Cellular heterogeneity and stem cells of vascular endothelial cells in blood vessel formation and homeostasis: Insights from single-cell RNA sequencing

Abstract

Vascular endothelial cells (ECs) that constitute the inner surface of blood vessels are essential for new vessel formation and organ homeostasis. ECs display remarkable phenotypic heterogeneity across different organs and the vascular tree during angiogenesis and homeostasis. Recent advances in single cell RNA sequencing (scRNA-seq) technologies have allowed a new understanding of EC heterogeneity in both mice and humans. In particular, scRNA-seq has identified new molecular signatures for arterial, venous and capillary ECs in different organs, as well as previously unrecognized specialized EC subtypes, such as the aerocytes localized in the alveolar capillaries of the lung. scRNA-seq has also revealed the gene expression profiles of specialized tissue-resident EC subtypes that are capable of clonal expansion and contribute to adult angiogenesis, a process of new vessel formation from the pre-existing vasculature. These specialized tissue-resident ECs have been identified in various different mouse tissues, including aortic endothelium, liver, heart, lung, skin, skeletal muscle, retina, choroid, and brain. Transcription factors and signaling pathways have also been identified in the specialized tissue-resident ECs that control angiogenesis. Furthermore, scRNA-seq has also documented responses of ECs in diseases such as cancer, age-related macular degeneration, Alzheimer's disease, atherosclerosis, and myocardial infarction. These new findings revealed by scRNA-seq have the potential to provide new therapeutic targets for different diseases associated with blood vessels. In this article, we summarize recent advances in the understanding of the vascular endothelial cell heterogeneity and endothelial stem cells associated with angiogenesis and homeostasis in mice and humans, and we discuss future prospects for the application of scRNA-seq technology.

Keywords: CD157/Bst1, pathological angiogenesis; angiogenesis; clonal expansion; heterogeneity; single-cell RNA sequencing; vascular endothelial cells (VECs); vascular endothelial stem cells.

FIGURE 1

Vascular endothelial cell heterogeneity within the vascular tree. Structural and molecular heterogeneity of vascular endothelial cells (ECs) within the vascular tree, including artery, arteriole, arterial capillary, capillary, venous capillary, post-capillary venule, and vein (i.e., intra-tissue heterogeneity).

FIGURE 2

Overview of single-cell RNA sequencing. (A) Isolation of target organ. (B) Creating single cell suspension by enzymatic digestion. (C) Flow cytometric sorting of CD31-positive CD45-negative vascular endothelial cells. (D) Single cell RNA sequencing of the single cell suspension. (E) Bioinformatic analysis of the single cell transcriptomes. (F) Validation of results obtained with single cell RNA sequencing by various approaches. Created with BioRender.com.

FIGURE 3

Vascular endothelial cell heterogeneity in the lung. (A) Vascular endothelial cells (ECs) in the lung can be classified into artery, vein, and capillary ECs based on their structure and molecular differences. (B) Alveolar capillary ECs are further classified into aerocytes and general capillary ECs.

FIGURE 4

Vascular endothelial cell heterogeneity in the kidney. Vascular endothelial cells (ECs) in the kidney show diverse heterogeneity based on their structure, location, complex function, and molecular signature.

FIGURE 5

Tip cell markers. Single-cell RNA sequencing has identified various different tip cell markers in the brain and retina.

FIGURE 6

Vascular endothelial cell heterogeneity in the liver. Vascular endothelial cells (ECs) in the liver show diverse heterogeneity. Liver sinusoidal ECs (LSECs) can be classified into periportal, midlobular, and pericentral ECs.

FIGURE 7

Schematic illustration of angiogenesis (i.e., new blood-vessel formation) from vascular endothelial stem cells (VESCs) by clonal expansion. (A) VESCs reside within the intima of the blood vessel. Mouse VESCs strongly express CD157 (Bst1), Myc, ATF3, Fosl2, Sox7, Foxp1, ABCB1a, and ABCG2. (B) Angiogenic stimuli activates VESCs following vascular injury, ischemia, and inflammation. (C) New sprouting occurs from nearby VESCs to form new blood vessels.

FIGURE 8

Vascular endothelial cell heterogeneity in pathological angiogenesis. Vascular endothelial heterogeneity exists within pathological vessels seen in (A) tumor (cancer) and (B) choroidal neovascularization (age-related macular degeneration). Potential therapeutic strategy to target endothelial cells responsible for pathological angiogenesis (C).

FIGURE 9

Future perspectives of single-cell RNA sequencing. Single-cell RNA sequencing is expected to elucidate vascular biological mechanisms and find new therapeutic targets.