The role of lung capillary endothelial cells in NFkB-directed alveolarization

University Of California, San Francisco, San Francisco CA

Investigators

Abstract

PROJECT SUMMARY Preterm birth affects 10% of pregnancies, with 40% of survivors developing bronchopulmonary dysplasia (BPD), a chronic lung disease marked by dysmorphic vascular growth and alveolar simplification. Despite advances in neonatal care, BPD incidence has not improved, and no therapies reverse its pathology. My long- term goal is to define critical mechanisms of lung intercellular crosstalk during alveolarization to develop therapies for BPD and other chronic lung diseases. Alveolarization is a key phase of lung development involving synchronized endothelial and alveolar epithelial cell (AT) expansion to establish the gas exchange surface. Endothelial cells (EC) influence lung development through angiogenesis and signaling to other cell types. Our data show EC-specific deletion of IKKβ, an essential NFκB regulator, impairs alveolarization by disrupting angiogenesis, reducing capillary EC and AT cell abundance, and altering cell-ECM interactions. However, mechanisms governing NFκB/IKKβ activity in specific EC subpopulations remain unclear. The central hypothesis of this proposal is that NFκB/IKKβ activity in capillary ECs promotes alveolarization through cell-autonomous mechanisms that drive angiogenesis and cell-nonautonomous mechanisms that regulate AT proliferation and alveolar niche organization. This will be tested in two aims: Aim 1: Identify cell-autonomous mechanisms downstream of NFκB/IKKβ that promote angiogenesis in specific EC subtypes. Single-nucleus RNA sequencing (snRNAseq) and CUT&RUN sequencing will define IKKβ-mediated proangiogenic gene expression in EC subpopulations. Candidate gene validation in vitro (Hmbox1, Fzd4, Glp1r and others determined from snRNAseq) will assess roles in EC proliferation and survival. Aim 2: Determine whether endothelial IKKβ promotes AT proliferation and ECM organization via cell-nonautonomous mechanisms. We will perform EdU proliferation assays, primary co-culture systems, and advanced imaging (transmission electron microscopy and confocal-generated 3D renderings from thick sections) to assess the impact of EC- IKKβ deletion on AT dynamics and alveolar niche architecture. Target genes among AT and identfied in Aim 1 will also be validated We will use the endothelial-IKKβ deletion mouse model we generated for in vitro and in vivo outcomes. These studies will provide novel insights into the role of EC subtypes and NFκB signaling in alveolarization and inform therapeutic strategies for BPD and other pulmonary diseases. Completion of this project will also equip me with the expertise to conduct rigorous, independent research on lung vascular and epithelial biology, and foster a career studying intercellular and ECM interactions within the alveolar niche.