Fig. 8: Impaired vascular response in hCD68–CreERT2;Lyve1^flox/flox infarcted hearts and a population shift toward inflammatory monocytes in Lyve1 KO CD45⁺ cells at day 7 after MI.

Identification of scar area at 7 dpi using wheat germ agglutinin (WGA) and Picrosirius red fibrosis stain. Visualization of vasculature and macrophages with CD31 (PECAM1) and IBA1, respectively. Representative images of littermate control (a) and hCD68–CreERT2;Lyve1^flox/flox (b) sections illustrating reduced neovascular response in Cre⁺ sections. Quantification of scar area revealed no significant difference between conditions (c). Quantification of discrete PECAM1 signal within the infarct zone relative to area as an indicator of vascular response revealed significantly reduced PECAM1-stained vasculature in Cre⁺ hearts (d). Quantification of macrophages by IBA1 stain revealed no significant difference between conditions (e). scRNA-seq was conducted, comparing CD45⁺ enriched cells from neonatal Lyve1 KO versus WT hearts at P2MI7dpi. The samples were FACS sorted using 7-AAD and CD45 to isolate live CD45⁺ cells, and libraries were prepared for sequencing using the 10x Genomics platform. UMAP plot of grouped WT and Lyve1 KO CD45⁺ cells (f). Comparison of CD45⁺ cell subset proportions between WT and Lyve1 KO conditions (g), including statistical analyses of quantified differences between subsets after deconvolution of individual heart samples by Vireo5 (ref. ³⁷) (h). Macrophage subset clustering in WT (i) and Lyve1 KO (j). Lyve1 gene expression within subclusters in WT (k) and Lyve1 KO (l). Dot plot illustrating relative expression of key pro-angiogenic, pro-inflammatory and pro-fibrotic genes in each macrophage subset and between WT and Lyve1 KO (m). Cumulative apoptotic marker expression scores between conditions and macrophage subsets (n). Representative LYVE1⁺ macrophage possessing an HA glycocalyx (o). Differential HA glycocalyx staining between WT and Lyve1^−/− macrophages (p). n = 4 control hearts, n = 3 hCD68–CreERT2;Lyve1^flox/flox hearts; four sections per heart for a–e, pooled samples from n = 5; five hearts for f–m. The box center in violin plots in n indicates the median; the lower and upper hinges correspond to the first and third quartiles; and the whiskers extend to values with a distance from the hinges that is at most the interquartile range multiplied by 1.5. Box plot parameters, including cell counts, are available in the Source Data. Unpaired Student’s t-tests were used to determine significance in c–e. Bonferroni-corrected pairwise Wilcoxon rank-sum test was used to determine significance in n. Qualitative observations in o and p were repeated across the scar and in a second infarcted heart. Scale bars, 200 μm for a, b and p; 50 μm for o. FDR, false discovery rate; HA, hyaluronic acid; macro, macrophage; NS, not significant; T_reg, regulatory T cell.

Source data