Dynamic Changes of Cardiac Lymphatics in Cardiac Tissue post-MI.

(A) Hematoxylin-eosin(H&E) stained and immunofluorescence(IF) stained tissue sections in sham and MI models in C57BL/6J mice(n=5∼7), LYVE1, lymphatic vessel endothelial hyaluronan receptor 1; WGA: wheat germ agglutinin; DAPI: 4’6-diamidino-2-phenylindole. The scale bar is in 10X:100μm, 40X:20μm. (B)Semi-quantitative fluorescence determination of specific regions using ImageJ (n=5 equally sized measurement areas/zone; mean±SEM).

Identificationg of infiltrated cell types in tissues.

(A) A Uniform Manifold Approximation and Projection (UMAP) plot of 44,860 cells 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 present work colored by endothelial cell types. (E) A UMAP plot of single cells profiled in the present 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 colour indicate the percentage of marker gene expression and the average expression level (Avg. Expression).

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

(A and B) Bar plots showing the proportion of cell types in different samples. (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, the color indicates the 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) The 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 showing the proportion of LEC 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 gene in LECs at each time point. (B) Hematoxylin-eosin(H&E) stained tissue section in sham and MI models of C57 mice(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 of 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, and F) Aqp1 RNA expression in mLEC cell line under CoCl2 hypoxic stimulation over different time points and concentrations. (G, H, I, and J) Aqp1 protein expression in mLEC cell line under CoCl2 hypoxic stimulation over different time points and concentrations.

Possibility of LECs Affecting the Infiltration of Macrophages through the Galectin9-CD44 Pathway.

(A and B) Expression of the gene Lgals9 and CD44 in LECs at each time point; (C) Gal-9 RNA expression in mLEC cell line under IFN-γ stimulation over different concentrations; (D and E) Gal-9 RNA expression in mLEC cell line under IFN-γ stimulation over different concentrations. (F) Mouse monocyte macrophage(J774A.1) wound healing assay under Gal-9 stimulation over different concentrations. (G and H) J774A.1 macrophage cultured in Transwell system under Gal-9 stimulation over different concentrations. (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) co-cultured 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-labeled collecting lymphatics with the lymphatic valves highlighted with white arrows. (E) IF-stained tissue section of the cardiac lymphatics in the remote zone

(A) The proportion of LEC cell types in each spot was determined via spatial transcriptome analysis in 1 h post-MI.

(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.