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><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>CO&lt;sub&gt;2&lt;/sub&gt; gas tank</td><td>Various suppliers</td><td>N/A</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Fisher Scientific</td><td>13998223</td><td>For 37 &amp;deg;C, 5% CO&lt;sub&gt;2&lt;/sub&gt; 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 (&lt;a href=&quot;https://imagej.net/Fiji/Downloads&quot; target=&quot;_blank&quot;&gt;https://imagej.net/Fiji/Downloads&lt;/a&gt;)</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

JoVE Journal

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

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Biology

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature maintenance and pathological malformations can result in severe ischemic diseases, while overly abundant vascular development is associated with cancer and inflammatory disorders. A promising form of pro-angiogenic therapy is the use of angiogenic cell sources, which can provide regulatory factors as well as physical support for newly developing vasculature.
Mesenchymal Stromal Cells (MSCs) are extensively investigated candidates for vascular regeneration due to their paracrine effects and their ability to detect and home to ischemic or inflamed tissues. In particular, first trimester human umbilical cord perivascular cells (FTM HUCPVCs) are a highly promising candidate due to their pericyte-like properties, high proliferative and multilineage potential, immune-privileged properties, and robust paracrine profile. To effectively evaluate potentially angiogenic regenerative cells, it is a requisite to test them in reliable and &#34;translatable&#34; pre-clinical assays. The aortic ring assay is an ex vivo angiogenesis model that allows for easy quantification of tubular endothelial structures, provides accessory supportive cells and extracellular matrix (ECM) from the host, excludes inflammatory components, and is fast and inexpensive to set up. This is advantageous when compared to in vivo models (e.g., corneal assay, Matrigel plug assay); the aortic ring assay can track the administered cells and observe intercellular interactions while avoiding xeno-immune rejection.
We present a protocol for a novel application of the aortic ring assay, which includes human MSCs in co-cultures with developing rat aortic endothelial networks. This assay allows for the analysis of the MSC contribution to tube formation and development through physical pericyte-like interactions and of their potency for actively migrating to sites of angiogenesis, and for evaluating their ability to perform and mediate ECM processing. This protocol provides further information on changes in MSC phenotype and gene expression following co-culture.

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Here, we present a novel application of the aortic ring assay where prelabelled mesenchymal cells are co-cultured with rat aorta-derived endothelial networks. This novel method allows visualization of Mesenchymal Stromal Cells (MSCs) homing and integration with endothelial networks, quantification of network properties, and evaluation of MSC immunophenotypes and gene expression.

Angiogenesis is a complex, highly regulated process responsible for providing and maintaining adequate tissue perfusion. Insufficient vasculature ...

Developmental Biology

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including embryogenesis and development, wound healing, tumor growth and metastasis, and ocular and cardiovascular diseases. Effective in vitro models that recapitulate the biology of angiogenesis are needed to appropriately study this process and identify mechanisms of regulation that can be ultimately targeted for novel therapeutic strategies. The bead angiogenesis assay has been previously demonstrated to recapitulate the multiple stages of endothelial sprouting in vitro. However, a limitation of this assay is a lack of endothelial &#8211; mural cell interactions, which are key to the molecular and phenotypic regulation of endothelial cell function in vivo. The protocol given here presents a methodology for the incorporation of mural cells into the bead angiogenesis assay and demonstrates a tight association of endothelial cells and pericytes during sprouting in vitro. The protocol also details a methodology for effective silencing of target genes using siRNA in endothelial cells for mechanistic studies. Altogether, this protocol provides an in vitro assay that more appropriately models the diverse cell types involved in sprouting angiogenesis, and provides a more physiologically-relevant platform for therapeutic assessment and novel discovery of mechanisms of angiogenesis regulation.

This protocol presents a novel in vitro bead assay that more appropriately models the process of in vivo sprouting angiogenesis by incorporating pericytes. This modification enables the bead assay to more faithfully recapitulate the heterotypic cellular interactions between endothelial cells and mural cells that are critical for angiogenesis.

Angiogenesis is the growth of new vessels from pre-existing vasculature and is an important component of many biological processes, including ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Neuroscience

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Bioengineering

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

This assay utilizes mouse embryonic stem cells differentiated into embryoid bodies cultured in 3D-collagen gel to analyze the biological processes that control sprouting angiogenesis in vitro. The technique can be applied for testing drugs, modeling diseases, and for studying specific genes in the context of deletions that are embryonically lethal.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for ...

Simplifying Angiogenesis Studies with a Modified Matrix Gel Plug Assay

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Author Spotlight: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

Versatile 2.5D Model for Analyzing Cellular Sprouting in Porcine Arteries

2.5D Model for Ex Vivo Mechanical Characterization of Sprouting Angiogenesis in Living Tissue

Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue growth and repair and pathological processes, including cancer and metastasis. The multistep process of sprouting angiogenesis is a molecularly and mechanically driven process. It consists of induction of cellular sprout by vascular endothelial growth factor, leader/follower cell selection through Notch signaling, directed migration of endothelial cells, and vessel fusion and stabilization. A variety of sprouting angiogenesis models have been developed over the years to better understand the underlying mechanisms of cellular sprouting. Despite advancements in understanding the molecular drivers of sprouting angiogenesis, the role of mechanical cues and the mechanical driver of sprouting angiogenesis remains underexplored due to limitations in existing models. In this study, we designed a 2.5D ex vivo model that enables us to mechanically characterize cellular sprouting from a porcine carotid artery using traction force microscopy. The model identifies distinct force patterns within the sprout, where leader cells exert pulling forces and follower cells exert pushing forces on the matrix. The model's versatility allows for the manipulation of both chemical and mechanical cues, such as matrix stiffness, enhancing its relevance to various microenvironments. Here, we demonstrate that the onset of sprouting angiogenesis is stiffness-dependent. The presented 2.5D model for quantifying cellular traction forces in sprouting angiogenesis offers a simplified yet physiologically relevant method, enhancing our understanding of cellular responses to mechanical cues, which could advance tissue engineering and therapeutic strategies against tumor angiogenesis.

Sprouting angiogenesis, fundamental for development and disease, involves complex molecular and mechanical processes. We present a versatile 2.5D ex vivo model that analyzes cellular sprouting from porcine carotid arteries, revealing stiffness-dependent angiogenesis and distinct leader-follower cell mechanics. This model aids in advancing tissue engineering strategies and cancer therapy approaches.

Sprouting angiogenesis is the formation of new blood vessels from pre-existing vasculature and is of great importance for physiological such as tissue ...

In Vitro Model of Coronary Angiogenesis

Summary

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Chapters in this video

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

The Aortic Ring Co-culture Assay: A Convenient Tool to Assess the Angiogenic Potential of Mesenchymal Stromal Cells In Vitro

Incorporating Pericytes into an Endothelial Cell Bead Sprouting Assay

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Aortic Ring Assay

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

In Vitro Three-Dimensional Sprouting Assay of Angiogenesis Using Mouse Embryonic Stem Cells for Vascular Disease Modeling and Drug Testing

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

2.5D Model for Ex Vivo Mechanical Characterization of Sprouting Angiogenesis in Living Tissue