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Summary

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.

Abstract

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.

Introduction

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 veins^1,2,3,4,5. These initial capillaries are built from endothelial progenitor cells found in the proepicardium, sinus venosus (SV), and endocardium (Endo) tissues^1,6,7,8. 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 vessels^9,10,11,12. 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.

Protocol

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

1. 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.
2. 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.
3. 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's weight will indicate a successful pregnancy, whereas no change in weight will indicate a failed pregnancy.

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.

1. 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.
2. 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.
3. 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).
4. 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.
5. 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.

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.

1. 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.
2. 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).
3. 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).
4. 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).
5. Using fine forceps, gently move the heart anteriorly (90°) 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.
6. Rinse the heart/lungs with cold 1x PBS to remove blood cells.
7. 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.

4. Isolating SVs and Ventricles from e11.5 Embryonic Mouse Hearts

1. 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.
2. 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.
3. 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.
4. 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).
5. 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.
6. To isolate the whole ventricles, remove the outflow tract (aorta and pulmonary trunk) from the heart after the SV is removed (Figure 5F,G).
7. 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.
8. Repeat steps 4.1-4.7 to isolate SVs and ventricles from the remaining hearts.

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 µM polyethylene terephthalate (PET) culture inserts, 24 well plates, 37 °C, 5% CO₂ incubator.

1. Let the ECM solution thaw on ice. Keep the ECM solution on ice to avoid solidification.
2. Place the PET membrane culture inserts (pore size = 8.0 µm, 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.
3. 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 µL/insert x number of inserts).
   NOTE: For instance, if there are six inserts, then the total volume will be 100 µL x 6 = 600 µL. Add 200 µL of ECM into 400 µL of basal medium.
4. Coat the inserts with 100 µL of freshly diluted ECM by adding it directly on top of the membrane. Incubate the plate at 37 °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.

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.

1. 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.
2. Sterilize the base of the stereomicroscope and surrounding working area with 70% ethanol.
3. 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.
4. 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.
5. Under a laminar flow tissue culture hood, add 100 µL of the prewarmed complete medium on top of the inserts and 200 µL 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.
6. Add 300 µL of PBS into the unused wells of the 24 well plates and cover with the lid. Incubate the plate in a 37 °C, 5% CO₂ incubator, and grow the cultures for 5 days.
7. 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.
8. After assessing the culture status, put the culture plate back into the incubator and continue to grow the culture for up to 5 days.

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.

1. Prepare the basal medium + 1% FBS and the basal medium + VEGF-A.
   1. 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 µL/well x 3 = 900 µL is the total volume needed. Add 9 µL of FBS into 891 µL of basal medium to make the basal medium + 1% FBS.
   2. 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 µL/well x 3 = 900 µL is the total volume and 50 ng/well x 3 = 150 ng VEGF-A. Add 150 ng of VEGF-A into 900 µL 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.
2. On day 2, remove the media from both chambers (the inserts and the wells). Wash cultures with 300 µL of 1x PBS by adding 100 µL to the inserts and 200 µL into the wells. Firmly swirl the plates a few times and remove the PBS.
3. Add 300 µL of basal medium + 1% FBS (100 µL into the insert and 200 µL into the wells) to starve the cultures for 24 h.
4. On day 3, after starvation, add 300 µL of basal medium + 1% FBS (100 µL into the insert and 200 µL into the wells) into the control wells and basal medium + VEGF-A (50 ng/well) into the treatment wells, respectively.
5. After treatment, continue to grow the cultures in the incubator.

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).

1. On the sixth day of culturing, remove the medium and wash cultures with 1x PBS at room temperature (RT).
   1. Fix the cultures by adding 200 µL of 4% PFA solution into the wells and 100 µL into the inserts. Fix cultures in 4 °C for 20 min while rocking.
   2. After 20 min fixation, remove PFA from the cultures in a fume hood and wash the cultures with 1x PBS by adding 200 µL into the wells and 100 µL into the inserts.
   3. 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.
2. Dilute primary antibodies (anti-VE-Cadherin, anti-ERG 1/2/3) in blocking solution (5% donkey serum, 0.5% PBT). Add 300 µL of primary antibody solution (200 µL in the bottom wells and 100 µL into the inserts). Incubate cultures in primary antibodies overnight at 4 °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.
3. The next day, wash and rock the culture plates 10x in 0.5% PBT, changing PBT every 10 min.
4. Dilute the secondary antibodies (donkey anti-rat Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 555) in blocking solution. Add 300 µL of the secondary antibody as in step 8.2 and incubate the cultures overnight at 4 °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.

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′,6-diamidino-2-phenylindole (DAPI), coverslips, and confocal microscope. After secondary antibody staining, mount the cultures onto slides for imaging using the following steps.

1. 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.
2. Seal off the edges of the slides with clear nail polish and let dry.
   NOTE: Slides can be stored in -20 °C for long-term storage.
3. Image slides using a confocal microscope.
4. 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.
   1. To perform quantification using FIJI/ImageJ, first download FIJI software.
   2. Open image files in FIJI: go to File | Open | Folder | Filename | Open.
   3. Go to Analyze | Set Measurements | Select Perimeter.
   4. Select the Straight Line tool from the main window.
   5. Draw a line across the length of a sprout as suggested in step 9.4.
   6. Go to Analyze | Measure.
      NOTE: Length measurements are displayed in the new window.
   7. Perform quantification in images that represent at least three randomly selected fields of view. Average the sprout length measurements and report them as mean ± standard deviation.

Representative Results

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 the myocardium. Surface vessels eventually become veins while invading vessels become arteries and capillaries^1,6,7. This distinction is preserved as the heart grows and the coronary vasculature expands.

To facilitate the discovery of the molecular underpinnings of coronary development, we have devised an in vitro model of SV sprouting that can be utilized for loss-of-function and gain-of-function experiments. SV tissue from embryonic mouse hearts are dissected, placed on the top of diluted ECM (which is commercially available, see Table of Materials), and maintained at the air-liquid interface in endothelial cell growth medium. The SV myocardium continues to beat throughout the culture period. After 2 days in culture, epicardial cells that line the tissue leave the explant and migrate out onto the matrix. After 5 days, SV endothelial cells sprout out and migrate onto the epicardial tissue (Figure 8A, black arrowheads). Collectively, this process recapitulates angiogenic aspects of coronary development.

Cultured SV sprouts also undergo venous dedifferentiation. As in the embryonic heart, COUP-TFII expression is reduced as the vessels migrate away from the SV (Figure 8B, compared to Figure 7). Control vessels such as umbilical or yolk sac arteries and veins can produce sprouting vessels. However, they are fewer in number and shorter in length and do not change their arterial or venous identity (not shown). Thus, venous reprogramming is specific to SV sprouting and not a general feature of angiogenesis or a response to ECM components. These data also indicate that the cell types contained within the SV itself are necessary and sufficient to induce dedifferentiation.

It is well known that vascular endothelial growth factor A (VEGF-A) is a potent inducer of angiogenesis^{7,13,14,15,16,17,18}. Previous studies have shown that myocardial VEGF-A stimulates endocardial angiogenesis to grow coronary vessels⁷. In addition, coronary endothelial cells have been shown to express VEGF-A receptor, and SV-derived coronary vessels in the myocardium might grow in response to VEGF-A in vivo². To show that our in vitro model of coronary angiogenesis faithfully responds to VEGF-A, we stimulated SV and Endo cultures with VEGF-A. As expected, our results showed that both SV and Endo are highly responsive to VEGF-A. Cultures stimulated with VEGF-A show increased growth of angiogenic sprouts both in density and length (Figure 9A,C). SV cultures stimulated by VEGF-A show an almost 3-fold increase in sprout length compared to the control (Figure 9B). These results suggest that endothelial sprouts from SV and Endo respond to similar cues that are known in vivo.

Taken together, our data suggest that these in vitro explant cultures share similar cellular and molecular events that occur during in vivo coronary development, including venous dedifferentiation and robust growth in response to VEGF-A stimulation. Therefore, our data suggest that these explant culture models are useful for studying coronary angiogenesis in vitro.

Figure 1: Vaginal plug identification. (A) A vaginal plug (white spot). (B) Magnified view of boxed region including the vaginal plug (indicated by arrow). Please click here to view a larger version of this figure.

Figure 2: Accessing the embryo string in the uterus of a pregnant mouse. A euthanized pregnant mouse is positioned with its ventral surface up and sprayed with 70% ethanol to wet skin for dissection. The skin is pulled up at the pelvic region using forceps and a small incision is made. The incision is extended laterally. An additional incision is made anteriorly up to the diaphragm. In the uterus, a string of embryos is visible (indicated by arrowheads). Please click here to view a larger version of this figure.

Figure 3: Dissection and removal of embryos from the uterus. (A) Image showing the uterus containing a string of embryos. The membrane on the dorsal side of the embryo (opposite of the side where the placenta is located) is peeled off. (B) The embryo in the yolk sac becomes visible after peeling off the uterus membrane. (C,D) Embryo attached to the placenta with the umbilical cord is visible after peeling off the yolk sac. Amnion is peeled off if still present. (E) Image showing an embryo attached to the placenta by the umbilical cord. (F) Image showing the detaching of the embryo from the placenta by pulling on the umbilical cord (Umb. Cord) using forceps. Please click here to view a larger version of this figure.

Figure 4: Removal of the heart/lungs pluck from embryos. (A,B) The head is removed from the embryo by capturing at the neck and scraping it off. (C) Image showing the removal of the head from the body. (D) The correct positioning of the embryo for heart dissection. The embryo is positioned with the ventral side up to have easy access to the thoracic cavity for the removal of the heart. Once the embryo is positioned as shown in D, the chest cavity is opened with forceps. (E) Image showing the open chest cavity with the heart exposed on its ventral side. (F) To remove the heart without damaging the SV, the heart is flipped anteriorly, and the dorsal aorta becomes visible. (G) Image showing the position at the base of the heart/lungs where the dorsal aorta/vein is captured with forceps and the heart/lungs pluck is pulled out anteriorly. (H) The image shows the e11.5 heart/lungs pluck removed from the embryo. V. heart = ventral heart; d. heart = dorsal heart; d. aorta = dorsal aorta; LA = left atrium; RA = right atrium; LV = left ventricle; RV = right ventricle. Please click here to view a larger version of this figure.

Figure 5: Isolating SV and ventricles from embryonic hearts. (A) Image showing the dorsal view of the isolated heart/lungs pluck from the e11.5 embryo. Location of the SV in the heart is indicated. (B) Image of the heart after the lung buds have been removed. (C) Atria and adjacent tissues surrounding the SV are removed, revealing the aorta. Location of the SV is labelled. (D) Image showing removal of the SV. Using forceps, the SV is gripped at its base and is scraped off from the heart. (E) SV removed from the heart is shown along with the remaining tissue of the heart. The aorta and pulmonary trunk (PT), collectively called the outflow tract (OFT), become visible in the heart after SV is removed. The SV will be used for in vitro culture, as described in Figure 6 (left panel). (F) Position of the OFT to be dissected is marked by a dashed white line. (G) Aorta/PT (outflow tract) is removed from the ventricles by dissection at the position shown in panel F. The remaining ventricles without the OFT are used for ventricle cultures as shown in Figure 6 (right panel). Please click here to view a larger version of this figure.

Figure 6: Schematic showing the workflow of SV and ventricle culture. Left: Procedure for SV culture. First, the SV is removed from e11.5 hearts and placed onto a culture insert. Insert contains porous membrane at the bottom (8 µm pore) and is coated with growth factor reduced ECM. Inserts are placed into the wells of a 24 well plate. Medium is added both in the bottom and top chamber without covering the explant. The culture is grown for 5 days. After 5 days, the membrane is removed from the insert and is mounted onto the slides for imaging and analysis. Right: Procedure for ventricle culture. Ventricles without the SV, atria, and OFT are removed from the e11.5 hearts and are cultured as described above for the SV. Please click here to view a larger version of this figure.

Figure 7: COUP-TFII expression is lost in the SV sprouts in vivo. (A) Dorsal view of e11.5 hearts. Confocal image of endothelial marker (red) that labels the sinus venosus (SV), coronary sprouts (cs, dotted line), and endocardium (endo) lining the heart lumen. (B) The venous maker COUP-TFII (green), a vein transcription factor (trx), expressed in the SV is progressively lost as sprouts leave the SV and grow onto the ventricle. Right: Higher magnification of the coronary sprout shown in the boxed region of panel A. Scale bar = 100 µm. Please click here to view a larger version of this figure.

Figure 8: In vitro models of venous identity and reprogramming. (A) Brightfield image of cultured SV tissue. Vascular sprouts (arrowheads) grow out from the original explant (black dotted line) over the ECM substrate. (B) Immunofluorescence of SV cultures labeled with antibodies that mark the endothelial cell surface (VE-Cadherin), endothelial cell nuclei (ERG 1/2/3), and vein and epicardial nuclei (COUP-TFII). Nuclei are positive for both ERG 1/2/3 and COUP-TFII at the SV, but ERG 1/2/3 only in sprouts migrating over the ECM. Scale bar = 50 µm. Please click here to view a larger version of this figure.

Figure 9: SV and endocardium respond to VEGF-A in vitro. (A) Confocal image of the 5-day-old control and VEGF-A treated SV explant culture. Endothelial cells are immunostained with VE-cadherin (green), endothelial cell nuclei stained with ERG 1/2/3 (red) antibodies, and cell nuclei with DAPI (blue). (B) Angiogenic outgrowth of endothelial cells is quantified by measuring the length of sprout extension (the distance migrated by ERG 1/2/3+ endothelial cells from the explant, indicated by white solid lines in the bottom panels of (A). (C) Confocal image of a 5-day-old culture of a control and VEGF-A treated ventricles. Endothelial cells are immunostained with VE-cadherin (green) and endothelial cell nuclei are stained with ERG 1/2/3 (blue) antibodies. Dots are individual measurements and error bars are mean ± SD. Scale bar = 200 µm (A,C). Please click here to view a larger version of this figure.

Discussion

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 e11−11.5 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.

First, isolation of SV tissue can be challenging. It is important to realize that the SV lies on the dorsal side of the heart between the left and right atria hidden within the lung lobules. Therefore, it is difficult to separate and isolate. Instead, the whole heart/lungs pluck must be extracted first. The SV is then isolated from the pluck by carefully removing the lung lobules and cleaning up the adjacent tissues with the help of fine forceps. Furthermore, one must be very careful while cleaning up the adjacent tissues to not lose the SV.

Second, for accurate endocardial angiogenesis, it is important to culture ventricles from embryos no older than e11.5. In e12.5 and older embryos, coronaries from the SV grow into a significant proportion of the ventricles. Therefore, the coronary vessels that grow from older ventricles can be comprised of both the SV- and Endo-derived coronary vessels^1,2,10,19,20. For this reason, to accurately assay the coronary vessel growth from the Endo, it is critical to culture ventricles (minus SV and outflow tract) from e11.5 embryos²⁰. In addition, the SV is relatively larger and is easier to isolate at e11.5.

Third, because the protocol involves a substantial amount of work outside the tissue culture hood, it is critical to maintain a sterile working environment. It is important to sterilize the dissection tools (forceps, scissors, etc.) and the tissue culture plates and avoid contact with unsterile areas at all times. It is important to spray the working area and dissection scope with 70% ethanol. To avoid contamination, it is important to perform the dissection procedure quickly and minimize work outside the tissue culture hood.

Fourth, to obtain healthy explant cultures, it is important that the explants remain attached to the base of the membrane at all times. Floating explants in the medium must be avoided to successfully grow the explant cultures. To avoid floating, it is critical to maintain an air-liquid interface where the basal surface of the insert is in contact with the medium, but the top surface is exposed to the air. Such an interface is obtained when the explant is sufficiently covered by the medium but is not fully submerged. It is important to monitor the volume of the medium daily, as volume can be lost to evaporation. To prevent this, the culture plate can be humidified by adding PBS into the unused wells of the culture plates.

Similar explant cultures of SV and Endo are described elsewhere²⁰. In these methods, the explants were cultured directly into the wells of tissue culture plates, which limits high-resolution confocal imaging. The images captured in this setting are relatively poor in quality compared to the method proposed here, limiting detailed microscopic analyses. To circumvent this, we utilized culture inserts from which the membranes containing the culture can be peeled off and mounted onto the glass slides for imaging. This allows for high-resolution confocal imaging where cellular details such as expression patterns and morphology can be viewed. In addition, culturing directly on plates requires a separate staining protocol for DAPI or other pan-nuclear marker staining. This protocol does not require separate staining steps because the slides can be mounted with mounting medium containing DAPI. Furthermore, mounting on slides allows for long-term storage without fluorescent fading, whereas cultures stored in wells with liquid medium will result in quick fading and are not amenable to long-term storage and imaging.

The in vitro culture models described here are ideal for interrogating cellular and molecular mechanisms of coronary angiogenesis in a high-throughput and accessible manner. This in vitro system can be used to screen for potential drugs or molecular targets to assess their effect on coronary angiogenesis. In addition, this system can also be used to study the gain-of-function and loss-of-function of various genes for their autonomous function in coronary angiogenesis. We observed that coronary sprouts from SV followed the epicardial migration in our in vitro cultures, suggesting an important interaction between coronary endothelial cells and epicardial cells. It is well known that the epicardium is a rich source of growth factors for coronary angiogenesis from the SV. For instance, epicardial-derived growth factors such as VEGF-C and ELABELA have been previously shown to regulate SV-derived coronary angiogenesis^2,19. We can use this SV culture system to further investigate the interaction between the epicardium and coronary endothelial cells and identify novel epicardial-derived molecular pathways of coronary angiogenesis. Additionally, our in vivo data suggest that myocardial hypoxia may be involved in endocardial angiogenesis¹⁹. Our in vitro culture system offers an excellent model to study the role of hypoxia in endocardial angiogenesis by incubating the cultures in hypoxic or normoxic conditions. These are difficult experiments to conduct in vivo because it requires a pregnant mouse to be placed in a controlled hypoxic chamber. From our own experience, it is very challenging to obtain consistent and reliable results from in vivo experiments. In summary, our in vitro model of coronary angiogenesis provides a reliable system to interrogate a wide range of questions relating to the cellular and molecular biology of coronary angiogenesis.

Materials

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