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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3785-3791
Noninflammatory Expression of E-Selectin Is Regulated by Cell Growth
By
Jianying Luo,
Gretchen Paranya, and
Joyce Bischoff
From the Surgical Research Laboratory, Children's Hospital and the
Department of Surgery, Harvard Medical School, Boston, MA.
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ABSTRACT |
E-selectin, an endothelial-specific adhesion molecule best known for
its role in leukocyte adhesion, is not detected in quiescent endothelial cells, but is induced by inflammatory stimuli. However, E-selectin is also expressed in proliferating endothelial cells under
noninflammatory conditions in vivo and in vitro, suggesting that
E-selectin is also regulated by growth signals. To investigate E-selectin expression in lipopolysaccharide-stimulated versus nonstimulated proliferating cells, we analyzed the distribution of
E-selectin-positive human microvascular endothelial cells in G0/G1, S, and G2/M phases of the
cell cycle under both conditions. Lipopolysaccharide treatment resulted
in uniformly increased E-selectin expression in cells in
G0/G1, S, and G2/M. In contrast,
levels of E-selectin in nonstimulated proliferating cells showed a
linear correlation with the percentage of cells in G2/M.
E-selectin in proliferating endothelial cells was not reduced by
addition of soluble tumor necrosis factor- -receptor or soluble
interleukin-1-receptor indicating that its expression was not due to
endogenous production of either cytokine. In addition, E-selectin was
increased in cells stimulated with basic fibroblast growth factor, a
well-known mitogen for endothelial cells. E-selectin in proliferating
endothelial cells is functional, as shown by E-selectin-dependent
adhesion of the promyelocytic leukemia cell line HL-60 to subconfluent human microvascular endothelial cells. In summary, these studies indicate that E-selectin can be regulated by a non-inflammatory pathway
that is related to the proliferative state of the endothelium.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE THREE MEMBERS of the selectin
family of cell adhesion molecules, E-, P-, and L-selectin, initiate
rolling of leukocytes along endothelium at sites of
inflammation.1 These carbohydrate-binding proteins share a
common structural domain organization and all three bind
sialylated fucosylated glycans such as sialyl Lewis-X (NeuAc 2,3Gal 1,4[Fuc1,3]GlcNAc) and sialyl Lewis-A
(NeuAc2,3Gal1,3[Fuc1,4]GlcNAc), as well as related
structures.2 The selectins promote leukocyte rolling by
binding to sialyl LewisX-containing counter receptors, the best
characterized being P-Selectin Glycoprotein Ligand-1 or
PSGL-1.3 P-, E-, and L-selectin-deficient mice have been generated to decipher the function of each selectin in different inflammatory settings.4-6 In endothelium, both E- and
P-selectin participate in leukocyte adhesion and have overlapping
functions.5 Analysis of E-selectin-deficient mice has
shown functional roles for E-selectin in granulocyte
rolling7 and stable adhesion of leukocytes to microvascular
endothelium.8 Mice deficient in both P- and E-selectin
exhibit dramatic leukocytosis, elevated levels of endogenous cytokines,
decreased leukocyte rolling, and increased susceptibility to
infections.9,10
Several studies indicate that E-selectin may also function in
angiogenesis. In vitro studies show that E-selectin is associated with
endothelial proliferation,11 migration,12 and
tube formation,13 all essential components of angiogenesis.
Furthermore, E-selectin is expressed in angiogenic tissues in
vivo. For example, E-selectin is present in proliferating endothelial
cells in vivo in noninflamed human hemangioma, a tumor of
endothelium that occurs in infants.14 The level of
E-selectin-positive blood vessels correlates with the proliferative
phase of hemangioma, indicating that E-selectin is expressed at the
right time and place to function in angiogenesis.14 E-selectin was also found to be significantly increased in human breast
carcinoma, with highest levels found in the most aggressive estrogen-receptor-negative tumors.15 E-selectin has been
detected in microvessels of many other human tumors16-19
and in normal noninflamed microvessels in human decidua,20
placenta, neonatal foreskin,14 and gingival
tissue.21 Its expression in noninflamed tissues suggests
that, in addition to its role in leukocyte adhesion, E-selectin may
participate in other important processes in the microvasculature.
In vitro analysis of E-selectin has been performed primarily using
human umbilical vein endothelial cells (HUVECs), which have served as a
model for endothelial activation and function.22-24 E-selectin is typically not detected in nonstimulated confluent HUVECs
but can be induced by treatment of the cells with cytokines such as
tumor necrosis factor- and interleukin-1 or lipopolysaccharide (LPS). E-selectin mRNA and polypeptide expression peaks 4 to 6 hours
after induction and then decreases over 24 hours to undetectable levels
even with continuous exposure to cytokine.23,24 This pattern of inducible E-selectin expression is consistent with its role
in leukocyte adhesion and its original identification as an endothelial
cell activation antigen. However, expression of E-selectin mRNA and
polypeptide has been observed in many types of endothelial cells
without addition of cytokines or LPS. For example, E-selectin mRNA has
been detected in bovine capillary endothelial (BCE) cells by Northern
blot analysis,13,25 in murine lung-derived microvascular
endothelial cells by ribonuclease protection analysis,26
and in human bone marrow-derived endothelial cells by reverse
transcription-polymerase chain reaction analysis.27 Constitutive E-selectin polypeptide expression has been reported in
human brain-derived endothelial cells28 and in BCE
cells.11 Cytokine-independent expression has also been
detected in low passage HUVECs by ribonuclease protection analysis and
enzyme-linked immunosorbent assay (ELISA).29 In each of
these studies, the level of E-selectin was upregulated by cytokine
treatment indicating that the pathway for cytokine stimulation was
functional in the endothelial cell cultures.
We and others have begun to examine the noninflammatory expression of
E-selectin. We showed that E-selectin polypeptide is expressed in
proliferating bovine endothelial cells and that E-selectin-positive cells are enriched in G2/M phases of the cell
cycle.11 Litwin et al30 investigated the role
of matrix and cell-cell contacts on cytokine-independent expression of
E-selectin in HUVECs and made several notable observations. First,
increased E-selectin in subconfluent endothelial cells was unaffected
by blocking antibodies against either tumor necrosis factor- or
interleukin-1, indicating that the increased E-selectin was not due
to autocrine stimulation by these cytokines. Second, cell-matrix and
cell-cell interactions were important regulators of the
cytokine-independent expression of E-selectin in that (1) cell
attachment to integrin ligands was required and (2) disruption of cell
contacts in confluent monolayers with anti-PECAM-1 antibody resulted
in increased E-selectin. They concluded that cytokine-independent
expression of E-selectin is regulated by endothelial cell density and
formation of endothelial cell junctions. Unpublished data were cited in
the report30 suggesting that the cytokine-independent
expression of human E-selectin was not related to cell proliferation.
In the present study, we investigated the noninflammatory expression of
E-selectin by examining the distribution of E-selectin-positive cells
in G0/G1, S, and G2/M phases of the
cell cycle in LPS-stimulated versus nonstimulated proliferating
endothelial cells. Our results indicate that noninflammatory expression
of E-selectin is regulated at least in part by cell proliferation.
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MATERIALS AND METHODS |
Isolation and culture of human dermal microvascular endothelial cells
(HDMEC).
HDMEC were isolated from newborn foreskin using Ulex europaeus
I-coated Dynabeads (Dynal, Inc, Oslo, Norway)31 with
some modifications.32 The HDMECs were grown in EBM 131 (Clonetics, San Diego, CA) supplemented with 10% heat-inactivated
fetal bovine serum (FBS) (Hyclone, Logan, UT), 1 × glutamine,
penicillin, streptomycin (GPS) (Irvine Scientific, Irvine, CA), 2 ng/mL
human recombinant basic fibroblast growth factor (bFGF) on 1%
gelatin-coated dishes in a 5% CO2 incubator. For
abbreviation, we designate this growth medium EBM-B. Cells were
passaged 1:3 every 4 to 6 days, and used at passages 3 to
12.32 The FBS used for these studies contained not more
than 0.06 EU/mL endotoxin, as determined by the manufacturer. Human
recombinant bFGF was kindly provided by Scios Inc (Mountain View, CA).
For comparison with other studies on HDMECs, cells were also grown in
EBM 131 supplemented with 20% FBS, 100 U/mL penicillin, 100 µg/mL
streptomycin sulfate, 0.25 µg/mL amphotericin B (ie, fungizone)
(GIBCO-BRL, Gaithersburg, MD), 2 mmol/L glutamine (Irvine Scientific),
0.5 mmol/L N6, 2'-O-dibutyryladenosine 3':5'-cyclic
monophosphate (Sigma Chemical Co, St Louis, MO), 1.0 µg/mL
hydrocortisone (Sigma). For abbreviation, we designate this medium
EBM-A.
Other cells used.
The human promyelocytic leukemia cell line HL-60 (ATCC cat. no.
CLL-240) was grown in suspension in RPMI medium-1640 (GIBCO-BRL) supplemented with 10% FBS, 1 × GPS and maintained at a density of
less than 1 × 106 cells/mL. BCE cells were grown in
Dulbecco's modified Eagle's medium supplemented with 10% calf serum,
GPS, and 3 ng/mL bFGF as described.11
FACS and cell-cycle analysis.
A gentle fixation and permeabilization method for simultaneous analysis
of cell surface antigens and quantification of DNA33 was
used. HDMECs were gently scraped from the dishes in phosphate-buffered saline (PBS) with 5 mmol/L EDTA, sedimented, resuspended in Hanks' balanced salt solution with calcium and magnesium (HBSS) and counted. The cells were centrifuged again and resuspended in HBSS, 2% calf serum, 0.02% NaN3 (Ab solution). The cells were incubated
with 5 µg/mL anti-human E-selectin monoclonal antibody
MoAb14 or an isotype-matched murine immunoglobulin G (IgG)
(Becton Dickinson, Mountain View, CA) for 30 minutes at 4°C on a
rotating platform. The cells were washed twice with Ab solution and
incubated with 1/200 dilution of horse anti-mouse IgG-fluorescein
isothiocyanate (FITC) conjugate as above. The cells were washed,
resuspended in 0.25% paraformaldehyde in PBS for 1 hour at 4°C, and
permeabilized with 0.2% Tween in HBSS for 15 minutes at 37°C. The
cells were sedimented again, resuspended in Ab solution at 1 × 106 cells/mL, and incubated with 200 µg/mL RNase A (11.25 Kunitz units/mL) for 90 minutes at room temperature. Propidium iodide was added to 10 µg/mL and the cells were stored at 4°C until
analyzed on a Becton Dickinson FACScan flow cytometer for FITC
(E-selectin) and propidium iodide (DNA). FITC-negative was defined as
the level of fluorescence observed in 99% of HDMECs incubated with
isotype-matched control IgG1. Cells stained with
anti-E-selectin with fluorescence intensity higher than this were
designated FITC+ or E-selectin-positive. The relative mean
fluorescence intensity ( MFI) was calculated by subtracting the MFI
of 10,000 HDMECs incubated with isotype-matched control antibody from
the MFI of 10,000 HDMECs incubated with anti-human E-selectin MoAb. For
the analysis of bFGF-stimulated cells, immunostaining was performed as
above, but after fixation in paraformaldehyde cells were washed once
with HBSS and resuspended in 0.5 mL of Krishan's reagent (1% sodium
citrate, 20 µg/mL RNAse A, 0.3% Nonidet-P 40, 50 µg/mL propidium iodide).
ELISA.
HDMEC were plated at a density of 3,000 cells per well in a 96-well
plate in EBM-B in the absence or presence of soluble TNF--receptor (1 µg/mL) or soluble interleukin (IL)-1-receptor (1 µg/mL) and incubated for 48 hours. After 48 hours, untreated parallel wells were
stimulated with 2 ng/mL TNF- in the presence or absence soluble
tumor necrosis factor (TNF)--receptor (1 µg/mL) or with 2 U/mL
IL-1 in the presence or absence of soluble IL-1-receptor (1 µg/mL) for 4 hours. The soluble recombinant dimeric human TNF receptor p80/IgG1 Fc fusion protein and soluble recombinant
human IL-1 receptor type I were kindly provided by Immunex Corp
(Seattle, WA). The cells were washed twice with PBS and fixed in
 20°C methanol for 10 minutes on ice. E-selectin was detected as
described previously.32 Absorbance at 410 nm obtained with
isotype-matched control IgG1 was subtracted as background.
Each assay point was performed in triplicate. Bar graphs represent mean ± standard deviation.
HL-60 adhesion assay.
HDMECs were plated on gelatin-coated 35-mm dishes and assayed 3 days
after plating (proliferating) or 7 days after plating (confluent). Cell
monolayers were fed with fresh medium with or without 0.2 µg/mL LPS 5 hours before the adhesion assay. For antibody blocking, the monolayers
were incubated in 1 mL of EBM-B with 20 µg/mL of anti-human
E-selectin MoAb H18/7 (Becton Dickinson) or with 20 µg/mL
isotype-matched control IgG2a (Becton Dickinson) for 30 minutes at room temperature on a rocking platform. The media was
removed and cell monolayers washed once with RPMI media with or without
2.5 mmol/L EGTA. HL-60 cells were washed once with RPMI media alone,
resuspended in the presence or absence of 2.5 mmol/L EGTA, and added to
the cell monolayers at 2 × 106 cells/0.6 mL. Cells were
incubated at 4°C on a rocking platform for 45 minutes, washed 5 times
with 2 mL of RPMI with or without 2.5 mmol/L EGTA, and fixed with 2.5%
glutaraldehyde. Bound HL-60 cells were counted in 10 randomly selected
fields. The number of bound cells is expressed as mean ± standard
error of the mean (SEM).
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RESULTS AND DISCUSSION |
Cell-cycle distribution of E-selectin-positive cells in LPS-stimulated
versus nonstimulated HDMEC.
We analyzed LPS-stimulated versus nonstimulated HDMEC to determine if
in response to an inflammatory stimulus the distribution of
E-selectin-positive cells in G0/G1, S, and
G2/M phases of the cell cycle differs from that observed in
the total cell population. Figure 1 shows
results from a representative experiment in which the distributions of
total and E-selectin-positive (FITC+) endothelial cells in
G0/G1, S, and G2/M phases of the
cell cycle were determined by flow cytometry using propidium iodide.
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| Fig 1.
Cell-cycle analysis of E-selectin-positive HDMECs.
Proliferating (A through D) and confluent (E through H) HDMECs were
treated without (Control; A, B, E, and F) or with 0.2 µg/mL LPS (LPS;
C, D, G, and H) for 4 hours. Cells were analyzed for E-selectin
expression and distribution in G0/G1 and S to
G2/M as described in Materials and Methods. (A, C, E, and
G) Histograms of DNA content (ie, propidium iodide staining) for the
total cell population. (B, D, F, and H) Histograms of DNA content in
E-selectin-positive cells (FITC+). The percent
distribution of cells in different phases of the cell cycle
(G0/G1, S, G2/M) is shown for each
panel.
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In LPS-treated HDMECs (Fig 1C, D, G, and H), the cell-cycle
distribution of E-selectin-positive cells (FITC+) was
similar or identical to that of the cell population as a whole (total
cells) indicating that in response to LPS, E-selectin was increased
uniformly in cells at each phase of the cell cycle. This occurred in
both proliferating (Fig 1C and D) and confluent (Fig 1G and H) HDMECs.
In contrast, the cell-cycle distribution of FITC+ cells
differed from that of the total cells in proliferating nonstimulated
HDMECs in that there was a 12% shift in cells from G0/G1 and S to G2/M (Fig 1A
v B). The enrichment of E-selectin-positive cells in
G2/M was more pronounced in confluent HDMECs wherein the
percentage of E-selectin-positive cells in G2/M was
increased 22% compared with the total cell population (Fig 1E
v F). As expected, fewer cells express E-selectin (ie,
FITC+) in confluent cells. The shape of the histogram
curves in Fig 1B and 1F are clearly different from the other 6 panels
and show the relative shift of E-selectin-positive cells into
G2/M in nonstimulated cells.
Table 1 presents the cell-cycle
distribution of the total and E-selectin-positive cell populations, as
determined by flow cytometry, determined in three additional
experiments. These data show that results presented in Fig 1 (A and B)
are reproducible and confirms the relative shift of
E-selectin-positive cells into G2/M in nonstimulated
cells.
Table 2 summarizes the levels of E-selectin
expression for the experiment presented in Fig 1. In absence of LPS
stimulation, E-selectin was well-expressed in proliferating cells (33%
FITC+), but very low in confluent cells (7%
FITC+). In the presence of LPS, E-selectin was increased in
both growing and confluent cells. The MFI under all four culture
conditions shows several important points (see Table 2). First, the
level of non-inflammatory E-selectin expression was 20-fold higher in proliferating HDMECs compared with confluent HDMECs. Second, because of
the high basal expression of E-selectin in proliferating HDMECs, LPS
caused only a twofold increase in E-selectin in proliferating HDMEC
compared with the 20-fold increase in confluent HDMEC. Third, the net
increase in E-selectin in LPS-treated cells, as measured by flow
cytometry, was similar in both proliferating and confluent endothelial
cells (ie, MFI ~ 190). In summary, our results indicate that LPS
increased E-selectin uniformly in cells in different phases of the cell
cycle while in nonstimulated cells, E-selectin-positive cells were
enriched in G2/M.
E-selectin expression in proliferating endothelial cells is not due
to endogenous TNF- or IL-1.
To determine if proliferating HDMECs produce endogenous TNF- or
IL-1 that could in turn induce E-selectin expression, we tested the
effect of neutralizing soluble receptor antagonists for these two
cytokines. As shown in Fig 2, excess
soluble TNF--receptor or soluble IL-1-receptor did not reduce
E-selectin in proliferating cells (Fig 2A). As a positive control, Fig
2B shows that the soluble receptor antagonists were effective in
blocking both TNF-- and IL-1-induced E-selectin expression.
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| Fig 2.
Noninflammatory expression of E-selectin in proliferating
HDMECs. (A) E-selectin was measured by ELISA in proliferating HDMECs
incubated for 2 days in the absence (C) or presence of soluble
TNF--receptor (sTNF-R) or soluble IL-1-receptor (sIL-1b-R). (B)
As a positive control, HDMECs were stimulated with either TNF- or
IL-1 for 4 hours in the absence (C) or presence of the corresponding
soluble receptor antagonist.
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Increased E-selectin in bFGF-treated HDMECs.
Our data presented thus far suggest that mitogens that stimulate
endothelial cell proliferation should stimulate E-selectin expression.
To test this directly, we examined the effect of bFGF on E-selectin
expression. To do this experiment, confluent HDMECs were serum-starved
and bFGF-starved by incubating in EBM 131, 0.5% FCS without bFGF for
24 hours. The cells were then trypsinized and replated in the EBM 131, 5% FCS at subconfluent density with or without 2 ng/mL bFGF for 46 hours. As seen in Fig 3A,
bFGF caused a dramatic shift of the cells out of
G0/G1. Coincident with this shift,
E-selectin-positive cells increased from 27% (Fig 3E) to 46% (Fig
3C). The percent FITC-positive cells (M1) detected with isotype-matched
control IgG (Fig 3B and D) was substracted from percent detected with
anti-human E-selectin (Fig 3C and E). In summary, the mitogenic effect
of bFGF on HDMECs corresponded with an increase in E-selectin as
measured by flow cytometry.
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| Fig 3.
Increased E-selectin in bFGF-stimulated HDMECs.
Cells were serum-starved and bFGF-starved for 24 hours, trypsinized,
and replated at subconfluent density in the absence (gray line)
or presence of 2 ng/mL bFGF (solid black region) for 46 hours, and
then analyzed by flow cytometry for cell-cycle distribution (A)
and E-selectin expression (C and E). Nonspecific antibody binding was
measured using isotype-matched control IgG1 (B and D). Forty-six
percent of the cells were E-selectin-positive (C) and 27% were
positive (E).
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Linear correlation of E-selectin expression with percentage of cells
in G2/M.
To further illustrate the relationship between E-selectin expression
and the cell cycle, we plotted the MFI for E-selectin versus
percentage of cells in G2/M that was determined in several different experiments. As seen in Fig 4,
there is a linear correlation between MFI and percentage of cells
in G2/M in nonstimulated HDMECs (+). Consistent with
results in Fig 1, there is no relationship between MFI and
percentage of cells in G2/M in LPS-treated cells ( ).
This analysis confirms the results in Fig 1 and, importantly, shows
that in the absence of inflammatory stimuli, E-selectin expression can
be used to assess the proliferative state of endothelial cells.
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| Fig 4.
Linear relationship between  MFI of cells incubated
with anti-E-selectin MoAb and percentage of cell population in
G2/M. MFI versus the percentage of HDMEC in
G2/M was plotted in a scatter graph format. Cells were
nonstimulated (+) or treated with 0.2 µg/mL LPS for 16 hours ().
Data from 17 experiments, including those shown in Fig 1 and Table 1,
were used for this analysis.
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Effect of growth medium on E-selectin expression.
For these studies, the HDMECs were grown and maintained in a relatively
simple growth medium (EBM-B). We have shown that cells grown in EBM-B
(EBM 131, 10% FBS, GPS, 2 ng/mL bFGF) maintain continued
responsiveness to growth factors and cytokines for at least 3 months
and up to 12 trypsin passages.32 More typically, human
microvascular endothelial cells are grown in media such as EBM-A, which
contains 20% FBS, fungizone, dibutryl cAMP, and hydrocortisone.34-36 To compare the effect of growth media
on levels of E-selectin polypeptide, we analyzed E-selectin expression
and the distribution of cells in G0/G1, S, and
G2/M in cells grown in EBM-B versus EBM-A. In three
separate experiments, the MFI was decreased 39% in cells grown in
EBM-A compared to cells grown in EBM-B with a concommitant decrease in
percentage of cells in G2/M (data not shown). This
indicates that growth media can affect the expression of E-selectin in
cultured cells.
E-selectin expressed in proliferating cells mediates adhesion of
HL-60 cells.
To determine if E-selectin expressed in proliferating HDMECs can
mediate leukocyte adhesion, we used a well-established cell-cell adhesion assay in which HL-60 cells are allowed to adhere to
endothelial cells grown on gelatin-coated culture dishes under normal
growth conditions. HL-60 is a human promyelocytic leukemia cell line that was used in the functional identification of E-selectin as an
endothelial-leukocyte adhesion molecule.23,24 HL-60 cell adhesion to proliferating versus confluent HDMECs was measured as
described in Materials and Methods (Fig
5A). HL-60 cells bound to proliferating but
not to confluent HDMECs (solid bars). As expected, LPS-stimulation
resulted in a dramatic increase in HL-60 cell adhesion to both cultures
(hatched bars). HL-60 cells did not adhere to gelatin-coated dishes
that did not have endothelial cells plated on them (data not shown).
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| Fig 5.
HL-60 cell adhesion to HDMEC. HL-60 cell adhesion to
endothelial cells treated for 5 hours with PBS (solid bars) or with 0.2 µg/mL LPS (hatched bars) was measured in HDMEC 3 days (proliferating)
or 7 days (confluent) after trypsin passage (A). (B) HL-60 adhesion to
proliferating HDMEC was measured in presence of 2.5 mmol/L EGTA, 20 µg/mL anti-E-selectin MoAb H18/7, or an isotype-matched
IgG2a control MoAb. Cells bound ± SEM was calculated from
10 randomly selected fields.
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The HL-60 cell adhesion to nonactivated proliferating HDMECs
was analyzed in more detail in Fig 5B. HL-60 cell adhesion to proliferating HDMECs was inhibited by 2.5 mmol/L EGTA, showing that the
adhesion was calcium-dependent, a feature expected for selectin-mediated binding. HL-60 cell adhesion to proliferating HDMECs
was inhibited by a function blocking anti-human E-selectin MoAb
designated H18/7,24 but not by an isotype-matched control Ig. This shows that a significant portion of the HL-60 cell adhesion to
HDMECs is mediated by E-selectin. This experiment also verifies that
E-selectin expressed in proliferating human microvascular endothelial
cells is on the cell surface and functional. If the same is true for
E-selectin expressed in proliferating endothelial cells in
vivo,14 E-selectin-mediated activities might have
functional significance in angiogenic tissues or diseases.
In summary, we show directly that noninflammatory expression of
E-selectin is correlated with cellular proliferation in human microvascular endothelial cells. Therefore, E-selectin expression may
be a useful parameter for monitoring the proliferative state of
endothelial cells. E-selectin polypeptide is enriched, but not
restricted to cells in G2/M. Its presence in other phases of the cell cycle may be related to the time required for turnover of
E-selectin or perhaps to other regulatory factors such as cell-cell and
cell-matrix interactions as suggested by Litwin et al.30 Our results are consistent with a previous study in which transforming growth factor-, a protein that inhibits endothelial cell progression through the cell cycle,37 was shown to downregulate
E-selectin in low passage HUVECs.29 The functional
significance of E-selectin expression in proliferating endothelial
cells is under investigation in our laboratory.
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ACKNOWLEDGMENT |
We thank Dr Debra Chao for help with flow cytometry analyses. We thank
Alan Flint and Marc Alande of the FACS Core Facility at Children's
Hospital and members of the FACS Core Facility at the Dana Farber
Cancer Institute for their technical assistance.
| |
FOOTNOTES |
Submitted May 18, 1998; accepted January 18, 1999.
Supported by grants from the National Institutes of Health (No. F32
HL09390 to J.L., No. P01 CA 45548 and R29 GM46757 to J.B.), and a grant
from the Charlotte Geyer Foundation to J.B.
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 Joyce Bischoff, PhD, Surgical Research
Laboratory, Children's Hospital, 300 Longwood Ave, Boston, MA 02115;
e-mail: bischoff{at}a1.tch.harvard.edu.
| |
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