12th Congress of the European Hematology ... - Haematologica

12 th Congress of the European Hematology Association
topenia model, the human platelet count was fully recovered. Interestingly,
the numbers of CD34 + CD41 + human megakaryoblasts and 4N to
128N human megakaryocytes in xenografted mice were not significantly
changed compared to the control mice after treatment with IFN for 7
weeks. We also analyzed the inhibitory effects of IFN on megakaryocytes
in vitro. Although IFN slightly reduced the number of CFU-MKs
[146±13 colonies/5000 cells (control), 130±13 colonies/5000 cells (10
ng/mL of IFN)], the percentage of megakaryocytes bearing PPT was significantly
reduced by IFN [20.4±3.0%(control), 12.5±3.6% (10 ng/mL of
IFN)]. Currently, we are investigating the inhibitory mechanisms of IFN
for PPT. Conclusion. These results indicated that NIP-004 is effective for
preventing IFN-induced thrombocytopenia in vivo. IFN might inhibit the
PPT formation rather than the proliferation of human megakaryocytopoiesis.
We expect that the small thrombopoietin molecule, NIP-004,
can be applied for the treatment of IFN-induced thrombocytopenia in
the patients with chronic hepatitis C.
0388
IN VITRO AND IN VIVO MICROPARTICLE-ASSOCIATED ENDOTHELIAL PROTEIN C RECEP-
TOR CAN REVERSE PRO-INFLAMMATORY AND APOPTOTIC CELL SIGNALS AND EFFECTS
T. Dutt, M. Perez-Casal, C.H. Toh
University of Liverpool, LIVERPOOL, United Kingdom
Background. We have described a novel mechanism of endothelial protein
C receptor (EPCR) release from cell surfaces in vitro and in vivo.
Induced by Activated Protein C (APC), EPCR is released in microparticulate
(MP) form. Unlike APC bound to truncated soluble EPCR, APC on
MP-EPCR retains proteolytic anticoagulant activity. We now hypothesise
that the MP EPCR-APC complex can also cleave endothelial protease
activated receptor-1 (PAR1) to modulate inflammation and cytoprotection.
Aims. 1. To compare the efficacy of MP EPCR-APC versus free-
APC, using two different pools of endothelial cells, following exposure
to a pro-inflammatory stimulus. 2. To determine if circulating MP EPCR-
APC from patients can regulate gene expression in a PAR1-dependent
manner. Methods. Using an in vitro system of human umbilical vein
endothelial cells (HUVECs) or human coronary artery endothelial cells
(HCAECs), TNFα, 10 ng/mL, was added to represent a cell model of sepsis.
After 4 hours the cells were treated with an equal concentration of
either free or MP-bound APC and left for a total of 24 hours. RNA was
extracted from the cells and used to make probes for hybridization of
GEArray Human Endothelial Cell Biology Gene Array (SuperArray).
Using untreated cells as a baseline, the specificity of APC and the role
of PAR1 were assessed by using the appropriate blocking antibodies and
antagonist. Circulating MPs were isolated from plasma of APC treated
patients by standard methods and platelet-derived MPs depleted using
a CD41a antibody and magnetic beads. MP-associated APC was estimated
from a standard curve and 17nM used for gene profiling. PAR1mediated
signalling was examined as above. Results. In TNFα stimulated
cells, APC, selectively induced anti-inflammatory and suppressed
pro-apoptotic gene regulation. In over 90% of these genes, MP-APC
had a 2-7 fold greater effect in relation to free-APC. These observations
were confirmed by Q-PCR for e-selectin, ICAM1, Bax, A20, caspase 10,
GM-CSF and IL8. This translated into increased GM-CSF and interleukin
8 secretion, and cytoprotection against staurosporine-induced apoptosis.
Protein C or PAR1 antagonism reversed these results to demonstrate
that the induced effects by MPs were APC specific and PAR1-dependent.
Further analysis using MP-associated EPCR from septic patients
during APC treatment also demonstrated PAR1-mediated anti-inflammatory
gene induction and anti-apoptotic function. Conclusions. APC on
MP-associated EPCR can ameliorate TNF induced inflammatory changes
and staurosporine induced apoptosis in both HUVECs and HCAECs. As
these effects are reproducible with MPs isolated from patients' plasma,
this may have clinical relevance in the septic patient treated with APC.
142 | haematologica/the hematology journal | 2007; 92(s1)
0389
ENDOTHELIAL COLONY FORMING CELLS FROM PATIENTS WITH CML,
POLYCYTEMIA VERA AND ESSENTIAL THROMBOCYTHEMIA, DERIVE FROM A STEM CELL
LACKING THE DISEASE SPECIFIC MARKER
F. Frassoni, 1 G. Piaggio, 2 M. Corselli, 1 F. Bertolotti, 1 S. Pozzi, 1
A. Parodi, 1 B. Chiavarina, 1 M. Sessarego, 3 G. Fugazza, 3 A. Garuti, 4
A. Ibatici, 1 A. Bacigalupo, 5 F. Frassoni1 1 Centro Cellule Staminali, GENOA; 2 Lab Diagnostica Emato e Colture in vitro,
GENOVA; 3 Laboratorio Citogenetica DIMI, GENOVA; 4 Terapie Cellulari
DIMI, GENOVA; 5 Ematologia e Trapianto AO S Martino, GENOVA, Italy
Background. the persistence of a common progenitor to haematopoietic
and vascular endothelial lineage in the adult haematopoietic tissue
appeared to be validated when Bcr/Abl fusion gene was found in a variable
proportion of endothelial like cells generated in vitro from the bone
marrow and the peripheral blood of CML patients. However, the different
colture conditions used to isolate and to expand endothelial cells
(EC) in vitro have generated some confusion. Only recently, the existence
of two EC populations has been clearly settled: early EPC, i.e.
colony forming unit-endothelial cells (CFU-EC), are haematopoietic
derived progeny committed to the myeloid lineage while late EPC,
namely ECFCs, are vessel-forming progenitors. Aims. we have reconsidered
the issue of endothelial cell origin (status) in CML and other MPDs
to verify whether circulating EPCs, isolated and expanded from the
peripheral blood of patients, bear the disease specific genetic marker of
the leukaemia clone or this is restricted to the haematopoietic lineage.
Methods. Forty-three patients and 12 normal controls were included in
this study. Twenty-four samples were Philadelphia and Bcr/Abl positive
CML, 19 were Philadelphia and Bcr/Abl negative MPDs, PV and ET with
JAK2-V617F mutation. Peripheral blood mononuclear cells were cultured
in vitro and expanded cells were searched for Bcr/Abl fusion gene
by RQ-PCR and FISH, and for JAK2-V617F mutation by nested PCR.
Flow cytometry and capillary formation assay in Matrigel were also performed.
Results. due to the low frequency of late EPCs in the peripheral
blood of patients, only 7/24 CML and 6/19 MPD samples gave rise in vitro
to a progeny of EC. None of them carried the disease specific genetic
marker. The expanded cells showed a complete outfit of endothelial
associated antigens and were able to form capillary like structures in vitro.
Conclusions. differently from previous reports and based on a clear distinction
between early and late EPCs, this study shows for the first time
that endothelial cells, derived in culture from Ph-positive, Bcr/Abl positive
CML patients, lack the disease marker in the late EPC. In addition
late EPCs from patients with PV or ET lack the JAK2-V617F mutation.
Thus it appears that in CML , PV and ET the cell able to give rise to
endothelial progenitors do not derive from the malignant clone. This
finding, per se, does not exclude the possibility of a common ancestor
cell for haematopoietic and endothelial lineage, and multiple explanations
could be hypothesized: the leukemogenetic event that starts the
CML involves a stem cells below the common ancestor cells. Indeed,
most T cells in CML do not belong to the leukemic clone. Alternatively,
since in the majority of newly diagnosed patients with CML, the
bone marrow contains a high number of normal Ph-negative early progenitor
cells (LTC-IC), one could argue that Ph-negative stem cells still
give rise to EPC therefore diluting those deriving from Ph-positive ones.
The same apply to MPD where it is now evident that part of
haematopoietic progenitors do not carry the JAK2-V617F mutation.