Dynamic modeling and control of extracellular ATP concentration on vascular endothelial cells via shear stress modulation

A new dynamic model for cell-deformation-induced adenosine triphosphate （ATP） release from vascular endothelial cells （VECs） is proposed in this paper to quantify the relationship between the ATP concentration at the surface of VECs and blood flow-induced shear stress. The simulation results demonstrate that ATP concentration at the surface of VECs predicted by the proposed new dynamic model is more consistent with the experimental observations than those by the existing static and dynamic models. Furthermore, it is the first time that a proportional-integral-derivative （PID） feedback controller is applied to modulate extracellular ATP concentration. Three types of desired ATP concentration profiles including constant, square wave and sinusoid are obtained by regulating the wall shear stress under this PID control. The systematic methodology utilized in this paper to model and control ATP release from VECs via adjusting external stimulus opens up a new scenario where quantitative investigations into the underlying mechanisms for many biochemical phenomena can be carded out for the sake of controlling specific cellular events.

A mathematical model for ATP-mediated calcium dynamics in vascular endothelial cells induced by fluid shear stress

In consideration of the mechanism for shear-stress-induced Ca^2＋ influx via ATP（adenosine triphosphate）-gated ion channel P2X4 in vascular endothelial cells, a modified model is proposed to describe the shear-stress-induced Ca^2＋ influx. It is affected both by the Ca^2＋ gradient across the cell membrane and extracellular ATP concentration on the cell surface. Meanwhile, a new static ATP release model is constructed by using published experimental data. Combining the modified intracellular calcium dynamics model with the new ATP release model, we establish a nonlinear Ca^2＋ dynamic system in vascular endothelial cells. The ATP-mediated calcium response in vascular endothelial cells subjected to shear stresses is analyzed by solving the governing equations of the integrated dynamic system. Numerical results show that the shear-stress-induced calcium response predicted by the proposed model is more consistent with the experimental observations than that predicted by existing models.

Effects of shear stress on differentiation of stem cells into endothelial cells

Stem cell transplantation is an appealing potential therapy for vascular diseases and an indispensable key step in vascular tissue engineering.Substantial effort has been made to differentiate stem cells toward vascular cell phenotypes,including endothelial cells(ECs)and smooth muscle cells.The microenvironment of vascular cells not only contains biochemical factors that influence differentiation but also exerts hemodynamic forces,such as shear stress and cyclic strain.More recently,studies have shown that shear stress can influence the differentiation of stem cells toward ECs.A deep understanding of the responses and underlying mechanisms involved in this process is essential for clinical translation.This review highlights current data supporting the role of shear stress in stem cell differentiation into ECs.Potential mechanisms and signaling cascades for transducing shear stress into a biological signal are proposed.Further study of stem cell responses to shear stress will be necessary to apply stem cells for pharmacological applications and cardiovascular implants in the realm of regenerative medicine.

The crosstalk between endothelial cells and vascular smooth muscle cells during low shear stress:a proteomic-based approach

Instruction Shear stress,caused by the parallel frictional drag force of blood flow,is a biomechanical force which plays an important role in the control of blood vessels growth and functions [1]. Clinical researches had found out that atherosclerotic le-

Converging Parallel Plate Flow Chambers for Studies on the Effect of the Spatial Gradient of Wall Shear Stress on Endothelial Cells

Many in vitro studies focus on effects of wall shear stress (WSS) and wall shear stress gradient (WSSG) on endothelial cells, which are linked to the initiation and progression of atherosclerosis in the arterial system. Limitation in available flow chambers with a constant WSSG in the testing region makes it difficult to quantify cellular responses to WSSG. The current study proposes and characterizes a type of converging parallel plate flow chamber (PPFC) featuring a constant gradient of WSS. A simple formula was derived for the curvature of side walls, which relates WSSG to flow rate (Q), height of the PPFC (h), length of the convergent section (L), its widths at the entrance (w0) and exit (w1). CFD simulation of flow in the chamber is carried out. Constant WSSG is observed in most regions of the top and bottom plates except those in close proximity of side walls. A change in Q or h induces equally proportional changes in WSS and WSSG whereas an alteration in the ratio between w0 and w1 results in a more significant change in WSSG than that in WSS. The current design makes possible an easy quantification of WSSG on endothelial cells in the flow chamber.

Shear stress effect on endothelial nitric oxide synthase in cultured human umbilical vein endothelial cells

Background: Low shear stress caused by disturbed or turbulent flow at arterial branch points is known to associate with atherosclerosis. However, shear stress at the venous valve location and its association with deep vein thrombosis are less understood due to the complex and poorly understood bi-directional flow in the valve pocket region. We investigated how venous endothelial cells respond to flow shear stress around the venous valve region using a novel in vitro system that mimics venous flow. Results: Human umbilical vein EAhy. 926 cells were cultured on a flexible silastic membrane that mimicked venous tissue. Confluent cells were exposed to sinusoidal uni-and bi-directional pulsatile shear stress (0.1 to 1 dyne/cm2) for up to 6 h. Western-blot analyses indicated that endothelial nitric oxide (eNOS) expression levels decreased regardless of all tested flow patterns, stress magnitude, and shearing time. In contrast, the expression levels of inhibitor of κB (kappa B) and α (alpha)-tubulin were unaffected by the shear stress. Conclusions: Our results indicate that shear stress causes a decrease specifically in eNOS expression, suggesting that it may play a significant role in regulating inflammation related protein expression in endothelial cells.

Effects of laminar shear stress versus resveratrol on the citrulline-NO cycle in endothelial cells

Laminar shear stress (LSS) due to pulsatile blood flow enhances endothelial function by multiple mechanisms including NO production. Red wine and its constituent, resveratrol, have also been postulated to provide vascular protective effects. The aim of the present study was to compare the effects of mechanical LSS and pharmacological resveratrol treatments on the endothelial citrulline-NO cycle. Human umbilical vein endothelial cells (HUVECs) were treated with LSS (12 dyn·cm-2) or resveratrol (25 - 100 μM). The expressions of argininosuccinate synthetase 1 (ASS1), argininosuccinate lyase (ASL), nitric oxide synthase 3 (NOS3) and cationic amino acid transporter 1 (CAT1), and the production of NO were determined. The expressions of Kruppel-like factor (KLF) 2 and KLF4 as upstream regulators of ASS1 and NOS3 were also analyzed. LSS strongly increased the mRNA levels of ASS1 (8.3 fold) and NOS3 (5.4 fold) without significant effects on ASL and CAT1 mRNAs. Resveratrol increased the ASS1 mRNA level in a dose-dependent manner up to 3.8 fold at 100 μM. The effects of resveratrol on the expressions of KLF2 and KLF4 mRNAs were smaller than those of LSS. Protein levels of ASS1 and NOS3, and NO production were markedly increased by LSS but resveratrol (50 μM) increased only ASS1 protein level. The results of the current study showed that LSS had greater effects on the citrulline-NO cycle activity leading to NO production, compared to resveratrol. Because resveratrol was not so effective at stimulating the endothelial citrulline-NO cycle, further studies are needed to find more potent drugs that increase the expression of ASS1 and NOS3 genes.

Phosphorothioate oligonucleotide inhibits tissue factor expression in endothelial cells induced by blood flow shear stress in rats

Objective:To determine the effect of antiparallel phosphorothioate triplex-forming oligonucleotide(apsTFO),which was designed according to shear stress response element(SSRE) in tissue factor(TF) gene promoter region,on the expression of endothelial TF in carotid artery stenosis rats.Methods:Rat model of severe carotid artery stenosis were inflicted by silica gel tube ligation.Half an hour before the model infliction,GT20-apsTFO,GT20-psTFO and GT21-apsTFO labeled with green fluorescence(FITC) were injected into the vena caudalis of rat at a dose of 0.5 mg/kg.Half an hour,4 or 9 h after the ligation,the distribution of TFO in the common carotid artery,the liver and the kidney was detected with aid of fluorescence microscopy.And the mRNA and protein expressions of TF,Egr-1 and Sp1 in the above-mentioned organs were determined with in situ hybridization and immunohistochemical assay respectively in 6 h after the model establishment,and the results were analyzed with an image analysis system.Results:Only in 1 h after TFO injection,fluorescent granules appeared in the liver,the kidney and the vascular wall and lumen of carotid artery,and then in 4.5 h,they still deposited in above sites except the vascular lumen.GT20-apsTFO and GT21-apsTFO significant down-regulated the mRNA and protein expressions of TF compared to the rats without treatment(P<0.05),and the former apsTFO had a more stronger effect than the later(P<0.05).GT20-psTFO had no such effect(P>0.05).The 3 TFOs had no inhibition on the mRNA and protein expressions of Egr-1 and Sp1.Conclusion:Pretreated apsTFO can partly come into the vascular endothelial cells,and inhibit TF expression induced by shear stress,but had no effect on Egr-1 and Sp1 gene expressions.

SHEAR STRESS RESISTANCE OF ENDOTHELIAL CELLS LINED ON PRETREATED PTFE VASCULAR GRAFTS

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The roles of focal adhesion and cytoskeleton systems in fluid shearstress-induced endothelial cell response

Focal adhesions are polyproteins linked to extracellular matrix and cytoskeleton,which play an important role in the process of transforming force signals into intracellular chemical signals and subsequently triggering related physiological or pathological reactions.The cytoskeleton is a network of protein fibers in the cytoplasm,which is composed of microfilaments,microtubules,intermediate filaments,and cross-linked proteins.It is a very important structure for cells to maintain their basic morphology.This review summarizes the process of fluid shear stress transduction mediated by focal adhesion and the key role of the cytoskeleton in this process,which focuses on the focal adhesion and cytoskeleton systems.The important proteins involved in signal transduction in focal adhesion are introduced emphatically.The relationship between focal adhesion and mechanical transduction pathways are discussed.In this review,we discuss the relationship between fluid shear stress and associated diseases such as atherosclerosis,as well as its role in clinical research and drug development.

Uptake of oxidative stress-mediated extracellular vesicles by vascular endothelial cells under low magnitude shear stress

Extracellular vesicles(EVs)are increasingly used as delivery vehicles for drugs and bioactive molecules,which usually require intravascular administration.The endothelial cells covering the inner surface of blood vessels are susceptible to the shear stress of blood flow.Few studies demonstrate the interplay of red blood cell-derived EVs(RBCEVs)and endothelial cells.Thus,the phagocytosis of EVs by vascular endothelial cells during blood flow needs to be elucidated.In this study,red blood cell-derived extracellular vesicles(RBCEVs)were constructed to investigate endothelial cell phagocytosis in vitro and animal models.Results showed that low magnitude shear stress including low shear stress(LSS)and oscillatory shear stress(OSS)could promote the uptake of RBCEVs by endothelial cells in vitro.In addition,in zebrafish and mouse models,RBCEVs tend to be internalized by endothelial cells under LSS or OSS.Moreover,RBCEVs are easily engulfed by endothelial cells in atherosclerotic plaques exposed to LSS or OSS.In terms of mechanism,oxidative stress induced by LSS is part of the reason for the increased uptake of endothelial cells.Overall,this study shows that vascular endothelial cells can easily engulf EVs in areas of low magnitude shear stress,which will provide a theoretical basis for the development and utilization of EVs-based nano-drug delivery systems in vivo.

In vitro fluidic systems: Applying shear stress on endothelial cells

Endothelial cells(ECs)that reside on the surface of blood vessels are constantly exposed to mechanical stimulation,including shear stress.Fluid shear stress(FSS)controls multiple physiological processes in ECs,regulating various pathways that maintain vascular tone and homeostasis function.The complexity of in vivo biological systems raises a demand for better in vitro techniques,which can generate FSS to closely mimic the cellular microenvironment.Through the rational design and use of flow chamber devices,in vitro fluidic systems are critical for a deeper understanding of endothelial responses to various shear conditions.The paper describes principal types of FSS systems,including functional attributes,development process and recent experiments on ECs.Finally,we prospect their possible contribution in the field of endothelial diseases.

Shear stress regulation of nanoparticle uptake in vascular endothelial cells

Nanoparticles(NPs)hold tremendous targeting potential in cardiovascular disease and regenerative medicine,and exciting clinical applications are coming into light.Vascular endothelial cells(ECs)exposure to different magnitudes and patterns of shear stress(SS)generated by blood flow could engulf NPs in the blood.However,an unclear understanding of the role of SS on NP uptake is hindering the progress in improving the targeting of NP therapies.Here,the temporal and spatial distribution of SS in vascular ECs and the effect of different SS on NP uptake in ECs are highlighted.The mechanism of SS affecting NP uptake through regulating the cellular ROS level,endothelial glycocalyx and membrane fluidity is summarized,and the molecules containing clathrin and caveolin in the engulfment process are elucidated.SS targeting NPs are expected to overcome the current bottlenecks and change the field of targeting nanomedicine.This assessment on how SS affects the cell uptake of NPs and the marginalization of NPs in blood vessels could guide future research in cell biology and vascular targeting drugs.

G3BP2 regulates oscillatory shear stress-induced endothelial dysfunction

GTPase-activating SH3 domain-binding protein 2(G3BP2)is a mediator that responds to environmental stresses through stress granule formation and is involved in the progression of chronic diseases.However,no studies have examined the contribution of G3BP2 in the oscillatory shear stress(OSS)-induced endothelial dysfunction.Here we assessed the effects of G3BP2 in endothelial cells(ECs)function and investigated the underlying mechanism.Using shear stress apparatus and partial ligation model,we identified that stress granulerelated genes in ECs could be induced by OSS with RNA-seq,and then confirmed that G3BP2 was highly and specifically expressed in athero-susceptible endothelia in the OSS regions.G3bp2e/eApoee/e mice had significantly decreased atherosclerotic lesions associated with deficiency of G3BP2 in protecting endothelial barrier function,decreasing monocyte adhesion to ECs and inhibiting the proinflammatory cytokine levels.Furthermore,loss of G3BP2 diminished OSS-induced inflammation in ECs by increasing YAP nucleocytoplasmic shuttling and phosphorylation.These data demonstrate that G3BP2 is a critical OSS regulated gene in regulating ECs function and that G3BP2 inhibition in ECs is a promising atheroprotective therapeutic strategy.

细胞骨架F-actin在层流剪切应力诱导EPCs内皮分化中的作用

目的探讨细胞骨架F-actin在层流剪切应力促内皮祖细胞(endothelial progenitor cells,EPCs)向内皮分化中的作用。方法对大鼠骨髓来源的EPCs施以层流剪切应力(1.2 Pa),以荧光定量RT-PCR及流式细胞术检测特异性内皮细胞标记分子vWF、CD31 mRNA及蛋白的表达来反映EPCs分化程度;以免疫荧光染色观测F-actin的排列情况;应用Ras GTPase Pull-Down方法检测Ras活性。结果层流剪切力处理后,EPCs分化标记vWF及CD31的基因及蛋白表达较静止组明显升高(P<0.05),细胞骨架F-actin发生重排,Ras活性明显增高。细胞骨架稳定剂Jasplakinolide(JAS)及细胞骨架松弛剂Cytochalasin D(CytoD)预处理均不同程度地抑制了层流剪切应力所致的细胞骨架的重排、Ras活性的上调及EPCs分化效应(P<0.05),而过表达Ras则对层流剪切应力诱导的EPCs分化有明显促进作用(P<0.05)。结论一定大小的层流剪切应力可促进EPCs向内皮细胞分化,其机制可能与层流剪切应力重塑细胞骨架F-actin,进而影响Ras活性有关;这对于揭示受损血管内皮修复的具体机制、阐明动脉粥样硬化等心血管疾病的发病机理及临床防治此类疾病具有一定的意义。

Mechanotransduction of liver sinusoidal endothelial cells under varied mechanical stimuli

Liver sinusoidal endothelial cells(LSECs)are the gatekeeper of liver to maintain hepatic homeostasis.They are formed into the highly specialized endothelium between vascular lumen and the space of Disse and are mechanosensitive to respond varied microenvironments.Shear stress and mechanical stretch induced by blood perfusion and substrate stiffness enhancement derived from deposition of extracellular matrix(ECM)are major mechanical stimuli that surround LSECs.This review introduces how LSECs respond to the external forces in both physiological and pathological cases and what is the interplay of LSECs with other hepatic cells.Molecular mechanisms that potentiate LSECs mechanotransduction are also discussed.

Role of endothelial cells in the regulation of mechanical microenvironment on tumor progression

Majority of cancer patients die from cancer metastases.The physical stimulation produced by microenvironment regulates invasive behavior of cancer cells.Blood vessel is one of the“pathways”for cancer to metastasize,in which tumor cells need to cross the endothelial barrier for intravasation and extravasation.Tumor vessels are arranged in untraditional hierarchies and characterized with rupture,bend,swell and high permeability that are beneficial to intravasation of cancer cell.Abnormal vessels are accompanied with uneven blood flow,increased compression and interstitial fluid pressure.Meanwhile,excessive proliferation of tumor leads to low oxygen pressure in solid tumor.The aberrant tumor mechanical microenvironment changes the biochemical and mechanical signal transduction of endothelial cells and participates in tumor progression.Many current researches focus on how chemical signals regulate endothelial cell function while the role of physical cues is unclear.In this review,the role of endothelial cells in the regulation of shear stress,intercellular force,extracellular matrix and pressure on tumor progression is summarized.

Trichostatin A and Shear Stress in Regulating Endothelium Differentiation of Bone Marrow Mesenchymal Stem Cells

Differentiation of bone marrow mesenchymal stem cells (MSCs) into endothelial cells (EC) is characterized by the expression of specific endothelial marker genes. Mechanical stimulations play potential effects in EC oriented differentiation of MSCs. However, molecular mechanisms of endothelial differentiation from MSCs have not been defined.Histone acetylations play important roles in regulating gene expression. Histone acetylation status is maintained by histone acetyltransferase (HAT) and histone deacetylases (HDACs). Our previous work described that VEGF and laminar shear stress (SS) work together in determining EC oriented differentiation of MSC. Trichostatin A (TSA) is one of the lustone deacetylase inhibitor. In this study, we found that both TSA and SS could induce EC oriented differentiation of MSCs. And TSA combined with SS showed more powerful influence on the EC oriented differentiation of MSCs.

ON WALL SHEAR STRESS OF ARTERY

In this paper, a method was proposed that the wall shear stress of arterycould be determined by measuring the centerline axial velocity and radial motion of arterial wallsimultaneously. The method is simple in application and can get higher precision when it is used todetermine the shear stress of arterial wall in vivo. As an example, the shear stress distribution inperiodic oscillatory flow of human carotid was calculated and discussed. The computed results showthat the shear stress distribution at any given instant is almost uniform and will be zero at thecenterline and tends to maximum at the vessel wall.