Single-Cell and Spatial Transcriptomic Analyses Reveals the Dynamic Transcript Profiles of Myocardial Lymphangiogenesis post Myocardial Infarction

Reviewer #1 (Public Review):

Summary:

Assessment of cardiac LEC transcriptomes post-MI may yield new targets to improve lymphatic function. scRNAseq is a valid approach as cardiac LECs are rare compared to blood vessel endothelial cells.

Strengths:

Extensive bioinformatics approaches employed by the group.

Weaknesses:

Too few cells are included in scRNAseq data set and the spatial transcriptomics data that was exploited has little relevance, or rather specificity, for cardiac lymphatics. This study seems more like a collection of preliminary transcriptomic data than a conclusive scientific report to help advance the field.

Reviewer #2 (Public Review):

Summary:

This study integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points post-MI. They identified four transcriptionally distinct subtypes of lymphatic endothelial cells and localized them in space. They observed that LECs subgroups are localized in different zones of infarcted heart with functions. Specifically, they demonstrated that LEC ca III may be involved in directly regulating myocardial injuries in the infarcted zone concerning metabolic stress, while LEC ca II may be related to the rapid immune inflammatory responses of the border zone in the early stage of MI. LEC ca I and LEC collection mainly participate in regulating myocardial tissue edema resolution in the middle and late stages post-MI. Finally, cell trajectory and Cell-Chat analyses further identified that LECs may regulate myocardial edema through Aqp1, and likely affect macrophage infiltration through the galectin9-CD44 pathway. The authors concluded that their study revealed the dynamic transcriptional heterogeneity distribution of LECs in different regions of the infarcted heart and that LECs formed different functional subgroups that may exert different bioeffects in myocardial tissue post-MI.

Strengths:

The study addresses a significant clinical challenge, and the results are of great translational value. All experiments were carefully performed, and their data support the conclusion.

Weaknesses:

(1) Language expression must be improved. Many incomplete sentences exist throughout the manuscript. A few examples: Lines 70-71: In order to further elucidate the effects and regulatory mechanisms of the lymphatic vessels in the repair process of myocardial injury following MI. Lines 71-73: This study, integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points after MI from publicly available data (E-MTAB-7895, GSE214611) in the ArrayExpress and gene expression omnibus (GEO) databases. Line 88-89: Since the membrane protein LYVE1 can present lymphatic vessel morphology more clearly than PROX1.

(2) The type of animal models (i.e., permeant MI or MI plus reperfusion) included in ArrayExpress and gene expression omnibus (GEO) databases must be clearly defined as these two models may have completely different effects on lymphatic vessel development during post-MI remodeling.

(3) Lines 119-120: Caution must be taken regarding Cav1 as a lymphocyte marker because Cav1 is expressed in all endothelial cells, not limited to LEC.

(4) Figure 1 legend needs to be improved. RZ, BZ, and IZ need to be labeled in all IF images. Day 0 images suggest that RZ is the tissue section from the right ventricle. Was RZ for all other time points sampled from the right ventricular tissue section?

(5) The discussion section needs to be improved and better focused on the findings from the current study.

Reviewer #3 (Public Review):

Summary:

It has been demonstrated that cardiac lymphatics are essential for cardiac health and function. Moreover, post-myocardial infarction, targeting lymphatics by stimulating lymphangiogenesis has been shown to improve cardiac inflammation, fibrosis, and function. Then, the aim of this study was to evaluate the transcriptomic changes of cardiac lymphatic endothelial cells (LECs) after a myocardial infarction, which could reveal new therapeutic targets targeting lymphatic function. Moreover, investigating the cell-cell communication between lymphatic and immune cells would give critical information for a better understanding of the disease.

Strengths:

The use of scRNAseq data to evaluate LECs is an effective strategy considering the small proportion of LECs compared to blood endothelial cells. The extensive bioinformatic analysis used by the authors for three different data sets.

Weaknesses:

Among a total of 44,860 cells, only 242 LECs and 5,688 endothelial cells were identified. This small number of LECs is not representative and is insufficient to reliably distinguish four different clusters. The bioinformatic analysis is not supported by significant results in their in vivo and in vitro experiments.