Barbara Ghinassi

Characterization of the TGF-β1 signaling abnormalities in the Gata1low mouse model of myelofibrosis.

Primary myelofibrosis (PMF) is characterized by fibrosis, ineffective hematopoiesis in marrow, and hematopoiesis in extramedullary sites and is associated with abnormal megakaryocyte (MK) development and increased transforming growth factor (TGF)-β1 release. To clarify the role of TGF-β1 in the pathogenesis of this disease, the TGF-β1 signaling pathway of marrow and spleen of the Gata1(low) mouse model of myelofibrosis (MF) was profiled and the consequences of inhibition of TGF-β1 signaling on disease manifestations determined. The expression of 20 genes in marrow and 36 genes in spleen of Gata1(low) mice was altered. David-pathway analyses identified alterations of TGF-β1, Hedgehog, and p53 signaling in marrow and spleen and of mammalian target of rapamycin (mTOR) in spleen only and predicted that these alterations would induce consequences consistent with the Gata1(low) phenotype (increased apoptosis and G1 arrest both in marrow and spleen and increased osteoblast differentiation and reduced ubiquitin-mediated proteolysis in marrow only). Inhibition of TGF-β1 signaling normalized the expression of p53-related genes, restoring hematopoiesis and MK development and reducing fibrosis, neovascularization, and osteogenesis in marrow. It also normalized p53/mTOR/Hedgehog-related genes in spleen, reducing extramedullary hematopoiesis. These data identify altered expression signatures of TGF-β1 signaling that may be responsible for MF in Gata1(low) mice and may represent additional targets for therapeutic intervention in PMF.

Biological function and clinical relevance of chromogranin A and derived peptides

Chromogranin A (CgA (CHGA)) is the major soluble protein co-stored and co-released with catecholamines and can function as a pro-hormone by giving rise to several bioactive peptides. This review summarizes the physiological functions, the pathogenic implications, and the recent use of these molecules as biomarkers in several pathological conditions. A thorough literature review of the electronic healthcare databases MEDLINE, from January 1985 to September 2013, was conducted to identify articles and studies concerned with CgA and its processing. The search strategies utilized keywords such as chromogranin A, vasostatins 1 and 2, chromofungin, chromacin, pancreastatin, catestatin, WE14, chromostatin, GE25, parastatin, and serpinin and was supplemented by the screening of references from included papers and review articles. A total of 209 English-language, peer-reviewed original articles or reviews were examined. The analysis of the retrospective literature suggested that CgA and its several bioactive fragments exert a broad spectrum of regulatory activities by influencing the endocrine, the cardiovascular, and the immune systems and by affecting the glucose or calcium homeostasis. As some peptides exert similar effects, but others elicit opposite responses, the regulation of the CgA processing is critical to maintain homeostasis, whereas an unbalanced production of peptides that exert opposing effects can have a pathogenic role in several diseases. These clinical implications entail that CgA and its derived peptides are now used as diagnostic and prognostic markers or to monitor the response to pharmacological intervention not only in endocrine tumors, but also in cardiovascular, inflammatory, and neuropsychiatric diseases.

The dominant negative β isoform of the glucocorticoid receptor is uniquely expressed in erythroid cells expanded from polycythemia vera patients

Glucocorticoid receptor (GR) agonists increase erythropoiesis in vivo and in vitro. To clarify the effect of the dominant negative GRβ isoform (unable to bind STAT-5) on erythropoiesis, erythroblast (EB) expansion cultures of mononuclear cells from 18 healthy (nondiseased) donors (NDs) and 16 patients with polycythemia vera (PV) were studied. GRβ was expressed in all PV EBs but only in EBs from 1 ND. The A3669G polymorphism, which stabilizes GRβ mRNA, had greater frequency in PV (55%; n = 22; P = .0028) and myelofibrosis (35%; n = 20) patients than in NDs (9%; n = 22) or patients with essential thrombocythemia (6%; n = 15). Dexamethasone stimulation of ND cultures increased the number of immature EBs characterized by low GATA1 and β-globin expression, but PV cultures generated great numbers of immature EBs with low levels of GATA1 and β-globin irrespective of dexamethasone stimulation. In ND EBs, STAT-5 was not phosphorylated after dexamethasone and erythropoietin treatment and did not form transcriptionally active complexes with GRα, whereas in PV EBs, STAT-5 was constitutively phosphorylated, but the formation of GR/STAT-5 complexes was prevented by expression of GRβ. These data indicate that GRβ expression and the presence of A3669G likely contribute to development of erythrocytosis in PV and provide a potential target for identification of novel therapeutic agents.

Gata1 expression driven by the alternative HS2 enhancer in the spleen rescues the hematopoietic failure induced by the hypomorphic Gata1low mutation.

Rigorously defined reconstitution assays developed in recent years have allowed recognition of the delicate relationship that exists between hematopoietic stem cells and their niches. This balance ensures that hematopoiesis occurs in the marrow under steady-state conditions. However, during development, recovery from hematopoietic stress and in myeloproliferative disorders, hematopoiesis occurs in extramedullary sites whose microenvironments are still poorly defined. The hypomorphic Gata1(low) mutation deletes the regulatory sequences of the gene necessary for its expression in hematopoietic cells generated in the marrow. By analyzing the mechanism that rescues hematopoiesis in mice carrying this mutation, we provide evidence that extramedullary microenvironments sustain maturation of stem cells that would be otherwise incapable of maturing in the marrow.

CXCR4-independent rescue of the myeloproliferative defect of the Gata1low myelofibrosis mouse model by Aplidin.

The discovery of JAK2 mutations in Philadelphia‐negative myeloproliferative neoplasms has prompted investigators to evaluate mutation‐targeted treatments to restore hematopoietic cell functions in these diseases. However, the results of the first clinical trials with JAK2 inhibitors are not as promising as expected, prompting a search for additional drugable targets to treat these disorders. In this paper, we used the hypomorphic Gata1low mouse model of primary myelofibrosis (PMF), the most severe of these neoplasms, to test the hypothesis that defective marrow hemopoiesis and development of extramedullary hematopoiesis in myelofibrosis is due to insufficient p27Kip1 activity and is treatable by Aplidin®, a cyclic depsipeptide that activates p27Kip1 in several cancer cells. Aplidin® restored expression of Gata1 and p27Kip1 in Gata1low hematopoietic cells, proliferation of marrow progenitor cells in vitro and maturation of megakaryocytes in vivo (reducing TGF‐β/VEGF levels released in the microenvironment by immature Gata1low megakaryocytes). Microvessel density, fibrosis, bone growth, and marrow cellularity were normal in Aplidin®‐treated mice and extramedullary hematopoiesis did not develop in liver although CXCR4 expression in Gata1low progenitor cells remained low. These results indicate that Aplidin® effectively alters the natural history of myelofibrosis in Gata1low mice and suggest this drug as candidate for clinical evaluation in PMF. J. Cell. Physiol. 225: 490–499, 2010.

The Expression of the Glucocorticoid Receptor in Human Erythroblasts is Uniquely Regulated by KIT Ligand: Implications for Stress Erythropoiesis

Studies in mice indicated that activation of the erythroid stress pathway requires the presence of both soluble KIT ligand (KITL) and the glucocorticoid receptor (GR). To clarify the relative role of KITL and GR in stress erythropoiesis in humans, the biological activities of soluble full length- (fl-, 26-190 aa), carboxy-terminus truncated (tr-, 26-162 aa) human (hKITL) and murine (mKITL) KITL in cultures of cord blood (CB) mononuclear cells (MNCs) and CD34(pos) cells that mimic either steady state (growth factors alone) or stress (growth factors plus dexamethasone [DXM]) erythropoeisis were investigated. In steady state cultures, the KITLs investigated were equally potent in sustaining growth of hematopoietic colonies and expansion of megakaryocytes (MK) and erythroid precursors (EBs). By contrast, under stress erythropoiesis conditions, fl-hKITL generated greater numbers of EBs (fold increase [FI]=140) than tr-hKITL or mKITL (FI=20-40). Flow cytometric analyses indicated that only EBs generated with fl-hKITL remained immature (>70% CD36(pos)/CD235a(neg/low)), and therefore capable to proliferate, until day 8-12 in response to DXM. Signaling studies indicated that all KITLs investigated induced EBs to phosphorylate signal transducer and activator of transcription 5 (STAT5) but that extracellular-signaling-regulated-kinases (ERK) activation was observed mainly in the presence of fl-hKITL. EBs exposed to fl-hKITL also expressed higher levels of GRα than those exposed to mKITL (and tr-hKITL) which were reduced upon exposure to the ERK inhibitor U0126. These data reveal a unique requirement for fl-hKITL in the upregulation of GRα and optimal EB expansion in cultures that mimic stress erythropoiesis.

Phenotypic Definition of the Progenitor Cells with Erythroid Differentiation Potential Present in Human Adult Blood

In Human Erythroid Massive Amplification (HEMA) cultures, AB mononuclear cells (MNC) generate 1-log more erythroid cells (EBs) than the corresponding CD34pos cells, suggesting that MNC may also contain CD34neg HPC. To clarify the phenotype of AB HPC which generate EBs in these cultures, flow cytometric profiling for CD34/CD36 expression, followed by isolation and functional characterization (colony-forming-ability in semisolid-media and fold-increase in HEMA) were performed. Four populations with erythroid differentiation potential were identified: CD34posCD36neg (0.1%); CD34posCD36pos (barely detectable-0.1%); CD34negCD36low (2%) and CD34negCD36neg (75%). In semisolid-media, CD34posCD36neg cells generated BFU-E and CFU-GM (in a 1 : 1 ratio), CD34negCD36neg cells mostly BFU-E (87%) and CD34posCD36pos and CD34negCD36low cells were not tested due to low numbers. Under HEMA conditions, CD34posCD36neg, CD34posCD36pos, CD34negCD36low and CD34negCD36neg cells generated EBs with fold-increases of ≈9,000, 100, 60 and 1, respectively, and maturation times (day with >10% CD36highCD235ahigh cells) of 10–7 days. Pyrenocytes were generated only by CD34neg/CD36neg cells by day 15. These results confirm that the majority of HPC in AB express CD34 but identify additional CD34neg populations with erythroid differentiation potential which, based on differences in fold-increase and maturation times, may represent a hierarchy of HPC present in AB.

Effects of ACE I/D polymorphism and aerobic training on the immune-endocrine network and cardiovascular parameters of postmenopausal women

CONTEXT Estrogen deficiency, systemic low-grade inflammation, and reduction of adrenal gland function have central roles in noncommunicable chronic disease (NCD) development. With angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism, the deletion variant (DD) is related to higher levels of circulating angiotensin II than I allele carriers (II/ID), which might interact with all of these molecular pathways to increase NCDs risk. On the other hand, physical exercise counteracts the occurrence of NCDs, potentially acting on the same pathways. OBJECTIVES The aim of the study was to investigate the effects of walking training on adrenal steroid and cytokine levels and on cardiovascular parameters in postmenopausal women with ACE I/D genotypes. METHODS Thirty-six (DD = 15, II/ID = 21) sedentary postmenopausal women (mean age, 56 ± 4 y) participated in a 13-week program of walking training at moderate intensity. Heart rate, blood pressure, double product, TNF-α, dehydroepiandrosterone sulfate (DHEA-S), and cortisol were evaluated before and after the intervention program. RESULTS Before walking training, the ACE DD genotype showed significantly higher TNF-α (P = .007) and lower DHEA-S concentrations (P = .022) than the ACE II/ID individuals. After walking training, both subgroups significantly decreased TNF-α plasma levels and cortisol/DHEA-S ratio (P = .001 and P = .016, respectively) and significantly increased DHEA-S levels (P < .001). Moreover, all the cardiovascular parameters were significantly reduced in the ACE DD participants (P ≤ .05), whereas the ACE I-allele carriers showed a decrease in heart rate (P ≤ .05) and the double product (P ≤ .05). CONCLUSION ACE I/D polymorphism is linked to different adrenal steroid and cytokine levels, and ACE I-allele carriers show a better adrenal activity and systemic inflammatory profile. The introduction of walking training positively influences the menopause immune-neuroendocrine changes, independent of ACE I/D genotype.

IL‐6 Activates PI3K and PKCζ Signaling and Determines Cardiac Differentiation in Rat Embryonic H9c2 Cells

Introduction: IL‐6 influences several biological processes, including cardiac stem cell and cardiomyocyte physiology. Although JAK‐STAT3 activation is the defining feature of IL‐6 signaling, signaling molecules such as PI3K, PKCs, and ERK1/2 are also activated and elicit different responses. Moreover, most studies on the specific role of these signaling molecules focus on the adult heart, and few studies are available on the biological effects evoked by IL‐6 in embryonic cardiomyocytes. Aim: The aim of this study was to clarify the biological response of embryonic heart derived cells to IL‐6 by analyzing the morphological modifications and the signaling cascades evoked by the cytokine in H9c2 cells. Results: IL‐6 stimulation determined the terminal differentiation of H9c2 cells, as evidenced by the increased expression of cardiac transcription factors (NKX2.5 and GATA4), structural proteins (α‐myosin heavy chain and cardiac Troponin T) and the gap junction protein Connexin 43. This process was mediated by the rapid modulation of PI3K, Akt, PTEN, and PKCζ phosphorylation levels. PI3K recruitment was an upstream event in the signaling cascade and when PI3K was inhibited, IL‐6 failed to modify PKCζ, PTEN, and Akt phosphorylation. Blocking PKCζ activity affected only PTEN and Akt. Finally, the overexpression of a constitutively active form of PKCζ in H9c2 cells largely mimicked the morphological and molecular effects evoked by IL‐6. Conclusions: This study demonstrated that IL‐6 induces the cardiac differentiation of H9c2 embryonic cells though a signaling cascade that involves PI3K, PTEN, and PKCζ activities. J. Cell. Physiol. 231: 576–586, 2016.

Thrombopoietin Inhibits Murine Mast Cell Differentiation

We have recently shown that Mpl, the thrombopoietin receptor, is expressed on murine mast cells and on their precursors and that targeted deletion of the Mpl gene increases mast cell differentiation in mice. Here we report that treatment of mice with thrombopoietin or addition of this growth factor to bone marrow‐derived mast cell cultures severely hampers the generation of mature cells from their precursors by inducing apoptosis. Analysis of the expression profiling of mast cells obtained in the presence of thrombopoietin suggests that thrombopoietin induces apoptosis of mast cells by reducing expression of the transcription factor Mitf and its target antiapoptotic gene Bcl2.