Renal tubular fluid shear stress promotes endothelial cell activation

Modified urinary fluid shear stress (FSS) induced by variations of urinary fluid flow and composition is observed in early phases of most kidney diseases. In this study, we hypothesized that changes in urinary FSS represent a tubular aggression that contributes to the development of inflammation, a key event in progression of nephropathies. Human renal tubular cells (HK-2) were exposed to FSS for 30 min at 0.01 Pa. Treatment of human endothelial cells (HMEC-1) with the resulting conditioned medium (FSS-CM) increased C-C chemokine ligand 2 (CCL2) and tumor necrosis factor (TNF)-α protein secretion, increased endothelial vascular adhesion molecule-1 (VCAM-1) mRNA expression and stimulated adhesion of human (THP-1) monocytes to the endothelial monolayer. These effects were TNF-α dependent as they were abolished by neutralization of TNF-α. Interestingly, the origin of TNF-α was not epithelial, but resulted from autocrine endothelial production. However, in contrast to short term FSS, long term FSS (5 h) induced the release of the key inflammatory proteins CCL2 and TNF-α directly from tubular cells. In conclusion, these results suggest for the first time that urinary FSS can contribute to the inflammatory state involved in initiation/perpetuation of renal diseases.     

G protein-coupled receptors serve as mechanosensors for fluid shear stress in neutrophils

Many cells respond to fluid shear stress but in a cell type-specific fashion. Fluid shear stress applied to leukocytes serves to control pseudopod formation, migration, and other functions. Specifically, fresh neutrophils or neutrophilic leukocytes derived from differentiated HL60 cells respond to fluid shear stress by cytoplasmic pseudopod retraction. The membrane elements that sense fluid shear and induce such a specific response are still unknown, however. We hypothesized that membrane receptors may serve as fluid shear sensors. We found that fluid shear decreased the constitutive activity of G protein-coupled receptors (GPCRs). Inhibition of GPCR constitutive activity by inverse agonists abolished fluid shear stress-induced cell area reduction. Among the GPCRs in neutrophils, the formyl peptide receptor (FPR) exhibits relatively high constitutive activity. Undifferentiated HL60 cells that lacked FPR formed few pseudopods and showed no detectable response to fluid shear stress, whereas expression of FPR in undifferentiated HL60 cells caused pseudopod projection and robust pseudopod retraction during fluid shear. FPR small interfering RNA-transfected differentiated HL60 cells exhibited no response to fluid shear stress. These results suggest that GPCRs serve as mechanosensors for fluid shear stress in neutrophils by decreasing its constitutive activity and reducing pseudopod projection. leukocyte; constitutive activity; mechanotransduction; formyl peptide receptor     

Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.     

Blood flow and vascular gene expression: fluid shear stress as a modulator of endothelial phenotype

Vascular endothelium, the cellular monolayer lining the entire cardiovascular system, is exposed to a variety of biochemical and biomechanical stimuli. Fluid shear stresses generated by blood flow in the vasculature can profoundly influence the phenotype of the endothelium by regulating the activity of certain flow-sensitive proteins (for example, enzymes), as well as by modulating gene expression. The finding that specific fluid mechanical forces can alter endothelial structure and function has provided a framework for a mechanistic understanding of flow-dependent processes, ranging from vascular remodeling in response to hemodynamic changes, to the initiation and localization of chronic vascular diseases such as atherosclerosis.     

A disk-type apparatus for applying fluid shear stress on cultured endothelial cell

To study the effect of fluid shear stress on cultured endothelial cells, we have developed an apparatus for the stress creation, which consists of a stainless steel disk driven by an electric DC motor and a stage to place a culture dish and to adjust the distance between the disk and the dish. When the disk is rotated, a concentric fluid movement occurs in the culture medium in the dish and exerts the shear stress on the endothelial cells cultured on the bottom of the dish. A theoretical analyses concerning the induced concentric flow velocity predicted that when the angular velocity of the disk rotation (omega) is slow enough to maintain a Reynolds' number of the order of 10, the exerted wall shear stress tau w on the endothelial cell monolayer is given for a constant as tau w = mu r omega/d where mu is the viscosity of the medium, d the distance from the plate to the monolayer and r the radial distance from the center of the dish. When omega is varied in a sinusoidal mode tau w also becomes sinusoidal, thus allowing to apply a pulsatile stress. In vitro experiments carried out to examine the validity of the theoretical results, using a suspension of polystyrene as a tracer with the ordinary culture medium and 99% ethanol, revealed excellent agreement of the measured velocity profiles with the predicted ones. The results demonstrated that the present apparatus can create both the steady and pulsatile wall shear stress on the culture cell layer as expected, unless Reynolds' number greatly exceeds the level of 10.     

Localization of VEGFR-2 and PLD2 in endothelial caveolae is involved in VEGF-induced phosphorylation of MEK and ERK

To clarify the role of caveolae in VEGF/VEGF receptor-2 (VEGFR-2)-mediated signaling cascades, primary cultured human umbilical vein endothelial cells (HUVECs) were fractionated to isolate caveolae-enriched cell membranes. Interestingly, VEGFR-2, phospholipase D2 (PLD2), and Ras were enriched in caveolae-enriched fractions. Moreover, VEGF increased PLD activity in a time- and dose-dependent manner in HUVECs, whereas a ligand specific for VEGFR-1 placental growth factor did not change PLD activity. A PLD inhibitor, 1-butanol, almost completely suppressed VEGF-induced ERK phosphorylation and cellular proliferation, whereas the negative control for 1-butanol, 3-butanol, did not produce significant changes. Addition of phosphatidic acid negated the 1-butanol-induced suppression. Pharmacological analyses using several inhibitors indicated that PKC-delta regulates the VEGF-induced activation of PLD/ERK. Thus PLD2 could be involved in MEK/ERK signaling cascades that are induced by the VEGF/VEGFR-2/PKC-delta pathway in endothelial cells. Pretreatment with the cholesterol depletion agent methyl-beta-cyclodextrin (MbetaCD) almost completely disassembled caveolar structures, whereas the addition of cholesterol to MbetaCD-treated cells restored caveolar structures. Pretreatment with MbetaCD largely abolished phosphorylation of MEK/ERK by VEGF, whereas the addition of cholesterol restored VEGF-induced MEK/ERK phosphorylations. These results indicate that intact caveolae are required for the VEGF/VEGFR-2-mediated MEK/ERK signaling cascade.     

Direct measurement of shear strain in adherent vascular endothelial cells exposed to fluid shear stress

Functional and morphological responses of endothelial cells (ECs) to fluid shear stress are thought to be mediated by several mechanosensitive molecules. However, how the force due to fluid shear stress applied to the apical surface of ECs is transmitted to the mechanosensors is poorly understood. In the present paper, we performed an analysis of an intracellular mechanical field by observation of the deformation behaviors of living ECs exposed to shear stress with a novel experimental method. Lateral images of human umbilical vein ECs before and after the onset of flow were obtained by confocal microscopy, and image correlation and finite element analysis were performed for quantitative analyses of subcellular strain due to shear stress. The shear strain of the cells changed from 1.06 ± 1.09% (mean ± SD) to 4.67 ± 1.79% as the magnitude of the shear stress increased from 2 to 10 Pa. The nuclei of ECs also exhibited shear deformation, which was similar to that observed in cytoplasm, suggesting that nuclei transmit forces from apical to intracellular components, as well as cytoskeletons. The obtained strain-stress relation resulted in a mean shear modulus of 213 Pa for adherent ECs. These results provide a mechanical perspective on the investigation of flow-sensing mechanisms of ECs.     

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

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

The role of endothelial glycocalyx components in mechanotransduction of fluid shear stress

The surface of endothelial cells is decorated with a wide variety of membrane-bound macromolecules that constitute the glycocalyx. These include glycoproteins bearing acidic oligosaccharides with terminal sialic acids (SA), and proteoglycans with their associated glycosaminoglycan that include: heparan sulfate (HS), chondroitin sulfate (CS), and hyaluronic acid (HA). In this study, enzymes were used to selectively degrade glycocalyx components from the surface of bovine aortic endothelial cells and the effects of these alterations on fluid shear-induced nitric oxide (NO) and prostacyclin (PGI(2)) production were determined. Depletion of HS, HA, and SA, but not CS, blocked shear-induced NO production. Surprisingly, the same enzyme depletions that blocked NO production had no influence on shear-induced PGI(2) production. The results may be interpreted in terms of a glypican-caveolae-eNOS mechanism for shear-induced NO transduction, with PGI(2) being transduced in basal adhesion plaques that sense the same reaction stress whether the glycocalyx is intact or not.     

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     

Differential regulation of vascular endothelial growth factor receptors (VEGFR) revealed by RNA interference: interactions of VEGFR-1 and VEGFR-2 in endothelial cell signaling

Vascular endothelial growth factor (VEGF) plays a central role in vascular homeostasis. VEGF receptors (VEGFRs) include several subtypes that may have a differential role in endothelial signal transduction, but interactions among these receptors are incompletely understood. In these studies, we designed small interfering RNA (siRNA) duplexes that targeted specific VEGFR subtypes in bovine aortic endothelial cells (BAEC). siRNA-mediated downregulation of VEGFR-2 by its cognate siRNA resulted in a significant attenuation of VEGF-mediated signaling. Compared to control siRNA-treated cells, VEGFR-2 siRNA markedly inhibited VEGF-mediated activation of PI3K/Akt/GSK3-beta as well as MAP kinase and PKC pathways. VEGFR-2 siRNA also blocked VEGF-stimulated phosphorylation and dephosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1179) and Ser(116), respectively. VEGFR-2-specific siRNA had no effect on the abundance of VEGFR-1 protein. By contrast, VEGFR-1-specific siRNA markedly not only downregulated the abundance of VEGFR-1 but also significantly reduced VEGFR-2 protein and mRNA abundance. VEGFR-1 siRNA had no effect on the stability of VEGFR-2 protein or mRNA. However, VEGFR-1 siRNA significantly inhibited VEGFR-2 promoter activity, as determined in luciferase assays using VEGFR-2 promoter fusion constructs in transfected BAEC. Deletion of either the 5' E box or the 3' E box and the GATA element in the VEGFR-2 promoter completely abolished the inhibition of VEGFR-2 promoter activity elicited by VEGFR-1 siRNA. Taken together, our data suggest that VEGFR-1 receptor is a critical determinant of VEGFR-2 abundance, while VEGFR-2 is the key receptor directly responsible for endothelial cell signaling stimulated by VEGF.     

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.     

Low density lipoprotein induces eNOS translocation to membrane caveolae: the role of RhoA activation and stress fiber formation

A decrease in the bioavailability of endothelium-derived nitric oxide (NO) is linked to hypercholesterolemia. However, the mechanism by which low density lipoprotein (LDL) mediates endothelial NO synthase (eNOS) dysfunction remains controversial. We investigate the effect of LDL on eNOS regulation in human endothelial cells (ECs). In cultured ECs, a high level of LDL increased the abundance of eNOS and caveolin-1 (Cav-1) in the membrane caveolae and the association of eNOS with Cav-1. Furthermore, it decreased the basal level of NO and blocked NO production stimulated by the calcium ionophore A23187. LDL exposure also increased the formation of stress fibers and the membrane translocation of eNOS. These effects can be blocked by cytochalasin D, an actin cytoskeleton disruptor. In revealing the mechanism underlying the translocation of eNOS, we found that a high level of LDL increased the level of membrane-associated and GTP-formed RhoA and activated the RhoA downstream kinase ROCK-1 activity. Y-27632, a specific inhibitor of ROCK-1, blocked LDL-induced stress fiber formation, eNOS translocation and NO production. In conclusion, a high level of LDL increases the movement of eNOS to membrane caveolae via the increased stress fibers. The RhoA-mediated pathway may play a crucial role in this process in vascular ECs.     

Measurements of strain on single stress fibers in living endothelial cells induced by fluid shear stress

Fluid shear stress (FSS) acting on the apical surface of endothelial cells (ECs) can be sensed by mechano-sensors in adhesive protein complexes found in focal adhesions and intercellular junctions. This sensing occurs via force transmission through cytoskeletal networks. This study quantitatively evaluated the force transmitted through cytoskeletons to the mechano-sensors by measuring the FSS-induced strain on SFs using live-cell imaging for actin stress fibers (SFs). FSS-induced bending of SFs caused the SFs to align perpendicular to the direction of the flow. In addition, the displacement vectors of the SFs were detected using image correlation and the FSS-induced axial strain of the SFs was calculated. The results indicated that FSS-induced strain on SFs spanned the range 0.01-0.1% at FSSs ranging from 2 to 10 Pa. Together with the tensile property of SFs reported in a previous study, the force exerted on SFs was estimated to range from several to several tens of pN.     

Effects of cold exposure and shear stress on endothelial nitric oxide synthase activation

Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37 °C and 28 °C, and a decrease at 4 °C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.     

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.     

The fluid shear stress distribution on the membrane of leukocytes in the microcirculation

Recent in-vivo and in-vitro evidence indicates that fluid shear stress on the membrane of leukocytes has a powerful control over several aspects of their cell function. This evidence raises a question about the magnitude of the fluid shear stress on leukocytes in the circulation. The flow of plasma on the surface of a leukocyte at a very low Reynolds number is governed by the Stokes equation for the motion of a Newtonian fluid. We numerically estimated the distribution of fluid shear stress on a leukocyte membrane in a microvessel for the cases when the leukocyte is freely suspended, as well as rolling along or attached to a microvessel wall. The results indicate that the fluid shear stress distribution on the leukocyte membrane is nonuniform with a sharp increase when the leukocyte makes membrane attachment to the microvessel wall. In a microvessel (10 microns diameter), the fluid shear stress on the membrane of a freely suspended leukocyte (8 microns diameter) is estimated to be several times larger than the wall shear stress exerted by the undisturbed Poiseuille flow, and increases on an adherent leukocyte up to ten times. High temporal stress gradients are present in freely suspended leukocytes in shear flow due to cell rotation, which are proportional to the local shear rate. In comparison, the temporal stress gradients are reduced on the membrane of leukocytes that are rolling or firmly adhered to the endothelium. High temporal gradients of shear stress are also present on the endothelial wall. At a plasma viscosity of 1 cPoise, the peak shear stresses for suspended and adherent leukocytes are of the order of 10 dyn/cm2 and 100 dyn/cm2, respectively.     

Fibroblast growth factor-2 binding to the endothelial basement membrane peaks at a physiologically relevant shear stress

Fibroblast growth factor-2 (FGF2) is produced and released by endothelial cells and binds to heparan sulfate proteoglycans in the endothelial basement membrane (BM), an important FGF2 storage reservoir. Experimental and computational models of FGF2 binding kinetics to both cells and BM under static conditions are well established in the literature but remain largely unexplored under flow. We now examine BM-FGF2 binding kinetics in fluid flow conditions. We hypothesized that FGF2 binding to the endothelial BM would decrease as fluid shear stress increased. To investigate this, BM-FGF2 equilibrium, associative, and dissociative bindings were measured at various shear stresses. Surprisingly, FGF2 binding increased up to a physiological arterial shear stress of 25 dynes/cm², after which it decreased to a level similar to the 1 dyne/cm² condition. Both BM-FGF2 dissociation and BM binding site availability increased with flow, while association remained constant. This suggests that force-dependent FGF2 equilibrium binding varies with shear stress due to a combination of an increase in binding site availability and FGF2 dissociation with flow. This improved understanding of BM-FGF2 binding with flow enriches current knowledge of FGF2 binding kinetics under physiologic conditions, which may contribute to improved growth factor therapy development.     

Differential responsiveness of vascular endothelial cells to different types of fluid mechanical shear stress

Early atherosclerotic lesions localize preferentially, in arterial regions exposed to low flow, oscillatory flow, or both; however, the cellular basis of this observation remains to be determined. Atherogenesis involves dysfunction of the vascular endothelium, the cellular monolayer lining the inner surfaces of blood vessels. How low flow, oscillatory flow, or both may lead to endothelial dysfunction remains unknown. Over the past two decades, fluid mechanical shear (or frictional) stress has been shown to intricately regulate the structure and function of vascular endothelial cells (ECs). Furthermore, recent data indicate that beyond being merely responsive to shear stress, ECs are able to distinguish among and respond differently to different types of shear stress. This review focuses on EC differential responses to different types of steady and unsteady shear stress and discusses the implications of these responses for the localization of early atherosclerotic lesions. The mechanisms by which endothelial differential responsiveness to different types of flow may occur are also discussed.     

Effects of different types of fluid shear stress on endothelial cell proliferation and survival

We attempted to clarify the effect of different types of shear stress on endothelial cell (EC) proliferation and survival. Bovine aortic ECs were subjected to either steady laminar, 1 Hz pulsatile, or 1 Hz to and fro shear at 14 dyne/cm(2). % of BrdU positive EC was 14.3 +/- 1.6% in steady, 21.5 +/- 3.2% in pulsatile, and 11.4 +/- 2.4% in to and fro after 4 h, respectively (P < 0.05). Pulsatile shear compared with static control. Rapamycin reduced BrdU incorporation in all shear regimens (P < 0.001). However, it was still higher in EC exposed to pulsatile shear than the other regimens (P < 0.005). PD98059 completely abolished the increased BrdU incorporation in all shear regimens, including pulsatile shear. Pulsatile shear had significantly elevated ERK1/2 phosphorylation at 5 min compared with steady (P < 0.05) and to and fro shear (P < 0.01) while there was no significant difference in pp70(S6k) phosphorylation between any shear regimen. The ratio of apoptotic cells in serum deprived EC in the presence of steady laminar, pulsatile and to and fro shear for 4 h were 2.7 +/- 0.78%, 2.7 +/- 0.42%, and 2.9 +/- 0.62%, respectively while after the addition of serum for 4 h, it was 4.3 +/- 0.73%. All shear regimens phosphorylated AKT in a time-dependent manner with no significant difference between regimens. Our results demonstrate that different types of shear stress regimens have different effects on EC and may account for the variable response of EC to hemodynamics in the circulation.