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Circulation Research. 1999;85:99-107

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(Circulation Research. 1999;85:99-107.)
© 1999 American Heart Association, Inc.

Rapid Communications

Cultured Porcine Coronary Artery Smooth Muscle Cells

A New Model With Advanced Differentiation

Thomas Christen, Marie-Luce Bochaton-Piallat, Pascal Neuville, Sander Rensen, Mireille Redard, Guillaume van Eys, Giulio Gabbiani

From the Department of Pathology (T.C., M.-L.B.-P., P.N., M.R., G.G.), University of Geneva-CMU, Geneva, Switzerland; Department of Molecular Cell Biology and Genetics (S.R., G.E.), University of Limburg, Maastricht, the Netherlands. The current affiliation for P.N. is Transgène S.A., Strasbourg, France.

Correspondence and reprints 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


*    Abstract
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 *Abstract
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Abstract—Arterial intimal thickening after endothelial injury induced in rodents has proven to be a relatively unreliable model of restenosis for testing clinically useful compounds. The same has been found for cultured rat or rabbit vascular smooth muscle cells (SMCs). To test alternative possibilities, we have studied several differentiation features of porcine coronary artery SMCs, cultured up to the 5th passage after enzymatic digestion of the media. The effects of heparin, transforming growth factor (TGF)-ß1 or TGF-ß2, and all-trans-retinoic acid (tRA) on proliferation, migration, and differentiation of these cells also were examined. Porcine arterial SMCs in culture not only express high levels of {alpha}-smooth muscle (SM) actin but, contrary to rodent SMCs, also maintain an appreciable expression of SM myosin heavy chain isoforms 1 and 2, desmin, and smoothelin, a recently described late differentiation marker of vascular SMCs. We demonstrate for the first time that smoothelin is colocalized with {alpha}-SM actin in these cells. Finally, we show that in the porcine model, heparin is more potent than TGF-ß1 or TGF-ß2 and tRA in terms of inhibition of proliferation and migration and of increasing the expression of differentiation markers. This model should be a useful complement to in vivo studies of SMC differentiation and of pathological situations such as restenosis and atheromatosis.


Key Words: atheromatosis • restenosis • actin • smoothelin • myosin


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The arterial intimal thickenings (IT) induced in the rat or rabbit after endothelial lesion are presently the most-studied models for atheroma formation and/or restenosis and have been useful for defining several biological features of smooth muscle cells (SMCs). However, these models have many important limitations (for review, see Reference 11 ), which could explain the clinical failure of substances that proved to be efficient inhibitors of IT formation in these animal models.

The biological features of SMCs in culture also have been systematically studied using cells derived from rat or rabbit arteries,2 3 4 5 6 7 but these, too, have shown limitations similar to those observed in in vivo experiments. Among the models in large animals, the pig coronary artery IT has been used more and more.8 9 Pigs may develop spontaneously coronary atheromatosis with age, and the induction of typical plaques is easily achieved by a cholesterol-rich diet.10 11 Furthermore, angioplasty and other interventions can be performed in porcine coronary arteries with the same instruments as in humans. Although pig aortic and coronary SMCs have already been studied in vitro, no systematic description of their differentiation features during culture has been published.

The present study describes the characterization of differentiation features and several biological properties of cultured porcine left anterior descending (LAD) coronary artery SMCs. We show that, contrary to rat or rabbit arterial SMCs,2 3 4 5 6 7 porcine SMCs maintain in culture a high level of differentiation marker expression, including the hitherto unreported expression of smoothelin, a recently described marker of late SMC differentiation. We describe the replicative and motile properties of these cells as well as the influence on these activities of heparin, TGF-ß1 or TGF-ß2, and tRA. Our findings suggest that porcine coronary artery SMCs retain a much higher level of differentiation and behave differently in vitro from the majority of SMCs previously studied and thus may represent a better model for the study of agents influencing SMC behavior in vitro and in vivo.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
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Cell Culture
Coronary arteries from domestic crossbred pigs (sus scrofa domesticus) were obtained from a nearby slaughterhouse at the age of {approx}8 months. The 40 mm of the LAD distal to the branching of the left circumflex coronary artery was chosen for dissection and enzymatic digestion. The vessel was opened by a longitudinal cut. The endothelium was gently scraped with a surgical blade, and the media was then stripped from the underlying adventitia with two ophthalmological tweezers and finely minced. The material of two coronary arteries was pooled and digested for 60 minutes at 37°C in 6 mL of DMEM (Gibco BRL, Life Technologies) with 2% HEPES (Life Technologies) containing 40 mg of collagenase (Sigma-blend collagenase, type F, 1800 U/mg; Sigma), 4.2 mg of elastase from hog pancreas (9.7 U/mg; Fluka), and 12 mg of soybean trypsin inhibitor (1 mg inhibiting 2.1 mg trypsin; Worthington). The enzymatic activity was stopped by FCS (Seromed, 10% final concentration; Biochrom KG), and the suspension was filtered through a sterile gauze to discard remnants. The filtrates of two preparations were pooled and plated on a 100-mm culture dish (Falcon, Becton-Dickinson) in DMEM without FCS. Two hours later, FCS was added up to a final concentration of 10%. The medium was changed after 24 hours, and the primary culture was confluent in {approx}6 days. Medium was always changed every 3 days. The plating efficiency (ratio of attached to plated cells) at 24 hours was found always to be {approx}70%. These cells were passed by a 1:2 split ratio and studied up to the 6th passage.

Cell Treatment
SMCs at the 5th passage were plated at a density of 60 cells/mm2 in 100-mm culture dishes containing DMEM supplemented with 10% FCS. In a first series of experiments, medium was changed 24 hours after plating, and SMCs were incubated alone or in combination with one of the following: 200 µg/mL heparin (H-9399; Sigma), 10 ng/mL TGF-ß1 (Sigma), 10 ng/mL TGF-ß2 (gift of Dr D.A. Cox, Novartis, Basel, Switzerland), or 10-6 mol/L tRA (Sigma). In a second series of experiments, medium was removed when cells reached subconfluence, and the cells were then incubated in DMEM supplemented with Monomed (a defined serum-free medium containing insulin, transferrin, sodium selenite, 2-mercaptoethanol, 2-aminoethanol, sodium pyruvate, glutamine, and a BSA–oleic acid complex; Commonwealth Serum Laboratories, Melbourne, Australia), containing the above-described agents. Vehicles (0.1% BSA in 4 mmol/L HCl for TGF-ß1 or TGF-ß2 and DMSO [Fluka] for tRA) were added in control cells at the same concentration as for treated cells. Medium was changed after 3 days, and cells were treated for 6 days.

Cell Proliferation and [3H]-Thymidine Incorporation
For evaluation of proliferation, SMCs were plated in DMEM supplemented with 10% FCS as described above and were counted after 7 days using a hemocytometer. The results were calculated, as the ratio of counted to seeded cells.

For [3H]-thymidine incorporation, SMCs were plated at a density of 60 cells/mm2 in DMEM supplemented with 10% FCS. At subconfluence, cells were synchronized for 3 days in DMEM containing Monomed. Fresh medium plus 10% FCS alone or containing one of the above-mentioned agents and 0.1 µCi/mL [3H]-thymidine (specific activity, 5 Ci · mmol-1 · L-1; Amersham) was then added for 24 hours. [3H]-thymidine incorporation was evaluated by trichloracetic acid and perchloric acid precipitation and counting in a liquid scintillation counter (Beckman Instruments).

Cell Migration
For evaluation of migratory capacity, SMCs were plated at a density of 60 cells/mm2 in DMEM in the presence of 10% FCS. Confluent cultures were scratched with a silicon-coated stick to obtain a 0.8-mm-wide in vitro wound12 and photographed in phase contrast using a Zeiss Axiovert 35 photomicroscope (Carl Zeiss, Oberkochen, Germany). Fresh medium plus FCS alone or containing one of the above-described agents was added. After 24 hours, nuclear staining with propidium iodide (0.05 mg/mL; Fluka) was performed, and migrating cells invading the empty space were counted using a Zeiss Axiovert 35 photomicroscope and a KS400 interactive image-analysis system (Carl Zeiss Vision). Six randomly preselected fields (length, 2.5 mm) were analyzed per condition. Results were calculated as the total number of migrated cells per field.

Immunohistochemical Techniques
Immunohistochemical stainings were performed on 4-µm-thick sections from formaldehyde-fixed paraffin-embedded blocks. Sections were deparaffinized and immersed in methanol containing 0.5% H2O2 for 10 minutes. Sections were then incubated for 1 hour at room temperature with the following first antibodies: mouse monoclonal immunoglobulin (Ig)G2a specific for {alpha}-smooth muscle (SM) actin at a dilution of 1:250,13 mouse monoclonal IgG1 anti-desmin at a dilution of 1:10 (Clone D33; Dako), affinity-purified rabbit polyclonal IgG specific for SM myosin heavy chain (MHC) at a dilution of 1:7,14 and undiluted mouse monoclonal IgG1 specific for smoothelin.15

Immunoreactivity of SMMHC and smoothelin staining was intensified by three microwave treatments for 5 minutes in citrate buffer (10 mmol/L, pH 6.0) before using the first antibody. Sections were incubated 30 minutes at room temperature with a secondary biotinylated goat anti-mouse or anti-rabbit antibody at a dilution of 1:250 (Dako). The presence of the specific protein was evaluated by means of the streptavidin-biotin-complex peroxidase method (Dako). Development of peroxidase activity was done with 3-amino-9-ethylcarbazole (Sigma). Slides were counterstained with hematoxylin, mounted in Eukit (Kindler, Freiburg, Germany), and observed using a Zeiss Axiophot photomicroscope (Carl Zeiss) equipped with a Plan-Neofluar x20/0.50 objective (Carl Zeiss). Pictures were acquired with a high-sensitivity Coolview color camera (Photonic Science, London, UK). Images were processed with Adobe Photoshop 5.0 (Adobe System, Mountain View, Calif) and printed with a digital Fujifilm Pictrography 4000 printer (Fujifilm, Tokyo, Japan).

For immunofluorescence, SMCs were cultured in the presence of 10% FCS, and simple or double stainings were performed directly in the culture dishes or on SMCs cytocentrifuged on glass slides.6 Cells were then fixed in methanol for 5 minutes at -20°C and stained with anti–{alpha}-SM actin, in ethanol for 30 seconds at room temperature and stained with anti-SMMHC or anti-desmin, or in methanol/acetone (1:1, vol/vol) for 5 minutes at –20°C and stained with anti-smoothelin. Anti-mouse IgG2a, anti-mouse IgG1, and anti-rabbit IgG coupled with either FITC or TRITC were used as secondary antibodies. Cell counts on cytocentrifuged cells were performed using an Axiophot photomicroscope (Carl Zeiss) at x40 magnification for at least three different fields for each sample. Each experiment was repeated at least three times. Photographs were taken with Tmax 400 film (Eastman Kodak, Rochester, NY) by using oil immersion Plan-Apochromat x40/1.3 and x63/1.4 objectives (Carl Zeiss).

Confocal Laser Scan Microscopy (CLSM) Analysis
Cells were grown in DMEM supplemented with 10% FCS on Labtec slides (Miles Scientific, Naperville, Ill), stimulated for 3 days with heparin at a concentration of 200 µg/mL, fixed in methanol/acetone (1:1, vol/vol) for 5 minutes at -20°C, and stained with anti–{alpha}-SM actin, anti-vimentin, or anti-smoothelin, followed by the above-described secondary antibodies.

Cell specimens were observed with a Zeiss confocal laser scan fluorescence-inverted microscope (LSM 410; Carl Zeiss) equipped with two lasers used simultaneously: a helium-neon laser (excitation wavelength at 543 nm) and an argon laser (excitation wavelength at 488 nm). The excitation spectra were separated by a dichroic beam splitter of 488/543 nm, and the emission spectra of the two fluorochromes were separated by a 560-nm dichroic beam splitter. Two detectors were used in parallel and were preceded with a 590- to 610-nm (rhodamine channel) or a 510- to 525-nm (fluorescein channel) narrow-band barrier filter. The partial superposition of the emission spectra of the two fluorochromes was negligible.16 Specimens were observed through an oil immersion Plan-Neofluar x63/1.4 objective. Between 20 and 40 optical sections of 512x512 pixels separated by 0.25 µm were performed in the z-axis. A three-dimensional image corresponding to the projection of all optical sections in one plan was reconstructed using the Imaris program (Bitplane, Zurich, Switzerland) running on an Octane Silicon Graphics Workstation (Mountain View, Calif). Colocalization of smoothelin and {alpha}-SM actin or vimentin was evaluated by counting the number of pixels containing both stainings (yellow color) in all optical sections using colocalization software developed by Bitplane.

Protein Extraction, Electrophoresis, and Western Blotting
Freshly isolated cells and confluent culture dishes after trypsinization and cell samples were washed in PBS supplemented with protease inhibitors: 10 mmol/L EGTA (Acros Organics), 1 mmol/L TAME (Fluka), 1 mmol/L PMSF (Fluka), 4 mg/100 mL aprotinin (Trasylol; Bayer AG), 0.5 mmol/L benzamidine (Sigma); and 1 mmol/L DFP (Fluka). Cells were resuspended in sample buffer containing all of the aforementioned inhibitors plus 1% SDS (Bio-Rad), 1% DTT (ICN), and 10% glycerol (Fluka), 80 mmol/L Tris-HCl at pH 6.8 (Gibco), and bromophenol blue (Merck), sonicated (Sonifier; Branson Sonic Power Co, Danbury, Conn), and boiled for 3 minutes. Protein concentration was determined according to Bradford.17

Proteins were separated by SDS-PAGE on a 5% to 20% gradient gel and stained with Coomassie brilliant blue (R250; Fluka). For Western blotting, 2 to 20 µg of total protein was electrophoresed and transferred to a nitrocellulose filter (Protran; Schleicher and Schuel, Dassel, Germany). Filters were incubated with either anti–{alpha}-SM actin at a dilution of 1:500, anti-desmin at a dilution of 1:500, or anti-smoothelin antibody at a dilution of 1:5 followed by a goat anti-mouse IgG at a dilution of 1:10 000 or anti-SMMHC at a dilution of 1:100 followed by a goat anti-rabbit IgG at a dilution of 1:10 000. Enhanced chemiluminescence was used for detection (Amersham). Signals were digitized by means of an Arcus II scanner (Agfa, Mortsel, Belgium) and analyzed using the Image Quant software (Image Quant Analysis; Molecular Dynamics, Sunnyvale, Calif). Results are shown as the sum of pixel values of the area of each band.

To separate the different MHC isoforms, samples of total protein were electrophoresed on 4% to 10% gradient gel containing 0.2% N, N'-methylenebisacrylamide. Gels were run at 150 V for 20 hours and were subsequently stained with Coomassie brilliant blue. For Western blotting, 50 µg of total protein was separated and transferred onto a nitrocellulose filter (Protran), which was incubated with a rabbit polyclonal IgG specific for SMMHC isoforms 1 and 2 at a dilution of 1:1000 (Biomedical Technologies Inc). This antibody does not recognize nonmuscle isoform B18 (Robert B. Low, personal communication, 1998). A goat anti-rabbit IgG at a dilution of 1:10 000 was used as second antibody. Enhanced chemiluminescence was used for detection (Amersham). Quantification of results was accomplished as described above.

{alpha}-SM Actin cDNA Probe, RNA Extraction, and Northern Blotting
Because the sequence of the porcine {alpha}-SM actin mRNA is not known, we selected a conserved sequence among mouse, rat, rabbit, and human in the 3' untranslated region (3'UTR). This region is specific for a given isoform19 but may not be species specific. Two primers selected at the extremities of this conserved sequence were used for polymerase chain reaction (PCR).

For practical reasons, PCR amplification was performed on an {alpha}-SM actin cDNA already cloned in a plasmid20 containing parts of the 3'UTR of the gene and yielded a DNA fragment of 121 bp. Two microliters of this plasmid was amplified in 30 cycles (94°C for 30 seconds, 55°C for 60 seconds, and 72°C for 60 seconds). PCR was performed in 1x reaction buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl [pH 9], and 0.1% Triton X-100) containing 25 pmol/µL sense and reverse primer, 25 mmol/L MgCl2 (Promega), 200 µmol/L each dNTP (Promega), and 1 U Taq DNA polymerase (Promega). All PCR reactions began with a hot start. PCR products were then electrophoresed on 2% to 4% agarose gel. DNA was stained with ethidium bromide (Sigma) and visualized in UV light. Elution and purification of specific DNA fragments on gels were performed with a Jetsorb gel extraction kit (Genomed, Research Triangle Park, NC) according to manufacturer information. Ligation and cloning of specific insert were accomplished by using pCR 2.1 Vector (Invitrogen). Identity and orientation of insert in recombinant plasmid were confirmed by sequencing (T7 sequencing kit; Pharmacia Biotech, Duebendorf, Switzerland) using a sequencing gel electrophoresis apparatus (model S2001; Gibco).

For yet-unexplained reasons, direct extraction of total RNA from the media of coronary arteries resulted in an important degree of RNA degradation. Therefore, total RNA was extracted from freshly isolated cells of the media according to manufacturer instructions as for confluent culture dishes. For Northern blot hybridization, 10 to 20 µg of total RNA was denatured with glyoxal, separated by electrophoresis on 1% agarose gel and blotted on Hybond N-filters (Amersham). After UV fixation, filters were stained with methylene blue to verify correct loading and transfer. Filters were then processed for hybridization by random priming (Megaprime DNA labeling; Amersham) of specific cDNA probes for {alpha}-SM actin. Prehybridization and hybridization were performed at 50°C for 3 and 15 hours, respectively, in 5x SSC, 5x Denhardt solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% BSA), 0.1% SDS, and 100 µg/mL salmon sperm DNA. After hybridization, filters were washed twice for 15 minutes at 50°C in 4x SSC and 0.1% SDS. They were then exposed to Kodak X-Omat SO-282 (Eastman Kodak) at -70°C for 24 hours to 3 days. Autoradiographic signals were digitized and processed as described above. {alpha}-SM actin mRNA levels were calculated taking into account differences in loading between lanes as assessed by scanning the 18S band of the methylene blue–stained membrane.

Statistical Analysis
Results are shown as mean±SEM. For statistical evaluation, the results were analyzed by Student t test. Differences were considered statistically significant at values of P<0.05.


*    Results
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 *Results
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Expression of SMC Differentiation Markers In Vivo
The porcine LAD has a mean diameter of {approx}4 mm just after branching away from the left circumflex artery, similar to the corresponding human vessel. Van Gieson elastin staining showed the typical architecture of a muscular artery with interrupted elastic fibers within the media (Figure 1aDown). Elastic fibers were abundant in the internal portion of the adventitia.



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  		Figure 1. Immunohistochemistry of normal porcine coronary artery. a, Van Gieson elastin staining showing collagen and elastin expression in a normal porcine coronary artery. Note the abundance of elastic fibers in the internal portion of the adventitia. Immunoperoxidase staining for {alpha}-SM actin (b), SMMHC (c), desmin (d), and smoothelin (e) shows that nearly all SMCs in the media express these markers whereas endothelial and adventitial cells do not. Bar=100 µm.

For immunohistochemical analysis of the coronary arteries, we chose a set of four well-accepted SMC differentiation markers: {alpha}-SM actin, desmin, SMMHC, and smoothelin, a protein recently described in arterial tissue, probably the most specific marker of well-differentiated SMCs.15 21 Almost all SMCs in the media expressed {alpha}-SM actin and SMMHC (Figure 1bUp and 1cUp) whereas a small proportion of cells in the media appeared negative for desmin and smoothelin (Figure 1dUp and 1eUp). Endothelial and adventitial cells were negative for all markers. All markers showed a similar staining distribution in proximal compared with distal segments of the same vessel (data not shown).

Features of SMCs In Vitro
Enzymatic digestion was preferred to explant method because it allows to include an SMC population as representative as possible of the in vivo setting.3 During primary culture and after subsequent passages, SMCs exhibited the typical spindle-shaped morphology with a hills-and-valleys pattern at confluence. SMCs displayed typical stress fibers after staining with anti–{alpha}-SM actin antibody (Figure 2aDown). The SMMHC staining demonstrated the classical interrupted distribution at the cell periphery (Figure 2bDown). The anti-desmin antibody showed a pattern irradiating from the nucleus to the cell surface (Figure 2cDown). The distribution of smoothelin has remained controversial up to now because this protein was not expressed spontaneously in cultured cells from conventional sources.15 22 In our SMCs, a stress fiber–like organization of smoothelin was observed (Figure 2dDown) with a strong intensity in the center of the cell and decreasing gradually toward the cell periphery.



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  		Figure 2. Morphological and cytoskeletal features of porcine coronary artery SMCs in vitro. Immunofluorescence staining of porcine SMCs at the 5th passage for {alpha}-SM actin (a), SMMHC with typical interrupted distribution of the protein (b), desmin (c), and smoothelin (d). Note the stress fiber pattern of smoothelin distribution. Bar=5 µm for panels a, c, and d; 5 µm for panel b.

CLSM analysis performed on cultured porcine SMCs showed that smoothelin was colocalized with {alpha}-SM actin (Figure 3aDown) and not with vimentin (Figure 3bDown). Colocalized pixels represented 85% of the total pixel number in the {alpha}-SM actin/smoothelin double staining and only 3% in the vimentin/smoothelin double staining.



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  		Figure 3. {alpha}-SM actin, vimentin, and smoothelin expression in porcine coronary artery SMCs examined by CLSM. Cells were double stained for {alpha}-SM actin (green, a) or vimentin (green, b) and smoothelin (red, a and b). Three-dimensional reconstruction by projection of optical sections in one plan shows a strong colocalization for {alpha}-SM actin and smoothelin along the stress fibers in panel a (yellow staining, corresponding to the overlay of red and green stainings) but not in panel b for vimentin and smoothelin. Bar=5 µm.

Immunofluorescence staining performed on cytocentrifuged SMCs showed that in accordance with in vivo observations, almost all freshly isolated SMCs (0 hours) were positive for {alpha}-SM actin and SMMHC (Figure 4Down). About 80% of the cells were positive for desmin and smoothelin. After 7 days of primary culture (P0), the proportion of positive cells decreased to {approx}70% for {alpha}-SM actin (P<0.01), to 40% for desmin (P<0.01), and to 35% for smoothelin (P<0.01). The proportion of SMMHC-expressing cells remained unchanged. The intensity of staining however clearly decreased in about half of SMMHC-positive SMCs; it remained at this level up to the 5th passage (P5). After one passage (P1), the percentage of {alpha}-SM actin– and smoothelin-positive SMCs remained at a level similar to that of primary culture whereas the percentage of desmin-positive SMCs decreased to {approx}30% (P1 versus P0, P<0.05). At the 5th passage, the percentage of {alpha}-SM actin–expressing cells increased to a level similar to that of 0 hours (P5 versus P0 and P1, P<0.01). The proportion of desmin-positive cells also increased to a level similar to that of primary culture (P5 versus P1, P<0.05). The percentage of smoothelin-positive cells further decreased to {approx}15% (P5 versus P0 and P1, P<0.01). These results are different from those previously described for adult rat aortic SMCs in which percentages of {alpha}-SM actin–, SMMHC-, and desmin-positive cells represent only {approx}80%, 45%, and 30%, respectively, at P0 and 70%, 3%, and 0%, respectively, at P5.3 6 23 Thus, porcine coronary artery SMCs maintain a high degree of differentiation in vitro.



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  		Figure 4. Percentage of porcine coronary artery SMCs expressing differentiation markers in vitro. Histogram showing the percentage of {alpha}-SM actin–, SMMHC-, desmin-, and smoothelin-positive cells just after enzymatic digestion (0 hr), in primary culture (P0), after 1 passage (P1), and after 5 passages (P5). The number of {alpha}-SM actin–positive cells decreases initially in culture and then increases at P5 to the value observed at 0 hours. The proportion of SMMHC-expressing cells does not change during subculture, whereas the percentage of desmin-positive cells and of smoothelin-positive cells decreases progressively during subculture. n=3 experiments for each condition. *P<0.05, **P<0.01 compared with 0 hours.

Immunoblotting results were in agreement with those of immunofluorescence studies (Figure 5Down). In primary culture, the content of {alpha}-SM actin was decreased to {approx}80% (P<0.01), that of SMMHC to 50% (P<0.01), that of desmin to 60% (P<0.05), and that of smoothelin to 40% (P<0.01) compared with 0 hours. Values further decreased at the 1st passage, to {approx}60% for {alpha}-SM actin and 30% for SMMHC (P<0.05) whereas desmin and smoothelin values did not change. At the 5th passage, {alpha}-SM actin content increased and reached a value close to that of primary culture (P5 versus P1, P<0.05). The SMMHC and desmin contents were maintained at the levels observed at the 1st passage, ie, 40% and 60% of the value at 0 hours, respectively (P1 versus P5, not significant, P5 versus 0 hours, P<0.05). Smoothelin expression at the 5th passage represented 10% of the value at 0 hours and was significantly smaller than in primary culture (P<0.05). All studied proteins, including desmin and smoothelin, remained clearly detectable up to the 9th passage (data not shown). In rat aortic SMCs, the decrease of {alpha}-SM actin, SMMHC, and desmin content during culture has been shown to be more pronounced than that observed in the pig model: at the 5th passage, {alpha}-SM actin content is only {approx}15% of the value at 0 hours whereas SMMHC and desmin proteins are not detectable.3 6 23



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  		Figure 5. Expression of differentiation markers in porcine coronary artery SMCs in vitro. A, SDS-PAGE of total protein extracts (a through d) and immunoblots (e through h) showing {alpha}-SM actin, SMMHC, desmin, and smoothelin expression in SMCs just after enzymatic digestion (a and e), in primary culture (b and f), after 1 passage (c and g), and after 5 passages (d and h). B, Densitometric scanning of immunoblots with values normalized to the pixel values measured in freshly isolated cells (0 hr). {alpha}-SM actin, SMMHC, and desmin content decreases in primary culture (P0) to a level that is maintained at P1 and P5. Smoothelin content progressively decreases up to P5. n=3 experiments for each condition. *P<0.05, **P<0.01 compared with 0 hours.

When separated by electrophoresis, the SMMHC isoforms 1 and 2 exhibited a similar expression profile. In subculture, they represented {approx}50% of the value detected at 0 hours (P<0.05, data not shown). In passaged rat or rabbit arterial SMCs, no expression of SMMHC 2 protein has been reported.24 25 26 27 The expression of nonmuscle MHC isoform B remained unchanged up to the 5th passage (data not shown). Confluence at the 5th passage did not affect the level of {alpha}-SM actin expression (data not shown), again different from previously reported rat data.3 28 Thus, porcine coronary artery SMCs remain relatively well differentiated in culture.

The expression of {alpha}-SM actin mRNA remained constant from freshly isolated cells up to the 5th passage (n=4 experiments for each condition; data not shown). Moreover the probe gave a specific signal also with rat and human SMC mRNA (data not shown).

Effect of Heparin, TGF-ß1 or TGF-ß2, and tRA
Several laboratories, including ours, have shown that heparin,29 30 TGF-ß1 or TGF-ß2,31 32 and, more recently, tRA33 are able to modulate cultured rat arterial SMC phenotype. Heparin, as well as TGF-ß1 or TGF-ß2, inhibits proliferation and induces differentiation of rat arterial SMCs, albeit through different mechanisms.29 30 31 32 tRA is also capable of affecting differentiation of these cells.33 To test whether porcine SMC features can be modulated in culture by these agents, we exposed cells to heparin (200 µg/mL), TGF-ß1 (10 ng/mL), TGF-ß2 (10 ng/mL), or tRA (10-6 mol/L) for 6 days.

When compared with control cells, the proliferation of SMCs stimulated during 7 days with 10% FCS was inhibited by 50% (P<0.01) in heparin-treated cells and 30% (P<0.01) in cells growing in TGF-ß1– or TGF-ß2–supplemented medium (Figure 6ADown). tRA had no effect on cell proliferation. The [3H]-thymidine incorporation rate, measured in synchronized SMCs 24 hours after addition of the different agents, was decreased to 40% (P<0.01) for heparin-treated cells and to only 60%, 75%, and 80% (P<0.01) for cells treated with TGF-ß1, TGF-ß2, and tRA, respectively (Figure 6BDown) when compared with control cells.



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  		Figure 6. Effect of heparin, TGF-ß1, TGF-ß2, and tRA on proliferative activity of cultured porcine coronary artery SMCs. A, Histogram showing the proliferation rate of SMCs treated during 7 days with 10% FCS alone (control) or supplemented 24 hours after plating with heparin (200 µg/mL), TGF-ß1 or TGF-ß2 (10 ng/mL), or tRA (10-6 mol/L). B, Histogram showing [3H]-thymidine incorporation rate of synchronized SMCs treated for 24 hours with 10% FCS alone (control) or supplemented with one of the different agents described above. The [3H]-thymidine incorporation (total cpm) is normalized to the control value. Heparin is the most efficient agent to inhibit proliferation and [3H]-thymidine incorporation of SMCs. n=5 experiments for each condition. *P<0.05, **P<0.01 compared with control.

Migratory activity was assessed by measuring the capability of SMCs to invade an in vitro wound when treated with FCS alone or supplemented with one of the different agents (Figure 7Down). After 24 hours of treatment, heparin decreased migration to {approx}40% and TGF-ß1 to {approx}60% when compared with control; TGF-ß2 and tRA had no significant effect under our conditions.



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  		Figure 7. Effect of heparin, TGF-ß1, TGF-ß2, and tRA on migratory activity of cultured porcine coronary artery SMCs. Histogram showing migratory capacity of cells cultured in the presence of 10% FCS without (control) or with heparin (200 µg/mL), TGF-ß1 or TGF-ß2 (10 ng/mL), or tRA (10-6 mol/L). Heparin is the strongest inhibitor of SMC migratory activity. n=3 experiments for each condition. *P<0.05, **P<0.01 compared with control.

Heparin increased the protein expression of SMMHC 2, desmin, and smoothelin by 3.5-fold (P<0.05), 4-fold (P<0.01), and 3-fold (P<0.05), respectively, whereas levels of {alpha}-SM actin and SMMHC 1 were not significantly changed (Figure 8Down). TGF-ß1 caused only a slight but significant decrease in SMMHC 2 (P<0.01) and a 2-fold increase in the protein expression of desmin (P<0.01). The expression of all other markers tested did not change after treatment with TGF-ß1. The only effect of TGF-ß2 was to cause a slight but significant decrease in SMMHC 2 (P<0.01). tRA decreased only the expression of desmin by 30% (P<0.01).



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  		Figure 8. Effect of heparin, TGF-ß1, TGF-ß2, and tRA on protein expression in cultured porcine coronary artery SMCs. A, Immunoblots showing protein content of cells cultured in the presence of 10% FCS without (control) or with heparin (200 µg/mL), TGF-ß1 or TGF-ß2 (10 ng/mL), or tRA (10-6 mol/L) at the 5th passage for {alpha}-SM actin, SMMHC isoform 1 and 2, desmin, and smoothelin. B, Histogram showing densitometric scanning of immunoblots with values normalized to the sum of the pixel values measured for control samples. Seven days of heparin treatment increases protein expression for SMMHC isoform 2, desmin, and smoothelin. TGF-ß1 treatment increases the expression of desmin only. n=3 experiments for each condition. *P<0.05, **P<0.01 compared with control.

The effects of the different agents on SMCs were also tested in absence of serum. As expected, SMCs did not grow in Monomed.12 The migration of cells was inhibited by treatment with heparin and not with the other agents. Serum withdrawal itself had no effect on {alpha}-SM actin expression but increased the expression of SMMHC 2 and to a lesser extent of desmin and smoothelin when compared with the 10% FCS condition, as assessed by Western blot analysis (data not shown). The expression of all differentiation markers was not affected by any of the above-mentioned agents compared with the Monomed condition (data not shown).


*    Discussion
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 *Discussion
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Our results show that SMCs cultured from porcine coronary artery retain a higher degree of differentiation than has been described in previous studies of SMCs cultured from animals such as the rat and rabbit, and that they maintain these properties even after several passages in culture. Thus, porcine cells represent a new model for examining the properties of well-differentiated SMCs, as well as regulation of the expression of differentiation markers such as SMMHC and smoothelin and their isoforms.

Porcine coronary arteries are very similar to human vessels in size and histology. Of all species examined to date, the pig is most similar to the human in its cardiovascular morphology and physiology, as well as its susceptibility to atheromatosis.34 35 Human arterial SMCs also have been reported to replicate relatively more slowly and to maintain a high degree of differentiation in terms of markers such as SMMHC36 compared with rodent SMCs.3 The fact that cultured porcine coronary artery SMCs also remain highly differentiated further suggests that the porcine culture model will be of importance in terms of improving our understanding of human vascular disease. It should be stressed that no well-established data on differentiation features of human vascular SMCs are present in the literature. The scarcity of data may be due to difficulties in obtaining standardized SMC populations from human donors.

Cytoskeletal proteins are well-accepted markers of the differentiation state of SMCs. Usually, a constellation of markers is necessary to assess SMC differentiation,37 and we have followed the same criterion for cultured porcine coronary artery SMCs. As in the rat model, almost all porcine SMCs express {alpha}-SM actin and SMMHC in vivo; but, differently from rat aortic SMCs where desmin is found only in half of the cells,38 most porcine coronary SMCs express this marker. Smoothelin, which cannot be studied in the rat with the presently available antibody, is present in practically all porcine SMCs, as previously described in human arteries.15

The expression of {alpha}-SM actin, desmin, and SMMHC is maintained at much higher levels in porcine SMCs compared with rat and rabbit SMCs when using such criteria as number of positive cells and protein or mRNA contents.4 6 23 In newborn rat arterial SMCs, the only situation in which SMMHC is expressed at a relatively high level, only 43% of the cells are positive at the 5th passage.6 In the present study, we additionally have been able to discriminate between SMMHC 1 and 2 protein expression. Both isoforms are markers of an advanced state of SMC differentiation, SMMHC 2 appears only at birth.27 37 39 At the 5th passage, >90% of the cells were positive for SMMHC 1 and 2 whereas only a very small percentage has been shown to be positive in adult rat arterial SMCs.40 Moreover, SMMHC 2 protein was expressed in passaged porcine SMCs, although it has been shown that its expression at the protein level is strictly limited to primary culture of adult rat arterial SMCs.24 It is noteworthy that {approx}50% of porcine coronary SMCs expressed desmin up to the 5th passage, whereas in rat aortic SMCs, desmin is not detectable in similar conditions.3 6

In general, the relationships between {alpha}-SM actin mRNA and protein expression correspond more closely in porcine coronary artery SMCs than they do for rat and rabbit.4 23 This suggests a tighter coupling between transcription and translation than is found in other species.

Smoothelin is a recently described cytoskeletal protein specific for SMCs; during development, it appears later than {alpha}-SM actin, desmin, and SMMHC.21 Smoothelin expression disappears during culture in all primary and passaged SMCs tested to date.15 Porcine coronary SMCs, however, maintain expression of smoothelin protein up to the 9th passage. Two smoothelin isoforms have been described: a small isoform of 59 kDa and a large isoform specific to vascular SMCs, to which the molecular mass of 94 kDa had been assigned previously.21 22 41 We have systematically tested cultured SMCs and arterial tissues, including those samples used for previous studies,21 22 41 and conclude that the molecular mass of the large isoform is 115 kDa, as assessed by electrophoretic migration.

The expression of smoothelin in cultured porcine SMCs allowed us to establish by CLSM that at least in cultured SMCs, smoothelin is colocalized with {alpha}-SM actin and not with intermediate filaments. In transfection experiments, smoothelin displayed a stress fiber–like pattern, an intermediate filament–like pattern, or both, depending on the cell type used.15 It is noteworthy in those regards that a putative actin-binding site has been suggested in the sequence of the smoothelin gene.15 Additional work using CLSM in tissues and assessing the binding capacity of smoothelin to {alpha}-SM actin will furnish more information on the possible interaction of the two proteins.

We have tested the effects of heparin, TGF-ß1 or TGF-ß2, and tRA that are known to modulate SMC differentiation, proliferation, and migration. Heparin, used at a concentration that has shown maximal effects on rat SMC proliferation,30 inhibited proliferation and increased differentiation marker expression only in the presence of serum whereas migration was inhibited by heparin also in the absence of serum. Similar findings have been described for rat and rabbit SMCs.30 42 43 44 45 46 Heparin did not affect the expression of {alpha}-SM actin and SMMHC 1 but increased the expression of SMMHC 2, desmin, and smoothelin, suggesting that a low level of protein expression is a prerequisite for heparin activity.

Both TGF-ß isoforms reduced proliferation in porcine coronary SMCs, but migration was inhibited by only TGF-ß1. It is noteworthy that proliferation of rat SMCs can be inhibited or stimulated by TGF-ß1 according to the concentration per cell used.47 48 49 Both TGF-ß isoforms had no inducing effect on {alpha}-SM actin or SMMHC and in fact decreased expression of SMMHC 2. This finding is different from what Hautmann et al50 have shown in rat arterial SMCs, for both {alpha}-SM actin and SMMHC. However, these investigators used a lower dose of TGF-ß and different growth conditions than we used in the present study. Interestingly, TGF-ß1 increased desmin expression in porcine SMCs, a marker that is rarely modulated in other models.30

tRA is considered to be a potential inducer of SMC differentiation.51 52 53 However, proliferation, migration, and differentiation marker expression were not changed in porcine SMCs when treated with tRA. Nevertheless tRA, similarly to heparin and TGF-ß1 or TGF-ß2, reduced serum-stimulated [3H]-thymidine incorporation. tRA inhibits rat arterial SMC proliferation,54 55 56 and [3H]-thymidine incorporation is decreased in only SMC whole populations and clones with an epithelioid phenotype.56 Moreover, IT development after endothelial injury is reduced in rats fed with a tRA-supplemented diet.56 57 On the contrary, tRA stimulates migration of cultured rat SMCs as well as the expression of tissue-type plasminogen activator, the main enzyme responsible for the degradation of extracellular matrix in this cell type.56 58 Interestingly, in cultured adult rat aortic SMCs56 and in the present model, {alpha}-SM actin expression was not affected by tRA. Taken together, these findings suggest that tRA acts mainly on poorly differentiated SMCs.

In conclusion, we have defined several biological and differentiation features of cultured porcine coronary artery SMCs. We have shown that they differ importantly from those established previously for rat and rabbit SMCs in terms of maintaining a more advanced degree of differentiation; in this respect, they appear to exhibit features reminiscent of those observed in human arterial SMCs. Our results indicate the potential value of these cells in the analysis of differentiation marker expression in SMCs. Porcine cells derived from the normal media or from the intimal thickening induced by angioplasty and/or stenting may provide further insight into the mechanisms involved in those processes as well as on the action of substances capable of influencing the biological behavior of SMCs in vitro and possibly in vivo.


*    Acknowledgments
 
This study was supported by the Swiss National Science Foundation, grant No. 31.50568.97, and by the Dutch Heart Foundation, grant No. D97.014. We are grateful to R.B. Low for a critical reading of the manuscript. We thank F. Gabbiani, P. Ropraz, and A. Geinoz for their expert technical assistance, J.-C. Rumbeli and E. Denkinger for photographic work, and M. Vitali for help in typing the manuscript.

Received February 19, 1999; accepted April 30, 1999.


*    References
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 *References
 

  1. Schwartz SM, O'Brien ER, de Blois D, Giachelli CM. Relevance of smooth muscle replication and development to vascular disease. In: Schwartz SM, Mecham RP, eds. The Vascular Smooth Muscle Cell. Molecular and Biological Responses to the Extracellular Matrix. San Diego, Calif: Academic Press Inc; 1995:81–140.
  2. Kocher O, Gabbiani G. Expression of actin mRNAs in rat aortic smooth muscle cells during development, experimental thickening, and culture. Differentiation. 1986;32:245–251.[Medline] [Order article via Infotrieve]
  3. Skalli O, Bloom WS, Ropraz P, Azzarone B, Gabbiani G. Cytoskeletal remodeling of rat aortic smooth muscle cells in vitro: relationships to culture conditions and analogies to in vivo situations. J Submicrosc Cytol. 1986;18:481–493.[Medline] [Order article via Infotrieve]
  4. Campbell JH, Kocher O, Skalli O, Gabbiani G, Campbell GR. Cytodifferentiation and expression of {alpha}-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells. Correlation with cell density and proliferative state. Arteriosclerosis. 1989;9:633–643.[Abstract/Free Full Text]
  5. Thyberg J, Hedin U, Sjölund M, Palmberg L, Bottger BA. Regulation of differentiated properties and proliferation of arterial smooth muscle cells. Arteriosclerosis. 1990;10:996–990.[Abstract/Free Full Text]
  6. Bochaton-Piallat M-L, Gabbiani F, Ropraz P, Gabbiani G. Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features. Differentiation. 1992;49:175–185.[Medline] [Order article via Infotrieve]
  7. Birukov KG, Frid MG, Rogers JD, Shirinsky VP, Koteliansky VE, Campbell JH, Campbell GR. Synthesis and expression of smooth muscle phenotype markers in primary culture of rabbit aortic smooth muscle cells: influence of seeding density and media and relation to cell contractility. Exp Cell Res. 1993;204:46–53.[Medline] [Order article via Infotrieve]
  8. Schwartz RS, Edwards WD, Huber KC, Antoniades LC, Bailey KR, Camrud AR, Jorgenson MA, Holmes DR. Coronary restenosis: prospects for solution and new perspectives from a porcine model. Mayo Clin Proc. 1993;68:54–62.[Medline] [Order article via Infotrieve]
  9. Zalewski A, Shi Y. Vascular myofibroblasts. Lessons from coronary repair and remodeling. Arterioscler Thromb Vasc Biol. 1997;17:417–422.[Free Full Text]
  10. Gal D, Rongione AJ, Slovenkai GA, DeJesus ST, Lucas A, Fields CD, Isner JM. Atherosclerotic Yucatan microswine: an animal model with high-grade, fibrocalcific, nonfatty lesions suitable for testing catheter-based interventions. Am Heart J. 1990;119:291–300.[Medline] [Order article via Infotrieve]
  11. Grinstead WC, Rodgers GP, Mazur W, French BA, Cromeens D, Van Pelt C, West SM, Raizner AE. Comparison of three porcine restenosis models: the relative importance of hypercholesterolemia endothelial abrasion, and stenting. Coron Artery Dis. 1994;5:425–434.[Medline] [Order article via Infotrieve]
  12. Bochaton-Piallat M-L, Ropraz P, Gabbiani F, Gabbiani G. Phenotypic heterogeneity of rat arterial smooth muscle cell clones. Implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol. 1996;16:815–820.[Abstract/Free Full Text]
  13. Skalli O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, Gabbiani G. A monoclonal antibody against {alpha}-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol. 1986;103:2787–2796.[Abstract/Free Full Text]
  14. Benzonana G, Skalli O, Gabbiani G. Correlation between the distribution of smooth muscle or nonmuscle myosins and {alpha}-smooth muscle actin in normal and pathological soft tissues. Cell Motil Cytoskeleton. 1988;11:260–274.[Medline] [Order article via Infotrieve]
  15. van der Loop FT, Schaart G, Timmer ED, Ramaekers FCS, van Eys GJ. Smoothelin, a novel cytoskeletal protein specific for smooth muscle cells. J Cell Biol. 1996;134:401–411.[Abstract/Free Full Text]
  16. Serini G, Bochaton-Piallat M-L, Ropraz P, Geinoz A, Borsi L, Zardi L, Gabbiani G. The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-ß1. J Cell Biol. 1998;142:873–881.[Abstract/Free Full Text]
  17. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.[Medline] [Order article via Infotrieve]
  18. Hirschi KK, Rohovsky SA, D'Amore PA. PDGF, TGF-ß, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate. J Cell Biol. 1998;141:805–814.[Abstract/Free Full Text]
  19. Ponte P, Gunning P, Blau H, Kedes L. Human actin genes are single copy for {alpha}-skeletal and {alpha}-cardiac actin but multicopy for ß-cytoskeletal genes: 3' untranslated regions are isotype specific but are conserved in evolution. Mol Cell Biol. 1983;3:1783–1791.[Abstract/Free Full Text]
  20. Kocher O, Gabbiani G. Analysis of {alpha}-smooth-muscle actin mRNA expression in rat aortic smooth-muscle cells using a specific cDNA probe. Differentiation. 1987;34:201–209.[Medline] [Order article via Infotrieve]
  21. van der Loop FTL, Gabbiani G, Kohnen G, Ramaekers FCS, van Eys GJJM. Differentiation of smooth muscle cells in human blood vessels as defined by smoothelin, a novel marker for the contractile phenotype. Arterioscler Thromb Vasc Biol. 1997;17:665–671.[Abstract/Free Full Text]
  22. van Eys GJJM, Völler MCW, Timmer EDJ, Wehrens XHT, Small JV, Schalken JA, Ramaekers FCS, van der Loop FTL. Smoothelin expression characteristics: development of a smooth muscle cell in vitro system and identification of a vascular variant. Cell Struct Funct. 1997;22:65–72.[Medline] [Order article via Infotrieve]
  23. Barja F, Coughlin C, Belin D, Gabbiani G. Actin isoform synthesis and mRNA levels in quiescent and proliferating rat aortic smooth muscle cells in vivo and in vitro. Lab Invest. 1986;55:226–233.[Medline] [Order article via Infotrieve]
  24. Rovner AS, Murphy RA, Owens GK. Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells. J Biol Chem. 1986;261:14740–14745.[Abstract/Free Full Text]
  25. Babij P, Kawamoto S, White S, Adelstein RS, Periasamy M. Differential expression of SM1 and SM2 myosin isoforms in cultured vascular smooth muscle. Am J Physiol. 1992;262:C607–C613.[Abstract/Free Full Text]
  26. Sartore S, Chiavegato A, Franch R, Faggin E, Pauletto P. Myosin gene expression and cell phenotypes in vascular smooth muscle during development, in experimental models, and in vascular disease. Arterioscler Thromb Vasc Biol. 1997;17:1210–1215.[Abstract/Free Full Text]
  27. Low RB, White SL. Lung smooth muscle differentiation. Int J Biochem Cell Biol. 1998;30:869–883.[Medline] [Order article via Infotrieve]
  28. Owens GK, Loeb A, Gordon D, Thompson MM. Expression of smooth muscle-specific {alpha}-isoactin in cultured vascular smooth muscle cells: relationship between growth and cytodifferentiation. J Cell Biol. 1986;102:343–352.[Abstract/Free Full Text]
  29. Clowes AW, Clowes MM. Kinetics of cellular proliferation after arterial injury, II: inhibition of smooth muscle growth by heparin. Lab Invest. 1985;52:611–616.[Medline] [Order article via Infotrieve]
  30. Desmoulière A, Rubbia-Brandt L, Gabbiani G. Modulation of actin isoform expression in cultured arterial smooth muscle cells by heparin and culture conditions. Arterioscler Thromb. 1991;11:244–253.[Abstract/Free Full Text]
  31. Björkerud S. Effects of transforming growth factor-ß1 on human arterial smooth muscle cells in vitro. Arterioscler Thromb. 1991;11:892–902.[Abstract/Free Full Text]
  32. Orlandi A, Ropraz P, Gabbiani G. Proliferative activity and {alpha}-smooth muscle actin expression in cultured rat aortic smooth muscle cells are differently modulated by transforming growth factor-ß1 and heparin. Exp Cell Res. 1994;214:528–536.[Medline] [Order article via Infotrieve]
  33. Neuville P, Geinoz A, Benzonana G, Redard M, Gabbiani F, Ropraz P, Gabbiani G. Cellular retinol-binding protein I is expressed by distinct subsets of rat arterial smooth muscle cells in vitro and in vivo. Am J Pathol. 1997;150:509–521.[Abstract]
  34. Schwartz RS, Murphy JG, Edwards WD, Camrud AR, Vlietstra RE, Holmes DR. Restenosis after balloon angioplasty: a practical proliferative model in porcine coronary arteries. Circulation. 1990;82:2190–2200.[Abstract/Free Full Text]
  35. Muller DW, Ellis SG, Topol EJ. Experimental models of coronary artery restenosis [erratum appears in J Am Coll Cardiol. 1992;19:1678]. J Am Coll Cardiol. 1992;19:418–432.
  36. Fager G, Hansson GK, Gown AM, Larson DM, Skalli O, Bondjers G. Human arterial smooth muscle cells in culture: inverse relationship between proliferation and expression of contractile proteins. In Vitro Cell Dev Biol. 1989;25:511–520.[Medline] [Order article via Infotrieve]
  37. Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiol Rev. 1995;75:487–517.[Abstract/Free Full Text]
  38. Gabbiani G, Schmid E, Winter S, Chaponnier C, de Chastonay C, Vanderkerckhove J, Weber K, Franke WW. Vascular smooth muscle cells differ from other smooth muscle cells: predominance of vimentin filaments and a specific {alpha}-type actin. Proc Natl Acad Sci U S A. 1981;78:298–302.[Abstract/Free Full Text]
  39. Borrione AC, Zanellato AMC, Scannapieco G, Pauletto P, Sartore S. Myosin heavy-chain isoforms in adult and developing rabbit vascular smooth muscle. Eur J Biochem. 1989;183:413–417.[Medline] [Order article via Infotrieve]
  40. Bochaton-Piallat M-L, Gabbiani F, Ropraz P, Gabbiani G. Age influences the replicative activity and the differentiation features of cultured rat aortic smooth muscle cell populations and clones. Arterioscler Thromb. 1993;13:1449–1455.[Abstract/Free Full Text]
  41. Wehrens XHT, Mies B, Gimona M, Ramaekers FCS, van Eys GJJM, Small JV. Localization of smoothelin in avian smooth muscle and identification of a vascular-specific isoform. FEBS Lett. 1997;405:315–320.[Medline] [Order article via Infotrieve]
  42. Chamley-Campbell JH, Campbell GR. What controls smooth muscle phenotype? Atherosclerosis. 1981;40:347–357.[Medline] [Order article via Infotrieve]
  43. Castellot JJ, Favreau LV, Karnovsky MJ, Rosenberg RD. Inhibition of vascular smooth muscle cell growth by endothelial cell-derived heparin. Possible role of a platelet endoglycosidase. J Biol Chem. 1982;257:11256–11260.[Abstract/Free Full Text]
  44. Weissberg PL, Grainger DJ, Shanahan CM, Metcalfe JC. Approaches to the development of selective inhibitors of vascular smooth muscle cell proliferation. Cardiovasc Res. 1993;27:1191–1198.[Free Full Text]
  45. Kohno M, Yokokawa K, Yasunari K, Minami M, Kano H, Mandal AK, Yoshikawa J. Heparin inhibits human coronary artery smooth muscle cell migration. Metabolism. 1998;47:1065–1069.[Medline] [Order article via Infotrieve]
  46. Koyama N, Kinsella MG, Wight TN, Hedin U, Clowes AW. Heparan sulfate proteoglycans mediate a potent inhibitory signal for migration of vascular smooth muscle cells. Circ Res. 1998;83:305–313.[Abstract/Free Full Text]
  47. Majack RA, Goodman LV, Dixit VM. Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation. J Cell Biol. 1988;106:415–422.[Abstract/Free Full Text]
  48. Battegay EJ, Raines EW, Seifert RA, Bowen-Pope DF, Ross R. TGF-ß induces bimodal proliferation of connective tissue cells via complex control of an autocrine PDGF loop. Cell. 1991;63:515–524.
  49. Janat MF, Liau G. Transforming growth factor ß1 is a powerful modulator of platelet-derived growth factor action in vascular smooth muscle cells. J Cell Physiol. 1992;150:232–242.[Medline] [Order article via Infotrieve]
  50. Hautmann MB, Madsen CS, Owens GK. A transforming growth factor ß (TGFß) control element drives TGFß-induced stimulation of smooth muscle {alpha}-actin gene expression in concert with two CArG elements. J Biol Chem. 1997;272:10948–10956.[Abstract/Free Full Text]
  51. Blank RS, Swartz EA, Thompson MM, Olson EN, Owens GK. A retinoic acid-induced clonal cell line derived from multipotential P19 embryonal carcinoma cells expresses smooth muscle characteristics. Circ Res. 1995;76:742–749.[Abstract/Free Full Text]
  52. Colbert MC, Kirby ML, Robbins J. Endogenous retinoic acid signaling colocalizes with advanced expression of the adult smooth muscle myosin heavy chain isoform during development of the ductus arteriosus. Circ Res. 1996;78:790–798.[Abstract/Free Full Text]
  53. Gollasch M, Haase H, Ried C, Lindschau C, Morano I, Luft FC, Haller H. L-type calcium channel expression depends on the differentiated state of vascular smooth muscle cells. FASEB J. 1998;12:593–601.[Abstract/Free Full Text]
  54. Miano JM, Topouzis S, Majesky MW, Olson EN. Retinoid receptor expression and all-trans retinoic acid-mediated growth inhibition in vascular smooth muscle cells. Circulation. 1996;93:1886–1895.[Abstract/Free Full Text]
  55. Chen SC, Gardner DG. Retinoic acid uses divergent mechanisms to activate or suppress mitogenesis in rat aortic smooth muscle cells. J Clin Invest. 1998;102:653–662.[Medline] [Order article via Infotrieve]
  56. Neuville P, Yan ZQ, Gidlöf A, Pepper MS, Hansson GK, Gabbiani G, Sirsjö A. Retinoic acid regulates arterial smooth muscle cell proliferation and phenotypic features in vivo and in vitro through an RAR{alpha}-dependent-signaling pathway. Arterioscler Thromb Vasc Biol. 1999;19:1430–1436.[Abstract/Free Full Text]
  57. Miano JM, Kelly LA, Artacho CA, Nuckolls TA, Piantedosi R, Blaner WS. all-Trans-retinoic acid reduces neointimal formation and promotes favorable geometric remodeling of the rat carotid artery after balloon withdrawal injury. Circulation. 1998;98:1219–1227.[Abstract/Free Full Text]
  58. Bochaton-Piallat ML, Gabbiani G, Pepper MS. Plasminogen activator expression in rat arterial smooth muscle cells depends on their phenotype and is modulated by cytokines. Circ Res. 1998;82:1086–1093.[Abstract/Free Full Text]



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