183 - Single Cell RNA Sequencing Reveals a Selective Induction of Venous Endothelial Cell Proliferation in Acute Hyperoxia and Durable Alterations in Venous Endothelial Cell Phenotype Upon Recovery.

Pulmonary angiogenesis drives distal lung growth. Disrupted angiogenesis impairs alveolarization and can lead to pathologic  vascular remodeling. Recent studies have shown that venous endothelial cells (VEC) are an important pool of vascular progenitors that contribute to both developmental and pathologic angiogenesis in other organs. However, the role of VEC in pulmonary vascular development and disease remains poorly defined.

Objective:

We hypothesized that single cell RNA-sequencing (sc-RNA-Seq) would identify alterations in the pulmonary VEC transcriptome during development, and elucidate how hyperoxia, an injury that impairs alveolarization and angiogenesis, alters VEC phenotype.

Design/Methods:

We performed deep, plate-based sc-RNA-Seq to profile pulmonary EC from E18.5 to P21 at baseline (normoxia), from P7 mice exposed to hyperoxia from birth (acute hyperoxia), and P21 mice maintained in hyperoxia until P14 and then recovered in air (hyperoxia recovery). We harmonized our data with an adult lung single cell dataset, and quantified proliferating VEC using multiplexed fluorescent in situ hybridization (ISH).

Results: Unsupervised clustering of neonatal and adult data identified VEC clusters in both groups. However,  immature VEC embedded far from their adult counterparts, highlighting a distinct transcriptome. Exploration of differentially expressed genes identified Peg3, a paternally imprinted gene that marks self-renewing progenitors, as highly expressed in immature VEC but minimally expressed in adult VEC (206 counts per million (cpm) vs. 0.06 cpm). Although acute hyperoxia decreased capillary EC proliferation, it induced a novel cluster of proliferating VEC, and ISH confirmed a 10-fold increase in proliferating VEC in hyperoxia-exposed P7 mice. This effect was associated with a more than 2-fold increase in Peg3 expression. Upon hyperoxia recovery, a subset of VEC maintained more than 10-fold higher expression of Peg3 than P21 controls (369 cpm vs. 14 cpm). A number of additional genes exhibited durable changes in recovery, including a down-regulation of the transcription factor (TF) Klf2, a TF that decreases vascular tone in quiescent vessels, and an increase in Edn1, a potent vasoconstrictor.

Conclusion(s):

These data highlight the developmental evolution and sensitivity to injury of immature pulmonary VEC.  Our results suggest that VEC may be key modulators of the vascular response to hyperoxia, and speculate that alterations in VEC proliferation and vascular tone may contribute to the pathologic vascular remodeling present in a subset of patients with disrupted alveolarization.