scholarly journals A Modified Parallel Plate Flow Chamber to Study Local Endothelial Response to Recirculating Disturbed Flow

2019 ◽  
Vol 142 (4) ◽  
Author(s):  
Jason Matthew Sedlak ◽  
Alisa Morss Clyne
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Parallel Plate ◽  
Flow Chamber ◽  
Monocyte Adhesion ◽  
Parallel Plate Flow Chamber ◽  
Disturbed Flow ◽  
Recirculating Flow ◽  
Flow Device

Abstract Atherosclerosis develops at arterial sites where endothelial cells (ECs) are exposed to low time-averaged shear stress, in particular in regions of recirculating disturbed flow. To understand how hemodynamics contributes to EC dysfunction in atheroma development, an in vitro parallel plate flow chamber gasket was modified with protruding baffles to produce large recirculating flow regions. Computational fluid dynamics (CFD) predicted that more than 60% of the flow surface area was below the 12 dynes/cm2 atheroprotective threshold. Bovine aortic endothelial cells (BAECs) were then seeded in the parallel plate flow chamber with either the standard laminar or the new disturbed flow gasket (DFG) and exposed to flow for 36 h. Cell morphology, nitric oxide (NO), proliferation, permeability, and monocyte adhesion were assessed by phase contrast and confocal microscopy. BAEC exposed to 20 dynes/cm2 shear stress in the laminar flow device aligned and elongated in the flow direction while increasing nitric oxide, decreasing permeability, and maintaining low proliferation and monocyte adhesion. BAEC in the recirculating flow and low shear stress disturbed flow device regions did not elongate or align, produced less nitric oxide, and showed higher proliferation, permeability, and monocyte adhesion than cells in the laminar flow device. However, cells in disturbed flow device regions exposed to atheroprotective shear stress did not consistently align or decrease permeability, and these cells demonstrated low nitric oxide levels. The new parallel plate DFG provides a means to study recirculating flow, highlighting the complex relationship between hemodynamics and endothelial function.

Wall Shear Stress Determination in a Small-Scale Parallel Plate Flow Chamber Using Laser Doppler Velocimetry Under Laminar, Pulsatile and Low-Reynolds Number Turbulent Flows

2018 ◽  
Vol 140 (6) ◽  
Author(s):  
Hamed Avari ◽  
Kem A. Rogers ◽  
Eric Savory
Keyword(s):  
Shear Stress ◽  
Wall Shear Stress ◽  
Laser Doppler Velocimetry ◽  
Parallel Plate ◽  
Flow Chamber ◽  
Laser Doppler ◽  
Small Scale ◽  
Doppler Velocimetry ◽  
Flow Conditions ◽  
Parallel Plate Flow Chamber

The parallel plate flow chamber (PPFC) has gained popularity due to its applications in fields such as biological tissue engineering. However, most of the studies using PPFC refer to theoretical relations for estimating the wall shear stress (WSS) and, hence, the accuracy of such quantifications remains elusive for anything other than steady laminar flow. In the current study, a laser Doppler velocimetry (LDV) method was used to quantify the flow in a PPFC (H = 1.8 mm × W = 17.5 mm, Dh = 3.26 mm, aspect ratio = 9.72) under steady Re = 990, laminar pulsatile (carotid Re0-mean = 282 as well as a non-zero-mean sinusoidal Re0-mean = 45 pulse) and low-Re turbulent Re = 2750 flow conditions. A mini-LDV probe was applied, and the absolute location of the LDV measuring volume with the respect to the wall was determined using a signal monitoring technique with uncertainties being around ±27 μm. The uniformity of the flow across the span of the channel, as well as the WSS assessment for all the flow conditions, was measured with the uncertainties all being less than 16%. At least two points within the viscous sublayer of the low-Re turbulent flow were measured (with the y+ for the first point < 3) and the WSS was determined using two methods with the differences between the two methods being within 5%. This paper for the first time presents the experimental determination of WSS using LDV in a small-scale PPFC under various flow conditions, the challenges associated with each condition, and a comparison between the cases. The present data will be useful for those conducting biological or numerical modeling studies using such devices.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity

2021 ◽  
Author(s):  
Sarah Basehore ◽  
Samantha Bohlman ◽  
Callie Weber ◽  
Swathi Swaminathan ◽  
Yuji Zhang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Laminar Flow ◽  
No Production ◽  
Disturbed Flow ◽  
Enos Activity ◽  
Enos Phosphorylation ◽  
Steady Laminar ◽  
In Cells

Rationale: In diabetic animals as well as high glucose cell culture conditions, endothelial nitric oxide synthase (eNOS) is heavily O-GlcNAcylated, which inhibits its phosphorylation and nitric oxide (NO) production. It is unknown, however, whether varied blood flow conditions, which affect eNOS phosphorylation, modulate eNOS activity via O-GlcNAcylation-dependent mechanisms. Objective: The goal of this study was to test if steady laminar flow, but not oscillating disturbed flow, decreases eNOS O-GlcNAcylation, thereby elevating eNOS phosphorylation and NO production. Methods and Results: Human umbilical vein endothelial cells (HUVEC) were exposed to either laminar flow (20 dynes/cm2 shear stress) or oscillating disturbed flow (4{plus minus}6 dynes/cm2 shear stress) for 24 hours in a cone-and-plate device. eNOS O-GlcNAcylation was almost completely abolished in cells exposed to steady laminar but not oscillating disturbed flow. Interestingly, there was no change in protein level or activity of key O-GlcNAcylation enzymes (OGT, OGA, or GFAT). Instead, metabolomics data suggest that steady laminar flow decreases glycolysis and hexosamine biosynthetic pathway (HBP) activity, thereby reducing UDP-GlcNAc pool size and consequent O-GlcNAcylation. Inhibition of glycolysis via 2-deoxy-2-glucose (2-DG) in cells exposed to disturbed flow efficiently decreased eNOS O-GlcNAcylation, thereby increasing eNOS phosphorylation and NO production. Finally, we detected significantly higher O-GlcNAcylated proteins in endothelium of the inner aortic arch in mice, suggesting that disturbed flow increases protein O-GlcNAcylation in vivo. Conclusions: Our data demonstrate that steady laminar but not oscillating disturbed flow decreases eNOS O-GlcNAcylation by limiting glycolysis and UDP-GlcNAc substrate availability, thus enhancing eNOS phosphorylation and NO production. This research shows for the first time that O-GlcNAcylation is regulated by mechanical stimuli, relates flow-induced glycolytic reductions to macrovascular disease, and highlights targeting HBP metabolic enzymes in endothelial cells as a novel therapeutic strategy to restore eNOS activity and prevent EC dysfunction in cardiovascular disease.

scholarly journals MicroRNA-128 Confers Anti-Endothelial Adhesion and Anti-Migration Properties to Counteract Highly Metastatic Cervical Cancer Cells’ Migration in a Parallel-Plate Flow Chamber

2020 ◽  
Vol 22 (1) ◽  
pp. 215
Author(s):  
Pei-Chin Chuang ◽  
Chun-Wun Lu ◽  
Ching-Chin Tsai ◽  
Shun-Hung Tseng ◽  
Wen-Hong Su
Keyword(s):  
Cervical Cancer ◽  
Endothelial Cells ◽  
Cancer Cells ◽  
Parallel Plate ◽  
Flow Chamber ◽  
Caski Cell ◽  
Parallel Plate Flow Chamber ◽  
Cervical Cancer Cells ◽  
Vascular Adhesion

Despite the distant metastasis of cervical cancer cells being a prominent cause of mortality, neither the metastasis capacity nor the in vitro conditions mimicking adhesion of cervical cancer cells to endothelial cells have been fully elucidated. Circulating metastatic cancer cells undergo transendothelial migration and invade normal organs in distant metastasis; however, the putative molecular mechanism remains largely uncertain. In this study, we describe the use of an in vitro parallel-plate flow chamber to simulate the dynamic circulation stress on cervical cancer cells and elucidate their vascular adhesion and metastasis. We isolate the viable and shear stress-resistant (SSR) cervical cancer cells for mechanistic studies. Remarkably, the identified SSR-HeLa and SSR-CaSki exhibited high in vitro adhesive and metastatic activities. Hence, a consistently suppressed miR-128 level was revealed in SSR cell clones compared to those of parental wild-type (WT) cells. Overexpressed miR-128 attenuated SSR-HeLa cells’ adherence to human umbilical cord vein endothelial cells (HUVECs); in contrast, suppressed miR-128 efficiently augmented the static adhesion capacity in WT-HeLa and WT-CaSki cells. Hence, amplified miR-128 modestly abolished in vitro SSR-augmented HeLa and CaSki cell movement, whereas reduced miR-128 aggravated the migration speed in a time-lapse recording assay in WT groups. Consistently, the force expression of miR-128 alleviated the SSR-enhanced HeLa and CaSki cell mobility in a wound healing assay. Notably, miR-128 mediated SSR-enhanced HeLa and CaSki cells’ adhesion and metastasis through suppressed ITGA5, ITGB5, sLex, CEACAM-6, MMP9, and MMP23 transcript levels. Our data provide evidence suggesting that miR-128 is a promising microRNA that prevented endothelial cells’ adhesion and transendothelial migration to contribute to the SSR-enhanced adhesion and metastasis progression under a parallel-plate flow chamber system. This indicates that the nucleoid-based miR-128 strategy may be an attractive therapeutic strategy to eliminate tumor cells resistant to circulation shear flow, prevent vascular adhesion, and preclude subsequent transendothelial metastasis.

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