From the sections of Cardiology (N.M., J.G., S.E.F., C.M.H.N., D.C.
Crossman) and Interventional Cardiology (N.M., J.G., C.M.H., L.S., D.C.
Cumberland), University of Sheffield, Clinical Sciences Centre, Northern
General Hospital, Sheffield, UK; and the Sheffield Kidney Institute (G.L.T.),
Northern General Hospital, Sheffield, UK.
Correspondence to Dr Nadim Malik, Cardiovascular Medicine, University of Sheffield, Clinical Sciences Centre, Northern General Hospital, Herries Road, Sheffield S5 7AU, UK. E-mail n.malik{at}sheffield.ac.uk
Methods and Results—Profiles of apoptosis and
proliferation were therefore examined in a porcine PTCA injury model
over a 28-day period. Forty-two arteries from 21 pigs, harvested at the
site of maximal injury at 1, 6, and 18 hours, and 3, 7, 14, and 28 days
after PTCA, were examined (n=3 animals per time point). Uninjured
arteries were used as controls. Apoptosis was demonstrated by
the terminal uridine nick-end labeling (TUNEL) method, transmission
electron microscopy (TEM), and DNA fragmentation. Cells traversing the
cell cycle were identified by immunostaining for
proliferating cell nuclear antigen (PCNA). Apoptosis was not
detected in control vessels at all time points nor at 28 days after
PTCA. Apoptotic cells were identified at all early time points
with a peak at 6 hours (5.1±0.26%; compared to uninjured artery,
P<0.001) and confirmed by characteristic DNA ladders
and TEM findings. Regional analysis showed apoptosis
within the media, adventitia, and neointima peaked at 18
hours, 6 hours, and 7 days after PTCA, respectively. In comparison,
PCNA staining peaked at 3 days after PTCA (7.16±0.29%; compared to
1.78±0.08% PCNA-positive cells in the uninjured artery,
P<0.001). Profiles of apoptosis and cell
proliferation after PTCA were discordant in all layers of the artery
except the neointima. These profiles also differed between
traumatized and nontraumatized regions of the arterial
wall. Immunostaining with cell-type specific markers
and TEM analysis revealed that apoptotic cells included
vascular smooth muscle cells (VSMCs), inflammatory cells, and
adventitial fibroblasts.
Conclusions—These results suggest that the profile of
apoptosis and proliferation after PTCA is regional and cell
specific, and attempts to modulate either of these events for
therapeutic benefit requires recognition of these differences.
Arterial injury during PTCA initiates vascular
healing including vascular smooth muscle cell (VSMC) migration and
proliferation and connective tissue
remodeling.12 13 14 This may lead to
restenosis.15 16 17 Because the total cell
number within any tissue depends on the rate of cell death and
proliferation, we hypothesize that apoptosis may be involved in
the genesis and/or modulation of restenosis. Apoptosis
peaks synchronously with proliferation in a rat carotid injury model 9
to 10 days after injury, but early time points after injury were not
examined.18 In a rat aortic
endothelial injury model, apoptosis is involved
in the regulation of intimal thickening.19
Apoptosis is seen in medial VSMCs as early as 30 minutes after
balloon injury in both rat and rabbit arterial models with
rapid elimination by 4 hours after PTCA.20 We
therefore investigated a complete time course of apoptosis in
the vascular response to injury and its relationship to cell
proliferation using a porcine coronary balloon injury
model.
Tissue Collection and Preparation
TUNEL and PCNA Immunostaining
Immunocolocalization With
Quantification of TUNEL and PCNA Staining
The arterial wall in each section was also subdivided into
regions of damage and normality. The damaged region was defined as the
cross-sectional area (CSA) of the artery adjacent to the breached IEL,
including remnants of the media, adventitia, and loose connective
tissue; the remainder was defined as undamaged (Figure 1
Quantification of Immunocolocalized TUNEL Cells
Transmission Electron Microscopy (TEM)
Electrophoretic Internucleosomal DNA Fragmentation
Statistical Analysis
The profiles of TUNEL and PCNA positivity were also characteristic for
individual layers of artery. Within the adherent thrombus, levels of
TUNEL and PCNA staining were low, with peak for TUNEL at 6 hours and
for PCNA at 3 days (Figure 2A
Within vessel wall, response to injury was different between sites of
damage, as defined by a breach in IEL, and the rest of the
arterial wall (undamaged region). TUNEL within the damaged
region of the arterial wall (Figure 3A
Verification of Apoptosis
TEM Features
DNA Fragmentation by Gel Electrophoresis
Immunocolocalization of VSMC and Inflammatory Cell Antigens With
Apoptotic Nuclei
At 1, 6, and 18 hours an inflammatory cell infiltrate was observed in
damaged regions and in overlying thrombus. Figure 7B
Immunocolocalization of
Phenotype of Apoptotic and Proliferating
Cells
Colocalization of PCNA with
Regional Differences in Apoptosis and Cell
Proliferation
Levels of Apoptosis and Proliferation After PTCA
Although our PCNA data was not greatly dissimilar from data concerning
cell proliferation with bromodeoxyuridine
(BrdU),14 PCNA expression occurs early in the
cell cycle, and may identify cells which fail to undergo mitosis but
will undergo apoptosis.26 27 This may
explain the observed 1.5-fold increase in PCNA positivity at 1 hour
compared with control artery. This possibility is supported by the
paucity of cells showing mitotic figures at early time points.
Furthermore, PCNA positivity at 6 and 18 hours remained similar to that
at 1 hour. This is in agreement with findings of a 1.5-fold increase in
PCNA positivity at 1 day after PTCA in the porcine carotid
artery.30 31 In contrast, the 4-fold increase in
PCNA positivity at 3 days, when apoptosis has returned to lower
levels, may reflect an increase in the number of cells traversing the
cell cycle. BrdU labeling would have been more reliable, but it
cannot be used on archival tissue.
VSMC Proliferation After PTCA: A Possible Role for In Vivo
Apoptosis
Study Limitations
Implications
Received January 5, 1998;
revision received May 7, 1998;
accepted May 20, 1998.
© 1998 American Heart Association, Inc.
Basic Science Reports
Apoptosis and Cell Proliferation After Porcine Coronary Angioplasty
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—Angioplasty initiates
a number of responses in the vessel wall including cellular migration,
proliferation, and matrix accumulation, all of which contribute to
neointima formation and restenosis. Cellular
homeostasis within a tissue depends on the balance between cell
proliferation and apoptosis.
Key Words: angioplasty • apoptosis • cell proliferation • restenosis
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Cell death may occur by engaging a specific program
(apoptosis) or necrosis. First identified in embryonic
tissue,1 apoptosis is now implicated in
many processes.2 3 Unlike necrosis,
apoptosis is an active process requiring protein synthesis and
gene transcription.4 It is associated with
characteristic morphologic changes involving both the nucleus and
cytoplasm.5 6 Apoptotic cells do not
cause an inflammatory response, and cell integrity is maintained until
removal by phagocytosis.7 8 9 A hallmark of
apoptosis is internucleosomal cleavage of genomic DNA into 200-
to 300- and/or 30- to 50-kb pieces, and eventual oligonucleosomal
fractions consisting of 180 to 200 base
pairs.6 10 This generates characteristic ladders
on DNA electrophoresis and is the basis of in situ detection of DNA
fragments with the use of the terminal uridine nick-end labeling
(TUNEL) method.11
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Porcine Coronary Angioplasty Model
Twenty-one Yorkshire white pigs weighing 20 to 25 kg were
premedicated with aspirin 150 mg by mouth, and we performed
coronary angioplasty as described
previously.21 The animals were killed with
phenobarbital (10 mL of 200 mg/mL, Vetdrug) at 1, 6, and 18 hours and
at 3, 7, 14, and 28 days after the procedure with 3 animals per time
point. All experiments conformed within UK Home Office regulations.
The porcine heart was explanted and coronary arteries
dissected with a minimum of surrounding tissue. After they were flushed
with saline, arteries were sectioned into 3-mm blocks and alternate
segments that were either snap-frozen for DNA analysis or
processed for histology and transmission electron microscopy (TEM).
Histology specimens were preserved in buffered formalin for 24 hours
and embedded in paraffin. Sequential 3- to 4-µm-thick transverse
sections were cut and affixed to
aminopropyltriethoxysilane-coated slides.
Representative sections from each block were stained
with hematoxylin and eosin and Van Gieson-elastin to determine location
and extent of injury. Arterial blocks with maximum balloon
injury, defined as maximum disruption in internal elastic lamina
(IEL),14 16 were selected for investigation of
apoptosis and proliferation.
The TUNEL method11 was used with minor
modifications. Specifically, optimal proteolysis required 100 µg/mL
proteinase K (Promega) for 30 minutes. An apoptosis detection
kit (Appligene Oncor) was used with the chromogen diaminobenzidine
(DAB) (Sigma). The counterstain was 0.5% methyl green. Biochemical
controls were performed with positive control slides treated with
DNase-1 (Pharmacia Biotech) and with staining in the absence of
terminal deoxynucleotidyl transferase enzyme as
negative control. Cell proliferation was detected immunohistochemically
with proliferating cell nuclear antigen (PCNA) labeling on
arterial sections immediately adjacent to those used for
TUNEL.22 
-SM Actin and
Monocyte/Granulocyte Antibody
For colocalization of apoptotic and proliferating cells
with specific cell-type markers, immunostaining was
combined with TUNEL and PCNA staining, respectively. TUNEL and PCNA
staining were performed up to DAB stage. Sections were then blocked
with 10% (vol/vol) normal horse serum (DAKO) and incubated overnight
at 4°C with primary antibodies, a monoclonal mouse anti-human -SM
actin (Sigma) at 1:150 dilution, and a monocyte/granulocyte (MAC 387,
DAKO) at 1:100 dilution. MAC 387 was not used with PCNA. A biotinylated
horse anti-mouse IgG (H+L) secondary antibody (Vector laboratories,
1:200 dilution) was added for 30 minutes and detected by streptavidin
alkaline phosphatase (DAKO) with New Fuchsin Red (DAKO). The
counterstain in both cases was methyl green. Appropriate negative
controls (without primary and/or secondary antibodies) were also
performed.
When viewed with a x40 objective, cells showing morphologic
features characteristic of apoptosis in addition to positive
TUNEL reaction were considered to be apoptotic. Nonspecific
cytoplasmic staining without nuclear involvement was considered
negative. PCNA-positive cells were also quantified with a x40
objective. The apoptotic and proliferating cells per high-power
field (HPF) were represented as a percentage of total cells
(ie, TUNEL or PCNA labeling index=TUNEL or PCNA-positive cells/total
cells per HPFx100).14 The TUNEL and PCNA indices
for each layer of artery (ie, thrombus, neointima, media,
adventitia, and loose connective tissue) were plotted against
time.14 Results are expressed as mean±SEM. 
). The undamaged region was further
subdivided into the region immediately adjacent to the fractured IEL
and the region opposite to the site of
damage.14 16 TUNEL and PCNA indices within these
regions were quantified and plotted against time. A number of randomly
selected slides were also quantified for TUNEL and PCNA indices by an
independent observer (C.M.H.) to assess interobserver variation.
View larger version (63K):
[in a new window]
Figure 1. Areas analyzed for TUNEL and PCNA
positivity by use of digital image analyser (SEESCAN) are described in
this cross-section of a porcine left anterior descending artery
obtained at site of maximum injury 6 hours after oversized balloon
angioplasty (PTCA). Damaged region was defined as CSA adjacent to the
breached IEL, and was measured by sketching two lines perpendicular to
broken ends of IEL (arrow). Total damaged region in section illustrated
included areas marked a, b, and c. Rest of arterial wall
CSA was defined as undamaged. M indicates media; A, adventitia; C,
clot; and L, loose connective tissue.
TUNEL-positive cells colocalized with -SM actin, and
macrophage/granulocyte antibodies were quantified as above.
These results are expressed as a percentage of total number of
apoptotic cells (mean±SEM of 3 animals per time point). Cells
that did not colocalize with either antibody were classed as "other
cells."
Three-millimeter segments of artery adjacent to those
analyzed by light microscopy were immersion-fixed in 2%
glutaraldehyde (final concentration), processed for
TEM,5 6 and examined with a Philips 400
transmission electron microscope at 60 kV.
Arterial segments immediately adjacent to site of
maximum injury were frozen in liquid nitrogen. Samples were
homogenized under liquid nitrogen and suspended in equal
volumes of 2x NTE (200 mmol/L NaCl, 20 mmol/L Tris, 2
mmol/L EDTA, pH 8.0) and 1% sodium dodecyl sulfate. DNA was
extracted.23 The DNA was resuspended in 10 µL
of distilled water with 5 µL of loading dye (0.25% bromphenol blue,
40% sucrose), and subjected to electrophoresis through a 2% agarose
gel (containing ethidium bromide) at 80 V for 1 hour. The gel was
visualized under UV transillumination and photographed with a Polaroid
camera.
TUNEL and PCNA results are expressed as mean±SEM (n=3 animals
per time point). The comparisons of TUNEL and PCNA indices at each time
point with their respective controls (uninjured arteries) were
performed by use of a 1-way ANOVA. A value of P<0.05 was
regarded as significant. All statistical work was done with SPSS
software (Microsoft).
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
TUNEL and PCNA Staining
The profiles of TUNEL and PCNA positivity over time were different
according to the area analyzed. When analyzed for the
whole arterial section, TUNEL-positive cells were
undetectable in control sections and at 28 days after PTCA. They were
detectable at 1 hour (4.47±0.30%; compared to uninjured artery,
P<0.001), maximal at 6 hours (5.39±0.23%,
P<0.001) and at low levels (0.32±0.09%,
P<0.21) at 14 days after PTCA. In contrast, PCNA staining
was maximal at 3 days (7.33±0.28%, compared with 1.78±0.08% in the
uninjured artery, P<0.001) and lower than baseline level
(P<0.01) at 28 days after PTCA. ). The
neointima was distinguishable from 7 days. Maximal PCNA and
TUNEL positivities were concordant at 7 days within this layer and
returned to control artery levels at 28 days (Figure 2B). Within the
media (Figure 2C), TUNEL was maximal at 18 hours with a decline to
baseline at 14 days, whereas PCNA positivity was maximal at 3 days,
returning to baseline at 28 days. Within the adventitia, TUNEL
positivity was maximal at 6 hours and basal at 14 days, whereas PCNA
was maximal at 3 days and basal at 28 days (Figure 2D). In loose
connective tissue (Figure 2E), relatively high levels of PCNA-positive
cells were found, whereas TUNEL positivity was low. In this layer,
TUNEL was maximal at 18 hours and basal at 14 days, whereas PCNA was
maximal at 3 days and basal at 28 days.
View larger version (22K):
[in a new window]
Figure 2. Profiles of TUNEL and PCNA positivity for
individual layers of artery are represented graphically
over a 28-day period. Results are expressed as mean±SEM (n=3 animals
per time point). TUNEL and PCNA labeling indices (%) for respective
layers are represented on y axis and time
(log) on x axis. A, Within adherent thrombus, there were
low levels of TUNEL and PCNA positivity (seen only at early time points
after PTCA). B, Neointima was only distinguishable at 7
days after PTCA with maximal TUNEL and PCNA positivity concordant at 7
days, returning toward baseline levels (for whole artery) at 28 days.
In all other layers of artery, maximal TUNEL and PCNA positivities were
discordant, with peak TUNEL positivity both earlier and shorter in
duration in media (C), adventitia (D), and loose connective tissue (E).
For comparison, labeling indices for media, adventitia, and loose
connective tissue in control (uninjured) artery are also
shown. ) was maximal at 18 hours, remained so
until 7 days, and returned to baseline at 28 days. The peak in PCNA
positivity was at 3 days and basal at 28 days. In the undamaged region
of the arterial wall, the region immediately adjacent to
the fractured IEL (Figure 3B) showed higher levels of TUNEL and PCNA
positivity than the region opposite to the site of injury (Figure 3C).
In both subregions of the undamaged wall, TUNEL peaked at 6 hours and
PCNA at 3 days.
View larger version (14K):
[in a new window]
Figure 3. Regional variations over 28-day period;
relationship to damaged and undamaged regions of arterial
wall. Results are expressed as mean±SEM (n=3 animals per time point).
TUNEL and PCNA labeling indices (%) for damaged and undamaged regions
are represented on y-axis and time (log) on
x-axis. A, In damaged region, TUNEL positivity is
maximal at 18 hours and PCNA positivity at 3 days after PTCA. For
comparison, labeling indices for control (uninjured) artery are also
shown. B, Within undamaged region of arterial wall, levels
of TUNEL and PCNA positivity are comparatively higher in region
adjacent to fractured ends of IEL compared to region opposite to site
damage (C). In addition, in all three regions of arterial
wall, profiles of TUNEL and PCNA are discordant.
Light Microscopic Features
The characteristic light microscopic features of apoptosis
were frequently associated with TUNEL staining in our
arterial sections. The features identified with a x100
objective included cell shrinkage, a preserved cell membrane, and a
small rounded nucleus (Figure 4D).
View larger version (178K):
[in a new window]
Figure 4. Detection of apoptosis. Porcine
coronary arterial sections showing
apoptosis by TUNEL method. TUNEL-positive cells appear brown
and counterstain is methyl green. A, TUNEL-positive cells (arrow) at 1
hour after PTCA are localized to site of damage (broken edge of media)
only. B, At 6 hours, TUNEL-positive cells are also identified in deeper
layers of media and at 18 hours after PTCA (C) in loose connective
tissue (arrows). D, At 7 days after PTCA TUNEL-positive cells (arrow)
are seen within neointima only. No TUNEL-positive cells
were seen in uninjured (control) artery (E) and biochemical negative
control (without terminal deoxynucleotidyl
transferase enzyme) (F) 6 hours after PTCA. Scale bars
represent 50 µm.
Apoptotic cells with characteristic nuclear morphology
were seen frequently at early time points, notably at 6 hours after
PTCA. These included VSMCs and inflammatory cells. Only a few
apoptotic cells were seen at 3 days. The ultrastructural
features of apoptotic VSMCs identified included cell shrinkage,
membrane blebbing, and chromatin condensation with collapse
against the nuclear membrane and apoptotic bodies (Figure 5A and 5B).
View larger version (144K):
[in a new window]
Figure 5. TEM. Electron micrographs from an artery 6 hours
after PTCA showing nuclear and cytoplasmic abnormalities characteristic
of apoptotic VSMCs. A, Condensed chromatin forming crescents in
apposition to nuclear envelope is characteristic of early stages of
apoptosis. Specialized regions of cell membrane known as motor
regions (arrow) are characteristic of VSMCs. B, Cytoplasmic shrinkage
giving this cell a ghostlike appearance, which together with chromatin
migration to periphery and probable apoptotic bodies (arrows)
suggests a more advanced stage of apoptosis in this VSMC.
Magnification x8000.
Electrophoresis of genomic DNA showed characteristic 180 to 200 bp
DNA fragmentation at 6 hours after PTCA, consistent with peak
level of apoptosis from our in situ data and TEM
analysis. The evidence for similar DNA cleavage at 1 and 18
hours was less definite. Only high-molecular-weight DNA was seen 3 days
after PTCA, similar to control (uninjured) arteries (Figure 6).
View larger version (146K):
[in a new window]
Figure 6. DNA ladders. Fragmentation of genomic DNA from
porcine coronary arteries after PTCA. Genomic DNA, isolated
from site of maximum damage (injury) 6 hours after PTCA (lane B) shows
characteristic nucleosomal DNA fragmentation at 6 hours after
PTCA. In uninjured (control) artery (lane A), high-molecular-weight DNA
is seen. DNA size markers (100 bp ladder) compose lane C.
Colocalization data are summarized in the
Table. Figure 7A
shows colocalization of TUNEL with SM actin. The TUNEL-positive
VSMCs at 1, 6, and 18 hours after PTCA constituted 52.0±7.9%,
43.0±8.4%, and 35.0±8.8% of total TUNEL-positive cells,
respectively. At 1 hour, these were mostly at the site of damage
(Figure 4A
), whereas at 6 and 18 hours they were distant from the
damage, including cells in deeper parts of the media (Figure 4B and 4C). The proportion of TUNEL-positive VSMCs remained fairly constant at
7 days (32.5±2.0%), 14 days (41.4±9.8%), and 28 days
(33.0±16.7%). Other TUNEL-positive cells, mainly in the adventitia
and loose connective tissue of the artery, which did not colocalize
with either antibody used were classified as "other cells"
(Table).
View this table:
[in a new window]
Table 1. Immunocolocalized Cells as Percentage of Total Apoptotic
Cells
View larger version (117K):
[in a new window]
Figure 7. Immunocolocalization: double
immunostaining to identify phenotype of
apoptotic cells in porcine coronary arteries obtained
from site of maximum damage after PTCA. A, Colocalization of TUNEL and
-SM actin (arrow) in media (M) 6 hours after PTCA. L
represents lumen; A, adventitia. VSMCs are labeled pink/red
with New Fuchsin Red whereas TUNEL end-product with DAB is brown.
B, Colocalization of TUNEL with monocyte/granulocyte (MAC 387) antibody
(arrow) in adventitia of artery 18 hours after PTCA. Inflammatory cells
appear pink/red whereas apoptotic nuclei are defined by their
brown nuclei. In both cases, counterstain is methyl green. Scale bars
represent 50 µm. shows
colocalization of TUNEL with inflammatory cell antibody. The
proportions of TUNEL-positive cells colocalizing with MAC387 at 1, 6,
and 18 hours were 5.1±0.4%, 8.9±1.7%, and 34.1±7.1%, respectively
(Table). At 3 days, inflammatory cells were seen in loose connective
tissue, especially in tissue adjacent to the breached IEL. The
TUNEL-positive inflammatory cells constituted 16.6±2.7% of total
TUNEL-positive cells at 3 days, 5.4±1.6% at 7 days, and 4.2% at 14
days. No inflammatory cells were seen at 28 days. -SM Actin With PCNA Staining
The majority of PCNA-positive cells in the media and
neointima were VSMCs. A few PCNA-positive cells in the
neoadventitia also colocalized for -SM actin. PCNA-positive cells in
the adventitia and loose connective tissue did not colocalize for
-SM actin, suggesting a different phenotype.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
In this study, we have demonstrated apoptosis in
porcine coronary arteries after balloon injury by use of TUNEL
assay with corroborating evidence from TEM and DNA cleavage. We have
also assessed cell proliferation in immediately adjacent sections using
PCNA immunostaining. Apoptotic cells appeared
early, reaching a peak at 6 hours and returning to a low level at 14
days. The maximum number of apoptotic cells in the damaged
region were at 18 hours, and remained at this level until 7 days,
whereas apoptosis declined more rapidly in all undamaged
regions of the arterial wall. Within the undamaged region,
there were more TUNEL and PCNA-positive cells adjacent to fractured
ends of IEL than in the region opposite to the site of injury. PCNA and
TUNEL positivity were temporally concordant in the
neointima, but discordant at all other sites.
Apoptotic cells within the neointima reached a peak
at 7 days. The majority of apoptotic cells, however, were seen
within the media and adventitia with a peak at 18 and 6 hours,
respectively.
The majority of apoptotic cells in the porcine
coronary artery were VSMCs (medial and neointimal
layers), inflammatory cells, and other cells (possibly adventitial
fibroblasts). At 1 hour, the majority of apoptotic VSMCs
in the media were close to the site of damage. At 6 and 18 hours, they
were circumferential and included cells in deeper layers of the media,
suggesting a delayed, progressive recruitment of VSMCs into
apoptosis. The inflammatory cells seen in damaged regions were
mostly confined to surrounding loose connective tissue by 3 days. Only
a few apoptotic inflammatory cells were identified at 1 or 6
hours, whereas at 18 hours they constituted a significant proportion of
total apoptotic population. At 18 hours they were seen mainly
in the loose connective tissue of damaged regions, and this may explain
peak TUNEL positivity at 18 hours in this layer. Apoptotic
cells within the adventitial layer did not colocalize with either of
the monoclonal antibodies (anti -SM actin and
anti-monocyte/granulocyte), suggesting a different phenotype
for these cells (possibly fibroblasts). -SM actin showed that cells traversing
the cell cycle in the media were VSMCs. PCNA-positive cells in the
adventitia and loose connective tissue, however, were not -SM actin
positive, which suggests a different phenotype. Weak -SM
actin positivity was seen within the neoadventitia, which implies that
these may be adventitial myofibroblasts, but there is currently no
specific antibody which can reliably identify this cell type. Resting
adventitial fibroblasts may be vimentin-positive but desmin- and -SM
actin negative. After injury, however, they may transform into
myofibroblasts,14 which exhibit some -SM-actin
and desmin positivity at varying time points after injury. The
induction of contractile and cytoskeletal proteins in these cells
is unpredictable and difficult to interpret. VSMCs may also undergo
dedifferentiation after vascular injury, resulting in decreased
expression of -SM actin and desmin
positivity.24 25 The changes in morphology and
expression of cytoskeletal proteins in the adventitial fibroblasts and
VSMCs in response to injury require full characterization before the
exact phenotype or origin of all cells contributing to the
neointimal hyperplasia in our model can be identified.
We used immunohistochemical methods to assess apoptosis
and cell proliferation in adjacent sections within the same model,
which allowed comparison of the timing and site of peak activity of
these two processes. We found regional differences in levels of
apoptosis and cell proliferation, as well as an important
influence of trauma on these biological events. Concordance of peak
activities of apoptosis and proliferation was seen only within
the neointima. In all other regions, apoptosis
peaked earlier than proliferation. The most plausible explanation for
separation in peak activities is that our TUNEL and PCNA data are
derived from arterial tissue sections that contain a
variety of cell types: VSMCs, inflammatory cells, and
fibroblasts. These have different properties and may undergo
apoptosis at different rates. For example, neutrophils are
terminally differentiated cells that undergo constitutive
apoptosis10 and are removed by
macrophage phagocytosis, prompted by specific cell-surface
changes.7 9 The kinetics of VSMC
apoptosis, in contrast, are less clear. Our data confirm the
importance of an inflammatory cell influx after PTCA, notably in the
outer adventitial and loose connective tissue layers. The discordance
in proliferation and apoptosis profiles in different wall
regions and layers may be explained by the distribution of
apoptotic neutrophils (which cannot divide). Furthermore, cells
in the cell cycle display a low but increased level of
apoptosis, presumably reflecting incorrect cell
division.26 27 This may explain the concordance
of apoptosis and proliferation in the neointima.
The influence of apoptotic inflammatory cells on
apoptosis in VSMCs is unknown and needs further
investigation.
A quantitative difference in levels of apoptosis and
proliferation after PTCA was seen, with proliferation greater than
apoptosis, except at early time points: 1, 6, and 18 hours.
This difference was evident within each layer and region of the artery.
In the neointima, proliferation was greater than
apoptosis but with both maximal at 7 days. Within the media,
adventitia, and loose connective tissue, a quantitative difference was
present but with a disparity in the timing of their maximal
activities (apoptosis peaking earlier than
proliferation). The higher levels of TUNEL and PCNA positivity
adjacent to fractured ends of IEL, relative to the site opposite to the
damage, is evidence of the contribution of this region toward
neointima formation. Although some difference in levels of
apoptosis and proliferation may be accounted for by the
different duration of these two biological events (proliferation
requires 14 to 18 hours, whereas apoptosis requires 2
hours28), the longer half-life of PCNA
positivity,22 29 and the possibility of different
rates of removal of different types of apoptotic cells, it may
partly explain the net accumulation of tissue.
In the vascular response to injury, VSMC apoptosis
preceded proliferation of remaining cells. This implies that cell death
may trigger cell migration and proliferation. The barotrauma of balloon
angioplasty may provide the initiating stimulus to propagate a cascade
of biochemical apoptotic responses in VSMCs similar to those
reported in cardiac myocytes,32 including gene
transcription, cytokines, calcium, platelet activating
factor, free radicals, and nitric oxide release. Although stretch may
initiate VSMC proliferation,33 34 35 we may
hypothesize that signals from apoptotic cells (VSMC and
inflammatory cells) may induce proliferation in surviving VSMCs.
Obviously, this point needs further investigation and is beyond the
scope of this article.
In our study, injury was induced in normal arterial
segments. The apoptotic response may differ in atherosclerotic
human coronary arteries exposed to PTCA. In vitro studies
showing that VSMCs derived from human coronary plaques undergo
apoptosis at a higher rate than normal VSMCs support this
concept.36 In situ detection of apoptosis
and proliferation within tissue sections concerns the kinetics of cell
cycle. Information on kinetics of in vivo apoptosis are less
clear than cell proliferation.24 31 35 There are
no data regarding duration of TUNEL positivity in vivo once cells are
committed to the apoptotic pathway. Furthermore, the total
number of cells exhibiting TUNEL positivity will be determined in part
by rate of clearance of apoptotic cells. This appears to be
very rapid for apoptotic neutrophils in the presence of primed
macrophages. The rate of clearance of apoptotic VSMCs
within the arterial wall is less clear. For these reasons,
total levels measured must be interpreted with caution. Rates of
apoptosis (and proliferation) cannot be measured in tissue
sections like we have used and, therefore, this term has been
avoided.
In conclusion, we have demonstrated different frequencies of
apoptosis and proliferation in different regions of the
arterial wall and involving different cell types after
porcine PTCA. Considering the similarities of porcine
arterial injury model to human coronary
restenotic lesion,34 37 we believe
these findings may provide reliable insights into the mechanisms and
interplay of apoptosis and proliferation after balloon injury.
A particular strength of this model is the comparison of damaged and
undamaged regions of the artery, which may allow novel therapeutic
interventions that target these regions to inhibit
neointimal hyperplasia. Previous studies have demonstrated
potential benefits of antiproliferative therapies to reduction of
neointimal hyperplasia with anti-sense
oligonucleotides to c-myc38
and c-myb,21 and transfection with
growth-inhibitory genes39 40 in a
porcine model. Local delivery of an agent to modulate apoptosis
after injury may reduce neointimal hyperplasia and thus
restenosis after PTCA. However, modulation of apoptosis
will require recognition of the fact that apoptosis after
injury occurs in a number of different cell types, at different levels,
and in different regions of the artery.
Acknowledgments
We would like to thank Ian R. Palmer, Electron Microscopy Unit,
Department of Pathology, University of Sheffield, UK, for his help with
electron microscopy work. We also thank the personnel of Medical
Illustration, Northern General Hospital, Sheffield, UK, for their
assistance with photographic work. We are grateful to the Northern
General Hospital Trust, Sheffield, UK, for providing funds for this
study (grant No. 57944).
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
This article has been cited by other articles:
 |
|
 |
|
  D. G. Sedding, M. Homann, U. Seay, H. Tillmanns, K. T. Preissner, and R. C. Braun-Dullaeus Calpain counteracts mechanosensitive apoptosis of vascular smooth muscle cells in vitro and in vivo FASEB J, February 1, 2008; 22(2): 579 - 589. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Guo, K.-H. Chen, Y. Guo, H. Liao, J. Tang, and R.-P. Xiao Mitofusin 2 Triggers Vascular Smooth Muscle Cell Apoptosis via Mitochondrial Death Pathway Circ. Res., November 26, 2007; 101(11): 1113 - 1122. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Sakao, L. Taraseviciene-Stewart, K. Wood, C. D. Cool, and N. F. Voelkel Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth Am J Physiol Lung Cell Mol Physiol, September 1, 2006; 291(3): L362 - L368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A K Mitra and D K Agrawal In stent restenosis: bane of the stent era. J. Clin. Pathol., March 1, 2006; 59(3): 232 - 239. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Cirillo, P. Golino, P. Calabro, G. Cali, M. Ragni, S. De Rosa, G. Cimmino, M. Pacileo, R. De Palma, L. Forte, et al. C-reactive protein induces tissue factor expression and promotes smooth muscle and endothelial cell proliferation Cardiovasc Res, October 1, 2005; 68(1): 47 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Lipskaia, F. del Monte, T. Capiod, S. Yacoubi, L. Hadri, M. Hours, R. J. Hajjar, and A.-M. Lompre Sarco/Endoplasmic Reticulum Ca2+-ATPase Gene Transfer Reduces Vascular Smooth Muscle Cell Proliferation and Neointima Formation in the Rat Circ. Res., September 2, 2005; 97(5): 488 - 495. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Ono, T. Ichiki, H. Ohtsubo, K. Fukuyama, I. Imayama, Y. Hashiguchi, J. Sadoshima, and K. Sunagawa Critical Role of Mst1 in Vascular Remodeling After Injury Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1871 - 1876. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. K. Jagadeesha, T. E. Lindley, J. DeLeon, R. V. Sharma, F. Miller, and R. C. Bhalla Tempol therapy attenuates medial smooth muscle cell apoptosis and neointima formation after balloon catheter injury in carotid artery of diabetic rats Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1047 - H1053. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Rupp, T. Hellwig-Burgel, V. Wobbe, U. Seitzer, E. Brandt, and M. Maass Chlamydia pneumoniae infection promotes a proliferative phenotype in the vasculature through Egr-1 activation in vitro and in vivo PNAS, March 1, 2005; 102(9): 3447 - 3452. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Schachner, A. Oberhuber, Y. Zou, A. Tzankov, H. Ott, G. Laufer, and J. Bonatti Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts Eur. J. Cardiothorac. Surg., February 1, 2005; 27(2): 302 - 306. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. C. Newby Dual Role of Matrix Metalloproteinases (Matrixins) in Intimal Thickening and Atherosclerotic Plaque Rupture Physiol Rev, January 1, 2005; 85(1): 1 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Blaschke, D. Bruemmer, F. Yin, Y. Takata, W. Wang, M. C. Fishbein, T. Okura, J. Higaki, K. Graf, E. Fleck, et al. C-Reactive Protein Induces Apoptosis in Human Coronary Vascular Smooth Muscle Cells Circulation, August 3, 2004; 110(5): 579 - 587. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Zohlnhofer, T. G. Nuhrenberg, F.-J. Neumann, T. Richter, A. E. May, R. Schmidt, K. Denker, M. A. Clauss, A. Schomig, and P. A. Baeuerle Rapamycin Effects Transcriptional Programs in Smooth Muscle Cells Controlling Proliferative and Inflammatory Properties Mol. Pharmacol., April 1, 2004; 65(4): 880 - 889. [Abstract] [Full Text]
|
|
|
|
|
|
J. Cornelissen, J. Armstrong, and C. M. Holt Mechanical Stretch Induces Phosphorylation of p38-MAPK and Apoptosis in Human Saphenous Vein Arterioscler. Thromb. Vasc. Biol., March 1, 2004; 24(3): 451 - 456. [Abstract] [Full Text]
|
|
|
|
|
|
A. Forte, S. Esposito, M. De Feo, U. Galderisi, C. Quarto, F. Esposito, A. Renzulli, L. Berrino, M. Cipollaro, L. Agozzino, et al. Stenosis progression after surgical injury in Milan hypertensive rat carotid arteries Cardiovasc Res, December 1, 2003; 60(3): 654 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Tulis, Z. H. Mnjoyan, R. L. Schiesser, H. S. Shelat, A. J. Evans, P. Zoldhelyi, and K. Fujise Adenoviral Gene Transfer of Fortilin Attenuates Neointima Formation Through Suppression of Vascular Smooth Muscle Cell Proliferation and Migration Circulation, January 7, 2003; 107(1): 98 - 105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Strotmann, J. Bauersachs, D. Fraccarollo, M. Kirchengast, P. A. Schnabel, J. Sykora, G. Ertl, and W. Voelker Trauma induced by nontraumatic coronary devices and its impact on vascular reactivity and morphology Am J Physiol Heart Circ Physiol, December 1, 2002; 283(6): H2356 - H2362. [Abstract] [Full Text] [PDF]
|
|
|