Model for the alignment of actin filaments in endothelial cells subjected to fluid shear stress

Cultured vascular endothelial cells undergo significant morphological changes when subjected to sustained fluid shear stress. The cells elongate and align in the direction of applied flow. Accompanying this shape change is a reorganization at the intracellular level. The cytoskeletal actin filaments reorient in the direction of the cells' long axis. How this external stimulus is transmitted to the endothelial cytoskeleton still remains unclear. In this article. we present a theoretical model accounting for the cytoskeletal reorganization under the influence of fluid shear stress. We develop a system of integro-partial-differential equations describing the dynamics of actin filaments, the actin-binding proteins, and the drift of transmembrane proteins due to the fluid shear forces applied on the plasma membrane. Numerical simulations of the equations show that under certain conditions, initially randomly oriented cytoskeletal actin filaments reorient in structures parallel to the externally applied fluid shear forces. Thus, the model suggests a mechanism by which shear forces acting on the cell membrane can be transmitted to the entire cytoskeleton via molecular interactions alone.     

Effect of the stress phase angle on the strain energy density of the endothelial plasma membrane

Endothelial cells are simultaneously exposed to the mechanical forces of fluid wall shear stress (WSS) imposed by blood flow and solid circumferential stress (CS) induced by the blood vessel's elastic response to the pressure pulse. Experiments have demonstrated that these combined forces induce unique endothelial biomolecular responses that are not characteristic of either driving force alone and that the temporal phase angle between WSS and CS, referred to as the stress phase angle, modulates endothelial responses. In this article, we provide the first theoretical model to examine the combined forces of WSS and CS on a model of the endothelial cell plasma membrane. We focus on the strain energy density of the membrane that modulates the opening of ion channels that can mediate signal transduction. The model shows a significant influence of the stress phase angle on the strain energy density at the upstream and downstream ends of the cell where mechanotransduction is most likely to occur.     

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.     

Molecular and mechanical bases of focal lipid accumulation in arterial wall

Mechanical forces such as shear stress can modulate gene and protein expressions and hence cellular functions by activating membrane sensors and intracellular signaling. Using cultured endothelial cells, we have shown that laminar shear stress causes a transient increase in monocyte chemotactic protein-1 (MCP-1) expression, which involves the Ras-MAP kinase signaling pathway. We have demonstrated that integrins and the vascular endothelial growth factor receptor Flk-1 can sense shear stress, with integrins being upstream to Flk-1. Other possible membrane components involved in the sensing of shear stress include G-protein coupled receptors, intercellular junction proteins, membrane glycocalyx, and the lipid bilayer. Mechano-transduction involves the participation of a multitude of sensors, signaling molecules, and genes. Microarray analysis has demonstrated that shear stress can upregulate and downregulate different genes. Sustained shear stress downregulates atherogenic genes (e.g., MCP-1 and the genes that facilitate lipid accumulation) and upregulates growth-arrest genes. In contrast, disturbed flow observed at branch points and simulated in step-flow channels causes sustained activation of MCP-1 and the genes facilitating cell turnover and lipid accumulation. These findings provide a molecular basis for the explanation of the preferential localization of atherosclerotic lesions at regions of disturbed flow, such as the arterial branch points. The combination of mechanics and biology (from molecules-cells to organs-systems) can help to elucidate the physiological processes of mechano-chemical transduction and improving the methods of the management of important clinical conditions such as coronary artery disease.     

Mechanical regulation of epigenetics in vascular biology and pathobiology

Vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) are constantly exposed to haemodynamic forces, including blood flow‐induced fluid shear stress and cyclic stretch from blood pressure. These forces modulate vascular cell gene expression and function and, therefore, influence vascular physiology and pathophysiology in health and disease. Epigenetics, including DNA methylation, histone modification/chromatin remodelling and RNA‐based machinery, refers to the study of heritable changes in gene expression that occur without changes in the DNA sequence. The role of haemodynamic force‐induced epigenetic modifications in the regulation of vascular gene expression and function has recently been elucidated. This review provides an introduction to the epigenetic concepts that relate to vascular physiology and pathophysiology. Through the studies of gene expression, cell proliferation, angiogenesis, migration and pathophysiological states, we present a conceptual framework for understanding how mechanical force‐induced epigenetic modifications work to control vascular gene expression and function and, hence, the development of vascular disorders. This research contributes to our knowledge of how the mechanical environment impacts the chromatin state of ECs and VSMCs and the consequent cellular behaviours.     

Cytoskeletal regulation of pulmonary vascular permeability.

The endothelial cell (EC) lining of the pulmonary vasculature forms a semipermeable barrier between the blood and the interstitium of the lung. Disruption of this barrier occurs during inflammatory disease states such as acute lung injury and acute respiratory distress syndrome and results in the movement of fluid and macromolecules into the interstitium and pulmonary air spaces. These processes significantly contribute to the high morbidity and mortality of patients afflicted with acute lung injury. The critical importance of pulmonary vascular barrier function is shown by the balance between competing EC contractile forces, which generate centripetal tension, and adhesive cell-cell and cell-matrix tethering forces, which regulate cell shape. Both competing forces in this model are intimately linked through the endothelial cytoskeleton, a complex network of actin microfilaments, microtubules, and intermediate filaments, which combine to regulate shape change and transduce signals within and between EC. A key EC contractile event in several models of agonist-induced barrier dysfunction is the phosphorylation of regulatory myosin light chains catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase and/or through the activity of the Rho/Rho kinase pathway. Intercellular contacts along the endothelial monolayer consist primarily of two types of complexes (adherens junctions and tight junctions), which link to the actin cytoskeleton to provide both mechanical stability and transduction of extracellular signals into the cell. Focal adhesions provide additional adhesive forces in barrier regulation by forming a critical bridge for bidirectional signal transduction between the actin cytoskeleton and the cell-matrix interface. Increasingly, the effects of mechanical forces such as shear stress and ventilator-induced stretch on EC barrier function are being recognized. The critical role of the endothelial cytoskeleton in integrating these multiple aspects of pulmonary vascular permeability provides a fertile area for the development of clinically important barrier-modulating therapies.     

The role of endothelial calcium and nitric oxide in the localisation of atherosclerosis

A mathematical model of endothelial cell calcium signalling and nitric oxide synthesis under flow conditions is presented. The model is coupled to two important environmental stimuli for endothelial cells: the frictional shear stress exerted on the cell membrane by the blood flow; and the binding of adenosine triphosphate in the bloodstream to cell surface receptors. These stimuli are closely linked to haemodynamic flow conditions and are, in general, spatially varying, allowing the cellular response in different regions of the endothelium to be evaluated. This is used to indicate which areas of the artery wall experience reduced bioavailability of nitric oxide, which is a major factor in the onset of atherosclerosis. The model thus directly addresses the key issue of the causative link, and its underlying biochemical mechanisms, between incidence of atherosclerosis and regions of low wall shear stress (WSS). Model results show that intracellular levels of free calcium and endothelial nitric oxide synthase are lower in endothelial cells adjacent to a region of recirculating flow than in cells adjacent to regions of fully developed arterial flow. This will lead to deficient levels of nitric oxide in the recirculation zone and hence a potentially elevated risk of developing atherosclerotic plaque. This is consistent with the observed spatial correlation between atherosclerosis and regions of disturbed blood flow and low WSS, and provides a mechanism for the localisation of the disease to sites such as arterial bifurcations and bends.     

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.     

On the preservation of vessel bifurcations during flow-mediated angiogenic remodelling

During developmental angiogenesis, endothelial cells respond to shear stress by migrating and remodelling the initially hyperbranched plexus, removing certain vessels whilst maintaining others. In this study, we argue that the key regulator of vessel preservation is cell decision behaviour at bifurcations. At flow-convergent bifurcations where migration paths diverge, cells must finely tune migration along both possible paths if the bifurcation is to persist. Experiments have demonstrated that disrupting the cells’ ability to sense shear or the junction forces transmitted between cells impacts the preservation of bifurcations during the remodelling process. However, how these migratory cues integrate during cell decision making remains poorly understood. Therefore, we present the first agent-based model of endothelial cell flow-mediated migration suitable for interrogating the mechanisms behind bifurcation stability. The model simulates flow in a bifurcated vessel network composed of agents representing endothelial cells arranged into a lumen which migrate against flow. Upon approaching a bifurcation where more than one migration path exists, agents refer to a stochastic bifurcation rule which models the decision cells make as a combination of flow-based and collective-based migratory cues. With this rule, cells favour branches with relatively larger shear stress or cell number. We found that cells must integrate both cues nearly equally to maximise bifurcation stability. In simulations with stable bifurcations, we found competitive oscillations between flow and collective cues, and simulations that lost the bifurcation were unable to maintain these oscillations. The competition between these two cues is haemodynamic in origin, and demonstrates that a natural defence against bifurcation loss during remodelling exists: as vessel lumens narrow due to cell efflux, resistance to flow and shear stress increases, attracting new cells to enter and rescue the vessel from regression. Our work provides theoretical insight into the role of junction force transmission has in stabilising vasculature during remodelling and as an emergent mechanism to avoid functional shunting.     

High Glucose Attenuates Shear-Induced Changes in Endothelial Hydraulic Conductivity by Degrading the Glycocalyx

Diabetes mellitus is a risk factor for cardiovascular disease; however, the mechanisms through which diabetes impairs homeostasis of the vasculature have not been completely elucidated. The endothelium interacts with circulating blood through the surface glycocalyx layer, which serves as a mechanosensor/transducer of fluid shear forces leading to biomolecular responses. Atherosclerosis localizes typically in regions of low or disturbed shear stress, but in diabetics, the distribution is more diffuse, suggesting that there is a fundamental difference in the way cells sense shear forces. In the present study, we examined the effect of hyperglycemia on mechanotranduction in bovine aortic endothelial cells (BAEC). After six days in high glucose media, we observed a decrease in heparan sulfate content coincident with a significant attenuation of the shear-induced hydraulic conductivity response, lower activation of eNOS after exposure to shear, and reduced cell alignment with shear stress. These studies are consistent with a diabetes-induced change to the glycocalyx altering endothelial response to shear stress that could affect the distribution of atherosclerotic plaques.     

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.     

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.     

Unusual transduction response of progenitor-derived and mature endothelial cells exposed to laminar pulsatile shear stress

Progenitor-derived endothelial cells (PDECs) isolated from human umbilical cord blood generate a great hope in the fields of vascular tissue engineering. Endothelial cells subjected to shear stress convert mechanical stimuli into intracellular signals that affect cellular functions. It is essential to ensure that PDECs are able to sense shear stress as mature endothelial cells from human saphenous veins (HSVECs) do with mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB signal transduction pathways. HSVECs and PDECs were seeded on glass slides coated with gelatin and exposed to 12dyn/cm(2) in a parallel-plate flow chamber. In both cell types, shear stress activated extracellular signal-related kinase (ERK)1/2 with a rapid time course (maximum 5min) followed by a reduced phosphorylation, and p38 pathway. c-Jun N-terminal protein kinase (JNK) phosphorylation is observed only in PDECs. With respect to NF-kappaB translocation to the nucleus, the NF-kappaB pathway is not activated by flow in HSVECs and PDECs although interleukin-1alpha (IL-1alpha) activates this pathway in both cell types. In our experimental conditions, shear stress does not modify the nuclear translocation of NF-kappaB in HSVECs after IL-1alpha stimulation. It can be stated that PDECs are shear stress sensitive and capable of signal transduction as mature HSVECs are, despite the unusual transduction response of both cell types.     

Fluid shear stress increases membrane fluidity in endothelial cells: a study with DCVJ fluorescence

Fluid shear stress (FSS) has been shown to be an ubiquitous stimulator of mammalian cell metabolism. Although many of the intracellular signal transduction pathways have been characterized, the primary mechanoreceptor for FSS remains unknown. One hypothesis is that the cytoplasmic membrane acts as the receptor for FSS, leading to increased membrane fluidity, which in turn leads to the activation of heterotrimetric G proteins (13). 9-(Dicyanovinyl)-julolidine (DCVJ) is a fluorescent probe that integrates into the cell membrane and changes its quantum yield with the viscosity of the environment. In a parallel-plate flow chamber, confluent layers of DCVJ-labeled human endothelial cells were exposed to different levels of FSS. With increased FSS, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of FSS caused an approximately linear drop of fluorescence within 5 s, showing fast and almost full recovery after shear cessation. A linear dose-response relationship between shear stress and membrane fluidity changes was observed. The average fluidity increase over the entire cell monolayer was 22% at 26 dyn/cm(2). This study provides evidence for a link between FSS and membrane fluidity, and suggests that the membrane is an important flow mechanosensor of the cell.     

Microvascular Endothelial Cells Migrate Upstream and Align Against the Shear Stress Field Created by Impinging Flow

At present, little is known about how endothelial cells respond to spatial variations in fluid shear stress such as those that occur locally during embryonic development, at heart valve leaflets, and at sites of aneurysm formation. We built an impinging flow device that exposes endothelial cells to gradients of shear stress. Using this device, we investigated the response of microvascular endothelial cells to shear-stress gradients that ranged from 0 to a peak shear stress of 9–210 dyn/cm². We observe that at high confluency, these cells migrate against the direction of fluid flow and concentrate in the region of maximum wall shear stress, whereas low-density microvascular endothelial cells that lack cell-cell contacts migrate in the flow direction. In addition, the cells align parallel to the flow at low wall shear stresses but orient perpendicularly to the flow direction above a critical threshold in local wall shear stress. Our observations suggest that endothelial cells are exquisitely sensitive to both magnitude and spatial gradients in wall shear stress. The impinging flow device provides a, to our knowledge, novel means to study endothelial cell migration and polarization in response to gradients in physical forces such as wall shear stress.     

Microvascular Endothelial Cells Migrate Upstream and Align Against the Shear Stress Field Created by Impinging Flow

At present, little is known about how endothelial cells respond to spatial variations in fluid shear stress such as those that occur locally during embryonic development, at heart valve leaflets, and at sites of aneurysm formation. We built an impinging flow device that exposes endothelial cells to gradients of shear stress. Using this device, we investigated the response of microvascular endothelial cells to shear-stress gradients that ranged from 0 to a peak shear stress of 9–210 dyn/cm². We observe that at high confluency, these cells migrate against the direction of fluid flow and concentrate in the region of maximum wall shear stress, whereas low-density microvascular endothelial cells that lack cell-cell contacts migrate in the flow direction. In addition, the cells align parallel to the flow at low wall shear stresses but orient perpendicularly to the flow direction above a critical threshold in local wall shear stress. Our observations suggest that endothelial cells are exquisitely sensitive to both magnitude and spatial gradients in wall shear stress. The impinging flow device provides a, to our knowledge, novel means to study endothelial cell migration and polarization in response to gradients in physical forces such as wall shear stress.     

Correspondence of low mean shear and high harmonic content in the porcine iliac arteries

BACKGROUND: Temporal variations in shear stress have been suggested to affect endothelial cell biology. To better quantify the range of dynamic shear forces that occur in vivo, the frequency content of shear variations that occur naturally over a cardiac cycle in the iliac arteries was determined. METHOD OF APPROACH: Computational fluid dynamic calculations were performed in six iliac arteries from three juvenile swine. Fourier analysis of the time-varying shear stress computed at the arterial wall was performed to determine the prevalence of shear forces occurring at higher frequencies in these arteries. RESULTS: While most of each artery experienced shear forces predominantly at the frequency of the heart rate, the frequency spectra at certain regions were dominated by shear forces at higher frequencies. Regions whose frequency spectra were dominated by higher harmonics generally experienced lower mean shear stress. The negative correlation between shear and dominant harmonic was significant (p=0.002). CONCLUSIONS: Since lesion development typically occurs in regions experiencing low time-average shear stress, this result suggests that the frequency content of the shear exposure may also be a contributing factor in lesion development. A better understanding of the vascular response to shear components of different frequencies might help rationalize the notion of "disturbed flow" as a hemodynamic entity.     

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm² shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.