Endoglin as a BMP9 co-receptor in vascular endothelial cells: prodomain displacement and TGFBRII recruitment

Domain: biology
Published: 2025-12-31
Editorial outcome: minor_revision

Abstract
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.

Introduction
Endoglin (ENG) is a single-pass transmembrane protein highly expressed in vascular endothelial cells (ECs). It has a large extracellular domain (ECD) and a small intracellular domain (ICD). Loss-of-function mutations in ENG cause type I hereditary haemorrhagic telangiectasia (HHT1)1; symptoms include telangiectasia in the nose, gastrointestinal tract and skin, as well as larger arteriovenous malformations in the brain, lungs and liver, which can cause life-threatening haemorrhage. ENG mutations are also found in patients with pulmonary arterial hypertension (PAH)2, a vascular disorder characterised by remodelling and occlusion of small pulmonary vessels, leading to increased right ventricular systolic pressure and ultimately right-sided heart failure.

ENG ECD can be shed from the cell surface and released into circulation as soluble ENG (sENG). Concentrations of sENG are markedly elevated by more than tenfold in the maternal circulation of women with preeclampsia (PE)3, and thought to contribute to its pathogenesis4. PE is a pregnancy-specific hypertensive disorder which increases the risk of poor outcomes for both mother and baby. Circulating sENG is also elevated in PAH (by about twofold) and has been proposed as a biomarker for the prognosis of PAH5. Despite the important roles of ENG and sENG in different cardiovascular diseases, the molecular mechanisms of ENG function and its role in the pathogenesis of HHT, PAH and PE remain elusive.

ENG was discovered initially as a component of the transforming growth factor-β (TGF-β) family signalling complex6. TGF-β family ligands, including bone morphogenetic proteins (BMPs), are primarily homodimers, initiating cellular responses by forming a signalling complex with two copies of a type I receptor and two copies of a type II receptor. TGF-β type I receptor (TGFBRI), also termed Activin receptor-like kinase 5 (ALK5), and TGF-β type II receptor (TGFBRII) mediate signals from TGF-β1, TGF-β2 and TGF-β3. Using cell surface labelling with radioactive ligands followed by crosslinking and immunoprecipitation in human umbilical vein ECs (HUVECs), ENG was found to associate with TGF-β1 and TGF-β3 but not TGF-β26. Hence, sENG was proposed as a ligand trap for TGF-β1 and TGF-β34. Moreover, it has been shown that TGF-β1 can signal through both ALK1 and ALK5 in ECs to regulate endothelial activation state and that its signalling through ALK1 requires ENG7–9. ENG was reported to interact with TGFBRII and ALK5 via both its ECD and ICD in COS-7 cells transiently transfected with plasmids containing the TGF-β receptors and ENG10,11. Of note, all these findings were published before 2007.

In 2007, ALK1 was found to be a specific type I receptor for BMP9 and BMP1012,13. In 2011, direct binding assays using recombinant sENG-Fc showed that ENG ECD binds with high affinities only to BMP9 and BMP10, not to any other TGF-β family receptors or ligands14. Thus, ENG is a co-receptor for BMP9 and BMP10 signalling, and sENG was thought to be a ligand trap for BMP9 and BMP10 but not for TGF-βs14. However, these findings cannot explain the reports prior to 2007, and a gap in knowledge remains and needs to be addressed to understand the function of ENG fully.

BMP9 and BMP10 are synthesised as pre-proproteins. Upon secretion, the prodomain is cleaved but remains non-covalently bound to the growth factor domain (GFD) forming pro:BMP9 and pro:BMP10 (Supplementary Fig. 1a,b). Crystal structures revealed that the prodomains bind to BMP9 and BMP10 overlapping with the type II receptor binding site15–18 (Supplementary Fig. 1b–d). Although pro:BMP9 and pro:BMP10 signal with equal potency as their respective GFDs in ECs17, signalling requires displacement of the prodomain. Unprocessed proBMP9 and proBMP10, where the prodomain cannot be displaced are not active19,20. The crystal structure of the ENG orphan domain (ENG-OR) bound to BMP9 further shows that its binding site on BMP9 also overlaps with both the prodomain and the type II receptor binding sites21 (Supplementary Fig. 1e). How ENG functions as a co-receptor to facilitate BMP9 and BMP10 signalling has remained unclear.

To fully appreciate the critical role of ENG in ECs and in vascular biology, it is important to understand its function at the molecular level. We first revisited sENG. We have previously shown that sENG in the circulation is mainly monomeric and does not inhibit BMP9 and BMP10 signalling in vascular ECs22. However, at the elevated concentrations found in PE plasma, sENG can inhibit BMP9 and BMP10 signalling in Eng knockout ECs or in non-ECs with low ENG22,23. Building on this, the present study investigates how ENG resolves the overlapping binding site with the prodomain and the type II receptors to facilitate BMP9 signalling. We show that ENG efficiently displaces the prodomain from both pro:BMP9 and pro:BMP10 at sub-molar ratios in solution, far more effectively than the type II receptors. Furthermore, ENG binds to and recruits TGFBRII into the BMP9-ALK1 complex, enabling TGFBRII to participate in BMP9 signalling. Comparative transcriptomics in Eng knockout and control cells identified Nog (encodes Noggin) and Adamtsl2 as BMP9 target genes that are more dependent on ENG, a finding confirmed in human dermal microvascular ECs (HDMECs), which express low levels of ENG. Importantly, mRNA expression of ENG, NOG and ADAMTSL2 is strongly correlated in human lung tissues and significantly reduced in PAH patients’ lungs compared to healthy subjects. Together, our results support a model in which ENG enables BMP9 signalling through alternative type II receptors, with TGFBRII participating in BMP9 signalling in an ENG-dependent manner. This model unifies earlier important findings of ENG’s role in TGF-β receptor complexes with later reports of ENG’s direct interaction with BMP9 and BMP10, establishing ENG as a key mediator of BMP9 and TGF-β crosstalk in ECs. This study reconciles the long-standing controversy of ALK1- and ALK5-mediated TGF-β signalling in controlling the activation states of vascular ECs. ENG mutations were identified in HHT and PAH over two decades ago; yet no curative therapies are available for these vascular disorders. Research progress on ENG function has been slow due to this knowledge gap. Our findings provide a mechanistic framework that can accelerate ENG research and guide therapeutic targeting ENG in vascular disease.

Results
ENG ECD, but not the type II receptor ECDs, can effectively displace BMP9 and BMP10 prodomains
The prodomains of BMP9 and BMP10 need to be displaced before a signalling complex can be assembled (Supplementary Fig. 1b–d). We have previously shown that binding of sENG to pro:BMP9 displaces its prodomain22. Here, we investigated this further by asking whether ENG could displace the prodomain from pro:BMP9 at a sub-molar ratio and whether this also applies to pro:BMP10.

We purified full-length ENG ECD in both dimeric (sENG(D)) and monomeric (sENG(M)) forms22 and performed native PAGE analysis, which separates proteins and protein complexes according to size and charge. Supplementary Fig. 2a summarises the molecular weight and the isoelectric point (pI) of all proteins and complexes used in this study.

Pro:BMP9 separated on native PAGE to yield a strong pro:BMP9 complex band (band 1) and a faint prodomain band (band 2) (Fig. 1a). When pre-mixed with increasing amounts of sENG, additional bands were observed representing sENG(M):BMP9 (bands 3, 4 and 5) and sENG(D):BMP9 (bands 6, 7 and 8) complexes. The identities of bands 1 to 8 were confirmed by excision and re-running on an SDS-PAGE (Supplementary Fig. 2b) and the relative abundance of the complexes was quantified (Supplementary Fig. 2c–e). Time-course experiment showed no evidence of prodomain reassociation after displacement (Supplementary Fig. 2f,g). Of note, sENG can displace the prodomain and form a complex with BMP9 GFD at molar ratios as low as 0.3, the lowest sENG concentration tested.

We then performed analytical gel filtration to confirm the native PAGE results (Fig. 1b, c). Under the identical running condition, pro:BMP9 eluted in fraction 18. When pre-mixing with 0.3-fold of sENG(M) (Fig. 1b) or 0.3-fold of sENG(D) (Fig. 1c), we detected additional peaks of sENG(M):BMP9 complex (fraction 16 in Fig. 1b) and sENG(D):BMP9 complex (fraction 13 in Fig. 1c), accompanied by the free prodomain (fraction 20 in Fig. 1b, c).

Similar results were obtained for pro:BMP10. On native PAGE (Fig. 1d), 0.3-fold sENG, either monomeric or dimeric, efficiently displaced the prodomain from pro:BMP10, resulting in reduced intensity of the pro:BMP10 band (band 2), the increased intensity of the prodomain band (band 3), and the appearance of new complex bands (bands 4–6 for sENG(M):BMP10 and bands 7–9 for sENG(D):BMP10). The identities of bands 1 to 9 were confirmed (Supplementary Fig. 3a), and complex abundance was quantified (Supplementary Fig. 3b–d). Time-course experiments showed no reassociation after prodomain displacement (Supplementary Fig. 3e, f). Similar to BMP9, 0.3-fold sENG(M) or sENG(D) displaced the prodomain and formed the corresponding complex with BMP10 GFD on gel filtration (Fig. 1e, f).

We next asked whether the type II receptor ECDs could displace the prodomain. It has been reported that BMP receptor type II (BMPRII)-Fc and activin receptor type IIB (ActRIIB)-Fc bind to BMP9 and BMP10 with the highest affinity15. Thus, we examined the ability of BMPRII ECD and ActRIIB ECD to displace the prodomain. Using untagged monomeric BMPRII ECD and ActRIIB ECD, we did not detect stable complexes of BMPRII or ActRIIB with BMP9 or BMP10 GFD on size exclusion chromatography under physiological buffer conditions such as PBS. We then performed native PAGE analysis under the same conditions as above. In the presence of BMPRII ECD (Fig. 1g), we did not detect any prodomain displacement for pro:BMP9, because pro:BMP9 bands remained at the same intensity across all experimental conditions (Supplementary Fig. 3g). In contrast, the pro:BMP10 band started to decrease when BMPRII was at a 1:1 ratio to pro:BMP10, suggesting partial displacement of the prodomain. Complete displacement was observed when BMPRII ECD was at threefold in excess. ActRIIB could not displace the prodomain of either pro:BMP9 or pro:BMP10 under identical experimental condition (Fig. 1h and Supplementary Fig. 3h).

These results confirm that both sENG(M) and sENG(D) displace the prodomain from pro:BMP9 and pro:BMP10 complexes far more efficiently than the type II receptors (Fig. 1i), suggesting that a significant function of ENG is to capture the circulating pro:BMP9 and pro:BMP10 at the cell surface.

The stoichiometry of ENG interactions with BMP9 and BMP10
To understand how ENG facilitates BMP9 and BMP10 signalling, it is important to elucidate the binding mode and stoichiometry of their interactions. Crystal structures were reported for ENG-OR alone, ENG-OR in complex with BMP9, and ENG zona pellucida (ZP) domain alone21. ENG-OR in complex with BMP9 shows that two copies of the orphan domain bind at the two knuckle areas of the BMP9 dimer, overlapping with the type II and prodomain binding sites (Supplementary Fig. 1e). Of note, to solve the ENG-OR and BMP9 structure, the complex was generated by co-expressing the maltose binding protein (MBP)-orphan domain fusion protein with BMP9 GFD in the same cells. Therefore, this crystal structure did not address how ENG displaced the prodomain.

As both sENG(D) and sENG(M) can completely displace the prodomain effectively and form complexes with BMP9 (and BMP10), there are a total of six possible forms of complexes after mixing pro:BMP9 (or pro:BMP10) with sENG(D) or sENG(M) (Fig. 2a). Based on the crystal structures21, it was modelled that one ENG dimer binds to one BMP9 dimer with BMP9 dimer being clamped by the two protomers of sENG(D) (Fig. 2a, form I). ENG monomer in complex with BMP9 was not detected in this report, probably due to the differentially tagged ENG employed and/or the limitation of the co-expression system.

We first assessed the size of the complexes by analytical gel filtration chromatography. sENG(M) was separately premixed with pro:BMP9 or pro:BMP10 at 6× molar excess, or sENG(D) premixed with pro:BMP9 or pro:BMP10 at 3× molar excess for 30 min before being loaded onto an S200 10/300 gel filtration column. As shown in Fig. 2b, c, control runs showed that sENG(M), sENG(D), pro:BMP9 and pro:BMP10 eluted at peak positions 1, 2, 3 and 7, respectively. Despite being at 6× molar excess, sENG(M) formed a complex with BMP9 or BMP10 GFD dimer at a 1:1 ratio (peaks 4 and 8) because no species that are close to or larger than sENG(D) was detected on the gel filtration, eliminating the possibility of two sENG(M) binding to the same GFD (Fig. 2a, form VI). Similarly, at 3× molar excess, the complexes between sENG(D) with BMP9 (peak 6) or BMP10 (peak 10) were only slightly shifted to the left of the sENG(D) peak, suggesting it is unlikely to have two copies of sENG(D) binding to the same GFD (Fig. 2a, form IV). Thus, gel filtration narrows down the possible forms of the complexes from six to four (Fig. 2a, forms I, II, III and V). Further experiments are required to ascertain whether forms I, II and III are all possible and whether one of them is a preferred form.

We also used crosslinking followed by SDS-PAGE to evaluate the stoichiometry. If two sENG(M) can bind to the same GFD dimer, we would expect a crosslinked species with a molecular weight larger than sENG(D) (Fig. 2d). Similarly, if two sENG(D) can bind to the same GFD dimer, the resulting species would be greater than 200 kDa (Fig. 2e). Experimental data revealed that crosslinked sENG(M) with BMP9 or BMP10 were detected at a molecular weight slightly bigger than sENG(M) but well below sENG(D) (Fig. 2f, g, black arrows), and crosslinked sENG(D) with BMP9 or BMP10 were detected at molecular weight below 200 kDa (Fig. 2f, g, arrowheads). Thus, the crosslinking experiments confirmed the gel filtration results that one molecule of sENG(M) or sENG(D) binds preferentially to only one BMP9 or BMP10 GFD dimer.

Binding of ENG to BMP9 and BMP10 reduces their binding affinity for ALK1
Since the ENG binding site on BMP9 partially overlaps with the type II but not the type I receptor binding site, we questioned whether ENG binding to BMP9 or BMP10 would affect their binding affinity for the type I receptor ALK1. To test this, surface plasmon resonance (SPR) experiments were performed by flowing BMP9 and BMP10 GFDs, with or without preincubation with sENG, over a CM5 chip immobilised with ALK1-Fc (Fig. 3). BMP9 bound strongly to ALK1, with a KD value of 58.6 pM (Fig. 3a, d), consistent with previous reports15,17. Preincubation with sENG(M) reduced BMP9 affinity to ALK1 by threefold, primarily due to a slower association rate (Fig. 3b, d). Incubation with sENG(D) reduced its affinity to ALK1 by 7.2-fold, due to a combination of a slower association rate and a slightly faster dissociation rate (Fig. 3c, d). A similar scenario was observed for BMP10. BMP10 bound to ALK1-Fc with a KD value of 183 pM (Fig. 3a, d). Binding to sENG(M) led to a ninefold decrease in affinity due to a slower association rate (Fig. 3b, d). Further reduction of the affinities (16-fold) was observed upon binding to sENG(D), due to both a slower association rate and a faster dissociation rate (Fig. 3c, d).

In conclusion, in the presence of ENG, BMP9 and BMP10 bind to ALK1 with slightly reduced affinity. This is likely due to steric hindrance because slower association rates primarily drive the reduced affinity. Of note, the presence of ALK1 ECD does not affect the binding affinities of BMP9 or BMP10 to ENG ECD (Supplementary Fig. 4).

Since BMP9 and BMP10 showed differences in prodomain displacement by BMPRII ECD (Fig. 1g and Supplementary Fig. 3g), and sENG:BMP10 complexes have different binding profiles to ALK1-Fc compared to sENG:BMP9 complexes (Fig. 3b, c), we reasoned that ENG and BMPRII may have different roles in BMP9 and BMP10 signalling. In this report, we focused the remainder of the mechanistic study on BMP9 only.

BMP type II receptors cannot efficiently displace ENG from ENG:BMP9 complex
We next investigated whether, once ENG has captured BMP9, the type II receptors are able to displace ENG to form a signalling complex. Using monomeric receptor ECDs, we did not detect displacement of ENG from its complex with BMP9 by BMPRII or ActRIIB ECDs in either native PAGE or gel filtration assays. We developed an ELISA-based competition assay to measure the displacement of ENG from sENG:BMP9 complex (Fig. 4a). Interestingly, BMPRII and ActRIIB showed displacement of sENG from sENG(M):BMP9 complex only at 125-fold excess, whereas BMPRII ECD displaced a small proportion of sENG from sENG(D):BMP9 complex from fivefold molar excess, reaching over 50% displacement when BMPRII ECD was at 125-fold molar excess. ActRIIB ECD displaced sENG from the sENG(D):BMP9 complex from 25-fold excess (Fig. 4b).

Type II receptors for ENG-dependent BMP9 signalling
We have previously shown that sENG:BMP9 complex can signal in wild-type ECs effectively, but not in Eng knockout ECs22. Since BMPRII and ActRIIB ECDs cannot effectively displace ENG from its complex with BMP9, the obvious other candidate to capture BMP9 from their complex with sENG is the cell surface ENG. This also suggests that after circulating pro:BMP9 is captured by cell surface ENG, it cannot effectively handover the ligand to cell surface BMPRII or ActRIIB for signalling. Another mechanism might be in place to allow ENG-bound BMP9 to signal.

Crystal structures revealed that all TGF-β family type II receptors interact with ligands at the knuckle epitope apart from TGFBRII, which binds TGF-β ligands at the fingertip15,18,24–26. Interestingly, ENG was initially discovered as a TGF-β co-receptor6 and was detected in a complex with TGFBRII in 125I-TGF-β ligand crosslinking assays27. Overlaying the ENG-OR:BMP9 structure21 onto the TGFBRII:TGF-β1 structure28 revealed that TGFBRII could potentially contact ENG:BMP9 complex without any clash (Fig. 5a). Therefore, we asked the question whether TGFBRII could participate in BMP9 signalling via ENG.

We performed siRNA knockdown of TGFBR2 in human pulmonary artery ECs (HPAECs) in parallel with knockdown of other type II receptors known to mediate BMP signalling and investigated pSmad1/5 signalling responses to BMP9 treatments. In agreement with previous findings29, knocking down the type II receptor individually did not completely abolish BMP9 signalling (Fig. 5b, c). However, knocking down TGFBR2 or ACVR2A resulted in partial reduction of the signalling, whereas knocking down BMPR2 did not affect BMP9-induced Smad1/5 phosphorylation, suggesting TGFBRII could be one of the type II receptors mediating BMP9 signalling.

We next asked whether TGFBRII can interact with BMP9 extracellularly to mediate BMP9 signalling. We did not detect any direct interaction between BMP9 GFD and TGFBRII ECD using either SPR or ELISA-based binding assays (Supplementary Fig. 5a–d). However, since it has been shown that ENG interacts with TGFBRII intracellularly by immunoprecipitation in transfected COS-7 cells10, we asked whether such intracellular interactions are present in human primary ECs, which could enable ENG to bring TGFBRII to ALK1 via BMP9. We have previously generated an anti-ENG affinity column, which pulled down sENG from ex vivo-cultured placenta-conditioned media22. Using this affinity column, we pulled down endogenous ENG and its interaction partners from total cell lysates of human primary aortic ECs (HAOECs), HUVECs as well as HPAECs and analysed total eluates by mass spectrometry. A BSA-coupled NHS column was used as the negative control to allow detection of those proteins that interact with the column non-specifically (Fig. 5d). ECs under three different culturing conditions were harvested and analysed: (1) medium with 0.1% Fetal Bovine Serum (FBS); (2) medium containing 10% FBS; (3) medium with 0.1% FBS and pro:BMP9. Of note, cells in 0.1% FBS medium do not have active BMP9 signalling, whereas 10% FBS contains high concentrations of BMP9 as well as other serum factors. Figure 5e summarises the mass spectrometry results, showing as the total number of unique peptide counts of ENG binders as well as controls. The detected peptides are highlighted on the protein sequences in Fig. 5f and Supplementary Fig. 6. The anti-ENG column pulled down many more ENG fragments from all ECs across three medium conditions (23–32 fragments) compared with the control BSA column (3–9 fragments). Interestingly, under 0.1% FBS condition, both TGFBRII and ALK1 were detected only in the anti-ENG column elutes for all the ECs examined. This indicates that endogenous ENG, TGFBRII and ALK1 are associated in human primary ECs under this condition. Under 10% FBS condition and upon BMP9 treatment, where Smad/1/5 phosphorylation is activated (Fig. 5g), we detected more peptides from TGFBRII, but no ALK1 peptide was detected. As a positive control, we detected a previously reported ENG binding protein TRIM21 in all anti-ENG column eluates30. For a negative control, RALA was pulled down from both anti-ENG and BSA columns at a similar level. Of note, under 10% FBS and BMP9-treated conditions, ENG may still interact with ALK1 indirectly via BMP9 (Fig. 3), but we will not be able to detect such interaction in this pull-down assay because the anti-ENG antibody used for the affinity column is TRC105, which blocks BMP9 binding to ENG21,31. Proteomic findings are summarised in schematics (Fig. 5h). These data support the idea that ENG can bring TGFBRII and ALK1 together, allowing TGFBRII to participate in BMP9 signalling in an ENG-dependent manner.

Model of ENG-dependent and ENG-independent BMP9 signalling
Our data strongly suggest that one aspect of ENG’s co-receptor function is to capture BMP9 and BMP10 from the circulation and deliver them to cell surface ALK1. ENG ECD monomer is over 60kDa (561 amino acids) with two distinct domains, whereas ALK1 ECDs is a much smaller (96 amino acids) single domain protein (Supplementary Fig. 7a). ENG clearly has the advantage of having a better reach into the circulation lumen for capturing BMP9 and BMP10, but at the same time, it will need to have a flexible structure to allow it to deliver the bound ligands to ALK1. We noticed that sENG(D) migrated as two distinct bands on the native PAGE (Fig. 1a, d), and both could bind to ligands because both bands shifted upon binding to BMP9 or BMP10 (Fig. 1a, d). This suggests that native sENG(D) may exist in two different forms with different net charge or shape. Although gel filtration could not separate the two forms and sENG(D) runs under a single peak (Fig. 2b), crosslinking experiments showed that only about 50% of the sENG(D) were crosslinked in solution, despite that four crosslinkers with different spacer arm lengths were used across different crosslinker concentrations (Fig. 6a, b and Supplementary Fig. 7b).

To further develop the model for cell surface ENG function, we need to know whether it exists in only dimeric form or both dimer and monomer forms. We noticed that in total cell lysates of human primary ECs from different vascular beds and different donors, a monomeric form of ENG can be consistently detected regardless of the culturing media (Supplementary Fig. 7c, d). To examine whether such monomeric ENG is at the cell surface or intracellular, we performed surface biotinylation coupled with immunoprecipitation by streptavidin beads (Fig. 6c) to detect ENG only at the cell surface. After surface biotinylation, both dimeric and monomeric ENG were pulled down by streptavidin agarose beads in both HPAECs and HUVECs (Fig. 6d, lane 7). For controls, no ENG was pulled down without biotinylation (Fig. 6d, lane 4); and the intracellular protein β-actin was not labelled by cell surface biotinylation, therefore only existed in the unbound fractions (Fig. 6d, lane 5), not in the elution (Fig. 6d, lane 7). These results support that both dimeric and monomeric ENG are present at the surface of these human primary ECs (Fig. 6e).

Our results thus support the following model of ENG co-receptor function for BMP9 signalling in ECs (Fig. 6f). Cell surface ENG can be in both monomeric and dimeric forms. Both can efficiently capture the BMP9 GFDs from circulating pro:BMP9, displace the prodomains, deliver the GFDs to cell surface ALK1 and form a ternary complex with BMP9 and ALK1. A proportion of this complex can dissociate to allow BMPRII or ActRIIA to bind; however, in the remaining complex, ENG-bound TGFBRII can phosphorylate ALK1 to initiate Smad-dependent or Smad-independent signalling.

The difference between ENG-dependent and ENG-independent signalling
We next questioned whether ENG-dependent and ENG-independent signalling paths would result in similar or different signalling outcomes; for example, different type II receptors may have different efficiency in phosphorylating ALK1. Previous efforts using microarray to compare sENG(M):BMP9 and pro:BMP9 signalling in HPAECs did not reveal any difference22, probably due to the significant overlap in signalling. Here, we isolated mouse lung ECs (MLECs) from Eng-floxed mice (Rosa26-CreERT2;Engfl/fl) and depleted Eng by 4-OH-tamoxifen treatment (Fig. 7a). Because only ENG-independent signalling occurs in Eng−/− (KO) cells, by comparing sENG(M):BMP9 signalling in Eng+/+ (Ctrl) cells versus pro:BMP9 signalling in KO cells, we anticipated better signals to tease out ENG-dependent signalling (Fig. 7b).

We confirmed the EC identity of Ctrl and KO MLECs by CD31 and VE-Cadherin staining (Fig. 7c and Supplementary Fig. 8a). The knockout of Eng was confirmed by ENG staining, immunoblotting and qPCR (Fig. 7c, d and Supplementary Fig. 8a, b). As we have shown previously, knocking out Eng led to reduced BMP9 signalling in the Smad1/5 phosphorylation assay (Fig. 7d)22. We treated Ctrl cells with sENG(M):BMP9 (or PBS) and KO cells with pro:BMP9 (or PBS) and then performed RNA sequencing to explore Eng-dependent and Eng-independent target genes. We first validated our RNA sequencing results by confirming the known differentially expressed genes (DEGs) induced by BMP9 in Ctrl cells and KO cells. Treatment of sENG(M):BMP9 in Ctrl cells (Fig. 7e) and pro:BMP9 in KO cells (Fig. 7f) induced canonical BMP9 target genes, such as Smad6, Smad7, Id1 and Atoh8. We then compared the DEGs in PBS-treated Ctrl and KO cells to examine the effect of knocking out Eng on MLECs at baseline. Only three genes were significantly downregulated after losing ENG, which are Col3a1, Elovl6 and Reln. However, the fold changes were minimal (Log2FC=0.45 to 0.69) (Fig. 7g). In contrast, when comparing DEGs from ligand-treated Ctrl cells and KO cells, we detected 76 significantly changed DEGs (Fig. 7h). Several DEGs had fold changes equal or bigger than 2, with the most significant fold changes observed for Nog and Adamtsl2, suggesting that the induction of these two genes was more dependent on ENG.

Significantly changed DEGs in Fig. 7h were subject to pathway analysis using String32. Three distinct clusters of protein-protein interaction network were detected (Supplementary Fig. 9a), centred around Irf9/Ifih1/Oasl1, Col3a1/Col6a2/Col6a1, and Bmp4, respectively. Gene ontology and KEGG pathway showed that significantly enriched terms include response to interferon-β, regulation of the viral processes, and TGF-β signalling pathways (Fig. 7i, j and Supplementary Fig. 9b, c).

Validation of ENG-dependent target genes in mouse and human primary ECs
RT-qPCR experiments were performed to validate the significantly changed DEGs (Fig. 7h). We first measured the basal expression of selected genes relative to the housekeeping gene Hprt and calculated the ratio of the basal expression in KO/Ctrl cells (Fig. 8a, dark grey bars). Most of the values are close to 1, indicating that the basal expressions of these genes were similar in KO and Ctrl cells without BMP9 treatment. We then measured the fold induction of these genes after ligand treatment (ENG(M):BMP9 in Ctrl cells and pro:BMP9 in KO cells, as shown in Fig. 7b), and calculated their ratio of KO/Ctrl (Fig. 8a, light grey bars). Interestingly, the ratios for most of the genes are below 1, indicating that the fold induction in KO cells is lower than in the Ctrl cells, consistent with the RNAseq data (Fig. 7h). When comparing the ratios in untreated cells (Fig. 8a, dark grey bars) with those in ligand-treated cells (Fig. 8a, light grey bars) for each investigated gene, the differences in Adamtsl2 and Nog were statistically significant. We confirmed that the lower fold induction in Adamtsl2 and Nog was not due to the difference in basal expression (Fig. 8b) but because of the lower fold induction in the KO cells (Fig. 8c).

It has been reported that HDMECs express significantly lower ENG than ECs from other vascular beds33. We hypothesised that HDMECs might be similar to the Eng KO MLECs in that they would have less fold induction of ENG-dependent BMP9 target genes. We first compared ENG expression in HDMECs with that in HAOECs and HUVECs at both protein and mRNA levels (Fig. 8d–f) and confirmed that ENG expression in HDMECs was much lower than that in HAOECs and HUVECs. We then performed BMP9 signalling assays in these cells, at both 0.1 and 1ng/ml BMP9 GFD concentrations. BMP9-induced the canonical BMP target gene ID1 at both concentrations in all three primary ECs, and fold induction was very similar across the three ECs (Fig. 8g). In contrast, BMP9 effectively induced NOG and ADAMTSL2 in both HAOECs and HUVECs, but not in HDMECs (Fig. 8g), and this was not due to the difference in the basal expression of these genes in different ECs (Fig. 8h). This is consistent with NOG and ADAMTSL2 being ENG-dependent genes, and the lack of induction in HDMECs is probably due to the very low ENG expression.

ENG, NOG and ADAMTSL2 in PAH patient samples
To assess the human relevance of our findings, we analysed a publicly available microarray dataset of human lung tissue34 and found the expression levels for ENG, NOG and ADAMTSL2 were all significantly reduced in PAH patients’ lungs compared to control (Fig. 9a). Importantly, the expression levels of both NOG and ADAMTSL2 were strongly and positively correlated with ENG, and with each other (Fig. 9b). This is consistent with our finding that NOG and ADAMTSL2 are ENG-dependent. We next examined these genes in the published whole blood RNAseq dataset from 363 PAH samples versus 126 controls35. The expression levels for ENG and NOG were both significantly reduced in PAH and significantly correlated with each other (Fig. 9c, d). Furthermore, lower levels of NOG mRNA were associated with poorer transplant-free survival in PAH patients (Fig. 9e) and worse REVEAL 2.0 mortality risk scores36 (Spearman test, Rho = −0.14, P = 0.0083). In a published proteomics dataset of whole blood plasma37,38, Noggin protein levels were also significantly reduced in PAH (Fig. 9f), consistent with our findings. Of note, we observed increased sENG protein in PAH patients’ plasma as previously reported5.

Methods
Proteinexpressionandpurification
cDNAsoffull-lengthhumanBMP9andBMP10,includingtheprodomain,sENG(Residue1-586)-(His) wereclonedintoapCEP4plasmid.Forproteinexpression,eachplasmidwastransfectedintoHEKEBNAcellsinDulbecco’smodifiedEagleMedium(DMEM,11965092,Gibco),5%FBS.ForBMP10expression,humanfull-lengthFURINcDNAinpCEP4wasco-transfectedtofacilitateprocessing.ThetransfectionmediumwasreplacedbyCDCHO(10743-029,ThermoFisherScientific)mediumonday2,andtheconditionedmediumwasharvestedtwiceaweekfor5harvests.

Topurifypro:BMP9andpro:BMP10,theconditionedmediumwasloadedontoa5-mlHiTrapQHPcolumn(17115401,Cytiva)andelutedwithaNaClgradient.ThiswasfollowedbyaHiLoadSuperdex200pg16/600gelfiltrationcolumnpre-equilibratedin20mMTris,50mMNaCl,pH7.4.PooledfractionswereloadedonaMonoP5/50GLcolumnandelutedwithaNaClgradient.TheelutedsamplewaspolishedusingaHiLoadSuperdex200pg16/600gelfiltrationcolumnpre-equilibratedin20mMTris,150mMNaCl,pH7.4.

sENG-HiswasexpressedasamixtureofsENG(D)andsENG(M)22.Theywereinitiallycapturedona5-mlHisTrapExcelcolumn(17371206,Cytiva)andtheelutedfractionswerepooledanddialysedin20mMTris,50mMNaCl,pH7.4.Thedialysedsamplewasthenloadedona5-mlHiTrapQHPcolumnandelutedwithaNaClgradient.sENG(D)andsENG(M)wereseparatedbyaHiLoadSuperdex200pg16/600gelfiltrationcolumnin20mMTris,150mMNaCl,pH7.4.

ThecDNAforhumanALK1(Residue22–118),BMPRII(Residue27–150)andActRIIB(Residue24–115)ECDsweredonedseparatelyintoapET-39bplasmid(70090,Novagen)tocreateafusionproteinDsbA-(His) -TEVsite-ECD.TheexpressionandpurificationforalltheECDswereidentical.Inbrief,theplasmidforeachproteinwastransformedintoE.coliRosettaDE3(70954,Novagen)forproteinexpression.Atmid-logphase,cellswereinducedby200μMisopropylβ-D-thiogalactopyranoside(IPTG)andthengrownat22°Covernight.TheECDswereextractedfromperiplasmicfractionsaccordingtothepETSystemManual(Novagen).TheHis-taggedfusionproteinswerecapturedandelutedfroma5-mlHisTrapexcelcolumn.ThiswasfollowedbyovernightTEVdigestionanddialysisinTBSat4°C.ThedigestedproteinswerepassedthroughtheHisTrapexcelcolumntoremovetheHis-taggedDsbAandTEVprotease.TheECDswereफिरpassedthroughaHiLoadSuperdex75pg16/600gelfiltrationcolumntopurifytheproteinsfurther.

Cellsurfacebiotinylation
HPAECsandHUVECswereseededin6-wellplatesovernightinendothelialgrowthmedium2(EGM2,C-22111,PromoCell)with10%FBS.Cellswerewashed3timeswithice-coldPBSpH8.0andthenincubatedwith0.33mg/mlThermoEZ-LinkSulfo-NHS-SS-Biotin(21328,ThermoFisherScientific,membraneimpermeable)inPBSfor30minat4°C.Thereactionwasquenchedwith2mlperwell35mMTrispH8.0for10minatroomtemperature,followedby2timeswashwithPBS.Thecellswerelysedwith200μlperwelllysisbuffer(125mMTrispH7.4,10%glycerol,2%SDS,2mMEDTAand1×proteaseinhibitor(11836170001,Sigma-Aldrich))onice.Thelysatewascentrifugedat20,000×gfor10minat4°Candthesupernatantwasincubatedwith150μlPierceAvidinAgarose(20219,ThermoFisherScientific)andincubatedwithmixingfor1hatroomtemperature.Theresinwasthenwashed5timeswithPBSandboiledin1×SDSloadingdye.ENGwasassessedbyimmunoblotting.Anti-ENG(555690,BDBioscience)andanti-β-Actin(A5441,Sigma-Aldrich)wereusedandrelatedinformationcanbefoundinSupplementaryData1.

Crosslinking
Proteinswereincubatedwith1mM(finalconcentration)ofDSG,BS3,BS(PEG) orBS(PEG) (A35392,A39266,A35396,21582,respectively,fromThermoFisherScientific)inPBSpH7.4–8.0for45minatroomtemperature.Thereactionwasthenquenchedwith40mMTris(finalconcentration)pH7.4–8.0for15minatroomtemperature.Concentrationsofthecrosslinkersweretitratedtooptimisethecrosslinkingefficiency.

NativeandSDS-PAGE
Pro:BMP9orpro:BMP10waspre-mixedwithsENG(D),sENG(M),BMPRIIorActRIIBECDsatratiosindicatedinthefigurelegendsinTBSinafinalvolumeof8μlandincubatedfor30minatroomtemperature.Thesampleswerethenrunona10%nativePAGEat4°C.Whereappropriate,theCoomassieInstantBlue(ab119211,Abcam)stainednativegelbandswerecutoutandinserteddirectlyintothewellsofa12%SDS-PAGEgelandrunundernon-reducingconditiontorevealtheiridentity.

Surfaceplasmonresonance
Surfaceplasmonresonance(SPR)bindingexperimentswereperformedusingaBiacoreT200system(Cytiva/GEHealthcare).Forallassays,ALK1-FcorsENG-FcwasimmobilisedontoaSeriesSCM5chip(BR100530,Cytiva)viaaminecoupling.TheSPRanalyteswereinjectedinduplicateataflowrateof40μl/min(inabuffercontaining10mMHEPESpH7.4,150mMNaCl,3.4mMEDTA,0.5mg/mlBSAand0.005%(v/v)surfactantP20)at25°C.Thesurfacewasregeneratedwith1–3injections(dependingonthebindingpartners)of4MGuanidineHydrochloride.Thekineticrateconstantswereobtainedbyfittingthecorrecteddatatoa1:1interactionmodelorsteady-statemodelusingBIAevaluationsoftware(Cytiva/GEHealthcare).

GeneratingEng-iKOECs
Rosa26-CreERT;Engfl/fl mice have been described previously22,51 and maintainedinaC57BL/6background.AllprocedureswereapprovedbytheUniversityofCambridgeAnimalWelfareEthicalReviewBoardundertheauthorityoftheUnitedKingdomHomeOfficeProjectLicencesPP7550697andPP3923706.Micewerehousedinapathogen-freebarrierfacilityundera12-hlightand12-hdarkcycleatambienttemperatureandhumiditywithstandardchowandwateradlibitum.PrimarymouselungmicrovascularECs(MLECs)wereisolatedfromtheRosa26-CreERT2;Engfl/fl mice carrying the Immorto mouse transgene using CD31 (553370, BD Biosciences)- and then ICAM2 (553326, BD Biosciences)-coated beads following the protocols as describe before22. MLECs were cultured at 33°C in MV2 media (C22121, PromoCell) with 10% FBS, 100μg/mlheparin,20U/mlinterferon-γ(315-05,PeproTech)and1μMSB431542(S4317-5MG,Sigma-Aldrich).Engknockoutwasinducedbytheadditionof2μM4-OH-tamoxifen(H7904,Sigma-Aldrich)intheculturemediafor72h.Cellswereseededintheculturemediawithoutinterferon-γ,SB431542and4-OH-tamoxifenat37°Cfor24hfollowedbyserumstarvationovernightinMV2mediacontaining0.1%FBSand100μg/mlheparinat37°Cbeforesignallingassays.AntibodyinformationcanbefoundinSupplementaryData1.

ENGdisplacementELISA
A96-wellplate(655001,Greiner)wascoatedwithanti-humanCD105antibody(555690,BDBiosciences,0.17μgperwell)inPBSandincubatedat4°Covernight.ThewellswerethenwashedwithPBS-Tandblockedwith1%BSA/PBSfor2hatroomtemperature.sENG:BMP9wasaddedtotheplateandincubatedfor2hatroomtemperature.AfterwashingwithPBS-T,BMPRIIorActRIIBECDs(at0,1,5,25and125×)wasaddedtothewellsfor30minatroomtemperature.Theplatewaswashedandincubatedwiththebiotinylatedanti-BMP9antibody(BAF3209,Bio-Techne,0.04μgperwell).Aftera2-hincubationatroomtemperature,theplatewaswashedandincubatedwith1mg/mlExtrAvidin-Alkalinephosphatase(E2636,Sigma-Aldrich)for30min.Theassaywasdevelopedwith4-Nitrophenylphosphate(pNPP)disodiumsalthexahydrate(S0942,Sigma-Aldrich)in1MDiethanolamine,0.5mMMgCl pH9.8andtheabsorbancewasmeasuredat405nm.

ENGpull-downfromprimaryECsfollowedbymassspectrometryanalysis
TRC105,achimericIgG1monoclonalantibodythatbindsENG,wasa kindgiftfromTRACONandwasusedtomaketheENGaffinitycolumnpreviously22.A1mlNHS-BSAcolumn(NHScolumnswerepurchasedfromCytiva,17-0716-01)wasgeneratedusingthesamemethodasanegetivecontrol.ThreehumanprimaryECs,HAOECs,HUVECsandHPAECsweregrowninT175flasksinEGM2with10%FBSfortwodays.Dependingontheexperiment,cellsweremaintainedinEGM2with10%FBS,orserum-starvedinEGM2with0.1%FBSfor20h,orserum-starvedinEGM2with0.1%FBSfor20hfollowedbytreatmentwith0.25ng/mlpro:BMP9for40min.Thecellswerethenharvestedandlysedin1%Triton-X100/PBSwith2mMEDTAandproteaseinhibitor(11873580001,Merck).Lysedcellswerecentrifugedat20,000×gfor10minat4°C.Thesupernatantfromeachexperimentwasdividedintotwoequalportions.Oneportionwaspassedthroughthe1mlENGaffinitycolumn,whiletheotherportionwaspassedthroughthe1mlNHS-BSAcolumnundertheidenticalcondition.ThecolumnswerewashedwithPBS,andboundproteinswereelutedwith0.15MglycinepH2.5.Theeluateswerecollected,TCAprecipitatedandrunonSDS-PAGEunderreducingconditionforabout1cm.ThestainedbandswerecutoutandsentforLC-MS/MSanalysisattheProteomicsCoreFacility,DepartmentofBiochemistry,UniversityofCambridge.Totalof18sampleswereanalysedacrossthree-differentculturingconditions,eachconditioncontainingN=3negativecontrolandN=3biologicalrepeats(threedifferentEClinesinthiscase).

Samplepreparationsformassspectrometryareasfollows:SDS-PAGEbandswerecutinto1–2mmpiecesandwashedin50%acetonitrile/100mMNH HCO solutionuntilclear.Reductionwasperformedwith10mMDTTin100mMNH HCO at37°Cfor1hfollowedbyalkylationwith55mMiodoacetamidein100mMNH HCO atroomtemperaturefor45mininthedark.Trypsin(V5111,Promega)wasaddedat5ng/ml50mMNH HCO ,digestionwasproceededovernightat37°C.

LC-MS/MSexperimentswereperformedusingaDionexUltimate3000RSLCnanoUHPLCsystemcoupledtoanOrbitrapFusionLumosmassspectrometer(ThermoFisherScientific).PeptideswerefirstloadedontoaPepMap100C18pre-column(5µm,100Å,300µm×5mm)with0.1%formicacidfor3minat15µl/min,thenseparatedonaPepMapC18analyticalcolumn(2µm,100Å,75µm×50cm)at300nl/min.ChromatographicseparationusedsolventA(0.1%formicacidinwater)andsolventB(80%acetonitrile,0.1%formicacid)witha2–30%Bgradientover30min.ElutingpeptideswereionizedusinganEasy-Spraysource.

MS1scanswereacquiredintheOrbitrapat120,000resolutionoverm/z380–1500.Data-dependentMS/MS(3s-cycle)usedhighenergycollisional-induceddissociation(HCD)(NormalisedCollisionEnergy(NCE)=32%)withfragmentdetectionat15,000resolution.Singlychargedions,ionswith>7charges,andunassignedchargestateswereexcluded;dynamicexclusionwassetto70s.

RawdatawereprocessedinProteomeDiscoverer2.5(ThermoFisherScientific)andexportedasmgffilesforMascot(v3.1,MatrixScience)searchingagainstacontaminantsdatabaseandtheCCP_Homo_sapiens_20221011database.Searchesemployedvariablemodificationsofoxidation(M)anddeamidation(N/Q),fixedcarbamidomethyl(C),andJ-28leucine/isoleucineambiguity,allowingtwomissedcleavages.Masstoleranceswere20ppm(precursors)and0.1Da(fragments).Proteinabundanceswerecalculatedfrompeptidepeakareas.

PeptideidentificationswerevalidatedinScaffold(v5.1.1)at≥95%probability(LocalFDR),andproteinsat≥99%probabilitywith≥2peptides,usingProteinProphet52.TheresultswereanalysedandviewedusingScaffold5(ProteomeSoftware.com).

RNAextractionandRT-qPCR
CellswerelysedandRNAwasextractedusingRNeasyMiniKitbuffers(74104,Qiagen)followingthemanufacturer’sprotocol.1μgRNAwasreversetranscribedusingaHigh-CapacitycDNAReversetranscriptasekit(4368814,ThermoFisherScientific)inaVeritithermalcycler(AppliedBiosystems).QuantitativePCRwasperformedon1/10dilutedcDNAsamplesusingPowerUPSYBRGreenMasterMix(A25777,AppliedBiosystems)inaMicroAmp®Optical384WellReactionplate(10005724,AppliedBiosystems)onaQuant-Studio6FlexReal-TimePCRsystem(AppliedBiosystems).TherelativeexpressionlevelswerecalculatedusingtheΔCtmethodandfoldchangesinexpressionlevelswerecalculatedusingtheΔΔCtmethod53.ThesequencesoftheprimersareinSupplementaryData1.AlltheprimersweresynthesisedbySigma-Aldrichandwerepreviouslyvalidatedinpublishedstudiesorinourassays.

SignallingassayinMLECs
Engknockoutwasinducedby2μM4-OH-tamoxifenwithvehicle-treatedcellsascontrols.Cellswereseededin6-wellplatesandserum-starvedovernightinMV2mediawith0.1%FBSand100μg/mlheparinat37°C.TheywerethentreatedwithPBS,pro:BMP9(0.2ng/mlGFDconcentration)orsENG(M):BMP9(0.2ng/mlGFDconcentration,sENGinlargeexcesstostabilise-thecomplex)for15minbeforebeingharvestedforanalysis.ThetotalproteinwasquantifiedusingtheDCproteinassay(5000112,Bio-Rad).Equalamountsoflysatewerefractionatedona10%SDS-PAGEandSmad1/5phosphorylation(9516S,CellSignalling)wasdetectedbyimmunoblotting.KnockoutefficiencyofENGwasconfirmedbyimmunoblotusingAF1320(Bio-Techne)andRT-qPCR.β-actin(A5441,Sigma-Aldrich)wasusedastheloadingcontrol.AntibodyinformationcanbefoundinSupplementaryData1.

Immunofluorescencestaining
MLECswereseededincellsofaMilicell®EZSlide(PEZGS0416,Merck)overnight.Theywerefixedthefollowingdaywithacetone+methanol(1:1)for5minatroomtemperature.Followingblockingwith1%BSAinPBS,thecellswerestainedwiththecorrespondingprimaryantibodies(CD31,553370,BDBioscience;VE-Cadherin,555289,BDBiosciences;ENG,14-1051-82,AffymetrixeBioscience,SupplementaryData1)for2hatroomtemperature.Theywerethenwashedandincubatedwithsecondaryantibodies(488,A-21208,Invitrogen;568,A78946,ThermoFisherScientific)for1hatroomtemperature.NucleiwerecounterstainedwithDAPI(40043-BT,Biotium)andimageswereacquiredusinganInvitrogenEVOSM5000ImagingSystem.Fluorescenceimageswereacquiredfromcellsisolatedfromthreedifferentmice.Foreachstainedcellline,4–5imagesweretakenfromdistinctregionsforeachprotein.Microscopesettingswerekeptconstantforagivenproteinacrossallimagesandcells.Totalintegratedfluorescenceintensity(IntDen)wasmeasuredusingFijisoftware(https://www.nature.com/articles/nmeth.2019).TheIntDenwasthennormalisedbythenumberofcellsineachimagetoobtaintheaverageintensitypercell(IntDen/cell).ThefinalIntDen/cellvalueforeachcelllinerepresentsthemeanofallimagescollectedforthatline.

RNAsequencingandbioinformaticanalysis
MLECsfromfourindependentisolationswereused.ForeachMLECline,Engknockoutwasinducedby2μM4-OH-tamoxifenwithvehicle-treatedcellsascontrols.Cellswereseededin6-wellplatesandserum-starvedovernightinMV2mediawith0.1%FBSand100μg/mlheparinat37°C.TheywerethentreatedwithPBS,pro:BMP9orsENG(M):BMP9(allat0.2ng/mlGFDconcentration,sENGinlargeexcesstostabilise-thecomplex)for1.5hbeforebeingharvestedandlysed.RNAextractionwasperformedasdescribedabove.TheintegrityandqualityoftheextractedRNAwereassessedusinganAgilentTapeStation4200System(agilent.com).LibrarypreparationwasconductedbyNovogene(www.novogene.com)usingtheNovogeneNGSStrandedRNALibraryPrepSet(PT044).RNAsequencingwascarriedoutbyNovogeneusinganIlluminaNovaSeqXPlussystemtogenerate150bp-pairedendreads.

RawdataprocessingandqualitycontrolwereperformedbyNovogeneusingin-housePerlscripts.ThereadswerealignedtotheMusmusculusGRCm39referencegenomeusingHisat2(v2.0.5).MappedreadsandsamplemetadatawereimportedintoR(v.4.0.3)fordownstreamanalysis.First,BiomaRt54(v.2.54.0)mappedtheensemblegeneIDs to HGNC symbols. After performing quantile normalisation, COMBAT55fromthesvapackagewasappliedtoremovebatcheffects.DifferentialexpressionanalysiswasconductedusingDESeq256(v.1.26.0)comparingthedifferentsamplegroups.TheBenjamini-Hochbergmethodadjustedtherawp-valuesfortheFalseDiscoveryRate(FDR).PathwayanalysiswasassessedusingStringserver(https://string-db.org/).

SignallinginhumanprimaryECs
HUVECs,HAOECs,HDMECsandHPAECswereplatedin6-wellplatesinEGM-2mediawith10%FBS.Thefollowingdaycellswereserum-starvedwith0.1%FBSEGM-2mediafor20hbeforebeingtreatedwithPBSorpro:BMP9at0.1or1ng/mlGFDconcentrationforthedurationasindicatedinthefigurelegends.CellswerethenharvestedforRNAextractionorimmunoblotting.

siRNAknockdownofreceptors
HPAECs(in60mmplate)weretransfectedusingDharmaFECT3transfectionreagentaccordingtoDharmaFECTprotocolwith50nM(finalconcentration)siRNAs(ON-TARGETplusSmartpoolhumansirnasfromDharmacon).Thefollowingdaycellswerere-seededpriortostimulation.Cellswereserum-starvedovernightbeforetreatmentwithBMP9GFDfor15minandharvestedinSDS-sampleloadingbufferorRNAextractionbuffer.RNAwasisolatedusingtheNucleoSpinRNAIIkit(BIOKE),followedbyreversetranscriptionPCR(RevertAidFirstStrandcDNASynthesisKits,Fermentas).ThecDNAswereanalysedbyqPCR,andcalculationswereperformedusingtheCFXManagersoftwareversion2.0(Bio-Rad).AllmRNAlevelswereanalysedintriplicateandnormalisedtoGAPDHexpression.

Bioinformaticsanalysisofhumansamples
ENG,NOG,andADAMTSL2expressionwasanalysedinapubliclyavailablehumanlungmicroarraydataset(GSE11343934containing15PAHpatientsand11controls).Differencesbetweennormalisedexpressionvalueswereassessedusingatwo-sidedMann–WhitneyUtest.InapreviouslypublishedwholebloodtranscriptomicdatasetofPAHpatients(n=363)andhealthycontrols(n=126)35,differencesingeneexpression,asmeasuredbytranscriptspermillion(TPM),wereassessedbetweencasesandcontrolsforNOGandENGusingatwo-sidedMann–WhitneyUtest.InthelungmicroarraydataandwholebloodRNAseq,transcriptomiccorrelationsbetweenENG,NOG,andADAMTSL2werecalculatedusingatwo-sidedSpearmantestforbothcasesandcontrolsusingLog2expressionlevelsorTPM.PlasmaproteomiclevelsofENGandNOGwerecomparedbetweenpatients(n=463)andcontrols(n=108)fromapreviouslypublisheddataset37,38.Differencesinnormalisedproteinlevelbetweencasesandcontrolswereassessedusingatwo-sidedMann–WhitneyUtest.

ToassesswhetherNOGexpressionlevelsareassociatedwithlong-termoutcomes,asurvivalanalysiswasperformed.NOGexpressionlevelsweresplitintwogroupsonitsmedianexpressionlevelinadult(diagnosedat≥18years)PAHpatientswithlong-termfollow-updataavailable(n=355).Subsequently,aKaplan–Meiercurvewasconstructedcomparingright-censoredtransplant-freesurvivalfromdiagnosisupto15yearspostdiagnosisusingtheSurvival(v3.8-3)andSurvminer(v0.5.0)packagesinR/Rstudio(version4.4.3).Alog-ranktestwasperformed.Subsequently,NOGexpressionlevelswerecorrelatedwithREVEAL2LitescoresusingaSpearmantest.

TheclinicaldatawereobtainedfromtheNationalCohortStudyofIdiopathicandHeritablePAH(UKcohort;NCT01907295,EastofEnglandEthicsCommittee:13/EE/0203)withagroup1PHdiagnosis.TheanalysesinthismanuscriptincludedpatientswithIPAHandHPAHdiagnosedat18yearsofageorolder.Clinicaldatawascollectedprospectivelyatannualoutpatientvisitsandthelaststudycensustookplaceon01/07/2022.Ifpatientswerediagnosedbeforethestudystartdate,diagnosticparameterswereincludedretrospectivelyinthestudy.

Statisticalanalysis
QuantitativedatafromqPCRandImmunoblotexperimentswerefromatleastthreeindependentrepeatsasindicatedinfigurelegend.ThestatisticalanalyseswereperformedusingGraphPadPrismversion10.2.3.Two-sidedunpairedT-test,one-wayANOVAortwo-wayANOVAwasusedasindicatedinthefigurelegends.P<0.05wasconsideredstatisticallysignificant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This study provides compelling evidence for the dual role of endoglin (ENG) as a co-receptor for BMP9/10 signaling, bridging the gap between its previously reported functions in TGF-β and BMP pathways. The authors demonstrate that ENG efficiently displaces prodomains from pro-BMP9/10, recruits TGFBRII to the signaling complex, and identifies Nog and Adamtsl2 as ENG-dependent BMP9 target genes. This manuscript presents a significant advance in understanding ENG’s role in endothelial signaling. The work is well-executed and addresses a long-standing controversy in endothelial biology. However, some methodological details and interpretations require clarification or additional validation to strengthen the conclusions. Addressing the following points will elevate the impact and clarity of the work. I recommend acceptance pending revision.\n1. Quantify prodomain displacement efficiency in Figure 1 using densitometric analysis of native PAGE bands, and perform kinetic assays to exclude potential prodomain reassociation post-displacement.\n2. Provide structural evidence (e.g., SEC-MALS) to substantiate the hypothesis that ENG adopts multiple conformations in Figure 2a.\n3. Label the analyte concentrations used in surface plasmon resonance (SPR) experiments in the legend of Figure 3.\n4. Include statistical comparisons between the displacement efficiencies of BMPRII and ActRIIB in Figure 4b.\n5. Validate the ENG-TGFBRII-ALK1 interaction in primary endothelial cells (ECs) by conducting co-immunoprecipitation (co-IP) assays with endogenous proteins under physiological conditions to support Figure 5e.\n6. Optimize the schematic diagram of Figure 6d, and list the small diagrams separately on one side, clearly marking the important proteins such as the receptors.\n7. Provide statistical analysis and comparison on the immunofluorescence intensity in Figure 7c.\n8. Perform functional rescue experiments, such as ENG overexpression or TGFBRII knockdown, to confirm the role of ENG in regulating Noggin and Adamtsl2 expression in Figure 8.\n9. Explore the clinical relevance of Noggin and Adamtsl2 dysregulation by analyzing patient samples to enhance the clinical significance, and elucidate the mechanistic link between soluble ENG (sENG) levels and the pathophysiology of preeclampsia (PE) or pulmonary arterial hypertension (PAH).\n10. Address spatial constraints of ENG’s extracellular domain (ECD) in recruiting TGFBRII and ALK1, and discuss potential ENG-independent BMP9 signaling pathways (e.g., via ActRIIA/BMPRII) in the Discussion.\n11. Compare the ENG-TGFBRII-ALK1 axis with other TGF-β/BMP receptor complexes (e.g., BMPRII-ALK3) in the Discussion to provide broader mechanistic context.\n12. Clarify in the Methods section whether crosslinker concentrations were titrated to optimize experimental conditions.", "reviewer_2": "In the study by Wei Li group, the authors elucidate a novel model of Endoglin (ENG) in the crosstalk of BMP9 and TGF-β signaling, as ENG firstly captures circulating BMP9 and BMP10 and efficiently displaces both their prodomains, secondly recruits TGFBRII to BMP9 and 10 signalling by binding to TGFBRII and ALK1 simultaneously. Their findings, in part, provide a long-standing question of how ENG regulates TBG-beta superfamily signaling. They also identify the target genes of ENG-dependent BMP9 signaling, which provides insight for how to dissect context-dependent ALK1 signaling to understand pathogenesis of, for example, pulmonary artrial hypertension. While the manuscript is well-structured and the experiments largely support the conclusions, several weaknesses should be addressed to strengthen the impact.\nMajor weaknesses:\n1. The study utilizes Rosa26-CreERT2; Engfl/fl; immorto mice to generate Eng knockout MLECs for in vitro RNA-seq and signaling assays. The immorto system’s potential interference raises concerns about whether MLECs used for this analysis truly reflect native endothelial behavior.\n2. The co-enrichment of SMAD1, SMAD2 in the ENG pulldown complexes (Figure 5e) is insufficiently addressed.\n3. The authors tested both BMP9 and BMP10 in some parts of the Results, however, they only tested BMP9 in other parts (Figure 4, 5 and 7). It would be helpful is the authors can explain the reason.\n4. The current results provide a new framework to facilitate ENG research, however, which is still not clear whether this new mechanism/function of ENG will help for therapeutic applications. While the in vitro data are robust, the absence of in vivo validation limits translational impact.\nSome minor issues in the text:\n0. “Displacement of prodomain” may need some expiations for general readers, i.e., the full length of the BMP9/10 are cleaved between the prodomain and the growth factor (GF) domain, and these domains form a loose complex to stay inactive. A releasing mechanism of the prodomain is one of the layers of signaling control, which is indeed studied in this manuscript.\n1. Line 39, 100, 217, 326, 399 and 471: the authors stated “BMP9 and 10”, which is easy to cause ambiguity. Please revised to “BMP9 and BMP10” to keep consistence.\n2. Line 75: The abbreviation “ICD” is introduced without its full term .\n3. Figure 1d: The unlabeled bands in the sENG(M) + pro: BMP10 condition (resembling bands 7-9) should be clarified in the figure legend or text.\n4. Figure 1g and 1h：Lane 11 (far right in each panel) is not annotated in the figure or legend.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Point-by-point Responses to Reviewers’ comments.\nWe thank the Editor and Reviewers for their thoughtful and constructive feedback. Below, we\nrespond point-by-point and describe all changes to the text, figures, and Supplementary\nInformation (Original comments from the reviewers are in black and our responses are in blue).\nReviewer #1 (Remarks to the Author):\nThis study provides compelling evidence for the dual role of endoglin (ENG) as a co-receptor for\nBMP9/10 signaling, bridging the gap between its previously reported functions in TGF-β and BMP\npathways. The authors demonstrate that ENG efficiently displaces prodomains from pro-\nBMP9/10, recruits TGFBRII to the signaling complex, and identifies Nog and Adamtsl2 as ENG-\ndependent BMP9 target genes. This manuscript presents a significant advance in understanding\nENG’s role in endothelial signaling. The work is well-executed and addresses a long-standing\ncontroversy in endothelial biology. However, some methodological details and interpretations\nrequire clarification or additional validation to strengthen the conclusions. Addressing the\nfollowing points will elevate the impact and clarity of the work. I recommend acceptance\npending revision.\n1. Quantify prodomain displacement efficiency in Figure 1 using densitometric analysis of native\nPAGE bands, and perform kinetic assays to exclude potential prodomain reassociation post-\ndisplacement.\nWe now provide densitometric quantification from N = 3 independent experiments for the\nprodomain, sENG:BMP9 (or sENG:BMP10) (revised Supplementary Figure 2c-e, 3b-d and 3g-\nh).\nTime-course assays up to 24 hour show no evidence of prodomain re-association for either\nproBMP9 or proBMP10 (revised Supplementary Figure 2f–g, 3e-f).\n2. Provide structural evidence (e.g., SEC-MALS) to substantiate the hypothesis that ENG adopts\nmultiple conformations in Figure 2a.\nFirstly, we would like to clarify that Figure 2a is not a hypothesis that ENG adopts multiple\nconformations, but to illustrate the theoretical “possible forms of complexes” (as marked on\nthe figure) from binding stoichiometry point of view to guide the analysis of the data in the rest\nof Figure 2. As shown in Figure 2 results, our experimental data can exclude the forms IV and VI.\nWe don’t have enough resolution to distinguish between forms I, II and III.\nTo acknowledge this and to avoid potential confusion (thank you for pointing this out), we\nadded the following sentence at the end of this paragraph. “Further experiments are required to\nascertain whether forms I, II and III are all possible and whether one of them is a preferred\nform.”(Page 8, line 212)\nIt is worth noting here that SEC-MALS of BMP9:ENG complex has been performed in Saito et al,\nCell Report, 2017, PMID: 28564608. This paper from Prof. Luca Jovine’s group reported crystal\nstructures of ENG-OR in complex with BMP9 as well as ENG ZP domain on its own. In Figure 4E\nfrom this paper, they detected sENG-ECD-BMP9 complex showing two peaks in SEC-MALS\nanalysis. In addition, the ZP domain alone secreted as both monomer and dimer, although\nmonomer preferentially crystallised. In this report, the authors also suggest that “Elucidation of\n1\nthe details of the signal transduction mechanism will require further structural studies of the\nintact ternary complex. This may adopt a range of conformations not recapitulated by our crystal\nstructure-based model as a result of flexibility at the ENG OR/ZP junction—possibly underlying\nthe two ECTO-BMP9 states observed by SEC-MALS (Figure 4E)—as well as membrane\nanchoring.” (page 1192 of the paper)\n3. Label the analyte concentrations used in surface plasmon resonance (SPR) experiments in\nthe legend of Figure 3.\nThank you. We have now added the analyte concentrations in the figure legend.\n4. Include statistical comparisons between the displacement efficiencies of BMPRII and ActRIIB\nin Figure 4b.\nStatistical comparisons at matched molar excesses are now included in Figure 4b and legend.\nNo significant digerences in displacement efficiency were detected, with a small ELISA signal\nenhancement at 1× ActRIIB for sENG(M):BMP9 noted in the legend and text.\n5. Validate the ENG-TGFBRII-ALK1 interaction in primary endothelial cells (ECs) by conducting\nco-immunoprecipitation (co-IP) assays with endogenous proteins under physiological\nconditions to support Figure 5e.\nThank you for your suggestion. To mimic the physiological condition, we performed further co-\nIP with cells cultured under (i) 10% FBS standard endothelial cell growth medium (EGM-2) and\n(ii) 0.1% FBS EGM-2 medium with 0.25 ng/ml BMP9 (within healthy human plasma range), in\nthree human primary endothelial cells as before.\nThis new data is now included in the revised Figure 5e&f, and we have added further western\nblots to report the Smad1/5 phosphorylation status of these cells (Figure 5g) as well as\ndiagrams to summarise the pull-down results (Figure 5h). We consistently recovered\nendogenous TGFBRII with ENG across conditions, whereas ALK1 was not detected under 10%\nFBS or BMP9 treatment (revised Figure 5e–h; Smad1/5 phosphorylation controls in Figure 5g).\nWe discuss that constitutive/ligand-induced ALK1 activation likely alters detectability\n(phosphorylated ALK1 may dissociate or be poorly recovered), and we note that our pulldown\nantibody (TRC105) competes with the ENG–BMP9 interface, precluding recovery of extracellular\nENG:BMP9:ALK1 complexes in this assay. The model in Figure 6f has been adjusted to avoid\nimplying an intracellular ALK1–TGFBRII interaction in the presence of BMP9. Overall, the data\nsupport the notion that ENG recruits TGFBRII into the BMP9–ALK1 signalling complex.\n6. Optimize the schematic diagram of Figure 6d, and list the small diagrams separately on one\nside, clearly marking the important proteins such as the receptors.\nMini-diagrams have been added adjacent to the relevant data (Figure 1i; Figure 3a–c; Figure\n5h; Figure 6b, e) and the final model (Figure 6f) was updated for consistency.\nProteins/receptors are clearly labelled throughout.\n7. Provide statistical analysis and comparison on the immunofluorescence intensity in Figure\n7c.\nThe quantification and statistical analysis are now added in Supplementary Figure 8a, from\nstaining of N=3 batches of cells isolated from 3 difference mice.\nexplained in the legend. Manuscript text has been modified accordingly.\n8. Perform functional rescue experiments, such as ENG overexpression or TGFBRII knockdown,\nto confirm the role of ENG in regulating Noggin and Adamtsl2 expression in Figure 8.\n1) We have performed siTGFBR2 in PAECs and did not observe any difference in NOG or\nADAMTSL2 induction by BMP9. We think the effect of ENG loss may not be fully captured by\nsiTGFBR2. This is because the effects from ENG loss can affect BMP9 signalling from at least\ntwo aspects: 1) less efficient capturing of BMP9 therefore less signalling strength (e.g. reduced\nSmad1/5 phosphorylation); 2) mediating TGFBRII to phosphorylate ALK1. siTGFBR2 can only\ntest the point 2, it will not capture point 1.\n2) We tried to transfect the human ENG-containing plasmid into the MLECs after Eng\nknockout, using Lipofectamine LTX Reagent with PLUS Reagent (Thermofisher 15338100) as\nwell as Neon NxT electroporation System with the transfection kit (ThermoFisher, N10025).\nThese cells were very susceptible to Lipofectamine LTX, and over 90% of the cells died after\ntransfection. We attempted various protocols and varying amounts of ENG and control vector\nplasmids. Approximately two-thirds of the cells died after electroporation in both the ENG- and\nthe control vector-transfected cells across all tested conditions. Although we can detect the\nsuccessful transfection of human ENG, these remaining cells were not healthy, as reflected by\nlower overall protein contents and lower RNA yield. When we treated remaining cells with BMP9,\nwe found that transfecting the vector control alone affected the basal expression of both Nog\nand Adamstl2. Therefore, it is not possible to do the rescue experiment using this approach.\n3) Although the gene manipulation approaches in the primary endothelial cells proved to\nbe challenging, we have managed to analyse some human datasets, especially microarray data\nfrom human lung tissues (both healthy controls and PAH patients). As lung tissues have large\nareas of blood vessels, especially small vessels and capillaries, they contain a large proportion\nof endothelial cells. The lung tissue microarray data would contain signals from endothelial\ncells without any further effects introduced by in vitro culturing, hence providing an even better\ntool to validate our findings. If our model is correct, we should find the mRNA expression of NOG\nand ADAMTSL2 to be positively correlated with the expression of ENG. Indeed, when we\nanalysed the published microarray data GSE113439 which contains all three genes, we found\nstrong positive correlation for both genes with ENG, using two different correlation analyses\nmethods (Pearson r=0.71 to 0.74 for ADAMTSL2 and NOG respectively, and p<0.0001 for both\ncases. If using Spearman test, Rho values for NOG and ADAMTSL2 are 0.66 and 0.76,\nrespectively). More interestingly, we found that the mRNA levels for ENG, NOG and ADAMTSL2\nare all reduced in PAH patient lungs. These novel exciting data, along with further analysis of\nPAH blood samples (more information on this in the next point), are now included in the new\nFigure 9.\nIn summary, although we could not add further rescue experiment data to the revised\nmanuscript, we believe these new human data provided more substantial evidence than in vitro\nfunctional rescue experiments to further support our findings.\n9. Explore the clinical relevance of Noggin and Adamtsl2 dysregulation by analyzing patient\nsamples to enhance the clinical significance, and elucidate the mechanistic link between\n3\nsoluble ENG (sENG) levels and the pathophysiology of preeclampsia (PE) or pulmonary arterial\nhypertension (PAH).\nThank you for the suggestion. We have now analysed the expression of Noggin and Adamtsl2 as\nwell as ENG in PAH patients’ samples by: 1) analysing a published microarray dataset which\ncompared lung mRNA between 15 PAH patients and 11 healthy controls; 2) analysing the total\nblood transcriptomics and proteomics, comparing PAH patients versus healthy controls. A new\nfigure (Figure 9) is added in the revision with accompanying texts in the results and discussion.\nWe show that in the GSE113439 microarray dataset, mRNA levels for ENG, NOG and ADAMTSL2\nare all significantly lower in PAH patients’ lung tissues compared with healthy controls (Figure\n9a). As mentioned above, there are strong positive correlations between the expression of NOG\nand ADAMTSL2 with the expression of ENG, consistent with our findings.\nIn the whole blood RNAseq data from 363 PAH samples versus 126 control samples, the\nexpression levels for both ENG and NOG are significantly lower in PAH patients (Figure 9c), and\nthe mRNA expression levels of ENG and NOG are also significantly correlated. ADAMTSL2\nexpression level was too low to be reliably analysed. We also found that lower levels of NOG\nmRNA are associated with poorer survival in PAH patients (Figure 9e). Proteomic data from a\npublished dataset containing 463 PAH patients and 108 healthy controls revealed a significant\ndecrease in the levels of Noggin protein (Figure 9f). Our data suggest that plasma NOG could be\nan important biomarker for PAH, and this may be linked to the ENG/BMP9 signalling axis, which\nwas known to be dysregulated from the human genetic studies.\n10. Address spatial constraints of ENG’s extracellular domain (ECD) in recruiting TGFBRII and\nALK1, and discuss potential ENG-independent BMP9 signaling pathways (e.g., via\nActRIIA/BMPRII) in the Discussion.\nIn the discussion, to address the spatial constraints of ENG ECD in recruiting TGFBRII and ALK1,\nwe added the following sentence (page 17, line 1) “Structural analysis shows that ENG, TGFBRII\nand ALK1 ECDs all bind to ligands at digerent sites and there will not be any structural constraint\nextracellularly for ENG to bring TGFBRII and ALK1 together.” This is also illustrated in new\nSupplementary Figure 10.\nTo address potential ENG-independent BMP9 signalling, the following sentence was added\n(page 17, line 477): “Interestingly, BMP9 signalling by reduced by around 50% in Eng-/- MLECs,\nsuggesting that both ENG-dependent and ENG-independent pathways play significant roles in\nBMP9 signalling.”\n11. Compare the ENG-TGFBRII-ALK1 axis with other TGF-β/BMP receptor complexes (e.g.,\nBMPRII-ALK3) in the Discussion to provide broader mechanistic context.\nWe have added a paragraph in the discussion (page 18, line 515) to compare the ENG-TGFBRII-\nALK1 axis with other TGF-β/BMP receptor complexes in non-endothelial context, highlighting\ncommon principles and unique features and putting our work in a broader mechanistic context.\n12. Clarify in the Methods section whether crosslinker concentrations were titrated to optimize\nexperimental conditions.\nRelated to Figure 6a, we agree that titrating the concentrations of the crosslinker is important to\nshow that the incomplete crosslinking of sENG(D) is not due to the sub-optimal crosslinker\nconcentrations. We now show that all crosslinkers at all tested concentrations can crosslink no\nmore than 50% of sENG(D), consistent with our initial findings. We now added this new data in\nthe Supplementary data 7b and cite it along with Figure 6a.\nReviewer #2 (Remarks to the Author):\nIn the study by Wei Li group, the authors elucidate a novel model of Endoglin (ENG) in the\ncrosstalk of BMP9 and TGF-β signaling, as ENG firstly captures circulating BMP9 and BMP10 and\nefficiently displaces both their prodomains, secondly recruits TGFBRII to BMP9 and 10 signalling\nby binding to TGFBRII and ALK1 simultaneously. Their findings, in part, provide a long-standing\nquestion of how ENG regulates TBG-beta superfamily signaling. They also identify the target\ngenes of ENG-dependent BMP9 signaling, which provides insight for how to dissect context-\ndependent ALK1 signaling to understand pathogenesis of, for example, pulmonary artrial\nhypertension. While the manuscript is well-structured and the experiments largely support the\nconclusions, several weaknesses should be addressed to strengthen the impact.\nMajor weaknesses:\n1. The study utilizes Rosa26-CreERT2; Engfl/fl; immorto mice to generate Eng knockout MLECs\nfor in vitro RNA-seq and signaling assays. The immorto system’s potential interference raises\nconcerns about whether MLECs used for this analysis truly reflect native endothelial behavior.\nWe clarify that the Immorto system is used strictly to enable discovery while minimising animal\nusage and passage-related drift; all functional assays are at 37 °C, where the ts T-antigen is\ninactive, and Eng deletion is induced immediately before experiments. Both control and KO carry\nthe same ts allele, so digerential phenotypes are attributable to ENG. MLECs are a powerful tool\nto facilitate the discovery of novel mechanisms which can then be further validated using other\nmore physiologically relevant cells.\nIn our case, using MLECs we identified Nog and Adamtsl2 to be preferentially dependent on Eng.\nWe recognise the importance of validating such data using human primary endothelial cells\nwhich is what we did in Figure 8g-h. We indeed found that in HDMECs, which express lowest\nlevels of ENG, both NOG and ADAMTSL2 are minimally induced by BMP9, despite ID1 being\ninduced at similar levels.\nIn the revision, we performed further validation using human lung tissue microarray data\n(GSE113439). Lung tissue has a significant proportion of endothelial cells; and these endothelial\ncells would all be under physiological or pathological conditions (such as PAH) without any ex\nvivo culturing. Because BMP9 circulate at active concentrations, BMP9 signalling should be\nconstitutively active in the lung ECs.\nIf our finding is correct that NOG and ADAMTSL2 expression is ENG-dependent, we would\nexpect the mRNA expression levels of NOG and ADAMTSL2 to correlate with the mRNA levels of\nENG. Indeed, we found strong positive correlation of ADAMTSL2 and NOG with the expression\n5\nof ENG, with Pearson r = 0.71 to 0.74 for ADAMTSL2 and NOG, respectively, both with P values\nless than 0.0001. Similarly using the Spearman test (which takes outlier into account), Rho\nvalues for NOG and ADAMTSL2 are 0.66 and 0.76, respectively, with P values of 0.0003 and\n0.00001 respectively. Such human data provide strong support to validate our findings from the\nMLECs. Of note, only Spearman test result was presented in the revised Figure 9.\n2. The co-enrichment of SMAD1, SMAD2 in the ENG pulldown complexes (Figure 5e) is\ninsufficiently addressed.\nThis is an interesting point. Of note, this was observed in the medium containing 0.1% FBS. As\nmentioned above in response to Reviewer 1, we have performed further pull-down/mass spec\nexperiments for cells harvested in more “physiological-like” conditions, such as 10% FBS and\n0.25 ng/ml pro:BMP9 in 0.1% FBS, where BMP9 signalling is constitutively activated.\nAs shown in the revised Figure 5e, under 10% FBS, we did not detect any SMADS in any of the\nthree cell types, whereas under BMP9+0.1% FBS condition, we detected SMAD1 and SMAD2 in\nHAOECs, SMAD1 and SMAD5 in HPAECs and none of the SMADS in HUVECs.\nWe have provided the information on the peptides detected in Supplementary Figure 6.\nR-SMADs are predicted to interact with the type I receptor under non-phosphorylated states\n(when signalling is not active) because after phosphorylation by the type I receptors, they will\ndissociate from the type I receptor and go into the nucleus to regulate transcription.\nMixed R-SMADs complex has been described before (PMID: 29376829), but its function in the\nphysiological responses is still not fully understood. Therefore, we cannot exclude the\npossibility that there is direct interaction between SMAD1 and SMAD2.\nWe have added a paragraph in the discussion to address the different observations on ALK1,\nSMAD1, SMAD2 and SMAD5 from the IP/mass spec experiment while avoiding mechanistic\nover-interpretation (Page 17, line 489).\n3. The authors tested both BMP9 and BMP10 in some parts of the Results, however, they only\ntested BMP9 in other parts (Figure 4, 5 and 7). It would be helpful is the authors can explain the\nreason.\nText (page 9, line 245) now explains that biochemical differences in prodomain displacement\n(BMPRII vs pro:BMP9 and Pro:BMP10) and sENG:ligand binding to ALK1-Fc motivated focusing\nmechanistic depth on BMP9. Because BMP10 signalling regulation digers from BMP9, role of\nENG in BMP10 will require separate in-depth investigation.\n4. The current results provide a new framework to facilitate ENG research, however, which is\nstill not clear whether this new mechanism/function of ENG will help for therapeutic\napplications. While the in vitro data are robust, the absence of in vivo validation limits\ntranslational impact.\nThank you for kindly pointing out that “The current results provide a new framework to facilitate\nENG research”.\nTo increase the translational impact, we have included additional data from analysing human\nPAH patients’ data in the revision (Figure 9) to enhance the clinical relevance and translational\nimpact. We show that NOG and ADAMTSL2 expression in human lungs has strong positive\n6\ncorrelations to ENG expression, and the expression of all three genes is reduced in PAH patients.\nWe also show that in whole blood transcriptomics, NOG and ENG expression are significantly\ncorrelated and reduced in PAH patients. Importantly, we identified that plasma protein levels of\nNOG are reduced in PAH. Together, our data strongly suggest plasma NOG and ENG gene and\nprotein levels could be helpful PAH biomarkers that are linked to the TGF-b/ BMP pathway, the\ndysregulated pathway in PAH identified by human genetics.\nSome minor issues in the text:\n0. “Displacement of prodomain” may need some expiations for general readers, i.e., the full\nlength of the BMP9/10 are cleaved between the prodomain and the growth factor (GF) domain,\nand these domains form a loose complex to stay inactive. A releasing mechanism of the\nprodomain is one of the layers of signaling control, which is indeed studied in this manuscript.\nThank you for this excellent suggestion. We added a schematic of BMP9/10 processing and\nclarified “prodomain displacement” for general readers, and explained specifically that the\nprodomain needs to be displaced because the unprocessed proBMP9 and proBMP10 are not\nactive with two reference papers cited (new Supp. Figure 1a; page 4, line 91-96)\n1. Line 39, 100, 217, 326, 399 and 471: the authors stated “BMP9 and 10”, which is easy to cause\nambiguity. Please revised to “BMP9 and BMP10” to keep consistence.\nThank you. We have revised all these to “BMP9 and BMP10” as suggested.\n2. Line 75: The abbreviation “ICD” is introduced without its full term .\nApologies for the oversight. “Intracellular domain (ICD)” is now introduced on first use.\n3. Figure 1d: The unlabeled bands in the sENG(M) + pro: BMP10 condition (resembling bands 7-\n9) should be clarified in the figure legend or text.\nWe have clarified the unlabelled bands in the figure/figure legends (that they are sENG(D) and\nits complex with BMP9 or BMP10). We have confirmed this by performing an additional test with\na purer version of sENG(M) containing less sENG(D) (see below, the additional bands are much\nfainter.\n4. Figure 1g and 1h：Lane 11 (far right in each panel) is not annotated in the figure or legend.\nApologies for the oversight. These are type II receptor-only controls without BMP9 or BMP10.\nBoth panels are now clearly annotated.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is\n7\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for reviewing our manuscript and providing constructive comments."}, {"round": 2, "reviewer_comments": {"reviewer_1": "I have no other concerns.", "reviewer_2": "The authors substantially and successfully addressed the critiques raised by this reviewer.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter