© 1999 American Heart Association, Inc.
Rapid Communications |
Cultured Porcine Coronary Artery Smooth Muscle Cells
A New Model With Advanced Differentiation
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
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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
-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 -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
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Introduction |
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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.
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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
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 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 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 -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–-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–-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 -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–-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.
-SM Actin cDNA Probe, RNA Extraction, and Northern
Blotting
Because the sequence of the porcine -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 -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 -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. -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.
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Expression of SMC Differentiation Markers In Vivo
The porcine LAD has a mean diameter of
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 1a
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Elastic fibers were abundant in the internal portion of the adventitia.
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For immunohistochemical analysis of the coronary
arteries, we chose a set of four well-accepted SMC differentiation
markers: -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 -SM actin and SMMHC (Figure 1b
and 1c)
whereas a small proportion of cells in the
media appeared negative for desmin and smoothelin (Figure 1d and 1e).
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–-SM actin antibody (Figure 2a).
The SMMHC staining demonstrated the classical interrupted distribution
at the cell periphery (Figure 2b). The anti-desmin antibody
showed a pattern irradiating from the nucleus to the cell surface
(Figure 2c). 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 2d) with a
strong intensity in the center of the cell and decreasing gradually
toward the cell periphery.
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CLSM analysis performed on cultured porcine SMCs showed that
smoothelin was colocalized with -SM actin (Figure 3a) and not with vimentin (Figure 3b).
Colocalized pixels represented 85% of the
total pixel number in the -SM actin/smoothelin double staining and
only 3% in the vimentin/smoothelin double staining.
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Immunofluorescence staining performed on
cytocentrifuged SMCs showed that in accordance with in vivo
observations, almost all freshly isolated SMCs (0 hours) were positive
for -SM actin and SMMHC (Figure 4).
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 70% for -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 -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 30% (P1 versus P0, P<0.05). At the 5th
passage, the percentage of -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 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 -SM actin–, SMMHC-, and
desmin-positive cells represent only 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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Immunoblotting results were in agreement with those of
immunofluorescence studies (Figure 5). In primary culture, the content of
-SM actin was decreased to 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 60% for -SM actin and 30% for SMMHC
(P<0.05) whereas desmin and smoothelin values did not
change. At the 5th passage, -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 -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, -SM actin content is only 15% of the
value at 0 hours whereas SMMHC and desmin proteins are not
detectable.3 6 23
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When separated by electrophoresis, the SMMHC isoforms 1 and 2 exhibited
a similar expression profile. In subculture, they
represented 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 -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 -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 6A). 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 6B) when compared with control
cells.
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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 7). After 24 hours of treatment, heparin
decreased migration to 40% and TGF-ß1 to
60% when compared with control; TGF-ß2 and
tRA had no significant effect under our conditions.
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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 -SM actin and SMMHC 1 were not significantly
changed (Figure 8).
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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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 -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).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 -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 -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 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 -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 -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 -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 -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
-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 -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 -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, -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.
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References |
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T. Christen, V. Verin, M.-L. Bochaton-Piallat, Y. Popowski, F. Ramaekers, P. Debruyne, E. Camenzind, G. van Eys, and G. Gabbiani Mechanisms of Neointima Formation and Remodeling in the Porcine Coronary Artery Circulation, February 13, 2001; 103(6): 882 - 888. [Abstract] [Full Text] [PDF]
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J. M. Miano and B. C. Berk Retinoids : Versatile Biological Response Modifiers of Vascular Smooth Muscle Phenotype Circ. Res., September 1, 2000; 87(5): 355 - 362. [Full Text] [PDF]
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S. Patel, Y. Shi, R. Niculescu, E. H. Chung, J. L. Martin, and A. Zalewski Characteristics of Coronary Smooth Muscle Cells and Adventitial Fibroblasts Circulation, February 8, 2000; 101(5): 524 - 532. [Abstract] [Full Text] [PDF]
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B. Su, S. Mitra, H. Gregg, S. Flavahan, M. A. Chotani, K. R. Clark, P. J. Goldschmidt-Clermont, and N. A. Flavahan Redox Regulation of Vascular Smooth Muscle Cell Differentiation Circ. Res., July 6, 2001; 89(1): 39 - 46. [Abstract] [Full Text] [PDF]
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