Leukemia (2002) 16, 911–919
 2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00
www.nature.com/leu
B-CLL cells are capable of synthesis and secretion of both pro- and anti-angiogenic
molecules
NE Kay1, ND Bone1, RC Tschumper2, KH Howell2, SM Geyer3, GW Dewald4, CA Hanson4 and DF Jelinek2
1
Department of Medicine, Division of Hematology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, MN, USA; 2Department
of Immunology, Mayo Graduate and Medical Schools, Mayo Clinic, Rochester, MN, USA; 3Department of Biostatistics, Mayo Graduate and
Medical Schools, Mayo Clinic, Rochester, MN, USA; and 4Department of Laboratory Medicine and Pathology, Mayo Graduate and Medical
Schools, Mayo Clinic, Rochester, MN, USA
Initial work has shown that clonal B cells from B-chronic lymphocytic leukemia (B-CLL) are able to synthesize pro-angiogenic molecules. In this study, our goal was to study the spectrum of angiogenic factors and receptors expressed in the CLL
B cell. We used ELISA assays to determine the levels of basic
fibroblast growth factors (bFGF), vascular endothelial growth
factor (VEGF), endostatin, interferon-␣ (IFN-␣) and thrombospondin-1 (TSP-1) secreted into culture medium by purified
CLL B cells. These data demonstrated that CLL B cells spontaneously secrete a variety of pro- and anti-angiogenic factors,
including bFGF (23.9 pg/ml ± 7.9; mean ± s.e.m.), VEGF (12.5
pg/ml ± 2.3) and TSP-1 (1.9 ng/ml ± 0.3). Out of these three factors, CLL B cells consistently secreted bFGF and TSP-1, while
VEGF was expressed in approximately two-thirds of CLL
patients. Of interest, hypoxic conditions dramatically upregulated VEGF expression at both the mRNA and protein levels. We
also employed ribonuclease protection assays to assay CLL B
cell expression of a variety of other angiogenesis-related molecules. These analyses revealed that CLL B cells consistently
express mRNA for VEGF receptor 1 (VEGFR1), thrombin receptor, endoglin, and angiopoietin. Further analysis of VEGFR
expression by RT-PCR revealed that CLL B cells expressed
both VEGFR1 mRNA and VEGFR2 mRNA. In summary, these
data collectively indicate that CLL B cells express both proand anti-angiogenic molecules and several vascular factor
receptors. Because of the co-expression of angiogenic molecules and receptors for some of these molecules, these data
suggest that the biology of the leukemic cells may also be
directly impacted by angiogenic factors as a result of autocrine
pathways of stimulation.
Leukemia (2002) 16, 911–919. DOI: 10.1038/sj/leu/2402467
Keywords: B-CLL; vascular factors; TSP-1; VEGF
Introduction
The process of angiogenesis in human tumors is now known
to play a critical role in many facets of tumor development.
The vascularization of solid tumors is linked to its subsequent
ability to disseminate with resultant more aggressive disease.1–3
The vascular phase of most tumors is linked to the ability of
a tumor cell to initiate and then become committed to the
production of pro-angiogenic factors.4,5 This latter component
is dominant to the simultaneous synthesis of negative regulators or anti-angiogenic factors by the same tumor. The shift
in balance between production of anti-angiogenic factors and
pro-angiogenic factors is a process that has been referred to
as an angiogenic switch. It has been suggested that the angiogenic switch marks the onset of neovascularization which is
probably a key step during tumor progression.4,5
The vast majority of the initial work in tumor-related angiogenesis has been conducted using solid tumor lines or models.
More recently, abnormal bone marrow angiogenesis has also
Correspondence: NE Kay, Mayo Clinic, 200 First Street SW, Rochester, Minnesota 55905, USA; Fax: 507-266-9277
Received 27 June 2001; accepted 16 January 2002
been observed in a number of diseases, including a variety of
leukemias. Thus, patients with acute lymphocytic leukemia,
acute myeloid leukemia, non-Hodgkin’s lymphoma, multiple
myeloma, myelofibrosis and acute promyelocytic leukemia
were shown to have increases in bone marrow microvessel
density.6–13 Recently, we and others14–18 have defined a variety of abnormalities in the angiogenic status of patients with
B-chronic lymphocytic leukemia (B-CLL). These abnormalities
have included increased microvessel density in marrow14 and
lymph nodes15 and increased levels of certain pro-angiogenic
vascular growth factors including basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF)
in blood and urine of B-CLL patients.14,17,18 There is evidence
that the tumor cells themselves may be an important source
of these pro-angiogenic factors. Indeed, these vascular growth
factors have been demonstrated in the cytoplasm of the CLL
B cell16,17 and are present in the culture medium of CLL B
cells (Kay, unpublished observations). Despite evidence that
B-CLL cells express and release vascular proteins, the precise
role that these factors play in the disease biology of B-CLL
remains unclear. Thus, it remains possible that bFGF and
VEGF may effect increased angiogenesis as well as have direct
effects on the leukemic cells. Indeed, a recent publication has
suggested that VEGF may play a role in an autocrine pathway
for B-CLL cells.15
The goal of this study was to obtain a more complete
characterization of the angiogenic status of the CLL B cell. In
particular, we were interested in determining if the CLL B cell
is capable of undergoing an angiogenic switch resulting in an
increased production rate of pro-angiogenic factors. Recent
insight into human angiogenesis mechanisms now permits a
detailed analysis of both the pro- and anti-angiogenic factors
as well as the array of vascular factor receptors expressed by
a tumor cell. This report presents evidence that the CLL B cell
clones from early stage and/or stable disease patients are capable of expressing and modulating their production of a wide
variety of both pro- and anti-angiogenic molecules. This information adds to the possibility that CLL B cell clones can
undergo an angiogenic switch. In addition, we analyzed the
vascular factor data in relation to the presence of germline
(GL) vs somatic mutation-type B cell clones (SM). The latter
was done since recent evidence including our own has suggested that GL-type patients have a worse clinical outcome
compared to SM-type patients.19–21
Methods
B-CLL patients
The cohorts of B-CLL patients studied were diagnosed using
the NCI Criteria.22 B-CLL patients included in this study were
either previously untreated patients (n = 30) and were in Rai
B-CLL cells express pro- and anti-angiogenic molecules
NE Kay et al
912
stage 0–1, or were treated patients who were in Rai stages 2–
4. Patients in the latter group were only studied in this protocol if distant from therapy by at least 3 weeks. Venous blood
and urine samples were only obtained from patients providing
informed consent.
erated in a NuAire incubator (NuAire, Plymouth, MN, USA)
with 2.0% O2, 5% CO2 and N2.
Isolation of CLL B cells
We used a ribonuclease protection assay (RPA) to measure
mRNA levels of relevant angiogenic factors and their receptors. Total RNA was isolated from cells by the Trizol method
(Life Technologies, Grand Island, NY, USA). RNase protection
assays were done using the RiboQuant Multi-Probe RNase
Protection Assay System from BD PharMingen (San Diego,
CA, USA). The Human Angiogenesis template set was used,
in conjunction with the In Vitro Transcription Kit (BD Pharmingen kit component), for the synthesis of the radiolabeled
anti-sense RNA probes. The probes used in this RPA were able
to detect mRNA for the vascular factor receptors Flt1
(VEGFR1), Flt4, Tie 1, Tie 2, and the thrombin receptor. In
addition, this RPA detected mRNA coding for CD31 (PECAM1), endoglin, angiopoietin, VEGF, VEGF C, and the housekeeping gene L32. The RPA was performed according to the
manufacturers’ recommended protocol.
Peripheral blood mononuclear cells (PBMC) were separated
from heparinized venous blood by density gradient centrifugation. To remove adherent cells, PBMC were suspended in
RPMI 1640 supplemented with 10% fetal calf serum, and cells
were incubated in plastic dishes at 37°C for 1 h prior to collection of non-adherent cells. In order to obtain at least 95%
purity of CLL B cells, non-adherent cells were depleted of T
cells by incubation with sheep erythrocytes. Following density
gradient centrifugation, the rosette-negative cells were isolated from the gradient interface, and these cells typically consisted of ⬎97% CD19+/CD5+ cells. In some cases we used an
alternative method for purification of CLL B cells. In brief,
highly purified CD19+ B cells (⬎95%) were obtained from
PBMCs by standard negative selection using a cocktail of subset-specific antibodies conjugated with magnetic beads
(Miltenyi Biotech, Auburn, CA, USA). These purified CLL B
cells were then used immediately for the laboratory studies
described below or were cryopreserved in RPMI 1640, 20%
fetal calf serum, and 10% DMSO and stored in liquid nitrogen
until use. These cells were then analyzed for mRNA and/or
protein expression of cytoplasmic or secreted vascular factors
as described below.
Detection of pro- and anti-angiogenic factors and
vascular factor receptors in CLL patients
Detection of pro- and anti-angiogenic factors in urine:
Urine samples were collected using sterile techniques and
stored at −70°C before analysis. Control urine samples were
also obtained from age-matched individuals. We then measured urine levels of the pro-angiogenic factors, bFGF and
VEGF as well as the anti-angiogenic factors TSP-1, endostatin,
and IFN-␣. Basic FGF, VEGF, TSP-1, endostatin, and IFN-␣
levels were quantified using Quantikine solid phase enzymelinked immunoabsorbent assays (ELISA; R & D Systems, Minneapolis, MN, USA). The lower limits of detection by ELISA
for these vascular factors were ⬍1–2 ng/ml (for TSP-1, interferon alpha, and endostatin) and 0.5–5 pg/ml (for bFGF and
VEGF). The data are presented as mean ± standard error of
the mean where replicates were done.
Assessment of CLL B cell culture supernatants for the presence
of pro- and anti-angiogenic factors:
Purified CLL B cells
were cultured in RPMI containing 10% FCS at 1 × 106 cells
per ml for 24 h prior to collection of cell-free supernatants.
We assessed the levels of pro-angiogenic factors (bFGF and
VEGF) and also anti-angiogenic factors (TSP-1, endostatin,
IFN-␣) in the medium obtained from purified, cultured CLL B
cells after a 24 h in vitro incubation. We used PBMC from
healthy donors as controls for the secreted CLL B cell culture
medium experiments. In some experiments we cultured CLL
B cells at 1 × 106 cells per ml for 24 h in a hypoxic (1% O2)
or normoxic environment before collecting culture medium
for assessment of VEGF levels. Hypoxic conditions were genLeukemia
Quantification of mRNA coding for angiogenic
factors/receptors
VEGFR-1, VEGFR-2 and TSP-1 RT-PCR
cDNA was synthesized in a 15 ␮l reaction using the FirstStrand cDNA Synthesis Kit (Pharmacia, Piscataway, NJ, USA)
2.0 ␮g of total RNA was dissolved in 8 ␮l RNase-free water
and incubated at 65°C for 10 min. While on ice, 5 ␮l of the
bulk first-strand mix, 1 ␮l DTT, and 1 ␮l pd(N)6 primer were
added to the RNA and then incubated for 60 min at 37°C.
VEGFR1 (FLT-1), VEGFR-2 (KDR), and TSP-1 RT-PCR was carried out in a 50 ␮l reaction containing the following reagents:
25 ␮l of the HotStarTaq Master Mix Kit (Qiagen); 18 ␮l sterile
water; 2.5 ␮l each of forward and reverse primers (10 ␮M); 2
␮l of cDNA preparation. The cycling conditions for the KDR
and FLT-1 primers were as follows: stage 1, 95°C for 15 min;
stage 2, 40 cycles of 94°C for 30 s, 55°C (KDR) or 58°C (FLT1) for 60 s, 72°C for 60 s; stage 3, 72°C for 10 min. The cycling conditions for the TSP-1 and ␤-actin primers were as follows: stage 1, 95°C for 15 min; stage 2, (20, 23, 25, 27, 30,
35 or 40 cycles) of 94°C for 30 s, 57°C for 60 s; 72°C for 60
s; stage 3, 72°C for 10 min. The primers used were as follows:
KDR forward 5⬘-GACTGCACAAACCAGCTTCTG-3⬘; reverse
5⬘-GACATCAATTGCTGAAAGC-3⬘, band size = 588 bp; FLT1 forward 5⬘-TGTCATCGTTTCCAGACCC-3⬘; reverse 5⬘-TGTT
TCTTCCCACAGTCCC-3⬘, band size = 339 bp; TSP-1 forward
5⬘-GCCTGATGACAAGTTCCAAGA-3⬘; reverse 5⬘-CTTTGCG
ATGCGGAGTCT-3⬘, band size = 394 bp; ␤-actin forward 5⬘GGATCCGACTTCGAGCAAGAGATGGCCAC-3⬘; reverse 5⬘CAATGCCAGGGTACATGGTGGTG-3⬘, band size = 299 bp.
HeLa cells and A549 cells were used as positive controls for
KDR and Flt-1 mRNA. RT-PCR products were separated by
agarose gel electrophoresis on a 2% gel and products were
visualized under ultraviolet illumination of ethidium bromide
stained bands.
Nucleotide sequencing of the immunoglobulin (Ig)
variable heavy (VH) chain region of clonal CLL B
cells
This was done to permit definition of CLL B cell clones that
were either germline or somatic mutation type clones.
B-CLL cells express pro- and anti-angiogenic molecules
NE Kay et al
Preparation of RNA and cDNA:
Total RNA was isolated
from using the Trizol reagent (GIBCO BRL, Life Technologies,
Grand Island, NY, USA) according to the manufacturer’s
instructions. Two ␮g of total RNA was converted into cDNA
using the Pharmacia First-Strand cDNA Synthesis Kit.
Analysis of Ig VH gene mutation status:
Following dilution
of the cDNA reaction with distilled water, up to 50 ␮l, 2 ␮l
of cDNA were amplified in eight separate reactions. Thus, for
each B-CLL sample, seven PCR reactions were set up using
sense VH family-specific framework region primers that are
specific for each of the seven human Ig VH gene families in
conjunction with an antisense C␮ primer. The primer
sequences were identical to those previously used by Fais et
al.23 The eighth PCR reaction was set up using primers specific
for ␤-actin as previously reported.24 The PCR reactions were
carried out using a Qiagen Hotstart PCR Kit (Qiagen, Valencia, CA, USA) in a total volume of 50 ␮l and included 20 pmol
of each primer, 200 ␮M of each dNTP, and 2.5 U Hotstart Taq.
A Perkin Elmer 9600 thermocycler was used as follows: initial
denaturation at 94°C for 15 min followed by 34 cycles of 30
s at 94°C, 1 min at 60°C, and 1 min at 72°C. A final cycle of
10 min at 72°C was used before holding PCR reactions at 4°C.
PCR products were electrophoresed on 1.5% agarose gels,
bands visualized by ethidium bromide staining, and PCR products were sequenced after purification with Wizard PCR
Preps (Promega, Madison, WI, USA) using an automated sequenator (Applied Biosystems, Foster City, CA, USA). In some
instances, PCR products were first subcloned into the TA cloning vector (Invitrogen, San Diego, CA, USA), processed using
Wizard minipreps (Promega), and sequenced using M13
forward and reverse primers.
experiments. These data demonstrate that CLL B cells spontaneously secrete a variety of pro- and anti-angiogenic factors.
We consistently observed CLL cell secretion of bFGF, albeit
a wide range of secretion was observed (Figure 1a). In
addition, a subset of CLL patients secreted endostatin and IFN␣. Only leukemic cells from one out of 35 patients secreted
endostatin at approximately 0.4 ng per ml, while 32 of 35
patients’ cells secreted IFN-␣ into the culture medium. Surprisingly, we also found that CLL B cells from all patients
tested secreted what appeared to be consistently detectable
levels of the anti-angiogenic protein TSP-1 into the culture
medium (Table 1 and Figure 1b). While all CLL B cell clones
secreted bFGF and TSP-1 into the media, we only detected
VEGF in the culture medium of the cultured CLL B cells from
17 of 24 patients. We also assessed the presence of TSP-1 in
the cytoplasm of B-CLL cells (n = 6) using flow cytometry as
previously described by us.25 All B cell clones were positive
for TSP-1 in their cytoplasm with a mean percent of 57.9 ±
14.8 (range of 2.1–89.6%). The mean fluorescence intensity
ranged from 59.3 to 386.8 for the six B-CLL clones tested.
The mean levels of bFGF, VEGF and TSP-1 were compared
for B-CLL patients (n = 29) when subdivided into low risk Rai
stage (stage 0, n = 18) vs high risk Rai stage (stage 3–4, n =
11) and also for B-CLL patients subdivided based on GL vs
SM type clones (Figure 2a and b). The mean levels (± standard
error of the mean) bFGF (pg/ml), VEGF (pg/ml), and TSP-1
(ng/ml) in low vs high risk B-CLL patients were 72.1 ± 25.6
vs 337.9 ± 254.5, 2.4 ± 0.7 vs 6.6 ± 3.2 and 222.3 ± 114.1
vs 1.3 ± 0.4, respectively. We also compared bFGF, VEGF
and TSP-1 for GL (n = 10) vs SM type clones (n = 19) in this
913
Analysis of Ig gene sequences:
Nucleotide sequences
were aligned with those in the V BASE sequence directory
using DNAPLOT software. In a manner consistent with other
reports, germline Ig sequences were defined as those with
⬍2% sequence deviation from the most similar germline Ig
VH gene and those that had acquired somatic mutations were
defined as those with ⭓2% sequence differences.
Results
CLL cells secrete both pro- and anti-angiogenic factors
We initially determined the secretion of pro- and anti-angiogenic vascular factors from clonal B cells isolated from CLL
patients (n = 35). The patients utilized for this were from all
Rai stages but not currently on therapy. We assessed the levels
of angiogenic factors in the medium of CLL B cells that had
been freshly isolated and incubated without in vitro stimulation for 24 h. Table 1 displays the data obtained from these
Table 1
Summary of mean values of pro- and anti-angiogenic factors secreted by CLL B cells
Mean ± s.e.
Range
bFGFa
VEGFa
TSP-1a
Interferon-␣a
23.9 ± 7.9
0.6–64
12.5 ± 2.3
0–33
1.9 ± 0.3
0.23–8.1
2.7 ± 0.3
1.2–6.48
a
Values presented are in picograms/ml for bFGF and VEGF while
TSP-1 and IFN-␣ are in nanograms/ml. These data represent the
mean values obtained for 35 B-CLL patients.
Figure 1
Both pro- and anti-angiogenic vascular factors are
secreted from CLL B cells. (a) bFGF (pg/ml) is secreted from CLL cells
albeit at variable levels. (b) CLL B cells from all patients tested secreted
high levels of the anti-angiogenic protein TSP-1 (ng/ml) into the culture medium.
Leukemia
B-CLL cells express pro- and anti-angiogenic molecules
NE Kay et al
914
Figure 2
Box plots of ELISA levels of BFGF, VEGF and TSP-1 for (a) low (L) risk vs high (H) risk and (b) GL- vs SM-type clones in a cohort
of 29 B-CLL patients.
same cohort of 29 patients. These values were 107.2 ± 54.1
vs 180.8 ± 48.3, 5.9 ± 1.9 vs 3.3 ± 1.8 and 202.7 ± 65.9 vs
413.8 vs 135.7 for bFGF, VEGF and TSP-1, respectively.
We also evaluated and compared the level of vascular factors detected in the urine from a cohort of 31 B-CLL patients.
The mean levels of bFGF, VEGF, and TSP-1 in urine were 1.7
pg/ml ± 0.7 (range 0.51–3.4), 166.6 pg/ml ± 102 (range 49.5–
454.1) and 0.15 ng/ml ± 0.21 (range 0.006–0.9), respectively.
CLL B cells have augmented VEGF mRNA and protein
production with exposure to hypoxia
We have previously demonstrated that VEGF is found in the
urine of CLL patients.14 However, since we did not detect
VEGF in the culture medium of all CLL B cells exposed to
ambient air, we then tested whether hypoxia, a known
inducer of VEGF production,26 could induce CLL cells to upregulate the production and/or secretion of VEGF. CLL cells
from three patients were cultured either under normoxic or
hypoxic conditions. The levels of VEGF in the culture medium
under normoxic conditions for each patient (31.8, 23.9 and 0
pg/ml, respectively) were found to increase (147.1, 36.8 and
23 pg/ml, respectively) when separate aliquots of the same
CLL B cells were cultured under hypoxic conditions. Although
RT-PCR analysis in a separate cohort of B-CLL patients (n =
Leukemia
8) indicated that freshly isolated CLL B cells expressed variable levels of mRNA for VEGF (data not shown), we next
wanted to determine whether hypoxic conditions could
induce a quantitative change in the level of VEGF mRNA
expressed in the CLL B cells. To accomplish this, we used a
ribonuclease protection assay (RPA) to assess VEGF mRNA
levels from the same three B cell clones. Figure 3 illustrates
the dramatic upregulation of VEGF mRNA for CLL cells
exposed to hypoxia using the RPA method. The ratio increase
in VEGF mRNA for each CLL patient under hypoxic compared
to normoxic conditions was 21.8, 8.4 and 5.0, respectively.
CLL B cells express mRNA for several vascular factors
and vascular factor receptors
Since the CLL B cell clones were capable of secreting a wide
variety of both pro- and anti-angiogenic factors, we then
decided to use the RPA method to screen for mRNA
expression of other known vascular factors and vascular
receptors in CLL B cells. Figures 3 and 4 illustrate representative RPA results from isolated blood B cells from CLL patients.
The eight CLL patients illustrated in Figure 3a and b were
chosen based on whether the B cell clones were somatic
mutation-type clones (n = 4) or germline-type clones (n = 4).
This latter feature was determined by nucleotide sequencing
B-CLL cells express pro- and anti-angiogenic molecules
NE Kay et al
915
Figure 3
Upregulation of VEGF mRNA for CLL cells (n = 3)
exposed to hypoxic vs normoxic conditions using the RPA method.
The fold induction ratios for the three patients were 21.8, 8.4 and
5.0, respectively.
across the immunoglobulin (Ig) heavy chain variable region.
In all cases, the CLL B cells expressed mRNA for VEGFR1
(Flt1), albeit at different levels. In addition, there was consistent expression of CD31 (PECAM-1) mRNA in all the CLL B
cell clones. Thrombin receptor (TR) mRNA expression was
also found in three of the four germline-type clones and all
four of the somatic mutation-type clones. mRNA for endoglin
was noted in all eight patients shown in Figure 3. We also
detected mRNA for the Tie receptor (Figure 4) in one of four
somatic mutation-type clones and two of four GL-type clones.
Angiopoietin mRNA was also present in all eight CLL patients.
An analysis for these same mRNA species by RPA in five
additional CLL B cell clones was also done. In these clones,
we detected VEGFR-1 and CD31 mRNA in all five CLL B cell
clones and thrombin receptor mRNA in three of the five CLL
clones (data not shown). The probes available in this commercial RPA did not allow assessment of VEGFR-2 (KDR), the
other major receptor known to bind and become activated by
VEGF.27 Thus, we designed primers specific for that receptor
and performed RT-PCR for both VEGFR-1 and VEGFR-2. Figure 5 illustrates the results of those experiments. We did detect
mRNA for VEGFR-2 (seven of seven clones) and VEGFR1 (four
of five CLL clones) in the CLL clones regardless of somatic or
germline origin. The five patients studied for VEGFR1 were
either GL (n = 2) or SM (n = 3) while the seven patients studied
for VEGFR2 were either GL (n = 3) or SM (n = 5).
Finally, we also designed probes for TSP-1 and conducted
RT-PCR assays which did show that mRNA for this anti-angiogenic molecule exists in clonal CLL B cells (Figure 6). We
studied two CLL B cell clones and normal peripheral blood B
cells. Of interest, normal peripheral blood B cells also
expressed TSP-1 mRNA. In order to see if hypoxia could alter
TSP-1 message as previously reported,28 we performed the
PCR reactions on RNA obtained from cells cultured under normoxic and hypoxic conditions. In one of two CLL clones, we
were able to detect a reduction in TSP-1 mRNA with hypoxic
exposure compared to normoxic conditions (Figure 6). This
Figure 4
RPA analysis of various angiogenesis-related molecules.
Using a Molecular Dynamics STORM Phosphorimager, mRNA levels
for each of the proteins indicated were quantitated and expressed relative to levels of the L32 housekeeping gene. Four GL-type CLL (panel
a) and four SM-type B-CLL (panel b) were assessed for expression levels of Flt-1, Tie, Tie-2, thrombin receptor (TR), CD31, endoglin (END)
and angiopoietin (ANG).
Figure 5
RT-PCR analysis of VEGFR-1 and -2 expression. Five BCLL patients were examined for VEGFR-1 in (a) (SM = 3, GL = 2) and
five separate B-CLL patients (SM = 3 and GL = 2) were examined for
VEGFR-2 in (b). A549 cells served as a positive control (C) for VEGFR1 and HeLa cells served as a positive control for VEGFR-2.
latter finding prompted us to do simultaneous assays for VEGF
and TSP-1 in the culture medium of CLL B cells from three
patients that have been exposed to normoxic or hypoxic conditions. These latter patients were not the same as the ones
tested for TSP-1 mRNA in Figure 6. In three experiments summarized in Table 2, all three B-CLL clones had an upregulation of VEGF with hypoxia and a diminution in their TSP-1
levels when cultured in hypoxia compared to normoxia. In
preliminary work not presented here, we find that normal peripheral blood B cells also secrete TSP-1 protein as detected
by ELISA.
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B-CLL cells express pro- and anti-angiogenic molecules
NE Kay et al
916
Figure 6
RT-PCR analysis of TSP-1 mRNA levels. Two B-CLL patients and purified blood B cells from a healthy control were assessed for
TSP-1 levels by employing different cycling conditions as noted on the bottom of each gel. Note the presence of mRNA for TSP-1 and actin
in all lanes for both blood B cells and the two CLL B cell clones. Also note that patient CLL1 has a downward modulation of TSP-1 mRNA
after hypoxic exposure. This is in contrast to the consistent levels of mRNA for both actin and TSP-1 in both the blood B cells and CLL B cells
from patient 2. N, normoxia; H, hypoxia.
Table 2
Levels of VEGF and TSP-1in the culture medium of CLL
B cells cultured under normoxic or hypoxic condtionsa
VEGF – normoxic
VEGF – hypoxic
TSP-1 normoxic
TSP-1 hypoxic
CLL 1
CLL 2
CLL 3
25.3
63.6
10.3
7.2
19.5
36.4
10
8.3
24.0
28.9
6.4
4.6
a
ELISA assays were done for VEGF or TSP-1 in the culture medium
obtained after a 24-h culture of purified CLL B cells (1 × 106 per
ml). CLL B cells from three separate patients were cultured under
both normoxic and hypoxic conditions. Data are shown in pg/ml for
VEGF and ng/ml for TSP-1.
Discussion
This investigation has detailed the array of angiogenic factors
that are expressed by the clonal leukemic B cells in B-CLL
patients. The major impetus for this study was our original
observation that the bone marrows of B-CLL patients exhibit
increased densities of blood vessels.14 This current study has
focused on the circulating blood cells of CLL patients and our
results indicate that unstimulated, circulating clonal B cells
are able to synthesize and secrete a variety of both pro- and
anti-angiogenic factors. While bFGF and VEGF proangiogenic
factors were detected, there was also evidence of secreted
anti-angiogenic factors endostatin, TSP-1 and IFN-␣, albeit at
varying levels. The anti-angiogenic factor TSP-129,30 was
secreted consistently and at higher levels than endostatin or
IFN-␣. Hypoxic conditions were found to dramatically alter
the low level of both mRNA and secreted levels of VEGF from
the blood CLL B cells. In addition, we detected the presence
of mRNA for the VEGF receptors; VEGFR-1 and VEGFR-2; the
adhesion molecules CD31 and endoglin (CD105); the proangiogenic factor, angiopoietin; and mRNA for the Tie1
receptor and thrombin receptor. Taken together, these results
strongly suggest that the blood B cells in CLL are malignant
cells with significant potential to interact with and/or be
modulated by angiogenic factors. In addition, the tissues infiltrated by these malignant B cell clones could have altered
Leukemia
development and growth of resident blood vessels based on
the angiogenic molecules expressed by CLL B cells.
The role of angiogenesis in the biology of B-CLL is as yet
unclear. However, our initial study of bone marrow tissue and
a more recent study of nodal sites in B-CLL14,15 adds to the
evidence that abnormal angiogenesis exists in this disease. In
both studies, either increased numbers of blood vessels in the
bone marrow or neovascularization of lymph node sites were
documented. Major questions from the B-CLL tissue-based
observations include what is the role if any for abnormal angiogenesis in the initiation or progression of this B cell leukemia
and do the CLL B cells themselves assist in the induction of
this angiogenesis? The latter study did find evidence for VEGF
protein in both clonal B cells circulating in the blood and
those cells resident in nodal tissue sites.15 Our own data on
circulating clonal B cells indicate that some, but not all, B
cell clones isolated from blood spontaneously release VEGF
protein into culture medium. Of interest, we always detected
VEGF in the urine of CLL patients indicating that in vivo there
is a consistent production of this pro-angiogenic factor.14
Since VEGF expression can be modulated by several factors
including hypoxia,26 activated oncogenes31,32 and various
cytokines,33–36 the exact angiogenic status of a CLL clone in
vivo in different sites is probably not easy to predict.
Our experiments confirm15 that CLL B cells exposed to
hypoxia have a brisk increase in the detectable levels of VEGF
protein secretion. The elevation of VEGF in hypoxic conditions was variable from clone to clone but does suggest that
under the right microenvironmental conditions CLL B cells are
capable of releasing increased levels of VEGF. In addition, the
increase in CLL VEGF protein release was paralleled by quantitative increases in mRNA for VEGF. This suggests that the
augmented VEGF secretion is related to increased synthesis of
new VEGF protein. The ELISA methods we used to evaluate
VEGF in CLL cells utilized a monoclonal antibody reactive for
VEGF isoform 165. VEGF 165 is the predominant molecular
type synthesized by both normal and malignant cells.37 This
VEGF isoform and VEGF isoform 121 have also been detected
from CLL cells stimulated by hypoxia.15 Interestingly, it is the
165 isoform that is a potent mitogen for vascular endothelial
cells. VEGF isoform 165 is able to bind both VEGFR1 and 2
B-CLL cells express pro- and anti-angiogenic molecules
NE Kay et al
receptors and subsequently generate angiogenesis and vasculogenesis.38,39 Indeed, the study by Chen et al15 did show that
CLL culture medium was able to induce angiogenesis in vitro.
Of interest, the other major biologic impact of VEGF is to
enhance the survival of endothelial cells.40 VEGF has been
shown to increase levels of Bcl-2 and A1, anti-apoptotic proteins in human endothelial cells.41 Since resistance to
apoptosis is also a critical feature of CLL B cells, this increases
the likelihood that VEGF could have important biologic effects
on CLL B cells by virtue of an autocrine pathway.
Our demonstration that CLL B cells consistently express
mRNA for VEGFR1 and VEGFR2 and express VEGFR1 and 2
by flow cytometry (results not shown) adds to the evidence
that a VEGF-based autocrine pathway in CLL B cells may
exist. Although we do not have direct evidence that such an
autocrine pathway exists in this disease, we do have preliminary data that suggest that incubating CLL cells with anti
VEGF-receptor antibody can increase CLL B cell apoptotic
rates. There are two high-affinity VEGF specific receptor tyrosine kinase (RTK) receptors (Flt-1 or VEGFR-1 and flk-1/KDR
or VEGFR-1) described (reviewed in Ref. 37) that bind VEGF
and generate cell signals that ultimately result in the modulation of angiogenesis and vasculogenesis. Activation of the
two VEGF RTK receptors by VEGF has been reported to
induce differential endothelial cell responses implying that
signal transduction pathways may be different for the two
receptors.42,43 The VEGFR-1 and -2 receptors are known to
be expressed predominantly in endothelial cells.38 However,
other cell types have also been shown to express angiogenictype receptors including both normal and tumor cells. The
VEGFR-1 receptor is found on renal mesangial cells, monocytes and trophoblasts.44–46 VEGFR-2 has been found on marrow stem cells, megakaryocytes and retinal progenitor
cells.47,48 Malignant melanoma cells have been found to
express VEGFR receptors.49,50 VEGFR-1, and to a lesser extent,
VEGFR-2, have been found by RT-PCR in acute leukemic
blasts.51–53 Flow cytometry and RT-PCR54 methods allowed
detection of Tie receptor expression in approximately 20% of
acute leukemia cases. Finally, a recent report has detected the
expression, albeit variable, of both Flt-1 and Tie1 in CLL B
cells using a Western blot and solid phase radioimmunoassay.55 Thus, there is ample evidence that human leukemic
cells and in particular, CLL B cells, can express these RTK
receptors. The potential interaction of VEGF and VEGFR1/VEGFR-2 in CLL cells is probably not the only way in which
angiogenesis factors might influence CLL B cells. The ELISA
data and RPA data indicate that the CLL B cell can generate
a surprisingly diverse repertoire of angiogenic molecules.
Thus, there are both pro- (bFGF, VEGF) and anti-angiogenic
(endostatin, TSP-1, IFN-␣) molecules secreted by the CLL B
cell clones. We have previously shown that CLL patients have
high levels of bFGF in the urine.14 Our current study indicates
that CLL B cells are spontaneously secreting this pro-angiogenic molecule and thus are probably the source of the high
levels of bFGF found in the urine. These findings have relevance to the potential for an angiogenic switch in CLL much
like that seen in solid tumors.4
The angiogenic switch in tumor cells is characterized at a
molecular level by an imbalance in the production of pro- vs
anti-angiogenic factors.4,5 In the case of CLL, this could be
defined by a variety of changes in the secretion profile of a
CLL clone including increased VEGF production and/or
decrease in TSP-1, endostatin, and/or IFN-␣. In order to determine if these vascular factors are associated with more
aggressive disease in B-CLL, we compared the vascular factor
levels secreted by CLL clones to the Rai risk and whether a
patient had a GL- or SM-type clone. We found that mean
VEGF levels were higher while TSP-1 levels were lower for
both high vs low risk Rai and GL- vs SM-type B cell clones.
Thus, the levels of these factors are indeed consistent with a
relative conversion from more anti-angiogenic factors (ie TSP1) to a pro-angiogenic (ie VEGF) state. Why this might occur
is not yet evident in B-CLL but critical features to be evaluated
include hypoxic microenvironments and/or genetic mutations
that might be occurring in the B cell clone. Hypoxia in marrow or nodal environments could be a factor since we were
able to find that hypoxic conditions generated both an
increase in mRNA for VEGF and VEGF protein while TSP
mRNA was decreased in CLL B cells. Two candidate genetic
loci known to regulate angiogenesis include Ras and the p53
tumor suppressor gene.33,56 While Ras mutations have not
been commonly found in CLL cells, mutations in p53 are
documented in progressive or transformed CLL.57 Since
mutations in this tumor suppressor gene are known to downregulate TSP-1,58 it is tempting to speculate that the high levels
of TSP-1 spontaneously secreted from CLL cells could be
decreased with the onset of p53 mutations in CLL clones. This
alteration may be sufficient for the angiogenic switch to occur
in CLL patients. Of interest, the normal p53 gene can also
increase the level of the pro-angiogenic factor VEGF.59 Future
work will need to evaluate the association of hypoxia and/or
alterations found in the CLL B cell clone with the production
of both pro- and anti-angiogenic molecules.
The relatively extensive profile of angiogenic molecules
found in CLL cells also included mRNA for CD31, endoglin,
Tie1 and angiopoietin. These expression patterns suggest that
multiple alternative pathways could exist for angiogenesisrelated functions in CLL B cells. Thus, CD31 or platelet endothelial cell adhesion molecule-1 (PECAM), is an endothelial
adhesion molecule known to be present on CLL cells.60 CD31
has been implicated as a pro-angiogenic molecule in human
endothelial cells.61 The presence of CD31 on the membrane
of CLL B cells may provide important signals for synthesis of
vascular growth factors. Endoglin is a receptor for TGF-␤ a
molecule with pro-angiogenic activity.62 CLL cells are also
known to secrete TGF-␤,63 thus providing another potential
autocrine circuit for modulation of angiogenic factors in the
CLL B cell. Of interest, TSP-1 has been implicated in the activation of TGF-␤64 suggesting an additional role for TSP-1 in
clonal B cell biology. Finally, we also detected low levels of
mRNA for angiopoietin, another protein that can increase
angiogenesis. There are two types of angiopoietin and both
can bind to Tie-2.65 However, since we saw no mRNA for
the Tie-2 receptor, this autocrine pathway is less likely to be
operative in CLL.
Taken together, we believe our data show that the leukemic
cells in B-CLL have a surprisingly diverse capacity to synthesize many pro- and anti-angiogenic factors. In addition, we
now know that the CLL B cells express mRNA and have membrane expression for several critical vascular receptors
(VEGFR-1, VEGFR-2 and TIE) along with CD31, an important
angiogenic adhesion molecule. The exact role of each one of
these vascular factors in the pathophysiology of the disease
remains to be determined. However, we believe these data
provide the foundation for the pursuit of novel autocrine pathways that could impact at least the apoptosis resistance known
to be a prominent feature of the clonal B cells.
917
Leukemia
B-CLL cells express pro- and anti-angiogenic molecules
NE Kay et al
918
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