PROJECT SUMMARY. Advances in neonatal critical care have greatly improved the survival of preterminfants but the long-term complications of prematurity including Bronchopulmonary dysplasia (BPD) causemortality and morbidity later in life. After premature birth transition of the lung to a non-aqueous environmentappears sufficient to disrupt subsequent alveolar growth and the attendant vascular structures required foreffective gas exchange. This is further exacerbated when the preterm lung is exposed to supplemental oxygenand positive pressure ventilation. Irreversible loss of alveolar capillaries and vascular remodeling after oxygenexposure cause pulmonary hypertension (PH) seen in patients with severe BPD (BPD-PH). There is an urgentneed for innovative therapeutic approaches to stimulate neonatal lung angiogenesis and preserve respiratoryfunction in BPD-PH infants. My laboratory recently created PEI600-MA5/PEG-OA/Cho nanoparticles that candeliver non-integrating expression plasmids with pro-angiogenic genes into pulmonary microvascularendothelial cells with the purpose of stimulating neonatal lung angiogenesis. We also identified a specializedsubpopulation of pulmonary endothelial progenitor cells (EPCs) FOXF1+ EPCs that are a subset of recentlydiscovered general capillary cells (gCAPs). Transplantation of FOXF1+ gCAPs increased neonatal lungangiogenesis and alveolarization in mice with congenital deficiency of alveolar capillaries. We propose to testthe hypothesis that increasing neonatal lung angiogenesis via the nanoparticle FOXF1 gene therapy or theFOXF1+ gCAP cell transplantation will prevent PH and improve lung function in mouse and rat models of BPD-PH. In Aim 1 we will determine whether the nanoparticle FOXF1 gene therapy has a long-term beneficialeffect in BPH-PH by preventing PH and right ventricular (RV) hypertrophy and accelerating lung regenerationafter neonatal hyperoxic lung injury. We will also identify novel downstream targets of FOXF1 in regeneratingendothelial cells and test whether FOXF1 recruits STAT3 to the chromatin to activate endothelial enhancers.Our studies will determine if the FOXF1-STAT3 protein-protein interactions are required for lung regenerationin BPH-PH models. In Aim 2 we will determine whether transplantation of donor FOXF1+ gCAPs has a long-term beneficial effect by preventing PH and RV hypertrophy in mouse model of BPH-PH. We will also testrequirements of the DLL4/NOTCH signaling pathway for the ability of donor FOXF1+ gCAPs to stimulateproliferation and tube formation in recipient endothelial cells during lung regeneration after hyperoxic injury.Finally we will produce mouse FOXF1+ gCAPs from embryonic stem cells (ESCs) in vitro (via directeddifferentiation of ESCs into FOXF1+ gCAPs) and in vivo (via interspecies mouse-rat chimeras). Mouse ESC-derived FOXF1+ gCAPs will be used for cell therapy to prevent or delay PH and RV hypertrophy in mouseBPD-PH model. Altogether the proposed preclinical studies will directly test whether endothelial delivery of theFOXF1 vector or cell therapy with FOXF1+ gCAPs have therapeutic potential in BPD-PH.