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Long non-coding RNAs are important regulators of biological processes, but their roles in the osteogenic differentiation of mesenchymal stem cells (MSCs) remain unclear. Here we investigated the role of murine HOX transcript antisense RNA (mHotair) in BMP9-induced osteogenic differentiation of MSCs using immortalized mouse adipose-derived cells (iMADs). Touchdown quantitative polymerase chain reaction analysis found increased mHotair expression in bones in comparison with most other tissues. Moreover, the level of mHotair in femurs peaked at the age of week-4, a period of fast skeleton development. BMP9 could induce earlier peak expression of mHotair during in vitro iMAD osteogenesis. Silencing mHotair diminished BMP9-induced ALP activity, matrix mineralization, and expression of osteogenic, chondrogenic and adipogenic markers. Cell implantation experiments further confirmed that knockdown of mHotair attenuated BMP9-induced ectopic bone formation and mineralization of iMADs, leading to more undifferentiated cells. Crystal violet staining and cell cycle analysis revealed that silencing of mHotair promoted the proliferation of iMAD cells regardless of BMP9 induction. Moreover, ectopic bone masses developed from mHotair-knockdown iMAD cells exhibited higher expression of PCNA than the control group. Taken together, our results demonstrated that murine mHotair is an important regulator of BMP9-induced MSC osteogenesis by targeting cell cycle and proliferation.

The etiology of nonimmune hydrops fetalis is extensive and includes genetic disorders. We describe a term-born female neonate with late onset extensive nonimmune hydrops, that is, polyhydramnios, edema, and congenital bilateral chylothorax. This newborn was successfully treated with repetitive thoracocentesis, total parenteral feeding, octreotide intravenously and finally surgical pleurodesis and corticosteroids. A genetic cause seemed plausible as the maternal history revealed a fatal nonimmune hydrops fetalis. A homozygous truncating variant inGDF2(c.451C>T, p.(Arg151*)) was detected with exome sequencing. Genetic analysis of tissue obtained from the deceased fetal sibling revealed the same homozygous variant. The parents and two healthy siblings were heterozygous for theGDF2variant. Skin and lung biopsies in the index patient, as well as the revised lung biopsy of the deceased fetal sibling, showed lymphatic dysplasia and lymphangiectasia. To the best of our knowledge, this is the first report of an association between a homozygous variant inGDF2with lymphatic dysplasia, hydrothorax and nonimmune hydrops fetalis.

Objective The study aimed to investigate the osteogenic ability of bioactive glass (bioglass) combined with recombinant human bone morphogenetic protein-9 (rhBMP-9) on rat bone marrow mesenchymal stem cells (BMSCs) in vitro. The study also compares bone regeneration using rhBMP9 soaked with different carrier systems, including bioglass or collagen membranes (BioGide, BG) in a rat alveolar bone site preservation model in vivo. Methods Scanning electron microscopy was employed to analyze bioglass surface. The absorption and release potential of rhBMP9 from bioglass were researched by ELISA. The cell viability, adhesion, proliferation, and differentiation were assessed for rhBMP9 soaked on bioglass by cck-8 kit, alkaline phosphatase (ALP) activity assay, alizarin red staining, and real-time PCR. Furthermore, prepared grafts (bioglass + BG, bioglass/rhBMP9+BG, and bioglass + BG/rhBMP9) were implanted into the maxillary right first incisor sockets of Sprague Dawley rats for 8 weeks, and new bone formation was quantified by micro-CT and histological analysis. Results Bioglass absorbed rhBMP9 dramatically and released it with a slow and stable speed within ten days by ELISA. When used with cck-8 kit detection, cell viability at 24 h, cell adhesion rate at 8 h, and cell proliferation at 1, 3, and 5 days were decreased in the bioglass alone group versus the control group but slightly increased with the addition of rhBMP9. Similarly, the effect of osteogenic differentiation on bioglass increased significantly when combined with rhBMP9 by upregulating the expression of ALP, mineralized matrix, and osteogenic related genes. Furthermore, both bioglass/rhBMP9+BG samples and bioglass + BG/rhBMP9 samples significantly improved several bone formation parameters compared with bioglass + BG samples. Interestingly, bioglass + BG/rhBMP9 samples demonstrated more bone regeneration in rat site preservation models. Conclusions Both bioglass and BG can be applied in GBR surgery as effective carriers of rhBMP9. However, BG may be more suitable than bioglass for investigating site preservation effect after tooth extraction when associated with rhBMP9 and provides a practical clinical solution to the problem of bone deficiency caused by alveolar bone atrophy., Bone regeneration; Bone morphogenetic proteins; BMP9; Osteogenesis; Bioglass tooth extraction site preservation.

Le recrutement des leucocytes dans les tissus est un processus courant dans toutes les maladies inflammatoires de l'adulte. Celui-ci est médié par des interactions de haute affinité entre les molécules d'adhésion endothéliales à la surface des vaisseaux sanguins et leurs ligands sur les cellules immunitaires circulantes. Ce processus implique une multitude de molécules telles que ICAM1, VCAM1 et les sélectines pour soutenir la « capture » des cellules immunitaires et leur extravasation à travers les cellules endothéliales vasculaires. Nos résultats préliminaires suggèrent que le récepteur ALK1, exprimé spécifiquement sur les cellules endothéliales, pourrait réguler l'expression de ces molécules d'adhésion. Pour examiner l'impact de la signalisation ALK1 sur le recrutement des cellules immunitaires, nous avons utilisé des lignées de cellules endothéliales siALK1 (HUVECs) traitées avec du lipopolysaccharide (LPS) pour induire un processus inflammatoire. Tout d'abord, l'expression des gènes, examinée par Ampliseq, a montré une diminution significative de l'expression de ICAM1, VCAM1 et E-sélectine dans les HUVECs siALK1 par rapport au contrôle. De plus, l'analyse par cytométrie en flux a démontré que l'expression des protéines d'adhésion augmentent dans les cellules traitées avec du LPS, mais que cet effet est atténué dans les cellules endothéliales dépourvues d'ALK1. De plus, nous avons observé que le p38 phosphorylé, un facteur qui joue un rôle clé dans le processus inflammatoire, est diminué dans les cellules siALK1. Enfin, pour étudier les conséquences fonctionnelles de la délétion d'ALK1 sur les interactions leucocytes/cellules endothéliales, un test d'adhésion en flux est réalisé, dans lequel les leucocytes circulent sur les cellules endothéliales et l'extravasation des cellules immunitaires est observée au microscope. Les résultats ont montré une diminution de l'infiltration des leucocytes dans les HUVECs siALK1 par rapport aux témoins. En conclusion, nos données démontrent qu'ALK1 a un impact majeur sur le recrutement des leucocytes vers l'endothélium par l'expression de protéines d'adhésion ainsi que par la normalisation vasculaire. Ce projet contribue à une meilleure compréhension de l'interaction entre les leucocytes et l'endothélium vasculaire., Leukocyte recruitment into tissues is a common process in all adult inflammatory diseases. This is mediated by high affinity interactions between endothelial adhesion molecules and their counter receptor ligands on circulating immune cells. This process involves a multitude of molecules such as ICAM1, VCAM1 and selectins to support the "capture’’ of immune cells and their extravasation through vascular endothelial cells. Our preliminary results suggest that ALK1 receptor, expressed specifically on endothelial cells, could regulate the expression of these adhesion molecules. To examine the impact of ALK1 signaling on immune cells recruitment, we used siALK1 endothelial cell lines (HUVECs) treated with LPS (Lipopolysaccharide) to induce an inflammatory process. First, gene expression, examined by Ampliseq, showed a significant decrease in ICAM1, VCAM1 and E-selectin expression in siALK1 HUVECs compared to control. Furthermore, flow cytometry analysis demonstrated that adhesion protein expression increases in control cells treated with LPS, but that this effect is lessened in endothelial cells lacking ALK1. Moreover, we observed that phosphorylated p38, a factor that plays a key role in inflammatory process, is decreased in siAlk1 cells. Finally, to study the functional consequences of ALK1 deletion on leukocyte/endothelial cells interactions, a flow adhesion assay is performed, in which leukocytes circulate on endothelial cells and extravasation of immune cells is observed under a microscope. The results showed decreased leukocyte infiltration in siALK1 HUVECs compared to controls. In conclusion, our data demonstrate that ALK1 has a major impact on leukocyte recruitment to the endothelium through the expression of adhesion proteins and the vascular normalization. This project contributes to a better understanding of the interaction of leukocytes and the vascular endothelium.

Background Bone morphogenetic protein 9 (BMP9) has been identified as a crucial inducer of osteoblastic differentiation in mesenchymal stem cells (MSCs). Although microRNAs (miRNAs) are known to play a role in MSC osteogenesis, the mechanisms of action of miRNAs in BMP9-induced osteoblastic differentiation remain poorly understood. Methods In this study, we investigate the possible role of the miR17-92 cluster in the BMP9-induced osteogenic differentiation of MSCs by using both in vitro and in vivo bone formation assays. Results The results show that miR-17, a member of the miR17-92 cluster, significantly impairs BMP9-induced osteogenic differentiation. This impairment is effectively rescued by a miR-17 sponge, an antagomiR sequence against miR-17. Using TargetScan and the 3′-untranslated region luciferase reporter assays, we show that the direct target of miR-17 is the retinoblastoma gene (RB1), a gene that is pivotal to osteoblastic differentiation. We also confirm that RB1 is essential for the miR-17 effects on osteogenesis. Conclusion Our results indicate that miR-17 expression impairs normal osteogenesis by downregulating RB1 expression and significantly inhibiting the function of BMP9.

In dit proefschrift begin ik met een algemene inleiding in Hoofdstuk 1 om kort de relevantie van EC-gedrag in vasculaire morfogenese en in angiogenese te presenteren. Bovendien bespreek ik hoe de EC-functie ingewikkeld wordt gereguleerd door positieve en negatieve factoren, en hoe hun functie kan worden gemanipuleerd voor therapeutische winst bij kanker en hart- en vaatziekten. In Hoofdstuk 2 bespreken we in detail de rol van de TGF-β-signaleringsroute in EndMT en bespreken we de bijdrage van dit proces aan de ontwikkeling van ziekten, evenals de mogelijke toepassingen ervan in weefselmanipulatie. In Hoofdstuk 3 onthullen we gedetailleerde werkprotocollen om TGF-β-geïnduceerde EndMT te onderzoeken en hoe de betrokkenheid van EndMT-effectoren te beoordelen met behulp van CRISPR/Cas9-genediting. In Hoofdstuk 4 hebben we de functie van EndMT transcriptiefactoren onderzocht en hun werkingsmechanisme opgehelderd. We ontdekten dat de EndMT-transcriptiefactoren (TF's) SNAIL en SLUG cruciaal zijn voor EndMT in endotheelcellen van muizen en dat de ID-eiwitten hun functie in EndMT compenseren. In Hoofdstuk 5 geven we een technisch overzicht van embryonale zebravis-xenotransplantaattesten om TGF-β-familiesignalering in de progressie van borstkanker bij de mens te onderzoeken, waaronder intravasatie/extravasatie van tumorcellen en tumorangiogenese. In Hoofdstuk 6 identificeren en onderzoeken we twee nieuwe BMP type I receptor macrocyclische kinaseremmers met therapeutisch potentieel om angiogenese in normale en tumorvatvorming bij zebravissen te normaliseren. In Hoofdstuk 7 vat ik alle studies in het proefschrift samen en geef ik enkele toekomstperspectieven met betrekking tot onze resultaten.

Considering the predominant role of rare variants of the Bone morphogenetic protein 9 (BMP9) gene in the occurrence of idiopathic pulmonary arterial hypertension (IPAH), here we conducted a case-control study, together with functional validation, to explore the relationships between variants of the BMP9 gene and development of IPAH. We found minor alleles of rs3740297 (OR: 0.72, 95% CI: 0.59-0.87, P=7.77×10-5) and rs7923671 (OR: 0.76, 95% CI: 0.62-0.93, P=0.009) were significantly associated with decreased risk of IPAH. Minor alleles of rs3740297 and rs7923671 were significantly associated with increased plasma level of BMP9 in both IPAH cases and controls (P

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.

Background Mesenchymal stem cells (MSCs) play a crucial role in maintaining the dynamic balance of bone metabolism. Melatonin may have a regulatory effect on bone metabolism by regulating the lineage commitment and differentiation signalling pathways of MSCs. Among the BMP families, the osteogenesis of BMP9 is considered to be one of the strongest in MSCs. Here, we explored whether melatonin and BMP9 act synergistically on MSC osteogenic differentiation. Methods The C3H10T1/2 osteogenic differentiation function induced by melatonin synergizes with BMP9, as detected by the expression of osteogenic markers at different periods. The result was further confirmed by foetal limb explant culture and in vivo stem cell implantation experiments. The effects of the AMPK/β-catenin pathway on the osteogenic differentiation of C3H10T1/2 cells were evaluated by Western blotting. Results Melatonin combined with BMP9 significantly enhanced the expression of osteogenic markers at different periods in C3H10T1/2 cells, effectively enhancing BMP9-induced bone formation in cultured foetal explants and ectopic bone formation in vivo in stem cell transplantation experiments. Melatonin increases the expression of BMP9 in C3H10T1/2 cells and induces Smad1/5/8 translocation from the cytoplasm to the nucleus. In addition, melatonin and BMP9 synergistically promote AMPK and β-catenin phosphorylation, which can be largely eliminated by AMPK siRNA pretreatment. Conclusions Melatonin and BMP9 in C3H10T1/2 cells synergistically promote osteogenic differentiation at least in part by activating the AMPK/β-catenin signalling pathway.

Bone tissue engineering requires a combination of cells, efficient biochemical and physicochemical factors, and biocompatible scaffolds. In this study, we evaluated the potential use of injectable Matrigel as a scaffold for the delivery of rat dental follicle stem/precursor cells (rDFSCs) transduced by bone morphogenetic protein (BMP) 9 to enhance osteogenic differentiation in vitro and promote ectopic bone formation in vivo. Recombinant adenovirus was used to overexpress BMP9 in rDFSCs. Alkaline phosphatase activity was measured using a histochemical staining assay and a chemiluminescence assay kit. Quantitative real-time polymerase chain reaction was used to determine mRNA expression levels of bone-related genes including distal-less homeobox 5 (DLX5), osteopontin (OPN), osterix (Osx), and runt-related transcription factor 2 (Runx2). Matrix mineralization was examined by Alizarin Red S staining. rDFSCs proliferation was analyzed using the Cell Counting Kit-8 assay. Subcutaneous implantation of rDFSCs-containing Matrigel scaffolds was used, and micro-computed tomography analysis, histological evaluation, and trichrome staining of implants extracted at 6 weeks were performed. We found that BMP9 enhanced alkaline phosphatase activity and mineralization in rDFSCs. The expression of bone-related genes (DLX5, OPN, Osx, and Runx2) was also increased as a result of BMP9 stimulation. Micro-computed tomography analysis and histological evaluation revealed that the bone masses retrieved from BMP9-overexpressing rDFSCs were significantly more pronounced in those with than in those without Matrigel. Our results suggest that BMP9 effectively promote osteogenic differentiation of rDFSCs, and Matrigel facilitate BMP9-induced osteogenesis of rDFSCs in vivo.

Background and Aims: It’s reported that bone morphogenetic protein 9 (BMP9) played an important role in lipid and glucose metabolism, but the role of BMP9 in nonalcoholic fatty liver disease (NAFLD) is unclear. Here, we evaluated the therapeutic efficacy of recombined BMP9 in NAFLD mice and investigated the potential mechanism.Methods: The effects of recombinant BMP9 on NAFLD were assessed in HFD-induced NAFLD mice. C57BL/6 mice were administrated with high-fat diet (HFD) for 12 weeks. In the last 4 weeks, mice were treated with PBS or recombined BMP9 once daily. Insulin sensitivity was evaluated by glucose tolerance test (GTT) and insulin tolerance test (ITT) at the end of the 12th week. Then NAFLD related indicators were assessed by a variety of biological methods, including histology, western blotting, real-time PCR, RNA-seq and assay for transposase-accessible chromatin using sequencing (ATAC-seq) analyses.Results: BMP9 reduced obesity, improved glucose metabolism, alleviated hepatic steatosis and decreased liver macrophages infiltration in HFD mice. RNA-seq showed that Cers6, Cidea, Fabp4 involved in lipid and glucose metabolism and Fos, Ccl2, Tlr1 involved in inflammatory response downregulated significantly after BMP9 treatment in HFD mouse liver. ATAC-seq showed that chromatin accessibility on promoters of Cers6, Fabp4, Ccl2 and Fos decreased after BMP9 treatment in HFD mouse liver. KEGG pathway analysis of dysregulated genes in RNA-seq and integration of RNA-seq and ATAC-seq showed that TNF signaling pathway and Toll-like receptor signaling pathway decreased in BMP9 treated HFD mouse liver.Conclusion: Our data revealed that BMP9 might alleviate NAFLD via improving glucose and lipid metabolism, decreasing inflammatory response and reshaping chromatin accessibility in HFD mouse liver. BMP9 downregulate genes related to lipid metabolism, glucose metabolism and inflammation expression, at least partially via decreasing promoter chromatin accessibility of Cers6, Fabp4, Fos and Tlr1. BMP9 may also reduce the expression of liver Ccl2, thereby changing the number or composition of liver macrophages, and ultimately reducing liver inflammation. The effect of BMP9 on NAFLD might be all-round, and not limit to lipid and glucose metabolism. Therefore, the underlying mechanism needs to be studied in detail further.

L’angiogenèse est définie comme la formation de nouveaux capillaires à partir des vaisseaux sanguins pré-existants. Elle contribue à l’extension du réseau vasculaire et assure ainsi l’efficacité des échanges gazeux et du transport des cellules, nutriments, métabolites et molécules de signalisation vers les tissus. L’angiogenèse par bourgeonnement passe par la spécification d’une cellule endothéliale en cellule meneuse et la formation d’un réseau de cellules suiveuses à la base du bourgeon vasculaire. Toute perturbation de ce mode de néovascularisation génère des vaisseaux fortement tortueux, immatures et non-étanches qui soit affectent les fonctions physiologiques des organes en causant ainsi des pathologies potentiellement fatales, ou accélèrent la progression des conditions telles que le cancer. Dans l’oeil, l’angiogenèse pathologique des vaisseaux choroïdiens et rétiniens cause une perte de la vue. Particulièrement, la dégénérescence maculaire liée à l’âge (DMLA) de type néovasculaire, une maladie oculaire caractérisée par le bourgeonnement anormal de la choroïde dans l’espace sous-rétinien, représente la cause majeure de cécité au sein des populations des pays industrialisés. Les thérapies conventionnelles contre la DMLA humide reposent sur l’usage des médicaments qui ciblent la signalisation du facteur de croissance de l’endothélium vasculaire (VEGF). Bien que démontrant des résultats cliniques, ces traitements anti-VEGFs sont invasifs et présentent multiples effets secondaires. Par ailleurs, ils n’induisent aucun effet chez une portion des patients traités. De ces faits, il existe présentement un grand besoin de thérapies alternatives aux anti-VEGFs. De façon intéressante, la protéine de morphogénèse osseuse 9 (BMP9), qui active son récepteur “activin receptor-like kinase 1” (ALK1), régule l’angiogenèse développementale des vaisseaux rétiniens de l’oeil de la souris. Par ailleurs, les mutations au sein du BMP9, de son récepteur ALK1 ou de ses intermédiaires de signalisation sont associées à la morphogénèse anormale des vaisseaux qui contribue ultimement à la pathogénèse de diverses maladies néovasculaires. De façon additionnelle, le récepteur ALK1 au BMP9 est restreint à la cellule endothéliale; contrairement à ceux des ligands angiogéniques tels que le VEGF, exprimés par une diversité de cellules. Par ailleurs, au sein de cette cellule, le BMP9 contribue à la régulation des phénotypes meneur et suiveur qui sont induits par le VEGF et requis pour le déroulement de l’angiogenèse par bourgeonnement. De ces faits qui précèdent, nous avons émis l’hypothèse du rôle du BMP9 dans la régulation de la néovascularisation pathologique relative à la DMLA humide. Ainsi, les travaux de la présente thèse déterminent spécifiquement l’effet du BMP9 sur l’angiogenèse pathologique à l’aide des modèles oculaires pertinents à la DMLA humide et examine aussi sa base mécanistique. Les travaux de cette thèse démontrent l’effet anti-angiogénique du BMP9 sous les conditions expérimentales de néovascularisation choroïdienne induite au laser (CNV) et de rétinopathie induite par l’oxygène (OIR). Ils montrent aussi les effets régulateurs de la signalisation du BMP9 sur les voies de signalisation endothéliales du VEGF et de Notch, respectivement de façon dépendante de VEGFR1 et de JAG1. En somme, les présentes études démontrent les effets anti-angiogéniques du BMP9 sur la néovascularisation pathologique relative à la DMLA humide et identifient les facteurs moléculaires qui contribuent à son action inhibitrice du bourgeonnement vasculaire induit par le VEGF., Angiogenesis is defined as the formation of new capillaries from existing blood vessels. It extends the vasculature and thereby sustains the efficient exchange of gases and transport of cells, nutrients, metabolites and signalling molecules to tissues. Sprouting angiogenesis proceeds through the selective specification of an endothelial cell into a leading tip cell and the formation of stalk cells at the base of the sprout. A disturbance in this modality of neovascularisation leads to highly tortuous, immature and leaky vessels that either impair the physiological functions of organs, thereby causing life-threatening diseases, or accelerate the progression of conditions such as cancer. In the eye, the pathological angiogenesis of choroidal and retinal vessels specifically results in vision loss. Particularly, the neovascular form of the age-related macular degeneration (AMD), an ocular disease characterized by the abnormal sprouting of the choroidal network into the subretinal space, represents the leading cause of blindness in populations of industrialized countries. Conventional therapies against wet AMD are based on drugs that target the signaling of the vascular endothelium growth factor (VEGF). Despite their clinical achievements, the anti-VEGFs treatments are invasive and show multiple adverse effects. Moreover, they are not effective in a portion of treated patients. Thus, there currently is a substantial need of therapy alternatives to anti-VEGFs. Interestingly, the bone morphogenetic protein 9 (BMP9), that activates its activin receptor-like kinase 1 (ALK1) transducer, regulates the developmental angiogenesis of the mouse eye retina vasculature. Moreover, mutations in BMP9, its receptor ALK1 or its signaling mediators correlate with the abnormal vessel morphogenesis that ultimately drives the pathogenesis of various neovascular diseases. Additionally, the BMP9-specific receptor ALK1 is restricted to endothelial cells; in contrast to those of neovascularisation-inducing ligands such as VEGF, expressed by a range of cells. Particularly within these cells, BMP9 contributes to regulate the VEGF-induced tip/stalk phenotypes required for sprouting angiogenesis. Given the aforementioned, we hypothesized the role of BMP9 in regulating the pathological angiogenesis associated with wet AMD. Thus, the studies from the current thesis specifically determine the effect of BMP9 on pathological NV using ocular models relevant to AMD and further investigate its mechanical basis. The current work demonstrates the antiangiogenic effects of BMP9 under experimentally induced oxygen-induced retinopathy (OIR) and laser-induced choroid neovascularisation (CNV) conditions. Moreover, this thesis shows the regulatory effects of BMP9 signaling on the VEGF and Notch endothelial pathways, respectively in VEGFR1 and JAG1 -dependent manners. Collectively, the current studies demonstrate the anti-angiogenic effects of BMP9 on pathological NV associated with wet AMD and identify the molecular players that mediate its inhibitory action on VEGF-mediated sprouting.

8 p.-2 fig. This entry is adapted from 10.3390/jcm9113571

Vascular smooth muscle cells (VSMCs) show a remarkable phenotypic plasticity reflecting their ability to acquire contractile or synthetic states. Numerous signaling pathways and conditions are known, which cause loss of contractility and attainment of a synthetic state but knowledge about signaling pathways promoting the contractile state is limited and in vivo evidence is critically missing. Here, we investigated the role of BMP9, BMP10, activin receptor-like kinase 1 (ALK1) and Smad7 for controlling formation of contractile VSMCs. Effects of Bmp9 and Bmp10 inactivation, conditional deletion of Alk1 in SMCs, overexpression of Bmp10 in endothelial cells (ECs) and overexpression of Smad7 in SMCs were evaluated in respect to coverage of vessels by VSMC, their contractile state and blood pressure. The molecular mechanisms underlying effects of BMP9/BMP10 were studied by treating dedifferentiated SMCs with BMP9/BMP10 in vitro. Concomitant genetic inactivation of BMP9/BMP10 in mice led to a dramatic diminution of VSMC layer, loss of contractile VSMCs and decreased systemic blood pressure. Consistently, overexpression of Bmp10 in ECs of adult mice dramatically enhanced VSMC contractility and increased formation of contractile VSMCs, indicating that BMP9/BMP10 play fundamental roles for differentiation and contractility of VSMCs. Importantly, treatment of BMP9/BMP10 induced an ALK1-dependent phenotypic switch from synthetic to contractile state in de-differentiated VSMCs. SMC-specific overexpression of inhibitory Smad7 completely suppressed differentiation and proliferation of VSMCs during embryonic development and reiterated the defects observed in BMP9/BMP10 double mutants in adults. Likewise, deletion of Alk1 in SMCs recapitulated attenuated VSMCs coverage and loss of contractile VSMCs seen in BMP9/BMP10 mutants but only in pulmonary and not in aortic or coronary arteries. Bulk RNA-seq and single molecule RNA-fluorescent in situ hybridization (RNA-FISH) uncovered heterogeneous expression of BMP type I receptors in distinct vessel beds, explaining phenotypic differences between Alk1 and Bmp9/Bmp10 inactivation. Furthermore, analysis of BMP9/BMP10 stimulated VSMCs by ATAC-seq combined with RNA-seq revealed that YAP/TEAD and BMP9/BMP10/Smad-signaling converge at promoters of several VSMC genes. Gain-and loss-of-function studies of the YAP-TEAD axis demonstrated that YAP-TEAD axis antagonized BMP9/BMP10 induced VSMCs differentiation and formation of contractile VSMCs. In conclusion, the study indicated that BMP9 and BMP10 are released into the blood stream by the liver and the right atrium, respectively and bypass the endothelial barrier to directly induce and maintain contractile VSMCs. Importantly, the effects of BMP9/BMP10 in VSMCs are modulated by different combination of BMP type one receptors in a vessel bed specific manner. Vaskuläre glatte Muskelzellen (VSMCs) verfügen über eine bemerkenswerte phänotypische Plastizität, die eine Umschaltung zwischen einem kontraktilen and einem synthetisch-proliferativen Zustand ermöglicht. Bis dato wurden zahlreiche Signalwege und Bedingungen beschrieben, welche den Übergang von VSMCs aus dem kontraktilen in den synthetischen Zustand fördern. Demgegenüber ist die Erkenntnis über Signalwege, die den kontraktilen Zustand induzieren sehr begrenzt und schlagende in-vivo Beweise fehlen. In der vorliegenden Arbeit wurde die Rolle von BMP9, BMP10, Activin-Receptor-like Kinase 1 (ALK1) und Smad7 hinsichtlich der Bildung und Erhaltung kontraktiler VSMCs untersucht. Im Fokus stand dabei die glatte Muskelzellschicht von Blutgefäßen aus konditionalen BMP9/10 Doppel-Knockout (dKO) Mutanten, aus glatten Muskelzell-spezifischen ALK1-KO Mutanten sowie aus endothel-spezifischen BMP10- bzw. VSMC-spezifische SMAD7-Überexpressionsmutanten. Die Abdeckung verschiedener Gefäße mit VSCMs wurde untersucht, ebenso der Blutdruck in den verschiedenen mutanten Mausstämmen, sowie die Kontraktilität der VSMC. Des Weiteren wurden primäre, dedifferenzierte VSMCs ex-vivo mit BMP9 / BMP10 stimuliert, um einen Einblick in molekularen Mechanismen zu erlangen, die der Wirkung von BMP9/10 zugrunde liegen. Eine kombinierte genetische Inaktivierung von BMP9 und BMP10 bei Mäusen führte zu einer dramatischen Verringerung der VSMC-Schicht, einem Verlust kontraktiler VSMCs und einem verringerten systemischen Blutdruck. Im Einklang hiermit führte eine Endothel-spezifische BMP10 Überexpression zu einer verstärkten Bildung von VSMCs, die zudem eine erhöhte Kontraktiliät aufwiesen, was auf eine grundlegende Funktion der beiden Liganden bei der Regulation der VSMC Differenzierung hinweist. Zudem induzierte eine BMP9 / BMP10 Behandlung von de-differenzierten VSMCs einen ALK1-abhängigen phänotypischen Wechsel vom synthetischen in den kontraktilen Zustand. Die VSMC-spezifische Überexpression von inhibitorischem Smad7 blockierte die Differenzierung und Proliferation von VSMCs während der Embryonalentwicklung vollständig und rekapitulierte die bei BMP9 / BMP10-Doppelmutanten beobachteten Defekte im Erwachsenenstadium. Ebenso phänokopierte die Deletion von ALK1 in SMCs die abgeschwächte VSMCs-Abdeckung und den Verlust kontraktiler VSMCs in Lungen-, jedoch nicht in Aorten- oder Koronararterien. Bulk-RNA-seq und Einzelmolekül-RNA-FISH enthüllte eine heterogene Expression von BMP-Typ-I-Rezeptoren in verschiedenen Gefäßbetten, was die phänotypischen Unterschiede der Ausschaltung von BMP9/10 gegenüber einer Inaktivierung von ALK1 erklärt. Die Analyse von BMP9 / BMP10-stimulierten VSMCs durch ATAC-seq in Kombination mit RNA-seq zeigte, dass YAP / TEAD- und BMP9 / BMP10 / Smad-Signale an Promotoren mehrerer VSMC-Gene konvergieren. Funktionsgewinn- und Funktionsverluststudien deuten weiterhin darauf hin, dass die YAP-TEAD-Achse eine BMP9/BMP10 induzierte Differenzierung von VSMCs und die Bildung kontraktiler VSMCs antagonisierte. Zusammenfassend zeigt die Studie, dass im Blutstrom zirkulierendes BMP9 und BMP10 die Endothelbarriere umgehen, um den Eigenschaften kontraktiler VSMCs direkt zu induzieren und aufrechtzuerhalten. Die Wirkungen von BMP9/BMP10 in VSMCs werden durch unterschiedliche Kombinationen von BMP-Typ-1-Rezeptoren in einer Gefäßbett-spezifischen Weise moduliert.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during 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. The 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 from endotoxemic mice. Measurements and 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 treatment (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 circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury.

BACKGROUND: The peptide apelin is expressed in human healthy livers and is implicated in the development of hepatic fibrosis and cirrhosis. Mutations in the bone morphogenetic protein receptor type II (BMPR-II) result in reduced plasma levels of apelin in patients with heritable pulmonary arterial hypertension. Ligands for BMPR-II include bone morphogenetic protein 9 (BMP9), highly expressed in liver, and BMP10, expressed in heart and to a lesser extent liver. However, it is not known whether reductions in BMP9 and/or BMP10, with associated reduction in BMPR-II signalling, correlate with altered levels of apelin in patients with liver fibrosis and cirrhosis. METHODS: Plasma from patients with liver fibrosis (n = 14), cirrhosis (n = 56), and healthy controls (n = 25) was solid-phase extracted using a method optimised for recovery of apelin, which was measured by ELISA. RESULTS: Plasma apelin was significantly reduced in liver fibrosis (8.3 ± 1.2 pg/ml) and cirrhosis (6.5 ± 0.6 pg/ml) patients compared with controls (15.4 ± 2.0 pg/ml). There was no obvious relationship between apelin and BMP 9 or BMP10 previously measured in these patients. Within the cirrhotic group, there was no significant correlation between apelin levels and disease severity scores, age, sex, or treatment with β-blockers. CONCLUSIONS: Apelin was significantly reduced in plasma of patients with both early (fibrosis) and late-stage (cirrhosis) liver disease. Fibrosis is more easily reversible and may represent a potential target for new therapeutic interventions. However, it remains unclear whether apelin signalling is detrimental in liver disease or is beneficial and therefore, whether an apelin antagonist or agonist have clinical use.

Teeth arise from the tooth germ through sequential and reciprocal interactions between immature epithelium and mesenchyme during development. However, the detailed mechanism underlying tooth development from tooth germ mesenchymal cells (TGMCs) remains to be fully understood. Here, we investigate the role of Wnt/β‐catenin signalling in BMP9‐induced osteogenic/odontogenic differentiation of TGMCs. We first established the reversibly immortalized TGMCs (iTGMCs) derived from young mouse mandibular molar tooth germs using a retroviral vector expressing SV40 T antigen flanked with the FRT sites. We demonstrated that BMP9 effectively induced expression of osteogenic markers alkaline phosphatase, collagen A1 and osteocalcin in iTGMCs, as well as in vitro matrix mineralization, which could be remarkably blunted by knocking down β‐catenin expression. In vivo implantation assay revealed that while BMP9‐stimulated iTGMCs induced robust formation of ectopic bone, knocking down β‐catenin expression in iTGMCs remarkably diminished BMP9‐initiated osteogenic/odontogenic differentiation potential of these cells. Taken together, these discoveries strongly demonstrate that reversibly immortalized iTGMCs retained osteogenic/odontogenic ability upon BMP9 stimulation, but this process required the participation of canonical Wnt signalling both in vitro and in vivo. Therefore, BMP9 has a potential to be applied as an efficacious bio‐factor in osteo/odontogenic regeneration and tooth engineering. Furthermore, the iTGMCs may serve as an important resource for translational studies in tooth tissue engineering.

Pulmonary arterial hypertension (PAH) is a rare disease initiated by dysfunction of the pulmonary vascular endothelium. Mutations in BMPR2, which encodes the type-II bone morphogenic protein (BMP) receptor BMPR-II, explain the majority of heritable PAH. BMPR-II forms a signalling complex, with ALK1 and endoglin, specific to endothelial cells which maintains vascular quiescence. BMP9 and BMP10 are the high affinity ligands for this signalling complex. Administering exogenous BMP9 has been shown to reverse disease in animal models of PAH, so the Morrell group is investigating the use of BMP9 as a therapy for PAH. Nonetheless, mutations in GDF2, which encodes BMP9, or BMP10 had not been conclusively associated with PAH. BMP9 and BMP10 are produced as inactive ProBMPs, which are processed by proteases to leave an active, non-covalent Pro:BMP complex of the prodomains and growth-factor domain. The circulating levels of processed and unprocessed BMP9 and BMP10 published in current reports are inconsistent. There is also some evidence circulating BMP9 and BMP10 may be co-complexed and otherwise coregulated. BMP9 is highly expressed in livers, and high doses can regulate lipogenesis and glucogenesis in vitro and in vivo. BMP9 deficiency is reported to protect against hepatic fibrosis and damage after inducement with severe surgical and chemical interventions. In contrast, BMP9 deficient animals do not appear to develop a phenotype reminiscent of PAH. Recently, the Morrell group discovered loss-of-function mutations in GDF2 were associated with PAH with genome-wide significance in a rare-variant analysis. Most of the identified mutations were missense. To validate their effect in vitro, I compared a selection of those predicted to be pathogenic in silico to those predicted to be benign. Pathogenic variants were poorly secreted due to destabilisation of the interaction between the prodomains and growth-factor domain, reducing their activity. Patients carrying loss-of-function GDF2 mutations had reduced circulating levels of both BMP9 and BMP10. Data from a combination of ELISAs, immunoprecipitations and activity assays suggested that active BMP9 circulates in healthy people, whilst inactive, unprocessed BMP10 circulates at higher levels. Circulating levels of BMP9 and BMP10 were tightly correlated, and data from immunoprecipitations and size-exclusion chromatography suggests they may circulate in macromolecular complexes. Exogenous BMP9 administered to mice was rapidly cleared from the circulation but was able to bind the lungs and activate signalling there, further demonstrating its therapeutic utility. Hepatic transcriptomes from Gdf2 knockout mice showed evidence of reduced lipogenesis and oxidative metabolism, confirming a role for endogenous BMP9 in metabolism. We developed a protocol to measure pulmonary vascular leak in BMP9 deficient mice.

Un endothélium vasculaire rétinien quiescent est essentiel dans l’approvisionnement et le maintien de l’homéostasie tissulaire afin d’assurer la fonction visuelle. L’hyperglycémie chronique dans le diabète peut mener à des complications reliées à plusieurs changements structuraux et fonctionnels de l’endothélium. Ces changements se manifestent souvent par des vaisseaux instables et hyperperméables, résultant en un tissu rétinien inadéquatement perfusé. Ces altérations sont rencontrées dans la pathophysiologie de l’œdème maculaire diabétique (OMD) qui affecte plus du quart des diabétiques. L’inhibition du facteur de perméabilité vasculaire VEGF-A a révolutionné le traitement de l’OMD, mais est aussi associée à des effets secondaires non négligeables reliés à leur non-spécificité et à une compréhension incomplète des mécanismes régulant la perméabilité vasculaire. Un ciblage spécifique de l’endothélium permettant la récupération de la quiescence et la stabilité physiologique réduisant l’hyperperméabilité pourrait fournir un nouvel outil thérapeutique. La BMP9 (Bone morphogenetic protein 9), qui est mise en circulation par le foie, est connue comme facteur de quiescence et de stabilité vasculaire ainsi que pour son effet sur l’homéostasie du glucose. Ces aspects étant peu documentés dans un contexte diabétique, nous avons évalué les capacités de la BMP9 sur la stabilisation de l’endothélium rétinien et ses effets paracrines/autocrines sur la gluconéogenèse hépatique. Nous avons démontré que la signalisation canonique de la BMP9/Smad1,5,9 via son récepteur spécifique à l’endothélium Alk1, était déficiente dans un modèle murin de diabète, ce qui exacerbe l’hyperperméabilité endothéliale rétinienne. À l’inverse, la surexpression de la BMP9 par un modèle murin de livraison adénovirale récupère la perméabilité physiologique, associée principalement à un renforcement des jonctions interendothéliales en limitant l’action du VEGF. De plus, nous avons trouvé que la BMP9 améliore le contrôle glycémique chez les souris diabétiques par l’inhibition de la gluconéogenèse hépatique, via la voie non-canonique Alk3/FOXO1. Fondamentalement, ce travail met en évidence les mécanismes régissant la perméabilité endothéliale dans un contexte diabétique, fournissant une alternative thérapeutique contre l’OMD. La régulation de la perméabilité rétinienne par la BMP9 s’effectue à plusieurs niveaux, indirectement par le contrôle glycémique, et directement par la solidification jonctionnelle de la barrière endothéliale rétinienne, réhabilitant ainsi la quiescence et la stabilité de l'endothélium physiologique., A quiescent retinal vascular endothelium is essential for the supply and maintenance of tissue homeostasis to ensure proper visual function. Chronic hyperglycemia in diabetes can lead to multiple complications related to several structural and functional changes in the endothelium, characterized by unstable and hyperpermeable vessels resulting in an inadequately perfused retinal tissue. These alterations are encountered in the pathophysiology of diabetic macular edema (DME), which affects more than a quarter of diabetics. Inhibition of the vascular permeability factor VEGF-A has revolutionized the treatments of DME but is associated with non-negligible side effects related to their non-specific action combined with an incomplete understanding of the mechanisms regulating vascular permeability. Specific endothelial targeting aiming to recover quiescence and reducing hyperpermeability could provide new therapeutic tools for the treatment or prevention of DME. BMP9 (Bone morphogenetic protein 9), which is produced by the liver, is known as a vascular quiescence and stability factor as well as for its effects on glucose homeostasis. Since these aspects are poorly documented in a diabetic context, we investigated BMP9’s capabilities on endothelium stabilization and its paracrine/autocrine effects on hepatic gluconeogenesis. In our studies, we found that the canonical BMP9/Smad1,5,9 signaling, via its physiological Alk1 endothelium-specific receptor, was deficient in a murine model of diabetes, which exacerbates retinal endothelial hyperpermeability. In contrast, adenoviral overexpression of BMP9 recovers physiological permeability, which was primarily associated with the enhancement of interendothelial junctions by limiting the action of VEGF. In addition, we found that BMP9 improves glycemic control in diabetic mice by inhibition of hepatic gluconeogenesis via the non-canonical ALK3/FOXO1 pathway. Fundamentally, this work highlights new insights of the mechanisms governing endothelial permeability in a diabetic context, providing a therapeutic alternative against DME. Regulation of retinal permeability by BMP9 occurs on several levels, indirectly, through glycemic control, and directly through the junctional solidification of the hyperglycemic retinal endothelial barrier, thus rehabilitating the quiescence and stability of the physiological endothelium.

Effective bone tissue engineering can restore bone and skeletal functions that are impaired by traumas and/or certain medical conditions. Bone is a complex tissue and functions through orchestrated interactions between cells, biomechanical forces, and biofactors. To identify ideal scaffold materials for effective mesenchymal stem cell (MSC)-based bone tissue regeneration, here we develop and characterize a composite nanoparticle hydrogel by combining carboxymethyl chitosan (CMCh) and amorphous calcium phosphate (ACP) (designated as CMCh-ACP hydrogel). We demonstrate that the CMCh-ACP hydrogel is readily prepared by incorporating glucono δ-lactone (GDL) into an aqueous dispersion or rehydrating the acidic freeze-dried nanoparticles in a pH-triggered controlled-assembly fashion. The CMCh-ACP hydrogel exhibits excellent biocompatibility and effectively supports MSC proliferation and cell adhesion. Moreover, while augmenting BMP9-induced osteogenic differentiation, the CMCh-ACP hydrogel itself is osteoinductive and induces the expression of osteoblastic regulators and bone markers in MSCs in vitro. The CMCh-ACP scaffold markedly enhances the efficiency and maturity of BMP9-induced bone formation in vivo, while suppressing bone resorption occurred in long-term ectopic osteogenesis. Thus, these results suggest that the pH-responsive self-assembled CMCh-ACP injectable and bioprintable hydrogel may be further exploited as a novel scaffold for osteoprogenitor-cell-based bone tissue regeneration.

Bone morphogenetic protein (BMP) 9 (BMP9) is one of most potent BMPs in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). Recently, evidence has shown that osteogenesis and angiogenesis are coupled, however, it is unclear whether BMP9 induces MSC differentiation into endothelial-like cells and further promotes blood vessel formation. In the present study, we explored the potential of BMP9-induced angiogenic differentiation of MSCs, and the relationship between BMP9-induced osteogenic and angiogenic differentiation of MSCs. Osteogenic activities and angiogenic differentiation markers were analyzed at mRNA and protein levels. In vivo osteogenic and angiogenic differentiation of MSCs were tested by the ectopic bone formation model. We identified that adenoviral vectors effectively transduced in immortalized mouse embryonic fibroblasts (iMEFs) and expressed BMP9 with high efficiency. We found that BMP9 induces early and late osteogenic differentiation, and it up-regulated osteogenic marker expression in MSCs. Meanwhile, BMP9 induces angiogenic differentiation of MSCs via the expression of vascular endothelial growth factor a (VEGFa) and CD31 at both mRNA and protein levels. CD31-positive cells were also increased with the stimulation of BMP9. The ectopic bone formation tests found that BMP9-induced trabecular bone formation was coupled with the expression of blood vessel formation markers and sinusoid capillary formation. These findings suggest that BMP9 exhibits dual and coupled roles in inducing osteogenic and angiogenic differentiation of MSCs.

International audience; Rationale: Recently, rare heterozygous mutations in GDF2 were identified in patients with pulmonary arterial hypertension (PAH). GDF2 encodes the circulating BMP (bone morphogenetic protein) type 9, which is a ligand for the BMP2 receptor.Objectives: Here we determined the functional impact of GDF2 mutations and characterized plasma BMP9 and BMP10 levels in patients with idiopathic PAH.Methods: Missense BMP9 mutant proteins were expressed in vitro and the impact on BMP9 protein processing and secretion, endothelial signaling, and functional activity was assessed. Plasma BMP9 and BMP10 levels and activity were assayed in patients with PAH with GDF2 variants and in control subjects. Levels were also measured in a larger cohort of control subjects (n = 120) and patients with idiopathic PAH (n = 260).Measurements and Main Results: We identified a novel rare variation at the GDF2 and BMP10 loci, including copy number variation. In vitro, BMP9 missense proteins demonstrated impaired cellular processing and secretion. Patients with PAH who carried these mutations exhibited reduced plasma levels of BMP9 and reduced BMP activity. Unexpectedly, plasma BMP10 levels were also markedly reduced in these individuals. Although overall BMP9 and BMP10 levels did not differ between patients with PAH and control subjects, BMP10 levels were lower in PAH females. A subset of patients with PAH had markedly reduced plasma levels of BMP9 and BMP10 in the absence of GDF2 mutations.Conclusions: Our findings demonstrate that GDF2 mutations result in BMP9 loss of function and are likely causal. These mutations lead to reduced circulating levels of both BMP9 and BMP10. These findings support therapeutic strategies to enhance BMP9 or BMP10 signaling in PAH.

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-&beta, 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.