Published online before print June 21, 2001, doi: 10.1161/hh1301.093631
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© 2001 American Heart Association, Inc.
Molecular Medicine |
Tumor Necrosis Factor-
Induces Fibronectin Synthesis in Coronary Artery Smooth Muscle Cells by a Nitric Oxide–Dependent Posttranscriptional Mechanism
From the Division of Cardiovascular Research, The Hospital for Sick Children and Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Correspondence to Dr Marlene Rabinovitch, Division of Cardiovascular Research, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, M5G 1X8, Canada. E-mail mr{at}sickkids.on.ca
Abstract
Abstract—Postcardiac
transplant coronary arteriopathy is associated with tumor
necrosis factor-
(TNF-) induction of fibronectin-dependent smooth
muscle cell (SMC) migration into the subendothelium,
resulting in occlusive neointimal formation. Because
expression of inducible nitric oxide synthase (iNOS) is elevated in
neointimal formation after transplantation and upregulated
in vascular SMCs by TNF-, we investigated whether TNF- induction
of fibronectin synthesis in coronary artery (CA) SMCs is
mediated by nitric oxide (NO). TNF- caused a dose-dependent increase
in reactive oxygen and nitrogen intermediates in CA SMCs
(P<0.05). This correlated with
increased NO production
(P<0.05) and fibronectin
synthesis (P<0.05). TNF-
induction of fibronectin synthesis was abrogated by the NOS
inhibitor
NG-monomethyl-L-arginine
(L-NMMA) (P<0.05) or the
flavonoid-containing enzyme inhibitor diphenyleneiodonium
(DPI) (P<0.05) and reproduced
with the NO donor
S-nitroso-N-acetyl-penicillamine
(SNAP) (P<0.05). Northern
blotting showed no effect of TNF- on steady-state fibronectin mRNA
levels. TNF- increased expression of light chain 3 (LC-3), a protein
shown previously to facilitate fibronectin mRNA translation through its
interaction with an adenosine-uracil rich element (ARE) in the
3'-untranslated region of fibronectin mRNA. RNA gel mobility shift and
UV cross-linking assays using CA SMC lysates revealed protein binding
complexes with radiolabeled oligonucleotide containing
the ARE, similar to those generated with recombinant LC-3. One of these
complexes increased after TNF- treatment, an effect inhibited with
L-NMMA or DPI. These data demonstrate a novel paradigm whereby
cytokines regulate mRNA translation of extracellular matrix
proteins through NO-dependent modulation of RNA binding protein
interaction with mRNA.
Key Words: nitric oxide • fibronectin • translation • atherosclerosis • cardiac transplant
Transplant
coronary artery disease, a major complication of cardiac
transplantation,1 is
characterized by the development of occlusive neointimal
lesions formed by smooth muscle cell (SMC) migration and proliferation
and extracellular matrix deposition in the
subendothelium.2
It appears to be initiated by an immune-inflammatory response involving
accumulation of activated T
cells,3 expression of the
cytokines interleukin-1ß
(IL-1ß)4 and tumor necrosis
factor-
(TNF-),5 6 and
upregulation of SMC fibronectin associated with SMC migration into the
subendothelium.7 8
In cultured porcine coronary artery (CA) SMCs, TNF-
and IL-1ß reciprocally coinduce synthesis of soluble
fibronectin.6 TNF-
induction of fibronectin has also been demonstrated in a rabbit
heterotopic cardiac transplant model, in which blockade of this effect
with soluble TNF- receptor reduced the number and severity of
allograft coronary artery lesions and was associated with a
decrease in subendothelial fibronectin expression and
in inflammatory cell
infiltrate.8 9
Furthermore, in coculture experiments, soluble fibronectin
produced by CA SMCs set up a gradient, stimulating
transendothelial lymphocyte
migration.8
The critical importance of fibronectin in SMC migration was demonstrated in the fetal ductus arteriosus (DA), a vessel in which neointimal formation begins as a normal developmental process at 100 days’ gestation in the fetal lamb (term 145 days) to form intimal cushions that facilitate closure of this vessel at birth.10 11 12 13 14 In 100-day-gestation DA SMCs, increased synthesis of soluble fibronectin was demonstrated12 relative to SMCs from the aorta and related to the enhanced migratory phenotype of the DA cells in 3-D collagen gels.10 Interestingly there was no difference in matrix-associated fibronectin synthesis between the neointima-forming DA SMCs and aortic SMCs.12
Increased synthesis of soluble fibronectin in 100-day-gestation sheep DA SMCs is regulated at a posttranscriptional level by the binding of light chain 3 (LC-3), a component of microtubule-associated protein complexes 1A and 1B,12 to an adenosine-uracil rich element (ARE) in the 3'-untranslated region (3'-UTR) of fibronectin mRNA.13 LC-3 facilitates fibronectin mRNA recruitment to membrane-bound polyribosomes.14 By sequestering LC-3 with a decoy RNA containing the fibronectin 3'-UTR ARE, fibronectin synthesis was inhibited and the migratory phenotype was not observed. Subsequent gene transfer experiments in fetal lambs using decoy RNA confirmed that fibronectin production and SMC migration were reduced, and neointimal formation was prevented.11
Although nitric oxide (NO) exhibits antiatherogenic properties by its inhibitory effects on platelet adhesion15 16 and activation17 as well as vascular SMC proliferation,18 there is increasing evidence that NO can also play a role in the development of neointimal lesions in inflammatory states in which superoxide levels are elevated.19 20 21 NO readily combines with superoxide (O·-) to form peroxynitrite anion (ONOO-), which has been shown to cause LDL and VLDL oxidation22 23 leading to lipid accumulation in macrophages of the vessel wall,24 25 26 27 as well as macrophage apoptosis.28 29
Because inducible NOS (iNOS) is expressed in SMCs during
neointimal formation, it has been proposed that NO may
regulate acquisition of the migratory SMC
phenotype.30
Expression of NO is elevated in atherosclerotic
lesions31 32 33
as well as in the coronary arteries of
human19 and animal
model20 34
cardiac allografts. Although iNOS was originally identified in
cytokine-activated macrophages, several reports
have demonstrated that vascular SMCs in culture also produce
iNOS35 36 37 38 39 40
in response to TNF- and
IL-1ß.35 36 37
NO-dependent TNF- induction of fibronectin expression was
demonstrated in a human umbilical vein endothelial cell
line.41 Elevated NO levels
in DA SMCs, associated with increased SMC expression of the
constitutive nitric oxide synthase (NOS) isoforms
endothelial NOS (ecNOS) and neuronal NOS (nNOS),
both in cultured cells and in sheep, regulate increased fibronectin
synthesis through enhanced expression and binding of LC-3 to the 3'-UTR
ARE of fibronectin mRNA.42
Thus, in this developmental paradigm of neointimal
formation, NO appears to play a central role in regulating
fibronectin-dependent SMC migration into the
neointima.
In the present study, we examine whether induction of
fibronectin synthesis in primary culture CA SMCs by the
cytokine TNF- is similarly regulated by NO. We show that
TNF- induction of fibronectin synthesis requires increased NO
production that modulates expression of LC-3 and binding of a
protein with properties consistent with LC-3 to the ARE in the
3'-UTR of fibronectin mRNA, a mechanism shown previously to facilitate
fibronectin mRNA
translation.13 14
These are the first experimental data demonstrating that modulation of
intracellular NO levels by cytokines can orchestrate specific
posttranscriptional mechanisms regulating extracellular matrix
expression.
Materials and Methods
Cell Culture
Porcine CA SMCs were isolated for primary cultures by
explant technique as previously
described43 44
and were used at passage 2.
Experimental Conditions
Semiconfluent cultures of CA SMCs were preincubated
for 1 hour in serum-free methionine/cysteine-free DMEM containing 1%
BSA. To assess the role of reactive oxygen or nitrogen intermediates
(ROI/RNI) and specifically NO in TNF- induction of fibronectin
synthesis, diphenyleneiodonium (DPI, Sigma Chemical
Co) (2 µmol/L) or
NG-monomethyl-L-arginine
(L-NMMA, Sigma) (250 µmol/L) was added during
the 1-hour preincubation. Culture medium was then replaced and
recombinant human TNF- (R&D Systems) (10, 30, and 100 ng/mL) was
added alone or in combination with DPI or L-NMMA for 4 hours. To assess
the effect of NO,
S-nitroso-N-acetyl-penicillamine
(SNAP) (BIOMOL Research Laboratories) was added
to the cells for 4 hours.
Fibronectin Synthesis
During the 4-hour incubation described above, 10
µCi/mL of [35S]-methionine
(Amersham) was added. Total protein synthesis
was assessed for each culture dish in triplicate by trichloroacetic
acid precipitation and liquid scintillation spectrometry. To
measure fibronectin synthesis, 1-mL aliquots of culture medium, cell
lysates, or extracted extracellular matrix were incubated with 100 µL
gelatin 4B-sepharose beads overnight at 4°C. The beads were washed in
PBS, and bound fibronectin was eluted into 100-µL SDS-PAGE sample
buffer by boiling for 5 minutes. The samples were standardized to total
protein synthesis and separated on 6% SDS polyacrylamide gels.
Identification of the 220-kDa band on the autoradiograph as fibronectin
was previously confirmed by
immunoblotting.45
FACS Analysis of ROI and RNI
To assess total ROI and RNI levels, dihydrorhodamine
123 (DHR) (2 µmol/L) was added to the cells during the 4-hour
incubation period. DHR is converted in the presence of ROI/RNI to the
fluorescent dye rhodamine 123 (500- to 540-nm emission
spectrum). After DHR loading, all manipulations of the cells were
carried out in the dark. CA SMCs were trypsinized (0.5% trypsin,
5.3 mmol/L EDTA · 4Na) (Gibco BRL),
washed in PBS, and fixed in 1.5% paraformaldehyde/PBS.
Fluorescence intensity was determined for 5000 cells from each
sample by fluorescence-activated cell sorting (FACS)
analysis using an argon laser with the excitation source
emitting at 488 nm as previously
described.46
Nitrate/Nitrite Analysis
A nitrate/nitrite fluorometric assay kit (Cayman
Chemical) was used to measure levels of the stable end products of
NO, nitrate and nitrite, in culture medium as
described.47 48
Cell-free culture medium was harvested from cells treated as described
above, and cell number was counted in triplicate for each condition
using a hemocytometer. Culture medium samples were incubated with
nitrate reductase and cofactors (Cayman Chemical) for 30 minutes at
room temperature to convert nitrate to nitrite. The samples were then
incubated with 2,3-diaminonaphthalene (DAN), which reacts with nitrite
to form the fluorescent compound
1(H)-naphthotriazole.
Fluorescence intensity was measured using a fluorometer
(excitation and emission wavelengths of 365 and 450
nm).
Northern Blotting
Cells were lysed in Trizol (Life
Technologies) (2 mL/100-mm dish extracted with chloroform
and total RNA precipitated according to the manufacturer’s
directions). RNA samples were separated in 1% agarose gels containing
6% formaldehyde transferred to nitrocellulose membranes and probed
with [32P]-dCTP
(106 cpm/mL)
(Amersham) labeled human fibronectin cDNA (1.4
kb) (Gibco BRL) and a control GAPDH cDNA probe
(1.2 kb) (ATCC).
Cell Extracts
Semiconfluent cultures of CA SMCs were harvested, and
the cells were resuspended in 2 volumes of hypotonic buffer (0.1
mmol/L EDTA, 25 mmol/L Tris-HCl [pH 7.9]) and lysed by 3 cycles
of freeze-thaw. Cytosolic proteins were extracted by pelleting the
lysed cells for 1 hour at
16 000g. Pellets were either
dissolved in Laemmli sample buffer for SDS-PAGE or subjected to 1
mol/L KCl extraction at 4°C to isolate membrane-associated proteins
for RNA gel mobility shift assays. Protein concentrations were
determined using a standard Bradford protein assay kit
(Bio-Rad) and spectrophotometry at 595
nm.
Immunoblotting
Protein extracts (30 µg per lane) from CA SMCs were
separated by SDS-PAGE under reducing conditions and transferred to
polyvinylidene difluoride membranes. Blots were blocked
for 1 hour in 5% nonfat dry milk/PBS containing 0.5% Tween-20 and
then probed with rabbit antiserum to LC-3 (1:3000) (prepared by Dr J.
Hammarback, Department of Neurobiology and Anatomy, Bowman Gray
School of Medicine, Winston-Salem, NC). Blots were washed three times
with PBS/0.5% Tween-20 followed by incubation with horseradish
peroxidase–conjugated goat anti-rabbit IgG (1:3000)
(Bio-Rad). The blots were washed three times as
above and developed using enhanced chemiluminescence (ECL) detection
reagents (Amersham).
RNA Gel Mobility Shift and UV Cross-Linking
Assays
Cytosol and KCl extracts from lysed cell pellets were
dialyzed against RNA binding buffer (5 mmol/L
MgCl2, 100 mmol/L KCl, 10% glycerol, and
15 mmol/L HEPES [pH 7.9] in diethyl pyrocarbonate–treated
water) overnight at 4°C in a Pierce System 100
microdialyzer. For each assay, 15 µg of protein extract from CA SMCs
was incubated with 104 cpm of wild-type or
mutant fibronectin ARE (underlined) containing RNA
oligonucleotide probe (wild type:
5'-ACCUGUUAUUUAUCAAUU-3';
mutant: 5'-ACCUGGGAGGGAGCAAUU-3') (synthesized by Biotechnology
Center, University of Calgary, Calgary, Alberta, Canada) in RNA binding
buffer containing 2 µg Escherichia
coli transfer RNA (Sigma) in a total
volume of 20 µL for 30 minutes at 30°C. Samples were separated on
6% native polyacrylamide gels in 0.25xTris-borate-EDTA (TBE)
buffer (90 mmol/L Tris, 90 mmol/L boric acid, and 2
mmol/L EDTA). For competition and specificity analysis of
protein-RNA binding excess (500:1), unlabeled RNA probe was incubated
with the cytoplasmic extracts and
[32P]-labeled probe. For UV cross-linking
assays, samples were cross-linked after incubation of protein extracts
with radiolabeled wild-type ARE probe by UV (254 nm) irradiation (120
mJ/cm2 in a UV 1800 Stratalinker
(Stratagene and separated in 12%
polyacrylamide gels by
SDS-PAGE.
Results
NO Mediates TNF- Induction of
Fibronectin Synthesis
We first examined whether TNF- induction of
fibronectin synthesis in CA SMCs is mediated by induction of ROI/RNI or
NO specifically. In CA SMCs, treatment with TNF- caused a
dose-dependent increase in intracellular ROI/RNI levels
(Figure 1A
). Consistent with previous studies in DA
SMCs that showed the importance of secreted rather than
matrix-associated fibronectin in SMC migration, in that the majority of
newly synthesized fibronectin within a 4-hour period was secreted
(57%), with 37.5% intracellular, and only 5.5% deposited in the
extracellular matrix. TNF- caused a dose-dependent increase in
secreted fibronectin synthesis in CA SMCs
(Figure 1B).
|
Subsequent studies showed that DPI, an inhibitor
of ROI and RNI producing flavonoid-containing enzymes such as NADPH
oxidase and NO synthase,49
abrogated the effect of TNF- on ROI/RNI induction and also caused a
decrease in basal levels of ROI/RNI in CA SMCs
(Figure 2A). TNF- induction of fibronectin synthesis was
also not observed in the presence of DPI
(Figure 2B). However, treatment with DPI alone did not cause
a decrease in fibronectin synthesis from basal levels
(Figure 2B) suggesting that constitutive expression of
fibronectin in cultured CA SMCs is not regulated by
ROI/RNI.
|
P<0.05 vs TNF; and 
P<0.05 vs CON.
Because TNF- increases iNOS expression in vascular
SMCs,38 we examined whether
the TNF-–induced increase in ROI/RNI levels was due to NO and
whether TNF- induction of fibronectin synthesis was mediated
specifically by NO. We first demonstrated that the NO donor SNAP
reproduced the effect of TNF- on CA SMC ROI/RNI levels
(Figure 2C
) and on fibronectin synthesis
(Figure 2D). Next we demonstrated that TNF- induction of
fibronectin synthesis could be abrogated by the specific NOS
inhibitor L-NMMA, whereas treatment with L-NMMA alone had
no significant effect on basal levels of fibronectin synthesis in CA
SMCs
(Figure 3). These results indicated that TNF- induction of
fibronectin synthesis is mediated by NOS production of
NO.
|
To confirm that TNF- was modulating NO in CA SMCs as
proposed, NO levels were assessed by measuring the accumulation of its
stable end products, nitrite and nitrate, in culture medium
(Figure 4). TNF- caused a 2-fold increase in
nitrate/nitrite accumulation, which was abrogated by L-NMMA and
partially inhibited by DPI
(Figure 4).
|
NO-Mediated TNF- Induction of
Fibronectin Is Posttranscriptional
Fibronectin mRNA levels did not change appreciably with
respect to GAPDH mRNA after treatment with TNF-, TNF- and L-NMMA
together, or L-NMMA alone (data not shown). These data suggest
that NO-mediated TNF- induction of fibronectin synthesis may be
regulated at a posttranscriptional level.
LC-3 Expression Is Increased in
TNF-–Treated CA SMCs
We have previously demonstrated NO-dependent
posttranscriptional upregulation of fibronectin
synthesis42 by
LC-3–mediated recruitment of fibronectin mRNA to the
polyribosomes in neointima-forming DA
SMCs.13 14 Two
forms of LC-3 of distinct molecular weights were identified in DA SMCs
by immunoblotting. These proteins could be separated by
isolating cytosolic fraction and cellular membrane fractions from
freeze-thawed, lysed SMCs. Cytosolic LC-3 has a molecular mass of 16
kDa and codistributes with
tubulin.42 However, a lower
molecular weight form of LC-3 was identified in the membrane fraction
and could be removed from DA SMC membranes by 1 mol/L KCl, suggesting
that it is a membrane-associated protein, a characteristic of RNA
binding proteins.50 This
form exhibited increased binding to the ARE of fibronectin mRNA,
suggesting that it is likely the form associated with fibronectin mRNA
at the polyribosomes where translation
occurs.42 We therefore
examined LC-3 expression in cell membrane fractions from CA SMCs
treated with TNF- alone or in combination with L-NMMA or with
L-NMMA alone
(Figure 5). Levels of membrane-associated LC-3 in CA SMCs
were increased by TNF- within 4 hours, and this increase was
partially inhibited by L-NMMA
(Figure 5).
|
TNF- Induces Protein Binding to the ARE of
Fibronectin mRNA
We previously demonstrated in DA SMCs that LC-3 binds
to an ARE in the 3'-UTR of fibronectin mRNA in RNA gel mobility shift
and UV cross-linking assays and that increased binding is associated
with increased recruitment of fibronectin mRNA to the
polyribosomes.13 14
Therefore, we used RNA gel mobility shift assays to examine protein
binding from KCl extracts of CA SMC membranes, to a
[32P]-labeled 18-mer
oligonucleotide containing the wild-type fibronectin
ARE (underlined) (5'-ACCUGUUAUUUAUCAAUU-3')
and to a control ARE-mutated 18-mer oligonucleotide
(5'-ACCUGGGAGGGAGCAAUU-3') shown previously to exhibit significantly
decreased LC-3 binding activity in DA SMCs.13
From KCl extracts of CA SMC membranes, two prominent,
shifted protein-fibronectin [32P]-ARE
complexes were identified (Figure 6A). We were unable to demonstrate a supershift of
these complexes using an antibody to LC-3, which was not unexpected
because this antibody also fails to immunoprecipitate LC-3 from cell
extracts. However, two complexes with the same migratory patterns on
polyacrylamide gels were detected when recombinant LC-3 was
incubated with the fibronectin [32P]-ARE
oligonucleotide probe
(Figure 6B).
|
Consistent with fibronectin synthesis data,
densitometric analyses revealed that formation of the faster
migrating complex was increased in CA SMCs treated with TNF-, an
effect that was inhibited by concomitant treatment with L-NMMA or DPI
(Figure 6B). In contrast, binding activity for the larger
complex did not appear to be altered appreciably with TNF- induction
of fibronectin synthesis. Furthermore, only the larger complex was
detectable after incubation with the ARE-mutated
oligonucleotide. This suggests that formation of the
smaller complex is more sensitive to the presence of the intact ARE
element and that formation of this ARE-specific complex is increased
with TNF- by a mechanism mediated through NO.
In UV cross-linking assays in which the 18-mer RNA
oligonucleotide containing the ARE was UV cross-linked
to bound proteins from CA SMC extracts, a band at 
22 kDa was
resolved on SDS-PAGE
(Figure 6D). Detection of the 22-kDa band is
consistent with the proposal that the protein-fibronectin ARE
complex contains the LC-3 monomer (15 kDa) bound to the 6 to 7
kDa [32P]-labeled fibronectin ARE
oligonucleotide. These UV cross-links also confirmed
that binding to the fibronectin ARE was enhanced by TNF- and
inhibited by concomitant treatment with L-NMMA
(Figure 6D). A protein with the same molecular weight was
previously detected in DA SMC extracts and purified as
LC-3.13
Discussion
We demonstrated that TNF- induction of fibronectin
synthesis in CA SMCs is mediated by NO and that the mechanism is
consistent with binding of the microtubule-associated RNA
binding protein LC-3 to an ARE in the 3'-UTR of fibronectin mRNA.
TNF- induction of NO production and NO-mediated induction of
soluble fibronectin synthesis in primary cultured porcine CA SMCs
correlates with increased expression of TNF-, iNOS, and fibronectin
in the neointima formed in restenosis,
atherosclerosis, and posttransplant coronary
arteriopathy.
Steady-state levels of fibronectin mRNA in CA SMCs cultured
in serum-free medium were not affected appreciably by a 4-hour exposure
to TNF-. Previous SMC culture studies have demonstrated that there
is reciprocal coinduction of fibronectin by TNF- and
IL-1ß.6 Because IL-1ß
induction of fibronectin synthesis is associated with increased
steady-state mRNA levels,43
it appears that TNF- and IL-1ß may regulate fibronectin synthesis
at transcriptional and posttranscriptional levels.
Increased elastase activity has also been linked to posttranscriptional regulation of increased fibronectin synthesis in post-cardiac transplant arteriopathy.51 52 Our recent data suggest that elastin peptides mediate an increase in fibronectin synthesis by a posttranscriptional mechanism, because the selective elastase inhibitor elafin reduces elastin-peptide–mediated induction of fibronectin synthesis without affecting steady-state mRNA levels (authors’ unpublished data, 1998). Because elafin also abrogates the development of postcardiac transplant coronary arteriopathy in experimental animals,52 posttranscriptional induction of fibronectin gene regulation may be critical in this model of occlusive neointimal formation.
We have demonstrated that a membrane-associated form of
LC-3, which facilitates fibronectin mRNA translation in
100-day-gestation DA SMCs, a vessel in which neointimal
formation occurs, also appears to be present in porcine CA SMCs and
increased by TNF-. In this report, we demonstrate by gel shift and
UV cross-linking assays that TNF- increases the formation of a
complex between the [32P]-labeled
fibronectin ARE and a membrane-associated protein from CA SMCs.
Evidence suggesting that the protein component is LC-3 was provided by
the following additional data: The complex could be reproduced by
incubating recombinant LC-3 with the fibronectin ARE. In
addition, UV cross-linking assays demonstrated that the molecular
weight of the protein binding to the ARE is the same as LC-3. Our
results with L-NMMA and DPI suggest that NO may mediate TNF-
induction of fibronectin synthesis by increasing binding of this
protein consistent with LC-3 to the fibronectin
ARE.
Interestingly, in DA SMCs, although several complexes were detectable in the cytosolic fraction of the cells, only the smaller complex was detectable in the membrane fraction of the cells,42 which is the site where LC-3 appears to act to facilitate mRNA translation at the polyribosomes.14 The membrane-associated smaller complex has been postulated to represent a phosphorylated form of LC-3.42 It is feasible that phosphorylation of LC-3 could result in its monomerization, hence a smaller complex size, with increased binding efficiency to the fibronectin 3'-UTR ARE, which in turn facilitates mRNA translation. From the present studies in CA SMCs, we have found that binding of this postulated phosphorylated/monomeric form of LC-3 appears to be ARE-sensitive, which would explain our previous gene transfer studies demonstrating the importance of the ARE in fibronectin upregulation and SMC migration in neointimal formation.11
Neointimal formation in the fetal ductus arteriosus can be abrogated by inhibiting SMC fibronectin synthesis by gene transfer of plasmid-encoding decoy RNA containing the 3'-UTR of fibronectin mRNA.11 Our demonstration that cytokine induction of fibronectin synthesis is likely mediated by a similar posttranscriptional mechanism suggests that the same gene transfer strategy might be useful in inhibiting occlusive neointimal formation of vascular disease.
Acknowledgments
This study was supported by a grant from the Canadian Institutes of Health Research (CIHR) PG-13920. M.R. is a Distinguished Scientist of the CIHR and C.O. was supported by a CIHR MD PhD studentship. M.R. was also supported by the Heart and Stroke Foundation of Ontario Research Chair. We are grateful to Joan Jowlabar and Judy Edwards for their assistance in preparing this manuscript and to Dr James Hammarback for providing the antiserum to LC-3.
Footnotes
Original received December 3, 1998; resubmission received March 27, 2001; revised resubmission received May 29, 2001; accepted May 29, 2001.
This manuscript was sent to Peter Libby, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
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