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. ### Competing Interest Statement Over the last three years, C.G. reports grants from Acceleron Pharma (Cambridge, MA, USA), a wholly-owned subsidiary of Merck & Co., Inc. (Rahway, NJ, USA), MSD, Corteria, Structure therapeutics (ex ShouTi), and Janssen, outside the submitted work. M.H. reports grants and personal fees from Acceleron, Aerovate, Altavant, AOP Orphan, Bayer, Chiesi, Ferrer, Janssen, Merck, MorphogenIX and United Therapeutics, outside the submitted work. ### Clinical Trial N/A ### Funding Statement This research was supported by grants from the French National Institute for Health and Medical Research (INSERM), the Université Paris-Saclay, the Marie Lannelongue Hospital, the Fondation pour la Recherche Médicale (FRM) grants no. EQU202203014670 and DEQ20180339158 (FRM), the French National Agency for Research (ANR) grants no ANR-16-CE17-0014 and ANR-17-CE14-0006, ANR-19-CE14?0027, ANR-19-P3IA-0002? 3IA and in part by the Assistance Publique-Hôpitaux de Paris (AP-HP), Service de Pneumologie, Centre de Référence de l'Hypertension Pulmonaire Sévère, the french Fonds de Dotation "Recherche en Santé Respiratoire" - (FRSR) - Fondation du Souffle (FdS), the Conseil Départemental des Alpes Maritimes (2016-294DGADSH-CV), The National Infrastructure France Génomique (Commissariat aux Grands Investissements) [ANR-10-INBS-09-03, ANR-10-INBS-09-02]; the 3IA@coted?azur [ANR-19-P3IA-0002], and the PPIA 4D-OMICS [21-ESRE-0052]. N. B. is a recipient of a PhD fellowship from the Ile-de-France region (ARDoc Health). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Animal experiments were approved by the Ethics Committee of the Université Paris-Saclay and carried out in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the National Institute of Health and Medical Research (Inserm) and were registered with ministerial numbers APAFiS #35319. The use of human tissues was approved by the local ethics committee (CPP EST-III no 18.06.06, Le Kremlin-Bicêtre, France) and all patients gave informed consent before the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The data utilized in this study is accessible upon request. In light of privacy and confidentiality concerns, we are unable to openly distribute the dataset. However, researchers interested in accessing the data can reach out to the corresponding author for requesting access. Each request will be thoroughly reviewed, and we will make every effort to provide the required data while adhering to relevant regulations and ethical considerations.