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

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

Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells

We tested the hypothesis that fluidshear stress () modifies the expression, function, and distributionof junctional proteins [connexin (Cx)43, Cx45, and zona occludens(ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to-values of 5 or 20 dyn/cm² for 1-3 h.Immunostaining indicated that at 5 dyn/cm², thedistribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cellmembranes; at 20 dyn/cm², disruption was more severe.Intercellular coupling was significantly decreased at both shear stresslevels. Western blots showed the downregulation of membrane-bound Cx43and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at differentlevels of shear stress. Similarly, Northern blots revealed thatexpression of Cx43, Cx45, and ZO-1 was selectively up- anddownregulated in response to different shear stress levels. Theseresults indicate that in cultured bone cells, fluid shear stressdisrupts junctional communication, rearranges junctional proteins, anddetermines de novo synthesis of specific connexins to an extent thatdepends on the magnitude of the shear stress. Such disconnection fromthe bone cell network may provide part of the signal whereby thedisconnected cells or the remaining network initiate focal bone remodeling.

     

Flow-induced expression of endothelial Na-K-Cl cotransport: dependence on K(+) and Cl(-) channels

Steady laminarshear stress has been shown previously to markedly increase Na-K-Clcotransporter mRNA and protein in human umbilical vein endothelialcells and also to rapidly increase endothelial K⁺ andCl channel conductances. The present study was done toevaluate the effects of shear stress on Na-K-Cl cotransporter activity and protein expression in bovine aortic endothelial cells (BAEC) and todetermine whether changes in cotransporter expression may be dependenton early changes in K⁺ and Cl channelconductances. Confluent BAEC monolayers were exposed in aparallel-plate flow chamber to either steady shear stress (19 dyn/cm²) or purely oscillatory shear stress (0 ± 19 dyn/cm²) for 6-48 h. After shearing, BAEC monolayerswere assessed for Na-K-Cl cotransporter activity or were subjected toWestern blot analysis of cotransporter protein. Steady shear stress ledto a 2- to 4-fold increase in BAEC cotransporter protein levels and a1.5- to 1.8-fold increase in cotransporter activity, increases thatwere sustained over the longest time periods studied. Oscillatory flow,in contrast, had no effect on cotransporter protein levels. In thepresence of flow-sensitive K⁺ and Cl channelpharmacological blockers, the steady shear stress-induced increase incotransporter protein was virtually abolished. These results suggestthat shear stress modulates the expression of the BAEC Na-K-Clcotransporter by mechanisms that are dependent on flow-activated ion channels.

     

Endothelium oriented differentiation of bone marrow mesenchymal stem cells under chemical and mechanical stimulations

Bone marrow mesenchymal stem cells (MSCs) have multi-differentiation capability. Their endothelial cell (EC) oriented differentiation is the key to vasculogenesis, in which both mechanical and chemical stimulations play important roles. Most previous studies reported individual effects of VEGF or fluid shear stress (SS), when MSCs were subjected to shear stress of 10–15 dyn/cm² over 24 hr. In this paper, we investigated responses of MSCs from young Sprague Dawley rats to shear stress, VEGF and the combination of the two stimuli. Our study showed that the combined stimulation of shear stress and VEGF resulted in more profound EC oriented differentiation of MSCs in comparison to any individual stimulation. Furthermore, we subjected MSCs to prolonged period of fluid shear stimulation, i.e. 48 hr rather than 24 hr, and increased the magnitude of the shear stress from 10 dyn/cm² to 15, 20 and 25 dyn/cm². We found that without VEGF, the endothelium oriented differentiation of MSCs that was seen following 24 hr of shear stimulation was largely abolished if we extended the shear stimulation to 48 hr. A similar sharp decrease in MSC differentiation was also observed when the magnitude of the shear stress was increased from 10–15 dyn/cm² to 20–25 dyn/cm² in 24 hr shear stimulation studies. However, with combined VEGF and fluid shear stimulation, most of the endothelial differentiation was retained following an extended period, i.e. at 48 hr, of shear stimulation. Our study demonstrates that chemical and mechanical stimulations work together in determining MSC differentiation dynamics.     

Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases

Blood vessels and blood cells are under continuous fluid shear. Studies on vascular endothelium and smooth muscle cells have shown the importance of this mechanical stress in cell signal transduction, gene expression, vascular remodeling, and cell survival. However, in circulating leukocytes, shear-induced signal transduction has not been investigated. Here we examine in vivo and in vitro the control of pseudopods in leukocytes under the influence of fluid shear stress and the role of the Rho family small GTPases. We used a combination of HL-60 cells differentiated into neutrophils (1.4% dimethyl sulfoxide for 5 days) and fresh leukocytes from Rac knockout mice. The cells responded to shear stress (5 dyn/cm²) with retraction of pseudopods and reduction of their projected cell area. The Rac1 and Rac2 activities were decreased by fluid shear in a time- and magnitude-dependent manner, whereas the Cdc42 activity remained unchanged (up to 5 dyn/cm²). The Rho activity was transiently increased and recovered to static levels after 10 min of shear exposure (5 dyn/cm²). Inhibition of either Rac1 or Rac2 slightly but significantly diminished the fluid shear response. Transfection with Rac1-positive mutant enhanced the pseudopod formation during shear. Leukocytes from Rac1-null and Rac2-null mice had an ability to form pseudopods in response to platelet-activating factor but did not respond to fluid shear in vitro. Leukocytes in wild-type mice retracted pseudopods after physiological shear exposure, whereas cells in Rac1-null mice showed no retraction during equal shear. On leukocytes from Rac2-null mice, however, fluid shear exerted a biphasic effect. Leukocytes with extended pseudopods slightly decreased in length, whereas initially round cells increased in length after shear application. The disruption of Rac activity made leukocytes nonresponsive to fluid shear, induced cell adhesion and microvascular stasis, and decreased microvascular density. These results suggest that deactivation of Rac activity by fluid shear plays an important role in stable circulation of leukocytes. microcirculation; mechanotransduction; actin polymerization; transgenic mouse; leukocyte     

In vitro system to study realistic pulsatile flow and stretch signaling in cultured vascular cells

We developed a novel real-timeservo-controlled perfusion system that exposes endothelial cells grownin nondistensible or distensible tubes to realistic pulse pressures andphasic shears at physiological mean pressures. A rate-controlled flowpump and linear servo-motor are controlled by digitalproportional-integral-derivative feedback that employspreviously digitized aortic pressure waves as a command signal. Theresulting pressure mirrors the recorded waveform and can be digitallymodified to yield any desired mean and pulse pressure amplitude,typically 0-150 mmHg at shears of 0.5-15 dyn/cm².The system accurately reproduces the desired arterial pressure waveformand cogenerates physiological flow and shears by the interaction ofpressure with the tubing impedance. Rectangular glass capillary tubes[1-mm inside diameter (ID)] are used for real-time fluorescentimaging studies (i.e., pH_i, NO, Ca²⁺), whereassilicon distensible tubes (4-mm ID) are used for more chronic (i.e.,2-24 h) studies regarding signal transduction and geneexpression. The latter have an elastic modulus of12.4 · 10⁶ dyn/cm² similar to in vivovessels of this size and are studied with the use of a benchtop system.The new approach provides the first in vitro application of realisticmechanical pulsatile forces on vascular cells and should facilitatestudies of phasic shear and distension interaction and pulsatile signal transduction.

     

Mechanical regulation of cancer cell apoptosis and autophagy: Roles of bone morphogenetic protein receptor,Smad1/5, and p38 MAPK

Mechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G₂/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12 dyn/cm² induced death of these four tumor cell lines. In contrast to LSS at 0.5 dyn/cm², oscillatory shear stress (OSS) at 0.5 ± 4 dyn/cm² cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V–FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells.     

Shear stress induces a time- and position-dependent increase in endothelial cell membrane fluidity

Blood flow-associatedshear stress may modulate cellular processes through its action on theplasma membrane. We quantified the spatial and temporal aspects of theeffects of shear stress () on the lipid fluidity of1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate [DiIC₁₆(13)]-stained plasma membranesof bovine aortic endothelial cells in a flow chamber. A confocalmicroscope was used to determine the DiI diffusion coefficient(D) by fluorescence recovery after photobleaching on cellsunder static conditions, after a step- of 10 or 20 dyn/cm², and after the cessation of . The methodallowed the measurements of D on the upstream and downstreamsides of the cell taken midway between the respective cell borders andthe nucleus. In <10 s after a step- of 10 dyn/cm²,D showed an upstream increase and a downstream decrease, and both changes disappeared rapidly. There was a secondary, larger increase in upstream D, which reached a peak at 7 min and decreased thereafter, despite the maintenance of .D returned to near control values within 5 s aftercessation of . Downstream D showed little secondarychanges throughout the 10-min shearing, as well as after its cessation.Further investigations into the early phase, with simultaneousmeasurements of upstream and downstream D, confirmed that astep- of 10 dyn/cm² elicited a rapid (5-s) but transientincrease in upstream D and a concurrent decrease indownstream D, yielding a significant difference between thetwo sites. A step- of 20 dyn/cm² caused D toincrease at both sites at 5 s, but by 30 s and 1 min theupstream D became significantly higher than the downstream D. These results demonstrate shear-induced changes inmembrane fluidity that are time dependent and spatially heterogeneous. These changes in membrane fluidity may have important implications inshear-induced membrane protein modulation.

     

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts

Osteoblasts subjected to fluid shearincrease the expression of the early response gene, c-fos, andthe inducible isoform of cyclooxygenase, COX-2, two proteins linked tothe anabolic response of bone to mechanical stimulation, in vivo. Theseincreases in gene expression are dependent on shear-induced actinstress fiber formation. Here, we demonstrate that MC3T3-E1osteoblast-like cells respond to shear with a rapid increase inintracellular Ca²⁺ concentration([Ca²⁺]_i) that wepostulate is important to subsequent cellular responses to shear. Totest this hypothesis, MC3T3-E1 cells were grown on glass slides coatedwith fibronectin and subjected to laminar fluid flow (12 dyn/cm²). Before application of shear, cells were treatedwith two Ca²⁺ channel inhibitors or various blockers ofintracellular Ca²⁺ release for 0.5-1 h. Althoughgadolinium, a mechanosensitive channel blocker, significantly reducedthe [Ca²⁺]_i response, neithergadolinium nor nifedipine, an L-type channel Ca²⁺ channelblocker, were able to block shear-induced stress fiber formation andincrease in c-fos and COX-2 in MC3T3-E1 cells. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraaceticacid-AM, an intracellular Ca²⁺ chelator, or thapsigargin,which empties intracellular Ca²⁺ stores, completelyinhibited stress fiber formation and c-fos/COX-2 production in shearedosteoblasts. Neomycin or U-73122 inhibition of phospholipase C, whichmediates D-myo-inositol 1,4,5-trisphosphate (IP₃)-induced intracellular Ca²⁺ release, alsocompletely suppressed actin reorganization and c-fos/COX-2 production.Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform ofU-73122, did not inhibit these shear-induced responses. These resultssuggest that IP₃-mediated intracellular Ca²⁺release is required for modulating flow-induced responses in MC3T3-E1 cells.

     

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease−2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm²) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm²) (P < 0.01). ³H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.     

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     

Fluid Shear Stress Increases Neutrophil Activation via Platelet-Activating Factor

Leukocyte exposure to hemodynamic shear forces is critical for physiological functions including initial adhesion to the endothelium, the formation of pseudopods, and migration into tissues. G-protein coupled receptors on neutrophils, which bind to chemoattractants and play a role in neutrophil chemotaxis, have been implicated as fluid shear stress sensors that control neutrophil activation. Recently, exposure to physiological fluid shear stresses observed in the microvasculature was shown to reduce neutrophil activation in the presence of the chemoattractant formyl-methionyl-leucyl-phenylalanine. Here, however, human neutrophil preexposure to uniform shear stress (0.1–2.75 dyn/cm²) in a cone-and-plate viscometer for 1–120 min was shown to increase, rather than decrease, neutrophil activation in the presence of platelet activating factor (PAF). Fluid shear stress exposure increased PAF-induced neutrophil activation in terms of L-selectin shedding, α_Mβ₂ integrin activation, and morphological changes. Neutrophil activation via PAF was found to correlate with fluid shear stress exposure, as neutrophil activation increased in a shear stress magnitude- and time-dependent manner. These results indicate that fluid shear stress exposure increases neutrophil activation by PAF, and, taken together with previous observations, differentially controls how neutrophils respond to chemoattractants.     

Shear stress regulates adhesion and rolling of CD44+ leukemic and hematopoietic progenitor cells on hyaluronan

Leukemic cells and human hematopoietic progenitor cells expressing CD44 receptors have the ability to attach and roll on hyaluronan. We investigated quantitatively the adhesion behavior of leukemic cell lines and hematopoietic progenitor cells on thin films of the polysaccharides hyaluronan and alginate in a microfluidic system. An applied flow enhances the interaction between CD44-positive cells and hyaluronan if a threshold shear stress of 0.2 dyn/cm² is exceeded. At shear stress ∼1 dyn/cm², the cell rolling speed reaches a maximum of 15 μm/s. Leukemic Jurkat and Kasumi-1 cells lacking CD44-expression showed no adhesion or rolling on the polysaccharides whereas the CD44-expressing leukemic cells KG-1a, HL-60, K-562, and hematopoietic progenitor cells attached and rolled on hyaluronan. Interestingly, the observations of flow-induced cell rolling are related to those found in the recruitment of leukocytes to inflammatory sites and the mechanisms of stem-cell homing into the bone marrow.     

Calpain activation in shear-induced platelet aggregation

Fluid shear stress has been known to activate platelet reaction such as aggregation, but the exact mechanism of shear-induced platelet aggregation (SIPA) has not been fully understood. Calpain, an intracellular calcium-activated cysteine protease, is abundant in platelets and is considered to be activated and involved in the proteolytic processes during platelet activation. A possible activation of calpain in SIPA was investigated, employing a newly developed aggregometer and specific monoclonal antibodies to detect activation of calpain. When a shear stress gradient varying between 6 and 108 dyn/cm² was applied to platelets, activation of μ-calpain was observed only in high-shear-stressed platelets, resulting in the proteolysis of talin. At 1 min after the onset of constant high shear stress of 108 dyn/cm², μ-calpain activation and proteolysis of talin were detected and increased in a time-dependent manner. Constant shear stress more than 50 dyn/cm², applied for 5 min, caused μ-calpain activation and proteolysis of talin, which were increased in a shear-force-dependent manner. Calpeptin, a calpain-specific peptide antagonist, caused the complete inhibition of both μ-calpain activation and proteolysis of talin, while SIPA profiles with calpeptin showed almost no change compared to those without calpeptin. These results suggest the possibility of calpain involvement in late phases of shear-induced platelet activation such as cytoskeletal reorganization. J. Cell. Biochem. 66:54–64, 1997. © 1997 Wiley-Liss, Inc.     

Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species

Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by K_ATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2^–/–, and gp91^phox–/– mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm² (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G₂/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G₂/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2^–/– and gp91^phox–/– cells. ANG II activation of NADPH oxidase was unaffected by K_ATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane K_ATP channel. cell signaling; ischemia; mechanotransduction; K_ATP channels; NADPH oxidase     

Reduced shear stress: A major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity

BHK-21 cells were cultured under various shear stress conditions in an Integrated Rotating-Wall Vessel (IRWV). Shear ranged from 0.5 dyn/cm² (simulated microgravity) to 0.92 dyn/cm². Under simulated microgravity conditions, BHK-21 cells complexed into three-dimensional cellular aggregates attaining 6 × 10⁶ cells/ml as compared to growth under 0.92 dyn cm² conditions. Glucose utilization in simulated microgravity was reduced significantly, and cellular damage at the microcarrier surface was kept to a minimum. Thus, the integrated rotating wall vessel provides a quiescent environment for the culture of mammalian cells. © 1993 Wiley-Liss, Inc.     

Experimental measurement of dynamic fluid shear stress on the ventricular surface of the aortic valve leaflet

Aortic valve (AV) calcification is a highly prevalent disease with serious impact on mortality and morbidity. The exact causes and mechanisms of AV calcification are unclear, although previous studies suggest that mechanical forces play a role. It has been clinically demonstrated that calcification preferentially occurs on the aortic surface of the AV. This is hypothesized to be due to differences in the mechanical environments on the two sides of the valve. It is thus necessary to characterize fluid shear forces acting on both sides of the leaflet to test this hypothesis. The current study is one of two studies characterizing dynamic shear stress on both sides of the AV leaflets. In the current study, shear stresses on the ventricular surface of the AV leaflets were measured experimentally on two prosthetic AV models with transparent leaflets in an in vitro pulsatile flow loop using two-component Laser Doppler Velocimetry (LDV). Experimental measurements were utilized to validate a theoretical model of AV ventricular surface shear stress based on the Womersley profile in a straight tube, with corrections for the opening angle of the valve leaflets. This theoretical model was applied to in vivo data based on MRI-derived volumetric flow rates and valve dimension obtained from the literature. Experimental results showed that ventricular surface shear stress was dominated by the streamwise component. The systolic shear stress waveform resembled a half-sinusoid during systole and peaks at 64–71 dyn/cm², and reversed in direction at the end of systole for 15–25?ms, and reached a significant negative magnitude of 40–51 dyn/cm². Shear stresses from the theoretical model applied to in vivo data showed that shear stresses peaked at 77–92 dyn/cm² and reversed in direction for substantial period of time (108–110?ms) during late systole with peak negative shear stress of 35–38 dyn/cm².     

流体剪切力对内皮细胞miR-21和miR-199a表达的影响

为探讨流体剪切力对内皮细胞micorRNAs表达的影响。采用旋转锥形圆盘剪切力系统对内皮细胞分别加载低(4dyn/cm2)、中(10 dyn/cm2)和高(15 dyn/cm2)3种不同梯度的剪切力作用24h。对照组未加载剪切力。采用高通量筛选芯片检测microRNAs表达变化,qRT-PCR验证,并进行生物信息学分析。与对照组比较,低剪切力组表达差异的microRNAs有33个(FC1.5或0.5倍,P0.05),其中28个上调,5个下调;中剪切力组表达差异的microRNAs有8个(FC1.5或0.5倍,P0.05),其中6个上调,2个下调;高剪切力组表达差异的microRNAs有31个(FC1.5或0.5倍,P0.05),其中25个上调,6个下调。miR-21在高剪切力组中上调最显著(FC=0.026),在低剪切力组中显著下调(FC=3.531)。miR-199a在低剪切力组中上调最显著(FC=0.075),在高剪切力组中显著下调(FC=3.031)。表达差异的microRNA的靶基因主要与内皮细胞的力学信号转导、细胞跨膜迁移、钙离子信号通路、细胞内吞作用等相关。流体剪切力可诱导内皮细胞miR-21和miR-199a表达发生改变。     

Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS

The shear-induced intracellular signal transduction pathway invascular endothelial cells involves tyrosine phosphorylation andactivation of mitogen-activated protein (MAP) kinase, which may beresponsible for the sustained release of nitric oxide. MAP kinase isknown to be activated by reactive oxygen species (ROS), such asH₂O₂,in several cell types. ROS production in ligand-stimulatednonphagocytic cells appears to require the participation of aRas-related small GTP-binding protein, Rac1. We hypothesized that Rac1might serve as a mediator for the effect of shear stress on MAP kinaseactivation. Exposure of bovine aortic endothelial cells to laminarshear stress of 20 dyn/cm² for5-30 min stimulated total cellular and cytosolic tyrosine phosphorylation as well as tyrosine phosphorylation of MAP kinase. Treating endothelial cells with the antioxidantsN-acetylcysteine and pyrrolidinedithiocarbamate inhibited in a dose-dependent manner theshear-stimulated increase in total cytosolic and, specifically, MAPkinase tyrosine phosphorylation. Hence, the onset of shear stresscaused an enhanced generation of intracellular ROS, as evidenced by anoxidized protein detection kit, which were required for theshear-induced total cellular and MAP kinase tyrosine phosphorylation. Total cellular and MAP kinase tyrosine phosphorylation was completely blocked in sheared bovine aortic endothelial cells expressing adominant negative Rac1 gene product (N17rac1). We concluded that theGTPase Rac1 mediates the shear-induced tyrosine phosphorylation of MAPkinase via regulation of the flow-dependent redox changes inendothelial cells in physiological and pathological circumstances.