doi: 10.1161/hq0701.092144
© 2001 American Heart Association, Inc.
Vascular Biology |
Phenotypic Heterogeneity Influences Apoptotic Susceptibility to Retinoic Acid and cis-Platinum of Rat Arterial Smooth Muscle Cells In Vitro
Implications for the Evolution of Experimental Intimal Thickening
From the Institute of Anatomic Pathology, Tor Vergata University of Rome (A.O., A.F., D.C., L.G.S.), and Institute of Pharmacological Sciences, University of Milan (A.C.), Italy.
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Abstract |
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Abstract—Rat aortic smooth muscle cells (SMCs) cultured from intimal thickening 15 days after endothelial injury (IT-15), unlike those of normal media, show a monolayered, epithelioid phenotype and high levels of cellular retinol binding protein-1 (CRBP). Epithelioid clones obtained from the normal media suggest a "mosaicism" of arterial SMCs. Intimal cell homeostasis from the balance of proliferation and apoptosis is critical for the progression of vascular lesions. All-trans retinoic acid (tRA) reduced [3H]thymidine incorporation and G1
S phase progression of
IT-15 and epithelioid clone but not of normal media and IT 60 days
after injury (IT-60) SMCs. Hoechst staining, flow cytometry, and
ligation-mediated polymerase chain reaction showed an increased
susceptibility of IT-15 and epithelioid clone to tRA and
cis-diaminedichloroplatinum II
(CDDP)–induced apoptosis and cytotoxicity compared with normal
media and IT-60 cells. The latter retained an increased susceptibility
to tRA-induced apoptosis compared with normal media SMCs.
tRA-induced apoptosis associated with an increased ratio of bax
to bcl-2 by bax overexpression and cleavage of caspase-3. Anti-CRBP but
not anti-IgG antibody prevented tRA-induced apoptosis and
changes in related signaling molecules but not CDDP effects. Our
findings support the relevant role of phenotypic
heterogeneity in the determining proliferative as well
as apoptotic behavior of arterial SMCs.
Key Words: atheromatosis • restenosis • flow cytometry • all-trans retinoic acid • cis-platinum
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Introduction |
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Intimal thickening (IT) after endothelial denudation has been widely used as a model for the development of atheromatous plaque and restenosis and has furnished useful information on smooth muscle cell (SMC) susceptibility to microenvironmental stimuli.1 2 3 4 It is well established that IT depends on migration and replication of SMCs from the media.1 Only a small proportion of medial SMCs migrate and replicate after endothelial denudation.5 SMC populations from different layers of arterial wall appear to be heterogeneous in their morphological and proliferative features.6 IT cells cultured 15 days after injury proliferate more6 and express different receptors, in particular cellular retinol-binding protein-1 (CRBP-1).7 Moreover, a small proportion of clones from normal media have the same phenotypic features as the SMC whole population from IT 15 days after injury.8 These findings are compatible with the possibility that the intimal SMC population derives from a small number of distinct SMCs resident in the normal media.9 Apoptosis plays a role in IT and plaque evolution,10 11 and the size of population in atherosclerotic and restenotic lesions relies on the balance between cell growth and apoptosis.11 This is also supported by evidence that apoptosis in IT takes place mainly in SMCs close to the lumen, as described for mitotic changes.10 Nevertheless, mechanisms regulating SMC apoptosis are still unclear. We compared the behavior of SMCs cultured from IT 15 and 60 days after endothelial injury, when they are active or arrested in their replicative activity, respectively,12 with normal media spindle-shaped and epithelioid cells cloned from the normal media SMCs. Epithelioid and spindle-shaped SMCs treated with all-trans-retinoic acid (tRA) and cis-diaminedichloroplatinum II (CDDP) demonstrate a different proliferative response and susceptibility to cytotoxicity and apoptosis independent of the layer of origin.
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Methods |
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Cells and Culture Conditions
Sixty male Wistar rats were used. A group of rats were preanesthetized with Ethrane (Abbott) for 60 seconds, then anesthetized with Nembutal sodium (Abbott, 35 mg/kg body wt IP), and the endothelium of the thoracic aorta was removed by ballooning.6 IT from the thoracic aorta was separated from the underlying media 15 and 60 days after the injury (IT-15 and IT-60).6 SMCs were isolated by enzymatic digestion,6 plated at a density of 3x103 cells/cm2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FCS (Biological Industries), and grown to the fifth passage. Cloning of normal media SMCs was performed by limiting elution,8 and clones were expanded up to the third passage, with epithelioid and monolayered cells up to the fifth passage. Cells were photographed with an Olympus inverted microscope (Olympus Optical Co).
Drug Treatments
Confluent SMC populations were trypsinized, counted
with a hemocytometer, seeded at a density of
5x103 cells/cm2
in 60-mm dishes, and synchronized in DMEM plus 0.1% FCS for 24 hours.
tRA (Sigma Chemical Co) was dissolved in DMSO at
10-2 mol/L, diluted in DMEM plus 10% FCS,
and added at various concentrations for 24 or 48 hours or 4 days. In
preliminary experiments, equimolar concentrations of DMSO did not
influence proliferative and apoptotic response of SMC
populations (data not shown). Apoptosis was also induced by
CDDP (Sigma Chemical Co) at equimolar concentrations for 24 and 48
hours. To verify the role of CRBP-1 in the apoptotic process,
SMCs were cultured in DMEM containing 10% FCS and a rabbit polyclonal
anti–CRBP-1 antibody (1:1000, gift from Prof G. Gabbiani, Department
of Pathology, Geneva,
Switzerland)7 for 3 hours,
and then tRA or CDDP was added at a concentration of
2.5x10-6 mol/L for 24 or 48 hours. As
control, we used rabbit polyclonal anti-IgG (Chemicon, 1:1000) or
anti–CRBP-1 antibody alone in the presence of serum. All experiments
were repeated in duplicate with 3 different subpopulations and
clones.
Cell Proliferation and Viability
Confluent SMCs were trypsinized, seeded at a density
of 5x103
cells/cm2 in 60-mm dishes, synchronized in
DMEM plus 0.1% FCS for 24 hours, and cultured for 4 days in the
presence of 10% FCS. After treatments, SMCs were trypsinized and
counted, and the seeded cell ratio was calculated. Cells were also
plated in triplicate at a concentration of
2x103 cells/cm2
in 60-mm dishes and synchronized in DMEM supplemented with 0.1% FCS.
After 24 hours, fresh medium plus 10% FCS containing 0.1 mCi/mL
[3H]thymidine [5 Ci/(mmol/L) specific
activity, Amersham] was added for 22 hours;
[3H]thymidine incorporation was
determined6 and expressed as
the ratio of cpm/cell numberx1000.
Cell viability was evaluated by 0.4% trypan blue exclusion in parallel to counting after 48 hours of culture in the presence of serum or plus various concentrations of tRA and CDDP. Small aliquots of cell suspensions were used for flow cytometry (see below). Experiments were repeated in 2 independent experiments using 3 different subpopulations and clones.
Flow Cytometry and Cell Cycle
Analysis
Flow cytometry was performed to analyze cell
cycle distribution. After treatments, SMCs were harvested by
trypsinization and centrifuged for 5 minutes at 1000 rpm.
Pellets were resuspended in 0.5 mL hypotonic fluorochrome solution of
propidium iodide (PI) 50 µg/mL in 0.1% sodium citrate containing
0.1% Triton X-100. Samples were placed in the
dark for 30 minutes, and the PI nuclear fluorescence was
measured. PI fluorescence signal was recorded on the FL2
channel of a FACScan flow cytometer
(Becton-Dickinson) with a Lysis II program. The
number of cells in sub-G1 (DNA content
<2N), G0/G1, S,
and G2/M phases was expressed as the percentage
of total events (10 000 cells).
Immunofluorescence Staining
and DNA Chromatin Morphology
Cells growing on glass slides were fixed for 5
minutes in cold methanol, rinsed twice in PBS, and subsequently
incubated with the anti–
-actin monoclonal
antibody13 and Hoechst 33342
(5 µg/mL in PBS)14 for 30
minutes at room temperature. Tetramethyl rhodamine–labeled goat
anti-mouse IgG (Nordic) was used as second antibody. Cells were
photographed with a Polyvar fluorescent microscope and DNA
chromatin morphology under UV visualization. The percentage of cells
showing nuclear features of apoptosis by Hoechst staining was
evaluated in 
1000 nuclei for each population with a Quantimet 920
image analyzer (Cambridge Instruments) connected to a Polyvar
microscope (Reichert Jung) by a Hamamatsu HC3077
camera.15 Three different
subpopulations and clones were analyzed in triplicate.
Assigning a serial number to each slide ensured the objectivity of
measurements.
SDS–Polyacrylamide Gel Electrophoresis
and Western Blotting
SDS–polyacrylamide gel
electrophoresis16 was
performed on a 5% to 20% gradient gel stained with Coomassie
brilliant blue (R 250, Fluka). For Western
blotting,17 2 to 50 µg of
proteins were electrophoresed; transferred to nitrocellulose filters
(0.45 mm, Schleicher & Schuell); and
incubated with anti-vimentin (YLEM, 1:200), anti–-actin
(DAKO, 1:500), and a goat anti-mouse IgG
(1:105) or alternatively with a rabbit
anti–CRBP-1 (1:200), anti–total actin (gifted from Prof G. Gabbiani,
Department of Pathology, Geneva, Switzerland, 1:100) or
anti–caspase-3 (Pharmingen, 1:100), a goat anti–nuclear factor-
B
p65 (NFBp65, Santa Cruz, 1:200), or anti–bax protein (Santa Cruz,
1:200), followed by a goat anti-rabbit or donkey anti-goat IgG antibody
(Santa Cruz, 1:105). Enhanced
chemiluminescence was used for detection (Amersham). Quantification of
Omat-x Kodak films was performed as previously
reported.15 To consider
protein loading, the densitometric value of each protein was normalized
to that of total actin; -actin content and effects of tRA and CDDP
were normalized to density values of normal media SMCs. Western
blottings were repeated in triplicate.
DNA Isolation and Ligation-Mediated Polymerase
Chain Reaction
Cells were scraped, pelleted, frozen in liquid
nitrogen, and suspended in 0.5 mL of digestion buffer (70 mmol/L
NaCl, 10 mmol/L Tris-HCl [pH 8.0], 25 mmol/L EDTA, and 1%
SDS) with 0.2 mg/mL proteinase K (Sigma) overnight at 37°C, and DNA
was extracted, quantified, and checked as previously
reported.15
Ligation-mediated polymerase chain reaction (PCR) of genomic DNA (1
µg)18 allows us to
distinguish apoptosis from smearing of necrosis-induced DNA
degradation by standard genomic DNA gel electrophoresis. The
nucleosomal ladder in PCR products (15
µL)15 was quantified in
duplicate in 1.2% electrophoretic agarose gels stained with ethidium
bromide (1 µg/mL).
Statistical Analysis
Results were expressed as arithmetic mean±SEM of
single experiments. For statistical evaluation, the results were
analyzed by means of Student’s
t test, and differences were
considered statistically significant at a value of
P<0.05.
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Results |
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Morphological Findings
In preconfluent cultures, IT-15 cells appeared characteristically epithelioid, with a tendency to grow in small groups. When confluent, IT-15 cells and epithelioid cells cloned from normal media conserved their appearance and grew in a single layer (Figure
I, see online at http://atvb.ahajournals. org). Cell size
analysis by means of flow cytometry did not show significant
differences between the cell volume of epithelioid clone and IT-15
cells (data not shown). IT-60 and normal media SMCs at confluence were
spindle-shaped, exhibited the characteristic hill-and-valley appearance
(Figure I, see online at http://atvb.ahajournals.org), and appeared
somewhat larger than IT-15 and epithelioid clone cells (data not
shown).
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Cell Proliferation and Viability
As reported in
Figure 1
, after 24 and 48 hours in the presence of serum,
proliferation of IT-15 and epithelioid clone was similar but greater
than IT-60 and normal media SMCs
(P<0.01). After 4 days, the
counted/seeded cell ratio of IT-15 cells was still similar to
epithelioid clone cells and higher than normal media and IT-60 cells
(P<001; Figure IIA, see online
at http://atvb.ahajournals.org). The 48-hour treatment with tRA
resulted in a dose-dependent reduction of IT-15 and epithelioid clone
but not IT-60 and normal media SMC counted/seeded cell ratio
(P<0.01).
[3H]thymidine incorporation correlated
with the growth curves of SMC populations. After 24 hours (Figure II,
see online at http://atvb.ahajournals.org), IT-15 and epithelioid clone
[3H]thymidine incorporation was double
that of IT-60 and normal media SMCs
(P<0.01). tRA reduced
[3H]thymidine incorporation in IT-15 and
epithelioid clone cells in a dose-dependent manner compared with
controls (P<0.01) but not in
IT-60 and normal media SMCs.
At a concentration of
2.5x10-6 mol/L, CDDP but not tRA reduced
cell viability
(Figure 1), more in IT-15 and epithelioid clone than in IT-60
and normal media SMCs
(P<0.01). At
5x10-6 mol/L, tRA reduced cell viability
(P<0.05) slightly, but less
than observed with equimolar treatment with CDDP
(P<0.01;
Figure 1). Higher concentrations of CDDP greatly reduced
cell viability. After 4 days of treatment with
5x10-6 mol/L of tRA, IT-15 and
epithelioid clone counted/seeded cell ratio did not differ from that of
IT-60 and normal media SMCs (Figure IIA, see online at
http://atvb.ahajournals.org).
Cell Cycle Analysis
In the presence of serum, the number of normal media
and IT-60 cells in G0/G1
was increased compared with IT-15 and epithelioid clone cells (Figure
III, see online at http://atvb.ahajournals.org). The treatment with
2.5x10-6 mol/L of tRA inhibited IT-15 and
epithelioid clone G1S progression, as
reflected by the higher percentage of cells in
G0/G1 phase. These
effects were not observed in tRA-treated normal media and IT-60 cells.
Flow cytometry also confirmed the higher percentage of
apoptotic (subdiploid) cells in IT-15 and epithelioid clone
than in IT-60 and normal media SMCs.
Cell Phenotype–Dependent Sensitivity
to Apoptosis
To quantify apoptotic cells, we calculated the
percentage of condensed or fragmented nuclei in sparse adherent
cultures stained with Hoechst 33342
(Figure 2). After 24 hours in the presence of
2.5x10-6 mol/L tRA
(Figure 3A), the percentage was slightly increased in IT-15
and epithelioid clones
(P<0.02) but not in IT-60 and
normal media SMCs. The equimolar treatment with CDDP resulted in a more
marked apoptotic stimulus of IT-15 and epithelioid cells than
IT-60 and medial SMCs
(P<0.01). After 48 hours
(Figure 3B), the percentage increased further in IT-15 and
epithelioid clone but not in normal media SMCs. In IT-60 cells, the
percentage of tRA-induced apoptotic cells was increased
compared with control and normal media SMCs
(P<0.05). The addition of
anti–CRBP-1 antibody to IT-15 and epithelioid clone cultures 3 hours
before treatment prevented the apoptotic stimulus of tRA but
not of CDDP
(Figure 3). Equimolar rabbit anti-IgG did not modify
tRA-induced levels of apoptosis. CDDP induced high levels of
apoptosis in IT-15 and epithelioid cells and lower levels in
IT-60 and control media SMCs
(P<0.01). The addition of
anti–CRBP-1 antibody did not prevent CDDP-induced apoptosis.
In 48-hour CDDP cultures, chromatin condensation was frequently
associated with cell shrinkage
(Figure 2c).
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Quantification of ligation-mediated PCR products
(Figure 4) showed that the integrated optical density
(IOD) value of IT-15 and epithelioid clone cells treated for 48 hours
with 2.5x10-6 mol/L of tRA was greater
than that of IT-60 and normal media SMCs
(P<0.01). IT-60 showed a
slight but significant increase of IOD value compared with normal media
SMCs (P<0.05;
Figure 4).
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X174 DNA marker from Sigma (D-0672).
Immunostaining and Western
Blotting
Immunofluorescence for -actin
showed that adherent IT-15 and epithelioid clone cells were slightly
positive or almost negative
(Figure 2a). Densitometric scanning confirmed that IT-15 and
epithelioid clone cells cultured in the presence of serum contained
less -actin (21.3±5.5% and 21.0±4.1%) than normal media SMCs. No
difference was observed between IT-60 (95.0±7.1%) and normal media
SMCs. The treatment with 2.5x10-6 mol/L
of tRA for 48 hours induced an increase of -actin expression in
IT-15 (46.2±6%) and epithelioid clone (59.5±5%) cells but not in
IT-60 and normal media SMCs
(Figure 5). The equimolar treatment with CDDP decreased
-actin expression in all populations, but more in IT-15
(20.8±1.5%) and epithelioid clone (20.1±2.5%) than in normal media
(60.0±4%) and IT-60 (53.8±5%) cells. DMSO-treated cells were
similar to controls in -actin expression (data not shown). tRA
increased CRBP-1 expression in IT-15 cells (135.0±6.8%,
P<0.03) compared with
controls, as previously
reported.19
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To verify the relationship between SMC phenotype and
the expression of apoptosis-related proteins, we investigated
the expression of bcl-2, bax, and NFBp65 and the cleavage of
caspase-3. IT-15 and normal media SMCs in the presence of 0.1% FCS
showed a low and similar expression of bcl-2; in the presence of 10%
FCS, bcl-2 increased significantly in both populations. As reported in
Figure 5, after 48 hours the expression of bcl-2 in IT-15
cells was not modified in the presence of
2.5x10-6 mol/L tRA but decreased with an
equimolar treatment of CDDP (42.0±12.1%,
P<0.03). Densitometric
scanning also showed that bax expression increased in IT-15 cells
(129.0±6%) after treatment with
2.5x10-6 mol/L tRA, whereas NFBp65 was
unchanged
(Figure 5). This was associated with an increase of the
density value of caspase-3 cleavage products (185±20%,
P<0.01) compared with
controls. No changes were observed in tRA-treated normal media SMCs.
Immunoblotting also demonstrated that the addition of
anti–CRBP-1 antibody to IT-15 prevents tRA-induced increase of bax and
cleavage of caspase-3 (data not shown). After 48 hours of treatment
with 2.5x10-6 mol/L CDDP, IT-15 cells
showed a decrease of NFBp65 (28.3±4.1%) and bax protein
(69.0±5.9%) and a marked increase of cleavage products of
caspase-3 (274±36%, P<0.01).
A similar but smaller effect was observed in normal media SMCs (data
not shown).
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Discussion |
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The size of the cell population in atherosclerotic and restenotic lesions relies on the balance between growth and apoptosis.11 20 The mechanisms regulating SMC replication have been considered essential for the understanding of lesion development21 22 and extensively studied in culture21 23 and in vivo, primarily in the model of arterial injury.4 24 25 Apoptosis of vascular SMCs has been identified in disease states such as human and experimental atherosclerosis and restenosis.10 11 20 Apoptotic cell death contributes to the regulation of SMC number within the arterial thickening after injury.10 11 Apoptosis takes place mainly in SMCs close to the lumen, similar to that described for mitotic changes,10 suggesting that highly proliferating SMCs undergo apoptosis. Previous works have shown that SMCs from normal and pathological arteries may differ in their growth properties and phenotypic features.24 26 27 In vitro, intimal SMCs obtained 15 days after the injury grow in monolayer and maintain an epithelioid phenotype,4 6 whereas those cultured after 60 days are spindle-shaped like normal media SMCs.6 Intimal and normal media SMCs react differently to heparin and transforming growth factor-ß.28 The finding of epithelioid clones from normal media8 is compatible with the possibility that the IT population derives mainly from a limited subpopulation of medial SMCs.9 We document now that (1) IT-15 cells are more susceptible to tRA- and CDDP-induced cytotoxicity and apoptosis than IT-60 and normal media SMCs, (2) the apoptotic response of epithelioid cells cloned from normal media is similar to IT-15 cells, and (3) the addition of anti–CRBP-1 antibody to epithelioid cultures results in a protection against tRA- but not CDDP-induced apoptosis. Unlike proliferation, IT-60 cells still retained a slightly increased susceptibility to tRA-induced apoptosis. It is well established that IT cell density decreases between 15 and 60 days after endothelial injury.12 24 The increased apoptotic susceptibility of IT-60 cells may be in agreement with a progressive but incomplete decrease of intimal epithelioid cells by apoptosis that accounts for the decrease of IT cell volume.24 Our results strengthen the hypothesis that epithelium-derived SMCs are mainly involved in the response of the arterial wall to damage,29 in agreement with the reported enhanced H2O2-induced cytotoxicity of epithelioid SMCs.30 In vivo, CRBP-1–positive cells proliferate early and disappear through apoptosis at later stages of neointimal evolution.7 In vitro, intimal cells retain high levels of CRBP-1 expression.7 Retinoids and their receptors have acquired a great relevance in the pathobiology of arterial wall (for review see Neuville et al31 ); tRA modulates proliferation with mechanisms varying according to culture conditions19 and SMC phenotype.7 Retinoids decrease neointimal area and cellularity and induce a favorable arterial remodeling without affecting the tunica media.7 CRBP-1 upregulation is a direct transcriptional effect of tRA, mediated through the binding of retinoic acid receptor (RAR)-
to the CRBP-1
promoter.32 Blocking
of CRBP-1 may result in a downregulation of RAR- transcriptional
effect and/or blockade of tRA-induced
apoptosis.32 Indeed,
during wound healing, myofibroblasts express CRBP-1 until they
disappear through
apoptosis.33 At the
same time, CRBP-1 blockage did not interfere with tRA-induced changes
of cell cycle, suggesting that tRA affects proliferation and
apoptosis through different pathways. CDDP induced an earlier and more marked decrease of number of epithelioid cells than equimolar doses of tRA. This suggests that CDDP-induced apoptosis rapidly progresses to secondary necrosis, with rupture of cell membrane, similar to that reported in human type A arteriosclerosis.34 CDDP is a DNA-reactive reagent widely used for chemotherapeutic treatment of human malignancies, and its action is related to cytotoxic activity.35 CDDP-induced DNA damage blocks replication as well as gene transcription, resulting in a triggering of apoptosis.35 36
Regulation of apoptosis is influenced by the
nature of the apoptotic stimulus as well as microenvironmental
changes.37 The prevalence of
apoptosis in epithelioid populations may be related to changes
in the expression of apoptosis-signaling molecules. Our results
indicate the relevant role of bax protein in the tRA-induced
apoptosis of epithelioid cells. This is confirmed by the
expression in vivo of high levels of bax protein in IT cells 15 days
after injury but not in normal medial SMCs (Orlandi et al,
unpublished data). Bax is a member of the bcl-2 family, and when
overexpressed, it accelerates cytokine-induced
apoptosis.38 Thus,
the ratio of bax to bcl-2 increased in IT-15 and epithelioid clone
cells especially by the overexpression of bax. This mechanism is
similar to that reported in SMCs within intimal hyperplasia of human
radial arteries.39 We also
observed that tRA- and CDDP-induced apoptosis in IT-15 cells
was associated with cleavage of caspase-3. The relevant role of
caspase-3 in the apoptotic process of vascular SMCs was
recently reported,40 also
associated with bax
overexpression.41 Cleavage
of caspase-3 depends on cell type and mechanism of induction of
apoptosis.42
Moreover, SMCs exhibit a constitutive activity of NF-B that
participates in dysregulation of vascular SMCs in human atherosclerotic
lesions.43 In particular,
its p65 subunit has a central role in the regulation of
apoptosis.44 In the
presence of serum, IT-15 expressed more NFBp65 than normal media
SMCs, according to that previously
reported.45 Anti–CRBP-1
antibody prevented IT-15 apoptosis and reverted bax to levels
similar to control, whereas NFBp65 remained less expressed,
supporting a main transcriptional role.
Our findings provide new data concerning the properties of SMCs isolated from different layers of the arterial wall. We do not know whether there is a relationship between the distinct apoptotic behavior of intimal epithelioid and normal media SMC populations in vitro and their activities in vivo, but these differences may help in understanding the adaptation of SMCs to different stimuli and the evolution of IT. Antihypertensive drugs induce a regression of aortic hypertrophy by stimulating SMC apoptosis in spontaneously hypertensive but not in Wistar rats.46 It will be of interest to verify whether drugs or cytokines may influence the evolution of IT by a modulation of apoptosis of intimal epithelioid cells in vivo.
In conclusion, our findings indicate differences in the proliferative response associated with changes in the susceptibility to tRA- and CDDP-induced apoptosis between epithelioid and spindle-shaped cells independent of the layer of origin. Further investigation of mechanisms influencing epithelioid cell apoptosis may be useful in understanding the pathobiology of the arterial cell population.
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Acknowledgments |
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This research was supported by a grant from MURST, protocol 995157119. We thank Sabrina Cappelli, Alfredo Colantoni, Renzo Bernabei, Antonio Volpe, and Angela Ortenzi for their excellent technical work.
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Footnotes |
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Reprint requests to Prof Augusto Orlandi, Institute of Anatomic Pathology, Department of Biopathology, Tor Vergata University of Rome, Via della Ricerca Scientifica, 00133, Rome, Italy.
Received November 10, 2000; accepted March 9, 2001.
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Retinoic acid regulates arterial smooth muscle cell
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