Endocardium-to-coronary artery differentiation during heart development and regeneration involves sequential roles of Bmp2 and Cxcl12/Cxcr4

Abstract

Endocardial cells lining the heart lumen are coronary vessel progenitors during embryogenesis. Re-igniting this developmental process in adults could regenerate blood vessels lost during cardiac injury, but this requires additional knowledge of molecular mechanisms. Here, we use mouse genetics and scRNA-seq to identify regulators of endocardial angiogenesis and precisely assess the role of CXCL12/CXCR4 signaling. Time-specific lineage tracing demonstrated that endocardial cells differentiated into coronary endothelial cells primarily at mid-gestation. A new mouse line reporting CXCR4 activity-along with cell-specific gene deletions-demonstrated it was specifically required for artery morphogenesis rather than angiogenesis. Integrating scRNA-seq data of endocardial-derived coronary vessels from mid- and late-gestation identified a Bmp2-expressing transitioning population specific to mid-gestation. Bmp2 stimulated endocardial angiogenesis in vitro and in injured neonatal mouse hearts. Our data demonstrate how understanding the molecular mechanisms underlying endocardial angiogenesis can identify new potential therapeutic targets promoting revascularization of the injured heart.

Keywords: BMP2 and Cxcl12/Cxc4 signaling; coronary vessels; endocardium; heart regeneration.

Figure 1.. Endocardial-derived coronary vessels arose early in development, first in the septum then inner free walls.

A) Experimental strategy for B-E. B) Whole-mount dorsal view of E11.5 BmxCreERT2;Rosa^tdTomato lineage-labeled heart. Solid line demarcates SV-derived ECs. Dotted line indicates section in B’. B’) Box highlights septum and first endo-derived CVs (arrowheads). C) Quantification from n=4 heart tissue sections. D) FISH labels endo (Npr3) and CV ECs (Cldn5). Arrows and arrowheads indicate IB4+; tdTomato+ endo and endo-derived CV ECs, respectively. E) Confocal images of BmxCreERT2;Rosa^tdTomato hearts. Arrowheads indicate endo-derived CVs on the ventral and dorsal sides. F) Quantification from n=6 whole-mount hearts. G) Experimental strategy for H and I. H) Whole-mount images of BmxCreERT2;Rosa^tdTomato hearts immunostained for Vegfr2 and the arterial marker Cx40. Boxed regions highlight a reduction of endo-derived CVs (tdTomato+, white cells) from early vs. later Cre inductions. I) Quantification where each symbol indicates an individual heart. J) Experimental strategy for K and L. K) Representative transverse heart sections. Boxed regions show endo contributions to septal and left ventricle (lv) CVs (tdTomato+, red cells). Dotted lines indicate border between inner (IM) and outer myocardium (OM). L) Quantification from tissues sections: E10.5, n = 6 hearts; E13.5, n = 4 hearts; E17.5, n = 4 hearts. Scale bar=200 μM in B, B’, D, E, H and K (full view). Scale bar=50 μM in B’, D, E, H and K (boxed regions). Data are mean ± s.d. ***P < 0.001, ****, P < 0.0001, by Student’s t-test.

Figure 2.. Cxcl12-Cxcr4 signaling was critical for artery formation rather than general endothelial migration.

A) Immunostaining of lineage-traced heart section. Arrowheads indicate endo-derived CV expressing Cxcr4. Endocardium (endo) lacks Cxcr4. B) In situ hybridization of Cxcl12 (blue) in E14.5 WT heart section. C and D) Whole-mount ventral views (C) and quantification (D) of BmxCreERT2;Rosa26tdTomato;Cxcr4^f/+ (controls, n = 4 hearts) and BmxCreERT2;Rosa26tdTomato;Cxcr4^//r (mutants, n = 3 hearts) with early endocardial deletion of Cxcr4. E and F) Whole-mount views (E) and quantification (F) of arteries in the hearts in C. G-J) Myocardial deletion of Cxc12 recapitulates phenotype seen in C-F. cTnTCre;CxcU2^f/+ are controls with n= 6 hearts and cTnTCre;Cxcl12^f/f are mutants with n = 6 hearts. K-M) Later endocardial deletion of Cxcr4 does not affect arterial development. Scale bar=200 μM in A, B, C, E, G, I, K and L (full view). Scale bar=50 μM in A, C, and G (boxed regions). Data are mean ± s.d. **P ≤ 0.01, ****, p ≤ 0.0001, n.s., not significant, by Student’s t-test.

Figure 3.. A new Cxcr4 activity reporter identified that most receptor activation is in artery endothelial cells.

A and B) Schematic representation of Cxcr4-Tango mouse system. B) Immunofluorescence on heart tissue sections. Cxcr4 expression in CV ECs was observed with (arrowheads) and without (arrows) Tango activity. C) Ventral and right lateral (rlv) view of whole mount, lineage labeled hearts. TdTomato+ endo-derived CV ECs colocalizedwith Tango activation (orange arrowheads) primarily in arteries (dotted lines) rather than plexus. White arrows indicate Cxcr4 activity in non-ECs. D) Quantification from n=3 hearts per stage. E) Experimental scheme for scRNAseq in F-L. F) Cxcr4 deletion does not affect the proportions of EC subtypes. G) UMAP plots of Cxcr4^fl/+ and Cxcr4^f/:fl integrated datasets subsetted for CV ECs cells. H and I) Feature plots (H) and quantification (I) showing reduced number of Cxcr4 expressing cells in fl/fl sample. J) Subtype percentages were not different among genotypes. K) Top marker genes allow more-specific identification of clusters based on published data (see text). L) Heatmaps showing normalized expression values (z-score) of the differentially expressed genes (DEG) between genotypes in Art2 (top) and Cap1 (bottom). Scale bar=200 μM in B and C (full view). Scale bar=50 μM in B and C (boxed regions). Data are mean ± s.d. n.s., not significant, by Student’s t-test.

Figure 4.. ScRNAseq of E12 cardiac ECs showed upregulation of Bmp2 signaling during endo- to-CV transition.

A) Experimental scheme for scRNAseq in Figures 4 and 5. B) UMAP plot of EC clusters grouped into the 4 indicated subtypes. C. UMAP with tdTomato expression in cells sorted as either tdTomato positive or negative. Dotted line colors are cluster identities from B. Percentages of total endo cells in each category are indicated. D) Bar plots indicating tdTomato+ and tdTomato− cells in clusters and their order based on similarity (top tree). Violin plots show gene expression used for cluster identification. E) Visualization of the trajectory inferred using slingshot on a dimensionality reduction of the cells (top panel) and as a 2D graph (bottom panel). F) Gene expression profile of 3 selected markers along the 2D trajectory from E (x axis=cells). G) Volcano plot showing differentially expressed genes between CV2 vs E1-E4 + CV1 clusters (cut-off for log2FC is >|2|; cut-off for P value is 10e-6). H) Bmp2 expression is enriched in CVs transitioning from endo. I) Expression of ligand-receptors genes related to BMP signaling pathway. J) Chord plot showing all significant interactions associated with BMP signaling pathway.

Figure 5.. Combined E12 and E17.5 scRNAseq indicated the endo-to-CV transition and Bmp2 expression is largely restricted to the early time point.

A) UMAP plot of EC clusters from E17.5 grouped into 5 subtypes: Endo; Valve (VE); Coronary Capillary (Cap1-Cap4); Coronary Veins (CVein); Coronary Arteries (CArt). B) UMAP with tdTomato expression in cells sorted as either tdTomato positive or negative. Dotted line colors are cluster identities from A. Percentages of total capillaries in each category are indicated. C) Bar plots indicating tdTomato+ and tdTomato− cells in clusters and their order based on similarity (top tree). Violin plots show gene expression used for cluster identification. D and E) UMAP of E12/17.5 integrated dataset (left panel) and visualization of the trajectory as a 2D graph structure (right panel), colored according to clusters of origin (D) or developmental stage (E). F) UMAP of integrated dataset colored by expression of the 3 indicated genes. G) Gene expression profile of 3 markers along the 2D trajectory of integrated dataset (x axis= cells). H) Expression at E17.5 of genes related to Ligand-Receptors pairs of BMP (left) and CXCL (right) signaling pathways. Bmp2 is down, and Cxcl12 is up. I) Chord plot showing all the significant interactions (L-R pairs) associated with CXCL signaling pathway.

Figure 6.. Bmp2 potentiates endo-derived CV angiogenesis in culture and neonatal hearts.

A) Experimental strategy for B and C. B and C) Immunostaining (B) and quantification (C) showed combining Vegfa and Bmp2 stimulates endo-derived CVs sprouting (arrowheads). (rBmp2, n = 5 explants; Vegfa, n = 6 explants; combination, n = 6 explants) D) Whole-mount ventral view of heart treated with Bmp signaling inhibitor (+ LDN 193189) or PBS (control). Magnifications highlight septal region where endo-derived CV ECs are sprouting. Dotted lines demarcate endocardium (endo) from endo-derived CV ECs within septum. E) Quantification of experiment in D showing the total number of lineage-labeled cells in the ventral septum and endo compartments (n = 4 Control; n =5 +LDN 193185). F) Experimental strategy for G and H. G) P18 tissue sections from lineage-labeled and injured hearts. Boxed regions show increased endo-derived CVs (arrowheads) in the left ventricles injected with AAV expressing Bmp2 injected hearts (n = 9 hearts, lower panels) compared with vehicle-treated hearts (n=8, upper panels). H) Quantification of experiment in G. Scale bar=200 pM in B, D and E (full view). Scale bar=50 pM in B, D and E (boxed regions). Data are mean ± s.d. ***P ≤ 0.001, ****, P ≤ 0.0001, by Student’s t-test.

Figure 7.. Model for sequential Bmp2-Cxcr4 signaling driving endo-to-CV ECs transition and artery formation during heart development and regeneration.

A) Endo cells (green) differentiate into CV ECs (light red) within the developing septum. CV ECs expressing Bmp2 signal to adjacent endo (dotted arrow) to favor endo-to-CV ECs transition. As development precedes, septal endo-derived CV ECs migrate ventrally into myocardial free wall. Cardiomyocytes expressing Cxcl8 guide a subset of Cxcr4+ endo-derived CV ECs (red) o form arteries. B) Early deletion of Cxcr4 in endo or deletion of myocardial Cxcl12 disrupts endo-derived artery formation in the ventral side of the heart. C) Exogenous administration of Bmp2 greatly enhances endo-to-CV ECs transition in subendocardial space in neonatal mice subjected to experimental MI.