scholarly journals Biomechanical Characterization of Endothelial Cells Exposed to Shear Stress Using Acoustic Force Spectroscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Giulia Silvani ◽  
Valentin Romanov ◽  
Charles D. Cox ◽  
Boris Martinac
Keyword(s):  
Mechanical Properties ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Actin Filament ◽  
Force Spectroscopy ◽  
Shear Stresses ◽  
Cell Periphery ◽  
Static Conditions ◽  
Cells Cultured ◽  
Cell Cell

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore the mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness of living endothelial cells was determined by Acoustic Force Spectroscopy (AFS), by pull parallel multiple functionalized microspheres located at the cell-cell periphery. The unique configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow for up to 48 h, with shear stresses in the physiological range (i.e., 6 dyn/cm2). Two different Endothelial cells lines, Human Aortic Endothelial Cells (HAECs) and Human Umbilical Vein Endothelial Cells (HUVECs), were investigated to show the potential of this tool to capture the change in cellular mechanical properties during maturation of a confluent endothelial monolayer. Immunofluorescence microscopy was exploited to follow actin filament rearrangement and junction formation over time. For both cell types we found that the application of shear-stress promotes the typical phenotype of a mature endothelium expressing a linear pattern of VE-cadherin at the cell-cell border and actin filament rearrangement along the perimeter of Endothelial cells. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was then applied in a single measurement revealing the force-dependent apparent stiffness of the membrane cortex in the kPa range. We also found that beads attached to cells cultured under dynamic conditions were harder to displace than cells cultured under static conditions, showing a stiffer membrane cortex at cell periphery. All together these results demonstrate that the AFS can identify changes in cell mechanics based on force measurements of adherent cells under conditions mimicking their native microenvironment, thus revealing the shear stress dependence of the mechanical properties of neighboring endothelial cells.

scholarly journals Biomechanical characterization of endothelial cells exposed to shear stress using acoustic force spectroscopy

2020 ◽  
Author(s):  
Giulia Silvani ◽  
Valentin Romanov ◽  
Charles D. Cox ◽  
Boris Martinac
Keyword(s):  
Mechanical Properties ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Cell Movement ◽  
Microfluidic Channel ◽  
Elastic Behavior ◽  
Shear Stresses ◽  
Cell Periphery ◽  
Experimental Conditions ◽  
Apparent Stiffness

AbstractCharacterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness and contractile prestress of living endothelial cells were determined by Acoustic Force Spectroscopy (AFS) focusing on the displacement of functionalized microspheres located at the cell-cell periphery. The specific configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow, reaching shear stresses in the physiological range (i.e. 8 dyne cm-2) within 48 hours of barrier function maturation. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was applied in a single measurement revealing a force-dependent apparent stiffness in the kPa range. Moreover, our results show that different degrees of stiffening, defining the elastic behavior of the cell under different experimental conditions, i.e. static and dynamic, are caused by different levels of contractile prestress in the cytoskeleton, and are modulated by shear stress-mediated junction development and stabilization at cell borders. These results demonstrate that the AFS is capable of fast and high-throughput force measurements of adherent cells under conditions mimicking their native microenvironment, and thus revealing the shear stress dependence of mechanical properties of neighbouring endothelial cells.

scholarly journals Visualization of three pathways for macromolecule transport across cultured endothelium and their modification by flow

2017 ◽  
Vol 313 (5) ◽  
pp. H959-H973 ◽  
Author(s):  
Mean Ghim ◽  
Paola Alpresa ◽  
Sung-Wook Yang ◽  
Sietse T. Braakman ◽  
Stephen G. Gray ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Density Lipoprotein ◽  
Paracellular Transport ◽  
Shear Stresses ◽  
Macromolecule Transport ◽  
Tracer Techniques ◽  
Static Conditions ◽  
Hemodynamic Stresses

Transport of macromolecules across vascular endothelium and its modification by fluid mechanical forces are important for normal tissue function and in the development of atherosclerosis. However, the routes by which macromolecules cross endothelium, the hemodynamic stresses that maintain endothelial physiology or trigger disease, and the dependence of transendothelial transport on hemodynamic stresses are controversial. We visualized pathways for macromolecule transport and determined the effect on these pathways of different types of flow. Endothelial monolayers were cultured under static conditions or on an orbital shaker producing different flow profiles in different parts of the wells. Fluorescent tracers that bound to the substrate after crossing the endothelium were used to identify transport pathways. Maps of tracer distribution were compared with numerical simulations of flow to determine effects of different shear stress metrics on permeability. Albumin-sized tracers dominantly crossed the cultured endothelium via junctions between neighboring cells, high-density lipoprotein-sized tracers crossed at tricellular junctions, and low-density lipoprotein-sized tracers crossed through cells. Cells aligned close to the angle that minimized shear stresses across their long axis. The rate of paracellular transport under flow correlated with the magnitude of these minimized transverse stresses, whereas transport across cells was uniformly reduced by all types of flow. These results contradict the long-standing two-pore theory of solute transport across microvessel walls and the consensus view that endothelial cells align with the mean shear vector. They suggest that endothelial cells minimize transverse shear, supporting its postulated proatherogenic role. Preliminary data show that similar tracer techniques are practicable in vivo. NEW & NOTEWORTHY Solutes of increasing size crossed cultured endothelium through intercellular junctions, through tricellular junctions, or transcellularly. Cells aligned to minimize the shear stress acting across their long axis. Paracellular transport correlated with the level of this minimized shear, but transcellular transport was reduced uniformly by flow regardless of the shear profile.

Tire Treadwear — A Comprehensive Evaluation of the Factors: Generic Type, Aspect Ratio, Tread Pattern, and Tread Composition Part IV: Laboratory Measurement of Mechanical Properties and Mechanical Actions of Tires Relating to Treadwear

1986 ◽  
Vol 14 (4) ◽  
pp. 264-291
Author(s):  
K. L. Oblizajek ◽  
A. G. Veith
Keyword(s):  
Mechanical Properties ◽  
Shear Stress ◽  
Contact Zone ◽  
Flexural Rigidity ◽  
Comprehensive Evaluation ◽  
Shear Stiffness ◽  
Shear Stresses ◽  
Lateral Shear ◽  
Band Bending ◽  
Tread Rubber

Abstract Treadwear is explained by specific mechanical properties and actions of tires. Rubber shear stresses in the contact zone between the tire and the road become large at large slip angles. When normal stresses are insufficient to prevent sliding at the rear of the footprint, wear occurs at a rate that depends on test severity. Two experimental approaches are described to relate treadwear to tire characteristics. The first uses transducers imbedded in a simulated road surface to obtain direct measurements of contact stresses on the loaded, freely-rolling, steered tires. The second approach is developed with the aid of a simple carcass, tread-band, tread-rubber tire model. Various tire structural configurations; characterized by carcass spring rate, edgewise flexural band stiffness, and tread rubber shear stiffness; are simulated and lateral shear stress response in the contact zone is determined. Tires featuring high band stiffness and low carcass stiffness generate lower lateral shear stress levels. Furthermore, coupling of tread-rubber stiffness and band flexural rigidity are important in determining level of shear stresses. Laboratory measurements with the described apparatus produced values of tread-band bending and carcass lateral stiffness for several tire constructions. Good correlation is shown between treadwear and a broad range of tire stiffness and test course severities.

scholarly journals Studying dynamic stress effects on the behaviour of THP-1 cells by microfluidic channels

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Semra Zuhal Birol ◽  
Rana Fucucuoglu ◽  
Sertac Cadirci ◽  
Ayca Sayi-Yazgan ◽  
Levent Trabzon
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Vascular System ◽  
Circulatory System ◽  
Disease Process ◽  
Microfluidic Channels ◽  
Shear Stresses ◽  
Stress Effects ◽  
Adhesion Behavior ◽  
Cycling System

AbstractAtherosclerosis is a long-term disease process of the vascular system that is characterized by the formation of atherosclerotic plaques, which are inflammatory regions on medium and large-sized arteries. There are many factors contributing to plaque formation, such as changes in shear stress levels, rupture of endothelial cells, accumulation of lipids, and recruitment of leukocytes. Shear stress is one of the main factors that regulates the homeostasis of the circulatory system; therefore, sudden and chronic changes in shear stress may cause severe pathological conditions. In this study, microfluidic channels with cavitations were designed to mimic the shape of the atherosclerotic blood vessel, where the shear stress and pressure difference depend on design of the microchannels. Changes in the inflammatory-related molecules ICAM-1 and IL-8 were investigated in THP-1 cells in response to applied shear stresses in an continuous cycling system through microfluidic channels with periodic cavitations. ICAM-1 mRNA expression and IL-8 release were analyzed by qRT-PCR and ELISA, respectively. Additionally, the adhesion behavior of sheared THP-1 cells to endothelial cells was examined by fluorescence microscopy. The results showed that 15 Pa shear stress significantly increases expression of ICAM-1 gene and IL-8 release in THP-1 cells, whereas it decreases the adhesion between THP-1 cells and endothelial cells.

Effect of glycocalyx on shear-dependent albumin uptake in endothelial cells

2004 ◽  
Vol 287 (5) ◽  
pp. H2287-H2294 ◽  
Author(s):  
Akinori Ueda ◽  
Manabu Shimomura ◽  
Mariko Ikeda ◽  
Ryuhei Yamaguchi ◽  
Kazuo Tanishita
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Laser Scanning ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Interface Barrier ◽  
Uptake Process ◽  
Static Conditions

The glycocalyx layer on the surface of an endothelial cell is an interface barrier for uptake of macromolecules, such as low-density lipoprotein and albumin, in the cell. The shear-dependent uptake of macromolecules thus might govern the function of the glycocalyx layer. We therefore studied the effect of glycocalyx on the shear-dependent uptake of macromolecules into endothelial cells. Bovine aorta endothelial cells were exposed to shear stress stimulus ranging from 0.5 to 3.0 Pa for 48 h. The albumin uptake into the cells was then measured using confocal laser scanning microscopy, and the microstructure of glycocalyx was observed using electron microscopy. Compared with the uptake into endothelial cells under static conditions (no shear stress stimulus), the albumin uptake at a shear stress of 1.0 Pa increased by 16% and at 3.0 Pa decreased by 27%. Compared with static conditions, the thickness of the glycocalyx layer increased by 70% and the glycocalyx charge increased by 80% at a shear stress of 3.0 Pa. The albumin uptake at a shear stress of 3.0 Pa for cells with a neutralized (no charge) glycocalyx layer was almost twice that of cells with charged layer. These findings indicate that glycocalyx influences the albumin uptake at higher shear stress and that glycocalyx properties (thickness and charge level) are involved with the shear-dependent albumin uptake process.

scholarly journals Mapping the endothelial cell S-Sulfhydrome highlights the crucial role of integrin sulfhydration in vascular function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
D R Bibli ◽  
D R Hu ◽  
D R Looso ◽  
D R Weigert ◽  
D R Wittig ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Vascular Disease ◽  
Disulfide Bond ◽  
Mesenteric Arteries ◽  
Human Endothelial Cells ◽  
Β3 Integrin ◽  
Static Conditions ◽  
Protein Disulfide

Abstract Background In vascular endothelial cells, cysteine metabolism by the cystathionine-γ lyase (CSE), generates hydrogen sulfide-related sulfane sulfur compounds (H2Sn), that exert their biological actions via cysteine S-sulfhydration of target proteins. This study set out to map the “S-sulfhydrome” i.e. the spectrum of proteins targeted by H2Sn in human endothelial cells. Methods LC-MS/MS was used to identify S-sulfhydrated cysteines in endothelial cell proteins and β3 integrin intra-protein disulfide bond rearrangement. Functional studies included endothelial cell adhesion, shear stress-induced cell alignment, blood pressure measurements and flow-induced vasodilatation in endothelial cell-specific CSE knock out mice and a small collective of patients with endothelial dysfunction. Results Three paired sample sets were compared: (1) native human endothelial cells isolated from plaque-free mesenteric arteries (CSE activity high) and plaque-containing carotid arteries (CSE activity low), (2) cultured human endothelial cells kept under static conditions or exposed to fluid shear stress to decrease CSE expression, and (3) cultured endothelial cells exposed to shear stress to decrease CSE expression and treated with solvent or the slow-releasing H2Sn donor, SG1002. The endothelial cell “S-sulfhydrome” consisted of 3446 individual cysteine residues in 1591 proteins. The most altered family of proteins were the integrins and focusing on β3 integrin in detail we found that S-sulfhydration affected intra-protein disulfide bond formation and was required for the maintenance of an extended-open conformation of the β leg. β3 integrin S-sulfhydration was required for endothelial cell mechanotransduction in vitro as well as flow-induced dilatation in murine mesenteric arteries. In cultured cells, the loss of S-sulfhydration impaired interactions between β3 integrin and Gα13, resulting in the constitutive activation of RhoA and impaired flow-induced endothelial cell realignment. In humans with atherosclerosis, endothelial function correlated with low H2Sn generation, impaired flow-induced dilatation and a failure to detect β3 integrin S-sulfhydration, all of which were rescued following the administration of an H2S supplement. Conclusions Vascular disease is associated with marked changes in the S-sulfhydration of endothelial cell proteins involved in mediating responses to flow. Short term H2Sn supplementation improved vascular reactivity in humans highlighting the potential of interfering with this pathway to treat vascular disease. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft

Identification of atheroprone shear stress responsive regulatory elements in endothelial cells

2019 ◽  
Vol 115 (10) ◽  
pp. 1487-1499 ◽  
Author(s):  
Olga Bondareva ◽  
Roman Tsaryk ◽  
Vesna Bojovic ◽  
Maria Odenthal-Schnittler ◽  
Arndt F Siekmann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Umbilical Vein ◽  
Regulatory Element ◽  
Hippo Pathway ◽  
Regulatory Elements ◽  
Binding Motif ◽  
Motif Analysis ◽  
Static Conditions

Abstract Aims Oscillatory shear stress (OSS) is an atheroprone haemodynamic force that occurs in areas of vessel irregularities and is implicated in the pathogenesis of atherosclerosis. Changes in signalling and transcriptional programme in response to OSS have been vigorously studied; however, the underlying changes in the chromatin landscape controlling transcription remain to be elucidated. Here, we investigated the changes in the regulatory element (RE) landscape of endothelial cells under atheroprone OSS conditions in an in vitro model. Methods and results Analyses of H3K27ac chromatin immunoprecipitation-Seq enrichment and RNA-Seq in primary human umbilical vein endothelial cells 6 h after onset of OSS identified 2806 differential responsive REs and 33 differentially expressed genes compared with control cells kept under static conditions. Furthermore, gene ontology analyses of putative RE-associated genes uncovered enrichment of WNT/HIPPO pathway and cytoskeleton reorganization signatures. Transcription factor (TF) binding motif analysis within RE sequences identified over-representation of ETS, Zinc finger, and activator protein 1 TF families that regulate cell cycle, proliferation, and apoptosis, implicating them in the development of atherosclerosis. Importantly, we confirmed the activation of EGR1 as well as the YAP/TAZ complex early (6 h) after onset of OSS in both cultured human vein and artery endothelial cells and, by undertaking luciferase assays, functionally verified their role in RE activation in response to OSS. Conclusions Based on the identification and verification of specific responsive REs early upon OSS exposure, we propose an expanded mechanism of how OSS might contribute to the development of atherosclerosis.

Effects of Fluid Shear Stress on Endothelial Cell Invasion Into Three-Dimensional Matrices

2007 ◽  
Author(s):  
Hojin Kang ◽  
Kayla J. Bayless ◽  
Roland Kaunas
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Cell Invasion ◽  
Fluid Shear Stress ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Shear Stresses ◽  
Collagen Gels ◽  
Fluid Shear

We have previously developed a cell culture model to study the effects of angiogenic factors, such as sphingosine-1-phosphate (S1P), on the invasion of endothelial cells into the underlying extracellular matrix. In addition to biochemical stimuli, vascular endothelial cells are subjected to fluid shear stress due to blood flow. The present study is aimed at determining the effects of fluid shear stress on endothelial cell invasion into collagen gels. A device was constructed to apply well-defined fluid shear stresses to confluent human umbilical vein endothelial cells (HUVECs) seeded on collagen gels. Fluid shear stress induced significant increases in cell invasion with a maximal induction at ∼5 dyn/cm2. These results provide evidence that fluid shear stress is a significant stimulus for endothelial cell invasion and may play a role in regulating angiogenesis.

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