Lucia Catani

Cyclooxygenase-2 expression is induced during human megakaryopoiesis and characterizes newly formed platelets

Cyclooxygenase (COX)-1 or -2 and prostaglandin (PG) synthases catalyze the formation of various PGs and thromboxane (TX) A2. We have investigated the expression and activity of COX-1 and -2 during human megakaryocytopoiesis. We analyzed megakaryocytes from bone marrow biopsies and derived from thrombopoietin-treated CD34+ hemopoietic progenitor cells in culture. Platelets were obtained from healthy donors and patients with high platelet regeneration because of immune thrombocytopenia or peripheral blood stem cell transplantation. By immunocytochemistry, COX-1 was observed in CD34+ cells and in megakaryocytes at each stage of maturation, whereas COX-2 was induced after 6 days of culture, and remained detectable in mature megakaryocytes. CD34+ cells synthesized more PGE2 than TXB2 (214 ± 50 vs. 30 ± 10 pg/106 cells), whereas the reverse was true in mature megakaryocytes (TXB2 8,440 ± 2,500 vs. PGE2 906 ± 161 pg/106 cells). By immunostaining, COX-2 was observed in <10% of circulating platelets from healthy controls, whereas up to 60% of COX-2-positive platelets were found in patients. A selective COX-2 inhibitor reduced platelet production of both PGE2 and TXB2 to a significantly greater extent in patients than in healthy subjects. Finally, we found that COX-2 and the inducible PGE-synthase were coexpressed in mature megakaryocytes and in platelets. We conclude that both COX-isoforms contribute to prostanoid formation during human megakaryocytopoiesis and that COX-2-derived PGE2 and TXA2 may play an unrecognized role in inflammatory and hemostatic responses in clinical syndromes associated with high platelet turnover.

Mobilization of Bone Marrow-Derived Hematopoietic and Endothelial Stem Cells After Orthotopic Liver Transplantation and Liver Resection

In animals, the bone marrow (BM) is a source of liver‐repopulating cells with therapeutic potential in case of tissue damage. However, the early response of human BM‐derived stem cells (SC) to liver injury is still unknown. Here, we studied 24 patients undergoing orthotopic liver transplantation (OLT) for end‐stage liver disease or hepatocellularcarcinoma, and 13 patients submitted to liver resection. The concentration of circulating BM‐derived SC was determined by phenotypic analysis and clonogenic assays. Moreover, we assessed the serum level of inflammatory and tissue‐specific cytokines. Reverse transcriptase‐polymerase chain reaction and fluorescence‐in situ hybridization were also used to characterize mobilized SC. At baseline, patients showed a significant lower concentration of circulating CD133+, CD34+ SC and clonogenic progenitors (colony‐forming unit cells) than healthy controls. However, the time‐course evaluation of peripheral blood cells after OLT demonstrated the significant early mobilization of multiple subsets of hematopoietic and endothelial stem/progenitor cells. Cytogenetic and molecular analyses of CD34+ cells showed the host origin of mobilized SC and the expression of transcripts for GATA‐4, cytokeratin 19, and α‐fetoprotein hepatocyte markers. In contrast with OLT, only total circulating CD34+ cells significantly increased after liver resection. Mobilization of BM cells after OLT or liver surgery was associated with increased serum levels of granulocyte‐colony stimulating factor, interleukin‐6, stem cell factor, hepatocyte growth factor, and vascular endothelial growth factor. In summary, we demonstrate that tissue damage after OLT and liver resection induces increased serum levels of multiple cytokines but only ischemia/reperfusion injury associated with OLT results in the remarkable mobilization of BM stem/progenitor cells.

In vivo and in vitro inhibitory effect of α-interferon on megakaryocyte colony growth in essential thrombocythaemia

Summary Megakaryocyte (MK) colony growth of bone marrow mononuclear non‐adherent cells was evaluated in 28 patients with essential thrombocythaemia (ET) and in 26 normal controls. The number of MK‐colony forming units (CFU‐MK per 3 × 105 plated cells) was similar in ET (68 ± 33) and in controls (63 ± 37), independently of bone marrow accessory cells. On the contrary, the size of the MK colonies was significantly (P < 0.01) greater in ET patients. Human recombinant α‐interferon 2a (α‐IFN), administered to 10 patients at a dose of 3 × 106 IU/d s.c. for 11 ± 3 weeks, was capable of inducing a significant (P < 0.01) decrease in the number (from 72 ± 16 to 31 ± 14) and size of bone marrow CFU‐MK, together with a significant reduction of the platelet count (from 1031 ± 325 to 378 ± 75 × 109/1). When added in vitro at time 0 to the culture dishes, α‐IFN inhibited the CFU‐MK growth of both normal and ET bone marrow samples, even at very low concentrations (1 and 10 IU/ml). This study demonstrates that α‐IFN, both in vivo and in vitro, exerts an inhibitory effect on the growth of MK progenitors, which appears to correlate with the clinically documented antiproliferative effect of this cytokine.

A novel model of CCl4-induced cirrhosis with ascites in the mouse

BACKGROUND/AIMS The current approaches to study the molecular mechanisms involved in the pathophysiology of liver diseases often rely on the use of transgenic mice. However, experimental models of decompensated cirrhosis have not been clearly established in mice. Thus, we aimed to set an efficient and well-tolerated protocol to induce cirrhosis in mice able to progress up to the ascitic stage. METHODS C57BL/6N mice received CCl(4) subcutaneously, intraperitoneally or by inhalation. In the latter group, gaseous CCl(4) was administered according to three different schedules: increasing exposure times, twice weekly (traditional protocol; TP), short inhalation cycles, twice or three times weekly. RESULTS Portal hypertension, sodium retention, and ascites developed in all groups between 11 and 15 weeks. Mortality reached 70% in the TP group, but it was only 0-10% with all other protocols. All the inhalation groups had significantly more ascites at sacrifice than those receiving CCl(4) subcutaneously and intraperitoneally. Extensive abdominal adhesions and evidence of enhanced hepatic inflammation, as suggested by the increased gene expression of pro-inflammatory cytokines in liver tissue, were found in the intraperitoneal group, while large granulomas at the injection site and marked neutrophil infiltration of lungs developed in the subcutaneous group. No extra-hepatic damage could be detected in mice inhaling CCl(4). CONCLUSIONS The use of short cycles of CCl(4) inhalation represents a novel, safe, and effective method to induce decompensated cirrhosis in mice. Intraperitoneal CCl(4) leads instead to abdominal adhesions precluding a correct evaluation of ascites, while subcutaneous CCl(4) causes an unwanted systemic inflammatory response.

Long-term follow-up of 386 consecutive patients with essential thrombocythemia: Safety of cytoreductive therapy†

Cytotoxic agents like Hydroxyurea, Busulfan and Interferon‐alpha are to date the most commonly used therapeutic approaches in Essential Thrombocythemia (ET). However, few data on the efficacy and safety of these agents in the long‐term are currently available. We report a retrospective analysis of the long‐term outcome of 386 consecutive ET patients, followed at single Institution for a median follow‐up of 9.5 years (range, 3–28.5). Cytoreductive therapy was administered to 338 patients (88%), obtaining a response in 86% of cases. Forty‐five patients (12%) experienced a thrombosis. Among baseline characteristics, only history of vascular events prior to ET diagnosis predicted a higher incidence of thrombosis. Evolution in acute leukemia/myelofibrosis occurred in 6 (1,5%) and 20 (5%) patients, and was significantly higher in patients receiving sequential cytotoxic agents. Overall survival was 38% at 19 years and was poorer for patients older than 60 years, with higher leukocytes count (>15 × 109/L), hypertension and mellitus diabetes at ET diagnosis and for patients experiencing a thrombotic event during follow‐up. Cytoreductive therapy was effective in decreasing platelet number with negligible toxicity; however, thrombocytosis control did not reduce the incidence of thrombosis and, for patients who received sequential therapies, the probability of disease evolution was higher and survival was poorer. Am. J. Hematol. 2009.

Impaired survival of bone marrow GPIIb/IIIa+ megakaryocytic cells as an additional pathogenetic mechanism of HIV-1-related thrombocytopenia

Glycoproteic (GP) IIb/IIIa+ megakaryocytic cells were purified from the bone marrow (BM) of 15 HIV‐1 seropositive thrombocytopenic patients, eight HIV‐1 seronegative patients affected by immune thrombocytopenic purpura (ITP) and 14 HIV‐1 seronegative normal donors. The presence of apoptosis was evaluated in freshly isolated GPIIb/IIIa+ cells by flow cytometry after propidium iodide staining and electron microscopy. GPIIb/IIIa+ cells from HIV‐1 seropositive thrombocytopenic patients showed a significant ( P < 0.001) increase of apoptosis with respect to both HIV‐1 seronegative ITP patients and normal donors. Moreover, the degree of apoptosis in bone marrow GPIIb/IIIa+ cells purified from HIV‐1 seropositive thrombocytopenic patients was inversely ( P < 0.01) related to the count of circulating platelets, whereas it did not show any significant correlation with the absolute number of circulating CD4 T cells, the CD4/CD8 ratio or the presence of proviral gag DNA sequences. Therefore neither an advanced stage of HIV‐1 disease nor a direct infection with HIV‐1 seems to play a primary role in the impaired survival of BM GPIIb/IIIa+ megakaryocytic cells. These findings strengthen the notion that, besides the immune targeting of peripheral platelets, an impairment of the bone marrow megakaryocyte compartment may also contribute to the pathogenesis of HIV‐1 related thrombocytopenia.

Stem cell mobilization and collection in patients with liver cirrhosis

Background  Bone marrow‐derived stem cells (BMSC) and granulocyte colony‐stimulating factor (G‐CSF) have been proved to contribute to tissue regeneration after liver injury.

Hypercoagulability during L-asparaginase treatment: the effect of antithrombin III supplementation in vivo.

To evaluate the occurrence of hypercoagulability during treatment with l‐asparaginase (l‐ase), thrombin‐antithrombin complex (TAT) and d‐dimer levels in plasma were serially measured in 15 consecutive adult patients with acute lymphoblastic leukaemia or lymphoblastic lymphoma who had recently completed a chemotherapy cycle with cytosine arabinoside and methotrexate. The first eight patients (group A) received i.v. l‐ase alone (20000 U/m2 on alternate days over 10 d); the last seven patients (group B) received, in addition to l‐ase, bolus injection of antithrombin concentrate (2000 U) on alternate days for a total of six administrations, beginning with the second l‐ase infusion. Increased levels of TAT (P<0·05) and d‐dimer (P<0·01) were observed prior to l‐ase, possibly related to inflammation and cytolysis secondary to previous chemotherapy. In patients treated with l‐ase alone, further elevation of TAT (P<0·05) and persistence of increased d‐dimer were observed, associated with marked reduction of the anticoagulant activities of protein C, protein S and antithrombin III. At variance, in patients receiving antithrombin III supplementation there was no increase of TAT and a normalization of d‐dimer levels occurred during l‐ase treatment. In these patients, mean plasma antithrombin III activity was maintained at levels higher than 70% of normal throughout the treatment. The rate of decline of fibrinogen, factor IX, protein C and protein S was unaffected by antithrombin III supplementation, indicating that hypercoagulability has little if any relevance for the reduction of coagulation factors and inhibitors induced by l‐ase treatment. The usefulness of antithrombin III concentrates in preventing thromboembolic complications in patients submitted to l‐ase treatment remains to be determined.

The Kinetic Status of Hematopoietic Stem Cell Subpopulations Underlies a Differential Expression of Genes Involved in Self‐Renewal, Commitment, and Engraftment

The gene expression profile of CD34− hematopoietic stem cells (HSCs) and the correlations with their biological properties are still poorly understood. To address this issue, we used the DNA microarray technology to compare the expression profiles of different peripheral blood hemopoietic stem/progenitor cell subsets, lineage‐negative (Lin−) CD34−, Lin−CD34+, and Lin+CD34+ cells. The analysis of gene categories differentially expressed shows that the expression of CD34 is associated with cell cycle entry and metabolic activation, such as DNA, RNA, and protein synthesis. Moreover, the significant upregulation in CD34− cells of pathways inhibiting HSC proliferation induces a strong differential expression of cyclins, cyclin‐dependent kinases (CDKs), CDK inhibitors, and growth‐arrest genes. According to the expression of their receptors and transducers, interleukin (IL)‐10 and IL‐17 showed an inhibitory effect on the clonogenic activity of CD34− cells. Conversely, CD34+ cells were sensitive to the mitogenic stimulus of thrombopoietin. Furthermore, CD34− cells express preferentially genes related to neural, epithelial, and muscle differentiation. The analysis of transcription factor expression shows that the CD34 induction results in the upregulation of genes related to self‐renewal and lineage commitment. The preferential expression in CD34+ cells of genes supporting the HSC mobilization and homing to the bone marrow, such as chemokine receptors and integrins, gives the molecular basis for the higher engraftment capacity of CD34+ cells. Thus, the different kinetic status of CD34− and CD34+ cells, detailed by molecular and functional analysis, significantly influences their biological behavior.

PMA-induced megakaryocytic differentiation of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level.

We investigated whether members of the protein kinase C (PKC) family of enzymes could play a role in the nuclear events involved in megakaryocytic differentiation. PKC activity was analysed using a serine substituted specific peptide, which enabled us to evaluate the whole catalytic activity in the pluripotent haemopoietic HEL cell line treated with 10−7 m phorbol myristate acetate (PMA) or haemin. In parallel, the subcellular distribution of different PKC isoforms (α, βI, βII, γ, δ, ε, θ, η, ζ) was evaluated by Western blot. PKC catalytic activity in the nuclei of HEL cells showed a peak after acute (30 min) treatment with PMA, followed by a significant (P < 0.05) decline after prolonged exposure (72 h) to the same agonist, when most HEL cells had acquired a differentiated megakaryocytic phenotype. Western blot analysis of nuclear lysates consistently showed a significant increase of PKC‐α, ‐βI, ‐ε, ‐θ and ‐ζ isoforms after 30 min of PMA treatment, followed by a drastic decline of all but PKC‐ζ isoforms. Moreover, PKC‐δ appeared in HEL nuclei only after 72 h of exposure to PMA. On the other hand, neither the catalytic activity nor the immunoreactivity of the different PKC isoforms showed remarkable variations in nuclei of HEL cells induced to differentiate along the erythroid lineage with 10−7 m haemin.