Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

YAP1 is amplified and up-regulated in hedgehog-associated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation.

Medulloblastoma is the most common solid malignancy of childhood, with treatment side effects reducing survivors' quality of life and lethality being associated with tumor recurrence. Activation of the Sonic hedgehog (Shh) signaling pathway is implicated in human medulloblastomas. Cerebellar granule neuron precursors (CGNPs) depend on signaling by the morphogen Shh for expansion during development, and have been suggested as a cell of origin for certain medulloblastomas. Mechanisms contributing to Shh pathway-mediated proliferation and transformation remain poorly understood. We investigated interactions between Shh signaling and the recently described tumor-suppressive Hippo pathway in the developing brain and medulloblastomas. We report up-regulation of the oncogenic transcriptional coactivator yes-associated protein 1 (YAP1), which is negatively regulated by the Hippo pathway, in human medulloblastomas with aberrant Shh signaling. Consistent with conserved mechanisms between brain tumorigenesis and development, Shh induces YAP1 expression in CGNPs. Shh also promotes YAP1 nuclear localization in CGNPs, and YAP1 can drive CGNP proliferation. Furthermore, YAP1 is found in cells of the perivascular niche, where proposed tumor-repopulating cells reside. Post-irradiation, YAP1 was found in newly growing tumor cells. These findings implicate YAP1 as a new Shh effector that may be targeted by medulloblastoma therapies aimed at eliminating medulloblastoma recurrence.

The TGF-beta co-receptor endoglin modulates the expression and transforming potential of H-Ras.

Endoglin is a coreceptor for transforming growth factor-ß (TGF-ß) that acts as a suppressor of malignancy during mouse skin carcinogenesis. Because in this model system H-Ras activation drives tumor initiation and progression, we have assessed the effects of endoglin on the expression of H-Ras in transformed keratinocytes. We found that TGF-ß1 increases the expression of H-Ras at both messenger RNA and protein levels. The TGF-ß1-induced H-Ras promoter transactivation was Smad4 independent but mediated by the activation of the TGF-ß type I receptor ALK5 and the Ras-mitogen-activated protein kinase (MAPK) pathway. Endoglin attenuated stimulation by TGF-ß1 of both MAPK signaling activity and H-Ras gene expression. Moreover, endoglin inhibited the Ras/MAPK pathway in transformed epidermal cells containing an H-Ras oncogene, as evidenced by the levels of Ras-guanosine triphosphate, phospho-MAPK kinase (MEK) and phospho-extracellular signal-regulated kinase (ERK) as well as the expression of c-fos, a MAPK downstream target gene. Interestingly, in spindle carcinoma cells, that have a hyperactivated Ras/MAPK pathway, endoglin inhibited ERK phosphorylation without affecting MEK or Ras activity. The mechanism for this effect is unknown but strongly depends on the endoglin extracellular domain. Because the MAPK pathway is a downstream mediator of the transforming potential of Ras, the effect of endoglin on the oncogenic function of H-Ras was assessed. Endoglin inhibited the transforming capacity of H-Ras(Q61K) and H-Ras(G12V) oncogenes in a NIH3T3 focus formation assay. The ability to interfere with the expression and oncogenic potential of H-Ras provides a new face of the suppressor role exhibited by endoglin in H-Ras-driven carcinogenesis.

Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1.

BACKGROUND: Activin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-beta (TGF-beta) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1. RESULTS: We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210) was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1. CONCLUSIONS: Our results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into the molecular mechanisms that contribute to the expression of ACVRL1 gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of ACVRL1 gene transcriptional activity.

TGF-beta regulates the expression of transcription factor KLF6 and its splice variants and promotes co-operative transactivation of common target genes through a Smad3-Sp1-KLF6 interaction.

KLF6 (Krüppel-like factor 6) is a transcription factor and tumour suppressor with a growing range of biological activities and transcriptional targets. Among these, KLF6 suppresses growth through transactivation of TGF-beta1 (transforming growth factor-beta1). KLF6 can be alternatively spliced, generating lower-molecular-mass isoforms that antagonize the full-length WT (wild-type) protein and promote growth. A key target gene of full-length KLF6 is endoglin, which is induced in vascular injury. Endoglin, a homodimeric cell membrane glycoprotein and TGF-beta auxiliary receptor, has a pro-angiogenic role in endothelial cells and is also involved in malignant progression. The aim of the present work was to explore the effect of TGF-beta on KLF6 expression and splicing, and to define the contribution of TGF-beta on promoters regulated by co-operation between KLF6 and Sp1 (specificity protein 1). Using co-transfection, co-immunoprecipitation and fluorescence resonance energy transfer, our data demonstrate that KLF6 co-operates with Sp1 in transcriptionally regulating KLF6-responsive genes and that this co-operation is further enhanced by TGF-beta1 through at least two mechanisms. First, in specific cell types, TGF-beta1 may decrease KLF6 alternative splicing, resulting in a net increase in full-length, growth-suppressive KLF6 activity. Secondly, KLF6-Sp1 co-operation is further enhanced by the TGF-beta-Smad (similar to mothers against decapentaplegic) pathway via the likely formation of a tripartite KLF6-Sp1-Smad3 complex in which KLF6 interacts indirectly with Smad3 through Sp1, which may serve as a bridging molecule to co-ordinate this interaction. These findings unveil a finely tuned network of interactions between KLF6, Sp1 and TGF-beta to regulate target genes.

Mutation study of Spanish patients with hereditary hemorrhagic telangiectasia.

BACKGROUND: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant and age-dependent vascular disorder characterised mainly by mutations in the Endoglin (ENG) or activin receptor-like kinase-1 (ALK1, ACVRL1) genes. METHODS: Here, we have identified 22 ALK1 mutations and 15 ENG mutations, many of which had not previously been reported, in independent Spanish families afflicted with HHT. RESULTS: We identified mutations in thirty-seven unrelated families. A detailed analysis of clinical symptoms was recorded for each patient analyzed, with a higher significant presence of pulmonary arteriovenous malformations (PAVM) in HHT1 patients over HHT2. Twenty-two mutations in ALK1 and fifteen in ENG genes were identified. Many of them, almost half, represented new mutations in ALK1 and in ENG. Missense mutations in ENG and ALK1 were localized in a tridimensional protein structure model. CONCLUSION: Overall, ALK1 mutations (HHT2) were predominant over ENG mutations (HHT1) in our Spanish population, in agreement with previous data from our country and other Mediterranean countries (France, Italy), but different to Northern Europe or North America. There was a significant increase of PAVM associated with HHT1 over HHT2 in these families.

Therapeutic action of tranexamic acid in hereditary haemorrhagic telangiectasia (HHT): regulation of ALK-1/endoglin pathway in endothelial cells.

Recurrent epistaxis is the most frequent clinical manifestation of hereditary haemorrhagic telangiectasia (HHT). Its treatment is difficult. Our objective was to assess the use of tranexamic acid (TA), an antifibrinolytic drug, for the treatment of epistaxis in HHT patients and to investigate in vitro the effects of TA over endoglin and ALK-1 expression and activity in endothelial cells. A prospective study was carried out on patients with epistaxis treated with oral TA in the HHT Unit of Sierrallana Hospital (Cantabria, Spain). Primary cultures of endothelial cells were treated with TA to measure the levels of endoglin and ALK-1 at the cell surface by flow cytometry. RNA levels were also measured by real-time PCR, and the transcriptional effects of TA on reporters for endoglin, ALK-1 and the endoglin/ALK-1 TGF-beta pathway were assessed. The results showed that the fourteen HHT patients treated orally with TA improved, and the frequency and severity of their epistaxis were decreased. No complications derived from the treatment were observed. Cultured endothelial cells incubated with TA exhibited increased levels of endoglin and ALK-1 at the protein and mRNA levels, enhanced TGF-beta signaling, and improved endothelial cell functions like tubulogenesis and migration. In summary, oral administration of TA proved beneficial for epistaxis treatment in selected patients with HHT. In addition to its already reported antifibrinolytic effects, TA stimulates the expression ofALK-1 and endoglin, as well as the activity of the ALK-1/endoglin pathway.

Mutation study of Spanish patients with hereditary hemorrhagic telangiectasia and expression analysis of Endoglin and ALK1.

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.

Blood outgrowth endothelial cells from Hereditary Haemorrhagic Telangiectasia patients reveal abnormalities compatible with vascular lesions.

OBJECTIVE: Hereditary haemorrhagic telangiectasia (HHT) is originated by mutations in endoglin (HHT1) and ALK1 (HHT2) genes. The purpose of this work was to isolate and characterize circulating endothelial cells from HHT patients. METHODS: Pure primary cultures of blood outgrowth endothelial cells (BOECs) were obtained from 50 ml of peripheral blood by selection on collagen plates with endothelial medium. RESULTS: The amount of endoglin in HHT1-BOECs is half the controls, but HHT2-BOECs are also endoglin-deficient. Since the TGF-beta/ALK1 pathway activates the endoglin promoter activity, these results suggest the involvement of ALK1 in endoglin gene expression. Endothelial TGF-beta pathways, mediated by ALK1 and ALK5, are impaired in HHT cells. HHT-BOECs show disorganized and depolymerized actin fibers, as compared to the organized stress fibers of healthy-BOECs. Functionally, HHT-BOECs have impaired tube formation, in contrast with the cord-like structures derived from normal donors. CONCLUSIONS: Decreased endoglin expression, impaired TGF-beta signalling, disorganized cytoskeleton, and failure to form cord-like structures are common characteristics of endothelial cells from HHT patients. These features may lead to fragility of small vessels and bleeding characteristic of the HHT vascular dysplasia and to a disrupted and abnormal angiogenesis, which may explain the clinical symptoms associated with this disease.

The Hippo in the room: a new look at a key pathway in cell growth and transformation.

During development and in cancer, tissue and cell growth control requires coordinated regulation of cell proliferation and apoptosis. The tumor suppressive Hippo pathway plays a key role in size regulation and cell-contact inhibition. During the past decade, this pathway has been delineated in Drosophila and now is starting to be better understood in mammals, where an increasing level of complexity and cell context specificity is becoming evident. As we discuss, dys-regulation of this pathway at any step can lead to uncontrolled growth and tumor formation. Indeed, a majority of the pathway components have been implicated in human cancers.

p27(Kip1), a double-edged sword in Shh-mediated medulloblastoma: Tumor accelerator and suppressor.

Medulloblastoma, a brain tumor arising in the cerebellum, is the most common solid childhood malignancy. the current standard of care for medulloblastoma leaves survivors with life-long side effects. Gaining insight into mechanisms regulating transformation of medulloblastoma cells-of-origin may lead to development of better treatments for these tumors. Cerebellar granule neuron precursors (CGNps) are proposed cells-of-origin for certain classes of medulloblastoma, specifically those marked by aberrant Sonic hedgehog (Shh) signaling pathway activation. CGNps require signaling by Shh for proliferation during brain development. In mitogen-stimulated cells, nuclear localized cyclin dependent kinase (cdk) inhibitor p27 (Kip1) functions as a checkpoint control at the G1- to S-phase transition by inhibiting cdk2. Recent studies have suggested cytoplasmically localized p27(Kip1) acquires oncogenic functions. Here, we show that p27(Kip1) is cytoplasmically localized in CGNps and mouse Shh-mediated medulloblastomas. transgenic mice bearing an activating mutation in the Shh pathway and lacking one or both p27(Kip1) alleles have accelerated tumor incidence compared to mice bearing both p27(Kip1) alleles. Interestingly, mice heterozygous for p27(Kip1) have decreased survival latency compared to p27(Kip1)-null animals. our data indicate that this may reflect the requirement for at least one copy of p27(Kip1) for recruiting cyclin D/cdk4/6 to promote cell cycle progression yet insufficient expression in the heterozygous or null state to inhibit cyclin E/cdk2. Finally, we find that mis-localized p27(Kip1) may play a positive role in motility in medulloblastoma cells. Together, our data indicate that the dosage of p27(Kip1) plays a role in cell cycle progression and tumor suppression in Shh-mediated medulloblastoma expansion.

ß-Arrestin-1 links mitogenic sonic hedgehog signaling to the cell cycle exit machinery in neural precursors.

Development of the cerebellum, a brain region regulating posture and coordination, occurs post-natally and is marked by rapid proliferation of granule neuron precursors (CGNPs), stimulated by mitogenic Sonic hedgehog (Shh) signaling. ß-Arrestin (ßArr) proteins play important roles downstream of Smoothened, the Shh signal transducer. However, whether Shh regulates ßArrs and what role they play in Shh-driven CGNP proliferation remains to be determined. Here, we report that Shh induces ßArr1 accumulation and localization to the nucleus, where it participates in enhancing expression of the cyclin dependent kinase (cdk) inhibitor p27, whose accumulation eventually drives CGNP cell cycle exit. ßArr1 knockdown enhances CGNP proliferation and reduces p27 expression. Thus, Shh-mediated ßArr1 induction represents a novel negative feedback loop within the Shh mitogenic pathway, such that ongoing Shh signaling, while required for CGNPs to proliferate, also sets up a cell-intrinsic clock programming their ultimate exit from the cell cycle.

Normal and oncogenic roles for microRNAs in the developing brain.

MicroRNAS (miRNAs) are small endogenous non-coding RNAs that play important roles in many different biological processes including proliferation, differentiation and apoptosis through silencing of target genes. Emerging evidence indicates that miRNAs are key players in mammalian development that, when altered, contribute to tumorigenesis. However, only a few studies to date have focused on the role of miRNAs in medulloblastoma, the most common malignant pediatric brain tumor. These tumors arise in the cerebellum and may attribute their origins to deregulated proliferation of neural progenitor cells during development. Understanding the interplay between normal brain development and medulloblastoma pathogenesis is necessary in order for more efficient, less toxic targeted therapies to be developed and implemented. MiRNA expression profiling of both mouse and human medulloblastomas has led to the identification of signatures correlating with the molecular subgroups of medulloblastoma, tumor diagnosis and response to treatment, as well as novel targets of potential clinical relevance. This review summarizes the recent miRNA literature in both medulloblastoma and normal brain development.

The miR-17/92 polycistron is up-regulated in sonic hedgehog-driven medulloblastomas and induced by N-myc in sonic hedgehog-treated cerebellar neural precursors.

Medulloblastoma is the most common malignant pediatric brain tumor, and mechanisms underlying its development are poorly understood. We identified recurrent amplification of the miR-17/92 polycistron proto-oncogene in 6% of pediatric medulloblastomas by high-resolution single-nucleotide polymorphism genotyping arrays and subsequent interphase fluorescence in situ hybridization on a human medulloblastoma tissue microarray. Profiling the expression of 427 mature microRNAs (miRNA) in a series of 90 primary human medulloblastomas revealed that components of the miR-17/92 polycistron are the most highly up-regulated miRNAs in medulloblastoma. Expression of miR-17/92 was highest in the subgroup of medulloblastomas associated with activation of the sonic hedgehog (Shh) signaling pathway compared with other subgroups of medulloblastoma. Medulloblastomas in which miR-17/92 was up-regulated also had elevated levels of MYC/MYCN expression. Consistent with its regulation by Shh, we observed that Shh treatment of primary cerebellar granule neuron precursors (CGNP), proposed cells of origin for the Shh-associated medulloblastomas, resulted in increased miR-17/92 expression. In CGNPs, the Shh effector N-myc, but not Gli1, induced miR-17/92 expression. Ectopic miR-17/92 expression in CGNPs synergized with exogenous Shh to increase proliferation and also enabled them to proliferate in the absence of Shh. We conclude that miR-17/92 is a positive effector of Shh-mediated proliferation and that aberrant expression/amplification of this miR confers a growth advantage to medulloblastomas.

Insulin receptor substrate 1 is an effector of sonic hedgehog mitogenic signaling in cerebellar neural precursors.

Sonic hedgehog (SHH) and insulin-like growth factor (IGF) signaling are essential for development of many tissues and are implicated in medulloblastoma, the most common solid pediatric malignancy. Cerebellar granule neuron precursors (CGNPs), proposed cells-of-origin for specific classes of medulloblastomas, require SHH and IGF signaling for proliferation and survival during development of the cerebellum. We asked whether SHH regulates IGF pathway components in proliferating CGNPs. We report that SHH-treated CGNPs showed increased levels of insulin receptor substrate 1 (IRS1) protein, which was also present in the germinal layer of the developing mouse cerebellum and in mouse SHH-induced medulloblastomas. Previous roles for IRS1, an oncogenic protein that is essential for IGF-mediated proliferation in other cell types, have not been described in SHH-mediated CGNP proliferation. We found that IRS1 overexpression can maintain CGNP proliferation in the absence of SHH. Furthermore, lentivirus-mediated knock down experiments have shown that IRS1 activity is required for CGNP proliferation in slice explants and dissociated cultures. Contrary to traditional models for SHH signaling that focus on gene transcription, SHH stimulation does not regulate Irs1 transcription but rather stabilizes IRS1 protein by interfering with mTOR-dependent IRS1 turnover and possibly affects Irs1 mRNA translation. Thus, we have identified IRS1 as a novel effector of SHH mitogenic signaling that may serve as a future target for medulloblastoma therapies. Our findings also indicate a previously unreported interaction between the SHH and mTOR pathways, and provide an example of a non-classical means for SHH-mediated protein regulation during development.

Gene expression fingerprinting for human hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) or Osler-Weber-Rendu syndrome is an autosomal dominant vascular disorder characterized by telangiectases and internal arteriovenous malformations. It is caused by mutations in elements of the transforming growth factor-beta (TGF-beta) receptor complex: endoglin, a co-receptor, responsible for HHT1, or ALK1 (activin receptor-like kinase 1), a type I receptor leading to HHT2. Recently, we have established cultures of HHT endothelial cells, primary targets of the disease. These cells showed deficient TGF-beta signaling and angiogenesis, representing a useful human model to study the molecular mechanism of this disease. To understand the pathogenic mechanism underlying HHT, we have used total RNA probes to compare HHT versus non-HHT cells by expression microarrays. This work represents a systematic study to identify target genes affected in HHT cells. Given the similarity of symptoms in HHT1 and HHT2, special interest has been put on the identification of common targets for both HHT types. As a result, 277 downregulated and 63 upregulated genes were identified in HHT versus control cells. These genes are involved in biological processes relevant to the HHT pathology, such as angiogenesis, cytoskeleton, cell migration, proliferation and NO synthesis. The type of misregulated genes found in HHT endothelial cells lead us to propose a model of HHT pathogenesis, opening new perspectives to understand this disorder. Moreover, as the disease is originated by mutations in proteins of the TGF-beta receptor complex, these results may be useful to find out targets of the TGF-beta pathway in endothelium.

Hereditary hemorrhagic telangiectasia, a vascular dysplasia affecting the TGF-beta signaling pathway.

Hereditary hemorrhagic telangiectasia (HHT) is caused by mutations in endoglin (ENG; HHT1) or ACVRL1/ALK1 (HHT2) genes and is an autosomal dominant vascular dysplasia. Clinically, HHT is characterized by epistaxis, telangiectases and arteriovenous malformations in some internal organs such as the lung, brain or liver. Endoglin and ALK1 proteins are specific endothelial receptors of the transforming growth factor (TGF)-beta superfamily that are essential for vascular integrity. Genetic studies in mice and humans have revealed the pivotal role of TGF-beta signaling during angiogenesis. Through binding to the TGF-beta type II receptor, TGF-beta can activate two distinct type I receptors (ALK1 and ALK5) in endothelial cells, each one leading to opposite effects on endothelial cell proliferation and migration. The recent isolation and characterization of circulating endothelial cells from HHT patients has revealed a decreased endoglin expression, impaired ALK1- and ALK5-dependent TGF-beta signaling, disorganized cytoskeleton and the failure to form cord-like structures which may lead to the fragility of small vessels with bleeding characteristic of HHT vascular dysplasia or to disrupted and abnormal angiogenesis after injuries and may explain the clinical symptoms associated with this disease.

Mutation analysis in Spanish patients with hereditary hemorrhagic telangiectasia: deficient endoglin up-regulation in activated monocytes.

BACKGROUND: Mutations in the endoglin (ENG) or ALK1 genes are responsible for hereditary hemorrhagic telangiectasia types 1 and 2 (HHT1 and HHT2), respectively, a dominant vascular dysplasia caused by haploinsufficiency. No formal mutation studies of patients with HHT have been conducted in Spain. METHODS: ENG and ALK1 mutation analyses were carried out in 13 Spanish HHT patients diagnosed according to the Curacao criteria. Because endoglin is up-regulated at the cell surface during the monocyte-macrophage transition, endoglin concentrations in activated monocytes were determined by immunofluorescence flow cytometry in a systematic analysis. As controls, 40 non-HHT volunteers were studied for up-regulation of endoglin in activated monocytes. RESULTS: The mutation responsible for HHT was identified in eight patients belonging to two unrelated families. One of the families has a nonsense mutation in exon 4 (c.511C>T; R171X) of the ENG gene, and accordingly the disorder was identified as HHT1. The other family has a missense mutation affecting exon 8 (c.1120C>T; R374W) of the ALK1 gene, and hence is a HHT2 family. Interestingly, endoglin up-regulation was deficient in activated monocytes of both HHT1 and HHT2 patients compared with controls. By contrast, endoglin up-regulation was age-independent in control donors across a broad range of ages. The extent of endoglin up-regulation in activated monocytes was most diminished in those patients with the most severe symptoms. CONCLUSIONS: Endoglin up-regulation in activated monocytes is impaired in HHT1 and HHT2 patients and is age-dependent in both HHT types. Endoglin expression may predict the clinical severity of HHT.

Characterization of a new subpopulation of mouse CD8alpha+ B220+ dendritic cells endowed with type 1 interferon production capacity and tolerogenic potential.

We describe a new B220+ subpopulation of immaturelike dendritic cells (B220+ DCs) with low levels of expression of major histocompatibility complex (MHC) and costimulatory molecules and markedly reduced T-cell stimulatory potential, located in the thymus, bone marrow, spleen, and lymph nodes. B220+ DCs display ultrastructural characteristics resembling those of human plasmacytoid cells and accordingly produce interferon-alpha after virus stimulation. B220+ DCs acquired a strong antigen-presenting cell capacity on incubation with CpG oligodeoxynucleotides, concomitant with a remarkable up-regulation of MHC and costimulatory molecules and the production of interleukin-12 (IL-12) and IL-10. Importantly, our data suggest that nonstimulated B220+ DCs represent a subset of physiological tolerogenic DCs endowed with the capacity to induce a nonanergic state of T-cell unresponsiveness, involving the differentiation of T regulatory cells capable of suppressing antigen-specific T-cell proliferation. In conclusion, our data support the hypothesis that B220+ DCs represent a lymphoid organ subset of immature DCs with a dual role in the immune system-exerting a tolerogenic function in steady state but differentiating on microbial stimulation into potent antigen-presenting cells with type 1 interferon production capacity.