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Circulation. 1996;93:1886-1895

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(Circulation. 1996;93:1886-1895.)
© 1996 American Heart Association, Inc.

Articles

Retinoid Receptor Expression and all-trans Retinoic Acid–Mediated Growth Inhibition in Vascular Smooth Muscle Cells

Joseph M. Miano, PhD; Stavros Topouzis, PhD; Mark W. Majesky, PhD; Eric N. Olson, PhD

From the Department of Biochemistry and Molecular Biology (J.M.M., E.N.O.), University of Texas MD Anderson Cancer Center; and Departments of Pathology (S.T., M.W.M.) and Cell Biology (M.W.M.), Baylor College of Medicine, Houston, Tex.

Correspondence to Joseph M. Miano, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Planck Rd, Milwaukee, WI 53226. E-mail jmiano@post.its.mcw.edu.


*    Abstract
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 *Abstract
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 down arrowResults
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Background Retinoids have been used in the successful treatment of a variety of human hyperproliferative diseases. Their role in smooth muscle cell (SMC) growth control, however, has not been clearly established. The present study was designed to assess the retinoid receptor mRNA expression profile in SMCs and to determine whether retinoids exert a growth-inhibitory effect in these cells.

Methods and Results Five of the six retinoid receptors were expressed in both cultured SMCs and aorta as determined by Northern blotting or reverse transcription-polymerase chain reaction. Receptor activity was demonstrated in SMCs with the use of a reporter assay with a retinoid receptor DNA binding sequence linked to a chloramphenicol acetyltransferase reporter gene. DNA synthesis and cell proliferation assays were performed to show that all-trans retinoic acid (atRA) antagonized platelet-derived growth factor-BB and serum-stimulated SMC growth. Growth inhibition was distal to early growth-signaling events because induction of c-fos, c-jun, and egr-1 mRNA was unaffected by atRA. However, with an activated protein-1–linked chloramphenicol acetyltransferase reporter, atRA was shown to inhibit the activity of activated protein-1–dependent transcription in a transient transfection assay.

Conclusions These results establish the presence of functional retinoid receptors in SMCs and document the growth-inhibitory action of atRA on these cells. Retinoid compounds, already in clinical use as antiproliferative agents for nonvascular indications, should be assessed further in in vivo models of intimal disease.


Key Words: retinoids • muscle, smooth • restenosis • genes


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Smooth muscle cell proliferation, migration, and extracellular matrix secretion account for a significant portion of the neointimal mass associated with atherosclerosis, cardiac transplant arteriopathy, iatrogenic restenosis, and hypertension.1 A number of pharmacological interventions have proved to be effective in limiting experimental neointimal formation, but all such therapies have failed in clinical trials.1 2 The inability to limit neointimal development in humans likely relates to its complex nature, which involves inflammatory cells and their mediators, angiogenesis, and hemostasis in addition to the SMC responses described above. Consequently, other, more multi-interventional, approaches must be considered for the management of human neointimal formation. Recently, vitamin E was shown to block neointimal thickening after balloon dilatation of the rat carotid artery.3 In addition, this natural nutrient scavenges reactive oxygen species that are known to produce minimally modified LDL, which is hyperatherogenic.4 Thus, vitamin E therapy may represent an approach to attack multiple processes eventuating in neointimal formation. Vitamin A is another essential vitamin that may have use in the treatment of vascular disease, yet it has attracted surprisingly little attention in this regard.

Vitamin A (retinol) and its natural and synthetic derivatives (retinoids) participate in a wide range of biological processes, including vision, embryonic development, regulation of epithelial and hematopoietic cellular differentiation, and normal reproductive physiology.5 With the exception of vision and some aspects of reproductive physiology, the bioactivity of vitamin A is ascribed to one of its many metabolites, atRA. This retinoid, together with its stereoisomer 9cRA, primarily functions by binding nuclear receptors belonging to the steroid receptor superfamily.6 Two families of retinoid receptors exist7 8 : the RAR family comprises three distinct genes (designated {alpha}, ß, and {gamma}), whose encoded proteins bind both atRA and 9cRA, and the RXR family, which also contains three members (also designated {alpha}, ß, and {gamma}) that preferentially bind 9cRA. These ligand-activated retinoid receptors act as transcription factors that bind to the promoters/enhancers of numerous target genes, leading to transcriptional stimulation or repression.9 The net action of a retinoid depends on the retinoid receptor composition of the cell as well as its growth and metabolic states (see "Discussion").

In general, retinoids such as atRA inhibit cell proliferation and promote differentiation.5 The recent genetic inactivation of retinoid receptors in mice supports this concept.8 10 11 More importantly, retinoids have been used clinically for the treatment of a variety of human cancers.12 13 The clinical use of retinoids is not limited to oncology; several dermatopathologies involving epidermal hyperproliferation (eg, psoriasis) are amenable to retinoid therapy.5 Given the successful management of these clinical hyperplasias with retinoid therapy, it is reasonable to consider whether retinoids may similarly be effective in controlling the growth of activated SMCs during neointimal formation. Here, we report the mRNA expression profile of retinoid receptors in cultured rat aortic SMCs and aorta and provide evidence that such receptors are biologically active in this cell type. We show that retinoids, principally atRA, potently antagonize growth factor– and serum-stimulated SMC proliferation at therapeutic doses that are tolerated in humans.14 Such growth inhibition is accompanied by reduced transcriptional activation of an AP-1 reporter construct. The decrease in AP-1–dependent transcription, however, is not the result of an atRA-mediated suppression of c-fos or c-jun mRNA expression. Our results clearly establish a role for retinoids in limiting SMC growth stimulation in vitro and provide a basis for the study of these bioactive agents in vivo after experimental balloon injury.


*    Methods
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 up arrowIntroduction
 *Methods
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Chemicals
atRA (lot No. 103H0701) was purchased from Sigma Chemical Co. 9cRA (Ro 04-4079) was a generous gift from Dr Michael Klaus (F. Hoffmann-La Roche Ltd, Basel, Switzerland). All retinoids were dissolved in DMSO to a working concentration of 2x10-3 mol/L and were made fresh for each experiment (apart from its known effect on enzymes that may metabolize retinoids,15 16 ethanol as a vehicle was found to poorly dissolve retinoic acid and to cause a dramatic reduction in baseline [3H]thymidine incorporation; data not shown). All experiments were executed under reduced lighting conditions to minimize photoisomerization of the parent retinoid used. The final concentration of DMSO (0.1%) did not have any effect on quiescent or growth-stimulated SMC responses (see below). Recombinant human PDGF (PDGF-BB; lot No. BW134081) was purchased from R&D Systems, Inc, reconstituted in 4 mmol/L HCl plus 0.2% BSA, and used at a final concentration of 10 ng/mL. [methyl-1',2,-3H]Thymidine (100 to 130 Ci/mmol) was purchased from Amersham Corp (No. TRK.565) and used at a final concentration of 2 µCi/mL.

cDNA Probes and Reporter Constructs
We cloned the following probes to SMC markers with the use of PCR (available on request): a 300-nt 3' UTR fragment of the rat SM-MHC cDNA,17 a 200-nt fragment of the rat SMC CALP cDNA,18 and a 275-nt fragment of the mouse SM22{alpha} cDNA.19 The mouse SM {alpha}-actin cDNA is a 157-nt fragment of the 3' UTR kindly provided by Dr Art Strauch (Department of Cell Biology, Ohio State University). The 2.1-kb RAR{alpha}, 1.95-kb RARß, and 2.0-kb RAR{gamma} probes20 were kindly provided by Dr Pierre Chambon (Laboratoire de Genetique Moleculaire des Eucaryotes, Strasbourg, France). The 4.8-kb RXR{alpha}, 2.2-kb RXRß, and 1.6-kb RXR{gamma} probes21 were generously provided by Dr Ron Evans (Salk Institute, San Diego, Calif). All immediate early gene probes have been described previously22 with the exception of egr-1, which is a 3.1-kb full-length cDNA kindly provided by Dr Vikas Sukhatme (Beth Israel Hospital, Boston, Mass). Antisera to SM {alpha}-actin and SMC CALP were purchased from Sigma. The tk TRE-CAT reporter construct originated in the laboratory of Dr Ron Evans, and was kindly provided by Phillipe Duprey (Universite Paris VII). The AP-1 CAT reporter was the generous gift of Dr Tom Curran (St Jude's, Memphis, Tenn).

Cell Culture
Cultured rat aortic SMCs were derived from aortas of six male Sprague-Dawley rats (300 to 350 g) by following an explant protocol in accordance with institutional guidelines for the handling of laboratory animals. Briefly, rats were anesthetized intraperitoneally with 50 mg/kg sodium pentobarbital. Aortas from the left subclavian branch to the renal bifurcation were then excised aseptically from the animal and placed into ice-cold PBS. After removal of the periaortic fat, the vessels were incubated at 37°C for 25 minutes in a cocktail of DMEM with penicillin (100 U/mL) and streptomycin (100 µg/mL), 2 mmol/L L-glutamine, 0.25% type I elastase (Sigma), 1% collagenase (Worthington Biochemicals), and 1% soybean trypsin inhibitor (Sigma), as described previously.23 This limited enzyme digestion allows for the complete removal of the adventitia and endothelial cells as assessed with the use of light microscopy and cell marker expression.23 The digested vessels were carefully cut into small rings and plated under sterile coverslips for at least 5 days in DMEM supplemented with 10% FBS (BRL), antibiotic/antimycotic, and 2 mmol/L L-glutamine. Cells were split 1:3 for the first passage and no more than 1:6 for all subsequent passages. We confirmed the identity of these cells by assaying for the mRNA expression of four SMC markers: SM-MHC, SMC CALP, SM {alpha}-actin, and SM22{alpha} (see below). In addition, Western blotting was performed with antisera to SMC CALP and SM {alpha}-actin (data not shown). Because the mRNA expression of all four markers was sustained over high passages (>40), we did not limit our studies to low-passaged SMCs. However, the results that we report were obtained from cultured SMCs between passages 10 and 30. All experiments were repeated at least once with one experiment from an independent cell isolate unless otherwise noted.

Northern Blotting/RT-PCR
Total RNA was isolated from duplicate 100-mm plates (Falcon) by following the guanidinium isothiocyanate/acid phenol method of Chomczynski and Sacchi.24 Approximately 20 µg of total RNA was fractionated through a 1.2% agarose gel containing 0.66 mol/L formaldehyde. The gel was washed in 10x SSC (1x SSC is 0.15 mol/L NaCl and 0.015 mol/L trisodium citrate, pH 7.0) and blotted to a nylon membrane (Zeta probe; Bio-Rad). After UV cross-linking of the RNA, the blot was prehybridized in 5x SSC, 5x Denhardt's, 6.5% dextran sulfate, 0.5% SDS, and 200 µg/mL salmon sperm DNA at 42°C for at least 6 hours. Random-primed radioactive-labeled cDNA probes (see above) were applied to the prehybridization solution, and the incubation was continued for an additional 16 hours. Blots were successively washed at 55°C in 2x SSC/1% SDS, 1x SSC/0.5% SDS, and 0.2x SSC/0.1% SDS for at least 30 minutes each. Blots were wrapped in plastic wrap and exposed to x-ray film (Kodak X-AR) at -80°C.

Where the mRNA abundance was below the level of detection by Northern blotting, we performed RT-PCR. Briefly, {approx}1 µg of total RNA was reverse-transcribed with AML RT (Boehringer Mannheim) at 42°C for 3 hours as described in the manufacturer's protocol. After RT, the cDNA templates were amplified with strand-specific primers to RXR{alpha} (forward primer, 5'-GCCCATCCCTCAGGAAATATG-3'; reverse primer, 5'-CAGAATCTTCTCTACAGGCAT-3'); RXRß (forward primer, 5'-CCAGTCATCAGTTCTTCCATG-3'; reverse primer, 5'-ACCTGGAGGGGGTGGACAGTG-3'), or RXR{gamma} (forward primer, 5'-TTGCCCACGGGGAAGCCAATG-3'; reverse primer, 5'-GCTGGTGGATGGGTAGTTCATA-3').21 The following PCR parameters were used: 94°C for 4 minutes and then 25 cycles of 1 minute at 95°C, 1 minute at 60°C, and 2 minutes at 72°C. The amplified product was visualized under UV light, excised from the gel, and column purified (QIAGEN Inc). The identity of the amplified band was verified with automated sequencing (Applied Biosystems, Inc) of the DNA with primers internal to those used for PCR. PCR primers to glyceraldehyde phosphate dehydrogenase (Clontech) were used as an internal standard.

[3H]Thymidine Assay
The growth assay outlined by Sudhir et al25 was followed. Cells (25 000/well) were cultured overnight in a 24-well plate (Corning, No. 25820) containing DMEM supplemented with 10% FBS, penicillin/streptomycin, and 2 mmol/L L-glutamine. Fresh medium was added the next day, and the cells were allowed to attain 80% to 90% confluence, at which time they were washed with PBS and growth-arrested for 72 hours in DMEM/F-12 supplemented with ITS (Sigma). Quiescent cells were pretreated with varying concentrations of retinoid or vehicle (0.1% DMSO) for 24 hours and then stimulated with PDGF-BB (10 ng/mL) or 10% FBS for 18 hours in the presence or absence of retinoid. In some experiments, retinoid treatment was simultaneous with PDGF-BB or delayed for 6 or 12 hours after PDGF-BB (see below). The cells were then pulsed with 2 µCi/mL [3H]thymidine, and the incubation was continued for an additional 6 hours. Cells were washed three times in assay buffer containing (mmol/L) NaCl 140, KCl 5, CaCl2 2, Na2HPO4 1, glucose 25, HEPES/NaOH 25, pH 7.2, and 0.5 mg/mL BSA. Cells were then extracted with 15% trichloroacetic acid for 30 minutes at 4°C. The supernatant was aspirated, and the cells were washed in distilled H2O. Cells were solubilized with 0.5 mL of 1 mol/L NaOH for 20 minutes at 37°C and then neutralized with 0.5 mL of 1 mol/L HCl. The solubilized radioactivity was counted in liquid scintillant and expressed as counts per minute per well.

Cell Proliferation Assay
A direct assessment of cell proliferation was made by counting cells exposed to growth factor or serum in the presence or absence of retinoids. Cells (35 000/well) were cultured in six-well dishes (Falcon) overnight in DMEM/10% FBS and allowed to spread and achieve a density of {approx}40 000 to 50 000 cells/well ({approx}20% confluence). Cells were then washed and incubated overnight in DMEM/F-12 with ITS and antibiotics. On day 0 of the growth assay, cells were fed fresh DMEM/F-12 medium with or without 2.5 to 10 ng/mL PDGF-BB (or DMEM/10% serum) in the presence or absence of retinoid (2x10-6 mol/L). Quiescent control plates received DMEM/F12 medium with ITS and DMSO (0.1%). The growth assay was carried out over a 6-day interval with fresh medium changes occurring every other day (both growth factor and retinoid were replenished). On day 6, cells were washed in PBS, trypsinized, and counted with a Coulter counter (Coulter Electronic).

Cellular Toxicity
We performed a number of tests to rule out retinoid-induced cellular toxicity. First, cell morphology was documented with phase-contrast microscopy. Second, cell viability was directly assessed at the termination of an experiment with trypan blue exclusion. Finally, reversibility studies were performed with [3H]thymidine. For reversibility studies, growth-arrested cells were stimulated with PDGF-BB or serum in the presence of retinoids for 24 hours. Cells were then washed and incubated with fresh DMEM/F12/ITS medium for a 24- or 48-hour recovery period to remove residual retinoids, after which PDGF-BB or 10% serum was added for 18 hours, and [3H]thymidine assays were carried out as described above.

CAT Assay
Cells were plated onto 100-mm dishes and allowed to reach {approx}50% confluence. The cells were washed with PBS and then incubated in DMEM/10% FBS in the presence or absence of retinoid. The next day, the media were aspirated, and fresh media were added for 6 hours. Approximately 10 µg of test plasmid (see below) was precipitated in HEPES-buffered saline/Ca3(PO4)2 and applied to the cells for 14 to 16 hours as described previously.26 Cells were then washed and incubated for an additional 48 hours, at which time they were scraped in PBS, briefly spun, and then sonicated in 0.1 mol/L Tris-HCl, pH 7.8. Total protein in the supernatant was measured by the Bradford method (Bio-Rad), and equivalent quantities of protein were assayed with CAT as described previously.26 Experiments were performed in duplicate for a minimum of three times. In some experiments, a plasmid containing the bacterial ß-galactosidase gene was cotransfected and used as an internal control for transfection efficiency. No difference was found between data normalized to ß-galactosidase or total protein.

Statistical Analysis
Where data were amenable to statistical analysis, a one-way ANOVA was performed, followed by Tukey's studentized test for significance between unpaired mean values as described previously.27 All data satisfied the Tukey requirements of normally distributed samples of equal number between groups. An {alpha} level of P<.05 was considered significant.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Fig 1Down shows the characteristic change in SMC morphology when quiescent cells (Fig 1ADown) are stimulated with PDGF-BB for 24 hours (Fig 1BDown). The addition of as much as 2x10-6 mol/L atRA did not alter the PDGF-induced change in cell morphology (Fig 1DDown). Higher doses of atRA, however, resulted in cell rounding and lifting due to cellular toxicity (Fig 1CDown). Cells treated with as much as 2x10-6 mol/L atRA never displayed any signs of toxicity (see below). Thus, our highest concentration of atRA was set at 2x10-6 mol/L.



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  		Figure 1. Effect of atRA on PDGF-stimulated SMC morphology. Cultures of rat aortic SMCs were growth arrested for 3 days in serum-free medium (A) and then treated with 10 ng/mL PDGF-BB in the absence (B) or presence of 2x10-5 (C) or 2x10-6 (D) mol/L atRA. Magnification x200.

RAR/RXR mRNA Expression Profile in Cultured SMCs and Aorta
No studies have reported the mRNA expression of retinoid receptors in SMCs. Thus, as a first step toward understanding retinoid action in SMCs, we measured the steady state mRNA levels of all six retinoid receptors in cultured atRA-treated SMCs and in rat aorta. Consistent with the assignment as a "housekeeping gene,"7 cultured SMCs and aorta express both transcripts corresponding to RAR{alpha}. The addition of atRA did not alter the levels of this transcript up to 72 hours (Fig 2Down). Low but reproducibly detectable transcripts to RARß were present in SMCs both in vitro and in vivo (Fig 2Down). The RARß promoter contains a retinoic acid–response element that binds and is activated by atRA-bound retinoid receptors.7 Results in Fig 2Down show that atRA similarly upregulates rat aortic SMC RARß mRNA by 6 hours. This suggests that SMC retinoid receptors are activated by atRA, bind the RARß promoter, and stimulate its transcription. Cultured SMCs and aorta also express high levels of RAR{gamma} mRNA, which does not appear to be modulated by atRA (Fig 2Down). The presence of RAR{gamma} transcripts in SMCs is noteworthy because this receptor has been shown to be highly restricted to skin in adult animals.20 Prolonged atRA treatment did not alter transcript levels of various SMC markers, including SM {alpha}-actin (Fig 2Down), SM-MHC, SMC CALP, and SM22{alpha} (data not shown).



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  		Figure 2. Steady state mRNA expression of RARs in rat aortic SMCs and aorta. Confluent quiescent cultures of SMCs were treated with 2x10-6 mol/L atRA for the indicated times and then harvested for Northern blotting as described in "Methods." Pooled aortas (n=5) were stripped of adventitia and endothelium and similarly processed for Northern blotting. FigureUp represents a single blot containing 20 µg/lane total RNA sequentially hybridized to the indicated cDNA probes. The relative abundance of each RAR can be appreciated visually because the probes were of similar length and specific activity. Transcript sizes and exposure times were RAR{alpha}, 3.6 and 2.8 kb (2 days); RARß, 3.4 kb (6 days); RAR{gamma}, 3.3 kb (18 hours); and SM {alpha}-actin 1.8 kb (18 hours). Representative of two independent experiments.

RXR transcripts are expressed at very low levels in cells and tissues.21 Thus, RT-PCR was necessary for the detection of these rare transcripts. Fig 3ADown shows that cultured SMCs and, to a lesser extent, aorta express the RXR{alpha} mRNA. Fig 3BDown shows that the widely expressed RXRß mRNA is also present in aorta and cultured rat aortic SMCs. We have been unable to reproducibly demonstrate RXR{gamma} mRNA in SMCs or aorta with the use of RT-PCR. Collectively, these results show that SMCs in culture and aortic media express all three RARs and two of the three RXRs.



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  		Figure 3. Steady state mRNA expression of RXRs in SMCs and aorta. Samples of RNA from selected time points in Fig 1Up were used in an RT-PCR assay as described in "Methods." A, Amplified 347-nt product corresponding to nucleotides 553–900 of the mouse RXR{alpha} cDNA.21 B, Amplified 158-nt product corresponding to nucleotides 169–327 of the mouse RXRß cDNA.21 An amplified 150-nt fragment of the rat glyceraldehyde phosphate dehydrogenase mRNA is included as an internal standard.

atRA Induces Retinoid Receptor–Mediated Transcription
The above results suggest that atRA might bind SMC retinoid receptors and activate the expression of at least one target gene (RARß). To assay the activity of SMC retinoid receptors more directly, we transfected SMCs with a CAT reporter gene under the control of a TRE in the presence of varying concentrations of atRA and then measured the corresponding CAT activity. Retinoid receptors have previously been shown to bind the TRE when occupied by atRA.28 Fig 4Down shows that atRA increases TRE CAT expression in a dose-dependent manner. Taken together with the results in Figs 2Up and 3Up, we conclude that SMCs express functional retinoid receptors that may mediate the effects of atRA.



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  		Figure 4. Dose-dependent increase in retinoid receptor activity with atRA treatment. Cultured SMCs were transfected with a TRE-linked CAT reporter plasmid as described in "Methods" and treated with varying concentrations of atRA for 48 hours before cell harvest. The quiescent (baseline) cells here and in all subsequent experiments received the same volume of vehicle (0.1% DMSO). Normalized CAT is expressed as fold increases over baseline, arbitrarily set at 1.

Retinoids Inhibit PDGF-Stimulated SMC DNA Synthesis and Mitogenesis
Having documented the expression and activity of retinoid receptors in SMCs, we next tested whether atRA could modulate PDGF-stimulated DNA synthesis. The results in Fig 5ADown show that atRA inhibits PDGF-stimulated [3H]thymidine uptake in a dose-dependent manner. This inhibitory effect was not specific for PDGF-BB because serum-stimulated SMC DNA synthesis was similarly inhibited (data not shown). Several lines of evidence suggest that the inhibition of SMC DNA synthesis was not attributable to cytotoxicity. First, SMCs treated with 2x10-6 mol/L atRA exhibited normal gross morphology (see Fig 1Up). Second, >95% of treated cells excluded trypan blue 24 hours after combined PDGF-BB and atRA treatment. Finally, the inhibition of SMC DNA synthesis by atRA was fully reversible on "washing out" of the atRA and restimulation with PDGF-BB (Fig 5ADown). We also considered the possibility that atRA induced apoptosis29 in treated SMCs. However, gel fragmentation assays revealed no evidence of atRA-mediated apoptosis (data not shown). Thus, therapeutic doses of atRA that clearly activate retinoid receptor activity also inhibit [3H]thymidine uptake in cultured rat aortic SMCs.



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  		Figure 5. atRA-mediated SMC growth inhibition. A, Confluent quiescent cultures of SMCs were pretreated with the indicated concentrations of atRA for 24 hours, washed, and then stimulated for 18 hours with PDGF-BB (10 ng/mL) plus atRA. Cells were then pulsed for 6 hours with [3H]thymidine and harvested for scintillation counting as described in "Methods" (n=4 per condition; bars here and in all subsequent figures represent the standard deviation from the mean of a single representative experiment). Second column, Cells treated with PDGF-BB and atRA at 2x10-6 mol/L for 24 hours followed by a 48-hour washout and then 18-hour PDGF-BB stimulation as above. Probability values are in relation to PDGF-BB only. B, Subconfluent (20%) SMCs were either continuously cultured in serum-free medium or PDGF-BB or simultaneously treated with PDGF-BB and either 2x10-6 mol/L atRA or 2x10-6 mol/L 9cRA over a 6-day interval as described in "Methods." No statistically significant differences existed between quiescent cultures vs retinoid cultures treated with or without PDGF-BB (n=6 per condition).

Fig 5BUp shows the effect of atRA and its stereoisomer, 9cRA, on PDGF-stimulated SMC proliferation as measured with direct cell counting. Over a 6-day period, SMC number increased nearly threefold with PDGF-BB stimulation (Fig 5BUp). This increase in cell number was effectively blocked with either retinoid. Similar inhibition was observed when SMCs were growth stimulated with 10% FBS and exposed to atRA (data not shown). We therefore conclude that atRA and 9cRA are potent inhibitors of PDGF and serum-stimulated SMC mitogenesis in vitro.

Full atRA-Mediated SMC Growth Inhibition Is Cell Cycle Dependent
The experiment in Fig 5AUp entailed the pretreatment of quiescent SMCs with atRA for 24 hours before PDGF stimulation. To begin understanding at what point in the cell cycle atRA exerts its growth-inhibitory effect, we administered the retinoid 24 hours before, simultaneous with, or 6 or 12 hours after PDGF-BB stimulation. The results depicted in Fig 6Down show complete inhibition of DNA synthesis regardless of whether the retinoid was administered 24 hours before or simultaneous with PDGF. This inhibition was significantly less effective if atRA administration was delayed 6 or 12 hours after PDGF stimulation. There was no statistically significant difference in [3H]thymidine uptake between the 6- or 12-hour delay conditions (Fig 6Down).



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  		Figure 6. Effect of delayed atRA administration on PDGF-BB–stimulated SMC [3H]thymidine uptake. Cultures of quiescent SMCs were treated with 2x10-6 mol/L atRA 24 hours before, simultaneous with, or 6 or 12 hours after PDGF-BB administration. Thymidine incorporation was then assayed exactly as described in the legend to Fig 5AUp (n=4 per condition). This experiment was repeated once with an independent isolate of SMC.

atRA Attenuates Serum-Stimulated AP-1 Activity
Previous studies have demonstrated that atRA inhibits the activity of c-Jun, which binds the AP-1 cis element.30 31 32 In light of these findings, coupled with the results in Fig 6Up, we hypothesized that atRA might inhibit SMC growth by attenuating the activities of immediate early transcription factors that bind AP-1. To assess whether SMC AP-1 activity was altered by atRA treatment, we transfected SMCs with a CAT reporter construct under the control of 73 nucleotides corresponding to the proximal human collagenase promoter/enhancer.33 This promoter/enhancer contains a single AP-1 site that is essential for normal collagenase transcription.33 In the absence of serum, very little CAT activity was detected (Fig 7Down, lane 1), which is consistent with the cells being quiescent and not expressing c-fos and c-jun transcripts. Serum stimulation, however, resulted in significant activation of the reporter gene (Fig 7Down, lane 3), which likely occurred because of the induction of c-fos and c-jun (see below). When serum-treated cells were simultaneously exposed to atRA, a consistent reduction in CAT activity was observed (Fig 7Down, lane 4; mean percent inhibition of 50%, P<.05, n=4 independent experiments). Thus, atRA attenuates AP-1 transcriptional activity in SMCs at concentrations that activate endogenous retinoid receptors and inhibit SMC proliferation.



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  		Figure 7. atRA-mediated repression of SMC AP-1 activity. Cultures of SMCs were transfected with an AP-1 CAT reporter in the absence or presence of 2x10-6 mol/L atRA. Quiescent cells received 0.1% DMSO in serum-free medium. Cells were then washed and replenished with either serum-free medium or 10% serum with or without atRA for an additional 48 hours, at which time the cells were harvested for CAT activity. Lane 1, quiescent cells; lane 2, EMSV CAT (positive control); lane 3, serum alone; and lane 4, serum plus atRA treatment. These results were repeated in four independent experiments with two different isolates of SMC (range of AP-1 inhibition was {approx}40% to 80%). The numbers at the bottom of each lane represent percent acetylation.

atRA-Mediated SMC Growth and AP-1 Inhibition Is Distal to Immediate Early Transcription Factor Expression
To determine whether atRA-mediated attenuation in AP-1–dependent transcription was due to an inhibition in expression of factors that bind AP-1, we measured c-fos and c-jun mRNA expression by Northern blotting. As seen in Fig 8Down, PDGF-BB evoked a rapid induction of c-fos, c-jun, and egr-1 transcripts in cultured SMCs. Pretreatment of cells with atRA 24 hours before and during growth stimulation, however, had no obvious inhibitory effects on the level or time of such gene induction. These results indicate that atRA does not interfere with the signaling pathways involved with immediate early transcription factor activation. They further demonstrate that the atRA-mediated suppression of AP-1 activity is not attributable to a decrease in the transcription of c-fos or c-jun mRNA.



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  		Figure 8. Effect of atRA on immediate early transcription factor expression. Duplicate plates of quiescent SMCs were pretreated with 2x10-6 mol/L atRA or 0.1% DMSO for 24 hours, washed, and then stimulated with 10 ng/mL PDGF-BB or 10% serum (data not shown) plus atRA for the indicated times before total RNA was isolated for Northern blotting. The quiescent cells received either 0.1% DMSO or atRA for 24 hours before RNA harvest. Panels represent the same blot sequentially hybridized to each indicated cDNA probe. This experiment was repeated in two additional experiments with an independent isolate of SMC.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The results of the present study document several retinoid-mediated bioactivities in cultured rat aortic SMCs. First, the presence of all three RARs and two of the three RXRs implies that SMCs can respond to exogenously administered retinoids in a receptor-mediated manner. This notion is supported by our data showing that atRA treatment stimulates expression of the RARß gene in SMCs, a finding consistent with other reports.5 Further support for retinoid receptor activity in SMCs arises from studies showing a dose-dependent increase in TRE CAT activity with atRA treatment. The second retinoid bioactivity identified in the present study relates to its potent growth-inhibitory properties when SMCs are stimulated with PDGF-BB or serum. This inhibition is dose dependent, reversible, and without cytotoxicity. Moreover, significant growth inhibition is observed when atRA is administered as late as 12 hours after PDGF-BB stimulation. This suggests that atRA exerts its growth-inhibitory effect at multiple points in the cell cycle. Finally, atRA elicits significant inhibition in AP-1 activity that appears to be distal to c-fos and c-jun mRNA expression. Such impaired transcriptional activity may in part explain the growth inhibition by atRA of SMCs as well as other activities that may be of significance during the progression of neointimal disease (see summary below).

SMCs Express Functionally Active Retinoid Receptors
The first indication that retinoid receptors might exist in SMCs occurred more than 25 years ago when it was found that vitamin A–deficient chick embryos failed to develop because of an incomplete circulatory system.34 Subsequent studies by Heine et al35 showed that this defect in vasculogenesis was due to the absence of omphalomesenteric veins, which bridge the embryonic and extraembryonic circulatory systems. Recently, the genetic inactivation of specific combinations of retinoid receptors has also revealed aberrations in the development of the elastic arteries.36 Taken together, these findings suggest an important role for retinoid signaling in vascular SMCs during development. That atRA evokes characteristics of SMC differentiation in vitro lends support to this idea.37 38 39

Our results documenting retinoid receptor expression and activity in cultured rat aortic SMCs provide the first direct evidence for the existence of retinoid receptor–mediated signaling in this cell type. The presence of RAR{alpha} and RXRß mRNA in vascular SMCs is not surprising given their widespread expression during development and throughout most adult tissues.7 21 High-level RAR{gamma} mRNA in SMCs and aorta, however, was not expected because previous work showed this receptor to be highly restricted to skin.20 The presence of RAR{gamma} and RXR{alpha} transcripts in SMCs raises interesting speculation pertaining to their recent genetic inactivation in mice.36 Mice deficient in both RAR{gamma} and RXR{alpha} display severe alterations in the large elastic arteries.36 Whether there are any functional defects in the SMCs investing these vessels remains unexplored. Finally, the induction of RARß mRNA in SMCs treated with atRA may be of mechanistic importance in the context of the growth inhibition by atRA of SMCs; there is evidence supporting RARß as a tumor-suppressor gene.40 It is important to emphasize, however, that no studies have yet assigned a retinoid receptor–mediated process to any action of retinoids in SMCs. Such studies will require dominant negative retinoid receptors,41 synthetic retinoids that bind specific receptors without activating them,42 or the cultivation of vascular SMCs from knockout mice deficient in specific retinoid receptors.

atRA-Mediated SMC Growth Inhibition
Few studies have addressed the influence of vitamin A derivatives in SMC growth control.43 44 45 The mechanism or mechanisms of retinoid-mediated SMC growth inhibition have not been previously addressed. Prior studies have shown that atRA inhibits the expression of various proto-oncogenes, including those belonging to the immediate early family of transcription factors.31 46 Our results, however, indicate that growth-stimulated c-fos, c-jun, and egr-1 activation is neither attenuated nor delayed with atRA administration. Similarly, Davis et al47 showed that atRA inhibition of PDGF-stimulated Ito cell growth was distal to Fos, Jun, and Egr-1 protein activation. Collectively, these data imply that the initial signaling pathways leading to such gene activation are not compromised by atRA.

Although SMC immediate early transcription factor induction is not affected by atRA, some early event or events must be because a delay in atRA administration 6 hours after PDGF treatment partially reversed the growth-inhibitory response of SMCs to atRA. We considered the possibility that SMC AP-1 activity might be negatively regulated by atRA because several reports have documented this phenomenon in other cell types.30 31 32 48 49 50 The theory postulates that atRA-activated retinoid receptors interfere with the binding of Jun/Jun homodimers or Fos/Jun heterodimers to the AP-1 cis element, thereby preventing transcriptional activation.32 Consistent with this model, we show a reduction in SMC AP-1–dependent transcription with atRA. Because we did not measure the levels of Fos or Jun protein, we cannot rule out translational suppression as a mechanism for reduced AP-1 activity, although it is clear that the growth induction of these genes is not diminished with atRA. We also do not know whether other members of the Fos (fra-1 and FosB) and Jun (JunB and JunD) families display reduced AP-1 binding in the presence of atRA. Regardless of the mechanism, the reduction in AP-1 activity by atRA could account for the difference in thymidine uptake between cultures of SMCs treated simultaneously or 6 hours after PDGF stimulation. Other cell cycle events are likely to be compromised (cell cycle–dependent kinases?) because significant SMC growth inhibition is noted when atRA is added up to 12 hours after PDGF cell stimulation. This finding is reminiscent of the report that heparin strongly inhibited SMC DNA synthesis in injured rat carotid arteries even when administered up to 18 hours after injury.51

Because the inhibition of SMC proliferation by atRA occurs at concentrations that stimulate retinoid receptor activity and repress AP-1–dependent transcription, it is tempting to invoke a receptor-mediated process for such growth inhibition. The concentration of atRA necessary to elicit such SMC responses, however, is orders of magnitude greater than its dissociation constant for different retinoid receptors. Two major factors may account for this frequently noted disparity between the EC50 of atRA and its receptor dissociation constant5 52 : the intracellular catabolism of atRA and the complexity of retinoid receptor expression and activity. Intracellular atRA concentrations are tightly governed by isomerization to other retinoids (eg, 13-cis RA and 9cRA)53 as well as by its uptake and catabolism to more polar (and excretable) retinoids by one of two cellular retinoic acid–binding proteins.54 A cell with high concentrations of cellular retinoic acid–binding proteins, for example, would be less sensitive to atRA and thus may require high atRA concentrations to elicit a response. Related to this point is the recent identification of a retinol-binding protein in adult but not newborn SMCs.55

The second issue to consider for the high atRA concentrations needed to evoke SMC responses relates to the retinoid receptors present in a given cell and their activities. SMCs express three RARs, each of which may exist as at least two isoforms due to alternative promoter use and splicing.7 Each RAR isoform can heterodimerize with any one of the three RXRs, of which two are present in SMCs and aorta. In addition to heterodimerizing with RAR, RXR may heterodimerize with a number of other receptors, including those to thyroid hormone, vitamin D3, and peroxisome proliferators.7 The complexity of retinoid receptor activity is further compounded by the multiplicity of DNA cis elements that they bind.7 56 Although SMCs express most of the retinoid receptors, only a handful of ligand-occupied heterodimers may be functionally important for the inhibition of SMC growth. Thus, the response of a given cell type to atRA is governed by the metabolic state of the cell, its constellation of retinoid receptors, and the accessibility of growth-related retinoid receptor DNA response elements. Such complexity in retinoid metabolism and receptor activity may account for the high concentrations of atRA necessary to stimulate receptor activity and SMC growth inhibition.

Although SMCs clearly display retinoid receptor activity that correlates with the inhibition of PDGF-stimulated growth by atRA, we must bear in mind that other receptor-independent mechanisms may contribute to such growth inhibition. One such mechanism involves the posttranslational modification of proteins through a process of retinoylation.5 57 The consequences of protein retinoylation have not been examined but could involve their inactivation. Thus, it is possible that key regulators of SMC DNA synthesis are retinoylated and rendered inactive. From this discussion, it is clear that much remains to be learned about the mechanisms underlying retinoid-mediated SMC growth inhibition.

Summary and Implications
Results of the present study provide the first detailed account of retinoid receptor activity and control of SMC growth by atRA. No clinically proven therapy exists for the successful management of neointimal formation. Several facts make retinoid therapy a potentially attractive approach for the treatment of neointimal disease. First, retinoids such as atRA have been approved and are comparatively well tolerated in humans at doses approaching those that we describe.14 Second, atRA is used for the successful treatment of a variety of human cancers, most notably, acute promyelocytic leukemia.13 Finally, atRA displays a wide spectrum of effects relevant to neointimal disease, including accelerated fibrinolysis,58 inhibition of the inflammatory response,59 60 and inhibition of SMC migration.61 Interestingly, inhibition of SMC migration by atRA was shown to be closely linked to the suppression of collagenase and stromelysin gene expression.61 Both of these proteases are regulated in an AP-1–dependent manner.30 33 Thus, atRA-mediated inhibition of SMC migration61 may occur due to the repression of AP-1 activity and, consequently, the inhibition of AP-1–dependent collagenase and stromelysin mRNA expression. Taken in aggregate, the available data point to a pleiotropic effect of atRA that could be exploited for the potential control of neointimal disease.


*    Selected Abbreviations and Acronyms
 
atRA = all-trans retinoic acid
AP-1 = activated protein–1
CALP = calponin
CAT = chloramphenicol acetyltransferase
9cRA = 9-cis retinoic acid
DMEM = Dulbecco's modified Eagle's medium
DMSO = dimethylsulfoxide
ITS = insulin/transferrin/selinium
PCR = polymerase chain reaction
PDGF = platelet-derived growth factor
RAR = retinoic acid receptor
RT = reverse transcription
RXR = retinoid X receptor
SMC = smooth muscle cell
SM-MHC = smooth muscle myosin heavy chain
TRE = thyroid hormone response element


*    Acknowledgments
 
Dr Olson is supported by grants from the National Institutes of Health, the Muscular Dystrophy Association, and the Robert A. Welch Foundation. Dr Majesky is supported by National Institutes of Health grant HL-47655 and the American Heart Association, Texas Affiliate, Inc, grant 91G-181. Dr Miano is supported by a National Research Service Award. We thank Dr Reuben Lotan for reading the manuscript and Alisha Tizenor for providing expert graphic assistance.

Received September 18, 1995; revision received November 1, 1995; accepted November 5, 1995.


*    References
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