© 1999 American Heart Association, Inc.
Cellular Biology |
Evidence From a Novel Human Cell Clone That Adult Vascular Smooth Muscle Cells Can Convert Reversibly Between Noncontractile and Contractile Phenotypes
From the Vascular Biology Group (S.L., L.H.C., J.G.P.), John P. Robarts Research Institute; Department of Medicine (Cardiology) (L.H.C., J.G.P.), London Health Sciences Centre; and Departments of Biochemistry (J.G.P.), Medical Biophysics (J.G.P.), and Physiology (S.S., Y.J.), University of Western Ontario, London, Ontario, Canada.
Correspondence to J. Geoffrey Pickering, MD, PhD, FRCP(C), London Health Sciences Centre, 339 Windermere Rd, London, Ontario N6A 5A5, Canada. E-mail gpickrng{at}rri.on.ca
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Abstract |
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Abstract—Smooth muscle cells (SMCs) perform diverse functions that can be categorized as contractile and synthetic. A traditional model holds that these distinct functions are performed by the same cell, by virtue of its capacity for bidirectional modulation of phenotype. However, this model has been challenged, in part because there is no physiological evidence that an adult synthetic SMC can acquire the ability to contract. We sought evidence for this by cloning adult SMCs from human internal thoracic artery. One clone, HITB5, expressed smooth muscle
-actin, smooth myosin heavy chains,
heavy caldesmon, and calponin and showed robust calcium transients in
response to histamine and angiotensin II, which confirmed
intact transmembrane signaling cascades. On serum withdrawal, these
cells adopted an elongated and spindle-shaped morphology, random
migration slowed, extracellular matrix protein production fell,
and cell proliferation and [3H]thymidine incorporation
fell to near 0. Cell viability was not compromised, however; in fact,
apoptosis rate fell significantly. In this state,
agonist-induced elevation of cytoplasmic calcium was even more
pronounced and was accompanied by SMC contraction. Readdition of 10%
serum completely returned HITB5 cells to a noncontractile,
proliferative phenotype. Contractile protein expression
increased after serum withdrawal, although modestly, which suggested
that the switch to contractile function involved reorganization or
sensitization of existing contractile structures. To our knowledge, the
physiological properties of HITB5 SMCs provide the
first direct demonstration that cultured human adult SMCs can convert
between a synthetic, noncontracting state and a contracting state.
HITB5 cells should be valuable for characterizing the basis of this
critical transition.
Key Words: muscle, smooth, vascular • differentiation • contraction • cell movement
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Introduction |
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The primary function of adult arterial smooth muscle cells (SMCs) is to contract. However, during vascular disease SMCs can be called on to perform noncontractile functions, including replication, migration, and elaboration and degradation of extracellular matrix (ECM) proteins. These noncontractile roles of SMCs are fundamental to the growth of atherosclerotic lesions, as well as lesions that develop after angioplasty/stent therapy and cardiac transplantation.1 2 Noncontractile functions of SMCs are also the means by which SMCs maintain the integrity of the fibrous cap that overlies atheromata, rupture of which can lead to arterial thrombosis and organ infarction.3 4
The basis by which adult SMCs can perform diverse functions has been an active area of inquiry over the past 2 decades. A classic view is that adult vascular SMCs have the capacity to modulate their phenotype, or state of differentiation, in a bidirectional manner. This concept was first hypothesized in 1967 by Wissler5 and was based on the assumptions that (1) SMCs were integral to the pathogenesis of atherosclerosis and (2) SMCs represented a single cell type in the artery wall. Subsequent support for the concept came from the finding that cells enzymatically dispersed from arterial media and placed in culture gradually assumed features of less-differentiated cells with characteristics of SMCs in vascular lesions.6 7 8 The paradigm thus holds that in response to vascular stress, SMCs specialized to contract can be induced to lose their contractile function and acquire synthetic functions; under different local conditions they can then revert to the more specialized, contractile state. This concept forms an important tenet of the response-to-injury hypothesis of atherosclerosis.9
Despite widespread appreciation of the concept of bidirectional SMC phenotype modulation, there remains no cell culture system whereby SMCs differentiate, reversibly or otherwise, to the extent that they can contract.10 Changes in various markers of differentiation have been identified (eg, myofibrillar content and smooth muscle [SM]–specific proteins),6 11 12 but there is no documentation to our knowledge that vascular SMCs in culture can acquire or reacquire the ability to contract. The absence of such a culture system has hindered efforts to dissect the molecular mechanisms that underlie the attainment and/or loss of contractile function of SMCs.
The lack of evidence for complete redifferentiation of cultured SMCs has also been suggested to pose a qualification to the concept of SMC phenotype modulation itself.13 Although it may be that the appropriate culture conditions for more complete differentiation have yet to be identified, the inability to drive cultured SMCs to a contractile state represents an apparent inconsistency to the SMC-shift paradigm. This is noteworthy in light of growing evidence that SMCs of the artery wall are not, as originally considered, a homogeneous population of cells,14 15 16 and thus there is no a priori need to invoke SMC phenotype shift to explain the emergence of a noncontractile phenotype. Instead, noncontractile or synthetic functions may arise by selective expansion of subpopulations of cells.13 This concept was supported by a recent observation that cells acutely dispersed from the canine carotid artery, and proven to be mature SMCs, did not have the capacity to dedifferentiate, proliferate, or migrate in culture. Instead, the migratory and proliferative functions seen in culture were performed by cells that, in the vessel wall, did not have markers of a mature SMC lineage.17 18
Ideally, to reliably determine whether a vascular SMC is capable of reversibly modulating to and from a contractile state and to exploit such a finding to model and better define the molecular basis of SMC differentiation, a homogeneous population of SMCs is required. This is because the heterogeneity inherent in primary cultures means that changes in individual cells cannot be easily distinguished from expansion of subpopulations with distinct phenotypes. Stable human SMC clones would overcome this limitation, but none have existed. We report here the generation and characterization of an adult human SMC clone that is capable of reversibly converting between a noncontractile state with migratory, proliferative, and synthetic properties and a well-differentiated state that contracts in response to vasomotor agonists.
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Cell Cloning
A primary culture of human SMCs was established by explant outgrowth of a segment of internal thoracic artery retrieved at the time of coronary artery bypass surgery.19 20 Endothelial cells were removed by scraping the luminal surface of the vessel with a scalpel blade, and the adventitia was mechanically stripped away. Primary cultures were maintained in medium 199 (M199) supplemented with 10% FBS. SMC identity was confirmed morphologically and by positive immunostaining for SM
-actin (1A4, Dako). Cloning of human SMCs was performed by a modified cloning ring approach. SMCs in the sixth subculture were dispersed with trypsin-EDTA and replated onto gelatin-coated culture dishes (100 µg/mL) at a density of 2 cells/cm2. These cultures were maintained in M199 that was conditioned by primary SMC cultures in log-phase growth and supplemented with 10% FBS. Individual colonies were identified microscopically, released by localized application of trypsin-EDTA, and subsequently expanded. We observed that 3 of the clones underwent striking morphological changes after serum withdrawal and displayed an unusually robust resilience to serum deprivation. Such clones were deemed to be uniquely informative with respect to differentiation of human SMCs. One of the clones, designated HITB5 (human internal thoracic B5) was characterized in this context and is the focus of this report. All experiments with HITB5 cells were performed with subcultures 14 to 19 after cloning. (HITB5 SMCs have been successfully passaged beyond the 40th subculture and have retained the properties described in this report.)
Cell Migration Assay
Motility of HITB5 cells was assessed by quantifying migration
path and migration speed using digital time-lapse video
microscopy.21 22 Cells were seeded onto culture dishes
precoated with 100 µg/mL type I collagen (Vitrogen, Collagen
Corp), and migration was monitored with an inverted microscope
(Zeiss Axiovert S100). A charge-coupled device video camera (Sony
XC-75) attached to the microscope was used to generate video images
that were digitally acquired over an 8-hour recording period
(Northern Eclipse, Empix Imaging, Inc.). Ambient temperature was
maintained at 37°C by placing the culture dish in a temperature
control cell (BC-500W, 20/20 Technology, Inc). Migration was measured
from digital images by tracking the location of cell centroids at
hourly intervals. Migration speed was determined as the sum of hourly
distances divided by the total time.
Cell Proliferation and DNA Synthesis Assay
To evaluate SMC replication, cells were seeded in 24-well plates
at a density of 3000 cells/cm2 and incubated in
M199 with 10% FBS for 48 hours. Cells were then washed with PBS and
incubated in serum-free M199 for 7 days, with a medium change on day 3.
Serum-supplemented conditions (M199 plus 10% FBS) were then restored,
and cells were cultured for 4 more days. On each of the 13 days, cells
from quadruplicate wells were dissociated with trypsin and counted with
a hemacytometer.
DNA synthesis rates were evaluated by pulse labeling SMCs with [3H]thymidine. HITB5 cells were seeded in 96-well plates at 5x103 cells/cm2 and incubated sequentially in serum-supplemented M199, serum-free medium, and serum-supplemented medium, as described for the proliferation assay. On designated days SMCs were incubated with [3H]thymidine (10 µCi/mL and 71 Ci/mmol, ICN) for 8 hours and then transferred onto glass fiber filters (Wallac) using an automated cell harvester (Tomtec). Incorporated [3H]thymidine was counted on a Trilax 1450 MicroBeta counter (Wallac).
Protein Synthesis Assay
Synthesis of proteins, including insoluble ECM proteins, was
studied by measuring incorporation of
[3H]leucine as previously
described.23 24 Briefly, SMCs were cultured in M199
supplemented, sequentially, with 10% FBS for 48 hours, with 0% FBS
for 3 days, and with 10% FBS for 2 more days. After each change of
growth medium, cells were pulsed with 4 µCi/mL
L-[4,5-3H]leucine (ICN) for 16
hours. Cells were then subjected to NH4OH lysis
and high-salt extraction. SMCs were washed with cold PBS and disrupted
with 0.25 mol/L NH4OH containing 1 mmol/L
PMSF and 1 mmol/L EDTA for 30 minutes. The lysate was withdrawn
and the remaining contents in the culture well washed with PBS
containing 1 mmol/L EDTA, followed by extraction with 50
mmol/L Tris (pH 7.4)–buffered NaCl (1 mol/L) for 15 minutes. The
culture well was then washed once more with PBS-EDTA. All lysis,
extraction, and wash solutions were combined to a single "cell
fraction," which was then precipitated in 10% trichloroacetic acid
(TCA). The acid-insoluble material was washed twice in 10% TCA and
solubilized in 1 mol/L NaOH followed by neutralization with an equal
volume of 1 mol/L acetic acid. Radioactivity was then determined by
scintillation counting (Cytoscint, ICN). The material remaining on the
culture surface after NH4OH and high-salt
extraction is referred to as the ECM fraction. This was solubilized in
NaOH (1 mol/L) and precipitated at 4°C by adding 0.1 volume of 100%
TCA. The TCA-insoluble fraction was washed and scintillation counted as
above. The protein concentration of the "cell fraction" was
determined with Bio-Rad DC protein assay reagents.
Apoptosis Assay
Apoptosis was assessed by terminal
deoxynucleotidyl transferase–mediated
dUTP-fluorescein nick end labeling (TUNEL) (Promega). SMCs
were washed with PBS, fixed for 25 minutes with 4% formaldehyde in
PBS, and permeabilized with 0.2% Triton X-100 in PBS.
End labeling of DNA fragments with 0.5 U/mL terminal
deoxynucleotidyl transferase and 5 mmol/L
fluorescein 12-dUTP was performed for 1 hour at 37°C.
Slides were then washed twice in 2x SSC followed by 3 washes in PBS.
Cells were counterstained with 5 mg/mL propidium iodide in PBS and
examined by fluorescence microscopy (Olympus BX100). For each
growth condition, 200 cells were counted and the percentage of
apoptotic cells (those with green fluorescence
localized to the nucleus) was determined.
Measurement of [Ca2+]i
Agonist-induced calcium transients were measured in single HITB5
cells loaded with fura-2–acetoxymethyl ester
(fura-2–AM).25 Trypsinized SMCs were loaded with 0.5
µmol/L fura-2–AM and transferred to a custom-made perfusion chamber
mounted on an inverted Nikon diaphot microscope. Cells were illuminated
with alternating 345- and 380-nm light and the 510-nm emission was
detected (Deltascan system, Photon Technology International).
Cytoplasmic free calcium concentration
([Ca2+]i) was calibrated
according to the methods of Grynkiewicz et al.26 Agonists
were applied to cells by pressure ejection from a micropipette. Data
were corrected for background fluorescence.
SMC Contraction
Contraction of sparsely seeded SMCs was evaluated at room
temperature using digital time-lapse video microscopy, as was used for
the migration assay. Cells cultured in M199 with 10% FBS or in
serum-free M199 for 3 days were imaged before and after addition of
either histamine or angiotensin II (Sigma Chemical Co). Two
approaches to quantifying contraction were used. With the first
approach, cells were maintained adherent to the culture dish and
changes in planar area were determined using image analysis
software (Empix). Acute contraction was evident within 1 minute of
adding agonist, and we recorded the maximum contraction for each
cell, expressed as the percentage reduction in cell area. In the second
approach, cell length was assessed. SMCs were gently loosened from the
substrate with trypsin-EDTA and then released with the addition of M199
with 10% FBS. This procedure maintained the elongated morphology of
the SMCs. Cells were then allowed to resettle on the culture substrate,
and agonist was added. Cell shortening was evident 
20 seconds after
agonist was added to the medium and contraction was expressed as the
maximal percentage reduction in cell length. Receptor
antagonists pyrilamine (Sigma Chemical Co) and
losartan (kindly provided by W. Henkler, Merck and Co, Inc,
Rahway, NJ) were added 10 minutes before agonists.
Western Blot Analysis
SMCs were washed with cold PBS and disrupted in lysis buffer
(in mmol/L, Tris [pH 7.4] 10, phenylmethylsulfonyl
fluoride 0.1, and EDTA 0.1, as well as 1% SDS and 10 µg/mL
leupeptin). For evaluation of SM myosin heavy chains (SM-MHC), the
lysis solution contained 40 mmol/L
Na4P2O7,
1 mmol/L EGTA, 0.1 mmol/L phenylmethylsulfonyl
fluoride, and 10 µg/mL leupeptin.27 Equal
amounts of proteins were separated on 12% (for SM -actin and
calponin), 6% (for caldesmon and metavinculin/vinculin), and 5% (for
SM-MHC) SDS-polyacrylamide gels. Proteins were
electrophoretically transferred to polyvinylidene difluoride
membranes (Millipore), which were then blocked overnight at 4°C with
5% nonfat dry milk in Tris-buffered saline containing 0.2% Tween 20.
Blots were then incubated for 2 hours at room temperature with
designated primary antibodies. Primary antibodies used were the
following: anti–SM -actin monoclonal antibody (1A4); anti–SM-MHC
monoclonal antibody that recognizes both SM1 and SM2 isoforms
(G4),28 anti-calponin monoclonal antibody (hCP, Sigma),
rabbit anti–chicken caldesmon polyclonal antibody that recognizes both
heavy and light caldesmon (kindly provided by Dr M.P. Walsh, University
of Calgary, Alberta, Canada),29 and anti-vinculin
monoclonal antibody (hVIN-1, Sigma) that recognizes both metavinculin
and vinculin. Horseradish peroxidase–conjugated anti-mouse and
anti-rabbit IgG F(ab')2 (Boehringer
Mannheim) were used as secondary antibodies. Blots were developed with
enhanced chemiluminescence detection reagents (Boehringer
Mannheim), exposed on Kodak X-Omat blue XB-1 film, and quantified by
laser scanning densitometry (GS-700 Imaging Densitometer, Bio-Rad).
Statistics
Results are expressed as mean±SEM. Differences between groups
were evaluated by ANOVA with the Scheffé post hoc test.
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Results |
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Serum Withdrawal Induces Reversible Morphological Changes in HITB5 SMCs
When cultured in M199 containing 10% FBS, HITB5 cells appeared as elongated cells that were well spread over the culture substrate (Figure 1a
and 1d). The appearance was
similar to that of primary cultures of human SMCs,19
although HITB5 cells were somewhat larger and demonstrably more
homogeneous. After serum withdrawal, HITB5 cells underwent
a striking change to a highly elongated and spindle-shaped cell that
was more refractive to phase-contrast light (Figure 1b). These
changes were evident after 12 hours and complete by 72 hours. After
restoration of serum, the cells reverted to their spread morphology,
which was indistinguishable from that of the cells before serum
withdrawal (Figure 1c).
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In addition to the morphological changes of individual cells, the
population of cells substantially reorganized after serum withdrawal.
Twelve hours after serum withdrawal, the elongating SMCs organized to
form dense, multilayered cell bundles that appeared as well-aligned
ridges reminiscent of the organization of SMCs in blood vessels (Figure 1e
). This morphology could be maintained for 2 weeks. Two days
after serum restoration, however, the cell bundles disassembled and the
cells lost the organized pattern (Figure 1f).
Serum Withdrawal Reversibly Suppresses Motility of HITB5
SMCs
Cell locomotion is a key feature that distinguishes SMCs
contributing to vascular disease from the quiescent SMCs of the normal
artery wall. Figure 2 illustrates
migration paths and migration speeds on type I collagen, for HITB5 SMCs
under serum-supplemented conditions (10% FBS), 3 days after serum
withdrawal, and 2 days after restoration of 10% FBS. To limit the
extent to which serum-deprived SMCs accumulated in multilayered
bundles, which would preclude quantitative analysis of
single-cell migration, cells were studied at low densities
(2x103 cells/cm2). As
illustrated, cells in serum-supplemented conditions demonstrated
considerable motility; however, the migration path was substantially
constrained after serum removal, with a 3.1-fold reduction in migration
speed (P<0.001). These changes were fully reversible on
readdition of serum. Migration on plastic or fibronectin (10 µg/mL)
showed similar profiles (data not shown).
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Serum Withdrawal Reversibly Inhibits Cell Proliferation and DNA
Synthesis in HITB5 SMCs
Low growth rate is another fundamental characteristic of
differentiated, contractile SMCs in the vessel wall. To determine
whether cell proliferation declined in concert with cell elongation and
the fall in random motility, we assessed cell number change by
hemocytometry. As shown in Figure 3A, growth arrest was induced 2 days after serum withdrawal. The cells
remained quiescent in serum-free medium, with no morphological evidence
for cell death and no change in cell number for 6 days. SMC
proliferation was promptly restimulated on readdition of 10% FBS.
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To determine whether the changes in cell number were related to
parallel changes in DNA synthesis rate,
[3H]thymidine incorporation was measured. As
shown in Figure 3B, [3H]thymidine
incorporation precipitously dropped to background levels after serum
withdrawal and was restored after reintroduction of serum to culture
medium 2 days later.
Serum Withdrawal Reversibly Inhibits ECM Protein Synthesis by
HITB5 SMCs
Increased protein synthesis, including synthesis of ECM proteins,
is a hallmark of SMCs with a synthetic phenotype in vascular
lesions. We assessed leucine incorporation into both cellular and ECM
fractions by incubating cultures with
[3H]leucine and isolating the cell fraction and
the insoluble ECM deposited by the cells. As shown in Figure 4, serum withdrawal reduced leucine
incorporation into the cellular fraction to 0.08±0.01 of basal levels
and reduced leucine incorporation into the ECM fraction to
0.10±0.01 of basal levels. Leucine incorporation into the cellular
fraction was fully restored to basal levels 3 days after serum
restoration. Leucine incorporation into the ECM fraction also reversed
3 days after serum replenishment, although it did not completely return
to basal levels.
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Serum Withdrawal Is Associated With Decreased Apoptosis
Despite prolonged periods of "starvation" conditions, HITB5
cells remained viable. In fact, we observed that cells in the
serum-starved state had less cytoplasmic granularity and a smoother
plasma membrane surface than did cells in serum-supplemented medium,
which suggests a healthier cell. There was also less cellular debris in
the medium. We reasoned, therefore, that the resilience in
serum-starved conditions might, in part, be due to a decreased rate of
apoptosis in the serum-starved cells. We tested this by
performing TUNEL with dUTP-fluorescein. As shown in Figure 5, 3 days after serum withdrawal the
percentage of apoptotic cells was reduced by half (4.3±0.6
versus 9.8±1.2%, P<0.001). In addition, nuclear
fragmentation, assessed from propidium iodide staining, was reduced in
serum-deprived cultures. It was also apparent that apoptosis
was inversely related to the attainment of the highly elongated,
spindle-shaped morphology; in microscopic fields in which cells were
most elongated, virtually no apoptosis was seen.
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HITB5 SMCs Possess Intact Receptors for Vasoactive Agonists That
Regulate Ca2+ Signaling
We next sought to determine whether HITB5 cells had functional
cell surface receptors for contractile hormones. Agonist-induced
Ca2+ mobilization was assessed because this had
the potential to establish both the presence of the receptor and
functional coupling to a signaling event central to SMC contraction.
Changes in [Ca2+]i were
quantified in individual SMCs loaded with fura-2 and stimulated with
histamine (20 µmol/L) or angiotensin II (1
µmol/L). As shown in Figure 6A, both
vasoactive substances induced robust Ca2+
transients in HITB5 cells. This was evident for HITB5 SMCs growing in
M199 with 10% FBS, as well as SMCs studied 3 days after serum
withdrawal. As shown in Figure 6A and 6B, the peak
[Ca2+]i stimulated by
histamine and angiotensin II was significantly higher after
serum deprivation, which suggests some upregulation of the
receptor signaling cascade in the contractile phenotype.
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To confirm that the responses were receptor mediated, cells were treated with the H1-receptor antagonist pyrilamine (1 µmol/L) or the AT1-receptor antagonist losartan (10 µmol/L). Agonist-induced Ca2+ transients were studied serially in the same cell before addition of the antagonist, immediately after its application, and after washout. Both antagonists were found to abrogate the rise of Ca2+ stimulated by the respective agonist (5 of 5 cells treated with pyrilamine and 3 of 3 cells treated with losartan).
Serum-Deprived HITB5 SMCs Contract in Response to Vasoactive
Hormones
The ability to contract in response to a
physiological stimulus is the only unequivocal
criterion for a "contractile" SMC phenotype. It is
noteworthy, therefore, that this has never been reported for
subcultured SMCs. Having established intact transmembrane signaling
cascades for vasoconstrictor peptides, we addressed this question in
HITB5 cells. Cells either in M199 with 10% FBS or after 3 days of
serum deprivation were stimulated with histamine or
angiotensin II. Between 7 and 15 cells were studied for
each experimental condition. Contraction was first assessed in adherent
cells by quantifying cell area. Administration of histamine (1
µmol/L) to cells that had been subjected to serum withdrawal caused a
reduction in 2-dimensional cell area of 27±3%, whereas the area of
vehicle-treated cells was reduced by 2±3% (P<0.001).
Preincubation of cells with 1 µmol/L pyrilamine inhibited
histamine-induced contraction (Figure 7A and 7B). Stimulation with angiotensin II (1 µmol/L)
to serum-deprived HITB5 SMCs also elicited a significant reduction in
cell area (21±2%), and this was blocked by losartan (1
µmol/L) (Figure 7B). In contrast, SMCs that were not serum
deprived showed no morphological change on agonist stimulation, and
this was independent of the culture substrate (cell area reduction
after angiotensin II stimulation of 0.4±1.2% for cells on
plastic, 1.4±1.6% for cells on fibronectin, and 0.1±1.3% for cells
on type I collagen). Similarly, SMCs that were serum deprived and then
subjected to serum restimulation for 48 hours no longer contracted. We
also evaluated contraction in serum-deprived HITB5 cells that had been
gently dispersed with trypsin-EDTA. These cells remained elongated and
retained this morphology for up to 2 hours. This was associated with
delayed readhesion to the substrate, which meant the cell was free to
shorten. As shown in Figure 7D, stimulation with histamine
shortened the cell by 25±2%, and this was inhibited by pretreatment
with pyrilamine. Angiotensin II reduced cell length by
23±3%, an effect blocked by losartan (Figure 7C and 7D), whereas vehicle alone caused an only 3±1% reduction in resting
cell length. Depolarizing the cell membrane with iso-osmotic Ringer's
solution containing 100 mmol/L KCl also resulted in cell
contraction (data not shown), which suggests the presence of
voltage-gated calcium channels.
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Contractile Apparatus Proteins Are Expressed in HITB5
SMCs and Modestly Increase as the Contractile State Is
Attained
To investigate the relationship between attainment of a
contractile phenotype and expression of biochemical markers of
SMC differentiation, Western blot analysis was performed on
cell lysates harvested before and 16 hours and 1, 2, 3, 5, and 7 days
after serum withdrawal. As shown in Figure 8, the contractile apparatus
proteins SM -actin, SM-MHC isoforms SM1 and SM2, calponin, and heavy
caldesmon were present in proliferating HITB5 SMCs before
withdrawal of serum. The muscle-specific isoform of vinculin,
metavinculin, was also detected. After serum withdrawal, there was a
gradual increase in expression of SM -actin, SM-MHC, calponin, and
metavinculin. There were also substantial increases in the ratios of
the SM to nonmuscle isoforms of caldesmon (heavy caldesmon/light
caldesmon) and vinculin (metavinculin/vinculin) (Figure 8B).
Additionally, the SM2 isoform of SM-MHC, which is expressed after the
SM1 isoform during development,30 appeared to increase in
expression to a greater extent than that of the SM1 isoform.
Interestingly, 3 days after serum withdrawal, at which time a
contraction-competent state had developed, the increases in
contractile-apparatus protein expression were relatively
modest. As quantified by scanning densitometry, there was a 1.5±0.1-,
1.5±0.1-, and 1.9±0.3-fold increase in abundance of SM -actin,
SM-MHC (SM1 plus SM2), and calponin, respectively.
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Discussion |
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The physiological properties of HITB5 cells, to our knowledge, provide the first direct demonstration that a cultured human adult SMC can bidirectionally convert from a contraction-incompetent state to a contraction-competent state. Conclusive demonstration of this phenomenon was facilitated by 3 unique aspects of the study. First, the human SMCs were clonal; observed changes in cell characteristics can thus be attributed to phenotype changes in individual cells rather than selective expansion of a subpopulation of cells. Second, we were able to establish that the transition between phenotypes was bidirectional, implicating an inherent cell property distinct from the commonly observed unidirectional dedifferentiation response to culture conditions. Third, SMC contraction per se was demonstrated and used to define the contractile phenotype, rather than surrogate features such as ultrastructure or marker proteins, which are not irrevocably linked to contraction.
A number of clonal SMC lines have been developed over the past several years in an effort to generate SMC populations that are longer lived and phenotypically more predictable than primary SMC cultures. This includes cell lines derived from rat embryonic thoracic aorta,31 a large T-transformed rat aortic SMC line,32 and a rat pulmonary artery SMC line.33 The latter cell line retains certain differentiated properties, including expression of SM-specific proteins and functional receptors for vasoactive hormones. However, none of these SMC lines have been shown to display substantial phenotype plasticity, and there is no evidence that they can be induced to contract. In contrast, HITB5 SMCs, a human clone derived from adult internal thoracic artery, underwent a striking functional change on withdrawal of serum from the culture medium. This was characterized by a decrease in random motility, cessation of cell proliferation and DNA synthesis, decreased ECM protein production, spatial alignment and organization of cells suggesting an attempt at organogenesis, and attainment of contractile competence. Each of these changes was fully reversible on readdition of serum, with the exception of ECM protein synthesis, which was partially reversible. ECM protein synthesis did not fully return to baseline levels, possibly because the ECM that had accumulated over the course of the experiment provided feedback inhibition, as has been described in fibroblasts.34
It is important to note that even in the presence of serum, HITB5 SMCs
expressed several contractile apparatus proteins, including
SM -actin, SM-MHC, calponin, and heavy caldesmon. It is well
established that during vascular development, SMCs sequentially express
specific contractile proteins.12 30 Importantly, these
developmental markers appear only after commitment to a SMC lineage
and, in the case of calponin and SM-MHC, only after a multilayered
artery wall has developed.35 36 Therefore, HITB5 cells
growing in serum are not primordial cells but are SMCs that are
relatively advanced along the differentiation program. This relative
maturity was also indicated by the expression of metavinculin and the
Ca2+ responses to vasoactive agonists, including
the fact that the AT1
antagonist, losartan, inhibited
angiotensin II–mediated calcium influx in
serum-supplemented, proliferating HITB5 SMCs. The
AT1 subtype has been found in adult rat aortic
SMCs, whereas the AT2 receptor subtype is
predominant in fetal aorta.37
At the same time, HITB5 SMCs in serum were unable to contract and
functioned in a nonspecialized and undifferentiated manner. Thus, when
in serum, HITB5 cells displayed an intermediate phenotype, in
that the cells were differentiated to the extent of being recognizable
as SMCs, but not to the extent of having specialized, contractile
function. Recent in vivo studies suggest that this intermediate
phenotype may be very relevant to the pathophysiology of
vascular disease. Neointimal cells of the injured rat
carotid artery have recently been reported to express various ECM genes
typical of synthetic SMCs at the same time as expressing the
SM-specific genes calponin and SM22.38 Similarly,
electron microscopic studies of the balloon-injured rat carotid artery
have demonstrated coexistence of a large endoplasmic reticulum,
implying synthetic function, and myofilaments in the same
cell.39 Furthermore, human atherosclerotic plaques have
been shown to have an abundance of SMCs with a well-developed
endoplasmic reticulum and Golgi apparatus, in addition to
having myofilament bundles and expressing SM myosin and
-actin.40 These data suggest that mesenchymal cells of
the vessel wall need not be of a primitive or fetal phenotype
to perform synthetic functions; these functions can be performed by
SMCs of an intermediate phenotype. The fact that HITB5 SMCs can
convert between an intermediate SMC phenotype and a contractile
SMC phenotype thus suggests that they may be a valuable model
for this aspect of vascular disease.
In addition to the functional changes observed after serum withdrawal,
there were biochemical changes consistent with SMC
maturation/differentiation. This included increases in the ratios of
heavy caldesmon to light caldesmon, metavinculin to vinculin, and SM2
to SM1. Expression of SM -actin and calponin also increased.
Although these changes biochemically confirm the process of
differentiation in HITB5 SMCs, we speculate that increases in
contractile apparatus proteins may not be a dominant
mechanism by which the contractile state was attained. This is because
the magnitude of change was relatively modest and because, with the
exception of SM2 expression, most of the increase in expression
occurred several days after the attainment of contractile competence.
Development of functional receptors for vasoactive proteins or
formation of an intact calcium-release mechanism were also unlikely to
significantly mediate the transition to a contractile state, as these
properties were already present in noncontractile SMCs. The
magnitude of agonist-mediated calcium transients did increase after
serum withdrawal; however, we have not yet determined which step(s) in
the receptor-Ca2+ signaling cascade is subject to
upregulation. Other mechanisms mediating the final stage of
differentiation to a contracting cell likely remain to be elucidated
and could include functional reorganization of existing contractile
structures or increased calcium sensitivity of myofilaments. The small
GTPase Rho and its target Rho-associated kinase have recently been
found to participate in the latter and are candidates in this
regard.41 42 It is also possible that observed shift in
phenotype is dependent on specific changes in the interaction
between the cell and ECM. Integrin-ligand interactions are known to
mediate cell differentiation, including myogenesis.43
Serum withdrawal precipitated a decline in apoptosis of HITB5 SMCs. This finding contrasts with the observation of increased apoptosis in cultured SMCs after serum withdrawal.44 However, an inverse association between death and differentiation has been well documented. In the developing human ductus arteriosus, for example, apoptosis was significantly more prevalent in the less-differentiated SMCs of the inner intima compared with well-differentiated SMCs of the media and outer intima.45 We speculate that the resistance to apoptosis in HITB5 SMCs that was induced by serum withdrawal was permissive to, and possibly a prerequisite for, the differentiation program.
The current study also adds to our understanding of SMC multifunctionality by establishing that conversion between contractile and noncontractile states is indeed a property of at least some human SMCs. This notion has recently been challenged. Although several studies have carefully charted changes of enzymatically dispersed arterial cells placed in culture,6 46 47 contractile SMCs isolated from the canine carotid artery were recently found not to dedifferentiate in culture. Instead, expansion of less-specialized cells accounted for the in vitro accumulation of cells with characteristics of synthetic SMCs.17 It is thus possible that selective emergence of noncontractile cells in culture might erroneously be interpreted as modulation of SMC phenotype. It must also be recognized that the SMC phenotype conversion hypothesis was initially based on the assumption that SMCs constitute a single cell type in the arterial media.5 This assumption is probably not valid, given the documented heterogeneity of cells in the normal adult artery,14 15 16 17 the diverse embryonic origins of arterial SMCs,48 49 50 and the diverse properties of cells that have been cloned from a given artery.51 52 The evidence for regions of monoclonal SMCs in atherosclerotic plaques and, occasionally, in arterial media likewise highlights the concept of selective cell expansion.53 The current findings do not preclude expansion of less mature cells as a pathophysiological feature of vascular disease, but they do suggest that the potential contribution of SMC phenotype conversion to vascular disease should not be dismissed, and efforts to understand the mechanism are of importance.
In summary, the physiological properties of HITB5 SMCs provide the first direct demonstration that cultured human adult SMCs can reversibly convert between a noncontracting state and a contracting state. This novel cell line may be valuable for clarifying our understanding of SMC phenotype switching and restructuring of the vessel wall.
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Acknowledgments |
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This research was supported by a grant from the Medical Research Council of Canada. J.G.P. was supported by a scholarship from the Medical Research Council of Canada and is a Career Investigator of the Heart and Stroke Foundation of Ontario. S.L. was supported by a Merck-Frosst-Robarts Research Institute fellowship in medical research. We thank Dr D. Boyd, London Health Sciences Center, for providing the surgical specimen.
Received March 17, 1999; accepted May 28, 1999.
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