Endothelial plasticity and signaling in development and regeneration of the lung

Division Of Basic Sciences - Nci

Investigators

Abstract

1. Established a functioning laboratory and trained and mentored fellows: The Niethamer laboratory opened in the Cancer and Developmental Biology Laboratory (CDBL) in the Center for Cancer Research (CCR), NCI, NIH, in January 2024. Since this time, I have set up a new laboratory space, including ordering equipment, re-establishing mouse lines after rederivation, cryo-recovery of several commercial mouse strains, and initiating a mouse breeding program to generate the animal models necessary for the laboratory's research projects. Several new fellows started in the laboratory late in FY2024, and I have spent time in FY2025 onboarding and training these individuals. I have also mentored the laboratory's first trainee, Arnav Sharma, through the application process to medical school. He will start at Central Michigan University in summer 2025. 2. Published work pertaining to lung development and regeneration: During his first months at NCI, post-baccalaureate fellow Arnav Sharma familiarized himself with the lung biology and vascular biology fields by writing a review article about specialized endothelial cells in lung development and disease. The review was published in the Annual Review of Physiology in February 2025. We hope this review will serve as a helpful reference for lung biologists, particularly those focused on the study of the lung endothelium, as it summarizes existing studies on lung endothelial heterogeneity in different developmental and disease contexts. In addition, Arnav and I completed revisions for a co-corresponding author manuscript that includes work completed during my postdoctoral fellowship at the University of Pennsylvania as well as work done in my laboratory at NCI. This manuscript details the longitudinal response of the lung to acute injury, spanning from homeostasis to one year post-injury, and was published in Cell Stem Cell in February 2025. 3. Developed an imaging system to visualize the gas exchange interface in the mouse lung at nanoscale resolution : To determine the role of endothelial cell subtypes in formation, maintenance, and repair of the gas exchange interface, we first needed to develop a high-resolution, three-dimensional imaging system capable of visualizing the interface between endothelial cells and the alveolar epithelium. Using mouse lines that specifically label capillary type 1 (CAP1) and capillary type 2 (CAP2) endothelial cells and alveolar type 1 epithelial cells, as well as fluorescent labeling techniques for the entire vascular tree, we are visualizing the gas exchange interface using confocal microscopy. In addition, in collaboration with Dr. Kedar Narayan at NCI, we are developing a volume electron microscopy strategy to visualize the gas exchange interface at nanoscale resolution in three dimensions. This will allow us to better understand the structure of the gas exchange interface and determine how it is perturbed and rebuilt after injury. 4. Developed a cell sorting strategy to isolate CAP1 and CAP2 endothelial cells for studies of transcriptional and epigenetic differences: To truly understand the function and regulation of CAP1 and CAP2 endothelial cells in the lung, it will be important to perform in-depth analysis of the transcriptional and chromatin accessibility states of each cell type. Comparison of CAP1s and CAP2s using bulk RNA sequencing (RNA-seq) and the Assay for Transposase Accessible Chromatin with next-generation sequencing (ATAC-seq) will permit our laboratory to determine differential expression of lowly-expressed transcripts that might not be revealed by single-cell RNA sequencing, identify differential transcription factor binding sites, and investigate alternative splicing of transcripts between cell types. We have therefore developed a fluorescence-activated cell sorting (FACS) strategy for isolating CAP1 and CAP2 endothelial cells and have isolated each cell type and generated libraries for bulk RNA-seq and ATAC-seq. We have just begun to process the sequencing data from these experiments and to delve into new insights into CAP1 and CAP2 regulation and function. 5. Established a collaboration with the Lewandoski laboratory to study the effects of hypoxia on lung endothelial cells: Our laboratory is interested in the role of transient and persistent endothelial cell transcriptional states in lung regeneration and in determining whether aberrant endothelial cell states contribute productively or negatively to lung tissue repair. To determine the effects of changes in oxygen tension on endothelial cell status in the lung, we have established a collaboration with the Lewandoski laboratory, as they study the role of hypoxia on early embryonic development. We are currently working to determine the effects of acute hypoxia on endothelial cells using flow cytometry, hybridization chain reaction, and single-cell RNA sequencing. We are also interested in determining whether endothelial cell states induced by hypoxia can be reversed. These studies will allow us to identify additional mechanistic regulators of aberrant endothelial cell states. Understanding how these states are induced and the mechanisms that permit reversal can then be applied to improve EC function and reverse tissue damage across many lung disease states.