© 1999 American Heart Association, Inc.
Original Contributions |
Generation and Characterization of Human Smooth Muscle Cell Lines Derived From Atherosclerotic Plaque
From the Fred Hutchinson Cancer Research Center (L.R.B., K.M., K.S., J.I., C.M.) and the Departments of Pathology (C.M., J.K.M., S.C.C.) and Surgery (S.A.), University of Washington, Seattle; and Department of Anatomic Pathology (N.P.-R.), Wm Beaumont Hospital, Royal Oak, Mich. Current address of Salim Aziz, Department of Cardiothoracic Surgery, University of Colorado Health Science Center, 4200 East 9th Avenue, Denver, CO 80262.
Correspondence to Lise R. Bonin, PhD, Fred Hutchinson Cancer Research Center, Mailstop C1-015, 1100 Fairview Ave N, Seattle, WA 98109. E-mail jmcdouga{at}fred.fhcrc.org
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Abstract—The study of atherogenesis in humans has been restricted by the limited availability and brief in vitro life span of plaque smooth muscle cells (SMCs). We describe plaque SMC lines with extended life spans generated by the expression of the human papillomavirus (HPV)-16 E6 and E7 genes, which has been shown to extend the life span of normal adult human aortic SMCs. Resulting cell lines (pdSMC1A and 2) demonstrated at least 10-fold increases in life span; pdSMC1A became immortal. The SMC identity of both pdSMC lines was confirmed by SM22 mRNA expression. pdSMC2 were generally diploid but with various structural and numerical alterations; pdSMC1A demonstrated several chromosomal abnormalities, most commonly -Y, +7, -13, anomalies previously reported in both primary pdSMCs and atherosclerotic tissue. Confluent pdSMC2 appeared grossly similar to HPV-16 E6/E7-expressing normal adult aortic SMCs (AASMCs), exhibiting typical SMC morphology/growth patterns; pdSMC1A displayed irregular cell shape/organization with numerous mitotic figures. Dedifferentiation to a synthetic/proliferative phenotype has been hypothesized as a critical step in atherogenesis, because rat neonatal SMCs and adult intimal SMCs exhibit similar gene expression patterns. To confirm that our pdSMC lines likewise express this apparent plaque phenotype, osteopontin, platelet-derived growth factor B, and elastin mRNA levels were determined in pdSMC1A, pdSMC2, and AASMCs. However, no significant increases in osteopontin or platelet-derived growth factor B expression levels were observed in either pdSMC compared with AASMCs. pdSMC2 alone expressed high levels of elastin mRNA. Lower levels of SM22 mRNA in pdSMC1A suggested greater dedifferentiation and/or additional population doublings in pdSMC1A relative to pdSMC2. Both pdSMC lines (particularly 1A) demonstrated high message levels for matrix Gla protein, previously reported to be highly expressed by human neointimal SMCs in vitro. These results describe 2 novel plaque cell lines exhibiting various features of plaque SMC biology; pdSMC2 may represent an earlier plaque SMC phenotype, whereas pdSMC1A may be representative of cells comprising an advanced atherosclerotic lesion.
Key Words: atherosclerosis • smooth muscle • plaque • platelet-derived growth factor B • osteopontin
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Introduction |
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Vascular remodeling is a critical yet successful adaptive feature for the maintenance of blood flow in vessels with thickened intimas. However, additional neointimal expansion may surpass the adaptive capabilities of a vessel, leading to lumenal narrowing (for review, see Schwartz et al1 ).Plaque rupture, vasospasm, and/or thrombus formation are common sequelae in the functionally compromised, diseased vessel. Smooth muscle cell (SMC) proliferation and migration from the arterial media into the intima have been described in the development of atherosclerotic disease in both human and animal models.2 3 In addition, these neointimal SMCs are believed to promote key processes of plaque formation including the accumulation of extracellular matrix and deposition of calcium.4 5 6
Several initiating mechanisms responsible for these events have been postulated including the response-to-injury theory (for review, see Ross7 ) and the theory of viral atherogenesis.8 9 10 The monoclonal nature of SMCs comprising the plaque supports the role of viral transformation in atherogenesis.11 12 13 Whether this altered SMC population is the progeny of a single activated/injured cell or represents the expansion of a developmentally unique population within the vessel wall is unknown.11 A compatible theory that is based on data obtained from the rat model suggests that the altered neointimal SMC represents the reversion of the adult medial SMC to an immature phenotype expressing genes characteristic of a synthetic/proliferative SMC.6 14 15 16 17 18 The previously mentioned hypotheses of atherogenesis have unfortunately remained largely unproven or are based on various animal models that appear to be of limited value in the clinical setting. For these reasons, a relevant and useful system is clearly needed to better understand this prevalent and deleterious pathological process.
One factor that has compounded this problem is the unavailability of normal adult human SMCs, and particularly plaque SMCs, that are easily propagated in culture. This laboratory has previously demonstrated that the in vitro life span of normal human SMCs derived from fetal aorta, adult myometrial artery, and adult aorta can be significantly increased by infection with retroviral constructs containing human papillomavirus type-16 (HPV-16) E6 and E7 open reading frames.19 20 At approximately passages 35 to 40, these SMCs entered a crisis period. The fetal and myometrial SMCs emerged as immortalized cells.19 In view of these previous results, the present studies have used this same strategy to extend the life span of human SMC lines derived from atherosclerotic plaque, thereby providing opportunities to better understand plaque SMC biology. In this study, we describe the generation and characterization of plaque-derived SMC lines with extended life spans.
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Methods |
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Tissue Culture
Cell culture was performed by using Dulbecco's modified essential medium (DMEM) (GibcoBRL) supplemented with 10% FBS (Hyclone), penicillin (100 U/mL; GibcoBRL), and streptomycin sulfate (100 µg/mL; GibcoBRL). pdSMC1A and pdSMC2 cells arose from explant cultures of surgical specimens obtained from 2 patients with grossly diseased atherosclerotic plaque present in an aortic aneurysm and a carotid artery, respectively.
The surgical specimens were washed with sterile PBS. A representative section of atherosclerotic tissue was retained from each for histological examination. (The entire pathological report is presented in Results.) The ablumenal surface of both specimens was lightly scraped to remove any connective tissue. The lumenal surface of both specimens was lightly scraped to remove endothelial cells. The lesions appeared to be quite advanced/developed such that no media was observed in either specimen. Pathological evaluation confirmed that media as well as elastic fibers typical of normal medial tissue were absent from both specimens. Grossly diseased areas of plaque were minced and washed with culture medium, followed by collection of tissue fragments and cells by centrifugation. Tissue resuspended in medium was plated on standard tissue culture plates. The medium was replaced every 4 days. After 30 days, halos of cell growth could be observed around the tissue fragments. The cells were harvested by trypsinization, and tissue fragments were removed by sedimentation for 30 seconds at 1g. The cell suspension was plated in a 100-mm plate in 10 mL of DMEM containing FBS and antibiotics and was designated passage 1. When confluent, this plate was split 1:4 and designated passage 2. Cells were harvested at confluence by trypsinization and passaged at a 1:3 split ratio. Other human cells used for these studies include the previously described fetal aortic SMCs expressing HPV-16 E6/E7 (early and late/immortal passage),19 20 adult aortic SMCs expressing HPV-16 E6/E7 (AASMCs),19 20 adult myometrial SMCs expressing HPV-16 E6/E7,19 20 and uninfected foreskin fibroblasts, all of which were grown in DMEM containing FBS and antibiotics.
Retroviral Infections
Twenty-four hours after plating, retroviral infections and G418
selection were performed on passage 2 cells as previously
described.19 21 One plate received the pLXSN-HPV-16 E6/E7
retrovirus, a second received the pLXSN control retrovirus, the third
was used to ensure adequate G418 selection, and the fourth was used to
monitor growth properties of the cells in the absence of retrovirus or
G418 selection. Selection was discontinued 48 hours after 100% cell
death was observed on the noninfected control plate (day 10 after
infection). On day 16 after infection, 200 to 300
G418-resistant colonies were harvested and replated as pooled
populations and designated passage 3. The plates were grown to
confluence and propagated via 1:3 splits until senescent to determine
the life span of the cells in vitro.
Proliferation Assays
SMCs were plated at an approximate density of
5x104 cells/60-mm plate in DMEM containing FBS
and antibiotics. Plating density was determined 24 hours later by
counting cells in a hematocytometer by using trypan blue exclusion as a
measure of viability. The medium was replenished every 24 hours
thereafter, and cells were counted that had been in culture for 48, 96,
and 120 hours.
Morphology/Immunocytochemistry
Indirect immunofluorescence was performed by
using antibodies directed against 
-smooth muscle actin (Sigma clone
1A4, 2.7 ng/mL) and vimentin (Dako clone V9, 1:25; data not shown).
Negative staining for von Willebrand factor eliminated the
possibility of endothelial cell contamination (Dako,
1:100, as previously described20 ; data not
shown).
Isolation of Poly(A)+ RNA From Cultured Cells
Poly(A)+ RNA was isolated from SMC
cultures by using a modification of the Fast Track mRNA Isolation Kit
(Invitrogen). Subconfluent or 2 days postconfluent SMC cultures were
lysed (0.2 mol/L NaCl, 0.2 mol/L Tris, pH 8, 1.5 mmol/L
MgCl2, and 2% SDS) and forced through a 22-gauge
needle to shear the DNA followed by treatment with proteinase K. Each
sample was adjusted to 0.5 mol/L NaCl, and hydrated oligo(dT) cellulose
was added. After incubation and several washes in binding buffer (0.5
mol/L NaCl, 0.01 mol/L Tris, pH 7.5), the final pellet was transferred
to a 0.45-µm filter unit (UFC30HV00, Millipore), centrifuged
for 20 seconds at 14 000g at room temperature, and washed
with binding buffer. Poly(A)+ RNA was eluted with
DEPC (Sigma Aldrich) H2O and precipitated
with 3 mol/L NaOAc, pH 7.0, and ethanol. RNA pellets were washed and
reconstituted in DEPC H2O.
Isolation of RNA From Tissue
Total RNA was isolated from snap-frozen tissue (
0.5 g) as
described by Chomczynski and Sacchi22 with a slight
modification. Tissue homogenate was subjected to 2
phenol:chloroform/isoamyl alcohol extractions after which precipitated
nucleic acids were redissolved in 0.01 mol/L Tris, pH 7.5, 0.005 mol/L
EDTA, pH 8.0, 1% SDS (TES) and purified by chloroform:butanol
extraction as described by Chirgwin et al.23
Northern Analysis
Eight to 10 µg of poly(A)+ RNA from
cultured SMCs or total RNA from heart transplants (human heart
transplant donor and recipient patients' surplus aortic tissue) was
electrophoresed on a 1.2% agarose gel according to standard methods
and transferred to Hybond N (Amersham) with 10x SSC (1x SSC=150
mmol/L NaCl, 15 mmol/L
Na3C6H5O7
· 2H2O). RNA was cross-linked by using a
Stratalinker (Stratagene), and the membrane was hydrated in 2x SSC for
5 minutes, prehybridized 1 hour in 10 mL hybridization solution
(0.5 mol/L Na2HPO4, pH 7.2,
5% SDS) at 68°C, and hybridized 18 hours with
2x107 cpm labeled probe. The membrane was also
hybridized with a cDNA probe for the ribosomal component 36B4 to
control for intraexperimental/gel loading error. cDNA probes were
labeled with [-32P]dCTP or
[-33P]dCTP (NEN) to a specific activity of
1x109 cpm/µg DNA (Random Primed Labeling
kit; Boehringer Mannheim). Hybridized membranes were rinsed in
50 mmol/L
Na2HPO4/0.2% SDS for 2x30
minutes at room temperature followed by ±10 minute wash in 50
mmol/L NaPO4/0.2% SDS at 68°C. Radioactive
signals were quantitated by using a Phosphorimager (Molecular Dynamics)
and ImageQuant software (Molecular Dynamics).
cDNA Probes
The rat SM22 cDNA (clone 3RF10; 1.0 kb)14
recognized a 1.3-kb transcript. The 600-bp human 36B4 probe recognized
a 1.38-kb transcript.24 The 3.3-kb rat tropoelastin (ELN)
cDNA (clone 56A3)17 recognized a 3.5-kb transcript. The
1493-kb human osteopontin (OP) cDNA (OP-10) recognized a 1.2-kb
transcript.25 The human platelet-derived growth factor
B (PDGF B) cDNA (2.5 kb) recognized a 3.5-kb transcript.26
The 0.7-kb human matrix Gla protein (MGP) cDNA recognized bands of
0.65 and 0.5 kb and was obtained from American Type Culture
Collection (ATCC no. 59694).27 The 1.3-kb human PDGF A
cDNA recognized transcripts of 1.7, 2.3, and 2.9 kb.28 The
1.84-kb human epidermal growth factor (EGF) receptor probe recognized a
5.8-kb transcript and was from ATCC (ATCC no. 57484).29
The 630-bp human heparin-binding epidermal growth factor (HB-EGF) cDNA
recognized a 2.5-kb transcript.30 The 1.11-kb human
AQP1/CHIP28 cDNA recognized a 3.1-kb transcript and was from ATCC (ATCC
no. 99538).
Statistical Analysis
Data from growth assays and northern analyses of mRNAs
expressed in vitro are reported as mean values of 3 experiments±SEM
values. Statistical analysis was determined by
nonparametric analysis, using the Mann–Whitney
U test with significant differences designated as having
P<0.05 unless otherwise noted.
Cytogenetics
Chromosomes were prepared from actively growing cultures by
using standard cytogenetic procedures. For pdSMC1A, both pre- and
postcrisis cultures were analyzed. To prepare metaphase
spreads, subconfluent cultures were arrested in 0.015 µg/mL colcemid
(deacetylmethyl colchicine; GibcoBRL) for 15 hours, harvested by the
addition of 0.075 mol/L KCl for 20 minutes, followed by fixation in
methanol/glacial acetic acid (3:1). G-bands were obtained in
0.125 mg/mL pancreatin (GibcoBRL) in Hanks' balanced salt solution, pH
7.5 (GibcoBRL), followed by staining in Gurr's Geimsa, 1:10 in Gurr's
buffer, pH 6.8. Resulting metaphase chromosome spreads were examined
for evidence of chromosomal abnormalities.
Comparative genome hybridization was performed according to the methods of Weber et al31 with minor modifications. In brief, test DNA (from pdSMC1A or 2) and reference DNA (from normal human foreskin fibroblasts) were labeled by nick translation with biotin-14-dATP and digoxigenin-11-dUTP (Boehringer Mannheim), respectively. Labeled DNAs were hybridized with normal human male chromosomes and then subjected to a series of stringent washes. Bound labeled DNAs were visualized by incubation with FITC-avidin (with the FITC signal amplified by prior incubation of hybridized chromosomes with FITC-avidin and then biotinylated goat anti-avidin antibodies) and rhodamine anti-digoxigenin antibodies. An average fluorescence ratio (test: reference or green:red and, hence a yellow signal) of 1.00 indicates equal representation of both DNAs, a ratio <1 (red signal) indicates underrepresentation of test or pdSMC1A DNA, and a ratio >1 (green signal) indicates overrepresentation of test or pdSMC1A DNA. To assure accurate results, the reverse experiment was also performed in which the test DNA and reference DNA were labeled with digoxigenin-11-dUTP and biotin-14-dATP, respectively.
Microscopy
Representative atherosclerotic plaque specimens
from carotid artery abdominal aorta were fixed in 10% buffered
formalin and paraffin embedded. Hematoxylin–eosin-stained sections
were prepared for light microscopic evaluation.
Telomerase Assay
The telomerase repeat amplification protocol (TRAP) assay was
performed as described,32 33 34 using 0.1 µg of
lyophilized CX primer ([5'-(CCCTTA)3CCCTAA-3'])
on the bottom of the tube sealed with a bead of Ampliwax
(Perkin-Elmer). The telomerase reaction above the wax involved a
TS primer (5'-AATCCGTCGAGCAGAGTT-3') labeled with
[
-32P]ATP using T4
polynucleotide kinase (Boehringer Mannheim). The TS
primer was used at 0.1 µg/reaction at 25x106
cpm/µg of primer. The reaction mix was made as previously
described,32 using 5 µg of protein. During the 25-minute
incubation at room temperature, the TS primer is used by telomerase, if
present, for the addition of TTAGGG repeats. After this incubation,
the sample was subjected to 3 minutes at 90°C to melt the wax barrier
and 27 cycles of PCR as described. One-tenth of the reaction was run on
an 8% nondenaturing polyacrylamide gel after which the gel was
dried and exposed to film for 24 hours at -80°C.
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Results |
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The atherosclerotic plaque material from both surgical specimens showed similar histological findings including marked thickening of the intima with collagen deposition, cholesterol clefts, dystrophic calcification, scattered macrophages, and SMCs (Figure 1
).
The SMCs were spindle-shaped with fine nuclear chromatin and showed no
cytological atypia. The SMCs were interspersed among dense collagen
bundles. The microscopic findings confirmed the diagnosis of
atherosclerotic plaque material. No media was present in either
specimen and Verhoeff–Van Gieson stains confirmed the absence of
elastic fibers typically seen in normal medial tissue of the aorta and
carotid arteries. It is presumed that neointimal formation
was quite progressed involving the region typically occupied by the
media. No adventitia was seen in either specimen.
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Figure 2 shows the in vitro life span of
HPV-16 E6/E7-expressing SMCs (designated pdSMC1A or pdSMC2 or, in
general, pdSMCs) compared with the life span of uninfected cells.
Vector control plates in all cases contained 200 to 300
G418-resistant small colonies, which were pooled and replated;
these cells senesced before reaching confluence at passage 3. The
uninfected cells reached confluence at passage 3 but could not be
further propagated. Both pdSMC lines entered a crisis at p30 to p33,
but only pdSMC1A emerged, continuing to proliferate at least 100
passages. Having undergone >75 population doublings, the pdSMC1A cell
line is apparently immortalized. Postcrisis pdSMC1A displayed
morphological and proliferative traits characteristic of immortalized
cells. The proliferative rate of these cells was increased relative to
the precrisis pdSMC1A. In addition, TRAP assays revealed that these
late-passage pdSMC1A exhibited telomerase activity, a feature common to
many immortalized cells (Figure 3).
Early-passage AASMCs, pdSMC1A, and pdSMC2 were telomerase
negative (Figure 3).
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Figure 4 shows the morphology of the
early-passage pdSMC1A, pdSMC2, and AASMCs, and the late-passage
(immortal) pdSMC1A. Both pdSMC2 and AASMC cultures were predominately
composed of elongated, spindle-shaped cells that grew to confluence,
forming the hill-and-valley pattern reminiscent of normal SMCs.
Early-passage pdSMC1A displayed more irregular morphology, less
hill-and-valley organization, and many mitotic figures in postconfluent
cultures relative to pdSMC2 and AASMCs. Late-passage pdSMC1A were
slightly smaller and more rounded in contrast to the more
spindle-shaped precrisis cells. Both pdSMC lines were dilution tolerant
and grew readily when plated at 1:10 split ratio. As shown in Figure 5, pdSMC1A and AASMCs demonstrated
similar growth rates in subconfluent cultures as well as in nearly
confluent cultures. In contrast, pdSMC2 exhibited higher rates of
proliferation than pdSMC1A after 4 days in culture
(P<0.10), with even greater increases becoming apparent
after 6 days in culture as the cells became confluent
(P<0.05). These proliferation assay data are particularly
interesting with regard to the increased numbers of mitotic figures
observed in postconfluent cultures of pdSMC1A but not pdSMC2 or AASMCs.
These results may indicate that confluent cultures of pdSMC1A are not
quiescent as are pdSMC2 but continue to proliferate, suggestive of
disregulated pdSMC1A replication. Further growth studies are necessary
to confirm this possibility.
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Immunohistochemical analysis revealed that 90% of the cells
in any given microscopic field stained strongly for -smooth
muscle-actin with staining localized to long filamentous cytoplasmic
structures consistent with the presence of thin filaments or
stress fibers (data not shown). The presence of -smooth muscle-actin
mRNA in pdSMCs was confirmed by northern analysis (data not
shown). High levels of mRNA encoding SM22 were also observed in both
pdSMC lines, confirming the SMCs identity of these cells (see Figure 7). Both pdSMC lines stained strongly for vimentin (data not
shown). No staining above background was observed when cells were
stained for von Willebrand factor, eliminating the possibility
of endothelial cell contamination (data not shown).
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Metaphase chromosome spreads from pdSMCs were analyzed for
numerical and structural chromosomal abnormalities
(Table). As expected, neither cell line
was clonal because primary cultures were generated from pooled
populations of G418-resistant colonies derived from large areas
of grossly diseased tissue, perhaps consisting of many overlapping foci
of disease. The pdSMC2 were essentially diploid with a variety of
apparently random abnormalities. The most consistent karyotypic
aberration was seen in the pdSMC1A line (46, X, -Y, +7). The -Y, +7
anomalies strongly emerged as predominant features in these cells as
metaphase spreads prepared from cells p12 to p22 were examined. These
results were confirmed by comparative genome hybridization (using
early-passage normal male fibroblasts as a control), which further
revealed the loss of 1 copy of chromosome 13 in the pdSMC1A cell line
(Figure 6). Karyotypic analysis
indicated that the -13 population likewise emerged in increasing
passages of pdSMC1A. Late-passage pdSMC1A were consistently
triploid, hypotriploid, and hypertriploid. A recurring feature in these
cells was the presence of 2 identical markers of unknown origin.
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To provide further evidence for the plaque origin of these SMC lines,
northern analysis was performed on pdSMCs and AASMCs for the
expression of OP, PDGF B, and ELN, genes implicated in atherogenesis
based on the rat "pup-intimal" or PI model of plaque
formation. According to this model, steady-state levels of these mRNAs
are highly expressed in rat pup SMCs and neointimal
(postinjury) SMCs but not in normal medial SMCs from adult
rat.6 14 15 16 17 18 For the following experiments, observed
differences in steady-state mRNA levels are expressed relative to
message levels expressed by AASMCs (AASMC 
"control").
Northern analysis for OP expression revealed similar steady
state message levels for AASMC and pdSMC1A whereas OP mRNA levels were
significantly decreased in pdSMC2 (14±6% of control) (Figure 7A
, and upper panel, Figure 7B).
For PDGF B mRNA, expression was decreased in both pdSMC1A and
pdSMC2 (73±10% and 73±6% of control, respectively) (Figure 7A, and middle panel, Figure 7B). pdSMC2 alone exhibited
high levels of ELN mRNA (1305±104% of control); pdSMC1A was not
significantly different from AASMCs (Figure 7A, and lower panel,
Figure 7B). Because these results did not clearly identify
pdSMC1A and pdSMC2 as "plaque-derived SMCs" based on published
characteristics and models,6 14 15 16 17 18 we considered several
other genes that have also been proposed to play a role in the
development of atherosclerosis. Two genes examined were
the EGF receptor35 36 and PDGF A,37 38 both
of which are located on chromosome 7. As previously mentioned, trisomy
7 has been observed in numerous plaques and plaque-derived SMC, as well
as the pdSMC1A described in the present studies. Although message
levels were not apparently elevated for the EGF receptor or its ligand
HB-EGF in pdSMCs (data not shown), steady-state levels of PDGF A mRNA
(all three transcripts) were increased in both pdSMC1A and pdSMC2
relative to AASMCs (240±50% and 310±58% of control, respectively)
(Figure 8A, showing the 2.3-kb
transcript, and upper panel, Figure 8B). Other genes considered
included the SMC markers MGP and SM22. Both pdSMC1A and pdSMC2
exhibited significantly higher levels of MGP mRNA (3056±1080% and
561±75% of control, respectively) with >5-fold greater increases
observed for pdSMC1A than for pdSMC2 (Figure 8A, and middle
panel, Figure 8B); results were similar for corresponding
subconfluent cultures. In contrast, northern analysis for SM22
indicated that expression of this gene is strongly decreased in
early-passage pdSMC1A (45±8% of AASMC control) but not in pdSMC2
(170±44% of control) of similar in vitro life span (Figure 8A, and lower panel, Figure 8B). Expression of SM22 was also
strongly decreased in late-passage (postcrisis) human fetal
SMCs19 (4±3% of control) but not in early-passage fetal
SMCs19 (92±15% of control) (Figure 7A, and lower
panel, Figure 8B).
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Examination of OP mRNA expression in normal and diseased aortic
tissue revealed high levels of message levels in an advanced
atherosclerotic specimen taken from heart transplant donor aortic
tissue but not in a grossly similar specimen from a heart transplant
recipient aortic tissue (Figure 9A, and upper panel, Figure 9B). It is noteworthy that higher levels of
MGP mRNA were observed in both of these diseased specimens and were
elevated relative to matched normal tissue for 1 of the diseased
specimens and normal tissue from 2 other heart transplant donors of
much younger age (Figure 9A, and lower panel, Figure 9B).
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Discussion |
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We relate the first generation of plaque SMC lines by the introduction of the HPV-16 E6 and E7 open reading frames into SMCs subcultured from aortic plaque tissue and an endarterectomy plaque specimen (cell lines designated pdSMC1A and pdSMC2, respectively). Microscopic and immunohistochemical analysis of the diseased tissue confirmed the pathological diagnosis of these specimens and the origin of SMCs used for these studies. Immunohistochemical and northern analysis confirmed the SMC identity of both pdSMC lines. The genotypic profile of these cells was further assessed by northern analysis for further confirmation of the plaque origin of the cells as described in the literature.6 14 15 16 17 18
Although pdSMC1A and pdSMC2 demonstrated extended life spans in vitro,
uninfected or vector-infected plaque SMCs senesced after 3 to 4
population doublings in culture. This observation is consistent
with published reports describing the limited viability of plaque SMCs
in vitro39 and excludes the possible contamination of
pdSMC cultures with normal medial SMCs typically having a longer in
vitro life span (10 passages). Karyotypic analysis revealed
genetic aberrations in these 2 pdSMC lines consistent with a
plaque-derived phenotype.40 41 42 43 pdSMC2 were
generally diploid with a variety of structural and numerical
alterations, yet with no consistent chromosomal losses or
gains; pdSMC1A were highly aneuploid, a -Y, +7, -13 population
strongly emerging with passage in culture. The -Y, +7 genotype
is a consistently reported feature of SMCs in atherosclerotic
lesions40 42 43 and has been observed in SMCs grown from
atherosclerotic lesions as well as in SMC-positive areas of
paraffin-embedded plaques.40 42 43 Whether the predominant
cytogenetic anomalies observed in the present studies contributed
to the outgrowth of this population or are the consequence of other
selective factors/stimuli is unknown. It is noteworthy that the
aneuploidy observed at early passage in the pdSMC1A reflects the plaque
origin of these cells rather than an artifact of tissue culture, since
cultured normal adult and fetal (early-passage) SMCs and pdSMC2 are not
aneuploid.19 20 44 Results reported by Perez-Reyes et
al19 as well as the present findings with pdSMC2
further indicate that the aneuploidy observed in pdSMC1A is not a
function of E6-mediated p53 inactivation. These observations support
the identification of these lines as derived from atherosclerotic
plaque and provide the foundation for further studies designed to
understand mechanisms involved in human atherogenesis.
Possible effects of the HPV-16 E6/E7 genes are unlikely because the pdSMC lines have been compared with their "normal" HPV-16 E6/E7-expressing counterparts (AASMCs). Furthermore, previous studies have demonstrated similar genotypic and phenotypic profiles for normal (uninfected) aortic SMCs and HPV-16 E6/E7-expressing SMCs derived from normal aorta (AASMCs).19 20 Of possible concern is the fact that AASMCs do not represent normal aortic medial SMCs having been derived from a 51-year-old male heart transplant recipient.19 However, morphological and genotypic characterizations19 20 of AASMCs suggest that these cells were isolated from nondiseased tissue and represent normal aortic medial SMCs.
Although both pdSMC lines present a typical SMC immunohistochemical phenotype, additional observations suggest that these 2 cell lines represent distinct pdSMC populations. pdSMC2 appeared spindle-shaped with hill-and-valley organization patterns reminiscent of SMCs; pdSMC1A displayed irregular cell shapes and organization as well as numerous mitotic figures in confluent cultures. Growth assays indicate a higher rate of proliferation in subconfluent cultures of pdSMC2 than in pdSMC1A. On reaching confluence, however, pdSMC2 became quiescent whereas pdSMC1A continue to divide. These distinguishing morphological/proliferative characteristics were found to be complemented with differing degrees of chromosomal aberrations. pdSMC1A consistently displayed specific chromosomal alterations (-Y, +7, -13) with increasing passage. pdSMC2 demonstrated only random losses and gains, although chromosomal abnormalities may be present that cannot be detected by karyotypic analysis. These data may suggest that pdSMC1A represent a more pathologically advanced population of plaque SMCs that proliferate in response to particular stimuli despite cell contact, whereas other plaque SMCs, typified by pdSMC2, remain quiescent. This possibility is supported by Belknap et al,6 who have hypothesized that the expression of a given neointimal SMC genotype/phenotype reflects the developmental state of the lesion or time after injury/activation rather than the reversion to a single specific immature phenotype. Because it is difficult to know the pathological history of a lesion, this hypothesis may explain the reported heterogeneity observed among subpopulations of injured SMCs18 45 as well as the differences between the 2 pdSMC lines under consideration in the present study. For this reason, it would be helpful to generate and characterize pdSMCs derived from other atherosclerotic lesions.
The possibility that pdSMC1A represent SMCs from a more advanced lesion is interesting with regard to the observation that pdSMC1A (but not pdSMC2) were able to escape senescence and become immortalized. The extensive chromosomal alterations observed in early-passage pdSMC1A may be important events leading to the transformation of these cells. Further evidence of genetic aberrations is provided by the observation that postcrisis pdSMC1A but not pdSMC2 expressed high levels of telomerase activity. Perez-Reyes et al19 reported that fetal human SMCs expressing HPV-16 E6/E7 also became immortal but not AASMCs, perhaps suggesting that a fetal-associated gene is somehow involved in escape from crisis. The presence of HPV-16 E6 and E7 in both pdSMC lines as well as AASMCs clearly makes viral intervention an unlikely mediator of the telomerase activation observed in pdSMC1A. However, the expression of telomerase activity does not always correlate with immortalization,46 indicating that other events are necessary for the continued proliferation observed in pdSMC1A. The present findings, however, do support the hypothesis that genetic instability, as seen in pdSMC1A, may contribute to the extended life span of these cells in vitro and, conceivably, SMCs comprising the neointimal lesion in vivo.
In an effort to genotypically confirm the plaque origin of pdSMC1A and
2, northern analysis was performed for genes expressed by
plaque SMCs.6 14 15 16 17 18 The increased expression of an
exclusive set of genes has been described in neonatal and injured adult
rat SMCs but not in uninjured or adult rat SMCs.6 14 15 16 17 18
This group of differentially expressed genes, known as the pup-intimal
(
) genes, includes OP, PDGF B, ELN, cytochrome P450IA1, collagen
type 1A, and the PDGF -receptor.6 14 15 16 17 18 We examined
the steady-state levels of OP, PDGF B, and ELN in pdSMCs relative to
AASMCs, their HPV-16 E6/E7-expressing "normal" counterparts, but
did not observe increased expression of these genes in our pdSMC lines
with the exception of ELN in pdSMC2. Having previously concluded that
our cell lines very likely represent true plaque SMCs, these
data indicate significant differences between rat and human
neointimal formation. Several possible factors may play a
role(s) in these apparently discrepant results. Basic anatomical
differences between the rat and the human vasculature have been
described that should be considered. Rats do not normally possess an
intima or exhibit vascularization of the media.1 In
addition, the rat model of neointimal formation is
dependent on balloon catheter–induced injury of a normal vessel
neointimal development over a relatively brief period of
time.1 17 47 48 49 50 Conversely, human atherosclerotic lesions
represent an accumulation of chronic inflammatory,
proliferative, adaptive, and necrotic processes. Although these 2 types
of lesions may share many features, very different processes may have
contributed to the resulting plaque, and key players such as plaque
SMCs may, therefore, present different phenotypes.
An alternate but not necessarily mutually exclusive hypothesis of
atherogenesis involves the dedifferentiation of medial SMCs to a
phenotype in which low levels of cytoskeletal/contractile
proteins (-actin, caldesmon, SM22, and calponin) are
expressed.5 In the present studies, SM22 mRNA levels
in fetal SMCs appear to be inversely related to the number of
population doublings (see Figure 7B). Furthermore, SM22 levels
were significantly lower in early-passage pdSMC1A compared with pdSMC2,
which were similar to those for AASMCs. These observations are
particularly interesting in regard to a possible mechanism of
atherogenesis such as the monoclonal expansion of a phenotypically
unique cell type. The decreased SM22 and calponin mRNA levels in
pdSMC1A relative to pdSMC2 may indicate a longer in vivo proliferative
history of the former and support the hypothesis that pdSMC1A
represent SMCs from a more advanced plaque. Whether the
dedifferentiated phenotype is accessory or consequential to
this extended life span is unknown.
The prevalence of the +7 genotype in plaque SMCs40 42 43 prompts some degree of consideration of a possible role of a gene(s) located on chromosome 7 in atherogenesis. Several of these genes' products have been associated with SMC mitogenic/chemoattractant activity, differentiation, or vascular tone/contractility14 17 36 47 54 55 56 57 58 and include PDGF A, tropoelastin, AQP1/CHIP28, nitric oxide synthase, and the EGF receptor. To this point, we have performed northern analysis for PDGF A, ELN, AQP1/CHIP28, and the EGF receptor (data not shown), and have evidence that message levels for PDGF A are quite elevated in the +7 pdSMC1A. However, PDGF A mRNA levels were also increased in pdSMC2 that did not display an extra copy of chromosome 7. In contrast, ELN mRNA levels were elevated in pdSMC2 rather than pdSMC1A (as previously discussed). Message levels for the EGF receptor and AQP1/CHIP28 were not significantly different in pdSMCs compared with AASMCs. Further investigation is therefore necessary to clarify a possible role of chromosome 7 in atherogenesis.
One characteristic identified in the pdSMC lines that appears to consistently typify human plaque-associated SMCs is the increased expression of MGP mRNA, the product of which correlates with the presence of SMC as well as areas of severe calcification within the neointimal lesion.5 The observation that mRNA levels for this gene were expressed at even greater levels in pdSMC1A than pdSMC2 again supports the hypothesized advanced lesion phenotype of pdSMC1A. We have also observed that MGP mRNA is highly expressed in plaques from 2 different heart transplant organ recipients but not in matched normal tissue or in normal tissue from heart transplant donors of much younger age. In contrast, OP mRNA was variably expressed in these plaques, possibly reflecting the stage of the lesion and macrophage involvement since other investigators have found that OP mRNA and protein are primarily expressed by macrophages rather than SMCs.5 49 51 52 53 These results may suggest that MGP rather than OP may be an accurate marker of SMCs associated with advanced plaques.
In conclusion, data have been presented describing the generation and characterization of 2 novel pdSMC lines. Results from these studies not only affirm the plaque-derived SMC origin of the pdSMCs, but have also revealed some important distinctions between rat and human plaque SMCs and suggest possible specifies-specific mechanisms of atherogenesis as well as heterogeneity among pdSMCs.
|
Acknowledgments |
|---|
This work was supported by National Institutes of Health grants HL47151 and CA42792 to J.K. McDougall.
Received March 18, 1998; accepted August 13, 1998.
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References |
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