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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4011-4019
Stromal Derived Factor-1-Induced Chemokinesis of Cord Blood
CD34+ Cells (Long-Term Culture-Initiating Cells) Through
Endothelial Cells Is Mediated by E-Selectin
By
Afzal J. Naiyer,
Deog-Yeon Jo,
Jongcheol Ahn,
Robert Mohle,
Mario Peichev,
George Lam,
Roy L. Silverstein,
Malcolm A.S. Moore, and
Shahin Rafii
From the Division of Hematology and Oncology, Weill Medical College
of Cornell University, New York, NY; the Laboratory of Developmental
Hematopoiesis, Sloan-Kettering Institute, New York, NY; and the
Tubingen Medical Center, University of Tubingen, Tu- bingen, Germany.
 |
ABSTRACT |
Homing of hematopoietic stem cells to the bone marrow (BM)
involves sequential interaction with adhesion
molecules expressed on BM endothelium (BMEC) and chemokine
stromal derived factor-1 (SDF-1). However, the mechanism whereby
adhesion molecules regulate the SDF-1-induced transendothelial
migration process is not known. E-selectin is an endothelial-specific
selectin that is constitutively expressed by the BMEC in vivo. Hence,
we hypothesized that E-selectin may mediate SDF-1-induced
transendothelial migration of CD34+ cells. We show that
CD34+ cells express both E-selectin ligand and
fucosyltransferase-VII (FucT-VII). Soluble E-selectin-IgG chimera
binds avidly to 75% ± 10% of CD34+ cells composed
mostly of progenitors and cells with long-term culture-initiating cell
(LTC-IC) potential. To assess the functional capacity of E-selectin to
mediate CD34+ cell migration in a transendothelial
migration system, CD34+ cells were placed on transwell
plates coated with interleukin-1 -activated BMEC. In the absence of
SDF-1, there was spontaneous migration of 7.0% ± 1.4% of
CD34+ cells and 14.1% ± 2.2% of LTC-IC. SDF-1 induced
migration of an additional 23.0% ± 4.4% of CD34+
cells and 17.6% ± 3.6% of LTC-IC. Blocking MoAb to E-selectin inhibited SDF-1-induced migration of CD34+ cells by
42.0% ± 2.5% and LTC-IC by 90.9% ± 16.6%. To define the
mechanism of constitutive expression of E-selectin by the BMEC in vivo,
we have found that vascular endothelial growth factor (VEGF165) induces E-selectin expression by cultured
endothelial cells. VEGF-stimulated endothelial cells support
transendothelial migration of CD34+ cells that could be
blocked by MoAb to E-selectin. These results suggest that trafficking
of subsets of CD34+ cells with LTC-IC potential is
determined in part by sequential interactions with E-selectin and
SDF-1.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
IN ADULTHOOD, HEMATOPOIESIS is restricted
to the extravascular compartment of the bone marrow (BM) separated by a
single layer of BM endothelial cells (BMEC). Thus, hematopoietic stem cells (HSC) arriving at the BM must first recognize or be recognized by
the luminal surface of the BMEC.1-5 Molecules that mediate adhesion of HSC to BMEC are likely to play a pivotal role in the phenomenon of HSC homing.1-3,6-8 Similar to leukocyte
trafficking,9,10 homing of HSC from the peripheral
circulation to the BM is a multistep process and involves sequential
interaction of CD34+ cells with adhesion molecules
expressed on BMEC and specific chemokine(s) expressed within the BM.
Chemokines orchestrate this process by providing directional cues for
CD34+ cells to migrate into the BM microenvironment.
Based on recent studies, the chemokine stromal derived factor-1 (SDF-1)
has been shown to play a key role in CD34+
trafficking.11-14 Targeted gene knockout of either
SDF-115 or its receptor CXCR416,17 resulted in
a defect in BM hematopoiesis, whereas fetal liver hematopoietic
activity remained intact. Recently, it was shown that CXCR4-dependent
migration in response to SDF-1 is essential for the homing and
engraftment of human stem cells into NOD/SCID mice.13
However, transplantation of fetal-derived hematopoietic cells from
CXCR4 knock out mice into lethaly irradiated syngenic mice resulted in
a profound but not complete decrease in the engraftment of
hematopoietic cells.18,19 These results suggest that homing
and long-term maintenance of hematopoietic cells within the BM is only
partially dependent on the expression of CXCR4 on the hematopoietic
stem and progenitor cells.19
However, the mechanism whereby SDF-1 promotes transmigration of
CD34+ cells and the role of adhesion molecules during the
SDF-1-induced transendothelial migration are not well defined. Similar
to leukocytes, transendothelial migration of CD34+ cells in
response to SDF-1 most likely is initiated by tethering of
CD34+ cells through interaction with selectins such as E-
or P-selectins followed by firm adhesion mediated through intercellular
adhesion molecule-1 (ICAM-1)/leukocyte function-associated antigen-1
(LFA-1), vascular adhesion molecule-1 (VCAM-1)/very late antigen 4 (VLA4) ligand pairs and engagement with junctional adhesion molecules such as platelet endothelial cell adhesion molecule
(PECAM).9,10
Among these adhesion molecules, E-selectin is an endothelial-specific
selectin that plays a critical role in tethering of leukocytes20 or hematopoietic progenitors21 to
endothelial cells at high shear stress. Although E-selectin is mostly
expressed by cytokine-activated endothelium, native BMEC in vivo
constitutively express E-selectin,22 suggesting that
E-selectin may play a role in the regulation of trafficking of
CD34+ cells. Frenette et al7 have shown that
recruitment of hematopoietic progenitor cells to the BM is reduced in
P- and E-selectin knock-out P/E( /) mice. In this study,
lethally irradiated recipient P/E(/) mice, transplanted
with minimal numbers (5 × 104) of wild-type BM cells,
engrafted poorly compared with the wild-type recipients. Based on these
results, we hypothesized that combinatorial interaction of HSC with
selectins and SDF-1 may promote selective homing of HSC to the BM.
E-selectin interacts with leukocyte counter-receptors E-selectin
ligand-1 (ESL-1) and P-selectin glycoprotein ligand-1
(PSGL-1).23-25 ESL-126 and
PSGL-127,28 are active as E-selectin counterreceptors only
when modified posttransitionally by fucosylated oligosaccharides represented by Sialyl Lewis x (sCD15, sLex) or its structural variants.
Synthesis of functional selectin ligands requires an ordered series of
glycosylation reactions, including addition of a fucose residue in an
 1,3-linkage, a reaction catalyzed by fucosyltransferase type VII
(FucT-VII), which is expressed at significant levels in
leukocytes.24,25,29-32 The targeted gene knockout of
FucT-VII results in impaired leukocyte extravasation and faulty
lymphocyte homing. Whether there is a dysregulation of HSC homing in
FucT-VII null mice24 is not known. In addition, the exact
nature of expression of various fully glycosylated functional E-selectin ligands on CD34+ cells is not well defined. The
majority of freshly isolated CD34+ cells do not express
CD15. Therefore, whether CD34+ cells with long-term
culture-initiating cell (LTC-IC) potential express functional
E-selectin ligands and their role in chemokine-induced transendothelial
migration is not known and is the subject of the present study.
In this report, we have evaluated the role of E-selectin in
SDF-1-induced transendothelial migration of CD34+ cells.
We show that subsets of CD34+ cells with LTC-IC potential
bind to the soluble E-selectin-IgG chimera. SDF-1-induced
transendothelial migration of subsets of CD34+ cells with
LTC-IC potential was partially dependent on the interaction with
E-selectin. In addition, vascular endothelial growth factor (VEGF)
induces the upregulation of E-selectin and promotes migration of
CD34+ cells through endothelial cells. These data
suggest that constitutive expression of E-selectin by BMEC may
facilitate SDF-1-mediated homing of CD34+ cells to
the BM.
| |
MATERIALS AND METHODS |
Purification of CD34+ cells.
Cord blood was obtained after informed consent was received through
approval of the Institutional Review Board. Mononuclear cells were
isolated from cord blood (CB) by Ficoll (Nycomed Pharmacia A.S., Oslo,
Norway) density gradient centrifugation. CD34+ cells were
isolated the same day by the standard Minimacs magnetic isolation
technique (Miltenyi Biotec, Auburn, CA). Isolated CD34+
cells were passaged up to 3 times through Minimacs column to achieve a
very highly enriched population of CB-derived CD34+ cells
(92% ± 3% purity).
Isolation of E-selectin ligand-positive CD34+ cells
with E-selectin-IgG2a chimera.
Freshly isolated CD34+ cells were incubated with (1 µg/mL) soluble E-selectin-(mouse)IgG2a chimera at 4°C for 30 minutes in Hanks' balanced salt solution (HBSS) supplemented with 2 mmol/L calcium and magnesium. After 3 washes, the cells were treated with sheep antimouse Fc-coated immunomagnetic beads (Dynal A.S., Oslo,
Norway) at 4°C for 30 minutes. CD34+ cells bound to
beads were obtained by magnetic separation.
RNA isolation and reverse transcription (RT) and polymerase chain
reaction (PCR).
Poly A RNA isolated from CD34+ cells, obtained from
umbilical CB, granulocyte colony-stimulating factor (G-CSF)-mobilized
peripheral blood (PB), fetal liver (FL), and BM, HL60 myeloid cell
line, and BMEC were used in RT-PCR reactions. Oligonucleotide primers spanning the insertion/deletion site of human ESL-1 and FucT-VII were
synthesized. The forward primer for ESL-1 was 5'-CTA CCT GTC CTT
GAG CTC-3' and the reverse primer was 5'-TCC AGT CTA TGT GCT GAA. RT-PCR of m-RNA encoding ESL-1 resulted in a PCR product 450 bp long. The forward primer for FucT-VII was 5'-CAC CTC CGA GGC
ATC TTC AAC TG-3' and the reverse primer was 5'-CGT TGG TAT CGG CTC TCA TTC ATG-3'. The expected RT-PCR mRNA product encoding FucT-VII was 497 bp long. After 35 cycles, the samples were analyzed by
gel electrophoresis.
Flow cytometry.
All fluorescein isothiocyanate (FITC)-and phycoerytherin
(PE)-conjugated monoclonal antibodies (MoAbs) used for flow cytometry experiments, including CD34-PE (HPCA2; Becton Dickinson, Mountain View,
CA), E-selectin-FITC (Biosource, Cama- rillo,
CA), CD15-FITC (Becton Dickinson), and IgG isotype control
FITC/PE (Immunotech, Miami, FL) were obtained from
commercially available sources. To quantify the number of cells that
have the capacity to bind to E-selectin, freshly isolated
CD34+ cells were incubated with human soluble
E-selectin-mouse IgG2a chimeric molecule (Texas Biotechnology,
Houston, TX) followed by goat antimouse FITC-conjugated secondary
antibody against mouse IgG2a in HBSS containing 2 mmol/L calcium and
magnesium with and without 2 mmol/L EDTA. Subsequently, the cells were
washed fixed with 1% formalin and analyzed by a Coulter Elite flow
cytometer (Coulter, Miami, FL).
Cell lines.
Murine stromal cell line MS-5 (kindly Provided by K. Mori)
were grown in -minimum essential medium (-MEM;
GIBCO-BRL Life Technologies, Grand Island, NY)
supplemented with 10% fetal bovine serum (FBS; HyClone, Logan, UT) and
passaged weekly. The cells support the proliferation of human HSC in
long-term culture. The myeloid cell line HL60 (American Type Culture
Collection, Rockville, MD) was cultured in RPMI 1640 medium (GIBCO) supplemented with 10% FBS.
Preparation of BMEC and human umbilical vein endothelial cells
(HUVEC) monolayers.
Primary HUVEC and BMEC were isolated by a standard method as described
previously4 and placed in medium 199 (M199; GIBCO-BRL) containing 20% FBS, 1 ng/mL VEGF, 5 ng/mL basic fibroblast growth factor (b-FGF; FGF-2), and heparin (5 U/mL). For transmigration assay,
5-mm pore transwells were plated with BMEC. After 3 days, the
monolayers reached full confluency. Before transmigration assay, the
integrity of the monolayer was confirmed by placing 100 µL medium
M199 and 20% FBS containing 14C albumin (American
Radiolabelled Chemicals, St Louis, MO) on the cells for 6 hours and
measuring the amount of radioactivity accumulating in the lower chamber.
Adhesion studies.
Freshly isolated CB CD34+ cells (105
cells/well) were incubated with interleukin-1 (IL-1; 10 U/mL)
-stimulated BMEC monolayers in HBSS supplemented with 2 mmol/L calcium
and magnesium in the presence of either 2 mmol/L EDTA or blocking MoAbs
to ICAM-1 (10 µg/mL; R&D Systems, Minneapolis, MN), E-selectin (10 µg/mL; R&D Systems), or VCAM-1 (10 µg/mL; Immunotech). After
removal of the nonadherent cells, the adherent population was
quantified by flow cytometry and phase contrast microscopy. After an
incubation period of 1 hour at 37°C, the nonadherent population was
removed and the number of attached CD34+ cells bound to
endothelium was quantified by flow cytometry.
Transmigration assay.
Freshly isolated CB CD34+ cells were washed once with HBSS
solution and resuspended with X-vivo-20 at a concentration of
106/mL. HUVEC monolayers were prestimulated with 10 U/mL of
IL-1 and 40 ng/mL of VEGF for 12 to 16 hours. Subsequently, after
washing the monolayer of IL-1 and VEGF, aliquots of CB
CD34+ cell suspension (100 µL) were applied on 5-mm pore
transwells covered with confluent monolayers of IL-1 and VEGF
activated BMEC or HUVEC in a 24-well plate (Costar Corp, Cambridge,
MA). Immediately, 600 µL of serum-free media containing SDF-1 (200 ng/mL) was placed in the lower chamber. After incubation for 24 hours
at 37°C in a CO2 incubator, the migrated and the
nonmigrated cells were counted on a hemacytometer. Transmigration
through both IL-1 VEGF-activated BMEC or HUVEC was inhibited by
preincubation with 10 µg/mL of anti-E-selectin (CD62E) in the upper
chamber for 15 minutes.
LTC-IC assay.
MS-5 cells were seeded on T-12.5 flasks in -MEM with 10% FBS. When
cells reached confluence, medium was replaced with LTC medium
consisting of -MEM, 12.5% FCS, 5 × 10 4
mol/L 2--mercaptoethanol (Fisher Scientific, Pittsburgh,
PA), and 106 mol/L hydrocortisone
(Sigma, St Louis, MO). The migrated population was plated at a density
of 104 cells on the stromal feeder layers for LTC. These
were then incubated for the first 3 to 4 days at 37°C and
subsequently at 33°C in 5% CO2. Cultures were weekly
demipopulated and replenished with fresh LTC medium. The cell
suspension as well as the adherent population from week-5 LTC were
plated at a density of 1 × 104 cells in
triplicate in 35-mm tissue culture dishes (Corning, Acton,
MA) containing 1 mL Iscove's modified Dulbecco's medium (IMDM; GIBCO), 0.36% agarose (FMC Bioproducts, Rockland, MD), and 20%
FBS together with human kit-ligand (KL; 20 ng/mL; Immunex, Seattle, WA)
and human granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 U/mL; Immunex). After 14 days of incubation at 37°C and 5%
CO2, colony-forming unit-granulocyte-macrophage (CFU-GM)
was scored.
Colony-forming cell (agarose/CFC) assays.
The cells from the migrated populations were plated at a cell
concentration of 1 × 103/mL per 35-mm dish (Corning)
in triplicate, containing 1 mL IMDM, 0.36% agarose, and 20% FBS
together with human KL (20 ng/mL), GM-CSF (100 U/mL), erythropoietin
(Epo; 6 U/mL; Amgen, Thousand Oaks, CA), IL-6 (20 ng/mL),
and IL-3 (50 ng/mL). After 14 days of incubation at 37°C and 5%
CO2, colonies were scored for burst-forming unit-erythroid
(BFU-E), CFU-GM, and CFU-Mix.
Stimulation of HUVEC monolayer with VEGF.
HUVEC or BMEC monolayers were placed in serum-free medium X-Vivo-20
(BioWhittaker, Walkersville, MD) for 18 hours with 40 ng/mL of VEGF and
stained with FITC-labeled MoAb to E-selectin (Biosource) to confirm
upregulation of E-selectin by fluorescence-activated cell sorting
(FACS) analysis.
Statistical analysis.
Data are expressed as the mean ± SEM of 3 to 5 independent
experiments. To detect differences between migrating and nonmigrating cells, the t-test for the paired samples was applied.
P < .05 was considered statistically significant.
| |
RESULTS |
CD34+ cells express ESL and FucT-VII by RT-PCR.
Creation of functional E-selectin ligands requires addition of a fucose
residue in a 1,3 linkage to 2,3-Sialyllactosamine precursor, a
reaction catalyzed by FucT-VII.24,25,29 The expression of
ESL and FucT-VII on CD34+ cells was analyzed by RT-PCR.
CD34+ cells isolated from CB, PB, BM, and FL express the
expected product of both ESL (Fig 1A) and
FucT-VII (Fig 1B). Expression of FucT-VII leads credence to the
possibility that ESL or its variants expressed on CD34+
cells is most likely a fucosylated, functional ligand capable of
binding to E-selectin expressed on native BMEC or activated endothelial
cells.
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| Fig 1.
Expression of ESL-1 and FucT-VII by CD34+
cells. RT-PCR analysis was performed to examine the capacity of
CD34+ cells to express ESL-1 and FucT-VII. (A) The
expected product of 450 bp for ESL-1 could be detected in BMEC (lane 1, EC), HL60 (lane 3, positive control), PB CD34 cells (lane 4, PB), BM
CD34 cells (lane 5, BM), FL CD34 cells (lane 6, FL), and CB CD34 cells
(lane 7, CB). RT (lane 2) is the PCR reaction without addition of RT.
(B) The expected product of 497 bp for FucT-VII could be detected in
HL60 (lane 3, positive control) and in PB- (lane 4), BM- (lane 5), FL-
(lane 6), CB-derived CD34(+) cells (lane 7). FucT-VII expression is
only limited to hematopoietic cells, and BMEC (EC, lane 1) did not
express FucT-VII. RT control (lane 2) is the PCR reaction without
addition of RT.
|
|
Chimeric E-selectin binds to CD34+ cells with LTC-IC
potential.
To explore the capacity of soluble E-selectin to bind to
CD34+ cells, a chimeric E-selectin-IgG2a molecule was
generated. Using flow cytometry, we demonstrated that the
E-selectin-IgG2a chimera binds to 75% ± 10% of CB-derived
CD34+ cells (CD34+ESL+ cells) in a
calcium-dependent manner, as demonstrated by complete abrogation of
binding in the presence of EDTA (Fig 2B).
Soluble E-selectin fails to bind to endothelial cells that lack
FucT-VII and therefore does not generate a functional ESL (Fig 2C).
Almost all freshly isolated CD34+ cells used in these
experiments did not express CD15 (Lewis X), suggesting that soluble
E-selectin chimera is interacting with a variant type of fucosylated
ESL on the surface of CD34+ cells (Fig 2A). Agarose and
LTC-IC assays showed that, when compared with the
CD34+ESL() cells, CD34+ESL+
cells composed of a population of cells that showed a 2.1- ± 0.6-fold higher progenitor (Fig 2D) and 14.9- ± 10.7-fold higher LTC-IC content (Fig 2E). In fact, CD34+ cells that failed
to bind to E-selectin were depleted of cells with LTC-IC potential (Fig
2E).
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| Fig 2.
Chimeric E-selectin-IgG2a binds to subsets of
CD34+ cells. Soluble E-selectin-IgG2a chimera was used
to quantify the number of ESL-expressing CD34+
immediately after purification from CB. Using FACS analysis, 75% ± 5% of CD34+ cells bind to E-selectin-IgG2a chimeric
molecule (B) (N = 3). The binding of E-selectin/IgG2a was dependent
on divalent cations and was inhibited in the presence of 2 mmol/L EDTA
(B). Endothelial cells that do not express FucT-VII showed no binding
to the soluble E-selectin-IgG chimera (C). Freshly isolated
CD34+ cells do not express CD15 (A). Agarose and LTC-IC
assays performed on CD34+ cells bound to soluble
E-selectin chimera showed a 2.09 ± 0.58-fold higher progenitor (D)
and 14.9- ± 10.7-fold higher LTC-IC content than the population that
did not bind to soluble E-selectin chimera (E). The CD34+
cells that failed to bind to E-selectin were virtually devoid of
CD34+ cells with LTC-IC potential (E) (N = 3, P < .05).
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Under static conditions, adhesion of CD34+ cells to
BMEC monolayers is independent of E-selectin.
Adhesion studies were performed under static conditions to assess the
role of E-selectin in the modulation of CD34+ progenitor
cell adhesion to BMEC. Given that primary cultured BMEC lose their
capacity to express E-selectin in vitro, BMEC monolayers were treated
with IL-1 to induce E-selectin expression. Freshly isolated
CD34+ cells from CB were incubated with IL-1-activated
BMEC monolayers in the presence of divalent cations and blocking MoAb
to various adhesion molecules. As shown in
Fig 3, after incubation for 1 hour at
37°C, under static conditions, only MoAb to VCAM-1 partially blocked the adhesion of CD34+ cells to BMEC monolayers. The
blocking MoAb to E-selectin did not inhibit adhesion even at high
concentrations (30 µg/mL). The binding of CD34+ cells to
the BMEC was completely inhibited by EDTA, suggesting that the binding
of CD34+ cells to adhesion molecules on BMEC is dependent
on divalent cations. These data suggest that, under static conditions,
E-selectin plays a minimal role in supporting the adhesion of
CD34+ cells to BMEC.
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| Fig 3.
Under static conditions, adhesion of CD34+
cells to BMEC monolayers is independent of E-selectin.
CD34+ cells derived from CB were plated on
IL-1-activated BMEC monolayers in the presence or absence of
blocking MoAb to either VCAM-1 (10 µg/mL), ICAM-1 (10 µg/mL),
E-selectin (10 µg/mL), or EDTA (2 mmol/L). After incubation for 1 hour, EDTA resulted in a complete inhibition of adhesion. However, only
MoAb to VCAM-1 partially blocked the adhesion of CD34+
cells by 44.0% ± 5.8%, whereas MoAb to E-selectin even at high
concentrations of 30 µg/mL had no effect on adhesion of
CD34+ cells to endothelium. MoAb to ICAM-1 also did not
have a significant effect on adhesion of CD34+ cells to
BMEC (N = 4, P < .01).
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SDF-1-induced transendothelial migration of CD34+
cells is dependent on E-selectin.
To assess the role of E-selectin in a more physiological
chemokine-driven transendothelial migration model, CD34+
cells isolated from CB were added to the upper chamber of 5-µm transwell plates that were coated with confluent monolayers of resting
or IL-1-activated BMEC in the presence or absence of a blocking
MoAb to E-selectin (Fig 4). Immediately,
200 ng/mL of SDF-1 was placed in the lower chamber, and after an
incubation period of 24 hours, the number of migrated cells was
examined. In the absence of SDF-1, there was spontaneous migration of
7.0% ± 1.4% of total CD34+ cells added. However,
SDF-1 induced migration of an additional 23.0% ± 4.4% of total
CD34+ cells through IL-1-activated endothelial cells.
In the presence of blocking MoAb to E-selectin, there was a 42.0% ± 2.5% inhibition of the SDF-1-induced migration of
CD34+ cells (Fig 4). In contrast, MoAbs to either VCAM-1 or
ICAM-1 did not significantly block SDF-1-induced migration of
CD34+ cells. Similar results were obtained with
IL-1-activated HUVEC monolayers (data not shown).
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| Fig 4.
Blocking MoAb to E-selectin inhibits SDF-1-induced
transmigration of CD34+ cells through IL-1-activated
endothelial cells. Freshly isolated CD34+ cells
(105 cells) from CB were placed in the upper chamber of
5-µm transwell plates coated with confluent monolayers of
IL-1-stimulated intact BMEC monolayers in the presence or absence
of 10 µg/mL blocking MoAb to E-selectin. Immediately, 200 ng/mL of
SDF-1 was placed in the lower chamber and the number of migrated cells
was evaluated by flow cytometry. After a period of 24 hours, in the
absence of SDF-1, there was migration of only 7.0% ± 1.4% of the
added CD34+ cells to the upper chamber. In the presence
of 200 ng/mL SDF-1, there was migration of an additional 23.0% ± 4.4% of the added CD34+ cells. In the presence of
blocking MoAb to E-selectin (10 µg/mL), there was a 42.0% ± 2.5%
inhibition of the SDF-1-induced migration of CD34+ cells
(N = 4, P < .003). Blocking MoAb to VCAM-1 and ICAM-1 did
not significantly influence the SDF-1-induced migration process.
|
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To determine the role of E-selectin in regulating the migration of
cells with progenitor potential, the migrating CD34+ cells
from each condition from the above experiments were also assayed in an
agarose assay to quantitate the number of CFC. In the absence of SDF-1,
there was spontaneous migration of 53% ± 1.5% of
progenitors (Fig 5D). SDF-1 induced
migration of an additional 41.8% ± 11.0% of CFC applied to the
upper wells. Pretreatment of the endothelial monolayer with MoAb to
E-selectin (10 µg/mL) resulted in virtually complete inhibition
(91.6% ± 13.0%) of SDF-1-induced transendothelial migration of
CFCs (Fig 5D). These results suggest that engagement of E-selectin with
its ligand on CD34+ cells plays a role in the
SDF-1-induced chemokinesis of CD34+ cells.
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| Fig 5.
MoAb to E-selectin blocks transendothelial migration of
primary CFU. CB-derived CD34+ cells (105
cells/plates) were plated in the upper chamber of 5-µm 24-well costar
plates coated with confluent monolayers of IL-1-activated BMEC.
Serum-free X-vivo medium was added to the control wells. After 24 hours
of incubation at 37°C, the number of migrating CFU were examined by
agarose assay. In the absence of SDF-1, there was spontaneous migration
of 29.6% ± 4.9% of added BFU-E (A), 22.3% ± 5% of CFU-GM (B),
and 1.4% ± 0.3% of CFU-Mix (C). In the presence of SDF-1, there was
an additional migration of 14.2% ± 9.3% of BFU-E (A), 19.1% ± 8.6% of CFU-GM (B), and 1.75% ± 0.37% of CFU-Mix (C) (N = 4, P < .01). However, in the presence of MoAb to
E-selectin (10 µg/mL), there was inhibition of the SDF-1-induced
migration of 61.8% ± 14.5% of CFU-GM (B) and 48.6% ± 15.0% of
CFU-MIX (C) (N = 4). In addition, MoAb to E-selectin inhibited the
SDF-1-induced BFU-E migration even below spontaneous levels to 23.2% ± 5.0% (A) (N = 4, P < .05). When taking the total CFU
(D) into account, there was spontaneous migration of 53% ± 7% of
the added CFC in the control samples. SDF-1 induced additional
migration of 41.8% ± 11.0% of the added CFU, and MoAb to E-selectin
completely blocked this SDF-1-induced migration by 91.6% ± 13.0%
(N = 4, P < .05).
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MoAb to E-selectin inhibits the SDF-1-induced transendothelial
migration of CD34+ cells with LTC-IC potential.
SDF-1 has been shown to induce the migration of
CD34+CD38-cells, which phenotypically can be considered as
HSC.14 However, whether SDF-1 also support the migration of
LTC-IC has not been evaluated. We demonstrate that, in the absence of
any added SDF-1, IL-1-activated endothelial monolayers induce
spontaneous migration of 14% ± 1.5% of added LTC-IC
(Fig 6). However, SDF-1 induced migration
of an additional 17.6% ± 3.6% of the added LTC-IC
through IL-1-activated endothelial monolayer. Blocking MoAb to
E-selectin almost completely inhibited (90.9% ± 16.6%) the
SDF-1-induced transendothelial migration of LTC-IC. These data suggest
that the forced SDF-1-induced migration of CD34+ cells
with LTC-IC potential is dependent on interaction with E-selectin.
However, LTC-IC populations migrating independently of SDF-1 do not
require interaction with E-selectin.
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| Fig 6.
MoAb to E-selectin inhibits the SDF-1-induced
transmigration of LTC-IC through IL-1-activated endothelial
monolayer. Ten thousand CB-derived CD34+ cells were
placed on the upper chamber of transwell plates that were covered with
confluent monolayers of BMEC in the presence or absence of SDF-1 in the
lower chamber and blocking MoAb to E-selectin in the upper chamber.
After 24 hours, migrating cells from each condition were placed on MS-5
feeder layers to quantitate the number of migrated cobblestone
area-forming cells (LTC-IC), which at week 5 were quantified by agarose
assay. In the absence of SDF-1, there was a spontaneous migration of
14.0% ± 1.5% LTC-IC that was independent of SDF-1 and E-selectin.
However, with the addition of SDF-1, an additional 17.6% ± 3.6% of
the added LTC-IC migrated through IL-1-activated endothelial cells.
Blocking MoAb to E-selectin (10 µg/mL) almost completely inhibited
(90.9% ± 16.6%) all SDF-1-induced migration of LTC-IC (N = 5, P < .007).
|
|
VEGF stimulation of HUVEC results in the induction of E-selectin
expression and enhanced migration of CD34+ cells.
BMEC constitutively express E-selectin in vivo. However, cultivation of
BMEC results in the downregulation of E-selectin expression. In fact,
adhesion molecule repertoire of in vitro-cultured unstimulated BMEC
monolayers simulates that of resting HUVECs.33 This
phenomenon is due to the finding that isolation and subsequent
propagation of BMEC monolayers results in the downregulation of
E-selectin, suggesting that factors released by the hematopoietic cells
within the BM microenvironment may confer organ specificity to BMEC by inducing upregulation of E-selectin. Most experiments described here
have been performed with IL-1-activated BMEC or HUVEC monolayers to
maintain the expression of E-selectin.
Among the factors that could potentially modulate endothelial cell
function is VEGF. We and others have shown that hematopoietic cells,
including megakaryocytes, secrete significant amount of VEGF165.34,35 Given that high levels of VEGF
are produced by hematopoietic cells, we explored the possibility that
VEGF may modulate adhesion molecule repertoire on endothelial cells.
Incubation of either BMEC or HUVEC monolayers under serum-free
conditions, in the presence of recombinant endotoxin-free
VEGF165 (40 ng/mL), resulted in upregulation of E-selectin
(Fig 7A) and, to a smaller degree, VCAM-1
and ICAM-1. This suggests that sustained E-selectin expression by BMEC
may be dependent on VEGF released by hematopoietic cells.
View larger version (1744K):
[in this window]
[in a new window]
| Fig 7.
VEGF stimulation of endothelial cells induces expression
of E-selectin and enhances SDF-1-induced CD34+ cell
transendothelial cell migration. (A) Confluent monolayers of either
BMEC or HUVEC in serum-free conditions were incubated with recombinant
endotoxin-free VEGF (40 ng/mL). After an incubation period of 16 hours,
the level of E-selectin expression was analyzed by FACS analysis. VEGF
upregulated E-selectin by BMEC monolayers (N = 5). Similar results
were obtained for HUVEC monolayers (data not shown).
IL-1-stimulated endothelium served as the positive control. (B)
Blocking MoAb to E-selectin inhibited SDF-1-induced transmigration of
CD34+ cells through VEGF-activated endothelial cells. To
determine if the E-selectin expression induced by VEGF supported the
SDF-1-induced migration, CB-derived CD34+ cells were
plated on VEGF-primed BMEC monolayers. In the absence of any
stimulation to the endothelial monolayers, SDF-1 induced migration of
only 1.5% ± 0.2% of the added CD34+ cells. SDF-1
induced migration of 15.5% ± 1.1% of CD34+ cells
through VEGF-activated endothelial monolayers. Blocking MoAb to
E-selectin (10 µg/mL) inhibited this migration of CD34+
cells by 31.6% ± 1.6% (N = 3, P < .05). In
addition, SDF-1 induced migration of 22% ± 2.8% of
CD34+ cells through IL-1-activated endothelial cells
(N = 3, P < .05), which was inhibited by MoAb to E-selectin
by 36.0% ± 2.8%. When the endothelial monolayers were treated with
both VEGF and IL-1, SDF-1 induced the migration of 24.8% ± 1.1%
of CD34+ cells through the endothelial monolayer; MoAb to
E-selectin inhibited this migration by 40.4% ± 0.4%.
|
|
To evaluate the capacity of VEGF-stimulated HUVEC monolayers to support
the migration of CD34+ cells, freshly isolated CB
CD34+ cells were placed on the upper chamber of transwell
plates that were coated with endothelial monolayers stimulated with
either IL-1 or VEGF165 or a combination of IL-1 or
VEGF165 for 16 hours. Subsequently, the number of migrating
CD34+ cells in response to SDF-1 was quantified with phase
contrast microscopy. In the absence of any stimulation to the
endothelial monolayer, SDF-1 induced migration of only 1.5% ± 0.2% of the added CD34+ cells. Both
VEGF165-activated and IL-1-activated endothelial cells
supported migration of 15.5% ± 1.1% and 22% ± 2.8% of the added CD34+ cells. MoAb to E-selectin
inhibited the SDF-1-induced migration by 31.6% ± 1.6% and 36.0% ± 2.8% through the VEGF165-activated and
IL-1-activated endothelial monolayers, respectively. These results
suggest that VEGF165 facilitates SDF-1-induced migration of CD34+ cells by induction of E-selectin expression.
| |
DISCUSSION |
Emerging data suggest that selective homing of transplanted HSC to the
BM is mediated in part through interaction of CD34+ cells
with adhesion molecules1-6 expressed on BMEC as well as chemokines expressed within the bone marrrow
microenvironment.12,13 BMEC exhibit unique features that
may convey organ specificity to the BM microenvironment.35
Although most microvascular beds require activation with inflammatory
cytokines to express the endothelial-specific adhesion molecule
E-selectin, BMEC constitutively express E-selectin.22
E-selectin has been shown to facilitate tethering of
leukocytes20 or hematopoietic progenitors21 on activated endothelial cells at high flow rates. Although the flow rate
within the BM microenvironment is relatively low, rapid forced transendothelial migration of hematopoietic cells in response to
chemokines may also necessitate tethering with selectins, including E-selectin.
Most studies related to the biology of E-selectin ligands have been
hampered by the lack of availability of specific antibodies that could
identify surface expression of fully glycosylated functional E-selectin
ligands on the hematopoietic cells. Therefore, we have used soluble
human E-selectin mouse IgG chimera to detect potential E-selectin
ligands that may bind to CD34+ cells. We demonstrate that
soluble E-selectin-IgG chimera binds to 75% ± 5% of
CD34+ cells in a calcium-dependent manner, suggesting that
functional ESL is expressed on a subset of CD34+ cells
that, when isolated, contain almost all of the cells with LTC-IC
potential (Fig 2). Furthermore, MoAb to E-selectin effectively blocked
the SDF-1-induced transendothelial migration of CD34+
cells with LTC-IC potential. Taken together, these data lend credence
to the possibility that CD34+ cells with LTC-IC potential
express some form of functional ESL.
As shown in Figs 5 and 6, there is spontaneous SDF-1-independent
migration of significant numbers of both primary CFU and LTC-IC. These
data suggest that subsets of LTC-IC or progenitors may respond to
another as yet unrecognized adhesion molecule(s) or chemokine(s)
produced by the IL-1-activated endothelial cells that mediate
SDF-1-independent transendothelial migration of CD34+
cells. These data are in agreement with other previously published reports demonstrating spontaneous migration of CD34+ cells
through a monolayer of BMEC-1 cell line.36,37
In the experiments reported here, E-selectin expression was induced on
BMEC and HUVEC by IL-1, which also stimulates the expression of
ICAM-1 and VCAM-1. However, blocking MoAb to VCAM-1 or ICAM-1 had a
minor effect on SDF-1-induced transendothelial migration of
CD34+ cells. VCAM-1, the inducible ligand for VLA4, has
been shown to be constitutively expressed in the BM stromal as well as
sinusoidal BMEC.39 Papayannopoulou et al1,6,8
have shown that homing of BM-derived HSC to the BM of the irradiated
mice was blocked by pretreatment of HSC with MoAb to VLA4. In addition,
MoAb to VLA4 or VCAM-1 induce significant mobilization of HSC,
suggesting that the VCAM-1/VLA4 ligand pair may modulate homing as well
as mobilization of HSC.1,40 We show that, under static
conditions, binding of human CB-derived CD34+ cells to BMEC
is dependent on VCAM-1/VLA4, but transendothelial migration of LTC-IC
in response to SDF-1 was not significantly inhibited by blocking MoAb
to VCAM-1. Therefore, it is possible that constitutive expression of
E-selectin may play a critical role in the initial phases of
SDF-1-induced transendothelial migration, whereas constitutive
expression of VCAM-1 by BM reticular and sinusoidal cells may support
long-term lodgment of HSC, once they have completed the transmigration
process. It is also conceivable that engagement of VCAM-1/VLA4 may be
more critical for SDF-1-independent transendothelial migration of HSC.
Pretreatment of CD34+ cells with cytokines such IL-3 has
been shown to enhance transendothelial migration of CD34+
cells.37 Imai et al38 have shown that SDF-1
induces migration of the murine IL-3-dependent HSC line through
VCAM-1-expressing murine endothelial cell lines. However, these
SV40-transformed cell lines do not express E-selectin, even with
IL-1 stimulation, and therefore the role of E-selectin could not
have been evaluated.38
Frenette et al7 have shown that recruitment of transplanted
hematopoietic progenitor cells to the BM of recipient P- and E-selectin
knock out mice P/E(/) was significantly reduced. In
addition, Zannettino et al41 have shown that primitive
hematopoietic cells bind to P-selectin. However, based on these
studies, the relative contribution of either P- or E-selectin in
supporting SDF-1-induced transendothelial migration of HSC has not
been studied. Resting or IL-1-activated BMEC do not express
P-selectin.33 However, given that tumor necrosis factor-
(TNF-) or thrombin could upregulate P-selectin on
BMEC,33 P-selectin may play a role in HSC trafficking
during inflammatory processes.
KL not only enhances SDF-1-induced chemokinesis of CD34+
progenitor cells,12 but also plays a role in
VCAM-1/VLA4-mediated mobilization of HSC.1 SDF-1 and KL
have also been shown to share common signaling pathways.42
Therefore, because endothelial cells express both soluble and membrane
bound KL,43 it is possible that KL may also augment
expression of functional ESL expression on CD34+ cells.
Isolation and cultivation of BMEC in vitro results in downregulation of
E-selectin expression, suggesting that factors released in the BM
microenvironment may confer organ specificity to BMEC and induce
upregulation of E-selectin. Among the factors modulating endothelial
cell function is VEGF. VEGF is not only the principal specific
mitogenic and survival factor for endothelial cells, but it may also
regulate the adhesion molecule repertoire on endothelial cells. We have
previously shown that one of the secreted isoforms of VEGF
(VEGF165) is expressed in large quantities by different hematopoietic cells, including megakaryocytes34 and
CD34+ cells (manuscript in preparation).
Hence, we explored the possibility that VEGF may influence E-selectin
expression by BMEC. Incubation of either unstimulated monolayers of
HUVEC or BMEC with endotoxin-free recombinant VEGF165
resulted in upregulation of E-selectin expression. In contrast to
unstimulated endothelial monolayers, there was substantial increase in
the number of SDF-1-induced CD34+ cell migration through
VEGF-stimulated endothelial monolayers. VEGF-induced migration was
partially mediated through interaction of CD34+ cells with
E-selectin.
These results suggest that reciprocal interaction between endothelial
cells and CD34+ cells is critical for homing of HSC and
maintenance of native BMEC phenotype. Whether combined interaction of
E-selectin, SDF-1, and KL with HSC conveys signals that modify adhesion
molecule expression and homing properties of HSC is the subject of
ongoing experiments.
| |
FOOTNOTES |
Submitted May 10, 1999; accepted August 12, 1999.
S.R. is supported by the American Heart Association Grant-In-Aid, NHLBI
Grants No. RO1 HL58707 and RO1 HL61849, the Dorothy Rodbell Foundation
for Sarcoma Research, and the Rich Foundation. M.A.S.M. is supported by
NHLBI Grant No. RO1 HL61401. R.L.S. is supported by NHLBI Grant No. PO1 HL46403.
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.
Address reprint requests to Shahin Rafii, MD, Weill Medical College of
Cornell University, Hematology-Oncology Division, 1300 York Ave, Room
C-606, New York, NY 10021; e-mail: srafii{at}mail.med.cornell.edu.
| |
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N. Sengupta, S. Caballero, R. N. Mames, J. M. Butler, E. W. Scott, and M. B. Grant
The Role of Adult Bone Marrow-Derived Stem Cells in Choroidal Neovascularization
Invest. Ophthalmol. Vis. Sci.,
November 1, 2003;
44(11):
4908 - 4913.
[Abstract]
[Full Text]
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G. B. Adams, K. T. Chabner, R. B. Foxall, K. W. Weibrecht, N. P. Rodrigues, D. Dombkowski, R. Fallon, M. C. Poznansky, and D. T. Scadden
Heterologous cells cooperate to augment stem cell migration, homing, and engraftment
Blood,
January 1, 2003;
101(1):
45 - 51.
[Abstract]
[Full Text]
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T. Netelenbos, S. Zuijderduijn, J. van den Born, F. L. Kessler, S. Zweegman, P. C. Huijgens, and A. M. Drager
Proteoglycans guide SDF-1-induced migration of hematopoietic progenitor cells
J. Leukoc. Biol.,
August 1, 2002;
72(2):
353 - 362.
[Abstract]
[Full Text]
[PDF]
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I. B. Mazo, E. J. Quackenbush, J. B. Lowe, and U. H. von Andrian
Total body irradiation causes profound changes in endothelial traffic molecules for hematopoietic progenitor cell recruitment to bone marrow
Blood,
May 13, 2002;
99(11):
4182 - 4191.
[Abstract]
[Full Text]
[PDF]
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S. Fruehauf, K. Srbic, R. Seggewiss, J. Topaly, and A. D. Ho
Functional characterization of podia formation in normal and malignant hematopoietic cells
J. Leukoc. Biol.,
March 1, 2002;
71(3):
425 - 432.
[Abstract]
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J. Cashman, I. Clark-Lewis, A. Eaves, and C. Eaves
Stromal-derived factor 1 inhibits the cycling of very primitive human hematopoietic cells in vitro and in NOD/SCID mice
Blood,
February 1, 2002;
99(3):
792 - 799.
[Abstract]
[Full Text]
[PDF]
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J. D. van Buul, C. Voermans, V. van den Berg, E. C. Anthony, F. P. J. Mul, S. van Wetering, C. E. van der Schoot, and P. L. Hordijk
Migration of Human Hematopoietic Progenitor Cells Across Bone Marrow Endothelium Is Regulated by Vascular Endothelial Cadherin
J. Immunol.,
January 15, 2002;
168(2):
588 - 596.
[Abstract]
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[PDF]
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T. Papayannopoulou, G. V. Priestley, B. Nakamoto, V. Zafiropoulos, and L. M. Scott
Molecular pathways in bone marrow homing: dominant role of {alpha}4{beta}1 over {beta}2-integrins and selectins
Blood,
October 15, 2001;
98(8):
2403 - 2411.
[Abstract]
[Full Text]
[PDF]
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C. J. Dimitroff, J. Y. Lee, S. Rafii, R. C. Fuhlbrigge, and R. Sackstein
Cd44 Is a Major E-Selectin Ligand on Human Hematopoietic Progenitor Cells
J. Cell Biol.,
June 11, 2001;
153(6):
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[Abstract]
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X.-F. Zhang, J.-F. Wang, E. Matczak, J. Proper, and J. E. Groopman
Janus kinase 2 is involved in stromal cell-derived factor-1{alpha}-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells
Blood,
June 1, 2001;
97(11):
3342 - 3348.
[Abstract]
[Full Text]
[PDF]
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K. Hattori, B. Heissig, K. Tashiro, T. Honjo, M. Tateno, J.-H. Shieh, N. R. Hackett, M. S. Quitoriano, R. G. Crystal, S. Rafii, et al.
Plasma elevation of stromal cell-derived factor-1 induces mobilization of mature and immature hematopoietic progenitor and stem cells
Blood,
June 1, 2001;
97(11):
3354 - 3360.
[Abstract]
[Full Text]
[PDF]
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F. Bautz, C. Denzlinger, L. Kanz, and R. Mohle
Chemotaxis and transendothelial migration of CD34+ hematopoietic progenitor cells induced by the inflammatory mediator leukotriene D4 are mediated by the 7-transmembrane receptor CysLT1
Blood,
June 1, 2001;
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[Abstract]
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O. Kollet, A. Spiegel, A. Peled, I. Petit, T. Byk, R. Hershkoviz, E. Guetta, G. Barkai, A. Nagler, and T. Lapidot
Rapid and efficient homing of human CD34+CD38{-}/lowCXCR4+ stem and progenitor cells to the bone marrow and spleen of NOD/SCID and NOD/SCID/B2mnull mice
Blood,
May 15, 2001;
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[Abstract]
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F. Prosper and C. M. Verfaillie
Regulation of hematopoiesis through adhesion receptors
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March 1, 2001;
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M. Majka, A. Janowska-Wieczorek, J. Ratajczak, M. A. Kowalska, G. Vilaire, Z. K. Pan, M. Honczarenko, L. A. Marquez, M. Poncz, and M. Z. Ratajczak
Stromal-derived factor 1 and thrombopoietin regulate distinct aspects of human megakaryopoiesis
Blood,
December 15, 2000;
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[Abstract]
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A. Peled, O. Kollet, T. Ponomaryov, I. Petit, S. Franitza, V. Grabovsky, M. M. Slav, A. Nagler, O. Lider, R. Alon, et al.
The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice
Blood,
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[Abstract]
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M. A. Simpson, J. Reiland, S. R. Burger, L. T. Furcht, A. P. Spicer, T. R. Oegema Jr., and J. B. McCarthy
Hyaluronan Synthase Elevation in Metastatic Prostate Carcinoma Cells Correlates with Hyaluronan Surface Retention, a Prerequisite for Rapid Adhesion to Bone Marrow Endothelial Cells
J. Biol. Chem.,
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TOP
ABSTRACT
INTRODUCTION
