Alterations of the BMP-9/ALK1/BMPR-II pathway play a critical role in pulmonary arterial hypertension (PAH) development. Paradoxically, previous work from our team showed that the loss of BMP-9 in the C57BL/6 strain does not lead to spontaneous pulmonary hypertension (PH) and confers reduced susceptibility to the remodeling of pulmonary vessels. However, the exact protective mechanisms remain unknown. We hypothesized that loss of BMP-9/ALK1/BMPR-II signaling induces endothelial cell activation and angiogenesis.

We created Bmp9 KO rats using the CRISPR-Cas9 technique. Then, we examined the pulmonary vascular network in these rats under basal conditions via computed micro-tomography scan. Using confocal immunofluorescence, we also assessed the pulmonary vascular density either under basal condition and after subjecting the rats to two severe and irreversible models of PH: the monocrotaline, and the Sugen/hypoxia model. In addition, we studied the susceptibility of Bmp9 KO rats to develop experimental PH through hemodynamic characterization and histological analyses. In vitro, we performed a clustering on control human pulmonary microvascular endothelial cells (PMECs) before and after BMP-9 treatment using single cell RNA sequencing (scRNAseq). We also studied the angiogenic functions of these cells upon the suppression of ALK1, the main receptor for BMP-9, and with BMP-9 stimulation.

Hemodynamic analyses revealed that Bmp9 KO rats do not develop spontaneous PH in room air. However, our data revealed the presence of vascular abnormalities in the lungs of Bmp9 KO rats with more pronounced vasodilation and vascularization of the pulmonary circulation relative to wild type littermates. Furthermore, we found that they displayed a partial protection against the experimental pulmonary vascular remodeling with less pronounced pulmonary arterial muscularization. In vitro with scRNAseq analyses, we identified for the first time two populations of cultured PMECs that differentially expressed ALK1: ALK1high and ALK1low. The ALK1high population expressed a specific pro-angiogenic gene signature relative to ALK1low PMECs. Consistent with our hypothesis, PMECs stimulation with exogenous BMP-9 reduced the pro-angiogenic gene signature. Conforming with these findings, functional studies showed a reduction of the proliferative and migratory abilities of human PMECs after the suppression of ALK1 expression. These in vitro results were replicated in vivo.

BMP-9/ALK1/BMPR-II axis plays a crucial role in the process of angiogenesis by stabilizing the functions of PMECs. This translates in vivo into an enhanced ability to maintain and produce new vessels, which might explain the lower susceptibility to pulmonary vascular remodeling in Bmp9 KO rats.