively high shear flow such as that characteristic of arterial circulation has been extensively studied. Much less is known about the responses of endothelial cells to slow shear flow such as that characteristic of venous circulation, early angiogenesis, atherosclerosis, intracranial aneurysm, or interstitial flow. Here we used a novel, to our knowledge, microfluidic technique to measure traction forces exerted by confluent vascular endothelial cell monolayers under slow shear flow. We found that cells respond to flow with rapid and pronounced increases in traction forces and cell-cell stresses. These responses are reversible in time and do not involve reorientation of the cell body. Traction maps reveal that local cell responses to slow shear flow are highly heterogeneous in magnitude and sign. Our findings unveil a low-flow regime in which endothelial cell mechanics is acutely responsive to shear stress.     

Mechanotransduction in embryonic vascular development

A plethora of biochemical signals provides spatial and temporal cues that carefully orchestrate the complex process of vertebrate embryonic development. The embryonic vasculature develops not only in the context of these biochemical cues, but also in the context of the biomechanical forces imparted by blood flow. In the mature vasculature, different blood flow regimes induce distinct genetic programs, and significant progress has been made toward understanding how these forces are perceived by endothelial cells and transduced into biochemical signals. However, it cannot be assumed that paradigms that govern the mature vasculature are pertinent to the developing embryonic vasculature. The embryonic vasculature can respond to the mechanical forces of blood flow, and these responses are critical in vascular remodeling, certain aspects of sprouting angiogenesis, and maintenance of arterial–venous identity. Here, we review data regarding mechanistic aspects of endothelial cell mechanotransduction, with a focus on the response to shear stress, and elaborate upon the multifarious effects of shear stress on the embryonic vasculature. In addition, we discuss emerging predictive vascular growth models and highlight the prospect of combining signaling pathway information with computational modeling. We assert that correlation of precise measurements of hemodynamic parameters with effects on endothelial cell gene expression and cell behavior is required for fully understanding how blood flow-induced loading governs normal vascular development and shapes congenital cardiovascular abnormalities.     

Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles

Bone cells subjected to mechanical loading by fluid shear stress undergo significant architectural and biochemical changes. The models of shear stress used to analyze the effects of loading bone cells in vitro include both oscillatory and unidirectional fluid shear profiles. Although the fluid flow profile experienced by cells within bone is most likely oscillatory in nature, to date there have been few direct comparisons of how bone cells respond to these two fluid flow profiles. In this study we evaluated morphologic and biochemical responses to a time course of unidirectional and oscillatory fluid flow in two commonly used bone cell lines, MC3T3-E1 osteoblasts and MLO-Y4 osteocytes. We determined that stress fibers formed and aligned within osteoblasts after 1 h of unidirectional fluid flow, but this response was not observed until greater than 5 h of oscillatory fluid flow. Despite the delay in stress fiber formation, oscillatory and unidirectional fluid flow profiles elicited similar temporal effects on the induction of both cyclooxygenase-2 (Cox-2) and osteopontin protein expression in osteoblasts. Interestingly, MLO-Y4 osteocytes formed organized stress fibers after exposure to 24 h of unidirectional shear stress, while the number of dendritic processes per cell increased along with Cox-2 protein levels after 24 h of oscillatory shear stress. Despite these differences, both flow profiles significantly altered osteopontin levels in MLO-Y4 osteocytes. Together these results demonstrate that the profile of fluid shear can induce significantly different responses from osteoblasts and osteocytes.     

Effects of extracellular fiber architecture on cell membrane shear stress in a 3D fibrous matrix

Interstitial fluid flow has been shown to affect the organization and behavior of cells in 3D environments in vivo and in vitro, yet the forces driving such responses are not clear. Due to the complex architecture of the extracellular matrix (ECM) and the difficulty of measuring fluid flow near cells embedded in it, the levels of shear stress experienced by cells in this environment are typically estimated using bulk-averaged matrix parameters such as hydraulic permeability. While this is useful for estimating average stresses, it cannot yield insight into how local matrix fiber architecture-which is cell-controlled in the immediate pericellular environment-affects the local stresses imposed on the cell surface. To address this, we used computational fluid dynamics to study flow through an idealized mesh constructed of a cubic lattice of fibers simulating a typical in vitro collagen gel. We found that, in such high porosity matrices, the fibers strongly affect the flow fields near the cell, with peak shear stresses up to five times higher than those predicted by the Brinkman equation. We also found that minor remodeling of the fibers near the cell surface had major effects on the shear stress profile on the cell. These findings demonstrate the importance of fiber architecture to the fluid forces on a cell embedded in a 3D matrix, and also show how small modifications in the local ECM can lead to large changes in the mechanical environment of the cell.     

Macrorheology and adaptive microrheology of endothelial cells subjected to fluid shear stress

Vascular endothelial cells (ECs) respond to temporal and spatial characteristics of hemodynamic forces by alterations in their adhesiveness to leukocytes, secretion of vasodilators, and permeability to blood-borne constituents. These physiological and pathophysiological changes are tied to adaptation of cell mechanics and mechanotransduction, the process by which cells convert forces to intracellular biochemical signals. The exact time scales of these mechanical adaptations, however, remain unknown. We used particle-tracking microrheology to study adaptive changes in intracellular mechanics in response to a step change in fluid shear stress, which simulates both rapid temporal and steady features of hemodynamic forces. Results indicate that ECs become significantly more compliant as early as 30 s after a step change in shear stress from 0 to 10 dyn/cm² followed by recovery of viscoelastic parameters within 4 min of shearing, even though shear stress was maintained. After ECs were sheared for 5 min, return of shear stress to 0 dyn/cm² in a stepwise manner did not result in any further rheological adaptation. Average vesicle displacements were used to determine time-dependent cell deformation and macrorheological parameters by fitting creep function to a linear viscoelastic liquid model. Characteristic time and magnitude for shear-induced deformation were 3 s and 50 nm, respectively. We conclude that ECs rapidly adapt their mechanical properties in response to shear stress, and we provide the first macrorheological parameters for time-dependent deformations of ECs to a physiological forcing function. Such studies provide insight into pathologies such as atherosclerosis, which may find their origins in EC mechanics. viscoelasticity; atherosclerosis; cell mechanics; particle tracking; mechanotransduction     

Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment

Natural cartilage remodels both in vivo and in vitro in response to mechanical forces and hence mechanical stimulation is believed to have a potential as a tool to modulate extra-cellular matrix synthesis in tissue-engineered cartilage. Fluid-induced shear is known to enhance chondrogenesis on animal cells. A well-defined hydrodynamic environment is required to study the biochemical response to shear of three-dimensional engineered cell systems. We have developed a perfused-column bioreactor in which the culture medium flows through chondrocyte-seeded porous scaffolds, together with a computational fluid-dynamic model of the flow through the constructs' microstructure. A preliminary experiment of human chondrocyte growth under static versus dynamic conditions is described. The median shear stress imposed on the cells in the bioreactor culture, as predicted by the CFD model, is 3 × 10⁻³ Pa (0.03 dyn/cm²) at a flow rate of 0.5 ml/min corresponding to an inlet fluid velocity of 44.2 μm/s. Providing a fluid-dynamic environment to the cells yielded significant differences in cell morphology and in construct structure. Received: 22 December 2001 / Accepted: 18 February 2002     

A new flow chamber for the study of shear stress and transmural pressure upon cells adhering to a porous biomaterial

Biomaterials used in some biomedical devices are porous and exposed to normal and tangential flow of biofluids. To examine the influence of flow induced forces on the morphology and the biochemical responses of cells adhering to such biomaterials, a Hele-Shaw cell with a porous bottom wall was designed and characterized experimentally. Theoretical predictions for the flow in the chamber are provided and allow to quantify the shear stress and/or transmural pressure exerted on cells. It is thus possible to follow up continuously the shape changes of cells that are adherent on a permeable membrane used in bioreactors.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Molecular basis of the effects of shear stress on vascular endothelial cells

Blood vessels are constantly exposed to hemodynamic forces in the form of cyclic stretch and shear stress due to the pulsatile nature of blood pressure and flow. Endothelial cells (ECs) are subjected to the shear stress resulting from blood flow and are able to convert mechanical stimuli into intracellular signals that affect cellular functions, e.g., proliferation, apoptosis, migration, permeability, and remodeling, as well as gene expression. The ECs use multiple sensing mechanisms to detect changes in mechanical forces, leading to the activation of signaling networks. The cytoskeleton provides a structural framework for the EC to transmit mechanical forces between its luminal, abluminal and junctional surfaces and its interior, including the cytoplasm, the nucleus, and focal adhesion sites. Endothelial cells also respond differently to different modes of shear forces, e.g., laminar, disturbed, or oscillatory flows. In vitro studies on cultured ECs in flow channels have been conducted to investigate the molecular mechanisms by which cells convert the mechanical input into biochemical events, which eventually lead to functional responses. The knowledge gained on mechano-transduction, with verifications under in vivo conditions, will advance our understanding of the physiological and pathological processes in vascular remodeling and adaptation in health and disease.     

Mechanotransduction in blood cells

Blood cells are subjected to various mechanical forces; including pressure, flow, shear force, gravity, and forces acting against them with varying stiffness (eg. blood vessel wall). Scientists have discovered that these forces have profound effects on cellular growth, differentiation, secretion of cytokines, cell death, and migration. These processes are called mechanotransduction, a conversion of mechanical forces to biochemical signals. In this article the author reviews biophysical forces that affect biological functions of blood cells and their responses in normal physiology and pathophysiology. Although input (forces) and output (cellular responses) have been well studied by utilizing recently developed various force-generating devices, the molecular mechanism of mechanotransudction is still a mystery. This is because reconstructing molecular interaction in the presence of mechanical forces in vitro is highly challenging and until now the molecular dynamics involved in structural changes caused by these forces are largely unknown. Nevertheless, the author has reviewed a few examples of potential structural effects on the molecular mechanism of mechanotransduction.     

Biorheological views of endothelial cell responses to mechanical stimuli

Vascular endothelial cells are located at the innermost layer of the blood vessel wall and are always exposed to three different mechanical forces: shear stress due to blood flow, hydrostatic pressure due to blood pressure and cyclic stretch due to vessel deformation. It is well known that endothelial cells respond to these mechanical forces and change their shapes, cytoskeletal structures and functions. In this review, we would like to mainly focus on the effects of shear stress and hydrostatic pressure on endothelial cell morphology. After applying fluid shear stress, cultured endothelial cells show marked elongation and orientation in the flow direction. In addition, thick stress fibers of actin filaments appear and align along the cell long axis. Thus, endothelial cell morphology is closely related to the cytoskeletal structure. Further, the dynamic course of the morphological changes is shown and the related events such as changes in mechanical stiffness and functions are also summarized. When endothelial cells were exposed to hydrostatic pressure, they exhibited a marked elongation and orientation in a random direction, together with development of centrally located, thick stress fibers. Pressured endothelial cells also exhibited a multilayered structure with less expression of VE-cadherin unlike under control conditions. Simultaneous loading of hydrostatic pressure and shear stress inhibited endothelial cell multilayering and induced elongation and orientation of endothelial cells with well-developed VE-cadherin in a monolayer, which suggests that for a better understanding of vascular endothelial cell responses one has to take into consideration the combination of the different mechanical forces such as exist under in vivo mechanical conditions.     

Fluid shear stress and the vascular endothelium: for better and for worse

As blood flows, the vascular wall is constantly subjected to physical forces, which regulate important physiological blood vessel responses, as well as being implicated in the development of arterial wall pathologies. Changes in blood flow, thus generating altered hemodynamic forces are responsible for acute vessel tone regulation, the development of blood vessel structure during embryogenesis and early growth, as well as chronic remodeling and generation of adult blood vessels. The complex interaction of biomechanical forces, and more specifically shear stress, derived by the flow of blood and the vascular endothelium raise many yet to be answered questions:How are mechanical forces transduced by endothelial cells into a biological response, and is there a "shear stress receptor"?Are "mechanical receptors" and the final signaling pathways they evoke similar to other stimulus-response transduction systems?How do vascular endothelial cells differ in their response to physiological or pathological shear stresses?Can shear stress receptors or shear stress responsive genes serve as novel targets for the design of diagnostic and therapeutic modalities for cardiovascular pathologies?The current review attempts to bring together recent findings on the in vivo and in vitro responses of the vascular endothelium to shear stress and to address some of the questions raised above.     

Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells

Numerous cellular biochemical responses to mechanical loading are transient, indicating a cell's ability to adapt its behavior to a new mechanical environment. Since load-induced cellular deformation can initiate these biochemical responses, the overall goal of this study was to investigate the adaptation of global, or whole-cell, mechanical behavior, i.e., cellular deformability, in response to mechanical loading for osteoblastic cells. Confluent cell cultures were subjected to 1 or 2 Pa flow-induced shear stress for 2 h. Whole-cell mechanical behavior was then measured for individual cells using an atomic force microscope. Compared to cells maintained under static conditions, whole-cell stiffness was 1.36-fold (p=0.006) and 1.70-fold (p<0.001) greater for cells exposed to 1 and 2 Pa shear loading, respectively. The increase in shear stress magnitude from 1 to 2 Pa also caused a statistically significant, 1.25-fold increase in cell stiffness (p=0.02). Increases in cell stiffness were not altered in either flow group for 70 min after flow was terminated (p=0.15). Flow-induced rearrangement of the actin cytoskeleton was also maintained for at least 90 min after flow was terminated. Taken together, these findings support the hypothesis that cells become mechanically adapted to their mechanical environment via cytoskeletal modifications. Accordingly, cellular mechanical adaptation may play a key role in regulation of cellular mechanosensitivity and the related effects on tissue structure and function.     

The shear stress of it all: the cell membrane and mechanochemical transduction

As the inner lining of the vessel wall, vascular endothelial cells are poised to act as a signal transduction interface between haemodynamic forces and the underlying vascular smooth-muscle cells. Detailed analyses of fluid mechanics in atherosclerosis-susceptible regions of the vasculature reveal a strong correlation between endothelial cell dysfunction and areas of low mean shear stress and oscillatory flow with flow recirculation. Conversely, steady shear stress stimulates cellular responses that are essential for endothelial cell function and are atheroprotective. The molecular basis of shear-induced mechanochemical signal transduction and the endothelium's ability to discriminate between flow profiles remains largely unclear. Given that fluid shear stress does not involve a traditional receptor/ligand interaction, identification of the molecule(s) responsible for sensing fluid flow and mechanical force discrimination has been difficult. This review will provide an overview of the haemodynamic forces experienced by the vascular endothelium and its role in localizing atherosclerotic lesions within specific regions of the vasculature. Also reviewed are several recent lines of evidence suggesting that both changes in membrane microviscosity linked to heterotrimeric G proteins, and the transmission of tension across the cell membrane to the cell-cell junction where known shear-sensitive proteins are localized, may serve as the primary force-sensing elements of the cell.     

Differentiation of stem/progenitor cells into vascular cells in response to fluid mechanical forces

Vascular functions are regulated not only by chemical mediators, such as hormones, cytokines, and neurotransmitters, but by mechanical hemodynamic forces generated by blood flow and blood pressure. The mechanical force-mediated regulation is based on the ability of vascular cells, including endothelial cells and smooth muscle cells, to recognize fluid mechanical forces, i.e., the shear stress produced by flowing blood and the cyclic strain generated by blood pressure, and to transmit the signals into the cell interior, where they trigger cell responses that involve changes in cell morphology, cell function, and gene expression. Recent studies have revealed that immature cells, such as endothelial progenitor cells (EPCs) and embryonic stem (ES) cells, as well as adult vascular cells, respond to fluid mechanical forces. Shear stress and cyclic strain promote the proliferation and differentiation of EPCs and ES cells into vascular cells and enhance their ability to form new vessels. Even more recently, attempts have been made to apply fluid mechanical forces to EPCs and ES cells cultured on polymer tubes and develop tissue-engineered blood vessel grafts that have a structure and function similar to that of blood vessels in vivo. This review summarizes the current state of knowledge concerning the mechanobiological responses of stem/progenitor cells and its potential applications to tissue engineering.     

Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport

Fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. In addition to membrane shear stress, loading-induced fluid flow will enhance chemotransport due to convection or mass transport thereby affecting the biochemical environment surrounding the cell. This study investigated the role of oscillating fluid flow induced shear stress and chemotransport in cellular mechanotransduction mechanisms in bone. Intracellular calcium mobilization and prostaglandin E(2) (PGE(2)) production were studied with varying levels of shear stress and chemotransport. In this study MC3T3-E1 cells responded to oscillating fluid flow with both an increase in intracellular calcium concentration ([Ca(2+)](i)) and an increase in PGE(2) production. These fluid flow induced responses were modulated by chemotransport. The percentage of cells responding with an [Ca(2+)](i) oscillation increased with increasing flow rate, as did the production of PGE(2). In addition, depriving the cells of nutrients during fluid flow resulted in an inhibition of both [Ca(2+)](i) mobilization and PGE(2) production. These data suggest that depriving the cells of a yet to be determined biochemical factor in media affects the responsiveness of bone cells even at a constant peak shear stress. Chemotransport alone will not elicit a response, but it appears that sufficient nutrient supply or waste removal is needed for the response to oscillating fluid flow induced shear stress.     

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering. Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes, including endothelial cells (ECs) and smooth muscle cells. The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces, such as shear stress and cyclic strain. More recently, studies have shown that shear stress can influence the differentiation of stem cells toward ECs. A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation. This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs. Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed. Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.     

Differential membrane potential and ion current responses to different types of shear stress in vascular endothelial cells

Vascular endothelial cells (ECs) distinguish among and respond differently to different types of fluid mechanical shear stress. Elucidating the mechanisms governing this differential responsiveness is the key to understanding why early atherosclerotic lesions localize preferentially in arterial regions exposed to low and/or oscillatory flow. An early and very rapid endothelial response to flow is the activation of flow-sensitive K⁺ and Cl^– channels that respectively hyperpolarize and depolarize the cell membrane and regulate several important endothelial responses to flow. We have used whole cell current- and voltage-clamp techniques to demonstrate that flow-sensitive hyperpolarizing and depolarizing currents respond differently to different types of shear stress in cultured bovine aortic ECs. A steady shear stress level of 10 dyn/cm² activated both currents leading to rapid membrane hyperpolarization that was subsequently reversed to depolarization. In contrast, a steady shear stress of 1 dyn/cm² only activated the hyperpolarizing current. A purely oscillatory shear stress of 0 ± 10 dyn/cm² with an oscillation frequency of either 1 or 0.2 Hz activated the hyperpolarizing current but only minimally the depolarizing current, whereas a 5-Hz oscillation activated neither current. These results demonstrate for the first time that flow-activated ion currents exhibit different sensitivities to shear stress magnitude and oscillation frequency. We propose that flow-sensitive ion channels constitute components of an integrated mechanosensing system that, through the aggregate effect of ion channel activation on cell membrane potential, enables ECs to distinguish among different types of flow. ion channels; atherosclerosis; mechanotransduction