This work was supported by the University of Central Florida start-up funds and the National Heart, Lung, And Blood Institute of the National Institute of Health under award K25HL132098.

1. Making polyacrylamide (PA) gels
<ol>
	<li>Preparation of Petri dish
	<ol>
		<li>Prepare bind silane solution by mixing 200 mL ultrapure water with 80 &#181;L acetic acid and 50 &#181;L of 3-(trimethoxysilyl)propyl methacrylate. Bind silane is a solution used to functionalize the glass bottom Petri dish surface for hydrogel attachment.</li>
		<li>Stir the bind silane solution on a stir plate for at least 1 h.</li>
		<li>Treat the center well of the Petri dish with bind silane solution for 45 min.</li>
		<li>Remove bind silane solution and rinse the Petri dish with ultra-pure water 2x-3x.</li>
		<li>Dry the Petri dish surface and store at room temperature for future use.</li>
	</ol>
	</li>
	<li>Preparation of hydrogel solution
	<ol>
		<li>Mix ultra-pure water, 40% acrylamide, and 2% bis-acrylamide in a 15 mL centrifuge tube according to Table 1.</li>
		<li>Add 80 &#181;L of fluorescent beads to the hydrogel solution.</li>
		<li>Gently shake the tube to mix beads with the gel solution.</li>
		<li>Lightly tighten the tube cap on the centrifuge tube and place in a vacuum chamber.</li>
		<li>Degas the gel solution for at least 45 min in vacuum chamber.</li>
	</ol>
	</li>
	<li>Hydrogel polymerization
	<ol>
		<li>First, add 75 &#181;L of 10% ammonium persulfate (dissolved in ultra-pure water) and then add 8 &#181;L of TEMED (N,N,N&#39;,N&#39;-tetramethylethane-1,2-diamine) to the hydrogel solution.</li>
		<li>Place 24 &#181;L of hydrogel solution at the center of the Petri dish (see Table 2).</li>
		<li>Use an 18 mm coverslip to flatten the hydrogel. This will give a height of ~100 &#181;m.</li>
		<li>Wait at least 30-40 min for gel polymerization.</li>
		<li>Submerge the polymerized hydrogel in ultra-pure water to prevent gel dehydration.</li>
		<li>Cover the Petri dish with aluminum foil to prevent photobleaching of fluorescent beads and store at 4 &#176;C. 
		NOTE: Hydrogels can be stored a period of up to 3 months, but it is suggested that these gels be used no longer than 1 week after fabrication to obtain the best results.</li>
	</ol>
	</li>
</ol>
2. Cell culture
<ol>
	<li>Culture human umbilical vein endothelial cells (HUVECs) in cell culture medium 200 (see Table of Materials) supplemented with 1% penicillin-streptomycin on 0.1% gelatin-coated flasks at 37 &#176;C and 5% CO2.</li>
</ol>
3. Micropattern stencil preparation
<ol>
	<li>Cure a thin layer of polydimethylsiloxane (PDMS) in a 100 mm Petri dish by mixing the silicone base with the silicone curing agent at a ratio of 20:1 (base: curing agent). 
	NOTE: It is possible to use other base to curing agent ratios (ex. 10:1 or 30:1). However, a lower base to curing agent ratio will yield a stiffer pattern, while a higher base to curing agent ratio will produce a softer pattern.</li>
	<li>Prepare a 20:1 (base:curing agent) PDMS solution and mix carefully in a 50 mL centrifuge tube. Invert the tube upside down and shake vigorously multiple times to ensure proper mixing of the PDMS solution, as nonuniform mixing will result in incomplete polymerization.</li>
	<li>Remove bubbles that have been introduced from the above step by centrifuging the PDMS solution for 1 min at 190 x g.</li>
	<li>Pour 5-6 mL of PDMS solution in the center of a 100 mm Petri dish and agitate the dish until the PDMS solution covers the entire Petri dish surface.</li>
	<li>Cure the PDMS solution overnight at 50-60 &#176;C. PDMS can also be cured at room temperature.</li>
	<li>Remove a circular, 16 mm diameter PDMS stencil with a hole puncher.</li>
	<li>Use a biopsy punch to create small holes in the PDMS stencil. This protocol uses a 1.25 mm diameter biopsy punch.</li>
	<li>Sterilize PDMS stencils by first submerging them in 70% ethanol for 2-3 min, aspirating off excess ethanol and then placing under a UV light for 5 min.</li>
</ol>
4. Collagen-I hydrogel coating
<ol>
	<li>Remove the coverslip from the hydrogel and aspirate any excess liquid.</li>
	<li>Place the PDMS stencil on the hydrogel surface. 
	NOTE: Tweezers can be used to apply light pressure to PDMS stencil to ensure a water-tight seal between the PDMS stencil and hydrogel surface. Our PDMS stencils are water-tight and thus prevent water access between the gel top surface and PDMS stencil bottom surface. Also, the hydrogel stiffness does not need to be adjusted with respect to the PDMS stiffness.</li>
	<li>Cover the hydrogel surface with sulfosuccinimidyl-6-(4-azido-2-nitrophenylamino) hexanoate (sulfo-SANPAH) dissolved in a 0.1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer solution at a concentration of 1 : 1000 and place under a UV lamp (power 36 W) for 8 min.</li>
	<li>Aspirate the excess sulfo-SANPAH and HEPES solution and rinse the hydrogel twice with 0.1 M HEPES followed by an additional two rinses with ultra-pure water.</li>
	<li>Aspirate excess ultra-pure water and coat hydrogels with 0.1 mg/mL collagen-I overnight at 4 &#176;C.</li>
	<li>Cover the dishes and protect fluorescent beads from photobleaching. 
	NOTE: PDMS stencils are used to create micropatterned monolayers. Micropatterned monolayers are utilized as they allow for multiple monolayers of the same geometry and dimensions to be observed simultaneously during each experiment. However, should micropatterns not be desired the above steps can be followed with the exception of step 4.2.</li>
</ol>
5. Creating HUVEC monolayers on hydrogels
<ol>
	<li>Use 1x trypsin to detach cells from tissue culture flasks for 3-5 min in the incubator.</li>
	<li>After trypsinization, add cell culture media to the trypsin solution and add to the 15 mL centrifuge tube.</li>
	<li>Centrifuge the cell solution for 3 min at 1710 x g. A small, white pellet of cells should be visible at the bottom of the centrifuge tube.</li>
	<li>Aspirate the supernatant and resuspend cells in media to a concentration of 50 x 104 cells/ mL.</li>
	<li>Remove collagen-I from the hydrogel and rinse 1x with PBS.</li>
	<li>Add 75 x 103 cells to the top of the PDMS stencil and allow cells to attach to the hydrogel surface for at least 1 h in the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Remove the PDMS stencil and add at least 2 mL of media to the Petri dish. Submerge the PDMS stencil in 10x trypsin to remove any attached cells and sterilize by spraying with 70% ethanol and then placing under the UV light for 5 min.</li>
	<li>Place the Petri dish in the incubator and wait at least 36 h or until a confluent monolayer is observed.</li>
</ol>
6. 2,5 dihydroxychalcone treatment for Cx43 disruption
<ol>
	<li>Dissolve 2,5 dihydroxychalcone (chalcone) in dimethylsulfoxide (DMSO) to make a 0.1875 mg/mL stock solution.</li>
	<li>Dilute the stock solution with cell culture media to make a low chalcone concentration (0.2 &#181;g/mL) aliquot and a high chalcone concentration (2 &#181;g/mL) aliquot.</li>
</ol>
7. Data acquisition
<ol>
	<li>Locate cell islands with a microscope.</li>
	<li>Acquire phase contrast and bead images to image cell morphology and hydrogel displacements, respectively. 
	NOTE: This protocol used a 10x objective for data acquisition.</li>
	<li>At the end of the experiment, detach cells with 10x trypsin and acquire an image of the gel surface without cells (reference image).</li>
</ol>
8. Immunostaining
<ol>
	<li>Fix monolayers with 4% formaldehyde and incubate at 37 &#176;C for 15 min.</li>
	<li>Remove 4% formaldehyde and add 0.2% Triton X-100 for 5 min at 37 &#176;C to permeabilize cells.</li>
	<li>Remove 0.2% Triton X-100 and rinse monolayers with PBS 2x-3x.</li>
	<li>Cover monolayer with 2% bovine serum albumin (BSA) solution for 45 min at 37 &#176;C.</li>
	<li>Remove the 2% BSA solution and rinse monolayers with PBS 2x-3x.</li>
	<li>Add primary Cx43 antibody at a concentration of 1:400 to the sample and incubate overnight at 4 &#176;C.</li>
	<li>Remove primary antibody and rinse sample with PBS 2x-3x.</li>
	<li>Add secondary antibody at a concentration of 3:200 and incubate for 2 h at 37 &#176;C. 
	NOTE: Samples should be covered to prevent photobleaching.</li>
	<li>Remove secondary antibody and rinse with PBS 2x-3x.</li>
	<li>Cover sample with mounting medium (Fluromount-G DAPI) and seal with an 18 mm cover slip.</li>
</ol>
9. Implementation of traction force microscopy (TFM) and monolayer stress microscopy (MSM)
<ol>
	<li>Hydrogel deformation calculation 
	NOTE: A step-by-step displacement calculation procedure using MATLAB is given below.
	<ol>
		<li>Open the main traction.m file in MATLAB (for all the MATLAB routines see Supplementary Materials). 
		NOTE: Follow instructions provided in the code to set the MATLAB directory.</li>
		<li>Define the following variables: image format, pixel-to-micron conversion, Young&#39;s modulus, Poisson&#39;s ratio, and microscope objective.</li>
		<li>Locate the bead, trypsin image, and phase image using the OpenFiles subroutine. 
		NOTE: For large data sets, it is best to name files in sequential order. For example, files should be named &#34;filename1.tif&#34;, &#34;filename2.tif&#34;, etc.</li>
		<li>Define a square ROI (region of interest) around the cell monolayer and execute the cell_cropper subroutine to crop the original image.</li>
		<li>Execute the displacement_finder subroutine to compute displacements.</li>
		<li>Execute the Dedrift subroutine to remove any additional non-cellular displacements that may be due to microscope stage drift.</li>
	</ol>
	</li>
	<li>Computation of tractions 
	NOTE: Here, unconstrained tractions are calculated using our custom-written MATLAB routine. Step by step traction computation using the MATLAB routine previously mentioned is given below.
	<ol>
		<li>Define the following variables within the traction.m routine: boundary condition, gel thickness, gel height, and dedrift.</li>
		<li>Execute the traction_finder subroutine to calculate tractions and execute the plot_traction subroutine to plot tractions. All the tractions in x-direction (Tx) and y-direction (Ty) along with their corresponding pixel locations can be found in a traction.dat file that will be generated by the code.</li>
	</ol>
	</li>
	<li>Computation of intercellular stresses 
	NOTE: Step by step instructions for intercellular stress computation using the MATLAB routine previously mentioned are given below.
	<ol>
		<li>Execute the mark_circular_domain subroutine to specify the monolayer boundary. This routine will prompt all cropped phase images sequentially for the user to manually draw a boundary around the monolayer. Keep a note of the nXPts generated in command window and use them later as grid parameters in both X axis and Y axis for FEM analysis (see step 9.3.2).</li>
		<li>Execute Run_StressCode to compute intercellular stresses. This subroutine directly reads parameters from &#34;model.in&#34; file and executes &#34;island.exe&#34; to perform FEM analysis. Before running this routine, make sure all parameters in the model.in file, i.e. grid parameters in X and Y, pixel to micron conversion, Young&#39;s modulus of the gel, Poisson&#39;s ratio, monolayer height, and monolayer pattern (strip or hole), are edited correctly.</li>
		<li>Plot all the FEM results using the plot_FEM_results subroutine. 
		NOTE: All results generated will automatically be stored in the Results folder in the MATLAB directory.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Our group, as well as others, has been successfully using TFM and MSM to probe the influence of cell-cell junctions in various pathological and physiological cellular processes in vitro7,15,18,27. For example, Hardin et al. presented a very insightful study that suggests intercellular stress transmission guides paracellular gap formation in endothelial cells15. While it ...

The field that refers to the study of the effects of physical forces and of mechanical properties on cellular and tissue physiology and pathology is known as mechanobiology1. A few useful techniques that have been utilized in mechanobiology are monolayer stress microscopy and traction force microscopy. Traction force microscopy allows for the computation of tractions generated at the cell-substrate interface, while monolayer stress microscopy allows for the computation of intercellular stresses generated between adjacent cells within a monolayer2,3,4,5,6. Results yielded from previous methods have suggested that cell-derived mechanical stresses play a crucial role in determining the fate of a host of cellular processes3,4,5. For example, upon exposure to an external mechanical force, a group of cells migrating as a collective can alter their morphology and polarize their shape to align and migrate along the direction of applied force by, in part, generating tractions7,8. Tractions provide a metric that can be used to evaluate cell contractility and are calculated using traction force microscopy (TFM). Traction force microscopy (TFM) begins with the determination of cell-induced substrate deformations followed by the calculation of the traction field using a mathematically rigorous, mechanics-based computational approach. Since the ability to calculate tractions has been around for quite some time, researchers have utilized TFM to reveal the impact tractions have on a host of processes, including cancer9, wound healing10 and assessment of engineered cardiac tissue11.
Implementation of TFM and MSM together can be divided into three essential steps that must be executed in the following order: first, the hydrogel deformations produced by the cells are determined; second, tractions are recovered from hydrogel deformations; and third, a finite element approach is used to compute normal and shear intercellular stresses within the entire monolayer. To compute gel displacements, fluorescence bead images with cells were compared with the reference bead image (without cells) by using a custom-written particle image velocimetry (PIV) routine. The cross-correlation window size and overlap for PIV analysis were chosen to be 32 x 32 pixels and 0.5, respectively. At this time, pixel shifts were converted into microns by multiplying with a pixel-to-micron conversion factor (for our microscope, this conversion factor is 0.65) to obtain in-plane displacements. Errors associated with ignoring out-of-plane displacements are negligible12,13. After computation of gel displacements, there are two types of traction measurements that can be utilized, constrained tractions and unconstrained tractions8,14. Unconstrained tractions provide the traction field for the entire field of view (including regions with and without cells), while constrained tractions provide the traction field only for regions that include cells14. Then, intercellular stresses are calculated using monolayer stress microscopy (MSM), which is an extension of traction force microscopy. Implementation of MSM is based off the assumption that local tractions exerted by a monolayer of cells at the cell-substrate interface must be balanced by mechanical forces transmitted between cells at the cell-cell interface as demanded by Newton&#39;s laws7,12,13. A key assumption here is that the cell monolayer can be treated as a thin elastic sheet because the traction distribution in the monolayer is known and the force balance does not depend on cell material properties. Another key assumption is that the traction forces are balanced by local intercellular stresses within the optical field of view (within the monolayer) and the influence of this force balance is minimal in the distal region (outside of the monolayer)13. Therefore, the boundary conditions defined by intercellular stresses, displacements, or a combination of both at the monolayer boundary are crucial to perform MSM13.Taking into account the above information, we utilize MSM to perform a finite element analysis (FEM) to recover the maximum principal stress (&#963;max) and minimum principal stress (&#963;min) by rotating the stress plane at every point within the monolayer. These principal stresses are subsequently used to compute the 2D average normal intercellular stress [(&#963;max + &#963;min) /2] and 2D maximum shear intercellular stress [(&#963;max - &#963;min) /2] within the entire monolayer12,13. This procedure is described in more detail by Tambe et al.12,13
Monolayer stress microscopy (MSM) allows for the calculation of cell-cell intercellular stresses generated within a monolayer6,7,8,12,13. These intercellular stresses have been suggested to be important for tissue growth and repair, wound healing, and cancer metastasis12,15,16,17. In addition, intercellular stresses have been suggested to also be important in endothelial cell migration and endothelial barrier function17,18. While cell-cell junctions such as tight junctions and adherens junctions have both been suggested to play a critical role in endothelial intercellular stress generation and transmission, the role of gap junctions remains elusive. Gap junctions physically connect adjacent cells and provide a pathway for electrical current and molecules (&#60;1 KDa) to pass between neighboring cells19,20,21. Although endothelial cells express Cx37, Cx40, and Cx43 gap junctions19,22, Cx43 is arguably the most important in terms of disease progression23. Evidence of Cx43&#39;s importance may be found in the fact that genetic deletion of Cx43 in mice results in hypotension24 and has adverse effects on angiogenesis25. In addition, Cx43 has been documented to be important for cell migration and proliferation and in the progression of atherosclerosis18,22,23,24,25.
In this protocol, we used TFM and MSM to investigate whether traction and intercellular stress generation within the confluent, endothelial monolayer would be impacted by the disruption of the endothelial gap junction Cx43. We disrupted Cx43 with 2,5-dihydroxychalcone (chalcone), a molecule documented to inhibit Cx43 expression26. Chalcone was used to disrupt Cx43 instead of siRNA as chalcone has been reported previously by Lee et al. to disrupt Cx43 expression26. In addition, we were particularly interested in chalcone&#39;s influence on the endothelium as it has also been reported to be an anti-inflammatory and anti-platelet compound that can potentially be used for the prevention and treatment of various vascular pathologies26. Chalcone treatments were performed an hour after the experiment onset, chalcone-treated monolayers were imaged for a total of six hours, and image processing was performed with a custom-written MATLAB code to determine tractions and subsequently intercellular stresses. Our results showed an overall decrease in tractions and intercellular stresses, suggesting Cx43 plays a key role in endothelial biomechanics.

<table><tbody><tr><td>18 mm coverslip</td><td>ThermoFisher</td><td>18CIR-1</td><td>Essential to flatten polyacrylamide gels</td></tr><tr><td>2% bis-acrylamide</td><td>BIO-RAD</td><td>1610143</td><td>Component of polyacrylamide gel</td></tr><tr><td>2&amp;prime;,5&amp;prime;-Dihydroxychalcone</td><td>SIGMA</td><td>IDF00046</td><td>To disrupt Cx43 structure</td></tr><tr><td>3-(Trimethoxysilyl)propyl methacrylate</td><td>SIGMA</td><td>2530-85-0</td><td>Stock solution to make bind silane mixture with acetic acid and ultra-pure water</td></tr><tr><td>40% Acrylamide</td><td>BIO-RAD</td><td>1610140</td><td>Component of polyacrylamide gel</td></tr><tr><td>Acetic acid</td><td>Fisher-Sceintific</td><td>64-19-7</td><td>Essential to make bind saline solution</td></tr><tr><td>Alexa Fluro 488 goat anti-mouse IgG;</td><td>ThermoFisher</td><td>Catalog # A-11001</td><td>Secondary antibody</td></tr><tr><td>Ammonium persulfate</td><td>BIO-RAD</td><td>1610700</td><td>Polyacrylamide gel polymerizing agent</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>SIGMA</td><td>9048-46-8</td><td>To make blocking solution</td></tr><tr><td>Bovine Type I Atelo-Collagen Solution, 3 mg/mL, 100 mL</td><td>Advance Biomatrix</td><td>5005-100ML</td><td>Use as a extracellular matrix</td></tr><tr><td>Corning Cell Culture Phosphate Buffered Saline (1x)</td><td>Fisher-Sceintific</td><td>21040CV</td><td>Buffer Saline needed for cell culture</td></tr><tr><td>Dimethyl Sulfoxide, Fisher BioReagents</td><td>Fisher-Sceintific</td><td>67-68-5</td><td>To dissolve chalcone and make stock solution</td></tr><tr><td>Fluoromount-G with DAPI</td><td>ThermoFisher</td><td>00-4959-52</td><td>Mounting medium for immunostaing used to stain for DAPI</td></tr><tr><td>Fluroscent microsphere Carboxylate-modified beads</td><td>ThermoFisher</td><td>F8812</td><td>0.5 micron carboxylate-modified beads (red), 2% solids</td></tr><tr><td>HEPES buffer solution 1 M</td><td>SIGMA</td><td>7365-45-9</td><td>Essential to</td></tr><tr><td>LVES</td><td>ThermoFisher</td><td>A1460801</td><td>Essential HUEVC media 200 supplement</td></tr><tr><td>Medium 200</td><td>ThermoFisher</td><td>M200500</td><td>Essential media for HUVEC cell culture</td></tr><tr><td>Mouse monoclonal Cx43 antibody (CX - 1B1)</td><td>ThermoFisher</td><td>Catalog #13-8300</td><td>Primary antibody for Cx43</td></tr><tr><td>Petri dish (35 mm dia)</td><td>CellVis</td><td>D35-20-1.5H</td><td>35 mm petri dish with a 20 mm center well</td></tr><tr><td>Sulfo-SANPAH Crosslinker 100 mg</td><td>Proteochem</td><td>102568-43-4</td><td>Essential to functionalize polyacrylamide gel surface</td></tr><tr><td>SYLGARD 184 Silicone Elastomer Kit</td><td>DOW corning</td><td>2646340</td><td>Silicon elastomer with curing agent to make PDMS</td></tr><tr><td>TEMED</td><td>BIO-RAD</td><td>1610801</td><td>Polyacrylamide gel polymerizing agent</td></tr><tr><td>Triton-X 100</td><td>SIGMA</td><td>9002-93-1</td><td>To permeabilize cells</td></tr><tr><td>Trypsin -EDTA</td><td>ThermoFisher</td><td>25300054</td><td>Used to detach cells</td></tr></tbody></table>

perturbing endothelial biomechanics via connexin 43 structural disruption

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 &#181;g/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

Phase contrast images of control, 0.2 &#181;g/mL, and 2 &#181;g/mL chalcone treated monolayers were taken 30 minutes before chalcone treatment (Figure 1A-C) and 2 hours after chalcone treatment (Figure 1D-F). Cell-induced bead displacements (&#181;m) were observed to decrease in both low dose chalcone and high dose chalcone conditions (Figure 2E,F) w...

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Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular ...

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Research

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Biochemistry

5.5K Views.  University of Central Florida. Here, we present a mechanics-based protocol to disrupt the gap junction connexin 43 and measure the subsequent impact this has on endothelial biomechanics via observation of tractions and intercellular stresses.

Video: Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

Bioengineering

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

Biology

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 ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Medicine

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

Immunology and Infection

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca2+-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca2+ that initiate the propagation of the Ca2+-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca2+-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca2+-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 &mu;m. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca2+-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Intercellular Ca2+-waves are driven by gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca2+-waves in cell monolayers in response to a local single-cell mechanical stimulus and its application to investigate the properties and regulation of gap junction channels and hemichannels.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the ...

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, ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

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 ...

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the reduced long-term patency compared to arterial conduits. The abrupt increase of hemodynamic stress associated with the graft arterialization results in vascular damage, especially the endothelium, that may influence the low patency of the saphenous vein graft (SVG). Here, we describe the isolation, characterization, and expansion of human saphenous vein endothelial cells (hSVECs). Cells isolated by collagenase digestion display the typical cobblestone morphology and express endothelial cell markers CD31 and VE-cadherin. To assess the mechanical stress influence, protocols were used in this study to investigate the two main physical stimuli, shear stress and stretch, on arterialized SVGs. hSVECs are cultured in a parallel plate flow chamber to produce shear stress, showing alignment in the direction of the flow and increased expression of KLF2, KLF4, and NOS3. hSVECs can also be cultured in a silicon membrane that allows controlled cellular stretch mimicking venous (low) and arterial (high) stretch. Endothelial cells' F-actin pattern and nitric oxide (NO) secretion are modulated accordingly by the arterial stretch. In summary, we present a detailed method to isolate hSVECs to study the influence of hemodynamic mechanical stress on an endothelial phenotype.

We describe a protocol to isolate and culture human saphenous vein endothelial cells (hSVECs). We also provide detailed methods to produce shear stress and stretch to study mechanical stress in hSVECs.

Coronary artery bypass graft (CABG) surgery is a procedure to revascularize ischemic myocardium. Saphenous vein remains used as a CABG conduit despite the ...

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

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In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

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

A Cell Culture Model of Resistance Arteries

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

Mechanical Stimulation-induced Calcium Wave Propagation in Cell Monolayers: The Example of Bovine Corneal Endothelial Cells

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

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca²⁺]_iand Nitric Oxide Production

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

Human Saphenous Vein Endothelial Cell Isolation and Exposure to Controlled Levels of Shear Stress and Stretch