Developmental Expression of Transforming Growth Factor Beta‐Induced Protein in the Alveolar Lung Promotes Nuclear Factor Kappa–B Dependent Pulmonary Endothelial Migration
Authors: Liu Min , Rao Shailaja P. , Fu Sha , Iosef Cristiana , Umbach Gray , Alvira Cristina M. 
Published: November 2023

BackgroundPulmonary angiogenesis is essential for alveolarization, and impaired angiogenesis contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). We previously demonstrated that nuclear factor kappa‐B (NFκB) is constitutively active in early alveolar, but quiescent in adult endothelium. Further, blocking NFκB disrupts angiogenesis of early alveolar primary pulmonary endothelial cells (PEC) in vitro, and impairs alveolarization in vivo. However, the upstream signals that activate NFκB in the developing pulmonary endothelium are not known.ObjectiveWe hypothesized that factors uniquely secreted from the early alveolar lung stimulate NFκB‐mediated pulmonary endothelial angiogenesis to promote alveolarization.MethodsProteomic analysis of the lung secretome at distinct stages of development was performed by 2D gel electrophoresis and tandem mass‐spectroscopy. Protein expression of secreted factors was validated by immunoblot analysis and immunostaining of lung tissue. NFκB activation was assessed by immunostaining and EMSA, and activation of mitogen‐activated protein kinases (MAPKs) that regulate angiogenic function determined by immunoblot analysis. Migration of early alveolar PEC was assessed using scratch and microfluidic chemotaxis assays in transforming growth factor beta induced protein (TGFBI)‐treated WT PEC, and in PEC obtained from mice with an EC‐specific deletion of the NFκB activator, IKKβ. In separate studies, TGFBI‐stimulated migration was assessed in PEC treated with; (i) BAY 11‐7082, a pharmacologic inhibitor of NFκB; (ii) antibodies to inhibit the αvβ3 integrin; or (iii) siRNA against the α5, αv, and β3 integrins.ResultsProteomic analysis identified TGFBI as a protein highly expressed in the early alveolar lung secretome. TGFBI immunoreactivity was intense throughout the early alveolar lung but completely absent in the adult, and TGFBI protein was high in the early alveolar lung but decreased to negligible levels in the adult lung (p<0.001). Recombinant TGFBI activated the MAPKs: ERK(p<0.01), p38(p<0.05) and Akt (p<0.05) within 5 minutes, and increased NFκB activation by 40% at 1hr. Although TGFBI promoted PEC migration in WT PEC (p<0.01), TGFBI‐mediated migration (p<0.05) was abrogated in WT PEC treated with BAY 11‐7082, and in PEC lacking IKKβ. TGFBI‐mediated migration was also blunted by antibodies to block αvβ3 integrin signaling (p<0.01), and by silencing αv (p<0.01) and β3 (p<0.05) integrins, but not affected by silencing α5 integrin.ConclusionsThese studies identify TGFBI as a developmentally regulated protein, highly expressed in the early alveolar lung, which functions to promote NFκB‐mediated pulmonary endothelial cell migration, perhaps via a mechanism involving either MAPK activation or αvβ3 integrin binding. We speculate that TGFBI may play a key role in regulating pulmonary angiogenesis during alveolarization, and could represent a new therapeutic target to treat lung disease characterized by impaired angiogenesis, such as BPD.Support or Funding InformationNIH Grant # R01 HL22918 and the Tashia and John Morgridge Faculty Scholar Fund, Stanford Child Health Research Institute.