Pericytes and postnatal alveolarization: Role of hypoxia inducible factors

During alveolarization, the final stage of lung development, angiogenesis drives the exponential, postnatal increase in gas-exchange surface area. Pericytes play a dual role in angiogenesis. Initially, pericytes stimulate and guide endothelial cells (EC) during early microvascular growth and subsequently constrain EC proliferation and migration during vessel stabilization. However, the molecular mechanisms that regulate pericyte phenotype to drive these distinct roles in vascular growth and stability remain poorly defined. Addressing this gap may motivate the development of therapies to treat neonatal lung diseases marked by compromised angiogenesis, including bronchopulmonary dysplasia. O2-sensitive transcription factors, termed hypoxia-inducible factors (HIF) are central regulators of angiogenesis. Our preliminary data suggest that HIF activity in select subsets of lung mesenchymal cells (MC) is required for postnatal angiogenesis and alveolarization. Genetic gain- and loss-of- function studies using Tagln promoter driven Cre-recombinase (a gene expressed by multiple MC including pericytes), demonstrated that Tagln-specific HIF stabilization preserved pulmonary vascular and alveolar growth in hyperoxia. Conversely, Tagln-specific Hif-1a deletion impaired pulmonary angiogenesis and alveolarization even in normoxia. However, the specific Tagln-expressing MC responsible was not identified. Our single cell transcriptomic studies point to a unique, hyperoxia-sensitive, developmental role for pericytes and HIF signaling in pericytes during postnatal lung development. These studies identified: (i) marked changes in the transcriptome of early (P7) versus late (P21) pericytes with blood vessel morphogenesis as the most enriched biologic pathway; (ii) a peak in both proliferating pericytes and microvascular EC at P7; (iii) persistent Hif-1a, -2a, and HIF downstream target expression in pericytes, including Rgs5, a gene that marks activated, angiogenic pericytes, and Lgals1, a gene encoding a secreted, pro-angiogenic, carbohydrate-binding protein; and (iv) hyperoxia-induced loss of ~90% of pericytes, all proliferating pericytes, and suppressed HIF-dependent, pericyte-EC interactions. These observations suggest the overall working hypothesis that HIF-mediated alterations in pericyte phenotype during postnatal development modulate the pulmonary angiogenesis that drives alveolarization. that will be tested in 3 specific aims. Aim 1 will use genetic mouse models, primary pericyte cultures, and ChIP-Seq to define the developmental role of HIF in pericytes on lung vascular growth. Aim 2 will use primary pericyte and EC co-cultures and loss of function strategies to determine if developmental regulation of select HIF-regulated targets in lung pericytes modulate pericyte phenotype and EC angiogenic function. Aim 3 will use HIF reporter mice, deep scRNA-Seq and genetic lineage tracing to determine if hyperoxia suppresses pericyte HIF-signaling, and disrupts pericytes fate and ontogeny during late lung development. By focusing on the lung pericyte, at single cell resolution, these studies will provide insight into pulmonary vascular development and identify strategies to promote lung growth and regeneration.