© 1998 American Heart Association, Inc.
Original Contributions |
Lipid Lowering Promotes Accumulation of Mature Smooth Muscle Cells Expressing Smooth Muscle Myosin Heavy Chain Isoforms in Rabbit Atheroma
Presented in part at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9–12, 1997, and published in abstract form (Circulation. 1997;96[suppl I]:I-232).
From the Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Departments of Medicine (M.A., S.J.V., P.L.) and Pathology (E.R., H.S., F.J.S.), Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, and the Second Department of Internal Medicine, Gunma University School of Medicine (R.N.), Gunma, Japan.
Correspondence to Dr Peter Libby, Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave, LMRC 307, Boston, MA 02115. E-mail plibby{at}rics.bwh.harvard.edu
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Abstract |
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Abstract—Smooth muscle cells (SMCs) in the atherosclerotic intima characteristically differ from those in the arterial media, for example, by reduced expression of SMC differentiation/maturation markers such as smooth muscle myosin heavy chain isoforms (SM1 and SM2). This study tested the hypothesis that lipid lowering promotes maturation of intimal SMCs in 33 rabbits subjected to balloon injury and cholesterol feeding (0.3%) for 4 months (Baseline group, n=15); some of which then were switched to a low-cholesterol diet for 8 months (Low group at 8 months, n=3) or 16 months (Low group at 16 months, n=10). The remaining rabbits continued to consume a high-cholesterol diet for 16 months (High group, n=5). We monitored SMC phenotype by expression of immunoreactive
-smooth muscle actin, SM1, and SM2. -Actin is an
early marker, and SM1 and SM2 are late markers for SMC
differentiation/maturation. Only fully differentiated or mature SMCs
express SM2. Data are reported as the percentage of the
-actin-positive intimal area occupied by smooth muscle
myosin–positive SMCs determined by color image analysis of
immunostained sections. Levels of SM1 and SM2, highly
expressed by SMCs in the normal aortic media (n=5) decreased in the
aortic intima of the Baseline and High groups, indicating a less mature
phenotype. In contrast, SM1 and SM2 increased in the Low (16
months) group, indicating that intimal SMCs exhibit a more mature
phenotype after lipid lowering. Electron microscopy also showed
the presence of mature intimal SMCs with abundant myofilaments.
Furthermore, lipid lowering reduced levels of platelet-derived
growth factor-B in the arterial intima, a factor known to
suppress smooth muscle myosin expression. These data demonstrate that
lipid lowering favors accumulation of mature SMCs in the
atherosclerotic intima in association with reduced levels of
platelet-derived growth factor-B expression. Intimal SMCs in the
Low group also displayed reduced expression of matrix
metalloproteinases-3 and -9 compared with the Baseline and High groups.
These findings shed new light on the effects of lipid lowering at the
level of the vascular wall, which may influence the biology of the
atheroma.
Key Words: atherosclerosis • smooth muscle cell • differentiation • hypercholesterolemia • lipid lowering
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Introduction |
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Smooth muscle cells (SMCs) in the normal aortic media express a series of genes encoding contractile proteins such as
-smooth muscle actin, SM-22, caldesmon, and 2 isoforms of smooth
muscle myosin heavy chain (SM1 and SM2). The expression of those
proteins serves as a marker for SMC differentiation and
maturation.1 -Actin appears at an earlier
stage of vascular development than other contractile proteins and
provides an early marker for SMC differentiation/maturation. Markers of
late stages of SMC differentiation/maturation include 2 isoforms of
smooth muscle myosin.2 3 Expression of smooth
muscle myosin isoforms is restricted highly to
SMCs.4 SM1 is first detected in SMCs at the late
fetal stage, and SM2 is expressed only after
birth.5 6 In particular, SM2 is a useful marker
for fully differentiated or mature SMCs. Thus, the combined assessment
of -actin, SM1, and SM2 expression permits monitoring of SMC
phenotypes.
In human coronary arteries and aortae, intimal SMCs have
decreased expression of smooth muscle myosin isoforms during aging and
the progression of atherosclerosis, first of SM2 and
then of SM1, whereas the expression of -actin is
maintained,6 indicating that intimal SMCs undergo
phenotypic modulation toward an immature state. The molecular and
cellular mechanisms underlying this phenotypic shift remain unknown.
Platelet-derived growth factor (PDGF)-B chain can suppress smooth
muscle myosin expression on cultured SMCs,7 but
the potential role of this factor in vivo is speculative.
Intimal SMCs of injured rabbit arteries5 8 9 and human coronary arteries after angioplasty10 also have reduced expression of smooth muscle myosin isoforms. Interestingly, these intimal SMCs appear to regain a more mature phenotype with time after injury,9 10 as determined by increased smooth muscle myosin expression.
Arterial SMCs also contain at least 2 types of nonmuscle myosin heavy chain isoforms. Kuro-o et al11 and Aikawa et al6 isolated cDNA clones encoding rabbit and human nonmuscle myosin (SMemb), which are predominantly expressed by embryonic SMCs. SMemb is identical to myosin heavy chain (MHC)-B.12 13 SMemb/MHC-B expression increases in neointimal SMCs after vascular injury in both animals and humans8 9 10 11 14 as well as in fibroblasts in culture,6 so-called myofibroblasts in rejected rat and monkey hearts after transplantation,15 and activated mesangial cells in rat models of various glomerular diseases.16 The other nonmuscle myosin expressed in SMCs is MHC-A. However, expression of this isoform does not change substantially during vascular development compared with SMemb/MHC-B (M.A. and R.N., unpublished observations, 1993).12 17
Intimal SMCs can overexpress certain genes that probably contribute to arterial lesion formation and coronary events. Expression of matrix metalloproteinases (MMPs) including MMP-1, MMP-3, and MMP-9 increases in the fibrous cap of unstable atheromatous plaques of human arteries,18 19 20 21 indicating a role for matrix-degrading proteinases in the pathogenesis of plaque vulnerability and rupture, resulting in acute coronary events.
Recent clinical trials have shown that lipid lowering reduces coronary events and mortality.22 23 24 These studies suggest that lipid lowering stabilizes the vulnerable plaque. Several animal studies have demonstrated decreased macrophage accumulation and lipid content and increased extracellular matrix content during lipid lowering.25 26 27 28 29 However, these previous studies have not addressed the molecular and cellular mechanisms underlying the stabilization of atheromatous plaque. We recently have demonstrated that dietary lipid lowering reduces macrophage accumulation and protease expression and activity and increases collagen content within the aortic lesions of hypercholesterolemic rabbits.30 However, the effects of lipid lowering on SMC phenotype has not been evaluated.
The present study investigated the hypothesis that lipid lowering can promote maturation of intimal SMCs in atherosclerotic rabbits. We report here that dietary lipid lowering promotes the accumulation of mature SMCs in the intima of aortic lesions in atherosclerotic rabbits as assessed by immunohistochemistry for smooth muscle and nonmuscle myosin isoforms and by transmission electron microscopy. Furthermore, we demonstrate reduced expression of PDGF-B chain in the arterial intima during lipid lowering, providing a potential mechanism of increased expression of smooth muscle myosin isoforms. To determine functional significance of maturation of intimal SMCs, we also examined expression of MMPs by and proliferative activity of SMCs.
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Materials and Methods |
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Animal Experimental Protocol and Diet
Thirty-six New Zealand White male rabbits (2.5 to 3 kg; Millbrook Farm, Amherst, Mass) were housed individually in stainless-steel cages. All experiments were performed in accordance with a protocol approved by the Standing Committee on Animals of Harvard Medical School. Thirty-three animals consumed an atherogenic diet (certified Purina Rabbit Chow, 5322, 95% with 0.3% cholesterol and 4.7% coconut oil, Research Diets) for 4 months to induce atheroma formation (Figure 1
). One week after initiation of the
atherogenic diet, we injured the thoracic aortae by withdrawal of a
4F-Fogarty embolectomy catheter introduced via the left iliac artery.
This procedure was performed under general anesthesia by
intramuscular injection of ketamine (35 mg/kg)/xylazine (7
mg/kg) and with local anesthesia of the inguinal region by
lidocaine. The balloon injury accelerates atheroma
formation, renders lesions more uniform in size and distribution, and
produces plaques with a smooth muscle–rich fibrous cap overlying a
layer of lipid-laden macrophages. Such lesions resemble the
atheroma of human coronary arteries more closely
than the typical foam cell lesions in rabbits produced by
hypercholesterolemia
alone.31 Fifteen rabbits were killed 4 months
after initiation of the atherogenic diet to evaluate the baseline
lesions (Baseline group). The remaining rabbits were divided into 2
groups with similar ranges of
hypercholesterolemia to avoid bias because of
variations in individual responsiveness to the atherogenic diet. In 1
group (n=13), we switched from the atherogenic diet to purified chow
with no added cholesterol and fat to reduce blood lipid
levels. Three of these rabbits were killed after an additional 8 months
(Low group at 8 months) and 10 were killed 16 months after changing
diet (Low group at 16 months). The remaining rabbits continued to
consume a cholesterol- and lipid-supplemented diet (0.05%
to 0.2% cholesterol and 4.95% to 4.8% coconut oil). The
amount of dietary cholesterol supplementation in this group
was adjusted during this period based on serial lipid determinations to
avoid levels of cholesterolemia (
<1000 mg/dL) that
would produce manifestations of liver disease. All of these rabbits on
a continued hypercholesterolemic diet were killed 16
months after assignment to the dietary condition (High group, n=5). The
collagen content, expression of MMPs, and characterization of cell
populations in these animals is the subject of a separate
report.30
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Plasma Cholesterol and Triglyceride Levels
Peripheral blood was collected from the ear artery
under local anesthesia for measurement of plasma
cholesterol and triglyceride concentrations by
enzymatic assays (Sigma Diagnostics).
Tissue Preparation
Rabbits were killed by intravenous sodium
pentobarbital (120 mg/kg). Heparin (100 U/kg) was injected
simultaneously to avoid blood clotting. The aortae were
excised and rinsed briefly with DMEM (BioWhittaker) without serum. The
proximal portion of the thoracic aorta (5 mm thick, 2 to 7 mm
below the ligamentum arteriosum) was excised and snap-frozen with OCT
Compound (Sakura Finetek, Inc) in isopentane prechilled with liquid
nitrogen for fresh-frozen sections for PDGF-B, MMP-3, and MMP-9
staining. An adjacent portion of the aorta (7 to 12 mm below the
ligamentum arteriosum) was fixed with 95% ethanol and 1% glacial
acetic acid for immunohistochemistry for myosin isoforms, -actin,
and macrophages. Ethanol-fixed tissues were embedded in
paraffin by conventional procedures.
Immunohistochemistry
Paraffin-embedded and fresh-frozen tissues were
sectioned in 6-µm slices from the proximal end. Sections were
preincubated with 0.3% hydrogen peroxide and Protein Block Serum-Free
(X0909, Dako Corp, Carpinteria, Calif). Mouse monoclonal antibodies
against rabbit SM1, rabbit SM2, rabbit SMemb,5 11
rabbit macrophages (RAM11, Dako Corp, for paraffin sections;
CD11b, Spring Valley Laboratories, Woodbine, Md, for frozen sections),
human -smooth muscle actin (1A4, Dako A/S, Glostrup, Denmark), human
PDGF-B (PGF 007, a gift of Mochida Pharmaceutical Co, Tokyo, Japan),
rabbit MMP-3 (148-1A3, Oncogene Research Products, Cambridge,
Mass), and human MMP-9 (7-11C, Oncogene Research Products) were
applied and incubated for 60 minutes at room temperature. Sections were
incubated with biotinylated anti-mouse immunoglobulins (E0354, Dako
A/S; or BA-2000, Vector Laboratories, Burlingame, Calif) for 30 minutes
and then incubated with horseradish peroxidase–labeled (for paraffin
sections) or alkaline phosphatase–labeled (for frozen sections)
streptavidin solution (Vectastain Standard, PK-6100 or AK-5000, Vector
Laboratories) for 30 minutes. Slides were rinsed in phosphate-buffered
saline (pH 7.4) after each incubation step. Peroxidase activity was
revealed by aminoethylcarbazole (Dako Corp), or Sigma Fast Red. Slides
were counterstained with hematoxylin and mounted.
In Situ Hybridization for Histone mRNA
To determine replication of intimal SMCs, in situ hybridization
for histone mRNA was performed using the Hyb-Probe Detection System
(Shandon/Lipshaw) according to the protocol recommended by the
provider. Briefly, fresh-frozen sections were fixed with 4% paraform
aldehyde for 5 minutes and hybridized with FITC-labeled
oligonucleotide cocktail for histone H2B, H3, and H4
mRNA (Shandon/Lipshaw) at 65°C for 10 minutes and then at 37°C for
2 hours. Sections were rinsed with Tris-buffered saline (pH 7.6)
containing 0.1% Triton X-100 and then incubated with alkaline
phosphatase–conjugated antibody against FITC at 37°C for 30 minutes.
Alkaline phosphatase activity was revealed by incubation with nitroblue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate chromogen at room
temperature for 12 hours. Sections were washed in running water to stop
the reaction. Then, immunohistochemistry for SMCs (1A4) was performed
as mentioned above. Sections from the Low group were counterstained
briefly with methyl green. Quality control was performed using
FITC-labeled random oligomers (as a negative control probe) and a
FITC-labeled poly d(T) probe (as a positive control). FITC-labeled
oligonucleotide cocktail for histone mRNAs were applied
for human cancer tissues as an additional positive control.
Quantitative Analysis for Histology and
Statistics
Analysis of immunohistochemistry for -actin, SM1,
SM2, and PDGF-B was performed using a personal computer-based
quantitative 24-bit (16.2 million unique combinations) color image
analysis system32 by S.J.V., who did not
know the background of each animal. Photographs were scanned into a 1
Kx1 K image buffer of the Optimas 5.2 image analysis system
(Optimas Co). A color threshold mask for immunostaining
was defined to detect the red color by sampling, and the same threshold
was applied to all specimens. The percentage of the -actin–positive
intimal area occupied by SM1- or SM2-positive SMCs for each section was
recorded to determine phenotype of intimal SMCs. The
percentage of the positive area of PDGF-B staining within the intima
also was measured. Statistical testing used 1-way ANOVA. The
differences between 3 groups were determined by post-hoc tests.
Transmission Electron Microscopic Study
Aortic rings were obtained from the thoracic aortae (12 to
15 mm below the ligamentum arteriosum) of 3 animals from each
group and divided into 2 pieces for electron microscopy and
immunohistochemistry for SM2. Few, if any, SMCs in the intima of 6
animals from the Baseline and High groups stained positively for SM2,
and the majority of intimal SMCs of the Low group were positive. Tissue
processed for electron microscopy was fixed with Karnovsky's fixative
(2.5% glutaraldehyde and 2.0%
paraformaldehyde in cacodylate buffer; pH 7.4) followed
by postfixation in 2.0% osmium tetroxide. Tissue was dehydrated in
ethanol, treated in propylene oxide, and embedded in Poly/Bed 812
medium (Polysciences). All specimens were rinsed and stained en bloc
with uranyl acetate before ethanolic dehydration. Five to six thick
sections per animal were examined. Thin sections were cut at 60 nm,
stained with lead citrate and uranyl acetate, and examined with a
JOEL-100CX transmission microscope (JOEL USA, Inc) at an accelerating
voltage of 80 kV.
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Results |
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Plasma Lipids
At the beginning of the experiment, the mean total cholesterol and triglyceride levels (in mg/dL) were 43±4 and 52±14, respectively and rose to 1562±123 and 244±49 after 4 months on the atherogenic diet (0.3% cholesterol and 4.7% coconut oil), as previously reported30 (see Table
, page 1023). The total plasma cholesterol and
triglycerides levels returned to baseline after 8 months on
the control diet lacking supplemental lipids (Low group at 8 months,
total cholesterol, 53±3, triglycerides,
53±14; Low group at 16 months, total cholesterol, 19±3,
triglycerides, 58±10) but remained elevated in the High
group (total cholesterol, 1108±158;
triglycerides, 224±87).
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Intimal SMCs in the Atheroma of
Hypercholesterolemic Rabbits Have Reduced Expression of
SM1 and SM2
The intimal lesions of the aortae after 4 months of
atherogenic diet (Baseline lesions) contained a superficial SMC layer
detected with anti-–actin antibody (1A4). This layer subtended
numerous macrophages identified with the RAM11 monoclonal
antibody, resembling the "fibrous cap" of human coronary
lesions (Figure 2, top). These intimal
SMCs exhibited decreased expression of SM1 and SM2 compared with medial
SMCs (Figure 2, bottom). In particular, expression of SM2, a marker for
mature SMCs, was nearly undetectable in the intima. After 16 months of
continued hypercholesterolemia, the intimal SMC
layer overlying macrophage accumulation showed suppression of
SM1 and SM2 expression (Figure 3).
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Intimal SMCs of the Rabbit Atheroma Regained Smooth
Muscle Myosin Expression During Lipid Lowering
After 8 months of dietary lipid lowering, the region of
SM2-positive SMCs was much smaller than that of -actin–positive
cells (Figure 4). After 16 months, many
intimal SMCs stained positively for both SM1 and SM2 (Figure 5). To quantitate these results, the
percent of the -actin-positive intimal area occupied by SM1- or
SM2-positive SMCs for all animals studied was determined by
computer-assisted color image analysis (Figure 6). SM1 and SM2 expression at 16 months
compared with baseline lesions increased significantly (SM1,
P<0.001; SM2, P<0.001).
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Nonmuscle Myosin Expression in the Atheroma Decreased
During Lipid Lowering
In all animals in the Baseline and High groups, some of the
-actin–positive intimal SMCs stained positively for SMemb/MHC-B,
whereas the tunica media contained few if any SMemb/MHC-B–positive
SMCs (Figure 7, top and middle).
Expression of SMemb/MHC-B was nearly undetectable in the intima of all
animals in the Low group at 16 months (Figure 7, bottom).
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Intimal SMCs in the Aorta of Treated Animals Show Features of a
Mature Phenotype With Abundant Myofilaments Detected by
Transmission Electron Microscopy
In addition to SMC-specific protein expression, we performed
transmission electron microscopy to examine SMC morphology. SMCs within
the intima of the Baseline and High groups showed characteristics of
modulated SMCs with well-developed rough endoplasmic reticulum and
Golgi apparatus, and scant myofilaments (Figure 8, left and middle). In contrast, many
intimal SMCs after 16 months of dietary lipid lowering contained
numerous myofilaments with focal density (dense body) and paucity of
synthetic organelles, characteristics of a well-differentiated
phenotype resembling the medial SMCs of mature animals (Figure 8, right).
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Lipid Lowering Reduced Lesional Expression of PDGF-B
Macrophages and SMCs within the lesions of the Baseline
and the High groups expressed PDGF-B chain, which is known to suppress
smooth muscle myosin expression in cultured SMCs (Figure 9, top).7 However,
expression of PDGF-B decreased in the Low group after 16 months of
dietary lipid lowering (Figure 9, bottom left). The double
immunostaining for PDGF-B (blue) and
macrophages (CD11b, red) demonstrates localization of
immunopositive PDGF-B in both macrophages and SMCs in the
intima of the Baseline animal. PDGF-B–expressing macrophages
stained purple as a result of the mixture of blue and red. The blue
spindle-shaped cells are PDGF-B–positive SMCs (Figure 9, bottom
right). Quantification of the percentage of positive areas of PDGF-B
immunostaining on all animals by computer-assisted
color image analysis showed a statistically significant
decrease with lipid lowering (Figure 10).
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Expression of MMP-3 and MMP-9 by and Proliferation of Intimal SMCs
Decreased During Lipid Lowering
To determine functional significance of maturation of
intimal SMCs, immunohistochemistry for MMP-3 and MMP-9 and in situ
hybridization for histone mRNA were performed. Intimal SMCs in both
Baseline (Figure 11, top) and High
(data not shown) groups with reduced SM2 expression stained positively
for anti–MMP-3 and anti–MMP-9 antibodies. However, in the Low (16
months) group, MMP-3 and MMP-9 were almost undetectable in SMCs, which
displayed high levels of SM2 expression (Figure 11, bottom). Based on
the expression of histone mRNA, which peaks in cells during the S phase
of the cell cycle and disappears rapidly during the G2
phase,33 34 SMCs replicate in
atheroma of the Baseline (Figure 12, top) and High (data not shown)
groups. However, in the Low group, few if any intimal cells contained
histone mRNA (Figure 12, bottom). Most cells stained intensely with a
poly d (T) probe (positive control) and no signals were detected with
random oligomers (negative control) (data not shown). In the sections
of human lung cancer, almost all cells were strongly positive for
histone mRNA probe (data not shown). The results regarding expression
of MMPs and histone are typical of 5 animals from each group in the
study.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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This study demonstrates the presence of mature SMCs in the arterial intima of hypercholesterolemic rabbits after dietary lipid lowering as determined by increased expression of smooth muscle myosin isoforms and typical ultrastructure for mature SMCs. Furthermore, we show that reduced expression of PDGF-B chain provides a potential mechanism for accumulation of mature SMCs. Decreased expression of MMP-3 and MMP-9 by SMCs after lipid lowering additionally affirms the functional significance of accumulation of mature SMCs in atheroma.
Intimal SMCs in the Atheroma Showed Immature
Phenotype Gauged by Decreased Expression of Smooth Muscle
Myosin Isoforms
Reduced expression of smooth muscle myosin isoforms has
demonstrated phenotypic modulation of SMCs toward an immature state in
culture and in vascular hyperplastic lesions. Smooth muscle myosin
expression decreases in cultured SMCs.1 35
Reduced myosin expression also occurs in the arterial SMCs
after mechanical injury and cholesterol-feeding of
rabbits,5 8 9 36 and in rat or monkey
coronary arteries after heart
transplantation.15 Aikawa et
al6 10 further demonstrated reduced expression of
human SM1 and SM2 within the intimal SMCs of the aortae and
coronary arteries and within the neointimal SMCs of
human coronary arteries after angioplasty.
In the present study, an SMC layer resembling the fibrous cap
of human coronary lesions overlaid the macrophage-rich
atheromatous core in the Baseline and High groups. The
percentage of SM1- or SM2-positive cells among the
-actin–positive population in these atheroma was
significantly lower compared with that of medial SMCs in normal control
animals, indicating that many lesional SMCs (-actin–positive and
smooth muscle myosin–negative) display features of immature SMCs at
early developmental stages. These findings regarding reduced expression
of smooth muscle myosin within -actin–positive SMCs in rabbit
atheroma match observations on chronic atherosclerotic
lesions of human coronary arteries.6
Continued Suppression of Smooth Muscle Myosin Isoforms in the
Rabbit Atheroma by High-Cholesterol Feeding
Okamoto et al9 previously demonstrated
that neointimal SMCs of the rabbit aorta created by balloon
injury without high-cholesterol feeding regain SM2
expression within 2 months. Aikawa et al10
recently reported that neointimal SMCs on human
coronary arteries 6 months after angioplasty express both SM1
and SM2. However, in the present study, intimal SMCs in the rabbit
atheroma of the High group continued to show features of
immaturity even at 20 months after balloon injury, probably because of
hypercholesterolemia.
PDGF-B chain is the only mediator currently known to suppress smooth
muscle myosin expression. Holycross et al7
demonstrated that PDGF-BB, but not PDGF-AA, suppresses smooth muscle
myosin expression by cultured SMCs. The ß-receptor for PDGF
likely mediates this effect of PDGF-B chain, because the SMCs used in
their study lack the -receptor. Lesional
macrophages37 and
SMCs38 in both human and experimental
atherosclerosis express PDGF-B detected
immunohistochemically using the same monoclonal antibody (PGF007)
employed here. We now report over- expression of PDGF-B in rabbit
atheroma. This result provides a potential mechanism for
the continued suppression of smooth muscle myosin protein expression in
atheroma of hypercholesterolemic
rabbits.
Intimal SMCs in the Intima of Hypercholesterolemic
Rabbits Regain Mature Phenotype During Lipid Lowering
The present study demonstrates that intimal SMCs in
atherosclerotic rabbits exhibit a mature phenotype during lipid
lowering. A reduction in serum cholesterol levels increased
the expression of smooth muscle myosin isoforms, first of SM1 and then
of SM2, in SMCs of the plaque's fibrous cap. Lipid lowering also
decreased nonmuscle myosin (SMemb/MHC-B) expression, which is augmented
in activated mesenchymal cells such as immature SMCs,
proliferating SMCs, and
myofibroblasts.10 11 14 15
In addition to myosin expression, features of mature SMCs in treated animals were determined by transmission electron microscopy as well. Kim et al39 and Kimura et al40 previously demonstrated that SMCs with reduced expression of smooth muscle myosin show features of the so-called "synthetic" phenotype with abundant cytoplasmic organelles such as rough endoplasmic reticulum and Golgi apparatus, and few myofilaments detected by transmission electron microscopy, as shown in the present study. We confirmed maturation of intimal SMCs during lipid lowering via the presence of abundant myofilaments, features typical of the so-called "contractile" state of SMCs.41
We recently demonstrated that long-term lipid lowering reduced macrophage accumulation in these rabbit atheroma.30 The present study shows a concomitant decrease in PDGF-B expression in the intima of the treated animals. This inverse relationship between smooth muscle myosin and PDGF-B expression suggests 1 possible mechanism of maturation of intimal SMCs in atheroma by lipid lowering. The recent isolation and characterization of the SM1/2 and SMemb/MHC-B genes42 43 44 45 46 47 should facilitate understanding the molecular mechanisms that regulate smooth muscle and nonmuscle myosin gene expression. Such studies may clarify the mechanisms not only of SMC differentiation/maturation but also of plaque stabilization.
The greater abundance of SMCs exhibiting a "mature" phenotype in the arterial intima after lipid lowering could result from several mechanisms. We favor the explanation that immature SMCs in the atherosclerotic rabbit intima originate from mature SMCs in the media and regain more mature phenotype by lipid lowering, by mechanisms discussed above. Alternatively, some intimal SMCs may originate from an immature subpopulation of medial SMCs and acquire mature phenotype during lipid lowering.48 49 The present data cannot distinguish between these and other possible origins of the population of mature SMCs in the intima after lipid lowering.
Maturation of SMCs in the Fibrous Cap of Rabbit
Atheroma as a Potential Mechanism of Stabilization of Human
Atherosclerotic Plaques
Acute coronary syndromes such as unstable angina or
myocardial infarction often result from rupture of vulnerable plaques.
"Vulnerable plaques" are characterized by a thin SMC–layer
(fibrous cap) overlying macrophage accumulation in the intima.
Intimal SMCs differ from medial SMCs with respect to gene expression,
function, and morphology.50 For example, SMCs in
the fibrous cap also overexpress matrix-degrading enzymes such as
MMP-1, MMP-3, and MMP-9 that may weaken the fibrous cap and promote
plaque rupture.18 19 20 21 In the present article,
we demonstrated decreased expression of MMP-3 and MMP-9 in intimal SMCs
after sustained lipid lowering. As in human
atheroma,51 proliferation in a small
population of intimal SMCs in rabbit atheroma was detected
by in situ hybridization for histone mRNA. However, after lipid
lowering, few if any intimal SMC displayed this sign of replication.
These results are consistent with recent observations by Kockx
et al52 demonstrating reduced proliferation of
vascular cells of hypercholesterolemic rabbits during
lipid lowering.
Residence in the intima of "mature" SMCs, gauged by increased expression of smooth muscle myosin isoforms and morphology, indicates a restoration of a more normal environment by lipid lowering. Elucidation of such alterations in the biology of the plaque will help us gain a mechanistic understanding of the reduced acute coronary events and death achieved by lipid lowering in patients at risk.
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Acknowledgments |
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This work was supported in part by National Heart, Lung, and Blood Institute grant PO1 HL48743 (P.L.). M.A. is an awardee of Research Fellowship Grant of the Japan Heart Foundation. We acknowledge Eugenia Shvartz and Elissa Simon-Morrisey for their skillful assistance and Andrea Dodge for her editorial expertise. We also thank Kevin Mullelly and his staff of the animal facility of Harvard Medical School for excellent management of experimental animals.
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Footnotes |
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This manuscript was sent to John T. Shepherd, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received February 24, 1998; accepted July 28, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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