Rationale:: Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized. Objective:: To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension. Method and Results:: The absence of BMP9 in Bmp9 ^−/− mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1 _ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9 ^−/− mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9 ^+/+ littermates. Conclusions:: Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.

Endothelial cells (ECs) are the primary sensors of variations in blood oxygen concentrations. They use the hypoxia-sensitive stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription factor to engage specific transcriptional programs in response to oxygen changes. The regulation of HIF-1α expression is well documented at the protein level, but much less is known about the control of its mRNA stability. Using small interfering RNA knockdown experiments, reporter gene analyses, ribonucleoprotein immunoprecipitations, and mRNA half-life determinations, we report a new regulatory mechanism of HIF-1α expression in ECs. We demonstrate that 1) sustained hypoxia progressively decreases HIF-1α mRNA while HIF-1α protein levels rapidly peak after 3 h and then slowly decay; 2) silencing the mRNA-destabilizing protein tristetraprolin (TTP) in ECs reverses hypoxia-induced down-regulation of HIF-1α mRNA; 3) the decrease in the half-life of Luciferase-HIF-1α-3′UTR reporter transcript that is observed after prolonged hypoxia is mediated by TTP; 4) TTP binds specifically to HIF-1α 3′UTR; and 5) the most distal AU-rich elements present in HIF-1α 3′UTR (composed of two hexamers) are sufficient for TTP-mediated repression. Finally, we bring evidence that silencing TTP expression enhances hypoxia-induced increase in HIF-1α protein levels with a concomitant increase in the levels of the carbonic anhydrase enzyme CA IX, thus suggesting that TTP physiologically controls the expression of a panel of HIF-1α target genes. Altogether, these data reveal a new role for TTP in the control of gene expression during the response of endothelial cell to hypoxia.

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo. First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9–BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad 1–5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9–BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9–BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible of most of the biological BMP activity found in plasma.

Bone morphogenetic proteins (BMPs) are dimeric transforming growth factor ß (TGFß) family cytokines that were first described in bone and cartilage formation but have since been shown to be involved in many pleiotropic functions. In human, there are 15 BMP ligands, which initiate their cellular signaling by forming a complex with two copies of type I receptors and two copies of type II receptors, both of which are transmembrane receptors with an intracellular serine/threonine kinase domain. Within this receptor family, ALK1 (activin receptor‐like kinase 1), which is a type I receptor mainly expressed on endothelial cells, and BMPRII (BMP Receptor type II), a type II receptor also highly expressed on endothelial cells, have been directly linked to two rare vascular diseases: hereditary hemorrhagic telangiectasia (HHT), and pulmonary arterial hypertension (PAH), respectively. BMP9 (gene name GDF2) and BMP10, two close members of the BMP family, are the only known ligands for the ALK1 receptor. This specificity gives them a unique role in physiological and pathological angiogenesis and tissue homeostasis. The aim of this current review is to present an overview of what is known about BMP9 and BMP10 on vascular regulation with a particular emphasis on recent results and the many questions that remain unanswered regarding the roles and specificities between BMP9 and BMP10.

BMP9 and BMP10 mutations were recently identified in patients with pulmonary arterial hypertension, but their specific roles in the pathogenesis of the disease are still unclear. We aimed to study the roles of BMP9 and BMP10 in cardiovascular homeostasis and pulmonary hypertension using transgenic mouse models deficient in Bmp9 and/or Bmp10. Single- and double-knockout mice for Bmp9 (constitutive) and/or Bmp10 (tamoxifen inducible) were generated. Single-knock-out (KO) mice developed no obvious age-dependent phenotype when compared with their wild-type littermates. However, combined deficiency in Bmp9 and Bmp10 led to vascular defects resulting in a decrease in peripheral vascular resistance and blood pressure and the progressive development of high-output heart failure and pulmonary hemosiderosis. RNAseq analysis of the lungs of the double-KO mice revealed differential expression of genes involved in inflammation and vascular homeostasis. We next challenged these mice to chronic hypoxia. After 3 weeks of hypoxic exposure, Bmp10-cKO mice showed an enlarged heart. However, although genetic deletion of Bmp9 in the single- and double-KO mice attenuated the muscularization of pulmonary arterioles induced by chronic hypoxia, we observed no differences in Bmp10-cKO mice. Consistent with these results, endothelin-1 levels were significantly reduced in Bmp9 deficient mice but not Bmp10-cKO mice. Furthermore, the effects of BMP9 on vasoconstriction were inhibited by bosentan, an endothelin receptor antagonist, in a chick chorioallantoic membrane assay. Our data show redundant roles for BMP9 and BMP10 in cardiovascular homeostasis under normoxic conditions (only combined deletion of both Bmp9 and Bmp10 was associated with severe defects) but highlight specific roles under chronic hypoxic conditions. We obtained evidence that BMP9 contributes to chronic hypoxia-induced pulmonary vascular remodelling, whereas BMP10 plays a role in hypoxia-induced cardiac remodelling in mice.