© 1997 American Heart Association, Inc.
Articles |
Differentiation of Smooth Muscle Cells in Human Blood Vessels as Defined by Smoothelin, a Novel Marker for the Contractile Phenotype
From the Department of Molecular Cell Biology and Genetics, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, Netherlands (F.T.L. van der L., F.C.S.R., G.J.J.M. van E.); University of Geneva (Switzerland), Department of Pathology (G.G.); and University of Glasgow, Department of Obstetrics and Gynecology, UK (G.K.).
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Abstract |
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Abstract Smoothelin is a constituent of the cytoskeleton specific for smooth muscle cells (SMCs) in a broad range of species. It has been postulated that smoothelin represents a marker of highly differentiated, contractile SMCs. Here, we present data on the presence of smoothelin in the human vascular system that support this hypothesis. For this purpose, smoothelin distribution was studied (1) during vasculogenesis of the placenta, (2) in normal adult blood vessels, and (3) in atherosclerotic lesions. Smoothelin was first observed in placental tissue at approximately week 10 to 11 of gestation. In full-term placenta, it was found in the SMCs of vessels in the large stem villi and in the chorionic plate. Furthermore, it was present in the fetal arteries of smaller stem villi, but it was not found in the veins. In adult blood vessels, a small population of aortic (
10%) and large muscular artery (30% to 50%) SMCs was
positive for smoothelin. In general, smoothelin and desmin were
coexpressed in the same SMCs, but expression of desmin appeared to be
less abundant. However, the majority of SMCs in these blood vessels
were smoothelin- and desmin-negative but expressed vimentin, whereas
-smooth muscle actin (-SMA) was present in all SMCs. The SMCs
in the media of small muscular arteries were positive for smoothelin
and desmin (>95%), whereas the vimentin-positive SMC type was scarce.
Smoothelin was absent in capillaries, pericytic venules, and small
veins but was occasionally observed in the SMCs of large veins. Thus,
the distribution of smoothelin in the SMCs of the vascular system
appears to be limited to blood vessels that are capable of pulsatile
contraction. In atherosclerotic femoral arteries, smoothelin-positive
cells were detected in the media, the atheromatous
plaque, and the intimal thickening. Smoothelin-positive cells were
present primarily at the luminal portion of advanced lesions. The
presence of a considerable number of such smoothelin-positive cells at
that location may indicate that these plaques are no longer expanding.
Key Words: smoothelin • smooth muscle cells • human blood vessels • atherosclerotic lesions • differentiation • placenta
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Introduction |
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Smooth muscle cells are the most abundant cell type in blood vessel walls. They occur in all vessels except capillaries and pericytic venules. In the media of blood vessels, SMCs are intermingled with elastic lamellae and laminae and appear as concentric, helically arranged bundles. Vascular SMCs have been divided into (at least) two distinct states of differentiation, usually referred to as synthetic and contractile phenotypes,1 2 the latter being predominant in the blood vessels of adult organisms. Contractile SMCs have a muscle-like morphology, contract in response to mechanical and biochemical stimuli, and are involved in the control of blood pressure and blood flow. Synthetic-type SMCs, with a fibroblast-like appearance, proliferate and produce extracellular matrix components. Between these two extremes, SMCs can acquire a broad spectrum of different phenotypes in response to changes in a variety of physiological or pathological factors.2 3 This potential is considered to play an important role in angiogenesis, maintenance of blood vessel wall integrity, and the development of atherosclerosis. Intimal thickening with myofilament-rich SMCs and without large accumulations of lipids is considered a physiological response to hemodynamic forces in specific arterial locations.4 During development of the atherosclerotic plaque, SMCs from the media migrate into the intima and thus participate in the formation of intermediate (transitional) and advanced lesions.5 6
The IFPs vimentin and desmin have been suggested as markers to
discriminate between cells of the synthetic and the contractile
phenotypes.1 2 The cellular content of vimentin,
desmin, -SMA, myosin, and tropomyosin changes when SMCs shift from
the synthetic to the contractile phenotype both in vivo and in
vitro.7 8 Also, differences of IFP content have been
observed among SMCs of different blood vessels.9 10 11
Recently, we identified a SMC protein, smoothelin, that appeared
to be useful to monitor SMC differentiation.12 This 59-kD
protein is expressed exclusively in fully differentiated (contractile)
SMCs. RNA and protein analysis revealed a broad species
distribution of this protein. Cells with SMC-like characteristics, such
as myofibroblasts and myoepithelial cells, as well as skeletal and
cardiac muscle did not contain smoothelin.12 Confocal
scanning laser microscopy of sections of human colon, myoma, and
arteries, as well as transfection studies, indicated a filamentous
organization of smoothelin that is different from those of desmin,
vimentin, or -SMA. This study elaborates on the distribution of
smoothelin in the vascular system, in embryonic and normal blood
vessels, and in atherosclerotic lesions. It provides additional
evidence that smoothelin expression is specific for contractile
SMCs.
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Methods |
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Tissues and Tissue Preparation
Human placenta. Human term placentas (n=8) and 4 term umbilical cords from clinically normal pregnancies (week 38 to 40 of gestation) were collected. In addition, 2 second-trimester placentas (week 25 of gestation) after preterm delivery and 8 first-trimester placentas (week 7 to 11 of gestation) were obtained after legal suction termination of pregnancy for psychosocial or medical reasons that were unlikely to affect placental structure and function. Informed consent was obtained from each patient, and the study was approved by the local ethics committee. Small blocks of placental and cord tissues were snap-frozen in liquid nitrogen.
Normal blood vessels and atherosclerotic lesions. Samples of human arteries, both normal (n=5; 3 patients) and atherosclerotic (n=12; 5 patients); abdominal aortic aneurysm biopsies (immediately after surgery; n=2; 2 patients); and aortas (thorax; 3 to 12 hours postmortem; n=4; 4 individuals) were either frozen in liquid nitrogen, mounted in Tissue-Tek (OCT compound; Miles Inc), and stored at -80°C or routinely embedded in paraffin. The use of the tissues was approved by the local ethics committee. We have adopted the following classification for normal muscular arteries: large muscular arteries (diameter, 2 to 10 mm) containing 10 to 40 concentric layers of SMCs, and small muscular arteries (diameter, 0.3 to 2 mm) containing <10 layers of SMCs.
Tissue sectioning and processing. Serial sections of the frozen placental and umbilical cord material (10 µm) were cut at -21°C, air-dried for 60 minutes, and stored at -70°C. Before use, cryostat sections were fixed in acetone at 4°C for 10 minutes. Sections 4 µm thick were cut from paraffin-embedded normal blood vessels and atherosclerotic plaques. From the frozen blood vessels and atherosclerotic plaques, sections 3 to 5 µm thick were cut at -25°C and immediately fixed in methanol (-20°C; 5 minutes) and acetone (-20°C; 30 seconds), and air-dried for 3 hours. Alternatively, sections were air-dried overnight at 20°C to 22°C (RT) and then treated for 5 minutes with 0.5% Triton X-100 (BDH Chemicals Ltd) in PBS (in mmol/L: sodium chloride 137, disodium hydrogen phosphate dihydrate 13, and potassium dihydrogen phosphate 3, pH 7.4; Merck), followed by a PBS washing step for 5 minutes.
Antibodies
Antibodies used in this study were (1) mouse MAb R4A
against smoothelin of the IgG1 subclass selected on the basis of its
reactivity pattern with SMCs of blood vessels of the human heart and
smooth muscle tissue of the human intestine (molecular characterization
of smoothelin as well as reactivity patterns, species, and tissue
specificity of the antibody have been described
elsewhere12 ); (2) polyclonal rabbit antisera to
desmin13 14 21 ; (3) MAb RD301 to desmin15 ;
(4) MAb anti–-Sm-1 to -SMA16 ; (5) polyclonal rabbit
antisera to vimentin14 17 21 ; (6) MAb RV203 to
vimentin18 ; (7) MAb to 
-SMA (clone B4)19 ;
and (8) MAb to smooth muscle myosin (clone hSM-V).20
Immunohistochemistry
Immunoperoxidase staining. After fixation in acetone,
the fixed cryostat sections were air-dried and rinsed in PBS for 10
minutes. The sections were preincubated for 20 minutes in PBS
supplemented with 1.5% BSA (Sigma Chemical Co). Primary and secondary
antibodies were diluted in 1.5% BSA/PBS, and washing steps were
carried out in PBS. All incubations were performed in a humidified
chamber at RT. The primary antibodies were applied for 60 minutes;
control sections revealed no immunostaining. After
three washings in PBS (5 minutes each), biotinylated secondary antibody
(DAKO A/S) was applied for 30 minutes. Sections were then incubated in
1% H2O2 in absolute methanol for 10 minutes to
inactivate endogenous peroxidase, followed by
incubation with streptavidin-peroxidase conjugate (streptAB
complex/HRP, DAKO) for 20 minutes. The streptavidin-biotin complex was
visualized with DAB (Serva). Sections were washed in distilled water,
counterstained with Harris hematoxylin, and mounted in
DPX.21
Immunofluorescence staining. Indirect immunofluorescence assays were performed according to standard procedures.12 15 18 In brief, the methanol/acetone–or Triton X-100–treated sections were incubated with the primary antibody for 30 minutes in a humidified chamber (RT) and washed three times (10 minutes each) with PBS. The appropriate FITC- or Texas Red–conjugated secondary antibodies (Southern Biotechnology Associates Inc) were applied for 30 minutes (RT). After washing with PBS, the immunofluorescence-stained tissues were mounted in Mowiol. For double-labeling studies, the incubation steps were repeated with primary and secondary antibodies of different subclasses or species. Sections were analyzed with a Zeiss Axiophot microscope.
Immunohistochemistry on paraffin-embedded tissue sections. To stain paraffin-embedded arteries with the smoothelin antibody R4A, sections had to be pretreated by microwave radiation. In brief, the sections were deparaffinized, treated with ethanol (100%), and incubated in methanol/hydrogen peroxide (5%) and demineralized water. The sections were placed in a 10 mmol/L citrate buffer (pH 6.0) and heated in a microwave (3 times 5 minutes).22 After cooling in the citrate buffer, the sections were incubated with undiluted culture supernatant of MAb R4A. After washing in PBS, antibody binding was monitored with the streptAB/HRP complex procedure performed according to the manufacturer's instructions (Vectastain ABC Kit, Vector). The peroxidase activity was visualized with DAB and H2O2. Sections were counterstained with Mayer's hematoxylin, dehydrated, and mounted in Eukitt.22
Gel Electrophoresis and Immunoblots
Approximately 40 cryostat sections (20 µm thick) of
fresh-frozen umbilical cord, sigmoid artery, aorta (thorax), aortic
aneurysm (abdomen), and colon (positive control tissue) were
collected, washed with 1 mL PBS, and centrifuged for 5 minutes
at 12 000g. After centrifugation, the
pellet was subjected to a Triton X-100 extraction step: the pellet was
suspended in 1% Triton X-100, 5 mmol/L EDTA/0.4 mmol/L PMSF
(Merck) in PBS, pH 7.4, and extracted for 5 minutes on
ice.18 After centrifugation for 5 minutes
at 12 000g, the pellet was washed in 1 mL PBS. After a
final centrifugation step (5 minutes,
12 000g), this cytoskeletal preparation was dissolved by
boiling for 4 minutes in sample buffer containing 2.3% SDS and 5%
ß-mercaptoethanol (Bio-Rad Laboratories).23
One-dimensional SDS-PAGE was performed with a Mini Protean II Electrophoresis Cell (Bio-Rad Laboratories). Polyacrylamide slab gels containing 0.1% SDS23 were loaded with the cytoskeletal preparations. After electrophoretic separation, the proteins were stained with Coomassie brilliant blue (PAGE blue 83, BDH Chemicals Ltd) or subjected to immunoblotting. The separated polypeptides were transferred from the slab gels to a nitrocellulose membrane (Schleicher and Schüll, Filter BA 85) by blotting for 1 hour at 100 V in a cold (4°C) buffer containing 25 mmol/L Tris (Merck), 192 mmol/L glycine (Merck), 0.02% SDS, and 20% methanol (Merck).18 24 For protein detection, two procedures using HRP-conjugated secondary antibodies (DAKO) were applied. The first procedure, using 4-chloro-1-naphthol (Merck) and 0.12% hydrogen peroxide (Merck) in PBS, was applied when large amounts of protein were expected or when blots had to be reincubated one or two times with other primary antibodies.18 For detection of low concentrations, the procedure using the chemiluminescence ECL-Kit (Amersham International pcl) was applied after an extensive blocking procedure according to the instructions of the manufacturer.
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Results |
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Smoothelin Distribution in Human Placenta and Umbilical Cord
Smoothelin could be detected immunohistochemically in placental tissue around week 10 to 11 of gestation. Earlier developmental stages (weeks 7 to 8 of gestation) did not show any immunoreactivity, although desmin,
-SMA, and SMC-specific myosin
were already present in some developing vessels. Smoothelin
expression seemed to be restricted to the SMCs of fetal arteries in
smaller stem villi (Fig 1a
through 1c). No
immunoreactivity was found in the veins (Fig 1a). In term placenta, the
presence of smoothelin was restricted to vessels in the large stem
villi and the chorionic plate, which represent the oldest part
in placental development (Fig 1d and 1e). In large stem villi, a few
extravascular stromal cells in close vicinity to the fetal vessels
coexpressed -SMA, smooth muscle myosin, and smoothelin. Embryonic
stromal cells have been shown to exhibit a complex cytoskeletal
composition and coexpress vimentin, desmin, - and -SMA, and
smooth muscle myosin.25 In term umbilical cords, smoothelin
was observed in the SMCs of all three vessels (two arteries, one vein),
whereas the surrounding stromal cells in the Wharton's jelly were
negative. The results of these immunohistochemical studies on placental
material are summarized in Table 1.
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Smoothelin Distribution in Adult Blood Vessels
Elastic arteries. The tunica media of elastic arteries,
eg, the aorta, contains up to 50 layers of elastic fibers. Interposed
between the (autofluorescent) elastic layers are SMCs and some
collagen. Immunohistochemistry on sections from four human individuals
revealed that the SMCs belong to different types, containing either
desmin (Fig 2b) or vimentin (Fig 2c) in addition to
-SMA. Only a limited number of media SMCs (5% to 10%) contained
smoothelin (Fig 2a). Although double staining of aorta sections is
hampered by the autofluorescence of the elastic fibers,
coexpression of smoothelin and desmin was detected in the SMCs of the
media of the aorta. No smoothelin-positive cells were detected in the
intima or in the adventitia. In sections of two biopsies of human
abdominal aortic aneurysm specimens, neither smoothelin- nor
desmin-positive SMCs were detected, but vimentin-containing SMCs were
present (not shown). Western blot analysis confirmed the
presence of vimentin and -SMA in aorta and aneurysm
biopsies, but smoothelin and desmin could not be demonstrated (Table 2;
Fig 4).
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Muscular arteries. In the media of five normal human large
muscular arteries, smoothelin was detected in a significant number of
SMCs (30% to 50%), either in frozen tissue sections (Fig 3b) or in sections of paraffin-embedded tissue (not
shown). In immunofluorescence, smoothelin appeared
to be more abundant than desmin. Both staining intensity and number of
smoothelin-positive cells appeared to be higher (Fig 3c). On Western
blots, however, the smoothelin signal was lower than the desmin signal,
most likely because of a decreased binding efficiency of the MAb R4A on
denatured antigen. The majority of the SMCs contained vimentin and did
not express smoothelin.
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In the media of small human muscular arteries, virtually all SMCs
(>95%) contained smoothelin (Fig 3a
). Coexpression of smoothelin and
desmin was also observed in these SMCs. All cells that contained
smoothelin also expressed -SMA. The SMC type expressing only
vimentin was hardly observed (not shown). The smoothelin antibody also
stained the SMCs of vasa vasorum, located in the adventitia of the
large muscular arteries. The presence of smoothelin, desmin, vimentin,
and -SMA was confirmed by Western blot analysis of
fresh-frozen human sigmoid artery biopsies (Table 2; Fig 4).
Capillaries, venules and veins. Smoothelin was not detected in capillaries, pericytic venules, and small veins, but the protein was observed in the concentric layers of SMCs of large veins. The thin layer of SMCs in the media of the wall of small veins consists of cells that do not contain smoothelin, whereas the walls of large veins contain SMCs that express this protein.
The data obtained from our immunohistochemical studies on normal
blood vessels are summarized in Table 1.
Western blot analysis. Immunoblot
experiments (summarized in Table 2) showed that smoothelin and desmin
were present in extracts of sigmoid artery (Fig 4), umbilical cord,
and colon but were not detected in extracts of either normal aorta
(thorax; Fig 4) or abdominal aortic aneurysm (Fig 4). Vimentin
and -SMA were detected in all tissues tested (Table 2), a result
that can be explained by the variety of cell types that are included in
the used homogenates.
Smoothelin Distribution in Atherosclerotic
Lesions
Atherosclerotic lesions were investigated for smoothelin
expression. Serial sections were stained with the antibody R4A in
combination with antibodies directed against other cytoskeletal
proteins. Smoothelin-positive cells were found in the majority of the
lesions. In advanced human atherosclerotic lesions, smoothelin
expression was observed in SMCs of demarcated areas. In general, the
luminal side of the lesion contained more positive cells than the
intimal side (Fig 5a and 5c). The staining pattern
indicated a more linear organization of smoothelin in the SMCs of the
luminal side, compared with the more globular organization on the
abluminal side. This finding indicates a more elongated morphology of
the SMCs in the luminal area of the lesion. Most cells in the
atherosclerotic lesions that contained smoothelin also coexpressed
desmin (although not as abundantly as smoothelin; Fig 5a and 5b),
vimentin (with an organization pattern resembling that of desmin; Fig 5c and 5d), and -SMA (not shown). In the luminal area of an intimal
thickening, 10% to 20% of the lesions consisted of cells that
expressed smoothelin abundantly (Fig 3b).
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Smoothelin Distribution in Placental and Adult Blood Vessels
Vascular SMCs can be subdivided into different classes on the basis of (1) their morphology, (2) their expression patterns of structural proteins (such as IFP or contractile proteins),9 10 11 26 or (3) their degree of differentiation.1 2 Cytoskeletal markers have been used to discriminate between synthetic and contractile arterial SMCs.1 2 Switches in actin and myosin isoforms2 27 or an increase of desmin and tropomyosin content in combination with a decrease in vimentin7 8 are indicative for SMC differentiation toward the contractile phenotype. Previously, we suggested that the presence of smoothelin is limited to the contractile phenotype of the SMCs because cultured, synthetic SMCs did not contain this constituent, and we hypothesized that smoothelin may be involved in the contractile activity of SMCs.12 Our present findings in the different types of vascular SMCs corroborate such a hypothesis.
The presence of smoothelin in term placenta was restricted to the fully developed vessels in the large stem villi and the chorionic plate, which represent the oldest part in placental development. In smaller stem villi, with smaller and less muscularized blood vessels, smoothelin-positive SMCs were present only in the fetal arteries. The vessels of placental stem villi, in particular the fetal arteries, are considered to play a major role in the regulation of the fetal blood flow in the placenta.
For adult elastic and large-diameter muscular arteries, blood vessel type–related differences in SMC types, as differentiated by their vimentin or desmin content, were described previously.9 10 In arteries of this type, which have to withstand tonically the high pressure generated by the heart, a SMC type predominates that contains vimentin but neither smoothelin nor desmin. SMCs of small muscular arteries generate contractions to continue the pulsatile movement of the blood flow and to regulate the blood pressure. These cells are mainly of the SMC type that contains smoothelin and desmin. Smoothelin is absent in small veins that have no continuous layers of SMCs and that do not generate contractions. In large veins that contain concentric layers of SMCs and that do slowly contract, smoothelin-positive SMCs are present. Also, the presence of smoothelin in vascular SMCs of placental and adult blood vessels appears to be correlated to isometric contractile activity. Filamentous smoothelin organization and interaction of smoothelin with actin filaments or stress fiberlike structures 12 may contribute to the contractile capacity or to the structural integrity of SMCs.
Smoothelin Distribution in Atherosclerotic Lesions
The development of experimental intimal thickening, as
described for the rat model, is characterized by a migration of SMCs
from the media into the intima and their modulation to a synthetic
phenotype.28 29 30 31 Some of these cells remain
quiescent, while others proliferate within the intima, thus increasing
the overall cellular mass of the lesion.31 32 The increase
in the number of SMCs takes place mainly at the luminal surface. The
number of SMCs decreases after regeneration of the
endothelium.14 30 In human atherosclerotic
plaque, the SMCs not only proliferate but also secrete extracellular
matrix components, thus forming a cap on the luminal side of the lipid
core.6 33 Babaev et al34 have shown that the
plaque cap of advanced lesions contains many cells that possess
ultrastructural features of contractile SMCs. These cells express
desmin and other cytoskeletal proteins characteristic of differentiated
SMCs. Our findings show that smoothelin is synthesized primarily in
cells at the luminal side of atherosclerotic lesions. The presence of
smoothelin-containing SMCs may be indicative of a reduced growth of the
atherosclerotic plaque. The capping of the atherosclerotic lesion by
nonproliferating cells containing desmin, -SMA, and smoothelin
suggests that growth of such intimal thickenings has ceased.
Furthermore, proteins such as desmin and smoothelin may contribute to
the integrity of the atherosclerotic cap, which prevents damage by the
shearing force of the blood flow.
Smoothelin as a Marker for Differentiated SMCs
Smoothelin appears to be a useful tool to discriminate
between the different SMC phenotypes, because
immunohistological and biochemical data indicate
that this cytoskeletal constituent is a specific marker for
differentiated SMCs. Smoothelin is absent in primary cultures (bovine
aorta allowed to settle on coverslips for 3 to 4 hours), and long-term
cultures of vascular SMCs (human uterine and mammary artery cultured
for at least five passages), whereas desmin and -SMA are present
in such cultures. The relatively late start of smoothelin expression in
chick embryos that already express -SMA in combination with the IFPs
vimentin and/or desmin is also in agreement with these observations:
preliminary results indicate that smoothelin expression in chick embryo
gizzard and femoral artery starts at Hamburger-Hamilton stage
35/3635 (ED 10; unpublished data). The expression of other
SMC differentiation markers, such as -SMA (ED 2.5), SM-22 (ED 4),
calponin (ED 6), h-caldesmon (ED 6), and smooth muscle -tropomyosin
(ED 6), starts earlier during development.2 A similar
finding was observed in the human placenta, in which expression of
smoothelin was observed at about week 10 to 11 of gestation in the
developing SMCs of the fetal blood vessels. In earlier material (week 7
to 8 of gestation), smoothelin was not expressed, although the
developing SMCs of the fetal vessel already coexpressed vimentin,
desmin, - and -SMA, and smooth muscle myosin (Table 1).
The inverse relationship between proliferation and SMC-specific
contractile protein expression was demonstrated in cultured human
arterial muscle cells by Fager et al.36 The
short time required to replace desmin by vimentin as the dominant IFP
(<24 hours), as well as the relatively fast shift from the - to the
ß-isoform of actin in vascular SMCs was reported
previously.37 Smoothelin expression and organization is
lost even faster than these constituents. Thus, smoothelin may be
useful to discriminate fully differentiated, contractile SMCs from
proliferative SMCs. Also, smoothelin provides a possibility of
discriminating SMCs from cells with smooth muscle–like
characteristics, such as myofibroblasts and myoepithelial cells. The
distribution of smoothelin-positive cells indicates a correlation
between smoothelin expression and smooth muscle tissue
contractility. Smoothelin may be a useful tool in the
evaluation of atherosclerotic lesions, because it can monitor the
proliferative nature of such lesions.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by the Swiss National Science Foundation (Drs van der Loop and Gabbiani, grant 31-40372.94) and the Dutch Heart Foundation (Dr van der Loop, grant D90.002). Dr Kohnen was a recipient of a European Community Human Capital and Mobility Fellowship (9030364). The contribution of the Obstetric and Midwifery colleagues at Glasgow Royal Infirmary and The Queen Mother's Hospital (Glasgow) in collecting placental and umbilical cord specimens is gratefully acknowledged. The authors would like to thank Prof Dr Peter Kaufmann (University of Aachen) for helpful discussions and are also indebted to M. Redard (University of Geneva) for the staining of paraffin-embedded tissues.
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Footnotes |
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Reprint requests to G.J.J.M van Eys, Department of Molecular Cell Biology and Genetics, Cardiovascular Research Institute Maastricht (CARIM), University of Limburg, PO Box 616, 6200 MD Maastricht, The Netherlands.
The Guest Editor for this article was Prof Göran K. Hansson.
Received June 11, 1996; accepted August 21, 1996.
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