This work was supported by CIHR MOP 142307 to P.A.M. H.S.J.M. is a recipient of a Canadian Institutes of Health Research Training Program in Regenerative Medicine Fellowship. H.S.J.M., A.N.S., K.H.K., and M.K.D. are recipients of the Queen Elizabeth II Graduate Scholarships in the Science and Technology.

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The authors have nothing to disclose.

Shear stress is a physiologic condition that modulates endothelial function, in part, by affecting steady-state gene expression2,5. Models of gene regulation in various shear stress conditions will contribute to a greater understanding of endothelial function. This pragmatic workflow includes a flow circuit using a parallel-plate flow chamber adapted from Lane et al.9 and represents laminar, non-pulsatile flow. The overall set-up was designed to facilitate experiments that require multiple flow chambers and minimize experimental variability in this setting.
The flow circuit assembly is a major component of this workflow and is adapted from Lane et al.9. Several adaptations of this assembly and protocol were made to reflect differences in the experimental systems. A large heated unit, the BEACH, is an adaptation that facilitates the simultaneous operation and monitoring of several flow circuits within the same environment. This system has been used successfully for the application of shear stress to endothelial cells for various time periods, from 1 h to 7 days, and at several levels of shear stress (e.g., 1.0, 1.5, and 2.0 Pa). This system was also used for gene knockdown studies to assess the function of a flow-responsive endothelial gene in the setting of shear stress (Figure 4)5. There is considerable variability between pumps and pump heads, which may also change over time due to normal wear and tear. To account for these differences, we use a flow sensor situated proximal to the flow chamber to continuously monitor flow rates. Various sizes of tubing and Luers are used to ensure a tight fit for each component and to prevent any leakage during experiments. We counted cells and seeded approximately 1,000,000 cells per glass slide, dropwise, and then let the cells incubate at 37 &#176;C for 15 min to increase adherence efficiency. Compared to endothelial progenitor cells, which can be seeded for a short time prior to the application of shear stress9, we seed human endothelial cells for at least 24 h prior to the application of shear stress. Shorter durations can lead to the dislodging of cells during flow, or a discontinuous layer of endothelial cells, even with the appropriate seeding density. We emphasize the inspection of the slides for a confluent monolayer of endothelial cells both prior to and after the application of shear stress. We find that slides coated with fibronectin, a natural extracellular matrix component18, maintain the endothelial monolayer more consistently compared to sides coated with gelatin (denatured fibrillar type I collagen). Finally, the flow dampeners in this protocol are optimized to use 30 mL of media, compared to 190 mL of media for other manufacturers.
Several steps in the flow circuit assembly require additional care. An even monolayer of endothelial cells should be established prior to the application of shear stress. It is important to seed cells onto the glass slide dropwise to increase the number of cells that adhere to the slide and, thus, the overall slide coverage. The wait time between cell seeding and adding additional media to the slide generally improves seeding efficiency as it provides sufficient time for cells to adhere to the slide rather than be washed away into the multi-slide tray. Inspect cells visually before, during, and after the application of shear stress. A microscope can be placed in the BEACH for this purpose. The flow sensor must be attached in the correct orientation and the target flow rate should be checked for each individual flow chamber. The flow loop system should be perfused without the chamber to ensure no media leakage or other problems, such as pressure build-up, to avoid perturbing the cells during the actual experiments. Inspect for and eliminate bubbles in the system. While testing the flow loop system, ensure the stopcocks are all open before turning on the peristaltic pump to allow uninterrupted, unidirectional flow. Ensure that all stopcocks are closed prior to attaching the flow chamber to the loop and fully opened prior to restarting the pump.
We find that the addition of an exogenous reference gene is helpful in a variety of scenarios. Endothelial cell media often contains heparin, which is an inhibitor of PCR17. While some RNA extraction protocols incorporate steps to remove heparin, trace amounts can cause differences in PCR efficiencies between samples. Potent treatments may also preclude the identification of an endogenous reference gene that is quantitatively stable. Our lab has created an efficient protocol to synthesize 5&#39;-capped and poly-A-tailed luciferase RNA for use as an exogenous reference RNA. This strategy has proved to be a cost-effective approach compared to purchasing a commercially available RNA. During the preparation of exogenous reference RNA, it is important to aliquot RNA into single-use aliquots to avoid multiple freeze-thaw cycles. Thorough mixing and accurate pipetting are critical to maintaining inter-aliquot consistency. Typical experiments show a luciferase efficiency in the range of 5% &#177; 2.5% but can range from 1% - 10%. It is prudent to correct RT-qPCR results for both the exogenous reference RNA efficiency and an endogenous reference (housekeeping) gene.
For the experiments we conduct, firefly luciferase sequences are used as a non-mammalian spike-in reference RNA in mammalian models. In experiments where firefly luciferase is expressed in cells, this would not be an appropriate reference gene. Other species-specific reference genes can be used, including the Caenorhabditis elegans miRNA cel-miR-3919 and ribulose bisphosphate carboxylase plant RNA20.
This flow circuit models a 2-D monolayer of endothelial cells grown on tissue culture plastic or glass, which is quite stiff. Matrix stiffness can influence the endothelial response to fluid shear stress21. This model system uses a relatively high oxygen concentration more similar to arterial than venous oxygen concentrations. This model closely resembles straight segments of larger vessels in a closed cardiovascular system and provides a relatively homogenous environment for endothelial cells on the slide. Other specific conditions in 3-D structures, such as bifurcations or curvatures of vessels, are not represented with this model. Other systems can model other flow patterns, including those in curved regions of the vasculature, but yield fewer cells than the system described in this protocol. Similarly, other assemblies may be more appropriate if &#62; 1 x 106 cells are required, or if a single-cell analysis is required. Our current application models non-pulsatile laminar flow. Yet, this model can be used to generate other waveforms, including pulsatile or oscillatory waveforms, with consistency, as the flow rates are monitored continuously.
Overall, this pragmatic workflow provides a system for the simultaneous application of shear stress to multiple flow chambers with monitored flow rates. Materials and procedures throughout this workflow are designed to minimize experimental variability between samples and conditions. This workflow has been successfully used for RNAi experiments in the setting of laminar flow and can be also used for any experiments requiring multiple conditions with laminar shear stress, or multiple laminar shear stress magnitudes and/or time points, including alternative waveforms.

Vascular endothelial cells form the inner cellular lining of blood vessels in the closed cardiovascular system of higher species. They form the interface between the blood and tissues and are characterized by luminal and abluminal surfaces. The endothelium is a diverse, active, and adaptive system that regulates blood flow, nutrient trafficking, immunity, and the growth of new blood vessels1. In the body, endothelial cells normally exist in an environment where they are exposed to the frictional force of circulation, shear stress2. Shear stress is an important regulator of endothelial cell gene expression3, and endothelial cells attempt to maintain shear stress within a given range2,4. Endothelial cells demonstrate angiogenic patterning in the absence of shear stress5 that can improve tissue perfusion. Regional patterns of disturbed flow and altered shear stress are associated with the expression of inflammatory genes6 and the development of atherosclerosis7,8. Thus, models that include shear stress are a major component of understanding endothelial gene regulation.
We describe a method for studying the gene regulation in vascular endothelial cells under shear stress. This system uses non-pulsatile flow and mimics fluid shear stress levels and oxygen concentration that model conditions for arterial endothelial cells. This protocol includes details of methods for the gene knockdown using RNA interference (RNAi), the set-up for the application of shear stress using the parallel-plate flow apparatus, and methods for the spike-in of an exogenous reference RNA prior to analysis by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR). This pipeline is used for studying gene regulation in endothelial cells in the presence and absence of laminar shear stress and includes an adaptation of the parallel-plate flow apparatus described by Lane et al.9. This particular set-up was designed to facilitate the simultaneous assessment of multiple experimental conditions that allows direct comparison of shear stress conditions, as well as the normalization of RNA analysis. A large heated unit with controlled humidity is utilized to allow multiple separate flow chambers and pumps to be running simultaneously with flow rates monitored for each flow chamber assembly in real-time. The application of this set-up is used for gene knockdown using RNAi in the setting of laminar flow/shear stress, but aspects of this protocol can be applied to any assessment of RNA expression.
Common approaches to the application of shear stress for endothelial cells include microfluidic systems10, a cone-and-plate viscometer11, and a parallel-plate flow chamber12. Microfluidic systems from various manufacturers have been useful in studying mechanobiology and mechanotransduction in multiple cell and tissue types and a variety of biophysical stimuli. For endothelial cells, they have been used to study endothelial cells in isolation, as well as the interaction of endothelial cells and the trafficking of immune or tumor cells10. However, these systems are less suitable for the recovery of large numbers of cells9. Both the cone-and-plate viscometer and parallel-plate flow chambers allow the recovery of large numbers of cells in confluent monolayers12. These systems can generate a range of shear forces and patterns12. The parallel-plate flow chamber assembly9 has the advantage that real-time imaging can be performed through the glass window to evaluate cellular morphology at any time point. Furthermore, the perfusate can be collected under sterile conditions. For the system presented here, the flow can also be monitored in real-time and in a multi-chamber set-up, which facilitates the maintenance of shear conditions between chambers.
For representative experiments, human umbilical vein endothelial cells (HUVEC), which represent a macrovascular endothelial cell type, are used, and the shear stress conditions we use (1 Pa) reflect arterial conditions (0.1 - 0.7 Pa). However, this protocol can be used with other endothelial cell types, and the shear stress conditions can be adjusted according to the experimental question. For example, the evaluation of human endothelial cells in conditions that model venous circulation would require lower levels of shear stress (1 - 6 Pa) and studies that model microvascular circulation have utilized shear stress levels of 0.4 - 1.2 Pa13,14. In addition, shear stress can vary even between endothelial cells within the same blood vessel6. In the current set-up, a single monitoring system is used that can simultaneously monitor four separate flow loops. For labs that need more flow loops, there is space in the dedicated environment for an additional monitoring system.
RT-qPCR is used for the absolute quantitation of gene expression in the setting of shear stress. The relative expression of target genes is often used to compare RNA expression across conditions. Some RNA species can exist at very low quantities or be absent, thus complicating relative measurements. For example, long noncoding RNAs in endothelial cells can exert potent effects at relatively low copy numbers per cell5. In addition, differences in primer efficiency can lead to an inaccurate interpretation from utilizing the delta-delta cycle threshold (Ct) method to analyze the data. To address this concern, we perform absolute quantitation by generating a standard curve using a known quantity of plasmid DNA. Furthermore, complementary DNA (cDNA) synthesis is an inefficient process, and differences in cDNA efficiency can account for differences in RNA expression between conditions and between samples15. The application of shear stress and/or transfection reagents can affect cell proliferation, apoptosis, and viability, or add components that may interfere with RNA isolation and/or cDNA synthesis. To account for the possibility of bias from RNA isolation and cDNA synthesis, we use a spike-in RNA control synthesized in the lab, added at the time of RNA extraction and measured with each cDNA synthesis via RT-qPCR. This allows not only the adjustment for technical differences in RNA extraction and cDNA synthesis but also allows the calculation of absolute quantities per cell, when the cell count is known.
This system uses additional steps to maintain similarity or account for technical differences between conditions. We particularly emphasize these steps because of the complex nature of these experiments, which involve multiple physical set-ups and experimental conditions that can lead to experimental variability.

<table><tbody><tr><td>0.05% Trypsin-EDTA</td><td>gibco</td><td>25300-062</td><td></td></tr><tr><td>10 mL Syringe</td><td>BD</td><td>302995</td><td></td></tr><tr><td>10 mm&lt;sup&gt;2&lt;/sup&gt; Culture Dish</td><td>Sarstedt</td><td>83.3902</td><td></td></tr><tr><td>30 mL Syringe</td><td>BD</td><td>302832</td><td></td></tr><tr><td>4-Way Stopcocks</td><td>Discofix</td><td>D500</td><td></td></tr><tr><td>Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>BEACH</td><td>Darwin Chambers Company</td><td>MN: HO85, SN: 4947549</td><td></td></tr><tr><td>Cell Scrapers</td><td></td><td></td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Meter</td><td>BioSphenix, Ltd.</td><td>MN: P120, SN: 0342</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Sensor</td><td>BioSphenix, Ltd.</td><td>MN: C700, SN: 52852</td><td></td></tr><tr><td>Distilled water</td><td>gibco</td><td>15230-170</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (DPBS) -/-</td><td>gibco</td><td>14190-144</td><td></td></tr><tr><td>Endothelial Cell Growth Medium 2</td><td>Promo Cell</td><td>C-22011</td><td></td></tr><tr><td>Endothelial Cell Growth Medium 2 Supplement Mix</td><td>Promo Cell</td><td>C-39216</td><td></td></tr><tr><td>Fibronectin (pure)</td><td>Sigma-Aldrich</td><td>11051407001</td><td></td></tr><tr><td>Filter (0.20 um)</td><td>Sarstedt</td><td>83.1826.001</td><td></td></tr><tr><td>Flow Dampener and Cap</td><td>U of T glass blowing shop</td><td></td><td></td></tr><tr><td>Flow Meter: 400 Series Console</td><td>Transonic Scisense Inc.</td><td>T402</td><td></td></tr><tr><td>Flow Meter: 400 Series Tubing</td><td>Transonic Scisense Inc.</td><td>TS410</td><td></td></tr><tr><td>Flow Reservoir and Cap</td><td>U of T glass blowing shop</td><td></td><td></td></tr><tr><td>Flow Sensor</td><td>Transonic Scisense Inc.</td><td>ME4PXL</td><td></td></tr><tr><td>Isotemp 737F Oven</td><td>Fisher Scientific</td><td>FI-737F</td><td></td></tr><tr><td>J cloth</td><td>J cloth</td><td></td><td></td></tr><tr><td>Microscope Slide (25 x 75 x 1 mm)</td><td>Fisherfinest</td><td>12-544-4</td><td></td></tr><tr><td>Paper sterilization pouch</td><td>Cardinal Health</td><td>92713</td><td></td></tr><tr><td>Pump (Masterflex L/S Economy Drive)</td><td>Cole-Parmer</td><td>7554-90</td><td></td></tr><tr><td>Pump Head (Masterflex L/S Easy Load)</td><td>Cole-Parmer</td><td>7518-00</td><td></td></tr><tr><td>Rectangular 4 Well Dish</td><td>Thermo Scientific</td><td>267061</td><td></td></tr><tr><td>Tweezers</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tubing&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Masterflex C-Flex L/S 25 Soft Tubing</td><td>Cole-Parmer</td><td>06424-25</td><td></td></tr><tr><td>Masterflex C-Flex L/S 14 Soft Tubing</td><td>Cole-Parmer</td><td>06424-14</td><td></td></tr><tr><td>Masterflex C-Flex L/S 16 Soft Tubing</td><td>Cole-Parmer</td><td>06424-16</td><td></td></tr><tr><td>Masterflex PharMed BPT L/S 13 Hard Tubing</td><td>Cole-Parmer</td><td>06508-13</td><td></td></tr><tr><td>Masterflex PharMed BPT L/S 14 Hard Tubing</td><td>Cole-Parmer</td><td>06508-14</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Luer &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3/16&quot; Male Luer</td><td>Cole-Parmer</td><td>45518-08</td><td>For #25 tubing</td></tr><tr><td>1/8&quot; Male Luer</td><td>Cole-Parmer</td><td>30800-24</td><td>For #16 tubing</td></tr><tr><td>1/8&quot; Female Luer</td><td>Cole-Parmer</td><td>30800-08</td><td>For #16 tubing</td></tr><tr><td>1/16&quot; Male Luer</td><td>Cole-Parmer</td><td>45518-00</td><td>For #14 tubing</td></tr><tr><td>1/16&quot; Female Luer</td><td>Cole-Parmer</td><td>45508-00</td><td>For #14 tubing</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Knockdown reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Oligofectamine Reagent</td><td>Invitrogen</td><td>12252-011</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>gibco</td><td>31985-070</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;In vitro&lt;/em&gt;&lt;/strong&gt;&lt;strong&gt; transcription &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Generuler 1kb+ DNA ladder</td><td>Thermo Scientific</td><td>SM1331</td><td></td></tr><tr><td>MEGAclear Kit</td><td>Ambion</td><td>AM1908</td><td></td></tr><tr><td>mMESSSEGE mMACHINE SP6 Transcription Kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>pSP-luc+</td><td>Promega</td><td>E4471</td><td></td></tr><tr><td>Supercoiled DNA Ladder</td><td>New England BioLabs Inc.</td><td>N0472S</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500-500</td><td></td></tr><tr><td>UltraPure Ethidium Bromide</td><td>Invitrogen</td><td>15585011</td><td></td></tr><tr><td>&lt;em&gt;XhoI&lt;/em&gt; Restriction Enzyme</td><td>New England BioLabs Inc.</td><td>R0146S</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;RNA extraction &lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>Sigma</td><td>M3148-100mL</td><td></td></tr><tr><td>RNeasy Mini Kit</td><td>Qiagen</td><td>74104</td><td></td></tr></tbody></table>

gene expression analysis of endothelial cells exposed to shear stress using multiple parallel-plate flow chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Successful linearization of luciferase plasmid using restriction enzymes was confirmed by running digested products on an agarose gel (Figure 1). The size of the linearized product was confirmed using DNA ladders and by comparison with uncut plasmid.
We have adapted the parallel-plate flow chamber set-up from Lane et al.9 for experiments that require multiple conditions/treatments with shear stress or multiple shear stress conditions. We use a dedicated environment, the BEACH, that can house multiple, fully-assembled flow circuits that all have monitored flow rates (Figure 2). The flow rate is monitored just upstream of the parallel-plate assembly (Figure 3). The flow circuits and rates can be monitored directly through glass doors without causing fluctuations in temperature, humidity, or gas content within the BEACH.
Manufacturing processes can lead to small variations in chamber height. Thus, flow rates must be calculated for each chamber to achieve the same shear stress (Table 1). In theory, chambers with identical heights can use identical flow rates to achieve the same shear stress and can be used in series. Typical experiments with endothelial cells use shear stress of 0 - 1.5 Pa. Laminar shear stress of 1 Pa was used in this workflow to model arterial endothelial shear stress. There can also be variations between pump head settings and within pump heads over time with use. Using the flow meter can account for these differences.
Experiments using the application of shear stress often involve multiple shear stress conditions, treatment conditions, and time points. Where possible, we use an endogenous reference RNA to account for any variabilities in the experimental set-up. For some experiments, finding an endogenous reference RNA with quantitative stability is not feasible16. Furthermore, quantitative stability or instability of endogenous reference genes between samples can be attributed to either stimulus-dependent effects on cellular expression levels or variations in efficiencies of RNA extractions or reverse transcription. To account for these inefficiencies, and in the setting where an endogenous reference gene is not quantitatively stable, we use a spike-in exogenous reference gene. For experiments incorporating laminar shear stress in mammalian endothelial cells, we use a firefly luciferase RNA as an exogenous RNA spike-in (Figure 4A).
Figure 4 shows analyzed RT-qPCR experimental data from shear stress experiments assessing Kr&#252;ppel-like factor 2 (KLF2) loss-of-function using siRNA. KLF2 is a transcription factor upregulated by laminar flow in endothelial cells and a major transcriptional mediator of endothelial gene expression in the setting of laminar flow2.
Figure 4A shows luciferase efficiencies for three separate experiments, each using two flow chambers. Luciferase efficiencies can be similar between samples of an experiment (Experiment 1) or show some variability (Experiments 2 and 3) (Figure 4A). These results are especially valuable in experimental systems where only small absolute changes are seen. The use of an exogenous reference gene may be particularly important in experiments where experimental treatments can interfere with the efficiency of reverse transcription or PCR17. The results depicted in Figure 4A are typical. A luciferase efficiency of 5% indicates that 5% of the luciferase RNA (i.e., the initial starting amount of RNA) added to the sample prior to RNA extraction is detected by RT-qPCR. Between samples or conditions within a single experiment, luciferase efficiencies are usually &#177; 50%. Results should be interpreted with caution if the variability of luciferase efficiencies is &#62; 50% and should include a review of the experimental procedures and conditions.
Figure 4B shows typical experimental results of RT-qPCR from repeated shear stress experiments, each using multiple parallel-plate flow chambers. Within each experiment, KLF2 mRNA expression is normalized in three ways. The first normalization uses an endogenous reference RNA, Cyclophilin A (CycA). The second normalization uses an exogenous reference RNA, firefly luciferase (Luc). The third normalization uses both the endogenous and exogenous reference RNA. Within each experiment shown in Figure 4B, all three normalization methods (normalization to the endogenous reference gene, the exogenous reference gene, and both endogenous and exogenous reference genes together) yields similar results. If the normalization method significantly changes the results (e.g., leads to &#62; 50% difference), the results should be interpreted with caution. When there is considerable variability between the methods of normalization, the endogenous reference gene(s) should be reviewed, as it may be a dependent variable in the experimental system. Similarly, the experimental procedures and conditions should be reviewed. In Figure 4B, KLF2 knockdown using siRNA yields similar knockdown efficiency between three separate experiments (Experiment 1, 2, and 3). We used three distinct biological samples for these experiments, using two simultaneously running flow chambers with shear stress at 1 Pa for each experiment.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58478/58478fig1.jpg" /> 
Figure 1: Agarose gel image of the linearization of exogenous luciferase plasmid. Supercoiled and 1 kb+ DNA ladders are used as markers to determine both uncut and cut luciferase plasmid sizes in kilobases (kb). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58478/58478fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58478/58478fig2.jpg" /> 
Figure 2: Schematic overview of multiple flow circuit assemblies within a dedicated environment (the BEACH). The flow rates in both flow circuits are easily monitored in real-time, without disturbing the environment, and both circuits can run simultaneously. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58478/58478fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58478/58478fig3.jpg" /> 
Figure 3: Schematic overview of a single flow circuit assembly. The tubing sizes and Luers used are indicated in this figure. Ensure that the flow meter is oriented in the direction of the flow and placed upstream of the flow chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58478/58478fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58478/58478fig4.jpg" /> 
Figure 4: Representative results from KLF2 loss-of-function experiments in human endothelial cells exposed to shear stress (1 Pa) for 24 h. (A) This panel shows the quantification of exogenous luciferase RNA in three separate flow experiments. Luciferase efficiencies can be similar between samples of an experiment (Experiment 1) or show some variability (Experiments 2 and 3). Luciferase (absolute copies) is the copy number of luciferase RNA detected by reverse-transcriptase quantitative PCR (RT-qPCR) by absolute quantitation using a standard curve. Luciferase efficiency is the experimental luciferase copies divided by the theoretical luciferase copies for each sample multiplied by 100 (see step 8 of the protocol). Relative luciferase efficiency is the luciferase efficiency of each sample divided by the reference condition (Flow + Ctlsi) within each experiment. (B) These panels show the normalization of gene expression in a set of sample shear stress experiments using both endogenous and exogenous reference genes. The results are from reverse-transcriptase quantitative PCR (RT-qPCR). Knockdown of KLF2 mRNA expression is shown in the presence of laminar flow with shear stress of 1 Pa for 24 h. FC = fold change; CycA = cyclophilin A, used as an endogenous reference RNA; Luc = luciferase, used as an exogenous reference RNA. The data adapted from Man et al.5. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58478/58478fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chamber Height 
			(microns; &#181;m) </td>
			<td>Chamber Width 
			(centimeters; cm)</td>
			<td>Flow Rate (mL/min) for 1 Pa shear stress </td>
			<td>Viscosity (centipoise; cP) </td>
		</tr>
		<tr>
			<td>303.80</td>
			<td>1.90</td>
			<td>19.48</td>
			<td>0.90</td>
		</tr>
		<tr>
			<td>326.10</td>
			<td>1.90</td>
			<td>22.45</td>
			<td>0.90</td>
		</tr>
		<tr>
			<td>344.84</td>
			<td>1.90</td>
			<td>25.10</td>
			<td>0.90</td>
		</tr>
		<tr>
			<td>319.06</td>
			<td>1.90</td>
			<td>21.49</td>
			<td>0.90</td>
		</tr>
	</tbody>
</table>
Table 1: Chamber heights and examples of flow rates for various flow chambers to achieve shear stress of 1 Pa.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>J cloths</td>
		</tr>
		<tr>
			<td>Tweezers</td>
		</tr>
		<tr>
			<td>Reservoir Bottle and Cap</td>
		</tr>
		<tr>
			<td>Dampener and Cap</td>
		</tr>
		<tr>
			<td>Flow Loop</td>
		</tr>
		<tr>
			<td>1/16&#34; Male Luer x 3</td>
		</tr>
		<tr>
			<td>1/16&#34; Female Luer x 3</td>
		</tr>
		<tr>
			<td>1/8&#34; Male Luer x 2</td>
		</tr>
		<tr>
			<td>1/8&#34; Female Luer x 4</td>
		</tr>
		<tr>
			<td>3/16&#34; Male Adaptor x 2</td>
		</tr>
		<tr>
			<td>14 L/S Hard Tube (2 inches) x 1</td>
		</tr>
		<tr>
			<td>14 L/S Soft Tube (5 inches) x 2</td>
		</tr>
		<tr>
			<td>16 L/S Soft Tube (3 inches) x 2</td>
		</tr>
		<tr>
			<td>25 L/S Soft Tube (3 inches) x 2</td>
		</tr>
		<tr>
			<td>13 L/S Hard Tube (10 inches) x 1</td>
		</tr>
		<tr>
			<td>Flow Chamber</td>
		</tr>
		<tr>
			<td>1/8&#34; Male Luer x 2</td>
		</tr>
		<tr>
			<td>1/8&#34; Female Luer x 2</td>
		</tr>
		<tr>
			<td>16 L/S Soft Tube (3 inches) x 3</td>
		</tr>
		<tr>
			<td>Top and Bottom Plates</td>
		</tr>
		<tr>
			<td>Screws</td>
		</tr>
	</tbody>
</table>
Table 2: Parts to be autoclaved in step 6.2 of the protocol.

Watch this Scientific Journal Video about Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers at JoVE.com

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

gene-expression-analysis-endothelial-cells-exposed-to-shear-stress

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.0K Views.  University of Toronto. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van endotheelbiologie, zoals waarom bloedvaten regionale verschillen hebben in de ontwikkeling van atherosclerose? Het belangrijkste voordeel van deze techniek is het mogelijk maakt de gelijktijdige studie van meerdere aandoeningen onder schuifspanning of meerdere afschuifspanning voorwaarden. De implicaties van deze techniek breiden zich uit tot het begrijpen van endotheel genregulatie in zowel de arteriële al...

Video: Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1.
Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3.
This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts.
The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (&gt; 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6.

We are describing a method to subject adherent cells to laminar flow shear stress in a sterile continuous flow circuit. The cells' adhesion, morphology can be studied through the transparent chamber, samples obtained from the circuit for metabolite analysis and cells harvested after shear exposure for future experiments or culture.

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Analysis of Physiologic E-Selectin-Mediated Leukocyte Rolling on Microvascular Endothelium

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone and inflamed tissues.  E-selectin expression is constitutive on dermal and bone microvessels and is inducible by pro-inflammatory cytokines, such as IL-1&#945;/  and TNF-&#945;, on microvessels in inflamed tissues.  This lectin receptor mediates weak binding interactions with carbohydrate counter-receptor ligands on circulating leukocytes, which results in a characteristic  rolling  behavior.  Because these interactions precede more stable adhesive events and diapedesis activity, characterization of leukocyte rolling activity and identification of leukocyte E-selectin ligands have been major goals in studies of leukocyte trafficking and inflammation and in the development of anti-inflammatory therapeutics (1-5).  The intent of this report is to provide a visual, comprehensive description of the most widely-used technology for studying E-selectin   E-selectin ligand interactions under physiologic blood flow conditions.  Our laboratory in conjunction with the Harvard Skin Disease Research Center uses a state-of-the-art parallel-plate flow chamber apparatus accompanied by digital visualization and new recording software, NIS-Elements.  This technology allows us to analyze adhesion events in real time for onscreen visualization as well as record rolling activity in a video format.  Cell adhesion parameters, such as rolling frequency, shear resistance and binding/tethering efficiency, are calculated with NIS-Elements software, exported to an Excel spreadsheet and subjected to statistical analysis.  In the demonstration presented here, we employed the parallel-plate flow chamber to investigate E-selectin-dependent leukocyte rolling activity on live human bone marrow endothelial cells (hBMEC).  Human hematopoietic progenitor KG1a cells, which express a high level of E-selectin ligand, were used as our leukocyte model, while an immortalized hBMEC cell line, HBMEC-60 cells, was used as our endothelial cell model (6).  To induce and simulate native E-selectin expression in the flow chamber, HBMEC-60 cells were first activated with IL-1 .  Our video presentation showed that parallel-plate flow analysis is a suitable method for studying physiologic E-selectin-mediated leukocyte rolling activities and that functional characterization of leukocyte E-selectin ligand(s) in the flow chamber can be ascertained by implementing protease or glycosidase digestions.

This report provides a visual depiction of parallel-plate flow chamber analysis for studying leukocyte endothelial interactions under physiologic shear stress. This method is particularly useful for investigating the role of endothelial (E)-selectin and leukocyte E-selectin ligands that trigger leukocyte rolling on endothelial cell surfaces.

E-selectin is a type-1 membrane protein on microvascular endothelial cells that helps initiate recruitment of circulating leukocytes to cutaneous, bone ...

Biology

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

Biochemistry

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Immunology and Infection

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood flow triggers a signaling cascade resulting in vasodilation thereby alleviating mechanical stress on the vascular wall. The pattern of shear stress is also well known to be a critical factor in the development of atherosclerosis with laminar shear stress being atheroprotective and disturbed shear stress being pro-atherogenic. While we have a detailed understanding of the various intermediate cell signaling pathways, the receptors that first translate the mechanical stimulus into chemical mediators are not completely understood. Mechanosensitive ion channels are critical to the response to shear and regulate shear-induced cell signaling thereby controlling the production of vasoactive mediators. These channels are among the earliest activated signaling components to shear and have been linked to shear-induced vasodilation through promoting nitric oxide production (e.g., inwardly rectifying K+ [Kir] and transient receptor potential [TRP] channels) and endothelium hyperpolarizing factor (e.g., Kir and calcium-activated K+ [KCa] channels) and shear-induced vasoconstriction through an undetermined mechanism that involves piezo channels. Understanding the biophysical mechanism by which these channels are activated by shear forces (i.e., directly or through a primary mechano-receptor) could provide potential new targets to resolve the pathophysiology associated with endothelial dysfunction and atherogenesis. It is still a major challenge to record flow-induced activation of ion channels in real time using electrophysiology. The standard methods to expose cells to well-defined shear stress, such as the cone and plate rheometer and closed parallel plate flow chamber do not allow real time study of ion channel activation. The goal of this protocol is to describe a modified parallel plate flow chamber that allows real time electrophysiological recording of mechanosensitive ion channels under well-defined shear stress.

The goal of this protocol is to describe a modified parallel plate flow chamber for use in investigating real time activation of mechanosensitive ion channels by shear stress.

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...

Studying Tissue Rigidity and Shear Stress Effects on Endothelial Cells

In Vitro Model Integrating Substrate Stiffness and Flow to Study Endothelial Cell Responses

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, which are crucial for understanding vascular health and the onset of diseases such as atherosclerosis. Traditionally, studies have explored the impacts of shear stress and substrate stiffness on ECs, independently. However, this integrated system combines these factors to provide a more precise simulation of the mechanical environment of the vasculature. The objective is to examine EC mechanotransduction across various tissue stiffness levels and flow conditions using human ECs. We detail the protocol for synthesizing gelatin methacrylate (GelMA) hydrogels with tunable stiffness and seeding them with ECs to achieve confluency. Additionally, we describe the design and assembly of a cost-effective flow chamber, supplemented by computational fluid dynamics simulations, to generate physiological flow conditions characterized by laminar flow and appropriate shear stress levels. The protocol also incorporates fluorescence labeling for confocal microscopy, enabling the assessment of EC responses to both tissue compliance and flow conditions. By subjecting cultured ECs to multiple integrated mechanical stimuli, this model enables comprehensive investigations into how factors such as hypertension and aging may affect EC function and EC-mediated vascular diseases. The insights gained from these investigations will be instrumental in elucidating the mechanisms underlying vascular diseases and in developing effective treatment strategies.

We synthesized and characterized a tunable gelatin-based substrate for culturing vascular endothelial cells (ECs) under relevant vascular flow conditions. This biomimetic surface replicates both physiological and pathological conditions, enabling the study of mechanical forces on EC behavior and advancing our understanding of vascular health and disease mechanisms.

We present an innovative in vitro model aimed at investigating the combined effects of tissue rigidity and shear stress on endothelial cell (EC) function, ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

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