© 1999 American Heart Association, Inc.
Vascular Biology |
Retinoic Acid Regulates Arterial Smooth Muscle Cell Proliferation and Phenotypic Features In Vivo and In Vitro Through an RAR
-Dependent Signaling Pathway
From the Departments of Pathology (P.N., G.G.) and Department of Morphology (M.S.P.), University of Geneva-CMU, Switzerland; and Center for Molecular Medicine (Z.-q Y., A.G., G.K.H., A.S.), Karolinska Hospital, Karolinska Institute, Stockholm, Sweden.
Correspondence to Prof Giulio Gabbiani, University of Geneva-CMU, Department of Pathology, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. E-mail giulio.gabbiani{at}medecine.unige.ch
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Abstract |
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Abstract—We have recently shown that all-trans retinoic acid (tRA) modulates arterial smooth muscle cell (SMC) morphologic features and biochemical composition in vitro. It has been proposed that different SMC phenotypes coexist in arteries, which may be retrieved in culture: hence, a differential action of tRA on distinct SMC subsets is conceivable. We have examined the effect of tRA on SMC proliferation, migration, plasminogen activator activity, and
-smooth muscle actin expression in 2 phenotypically different rat
SMC populations, cultured respectively from the normal aortic media and
from the intimal thickening (IT) after endothelial
injury. tRA inhibited proliferation and increased migration and
tissue-type plasminogen activator activity in
both SMC populations, but decreased -smooth muscle actin only in SMC
cultured from the IT. The action of tRA is mediated by 2 families of
nuclear receptors, RAR and RXR, each containing 3 isoforms, , ß,
and 
. RAR and RAR- agonists, but not RXR agonists, inhibited SMC
proliferation in both cell populations and -smooth muscle actin
expression only in IT SMC. When administered
intraperitoneally to balloon-injured rats, tRA and
RAR- agonists reduced the intimal hyperplasia in the carotid artery.
Our results show that tRA and synthetic retinoids can affect the
proliferation, migration, and differentiation of SMC in vitro.
Furthermore, retinoids are able to reduce the IT induced by
endothelial injury in vivo.
Key Words: smooth muscle cell heterogeneity • smooth muscle cell motility • smooth muscle cell differentiation • intimal thickening
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Introduction |
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Smooth muscle cell (SMC) differentiation, migration, and proliferation are key factors in the development of atherosclerosis and restenosis after angioplasty.1 2 These phenomena involve an SMC phenotypic modulation characterized by modification of gene expression.3 It has been suggested that SMC subsets present in the normal media are particularly prone to undergoing phenotypic modulation, and the concept of SMC heterogeneity is gaining wider acceptance (for review see Reference 44 ). For instance, SMC populations cultured from the rat aortic media and from the intima 15 days after endothelial injury are characterized by a spindle-shaped and an epithelioid morphology, respectively (for discussion of this point see Reference 55 ). These 2 phenotypes are also obtained when SMC are cloned from the aortic media or the intima, albeit in different proportions.6
The regulation of SMC phenotype is thought to be exerted by cytokines, growth factors, and agents regulating differentiation.7 Within the last category, some agents are known to govern SMC differentiation during embryogenesis, and there is a growing body of evidence suggesting that the genetic programs used during embryogenesis may act also during arterial disease processes.8 9
Vitamin A plays a crucial role in the regulation of cell growth and
differentiation, and its active form, retinoic acid, is involved in
signal transduction pathways regulating embryonic
development.10 These effects are mediated by 2 families of
nuclear receptors that are ligand-dependent transcription factors, the
RARs and the RXRs, each with 3 isoforms, , ß, and
.11 Recently, we have observed that a protein involved
in the retinoid pathway, cellular retinol-binding protein-1 (CRBP-1),
is expressed selectively in the epithelioid SMC populations and
clones.12 13
The aim of the present study was to explore the possibility that
retinoids influence differentially the features of distinct SMC
phenotypes. We show that all-trans retinoic acid
modulates proliferation, migration, plasminogen
activator (PA) activity, and -smooth muscle (-SM)
actin expression in SMC. By using a series of RAR and RXR agonists, we
present evidence that these effects are mediated through an
RAR-dependent signaling pathway, the stimulation of which inhibits
the development of intimal thickening (IT) after arterial
injury in vivo.
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Methods |
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In Vivo Experimental Procedures
Adult male Wistar and Sprague-Dawley (body weight 300 to 400 g) rats were used for the experiments. Anesthesia was induced with enflurane (Ethrane, Abbott Laboratories) and then maintained with an intraperitoneal injection of pentobarbital sodium (Nembutal sodium, Abbott Laboratories). Angioplasty of rat carotid artery and thoracic aorta was achieved by removal of the endothelium using an inflated embolectomy catheter according to Baumgartner and Studer.14 Animals were sacrificed with an overdose of pentobarbital sodium 14 days after injury. The carotid arteries were excised, fixed in 4% buffered formaldehyde, and embedded in OCT. The thoracic aortas were excised for cell isolation. Procedures involving animals were approved by the Ethical Committee of Geneva Medical Faculty and the Swedish Animal Ethics Committee.
In Vivo Drug Treatments
All-trans retinoic acid (tRA, Sigma Chemical Co) and
CD336 (CIRD Galderma) were dissolved in 70% Intralipid (200
mg/mL; Pharmacia), 25% saline, and 5% ethanol, and injected
intraperitoneally at a dose of 0.5 mg/kg
immediately after rat carotid deendothelialization and
once a day thereafter for the following 14 days. The controls were
injected with the identical volume of 70% Intralipid, 25% saline, and
5% ethanol. Body weight was measured every day. Vessels were sectioned
and stained with hematoxylin and eosin. The media and IT area were
tracked and digitized by image analysis.
Cells and Culture Conditions
The aortic media from normal rats and the IT from injured rats
were enzymatically digested.5 Isolated SMCs were plated at
a density of 3x103
cells/cm2 in Dulbecco's modified Eagle's medium
(DMEM, HyQ, HyClone Europe) supplemented with 10% FCS (Seromed,
Biochrom KG). SMC populations were grown up to the 10th passage in the
same medium. Cells were used between the 5th and the 10th passages.
Measurement of DNA Synthesis
Incorporation of [3H]thymidine was
measured to determine the effect of tRA on DNA synthesis. Rat SMCs were
grown on 24-well plates in DMEM/F12 plus 10% FCS to 70% to 80%
confluence and made quiescent by starvation for 24 hours in DMEM/F12
plus 0.1% FCS. Quiescent cells were labeled with 1 µCi/well of
[3H]thymidine (Amersham) and stimulated with
10% FCS in the presence or absence of retinoids or vehicle (0.1%
DMSO) for the next 24 hours. The cells were then washed twice with PBS
and extracted with 5% trichloroacetic acid. The supernatant was
aspirated, and the cells were washed twice with 5% trichloroacetic
acid. Cells were lysed with 0.1 mol/L KOH. Radioactivity was counted in
a 2500TR scintillation counter (LKB Instruments).
In Vitro Drug Treatments
All-trans retinoic acid, Ro13-7410 (TTNPB, RAR
agonist), Ro44-4753 (RAR agonist), Ro47-5944 and Ro48-2250 (RXR
agonists), Ro25-7386 (RXR agonist), Ro61-8431 (RAR
antagonist), Ro41-5253 (RAR antagonist; F.
Hoffmann-LaRoche Ltd), Am80 and Am580 (RAR agonists; F.
Hoffmann-LaRoche Ltd and Sumitomo Pharmaceuticals Co, Ltd), BMS 753
(RAR agonist), and BMS 961 (RAR agonist; Bristol-Myers Squibb,
kindly provided by Prof. P. Chambon, IGBMC) were dissolved in DMSO at
10-2 mol/L, then diluted in DMEM and added to
the cells at 10-6 mol/L final concentration.
SMCs were plated at a density of 5x103
cells/cm2, and retinoids were added every 2 days
when medium was changed. Cells treated with the same concentrations of
DMSO were used as control. Cells were counted and harvested after 7
days.
RNA Extraction and Northern Blot Hybridization
Cultured cells were scraped from tissue culture dishes using a
rubber policeman in 0.8 mL of Tri reagent (Sigma) per dish. Total RNA
extraction and purification were performed according to the protocol of
the manufacturer. Ten micrograms of total RNA was denatured with
glyoxal, separated by electrophoresis on 1% agarose gel, and blotted
on hybond-N filters (Amersham Corp). After UV fixation, filters were
stained with 0.04% methylene blue in 0.5 mol/L sodium acetate to
verify correct loading and transfer. Prehybridization and hybridization
were performed for 3 and 16 hours, respectively, at 55°C in 5x SSC,
5x Denhardt solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone,
0.02% BSA), 0.01% SDS, and 400 µg/mL salmon sperm DNA. After
hybridization, filters were washed twice for 15 minutes at 55°C in
2x SSC, 0.1% SDS. They were then exposed to Kodak X-Omat SO-282 film
(Eastman Kodak) at -70°C for 20 hours. Hybridizations were performed
with a random priming-labeled GAPDH probe and an -SM actin probe
(prepared in our laboratory) corresponding to 120 bp of the 3'
untranslated region of the -SM actin gene.
Western Blotting
Cultured SMCs were trypsinized and mixed in buffer according to
Laemmli.15 The samples were immediately sonicated and
boiled for 3 minutes. The loading of the same amounts of protein was
determined according to Bradford.16 For Western blotting,
3 µg for -SM actin and 30 µg for CRBP-1 of total protein was
loaded on a 5% to 20% gradient gel and transferred to nitrocellulose
papers. -SM actin and CRBP-1 detection were performed essentially as
previously described.13 17
Cell Migration
For evaluation of cell migration, SMCs were plated at a
concentration of 5x103
cells/cm2 in DMEM in the presence of 10% FCS.
Subconfluent cultures were treated for 2 days with tRA and Am80 at
10-6 mol/L final concentration or with 10% FCS
plus DMSO as control, and then scratched with a silicone rubber to
obtain an 0.8-mm wide in vitro wound.6 18 Four fields were
randomly selected, and photomicrographs were taken.
5-Bromo-2'-deoxyuridine (BrdU, Fluka) and 5-fluoro-2'-deoxyuridine
(FldU, Fluka) were added at 25 and 2 µmol/L, respectively, for
24 hours. Then cells were fixed in methanol, and the extent of
incorporation was determined by using a monoclonal antibody for BrdU
(Dako). Nuclei were stained with propidium iodide (Sigma).
Photomicrographs of the same fields were taken after 24 hours, and the
proportion of migrating cells stained by BrdU antibody was evaluated by
image analysis using the KS400 system (Kontron Elektronik).
Zymographic and Reverse Zymographic Assays
Cells were seeded into 35-mm tissue culture dishes at
104 cells/dish in DMEM plus 10% FCS and grown to
subconfluence. Twenty-four hours after the last medium change, culture
dishes were washed twice with serum-free DMEM, and tRA was then added
for 15 hours at 10-6,
10-7, or 10-8 mol/L in
1.5 mL serum-free DMEM containing Trasylol (200 KIU/mL; Bayer). For
zymographic and reverse zymographic assays, culture media and cell
lysates were analyzed as previously described.19
For reverse zymographic assays, samples were preincubated with 0.5%
SDS and 0.5% ß-mercaptoethanol for 1 hour at 37°C to neutralize PA
activity. Twenty-microliter aliquots were subjected to SDS-PAGE and
zymographic analysis using a casein- and
plasminogen-containing substrate gel. Human urokinase (0.05
U/mL; Serono) was added to substrate gels for reverse zymographic
analysis. Zymograms and reverse zymograms were photographed
under dark-field illumination. In each experiment, cell numbers were
determined in a duplicate series of dishes incubated in parallel;
samples were then analyzed on the basis of cell number
equivalents.
Statistical Analysis
Data are given as mean±SEM. A Student's t test for
unpaired samples was used for statistical analysis. Significant
differences were accepted for P<0.05.
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Results |
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Effect of tRA Treatment on IT and Medial SMC
We examined the effect of tRA on SMCs cultured from normal rat aortic media and IT that are respectively spindle-shaped and epithelioid. The growth inhibitory or stimulatory activity of tRA was analyzed by DNA synthesis measurements. tRA, ranging from 0.125 to 2.0 µmol/L, induced a dose-dependent inhibition of serum-stimulated [3H]thymidine uptake in IT cells (5% to 40%) but did not affect medial SMCs at any dose tested (Figure 1A
). However, after a 7-day
treatment, SMC proliferation was inhibited by tRA to the same extent in
medial SMCs (62.50±3.51% of control values) and in IT SMCs
(62.75±1.25%) (Figure 1B). These data indicate that IT SMCs
are more sensitive to tRA treatment than medial cells at early times,
but that after 7 days, selectivity is lost.
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We also observed an effect of tRA on SMC migration. Medial and IT SMCs
were cultured until subconfluence, and were treated for 2 days with tRA
at 10-6 mol/L before wounding. After 24 hours,
numerous SMCs invaded the empty space in both populations. SMC
migration was increased by tRA treatment in both IT (209.88±11.31%)
and medial cells (202.78±6.53%) compared with control cultures
(Figure 2A). Despite the fact that, in
absolute terms, IT SMCs migrate more actively than medial
cells,6 no significant difference in the percentage level
of induction was observed between the 2 cell populations. To assess the
effect of tRA on cell proliferation in this experiment, we have
analyzed the proportion of migrating cells that had
incorporated BrdU. Results demonstrated that BrdU-incorporating cells
were present in the same proportion in IT (92.23±4.89%) and
medial cells (98.03±5.27%) treated by tRA compared with control
cultures, indicating that the effect of tRA is essentially exerted on
SMC migratory activity (Figure 2B).
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The effect of tRA on the proteolytic activity of IT and medial SMCs
caused by PA was investigated by zymographic and reverse zymographic
assays. Tissue-type PA (tPA) and urokinase PA (uPA) activities were
present in the culture supernatants of both SMC populations as
previously reported.20 After tRA treatment, tPA increased
and uPA decreased in a dose-dependent manner in both IT and medial SMCs
(Figure 3, top), whereas
plasminogen activator inhibitor-1
(PAI-1) was not significantly affected (Figure 3, bottom).
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We studied the effect of tRA on -SM actin expression by means of
Western blot analysis. In medial SMCs a 7-day tRA treatment did
not affect -SM actin expression (107.12±1.39%) whereas it induced
a significant decrease in IT SMC (63.66±0.46%) (Figures 4 and 5). This effect was not observed
at the mRNA level after a 7-day treatment with retinol or tRA, or
during a time course of 48 hours (data not shown).
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Effects of RAR and RXR agonists on IT and Medial SMCs
tRA effects are mediated by 2 families of nuclear receptors, the
RARs and the RXRs. To identify the receptors responsible for the action
of tRA on SMCs, we examined the effects of synthetic retinoid
derivatives, an RAR general agonist (Ro13-7410/TTNPB) and 2 RXR general
agonists (Ro47-5944 and Ro48-2250). These analogs induced growth
inhibition of both IT and medial SMCs, whereas -SM actin content was
reduced to the same level as tRA-treated cells only after Ro13-7410
treatment (data not shown). An RAR-selective antagonist
(Ro61-8431) that had no effect by itself on proliferation and -SM
actin expression inhibited those of the RAR agonist Ro13-7410 when
added simultaneously to SMCs.
Each receptor subfamily contains 3 members: RAR-, RAR-ß, and
RAR-; and RXR-, RXR-ß, and RXR-. Therefore, we used
different derivatives to examine whether one of these receptor isotypes
could account for the effect of tRA on SMC proliferation and -SM
actin expression. Am80 (Ro40-6055) is an RAR--selective agonist that
inhibited SMC proliferation in medial (54.22±1.16%) and IT
(54.16±1.06%) SMC populations (Figure 6), induced SMC migration in medial
(240±12.22%) and IT (172.33±7.38%) SMC populations (Figure 2A
) without affecting significantly BrdU incorporation in medial
(97.79±4.38%) and IT (99.78±3.30%) cells (Figure 2B), and
lowered -SM actin only in IT (62.43±2.08%) cells (Figures 4 and 5) as tRA and RAR agonists. The same results were obtained
with 2 other RAR- agonists, Am580 and BMS 753, whereas RAR-
agonists (Ro44-4753 and BMS 961) had no effect (data not shown).
Moreover, Ro41-5253, an RAR--specific antagonist,
abolished the effect of both RAR- agonists on -SM actin
protein expression (Figures 4 and 5) and to a lesser
extent on SMC proliferation (Figure 6). As control, we showed
that CRBP-1 induction by Am80 in medial SMCs was abolished by the
addition of the RAR- antagonist (Figure 4B).
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Retinoic Acid and RAR- Agonist Inhibit Intimal Thickening After
Balloon Injury in Rat Carotid Arteries
We examined the effect of retinoids on the
arterial wall in vivo using the classic
endothelial injury model. tRA and CD336 (both at 0.5
mg/kg body weight) were injected intraperitoneally
daily for 14 days subsequent to balloon catheter injury of the carotid
artery. In vehicle-treated control rats, extensive IT
(0.114±0.014 mm2 was observed in all
injured vessels 14 days after injury (Figure 7). In contrast, tRA-treated rats showed
a 76% (0.028±0.018 mm2) reduction in the
cross-sectional area of the carotid neointima (Figure 7). The media itself was not affected by tRA (0.061±0.004
mm2 versus 0.070±0.005
mm2 for the controls). As a consequence of the
reduction of the IT, the diameter of the lumen was increased by 33% in
the tRA-treated rats (0.677±0.0228 mm versus 0.508±0.0328
mm for the controls).
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The RAR- agonist, CD336, also reduced neointima
formation after balloon catheter injury (0.035±0.021
mm2) but the effects were less pronounced than
those observed using tRA (Figure 7). Inhibition of IT by tRA and
RAR- agonists was obtained also after balloon-induced lesion of the
rat aorta (data not shown).
There was no difference in body weight between animals treated with retinoids and vehicle, and no signs of toxicity were observed in the animals.
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Discussion |
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We have previously reported that CRBP-1, a protein involved in retinoid metabolism, is selectively expressed by a subpopulation of rat SMCs prone to migrate, proliferate, and give rise to intimal hyperplasia. Moreover, we have shown that tRA induces morphological changes in this SMC subset.13 Retinoids are known for their ability to inhibit cell growth and promote differentiation of many cell types.21 They are used for the treatment of several disease processes involving cell hyperproliferation or dedifferentiation such as psoriasis and cancer.22 23
The action of retinoids on SMC growth has been the subject of contradictory reports indicating that tRA either increases or decreases SMC proliferation.24 25 It is now considered that tRA activates mitogenesis in quiescent SMCs and decreases growth-stimulated SMC proliferation, suggesting that more than a single mechanism accounts for the growth-regulatory activity of tRA.26 In this study, we demonstrate that, in serum-stimulated conditions, tRA inhibits both medial and IT SMC proliferation in vitro and that this effect is more rapid on IT SMCs. The difference in retinoid sensitivity can be explained by a difference in growth rate of the 2 populations, because IT SMCs grow more rapidly than medial cells.5 Indeed, when both populations reached confluence, the tRA-mediated growth-inhibition was identical. IT SMCs may also be more sensitive to tRA because of their ability to metabolize retinoids as indicated by the presence of CRBP-1.13 A positive correlation exists between the level of CRBP-1 and retinoid responsiveness, and cells that metabolize retinoic acid generally are growth-inhibited by tRA.27 28
We examined the effect of retinoids on SMC migration in vitro and found that tRA stimulated medial and IT SMC migration to the same extent. This effect is not caused by increase proliferation, because in serum-stimulated conditions, tRA inhibits SMC growth. Moreover, BrdU incorporation indicates that proliferation is not significantly modified by tRA treatment during the migration test.
Migration, which is essential for neointima formation, requires degradation of extracellular matrix components. Enzymes involved in matrix degradation belong either to the matrix metalloproteinases (MMPs) or to the PA system. Many of them are tRA-regulated genes such as stromelysin, collagenase, or tPA.29 30 31 MMPs are negatively regulated by tRA, and this inhibition is accompanied by a tRA-mediated activation of genes encoding extracellular matrix components such as fibronectin, laminin, and collagen IV.32 33 In SMCs, the production of pro-MMP-1 is downregulated by tRA.34 These observations suggest that tRA may reduce SMC migration. However we observed that this process was stimulated by tRA. On the basis of this finding, we investigated whether tRA modulates the PA system in both SMC populations. These enzymes, mainly uPA and tPA, are activators of the plasminogen from which plasmin is derived. In turn, plasmin may induce the degradation of many extracellular proteins either directly or through the activation of latent MMPs.35 36 We have recently reported that tPA is the main PA involved in the proteolytic activities of both SMC populations and that IT SMCs exhibit a higher proteolytic activity, mainly because of increased expression of tPA.20 The results presented herein confirm and extend these previous findings. We report that tPA and uPA, expressed at different levels in the 2 populations, are respectively increased and decreased by tRA. Similar results were obtained when IT and medial SMCs were treated by basic fibroblast growth factor and platelet-derived growth factor, 2 cytokines that induce SMC migration.20 It is noteworthy that a stimulation of tPA expression by tRA has been described in endothelial cells.37 Finally, an activation of tPA expression has been reported, during SMC migration from the media to the intima, by cells located in the luminal layer, where CRBP-1, and consequently retinoid activity, is maximal.13 38 39
Taken together, these observations suggest that epithelioid IT SMCs expressing CRBP-1 are more prone than spindle-shaped medial SMCs to respond to retinoids by decreasing their growth rate and by increasing their migrating activity through tPA activation. It also is tempting to speculate that retinoids may have distinct roles depending on the retinoid sensitivity of SMCs: they might decrease migrating activities mediated by MMPs and enhance extracellular matrix deposition,34 or they might increase migrating activities mediated by the PA system.
We further investigated the effect of tRA on expression of -SM
actin, one of the SMC differentiation markers. We found that -SM
actin expression was selectively inhibited by tRA in IT SMCs, whereas
it remained unchanged in medial cells. The observation that tRA
decreases -SM actin expression is surprising because tRA is
considered as a potential inducer of SMC differentiation, at least
during development.40 It has also been shown that P19
embryonal carcinoma cells and dedifferentiated rat aortic SMCs (A7r5)
exposed to tRA express -SM actin, suggesting tRA-induced SMC
differentiation.41 42 43 44 The apparent discrepancy between
these findings and our data may have at least 2 explanations. First,
the level of differentiation of cultured SMCs may influence their
sensitivity to tRA. It is conceivable that tRA exerts differentiating
effects only on poorly differentiated SMCs (eg, containing low levels
of -SM actin); this is not the case for medial and IT SMCs in
culture that express -SM actin at comparably relatively high levels.
Second, tRA may increase or decrease -SM actin expression acting at
different metabolic steps. Many genes, such as CRBP-1, are
regulated by tRA at the transcriptional level through interactions
between RA receptors and an RA response element (RARE) located in the
promoter region.45 46 This is not the case for -SM
actin, and the tRA-mediated downregulation that we observed in
differentiated SMCs is certainly posttranscriptional, because no
variation of -SM actin mRNA was observed in tRA-treated SMCs. This
suggests that a hitherto unknown mediator of retinoid action acts at
the -SM actin translational level.
We also tried to shed light on the receptor(s) involved in
retinoid signaling events. It has been previously reported that 5 of
the 6 nuclear receptors are expressed in rat SMCs in vitro and in
vivo.25 We used agonists specific for the different RA
receptors and found that RAR- agonists modulate SMC growth, SMC
migration, and -SM actin expression in IT cells similarly to tRA. In
addition, the RXR-selective retinoids were unable to elicit these
events. Moreover, the action of tRA, at least on SMC proliferation and
-SM actin expression, was inhibited by the RAR-
antagonist Ro41-5253. This compound competes with tRA for
binding to RAR-.47 Taken together, these results
provide evidence for the involvement of RAR- in the transduction of
the retinoid signal in SMC. This pathway may also apply to the
tRA-mediated SMC differentiation of embryonal carcinoma cells, because
a subclone of P19 cells (RAC65) that fails to differentiate carries a
mutation in the RAR- gene.48 49 We did not investigate
the action of RAR- agonists on the PA system, but it is of
particular interest to note that tRA-induced tPA gene expression in
endothelial cells is specifically mediated by
RAR-.50
Finally we investigated the action of tRA and CD336, an RAR-
agonist, on the formation of IT after endothelial
lesion in rat carotid arteries. We observed that both tRA and RAR-
agonist reduced the IT and increase the size of the lumen diameter
without affecting the media. Our results confirm the observations of
Miano et al,51 published while this manuscript was under
review process. In addition, we show here that RAR- is responsible
for tRA actions. Thus, our results indicate that tRA and selective
retinoids are capable of blocking intima formation. Moreover, the
effect of tRA on IT SMCs, without interfering with the
physiological properties of medial SMCs, suggests
that tRA action is exerted preferentially on this SMC subset.
Therefore, identification of the target genes of tRA in IT SMCs may
help to define the mechanisms underlying the sensitivity of IT cells to
tRA. This knowledge may be useful for the design and evaluation of
drugs capable of inhibiting intima SMC replication in vivo.
The finding that the antiproliferative role of tRA is efficient
both in vitro and in vivo suggests a potential therapeutic use of tRA
and analogs during hyperproliferative vascular disease. Clinical
applications of tRA have had some success in the treatment of human
diseases such as cancer, psoriasis, and leukemia, but its therapeutic
use has been precluded by serious adverse effects such as skin or liver
toxicity and teratogenesis.52 Moreover, the biological
efficacy of tRA is greatly impaired by a hypercatabolism of the drug
accompanied by rapid development of resistance.53 The
development of new retinoic acid derivatives targeted against RAR-
exhibiting higher activity and lower toxicity than tRA could be one of
the promising strategies for therapeutic intervention in
arterial disease.
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Acknowledgments |
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Supported in part by the Swiss National Science Foundation, grants 3100-50568.97 and 3100-43364.95; Sumitomo Pharmaceuticals Co, Ltd, Osaka, Japan; the Swedish Medical Research Council, grants 6816 and 2042; the Swedish Heart-Lung Foundation; and the Swedish Cancer Society. The authors thank Drs P. Chambon, M. Klaus, and T. Noguchi for providing retinoids and Dr R.B. Low for critically reading this manuscript. We thank C. Di Sanza, F. Gabbiani, M. Quayzin, and P. Ropraz for technical assistance, J.C. Rumbeli and E. Denkinger for photographic work, and M. Vitali for typing the manuscript.
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Footnotes |
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Elisabetta Dejana, Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy, served as guest editor for the decision making process of this article.
Received July 7, 1998; accepted November 25, 1998.
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