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Atherosclerosis preferentially develops at branches and curvatures of the arterial tree, where blood flow is disturbed from a laminar pattern, and wall shear stress is non-uniform and has an irregular distribution. Vascular endothelial cells (ECs), which form an interface between the flowing blood and the vessel wall, are exposed to blood flow-induced shear stress. There is increasing evidence suggesting that laminar blood flow and sustained high shear stress modulate the expression of EC genes and proteins that function to protect against atherosclerosis; in contrast, disturbed blood flow and the associated low and reciprocating shear stress upregulate proatherosclerotic genes and proteins that promote development of atherosclerosis. Understanding of the effects of shear stress on ECs will provide mechanistic insights into its role in the pathogenesis of atherosclerosis. The aim of this review article is to summarize current findings on the effects of shear stress on ECs, in terms of their signal transduction, gene expression, structure, and function. These endothelial cellular responses have important relevance to understanding the pathophysiological effects of altered shear stress associated with atherosclerosis and thrombosis and their complications.

Cyclic mechanical stretch associated with pulsatile blood pressure can modulate cytoskeletal remodeling and intracellular signaling in vascular endothelial cells. The aim of this study was to evaluate the role of stretch-induced actin stress fiber orientation in intracellular signaling involving the activation of c-jun N-terminal kinase (JNK) in bovine aortic endothelial cells. A stretch device was designed with the capability of applying cyclic uniaxial and equibiaxial stretches to cultured endothelial cells, as well as changing the direction of cyclic uniaxial stretch. In response to 10% cyclic equibiaxial stretch, which did not result in stress fiber orientation, JNK activation was elevated for up to 6 h. In response to 10% cyclic uniaxial stretch, JNK activity was only transiently elevated, followed by a return to basal level as the actin stress fibers became oriented perpendicular to the direction of stretch. After the stress fibers had aligned perpendicularly and the JNK activity had subsided, a 90-degree change in the direction of cyclic uniaxial stretch reactivated JNK, and this activation again subsided as stress fibers became re-oriented perpendicular to the new direction of stretch. Disrupting actin filaments with cytochalasin D blocked the stress fiber orientation in response to cyclic uniaxial stretch and it also caused the uniaxial stretch-induced JNK activation to become sustained. These results suggest that stress fiber orientation perpendicular to the direction of stretch provides a mechanism for both structural and biochemical adaptation to cyclic mechanical stretch.

Transforming growth factor-beta1 (TGF-beta1) is known to induce phenotypic modulation of mesenchymal cells to SMCs. However, the intracellular signals regulating induction of the SMC phenotype of mesenchymal cells have not been fully clarified. In the present study, we examined the role of the mitogen-activated protein kinase (MAPK) superfamily and phosphatidylinositol 3-kinase (PI3K)/Akt in the TGF-beta1-mediated phenotypic modulation of 10T1/2 mesenchymal cells to SMCs characterized by the expression of SMC-specific markers, including smooth muscle alpha-actin (SMalpha-actin), myosin heavy chain (SM-MHC), and protein 22-alpha (SM22alpha). The results showed the following: (1) TGF-beta1 induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells in a time-dependent manner. (2) TGF-beta1 induced biphasic increases in extracellular signal-regulated kinase (ERK), p38 MAPK, c-Jun-NH2-terminal kinase (JNK), and Akt phosphorylation. (3) The inhibitor for PI3K/Akt (i.e., LY294002), but not those for MAPKs (i.e., SB203580, PD98059, and SP600125), attenuated the TGF-beta1-induced SMalpha-actin and SM-MHC expressions in 10T1/2 cells; in addition, transfection of 10T1/2 cells with the Akt-specific small interfering RNA (siRNA) significantly reduced their SMalpha-actin and SM-MHC expressions. (4) LY294002 and the Akt-specific siRNA inhibited the TGF-beta1-induced SM22alpha gene expression and promoter activity, suggesting that the TGF-beta1-induced gene expression was mediated by PI3K/Akt at the transcriptional level. (5) LY294002 inhibited the TGF-beta1-induced gene expression and DNA binding activity of serum response factor (SRF). These results indicate that TGF-beta1 is capable of inducing the SMC phenotype of 10T1/2 cells and that this induction is mediated through the PI3K/Akt signaling pathway.

We investigate the role of shear stress in regulating the gene expression in endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNF-alpha). ECs were kept in static condition or pre-exposed to a high level (HSS, 20 dynes/cm2) or a low level of shear stress (LSS, 0.5 dynes/cm2) for 24 h, and TNF-alpha was added under static condition for 4 h. In static ECs, DNA microarray showed that TNF-alpha caused a significant increase in expression of 102 genes and a significant decrease in expression of 12 genes. Pre-shearing of ECs decreased the TNF-alpha-responsiveness of many pro-inflammatory, pro-coagulant, proliferative, and pro-apoptotic genes, whereas it increased the responsiveness of some antioxidant, anti-coagulant, and anti-apoptotic genes. LSS showed less regulatory effects than HSS on EC gene expression in response to TNF-alpha. The microarray data were confirmed by reverse-transcription polymerase chain reaction for 64 selected genes. Pre-shearing of ECs at HSS significantly inhibited the TNF-alpha-induced p65 and p50 mRNA expressions and nuclear factor-kappaB (NF-kappaB)-DNA binding activity. Inhibition of NF-kappaB activity with the p65-antisense or lactacystin under static condition blocked the expression of most of the genes that are TNF-alpha-inducible and shear stress-down-regulated. Our findings suggest that laminar shear stress serves protective functions against atherogenesis.

Objectives— Vascular endothelial cells (ECs) are influenced by shear stress and neighboring smooth muscle cells (SMCs). We investigated the inflammation-relevant gene expression in EC/SMC cocultures under static condition and in response to shear stress. Materials and Methods— Under static condition, DNA microarrays and reverse-transcription polymerase chain reaction identified 23 inflammation-relevant genes in ECs whose expression was significantly affected by coculture with SMCs, with 18 upregulated and 5 downregulated. Application of shear stress (12 dynes/cm 2 ) to the EC side of the coculture for 6 hours inhibited most of the proinflammatory gene expressions in ECs induced by coculture with SMCs. Inhibition of nuclear factor-κB (NF-κB) activation by the p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced EC expression of proinflammatory genes, indicating that the NF-κB binding sites in the promoters of these genes play a significant role in their expression as a result of coculture with SMCs. Chromatin immunoprecipitation assays demonstrated the in vivo regulation of NF-κB recruitment to selected target promoters. Shear stress inhibited the SMC coculture-induced NF-κB activation in ECs and monocytic THP-1 cell adhesion to ECs. Conclusions— Our findings suggest that shear stress plays an inhibitory role in the proinflammatory gene expression in ECs located in close proximity to SMCs.

Vascular endothelial cells (ECs) are subjected to shear stress and cytokine stimulation. We studied the interplay between shear stress and cytokine in modulating the expression of adhesion molecule genes and the adhesive function of ECs. Shear stress (20 dynes/cm2) was applied to ECs prior to or following the addition of tumor necrosis factor (TNF)-α. Shear stress increased the TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-α-induced expression of vascular adhesion molecule-1 (VCAM-1). The TNF-α-induced increase in EC adhesiveness for monocytic THP-1 cells was reduced by shear stress. After 24-h pre-shearing followed by 1 h of static incubation, the effect of pre-shearing on TNF-α-induced ICAM-1 mRNA expression vanished. The recovery of the TNF-α-induced VCAM-1 mRNA expression following pre-shearing, however, required a static incubation time of >6 h (completely recovery at 24 h). Pre- and post-shearing caused a reduction in the nuclear factor (NF)-κB-DNA binding activity induced by TNF-α in the EC nucleus. Our findings suggest that shear stress plays differential roles in modulating the TNF-α-induced EC expressions of ICAM-1 and VCAM-1 genes, which serve similar functions in vascular biology.

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.

Objective—Vascular endothelial cells (ECs) are subjected to shear stress and cytokine stimulation. We studied the interplay between shear stress and cytokine in modulating the expression of adhesion molecule genes in ECs.Methods and Results—Shear stress (20 dynes/cm2) was applied to ECs prior to and/or following the addition of tumor necrosis factor (TNF)-α. Shear stress increased the TNF-α–induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-α–induced expression of vascular adhesion molecule-1 (VCAM-1) and E-selectin. Transfection studies using promoter reporter gene constructs of ICAM-1, VCAM-1, and E-selectin demonstrated that these shear stress modulations of gene expression occur at the transcriptional levels. After 24-hour preshearing followed by 1 hour of static incubation, the effect of preshearing on TNF-α–induced ICAM-1 mRNA expression vanished. The recovery of the TNF-α–induced VCAM-1 and E-selectin mRNA expressions following preshearing, however, required a static incubation time of >6 hours (complete recovery at 24 hours). Pre- and postshearing caused a reduction in the nuclear factor-κB-DNA binding activity induced by TNF-α in the EC nucleus.Conclusions—Our findings suggest that shear stress plays differential roles in modulating the TNF-α–induced expressions of ICAM-1 versus VCAM-1 and E-selectin genes in ECs.

The preferential adhesion of monocytes to vascular endothelial cells (ECs) at regions near branches and curvatures of the arterial tree, where flow is disturbed, suggests that hemodynamic conditions play significant roles in monocyte adhesion. The present study aims to elucidate the effects of disturbed flow on monocyte adhesion to ECs and the adhesive properties of ECs. We applied, for the first time, the micron-resolution particle image velocimetry (microPIV) technique to analyze the characteristics of the disturbed flow produced in our vertical-step flow (VSF) chamber. The results demonstrated the existence of a higher near-wall concentration and a longer residence time of the monocytic analog THP-1 cells near the step and the reattachment point. THP-1 cells showed prominent adhesion to ECs pretreated with TNFalpha in the regions near the step and the reattachment point, but they showed virtually no adhesion to un-stimulated ECs. Pre-incubation of the TNFalpha-treated ECs with antibodies against intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin inhibited the THP-1 adhesion; the maximal inhibition was observed with a combination of these antibodies. Pre-exposure of ECs to disturbed flow in VSF for 24 h led to significant increases in their surface expressions of ICAM-1 and E-selectin, but not VCAM-1, and in the adhesion of THP-1 cells. Our findings demonstrate the importance of complex flow environment in modulating the adhesive properties of vascular endothelium and consequently monocyte adhesion in regions of prevalence of atherosclerotic lesions.

Vascular endothelial cells (ECs), which exist in close proximity to vascular smooth muscle cells (SMCs), are constantly subjected to blood flow–induced shear stress. Although the effect of shear stress on endothelial biology has been extensively studied, the influence of SMCs on endothelial response to shear stress remains largely unexplored. We examined the potential role of SMCs in regulating the shear stress–induced gene expression in ECs, using a parallel-plate coculture flow system in which these 2 types of cells were separated by a porous membrane. In this coculture system, SMCs tended to orient perpendicularly to the flow direction, whereas the ECs were elongated and aligned with the flow direction. Under static conditions, coculture with SMCs induced EC gene expression of intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin, while attenuating EC gene expression of endothelial nitric oxide synthase (eNOS). Shear stress significantly inhibited SMC-induced adhesion molecule gene expression. These EC responses under static and shear conditions were not observed in the absence of close communication between ECs and SMCs, and they were also not observed when ECs were cocultured with fibroblasts instead of SMCs. Our findings indicate that under static conditions, coculture with SMCs induces ICAM-1, VCAM-1, and E-selectin gene expression in ECs. These coculture effects are inhibited by shear stress and require specific interaction between ECs and SMCs in close contact.

The interaction of vascular smooth muscle cells (SMCs) and extracellular matrix plays important roles in vascular remodeling. We investigated the signaling pathways involved in SMC-induced matrix contraction and SMC migration in three-dimensional (3D) collagen matrix. Matrix contraction is inhibited by the disruption of actin filaments but not microtubules. Therefore, we investigated the roles of signaling pathways related to actin filaments in matrix contraction. SMC-induced matrix contraction was markedly blocked (–80%) by inhibiting the Rho-p160ROCK pathway and myosin light chain kinase, and was decreased to a lesser extent (30–40%) by a negative mutant of Rac and inhibitors of phosphatidylinositol 3-kinase (PI 3-kinase) or p38 mitogen-activated protein kinase (MAPK), but it was not affected by the inhibition of Ras and Cdc42-Wiskott-Aldrich syndrome protein (WASP) pathways. Inhibition of extracellular-signal-regulated kinase (ERK) decreased SMC-induced matrix contraction by only 15%. The migration speed and persistence of SMCs in the 3D matrix were decreased by the inhibition of p160ROCK, PI 3-kinase, p38 MAPK or WASP to different extents, and p160ROCK inhibitor had the strongest inhibitory effect. Our results suggest that the SMC-induced matrix contraction and the migration of SMCs in 3D matrix share some signaling pathways leading to force generation at cell-matrix adhesions and that various signaling pathways have different relative importance in the regulations of these processes in SMCs.

The migration of vascular endothelial cells (ECs) is critical in vascular remodeling. We showed that fluid shear stress enhanced EC migration in flow direction and called this “mechanotaxis.” To visualize the molecular dynamics of focal adhesion kinase (FAK) at focal adhesions (FAs), FAK tagged with green fluorescence protein (GFP) was expressed in ECs. Within 10 min of shear stress application, lamellipodial protrusion was induced at cell periphery in the flow direction, with the recruitment of FAK at FAs. ECs under flow migrated with polarized formation of new FAs in flow direction, and these newly formed FAs subsequently disassembled after the rear of the cell moved over them. The cells migrating under flow had a decreased number of FAs. In contrast to shear stress, serum did not significantly affect the speed of cell migration. Serum induced lamellipodia and FAK recruitment at FAs without directional preference. FAK(Y397) phosphorylation colocalized with GFP-FAK at FAs in both shear stress and serum experiments. The total level of FAK(Y397) phosphorylation after shear stress was lower than that after serum treatment, suggesting that the polarized change at cell periphery rather than the total level of FAK(Y397) phosphorylation is important for directional migration. Our results demonstrate the dynamics of FAK at FAs during the directional migration of EC in response to mechanical force, and suggest that mechanotaxis is an important mechanism controlling EC migration.

Vascular endothelium transduces the temporal gradients in shear stress (tau) originating from unsteady blood flow into functional responses. We measured the effects of step-tau and ramp-tau (i.e., t with different temporal shear gradients) on the lipid lateral diffusion coefficient (D) in the apical membranes of confluent cultured bovine aortic endothelial cells by using fluorescence recovery after photobleaching. A step-tau of 10 dynes/cm2 elicited a rapid (5 s) increase of D in the portion of the cell upstream of the nucleus and a concomitant decrease in the downstream portion. A ramp-tau with a rate of 20 dynes/cm2 per min elicited a rapid (5 s) decrease of D in both the upstream and the downstream portions. The mitogen-activated protein kinases (MAPKs) ERK and JNK were activated by step-tau but not by ramping to the same tau level. Benzyl alcohol, which increases D, enhanced the activities of both MAPKs; cholesterol, which reduces D, diminished these activities. We conclude that the lipid bilayer can sense the temporal features of the applied tau with spatial discrimination and that the tau-induced membrane perturbations can be transduced into MAPK activation. These results have implications for understanding the role of t in modulating vascular functions in health and disease.

This study was designed to elucidate the mechanism underlying the inhibition of endothelial cell growth by laminar shear stress. Tumor suppressor gene p53 was increased in bovine aortic endothelial cells subjected to 24 h of laminar shear stress at 3 dynes (1 dyne = 10 μN)/cm 2 or higher, but not at 1.5 dynes/cm 2 . One of the mechanisms of the shear-induced increase in p53 is its stabilization after phosphorylation by c-Jun N-terminal kinase. To investigate the consequence of the shear-induced p53 response, we found that prolonged laminar shear stress caused increases of the growth arrest proteins GADD45 (growth arrest and DNA damage inducible protein 45) and p21 cip1 , as well as a decrease in phosphorylation of the retinoblastoma gene product. Our results suggest that prolonged laminar shear stress causes a sustained p53 activation, which induces the up-regulation of GADD45 and p21 cip1 . The resulting inhibition of cyclin-dependent kinase and hypophosphorylation of retinoblastoma protein lead to endothelial cell cycle arrest. This inhibition of endothelial cell proliferation by laminar shear stress may serve an important homeostatic function by preventing atherogenesis in the straight part of the arterial tree that is constantly subjected to high levels of laminar shearing.

The objective of this study is to design a new apparatus to allow the control of the magnitude and frequency of dynamic stretch applied uniformly to cells cultured on a silicon elastic membrane. The apparatus is designed to produce equi-biaxial dynamic stretches with area changes ranging from 0% to 55% and frequencies ranging from 0 to 2 Hz. Homogeneous finite strain analysis using triangles of markers was performed to compute the symmetric two-dimensional Lagrangian strain tensor on the membrane. Measurements of strain in both static and dynamic conditions showed that the shear component of the strain tensor (Erc) was near zero, and that there was no significant difference between radial (Err) and circumferential (Ecc) components, indicating the attainment of equi-biaxial strain. Bovine aortic endothelial cells were transiently transfected with a chimeric construct in which the luciferase reporter is driven by TPA-responsive elements (TRE). The transfected cells cultured on the membrane were stretched. The luciferase activity increased significantly only when the cells were stretched by 15% or more in area. Cells in different locations of the membrane showed similar induction of luciferase activities, confirming that strain is uniform and equi-biaxial across the membrane. © 1998 Biomedical Engineering Society.

Atherosclerotic lesions tend to localize at curvatures and branches of the arterial system, where the local flow is often disturbed and irregular (e.g., flow separation, recirculation, complex flow patterns, and nonuniform shear stress distributions). The effects of such flow conditions on cultured human umbilical vein endothelial cells (HUVECs) were studied in vitro by using a vertical-step flow channel (VSF). Detailed shear stress distributions and flow structures have been computed by using the finite volume method in a general curvilinear coordinate system. HUVECs in the reattachment areas with low shear stresses were generally rounded in shape. In contrast, the cells under higher shear stresses were significantly elongated and aligned with the flow direction, even for those in the area with reversed flow. When HUVECs were subjected to shearing in VSF, their actin stress fibers reorganized in association with the morphological changes. The rate of DNA synthesis in the vicinity of the flow reattachment area was higher than that in the laminar flow area. These in vitro experiments have provided data for the understanding of the in vivo responses of endothelial cells under complex flow environments found in regions of prevalence of atherosclerotic lesions.

Observing microscopic specimens is often useful in studies of cellular interaction with a vascular wall. We have developed an in vitro side-view flow chamber that permits observations from the side of the cell's contact with various adhesive surfaces under dynamic flow conditions. This side-view flow chamber consists of two precision rectangular glass tubes called microslides. A smaller microslide is inserted into a larger one to create a flow channel with a flat surface on which either cultured vascular endothelium can be grown or purified adhesion molecules can be coated. Two optical prisms with a 45 degrees chromium-coated surface are used along the flow channel to generate light illumination and observation pathways. The side-view images of cell-substrate contact can be obtained using a light microscope. This design allows us not only to measure the effects of flow on cell-surface adhesion strength, but also to have close observation of cell deformation and adhesive contact to various surfaces in shear flow. In addition, this chamber can readily serve for a conventional top-view flow channel, similar to the parallel-plate flow chambers used in many areas. The development of such a side-view flow chamber can be beneficial to various in vitro applications in cellular studies that require an edge view, especially for various cell interactions with cultured vascular endothelium or surfaces containing single-type adhesive molecules under flow conditions.

Changes in intracellular calcium concentration [Ca2+]i of fura-2-loaded human platelet during its adhesion to a fibrinogen-coated surface were studied, using a flow chamber mounted on an epifluorescence microscope equipped with digital-ratio imaging. Adherent platelets were individually mapped under a scanning electron microscope to establish the possible correlation between adhesion-associated shape alterations and [Ca2+]i changes. We found that 1) there was no immediate [Ca2+]i elevation on platelet adhesion; 2) [Ca2+]i changes varied drastically platelets with a lag time ranging 10 to 200 s, averaging about 1 minute; 3) the pattern of [Ca2+]i changes varied drastically among individual adherent platelets; 4) the degree of [Ca2+]i elevation appeared to correlate with the extent of morphology change, with the vast majority ( > 90%) of spread platelets showed detectable [Ca2+]i changes; 5) neither morphological nor [Ca2+]i changes correlated with the lag time; 6) platelets treated with dimethyl-BAPTA (15 mumol/L) underwent normal shape change without [Ca2+]i elevation; 7) cytochalasin D (10 mumol/L) inhibited both shape change and [Ca2+]i elevation; 8) colchicine (1 mmol/L) was ineffective in both regards. We conclude that although platelet adhesion-associated shape changes may be accompanied with heterogeneous [Ca2+]i changes that are microfilament-dependent, [Ca2+]i changes do not happen immediately after platelet-surface contact and they are not required for adherent platelets to undergo postcontact morphological changes.

To assess the role of intracellular hemoglobin concentration in the deformability of sickle (HbSS) cells after deoxygenation, rheologic coefficients (static rigidity E and dynamic rigidity eta) of density-fractionated individual sickle erythrocytes (SS cells) were determined as a function of oxygen tension (pO2) using the micropipette technique in a newly developed experimental chamber. With stepwise deoxygenation, E and eta values showed no significant increase before morphologic sickling but rose sharply after sickling. In denser cells, continued deoxygenation led to steep rises of E and eta toward infinity, as the cell behaved as a solid. The pO2 levels at which rheologic and morphologic changes occurred for individual SS cells during deoxygenation varied directly with the cell density. The extent of recovery in E and eta during reoxygenation varied inversely with the cell density. These results provide direct evidence that the intracellular sickle hemoglobin (HbS) concentration of SS cells plays an important role in their rheologic heterogeneity in deoxygenation and reoxygenation. The elevations of eta during pO2 alteration were greater than those of E, especially for the denser cells, suggesting the importance of the elevated dynamic rigidity in initiating microcirculatory disturbances in sickle cell disease.

The focal distribution of atherosclerotic lesions in the arterial tree is related to the local shear stress generated by blood flow, but the molecular basis of the atherogenic response of endothelial cells in these lesion-prone areas is still unclear. We report that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells (EC). Northern blot analysis indicated that the level of MCP-1 mRNA in human umbilical vein EC (HUVEC) subjected to a shear stress of 16 dynes/cm2 (1 dyne = 10 microN) for 1.5 hr increased by 2- to 3-fold when compared with static cells. The MCP-1 gene expression decreased to the basal level at 4 hr and then declined further to become completely quiescent at 5 hr after the onset of shear. Once the gene expression was fully suppressed, it remained quiescent even after static incubation for 1.5 hr and would not respond to reshearing after this static incubation. However, if the postshearing incubation extended from 1.5 to 24 hr, the MCP-1 mRNA returned to the basal level and was then able to increase after the reapplication of shear stress. Nuclear run-on experiments showed that the shear-induced increased MCP-1 mRNA in HUVEC was regulated at the transcriptional level. By using cycloheximide, it was shown that de novo protein synthesis was not necessary for the induction of MCP-1 by shear stress. The biphasic response of MCP-1 gene expression was found in experiments in which the applied shear stress was 6, 16, or 32 dynes/cm2, and it was observed not only in HUVEC but also in HeLa cells, glioma cell lines, and skin fibroblasts. This in vitro study demonstrates that the response of MCP-1 gene to shear stress represents an immediate early gene activation and suggests that this gene is probably suppressed in EC that have been exposed to a constant shear stress.

Atherosclerosis is prone to develop at branches and bends of the arterial tree, where laminar blood flow is disturbed by recirculation, with a non-uniform and irregular distribution of wall shear stress. Vascular endothelial cells (ECs) form an interface between the flowing blood and the vessel wall, and are exposed to blood flow-induced shear stress. Recent evidence suggests that laminar blood flow and sustained high shear stress modulate the expression of EC genes and proteins that function to protect against atherosclerosis, whereas disturbed flow and the associated oscillatory and low shear stress up-regulate pro-atherosclerotic genes and proteins that promote development of atherosclerosis. Understanding of the effects of disturbed flow on ECs not only provides mechanistic insights into the role of complex flow patterns in the pathogenesis of atherosclerosis, but also helps to define the differences between quiescent (non-atherogenic) and activated (atherogenic) ECs, which may lead to the discovery and identification of new therapeutic strategies. In this chapter, we summarize the current experimental and theoretical knowledge on the effects of disturbed flow on ECs, in terms of their signal transduction, gene expression, structure, and function. Our purpose is to provide the basic information on the effects of disturbed flow on ECs that is necessary to understand the etiology of lesion development in the disturbed flow-regions of the arterial tree.

Red blood cell (RBC) rouleaux were formed in a flow channel in the presence of 2 g/dl dextran (molecular weight 76,000). The partial separation of RBC rouleau doublets adhering to the floor of the flow channel in response to small oscillatory shear stresses was observed experimentally. Theoretical analyses on displacement and drag force were performed to determine whether the motion of the cell involves membrane rotation (i.e., rolling) or sliding. From the experimental data and the results of theoretical analyses, it is concluded that, under the conditions of the experiments, the RBCs in a doublet separate from each other by rolling, rather than sliding of the sheared cell.

E-selectin is a major adhesion molecule expressed by endothelial cells (ECs), which are exposed to shear stress and neighboring smooth muscle cells (SMCs). We investigated the mechanisms underlying the modulation of EC E-selectin expression by SMCs and shear stress. SMC coculture induced rapid and sustained increases in expression of E-selectin and phosphorylation of interleukin-1 (IL-1) receptor-associated kinase glycoprotein-130, as well as the downstream mitogen-activated protein kinases (MAPKs) and Akt. By using specific inhibitors, dominant-negative mutants, and small interfering RNA, we demonstrated that activations of c-Jun-NH2-terminal kinase (JNK) and p38 of the MAPK pathways are critical for the coculture-induced E-selectin expression. Gel shifting and chromatin immunoprecipitation assays showed that SMC coculture increased the nuclear factor-κB (NF-κB)–promoter binding activity in ECs; inhibition of NF-κB activation by p65-antisense, lactacystin, and N-acetyl-cysteine blocked the coculture-induced E-selectin promoter activity. Protein arrays and blocking assays using neutralizing antibodies demonstrated that IL-1β and IL-6 produced by EC/SMC cocultures are major contributors to the coculture induction of EC signaling and E-selectin expression. Preshearing of ECs at 12 dynes/cm2 inhibited the coculture-induced EC signaling and E-selectin expression. Our findings have elucidated the molecular mechanisms underlying the SMC induction of EC E-selectin expression and the shear stress protection against this SMC induction.

To study the roles of anisotropic cell morphology and directionality of mechanical force in apoptosis, the spreading of human umbilical vein endothelial cells (HUVECs) was constrained by growing on micropatterned (MP) strips of fibronectin (FN, 20 μg/cm 2 ) with widths of 15, 30, and 60 μm on silicone membrane. Cells on 30- and 60-μm strips, like cells on a nonpatterned (NP) surface coated with FN, showed clear actin stress fibers with anchoring spots of phosphorylated focal adhesion kinase ( p -FAK) and no significant apoptosis. On 15-μm strips, cells had few stress fibers, no p -FAK, and significant apoptosis. After seeding for 12 h, the cells were subjected to pulsatile shear stress (12 ± 4 dyn/cm 2 ) parallel or perpendicular to MP strips, or kept under static condition. Parallel flow caused cell elongation with enhanced stress fibers and p -FAK, and a reduction in apoptosis, but perpendicular flow did not. The Rho inhibitory C3 exoenzyme abolished the effects of parallel flow. RhoV14, the constitutively active Rho, enhanced stress fibers and p -FAK, and prevented apoptosis of HUVECs on 15-μm strips under static condition. RhoV14 also reduced cell apoptosis under both parallel and perpendicular flows. Our results indicate that cell apoptosis can be modulated by changes in ECM micropatterning, anisotropic cell morphology, and mechanical forces. These extracellular microenvironment factors affect cell survival through alterations in Rho GTPase activity, stress fiber organization, and FAK phosphorylation.

The phenotype of smooth muscle cells (SMCs) plays an important role in vascular function in health and disease. We investigated the mechanism of modulation of SMC phenotype (from contractile to synthetic) induced by the synergistic action of a growth factor (platelet-derived growth factor, PDGF-BB) and a cytokine (interleukin, IL-1beta). Human aortic SMCs grown on polymerized collagen showed high expression levels of contractile markers (smooth muscle alpha-actin, myosin heavy chain, and calponin). These levels were not significantly affected by PDGF-BB and IL-1beta individually, but decreased markedly after the combined usage of PDGF-BB and IL-1beta. PDGF/IL-1beta costimulation also induced a sustained phosphorylation of Akt and p70 ribosomal S6 kinase (p70S6K). The effects of PDGF/IL-1beta costimulation on contractile marker expression and Akt and p70S6K phosphorylation were blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 and by adenovirus expressing a dominant-negative Akt, and they were mimicked by constitutively active Akt. PDGF-BB/IL-1beta induced a sustained phosphorylation of PDGF receptor (PDGFR)-beta and its association with IL-1 receptor (IL-1R1). Such activation and association of receptors were blocked by a PDGFR-beta neutralizing antibody (AF385), an IL-1R1 antagonist (IL-1ra), as well as a specific inhibitor of PDGFR-beta phosphorylation (AG1295); these agents also eliminated the PDGF-BB/IL-1beta-induced signaling and phenotypic modulation. PDGF-BB/IL-1beta inhibited the polymerized collagen-induced serum response factor DNA binding activity in the nucleus, and this effect was mediated by the PDGFR-beta/IL-1R1 association and phosphatidylinositol 3-kinase/Akt/p70S6K pathway. Our findings provide insights into the mechanism of SMC phenotypic modulation from contractile to synthetic, e.g., in atherosclerosis.

Adhesions of cells to extracellular matrix and adjacent cells are mediated by integrins and VE-cadherin, respectively. Although these adhesion processes play crucial roles in vascular cell migration and angiogenesis, it remains unclear as to how they are coordinated to regulate cellular functions. We report here that integrin engagement by treating bovine endothelial aortic cell monolayers with beads coated with fibronectin (Fn) led to disruption of the VE-cadherin-containing adherens junctions. This disruption was accompanied by increases of tyrosine phosphorylation of β-catenin, γ-catenin, and p120 ctn , as well as the dissociation of α-catenin and γ-catenin from VE-cadherin. We applied a membrane-targeted Src reporter based on the fluorescence resonance energy transfer technique to visualize the dynamic Src activation at subcellular levels in live cells. The integrin engagement induced by Fn-coated beads caused the activation of Src around the beads and at adherens junctions, which are subsequently disrupted. The inhibition of Src with PP1 blocked the effects of integrin engagement on adherens junctions. Although Ras can also modulate adherens junctions, the resulting patterns of phosphorylation and association of junction proteins were distinct from those induced by integrin engagement. The inhibition of Ras by RasN17 did not rescue the disruption of adherens junctions induced by integrin engagement or by Src activation. Integrin engagement by Fn-coated beads also induced a significant alteration of cortical actin filaments at adherens junctions. The results indicate that integrin engagement disrupts VE-cadherin-containing adherens junctions via the activation of Src, but not Ras, possibly as a result of modulation of the actin network.

We investigated the effects of oscillatory flow in regulating the gene expressions of type I collagen (COL1, the main component of human bone tissues) and osteopontin (OPN, the key gene for calcium deposition) in human osteoblast-like (MG-63) cells, and the roles of mitogen-activated protein kinases (MAPKs) in this regulation. The cells were subjected to oscillatory flow (0.5 +/- 4 dyn/cm(2)) or kept under static condition for various time periods (15 min, 30 min, 1 h, 2 h, 4 h, 8 h, and 16 h). Oscillatory flow caused significant up-regulations of both COL1 and OPN gene expressions over the 16 h of study, and a transient activation of MAPKs was starting at 15 min and declining to basal level in 2 h. The flow-induction of COL1 was blocked by an ERK inhibitor (PD98059) and reduced by a JNK inhibitor (SP600125), whereas that of OPN was abolished by PD98059. Analysis of the cis-elements in the COL1 and OPN promoters suggests the involvement of transacting factors Elk-1 and AP-1 in the transcription regulation. The ERK inhibitor (PD98059) blocked Elk-1 phosphorylation, as well as COL1 and OPN gene expression. The JNK inhibitor (SP600125) abolished c-jun phosphorylation and COL1 expression. These results suggest that the flow-induction of OPN was mediated through the ERK-Elk1-OPN pathway, and that COL1 was regulated by both the ERK-Elk1-COL1 and JNK-c-JUN-COL1 pathway.

The small GTPase Rho regulates the formation of actin stress fibers in adherent cells through activation of its effector proteins Rho-kinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Delta1, respectively) suppressed stress fiber formation, but fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of stress fibers to the stretch direction in normal cells, the stress fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of stress fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced stress fiber orientation by an extent equivalent to an additional approximately 3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2Delta1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced stress fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced stress fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.

The mechanical environment crucially influences many cell functions. However, it remains largely mysterious how mechanical stimuli are transmitted into biochemical signals. Src is known to regulate the integrin-cytoskeleton interaction, which is essential for the transduction of mechanical stimuli. Using fluorescent resonance energy transfer (FRET), here we develop a genetically encoded Src reporter that enables the imaging and quantification of spatio-temporal activation of Src in live cells. We introduced a local mechanical stimulation to human umbilical vein endothelial cells (HUVECs) by applying laser-tweezer traction on fibronectin-coated beads adhering to the cells. Using the Src reporter, we observed a rapid distal Src activation and a slower directional wave propagation of Src activation along the plasma membrane. This wave propagated away from the stimulation site with a speed (mean +/- s.e.m.) of 18.1 +/- 1.7 nm s(-1). This force-induced directional and long-range activation of Src was abolished by the disruption of actin filaments or microtubules. Our reporter has thus made it possible to monitor mechanotransduction in live cells with spatio-temporal characterization. We find that the transmission of mechanically induced Src activation is a dynamic process that directs signals via the cytoskeleton to spatial destinations.

Atherosclerosis occurs preferentially at vascular curvature and branch sites where the vessel walls are exposed to fluctuating shear stress and have high endothelial permeability. Endothelial permeability is modulated by intercellular adhesion molecules such as VE-cadherin. This study was designed to elucidate the effects of different flow patterns on the localization and expression of VE-cadherin in endothelial cells (ECs) both in vivo and in vitro. VE-cadherin staining at EC borders was much stronger in the descending thoracic aorta and abdominal aorta, where the pulsatile flow has a strong net forward component than in the aortic arch and the poststenotic dilatation site beyond an experimental constriction, where the flow near the wall is complex and reciprocating with little net flow. With the use of flow chambers the effects of pulsatile flow (12 ± 4 dyn/cm2 at 1 Hz) and reciprocating flow (0.5 ± 4 dyn/cm2 at 1 Hz) on VE-cadherin organization in endothelial monolayers were studied in vitro. VE-cadherin staining was continuous along cell borders in static controls. Following 6 h of either pulsatile or reciprocating flow, the VE-cadherin staining at cell borders became intermittent. When the pulsatile flow was extended to 24, 48 or 72 h the staining around the cell borders became continuous again, but the staining was still intermittent when the reciprocating flow was similarly extended. Exposure to pulsatile or reciprocating flow for 6 and 24 h neither change the expression level of VE-cadherin nor its distribution between membrane and cytosol fractions as determined by Western blot and compared with static controls. These findings suggest that the cell junction remodeling induced by different flow patterns may result from a redistribution of VE-cadherin within the cell membrane. Both the in vivo and in vitro data indicate that pulsatile and reciprocating flow patterns have different effects on cell junction remodeling. The lack of junction reorganization in regions of reciprocating flow in vivo and in vitro may provide a mechanistic basis for the high permeability and the preferential localization of atherosclerosis in regions of the arterial stress with complex flow patterns and fluctuating shear stress.

Aortic endothelial cells lining the inner surface of the aorta are exposed to pulsatile strain due to pulsatile changes in blood pressure. We studied the effects of directionality of pulsatile strain on c-Jun N-terminal kinase (JNK) activation and cell shape. Uniaxial pulsatile strain (10%, 1 Hz) results in cell elongation and alignment perpendicular to the direction of strain. Equibiaxial pulsatile strain (10%, 1 Hz), which has no preferential direction, does not result in significant changes in cell shape or orientation. JNK activation is transient following uniaxial pulsatile strain, but sustained following equibiaxial pulsatile strain. After the JNK activation in response to uniaxial pulsatile strain has subsided, changing the direction of uniaxial strain by 90 results in a reactivation of JNK. These results suggest that the perpendicular alignment to the direction of uniaxial strain is an adaptive response of the cell, which is accompanied by a subsidence of JNK activation. In contrast, cells exposed to equibiaxial pulsatile strain do not undergo directional alignment, and there is a sustained JNK activation.

The migration of vascular endothelial cells (ECs) is critical in vascular remodeling. We showed that fluid shear stress enhanced EC migration in flow direction (defined as mechanotaxis). By expressing focal adhesion kinase (FAK) tagged with green fluorescence protein in ECs, we showed that shear stress induced lamellipodial protrusion in the flow direction, with the recruitment of FAK at FAs in lamellipodia. The newly formed FAs subsequently disassembled after the rear of the cell moved over them. The cells migrating under flow had decreased number of FAs. In contrast to shear stress, serum induced lamellipodia and FAK recruitment at FAs without directional preference. The dynamics of FAs was tracked and quantified. Our results demonstrate the dynamics of FAK at FAs during the directional migration of ECs in response to mechanical force, and suggest that mechanotaxis is an important mechanism controlling EC migration.

Vascular endothelial cells (ECs) in vivo are subject to different flow conditions due to the variation in vessel geometry. The aim of this study is to elucidate the effects of different flow conditions on EC monolayer migration into a mechanically denuded zone and their underlying mechanisms. Both laminar and disturbed flows significantly enhanced EC migration. EC migration speed was the fastest under laminar flow, which preferentially promoted directional EC migration from the upstream side of the wounded monolayer. C3 exoenzyme (a Rho inhibitor) inhibited EC migration under static and flow conditions, and markedly reduced the effects of flow on EC migration. These results indicate that flow promotes EC migration through the Rho signaling pathway. Genistein (a tyrosine kinase inhibitor) selectively retarded EC migration under disturbed flow, suggesting that tyrosine phosphorylation may play a role in EC migration under disturbed flow. This study has demonstrated that different flow patterns differentially affect EC monolayer migration into the denuded zone involving multiple mechanisms.

In the body, vascular endothelial cells (ECs) are constantly exposed to shear stress, which is the tangential component of hemodynamic forces acting on the vessel wall. While shear stress plays important roles in maintaining vascular homeostasis, it can also be pathophysiological factors in vascular disorders such as atherosclerosis. Flow channels with cultured ECs such as human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) have been used as in vitro models to study the endothelial responses to applied shear stress. Such studies have demonstrated that the application of shear stress leads to the phosphorylation of multiple cellular proteins in ECs, resulting in the activation of signaling pathways to modulate geneexpression, cytoskeletal organization, vessel dilation/constriction, and cell proliferation/ apoptosis. These phosphorylation cascades have been shown to result from the activation of cellular pr otein tyrosine kinases (PTKs) and Ser/Thr kinases.

Abstract Monocyte chemotactic protein-1 (MCP-1), a potent monocyte chemoattractant secreted by endothelial cells (ECs), is believed to play a key role in the early events of atherogenesis. Since vascular ECs are constantly subjected to mechanical stresses, we examined how cyclic strain affects the expression of the MCP-1 gene in human ECs grown on a flexible membrane base deformed by sinusoidal negative pressure (peak level, −16 kPa at 60 cycles per minute). Northern blot analysis demonstrated that the MCP-1 mRNA levels in ECs subjected to strain for 1, 5, or 24 hours were double those in control ECs ( P P P 2+ chelator BAPTA/AM strongly suppressed the strain-induced MCP-1 mRNA. Verapamil, a Ca 2+ channel blocker, greatly reduced MCP-1 mRNA levels in both strained and unstrained ECs. These results indicate that mechanical strain can stimulate monocyte chemotaxis and adhesion by increasing MCP-1 gene expression in ECs. This increased gene expression is predominantly mediated via the protein kinase C pathway and requires Ca 2+ influx. Such strain-induced MCP-1 expression might contribute to the trapping of monocytes in the subendothelial space. Strain-induced gene expression might provide a molecular mechanism for the role of hypertension in atherogenesis.

Neutrophils are defensive cells that are recruited from the blood into damaged or inflamed tissues by chemotactic agents. Ligand-receptor interactions at the cell surface elicit adhesion, locomotion, granule exocytosis, and the respiratory burst [Baggiolini and Kernen, 1992]. In vitro analysis shows that these responses are controlled at several levels of intracellular signal transduction processes. In this introduction we first review some of the biochemical events associated with these responses.

A novel experimental method of producing and observing the active motion of polymorphonuclear leukocytes (PMNs) using a micropipette technique has been recently developed (Usami et al., 1992). The present paper develops a quantitative theory for the chemoattractant gradients and cell locomotion observed in these experiments. In previous experimental methods (e.g., the Boyden chamber, the Zygmond chamber and the Dunn chamber) for study chemotaxis of leukocytes, fibroblasts, and PMNs, the exact nature of the concentration gradient of the chemoattractant is unknown. The cells may themselves modify the local gradient of the chemoattractant. In experiments using the micropipette, an internal source of chemoattractant provides well-defined boundary and initial conditions which allow the computation of the chemoattractant concentration gradient during the active locomotion of the PMNs. Since the cell completely fills the pipette lumen, convection is limited to the motion of the cells themselves. In coordinates moving with cell, it is assumed that diffusion is the only mechanism of mass transport of the chemoattractant (fMLP). Computations of the fMLP concentration during locomotion of the cell were carried out for a range of rates of fMLP binding by the receptors expressed on the front face of the cell membrane. The results show that the front face of the cell is subjected to increasing fMLP concentration during the cell motion. The sequence of events involve receptor binding of fMLP, signal transduction, polymerization of the cell cytoskeleton at the membrane of the front face, spatially dependent adhesion to the pipette wall, and localized contraction of the cytoskeleton. This sequence of events leads to the steady locomotion of the leukocytes in the micropipette. The computation of the distribution of the fMLP concentration during cell locomotion with constant velocity in micropipette experiments shows that the cell is exposed to increasing concentration of fMLP. This suggests that chemotaxis maybe induced by temporal gradient of an attractant.

A new paralle plate flow chamber that has a linear variation of shear stress, starting from a predetermined maximum value at the entrance and falling to zero at the exit, has been designed and tested. This is in contrast to the usual rect-angular channel plan which produces a constant shear stress over the entire length. The new design is based on the theory of Hele-Shaw flow between parallel plates. To verify the efficacy of the flow channel, the effect of fluid shear stress on platelet adhesion to a fibrinogen-coated glass surface was tested. The percentage of attached platelets after 5 min of shear stress is shown to be a function of shear stress. With this new flow chamber, cell-cell interactions can be studied efficiently over a wide range of shear stress using a single run at constant discharge.

Adhesion of blood cells to vascular endothelial cells may contribute to microcirculatory obstruction and ischemia in a variety of pathological conditions. Studies of such interactions in vitro are best carried out using assays that mimic flow in vivo. It is also widely accepted that hemodynamic flow forces influence the structure and regulate the function of the endothelial cells themselves.

We have developed a simplified technique for culturing human umbilical vein endothelial cells under shear flow conditions, using prefabricated glass microcapillary tubes ("microslides") with a well-defined rectangular cross section and good optical quality. These microslides have been incorporated into a controlled flow system for quantitative video-microscopic analysis of the adhesion of blood cells to endothelial cells. Microslides were pretreated with 3-aminopropyltriethoxy-silane, or gelatin, and then loaded with a suspension of endothelial cells. After the cells had settled and attached to the substrate, the microslides were inserted into a flow-based culture system. Medium was drawn through them at intervals or continuously until confluency was reached (approximately 24 hr). Cells were cultured at wall shear stresses over a range 0.06 to 2.2 Pa. For adhesion assays, the endothelialized microslides were attached to microscope slides, and suspensions of blood cells were drawn through at desired wall shear stresses (0.02-0.5 Pa). Adhesion of malarial-infected red blood cells and of neutrophilic granulocytes was quantitated by direct microscopic observation. The adhesive behavior of both cell types closely resembled that previously described by ourselves and others using flow chambers incorporating endothelial-coated glass coverslips. The use of microslides represents a significant simplification of methodology for endothelial cell growth and adhesion studies under flow conditions.

Although the rheologic behavior of sickle erythrocytes (SS cells) is highly dependent on oxygen tension (pO2) and temperature, very little data exist regarding the effects of deoxygenation and reoxygenation on the rheology of “individual” SS cells at body temperature. We have devised and assessed a new experiment system, in which micropipette aspiration can be performed on individual cells in a constant- temperature chamber that has ports for changing media with different pO2 (effected in 30 to 120 seconds) and sensing probes for monitoring pO2 and temperature. This system enabled us to simultaneously alter and monitor pO2 at 37 +/- 0.5 degrees C, and to monitor and study a single cell under microscopic observation. The static rigidity (E) and dynamic rigidity (eta) of individual SS cells were determined by repeated aspirations of the same cell under various pO2. With stepwise reductions in pO2, E and eta showed no significant changes before sickling, but once sickled, their values markedly increased by 10(2)- to 10(3)-fold concomitantly with morphologic alteration of the cell. Thus, the deformability of a single SS cell behaves in an “all or none” manner at a critical pO2, and earlier studies on the effect of deoxygenation on the rheology of SS cell suspensions probably reflect the overall behavior of SS cells with widely distributed critical pO2.