Mice with a deficiency in CLEC-2 are protected against deep vein thrombosis.

Deep vein thrombosis (DVT) with its major complication, pulmonary embolism, is a global health problem. Mechanisms of DVT remain incompletely understood. Platelets play a role in DVT, but the impact of specific platelet receptors remains unclear. Platelet C-type lectin-like receptor 2 (CLEC-2) is known to maintain the physiological state of blood vasculature under inflammatory conditions. DVT is a thromboinflammatory disorder developing largely as sterile inflammation in the vessel wall. We hypothesized therefore that CLEC-2 might play a role in DVT. Here, using a murine DVT model of inferior vena cava (IVC) stenosis, we demonstrate that mice with general inducible deletion of CLEC-2 or platelet-specific deficiency in CLEC-2 are protected against DVT. No phenotype in the complete stasis model was observed. Transfusion of wild-type platelets into platelet-specific CLEC-2 knockout mice restored thrombosis. Deficiency in CLEC-2 as well as inhibition of podoplanin, a ligand of CLEC-2, was associated with reduced platelet accumulation at the IVC wall after 6 hours of stenosis. Podoplanin was expressed in the IVC wall, where it was localized in the vicinity of the abluminal side of the endothelium. The level of podoplanin in the IVC increased after 48 hours of stenosis to a substantially higher extent in mice with a thrombus vs those without a thrombus. Treatment of animals with an anti-podoplanin neutralizing antibody resulted in development of smaller thrombi. Thus, we propose a novel mechanism of DVT, whereby CLEC-2 and upregulation of podoplanin expression in the venous wall trigger thrombus formation.

Mast Cells Granular Contents Are Crucial for Deep Vein Thrombosis in Mice.

RATIONALE: Deep vein thrombosis (DVT) and its complication pulmonary embolism have high morbidity reducing quality of life and leading to death. Cellular mechanisms of DVT initiation remain poorly understood. OBJECTIVE: We sought to determine the role of mast cells (MCs) in DVT initiation and validate MCs as a potential target for DVT prevention. METHODS AND RESULTS: In a mouse model, DVT was induced by partial ligation (stenosis) of the inferior vena cava. We demonstrated that 2 strains of mice deficient for MCs were completely protected from DVT. Adoptive transfer of in vitro differentiated MCs restored thrombosis. MCs were present in the venous wall, and the number of granule-containing MCs decreased with thrombosis. Pharmacological depletion of MCs granules or prevention of MC degranulation also reduced DVT. Basal plasma levels of von Willebrand factor and recruitment of platelets to the inferior vena cava wall after DVT induction were reduced in MC-deficient mice. Stenosis application increased plasma levels of soluble P-selectin in wild-type but not in MC-deficient mice. MC releasate elevated ICAM-1 (intercellular adhesion molecule-1) expression on HUVEC (human umbilical vein endothelial cells) in vitro. Topical application of compound 48/80, an MC secretagogue, or histamine, a Weibel-Palade body secretagogue from MCs, potentiated DVT in wild-type mice, and histamine restored thrombosis in MC-deficient animals. CONCLUSIONS: MCs exacerbate DVT likely through endothelial activation and Weibel-Palade body release, which is, at least in part, mediated by histamine. Because MCs do not directly contribute to normal hemostasis, they can be considered potential targets for prevention of DVT in humans.

Neutrophil extracellular traps and inflammasomes cooperatively promote venous thrombosis in mice.

Deep vein thrombosis (DVT) is linked to local inflammation. A role for both neutrophil extracellular traps (NETs) and the assembly of inflammasomes (leading to caspase-1-dependent interleukin-1ß activation) in the development of DVT was recently suggested. However, no link between these 2 processes in the setting of thrombosis has been investigated. Here, we demonstrate that stimulation of neutrophils induced simultaneous formation of NETs and active caspase-1. Caspase-1 was largely associated with NETs, suggesting that secreted active caspase-1 requires NETs as an adhesive surface. NETs and their components, histones, promoted robust caspase-1 activation in platelets with the strongest effect exerted by histones 3/4. Murine DVT thrombi contained active caspase-1, which peaked at 6 hours when compared with 48-hour thrombi. Platelets constituted more than one-half of cells containing active caspase-1 in dissociated thrombi. Using intravital microscopy, we identified colocalized NETs and caspase-1 as well as platelet recruitment at the site of thrombosis. Pharmacological inhibition of caspase-1 strongly reduced DVT in mice, and thrombi that still formed contained no citrullinated histone 3, a marker of NETs. Taken together, these data demonstrate a cross-talk between NETs and inflammasomes both in vitro and in the DVT setting. This may be an important mechanism supporting thrombosis in veins.

A Transposon Screen Identifies Loss of Primary Cilia as a Mechanism of Resistance to SMO Inhibitors.

Drug resistance poses a great challenge to targeted cancer therapies. In Hedgehog pathway-dependent cancers, the scope of mechanisms enabling resistance to SMO inhibitors is not known. Here, we performed a transposon mutagenesis screen in medulloblastoma and identified multiple modes of resistance. Surprisingly, mutations in ciliogenesis genes represent a frequent cause of resistance, and patient datasets indicate that cilia loss constitutes a clinically relevant category of resistance. Conventionally, primary cilia are thought to enable oncogenic Hedgehog signaling. Paradoxically, we find that cilia loss protects tumor cells from susceptibility to SMO inhibitors and maintains a "persister" state that depends on continuous low output of the Hedgehog program. Persister cells can serve as a reservoir for further tumor evolution, as additional alterations synergize with cilia loss to generate aggressive recurrent tumors. Together, our findings reveal patterns of resistance and provide mechanistic insights for the role of cilia in tumor evolution and drug resistance.Significance: Using a transposon screen and clinical datasets, we identified mutations in ciliogenesis genes as a new class of resistance to SMO inhibitors. Mechanistically, cilia-mutant tumors can either grow slowly in a "persister" state or evolve and progress rapidly in an "aggressive" state. Cancer Discov; 7(12); 1436-49. ©2017 AACR.See related commentary by Goranci-Buzhala et al., p. 1374This article is highlighted in the In This Issue feature, p. 1355.

Corrigendum: Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

This corrects the article DOI: 10.1038/ncomms11208.

Large-scale production of megakaryocytes from human pluripotent stem cells by chemically defined forward programming.

The production of megakaryocytes (MKs)--the precursors of blood platelets--from human pluripotent stem cells (hPSCs) offers exciting clinical opportunities for transfusion medicine. Here we describe an original approach for the large-scale generation of MKs in chemically defined conditions using a forward programming strategy relying on the concurrent exogenous expression of three transcription factors: GATA1, FLI1 and TAL1. The forward programmed MKs proliferate and differentiate in culture for several months with MK purity over 90% reaching up to 2 × 10(5) mature MKs per input hPSC. Functional platelets are generated throughout the culture allowing the prospective collection of several transfusion units from as few as 1 million starting hPSCs. The high cell purity and yield achieved by MK forward programming, combined with efficient cryopreservation and good manufacturing practice (GMP)-compatible culture, make this approach eminently suitable to both in vitro production of platelets for transfusion and basic research in MK and platelet biology.

RAS/MAPK Activation Drives Resistance to Smo Inhibition, Metastasis, and Tumor Evolution in Shh Pathway-Dependent Tumors.

Aberrant Shh signaling promotes tumor growth in diverse cancers. The importance of Shh signaling is particularly evident in medulloblastoma and basal cell carcinoma (BCC), where inhibitors targeting the Shh pathway component Smoothened (Smo) show great therapeutic promise. However, the emergence of drug resistance limits long-term efficacy, and the mechanisms of resistance remain poorly understood. Using new medulloblastoma models, we identify two distinct paradigms of resistance to Smo inhibition. Sufu mutations lead to maintenance of the Shh pathway in the presence of Smo inhibitors. Alternatively activation of the RAS-MAPK pathway circumvents Shh pathway dependency, drives tumor growth, and enhances metastatic behavior. Strikingly, in BCC patients treated with Smo inhibitor, squamous cell cancers with RAS/MAPK activation emerged from the antecedent BCC tumors. Together, these findings reveal a critical role of the RAS-MAPK pathway in drug resistance and tumor evolution of Shh pathway-dependent tumors.

Neural protein Olig2 acts upstream of the transcriptional regulator Sim1 to specify diencephalic dopaminergic neurons.

Neural factors are expressed in neural progenitors and regulate neurogenesis and gliogenesis. Recent studies suggested that these factors are also involved in determining specific neuronal fates by regulating the expression of their target genes, thereby creating transcriptional codes for neuronal subtype specification. In the present study, we show that in the zebrafish the neural gene Olig2 and the transcriptional regulator Sim1 are co-expressed in a subset of diencephalic progenitors destined towards the dopaminergic (DA) neuronal fate. While sim1 mRNA is also detected in mature DA neurons, the expression of olig2 is extinguished prior to terminal DA differentiation. Loss of function of either Olig2 or Sim1 leads to impaired DA development. Finally, Olig2 regulates the expression of Sim1 and gain of function of Sim1 rescues the deficits in DA differentiation caused by targeted knockdown of Olig2. Our findings demonstrate for the first time that commitment of basal diencephalic DA neurons is regulated by the combined action of the neural protein Olig2 and its downstream neuronal specific effector Sim1.