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 10×-100μm and 40×-25μm;(B)Semi-quantitative fluorescence determination of Lymphatic vessel area ratio under 100-fold magnification by ImageJ(n=5).

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 10×-100μm, 40×-25μm. (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.