Current Research Projects
Developmental Diversity of Pulmonary Endothelial Cell Phenotype During Postnatal Lung Growth
Postnatal lung growth during alveolarization markedly increases gas exchange surface area. Active growth of the pulmonary vasculature during early alveolarization drives distal lung growth. However, as alveolarization slows, the vasculature transitions from a phase of angiogenic growth to quiescence, however the molecular mechanisms regulating this transition remain poorly defined. This gap in knowledge confounds efforts to develop targeted therapies to treat diseases of dysregulated angiogenesis and impaired alveolarization, including bronchopulmonary dysplasia, the most common complication of preterm birth. We recently employed single cell transcriptomics to define endothelial cell (EC) diversity during postnatal lung development and to identify novel mechanisms regulating pulmonary angiogenesis and quiescence.
Our preliminary data identified a tremendous increase in EC diversity after birth, marked by the appearance of numerous transcriptionally distinct clusters. The microvascular EC (MEC) broadly separated into Car4 expressing (Car4+) and Car4- MEC. In contrast with gradual changes in gene expression in the Car4+ MEC over time, gene expression changed dramatically in the Car4- MEC, with separation of this population into two transcriptionally distinct clusters of “early” (Postnatal day 1-7) and “late” (Postnatal day 21) Car4- MEC. High expression of the paternally imprinted gene-3 (Peg3), a gene expressed by self-renewing progenitor cells, distinguished the “early” from the “late” Car4- MEC. Peg3 also enhances NFkB signaling, a pathway we previously identified as essential for pulmonary angiogenesis during early alveolarization. Our new project aims to determine if the early Car4- MEC represent a specialized, highly proliferative and angiogenic EC population required for the rapid growth of the pulmonary vasculature during early alveolarization.
Homeostatic Roles for Distinct Macrophages in the Developing Lung Vasculature
Rapid growth of the pulmonary vasculature by angiogenesis during early alveolarization drives distal lung growth, and disrupted angiogenesis impairs alveolarization. In other organs, specialized macrophages support angiogenesis by promoting blood vessel formation, providing survival and migratory cues to EC, and facilitating vascular anastomoses. However, the role of macrophages in the developing pulmonary vasculature remains entirely unknown. Recently, we embarked on a project employing single cell RNA-sequencing to define macrophage diversity during late embryonic and early postnatal lung development. Macrophages are extremely heterogenous with diverse phenotypes that are lineage- and tissue-specific, and highly influenced by the microenvironment. Our preliminary data found a tremendous increase in macrophage diversity after birth. Specialized, highly proliferative macrophages present before birth are replaced after birth by a complex and dynamic mixture of diverse macrophage subtypes exhibiting unique gene signatures, developmental gradients in gene expression, and specific locations within the lung suggesting distinct functions in tissue remodeling, angiogenesis, and immunity. Interestingly, a subset of embryonic macrophages was found to completely encircle small arterioles and express numerous genes that regulate lung branching, angiogenesis, and EC phenotype. After birth, these cells transitioned to an intermediate subset present only during the first few weeks of postnatal life that expressed additional tissue remodeling genes. Taken together, our data suggest that distinct macrophage populations support alveolarization by regulating pulmonary vascular development through the expression of factors that influence vascular growth and remodeling.
Our current work combines multiplex in situ hybridization, lineage tracing, studies in primary EC and macrophages, and advanced imaging in transgenic and knock-out mice to define the role of specific macrophage subsets in modulating EC phenotype and regulating lung parenchymal and vascular growth. In addition, we will utilize conditional knock out mouse models, and ligand-receptor profiling of single cell datasets from pulmonary EC and macrophages to probe pathways mediating macrophage-EC communication. Finally, we are interested in defining whether chronic hyperoxia alters diversity and phenotype of the lung macrophages during acute injury and after recovery, and specifically impairs developmental and homeostatic functions of lung macrophages.
The Role of IKK-beta in Mediating Pulmonary Endothelial Angiogenesis
The transcription factor, nuclear factor kappa- B (NFκB) plays a key role in cell proliferation, survival, and inflammation. NFκB is also a key regulator of angiogenesis in wound healing and cancer. While extensive evidence has implicated activation of NFκB in the pathogenesis of lung disease, until recently, a role for NFκB in lung development had not been previously described. Prior work from the Alvira Lab has shown that NFκB is constitutively active in the early alveolar pulmonary endothelium, and that systemic administration of a pharmacologic inhibitor of the NFκB activating kinases, IKKa and IKKβ, durably disrupts alveolarization and impairs primary pulmonary endothelial cells (PEC) survival, proliferation, and migration.
However, definitive evidence confirming a role for IKKb-mediated NFκB activation in lung angiogenesis and alveolarization has been hindered by the embryonic lethality of mice containing constitutive and conditional deletions of key NFκB family members. Moreover, whether IKKa or IKKβ is the primary activating kinase mediating the pro-angiogenic functions of NFκB remains debated. Therefore, in this application we plan to utilize a novel mouse model, recently created in the Alvira lab, containing an inducible, endothelial cell (EC)-specific deletion of IKKβ to determine if EC-specific deletion of IKKβ impairs pulmonary angiogenesis and alveolarization in vivo, and assess whether these IKKβ-dependent effects are mediated via VEGFR2.
How the Lung Microenvironment Alters the Pulmonary Endothelial Angiogenic Phenotype
In contrast to many other organs which complete their development during early embryonic development, a significant component of lung development occurs postnatally during alveolarization, the final stage of lung development that likely extends through the first decade of life. During alveolarization, the gas exchange surface area of the lung increases 35-fold and the pulmonary capillary network by 20-fold. Growth of the pulmonary vascular network by angiogenesis during early alveolarization is an essential component of this process, and impaired angiogenesis disrupts alveolar development, resulting in large, simplified alveoli and decreased surface area to allow efficient gas exchange.
The molecular mechanisms that allow for rapid pulmonary vascular growth during this discrete window of time are not know. Our prior work has shown that primary endothelial cells derived from the early alveolar lung are much more angiogenic than endothelial cells derived from the adult lung. Interestingly, angiogenic signaling pathways can be induced, and angiogenic function enhanced in adult pulmonary endothelial cells exposed to secreted factors found in the lung microenvironment. Current studies in our laboratory are using proteomic and transcriptomic approaches to identify novel angiogenic factors present in the early alveolar lung microenvironment.
Identifying Novel Targets to Treat Preterm Birth
Premature birth is the primary worldwide cause of morbidity and mortality in newborns and now the top cause of child mortality among those younger than 5 years. Chronic lung disease of infancy (bronchopulmonary dysplasia remains the most common complication of premature birth. Economic consequences of prematurity include lengthy neonatal hospitalizations, costs of special education, maternal health care, and diminished work productivity of adult survivors of prematurity. To date, there are no therapeutic strategies that reliably prevent preterm labor and delivery.
During pregnancy, the uterus must first remain quiescent to allow growth of the developing fetus, and then powerfully contract at birth to deliver the infant. Though myometrial quiescence and activation are central to mammalian life, efforts to prevent premature birth have been confounded by an incomplete understanding of the fundamental biology of myometrial contractility.
We recently identified the transient receptor potential vanilloi-4 (TRPV4) channel as a previously unrecognized modulator of myometrial contractility, and a potential new drug target to address preterm labor. Current studies are underway to better understand how TRPV4 expression and activity are regulated in the pregnant and non-pregnant myometrium, and to develop strategies to specifically target TRPV4 inhibitors to the myometrium as a new therapeutic strategy to treat preterm labor.