Small molecule inhibitors, such as imatinib, are effective therapies for tyrosine kinase fusions BCR-ABL–TEL-PDGFβR–mediated human leukemias, but resistance may develop. The unique fusion junctions of these molecules are attractive candidates for molecularly targeted therapeutic intervention using RNA interference (RNAi), which is mediated by small interfering RNA (siRNA). We developed a retroviral system for stable expression of siRNA directed to the unique fusion junction sequence of TEL-PDGFβR in transformed hematopoietic cells. Stable expression of the siRNA resulted in approximately 90% inhibition of TEL-PDGFβR expression and its downstream effectors, including PI3K and mammalian target of rapamycin (mTOR). Expression of TEL-PDGFβR–specific siRNA (TPsiRNA) significantly attenuated the proliferation of TEL-PDGFβR–transformed Ba/F3 cells or disease latency and penetrance in mice induced by intravenous injection of these Ba/F3 cells. Although a 90% reduction in TEL-PDGFβR expression was insufficient to induce cell death, stable siRNA expression sensitized transformed cells to the PDGFβR inhibitor imatinib or to the mTOR inhibitor rapamycin. TPsiRNA also inhibited an imatinib-resistant TEL-PDGFβR mutant, and the inhibition was enhanced by siRNA in combination with PKC412, another PDGFβR inhibitor. Although siRNA delivery in vivo is a challenging problem, stable expression of siRNA, which targets oncogenic fusion genes, may potentiate the effects of conventional therapy for hematologic malignancies.
Jing Chen, Nathan R. Wall, Kerry Kocher, Nicole Duclos, Doriano Fabbro, Donna Neuberg, James D. Griffin, Yang Shi, D. Gary Gilliland
The leukocyte integrin αMβ2/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-αMβ2 interaction in vivo, gene-targeted mice were generated in which the αMβ2-binding motif within the fibrinogen γ chain (N390RLSIGE396) was converted to a series of alanine residues. Mice carrying the Fibγ390–396A allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support αMβ2-mediated adhesion of primary neutrophils, macrophages, and αMβ2-expressing cell lines. The elimination of the αMβ2-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for αMβ2, integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.
Matthew J. Flick, XinLi Du, David P. Witte, Markéta Jiroušková, Dmitry A. Soloviev, Steven J. Busuttil, Edward F. Plow, Jay L. Degen
Hypoferremia is a common response to systemic infections or generalized inflammatory disorders. In mouse models, the development of hypoferremia during inflammation requires hepcidin, an iron regulatory peptide hormone produced in the liver, but the inflammatory signals that regulate hepcidin are largely unknown. Our studies in human liver cell cultures, mice, and human volunteers indicate that IL-6 is the necessary and sufficient cytokine for the induction of hepcidin during inflammation and that the IL-6–hepcidin axis is responsible for the hypoferremia of inflammation.
Elizabeta Nemeth, Seth Rivera, Victoria Gabayan, Charlotte Keller, Sarah Taudorf, Bente K. Pedersen, Tomas Ganz
The pituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide of the vasoactive intestinal peptide/secretin/glucagon superfamily. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from severe mental retardation and have a bleeding tendency with mild thrombocytopenia, and their fibroblasts show increased PACAP mRNA levels. The PACAP receptor (vasoactive intestinal peptide/pituitary adenylate cyclase–activating peptide receptor 1 [VPAC1]) in platelets and fibroblasts is coupled to adenylyl cyclase activation. Accordingly, we found increased basal cAMP levels in patients’ platelets and fibroblasts, providing a basis for the reduced platelet aggregation in these patients. Megakaryocyte-specific transgenic overexpression of PACAP in mice correspondingly increased PACAP release from platelets, reduced platelet activation, and prolonged the tail bleeding time. In contrast, the PACAP antagonist PACAP(6-38) or a monoclonal PACAP antibody enhanced the collagen-induced aggregation of normal human platelets, and in PACAP knockout mice, an increased platelet sensitivity toward collagen was found. Thus, we found that PACAP modulates platelet function and demonstrated what we believe to be the first hemostatic defect associated with PACAP overexpression; our study suggests the therapeutic potential to manage arterial thrombosis or bleeding by administration of PACAP mimetics or inhibitors, respectively.
Kathleen Freson, Hitoshi Hashimoto, Chantal Thys, Christine Wittevrongel, Sophie Danloy, Yoshiko Morita, Norihito Shintani, Yoshiaki Tomiyama, Jos Vermylen, Marc F. Hoylaerts, Akemichi Baba, Chris Van Geet
The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-β1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-β1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-β1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-β1. Active TGF-β1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-β1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti–TGF-β1 antibodies. These results show, for the first time to our knowledge, that TGF-β1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.
Medhat Shehata, Josef D. Schwarzmeier, Martin Hilgarth, Rainer Hubmann, Markus Duechler, Heinz Gisslinger
A central tenet of fibrinolysis is that tissue plasminogen activator–dependent (t-PA– dependent) conversion of plasminogen to active plasmin requires the presence of the cofactor/substrate fibrin. However, previous in vitro studies have suggested that the endothelial cell surface protein annexin II can stimulate t-PA–mediated plasminogen activation in the complete absence of fibrin. Here, homozygous annexin II–null mice displayed deposition of fibrin in the microvasculature and incomplete clearance of injury-induced arterial thrombi. While these animals demonstrated normal lysis of a fibrin-containing plasma clot, t-PA–dependent plasmin generation at the endothelial cell surface was markedly deficient. Directed migration of annexin II–null endothelial cells through fibrin and collagen lattices in vitro was also reduced, and an annexin II peptide mimicking sequences necessary for t-PA binding blocked endothelial cell invasion of Matrigel implants in wild-type mice. In addition, annexin II–deficient mice displayed markedly diminished neovascularization of fibroblast growth factor–stimulated cornea and of oxygen-primed neonatal retina. Capillary sprouting from annexin II–deficient aortic ring explants was markedly reduced in association with severe impairment of activation of metalloproteinase-9 and -13. These data establish annexin II as a regulator of cell surface plasmin generation and reveal that impaired endothelial cell fibrinolytic activity constitutes a barrier to effective neoangiogenesis.
Qi Ling, Andrew T. Jacovina, Arunkumar Deora, Maria Febbraio, Ronit Simantov, Roy L. Silverstein, Barbara Hempstead, Willie H. Mark, Katherine A. Hajjar
Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.
Myriam Alcalay, Natalia Meani, Vania Gelmetti, Anna Fantozzi, Marta Fagioli, Annette Orleth, Daniela Riganelli, Carla Sebastiani, Enrico Cappelli, Cristina Casciari, Maria Teresa Sciurpi, Angela Rosa Mariano, Simone Paolo Minardi, Lucilla Luzi, Heiko Muller, Pier Paolo Di Fiore, Guido Frosina, Pier Giuseppe Pelicci
Basic and clinical observations suggest that thrombosis and inflammation are closely related. Here we addressed the role played by TNF-α in thrombus formation and growth in an in vivo mouse model. Using intravital microscopy, we show that systemic administration of TNF-α at doses found in sepsis transiently inhibits thrombus formation and delays arterial occlusion upon vascular injury. These results were reflected in a prolonged bleeding time. Platelets isolated from the TNF-α–treated mice showed a marked decrease in fibrinogen binding and P-selectin expression as well as reduced platelet aggregation in response to various agonists. In contrast, in vitro treatment of platelets with TNF-α did not affect their function. TNF receptor 1– and 2–deficient mice exhibited normal thrombogenesis in the presence of TNF-α. Additionally, the inhibitory effect of TNF-α was lost either after treatment with NG-monomethyl-L-arginine, an inhibitor of NO production, or in mice deficient for iNOS. These results indicate that under inflammatory conditions, when leukocytes need free passage to transmigrate into tissues, TNF-α decreases platelet activation and inhibits thrombi formation. This effect is not exerted directly on platelets but mediated through the rapid generation of NO in the vessel wall.
Beatrice Cambien, Wolfgang Bergmeier, Simin Saffaripour, Heather A. Mitchell, Denisa D. Wagner
Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow–derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP→WT, A2A-KO→WT, or WT→WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO→WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT→WT chimera. ATL146e reduced the induction of IL-6, IL-1β, IL-1ra, and TGF-α mRNA in WT→WT mice but not in A2A-KO→WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells.
Yuan-Ji Day, Liping Huang, Marcia J. McDuffie, Diane L. Rosin, Hong Ye, Jiang-Fan Chen, Michael A. Schwarzschild, J. Stephen Fink, Joel Linden, Mark D. Okusa
Trousseau described spontaneous, recurrent superficial migratory thrombophlebitis associated with occult cancers, and this was later correlated with disseminated microangiopathy (platelet-rich clots in small blood vessels). Trousseau syndrome often occurs with mucinous adenocarcinomas, which secrete abnormally glycosylated mucins and mucin fragments into the bloodstream. Since carcinoma mucins can have binding sites for selectins, we hypothesized that selectin-mucin interactions might trigger this syndrome. When highly purified, tissue-factor free carcinoma mucin preparations were intravenously injected into mice, platelet-rich microthrombi were rapidly generated. This pathology was markedly diminished in P- or L-selectin–deficient mice. Heparin (an antithrombin-potentiating agent that can also block P- and L-selectin recognition of ligands) ameliorated this platelet aggregation, but had no additional effect in P- or L-selectin–deficient mice. Inhibition of endogenous thrombin by recombinant hirudin also did not block platelet aggregation. Mucins generated platelet aggregation in vitro in hirudinized whole blood, but not in platelet-rich leukocyte-free plasma nor in whole blood from L-selectin–deficient mice. Thus, Trousseau syndrome is likely triggered by interactions of circulating carcinoma mucins with leukocyte L-selectin and platelet P-selectin without requiring accompanying thrombin generation. These data may also explain why heparin ameliorates Trousseau syndrome, while vitamin K antagonists that merely depress thrombin production do not.
Mark Wahrenbrock, Lubor Borsig, Dzung Le, Nissi Varki, Ajit Varki