Real-time quantification of endothelial response to shear stress and vascular modulators

DeStefano, Jackson G.; Williams, Ashley J.; Wnorowski, Alexa; Yimam, Nahom; Searson, Peter C.; Wong, Andrew D.

doi:10.1039/c7ib00023e

Cited by 33 publications

(34 citation statements)

References 62 publications

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“…To assess cell motility, we measured the average cell speed, the RMS displacement, and the directionality. The average cell speed, a measure of cell activity [ 11 , 26 , 33 ], was calculated by automated particle image velocimetry (PIV) analysis [ 19 ]. The average speed within the monolayers decreased from a maximum of approximately 0.2 µm min −1 during the 6-h conditioning period, to a steady state value of about 0.1 µm min −1 under static conditions and under 4 and 12 dyne cm −2 shear stress (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…To determine the net rate of change in cell number (% h −1 ), the apoptosis rate was subtracted from the division rate. Identification of apoptosis and proliferation events from phase contrast movies allows quantitative analysis of the dynamic behavior of the monolayer as a function of time [ 33 , 34 ]. Furthermore, direct observation ensures that we include apoptosis events associated with cell loss and removal from the monolayer by shear flow, which may not be detected by labeling methods.…”

Section: Methodsmentioning

confidence: 99%

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Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs)

DeStefano

Williams

et al. 2017

Fluids Barriers CNS

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129

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BackgroundThe endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood–brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells.MethodsTo assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm−2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood–brain barrier were performed.ResultsHuman brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood–brain barrier markers.ConclusionsThe characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood–brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation of tight junctions decreases cell motility and prevents any morphological transitions, (3) flow serves to increase the contact area between cells, resulting in very low cell displacement in the monolayer, (4) since tight junctions are already formed under static conditions, increasing the contact area between cells does not cause upregulation in protein and gene expression of BBB markers, and (5) the increase in contact area induced by flow makes barrier function more robust.Electronic supplementary materialThe online version of this article (doi:10.1186/s12987-017-0068-z) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs)

DeStefano

Williams

et al. 2017

Fluids Barriers CNS

Self Cite

129

125

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“…While these effects have been broadly documented across ECs of other organs, the response of BMECs to shear stress appears unique. While human umbilical vein endothelial cells (HUVECs) elongate in the direction of flow, BMECs instead remain rounded [ 41 , 42 ]. Other physical characteristics of capillaries, such as the degree of vessel curvature, have been shown to elicit elongation and alignment from HUVECs but not from immortalized BMECs, providing further evidence of their unique phenotype [ 43 ].…”

Section: Introductionmentioning

confidence: 99%

Engineering the human blood-brain barrier in vitro

2017

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The blood-brain barrier (BBB) is the interface between the vasculature and the brain, regulating molecular and cellular transport into the brain. Endothelial cells (ECs) that form the capillary walls constitute the physical barrier but are dependent on interactions with other cell types. In vitro models are widely used in BBB research for mechanistic studies and drug screening. Current models have both biological and technical limitations. Here we review recent advances in stem cell engineering that have been utilized to create innovative platforms to replicate key features of the BBB. The development of human in vitro models is envisioned to enable new mechanistic investigations of BBB transport in central nervous system diseases.

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“…9 DeStefano et al observed lower cell proliferation rates and higher apoptosis rates of EC at lower shear stress levels (4 dyn/cm 2 ) in comparison to higher WSS levels (16 dyn/cm 2 ). 49 Our results show cell proliferation between day 3 and day 21, where cell proliferation must overcome apoptosis or cell detachment because density is significantly higher on day 21 in comparison to days 3, 7, and 14.…”

Section: Discussionmentioning

confidence: 55%

EndOxy: Mid‐term stability and shear stress resistance of endothelial cells on PDMS gas exchange membranes

et al. 2020

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Endothelialized oxygenator devices (EndOxy) with a physiological, nonthrombogenic, and anti‐inflammatory surface offer the potential to overcome current shortcomings of conventional extracorporeal membrane oxygenation such as complications like thromboembolism and bleeding that deteriorate adequate long‐term hemocompatibility. The approach of endothelialization of gas exchange membranes, and thus the formation of a nonthrombogenic and anti‐inflammatory surface, is promising. In this study, we investigated the mid‐term shear stress resistance as well as gas transfer rates and cell densities of endothelial cells seeded on RGD‐conjugated polydimethylsiloxane (RGD‐PDMS) gas exchange membranes under dynamic conditions. Human umbilical vein endothelial cells were seeded on RGD‐PDMS and exposed to defined shear stresses in a microfluidic bioreactor. Endothelial cell morphology was assessed by bright field microscopy and immunocytochemistry. Furthermore, gas transfer measurement of blank, RGD‐conjugated, and endothelialized PDMS oxygenator membranes was performed. RGD‐PDMS gas exchange membranes proved suitable for the dynamic culture of endothelial cells for up to 21 days at a wall shear stress of 2.9 dyn/cm2. Furthermore, the cells resisted increased wall shear stresses up to 8.6 dyn/cm2 after a previous dynamic preculture of each one hour at 2.9 dyn/cm2 and 5.7 dyn/cm2. Also, after a longer dynamic preculture of three days at 2.9 dyn/cm2 and one hour at 5.7 dyn/cm2, increased wall shear stresses of 8.6 dyn/cm2 were tolerated by the cells and cell integrity could be remained. Gas transfer (GT) tests revealed that neither RGD conjugation nor endothelialization of RGD‐PDMS significantly decrease the gas transfer rates of the membranes during short‐term trials. Gas transfer rates are stable for at least 72 hours of dynamic cultivation of endothelial cells. Immunocytochemistry showed that the cell layer stained positive for typical endothelial cell markers CD31 and von Willebrand factor (VWF) after all trials. Cell density of EC on RGD‐PDMS increased between 3 and 21 days of dynamic culture. In this study, we show the suitability of RGD‐PDMS membranes for flow resistant endothelialization of gas‐permeable membranes, demonstrating the feasibility of this approach for a biohybrid lung.

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Real-time quantification of endothelial response to shear stress and vascular modulators

Cited by 33 publications

References 62 publications

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs)

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs)

Engineering the human blood-brain barrier in vitro

EndOxy: Mid‐term stability and shear stress resistance of endothelial cells on PDMS gas exchange membranes

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