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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4247-4254
Role of Vascular Endothelial Growth Factor/Vascular Permeability
Factor in the Pathogenesis of Kaposi's Sarcoma-Associated
Herpesvirus-Infected Primary Effusion Lymphomas
By
Yoshiyasu Aoki and
Giovanna Tosato
From the Division of Hematologic Products, Center for Biologics
Evaluation and Research, Food and Drug Administration, Bethesda, MD.
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ABSTRACT |
Primary effusion lymphomas (PELs), which are rare lymphomas
associated with Kaposi's sarcoma-associated herpesvirus (or human herpesvirus-8) infection, present as malignant lymphomatous effusions in body cavities. Because PELs prefer liquid growth, we hypothesized that increased vascular permeability would be required for effusions to
form. We found that the PEL cell lines BC-1, BCP-1, and BCBL-1 produce
high levels of vascular endothelial growth factor/vascular permeability
factor (VEGF/VPF). Reverse transcriptase-polymerase chain reaction
analysis of RNA from the PEL cell lines amplified the 3 VEGF-secreted
isoforms: VEGF/VPF121, VEGF/VPF145, and
VEGF/VPF165. Two of the PEL cell lines expressed the
VEGF/VPF receptor Flt-1, but VEGF/VPF did not stimulate proliferation
in these cells. Most (13/14) control SCID/beige mice inoculated
intraperitoneally with BCBL-1 cells and subsequently observed or
treated with control antibodies developed effusion lymphoma of human
cell origin with prominent bloody ascites. In contrast, none (0/9) of
the mice treated with a neutralizing antihuman VEGF/VPF antibody
developed ascites and effusion lymphoma. These results demonstrate that VEGF/VPF is critical to BCBL-1 growth as effusion lymphoma in mice and
suggest that VEGF/VPF stimulation of vascular permeability may be
critical to the pathogenesis of PELs.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
KAPOSI'S SARCOMA-associated herpesvirus
(KSHV; also known as human herpesvirus-8 [HHV-8]) is a herpesvirus
linked to the development of Kaposi's sarcoma (KS), primary effusion
lymphoma (PEL), and a proportion of Castleman's
disease.1-4 KSHV-associated malignancies arise
predominantly, but not exclusively, in human immunodeficiency virus
(HIV)-positive individuals.5 PEL, otherwise termed body
cavity-based lymphoma, is a peculiar and infrequent type of
non-Hodgkin's lymphoma that displays a marked preference for liquid
growth in the serous cavities of the body, usually in the absence of an
identifiable tumor mass.6 PEL consistently originates from
B cells, but the vast majority of cases exhibit a non-B, non-T
phenotype, lacking expression of surface Igs and B-cell-associated
antigens. At the molecular level, PEL cells are characterized by clonal
Ig gene rearrangement, without c-myc rearrangement or
alterations of the bcl-2, ras, and p53 genes.6 Most cases
of PEL are dually infected with Epstein-Barr virus (EBV) and
KSHV,7,8 but occasional cases of EBV-negative/KSHV-positive PEL have been reported.9-11 Thus, KSHV is likely to be
involved in the pathogenesis of PEL, but its role is currently unclear.
Prior studies of KS-derived cell lines have shown that these cells can
produce several cytokines, including granulocyte-macrophage colony-stimulating factor, tumor necrosis factor, transforming growth
factor- , interleukin-1 (IL-1), IL-6, oncostatin M, and KSHV-encoded chemokine homologues.12-14 Some of these have
been reported to modulate KS cell growth in an autocrine/paracrine fashion. Of particular interest is vascular endothelial growth factor
(VEGF), originally discovered as vascular permeability factor
(VPF),15 and its tyrosine kinase receptors, which have been
proposed to play an important role in KS pathogenesis.16-20 VEGF/VPF is a homodimeric glycoprotein that promotes endothelial cell
proliferation, angiogenesis, and increased vascular
permeability.21-23 VEGF/VPF binds to 1 of 2 tyrosine kinase
receptors: fms-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1
(Flk-1/KDR). These receptors are found predominantly on endothelial
cells, and their activation leads not only to cell proliferation, but
also to increased vascular permeability and vasodilatation. When
compared with histamine, VEGF/VPF is approximately 50,000 times more
potent on a molar basis at increasing the permeability of microvessels
to plasma macromolecules.15 In addition to playing a
central role in promoting tumor neovascularization,24,25
VEGF/VPF is directly associated with hyperpermeability of
tumor vessels and is commonly detected in rodent and human tumor
effusions.15,26,27 Recent studies have suggested that
VEGF/VPF may play a role in the pathogenesis of certain experimental
ascites tumors.28,29 In this study, we investigated a
potential role of VEGF/VPF in the pathogenesis of PELs.
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MATERIALS AND METHODS |
Cell lines.
The PEL cell lines BC-1 and BCP-1 were kind gifts from Dr Y. Chang
(Columbia University, New York, NY), and the BCBL-1 cell line was a
kind gift from Dr R. Yarchoan (National Cancer Institute, Bethesda,
MD). All of these cell lines are positive for KSHV. BC-1 cells also
harbor EBV, whereas BCBL-1 and BCP-1 cells are EBV-negative. The
KSHV/EBV-negative effusion type lymphoma cell line DS-1 was a kind gift
from Dr D. Nelson (National Cancer Institute). EW36, BL41, CA46, and
JLP119 are EBV-negative Burkitt's lymphoma cell lines, whereas
Eubanks, Raji, and Namalwa are EBV-positive Burkitt's lymphoma cell
lines.30 These cells were maintained as suspension cultures
in RPMI1640 (Biowhittaker, Walkersville, MD) supplemented with 10% or
20% (for BCP-1) heat-inactivated fetal bovine serum (FBS) at 37°C
in 5% CO2. DS-1 was maintained in RPMI1640 with 10% FBS
and 10 U/mL of recombinant human IL-6. Primary cultures of human
umbilical vein endothelial cells (HUVECs) and the human cell lines Hs68
(skin fibroblast), DU145 (prostate carcinoma), SK-N-MC (neuroblastoma),
A-375 (malignant melanoma), MDA-MB-468 (breast carcinoma), and SW-480
(colon carcinoma) were purchased from the American Type Culture
Collection (Manassas, VA). Hs68 and A-375 cell lines were maintained in
Dulbecco's modified Eagles medium (Biofluids, Rockville, MD) with 10%
FBS. DU145 cell line was maintained in RPMI1640 with 10% FBS. HUVECs
were maintained in RPMI1640 with 15% FBS, 20 U/mL porcine heparin
(Sigma, St Louis, MO), and 100 µg/mL endothelial cell growth
supplement (Calbiochem-Novabiochem, La Jolla, CA). SK-N-MC cell line
was maintained in Eagle minimum essential medium and Earle's BSS
(Biowhittaker) with 10% FBS. DMA-MB-468 and SW-480 cell lines were
maintained in Leibovitz's L-15 medium (Life Technologies,
Gaithersburg, MD) with 10% FBS.
Preparation of conditioned media.
Suspension cells were seeded in 12-well plates (Becton Dickinson,
Franklin Lakes, NJ) at 1 × 106 cells/well in 2.5 mL
of RPMI1640 medium supplemented with 10% FBS and cultured with or
without 20 ng/mL phorbol 12-myristate 13-acetate (TPA;
Sigma) for 72 hours. Adherent cells were seeded in 6-well plates at 1 × 106 cells/well in 2.5 mL culture media (described
above) and cultured for 72 hours.
Quantification of VEGF/VPF.
VEGF/VPF was measured by enzyme-linked immunosorbent assay (ELISA)
using a human VEGF Quantikine kit (R&D, Minneapolis, MN), following the
manufacturer's instructions.
Cell proliferation assays.
PEL cells were seeded in 96-well plates (Costar, Cambridge, MA) at 1 × 104 cells/well in RPMI1640 with 1% or 10% FBS and
cultured with or without 2 to 50 ng/mL human VEGF/VPF (PeproTech, Rocky
Hill, NJ) for 48 hours. In some experiments, the same number of PEL
cells were cultured in autologous conditioned medium that was
preincubated with 10 µg/mL of mouse monoclonal antihuman VEGF/VPF
antibody (Ab; A4.6.1; a generous gift from Genentec Inc, South San
Francisco, CA) to neutralize endogenous VEGF/VPF.31
Proliferation was measured by a 6-hour pulse with 1 µCi/well of
[3H]-thymidine (Amersham, Arlington Heights, IL).
[3H]-thymidine incorporation was determined after
harvesting cells onto glass fiber filters.
Reverse transcriptase-polymerase chain reaction (RT-PCR).
Total RNA was isolated using the Tri-zol reagent (Life Technologies)
according to the manufacturer's instructions. RNA (0.25 µg for each
reaction) was treated with RNase-free DNase I (Promega, Madison, WI) at
37°C for 30 minutes, heated at 75°C for 5 minutes, reverse-transcribed using an RNase H-RT (SuperScript; Life
Technologies), and suspended in 100 µL of
Tris-Ethylenediaminetetraacetic acid (TE). The resultant
cDNA was amplified by PCR using primers specific to human Flt-1,
Flk-1/KDR, and VEGF/VPF.17,18 PCR amplification was
performed in 50 µL containing 20 mmol/L Tris-HCl, 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L dNTP mixture (Life Technologies), 0.4 µmol/L of each primer, and 2.5 U Taq DNA polymerase (Life Technologies). An initial denaturation step at 94°C was followed by
35 cycles of denaturation at 94°C for 1 minute, annealing at 59°C for 1 minute, and extension at 72°C for 1 minute, followed by a final extension step at 72°C for 5 minutes. Each amplified product was electrophoresed through a 1.8% agarose gel prestained with
1 µg/mL of ethidium bromide and was visualized under UV light. The
quality of RNA was confirmed in all samples by parallel RT-PCR for
GAPDH.32
Establishment of PEL tumors and treatment with anti-VEGF/VPF Ab.
All animal experiments were performed according to National Institute
of Health guidelines for the care and handling of mice. Groups of
6-week-old male C.B-17 SCID/beige mice received 400 rad of total body
irradiation. The following day, mice were injected intraperitoneally
(IP) with BCBL-1 cells (1 × 107 cells/mouse suspended
in 0.2 mL phosphate-buffered saline [PBS]). As controls, mice were
injected IP with the same number of Eubanks, Raji, or Namalwa cells.
The neutralizing antihuman VEGF/VPF Ab or an isotype control Ab (mouse
IgG1; Cappel ICN, Aurora, OH) was injected IP at a dose of
100 µg twice weekly in a volume of 0.2 mL. Treatment was initiated 1 day after BCBL-1 cell injection.
Histopathology and immunohistochemistry.
Histochemical staining of ascites tumor samples, peripheral blood
smears, and formalin-fixed paraffin-embedded tissues was performed
using Diff-Quick (Baxter Scientific Products, McGaw Park, IL). Cytospin
samples of ascites tumor cells were fixed in acetone at  20°C
for 1 minute and stained for human CD38 and HLA-DR by the
avidin-biotin-peroxidase method using Vectastain Elite ABC kit (Vector
Laboratories, Burlingame, CA), mouse antihuman CD38 Ab (Becton
Dickinson), or rat anti-HLA-DR Ab (Serotec Inc, Raleigh, NC),
according to the manufacturer's instructions. Mouse IgG and rat IgG
(Cappel ICN, Aurora, OH) were used as negative controls.
Flow cytometric analysis.
BCBL-1 cells that had been cultured in complete medium were stained
with mouse antihuman CD38 Ab or rat anti-HLA-DR Ab, followed by
fluorescein isothiocyanate-labeled antimouse IgG (Becton Dickinson) or
phycoerythrin-labeled anti-rat IgG (Becton Dickinson). Single-color analysis was performed using FACScan and CELLQuest (Becton Dickinson). After gating on live cells by forward and side light scattering, the
percentage of positive cells was determined by integrating profiles on
the basis of 2 × 104 live cells.
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RESULTS |
VEGF/VPF production from PEL cells.
VEGF/VPF is commonly detected in solid tumor tissues, malignant ascites
fluid, lymphoma, and KS tissues.15-20 Malignant and transformed cells are known to express VEGF/VPF.21-23 Using
a specific ELISA, we found that 3 KSHV-positive PEL cell lines (BC-1,
BCP-1, and BCBL-1) constitutively express high levels of VEGF/VPF
(Fig 1). DS-1, an IL-6-dependent
KSHV/EBV-negative primary effusion lymphoma cell line,33
also produced large amounts of VEGF/VPF. Levels of VEGF/VPF in the
culture supernatants of the KSHV-positive PEL cells and the DS-1 cells
were comparable or higher than those in the culture supernatants of 7 Burkitt's lymphoma and 5 solid tumor-derived cell lines (Fig 1). In
previous studies, we demonstrated that these solid tumor-derived cell
lines consistently give rise to progressively growing subcutaneous
tumors in athymic mice.34
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| Fig 1.
VEGF/VPF production by PEL, Burkitt's lymphoma, and
solid tumor cells. PEL cells, Burkitt's lymphoma cells, and solid
tumor-derived cell lines were cultured (1 × 106
cells/well) for 72 hours in 6-well plates (2.5 mL/well). A-375,
melanoma; MDA-MB-486, breast cancer; SK-N-MC, neuroblastoma; SW-480,
colon carcinoma; DU145, prostate carcinoma. VEGF/VPF levels in the
culture supernatants were determined by ELISA. Results represent the
mean (±SD) of 3 independent experiments.
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VEGF/VPF isoforms produced by PEL cells.
Genomic and cDNA analyses of the human VEGF/VPF gene showed the
occurrence of at least 5 subtypes: VEGF/VPF121,
VEGF/VPF145, VEGF/VPF165,
VEGF/VPF189, and VEGF/VPF206
(Fig 2A).17 These isoforms are
generated by alternative splicing from a single gene. We performed
RT-PCR analysis to identify the VEGF/VPF subtypes expressed in PEL
cells. By this method, 5 bands were amplified from the control prostate
carcinoma DU145 cell line (Fig 2B). By contrast, only 3 predominant
bands corresponding to VEGF/VPF121, VEGF/VPF145, and VEGF/VPF165 were amplified
from the PEL cell lines BC-1, BCP-1, and BCBL-1. These VEGF/VPF
isoforms correspond to secreted forms of the protein.
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| Fig 2.
Expression of VEGF/VPF isoforms by PEL cell lines. (A)
Schematic representation of the VEGF/VPF mRNA splice variants together
with the expected length of the respective RT-PCR amplification
products. aa, amino acids. (B) Expression of VEGF/VPF isoforms in PEL
(BC-1, BCP-1, and BCBL-1), skin fibroblast (Hs68), and prostate
carcinoma (DU145) cells. Total RNA was translated into cDNA and
amplified by PCR. The 3 splice variants coding for the secreted
forms of VEGF/VPF were amplified from all 3 PEL cell lines.
Signals for all 5 isoforms were amplified from DU145 cells and none
from Hs68 cells.
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Detection of the VEGF/VPF receptor Flt-1 on PEL cells.
The high-affinity VEGF/VPF receptors Flt-1 and Flk-1/KDR are expressed
predominantly on vascular endothelial cells. Recent studies have also
described expression of the VEGF/VPF receptor Flt-1 on a subset of
primary hematopoietic cells,35 but not in most human tumor
cell lines.21 We looked for expression of VEGF/VPF
receptors on PEL cell lines. By RT-PCR, Flt-1 message was amplified
from BCBL-1 and BC-1 cell lines, but not from the BCP-1 cell line
(Fig 3). Flk-1/KDR was not detectable in
these 3 PEL cell lines (Fig 3). Despite their expression of Flt-1 mRNA, BC-1 and BCBL-1 cell lines did not display enhanced proliferation in
response to recombinant VEGF/VPF (2 to 50 ng/mL;
Table 1). This result is consistent with
the reported failure of VEGF/VPF to promote the proliferation in NIH3T3
cells transfected with the Flt-1 gene.21 We tested whether
VEGF/VPF could contribute to the autocrine growth factor activity
displayed by conditioned media from PEL cells. However, a neutralizing
Ab directed at VEGF/VPF had minimal effect on the proliferation of PEL
cells (Table 1). Based on these results, we conclude that VEGF/VPF does
not act as an autocrine growth factor for PEL cells.
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| Fig 3.
Expression of VEGF/VPF receptors in PEL cells detected by
RT-PCR. cDNAs from HUVECs, PEL cells (BC-1, BCP-1, and BCBL-1), and
skin fibroblast (Hs68) cells were subjected to PCR amplification for
Flt-1 and Flk-1/KDR (498- and 709-bp products, respectively). The
quality of RNA was confirmed by parallel RT-PCR amplification for
GAPDH. MWM, molecular weight marker.
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Effects of a neutralizing antihuman VEGF/VPF Ab on experimental
effusion lymphoma.
Previous studies have shown that the PEL cell lines KS-1, BCP-1, HBL-6,
and BCBL-1 are transplantable IP into certain immunodeficient mice,
giving rise to lymphomatous effusions that resemble PEL occurring in
humans.5,11,36 Using a specific neutralizing Ab, we looked
for a potential role of VEGF/VPF in PEL cell growth in vivo. To this
end, 23 SCID/beige mice were injected IP with BCBL-1 cells (1 × 107 cells/mouse) after  -irradiation with 400 rad. Beginning 2 days after cell inoculation and continuing twice per
week for 4 weeks, 8 mice received IP injections of PBS (0.2 mL), 9 mice
received IP injections of a monoclonal antihuman VEGF/VPF Ab (A4.6.1,
0.1 mg in 0.2 mL PBS), and 6 mice received IP injections of murine IgG1 (0.1 mg in 0.2 mL PBS). After 24 or 25 days, 7 of 8 mice inoculated with PBS alone and 6 of 6 mice inoculated with control murine IgG1 developed abdominal distention attributable to
the presence of ascites. A test puncture on day 29 after cell
inoculation showed the presence of bloody ascites in 5 of 5 tested
animals. By contrast, none of the mice (0/9) treated with anti-VEGF/VPF Ab developed abdominal distention (Table
2). All mice were killed on day 30 after cell inoculation, at which
time gross and histological examinations of the peritoneal cavity,
liver, spleen, kidneys, heart, lung, and lymph nodes were performed.
The PEL-derived BCBL-1 cell line injected IP into these mice expresses
CD38 and HLA-DR detectable by fluorescence-activated cell sorting
(FACS) analysis (Fig 4). By
immunohistochemistry, most of the nucleated cells in the ascites fluids
stained positively for human CD38 and HLA-DR (Fig 4). Morphologically,
these lymphoid cells were similar to human PEL
(Fig 5A). In most cases, the volume of
maligant effusions was greater than 2 mL. Human VEGF/VPF was detected
in ascites fluids from 5 of 5 tested animals at concentrations ranging
between 5.7 and 15.1 ng/mL. Peripheral blood smears and organ sections
showed the presence of large lymphoid cells in 6 of 7 control mice
tested (4 of 5 mice treated with PBS and 2 of 2 treated with murine
IgG), suggesting the presence of BCBL-1 cells in the circulation (Fig
5B and C). In 8 of 9 control mice (4 of 5 treated with PBS and 4 of 4 treated with IgG), solid lymphoma-like tissue was observed under the
renal capsule (Fig 5D) or in the mesenteric fat tissue proximal to the
spleen. By immunohistochemistry, these lymphomatous lesions were
positive for HLA-DR (not shown), suggesting that the mice had developed
BCBL-1 lymphoma. Despite the absence of perceptible ascites in 9 mice
treated with anti-VEGF/VPF Ab, microscopic analysis showed that 1 mouse
had a similar lymphomatous nodule under the renal capsule and lymphoma
cells in peripheral blood. Gross and microscopic examination of other
organs showed no abnormalities, except for the presence of mild to
moderate hepatosplenomegaly in mice with peritoneal effusions. These
results demonstrate that, in mice, neutralizing Ab against VEGF/VPF are effective in preventing the development of malignant PEL ascites and
reducing PEL dissemination to distant sites.
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| Fig 4.
Expression of human CD38 and HLA-DR in lymphoid cells
from the ascites fluid of SCID/beige mice inoculated IP with BCBL-1
cells. (Left panels) FACS analysis of CD-38 and HLA-DR expression by
BCBL-1 cells cultured in vitro. (Middle panels) Immunohistochemical
staining for human CD-38 and HLA-DR of cytospin preparations from
ascites of mice injected IP with BCBL-1 cells. (Right panels) Control
staining with mouse IgG and rat IgG of cytospin preparations from
ascites of mice injected IP with BCBL-1 cells.
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| Fig 5.
Identification of lymphoma-like cells in the peritoneal
cavity, circulation, and kidney of mice inoculated IP with BCBL-1
cells. (A) Representative cytospin preparation of ascites fluid stained
with Diff-Quick showing the presence of large lymphoid cells (original
magnification × 1,000). (B and C) Representative peripheral blood
smears depicting the presence of large lymphoid cells in the
circulation (hematoxylin-eosin stain; original magnification × 400).
(D) Representative lymphoma tissue localized under the renal capsule
(hematoxylin-eosin stain; original magnification × 200).
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Association between VEGF/VPF production and effusion development.
To explore further the potential role of VEGF/VPF in the pathogenesis
of effusion lymphoma, the Burkitt's lymphoma cell lines Eubanks, Raji,
and Namalwa were tested for their ability to induce malignant ascites
formation. As shown in Fig 1, Eubanks cells produced 108 pg/mL VEGF/VPF
in the culture supernatant, Raji cells produced 178 pg/mL VEGF/VPF, and
Namalwa cells produced 802 pg/mL VEGF/VPF. Groups of 8 SCID/beige mice
were -irradiated with 400 rad and subsequently inoculated IP with 1 × 107 cells from each of the cell lines. Four mice of
each group were killed on day 30, the remaining mice were killed on day
40 after cell injection, and gross pathology and tissue histology were obtained. Consistent with Eubanks cells failure to induce subcutaneous tumors in athymic mice,30 none of 4 SCID/beige mice
inoculated IP with Eubanks cells presented any abnormality. In
contrast, 2 of 4 mice inoculated with Raji cells and 3 of 4 mice
inoculated with Namalwa cells showed the presence of multiple tumor
nodules on the peritoneum, diaphragm, and mesentery, ranging in size
between 3 and 20 mm in diameter (Table 3).
Macroscopic and microscopic examination showed mild hepatosplenomegaly
and the occurrence of infiltration with lymphoma cells. Histological
examination of these nodules showed the presence of lymphoma consistent
with Burkitt's lymphoma. During the initial 30 days of observation, none of the mice developed ascites. In the subsequent 10 days, abdominal distention appeared in 2 of 4 mice injected with Namalwa cells. The peritoneal cavities of these mice were occupied by large
tumor nodules (35 mm in maximum diameter) associated with small amounts
(<0.3 mL) of bloody ascites. Histological examination of the cells in
the ascites fluid showed the presence of red blood cells and large
cells consistent morphologically with Burkitt's lymphoma cells. By
contrast, neither Raji nor Eubanks cells developed visible effusions.
These results are consistent with the notion that VEGF/VPF is important
to the development of lymphomatous effusions and suggest that threshold
amounts of VEGF/VPF may be required for the formation of malignant
lymphoma effusion. Many of the animals developed nodules of lymphoma in
the absence of malignant effusion, suggesting that factors other than
VEGF/VPF play a critical role in the formation of solid lymphoma.
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DISCUSSION |
In this study, we found that 3 KSHV-associated PEL cell lines (BC-1,
BCBL-1, and PCP-1) produce high levels of VEGF/VPF in the culture
supernatant. RT-PCR analysis of RNA from these PEL cell lines amplified
predominantly the message for the secreted VEGF/VPF isoforms
VEGF/VPF121, VEGF/VPF145, and
VEGF/VPF165. The cell lines BC-1 and BCBL-1, but not
PCP-1, expressed the tyrosine kinase VEGF/VPF receptor Flt-1, but
neither exogenous nor endogenous VEGF/VPF stimulated PEL cell
proliferation in culture. When inoculated into the peritoneal cavity of
SCID/beige mice, the PEL cell line BCBL-1 produced effusion lymphomas
with bloody ascites in 7 of 8 animals. Administration of a neutralizing
antihuman VEGF/VPF Ab, but not control IgG1, to these mice
inhibited the formation of effusion lymphomas and the associated
ascites in all 9 animals. Despite the absence of identifiable effusion
tumor in the peritoneal cavity, 1 of 9 mice had evidence of PEL-derived
lymphoma under the renal capsule. Other mice were inoculated IP with 1 of 3 Burkitt's lymphoma cell lines secreting varying amounts of
VEGF/VPF. The Namalwa cell line that secreted 802 pg/mL VEGF/VPF gave
rise to malignant ascites in 2 of 8 animals, whereas the cell lines
Eubanks and Raji that secreted 109 and 178 pg/mL VEGF/VPF,
respectively, did not produce ascites. A proportion of the animals
injected with Raji (3/8) and Namalwa (5/8) developed solid lymphoma
nodules on the peritoneum, diaphragm, and mesentery. In the peritoneal cavity of SCID/beige mice, Burkitt's lymphoma cells expanded primarily as neoplastic nodules, whereas PEL cells displayed suspension growth.
These results demonstrate that VEGF/VPF is critical to the development
of experimental PEL in mice and point to an association between
VEGF/VPF production by lymphoma cells and the development of effusion lymphoma.
VEGF/VPF is a highly conserved, disulfide-bonded, dimeric glycoprotein
of 34 to 42 kD that displays a limited structural similarity to
platelet-derived growth factor.23 The domain encoded by
exon 1, which is common to all VEGF/VPF isoforms, contains information required for the recognition of the known VEGF/VPF receptors Flk-1/KDR and Flt-1 (Fig 1A).21 The amino acids encoded by exon 8 are also present in all of the VEGF/VPF splice variants. Peptides encoded
by exons 6 and 7 distinguish various VEGF/VPF isoforms. VEGF/VPF121, VEGF/VPF145, and
VEGF/VPF165 are secreted forms of VEGF/VPF, whereas
VEGF/VPF189 and VEGF/VPF206 are sequestered by
cell surface heparan sulfates.21 The most abundant product of the VEGF/VPF gene appears to be
VEGF/VPF165.21 Consistent with the detection of
VEGF/VPF in the culture supernatant of PEL cells and in PEL ascites, we
found that PEL cells express predominantly the secreted VEGF/VPF
isoforms VEGF/VPF121, VEGF/VPF145, and
VEGF/VPF165.
The actions of VEGF/VPF are mediated by 2 cell surface receptors: Flt-1
and Flk-1/KDR. In the current study, we found that the PEL cell lines
BC-1 and BCBL-1 express Flt-1 mRNA but do not proliferate in response
to exogenous or endogenous VEGF/VPF. Flt-1 shows at least a 10-fold
higher affinity to but approximately 10-fold lower kinase activity than
Flk-1/KDR.21 When overexpressed in NIH3T3 cells or bovine
endothelial cells, Flt-1 did not support cell proliferation in the
presence of VEGF/VPF, whereas Flk-1/KDR overexpressed in these cells
did.21 Flt gene transcripts are present in vascular
endothelial cells starting from early stages of
embryogenesis.21 Flt-1-deficient homozygous mice
showed a marked disorganization of blood vessels and died at embryonic day 8.5 to 9.37 However, Flt-1 tyrosine kinase-deficient
homozygous mice developed normal vessels with normal vascular
permeability and survived.38 In adult mice, Flt-1 mRNA is
detectable in most normal tissues, particularly in placenta, lung,
kidney, and brain.21 No cells or cell lines other than
endothelial cells are known to express both Flt-1 and Flk-1/KDR
receptors and to proliferate in response to VEGF/VPF, strongly
suggesting the existence of endothelial-specific signaling mechanisms.
The role of Flt-1 on PEL cells is currently unknown. Like PEL cells,
monocytes can express Flt-1 in the absence of Flk-1/KDR and through
Flt-1 VEGF/VPF can stimulate tissue factor production and chemotaxis in
monocytes.35
Functionally, VEGF/VPF is a potent inducer of angiogenesis attributed
to direct stimulation of endothelial cell growth and of vascular
permeability attributed to stimulation of nitric oxide release.39 Angiogenesis is required for a wide variety of
physiological and pathological processes, including tumor
growth.40 The expression of VEGF/VPF is necessary for the
formation of blood vessels in mouse and rat embryos.41,42
Many tumor cell lines secrete VEGF/VPF, and a variety of solid tumor
tissues express high levels of VEGF/VPF.40 Transfection of
VEGF/VPF renders Chinese hamster ovary cells tumorigenic in nude
mice,43 and treatment with a neutralizing monoclonal Ab
directed against VEGF/VPF inhibited the growth of a variety of
experimental tumors.25,31,44 Stimulation of vascular
permeability by VEGF/VPF is believed to be critical to the pathogenesis
of certain malignant effusions.15 It is well documented
that the formation of malignant ascites results largely from increased permeability of peritoneal lining vessels and the accumulation of a
plasma exudate in the peritoneal cavity.27,45,46 A temporal and spatial correlation was noted between the appearance of malignant ascites and the presence of VEGF/VPF in the peritoneal
cavity.15 Recently, a neutralizing Ab against VEGF/VPF was
reported to significantly reduce the accumulation of malignant ascites
in mice bearing intraperitoneal human ovarian tumors or syngeneic
breast adenocarcinoma tumors.29,44 However, levels of
VEGF/VPF in malignant ascites were found to vary widely depending upon
the nature of the malignancy; tumors of sarcoma- and carcinoma-origin
produced substantially more VEGF/VPF than did tumors of hematological
derivation.28 In addition, anti-VEGF/VPF Ab completely
inhibited ascites production in mice bearing ovarian tumors, whereas it
only partially inhibited intraperitoneal tumor growth.29
Also, VEGF/VPF neutralization had a minimal effect on peritoneal fluid
and tumor cell accumulation in mice bearing a syngeneic
lymphoma.44
In the present study, PEL cell lines secreted VEGF/VPF at levels
comparable to those produced by solid tumor-derived cell lines. In
addition to secreting VEGF/VPF, PEL cells lack expression of several
adhesion and homing molecules, including intercellular adhesion
molecule-1, L- and E-selectin, CD19, CD31, CD44, CD11a, and
CD11c.11 The absence of certain adhesion molecules could impair anchorage of PEL cells to the peritoneal wall. This would explain why PEL cells inoculated IP did not form tumor nodules on the
peritoneal serous membrane. However, most mice inoculated IP with PEL
cells, either alone or in conjunction with control IgG, developed a
solid lymphoma under the renal capsule or proximal to the spleen,
suggesting the occurrence of transmigration through the capillary
vessel wall. Only one of the animals treated with anti-VEGF/VPF Ab displayed one of these lymphomas. The receptor CX3CR1 and its ligand fractalkine, a transmembrane molecule with a
CX3C-motif, can promote cell adhesion in the absence of
other adhesion molecules, raising the possibility that PEL cell
trafficking to the endothelium could occur through this or other
pathways.47
KSHV can promote VEGF/VPF secretion through the virally encoded
G-coupled protein receptor and the viral cytokine
vIL-6.48,49 It can also promote angiogenesis directly,
through the expression of the chemokines viral macrophage
inflammatory protein-I (vMIP-I) and vMIP-II.14 This
redundancy of tools for induction of angiogenesis and VEGF/VPF
secretion has suggested that neovascularization is important to KSHV
survival and spread. However, it is unclear how these effects might be
mediated.50 It was proposed that VEGF/VPF may stimulate
KSHV replication. It was also proposed that, by inducing the expression
of VEGF/VPF and other angiogenic proteins, KSHV may favor
vascularization and nutrients supply of virally infected cells,
ensuring their growth. Enhanced angiogenesis, vascular permeability,
and the presence of VEGF/VPF are characteristic features of KS
tissues.16-20 In Castleman's disease, lymphoid hyperplasia is often associated with evidence of excessive vascularization in the
germinal centers, which has been attributed to local VEGF/VPF expression by nonlymphoid cells.51 The current study
provides evidence that the VEGF/VPF that PEL cells secrete contributes to lymphoma growth essentially by accelerating vascular permeability rather than tumor vascularization. The study also shows that VEGF/VPF can play a critical role in the pathogenesis of PEL, because VEGF/VPF neutralization prevented PEL formation in mice. Patients with PEL
usually have advanced acquired immunodeficiency syndrome (AIDS) and are
poor candidates for aggressive chemotherapy, which has resulted in a
median survival of only 3 months.2,52,53 Based on the
results presented here, anti-VEGF/VPF neutralizing Ab represents a
rational approach to the investigational treatment of PEL.
| |
ACKNOWLEDGMENT |
The authors thank Drs Y. Chang for providing us BC-1 and BCP-1 cells,
R. Yarchoan for BCBL-1 cells, D. Nelson for DS-1 cells, L. Yao for
control HUVEC RNA, and J. Teruya for help in reviewing sections.
| |
FOOTNOTES |
Submitted April 30, 1999; accepted August 11, 1999.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Presented in part at the 3rd National AIDS Malignancy Conference, May
27, 1999, in Bethesda, MD.
Address reprint requests to Yoshiyasu Aoki, MD, PhD, Division of
Hematologic Products, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bldg 29A, Room 2D06, HFM-535, 8800 Rockville Pike, Bethesda, MD 20892; e-mail: AOKI{at}CEBR.FDA.GOV.
| |
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ABSTRACT
INTRODUCTION