Defining the role of pulmonary endothelial cell heterogeneity in the response to acute lung injury

Abstract

47Pulmonary endothelial cells (ECs) are an essential component of the gas exchange 48 machinery of the lung alveolus. Despite this, the extent and function of lung EC heterogeneity 49 remains incompletely understood. Using single-cell analytics, we identify multiple EC 50 populations in the mouse lung, including macrovascular endothelium (maEC), microvascular 51 endothelium (miECs), and a new population we have termed Car4-high ECs. Car4-high ECs 52 express a unique gene signature, and ligand-receptor analysis indicates they are primed to 53 receive reparative signals from alveolar type I cells. After acute lung injury, they are 54 preferentially localized in regenerating regions of the alveolus. Influenza infection reveals the 55 emergence of a population of highly proliferative ECs that likely arise from multiple miEC 56 populations and contribute to alveolar revascularization after injury. These studies map EC 57 heterogeneity in the adult lung and characterize the response of novel EC subpopulations 58 required for tissue regeneration after acute lung injury. 59 Significance 60Using transcriptional profiling of the pulmonary vascular endothelium and confirmation at 61 the RNA and protein levels, we have revealed extensive EC heterogeneity throughout the 62 vasculature of the lungs. We show that a subpopulation of endothelium re-enters the cell cycle 63 and proliferates in response to acute injury, whereas another subpopulation is enriched in 64 vasculogenic gene expression. These data provide foundational information regarding the 65 biological complexity of lung ECs, which will contribute to the development of novel tools to 66 enhance regeneration of the lung following injury. 67 whether such EC subpopulations contribute to in vivo tissue homeostasis and response to injury 93 in the adult lung remains unknown. 94To address these questions and to define pulmonary EC heterogeneity at homeostasis 95 and during regeneration, we utilized single cell RNA sequencing (scRNA-seq) analysis of the 96 adult mouse lung, both uninjured and after acute influenza-induced viral injury. In addition to 97 identifying microvascular (miEC) and both arterial and venous macrovascular (maEC) 98 populations, we identified a new population we have termed Car4-high ECs that possess a 99 unique transcriptome. Car4-high ECs express high levels of Car4 and Cd34, are found 100 throughout the lung periphery at homeostasis, and are primed to respond to Vegfa signaling 101 based on their high expression of Vegf receptors, which corresponds to a receptor-ligand 102 interaction analysis between Car4-high ECs and AT1 cells, their epithelial co-partners in gas 103 exchange. Car4-high ECs are enriched in the regenerating zones surrounding the most 104 damaged regions of the lung following influenza-or bleomycin-induced lung injury during the 105 subsequent tissue regeneration process. Influenza injury revealed the emergence of a unique 106 population of highly proliferative ECs, which are closely related to Car4-low miECs in gene 107 expression ...

Deciphering cell–cell interactions and communication from gene expression

“…To date, this approach has been applied to brain 31 , 32 , heart 33 , 34 , kidney 35 – 37 , liver 38 , 39 , lungs 40 – 43 , placenta 29 , 30 , 44 , retina 45 and visual cortex 46 . Remarkably, this approach revealed new roles of cells within a tissue 41 and helped explain how ageing 32 , 47 , diseases 34 – 36 , 48 – 51 , infections 52 and injuries 31 , 40 , 43 shape multicellular organization. Intercellular crosstalk has been investigated in healthy and diseased liver 50 , kidneys during homeostasis and rejected kidney transplants 35 , 36 , heart during failure and recovery conditions 34 , and healthy and asthmatic lungs 51 .…”

“…Several studies have incorporated imaging to spatially map cells and integrated transcriptomics to measure CCC. For example, researchers interrogated the communication and response of lung cells to tissue injury 40 , 43 ; spatial information aided the identification of a new population of lung endothelial cells ( Car4 high) and elucidated how these cells communicate with neighbouring alveolar type 1 cells through VEGF signalling given both their proximity and their expression of the cognate genes 43 . Spatial maps have also helped investigate the communication of T cells during their development in the human thymus 13 .…”

Single cell transcriptomic profiling identifies molecular phenotypes of newborn human lung cells

“…The epithelial cell population was not large enough to separate known distinct lineages. Among the other cellular populations, the extent of endothelial cell diversity (e.g., large vs. small vessel), has been well documented [40].…”

Mitigating Coronavirus-Induced Acute Respiratory Distress Syndrome by Radiotherapy

“…Teijaro et al have revealed that blocking S1P(1) receptor that is expressed on both endothelial cells and lymphocytes in the lung can suppress CS with reduced mortality in mice infected with human influenza virus (Teijaro et al, 2011). In addition, the lung microvasculature is shown to contain heterogenic endothelial subpopulations with various vasculogenic capacities coordinately contributing to lung injury response (Niethamer et al, 2020;Stevens et al, 2008). Notably, proliferation of the lung endothelial cell subpopulation is enhanced during influenza-mediated lung injury (Basil et al, 2020;Niethamer et al, 2020).…”

“…In addition, the lung microvasculature is shown to contain heterogenic endothelial subpopulations with various vasculogenic capacities coordinately contributing to lung injury response (Niethamer et al, 2020;Stevens et al, 2008). Notably, proliferation of the lung endothelial cell subpopulation is enhanced during influenza-mediated lung injury (Basil et al, 2020;Niethamer et al, 2020). On the other hand, cutaneous low-dose radiation has been shown to increase tissue vascularity through upregulation of angiogenic and vasculogenic pathways (Thanik et al, 2010).…”