Adrenomedullin in the growth modulation and differentiation of acute myeloid leukemia cells
- Authors:
- Published online on: February 2, 2016 https://doi.org/10.3892/ijo.2016.3370
- Pages: 1659-1669
Abstract
Introduction
Acute myeloid leukemias are a heterogeneous group of clonal neoplastic diseases due to genetic and epigenetic alterations of hematopoietic stem cells (HSCs) or committed progenitors, causing an aberrant growth of myeloid precursor cells.
Acute promyelocytic leukemia (APL), a subtype of AML, is characterized by an abnormal accumulation of promyelocytes following specific translocation t(15;17) (q21;12) and formation of promyelocytic leukemia (PML)/retinoic acid receptor α (RARα) fusion gene. It has been demonstrated that the PML-RARα fusion protein is involved in the pathogenesis of APL through the recruitment of a complex that, composed of nuclear co-repressor molecule (NCOR) and histone deacetylase (HDAC), inhibits the expression of myeloid differentiation genes (1). Under treatment with all-trans-retinoic acid (ATRA), the NCOR/HDAC complex is dissociated from RAR and the maturation of promyelocytes is restored. Standard protocols combining ATRA with anthracycline-based chemotherapy are demonstrated to guarantee ~70% cure rate (2).
Originally isolated from human pheochromocytoma, adrenomedullin is a 52 aa peptide belonging to the calcitonin gene-related peptide family (3). Its maturation involves the proteolytic cleavage of preproadrenomedullin, a precursor of 185 amino acid residues that includes at amino terminal end the so-called proadrenomedullin N-terminal 20 peptide (PAMP), a peptide with known transient hypotensive activity (4,5).
The biological effects of ADM are mainly mediated by its interaction with two cell-surface receptors, multimeric complexes of calcitonin receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMPs). It is known that the RAMP family includes three members (RAMP1, RAMP2 and RAMP3) and regulates both transport and ligand specificity of CRLR. When RAMP2 or RAMP3 is associated with CRLR, adrenomedullin receptors 1 (AMR1) and 2 (AMR2) are generated, respectively, while RAMP1 is included in calcitonin gene-related peptide (CGRP) receptors (6,7).
Although ADM was first described as a potent vasodilator and hypotensive factor, several studies reported that it exerts various biological activities acting in an autocrine/paracrine manner (8). ADM is produced and secreted by several cell types, such as neurons, epithelial and endothelial cells supporting their survival and/or proliferation (9). Compelling evidence has shown that adrenomedullin can contribute to the pathogenesis of solid tumors in several ways (10). Firstly, the hypoxic conditions induce an upregulation of the peptide in the tumor cells leading to stimulation of cell growth and inhibition of apoptosis (11). Secondly, ADM exerts proangiogenic effects thus providing nutrients and oxygen to the tumor and allowing the spreading of tumor cells (12). Thirdly, it decreases the expression of proinflammatory cytokines, thus, inhibiting the immune system (13).
ADM is involved in the regulation of hematopoietic compartment as demonstrated by its expression in peripheral blood granulocytes, lymphocytes, monocytes and monocyte-derived macrophages under homeostatic and lipopolysaccharide-induced inflammation (14–16). Notably, ADM stimulates the proliferation of human cord blood hematopoietic stem cells through autocrine mechanism (17,18).
Based on the above evidence and considering that ADM plays a critical role in cancer cell proliferation, the aim of the present study was to evaluate whether ADM signaling is involved in the maintenance of impaired differentiation of APL cells to mature granulocytes or monocytes. Thus, the proliferative and differentiative effects induced by ADM and its inhibitor, ADM22-52, were evaluated in vitro at different time-points in HL60 cells, a human promyelocytic leukaemia cell line widely used to study granulocyte differentiation (19).
Materials and methods
Cell culture
Human promyelocytic leukaemia cell line HL60 (kindly provided by CRO, Aviano, Italy) was grown in Iscove's medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% antibiotic antimycotic solution (Sigma-Aldrich) at 37°C in a humidified atmosphere containing 5% CO2.
Flow cytometry (FCM)
The expression level of endogenous ADM (ADMendo), RAMP1, RAMP2, RAMP3 and CRLR was studied in HL60 cells cultured under resting or primed conditions with exogenous ADM (ADMexo) and ADM22-52. After washing with 0.5% BSA in PBS, the samples were incubated with primary antibodies (Table I) for 15 min at room temperature (RT). For indirect staining, the detection of specific binding sites was carried out by staining for 15 min at RT with Alexa Fluor® 488- or PE-conjugated secondary antibodies. For intracellular staining, HL60 cells were pre-fixed with BD Cytofix/Cytoperm solution (BD Biosciences, San Josè, CA, USA) for 20 min at 4°C. Data were analyzed by FACSCanto II flow cytometer (BD Biosciences) and expressed as percentage (%) ± standard deviation (SD) of positive cells compared with II antibody (Ab)- or isotype-matched controls. Statistical significance was calculated by Student's t-test.
Gene expression study of PAM
Total mRNA of HL60 cells was isolated using TRIzol® reagent (Sigma-Aldrich), according to the manufacturer's protocol. From each sample, 1 μg total RNA was reverse transcribed into cDNA by using M-MLV reverse transcriptase (Sigma-Aldrich), and oligo (dT)23 primers (Invitrogen-Life Technologies, Paisley, UK). The amplification reaction was performed by PTC-100 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA) using ReadyMix™ Taq PCR Reaction Mix with MgCl2 (Sigma-Aldrich), primer pairs designed to detect peptidylglycine α-amidating monooxygenase (PAM) and housekeeping gene GAPDH (Table II). PCR products were electrophoresed on 2% agarose gel (Sigma-Aldrich) pre-stained with GelRed™, and then visualized by UV transilluminator Gel Doc 2000 Gel Documentation system (Bio-Rad Laboratories).
ADM secretion assay
The constant release of endogenous ADM into culture medium was studied in HL60 cells using protein transport inhibition. Cells were seeded at density of 1×105 cells/ml and then cultured for 12, 36 and 60 h in proliferation medium before incubation for 12 h at 37°C with GolgiPlug™ (BD Biosciences). At 24, 48 and 72 h after plating, the samples were collected and then submitted to permeabilization with BD Cytofix/Cytoperm solution. The analysis was performed by intracellular ADM staining followed by flow cytometric analysis, as above reported. In the present study, cultures untreated with GolgiPlug™ were used as positive control of ADM secretion. The acquired data were analyzed by the overton substraction tool of Summit 4.3 software (Beckman Coulter, Inc., Brea, CA, USA) and were reported as percentage (%) ± SD of ADM positive cells compared with relative II Ab-matched control.
Treatment of HL60 cells with exogenous ADM and ADM22-52
Cells were seeded in 25-cm2 flasks at a density of 1×105 cells/ml and then cultured for 72 h with different concentrations of ADM (ranging from 0.625×10−8 M to 5×10−8 M) (Sigma-Aldrich) or 5×10−7 M ADM22-52 (Sigma-Aldrich) (13,18,20). In parallel, untreated cultures were used as reference. At the end of the treatment, growth assay and FCM analysis of endogenous ADM system were carried out. In parallel, cytospin preparations for May-Grunwald Giemsa staining were made to detect morphological changes of the nuclei.
Growth assay
At 72 h, cell suspensions were collected and then centrifuged at 1,200 rpm for 5 min at 4°C. After resuspension of pellets in 1 ml of Iscove's medium, HL60 cells were counted by Nexcelom Bioscience-Auto T4 Cellometer (Invitrogen-Life Technologies). Data are expressed as mean of total cell number ± SD of three independent experiments performed in triplicate. Statistical analysis was performed using the Student's t-test.
Investigation of PI3K/Akt and MAPK/ERK signaling pathways
At different time-points (5, 15 and 30 min) after stimulation, the activation of PI3K/Akt and MAPK/ERK signaling pathways was evaluated in HL60 cells treated with 5×10−8 M ADM and 5×10−7 M ADM22-52. The analysis was performed by FCM as previously described, using antibodies specific for phosphorylated and unphosphorylated forms of Akt and MAPK enzymes (Table I). In parallel, untreated samples were used as reference. For each marker and its corresponding II Ab-matched control, data were reported as geometric mean fluorescence intensity (MFI) ± SD. Statistical significance was calculated using the Student's t-test comparing primed cells with resting samples.
Differentiative response of HL60 cells to ADMexo and ADM22-52 Quantitative real-time polymerase chain reaction (qPCR)
The quantitative analysis of Cullin 5 (Cul5) gene expression was performed using Platinum® SYBR® Green qPCR SuperMix-UDG (Invitrogen-Life Technologies) and oligo primers (Invitrogen-Life Technologies) designed for the detection of Cul5 and hypoxanthine-guanine phosphoribosyltransferase (HPRT) housekeeping gene (Table II). The amplification reactions were performed using a DNA Engine Opticon Real-Time Thermal Cycler (Bio-Rad Laboratories). The relative expression of Cul5 mRNA was determined using the ΔΔCT Livak method (21) and data were reported as fold increase calculated using the 2−ΔΔCT ± SD equation.
FCM
At 72 h after stimulation with ADMexo and ADM22-52, the samples were collected and then analysed by flow cytometry to measure the expression of myeloid differentiation markers and adhesion molecules (CD45, CD34, CD66b, CD11b, CD11c, CD14, CD31 and CD38). The samples were prepared as described above using antibodies listed in Table I. For each marker, data were expressed as the ratio of geometric MFI (rMFI) ± SD derived from primed cells and relative resting samples. Statistical significance was calculated using the Student's t-test.
Histochemistry
Air-dried slides of cells were prepared using a Cytospin 4 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 500 rpm for 5 min. The slides were fixed in methanol (Sigma-Aldrich) for 5 min and then stained for 5 min in 50% (v/v) May-Grunwald stain (Merck Millipore, Billerica, MA, USA) followed by 15 min in 10% Giemsa stain (Merck Millipore). Both stain solutions were freshly made using Sorenson's phosphate buffer (133 mM, pH 6.6). After staining, the slides were washed and destained for 5 min in the phosphate buffer.
Results and Discussion
Immunophenotype of HL60 cells
Before priming with ADMexo and ADM22-52, the immunophenotype of HL60 cells was explored by FCM evaluating the expression level of undifferentiated and differentiated state markers (Fig. 1). The myeloid phenotype was confirmed by the expression of CD33 while the presence of stem cell compartment was suggested by the absent expression of CD117/CKIT, Lin 1 and CD34 (22,23). Moreover, the undifferentiated state of cells was demonstrated by the negative or low fluorescence intensity of HLA-DR, CD45, CD11b, CD11c and CD66b (24–27). Among adhesion molecules expressed by acute myelogenous leukemia blasts, CD31 (28,29) and CD38 (30) are reported to play pivotal role in the interaction of tumor cells with microenviromental elements, i.e. CD31 on the surface of marrow endothelial cells (CD31/CD31 and CD38/CD31 interactions) and hyaluronate (CD38/hyaluronate interactions). In the present study, an excess of CD31 relative to CD38 was detected (CD31/CD38 MFI ratio >1) suggesting that HL60 cells have a high potential of transendothelial migration (31–33). Similar expression level of CD31, CD33, CD38 in HL60 and APL cells was considered important to define a prospective correlation between in vitro and in vivo conditions.
HL60 cells express ADM and its receptors
As reported in Fig. 2A, HL60 cells were shown to express ADMendo (94.7±2.8%), RAMP2 (20.0±2.3%), RAMP3 (31.5±2.8%) and CRLR (14.3±1.8%). In contrast, RAMP1 was not detected. The gene expression of peptidylglycine α-amidating monooxygenase (PAM), that is the enzyme producing α-amidated bioactive peptide from the inactive precursor (34), confirmed the potential of cells to synthesize the active form of ADM (Fig. 2B). Produced by peripheral blood mononuclear and polymorphonuclear cells, ADM is known to regulate cell growth or differentiation through an autocrine mode of action (15). As shown in Fig. 3, the release of ADM endogenously synthetized by HL60 cells was evaluated using Brefeldin A/GolgiPlug™ kit, a protein transport inhibitor that is able to prevent the secretion process and to trap peptides into intra-cellular compartments. The increased immunoreactivity for ADM in GolgiPlug™-treated samples (24 h, 77.4±8.3%; 48 h, 50.5±8.3%; and 72 h, 68.5±7.3%) compared to untreated cells (24 h, 33.4±2.8%; 48 h, 32.3±4.8%; and 72 h, 59.0±6.1%) was interpreted as indicative of the cytoplasmic accumulation of ADMendo while the decreased fluorescent signal suggested that HL60 constitutively secrete ADM into culture medium after synthesis, as previously reported (15).
ADMexo enhances the proliferation of HL60 cells
It is known that ADM is produced not only by cancer cells but also by endothelial, macrophages, and mast cells of tumor microenvironment (35), where it contributes to cancer pathogenesis both directly stimulating cancer cell growth or indirectly inducing angiogenesis and reducing the effectiveness of the immune system.
Our data (Fig. 4) show that exogenous ADM exerted a strong proliferative effect on HL60 cells, as already observed in several cell types (36–39). Indeed, after a 72 h incubation period, significant increases in cell number were detected in cultures treated with ADMexo compared to resting cells. In particular, 5×10−8 M ADM was shown to be the most effective concentration. In contrast, ADM22-52 demonstrated not to affect the proliferation of HL60 cells, since total cell number in primed cultures was comparable to that of untreated samples.
ADMexo and ADM22-52 affect the ADM system
In the present study, we explored by FCM the modulation of adrenomedullin system (ADMendo and its receptors) in HL60 cells treated with exogenous 5×10−8 M ADM or 5×10−7 M ADM22-52. When HL60 cells were in vitro cultured with ADMexo (Fig. 5A) and ADM22-52 (Fig. 5B), a different expression profile of ADMendo was detected in comparison to resting samples. In particular, at 24 h, a decreased immunoreactivity for ADM was observed in primed samples suggesting that, probably due to an increased level of intracellular Ca2+ (40), a major release of endogenous ADM was promoted. At 72 h, the secretion rate of ADM was negatively controlled by ADMexo (Fig. 5A) but not under treatment with ADM22-52 (Fig. 5B). It is known that the expression of RAMP isoforms may change between physiological and pathological conditions (41), determining the degree of cell response to ADM. The cell sensitivity to ADM stimulation is defined both by a balanced expression of RAMPs and CRLR at cell membrane and their rapid rate of recycling. It is reported that RAMPs play pivotal roles in the transport of CRLR to plasma membrane (42). At physiological conditions, the most abundant isoform is RAMP2. Under pathological conditions, significant changes in RAMP expression levels are active under a concomitant increase in plasma ADM level (43). In the present study, the expression of AMR1 (CRLR/RAMP2), AMR2 (CRLR/RAMP3) and CGRP (CRLR/RAMP1) components was demonstrated to be altered following the treatment with exogenous ADM and ADM22-52. Indeed, at 24 h, ADMexo upregulated RAMP1, RAMP3 and CRLR (Fig. 5A) while ADM22-52 was effective in increasing the expression of RAMP3, but drastically reduced CRLR (Fig. 5B). In both primed groups, probably due to a receptor degradation after internalization (43), RAMP2 significantly decreased (Fig. 5), suggesting that ADMexo and ADM22-52 mediated their activity at early phase by AMR1. These data were in accordance with the hypothesis that an excess of ADM in bone marrow matrix could increase the responsiveness of leukemia cells to ADM upregulating CRLR, RAMP1 and RAMP3 (44), while the inhibition of ADM signaling could raise the cell insensitivity to stimulation by a significant reduction of ADM receptor system. At 72 h, a correlation between the increased expression level of RAMP1/2 and the release of ADM was observed in resting samples suggesting that HL60 cells could combine the release of ADM with the exposure of ADM receptors at cell membrane to control their growth. Notably, the treatment of HL60 cells with exogenous ADM reduced the release of ADMendo and the expression of RAMP1 and RAMP2 while no change was induced by ADM22-52 compared to resting cells. The expression of RAMP3 and CRLR was drastically reduced at 72 h in all experimental groups. Unlike previously reported by Poyner et al (42), ADM22-52 exerted inhibitory activity of ADM signaling in leukemia cells through a negative regulation of CRLR expression, either by an increased receptor internalization followed by degradation or a block of receptor transport to plasma membrane.
AKT and MAPK signaling pathways were differently modulated by ADMexo and ADM22-52
Pleiotropic effects of ADM are reported to be mediated through several intracellular signal transduction pathways (43). Using HL60 leukemia cells, we explored by FCM the modulation exerted by 5×10−8 M ADM and 5×10−7 M ADM22-52 on PI3K/Akt and ERK/MAPK signaling pathways, that are involved in several haematopoiesis malignancies, including AML (45–49). As shown in Fig. 6, the simultaneous detection of p(Ser473)-Akt1/2/3 and phospho(Thr202/Tyr204)-p44/42 MAPK in resting cells (ADMendo) suggested a constitutive activity of both pathways in HL60 cells. Hayashi and colleagues (50) reported that PI3K/Akt positively regulates the MAPK cascade, facilitating maximal ERK responses to physiological stimuli, whereas activated ERK, in turn, negatively controls the PI3K/Akt pathway. In the present study (Fig. 6), the phosphorylation level of Akt in ADMendo reached the maximum value at 5 to 15 min and decreased at 30 min. In parallel, the upregulation in MAPK activity from 15 to 30 min could be indicative of a reciprocal regulation loop between Akt and MAPK. Notably, the stimulation of HL60 with ADMexo and ADM22-52 differently affected the activity of both pathways. Under stimulation with ADMexo, an increase in the expression level of p(Ser473)-Akt and phospho(Thr202/Tyr204)-p44/42 MAPK was observed at 5 min indicating that HL60 cells were responsive to exogenous ADM via the PI3K/Akt and ERK/MAPK cascade. In samples treated with ADMexo compared to ADMendo group, the activation level of MAPK unchanged from 5 to 15 min while a significant decrease in phosphorylated Akt expression was observed. In contrast, the stimulation with ADM22-52 promoted the maximum activation of Akt at 5 to 30 min and a downregulation of MAPK activity at 30 min. Based on this evidence, we hypothesized that the activity of ADMexo was mainly mediated by the ERK/MAPK pathway while that of ADM22-52 was especially involved in PI3K/Akt signaling.
ADM22-52 induces a strong differentiative induction on HL60 cells
qPCR
To verify the differentiative induction after the inhibition of ADM system, the expression of Cul5 was assessed by quantitative RT-PCR (Fig. 7). Cul5 belongs to the Cullins gene family, including 7 isoforms identified in mammalians. Cul5, Asb-2 (ankyrin repeat SOCS box 2), a RING finger protein (ROC/Rbx), and an adapter protein complex (elongin B/C) form an E3 ubiquitin ligase complex that, due to an E2 ubiquitin-conjugating enzyme, leading to the degradation of target proteins by 26S proteasome (51). As component of this complex, Cul5 brings the enzyme the substrate recognition components (52). Furthermore, it seems to act as tumor suppressor in breast cancer (53) and its expression decreases in B-cell chronic lymphocytic leukemia (54). At 24 and 72 h after stimulation with ADM22-52, Cul5 mRNA increased 1.35- and 4.18-fold, respectively, in primed cells vs. resting cultures. Our data were in agreement with the increase of Cul5 mRNA and protein detected by Baxter et al (19) during ATRA-mediated differentiation of HL60 cells.
FCM
Several reports highlighted that PI3K/Akt and ERK/MAPK signaling pathways control the self-renewal and differentiation of leukemia cells. Akt was demonstrated to exert its activity (55) activating nuclear factor-kappa B (NF-κB) (56), phosphorylating GSK-3 (57) and inhibiting transcription factors of FOXO family (58). Marcinkowska et al (59) demonstrated that ERK/MAPK pathway stimulates the monocytic differentiation of HL60 cells through the phosphorylation of transcription factor C/EBPβ. As shown in Fig. 8A, the treatment with 5×10−8 M ADMexo was accompanied by an upregulation in the expression level of CD14 and CD31, which are associated with monocytic differentiation of HL60 cells (59,60). Interestingly, a decreased (rMFI<1) or unchanged (rMFI=1) expression level of other markers was observed in comparison to resting samples (ADMendo), suggesting that ADMexo could exert a negative control on the granulocytic differentiation process. In samples treated for 72 h with ADM22-52, FCM analysis evidenced a significant increase in the expression level of CD11b, a subunit of αMβ2 integrin, CD11c, an integrin αX, and CD66b, a granulocyte-specific activation antigen expressed on secondary granule membranes, that are all associated with granulocytic differentiation of HL60 cells (21,61,62). Moreover, we observed the upregulation of CD45 and CD38, that are reported to increase during myeloid maturation (63,64) and ATRA-induced granulocytic/monocytic differentiation (24,64). The increased MFI value of CD14 in ADM22-52 compared to that of ADMendo-samples further confirmed the granulocytic/monocytic maturation of HL60 cells (65). It is known that the expression of CD31 and CD38 on leukemic cells could exert a balanced control between extramedullary dissemination and marrow retention of leukemic cells. As previously demonstrated in ATRA-induced HL60 differentiation (66), an excess of CD38 relative to CD31 (CD31/CD38 MFI ratio<1) was detected in ADM22-52-treated cells, suggesting a higher retention of cells in bone marrow through the interaction with hyaluronate. In contrast, the excess of CD31 relative to CD38 (CD31/CD38 MFI ratio >1) in samples treated with ADMexo evidenced an increased ability of transendothelial migration.
Histochemistry
To investigate HL60 cell differentiation, nuclear morphology of cells stained with May-Grunwald-Giemsa was performed (Fig. 8B). In ADMexo-treated cells, only ovoid and intended nuclei were detected, whereas ADM22-52 determined the appearance of differentiated cells characterized by several multilobulated segmented nuclei.
Conclusions
Understanding the aberrant expression of self-renewal pathways in myeloid malignancies is an emerging area of investigation for the development of novel treatment strategies. Extrinsic factors driven by bone marrow microenvironment are reported to regulate the growth of hematopoietic stem cells (67) within supportive osteoblastic and vascular niches (68). Among the soluble factors secreted in BM niches by stem cells (69) and stromal compartment (10), ADM synergizes with stem cell factor and Flt-3 (FMS-like tyrosine kinase-3) ligand to induce the proliferation of primitive human CD34+CD38−lin−cells and to promote the expansion of CD34+ progenitors in culture (70). The development of hematopoietic malignancies causes a remodeling of BM microenvironment followed by the alteration of HSC function, leukemia survival, protection of cancer cells from apoptotic stimuli and development of drug-resistant phenotype (67,71). Hypoxic conditions in leukemia niche are demonstrated to support the progression of cancer and to upregulate ADM (72), thus, suggesting a correlation between the survival of leukemia cells and the increased release of ADM in tumor microenviroment (10). Consistent with the above evidence, and studies in several human tumor cell lines, including HL60 and chronic monocytic leukemia (U937) (73), our results suggest for ADM a regulatory role in the proliferation of APL by paracrine effects, as already demonstrated for ADM in modulating the activity of the hypothalamo-pituitary-adrenal axis (74), the growth of cardiac and vascular smooth muscle cells (75), the hemodynamics of brain, lungs, and kidneys (76), physiological and pathological angiogenesis (12). Using the in vitro model of HL60 cells, we demonstrated that exogenously administered ADM preserves promyelocytic leukemia cells from differentiation as shown under ADM signaling inhibition with ADM22-52 by the detection of typical differentiation features such as multilobulated segmented nuclei, Cul5 expression and increased expression level of granulocytic and monocytic antigens. As the treatment with exogenous ADM significantly stimulated the growth of HL60 cells, we speculated that paracrine ADM acts in APL niche supporting the survival of leukemia cells and promoting their transendothelial migration upregulating the cellular expression of CD31 (64). Based on the consideration that ADM22-52 stimulates both the maturation and the retention in the bone marrow of leukemia cells through an increased expression of CD38, as previously demonstrated in HL60 and human primary APL cells treated with ATRA (64), our data provide strong evidence for the therapeutical potential that the inhibition of ADM signaling could have in the treatment of APL. A potentially important therapeutic effect of the blockade of ADM22-52-sensitive receptors has been already demonstrated for endotoxic shock (77) and expansion of keratinocytes, fibro-blasts (78) and adrenal cortical cells (79).
Further studies will be necessary to verify ADM expression in bone marrow microenvironment and its involvement in the induction and progression of hematopoietic malignancy.
Acknowledgements
The authors are grateful to Teresa Mariotto Pelos and Italian Leukemia-Lymphoma-Myeloma ONLUS Association, Section of Treviso (Italy) for the financial support.
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