(Hypertension. 1998;32:1003-1010.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Reactive Oxygen Species Are Critical in the Oleic Acid–Mediated Mitogenic Signaling Pathway in Vascular Smooth Muscle Cells
From the Departments of Pharmacology (G.L., B.M.E.) and Medicine (E.L.G., T.N., B.M.E.), Medical University of South Carolina, Charleston.
Correspondence to Brent M. Egan, MD, Division of Clinical Pharmacology, Medical University of South Carolina, 171 Ashley Ave, CSB 826, Charleston, SC 29425.
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Abstract |
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Abstract—Obese hypertensive patients with cardiovascular risk factor clustering have increased plasma nonesterified fatty acid levels and are at high risk for atherosclerotic events. Our previous studies demonstrated that oleic acid induces a mitogenic response in rat aortic smooth muscle cells (RASMCs) through protein kinase C (PKC)– and extracellular signal–regulated kinase (ERK)–dependent pathways. In the present study we investigated the possibility that the generation of reactive oxygen species (ROS) constitutes a critical component of the oleic acid–induced mitogenic signaling pathway in RASMCs. We studied the effect(s) of oleic acid on the generation of ROS using the oxidant-sensitive fluoroprobe 2',7'-dichlorofluorescin diacetate. Relative fluorescence intensity and fluorescent images were obtained with laser confocal scanning microscopy from 1 to 5 minutes, since preliminary studies demonstrated that the peak fluorescence intensity occurred within 5 minutes. Oleic acid (100 µmol/L) induced a time-dependent increase of cell fluorescence that was >8-fold of that seen in control cells at 5 minutes. This was blocked by catalase, which suggests that H2O2 was the principal ROS. The oleic acid–induced increases in H2O2 were blocked when PKC was inhibited with the use of bisindolylmaleimide and when PKC activity was downregulated by exposing RASMCs to phorbol 12–myristate 13–acetate for 24 hours. Stearic and elaidic acids, which are weak PKC activators, did not significantly increase H2O2 production. The increase of H2O2 in response to oleic acid was inhibited by the antioxidant N-acetylcysteine. N-Acetylcysteine also completely blocked ERK activation and the increase of thymidine incorporation in response to oleic acid. The data suggest that generation of H2O2 in RASMCs exposed to oleic acid is PKC dependent. Moreover, H2O2 production emerges as a critical intermediary event in the oleic acid–mediated mitogenic signaling pathway between the activation of PKC and ERK. These observations raise the possibility that the elevated plasma nonesterified fatty acids, including oleic acid, in obese hypertensive patients contribute to vascular growth and remodeling by a PKC-dependent mechanism to generate ROS that subsequently activate ERK.
Key Words: muscle, smooth, vascular • oleic acid • kinase • reactive oxygen species • hydrogen peroxide
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Introduction |
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Obese hypertensive patients have insulin resistance and the associated cardiovascular risk factor cluster.1 2 3 4 While abdominal obesity and insulin resistance emerge as independent risk factors for atherosclerotic disease, the intermediary mechanisms are not well defined. Evidence suggests that resistance to the fatty acid–lowering actions of insulin among insulin-resistant individuals may contribute to structural and functional vascular changes.5 6 7 8 We previously reported that obese hypertensive patients have elevated plasma nonesterified fatty acids (NEFAs), including oleic acid, which reflect resistance to the antilipolytic action of insulin.5 We demonstrated that oleic acid induced a protein kinase C (PKC)– and extracellular signal–regulated kinase (ERK)–dependent mitogenic response in rat aortic smooth muscle cells (RASMCs).6 7 Activation of PKC and ERK has been linked to vascular smooth muscle cell (VSMC) proliferation and vascular contraction.9 10 11 12 In addition, activation of ERK has been linked to VSMC migration.13 Thus, the activation of PKC and ERK is linked to events associated with hypertension and vascular remodeling.
A better understanding of early events in the oleic acid–induced signaling pathway in VSMCs could shed light on intermediary mechanisms by which insulin resistance leads to functional and structural vascular changes. Of note, NEFAs, including oleic acid, induce a PKC-dependent activation of the respiratory burst in white blood cells.14 Recently, reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radical (O2-) generated in response to hormone or ligand binding have been implicated as participants in signaling pathways stimulating VSMC growth and DNA synthesis.15 16 17 18 Moreover, ROS have been associated with the activation of ERKs,19 transcription factors,20 phospholipase A2,21 collagenases, and protooncogene expression.22 23 Collectively, the extant literature supports the hypothesis that oleic acid induces a PKC-dependent generation of oxygen radicals that, in turn, activate ERK in VSMC. The present study was undertaken to determine whether ROS are major participants in critical intermediary signaling events between the activation of PKC and ERK in RASMCs. We used the following criteria to establish that oleic acid induces the generation of ROS and that activation of PKC is a critical upstream event and activation of ERK is a downstream event: (1) Stimulation of VSMCs by oleic acid should lead to the generation of measurable quantities of ROS. (2) PKC inhibition and downregulation should reduce the generation of ROS in VSMCs stimulated with oleic acid. (3) Controlling for nonspecific effects of fatty acids and elaidic and stearic acids, which are poor activators of PKC compared with oleic acid, should induce a lesser amount of ROS than oleic acid. (4) Antioxidants should attenuate ERK activation and thymidine incorporation induced by oleic acid. Observations from these experiments could help to define signaling mechanism(s) by which insulin resistance and abnormal fatty acid metabolism could accelerate vascular remodeling. This information could help to foster novel approaches to the prevention and treatment of cardiovascular disease in patients with the risk factor cluster.
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Methods |
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Materials
Individual fatty acids, catalase, and N-acetylcysteine (NAC) were purchased from Sigma Chemical Co. Sodium salts were prepared as previously described.6 7 Cell culture materials were purchased from GIBCO BRL. Fatty acid–free albumin was obtained from ICN Biomedical Inc.
Cell Culture
RASMCs were cultured by procedures previously
described.6 7
Assay for Intracellular Oxidant Production
RASMCs were plated in 35-mm cell culture plates and grown until
confluent. Cells were placed in DMEM containing 0.1% fetal bovine
serum for 48 hours to induce growth arrest. Quiescent cells were
incubated with the
H2O2-sensitive
fluorescent probe 2',7'-dichlorofluorescin diacetate (DCF-DA)
(Molecular Probes) to assess the generation of intracellular
ROS.24 25 Nonfluorescent DCF-DA diffuses
through the plasma membrane, where it is subsequently
deacetylated enzymatically by cellular esterases to the polar
compound 2',7'-dichlorofluoroscein (DCF), which remains trapped in the
cell and fluoresces in the presence of intracellular peroxides
(H2O2 and lipid
hydroperoxides). Cells were incubated with DCF-DA for 30 minutes at
37°C before stimulation with 100 µmol/L oleic acid as well as
100 µmol/L stearic acid and 100 µmol/L elaidic acid. In
other experiments, 100 µmol/L oleic acid was added to DCF-loaded
RASMCs that were pretreated for 30 minutes with 4 µmol/L
bisindolylmaleimide to inhibit PKC or pretreated for 24 hours
with 200 nmol/L phorbol 12–myristate 13–acetate (PMA) to
deplete PKC immunoreactivity and activity.6 To
verify that H2O2 was the
main ROS generated in response to oleic acid, experiments were
performed in DCF-loaded RASMCs pretreated with either 1000 or 2000
IU/mL catalase for 30 minutes. Relative fluorescence intensity
and fluorescent images were obtained over time (1 to 20
minutes) after addition of different fatty acids with laser confocal
scanning microscopy (LSMGB-200, Olympus Optical Co Ltd) at an
excitation wavelength of 485 nm and an emission wavelength of 530 nm.
Densitometric analysis of the confocal microscopic images was
performed with Adobe Photoshop (Adobe Systems Inc).
Immunoblot Assay for Phosphorylation of
Mitogen-Activated Protein Kinase
Confluent RASMC in 100-mm cell culture plates were incubated in
media containing 0.1% fetal bovine serum and DMEM for 48 hours. Oleic
acid (100 µmol/L) was added to control cells and also cells
pretreated for 10 to 20 minutes with NAC (30 mmol/L). The
incubation was stopped by adding ice-cold PBS. Cells were scraped into
PBS and then pelleted by centrifugation for 20 seconds
at 2000g. The cell pellet was suspended in cold lysis buffer
containing 10 mmol/L Tris, 10 mmol/L NaCl, 3 mmol/L
MgCl2, 500 µmol/L sodium vanadate, 2
mmol/L phenylmethylsulfonyl fluoride, and 10 000 U/mL
aprotinin. The suspension was maintained at 4°C with occasional
vortexing, followed by centrifugation at
5000g at 4°C for 10 minutes. The supernatant was collected
as the whole cell lysate. Proteins in the whole cell lysate were
resolved by SDS-PAGE with 10% Laemmli gels. Proteins were
electrophoretically transferred to polyvinylidene membranes (Millipore)
and then immunoblotted with antibodies against anti-ACTIVE
mitogen-activated protein kinase polyclonal antibody (Promega
Co) and anti-ERK monoclonal antibody (Transduction
Laboratories). IgG horseradish peroxidase–conjugated antibody
(Amersham) was used as the secondary antibody. Visualization of the
blot was performed with the ECL Western blotting system.
3H-Thymidine Incorporation
Oleic acid (100 µmol/L) was added to quiescent RASMCs
that were pretreated for 30 minutes with either 30 mmol/L NAC or
vehicle. Cells were pulse-labeled with3H-thymidine (0.5 µCi/well) 6 hours before completion of
the 24-hour incubation period. 3H-thymidine
incorporation was measured as described.6 7
Statistical Analysis
Data are presented as mean±SD. Data were
analyzed with SPSS 6.0 (SPSS Inc). The time-dependent changes
in fluorescence intensity within and across the various
treatment conditions were analyzed with 1-way ANOVA followed by
Duncan's multiple range test or 2-factor ANOVA, respectively.
P values <0.05 were considered statistically
significant.
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Results |
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Effect of Oleic Acid on Production of H2O2
To fulfill the first criterion, quiescent RASMCs were pretreated with DCF-DA for 30 minutes and then treated with 100 µmol/L oleic acid for up to 20 minutes. We postulated that oleic acid would increase the generation of ROS. Relative fluorescence intensity and microscopic fluorescent images of the cells were obtained from 1 to 20 minutes after exposure to oleic acid with laser confocal scanning microscopy. Initial studies indicated that the increase in fluorescence intensity peaked after 5 minutes and persisted at the same levels over the next 15 minutes. Therefore, subsequent imaging studies were limited to measuring change in fluorescence intensity from 1 to 5 minutes. As shown in Figure 1A
, oleic acid induced a time-dependent
increase in fluorescence in RASMCs from 1 minute to 5 minutes,
which presumably reflects an increase of intracellular
H2O2 production. In
the control RASMCs, fluorescence intensity increased minimally
over 5 minutes, which suggests a low basal level of
H2O2 production.
Quantitative analysis of the confocal images indicated that the
increase in fluorescence intensity in cells treated with oleic
acid reached levels 
8 times that observed in control cells at 5
minutes (Figure 1B). To prove that fluorescence in DCF-loaded
cells was specific for
H2O2, cells were
preincubated with catalase. As shown in Figure 2, 1000 IU/mL catalase inhibited
H2O2 production,
while 2000 IU/mL catalase completely blocked the response to oleic
acid.
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Effect of PKC Inhibition and Depletion on
H2O2 Production in Response to
Oleic Acid
We postulated (second criterion) that if the generation of ROS in
RASMCs treated with oleic acid was PKC dependent, then ROS
production would be inhibited by (1) using the PKC
inhibitor bisindolylmaleimide to directly inhibit PKC and
(2) pretreating cells with 200 nmol/L PMA for 24 hours to deplete PKC.
As shown in Figure 3, in cells pretreated
with 4 µmol/L bisindolylmaleimide for 30 minutes to block PKC or
200 nmol/L PMA for 24 hours to deplete PKC, the increase in
H2O2 after treatment with
oleic acid was completely blocked. Basal
H2O2 production in
cells treated with bisindolylmaleimide or PMA pretreatment for 24 hours
was not significantly altered (data not shown).
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Effect of Elaidic and Stearic Acids on
H2O2 Production
To ensure that the effect(s) of oleic acid did not
represent nonspecific effects of fatty acids (criterion 3),
RASMCs were treated with 100 µmol/L concentrations of elaidic
acid, the trans isomer of oleic acid, and stearic acid, an
18-carbon saturated fatty acid. RASMCs treated with stearic and elaidic
acids did not show significant increases in fluorescence
intensity, which raises the possibility of a selective effect of oleic
acid on cell function (Figure 4).
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Effect of NAC on ERK Activation and 3H-Thymidine
Incorporation in Response to Oleic Acid
To fulfill the fourth criterion, ie, that antioxidants should
inhibit ERK activation and thymidine incorporation, we treated cells
with NAC. Treatment of quiescent RASMCs with 100 µmol/L oleic
acid for 10 minutes induced a marked increase in the
phosphorylation of ERK-1 and ERK-2 (Figure 5A, top panel). Pretreatment of cells
with 30 mmol/L NAC completely blocked
phosphorylation of ERKs. In the bottom panel, an
immunoblot with anti-ERK antibody confirms that equal
amounts of proteins were loaded in each lane. Treatment of cells with
NAC alone did not change either ERK protein expression or the
phosphorylation of ERK-1 and ERK-2. NAC (30
mmol/L) also completely blocked the increase of thymidine incorporation
in response to oleic acid (Figure 5B). To support the notion that NAC
blocked ERK activation and thymidine incorporation by inhibiting ROS,
we examined whether NAC would limit the increase of DCF
fluorescence intensity in cells stimulated with oleic acid.
When RASMCs were pretreated with 30 mmol/L NAC for 30 minutes, the
increase in fluorescence after stimulation with 100
µmol/L oleic acid was blocked (Figure 6).
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Discussion |
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Oxidative stress and the generation of ROS in vascular cells have been postulated as contributors to atherosclerosis and related cardiovascular complications. Oxidants could conceivably participate in vascular injury through several mechanisms, which include (1) effects on cellular function through lipid peroxidation of plasma membrane and subcellular membrane lipids, (2) oxidization of critical cellular proteins and enzymes, and (3) direct cytotoxicity. However, oxidants are not always generated in quantities that are immediately cytotoxic or injurious. Moreover, limited quantities of ROS can be generated in response to hormone or ligand binding to cell surface receptors and function as second messengers involved in cell growth and proliferation.15 16 17 18 ROS fulfill several criteria as possible signaling molecules. For example, ROS are small diffusible molecules that are ubiquitously present and can be rapidly synthesized and destroyed.26
The findings of the present study suggest that oleic acid induces a
PKC-dependent increase in the generation of ROS leading to ERK
activation in cultured VSMCs. Fluorescence intensity in
DCF-loaded RASMCs treated with oleic acid increased rapidly as a
function of time up to 5 minutes (Figure 1
). Furthermore, the increase
in fluorescence was inhibited by catalase (Figure 2). These
data suggested that the oleic acid–induced increases in intracellular
fluorescence intensity principally reflect a rise in
H2O2.24 25
Of note, the 100 µmol/L concentration of oleic acid used in our
experiments falls within the range of plasma levels measured in healthy
normotensives, which are roughly two thirds the values obtained in
obese hypertensives. Interstitial fluid concentrations of
fatty acids are likely approximately one fourth those in
plasma.7 Thus, the 100 µmol/L
concentration of oleic acid probably lies at the upper end of
interstitial fluid values observed in
insulin-resistant patients, which includes obese
hypertensives.
Both PKC inhibition with bisindolylmaleimide and downregulation by
24-hour pretreatment with PMA blocked the increase in cellular
fluorescence in response to oleic acid (Figure 3). We have
shown that PKC inhibition with bisindolylmaleimide and downregulation
with PMA prevent agonist-induced increases of PKC activity. However,
neither of these treatments adversely affected cell morphology or the
increase of 3H-thymidine uptake in response to
10% fetal bovine serum.6 These findings suggest
that oleic acid induces a PKC-dependent increase in the generation of
H2O2 in VSMCs.
To control for possible nonspecific effects of fatty acids, additional
experiments were performed with elaidic and stearic acids. In contrast
to 100 µmol/L oleic acid, an 18-carbon,
cis-monounsaturated fatty acid,
identical concentrations of elaidic acid, the trans isomer
of oleic acid, and stearic acid, an 18-carbon saturated fatty acid,
caused minimal changes in cell fluorescence (Figure 4). In
comparison to oleic acid, stearic and elaidic acids are weak
activators of PKC. Thus, the generation of
H2O2 in response to these
three 18-carbon NEFAs corresponds to their known capacity to
activate PKC.26 27 28 These data are
consistent with the notion that oleic acid induces a specific
PKC-dependent increase of ROS in VSMCs.
We reported that oleic acid induced a PKC-dependent activation of ERK
in RASMCs6 and subsequently demonstrated that the
mitogenic response to oleic acid was also ERK
dependent.7 As noted above, other studies have
shown that ROS activate ERK.17 Activation
of ERK has emerged as an important signaling event in VSMC migration,
growth, and vascular tone.8 9 10 11 12 13 Therefore, our
studies of ROS and oleic acid–mediated activation of ERK complement
the existing literature. NAC, an antioxidant, blocked the generation of
ROS in oleic acid–treated cells (Figure 6). NAC also blocked the
ability of oleic acid to induce phosphorylation of
ERK-1 and ERK-2 and DNA synthesis in cells stimulated with oleic acid
(Figure 5). These observations suggest that the generation of ROS is a
critical event in the activation of ERK and cell proliferation by oleic
acid. Collectively, our data suggest that the generation of ROS
represents a critical intermediary event between the activation
of PKC and ERK and cell growth in VSMCs stimulated with oleic acid.
A large body of literature has established activation of both PKC and ERK as signaling events that may play an important role in vascular remodeling.5 6 9 10 11 12 13 A more recent and rapidly growing number of studies implicates oxygen radicals in vascular pathophysiology. ROS trigger several events linked with vascular remodeling and cardiovascular complications, including activation of ERKs, transcription factors, phospholipase A2, insulin-like growth factor 1, DNA synthesis, and metalloproteinases.19 20 21 22 23 ROS have also been associated with apoptosis and impairment of endothelium-dependent vasodilation.5 29 Thus, oleic acid is now associated with 3 steps of the signaling cascade in VSMCs (ie, PKC, ROS, and the ERKs) that are implicated in vascular pathophysiology.
In summary, the cardiovascular risk factor cluster associated with insulin resistance includes resistance to the fatty acid–lowering action of insulin. Obese hypertensives, in particular, are highly resistant to the antilipolytic effect of insulin and have elevated plasma oleic acid concentrations. Oleic acid induces a PKC-dependent increase in the generation of ROS, which, in turn, activate ERK in VSMCs. Therefore, the study data suggest that ROS represent a critical event in the oleic acid–mediated mitogenic signaling pathway between PKC and ERK. These findings raise the possibility that oleic acid participates in the vascular remodeling and complications associated with the risk factor cluster and may shed further light on intermediary events by which insulin resistance accelerates vascular remodeling.
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Acknowledgments |
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This research was supported by National Institutes of Health R01 HL-58794 (Dr Egan), K01 HL-03710 (Dr Greene), and grants-in-aid from the American Heart Association, South Carolina affiliate (Dr Egan) and Dialysis Clinics Incorporated (Dr Greene). Dr Lu is supported by National Institutes of Health cardiovascular training grant HL-07260. The authors appreciate the expert assistance of Ainslie Lawrence in preparing this manuscript.
Received April 20, 1998; first decision May 29, 1998; accepted August 6, 1998.
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 R. M. Touyz and E. L. Schiffrin Signal Transduction Mechanisms Mediating the Physiological and Pathophysiological Actions of Angiotensin II in Vascular Smooth Muscle Cells Pharmacol. Rev., December 1, 2000; 52(4): 639 - 672. [Abstract] [Full Text] [PDF]
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E. L. Greene, V. Velarde, and A. A. Jaffa Role of Reactive Oxygen Species in Bradykinin-Induced Mitogen-Activated Protein Kinase and c-fos Induction in Vascular Cells Hypertension, April 1, 2000; 35(4): 942 - 947. [Abstract] [Full Text] [PDF]
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R. M. Touyz and E. L. Schiffrin Ang II-Stimulated Superoxide Production Is Mediated via Phospholipase D in Human Vascular Smooth Muscle Cells Hypertension, October 1, 1999; 34(4): 976 - 982. [Abstract] [Full Text] [PDF]
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