OTULIN coordinates with LUBAC to edit linear polyubiquitin chains in embryonic development, autoimmunity, and inflammatory diseases. However, the mechanism by which angiogenesis, especially that of endothelial cells (ECs), is regulated by linear ubiquitination remains unclear. Here, we reveal that constitutive or EC-specific deletion of Otulin resulted in arteriovenous malformations and embryonic lethality. LUBAC conjugates linear ubiquitin chains onto Activin receptor-like kinase 1 (ALK1), which is responsible for angiogenesis defects, inhibiting ALK1 enzyme activity and Smad1/5 activation. Conversely, OTULIN deubiquitinates ALK1 to promote Smad1/5 activation. Consistently, embryonic survival of Otulin-deficient mice was prolonged by BMP9 pretreatment or EC-specific ALK1^Q200D (constitutively active) knockin. Moreover, mutant ALK1 from type 2 hereditary hemorrhagic telangiectasia (HHT2) patients exhibited excessive linear ubiquitination and increased HOIP binding. As such, a HOIP inhibitor restricted the excessive angiogenesis of ECs derived from ALK1^G309S-expressing HHT2 patients. These results show that OTULIN and LUBAC govern ALK1 activity to balance EC angiogenesis.

Exogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies.

Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-β. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-β and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

Reconstruction of bone defects is one of the most substantial and difficult clinical challenges in orthopedics. Transforming growth factor beta 1 (TGFβ1) might play an important role in stimulating osteogenic differentiation of bone morphogenetic protein 9 (BMP9)-induced C3H10T1/2 mesenchymal stem cells. In our current study, we examined the potential synergy between TGFβ1 and BMP9 in promoting the osteogenesis of C3H10T1/2 cells, and whether such effects could contribute to bone formation in vivo. Our experiment data indicated that TGFβ1 could increase the expression of osteogenic markers and the formation of mineralized calcium nodules in, while suppressing the proliferation of, BMP9-induced C3H10T1/2 cells. Furthermore, mice intramuscularly injected with BMP9/TGFβ1-transduced C3H10T1/2 cells into the gastrocnemius muscle on their tibiae developed ectopic bone masses with more mature osteoid structures, compared to those grafted with cells expressing BMP9/RFP. Subsequent mechanistic studies found that TGFβ1-induced enhancement of osteogenesis in BMP9-overexpressing C3H10T1/2 cells was accompanied by augmented expression of heat shock protein 47 (HSP47), a collagen-specific molecular chaperone essential for collagen biosynthesis, and can be attenuated by pirfenidone, a known anti-fibrotic inhibitor. Interestingly, protein microarray analysis suggested that TGFβ1/BMP9-dependent osteogenesis of C3H10T1/2 cells seemed to involve several non-canonical signaling pathways such as Janus kinase-signal transducer and activator of transcription, phosphoinositide-3-kinase-protein kinase B, and mitogen-activated protein kinase. These results provided further evidence that TGFβ1 could promote bone formation from BMP9-induced C3H10T1/2 cells and shed important light on the underlying molecular mechanisms.

Keywords: BMP9; Bone mesenchymal stem cell; HSP47; Osteogenesis; TGFβ1.

Bone morphogenetic protein 9 (BMP9) is one of the most efficacious osteogenic cytokines. PTEN and Wnt10b are both implicated in regulating the osteogenic potential of BMP9, but the potential relationship between them is unknown. In this study, we determined whether PTEN could reduce the expression of Wnt10b during the osteogenic process initialized by BMP9 in mesenchymal stem cells (MSCs) and the possible molecular mechanism. We find that PTEN is inhibited by BMP9 in MSCs, but Wnt10b is increased simultaneously. The BMP9-induced osteogenic markers are reduced by PTEN but increased by silencing PTEN. The effects of knockdown PTEN on elevating BMP9-induced osteogenic markers are almost abolished by knockdown of Wnt10b. On the contrary, the BMP9-increased ALP activities and mineralization are both inhibited by PTEN but almost reversed by the combination of Wnt10b. Bone masses induced by BMP9 are enhanced by knockdown of PTEN, which is reduced by knockdown of Wnt10b. The BMP9-increased Wnt10b is decreased by PTEN but enhanced by knockdown of PTEN. Meanwhile, the BMP9-induced Wnt10b is also reduced by a PI3K-specific inhibitor (Ly294002) or rapamycin, respectively. The BMP9-induced phosphorylation of CREB or Smad1/5/9 is also reduced by PTEN, but enhanced by PTEN knockdown. In addition, p-CREB interacts with p-Smad1/5/9 in MSCs, and p-CREB or p-Smad1/5/9 are both enriched at the promoter region of Wnt10b. Our findings indicate that inhibitory effects of PTEN on BMP9's osteogenic potential may be partially mediated through decreasing the expression of Wnt10b via the disturbance of interaction between CREB and BMP/Smad signaling.

Keywords: BMP9; WNT10B; mesenchymal stem cell; osteogenic differentiation; phosphatase and tensin homolog deleted on chromosome 10.

Despite advances in screening and treatment, colon cancer remains one of the leading causes of cancer-related death. Finding novel and useful drug treatment targets is also an urgent need for clinical applications. Tetrandrine (Tet) is extracted from the Chinese medicinal herbal medicine, which is a well-known calcium blocker with a variety of pharmacological activities, including anti-cancer. In this study, we recruited cell viability assay, flow cytometry analysis, cloning formation to confirm that Tet can inhibit the proliferation of SW620 cells, and induce apoptosis. Mechanically, we confirmed that Tet up-regulates the mRNA and protein level of BMP9 in SW620 cells. Over-expression BMP9 enhances the anti-cancer effects of Tet in SW620 cells, but these effects can be partly reversed by silencing BMP9. Also, Tet reduces phosphorylation of Aktl/2/3 in SW620 cells, which could be elevated by overexpressed BMP9 and impaired by silencing BMP9. Furthermore, we demonstrated that Tet reduces phosphorylated PTEN, which can be promoted by overexpressed BMP9, analogously also be attenuated through silencing BMP9. Finally, we introduced a xenograft tumor model to investigate the anti-proliferative effect of Tet, further to explore the effects of BMP9 and PTEN in SW620 cells. Our findings suggested that the anti-cancer activity of Tet in SW620 cells may be mediated partly by up-regulating BMP9, followed by inactivation PI3K/Akt through up-regulating PTEN at least.

Keywords: Akt1/2/3; BMP9; Colon cancer; PTEN; Tetrandrine (Tet).

The aim of the present study was to investigate the expression and function mechanism of bone morphogenetic protein 9 (BMP9) in cerebral ischemia‑reperfusion (I/R) injuries in vivo and in vitro. A total of 40 Sprague‑Dawley rats were randomly divided into four groups (n=10): i) Normal control; ii) sham surgery group, the procedure without occlusion; iii) I/R group, right middle cerebral artery occlusion (MCAO) followed by reperfusion; and iv) adenoviral vector (Ad)‑BMP9 + I/R group, Ad‑BMP9 intracerebroventricular injection was performed 2 days prior to MCAO. Neurological deficit score and infarct volume were measured at 24 h following reperfusion. To further test the mechanism of BMP9, astrocytes were isolated and treated with Ad‑BMP9, Ad‑BMP9 + extracellular signal‑regulated kinase (ERK) inhibitor PD098059, Ad‑BMP9 + c‑Jun N‑terminal kinase inhibitor SP600125 and Ad‑BMP9 + p38 inhibitor SB203580 for 24 h, followed by undergoing oxygen‑glucose deprivation and reoxygenation (OGD/R) treatment. Cell viability and death were assessed by 3‑(4,5‑dimethylthiazol‑2yl)‑5‑(3‑carboxymethoxyphenyl)‑(4‑sulfophenyl)‑2H‑tetrazolium and lactate dehydrogenase release, respectively. Gene expression was determined by quantitative polymerase chain reaction and western blotting. BMP9 was identified to be upregulated at mRNA and protein levels in cerebral I/R animal and cell models. BMP9 pretreatment significantly reduced the neurological score and infarct volume compared with I/R rats. In astrocytes, overexpression of BMP9 significantly decreased cell death and improved cell viability, an effect which may be mediated by the ERK signaling pathway, as ERK was activated by BMP9 and the use of PD098059 partially reversed the protective effect of BMP9. Pretreatment with BMP‑9 may be a promising treatment option for prevention of cerebral I/R injuries.

Venous malformation (VM) is a type of vascular morphogenic defect in humans with an incidence of 1%. Although gene mutation is considered as the most common cause of VM, the pathogenesis of those without gene mutation remain to be elucidated. Here, we aimed to explore the relation of bone morphogenetic protein 9 (BMP9) and development of VM. At first, we found serum and tissue BMP9 expression in VM patients was significantly lower than that in healthy subjects, detected via enzyme-linked immunosorbent assay. Next, with wound healing assay, transwell assay and tube formation assay, we discovered BMP9 could inhibit migration and enhance tube formation activity of human umbilical vein endothelial cells (HUVECs) via receptor activin receptor-like kinase 1 (ALK1). Besides, BMP9 improved the expression of structural proteins alpha-smooth muscle actin (α-SMA) and Desmin in human umbilical vein smooth muscle cells (HUVSMCs) via activation of the SMAD1/5-ID1 pathway, determined by RNA-based next-generation sequencing, qPCR, immunofluorescence and western blotting. Intriguingly, this effect could be blocked by receptor ALK1 inhibitor, SMAD1/5 inhibitor and siRNAs targeting ID1, verifying the BMP9/ALK1/SMAD1/5/ID1/α-SMA pathway. Meanwhile, knocking out BMP9 in C57BL/6 mice embryo led to α-SMA scarcity in walls of lung and mesenteric vessels, as well as walls of small trachea. BMP9-/- zebrafish also exhibited abnormal vascular maturity, indicating a critical role of BMP9 in vascular maturity and remodeling. Finally, a VM mice model revealed that BMP9 might has therapeutic effect in VM progression. Our study discovered that BMP9 might inhibit the occurrence of VM by strengthening the vessel wall and maintaining endothelium quiescence. These findings provide promising evidences of new therapeutic targets that might be used for the management of VM.

Keywords: Alpha-smooth muscle actin; Bone morphogenetic protein 9; Orbital venous malformation; Vascular remodeling; Venous malformation.

RNA interference (RNAi) is mediated by an ∼21-nt double-stranded small interfering RNA (siRNA) and shows great promise in delineating gene functions and in developing therapeutics for human diseases. However, effective gene silencing usually requires the delivery of multiple siRNAs for a given gene, which is often technically challenging and time-consuming. In this study, by exploiting the type IIS restriction endonuclease-based synthetic biology methodology, we developed the fast assembly of multiplex siRNAs (FAMSi) system. In our proof-of-concept experiments, we demonstrated that multiple fragments containing three, four, or five siRNA sites targeting common Smad4 and/or BMPR-specific Smad1, Smad5, and Smad8 required for BMP9 signaling could be assembled efficiently. The constructed multiplex siRNAs effectively knocked down the expression of Smad4 and/or Smad1, Smad5, and Smad8 in mesenchymal stem cells (MSCs), and they inhibited all aspects of BMP9-induced osteogenic differentiation in bone marrow MSCs (BMSCs), including decreased expression of osteogenic regulators/markers, reduced osteogenic marker alkaline phosphatase (ALP) activity, and diminished in vitro matrix mineralization and in vivo ectopic bone formation. Collectively, we demonstrate that the engineered FAMSi system provides a fast-track platform for assembling multiplexed siRNAs in a single vector, and thus it may be a valuable tool to study gene functions or to develop novel siRNA-based therapeutics.

Keywords: BMP9/Smad signaling; RNA interference; RNAi; double-stranded small interfering RNA; mesenchymal stem cells; multiplex siRNA expression; osteoblastic differentiation; siRNA.

During chronic liver disease, hepatic progenitor cells (HPC, oval cells in rodents) become activated, proliferate, and differentiate into cholangiocytes and/or hepatocytes contributing to the final outcome of the regenerative process in a context-dependent fashion. Here, we analyze the crosstalk between the hepatocyte growth factor (HGF)/c-Met signaling axis, key for liver regeneration, and bone morphogenetic protein (BMP)9, a BMP family ligand that has emerged as a critical regulator of liver pathology. Our results show that HGF/c-Met signaling blocks BMP9-mediated apoptotic cell death, while it potentiates small mothers against decapentaplegic (SMAD)1 signaling triggered by BMP9 in oval cells. Interestingly, HGF-induced overactivation of SMAD1, -5, -8 requires the upregulation of TGF-β type receptor activin receptor-like kinase (ALK)1, and both ALK1 and SMAD1 are required for the counteracting effect of HGF on BMP9 apoptotic activity. On the other hand, we also prove that BMP9 triggers the activation of p38MAPK in oval cells, which drives BMP9-apoptotic cell death. Therefore, our data support a model in which BMP9 and HGF/c-Met signaling axes establish a signaling crosstalk via ALK1 that modulates the balance between the two pathways with opposing activities, SMAD1 (pro-survival) and p38 mitogen-activated protein kinases (p38MAPK; pro-apoptotic), which determines oval cell fate. These data help delineate the complex signaling network established during chronic liver injury and its impact on the oval cell regenerative response.

Our previous study confirmed that bone morphogenetic protein 9 (BMP9) participated in the development of nonalcoholic steatohepatitis (NASH) by affecting macrophage polarization. The focus of this study was to further confirm the role of macrophages in BMP9-mediated NASH and to analyze the underlying mechanism. In vivo, mice that were administered adeno-associated viral (AAV) vectors containing a null transgene (AAV-null) or the BMP9 transgene (AAV-BMP9) were divided into methionine- and choline-deficient (MCD) and control diet (CD) groups, and they were administered either control liposomes or clodronate liposomes via tail vein injection, the latter to deplete macrophages. The mice were sacrificed after 4 weeks of MCD diet feeding. In vitro, RAW264.7 cells were pretreated with or without BAY11-7085 (an NF-κB inhibitor) and stimulated with recombinant human BMP9 (rh-BMP9). To explore the underlying mechanism of action of BMP9, primary human monocyte-derived macrophages were additionally investigated and immunohistochemistry, biochemical assays, qRT-PCR, and Western blotting were used. The characteristics of NASH-related inflammation were assessed by hepatic histological analysis. Serum AST and ALT and hepatic triglyceride were examined by biochemical assays. We found that the expression of M1 macrophage genes (including CD86, IL1β, IL6, MCP-1 and TNFα) and the number of M1 macrophages (iNOS⁺ macrophages) in the liver were significantly elevated after BMP9 overexpression and BMP9 directly upregulated TLR4 expression in MCD-induced NASH. These effects were eliminated by macrophage depletion. In vitro, we discovered that BMP9 enhanced the nuclear translocation of NF-κB to induce macrophage M1 polarization in RAW264.7 cells and it promoted LPS-mediated activation of the NF-κB pathway in primary human macrophages. Taken together, this study demonstrates that BMP9 promotes NASH development by directly acting on macrophages.

Keywords: Bone morphogenetic protein 9 (BMP9); Inflammation; NF-κB; Nonalcoholic fatty liver disease (NAFLD); Nonalcoholic steatohepatitis (NASH).

The improvement of cancer therapy efficacy, the extension of patient survival and the reduction of adverse side effects are major challenges in cancer research. Targeting blood vessels has been considered a promising strategy in cancer therapy. Since the tumor vasculature is disorganized, leaky and triggers immunosuppression and tumor hypoxia, several strategies have been studied to modify tumor vasculature for cancer therapy improvement. Anti-angiogenesis was first described as a mechanism to prevent the formation of new blood vessels and prevent the oxygen supply to tumor cells, showing numerous limitations. Vascular normalization using low doses of anti-angiogenic drugs was purposed to overcome the limitations of anti-angiogenic therapies. Other strategies such as vascular promotion or the induction of high endothelial venules are being studied now to improve cancer therapy. Bone morphogenetic protein 9 (BMP9) exerts a dual effect through the activin receptor-like kinase 1 (ALK1) receptor in blood vessel maturation or activation phase of angiogenesis. Thus, it is an interesting pathway to target in combination with chemotherapies or immunotherapies. This review manuscript explores the effect of the BMP9-ALK1 pathway in tumor angiogenesis and the possible usefulness of targeting this pathway in anti-angiogenesis, vascular normalization or vascular promotion therapies.

Keywords: ALK1; bone morphogenetic protein 9; cancer therapy; tumor angiogenesis.

A pedigree of subjects presented with frontonasal dysplasia (FND). Genome sequencing and analysis identified a p.L165F missense variant in the homeodomain of the transcription factor ALX1 which was imputed to be pathogenic. Induced pluripotent stem cells (iPSC) were derived from the subjects and differentiated to neural crest cells (NCC). NCC derived from ALX1^L165F/L165F iPSC were more sensitive to apoptosis, showed an elevated expression of several neural crest progenitor state markers, and exhibited impaired migration compared to wild-type controls. NCC migration was evaluated in vivo using lineage tracing in a zebrafish model, which revealed defective migration of the anterior NCC stream that contributes to the median portion of the anterior neurocranium, phenocopying the clinical presentation. Analysis of human NCC culture media revealed a change in the level of bone morphogenic proteins (BMP), with a low level of BMP2 and a high level of BMP9. Soluble BMP2 and BMP9 antagonist treatments were able to rescue the defective migration phenotype. Taken together, these results demonstrate a mechanistic requirement of ALX1 in NCC development and migration.

Keywords: iPSC; ALX1; frontonasal dysplasia; neural crest cells; zebrafish.

Although a number of experimental models have been developed for liver research, each has its own advantages and disadvantages. The present study attempted to develop a simple and effective 3‑dimensional mouse liver microsphere tissue culture (LMTC) model in vitro for the analysis of hepatic functions. Hepatic characteristics and potential applications of this model were compared with that of mouse model in vivo and mouse primary hepatocytes in vitro. Using freshly‑perfused mouse liver tissue passed through 80‑mesh sift strainer (sift80), it was demonstrated that under the optimal culture conditions, the sift80 microsphere tissue cultured in 2% bovine calf serum medium remained viable with marked proliferating cell nuclear antigen and anti‑Myc proto‑oncogene protein expression, exhibited normal hepatic functions including indocyanine green (ICG) uptake/release and periodic acid‑Schiff staining, and expressed hepatocyte‑specific genes for up to 2 weeks. The microsphere tissue was responsive to bone morphogenic protein 9 (BMP9) stimulation leading to upregulation of downstream targets of BMP9 signaling. Furthermore, 3 commonly‑used liver‑damaging drugs were indicated to effectively inhibit hepatic ICG uptake, and induce the expression of hepatotoxicity‑associated genes. Therefore, this simplified LMTC model may be a useful in vitro tissue culture model to investigate drug‑induced liver injury and metabolism, and hepatocyte‑based cell singling.

Articular cartilage contains a subpopulation of tissue-specific progenitors that are an ideal cell type for cell therapies and generating neo-cartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium employing transforming growth factor-β (TGFβ) isoforms is optimal to differentiate these cells. We therefore used pellet culture to screen progenitors from immature bovine articular cartilage with a number of chondrogenic factors and discovered that bone morphogenetic factor-9 (BMP9) precociously induces their differentiation. This difference was apparent with toluidine blue staining and confirmed by biochemical and transcriptional analyses with BMP9 treated progenitors exhibiting 11-fold and 5-fold greater aggrecan and collagen type II gene expression than TGFβ1 treated progenitors. Quantitative gene expression analysis over 14 days highlighted the rapid and phased nature of BMP9 induced chondrogenesis with sequential activation of aggrecan then collagen type II, and negligible collagen type X gene expression. The extracellular matrix of TGFβ1treated progenitors analysed using atomic force microscopy was fibrillar and stiff whist BMP9-induced matrix of cells more compliant and correspondingly less fibrillar. Polarised light microscopy revealed an annular pattern of collagen fibril deposition typified by TGFβ1 treated pellets, whereas BMP9 treated pellets displayed a birefringence pattern that was more anisotropic. Remarkably, differentiated immature chondrocytes incubated as high-density cultures in vitro with BMP9 generated a pronounced anisotropic organisation of collagen fibrils indistinguishable from mature adult articular cartilage, with cells in deeper zones arranged in columnar fashion. This contrasted with cells grown with TGFβ1 where a concentric pattern of collagen fibrils was visualised within tissue pellets. In summary, BMP9 is a potent chondrogenic factor for articular cartilage progenitors and is also capable of inducing morphogenesis of adult-like cartilage, a highly desirable attribute for in vitro tissue-engineered cartilage.

This study aimed to investigate the transdifferentiation of human pulmonary arterial endothelial cells (HPAECs) into smooth muscle like (SM-like) cells under hypoxic conditions and reveal the role of endogenous small molecular compound 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylicacid methyl ester (ITE) in this process. HPAECs were treated by hypoxia and hypoxia + ITE with different durations. The endothelial markers (CD31 and VE-cad) and smooth muscle markers (α-SMA, SM22α, and OPN) were investigated by immunofluorescence double staining, and their expressions, along with the differentiation regulators transforming growth factor-β (TGF-β) ligands and downstream signals including TGF-β1, bone morphogenetic protein (BMP2), BMP9, Samd2/3, ERK, and p38 MAPK, were determined by Western blot analysis. The viability and proliferation of HPAECs were detected by Cell Counting Kit-8 (CCK-8) method and bromodeoxyuridine (BrdU) assays. As a result, hypoxia induced HPAECs transdifferentiation from paving-stone-like into polygonal or spindle cells, whose number increased greatly after additional ITE stimulation for 7 days. Compared with the normoxic HPAECs, the expression of endothelial markers reduced and smooth muscle markers were enhanced with the extension of hypoxia + ITE treatment, and meanwhile the cell viability increased significantly. Hypoxia could promote expression of TGF-β1 protein rather than BMP2 and BMP9, and regulate phosphorylation levels of Samd2/3, ERK and p38 MAPK in different manners. In conclusion, ITE can promote the hypoxia-induced transdifferentiation of HPAECs into SM-like cells via TGF-β/Smads and MAPK/ERK pathways.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome (ARDS). Circulating bone morphogeneic protein 9 (BMP9) is emerging as an important regulator of pulmonary vascular homeostasis.

Objective: To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity, and whether loss of endogenous BMP9 occurs during lipopolysacharride (LPS) challenge.

Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. Impact of BMP9 administration 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 results: Subacute neutralization of endogenous BMP9 in mice (N=12) resulted in increased lung vascular permeability (P=0.022), interstitial edema (P=0.0047) and neutrophil extravasation (P=0.029) compared with IgG control (N=6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell membrane integrity. Augmentation of BMP9 signaling in mice (N=8) prevented inhaled LPS-induced lung injury (P=0.0027) and edema (P<0.0001). In endotoxemic mice (N=12), 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 (N=10), circulating levels of BMP9 were also markedly reduced (P<0.0001).

Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial protective factor, down-regulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

Keywords: BMP9, BMP signalling in endothelial cells, Pulmonary endothelium, Lung injury.

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

Complex formation and endocytosis of transforming growth factor-β (TGF-β) receptors play important roles in signaling. However, their inter-dependence remained unexplored. Here, we demonstrate that ALK1, a TGF-β type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-β receptors (TβRIII).We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-β receptor (TβRII), endoglin or TβRIII. These complexes regulate the endocytosis of the TGF-β receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TβRIII and endoglin, while ALK5 and TβRII mildly enhance endoglin, but not TβRIII, internalization. Conversely, the slowly-endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5 or TβRII, while TβRIII has a differential effect, slowing down the internalization of ALK5 and TβRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-β receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications on signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.

The liver is the largest solid organ in the body, yet it remains incompletely characterized. Here we present a spatial proteogenomic atlas of the healthy and obese human and murine liver combining single-cell CITE-seq, single-nuclei sequencing, spatial transcriptomics, and spatial proteomics. By integrating these multi-omic datasets, we provide validated strategies to reliably discriminate and localize all hepatic cells, including a population of lipid-associated macrophages (LAMs) at the bile ducts. We then align this atlas across seven species, revealing the conserved program of bona fide Kupffer cells and LAMs. We also uncover the respective spatially resolved cellular niches of these macrophages and the microenvironmental circuits driving their unique transcriptomic identities. We demonstrate that LAMs are induced by local lipid exposure, leading to their induction in steatotic regions of the murine and human liver, while Kupffer cell development crucially depends on their cross-talk with hepatic stellate cells via the evolutionarily conserved ALK1-BMP9/10 axis.

Keywords: CITE-seq; Kupffer cell; NAFLD; across species; atlas; lipid-associated macrophage; liver; multi-omic; proteogenomic; spatial transcriptomics.