VEGF-C and aortic cardiomyocytes guide coronary artery stem development

Abstract

Coronary arteries (CAs) stem from the aorta at 2 highly stereotyped locations, deviations from which can cause myocardial ischemia and death. CA stems form during embryogenesis when peritruncal blood vessels encircle the cardiac outflow tract and invade the aorta, but the underlying patterning mechanisms are poorly understood. Here, using murine models, we demonstrated that VEGF-C-deficient hearts have severely hypoplastic peritruncal vessels, resulting in delayed and abnormally positioned CA stems. We observed that VEGF-C is widely expressed in the outflow tract, while cardiomyocytes develop specifically within the aorta at stem sites where they surround maturing CAs in both mouse and human hearts. Mice heterozygous for islet 1 (Isl1) exhibited decreased aortic cardiomyocytes and abnormally low CA stems. In hearts with outflow tract rotation defects, misplaced stems were associated with shifted aortic cardiomyocytes, and myocardium induced ectopic connections with the pulmonary artery in culture. These data support a model in which CA stem development first requires VEGF-C to stimulate vessel growth around the outflow tract. Then, aortic cardiomyocytes facilitate interactions between peritruncal vessels and the aorta. Derangement of either step can lead to mispatterned CA stems. Studying this niche for cardiomyocyte development, and its relationship with CAs, has the potential to identify methods for stimulating vascular regrowth as a treatment for cardiovascular disease.

Figure 9. Proposed model for the role of VEGF-C and aortic cardiomyocytes in CA stem formation.

(A) VEGF-C stimulates vascular growth near the outflow tract, while a vessel-free zone directly surrounds the aorta and pulmonary artery. (B) Peritruncal vessels 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 peritruncal vessels and lumen endothelium. (C) The result is a correctly positioned CA stem (ca) on the aorta.

Figure 8. Ectopic vessel connections with the pulmonary artery lumen can form in the presence of cardiomyocytes.

(A–C) Confocal images of pulmonary artery outflow tract explants cultured alone or adjacent to other tissues. Endothelial cells are labeled in blue (VE-cadherin⁺), and cardiomyocytes are labeled in red (cTnT⁺). Schematics summarizing experimental setup and results are shown. (A) Pulmonary artery explants retain a luminal endothelial layer that does not sprout into the vessel-free zone (dotted line). (B) Endothelial cells within lung tissue do not connect with the pulmonary artery lumen but do migrate into the myocardial (myo) region at its base (arrow), which contains cardiomyocytes. (C) When cardiomyocytes from ventricular myocardium (ven myo) are placed alongside artery explants, ventricular coronary vessels frequently form connections (arrowhead) with the pulmonary artery endothelium. (D) Percentage of explant samples that did or did not form connections with the pulmonary artery lumen when cultured beside lung or ventricular myocardium. Scale bars: 100 μm (left and center panels in A–C); 25 μm (right panels in A–C).

Figure 7. Misplaced CA stems are correlated with aortic cardiomyocytes in hearts from Pax3-null embryos.

(A and B) The side-by-side positioning of the aorta and pulmonary artery in hearts from (B) Pax3-null embryos compared with (A) wild-type embryos. Blue lines indicate the locations of CA stems. (C–H) Confocal images of hearts immunofluorescently labeled for VE-cadherin (endothelium) and cTnT (cardiomyocytes). CA stems (arrowheads) associate with aortic cardiomyocytes (brackets) in wild-type and in Pax3 knockouts. CA stems are outlined in solid lines in D–F. Outflow tracts are outlined with dotted lines in C–F. Images in G and H are maximum projections of optical sections through the aortic lumen captured from the right lateral side of the heart. Scale bars: 100 μm.

Figure 6. ISL1 heterozygosity decreases aortic cardiomyocytes and disrupts normal stem development and positioning.

(A) Second heart field lineage tracing (Mef2c-AHF-Cre, Rosa^mTmG) labels aortic cardiomyocytes (CMs, arrows). (B) Confocal images of wild-type and Isl1 heterozygous embryos showing decreased numbers of cardiomyocytes within the aorta (dotted line). The pulmonary artery is also outlined (solid lines). Cardiomyocytes (cTnT⁺) are shown in red; peritruncal vessels (VE-cadherin⁺) are shown in blue. The bottom panel shows only the cTnT channel where red lines indicate the stem position. (C) Boxed regions in A showing only the cTnT channel. (D) Quantification of the length distal to the valve occupied by aortic cardiomyocytes shows a significant decrease in Isl1 mutant hearts. (E and F) Other developmental parameters, (E) smooth muscle cell development and (F) coronary vessel growth, are unchanged in mutant hearts. (D–F) Data represent mean ± SD. Each dot represents a value obtained from one sample. (G) Isl1 heterozygous hearts have abnormally low stem positioning with respect to the valves (lines) in comparison to wild-type hearts. (H) Distribution of stem phenotypes observed in Isl1 mutant and wild-type hearts. n values are shown on the right. (I) The height of CA stems does not significantly rise above the level of aortic cardiomyocytes in both wild-type and mutant hearts, as evidenced by the paucity of points in the pink area (y > x) of the graph. These data were measured at E14.5. A linear regression for the data set (dotted line, y = 0.5999x – 36.39) indicates a positive correlation. ****P < 0.0001; NS ≥ 0.05. Scale bars: 100 μm.

Figure 5. Aortic cardiomyocytes do not migrate up from the ventricle but differentiate in situ during explant culture.

Images taken from time-lapse movies of E12.5 Myh6-Cre, Rosa^mTmG heart cultures. Cardiomyocyte migration is not observed (arrows) at the junction of the ventricle and aorta. Instead, there is the appearance of a new cardiomyocyte (arrowheads), which seems to have differentiated in situ (images are shown at higher magnification in bottom panels). Scale bars: 100 μm (top panels); 20 μm (bottom panels).

Figure 4. Aorta-specific cardiomyocyte development precedes CA stem formation.

(A) Confocal image of the right lateral side of an E13.5 heart showing that cardiomyocytes (red, arrowheads) develop on the aorta distal to the valves but are absent in the analogous region of the pulmonary artery. The right coronary sinus (rcs) and noncoronary sinus (ncs) regions are indicated by square and curly brackets, respectively. (B) Quantification of cardiomyocytes within the aorta and pulmonary artery. (C) Quantification of cardiomyocytes among the different regions of the aorta. (D) Right and left lateral views of the aorta showing concentration of cardiomyocytes specifically around the right and left coronary sinuses (lcs) at the maturing stem sites (arrowheads). (E) Cardiomyocytes (arrowheads) are present in the aortic wall around the right and left coronary sinuses at E11.5 before stem formation. Aortic and pulmonary valve sinuses are schematized with dotted lines. (F) Cardiomyocytes directly contact the aortic endothelium (arrowhead) where aortic sprouts (sp) form — (G) but not the pulmonary artery (arrow), which never sprouts — as quantified in H. High-magnification views of the boxed regions are shown. (I) Developing CA stems (arrowhead) are closely associated with aortic cardiomyocytes. (J) Aortic cardiomyocytes are no longer present in adult hearts. (K) Developing human CA stems (arrowheads, dotted lines) are associated with aortic cardiomyocytes. OFT, outflow tract. All data represent mean ± SD. Each dot represents a value obtained from one sample. ****P < 0.0001; ***P = 0.0001 to 0.001; **P = 0.001 to 0.01. Scale bars: 100 μm (A, D, E, left panel in F, right panel in G, J, and K); 25 μm (right panel in F and left panel in G); 50 μm (I).

Figure 3. VEGF-C is required for peritruncal vessel growth and CA stem formation and patterning.

(A) Tissue sections through Vegfc–lacZ hearts showing expression (blue) in the aorta and pulmonary artery. (B) Boxed region in A showing expression throughout the vessel wall (brackets) and base of the outflow tract. (C) Schematic of VEGF-C expression (blue). (D) VEGFR2 is expressed in all cardiac endothelial cell types while VEGFR3 is expressed in coronary and ASV endothelium and in the endocardium (endo). Right panels are high-magnification views of the boxed regions and show expression in coronary vessels that share cell-cell junctions with endocardial cells (dotted lines). (E) Confocal images showing the absence of peritruncal vessels (VE-cadherin⁺, blue) around the outflow tract in VEGF-C–deficient hearts at E12.5 and E14.5. Cardiomyocytes are shown in red (cTnT). (F) Heterozygous (het) hearts have hypoplastic (hypo) peritruncal vessels. (G and H) CA stems (arrowheads) are present in wild-type but absent (arrows) or abnormal (arrowheads) in knockout (ko) hearts. The trough of each aortic valve sinus is traced with a dotted line for reference in comparison of stem location. (G) In many of the mutant hearts, sprouts connected to the endocardium (open arrowhead) appear to be extending up toward the aorta, even when other peritruncal vessels are absent. (I) Distribution of CA stem phenotypes in VEGF-C knockout embryos. n values are shown on the right. (J) Schematics of representative CA stems in VEGF-C–deficient outflow tracts. ao lum, aortic lumen; val, valve; ven, ventricle. Scale bars: 100 μm (A, B, left panel in D, and E–H); 50 μm (right panel in D).

Figure 2. The sinus venosus and endocardium both contribute to CA stems.

Tissue sections of hearts immunostained with endothelial markers (VE-cadherin or CD31) and a nuclear dye (DAPI). Lineage-traced cells (arrowheads) are present in CA stems from (A) Apj-CreER, Rosa^mTmG and (B) Nfatc1-CreER, Rosa^tdTomato embryos. Right panels are high-magnification views of the boxed regions. Scale bars: 100 μm.

Figure 1. CA stem development in mice.

(A) Schematics showing top and right lateral views of the heart, including CA stem locations (black arrows). (B) Confocal images of the right lateral side of hearts from the indicated embryonic days labeled for VE-cadherin. Coronary vessels (cv) and ASVs (arrowheads) grow toward the future right CA stem site (red asterisk) on the aorta (ao, dotted lines). (C) Tissue section through the aorta showing the presence of ASVs (red, arrowheads) directly beneath the epicardium (WT1⁺, green) and a vessel-free region around the aorta. The right panel is a high-magnification view of the boxed region. (D) Luminal endothelial cells lining the aorta, but not the pulmonary artery (pa), form sprouts (arrow). Inset is a high-magnification view of the boxed region. (E) Apelin-nlacZ expression (green, arrows) in VE-cadherin⁺ (red) aortic endothelium and adjacent sprouts. (F–I) Image projections of the CA stem site. (F) Small endothelial extensions from the aorta (arrow). (G) Initial interactions between coronary vessels and the aorta consist of thin, single-celled connections (arrowhead). (H) Multiple, lumenized connections that receive blood flow (arrowheads) develop at the future stem site and begin to acquire smooth muscle cell (SMC) coverage. (I) The mature pattern is a single, larger bore vessel stemming from the right coronary sinus (arrowheads) directly distal to the valves. (J) CA stem development. epi, epicardium; EC, endothelial cell; L atrium, left atrium; lca, left CA; ot, outflow tract; ra, right atrium; R atrium, right atrium; r ven, right ventricle; RCA, right CA. Scale bars: 100 μm (B–D); 50 μm (F–I); 25 μm (E, right panel in C, and inset in D).