Endothelial cells play a critical role in multiple aspects of lung development and homeostasis, yet the precise mechanisms that regulate vasculogenesis and how endothelial cells signal to other cell types to orchestrate lung development have not been well defined. Accurate regulation of these processes is critical to ensure proper lung development and prevent bronchopulmonary dysplasia (BPD), a lung disease of premature infants that often results in life-long complications. Hallmarks of BPD are impaired vasculature and arrested lung development. We created three-dimensional (3D) co-culture systems in which lung endothelial cells (LuMECs) drive the putative lung stem cells, BASCs, to produce bronchiolar or alveolar organoids. These organoid cultures provide an opportunity to model interactions that occur between endothelial cells and alveolar cells during lung development. Using this system, we discovered a novel BMP4-NFATc1- Thrombospondin (TSP1) pathway in endothelial and epithelial cells in the adult lung. We showed that TSP1, known as an anti-angiogenic factor, is required for the development of alveolar organoids. We also identified R-spondin2 (Rspo2), which regulates Wnt signaling in other systems, as a BMP4-regulated protein that may be downstream of TSP1 in endothelial cells. The mechanisms underlying TSP1 regulation of lung vascular development and alveolarization are unknown. We hypothesize that endothelial-derived TSP1 induced by BMP4-NFATc1 signaling inhibits pulmonary vascular development and is required for proper lung development. Further, we posit that vascular lung injury, such as that seen with hyperoxia exposure, impairs TSP1 signaling, thereby altering vasculogensis and blocking alveolar development.
In Aim 1, we will determine how TSP1- CD47 interactions regulate vasculogenesis and lung development. Defective lung endothelial cells from mice exposed to hyperoxia will be used in lung organoid co-cultures to test the impact of impaired endothelial cell signals on alveolarization. Similarly, cells from knockout mice will be used in neonatal lung organoid cultures to determine the effect of CD47 deficiency on alveolar development. CD47-knockout mice and endothelial- specific TSP1-knockout mice will be used to test the role of TSP1-CD47 interactions in the response to neonatal hyperoxia-mediated lung vascular injury in vivo.
In Aim 2, we will define the mechanisms by which TSP1-dependent Rspo2 mediates pulmonary vascular development by evaluating ligand response in vitro and after neonatal hyperoxic injury in vivo. Recombinant Rspo2 will be used in neonatal lung organoid cultures and in assays for endothelial cell proliferation, migration and tube formation. We will determine if lung endothelial cell secreted factors, and more specifically, Rspo2, is sufficient for prevention of neonatal hyperoxia-mediated lung injury. These studies will elucidate mechanisms by which lung endothelial cells drive critical outcomes in development. This work will determine if defective TSP1-dependent signaling is a mechanism of interrupted lung development in premature infants with BPD, leading to new ways to direct therapy for BPD.

Public Health Relevance

Bronchopulmonary dysplasia (BPD), a lung disease of premature infants that often results in life-long complications, involves impaired lung endothelial cells. These studies will elucidate mechanisms by which lung endothelial cells drive critical outcomes during development, building on innovative 3D lung organoid assays we developed. This work will reveal new strategies for therapeutic intervention in lung diseases involving impaired vasculogenesis such as BPD.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL132266-02
Application #
9394032
Study Section
Respiratory Integrative Biology and Translational Research Study Section (RIBT)
Program Officer
Fessel, Joshua P
Project Start
2016-12-15
Project End
2020-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Boston Children's Hospital
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
Garcia-de-Alba, Carolina; Pessina, Patrizia; Kim, Carla F (2018) A New ""Age""r for Lung Research Arrives: Genetic Targeting of Alveolar Type 1 Epithelial Cells. Am J Respir Cell Mol Biol 59:661-662
Tan, Jean L; Lau, Sin N; Leaw, Bryan et al. (2018) Amnion Epithelial Cell-Derived Exosomes Restrict Lung Injury and Enhance Endogenous Lung Repair. Stem Cells Transl Med 7:180-196
Sinkevicius, Kerstin W; Bellaria, Kelly J; Barrios, Juliana et al. (2018) E-Cadherin Loss Accelerates Tumor Progression and Metastasis in a Mouse Model of Lung Adenocarcinoma. Am J Respir Cell Mol Biol 59:237-245
Rowbotham, S P; Li, F; Dost, A F M et al. (2018) H3K9 methyltransferases and demethylases control lung tumor-propagating cells and lung cancer progression. Nat Commun 9:4559
Zhu, Dandan; Tan, Jean; Maleken, Amina S et al. (2017) Human amnion cells reverse acute and chronic pulmonary damage in experimental neonatal lung injury. Stem Cell Res Ther 8:257
Lee, Joo-Hyeon; Tammela, Tuomas; Hofree, Matan et al. (2017) Anatomically and Functionally Distinct Lung Mesenchymal Populations Marked by Lgr5 and Lgr6. Cell 170:1149-1163.e12
Agrawal, Pankaj B; Wang, Ruobing; Li, Hongmei Lisa et al. (2017) The Epithelial Sodium Channel Is a Modifier of the Long-Term Nonprogressive Phenotype Associated with F508del CFTR Mutations. Am J Respir Cell Mol Biol 57:711-720
Zhang, Haikuo; Fillmore Brainson, Christine; Koyama, Shohei et al. (2017) Lkb1 inactivation drives lung cancer lineage switching governed by Polycomb Repressive Complex 2. Nat Commun 8:14922