Anaplastic large cell lymphoma (ALCL) is associated with the t(2;5)(p23;q35) translocation involving the anaplastic lymphoma kinase gene (ALK) and the nucleophosmin gene (NPM), which result in expression of a novel fusion protein, NPM-ALK (p80). Clinicopathologic studies have shown that ALK expression in ALCL is associated with improved 5-year survival rates when compared with ALCL lacking ALK expression. This study used paraffin-embedded tissue to compare interphase fluorescence in situ hybridization (FISH) and reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of t(2;5) with immunohistochemical analysis for the detection of ALK protein expression in 27 patients with CD30-positive ALCLs. ALK protein expression was detected with ALK1 antibody in 14 of the 27 patients. The neoplastic cells in 13 of these 14 lymphomas reacted with the p80NPM/ALK antibody. FISH, using a two-color ALK DNA probe, correlated 100% with the immunohistochemical results: a translocation involving the ALK gene was detected in all 14 lymphomas that reacted with anti-ALK1. RT-PCR, performed on 21 lymphomas, detected NPM-ALK mRNA in five of the lymphomas, all of which reacted with anti-ALK1 and showed ALK gene rearrangement by FISH. Lymphomas showing ALK1 reactivity occurred in a younger patient population (median age, 19.5 years) and were associated with improved 5-year survival rates (84%), as compared with lymphomas lacking ALK1 reactivity (median age, 68.0 years; 5-year survival rate, 35%; p = 0.008). We conclude that immunohistochemical studies, using antibody ALK1. and FISH for ALK gene rearrangement are equally effective for identifying patients with ALCL who have a favorable clinical outcome.

Mutations affecting transforming growth factor-beta (TGF-β) superfamily receptors, activin receptor-like kinase (ALK)-1, and endoglin (ENG) occur in patients with pulmonary arterial hypertension (PAH). To determine whether the TGF-β/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-β, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs) and pulmonary endothelial cells (PECs) from 14 patients with idiopathic PAH (iPAH) and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-β on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (Eng+/-) and wild-type (Eng+/+) mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-β levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-β led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, Eng+/- mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-β/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.

OBJECTIVE

In anaplastic large cell lymphoma (ALCL), the site of origin has been described as an important prognostic factor. Recently, a fusion protein containing anaplastic lymphoma kinase (ALK) was described in systemic nodal ALCL, and shown to be associated with a good prognosis. The aims of this study were to investigate whether the presence of ALK protein differs between ALCL of different sites of origin; to determine whether ALK expression occurs before dissemination to other sites; and, finally, to investigate whether the site of origin remains a prognostic parameter in ALK negative ALCL.

METHODS

ALK expression, as detected by immunohistochemistry using the monoclonal antibodies ALK1 and ALKc, was studied in 85 ALCLs from different sites of origin. In 22 patients, ALK expression was studied in multiple biopsies from different sites (including 13 skin, 16 lymph node, and nine other). Overall survival time was analysed using the Kaplan Meier method.

RESULTS

ALK expression was found in 20 of 51 systemic ALCLs with (primary) nodal involvement. No ALK expression was found in 15 primary cutaneous, 14 gastrointestinal, and five nasal ALCLs. Multiple and subsequent biopsies of patients showed ALK expression to be identical to that seen in the primary diagnostic biopsy. Kaplan Meier survival curves showed that in ALK negative ALCLs originating from different sites, primary cutaneous cases are associated with an excellent overall survival, whereas the other cases show a comparable five years survival of less than 40%.

CONCLUSIONS

If present, ALK expression favours systemic ALCL with (primary) nodal involvement, and can be used in differentiating between extranodal involvement of systemic (nodal) ALCL and primary extranodal ALCL. ALK is expressed consistently in multiple biopsies of a given patient, indicating that the chromosomal abnormality leading to aberrant ALK expression occurs before dissemination to other sites. Finally, in ALK negative non-cutaneous ALCLs, different sites of origin show comparable poor survival.

The ability of inflammatory cytokine TGF-beta1 to alter endothelial cell phenotype suggests its role in the regulation of vascular endothelial cell permeability. We demonstrate that depletion of TGF-beta1 receptor ALK5 and regulatory protein Smad4, but not ALK1 receptor attenuates TGF-beta1-induced permeability increase and significantly inhibits TGF-beta1-induced EC contraction manifested by actin stress fiber formation and increased MLC and MYPT1 phosphorylation. Consistent with these results, EC treatment with SB 431542, an inhibitor of ALK5 but not ALK1 receptor, significantly attenuates TGF-beta1-induced permeability. Thus, our data demonstrate for the first time direct link between TGF-beta1-mediated activation of ALK5/Smad and EC barrier dysfunction.

Lysyl hydroxylase 2b (LH2b) is known to increase pyridinoline cross-links, making collagen less susceptible to enzymatic degradation. Previously, we observed a relationship between LH2b and osteoarthritis-related fibrosis in murine knee joint. For this study, we investigate if transforming growth factor-beta (TGF-ß) and connective tissue growth factor (CTGF) regulate procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) (gene encoding LH2b) and LH2b expression differently in osteoarthritic human synovial fibroblasts (hSF). Furthermore, we investigate via which TGF-ß route (Smad2/3P or Smad1/5/8P) LH2b is regulated, to explore options to inhibit LH2b during fibrosis. To answer these questions, fibroblasts were isolated from knee joints of osteoarthritis patients. The hSF were stimulated with TGF-ß with or without a kinase inhibitor of ALK4/5/7 (SB-505124) or ALK1/2/3/6 (dorsomorphin). TGF-ß, CTGF, constitutively active (ca)ALK1 and caALK5 were adenovirally overexpressed in hSF. The gene expression levels of PLOD1/2/3, CTGF and COL1A1 were analyzed with Q-PCR. LH2 protein levels were determined with western blot. As expected, TGF-ß induced PLOD2/LH2 expression in hSF, whereas CTGF did not. PLOD1 and PLOD3 were not affected by either TGF-ß or CTGF. SB-505124 prevented the induction of TGF-ß-induced PLOD2, CTGF and COL1A1. Surprisingly, dorsomorphin completely blocked the induction of CTGF and COL1A1, whereas TGF-ß-induced PLOD2 was only slightly reduced. Overexpression of caALK5 in osteoarthritic hSF significantly induced PLOD2/LH2 expression, whereas caALK1 had no effect. We showed, in osteoarthritic hSF, that TGF-ß induced PLOD2/LH2 via ALK5 Smad2/3P. This elevation of LH2b in osteoarthritic hSF makes LH2b an interesting target to interfere with osteoarthritis-related persistent fibrosis.

OBJECTIVE

In humans, activin receptor-like kinase 1 (Alk1) deficiency causes arteriovenous malformations (AVMs) in multiple organs, including the brain. Focal Alk1 pan-cellular deletion plus vascular endothelial growth factor stimulation induces brain AVMs in the adult mouse. We hypothesized that deletion of Alk1 in endothelial cell (EC) alone plus focal vascular endothelial growth factor stimulation is sufficient to induce brain AVM in the adult mouse.

METHODS

Focal angiogenesis was induced in the brain of 8-week-old Pdgfb-iCreER;Alk1(2f/2f) mice by injection of adeno-associated viral vectors expressing vascular endothelial growth factor. Two weeks later, EC-Alk1 deletion was induced by tamoxifen treatment. Vascular morphology was analyzed, and EC proliferation and dysplasia index (number of vessels with diameter>15 μm per 200 vessels) were quantified 10 days after tamoxifen administration.

RESULTS

Tangles of enlarged vessels resembling AVMs were present in the brain angiogenic region of tamoxifen-treated Pdgfb-iCreER;Alk1(2f/2f) mice. Induced brain AVMs were marked by increased dysplasia index (P<0.001) and EC proliferation clustered within the dysplastic vessels. AVMs were also detected around the ear tag-wound and in other organs.

CONCLUSIONS

Deletion of Alk1 in EC in adult mice leads to an increased local EC proliferation during brain angiogenesis and de novo brain AVM.

Senescence of endothelial cells (ECs) may contribute to age-associated cardiovascular diseases, including atherosclerosis and hypertension. The functional and gene expression changes associated with cellular senescence are poorly understood. Here, we have analyzed the expression, during EC senescence, of 2 different isoforms (L, long; S, short) of endoglin, an auxiliary transforming growth factor (TGF)-beta receptor involved in vascular remodeling and angiogenesis. As evidenced by RT-PCR, the S/L ratio of endoglin isoforms was increased during senescence of human ECs in vitro, as well as during aging of mice in vascularized tissues. Next, the effect of S-endoglin protein on the TGF-beta receptor complex was studied. As revealed by coimmunoprecipitation assays, S-endoglin was able to interact with both TGF-beta type I receptors, ALK5 and ALK1, although the interaction with ALK5 was stronger than with ALK1. S-endoglin conferred a lower proliferation rate to ECs and behaved differently from L-endoglin in relation to TGF-beta-responsive reporters with ALK1 or ALK5 specificities, mimicking the behavior of the endothelial senescence markers Id1 and plasminogen activator inhibitor-1. In situ hybridization studies demonstrated the expression of S-endoglin in the endothelium from human arteries. Transgenic mice overexpressing S-endoglin in ECs showed hypertension, decreased hypertensive response to NO inhibition, decreased vasodilatory response to TGF-beta(1) administration, and decreased endothelial nitric oxide synthase expression in lungs and kidneys, supporting the involvement of S-endoglin in the NO-dependent vascular homeostasis. Taken together, these results suggest that S-endoglin is induced during endothelial senescence and may contribute to age-dependent vascular pathology.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by a multi-systemic vascular dysplasia and hemorrhage. The precise factors leading to these vascular malformations are not yet understood and robust animal models of HHT are essential to gain a detailed understanding of the molecular and cellular events that lead to clinical symptoms, as well as to test new therapeutic modalities. Most cases of HHT are caused by mutations in either endoglin (ENG) or activin receptor-like kinase 1 (ACVRL1, also known as ALK1). Both genes are associated with TGFβ/BMP signaling, and loss of function mutations in the co-receptor ENG are causal in HHT1, while HHT2 is associated with mutations in the signaling receptor ACVRL1. Significant advances in mouse genetics have provided powerful ways to study the function of Eng and Acvrl1 in vivo, and to generate mouse models of HHT disease. Mice that are null for either Acvrl1 or Eng genes show embryonic lethality due to major defects in angiogenesis and heart development. However mice that are heterozygous for mutations in either of these genes develop to adulthood with no effect on survival. Although these heterozygous mice exhibit selected vascular phenotypes relevant to the clinical pathology of HHT, the phenotypes are variable and generally quite mild. An alternative approach using conditional knockout mice allows us to study the effects of specific inactivation of either Eng or Acvrl1 at different times in development and in different cell types. These conditional knockout mice provide robust and reproducible models of arteriovenous malformations, and they are currently being used to unravel the causal factors in HHT pathologies. In this review, we will summarize the strengths and limitations of current mouse models of HHT, discuss how knowledge obtained from these studies has already informed clinical care and explore the potential of these models for developing improved treatments for HHT patients in the future.

Hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber disease, is an autosomal dominant disorder of the fibrovascular tissue. It is characterized by the classic triad of (muco-)cutaneous telangiectases, arteriovenous malformations with recurrent epistaxis and hemorrhages, and inheritance. A wide variety of clinical manifestations in HHT have been described. In more than 90% of the patients, nosebleeds are the first predominant symptom, therefore ENT physicians often play a key role as far as diagnosis and management of the disease are concerned. In spite of recent diagnostic and therapeutic progress, a cure for this often burdening and handicapping disease is still not available. Apart from affecting the nose, arteriovenous malformations (AVMs) may also affect the skin, lungs, brain, liver and gastrointestinal tract. The two known genes that are implicated in HHT are endoglin (ENG) located on chromosome 9q33-q34 and activin-receptor-like kinase (ALK1) located on chromosome 12q13. Mutations of ENG are observed in HHT type 1 with an incidence up to 40% for pulmonary AVMs, whereas mutations of ALK1 are observed in HHT type 2 with an incidence of only 14% for pulmonary AVMs, which clinically distinguishes these two types of mutation. The emphasis of this paper is mainly on the clinical manifestation, molecular genetics and diagnosis of HHT, taking account of current literature on HHT in order to better understand the complexity of the disease. Recent therapeutic options in the treatment of HHT have been omitted from this paper as they are subject of a following paper. HHT is more common than previously thought and shows a broad range of different clinical organ manifestations that can be sources of substantial morbidity and mortality, making HHT a continuing challenge for many sub-specialties where interdisciplinary diagnostic screening is mandatory in the management of the disease.

The Janus kinase 2 (JAK2)-signal transducers and activators of transcription (STAT) pathway plays an important role in hematological malignancies. Mutations and translocations of the JAK2 gene, mapped at 9p24, lead to constitutive activation of JAK2 and its downstream targets. The presence of JAK2 mutations in lymphomas has been addressed in larger cohorts, but there are little systemic data on numerical and structural JAK2 aberrations in lymphoid neoplasms. To study the molecular epidemiology of these aberrations and the consecutive activation of the JAK2-STAT pathway in lymphomas, we examined 527 cases, covering the most common entities, in a tissue microarray by fluorescent in situ hybridization with breakable JAK2 probes, and immunohistochemistry for phosphorylated JAK2 (pJAK2) and its preferred downstream pSTAT3 and pSTAT5. 9p24 gains were detected in 6/17 (35%) primary mediastinal B-cell lymphomas (PMBCLs), 25/77 (33%) Hodgkin's lymphomas (HLs), 3/16 (19%) angioimmunoblastic T-cell lymphomas (AILTs) and 1/5 ALK1(+) anaplastic large cell lymphomas (ALCLs); breaks were observed only in three cases. pJAK2 expression was most prevalent in PMBCL, peripheral T-cell lymphomas and HL. pSTAT3 predominated in ALCLs, HLs, AILTs, PMBCLs and peripheral T-cell lymphomas. pSTAT5 expression was detected frequently in follicular lymphomas, diffuse large B-cell lymphomas and AILTs. 9p24 gains correlated with increased proportions of tumor cells expressing pJAK2 (P=0.002) and pSTAT3 (P=0.001). In follicular lymphomas, concomitant expression of pJAK2 and pSTAT5 was linked to better prognosis, whereas expression of pSTAT3 in nongerminal center-like diffuse large B-cell lymphomas could identify a patient group with an inferior outcome. Our findings stress that despite the rarity of activating JAK2 mutations in lymphomas, JAK2 is recurrently targeted by numerical, and rarely by structural, genetic aberrations in distinct lymphoma subtypes and that JAK2-STAT pathway may play a role in lymphomagenesis.

BACKGROUND

Age is the most important risk factor for primary osteoarthritis (OA). Members of the TGF-β superfamily play a crucial role in chondrocyte differentiation and maintenance of healthy articular cartilage.

OBJECTIVE

We have investigated whether age-related changes in TGF-β superfamily signaling components play a role in the relationship between OA-related cartilage degradation and aging.

METHODS

The relationship between age, OA and TGF-β superfamily signaling was studied using murine experimental OA models, aging mice, bovine articular cartilage and human OA cartilage. The effects of TGF-β on cartilage homeostasis was studied with immunohistochemistry, Q-RT-PCR and signaling pathway analysis with Western blotting and the application of specific TGF-β inhibitors.

RESULTS

We have found that TGF-β loses its protective effects in old cartilage. Moreover, we found that on chondrocytes, TGF-β not only signals via the canonical type I receptor ALK5 (TGFBR1) but also via the ALK1 (ACVRL1) receptor. Remarkably, signaling via ALK5 (Smad2/3 route) results in protective while ALK1 signaling (Smad1/5/8 route) results in deleterious responses in articular chondrocytes. In cartilage of aging mice it was detected that the ALK1/ALK5 ratio is significantly increased, favoring TGF-β signaling via the Smad1/5/8 route, inducing changes in chondrocyte differentiation and matrix metalloproteinase-13 (MMP-13) expression. Moreover, human OA cartilage showed a significant correlation between ALK1 and MMP-13 expression. Since in mice aging and OA in often goes hand in hand, we also analyzed age-related expression of TGF-β superfamily related signaling molecules in healthy bovine cartilage in an age range from 6 months to 14 years. In this cohort of aging cartilage, we found that mainly signaling receptors determining the Smad2/3 pathway were decreased with age while Smad1/5/8-related signaling molecules did not alter, confirming our findings in aging mice.

CONCLUSIONS

Old cartilage appears to be less protected by TGF-β and shows significant alterations in TGF-β signaling pathways. Loss of the protective Smad2/3 pathway during aging can provide an explanation for the relationship between OA and aging.

BACKGROUND

Bone morphogenetic proteins (BMPs) and their receptors, such as bone morphogenetic protein receptor (BMPR) II, have been implicated in a wide variety of disorders including pulmonary arterial hypertension (PAH). Similarly, endothelin-1 (ET-1), a mitogen and vasoconstrictor, is upregulated in PAH and endothelin receptor antagonists are used in its treatment. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof.

RESULTS

Using western blot, real time RT-PCR, ELISA and small RNA interference methods we provide evidence that in HPAECs BMP-9, but not BMP-2, -4 and -6 significantly stimulated ET-1 release under physiological concentrations. This release is mediated by both Smad1 and p38 MAPK and is independent of the canonical Smad4 pathway. Moreover, knocking down the ALK1 receptor or BMPR II attenuates BMP-9 stimulated ET-1 release, whilst causing a significant increase in prepro ET-1 mRNA transcription and mature peptide release. Finally, BMP-9 induced ET-1 release is involved in both inhibition of endothelial cell migration and promotion of tubule formation.

CONCLUSIONS

Although our data does not support an important role for BMP-9 as a source of increased endothelial ET-1 production seen in human PAH, BMP-9 stimulated ET-1 production is likely to be important in angiogenesis and vascular stability. However, increased ET-1 production by endothelial cells as a consequence of BMPR II dysfunction may be clinically relevant in the pathogenesis of PAH.

BACKGROUND

TGF-beta1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-beta signalling is mediated by the TbetaRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-beta utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TbetaRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT).

METHODS

The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis.

RESULTS

After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPeta and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-beta1 induced gene expression in HMEC-1 cells and primary HUVECs was observed.

CONCLUSIONS

Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-beta signalling.

Acetylcholine (ACh) synthesis and release from basal forebrain cholinergic neurons (BFCN) innervating the cerebral cortex and hippocampus are essential processes for normal learning, memory and attention. Bone morphogenetic protein (BMP) 9 is a cholinergic differentiation factor in the developing septum that increases ACh synthesis and choline acetyltransferase (Chat) gene expression both in vivo and in vitro. We investigated the possible induction of cholinergic trophic factors by BMP9 in murine septal cells. Nerve growth factor (NGF) protein expression and secretion into the medium was increased in cultured embryonic septal cells treated with BMP9, and partially mediated BMP9-induced acetylcholine production and Chat gene expression. BMP9-induced Ngf gene expression was detected in postmitotic cells, required new protein synthesis and was blocked by BMP type I receptor inhibition. Cholinergic neurons were isolated by fluorescence-activated cell sorting based on either transgenic expression of green fluorescent protein driven by the Chat promoter or NGF receptor (p75) immunostaining. Although both noncholinergic and cholinergic neurons in untreated cultures expressed similar low levels of Ngf, increased Ngf gene expression was restricted to Chat-positive neurons in BMP9-treated cultures. Likewise, similar levels of Ngf mRNA were detected in p75-negative and p75-positive septal cells, yet only p75-positive BFCN increased their Ngf gene expression when treated with BMP9, and only these cells expressed the Alk1 BMP receptor. The data suggest an autocrine/paracrine role for NGF in the development and/or maintenance of BFCN and imply that the stimulation of NGF production and release contributes to the cholinergic-supportive properties of BMP9.

Anaplastic lymphoma kinase (ALK)-positive large B-cell lymphoma (ALK-positive LBCL) is an extremely rare distinct clinicopathological subtype of LBCL, characterized by the presence of ALK-positive monomorphic large immunoblast-like neoplastic B cells. Herein, we describe the first cytological report on ALK-positive LBCL in the pleural effusion. A 69-year-old Japanese male with a past history of malignant lymphoma of the cecum presented with progressive dyspnea and pleural effusion. Removal of the pleural effusion and aspiration of bone marrow were performed. May-Grünwald-Giemsa stain of the pleural fluid revealed abundant single or small aggregates of large-sized round cells. These cells had centrally-located large round to oval nuclei. The peculiar finding was the presence of pseudopodial cytoplasmic projections, and some neoplastic cells had eosinophilic pseudopodial cytoplasmic projections, which resembled "flaming plasma cells". Histopathological and immunohistochemical studies of the bone marrow demonstrated CD138(+), ALK1(+), CD20(-), CD79a(-), CD30(-), and IgA(+) large-sized neoplastic cells. Therefore, a diagnosis of ALK-positive LBCL was made. The peculiar finding of the present case was that most of the neoplastic cells had pseudopodial cytoplasmic projections, and some of them had eosinophilic pseudopodial cytoplasmic projections that resembled "flaming plasma cells", which has been recognized as the characteristic finding of IgA myeloma. Therefore, tumor cells that resembled "flaming plasma cells" in the pleural effusion may have had IgA in the cytoplasm. Albeit extremely rare, ALK-positive LBCL shows aggressive clinical course, thus, recognition of the cytomorphological features of this type of malignant lymphoma is important for early and correct diagnosis.

OBJECTIVE

The angiogenesis inhibitor dalantercept (formerly ACE-041) is a soluble form of activin receptor-like kinase-1 (ALK1) that prevents activation of endogenous ALK1 by bone morphogenetic protein-9 (BMP9) and BMP10 and exhibits antitumor activity in preclinical models. This first-in-human study of dalantercept evaluated its safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity in adults with advanced solid tumors.

METHODS

Patients in dose-escalating cohorts received dalantercept subcutaneously at one of seven dose levels (0.1-4.8 mg/kg) every 3 weeks until disease progression. Patients in an expansion cohort received dalantercept at 0.8 or 1.6 mg/kg every 3 weeks until disease progression.

RESULTS

In 37 patients receiving dalantercept, the most common treatment-related adverse events were peripheral edema, fatigue, and anemia. Edema and fluid retention were dose-limiting toxicities and responded to diuretic therapy. No clinically significant, treatment-related hypertension, proteinuria, gross hemorrhage, or gastrointestinal perforations were observed. One patient with refractory squamous cell cancer of the head and neck had a partial response, and 13 patients had stable disease according to RECISTv1.1, eight of whom had prolonged periods (≥12 weeks) of stable disease. Correlative pharmacodynamic markers included tumor metabolic activity and tumor blood flow, which decreased from baseline in 63% and 82% of evaluable patients, respectively, and telangiectasia in eight patients.

CONCLUSIONS

Dalantercept was well-tolerated at doses up to 1.6 mg/kg, with a safety profile distinct from inhibitors of the VEGF pathway. Dalantercept displayed promising antitumor activity in patients with advanced refractory cancer, and multiple phase II studies are underway.

Pathological features and genomic basis of a rare case of ALK(+), CD30(-), CD20(-) large B-cell lymphoma were analyzed. A 36-year-old Japanese female was admitted because of lumbago and constitutional symptoms. Physical examination and laboratory tests showed anemia (hemoglobin, 7.5 g/dL), mild hepatosplenomegaly, and immunoglobin G (IgG) lambda-type monoclonal gammopathy (IgG, 2782 mg/dL). The lymphoma spread exclusively in extranodal sites such as bone marrow, liver, spleen, ovary, and muscle. Biopsy specimens obtained from the ovary showed monomorphic proliferation of large immunoblastic cells with basophilic cytoplasm, round-shaped nuclei with a high nuclear to cytoplasmic ratio, and prominent single nucleolus. Immunostaining with anti-anaplastic lymphoma kinase (ALK) antibody, ALK1, showed finely granular cytoplasmic staining pattern. These cells were also positive for epithelial membrane antigen, CD4, CD19, CD38, CD138, cytoplasmic IgG, and lambda chain, but negative for CD30 (Ber-H2), CD56, CD57, and other T- and B-cell markers. Southern blot analyses revealed that Ig heavy and lambda light chain genes, but not T-cell receptor (TCR) beta gene, were clonally rearranged. Chromosomal analyses by conventional G-banding, spectral karyotyping, and fluorescence in situ hybridization showed complex abnormality involving 2p23, and chromosome 2 was translocated to chromosome 17. As 2;17 translocation resulting in the fusion of clathrin heavy chain (CLTC) gene with ALK was previously reported in inflammatory myofibroblastic tumor, we performed reverse transcriptase-polymerase chain reaction and demonstrated that the lymphoma cells contained CLTC-ALK fusion transcript. Under the diagnosis of ALK(+), CD30(-), CD20(-) large B-cell lymphoma, she was treated with conventional combination chemotherapies. However, the lymphoma was primarily chemotherapy resistant, and the patient died 11 months after admission. We consider that this case confirms the existence of ALK(+), CD30(-), CD20(-) large B-cell lymphomas proposed by Delsol et al. (16) and further provides relevant information regarding their clinicopathological features and cytogenetics.

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease characterized by arteriovenous malformations and resulting from mutations in two major genes: ENG and ACVRL1. The aim of the present study was to estimate the prevalence of the mutations of ENG and ACVRL1 in HHT, based on the largest series of patients reported so far, recruited through a national network. We previously reported the first mutation screening of both genes, in French HHT patients, using heteroduplex analysis. This previous study, bringing 60 novel mutations, provided a significant improvement to the knowledge of molecular pathology in HHT. However, 32% (n=48) of the patients with a confirmed clinical diagnosis remained without mutation. In these patients, we performed an extensive molecular analysis that included the sequencing of the whole coding sequence, the search for large rearrangements, and screening of the potential 5' regulatory regions. Additionally, due to the lack of large pedigrees suitable for linkage analysis, and since SMAD4 germline mutations have been reported in families with combined HHT and juvenile polyposis, we screened this gene and five other genes involved in the TGF-beta/BMP pathway in the patients without mutation of ENG or ACVRL1. Only a novel SMAD1 non-conservative substitution was found in one patient, changing a poorly conserved methionine to an isoleucin. Twenty-three mutations were found in ACVRL1 and 8 in ENG (including a duplication of exons 4 to 8 and deletions of exons 1 to 3 and 9 to 14). Our results, combined with our previous data, increase the mutation rate to 88% (n=119/136) in French patients with a confirmed clinical diagnosis. Our results also emphasize the higher prevalence of large insertions/deletions in ENG and the predominance of ACVRL1 over ENG mutations.

Transforming growth factor-beta (TGF-beta) superfamily signaling has been implicated in many developmental processes, including pancreatic development. Previous studies are conflicting with regard to an exact role for TGF-beta signaling in various aspects of pancreatic organogenesis. Here we have investigated the role of TGF-beta isoform signaling in embryonic pancreas differentiation and lineage selection. The TGF-beta isoform receptors (RI, RII and ALK1) were localized mainly to both the pancreatic epithelium and mesenchyme at early stages of development, but then with increasing age localized to the pancreatic islets and ducts. To determine the specific role of TGF-beta isoforms, we functionally inactivated TGF-beta signaling at different points in the signaling cascade. Disruption of TGF-beta signaling at the receptor level using mice overexpressing the dominant-negative TGF-beta type II receptor showed an increase in endocrine precursors and proliferating endocrine cells, with an abnormal accumulation of endocrine cells around the developing ducts of mid-late stage embryonic pancreas. This pattern suggested that TGF-beta isoform signaling may suppress the origination of secondary transition endocrine cells from the ducts. Secondly, TGF-beta isoform ligand inhibition with neutralizing antibody in pancreatic organ culture also led to an increase in the number of endocrine-positive cells. Thirdly, hybrid mix-and-match in vitro recombinations of transgenic pancreatic mesenchyme and wild-type epithelium also led to increased endocrine cell differentiation, but with different patterns depending on the directionality of the epithelial-mesenchymal signaling. Together these results suggest that TGF-beta signaling is important for restraining the growth and differentiation of pancreatic epithelial cells, particularly away from the endocrine lineage. Inhibition of TGF-beta signaling in the embryonic period may thus allow pancreatic epithelial cells to progress towards the endocrine lineage unchecked, particularly as part of the secondary transition of pancreatic endocrine cell development. TGF-beta RII in the ducts and islets may normally serve to downregulate the production of beta cells from embryonic ducts.

Genetic causes of pulmonary arterial hypertension (PAH) and pulmonary veno-occlusive disease (PVOD) have been identified, leading to a growing need for genetic counselling.Between 2003 and 2014, genetic counselling was offered to 529 PAH and 100 PVOD patients at the French Referral Centre for Pulmonary Hypertension.Mutations in PAH-predisposing genes were identified in 72 patients presenting as sporadic PAH (17% of cases; 62 mutations in BMPR2, nine in ACVRL1 (ALK1) and one in ENG) and in 94 patients with a PAH family history (89% of cases; 89 mutations in BMPR2, three in ACVRL1 (ALK1) and two in KCNK3). Bi-allelic mutations in EIF2AK4 were identified in all patients with a family history of PVOD (n=19) and in seven patients (8.6%) presenting as sporadic PVOD. Pre-symptomatic genetic diagnosis was offered to 272 relatives of heritable PAH patients, identifying mutations in 36.4% of them. A screening programme is now offered to asymptomatic mutation carriers to detect PAH in an early phase and to identify predictors of outcomes in asymptomatic BMPR2 mutation carriers. BMPR2 screening allowed us to offer pre-implantation diagnosis to two couples with a BMPR2 mutation.Genetic counselling can be implemented in pulmonary hypertension centres.

OBJECTIVE

To review the literature on modulation of chondrocyte activities in the osteoarthritic joint, and to discuss these changes in relation to established hard and soft tissue repair paradigms, with an emphasis on transforming growth factor beta (TGFβ1)-mediated signaling which can promote either a chondrogenic or fibrogenic phenotype.

METHODS

Papers addressing the close relationship between repair in general, and the specific post-injury response of joint tissues are summarized. Different interpretations of the role of TGFβ1 in the emergence of an "osteoarthritic" chondrocyte are compared and the phenotypic plasticity of "reparative" progenitor cells is examined. Lastly, emerging data on a central role for A-Disintegrin-And-Metalloproteinase-with-Thrombospondin-like-Sequences-5 (ADAMTS5) activity in modulating TGFβ1 signaling through activin receptor-like kinase 1 (ALK1) and activin receptor-like kinase 5 (ALK5) pathways is discussed.

RESULTS

The review illustrates how a transition from ALK5-mediated fibrogenic signaling to ALK1-mediated chondrogenic signaling in joint cells represents the critical transition from a non-reparative to a reparative cell phenotype. Data from cell and in vivo studies illustrates the mechanism by which ablation of ADAMTS5 activity allows the transition to reparative chondrogenesis. Multiple large gene expression studies of normal and osteoarthritis (OA) human cartilages (CAs) also support an important role for TGFβ1-mediated pro-fibrogenic activities during disease progression.

CONCLUSIONS

We conclude that progressive articular CA damage in post-injury OA results primarily from biomechanical, cell biologic and mediator changes that promote a fibroblastic phenotype in joint cells. Since ADAMTS5 and TGFβ1 appear to control this process, agents which interfere with their activities may not only enhance endogenous CA repair in vivo, but also improve the properties of tissue-engineered CA for implantation.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder originated by mutations in Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes. The first large series HHT analysis in Spanish population has identified mutations in 17 unrelated families. Ten different mutations in ALK1 and six in ENG genes were found. Six unrelated families had a mutation in ENG gene, four representing new mutations, p.Y258fs, pV323fs, p.F279fs (c.834_837del CTTC), and p.F279fsdupC. Eleven unrelated families harboured mutations in ALK1; ten were new mutations identified as p.H328P, p.R145fs, p.G68C, p.A377T, p.H297R, p.M376T, p.C36Y, p.H328P, p.T82del and p.R47P. Overall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1), in agreement with data reported for other Mediterranean countries (France, Italy), but at variance with Northern Europe or North America. Endoglin expression in HHT1 or HHT2 activated monocytes and blood outgrowth endothelial cells (BOECs) from older patients was well below the theoretical 50% level expected from the HHT1 haploinsufficiency model, suggesting that the pathogenic endoglin haploinsufficiency leading to the HHT phenotype is age-dependent. Interestingly, ALK1 protein levels of HHT BOECs in some missense ALK1 mutants were similar to controls. In vitro expression of these ALK1 constructs suggests that, in addition to the haploinsufficiency model, certain ALK1 mutants may inhibit the function of the wild type allele.

OBJECTIVE

Transforming growth factor beta (TGFbeta)-activated kinase 1 (TAK1) is a MAP kinase kinase kinase involved in numerous signalling pathways and is strongly implicated in cardiac hypertrophy and heart failure. TGFbeta is also associated with hypertension and heart disease, and evidence suggests that TGFbeta1 and TAK1 act together in a cardiac stress signalling pathway. Canonical TGFbeta signalling is mediated through Smad transcription factors, but TGFbeta can also rapidly activate TAK1. The activation of the Smad cascade is well characterised, but little is known about how TAK1 is activated in response to TGFbeta, and no direct link between any MAPK kinase pathway and the TGFbeta receptors has yet been established. Since TAK1 is activated by TGFbeta within 1 min in cardiomyocytes, we hypothesised there might be a direct interaction between TAK1 and one of the TGFbeta receptors.

METHODS

We used a combination of in vitro binding assays and co-immunoprecipitation (IP) experiments to investigate whether TAK1 interacted with the type I (ALK1 or ALK5) or type II (TBRII) TGFbeta receptors. Interactions between endogenous proteins were tested using mouse myoblast and rat cardiomyocyte cells.

RESULTS

Immunoprecipitation and in vitro binding assays show that TAK1 binds directly to TBRII. Precipitation of endogenous TAK1 protein in rat cardiomyocytes shows that, in addition to a direct association with TBRII, it also interacts indirectly with ALK5.

CONCLUSIONS

We describe a novel and specific interaction between TAK1 and TBRII which, for the first time, directly links TAK1 to the TGFbeta signalling cascade and potentially explains how TGFbeta signalling in cardiomyocytes mediates a hypertrophic response.

The process of vascular calcification shares many similarities with that of physiological skeletal mineralization, and involves the deposition of hydroxyapatite crystals in arteries. However, the cellular mechanisms responsible have yet to be fully explained. Bone morphogenetic protein (BMP-9) has been shown to exert direct effects on both bone development and vascular function. In the present study, we have investigated the role of BMP-9 in vascular smooth muscle cell (VSMC) calcification. Vessel calcification in chronic kidney disease (CKD) begins pre-dialysis, with factors specific to the dialysis milieu triggering accelerated calcification. Intriguingly, BMP-9 was markedly elevated in serum from CKD children on dialysis. Furthermore, in vitro studies revealed that BMP-9 treatment causes a significant increase in VSMC calcium content, alkaline phosphatase (ALP) activity and mRNA expression of osteogenic markers. BMP-9-induced calcium deposition was significantly reduced following treatment with the ALP inhibitor 2,5-Dimethoxy-N-(quinolin-3-yl) benzenesulfonamide confirming the mediatory role of ALP in this process. The inhibition of ALK1 signalling using a soluble chimeric protein significantly reduced calcium deposition and ALP activity, confirming that BMP-9 is a physiological ALK1 ligand. Signal transduction studies revealed that BMP-9 induced Smad2, Smad3 and Smad1/5/8 phosphorylation. As these Smad proteins directly bind to Smad4 to activate target genes, siRNA studies were subsequently undertaken to examine the functional role of Smad4 in VSMC calcification. Smad4-siRNA transfection induced a significant reduction in ALP activity and calcium deposition. These novel data demonstrate that BMP-9 induces VSMC osteogenic differentiation and calcification via ALK1, Smad and ALP dependent mechanisms. This may identify new potential therapeutic strategies for clinical intervention.