The authors thank the members of Sharma laboratory for providing a supportive research environment. We like to extend special thank you to Diane (Dee) R. Hoffman who maintains and cares for our mouse colony. We also would like to thank Drs. Philip J. Smaldino and Carolyn Vann for thoroughly proofreading the manuscript and providing helpful comments. This work was supported by funds from Ball State University Provost Office and Department of Biology to B.S, Indiana Academy of Sciences Senior Research Grant funds to B.S, and NIH (RO1-HL128503) and The New York Stem Cell Foundation funds to K.R.

Use of all the animals in this protocol followed Ball State University Institutional Animal Care and Use Committee (IACUC) guidelines.
1. Establishing Mouse Breeders and Detecting Vaginal Plugs for Timed Pregnancies
<ol>
	<li>Set up a mouse breeding cage with wild type male and female mice. Ensure that the age of the breeding mice is between 6-8 weeks. Set up either a pair (1 male and 1 female) or as a trio (1 male and 2 female) for breeding.</li>
	<li>Check for a vaginal plug the following morning. Use an angled metal probe to detect a deep plug by inserting it into the vaginal opening. Designate the morning of a positive vaginal plug to be embryonic day 0.5 (e0.5). 
	NOTE: A vaginal plug can be either superficial (which is easily visible, see Figure 1) or deep (which is not easily visible). Presence of a deep plug will block full insertion of the probe whereas the absence of a plug will allow full insertion without resistance.</li>
	<li>Maintain timed pregnancy until the embryos reach e11.5 at which they will be harvested. To confirm pregnancy before harvesting embryos, record the weight of female mice between e7.5 and e11.5. 
	NOTE: Daily increase in the mother&#39;s weight will indicate a successful pregnancy, whereas no change in weight will indicate a failed pregnancy.</li>
</ol>
2. Harvesting Embryos from Pregnant Mice
NOTE: Before beginning, make sure to have the following equipment and reagents: a CO2 euthanasia chamber, 70% ethanol, paper towels, regular forceps, fine forceps, scissors, 1x sterile phosphate-buffered saline (PBS), 10 cm sterile Petri dishes, container with ice, perforated spoon, dissection stereomicroscope.
<ol>
	<li>Place an e11.5 pregnant mouse in a clean CO2 euthanasia chamber to sacrifice it. Close the lid of the chamber to prevent the mouse from escaping.</li>
	<li>After the mouse is secured in the euthanasia chamber, turn on CO2. Make sure to regulate the flow rate of CO2 per IACUC recommendations (i.e., 10-30% displacement per minute). After the mouse is completely euthanized, perform cervical dislocation to ensure death.</li>
	<li>Spray the mouse with 70% ethanol. Lift the skin over the belly using forceps, make a small incision using a pair of scissors and extend the incision laterally. Enlarge the incision anteriorly up to the diaphragm and expose the uterine horn containing the embryos (Figure 2).</li>
	<li>Pull out the string of embryos (uterine horn + embryos) by grasping the uterus and cutting it free. Place the string of embryos in ice-cold sterile 1x PBS.</li>
	<li>Dissect out the individual embryos from the uterus horn by peeling off the uterine muscle, yolk sac, and amnion one by one (Figure 3A-F) under a stereomicroscope. Transfer the cleaned embryos using a perforated spoon to a Petri dish containing sterile 1x PBS on ice. Make sure to keep the embryos cold.</li>
</ol>
3. Isolating Hearts from e11.5 Embryos
NOTE: Before beginning, make sure to have the following equipment and reagents: regular forceps, fine forceps, 1x sterile PBS, 10 cm sterile Petri dish, 6 cm sterile Petri dish, container with ice, perforated spoon, dissection stereomicroscope.
<ol>
	<li>Set up a new Petri dish with ice-cold sterile 1x PBS under a stereomicroscope. Transfer an embryo from step 2.5 into the Petri dish to dissect out the heart.</li>
	<li>Remove the head of the embryo using forceps. First, squeeze the head between the forceps with one hand and then remove the head by scraping it away with the other hand using closed forceps (Figure 4A-C).</li>
	<li>After removing the head, orient the embryo with its ventral side up by holding the embryo with forceps at its belly with one hand (Figure 4D).</li>
	<li>With the other hand, open the chest wall of the embryo by first making a small incision in the chest slightly above the diaphragm using fine forceps. Then, enlarge the incision very carefully by inserting closed forceps and tearing the chest wall by opening the forceps. Make sure to not thrust too deep, which can damage the heart. With the help of the forceps, keep the chest wall wide open to expose the heart and lungs in the thoracic cavity (Figure 4E).</li>
	<li>Using fine forceps, gently move the heart anteriorly (90&#176;) and expose the dorsal aorta/vein. Pull out the heart/lungs anteriorly by capturing the dorsal aorta/vein at the base of the heart (Figure 4F-H). 
	NOTE: Be gentle while pulling out the heart/lungs to avoid tearing of the SV, which is located at the dorsal side of the heart.</li>
	<li>Rinse the heart/lungs with cold 1x PBS to remove blood cells.</li>
	<li>Repeat steps 3.1-3.6 to remove the heart/lungs from the remaining embryos. Make sure to keep the isolated heart/lungs on ice.</li>
</ol>
4. Isolating SVs and Ventricles from e11.5 Embryonic Mouse Hearts
<ol>
	<li>Place the Petri dish with heart/lungs from step 3.7 under a stereomicroscope to isolate the SVs and whole ventricles. Peel off the attached lobes of the lungs one-by-one from their root using fine forceps.</li>
	<li>Orient the heart on its dorsal side and remove atria and the adjacent tissue that surrounds the SV anteriorly without tearing the SV. Remove the left and right atria from the heart by holding at its base and scraping it off using fine forceps (Figure 5B). Remove the adjacent tissue surrounding the SV using a similar technique (Figure 5C). 
	NOTE: Keep in mind that the right atrium is attached to the SV, so be careful to only remove the atrium.</li>
	<li>To isolate the SV, first orient the heart with its dorsal side facing up (because the SV is on the dorsal side) and keep the heart still in this position by gently holding the heart at its ventricles with forceps. 
	NOTE: The SV is an inflow organ of an embryonic heart that lies in between the atria on the dorsal side of the heart.</li>
	<li>Remove the SV by carefully peeling it off the heart where it is attached or by holding the SV at the base of its attachment with fine forceps and scraping it off with closed forceps (Figure 5D,E).</li>
	<li>Transfer the isolated SV into a new 6 cm Petri dish with ice-cold sterile 1x PBS on ice using a sterile transfer pipette and label the Petri dish as SV.</li>
	<li>To isolate the whole ventricles, remove the outflow tract (aorta and pulmonary trunk) from the heart after the SV is removed (Figure 5F,G).</li>
	<li>Transfer the whole ventricles into a new 6 cm Petri dish containing sterile 1x PBS on ice using a sterile transfer pipette and label the Petri dish as ventricles. Keep the isolated SV and ventricles on ice.</li>
	<li>Repeat steps 4.1-4.7 to isolate SVs and ventricles from the remaining hearts.</li>
</ol>
5. Setting Up Tissue Culture Plates with Inserts and Extracellular Matrix Coating
NOTE: Before beginning, make sure to have the following equipment and reagents: commercial extracellular matrix solution (ECM; e.g., Matrigel), 8.0 &#181;M polyethylene terephthalate (PET) culture inserts, 24 well plates, 37 &#176;C, 5% CO2 incubator.
<ol>
	<li>Let the ECM solution thaw on ice. Keep the ECM solution on ice to avoid solidification.</li>
	<li>Place the PET membrane culture inserts (pore size = 8.0 &#181;m, filtration area = 0.3 cm2, filter diameter = 6.5 mm) into the wells of non-tissue culture treated 24 well plates. Label the plates as SV or ventricles for the SV or the endocardial angiogenesis assays, respectively. 
	NOTE: Set up the inserts in separate plates for the SV and the ventricles when performing both cultures simultaneously. Make sure to set up enough wells for all the experimental samples and controls.</li>
	<li>After the ECM solution is thawed, immediately dilute ECM 1:2 in precooled basal medium (i.e., EBM-2 basal medium, see Table of Materials) to a sufficient volume (100 &#181;L/insert x number of inserts). 
	NOTE: For instance, if there are six inserts, then the total volume will be 100 &#181;L x 6 = 600 &#181;L. Add 200 &#181;L of ECM into 400 &#181;L of basal medium.</li>
	<li>Coat the inserts with 100 &#181;L of freshly diluted ECM by adding it directly on top of the membrane. Incubate the plate at 37 &#176;C for at least 30 min to allow the ECM to solidify. 
	NOTE: This must be performed under a laminar flow tissue culture hood to avoid contamination.</li>
</ol>
6. SVs and Whole Ventricles Cultures
NOTE: Before beginning, make sure to have the following equipment and reagents: 70% ethanol, transfer pipette, stereomicroscope, forceps, laminar flow tissue culture hood, microvascular endothelial cell supplement kit (Table of Materials), basal medium, 1x sterile PBS). Figure 6 shows the workflow of SV and ventricle culture.
<ol>
	<li>Thaw out the contents of the supplement kit on ice. Prepare the complete medium by adding all the contents of the supplement kit into 500 mL of basal medium under a certified laminar flow tissue culture hood. Mix the medium well and distribute into 50 mL aliquots.</li>
	<li>Sterilize the base of the stereomicroscope and surrounding working area with 70% ethanol.</li>
	<li>Obtain the tissue culture plates from step 5.4. With the aid of a transfer pipette, carefully transfer the explants from step 4.7 on top of the insert membrane. Under a stereomicroscope and with the aid of clean forceps, position the explants at the center of the inserts to ensure they are not stuck in the corner of the inserts or attached to the side walls.</li>
	<li>After the explants are placed and centered on the inserts, carefully remove any extra PBS from the inserts and close the lids of the plates.</li>
	<li>Under a laminar flow tissue culture hood, add 100 &#181;L of the prewarmed complete medium on top of the inserts and 200 &#181;L into the wells to culture the explants at the air-liquid interface such that the basal surface of the insert is in contact with the medium, but the top surface is exposed to the air. 
	NOTE: Make sure to adjust the volume to obtain an air-liquid interface if using different size inserts/well plates.</li>
	<li>Add 300 &#181;L of PBS into the unused wells of the 24 well plates and cover with the lid. Incubate the plate in a 37 &#176;C, 5% CO2 incubator, and grow the cultures for 5 days.</li>
	<li>In the following days, routinely observe the cultures under an inverted light microscope to assess the status of the explant cultures. Make sure that the explants exhibit contractile beating and that all the explants are attached to the bottom of the membrane embedded with ECM. Take note of any floating explants. 
	NOTE: The periodic contraction of the explants indicates that they are alive. Floating explants should be omitted from the analysis.</li>
	<li>After assessing the culture status, put the culture plate back into the incubator and continue to grow the culture for up to 5 days.</li>
</ol>
7. Treatment of Cultures with VEGF-A (Positive Control)
NOTE: Before beginning, make sure to have the following equipment and reagents: laminar flow tissue culture hood, 1x PBS, basal medium + 1% fetal bovine serum (FBS), basal medium + VEGF-A, pipettes, and pipette tips.
<ol>
	<li>Prepare the basal medium + 1% FBS and the basal medium + VEGF-A.
	<ol>
		<li>To prepare the basal medium + 1% FBS, first determine the number of control wells needed. For instance, if there are three control wells, then 300 &#181;L/well x 3 = 900 &#181;L is the total volume needed. Add 9 &#181;L of FBS into 891 &#181;L of basal medium to make the basal medium + 1% FBS.</li>
		<li>To prepare the basal medium + VEGF-A, first determine the total number of wells needing VEGF-A medium. If there are three wells, then 300 &#181;L/well x 3 = 900 &#181;L is the total volume and 50 ng/well x 3 = 150 ng VEGF-A. Add 150 ng of VEGF-A into 900 &#181;L of basal medium to make the basal medium + VEGF-A. 
		NOTE: Assemble this solution at a larger volume than calculated to insure a sufficient number of smaller aliquots for each experiment.</li>
	</ol>
	</li>
	<li>On day 2, remove the media from both chambers (the inserts and the wells). Wash cultures with 300 &#181;L of 1x PBS by adding 100 &#181;L to the inserts and 200 &#181;L into the wells. Firmly swirl the plates a few times and remove the PBS.</li>
	<li>Add 300 &#181;L of basal medium + 1% FBS (100 &#181;L into the insert and 200 &#181;L into the wells) to starve the cultures for 24 h.</li>
	<li>On day 3, after starvation, add 300 &#181;L of basal medium + 1% FBS (100 &#181;L into the insert and 200 &#181;L into the wells) into the control wells and basal medium + VEGF-A (50 ng/well) into the treatment wells, respectively.</li>
	<li>After treatment, continue to grow the cultures in the incubator.</li>
</ol>
8. Fixation and Immunostaining
NOTE: Before beginning, make sure to have the following equipment and reagents: 4% paraformaldehyde (PFA), 1x PBS, primary and secondary antibodies, a shaker, 0.5% nonionic surfactant in PBS (PBT).
<ol>
	<li>On the sixth day of culturing, remove the medium and wash cultures with 1x PBS at room temperature (RT).
	<ol>
		<li>Fix the cultures by adding 200 &#181;L of 4% PFA solution into the wells and 100 &#181;L into the inserts. Fix cultures in 4 &#176;C for 20 min while rocking.</li>
		<li>After 20 min fixation, remove PFA from the cultures in a fume hood and wash the cultures with 1x PBS by adding 200 &#181;L into the wells and 100 &#181;L into the inserts.</li>
		<li>Repeat washes 3x, 10 min each, while rocking. Then proceed to perform immunostaining. 
		NOTE: All the wash steps are performed on a benchtop at RT.</li>
	</ol>
	</li>
	<li>Dilute primary antibodies (anti-VE-Cadherin, anti-ERG 1/2/3) in blocking solution (5% donkey serum, 0.5% PBT). Add 300 &#181;L of primary antibody solution (200 &#181;L in the bottom wells and 100 &#181;L into the inserts). Incubate cultures in primary antibodies overnight at 4 &#176;C while rocking. 
	NOTE: Anti-VE-Cadherin is used to label the endothelial cell membrane and anti-ERG 1/2/3 is used to label the endothelial cell nucleus in order to visualize the angiogenic sprouts of endothelial cells.</li>
	<li>The next day, wash and rock the culture plates 10x in 0.5% PBT, changing PBT every 10 min.</li>
	<li>Dilute the secondary antibodies (donkey anti-rat Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 555) in blocking solution. Add 300 &#181;L of the secondary antibody as in step 8.2 and incubate the cultures overnight at 4 &#176;C while rocking. The next day, wash the secondary antibodies 10x in PBT, changing PBT every 10 min. 
	NOTE: Wash a minimum of 10x but more washes are better. After the washes are complete, the cultures can be stored with 1x PBS until they are mounted onto slides.</li>
</ol>
9. Mounting Cultures Onto Slides, Imaging, and Analysis
NOTE: Before beginning, make sure to have the following equipment and reagents: fine forceps, slides, mounting medium with 4&#8242;,6-diamidino-2-phenylindole (DAPI), coverslips, and confocal microscope. After secondary antibody staining, mount the cultures onto slides for imaging using the following steps.
<ol>
	<li>Peel off the membrane carefully from the insert using fine forceps and transfer it onto the slides by putting the membrane side down and placing the explant cultures upward. Place the replicate samples into the same slides and label the slides as control or VEGF-A. Add a few drops of mounting medium with DAPI directly onto the membrane and cover the slides with cover slips. 
	NOTE: Make sure to avoid air bubbles while placing the coverslips.</li>
	<li>Seal off the edges of the slides with clear nail polish and let dry. 
	NOTE: Slides can be stored in -20 &#176;C for long-term storage.</li>
	<li>Image slides using a confocal microscope.</li>
	<li>Perform analysis to measure the length of angiogenic outgrowth. Quantify angiogenic outgrowth length by measuring the distance of the endothelial cells (Ve-Cadherin+/ERG 1/2/3+) extended from the inside boundary of the ERG 1/2/3+ cells in the ventricle cultures and from the center of the SV explants in the SV cultures.
	<ol>
		<li>To perform quantification using FIJI/ImageJ, first download FIJI software.</li>
		<li>Open image files in FIJI: go to File | Open | Folder | Filename | Open.</li>
		<li>Go to Analyze | Set Measurements | Select Perimeter.</li>
		<li>Select the Straight Line tool from the main window.</li>
		<li>Draw a line across the length of a sprout as suggested in step 9.4.</li>
		<li>Go to Analyze | Measure. 
		NOTE: Length measurements are displayed in the new window.</li>
		<li>Perform quantification in images that represent at least three randomly selected fields of view. Average the sprout length measurements and report them as mean &#177; standard deviation.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

Some of the most critical steps for successfully growing coronary vessels from the SV and Endo progenitor tissues are: 1) Correctly identifying and isolating the SV tissue for SV culture; 2) using ventricles from embryos between the ages of e11&#8722;11.5 for accurate Endo culture; 3) maintaining sterile conditions throughout the dissection period and keeping the tissues cold at all times; and 4) keeping the explants attached to the ECM coated membrane to avoid tissue floating in the medium.
F...

Blood vessels of the heart are commonly called coronary vessels. These vessels are comprised of arteries, veins, and capillaries. During development, highly branched capillaries are established first, which then remodel into coronary arteries and veins1,2,3,4,5. These initial capillaries are built from endothelial progenitor cells found in the proepicardium, sinus venosus (SV), and endocardium (Endo) tissues1,6,7,8. SV is the inflow organ of embryonic heart and Endo is the inner lining of the heart lumen. Endothelial progenitor cells found in the SV and Endo build the majority of coronary vasculature, whereas the proepicardium contributes to a relatively small portion of it2. The process by which the capillary network of coronary vessels grow in the heart from its preexisting precursor cells is called coronary angiogenesis. Coronary artery disease is one of the leading causes of death worldwide and yet an effective treatment for this disease is lacking. Understanding the detailed cellular and molecular mechanisms of coronary angiogenesis can be useful in designing novel and effective therapies to repair and regenerate damaged coronary arteries.
Recently, a surge in our understanding of how coronary vessels develop has been in part achieved through the development of new tools and technologies. In particular, in vivo lineage labelling and advanced imaging technologies have been very useful in uncovering the cellular origin and differentiation pathways of coronary vessels9,10,11,12. Despite the advantages of these in vivo tools, there are limitations in terms of speed, flexibility, and accessibility. Therefore, robust in vitro model systems can complement in vivo systems to elucidate the cellular and molecular mechanisms of coronary angiogenesis in a high-throughput manner.
Here, we describe an in vitro model of coronary angiogenesis. We have developed an in vitro explant culture system to grow coronary vessels from two progenitor tissues, SV and Endo. With this model, we show that the in vitro tissue explant cultures grow coronary vessel sprouts when stimulated by growth medium. Additionally, the explant cultures grow rapidly compared to control when stimulated by vascular endothelial growth factor A (VEGF-A), a highly potent angiogenic protein. Furthermore, we found that the angiogenic sprouts from the SV culture undergo venous dedifferentiation (loss of COUP-TFII expression), a mechanism similar to SV angiogenesis in vivo1. These data suggest that the in vitro explant culture system faithfully reinstates angiogenic events that occur in vivo. Collectively, in vitro models of angiogenesis that are described here are ideal for probing cellular and molecular mechanisms of coronary angiogenesis in a high-throughput and accessible manner.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 x 20 MM Tissue Culture Dish</td>
			<td>Fisher Scientific</td>
			<td>877222</td>
			<td>Referred in the protocol as Petri dish</td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>Fisher Scientific</td>
			<td>08-772-51</td>
			<td></td>
		</tr>
		<tr>
			<td>8.0 uM PET membrane culture inserts</td>
			<td>Millipore Sigma</td>
			<td>MCEP24H48</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-rabbit 555</td>
			<td>Fisher Scientific</td>
			<td>A31572</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Alexa Fluor Donkey anti-rat 488</td>
			<td>Fisher Scientific</td>
			<td>A21206</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Angled Metal Probe</td>
			<td>Fine science tools</td>
			<td>10088-15</td>
			<td>Angled 45 degree, used for detecting deep plugs</td>
		</tr>
		<tr>
			<td>Anti- ERG 1/2/3 antibody</td>
			<td>Abcam</td>
			<td>Ab92513</td>
			<td>Primary antibody</td>
		</tr>
		<tr>
			<td>Anti- VE-Cadherin antibody</td>
			<td>Fisher Scientific</td>
			<td>BDB550548</td>
			<td>Primary antibody, manufacturer BD BioSciences</td>
		</tr>
		<tr>
			<td>CO2 gas tank</td>
			<td>Various suppliers</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Fisher Scientific</td>
			<td>13998223</td>
			<td>For 37 &#176;C, 5% CO2 incubation</td>
		</tr>
		<tr>
			<td>Dissection stereomicrosope</td>
			<td>Leica</td>
			<td>S9i</td>
			<td>Leica S9i Stereomicroscope</td>
		</tr>
		<tr>
			<td>EBM-2 basal media</td>
			<td>Lonza</td>
			<td>CC-3156</td>
			<td>Endothelial cell growth basal media</td>
		</tr>
		<tr>
			<td>ECM solution</td>
			<td>Corning</td>
			<td>354230</td>
			<td>Commercially known as Matrigel</td>
		</tr>
		<tr>
			<td>EGM-2 MV Singlequots Kit</td>
			<td>Lonza</td>
			<td>CC-4147</td>
			<td>Microvascular endothelial cell supplement kit; This is mixed into the EBM-2 to make the EGM-2 complete media</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fisher Scientific</td>
			<td>SH3007003IR</td>
			<td></td>
		</tr>
		<tr>
			<td>FiJi</td>
			<td>NIH</td>
			<td>NA</td>
			<td>Image processing software (<a href="https://imagej.net/Fiji/Downloads" target="_blank">https://imagej.net/Fiji/Downloads</a>)</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine science tools</td>
			<td>11412-11</td>
			<td>Used for embryo dissection</td>
		</tr>
		<tr>
			<td>Fisherbrand Straight-Blade operating scissors</td>
			<td>Fisher Scientific</td>
			<td>13-808-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyclone Phosphate Buffered Saline (1X)</td>
			<td>Fisher Scientific</td>
			<td>SH-302-5601LR</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar flow tissue culture hood</td>
			<td>Fisher Scientific</td>
			<td>various models available</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Vectashield with DAPI</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Electron Microscopy/Fisher</td>
			<td>50-980-494</td>
			<td>This is available at 32%; needs to be diluted to 4%</td>
		</tr>
		<tr>
			<td>Perforated spoon</td>
			<td>Fine science tools</td>
			<td>10370-18</td>
			<td>Useful in removing embryo/tissues from a solution</td>
		</tr>
		<tr>
			<td>Recombinant Murine VEGF-A 165</td>
			<td>PeproTech</td>
			<td>450-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard forceps, Dumont #5</td>
			<td>Fine science tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Sure-Seal Mouse/Rat chamber</td>
			<td>Easysysteminc</td>
			<td>EZ-1785</td>
			<td>Euthanasia chamber</td>
		</tr>
	</tbody>
</table>

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.

One of the most striking features of SV angiogenesis in vivo is that it follows a specific pathway and involves cell dedifferentiation and redifferentiation events that occur at stereotypical times and positions1. As initial SV cells grow onto the heart ventricle, they stop producing venous markers such as COUP-TFII (Figure 7). Subsequently, coronary sprouts take two migration paths, either over the surface of the heart or deep within ...

Watch this Scientific Journal Video about In Vitro Model of Coronary Angiogenesis at JoVE.com

In Vitro Model of 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 ...

Research

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Medicine

7.4K Views.  Ball State University. 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.

Video: In Vitro Model of Coronary Angiogenesis

A Mouse Model of the Cornea Pocket Assay for Angiogenesis Study

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are administered 1. This uniqueness has made the cornea pocket assay one of the most used models for angiogenesis studies. The cornea composes multiple layers of cells. It is therefore possible to embed a pellet containing the angiogenic factor of interest in the cornea to investigate its angiogenic effect 2,3. Here, we provide a step by step demonstration of how to (I) produce the angiogenic factor-containing pellet (II) embed the pellet into the cornea (III) analyze the angiogenesis induced by the angiogenic factor of interest. Since the basic fibroblast growth factor (bFGF) is known as one of the most potent angiogenic factors 4, it is used here to induce angiogenesis in the cornea.

The cornea is unique in that it lacks vascular tissues. However, robust blood vessel growth and survival can be induced in the cornea by potent angiogenic factors. Therefore, the cornea can provide with us a valuable tool for angiogenic studies. This protocol demonstrates how to perform the mouse model of cornea pocket assay and how to assess the angiogenesis induced by angiogenic factors using this model.

A normal cornea is clear of vascular tissues. However, blood vessels can be induced to grow and survive in the cornea when potent angiogenic factors are ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.

Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ...