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An important goal of developmental biology is to understand how millions of cells unite to produce a complex organ. For this process to be successful, the cells must take on proper fates at the appropriate times, in the correct proportions, and organize themselves so that the organ functions effectively. We are using the cardiovascular system as a model to understand the rules of these developmental events. Our laboratory is specifically focused on how the coronary vasculature of the heart is constructed from start to finish (Fig. 1). We have established a three-dimensional, single-cell resolution map of coronary vessel development and created a novel tool for genetically manipulating these structures in vivo. This allows us to study coronary development at an unprecedented level of detail and has already led to important discoveries and contributions about how this organ system is assembled during embryogenesis (1-4)(Volz et al., 2015).
Coronary vessel development
Early in development, the heart lacks its own vascular bed. However, as the ventricular wall grows beyond the oxygen diffusion limit, nearby endothelial cells are induced to sprout and form the coronary vasculature. Most sprouting occurs from structures call the sinus venosus (the venous inflow vessel to the heart) and endocardium (cells that line the heart lumen)(Fig. 1Bi and Bii)(2-5). Endothelial sprouts extend and branch, much like roots of a plant, eventually covering the entire heart and filling the cardiac muscle layer, or myocardium. This sprouting angiogenesis establishes an immature coronary plexus comprised of a network of similarly sized vessels that, at this stage, are not yet connected to the circulation. Blood flow is initiated once the plexus invades and connects specifically with the aorta, a large artery exiting the heart (Fig. 1Bii). Subsequent remodeling occurs to transform the plexus into a mature vascular bed with large coronary arteries and veins interconnected by capillaries. Surprisingly little is known about each of these developmental events. We hope that understanding coronary vessel formation will provide deeper understanding for organ-specific developmental pathways and inform cardiovascular regenerative medicine.  
Figure 1
Figure 1. Coronary vessel development. (A) Blood vessels are tubes lined by a polarized endothelial cell layer through which blood flows. Arteries are further covered by a smooth muscle cell layer and adventitial fibroblasts. (B) Schematic sagittal sections of the developing heart as it acquires a coronary blood circulation (altered from Red-Horse et al., 2010). (i) Sinus venosus (sv) sprouts migrate toward the heart ventricle. (ii) Sv and endocardial sprouts travel beneath the epicardial layer (sub epi) and into the myocardium (intra myo) to form the coronary plexus. Plexus vessels connect with the aorta (ao) to established blood flow. (iii) Subsequently, plexus remodeling and maturation occurs. Numbers indicate steps currently being investigated in our laboratory.
Defining progenitor cell contributions to the coronary vasculature
Previous work from our lab and others identified the sinus venosus and endocardium as coronary vessel endothelial cell progenitors in the developing mammalian heart (Fig 1B)(4,5). In addition, the epicardium was shown to contribute to a minor portion of the coronary endothelium (7). Together, these studies elicited significant questions for the field regarding which of the three progenitors is the primary source of coronary vessels, whether each source develops according to similar or distinct molecular programs, and why different pathways exist at all. To address these questions, we created an ApjCreER mouse line to allow us to lineage-trace the entire sinus venosus-derived coronary population. We used this tool to map sinus venosus-derived vessels onto the whole heart and compare this population to the whole heart lineage pattern from endocardial and epicardial progenitors. The data showed a striking compartmentalization to coronary vessel development where complementary regions were populated by different progenitor sources (Fig. 2). Sinus venosus-derived vessels contributed to a large number of arteries, capillaries, and veins on the dorsal (back) and lateral sides of the heart. In contrast, coronary vessels in the central portion of the ventral (front) side and ventricular septum were derived primarily from the endocardium. These data demonstrate that coronary vessels arise largely from the sinus venosus and endocardium at opposite sides of the heart—a spatial mechanism that may have evolved as the fastest way to vascularize the myocardium during a period of rapid growth and expansion.
Given the prominent roles of the sinus venosus and endocardial pathways, we next sought to determine whether the two sources are triggered to sprout onto the heart in response to similar or different signals. We discovered that VEGF-C is critical for the migration of sinus venosus sprouts over the heart (Fig. 1Bi, circled 1)(2). VEGF-C is an endothelial growth factor best described for its role in lymphatic vessel development. However, our data reveal for the first time that VEGF-C can also function as a tissue-specific mediator of blood vessel development. In contrast, VEGF-A was reported to be important specifically for endocardial-derived coronary vessels (5). Together, these findings indicate that the sinus venosus and endocardium populate complementary regions in response to distinct molecular signals. Our research continues to delineate the mechanisms utilized by the two progenitor populations (Fig. 1B) and explores how they could be utilized to induce new coronary vessels in the context of heart disease. 
Figure 2
Figure 2. The sinus venosus and endocardium give rise to complementary regions of the coronary vasculature. (A) Whole mount hearts subjected to the indicated lineage tracing experiments. Lineage-labeled coronary vessels are green; Dach-1 (red) marks coronary vessels. (B) Quantification of the percent of coronary vessels from different sources. Scale bars, 100 μm.
Targeting coronary arteries to the aorta
A critical event in the development of coronary vessels is their proper attachment to the aorta (Fig. 1Bii, circled 2). This process results in coronary arteries that stem from the aorta (carrying oxygenated blood) but not from the adjacent pulmonary artery (carrying deoxygenated blood postnatally). Although this targeting event has long fascinated cardiovascular biologists, the underlying patterning mechanisms are poorly understood. Our work has shown that VEGF-C and cardiomyocytes collaborate to guide proper placement of the coronary artery stem (1). VEGF-C is widely expressed in the outflow arteries (aorta and pulmonary artery), and its deficiency inhibits angiogenesis in the region, resulting in delayed and abnormally positioned coronary artery stems. VEGF-C induces vessel growth to both outflow vessels, but we found that cardiomyocytes, the muscle cells of the heart, develop specifically on the aorta at prospective stem sites. Islet-1 is a gene required for cardiomyocyte progenitor proliferation, and we discovered that Islet-1 heterozygous mice have decreased numbers of aortic cardiomyocytes, a phenotype that was also associated with delayed and abnormally low coronary artery stems. Furthermore, in hearts with outflow tract rotation defects, misplaced stems remain associated with shifted aortic cardiomyocytes, and myocardium can induce ectopic connections with the pulmonary artery in culture. Our data support a model where coronary artery stem development first requires VEGF-C to stimulate vessel growth around the aorta. Then, aortic cardiomyocytes act as chaperone cells that facilitate interactions between coronary vessels and the aorta (Fig. 3). Studying this new niche for cardiomyocyte development, and its relationship with coronary arteries, should reveal how chaperone cells are used to pattern development and may lead to new methods for stimulating vascular regrowth as a treatment for cardiovascular disease. Ongoing experiments are aimed at identifying the molecules that inhibit vessel growth around most of the aorta and pulmonary artery (Fig. 3A, yellow zone) and exactly how cardiomyocytes alleviate this inhibition at the stem sites (Fig. 3B and C).
Figure 3
Figure 3. Model for the role of VEGF-C and aortic cardiomyocytes in coronary artery stem formation. (A) VEGF-C stimulates vascular growth near the outflow tract while a vessel-free zone (yellow) directly surrounds the aorta (ao) and pulmonary artery (pa). (B) Coronary vessels (red lines) develop around the outflow tract, but do not invade the vessel-free zone. Wild-type Isl1 expression levels in the embryo allow cardiomyocytes to differentiate specifically in the aortic wall where they support vessel growth and facilitate connections between coronary vessels and luminal endothelium. (C) The result is a correctly positioned coronary artery stem (ca) on the aorta.
Selected References
  1. Volz KS, Jacobs AH, Chen HI, Poduri A, McKay AS, Riordan DP, Kofler N, Kitajewski J, Weissman I, Red-Horse K. (2015). Pericytes are progenitors for coronary artery smooth muscle. Elife;2015 Oct 19; doi: 10.7554/eLife.10036. Epub 2015 Oct 19. PMID: 26479710.
  2. Chen HI, Poduri A, Numi  H, Kivela R, Saharinen P, McKay, AS, Raftrey B, Churko J, Tian X, Zhou B, Wu JC,  Alitalo K,  Red-Horse K. (2014). VEGF-C and aortic cardiomyocytes guide coronary artery stem development. J Clin Invest, 2014 Nov 3;124(11):4899-914. doi: 10.1172/JCI77483. Epub 2014 Oct 1. PMID: 25271623.
  3. Chen HI, Sharma B, Akerberg BN, Numi HJ, Kivela R., Saharinen P, Aghajanian H, McKay AS, Bogard PE,  Chang AH,  Jacobs AH, Epstein JA, Stankunas K, Alitalo K,  Red-Horse K. (2014). The sinus venosus contributes to coronary vasculature through VEGFC-stimulated angiogenesis. Development, Dec;141(23):4500-12. doi: 10.1242/dev.113639. Epub 2014 Nov 5. PMID: 25377552.
  4. Greif DM, Kumar M, Lighthouse JK, Hum J, An A, Ding L, Red-Horse K, Espinoza FH, Olson L, Offermanns S, Krasnow MA. (2012). Radial construction of an arterial wall. Dev Cell, 23(3), 482–93. PMCID: PMC3500096
  5. Red-Horse K, Ueno H, Weissman IL, Krasnow MA. (2010). Coronary arteries form by developmental reprogramming of venous cells. Nature, 464(7288), 549–53. PMCID: PMC2924433
  6. Wu B,  Zhang Z, Lui W, Chen, X, Wang Y, Chamberlain AA, Moreno-Rodriguez RA, Markwald RR,  O'Rourke BP, Sharp, DJ, Zheng D, Lenz J, Baldwin HS, Chang CP,  Zhou, B. (2012). Endocardial cells form the coronary arteries by angiogenesis through myocardial-endocardial VEGF signaling. Cell, 151(5), 1083–96. PMCID: PMC3508471
  7. Tian X, Hu T, Zhang H, He L, Huang X, Liu Q, Yu W,  He L, Yang Z,  Zhang Z, Zhong TP, Yang X, Yang Z, Yan Y, Baldini A, Sun Y, Lu J,  Schwartz RJ,  Evans SM, Gittenberger-de Groot AC,  Red-Horse K, Zhou, B. (2013). Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries. Cell Res., 23(9), 1075–90. PMCID: PMC3760626 
  8. Katz TC, Singh MK, Degenhardt K, Rivera-Feliciano J, Johnson RL, Epstein JA, Tabin CJ. (2012). Distinct compartments of the proepicardial organ give rise to coronary vascular endothelial cells. Dev Cell, 22(3), 639–50. PMCID: PMC3306604


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