Name: a cell culture model of resistance arteries
Uploaded: 2017-09-08T12:00:00.000+00:00
Duration: 10 min 54 s
Description: Watch this Scientific Journal Video about A Cell Culture Model of Resistance Arteries at JoVE.com

This work was supported by National Heart, Lung and Blood Institute grants R01088554, T32HL007284 and American Heart Association predoctoral fellowship 14PRE20420024. We especially thank the St. Jude Cellular Imaging Shared Research Core, including Randall Wakefield and Linda Horner for the electron microscopy images, Adam Straub for assistance with representative immunofluorescence images,&#160;and Pooneh Bagher for her valuable feedback on the manuscript protocol.

1. Plating Cells for the VCCC
<ol>
	<li>Culture large 225 cm2 flasks of EC and SMC under sterile conditions at 37 &#176;C, until 70-90% confluent. Ensure that more EC than SMC are used; if using primary human EC and SMC, typically 3 large flasks of EC to 1 large flask of SMC.
	<ol>
		<li>Use primary cells at lower passages and do not use past passage 10. Choose the culture media based upon cells to be co-cultured. For primary human coronary artery EC, use EBM-2 EC basal media with EGM2 MV growth factors. For primary human coronary SMC, use SmBM SMC basal media with SmGM-2 growth factors. Prior to use with cultured cells, add growth factors to the media.</li>
	</ol>
	</li>
	<li>Construction of the VCCC Day 1: Spray large (150 mm) Petri dishes and lids with disinfectant (e.g., Cavicide) and wipe away with a paper towel or lint-free wipes. Then spray the Petri dishes with 70% ethanol (EtOH) and place them in the hood to air dry.</li>
	<li>Open the plate of filter inserts (see the Table of Materials) under sterile conditions, and coat the SMC side (bottom side of filter) of the insert with fibronectin solution. Keeping the insert in the provided 6-well plate, pipet the fibronectin solution through the side slits to the bottom of the plate, making sure that the bottom half of the filter is covered (no more than 1 mL).</li>
	<li>After 30 min of treatment with fibronectin solution at room temperature in the cell culture hood, take inserts out of the plate, vacuum any excess solution, and invert, placing into the bottom half of the clean Petri dish. Set aside. Be sure not to disturb the filter insert membrane when suctioning excess solution; this can be avoided by gently tilting the plate, and vacuuming the solution off the edge of the insert.</li>
	<li>Trypsinize a 225 cm2 flask of SMC with 3 mL of pre-warmed 2X Trypsin-EDTA solution, and once the cells have lifted off the plate, add 9 mL SMC media to neutralize the trypsin (3:1, media:trypsin). Transfer to a conical tube, mix well, and pipet 10 &#181;L onto a hemocytometer to determine cell number.
	<ol>
		<li>Count the number of cells in 5 x 5 boxes (i.e., 18), multiply this by 10,000 to get the number of cells in 1 mL (180,000). Then multiply by 12 mL (total volume of cell suspension, 2.16 million SMC). Ensure that there are enough SMC to plate the desired number of wells. 
		NOTE: For example, 1 well should have 75,000 SMC and thus 18 wells would have 1.35 million total SMC. Divide 1.35 million cells needed by 180,000 cells per 1 mL and this results in 7.5 mL of the cell suspension that will be needed for plating. Then calculate the final volume needed (18 wells x 750 &#181;L = 13.5 mL). Thus, take 7.5 mL of cell suspension, bring the volume up to 13.5 mL with pre-warmed SMC media, and mix well.</li>
	</ol>
	</li>
	<li>Plate 750 &#181;L of the cell suspension containing approximately 75,000 SMC onto the bottom side of each filter, being careful to not spill any of the media and cell solution. Place the lid on top and carefully transfer the Petri dish with the inserts into the incubator at 37 &#176;C overnight. 
	NOTE: The optimum cell density for other cell types will need to be empirically determined.</li>
	<li>On day 2, fill clean 6-well dishes with 2 mL of fresh, pre-warmed SMC media. Remove the plates from the incubator. Take the inserts out one at a time and remove the media by suction, then place into the 6-well dish with SMC media.</li>
	<li>Coat the opposite side of the filter (EC side) with 1 mL of a 0.5% bovine gelatin solution for at least 30 min at 37 &#176;C.</li>
	<li>Trypsinize the 225 cm2 flask(s) of EC with 3 mL of pre-warmed 2X Trypsin-EDTA (10x diluted in sterile Dulbecco&#39;s PBS) solution, with gentle tapping of the flask in order to lift the cells off the plate. 
	NOTE: It may be necessary to place the flask back into the incubator at 37 &#176;C for 1-3 min.
	<ol>
		<li>Once the cells have lifted off the plate, add 9 mL EC media to neutralize the trypsin (3:1). Transfer to a conical tube, pool together, mix well, and pipet 10 &#181;L onto a hemocytometer to determine cell number.
		<ol>
			<li>Count the number of cells in 5 x 5 boxes (i.e., 60), multiply this by 10,000 to get the number of cells in 1 mL (600,000). Then multiply by 12 mL (total volume of cell suspension, 7.2 million EC). Ensure that there are enough EC to plate the desired number of wells. 
			NOTE: Approximately 360,000 EC are required per well. For example, 18 wells require 6.48 million EC. Divide 6.48 million cells needed by 600,000 EC per 1 mL and this results in 10.8 mL of the cell suspension that will be needed for plating. Then calculate the final volume needed (18 wells x 1 mL = 18 mL). Thus, take 10.8 mL of cell suspension, bring the volume up to 18 mL with pre-warmed EC media, and gently resuspend.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Plate 1 mL of 360,000 EC onto the top side of each insert filter. Let the cells incubate undisturbed at 37 &#176;C for 24 h. Replace medium on Day 4. Harvest on Day 5.</li>
</ol>
2. Harvesting Fractions from the VCCC
<ol>
	<li>Perform the isolation in a 4 &#176;C room and keep all instruments on ice.</li>
	<li>Keep in mind the number of individual inserts being isolated and add lysis buffer to a 50 mL tube labeled MEJ (for the isolated filters). Then label two 10 mm dishes; one for EC and one for SMC.</li>
	<li>Adjust the volume of lysis buffer depending on how concentrated the lysate needs to be; for 15 inserts, 500 &#181;L of lysis buffer for the MEJ tube and 250 &#181;L per Petri dish is recommended.</li>
	<li>Work with one 6-well plate of inserts at a time. Suction off the medium in the cell culture hood and then transport to a cold room on ice. 
	NOTE: The following instructions are for 1 insert isolation.</li>
	<li>Pipet 10 &#181;L of 1x PBS onto the SMC side of the insert.</li>
	<li>Use the cell lifter to scrape down the cells to one end of the insert, and transfer the cells from the lifter to the SMC Petri dish that has lysis buffer in it.</li>
	<li>Once the cell lifter has touched the lysis buffer in the dish, do not allow it to touch the insert filter until it has been completely wiped and dried off on paper towels. Repeat the scraping of the SMC filter at least one to two times to fully remove the remaining SMC cell debris.</li>
	<li>Turn the insert over and pipet 10 &#181;L of 1x PBS into the upper/EC side of the insert filter. Repeat steps 2.6 and 2.7 with a cell scraper and the EC Petri dish.</li>
	<li>Collect the remaining cell and PBS slurry from the EC side of the filter with a pipette and transfer to the EC Petri dish with lysis buffer. Be very thorough in scraping the cells off both sides of the filter as to leave minimal EC/SMC contamination in the MEJ fraction.</li>
	<li>Carefully use the scalpel to cut out the filter from the plastic insert. Do this by cutting 70-80% of it away from the plastic and use forceps to pull the filter completely off the plastic insert structure. Put the filter in the 50 mL conical tube labeled MEJ with lysis buffer, and ensure it sits in the lysis buffer and remains wet.</li>
	<li>Repeat the isolation steps, in groups of 6, until all of the inserts have been processed.</li>
	<li>Ensure that different treatment groups are kept in separate tubes and dishes.</li>
	<li>Vortex the 50 mL MEJ conical tube on full strength for 15 s or until mixed well. Remove the filters from the MEJ conical with forceps, dragging them along the side walls of the tube as to leave the maximum amount of proteins and liquid in the tube. After this point, discard the filters.</li>
	<li>Once all the filters have been removed from the MEJ conical tube, do a quick spin (10-15 s at max speed ~16,000 x g) in a centrifuge to pull all the liquid and proteins to the bottom of the tube. Transfer the contents of the MEJ tube and the EC and SMC Petri dishes into separate, smaller centrifuge tubes.</li>
	<li>Optional: Sonicate the contents of the MEJ/SMC/EC tubes on setting 4 for three 10 s pulses on ice or homogenize gently by hand.</li>
	<li>Centrifuge at max speed (~16,000 x g) 15 min at 4 &#176;C. Pipet out the supernatant and assay the lysate for protein content using the bicinchoninic assay method.</li>
</ol>
3. Harvesting the VCCC for En Face Immunofluorescence
<ol>
	<li>On Day 5 of VCCC incubation, remove the inserts from the incubator. Working in the cell culture hood, suction off the SMC media from each of the lower wells of the insert filter and rinse two times with 1x PBS. Repeat with the EC side.</li>
	<li>Keeping the inserts intact and in the plate, add 4% paraformaldehyde (PFA) for 10-20 min in a walk-in 4 &#176;C room on a gentle shaker. Wash both sides of the insert filter with 1x PBS for 5 min and repeat two times. 
	CAUTION: PFA is toxic and must be handled carefully.</li>
	<li>Perform the blocking, primary and secondary antibody incubations in the plate, ensuring that both sides of the filter are kept in the antibody + blocking solution (9 mL PBS, 25 &#181;L Triton X100, 50 mg bovine serum albumin, 500 &#181;L normal serum)</li>
	<li>To mount the filter on a slide, cut the filter out of the plastic insert with a scalpel mounted on a handle and place on a microscope slide with mounting medium.</li>
</ol>
4. Harvesting the VCCC for Transverse Immunofluorescence
<ol>
	<li>On Day 5 of VCCC incubation, remove the inserts from the incubator. Suction off the media from both sides of the insert filter in the cell culture hood and rinse each side of the filter two times with 1x PBS.</li>
	<li>Keeping the inserts intact and in the 6-well plate, add 4% PFA and incubate overnight in a walk-in 4 &#176;C room on a gentle shaker. 
	CAUTION: PFA is toxic and must be handled carefully.</li>
</ol>
5. Embedding the VCCC for Transverse Immunofluorescence
<ol>
	<li>After filter inserts have been fixed approximately for 24 h in 4% PFA, transfer to 70% EtOH for at least 24 h, but up to several weeks.</li>
	<li>Process the filters on a long (overnight) run on an automated tissue processor. Use the program as follows: 70% EtOH for 30 min, 85% EtOH 30 min, 95% EtOH 30 min, 100% EtOH 30 min, 100% EtOH 45 min, 100% EtOH 45 min, Xylene 30 min, Xylene 30 min, Xylene 45 min, 60 &#176;C paraffin 30 min, 60 &#176;C paraffin 30 min, 60 &#176;C paraffin 45 min.</li>
	<li>After processing, transfer the harvested filters to the embedding station into liquid paraffin, keeping the filter in the cassette.</li>
	<li>Remove the filter from the cassette, carefully using forceps to hold it just at the edge. With scissors, cut the filter in half, forming two semi-circular shaped halves. Lay each of the 2 halves on the cold plate portion of the embedding station just long enough to fill the embedding mold with liquid paraffin.</li>
	<li>Once filled with liquid paraffin, very briefly touch the embedding mold to the cold plate, then embed each half of the filter into embedding mold (cut side down); this ensures that during sectioning, one will be cutting from the center of the filter towards the edge.</li>
	<li>During embedding, use forceps to hold the filter in place to prevent the halves from falling into each other until the paraffin is cool enough for them to stand alone. Move the embedding mold to a cold plate until completely solidified. 
	NOTE: Cooling blocks on ice trays helps to prevent wrinkled sections.</li>
</ol>
6. Sectioning and Staining the VCCC for Transverse Immunofluorescence
<ol>
	<li>For the paraffin blocks sectioning and slide mounting: cut 5 &#181;m sections, place the sections into a 42 &#176;C water bath, and pick up the sections using positively charged slides. Then keep slides overnight in a 42 &#176;C oven to dry.</li>
	<li>Before beginning the rehydration steps, bake the slides at 60 &#176;C to melt the paraffin and help adhere the VCCC sections to the slide. Cool briefly, then rehydrate the slides through two changes of xylene, 100% EtOH, 95% EtOH, one change of 70% EtOH, and two changes of distilled water.</li>
	<li>Avoid antigen retrieval methods that involve microwaving or heating as sections may fall off the slide.</li>
	<li>Perform the standard blocking for 1 h at room temperature, then the overnight incubation with the primary antibody at 4 &#176;C, followed by washing steps and secondary antibody for 1 h at room temperature. Mount with the mounting media and coverslip.</li>
</ol>

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The authors have nothing to disclose.

The VCCC has a number of advantages in terms of recapitulating MEJs in vitro, but there are points of discussion when determining if the VCCC can be utilized for specific application. For example, if different cell types are to be used with this model, it will need to be optimized. We have used primary human cells but it is possible to isolate cells from bovine aortas24, or wildtype or knockout mice, and use them in the VCCC1,5<su...

In small diameter resistance arteries, a thin internal elastic lamina (IEL) separates endothelial (EC) and smooth muscle cells (SMC). The cells can project through holes in this elastic matrix and make direct cytoplasmic connections via gap junction channels1,2. This unique structure is termed the myoendothelial junction (MEJ). The MEJ is a small (approximately 0.5 &#181;m x 0.5 &#181;m, depending on the vascular bed), cellular projection composed predominantly of endothelial cells but may originate from smooth muscle as well3,4. A number of investigators have demonstrated the immense complexity of signaling networks that occur selectively at the MEJ, rendering it an especially important location for facilitating bi-directional signaling between endothelium and smooth muscle5,6,7,8,9,10.
However, a mechanistic dissection of signaling pathways at the MEJ is difficult in an intact artery. Because the MEJ is a cellular projection, it is not currently possible to isolate an in vivo MEJ from the vascular wall. For this reason, the VCCC model1 was developed. Importantly, the VCCC replicates physiological endothelial morphology11 and polarization of signaling between the apical and MEJ portions of the cell12. It was this unique model that facilitated the discovery that alpha hemoglobin is in the endothelial cell, polarized to the MEJ. This is in contrast to conventionally cultured endothelial cells which do not express alpha hemoglobin13. For researchers interested in microvascular endothelium, it may be more appropriate to use endothelial cells that have been cultured in the VCCC, particularly if the intent is to dissect signaling pathways that occur in small diameter resistance arteries.
Using a sturdy plastic insert containing a filter with small diameter pores (0.4 &#181;m in diameter) to co-culture two distinct cell types prevents the cells from migrating between layers. It results in a 10 &#181;m distance between the cells, which is significantly longer than in vivo, but still replicates many of the in vivo characteristics of MEJs, including protein localization and second messenger signaling1,14. In addition, the VCCC allows for targeting of cell type-specific signaling via the addition of an agonist or antagonist to the specific cellular compartment. For example, loading the EC with BAPTA-AM to chelate calcium, and stimulating the SMC with phenylephrine14. In contrast to other descriptions of co-culture models15,16,17,18,19,20,21, this provides instructions on isolating the distinct fractions for mRNA and protein, including the distinct MEJ fraction within the filter pores. The addition of this technique to the VCCC allows for specific investigation of changes in mRNA localization or transcription22, protein phosphorylation12,23, and protein activity12. This article will describe plating of EC on top of the filter and SMC on the bottom, although it is possible to culture the two cell types in different conformations11,18,24.

<table><tbody><tr><td>24 mm diameter, 0.4 &amp;micro;m Transwell Polyester membrane filter insert</td><td>Corning</td><td>3450</td><td>This is one 6 well plate of inserts</td></tr><tr><td>225 cm flask</td><td>Corning</td><td>431082</td><td></td></tr><tr><td>6 well plates (without filter inserts)</td><td>Corning</td><td>3506</td><td></td></tr><tr><td>Primary human coronary artery endothelial cells</td><td>Lonza</td><td>CC-2585</td><td></td></tr><tr><td>Primary human coronary artery smooth muscle cells</td><td>Lonza</td><td>CC-2583</td><td></td></tr><tr><td>EBM-2 EC Basal media</td><td>Lonza</td><td>CC-3156</td><td></td></tr><tr><td>EC growth factors (EGM2 MV Single quots)</td><td>Lonza</td><td>CC-4147</td><td>Add to basal media before use</td></tr><tr><td>SmBM SMC basal media</td><td>Lonza</td><td>CC-3181</td><td></td></tr><tr><td>SMC growth factors (SmGM-2 SingleQuot)</td><td>Lonza</td><td>CC-4149</td><td>Add to basal media before use</td></tr><tr><td>0.5% Trypsin-EDTA 10X</td><td>Gibco</td><td>15400-054</td><td>Dilute to 2X with sterile dPBS</td></tr><tr><td>Hemocytometer</td><td>VWR</td><td>15170-208</td><td></td></tr><tr><td>large petri dishes for SMC Plating (150mm)</td><td>Corning</td><td>353025</td><td></td></tr><tr><td>Bovine gelatin</td><td>Sigma</td><td>G-9382</td><td></td></tr><tr><td>Human fibronectin</td><td>Corning</td><td>356008</td><td>5&amp;micro;g/ml of fibronectin and store at -20</td></tr><tr><td>10x Hanks&#039; Buffered salt solution (HBSS)</td><td>Gibco</td><td>14065-056</td><td>Dilute to 1x with sterile water</td></tr><tr><td>10x Dulbecco&#039;s Phosphate buffered saline (dPBS)</td><td>Gibco</td><td>14200-075</td><td>Dilute to 1x with sterile water</td></tr><tr><td>Disposable Curved Scalpel (30mm cutting edge) with handle</td><td>Amazon</td><td>MDS15210</td><td></td></tr><tr><td>Forceps</td><td>Fisher</td><td>17-467-201</td><td></td></tr><tr><td>Cell lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cell scraper</td><td>BD Falcon</td><td>353085</td><td></td></tr><tr><td>50 mL conical tube</td><td>Corning</td><td>352070</td><td></td></tr><tr><td>10 mm petri dishes</td><td>Falcon</td><td>35-1008</td><td></td></tr><tr><td>Rneasy mini kit</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>RNA lysis reagent</td><td>Qiagen</td><td>79306</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>158127</td><td>Make 4% solution with dPBS</td></tr><tr><td>70% ethanol</td><td>Fisher</td><td>04-355-305</td><td></td></tr><tr><td>Cavicide disinfectant</td><td>Fisher</td><td>131000</td><td></td></tr><tr><td>RIPA protein lysis buffer</td><td>Sigma</td><td>R0278</td><td></td></tr><tr><td>Sonic dismembranator</td><td>Fisher</td><td>Model 50</td><td></td></tr><tr><td>1x PBS for harvesting EC/SMC and immunocytochemistry</td><td>Gibco</td><td>10010023</td><td>does not need to be sterile</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;Embedding/Sectioning/Staining&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Paraffin</td><td>Fisher</td><td>P31-500</td><td></td></tr><tr><td>Automated tissue processer</td><td>Excelsior</td><td>ES</td><td></td></tr><tr><td>Embedding station + cold plate</td><td>Leica</td><td>HistoCore Arcadia</td><td></td></tr><tr><td>Tissue Tek Accu-Edge Microtome blade</td><td>VWR</td><td>25608-961 or 25608-964</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Fisher</td><td>22-230-900</td><td></td></tr><tr><td>Coverslips</td><td>Thermo Fisher</td><td>12-548-5E</td><td></td></tr><tr><td>Prolong Gold mounting medium</td><td>Thermo Fisher</td><td>P36931</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;Other Solutions&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;**Perform all steps in sterile conditions if using the solution on live cells&lt;/strong&gt;</td></tr><tr><td>Sterile dPBS</td><td></td><td></td><td>Autoclave water to sterilize. Using the sterile water make DPBS (450mL sterile water: 50mL 10X DPBS), mix and filter sterilize (0.2&amp;micro;m filter).&lt;br/&gt; Final concentration: 1X</td></tr><tr><td>sterile HBSS</td><td></td><td></td><td>Autoclave water to sterilize. Using the sterile water make HBSS (450mL sterile water: 50mL 10X HBSS), mix and filter sterilize (0.2&amp;micro;m filter).&lt;br/&gt; Final concentration: 1X</td></tr><tr><td>Fibronectin stock solution</td><td></td><td></td><td>Use 50mL of sterile water to fibronectin to make stock of 1mL aliquots, then freeze until needed&lt;br/&gt; Final concentration: 100&amp;micro;g/mL</td></tr><tr><td>Fibronectin to coat SMC side of VCCC</td><td></td><td></td><td>thaw one aliquot and add 1mL of fibronectin (100&amp;micro;g/mL) to 19mLs of 1X HBSS to reach a final concentration of 5&amp;micro;g/mL.&lt;br/&gt; Final concentration: 5&amp;micro;g/mL</td></tr><tr><td>2x Trypsin EDTA solution</td><td></td><td></td><td>Dilute the 10X 0.5% Trypsin-EDTA to 2X with 1X dPBS (40mL DPBS: 10mL 10X Trypsin) .&lt;br/&gt; Final concentration: 2X</td></tr><tr><td>Blocking/antibody solution</td><td></td><td></td><td>(9mL 1X PBS, 25mL Triton X100, 50mg bovine serum albumin, 500mL normal serum)</td></tr><tr><td>Bovine gelatin to coat EC VCCC</td><td></td><td></td><td>Mix 0.5g bovine gelatin into 100mL distilled water. Autoclave prior to first use and store at room temperature. Keep in sterile conditions.&lt;br/&gt; Final concentration: 0.50%</td></tr><tr><td>1x PBS for harvesting cells</td><td></td><td></td><td>does not need to be sterile</td></tr></tbody></table>

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.

The filter inserts used for the VCCC have small, 0.4 &#181;m holes. Figure 1A, an en face view of the VCCC filter inset, shows the pores in which the MEJ can form in vitro. The schematic depicts the smooth muscle cells plated on the lower side of the filter one day before the endothelial cells are plated on the opposite side (Figure 1B). Once the cells are plated, the EC, MEJ, and SMC fractions can be isolated t...

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A Cell Culture Model of Resistance Arteries

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 ...

a-cell-culture-model-of-resistance-arteries

Research

JoVE Journal

Medicine

7.8K Views.  University of Virginia School of Medicine. 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.

Video: A Cell Culture Model of Resistance Arteries

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated blood is the aorta. The aorta may become affected in diseases such as atherosclerosis and aneurysm. Researchers investigating these diseases would benefit from direct observation of the aorta to characterize disease progression as well as to evaluate efficacy of potential therapeutics. The goal of this protocol is to describe proper isolation and excision of the aorta to aid investigators researching cardiovascular disease. Isolation and excision of the aorta allows investigators to look at gross morphometric changes as wells as allowing them to preserve and stain the tissue to look at histologic changes if desired. The aorta may be used for molecular studies to evaluate protein and gene expression to discover targets of interest and mechanisms of action. This technique is superior to imaging modalities as they have inherent limitations in technology and cost. Additionally, primary isolated cells from a freshly isolated and excised aorta can allowing researchers to perform further in situ and in vitro assays. The isolation and excision of the aorta has the limitation of having to sacrifice the animal however, in this case the benefits outweigh the harm as it is the most versatile technique in the study of aortic disease.

Pathology of the aorta can lead to severe morbidity and mortality, therefore research of disease progression and potential therapies is warranted. Here, we present a protocol to isolate and excise the murine aorta to aid researchers in their investigation of cardiovascular disease.

Cardiovascular disease is a broad term describing disease of the heart and/or blood vessels. The main blood vessel supplying the body with oxygenated ...

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations and development of microstructurally motivated mathematical models for understanding the mechanical properties of human resistance arteries in health and disease have the potential to aid understanding how disease and medical treatments affect the human microcirculation. To develop these mathematical models, it is essential to decipher the relationship between the mechanical and microarchitectural properties of the microvascular wall. In this work, we describe an ex vivo method for passive mechanical testing and simultaneous label-free three-dimensional imaging of the microarchitecture of elastin and collagen in the arterial wall of isolated human resistance arteries. The imaging protocol can be applied to resistance arteries of any species of interest. Image analyses are described for quantifying i) pressure-induced changes in internal elastic lamina branching angles and adventitial collagen straightness using Fiji and ii) collagen and elastin volume densities determined using Ilastik software. Preferably all mechanical and imaging measurements are performed on live, perfused arteries, however, an alternative approach using standard video-microscopy pressure myography in combination with post-fixation imaging of re-pressurized vessels is discussed. This alternative method provides users with different options for analysis approaches. The inclusion of the mechanical and imaging data in mathematical models of the arterial wall mechanics is discussed, and future development and additions to the protocol are proposed.

We describe simultaneous mechanical testing and 3D-imaging of the arterial wall of isolated, live human resistance arteries, and Fiji and Ilastik image analyses for the quantification of elastin and collagen spatial organization and volume densities. We discuss the use of these data in mathematical models of arterial wall mechanics.

The pathogenic contribution of resistance artery remodeling is documented in essential hypertension, diabetes and the metabolic syndrome. Investigations ...

Bioengineering

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of various important processes in the cardiovascular system. Recently, although several biological studies have been performed using such 3D models of human arteries, they have not been applied to study vascular targeting. This paper presents a new method to fabricate real-sized, reconstructed human arterial models using a 3D printing technique, line them with human endothelial cells (ECs), and study particle targeting under physiological flow. These models have the advantage of replicating the physiological size and conditions of blood vessels in the human body using low-cost components. This technique may serve as a new platform for studying and understanding drug targeting in the cardiovascular system and may improve the design of new injectable nanomedicines. Moreover, the presented approach may provide significant tools for the study of targeted delivery of different agents for cardiovascular diseases under patient-specific flow and physiological conditions.

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Here, we present a new protocol to study and map the targeted deposition of drug carriers to endothelial cells in fabricated, real-sized, three-dimensional human artery models under physiological flow. The presented method may serve as a new platform for targeting drug carriers within the vascular system.

The use of three-dimensional (3D) models of human arteries, which are designed with the correct dimensions and anatomy, enables the proper modeling of ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection (TAAD). However, human TAAD sometimes cannot be characterized by animal models. There is an apparent species gap between clinical human studies and animal experiments that may hinder the discovery of therapeutic drugs. In contrast, the conventional cell culture model is unable to simulate in vivo biomechanical stimuli. To this end, microfabrication and microfluidic techniques have developed greatly in recent years, providing novel techniques for establishing organoids-on-a-chip models that replicate the biomechanical microenvironment. In this study, a human aorta smooth muscle cell organ-on-a-chip (HASMC-OOC) model was developed to simulate the pathophysiological parameters of aortic biomechanics, including the amplitude and frequency of cyclic strain experienced by human aortic smooth muscle cells (HASMCs) that play a vital role in TAAD. In this model, the morphology of HASMCs became elongated in shape, aligned perpendicularly to the strain direction, and presented a more contractile phenotype under strain conditions than under static conventional conditions. This was consistent with the cell orientation and phenotype in native human aortic walls. Additionally, using bicuspid aortic valve-related TAAD (BAV-TAAD) and tricuspid aortic valve-related TAAD (TAV-TAAD) patient-derived primary HASMCs, we established BAV-TAAD and TAV-TAAD disease models, which replicate HASMC characteristics in TAAD. The HASMC-OOC model provides a novel in vitro platform that is complementary to animal models for further exploring the pathogenesis of TAAD and discovering therapeutic targets.

Here, we developed a human aorta smooth muscle cell organ-on-a-chip model to replicate the in vivo biomechanical strain of smooth muscle cells in the human aortic wall.

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Biology

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

A Cell Culture Model of Resistance Arteries

Summary

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Chapters in this video

Isolation and Excision of Murine Aorta; A Versatile Technique in the Study of Cardiovascular Disease

Assessing Collagen and Elastin Pressure-dependent Microarchitectures in Live, Human Resistance Arteries by Label-free Fluorescence Microscopy

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

In Vitro 3D Cell-Cultured Arterial Models for Studying Vascular Drug Targeting Under Flow

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

In Vitro Model of Coronary Angiogenesis

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function