Bone Morphogenetic Protein-9 Controls Pulmonary Vascular Growth and Remodeling

Background Pulmonary arterial hypertension (PAH), a life-limiting condition characterized by dysfunction of pulmonary microvascular endothelium, is predisposed by mutations in several genes that are critical for the proper activation of specific bone morphogenetic protein (BMP) receptor complexes that phosphorylate intracellular Smad1/5/8 in endothelial cells. However, the functional importance of BMP-9 ( GDF2 ), one of the high affinity ligands for ALK1 ( ACVRL1 ) and BMPR-II ( BMPR2 ), for the pulmonary microvasculature remains imperfectly understood. Objective The aim of this study was first to determine the in vivo impact of BMP-9 deficiency on pulmonary vascular growth and remodeling, then to assess whether ALK1 expression can alter BMP-9 transcriptional signatures in human pulmonary microvascular endothelial cells (PMECs). Methods CRISPR-Cas9 gene editing was used to create Gdf2 knockout rats in Sprague Dawley background. Computed micro-tomography (Micro-Ct) scan after Microfil perfusion was performed to generate high-resolution 3D-images of the pulmonary arterial tree. The influence of ALK1 abundance on the transcriptional signatures of BMP-9 responses in human PMECs was assessed by single cell (sc)-RNAseq. Functional studies were performed using human PMECs exposed to BMP-9, the ALK1/2 inhibitor ML347, and ALK1-Fc fusion protein that neutralizes BMP9/10 and two animal models of severe pulmonary hypertension (PH). Results Micro-Ct angiography revealed structural and functional remodeling along the pulmonary vascular tree in BMP-9 deficient rats, resulting in vasodilation and increase in vascular density. scRNA-seq experiments identified distinct transcriptional signatures in human PMECs in response to BMP-9 responses. ALK1 expression had a direct impact on both proangiogenic capacities and transcriptional responses of PMECs to BMP-9. Functional studies performed in human PMECs confirmed that abundance of BMP-9 and ALK1 acted as modulators of PMEC tube formation, migration and proliferation, and also of vascular endothelial growth factor (VEGF)/VEGFR activities. The structural and functional remodeling observed in Gdf2 knockout rats coincided with a lower susceptibility to develop severe PH induced by monocrotaline (MCT) and SU5416+hypoxia (SuHx). Conclusion BMP-9 and ALK1 are critical modulators of pulmonary vascular growth and remodeling. Our results provide potential mechanisms explaining why BMP-9 deficient animals are less susceptible to the rise in pulmonary vascular resistance in experimental models of PH.