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MYELOID NEOPLASIA
Endothelial progenitor cells are clonal and exhibit the JAK2V617F mutation in a
subset of thrombotic patients with Ph-negative myeloproliferative neoplasms
*Luciana Teofili,1 *Maurizio Martini,2 Maria Grazia Iachininoto,1 Sara Capodimonti,1 Eugenia Rosa Nuzzolo,1 Lorenza Torti,1
Tonia Cenci,2 Luigi Maria Larocca,2 and Giuseppe Leone1
Departments of 1Hematology and 2Pathology, Catholic University, Rome, Italy
In this study we investigated whether
neoplastic transformation occurring in
Philadelphia (Ph)–negative myeloproliferative neoplasms (MPNs) could involve
also the endothelial cell compartment.
We evaluated the level of endothelial
colony-forming cells (E-CFCs) in 42 patients (15 with polycythemia vera, 12 with
essential thrombocythemia, and 15 with
primary myelofibrosis). All patients had
1 molecular abnormality (JAK2V617F or
MPLW515K mutations, SOCS gene hypermethylation, clonal pattern of growth) de-
tectable in their granulocytes. The growth
of colonies was obtained in 22 patients
and, among them, patients with primary
myelofibrosis exhibited the highest
level of E-CFCs. We found that E-CFCs
exhibited no molecular abnormalities
in12 patients, had SOCS gene hypermethylation, were polyclonal at human
androgen receptor analysis in 5 patients,
and resulted in JAK2V617F mutated and clonal
in 5 additional patients, all experiencing
thrombotic complications. On the whole,
patients with altered E-CFCs required anti-
proliferative therapy more frequently than
patients with normal E-CFCs. Moreover
JAK2V617F-positive E-CFCs showed signal
transducer and activator of transcription 5
and 3 phosphorylation rates higher than
E-CFCs isolated from healthy persons and
patients with MPN without molecular abnormalities. Finally, JAK2V617F-positive E-CFCs
exhibited a high proficiency to adhere to
normal mononuclear cells. This study highlights a novel mechanism underlying the
thrombophilia observed in MPN. (Blood.
2011;117(9):2700-2707)
Introduction
During the embryonic development hematopoietic and endothelial
cells arise from a mesoderm-derived common precursor called
hemangioblast.1 The existence of an exceedingly rare hemangioblast progenitor has been shown also in the postnatal life in the
CD34/KDR-positive cell subset.2 This cell appears to be endowed
with long-term proliferative potential and with both hematopoietic
and endothelial differentiation capacity.2 Because Philadelphia
(Ph)–negative myeloproliferative neoplasms (MPNs) show a high
incidence of vascular complications3 and an endothelial cell
dysfunction has been evidenced in patients with polycythemia vera
(PV),4 several studies explored the possibility that the neoplastic
transformation in MPN could involve also the endothelial cell
compartment.5-9 On the whole, studies that were based on the in
vitro assays for endothelial progenitors have identified JAK2V617F
mutation or specific chromosome alterations only in endothelial
progenitors derived from the hematopoietic lineage (the so-called
colony forming unit-endothelial cells; CFU-ECs),5-7 whereas the
true endothelial colony-forming cells, (E-CFCs) do not harbor
genetic abnormalities.6,7 In conflict with these findings, Sozer et al8
found that endothelial cells isolated by microdissection from liver
biopsies of patients with PV with Budd-Chiari Syndrome (BCS)
exhibit the JAK2V617F mutation. In reality, granulocytes isolated
from patients with MPN can harbor several genetic defects in
addition to the JAK2V617F mutation, such as the hypermethylation
of suppressor of cytokine signaling (SOCS) genes10-12 or the
presence of a clonal pattern of proliferation.13 Importantly, in some
circumstances, these defects can be detected in the absence10,11 or
even before the neoplastic clone acquired the JAK2V617F muta-
tion.14-16 For these reasons, in this study we investigated endothelial progenitor cells isolated from patients with MPN for a large
panel of molecular markers. Our results provided evidence that a
subset of patients with MPN shows in endothelial progenitors the
same molecular signatures detectable in the hematopoietic clone.
Moreover, we found that the presence of JAK2V617F mutation into
endothelial progenitors is associated with the hyper-phosphorylation of signal transducer and activator of transcription 3 (pSTAT-3)
and STAT-5 (pSTAT-5), with an increased adhesion to normal
mononuclear cells and, from a clinical point of view, with an
increased risk of thrombosis.
Submitted July 21, 2010; accepted December 12, 2010. Prepublished online as
Blood First Edition paper, January 6, 2011; DOI 10.1182/blood-201007-297598.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
*L. Teofili and M.M. contributed equally to the study.
© 2011 by The American Society of Hematology
2700
Methods
Patients
This observational single-center study was carried out in accordance with
the Declaration of Helsinki and was approved by the institutional ethic
committee of Catholic University. The cohort consisted of 42 patients with
MPN (20 men and 22 women; median age, 58.5 years; range, 25-76; mean
disease duration, 52 months; range, 6-140 months), recruited on an outpatient basis during the period of March 2008 to April 2010. The study
inclusion criteria were (1) presence of detectable molecular markers in
granulocytes, (2) absence of antiproliferative therapy at the time of the
investigation (only phlebotomy or antiplatelet therapy or both were
admitted). Seven patients were evaluated at diagnosis and 35 during the
follow-up. Fifteen patients were affected by PV, 12 by essential thrombocythemia (ET) and 15 by primary myelofibrosis (PMF). All diagnoses were
made according to criteria used at the time of the first evaluation and were
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
ENDOTHELIAL PROGENITORS AND MPN
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Table 1. Hematological and molecular findings of patients at their first evaluation
Polycythemia vera (n ⴝ 15)
Males/females, n
9/6
Essential thrombocythemia (n ⴝ 12)
4/8
Primary myelofibrosis (n ⴝ 15)
9/6
Age, y
Mean
54.9
54.3
55
Median (range)
59 (27-70)
56 (25-76)
55 (31-72)
WBC count, ⴛ 109/L
Mean
11.83
8.11
11.28
Median (range)
10.7 (6.70-18.00)
7.50 (4.18-14.06)
10.5 (4.3-24.1)
Hb, g/dL
Mean
18.1
14
12.4
Median (range)
17.1 (14.8-24.8)
14 (12.6-15.6)
12.8 (7.9-19.4)
PLT count, ⴛ 109/L
Mean
504
672
621
Median (range)
518 (205-849)
604 (456-970)
529 (58-2.696)
JAK2V617F or MPLW515K positive, n (%)
SOCS methylation, n (%)
13 (87)
7 (58)
11 (73)
5 (28)
3 (27)
8 (47)
WBC indicates white blood cell; Hb, hemoglobin; and PLT, platelet.
revised according to World Health Organization 2008 diagnostic criteria.17
On the whole, 22 patients were JAK2V617F positive, 10 patients had both
JAK2 mutation and SOCS gene hypermethylation, 5 patients had SOCS
gene hypermethylation, and 1 patient had MPLW515K mutation and SOCS
hypermethylation. Clinical and hematologic features of investigated patients are shown in Table 1. Twenty-five healthy blood donors (16 men and
9 women; median age, 37 years; range, 19-62 years) were investigated as
the control group. Blood samples (30 mL) were collected after written
informed consent.
35 cycles at 94°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute, and a
final step at 72°C for 5 minutes. Representative CFU-EC and E-CFC
colonies and their RT-PCR characterization are shown in Figure 1.
Capillary formation assay was performed as described19,20 with the use of
E-CFCs at passage 2. When appropriate, single E-CFC–derived colonies
were expanded for 2 or 3 passages to obtain an amount of cells suitable for
adhesion assays and for Western blot analysis.
CFU-EC and E-CFC colony assay
The presence of the JAK2V617F mutation was investigated according to the
method of Baxter et al21 as previously described.22,23 Briefly, after DNA
extraction with DNAzol (Invitrogen), following the product’s protocol,
80 ng of DNA was used to assess the presence of the JAK2V617F mutation in
allele-specific PCR. PCR products were separated onto 3% agarose gel,
stained with ethidium bromide, and visualized under UV light. A 203-base
pair (bp) fragment indicated the presence of 1849G ⬎ T mutation. To
determine whether the mutation was carried in the homozygous state,
digestion of PCR products with BsaXI restriction enzyme was performed.22
Other functionally similar JAK2 exon 12 mutations were investigated by
sequencing, according to the method of Scott et al24 in all JAK2V617F-
CFU-EC and E-CFC assays were performed according to the method of
Hill et al18 and of Ingram et al,19 respectively, as previously described.20
Briefly, for CFU-EC colonies, Ficoll density gradient–isolated mononuclear
cells were suspended in EndoCult medium (EndoCult Liquid Medium Kit,
Stem Cell Technologies) and plated on 6-well dishes coated with human
fibronectin (Sigma-Aldrich). After 48 hours, nonadherent cells were recovered and replated in 24-well human fibronectin-coated dishes (SigmaAldrich) in the same culture medium, at 106/mL cell concentration. After an
additional 3-5 days, aggregates consisting of multiple thin, flat cells
emanating from a central cluster of rounded cells were counted as
CFU-ECs. For the E-CFC colonies, mononuclear cells were suspended in
endothelial cell growth medium-2 (EGM-2 Bulletkit; Lonza) on human
fibronectin-coated 6-well dishes. At day 2 nonadherent cells were discharged, and residual adherent cells were grown in endothelial cell growth
medium-2 for 28 days, with medium replacement every 3 days. Wellcircumscribed monolayer of cobblestone-appearing cells growing from day
9 to day 28 were counted as E-CFCs and singly recovered. The true
hematologic or endothelial nature of CFU-ECs and E-CFCs was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and the
expression of von Willebrand factor, CD146, CD45, KDR, CD11b, and
CD115, as described.9 The following primers were used: KDR, forward
5⬘-CCC ACG TTT TCA GAG TTG GT-3⬘ and reverse 5⬘-CTA CCG GTT
TGC ACT CCA AT-3⬘; GAPDH (glyceraldehyde-3-phosphate dehydrogenase), forward 5⬘-AGG TGA AGG TCG GAG TCA ACG-3⬘ and reverse
5⬘-GCT CCT GGA AGA TGG TGAT GG-3⬘; von Willebrand factor,
forward 5⬘-TAA GTC TGA AGT AGA GGT GG-3⬘ and reverse 5⬘-AGA
GCA GCA GGA GCA CTG GT-3⬘; CD146, forward 5⬘-CCA AGG CAA
CCT CAG CCA TGT C-3⬘ and reverse 5⬘-CTC GAC TCC ACA GTC TGG
GAC GAC T-3⬘; CD34, forward 5⬘-TGA AGC CTA GCC TGT CAC CT
and reverse 5⬘-CGC ACA GCT GGA GGT CTT AT; CD45, forward
5⬘-AAT GAG AAT GTG GAA TGT GG and reverse 5⬘-TTG CGT TAG
TAA ACT TGT GG; CD115, forward 5⬘-CAC CAA GCT CGC AAT
CCC-3⬘ and reverse 5⬘-CTC TAC CAC CCG GAA GAA CA-3⬘; CD11b,
forward 5⬘-GCC GGT GAA ATA TGC TGT CT-3⬘ and reverse 5⬘-GCG
GTC CCA TAT GAC AGT CT-3⬘. PCR conditions for amplification
consisted in initial denaturation step at 94°C for 3 seconds, followed by
JAK2 and MPL mutations
Figure 1. Representative images of CFU-EC and E-CFC colonies and the typical
RNA transcript, assessing the hematologic derivation of CFU-ECs and the true
endothelial origin of the E-CFCs. GAPDH indicates glyceraldehyde-3-phosphate
dehydrogenase; VWF, von Willebrand factor; HUVEC, human umbilical vein endothelial cell.
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
TEOFILI et al
Table 2. E-CFC frequency in healthy blood donors and in patients with MPN grouped according to the diagnosis
CFU-EC,
106
cells, median (range)
E-CFC, 107 cells, median (range)
Samples with ⱖ 1 E-CFC, n/N
Controls
PV
ET
PMF
4.9 (0-42.5)
0.75 (0-15.8)
1.2 (0-6.5)
0.7 (0-20.7)
0.1 (0-0.8)
0.1 (0-6.7)
0 (0-9.6)
0.8 (0-8)
14/25
6/15
6/12
10/15
Patients with PMF showed a higher E-CFC frequency than controls (P ⫽ .0078).
negative patients. The presence or the absence of the JAK2 mutation was
confirmed by direct sequencing with the use of the external primers
described in the method of Baxter et al12. The presence of MPL mutations
(MPLW515L and MPLW515K) was investigated by sequencing, using the same
primers and PCR conditions previously described.23
JAK2V617F allele burden
The mutant allele burden was measured by a quantitative real-time PCR
assay, following the methods previously reported.25 All samples were
analyzed in triplicate.
Human androgen receptor analysis
The clonal pattern of growth was assessed by the HUMARA polymorphism
assay and by the HUMARA methylation-specific PCR (MS-PCR) in
granulocytes and in pooled E-CFCs isolated from female patients, as
previously described.22 T lymphocytes were used as control. Briefly, 1 ␮g
of DNA was incubated with and without 20 U of HpaII (New England
Biolabs) at 37°C for 12 hours. After 10 minutes of incubation at 95°C, 3 ␮L
of all samples was amplified with specific primers as previously described.22 Results obtained by the HUMARA polymorphism assay were
confirmed by HUMARA MS-PCR analysis. Briefly, after treatment of 1 ␮g
of DNA with sodium bisulfate, purification, and desulfonation with sodium
hydroxide, DNA was amplified with specific primers for the methylated and
unmethylated HUMARA gene.23 PCR amplification and correction of the
ratio of peaks for an appropriate X-inactivation pattern were performed
with the ABI PRISM 3100Avant Genetic Analyzer and GeneMapper ID
software (Applied Biosystems).
MS-PCR for SOCS-1, SOCS-2, and SOCS-3
cytosine-phosphate-guanosine islands
Methylation status of SOCS-1, SOCS-2 and SOCS-3 cytosine-phosphateguanosine islands was investigated by MS-PCR as described.11 Genomic
DNA (1 ␮g) was subjected to bisulfite modification with the use of EpiTect
Bisulphite kit (QIAGEN), according to the manufacturer’s protocol, and
then amplified in a mixture containing PCR buffer containing primers
(20pM each) and 0.25 U of GoTaq polymerase (Promega).11 PCR products
were electrophoresed in a 2.5% agarose gel, stained with ethidium bromide,
and visualized under UV light. DNA isolate from normal lymphocytes,
hypermethylated with SssI methyltransferase (New England Biolabs), and
subsequently modified with bisulfite was used as unmethylated and
methylated controls; water was used as negative control; unmodified DNA
was used as internal control of MS-PCR. All samples that resulted in
methylation were subjected to sequencing, either directly or after subcloning.11 Briefly, the PCR products were cloned into a pGEM-T easy vector
system (Promega) with the use of JM109 high-efficiency competent cells.
Five randomly picked clones were sequenced on an ABI PRISM 3100
Genetic Analyzer (Applied Biosystems).
Western blot analysis of pSTAT-3 and pSTAT-5
In selected experiments, E-CFCs recovered in each patient were pooled and
evaluated for the expression of pSTAT-3 and pSTAT-5, as described.22
Briefly, after cell lysis, supernatant proteins were recovered, dissolved in
sodium dodecylsulfate sample buffer, boiled for 5 minutes, and then separated on
sodium dodecylsulfate–polyacrylamide gel electrophoresis. Gels were blotted
with transfer buffer directly on pure nitrocellulose membrane (Bio-Rad) at
330 mA for 1 hour. After not specific binding sites blocking, blots were probed
with an anti–pSTAT-3 (1:500; Santa Cruz Biotechnology), anti–pSTAT-5 (1:500;
BD Biosciences), and anti-actin (Ab-5, 1:5000; BD Biosciences) mouse monoclonal antibodies. Blots were the incubated with goat anti–mouse horseradish
peroxidase (HRP)–conjugated antiserum (1:1000; BD Biosciences), covered
with enhanced chemiluminescence solutions for 1 minute (Western blotting
analysis system; GE Healthcare) and exposed to Kodak XAR-5 x-ray film
(Kodak). Precipitates were subjected to densitometric analysis with the use of the
Gel-Doc 2000 Quantity One program (Bio-Rad), after normalization with the
actin intensity.
Mononuclear and endothelial cell adhesion assay
E-CFCs obtained from healthy donors and from patients were grown to
confluence and were assayed for the adhesion to normal mononuclear cells.
Briefly, normal mononuclear cells isolated from healthy donors were
divided in 2 aliquots; one of them was labeled with 0.2␮M/106 cells of
CellTrace carboxyfluorescein diacetate succynimidyl ester (CFSE; Invitrogen) in phosphate-buffered saline (PBS) containing 2% of fetal bovine
serum, for 15 seconds at 37°C in darkness. The surplus of CFSE was
discharged by repeated washes with PBS. Mononuclear cells exposed or not
(as negative control) to CFSE staining were then plated over confluent
E-CFCs in 6-well plates, at a concentration of 106 cells/well and incubated
at 37°C for 1 hour. Nonadherent cells were then removed by 3 courses of
gentle PBS washing. Mononuclear cells adherent to E-CFCs were then
detached from the well bottom together with E-CFCs by trypsin (Lonza).
The cell suspension was then analyzed by flow cytometry at a 488-nm
excitation source (FACSCanto 4; BD Biosciences). CFSE-positive cells
were defined after detracting the autofluorescence of the negative control.
Results are expressed as the percentage of CFSE-positive cells over total
counted events. To exclude that enhanced cell-to-cell adhesion properties in
vivo may have led to hematopoietic-endothelial cell fusion in vitro, after the
adhesion assay, aliquots of E-CFCs were analyzed for the mRNA expression of hematopoietic-specific antigens (CD45 and CD11b), both at early
and late passages.
Statistical analysis
Statistical analysis was performed with using GraphPad Prism (Version
5.00 for Windows; GraphPad Software Inc). All P values were 2-tailed, and
statistical significance was set at the level of P ⬍ .05. Comparison between
categorical variables was performed by ␹2 statistics. Comparison between
continuous variables was performed by either the Mann-Whitney U test or
the Kruskal-Wallis test. Survival curves were obtained by the Kaplan-Meier
method and then compared through the log-rank test.
Results
CFU-EC and E-CFC recovery
Table 2 shows the frequency of CFU-ECs and E-CFCs in patients
with MPN in comparison with healthy persons. In general, patients
with MPN exhibited a low level of CFU-ECs (P ⫽ .048 in
comparison with healthy controls at the Kruskal-Wallis test).
Moreover, the level of CFU-ECs in patients with MPN experiencing thrombosis was significantly lower than in patients without
thrombosis (median value/106 plated cells, 0 ⫾ 0.5 versus 3 ⫾ 1,
respectively; P ⫽ .027). Conversely, the frequency of E-CFCs was
higher in patients with patients than in healthy controls, although
the differences were statistically significant only for the PMF group
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ENDOTHELIAL PROGENITORS AND MPN
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Table 3. Myeloproliferative molecular markers detected in granulocytes and in E-CFCs isolated from patients with MPN
Granulocytes
Patient
Diagnosis
Thrombosis
JAK2
SOCS methylation
E-CFCs
HUMARA
Colonies assayed
JAK2
SOCS methylation
HUMARA
1
PMF
No
wt
Yes
Polyclonal
20
wt
No
Polyclonal
2
PMF
No
V617F
No
NE
7
wt
No
NE
3
PMF
No
V617F
Yes
NE
2
wt
No
NE
4
PV
No
V617F
Yes
NE
3
wt
No
NE
5
ET
No
wt
Yes
NE
10
wt
No
NE
6
PMF
No
wt
Yes
NE
2
wt
No
NE
7
PMF
No
V617F
Yes
NE
14
wt
No
NE
8
ET
No
V617F
No
NE
3
wt
No
NE
9
ET
No
V617F
No
Clonal
7
wt
No
Polyclonal
10
PV
No
V617F
No
Clonal
Polyclonal
11
PV
No
V617F
No
NE
12
PV
No
V617F
No
Clonal
13
ET
No
V617F
Yes
NE
1
14
PV
No
wt
Yes
NE
3
15
ET
No
V617F
Yes
Polyclonal
2
16
PMF
No
wt*
Yes
Clonal
17
PMF
No
V617F
Yes
NE
18
PMF
PT
V617F
No
Clonal
19
ET
BCS
V617F
No
Clonal
20
PMF
PE, DVT
V617F
No
NE
21
PMF
DVT
V617F
No
22
PV
Stroke
V617F
No
5
wt
No
11
wt
No
NE
2
wt
No
NE
wt
Yes
NE
wt
Yes
NE
wt
Yes
NE
5
wt*
Yes
Polyclonal
4
wt
Yes
NE
21
V617F
No
Clonal
10
V617F
No
Clonal
2
V617F
No
NE
Clonal
6
V617F
No
Clonal
Clonal
10
V617F
No
Clonal
wt indicates wild type; NE, not evaluable; PT, portal vein thrombosis; BCS, Budd-Chiari syndrome; PE, pulmonary embolism; and DVT, deep vein thrombosis of the legs.
*This patient carried the MPLK515L mutation.
(P ⫽ .0078). Among patients with MPN, 6 of 15 patients with PV,
6 of 12 patients with ET, and 10 of 15 patients with PMF showed
E-CFC growth (Table 2). The levels of CFU-ECs and E-CFCs that
we observed in our series of patients and in healthy controls and the
percentage of samples producing ⱖ 1 E-CFC colony are in good
agreement with those reported by other investigators that used the
same methods.6
MPN molecular markers in CFU-ECs and E-CFCs
Among the 22 patients showing the growth of ⱖ 1 E-CFC, 11 were
JAK2V617F mutated, 5 presented with both JAK2V617F mutation and
SOCS gene hypermethylation, 1 patient had MPLW515K mutation
and SOCS gene hypermethylation, and 5 patients had SOCS gene
hypermethylation (Table 3). Ten patients were women and could
be evaluated also for the human androgen receptor analysis
(HUMARA) assay. According to their hematologic signature and to
confirm previous findings from other groups,6,7 we found that
CFU-ECs in these patients always exhibited the same molecular
markers as granulocytes (data not shown). In contrast, the behavior
of E-CFCs was variable, and we could identify 3 groups of patients
(Table 3). On the whole, the mean number of assayed E-CFCs for
each patient was 7 in the first group, 5 in the second group, and 9 in
the third group (P ⫽ .14). The first group included 12 patients who
had normal E-CFCs, lacking those MPN molecular alterations
observed in the respective granulocyte samples (Table 3). In 3 of
these patients, we evidenced that the pattern of growth of E-CFCs
was polyclonal at the HUMARA assay. Conversely, E-CFC
progenitors obtained from the remaining 10 patients exhibited
molecular myeloproliferative signatures. In particular, E-CFCs
collected from 5 patients (13-17; Table 3) showed a pattern of
SOCS gene hypermethylation overlapping that found in the respective granulocytes; in particular SOCS-1 gene was involved in
3 cases (patients 14, 15, and 17; Table 3), SOCS-2 in 1 case (patient
13, Table 3), and both SOCSC-1 and SOCS-2 in 1 case (patient 16;
Table 3). Interestingly, although 3 of them were JAK2V61F positive
and 1 was MPLK515L positive, these mutations were not detected in
E-CFCs. In one case (patient 13; Table 3) we found E-CFCs both
unmethylated (1 colony) and methylated (4 colonies). When these
colonies were pooled and assayed for the HUMARA, they appeared
polyclonal. Indeed, these observations show evidence that in this
patient normal endothelial progenitors may persist and circulate.
Finally, the third group included 5 patients exhibiting the growth of
JAK2V61F-positive E-CFCs (patients 18-22; Table 3). All the
examined E-CFCs were JAK2V61F-positive, and no JAK2 wild-type
colonies were observed. The burden of the JAK2V61F allele in each
examined E-CFC was variable from colony to colony and ranged from
30% to 74% (median value, 52%). Moreover, in 4 patients, the
HUMARA assay performed on pooled E-CFCs documented that
endothelial progenitors were clonally related (Table 3). These findings
suggest that, in this subgroup of patients, the endothelial progenitor cell
compartment is entirely involved by the neoplastic transformation,
whereas each progenitor might exhibit variable proportions of wild-type
and mutated alleles. The detection of SOCS gene hypermethylation and
Figure 2. RNA expression (RT-PCR) of myeloid lineage–associated antigens
(CD45, CD115, and CD11b) and GAPDH (internal control). Lane 1, peripheral
blood mononuclear cells (positive control); lane 2, CFU-ECs from healthy control;
lanes 3-5, E-CFCs at passages I obtained in 3 different patients with JAK2V617F
E-CFCs; lane 6-8, E-CFCs at passages IV obtained from the same 3 patients. MW
indicates molecular weight marker (50 base pair); GAPDH, glyceraldehyde-3phosphate dehydrogenase.
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TEOFILI et al
BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
Figure 3. Antiproliferative therapy–free survival.
(A) Comparison between patients showing or not the
E-CFC growth (P ⫽ .75). (B) Comparison among the
different groups of patients showing the E-CFC growth
according to the molecular signatures found in E-CFC
progenitors.
of JAK2V617F mutation in E-CFCs was confirmed after expanding
additional colonies for 2 or 3 passages. Finally, we rule out the
possibility that the detection of MPN molecular signatures in E-CFCs
could result from the in vivo hematopoietic-endothelial cell fusion.
Actually, we demonstrated that JAK2V617F E-CFCs isolated from 3
patients with MPN, evaluated at both early and late passages, do not
express myeloid lineage–associated antigens (CD45, CD115, and CD11b)
(Figure 2).
Clinical characteristics of patients grouped according to
E-CFC findings
Overall, among 42 patents investigated, 10 experienced thrombosis
(2 portal thrombosis, 4 deep vein thrombosis, 1 deep vein
thrombosis with pulmonary embolism, 1 transient ischemic attack,
1 stroke, 1 BCS) and 23 required antiproliferative therapy (hydroxyurea or interferon). The criteria for starting therapy were
history of thrombosis (10 patients), splenomegaly (4 patients),
failure of phlebotomy in lowering hematocrit (6 patients), age
⬎ 65 years (2 patients), platelet count ⬎ 1500 ⫻ 109/L (1 patient).
First, we evaluated if the ability to produce E-CFCs could identify
a particular subset of patients with MPN, having peculiar clinical
or hematologic features. The 2 groups of patients (22 with and
20 without E-CFCs) were comparable for age, diagnosis, and sex
distribution; hematologic findings at the first observation (data not
shown); and disease duration (mean, 49 ⫾ 9 months vs 54 ⫾ 9 in
patients with and without E-CFC growth, respectively; P ⫽ .52).
No differences were found for the presence of SOCS gene
hypermethylation (P ⫽ .12), of JAK2V617F mutation (P ⫽ .72), and
of JAK2V617F allele burden (P ⫽ .79); we did not observe a
different incidence of thrombosis (P ⫽ .53). Moreover, the proportion of patients requiring antiproliferative therapy (P ⫽ .72) and
the antiproliferative therapy–free survival were similar in both
groups (P ⫽ .75; Figure 3A). Indeed, we focused on the 22 patients
showing the E-CFC growth, grouped as having normal E-CFC,
hypermethylated E-CFCs, or JAK2V617F-positive E-CFCs. We did
not find differences among the 3 groups for age, diagnosis, and sex
distribution (data not shown); disease duration (mean,
59 ⫾ 14 months in patients with normal E-CFCs, 45 ⫾ 15 in
patients with hypermethylated E-CFCs, and 53 ⫾ 17 months in
Figure 4. Expression of pSTAT-3 and pSTAT-5 in
E-CFCs isolated from 2 healthy persons (1 and 2),
from 2 patients with JAK2 wild-type E-CFCs (3 and 4),
and in JAK2V627F-mutated E-CFCs obtained from 3 patients (5-7). Values are expressed in arbitrary units as the
ratio of the densitometric analysis between pSTAT-3 and
pSTAT-5 and the respective ␤-actin bands.
patients with JAK2V617F E-CFCs, respectively; P ⫽ .98); and
percentage of JAK2-mutated subjects (P ⫽ .32). Nevertheless,
compared with patients with JAK2 wild-type E-CFCs, those with
JAK2-mutated E-CFCs exhibited higher incidence of thrombosis
(P ⬍ .0001), higher JAK2V61F allele burden (mean, 70 ⫾ 6 vs
39 ⫾ 8, respectively; P ⫽ .022), and higher leukocyte count
(15.95 ⫾ 2.43 ⫻ 109/L vs 8.64 ⫾ 0.8 ⫻ 109/L, respectively;
P ⫽ .007). Among patients requiring antiproliferative therapy
(13 cases), 5 showed JAK2-mutated E-CFCs and 4 showed
SOCS-methylated E-CFCs (P ⫽ .011). Finally, the median antiproliferative drug–free survival was 2 months for patients exhibiting
JAK2-mutated E-CFCs, 18 months for those with hypermethylated
E-CFCs, whereas it was not reached in patients without molecular
abnormalities (P ⫽ .013 at the log-rank test in comparison with
patients with JAK2-mutated E-CFCs; Figure 3B).
Functional characterization of altered endothelial progenitors
The presence of JAK2 mutation and clonality (but not of SOCS
gene hypermethylation) in E-CFCs was associated with an increased incidence of vascular complications. Indeed, we first
evaluated if the JAK2V617F mutation in these cells induced an
increased phosphorylation of the downstream STAT pathway in
respect to endothelial progenitors with wild-type JAK2. To this
purpose, Western blot analysis for pSTAT-3 and pSTAT-5 was
carried out in endothelial progenitors expanded from 2 healthy
persons, from 2 patients with MPN showing no abnormalities in
their E-CFCs, and from 3 patients exhibiting the growth of
JAK2V617F-mutated colonies. Results obtained are reported in
Figure 4 and show that the JAK/STAT pathway is exaggeratedly
activated in cells originating from abnormal progenitors, whereas
both STAT-3 and STAT-5 exhibited a low phosphorylation rate in
E-CFCs isolated from healthy persons and in patients with MPN
with E-CFCs showing no molecular abnormalities. For this reason
we investigated by the Matrigel assay the ability of JAK2V617Fmutated E-CFCs to form tubes in vitro, but, as previously reported
for JAK2 wild-type E-CFCs isolated from patients with MPN,6
we did not find differences in respect to E-CFCs obtained from
healthy controls (data not shown), suggesting that molecular
abnormalities do not affect their angiogenetic ability. To further
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BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
ENDOTHELIAL PROGENITORS AND MPN
2705
the coculture adhesion assay, we evaluated the expression of
CD45 and CD11b either at first passage after the adhesion assay
and thereafter at late passages, and we found that it was always
absent (Figure 5D).
Discussion
Figure 5. CFSE-labeled mononuclear cell adhesion to E-CFCs. Representative
experiments showing (left) the cytofluorimetric analysis of unstained peripheral blood
mononuclear cells (negative control) and (right) the number of CFSE-labeled
mononuclear cells bound by E-CFCs (A) in a healthy control and (B) in a patient with
JAK2V617F-positive E-CFCs. (C) Percentages of mononuclear cells adherent to
normal E-CFCs (plot 1, mean values ⫾ SEM of 4 healthy controls), to JAK2 wild-type
E-CFC (plot 2, mean values ⫾ SEM of 2 patients), and to JAK2V627F-mutated E-CFCs
(plot 3, mean values ⫾ SEM of 4 patients). (D) RNA expression (RT-PCR) of CD45,
CD11b, and GAPDH (internal control) in E-CFCs recovered from the adhesion assay,
at early or late passages. Lane 1 shows peripheral blood mononuclear cells (positive
control); lanes 2 and 3, JAK2 wild-type E-CFCs isolated from a patient with MPN at
passages I and IV; lanes 4 and 5, JAK2V617F E-CFCs at passages I and IV. FITC,
fluorescein isothiocyanate; MW, molecular weight marker (50 base pair); GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
explore the effect of JAK2V617F mutation on endothelial cell
function, we evaluated the ability of endothelial cells to adhere to
normal mononuclear cells. The representative experiments performed in a healthy control and in a patient with JAK2V617F-positive
E-CFCs are shown in Figure 5A and B, respectively. The percentages of mononuclear cells adherent to E-CFCs acquired from
healthy controls and from patients are shown in Figure 5C (mean
values of experiments performed with 2 JAK2 wild-type E-CFCs
and 4 JAK2V617F E-CFCs). Actually, JAK2V617F-mutated E-CFCs
showed a significantly higher adhesion proficiency to mononuclear
cells than normal E-CFCs (P ⫽ .033). Finally, to evaluate whether
the JAK2V617F E-CFCs might have bound mononuclear cells in
The pathophysiologic mechanisms linking JAK2V617Fmutation and
the risk of thrombosis in patients with Ph-negative MPNs remain
largely speculative. This study explored the hypothesis that the
oncogenic lesion could hit a common endothelial and hematopoietic progenitor cell, inducing an endothelial cell dysfunction
primarily responsible for the pathogenesis of the vascular damage.
Either among healthy persons or patients with MPN, endothelial
progenitor cells are detectable through cell culture assay only in a
part of subjects, and we are not aware of biologic causes underlying
this finding. Overall, our observations suggest that the ability to
give rise to endothelial progenitor colonies does not identify a
particular subset of patients with MPN having different hematologic or clinical peculiarities. Moreover, we show in this study that
endothelial progenitor colonies isolated from patients with MPN
display different profiles in respect to disease-specific molecular
markers.
In general population, the reduced number of circulating
endothelial progenitor cells predicts future cardiovascular events,18,26
and it is has been hypothesized that also in patients with PV this
mechanism could account for the increased thrombotic risk.4 The
first finding of our study is that patients with PV and ET have a
level of true circulating endothelial progenitors in the normal
range, whereas patients with PMF have an increased number of
E-CFCs. Our data well agree with those reported in another study
using the same rigorous cell culture procedures.6 Dissimilar results
have been reported by other groups, but the discrepancy can be
explained by the use of different culture time and conditions, which
evaluated hematopoietic-derived endothelial-like colonies (ie,
CFU-ECs) rather than the true endothelial ones (E-CFCs).4,7
Accordingly, we found that in our series of patients, and in
particular in those patients with history of thrombosis, the CFU-EC
level was significantly lower than in healthy subjects. In addition,
the increased level of E-CFCs found in patients with PMF appears
to be an interesting finding, considering that a defective stem cell
niche would take part in the pathogenesis of this disease.27
Actually, the development of the pathologic clone in PMF is deeply
influenced by alterations of the microenvironmental niches;
thus, the imbalance between endosteum and vascular niches within
the bone marrow could favor proliferation of hematopoietic,
mesenchymal, and endothelial progenitors and their mobilization
through the blood.27
The second finding of this study is that, in a subset of patients
with MPN, molecular alterations usually observed in hematopoietic cells may be detected also in the circulating endothelial
progenitors. Several groups investigated the relationship between
endothelial cells and hematologic neoplasms,27-32 but the search for
clonal markers in endothelial cells isolated from patients with
Ph-negative MPN produced different and conflicting results. On
the one hand, Piaggio et al6 firmly excluded that E-CFCs obtained
from patients with chronic myeloproliferative diseases (including
patients with chronic myeloid leukemia) harbored the diseasespecific molecular clonality marker (JAK2V617F mutation or BCRABL rearrangement). On the other hand, Sozer et al8 not only
reported the presence of JAK2V617F mutation in the liver endothelial
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2706
BLOOD, 3 MARCH 2011 䡠 VOLUME 117, NUMBER 9
TEOFILI et al
cells of patients with BCS but also demonstrated that CD34⫹ cells
isolated from peripheral blood of patients with JAK2V617F PV or
PMF are capable of generating both wild-type and JAK2-mutated
endothelial-like cells when transplanted into nonobese diabetic/
severe combined immunodeficient mice. In addition, Yoder et al7
reported that a minority of E-CFCs derived from a patient with
vascular thrombosis and subsequently developing JAK2V617F PV,
carried the JAK2V617F mutation. Finally, several studies established that hemangioblasts are present in chronic myeloid
leukemia and contribute to both malignant hematopoiesis and
endotheliopoiesis.28,33
In this study the pathologic involvement of endothelial progenitor compartment was explored by evaluating additional molecular
markers of disease other than JAK2 or MPL mutations, such as
clonality and hypermethylation of SOCS genes. We demonstrated
that in patients with MPN a part of molecular signatures found in
hematopoietic cells (both granulocytes and CFU-EC colonies) can
be detected also in endothelial progenitors. Indeed, our observations add evidence to the existence of a common hematopoietic and
endothelial bi-lineage progenitor also in Ph-negative MPNs. The
third finding of this study is that patients with molecular abnormalities in E-CFCs need antiproliferative therapy more frequently and
earlier than others. Actually, a significantly shorter antiproliferative
therapy–free survival was observed in patients with JAK2V617Fpositive E-CFCs.
Finally, we found that the detection of clonality and JAK2
mutation in E-CFCs was associated with a thrombotic proficiency.
Interestingly, these patients have at diagnosis high JAK2 allele
burden and high leukocyte count, 2 characteristics that have been
associated with an increased thrombotic risk.34 Actually, the
increased number of neutrophils has emerged as one of the most
relevant factors contributing to the thrombophilic state of patients
with MPN, and neutrophil activation occurs in these patients in
parallel with the appearance of laboratory signs of hemostatic
system activation.34 Moreover, the presence of high JAK2-mutated
allele burden has been variably associated with both thrombosis
and high leukocyte count.34 Our study supports the hypothesis that
the pathogenesis of thrombosis in MPN may result from an
endothelial progenitor cell dysfunction, as well. In fact, we show
that the JAK2 mutation in endothelial cells abnormally activates the
JAK/STAT pathway, which is an important regulator of the
response of endothelial cells to injury35,36 and increases their
proficiency to adhere to normal mononuclear cells. Interestingly,
2 of the 3 patients exhibiting clonal and mutated E-CFCs had
thrombosis in the splanchnic veins (1 portal thrombosis and
1 BCS), whereas a third patient presenting with massive bleeding
from gastric varices had severe portal hypertension, albeit in the
absence of evident previous portal or suprahepatic vein thrombosis.
Indeed, these findings well agree with previous observations that
JAK2-mutated endothelial cells could be isolated from the liver of
patients with BCS.8,9
In conclusion, this study suggests that endothelial progenitor
cell dysfunction might sustain the thrombophilic state observed in
patients with MPN and unravels an additional mechanism by which
antiproliferative therapies could reduce the thrombotic risk. The
main limit of this study resides in its retrospective nature, and only
prospective investigations may definitely confirm our observations.
Nevertheless, the definition of molecular profile of endothelial
progenitors appears to be an attracting topic, because it could
represent a useful tool for evaluating the individual thrombotic risk
in each patient with MPN.
Acknowledgments
This work was supported by a grant from Fondazione Roma
“Progetto cellule staminali. Una nuova frontiera nella ricerca
biomedica” and by PRIN 2008, Ministero Università e Ricerca
Scientifica (Rome, Italy).
Authorship
Contribution: L. Teofili and M.M. designed the study, analyzed
data, and wrote the manuscript; M.G.I. and E.R.N. carried out cell
cultures and analyzed data; L. Torti enrolled patients and recorded
clinical data of patients; S.C. and T.C. performed molecular
analysis; and L.M.L. and G.L. designed the study and critically
reviewed the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Luigi M. Larocca, Istituto di Anatomia Patologica, Università Cattolica, Largo Gemelli 8, 00168 Roma, Italy;
e-mail: [email protected]
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From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
2011 117: 2700-2707
doi:10.1182/blood-2010-07-297598 originally published
online January 6, 2011
Endothelial progenitor cells are clonal and exhibit the JAK2V617F
mutation in a subset of thrombotic patients with Ph-negative
myeloproliferative neoplasms
Luciana Teofili, Maurizio Martini, Maria Grazia Iachininoto, Sara Capodimonti, Eugenia Rosa
Nuzzolo, Lorenza Torti, Tonia Cenci, Luigi Maria Larocca and Giuseppe Leone
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