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.

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.

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.

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.

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.

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 Spl 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 Spl. 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 Spl gene expressions.

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-

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.

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

he potential benefits of endothelial cell seeding depend not only on effective cell attachment, but also on the ability of the cells to resist the shear stresses of blood flow. The shear stressresistance of cultured adult human endothelial cells was investigated on 6mm polytetrafluoroethylene vascular grafts (made in China). Endothelial cells were sodded onto pretreated vascular grafts at a high density. Grafts were then cultivated for 9 days to enable the maturation of thecytoskeleton, before they were exposed to pulsatile flow simulating the flow patterns and the wallshear forces of the small srtery. After 1 hour of per fusion,a cell loss of 11% in grafts pretreatedwith 20μ g/ ml fibronectin and of 12% in grafts pretreated with 5μ g /ml fibronectin coated human platelet- poor- plasma (p>0. 50 n = 10). Therefore we conclude that the endothelial celllining on vascular grafts pretreated with suitable substrates can form a shear stress-resistant endothelial cell monolayer on polytetrafluoroethylene vascular grafts.

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.

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.

人脐静脉内皮细胞在RGD肽聚酯材料表面的黏附稳定性研究

研究RGD肽对内皮细胞(Endothelialcell,EC)在生物材料表面黏附稳定性的影响。实验材料(聚酯)分为三组:RGD组(表面共价接枝人工合成的RGD三肽)、对照组(表面预衬纤维粘连蛋白)和空白组(表面未作任何处理),然后在三组材料表面种植体外培养的人脐静脉内皮细胞,并在定常流条件下观察比较RGD肽和纤维粘连蛋白对材料表面细胞黏附稳定性的影响。结果显示随着剪切力作用时间延长和剪切力加大,三组材料表面黏附的细胞脱落逐渐增多。空白组PET表面细胞脱落最为明显,8.19dyne/cm2作用4h后,材料表面细胞残留率仅为13.73%。PET表面结合RGD或纤维粘连蛋白后,细胞残留率明显增加,同样剪切力作用下细胞残留率分别为43.33%和40.75%,两组之间无显著性差异。由此得出结论,RGD可以提高EC在材料表面的黏附稳定性。本结果仅是一个体外实验的初步结果,需要进一步的体内实验加以证实。

流体剪应力对人脐静脉内皮细胞葡萄糖调节蛋白78和C/EBP同源蛋白表达的影响

目的探索流体剪应力对人脐静脉内皮细胞(HUVECs)葡萄糖调节蛋白78(GRP78)和C/EBP同源蛋白(CHOP)表达的影响。方法以HUVECs作为实验细胞,设计并构建流体动力学模拟实验系统,控制流体动力学模拟实验系统中灌流液的流速,以实现对实验细胞施加不同的流体剪应力。按实验细胞在实验系统中所承受的不同流体剪应力,将实验细胞分为低剪应力组(A组;0.4 Pa)、中剪应力组(B组;0.8 Pa)和高剪应力组(C组;1.2 Pa)。每组HUVECs包含3个细胞玻片,每个玻片经实验系统灌流液反复循环流经12 h。采用蛋白质印迹法对各组细胞中GRP78和CHOP蛋白水平进行检测,采用实时荧光定量逆转录聚合酶链反应技术测定各组细胞GRP78和CHOP蛋白及其信使RNA(mRNA)相对水平。应用GraphPad Prism 8.0软件对数据进行统计学分析。结果(1)A、B、C组HUVECs中GRP78蛋白相对表达水平分别为1.33±0.46、0.93±0.34、0.64±0.30;多组间比较差异有统计学意义(F=36.17,P<0.05)。A组GRP78蛋白相对表达水平高于B组、C组(均P<0.01),B组GRP78蛋白相对表达水平高于C组(P=0.0013)。3组HUVECs中CHOP蛋白相对表达水平分别为:A组1.29±0.38,B组0.90±0.34,C组0.59±0.29;多组间比较差异有统计学意义(F=41.27,P<0.05)。A组CHOP蛋白相对表达水平高于B组、C组(均P<0.01),B组CHOP蛋白相对表达水平高于C组(P=0.0004)。(2)A、B、C组HUVECs中GRP78 mRNA相对表达水平分别为18.3±3.4、11.3±1.8、5.4±2.2;多组间比较差异有统计学意义(F=189.20,P<0.05)。A组GRP78 mRNA相对表达水平高于B组、C组(均P<0.01),B组GRP78 mRNA相对表达水平高于C组(P<0.01)。3组HUVECs中CHOP mRNA相对表达水平分别为:A组20.4±3.8,B组14.2±2.1,C组7.8±1.3;多组间比较差异有统计学意义(F=171.80,P<0.05)。A组CHOP mRNA相对表达水平高于B组、C组(均P<0.01),B组CHOP mRNA相对表达水平高于C组(P<0.01)。结论低流体剪应力可能增加HUVECs中GRP78、CHOP的蛋白及其mRNA表达水平。

具有内表面拓扑结构的仿生血管设计与制备

针对临床人工血管不具备诱导内皮化的内表面微结构,无法为内皮细胞生长提供合适的力学环境的问题,设计并制备了一种具有内表面拓扑结构的仿生血管(BVST),实现了壁面剪切力的优化分布。基于内皮细胞的形态分布,设计了具有正交、菱形、圆弧三种内表面拓扑结构的仿生血管。通过仿真分析,发现正交与菱形结构的BVST对血液流速影响较小,周期波动相近,且BVST的内表面拓扑结构能减缓细胞流速、增加细胞滞留时间,有利于内皮细胞在进行体外培养时沉积、黏附在BVST内表面。通过对比BVST与无拓扑结构仿生血管的剪切力分布,发现BVST整体剪切力分布更加均匀。分析单个拓扑单元内的剪切力分布,发现菱形拓扑单元内部剪切力分布均匀且连续,剪切力变化较小,有利于内皮细胞的生长。将聚己内酯(PCL)溶于二氯甲烷(DCM)配制质量分数35%的PCL-DCM溶液,采用内径为0.2 mm的针头,在移动速度40 mm/s、电压5.8 kV、针头高度1 mm、挤出气压0.05 MPa的条件下,基于电纺直写技术制备具有拓扑结构的仿生血管内膜;将拓扑结构浸水黏附在纺丝收集柱,基于静电纺丝技术制备仿生血管外膜,获得具有内表面拓扑结构的双层仿生血管。提出的仿生血管设计与制备工艺可为血管内皮化研究提供参考。

流体剪切力对RGD肽修饰纯钛表面细胞粘附稳定性影响的研究

目的研究精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)肽修饰的纯钛表面是否有助于提高成骨细胞的抗剪切能力,比较剪切力大小对细胞粘附的影响。方法实验分为空白处理纯钛组(CpTi组)、对照处理碱-热陈化组(AWTi组)、对照处理溶胶涂层组(ATTi组)和实验处理粘附肽修饰组(RGDTi组)。利用流体应力加力系统,定常流下作用于4组纯钛表面的成骨细胞,计算平均剪切力分别为2.05、3.12、4.20Pa时4组纯钛表面细胞残留率。结果细胞脱落量与剪切力的大小成正比,RGDTi组在2.05、3.12、4.20Pa的流体剪切力作用下,表面细胞残留率分别为92.8%、70.1%、66.8%,多于其他3组。结论RGD有利于提高成骨细胞在纯钛表面粘附稳定性,增加成骨细胞的抗剪切力。

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.

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.