Transendothelial flow inhibits neutrophil transmigration through a nitric oxide-dependent mechanism: potential role for cleft shear stress

Endothelial cells in vivo are well known to respond to parallel shear stress induced by luminal blood flow. In addition, fluid filtration across endothelium (transendothelial flow) may trigger nitric oxide (NO) production, presumably via shear stress within intercellular clefts. Since NO regulates neutrophil-endothelial interactions, we determined whether transendothelial flow regulates neutrophil transmigration. Interleukin-1beta-treated human umbilical vein endothelial cell (HUVEC) monolayers cultured on a polycarbonate filter were placed in a custom chamber with or without a modest hydrostatic pressure gradient (DeltaP, 10 cm H(2)O) to induce transendothelial flow. In other experiments, cells were studied in a parallel plate flow chamber at various transendothelial flows (DeltaP = 0, 5, and 10 cm H(2)O) and luminal flows (shear stress of 0, 1, and 2 dyn/cm(2)). In the absence of luminal flow, transendothelial flow reduced transmigration of freshly isolated human neutrophils from 57% to 14% (P < 0.05) and induced an increase in NO detected with a fluorescent assay (DAF-2DA). The NO synthase inhibitor L-NAME prevented the effects of transendothelial flow on neutrophil transmigration, while a NO donor (DETA/NO, 1 mM) inhibited neutrophil transmigration. Finally, in the presence of luminal flow (1 and 2 dyn/cm(2)), transendothelial flow also inhibited transmigration. On the basis of HUVEC morphometry and measured transendothelial volume flow, we estimated cleft shear stress to range from 49 to 198 dyn/cm(2). These shear stress estimates, while substantial, are of similar magnitude to those reported by others with similar analyses. These data are consistent with the hypothesis that endothelial cleft shear stress inhibits neutrophil transmigration via a NO-dependent mechanism.     

Shear stress enhances human endothelial cell wound closure in vitro

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm(2) to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm(2) under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm(2) closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.     

Vascular endothelial wound closure under shear stress: role of membrane fluidity and flow-sensitive ion channels.

Sufficiently rapid healing of vascular endothelium following injury is essential for preventing further pathological complications. Recent work suggests that fluid dynamic shear stress regulates endothelial cell (EC) wound closure. Changes in membrane fluidity and activation of flow-sensitive ion channels are among the most rapid endothelial responses to flow and are thought to play an important role in EC responsiveness to shear stress. The goal of the present study was to probe the role of these responses in bovine aortic EC (BAEC) wound closure under shear stress. BAEC monolayers were mechanically wounded and subsequently subjected to either "high" (19 dyn/cm(2)) or "low" (3 dyn/cm(2)) levels of steady shear stress. Image analysis was used to quantify cell migration and spreading under both flow and static control conditions. Our results demonstrate that, under static conditions, BAECs along both wound edges migrate at similar velocities to cover the wounded area. Low shear stress leads to significantly lower BAEC migration velocities, whereas high shear stress results in cells along the upstream edge of the wound migrating significantly more rapidly than those downstream. The data also show that reducing BAEC membrane fluidity by enriching the cell membrane with exogenous cholesterol significantly slows down both cell spreading and migration under flow and hence retards wound closure. Blocking flow-sensitive K and Cl channels reduces cell spreading under flow but has no impact on cell migration. These findings provide evidence that membrane fluidity and flow-sensitive ion channels play distinct roles in regulating EC wound closure under flow.     

Dynamic contact forces on leukocyte microvilli and their penetration of the endothelial glycocalyx

We develop a theoretical model to examine the combined effect of gravity and microvillus length heterogeneity on tip contact force (F(m)(z)) during free rolling in vitro, including the initiation of L-, P-, and E-selectin tethers and the threshold behavior at low shear. F (m)(z) grows nonlinearly with shear. At shear stress of 1 dyn/cm(2), F(m)(z) is one to two orders of magnitude greater than the 0.1 pN force for gravitational settling without flow. At shear stresses > 0.2 dyn/cm(2) only the longest microvilli contact the substrate; hence at the shear threshold (0.4 dyn/cm(2) for L-selectin), only 5% of microvilli can initiate tethering interaction. The characteristic time for tip contact is surprisingly short, typically 0.1-1 ms. This model is then applied in vivo to explore the free-rolling interaction of leukocyte microvilli with endothelial glycocalyx and the necessary conditions for glycocalyx penetration to initiate cell rolling. The model predicts that for arteriolar capillaries even the longest microvilli cannot initiate rolling, except in regions of low shear or flow reversal. In postcapillary venules, where shear stress is approximately 2 dyn/cm(2), tethering interactions are highly likely, provided that there are some relatively long microvilli. Once tethering is initiated, rolling tends to ensue because F(m)(z) and contact duration will both increase substantially to facilitate glycocalyx penetration by the shorter microvilli.     

Shear stress-induced detachment of human polymorphonuclear leukocytes from endothelial cell monolayers

We employed a static-incubation assay to determine the intensity of wall shear stress (tau) needed to detach human polymorphonuclear leukocytes (HPMNs) from human umbilical vein endothelial cell (HUVE) monolayers. Confluent monolayers of HUVE were placed in a parallel-plate flow chamber which was mounted on the stage of an inverted tissue culture microscope, attached to a perfusion system and maintained at 37 degrees C. All events in the selected fields were recorded using videomicroscopy. HPMNs were co-incubated for 15 minutes with the HUVE monolayers under control conditions or in the presence of 10(-7) M formyl-methionyl-leucyl-phenylalanine (FMLP). Following this static incubation, a series of five individual flows, each 1 minute in duration, were driven through the flow channel, exposing the cells to 1.0, 2.0, 3.8, 7.6 and 14.8 dyn/cm2 wall shear stresses. Under control conditions, the percentage of HPMNs remaining attached to the HUVE monolayers following exposure to each shear stress was 61, 38, 25, 12 and 5, respectively. In the FMLP-treated condition, the percentage of HPMNs remaining attached to the monolayers was significantly greater than control at all five levels of tau. Thus, under control conditions, adherent HPMNs can be detached from endothelial cell monolayers in vitro with levels of shear stress normally found in the microcirculation (18). In the presence of FMLP, the level of shear stress needed to overcome the adhesions is increased significantly.     

Biomechanics of cell rolling: shear flow, cell-surface adhesion, and cell deformability

The mechanics of leukocyte (white blood cell; WBC) deformation and adhesion to endothelial cells (EC) has been investigated using a novel in vitro side-view flow assay. HL-60 cell rolling adhesion to surface-immobilized P-selectin was used to model the WBC-EC adhesion process. Changes in flow shear stress, cell deformability, or substrate ligand strength resulted in significant changes in the characteristic adhesion binding time, cell-surface contact and cell rolling velocity. A 2-D model indicated that cell-substrate contact area under a high wall shear stress (20 dyn/cm2) could be nearly twice of that under a low stress (0.5 dyn/cm2) due to shear flow-induced cell deformation. An increase in contact area resulted in more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy that inputs to a cell decreased due to a flattened cell shape. The model also predicted a plateau of WBC rolling velocity as flow shear stresses further increased. Both experimental and computational studies have described how WBC deformation influences the WBC-EC adhesion process in shear flow.     

Influence of cell deformation on leukocyte rolling adhesion in shear flow

Blood cell interaction with vascular endothelium is important in microcirculation, where rolling adhesion of circulating leukocytes along the surface of endothelial cells is a prerequisite for leukocyte emigration under flow conditions. HL-60 cell rolling adhesion to surface-immobilized P-selectin in shear flow was investigated using a side-view flow chamber, which permitted measurements of cell deformation and cell-substrate contact length as well as cell rolling velocity. A two-dimensional model was developed based on the assumption that fluid energy input to a rolling cell was essentially distributed into two parts: cytoplasmic viscous dissipation, and energy needed to break adhesion bonds between the rolling cell and its substrate. The flow fields of extracellular fluid and intracellular cytoplasm were solved using finite element methods with a deformable cell membrane represented by an elastic ring. The adhesion energy loss was calculated based on receptor-ligand kinetics equations. It was found that, as a result of shear-flow-induced cell deformation, cell-substrate contact area under high wall shear stresses (20 dyn/cm2) could be as much as twice of that under low stresses (0.5 dyn/cm2). An increase in contact area may cause more energy dissipation to both adhesion bonds and viscous cytoplasm, whereas the fluid energy input may decrease due to the flattened cell shape. Our model predicts that leukocyte rolling velocity will reach a plateau as shear stress increases, which agrees with both in vivo and in vitro experimental observations.     

Fluid shear stress modulates endothelial cell invasion into three-dimensional collagen matrices

Endothelial cells are subjected to biochemical and mechanical stimuli, which regulate their angiogenic potential. We determined the synergistic effects of sphingosine-1-phosphate (S1P) and fluid wall shear stress (WSS) on a previously established model of human umbilical vein endothelial cell invasion into three-dimensional collagen matrices. Collagen matrices were incorporated into a parallel-plate flow chamber to apply controlled WSS to the surface of endothelial monolayers over a period of 24 h. Cell invasion required the presence of S1P, with the effects of S1P being enhanced by shear stress to an extent comparable with S1P combined with angiogenic growth factor stimulation. The number of invading cells depended on the magnitude of shear stress, with a maximal induction at a shear stress of approximately 5 dyn/cm2, whereas the invasion distance was proportional to the magnitude of shear stress. The enhancement of invasion by 5.3 dyn/cm2 shear stress coincided with elevated phosphorylation of Akt and matrix metalloproteinase (MMP)-2 activation. Furthermore, invasion induced by the combined application of WSS and S1P was attenuated by inhibitors of MMPs (GM6001) and the phosphatidylinositol 3-kinase/Akt signaling pathway (wortmannin). These results provide evidence that shear stress is a positive modulator of S1P-induced endothelial cell invasion into collagen matrices through enhanced Akt and MMP-2 activation.     

Shear-stress effect on mitochondrial membrane potential and albumin uptake in cultured endothelial cells

Endothelial cells (ECs) that line the inner surface of blood vessels are continuously exposed to shear stress induced by blood flow in vivo, and shear stress affects ATP-dependent macromolecular transport in ECs. However, the relationship between the ATP production and shear stress is still unclear. We, therefore, evaluated mitochondrial ATP synthesis activity in cultured endothelial cells exposed to shear stress, using a confocal laser scanning microscope (CLSM) and a mitochondrial membrane potential probe (5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethyl-benzimidazolycarbocyanine iodide, JC-1). Low shear stress (10 dyn/cm(2)) increased mitochondrial membrane potential by 30%. On the contrary, high shear stress (60 dyn/cm(2)) decreased it by 20%. This observation was consistent with the ATP-dependent albumin uptake into endothelial cells. Our results indicate that ATP synthetic activity is related to the albumin uptake into endothelial cells.     

Microtopography and flow modulate the direction of endothelial cell migration

The migration of vascular endothelial cells under flow can be modulated by the addition of chemical or mechanical stimuli. The aim of this study was to investigate how topographic cues derived from a substrate containing three-dimensional microtopography interact with fluid shear stress in directing endothelial cell migration. Subconfluent bovine aortic endothelial cells were seeded on fibronectin-coated poly(dimethylsiloxane) substrates patterned with a combinatorial array of parallel and orthogonal microgrooves ranging from 2 to 5 microm in width at a constant depth of 1 microm. During a 4-h time-lapse observation in the absence of flow, the majority of the prealigned cells migrated parallel to the grooves with the distribution of their focal adhesions (FAs) depending on the groove width. No change in this migratory pattern was observed after the cells were exposed to moderate shear stress (13.5 dyn/cm(2)), irrespective of groove direction with respect to flow. After 4-h exposure to high shear stress (58 dyn/cm(2)) parallel to the grooves, the cells continued to migrate in the direction of both grooves and flow. By contrast, when microgrooves were oriented perpendicular to flow, most cells migrated orthogonal to the grooves and downstream with flow. Despite the change in the migration direction of the cells under high shear stress, most FAs and actin microfilaments maintained their original alignment parallel to the grooves, suggesting that topographic cues were more effective than those derived from shear stress in guiding the orientation of cytoskeletal and adhesion proteins during the initial exposure to flow.     

Short-Term Shear Stress Induces Rapid Actin Dynamics in Living Endothelial Cells

Hemodynamic shear stress guides a variety of endothelial phenotype characteristics, including cell morphology, cytoskeletal structure, and gene expression profile. The sensing and processing of extracellular fluid forces may be mediated by mechanotransmission through the actin cytoskeleton network to intracellular locations of signal initiation. In this study, we identify rapid actin-mediated morphological changes in living subconfluent and confluent bovine aortic endothelial cells (ECs) in response to onset of unidirectional steady fluid shear stress (15 dyn/cm²). After flow onset, subconfluent cells exhibited dynamic edge activity in lamellipodia and small ruffles in the downstream and side directions for the first 12 min; activity was minimal in the upstream direction. After 12 min, peripheral edge extension subsided. Confluent cell monolayers that were exposed to shear stress exhibited only subtle increases in edge fluctuations after flow onset. Addition of cytochalasin D to disrupt actin polymerization served to suppress the magnitude of flow-mediated actin remodeling in both subconfluent confluent EC monolayers. Interestingly, when subconfluent ECs were exposed to two sequential flow step increases (1 dyn/cm² followed by 15 dyn/cm² 12 min later), actin-mediated edge activity was not additionally increased after the second flow step. Thus, repeated flow increases served to desensitize mechanosensitive structural dynamics in the actin cytoskeleton.     

Synergism of biochemical and mechanical stimuli in the differentiation of human placenta-derived multipotent cells into endothelial cells

There have been intensive studies on the differentiation of endothelial progenitor cells (EPCs) into endothelial cells. We investigated the endothelial differentiation of placenta-derived multipotent cells (PDMCs), a population of CD34(-)/CD133(-)/Flk-1(-) cells. PDMCs were cultured in basal media or media containing endothelial growth factors (EGM), including vascular endothelial growth factor (VEGF), for 3 days and then subjected to shear stress of 6 or 12dyn/cm(2) for 24h. Culture of PDMCs in EGM under static conditions resulted in significant increases in VEGF receptor-1 (Flt-1) and receptor-2 (Flk-1) expression. Application of shear stress at 12dyn/cm(2) to these cells led to significant increases in their expression of von Willebrand Factor and platelet-endothelial cell adhesion molecule-1 at both the gene and protein levels. Shear stress at 6dyn/cm(2) had lesser effects. Uptakes of acetylated low-density lipoproteins as well as formation of tube-like structures on Matrigel were significantly increased after subjecting to shear stress of 12dyn/cm(2) for 24h. Our findings suggest that the combined use of endothelial growth factors and high shear stress is synergistic for the endothelial differentiation of PDMCs.     

Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies.

The parallel plate flow chamber provides a controlled environment for determinations of the shear stress at which cells in suspension can bind to endothelial cell monolayers. By decreasing the flow rate of cell-containing media over the monolayer and assessing the number of cells bound at each wall shear stress, the relationship between shear force and binding efficiency can be determined. The rate of binding should depend on the delivery of cells to the surface as well as the intrinsic cell-surface interactions; thus, only if the cell flux to the surface is known can the resulting binding curves be interpreted correctly. We present the development and validation of a mathematical model based on the sedimentation rate and velocity profile in the chamber for the delivery of cells from a flowing suspension to the chamber surface. Our results show that the flux depends on the bulk cell concentration, the distance from the entrance point, and the flow rate of the cell-containing medium. The model was then used in a normalization procedure for experiments in which T cells attach to TNF-alpha-stimulated HUVEC monolayers, showing that a threshold for adhesion occurs at a shear stress of about 3 dyn/cm2.     

Acute laminar shear stress reversibly increases human glomerular endothelial cell permeability via activation of endothelial nitric oxide synthase

Laminar shear stress is a key determinant of systemic vascular behavior, including through activation of endothelial nitric oxide synthase (eNOS), but little is known of its role in the glomerulus. We confirmed eNOS expression by glomerular endothelial cells (GEnC) in tissue sections and examined effects of acute exposure (up to 24 h) to physiologically relevant levels of laminar shear stress (10-20 dyn/cm(2)) in conditionally immortalized human GEnC. Laminar shear stress caused an orientation of GEnC and stress fibers parallel to the direction of flow and induced Akt and eNOS phosphorylation along with NO production. Inhibition of the phophatidylinositol (PI)3-kinase/Akt pathway attenuated laminar shear stress-induced eNOS phosphorylation and NO production. Laminar shear stress of 10 dyn/cm(2) had a dramatic effect on GEnC permeability, reversibly decreasing the electrical resistance across GEnC monolayers. Finally, the laminar shear stress-induced reduction in electrical resistance was attenuated by the NOS inhibitors l-N(G)-monomethyl arginine (l-NMMA) and l-N(G)-nitroarginine methyl ester (l-NAME) and also by inhibition of the PI3-kinase/Akt pathway. Hence we have shown for GEnC in vitro that acute permeability responses to laminar shear stress are dependent on NO, produced via activation of the PI3-kinase/Akt pathway and increased eNOS phosphorylation. These results suggest the importance of laminar shear stress and NO in regulating the contribution of GEnC to the permeability properties of the glomerular capillary wall.     

Vascular smooth muscle cell glycocalyx modulates shear-induced proliferation, migration, and NO production responses

The endothelial cell glycocalyx, a structure coating the luminal surface of the vascular endothelium, and its related mechanotransduction have been studied by many over the last decade. However, the role of vascular smooth muscle cells (SMCs) glycocalyx in cell mechanotransduction has triggered little attention. This study addressed the role of heparan sulfate proteoglycans (HSPGs), a major component of the glycocalyx, in the shear-induced proliferation, migration, and nitric oxide (NO) production of the rat aortic smooth muscle cells (RASMCs). A parallel plate flow chamber and a peristaltic pump were employed to expose RASMC monolayers to a physiological level of shear stress (12 dyn/cm(2)). Heparinase III (Hep.III) was applied to selectively degrade heparan sulfate on the SMC surface. Cell proliferation, migration, and NO production rates were determined and compared among the following four groups of cells: 1) untreated with no flow, 2) Hep.III treatment with no flow, 3) untreated with flow of 12 dyn/cm(2) exposure, and 4) Hep.III treatment with flow of 12 dyn/cm(2) exposure. It was observed that flow-induced shear stress significantly suppressed SMC proliferation and migration, whereas cells preferred to aligning along the direction of flow and NO production were enhanced substantially. However, those responses were not found in the cells with Hep.III treatment. Under flow condition, the heparinase III-treated cells remained randomly oriented and proliferated as if there were no flow presence. Disruption of HSPG also enhanced wound closure and inhibited shear-induced NO production significantly. This study suggests that HSPG may play a pivotal role in mechanotransduction of SMCs.     

T Lymphocytes Orient against the Direction of Fluid Flow during LFA-1-Mediated Migration

As they leave the blood stream and travel to lymph nodes or sites of inflammation, T lymphocytes are captured by the endothelium and migrate along the vascular wall to permissive sites of transmigration. These processes take place under the influence of hemodynamic shear stress; therefore, we investigated how migrational speed and directionality are influenced by variations in shear stress. We examined human effector T lymphocytes on intercellular adhesion molecule 1 (ICAM-1)-coated surfaces under the influence of shear stresses from 2 to 60 dyn.cm⁻². T lymphocytes were shown to respond to shear stress application by a rapid (30 s) and fully reversible orientation of their migration against the fluid flow without a change in migration speed. Primary T lymphocytes migrating on ICAM-1 in the presence of uniformly applied SDF-1α were also found to migrate against the direction of shear flow. In sharp contrast, neutrophils migrating in the presence of uniformly applied fMLP and leukemic HSB2 T lymphocytes migrating on ICAM-1 alone oriented their migration downstream, with the direction of fluid flow. Our findings suggest that, in addition to biochemical cues, shear stress is a contributing factor to leukocyte migration directionality.     

Alterations by leukocytes of erythrocyte flow in microchannels

The effects of leukocytes on blood rheology in the microcirculation may be predicted to result from the rheological characteristics of the individual white blood cell types and their behavior at entrances of small vessels; their influence on flow resistance of blood due to their disturbance of erythrocyte flow; and the obstruction caused by their adherence to endothelial cells or geometrical hindrance of their flow at irregular or narrow regions. Deformation of leukocytes in micropipettes indicates the relative rigidity of lymphocytes and blast cells from leukemias in contrast to granulocyte viscoelastic properties, and entry times for lymphocytes in 2.6-4.6-microns channels were 11-151 s but milliseconds in 9.1-micron tubes. Lymphocytes and erythrocytes rarely (less than 1%) adhered to cultured endothelium; however, granulocytes adhered avidly at shear stresses of 10-100 dyn/cm2, typical of microcirculation. In the 9.1-microns flow tube at Hct = 17.7 +/- 2.6%, increasing [WBC] caused a linear increase in flow resistance, but above [WBC] = 1000/mm3 resistance tended to plateau. These data support the interpretation that granulocyte adherence to endothelial cells with the potential for obstruction of flow may be a more significant rheological consequence of leukocytes in blood than their resistance to flow because of their relative rigidity or their influence on flow resistance by perturbation of the erythrocyte flow.     

Threshold Levels of Fluid Shear Promote Leukocyte Adhesion through Selectins (CD62L,P,E)

Leukocyte adhesion through L-selectin to peripheral node addressin (PNAd, also known as MECA-79 antigen), an L-selectin ligand expressed on high endothelial venules, has been shown to require a minimum level of fluid shear stress to sustain rolling interactions (Finger, E.B., K.D. Puri, R. Alon, M.B. Lawrence, V.H. von Andrian, and T.A. Springer. 1996. Nature (Lond.). 379:266–269). Here, we show that fluid shear above a threshold of 0.5 dyn/cm² wall shear stress significantly enhances HL-60 myelocyte rolling on P- and E-selectin at site densities of 200/μm² and below. In addition, gravitational force is sufficient to detach HL60 cells from P- and E-selectin substrates in the absence, but not in the presence, of flow. It appears that fluid shear–induced torque is critical for the maintenance of leukocyte rolling. K562 cells transfected with P-selectin glycoprotein ligand-1, a ligand for P-selectin, showed a similar reduction in rolling on P-selectin as the wall shear stress was lowered below 0.5 dyn/cm². Similarly, 300.19 cells transfected with L-selectin failed to roll on PNAd below this level of wall shear stress, indicating that the requirement for minimum levels of shear force is not cell type specific. Rolling of leukocytes mediated by the selectins could be reinitiated within seconds by increasing the level of wall shear stress, suggesting that fluid shear did not modulate receptor avidity. Intravital microscopy of cremaster muscle venules indicated that the leukocyte rolling flux fraction was reduced at blood centerline velocities less than 1 mm/s in a model in which rolling is mediated by L- and P-selectin. Similar observations were made in L-selectin–deficient mice in which leukocyte rolling is entirely P-selectin dependent. Leukocyte adhesion through all three selectins appears to be significantly enhanced by a threshold level of fluid shear stress.     

CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced endothelial cell migration

The migration of endothelial cells (ECs) plays critical roles in vascular physiology and pathology. The receptors CXCR1 and CXCR2, known as G protein-coupled receptors which are essential for migratory response of ECs toward the shear stress-dependent CXCL8 (interleukin-8), are potential mechano-sensors for mechanotransduction of the hemodynamic forces. In present study, the mRNA and protein expression of CXCR1 and CXCR2 in EA.hy926 cells was detected by RT-PCR and Western blot analysis under three conditions of laminar shear stress (5.56, 10.02 and 15.27 dyn/cm(2)) respectively. Using a scratched-wound assay, the effects of CXCR1 and CXCR2 were assessed by the percentage of wound closure while CXCR1 and CXCR2 were functional blocked by the CXCL8 receptor antibodies. The results showed that the mRNA and protein expression of CXCR1 and CXCR2 was both upregulated by 5.56 dyn/cm(2) laminar shear stress, but was both downregulated by 15.27 dyn/cm(2). The wound closure was inhibited significantly while cells were treated with those antibodies in all the conditions. It was suggested that CXCR1 and CXCR2 are involved in mediating the laminar shear stress-induced EC migration. Taken together, these findings indicated that CXCR1 and CXCR2 are novel mechano-sensors mediating laminar shear stress-induced EC migration. Understanding this expanded mechanism of laminar shear stress-induced cell migration will provide novel molecular targets for therapeutic intervention in cancer and cardiovascular diseases.     

Recruitment of endothelial caveolae into mechanotransduction pathways by flow conditioning in vitro

The luminal surface of rat lung microvascular endothelial cells in situ is sensitive to changing hemodynamic parameters. Acute mechanosignaling events initiated in response to flow changes in perfused lung microvessels are localized within specialized invaginated microdomains called caveolae. Here we report that chronic exposure to shear stress alters caveolin expression and distribution, increases caveolae density, and leads to enhanced mechanosensitivity to subsequent changes in hemodynamic forces within cultured endothelial cells. Flow-preconditioned cells expressed a fivefold increase in caveolin (and other caveolar-residing proteins) at the luminal surface compared with no-flow controls. The density of morphologically identifiable caveolae was enhanced sixfold at the luminal cell surface of flow-conditioned cells. Laminar shear stress applied to static endothelial cultures (flow step of 5 dyn/cm2), enhanced the tyrosine phosphorylation of luminal surface proteins by 1.7-fold, including caveolin-1 by 1.3-fold, increased Ser1179 phosphorylation of endothelial nitric oxide synthase (eNOS) by 2.6-fold, and induced a 1.4-fold activation of mitogen-activated protein kinases (ERK1/2) over no-flow controls. The same shear step applied to endothelial cells preconditioned under 10 dyn/cm2 of laminar shear stress for 6 h and induced a sevenfold increase of total phosphotyrosine signal at the luminal endothelial cell surface enhanced caveolin-1 tyrosine phosphorylation 5.8-fold and eNOS phosphorylation by 3.3-fold over static control values. In addition, phosphorylated caveolin-1 and eNOS proteins were preferentially localized to caveolar microdomains. In contrast, ERK1/2 activation was not detected in conditioned cells after acute shear challenge. These data suggest that cultured endothelial cells respond to a sustained flow environment by directing caveolae to the cell surface where they serve to mediate, at least in part, mechanotransduction responses.