1 Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095; and 2 Department of Biochemistry and Molecular Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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The objectives of
this study were to determine whether rat aortic smooth muscle cells
(RASMC) express arginase and to elucidate the possible mechanisms
involved in the regulation of arginase expression. The results show
that RASMC contain basal arginase I (AI) activity, which is
significantly enhanced by stimulating the cells with either interleukin
(IL)-4 or IL-13, but arginase II (AII) expression was not detected
under any condition studied here. We further investigated the signal
transduction pathways responsible for AI induction. AI mRNA and protein
levels were enhanced by addition of forskolin (1 µM) and inhibited by
H-89 (30 µM), suggesting positive regulation of AI by a
protein kinase A pathway. Genistein (10 µg/ml) and sodium
orthovanadate (Na3VO4; 10 µM) were used to
investigate the role of tyrosine phosphorylation in the control of AI
expression. Genistein inhibited, whereas Na3VO4
enhanced the induction of AI by IL-4 or IL-13. Along with immunoprecipitation and immunoblot analyses, these data implicate the
JAK/STAT6 pathway in AI regulation. Dexamethasone (Dex) and interferon
(IFN)- were investigated for their effects on AI induction. Dex (1 µM) and IFN-
(100 U/ml) alone had no effect on basal AI expression
in RASMC, but both reduced AI induction by IL-4 and IL-13. In
combination, Dex and IFN-
abolished AI induction by IL-4 and IL-13.
Finally, both IL-4 and IL-13 significantly increased RASMC DNA
synthesis as monitored by [3H]thymidine incorporation,
demonstrating that upregulation of AI is correlated with an increase in
cell proliferation. Blockade of AI induction by IFN-
, H-89, or
genistein also blocked the increase in cell proliferation. These
observations are consistent with the possibility that upregulation of
AI might play an important role in the pathophysiology of vascular
disorders characterized by excessive smooth muscle growth.
interleukin; rat aortic smooth muscle cells; cell proliferation; cytokines
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INTRODUCTION |
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THERE ARE TWO ISOFORMS of arginase in vertebrates, both of which catalyze the conversion of arginine to ornithine and urea. They differ with regard to subcellular localization, tissue distribution, and certain enzymatic properties, reflecting the fact that they are encoded by different genes (reviewed in Ref. 20). Arginase I (AI) is expressed almost exclusively in the cytosol of liver cells where it serves as the fifth and final enzyme of the urea cycle. Expression of AI in liver is regulated by dietary protein (33) and by hormones such as glucagon and glucocorticoids (15, 36). Recently, however, AI has been found to be induced also by lipopolysaccharide (LPS), interleukin (IL)-4, cAMP, and hypoxia in macrophages (26, 27, 32, 58) and by LPS in various tissues of rats and mice (43, 47). In contrast to AI, however, arginase II (AII) is located within the mitochondrial matrix and is expressed at low levels in many tissues (13, 31). It has recently been demonstrated that AII expression can be induced by cAMP in murine macrophages (32) and by LPS in macrophages and various tissues of mice (27, 32, 43), but LPS induction of AII has not been observed for rat tissue (47). Unlike AI in liver, the physiological role of AII has not been established.
Cloned cDNA for AI and AII have been isolated for rat, mouse, human, and Xenopus laevis (summarized in Ref. 39), and the genes encoding rat and human AI (37, 52) and murine AII (45) also have been cloned. The genes for AI and AII are structurally similar and consist of eight exons, reflecting the fact that the two genes are probably the consequence of an ancient gene duplication event. Some promoter and enhancer elements in the rat AI gene that are required for expression in hepatocytes have been characterized (6, 12, 51).
With the exception of enterocytes (59), extrahepatic organs lack a complete urea cycle, and it has been suggested that arginase in tissues other than liver may be involved in the regulation of cell growth and tissue repair (1, 4, 26, 43). L-Ornithine, the enzymatic product of arginase, is a precursor for the synthesis of polyamines (putrescine, spermidine, and spermine). Polyamines have been shown to stimulate DNA synthesis, enhance transcription of growth-related genes, and are essential for cell proliferation (19, 50). Furthermore, activity of ornithine decarboxylase, which is required for polyamine synthesis, parallels the intensity of the repair processes in injured tissues (11).
IL-4 is a pleiotropic cytokine that plays an important role in
immune-inflammatory reactions by modulating growth, differentiation, cytokine production, and major histocompatibility complex class II
expression (23). IL-4 has also been reported to induce
morphological changes in human umbilical vein endothelial cells, which
may be important in angiogenesis (24). IL-4 induces
arginase activity in cultured macrophages (7,
29), and this has been identified as the AI isoform
(26). IL-13 is a cytokine that elicits biological responses similar to IL-4 (2, 22). The
purpose for the present study was to determine whether rat aortic
smooth muscle cells (RASMC) express arginase, if arginase expression
can be modulated by IL-4 and IL-13, and if arginase activity correlates
with RASMC proliferation. In the present study, IL-4 and IL-13 were
shown to significantly increase AI activity in RASMC. The signal
transduction pathways regulating this induction were investigated.
Janus kinases (JAK) and signal transducer and activator of
transcription (STAT) and cAMP-dependent pathways were all shown to
modulate AI expression and activity. Dexamethasone and interferon
(IFN)- abrogated the response of RASMC to IL-4 and IL-13.
Upregulation of AI was accompanied by, and may have caused, enhanced
RASMC proliferation. These results suggest that elevated arginase
activity may have physiological and/or pathophysiological roles in the
cardiovascular system.
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MATERIALS AND METHODS |
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Reagents.
Forskolin, dexamethasone, and genistein were purchased from Sigma.
Sodium orthovanadate (Na3VO4) and H-89 were
purchased from Calbiochem. Recombinant rat IL-4 and recombinant human
IL-13 were purchased from R & D Systems. Rat recombinant IFN- was
purchased from GIBCO. Rabbit anti-STAT6 polyclonal antibody, mouse
anti-p-Tyr (epitope, phospho-Y99) monoclonal antibody, and protein
A-coupled agarose beads were purchased from Santa Cruz Biotechnology.
Cell culture of RASMC. RASMC was a generous gift from Dr. Steven Gross. Cells were plated in high-glucose DMEM-HEPES supplemented with 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 mg/ml amphotericin B and were grown until confluent at which time cells were subcultured by trypsinization. Cell cultures were performed at 37°C in a humidified atmosphere of 5% CO2-95% air. Subcultured strains were used between passages 20 and 28. Cells were plated at a density of 106 cells/100-mm dish. When cells reached 80% confluence, the culture medium was replaced with fresh DMEM-HEPES and experiments were started.
Arginase assay. Arginase activity was determined by methods that we have previously described for endothelial cells (4). Briefly, RASMC (4 × 106 cells/sample) were washed twice with PBS, harvested, pelleted by centrifugation, then lysed. Supernatant fractions were assayed for arginase activity under optimal conditions of pH (9.6) and arginase concentration (20 mM) by monitoring the conversion of L-[guanido-14C]arginine to [14C]urea during 10-min incubation.
Measurements of cell proliferation. Cell proliferation was assessed by monitoring rates of DNA synthesis as determined by the incorporation of [3H]thymidine into DNA. RASMC were seeded in six-well plates at a density of ~3,000 cells/cm2 in DMEM-HEPES containing 10% FBS and incubated for 4 h to allow cells to adhere to the plates. Cell synchronization was achieved by arresting cell growth by incubation in serum- and arginine-free DMEM-HEPES for 48 h. The effects of IL-4 and IL-13 on cell proliferation were examined in DMEM-HEPES containing 10% FBS. IL-4 or IL-13 was added to cell cultures and incubated for 24 h at 37°C. After this incubation period, 0.1 µCi [3H]thymidine was added to each well and incubated for an additional 24 h. The cells were collected for determining rates of DNA synthesis according to procedures described previously (4).
Immunoprecipitation and immunoblot analysis. For STAT6 activation assays, RASMC (107 cells/sample) were incubated with IL-4 or IL-13 at 37°C for 10 min. Cells were washed twice with PBS containing 100 µM Na3VO4, followed by addition of 1.5 ml lysis buffer (50 mM Tris·HCl, pH 7.5, 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 5 mM EDTA, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin). After 10 min on ice, lysates were cleared of insoluble materials by centrifugation for 10 min at 12,000 g. The cleared lysates were immunoprecipitated with STAT6 antiserum (1:500) plus 40 µl of protein A-coupled agarose beads overnight at 4°C. Immunoprecipitates were collected by centrifugation at 2,500 rpm for 5 min at 4°C. Immunoprecipitates were then washed four times with lysis buffer, solubilized with Laemmli buffer, boiled 5 min, and resolved by 8% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 25 mM Tris·HCl, pH 7.2, 150 mM NaCl, and 0.2% (wt/vol) Tween (TBST) buffer supplemented with 2% BSA. The membranes were subsequently probed with the following antiserum diluted in TBST: anti-p-Tyr (PY 99, 1:1,000) and anti-STAT6 (1:2,000). The membranes were then washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody. AI protein expression was determined in the same cell lysates used for the arginase activity assays. Equal samples (20 µg of total protein from each sample) were subjected to SDS-PAGE (10%) then transferred to a nitrocellulose membrane. The membrane was probed with chicken anti-rat AI antibody (1:50,000 dilution) (32). The secondary antibody for AI immunoblots was peroxidase-conjugated rabbit anti-chicken IgG (Jackson ImmunoResearch Laboratories). The immunoblots were developed with enhanced chemiluminescence Western blotting detection kits (Amersham) according to the manufacturer's directions.
Isolation and analysis of RNA. RNA was isolated using a commercially available kit (QIAshredder and RNeasy total RNA; Qiagen). Northern blot analysis was performed according to a standardized protocol (44). Full-length cDNA probes encoding rat AI (21) or murine AII (31) were used for Northern blots. A human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probe was used as an internal control (Ambion). Densitometry of the bands was performed on Fuji RX autoradiography film using a Hewlett-Packard flatbed scanner and NIH Image densitometry software.
Statistical analyses. Data were statistically analyzed using the Student's t-test for unpaired values. Probability values of <0.05 were taken to indicate statistical significance.
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RESULTS |
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The protein kinase A signal transduction pathway regulates arginase expression and activity in RASMC.
RASMC contain a basal arginase activity that is markedly increased
(4- to 5-fold) by incubation of the cells with IL-4 (10 ng/ml) or IL-13
(10 ng/ml). A time course for the induction of arginase activity by
IL-4 and IL-13 is illustrated in Fig.
1. Arginase activity was elevated
at 12 h, peaked at 24-48 h, and started to decline at 72 h. These data are consistent with the results of a Western blot
analysis of AI protein (Fig. 1), indicating that the induced
arginase protein and catalytic activity are maintained through at least
72 h after cytokine induction. The 24-h time point was selected
for further experiments involving test agents. Basal arginase activity
is slightly increased by incubation cells with the adenylate cyclase
activator forskolin (1 µM) (Fig. 1). Forskolin potentiates arginase
induction by IL-4 and IL-13 about twofold. In contrast, basal arginase
activity is reduced by incubating cells with the protein kinase A
inhibitor H-89 (30 µM) (Fig. 2). Preincubation of RASMC with H-89 for 1 h markedly attenuates the increase in arginase activity caused by IL-4 and IL-13.
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To identify the arginase isoform(s) expressed in RASMC, Northern and
Western blot analyses were employed to monitor mRNA and protein
expression, respectively. Untreated RASMC express AI mRNA and protein
(Fig. 3). Both IL-4 and IL-13 markedly
enhance AI mRNA and protein expression in comparison with the basal
level. In contrast, mRNA for the AII isoform was not detected in
control cells, and neither IL-4 nor IL-13 induced AII mRNA (data not
shown). Coincubation of cells with forskolin enhances both the basal
and IL-4- and IL-13-induced AI protein and mRNA levels. Incubation of
RASMC with H-89 inhibits basal AI mRNA and protein expression. Moreover, preincubation with H-89 for 1 h significantly diminishes IL-4- and IL-13-induced AI protein and mRNA levels. There is a direct
correlation between the changes in AI protein and mRNA levels with
changes observed in arginase activity. These data implicate protein
kinase A in the regulation of both basal and induced AI expression in
RASMC.
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Tyrosine phosphorylation may be involved in the regulation of basal
and induced arginase activity in RASMC.
To determine if tyrosine phosphorylation may be involved in the
regulation of arginase, RASMC were incubated with either genistein, an
inhibitor of protein tyrosine kinases, or
Na3VO4, a tyrosine phosphatase inhibitor.
Incubation of cells with genistein (10 µg/ml) significantly reduces
basal arginase activity in RASMC (Fig.
4). Furthermore,
preincubation of cells with genistein for 1 h diminishes IL-4- and
IL-13-induced arginase activity by ~50%. In contrast, incubation of
cells with Na3VO4 (10 µM) slightly increases
basal arginase activity. In combination with either IL-4 or IL-13,
Na3VO4 enhances arginase activity two- to
threefold. These data for arginase activity closely agree with the
changes observed in mRNA and protein expression. These data suggest
that a signal transduction pathway involving phosphotyrosine formation is involved in the regulation of AI expression in RASMC.
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STAT6 activation by IL-4 and IL-13 correlates with upregulation of
arginase I expression.
STAT6 is a cytoplasmic transcription factor regulated by its
phosphorylation state and is activated in response to IL-4 and IL-13
(8). After phosphorylation of a critical tyrosine residue, STAT6 dimerizes, translocates from the cytoplasm to the nucleus, and
binds to its cognate DNA, leading to a specific transcriptional upregulation of target genes (18). Because we have
demonstrated in this report that phosphotyrosine is essential for the
regulation of AI by IL-4 and IL-13, we investigated the status of STAT6
phosphorylation following stimulation with either IL-4 or IL-13.
Unstimulated RASMC have no detectable phosphorylated STAT6 (p-STAT6)
protein (Fig. 5). Addition of IL-4 or
IL-13 promotes a rapid and intense phosphorylation of STAT6. The
appearance of p-STAT6 correlates with the upregulation of AI expression
by IL-4 and IL-13 and supports the hypothesis for a cytokine-JAK/STAT
pathway for AI regulation. Studies are in progress to test the
hypothesis that activation of the JAK/STAT6 pathway is involved in the
induction of AI by IL-4 and IL-13 in RASMC.
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Dexamethasone and IFN- abrogate the stimulatory effect of IL-4
and IL-13 on AI.
The glucocorticoid dexamethasone and the cytokine IFN-
have been
well documented to inhibit the proliferation of vascular smooth muscle
(25). The mechanism of this cytostatic action has not been
completely defined for either agent. Therefore, the effects of
dexamethasone and IFN-
on arginase induction were examined.
Incubation of RASMC with either dexamethasone (1 µM) or IFN-
(100 U/ml) alone has no measurable influence on basal arginase
activity (Figs. 6 and
7). However, dexamethasone or IFN-
each moderately attenuates arginase activity induced by IL-4 or IL-13.
Moreover, the combination of dexamethasone plus IFN-
abolishes the
stimulatory effect of IL-4 and IL-13 on arginase induction. In all
cases, changes in arginase activity were correlated with relative
changes in AI mRNA and protein expression (Fig.
8).
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Upregulation of AI expression by IL-4 and IL-13 correlates with
cytokine-stimulated RASMC proliferation.
A recent study from this laboratory revealed that inhibiting arginase
activity in the human adenocarcinoma Caco-2 cell line resulted in the
inhibition of cell proliferation, thereby implicating arginase in the
regulation of cell growth (4). In the present study, we
investigated the relationship between AI induction and cell
proliferation. DNA synthesis, as monitored by
[3H]thymidine incorporation, increases by ~50% in
RASMC exposed to IL-4 (10 ng/ml) or IL-13 (10 ng/ml) for 48 h
(Fig. 7). This increase in cell proliferation correlates with
the increase in AI expression and activity observed in response to IL-4
and IL-13, supporting our hypothesis that AI is involved in regulating
the growth of RASMC. Moreover, IFN-, H-89, and genistein each
markedly inhibited the increase in cell proliferation caused by IL-4 or IL-13. Addition of these test agents alone to cells in the absence of
added cytokine caused only little or no significant decrease in cell
proliferation (data not shown). Although dexamethasone markedly
inhibited the increase in cell proliferation caused by IL-4 and IL-13,
dexamethasone alone significantly inhibited RASMC proliferation, and
additional experiments are required to elucidate the mechanism of this effect.
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DISCUSSION |
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For many years, AI had been thought to be expressed exclusively in the liver. However, it was recently reported that rat endothelial cells (4) and human neutrophils (56) express AI. Of relevance to the present study are reports that AI can be induced in macrophages (26, 32) and mesangial cells (57) by either 8-bromo-cAMP or IL-4. This study provides the first demonstration that RASMC express AI; that IL-4 and IL-13 both significantly upregulate AI mRNA, protein, and activity; and that cAMP is involved in AI expression in those cells.
Our results demonstrate that the PKA inhibitor H-89 inhibits both basal and IL-4- and IL-13-induced AI expression in RASMC. Moreover, forskolin, an activator of adenylate cyclase, significantly enhances IL-4- and IL-13-induced AI levels and activity. These data indicate that the cAMP signal transduction pathway is involved in the regulation of basal and induced AI expression in RASMC. Interestingly, the AI promoter region contains a putative cAMP response element (CRE) (52), although a functional CRE in the AI has yet to be identified.
IL-4, a pleiotropic modulator of the immune system, exerts its activity
on target cells through IL-4 receptor (IL-4R), which consists of the
IL-4R -chain (IL-4R
) and the common
-receptor chain (
c),
also called the type I receptor (22). IL-4R
also forms
a functional IL-4R in conjunction with the IL-13 receptor (IL-13R
)
instead of
c. This type of IL-4R, called the type II receptor, is
found particularly in nonimmune cells (5). Treatment of
cells with IL-4 activates cytoplasmic tyrosine kinases (8, 17). The present study reveals that genistein, an
inhibitor of tyrosine kinases, inhibits both basal and IL-4- and
IL-13-induced AI in RASMC. Also, Na3VO4, a
protein tyrosine phosphatase inhibitor, markedly increases both basal
and IL-4- and IL-13-induced AI expression in RASMC. These data
illustrate, albeit indirectly, that a tyrosine kinase pathway may be
involved in the regulation of basal and induced AI expression in RASMC.
Activation of the IL-4R triggers the function of intracellular signal mediators, such as the JAK/STAT pathway, which involves the IL-4- and IL-13-specific transcription factor STAT6 (8, 40). STAT proteins are phosphorylated in response to ligand stimulation through receptor-associated JAK proteins. Tyrosine phosphorylation of STAT proteins induce dimerization. The dimerized STAT then translocate to the nucleus and modulate transcription of target genes (18). We demonstrate that untreated RASMC contain no detectable phosphorylated STAT6 protein. Phosphorylated STAT6 was readily detected, however, after stimulating the cells with IL-4 or IL-13 for only 10 min at 37°C. The correlation of AI induction with STAT6 phosphorylation suggests that the JAK-STAT6 pathway may be involved in the regulation of AI expression in RASMC. Alternative pathways, however, cannot be ruled out.
Dexamethasone induces arginase in hepatocytes and hepatoma cells
(9, 15, 36). In contrast, our
results show that dexamethasone alone has no effect on basal AI
expression in RASMC but can reduce the AI induction by IL-4 and IL-13.
This observation is similar to that in a report showing that
dexamethasone blocks AI induction caused by 8-bromo-cAMP in RAW 264.7 cells (32). We also found that dexamethasone plus IFN-
completely blocks AI induction by IL-4 and IL-13. Why dexamethasone
elicits opposite effects on arginase expression in different cell type
is unknown.
In the present study, IFN- alone had no effect on basal AI
expression in RASMC but significantly inhibited the induction of AI
mediated by IL-4 and IL-13, and the combination of dexamethasone with
IFN-
inhibited the induction of AI even more. These results support
the general impression that IFN-
inhibits induction of arginase. For
example, IFN-
completely blocks the induction of AI expression by
8-bromo-cAMP in RAW 264.7 cells (32). Moreover, IFN-
inhibits LPS-mediated induction of AII in RAW 264.7 cells (58) and may inhibit the same response in rat aorta
endothelial cells (4). In contrast to these inhibitory
effects, however, is the finding that IFN-
synergizes with IL-4 in
induction of AI expression in RAW 264.7 cells (46). Thus,
like dexamethasone, IFN-
has varying effects on AI expression in
different cell types.
A family of cytokine-inducible SH2 proteins (CIS) has recently been
identified (54, 61). Most CIS appear to be
induced by several cytokines, and at least three of them [CIS1, CIS3
and JAB (JAK-binding protein)] negatively regulate cytokine signal transduction (10, 34, 49). CIS3
and JAB directly bind to the kinase domain of JAK, thereby inhibiting
kinase activity (28). These CIS family members appear to
function in a classical negative-feedback loop of cytokine signaling.
Because IFN- is the most potent inducer of JAB in a wide variety of
cell lines (60), it is possible that IFN-
-activated JAB
could be a part of the mechanism of preventing the AI induction by IL-4
and IL-13.
Another novel aspect of this study is that the upregulation of AI by
IL-4 and IL-13 correlates with enhanced RASMC proliferation. Although
these data do not prove that the stimulatory effects of IL-4 and IL-13
on RASMC proliferation are attributed to arginase induction, or are
mediated by cAMP, they are consistent with these possibilities. IL-4
and other vasoactive and inflammatory cytokines have been shown to
promote the growth of other cell types including porcine aorta smooth
muscle cells (35), human adrenal capillary endothelial
cells (53), and mouse skin fibroblasts (30).
On the other hand, IL-4 and cAMP have been reported to inhibit
proliferation of vascular smooth muscle cells from other sources
(38, 48, 55). The reasons for
these differences in results are not apparent but may be attributed to
differences in cell types and/or cell culture conditions. Our findings
that IFN- and dexamethasone inhibit the induction of AI in RASMC are
consistent with the well-documented cytostatic actions of
glucocorticosteroids and IFN-
in vascular smooth muscle
(16, 25).
Increased proliferation of vascular smooth cells contributes to intimal hyperplasia during atherosclerosis and restenosis (41, 42). The endogenous factors regulating vascular smooth muscle cell growth in response to arterial injury are not well understood. Both arginase and ornithine decarboxylase are intimately involved in cell proliferation. Arginase catalyzes the conversion of arginine to ornithine, and ornithine decarboxylase catalyzes the subsequent conversion of ornithine to putrescine. The production of polyamines plays an essential role in cell proliferation (19, 50). We hypothesize that at least one consequence of upregulating AI expression in RASMC may be the enhanced synthesis of polyamines, which may explain the enhancement of cell proliferation observed in this study. Accordingly, upregulation of AI expression caused by IL-4 and IL-13 might play an important role in the pathophysiology of atherosclerosis, angiogenesis, and restenosis, which result from a complex cascade of events involving abnormal proliferation of vascular smooth muscle cells.
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ACKNOWLEDGEMENTS |
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We thank Dr. Steven Gross for the kind gift of RASMC. We also thank Dr. Georgette M. Buga for providing expert technical support on the [3H]thymidine incorporation assay.
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FOOTNOTES |
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This research was supported by National Institutes of Health Grants HL-35014, HL-40922, and HL-58433 (to L. J. Ignarro) as well as GM-57384 (to S. M. Morris).
Address for reprint requests and other correspondence: L. J. Ignarro, Dept. of Molecular and Medical Pharmacology, UCLA School of Medicine, 23-120 CHS, Box 951735, Los Angeles, CA 90095-1735 (E-mail: lignarro{at}mednet.ucla.edu).
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. §1734 solely to indicate this fact.
Received 25 August 1999; accepted in final form 25 January 2000.
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