Giorgio Zauli

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.

microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early survival

Aberrant expression of microRNAs (miRNAs) has been associated with clinical outcome in patients with chronic lymphocytic leukemia (CLL). To identify a powerful and easily assessable miRNA bio-marker of prognosis and survival, we performed quantitative reverse-transcription polymerase chain reaction (qRT-PCR) profiling in 104 CLL patients with a well-defined chromosome 17p status, and we validated our findings with miRNA microarray data from an independent cohort of 80 patients. We found that miR-15a, miR-21, miR-34a, miR-155, and miR-181b were differentially expressed between CLLs with chromosome 17p deletion and CLLs with normal 17p and normal karyotype, and that miR-181b was down-regulated in therapy-refractory cases. miR-21 expression levels were significantly higher in patients with poor prognosis and predicted overall survival (OS), and miR-181b expression levels significantly predicted treatment-free survival. We developed a 21FK score (miR-21 qRT-PCR, fluorescence in situ hybridization, Karyotype) to stratify patients according to OS and found that patients with a low score had a significantly longer OS time. When we evaluated the relative power of the 21FK score with the most used prognostic factors, the score was the most significant in both CLL cohorts. We conclude that the 21FK score represents a useful tool for distinguishing between good-prognosis and poor-prognosis CLL patients.

Systemic Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Delivery Shows Antiatherosclerotic Activity in Apolipoprotein E-Null Diabetic Mice

Background— Although in vitro studies have suggested that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) might be involved in vascular biology, its potential role in the pathogenesis and/or treatment of atherosclerosis has not been investigated. Methods and Results— Both recombinant human TRAIL and an adeno-associated virus vector expressing human TRAIL were used to deliver TRAIL in apolipoprotein E (apoE)–null mice in which diabetes mellitus was induced by destruction of islet cells with streptozotocin. Diabetes in apoE-null mice was associated with a significant increase in atherosclerotic plaque area and complexity in the aorta as assessed by a marked increase in interstitial collagen, cellular proliferation, and macrophage infiltration and a focal loss of endothelial coverage. Repeated intraperitoneal injections of recombinant human TRAIL and a single intravenous injection of adeno-associated virus–human TRAIL significantly attenuated the development of atherosclerotic plaques in apoE-null animals. TRAIL also markedly affected the cellular composition of plaque lesions by inducing apoptosis of infiltrating macrophages and increasing the vascular smooth muscle cell content. Moreover, TRAIL promoted the in vitro migration of cultured human aortic vascular smooth muscle cells but not of monocytes or macrophages. Conversely, TRAIL selectively induced apoptosis of human cultured macrophages but not of vascular smooth muscle cells. Conclusions— Overall, data from the present study indicate that atherosclerosis in diabetic apoE-null mice is ameliorated by systemic TRAIL administration and that adeno-associated virus–mediated TRAIL gene delivery might represent an innovative method for the therapy of diabetic vascular diseases.

Effect of antibody to HIV-1 Tat protein on viral replication in vitro and progression of HIV-1 disease in vivo

In HIV-1-infected cell cultures, a relatively low concentration (5 micrograms/ml) of monoclonal antibody (mAb) against HIV-1-transactivating Tat protein was an efficient inhibitor of HIV-1 replication both in HIV-1(IIIB)-infected Jurkat cell and peripheral blood mononuclear cell (PBMC) cultures and significantly reduced the expression of a Tat-responsive CAT-reporter construct in HIV-1(IIIB)-infected Jurkat cells. Anti-Tat mAb also caused a significant reduction and a consistent delay in HIV-1 replication when added to PBMCs from HIV-1-infected patients cocultivated with phytohemagglutinin (PHA)-stimulated normal PBMCs. These data indicate that an autocrine-paracrine loop sustained by extracellular Tat protein, which is actively released by HIV-1-infected cells, may affect HIV-1 replication in cell cultures in vitro. An inverse relationship between natural anti-Tat antibody levels and p24 antigenemia was demonstrated by retrospective analysis of serial serum samples obtained from 10 HIV-1-seropositive hemophiliac patients followed over a 7-9-year period. This datum points to a possible influence of anti-Tat antibody on the progression of HIV-1 disease in vivo. These findings have strong implications for Tat protein as a possible target for specific immunotherapy in HIV-1-infected patients.

Emerging Non-Apoptotic Functions of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)/Apo2L

Tumor necrosis factor (TNF) is a cytokine that mediates tumor necrosis. To date, 20 different members of the TNF super‐family and 21 different receptors have been identified. All ligands of the TNF super‐family have been found to activate transcription factor NF‐κB and c‐Jun kinase. Members of this family have diverse biological effects, including induction of apoptosis, promotion of cell survival, and regulation of the immune system and hematopoiesis. The current review focuses on the biological effects of TNF‐related apoptosis‐inducing ligand (TRAIL), a TNF super‐family member which, a few years ago, generated considerable enthusiasm for its anticancer activity, not accompanied by general toxicity in most normal tissues and organs.

TRAIL promotes the survival, migration and proliferation of vascular smooth muscle cells

AbstractHuman and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor-α + interleukin-1β + interferon-γ or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.

Recent Advances in the Therapeutic Perspectives of Nutlin-3

Nutlin-3 is a small molecule inhibitor of the MDM2/p53 interaction, which leads to the non-genotoxic p53 stabilization, activation of cell cycle arrest and apoptosis pathways. A series of recent studies have strengthened the concept that selective, non-genotoxic p53 activation by Nutlin-3 might represent an alternative to the current cytotoxic chemotherapy, in particular for pediatric tumors and for hematological malignancies, which retain a high percentage of p53(wild-type) status at diagnosis. Like most other drugs employed in cancer therapy, it will be unlikely that Nutlin-3 will be used as a monotherapy. In this respect, Nutlin-3 shows a synergistic cytotoxic effect when used in combination with innovative drugs, such as TRAIL or bortozemib. Although Nutlin-3 is currently in phase I clinical trial for the treatment of retinoblastoma, its effects on normal tissues and cell types remain largely to be determined and will require further investigation in the future years.

Antiangiogenic Activity of the MDM2 Antagonist Nutlin-3

Nutlin-3, a nongenotoxic activator of the p53 pathway, dose-dependently (range 0.1 to 10 &mgr;mol/L) inhibited the formation of capillaries in an in vivo matrigel assay, as well as the formation of capillary-like structures in an in vitro coculture system composed of endothelial cells surrounded by fibroblasts. In contrast to the chemotherapeutic agent doxorubicin, nutlin-3 showed no induction of apoptosis in vitro either in the cocultures or in isolated vascular endothelial cells, even when used at the highest concentration (10 &mgr;mol/L). However, treatment with pharmacological inhibitors of the nuclear factor &kgr;B and phosphatidylinositol 3-kinase/Akt pathways sensitized endothelial cells to nutlin-3–induced apoptosis. Although nutlin-3 and doxorubicin induced a comparable p53 accumulation in endothelial cells, nutlin-3 was significantly more efficient than doxorubicin in upregulating the p53 target genes CDKN1A/p21, MDM2, and GDF-15, as well as in inhibiting cell cycle progression. However, the predominant in vitro effect of nutlin-3 was its strong antimigratory activity observed at concentrations significantly lower (0.1 &mgr;mol/L) than those required to inhibit endothelial cell cycle progression. Taken together, our data suggest that the antiangiogenic activity of nutlin-3 observed in vivo was mainly attributable to inhibition of endothelial cell migration, to some extent attributable to cell cycle arrest, and to a lesser extent attributable to induction of apoptosis.

Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (TRAIL) Sequentially Upregulates Nitric Oxide and Prostanoid Production in Primary Human Endothelial Cells

&NA; —Endothelial cells express tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) receptors, but the function of TRAIL in endothelial cells is not completely understood. We explored the role of TRAIL in regulation of key intracellular signal pathways in endothelial cells. The addition of TRAIL to primary human endothelial cells increased phosphorylation of endothelial nitric oxide synthase (eNOS), NOS activity, and NO synthesis. Moreover, TRAIL induced cell migration and cytoskeleton reorganization in an NO‐dependent manner. TRAIL did not activate the NF‐&kgr;B or COX‐2 pathways in endothelial cells. Instead, TRAIL increased prostanoid production (PGE2=PGI2>TXA2), which was preferentially inhibited by the COX‐1 inhibitor SC‐560. Because NO and prostanoids play a crucial role in the state of blood vessel vasodilatation and angiogenesis, our data suggest that TRAIL might play an important role in endothelial cell function. (Circ Res. 2003;92:732–740.)

Human Bone Marrow Mesenchymal Stem Cells Display Anti-Cancer Activity in SCID Mice Bearing Disseminated Non-Hodgkin's Lymphoma Xenografts

Background Although multimodality treatment can induce high rate of remission in many subtypes of non-Hodgkins lymphoma (NHL), significant proportions of patients relapse with incurable disease. The effect of human bone marrow (BM) mesenchymal stem cells (MSC) on tumor cell growth is controversial, and no specific information is available on the effect of BM-MSC on NHL. Methodology/Principal Findings The effect of BM-MSC was analyzed in two in vivo models of disseminated non-Hodgkins lymphomas with an indolent (EBV− Burkitt-type BJAB, median survival = 46 days) and an aggressive (EBV+ B lymphoblastoid SKW6.4, median survival = 27 days) behavior in nude-SCID mice. Intra-peritoneal (i.p.) injection of MSC (4 days after i.p. injection of lymphoma cells) significantly increased the overall survival at an optimal MSC∶lymphoma ratio of 1∶10 in both xenograft models (BJAB+MSC, median survival = 58.5 days; SKW6.4+MSC, median survival = 40 days). Upon MSC injection, i.p. tumor masses developed more slowly and, at the histopathological observation, exhibited a massive stromal infiltration coupled to extensive intra-tumor necrosis. In in vitro experiments, we found that: i) MSC/lymphoma co-cultures modestly affected lymphoma cell survival and were characterized by increased release of pro-angiogenic cytokines with respect to the MSC, or lymphoma, cultures; ii) MSC induce the migration of endothelial cells in transwell assays, but promoted endothelial cell apoptosis in direct MSC/endothelial cell co-cultures. Conclusions/Significance Our data demonstrate that BM-MSC exhibit anti-lymphoma activity in two distinct xenograft SCID mouse models of disseminated NHL.