Effect of pressure-overload on cardiac endothelial cells in Balb/c mice

We performed single-cell RNA-seq analysis of FACS-sorted cardiac blood vascular and lymphatic endothelial cells in the heart of Balb/c female mice in a model of pressure-overload. Overall design: Cardiac CD31+ CD45neg endothelial cells, and Lyve1/Podoplanin-expressing lymphatic endothelial cells (LEC) were extracted from hearts of mice 8 weeks after pressure-overload or sham surgery in a murine model of transaortic constriction, and analyzed by single-cell RNA-sequencing. Briefly, cardiac single cell suspensions were sorted by FACS (ARIA II, BD Biosciences). BECs were defined as live CD45-/CD31+/Lyve1- cells, whereas LECs were identified as CD45-/CD31+/Lyve1+/Pdpn+ cells. A 1:3 mix of cardiac LECs and BECs were included in each sample prepared using the 10X Genomics Chromium Next Gem Single Cell 3' kit. For each group 1-2 reactions were performed using each time pooled cells from 10 mouse hearts. Samples were sequenced using Illumina NextSeq550 at an estimated sequencing depth of 20 000 reads per cell. Reads were aligned to the mouse reference genome (GRCm39.111) using STARsolo RNA-seq aligner (v2.7) to produce raw UMI count matrix. Data filtering, normalization, integration, principal component-based unsupervised clustering, and differential analysis were performed using the Seurat package (version 5.0.2) in R (version 4.1.2). For low-quality data filtering, genes expressed by <10 cells, and cells expressing <500 genes, were excluded, as were cells with UMI numbers <500 or cells with >10% mitochondria-derived UMI counts. Among 3908 total sequenced cells, 3436 cardiac cells remained post-filtering, including 1932 cells from sham-operated BALB/c controls and 1504 from post-TAC mice.