466 - Phenotypic Diversity and Sensitivity to Injury of the Pulmonary Endothelium During a Period of Rapid Postnatal Growth

Poster Number: 466
Publication Number: 466.432

Fabio Zanini, UNSW Sydney, Sydney, New South Wales, Australia; Xibing Che, Stanford University School of Medicine, Palo Alto, CA, United States; Carsten Knutsen, Lucile Packard Children's Hospital Stanford, Palo Alto, CA, United States; Min Liu, Stanford University School of Medicine, Stanford, CA, United States; Nina E. Suresh, Stanford University School of Medicine, Redwood City, CA, United States; David N. Cornfield, Stanford University, Stanford, CA, United States; Cristina M. Alvira, Stanford University School of Medicine, Stanford, CA, United States

Background: At birth, marked changes in pulmonary endothelial cell (EC) function drive blood from the placenta to the lungs and expand the microvasculature to promote alveolarization. As alveolarization ends, the EC switch from an angiogenic to quiescent phenotype, however the mechanisms regulating this transition remain elusive.

Objective: We hypothesized that single cell RNA-sequencing would identify distinct pulmonary EC with unique roles in angiogenesis and remodeling during postnatal development.

Design/Methods: We isolated 3000 pulmonary EC from mice at E18.5, P1, P7 and P21, and from P7 mice exposed to hyperoxia from birth. Single EC were sequenced at a depth of 1 million reads per cell. EC populations and genes of interest were validated with fluorescent in situ hybridization.

Results: Unsupervised clustering identified 9 unique EC clusters, and a marked increase in EC diversity after birth. Harmonization with data from the adult showed that perinatal lung EC are transcriptionally distinct from adult counterparts, and include a unique arterial subtype enriched for genes regulating vascular growth and patterning. Birth heightened microvascular EC (MEC) diversity, with a single embryonic MEC cluster replaced postnatally by 3 clusters: aerocytes (Car4+ MEC), “early” Car4- MEC comprised of EC from P1 and P7, and “late” Car4- MEC comprised of EC from P21. “Early” Car4- MEC highly expressed Peg3, a transcription factor that marks self-renewing progenitor cells. Across postnatal development, pathways promoting proliferation, migration and immunity were up-regulated, followed by global down-regulation of gene expression as the EC transitioned to quiescence.  Highly proliferative EC were prevalent at E18.5, and peaked again at P7, corresponding to a time of rapid pulmonary angiogenesis. Hyperoxia induced both common gene signatures across all EC, as well as unique alterations in aerocytes, Car4- MEC, and proliferative EC. Hyperoxia also induced a 7-fold (p < 0.0001) increase in proliferating venous EC, cells recently described to play a key role in developmental angiogenesis.  Conclusion(s): Our data reveal tremendous diversity of pulmonary EC during a critical window of postnatal vascular development, and distinction from mature pulmonary EC. Hyperoxia generally disrupts the transcriptome of EC, but induces specific alterations in key subsets, including a previously unreported increase in venous EC proliferation. Our results provide a detailed molecular map of mechanisms regulating EC activation and quiescence, with broad implications for lung diseases marked by aberrant vascular growth and remodeling.