1 Molecular Immunology Group, Department of Clinical and Surgical Sciences, University of Edinburgh, Edinburgh EH3 9YW, United Kingdom; and 2 Cardiovascular and Gastrointestinal Discovery Department, AstraZeneca, Macclesfield SK10 4TG, United Kingdom
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ABSTRACT |
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Proteolysis-inducing factor
(PIF) is a novel sulfated glycoprotein initially identified as a
protein capable of triggering muscle proteolysis during the process of
cancer cachexia. Only skeletal muscle and liver exhibit substantial
binding of PIF in adult tissue. Here, we demonstrate that PIF induces
transcriptional regulation in both the liver endothelial cell line
SK-HEP-1 and in human umbilical vein endothelial cells (HUVECs) but not
in pulmonary artery endothelial cells. PIF differentially induces activation of nuclear factor-B, resulting in the induction of proinflammatory cytokines [interleukin (IL)-8 and IL-6] and increased expression of the cell surface proteins intercellular adhesion molecule-1 and vascular cell adhesion molecule in SK-HEP-1 and HUVECs
only. In addition, PIF induces the shedding of syndecans from the cell
surface. Syndecans are involved in wound repair, metastasis of cancers,
and embryonic development. These results suggest that PIF may play
additional roles in the proinflammatory response observed in cancer
cachexia but may also have a role without the cachectic process.
human umbilical vein endothelial cells; human pulmonary arterial endothelial cells; proteolysis-inducing factor; cachexia; inflammation
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INTRODUCTION |
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PROTEOLYSIS-INDUCING FACTOR (PIF), a novel protein of 24 kDa, was initially identified as sulfated glycoprotein capable of triggering muscle proteolysis during the process of cancer cachexia (24). PIF is capable of both inducing muscle protein degradation in isolated gastrocnemius muscle preparations and inducing weight loss (5, 23). In addition, the presence of PIF in the urine of cancer cachexia patients correlates with weight loss of >2.8 kg/mo (29). Comprehensive tissue screening in adults demonstrated that only skeletal muscle and liver exhibited substantial binding of PIF, leading us to explore the effects of PIF on transcriptional regulation in liver parenchymal cells.
We have recently demonstrated (26) that PIF is able to
induce both the proinflammatory response and the acute-phase response in human hepatocytes, suggesting additional roles of PIF other than the
degradation of skeletal muscle. PIF induced hepatocyte production of
the proinflammatory cytokines interleukin (IL)-8 and IL-6, postulated
as signals for the increase in muscle proteolysis during cachexia
(3, 20), and an induction of the acute-phase protein
response (8, 21). Induction of proinflammatory cytokines and acute-phase proteins is the result of increased gene transcription, mediated by cis-activating promoter elements that are
binding sites for cytokine-activating nuclear transcription factors,
including nuclear factor (NF)-B and STAT3.
Because liver comprises a variety of cell types, and to further
ascertain the function and signaling capacity of PIF, we have investigated the biological effect of this molecule on gene expression in a variety of endothelial cells. Liver endothelial cells were of
initial interest, but these could not be obtained in sufficient purity
from human liver isolations. We therefore employed endothelial cells
from a variety of sources to test the hypothesis that PIF could
influence transcriptional regulation and gene expression in endothelial
cells from different sites. These cells included the liver endothelial
cell line SK-HEP-1, primary human umbilical vein endothelial cells
(HUVECs), and human pulmonary artery endothelial cells (HPAECs). We
have investigated the effect of PIF on the expression of several
proteins regulated by NF-B and have thus examined the effect of PIF
on the activation of the NF-
B transcriptional pathway. The
NF-
B/Rel family consists of five members and can form various homo-
or heterodimer complexes in the cytosol (17), where it
remains inactive because of its association with inhibitory proteins of
the inhibitory (I)
B family, particularly I
B
. NF-
B is
activated by the phosphorylation, ubiquitination, and degradation of
I
B
, allowing NF-
B to translocate to the nucleus. Activation of
NF-
B is central to the regulation of many genes, including proinflammatory cytokines and cell surface markers (4).
Here we describe the effect of PIF on the induction of the
proinflammatory cytokines IL-8 and IL-6, the cell surface adhesion
molecules intercellular adhesion molecule (ICAM)-1 and vascular cell
adhesion molecule (VCAM), and the reduction in the expression of the
transmembrane proteolglycans syndecan-1 and syndecan-2 in endothelial cells.
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MATERIALS AND METHODS |
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Reagents.
DMEM, penicillin/streptomycin, L-glutamine, and FCS were
obtained from Life Technologies (Life Technologies, Inchinan, UK). Tumor necrosis factor (TNF)- and IL-6 were obtained from R&D Systems
(Oxon, UK). Protease inhibitor tablets were obtained from Boehringer
Mannheim (Lewes, UK). Enzyme-linked immunosorbent assay (ELISA) kits
were obtained from CLB (Eurogenetics UK, Hampton, UK). Antibodies for
flow cytometry were obtained from the endothelial section of the Sixth
International Workshop on Human Leukocyte Differentiation Antigens with
the following clone names: B-B42, B-B4, 10H4, 2G7Hu 8/4, and
51-10C9. ICAM-1 antibody was obtained from Beckman Coulter
(Beckman Coulter, High Wycombe, UK). Lipopolysaccharide (LPS) was
derived from Escherichia coli 0127:B8 Sigma (Sigma, Poole, Dorset, UK). SK-HEP-1 cells were obtained from ECACC (Porton Down, UK). HUVECs, HPAECs, and endothelial cell basal medium were obtained from Biowhittaker. All other reagents were purchased from
Sigma except the Pierce chemiluminescent system (Perbio, Cheshire, UK).
PIF was purified at Astra-Zeneca (Macclesfield, UK) from the urine of
pancreatic cancer patients with cachexia, as described previously
(23, 24). Column purification procedures were employed to
avoid endotoxin contamination, and purified material was checked at
each stage for endotoxin contamination. A Western blot showing native,
purified PIF alongside recombinant PIF from a mammalian expression
system is shown in Fig. 1.
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Cell culture.
Cells were grown in tissue culture flasks until 95% confluency was
reached. They were harvested using trypsin-EDTA, centrifuged at 500 g for 5 min, and resuspended in cell medium. Cells were then
seeded at 1 × 106/ml into 6- or 96-well culture
plates in medium containing 10% FCS and allowed to rest for 24 h
before commencing experiments. Cells were treated in these wells with
either TNF-, LPS, or PIF for 30 min or 24 h. Supernatants were
stored for ELISA analysis, and cells were used for either nuclear and
cytoplasmic protein extraction or staining with antibodies for flow
cytometric analysis.
Nuclear and cytoplasmic protein extraction.
Extracts were prepared according to the method of Johnson et al.
(15), and all buffers contained protease inhibitor
cocktail tablets (Boehringer Mannheim). All extraction
procedures were performed on ice with ice-cold reagents. Briefly, cell
monolayers in six-well tissue culture plates were washed two times with
150 mM PBS, pH 7.2. Cells were then scraped into 200 µl of
buffer A (10 mM HEPES, pH 7.9; 10 mM KCl; and 1.5 mM
MgCl2) and incubated for 15 min on ice. Lysis was achieved
by the addition of 25 µl of Nonidet P-40 in buffer A.
Nuclei were pelleted by centrifugation at 500 g for 4 min,
and the cytoplasmic supernatant was stored at 70°C. The pellet was
extracted with 50 µl of lysis buffer (30 mM HEPES, pH 7.9; 0.45 M
NaCl; and 1 mM EDTA). After a 15-min incubation on a mixer at 4°C,
the extract was centrifuged at 14,000 g at 4°C. The
supernatant was diluted 1:1 with 20 mM HEPES (pH 7.9), 0.1 M KCl, 0.2 mM EDTA, and 20% glycerol. Protein was estimated by the Bio-Rad DC
protein assay kit according to the manufacturer's instructions
(Bio-Rad, Hercules, CA), and nuclear supernatants were stored at
70°C until used for electrophoretic mobility shift assays (EMSA).
EMSA.
The transcription factor consensus oligonucleotides for the NF-B
responsive element (5'-AGT TGA GGG GAC TTT CCC AGG C-3') and the
surfactant protein (SP)-1-responsive element (5'-ATT CGA TCG GGG CGG
GGC GAG-3') were purchased from Promega (Southampton, UK). The probes
were labeled with [
-32P]ATP using T4 polynucleotide
kinase (Promega) and purified on Sephadex G-25 (Pharmacia) spin
chromatography columns. For the binding reaction, nuclear extract (1 µg of protein) was incubated in a 10-µl volume with 2 µl of EMSA
binding buffer [20% glycerol, 5 mM MgCl2, 250 mM NaCl,
2.5 mM EDTA, 2.5 mM dithiothrietol, 50 mM Tris · HCl (pH 7.5),
and 0.25 mg/ml poly(dI-dC) · poly(dI-dC)] and 1 µl
-32P-labeled probe according to manufacturer's
instructions (Promega). The specificity of the binding reaction was
determined by coincubating duplicate samples with either unlabeled
NF-
B or the nonspecific SP-1 oligonucleotide probe or an
anti-NF-
B p65 antibody (Insight Biotechnology, Santa Cruz, CA).
Protein-nucleic acid complexes were resolved using a nondenaturing
polyacrylamide gel consisting of 4% acrylamide gel run in 5 mM Tris
(pH 8.3) for 3 h at 200 V. Gels were transferred to Whatman 3M
paper (Whatman, Clifton, NJ), dried under a vacuum at 80°C for 1 h, and exposed to photographic film at
70°C with an intensifying screen.
Measurement of cytokines. IL-8 and IL-6 were measured using sandwich ELISA, as described previously (27). Briefly, immunoplates were coated overnight with rabbit anti-human antibody to the specific protein. Plates were washed four times, sample supernatants were added to the wells, and the plates were incubated for 1 h at room temperature. Plates were washed four times, and peroxidase-conjugated rabbit anti-human secondary antibody was added. Plates were incubated for 1 h at room temperature and then washed four times. The substrate 3,3',5,5'-tetramethylbenzidine was added for 30 min, and the reaction was stopped with 1 M sulfuric acid. The plates were read using a MR5000 ELISA plate reader (Dynatech, Billinghurst, UK), and sample concentrations were calculated using AssayZap (Biosoft, Cambridge, UK).
Flow cytometry. Cells were harvested from tissue culture plastic using Cell Dissociation solution (Sigma) and flow cytometry analysis performed as described previously (12). Briefly, cells were washed two times in PBS and incubated at room temperature for 20 min in rabbit serum. Cells were washed again two times in PBS and incubated for 30 min at room temperature with the appropriate antibody. Cells were washed again two times in PBS and incubated for 30 min at room temperature with a secondary antibody conjugated with fluorescein isothiocyanate (Sigma). Cells were then analyzed by flow cytometry.
Statistical analysis. Unless otherwise stated, values are reported as means ± SE. Data were analyzed by ANOVA followed by the Student's t-test where appropriate. Two-tailed tests were used on all occasions, and P values <0.05 were considered statistically significant.
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RESULTS |
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NF-B activation.
SK-HEP-1 cells, HUVECs, and HPAECs were grown to subconfluency, and all
cell types were treated with TNF-
(10 ng/ml) or PIF (10 and 100 ng/ml) in medium containing 10% serum. It has been postulated that PIF
binds to albumin, and parallel experiments have been carried out in
which cells were treated in the absence of FCS with no differences
observed (data not shown). Cells were treated for 15, 30, and 60 min,
and nuclear extracts were prepared to determine if NF-
B activation
occurred. Figure 2 shows that, in both
SK-HEP-1 cells and HUVECs, NF-
B was activated by both TNF-
and
PIF at 30 min. However, PIF had no effect on NF-
B activation in
HPAECs (Fig. 2C). Figure 2D shows a control EMSA
to determine the specific NF-
B band.
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Proinflammatory cytokine release.
The effect of PIF on the production of the NF-B-regulated cytokines
IL-8 and IL-6 was investigated in SK-HEP-1 cells, HUVECs, and HPAECs
after 24 h of exposure to PIF. Figure
3A shows the effects of
TNF-
(10 ng/ml) and PIF (10 or 100 ng/ml) on IL-8 release in the
cell supernatant in SK-HEP-1 cells, HUVECs, and HPAECs. TNF-
significantly induces IL-8 in all three cell types. IL-8 was also
induced in SK-HEP-1 cells and HUVECs after treatment with PIF. PIF did
not, however, induce IL-8 in HPAECs. Previous studies indicated that
TNF-
had a minimal effect on IL-6 release from hepatocytes; thus,
LPS was used as a positive control in endothelial cells. Figure 3,
B and C, shows the effects of LPS (10 µg/ml)
and PIF (10 or 100 ng/ml) on IL-6 release from SK-HEP-1 cells and
HUVECs. PIF significantly induces the production of IL-6 in both cell
types, and, although LPS (data not shown) was able to induce IL-6 in
HPAECs, PIF had no effect.
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Expression of adhesion molecules.
The effect of PIF on ICAM-1 and VCAM expression was investigated in
SK-HEP-1 cells, HUVECs, and HPAECs after 24 h of exposure to PIF
(10 ng/ml). Figure 4 shows the effects of
PIF on ICAM-1 and VCAM induction in SK-HEP-1 cells and HUVECs. PIF
significantly increases ICAM-1 expression in both SK-HEP-1 cells and
HUVECs but has no effect on HPAECs (data not shown). PIF also
significantly increases VCAM expression in the SK-HEP-1 cells but has
no effect on VCAM expression in either the HUVECs or HPAECs.
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Syndecan expression.
SK-HEP-1 cells, HUVECs, and HPAECs were exposed to PIF (10 ng/ml) for
24 h to determine the effect of PIF on cell surface expression of
a number of molecules. One of the most significant observations was
that syndecan-1 and syndecan-2 were decreased in both SK-HEP-1 cells
(Fig. 5A) and HUVECs (Fig.
5B) exposed to PIF. LPS had no effect on the expression of
syndecans in either SK-HEP-1 cells or HUVECs (data not shown). Syndecan
levels were not altered by PIF in HPAEC cells.
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DISCUSSION |
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PIF has no homology with known cytokine families and is
distinguished from other cytokines by the ability to accelerate the breakdown of skeletal muscle both in vitro and in vivo (5, 24). There is some evidence that the primary role of PIF is in
developmental regulation (26), and the molecule does not appear to be expressed in the normal adult, although this requires further investigation. However, the glycoprotein appears to be aberrantly reexpressed by some tumor cells and has been shown to be
present in the urine of pancreatic cancer patients with a weight loss
of >2.8 kg/mo (29). These patients suffer from cachexia,
characterized by massive weight loss resulting from the degradation of
skeletal muscle, loss of lean and fat body mass, increased energy
expenditure, and an increase in the proinflammatory and acute-phase
response (8, 28). Moreover, screening of adult tissues
suggested that the major binding sites for PIF in adult tissue were
skeletal muscle and liver. We have previously shown (26)
that PIF may contribute to the acute-phase response and the
proinflammatory response because of its ability to induce NF-B and
STAT3 activation in human hepatocytes. This induction is associated
with an increase in IL-8, IL-6, and C-reactive protein production, an
increase in ICAM-1 expression, and a decrease in transferrin
production. This suggests that PIF may play a crucial role in
the inflammatory state associated with some types of cancer in addition
to its postulated role in the degradation of skeletal muscle. Here we
show that PIF is able to activate both liver endothelial cells
(SK-HEP-1 cells) and HUVECs but not HPAECs. Endothelial cells form a
physical and interactive barrier between the blood and tissues and are
important in the pathophysiology of many conditions. Circulating
factors such as TNF-
are able to alter endothelial function,
including the synthesis and release of cytokines, such as IL-8 and
IL-6, and expression of cell surface adhesion molecules (16). Our data show that PIF induces the activation of
NF-
B in both SK-HEP-1 cells and HUVECs (but not the HPAECs),
resulting in the release of the proinflammatory cytokines IL-8 and
IL-6. NF-
B activation is central to the regulation of many genes,
including those involved in growth control (18) and
inflammatory processes (4). IL-8 and IL-6 influence the
inflammatory response, resulting in local activation of cells and
chemoattraction, and these results suggest that PIF may play a role in
endothelial gene regulation.
In addition to the induction of proinflammatory cytokine production,
this study demonstrates that PIF can induce the expression of both
ICAM-1 in SK-HEP-1 cells and HUVECs and VCAM in SK-HEP-1 cells. Neither
ICAM-1 nor VCAM expression was increased in the HPAECs after exposure
to PIF. Both ICAM-1 and VCAM are cell surface expression molecules that
can be upregulated by proinflammatory cytokines such as TNF- to
mediate the interaction between cells (7). One of the key
features of the inflammatory response is the local accumulation of
leukocytes, the type of which depends on the nature of the inflammatory
stimuli and the expression of adhesion molecules, such as ICAM and VCAM
on the endothelial surface. Migrating monocytes express leukocyte
function-associated antigen-1 and
4-integrin (CD49d),
which are ligands for expression molecules such as ICAM-1 and VCAM, and
the presence of these two adhesion molecules allows selective adherence
and accumulation of monocytes on the endothelial cell layer
(7). Accumulation of monocytes in the liver would
further enhance the proinflammatory response, especially since binding
of monocytes to the endothelium results in further activation of
endothelial cells (16). In addition, preliminary data show
that PIF has an effect on NF-
B activation and, hence, the
proinflammatory response in both Kupffer cells and circulating
monocytes. These results therefore suggest that PIF produced by some
cancers plays a role in the proinflammatory response that accompanies
the cancer. The results also suggest that there is some differential
specificity of action of PIF on endothelial cells at different sites.
Although the present work has examined resting endothelial cells, the
effect of shear stress on the production of cytokines is important.
Shear stress can profoundly influence cytokine expression by the
endothelium, but the effect is highly dependent on the magnitude of the
shear stress exerted. Normal fluid shear stress appears to be
protective against atherosclerosis and suppresses, for example, IL-8
production in HUVECs (25). However, high shear stress
involves the generation of platelet-derived microparticles and causes
activation of endothelial cells with the production of IL-8, IL-1,
and IL-6 (19). It is difficult to predict the effect of
PIF on endothelium in the presence of normal or abnormal shear stress,
and the effects may also vary depending on the origin of the
endothelial cells.
It is of considerable interest that PIF induces syndecan shedding from the SK-HEP-1 cell line and HUVECs, but not from the HPAECs, whereas LPS has no effect on syndecan expression. The syndecans are a family of transmembrane proteoglycans that are important in several signal transduction cascades, particularly in the regulation of cell proliferation (30). Expression of syndecans is highly regulated, and there are distinct patterns of expression in individual cell types and tissues and during developmental stages. Expression of the syndecans can be modified in pathological situations such as wound healing. Syndecans are able to interact with a number of molecules, including adhesion molecules, growth factors, lipid metabolism proteins, and pathogens, and can be involved in proliferation, differentiation, adhesion, migration, lipid metabolism, and infection (30).
Although little is known about syndecan shedding, it is thought to be highly regulated, stimulated by receptor activation and cell stress. Cleavage of the core protein from the cell surface appears to be controlled by the intracellular signaling pathway. The shed syndecan is thus rendered soluble and can exert extracellular effects, such as modulating the activity of heparin-binding molecules, like growth factors and cytokines, to promote tissue repair. However, excess shedding can interfere with repair and also with the host defense mechanism. This indicates that the shedding of syndecan is facultative in tissue repair and host defense (22, 30). In addition, it has been reported (14) that syndecan-1 can be used as a prognostic marker in cancer patients, since there appears to be a correlation between the loss (shedding) of syndecan-1 expression and mortality. Development of malignant epithelial tumors is associated with reduced intracellular adhesion, distribution, differentiation, and changes in the composition of the basement membrane, suggesting that expression and function of cell adhesion molecules could also change during malignant transformation. Loss of syndecan-1 is involved in uncontrolled proliferation, decreased adhesion, and disturbed differentiation of tumor cells. Hence. secretion of PIF may contribute to the pathogenesis/metastasis of a tumor by inducing a decrease in the expression of syndecans.
The normal role of PIF is unknown, but it is unlikely to be a protein produced only by tumor cells. In this study, it is clear that PIF does not affect transcriptional regulation in all endothelial subtypes. PIF induced responses in both SK-HEP-1 cells and HUVECs but not in HPAECs, indicating there may be some specialization in receptor expression by endothelial subtypes, possibly because of the normal role of PIF. In addition, VCAM expression was only upregulated in SK-HEP-1 cells. One clue to the normal role of PIF arises from the observation (unpublished data; see Ref. 5) that there is a peak of expression in the mouse embryo at embryonic day 8.5, suggesting an involvement in development. In addition, syndecan-1 is expressed at embryonic day 8.5 (2), apparently concurrent with the peak of PIF expression. The most dramatic changes in syndecan expression occur during development, and these are associated with morphological transitions, cell differentiation, and changes in tissue organization (2, 14, 22, 30). Embryogenesis is a dynamic process, and tissue-specific expression of PIF and syndecans during development may be coordinated through expression of ligands and coreceptors. In this study, the addition of PIF to endothelial cells results in a decrease in syndecan expression, suggesting a possible role in development through the induction of shedding of the functioning ectoderm portion of syndecan, allowing movement to the areas necessary for growth and development.
Furthermore, vascular development, in particular umbilical arteries, occurs at embryonic day 8.5 (10), again concurrent with PIF expression, and this is followed by organization of endothelial cells at embryonic day 9.25. It has been demonstrated that organization of endothelial cells to form into vessels requires cell-to-cell adhesion, requiring cell surface proteins such as cadherin and ICAM. ICAM is also reported to be expressed in cells during development, being involved in morphogenesis (1).
Activation of NF-B is also vital during development and cell growth.
NF-
B has been shown to be required for growth of tissues, particularly in the developmental phases, and its ability to regulate growth control through the regulation of cyclin D may be related to its
cell survival properties (11). NF-
B is involved in cell cycle regulation (13) by its ability to promote transition
from the G1 phase to the S phase in mouse embryonic
fibroblasts. NF-
B activation during the early phases of the cell
cycle is therefore necessary for regulation of both growth and
differentiation (9). In addition, preliminary data
indicate that PIF induces activation of the transcription factor
hepatocyte nuclear factor (HNF)-4. HNF-4 belongs to a family of
transcription factors that are involved in cell proliferation and
differentiation; HNF-4 is observed during morphogenesis and development
of the liver, at embryonic day 8.5 (6). Again,
this is concurrent with PIF expression. The identity of the receptor
for PIF remains undefined, and the reasons for continued expression of
the receptor in adult tissues are still unclear. The heavily
glycosylated nature of PIF may suggest that it can interact with a
number of cell surface structures to induce signaling. This is clearly
an area for future investigation.
In conclusion, PIF appears to have several important roles, including
skeletal muscle proteolysis during cachexia. We have demonstrated that
PIF is also able to induce the proinflammatory and acute-phase response
in human hepatocytes. Here we show that the action of PIF on liver
endothelial cells and HUVECs is to activate NF-B, resulting in both
the production of proinflammatory cytokines and modulation of the
expression of adhesion molecules. It is therefore probable that, during
cachexia, PIF induces a local inflammatory response in the liver by
increasing hepatocyte and endothelial proinflammatory production, which
attracts monocytes. An increase in endothelial adhesion molecules
allows leukocytes, such as monocytes, to adhere and thus contribute to
inflammation. In addition, the combined increase in IL-8 and IL-6
release from both the endothelial cells and hepatocytes may contribute
to the acute-phase response. PIF may therefore be a therapeutic target in the treatment of cancer cachexia. Perhaps more interesting, however,
are the differential effects of PIF on endothelial subtypes in view of
the putative role of this molecule in embryonic development.
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ACKNOWLEDGEMENTS |
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We thank Kathryn Sangster, Jean Maingay, and Ian Ansell for technical assistance.
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FOOTNOTES |
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This work was supported by the Biotechnology and Biological Sciences Research Council through Grant 15/G14945.
Address for reprint requests and other correspondence: J. A. Ross, Lister Research Laboratories, RIE, Lauriston Pl., Edinburgh EH3 9YW, United Kingdom (E-mail: J.A.Ross{at}ed.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpendo.00408.2001
Received 14 September 2001; accepted in final form 22 November 2001.
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