1. No category

Cardiovascular Research 61 (2004) 152 – 158
www.elsevier.com/locate/cardiores
YC-1 prevents sodium nitroprusside-mediated apoptosis in vascular
smooth muscle cells
Shiow L. Pan a, Jih H. Guh b, Ya L. Chang a, Sheng C. Kuo c, Fang Y. Lee d, Che M. Teng a,*
a
Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section 1, 100 Taipei, Taiwan
b
School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan
c
Graduate Institute of Pharmaceutical Chemistry, China Medical College, Taichung, Taiwan
d
Yung-Shin Pharmaceutical Industry Co., Ltd., Taichung, Taiwan
Received 25 April 2003; received in revised form 27 August 2003; accepted 9 September 2003
Time for primary review 20 days
Abstract
Keywords: YC-1; Vascular smooth muscle cells; Apoptosis; Nitric oxide
1. Introduction
Apoptosis, programmed cell death, is an important
mechanism in the formation of vascular lesion [1]. In recent
years, apoptosis of vascular smooth muscle cells (VSMC)
has been implicated in both development and outcome of
atherosclerosis. The altered balance between apoptosis and
proliferation appears to promote disease development [2].
Identification of both the negative and positive modulators
of apoptosis may lead to novel therapeutic approaches in
treating vascular disease. NO inhibits key events that
promote atherogenesis, including alterations in endothelial
redox state, platelet and monocyte adhesion to the vessel
* Corresponding author. Tel./fax: +886-2-2322-1742.
E-mail address: [email protected] (C.M. Teng).
wall and migration and proliferation of VSMC [3,4]. The
small amount of NO has shown antiapoptotic effects via
cGMP (guanosine 3V:5V-cyclic monophosphate)-mediated
interruption of apoptotic signaling pathways and direct
inhibition of caspase activity [5,6]. Higher NO concentrations promote apoptosis, which has been studied in a variety
of cell types including macrophages, chondrocytes, fibroblasts, and smooth muscle cells [7– 10]. Under these circumstances, the proapoptotic effects of NO seem to be
independent of cGMP accumulation [10].
There are numerous lines of evidence suggesting that the
lipid kinase phosphatidylinositol (PI) 3-kinase plays an
important role on the regulation of cell death in a lot of
types of cells and it becomes clear that PI 3-kinase is also a
determinant of VSMC fate [11]. Recent studies provide
evidence that Akt/PKB (protein kinase B) is a critical
downstream effector of PI 3-kinase [12]. Furthermore, it
0008-6363/$ - see front matter D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2003.09.013
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Objective: Nitric oxide signaling pathways are of central importance in both the maintenance of vascular homeostasis and the progression
of vascular disease. Since smooth muscle cell apoptosis is associated with numerous vascular disorders, the authors investigated whether YC1, a soluble guanylyl cyclase (sGC) activator, regulates apoptosis in vascular smooth muscle cells (VSMC). Methods and results: Sodium
nitroprusside (SNP) (1 mM) induced cGMP (guanosine 3V:5V-cyclic monophosphate)-independent apoptosis in rat vascular smooth muscle
cells using MTT assay and TUNEL-reaction techniques. Furthermore, sodium nitroprusside induced apoptosis via Bcl-2 down-regulation,
cytochrome c release reaction, and caspase-3 activation by Western blotting analysis and enzymatic assay methods. YC-1 abolished these
apoptotic signaling cascades and prevented apoptosis through a cGMP-involved pathway, and phosphatidylinositol (PI) 3-kinase behaved a
downstream event in this pathway. Conclusions: These results suggest that YC-1 inhibits sodium nitroprusside-induced vascular smooth
muscle cells apoptosis via a cGMP- and phosphatidylinositol 3-kinase-involved inhibition on Bcl-2 down-regulation/cytochrome c release/
caspase-3 activation cascades. The ability of YC-1 to prevent smooth muscle cell apoptosis may play an important role in blocking lesion
formation at sites of vascular injury.
D 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
S.L. Pan et al. / Cardiovascular Research 61 (2004) 152–158
has been defined that VSMC fate is dependent on a cellsurvival signaling cascade regulated by PI 3-kinase and the
activation of Akt/PKB [11].
YC-1 (3-(5V-hydroxymethyl-2V-furyl)-1-benzyl-indazol)
was discovered in our laboratory as a novel NO-independent
type of soluble guanylyl cyclase (sGC) activator [13]. It is
now recognized that YC-1 may have beneficial effects on
cardiovascular function [14]. YC-1 inhibits cell proliferation
of VSMC [15], inhibits glucose transport of cardiomyocytes
[16], and attenuates the development of intimal hyperplasia
in animal models of balloon-catheter vascular injury [15]. In
the current study, we investigated the role of YC-1 in
VSMC apoptosis induced by high concentrations of sodium
nitroprusside (SNP) in vitro.
2. Methods
2.1. Cell culture
2.2. Cytotoxicity assay
The cytotoxicity assay was carried out using the mitochondrial reduction activity assay. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma,
St. Louis, MO) was dissolved in phosphate-buffered saline
(PBS) at a concentration of 5 mg/ml and filtered (Millipore, Bedford, MA). From this stock solution, 10 Al/100
Al of medium was added to each well, and plates was
gently shaken and incubated at 37 jC for 2 h. After the
incubation period, the cells were lysed with dimethylsulphoxide (DMSO) and quantified by the measurement of
OD 550 with an enzyme-linked immunosorbent assay
(ELISA) reader.
2.3. In situ labeling of apoptotic cells
In situ detection of apoptotic cells was carried out by
using a terminal deoxynucleotidyl transferase (TdT) dUTP
nick-end labeling (TUNEL) method with an apoptotic
detection kit (Promega, Madison, WI, USA) as described
previously [18] and photomicrographs were obtained with a
fluorescence microscope (Nikon).
2.4. Assay of cGMP contents
At confluence, monolayer cells were incubated with
indicated agents for 10 min. Then, cells were washed twice
with ice-cold PBS and lysed with 0.5 ml NaOH (0.1 M). A
0.5 ml HCl (0.1 M) was then added to neutralize the assay
solution. After the centrifugation (3000 g for 3 min), the
supernatant was used for the detection of cGMP content by
using a cGMP ELISA kit.
2.5. Determination of caspase-3 activity
The caspase-3 activity was assayed with the caspase-3
colorimetric assay kit (R&D Systems, Minneapolis, MN).
After the treatment of cells with indicated agents for 10 h,
cells were harvested and the cell pellet was re-suspended in
pre-cooled lysis buffer. After a 10-min incubation on ice,
cell homogenates were centrifuged at 10,000 g for 1 min
and supernatants were removed for the determination of
caspase-3 activity. Proteolytic reactions were performed in a
total volume of 100 Al reaction buffer containing 50 Al of
cytosolic extracts and 5 Al DEVD-pNA. The reaction
mixture was incubated at 37 jC for 1 to 2 h and the
formation of p-nitroaniline was measured at 405 nm by an
ELISA reader.
2.6. Preparation of cellular cytosol fraction
Cells were harvested and the cell pellet was re-suspended in 50 Al of extraction buffer (20 mM HEPES, pH
7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM
EGTA, 1 mM dithithretol, and 1 mM PMSF) and incubated for 3 min on ice. Cells were managed with 30
strokes and centrifuged at 15,000 g for 15 min at 4 jC.
The supernatant was obtained for the detection of cytochrome c.
2.7. Western blot analysis
After the exposure of cells to the indicated agents and
time courses, cells were washed twice with ice-cold PBS
and reaction was terminated by the addition of 100 Al icecold lysis buffer (10 mM Tris –HCl, pH 7.4, 150 mM
NaCl, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 Ag/ml aprotinin, 10 Ag/ml leupeptin, and
1% Triton X-100). For the detection of Akt and phosphorylated Akt, 1 mM Na3VO4, 1 mM NaF, 50 mM tetrasodium pyrophosphate, 10 nM okadaic acid, 0.25% sodium
deoxycholate were included in the lysis buffer. The cell
lysates (25 Ag/lane) were electrophoresized on 10– 15%
SDS-polyacrylamide gels and the Western blot analysis
was carried out as we previously described [18]. Detection
of signal was performed with an enhanced chemilumines-
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The investigation conforms with Guide for the Care
and Use of Laboratory Animals published by the US
National Institutes of Health (NIH Publication No. 85-23,
revised 1996). Wistar rats were euthanatized with intraperitoneal administration of pentobarbital following the guidelines for animal studies at our institution. VSMC were
prepared and cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% FBS, 100
units/ml penicillin and 100 Ag/ml streptomycin (Gibco,
Grand Island, NY) as previously described [17]. Cells from
passages 3 through 7 were used for all studies. The cells
were characterized as smooth muscle cells by morphology
and immunostaining with monoclonal antibody specific for
smooth muscle a-actin.
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S.L. Pan et al. / Cardiovascular Research 61 (2004) 152–158
AM) was added to the cells and incubated for the last 30 min
at 37 jC. Then, cells were harvested for the detection of
ROS accumulation using FACS analysis.
2.9. Statistical analysis
Data are presented as the mean F S.E.M. for the indicated
number of separate experiment. Statistical analysis of data
was performed with one-way analysis of variance (ANOVA)
followed by a t-test and P-values less than 0.05 were
considered significant.
3. Results
3.1. YC-1 prevents SNP-induced apoptosis in VSMCs
Fig. 1. Effects of SNP and YC-1 on VSMC cell survival. Cells were
pretreated with or without YC-1 (A, 30 AM; B, 10 and 30 AM) for 30 min.
Then vehicle, SNP (1 mM, A), SIN-1 (100 AM, B) or SNAP (100 AM, B)
was added for another 24 h. After the incubation period, the cell viability
was assayed using MTT assay method and TUNEL-techniques (as showed
in the photographs) as described in the Methods section. Data are expressed
as mean F S.E.M. of five determinations (each performed in triplicate).
cence detection kit (ECL; Amersham International, Little
Chalfont, UK).
2.8. Measurement of reactive oxygen species (ROS)
Cells were incubated in the absence or presence of SNP
and/or YC-1 for the indicated time courses. Thirty minutes
before the termination of incubation period, DCFH-DA (10
Table 1
Effects of sodium nitroprusside, YC-1, and ODQ on the regulation of cell
survival in rat aortic smooth muscle cells
Treatment
Cell survival (%)
Control
SNP
YC-1
SNP + YC-1
ODQ
ODQ + YC-1
ODQ + SNP + YC-1
100 F 0
49.3 F 4.4*
111.3 F 8.8
102.9 F 8.1+
99.5 F 5.7
96.1 F 4.2
69.6 F 4.0#
Data are expressed as mean F S.E.M. of five determinations.
SNP, sodium nitroprusside.
* P < 0.001 vs. control.
+
P < 0.001 vs. SNP.
#
P < 0.01 vs. SNP plus YC-1.
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We examined the effect of YC-1 on SNP-induced apoptosis in cultured VSMC. MTT assay method and TUNELreaction technique showed that a high concentration of SNP
(1 mM) induced profound cell apoptosis, but YC-1 (30 AM)
completely abolished these SNP-induced effects (Fig. 1A).
ODQ, an inhibitor of sGC, had no influence on SNPinduced apoptosis but significantly reversed YC-1-mediated
action (Table 1), suggesting that the activation of sGC is
involved in YC-1-mediated antiapoptotic activity other than
SNP-induced apoptotic reaction. Additionally, YC-1 is able
to reverse the apoptosis induced by other NO generating
systems, such as 3-morpholino-sydnonimine (SIN-1) and Snitroso-N-acetylpenicillamine (SNAP) (Fig. 1B).
Intracellular cGMP levels were also assayed in this study.
As shown in Fig. 2, YC-1 alone induced a marked increase
in cGMP synthesis (4.2 F 0.8 vs. baseline value of 2.3 F 0.3
fmol/well). Additionally, the combination of SNP and YC-1
synergistically evoked more than 60-fold increase of this
cyclic nucleotide formation. However, ODQ significantly
inhibited the effects of YC-1 alone and the combination
action of SNP and YC-1 (Fig. 2). Furthermore, the cellpermeable cGMP analogue dibutyl-cGMP (300 AM) effec-
S.L. Pan et al. / Cardiovascular Research 61 (2004) 152–158
155
Fig. 4. Effects of several agents on the expression of phosphorylated Akt.
After the treatment of the indicated agents for 15 min, cells were harvested
and cell lysates (25 Ag/lane) were prepared for the detection of Akt (internal
control) and phosphorylated Akt expression. The proteins were separated
and detected using Western blotting method.
3.2. YC-1 action involves the activation of PI 3-kinase
tively reversed SNP-induced apoptosis (52.6% and 21.4%
reduction of 100 AM and 1 mM SNP-induced effect,
respectively). Taken together, these data suggest that SNP
induces a cGMP-independent apoptosis, while YC-1 prevents the SNP action through a cGMP-involved signaling
pathway.
3.3. YC-1 prevents SNP-induced Bcl-2 down-regulation and
cytochrome c release reaction
Exposure of cells to SNP (1 mM) caused a profound
down-regulation of Bcl-2 expression and increased cytochrome c release into the cytosol. The SNP-induced effects
were completely prevented by YC-1. However, ODQ and
wortmannin significantly reversed YC-1-mediated effects
(Fig. 5). These data suggest that the prevention of SNP-
Fig. 3. Effects of several agents on VSMC cell survival. Cells were
pretreated with or without YC-1 (30 AM) in the absence or presence of the
indicated agent for 30 min, and vehicle or SNP (1 mM) was added for 24 h.
After the incubation period, the cell viability was assayed using the MTT
assay method as described in the Methods section. Data are expressed as
mean F S.E.M. of five determinations (each performed in triplicate).
Fig. 5. Effect of several agents on the expressions of Bcl-2 and cytosolic
cytochrome c. After the treatment of the indicated agents, cells were
harvested and prepared for the detection of Bcl-2 and cytosolic cytochrome
c expressions. The proteins were separated and detected using Western
blotting method. a-Tubulin was used as the internal standard.
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Fig. 2. Effects of SNP, YC-1, and ODQ on cGMP synthesis. Cells were
treated with the indicated agents for 10 min, and intracellular cGMP was
detected as described in the Methods section. Data are expressed as
mean F S.E.M. of six determinations. *P < 0.05 and **P < 0.001 vs.
control; #P < 0.05 vs. YC-1; + P < 0.01 vs. SNP plus YC-1.
As demonstrated in Fig. 3, wortmannin (a PI 3-kinase
inhibitor) but not PD98059 (a MEK specific inhibitor)
significantly reversed the YC-1 action indicating that the
activation of PI 3-kinase might be involved in YC-1mediated effects. In a further identification, the results
showed that YC-1 alone and in combination with SNP
induced a profound increase in phosphorylated Akt expression. These effects were significantly inhibited by ODQ and
wortmannin (Fig. 4), suggesting that the YC-1-induced
activation of PI 3-kinase is a downstream event of cGMP
synthesis, and that the activation of PI 3-kinase might play a
central role in this YC-1-induced antiapoptotic effect.
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S.L. Pan et al. / Cardiovascular Research 61 (2004) 152–158
induced Bcl-2 down-regulation and cytochrome c release
reaction contributed to the cGMP- and PI 3-kinase-involved signaling pathways to YC-1 action. Furthermore,
we also examined the involvement of Bad, a pro-apoptotic
Bcl-2 family member, on SNP-mediated apoptotic pathway. However, SNP as well as YC-1 had little effect on
Bad protein expression suggesting that Bad did not play a
role on the apoptotic signaling in this study (data not
shown).
3.4. YC-1 inhibits SNP-induced caspase-3 activation
Apoptotic processes are stimulated by a variety of stimuli
converge on the activation of the caspase family. Among
these caspases, the activation of caspase-3 is the crucial
event that leads to the apoptosis in a variety of cells. We
measured the caspase-3 activity after the exposure of cells to
SNP (1 mM), and found that although SNP significantly
increased the caspase-3 activity in VSMC, YC-1 completely
inhibited this response (Fig. 6). Moreover, this YC-1-mediated inhibitory effect was significantly reversed by ODQ
and wortmannin (Fig. 6).
To determine whether SNP-induced cytotoxicity is
mediated by ROS, two experiments have been carried
Fig. 6. Effects of several agents on the caspase-3 activities and their
correlation with cytotoxic effects. After the treatment of cells with indicated
agents, cells were washed, trypsinized, and lysed for the determination of
caspase-3 activity as described in the Methods section. Data are expressed
as mean F S.E.M. of five determinations.
4. Discussion
The results of the current study strongly suggest that
VSMC apoptosis, an event that has been well studied in
atherosclerosis and neointimal formation postinjury [19], is
environmentally dependent. The actions of NO on apoptosis
are dependent on cell type, concentration, radical circumstances, and the redox state of cells [9,20]. Our data indicate
that the NO donor SNP induces VSMC apoptosis through a
cGMP-independent pathway, since ODQ did not reverse
and dibutyl-cGMP did not mimic the SNP-evoked effects.
These findings supported similar studies of VSMC [9]. In
the current study, YC-1 prevented an SNP-induced apoptotic effect in a cGMP-dependent manner, in that YC-1 in
combination with SNP synergistically increased cGMP
synthesis, dibutyl-cGMP efficiently mimicked YC-1-mediated effect, and ODQ significantly reversed the YC-1 action.
However, approximately 20% of ODQ-irresponsible action
remained in the YC-1-mediated effect. It has been suggested
that high concentrations of YC-1 could also inhibit phosphodiesterase activity [21], but further investigations are
needed to determine if the ODQ-resistant response to YC-1
action results from its regulation of phosphodiesterase
activity.
The mechanisms suggested for NO-induced cytotoxicity
include inactivation of the mitochondrial respiratory chain,
DNA damage, and Bcl-2 down-regulation/Bax up-regulation [22,23]. The balance between prosurvival and proapoptotic members of Bcl-2 family can regulate significant
events in apoptosis, such as cytochrome c release into the
cytosol and the activation of caspase-9 and downstream
caspases (especially caspase-3). These events account for
most of the apoptotic mechanisms in various cell types
[24]. In this study, SNP induced a significant downregulation of Bcl-2 proteins other than the influence on
Bax expression (data not shown), and stimulated the
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3.5. YC-1 inhibits SNP-induced ROS production
out during the revised presses. At first, we examined the
generation of ROS by means of the fluorescent probe
DCFH-DA and flow cytometric analysis. This cell-permeable dye DCFH-DA, once inside the cells, is cleaved by
endogenous esterase into DCFH. The intracellular nonfluorescent form of DCFH is oxidized, commonly by hydrogen peroxide, into the fluorescent form, DCF. The
fluorescence intensity was measured after the exposure
of cells to the indicated agents for 4 h. The data showed
that SNP (1 mM) induced a significant increase of
fluorescence intensity (3.16 F 0.05, n = 3, P < 0.001 compared with vehicle control of 1 F 0). YC-1 alone (30 AM)
had little effect on ROS production (0.94 F 0.24, n = 3)
but completely abolished SNP-mediated effect. These data
demonstrate that SNP-induced cytotoxic effect might involve the production of ROS and the ROS-reducing effect
of YC-1 might explain its inhibition on SNP-induced
cytotoxicity.
S.L. Pan et al. / Cardiovascular Research 61 (2004) 152–158
Acknowledgements
This work was supported by a research grant of the
National Science Council of the Republic of China (NSC
91-2320-B-002-157).
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release reaction of cytochrome c into the cytosol and the
activation of caspase-3 activity. These data demonstrate
the regulation of Bcl-2/cytochrome c/caspase-3 signaling
pathways in the SNP-mediated apoptotic mechanism in
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in VSMC (Table 1 and Fig. 3). Both ODQ and PD98059
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in Rat VSMCs while YC-1 completely abolished SNPmediated effect, indicating that SNP-induced cytotoxic effect
might involve the production of ROS and the ROS-reducing
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SNP-induced cytotoxicity.
We conclude that SNP induces VSMC apoptosis via a
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activation. YC-1 produces its antiapoptotic effect through
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