Although interaction between BMP and Notch signaling has been demonstrated to be crucial for osteogenic differentiation of mesenchymal stem cells (MSCs), the precise molecular mechanism remains unknown. Here, we show that Notch intracellular domain (NICD) overexpression inhibits BMP9-induced C3H10T1/2 cell osteogenesis in vivo and in vitro. Our results show that activated Notch signaling results in down-regulation of Runx2 and early osteogenesis differentiation factors, without affecting p-Smad1/5/8 expression, and that blocking Notch signaling with DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester) significantly increases p-Smad1/5/8 expression. Interestingly, Notch signaling also regulates the cell cycle by increasing PCNA (proliferation cell nuclear antigen) and CyclinD1 expression. Furthermore, similar results were obtained by ectopic bone formation and histological analyses, indicating that Notch signaling activation significantly inhibits BMP9-induced MSC osteogenic, cartilage and adipogenic differentiation. Moreover, we are the first to show that Notch regulates by suppressing JunB synthesis and that the negative effect of Notch is partially reversed by treatment with the JunB activator TPA (12-O-tetradeca-noylphorbol-13-acetate). Our findings demonstrate that Notch signaling significantly enhances cell proliferation but inhibits MSC osteogenic differentiation induced by BMP9 via JunB protein suppression rather than by BMP/Smad signaling regulation.

Prostate cancer is the second most common cancer in men and lethality is normally associated with the consequences of metastasis rather than the primary tumor. Therefore, targeting the molecular pathways that underlie dissemination of primary tumor cells and the formation of metastases has a great clinical value. Bone morphogenetic proteins (BMPs) play a critical role in tumor progression and this study focuses on the role of BMP9- Activin receptor-Like Kinase 1 and 2 (ALK1 and ALK2) axis in prostate cancer. In order to study the effect of BMP9 in vitro and in vivo on cancer cells and tumor growth, we used a soluble chimeric protein consisting of the ALK1 extracellular domain (ECD) fused to human Fc (ALK1Fc) that prevents binding of BMP9 to its cell surface receptors and thereby blocks its ability to activate downstream signaling. ALK1Fc sequesters BMP9 and the closely related BMP10 while preserving the activation of ALK1 and ALK2 through other ligands. We show that ALK1Fc acts in vitro to decrease BMP9-mediated signaling and proliferation of prostate cancer cells with tumor initiating and metastatic potential. In line with these observations, we demonstrate that ALK1Fc also reduces tumor cell proliferation and tumor growth in vivo in an orthotopic transplantation model, as well as in the human patient derived xenograft BM18. Furthermore, we also provide evidence for crosstalk between BMP9 and NOTCH and find that ALK1Fc inhibits NOTCH signaling in human prostate cancer cells and blocks the induction of the NOTCH target Aldehyde dehydrogenase member ALDH1A1, which is a clinically relevant marker associated with poor survival and advanced-stage prostate cancer. Our study provides the first demonstration that ALK1Fc inhibits prostate cancer progression, identifying BMP9 as a putative therapeutic target and ALK1Fc as a potential therapy. Altogether, these findings support the validity of ongoing clinical development of drugs blocking ALK1 and ALK2 receptor activity.

Background: CD105 (endoglin, ENG) is a membranous protein that is overexpressed in tumor-associated endothelial cells and some actual tumor cells and is associated with poor prognosis. However, the association between CD105 and response to chemoresistance in choriocarcinoma cells has not been clearly defined. The present study aimed to investigate the effects of targeting CD105 in drug-resistant choriocarcinoma. Methods: CD105 expression was evaluated in drug-resistant and parental choriocarcinoma cells by qRT-PCR, western blotting, and immunofluorescence. CD105 overexpressing and knockdown cells were established by lentiviral transfection. CCK8, transwell, and flow cytometric assays were used to measure changes in drug-sensitivity, invasion, migration, and apoptosis. Drug-sensitivity and Smad1/5/8, Smad2, and Smad3 expression were also detected after BMP9 treatment. Immunohistochemical staining for CD105 and BMP9 was performed on choriocarcinoma tissues and the relationships between clinical and pathological characteristics were analysed. Results: Data demonstrated that CD105 overexpression could decrease drug sensitivity, promote invasion and migration, and inhibit apoptosis in choriocarcinoma cells, and this protein was confirmed to mediate drug resistance through the BMP9/Smad pathway. Further experiments showed that the expression of CD105 and BMP9 was consistent in choriocarcinoma tissues and significantly associated with disease recurrence. Conclusions: This study provides evidence suggesting that CD105 is critical for the development of drug-resistance in choriocarcinoma and might serve as a therapeutic target for reversing chemoresistance in choriocarcinoma patients.

The Rendu-Osler disease, also called Hereditary Hemorrhagic Telangiectasia (HHT) affects 1 in -5-8000 people. A french epidemiological study pointed out that it was particularly high in the Haut-Jura mountains in France. This pathology is characterized by frequent nosebleeds, mucocutaneous and visceral telangiectasia and hereditary autosomal-dominant trait. The mucocutaneous telangiectasia are hemorrhagic while the visceral telangiectasia, less frequent, lead to arteriovenous fistula in the lungs, the liver and the brain. HHT disease-causing genes (ENG, ACVRL1 and MADH4) encode proteins that modulate TGFβ superfamilly signaling in vascular endothelial cells. The recent discovery that BMP9 acts as the specific ligand of the receptor ALK1 and endoglin as its co-receptor shows that this signaling pathway is involved in the maturation phase of angiogenesis. Mice heterozygous for endoglin or ALK1 defects reproduce the HHT phenotype and further support the involvement of endothelial hyper proliferation in the pathogenesis of the disease. The medical management of patients remains mainly symptomatic, however the angiogenic trait of this disease should allow us to consider in the future new -therapeutic approaches using anti-angiogenic drugs.

The present study aimed to evaluate the effect of bone morphogenetic protein 9 (BMP9) and BMP13 on cardiac differentiation of C3H10T1/2 cells in vitro and to characterize the differentiated cells on their ultrastructure and transmembrane electrophysiological features. C3H10T1/2 cells were transfected with the vectors for BMP9 or BMP13 and differentiated into cardiomyocytes in vitro for up to 28 days. The expression of cardiac-specific genes Gata4 and Mef2c and proteins troponin T (cTnT) and connexin 43 (Cx43) was significantly increased in the cells transfected with BMP9 or BMP13 after differentiation over the controls as evaluated using quantitative RT-PCR, Western blotting, and immunofluorescence staining. Transmission electron microscopy and Masson trichrome staining showed that the specific myocardial leap dish and myofilament-like structure were present in the cells overexpressing BMP9 or BMP13, not in the control cells. Whole-cell patch-clamping study demonstrated the presence of delayed rectifier potassium current, inward rectifier potassium current, and T-type calcium current in the cells overexpressing BMP9 or BMP13. Sodium current was detected in a small number of cells overexpressing BMP9, not in the BMP13-transfected cells or the control cells. The expression of Mef2c gene and Cx43 and cTnT proteins was also significantly higher in the cells overexpressing BMP9 than those overexpressing BMP13. Our data indicate that BMP9 and BMP13 (BMP9 might be more effective) promoted the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells with cellular ultrastructures and ion channel currents similar to mature cardiomyocytes in vitro.

The role of bone morphogenic protein 9 (BMP9) signaling in angiogenesis has been controversial, with a number of studies showing that it acts either as a pro-angiogenic or, conversely, as an anti-angiogenic factor in a context-dependent manner. Notably, BMP9 was also reported to function in both pro- or anti-tumorigenic roles during tumor progression. It has therefore remained unclear, whether selective BMP9 inhibition is a useful target for antibody therapy of cancer. To shed light on these questions, we characterized BMP9 expression in plasma of patients with different cancer indications and found elevated levels of pro-domains and precursor BMP9 with a strong response in renal cell carcinoma (RCC). These studies prompted us to evaluate the potential of selective anti-BMP9 cancer therapy in RCC. We generated a novel monoclonal therapeutic antibody candidate, mAb BMP9-0093, that selectively targets all different BMP9 variants but does not bind to the closest homolog BMP10. In vitro, mAb BMP9-0093 treatment inhibited signaling, endothelin-1 (ET-1) production and spreading of endothelial cells and restored BMP9-induced decrease in pericyte migration and attachment. Furthermore, BMP9-mediated epithelial-mesenchymal transition of renal cell carcinoma cells was reversed by mAb BMP9-0093 treatment in vitro. In vivo, mAb BMP9-0093 showed significant anti-tumor activity that was associated with an increase in apoptosis as well as a decrease in tumor cell proliferation and ET-1 release. Furthermore, mAb BMP9-0093 induced mural cell coverage of endothelial cells, which was corroborated by a reduction in vascular permeability, demonstrated by a diminished penetration of omalizumab-Alexa 647 into tumor tissue. Our findings provide new evidence for a better understanding of BMP9 contribution in tumor progression and angiogenesis that may result in the development of effective targeted therapeutic interventions.

Background: BMP9, originating from the liver, and BMP10 are circulating BMPs that preserve vascular endothelial integrity. We assessed BMP9, BMP10 and soluble endoglin (sEng) levels and their relationships to liver disease severity and associated pulmonary vascular syndromes in a cohort of well-characterised liver disease patients.

Methods: Plasma samples from patients with liver disease (n = 83) and non-disease controls (n = 21) were assayed for BMP9, BMP10 and sEng. Levels were also assessed in a separate cohort of controls (n = 27) and PoPH patients (n = 8). Expression of mRNA and immunohistochemical staining was undertaken in liver biopsy specimens. Plasma BMP activity was assessed using an endothelial cell bioassay.

Findings: Plasma BMP9 and BMP10 levels were normal in patients with compensated cirrhosis or fibrosis without cirrhosis, but markedly reduced in patients with decompensated cirrhosis, including those with hepatopulmonary syndrome (HPS) or portopulmonary hypertension (PoPH). Liver biopsy specimens revealed reduced mRNA expression and immunostaining for these ligands. Patient plasma samples with reduced BMP9 and BMP10 levels exhibited low BMP activity that was restored with exogenous BMP9. Endoglin mRNA expression was increased in cirrhotic livers and elevated circulating sEng levels in PoPH and HPS patients suggested increased endothelial sEng shedding in these syndromes.

Interpretation: Plasma BMP9 and BMP10 levels are reduced in decompensated cirrhosis, leading to reduced circulating BMP activity on the vascular endothelium. The pulmonary complications of cirrhosis, PoPH and HPS, are associated with markedly reduced BMP9 and BMP10 and increased sEng levels, suggesting that supplementation with exogenous ligands might be a therapeutic approach for PoPH and HPS.

Keywords: BMP10; Bone morphogenetic protein 9; Cirrhosis; ELISA; Endoglin.

OBJECTIVE

To study the interactive effects of Type II glutamine transaminase (TG2) and bone morphogenetic protein-9 (BMP-9) in the induction of osteogenesis in mice mesenchymal stem cells (MSCs) C3H10T1/2 model.

METHODS

Batches of MSCs C3H10T1/2, divided into two groups, were treated with BMP-9 (control group) or BMP-9 and TG2 (experimental group) under oxygen deficient conditions. The secreted alkaline phosphatase (SEAP) chemiluminescence and the histochemical staining methods were used to detect the alkaline phosphatase (ALP) expression. The alizarin red S staining was used to detect the calcium salt precipitation and the caspase-3 protein expression was monitored using Western blot. Flow cytometry was employed to identify cell cycle, and trypan blue exclusion method to count the living cells and monitor cell proliferation.

RESULTS

The levels of ALP expression in the experimental group were much higher than that of the control group. The level of expression of advanced caspase-3 protein was significantly lower (p < 0.05) in the experimental group than in the control group. The highest fraction of cells in the experimental group was in the phase M while cells in the control group were in the interphase. Moreover, cell number in the experimental group was significantly increased (p < 0.05) relatively to the control group.

CONCLUSIONS

BMP-9 interacts with TG2 in osteogenesis of MSCs C3H10T1/2 cells. Further studies are needed to understand the exact mechanism of BMP9/PG2 interactions in osteogenesis.

OBJECTIVE

To construct a recombinant adenovirus co-expressing bone morphogenic protein (BMP) 9 and BMP6 and observe its effect on the osteogenesis in C3H10 cells.

METHODS

The full-length sequences of BMP9 and BMP6 were amplified from AdEasy vector by PCR and cloned into the shuttle plasmid pASG2 vector to construct the co-expression shuttle plasmid pASG2-BMP9, 6 followed by homologous recombination with plasmid pAdeasy-1 in BJ5183. After confirmation by restriction endonuclease digestion, the recombinant vector was transfected into HEK293 cells, and high-titer recombinant adenovirus (Ad-BMP9, 6) was collected after amplification. Ad-BMP9, 6 was then transduced into C3H10 cells in vitro, and the mRNA expression of BMP9 and BMP6 was detected by RT-PCR. The osteogenic capability of the transfected cells was observed by alkaline phosphatase staining and calcium-alizarin red staining.

RESULTS

AdBMP9,6 was constructed successfully and effectively infected in C3H10 cells, in which high expressions of BMP6 and BMP9 were detected. C3H10 cells infected with Ad-BMP9,6 showed stronger alkaline phosphatase and calcium-alizarin red staining than the cells transfected by either BMP9 or BMP6 alone.

CONCLUSIONS

The recombinant adenovirus co-expressing BMP9 and BMP6 we constructed shows a more potent effect than the adenoviruses expressing either BMP9 or BMP6 alone in inducing the osteogenic differentiation of C3H10 cells into osteoblasts.

Bone morphogenetic protein (BMP)-9 and BMP10 are circulating ligands that mediate endothelial cell (EC) protection via complexes of the type I receptor, ALK1, and the type II receptors, the activin type-IIA and bone morphogenetic type II receptors. We previously demonstrated that BMP9 induces the expression of interleukin-6, interleukin-8 and E-selectin in ECs and may influence their interactions with monocytes and neutrophils. We asked whether BMP9 and BMP10 regulate the expression of Chemokine (C-C motif) ligand 2 (CCL2), a key chemokine involved in monocyte-macrophage chemoattraction. Here, we show that BMP9 and BMP10 repress basal CCL2 expression and release from human pulmonary artery ECs and aortic ECs. This was dependent on ALK1 and co-dependent on ACTR-IIA and BMPR-II. Assessment of canonical Smad signalling indicated a reliance of this response on Smad4. Of note, Smad1/5 signalling contributed only at BMP9 concentrations similar to those in the circulation. In the context of inflammation, BMP9 did not alter the induction of CCL2 by TNF-α. As CCL2 promotes monocyte/macrophage chemotaxis and endothelial permeability, these data support the concept that BMP9 preserves basal endothelial integrity.

Keywords: BMP10; BMP9; CCL2; Endothelial; TNF.

Introduction: The differentiation of dental pulp cells (DPCs) plays an important role in the repair of dental pulp injury. Bone morphogenetic protein 9 (BMP9) is one of the most effective BMPs to induce the differentiation tem cells. However, the role of BMP9 in promoting the odontogenic differentiation of DPCs and dentinogenesis is worth to know.

Methods: Fluorescence in situ hybridization and immunohistochemistry staining were performed to detect the BMP9 expression in human dental pulp. BMP9 was overexpressed in human DPCs (hDPCs), and the mineralization of hDPCs was tested by alkaline phosphatase staining and alizarin red staining. The expression of odontogenic differentiation related genes was examined by quantitative real-time PCR and western blotting. The subcutaneous transplantation experiment was performed to test the odonto-induction ability of BMP9 in vivo. The rat direct pulp capping experiment was performed to test the function of BMP9 in promoting the dentin formation.

Results: BMP9 showed an increased expression in odontoblast layer both at mRNA and protein level. BMP9 enhanced the mineralization and induced the expression of odontogenic differentiation related genes in hDPCs. More mineralized nodules, and increased expression of dentin sialophosphoprotein (DSPP) and dentin matrix protein-1 (DMP1) were detected in the beta-tricalcium phosphate scaffold/cells composites of BMP9 group compared with the control group. Meanwhile, there was thicker reparative dentin formation in the BMP9 group in the rat pulp exposure experiment.

Conclusions: BMP9 participates in the process of DPCs differentiation and promotes DPCs mineralization and dentinogenesis. BMP9 might be a potential therapeutic target in the repair of dental pulp injury.

Keywords: BMP9; dental pulp cells; dentinogenesis; odontogenic differentiation.

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.

Keywords: BMP9; HOTAIR; long non-coding RNA; mesenchymal stem cell; osteogenic differentiation.

Colon cancer is one of the most common malignancies. Although there has been great development in treatment regimens over the last few decades, its prognosis remains poor. There is still a clinical need to find new drugs for colon cancer. Evodiamine (Evo) is a quinolone alkaloid extracted from the traditional herbal medicine plant Evodia rutaecarpa. In the present study, CCK‑8, flow cytometry, reverse transcription quantitative polymerase chain reaction, western blot analysis and a xenograft tumor model were used to evaluate the anti‑cancer activity of Evo in human colon cancer cells and determine the possible mechanism underlying this process. It was revealed that Evo exhibited prominent anti‑proliferation and apoptosis‑inducing effects in HCT116 cells. Bone morphogenetic protein 9 (BMP9) was notably upregulated by Evo in HCT116 cells. Exogenous BMP9 potentiated the anti‑cancer activity of Evo, and BMP9 silencing reduced this effect. In addition, HIF‑1α was also upregulated by Evo. The anticancer activity of Evo was enhanced by HIF‑1α, but was reduced by HIF‑1α silencing. BMP9 potentiated the effect of Evo on the upregulation of HIF‑1α, and enhanced the antitumor effect of Evo in colon cancer, which was clearly reduced by HIF‑1α silencing. In HCT116 cells, Evo increased the phosphorylation of p53, which was enhanced by BMP9 but reduced by BMP9 silencing. Furthermore, the effect of Evo on p53 was potentiated by HIF‑1α and reduced by HIF‑1α silencing. The present findings therefore strongly indicated that the anticancer activity of Evo may be partly mediated by BMP9 upregulation, which can activate p53 through upregulation of HIF‑1α, at least in human colon cancer.

Our previous study revealed that 3T3-L1 preadipocytes can differentiate to either osteoblasts or adipocytes in response to bone morphogenic protein 9 (BMP9). In the present study, we try to further investigate whether the Wnt/β-catenin signaling plays a crucial role in this process. It was found that BMP9 effectively activated the Wnt/β-catenin signaling, and induced the expression levels of certain canonical Wnt ligands and their receptors in preadipocytes. Exogenous expression of β-catenin, Wnt1, Wnt3a, and Wnt10b potentiated BMP9-induced alkaline phosphatase (ALP) activity, while β-catenin knockdown or Dickkopf 1 (Dkk1) diminished BMP9-induced ALP activity. Moreover, it was demonstrated that β-catenin overexpression promoted BMP9-induced mineralization, and increased the expression levels of late osteogenic markers osteopontin and osteocalcin. Furthermore, β-catenin inhibited BMP9-induced lipid accumulation and the adipogenic marker adipocyte fatty acid binding protein (aP2). The cell-implantation assay results identified that β-catenin not only augmented BMP9-induced ectopic bone formation, but also blocked adipogenesis in vivo. Mechanistically, it was found that β-catenin and BMP9 synergistically stimulated the osteogenic transcription factors runt-related transcription factor 2 (Runx2) and Osterix (OSX). However, BMP9-induced adipogenic transcription factors, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT enhancer-binding protein α (C/EBPα), were inhibited by β-catenin. Therefore, these findings suggested that the Wnt/β-catenin signaling, potentially via the modulation of osteogenic and adipogenic transcriptional factors, exerts an opposite effect on BMP9-induced osteogenic and adipogenic differentiation in preadipocytes.

Keywords: Adipogenesis; Bone morphogenic protein 9; Osteogenesis; Preadipocytes; Wnt/β-catenin signaling.

The osteogenic differentiation capacity of senescent bone marrow mesenchymal stem cells (MSCs) is reduced. p53 not only regulates cellular senescence but also functions as a negative regulator in bone formation. However, the role of p53 in MSCs senescence and differentiation has not been extensively explored. In the present study, we investigated the molecular mechanism of p53 in MSCs senescence and osteogenic differentiation. We found that p53 was upregulated during cellular senescence and osteogenic differentiation of MSCs respectively induced by H₂O₂ and BMP9. Similarly, the expression of p53-induced miR-145a was increased significantly. Furthermore, Overexpression of miR-145a in MSCs promoted cellular senescence and inhibited osteogenic differentiation. Then, we identified that p53-induced miR-145a inhibited osteogenic differentiation by targeting core binding factor beta (Cbfb), and the restoration of Cbfb expression rescued the inhibitory effects of miRNA-145a. In summary, our results indicate that p53/miR-145a axis exert its functions both in promoting senescence and inhibiting osteogenesis of MSCs, and the novel p53/miR-145a/Cbfb axis in osteogenic differentiation of MSCs may represent new targets in the treatment of osteoporosis.

Keywords: Cbfb; mesenchymal stem cells; microRNAs; osteogenic differentiation; p53; senescence.

This study evaluates diabetic macular-edema (DME) patients for the effect of intravitreal bevacizumab (IVB) injection on macular thickness and proangiogenic biomarkers in serum and vitreous.

Forty DME patients were analyzed for macular thickness (MT). Twelve proangiogenic biomarkers in serum and vitreous were analyzed before and after IVB.

Significant decrease in MT with vitreal vascular endothelial growth factor-A (VEGF-A) was observed as expected after IVB, while serum VEGF-A did not follow a decreasing trend in contrast to VEGF-C, which decreased both in serum and vitreous. Other vitreal factors like bone morphogenetic protein-9 (BMP9) and fibroblast growth factor (FGF) were also significantly decreased, while endothelial growth factor (EGF) increased following IVB. Before IVB, significant negative correlations were vitreous BMP9 with serum FGF, vitreous human growth factor (HGF) and interleukin-8 (IL-8) with serum endothelin, and vitreous and serum FGF and serum placental growth factor (PLGF) with EGF. After IVB, negative correlations in serum vs. vitreous were found for both HGF and PLGF with BMP9, and angiopoietin with FGF. Cube average thickness was negatively correlated with serum FGF and positively correlated with vitreous PLGF and endothelin.

Vascular endothelial growth factors are not the only factors that cause macular edema in diabetic patients. The effect of IVB on different proangiogenic biomarkers indicated a complex interplay of other factors in DME.

Notch signaling pathway is one of the most important pathways to regulate intercellular signal transduction and is crucial in the regulation of bone regeneration. Nephroblastoma overexpressed (NOV or CCN3) serves as a non-canonical secreted ligand of Notch signaling pathway and its role in the process of osteogenic differentiation of mesenchymal stem cells (MSCs) was undefined. Here we conducted a comprehensive study on this issue. In vivo and in vitro studies have shown that CCN3 significantly inhibited the early and late osteogenic differentiation of mouse embryonic fibroblasts (MEFs), the expression of osteogenesis-related factors, and the subcutaneous ectopic osteogenesis of MEFs in nude mice. In mechanism studies, we found that CCN3 significantly inhibited the expression of BMP9 and the activation of BMP/Smad and BMP/MAPK signaling pathways. There was also a mutual inhibition between CCN3 and DLL1, one of the classic membrane protein ligands of Notch signaling pathway. Additionally, we further found that Hey1, the target gene shared by BMP and Notch signaling pathways, partially reversed the inhibitory effect of CCN3 on osteoblastic differentiation of MEFs. In summary, our findings suggested that CCN3 significantly inhibited the osteogenic differentiation of MEFs. The inhibitory effect of CCN3 was mainly through the inhibition of BMP signaling and the mutual inhibition with DLL1, so as to inhibit the expression of Hey1, the target gene shared by BMP and Notch signaling pathways.

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 in GDF2 (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 the GDF2 variant. 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 in GDF2 with lymphatic dysplasia, hydrothorax and nonimmune hydrops fetalis.

Keywords: BMP9; GDF2; hereditary hemorrhagic telangiectasia; lymphatic dysplasia; nonimmune hydrops fetalis; pulmonary arterial hypertension.

Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in TGFβ/BMP9 pathway genes and characterized by vascular malformations (VM) including arteriovenous malformations (AVM) in lung, liver, and brain, which lead to severe complications including intracranial hemorrhage (ICH) from brain VM. The clinical heterogeneity of HHT suggests a role for genetic modifier effects. Common variants in loci that modify phenotype severity in Tgfb knockout mice were previously reported as associated with lung AVM in HHT. Common variants in candidate genes were reported as associated with sporadic brain AVM and/or ICH. We investigated whether these variants are associated with HHT organ VM or with ICH from brain VM in 752 Caucasian HHT patients enrolled by the Brian Vascular Malformation Consortium.

We genotyped 11 candidate variants: four variants reported as associated with lung AVM in HHT (PTPN14 rs2936018, USH2A rs700024, ADAM17 rs12474540, rs10495565), and seven variants reported as associated with sporadic BAVM or ICH (APOE ε2, ANGPTL4 rs11672433, EPHB4 rs314308, IL6 rs1800795, IL1B rs1143627, ITGB8 rs10486391, TNFA rs361525). Association of genotype with any VM, lung AVM, liver VM, brain VM or brain VM ICH was evaluated by multivariate logistic regression adjusted for age, gender, and family clustering.

None of the 11 variants was significantly associated with any phenotype. There was a trend toward association of USH2A rs700024 with ICH (OR = 2.77, 95% CI = 1.13-6.80, p = .026).

We did not replicate previously reported associations with HHT lung AVM and variants in Tgfb modifier loci. We also did not find significant associations between variants reported in sporadic brain AVM and VM or ICH in HHT.

We synthesized a generation of water-soluble, atomically precise gold nanoclusters (Au NCs) with anisotropic surface containing short dithiol pegylated chain (AuMHA/TDT). These AuMHA/TDT exhibit a high brightness (QY∼6%) in the shortwave infrared (SWIR) spectrum with a detection above 1250 nm. Furthermore, they show an extended half-life in blood (t_1/2ß = 19.54 ±0.05 h) and a very weak accumulation in organs. We also developed a non-invasive, whole-body vascular imaging system in the SWIR window with high-resolution, benefiting from a series of Monte Carlo image processing of the images. The imaging process enabled to improve contrast by one order of magnitude and enhance by 59% the spatial resolution. After systemic administration of these nanoprobes in mice, we can quantify vessel complexity in depth (>4mm) allowing to detect very subtle vascular disorders non-invasively in bone morphogenetic 9 (Bmp9) deficient mice. The combination of these anisotropic surface charged gold nanoclusters plus an improved SWIR imaging device allows then a precise mapping at high resolution and in depth of the organization of the vascular network in live animals.

Oral and maxillofacial surgery is often challenging due to defective bone healing owing to the microbial environment of the oral cavity, the additional involvement of teeth and esthetic concerns. Insufficient bone volume as a consequence of aging and some oral and maxillofacial surgical procedures, such as tumor resection of the jaw, may further impact facial esthetics and cause the failure of certain procedures, such as oral and maxillofacial implantation. Bone morphogenetic protein (BMP) 9 (BMP9) is one of the most effective BMPs to induce the osteogenic differentiation of different stem cells. A large cross-talk network that includes the BMP9, Wnt/β, Hedgehog, EGF, TGF-β and Notch signaling pathways finely regulates osteogenesis induced by BMP9. Epigenetic control during BMP9-induced osteogenesis is mainly dependent on histone deacetylases (HDACs), microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), which adds another layer of complexity. As a result, all these factors work together to orchestrate the molecular and cellular events underlying BMP9-related tissue engineering. In this review, we summarize our current understanding of the SMAD-dependent and SMAD-independent BMP9 pathways, with a particular focus on cross-talk and cross-regulation between BMP9 and other major signaling pathways in BMP9-induced osteogenesis. Furthermore, recently discovered epigenetic regulation of BMP9 pathways and the molecular and cellular basis of the application of BMP9 in tissue engineering in current oral and maxillofacial surgery and other orthopedic-related clinical settings are also discussed.

Keywords: BMP9; crosstalk; non-coding rna; osteogenesis; therapeutics.

Synovial mesenchymal stem cells (SMSCs) with high proliferation and multi differentiation ability, and low immunogenicity have attracted research attention for their potential application in tissue engineering. Once their ability of osteogenesis is strengthened, it will be of practical value to apply the SMSCs in the field of bone regeneration. The current study aimed to investigate the osteogenic characteristics of SMSCs induced by bone morphogenetic protein 9 (BMP9) both in vitro and in vivo and to elucidate the mechanism underlying these characteristics. Specifically, different BMPs were assessed to determine the protein that would be the most favorable for stimulating osteogenic differentiation of SMSCs following their separation. The BMP9-enhanced osteogenesis of SMSCs was fully investigated in vitro and in vivo, and the c-Jun N-terminal kinase (JNK)/Smad2/3 signaling pathway stimulated by BMP9 was further explored. Our data suggested that BMP9 could significantly promote gene and protein expression of runt-related transcription factor 2, alkaline phosphatase, osteopontin, and osteocalcin, and SP600125, a JNK-specific inhibitor, could effectively decrease this tendency. Similar results were also confirmed in rats with cranial defects. In conclusion, our study indicated that BMP9 promotes bone formation both in vitro and in vivo possibly by activating the JNK/Smad2/3 signaling pathway.