Bone morphogenetic proteins (BMPs) are secreted signaling proteins - they transduce their signals by assembling complexes comprised of one of three known type II receptors and one of four known type I receptors. BMP-9 binds and signals through the type I receptor Alk1, but not other Alks, while BMP-2, -4, and -7 bind and signal through Alk3, and the close homologue Alk6, but not Alk1. The present results, which include the determination of the Alk1 structure using NMR and identification of residues important for binding using SPR, show that the β-strand framework of Alk1 is highly similar to Alk3, yet there are significant differences in loops shown previously to be important for binding. The most pronounced difference is in the N-terminal portion of the β4-β5 loop, which is structurally ordered and includes a similarly placed but shorter helix in Alk1 compared to Alk3. The altered conformation of the β4-β5 loop, and to lesser extent β1-β2 loop, cause clashes when Alk1 is positioned onto BMP-9 in the manner that Alk3 is positioned onto BMP-2. This necessitates an alternative manner of binding, which is supported by a model of the BMP-9/Alk1 complex constructed using the program RosettaDock. The model shows that Alk1 is positioned similar to Alk3 but is rotated by 40 deg. The alternate positioning allows Alk1 to bind BMP-9 through a large hydrophobic interface, consistent with mutational analysis that identified several residues in the central portion of the β4-β5 loop that contribute significantly to binding and are nonconservatively substituted relative to the corresponding residues in Alk3.

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9-KO mice, we generated a conditional Bmp10-KO mouse strain. The plasma from Bmp10-KO mice, similarly to that of Bmp9-KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma.

Crosstalk between subchondral bone and articular cartilage is considered a central feature of osteoarthritis (OA) initiation and progression, but its underlying molecular mechanism remains elusive. Meanwhile, specific administration of drugs in subchondral bone is also a great challenge during investigation of the process. We here explore the role of stromal cell-derived factor 1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis in the crosstalk between subchondral bone and articular cartilage in OA pathogenesis, using osmotic infusion pumps implanted in tibial subchondral bone directly to ensure quantitative, continuous and steady drug delivery over the entire experiment. We found that increased SDF-1 in subchondral bone firstly induced subchondral bone deterioration by erroneous Mesenchymal Stem Cells (MSCs) recruitment and excessive bone resorption in anterior cruciate ligament transection (ACLT) mice. Deterioration of subchondral bone then led to the traverse of SDF-1 from subchondral bone to overlying cartilage. Finally, SDF-1 from underlying subchondral bone combined with CXCR4 in chondrocytes to induce articular cartilage degradation by promoting the shift of transforming growth factor-β receptor type I (TβRI) in chondrocytes from activin receptor-like kinase 5 (ALK5) to activin receptor-like kinase 1 (ALK1). More importantly, specific inhibition of SDF-1/CXCR4 axis in ACLT rats attenuated OA by stabilizing subchondral bone microarchitecture, reducing SDF-1 in cartilage and abrogating the shift of TβRI in chondrocytes. Our data demonstrate that the SDF-1/CXCR4 axis may coordinate the crosstalk between subchondral bone and articular cartilage in OA pathogenesis. Therefore, specific inhibition of SDF-1/CXCR4 axis in subchondral bone or intervention in SDF-1 traverse may be therapeutic targets for OA.

The authors report on a case of spinal inflammatory myofibroblastic tumor (IMT) in a 22-year-oldwoman. Neuroradiological features of this intradural extramedullary mass were suggestive of a meningioma or neurinoma. The lesion was easily resected following a T-9 laminectomy. Light microscopy showed a proliferation of spindle cells with prominent nucleoli on a fibrous or edematous background with infiltration of numerous lymphocytes and plasma cells. Some spindle cells immunostained positively for ALK1. This led to the diagnosis of IMT. The patient's postoperative course was complicated by a multifocal local recurrence requiring a second surgery, which was followed by radio- and chemotherapy. The occurrence of IMT in the spinal cord has rarely been reported. In this case, ALK overexpression was associated with early multifocal recurrence. This has been recently reported in this tumor typein other locations.

In the alkane-assimilating yeast Yarrowia lipolytica, the expression of ALK1, a gene encoding cytochrome P450 that catalyzes the first step of n-alkane oxidation, is induced by n-alkanes. We previously demonstrated that two basic helix-loop-helix proteins, Yas1p and Yas2p, activate the transcription of ALK1 in an alkane-dependent manner by forming a heterocomplex and binding to alkane-responsive element 1 (ARE1), a cis-acting element in the ALK1 promoter. Here we identified an Opi1 family transcription factor, Yas3p, involved in the alkane-dependent transcription regulation of ALK genes. Deletion of YAS3 caused a significant increase in ALK1 mRNA in cells grown on glucose, glycerol, and n-alkanes. The YAS3 deletion also resulted in a marked elevation of reporter gene expression driven by an ARE1-containing promoter on glycerol and n-decane. Bacterially expressed Yas3p bound specifically to Yas2p, but not to Yas1p, in vitro. In addition, although green fluorescent protein-tagged Yas3p was localized in the nucleus in glucose-containing medium, it changed its localization to an endoplasmic reticulum-like compartment upon transfer to medium containing n-decane. These findings suggest that Yas3p functions as a master regulator of transcriptional response, which changes its localization between the nucleus and endoplasmic reticulum membrane in response to different carbon sources. Furthermore, quantitative real time PCR analysis of 12 ALK genes in YAS1, YAS2, and YAS3 deletion mutants suggested that Yas3p is involved in the transcriptional repression of a variety of ALK genes, including ALK1. In contrast, YAS3 deletion did not affect the mRNA level of an INO1 ortholog in Y. lipolytica, indicating functional diversity of Opi1 family transcription factors.

The vascular endothelial growth factor (VEGF) pathway is critical for tumor angiogenesis. However, VEGF pathway inhibition has been limited by intrinsic and acquired resistance. Simultaneously targeting multiple steps involved in tumor angiogenesis is a potential means of overcoming this resistance. Activin like kinase 1 (ALK1) and endoglin (ENG) have effects on angiogenesis that are distinct from those of VEGF. Whereas VEGF is important for vessel initiation, ALK1 and endoglin are involved in vessel network formation. Thus, ALK1 and endoglin pathway inhibitors are attractive partners for VEGF-based combination antiangiogenic therapy. Genetic evidence supports a role for this receptor family and its ligands, bone morphogenetic proteins (BMP) 9 and 10, in vascular development. Patients with genetic alterations in ALK1 or endoglin develop hereditary hemorrhagic telangiectasia, a disorder characterized by abnormal vessel development. There are several inhibitors of the ALK1 pathway advancing in clinical development for treatment of various tumor types, including renal cell and ovarian carcinomas. Targeting of alternate angiogenic pathways, particularly in combination with VEGF pathway blockade, holds the promise of optimally inhibiting angiogenically driven tumor progression. Clin Cancer Res; 20(11); 2838-45. ©2014 AACR.

Primary non-Hodgkin's lymphoma of the thyroid gland are infrequent tumors. They almost exclusively derive from B cells of mucosa-associated lymphatic tissue and only a very small minority of them is represented by T cell lymphomas. CD30 molecule, other than in Hodgkin's and Redd-Sternberg' cells, is strictly associated with anaplastic large cell lymphoma and ALK lymphomas, the latter being identified by the monoclonal antibody ALK1. We report a case of CD30-positive non-anaplastic (ALK1-negative) peripheral T cell lymphoma of the thyroid gland and speculate on aspects concerning diagnosis and the morphologic and immunohistochemical findings.

Lung cancer is often diagnosed by molecular markers for prediction and treatment. To date, the golden standard for detection of anaplastic lymphoma kinase (ALK) rearrangements is fluorescence in situ hybridization (FISH). We performed a pooled-data analysis on the diagnostic operating characteristics of immunohistochemistry (IHC) assay on non-small cell lung cancer (NSCLC). We searched Embase, Pubmed, and Springer databases. The results of IHC were evaluated using a modified H-score. We used a 2-level bivariate meta-analysis following a random effect model to summarize sensitivity and specificity and fit hierarchical summary receiver-operating characteristic curves. We also performed sensitivity analysis using different antibodies to investigate potential heterogeneity. Twelve studies consisting of a total of 3754 NSCLC specimens were analyzed. When we defined 1+/2+/3+, 2+/3+, and 3+ as ALK positive, we found the sensitivities to be 99% (95% confidence interval [CI], 97%-100%), 86% (95% CI, 73%-93%), and 56% (95% CI, 36%-74%) and the specificities to be 98% (95% CI, 95%-99%), 99% (95% CI, 99%-100%), and 100% (95% CI, 100%-100%), respectively. We demonstrated that when defining 3+ as positive and 0 as negative the sensitivity was 99% and specificity was 100%. In our sensitivity analysis, we found the sensitivity of D5F3 and 5A4 antibodies to be much higher than that of ALK1. We concluded that IHC scores 0 and 3+ were nearly 100% concordant with FISH-negative and FISH-positive status, respectively. However, IHC scores 1+ and 2+ might require further confirmatory testing by FISH assay. IHC assay using D5F3 and 5A4 antibodies reliably detected NSCLC with ALK rearrangement and may be useful as a screening method to identify these tumors.

Angiogenesis is a crucial event during cancer progression that regulates tumor growth and metastasis. Activin receptor-like kinase 1 (ALK1), predominantly expressed in endothelial cells, plays a key role in the organization of neo-angiogenic vessels. Therapeutic targeting of ALK1 has been proposed as a promising strategy for cancer treatment, and microRNAs (miRNAs) are increasingly being explored as modulators of angiogenesis. However, the regulation of ALK1 by miRNAs is unclear. In this study, we identified that ALK1 is directly targeted by miR-199b-5p, which was able to inhibit angiogenesis in vitro and in vivo. Moreover, it was found that miR-199b-5p was repressed in breast cancer cells and its expression was decreased during the VEGF-induced angiogenesis process of human umbilical vein endothelial cells (HUVECs). Overexpression of miR-199b-5p inhibited the formation of capillary-like tubular structures and migration of HUVECs. Furthermore, overexpression of miR-199b-5p inhibited the mRNA and protein expression of ALK1 in HUVECs by directly binding to its 3'UTR. Additionally, overexpression of miR-199b-5p attenuated the induction of ALK1/Smad/Id1 pathway by BMP9 in HUVECs. Finally, overexpression of miR-199b-5p reduced tumor growth and angiogenesis in in vivo. Taken together, these findings demonstrate the anti-angiogenic role of miR-199b-5p, which directly targets ALK1, suggesting that miR-199b-5p might be a potential anti-angiogenic target for cancer therapy.

Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation.

The formation of new blood vessels from existing vasculature, angiogenesis, is facilitated through a host of different signaling processes. Members of the TGF-β superfamily, TGF-β1, TGF-β3, and BMP9, are key propagators of both inhibition and initiation of angiogenesis. HHT, characterized by AVM and capillary bed defects, is caused by germline mutations in the ENG and ACVRL1/ALK1 genes, respectively. Clinical symptoms include epistaxis and GI hemorrhage. The membranous receptors endoglin and ALK1 activate proliferation and migration of endothelial cells during the angiogenic process via the downstream intracellular SMAD signaling pathway. Endothelial cell senescence or activation is dependent on the type of cytokine, ligand concentration, cell-cell interaction, and a multitude of other signaling molecules. Endoglin and ALK1 receptor levels in tumor vasculature correlate inversely with prognosis in humans, whereas in mice, endoglin deficiency decelerates tumor progression. Therefore, endoglin and ALK1 have been identified as potential therapeutic targets for antibody treatment in various cancers. Early phase clinical trials in humans are currently underway to evaluate the efficacy and safety of biological therapy targeting endoglin/ALK1-mediated cells signaling.

BACKGROUND

Fibroinflammatory biliary stricture (FIBS) is a rare benign tumor-like process of the extrahepatic bile duct that masquerades as cholangiocarcinoma.

METHODS

In order to distinguish this unusual entity from cancer, we performed a systematic analysis of 11 patients with FIBS. All patients presented with jaundice; six patients had coexisting autoimmune disease. Preoperative evaluation included computed tomography scan and endoscopic retrograde cholangiopancreatography with benign brush cytology. Surgical treatment included nine bile duct resections with five concurrent liver resections and two incisional biopsies. Light microscopy demonstrated fibrous lesions admixed with chronic inflammation.

CONCLUSIONS

Immunohistochemistry demonstrated smooth muscle actin expression in all lesions except one; five tumors exhibited IgG4 positive plasma cells. The lesions were negative for cytokeratin, ALK1, CD21, S100, Ki67, and p53. Six patients received postoperative immunosuppression. At 41 month median follow-up (range 15-58 months), there was no evidence of recurrent FIBS in ten patients, while one was lost to follow-up.

CONCLUSIONS

FIBS is a rare myofibroblastic lesion with an immunohistochemical profile distinct from other epithelial and stromal neoplasms of the extrahepatic bile duct. A subset of these cases appear to represent IgG4-related sclerosing cholangitis. Because preoperative cytology is not diagnostic of FIBS, surgical resection remains the mainstay of diagnosis and treatment, while immunosuppression may reduce the risk of recurrence.

Anti-VEGF therapy has been proven to be effective in the treatment of pathological angiogenesis. However, therapy resistance often occurs, leading to development of alternative approaches. The present study examines if AMPK negatively regulates ALK1-mediated signaling events and associated angiogenesis. Thus, we treated human umbilical vein endothelial cells with metformin as well as other pharmacological AMPK activators and showed that activation of AMPK inhibited Smad1/5 phosphorylation and tube formation induced by BMP9. This event was mimicked by expression of the active mutant of AMPKα1 and prevented by the dominant negative AMPKα1. Metformin inhibition of BMP9 signaling is possibly mediated by upregulation of Smurf1, leading to degradation of ALK1. Furthermore, metformin suppressed BMP9-induced angiogenesis in mouse matrigel plug. In addition, laser photocoagulation was employed to evaluate the effect of metformin. The data revealed that metformin significantly reduced choroidal neovascularization to a level comparable to LDN212854, an ALK1 specific inhibitor. In conjunction, metformin diminished expression of ALK1 in endothelium of the lesion area. Collectively, our study for the first time demonstrates that AMPK inhibits ALK1 and associated angiogenesis/neovascularization. This may offer us a new avenue for the treatment of related diseases using clinically used pharmacological AMPK activators like metformin in combination with other strategies to enhance the treatment efficacy or in the case of anti-VEGF resistance.

Liver fibrosis is a common phenomenon that is associated with several pathologies and is characterized by excessive extracellular matrix deposition that leads to progressive liver dysfunction. Bone morphogenetic protein 9 (BMP9) is the most recently discovered member of the BMP family. BMP9 bound with high affinity to activin receptor-like kinase 1 (ALK1) and endoglin in non-parenchymal liver cells. In addition, BMP9 activated Smad1/Smad5/Smad8 and induced the expression of the target genes inhibitor of differentiation 1 (Id1), hepcidin, Snail and the co-receptor endoglin in liver cells. Although the role of BMP9 in liver fibrosis is currently poorly understood, the presence of BMP9-activated proteins and its target genes have been reported to be associated with liver fibrosis development. This review summarizes the indirect connection between BMP9 and liver fibrosis, with a focus on the BMP9 signaling pathway members ALK1, endoglin, Id1, hepcidin and Snail. The observations on the role of BMP9 in regulating liver fibrosis may help in understanding the pathology mechanisms of liver disease. Furthermore, BMP9 could be served as a potent biomarker and the target of potential therapeutic drugs to treat hepatocytes fibrosis.

OBJECTIVE

Notch and activin receptor-like kinase 1 (ALK1) have been implicated in arterial specification, angiogenic tip/stalk cell differentiation, and development of arteriovenous malformations (AVMs), and ALK1 can cooperate with Notch to up-regulate expression of Notch target genes in cultured endothelial cells. These findings suggest that Notch and ALK1 might collaboratively program arterial identity and prevent AVMs. We therefore sought to investigate the interaction between Notch and Alk1 signalling in the developing vertebrate vasculature.

RESULTS

We modulated Notch and Alk1 activities in zebrafish embryos and examined effects on Notch target gene expression and vascular morphology. Although Alk1 is not necessary for expression of Notch target genes in arterial endothelium, loss of Notch signalling unmasks a role for Alk1 in supporting hey2 and ephrinb2a expression in the dorsal aorta. In contrast, Notch and Alk1 play opposing roles in hey2 expression in cranial arteries and dll4 expression in all arterial endothelium, with Notch inducing and Alk1 repressing these genes. Although alk1 loss increases expression of dll4, AVMs in alk1 mutants could neither be phenocopied by Notch activation nor rescued by Dll4/Notch inhibition.

CONCLUSIONS

Control of Notch targets in arterial endothelium is context-dependent, with gene-specific and region-specific requirements for Notch and Alk1. Alk1 is not required for arterial identity, and perturbations in Notch signalling cannot account for alk1 mutant-associated AVMs. These data suggest that AVMs associated with ALK1 mutation are not caused by defective arterial specification or altered Notch signalling.

IL-34 is a proinflammatory cytokine implicated in rheumatoid arthritis (RA). The current study aimed to assess the IL-34 expression in response to two members of the transforming growth factor (TGF)-β family, TGF-β1 and bone morphogenetic protein (BMP)-2, in synovial fibroblasts from RA patients. IL-34, TGF-β1, and BMP-2 productions were measured in patient synovial fluids by enzyme-linked immunosorbent assay. IL-34 mRNA levels were quantified by real-time quantitative PCR in human synovial fibroblasts and murine mesenchymal stem cells. Pharmacologic inhibitions were used to determine the involvement of activin receptor-like kinase 1 (ALK1) and ALK5 downstream TGF-β1 and BMP-2. IL-34, TGF-β1, and BMP-2 were expressed in synovial fluids from RA patients. We found a significant correlation between IL-34 and TGF-β1 expressions. Levels of both IL-34 and TGF-β1 were thus correlated with the total leukocyte counts in the synovial fluids. TGF-β1 and BMP-2 decreased IL-34 expression in the synovial fibroblasts or in murine mesenchymal stem cells in a dose- and time-dependent manner through ALK5 and ALK1 pathways, respectively. In addition, TGF-β1 and BMP-2 antagonized tumor necrosis factor α-induced IL-34 gene expression. This work identifies TGF-β1 and BMP-2 as potent inhibitors of IL-34 expression in RA synovial fibroblasts. These cytokines, as upstream inhibitors of IL-34, may thus contribute to antagonize inflammation and bone erosions in RA.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder. Circulating angiogenic cells (CACs) play an important role in vascular repair and regeneration. This study was designed to examine the function of CACs derived from patients with HHT. Peripheral blood mononuclear cells (PBMNCs) isolated from patients with HHT and age- and gender-matched healthy volunteers were assessed for expression of CD34, CD133 and VEGF receptor 2 by flow cytometry. PBMNCs were cultured to procure early outgrowth CACs. Development of endothelial cell (EC) phenotype in CACs was analyzed by fluorescence microscopy. CAC apoptosis was assayed with Annexin V staining, and CAC migration assessed by a modified Boyden chamber assay. mRNA expression of endoglin (ENG), activin receptor-like kinase-1 (ACVLR1 or ALK1) and endothelial nitric oxide synthase (eNOS) in CACs was measured by real time RT-PCR. The percentage of CD34+ cells in PBMNCs from HHT patients was significantly higher than in PBMNCs of healthy controls. CACs derived from patients with HHT not only showed a significant reduction in EC-selective surface markers following 7-day culture, but also a significant increase in the rate of basal apoptosis and blunted migration in response to vascular endothelial growth factor and stromal cell-derived factor-1. CACs from HHT patients expressed significantly lower levels of ENG, ALK1 and eNOS mRNAs. In conclusion, CACs from patients with HHT exhibited various functional impairments, suggesting a reduced regenerative capacity of CACs to repair the vascular lesions seen in HHT patients.

OBJECTIVE

In osteoarthritic cartilage, expression of the receptor ALK1 correlates with markers of deleterious chondrocyte hypertrophy. Recently, bone morphogenetic protein 9 (BMP9) was identified as a high affinity ligand for ALK1. Therefore, we studied if BMP9 signaling results in expression of hypertrophy markers in chondrocytes. Furthermore, because transforming growth factorß1 (TGFβ1) is a well known anti-hypertrophic factor, the interaction between BMP9 and TGFβ1 signaling was also studied.

METHODS

Primary chondrocytes were isolated from bovine cartilage and stimulated with BMP9 and/or TGFβ1 to measure intracellular signaling via pSmads with the use of Western blot. Expression of Smad-responsive genes or hypertrophy-marker genes was measured using qPCR. To confirm observations on TGFβ/Smad3 responsive genes, a Smad3-dependent CAGA12-luc transcriptional reporter assay was performed in the chondrocyte G6 cell line.

RESULTS

In primary chondrocytes, BMP9 potently induced phosphorylation of Smad1/5 and Smad2 to a lesser extent. BMP9-induced Smad1/5 phosphorylation was rapidly (2 h) reflected in gene expression, whereas Smad2 phosphorylation was not. Remarkably, BMP9 and TGFβ1 dose-dependently synergized on Smad2 phosphorylation, and showed an additive effect on expression of Smad3-dependent genes like bSerpine1 after 24 h. The activation of the TGFβ/Smad3 signaling cascade was confirmed using the CAGA12-luc transcriptional reporter. BMP9 selectively induced bAlpl and bColX expression, which are considered early markers of cellular hypertrophy, but this was potently antagonized by addition of a low dose of TGFβ1.

CONCLUSIONS

This study shows that in vitro in chondrocytes, BMP9 potently induces pSmad1/5 and a chondrocyte hypertrophy-like state, which is potently blocked by TGFβ1. This observation underlines the importance of TGFβ1 in maintenance of chondrocyte phenotype.

BACKGROUND

Systemic anaplastic large cell lymphoma (ALCL) is predominantly a nodal disease, but extranodal involvement can occur during the disease course or as the primary presentation. We report two rare cases of ALCL presenting with a pleural effusion, mimicking primary effusion lymphoma (PEL).

METHODS

Two patients, a 47-year-old woman and an 81-year-old man, presented with a pleural effusion for investigation. The pleural fluid contained abundant, large, lymphoid cells with marked nuclear atypia. These neoplastic cells strongly expressed CD30 and EMA and showed a T-cell phenotype (CD3+CD45RO+ for case 1 and CD4+ for case 2). Case 1, in addition, showed ALK1 expression. The tumor cells in both cases were negative for human herpes virus type 8 (HHV8) and Epstein-Barr virus (EBV). ALCL shows overlapping cytologic features with PEL, but the T-cell phenotype, ALK1 expression in case 1, lack of association with HHV8 and EBV, HIV seronegativity and subsequent discovery of nodal disease in case 2 were all in favor of ALCL over PEL.

CONCLUSIONS

In rare cases a pleural effusion is the presenting feature of ALCL, and distinction from PEL depends on correlation with clinical findings, detailed immunophenotyping and study of the status of HHV8 and EBV.

Endoglin is part of the TGF-β receptor complex and has a crucial role in fibrogenesis and angiogenesis. It is also an important protein for tumor growth, survival, and cancer cell metastasis. In a previous study, we have shown that hepatitis C virus (HCV) infection induces epithelial-mesenchymal transition (EMT) state and cancer stem-like cell (CSC) properties in human hepatocytes. Our array data suggested that endoglin (CD105) mRNA is significantly upregulated in HCV-associated CSCs. In this study, we have observed increased endoglin expression on the cell surface of an HCV core-expressing hepatocellular carcinoma (HepG2) cell line or immortalized human hepatocytes (IHH) and activation of its downstream signaling molecules. The status of phospho-SMAD1/5 and the expression of inhibitor of DNA binding protein 1 (ID1) were upregulated in HCV-infected cells or viral core gene-transfected cells. Additionally, we observed upregulation of endoglin/ID1 mRNA expression in chronic HCV patient liver biopsy samples. CSC generation by HCV core protein was dependent on the endoglin signaling pathway using activin receptor-like kinase 1 (ALK1) Fc blocking peptide and endoglin small interfering RNA (siRNA). Further, follow-up from in vitro analysis suggested that the antiapoptosis Bcl2 protein, proliferation-related cyclin D1 protein, and CSC-associated Hes1, Notch1, Nanog, and Sox2 proteins are enhanced during infection or ectopic expression of HCV core protein.IMPORTANCE Endoglin plays a crucial role in fibrogenesis and angiogenesis and is an important protein for tumor growth, survival, and cancer cell metastasis. Endoglin enhances ALK1-SMAD1/5 signaling in different cell types, leading to increased proliferation and migration responses. We have observed endoglin expression on the HCV core-expressing cell surface of human hepatocyte origin and activation of phospho-SMAD1/5 and ID1 downstream signaling molecules. ID1 protein plays a role in CSC properties, and we found that this pathway is important for antiapoptotic and cell proliferation signaling. Blocking of endoglin-ALK1-SMAD1/5 might be a good candidate for therapy for liver cancer stem cells together with liver cirrhosis.

Objective To assess the phenotype of human articular chondrocytes cultured in normoxia (21% O2) or continuous hypoxia (2% O2). Design Chondrocytes were extracted from patients undergoing total knee replacement ( n = 5) and cultured in ~21% (normoxic chondrocytes, NC) and 2% (hypoxic chondrocytes, HC) oxygen in both monolayer and 3-dimensional (3D) pellet culture and compared with freshly isolated chondrocytes (FC). Cells were assessed by flow cytometry for markers indicative of mesenchymal stromal cells (MSCs), chondrogenic-potency and dedifferentiation. Chondrogenic potency and immunomodulatory gene expression was assessed in NC and HC by reverse transcription quantitative polymerase chain reaction. Immunohistochemistry was used to assess collagen II production following 3D pellet culture. Results NC were positive (>97%, n = 5) for MSC markers, CD73, CD90, and CD105, while HC demonstrated <90% positivity ( n = 4) and FC ( n = 5) less again (CD73 and CD90 <20%; CD105 <40%). The markers CD166 and CD151, indicative of chondrogenic de-differentiation, were significantly higher on NC compared with HC and lowest on FC. NC also produced the highest levels of CD106 and showed the greatest levels of IDO expression, following interferon-γ stimulation, indicating immunomodulatory potential. NC produced the highest levels of CD49c (>60%) compared with HC and FC in which production was <2%. Hypoxic conditions upregulated expression of SOX9, frizzled-related protein ( FRZB), fibroblast growth factor receptor 3 ( FGFR3), and collagen type II ( COL2A1) and downregulated activin receptor-like kinase 1 ( ALK1) in 3 out of 4 patients compared with normoxic conditions for monolayer cells. Conclusions Hypoxic conditions encourage retention of a chondrogenic phenotype with some immunomodulatory potential, whereas normoxia promotes dedifferentiation of chondrocytes toward an MSC phenotype with loss of chondrogenic potency but enhanced immunomodulatory capacity.