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Department of Pathology and Laboratory Medicine (B.Z., D.Y.H.), Division of Endocrinology and Metabolism (G.Z., J.A.F.), University of Cincinnati, and Department of Pathology, Children’s Hospital Medical Center (D.P.W.), Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: James A. Fagin, M.D., Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, Ohio 45267. E-mail: faginja{at}uc.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin promoter) and
wild-type controls were injured mechanically with an epon resin probe.
After 7 and 14 d, a progressive increase in medial area was seen
in both SMP8-IGF-I and wild-type mice, but they were not significantly
different from each other. However, by 14 d there was a more than
4-fold increase in neointimal area in transgenic vs.
wild-type. The intima/media ratios were also strikingly increased at
14 d in the IGF-I-overexpressing animals. The mitotic index,
determined in animals injected daily with bromodeoxyuridine for 3
d before death, was markedly elevated in both the media and neointima
7 d after injury in SMP8-IGF-I mice, but the effect had subsided
by 14 d. Despite a higher rate of cell division, the relative
increase in medial area was less in the SMP8-IGF-I mice than in
wild-type mice at both 7 and 14 d, consistent with a stimulation
of cell migration to the neointima. The experiments reported here
provide compelling evidence that paracrine expression of IGF-I is a
powerful stimulus for smooth muscle cell proliferation and migration
in vivo. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGF-I, a small polypeptide with structural homology to IGF-II and
proinsulin, is produced by many cell types and acts as an
autocrine/paracrine growth factor. It has been postulated to play a
role in the regulation of growth of SMC of the bladder, uterus, and
vasculature. The cooperative effects of platelet-derived growth factor
(PDGF) and IGF-I in inducing aortic SMC proliferation have been
demonstrated (6). Using embryonic fibroblast cell lines
derived from mice with targeted disruption of the type I IGF receptor
(IGF-IR), Sell et al. observed that about 60% of the
proliferative capacity of serum or PDGF is dependent on autocrine
production of IGF-I (7). Furthermore, integrity of the
IGF-IR is obligatory for the PDGF-dependent increase in proliferating
cell nuclear antigen mRNA levels in replicating cells (8).
The critical need for IGF-IR signaling was further demonstrated by the
fact that IGF-IR-null fibroblasts could not be transformed by the
simian virus 40 (SV40) large T antigen (9). Until
recently, information on the paracrine effects of IGF-I in SMC tissue
beds in vivo was conjectural and based primarily on
descriptions of the regulation of IGF-I gene expression in association
with events that trigger smooth muscle hyperplasia or hypertrophy. For
instance, there is a marked induction of IGF-I gene expression in the
medial layer of the rat aorta after balloon arterial injury, coincident
with the peak time of SMC DNA synthesis (3, 4, 5). Smooth
muscle hypertrophy secondary to partial urethral ligation is associated
with increased IGF-I biosynthesis in the bladder wall
(10). Estrogen and progesterone induce IGF-I and IGF-II
mRNA levels in the uterus, consistent with the idea that these growth
factors may help mediate sex steroid-dependent endometrial
proliferation and perhaps myometrial hypertrophy (11, 12, 13).
Direct evidence for a role of paracrine expression of IGF-I in SMC
growth was shown in transgenic (TG) mice selectively overexpressing
IGF-I in SMC by means of a mouse smooth muscle -actin promoter
(14). These mice exhibit marked overexpression of IGF-I in
all smooth muscle-rich tissues (i.e. bladder, stomach,
intestine, uterus, and aorta), where the growth factor promotes a
striking degree of hyperplasia without affecting plasma IGF-I levels or
total body weight.
A functional role for IGF-I in vascular injury has been proposed based on several lines of evidence. Treatment of rats with the long-acting somatostatin analog octreotide evoked a dose-dependent decrease in neointima/media ratios after balloon injury of femoral arteries (15). Octreotide acts primarily by inhibiting GH secretion and, hence, IGF-I gene expression in liver and peripheral tissues. However, at the doses and schedules used in these experiments, plasma GH and IGF-I (and glucagon) levels were not affected by octreotide. By contrast, femoral artery IGF-I mRNA levels were markedly decreased and failed to increase after arterial injury. The impairment of arterial IGF-I gene expression was selective, as induction of PDGF-A mRNA was unaffected by octreotide treatment (15). Hayry et al. (5) demonstrated that treatment of rats with a stable peptide analog of IGF-I designed to serve as an IGF-IR antagonist decreased [3H]thymidine labeling and intimal thickness after carotid injury. Taken together, these studies indicate that IGF-I may be a significant mediator of SMC proliferation after arterial injury. However, there are alternative explanations for the results described above, which provided the impetus to test the hypothesis that paracrine overexpression of IGF-I in the arterial wall is sufficient to modulate the way in which vascular SMC respond to mechanical injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-actin promoter (SMP8) cloned
upstream of a rat IGF-I cDNA. Expression of the transgene was robust
and was found exclusively in smooth muscle-rich tissues postnatally
(artery, vein, bladder, uterus, and myocyte layer of the
gastrointestinal tract). IGF-I mRNA levels in these tissues
(i.e. aorta and bladder) were equivalent or greater than
endogenous IGF-I mRNA levels in liver. IGF-I expression in SMP8-IGF-I
mice remained entirely paracrine, as there was no increase in plasma
IGF-I levels. SMP8-IGF-I mice had arterial SMC hyperplasia, and the
medial area was increased by about 20% (14, 16). All
animal experimentation protocols were performed under the guidelines of
animal welfare by the University of Cincinnati, in accordance with NIH
guidelines.
Carotid artery injury
Mechanically induced endothelial denudation was performed
as described previously (17). Briefly, an epon resin probe
made by forming epon beads slightly larger than the diameter of the
carotid artery (0.45 mm) on a 3-0 nylon suture was used for the
arterial injury. The animals were anesthetized by ip injection with a
solution composed of ketamine (80 mg/kg; Fort Dodge Laboratories, Inc.,
Fort Dodge, IA) and xylazine (16 mg/kg; The Butler Co., Columbus, OH)
diluted in 0.9% NaCl. The mice were immobilized, and the fur covering
the neck from sternum to chin was removed with lotion hair remover.
Surgery was carried out using a dissection microscope (GZ6, Leica Corp., Buffalo, NY). The entire length of the left carotid
artery was exposed. The distal bifurcation of the carotid artery was
looped proximally and ligated distally with 7-0 silk suture (Ethicon,
Somerville, NJ). A transverse arteriotomy was made between the 7-0 silk
sutures, and the resin probe was inserted, advanced toward the
aortic arch, and withdrawn five times. The probe was removed, the
proximal 7-0 suture was ligated, a 6-0 suture was secured, and the
incision was closed with 5-0 sterile surgical gut (Ethicon). All of
these procedures were performed within 20 min. Animals were allowed to
recover in a 37 C heat box. An identical surgical procedure was applied
by the same operator to each animal to assure reproducibility of the
results.
Tissue preparation and histological staining
After the indicated times after arterial injury, animals
were anesthetized and perfused with 0.9% NaCl by placement of a
22-gauge butterfly angiocatheter in the left ventricle. The mice were
subsequently perfusion fixed in situ by infusion with 10%
buffered formalin (pH 7.0) for 20 min at a constant pressure of 100 mm
Hg. The entire neck was dissected from each mouse and fixed in 10%
buffered formalin for an additional 48 h. The whole neck was
decalcified for 48 h before embedding in paraffin. Identical
whole-neck cross sections of 5 µm were made from the distal side of
the neck beginning at the point of the distal 7-0 ligature. Whole neck
sections were used to evaluate both the injured and the uninjured
control vessels on the same section. For each mouse, four levels of
serial sections were taken at 500-µm intervals (Fig. 1A
). Parallel sections were subjected to
routine hematoxylin and eosin staining as well as to Verhoeff
Van-Gieson (VVG) staining of the elastic lamina. Four unstained
sections from each level were used for immunohistochemistry.
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Immunohistochemistry
All sections were deparaffinized with xylene by incubating for
10 min three times and then were dehydrated with a series of graded
ethanol baths from 70–100% for 10 min each time. Slides were washed
in distilled water for 5 min, and endogenous peroxidase activities were
blocked by incubation for 30 min with 0.5% hydrogen peroxide in PBS
containing Triton X-100. Slides were washed three times in the same
solution without H2O2 for
15 min each. Nonspecific binding sites were blocked by incubation for
30 min with 1.5% serum in PBS containing Triton X-100. For anti-smooth
muscle -actin staining, sections were then incubated overnight at 4
C with anti-smooth muscle -actin (clone 1A4, Sigma, St.
Louis, MO) at 1:3000 dilution. The slides were washed three times for
15 min each time with PBS containing Triton X-100 and then were
incubated for 1 h at room temperature with 0.5% biotinylated
antimouse IgG (Vector Laboratories, Inc., Burlingame, CA)
in the same solution containing 1.5% normal serum. Slides were washed
as described above, and then incubated with the avidin-peroxidase
complex reagent (peroxidase Vectastain Elite ABC Kit,
Vector Laboratories, Inc.) for 1 h at room
temperature. The sections were visualized with 3,3'-diaminobenzidine.
The slides were counterstained with Nuclear Fast Red (Zymed Laboratories, Inc., South San Francisco, CA). Two different
monoclonal antibodies, HUC-1 and B4 (provided by Dr. James Lessard,
Children’s Hospital, Cincinnati, OH), directed against separate
epitopes of -actin and one monoclonal antibody against smooth muscle
myosin heavy chain (courtesy of Dr. Muthu Periasamy) were also used in
the staining experiments described above. For mouse monoclonal
antibromodeoxyuridine (anti-BrdU) staining (clone BU33,
Sigma; diluted 1:300), sections were pretreated by
incubation with 4 M HCl for 30 min at 37 C and neutralized
in 0.2 M borate buffer, pH 9.0. After a 15-min PBS/Triton
X-100 wash, sections were further incubated with 0.1% trypsin for 30
min at 37 C, followed by blocking endogenous peroxidase and nonspecific
binding sites as described above. The reaction was visualized using the
Vectastain Elite ABC kit as described above.
In situ hybridization
In situ hybridization was performed as
previously described (14). Briefly, injured and uninjured
carotid arteries dissected from mice 2 or 14 d after injury were
fixed in 4% paraformaldehyde, saturated overnight with 30% sucrose in
PBS, and frozen in OCT (Miles, Elkhart, IN). Cryostat sections (7 µm)
were mounted on silane-coated slides. The following sense and antisense
cRNA probes were used: rat IGF-I, which hybridizes to both endogenous
mouse and TG rat IGF-I mRNA; transgene-specific SV40 3'-untranslated
region/poly(A) signal sequence, that recognizes only the
transgene-derived IGF-I mRNA, and smooth muscle -actin. Probes were
labeled with [35S] UTP, using a
commercially available kit (Stratagene, La Jolla, CA). The
strategy for generation of rat IGF-I and SV40 probes was previously
described (14). The smooth muscle -actin sense and
antisense probes were obtained from pSMAA-1 and pSMAA-6 plasmids,
respectively, which were provided by Dr. James Lessard (Children’s
Hospital of Cincinnati, OH). Both plasmids were linearized with
BamHI and transcribed with T7 RNA polymerase to generate the
sense and antisense probes. Hybridization was performed with a total of
5 x 105 to 1 x
106 cpm in a final volume of 30 µl/slide. The
sections were hybridized overnight at 42 C, treated with 50 µg/ml
ribonculease A and 100 U/ml of RNase T1 for 30 min at 37 C, and washed
with 0.1 x SSC (standard saline citrate) at 50 C. Slides were
dipped in NTB2 emulsion, diluted 1:1 with 0.6 mol/liter ammonium
acetate, exposed for 10–14 d, and developed in D19 developer. Sections
were counterstained in hematoxylin and eosin and photographed under
dark- and brightfield illumination.
Statistical analysis
All values were expressed as the mean ± SEM.
When only two groups (injured arteries and contralateral control
arteries) were compared, differences were assessed by paired
t test. Multiple comparisons were first tested by ANOVA.
When the ANOVA demonstrated significant differences, individual mean
differences were analyzed by the Student-Newman-Keuls test. The
statistical software SigmaStat (version 2.0, Jandel Co., San Rafael,
CA) was used. For all statistical analyses, P < 0.05
was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Moreover, the time course of
reendothelialization was comparable in both WT and TG mice (91.11
± 2.41% vs. 94.40 ± 2.16% 10 d after injury,
WT vs. TG, n = 6 and 4, respectively). As arterial
smooth muscle cells are normally quiescent, their proliferation rate is
very low and barely detectable in intact mice. Moreover, even in
SMP8-IGF-I mice, the basal mitotic index was no different from that in
WT controls (Fig. 2). We took advantage
of the carotid artery injury model to study the effects of paracrine
IGF-I on SMC proliferation in vivo. BrdU was injected into
mice daily for 3 d before death in 7-d experiments and for 5
d in animals killed at the 14-d point. Seven or 14 d after injury,
mice were killed, and carotid arteries were prepared and sectioned.
BrdU staining was then performed (Fig. 2A). Total and BrdU-positive
cells in the medial and neointimal areas were counted under the
microscope by a blinded operator. As shown in Fig. 2, the mitotic index
was markedly elevated in both the media and neointima 7 d after
injury in SMP8-IGF-I TG mice compared with WT controls, but the effects
had subsided by 14 d. This supports the notion that locally
expressed IGF-I increases the initial proliferative wave secondary to
arterial injury.
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). After injury, a progressive increase
in medial area was observed in both WT and SMP8-IGF-I TG mice, which
were not significantly different from each other (Fig. 3A). However,
the relative increase in medial area in the injured arteries compared
with that in uninjured vessels was greater in WT mice, especially
14 d after injury (Fig. 3B). Thus, although IGF-I increased smooth
muscle cell proliferation in the media, this did not result in a
greater expansion of this vascular compartment. This can be explained
if IGF-I simultaneously stimulated the migration of smooth muscle cells
from the media to the neointima. Indeed, as shown in Fig. 3C, the
neointimal area was increased by about 4-fold in SMP8-IGF-I TG mice
compared with controls 14 d after injury. The intima to media
ratio was also markedly increased in IGF-I-overexpressing mice 14
d after injury compared with that in controls, whereas at 7 d
there was no significant difference (Fig. 3D). In addition to the
increase in neointimal formation after arterial injury, luminal
stenosis was more severe in the SMP8-IGF-I TG mice 14 d after
injury (Fig. 3E).
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-actin
expression after injury. A greater accumulation of cells in the
neointima 14 d after injury was seen in SMP8-IGF-I compartments
with WT vessels (Fig. 4, top two panels). VVG staining of
the elastin layer helps to demarcate the vascular components and
confirms the profound increase in neointimal formation in the
IGF-I-overexpressing animal (Fig. 4, bottom two panels).
Immunohistochemical analysis with smooth muscle-specific -actin
antibodies and smooth muscle myosin heavy chain antibodies confirms
that the cells accumulating in the neointima in both WT and TG mice
after injury are smooth muscle cells (Fig. 5; data not shown). However, the medial
layer of the carotid artery from the TG mice showed almost complete
absence of -actin staining compared with WT controls after injury
(Fig. 5). A decrease in smooth muscle -actin expression has been
previously observed after injury (18, 19, 20). To determine
whether smooth muscle -actin gene expression is also decreased in
injured TG vessels, in situ hybridization was performed on
injured and uninjured carotid arteries at 2 and 14 d using a
smooth muscle -actin cRNA probe. Smooth muscle -actin mRNA was
dramatically reduced in the medial layer of the vessels 14 d after
injury compared with vessels 2 d after injury in TG mice (Fig. 6, G and H). By contrast, cells in the
neointima showed abundant smooth muscle actin mRNA at 14 d
(Fig. 6H). Our immunohistochemical data support the idea that IGF-I
results in a more profound loss of differentiated gene expression in
these injured vessels. Indeed, staining with monoclonal antibodies
directed against separate epitopes of -actin and with an antibody
against smooth muscle myosin heavy chain supported these data (not
shown).
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-actin promoter, we examined whether the decrease in smooth muscle
-actin gene expression in the medial layer would also result in
lower transgene expression. Using both a probe for total IGF-I mRNA as
well as a transgene-specific probe complementary to the SV40 poly(A)
signal sequence, we found that IGF-I mRNA was dramatically reduced in
the medial layer of the carotid arteries from TG mice 14 d after
injury (Fig. 6, D and F) compared with vessels from TG mice 2 d
after injury (Fig. 6C). Thus, expression of the IGF-I transgene was
absent in the media, but was highly abundant in the neointima after
14 d, coincident with the changes in expression of smooth muscle
-actin and presumably with the activity of the smooth muscle
-actin promoter. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we used an epon resin probe on a 3-0 nylon suture to induce arterial injury in mice. By Evans blue staining, we demonstrated that the resin probe introduced a consistent denudation of endothelium without damage to the elastic lamina. Thus, this procedure is appropriate to evaluate neointimal hyperplasia as a consequence of endothelial denudation with minimal trauma to underlying medial SMC. Furthermore, the increase in the proliferation of SMC in both media and neointima 7 or 14 d after resin probe injury is consistent with that reported in other murine vascular injury models (5, 18).
The present study demonstrated that overexpression of IGF-I in SMC
evoked greater neointimal formation after carotid artery injury. The
mitotic rate of both medial and neointimal SMC was markedly increased
in SMP8-IGF-I mice 7 d after injury, consistent with enhancement
of SMC proliferation by IGF-I in vivo. Notably, the effect
of IGF-I on SMC proliferation subsided by 14 d. We excluded the
possibility that an accelerated rate of reendothelialization may have
taken place in the TG mice, which could have potentially explained the
decline in the SMC mitotic index at 2 wk (24). It is
possible that augmentation of the initial wave of replication in
SMP8-IGF-I mice is due to the release of high levels of IGF-I stored in
the vessel wall at the time of injury. We reported that the tissue
IGF-I concentration in the aorta of SMP8-IGF-I TG mice is about 4-fold
greater than that in WT controls (14). Most arterial IGF-I
is probably bound to one of the IGF-binding proteins, in rodents mainly
IGF-binding protein-4, and is liberated at the time of injury through
activation of a specific IGFBP protease (Smith, E. P.,
submitted). An alternative interpretation is that the down-regulation
of SM -actin expression after injury through decreased activity of
the exogenous -actin promoter within the media (25)
resulted in lower IGF-I transgene expression at later time points after
intervention in this vascular compartment, resulting in a lower mitotic
rate.
Smooth muscle myosin heavy chain immunostaining was also markedly
decreased in the media, which may indicate a general effect on the
differentiated state of these cells. Indeed, down-regulation of
endogenous -actin abundance appears to be more pronounced in
arteries from SMP8-IGF-I mice. Thus, although paracrine overexpression
of IGF-I in the artery does not by itself diminish the expression of
-actin (16), in the setting of injury, it appears to
accentuate the effects of other dedifferentiating stimuli. Notably,
cells in the neointima regained the expression of both smooth muscle
-actin (as demonstrated by in situ hybridization and
immunohistochemical staining) and smooth muscle myosin heavy chain.
These cells are therefore phenotypically smooth muscle-like. Whether
they migrated from the media or regained differentiated function after
migration cannot be addressed by this study.
Despite a higher mitotic rate in the media of SMP8-IGF-I arteries, the
relative increase in medial area after injury was not significantly
greater in the TG animals compared with the WT controls. A likely
explanation is that IGF-I, besides stimulating a wave of cell
proliferation, also induced cells to migrate to the neointima. IGF-I
has been reported to increase vascular SMC migration in
vitro through mechanisms that require ligand occupancy of the
Vß3 integrin receptor.
This phenomenon may be operating in vivo, as
Vß3 inhibitors reduce
the size of atherosclerotic lesions in pigs through a mechanism that
may include inhibition of IGF-I-induced migration of SMC (26, 27).
The observation that the stimulation of DNA synthesis by paracrine IGF-I expression is not associated with a disproportionate increase in the medial compartment after injury could also be accounted for, at least in theory, through concomitant activation of cell death. Apoptotic cell death of SMC appears to play a significant role in vascular remodeling during atherogenesis and arterial injury (28, 29, 30, 31). However, IGF-I functions as a survival factor for many cell types in vitro, including neurons (32), skeletal myoblasts (33), fibroblasts (34), and arterial SMC (35). IGF-I and PDGF inhibit cell death induced by c-myc in serum-deprived fibroblasts (28) and by serum deprivation in human arterial SMC (36). The contribution of individual growth factors to the modulation of apoptosis in SMC in vivo has not been tested. Moreover, terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling assays of sections of injured arteries from SMP8-IGF-I and WT mice 14 d after injury failed to show any significant differences between the groups (data not shown) (29). Finally, we cannot exclude that physical differences in the baseline characteristics of the respective vessels (elasticity and changes in luminal area) resulted in altered responses to injury.
In conclusion, targeted overexpression of IGF-I in arterial SMCs of TG mice is associated with increased neointimal formation after carotid injury. This results from an IGF-I-induced increase in SMC proliferation and, in all likelihood, in cell migration. These effects take place despite the fact that IGF-I in plasma is equally abundant in WT and SMP8-IGF-I animals. Although the endothelial layer is disrupted by the intervention, presumably allowing circulating IGF-I to access the arterial media, local overproduction of IGF-I within the vessel wall is sufficient to markedly modify the response to injury, supporting a direct role of paracrine IGF-I in this process.
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Acknowledgments |
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Footnotes |
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1 These two investigators contributed equally to this study and should
be considered as joint first authors. 
Abbreviations: BrdU, Bromodeoxyuridine; IA, intimal area; IELA, area inside the internal elastic lamina; IGF-IR, type I IGF receptor; PDGF, platelet-derived growth factor; SMC, smooth muscle cells; SV40, simian virus; VVG, Verhoeff Van-Gieson; WT, wild-type.
Received December 29, 2000.
Accepted for publication April 16, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Vß3 inhibitors is
associated with inhibition of insulin-like growth factor-I-mediated
signaling. Circ Res 85:1040–1045vß3 integrin
blockade potently limits neointimal hyperplasia and lumen stenosis
following deep coronary arterial stent injury: evidence for the
functional importance of integrin
vß3 and osteopontin
expression during neointima formation. Cardiovasc Res 36:408–428This article has been cited by other articles:
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 L. A. Maile, J. Badley-Clarke, and D. R. Clemmons The Association between Integrin-associated Protein and SHPS-1 Regulates Insulin-like Growth Factor-I Receptor Signaling in Vascular Smooth Muscle Cells Mol. Biol. Cell, September 1, 2003; 14(9): 3519 - 3528. [Abstract] [Full Text] [PDF]
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 H. Li, P. Dimayuga, M. Yamashita, J. Yano, M. Fournier, M. Lewis, and B. Cercek Arterial Injury in Mice with Severe Insulin-Like Growth Factor-1 (IGF-1) Deficiency Journal of Cardiovascular Pharmacology and Therapeutics, December 1, 2002; 7(4): 227 - 233. [Abstract] [PDF]
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 J. L. Hall, G. H. Gibbons, and J. C. Chatham IGF-I promotes a shift in metabolic flux in vascular smooth muscle cells Am J Physiol Endocrinol Metab, September 1, 2002; 283(3): E465 - E471. [Abstract] [Full Text] [PDF]
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D. G. Kuhel, B. Zhu, D. P. Witte, and D. Y. Hui Distinction in Genetic Determinants for Injury-Induced Neointimal Hyperplasia and Diet-Induced Atherosclerosis in Inbred Mice Arterioscler. Thromb. Vasc. Biol., June 1, 2002; 22(6): 955 - 960. [Abstract] [Full Text] [PDF]
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B. Zhu, C. A. Reardon, G. S. Getz, and D. Y. Hui Both Apolipoprotein E and Immune Deficiency Exacerbate Neointimal Hyperplasia After |
) and injured (
) carotid arteries from SMP8-IGF-I and WT mice.
The medial area (A) was calculated as the area encircled by the
external elastic lamina minus the area encircled by the internal
elastic lamina. The fold increase in medial area (B) was calculated as
the ratio of the medial area from injured to uninjured vessels. The
neointimal area (C) was determined by subtracting the luminal area from
the area encircled by the internal elastic lamina. The intima to media
ratio (D) was determined based on the data in B and C. Luminal stenosis
(E) was calculated as the percentage of the area inside the internal
elastic lamina occupied by the intimal area in the injured carotid
arteries. n = 5 at the 7-d point for each group; n = 8 at the
14-d point for each group. *, P < 0.05.
