(Received for publication, August 10, 1995; and in revised form, November 22, 1995)
From the
Pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) have been used as antioxidants to prevent apoptosis in lymphocytes, neurons, and vascular endothelial cells. We report here that PDTC and NAC induce apoptosis in rat and human smooth muscle cells. In rat aortic smooth muscle cells, PDTC induced cell shrinkage, chromatin condensation, and DNA strand breaks consistent with apoptosis. In addition, overexpression of Bcl-2 suppressed vascular smooth muscle cell death caused by PDTC and NAC. The viability of rat aortic smooth muscle cells decreased within 3 h of treatment with PDTC and was reduced to 30% at 12 h. The effect of PDTC and NAC on smooth muscle cells was not species specific because PDTC and NAC both caused dose-dependent reductions in viability in rat and human aortic smooth muscle cells. In contrast, neither PDTC nor NAC reduced viability in human aortic endothelial cells. The use of antioxidants to induce apoptosis in vascular smooth muscle cells may help prevent their proliferation in arteriosclerotic lesions.
Apoptosis or programmed cell death is characterized by cell
shrinkage, membrane blebbing, and chromatin condensation that culminate
in cell fragmentation(1) . Stimuli as diverse as hyperthermia,
growth factor withdrawal, chemotherapeutic agents, radiation, and
oxidative stress induce apoptosis in many cell
types(2, 3, 4, 5) , and several
cellular proteins have been identified that activate or suppress
it(6, 7, 8, 9, 10, 11, 12, 13) .
The B-cell leukemia/lymphoma-2 (Bcl-2) ()protein has been
shown to prevent apoptosis induced by diverse stimuli (14, 15, 16, 17) , perhaps by acting
as an antioxidant(14, 18) . This hypothesis is
consistent with observations that antioxidants such as
pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC)
prevent apoptosis in
lymphocytes(14, 19, 20, 21) ,
neurons(18, 22) , and vascular endothelial
cells(23) .
Proliferation of vascular smooth muscle cells is
one of the most important features of arteriosclerosis(24) .
Rao and Berk (25) have shown that hydrogen peroxide stimulates
proliferation of vascular smooth muscle cells but inhibits
proliferation of vascular endothelial cells. However, the effect of
antioxidants on smooth muscle cells has been unclear. PDTC and NAC are
two structurally different thio-containing agents. Although NAC at low
concentrations (<1 mM) has been reported to cause oxidative
stress(5) , both agents have been shown to function as
effective antioxidants and to prevent oxidant-induced apoptosis (14, 19, 20, 21) and activation of
the transcription factors NF-B and AP-1 (26, 27, 28) and vascular cell adhesion
molecule-1(29) . In this study, we tested the effects of PDTC
and NAC on vascular smooth muscle and endothelial cells and show that
both agents, in dose ranges at which they are used as antioxidants,
induced apoptosis in rat and human aortic smooth muscle cells but not
in human aortic endothelial cells. This induction of apoptosis in
smooth muscle cells occurred in a dose- and time-dependent manner.
Furthermore, overexpression of Bcl-2 blocked PDTC- and NAC-induced
apoptosis in rat aortic smooth muscle cells.
After 6 h of exposure to 150 µM PDTC, normal RASMC (Fig. 1A) underwent cell shrinkage characteristic of apoptosis (Fig. 1B); fluorescent staining of the DNA revealed chromatin condensation in PDTC-treated RASMC (Fig. 1D) but not in untreated RASMC (Fig. 1C). Another hallmark of apoptosis is DNA strand breaks caused by endonuclease, which can be detected in situ by nick end labeling tissue sections with dUTP-biotin by terminal deoxynucleotidyl transferase(34, 38) . In contrast with untreated cells (Fig. 1E), positive staining was visible in most of the nuclei in RASMC that had been treated with PDTC (Fig. 1F). Finally, electron microscopy revealed highly condensed chromatin localized to the inner side of an intact nuclear membrane in PDTC-treated RASMC. The state of the treated nucleus (Fig. 1H) was in sharp contrast with that of the normal (untreated) nucleus (Fig. 1G). In addition to being a thio-containing antioxidant, PDTC is a metal chelator. To exclude the possibility that the effect of PDTC depended solely on its ability to chelate metals, we treated RASMC with NAC. NAC is another thio-containing antioxidant that does not have the ability to chelate metals. RASMC treated with 10 mM NAC manifested morphologic changes identical to those observed in PDTC-treated RASMC (not shown), indicating that two different antioxidants induce apoptosis in vascular smooth muscle cells.
Figure 1:
Morphology and
DNA fragmentation after PDTC-induced apoptosis in RASMC. RASMC were not
treated (left) or treated (right) with 150 µM PDTC. A and B, light micrographs of growing
RASMC (100). After 6 h of treatment, cytoplasmic condensation
and cell shrinkage are visible in some RASMC (B). Arrow indicates a representative apoptotic cell. C and D, micrographs of fluorescent-stained RASMC DNA (600
).
The homogeneous, lightly stained nuclear chromatin in untreated cells (C) is in sharp contrast to the chromatin condensation
accompanying PDTC-induced apoptosis (D). Arrow indicates an apoptotic body. E and F, DNA breaks
stained in situ by the TdT-mediated dUTP-biotin nick end
labeling method (34) (200
). Arrow (F)
indicates a representative apoptotic nucleus. G and H, electron micrographs of RASMC (3000
). RASMC in 60-mm
Petri dishes were treated with PDTC for 6 h and processed as
described(33) . The untreated RASMC (G) has large
nucleoli, and the heterochromatin is scanty, whereas the PDTC-treated
RASMC (H) shows marked chromatin condensation within an intact
nuclear envelope. Arrow (H) marks dense aggregation
of chromatin in the periphery of the
nucleus.
We also used a modified MTT assay of cell viability (35, 39) to measure antioxidant-induced apoptosis. The viability of RASMC decreased within 3 h of treatment with PDTC (Fig. 2A) and was reduced to approximately 30% of base line at 12 h. PDTC also decreased the viability of RASMC in a dose-dependent manner (Fig. 2B). As little as 25 µM PDTC reduced rat aortic smooth muscle cell viability by 25%, whereas 150 µM PDTC reduced viability by 73%. This decrease in vascular smooth muscle cell survival was not specific to rats; PDTC (Fig. 2C) and NAC (Fig. 2D) both caused dose-dependent reductions in survival in HASMC at 24 h. In contrast, neither PDTC nor NAC reduced survival in HAEC (Fig. 2, C and D).
Figure 2: Viability of vascular cells after treatment with PDTC and NAC. Subconfluent, exponentially growing RASMC, HASMC, and HAEC in 24-well plates were incubated with PDTC or NAC for the indicated times. Cell viability was determined by a modified MTT assay(35) . A, time course of the effect of PDTC on rat aortic smooth muscle cell viability. B, dose response of the effect of PDTC on rat aortic smooth muscle cell viability. C, Differential effect of PDTC on the viability of HASMC and HAEC. D, differential effect of NAC on the viability of HASMC and HAEC. Values represent mean ± S.E. from four samples. A factorial analysis of variance was applied to the values, followed by Fisher's least significant difference test. Significance was accepted at p < 0.05. *, treated group different from control group. , HASMC group different from HAEC group.
The concentrations of NAC that induced apoptosis in vascular smooth muscle cells (Fig. 2D) have been shown to prevent apoptosis in lymphocytes, neurons, and endothelial cells(14, 19, 20, 21) . To confirm that an antioxidant could prevent apoptosis under our culture conditions, we performed experiments in PC-12 neuronal cells. Serum deprivation induced apoptosis in PC-12 cells (Fig. 3), as Greene (40) has also shown. PDTC inhibited this apoptosis in a dose-dependent manner (Fig. 3), and 100 µM PDTC completely prevented apoptosis in PC-12 cells. This observation suggests that the induction of apoptosis by antioxidants in RASMC and HASMC is cell type specific.
Figure 3:
Prevention by PDTC of apoptosis induced by
serum deprivation in PC-12 cells. PC-12 cells were plated at a density
of 1 10
cells/well in 24-well, collagen-coated
plates and cultured in medium containing 10% horse serum and 5% fetal
bovine serum. After 24 h, cells were extensively washed and placed in
serum-free medium. Cell viability was determined as described for Fig. 2. Black bar represents control cells maintained
for 36 h in medium containing serum. Shaded bars represent
cells maintained for 36 h in serum-free medium and treated with the
indicated concentrations (in µM) of PDTC. Values represent
mean ± S.E. from three samples. Comparisons were subjected to
ANOVA followed by Fisher's least significant difference test, and
significance was accepted at p < 0.05. *, significant
decrease in survival in cells depleted of serum versus those
replete with serum.
To determine whether Bcl-2 inhibited antioxidant-induced apoptosis in vascular smooth muscle cells, we transfected into fetal RASMC expression plasmids that did or did not contain the human Bcl-2 coding region. Several stably transfected clones were isolated, and Bcl-2 expression was confirmed by Western blotting with an antibody against human Bcl-2 (Fig. 4A). As in adult RASMC (Fig. 2A), PDTC (Fig. 4B) and NAC (not shown) both induced dose-dependent apoptosis in fetal RASMC. Cells that overexpressed Bcl-2, however, were resistant to apoptosis induced by PDTC (Fig. 4B) and NAC (data not shown).
Figure 4:
Overexpression of Bcl-2 rescues RASMC from
PDTC-induced apoptosis. Protein overexpression was obtained by stably
transfecting fetal RASMC (A7r5 cells, ATCC) with a Bcl-2 expression
plasmid (36) or control plasmid. Clones were selected in medium
containing G418 (Geneticin) (500 µg/ml). A, Bcl-2 protein
expression was assessed by Western blot analysis as described (50) . Protein was extracted from cells transfected with
plasmid pCj-SV2 (control) and pC
j-bcl-2 (Bcl-2; two
different cell lines), and 20 µg of protein was loaded in each
lane. A polyclonal antibody to human Bcl-2 (1:800 dilution, Pharmingen)
identified a 26-kDa protein in the Bcl-2 lanes but not in the control
lane. B, viability of control and Bcl-2 cell lines exposed to
PDTC. Because fetal RASMC are very sensitive to antioxidants, they were
treated with 25-75 µM PDTC for 24 h. Viability was
determined by the MTT assay. PDTC reduced the viability of control
RASMC (black bars) in a dose-dependent manner. Viability in
the two lines of RASMC that overexpressed Bcl-2 (hatched bars)
was significantly greater than that in the control line. *, p < 0.05, treated versus untreated control. , p < 0.05, Bcl-2 groups versus control.
Reactive oxygen
species (superoxide anion (O
),
hydroxyl radical (
OH), and hydrogen peroxide
(H
O
)) have been implicated in causing cell
damage and cell death(14, 18, 41) . Yet, we
have observed that PDTC and NAC, two structurally different
antioxidants that prevent apoptosis in other cell types in
vitro, induced apoptosis in human and rat aortic smooth muscle
cells ( Fig. 1and Fig. 2). The unique susceptibility to
antioxidants of vascular smooth muscle cells indicates that they
respond differently than other cell types to changes in the
reduction-oxidation state. Consistent with this view, Rao and Berk (25) have shown that H
O
increases
proliferation of vascular smooth muscle cells but inhibits
proliferation of vascular endothelial cells.
In the presence of iron
or copper, antioxidants such as ascorbic acid act as prooxidants via
the Fenton reaction(42, 43) . To rule out the
possibility that the antioxidant-induced apoptosis we observed was
caused simply by autooxidation and subsequent generation of free
radicals, we treated RASMC with PDTC in the presence or absence of 50
µM deferoxamine, D-penicillamine, or BPSA (this
concentration was chosen because it was sufficient to allow chelation
of 100 times the iron (deferoxamine) or copper (D-penicillamine and BPSA) actually present in the culture
medium). Neither deferoxamine nor D-penicillamine affected
PDTC-induced apoptosis, and BPSA prevented only 20% of
PDTC-induced apoptosis. Thus, the antioxidant-induced apoptosis we
observed in RASMC cannot be ascribed simply to autooxidation by the
thio compounds added during the experiment.
Although the dose-response curve for the PDTC-induced decrease in RASMC survival is rather linear (Fig. 2B), this pattern is not observed universally for antioxidants in other cell types. For example, McCord and co-workers (44, 45, 46) published a bell-shaped dose-response curve for the protective effect of superoxide dismutase against ischemia-reperfusion injury in rat and rabbit hearts. High doses of superoxide dismutase were less effective than low doses, and very high doses had a deleterious effect.
Bcl-2 has been shown to protect cell types of diverse lineage from apoptosis induced by many stimuli(14, 15, 16, 17) . Our finding that Bcl-2 suppresses antioxidant-induced cell death (Fig. 4B) supports our conclusion from morphological studies that vascular smooth muscle cell death caused by PDTC and NAC is apoptosis. Bcl-2 has been shown to prevent apoptosis in lymphocytes and neurons by regulating an antioxidant pathway(14, 18) . However, Bcl-2 has also been shown to function as a prooxidant(47) . Furthermore, recent reports indicate that Bcl-2 prevents apoptosis induced by hypoxia (48) or by staurosporine under very low oxygen conditions(49) . Thus, Bcl-2 may also prevent apoptosis via a mechanism unrelated to its effect on reactive oxygen species. Our observation that Bcl-2 overexpression rescues RASMC from apoptosis induced by PDTC (Fig. 4B) and NAC also suggests that Bcl-2 may prevent apoptosis in vascular smooth muscle cells through a pathway unrelated to its antioxidant activity.
Arteriosclerosis and its complications, heart attack and stroke, are the major causes of death in developed countries(24) . Since proliferation of vascular smooth muscle cells is one of the key features of arteriosclerosis (atherosclerosis, restenosis after balloon angioplasty or coronary bypass surgery, and transplant arteriosclerosis), our finding that antioxidants promote apoptosis in vascular smooth muscle cells, but not in vascular endothelial cells, may provide a new therapeutic strategy for the treatment of arteriosclerosis.