© 1997 American Heart Association, Inc.
Articles |
Basic Fibroblast Growth Factor (bFGF) Regulates the Expression of the CC Chemokine Monocyte Chemoattractant Protein-1 (MCP-1) in Autocrine-Activated Endothelial Cells
From the Cell Biology Laboratory, Department of Gynecology and Obstetrics, University of Göttingen Medical School, Germany; and the Department of Surgery, Maine Medical Center Research Institute, South Portland (V.L.).
Correspondence to Dr Hellmut G. Augustin, Cell Biology Laboratory, Department of Gynecology and Obstetrics, University of Göttingen Medical School, Robert-Koch-Str 40, 37075 Göttingen, Germany. E-mail haugust{at}med.uni-goettingen.de
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Abstract The CC chemokine monocyte chemoattractant protein (MCP)-1 is induced by inflammatory cytokines and acts as a potent regulator of monocyte trafficking. Monocytes adhere preferentially to migrating endothelial cells in vitro and to endothelial cells at the migration front in vivo after aortic balloon denudation injury. Based on these findings, we analyzed MCP-1 expression in migrating and resting bovine aortic endothelial (BAE) cells and identified prominently upregulated levels of MCP-1 expression in migrating BAE cells. Stimulation of resting BAE cells with 5 ng/mL bFGF resulted in a fourfold induction of MCP-1 mRNA expression. The time course of bFGF-induced MCP-1 mRNA expression indicated a rapid and direct stimulation of MCP-1 expression that was detectable 30 minutes after stimulation. Levels of basal MCP-1 expression, as well as upregulated levels of MCP-1 in migrating BAE cells, were downregulated by addition of a neutralizing anti-bFGF monoclonal antibody (1.0 µg/mL). Digestion of conditioned media of resting BAE cells with collagenase led to a dose-dependent induction of MCP-1 expression in resting BAE cells, which was inhibited >50% by addition of neutralizing anti-bFGF antibody. Confirmation of the Northern blot experiments by ELISA-based quantitation of MCP-1 protein levels identified threefold to sixfold higher levels of MCP-1 in the supernatants of bFGF-stimulated BAE cells than in unstimulated resting BAE cells. Finally, analysis of MCP-1 expression by in situ hybridization carried out on en face preparations of aortas demonstrated that MCP-1 expression is dramatically upregulated in regenerating endothelial cells in vivo after balloon denudation. Though not establishing a direct causal relation between the preferential adhesion of monocytes to migrating endothelial cells, these findings strongly suggest that autocrine-activated endothelial cell–derived MCP-1 may play a critical role in recruiting monocytes. They furthermore support the concept that bFGF acts as an autocrine regulator of endothelial cell activity and may imply an involvement of bFGF as a mediator of inflammatory cell trafficking.
Key Words: endothelial cells • chemokines • MCP-1 • bFGF
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Introduction |
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Chemokines are a family of polypeptides (8 to 10 kD) that act as potent chemoattractants on leukocytes. They are structurally grouped into two subfamilies, the
- or C-X-C subfamily and the ß- or CC subfamily,
based on the characteristic presence of four conserved cysteine
residues.1 Lymphotactin has been identified as a C
chemokine that lacks two of the four conserved cysteine
residues.2 Additionally, the recently identified
fractalkine appears to represent a novel subfamily of
CX3C chemokines.3 Chemokines are produced by a
large number of different cell types and have distinct but overlapping
target cell specificities. Members of the -chemokine subfamily act
predominately on neutrophils, whereas ß-chemokines attract monocytes,
eosinophils, and basophils. Members of both subfamilies together with
lymphotactin attract specific lymphocyte subpopulations. Chemokines act in concert with endothelial cell adhesion molecules to recruit leukocytes to sites of inflammation.4 These synergistic activities of adhesion molecules and chemokines are coordinately regulated and involve functional cross talk between the two classes of molecules. In addition to their direct chemotactic activity on distinct target cells, chemokines, possibly presented by proteoglycans on the surface of endothelial cells,5 regulate adhesion molecule expression,6 7 avidity of cell surface integrins,8 and surface distribution of adhesion molecules.9 In turn, adhesive interactions between leukocytes and endothelial cells trigger the expression of specific chemokines.10 11 12 It thus appears that chemokine functions are involved in several steps of the adhesion and recruitment cascade that include the switch from leukocyte rolling to firm adhesion, activation of the adhering leukocyte, and diapedesis and migration into the perivascular tissue.1
MCP-1 is one of the best-studied members of the ß- or CC chemokine
subfamily.13 14 It is expressed by a wide variety of
normal and malignant cells, including endothelial
cells, monocytes, vascular smooth muscle cells, fibroblasts, and
glioma, sarcoma, and melanoma cells.14 Originally
identified as a platelet-derived growth factor–inducible
gene,15 MCP-1 was soon characterized as chemoattractant
for monocytes16 and T lymphocytes17 and was
found to be primarily regulated by inflammatory cytokines such
as TNF- and IL-1.18 19 20 21 Its primary function appears to
be the recruitment of monocytes, as suggested by the phenotype
of transgenic mice overexpressing MCP-1 (perivascular cuffs of
monocytes).22
Of the different cell types that express MCP-1 on stimulation by inflammatory cytokines, induced expression of MCP-1 by vascular endothelial cells is probably most important for the initial recruitment of monocytes to sites of inflammation. The monocytic cell line U937 has previously been shown to adhere preferentially to migrating endothelial cells that had not been stimulated by inflammatory cytokines.23 These observations suggested that the expression of cell adhesion molecules involved in monocyte adhesion to endothelial cells might be regulated by autocrine activity of the endothelial cells that were activated by simply the release from growth arrest. Analogously, we hypothesized that migrating endothelial cells might contribute to the recruitment of monocytes by autocrine-regulated expression of MCP-1. Here we have analyzed the differential expression of MCP-1 in resting and migrating endothelial cells in vitro and in vivo. The data demonstrate that endogenous bFGF regulates MCP-1 expression in autocrine-activated endothelial cells. Upregulation of MCP-1 expression at the migrating front of regenerating endothelial cells after aortic balloon denudation suggests that similar mechanisms govern endothelial cell–regulated monocyte trafficking in vivo.
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Methods |
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Cytokines, Antibodies, and Reagents
bFGF, aFGF, and TNF
were obtained from Promega. transforming
growth factor-ß1 was purchased from Life Technologies.
Neutralizing monoclonal mouse anti-bovine bFGF antibody was purchased
from Upstate Biotechnology (Biomol). Crude bacterial
collagenase (CLS2) was obtained from Worthington
(CellSystems). Endothelial cell growth medium and
endothelial cell growth supplement (human umbilical
vein endothelial cell culture) were purchased from
PromoCell. Dulbecco's modified Eagle's medium (DMEM) and other
cell-culture media were from Life Technologies. Fetal bovine serum was
obtained from Biochrom.
Cells
BAE cells were isolated from thoracic aortas of healthy cattle
by collagenase digestion following standard
protocols.24 Cells were cultured at 37°C in
75-cm2 tissue-culture dishes in Dulbecco's modified Eagle
medium containing 10% heat-inactivated fetal calf serum
and frozen in liquid nitrogen at passage 2 or 3. Cells were routinely
split at a 1:5 ratio and cultured up to 50 passages. Using these
culture conditions, BAE cells express high levels of
endogenous bFGF up to passage 20 that will then gradually
decline to undetectable levels during in vitro
senescence.24 U937 cells were from American Type Culture
Collection and cultured under standard culture conditions.
Cell-Culture Assays
Two-dimensional lateral sheet migration of
endothelial cells was studied using a silicon template
fencing technique, which allows controlled release from growth arrest
without wounding the cells at the migration front.25
Populations of migrating cells (Northern blot analysis) were
produced by seeding cells at low density (1:10) and harvesting them 24
hours later as subconfluent monolayers. These subconfluent cells
express the same phenotypic properties as the cells at the migration
front.26 Monolayers of resting confluent cells were refed
24 hours before harvesting. For cytokine stimulation
experiments, cells were grown to confluence and stimulated with
different recombinant human cytokines (5 ng/mL) in fresh
media (BAE cells [P10-P25]: Dulbecco's modified Eagle's medium, low
glucose, with 3% fetal calf serum). For collagenase
treatment, BAE cells (P20-P30) were grown to confluence for 3 days. The
cells were refed and cultured for another 2 days, after which varying
concentrations of crude bacterial collagenase were added to
the medium for 2.5 hours. Where indicated, anti-bFGF monoclonal
antibody (1 µg/mL) was added to the medium 30 minutes before
the addition of collagenase. Anti-bFGF antibody experiments
involving migrating BAE cells were performed by passaging BAE cells at
a 1:5 ratio, allowing them to adhere for 3 hours, adding 1
µg/mL neutralizing antibody, and harvesting them as
subconfluent monolayers 2.5 hours later.
For BAEC and U937 coculture experiments, confluent BAE cells (in 12-well plates) were released from the silicon ring25 and allowed to migrate for 24 hours, after which 5x105 U937 cells were added. The monocytic U937 cells were allowed to adhere for 2 hours at 37°C, after which the monolayer was washed, fixed with 4% paraformaldehyde, and stained with hematoxylin.
RNA Isolation and Northern Blot Analysis
Cells were washed twice with PBS and harvested with a cell
scraper. Total RNA was isolated according to the single-step
guanidinium thiocyanate-phenol-chloroform extraction
procedure.24 For Northern blot analysis of MCP-1
expression, 5 to 10 µg of total RNA was electrophoresed in a 1%
agarose gel, capillary transferred onto nylon membranes, and used for
hybridization with the bovine MCP-1 cDNA, pH 42.27
Hybridization with an 18S rRNA oligonucleotide was
performed to confirm equal loading of the different
lanes.24 Hybridization signals were quantitated by
PhosphorImager analysis.
Quantitation of MCP-1 Protein
MCP-1 protein concentrations in the different cell populations
were determined using a sandwich ELISA technique according to the
manufacturer's instructions (R&D Systems). Supernatants of resting
confluent monolayers, as well as 6 hours' cytokine-stimulated
monolayers, were harvested, centrifuged at 1000g,
and directly used for ELISA quantitation. Concentrations of MCP-1
protein in the supernatants of human cells were determined using the
provided standards. The same human ELISA kit was also used to
quantitate MCP-1 protein in the supernatants of bovine cells. The
anti-human antibodies cross-reacted with bovine MCP-1 but identified
bovine MCP-1 with a 30-fold lower sensitivity than the human MCP-1.
Bovine MCP-1 used as standard for these experiments was purified from
seminal vesicle fluid as described.27
Arterial Injury Model
Aortic endothelium of male Sprague-Dawley rats
(400 g, 3 to 4 months old) was partially denuded with an uninflated 2
French balloon catheter.28
Deendothelialized segments of aorta were
identified by intravenous injection of Evans blue (0.3 mL
in 5% saline solution) 10 minutes before killing. The rats (three
animals per time point) were perfusion-fixed with phosphate buffered
4% paraformaldehyde. Denuded aortas and control aortas
were cut open longitudinally, and the corresponding segments were
trimmed and used for in situ hybridizations. For detection of adherent
monocytes, en face preparations were processed by the Häutchen
procedure29 and stained with a 1:200 dilution of a mouse
monoclonal antibody recognizing rat monocyte/macrophages (ED-1;
Serotec).
In Situ Hybridization
Vessel segments were treated with proteinase K (1
µg/mL, 37°C, 15 minutes), prehybridized for 2 hours at
55°C in 0.3 mol/L NaCl, 20 mmol/L Tris (pH 7.5),
5 mmol/L EDTA, 1x Denhardt's solution, 10
mmol/L dithiothreitol, and 50% formamide, and incubated with
35S-UTP-labeled sense and antisense MCP-1 riboprobes for 16
hours. A 364-bp fragment of rat MCP-1 cDNA containing 348 bp of coding
sequence was cloned into pCRII (Invitrogen). Sense and antisense
riboprobes were generated after linearization with Bam HI
and Not I using Sp6 and T7 RNA polymerase. After
hybridization (at 55°C overnight), the specimens were washed with 2x
standard saline citrate (SSC), 10 mmol/L
ß-mercaptoethanol, 1 mmol/L EDTA (twice for 10 minutes
each), treated with RNase A (20 µg/mL, 30 minutes, 37°C),
and washed in 2x SSC (as above) followed by a high-stringency wash at
55°C for 2 hours (0.1xSSC, 10 mmol/L
ß-mercaptoethanol, 1 mmol/L EDTA). The Häutchen
procedure for en face preparations was carried out after
hybridization.29 Slides were coated with
autoradiographic emulsion (Kodak, NTB2), exposed for 3
weeks, and then developed (Kodak, D-19). Slides were evaluated and
photographed by light microscopy using dark-field and bright-field
illumination.
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Results |
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MCP-1 Expression Is Upregulated in Migrating BAE Cells
Monocyte recruitment is a consequence of adhesive and chemoattractant interactions. Monocytic U937 cells have previously been shown to adhere preferentially to migrating BAE cells, which appears to be related to upregulated expression of fibronectin and/or vascular cell adhesion molecule-1 by migrating endothelial cells.23 We have confirmed these findings (Fig 1A
) and could correspondingly demonstrate
that the same preferential adhesion of monocytic cells to the
endothelial cells at the migration front is also
detectable in vivo after balloon denudation injury of rat aortas (Fig 1B).
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On the basis of the differential adhesion of monocytes to
endothelial cells in vitro and in vivo, we decided to
study the expression of the primary chemoattractant molecule of
monocytic cells MCP-1 in migrating and quiescent resting BAE cells.
Quiescent, resting BAE cells expressed barely detectable levels of
baseline MCP-1 expression (Fig 2A). In
contrast, steady state mRNA levels of MCP-1 were prominently
upregulated in subconfluent migrating BAE cells (Fig 2A).
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bFGF Induces MCP-1 Expression in BAE Cells
MCP-1 expression is induced by the proinflammatory
cytokines IL-1 and TNF, as well as lipopolysaccharide (LPS).
To identify cytokines that could account for the
autocrine-regulated expression in migrating BAE cells, we stimulated
resting BAE cells with bFGF, which has been implicated in regulating
autocrine activity of migrating cells.30 Comparative
Northern blot analysis of resting and bFGF-stimulated BAE cells
identified MCP-1 as a bFGF-inducible gene of
endothelial cells (Fig 2B
). Analysis of the
time course of MCP-1 expression in BAE cells after bFGF stimulation
identified a rapid induction of MCP-1 expression after bFGF stimulation
that was detectable as early as 30 minutes after stimulation (Fig 3). Levels of expression increased for up
to 7 hours.
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bFGF Stimulates Synthesis and Secretion of MCP-1 Protein
Confirming and extending the results of the Northern blot
analysis, both bFGF and aFGF stimulation of BAE cells induced
approximately threefold higher levels of MCP-1 protein in the
supernatants of stimulated BAE cells compared with unstimulated control
cells (1.5 ng/mL versus 0.5 ng/mL; Fig 4). Control experiments with TNF
identified fivefold to sixfold higher levels of MCP-1 protein in the
supernatants of TNF-stimulated BAE cells. Stimulation of bovine
coronary venular endothelial cells with bFGF
led to similarly elevated levels of MCP-1 protein in the supernatants
of stimulated cells, whereas human umbilical vein
endothelial cells responded to exogenous bFGF
stimulation with elevated levels of MCP-1 protein only after prior
overnight starvation with growth factor–deprived medium (data not
shown).
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Collagenase Treatment Induces MCP-1 Expression in
Resting BAE Cells
On the basis of the differential expression of MCP-1 by migrating
and resting BAE cells and the induction of MCP-1 expression by bFGF, we
looked for alternate mechanisms of MCP-1 induction in
endothelial cells. Treatment of resting BAE cells with
increasing concentrations of bacterial collagenase resulted
in a dose-dependent induction of MCP-1 expression (Fig 5A). Experiments were performed in the
presence of 10% fetal calf serum. Monolayer integrity was not
disturbed by the collagenase concentrations used to induce
MCP-1 expression (0.8 µg/mL to 20 µg/mL) and was
confirmed by the ultrastructural analysis of
collagenase-treated BAE cell monolayers (data not shown).
Collagenase concentrations in excess of 200 µg/mL
led to partial detachment of endothelial cells from the
monolayer. Addition of neutralizing anti-bFGF monoclonal antibodies
(1.0 µg/mL) to the collagenase-treated BAE cell
monolayers inhibited the collagenase-mediated induction of
MCP-1 gene expression by >50% (Fig 5A). These experiments suggested
that collagenase treatment of BAE cell monolayers led to
the liberation of bioactive bFGF, which in turn upregulated MCP-1 gene
expression in the collagenase-treated BAE cell
monolayers.
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MCP-1 Expression in BAE Cells Is Regulated by Endogenous
bFGF
Experiments performed so far suggested that MCP-1 expression in
BAE cells may be regulated by endogenous bFGF. As shown in
Fig 5A, neutralizing anti-bFGF monoclonal antibodies (1.0
µg/mL) did not just suppress the
collagenase-mediated induction of MCP-1 gene expression but
also downregulated basal levels of MCP-1 expression in untreated
resting BAE cell monolayers (lane 2 versus lane 1). It is noteworthy
that an antibody concentration as little as 1.0 µg/mL was able
to suppress bFGF-mediated induction of MCP-1 expression, which may also
explain the only partial inhibition of bFGF-mediated MCP-1 expression
at higher collagenase concentrations (4 µg/mL and
20 µg/mL). Likewise, when anti-bFGF monoclonal antibodies were
added to cultures of subconfluent migrating BAE cells with upregulated
levels of MCP-1 gene expression (Fig 5B, lane 2 versus lane 1),
expression of MCP-1 gene expression was significantly downregulated
(Fig 5B, lane 3 versus lane 2). Isotype-matched control antibodies did
not affect MCP-1 expression. These experiments confirmed the role of
bFGF as an autocrine regulator of the migratory phenotype of
BAE cells in general and the role of endogenous bFGF in
regulating endothelial cell MCP-1 expression in
particular.
MCP-1 Is Expressed by Aortic Endothelial Cells
During Reendothelialization In Vivo
We next wanted to determine whether the differential expression of
MCP-1 by endothelial cells as observed in cultured BAE
cell monolayers is also present in activated
endothelial cells in vivo. For this purpose, MCP-1 mRNA
expression by endothelial cells in rat aortas was
visualized by in situ hybridization at different time points after
balloon denudation injury. Sense and antisense hybridizations of en
face preparations of undenuded control carotid arteries revealed
similar intensities of background hybridization, indicating that MCP-1
was not detectable in intact endothelium in vivo (Fig 6A and 6B). After balloon denudation,
however, a strong MCP-1 hybridization signal was detected at the
migrating front of the regenerating endothelium as
early as 2 hours after denudation (Fig 6C). Upregulation of MCP-1 gene
expression at the migrating front was intense and sustained, as
evidenced by the prominent MCP-1 hybridization signal still present
8 days after balloon denudation (Fig 6D). It should be noted that MCP-1
gene expression was spatially tightly restricted to the first two to
three rows of cells at the migration front.
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Adhesion molecules of activated endothelial cells act in concert with a number of chemoattractant molecules to regulate the trafficking of circulating leukocytes into the different body compartments. The CC chemokine MCP-1 has been most extensively studied for its ability to recruit monocytes to sites of inflammation.3 14 31 Monocytes appear to be the primary target cell population of the chemotactic activity of MCP-1, as evidenced by the presence of prominent perivascular cuffs of monocytes in transgenic mice overexpressing MCP-1.22 Reflecting its role in controlling inflammatory cell trafficking, MCP-1 expression is regulated by the inflammatory cytokines IL-1, TNF
, interferon-
, and LPS,19 21 32 as well as by
platelet-derived growth factor,15 IL-4,33
thrombin,34 minimally modified-LDL,35 and
MCSF.36 We have studied MCP-1 expression in endothelial cells and found prominently elevated steady state levels of MCP-1 mRNA in subconfluent, migrating BAE cells that have not been stimulated by exogenous cytokine exposure. Several molecules have been implicated in regulating autocrine, and possibly intracrine endothelial cell activity, including the heparin-binding growth factor bFGF.35 37 38 39 Detailed analysis of the regulation of MCP-1 expression in BAE cells identified MCP-1 as a bFGF-inducible gene, as shown by the increase of MCP-1 mRNA levels and secreted protein after stimulation with exogenous bFGF. Several lines of evidence suggested that MCP-1 expression in BAE cells is regulated by autocrine bFGF: Subconfluent, migrating endothelial cells, known to express elevated levels of endogenous bFGF in vitro40 and in vivo,28 express elevated levels of MCP-1 mRNA. Both basal MCP-1 expression in resting BAE cells and upregulated MCP-1 expression in migrating BAE cells are downregulated by addition of neutralizing anti-bFGF antibodies. Likewise, induction of endothelial cell MCP-1 expression by treatment with collagenase and its inhibition by neutralizing anti-bFGF antibodies suggested that proteolytic treatment of BAE cell monolayers led to the release of bioactive endothelial cell–derived bFGF. Conditions of collagenase treatment in these experiments were such that monolayer integrity of BAE cells was not disturbed, as determined by electron microscopy, suggesting that proteolytic treatment of cells led to the specific liberation of bioactive bFGF and not to release as a consequence of cellular damage.
The heparin-binding growth factor bFGF stimulates a number of endothelial cell functions. It induces endothelial cell migration and proliferation in vitro and stimulates angiogenesis41 and reendothelialization in vivo.42 43 bFGF regulates the expression of a number of endothelial cell genes, such as molecules of the proteolytic balance (plasminogen activator [tPA and uPA]),44 plasminogen activator inhibitor-1,45 adhesion molecules (ICAM-1,46 integrins47 ), and extracellular matrix molecules.48 Endothelial cells from a number of vascular beds have been shown to synthesize bFGF.49 50 51 Its expression is induced by exogenous stimulation as well as autocrine control after injury or mechanical release from growth arrest.28 40 Interestingly, exogenous bFGF stimulates transcription of its own gene, acting through a positive feedback loop.52 53
Endothelial cell–derived bFGF has not yet been related to the regulation of inflammatory processes. The demonstration of autocrine-activated endothelial cell–derived bFGF as an inducer of endothelial cell MCP-1 expression, however, raises the possibility that bFGF, in addition to its capacity to act as a growth factor, may act as a regulator of inflammatory processes. bFGF is a potent inducer of tumor and wound healing angiogenesis, and both tumor growth and wound healing are most frequently associated with variable numbers of macrophages,54 which themselves stimulate angiogenesis.55
Analysis of MCP-1 expression in vivo by in situ hybridization studies of en face preparations of rat aortic endothelial cells identified dramatically upregulated levels of MCP-1 mRNA in the regenerating migrating endothelial cells after aortic balloon denudation injury. Induction was rapid and sustained, being detectable within 2 hours of denudation and still present at the regenerating front 8 days after denudation. Regenerating endothelial cells express elevated levels of endogenous bFGF after aortic denudation injury,28 suggesting that similar autocrine and/or local paracrine mechanisms of bFGF-mediated MCP-1 induction act on endothelial cell MCP-1 expression in vivo as identified in vitro.
Preferentially, adhesion of monocytic cells to migrating endothelial cells23 and autocrine-regulated increased expression of MCP-1 by migrating endothelial cells in vivo and in vitro suggest a critical role of activated or perturbed endothelial cells in recruiting monocytes. Monocyte accumulation plays a critical role in the early pathogenesis of atherosclerosis.56 Monocytes adhere preferentially to atherosclerotic plaque endothelial cells,57 58 and both bFGF59 60 and MCP-161 62 have been found to be expressed at high levels in atherosclerotic plaques. Identification of the regulation of MCP-1 by bFGF may thus have direct implications for the pathogenesis of atherosclerosis.
In summary, autocrine-activated migrating endothelial cells express both the adhesion molecule(s) (most likely vascular cell adhesion molecule-1 and/or others) and the chemoattractant molecule MCP-1 required to attract circulating monocytes. Though this study did not demonstrate a causal relationship between the preferential adhesion of monocytic cells to migrating endothelial cells and expression of MCP-1 by autocrine-activated endothelial cells, it appears likely that adhesion and chemoattraction act in synergy to recruit monocytic cells to sites of activated endothelial cells. Furthermore, the regulation of MCP-1 expression through endothelial cell–derived bFGF supports the concept that bFGF acts as a major autocrine regulator of effector functions of activated endothelial cells. The regulation of MCP-1 expression by bFGF suggests that bFGF may also act as an inflammation-regulating cytokine that contributes to regulating inflammatory cell trafficking. The demonstration of upregulated MCP-1 expression in endothelial cells after denudation injury may be most relevant for the elucidation of the mechanisms that regulate monocyte recruitment during early atherogenesis. The implications of these findings for other situations involving bFGF activation of endothelial cells, such as specific forms of angiogenesis, deserves further analysis and may well help to shed further light into the mechanisms by which bFGF stimulates angiogenesis.
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Acknowledgments |
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This work was supported by grants from the Deutsche Krebshilfe/Mildred Scheel-Stiftung (10-0986-Au2, to Dr Augustin) and the Deutsche Forschungsgemeinschaft (SFB 500, C3, to Dr Augustin) and a grant-in-aid from the American Heart Association (to Dr Lindner). The authors would like to acknowledge the excellent technical assistance of Renate Dietrich, Cathleen Lakoma, and Leslie Couper.
Received April 2, 1997; accepted June 13, 1997.
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