Redox Regulation of Vascular Smooth
Muscle Cell Differentiation
B. Su,* S. Mitra,* H. Gregg, S. Flavahan, M.A. Chotani, K.R. Clark,
P.J. Goldschmidt-Clermont, N.A. Flavahan
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Abstract—Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular
smooth muscle cell (VSMC) phenotype. After quiescence, cultured human VSMCs increased their expression of
differentiation proteins (␣-actin, calponin, and SM1 and SM2 myosin), but not ␤-actin. ROS activity, determined using
the H2O2-sensitive probe dichlorodihydrofluorescein (DCF), remained high in quiescent cells and was inhibited by
catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A superoxide dismutase mimic (SOD;
MnTMPyP, 25 ␮mol/L) or SOD plus low concentrations of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence,
which was inhibited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity (by catalase or NAC)
decreased the baseline expression of differentiation proteins, whereas elevation of ROS (by SOD or SODNAC2)
increased expression of the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to
20 mmol/L). None of the treatments altered ␤-actin expression. SODNAC2-treated cells demonstrated contractions to
endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased
when ROS activity was reduced (NAC, 10 mmol/L) and was augmented when ROS activity was increased (SODNAC2).
Inhibition of p38 MAPK with pyridyl imidazole compound (SB202190, 2 to 10 ␮mol/L) reduced expression of
differentiation proteins occurring under basal conditions and in response to SODNAC2. Transduction of VSMCs with
an adenovirus encoding constitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation
proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the
differentiation proteins. These findings demonstrate that ROS can increase VSMC differentiation through a p38
MAPK– dependent pathway. (Circ Res. 2001;89:39-46.)
Key Words: reactive oxygen species 䡲 p38 MAPK 䡲 myosin 䡲 calponin
V
ascular smooth muscle cells (VSMCs) exist in a diverse
range of phenotypes.1–5 In normal mature blood vessels,
the predominant phenotype is the contractile or differentiated
VSMC, which has as its major function the regulation of
blood vessel diameter and blood flow. During protective (ie,
arteriogenesis) or pathogenic (ie, arteriosclerosis) vascular
remodeling, VSMCs with a noncontractile or synthetic phenotype generate intimal vascular lesions.6 –9 These VSMCs,
termed dedifferentiated cells, have reduced expression of
proteins required for normal regulation of contractile function
(eg, smooth muscle–specific isoforms of myosin, actin, and
calponin) and have increased capacity to generate extracellular matrix proteins.1–5 Dedifferentiated cells do not regulate
contraction but instead control vascular construction.
The mechanisms regulating vascular smooth muscle differentiation and phenotypic modulation have not been fully
defined. Indeed, the relationship between differentiated and
dedifferentiated VSMCs is controversial. Although intimal
dedifferentiated cells may derive from contractile cells,5,10
they also may result from a progenitor cell that becomes
activated during vascular stress or injury.1,11 The widespread
destruction of medial VSMCs that occurs during vascular
remodeling6 –9 is consistent with a terminal role for medial,
contractile cells. During vascular remodeling, dedifferentiated VSMCs are capable of maturing into fully differentiated
contractile VSMCs,6 – 8,12 and this process can mimic the
maturation process that occurs normally during vascular
development.5,13–15
Previous studies have demonstrated that reactive oxygen
species (ROS) play an important permissive role in a number
of responses involved in vascular remodeling, including
proliferation, migration, and hypertrophy.16 –18 The aims of
the present study were to determine whether ROS have a
modulatory role in regulating VSMC differentiation in vitro
using cultured cells and to analyze the underlying signal
transduction processes responsible for the modulation.
Original received October 13, 2000; revision received May 1, 2001; accepted May 25, 2001.
From the Heart and Lung Institute (B.S., S.M., H.G., S.F., M.A.C., P.J.G.-C., N.A.F.) and the Department of Pediatrics, Ohio State University (K.R.C.),
Columbus, Ohio.
*Both authors contributed equally to this work.
Correspondence to N.A. Flavahan, PhD, Heart and Lung Institute, Room 110E, 473 W 12th Ave, Columbus OH 43210. E-mail [email protected]
© 2001 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
39
40
Circulation Research
July 6, 2001
Materials and Methods
Cell Culture
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Human aortic VSMCs (Clonetics Human Cell Systems, BioWhittaker, Inc, Walkersville, Md) were grown in Clonetics SMGM and
studied from passage 7 through 11. For most experiments, cells were
plated at 50 cells/mm2, so that by the next day they had attained
⬇30% confluence. At that point, the cells were either harvested as
proliferating cells or made quiescent by exchanging the growth
media for quiescent media (50:50 mixture of DMEM and Ham’s F12
media with glutamine [200 ␮g/mL], penicillin/streptomycin [100
U/mL each], and ITS). When VSMCs are placed in culture, the
expanding cell population represents immature, dedifferentiated
cells, and this quiescent media is often used to induce a more
differentiated phenotype.19 –22 The influence of agents on VSMC
phenotype was assessed after 3 days in the quiescent media, when
the VSMCs had become quiescent. Preliminary experiments revealed that the optimal time for analyzing phenotypic modulation
was after an additional 3-day treatment. Therefore, most experiments
were performed after a 6-day quiescent period, with the first 3 days
in quiescent media and the last 3 days in quiescent media with the
test agent.
Western Blot Analysis of VSMC
Phenotypic Modulation
Expression of VSMC differentiation proteins and ␤-actin was
assessed by Western blot analysis of VSMC lysates, using mouse
monoclonal antibodies that recognize the human proteins. Full
details of the procedure are contained in an online data supplement
available at http://www.circresaha.org.
p38 Mitogen-Activated Protein Kinase Assays
Aortic cells were rinsed with ice-cold PBS. The cells were lysed in
buffer (New England BioLabs) containing antiproteases (as above),
incubated for 5 minutes on ice, then scraped, sonicated on ice (4
times for 5 seconds each), and cleared by centrifugation (14 000g)
for 10 minutes at 4°C. Supernatants were used to assess p38
mitogen-activated protein kinase (MAPK) enzyme activity using the
immunoprecipitation/ATF-2 phosphorylation assay kit from New
England BioLabs, according to the manufacturer’s instructions.
Assessment of ROS Activity
Aortic cells were treated with the H2O2 -sensitive probe, 5-(and-6)chloromethyl-2⬘, 7⬘-dichlorodihydrofluorescein diacetate (DCF, Molecular Probes), 5 ␮g/mL, for 30 minutes at 37°C in Krebs-Ringer
bicarbonate solution (in mmol/L: 118.3 NaCl, 4.7 KCl, 1.2 MgSO4,
1.2 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose). When
analyzing the effect of ROS inhibitors, the cells were incubated with
the agents before DCF: 4 hours for catalase (to enable intracellular
accumulation of the enzyme,17 2 hours for MnTMPyP (SOD), or 2.5
hours for N-acetylcysteine (NAC). After DCF incubation, in order to
reduce stress-induced oxidant activation, the attached cells were
cooled and harvested by trypsinization at 4°C. They were then
collected by centrifugation (4°C, 500g), washed once in cold
Krebs-Ringer solution, and analyzed by flow cytometry (FACSCalibur, Becton Dickinson).
Taqman/Realtime PCR
For quantitative measurement of mRNA, we used Realtime PCR
with 18S ribosomal RNA as an internal control. For further detail,
see the online data supplement.
Cell Cycle Analysis
Cells (5 ⫻ 10 ) were trypsinized, pelleted, and resuspended in 0.5
mL cold PBS. While vortexing (at setting 4), 4.5 mL of cold ethanol
was slowly added to the cells. Cell samples were stored at ⫺20°C for
ⱖ30 minutes before proceeding to DNA staining. The cells were
pelleted by centrifugation (200g), washed twice with PBS, and
resuspended in 1 mL PBS containing 50 ␮g/mL RNase A. After 20
minutes’ incubation at 37°C, propidium iodide was added to a final
6
Figure 1. Generation of ROS in VSMCs. Oxidant levels were
determined by FACS analysis of DCF fluorescence. Cells were
analyzed under proliferating conditions and after 3 days (Q3) or
6 days (Q6) of culture in quiescent media, as described in Methods. At each stage, the effect of acute administration of NAC
(10 mmol/L, 2.5 hours) was determined. Results are expressed
as absolute level of fluorescence (arbitrary units) and are presented as mean⫾SEM for n⫽3.
concentration of 5 ␮g/mL and incubated in darkness for ⱖ2 hours.
Cell fluorescence was then determined using the FACSCalibur
System. For each sample, 100 000 gated events were collected, and
results were analyzed using ModFit LT (Verity Software House).
Silicon Gel Preparation and Cell Contraction
Preparation of the silicon gel substrate and analysis of cell contraction were performed as previously described23 and are detailed fully
in the online data supplement.
Adenoviral Transduction
Cells were plated in a 6-well plate (50 cells/mm2) 1 day before
transduction. The next day, growth medium (SMGM) was switched
to quiescent medium, and cells were infected with replicationincompetent adenovirus at a multiplicity of infection of 250, optimized using Ad.␤-gal and Ad.GFP viruses. After 16 hours, cells
were recovered for 24 hours in SMGM followed by 72 hours in
quiescent medium in the absence or presence of pyridyl imidazole
compound (SB202190). Cells were then harvested for Western
analysis. Dr J. Han (The Scripps Research Institute, La Jolla, Calif)
kindly provided the adenoviruses Ad.MKK6 (encoding a constitutively active mutant of MKK6), Ad.P38DN (encoding a dominant
negative mutant of p38 MAPK), and Ad.GFP (encoding green
fluorescent protein).
Statistical Analysis
Statistical evaluation of the data was performed by Student’s t test
for either paired or unpaired observations. When ⬎2 means were
compared, analysis of variance was used. If a significant F value was
found, Scheffe’s test for multiple comparisons was used to identify
differences among groups. Values were considered to be statistically
different when P⬍0.05.
An expanded Materials and Methods section can be found in the
online data supplement available at http://www.circresaha.org.
Results
Modulation of ROS Activity in VSMCs
When human VSMCs were cultured in quiescent media,
oxidant activity decreased by ⬇35% (Figure 1). Treatment
with the antioxidant NAC (2 to 20 mmol/L) reduced ROS
activity in a concentration-dependent manner in quiescent
VSMCs (Figure 2), whereas a cell-permeable mimic of
superoxide dismutase (SOD; MnTMPyP, 25 ␮mol/L) increased oxidant activity (Figure 2). Because DCF is sensitive
Su et al
Redox Control of VSMC Differentiation
41
Exogenous H2O2, at 10, 30, and 100 ␮mol/L increased
DCF fluorescence to 166.8⫾15.7%, 200.6⫾15.8%, and
277.3⫾59.9% of control cells, respectively (n⫽4, P⬍0.05).
Redox Modulation of VSMC Phenotype
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Figure 2. Regulation of oxidant activity in VSMCs, determined
by flow cytometry analysis of DCF fluorescence. A, Effect of the
antioxidant NAC (2, 10, and 20 mmol/L), the superoxide dismutase mimic MnTMPyP (SOD, 25 ␮mol/L), or SOD (25 ␮mol/L)
⫹ NAC (2, 10, and 20 mmol/L)) on the oxidant activity of quiescent VSMCs. Results are expressed as percentage of the control response and are presented as mean⫾SEM for 5 experiments. Inset shows FACS output for a single representative
experiment. B, Effect of low concentrations of NAC (0.5, 1, 2
and 10 mmol/L) in the presence and absence of MnTMPyP
(SOD, 25 ␮mol/L) on the oxidant activity of VSMCs. Results are
expressed as percent of the control response and are presented
as mean⫾SEM for 4 experiments.
to H2O2, but insensitive to superoxide, this effect is consistent
with SOD-mediated dismutation of endogenous superoxide to
generate H2O2. Indeed, catalase (3000 U/mL), which inactivates H2O2, reduced the basal oxidant activity and the
increased activity after SOD (25 ␮mol/L) (Figure 3C). In a
similar manner, NAC (at 10 and 20 mmol/L) also reduced the
increased ROS activity in response to SOD (Figure 2).
However, low concentrations of NAC (0.5 to 2 mmol/L)
amplified the effect of SOD and further increased oxidant
activity (Figure 2). Superoxide dismutase mimics such as
MnTMPyP have higher activity in the presence of reductants
because they maintain Mn in the reduced state.24 Therefore,
in the presence of SOD, the effect of NAC appears to be
balanced between a direct inhibitory effect (observed more at
high concentrations) and an indirect action to amplify SODmediated dismutation of superoxide to H2O2 (observed only at
low concentrations).
Consistent with their characterization as dedifferentiated
VSMCs, proliferating cells expressed minimal levels of
VSMC differentiation marker proteins (Figure 3). As previously reported,19 –22 quiescence of VSMCs increased the
differentiation characteristics of the cells, with increased
expression of basic calponin, SM1 and SM2 myosins, and
␣-actin, but no change in the levels of ␤-actin (Figures 3A,
3B). When oxidant activity was decreased by NAC (2 to
10 mmol/L) or catalase (3000 U/mL), expression of the
differentiation proteins was reduced, whereas expression of
␤-actin was unchanged (Figure 3). Furthermore, when oxidant activity was increased by SOD (25 ␮mol/L) or by NAC
(2 mmol/L) plus SOD (25 ␮mol/L) (SODNAC2), there was a
marked increase in expression of the differentiation proteins,
but again the expression of ␤-actin remained unchanged
(Figure 3). The effect of SODNAC2 to increase expression of
the differentiation proteins was blocked by inhibiting oxidant
activity with NAC (10 or 20 mmol/L) or catalase (Figure 3).
In the presence of SOD (25 ␮mol/L), exogenous H2O2
(30 ␮mol/L) increased the expression of calponin (by
52.1⫾10.8%, n⫽4, P⬍0.05) and SM2 myosin (by
33.5⫾6.3%, n⫽4, P⬍0.05), but not ␤-actin (decreased
by 9.2⫾2.6%, n⫽4, P⬍0.05).
The changes occurring in the expression of differentiation
proteins was associated with altered levels of mRNA for the
differentiation markers, determined by Realtime reverse
transcriptase-PCR (Figure 4).
To determine the extent of the differentiation process, the
contractile activity of cultured vascular smooth muscle cells
was assessed using cells cultured on a silicone substrate. Cells
cultured in this manner and treated with SODNAC2 contracted in response to endothelin (30 nmol/L), shortening by
33.6⫾6.7% (mean⫾SEM, n⫽29). Untreated quiescent cells
also contracted in response to the agonist (shortening by
4.5⫾2.0%, n⫽14, P⬍0.05) but significantly less than did
SODNAC2 cells. In proliferating cells, endothelin (30
nmol/L) did not evoke contraction but did stimulate calcium
mobilization, assessed using fura-2–loaded cells (data not
shown). By comparing lysates of cultured cells and of medial
extracts of human aorta, expression of calponin and SM2
myosin in SODNAC2-treated cultured cells was estimated to
be 18.7⫾2.1% and 17.9⫾2.3% (n⫽15), respectively, of
expression in native smooth muscle cells.
Role of p38 MAPK in Redox Regulation of
VSMC Differentiation
Experiments were performed to assess the downstream signaling mechanisms involved in the ROS-dependent increase
in VSMC differentiation. The p38 and p42/44 MAPK signaling pathways in VSMCs are redox-sensitive, with p42/44
MAPK being highly sensitive to superoxide and p38 MAPK
having increased sensitivity to H2O2.25,26 In quiescent
VSMCs, the activity of p38 MAPK was decreased by NAC
(10 mmol/L) and increased by SODNAC2 (Figure 5A). In
42
Circulation Research
July 6, 2001
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Figure 3. Role of oxidants in regulating expression of differentiation proteins (basic calponin, SM1 and SM2 myosin, ␣-actin) and of
␤-actin in quiescent VSMCs. Expression was assessed after 6 days in quiescent media, with the last 3 days in the absence (control,
QC) or presence of the oxidant modulators. A, Oxidant activity was modulated using NAC (2 to 10 mmol/L) in the presence and
absence of MnTMPyP (SOD, 25 ␮mol/L) to regulate endogenous levels of oxidants. A representative Western blot is shown. B, Combined data from 6 to 10 experiments demonstrating effect of oxidant modulators on expression of calponin, SM2 myosin, and ␤-actin.
Data are expressed as percentage of the response in control quiescent cells (Q) and are presented as mean⫾SEM. Expression in proliferating cells (P) shown for comparison. C, Influence of catalase (CAT, 3000 U/mL) on the oxidant activity (determined by flow cytometry analysis of DCF fluorescence, upper panel) and expression of calponin (determined by Western blot analysis, lower panel) in quiescent VSMCs. The effect of catalase was assessed in the absence and presence of SODNAC2. Catalase was administered 4 hours
before SODNAC2 to optimize intracellular accumulation of the enzyme.17 Data are expressed as percentage of the response in control
quiescent cells and are presented as mean⫾SEM for 3 experiments.
contrast, neither NAC (10 mmol/L) nor SODNAC2 had any
effect on the activity of p42/44 MAPK (data not shown).
Inhibition of the p38 MAPK pathway by SB202190 (2 and 10
␮mol/L) decreased the expression of differentiation proteins
under normal quiescent conditions and the elevated levels
after SODNAC2 treatment (Figure 5B) but did not affect
expression of ␤-actin (data not shown). Inhibition of the
p42/44 MAPK pathway by 2⬘-amino-3⬘methoxyflavone
(PD98059, 3 to 30 ␮mol/L) did not affect expression of the
differentiation proteins (data not shown).
Transduction of VSMCs with a replication-incompetent
adenovirus encoding a constitutively active form of MKK6
(MKK6-CA), an activator of p38 MAPK, increased expression of calponin and SM2 myosin (Figure 6), an effect that
was inhibited by SB202190 (10 ␮mol/L) (Figure 6). An
adenovirus encoding a dominant-negative form of p38
MAPK (p38DN) decreased expression of the proteins,
whereas transduction with control adenovirus had no effect
(Figure 6). Expression of ␤-actin was not affected by
MKK6-CA or p38DN (Figure 6).
Su et al
Redox Control of VSMC Differentiation
43
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Figure 4. Role of oxidants in regulating expression of differentiation proteins in quiescent VSMCs. The effects of quiescence
and of SODNAC2 (in quiescent cells) on the transcript levels of
calponin, SM2 myosin, and GAPDH were determined by Taqman/Realtime PCR. Levels of transcripts, normalized with 18S
ribosomal RNA, are expressed relative to expression in proliferating cells. Data are presented as mean⫾SEM for 3 experiments. P indicates proliferating cells; Q, cells cultured in quiescent media for 6 days; SODNAC2, cells cultured in quiescent
media for 6 days and exposed to SODNAC2 for the last 3 days.
ROS activity of VSMCs was not influenced by modulation
of p38 MAPK activity. For example, inhibition of p38 MAPK
by SB202190 (2 ␮mol/L) did not affect ROS activity under
quiescent conditions (DCF fluorescence: control, 100%;
SB202190, 91.4⫾7.8%, n⫽5, P⫽NS) or after SODNAC2
(percent of control: SODNAC2, 248.7⫾54.7%; SODNAC2
⫹ SB202190, 241.0⫾64.4%, n⫽5, P⫽NS). Similarly, activation of p38 MAPK after transduction of VSMCs with
adenovirus encoding MKK6-CA did not alter ROS activity
(control, 100%; MKK6-CA, 109.3⫾5.26%, n⫽3, P⫽NS).
Vascular Smooth Muscle Growth Responses
Oxidants have been implicated in growth and remodeling
responses of VSMCs, including smooth muscle proliferation
and cellular hypertrophy.16,17,25,27,28 Therefore, experiments
were performed to determine whether the increased oxidant
activity caused by SODNAC2 treatment was associated with
smooth muscle growth. VSMCs became quiescent when
cultured in the quiescent media and did not re-enter the cell
cycle after SODNAC2 treatment (Figure 7A). VSMC hypertrophy refers to an increase in VSMC size and occurs during
vascular development, hypertensive remodeling, or in cultured cells after exposure to hypertrophic stimuli.29 –31 The
size of VSMCs, assessed by forward-angle light scatter31 did
not increase after SODNAC2 treatment (Figure 7B).
Discussion
In the present study, proliferating human VSMCs displayed
the characteristics of immature or dedifferentiated VSMCs.
Figure 5. Role of p38 MAPK signaling pathway in VSMC differentiation. A, Activity of p38 MAPK was assessed by immunoprecipitation assay after 30 minutes incubation with NAC
(10 mmol/L) or SODNAC2, as described in Methods. In quiescent VSMCs, p38 MAPK activity was increased by SODNAC2
and decreased by NAC (10 mmol/L). Data are expressed as a
percentage of activity in the time-control (Control) and are presented as mean⫾SEM for 4 experiments. B, Effect of the p38
MAPK inhibitor, SB202190 (2 and 10 ␮mol/L [SB2 and SB10,
respectively]), on expression of the differentiation proteins calponin and SM2 myosin occurring under basal conditions or after
elevation by SODNAC2. Data are expressed as a percentage of
the expression under control conditions and are presented as
mean⫾SEM for 3 experiments. C indicates cells cultured in quiescent media for 6 days; SODNAC2, cells cultured in quiescent
media for 6 days and exposed to SODNAC2 for the last 3 days.
Where indicated, SB202190 was also present in the media for
the last 3 days and was administered to the cells 30 minutes
before SODNAC2. SB202190 (2 and 10 ␮mol/L) dramatically
reduced expression of the differentiation proteins. Inset shows a
representative Western blot.
They had minimal expression of differentiation marker proteins and failed to contract to endothelin when cultured on a
silicone substrate. Previous reports have demonstrated that
proliferating cultured VSMCs can be converted to a more
mature phenotype by placing the cells in quiescent me-
44
Circulation Research
July 6, 2001
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Figure 6. Role of the p38 MAPK signaling pathway in VSMC
differentiation. Effect of adenoviral transduction with replicationincompetent viruses encoding constitutively active MKK6, an
upstream activator of p38 MAPK, or a dominant-negative form
of p38 MAPK (p38DN), on the expression of the differentiation
proteins SM2 myosin and calponin and of nonmuscle ␤-actin.
Expression of the proteins is also presented for nontransduced
cells (⫺) and for cells that were transduced with control viral
vectors (Control, replication-incompetent viruses encoding
Ad.GFP or containing no insert). A representative Western blot
is presented; similar results were obtained in 2 other experiments. Expression of calponin and SM2 myosin was increased
by adenovirus for MKK6 and decreased by adenovirus for
p38DN, whereas the expression of ␤-actin was not modulated.
In each case, the expression of calponin and SM2 myosin was
suppressed by SB202190 (10 ␮mol/L), the inhibitor of p38
MAPK.
dia.10,19 –22,32 This was demonstrated in the present study by a
selective increase in the expression of the differentiation
proteins ␣-actin, SM1 myosin, SM2 myosin, and calponin,
with no change in expression of ␤-actin. Although ROS are
thought to play a key role in the reparative functions of
dedifferentiated cells,16 –18,33 VSMC quiescence was associated with only a small decrease in oxidant activity. When
oxidant activity was reduced in these quiescent VSMCs,
either by NAC or by catalase, the expression of the differentiation proteins decreased, whereas expression of nonmuscle
␤-actin was unaffected. These results indicate that increased
expression of differentiation proteins after quiescence of
VSMCs requires oxidant activity. Indeed, when oxidant
activity was further increased by SOD or SODNAC2 there
was a further selective increase in the expression of the
differentiation proteins, with no change in the expression of
␤-actin. Again, this response was inhibited by catalase or by
higher concentrations of NAC. Therefore, oxidant activity
induced a more mature phenotype in VSMCs, which culminated in the resumption of contractile activity to endothelin-1.
The oxidant species responsible for increasing smooth
muscle differentiation is likely to be H2O2. This is supported
by the following observations: (1) Expression of the differentiation proteins closely mimicked the fluorescent activity of
the H2O2-sensitive probe DCF; (2) SOD (or SODNAC2),
which catalyzes the dismutation of superoxide to H2O2,
increased H2O2 levels and increased expression of the differentiation proteins, but did not affect expression of ␤-actin;
(3) catalase, which inactivates H2O2, inhibited expression of
differentiation proteins occurring under basal conditions or
after SODNAC2 treatment, but did not affect expression of
␤-actin; and (4) exogenous H2O2 increased expression of the
differentiation markers. Therefore, basal production of H2O2
Figure 7. Effect of SODNAC2 on growth responses of VSMCs.
A, Cell cycle analysis. Quiescence of VSMCs was associated
with a decrease in S phase and a corresponding increase in
cells in G0/G1 phase. Culture of cells in the presence of SODNAC2 did not affect cell cycle distribution. P indicates proliferating cells; Q, cells cultured in quiescent media for 6 days; SODNAC2, cells cultured in quiescent media for 6 days and exposed
to SODNAC2 for the last 3 days. B, Cell size analysis. Cell size
was assessed by flow cytometry analysis of forward-angle light
scatter (FALS).31 FALS was assessed in quiescent cells that had
been cultured in the absence (Control, solid line) and presence
(dashed line) of SODNAC2. SODNAC2 did not affect VSMC size.
is important in the expression of a more differentiated
phenotype after quiescence of VSMCs, and increased activity
of endogenous H2O2 is associated with a further increase in
the differentiated characteristics of the cells.
MAPK signaling pathways previously have been demonstrated to be oxidant-sensitive in VSMCs, with preferential
activation of p38 MAPK by H2O2.25,26 Indeed, in human aortic
VSMCs, the activity of the p38 MAPK, but not the p42/44
MAPK pathway, was decreased by NAC and increased by
SODNAC2. Inhibition of the p38 MAPK signaling pathway
by SB202190 decreased expression of the differentiation
markers occurring under baseline conditions or in response to
SODNAC2. This suggests that basal production of oxidants
and subsequent activation of the p38 MAPK signaling pathway mediate the expression of differentiated characteristics in
quiescent VSMCs. Further increases in oxidant activity (by
SODNAC2) can further increase oxidant-mediated activation
of p38 MAPK and the differentiated characteristics of
VSMCs. Consistent with this proposal, expression of a
dominant-negative mutant of p38 MAPK decreased expression of differentiation proteins, whereas activation of the p38
MAPK pathway using the constitutively active mutant of
MKK6, an upstream stimulus for p38 MAPK, increased the
expression of differentiation markers. Neither inhibition (with
SB202190) nor activation (with MKK6-CA) of p38 MAPK
Su et al
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
altered oxidant activity in VSMCs. Therefore, these results
indicate that activation of p38 MAPK, in either an oxidantdependent or oxidant-independent fashion, increases the differentiation characteristics of VSMCs.
In VSMCs, CArG [CC(A/T)6GG] or CArG-like motifs,
located in the promoter regions of smooth muscle differentiation proteins, are thought to play a key role in regulating
these genes during differentiation.5,15,34,35 A key step in
transcriptional activation is the formation of a multiprotein
complex containing serum response factor (SRF).35 Indeed,
the CArG-binding activity of SRF is higher in differentiated
compared with dedifferentiated cells, despite similar expression of SRF in the 2 cell types.34 Because p38 MAPK can
increase SRF-mediated transcriptional activation,36,37 this
may account for the ROS/p38 MAPK– dependent increase in
smooth muscle differentiation observed in the present study.
Although oxidants and oxidant-mediated activation of p38
MAPK played an important role in determining VSMC
phenotype, the influence of oxidants was not evident in
proliferating cells. In those cells, oxidant activity was high
relative to that in quiescent cells, but there was no stimulation
of cellular differentiation. This may reflect the activity of
serum factors, such as platelet-derived growth factor (PDGF),
which are known to inhibit the expression of differentiation
proteins.38 – 40 PDGF also increases oxidant activity,17 suggesting that its inhibitory effect on differentiation can occur
despite elevated ROS levels. Indeed, the overall effect of
oxidants in VSMCs likely will be determined by the activity
of other signaling pathways. Therefore, although oxidants
and p38 MAPK can increase the differentiation characteristics of VSMCs, activation of other signaling pathways may
suppress or redirect these signaling pathways to alternate
VSMC responses. Indeed, ROS have been implicated in
regulating a diverse range of cellular responses, including
migration, proliferation, apoptosis, senescence, and
hypertrophy.16,18,27,28,33,41,42
VSMCs display 2 distinct growth responses: hyperplasia,
characterized by increased DNA/protein synthesis and cell
division, and hypertrophy, characterized by increased cell
size and protein content without DNA synthesis or
cell division. Increased oxidant activity is thought to play a
key role in the proliferative response of VSMCs16,17,25,33 and
in the hypertrophic response to angiotensin II.26,31,42,43 However, SODNAC2 did not influence VSMC size and did not
stimulate VSMC proliferation. Furthermore, unlike hypertrophy, which may result in large part from a generalized
increase in protein synthesis,44,45 the effects of SODNAC2 in
increasing expression of differentiation proteins was specific
and was associated with increased transcript levels for the
differentiation proteins. Therefore, the increased oxidant
activity associated with SODNAC2 was not associated either
with VSMC hypertrophy or hyperplasia. This observation
appears to contrast with the proposed role of cellular H2O2
and p38 MAPK signaling in the hypertrophic effects of
angiotensin II. Angiotensin II increased the size and protein
synthesis of quiescent VSMCs, with the latter effect being
reduced by antioxidants (NAC) or by inhibition of p38
MAPK signaling.26,31,42,43 This may reflect an interaction of
these signaling systems with distinct signaling pathways
Redox Control of VSMC Differentiation
45
activated by angiotensin II. Indeed, the p38 MAPK signaling
pathway was identified as only one of multiple interacting
components in the hypertrophic signal transduction
pathway.26
Conclusions
The results of the present study demonstrate that endogenous
ROS can increase VSMC maturation and differentiation
through a p38 MAPK– dependent pathway. Previous studies
have demonstrated independently that oxidant activity46,47 or
p38 MAPK48 –50 can induce differentiation of other cell types.
Indeed, oxidant activity has been proposed as a generalized
stimulus for cell differentiation during development.46,47
Therefore, this pathway may contribute to regulating smooth
muscle maturation during development and vascular
remodeling.
Acknowledgments
This work was supported by grants AR46126, HL67331, and
HL56091 from the National Institutes of Health (Dr Flavahan) and
by grants from the Scleroderma Research Foundation (Dr Flavahan)
and the American Heart Association, Ohio Valley Affiliate (Dr
Chotani). The authors thank Mark Kotur for technical assistance with
FACS analysis.
References
1. Frid MG, Dempsey EC, Durmowicz AG, Stenmark KR. Smooth muscle
cell heterogeneity in pulmonary and systemic vessels: importance in
vascular disease. Arterioscler Thromb Vasc Biol. 1997;17:1203–1209.
2. Bochaton-Piallat ML, Ropraz P, Gabbiani F, Gabbiani G. Phenotypic
heterogeneity of rat arterial smooth muscle cell clones: implications for
the development of experimental intimal thickening. Arterioscler Thromb
Vasc Biol. 1996;16:815– 820.
3. Gittenberger-de Groot AC, DeRuiter MC, Bergwerff M, Poelmann RE.
Smooth muscle cell origin and its relation to heterogeneity in development and disease. Arterioscler Thromb Vasc Biol. 1999;19:1589 –1594.
4. Majesky MW, Giachelli CM, Reidy MA, Schwartz SM. Rat carotid
neointimal smooth muscle cells reexpress a developmentally regulated
mRNA phenotype during repair of arterial injury. Circ Res. 1992;71:
759 –768.
5. Owens GK. Molecular control of vascular smooth muscle cell differentiation. Acta Physiol Scand. 1998;164:623– 635.
6. Wolf C, Cai WJ, Vosschulte R, Koltai S, Mousavipour D, Scholz D,
Afsah-Hedjri A, Schaper W, Schaper J. Vascular remodeling and altered
protein expression during growth of coronary collateral arteries. J Mol
Cell Cardiol. 1998;30:2291–2305.
7. Schaper W, Ito WD. Molecular mechanisms of coronary collateral vessel
growth. Circ Res. 1996;79:911–919.
8. Schaper W, Schaper J, eds. Collateral Circulation: Heart, Brain, Kidney,
Limbs. Boston, Mass: Kluwer Academic Publishers; 1993.
9. Moldovan NI, Qian Z, Chen Y, Chen C-L, Mouton P, Hruban RH,
Flavahan NA, Baldwin WM, Sanfilippo F, Goldschmidt-Clermont PJ.
Fas-mediated apoptosis and transplant vasculopathy. Angiogenesis. 1999;
2:245–254.
10. Li S, Sims S, Jiao Y, Chow LH, Pickering JG. Evidence from a novel
human cell clone that adult vascular smooth muscle cells can convert
reversibly between noncontractile and contractile phenotypes. Circ Res.
1999;85:338 –348.
11. Holifield B, Helgason T, Jemelka S, Taylor A, Navran S, Allen J, Seidel
C. Differentiated vascular myocytes: Are they involved in neointimal
formation? J Clin Invest. 1996;97:814 – 825.
12. Aikawa M, Sakomura Y, Ueda M, Kimura K, Manabe I, Ishiwata S,
Komiyama N, Yamaguchi H, Yazaki Y, Nagai R. Redifferentiation of
smooth muscle cells after coronary angioplasty determined via myosin
heavy chain expression. Circulation. 1997;96:82–90.
13. Hungerford JE, Little CD. Developmental biology of the cell: building a
multilayered vessel wall. J Vasc Res. 1999;36:2–27.
46
Circulation Research
July 6, 2001
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
14. Hungerford JE, Owens GK, Argraves WS, Little CD. Development of the
aortic vessel wall as defined by vascular smooth muscle and extracellular
matrix markers. Dev Biol. 1996;178:375–392.
15. Landerholm TE, Dong XR, Lu J, Belaguli NS, Schwartz RJ, Majesky
MW. A role for serum response factor in coronary smooth muscle differentiation from proepicardial cells. Development. 1999;126:2053–2062.
16. Griendling KK, Ushio-Fukai M. Redox control of vascular smooth
muscle proliferation. J Lab Clin Med. 1998;132:9 –15.
17. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for
generation of H2O2 for platelet-derived growth factor signal transduction.
Science. 1995;270:296 –299.
18. Doanes AM, Irani K, Goldschmidt-Clermont PJ, Finkel T. A requirement
for rac1 in the PDGF-stimulated migration of fibroblasts and vascular
smooth cells. Biochem Mol Biol Int. 1998;45:279 –287.
19. Turla MB, Thompson MM, Corjay MH, Owens GK. Mechanisms of
angiotensin II– and arginine vasopressin-induced increases in protein
synthesis and content in cultured rat aortic smooth muscle cells: evidence
for selective increases in smooth muscle isoactin expression. Circ Res.
1991;68:288 –299.
20. Duplaa C, Couffinhal T, Dufourcq P, Llanas B, Moreau C, Bonnet J. The
integrin very late antigen-4 is expressed in human smooth muscle cell:
involvement of alpha 4 and vascular cell adhesion molecule-1 during
smooth muscle cell differentiation. Circ Res. 1997;80:159 –169.
21. Lavie J, Dandre F, Louis H, Lamaziere JM, Bonnet J. Vascular cell
adhesion molecule-1 gene expression during human smooth muscle cell
differentiation is independent of NF-␬B activation. J Biol Chem. 1999;
274:2308 –2314.
22. Halayko AJ, Camoretti-Mercado B, Forsythe SM, Vieira JE, Mitchell
RW, Wylam ME, Hershenson MB, Solway J. Divergent differentiation
paths in airway smooth muscle culture: induction of functionally contractile myocytes. Am J Physiol. 1999;276:L197–L206.
23. Harris AK, Wild P, Stopak D. Silicone rubber substrata: a new wrinkle in
the study of cell locomotion. Science. 1980;208:177–179.
24. Patel M, Day BJ, Crapo JD, Fridovich I, McNamara JO. Requirement for
superoxide in excitotoxic cell death. Neuron. 1996;16:345–355.
25. Baas AS, Berk BC. Differential activation of mitogen-activated protein
kinases by H2O2 and O2⫺ in vascular smooth muscle cells. Circ Res.
1995;77:29 –36.
26. Ushio-Fukai M, Alexander RW, Akers M, Griendling KK. p38 Mitogenactivated protein kinase is a critical component of the redox-sensitive
signaling pathways activated by angiotensin II: role in vascular smooth
muscle cell hypertrophy. J Biol Chem. 1998;273:15022–15029.
27. Finkel T. Oxygen radicals and signaling. Curr Opin Cell Biol. 1998;10:
248 –253.
28. Griendling KK, Sorescu D, Ushio-Fukai M. NAD(P)H oxidase: role in
cardiovascular biology and disease. Circ Res. 2000;86:494 –501.
29. Miller BG, Overhage JM, Bohlen HG, Evan AP. Hypertrophy of arteriolar smooth muscle cells in the rat small intestine during maturation.
Microvasc Res. 1985;29:56 – 69.
30. Owens GK, Rabinovitch PS, Schwartz SM. Smooth muscle cell hypertrophy versus hyperplasia in hypertension. Proc Natl Acad Sci U S A.
1981;78:7759 –7763.
31. Braun-Dullaeus RC, Mann MJ, Ziegler A, von der Leyen HE, Dzau VJ.
A novel role for the cyclin-dependent kinase inhibitor p27(kip1) in
angiotensin II–stimulated vascular smooth muscle cell hypertrophy.
J Clin Invest. 1999;104:815– 823.
32. Christen T, Bochaton-Piallat ML, Neuville P, Rensen S, Redard M, van
Eys G, Gabbiani G. Cultured porcine coronary artery smooth muscle
cells: a new model with advanced differentiation. Circ Res. 1999;85:
99 –107.
33. Anand-Apte B, Zetter BR, Viswanathan A, Qiu RG, Chen J, Ruggieri R,
Symons M. Platelet-derived growth factor and fibronectin-stimulated
migration are differentially regulated by the Rac and extracellular signal–regulated kinase pathways. J Biol Chem. 1997;272:30688 –30692.
34. Sobue K, Hayashi K, Nishida W. Expressional regulation of smooth
muscle cell–specific genes in association with phenotypic modulation.
Mol Cell Biochem. 1999;190:105–118.
35. Mack CP, Thompson MM, Lawrenz-Smith S, Owens GK. Smooth
muscle ␣-actin CArG elements coordinate formation of a smooth muscle
cell-selective, serum response factor-containing activation complex. Circ
Res. 2000;86:221–232.
36. Heidenreich O, Neininger A, Schratt G, Zinck R, Cahill MA, Engel K,
Kotlyarov A, Kraft R, Kostka S, Gaestel M, Nordheim A. MAPKAP
kinase 2 phosphorylates serum response factor in vitro and in vivo. J Biol
Chem. 1999;274:14434 –14443.
37. Thuerauf DJ, Arnold ND, Zechner D, Hanford DS, DeMartin KM,
McDonough PM, Prywes R, Glembotski CC. p38 Mitogen-activated
protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element: a potential role for
the transcription factor Atf6. J Biol Chem. 1998;273:20636 –20643.
38. Blank RS, Owens GK. Platelet-derived growth factor regulates actin
isoform expression and growth state in cultured rat aortic smooth muscle
cells. J Cell Physiol. 1990;142:635– 642.
39. Owens GK, Loeb A, Gordon D, Thompson MM. Expression of smooth
muscle-specific alpha-isoactin in cultured vascular smooth muscle cells:
relationship between growth and cytodifferentiation. J Cell Biol. 1986;
102:343–352.
40. Holycross BJ, Blank RS, Thompson MM, Peach MJ, Owens GK. Platelet-derived growth factor-BB–induced suppression of smooth muscle cell
differentiation. Circ Res. 1992;71:1525–1532.
41. Tsai JC, Jain M, Hsieh CM, Lee WS, Yoshizumi M, Patterson C, Perrella
MA, Cooke C, Wang H, Haber E, Schlegel R, Lee ME. Induction of
apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular
smooth muscle cells. J Biol Chem. 1996;271:3667–3670.
42. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG,
Taylor WR, Griendling KK. Role of NADH/NADPH oxidase– derived
H2O2 in angiotensin II–induced vascular hypertrophy. Hypertension.
1998;32:488 – 495.
43. Puri PL, Avantaggiati ML, Burgio VL, Chirillo P, Collepardo D, Natoli
G, Balsano C, Levrero M. Reactive oxygen intermediates mediate angiotensin II–induced c-Jun: c-Fos heterodimer DNA binding activity and
proliferative hypertrophic responses in myogenic cells. J Biol Chem.
1995;270:22129 –22134.
44. Patton WF, Erdjument-Bromage H, Marks AR, Tempst P, Taubman MB.
Components of the protein synthesis and folding machinery are induced
in vascular smooth muscle cells by hypertrophic and hyperplastic agents:
identification by comparative protein phenotyping and microsequencing.
J Biol Chem. 1995;270:21404 –21410.
45. Hershey JC, Hautmann M, Thompson MM, Rothblum LI, Haystead TA,
Owens GK. Angiotensin II–induced hypertrophy of rat vascular smooth
muscle is associated with increased 18 S rRNA synthesis and phosphorylation of the rRNA transcription factor, upstream binding factor. J Biol
Chem. 1995;270:25096 –25101.
46. Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radic
Biol Med. 2000;28:463– 499.
47. Allen RG. Oxygen-reactive species and antioxidant responses during
development: the metabolic paradox of cellular differentiation. Proc Soc
Exp Biol Med. 1991;196:117–129.
48. Zetser A, Gredinger E, Bengal E. p38 Mitogen-activated protein kinase
pathway promotes skeletal muscle differentiation. J Biol Chem. 1999;
274:5193–5200.
49. Morooka T, Nishida E. Requirement of p38 mitogen-activated protein
kinase for neuronal dfferentiation in PC12 cells. J Biol Chem. 1998;273:
24285–24288.
50. Davidson SM, Morange M. Hsp25 and the p38 MAPK pathway are
involved in differentiation of cardiomyocytes. Dev Biol. 2000;218:
146 –160.
Redox Regulation of Vascular Smooth Muscle Cell Differentiation
B. Su, S. Mitra, H. Gregg, S. Flavahan, M. A. Chotani, K. R. Clark, P. J. Goldschmidt-Clermont
and N. A. Flavahan
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 2001;89:39-46; originally published online June 21, 2001;
doi: 10.1161/hh1301.093615
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2001 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/89/1/39
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2001/06/20/hh1301.093615.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/