© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Fish Oil Supplementation Prevents Neointima Formation in Nonhypercholesterolemic Balloon-Injured Rabbit Carotid Artery by Reducing Medial and Adventitial Cell Activation
From the Department of Experimental and Clinical Medicine (E.F., M.P., P.P.) and the Department of Biomedical Sciences (A.C., R.F., S.S.), University of Padua, and the CNR Unit for Muscle Biology and Physiopathology (S.S.), Padua, Italy.
Correspondence to Saverio Sartore, PhD, Department of Biomedical Sciences, University of Padua, Viale G. Colombo, 3, I-35121 Padua, Italy. E-mail sartore{at}civ.bio.unipd.it
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Abstract—We asked whether balloon-injured neointima formation in the presence of high/low serum cholesterol (CT) levels might be affected by dietary supplementation with fish oil (FO). To test this hypothesis, we examined the differentiation, proliferation, or apoptosis profile of smooth muscle cell (SMC) and adventitial cell response to a mild injury induced via a Fogarty catheter in the carotid artery of adult rabbits that had been fed a standard chow or 0.5% CT-enriched diet starting 4 weeks before the lesion. One week before surgery, animals received FO supplementation. This regimen was continued for the following 3 weeks. The effect of FO on the early proliferative/migratory response of carotid SMCs was also examined in 2- and 7-day–injured normocholesterolemic rabbits. As controls, animals subjected to 3-week endothelial injury and animals kept on a 7-week CT diet were used. Carotid cryosections from the various animal groups were evaluated for morphometry (image analysis), differentiation (immunofluorescence with monoclonal antibodies specific for smooth muscle markers, ie, myosin isoforms, SM22, and fibronectin), proliferation (bromodeoxyuridine incorporation), and apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling). FO treatment significantly reduced the development of intimal thickening in normocholesterolemic rabbits but had no efficacy in the presence of relatively higher serum CT levels. At day 2 (adventitia) and day 7 (neointima, media, and adventitia), the proliferation index of SMCs in FO-treated injured rabbits was markedly lower than in untreated injured controls. Concomitantly with the antiproliferative effect, FO was able to decrease the size of 2 cell types involved in the cell growth response to endothelial injury, namely, the "fetal-type" medial SMC subpopulation and the fibroblast-derived adventitial myofibroblasts. Thus, in our experimental conditions, a low CT level is a permissive condition for FO to prevent neointima formation to a considerable extent. This event is attributable to the early postinjury effect of FO on SMC/adventitial cell proliferation/differentiation patterns.
Key Words: smooth muscle cells • adventitia • fish oil • endothelial injury • atherogenesis
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Introduction |
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The low occurrence rate of coronary heart disease in fish-consuming populations has raised the possibility that an increased consumption of the n (
)-3 polyunsaturated fatty acids
(PUFAs) might be able to decrease the development of
atherosclerosis in the vascular
system.1 2 3 4 5 Despite the expectancy raised by
epidemiological studies,3 5 contrasting results have been
reported on the efficacy of FO as an antiatherogenic agent (in
experimental animals6 7 8 9 10 11 12 13 14 15 16 17 ) and antirestenotic drug
(in clinical trials18 19 20 21 22 23 24 25 26 ). The experimental studies
published so far focused almost exclusively on the regressive capacity
of PUFA/FO on atherosclerotic lesions in different models (rabbit,
swine, nonhuman primates) and conditions (composition, dose, time of
application, duration of PUFA/FO treatment, different ratio of
polyunsaturated versus saturated fatty acids, isocaloric or
nonisocaloric fatty acid substitutions). There are few data concerning the preventive effect of FO on neointima formation after endothelial lesion.27 Given that the wide array of effects consequent to PUFA/FO supplementation are all pertinent to the initial phase of atherogenesis, we examined the effect of FO dietary supplementation on the early response of vessel wall to endothelial injury. In this investigation, particular emphasis is given to the parietal cell types to which previous and recent studies28 29 30 assign a key role in neointima formation and whose activation occurs soon after lesion: SMCs28 29 30 and adventitial cells.31 32 33 Because medial SMCs are a structurally and functionally heterogeneous population,34 35 36 it might be that FO action is carried out selectively on distinct SMC subtypes, possibly influencing the specific differentiation profile of these cells. It is also known that medial SMCs are phenotypically unstable34 35 36 and are able to modify their structural and functional commitment soon after lesion, in association with the alteration in the proliferation and apoptosis profile and before the migration "wave" to the subendothelial space occurs.29 35 If FO may potentially be able to influence these processes in the media/neointima, it is reasonable to assume that the marine oils may also affect the phenotypic stability of adventitial fibroblasts (ie, the inherent property of these cells of becoming myofibroblasts31 32 33 ) once the wall is damaged. These cells are considered to be a hybrid fibroblast-SMC phenotype or an intermediate cell phenotype between the 2 cell types.32
We have also sought to compare the potential efficacy of FO treatment on injured carotid arteries in the presence of high or low serum CT levels. Because lipid accumulation in the vascular wall is preceded by intimal SMC proliferation37 38 and LDL-CT can alter the in vitro phenotypic39 and the proliferative40 level of SMCs, the possibility exists that this atherogenic risk factor may interfere with the putative antineointima action of FO.27
Using a combined morphometric and immunocytochemical approach, we determined that the efficacy of FO in preventing neointima formation is restricted to the normocholesterolemic rabbit. A possible mechanism through which FO may exert its effect is also delineated in this study, ie, by reducing the 2 arterial wall cell populations that are markedly increased in cell growth response to endothelial injury, the "fetal"-type medial SMCs and the adventitial myofibroblasts.
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Methods |
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Animals
Sixty-two male 3- to 4-month-old New Zealand White rabbits were divided into 6 animals groups (see Figure 1
). A different feeding protocol
(duration, 7 weeks) was applied for the various groups: the standard
chow diet and standard chow diet containing 0.5% CT (CT feeding). A
low-calorie 2RB15 "certificate" standard diet chow containing
2.47% fat and 13.16% protein was purchased from Mucedola. Animals
received FO supplementation starting 1 week before surgery up to the
end of the experiment (week 7). FO supplement (Esapent; Pharmacia &
Upjohn) containing 85% of eicosapentaenoic and
docosahexaenoic acids in the ratio of 
0.9% to 1.5% to 84%,
respectively, was administered by an orogastric gavage device at a
dosage of 0.35 g · kg-1 ·
d-1. At 4 weeks from the beginning of the
experiments, some groups of animals were subjected to
endothelial lesion. To study the early response to wall
damage in the presence or absence of FO supplementation, 2 subgroups of
animals were euthanized on days 2 and 7 after lesion. A control group
was composed of rabbits with an intact carotid artery subjected to a
standard diet and without FO treatment. The rabbits were maintained in
accordance with the recommendations stated in Principles of
Animal Care (NIH publication No. 85-23, revised 1985) and the
Guidelines of the Animal Care Advisory Committee of the
Italian Ministry of Public Health.
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Endothelial Lesion
Animals were anesthetized with sodium pentobarbital (30
mg/kg; sodium pentothal; Abbott Laboratories) and urethan (70 mg/kg)
administered via a marginal ear vein. A 2F Fogarty embolectomy catheter
(Baxter Healthcare Co) was introduced through an aseptic neck incision
produced in the facial branch of the external left carotid artery and
positioned approximately at the origin of the common carotid artery. An
acute balloon injury was performed by inflating the balloon with 0.2 mL
saline solution and then gently pulling it back along the entire length
of the common carotid artery with constant rotation. The procedure was
repeated 3 times. The catheter was then removed, the artery branch
ligated, and the surgical wound closed. The animals were allowed to
recover under observation before being placed in their cages. Injection
of Evans blue dye 3 hours after surgery confirmed the
homogeneous removal of endothelium from
injured carotid artery. Histological examination of
this area revealed a marked structural alteration of SMCs in the
presence of a substantially intact wall organization (not shown).
Because the lesion apparently did not involve disruption of the medial
integrity and a direct contact of the blood with the adventitia, we
define the impact of the lesion on the carotid wall as "mild"
injury.41
At the end of the study period (days 2, 7, and 21), animals were killed under pentobarbital anesthesia. The left common carotid artery and the contralateral vessel were excised with great care to avoid any damage to the adventitial layer. The vessel segments were then perfused with OCT mounting medium (Tissue Tek, Miles Inc) under constant pressure and then inserted into the lumen of the thoracic aorta. The mounted block was immersed in liquid nitrogen and stored at -80°C. The contralateral carotid artery was used as control.
Determination of Serum Lipoproteins
Total serum CT and triglycerides were determined by
the CHOD-PAP method and analyzed with an automatic Hitachi 717
analyzer (Hitachi/Boehringer Mannheim).
Bromodeoxyuridine Labeling
Bromodeoxyuridine (BrdU; 30 mg/kg IP; Boehringer
Mannheim) dissolved in PBS, pH 7.2, was injected 12 and 24 hours before
euthanasia (at 2 and 7 days and 3 weeks).
Morphometric Measurements and Image Analysis
Calculation of tissue areas as well as counting of
BrdU-incorporating cells, fetal-type medial SMCs, adventitial
myofibroblasts, and total cell number were carried out by computerized
image analysis systems. For tissue area calculations,
transverse cryosections 10 µm thick prepared from each carotid
segment were fixed in 1.5% formaldehyde in PBS and then stained with
hematoxylin-eosin. Morphometric analysis of the medial and
intimal areas was assessed in carotid specimens from each animal with a
computerized planimetry system (VIDS V). Measurements were carried out
blinded on 6 sections per animal. The ratio of intimal area/medial area
was taken as index of carotid neointima formation. Data
expressed as mean±SD were compared by 2-way ANOVA. Then statistically
significant data were corrected with the Bonferroni unpaired
t test for direct post hoc comparisons. A probability
of P<0.001 was considered statistically significant.
A detailed blinded quantification of fetal-type medial
SMCs35 and adventitial myofibroblasts32 from
carotid arteries injured on days 2 and 7 (treated or not treated with
FO) (6 sections per animal, 3 microscopic fields for each cryosection)
was performed by counting immunoperoxidase-positive cells for nonmuscle
(NM)-F6 anti–NM myosin and SM-type 
-actin antibody, respectively,
in comparison with the total hematoxylin-positive cell nuclei.
Cryosections were viewed with a high-resolution, charged-coupled device
(CCD), TV video camera (Sony DXC-102P) attached to a Zeiss microscope
(UMSP80) fitted with an x25 objective. The resulting images were
contrast-enhanced, digitalized, and measured with an AT-IBAS Image
Analysis System (Kontron). According to these settings, the
size of the image analyzed was 26 000 µm.2
After image acquisition and contrast enhancement, the thresholding
procedure was applied to measure in each field the total area covered
by negative and positive cytoplasm, respectively. The threshold
cytoplasm antigen level for medial and adventitial cells was evaluated
by use of a control slide in which the primary antibody was omitted in
the immunoperoxidase processing of cryosections. The mean nuclear area
of both positive and negative nuclei was also estimated by measuring
for each sample 15 randomly chosen nuclear profiles. The mean number of
nuclei was then counted for each sample by calculation of the ratio
between the mean total area covered by positive or negative nuclei and
the corresponding mean nuclear area. The mean number of cells was
determined by the same procedure using the mean cellular area
value.
The BrdU-incorporation profile in the various types of carotid arteries was studied by use of an immunoperoxidase protocol and the procedure described above for the AT-IBAS Image Analysis System. Twenty-four separate microscopic fields were measured for each sample. Counting of red (BrdU-incorporating) and of blue-violet+red (total number) nuclei was expressed as mean±SD and normalized to the area unit (mm2). Labeling index (per mm2) expresses the ratio between BrdU-positive nuclei and the total number of nuclei for each animal group.
Statistical analysis of data was carried out by 2-way ANOVA followed, when statistically significant, by Bonferroni’s correction performed with the unpaired t test for direct post hoc comparisons. A probability of P<0.05 was considered statistically significant.
Semiquantitative evaluation of apoptotic cells in the carotid arteries from normocholesterolemic, hypercholesterolemic, and injured animals was determined by counting the yellow terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL)-positive versus the TUNEL-negative nuclei as they appeared after fluorescein labeling. Cryosections were observed with a Zeiss Axioplan microscope equipped with a Hamamatsu CCD camera coupled with a Power Macintosh computer. Images were captured by a Hamamatsu frame grabber and analyzed with Optilab 2.5.1 software.42
Antibodies
The following monoclonal antibodies were used in this study:
SM-E7 anti–SM-type myosin heavy chain (MyHC) and NM-F6 and NM-G2
anti-NM MyHC.35 As previously reported, the SM-E7 reacts
with both SM1 and SM2 SM-type MyHC, whereas NM-G2 and NM-F6 are able to
differently recognize an antigenic epitope localized in the
platelet-type MyHC isoforms MyHC-Apla2 and
MyHC-Apla1,35 respectively. IST-9
anti-fibronectin EIIIA isoform43 (EIIIAFn; a generous gift
of Dr L. Zardi, Istituto Nazionale per la Ricerca sal Cancro, Genoa,
Italy), RAM anti–monocyte-macrophage (Dako, Dakopatts),
and anti–SM-specific E-11 anti-SM2233 44 antibodies.
SM-type -actin was purchased from Sigma Chemical Co, and
anti-vimentin was from Boehringer Mannheim.
Immunocytochemistry
Primary antibodies (except for the E-11) were applied to freshly
cut unfixed cryosections as previously described.33 44 For
anti-SM22 antibodies, cryosections were first fixed in 1.5%
formaldehyde in PBS, pH 7.2.33 44 In these procedures, the
secondary antibody was the anti-mouse IgG coupled with rhodamine
isothiocyanate or horseradish peroxidase. In the latter circumstance,
bound IgGs were revealed by incubation in aminoethylcarbazole solution.
Counterstaining was performed with Mayer’s hematoxylin. Controls were
made by omitting the primary antibody and using nonimmune IgG followed
by the secondary antibody. Nuclei were stained with bis-benzimide
(Hoechst 33258).
BrdU-incorporating nuclei in normal and injured vascular tissue from end-labeling experiments were detected with a specific anti-BrdU antibody (Dako) according to the procedure reported in Reference 4545 . Negative and positive BrdU-incorporating tissues (myocardium and small intestine, respectively) from the same animals as used for BrdU labeling of injured vessels were used as controls.
TUNEL Analysis
Freshly cut cryosections from normal and injured carotid wall
were processed for apoptosis by the TUNEL procedure with the
Boehringer Mannheim kit. The experiments and the negative
control were performed according to the manufacturer’s instructions,
including an incubation with 3% citric acid solution and omitting the
proteinase K pretreatment as recommended by some
authors.46 47 In addition, the cardiac muscle
tissue and a leiomyosarcoma48 were used as alternative
negative and positive controls, respectively.
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Results |
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Effect of FO on Serum Lipids
Rabbits belonging to the various groups examined remained healthy throughout the study period. At the end of the experiments, there was no significant difference among the groups with respect to body weight (average, 3 kg/animal).
The total serum CT and triglyceride levels present in
injured rabbits subjected to the various treatments/feedings is shown
in the Table. There was no
difference between the injured and injured+FO groups with regard to the
CT level, whereas there was a slight, not significant, decrease in the
triglyceride content after FO treatment. Total
CT/triglyceride values in CT-fed animals and injured
animals fed a CT-enriched diet were slightly lower than the
corresponding values obtained after FO supplementation (B+CT+FO group).
As expected, the values shown by the injured animals kept on standard
chow diet are very similar to those of uninjured
normocholesterolemic rabbits.
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Effect of FO on Carotid Lesion Area
Figure 2 shows the results obtained
with the morphometry measurements on cryosections from the various
carotid arteries obtained from the 6 scrutinized animal groups.
Importantly, the segment of carotid artery used in this study did not
show, under normal cholesterol diet regimens,
morphometrically detectable tunica intima (Figures 2 and 3); it
was also selected because it is hardly susceptible to developing
histologically visible atherosclerotic lesions at the
CT feeding condition used here and despite a relatively high serum CT
level (Figure 2 and Reference 4949 ). The intima/media ratio is
significantly lower in injured+FO-treated rabbits than in the injured
animals (0.229±0.032 versus 0.402±0.076). If injured rabbits were fed
a CT diet, the values obtained were very close to those of injured
animals (0.382±0.090 versus 0.402±0.076). In this case, FO treatment
was not able to reverse the higher intima/media ratio (0.400±0.142) to
that obtained with standard diet in the injured animal group. Thus, the
efficacy of FO supplementation on intimal thickening development is
restricted to the animal group kept on a standard diet.
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Effect of FO on SMC Composition 3 Weeks After Lesion
The potential effect of FO on the differentiation pattern of
carotid SMCs was studied by immunophenotyping the expression of SM- and
NM-type MyHC isoforms in the injured vessel wall from
normocholesterolemic rabbits. The combined use of
SM-E7, NM-G2, and NM-F6 anti-myosin antibodies in developing and adult
rabbit arterial SMCs has allowed us to identify 3
categories of SMCs, namely, "fetal" (labeled with SM-E7+NM-F6, ie,
containing SM+MyHC-Apla1 myosin), "postnatal"
(labeled with SM-E7+NM-G2, ie, containing
SM+MyHC-Apla2 myosin), and "adult" (labeled
with SM-E7 only, ie, containing SM myosin exclusively). In this
investigation, we also included 2 other antibodies: IST-9, whose
recognized antigenic target (EIIIA fibronectin), in our model, permits
the identification of "dedifferentiated" SMCs; and E-11, specific
for a SMC lineage–specific marker (the SM22 isoform
complex33 50 51 ).
Figures 3
and 4 show the results obtained with the
immunophenotyping study performed on cryosections from the
normocholesterolemic intact (N group; Figure 3)
and 3 weeks–injured/injured+FO treatment (B/B+FO group) (Figure 4) rabbits. In uninjured carotid artery (see Figure 3),
the majority of cells are adult (Figure 3A), some are postnatal
(Figure 3B), and none are fetal (Figure 3C). SM22 labels
all medial SMCs, although with different intensities (Figure 3D). IST-9 is localized exclusively to the
subendothelial cells (Figure 3E), where the
majority of postnatal-type SMCs are accumulated (Figure 3B).
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In injured carotid arteries, monocyte/macrophage contaminants
are absent from both the neointima and the underlying
media.33 Neointimal SMCs in the injured
vessels are represented by postnatal SMCs (Figure 4A and 4B), being almost negative or weakly positive with NM-F6 (Figure 4C) or E-11 (Figure 4D), respectively. Fetal-type
fibronectin is expressed exclusively in the neointima
(Figure 4E). Compared with the uninjured group, in the media
subjacent to the neointima of carotids from the injured
group there is a marked decrease in the SM22 content (Figure 4D)
and a weaker postnatal-type immunoreactivity (Figure 4B). FO
treatment of the injured carotid does not substantially modify the SMC
immunophenotypic patterns in the media/neointima compared
with the injured vessels.
Effect of FO on SMC Composition 2 and 7 Days After Lesion
Because the possibility exists that the anti–intimal thickening
action of FO may be exerted earlier in the postinjury period, ie, in
close relationship with the proliferation and/or migratory phase of SMC
response to the endothelial injury,52 53
we studied in greater detail the SMC phenotypic pattern at days 2 and 7
from the induction of the lesion. Figure 5 shows the results obtained with
SMC immunophenotyping on carotid cryosections from day 2 injured and
injured+FO-treated rabbits. At this time, a neointima layer
is not yet visible, and the major changes occur at the level of the
media and adventitia (see also Reference 3333 ). In addition, very rare
monocytes/macrophages are present near the internal elastic
lamina, which disappeared after FO treatment (not shown). In injured
media, a large number of fetal SMCs are present (Figure 5A, 5C, and 5E), whereas with FO treatment (Figure 5B, 5D, and 5F),
most SMCs now express the postnatal phenotype. The rare strong
subendothelial SM22-positive cells (Figure 5G and 5H) and the fibronectin-containing cells (Figure 5I and 5J)
have almost disappeared. Similar data are obtained for day 7–injured
animals (not shown). The fact that FO treatment induces a decrease in
the fetal SMCs is documented by the bar graph shown in Figure 6. Quantification of this medial cell
population indicates that this phenotypic change occurs in a
statistically significant manner at approximately day 2 after injury.
These data, along with those presented above, would indicate
that 1 effect of FO treatment is the shift toward a relatively more
differentiated SMC phenotype in the medial layer.
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Effect of FO on Adventitial Cells
Considering the role that adventitial cells might play in our
model, we studied whether the locally activated cells, ie, the
myofibroblasts31 32 33 41 (identified on the basis of
SM-type -actin and vimentin) are distributed differently in injured
FO-treated versus untreated animals. Figure 7 shows that the FO supplementation
induces a marked decrease in the actin-positive myofibroblasts in the
adventitia. Vimentin immunostaining resembles the actin
pattern very closely (not shown). Cell counting of
VD-myofibroblasts54 performed on carotid cryosections from
rabbits injured on days 2 and 7 and injured+FO rabbits confirmed that a
considerable decrease of SM-type -actin–containing cells occurs
after FO treatment in the carotid adventitia (Figure 8).
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Effect of FO on BrdU Incorporation Level
To establish whether the early (2/7 days) phenotypic changes and
the late (3 weeks) morphometric gain occurring with FO treatment in
endothelium-injured carotid wall is related to a
proliferation profile of cells in the carotid media and adventitia, we
performed a BrdU-incorporation study. Figure 9 shows that FO treatment is able to
significantly reduce the number of BrdU-positive cells in adventitia at
both day 2 and day 7 postinjury (from 73.2±38.2 to 27.7±24.5 at day
2; from 9.7±11.4 to 0.0±0.0 at day 7). FO can also decrease the
number of BrdU-reactive cells in the media (from 18.7±13.1 to
1.6±3.1) and neointima (from 48.9±17.5 to 16.3±8.8) at
day 7 after the induction of lesion. The BrdU incorporation levels
attained by neointima and media at 3 weeks after injury is
similar to the control rabbit (N group) reported in Reference 5555 .
Figure 10 shows the tissue-specific
distribution of BrdU-positive cells at days 2, 7, and 21 after injury.
The majority of BrdU-incorporating cells are localized to the
adventitia soon after lesion, whereas at day 7, adventitia is almost
deprived of positive cells in favor of the innermost layer of the media
and the developing neointima. FO treatment markedly reduces
the presence of BrdU-positive cells in all the wall compartments. By 3
weeks after injury, no positive cells can be detected, irrespective of
the experimental condition.
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Effect of FO on Apoptosis Distribution Pattern
Apoptosis distribution in carotid cryosections from
uninjured, injured, and injured+FO-treated animals is shown in Figure 11. In the intact vessel,
TUNEL-positive cells are present almost exclusively in the
adventitia (Figure 11A), whereas at day 2, in both FO-treated
and untreated animals, numerous positive cells are found in the
endothelial layer but not in the medial or adventitial
layer (Figure 11B and 11C). At days 7 (Figure 11D and 11E) and 21 (Figure 11F and 11G) after surgery, <1% of SMCs
are apoptotic, irrespective of the tissue layer or the
treatment.
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Discussion |
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The results of this study indicate that lesion area development is significantly reduced after 3 weeks of FO only in the animal group fed a low-cholesterol diet. In fact, in the injured hypercholesterolemic animal group, the efficacy of FO treatment as lesion antagonist is dramatically lost. We are in concordance with Chen et al,27 who reported that a more severe effect of FO on the myointimal growth in the injured rabbit abdominal aorta is achieved in the presence of low CT levels. Our data are also in keeping with the results obtained by Sassen et al14 and Fincham et al,13 who found that in porcine injured arteries, the retardation in the progression or the lack of regression (ie, stabilization) of atherosclerotic lesions is related to the plasma CT content.
The serum CT-dependent efficacy of FO treatment in
endothelium-injured arterial wall might be
related to the differential cellular assortment of the lesion at low or
high CT level, ie, the number of
monocytes-macrophages/lymphocytes1 2 versus
SMCs33 and/or the amounts of native versus oxidized LDL.
Although in our experimental conditions, FO treatment in injured
rabbits subjected to short-term CT feeding and FO supplementation was
unable to substantially alter the total serum CT level (Table),
it might be that LDL particles accumulated at the site of the lesion
were present in an abnormal quantity or were qualitatively prone to
be chemically modified.56 Oxidized LDL can stimulate
arterial SMC growth indirectly, via induction of growth
factors (eg, platelet-derived growth factor) from the same or other
cells (macrophages and endothelial
cells28 29 ), or directly, via activation of the pathway of
mitogen-activated protein kinases.57
Triglyceride levels did not show appreciable changes in the
animal group treated with FO, in keeping with other
reports.27 58
The study of SMC differentiation has furnished some indications
about the mechanism through which FO acts at low CT concentrations. We
purposely selected a segment of the rabbit common carotid artery that
is histologically unable to develop an atherosclerotic
plaque when subjected to the diet regimen used in this
study.49 In addition, this vessel is relatively enriched
with postnatal-type SMCs35 37 and thus is potentially at
risk for developing SMC growth and migration.35 37
Twenty-one days after injury, in the FO-treated group, there was a
slight increase in the differentiation pattern of both
neointimal and medial SMCs, although a complete maturation
was not achieved (high signal for fetal fibronectin59 and
scarce presence of SM2260 ; see Figure 4). In the
early phase of response to injury (days 2 and 7), the effect of FO
treatment became more evident. Here, both the general organization of
arterial wall and the degree of SMC differentiation were
markedly improved. In particular, the media contained fewer fetal-type
SMCs (Figure 5), which in the injured carotid artery are
particularly high and are directly implicated in neointima
formation35 (Figure 5A, 5C, and 5E). Similarly, the
adventitia from injured–FO-treated artery contained fewer vimentin-
and SM -actin–containing myofibroblasts (the VA
myofibroblasts54 ), which are known to participate in the
medial cell incorporation/neointima
formation.31 32 33
Taken together, these findings are fairly consonant with our previous data about the correlation existing between the reduction of plaque formation induced by calcium channel blockers and the shift toward more differentiated SMCs61 62 63 (both decrease in the fetal-type and increase in the adult-type SMCs).
The change in the phenotypic pattern of medial SMCs induced by FO treatment soon after injury is strictly linked to the modification of proliferation indices in the adventitia and media, but not the apoptotic cell distribution, which remains substantially unaffected compared with the injured carotid artery. We cannot rule out completely the possibility that FO may influence apoptosis, inasmuch as the burst of this event seems to occur within hours after injury.47 Interestingly, a decrease of the adventitial myofibroblast content in FO-treated day 2–injured carotids is mirrored by a marked decrease in the proliferation ability of these cells, whereas the BrdU-incorporation level of the medial SMCs remains unaffected. It is at day 7 postinjury that adventitia, media, and neointima all display a much lower propensity to proliferate on FO treatment. Recent experiments indicate that neointimal SMCs may derive from the dedifferentiated or phenotypically modulated medial SMCs and by recruitment and incorporation of adventitial myofibroblasts (porcine coronary arteries and rabbit carotid artery).28 29 30 31 32 33 Thus, the antiproliferative effect of FO treatment on the injured carotid seems to be dual, affecting both medial and adventitial cells. In particular, the reduction of proliferative index exerted by FO on adventitial cells suggests that the local cell conversion pathway from fibroblast to myofibroblast,31 32 33 41 as part of the response-to-injury process, may be controlled by FO.
Although the results of this study might have important implications for the still-debated use of FO as an antirestenotic agent, some caution must be taken. First, the dose used is certainly not in the range of that used up to now in human studies. Second, the different lipid metabolism in rabbit versus human does not allow any firm conclusions about the real efficacy of FO as an antiatherogenic or antistenotic agent. Third, the relatively short period of FO pretreatment that precedes the cellular wall response triggered by the endothelial lesion might have led to an underestimation of FO efficacy. However, on the basis of the data presented here and those of Chen et al,27 we are in a position to suggest that relatively high doses of FO might have a short-term beneficial effect on restenosis soon after angioplasty. Certainly, this hypothesis needs to be preliminarily tested in experimental settings in which FO treatment is applied to animals with a well-developed atherosclerotic plaque at the time of angioplastic intervention.
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Acknowledgments |
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This work was done in part under grants from Ministero dell Universita edella Ricerca Scientifica Technologica and the Biomedical Association for Vascular Research of Padua, Italy. We wish to thank Marissa Galliani Sanavio for her excellent editorial assistance and Dr Diego Guidolin (Fidia Research Laboratories, Abano) for his help with the image analysis measurements.
Received September 28, 1998; accepted April 15, 1999.
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References |
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