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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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

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 .......

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 .......

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.

F.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
100 x 20 MM Tissue Culture DishFisher Scientific877222Referred in the protocol as Petri dish
24-well platesFisher Scientific08-772-51
8.0 uM PET membrane culture insertsMillipore SigmaMCEP24H48
Alexa Fluor Donkey anti-rabbit 555Fisher ScientificA31572Secondary antibody
Alexa Fluor Donkey anti-rat 488Fisher ScientificA21206Secondary antibody
Angled Metal ProbeFine science tools10088-15Angled 45 degree, used for detecting deep plugs
Anti- ERG 1/2/3 antibodyAbcamAb92513Primary antibody
Anti- VE-Cadherin antibodyFisher ScientificBDB550548Primary antibody, manufacturer BD BioSciences
CO2 gas tankVarious suppliersN/A
CO2 IncubatorFisher Scientific13998223For 37 °C, 5% CO2 incubation
Dissection stereomicrosopeLeicaS9iLeica S9i Stereomicroscope
EBM-2 basal mediaLonzaCC-3156Endothelial cell growth basal media
ECM solutionCorning354230Commercially known as Matrigel
EGM-2 MV Singlequots KitLonzaCC-4147Microvascular endothelial cell supplement kit; This is mixed into the EBM-2 to make the EGM-2 complete media
Fetal Bovine Serum (FBS)Fisher ScientificSH3007003IR
FiJiNIHNAImage processing software (https://imagej.net/Fiji/Downloads)
Fine ForcepsFine science tools11412-11Used for embryo dissection
Fisherbrand Straight-Blade operating scissorsFisher Scientific13-808-4
Hyclone Phosphate Buffered Saline (1X)Fisher ScientificSH-302-5601LR
Laminar flow tissue culture hoodFisher Scientificvarious models available
Mounting MediumVector LaboratoriesH-1200Vectashield with DAPI
Paraformaldehyde (PFA)Electron Microscopy/Fisher50-980-494This is available at 32%; needs to be diluted to 4%
Perforated spoonFine science tools10370-18Useful in removing embryo/tissues from a solution
Recombinant Murine VEGF-A 165PeproTech450-32
Standard forceps, Dumont #5Fine science tools11251-30
Sure-Seal Mouse/Rat chamberEasysystemincEZ-1785Euthanasia chamber

References

  1. Red-Horse, K., et al. Coronary arteries form by developmental reprogramming of venous cells. Nature. 464 (7288), 549-553 (2010).
  2. Chen, H. I., et al. The sinus venosus contributes to coronary ....

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