Endoglin (ENG) is a single-pass transmembrane protein highly expressed in vascular endothelial cells (ECs), where it plays fundamental roles in EC functions. ENG is implicated in several cardiovascular disorders including hereditary haemorrhagic telangiectasia, pulmonary arterial hypertension (PAH) and preeclampsia. However, molecular mechanisms underlying ENG function are not fully understood. Initially identified as a co-receptor for TGF-β signalling, ENG's extracellular domain was later found to only bind BMP9 and BMP10 with high affinity. The relationship between these two observations is unclear. Here, we provide evidence for two primary functions of co-receptor ENG. First, ENG efficiently displaces prodomains from BMP9 and BMP10, enabling effective capturing of both ligands from the circulation. Second, ENG binds to and recruits TGFBRII into the BMP9 signalling complex, thereby explaining ENG's involvement in both TGF-β and BMP9 pathways. We identify BMP9 target genes NOG and ADAMTSL2 as preferentially dependent on ENG and show that their transcript levels have strong positive correlation with ENG in human lung tissues; the expression levels of all three genes are significantly reduced in PAH. Our findings address an important gap in our understanding on ENG biology and provide crucial insight for therapeutic targeting these pathways in vascular diseases.

Background Non-communicable diseases such as coronary artery disease, atrial fibrillation, type 2 diabetes, hypertension, and others share endothelial dysfunction as one of their underlying features. The endothelium, as the interface between blood and vasculature, shapes disease onset and progression through its response to environmental cues. However, while the genetic component of these diseases has been captured by genome wide association studies (GWAS), which also highlighted a shared immune component, it remains unclear which of these disease loci exerts their effects through endothelial cells. This study identifies, and quantifies, the genetic determinants of endothelial cells molecular traits and their overlap to the common genetic variation component of these diseases. Methods We generated genotype, RNA-sequencing, H3K27ac ChIP-sequencing, ATAC-sequencing, and endothelial cells barrier stimuli response measurements for 100 samples of human umbilical vein endothelial cells. These were used to identify quantitative trait loci (QTL) for gene expression, transcriptional isoform usage, splice junction usage, chromatin activity and barrier response. We applied statistical colocalisation to identify the overlap between data layers, and to explain molecular QTLs contribution to GWAS disease loci. Results We used molecular QTLs to identify the regulatory features of 8,214 genes, representing 36% of all expressed genes in endothelial cells. We also identified the molecular mechanisms underlying 815 loci across 16 disease GWAS. These represent between 29% and 40% of all loci for each disease, compared to the previous average of 23%. This is due to the choice of a cell type often underrepresented in tissue level data, and the inclusion of isoform, splicing and chromatin activity datasets. Furthermore, we compared the endothelial cells molecular QTLs with similar datasets in monocytes, neutrophils and CD4 T lymphocytes to shed light on the interplay between the endothelial and the immune compartments in these diseases. We identified loci acting through both the endothelial and the immune compartment, mostly with the same directionality of effect, and endothelial specific ones. Conclusions This work expands the knowledge of the mechanisms and genes underlying the effect of common genetic variation on non-communicable diseases having endothelial dysfunction as a shared feature. It also illustrates the interplay between endothelial cells and immune cell types in these diseases, highlighting shared and unique pathways. ### Competing Interest Statement The authors have declared no competing interest.

Activin-A is elevated in pulmonary arterial hypertension (PAH) patients, and reportedly suppresses BMPR-II. This suggests one mechanism of action for PAH drug, sotatercept, an activin-ligand trap. However, we were unable to confirm that activin-A reduces BMPR-II in pulmonary endothelial cells. Thus, it seems unlikely that sotatercept influences BMPR-II or PAH via this mechanism.

Deleterious mutations in the GDF2 gene, encoding BMP9, are causative of pulmonary arterial hypertension and hereditary haemorrhagic telangiectasia. Paradoxically, BMP9 germ-line knockout (Gdf2−/−; Bmp9 KO) and double Bmp9 KO/conditional Bmp10 cKO (dKO) mice exhibit an attenuated response to PAH-inducing stimuli. We asked whether this contradiction is due to the pathological, physiological, or genetic consequences of BMP9 knockout. In Bmp9 KO mice we observed reduced pulmonary vascular smooth muscle cell (SMC) coverage and using RNA-seq analysis of Bmp9 KO mouse lungs identified two novel genes, COLQ and ITGA6, which were differentially regulated in a human PAH RNA-seq dataset. In order to study BMP10 loss, postnatal tamoxifen treatment was required to induce Bmp10 cKO. As previously reported, in dKO mice we observed cardiomegaly and splenomegaly, as well as hyperplasia and hemosiderosis in the pulmonary vasculature. Surprisingly, tamoxifen treated Bmp9 KO control mice phenocopied these pathological changes in dKO mice and downregulated SMC marker gene transcription. Loss of BMP10 is not critical for severe tissue remodelling in the lung, heart, and spleen, rather Bmp9 KO mice appear sensitive to tamoxifen and BMP9 loss is the primary cause of mild vessel remodelling due to a basal reduction of smooth muscle cell coverage. This study suggests that interaction of the BMP pathway with tamoxifen needs to be carefully considered when studying Bmp9 KO mice and urges caution in the context of tamoxifen use when studying cardiovascular and pulmonary disease models.

Adipocyte dysfunction contributes to lipotoxicity and cardiometabolic diseases. Bone morphogenetic protein 4 (BMP4) is expressed in white adipocytes and remodels white adipose tissue, while liver-derived BMP9, a key circulating BMP, influences adipocyte lipid metabolism. The gene sets regulated by BMP4 and BMP9 signalling in mature adipocytes remain unclear. Here, we directly compare BMP4 and BMP9 signalling in mature brown and white adipocytes. While both BMPs showed comparable potency across adipocyte types, RNA sequencing analysis revealed extensive gene regulation, with many more differentially expressed genes and suppression of critical metabolic pathways in white adipocytes. Although BMP4 and BMP9 induced inhibitors of BMP and GDF signalling in both adipocytes, they selectively upregulated several TGF-β family receptors and BMP4 expression only in white adipocytes. These findings underscore a central role of BMP signalling in adipocyte homeostasis and suggest both BMP4 and BMP9 as regulators of white adipocyte plasticity with potential therapeutic implications.

Pulmonary arterial hypertension (PAH) is a rare and life-threatening vascular disorder, characterised by abnormal remodelling of the pulmonary vessels and elevated pulmonary artery pressure, leading to right ventricular hypertrophy and right-sided heart failure. The importance of bone morphogenetic protein (BMP) signalling in the pathogenesis of PAH is demonstrated by human genetic studies. Many PAH risk genes are involved in the BMP signalling pathway and are highly expressed or preferentially act on vascular endothelial cells. Endothelial dysfunction is recognised as an initial trigger for PAH, and endothelial BMP signalling plays a crucial role in the maintenance of endothelial integrity. BMPR2 is the most prevalent PAH gene, found in over 80% of heritable cases. As BMPRII protein is the major type II receptor for a large family of BMP ligands and expressed ubiquitously in many tissues, dysregulated BMP signalling in other cells may also contribute to PAH pathobiology. Sotatercept, which contains the extracellular domain of another transforming growth factor-β family type II receptor ActRIIA fused to immunoglobin Fc domain, was recently approved by the FDA as a treatment for PAH. Neither its target cells nor its mechanism of action is fully understood. This review will revisit BMPRII function and its extracellular regulation, summarise how dysregulated BMP signalling in endothelial cells and smooth muscle cells may contribute to PAH pathogenesis, and discuss how novel therapeutics targeting the extracellular regulation of BMP signalling, such as BMP9 and Sotatercept, can be related to restoring BMPRII function.

Endoglin (ENG) is a single-pass transmembrane protein highly expressed on vascular endothelial cells, although low expression levels can be detected in many other cell types. Its extracellular domain can be found in circulation known as soluble endoglin (sENG). Levels of sENG are elevated in many pathological conditions, in particular preeclampsia. We have shown that while loss of cell surface ENG decreases BMP9 signaling in endothelial cells, knocking down ENG in blood cancer cells enhances BMP9 signaling. Despite sENG binding to BMP9 with high affinity and blocking the type II receptor binding site on BMP9, sENG did not inhibit BMP9 signaling in vascular endothelial cells, but the dimeric form of sENG inhibited BMP9 signaling in blood cancer cells. Here we report that in non-endothelial cells such as human multiple myeloma cell lines and the mouse myoblast cell line C2C12, both monomeric and dimeric forms of sENG inhibit BMP9 signaling when present at high concentrations. Such inhibition can be alleviated by the overexpression of ENG and ACVRL1 (encoding ALK1) in the non-endothelial cells. Our findings suggest that the effects of sENG on BMP9 signaling is cell-type specific. This is an important consideration when developing therapies targeting the ENG and ALK1 pathway.

Hereditary Haemorrhagic Telangiectasia (HHT) is an autosomal dominant disorder characterised by arteriovenous malformations (AVMs), capillary telangiectases, and recurrent nosebleeds. About 80% of HHT cases are caused by mutations in ENG (endoglin) or ACVRL1 (encoding ALK1), yet the molecular and cellular mechanisms of how these mutations drive the disease are not well understood. Endoglin (ENG) is highly expressed on the surface of vascular endothelial cells. It is a coreceptor for the bone morphogenetic protein 9 (BMP9) and BMP10, promoting their signalling activity. Endoglin mutations lead to loss-of-function in HHT1 patients. Endothelial-specific, homozygous deletion of endoglin in adult mice has been shown to model the HHT phenotype, promoting high-output heart failure (HOHF) through AVM formation in the pubic symphysis driven by VEGF signalling. We hypothesised that treatment with recombinant BMP10 protein would restore BMP signalling and rescue disease phenotype. Method Endotoxin-free, recombinant human prodomain-bound BMP10 was generated using the HEK-EBNA cell expression system and three chromatography column steps. Adult Cdh5(PAC)-CreERT2; Engfl/fl mice were rederived from the University of Newcastle. After tamoxifen treatment by intraperitoneal injection for five days to induce endothelial-specific endoglin knockdown, BMP10 or PBS was administered intraperitoneally every weekday for two weeks (for latex blue measurement) or five weeks for monitoring HOHF. Tamoxifen-treated Engfl/fl mice were used as pseudo-wild-type (WT) controls. AVM formation was monitored using latex blue perfusion as the medium is too large to pass through capillaries, HOHF was evaluated by measuring heart/body weight ratio and histochemistry. Results Prodomain-bound BMP10 was purified, quantified, and its endotoxin levels and signalling activity measured. In two pilot studies, we observed successful endoglin knockdown, as measured by RNA expression using qPCR. The endoglin knockdown resulted in AVM formation in the pubic symphysis and HOHF phenotype in the disease cohort. The PBS-treated disease cohort showed increased average cardiomyocyte size and increased heart-to-body weight ratio compared to the control group. A greater percentage of the pubic symphysis vascular area was perfused with latex blue indicating AVM formation. There was also reduced expression of BMP target genes such as Id1. The BMP10-treated cohort showed a trend of reduction in average cardiomyocyte size and percentage of latex blue perfused vessel area in the pubic symphysis but was not statistically significantly different from the disease group in this small pilot study. BMP10 treatment showed a decrease in the expression of VEGF target genes such as Kdr, and Flt1 when compared with the WT group. A full reversal study is currently ongoing, with 24 mice in each cohort with expected results in late March. Conflict of Interest none

Rationale: Despite the increasing number of TBX4 mutations underlying pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Methods: We assembled a multi-center cohort of 137 patients harboring TBX4 sequence variants and investigated the effect of missense variants (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with PAH patients genotyped via the NIHR BioResource - Rare Diseases (NBR) Study harboring BMPR2 mutations (n = 162) or without any identified mutations in PAH risk genes (n = 741). Results: Functional assessment of TBX4 missense variants revealed gain- (n=8) or loss-of-function effects (n=23) with a similar wide phenotypic spectrum ranging from early-onset developmental lung disease to adult-onset PAH. Variants located in the T-BOX and nuclear localization domains associated with earlier presentation (p = 0.002) and increased incidence of interstitial lung disease (p = 0.001). Event-free survival (death or transplantation) was marginally lower in the T-BOX group (p = 0.049) with adjustment for age and sex. Compared to patients with BMPR2 and no mutations, TBX4 mutation carriers were diagnosed at a younger age (p < 0.001) and had worse baseline lung function (FEV1, FVC) (p < 0.001). Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain-of-function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.

Pulmonary arterial hypertension (PAH) is an often fatal condition, the primary pathology of which involves loss of pulmonary vascular perfusion due to progressive aberrant vessel remodeling. The reduced capacity of the pulmonary circulation places increasing strain on the right ventricle of the heart, leading to death by heart failure. Currently, licensed therapies are primarily vasodilators, which have increased the median post-diagnosis life expectancy from 2.8 to 7 years. Although this represents a substantial improvement, the search continues for transformative therapeutics that reverse established disease. The genetics of human PAH heavily implicates reduced endothelial bone morphogenetic protein (BMP) signaling as a causal role for the disease pathobiology. Recent approaches have focused on directly enhancing BMP signaling or removing the inhibitory influence of pathways that repress BMP signaling. In this critical commentary, we review the evidence underpinning the development of two approaches: BMP-based agonists and inhibition of activin/GDF signaling. We also address the key considerations and questions that remain regarding these approaches.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) experience excess cardiovascular morbidity and mortality, and exacerbations further increase the risk of such events. COPD is associated with persistent blood and airway neutrophilia and systemic and tissue hypoxia. Hypoxia augments neutrophil elastase release, enhancing capacity for tissue injury. Objective: To determine whether hypoxia-driven neutrophil protein secretion contributes to endothelial damage in COPD. Methods: The healthy human neutrophil secretome generated under normoxic or hypoxic conditions was characterized by quantitative mass spectrometry, and the capacity for neutrophil-mediated endothelial damage was assessed. Histotoxic protein concentrations were measured in normoxic versus hypoxic neutrophil supernatants and plasma from patients experiencing COPD exacerbation and healthy control subjects. Measurements and Main Results: Hypoxia promoted PI3Kγ-dependent neutrophil elastase secretion, with greater release seen in neutrophils from patients with COPD. Supernatants from neutrophils incubated under hypoxia caused pulmonary endothelial cell damage, and identical supernatants from COPD neutrophils increased neutrophil adherence to endothelial cells. Proteomics revealed differential neutrophil protein secretion under hypoxia and normoxia, and hypoxia augmented secretion of a subset of histotoxic granule and cytosolic proteins, with significantly greater release seen in COPD neutrophils. The plasma of patients with COPD had higher content of hypoxia-upregulated neutrophil-derived proteins and protease activity, and vascular injury markers. Conclusions: Hypoxia drives a destructive "hypersecretory" neutrophil phenotype conferring enhanced capacity for endothelial injury, with a corresponding signature of neutrophil degranulation and vascular injury identified in plasma of patients with COPD. Thus, hypoxic enhancement of neutrophil degranulation may contribute to increased cardiovascular risk in COPD. These insights may identify new therapeutic opportunities for endothelial damage in COPD.

Heterozygous mutations in BMPR2 (bone morphogenetic protein (BMP) receptor type II) cause pulmonary arterial hypertension. BMPRII is a receptor for over 15 BMP ligands, but why BMPR2 mutations cause lung-specific pathology is unknown. To elucidate the molecular basis of BMP:BMPRII interactions, we report crystal structures of binary and ternary BMPRII receptor complexes with BMP10, which contain an ensemble of seven different BMP10:BMPRII 1:1 complexes. BMPRII binds BMP10 at the knuckle epitope, with the A-loop and β4 strand making BMPRII-specific interactions. The BMPRII binding surface on BMP10 is dynamic, and the affinity is weaker in the ternary complex than in the binary complex. Hydrophobic core and A-loop interactions are important in BMPRII-mediated signalling. Our data reveal how BMPRII is a low affinity receptor, implying that forming a signalling complex requires high concentrations of BMPRII, hence mutations will impact on tissues with highest BMPR2 expression such as the lung vasculature.

Rationale: Autoimmunity is believed to play a role in idiopathic pulmonary arterial hypertension (IPAH). It is not clear whether this is causative or a bystander of disease and if it carries any prognostic or treatment significance. Objectives: To study autoimmunity in IPAH using a large cross-sectional cohort. Methods: Assessment of the circulating immune cell phenotype was undertaken using flow cytometry, and the profile of serum immunoglobulins was generated using a standardized multiplex array of 19 clinically validated autoantibodies in 473 cases and 946 control subjects. Additional glutathione S-transferase fusion array and ELISA data were used to identify a serum autoantibody to BMPR2 (bone morphogenetic protein receptor type 2). Clustering analyses and clinical correlations were used to determine associations between immunogenicity and clinical outcomes. Measurements and Main Results: Flow cytometric immune profiling demonstrates that IPAH is associated with an altered humoral immune response in addition to raised IgG3. Multiplexed autoantibodies were significantly raised in IPAH, and clustering demonstrated three distinct clusters: "high autoantibody," "low autoantibody," and a small "intermediate" cluster exhibiting high concentrations of ribonucleic protein complex. The high-autoantibody cluster had worse hemodynamics but improved survival. A small subset of patients demonstrated immunoglobulin reactivity to BMPR2. Conclusions: This study establishes aberrant immune regulation and presence of autoantibodies as key features in the profile of a significant proportion of patients with IPAH and is associated with clinical outcomes.

Background: Alzheimer’s disease (AD) and vascular dementia (VaD) are responsible for at least 20 % of diagnosed cases of dementia. It has been established that hearing loss is a risk factor for cognitive impairment and dementia. In this study, the association between hearing impairment and AD/VaD was determined in patients, as well as the characteristics of hearing loss in the two types of dementia.Methods: 70 participants comprising 30 subjects in control group, 20 patients in AD group, and 20 patients in VaD group, were recruited and assessed with respect to pure-tone audiometric test for peripheral auditory system. On admission, age, gender, body mass index (BMI), smoking and drinking habits, diabetes, stroke, family history, as well as levels of cholesterol (TC), triglycerides (TG), fasting blood glucose (FBG), low-density lipoprotein (LDL), and glycosylated hemoglobin A1c (HbA1c) were measured. Hearing loss results and other clinical indexes were analyzed, and their correlations with severity of dementia were determined.Results: Degree of hearing impairment and HbA1c level differed significantly between the AD and control groups. However, only age and degree of hearing impairment were positively correlated with grades of cognitive impairment in AD and control groups. Body mass index (BMI) and smoking history; HbA1c level, grades of cognitive impairment, and degree of hearing impairment, as well as related cognitive impairment, differed significantly between VaD and control groups. Results of logistic regression analysis indicated that the degree of hearing impairment could be a predictor for grades of cognitive impairment in AD and VaD. Moreover, the degree of hearing loss in full band hearing was significantly higher in VaD group than in AD group.Conclusion: Hearing loss may be a potential risk factor for cognitive impairment in AD and VaD, two major types of dementia with different clinical characteristics. The clinical features of hearing loss in the two types dementia might be helpful in their diagnosis and treatment.

Non-alcoholic steatohepatitis (NASH) is characterized by lipotoxicity, inflammation and fibrosis, ultimately leading to end-stage liver disease. The molecular mechanisms promoting NASH are poorly understood, and treatment options are limited. Here, we demonstrate that hepatic expression of bone morphogenetic protein 8B (BMP8B), a member of the transforming growth factor beta (TGFβ)-BMP superfamily, increases proportionally to disease stage in people and animal models with NASH. BMP8B signals via both SMAD2/3 and SMAD1/5/9 branches of the TGFβ-BMP pathway in hepatic stellate cells (HSCs), promoting their proinflammatory phenotype. In vivo, the absence of BMP8B prevents HSC activation, reduces inflammation and affects the wound-healing responses, thereby limiting NASH progression. Evidence is featured in primary human 3D microtissues modelling NASH, when challenged with recombinant BMP8. Our data show that BMP8B is a major contributor to NASH progression. Owing to the near absence of BMP8B in healthy livers, inhibition of BMP8B may represent a promising new therapeutic avenue for NASH treatment.

Activin receptor-like kinase 1 (ALK1)-mediated endothelial cell signalling in response to bone morphogenetic protein 9 (BMP9) and BMP10 is of significant importance in cardiovascular disease and cancer. However, detailed molecular mechanisms of ALK1-mediated signalling remain unclear. Here, we report crystal structures of the BMP10:ALK1 complex at 2.3 Å and the prodomain-bound BMP9:ALK1 complex at 3.3 Å. Structural analyses reveal a tripartite recognition mechanism that defines BMP9 and BMP10 specificity for ALK1, and predict that crossveinless 2 is not an inhibitor of BMP9, which is confirmed by experimental evidence. Introduction of BMP10-specific residues into BMP9 yields BMP10-like ligands with diminished signalling activity in C2C12 cells, validating the tripartite mechanism. The loss of osteogenic signalling in C2C12 does not translate into non-osteogenic activity in vivo and BMP10 also induces bone-formation. Collectively, these data provide insight into ALK1-mediated BMP9 and BMP10 signalling, facilitating therapeutic targeting of this important pathway.

ABSTRACTRationalePulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome (ARDS), which carries a high mortality. Circulating bone morphogeneic protein 9 (BMP9) is emerging as an important regulator of pulmonary vascular homeostasis.ObjectiveTo determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity, and whether loss of endogenous BMP9 occurs during lipopolysacharride (LPS)-induced lung inflammation and permeability.MethodsA BMP9-neutralizing antibody was administrated to healthy adult mice and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human primary lung endothelial cells. Impact of BMP9 was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and endotoxemic mice.Main ResultsSubacute neutralization of endogenous BMP9 in mice resulted in increased lung vascular permeability, interstitial edema and neutrophil extravasation. In lung endothelial cells, BMP9 regulated a programme of gene expression and pathways controlling vascular permeability and cell membrane integrity. Augmentation of BMP9 signalling in mice with exogenous BMP9 prevented inhaled LPS-caused lung injury and edema. In endotoxemic mice, endogenous BMP9 levels were markedly reduced, due to a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human sepsis patients, circulating levels of BMP9 were also markedly reduced.ConclusionsEndogenous circulating BMP9 is a pulmonary endothelial protective factor, down-regulated during inflammation. Supplementation with exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in the setting of lung injury.Short summaryScientific Knowledge on the SubjectIncreased pulmonary endothelial permeability is a major factor in the development of acute respiratory distress syndrome (ARDS). Evidence is emerging that circulating BMP9, secreted from the liver, might protect the pulmonary endothelium from injury. For example, loss of BMP9 levels or signalling receptor contributes to the development of pulmonary arterial hypertension. The role of endogenous BMP9 in endothelial permeability remains unclear.What This Study Adds to the FieldHere we show that subacute neutralization of endogenous BMP9 leads to lung vascular injury, including enhanced permeability and neutrophil extravasation. BMP9 levels were markedly reduced in the setting of inflammation in mice and humans. Conversely, exogenous supplementation of BMP9 protected the lung from LPS-induced injury. This study suggests that exogenous BMP9 could offer a novel approach to prevent increased pulmonary endothelial permeability in the setting of lung injury and ARDS.