Our protocol is significant because it allows researchers to investigate the molecular mechanism of coronary angiogenesis outside of an organism. The main advantage of this technique is that it accurately models the process of coronary angiogenesis inside an organism, facilitating the study of the mechanisms of coronary angiogenesis. Begin by spraying a pregnant mouse carrying embryonic day 11.5 embryos with 70%ethanol.
Lifting the skin over the belly with forceps, use scissors to make an abdominal skin and peritoneal incision laterally and anteriorly up to the diaphragm to expose the uterine horn. Grasping the uterus, cut the tissue free and pull out the uterine horn. Place the string of embryos into ice cold sterile PBS under a stereo microscope, and peel off the uterine muscle, yolk sac, and amnion cover from each embryo.
As each embryo is isolated, use a perforated spoon to transfer the tissues into a new Petri dish containing sterile PBS on ice. To harvest the embryonic heart tissue, transfer the first embryo to be dissected into a new Petri dish under the microscope, and place the embryo in ventral side up. Use fine forceps to make a small incision in the chest, slightly above the diaphragm, and insert the closed forceps into the incision to enlarge the incision.
Using the forceps to hold the chest wall open to expose the heart and lungs, use a second pair of forceps to gently move the heart anteriorly 90 degrees to expose the dorsal aorta and vein. Then, grasping these vessels at the base of the heart, carefully pull out the heart and lungs anteriorly and rinse the tissues with cold PBS. When all of the cardiac tissue has been harvested, place the heart samples under the microscope and remove the lung lobes from each heart.
Orient the first heart on its dorsal side, and holding the heart at its base, use fine forceps to scrape the left and right atria in the adjacent tissue surrounding the SV from the heart. To isolate the SV, orient the heart dorsal side up. Carefully remove the SV from the heart and use a sterile transfer pipette to place the isolated tissue into a new six centimeter Petri dish containing ice cold sterile PBS on ice.
To isolate the whole ventricles, remove the outflow tract and use a new sterile transfer pipette to place the ventricles into a separate six centimeter Petri dish containing ice cold sterile PBS on ice. To set up SV and whole ventricle cultures, first coat the membranes of one eight micrometer pore PET culture insert per well of a 24 well culture plate, with 100 microliters of freshly diluted extracellular matrix per culture for at least 30 minutes at 37 degrees Celsius. When the matrix has solidified, use a transfer pipette to transfer one explant onto each insert, and move the plate under the microscope.
Using clean forceps, position the explants at the center of each insert to ensure that they are not attached to the side walls, and carefully remove any extra PBS. Next, transfer the plate with the lid closed under a laminar flow tissue culture hood, and add 100 microliters of pre-warmed complete medium to each insert, and 200 microliters to the well below each insert. Then add 300 microliters of PBS to any unused wells, and place the plate into the cell culture incubator for five days.
For VEGF-A treatment on day two of culture, remove the medium from both chambers of each culture and add 100 microliters of PBS to each insert, and 200 microliters of PBS to each well. After swirling the plate a few times, replace the PBS with 300 microliters of basal medium supplemented with 1%fetal bovine serum to start the cultures, and return the plate to the incubator. On day three after starvation, add 300 microliters of fresh basal medium plus serum to the control wells and basal medium plus 50 nanograms per milliliter of VEGF-A to the treatment wells, and return the plate to the cell culture incubator.
On the sixth day of culture, wash the chambers with PBS as demonstrated, and fix the cultures with 200 microliters of 4%paraformaldehyde in each well, and 100 microliters of fixative in each insert. After 20 minutes at 4 degrees Celsius with rocking, wash the cultures with 300 microliters of PBS for 10 minutes per wash with rocking at room temperature. After the last wash, add 300 microliters of primary antibody solution to the wells and inserts for an overnight culture at four degrees Celsius with rocking.
The next morning, wash the cultures 10 times in non-ionic surfactant in PBS with rocking, changing the wash solution every 10 minutes before labeling the cultures with 300 microliters of an appropriate fluorophore conjugated secondary antibody at four degrees Celsius overnight with rocking. The next day, wash the cultures 10 times in fresh PBS with non-ionic surfactant as demonstrated. After the last wash, use fine forceps to carefully peel the membrane from each insert and place the membranes onto individual glass microscope slides, explant side up.
Cover the samples with DAPI supplemented mounting medium in a cover slip, and seal the edges of the cover slips with clear nail polish. When the nail polish has dried, image the slides by confocal microscopy. As initial SV cells grow onto the heart ventricle, they stop producing venous markers such as Coup-TF2.
After five days of culture, SV endothelial cells sprout and migrate onto the membrane. As in the embryonic heart, Coup-TF2 expression is reduced as the vessels migrate away from the SV.Cultures stimulated with VEGF-A exhibit an increased growth of angiogenic sprouts both in density and length. Further SV cultures stimulated by VEGF-A demonstrate an almost three-fold increase in sprout length compared to controls, suggesting that endothelial sprouts from SV and endocardium respond to VEGF-A.
It is important not to lose the SV tissue while pulling out the heart and lungs, removing the atria, and cleaning the adjacent tissue surrounding the SV.Live imaging experiments can be performed to visualize coronary angiogenesis and to capture the details of cellular and molecular dynamics during the process. This technique is extremely useful for assessing potential molecular mechanisms of coronary angiogenesis and as a model system for studying angiogenesis in general.
