Dynamic Changes of Cardiac Lymphatics in Cardiac Tissue after MI.

(A) Hematoxylin-eosin (H&E) staining and immunofluorescence (IF) co-staining with LYVE1 and PDPN of tissue sections in sham and MI mice model at d3, d7, d14, and d28 post-MI (n=5∼7/group). LYVE1: lymphatic vessel endothelial hyaluronan receptor 1; PDPN: Podoplanin; DAPI: 4’6-diamidino-2-phenylindole; IZ: Infarct Zone; BZ: Border Zone; the scale bars are in 10X-100μm and 40X-20μm; (B) Semi-quantitative fluorescence determination of specific regions using ImageJ(n=5 equally sized measurement areas/zone; mean±SEM).

Identifying infiltrated cell types in tissues.

(A). A Uniform Manifold Approximation and Projection (UMAP) plot of 44,860 showing five identified non-myocardium cell types in infarcted heart, cell types are coded with different colors; (C). A UMAP plot of single cells profiled in the presenting work colored by endothelial cell types; (E). A UMAP plot of single cells profiled in the presenting work colored by LEC cell types; (B, D, and F). Dot plot showing the average expression of selected cell marker genes. The dots’ size and color indicate the percentage of marker gene expression and the average expression level (Avg. Expression).

The Relationship Between the Spatial Localization and Function of LEC Subpopulations post MI.

(A) Bar plots showing total numbers and subtypes of LECs at different time points post-MI. The y-axis represents the percentage of the total number of LECs at d1, d3, d7, d14, and d28 post-MI, relative to the number of LECs at d0, which is used as the reference value set at 100%. (B) Bar plots showing total cell counts of each subcluster at different time points. (C) The proportion of LEC cell types in each spot determined by spatial transcriptome in d3 and d7 post-MI; (D) Enriched KEGG pathways of upregulated genes in different LEC cell types, color indicate different cell types.

Single-Cell Trajectories of LECs Subsets post MI.

(A, B, and C). Pseudo-time analysis by Monocle2 shows the potential evolutionary trajectory of LECs, colored by cell types; (D). Heatmap shows the set of genes with altered expression levels along the pseudo-time; (E) Enriched KEGG pathways in four different gene sets, color indicate different cell types; (F) Bar plots show the proportion of LECs subgroups at different time points.

Cell-cell interactions between LECs and immune cells.

(A) Interaction net count plot of LECs and immune cells. The thickness of the line represents the number of interactions. (B) Bubble plots showing the ligand-receptor pairs originating from LEC ca I and ca III that target monocytes and macrophages, the color indicates the probability of interaction and the size indicates the p-value. (C) Bubble plots showing the pathways originating from LEC ca I and LEC ca III that target monocytes and macrophages, the color indicates the probability of interaction and the size indicates the p-value.

Aqp1 expression on LECs in mouse MI model and on LEC cell line.

(A) Expression of Aqp1 in LECs at different time points post-MI from the Sc-RNA sequence data. Kruskal□Wallis analysis was performed to compare differences among groups; (B)Hematoxylin-eosin(H&E) stained tissue section in sham and MI modle in C57 mouse(n=5); (C)Quantification of the edema in infarcted hearts,(n=5); (D)H&E stained and immunofluorescence(IF) stained tissue section in sham and MI models in C57 mice(n=5∼7), LYVE1:lymphatic vessel endothelial hyaluronan receptor 1; AQP1: aquaporin1; DAPI: 4’6-diamidino-2-phenylindole; the scale bar is in 10X-100μm, 40X-20μm. (E,F)Aqp1 RNA expression in mLEC cell line under CoCl2 hypoxic stimulation over different time and different concentrations; (G,H,I,J) Aqp1 protein expression in mLEC cell line under CoCl2 hypoxic stimulation over different time pointss and concentrations.

LEC Affect Infiltration of Macrophages Through the Galectin9-CD44 Pathway.

(A and B) Expression of Lgals9 in LECs and CD44 in macrophages at different time points post-MI from the Sc-RNA sequence data. Kruskal□Wallis analysis was performed to compare differences among groups; (C) Gal-9 RNA expression in mLEC cell line under IFN-γ stimulation over different concentration; (D and E) Gal-9 RNA expression in mLEC cell line under IFN-γ stimulation in different concentration; (F) Mouse monocyte macrophage(J774A.1) wound healing assay under Gal-9 stimulation over different concentration; (G and H) J774A.1 macrophage cultured in Transwell system under Gal-9 stimulation over different concentration; (I, J, and K) Gal-9 RNA and protein expression after Lgals9 gene.silenced; (L and M)J774A.1(in upper cabinet) and LEC (in lower cabinet) cocultured in a transwell system under INF-γ stimulation with or without SiGal-9.

(A) Time points for heart harvest after the establishment of the MI models; (B) Transthoracic echocardiography confirmation of the MI mouse model; (C) Prox1 and Lyve1 labeled cardiac lymphatics in IF stained frozen tissue section; (D) LYVE1&PDPN labeled collecting lymphatics with the lymphatic valves highlighted with white arrows; (E) IF-stained tissue section of Cardiac lymphatics in remote zone; (F) CD68 and Pdpn co-stained the BZ in different time point.

(A) The proportion of LEC cell types in each spot was determined by spatial transcriptome in 1 h post MI(Because we could not download spatial transcriptome data for day d0 in the public database (GSE214611) or from the authors, we have used data of 1 h after IR as a reference for approximating the physiological state).

(A) A Uniform Manifold Approximation and Projection (UMAP) plot of 16373 cells showing seven identified cell types in immune cells, cell types have been coded with different colors. (B) A dot plot showing the average expression of the selected cell marker genes. Dot size and color indicate the percentage of marker gene expression and average expression level (Avg. Expression). (C) Bubble plots showing the pathway originating from LECs targeting immune cells; the color indicates the probability of interaction. (D) Bubble plots showing the ligand-receptor pairs originating from dendritic cells (DCs) targeting immune cells; the color indicates the probability of interaction and the size indicates the p-value. (E) Bubble plots showing the pathways originating from dendritic cells (DCs) targeting other immune cells; color indicates the probability of interaction. (F) Bubble plots showing the ligand-receptor pairs originating from dendritic cells (DCs) targeting other immune cells; the color indicates the probability of interaction and the size indicates the p-value.

(A) Bubble plots showing the pathways originating from the macrophages (Mφ) targeting other immune cells; the color indicates the probability of interaction. (B) Bubble plots showing the ligand-receptor pairs originating from Mφ targeting other immune cells; the color indicates the probability of interaction and the size indicates the p-value. (C) Bubble plots showing the pathway originating from B cell targeting other immune cells; the color indicates the probability of interaction. (D) Bubble plots showing the ligand-receptor pairs originating from B cells targeting other immune cells; the color indicates the probability of interaction and the size indicates the p-value.

(A) Correlation between Aqp1 expression intensity and edema score.; (B)Semi-quantification of AQP1 protein expression in LECs by measure AQP1&LYVE1 co-staining fluorescence intensity.

(A and B) Expression of Gal-9 protein of LEC under 1% oxygen concentration stimulation over different times; (C and D)Expression of Gal-9 protein of LEC under CoCl2 intervention over different concentration. By one-way ANOVA test, the protein expression of Gal-9 in LECs stimulated by hypoxia over different times or different concentrations of CoCl2 were all not statistically different from the control group(P>0.1).