Our study attempted to compare the efficacies of bone morphogenetic protein (BMP) 2, 6, and 9 in inducing osteogenic differentiation of preodontoblasts (PDBs). We immortalized PDBs by introducing a reversible SV40 T antigen-based immortalization system. Cell proliferation capability was examined by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The effects of BMP2, 6, and 9 on the osteogenic differentiation of immortalized preodontoblasts (iPDBs) were measured by alkaline phosphatase (ALP) activity assays and alizarin red S staining. The expression of osteogenic markers was evaluated by semiquantitative real-time polymerase chain reaction analysis. To assess ectopic bone formation, rat-derived iPDBs were transfected in culture with adenoviral vectors designated Ad-BMP2, 6, and 9 and subcutaneously or intramuscularly injected into mice. Several BMPs retained endogenous expression in PDBs and regulated the mRNA expression of mineralized tissue-associated proteins. ALP activity and mineralized nodule formation were significantly increased in the Ad-BMP9-transfected group relative to the control group. In addition, the most significant hard tissue formation was in this group. The results indicated that BMP signaling was involved in the osteogenic differentiation of iPDBs. BMP9 could be an efficacious accelerant of the osteogenic differentiation of iPDBs.

Hematoma and skeletal muscles play a crucial role in bone fracture healing. The muscle resident mesenchymal stromal cells (mrSCs) can promote bone formation by differentiating into osteoblasts upon treatment by bone morphogenetic proteins (BMP), such as BMP9. However, the influence of hematoma fracture extracts (Hema) on human mrSC (hmrSC) response to BMP9 is still unknown. We therefore determined the influence of Hema, human healthy serum (HH), and fetal bovine serum (FBS, control) on BMP9-induced osteoblast commitment of hmrSC by measuring alkaline phosphatase activity. Multiplex assays of 90 cytokines were performed to characterize HH and Hema composition and allow their classification by a multivariate statistical approach depending on their expression levels. We confirmed that BMP9 had a greater effect on osteoblastic differentiation of hmrSCs than BMP2 in presence of FBS. The hmrSCs response to BMP9 was enhanced by both Hema and HH, even though several cytokines were upregulated (IL-6, IL-8, MCP-1, VEGF-A and osteopontin), downregulated (BMP9, PDGF) or similar (TNF-alpha) in Hema compared with HH. Thus, hematoma may potentiate BMP9-induced osteogenic differentiation of hmrSCs during bone fracture healing. The multivariate statistical analyses will help to identify the cytokines involved in such phenomenon leading to normal or pathological bone healing.

OBJECTIVE

To screen specific small interfering RNA (siRNA) targeting mouse BMP9 gene and identify its function in BNLCL.2 fetal liver cells and C3H10 cells.

METHODS

Four pairs of double-stranded DNA fragments for silencing mouse BMP9 were annealed in vitro and cloned into pSOS-BMP9 vector with BMP9 gene to construct pSOS-simBMP9 plasmid. The 4 pSOS-simBMP9 plasmids were separately transfected in HEK293 cells via Lipofectamine, and the gene silencing efficiency was assessed by GFP detection. BNLCL.2 fetal liver cells were infected with the constructed adenovirus simBMP9s, and their BMP9 expression was detected with RT-PCR and Western blotting. C3H10 cells were co-infected with Ad-simBMP9 and Ad-BMP9, and the inhibitory effect on BMP9-induced osteoblasts was evaluated by alkaline phosphatase (ALP) activity.

RESULTS

GFP expression in the two simBMP9 groups was significantly decreased in HEK293 cells, and the endogenous expression of BMP9 was reduced by 50%-70% by adenovirus-mediated simBMP9 in the fetal liver cells. ALP activity in C3H10 cells was significantly higher in BMP9 group than in the control group (P<0.01), while the activity of the two Ad-simBMP9-infected groups was significantly lower than that in Ad-BMP9-infected group (P<0.01).

CONCLUSIONS

Two specific siRNA targeting mouse BMP9 gene have been obtained, which can effectively inhibit both endogenous and exogenous expressions of BMP9 to facilitate the study of the mechanisms of BMP9 in liver cell differentiation.

Periodontitis is a chronic infectious disease that alters the cellular microenvironment and promotes bone absorption. Bone morphogenetic protein 9 (BMP9) serves an important role in proliferation and differentiation, and tumor necrosis factor‑alpha (TNF‑α) is an important contributor to bone resorption. The present study aimed to investigate the effect of osteogenic differentiation in the presence of BMP9 and TNF‑α in rat follicle stem cells (rDFCs). rDFCs were transfected with adenoviruses expressing BMP9 (AdBMP9) and the expression levels of important proteins [BMP9, β‑catenin, glycogen synthase kinase 3β (GSK3β), phosphorylated‑GSK3β, calcium/calmodulin dependent protein kinase II and nemo like kinase] were determined using western blotting. The effect of osteogenesis was analyzed using reverse transcription‑quantitative PCR, in addition to alkaline phosphatase, Alizarin Red S, and hematoxylin and eosin staining methods. The results of the present study revealed that TNF‑α activated the canonical Wnt signaling pathway and suppressed osteogenesis. High concentrations of Dickkopf 1 (DKK1) reduced the osteogenic differentiation of AdBMP9‑transduced rDFCs, whereas low concentrations of DKK1 promoted BMP9‑induced bone formation, which was discovered to partially act via the canonical and non‑canonical Wnt signaling pathways. In conclusion, the findings of the present study suggested that the enhanced promoting effect of BMP9 alongside the treatment with low concentrations of DKK1 may be useful for treating periodontitis bone absorption.

Thyroid hormone (TH), triiodothyronine (T3), and thyroxine (T4), which are released from the thyroid, control many cellular processes in various cell types. It is worth noting that TH plays a complex role in skeletal metabolic balance, and few studies have investigated whether TH exerts any effects on osteogenesis in bone mesenchymal stem cells (MSCs). We explored the effects of T3 on bone morphogenetic protein 9 (BMP9)-induced osteogenesis, which process is considered the most important in the osteogenic differentiation of C3H10T1/2 cells. In vitro osteogenesis was analyzed by alkaline phosphatase (ALP) activity and staining, bone mineralisation, and osteocalcin and osteopontin expression. Fetal limb explant cultures and ectopic MSC implantation further confirmed the role of T3. Finally, we examined the effect of AMPK/p38 signaling on the osteoblastic differentiation. T3 synergizes with BMP9 to enhance osteogenic marker expression induced by BMP9. Furthermore, T3 promotes BMP9-induced bone formation by fetal limb explant cultures and ectopic MSC implantation. Co-treatment with BMP9 and T3 can promote AMPK and p38 phosphorylation, and pretreatment with the AMPK inhibitor compound C and siRNA can abolish phosphorylation of p38 and BMP9+T3-induced ALP activity. Our results suggest that BMP9 and T3 promote osteogenic differentiation at least partially via the activation of the AMPK/p38 signaling pathway.

Keywords: AMPK; Bone morphogenetic protein 9; mesenchymal stem cells; osteogenesis; triiodothyronine.

Within the past years, BMP9 has been characterized as one of the most osteogenic bone-inducers among the BMP family, however up until recently, BMP9 has only been available through adenovirus transfection experiments (gene therapy) not approved for clinical use. The aim of this study was to investigate recombinant rhBMP9 versus rhBMP2 at 2 concentrations (10 and 100 ng/mL) in combination with 2 bone grafts: (1) a natural bone mineral (NBM) without collagen versus (2) a novel NBM integrated with atelo-collagen type I (NBM-Col). Scanning electron microscopy revealed that while NBM demonstrated a mineralized roughened surface morphology, NBM-Col particles contained many more visible collagen fibrils throughout the scaffold surface significantly increasing rhBMP adsorption from 8 h to 10 days (as quantified by ELISA). Thereafter, ST2 preosteoblasts were used to investigate cell attachment, proliferation, and differentiation. While little change was observed for cell attachment/proliferation, osteoblast differentiation demonstrated a significant increase in alkaline phosphatase (ALP) activity when scaffolds were loaded with rhBMP9 when compared to rhBMP2. Furthermore, a 2-3 fold increase in alizarin red staining, and in mRNA levels of osteoblast differentiation markers Runx2, Collagen1α2, ALP, and osteocalcin was observed when rhBMP9 was combined with NBM-Col when compared to NBM without collagen at equivalent doses and when compared to rhBMP2. The results from this study demonstrate that (1) the use of rhBMP9 significantly and markedly induced osteoblast differentiation when compared to rhBMP2 and (2) the incorporation of atelo-collagen type I into NBM bone grafts markedly improved these findings by serving as a scaffold capable of improving growth factor adsorption and osteoblast behavior. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 408-418, 2017.

Background: DNA polymorphisms in PTGIS and TFAP2B have been identified as risk factors for patent ductus arteriosus (PDA) in a population composed of preterm infants with European genetic ancestry but not in more genetically diverse populations.

Goal: To determine if the effects of TFAP2B and PTGIS polymorphisms on ductus arteriosus (DA) gene expression differ based on genetic ancestry.

Methods: DA from 273 human second trimester fetuses were genotyped for TFAP2B and PTGIS polymorphisms and for polymorphisms distributing along genetic ancestry lines. RT-PCR was used to measure the RNA expression of 49 candidate genes involved with DA closure.

Results: Seventeen percent of the DA analyzed were of European ancestry. In multivariable regression analyses we found consistent associations between four PDA-related TFAP2B polymorphisms (rs2817399(A), rs987237(G), rs760900(C), and rs2817416(C)) and expression of the following genes: EPAS1, CACNB2, ECE1, KCNA2, ATP2A3, EDNRA, EDNRB, BMP9, and BMP10, and between the PTGIS haplotype rs493694(G)/rs693649(A) and PTGIS and NOS3. These changes only occurred in DA with European ancestry. No consistent positive or negative associations were found among DA samples unless an interaction between the polymorphisms and genetic ancestry was taken into account.

Conclusion: PTGIS and TFAP2B polymorphisms were associated with consistent changes in DA gene expression when present in fetuses with European ancestry.

Impact: DNA polymorphisms in PTGIS and TFAP2B have been identified as risk factors for patent ductus arteriosus (PDA) in a population composed primarily of preterm infants with European genetic ancestry but not in more genetically diverse populations. The same PTGIS and TFAP2B polymorphisms are associated with changes in ductus gene expression when present in ductus from fetuses with European genetic ancestry. No consistent associations with gene expression can be found unless an interaction between the polymorphisms and genetic ancestry is taken into account.

BMP9 is a cytokine involved in the maturation phase of the angiogenic process that signals through its serine/threonine receptor ALK1 and its coreceptor endoglin. In this paper, we explain how BMP9 directs the regulation of endothelial cell proliferation blockage while in turn stimulating protein synthesis. To achieve this, BMP9 promotes SGK1 synthesis and activation through mTORC2 in order to stimulate the mTORC1/S6K/S6 axis. Moreover, BMP9 blocks proliferation also through SGK1 by reducing the activity of the MEK/ERK signalling pathway. Inhibition of SGK1 activity is sufficient to prevent BMP9-mediated inhibition of ERK, leading to an increase in endothelial cell proliferation. Overall, our findings reveal that SGK1 is a key player during angiogenesis, mediating the pro-quiescent and maturation effects of BMP9/ALK1.

Keywords: ALK1; BMP9; ERK; HHT; SGK1; angiogenesis; endothelial cells; protein synthesis.

Rationale: The bone morphogenetic proteins (BMPs) are essential morphogens in angiogenesis and vascular development. Disruption of BMP signaling can trigger cardiovascular disease such as arteriovenous malformations. Objective: A computational model predicted that BMP4 and BMP9 and their inhibitors matrix Gla protein (MGP) and Crossveinless-2 (CV2) would form a regulatory system consisting of negative feedback loops with time delays, and that BMP9 would trigger oscillatory expression of the two inhibitors. The goal was to investigate this regulatory system in endothelial differentiation and vascular growth. Methods and Results: Oscillations in the expression of MGP and CV2 were detected in endothelial cells (ECs) in vitro, using qPCR and immunoblotting. These organized temporally downstream BMP-related activities including expression of stalk cell markers and cell proliferation, consistent with an integral role of BMP9 in vessel maturation. In vivo, the inhibitors were located in distinct zones in relation to the front of the expanding retinal network, as determined by immunofluorescence. Time-dependent changes of the CV2 location in the retina, and the existence of an endothelial population with signs of oscillatory MGP expression in developing vasculature supported the in vitro findings. Loss of MGP or its BMP4-binding capacity disrupted the retinal vasculature, resulting in poorly formed networks, especially in the venous drainage areas, and arteriovenous malformations as determined by increased cell coverage and functional testing. Conclusions: Our results suggest a previously unknown mechanism of temporal orchestration of BMP4 and BMP9 activities that utilizes the tandem actions of the extracellular antagonists MGP and CV2. Disruption of this mechanism may contribute to vascular malformations and disease.

Keywords: BMP antagonist; bone morphogenetic protein (BMP); crossveinless-2; matrix Gla protein (MGP).

This study investigated the effects of transforming growth factor-β1 (TGF-β1) and cyclooxygenase-2 (COX-2) on bone morphogenetic protein 9 (BMP9) in mesenchymal stem cells (MSCs). We found that BMP9 increased mRNA levels of TGF-β1 and COX-2 in C3H10T1/2 cells. BMP9-induced osteogenic markers were enhanced by TGF-β1 and reduced by TGF-βRI-specific inhibitor LY364947. BMP9 increased level of p-Smad2/3, which were either enhanced or reduced by COX-2 and its inhibitor NS398. BMP9-induced osteogenic markers were decreased by NS398 and it was partially reversed by TGF-β1. COX-2 increased BMP9-induced osteogenic marker levels, which almost abolished by LY364947. BMP9-induced bone formation was enhanced by TGF-β1 but reduced by silencing TGF-β1 or COX-2. BMP9's osteogenic ability was inhibited by silencing COX-2 but partially reversed by TGF-β1. TGF-β1 and COX-2 enhanced activation of p38 signaling, which was induced by BMP9 and reduced by LY364947. The ability of TGF-β1 to increase the BMP9-induced osteogenic markers was reduced by p38-specific inhibitor, while BMP9-induced TGF-β1 expression was reduced by NS398, but enhanced by COX-2. Furthermore, CREB interacted with Smad1/5/8 to regulate TGF-β1 expression in MSCs. These findings suggest that COX-2 overexpression leads to increase BMP9's osteogenic ability, resulting from TGF-β1 upregulation which then activates p38 signaling in MSCs.

Keywords: BMP9; COX-2; TGF-β1; osteogenic differentiation; p38 MAPK.

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 = 26) 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.

Angiogenesis occurs early in tumor development, sustains primary tumor growth and provides a route for metastatic escape. The TGF-β family receptors modulate angiogenesis via endothelial-cell specific pathways. Here we investigate the interaction of two such receptors, ALK1 and endoglin, in pancreatic neuroendocrine tumors (PanNET). Independently, ALK1 and endoglin deficiencies exhibited genetically divergent phenotypes, while both highly correlate to an endothelial metagene in human and mouse PanNETs. A concurrent deficiency of both receptors synergistically decreased tumor burden to a greater extent than either individual knockdown. Furthermore, the knockout of Gdf2 (BMP9), the primary ligand for ALK1 and endoglin, exhibited a mixed phenotype from each of ALK1 and endoglin deficiencies; overall primary tumor burden decreased, but hepatic metastases increased. Tumors lacking BMP9 display a hyperbranching vasculature, and an increase in vascular mesenchymal-marker expression, which may be implicit in the increase in metastases. Taken together, our work cautions against singular blockade of BMP9 and instead demonstrates the utility of dual blockade of ALK1 and endoglin as a strategy for anti-angiogenic therapy in PanNET.

Transgenic animals incorporating human antibody genes are extremely attractive for drug development because they obviate subsequent antibody humanization procedures required for therapeutic translation. Transgenic platforms have previously been established using mice, but also more recently rats, chickens, and cows and are now in abundant use for drug development. However, rabbit-based antibody generation, with a strong track record for specificity and affinity, is able to include gene conversion mediated sequence diversification, thereby enhancing binder maturation and improving the variance/selection of output antibodies in a different way than in rodents. Since it additionally frequently permits good binder generation against antigens that are only weakly immunogenic in other organisms, it is a highly interesting species for therapeutic antibody generation. We report here on the generation, utilization, and analysis of the first transgenic rabbit strain for human antibody production. Through the knockout of endogenous IgM genes and the introduction of human immunoglobulin sequences, this rabbit strain has been engineered to generate a highly diverse human IgG antibody repertoire. We further incorporated human CD79a/b and Bcl2 (B-cell lymphoma 2) genes, which enhance B-cell receptor expression and B-cell survival. Following immunization against the angiogenic factor BMP9 (Bone Morphogenetic Proteins 9), we were able to isolate a set of exquisitely affine and specific neutralizing antibodies from these rabbits. Sequence analysis of these binders revealed that both somatic hypermutation and gene conversion are fully operational in this strain, without compromising the very high degree of humanness. This powerful new transgenic strategy will allow further expansion of the use of endogenous immune mechanisms in drug development.

Keywords: IgG; Rabbit; affinity; diversity; drug discovery; functionality; gene conversion; human immunoglobulins; humanness; immune repertoire; somatic hypermutation; specificity; transgenic.

Hepatopulmonary syndrome (HPS) is a serious pulmonary vascular disease derived from chronic liver disease, and its key pathogenesis is angiogenesis. Krüppel-like factor 6 (KLF6) mediates physiological repair and remodeling during vascular injury. However, the role of KLF6 in pulmonary microvascular endothelial cells (PMVECs) during angiogenesis of HPS and its underlying mechanism in HPS have not been investigated. Common bile duct ligation (CBDL) in rats can replicate pulmonary vascular abnormalities of human HPS. Here, we found that advanced pulmonary angiogenesis and pulmonary injury score coincided with the increase of KLF6 level in PMVECs of CBDL rat; KLF6 in PMVECs was also induced while cultured with CBDL rat serum in vitro Inhibition of KLF6 dramatically suppressed PMVEC-mediated proliferation, migration and tube formation in vivo; this may be related to the downregulation of activin receptor-like kinase-1 (ALK1) and endoglin (ENG), which are transacted by KLF6. Bone morphogenetic protein 9 (BMP9) enhanced the expression of KLF6 in PMVECs and was involved in the angiogenesis of HPS. These results suggest that KLF6 triggers PMVEC-mediated angiogenesis of HPS and is aggravated by BMP9, and the inhibition of the BMP9/KLF6 axis may be an effective strategy for HPS treatment.

Bone morphogenetic proteins (BMPs) belong to TGF-β superfamily and are a group of important cytokines involved in cell differentiation, proliferation and embryonic development. Multiple BMPs play important roles in several functions of vertebrates. Signaling pathway of BMPs is known to be mediated by Smad proteins, which include 8 members while Smad1, Smad5 and Smad8 are involved in BMPs signal transduction while Smad2 and Smad3 are mediated TGF-β signal transduction. Although several BMPs such as BMP4 and BMP9 have been documented in the liver, BMP13 has not been examined in the liver. BMP13 also known as growth differentiation factor (GDF)-6 or cartilage-derived morphogenetic protein (CDMP)-2 is one of the BMPs family members. Function of BMP13 has been investigated in bone and tendon repair. It can stimulate tendon-like cell proliferation. However, our recent findings revealed that there was expression of BMP13 in the liver and its expression was modulated during metabolic disorders. The current article is to understand biological function of BMP13 especially in the liver.

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.

This work focuses on the development of a system to control the formation of bone to complement developments that have enabled potent regeneration of bony tissue. Scaffolds were fabricated with chemically modified RNA encoding for bone morphogenetic protein-9 (cmBMP9) and capped with salicylic acid (SA)-containing polymer (SAPAE). The goal was to determine if SAPAE could inhibit the formation of bone in a pilot animal study since cmBMP9 has been demonstrated to be highly effective in regenerating bone in a rat calvarial defect model. The results indicated that cmBMP9 increased bone formation (30% increase in area covered compared to control) and that SAPAE trended toward reducing the bone formation. These results suggest SAPAE could be useful as a chemical agent in reducing unwanted bone formation in implants loaded with cmBMP9.

Bone morphogenetic protein 9 (BMP9) has recently been described as a crucial regulator in modulating fibroblast-type cell activation. Activin receptor-like kinase 1 (ALK1) is a high affinity receptor for BMP9 that exerts its role via Smad1/5. However, the functional roles of BMP9 in activating lung fibroblasts and the underlying signaling pathway are not completely understood. The present study aimed to explore the effect of exogenous BMP9 on human lung fibroblast HFL-1 cell proliferation and differentiation, as well as the potential role of the ALK1/Smad1/5 signaling pathway. In the present study, fibroblast proliferation was assessed using Cell Counting Kit-8 and colony formation assays, and the mRNA and protein expression of target genes was examined using reverse transcription-quantitative PCR and western blot assays, respectively. Compared with the control group, BMP9 treatment increased HFL-1 cell proliferation, mRNA and protein expression of differentiated markers, including α-smooth muscle actin, type I collagen and type III collagen, and the expression of ALK1 and phosphorylated Smad1/5 expression. Furthermore, the effects of BMP9 were partially rescued by dorsomorphin-1, an inhibitor of ALK1. The results indicated that BMP9 may serve as a key inducer of lung fibroblast activation and ALK1/Smad1/5 signaling might be associated with BMP9-mediated effects in HFL-1 cells. Therefore, the present study highlighted that the potential role of the BMP9/ALK1/Smad1/5 signaling pathway in the development of pulmonary fibrosis requires further investigation.

Keywords: activin receptor-like kinase 1; bone morphogenic protein 9; differentiation; in vitro; lung fibroblast; proliferation; signaling pathway.

Pulmonary Arterial Hypertension (PAH) is a severe complication of Connective Tissue Disease (CTD), with remarkable morbidity and mortality. However, the molecular and genetic basis of CTD-PAH remains incompletely understood. This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH, using a PAH-specific panel of 35 genes. During recruitment, 79 patients were studied, including 59 Systemic Sclerosis patients (SSc) and 69 females. Disease-associated variants were observed in nine patients: 4 pathogenic/likely pathogenic variants in 4 different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9) and 5 Variants of Unknown Significance (VUS) in 4 genes (ABCC8, NOTCH3, TOPBP1 and CTCFL). One patient with mixed CTD had a frameshift pathogenic variant in TBX4. Two patients with SSc-PAH carried variants in ABCC8. A patient diagnosed with Systemic Lupus Erythematous (SLE) presented a pathogenic nonsense variant in GDF2/BMP9. Another patient with SSc-PAH presented a pathogenic variant in KCNA5. Four patients with SSc-PAH carried a VUS in NOTCH1, CTCFL, CTCFL and TOPBP1, respectively. These findings suggest that genetic factors may contribute to Pulmonary Vascular Disease (PVD) in CTD patients.

Keywords: BMP signalling; PAH; genetics; immunity.

[Erratum to: BMB Reports 2012; 45(9): 509-514, PMID: 23010171] The BMB Reports would like to correct in the Figure 2 of BMB Rep. 2012; 45(9): 509-514 titled "Biphasic effects of TGFβ1 on BMP9-induced osteogenic differentiation of mesenchymal stem cells." The original version of this article unfortunately contained image assembling error in the Figure 2. The image for "GFP-Day13" group was inadvertently duplicated from that for "BT20-Day 5" group, and an incorrect image was used for "GFP-Day 17" group. This article has been updated to correct this error in Figure 2.

Mesenchymal stem cells (MSCs) are capable of differentiating into bone, cartilage and adipose tissues. We identified BMP9 as the most potent osteoinductive BMP although detailed mechanism underlying BMP9-regulated osteogenesis of MSCs is indeterminate. Emerging evidence indicates that autophagy plays a critical role in regulating bone homeostasis. We investigated the possible role of autophagy in osteogenic differentiation induced by BMP9. We showed that BMP9 upregulated the expression of multiple autophagy-related genes in MSCs. Autophagy inhibitor chloroquine (CQ) inhibited the osteogenic activity induced by BMP9 in MSCs. While overexpression of ATG5 or ATG7 did not enhance osteogenic activity induced by BMP9, silencing Atg5 expression in MSCs effectively diminished BMP9 osteogenic signaling activity and blocked the expression of the osteogenic regulator Runx2 and the late marker osteopontin induced by BMP9. Stem cell implantation study revealed that silencing Atg5 in MSCs profoundly inhibited ectopic bone regeneration and bone matrix mineralization induced by BMP9. Collectively, our results strongly suggest a functional autophagy pathway may play an essential role in regulating osteogenic differentiation induced by BMP9 in MSCs. Thus, restoration of dysregulated autophagic activity in MSCs may be exploited to treat fracture healing, bone defects or osteoporosis.

Keywords: BMP9; autophagy; bone formation; lineage-specific differentiation; mesenchymal stem cells; osteogenic signaling.

Macrophages are highly heterogeneous immune cells that fulfill tissue-specific functions. Tissue-derived signals play a critical role in determining macrophage heterogeneity. However, these signals remain largely unknown. BMP receptor ALK1 is well-known for its role in blood vessel formation; however, its role within the immune system has never been revealed. Here, we found that BMP9/BMP10-ALK1 signaling controlled the identity and self-renewal of Kupffer cells (KCs) through a Smad4-dependent pathway. In contrast, ALK1 was dispensable for the maintenance of macrophages located in the lung, kidney, spleen and brain. Following ALK1 deletion, KCs were lost over time and were replaced by monocyte-derived macrophages. These hepatic macrophages displayed significantly reduced expression of the complement receptor VSIG4 and alterations in immune zonation and morphology, which is important for the tissue-specialized function of KCs. Furthermore, we found that this signaling pathway was important for KC-mediated Listeria monocytogenes capture, as the loss of ALK1 and Smad4 led to a failure of bacterial capture and overwhelming disseminated infections. Thus, ALK1 signaling instructs a tissue-specific phenotype that allows KCs to protect the host from systemic bacterial dissemination.

Keywords: Immunology; Macrophages.

Background: Rheumatoid arthritis (RA) increases the risk for abnormalities of the cardiac structure and function, which may lead to heart failure (HF). Studying the association between circulating biomarkers and echocardiographic parameters is important to screen patients with RA with a higher risk of cardiac dysfunction. Aim: To study the association between circulating biomarkers and echocardiographic parameters in patients with RA. Methods: Echocardiography was performed in 355 patients with RA from RA Porto cohort and the associations between echocardiographic characteristics and 94 circulating biomarkers were assessed. These associations were also assessed in the Metabolic Road to Diastolic Heart Failure (MEDIA-DHF) [392 patients with HF with preserved ejection fraction (HFpEF)] and the Suivi Temporaire Annuel Non-Invasif de la Santé des Lorrains Assurés Sociaux (STANISLAS) (1,672 healthy population) cohorts. Results: In the RA Porto cohort, mean age was 58 ± 13 years, 23% were males and mean RA duration was 12 ± 10 years. After adjustment and multiple testing correction, left ventricular mass index (LVMi), left atrial volume index (LAVi), and E/e' were independently associated with biomarkers reflecting inflammation [i.e., bone morphogenetic protein 9 (BMP9), pentraxin-related protein 3 (PTX3), tumor necrosis factor receptor superfamily member 11a (TNFRSF11A)], extracellular matrix remodeling [i.e., placental growth factor (PGF)], congestion [i.e., N-terminal pro-brain natriuretic peptide (NT-proBNP), adrenomedullin (ADM)], and myocardial injury (e.g., troponin). Greater LVMi [hazard ratio (HR) (95% CI) per 1 g/m² = 1.03 (1.02-1.04), p < 0.001], LAVi [HR (95% CI) per 1 ml/m² = 1.03 (1.01-1.06), p < 0.001], and E/e' [HR (95% CI) per 1 = 1.08 (1.04-1.13), p < 0.001] were associated with higher rates of cardiovascular events. These associations were externally replicated in patients with HFpEF and asymptomatic individuals. Conclusion: Circulating biomarkers reflecting inflammation, extracellular matrix remodeling, congestion, and myocardial injury were associated with underlying alterations of cardiac structure and function. Biomarkers might be used for the screening of cardiac alterations in patients with RA.

Keywords: circulating biomarkers; echocardiogram; heart failure with preserved ejection fraction; prognosis; rheumatoid arthritis.