Abstract
New coronary vessels are added to the heart around birth to support postnatal cardiac growth. Here we show that, in late fetal development, the embryonic coronary plexus at the inner myocardium of the ventricles expresses the angiogenic signalling factors VEGFR3 and DLL4 and generates new coronary vessels in neonates. Contrary to a previous model in which the formation of new coronary vessels in neonates from ventricular endocardial cells was proposed, we find that late fetal and neonatal ventricular endocardial cells lack angiogenic potential and do not contribute to new coronary vessels. Instead, we show using lineage-tracing as well as gain- and loss-of-function experiments that the pre-existing embryonic coronary plexus at the inner myocardium undergoes angiogenic expansion through the DLL4–NOTCH1 signalling pathway to vascularize the expanding myocardium. We also show that the pre-existing coronary plexus revascularizes the regenerating neonatal heart through a similar mechanism. These findings provide a different model of neonatal coronary angiogenesis and regeneration, potentially informing cardiovascular medicine.
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Data availability
Additional images supporting the findings reported in the representative images in Figs. 1–8 have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.15047106). All raw data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.
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Acknowledgements
We thank R. H. Adams for sharing the BmxcreER mouse line and K. Red-Horse for sharing the ApjcreER mouse line in this study. This work was support by the American Heart Association (AHA-17POST33410599, to P.L.) and The National Heart, Lung, and Blood Institute (NHLBI) of the United States (HL133120, to B.Z.). R.P.H. received salary support from the National Health and Medical Research Council (NHMRC) of Australia (1118576) and the Victor Chang Cardiac Research Institute.
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Contributions
P.L., R.P.H. and B.Z. conceived the concept. P.L. designed and generated conditional knockin mice. P.L., Yidong Wang, Yifeng Wang and B.W. conducted experiments. P.L., Yidong Wang, Y.L., B.W., D.Z., R.P.H. and B.Z. analysed the data. P.L., R.P.H. and B.Z. wrote the manuscript. B.Z. supervised experiments and provided grant support.
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Peer review information Nature Cell Biology thanks Kristy Red-Horse and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Generation of an inducible Npr3CreERT2 mouse line for fate mapping of perinatal ventricular endocardial cells.
a, RNAscope ISH and immunofluorescence images showing Npr3 transcripts (red) and EMCN (green) in ventricular endocardial cells of P0 hearts. The images are representative of three individual hearts. tb, trabecular myocardium; com, compact myocardium; pm, papillary muscle. b, Schematic showing the strategy for generating a tamoxifen-inducible Npr3CreERT2 mouse line for fate mapping of perinatal ventricular endocardial cells. c, Co-immunofluorescence images showing expression of estrogen receptor (ER, indicative of CreERT2, red) and EMCN (green) in ventricular endocardium (en), but not coronary plexus, of E17.5 Npr3CreERT2 heart. The images are representative of four individual hearts. d, Co-immunofluorescence showing comparable expression level and pattern of NPR3 between wild-type and Npr3CreERT2 neonatal heart at P0. Some small red peppered background signals are associated with the NRP3 antibody staining. Arrows indicate coronary artery. The images are representative of 4 individual hearts. e, RT-qPCR analysis indicating comparable expression of Npr3 transcripts between wild type and Npr3CreERT2 perinatal heart at E17.5 (n=5 hearts for each group) and P0 (n=4 hearts for each group). Data are presented as mean ± SEM and two-tailed unpaired Student’s t-test. All samples were derived from biologically independent experiments. Scale bar: 500 µm in a, c, d.
Extended Data Fig. 2 Npr3CreERT2-labelled progenitor cells generate the embryonic coronary vessels.
a, Co-immunofluorescence for PECAM1 (red) and GFP (green) showing the presence of the progenies from the Npr3+ cells labelled by tamoxifen induction at E8.5 (iE8.5) in ventricular endocardium, atrial myocardium and sinoatrial myocardium including sinoatrial valve (arrows) of E10.5 heart. b, Co-immunofluorescence for PECAM1 (red) and GFP (green) showing the presence of the progenies from the Npr3+ cells labelled at E8.5 (iE8.5) in ventricular endocardium and coronary plexus at the left and right (to a lesser degree) ventricular wall as well as interventricular septum of E16.5 heart. Dashed line separates trabeculae (tb) from compact myocardium (cm). Note that the Npr3+ progenitors labelled at E8.5 also contributes to atrial myocardium, epicardium (arrow), and cardiac valves of E16.5 heart. a, atrium. v, ventricle. ep, epicardium. sv, sinus venosus. ra/la, right/left atrium. rv/lv, right/left ventricle. ivs, interventricular septum. All samples were derived from biologically independent experiments. The images in a and b are representative of four individual hearts for each staining. Scale bar: 100 µm in a and 200 µm in b.
Extended Data Fig. 3 Effective and specific labelling of perinatal ventricular endocardial cells in Npr3CreERT2.
a,b, Co-immunofluorescence for PECAM1 (red) and GFP (green) showing that Npr3CreERT2 activated by tamoxifen at P0 (iP0) and E17.5 (iE17.5) effectively drives GFP expression specifically in ventricular endocardium at P2 and P0, respectively. Quantitative analysis indicating 85-90% of ventricular endocardial cells (VEC) were marked by GFP at either stage, two days after tamoxifen indication. n=5 hearts for a and b. Data are presented as mean ± SEM and two-tailed unpaired Student’s t-test. Scale bar: 500 µm in a and b.
Extended Data Fig. 4 Perinatal ventricular endocardial cells do not generate coronary vessels.
a, Quantitative immunofluorescence studies showing rare, if any, contribution of Npr3 lineage-positive ventricular endocardial cells (VEC, green) to PECAM1-positive coronary endothelial cells (CEC, red) at P7. Note that ~90% of ventricular endocardial cells were labelled by GFP after tamoxifen induction at P0 (iP0). n=5 hearts, p<0.0001. Data are presented as mean ± SEM and two-tailed unpaired Student’s t-test. b and c, Immunofluorescence images showing co-localized expression of GFP and PECAM1 or NPR3 in ventricular endocardial cells, but not coronary endothelial cells, in P7 hearts after tamoxifen induction at E17.5 (iE17.5), two days before birth. All samples were derived from biologically independent experiments. The images in b and c are representative of four hearts for each staining. Scale bar: 500 µm in a-c.
Extended Data Fig. 5 VEGFR3-expressing angiogenic cells are highly proliferative.
a,b, Quantitative EdU assays show increased number of EdU (white) incorporation among ERG-expressing coronary endothelial cells expressing a high level of VEGFR3 (red, arrows), compared to those expressing a low level of VEGFR3. en, endocardium; ep, epicardium. n=5 hearts, p<0.0001. Data are presented as mean ± SEM and two-tailed unpaired Student’s t-test. Scale bar: 100 µm.
Extended Data Fig. 6 Opposing gradients of gene expression in inner versus outer preexisting coronary vessels, and generation of Dll4+ lineage tracing mice.
a,b, Multicolor RNAscope for Dll4 (red) or Vegfr3 (red) with Aplnr (green) showing that Dll4 or Vegfr3 expressing angiogenic cells are mainly localized to the inner myocardium, whereas Aplnr-expressing cells are present in the outer myocardium at E16.5. c,d, RNAscope for Dll4, Vegfr3 or Aplnr (red) and co-immunofluorescence for EMCN (green) showing that Dll4 or Vegfr3 expressing angiogenic cells are mainly localized in the inner myocardium expressing low-to-negative levels of EMCN, whilst Aplnr-expressing cells are present in the outer myocardium expressing high levels of EMCN in the perinatal heart between E17.5 and P0. ca: coronary artery. Scale bar: 500 µm. e,f, Strategy for generating an inducible Dll4CreERT lineage-tracing mouse line and lineage tracing workflow. All samples were derived from biologically independent experiments. The images in a, b, c, and d are representative of three hearts in each experiment. Scale bar: 500 µm in a-d.
Extended Data Fig. 7 Lineage tracing of preexisting AplnrCreER cells.
a,b, Lineage tracing analysis showing that AplnrCreER lineage-positive cells (GFP, green) labelled at P0 (iP0) and expressing PECAM1 (red) are located in the outer myocardium in P2 hearts (a), whereas VEGFR3-expressing angiogenic cells (red) are mainly present in the inner myocardium and negative for GFP (b). pm, papillary muscle. All samples were derived from biologically independent experiments. The images in a and b are representative of six hearts in each experiment. Scale bar: 500 µm.
Extended Data Fig. 8 Regional expansion of preexisting coronary vessels in inner and outer ventricular wall after birth.
a,b, Co-immunofluorescence for PECAM1 (red) and GFP (green) showing dominant contribution of Dll4 and Aplnr lineage-positive cells (green) labelled by tamoxifen indication at P0 (iP0) to inner and outer coronary plexus, respectively, of P7 heart. Dashed line separates inner from outer myocardium. All samples were derived from biologically independent experiments. The images in a and b are representative of six hearts for each staining. Scale bar: 500 µm.
Extended Data Fig. 9 Time course analysis of VEGFR3 expression in postnatal coronary vessels.
a,b, Immunofluorescence images showing downregulation of VEGFR3 expression (red) in the Dll4 lineage-positive coronary endothelial cells (GFP, green) labelled at P0 between P7 and P14. All samples were derived from biologically independent experiments. The images in a and b are representative of three hearts for each stage. Scale bars: 500 µm.
Extended Data Fig. 10 DLL4-NOTCH1 signalling is required for neonatal angiogenic expansion of the preexisting coronary vasculature.
a,b, EdU assays showing the increased number of VEGFR3-expressing angiogenic cells (red) incorporating EdU (green) in P5 hearts after endothelial-specific Dll4 deletion at P2. n=5 hearts, p<0.0001. Data are presented as mean ± SEM and two-tailed unpaired Student’s t-test. Scale bar: 100 µm. c,d, Immunofluorescence images and quantification showing significantly decreased density of PECAM1-expressing cells (red) in the inner myocardium resulting from overexpression of NICD. n=5 hearts for each group, p<0.0001. Data are presented as mean ± SEM and two-tailed unpaired Student’s t-test. Scale bar: 100 µm in a, 500 µm in c.
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Lu, P., Wang, Y., Liu, Y. et al. Perinatal angiogenesis from pre-existing coronary vessels via DLL4–NOTCH1 signalling. Nat Cell Biol 23, 967–977 (2021). https://doi.org/10.1038/s41556-021-00747-1
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DOI: https://doi.org/10.1038/s41556-021-00747-1
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