Jack D. Stopa, Donna Neuberg, Maneka Puligandla, Bruce Furie, Robert Flaumenhaft, Jeffrey I. Zwicker
In patients with sickle cell disease (SCD), the polymerization of intraerythrocytic hemoglobin S promotes downstream vaso-occlusive events in the microvasculature. While vaso-occlusion is known to occur in the lung, often in the context of systemic vaso-occlusive crisis and the acute chest syndrome, the pathophysiological mechanisms that incite lung injury are unknown. We used intravital microscopy of the lung in transgenic humanized SCD mice to monitor acute vaso-occlusive events following an acute dose of systemic lipopolysaccharide sufficient to trigger events in SCD but not control mice. We observed cellular microembolism of precapillary pulmonary arteriolar bottlenecks by neutrophil-platelet aggregates. Blood from SCD patients was next studied under flow in an in vitro microfluidic system. Similar to the pulmonary circulation, circulating platelets nucleated around arrested neutrophils, translating to a greater number and duration of neutrophil-platelet interactions compared with normal human blood. Inhibition of platelet P-selectin with function-blocking antibody attenuated the neutrophil-platelet interactions in SCD patient blood in vitro and resolved pulmonary arteriole microembolism in SCD mice in vivo. These results establish the relevance of neutrophil-platelet aggregate formation in lung arterioles in promoting lung vaso-occlusion in SCD and highlight the therapeutic potential of targeting platelet adhesion molecules to prevent acute chest syndrome.
Margaret F. Bennewitz, Maritza A. Jimenez, Ravi Vats, Egemen Tutuncuoglu, Jude Jonassaint, Gregory J. Kato, Mark T. Gladwin, Prithu Sundd
Monique A.J. van Eijndhoven, Josée M. Zijlstra, Nils J. Groenewegen, Esther E.E. Drees, Stuart van Niele, S. Rubina Baglio, Danijela Koppers-Lalic, Hans van der Voorn, Sten F.W.M. Libregts, Marca H.M. Wauben, Renee X. de Menezes, Jan R.T. van Weering, Rienk Nieuwland, Lydia Visser, Anke van den Berg, Daphne de Jong, D. Michiel Pegtel
von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein’s multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine
Alexandre Kauskot, Sonia Poirault-Chassac, Frédéric Adam, Vincent Muczynski, Gabriel Aymé, Caterina Casari, Jean-Claude Bordet, Christelle Soukaseum, Chantal Rothschild, Valérie Proulle, Audrey Pietrzyk-Nivau, Eliane Berrou, Olivier D. Christophe, Jean-Philippe Rosa, Peter J. Lenting, Marijke Bryckaert, Cécile V. Denis, Dominique Baruch
Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645–1648) in the design of B-domain–deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector–based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.
Joshua I. Siner, Benjamin J. Samelson-Jones, Julie M. Crudele, Robert A. French, Benjamin J. Lee, Shanzhen Zhou, Elizabeth Merricks, Robin Raymer, Timothy C. Nichols, Rodney M. Camire, Valder R. Arruda
Transgenic expression of key myelosupportive human cytokines in immune-deficient mice corrects for the lack of cross-species activities of stem cell factor (SCF), IL-3, and GM-CSF. When engrafted with human umbilical cord blood (UCB), these triple-transgenic mice produce BM and spleen grafts with much higher myeloid composition, relative to nontransgenic controls. Shortly after engraftment with UCB, these mice develop a severe, fatal macrophage activation syndrome (MAS) characterized by a progressive drop in rbc numbers, increased reticulocyte counts, decreased rbc half-life, progressive cytopenias, and evidence of chronic inflammation, including elevated human IL-6. The BM becomes strikingly hypocellular, and spleens are significantly enlarged with evidence of extramedullary hematopoiesis and activated macrophages engaged in hemophagocytosis. This manifestation of MAS does not respond to lymphocyte-suppressive therapies such as steroids, i.v. immunoglobulin, or antibody-mediated ablation of human B and T cells, demonstrating a lymphocyte-independent mechanism of action. In contrast, elimination of human myeloid cells using gemtuzumab ozogamicin (anti-CD33) completely reversed the disease. Additionally, the IL-6R antibody tocilizumab delayed progression and prolonged lifespan. This new model of MAS provides an opportunity for investigation of the mechanisms driving this disease and for the testing of directed therapies in a humanized mouse.
Mark Wunderlich, Courtney Stockman, Mahima Devarajan, Navin Ravishankar, Christina Sexton, Ashish R. Kumar, Benjamin Mizukawa, James C. Mulloy
Metastatic dissemination of cancer cells, which accounts for 90% of cancer mortality, is the ultimate hallmark of malignancy. Growing evidence suggests that blood platelets have a predominant role in tumor metastasis; however, the molecular mechanisms involved remain elusive. Here, we demonstrate that genetic deficiency of integrin α6β1 on platelets markedly decreases experimental and spontaneous lung metastasis. In vitro and in vivo assays reveal that human and mouse platelet α6β1 supports platelet adhesion to various types of cancer cells. Using a knockdown approach, we identified ADAM9 as the major counter receptor of α6β1 on both human and mouse tumor cells. Static and flow-based adhesion assays of platelets binding to DC-9, a recombinant protein covering the disintegrin-cysteine domain of ADAM9, demonstrated that this receptor directly binds to platelet α6β1. In vivo studies showed that the interplay between platelet α6β1 and tumor cell–expressed ADAM9 promotes efficient lung metastasis. The integrin α6β1–dependent platelet-tumor cell interaction induces platelet activation and favors the extravasation process of tumor cells. Finally, we demonstrate that a pharmacological approach targeting α6β1 efficiently impairs tumor metastasis through a platelet-dependent mechanism. Our study reveals a mechanism by which platelets promote tumor metastasis and suggests that integrin α6β1 represents a promising target for antimetastatic therapies.
Elmina Mammadova-Bach, Paola Zigrino, Camille Brucker, Catherine Bourdon, Monique Freund, Adèle De Arcangelis, Scott I. Abrams, Gertaud Orend, Christian Gachet, Pierre Henri Mangin
Ashutosh Lal, Esteban Gomez, Cassandra Calloway
Within the CNS, a dysregulated hemostatic response contributes to both hemorrhagic and ischemic strokes. Tissue factor (TF), the primary initiator of the extrinsic coagulation cascade, plays an essential role in hemostasis and also contributes to thrombosis. Using both genetic and pharmacologic approaches, we characterized the contribution of neuroectodermal (NE) cell TF to the pathophysiology of stroke. We used mice with various levels of TF expression and found that astrocyte TF activity reduced to ~5% of WT levels was still sufficient to maintain hemostasis after hemorrhagic stroke but was also low enough to attenuate inflammation, reduce damage to the blood-brain barrier, and improve outcomes following ischemic stroke. Pharmacologic inhibition of TF during the reperfusion phase of ischemic stroke attenuated neuronal damage, improved behavioral deficit, and prevented mortality of mice. Our data demonstrate that NE cell TF limits bleeding complications associated with the transition from ischemic to hemorrhagic stroke and also contributes to the reperfusion injury after ischemic stroke. The high level of TF expression in the CNS is likely the result of selective pressure to limit intracerebral hemorrhage (ICH) after traumatic brain injury but, in the modern era, poses the additional risk of increased ischemia-reperfusion injury after ischemic stroke.
Shaobin Wang, Brandi Reeves, Erica M. Sparkenbaugh, Janice Russell, Zbigniew Soltys, Hua Zhang, James E. Faber, Nigel S. Key, Daniel Kirchhofer, D. Neil Granger, Nigel Mackman, Rafal Pawlinski
The efficacy of allogeneic hematopoietic stem cell transplantation for hematologic malignancies is limited by the difficulty in suppressing graft-versus-host disease (GVHD) without compromising graft-versus-tumor (GVT) effects. We previously showed that RAS/MEK/ERK signaling depends on memory differentiation in human T cells, which confers susceptibility to selective inhibition of naive T cells. Actually, antineoplastic MEK inhibitors selectively suppress alloreactive T cells, sparing virus-specific T cells in vitro. Here, we show that trametinib, a MEK inhibitor clinically approved for melanoma, suppresses GVHD safely without affecting GVT effects in vivo. Trametinib prolonged survival of GVHD mice and attenuated GVHD symptoms and pathology in the gut and skin. It inhibited ERK1/2 phosphorylation and expansion of donor T cells, sparing Tregs and B cells. Although high-dose trametinib inhibited myeloid cell engraftment, low-dose trametinib suppressed GVHD without severe adverse events. Notably, trametinib facilitated the survival of mice transplanted with allogeneic T cells and P815 tumor cells with no residual P815 cells observed in the livers and spleens, whereas tacrolimus resulted in P815 expansion. These results confirm that trametinib selectively suppresses GVHD-inducing T cells while sparing antitumor T cells in vivo, which makes it a promising candidate for translational studies aimed at preventing or treating GVHD.
Hidekazu Itamura, Takero Shindo, Isao Tawara, Yasushi Kubota, Ryusho Kariya, Seiji Okada, Krishna V. Komanduri, Shinya Kimura
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